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Translocation of Connexin 43 to the Inner Mitochondrial Membrane of Cardiomyocytes Through the Heat Shock Protein 90–Dependent TOM Pathway and Its Importance for Cardioprotection
Abstract
We have previously shown that connexin 43 (Cx43) is present in mitochondria, that its genetic depletion abolishes the protection of ischemia- and diazoxide-induced preconditioning, and that it is involved in reactive oxygen species (ROS) formation in response to diazoxide. Here we investigated the intramitochondrial localization of Cx43, the mechanism of Cx43 translocation to mitochondria and the effect of inhibiting translocation on the protection of preconditioning. Confocal microscopy of mitochondria devoid of the outer membrane and Western blotting on fractionated mitochondria showed that Cx43 is located at the inner mitochondrial membrane, and coimmunoprecipitation of Cx43 with Tom20 (Translocase of the outer membrane 20) and with heat shock protein 90 (Hsp90) indicated that it interacts with the regular mitochondrial protein import machinery. In isolated rat hearts, geldanamycin, a blocker of Hsp90-dependent translocation of proteins to the inner mitochondrial membrane through the TOM pathway, rapidly (15 minutes) reduced mitochondrial Cx43 content by approximately one-third in the absence or presence of diazoxide. Geldanamycin alone had no effect on infarct size, but it ablated the protection against infarction afforded by diazoxide. Geldanamycin abolished the 2-fold increase in mitochondrial Cx43 induced by 2 preconditioning cycles of ischemia/reperfusion, but this effect was not associated with reduced protection. These results demonstrate that Cx43 is transported to the inner mitochondrial membrane through translocation via the TOM complex and that a normal mitochondrial Cx43 content is important for the diazoxide-related pathway of preconditioning.
... Cx43 has been described to be also located at the inner mitochondrial membrane of cardiomyocyte subsarcolemmal mitochondria (SSM), but not in interfibrillar mitochondria (IFM) [6,10,37], where it forms hemichannels that control K + fluxes [11,33] and modulate respiratory complex I activity and, importantly, ROS production [9,25]. The presence of Cx43 at SSM is suggested to be crucial for preconditioning protection [42,45], an effect that might be influenced by casein kinase 1-dependent phosphorylation of the protein [28]. ...
... The protective effect of pharmacological preconditioning with diazoxide is very sensitive to the endogenous levels of Cx43; thus, it turned out to be ineffective also in hearts from heterozygous Cx43KI32 mice [38] and Cx43 Cre−ER(T)/ fl animals [43], in which ischemic preconditioning still had a protective effect, whereas both pharmacologic and ischemic preconditioning failed to show protection in homozygous animals [38,43]. Further supporting a role for mitochondrial Cx43 in preconditioning protection, it was demonstrated that the pharmacological inhibition of Cx43 translocation to the mitochondria ablated protection by diazoxide [37]. Similarly, the age-dependent reduction of Cx43 content (both in sarcolemma and mitochondria) could underlie the loss of preconditioning protection in aged mouse hearts [7,8]. ...
... Considering that Cx43 is located at the inner mitochondrial membrane of SSM [6,10,37], where it significantly influences ROS production [9,25], along with evidence demonstrating that an almost complete deletion of the protein provides an extensive protection against IR injury [43], difficult to attribute exclusively to reduced cell-to-cell propagation of injury through gap junctions [20], here we aimed to assess whether additional mechanisms, and specifically RET, could be modulated by the presence of Cx43 at the mitochondria. Moreover, we also investigated the potential mechanism involved in RET modulation, including changes in succinate concentrations at the end of ischemia, as well as mitochondrial membrane potential Δψ, coenzyme Q (CoQ) levels and its redox state. ...
Succinate accumulates during myocardial ischemia and is rapidly oxidized during reperfusion, leading to reactive oxygen species (ROS) production through reverse electron transfer (RET) from mitochondrial complex II to complex I, and favoring cell death. Given that connexin 43 (Cx43) modulates mitochondrial ROS production, we investigated whether Cx43 influences RET using inducible knock-out Cx43Cre−ER(T)/fl mice. Oxygen consumption, ROS production, membrane potential and coenzyme Q (CoQ) pool were analyzed in subsarcolemmal (SSM, expressing Cx43) and interfibrillar (IFM) cardiac mitochondria isolated from wild-type Cx43fl/fl mice and Cx43Cre−ER(T)/fl knock-out animals treated with 4-hydroxytamoxifen (4OHT). In addition, infarct size was assessed in isolated hearts from these animals submitted to ischemia–reperfusion (IR), and treated or not with malonate, a complex II inhibitor attenuating RET. Succinate-dependent ROS production and RET were significantly lower in SSM, but not IFM, from Cx43-deficient animals. Mitochondrial membrane potential, a RET driver, was similar between groups, whereas CoQ pool (2.165 ± 0.338 vs. 4.18 ± 0.55 nmol/mg protein, p < 0.05) and its reduction state were significantly lower in Cx43-deficient animals. Isolated hearts from Cx43Cre−ER(T)/fl mice treated with 4OHT had a smaller infarct size after IR compared to Cx43fl/fl, despite similar concentration of succinate at the end of ischemia, and no additional protection by malonate. Cx43 deficiency attenuates ROS production by RET in SSM, but not IFM, and was associated with a decrease in CoQ levels and a change in its redox state. These results may partially explain the reduced infarct size observed in these animals and their lack of protection by malonate.
... Several members of the connexin family have been reported in mitochondrial membranes, which include connexin 40 (Cx40) in coronary endothelial cells [37], Cx32 and connexin 26 (Cx26) in liver mitochondria [3,38,39,89,91] and Cx43 in a wide variety of different cells [38,55]. Proof of the presence of connexin in mitochondria has been provided by researchers who used a combination of imaging and molecular biological approaches. ...
... The imaging techniques used were light and super-resolution immunocytochemistry and immunogold transmission electron microscopy [33]. Flow cytometry, Western blot analysis [33,38,91] and subcellular fractionation combined with immunoprecipitation-high throughput mass spectrometry and reciprocal co-immunoprecipitation (co-IP) [38,92] were molecular approaches used to provide proof for the presence of connexin within mitochondria. The most evidence for connexin in the mitochondria, however, comes from the studies of Cx43, the most widely expressed member of the connexin family [93]. ...
... Within the subsarcolemmal mitochondria, Cx43 delivery to the mitochondria requires heat shock protein 90 (Hsp90) and the translocase of the outer membrane 20 (TOM 20) [39,91]. While the orientation of the Cx43 in the outer mitochondrial membrane has not been demonstrated, the orientation of the Cx43 carboxyl-terminal tail and extracellular loop relative to the mitochondrial matrix has been demonstrated. ...
Mitochondria contain connexins, a family of proteins that is known to form gap junction channels. Connexins are synthesized in the endoplasmic reticulum and oligomerized in the Golgi to form hemichannels. Hemichannels from adjacent cells dock with one another to form gap junction channels that aggregate into plaques and allow cell–cell communication. Cell–cell communication was once thought to be the only function of connexins and their gap junction channels. In the mitochondria, however, connexins have been identified as monomers and assembled into hemichannels, thus questioning their role solely as cell–cell communication channels. Accordingly, mitochondrial connexins have been suggested to play critical roles in the regulation of mitochondrial functions, including potassium fluxes and respiration. However, while much is known about plasma membrane gap junction channel connexins, the presence and function of mitochondrial connexins remain poorly understood. In this review, the presence and role of mitochondrial connexins and mitochondrial/connexin-containing structure contact sites will be discussed. An understanding of the significance of mitochondrial connexins and their connexin contact sites is essential to our knowledge of connexins’ functions in normal and pathological conditions, and this information may aid in the development of therapeutic interventions in diseases linked to mitochondria.
... Although other transport mechanisms exist , many mitochondrial IMS proteins are translocated through the TOM complex across the OMM (Kulawiak et al., 2013). Non-mitochondrial proteins are also translocated into mitochondria via the TOM complex pathway (Budas et al., 2010;Fu et al., 2012;Rodriguez-Sinovas et al., 2006). Given that Mb inside mitochondria is localized in the IMS (Koma et al., 2021), it is a possible that Mb uses the TOM complex to transport into mitochondria. ...
... Since Mb is not a mitochondrial protein, it is not certain whether it is recognized by Tom20 or Tom70. When investigating whether a protein is imported into the mitochondria via the TOM complex, the IP technique is initially used to ascertain whether a protein interacts with the receptors (Frank et al., 2015;Mackenzie et al., 2013;Rodriguez-Sinovas et al., 2006;Santos & Kowluru, 2013). Therefore, we performed IP analysis of the mitochondrial fraction to determine if Mb is recognized by Tom20 and Tom70. ...
... Since Tom20 interacts with Tom70 (Fan et al., 2011), it is logical that Mb was detected following IP with either Tom20 or Tom70. Furthermore, several previous studies have suggested that interactions with TOM complex receptors reflect the involvement of the TOM complex in the mitochondrial import of a protein (Mackenzie et al., 2013;Rodriguez-Sinovas et al., 2006;Santos & Kowluru, 2013). Accordingly, we hypothesized that Mb utilizes the TOM complex to enter mitochondria. ...
Recently, we found that myoglobin (Mb) localizes in both the cytosol and mitochondrial intermembrane space in rodent skeletal muscle. Most proteins of the intermembrane space pass through the outer mitochondrial membrane via the translocase of the outer membrane (TOM) complex. However, whether the TOM complex imports Mb remains unknown. The purpose of this study was to investigate the involvement of the TOM complex in Mb import into the mitochondria. A proteinase K protection assay of mitochondria from C2C12 myotubes confirmed that Mb integrated into the mitochondria. An immunoprecipitation assay verified the interaction of Mb and TOM complex receptors (Tom20, Tom70) in isolated mitochondria. The assay showed a clear interaction of Mb with Tom20 and Tom70. A knockdown experiment using siRNA for TOM complex receptors (Tom20, Tom70) and TOM complex channel (Tom40) did not alter the amount of Mb expression in the mitochondrial fraction. These results suggested that Mb does not necessarily require the TOM complex for mitochondrial import of Mb. Although the physiological role of Mb interactions with TOM complex receptors remains unclear, further studies are needed to clarify how Mb enters the mitochondria independently of the TOM complex.
... As a protein containing transmembrane domains it is likely that Cx43 is also present in mitochondrial membranes and the detailed analysis of mitochondria sub-fractionated using digitonin shows the protein to be present in the inner membrane of mitochondria from cardiac, endothelial and stem cell antigen-1 + cells (Rodriguez-Sinovas et al., 2006;Lu et al., 2010;Trudeau et al., 2012;Srisakuldee et al., 2014). However, the use of a different protocol for the mitochondrial sub-fractionation (sonication, freeze/thaw cycles and sucrose gradient centrifugation) identifies Cx43 predominantly in the outer mitochondrial membrane (Goubaeva et al., 2007). ...
... The chaperone heat shock protein 90 (Hsp90) binds unfolded proteins and delivers them to the TOM complex (Zanphorlin et al., 2016). Coimmunoprecipitation experiments demonstrate an interaction between Cx43 and Hsp90 (Rodriguez-Sinovas et al., 2006) and accordingly the inhibition of HSP90 using siRNA or pharmacological agents such as geldanamycin or radicicol reduce the mitochondrial Cx43 content (Rodriguez-Sinovas et al., 2006;Tu et al., 2017;Pecoraro et al., 2021). The interaction between Cx43 and Tom20 suggests the involvement of the TOM complex in the import of Cx43 into mitochondria (Rodriguez-Sinovas et al., 2006). ...
... The chaperone heat shock protein 90 (Hsp90) binds unfolded proteins and delivers them to the TOM complex (Zanphorlin et al., 2016). Coimmunoprecipitation experiments demonstrate an interaction between Cx43 and Hsp90 (Rodriguez-Sinovas et al., 2006) and accordingly the inhibition of HSP90 using siRNA or pharmacological agents such as geldanamycin or radicicol reduce the mitochondrial Cx43 content (Rodriguez-Sinovas et al., 2006;Tu et al., 2017;Pecoraro et al., 2021). The interaction between Cx43 and Tom20 suggests the involvement of the TOM complex in the import of Cx43 into mitochondria (Rodriguez-Sinovas et al., 2006). ...
Connexins are known for their ability to mediate cell-cell communication via gap junctions and also form hemichannels that pass ions and molecules over the plasma membrane when open. Connexins have also been detected within mitochondria, with mitochondrial connexin 43 (Cx43) being the best studied to date. In this review, we discuss evidence for Cx43 presence in mitochondria of cell lines, primary cells and organs and summarize data on its localization, import and phosphorylation status. We further highlight the influence of Cx43 on mitochondrial function in terms of respiration, opening of the mitochondrial permeability transition pore and formation of reactive oxygen species, and also address the presence of a truncated form of Cx43 termed Gja1-20k. Finally, the role of mitochondrial Cx43 in pathological conditions, particularly in the heart, is discussed.
... It has been observedthat the C-terminal tail of Cx43, which includes GJA1-20k, has several phosphorylation sites and that Cx43 phosphorylation regulates Cx43 gap junction formation and stabilization (Laird, 2005;Moreno, 2005;Procida et al., 2009;Rodriguez-Sinovas et al., 2021). In addition, post-translational modification such as phosphorylation and dephosphorylation occurs during ischemia or reperfusion stress (Beardslee et al., 2000;Rusiecka et al., 2020). ...
... It has been reported that full-length Cx43 is localized not only at surrounding cell membrane for formation of intercellular gap junctions but also at the mitochondrial inner membrane, mediated by heat shock protein 90 (HSP90) and TOM pathway (Rodriguez-Sinovas et al., 2006;Rodriguez-Sinovas et al., 2018). At mitochondrial inner membrane, Cx43 could form hemichannels and regulate mitochondrial respiration by modulating potassium ion flux and respiratory complex activity (Miro-Casas et al., 2009;Boengler et al., 2012;Rodriguez-Sinovas et al., 2018). ...
... It has been reported that full-length Cx43 is localized not only at surrounding cell membrane for formation of intercellular gap junctions but also at the mitochondrial inner membrane, mediated by heat shock protein 90 (HSP90) and TOM pathway (Rodriguez-Sinovas et al., 2006;Rodriguez-Sinovas et al., 2018). At mitochondrial inner membrane, Cx43 could form hemichannels and regulate mitochondrial respiration by modulating potassium ion flux and respiratory complex activity (Miro-Casas et al., 2009;Boengler et al., 2012;Rodriguez-Sinovas et al., 2018). ...
Connexin 43 (Cx43) is the primary gap junction protein of mammalian heart ventricles and is encoded by the gene Gja1 which has a single coding exon and therefore cannot be spliced. We previously identified that Gja1 mRNA undergoes endogenous internal translation initiated at one of several internal AUG (M) start codons, generating N-terminal truncated protein isoforms that retain the C-terminus distal to the start site. GJA1-20k, whose translation initiates at mRNA M213, is usually the most abundant isoform in cells and greatly increases after ischemic and metabolic stress. GJA1-20k consists of a small segment of the last transmembrane domain and the complete C-terminus tail of Cx43, with a total size of about 20 kDa. The original role identified for GJA1-20k is as an essential subunit that facilitates the trafficking of full-length Cx43 hexameric hemichannels to cell-cell contacts, generating traditional gap junctions between adjacent cells facilitating, in cardiac muscle, efficient spread of electrical excitation. GJA1-20k deficient mice (generated by a M213L substitution in Gja1) suffer poor electrical coupling between cardiomycytes and arrhythmogenic sudden death two to 4 weeks after their birth. We recently identified that exogenous GJA1-20k expression also mimics the effect of ischemic preconditioning in mouse heart. Furthermore, GJA1-20k localizes to the mitochondrial outer membrane and induces a protective and DRP1 independent form of mitochondrial fission, preserving ATP production and generating less reactive oxygen species (ROS) under metabolic stress, providing powerful protection of myocardium to ischemic insult. In this manuscript, we focus on the detailed roles of GJA1-20k in mitochondria, and its interaction with the actin cytoskeleton.
... Evidence indicates that mtCx43 plays a critical role in regulating mitochondrial function [8][9][10] and apoptosis mediated through the mitochondrial intrinsic pathway via release of cytochrome c [11][12][13] and initiation of caspase-3 dependent cascade [14]. Several studies have reported the presence of Cx43 in the inner mitochondrial membrane (IMM) [10,[15][16][17][18][19], however, their exact function is unclear. Previously we have observed that HG downregulates mtCx43 level concomitant with increased mitochondrial fragmentation and apoptosis in retinal endothelial cells [10]. ...
... Presence of Cx43 in the mitochondria and the subsequent maintenance of mtCx43 level play a critical role in regulating cell death [30]. Studies demonstrate that the full-length Cx43 is present in the IMM where it is translocated from the cytosol [17] and that inhibition of Cx43 translocation to mitochondria and subsequent reduced mtCx43 level accelerates cell death [16] by reducing cellular respiration [17], promoting mitochondrial energy deprivation and triggering apoptosis [18]. Additionally, evidence suggests that reduced mtCx43 level results in cytochrome c release and apoptosis, indicating that maintenance of mtCx43 level could be useful in preventing apoptosis [14]. ...
... Presence of Cx43 in the mitochondria and the subsequent maintenance of mtCx43 level play a critical role in regulating cell death [30]. Studies demonstrate that the full-length Cx43 is present in the IMM where it is translocated from the cytosol [17] and that inhibition of Cx43 translocation to mitochondria and subsequent reduced mtCx43 level accelerates cell death [16] by reducing cellular respiration [17], promoting mitochondrial energy deprivation and triggering apoptosis [18]. Additionally, evidence suggests that reduced mtCx43 level results in cytochrome c release and apoptosis, indicating that maintenance of mtCx43 level could be useful in preventing apoptosis [14]. ...
Diabetic retinopathy (DR) is one of the most common causes of vision loss and blindness among the working-age population. High glucose (HG)-induced decrease in mitochondrial connexin 43 (mtCx43) level is known to promote mitochondrial fragmentation, cytochrome c release, and apoptosis in retinal endothelial cells associated with DR. In this study, we investigated whether counteracting HG-induced decrease in mtCx43 level would preserve mitochondrial integrity and prevent apoptosis. Rat retinal endothelial cells (RRECs) were grown in normal (N; 5 mM glucose) or HG (30 mM glucose) medium for 7 days. In parallel, cells grown in HG were transfected with Cx43 plasmid, or empty vector (EV), as control. Western blot (WB) analysis showed a significant decrease in mtCx43 level concomitant with increased cleaved caspase-3, Bax, cleaved PARP, and mitochondrial fragmentation in cells grown in HG condition compared to those grown in N medium. When cells grown in HG were transfected with Cx43 plasmid, mtCx43 level was significantly increased and resulted in reduced cleaved caspase-3, Bax, cleaved PARP and preservation of mitochondrial morphology with a significant decrease in the number of TUNEL-positive cells compared to those grown in HG alone. Findings from the study indicate a novel role for mtCx43 in regulating apoptosis and that maintenance of mtCx43 level could be useful in preventing HG-induced apoptosis by reducing mitochondrial fragmentation associated with retinal vascular cell loss in DR.
... However, cells respond to hypoxia by complex metabolic reprogramming and molecular mechanisms aimed to minimize the detrimental consequences of oxygen deprivation on mitochondria [2]. Cytoprotective programs activated during hypoxia injury integrate several processes, including the hypoxiainducible factor 1/hypoxia response element pathway [3], translocation of connexin43 (Cx43) to the inner mitochondrial membrane [4] and opening of the mitochondrial ATPregulated potassium (mitoKATP) channels [5]. ...
... Transmembrane Cx43 contributes to cell-cell communication and electrical coupling by the formation of gap junction channels, while on the mitochondrial membrane Cx43 exists as a hemichannel and is involved in mitochondrial volume regulation and respiration [6]. During stress conditions, Cx43 translocates to the mitochondria with a mechanism that involves the Hsp90/Tom20 machinery system [4,7], and many studies conducted using the Hsp90 inhibitor radicicol supported this theory [8][9][10]. Mitochondrial Cx43 (mCx43) acts as an important regulator of apoptosis, allowing the passage of molecules that induce apoptosis such as Ca 2+ , IP3 and cAMP ions [11] and influencing mitochondrial respiration, matrix ion fluxes and ROS production [9]. ...
... The opening of mitoKATP channels reduces the expression of apoptotic markers induced by the ischemia/reperfusion injury [17,18], while the inverse occurs when the same channels are closed [16]. A previous study reports that the pharmacological protection exerted by dzx is abolished when Cx43 translocation on mitochondria is inhibited [4], however, the functional link between mCx43 and KATP channels is not fully established. ...
Hypoxia is the leading cause of death in cardiomyocytes. Cells respond to oxygen deprivation by activating cytoprotective programs, such as mitochondrial connexin43 (mCx43) overexpression and the opening of mitochondrial KATP channels, aimed to reduce mitochondrial dysfunction. In this study we used an in vitro model of CoCl2-induced hypoxia to demonstrate that mCx43 and KATP channels cooperate to induce cytoprotection. CoCl2 administration induces apoptosis in H9c2 cells by increasing mitochondrial ROS production, intracellular and mitochondrial calcium overload and by inducing mitochondrial membrane depolarization. Diazoxide, an opener of KATP channels, reduces all these deleterious effects of CoCl2 only in the presence of mCx43. In fact, our results demonstrate that in the presence of radicicol, an inhibitor of Cx43 translocation to mitochondria, the cytoprotective effects of diazoxide disappear. In conclusion, these data confirm that there exists a close functional link between mCx43 and KATP channels.
... For example, Wattamon recently reported that the protective role of fibroblast growth factor 2 (FGF-2) against calcium overload was partially mediated by mitochondrial connexin 43 (Cx43) (introduced below), probably in a Tom20-dependent manner (209). Cx43 was imported into mitochondria via a Tom20-dependent pathway (257). In another study, Tom20 was reported to be responsible for the direct insertion of VDAC, a protein crucial in regulating cardiac calcium homeostasis (210) through mitochondrial permeability transition pore (mPTP) (294) and mitochondriaassociated endoplasmic reticulum membranes (MAMs), into the OM of heart mitochondria in rats (295). ...
... HSP90, a chaperone mainly located in the cytoplasm, also played a beneficial role against I/R injury (258,259) through translocation of PKCε, (208,217) Cx43, (257,260) AKT, (261), and Pim1/Lon (262) into mitochondria, potentially via the HSP-TOM mitochondrial import pathway. ...
... The translocation of Cx43 to the IM was TOM-HSP90-dependent and was enhanced by ischemic preconditioning (IP). The beneficial role of mitochondrial Cx43 in I/R stress was associated with its regulation of mitochondrial potassium influx and ROS production (257,(313)(314)(315). The cardioprotection of IP was abolished by genetic ablation of Cx43, blockade of mitochondrial Cx43 import, or age-related loss of mitochondrial Cx43 (257,(316)(317)(318). ...
Mitochondria are essential organelles for cellular energy production, metabolic homeostasis, calcium homeostasis, cell proliferation, and apoptosis. About 99% of mammalian mitochondrial proteins are encoded by the nuclear genome, synthesized as precursors in the cytosol, and imported into mitochondria by mitochondrial protein import machinery. Mitochondrial protein import systems function not only as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles. Mitochondrial protein import deficiency is linked to various diseases, including cardiovascular disease. In this review, we describe an emerging class of protein or genetic variations of components of the mitochondrial import machinery involved in heart disease. The major protein import pathways, including the presequence pathway (TIM23 pathway), the carrier pathway (TIM22 pathway), and the mitochondrial intermembrane space import and assembly machinery, related translocases, proteinases, and chaperones, are discussed here. This review highlights the importance of mitochondrial import machinery in heart disease, which deserves considerable attention, and further studies are urgently needed. Ultimately, this knowledge may be critical for the development of therapeutic strategies in heart disease.
... In addition to their location at the plasma membrane, some connexins have been described in other less conventional cell structures, including the nucleus [22] and the mitochondria [23][24][25]. In this review, we will, first, provide an overview on general aspects of connexin biology, including synthesis and degradation, regulation and interac- Intercellular channels put into contact the cytoplasms of connected cells, allowing the transfer of ions and small intracellular molecules between them, in a process known as gap junctional intercellular communication (GJIC) [16]. ...
... In addition to their location at the plasma membrane, some connexins have been described in other less conventional cell structures, including the nucleus [22] and the mitochondria [23][24][25]. In this review, we will, first, provide an overview on general aspects of connexin biology, including synthesis and degradation, regulation and interactions, with especial interest in those isoforms expressed in the heart, followed by an in-depth examination of their involvement in cardiac pathophysiology. ...
... Cx43 at cardiomyocyte mitochondria has been shown to immunoprecipitate and/or colocalize with proteins involved in its translocation to the inner mitochondrial membrane, including Tom20, a member of the TOM/TIM (translocase of the outer membrane/translocase of the inner membrane) import system, and with heat shock protein 90 (Hsp90) [24], but also with others that are essential for mitochondrial function, such as ANT (adenine nucleotide transporter) or the mitochondrial respiratory complex II ( Figure 6) [24,25]. In addition, it interacts with apoptosis-inducing factor (AIF) and with the beta subunit of the electron transfer protein (ETFB), two proteins involved in oxidative phosphorylation and redox control [274]. ...
Connexins are a family of transmembrane proteins that play a key role in cardiac physiology. Gap junctional channels put into contact the cytoplasms of connected cardiomyocytes, allowing the existence of electrical coupling. However, in addition to this fundamental role, connexins are also involved in cardiomyocyte death and survival. Thus, chemical coupling through gap junctions plays a key role in the spreading of injury between connected cells. Moreover, in addition to their involvement in cell-to-cell communication, mounting evidence indicates that connexins have additional gap junction-independent functions. Opening of unopposed hemichannels, located at the lateral surface of cardiomyocytes, may compromise cell homeostasis and may be involved in ischemia/reperfusion injury. In addition, connexins located at non-canonical cell structures, including mitochondria and the nucleus, have been demonstrated to be involved in cardioprotection and in regulation of cell growth and differentiation. In this review, we will provide, first, an overview on connexin biology, including their synthesis and degradation, their regulation and their interactions. Then, we will conduct an in-depth examination of the role of connexins in cardiac pathophysiology, including new findings regarding their involvement in myocardial ischemia/reperfusion injury, cardiac fibrosis, gene transcription or signaling regulation.
... In addition to its localization at the sarcolemma, Cx43 is also present at the inner membrane of mitochondria isolated from ventricular tissue [55,73], especially in subsarcolemmal mitochondria (SSM) and to a far lesser extent in interfibrillar mitochondria (IFM), which are located between the myofibrils [9,75]. Compared to the detailed analysis of the phosphorylation of gap junctional Cx43, the phosphorylation of mitochondrial Cx43 is relatively unknown; however, phosphorylation at S262 and S368 has been detected [73,76]. ...
... Following I/R, phosphorylation of mitochondrial Cx43 is decreased [76]. Mitochondrial Cx43 is also involved in IPC, since a specific reduction of mitochondrial Cx43 abrogates preconditioning [55] and overexpression of mitochondrial Cx43 is sufficient to induce cell protection [43]. ...
... Data showing that ischemia induces dephosphorylation of Cx43 lead to the hypothesis that the cardioprotection by IPC may prevent such dephosphorylation, and indeed, IPC enhances Cx43 phosphorylation (S262 and S368) both in vitro and in vivo [56,61,72]. It is suggested that the cardioprotection by IPC is mediated at least in part via suppressing chemical gap junctional communication [46,60], but also mitochondrial Cx43 seems to play a role [40,55]. A reduction of Cx43 to about 50% in constitutive knockout mice is sufficient to abrogate IPC's cardioprotection in vivo [62] and in vitro (present study with Cx43 Cre−ER(T)/fl mice); however, others demonstrate that IPC reduces infarct size in vitro in inducible Cx43 knockout mice expressing half the level of Cx43 [59]. ...
Myocardial connexin 43 (Cx43) forms gap junctions and hemichannels, and is also present within subsarcolemmal mitochondria. The protein is phosphorylated by several kinases including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and casein kinase 1 (CK1). A reduction in Cx43 content abrogates myocardial infarct size reduction by ischemic preconditioning (IPC). The present study characterizes the contribution of Cx43 phosphorylation towards mitochondrial function, hemichannel activity, and the cardioprotection by IPC in wild-type (WT) mice and in mice in which Cx43-phosphorylation sites targeted by above kinases are mutated to non-phosphorylatable residues (Cx43 MAPKmut , Cx43 PKCmut , and Cx43 CK1mut mice). The amount of Cx43 in the left ventricle and in mitochondria was reduced in all mutant strains compared to WT mice and Cx43 phosphorylation was altered at residues not directly targeted by the mutations. Whereas complex 1 respiration was reduced in all strains, complex 2 respiration was decreased in Cx43 CK1mut mice only. In Cx43 epitope-mutated mice, formation of reactive oxygen species and opening of the mitochondrial permeability transition pore were not affected. The hemichannel open probability was reduced in Cx43 PKCmut and Cx43 CK1mut but not in Cx43 MAPKmut cardiomyocytes. Infarct size in isolated saline-perfused hearts after ischemia/reperfusion (45 min/120 min) was comparable between genotypes and was significantly reduced by IPC (3 × 3 min ischemia/5 min reperfusion) in WT, Cx43 MAPKmut , and Cx43 PKCmut , but not in Cx43 CK1mut mice, an effect independent from the amount of Cx43 and the probability of hemichannel opening. Taken together, our study shows that alterations of Cx43 phosphorylation affect specific cellular functions and highlights the importance of Cx43 phosphorylation by CK1 for IPC’s cardioprotection.
... Most studies have shown that Cx43 is almost exclusively present in SSM [100,104,105]. Cx43 is encoded by the nuclear genome and the rapid increase in Cx43 in mitochondria in response to IPC is mediated by the translocase of the outer membrane (TOM) pathway [95,106]. Indeed, interaction between Cx43, TOM20 and heat shock protein 90 (Hsp90) was shown by co-immunoprecipitation in the cardiac mitochondria of both pig and rat [106]. ...
... Cx43 is encoded by the nuclear genome and the rapid increase in Cx43 in mitochondria in response to IPC is mediated by the translocase of the outer membrane (TOM) pathway [95,106]. Indeed, interaction between Cx43, TOM20 and heat shock protein 90 (Hsp90) was shown by co-immunoprecipitation in the cardiac mitochondria of both pig and rat [106]. Moreover, the treatment of isolated rat heart with geldanamycin, a drug which blocks mitochondrial import by destabilizing protein complexes with Hsp90, demonstrated the involvement of Hsp90 chaperone protein in the import of Cx43 to the SSM [106]. ...
... Indeed, interaction between Cx43, TOM20 and heat shock protein 90 (Hsp90) was shown by co-immunoprecipitation in the cardiac mitochondria of both pig and rat [106]. Moreover, the treatment of isolated rat heart with geldanamycin, a drug which blocks mitochondrial import by destabilizing protein complexes with Hsp90, demonstrated the involvement of Hsp90 chaperone protein in the import of Cx43 to the SSM [106]. Once Cx43 reaches the outer mitochondrial membrane (OMM), it is further recruited to the inner mitochondrial membrane (IMM) through the translocase of the inner membrane (TIM) complex [106,107], where it locates with its CT directed towards the intermembrane space [106]. ...
Since the mid-20th century, ischemic heart disease has been the world’s leading cause of death. Developing effective clinical cardioprotection strategies would make a significant impact in improving both quality of life and longevity in the worldwide population. Both ex vivo and in vivo animal models of cardiac ischemia/reperfusion (I/R) injury are robustly used in research. Connexin43 (Cx43), the predominant gap junction channel-forming protein in cardiomyocytes, has emerged as a cardioprotective target. Cx43 posttranslational modifications as well as cellular distribution are altered during cardiac reperfusion injury, inducing phosphorylation states and localization detrimental to maintaining intercellular communication and cardiac conduction. Pre- (before ischemia) and post- (after ischemia but before reperfusion) conditioning can abrogate this injury process, preserving Cx43 and reducing cell death. Pre-/post-conditioning has been shown to largely rely on the presence of Cx43, including mitochondrial Cx43, which is implicated to play a major role in pre-conditioning. Posttranslational modifications of Cx43 after injury alter the protein interactome, inducing negative protein cascades and altering protein trafficking, which then causes further damage post-I/R injury. Recently, several peptides based on the Cx43 sequence have been found to successfully diminish cardiac injury in pre-clinical studies.
... Emerging evidence has indicated that Cx43 is present in cardiomyocyte subsarcolemmal mitochondria (SSM, one major population of mitochondria that locate directly beneath the sarcolemma and are in contrast to another population of mitochondria-interfibrillar mitochondria [IFM] that are aligned among the myofibrils) [11,71,72]. Mitochondrial Cx43 significantly contributes to protecting myocardial function during I/R [7,11,57,62,72,93]. Cardiac protective approaches to increasing myocardial SSM Cx43 mediate mitochondriaderived protection [7,11,62,65,71,72]. ...
... Mitochondrial Cx43 significantly contributes to protecting myocardial function during I/R [7,11,57,62,72,93]. Cardiac protective approaches to increasing myocardial SSM Cx43 mediate mitochondriaderived protection [7,11,62,65,71,72]. Interestingly, total and mitochondrial Cx43 content is reduced in aged mouse hearts, likely contributing to the age-associated loss of cardiac protection [8]. ...
... Given better myocardial functional recovery in female hearts than in male hearts following I/R from our previous studies [81,83,85,86] and the importance of mitochondrial Cx43 in cardiac protection [7,11,57,62,72,93], we next determined myocardial levels of Cx43 between male and female. Without I/R there was no sex-specific difference in Cx43 expression in mouse hearts and cardiac mitochondria (Fig. 4a, b, S4A). ...
Preserving mitochondrial activity is crucial in rescuing cardiac function following acute myocardial ischemia/reperfusion (I/R). The sex difference in myocardial functional recovery has been observed after I/R. Given the key role of mitochondrial connexin43 (Cx43) in cardiac protection initiated by ischemic preconditioning, we aimed to determine the implication of mitochondrial Cx43 in sex-related myocardial responses and to examine the effect of estrogen (17β-estradiol, E2) on Cx43, particularly mitochondrial Cx43-involved cardiac protection following I/R. Mouse primary cardiomyocytes and isolated mouse hearts (from males, females, ovariectomized females, and doxycycline-inducible Tnnt2-controlled Cx43 knockout without or with acute post-ischemic E2 treatment) were subjected to simulated I/R in culture or Langendorff I/R (25-min warm ischemia/40-min reperfusion), respectively. Mitochondrial membrane potential and mitochondrial superoxide production were measured in cardiomyocytes. Myocardial function and infarct size were determined. Cx43 and its isoform, Gja1-20k, were assessed in mitochondria. Immunoelectron microscopy and co-immunoprecipitation were also used to examine mitochondrial Cx43 and its interaction with estrogen receptor-α by E2 in mitochondria, respectively. There were sex disparities in stress-induced cardiomyocyte mitochondrial function. E2 partially restored mitochondrial activity in cardiomyocytes following acute injury. Post-ischemia infusion of E2 improved functional recovery and reduced infarct size with increased Cx43 content and phosphorylation in mitochondria. Ablation of cardiac Cx43 aggravated mitochondrial damage and abolished E2-mediated cardiac protection during I/R. Female mice were more resistant to myocardial I/R than age-matched males with greater protective role of mitochondrial Cx43 in female hearts. Post-ischemic E2 usage augmented mitochondrial Cx43 content and phosphorylation, increased mitochondrial Gja1-20k, and showed cardiac protection.
... Cx43 is encoded by the nuclear genome, and the rapid increase in Cx43 in mitochondria, in response to IPC, is achieved by shuffling cytosolic Cx43 to the mitochondria via heat shock protein 90 (Hsp90) [126]. Once Cx43 reaches the outer mitochondrial membrane, it is imported through the translocase of the outer membrane (TOM) complex and subsequently inserted into the inner mitochondrial membrane through the translocase of the inner membrane complex (TIM) [126]. ...
... Cx43 is encoded by the nuclear genome, and the rapid increase in Cx43 in mitochondria, in response to IPC, is achieved by shuffling cytosolic Cx43 to the mitochondria via heat shock protein 90 (Hsp90) [126]. Once Cx43 reaches the outer mitochondrial membrane, it is imported through the translocase of the outer membrane (TOM) complex and subsequently inserted into the inner mitochondrial membrane through the translocase of the inner membrane complex (TIM) [126]. Intriguingly, cardiomyocytes contain the following two mitochondrial subpopulations: the subsarcolemmal (SSM) and the interfibrillar (IFM) mitochondria, which have different morphology and functions [127]. ...
For the first time in animal evolution, the emergence of gap junctions allowed direct exchanges of cellular substances for communication between two cells. Innexin proteins constituted primordial gap junctions until the connexin protein emerged in deuterostomes and took over the gap junction function. After hundreds of millions of years of gene duplication, the connexin gene family now comprises 21 members in the human genome. Notably, GJA1, which encodes the Connexin43 protein, is one of the most widely expressed and commonly studied connexin genes. The loss of Gja1 in mice leads to swelling and a blockage of the right ventricular outflow tract and death of the embryos at birth, suggesting a vital role of Connexin43 gap junction in heart development. Since then, the importance of Connexin43-mediated gap junction function has been constantly expanded to other types of cells. Other than forming gap junctions, Connexin43 can also form hemichannels to release or uptake small molecules from the environment or even mediate many physiological processes in a gap junction-independent manner on plasma membranes. Surprisingly, Connexin43 also localizes to mitochondria in the cell, playing important roles in mitochondrial potassium import and respiration. At the molecular level, Connexin43 mRNA and protein are processed with very distinct mechanisms to yield carboxyl-terminal fragments with different sizes, which have their unique subcellular localization and distinct biological activities. Due to many exciting advancements in Connexin43 research, this review aims to start with a brief introduction of Connexin43 and then focuses on updating our knowledge of its gap junction-independent functions.
... Later, functional hemichannels of Cx43 were located at the IMM of rat cardiomyocytes, with their C-terminal oriented towards the intermembrane space (53). Cx43 association with OXPHOS Complex II was increased by an ischemia preconditioning (IP) protocol and its translocation to mitochondria was mediated by the Hsp90-dependent TOM protein import system (54). ...
... The mechanism responsible for PM molecule translocation to mitochondria has not been fully elucidated. In the case of Cx43, the mechanism involved the Hsp90-dependent TOM protein import system (54), and vesicles seem to play a role. ...
Mitochondria are known as the powerhouses of eukaryotic cells; however, they perform many other functions besides oxidative phosphorylation, including Ca²⁺ homeostasis, lipid metabolism, anti-viral response, and apoptosis. Although other hypotheses exist, mitochondria are generally thought as descendants of an α-proteobacteria that adapted to the intracellular environment within an Asgard archeobacteria, that have been studied for decades as an organelle subdued by the eukaryotic cell. Nevertheless, several early electron microscopy observations hinted that some mitochondria establish specific interactions with certain plasma membrane (PM) domains in mammalian cells. Furthermore, recent findings have documented the direct physical and functional interaction of mitochondria and the PM, the organization of distinct complexes, and their communication through vesicular means. In yeast, some molecular players mediating this interaction have been elucidated, but only a few works have studied this interaction in mammalian cells. In addition, mitochondria can be translocated among cells through tunneling nanotubes or by other mechanisms, and free, intact, functional mitochondria have been reported in the blood plasma. Together, these findings challenge the conception of mitochondria as organelles subdued by the eukaryotic cell. This review discusses the evidence of the mitochondria interaction with the PM that has been long disregarded, despite its importance in cell function, pathogenesis, and evolution. It also proposes a scheme of mitochondria-PM interactions with the intent to promote research and knowledge of this emerging pathway that promises to shift the current paradigms of cell biology.
... We first applied geldanamycin, an HSP90 ATPase inhibitor for both HSP90α and HSP90β, which is widely used in studies of mitochondrial precursor import. 20,21,46,47 In the presence of 18 μM geldanamycin, mitochondria from PS120-hSLC4A11-HA cells (Fig. 4a) and WT MCECs (Supplementary Fig. S3a) showed gradually diminishing levels of SLC4A11 (HA tagged or endogenous, respectively) in the mitochondria over time, suggesting there is an impairment of the import of SLC4A11 into the mitochondria. For verification of HSP90 inhibitor specificity on mitochondrial SLC4A11 translocation, another selective HSP90 inhibitor, novobiocin, 20,48 was tested in both Slc4a11 WT MCECs (Fig. 4c) and PS120-hSLC4A11-HA cells ( Supplementary Fig. S3b), and purified mitochondria were analyzed by Western blotting. ...
... 21,25 The mechanism of HSP90 action in the context of TOM70-dependent mitochondrial precursor trafficking has been demonstrated by using two HSP90 inhibitors that have different inhibitory mechanisms: (1) novobiocin targets the C-terminal domain of HSP90, causing degradation of the preprotein-chaperone complex in the cytosol, and (2) geldanamycin binds to the ADP/ATP binding pocket on the N-terminal domain of HSP90, inhibiting targeting of precursors to the outer mitochondrial membrane. 20,21,46,47 Consistent with this observation, both pharmacologic inhibitors of HSP90 reduced the mitochondrial SLC4A11 translocation, which parallels the disruption of mitochondrial SLC4A11 import by siRNA knockdown of the chaperones HSP90 and HSC70. What is less clear is whether the chaperones act together in trafficking SLC4A11 or can work alone. ...
Purpose: SLC4A11, an electrogenic H+ transporter, is found in the plasma membrane and mitochondria of corneal endothelium. However, the underlying mechanism of SLC4A11 targeting to mitochondria is unknown.
Methods: The presence of mitochondrial targeting sequences was examined using in silico mitochondrial proteomic analyses. Thiol cross-linked peptides binding to SLC4A11 were screened by untargeted LC-MS/MS analysis. Direct protein interactions between SLC4A11 and chaperones was examined using co-immunoprecipitation analysis and Proximity Ligation Assay. Knockdown or pharmacological inhibition of chaperones in human (HCEC) or mouse corneal endothelial cells, ex vivo kidney, or HA-SLC4A11-transfected fibroblasts was performed to investigate the functional consequences of interfering with mitochondrial SLC4A11 trafficking.
Results: SLC4A11 does not contain canonical N-terminal mitochondrial targeting sequences. LC-MS/MS analysis showed that HSC70 and/or HSP90 are bound to HA-SLC4A11-transfected PS120 fibroblast whole cell lysates or isolated mitochondria, suggesting trafficking through the chaperone-mediated carrier-pathway. SLC4A11 and either HSP90 or HSC70 complexes are directly bound to the mitochondrial surface receptor, TOM70. Interference with this trafficking leads to dysfunctional mitochondrial glutamine catabolism and increased ROS production. In addition, Gln use upregulated SLC4A11, HSP70 and HSP90 expression in whole cell lysates or purified mitochondria of HCEC and HA-SLC4A11-transfected fibroblasts.
Conclusions: HSP90 and HSC70 are critical in mediating mitochondrial SLC4A11 translocation in corneal endothelial cells and kidney. Gln promotes SLC4A11 import to the mitochondria, and the continuous oxidative stress derived from Gln catabolism induced HSP70 and HSP90, protecting cells against oxidative stress.
... The mechanism by which Rictor regulates the translocation of Cx43 to mitochondria Cx43 is translocated to the inner mitochondrial membrane through the Hsp90-dependent TOM20 pathway [30]. Knockdown of Rictor led to a significant decrease in the interactions of Hsp90 and TOM20 with Cx43 in ESC-CMs (Fig. 4a). ...
... The expression of mtCx43 in the rat cortex was reduced by treatment with the PI3K/Akt pathway inhibitor LY 294002 (LY) [35]. Cx43 transport to the mitochondrial inner membrane is dependent on the Hsp90-TOM20 transport system [30]. In addition, the deacetylation of Hsp90, mediated by HDAC6, has been shown to be involved in the mitochondrial transport of proteins [36,37]. ...
Rictor is a key component of the mammalian target of rapamycin complex 2 (mTORC2) and is required for Akt phosphorylation (Ser473). Our previous study shows that knockdown of Rictor prevents cardiomyocyte differentiation from mouse embryonic stem (ES) cells and induces abnormal electrophysiology of ES cell-derived cardiomyocytes (ESC-CMs). Besides, knockdown of Rictor causes down-expression of connexin 43 (Cx43), the predominant gap junction protein, that is located in both the sarcolemma and mitochondria in cardiomyocytes. Mitochondrial Cx43 (mtCx43) plays a crucial role in mitochondrial function. In this study, we used the model of cardiomyocyte differentiation from mouse ES cells to elucidate the mechanisms for the mitochondrial damage in ESC-CMs after knockdown of Rictor. We showed swollen and ruptured mitochondria were observed after knockdown of Rictor under transmission electron microscope. ATP production and mitochondrial transmembrane potential were significantly decreased in Rictor-knockdown cells. Furthermore, knockdown of Rictor inhibited the activities of mitochondrial respiratory chain complex. The above-mentioned changes were linked to inhibiting the translocation of Cx43 into mitochondria by knockdown of Rictor. We revealed that knockdown of Rictor inactivated the mTOR/Akt signalling pathway and subsequently decreased HDAC6 expression, resulted in Hsp90 hyper-acetylation caused by HDAC6 inhibition, thus, inhibited the formation of Hsp90-Cx43-TOM20 complex. In conclusion, the mitochondrial Cx43 participates in shRNA-Rictor-induced mitochondrial function damage in the ESC-CMs.
... This study highlights a cell protective role of Cx HCs against oxidative damage. In cardiomyocytes, Cx43 can be translocated to the mitochondrial inner membrane, exerting cardioprotection during ischemic preconditioning as well as during hypoxic postconditioning, a process involving ROS as a critical mediator to heart damage [33][34][35]. The translocation of Cx43 is initiated by heat shock protein 90 as a molecular chaperone and associates with the interaction of a mitochondrial protein, TOM20 [33]. ...
... In cardiomyocytes, Cx43 can be translocated to the mitochondrial inner membrane, exerting cardioprotection during ischemic preconditioning as well as during hypoxic postconditioning, a process involving ROS as a critical mediator to heart damage [33][34][35]. The translocation of Cx43 is initiated by heat shock protein 90 as a molecular chaperone and associates with the interaction of a mitochondrial protein, TOM20 [33]. Recent studies show that ATP-sensitive potassium channels in mitochondria (mK ATP ) also interact with mitochondrial Cx43 HCs and protect cardiomyocytes under mild hypoxic conditions [32,36,37]. ...
Purpose of Review
The goal of this review is to provide an overview of the impact and underlying mechanism of oxidative stress on connexin channel function, and their roles in skeletal aging, estrogen deficiency, and glucocorticoid excess associated bone loss.
Recent Findings
Connexin hemichannel opening is increased under oxidative stress conditions, which confers a cell protective role against oxidative stress-induced cell death. Oxidative stress acts as a key contributor to aging, estrogen deficiency, and glucocorticoid excess-induced osteoporosis and impairs osteocytic network and connexin gap junction communication.
Summary
This paper reviews the current knowledge for the role of oxidative stress and connexin channels in the pathogenesis of osteoporosis and physiological and pathological responses of connexin channels to oxidative stress. Oxidative stress decreases osteocyte viability and impairs the balance of anabolic and catabolic responses. Connexin 43 (Cx43) channels play a critical role in bone remodeling, mechanotransduction, and survival of osteocytes. Under oxidative stress conditions, there is a consistent reduction of Cx43 expression, while the opening of Cx43 hemichannels protects osteocytes against cell injury caused by oxidative stress.
... In the context of cardiac HCs, attention should be paid to putative mitochondrial HCs that are formed upon the translocation of Cx43 from the cytosol to the mitochondria [82,184,185]. Translocation can be mediated by heat shock protein 90 and translocase of the outer mitochondria membrane [186] following PKCε activation with subsequent Cx43 phosphorylation [185]. An enhanced mito-Cx43 level has been induced by ischemic preconditioning, and this implies cardioprotective effects against ischemic heart injury [82,184,[187][188][189], ischemia-reperfusion injury [20,186], and the regulation of apoptosis [190] and energy metabolism [122,191,192]. ...
... Translocation can be mediated by heat shock protein 90 and translocase of the outer mitochondria membrane [186] following PKCε activation with subsequent Cx43 phosphorylation [185]. An enhanced mito-Cx43 level has been induced by ischemic preconditioning, and this implies cardioprotective effects against ischemic heart injury [82,184,[187][188][189], ischemia-reperfusion injury [20,186], and the regulation of apoptosis [190] and energy metabolism [122,191,192]. ...
Cardiac connexin-43 (Cx43) creates gap junction channels (GJCs) at intercellular contacts and hemi-channels (HCs) at the peri-junctional plasma membrane and sarcolemmal caveolae/rafts compartments. GJCs are fundamental for the direct cardiac cell-to-cell transmission of electrical and molecular signals which ensures synchronous myocardial contraction. The HCs and structurally similar pannexin1 (Panx1) channels are active in stressful conditions. These channels are essential for paracrine and autocrine communication through the release of ions and signaling molecules to the extracellular environment, or for uptake from it. The HCs and Panx1 channel-opening profoundly affects intracellular ionic homeostasis and redox status and facilitates via purinergic signaling pro-inflammatory and pro-fibrotic processes. These conditions promote cardiac arrhythmogenesis due to the impairment of the GJCs and selective ion channel function. Crosstalk between GJCs and HCs/Panx1 channels could be crucial in the development of arrhythmogenic substrates, including fibrosis. Despite the knowledge gap in the regulation of these channels, current evidence indicates that HCs and Panx1 channel activation can enhance the risk of cardiac arrhythmias. It is extremely challenging to target HCs and Panx1 channels by inhibitory agents to hamper development of cardiac rhythm disorders. Progress in this field may contribute to novel therapeutic approaches for patients prone to develop atrial or ventricular fibrillation.
... by IPC [15,17]. Mechanistically, IPC induces Cx43 translocation to the IMM via the translocase of the outer membrane (TOM) complex [123]. However, the precise mechanisms by which mitochondrial translocation of Cx43 protects the heart remain unclear. ...
Mitochondrial calcium (Ca²⁺) signals play a central role in cardiac homeostasis and disease. In the healthy heart, mitochondrial Ca²⁺ levels modulate the rate of oxidative metabolism to match the rate of adenosine triphosphate consumption in the cytosol. During ischemia/reperfusion (I/R) injury, pathologically high levels of Ca²⁺ in the mitochondrial matrix trigger the opening of the mitochondrial permeability transition pore, which releases solutes and small proteins from the matrix, causing mitochondrial swelling and ultimately leading to cell death. Pharmacological and genetic approaches to tune mitochondrial Ca²⁺ handling by regulating the activity of the main Ca²⁺ influx and efflux pathways, i.e., the mitochondrial Ca²⁺ uniporter and sodium/Ca²⁺ exchanger, represent promising therapeutic strategies to protect the heart from I/R injury.
... 15 Rodriguez-Sinovas et al. found that HSP90 bound to Cx43 and promoted the mitochondrial translocation of Cx43 by TOM20 pathway to exert its cardioprotective functions. 16 In addition, our pilot study predicted a possible interaction between stress-induced phosphoprotein 1 (STIP1) and HSP90 through the string database. STIP1 is an adaptor protein that assists in the transfer of client proteins from HSP70 to HSP90 by binding both HSP90 and substrate-bound HSP70, which was identified to be a widely studied helper molecular chaperone. ...
Objective
Connexin 43 (Cx43) is a critical gene for maintaining myocardial homeostasis. Interestingly, Cx43 and stress‐induced phosphoprotein 1 (STIP1) were recorded to be lowly expressed in ischemia/reperfusion (I/R). However, their impacts on reperfusion arrhythmia (RA) remain to be explored. Our study aimed to find out the related underlying mechanisms.
Methods
After the establishment of an isolated heart model through Langendorff perfusion, the heart rate, conduction activation time, conduction velocity, and conduction direction of the left ventricle were evaluated, along with the apoptotic rate detection in the collected myocardial tissues. After the construction of a hypoxia/reoxygenation (H/R)‐induced cellular model, cell apoptosis, intercellular communication, cell viability, and the content of reactive oxygen species, superoxide dismutase, malondialdehyde, and lactic dehydrogenase were measured. The expression of Cx43 and STIP1 was determined in both rat heart and cell models. The bindings of STIP3 and Cx43 to heat shock protein 90 (HSP90) and heat shock protein 70 (HSP70) were verified.
Results
Relative to the corresponding controls, Cx43 and STIP1 were decreased in myocardial tissues of RA rats and H/R‐stimulated H9C2 cells, where Cx43‐binding HSP70 and HSP90 were respectively increased and decreased, and ubiquitination level of Cx43 was enhanced. STIP1 overexpression promoted protein expression of Cx43, intercellular communication, and cell viability, and reduced cell apoptosis and oxidative stress in H/R‐stimulated H9C2 cells.
Conclusion
STIP1 promoted Cx43 expression to improve intercellular communication and reduce oxidative stress in H/R‐stimulated H9C2 cells.
... The role of mitochondrial Cx43 has been linked to both the inner membrane (142) and outer mitochondrial membranes (136,143) where it might serve distinctly different functions. As a full-length integral membrane protein, it has been proposed that the translocase of the outer membrane (TOM) and heat shock protein (HSP) 90 participate in Cx43 import across the outer J o u r n a l P r e -p r o o f mitochondrial membrane as it seeks to insert into the inner mitochondrial membranes (142). ...
Over 35 years ago the cell biology community was introduced to connexins as the subunit employed to assemble semicrystalline clusters of intercellular channels that had been well described morphologically as gap junctions. The decade that followed would see knowledge of the unexpectedly large 21-member human connexin family grow to reflect unique and overlapping expression patterns in all organ systems. While connexin biology initially focused on their role in constructing highly regulated intercellular channels, this was destined to change as discoveries revealed that connexin hemichannels at the cell surface had novel roles in many cell types, especially when considering connexin pathologies. Acceptance of connexins as having bifunctional channel properties was initially met with some resistance, which has given way in recent years to the premise that connexins have multifunctional properties. Depending on the connexin isoform and cell of origin, connexins have wide-ranging half-lives that vary from a couple of hours to the life expectancy of the cell. Diversity in connexin channel characteristics and molecular properties were further revealed by X-ray crystallography and single-particle cryo-EM. New avenues have seen connexins or connexin fragments playing roles in cell adhesion, tunneling nanotubes, extracellular vesicles, mitochondrial membranes, transcription regulation, and in other emerging cellular functions. These discoveries were largely linked to Cx43, which is prominent in most human organs. Here, we will review the evolution of knowledge on connexin expression in human adults and more recent evidence linking connexins to a highly diverse array of cellular functions.
... Once ATP rebinds, HSP70 reverts back to the fully closed conformation, and the substrate is squeezed out of HSP70. It is reported that HSP90 mediates the translocation of Cx43 to the inner mitochondrial membrane of cardiomyocytes(Rodriguez- Sinovas et al. 2006). However, little is known about the function of the HSP40-HSP70-STIP1-HSP90 machinery in the regulation of Cx43 stability. ...
To investigate the involvement of stress induced phosphoprotein 1 (STIP1), heat shock protein (HSP) 70, and HSP90 in ubiquitination of connexin 43 (Cx43) in rat H9c2 cardiomyocytes. Co-immunoprecipitation was used to detect protein-protein interactions and Cx43 ubiquitination. Immunofluorescence was used for protein co-localization. The protein binding, Cx43 protein expression, and Cx43 ubiquitination were reanalyzed in H9c2 cells with modified STIP1 and/or HSP90 expression. STIP1 bound to HSP70 and HSP90, and Cx43 bound to HSP40, HSP70, and HSP90 in normal H9c2 cardiomyocytes. Overexpression of STIP1 promoted the transition of Cx43-HSP70 to Cx43-HSP90 and inhibited Cx43 ubiquitination; knockdown of STIP1 resulted in the opposite effects. Inhibition of HSP90 counteracted the inhibitory effect of STIP1 overexpression on Cx43 ubiquitination. STIP1 suppresses Cx43 ubiquitination in H9c2 cardiomyocytes by promoting the transition of Cx43-HSP70 to Cx43-HSP90.
... Connexin43 (Cx43) is a major connexin that forms gap junction (GJ) channels in cardiac ventricular muscle [44] and is present as unopposed/nonjunctional hemichannels in the sarcolemmal membrane [12,35] and as vesicles within the muscle [27]. Recently, Cx43 is also found in the inner mitochondrial membrane as hemichannels [4,32], and this mitochondrial Cx43 is associated with ADP-stimulated complex 1 respiration, ATP generation [3], mitochondrial K + influx, and the formation of reactive oxygen species (ROS) [39]. In addition, the presence of Cx43 is necessary for ischemic and pharmacological preconditioning [14,34,38] and is also necessary for the activation of mitochondrial K ATP channels [21,29] to reduce ventricular arrhythmias induced by ischemia [20]. ...
Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43−/−) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/− mice. The resulting offspring, Cx43flox/flox/Myh6-cre+/− mice (cCx43−/− mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2′,7′-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most of cCx43−/− mice died suddenly within 8 weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+ mice but not in cCx43−/− mice. In cCx43−/− mice, the [Ca2+]o,min was lower, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43−/− mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43−/− mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts.
... As for their subcellular location, subsarcolemmal mitochondria have a greater contribution to ROS production (Crochemore et al., 2015) and IRI (Lesnefsky et al., 1997) and are more sensitive toward pharmacological and ischemic conditioning than interfibrillar mitochondria (Holmuhamedov et al., 2012;Sun et al., 2015). Moreover, only subsarcolemmal mitochondria contain connexin 43 at their inner membrane ), a protein involved in the ischemic PreC cardioprotection (Rodriguez-Sinovas et al., 2006;Ruiz-Meana et al., 2014) that has recently been identified as one of the interactors of the FoF1-ATP synthase . Also, STAT 3 activation impacts on mitochondrial function; it increases respiration, ATP formation, and calcium retention capacity and decreases ROS formation in rat and mouse mitochondria of myocardium undergoing IRI with ischemic PreC or PostC (Boengler et al., 2008;Boengler et al., 2010;Heusch et al., 2011;Skyschally et al., 2018). ...
Preconditioning, postconditioning, and remote conditioning of the myocardium enhance the ability of the heart to withstand a prolonged ischemia/reperfusion insult and the potential to provide novel therapeutic paradigms for cardioprotection. While many signaling pathways leading to endogenous cardioprotection have been elucidated in experimental studies over the past 30 years, no cardioprotective drug is on the market yet for that indication. One likely major reason for this failure to translate cardioprotection into patient benefit is the lack of rigorous and systematic preclinical evaluation of promising cardioprotective therapies prior to their clinical evaluation, since ischemic heart disease in humans is a complex disorder caused by or associated with cardiovascular risk factors and comorbidities. These risk factors and comorbidities induce fundamental alterations in cellular signaling cascades that affect the development of ischemia/reperfusion injury and responses to cardioprotective interventions. Moreover, some of the medications used to treat these comorbidities may impact on cardioprotection by again modifying cellular signaling pathways. The aim of this article is to review the recent evidence that cardiovascular risk factors as well as comorbidities and their medications may modify the response to cardioprotective interventions. We emphasize the critical need for taking into account the presence of cardiovascular risk factors as well as comorbidities and their concomitant medications when designing preclinical studies for the identification and validation of cardioprotective drug targets and clinical studies. This will hopefully maximize the success rate of developing rational approaches to effective cardioprotective therapies for the majority of patients with multiple comorbidities.
Significance Statement
Ischemic heart disease is a major cause of mortality; however, there are still no cardioprotective drugs on the market. Most studies on cardioprotection have been undertaken in animal models of ischemia/reperfusion in the absence of comorbidities; however, ischemic heart disease develops with other systemic disorders (e.g., hypertension, hyperlipidemia, diabetes, atherosclerosis). Here we focus on the preclinical and clinical evidence showing how these comorbidities and their routine medications affect ischemia/reperfusion injury and interfere with cardioprotective strategies.
... It is not understood how Cx43, which is a hexameric ion channel each with four transmembrane domains and thus 24 transmembrane domains for each channel (Unwin and Zampighi 1980), either arrives at mitochondria or inserts in outer mitochondrial membrane. If Cx43 hemichannels translocate to the inner mitochondrial membrane via the TOM transporter to achieve IPC (Rodriguez-Sinovas et al., 2006), it is not clear how the hexameric 24 transmembrane domain channel is able to disassemble and unfold only to reconstitute as a functional ion channel in the inner membrane. It should be noted that antibody-based selection of Cx43 for the purposes of immunofluorescence and biochemistry involved Cx43 epitopes on the C-terminus which remains intact for many internally translated isoforms of Cx43 including GJA1-20k (Smyth and Shaw 2013). ...
Internal translation is a form of post-translation modification as it produces different proteins from one mRNA molecule by beginning translation at a methionine coding triplet downstream of the first methionine. Internal translation can eliminate domains of proteins that otherwise restrict movement or activity, thereby creating profound functional diversity. Connexin43 (Cx43), encoded by the gene Gja1, is the main gap junction protein necessary for propagating action potentials between adjacent cardiomyocytes. Gja1 can be internally translated to produce a peptide 20 kD in length named GJA1-20k. This review focuses on the role of GJA1-20k in maintaining cardiac electrical rhythm as well as in ischemic preconditioning (IPC). Connexin43 is the only ion channel we are aware that has been reported to be subject to internal translation. We expect many other ion channels also undergo internal translation. The exploration of post-translational modification of ion channels, and in particular of internal translation, has the potential to greatly increase our understanding of both canonical and non-canonical ion channel biology.
... Furthermore, in another study, it has been demonstrated that the reduction in mitochondrial Cx43 by geldanamycin is associated with the abrogation of the cardioprotective agent of diazoxide against ischemia-reperfusion-induced cell death [160]. It has also been found that one of the major cellular responses elicited by geldanamycin is the degradation of HSP90 client proteins and the induction of molecular chaperones such as HSP70 [161]. Among them, the abundant HSP70 and HIF-1α and their metabolic target genes are related to cell protection [162]. ...
Cardiovascular disease is the result of complicated pathophysiological processes in the tissues that make up the blood vessels and heart. Heat shock protein 90 (HSP90) can interact with 10% of the proteome and is the most widely studied molecular chaperone in recent years. HSP90 is extensively involved in the regulation of protein folding and intracellular protein stability, making HSP90 a hopeful target for the treatment of multiple cardiovascular diseases. Numerous client proteins of HSP90 have been identified in known cardiac disease pathways, including MAPK signaling, PI3K/AKT (PKB)/mTOR, and TNF-α signaling. Therefore, these pathways can be controlled by regulating HSP90. Among them, the activity of HSP90 can be regulated via numerous inhibitors. In this review, first, we will discuss the function of HSP90 and its role in pathological pathways. In addition, HSP90 plays a significant role in most cardiovascular diseases, including hypertension, pulmonary venous hypertension, atherosclerosis, and heart failure; next we will focus on this part. Finally, we will summarize the currently known HSP90 inhibitors and their potential in the treatment of heart disease.
... Intercellular communication between cardiomyocytes through Cx43 allows for rapid electrical signal diffusion and synchronous ventricular contraction. Myocardial Cx43 is trafficked via three main pathways: (i) autophagosomal degradation via direct fusion with lysosomes (25) or phagosomes (26,27); (ii) lateralization to the lateral membrane; and (iii) translocation to the inner mitochondrial membrane (IMM) through heat shock protein (HSP) 90-dependent translocase (28). Previously, we have demonstrated that hypoglycemic challenge resulted in decreased Cx43 expression in hyperglycemiacultured H9c2 cells (29), suggestive an active role of Cx43 in DCM progression. ...
Background
Diabetic cardiomyopathy (DCM) is a complex multifaceted disease responsible for elevated heart failure (HF) morbidity and mortality in patients with diabetes mellitus (DM). Patients with DCM exhibit subclinical diastolic dysfunction, progression toward systolic impairment, and abnormal electrophysiology. Hypoglycemia events that occur spontaneously or due to excess insulin administration threaten the lives of patients with DM—with the increased risk of sudden death. However, the molecular underpinnings of this fatal disease remain to be elucidated.
Methods and Results
Here, we used the established streptozotocin-induced DCM murine model to investigate how hypoglycemia aggravates DCM progression. We confirmed connexin 43 (Cx43) dissociation from cell–cell interaction and accumulation at mitochondrial inner membrane both in the cardiomyocytes of patients with DM and DCM murine. Here, we observed that cardiac diastolic function, induced by chronic hyperglycemia, was further aggravated upon hypoglycemia challenge. Similar contractile defects were recapitulated using neonatal mouse ventricular myocytes (NMVMs) under glucose fluctuation challenges. Using immunoprecipitation mass spectrometry, we identified and validated that hypoglycemia challenge activates the mitogen-activated protein kinase kinase (MAPK kinase) (MEK)/extracellular regulated protein kinase (ERK) and inhibits phosphoinositide 3-kinase (PI3K)/Akt pathways, which results in Cx43 phosphorylation by Src protein and translocation to mitochondria in cardiomyocytes. To determine causality, we overexpressed a mitochondrial targeting Cx43 (mtCx43) using adeno-associated virus serotype 2 (AAV2)/9. At normal blood glucose levels, mtCx43 overexpression recapitulated cardiac diastolic dysfunction as well as aberrant electrophysiology in vivo . Our findings give support for therapeutic targeting of MEK/ERK/Src and PI3K/Akt/Src pathways to prevent mtCx43-driven DCM.
Conclusion
DCM presents compensatory adaptation of mild mtCx43 accumulation, yet acute hypoglycemia challenges result in further accumulation of mtCx43 through the MEK/ERK/Src and PI3K/Akt/Src pathways. We provide evidence that Cx43 mislocalization is present in hearts of patients with DM hearts, STZ-induced DCM murine model, and glucose fluctuation challenged NMVMs. Mechanistically, we demonstrated that mtCx43 is responsible for inducing aberrant contraction and disrupts electrophysiology in cardiomyocytes and our results support targeting of mtCx43 in treating DCM.
... Chi et al. [115] prove that efflux of GSH via Cx43 HCs contributes to the Ca 2+ depletion-elicited disassembly of cell junctions. In cardiomyocytes, the translocation of Cx43 to the mitochondrial inner membrane exerts cardioprotection ischemic and hypoxic postconditioning [114,116]. Recent studies show that Cx43 HCs in cardiomyocyte mitochondria interact with ATP-sensitive potassium channels (mKATP) and protect cardiomyocytes against hypoxia/ischemia stress [117,118]. In contrast to the cardioprotection, mitochondrial Cx43 HCs facilitate mitochondrial Ca 2+ entry and may trigger permeability transition and cell injury/death by using connexin-targeting peptides interacting with extracellular (Gap26) and intracellular (Gap19, RRNYRRNY) Cx43 domains [119,120], indicating a detrimental role of Cx43 HCs in cardiomyocytes. ...
The lens is continuously exposed to oxidative stress insults, such as ultraviolet radiation and other oxidative factors, during the aging process. The lens possesses powerful oxidative stress defense systems to maintain its redox homeostasis, one of which employs connexin channels. Connexins are a family of proteins that form: (1) Hemichannels that mediate the communication between the intracellular and extracellular environments, and (2) gap junction channels that mediate cell-cell communication between adjacent cells. The avascular lens transports nutrition and metabolites through an extensive network of connexin channels, which allows the passage of small molecules, including antioxidants and oxidized wastes. Oxidative stress-induced post-translational modifications of connexins, in turn, regulates gap junction and hemichannel permeability. Recent evidence suggests that dysfunction of connexins gap junction channels and hemichannels may induce cataract formation through impaired redox homeostasis. Here, we review the recent advances in the knowledge of connexin channels in lens redox homeostasis and their response to cataract-related oxidative stress by discussing two major aspects: (1) The role of lens connexins and channels in oxidative stress and cataractogenesis, and (2) the impact and underlying mechanism of oxidative stress in regulating connexin channels.
... In addition to the plasma membrane, functional Cx43 HCs are also present at the mitochondrial inner membrane of cardiomyocytes [7,[154][155][156], and phosphorylation by mitochondrial PKC and probably other posttranslational modifications seem to affect their function [7,157]. Decrease in the number of active mitochondrial Cx43 HCs is associated to reductions in the production of reactive oxygen species [7,158,159], and overall, the evidence suggest that Cx43 HCs in cardiac subsarcolemmal mitochondria have a protective role in ischemic preconditioning, but increased HC activity contributes to the ischemic injury in the absence of preconditioning [7,[160][161][162]. ...
Connexins are membrane proteins involved directly in cell-to-cell communication through the formation of gap-junctional channels. These channels result from the head-to-head docking of two hemichannels, one from each of two adjacent cells. Undocked hemichannels are also present at the plasma membrane where they mediate the efflux of molecules that participate in autocrine and paracrine signaling, but abnormal increase in hemichannel activity can lead to cell damage in disorders such as cardiac infarct, stroke, deafness, cataracts, and skin diseases. For this reason, connexin hemichannels have emerged as a valid therapeutic target. Know small molecule hemichannel inhibitors are not ideal leads for the development of better drugs for clinical use because they are not specific and/or have toxic effects. Newer inhibitors are more selective and include connexin mimetic peptides, anti-connexin antibodies and drugs that reduce connexin expression such as antisense oligonucleotides. Re-purposed drugs and their derivatives are also promising because of the significant experience with their clinical use. Among these, aminoglycoside antibiotics have been identified as inhibitors of connexin hemichannels that do not inhibit gap-junctional channels. In this review, we discuss connexin hemichannels and their inhibitors, with a focus on aminoglycoside antibiotics and derivatives of kanamycin A that inhibit connexin hemichannels, but do not have antibiotic effect.
... The translocase of the outer membrane (TOM) complex is responsible for the initial translocation of approximately 90% of mitochondrial precursors from the cytosol to the IMS. Indeed, connexin 43, which is mainly localized at the sarcolemma, is translocated to the IMS side of the IMM via the TOM complex (Boengler et al., 2009;Rodriguez-Sinovas et al., 2006). Thus, it is possible that Mb is also imported into mitochondria through the TOM complex. ...
Mitochondria play a principal role in metabolism, and mitochondrial respiration is an important process for producing adenosine triphosphate. Recently, we showed the possibility that the muscle-specific protein myoglobin (Mb) interacts with mitochondrial complex IV to augment the respiration capacity in skeletal muscles. However, the precise mechanism for the Mb-mediated upregulation remains under debate. The aim of this study was to ascertain whether Mb is truly integrated into the mitochondria of skeletal muscle and to investigate the submitochondrial localization. Isolated mitochondria from rat gastrocnemius muscle were subjected to different proteinase K (PK) concentrations to digest proteins interacting with the outer membrane. Western blotting analysis revealed that the PK digested translocase of outer mitochondrial membrane 20 (Tom20), and the immunoreactivity of Tom20 decreased with the amount of PK used. However, the immunoreactivity of Mb with PK treatment was better preserved, indicating that Mb is integrated into the mitochondria of skeletal muscle. The mitochondrial protease protection assay experiments suggested that Mb localizes within the mitochondria in the inner membrane from the intermembrane space side. These results strongly suggest that Mb inside muscle mitochondria could be implicated in the regulation of mitochondrial respiration via complex IV.
... Il est intéressant de noter que la cardioprotection induite par l'PI est abolie chez les souris hétérozygotes pour la Cx43, où lorsqu'on bloque les JG 275,276 . Enfin, notons que de nombreuses études mettent en évidence également la Cx43 mitochondriale dans le PI cardiaque, part son implication dans la production de ROS et la survie cellulaire [277][278][279][280] . ...
Les insuffisances rénales et cardiaques demeurent des problèmes majeurs de santé publique. Ainsi, mes recherches sont articulées sur 2 axes, l’un cardiaque et l’autre rénal. Notch3 joue un rôle majeur dans la physiopathologie vasculaire: en contrôlant la prolifération et la maturation des cellules musculaires lisses vasculaires (CMLV), ce récepteur est nécessaire à l’adaptation cardiaque lors d’une hypertension artérielle (HTA). Mes objectifs étaient d’étudier les effets de la suractivation de la signalisation de Notch3 dans les CMLV lors d’une HTA et son implication lors d’un remodelage cardiaque physiologique induit par un entrainement physique modéré (EP). Les souris surexprimant Notch3 dans les CMLV développent une HTA, mais présentent une hypertrophie cardiaque et une fibrose moindres en réponse à l’AngII. De plus, l’EP permet de contrecarrer les défauts liés à l’absence de Notch3 car la cardiopathie des souris Notch3-/- régresse après 5 semaines d’EP. La connexine 43 (Cx43), protéine constitutive des jonctions gap, est anormalement exprimée dans la néphropathie chronique et sa réduction génétique protège contre la maladie. Ainsi, nous avons évalué son rôle dans la l’insuffisance rénale aigüe (IRA). Nous avons montré que les souris Cx43+/- avaient une fonction et une structure rénales améliorées par rapport aux WT après IRA. Cette protection semble liée à une réponse inflammatoire modérée. Cependant, la délétion ciblée de Cx43 dans les cellules endothéliales ou les tubules rénaux ne semble pas reproduire le phénotype observé chez des Cx43+/-. Ainsi, la réduction simultanée de Cx43 dans plusieurs types cellulaires est indispensable contre la progression de l’IRA.
... In addition to the plasma membrane, Cx43 is localized at the inner membrane of mitochondria isolated from ventricular myocardium, specifically in subsarcolemmal mitochondria (SSM) [7,8]. Cx43, which is encoded in the nucleus, is imported into the SSM in an Hsp90 (heat shock protein 90)/TOM (translocase of the outer membrane)-dependent pathway [9]. Only limited amounts of Cx43 are detected within interfibrillar mitochondria (IFM), which differ from the subsarcolemmal mitochondria in terms of respiration and calcium handling [10,11]. ...
In the heart, connexins form gap junctions, hemichannels, and are also present within mitochondria, with connexin 43 (Cx43) being the most prominent connexin in the ventricles. Whereas the role of Cx43 is well established for the healthy and diseased left ventricle, less is known about the importance of Cx43 for the development of right ventricular (RV) dysfunction. The present article focusses on the importance of Cx43 for the developing heart. Furthermore, we discuss the expression and localization of Cx43 in the diseased RV, i.e., in the tetralogy of Fallot and in pulmonary hypertension, in which the RV is affected, and RV hypertrophy and failure occur. We will also introduce other Cx molecules that are expressed in RV and surrounding tissues and have been reported to be involved in RV pathophysiology. Finally, we highlight therapeutic strategies aiming to improve RV function in pulmonary hypertension that are associated with alterations of Cx43 expression and function.
... Connexin hemichannels have been shown to release NAD + , a co-enzyme involved in many metabolic pathways [21,207], suggesting that hemichannels may work to support high levels of glycolysis in surrounding cells while potentially decreasing glycolysis in cancer cells themselves. Additionally, while it remains to be seen whether this is also the case in cancer cells, Cx43 has been shown to localize to mitochondria, in particular the inner mitochondrial membrane [208,209], in cardiomyocytes. It appears likely that the protein exists as part of a functional hemichannel structure there, as cross-linking studies suggest the presence of connexin hexamers that are capable of transfer of Lucifer yellow and can be inhibited by CBX and heptanol [208]. ...
The expression, localization, and function of connexins, the protein subunits that comprise gap junctions, are often altered in cancer. In addition to cell–cell coupling through gap junction channels, connexins also form hemichannels that allow communication between the cell and the extracellular space and perform non-junctional intracellular activities. Historically, connexins have been considered tumor suppressors; however, they can also serve tumor-promoting functions in some contexts. Here, we review the literature surrounding connexins in cancer cells in terms of specific connexin functions and propose that connexins function upstream of most, if not all, of the hallmarks of cancer. The development of advanced connexin targeting approaches remains an opportunity for the field to further interrogate the role of connexins in cancer phenotypes, particularly through the use of in vivo models. More specific modulators of connexin function will both help elucidate the functions of connexins in cancer and advance connexin-specific therapies in the clinic.
... 124 Inhibition of the mitochondrial import of Cx43 abolished MI size reduction by pharmacological pre-conditioning. 125 Although mitochondrial Cx43 increased after I/R, such an increase was absent in hearts undergoing a IPC protocol, 126 and the phosphorylation status of mitochondrial Cx43 was preserved. 127 Although isolated mitochondria can be protected by IPC, such beneficial effects depend on the presence of Cx43. ...
Acute myocardial infarction (AMI) and the heart failure (HF) that often result remain the leading causes of death and disability worldwide. As such, new therapeutic targets need to be discovered to protect the myocardium against acute ischaemia/reperfusion (I/R) injury in order to reduce myocardial infarct (MI) size, preserve left ventricular function and prevent the onset of HF. Mitochondrial dysfunction during acute I/R injury is a critical determinant of cell death following AMI, and therefore, ion channels in the inner mitochondrial membrane, which are known to influence cell death and survival, provide potential therapeutic targets for cardioprotection. In this article, we review the role of mitochondrial ion channels, which are known to modulate susceptibility to acute myocardial I/R injury, and we explore their potential roles as therapeutic targets for reducing MI size and preventing HF following AMI.
... In this study, pharmacological inhibition of mtCx43 induced opening of mPTP in SSM by increased levels of Ca 2+ [123]. Another study with IPC abolishment in which reduction of mtCx43 was induced by geldanamycin (which prevents translocation of Cx43 to mitochondria by blocking the HSP90 dependent pathway) confirmed that only mtCx43 is implicated in this cardioprotection [124]. ...
Therapies intended to mitigate cardiovascular complications cannot be applied in practice without detailed knowledge of molecular mechanisms. Mitochondria, as the end-effector of cardioprotection, represent one of the possible therapeutic approaches. The present review provides an overview of factors affecting the regulation processes of mitochondria at the level of mitochondrial permeability transition pores (mPTP) resulting in comprehensive myocardial protection. The regulation of mPTP seems to be an important part of the mechanisms for maintaining the energy equilibrium of the heart under pathological conditions. Mitochondrial connexin 43 is involved in the regulation process by inhibition of mPTP opening. These individual cardioprotective mechanisms can be interconnected in the process of mitochondrial oxidative phosphorylation resulting in the maintenance of adenosine triphosphate (ATP) production. In this context, the degree of mitochondrial membrane fluidity appears to be a key factor in the preservation of ATP synthase rotation required for ATP formation. Moreover, changes in the composition of the cardiolipin’s structure in the mitochondrial membrane can significantly affect the energy system under unfavorable conditions. This review aims to elucidate functional and structural changes of cardiac mitochondria subjected to preconditioning, with an emphasis on signaling pathways leading to mitochondrial energy maintenance during partial oxygen deprivation.
... Cx43 exists not only in cell membrane [29], but also in the mitochondrial intima [30]. Cx43 was transported to the inner membrane of mitochondria through the general outer membrane transporter/intimal transporter pathway, and the transport pathway depended on the cytoplasmic chaperone heat shock protein 90 [31]. Our results show that the deletion of mitochondrial DNA leads to the increase of the expression level of gap junction protein in SK-Hep1 hepatocellular carcinoma cells. ...
... Presence of Cx43 in the mitochondria is a relatively recent discovery and has gained significant attention [55][56][57][58][59][60][61][62][63][64][65][66][67][68][69][70][71]. A study showed that inhibition of mtCx43 activity can promote cytochrome c, and thereby promote apoptosis [60]. ...
At the core of proper mitochondrial functionality is the maintenance of its structure and morphology. Physical changes in mitochondrial structure alter metabolic pathways inside mitochondria, affect mitochondrial turnover, disturb mitochondrial dynamics, and promote mitochondrial fragmentation, ultimately triggering apoptosis. In high glucose condition, increased mitochondrial fragmentation contributes to apoptotic death in retinal vascular and Müller cells. Although alterations in mitochondrial morphology have been detected in several diabetic tissues, it remains to be established in the vascular cells of the diabetic retina. From a mechanistic standpoint, our current work supports the notion that increased expression of fission genes and decreased expression of fusion genes are involved in promoting excessive mitochondrial fragmentation. While mechanistic insights are only beginning to reveal how high glucose alters mitochondrial morphology, the consequences are clearly seen as release of cytochrome c from fragmented mitochondria triggers apoptosis. Current findings raise the prospect of targeting excessive mitochondrial fragmentation as a potential therapeutic strategy for treatment of diabetic retinopathy. While biochemical and epigenetic changes have been reported to be associated with mitochondrial dysfunction, this review focuses on alterations in mitochondrial morphology, and their impact on mitochondrial function and pathogenesis of diabetic retinopathy.
... it forms gap junction channels that are essential for the propagation of electrical depolarization in cardiomyocytes (Rodriguez-Sinovas et al., 2006;Soetkamp et al., 2014). Inhibition of this gap junction channel formation leads to an increase in mPTP calcium sensitivity and eliminates cardioprotection (Srisakuldee et al., 2014). ...
It is now firmly established that an important event in the formation of reperfusion injury of the heart is the opening of mitochondrial permeability transition pores (mPTPs), which changes the permeability of the mitochondria. mPTP opening results in the death of cardiomyocytes through activation of apoptosis and necroptosis. Experimental studies have shown that pharmacological inhibition of mPTP opening promotes the reduction in the infarct size and the suppression of apoptosis. Indeed, studies have shown the efficacy of mPTP inhibitors in animal models of myocardial reperfusion and isolated human myocardial trabeculae. However, clinical trials of cyclosporin A and TRO40303 have not provided a clear answer to the question of the effectiveness of mPTP inhibitors in patients with acute myocardial infarction. This article presents an analysis of possible approaches for the pharmacological regulation of mPTP during reperfusion injury of the heart.
On the one hand, reactive oxygen species (ROS) are involved in the onset and progression of a wide array of diseases. On the other hand, these are a part of signaling pathways related to cell metabolism, growth and survival. While ROS are produced at various cellular sites, in cardiomyocytes the largest amount of ROS is generated by mitochondria. Apart from the electron transport chain and various other proteins, uncoupling protein (UCP) and monoamine oxidases (MAO) have been proposed to modify mitochondrial ROS formation. Here, we review the recent information on UCP and MAO in cardiac injuries induced by ischemia-reperfusion (I/R) as well as protection from I/R and heart failure secondary to I/R injury or pressure overload. The current data in the literature suggest that I/R will preferentially upregulate UCP2 in cardiac tissue but not UCP3. Studies addressing the consequences of such induction are currently inconclusive because the precise function of UCP2 in cardiac tissue is not well understood, and tissue- and species-specific aspects complicate the situation. In general, UCP2 may reduce oxidative stress by mild uncoupling and both UCP2 and UCP3 affect substrate utilization in cardiac tissue, thereby modifying post-ischemic remodeling. MAOs are important for the physiological regulation of substrate concentrations. Upon increased expression and or activity of MAOs, however, the increased production of ROS and reactive aldehydes contribute to cardiac alterations such as hypertrophy, inflammation, irreversible cardiomyocyte injury, and failure.
Metabolic reprogramming of macrophages initiates the polarization of pro-inflammatory macrophages that exacerbates adipocyte dysfunction and obesity. The imbalance of mitochondrial Ca2+ homeostasis impairs mitochondrial function and promotes inflammation. Connexin 43 (Cx43), a ubiquitous gap junction protein, has been demonstrated to regulate intracellular Ca2+ homeostasis. Here we explored whether macrophage Cx43 affects the obesity process by regulating the polarization of macrophage. HFD treatment induced obesity and exacerbated macrophages infiltration with upregulation of macrophages Cx43. Macrophage-specific knockout of Cx43 reduced HFD-induced obesity by alleviating inflammation in adipose tissue, with less pro-inflammatory M1 macrophage infiltration. Consistently, inhibition or knockdown of Cx43 improved palmitic acid (PA) induced mitochondrial dysfunction, as indicated by improved oxidative phosphorylation (OXPHOS), reduced formation of mitochondria-associated membranes (MAM) and mitochondrial Ca2+ overload. Mechanistically, Cx43 interacted with the mitochondrial Ca2+ uniporter (MCU) and knockdown of Cx43 alleviated PA-induced succinate dehydrogenase (SDH) oxidation by lowering MCU-mediated mitochondrial Ca2+ uptake, which then, promoting the polarization of pro-inflammatory M1 macrophages. Thus, this study identified Cx43 as a mitochondrial Ca2+ regulator that aggravates obesity via promoting macrophages polarized to M1 pro-inflammatory phenotype and suggests that Cx43 might be a promising therapeutic target antagonizing obesity.
Traumatic brain injury (TBI) can lead to neurodegenerative diseases such as Alzheimer’s disease (AD) through mechanisms that remain incompletely characterized. Similar to AD, TBI models present with cellular metabolic alterations and modulated cleavage of amyloid precursor protein (APP). Specifically, AD and TBI tissues display increases in amyloid-β as well as its precursor, the APP C-terminal fragment of 99 a.a. (C99). Our recent data in cell models of AD indicate that C99, due to its affinity for cholesterol, induces the formation of transient lipid raft domains in the ER known as mitochondria-associated endoplasmic reticulum (ER) membranes (“MAM” domains). The formation of these domains recruits and activates specific lipid metabolic enzymes that regulate cellular cholesterol trafficking and sphingolipid turnover. Increased C99 levels in AD cell models promote MAM formation and significantly modulate cellular lipid homeostasis. Here, these phenotypes were recapitulated in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, the injured cortex and hippocampus displayed significant increases in C99 and MAM activity, as measured by phospholipid synthesis, sphingomyelinase activity and cholesterol turnover. In addition, our cell type-specific lipidomics analyses revealed significant changes in microglial lipid composition that are consistent with the observed alterations in MAM-resident enzymes. Altogether, we propose that alterations in the regulation of MAM and relevant lipid metabolic pathways could contribute to the epidemiological connection between TBI and AD.
Graphical Abstract
Cardiomyocyte death caused by hypoxia is one of the main causes of myocardial infarction or heart failure, and mitochondria play an important role in this process. Agrimonolide (AM) is a monomeric component extracted from Agrimonia pilosa L. and has antioxidant, antitumor, and anti‐inflammatory effects. This study aimed to investigate the role and mechanism of AM in improving hypoxia‐induced H9c2 cell damage. The results showed that low AM concentrations promote H9c2 cell proliferation and increase cellular ATP content. Transcriptome sequencing showed that AM induces differential expression of genes in H9c2 cells. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that these genes were concentrated in mitochondrial function. Subsequent experiments confirmed that AM regulates hypoxia‐induced cell cycle arrest. AM inhibited the rate of apoptosis by regulating the expression of apoptosis‐related proteins, reducing the level of cleaved Caspase 3 and Bax, and increasing the level of Bcl2, thereby protecting H9c2 cells from hypoxia‐induced apoptosis. AM restored the mitochondrial membrane potential, inhibited the generation of ROS, maintained the normal shape of the mitochondria, improved the level of the mitochondrial functional proteins OPA1, MFN1, MFN2, Tom20, and increased the level of ATP. In conclusion, AM protects H9c2 cells from hypoxia‐induced cell damage.
Hepatic ischemia reperfusion injury (IRI) occurs in liver transplantation, complex liver resection, and hemorrhagic shock, which causes donor organ shortage and hepatic damage. The burst of reactive oxygen species (ROS) during reperfusion leads to cell apoptosis and necroptosis. It has been reported that estrogen could attenuate hepatic IRI. G protein estrogen receptor (GPER) mediates estrogen effects via nonclassic receptor systems. Here, we investigate whether estrogen protecting liver from hepatic IRI depends on GPER and the influence of GPER activation on hepatocyte necroptosis. We proved that estrogen had a protective effect on both hepatocyte hypoxia re-oxygen (H/R) challenge and mouse hepatic ischemia reperfusion model. However, the application of GPER specific antagonist G15 before estrogen inhibited this beneficial effect. The results of mitochondria functional measurement revealed that estrogen improved hepatocyte mitochondria function by activating GPER, which might benefit from the increased expression of connexin 43 (Cx43) in mitochondria. To investigate the relationship between GPER activation and necroptosis, we used caspase-3/7 inhibitor benzyloxycarbonyl-Asp(OMe)-Glu(OMe)-Val-Asp(OMe)-chloromethylketone (Z-DEVD-FMK) to eliminate the interference of apoptosis. Estrogen showed a protective effect on hepatic IRI after using Z-DEVD-FMK, which could be suppressed by G15. GPER activation decreased the level of receptor interacting protein kinase (RIPK) 3, phosphorylated (p-) RIPK1, and p-mixed lineage kinase domain-like (MLKL). The co-immunoprecipitation result indicated that GPER could bind with RIPK3. GPER is indispensable in estrogen protecting liver from IRI. GPER activation attenuates hepatocyte necroptosis by decreasing the level of RIPK3, p-RIPK1, and p-MLKL.
A well-balanced intercellular communication between the different cells within the heart is vital for the maintenance of cardiac homeostasis and function. Despite remarkable advances on disease management and treatment, acute myocardial infarction remains the major cause of morbidity and mortality worldwide. Gold standard reperfusion strategies, namely primary percutaneous coronary intervention, are crucial to preserve heart function. However, reestablishment of blood flow and oxygen levels to the infarcted area are also associated with an accumulation of reactive oxygen species (ROS), leading to oxidative damage and cardiomyocyte death, a phenomenon termed myocardial reperfusion injury. In addition, ROS signaling has been demonstrated to regulate multiple biological pathways, including cell differentiation and intercellular communication. Given the importance of cell-cell crosstalk in the coordinated response after cell injury, in this review, we will discuss the impact of ROS in the different forms of inter- and intracellular communication, as well as the role of gap junctions, tunneling nanotubes and extracellular vesicles in the propagation of oxidative damage in cardiac diseases, particularly in the context of ischemia/reperfusion injury.
Excessive mitochondrial ROS production has been causally linked to the pathophysiology of aging in the heart and other organs, and plays a deleterious role in several age-related cardiac pathologies, including myocardial ischemia-reperfusion injury and heart failure, the two worldwide leading causes of death and disability in the elderly. However, ROS generation is also a fundamental mitochondrial function that orchestrates several signaling pathways, some of them exerting cardioprotective effects. In cardiac myocytes, mitochondria are particularly abundant and are specialized in subcellular populations, in part determined by their relationships with other organelles and their cyclic calcium handling activity necessary for adequate myocardial contraction/relaxation and redox balance. Depending on their subcellular location, mitochondria can themselves be differentially targeted by ROS and display distinct age-dependent functional decline. Thus, precise mitochondria-targeted therapies aimed at counteracting unregulated ROS production are expected to have therapeutic benefits in certain aging-related heart conditions. However, for an adequate design of such therapies, it is necessary to unravel the complex and dynamic interactions between mitochondria and other cellular processes.
Traumatic brain injury (TBI) is a major cause of death and disability in the United States. A history of TBI can lead to neurodegenerative diseases such as Alzheimer's disease (AD) in a severity-and frequency-dependent manner. We previously reported that early stages of AD are characterized by alterations in lipid metabolism due to upregulated functionality of mitochondria-associated ER membranes ("MAM" domains of the ER), a cellular hub of lipid metabolic regulation. This can be caused by increased localization of amyloid precursor protein (APP)'s C-terminal fragment of 99 a.a. (C99) at MAM, which promotes cellular cholesterol uptake and trafficking to the ER. Through this mechanism, MAM-localized C99 can stimulate MAM functionality. In this study, we recapitulate these phenotypes in the controlled cortical impact (CCI) model of TBI in adult mice. Specifically, we observed increased phospholipid synthesis, sphingomyelinase activity and cholesterol esterification in the cortex and hippocampus 1, 3 and 7 days after injury. These responses were predominant in microglia, and coincided with increased levels of MAM-localized C99. Altogether, we propose that upregulation of MAM functionality could contribute, in part, to the epidemiological connection between TBI and AD.
Despite the known risk of cardiotoxicity, anthracyclines are widely prescribed chemotherapeutic agents. They are broadly characterized as being a robust effector of cellular apoptosis in rapidly proliferating cells through its actions in the nucleus and formation of reactive oxygen species (ROS). And, despite the early use of dexrazoxane, no effective treatment strategy has emerged to prevent the development of cardiomyopathy, despite decades of study, suggesting that much more insight into the underlying mechanism of the development of cardiomyopathy is needed. In this review, we detail the specific intracellular activities of anthracyclines, from the cell membrane to the sarcoplasmic reticulum, and highlight potential therapeutic windows that represent the forefront of research into the underlying causes of anthracycline-induced cardiomyopathy.
The Atypical Chemokine Receptor 3 (ACKR3) and CXCR4 are two G protein-coupled receptors (GPCR) belonging to the CXC chemokine receptor family. Both receptors are activated upon CXCL12 binding and are over-expressed in various tumours, including glioma, where they have been found to promote proliferation and invasive behaviours. Upon CXCL12 binding, CXCR4 activates canonical GPCR signalling pathways involving Gαi protein and β-arrestins. In addition, CXCR4 was found to interact with several proteins able to modify its signalling, trafficking and localization. In contrast, the cellular pathways underlying ACKR3-dependent effects remain poorly characterized. Several reports show that ACKR3 engages β-arrestin-dependent signalling pathways, but its coupling to G proteins is restricted to either specific cellular populations, including astrocytes, or occurs indirectly via its interaction with CXCR4. ACKR3 also associates with the epidermal growth factor receptor to promote proliferation of tumour cells in an agonist-independent manner. These examples suggest that the extensive characterization of ACKR3 and CXCR4 interactomes might be a key step in understanding or clarifying their roles in physiological and pathological contexts. This thesis addressed this issue employing an affinity purification coupled to high-resolution mass spectrometry proteomic strategy that identified 19 and 151 potential protein partners of CXCR4 and ACKR3 transiently expressed in HEK-293T cells, respectively. Amongst ACKR3 interacting proteins identified, we paid particular attention on the gap junction protein Connexin-43 (Cx43), in line with its overlapping roles with the receptor in the control of leukocyte entry into the brain, interneuron migration and glioma progression. Western blotting and BRET confirmed the specific association of Cx43 with ACKR3 compared to CXCR4. Likewise, Cx43 is co-localized with ACKR3 but not CXCR4 in glioma initiating cell lines, and ACKR3 and Cx43 are co-expressed in astrocytes of the sub-ventricular zone and surrounding blood vessels in adult mouse brain, suggesting that both proteins form a complex in authentic cell or tissue contexts. Further functional studies showed that ACKR3 influences Cx43 trafficking and functionality at multiple levels. Transient expression of ACKR3 in HEK-293T cells to mimic ACKR3 overexpression detected in several cancer types, induces Gap Junctional Intercellular Communication (GJIC) inhibition in an agonist-independent manner. In addition, agonist stimulation of endogenously expressed ACKR3 in primary cultured astrocytes inhibits Cx43-mediated GJIC through a mechanism that requires activation of Gαi protein, and dynamin- and β-arrestin2-dependent Cx43 internalisation. Therefore, this thesis work provides the first functional link between the CXCL11/CXCL12/ACKR3 axis and gap junctions that might underlie their critical role in glioma progression.
Acute ST-segment elevation myocardial infarction (STEMI) remains a leading cause of morbidity and mortality around the world. A large number of STEMI patients after the infarction gradually develop heart failure due to the infarcted myocardium. Timely reperfusion is essential to salvage ischemic myocardium from the infarction, but the restoration of coronary blood flow in the infarct-related artery itself induces myocardial injury and cardiomyocyte death, known as ischemia/reperfusion injury (IRI). The factors contributing to IRI in STEMI are complex, and microvascular obstruction, inflammation, release of reactive oxygen species, myocardial stunning, and activation of myocardial cell death are involved. Therefore, additional cardioprotection is required to prevent the heart from IRI. Although many mechanical conditioning procedures and pharmacological agents have been identified as effective cardioprotective approaches in animal studies, their translation into the clinical practice has been relatively disappointing due to a variety of reasons. With new emerging data on cardioprotection in STEMI over the past few years, it is mandatory to reevaluate the effectiveness of “old” cardioprotective interventions and highlight the novel therapeutic targets and new treatment strategies of cardioprotection.
The epsilon isoform of protein kinase C (PKCepsilon) is a member of the PKC family of serine/threonine kinases and plays a critical role in protection against ischemic injury in multiple organs. Functional proteomic analyses of PKCepsilon signaling show that this isozyme forms multiprotein complexes in the heart; however, the precise signaling mechanisms whereby PKCepsilon orchestrates cardioprotection are poorly understood. Here we report that Lck, a member of the Src family of tyrosine kinases, forms a functional signaling module with PKCepsilon. In cardiac cells, PKCepsilon interacts with, phosphorylates, and activates Lck. In vivo studies showed that cardioprotection elicited either by cardiac-specific transgenic activation of PKCepsilon or by ischemic preconditioning enhances the formation of PKCepsilon-Lck modules. Disruption of these modules, via ablation of the Lck gene, abrogated the infarct-sparing effects of these two forms of cardioprotection, indicating that the formation of PKCepsilon-Lck signaling modules is required for the manifestation of a cardioprotective phenotype. These findings demonstrate, for the first time to our knowledge, that the assembly of a module (PKCepsilon-Lck) is an obligatory step in the signal transduction that results in a specific phenotype. Thus, PKCepsilon-Lck modules may serve as novel therapeutic targets for the prevention of ischemic injury.
Gap junction channels are major determinants of intercellular resistance to current flow between cardiac myocytes. Alterations in gap junctions may contribute to development of arrhythmia substrates in patients. However, there is significant interspecies variation in the types and amounts of gap junction subunit proteins (connexins) expressed in disparate regions of mammalian hearts. To elucidate determinants of conduction properties in the human heart, we characterized connexin phenotypes of specific human cardiac tissues with different conduction properties.
The distribution and relative abundance of Cx37, Cx40, Cx43, Cx45, and Cx46 were studied immunohistochemically using monospecific antibodies and frozen sections of the sinoatrial node and adjacent atria. AV node and His bundle, the bundle branches, and the left and right ventricular walls. Patterns of expression of these connexins in the human heart differed from those in previous animal studies. Sinus node gap junctions were small and sparse and contained Cx45 and apparently smaller amounts of Cx40 but no Cx43. AV node gap junctions were also small and contained mainly Cx45 and Cx40 but, unlike the sinus node, also expressed Cx43. Atrial gap junctions were larger than nodal junctions and contained moderate amounts of Cx40, Cx43, and Cx45. Junctions in the bundle branches were the largest in size and contained abundant amounts of Cx40, Cx43, and Cx45. Gap junctions in ventricular myocardium contained mainly Cx43 and Cx45; only a very small and amount of ventricular Cx40 was detected in subendocardial myocyte junctions and endothelial cells of small to medium sized intramural coronary arteries. Minimal Cx37 and Cx46 immunoreactivity was detected between occasional atrial or ventricular myocytes.
The relative amounts of individual connexins and the number and size of gap junctions vary greatly in specific regions of the human heart with different conduction properties. These differences likely play a role in regulating cardiac conduction velocity. Differences in the connexin phenotypes of specific regions of the human heart and experimental animal hearts must be considered in future experimental or modeling studies of cardiac conduction.
Preproteins destined for mitochondria either are synthesized with amino-terminal signal sequences, termed presequences, or possess internal targeting information within the protein. The preprotein translocase of the outer mitochondrial membrane (designated Tom) contains specific import receptors. The cytosolic domains of three import receptors, Tom20, Tom22, and Tom70, have been shown to interact with preproteins. Little is known about the internal targeting information in preproteins and the distribution of binding sequences for the three import receptors. We have studied the binding of the purified cytosolic domains of Tom20, Tom22, and Tom70 to cellulose-bound peptide scans derived from a presequence-carrying cleavable preprotein, cytochrome c oxidase subunit IV, and a non-cleavable preprotein with internal targeting information, the phosphate carrier. All three receptor domains are able to bind efficiently to linear 13-mer peptides, yet with different specificity. Tom20 preferentially binds to presequence segments of subunit IV. Tom22 binds to segments corresponding to the carboxyl-terminal part of the presequence and the amino-terminal part of the mature protein. Tom70 does not bind efficiently to any region of subunit IV. In contrast, Tom70 and Tom20 bind to multiple segments within the phosphate carrier, yet the amino-terminal region is excluded. Both charged and uncharged peptides derived from the phosphate carrier show specific binding properties for Tom70 and Tom20, indicating that charge is not a critical determinant of internal targeting sequences. This feature contrasts with the crucial role of positively charged amino acids in presequences. Our results demonstrate that linear peptide segments of preproteins can serve as binding sites for all three receptors with differential specificity and imply different mechanisms for translocation of cleavable and non-cleavable preproteins.
The critical time for opening mitochondrial (mito) K(ATP) channels, putative end effectors of ischemic preconditioning (PC), was examined. In isolated rabbit hearts 29+/-3% of risk zone infarcted after 30 minutes of regional ischemia. Ischemic PC or 5-minute exposure to 10 micromol/L diazoxide, a mito K(ATP) channel opener, reduced infarction to 3+/-1% and 8+/-1%, respectively. The mito K(ATP) channel closer 5-hydroxydecanoate (200 micromol/L), bracketing either 5-minute PC ischemia or diazoxide infusion, blocked protection (24+/-3 and 28+/-6% infarction, respectively). However, 5-hydroxydecanoate starting 5 minutes before long ischemia did not affect protection. Glibenclamide (5 micromol/L), another K(ATP) channel closer, blocked the protection by PC only when administered early. These data suggest that K(ATP) channel opening triggers protection but is not the final step. Five minutes of diazoxide followed by a 30-minute washout still reduced infarct size (8+/-3%), implying memory as seen with other PC triggers. The protection by diazoxide was not blocked by 5 micromol/L chelerythrine, a protein kinase C antagonist, given either to bracket diazoxide infusion or just before the index ischemia. Bracketing preischemic exposure to diazoxide with 50 micromol/L genistein, a tyrosine kinase antagonist, did not affect infarction, but genistein blocked the protection by diazoxide when administered shortly before the index ischemia. Thus, although it is not protein kinase C-dependent, the protection by diazoxide involves tyrosine kinase. Bracketing diazoxide perfusion with N:-(2-mercaptopropionyl) glycine (300 micromol/L) or Mn(III)tetrakis(4-benzoic acid) porphyrin chloride (7 micromol/L), each of which is a free radical scavenger, blocked protection, indicating that diazoxide triggers protection through free radicals. Therefore, mito K(ATP) channels are not the end effectors of protection, but rather their opening before ischemia generates free radicals that trigger entrance into a preconditioned state and activation of kinases.
There is recent evidence that Ca(2+) influx via reverse mode Na(+)/Ca(2+) exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca(2+)](i) during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability.
The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca(2+)](i) and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion.
In myocytes, KB-R7943 at concentrations up to 15 microM reduced [Ca(2+)](i) rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3-30 microM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 microM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations > or = 15 microM, which increased LDH. KB-R7943 at 5 microM given during the first 10 min of reflow reduced contractile dysfunction (P=0.011), LDH release (P=0.019) and contraction band necrosis (P=0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P=0.033) in pigs submitted to 48 min of coronary occlusion.
These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca(2+) overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca(2+) extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.
Recent evidence suggests that opening of mitochondrial K(ATP) channels in cardiac muscle triggers the preconditioning phenomenon through free radical production. The present study tested the effects of K(ATP) channel openers in a vascular smooth muscle cell model using the fluorescent probe MitoTracker (MTR) Red trade mark for detection of reactive oxygen species (ROS). Rat aortic smooth muscle cells (A7r5) were incubated with 1 micro M reduced MTR (non-fluorescent) and the MTR oxidation product (fluorescent) was quantified. Thirty-minute pretreatment with either diazoxide (200 micro M) or pinacidil (100 micro M), both potent mitochondrial K(ATP) channel openers, increased fluorescent intensity (FI) to 149 and 162 % of control (p < 0.05 for both), respectively, and the K(ATP) channel inhibitor 5-hydroxydecanoate (5 HD) blocked it. Valinomycin, a potassium-selective ionophore, raised FI to 156 % of control (p <: 0.05). However, 5 HD did not affect the valinomycin-induced increase in FI. Inhibition of mitochondrial electron transport (myxothiazol) or uncoupling of oxidative phosphorylation (dinitrophenol) also blocked either valinomycin- or diazoxide-induced increase in FI, and free radical scavengers prevented any diazoxide-mediated increase in fluorescence. Finally the diazoxide-induced increase in fluorescence was not blocked by the PKC inhibitor chelerythrine, but was by HMR 1883, a putative surface K(ATP) channel blocker. Thus opening of K(ATP) channels increases generation of ROS via the mitochondrial electron transport chain in vascular smooth muscle cells. Furthermore, a potassium-selective ionophore can mimic the effect of putative mitochondrial KATP channel openers. We conclude that potassium movement through KATP directly leads to ROS production by the mitochondria.
Studies with different ATP-sensitive potassium (K(ATP)) channel openers and blockers have implicated opening of mitochondrial K(ATP) (mitoK(ATP)) channels in ischaemic preconditioning (IPC). It would be predicted that this should increase mitochondrial matrix volume and hence respiratory chain activity. Here we confirm this directly using mitochondria rapidly isolated from Langendorff-perfused hearts. Pre-ischaemic matrix volumes for control and IPC hearts (expressed in microl per mg protein +/- S.E.M., n = 6), determined with (3)H(2)O and [(14)C]sucrose, were 0.67 +/- 0.02 and 0.83 +/- 0.04 (P < 0.01), respectively, increasing to 1.01 +/- 0.05 and 1.18 +/- 0.02 following 30 min ischaemia (P < 0.01) and to 1.21 +/- 0.13 and 1.26 +/- 0.25 after 30 min reperfusion. Rates of ADP-stimulated (State 3) and uncoupled 2-oxoglutarate and succinate oxidation increased in parallel with matrix volume until maximum rates were reached at volumes of 1.1 microl ml(-1) or greater. The mitoK(ATP) channel opener, diazoxide (50 microM), caused a similar increase in matrix volume, but with inhibition rather than activation of succinate and 2-oxoglutarate oxidation. Direct addition of diazoxide (50 microM) to isolated mitochondria also inhibited State 3 succinate and 2-oxoglutarate oxidation by 30 %, but not that of palmitoyl carnitine. Unexpectedly, treatment of hearts with the mitoK(ATP) channel blocker 5-hydroxydecanoate (5HD) at 100 or 300 microM, also increased mitochondrial volume and inhibited respiration. In isolated mitochondria, 5HD was rapidly converted to 5HD-CoA by mitochondrial fatty acyl CoA synthetase and acted as a weak substrate or inhibitor of respiration depending on the conditions employed. These data highlight the dangers of using 5HD and diazoxide as specific modulators of mitoK(ATP) channels in the heart.
The role of cytosolic factors in protein targeting to mitochondria is poorly understood. Here, we show that in mammals, the cytosolic chaperones Hsp90 and Hsp70 dock onto a specialized TPR domain in the import receptor Tom70 at the outer mitochondrial membrane. This interaction serves to deliver a set of preproteins to the receptor for subsequent membrane translocation dependent on the Hsp90 ATPase. Disruption of the chaperone/Tom70 recognition inhibits the import of these preproteins into mitochondria. In yeast, Hsp70 rather than Hsp90 is used in import, and Hsp70 docking is required for the formation of a productive preprotein/Tom70 complex. We outline a novel mechanism in which chaperones are recruited for a specific targeting event by a membrane-bound receptor.
To examine the participation of HSP90 in portal hypertensive rat mesentery in vitro.
Immunohistochemistry and Western-blot were used to examine the expression of HSP90 in mesenteric vasculature. HSP90 mRNA was detected by RT-PCR, and the role of HSP90 in hyperdynamic circulation was examined by in vitro mesenteric perfusion studies.
HSP90 was overexpressed in endothelium of mesentery vasculature in animals with experimental portal hypertension induced by partial portal vein ligation (PVL) compared with normal animals. Geldanamycin (GA), a special inhibitor of HSP90 signaling, attenuated ACh-dependent vasodilation but did not affect vasodilation in response to sodium nitroprusside in normal rats. In PVL animals, the perfused mesentery was hyporesponsive to vasoconstrictor methoxamine. GA significantly potentiated methoxamine-induced vasoconstrictor after PVL.
HSP90 plays a key role in NO-dependent hyperdynamic circulation in portal hypertension and provides a novel method for future treatment of portal hypertension.
Reperfusion of the heart after a period of ischaemia leads to the opening of a nonspecific pore in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore (MPTP). This transition causes mitochondria to become uncoupled and capable of hydrolysing rather than synthesising ATP. Unrestrained, this will lead to the loss of ionic homeostasis and ultimately necrotic cell death. The functional recovery of the Langendorff-perfused heart from ischaemia inversely correlates with the extent of pore opening, and inhibition of the MPTP provides protection against reperfusion injury. This may be mediated either by a direct interaction with the MPTP [e.g., by Cyclosporin A (CsA) and Sanglifehrin A (SfA)], or indirectly by decreasing calcium loading and reactive oxygen species (ROS; key inducers of pore opening) or lowering intracellular pH. Agents working in this way may include pyruvate, propofol, Na+/H+ antiporter inhibitors, and ischaemic preconditioning (IPC). Mitochondrial KATP channels have been implicated in preconditioning, but our own data suggest that the channel openers and blockers used in these studies work through alternative mechanisms. In addition to its role in necrosis, transient opening of the MPTP may occur and lead to the release of cytochrome c and other proapoptotic molecules that initiate the apoptotic cascade. However, only if subsequent MPTP closure occurs will ATP levels be maintained, ensuring that cell death continues down an apoptotic, rather than a necrotic, pathway.
Both estrogens and bisphosphonates attenuate osteocyte apoptosis by activating the extracellular signal-regulated kinases (ERKs). However, whereas estrogens activate ERKs via an extranuclear function of the estrogen receptor, bisphosphonates do so by opening connexin 43 hemichannels. Here, we demonstrated that the signaling events downstream of ERKs induced by these two stimuli are also distinct. Inhibition of osteocyte apoptosis by estrogens requires nuclear accumulation of ERKs and activation of downstream transcription factors. On the other hand, anti-apoptosis induced by bisphosphonates requires neither transcription nor ERK-dependent transcription factors. Instead, the effect of bisphosphonates is abolished when ERKs are restricted to the nucleus by blocking CRM1/exportin1-mediated nuclear protein export or by expressing nuclear-anchored ERKs, but it is unaffected in cells expressing cytoplasmic-anchored ERKs. Connexin 43/ERK-mediated anti-apoptosis induced by bisphosphonates requires the kinase activity of the cytoplasmic target of ERKs, p90(RSK), which in turn phosphorylates the pro-apoptotic protein BAD and C/EBPbeta. Phosphorylation of BAD renders it inactive, whereas phosphorylation of C/EBPbeta leads to binding of pro-caspases, thus inhibiting apoptosis independently of the transcriptional activity of this transcription factor. Consistent with the evidence that estrogens and bisphosphonates phosphorylate diverse targets of ERKs, probably resulting from activation of spatially distinct pools of these kinases, the two agents had additive effects on osteocyte survival.
Several signaling pathways that monitor the dynamic state of the cell converge on the tumor suppressor p53. The ability of p53 to process these signals and exert a dynamic downstream response in the form of cell cycle arrest and/or apoptosis is crucial for preventing tumor development. This p53 function is abrogated by p53 gene mutations leading to alteration of protein conformation. Hsp90 has been implicated in regulating both wild-type and mutant p53 conformations, and Hsp90 antagonists are effective for the therapy of some human tumors. Using cell lines that contain human tumor-derived temperature-sensitive p53 mutants we show that Hsp90 is required for both stabilization and reactivation of mutated p53 at the permissive temperature. A temperature decrease to 32 degrees C causes conversion to a protein conformation that is capable of inducing expression of MDM2, leading to reduction of reactivated p53 levels by negative feedback. Mutant reactivation is enhanced by simultaneous treatment with agents that stabilize the reactivated protein and is blocked by geldanamycin, a specific inhibitor of Hsp90 activity, indicating that Hsp90 antagonist therapy and therapies that act to reactivate mutant p53 will be incompatible. In contrast, Hsp90 is not required for maintaining wild-type p53 or for stabilizing wild-type p53 after treatment with chemotherapeutic agents, indicating that Hsp90 therapy might synergize with conventional therapies in patients with wild-type p53. Our data demonstrate the importance of the precise characterization of the interaction between p53 mutants and stress proteins, which may shed valuable information for fighting cancer via the p53 tumor suppressor pathway.
Pretreatment with D-myo-inositol-1,4,5-trisphosphate hexasodium (D-myo-IP(3)), the sodium salt of the second messenger inositol 1,4,5-trisphosphate (IP(3)), is cardioprotective and triggers a reduction of infarct size comparable in magnitude to that obtained with ischemic preconditioning. However, this observation is enigmatic; whereas IP(3) signaling is conventionally initiated by receptor binding, IP(3) receptors are typically considered to be intracellular, and D-myo-IP(3) is membrane-impermeable. We propose that this paradox is explained by the presence of poorly characterized external IP(3) receptors and hypothesize that: 1) infarct size reduction with D-myo-IP(3) is receptor-mediated; and 2) communication via gap junctions and/or hemichannels is required to initiate this protection. To investigate the role of receptor binding, isolated buffer-perfused rabbit hearts underwent 30 min of coronary occlusion (CO) and 2 h of reflow. Prior to CO, hearts received no treatment (controls), D-myo-IP(3), L-myo-IP(3) (enantiomer not recognized by the IP(3) receptor), D-myo-IP(3) + the IP(3) receptor inhibitor xestospongin C (XeC), or XeC alone. Infarct size, assessed by tetrazolium staining, was reduced with D-myo-IP(3) treatment, whereas hearts that received L-myo-IP(3) or D-myo-IP(3) + XeC showed no protection. To evaluate the contribution of gap junctions/hemichannels, additional control and D-myo-IP(3)-treated cohorts received a 5-min infusion of heptanol or Gap 27, two structurally distinct gap junction inhibitors, administered at doses confirmed to attenuate intercellular transmission of a gap junction-permeable fluorescent dye. There was no infarct-sparing effect of D-myo-IP(3) in inhibitor-treated hearts. These data support the concepts that infarct size reduction with D-myo-IP(3) is triggered by receptor binding and that communication via gap junctions/hemichannels is involved in initiating this protection.
The end-effectors of ischemic preconditioning (IPC) are not well known. It has been recently shown that transgenic mice underexpressing the gap junction protein connexin43 (Cx43) cannot be preconditioned. Because gap junctions allow spreading of cell death during ischemia-reperfusion in different tissues, including myocardium, we hypothesized that the protection afforded by IPC is mediated by effects on gap junction-mediated intercellular communication. To test this hypothesis, we analyzed the effect of IPC (5 min ischemia-5 min reperfusion x 2) on the changes in electrical impedance (four electrode probe) and impulse propagation velocity (transmembrane action potential) induced by ischemia (60 min) and reperfusion (60 min) in isolated rat hearts. IPC (n = 8) reduced reperfusion-induced lactate dehydrogenase release by 65.8% with respect to control hearts (n = 9) (P = 0.04) but had no effect on the time of onset of rigor contracture (increase in diastolic tension), electrical uncoupling (sharp changes in tissue resistivity and phase angle in impedance recordings), or block of impulse propagation during ischemia. Normalization of electrical impedance during reperfusion was also unaffected by IPC. The lack of effect of IPC on ischemic rigor contracture and on changes in tissue impedance during ischemia-reperfusion were validated under in vivo conditions in pigs submitted to 48 min of coronary occlusion and 120 min of reperfusion. IPC (n = 12) reduced infarct size (triphenyltetrazolium) by 64.9% (P = 0.01) with respect to controls (n = 17). We conclude that the protection afforded by IPC is not mediated by effects on electrical coupling. This result is consistent with recent findings suggesting that Cx43 could have effects on cell survival independent on changes in cell-to-cell communication.
Discovered in the cardiac sarcolemma, ATP-sensitive K+ (KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+ channel openers to examine the effect of mitochondrial KATP channel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+ transport, and matrix volume. From a mitochondrial membrane potential of -180 ± 15 mV, K+ channel openers, pinacidil (100 μM), cromakalim (25 μM), and levcromakalim (20 μM), induced membrane depolarization by 10 ± 7, 25 ± 9, and 24 ± 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+ or application of a KATP channel blocker. K+ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+ channel opener released Ca2+ from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+ concentration and sensitive to KATP channel blockade. In addition, K+ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome c and adenylate kinase. Thus, in isolated cardiac mitochondria, KATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATP channels in regulating functions vital for the cardiac mitochondria.
Time for primary review 25 days.
In clinical therapy of evolving acute myocardial infarction, coronary reperfusion has proven to be the only way to limit infarct size, provided it occurs soon enough after coronary artery occlusion. However, there is also evidence that reperfusion is accompanied by detrimental manifestations known as ‘reperfusion injury’. Reperfusion injury refers to a causal event associated with reperfusion that had not occurred during the preceding ischemic period and can be entirely attenuated by an intervention given only at the time of reperfusion. It classically includes myocardial stunning, reperfusion arrhythmias and lethal reperfusion injury. Clearly, reperfusion arrhythmias do not represent an important problem for the clinician because their incidence is very low and they can be quite easily treated. Myocardial stunning is usually not a major clinical problem in the context of acute myocardial infarction because it disappears spontaneously and is very sensitive to inotropic agents. Myocardial stunning becomes of serious concern only if the affected portion of the myocardium is very large.
Lethal reperfusion injury could be an interesting target for the clinician who is now able to promptly revascularize acutely ischemic myocardium. However, the existence of lethal reperfusion injury has been debated for years by scientists and is still controversial [1–4]. One of the problems is that the development of necrosis cannot be accurately followed in time, both in experimental animal preparations and in man. Instead, scientists have used an indirect approach to deal with this issue; the principle is to modify the nature of reperfusion and then assess whether the extent of necrosis is reduced. Numerous studies have been performed according to this general design. Unfortunately, many of them remained inconclusive, partly for technical reasons [5]. In most, actually hundreds of studies on lethal reperfusion injury, the effects of antioxidants were investigated. …
* Corresponding author. Tel.: +49 (641) 994-7241; Fax: +49 (641) 994-7239.
Objective: There is recent evidence that Ca2+ influx via reverse mode Na+/Ca2+ exchange (NCX) at the time of reperfusion can contribute to cardiomyocyte hypercontracture. However, forward NCX is essential for normalization of [Ca2+]i during reperfusion, and its inhibition may be detrimental. This study investigates the effect of NCX inhibition with KB-R7943 at the time of reperfusion on cell viability. Methods: The effect of several concentrations of KB-R7943 added at reperfusion was studied in Fura-2 loaded quiescent cardiomyocytes submitted to 40 min of simulated ischemia (NaCN 2 mM, pH 6.4), and in rat hearts submitted to 60 min of ischemia. [Ca2+]i and cell length were monitored in myocytes, and functional recovery and LDH release in isolated hearts. From these experiments an optimal concentration of KB-R7943 was identified and tested in pigs submitted to 48 min of coronary occlusion and 2 h of reperfusion. Results: In myocytes, KB-R7943 at concentrations up to 15 μM reduced [Ca2+]i rise and the probability of hypercontracture during re-energization (P<0.01). Nevertheless, in rat hearts, the effects of KB-R7943 applied during reperfusion after 60 min of ischemia depended on concentration and timing of administration. During the first 5 min of reperfusion, KB-R7943 (0.3–30 μM) induced a dose-dependent reduction in LDH release (half-response concentration 0.29 μM). Beyond 6 min of re-flow, KB-R7943 had no effect on LDH release, except at concentrations ≥15 μM, which increased LDH. KB-R7943 at 5 μM given during the first 10 min of reflow reduced contractile dysfunction (P = 0.011), LDH release (P = 0.019) and contraction band necrosis (P = 0.014) during reperfusion. Intracoronary administration of this concentration during the first 10 min of reperfusion reduced infarct size by 34% (P = 0.033) in pigs submitted to 48 min of coronary occlusion. Conclusions: These results are consistent with the hypothesis that during initial reperfusion NCX activity results in net reverse mode operation contributing to Ca2+ overload, hypercontracture and cell death, and that NCX inhibition during this phase is beneficial. Beyond this phase, NCX inhibition may impair forward mode-dependent Ca2+ extrusion and be detrimental. These findings may help in the design of therapeutic strategies against lethal reperfusion injury, with NCX as the target.
Connexins and Propagation. Gap junction channels are essential for normal cardiac impulse propagation. Three gap Junction proteins, known as connexins, are expressed in the heart: Cx40, Cx43, and Cx45. Each of these proteins forms channels with unique biophysical and electrophysiologic properties, as well as spatial distribution of expression throughout the heart. However, the specific functional role of the individual connexins in normal and abnormal propagation is unknown. The availability of genetically engineered mouse models, together with new developments in optical mapping technology, makes it possible to integrate knowledge about molecular mechanisms of intercellular communication and its regulation with our growing understanding of the microscopic and global dynamics of electrical impulse propagation during normal and abnormal cardiac rhythms. This article reviews knowledge on the mechanisms of cardiac impulse propagation, with particular focus on the role of cardiac connexins in electrical communication between cells. It summarizes results of recent studies on the electrophysiologic consequences of defects in the functional expression of specific gap junction channels in mice lacking either the Cx43 or Cx40 gene. It also reviews data obtained in a transgenic mouse model in which cell loss and remodeling of gap junction distribution leads to increased susceptibility to arrhythmias and sudden cardiac death. Overall, the results demonstrate that these are potentially powerful strategies for studying fundamental mechanisms of cardiac electrical activity and for testing the hypothesis that certain cardiac arrhythmias involve gap junction or other membrane channel dysfunction. These new approaches, which permit one to manipulate electrical wave propagation at the molecular level, should provide new insight into the detailed mechanisms of initiation, maintenance, and termination of cardiac arrhythmias, and may lead to more effective means to treat arrhythmias and prevent sudden cardiac death.
Blockers of ATP-sensitive K+ channels (KATP) abolish preconditioning in several species. Glyburide does not abolish preconditioning in rat hearts, but this may be due to a loss of its activity during ischemia. We determined the effect of a KATP blocker, which is more active during ischemia (sodium 5-hydroxydecanoate, 5-HD), on preconditioning in isolated rat hearts. Rat hearts were subjected to 4 periods of 5 min global ischemia followed by 30 min of global ischemia and reperfusion. Preconditioning significantly enhanced post-ischemic recovery of function and reduced lactate dehydrogenase (LDH) release vs. sham. 5-HD (100 microM) did not abolish preconditioning. Cromakalim (20 microM) was protective in this ischemic model and this was abolished by 5-HD. This is further evidence that KATP opening is not the mechanism of preconditioning in rats.
The Hsp90 chaperone is required for the activation of several families of eukaryotic protein kinases and nuclear hormone receptors, many of which are protooncogenic and play a prominent role in cancer. The geldanamycin antibiotic has antiproliferative and antitumor effects, as it binds to Hsp90, inhibits the Hsp90-mediated conformational maturation/refolding reaction, and results in the degradation of Hsp90 substrates. The structure of the geldanamycin-binding domain of Hsp90 (residues 9-232) reveals a pronounced pocket, 15 A deep, that is highly conserved across species. Geldanamycin binds inside this pocket, adopting a compact structure similar to that of a polypeptide chain in a turn conformation. This, and the pocket's similarity to substrate-binding sites, suggest that the pocket binds a portion of the polypeptide substrate and participates in the conformational maturation/refolding reaction.
Ischemic preconditioning is a phenomenon in which exposure of the heart to a brief period of ischemia causes it to quickly adapt itself to become resistant to infarction from a subsequent ischemic insult. The mechanism is not fully understood but, at least in the rabbit, it is known to be triggered by occupation of adenosine receptors, opioid receptors, bradykinin receptors and the generation of free radicals during the preconditioning ischemia. All of these are thought to converge on and activate protein kinase C (PKC), which in turn activates a tyrosine kinase. This kinase cascade eventually terminates on some unknown effector, possibly a potassium channel or a cytoskeletal protein, which makes the cells resistant to infarction. If this process can be understood, it should be possible to devise a method for conferring this protection to patients with acute myocardial infarction.
The objective of this study was to test the hypothesis that chemical interaction through gap junctions may result in cell-to-cell progression of hypercontracture and that this phenomenon contributes to the final extent of reperfused infarcts.
Cell-to-cell transmission of hypercontracture was studied in pairs of freshly isolated adult rat cardiomyocytes. Hypercontracture induced by microinjection of a solution containing 1 mmol/L Ca2+ and 2% lucifer yellow (LY) was transmitted to the adjacent cell (11 of 11 pairs), and the gap junction uncoupler heptanol (2 mmol/L) prevented transmission in 6 of 8 pairs (P=.003), with a perfect association between passage of the LY and transmission of hypercontracture. In the isolated, perfused rat heart submitted to 30 minutes of hypoxia, addition of heptanol to the perfusion media during the first 15 minutes of reoxygenation had a dose-related protective effect against the oxygen paradox, as demonstrated by a reduction of diastolic pressure and marked recovery of developed pressure (P<.001), as well as less lactate dehydrogenase release during reoxygenation (P<.001) and less contraction band necrosis (P<.001) than controls. In the in situ pig heart submitted to 48 minutes of coronary occlusion, the intracoronary infusion of heptanol during the first 15 minutes of reperfusion at a final concentration of 1 mmol/L limited myocardial shrinkage, reflecting hypercontracture (P<.05), reduced infarct size after 5 hours of reperfusion by 54% (P=.04), and modified infarct geometry with a characteristic fragmentation of the area of necrosis. Heptanol at 1 mmol/L had no significant effect on contractility of nonischemic myocardium.
These results demonstrate that hypercontracture may be transmitted to adjacent myocytes through gap junctions and that heptanol may interfere with this transmission and reduce the final extent of myocardial necrosis during reoxygenation or reperfusion. These findings are consistent with the hypothesis tested and open a new approach to limitation of infarct size by pharmacological control of gap junction conductance.
The aim of this study was to investigate the role of mitochondrial ionic homeostasis in promoting reoxygenation-induced hypercontracture in cardiac muscle. Mitochondrial membrane potential and intramitochondrial Ca2+ concentration ([Ca2+]) were measured using confocal imaging in guinea pig ventricular myocytes exposed to anoxia and reoxygenation. Anoxia produced a variable, but often profound, mitochondrial depolarization. Some cells mounted a recovery of their mitochondrial membrane potential during reoxygenation; the depolarization was sustained in other cells. Recovery of the mitochondrial membrane potential seemed essential to avoid reoxygenation-induced hypercontracture. Reoxygenation also caused a sizable elevation in intramitochondrial [Ca2+], the amplitude of which was correlated with the likelihood of a cell undergoing hypercontracture. A sustained Ca2+ load analogous to that seen during reoxygenation was imposed on cardiac mitochondria through permeabilization of the plasma membrane. Elevation of intracellular [Ca2+] to 800 nM caused a substantial mitochondrial depolarization. We propose that the conditions seen in guinea pig ventricular myocytes during reoxygenation are well suited to produce Ca2+-dependent mitochondrial depolarization, which may play a significant role in promoting irreversible cell injury.
Discovered in the cardiac sarcolemma, ATP-sensitive K+ (KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+ channel openers to examine the effect of mitochondrial KATP channel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+ transport, and matrix volume. From a mitochondrial membrane potential of -180 +/- 15 mV, K+ channel openers, pinacidil (100 microM), cromakalim (25 microM), and levcromakalim (20 microM), induced membrane depolarization by 10 +/- 7, 25 +/- 9, and 24 +/- 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+ or application of a KATP channel blocker. K+ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+ channel opener released Ca2+ from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+ concentration and sensitive to KATP channel blockade. In addition, K+ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome c and adenylate kinase. Thus, in isolated cardiac mitochondria, KATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATP channels in regulating functions vital for the cardiac mitochondria.
Gap junction channels are essential for normal cardiac impulse propagation. Three gap junction proteins, known as connexins, are expressed in the heart: Cx40, Cx43, and Cx45. Each of these proteins forms channels with unique biophysical and electrophysiologic properties, as well as spatial distribution of expression throughout the heart. However, the specific functional role of the individual connexins in normal and abnormal propagation is unknown. The availability of genetically engineered mouse models, together with new developments in optical mapping technology, makes it possible to integrate knowledge about molecular mechanisms of intercellular communication and its regulation with our growing understanding of the microscopic and global dynamics of electrical impulse propagation during normal and abnormal cardiac rhythms. This article reviews knowledge on the mechanisms of cardiac impulse propagation, with particular focus on the role of cardiac connexins in electrical communication between cells. It summarizes results of recent studies on the electrophysiologic consequences of defects in the functional expression of specific gap junction channels in mice lacking either the Cx43 or Cx40 gene. It also reviews data obtained in a transgenic mouse model in which cell loss and remodeling of gap junction distribution leads to increased susceptibility to arrhythmias and sudden cardiac death. Overall, the results demonstrate that these are potentially powerful strategies for studying fundamental mechanisms of cardiac electrical activity and for testing the hypothesis that certain cardiac arrhythmias involve gap junction or other membrane channel dysfunction. These new approaches, which permit one to manipulate electrical wave propagation at the molecular level, should provide new insight into the detailed mechanisms of initiation, maintenance, and termination of cardiac arrhythmias, and may lead to more effective means to treat arrhythmias and prevent sudden cardiac death.
The molecular chaperone Hsp90 binds and hydrolyses ATP, but how this ATPase activity regulates the interaction of Hsp90 with a polypeptide substrate is not yet understood. Using the glucocorticoid receptor ligand binding domain as a substrate, we show that dissociation of Hsp90 from bound polypeptide depends on the Hsp90 ATPase and is blocked by geldanamycin, a specific ATPase inhibitor. The co-chaperone p23 greatly stimulates Hsp90 substrate release with ATP, but not with the non-hydrolysable nucleotides ATPgammaS or AMP-PNP. Point mutants of Hsp90 with progressively lower ATPase rates are progressively slower in ATP-dependent substrate release but are still regulated by p23. In contrast, ATPase-inactive Hsp90 mutants release substrate poorly and show no p23 effect. These results outline an ATP-driven cycle of substrate binding and release for Hsp90 which differs from that of other ATP-driven chaperones. Conversion of the ATP state of Hsp90 to the ADP state through hydrolysis is required for efficient release of substrate polypeptide. p23 couples the ATPase activity to polypeptide dissociation and thus can function as a substrate release factor for Hsp90.
It has been assumed that all G(i)-coupled receptors trigger the protective action of preconditioning by means of an identical intracellular signaling pathway. To test this assumption, rabbit hearts were isolated and perfused with Krebs buffer. All hearts were subjected to a 30-minute coronary artery occlusion followed by 120 minutes of reperfusion. Risk area was measured with fluorescent particles and infarct size with triphenyltetrazolium chloride staining. Control hearts showed 29.1+/-2.8% infarction of the risk zone. A 5-minute infusion of acetylcholine (0.55 mmol/L) beginning 15 minutes before the 30-minute occlusion resulted in significant protection (9.2+/-2.7% infarction). This protection could be blocked by administration of 300 micromol/L N-2-mercaptopropionyl glycine (MPG), a free radical scavenger, or by 200 micromol/L 5-hydroxydecanoate (5-HD), a mitochondrial K(ATP) antagonist, for 15 minutes beginning 5 minutes before the acetylcholine infusion (35.2+/-3.9% and 27.8+/-2.4% infarction, respectively). Similar protection was observed with other known triggers, ie, bradykinin (0.4 micromol/L), morphine (0.3 micromol/L), and phenylephrine (0.1 micromol/L), and in each case protection was completely abrogated by either MPG or 5-HD. In contrast, protection by adenosine or its analog N(6)-(2-phenylisopropyl) adenosine could not be blocked by either MPG or 5-HD. Therefore, whereas most of the tested agonists trigger protection by a pathway that requires opening of mitochondrial K(ATP) channels and production of free radicals, the protective action of adenosine is not dependent on either of these steps. Hence, it cannot be assumed that all G(i)-coupled receptors use the same signal transduction pathways to trigger preconditioning.
Mitochondria play a critical role in initiating both apoptotic and necrotic cell death. A major player in this process is the mitochondrial permeability transition pore (MPTP), a non-specific pore, permeant to any molecule of < 1.5 kDa, that opens in the inner mitochondrial membrane under conditions of elevated matrix [Ca(2+)], especially when this is accompanied by oxidative stress and depleted adenine nucleotides. Opening of the MPTP causes massive swelling of mitochondria, rupture of the outer membrane and release of intermembrane components that induce apoptosis. In addition mitochondria become depolarised causing inhibition of oxidative phosphorylation and stimulation of ATP hydrolysis. Pore opening is inhibited by cyclosporin A analogues with the same affinity as they inhibit the peptidyl-prolyl cis-trans isomerase activity of mitochondrial cyclophilin (CyP-D). These data and the observation that different ligands of the adenine nucleotide translocase (ANT) can either stimulate or inhibit pore opening led to the proposal that the MPTP is formed by a Ca-triggered conformational change of the ANT that is facilitated by the binding of CyP-D. Our model is able to explain the mode of action of a wide range of known modulators of the MPTP that exert their effects by changing the binding affinity of the ANT for CyP-D, Ca(2+) or adenine nucleotides. The extensive evidence for this model from our own and other laboratories is presented, including reconstitution studies that demonstrate the minimum configuration of the MPTP to require neither the voltage activated anion channel (VDAC or porin) nor any other outer membrane protein. However, other proteins including Bcl-2, BAX and virus-derived proteins may interact with the ANT to regulate the MPTP. Recent data suggest that oxidative cross-linking of two matrix facing cysteine residues on the ANT (Cys(56) and Cys(159)) plays a key role in regulating the MPTP. Adenine nucleotide binding to the ANT is inhibited by Cys(159) modification whilst oxidation of Cys(56) increases CyP-D binding to the ANT, probably at Pro(61).
Diazoxide and 5-hydroxydecanoate (5-HD; C10:0) are reputed to target specifically mitochondrial ATP-sensitive K(+) (K(ATP)) channels. Here we describe K(ATP) channel-independent targets of diazoxide and 5-HD in the heart. Using submitochondrial particles isolated from pig heart, we found that diazoxide (10-100 microM) dose-dependently decreased succinate oxidation without affecting NADH oxidation. Pinacidil, a non-selective K(ATP) channel opener, did not inhibit succinate oxidation. However, it selectively inhibited NADH oxidation. These direct inhibitory effects of diazoxide and pinacidil cannot be explained by activation of mitochondrial K(ATP) channels. Furthermore, application of either diazoxide (100 microM) or pinacidil (100 microM) did not decrease mitochondrial membrane potential, assessed using TMRE (tetramethylrhodamine ethyl ester), in isolated guinea-pig ventricular myocytes. We also tested whether 5-HD, a medium-chain fatty acid derivative which blocks diazoxide-induced cardioprotection, was 'activated' via acyl-CoA synthetase (EC 6.2.1.3), an enzyme present both on the outer mitochondrial membrane and in the matrix. Using analytical HPLC and electrospray ionisation mass spectrometry, we showed that 5-HD-CoA (5-hydroxydecanoyl-CoA) is indeed synthesized from 5-HD and CoA via acyl-CoA synthetase. Thus, 5-HD-CoA may be the active form of 5-HD, serving as substrate for (or inhibiting) acyl-CoA dehydrogenase (beta-oxidation) and/or exerting some other cellular action. In conclusion, we have identified K(ATP) channel-independent targets of 5-HD, diazoxide and pinacidil. Our findings question the assumption that sensitivity to diazoxide and 5-HD implies involvement of mitochondrial K(ATP) channels. We propose that pharmacological preconditioning may be related to partial inhibition of respiratory chain complexes.
Protein kinase Cepsilon (PKCepsilon) plays a central role in ischemic preconditioning (IP) in mice and rabbits, and activated PKCepsilon colocalizes with and phosphorylates connexin43 (Cx43) in rats and humans. Whether or not Cx43 contributes to the mechanism(s) of IP in vivo is yet unknown. Therefore, wild-type (n = 8) and heterozygous Cx43-deficient mice (n = 8) were subjected to 30 min occlusion and 120 min reperfusion of the left anterior descending coronary artery. IP was induced by one cycle of 5 min occlusion and 10 min reperfusion (n = 8/8 mice) before the sustained occlusion. Infarct size was reduced by IP in wild-type mice [11.3 +/- 3.4% vs. 23.7 +/- 7.2% of the left ventricle (LV), P < 0.05] but not in Cx43-deficient mice (26.0 +/- 6.0% vs. 25.1 +/- 3.8% of LV). Also, three cycles of 5 min occlusion and 10 min reperfusion (n = 5) did not induce protection in Cx43-deficient mice (27.6 +/- 5.5 % of LV). Thus Cx43 contributes to the protection of IP in mice in vivo.
Apart from a handful of proteins encoded by the mitochondrial genome, most proteins residing in this organelle are nuclear-encoded and synthesised in the cytosol. Thus, delivery of proteins to their final destination depends on a network of specialised import components that form at least four main translocation complexes. The import machinery ensures that proteins earmarked for the mitochondrion are recognised and delivered to the organelle, transported across membranes, sorted to the correct compartment and assisted in overcoming energetic barriers.
The mitochondrial outer membrane contains a multi-subunit machinery responsible for the specific recognition and translocation of precursor proteins. This translocase of the outer membrane (TOM) consists of three receptor proteins, Tom20, Tom22 and Tom70, the channel protein Tom40, and several small Tom proteins. Single-particle electron microscopy analysis of the Neurospora TOM complex has led to different views with two or three stain-filled centers resembling channels. Based on biochemical and electron microscopy studies of the TOM complex isolated from yeast mitochondria, we have discovered the molecular reason for the different number of channel-like structures. The TOM complex from wild-type yeast contains up to three stain-filled centers, while from a mutant yeast selectively lacking Tom20, the TOM complex particles contain only two channel-like structures. From mutant mitochondria lacking Tom22, native electrophoresis separates an approximately 80 kDa subcomplex that consists of Tom40 only and is functional for accumulation of a precursor protein. We conclude that while Tom40 forms the import channels, the two receptors Tom22 and Tom20 are required for the organization of Tom40 dimers into larger TOM structures.
The phenomenon of ischemic preconditioning, in which a period of sublethal ischemia can profoundly protect the cell from infarction during a subsequent ischemic insult, has been responsible for an enormous amount of research over the last 15 years. Ischemic preconditioning is associated with two forms of protection: a classical form lasting approximately 2 h after the preconditioning ischemia followed a day later by a second window of protection lasting approximately 3 days. Both types of preconditioning share similarities in that the preconditioning ischemia provokes the release of several autacoids that trigger protection by occupying cell surface receptors. Receptor occupancy activates complex signaling cascades which during the lethal ischemia converge on one or more end-effectors to mediate the protection. The end-effectors so far have eluded identification, although a number have been proposed. A range of different pharmacological agents that activate the signaling cascades at the various levels can mimic ischemic preconditioning leading to the hope that specific therapeutic agents can be designed to exploit the profound protection seen with ischemic preconditioning. This review examines, in detail, the complex mechanisms associated with both forms of preconditioning as well as discusses the possibility to exploit this phenomenon in the clinical setting. As our understanding of the mechanisms associated with preconditioning are unravelled, we believe we can look forward to the development of new therapeutic agents with novel mechanisms of action that can supplement current treatment options for patients threatened with acute myocardial infarction.
Connexin 43 (Cx 43) has recently been implicated in protection of ischemic preconditioning. Cx 43 colocalization with protein kinase C and p38 mitogen-activated protein kinase is increased in preconditioned myocardium, Cx 43 phosphorylation is preserved in preconditioned myocardium, and hearts from Cx 43-deficient mice cannot be preconditioned. It is, however, unclear whether the important role of Cx 43 relates to intercellular communication through gap junctions or its function in volume homeostasis. To address this issue, we used isolated cardiomyocytes, which no longer-form gap junctions, from wild-type (n = 5) and heterozygous Cx 43-deficient mice (n = 8) and subjected them to 2 h simulated ischemia (hypoxia, acidosis) and an additional challenge by extracellular hypo-osmolarity (from 310 to 250 mOsm/l). Viability (trypan blue exclusion) was well maintained in normoxic wild-type cardiomyocytes (54 +/- 5% at baseline vs. 46 +/- 4 (mean +/- S.D.) % at 2 h). With simulated ischemia, viability was reduced to 17 +/- 5%. Preconditioning by a preceding exposure to 10 min simulated ischemia and 15 min reoxygenation preserved viability after 2 h simulated ischemia (36 +/- 1%, P < 0.001 vs. simulated ischemia). In Cx 43-deficient cardiomyocytes, viability was also well maintained in normoxia (56 +/- 10% vs. 44 +/- 10%). Viability was also reduced to 17 +/- 6% with 2 h simulated ischemia. In contrast to wild-type cells, preconditioning did not prevent the reduction in viability (18 +/- 8%). In conclusion, Cx 43 is essential for preconditioning in the absence of gap junctions, supporting its function through improved volume regulation.
Gap junction-mediated intercellular communication (GJMIC) has been known for a long time to be essential in propagation of electrical impulse in the heart, and the contribution of altered GJMIC to the genesis of arrhythmias has been extensively investigated. However, although it is well known that GJMIC allows the exchange of biologically important molecules between adjacent cells, the pathophysiological significance of such chemical coupling during myocardial ischemia and reperfusion is much less known. It has been solidly established that ischemia impairs GJMIC and eventually leads to electrical uncoupling, but recent studies suggest that GJMIC may still allow synchronization of the onset of ischemic rigor contracture and of the progression of ischemic injury beyond rigor onset. During reperfusion, GJMIC has been shown to mediate cell-to-cell propagation of hypercontracture and cell death, and there is evidence that this phenomenon explains the continuity of areas of contraction band necrosis and contributes to final infarct size. Finally, there is increasing evidence that GJ or their protein components are involved in the genesis of the protective effect of ischemic preconditioning, although probably through mechanisms independent from modulator of GJMIC. GJ play an important role in the pathophysiology of cell injury and death during myocardial ischemia-reperfusion and are potential targets for new cardioprotective therapeutic strategies.
During the past decade, the understanding has grown that control of the conditions of reperfusion is critical for salvaging ischemic-reperfused myocardium. The first few minutes of reperfusion constitute a critical phase, as here lethal tissue injury in addition to that already developed during ischemia may be initiated. The identification of the mechanisms of reperfusion-induced cell death opens a new window of opportunity for cardioprotection in the clinic. Development of cardiomyocyte hypercontracture is a predominant feature of reperfusion injury. We and others have shown that control of hypercontracture in reperfusion reduces the extent of tissue injury. On the cellular level, it was shown that reperfusion-induced hypercontracture might either originate from a rigor-type mechanism, when energy recovery proceeds very slowly, or from Ca2+ overload, when energy recovery is rapid but cytosolic Ca2+ load is high. These two mechanisms can be influenced by various interventions that either connect with cytosolic Ca2+ control or myofibrillar Ca2+ sensitivity or with mitochondrial energy production. These experimental approaches will hopefully lead to novel strategies for clinical cardioprotection during the early phase of reperfusion.
Connexin 43 (Cx43) is the essential protein to form hemichannels and gap junctions in the myocardium. The phosphorylation status of Cx43 which is regulated by a variety of protein kinases and phosphatases determines hemichannel and/or gap junction conductance and permeability. Gap junctions are involved in cell-cell coupling while hemichannels contribute to cardiomyocyte volume regulation. Cx43-formed channels are involved in ischemia/reperfusion injury, since blockade of a large portion of Cx43-formed channels attenuates ischemic hypercontracture, infarct development and post myocardial infarction remodeling. Ischemic preconditioning's protection also depends on functional Cx43-formed channels, since uncoupling of channels or genetic Cx43 deficiency abolishes infarct size reduction by ischemic preconditioning. The exact underlying mechanism(s) how Cx43 mediates protection remain to be established.
Transient inhibition of gap junction (GJ)-mediated communication with heptanol during myocardial reperfusion limits infarct size. However, inhibition of cell coupling in normal myocardium may be arrhythmogenic. The purpose of this study was to test the hypothesis that the consequences of GJ inhibition may be magnified in reperfused myocardium compared with normal tissue, thus allowing the inhibition of GJs in reperfused tissue while only minimally modifying overall macroscopic cell coupling in normal myocardium. Concentration-response curves were defined for the effects of heptanol, 18alpha-glycyrrhetinic acid, halothane, and palmitoleic acid on conduction velocity, tissue electrical impedance, developed tension and lactate dehydrogenase (LDH) release in normoxically perfused rat hearts (n= 17). Concentrations lacking significant effects on tissue impedance were added during the initial 15 min of reperfusion in hearts submitted to 60 min (n= 43) or 30 min (n= 35) of ischaemia. These concentrations markedly increased myocardial electrical impedance (resistivity and phase angle) in myocardium reperfused after either 30 or 60 min of ischaemia, and reduced reperfusion-induced LDH release after 1 h of ischaemia by 83.6, 57.9, 51.7 and 52.5% for heptanol, 18alpha-glycyrrhetinic acid, halothane and palmitoleic acid, respectively. LDH release was minimal in hearts submitted to 30 min of ischaemia, independently of group allocation. In conclusion, the present results strongly support the hypothesis that intercellular communication in postischaemic myocardium may be effectively reduced by concentrations of GJ inhibitors affecting only minimally overall electrical impedance in normal myocardium. Reduction of cell coupling during initial reperfusion was consistently associated with attenuated lethal reperfusion injury.
The mitochondrial inner membrane is rich in multispanning integral membrane proteins, most of which mediate the vital transport of molecules between the matrix and the intermembrane space. The correct transport and membrane insertion of such proteins is essential for maintaining the correct exchange of molecules between mitochondria and the rest of the cell. Mitochondria contain several specific complexes — known as translocases — that translocate precursor proteins. Recent analysis of the inner-membrane, twin-pore protein translocase (TIM22 complex) allows a glimpse of the molecular mechanisms by which this machinery triggers protein insertion using the membrane potential as an external driving force.
It has been shown that sarcolemmal rupture can occur during reenergization in cardiomyocytes in which previous ischemia has induced sarcolemmal fragility by calpain-dependent hydrolysis of structural proteins. We tested the hypothesis that attenuated calpain activation contributes to the protection against reperfusion-induced cell death afforded by ischemic preconditioning (IPC), and investigated the involvement of protein kinase A (PKA) in this effect.
Calpain activity and degradation of different structural proteins were studied along with the extent of necrosis in isolated rat hearts submitted to 60 min of ischemia and 30 min of reperfusion with or without previous IPC (two cycles of 5 min ischemia-5 min reperfusion), and the ability of different treatments to mimic or blunt the effects of IPC were analyzed.
IPC accelerated ATP depletion and rigor onset during ischemia but reduced LDH release during reperfusion by 69% (P<0.001). At the end off reperfusion, calpain activity was reduced by 66% (P<0.001) in IPC, and calpain-dependent degradation of sarcolemmal proteins was attenuated. Addition of the calpain inhibitor MDL-28170 mimicked the effects of IPC on protein degradation and reduced LDH release by 48% (P<0.001). The effects of IPC on calpain, alpha-fodrin, and LDH release were blunted by the application of the PKA inhibitor H89 or alprenolol during IPC, while transient stimulation of PKA with CPT-cAMP or isoproterenol before ischemia attenuated calpain activation, alpha-fodrin degradation, and markedly reduced LDH release (P<0.001). In hearts exposed to Na(+)-free perfusion, IPC attenuated calpain activation by 67% (P<0.001) and reduced by 56% (P<0.001) LDH release associated to massive edema occurring during Na(+) readmission without modifying its magnitude.
These results are consistent with PKA-dependent attenuation of calpain-mediated degradation of structural proteins being an end-effector mechanism of the protection afforded by IPC.
Connexin 43 (Cx43) is involved in infarct size reduction by ischemic preconditioning (IP); the underlying mechanism of protection, however, is unknown. Since mitochondria have been proposed to be involved in IP's protection, the present study analyzed whether Cx43 is localized at mitochondria of cardiomyocytes and whether such localization is affected by IP.
Western blot analysis on mitochondrial preparations isolated from rat, mouse, pig, and human hearts showed the presence of Cx43. The preparations were not contaminated with markers for other cell compartments. The localization of Cx43 to mitochondria was also confirmed by FACS sorting (double staining with MitoTracker Red and Cx43) and immuno-electron and confocal microscopy. To study the role of Cx43 in IP, mitochondria were isolated from the ischemic anterior wall (AW) and the control posterior wall (PW) of pig myocardium at the end of 90 min low-flow ischemia without (n=13) or with (n=13) a preceding preconditioning cycle of 10 min ischemia and 15 min reperfusion. With IP, the mitochondrial Cx43/adenine nucleotide transporter ratio was 3.4+/-0.7 fold greater in AW than in PW, whereas the ratio remained unchanged in non-preconditioned myocardium (1.1+/-0.2, p<0.05). The enhancement of the mitochondrial Cx43 protein level occurred rapidly, since an increase of mitochondrial Cx43 was already detected with two cycles of 5 min ischemia/reperfusion in isolated rat hearts to 262+/-63% of baseline.
These data demonstrate that Cx43 is localized at cardiomyocyte mitochondria and that IP enhances such mitochondrial localization.
Since the discovery of the mitochondrial ATP-sensitive potassium channel (mitoK(ATP)) more than 13 years ago, it has been implicated in the processes of ischemic preconditioning (IPC), apoptosis and mitochondrial matrix swelling. Different approaches have been employed to characterize the pharmacological profile of the channel, and these studies strongly suggest that cellular protection well correlates with the opening of mitoK(ATP). However, there are many questions regarding mitoK(ATP) that remain to be answered. These include the very existence of mitoK(ATP) itself, its degree of importance in the process of IPC, its response to different pharmacological agents, and how its activation leads to the process of IPC and protection against cell death. Recent findings suggest that mitoK(ATP) may be a complex of multiple mitochondrial proteins, including some which have been suggested to be components of the mitochondrial permeability transition pore. However, the identity of the pore-forming unit of the channel and the details of the interactions between these proteins remain unclear. In this review, we attempt to highlight the recent advances in the physiological role of mitoK(ATP) and discuss the controversies and unanswered questions.
Na+ overload and secondary Ca2+ influx via Na+/Ca2+ exchanger are key mechanisms in cardiomyocyte contracture and necrosis during reperfusion. Impaired Na+/K+-ATPase activity contributes to Na+ overload, but the mechanism has not been established. Because Na+/K+-ATPase is connected to the cytoskeleton protein fodrin through ankyrin, which are substrates of calpains, we tested the hypothesis that calpain mediates Na+/K+-ATPase impairment in reperfused cardiomyocytes. In isolated rat hearts reperfused for 5 minutes after 60 minutes of ischemia, Na+/K+-ATPase activity was reduced by 80%, in parallel with loss of alpha-fodrin and ankyrin-B and detachment of alpha1 and alpha2 subunits of Na+/K+-ATPase from the membrane-cytoskeleton complex. Calpain inhibition with MDL-7943 during reperfusion prevented the loss of these proteins, increased Na+/K+-ATPase activity, attenuated lactate dehydrogenase release, and improved contractile recovery, and these beneficial effects of MDL-7943 were reverted by ouabain. The impairment of Na+/K+-ATPase was not a mere consequence of cell death because it was not altered in hearts in which contracture and cell death had been prevented by contractile blockade with 2,3-butanedione monoxime. In these hearts, concomitant calpain inhibition preserved Na+/K+-ATPase content and function and attenuated cell death occurring on withdrawal of 2,3-butanedione monoxime. In vitro assay showed no detectable degradation of Na+/K+-ATPase subunits after 10 minutes of incubation with activated calpain. Thus, we conclude that calpain activation contributes to the impairment of Na+/K+-ATPase during early reperfusion and that this effect is mainly mediated by degradation of the anchorage of Na+/K+-ATPase to the membrane cytoskeleton.
Protection by ischemic preconditioning is lost in cardiomyocytes and hearts of heterozygous connexin 43 deficient (Cx43+/-) mice. Because connexin 43 (Cx43) is localized in cardiomyocyte mitochondria and mitochondrial Cx43 content is increased with ischemic preconditioning, we now tried to identify a functional defect at the level of the mitochondria in Cx43+/- mice by use of diazoxide and menadione. Diazoxide stimulates the mitochondrial formation of reactive oxygen species (ROS) and menadione generates superoxide at multiple intracellular sites; both substances elicit cardioprotection through increased ROS formation. ROS formation in response to the potassium ionophore valinomycin was also measured for comparison. Menadione (2 micromol/L) and valinomycin (10 nmol/L) induced similar ROS formation in wild-type (WT) and Cx43+/- cardiomyocytes. In contrast, diazoxide (200 micromol/L) increased ROS formation by 43+/-10% versus vehicle in WT, but only by 18+/-4% in Cx43+/- cardiomyoctes (P<0.05). Two hour-simulated ischemia and oxygenated, hypo-osmolar reperfusion reduced viability as compared with normoxia (WT: 7+/-1% versus 39+/-2%, (Cx43+/-): 8+/-1% versus 40+/-3%, P<0.01). Although menadione protected WT and Cx43+/- cardiomyocytes, diazoxide increased viability (17+/-2%, P<0.01) in WT, but not in Cx43+/- (9+/-1%). Menadione (37 microg/kg i.v.) before 30 minutes coronary occlusion and 2 hour reperfusion reduced infarct size in WT and Cx43+/- mice (24+/-4% versus 24+/-5%). In contrast, diazoxide (5 mg/kg i.v.) reduced infarct size in WT (35+/-4% versus 55+/-3% of area at risk, P<0.01), but not in Cx43+/- mice (56+/-2% versus 54+/-3%). Cardiomyocytes of Cx43+/- mice have a specific functional deficit in ROS formation in response to diazoxide and accordingly less protection.
The aim of the present study was to examine the hypothesis that acceleration of gap junction (GJ) closure during ischemia contributes to anti-infarct tolerance afforded by preconditioning (PC). First, the effects of PC on GJ communication during ischemia were assessed. Isolated buffer-perfused rabbit hearts were subjected to 5-min global ischemia with or without PC with two cycles of 5-min ischemia/5-min reperfusion or a GJ blocker (2 mM heptanol), and then the tissue excised from the ischemic region was incubated in anoxic buffer containing lucifer yellow (LY; 2.5 mg/ml), a tracer of GJ permeability, for 20 min at 37 degrees C. PC and heptanol significantly reduced the area to which LY was transported in the ischemic myocardium by 39% and by 54%, respectively. In the second series of experiments, three GJ blockers (heptanol, 18beta-glycyrrhetinic acid, and 2,3-butanedione monoxime) infused after the onset of ischemia reduced infarct size after 30-min ischemia/2-h reperfusion to an extent equivalent to that in the case of PC. In the third series of experiments, Western blotting for connexin43 (Cx43) showed that PC shortened the time to the onset of ischemia-induced Cx43 dephosphorylation but reduced the extent of Cx43 dephosphorylation during a 30-min period of ischemia. Calphostin C, a protein kinase C (PKC) inhibitor, abolished preservation of phosphorylated Cx43 but not the early onset of Cx43 dephosphorylation after ischemia in the preconditioned myocardium. These results suggest that PC-induced reduction of GJ permeability during ischemia, presumably by PKC-mediated Cx43 phosphorylation, contributes to infarct size limitation.
Mitochondrial K(ATP) channels in cell survival and death
- H Ardehali
- O Rourke
Ardehali H, O'Rourke B. Mitochondrial K(ATP) channels in cell survival
and death. J Mol Cell Cardiol. 2005;39:7–16.
Myocardial cell protection in acute coronary syndromes
- D Garcia-Dorado
Garcia-Dorado D. Myocardial cell protection in acute coronary syndromes. In: Theroux P, ed. Acute Coronary Syndromes. A Companion to
Braunwald's Heart Disease. Philadelphia: Saunders; 2003.