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Gap junction alterations in human cardiac disease
Abstract
Gap junctions, assembled from connexins, form the cell-to-cell pathways for propagation of the precisely orchestrated patterns of current flow that govern the regular rhythm of the healthy heart. As in most tissues and organs, multiple connexin types are expressed in the heart; connexin43, connexin40 and connexin45 are found in distinctive combinations and relative quantities in different, functionally specialized subsets of cardiomyocyte. Alterations of gap junction organization and connexin expression are now well established as a consistent feature of human heart disease in which there is an arrhythmic tendency. These alterations may take the form of structural remodelling, involving disturbances in the distribution of gap junctions and/or alteration of the amount or type of connexin(s) expressed. In the diseased ventricles, the most consistent quantitative alteration involves heterogeneous reduction in connexin43 expression. In the atria, features of gap organization and connexin expression have been implicated in the initiation of atrial fibrillation and, once the condition becomes chronic, gap junction alterations associated with remodelling may contribute to persistence of the condition. By correlating data from studies on the human patient with those from animal and cell models, alterations in gap junctions and connexins have emerged as important factors to be considered in understanding the pro-arrhythmic substrate found in a variety of forms of heart disease.
... These connexins function as pathways enabling electrical current propagation, which controls the rhythm of the heart. As is the case in most tissues and organs, multiple connexins are expressed in the heart, specifically connexin43, connexin 40 and connexin45 [96,97]. The presence of each connexin type varies in relative to quantities depending on the functional specialization of each subset of cardiomyocytes. ...
... The presence of each connexin type varies in relative to quantities depending on the functional specialization of each subset of cardiomyocytes. The most predominant gap junction protein in the adult heart is connexin43, expressed highly in all cardiomyocytes subsets of the heart [96,97]. In the sinoatrial node, the site of impulse generation, and the atrioventricular node, the site where impulse is slowed before being routed to the ventricles, cardiomyocyte gap junctions are formed by connexin43 and connexin45, associated with slow conductance [97]. ...
... The most predominant gap junction protein in the adult heart is connexin43, expressed highly in all cardiomyocytes subsets of the heart [96,97]. In the sinoatrial node, the site of impulse generation, and the atrioventricular node, the site where impulse is slowed before being routed to the ventricles, cardiomyocyte gap junctions are formed by connexin43 and connexin45, associated with slow conductance [97]. Cardiomyocytes of the His-Purkinje conduction systems are mainly characterized by the expression of connex-in40, a connexin associated with high conductance, which facilitates rapid distribution of the impulse throughout the working ventricular myocardium [97]. ...
As the heart matures during embryogenesis from its foetal stages, several structural and functional modifications take place to form the adult heart. This process of maturation is in large part due to an increased volume and work load of the heart to maintain proper circulation throughout the growing body. In recent years, it has been observed that these changes are reversed to some extent as a result of cardiac disease. The process by which this occurs has been characterized as cardiac foetal reprogramming and is defined as the suppression of adult and re‐activation of a foetal genes profile in the diseased myocardium. The reasons as to why this process occurs in the diseased myocardium are unknown; however, it has been suggested to be an adaptive process to counteract deleterious events taking place during cardiac remodelling. Although still in its infancy, several studies have demonstrated that targeting foetal reprogramming in heart failure can lead to substantial improvement in cardiac functionality. This is highlighted by a recent study which found that by modulating the expression of 5‐oxoprolinase (OPLAH, a novel cardiac foetal gene), cardiac function can be significantly improved in mice exposed to cardiac injury. Additionally, the utilization of angiotensin receptor neprilysin inhibitors (ARNI) has demonstrated clear benefits, providing important clinical proof that drugs that increase natriuretic peptide levels (part of the foetal gene programme) indeed improve heart failure outcomes. In this review, we will highlight the most important aspects of cardiac foetal reprogramming and will discuss whether this process is a cause or consequence of heart failure. Based on this, we will also explain how a deeper understanding of this process may result in the development of novel therapeutic strategies in heart failure.
Abstract
... For instance, channel proteins connexin 40 and 43 (CX40/ GJA5 and CX43/GJA1, respectively) mediate gap junction intercellular communication (GJIC) that is essential for proper cardiac development and function (Figure 3). Mutations in either CX40/ Cx40/cx40 or CX43/Cx43/cx43, both of which are transcriptionally dependent on cohesins (Figure 3), result in a variety of cardiac phenotypes that include both morphological malformations (septal and looping abnormalities) and functional defects (arrhythmias and cardiomyopathies; Huang et al., 1998;Lo and Wessels, 1998;Alcoléa et al., 1999;Dasgupta et al., 1999;Lo et al., 1999;Nishii et al., 2001;Li et al., 2002;Sohl and Willecke, 2003;Severs et al., 2004Severs et al., , 2006Duffy et al., 2006;Delmar and Makita, 2012;Salameh et al., 2013;Ahir and Pratten, 2014;Molica et al., 2014;Boengler and Schulz, 2017;Hyland et al., 2021). ...
... In fact, numerous noncohesin factors are targeted by ESCO2/Eco1-dependent acetylation (PCNA, MPS3, Rad30; Moldovan et al., 2006;Ghosh et al., 2012;Billon et al., 2017. Regardless of the mechanism, dysregulation of gap junction levels or function can produce cardiomyopathies, arrhythmias, heart malformations, and/or ischemia (Severs, 1994(Severs, , 2004(Severs, , 2008Gros and Jongsma, 1996;Thomas et al., 1998;van der Velden and Jongsma, 2002;Dhein, 2006;van Rijen et al., 2006;Chaldoupi et al., 2009;Delmar and Makita, 2012;Kato et al., 2012;Salameh et al., 2013;Ahir and Pratten, 2014;Gemel et al., 2014;Molica et al., 2014;Lambiase and Tinker, 2015;Michela et al., 2015;Boengler and Schulz, 2017;Leybaert et al., 2017;Delmar et al., 2018;Zu et al., 2018;Hyland et al., 2021), all of which are likely to contribute to cohesinopathic lethality. ...
Cohesins are ATPase complexes that play central roles in cellular processes such as chromosome division, DNA repair, and gene expression. Cohesinopathies arise from mutations in cohesin proteins, or cohesin complex regulators, and encompass a family of related developmental disorders that present with a range of severe birth defects, affect many different physiological systems, and often lead to embryonic fatality. Treatments for cohesinopathies are limited, in large part due to the lack in understanding of cohesin biology. Thus, characterizing the signaling networks that lie upstream and downstream of cohesin-dependent pathways remains clinically relevant. Here, we highlight alterations in cohesins, and cohesin regulators, that result in cohesinopathies with a focus on cardiac defects. In addition, we suggest a novel and more unifying view regarding the mechanisms through which cohesinopathy-based heart defects may arise.
... Cardiac conduction often gets obstructed under pathological conditions where it could result in arrhythmias and death sometimes when normal propagation is not restored properly [1]. In cardiac tissues, The quick flow of ions via the cytoplasm of cardiac cells facilitate the electrical communication at the subcellular level and the gap junctions made up by hemichannels of specialised proteins known as connexins included into the intercalated discs control and mediate the slow intracellular flow [2]. ...
Gap junctions are important intercellular communication mechanisms in heart tissue, and their function is critical to maintaining normal cardiac electrical signals. Gap junctions allow direct electrical connectivity between cardiac myocytes with every beating, allowing for the fast and synchronized spread of cardiac excitement. Proper gap junction communication results in the relatively close start of all cardiomyocyte action potentials as well as an ordered contraction. Many types of cardiac illness cause changes in gap junction coupling. It is understood that the connexin (Cx) component of gap junctions has both direct and indirect functions in the transmission of electrical impulses from the cardiac pacemaker to functioning myocytes through the cardiac conduction system (CCS). In this work, the single cardiac cell of human Purkinjie Fibre and Ventricular Cells are modelled. The modelled cells are coupled via gap junction channels. The computational research intends to investigate the electrotonic function of gap junction conduits in the transmission of electrical impulses between heart cells. It is also studied the effect of the gap junction role between pairs of cells and extrapolate these findings at the tissue level.
... The cardiac milieu is critically dependent on gap junctions for the electrical coupling of cardiomyocytes, which facilitates the synchronized contraction of heart muscle [28,29]. Beyond their role in electrical propagation, gap junctions are integral to autocrine and paracrine signaling cascades that underpin cellular metabolism, growth, and tissue equilibrium [12,30,31]. ...
Anthracycline chemotherapeutics like doxorubicin (DOX) are widely used against various cancers but are accompanied by severe cardiotoxic effects that can lead to heart failure. Through whole transcriptome sequencing and pathological tissue analysis in a murine model, our study has revealed that DOX impairs collagen expression in the early phase, causing extracellular matrix anomalies that weaken the mechanical integrity of the heart. This results in ventricular wall thinning and dilation, exacerbating cardiac dysfunction. In this work, we have identified 5-hydroxytryptophan (5-HTP) as a potent inhibitor of gap junction communication. This inhibition is key to limiting the spread of DOX-induced cardiotoxicity. Treatment with 5-HTP effectively countered the adverse effects of DOX on the heart, preserving ventricular structure and ejection fraction. Moreover, 5-HTP enhanced mitochondrial respiratory function, as shown by the O2k mitochondrial function assay, by improving mitochondrial complex activity and ATP production. Importantly, the cardioprotective benefits of 5-HTP did not interfere with DOX’s ability to combat cancer. These findings shed light on the cardiotoxic mechanisms of DOX and suggest that 5-HTP could be a viable strategy to prevent heart damage during chemotherapy, offering a foundation for future clinical development. This research opens the door for 5-HTP to be considered a dual-purpose agent that can protect the heart without compromising the oncological efficacy of anthracycline chemotherapy.
... During pregnancy, scientists observed a pronounced expression of genes encoding tight junction proteins -claudin-1 and 2, as well as the protein of intercellular gap junctions (IGJ) -connexin 43, in the myometrium [19], [41]. Toward full-term pregnancy, the number of IGJs between SMCs increases, and during childbirth, their number and the content of Connexin-43 sharply increase but it is not detected 24 hours after birth [42]. ...
In this paper, the authors conduct a system analysis of the outcomes from contemporary scientific research. They describe the natural system that regulates parturition as a genetic-psychosomatic phenomenon, wherein all the major systems in the bodies of both the mother and child are aligned towards the central goal —the birthing of a person.Approximately 2-3 weeks before delivery, notable processes commence in the uterus. These include desympathization and the formation of an acupuncture network. Along the channels of this network, wave flows of biologically active substances, possessing both contractile and inhibitory properties, move. These substances are transported by the bloodstream and blood cells. Importantly, these substances also exhibit psychotropic properties, thereby enhancing their impact on the brain and inducing a state of altered consciousness in both the woman and the child.As childbirth approaches, researchers have identified the activation of a locus on chromosome-2 in the prenate. This discovery leads them to assert that it is the prenate that initiates the birthing process.
... 55 Notably, these changes differ quantitatively between the right and left atria, potentially explaining their varied susceptibilities to sustain stable re-entry at different frequencies. Gap junctions, crucial for atrial conduction, undergo remodeling in terms of altered distribution, orientation, and expression of proteins, contributing to sustained AF. 56,57 Abnormal expression and distribution of connexins (Cx), particularly Cx40 and Cx43, are strongly associated with AF development in patients and animal models. [58][59][60] In a dilated left atrium with chronic pressure overload, reduced expression and irregular distribution of Cx43, combined with interstitial fibrosis, lead to conduction abnormalities and increased AF susceptibility. ...
... Therefore, improving GJ function may help treat depression. GJs are also crucial in the treatment of heart disease, ischemic stroke, cancer and other diseases (Severs et al. 2004;Kandouz and Batist 2010;Yang et al. 2020a, b), which indirectly indicates that the roles of connexins are different in different tissues. Connexins and GJ function are sensitive to chemical pollutants, which disrupt GJICs through the BBB, thus damaging the physiological function of the brain (Mesnil et al. 2020). ...
Major depression disorder (MDD) is a neuropsychiatric disorder associated with a high suicide rate and a higher disability rate than any other disease. Evidence suggests that the pathological mechanism of MDD is related to astrocyte dysfunction. Depression is mainly associated with the expression of connexin 43 (Cx43) and the function of Cx43-mediated gap junctions and hemichannels in astrocytes. Moreover, neuroinflammation has been a hotspot in research on the pathology of depression, and Cx43-mediated functions are thought to be involved in neuroinflammation-related depression. However, the specific mechanism of Cx43-mediated functions in neuroinflammation-related depression pathology remains unclear. Therefore, this review summarizes and discusses Cx43 expression, the role of gap junction intercellular communication, and its relationship with neuroinflammation in depression. This review also focuses on the effects of antidepressant drugs (e.g., monoamine antidepressants, psychotropic drugs, and N-methyl-d-aspartate receptor antagonists) on Cx43-mediated function and provides evidence for Cx43 as a novel target for the treatment of MDD.
The pathogenesis of MDD is related to astrocyte dysfunction, with reduced Cx43 expression, GJ dysfunction, decreased GJIC and reduced BDNF expression in the depressed brain. The effect of Cx43 on neuroinflammation-related depression involving inflammatory cytokines, glutamate excitotoxicity, and HPA axis dysregulation. Antidepressant drugs targeting Cx43 can effectively relieve depressive symptoms.
... Gap junctions, a unique membrane structure made of connexin (Cx), are the structural basis for electrical, metabolic, and mechanical coupling between neighboring cells. They are also an important channel for electrochemical signaling, maintenance of inter-cardiac communication, regulation, and assurance of normal cardiac rhythmicity by neighboring cardiomyocytes, which plays a crucial role in the development of cardiac arrhythmias [11][12][13].Cx43, the primary channel protein produced by cardiac working cells, is nearly exclusively present in the atrial and ventricular myocardium as well as the distal conduction system of all adult mammalian hearts. Its primary role is to in uence the transmission of electrical impulses between cardiac myocytes. ...
High mortality due to hygrothermal stress (high temperature and humidity, HHS) during heat waves is mostly linked to cardiovascular malfunction, the most serious of which are malignant arrhythmias. However, the mechanism associated with HHS leading to malignant arrhythmias remains unclear. Here, SD rats were exposed to 40°C and 85% humidity for constructing the HHS model, and the incidence of arrhythmic events, as well as the expression, phosphorylation, and distribution of Cx43 in the myocardium, were examined. The adenosine monophosphate-activated protein kinase (AMPK) activator, AICAR, was also administered to investigate the role played by AMPK in the process. Our results showed that HHS could induce malignant arrhythmias such as ventricular tachycardia (VT), ventricular fibrillation (VF), and severe atrioventricular block (AVB). Besides, HHS increased the distribution of "side-to-side" connections, decreased the phosphorylation of Cx43, and enhanced myocardial fibrosis. Furthermore, HHS also caused LKB1 and p-AMPK expression to be less abundant. While, pretreatment with AICAR could significantly ameliorate Cx43 remodeling and malignant arrhythmias, indicating that the HHS-induced arrhythmias is associated with the redistribution of gap junctions in cardiomyocytes, the dysfunction of intercellular conduction, and the organism's energy metabolism.
... The connexin 43 (Cx43) gap junction-forming protein is the most abundant connexin in the heart and is found in the working myocardium of the atrium and ventricle as well as the more distal regions of the Purkinje network (Gutstein et al., 2001;Oxford et al., 2007;Kleber and Saffitz, 2014). Multiple inherited or acquired cardiomyopathies, including dystrophic muscle dysfunction, arrhythmogenic right ventricular cardiomyopathy (ARVC), ischemia/reperfusion, and hypertension, show an abnormal expression and remodeling of Cx43 (Severs et al., 2004a(Severs et al., , 2004b(Severs et al., , 2006Oxford et al., 2007;Wang et al., 2013;Gonzalez et al., 2015Gonzalez et al., , 2018Kim et al., 2019;Himelman et al., 2020). This dysregulation is thought to play a meaningful mechanistic role in the evolution of lethal cardiac arrhythmias (Gonzalez et al., 2015;Kim et al., 2019;Lillo et al., 2019;Himelman et al., 2020), likely by affecting the appropriate generation and spread of cardiac action potentials (APs; Severs et al., 2006;Remo et al., 2011Remo et al., , 2012Lillo et al., 2019) A common pathological feature of Cx43 cardiac remodeling is the dephosphorylation of a triplet of serine residues S325/S328/ S330 at the C-terminal domain of Cx43. ...
Connexin-43 (Cx43) is the most abundant protein forming gap junction channels (GJCs) in cardiac ventricles. In multiple cardiac pathologies, including hypertrophy and heart failure, Cx43 is found remodeled at the lateral side of the intercalated discs of ventricular cardiomyocytes. Remodeling of Cx43 has been long linked to spontaneous ventricular arrhythmia, yet the mechanisms by which arrhythmias develop are still debated. Using a model of dystrophic cardiomyopathy, we previously showed that remodeled Cx43 function as aberrant hemichannels (non-forming GJCs) that alter cardiomyocyte excitability and, consequently, promote arrhythmias. Here, we aim to evaluate if opening of remodeled Cx43 can serve as a general mechanism to alter cardiac excitability independent of cellular dysfunction associated with a particular cardiomyopathy. To address this issue, we used a genetically modified Cx43 knock-in mouse (S3A) that promotes cardiac remodeling of Cx43 protein without apparent cardiac dysfunction. Importantly, when S3A mice were subjected to cardiac stress using the β-adrenergic agonist isoproterenol (Iso), they displayed acute and severe arrhythmias, which were not observed in WT mice. Pretreatment of S3A mice with the Cx43 hemichannel blocker, Gap19, prevented Iso-induced abnormal electrocardiographic behavior. At the cellular level, when compared with WT, Iso-treated S3A cardiomyocytes showed increased membrane permeability, greater plasma membrane depolarization, and Ca²⁺ overload, which likely caused prolonged action potentials, delayed after depolarizations, and triggered activity. All these cellular dysfunctions were also prevented by Cx43 hemichannel blockers. Our results support the notion that opening of remodeled Cx43 hemichannels, regardless of the type of cardiomyopathy, is sufficient to mediate cardiac-stress-induced arrhythmogenicity.
... Heart failure patients have reduced levels of Cx43 in the gap junction plaques found at the intercalated discs and an increase in the number of Cx43 in the lateral walls of cardiomyocytes (208,209). Lateralization of Cx43 is in contrast with the normal distribution of Cx43 which is concentrated at the intercalated disc in the ventricular myocardium (210). Cx43 hemichannels require positive membrane potential of >+50 mV to open. ...
Intercellular communication mediated by gap junction channels and hemichannels composed of Connexin 43 (Cx43) is vital for the propagation of electrical impulses through cardiomyocytes. The carboxyl terminal tail of Cx43 undergoes various post-translational modifications including phosphorylation of its Serine-368 (S368) residue. Protein Kinase C isozymes directly phosphorylate S368 to alter Cx43 function and stability through inducing conformational changes affecting channel permeability or promoting internalization and degradation to reduce intercellular communication between cardiomyocytes. Recent studies have implicated this PKC/Cx43-pS368 circuit in several cardiac-associated diseases. In this review, we describe the molecular and cellular basis of PKC-mediated Cx43 phosphorylation and discuss the implications of Cx43 S368 phosphorylation in the context of various cardiac diseases, such as cardiomyopathy, as well as the therapeutic potential of targeting this pathway.
... Tubulin acetylation is known to influence the binding of motor proteins to microtubules and to play a role in intracellular Cx43 trafficking 65,87 . Changes in the localization and regulation of Cx43 have been described in many forms of cardiac diseases including heart failure [88][89][90] . However, the molecular mechanisms of gap junction remodeling are not yet known, and their elucidation is of paramount importance for the development of therapies aimed at improving gap junction coupling during disease. ...
Mutations in the lamin A/C gene ( LMNA ) cause dilated cardiomyopathy associated with increased activity of ERK1/2 in the heart. We recently showed that ERK1/2 phosphorylates cofilin-1 on threonine 25 (phospho(T25)-cofilin-1) that in turn disassembles the actin cytoskeleton. Here, we show that in muscle cells carrying a cardiomyopathy-causing LMNA mutation, phospho(T25)-cofilin-1 binds to myocardin-related transcription factor A (MRTF-A) in the cytoplasm, thus preventing the stimulation of serum response factor (SRF) in the nucleus. Inhibiting the MRTF-A/SRF axis leads to decreased α-tubulin acetylation by reducing the expression of ATAT1 gene encoding α-tubulin acetyltransferase 1. Hence, tubulin acetylation is decreased in cardiomyocytes derived from male patients with LMNA mutations and in heart and isolated cardiomyocytes from Lmna p.H222P/H222P male mice. In Atat1 knockout mice, deficient for acetylated α-tubulin, we observe left ventricular dilation and mislocalization of Connexin 43 (Cx43) in heart. Increasing α-tubulin acetylation levels in Lmna p.H222P/H222P mice with tubastatin A treatment restores the proper localization of Cx43 and improves cardiac function. In summary, we show for the first time an actin-microtubule cytoskeletal interplay mediated by cofilin-1 and MRTF-A/SRF, promoting the dilated cardiomyopathy caused by LMNA mutations. Our findings suggest that modulating α-tubulin acetylation levels is a feasible strategy for improving cardiac function.
... 5,9 Signal-Averaged Electrocardiography, Electrocardiography, and Ultrasound Cardiography In vivo measurements were performed at the end of the 14-week feeding period (2 weeks after irradiation in the propagation depends on intercellular current flow passing through gap junctions. 4 The principal gap junction proteins expressed in the ventricle and atrium are connexin (Cx) 43 and Cx40, respectively. Previously, we reported that targeted heavy ion irradiation (THIR; 15 Gy) to rabbit hearts upregulated Cx43 in the ventricles after 2 weeks. ...
Background: Low-invasive stereotactic body radiation therapy is a novel anti-arrhythmic strategy. The mechanisms underlying its effects against ventricular tachycardia/fibrillation (VT/VF) are gradually becoming clear, whereas those underlying atrial tachycardia/fibrillation (AT/AF) remain unknown. This study investigated the effects of carbon ion beam on gap junction expression and sympathetic innervation.
Methods and Results: Atrial and ventricular tachyarrhythmia models was established in 26 hypercholesterolemic (HC) 3-year-old New Zealand white rabbits; 12 rabbits were irradiated with a single 15-Gy carbon ion beam (targeted heavy ion irradiation [THIR]) and 14 were not (HC group). Eight 3-month-old rabbits (Young) were used as a reference group. In vivo induction frequencies in the Young, HC, and HC+THIR groups were 0%, 9.9%, and 1.2%, respectively, for AT/AF and 0%, 7.8%, and 1.2%, respectively, for VT/VF (P<0.01). The conduction velocity of the atria and ventricles on optical mapping was significantly reduced in the HC group; this was reversed in the HC+THIR group. Connexin-40 immunolabelling in the atria was 66.1–78.7% lower in the HC than Young group; this downregulation was less pronounced in the HC+THIR group (by 23.1–44.4%; P<0.01). Similar results were obtained for ventricular connexin-43. Sympathetic nerve densities in the atria and ventricles increased by 41.9–65.3% in the HC vs. Young group; this increase was reversed in the HC+THIR group.
Conclusions: Heavy ion radiation reduced vulnerability to AT/AF and VT/VF in HC elderly rabbits and improved cardiac conductivity. The results suggest involvement of connexin-40/43 upregulation and suppression of sympathetic nerve sprouting.
... Numerous investigations have established that Cx43 localization is essential for cardiac cell impulse propagation and thus normal cardiac function in the healthy human heart. 64,65 Therefore, variations in the localization of Cx43 can be a prominent mechanism for arrhythmias. 66 Since both AFM results and calcium transient highlighted an arrhythmic profile of HGPS NRVM, we investigated the levels and localizations of the gap junctions through immunostaining for Cx43. Figure 8B shows characteristic images of NRVM stained for the cardiac marker, α-actinin (gray), the gap junction marker Cx43 (red), the GFP virus (green), and the cell nuclei marker DAPI (blue). ...
Given the clinical effect of progeria syndrome, understanding the cell mechanical behavior of this pathology could benefit the patient's treatment. Progeria patients show a point mutation in the lamin A/C gene (LMNA), which could change the cell's biomechanical properties. This paper reports a mechano-dynamic analysis of a progeria mutation (c.1824 C > T, p.Gly608Gly) in neonatal rat ventricular myocytes (NRVMs) using cell indentation by atomic force microscopy to measure alterations in beating force, frequency, and contractile amplitude of selected cells within cell clusters. Furthermore, we examined the beating rate variability using a time-domain method that produces a Poincaré plot because beat-to-beat changes can shed light on the causes of arrhythmias. Our data have been further related to our cell phenotype findings, using immunofluorescence and calcium transient analysis, showing that mutant NRVMs display changes in both beating force and frequency. These changes were associated with a decreased gap junction localization (Connexin 43) in the mutant NRVMs even in the presence of a stable cytoskeletal structure (microtubules and actin filaments) when compared with controls (wild type and non-treated cells). These data emphasize the kindred between nucleoskeleton (LMNA), cytoskeleton, and the sarcolemmal structures in NRVM with the progeria Gly608Gly mutation, prompting future mechanistic and therapeutic investigations.
... [79][80][81][82][83] Disruption of cell-cell adhesion molecules results in abnormal cardiac conduction, arrythmias, and cardiomyopathies. 5,84,85 Zebrafish hearts lacking TAZ (WWTR1) were shown to exhibit abnormal cell-cell junctions. 86 Yap CUT&Tag sequencing data indicated that many genes that regulate cell-cell communication and adhesion are potentially regulated by Yap. ...
Background:
The sinoatrial node (SAN) functions as the pacemaker of the heart, initiating rhythmic heartbeats. Despite its importance, the SAN is one of the most poorly understood cardiac entities because of its small size and complex composition and function. The Hippo signaling pathway is a molecular signaling pathway fundamental to heart development and regeneration. Although abnormalities of the Hippo pathway are associated with cardiac arrhythmias in human patients, the role of this pathway in the SAN is unknown.
Methods:
We investigated key regulators of the Hippo pathway in SAN pacemaker cells by conditionally inactivating the Hippo signaling kinases Lats1 and Lats2 using the tamoxifen-inducible, cardiac conduction system-specific Cre driver Hcn4CreERT2 with Lats1 and Lats2 conditional knockout alleles. In addition, the Hippo-signaling effectors Yap and Taz were conditionally inactivated in the SAN. To determine the function of Hippo signaling in the SAN and other cardiac conduction system components, we conducted a series of physiological and molecular experiments, including telemetry ECG recording, echocardiography, Masson Trichrome staining, calcium imaging, immunostaining, RNAscope, cleavage under targets and tagmentation sequencing using antibodies against Yap1 or H3K4me3, quantitative real-time polymerase chain reaction, and Western blotting. We also performed comprehensive bioinformatics analyses of various datasets.
Results:
We found that Lats1/2 inactivation caused severe sinus node dysfunction. Compared with the controls, Lats1/2 conditional knockout mutants exhibited dysregulated calcium handling and increased fibrosis in the SAN, indicating that Lats1/2 function through both cell-autonomous and non-cell-autonomous mechanisms. It is notable that the Lats1/2 conditional knockout phenotype was rescued by genetic deletion of Yap and Taz in the cardiac conduction system, and these rescued mice had normal sinus rhythm and reduced fibrosis of the SAN, indicating that Lats1/2 function through Yap and Taz cleavage under targets and tagmentation sequencing data showed that Yap potentially regulates genes critical for calcium homeostasis such as Ryr2 and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as Tgfb1 and Tgfb3. Consistent with this, Lats1/2 conditional knockout mutants had decreased Ryr2 expression and increased Tgfb1 and Tgfb3 expression compared with control mice.
Conclusions:
We reveal, for the first time to our knowledge, that the canonical Hippo-Yap pathway plays a pivotal role in maintaining SAN homeostasis.
... At first glance, they revealed that the CMs cultured on the PPy/CNT and PEDOT/ CNT substrates presented an increased area of Cx43 with a more organized localization when compared with the gelatin-coated glass substrates. Furthermore, multiple studies suggested that the organization of Cx43 is crucial for a healthy cardiac function and impulse propagation, and the alteration of such organization may lead to arrythmias (Kostin et al. 2004;Severs et al. 2004). Thus, we can conclude that both PPy/CNT and PEDOT/CNT systems induce a more organized Cx43 localization which correlate with our calcium imaging analysis results described above and thus, this indicate the great potential of CNT to be Fig. 5 Fluorescence imaging staining of Cx43 gap junctions (in red) to evaluate the intercellular communication after 14 days of cocultured CMs (green) and fibroblasts (pink) on gelatin, PPy/CNT, and PEDOT/CNT bidimensional films used in scaffolds to improve CMs function and better survival in cardiac tissue engineering applications (Ballerini 2013). ...
Carbon nanotubes (CNTs) have become promising advanced materials and a new tool to specifically interact with electroresponsive cells. Likewise, conductive polymers (CP) appear promising electroactive biomaterial for proliferation of cells. Herein, we have investigated CNT blends with two different conductive polymers, polypyrrole/CNT (PPy/CNT) and PEDOT/CNT to evaluate the growth, survival, and beating behavior of neonatal rat ventricular myocytes (NRVM). The combination of CP/CNT not only shows excellent biocompatibility on NRVM, after 2 weeks of culture, but also exerts functional effects on networks of cardiomyocytes. NRVMs cultured on CNT-based substrates exhibited improved cellular function, i.e., homogeneous, non-arrhythmogenic, and more frequent spontaneous beating; particularly PEDOT/CNT substrates, which yielded to higher beating amplitudes, thus suggesting a more mature cardiac phenotype. Furthermore, cells presented enhanced structure: aligned sarcomeres, organized and abundant Connexin 43 (Cx43). Finally, no signs of induced hypertrophy were observed. In conclusion, the combination of CNT with CP produces high viability and promotes cardiac functionality, suggesting great potential to generate scaffolding supports for cardiac tissue engineering.
Graphical abstract
... The disorder of Cx43 distribution and abnormal expression are an important pathogenic mechanism to arrhythmia caused by various heart diseases [29]. Cx43 is the main connexin of ventricular myocytes. ...
Background:
Dapagliflozin can significantly improve heart failure, and Cx43 is one of the molecular mechanisms of heart failure. This study investigated the effect of dapagliflozin on Cx43 and Akt/mTOR signaling pathway in ventricular myocytes.
Methods:
A rat model of type 2 diabetes mellitus was established by high-fat diet combined with streptozotocin, and the animals were treated randomly with dapagliflozin. The morphological changes of the myocardium were observed by hematoxylin eosin staining, and the expression and distribution of Cx43 in ventricular myocytes were detected by immunohistochemistry. And Western blot determined the expressions of Cx43, Akt, mTOR, p62, and LC3 proteins in rat myocardium.
Results:
Compared with the normal control group, the heart rate of diabetic rats decreased significantly (p < 0.05), QRS wave of ECG widened, and QT interval prolonged (p < 0.05). Dapagliflozin treatment in diabetic rats resulted in improvements in these ECG indexes (p < 0.05) with early administration group obtaining greater efficacy than the late administration group (p < 0.05). In the normal control group, the cardiomyocytes were arranged orderly, and the expression of Cx43 was dense, uniform, and regular, which was higher than that in the intercalated disc. In the diabetic control model group, the cardiomyocytes were enlarged and presented disorderly with detection of Cx43 in the cytoplasm. Early use of dapagliflozin better improved these myocardial tissue lesions. Of note, as diabetic rats exhibited decreased expression of Cx43, Akt, and mTOR (p < 0.05), increased p62 expression (p < 0.05), and decreased LC3-II/I ratio (p < 0.05), administration of dapagliflozin partially reversed the expression of the above proteins (p < 0.05) with greater improvement in the early administration group compared with the late administration group (p < 0.05).
Conclusions:
Dapagliflozin increases the expression of Cx43 in cardiomyocytes of diabetic rats and thereby alleviates heart failure partly through regulating the Akt/mTOR signaling pathway.
... Cx45 is the first connexin expressed during early stages of cardiovascular development [30]. However, in the adult heart, it is expressed predominantly in the conduction system while expressed in low quantities in both ventricles and atria with a slightly higher level in the atrium than the ventricle [32]. Cx37 is expressed mainly in the vascular endothelium [31]. ...
Atrial fibrillation (AF) represents the most common type of clinical cardiac arrhythmia worldwide and contributes to substantial morbidity, mortality and socioeconomic burden. Aggregating evidence highlights the strong genetic basis of AF. In addition to chromosomal abnormalities, pathogenic mutations in over 50 genes have been causally linked to AF, of which the majority encode ion channels, cardiac structural proteins, transcription factors and gap junction channels. In the heart, gap junctions comprised of connexins (Cxs) form intercellular pathways responsible for electrical coupling and rapid coordinated action potential propagation between adjacent cardiomyocytes. Among the 21 isoforms of connexins already identified in the mammal genomes, 5 isoforms (Cx37, Cx40, Cx43, Cx45 and Cx46) are expressed in human heart. Abnormal electrical coupling between cardiomyocytes caused by structural remodeling of gap junction channels (alterations in connexin distribution and protein levels) has been associated with enhanced susceptibility to AF and recent studies have revealed multiple causative mutations or polymorphisms in 4 isoforms of connexins predisposing to AF. In this review, an overview of the genetics of AF is made, with a focus on the roles of mutant myocardial connexins and gap junctions in the pathogenesis of AF, to underscore the hypothesis that cardiac connexins are a major molecular target in the management of AF.
... However, Hsieh et al. (2011) reported that TH decreases NP-Cx43, which coincided with conduction disturbance in normal rabbit hearts. Cx43 dephosphorylation has been shown to promote conduction abnormalities and facilitate arrhythmias (Severs et al., 2004). Why a decrease in NP-Cx43 results in conduction slowing during TH remains unclear. ...
Aims: Whether therapeutic hypothermia (TH) is proarrhythmic in preexisting failing hearts with acute ischemia–reperfusion (IR) injury is unknown. Additionally, the effectiveness of rotigaptide on improving conduction slowing in hearts with IR injury is ambiguous. We investigated the electrophysiological effects of TH and rotigaptide in failing rabbit hearts with acute IR injury and determined the underlying molecular mechanisms.
Methods and Results: Heart failure was induced by right ventricular pacing (320 beats/min, 4 weeks). Rabbits with pacing-induced heart failure were randomly divided into TH (n = 14) and non-TH (n = 7) groups. The IR rabbit model was created by ligating the coronary artery for 60 min, followed by reperfusion for 15 min in vivo. Then, the hearts were excised quickly and Langendorff-perfused for simultaneous voltage and intracellular Ca²⁺ (Cai) optical mapping. Electrophysiological studies were conducted, and vulnerability to ventricular fibrillation (VF) was evaluated using pacing protocols. TH (33°C) was instituted after baseline studies, and electrophysiological studies were repeated. Rotigaptide (300 nM) was infused for 20 min, and electrophysiological studies were repeated under TH. Cardiac tissues were sampled for Western blotting. TH increased the dispersion and beat-to-beat variability of action potential duration (APD), aggravated conduction slowing, and prolonged Cai decay to facilitate spatially discordant alternans (SDA) and VF induction. Rotigaptide reduced the dispersion and beat-to-beat variability of APD and improved slowed conduction to defer the onset of arrhythmogenic SDA by dynamic pacing and elevate the pacing threshold of VF during TH. However, the effect of rotigaptide on TH-enhanced VF inducibility was statistically insignificant. TH attenuated IR-induced dysregulation of protein expression, but its functional role remained uncertain.
Conclusion: Therapeutic hypothermia is proarrhythmic in failing hearts with acute IR injury. Rotigaptide improves TH-induced APD dispersion and beat-to-beat variability and conduction disturbance to defer the onset of arrhythmogenic SDA and elevate the VF threshold by dynamic pacing, but these beneficial electrophysiological effects are unable to suppress TH-enhanced VF inducibility significantly.
... This facilitates the passage of potential through a tissue. For example, moving action potential in heart muscles flows across cells, causing the heart to pulse rhythmically (13). ...
Cell-cell adhesion complexes are macromolecular adhesive organelles that integrate cells into tissues. Perturbations of the cell-cell adhesion structure or relatedmechanotransduction pathways lead to pathological conditions such as skin and heart diseases, arthritis, and cancer. Mechanical stretching has been used to stimulate the mechanotransduction process originating from the cell-cell adhesion and cell-extracellular matrix (ECM) complexes. The current techniques, however, have limitations on their ability to measure the cell-cell adhesion force directly and quantitatively. These methods use a monolayer of cells, which makes it impossible to quantify the forces within a single cell-cell adhesion complex. Other methods using single cells or cell pairs rely on cell-ECM adhesion to find the cell-cell adhesion forces and consequently, they indirectly measure the junctional forces. In the current study, we designed and developed a single cell-cell adhesion interrogation and stimulation platform based on nanofabricated polymeric structures. The platform employs microstructures fabricated from biocompatible materials using two photon polymerization (TPP), a process that enables direct 3D structure writing with nanometer precision. The microdevice allows a pair of epithelial cells to form a mature cell junction. The single matured cell junction is stretched with controlled strain until cell-cell junction ruptures while the forces within the cell-junction-cell system are recorded. Using this platform, we have conducted mechanical characterization of a single cell junction with strain-stress analysis. The strain dependency of the junction has been investigated through the stretch test with four different strain rates. The results showed that the junction behaves in a strain-rate dependent manner, where high strain-rates lead to decreased viscosity property, a characteristic for a shear-thinning viscoelastic material. This also confirms our hypothesis that strain-rate plays an important role in the cell mechanical behavior, particularly the cytoskeleton dominant cell mechanics. The maturation of this technology can pave the way for the in situ investigation of mechano-chemical signaling pathways mediated by cell-cell junctions and potentially reveal novel disease mechanisms in which defects in cell-cell adhesion play a significant role in the disease pathology. Advisor: Ruiguo Yang
... Cx43 lh10 zebrafish exhibit severe defects of the cardiovascular system including malformed, elongated hearts, decreased heart rate, disorganized and malformed vasculature, and impaired blood flow, indicating that undisturbed gap junction endocytosis is crucial for normal organ development. Cardiovascular phenotypes in the cx43 lh10 mutant fish correlates with the well-established and documented role GJs play in heart development and function (Reaume et al., 1995, Ya et al., 1998, Severs, 1999, Jongsma et al., 2000, Gutstein et al., 2001, Inoguchi et al., 2001, Severs et al., 2004, Bruce et al., 2008, Maass et al., 2009, Bakkers, 2011, Martins-Marques et al., 2015a-d, Michela et al., 2015. In addition, altered Cx43 function and GJIC is known to be associated with cardiomyopathies that include heart failure, myocardial ischemia, and cardiac arrhythmias (Beardslee et al., 2000;Bruce et al., 2008;Duffy, 2012;Martins-Marques et al., 2015d). ...
Gap junctions mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting the exchange of molecules, ions, and electrical impulses. Gap junctions are assembled from connexin (Cx) proteins, with connexin 43 (Cx43) being the most ubiquitously expressed and best studied. While the molecular events that dictate the Cx43 life cycle have largely been characterized, the unusually short half-life of connexins of only 1-5 hours, resulting in constant endocytosis and biosynthetic replacement of gap junction channels has remained puzzling. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43 ∆256-289 ) and a zebrafish model ( cx43 lh10 ). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx 43 lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired development. Our findings demonstrate for the first time that Cx43 gap junction endocytosis is an essential aspect of gap junction function and when impaired, gives rise to significant physiological problems as revealed here for cardiovascular development and function.
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... In fact, gap junctional intercellular communication (GJIC) is actively involved in virtually all aspects of the cellular life cycle, ranging from cell growth to cell death, such as cell proliferation, migration, and apoptosis [60]. They are also related to a wide variety of diseases and pathological processes, including congenital [61] or acquired disorders related, including some related to the heart [62][63][64], brain [65,66], kidney [67,68], among many others. It highlights the fact that gap junctions have been described associated in various ways with cancer [69][70][71][72], either as a cause or consequence [73]. ...
Ouabain is a cardiac glycoside that has been described as a hormone, with interesting effects on epithelial physiology. We have shown previously that ouabain induces gap junctional intercellular communication (GJIC) in wild, sensitive cells (MDCK-S), but not in cells that have become insensitive (MDCK-I) by modifying their Na+-K+-ATPase. We have also demonstrated that prostaglandin E2 (PGE2) is able to induce increased GJIC by a mechanism other than ouabain, that does not depend on Na+-K+-ATPase. In this work we show, by dye transfer assays, that when MDCK-S and MDCK-I are randomly mixed, to form monolayers, the latter stablish GJIC, because of stimulation by a compound released to the extracellular media, by MDCK-S cells, after treatment with ouabain, as evidenced by the fact that monolayers of only MDCK-I cells, treated with a conditioned medium (CM) that is obtained after incubation of MDCK-S monolayers with ouabain, significantly increase their GJIC. The further finding that either (1) pre-treatment with COX-2 inhibitors or (2) addition to CM of antagonists of EP2 receptor abolish CM’s ability to induce GJIC in MDCK-I monolayers indicate that PGE2 is the GJIC-inducing compound. Therefore, these results indicate that, in addition to direct stimulation, mediated by Na+-K+-ATPase, ouabain enhances GJIC indirectly through the paracrine production of PGE2.
... Both the normal distribution of connexins and their phosphorylation state are often disturbed under pathological conditions. This would be the case of myocardial ischemia, where gap junction remodeling occurs rapidly after coronary occlusion ( Figure 5) [298,299]. Redistribution of gap junctions outside intercalated discs was first reported in the ventricular myocardium bordering the infarct scar in patients with advanced ischemic heart disease [300]. This was later confirmed in the epicardial border zone of healing myocardial infarctions in dogs, where Cx43 was shown to be mainly distributed, 4 days after coronary occlusion, along the lateral surface of cardiomyocytes [301]. ...
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.
... The second mechanism of arrhythmogenesis involves a decrease in coupling strength. The role of reduced gap junction coupling in promoting arrhythmogenesis is very well documented in experimental systems [23,24]. A reduction in cell-to-cell coupling diminishes the electrotonic currents that couple cells so relives the dampening effect of neighbouring tissue in limiting the emergence of ectopy (i.e. ...
Contemporary accounts of the initiation of cardiac arrhythmias typically rely on after-depolarizations as the trigger for reentrant activity. The after-depolarizations are usually triggered by calcium entry or spontaneous release within the cells of the myocardium or the conduction system. Here we propose an alternative mechanism whereby arrhythmias are triggered autonomously by cardiac cells that fail to repolarize after a normal heartbeat. We investigated the proposal by representing the heart as an excitable medium of FitzHugh-Nagumo cells where a proportion of cells were capable of remaining depolarized indefinitely. As such, those cells exhibit bistable membrane dynamics. We found that heterogeneous media can tolerate a surprisingly large number of bistable cells and still support normal rhythmic activity. Yet there is a critical limit beyond which the medium is persistently arrhythmogenic. Numerical analysis revealed that the critical threshold for arrhythmogenesis depends on both the strength of the coupling between cells and the extent to which the abnormal cells resist repolarization. Moreover, arrhythmogenesis was found to emerge preferentially at tissue boundaries where cells naturally have fewer neighbors to influence their behavior. These findings may explain why atrial fibrillation typically originates from tissue boundaries such as the cuff of the pulmonary vein.
... Gap junctions in the heart provide low resistance pathways for propagating the action potential across the myocardium, contributing to electrical coupling and signal propagation [10]. Alteration of cardiomyocyte gap junctions and their main components, connexins (Cx), has been suggested to contribute to the formation of arrhythmias, including atrial fibrillation [11][12][13]. ...
Aging leads to structural and electrophysiological changes that increase the risk of postoperative atrial arrhythmias; however, noninvasive preoperative markers of atrial proarrhythmic conditions are still needed. This study is aimed at assessing whether interatrial dyssynchrony determined using two-dimensional speckle tracking echocardiography relates to proarrhythmic structural and functional remodeling. A cohort of 45 patients in sinus rhythm referred for cardiac surgery was evaluated by echocardiography and surface electrocardiogram the day before the intervention. Transmembrane potential, connexin, and potassium channel distribution, inflammatory, and nitrooxidative markers were measured from right atrial tissue obtained from patients. A difference greater than 40 milliseconds between right and left atrial free wall contraction confirmed the presence of interatrial dyssynchrony in 21 patients. No difference in relation with age, previous diseases, and 2-dimensional echocardiographic findings as well as average values of global longitudinal right and left atrial strain were found between synchronic and dyssynchronic patients. Postoperative atrial fibrillation incidence increased from 8.3% in the synchronic group to 33.3% in the dyssynchronic ones. P wave duration showed no difference between groups. Action potentials from dyssynchronous patients decreased in amplitude, maximal rate of depolarization, and hyperpolarized. Duration at 30% of repolarization increased, being markedly shorter at 90% of repolarization. Only the dyssynchronous group showed early and delayed afterdepolarizations. Atrial tissue of dyssynchronous patients displayed lateralization of connexin 40 and increased connexin 43 expression and accumulation of tumor necrosis factor-α in the intercalated disc. Tumor necrosis factor-α did not colocalize, however, with lateralized connexin 40. Nitroxidative marks and KATP channels increased perivascularly and in myocytes. Our results demonstrate that, as compared to a traditional surface electrocardiogram, the novel noninvasive echocardiographic evaluation of interatrial dyssynchrony provides a better identification of nonaged-related proarrhythmic atrial remodeling with increased susceptibility to postoperative atrial fibrillation.
1. Introduction
Arrhythmias usually complicate cardiovascular surgery, and aging is the main risk factor. Atrial fibrillation is the most frequent sustained arrhythmia with a peak of appearance between the second and fifth days of the postoperative stay [1]. Its incidence progressively increases from 18% in sexagenarians to 50% in octogenarians [2]. Arrhythmic events are usually self-limited, and treatment frequently restores sinus rhythm. However, postoperative atrial fibrillation (POAF) prolongs hospital stay and increases the risk of stroke and mortality [1].
Atrial aging is an elusive prooxidative and proarrhythmic condition. Oxidative stress, hyperadrenergic states, and inflammation contribute to age-related atrial remodeling, but these tissue alterations are challenging to identify noninvasively [3, 4]. In this context, atrial enlargement, stiffness, and conduction blockade are known risk factors, but they are present in only a few patients. Therefore, the mechanisms involved in the onset and perpetuation of POAF are difficult to foresee [5]. Additionally, surgery per se facilitates arrhythmias due to ischemia-reperfusion injury, which increases the preexisting oxidative stress state. The lack of tools to estimate the proarrhythmic substrate evidences the absence of preventive interventions.
Structure and function of the beating heart can reveal atrial hidden oxidation and inflammation. Signs of tissue remodeling, before dilatation, arise from atrial dynamic cyclic changes. New echocardiographic techniques like strain and strain rate represent the magnitude and rate of myocardial deformation. Strain can reflect distensibility and atrial contractility [6]. Nonmyocytic cells and extracellular matrix mainly affect distensibility [7]. Cardiomyocyte structure and intercellular communication determine contractile function. Each atrial segment follows a trajectory during the cardiac cycle representative of the tissue physiology [8]. Electrical remodeling involves alterations in both myocytes and nonmyocytic cells, which manifest as contractile dyssynchrony in the echocardiogram, in a similar way as reported for the ventricles [9].
Gap junctions in the heart provide low resistance pathways for propagating the action potential across the myocardium, contributing to electrical coupling and signal propagation [10]. Alteration of cardiomyocyte gap junctions and their main components, connexins (Cx), has been suggested to contribute to the formation of arrhythmias, including atrial fibrillation [11–13]. Accumulating evidence also suggests that inflammation and oxidative stress are involved in atrial remodeling. Detection of protein 3-nitrotyrosine is regarded as a marker of nitrooxidative stress and is observed especially in inflammatory processes. The reaction of peroxynitrite with tyrosine leads to the formation of 3-nitrotyrosine and promotes protein, lipid, and DNA damage [14, 15]. ATP-regulated potassium channels (KATP) are well-characterized metabolic and oxidative sensors in ischemia/reperfusion arrhythmias and here postulated as an interesting substrate of POAF [16].
This study is aimed at assessing whether interatrial dyssynchrony, determined by using two-dimensional speckle tracking echocardiography, relates to proarrhythmic structural and functional remodeling of the atria and whether this increases the susceptibility for POAF.
2. Materials and Methods
2.1. Subjects and Ethical Considerations
Patients with coronary artery disease, aortic stenosis, or the combination of both pathologies, scheduled for surgery at the Department of Cardiac Surgery (Clinic of Cuyo, Mendoza, Argentina), were prospectively enrolled between January 2018 and March 2020. Coronary disease and aortic valve stenosis severity were defined according to current ESC Guidelines to determine surgery indication [17, 18]. All subjects provided written informed consent under the research protocol approved by the Ethics Committee of the National University of Cuyo (Exp-Cuy: 22959/2017).
Clinical data including age, gender, and history of previous myocardial infarction and heart failure was collected. The presence of preoperative atrial fibrillation was determined according to previous electrocardiographic reports or diagnosis in medical history. Information regarding the following cardiovascular risk factors was collected: hypertension, dyslipidemia (low-density lipoprotein cholesterol above 100 mg/dL or the use of lipid-lowering drugs), smoking (current or any smoking habit in the past ten years), and diabetes mellitus (previous diagnosis of diabetes mellitus or glycated hemoglobin greater than 6.5%). The preoperative use of medications was also documented.
2.2. Inclusion and Exclusion Criteria
Patients over 18 years of age in sinus rhythm with an indication of cardiovascular surgery that gave written informed consent were included in the study. Exclusion criteria were as follows: indication of mitral or tricuspid valve repair or replacement, being older than 80 years, history of previous atrial fibrillation, presence of moderate valvular disease or valvular prosthesis, history of congenital cardiac abnormalities or cardiac tumors, emergency surgery, inability to provide informed consent, and a not entirely detectable left and right atrial profile from the apical four-chamber view during preoperative echocardiography.
2.3. Preoperative Electrocardiogram
Before surgery, patients underwent a 12-lead electrocardiogram (ECG) using the Synchronous ECG software V1.3.5. Measurement in milliseconds of the P wave and the PR segment was performed with the software caliper. According to the current classification, these patients were evaluated for the presence of an interatrial conduction disturbance called Bayes syndrome [19]. There are two major categories for this syndrome: complete and incomplete, both based on P wave duration and morphology in the 12-lead ECG.
2.4. Echocardiography and Atrial Strain
All patients were imaged in a left lateral decubitus position using ESAOTE ultrasound system equipment (MyLab30Gold Cardiovascular) with a 2-4 MHz/PA240 probe. Two-dimensional speckle tracking strain imaging was performed from the apical position by an experienced technician. The average frame rate for analysis was 60-80 frames/s. During a single breath-hold, three consecutive cardiac cycles were stored digitally for off-line analysis in the four-, two-, and three-chamber view. The entire right and left atriums were carefully visualized to prevent walls’ dropout.
Measurements focused on evaluating the indexed volume of the left atrium and the area of the right atrium. In the parasternal long-axis or short-axis view, M-mode of the left ventricle chamber was measured for diameter and wall thickness. In the apical 4-chamber view, the left ventricle ejection fraction was determined using the Simpson measurement. Mitral and tricuspid inflows were recorded at the tip of the valve leaflets. The peak velocities of early and late diastolic filling waves (E wave and A wave) and the E/A velocity ratio were measured. The e velocity was obtained by tissue Doppler averaging the lateral and septal mitral annulus values and the e wave of the tricuspid lateral wall. E/e’ values were obtained from both ventricles.
Strain and strain rate datasets were analyzed using a wall motion tracking software (ESAOTE MyLab). In apical views (4-chamber, 3-chamber, and 2-chamber), left atrial endocardial boundaries were manually measured at the end-diastolic phase. Right atria were only registered in the 4-chamber view. The values of the reservoir, conduit, and atrial contraction strain were recorded according to the EACVI/ASE/Industry Task Force to standardize deformation imaging [20]. The values of strain rate for the reservoir phase, the conduit, and contraction phases were also recorded (Figure 1). The same was also done in 4 chambers for the right atrium. The left atrium was subsequently divided into basal, medial, and roof atrial segments. This determined a total of 15 segments for the left atria when the 3 apical views were added. The right atrium was evaluated similarly, but only the lateral segments were taken into account, adding 3 more segments, thus making a total of 18 segments. Septal segments were considered as left atria.
(a)
... The mutation was also presented in the proband's father with lone AF but it was not found in the unaffected family members [52]. GJA5 remodeling may lead to abnormal electrical coupling to exert an effect on potential arrhythmogenic [55]. AF risk variants, rs10824026 and rs3740293, were associated with the expression of MYOZ1 [56,57]. ...
The biological features of the valvular heart disease with atrial fibrillation (AF-VHD) remain unknown when involving long non-coding RNAs (lncRNAs). This study performed system analysis on lncRNA and messenger RNA (mRNA) expression profiles constructed by using bioinformatics methods and tools for biological features of AF-VHD. Fold change and t-test were used to identify differentially expressed (DE) lncRNAs and mRNAs. The enrichment analysis of DE mRNAs was performed. The subgroups formed by lncRNAs and nearby mRNAs were screened, and a transcriptional regulation network among lncRNAs, mRNAs, and transcription factors (TFs) was constructed. The interactions between mRNAs related to lncRNAs and drugs were predicted. The 620 AF-VHD-related DE lncRNAs and 452 DE mRNAs were identified. The 3 lncRNA subgroups were screened. The 665 regulations mediated by lncRNAs and TFs were identified. The 9 mRNAs related to lncRNAs had 1 or more potential drug interactions, totaling 37 drugs. Of these, 9 drugs targeting 3 genes are already known to be able to control or trigger atrial fibrillation (AF) or other cardiac arrhythmias. The found biological features of AF-VHD provide foundations for further biological experiments to better understand the roles of lncRNAs in development from the valvular heart disease (VHD) to AF-VHD.
... 26,46 Relative expression of the different types of Cxs also depend on healthy versus diseased conditions, as described in a previously published review paper. 88 In brief, healthy ECs mostly express Cx37 and Cx40. During the initiation of atherosclerotic lesions, Cx43 begins to be expressed in addition to Cx37 and Cx40. ...
Purpose:
The endothelial glycocalyx (GCX) plays a critical role in the health of the vascular system. Degradation of the GCX has been implicated in the onset of diseases like atherosclerosis and cancer because it disrupts endothelial cell (EC) function that is meant to protect from atherosclerosis and cancer. Examples of such EC function include interendothelial cell communication via gap junctions and receptor-mediated interactions between endothelial and tumor cells. This review focuses on GCX-dependent regulation of these intercellular interactions in healthy and diseased states. The ultimate goal is to build new knowledge that can be applied to developing GCX regeneration strategies that can control intercellular interaction in order to combat the progression of diseases such as atherosclerosis and cancer.
Methods:
In vitro and in vivo studies were conducted to determine the baseline expression of GCX in physiologically relevant conditions. Chemical and mechanical GCX degradation approaches were employed to degrade the GCX. The impact of intact versus degraded GCX on intercellular interactions was assessed using cytochemistry, histochemistry, a Lucifer yellow dye transfer assay, and confocal, intravital, and scanning electron microscopy techniques.
Results:
Relevant to atherosclerosis, we found that GCX stability determines the expression and functionality of Cx43 in gap junction-mediated EC-to-EC communication. Relevant to cancer metastasis, we found that destabilizing the GCX through either disturbed flow-induced or enzyme induced GCX degradation results in increased E-selectin receptor-mediated EC-tumor cell interactions.
Conclusion:
Our findings lay a foundation for future endothelial GCX-targeted therapy, to control intercellular interactions and limit the progression of atherosclerosis and cancer.
... The role of reduced gap junction coupling in promoting arrhythmogenesis is very well 352 documented in experimental systems [25,26]. A reduction in cell-to-cell coupling 353 diminishes the electrotonic currents that couple cells so relives the dampening effect of 354 neighbouring tissue in limiting the emergence of ectopy (i.e. ...
Contemporary theories of cardiac fibrillation typically rely on the emergence of rotors to explain the transition from regular sinus rhythm to disordered electrophysiological activity. How those rotors spontaneously arise in the absence of re-entrant anatomical circuits is not fully understood. Here we propose a novel mechanism where arrhythmias are initiated by cardiac cells that fail to repolarize following a normal heartbeat. Those cells subsequently act as a focal ectopic source that drive the ensuing fibrillation. We used a simple computational model to investigate the impact of such cells in both homogeneous and heterogeneous excitable media. We found that heterogeneous media can tolerate a surprisingly large number of abnormal cells and still support normal rhythmic activity. At a critical limit the medium becomes chronically arrhythmogenic. Numerical analysis revealed that the critical threshold for arrhythmogenesis depends on both the strength of the coupling between cells and the extent to which the abnormal cells resist repolarization. Arrhythmogenesis was also found to emerge first at tissue boundaries where cells naturally have fewer neighbors to influence their behavior. These findings may explain why atrial fibrillation typically originates from the cuff of the pulmonary vein.
Author summary
Cardiac fibrillation is a medical condition where normal heart function is compromised as electrical activity becomes disordered. How fibrillation arises spontaneously is not fully understood. It is generally thought to be triggered by premature depolarization of the cardiac action potential in one or more cells. Those premature beats, known as early-afterdepolarizations, subsequently initiate a self-sustaining rotor in the otherwise normal heart tissue. In this study, we propose an alternative mechanism whereby arrhythmias are initiated by cardiac cells that fail to repolarize of their own accord but still operate normally when embedded in functional heart tissue. We find that such cells can act as focal ectopic sources under appropriate conditions of inter-cellular coupling. Moreover, cells on natural tissue boundaries are more susceptible to arrhythmia because they are coupled to fewer cells. This may explain why the pulmonary vein is often implicated as a source of atrial fibrillation.
... This study indicates that LVAD therapy does not reverse many of the transcriptional changes associated with heart failure and increased the expression of PDK4, a key regulator of glycolysis. Heart failure is a complex disease process where transcriptional changes contribute to contractile dysfunction, arrhythmias, and ultimately cell death [20]. These ongoing changes in gene expression likely contribute to poor rates of myocardial recovery post-LVAD therapy and ongoing issues with symptomatic heart failure as well as arrhythmias. ...
Background
Myocardial recovery with Left ventricular assistant device (LVAD) therapy is dichotomous with some patients obtaining remission from end-stage heart failure whereas most require transplantation or remain on pump support long term. Our goal was to determine transcriptional and free radical responses to LVAD treatment.
Methods
Tissues were collected from patients before and after LVAD placement in non-ischemic dilated cardiomyopathy patients (n = 14) along with controls (n = 3). RNA sequencing (RNASeq) analysis quantified transcriptional profiles by using a custom targeted panel of heart failure related genes on the PGM sequencer. The differential expression analysis between groups was conducted using edgeR (Empirical analysis of digital gene expression data in R) package in Bioconductor. Ingenuity Pathway Analysis (IPA) was carried out on differentially expressed genes to understand the biological pathways involved. Electron Paramagnetic Resonance (EPR) Spectroscopy was utilized to measure levels of free radicals in whole blood collected pre- and post-LVAD implantation (n = 16).
Results
Thirty-five genes were differentially expressed in pre-LVAD failing hearts compared to controls. In response to LVAD therapy, only Pyruvate dehydrogenase kinase 4 (PDK4) and period circadian protein homolog 1 (PER1) were altered with 34 heart failure related genes still differentially expressed post-LVAD compared to controls. IPA showed that DNA methylation-related genes were upregulated in both pre- and post-LVAD and was persistent with a Z-score of 2.00 and 2.36 for DNA Methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B), respectively. Inhibition of micro RNA21 (mir21) was also significant on pathway analysis in the post-LVAD population with a Z-score of − 2.00. Levels of free radicals in blood of pre- and post-LVAD patients did not change significantly.
Conclusion
LVAD therapy does not reverse many of the transcriptional changes associated with heart failure. Persistent changes in gene expression may be related to ongoing oxidative stress, continued DNA methylation, or changes in metabolism. PDK4 is a key regulator of glucose metabolism and its increased expression by LVAD therapy inhibited pyruvate metabolism.
... Furthermore, the disturbances in the gap junctions may contribute to the development of cardiovascular abnormalities, including myocardial infarction (30,31). Thus, it may be hypothesized that diabetes-mediated changes in the myocardial expression of connexin 43 ...
The present study was designed to investigate the role of amylin, H2S, and connexin 43 in vascular dysfunction and enhanced ischemia-reperfusion (I/R)-induced myocardial injury in diabetic rats. A single dose of streptozotocin (65 mg/kg) was employed to induce diabetes mellitus. After eight weeks, there was a significant decrease in the plasma levels of amylin, an increase in I/R injury to isolated hearts (increase in CK-MB and cardiac troponin release) on the Langendorff apparatus. Moreover, there was a significant impairment in vascular endothelium function as assessed by quantifying acetylcholine-induced relaxation in norepinephrine-precontracted mesenteric arteries. There was also a marked decrease in the expression of H2S and connexin 43 in the hearts following I/R injury in diabetic rats. Treatment with amylin agonist, pramlintide (100 and 200 µg/kg), and H2S donor, NaHS (10 and 20 μmol/kg) for two weeks improved the vascular endothelium function, abolished enhanced myocardial injury and restored the levels of H2S along with connexin 43 in diabetic animals. However, pramlintide and NaHS failed to produce these effects the presence of gap junction blocker, carbenoxolone (20 and 40 mg/kg). Carbenoxolone also abolished the myocardial levels of connexin 43 without affecting the plasma levels of amylin and myocardial levels of H2S. The decrease in the amylin levels with a consequent reduction in H2S and connexin 43 may contribute to inducing vascular dysfunction and enhancing I/R-induced myocardial injury in diabetic rats.
... This study indicates that LVAD therapy does not reverse many of the transcriptional changes associated with heart failure and increased the expression of PDK4, a key regulator of glycolysis. Heart failure is a complex disease process where transcriptional changes contribute to contractile dysfunction, arrhythmias, and ultimately cell death [20]. These ongoing changes in gene expression likely contribute to poor rates of myocardial recovery post-LVAD therapy and ongoing issues with symptomatic heart failure as well as arrhythmias. ...
Background: Myocardial recovery with Left ventricular assistant device (LVAD) therapy is dichotomous with some patients obtaining remission from end-stage heart failure whereas most require transplantation or remain on pump support long term. Our goal was to determine transcriptional and free radical responses to LVAD treatment.
Methods: Tissues were collected from patients before and after LVAD placement in non-ischemic dilated cardiomyopathy patients (n=14) along with controls (n=3). RNA sequencing (RNASeq) analysis quantified transcriptional profiles by using a custom targeted panel of heart failure related genes on the PGM sequencer. The differential expression analysis between groups was conducted using edgeR (Empirical analysis of digital gene expression data in R) package in Bioconductor. Ingenuity Pathway Analysis (IPA) was carried out on differentially expressed genes to understand the biological pathways involved. Electron Paramagnetic Resonance (EPR) Spectroscopy was utilized to measure levels of free radicals in whole blood collected pre- and post-LVAD implantation (n=16).
Results: 35 genes were differentially expressed in pre-LVAD failing hearts compared to controls. In response to LVAD therapy, only Pyruvate dehydrogenase kinase 4 (PDK4) and period circadian protein homolog 1 (PER1) were altered with 34 heart failure related genes still differentially expressed post-LVAD compared to controls. IPA showed that DNA methylation-related genes were upregulated in both pre- and post-LVAD and was persistent with a Z-score of 2.00 and 2.36 for DNA Methyltransferase 3A (DNMT3A) and DNA methyltransferase 3B (DNMT3B), respectively. Inhibition of micro RNA21 (mir21) was also significant on pathway analysis in the post-LVAD population with a Z-score of -2.00. Levels of free radicals in blood of pre- and post-LVAD patients did not change significantly.
Conclusion: LVAD therapy does not reverse many of the transcriptional changes associated with heart failure. Persistent changes in gene expression may be related to ongoing oxidative stress, continued DNA methylation, or changes in metabolism. PDK4 is a key regulator of glucose metabolism and its increased expression by LVAD therapy inhibited pyruvate metabolism.
... Electrical activity initiated at the SA node must propagate through atrial myocardium, the AV node, His-Purkinje system, and into ventricular myocardium. Disruption of cell-cell conduction results in cardiac arrhythmias and cardiomyopathies, a leading cause of morbidity and mortality worldwide [50][51][52][53] . Intercalated disc proteins have been recognized as important regulators of cardiac conduction due to their function in maintaining electrical and physical coupling between cells. ...
Rationale:
ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 (TJP1) gene, is a regulator of paracellular permeability in epithelia and endothelia. ZO-1 interacts with the actin cytoskeleton, gap, and adherens junction proteins and localizes to intercalated discs in cardiomyocytes. However, the contribution of ZO-1 to cardiac physiology remains poorly defined.
Objective:
We aim to determine the role of ZO-1 in cardiac function.
Methods and results:
Inducible cardiomyocyte-specific Tjp1 deletion mice (Tjp1
fl/fl
; Myh6
Cre/Esr1*
) were generated by crossing the Tjp1 floxed mice and Myh6
Cre/Esr1*
transgenic mice. Tamoxifen-induced loss of ZO-1 led to atrioventricular (AV) block without changes in heart rate, as measured by ECG and ex vivo optical mapping. Mice with tamoxifen-induced conduction system-specific deletion of Tjp1 (Tjp1
fl/fl
; Hcn4
CreERt2
) developed AV block while tamoxifen-induced conduction system deletion of Tjp1 distal to the AV node (Tjp1
fl/fl
; Kcne1
CreERt2
) did not demonstrate conduction defects. Western blot and immunostaining analyses of AV nodes showed that ZO-1 loss decreased Cx (connexin) 40 expression and intercalated disc localization. Consistent with the mouse model study, immunohistochemical staining showed that ZO-1 is abundantly expressed in the human AV node and colocalizes with Cx40. Ventricular conduction was not altered despite decreased localization of ZO-1 and Cx43 at the ventricular intercalated disc and modestly decreased left ventricular ejection fraction, suggesting ZO-1 is differentially required for AV node and ventricular conduction.
Conclusions:
ZO-1 is a key protein responsible for maintaining appropriate AV node conduction through maintaining gap junction protein localization.
BACKGROUND
Kawasaki disease (KD) is an acute febrile illness and systemic vasculitis often associated with cardiac sequelae, including arrhythmias. Abundant evidence indicates a central role for IL (interleukin)-1 and TNFα (tumor necrosis factor-alpha) signaling in the formation of arterial lesions in KD. We aimed to investigate the mechanisms underlying the development of electrophysiological abnormalities in a murine model of KD vasculitis.
METHODS
Lactobacillus casei cell wall extract–induced KD vasculitis model was used to investigate the therapeutic efficacy of clinically relevant IL-1Ra (IL-1 receptor antagonist) and TNFα neutralization. Echocardiography, in vivo electrophysiology, whole-heart optical mapping, and imaging were performed.
RESULTS
KD vasculitis was associated with impaired ejection fraction, increased ventricular tachycardia, prolonged repolarization, and slowed conduction velocity. Since our transcriptomic analysis of human patients showed elevated levels of both IL-1β and TNFα, we asked whether either cytokine was linked to the development of myocardial dysfunction. Remarkably, only inhibition of IL-1 signaling by IL-1Ra but not TNFα neutralization was able to prevent changes in ejection fraction and arrhythmias, whereas both IL-1Ra and TNFα neutralization significantly improved vasculitis and heart vessel inflammation. The treatment of L casei cell wall extract–injected mice with IL-1Ra also restored conduction velocity and improved the organization of Cx43 (connexin 43) at the intercalated disk. In contrast, in mice with gain of function of the IL-1 signaling pathway, L casei cell wall extract induced spontaneous ventricular tachycardia and premature deaths.
CONCLUSIONS
Our results characterize the electrophysiological abnormalities associated with L casei cell wall extract–induced KD and show that IL-1Ra is more effective in preventing KD-induced myocardial dysfunction and arrhythmias than anti-TNFα therapy. These findings support the advancement of clinical trials using IL-1Ra in patients with KD.
A basic phenomenon of the heart is its regular beating and a directed propagation of the electrical impulse which, during the plateau phase of the action potential, initiates contraction. The heart is a network of inter-communicating cells, allowing such a directed spread of activation. Cardiomyocytes communicate among each other and to a certain degree with non-cardiomyocytes such as fibroblasts. Action potentials are transferred from one cell to the next by intercellular communication channels, the gap junction channels, formed as dodecameric channels from protein subunits called connexins. In the heart, the main connexin isoforms are Cx43 (the 43 kDa connexin; ubiquitous), Cx40 (mostly in the atrium and specific conduction system) and Cx45 (in early developmental states and between fibroblasts and cardiomyocytes). Gap junction channels allow the intercellular transfer of current and small molecules (<1000 Da). Regarding arrhythmia, a specific feature of gap junctions is that they are normally found at the cell poles, thereby contributing to the heart’s anisotropic properties, while in cardiac disease, these channels are often found at the lateral borders of the cells. Moreover, a broad number of stimuli can regulate the channels. Thus, ions like H+, Ca++ and Na+, as well as ATP-loss, acylcarnitines, and lysophosphoglycerides, among others, can close the channels. Since these factors occur during cardiac ischemia, they lead to electrical isolation and silencing of the ischemic area. This alters the current source/sink ratio at the border, which will affect successive conduction.
Antiarrhythmic peptides, such as AAP10 enhance the Cx43-gap junction current by preventing the channels from uncoupling. This effect of AAPs is mediated via PKCα-activation and PKC-dependent phosphorylation of Cx43. The effect of AAP10 is pronounced in areas of increased de-phosphorylation and thereby shows a preference for ischemic tissue. These new agents open novel pharmacological options for prevention of ischemia-associated ventricular fibrillation.
Large-cohort studies using cardiovascular imaging and diagnostic datasets have assessed cardiac anatomy, function, and outcomes, but typically do not reveal underlying biological mechanisms. Cardiac digital twins (CDTs) provide personalized physics- and physiology- constrained in-silico representations, enabling inference of multi-scale properties tied to these mechanisms.
We constructed 3464 anatomically-accurate CDTs using cardiac magnetic resonance images from UK biobank and personalised their myocardial conduction velocities (CVs) from electrocardiograms (ECG), through an automated framework.
We found well-known sex-specific differences in QRS duration were fully explained by myocardial anatomy, as CV remained consistent across sexes. Conversely, significant associations of CV with ageing and increased BMI suggest myocardial tissue remodelling. Novel associations were observed with left ventricular ejection fraction and mental-health phenotypes, through a phenome-wide association study, and CV was also linked with adverse clinical outcomes.
Our study highlights the utility of population-based CDTs in assessing intersubject variability and uncovering strong links with mental health.
Mathematical modeling and simulation are well-established and powerful tools to integrate experimental data of individual components of cardiac electrophysiology, excitation-contraction coupling, and regulatory signaling pathways, to gain quantitative and mechanistic insight into pathophysiological processes and guide therapeutic strategies. Here, we briefly describe the processes governing cardiac myocyte electrophysiology and Ca2+ handling and their regulation, as well as action potential propagation in tissue. We discuss the models and methods used to describe these phenomena, including procedures for model parameterization and validation, in addition to protocols for model interrogation and analysis and techniques that account for phenotypic variability and parameter uncertainty. Our objective is to provide a summary of basic concepts and approaches as a resource for scientists training in this discipline and for all researchers aiming to gain an understanding of cardiac modeling studies.
A brand-new class of interstitial cells, called Tlocytes(TCs), has been detected in the heart. TCs can connect and transmit signals to almost all cardiomyocytes; this is highly interrelated with the occurrence and development of heart diseases. Modern studies have shown that berberine has a therapeutic effect on cardiovascular health. However, berberine’s mechanism of action on the cardiovascular system through cardiac TCs is unclear. Interestingly, 5 μm of berberine remarkably decreased the concentration of intracellular calcium and the membrane depolarization in cultured TCs, upregulated the expression of CX43 and β-catenin, and downregulated the expressions of TRPV4 and TRPV1. Here, TCs were identified in the vascular adventitia and intima, endocardium, myocardium, adventitia, and heart valves. Moreover, TCs were broadly dispersed around cardiac vessels and interacted directly through gap junctions and indirectly through extracellular vesicles. Together, Cardiac TCs interact with berberine and then deliver drug information to the heart. TCs may be an essential cellular target for drug therapy of the cardiovascular system.
Background:
Propagation of action potentials through the heart coordinates the heartbeat. Thus, intercalated discs, specialized cell-cell contact sites that provide electrical and mechanical coupling between cardiomyocytes, are an important target for study. Impaired propagation leads to arrhythmias in many pathologies, where intercalated disc remodeling is a common finding, hence the importance and urgency of understanding propagation dependence on intercalated disc structure. Conventional modeling approaches cannot predict changes in propagation elicited by perturbations that alter intercalated disc ultrastructure or molecular organization, because of lack of quantitative structural data at subcellular through nano scales.
Objectives:
This study sought to quantify intercalated disc structure at these spatial scales in the healthy adult mouse heart and relate them to chamber-specific properties of propagation as a precursor to understanding the effects of pathological intercalated disc remodeling.
Methods:
Using super-resolution light microscopy, electron microscopy, and computational image analysis, we provide here the first ever systematic, multiscale quantification of intercalated disc ultrastructure and molecular organization.
Results:
By incorporating these data into a rule-based model of cardiac tissue with realistic intercalated disc structure, and comparing model predictions of electrical propagation with experimental measures of conduction velocity, we reveal that atrial intercalated discs can support faster conduction than their ventricular counterparts, which is normally masked by interchamber differences in myocyte geometry. Further, we identify key ultrastructural and molecular organization features underpinning the ability of atrial intercalated discs to support faster conduction.
Conclusions:
These data provide the first stepping stone to elucidating chamber-specific effects of pathological intercalated disc remodeling, as occurs in many arrhythmic diseases.
We aimed to investigate the effect of chronic D-galactose exposure on the mimicking of natural aging processes based upon the hallmarks of aging. Seven-week-old male Wistar rats (n = 12) were randomly assigned to receive either normal saline solution as a vehicle (n = 6) or 150mg/kg/day of D-galactose subcutaneously for 28 weeks. Seventeen-month-old rats (n = 6) were also included as the chronologically aged controls. At the end of week 28 of the experiment (when the rats reach 35 weeks old and 24 months old), all rats were sacrificed for brain and heart collection. Our results showed that chronic D-galactose exposure mimicked natural aging characteristics of the brain and the heart in terms of deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, altered intercellular communication, and functional impairment. All of which highlight the potential of D-galactose as a substance for inducing brain and cardiac aging in animal experiments. DATA AVAILABILITY: The data that support the findings of this study are available from the corresponding author upon reasonable request.
During each heartbeat, the propagation of action potentials through the heart coordinates the contraction of billions of individual cardiomyocytes and is thus, a critical life process. Unsurprisingly, intercalated discs, which are cell-cell contact sites specialized to provide electrical and mechanical coupling between adjacent cardiomyocytes, have been the focus of much investigation. Slowed or disrupted propagation leads to potentially life-threatening arrhythmias in a wide range of pathologies, where intercalated disc remodeling is a common finding. Hence, the importance and urgency of understanding intercalated disc structure and its influence on action potential propagation. Surprisingly, however, conventional modeling approaches cannot predict changes in propagation elicited by perturbations that alter intercalated disc ultrastructure or molecular organization, owing to lack of quantitative structural data at subcellular through nano scales. In order to address this critical gap in knowledge, we sought to quantify intercalated disc structure at these finer spatial scales in the healthy adult mouse heart and relate them to function in a chamber-specific manner as a precursor to understanding the impacts of pathological intercalated disc remodeling. Using super-resolution light microscopy, electron microscopy, and computational image analysis, we provide here the first ever systematic, multiscale quantification of intercalated disc ultrastructure and molecular organization. By incorporating these data into a rule-based model of cardiac tissue with realistic intercalated disc structure, and comparing model predictions of electrical propagation with experimental measures of conduction velocity, we reveal that atrial intercalated discs can support faster conduction than their ventricular counterparts, which is normally masked by inter-chamber differences in myocyte geometry. Further, we identify key ultrastructural and molecular organization features underpinning the ability of atrial intercalated discs to support faster conduction. These data provide the first stepping stone to elucidating chamber-specific impacts of pathological intercalated disc remodeling, as occurs in many arrhythmic diseases.
Ventricular tachycardia (VT) and ventricular fibrillation are most causes of early death in patients with acute myocardial infarction (AMI). Conditional cardiac-specific low-density lipoprotein receptor-related protein 6 (LRP6)-knockout mice with connexin 43 (Cx43) reduction triggered the lethal ventricular arrhythmias. Thus, it is necessary for exploring whether LRP6 and its upstream genes circRNA1615 mediate the phosphorylation of Cx43 in VT of AMI. Here, we showed that circRNA1615 regulated the expression of LRP6 mRNA through sponge adsorption of miR-152-3p. Importantly, LRP6 interference fragments aggravated hypoxia injury of Cx43, while overexpression of LRP6 improved the phosphorylation of Cx43. Subsequently, interference with G-protein alpha subunit (Gαs) downstream of LRP6 further inhibited the phosphorylation of Cx43, along with increasing VT. Our results demonstrated that LRP6 upstream genes circRNA1615 controlled the damage effect and VT in AMI, and LRP6 mediated the phosphorylation of Cx43 via Gαs which played a role in VT of AMI.
Aim: Biologically engineered pacemaker, or bio-pacemaker, is a promising replacement for electronic pacemakers for treating cardiac dysfunction. Previous animal experimental studies, however, have not been able to accurately demonstrate the stability and efficiency of the bio-pacemaker yet. This study aimed to elucidate the underlying factors that affect bio-pacemaker's performance and to discover possible optimising solutions to enable the potential use of bio-pacemaker therapy.
Methods and results: The human ventricular myocytes model in this study followed the ten Tussucher's model in 2006, and the bio-pacemaker single cell model was modified based on it as what has been expatiated in our previous work. In tissue model, two factors were primarily evaluated for their effects on bio-pacemakers to pace and drive surrounding cardiac tissue: gap junction between bio-pacemaker cells (PMs) and adjacent ventricular myocytes (VMs) and the spatial distribution of bio-pacemakers. A suppressed gap junctional electrical coupling between and heterotypic gap junctions were simulated and a combination of them led to the best performance of the bio-pacemaker. Then, the pacemaking behaviours of three kinds of idealised PM-VM slices were simulated, in which an electrically isolated distribution of bio-pacemaker showed optimal drive capacities. Finally, a real human ventricular slice model was used to verified the conclusions in idealized tissues.
Conclusion: This study develops a theory that weak-rectified electrical coupling and electrically isolated distribution can enhance the pacemaking efficiency of bio-pacemakers, which lays the groundwork for future research into therapeutic applications of bio-pacemakers.
Objectives
Secondhand smoke exposure (SHSE) is associated with increased risk of cerebrovascular accident (CVA). Abnormal P-wave axis (aPWA) is a marker for atriopathy that is also associated with CVA risk. We hypothesized that SHSE is associated with aPWA.
Methods
This analysis included 5986 non-smokers (age 61.7 ± 13.8 years, 45.8% men, 77.4% Whites) from the Third National Health and Nutrition Examination Survey. SHSE was defined as serum cotinine ≥1 ng/ml aPWA was defined as any P-wave axis outside of 0–75°. Multivariable logistic regression was used to examine the association between SHSE and aPWA, overall and among subgroups stratified by demographics and comorbidities.
Results
About 18.5% (n = 1109) of the participants had SHSE. aPWA was more prevalent among those with SHSE than those without (23.9% versus 19.8%, respectively, P-value = 0.003). In a model adjusted for sociodemographic and potential confounders, presence (versus absence) of SHSE was associated with increased odds of aPWA (odds ratio [95% confidence interval]: 1.28 [1.09, 1.50]; P-value = 0.003). This association was stronger among Whites vs non-Whites (interaction P-value = 0.04) and non-obese versus obese (interaction P-value = 0.04). Higher levels of serum cotinine were associated with increased odds of aPWA. Compared with serum cotinine level <1 ng/ml, serum cotinine ≥3 ng/ml and ≥6 ng/ml were associated with 35% (P-value = 0.002) and 38% (P-value = 0.002) increased odds of aPWA, respectively.
Conclusions
SHSE is associated with abnormal atrial conduction, measured as aPWA, with possible effect modification by ethnicity and obesity. These findings underscore the harmful effects of SHSE on cardiovascular health which merits a personalized risk assessment when counseling patients on SHSE.
Cell therapy offers a promising paradigm for heart tissue regeneration. Human induced pluripotent stem cells (hiPS) and their cardiac derivatives are emerging as a novel treatment for post-myocardial infarction repair. However, the immature phenotype and function of hiPS-derived cardiomyocytes (hiPS-CMs), particularly poor electrical coupling, limit their potential as a therapy. Herein, we developed a hybrid gold nanoparticle (AuNP)-hyaluronic acid (HA) hydrogel matrix encapsulating hiPS-CMs to overcome this limitation. Methacrylate-modified-HA was used as the backbone and crosslinked with a matrix metalloproteinase-2 (MMP-2) degradable peptide to obtain a MMP-2-responsive hydrogel; RGD peptide was introduced as an adhesion point to enhance biocompatibility; AuNPs were incorporated to regulate the mechanical and topological properties of the matrix by significantly increasing its stiffness and surface roughness, thereby accelerating gap junction formation in hiPS-CMs and orchestrating calcium handling via the αnβ1integrin-mediated ILK-1/p-AKT/GATA4 pathway. Transplanted AuNP-HA-hydrogel-encapsulated-hiPS-CMs developed more robust gap junctions in the infarcted mice heart and resynchronized electrical conduction of the ventricle post-myocardial infarction. The hiPS-CMs delivered by the hydrogels exerted stronger angiogenic effects, which also contributed to the recovery process. This study provides insight into constructing an injectable biomimetic for structural and functional renovation of the injured heart.
Background
Patients with atrial fibrillation (AF) exhibit decreased atrial expression of connexin (Cx), which has been causally linked to a pro-arrhythmogenic substrate. Interestingly, patients with sleep-disordered-breathing (SDB) are at increased risk for AF, but the mechanisms remain unclear.
Objective
We tested the hypothesis that patients with SDB have reduced atrial connexin expression independent of important comorbidities.
Methods
We analyzed right atrial appendage biopsies from 77 patients undergoing coronary artery bypass grafting. Patients were tested for SDB by polygraphy before surgery. Expression of Cx40 and Cx43 mRNA were quantified using real-time qPCR and Western blot (Cx43). Structural atrial remodeling was investigated histologically and by qPCR. Postoperative AF was assessed by 12-lead ECG.
Results
Patients were stratified according to apnea-hypopnea-index (SDB if AHI≥15/h, 32 vs. 45). Patients with SDB had significantly lower atrial Cx43 expression, which was negatively correlated with AHI and oxygen-desaturation-index. No significant increase in atrial fibrosis or expression of hypertrophy and inflammatory markers was observed. Interestingly, SDB remained the strongest independent predictor of decreased atrial Cx43 expression in a multivariate logistic regression model including age, gender, diabetes, and HFrEF, (odds ratio, OR, and confidence interval, CI: 7.58 (1.891-30.375), p=0.004). Moreover, the reduced atrial Cx43 expression was strongly associated with the occurrence of postoperative AF (OR 15.749 with CI 1.072-231.472, p=0.044).
Conclusion
Patients with SDB exhibited decreased atrial Cx43 expression, which correlated with the severity of SDB. This correlation was independent of several concomitant diseases and may be linked to an increased risk of AF after cardiac surgery.
Histologically, the cardiac conduction network is formed of electrically isolated subendocardial fibers that comprise specialized cells with fewer myofibrils and mitochondria than cardiomyocytes. Our aim is to uncover regional variations of cardiac conduction fibers through histological and morphometric study in a porcine and human model. We analyzed five male adult human hearts and five male pig hearts. The left ventricles were dissected and sectioned in the axial plane into three parts: basal, middle third and apex regions. Cardiac conduction fibers study was carried out using hematoxylin-eosin and Masson's trichrome staining, and cardiac conduction cells and their junctions were identified using desmin, connexin 40 and a PAS method. Cardiac conduction fibers were difficult to pinpoint in humans, mostly showing a darker color or equal to cardiomyocytes. Cardiac conduction fibers in humans were in the subendocardium and in pigs in the myocardium and subendocardium. Cardiac conduction fibers were located mainly in the septal region in both humans and pigs. In our morphometric analysis, we were able to determine that cardiac conduction cells in humans (18.52 +/− 5.41 μm) and pigs (21.32 +/− 6.45 μm) were large, compared to cardiomyocytes. Conduction fiber-myocardial junctions were present in 10% in humans and 24.2% in pigs. The performance of immunohistochemical methods made it possible to improve the identification of cardiac conduction cells in the species studied. Study of cardiac conduction fibers and cells and their myocardial junctions is vital to gain insight into their normal distribution in the species analyzed, and thus advance the use of pigs in experimental models of the cardiac conduction system in humans.
Aim: Vildagliptin (vild) improves diastolic dysfunction and is associated with a lower relative risk of major adverse cardiovascular events in younger patients. The present study aimed to evaluate whether vild prevents the development of diabetic cardiomyopathy in type 2 diabetic mice and identify its underlying mechanisms.
Methods: Type 2 diabetic mouse model was generated using wild-type (WT) (C57BL/6J) and miR-21 knockout mice by treatment with HFD/STZ. Cardiomyocyte-specific miR-21 overexpression was achieved using adeno-associated virus 9. Echocardiography was used to evaluate cardiac function in mice. Morphology, autophagy, and proteins levels in related pathway were analyzed. qRT-PCR was used to detect miR-21. Rat cardiac myoblast cell line (H9c2) cells were transfected with miR-21 mimics and inhibitor to explore the related mechanisms of miR-21 in diabetic cardiomyopathy.
Results: Vild restored autophagy and alleviated fibrosis, thereby enhancing cardiac function in DM mice. In addition, miR-21 levels were increased under high glucose conditions. miR-21 knockout DM mice with miR-21 knockout had reduced cardiac hypertrophy and cardiac dysfunction compared to WT DM mice. Overexpression of miR-21 aggravated fibrosis, reduced autophagy, and attenuated the protective effect of vild on cardiac function. In high-glucose-treated H9c2 cells, the downstream effectors of sprouty homolog 1 (SPRY1) including extracellular signal-regulated kinases (ERK) and mammalian target of rapamycin showed significant changes following transfection with miR-21 mimics or inhibitor.
Conclusion: The results of our study indicate that vild prevents DCM by restoring autophagy through the miR-21/SPRY1/ERK/mTOR pathway. Therefore, miR-21 is a target in the development of DCM, and vild demonstrates significant potential for clinical application in prevention of DCM.
Gap junction proteins, termed connexins (Cx), mediate direct cell-to-cell communication by forming channels that physically couple cells, thereby linking their cytoplasm, permitting exchange of molecules, ions, and electrical impulses. The most ubiquitously expressed gap junction protein, connexin43 (Cx43) has been implicated in cardiovascular diseases including arrhythmias, cardiomyopathies, hypertension and diabetes. The Cx43 C-terminal (CT) domain serves as the regulatory hub of the protein affecting all aspects of gap junction function. Here, deletion within the Cx43 CT (amino acids 256-289), a region known to encode key residues regulating gap junction turnover is employed to examine the effects of dysregulated Cx43 gap junction endocytosis using cultured cells (Cx43∆256-289) and zebrafish model (cx43lh10). We report that this CT deletion causes defective gap junction endocytosis as well as increased gap junction intercellular communication (GJIC). Increased Cx43 protein content in cx43lh10 zebrafish, specifically in the cardiac tissue, larger gap junction plaques and longer Cx43 protein half-lives coincide with severely impaired cardiovascular development. These findings suggest that normal, unimpaired Cx43 gap junction endocytosis and turnover is an essential aspect of gap junction function as demonstrated here for cardiovascular development that when impaired can give rise to arrhythmias, heart malformations and aberrant vasculature structure and function.
The principal function of the ventricular conduction system is rapid electrical activation of the ventricles. The aim of this study is to conduct a morphometric study to pinpoint the morphological parameters that define cardiac conduction cells, allowing us to distinguish them from other cells.
Five male horse hearts and five male dog hearts were used in the study. The hearts were fixed in a 5% formaldehyde solution. Histological sections of 5 μm thickness were acquired and stained with hematoxylin-eosin and Masson's trichrome and cardiac conduction cells and their junctions were identified by desmin, connexin 40 and a PAS method.
We found statistically significant differences in cardiac conduction fibers density and thickness, which was much higher in horses than in dogs (p = 0.000 for both values). By comparing the measured parameters of the cells in both species, we determined that cardiac conduction cells area and diameters were greater in horses than in dogs (p = 0.000 for all values). In dogs there are more junctions (30.8%) than in horses (26.1%), a statistically significant difference (p = 0.041). Our findings regarding the cardiac conduction fibers distribution in the animal species studied becomes new knowledge that contributes to the morphological study of this component of the cardiac conduction system and also makes it possible to locate exactly the site with the highest density of cardiac conduction fibers as a contribution to the cardiological study of these structures that lead to the prevention of ventricular arrhythmias and the identification of their treatment site.
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.
Proinflammatory molecule tumor necrosis factor alpha (TNF-α) is predominantly elevated in cytokine storm as well as worsening cardiac function. Here we model the molecular and functional effects of TNF-α in cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSC). We found that treatment of hiPSC-CMs with TNF-α increased reactive oxygen species (ROS) and caspase 3/7 activity and caused cell death and apoptosis. TNF-α treatment also resulted in dysregulation of cardiomyocyte function with respect to the increased abnormal calcium handling, calcium wave propagation between cells and excitation-contraction coupling. We also uncovered significant changes in gene expression and protein localization caused by TNF-α treatment. Notably, TNF-α treatment altered the expression of ion channels, dysregulated cadherins, and affected the localization of gap-junction protein connexin-43. In addition, TNF-α treatment up-regulated IL-32 (a human specific cytokine, not present in rodents and an inducer of TNF-α) and IL-34 and down-regulated glutamate receptors and cardiomyocyte contractile proteins. These findings provide insights into the molecular and functional consequences from the exposure of human cardiomyocytes to TNF-α. Our study provides a model to incorporate inflammatory factors into hiPSC-CM-based studies to evaluate mechanistic aspects of heart disease.
To examine the spatial pattern of labelling for the gap junctional protein, connexin45, in relation to that of the other two cardiac connexins, connexin40 and connexin43, during the development of the central conduction system in mouse heart.
Hearts from Balb-c mice at stages from embryonic day (E) 12.5 to adult were frozen and sectioned. The sections were immunolabelled for connexins 45, 40 and 43 using fully characterized connexin-specific antibodies. Labelled sections were observed using confocal microscopy. Single, double and triple labelling were employed with sequential scanning to record images from multiple-labelled sections for the analysis of the spatial distribution of the three connexin types in relation to each other.
High levels of connexin45 label were detected in specific regions within the developing mouse heart. These regions corresponded to the conus myocardium, developing interatrial septum and other developing conduction tissues of the heart. Connexin40 label was initially absent from these tissues but by E15.5 was present in the more distal regions of the conduction system. However, by E17.5, connexin45 and 40 labelling was similar to the pattern observed in the adult heart, with both connexins present in most regions of the conduction system, though they were not completely colocalized. Connexin43 label was not observed in the regions of high connexin45 labelling.
These results show connexin45 to be the earliest detectable connexin in the central conduction system and to be the only connexin present throughout the whole conduction system. A distinct temporal pattern of connexin expression was also shown to occur during the development of the conduction tissues of the mouse heart.
Arrhythmias are a common and potentially life-threatening complication of myocardial ischemia and infarction in humans. The structural pathways for the rapid intercellular conduction of the electrical impulse that stimulates coordinated contraction in the myocardium are formed by the gap junctions situated at intercalated disks. By raising antibodies to cardiac gap-junctional protein, and using these antibodies in an immunohistochemical procedure in combination with the technique of laser scanning confocal microscopy, we have succeeded in localizing gap junctions, with a clarity not previously possible, through thick volumes of human myocardial tissue. To explore the structural basis for ischemia and infarction-related arrhythmogenesis, antibody labeling and laser scanning confocal microscopy were applied to study the organization, distribution, and other characteristics of gap junctions in the explanted hearts of patients undergoing cardiac transplantation for advanced ischemic heart disease. In areas of myocardium free from histologically detectable structural damage, there was no significant difference in the size of distribution of labeled gap junctions, or in their number per intercalated disk, between left ventricular tissue (in which functional impairment was severe) and right ventricular tissue (in which functional impairment was minimal). However, in myocytes at the border of healed infarcts--zones to which the slow conduction responsible for reentry arrhythmias has been localized--the organization of gap junctions was markedly disordered; instead of being aggregated into discrete intercalated disks, gap-junctional immunostaining was spread extensively over myocyte surfaces. Some infarct zones were bridged by continuous strands of myocytes, coupled to one another by gap junctions, thereby linking healthy myocardium on either side. At their thinnest, these bridges were in some instances no wider than a single attenuated myocyte. The conclusions are 1) a widespread, generalized derangement of gap junction organization does not appear to underlie functional impairment in the ischemic heart, 2) a disorderly arrangement typifies gap junctions in myocytes of the infarct border zone, and this may contribute to alterations in conduction that are capable of precipitating reentry arrhythmias, and 3) delicate chains of myocytes traverse some healed infarcts, apparently forming electrically coupled bridges across what would otherwise constitute blocked zones. The weakest link in this chain can be a single, degenerating myocyte; avoidance of arrhythmia may therefore depend on the continued survival of this single cell.
A polyclonal antiserum, raised against a synthetic peptide matching part of the sequence of connexin43 (a rat cardiac gap-junctional protein), was used in combination with laser scanning confocal microscopy to investigate gap junction distribution in cardiac tissues from a range of mammalian species. Comparison of the localised punctate staining patterns obtained in ventricular tissue with the distribution of intercalated disks as viewed by conventional light microscopy and electron microscopy, and with the staining observed by standard light-microscope immunofluorescence using the same anti-serum, demonstrated highly specific labelling of clearly resolved individual gap junctions. Laser scanning confocal microscopy of ventricular myocardium showed the immunostained gap junctions to be confined to well-defined intercalated disks bisecting the long axis of the muscle fibre, whereas in the atrial myocardium, gap junctions were commonly distributed widely over the lateral surfaces of the myocyte body. Rat atrial gap junctions were significantly larger (as measured by the longest axial lengths of fluorescent spots), and showed a narrower spread of sizes, than their counterparts in the ventricle. Ventricular myocardium from six mammalian species including man gave similar immunostaining patterns, indicating conservation both of the epitope(s) detected by the antiserum, and of the general organisation of the cell-to-cell pathways for electrical propagation, in the mammalian heart. Optical section series obtained by laser scanning confocal microscopy permitted the quantification and mapping of the three-dimensional distribution of gap junctions in ventricular intercalated disks with high clarity over substantial specimen depths. A consistent feature of gap junction organisation within disks of ventricular myocardium in all species studied was the presence of a conspicuous ring of large gap junctions around the periphery of the disk. Immunostained gap junctions lying within the interior zone delineated by the peripheral junctions generally occurred at lower numerical densities and were significantly smaller. In all species, less than 3% of all immunolabelled gap junctions measured were greater than 2 microns in maximal length, though a small proportion (0.06%) exceeded 4 microns. The numerical density of immunolabelled gap junctions in the disk was similar between species; however, within species there was a significant decrease in numerical density with increasing disk size. The new features of intercalated disk structure revealed in this study may have an important part to play in the intercellular communication and electrical propagation properties of the mammalian heart.
We have evaluated the voltage dependence and unitary conductance of gap junctional channels that were recorded in a clone isolated from the hepatoma cell line SKHep1. In this clonal population (designated SKHep1A), Northern blots, immunoprecipitation, and immunohistochemical staining demonstrated the expression of connexin (Cx) 45; no other gap junction protein was identified by these techniques, although weak hybridization with Cx40 was detected. Macroscopic junctional conductance (gj) in these cells was low, averaging 1.3 nS, and was steeply voltage dependent. Parameters of voltage sensitivity were as follows: voltage at which voltage-sensitive conductance is reduced by 50%, 13.4 mV; steepness of relation, 0.115 (corresponding to 2.7 gating charges), and voltage-insensitive fraction of residual to total conductance approximately 0.06. Unitary conductance (gamma j) of these junctional channels averaged 32 +/- 8 pS; although gamma j was independent of transjunctional voltage (Vj), at high Vj values (> 50 mV), smaller conductance values were also detected. Open probabilities of the 30-pS channels at various Vj values closely matched the predicted voltage-dependent component of macroscopic gj, the residual conductance at high Vj might be attributable to the smaller conductance events. The voltage dependence of human Cx45 gap junction channels is as steep as that seen for channels formed by Xenopus Cx38 and is much steeper than that previously reported for channels formed of the highly homologous chick Cx45 and for other mammalian connexins expressed either endogenously or exogenously.
Gap junction channels consisting of connexin protein mediate electrical coupling between cardiac cells. Expression of two connexins, connexin40 (Cx40) and connexin43 (Cx43), has been studied in ventricular myocytes from normal and hypertensive rats. Polyclonal affinity-purified rabbit antibodies to Cx43 and Cx40 have been used for immunohistochemical analysis on frozen sections from rat heart. These studies revealed coexpression of Cx43 and Cx40 in ventricular myocytes. In addition, Cx40 is preferentially expressed in three distinct regions: first, in the endothelial layer of the heart blood vessels but not in the smooth muscle layer of the arteries; second, in the ventricular conductive myocardium, particularly in the atrioventricular bundle and bundle branches, where Cx43 is not observed; and third, in the myocyte layers close to the ventricular cavities. These results suggest that Cx40 is preferentially expressed in the fast conducting areas of myocardial tissue. Expression of both Cx40 and Cx43 was also found in immunoblots from normal and hypertensive rat myocardiocytes. Under hypertensive conditions (ie, in spontaneous hypertensive rats and in transgenic rats that exhibit hypertension due to expression of an exogenous renin gene), we found a 3.1-fold increase in Cx40 expression, compared with normal myocardium. Furthermore, we detected a 3.3-fold decrease in Cx43 protein level in transgenic hypertensive rats. The coexpression of Cx40 and Cx43 proteins in rat myocytes, their spatial distribution, and the increased amount of Cx40 protein during cardiac hypertrophy suggest that Cx40 may be involved in mediating fast conduction under normal and pathological conditions. The increased expression of Cx40 in hypertrophic heart may be a compensatory mechanism to increase conduction velocity.
Electrical coupling between heart muscle cells is mediated by specialised regions of sarcolemmal interaction termed gap junctions. In previous work, we have demonstrated that connexin42, a recently identified gap-junctional protein, is present in the specialised conduction tissues of the avian heart. In the present study, the spatial distribution of the mammalian homologue of this protein, connexin40, was examined using immunofluorescence, confocal scanning laser microscopy and quantitative digital image analysis in order to determine whether a parallel distribution occurs in rat. Connexin40 was detected by immunofluorescence in all main components of the atrioventricular conduction system including the atrioventricular node, atrioventricular bundle, and Purkinje fibres. Quantitation revealed that levels of connexin40 immunofluorescence increased along the axis of atrioventricular conduction, rising over 10-fold between atrioventricular node and atrioventricular bundle and a further 10-fold between atrioventricular bundle and Purkinje fibres. Connexin40 and connexin43, the principal gap-junctional protein of the mammalian heart, were co-localised within atrioventricular nodal tissues and Purkinje fibres. By applying a novel photobleach/double-labelling protocol, it was demonstrated that connexin40 and connexin43 are co-localised in precisely the same Purkinje fibre myocytes. A model, integrating data on the spatial distribution and relative abundance of connexin40 and connexin43 in the heart, proposes how myocyte-type-specific patterns of connexin isform expression account for the electrical continuity of cardiac atrioventricular conduction.
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.
To examine the distribution pattern of intercellular junctions (the mechanically coupling desmosomes and the electrically coupling gap junctions) in hypertrophic cardiomyopathy (HCM) hearts showing myofibre disarray.
Samples from six necropsied hearts were studied, representing the interventricular septum and the free walls of the left and right ventricles. Immunohistochemical labelling of desmoplakin was used as a marker for desmosomes, and of connexin43 as a marker for gap junctions, in single and double stainings. The slides were examined by confocal laser scanning microscopy.
Marked disorganisation of intercalated discs was observed in areas featuring myofibre disarray. Besides overall derangement, localised abnormalities in desmosome organisation were evident, which included: (1) the formation of abnormally enlarged megadiscs; (2) the presence of intersecting disc structures; and (3) aberrant side to side desmosomal connections. Gap junctional abnormalities included: (1) random distribution of gap junctions over the surface of myocytes, rather than localisation to intercalated discs; (2) abundant side to side gap junction connections between adjacent myocytes; and (3) formation of abnormally shaped gap junctions. Circles of myocytes continuously interconnected by gap junctions were also observed. Regions of the diseased hearts lacking myofibre disarray, and control hearts of normal patients and patients with other cardiac diseases, did not show these alterations.
The disorganisation of the intercellular junctions associated with myofibre disarray in HCM may play an important role in the pathophysiological manifestations of the disease. The remodelling of gap junction distribution may underlie the formation of an arrhythmogenic substrate, thereby contributing to the generation and maintenance of cardiac arrhythmias associated with HCM.
Nonuniformity in the spatial patterning of gap junctions between heart muscle cells is now recognized as an important determinant of electromechanical function in working myocardium. Breakdown of the normal geometry of electrical intercellular connectivity in diseased myocardium correlates with reentry, arrhythmia, and conduction disturbance. The developmental mechanism(s) that determines this precise spatial order in gap junction organization in normal myocardium is at present unknown. To examine this question, we have used immunoelectron and immunoconfocal microscopy to analyze the spatial distributions of gap junctional (connexin43), desmosomal (desmoplakin), and adherens junctional (N-cadherin) components during maturation of rodent and canine left ventricular myocardium. In rats, a striking divergence in the distribution of gap junctions and cell adhesion junctions emerged within the first 20 days of postnatal life. It was found that although gap junctions initially demonstrated dispersed distributions across myocyte cell membranes, desmosomes and adherens junctions showed more rapid polarization toward cell termini (ie, nascent intercalated disks) after birth. Over subsequent postnatal development (20 to 90 postnatal days), gap junctions became progressively concentrated in these cell adhesion junction-rich zones of membrane. Quantitative analyses of this process in a series of rats aged 15 embryonic and 1, 5, 10, 20, 40, 70, and 90 postnatal days indicated that significantly higher levels (P < .01) of N-cadherin and desmoplakin than of connexin43 were immunolocalized to cell termini by as early as postnatal day 5. Although all three junctions types showed increasing polarization to myocyte termini with development, variation between junctions remained significant (P < .05) at all times points between 5 and 70 postnatal days. Only at 90 postnatal days, when the animals were nearly full grown, did the proportions of gap junction, desmosome, and adherens junction at intercalated disks become statistically similar (P > .05). Examination of myocardium from 1- and 3-month-old canines revealed that related differential changes to the spatiotemporal distribution of intercellular junctions occurred during postnatal maturation of the dog heart, suggesting that the process was not rodent specific. It is concluded that this progressive change in the organization and pattern of association between gap junctions and cell adhesion junctions is likely to be an important factor in maturation of electromechanical function within the mammalian heart.
To characterize the role of the gap junction protein connexin43 (Cx43) in ventricular conduction, we studied hearts of mice with targeted deletion of the Cx43 gene. Mice homozygous for the Cx43 null mutation (Cx43 -/-) die shortly after birth. Attempts to record electrical activity in neonatal Cx43 -/- hearts (n = 5) were unsuccessful. Ventricular epicardial conduction of paced beats, however, was 30% slower in heterozygous (Cx43 -/+) neonatal hearts (0.14+/-0.04 m/s, n = 27) than in wild-type (Cx43 +/+) hearts (0.20+/-0.07 m/s, n = 32; P < 0.001). This phenotype was even more severe in adult mice; ventricular epicardial conduction was 44% slower in 6-9 mo-old Cx43 -/+ hearts (0.18+/-0.03 m/s, n = 5) than in wild-type hearts (0.32+/-0.07 m/s, n = 7, P < 0.001). Electrocardiograms revealed significant prolongation of the QRS complex in adult Cx43 -/+ mice (13.4+/-1.8 ms, n = 13) compared with Cx43 +/+ mice (11.5+/-1.4 ms, n = 12, P < 0.01). Whole-cell recordings of action potential parameters in cultured disaggregated neonatal ventricular myocytes from Cx43 -/+ and +/+ hearts showed no differences. Thus, reduction in the abundance of a major cardiac gap junction protein through targeted deletion of a Cx43 allele directly leads to slowed ventricular conduction.
Sequential contraction of the cardiac chambers depends on orderly spread of the wave of electrical excitation from one cardiomyocyte to the next, throughout the heart. As discussed in earlier chapters of this volume, the pathways enabling this cell-to-cell current flow are formed by the gap junctions that link individual cardiomyocytes into a functional syncytium. Gap junctions are essentially clusters of transmembrane channels that span the paired plasma membranes of neighboring cells, linking their cytoplasmic compartments together to form pathways for direct cell-to-cell communication. The component proteins of the gap-junctional channel, connexins, are assembled into hexamers which form hemi-channels termed connexons, the complete channel being formed by the docking of a pair of connexons across the adjacent plasma membranes. Twenty different connexin genes have now been identified in the human (Willecke et al., 2001), and most tissues, including those of the cardiovascular system, express two or more connexin isoforms. Three principal isoforms — connexin43, connexin40 and connexin45 — are expressed in cardiomyocytes (reviews, Beyer et al., 1997; Severs, 1999; Severs et al., 2001), and further isoforms such as connexin46 (Paul et al., 1991) and connexin57 (Manthey et al., 1999) may also be present in trace amounts. Gapjunctional channels composed of different connexins exhibit distinctive biophysical properties in vitro (review, Bruzzone et aI., 1996), and studies on transgenic mice demonstrate that the precise functional properties of gap junctions in vivo may depend in part on the specific connexins from which they are constructed, though there is also considerable capacity for functional compensation of one connexin isoform by another (Kirchhoff et al., 2000; KrUger et al., 2000; Plum et al., 2000; Tamaddon et al., 2000; van ijen et al., 2001). Different subsets of cardiomyocyte express different combinations and relative quantities of connexins 43, 40 and 45, potentially providing for regional differentiation of electrophysiological properties. The concept has thus developed that gap junction organization and spatially defined patterns of connexin expression may preside over the precisely or chestrated patterns of current flow that govern the normal heart rhythm.
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Generally, impulse propagation in cardiac tissue is assumed to be impaired by a reduction of intercellular electrical coupling or by the presence of structural discontinuities. Contrary to this notion, the spatially uniform reduction of electrical coupling induced successful conduction in discontinuous cardiac tissue structures exhibiting unidirectional conduction block. This seemingly paradoxical finding can be explained by a nonsymmetric effect of uncoupling on the current source and the current sink in the preparations used. It suggests that partial cellular uncoupling might prevent the initiation of cardiac arrhythmias that are dependent on the presence of unidirectional conduction block.
In this study we report about the modulation of connexin45 (Cx45) gap junction channel properties by phosphorylation of the connexin molecules through different protein kinases. Phosphorylation of Cx45 was studied in HeLa cells transfected with mouse Cx45 (mCx45). Using Western blotting (WB) and immunocytochemistry, these cells were found exclusively positive for Cx45 and the protein was separated as a doublet of bands with a calculated mass of 46 and 48 kD. After dephosphorylation using calf intestine phosphatase (CIP), the 48 kD band disappeared almost completely leaving a single band at 46 kD. This effect can be prevented by including phosphatase inhibitors during CIP treatment. These results indicate that the 48 kD signal represents a phosphorylated form of Cx45. To investigate the effects of (de)phosphorylation of Cx45 on the conductive properties of gap junction channels built of this connexin, cell pairs were subjected to dual voltage clamp experiments and coupling was determined before and after addition of PMA, 4α-PDD, cAMP, cGMP, and pervanadate to the superfusate. 100 nM of the PKC activating phorbol ester PMA increased normalized junctional conductance by 50.9±28%. 100 nM of the inactive phorbol ester 4α-PDD had no significant effect. Activation of PKA with 1 mM 8-Br-cAMP decreased coupling by 20.9±5.7% while 1 mM 8-Br-cGMP (PKG-activation) was ineffective. 100 μM pervanadate, a tyrosine phosphatase inhibitor, reduced coupling by 43.7±11.1%. Single channel measurements, under identical phosphorylating conditions, were not significantly different from each other and all frequency histograms exhibited two conductance peaks at approximately 20 and 40 pS. WB analysis revealed, as compared to control conditions, a relative increase of the 48 kD signal upon stimulation with pervanadate (142±42%) and 8-Br-cAMP (50±23%) whereas neither stimulation with PMA nor 8-Br-cGMP had a significant effect. These experiments show that electrical intercellular conductance via Cx45 gap junction channels is differentially regulated by phosphorylation. However, regulation does not act by changing single channel conductance, but most likely by modulation of the open probability of Cx45 gap junction channels.
Objective: We tested the hypothesis that structural remodeling of cellular connections, alterations in the expression of connexins (Cx), and an increase in fibrosis represent anatomic substrates of atrial fibrillation (AF). Methods: In 31 patients with AF undergoing a Maze procedure and 22 patients in sinus rhythm (SR), biopsies were taken intraoperatively from the right atrial (RA) free wall and appendages and investigated with immunoconfocal and electron microscopy. Results: All patients with AF exhibited a concomitant lateralization of gap junctional proteins Cx43 and Cx40, and N-cadherin (the major mechanical junction protein), instead of being confined to the intercalated discs, as observed in SR. These results were confirmed by quantitative immunoconfocal analysis and electron microscopy. Among diverse junctional proteins, in AF, Cx40 was markedly heterogenous in distribution. As compared with the SR group, Cx43 was significantly decreased in AF by 57% in RA appendages and by 56% in RA free wall. Cx40 was reduced by 54% in appendages, but had a tendency to be increased in the RA free wall. Collagen I was significantly higher in AF than in SR by 48% in RA appendages and by 69% in the RA free wall tissues. Conclusions: The structural correlate of AF comprises extensive concomitant remodeling of mechanical and electrical junctions, reduction of Cx43, heterogenous distribution of Cx40 in terms of different amounts of Cx40 in different RA tissues or in spatially adjacent regions of atrial myocardium. These changes, together with augmentation of fibrosis, may underlie localized conduction abnormalities and contribute to initiation and self-perpetuation of re-entry pathways and AF.
Heart Cell Coupling and Impulse Propagation in Health and Disease includes an up-to-date review on how heart cells communicate and impulse propagation under normal as well as under pathological conditions. The complexity of intercellular coupling and impulse propagation is discussed, providing the reader with a broad view of the importance of these processes and how they contribute to the generation of cardiac arrhythmias and heart failure. The different aspects and intricacies of heart cell communication is discussed by different authors, each one an expert in their own field.
The present publication will be of interest to cardiologists, electrophysiologists, heart physiologists, cardiac pharmacologists, biophysicists, and cell or molecular biologists.
Gap junctions are plasma membrane specializations containing channels which permit the intercellular exchange of ions and
small molecules. Gap junction channels are of central importance in electrically excitable tissues such as myocardium where
cell-to-cell passage of ions allows propagation of action potentials. Gap junctions are also present in many non-excitable
cells (for example endothelial cells) where they may facilitate intercellular exchange of nutrients, metabolites, and signaling
molecules as well as ions. The present review will focus on molecular biological and biochemical studies that have enhanced
our understanding of the molecular composition of cardiac and vascular gap junction channels and the regulation of the subunit
proteins that form them.
The mouse is currently widely used as a model organism in the analysis of gene function but how developmentally regulated patterns of connexin gene expression in the mouse compare with those in the human is unclear. Here we compare the patterns of connexin expression in the heart during the development of the mouse (from embryonic day 12.5 to 6 weeks postpartum) and the human (at 9 weeks gestation and adult stage). The extent of connexin43 expression in the ventricles progressively increased during development of the mouse heart. The developmental pattern of expression for connexins 40 and 45 in the mouse heart was similar, but not identical, and in the ventricles showed a progressive and preferential expression in the conduction system. In general, these dynamic changes of connexins 43, 40 and 45 during mouse cardiac development appear to be mirrored in the human.
OBJECTIVES
To elucidate the structural basis for the electrophysiologic remodeling induced by chronic atrial fibrillation (AF), we investigated connexin40 and connexin43 (Cx40 and Cx43) expression and distribution in atria of patients with and without chronic AF and in an animal model of AF with additional electrophysiologic investigation of anisotropy (ratio of longitudinal and transverse velocities).BACKGROUND
Atrial fibrillation is a common arrhythmia that has a tendency to become persistent. Since gap junctions provide the syncytial properties of the atrium, changes in expression and distribution of intercellular connections may accompany the chronification of AF.METHODS
Atrial tissues isolated from 12 patients in normal sinus rhythm at the time of cardiac surgery and from 12 patients with chronic AF were processed for immunohistology and immunoblotting for the detection of the gap junction proteins. The functional study of the cardiac tissue anisotropy was performed in rat atria in which AF was induced by 24 h of rapid pacing (10 Hz).RESULTSImmunoblotting revealed that AF did not induce any significant change in Cx43 content in human atria. In contrast, a 2.7-fold increase in expression of Cx40 was observed in AF. Immunohistologic analysis indicated that AF resulted in an increase in the immunostaining of both connexins at the lateral membrane of human atrial cells. A similar spatial redistribution of the Cx43 signal was seen in isolated rat atria with experimentally-induced AF. In addition, AF in rat atria resulted in decreased anisotropy with slightly enhanced transverse conduction velocity.CONCLUSIONS
This experimental study showed that AF is accompanied by spatial remodeling of gap junctions that might induce changes in the biophysical properties of the tissue.
Gap junctions play essential roles in the normal function of the heart and arteries, mediating the spread of the electrical impulse that stimulates synchronized contraction of the cardiac chambers, and contributing to co-ordination of activities between cells of the arterial wall. In common with other multicellular systems, cardiovascular tissues express multiple connexin isotypes that confer distinctive channel properties. This review highlights how state-of-the-art immunocytochemical and cellular imaging techniques, as part of a multidisciplinary approach in gap junction research, have advanced our understanding of connexin diversity in cardiovascular cell function in health and disease. In the heart, spatially defined patterns of expression of three connexin isotypes—connexin43, connexin40, and connexin45—underlie the precisely orchestrated patterns of current flow governing the normal cardiac rhythm. Derangement of gap junction organization and/or reduced expression of connexin43 are associated with arrhythmic tendency in the diseased human ventricle, and high levels of connexin40 in the atrium are associated with increased risk of developing atrial fibrillation after coronary by-pass surgery. In the major arteries, endothelial gap junctions may simultaneously express three connexin isotypes, connexin40, connexin37, and connexin43; underlying medial smooth muscle, by contrast, predominantly expresses connexin43, with connexin45 additionally expressed at restricted sites. In normal arterial smooth muscle, the abundance of connexin43 gap junctions varies according to vascular site, and shows an inverse relationship with desmin expression and positive correlation with the quantity of extracellular matrix. Increased connexin43 expression between smooth muscle cells is closely linked to phenotypic transformation in early human coronary atherosclerosis and in the response of the arterial wall to injury. Current evidence thus suggests that gap junctions in both their guises, as pathways for cell-to-cell signaling in the vessel wall and as pathways for impulse conduction in the heart, contribute to the initial pathogenesis and eventual clinical manifestation of human cardiovascular disease. Microsc. Res. Tech. 52:301–322, 2001. © 2001 Wiley-Liss, Inc.
In the heart, individual cardiac muscle cells are linked by gap junctions. These junctions form low resistance pathways along which the electrical impulse flows rapidly and repeatedly between all the cells of the myocardium, ensuring their synchronous contraction. To obtain probes for mapping the distribution of gap junctions in cardiac tissue, polyclonal antisera were raised to three synthetic peptides, each matching different cytoplasmically exposed portions of the sequence of connexin43, the major gap-junctional protein reported in the heart. The specificity of each antiserum for the peptide to which it was raised was established by dot blotting. New methods were developed for isolating enriched fractions of gap junctions from whole heart and from dissociated adult myocytes, in which detergent-treatment and raising the temperature (potentially damaging steps in previously described techniques) are avoided. Analysis of these fractions by SDS-polyacrylamide gel electrophoresis revealed major bands at 43 kDa (matching the molecular mass of connexin43) and at 70 kDa. Western blot experiments using our antisera indicated that both the 43-kDa and the 70-kDa bands represent cardiac gap-junctional proteins. Pre-embedding immunogold labelling of isolated gap junctions and post-embedding immunogold labelling of Lowicryl-embedded whole tissue demonstrated the specific binding of the antibodies to ultrastructurally defined gap junctions. One antiserum (raised to residues 131–142) was found to be particularly effective for cytochemical labelling. Using this antiserum for immunofluorescence labelling in combination with confocal scanning laser microscopy enabled highly sensitive detection and three-dimensional mapping of gap junctions through thick slices of cardiac tissue. By means of the serial optical sectioning ability of the confocal microscope, images of the entire gap junction population of complete en face-viewed disks were reconstructed. These reconstructions reveal the presence of large junctions arranged as a peripheral ring around the disk, with smaller junctions in an interior zone: an arrangement that may facilitate efficient intercellular transfer of current. By applying our immunolabelling techniques to tissue from hearts removed from transplant patients with advanced ischaemic heart disease, we have demonstrated that gap junction distribution between myocytes at the border zone of healed infarcts is markedly disordered. This abnormality may contribute to the genesis of reentrant arrhythmias in ischaemic heart disease.
Communicating junctions, commonly known as gap junctions, comprise clusters of transmembrane channels that mediate electrical
coupling between cardiomyocytes. The component proteins of these channels are termed connexins. Different combinations and relative quantities of three connexins—connexin43, connexin40 and connexin45—are expressed in
different subsets of cardiomyocyte, providing potential for regional differentiation of electrophysiological properties. In
the healthy heart, gap junction organization and spatially defined patterns of connexin expression form the cell-to-cell pathways
for the flow of the precisely orchestrated patterns of electrical excitation that govern the normal heart rhythm. Remodeling
of gap junction organization and connexin expression is a conspicuous feature of human heart disease in which there is an
arrhythmic tendency. This remodeling may take the form of structural remodeling, involving disturbances in the distribution
of gap junctions (i.e., disruption of the normal ordered pathways for cell-to-cell conduction), or remodeling of connexin
expression, involving alteration in the amount or type of connexin(s) present. Most notable among quantitative alterations
in connexin expression is a reduction in ventricular connexin43 levels in human congestive heart failure. By correlating data
from studies in experimental animal models, gap junction and connexin remodeling emerges as a key factor to be considered
in understanding the pro-arrhythmic substrate characteristic of many forms of heart disease.
Key wordsgap junctions–connexins–intercalated disk–cell structure–intercellular communication
Congestive heart failure is associated with a high risk of life-threatening ventricular re-entrant arrhythmias. Down-regulation of the principal gap-junctional protein of the ventricular myocytes, connexin43, has previously been implicated in arrhythmia in ischaemic heart disease, but it is not known whether connexin43 is similarly reduced in heart failure due to idiopathic dilated cardiomyopathy, whether disease-related connexin43 down-regulation occurs at the level of transcription or translation, or whether the expression of other connexin isotypes is altered in congestive heart failure. We therefore investigated the expression of the four connexins expressed in the heart—connexins 43, 40, 45 and 37—at the mRNA and protein levels in explanted hearts from transplant patients with end-stage heart failure (NYHA class 4) by immunoconfocal analysis, and northern and western blotting. Connexin43 mRNA and protein were markedly downregulated in the left ventricle in end-stage heart failure due both to ischaemic cardiomyopathy and idiopathic dilated cardiomyopathy. Connexin43 content was spatially heterogeneous in the diseased ventricle. Connexin40 mRNA was increased in the ischaemic group, more so in the left ventricle than the right. This correlated with an increased depth of connexin40 protein expression in myocytes at the endocardial surface. Connexin45 mRNA and protein, present only in very low quantities, followed a similar trend to connexin43, while connexin37 (exclusively expressed in endothelium) showed no change. Our findings show that congestive heart failure is associated with significantly reduced levels of the principal gap junction protein, connexin43, in the left ventricle, potentially contributing to enhanced arrhythmogenicity and contractile dysfunction. This down-regulation is due predominantly to a reduced transcript steady-state level. Elevated connexin40 may represent a compensatory response that improves the spread of depolarization in the otherwise compromised ischaemic ventricle.
Remodeling of myocyte interconnections may be an important determinant of ventricular tachycardia in regions bordering healed infarcts. We used quantitative electron microscopy to characterize the distribution of gap junctions in 10 canine left ventricles 3-10 wk after coronary occlusion. In three normal canine left ventricles analyzed ultrastructurally, myocardial gap junctions were distributed anisotropically; gap junction profile length was significantly greater in the transverse than in longitudinal planes of section. In infarct border zone tissues, the normal anisotropic distribution was completely abolished and fewer gap junctions per unit intercalated disk length were observed. Analysis of individual gap junction profile length distributions revealed selective disruption of the largest gap junctions that collectively comprised only 9.6% of total junction profiles, but encompassed nearly 40% of aggregate gap junction length in the transverse plane of section. Three-dimensional reconstructions of myocyte interconnections by high resolution quantitative light microscopy of serial sections demonstrated a reduction in the number of cells connected by intercalated disks to a single myocyte from 11.2 +/- 1.0 in normal myocardium to 6.5 +/- 1.3 in border zone tissues (P less than 0.001). Connections of cells in primarily side-to-side apposition were reduced by 75%, whereas primarily end-to-end connections were reduced by only 22% (P less than 0.05). These alterations would disproportionately enhance axial resistivity in the transverse direction, potentially contributing to development of reentrant arrhythmias.
This review sets out to explore, with the pictorial support of electron microscopy, the nature of the intercalated disc, focussing in particular on the structure and function of the cardiac gap junction
Electrotonic coupling of cardiac myocytes at gap junctions may influence patterns of conduction in myocardium. To delineate the three-dimensional structure and distribution of intercellular junctions, we analyzed serial ultrathin sections of canine myocardium with transmission electron microscopy and disaggregated myocytes with scanning electron microscopy. Morphometric analysis of left ventricular myocardium sectioned in three orthogonal planes revealed that 80% of total gap junctional membrane occurred in large, ribbon-like gap junctions oriented transversely at cell end processes. The remaining 20% of gap junctional membrane was contained in small gap junctions located within plicate segments (interdigitating regions of cell-to-cell adhesion) of intercalated disks. In serial ultrathin sections, all gap junctions were contiguous with plicate segments. Thus, true "lateral" gap junctions do not exist in working ventricular myocytes and would not likely be able to withstand shear forces created by laterally sliding cells. Examination of serial plastic sections with light microscopy revealed complex overlapping of myocytes such that individual myocytes were connected at intercalated disks to an average of 9.1 +/- 2.2 other myocytes. These observations provide an improved understanding of the extent and distribution of cell junctions and should facilitate experimental and model studies of conduction in myocardium.
Cardiac muscle cells are equipped with three distinct types of intercellular junction--gap junctions, "spot" desmosomes, and "sheet" desmosomes (or fasciae adherentes)--located in a specialized portion of the plasma membrane, the intercalated disk. Gap junctions are responsible for electrical coupling and the transfer of small molecules between cells, whereas the desmosomelike junctions (also known as adherens junctions) provide strong intercellular adhesion. The adhesion sites formed by the "spot" desmosome anchor the intermediate-filament cytoskeleton of the cell; those formed by the fascia adherens anchor the contractile apparatus. An understanding of the ultrastructure of these junctions helps explain how they carry out their functions, and new observations in this field have been made through the application of ultrarapid freezing techniques in conjunction with freeze-fracture electron microscopy. With recent findings from biochemical and immunocytochemical studies, this understanding is now being extended to the molecular level.
Gap junctions are thought to mediate the direct intercellular coupling of adjacent cells by the gating of an aqueous pore permeable to ions and molecules of up to 1 kD or 8 to 14 A in diameter. We performed ion-substitution and dye-transfer experiments to determine the relative Cl-/K+ conductance and dye permeability of anionic fluorescein derivatives in chick connexin45 (Cx45) channels. We demonstrate that Cx45 forms a 26 +/- 6-picosiemen (pS) channel with a maximum detectable Cl- permeability of 0.2 relative to K+ or Cs+. Although homogeneous channel conductances were observed in multichannel recordings, the open probability estimates were indicative of nonhomogeneous gating behavior and occasional cooperativity. A second conductance state of 19 +/- 4 pS begins to predominate at higher voltages. Cx45 gap junctions are permeable to 2',7'-dichlorofluorescein but are not permeable to the more polar 6-carboxyfluorescein dye. These observations suggest that the Cx45 pore diameter is approximately 10 A and is associated with a fixed negative charge within the junctional channel.
A clone of human HeLa cells stably transfected with mouse connexin40 DNA was used to examine gap junctions. Two separate cells were brought into physical contact with each other ("induced cell pair") to allow insertion of gap junction channels and, hence, formation of a gap junction. The intercellular current flow was measured with a dual voltage-clamp method. This approach enabled us to study the electrical properties of gap junction channels (cell pairs with a single channel) and gap junctions (cell pairs with many channels). We found that single channels exhibited multiple conductances, a main state (gamma j(main state)), several substates (gamma j(substates)), a residual state (gamma j (residual state)), and a closed state (gamma j(closed state)). The gamma j(main state) was 198 pS, and gamma j(residual state) was 36 pS (temperature, 36-37 degrees C; pipette solution, potassium aspartate). Both properties were insensitive to transjunctional voltage, Vj. The transitions between the closed state and an open state (i.e., residual state, substate, or main state) were slow (15-45 ms); those between the residual state and a substate or the main state were fast (1-2 ms). Under steady-state conditions, the open channel probability, Po, decreased in a sigmoidal manner from 1 to 0 (Boltzmann fit: Vj,o = -44 mV; z = 6). The temperature coefficient, Q10, for gamma j(main state) and gamma j(residual state) was 1.2 and 1.3, respectively (p < 0.001; range 15-40 degrees C). This difference suggests interactions between ions and channel structure in case of gamma j(residual state). In cell pairs with many channels, the gap junction conductance at steady state, gj, exhibited a bell-shaped dependency from Vj (Boltzmann fit, negative Vj, Vj,o = -45 mV, gj(min) = 0.24; positive Vj, Vj,o = 49 mV, gj(min) = 0.26; z = 6). We conclude that each channel is controlled by two types of gates, a fast one responsible for Vj gating and involving transitions between open states (i.e., residual state, substates, main state), and a slow one involving transitions between the closed state and an open state.
In this study we tested the hypothesis that atrial fibrillation (AF) causes electrophysiological changes of the atrial myocardium which might explain the progressive nature of the arrhythmia.
Twelve goats were chronically instrumented with multiple electrodes sutured to the epicardium of both atria. Two to 3 Weeks after implantation, the animals were connected to a fibrillation pacemaker which artificially maintained AF. Whereas during control episodes of AF were short lasting (6 +/- 3 seconds), artificial maintenance of AF resulted in a progressive increase in the duration of AF to become sustained (> 24 hours) after 7.1 +/- 4.8 days (10 of 11 goats). During the first 24 hours of AF the median fibrillation interval shortened from 145 +/- 18 to 108 +/- 8 ms and the inducibility of AF by a single premature stimulus increased from 24% to 76%. The atrial effective refractory period (AERP) shortened from 146 +/- 19 to 95 +/- 20 ms (-35%) (S1S1, 400 ms). At high pacing rates the shortening was less (-12%), pointing to a reversion of the normal adaptation of the AERP to heart rate. In 5 goats, after 2 to 4 weeks of AF, sinus rhythm was restored and all electrophysiological changes were found to be reversible within 1 week.
Artificial maintenance of AF leads to a marked shortening of AERP, a reversion of its physiological rate adaptation, and an increase in rate, inducibility and stability of AF. All these changes were completely reversible within 1 week of sinus rhythm.
The growing postnatal human heart maintains electromechanical function while undergoing substantial changes of cellular topology and myocardial architecture. The capacity for growth and remodeling of ventricular myocardium in adaptation to the hemodynamic changes of early infancy later declines. This decline is associated with changes in electromechanical properties of the myocardium, which suggest that the electrical and mechanical interactions between the myocytes may change in an age-dependent manner. Thus, reduction in the capacity for myocardial growth and adaptability may relate to age-dependent alterations in the patterns of the intercellular junctions that mediate electrical and mechanical coupling. We therefore examined the hypotheses that (1) age-dependent changes in the distribution patterns of gap junctions and fasciae adherentes, the intercellular junctions responsible, respectively, for electrical and mechanical coupling, accompany postnatal development in the human heart and that (2) such changes continue into the first few years of childhood. Further, the spatial relation between the two types of junction, for which a close association has been hypothesized as necessary, was explored.
Ventricular myocardial gap-junction distribution was investigated in 23 pediatric surgical patients (4 weeks to 15 years old) by quantitative immunohistochemical localization of the principal cardiac gap-junctional protein, connexin43, using confocal microscopy. Immunolocalization of fascia adherens junctions by labeling N-cadherin, and correlative immunogold and standard electron microscopy, were performed in parallel. In the neonate, connexin43 gap junctions have a punctate distribution over the entire surface of the ventricular myocytes. With advancing age, gap junctions become progressively confined to the transverse terminals of the cell, ie, toward the distribution within the intercalated disk characteristic of the adult ventricle. The transversely arrayed proportion of gap-junctional label showed a linear increase with age (R = .88, P < .001), reaching the adult pattern at about 6 years, and the fascia adherens junctions showed a similar progression. Electron microscopy confirmed the changing pattern of junctional contacts and demonstrated that initially gap junctions and adhering junctions are frequently not closely adjacent but become increasingly so with maturation of the intercalated disk.
Changes in the spatiotemporal patterns of the intercellular junctions responsible for electrical and mechanical coupling are closely coordinated in postnatal human ventricular myocardium and continue to about 6 years of age. Over this period there is a close and increasing association between the gap junctions and fascia adherens junctions. These changes in the distribution of intercellular electrical and adhering junctions may parallel the changing functional requirements of the ventricle, from a distribution that facilitates the remodeling necessitated by rapid growth and changing hemodynamics to that of the relatively stable and rapidly conducting adult myocardium. These age-related changes may also diminish the ability for appropriate myocardial remodeling in response to physiological, pathological, or surgical hemodynamic alterations.
Connexin40 (Cx40) is a member of the connexin family of gap junction proteins. Its mRNA, abundant in lung, is also present in mammalian heart, although in lower amount. Rabbit antipeptide antibodies directed to the COOH terminus (residues 335 to 356) of rat Cx40 were characterized to investigate the distribution of Cx40 in rat and guinea pig cardiac tissues. The affinity-purified antibodies detect specifically a major protein (M(r), 40,000) in immunoblots of total extracts from rat lung and rat and guinea pig heart. In sections of guinea pig atrial tissue treated for immunofluorescence, a strong labeling associated with myocytes was seen with a distribution consistent with that of intercalated disks. The results of immunoelectron microscopy carried out with guinea pig atrial tissue showed that epitopes recognized by these antibodies were exclusively associated with gap junctions. These results, added to those of control experiments, demonstrate that antibodies 335-356 are specific for Cx40. Double-labeling experiments carried out with lung sections using anti-factor VIII and anti-Cx40 antibodies suggest that Cx40 is expressed in blood vessel endothelial cells. In guinea pig and rat heart sections, investigated using both immunofluorescence and immunoperoxidase techniques, a signal was also found to be associated with vascular walls. In guinea pig heart, only atrial myocytes are Cx40-positive. No labeling was detected in ventricular myocytes, including those of the His bundle and the bundle branches, which otherwise do express connexin43 (Cx43). In rat heart Cx40-expressing myocytes are localized in branches, and the Purkinje fibers. Cx43 is not detected either in the His bundle or in the proximal parts of the bundle branches, and consequently, Cx40 is the first connexin demonstrated in this region of the rat conduction system. Cx40 was not detected in the working ventricular myocytes. Double-labeling experiments carried out with hen anti-Cx43 antibodies and rabbit anti-Cx40 antibodies demonstrated that, in tissues expressing both Cx43 and Cx40, these two connexins were localized in the same immunoreactive sites. A few sites, however, appear to contain only one or the other of these two connexins.
Gap junctions are a determinant of myocardial conduction. Disturbances of gap-junctional content may account for abnormalities of impulse propagation, contributing to the arrhythmic tendency and mechanical inefficiency of ischemic and hypertrophied myocardium. The aim of this study was to characterize gap junction organization in normal human ventricular myocardium and to establish whether abnormalities exist in myocardium of chronically ischemic and hypertrophied hearts.
Cardiac gap-junctional connexin43 antibodies and confocal microscopy were used in a quantitative immunohistochemical study of surgical myocardial samples to explore the structural basis of electromechanical ventricular dysfunction in chronic ischemic and hypertrophic heart diseases. Normal adult human left ventricular myocardium had a gap-junctional surface area of 0.0051 micron2/micron3 myocyte volume; gap junctions were confined to intercalated disks, of which there was a mean of 11.6 per cell. The right ventricle showed similar gap junction surface area. Left ventricular myocardium from ischemic hearts (distant from any fibrotic scarring), despite normal numbers of intercalated disks per cell, had a reduced gap junction surface area (0.0027 micron2/micron3; P = .02), as did hypertrophied myocardium (0.0031 micron2/micron3; P = .05). The cardiac myocytes in the pathological tissues were larger than normal, and estimated gap-junctional content per cell was reduced in ischemic ventricle (P = .02) compared with normal.
Gap junctions in normal adult human working ventricular myocardium occupy an area of 0.0051 micron2/micron3 myocyte volume. This surface area is reduced in ventricular myocardium from hearts subject to chronic hypertrophy and ischemia, despite a normal number of intercellular abutments, and this alteration may contribute to abnormal impulse propagation in these hearts.
Using immunohistochemical staining, the distribution of connexin40 (Cx40) and connexin43 (Cx43) was studied in rat, guinea pig, porcine, bovine and human hearts. These species display differences in the degree of morphological differentiation of the conduction system. This study was performed in the anticipation that comparison of the distributions of Cx40 and Cx43 in young and adult specimens may provide clues as to the physiological role of connexins in the heart. To a large extent, the distribution patterns of Cx40 and Cx43 are comparable between species. In neonates and adults, Cx43 was immunolocalized throughout the working myocardium, but in the conduction system Cx43 was detected only after birth. Cx40 was found to appear slightly earlier in development than Cx43 and to disappear when levels of Cx43 became more abundant. This time course was seen in working myocardium and in the ventricular conduction system. Together these data suggest that expression of Cx40 induces or facilitates expression of Cx43, while abundant expression of Cx43 in turn leads to suppression of Cx40 expression. The exceptions to this may represent blocks in this potential regulatory sequence. A second conclusion is that Cx40 and Cx43 containing gap junctions appear in the ventricular conduction system from distal to proximal and only after birth. This indicates that terminal differentiation of the conduction system occurs unexpectedly late in development.
Generally, impulse propagation in cardiac tissue is assumed to be impaired by a reduction of intercellular electrical coupling or by the presence of structural discontinuities. Contrary to this notion, the spatially uniform reduction of electrical coupling induced successful conduction in discontinuous cardiac tissue structures exhibiting unidirectional conduction block. This seemingly paradoxical finding can be explained by a nonsymmetric effect of uncoupling on the current source and the current sink in the preparations used. It suggests that partial cellular uncoupling might prevent the initiation of cardiac arrhythmias that are dependent on the presence of unidirectional conduction block.
Slow, nonuniform conduction caused by abnormal gap-junctional coupling of infarct-related myocardium is thought to be a component of the arrhythmogenic substrate. The hypothesis that changes in gap-junctional distribution in the epicardial border zone (EBZ) of healing canine infarcts define the locations of reentrant ventricular tachycardia (VT) circuits was tested by correlating activation maps of the surviving subepicardial myocardial layer with immunolocalization of the principal gap-junctional protein, connexin43 (Cx43).
The EBZ overlying 4-day-old anterior infarcts in three dogs with inducible VT and three noninducible dogs was mapped with a high-resolution electrode array and systematically examined by standard histology and confocal immunolocalization of Cx43. The thickness of the EBZ was significantly less in the hearts with (538 +/- 257 microns) than without (840 +/- 132 microns; P < .05) VT. At the interface with the underlying necrotic cells, the EBZ myocardium showed a marked disruption of gap-junctional distribution, with Cx43 labeling abnormally arrayed longitudinally along the lateral surfaces of the cells. In the EBZ of all hearts, the disrupted Cx43 labeling extended part of the way to the epicardial surface, with the most superficial epicardial myocytes having the normal transversely orientated pattern. Only in the hearts with inducible VT did the disorganization extend through the full thickness of the surviving layer at sites correlating with the location of the central common pathways of the figure-of-8 reentrant VT circuits.
Altered gap-junctional distribution is part of the early remodeling of myocardium after infarction, and by defining the location of the common central pathway of the reentrant VT circuits, it may be a determinant of VT susceptibility.
Theoretical simulations were performed to study the interplay between membrane ionic currents and gap-junction coupling in determining cardiac conduction. Results demonstrate that a much slower conduction velocity can be achieved with reduced gap-junction coupling than with reduced membrane excitability. Also, uniform reduction in intercellular coupling increases spatial asymmetries of excitability and, consequently, the vulnerability to unidirectional block.
In cardiac tissue, reduced membrane excitability and reduced gap junction coupling both slow conduction velocity of the action potential. However, the ionic mechanisms of slow conduction for the two conditions are very different. We explored, using a multicellular theoretical fiber, the ionic mechanisms and functional role of the fast sodium current, INa, and the L-type calcium current, ICa(L), during conduction slowing for the two fiber conditions. A safety factor for conduction (SF) was formulated and computed for each condition. Reduced excitability caused a lower SF as conduction velocity decreased. In contrast, reduced gap junction coupling caused a paradoxical increase in SF as conduction velocity decreased. The opposite effect of the two conditions on SF was reflected in the minimum attainable conduction velocity before failure: decreased excitability could reduce velocity to only one third of control (from 54 to 17 cm/s) before failure occurred, whereas decreased coupling could reduce velocity to as low as 0.26 cm/s before block. Under normal conditions and conditions of reduced excitability, ICa(L) had a minimal effect on SF and on conduction. However, ICa(L) played a major role in sustaining conduction when intercellular coupling was reduced. This phenomenon demonstrates that structural, nonmembrane factors can cause a switch of intrinsic membrane processes that support conduction. High intracellular calcium concentration, [Ca]i, lowered propagation safety and caused earlier block when intercellular coupling was reduced. [Ca]i affected conduction via calcium-dependent inactivation of ICa(L). The increase of safety factor during reduced coupling suggests a major involvement of uncoupling in stable slow conduction in infarcted myocardium, making microreentry possible. Reliance on ICa(L) for this type of conduction suggests ICa(L) as a possible target for antiarrhythmic drug therapy.
The long-term consequences of CAD remain a prominent clinical problem. Particularly with new therapeutic strategies that reduce the mortality associated with acute coronary syndromes, more patients suffer from the long-term sequelae of this condition. In this setting, the identification of those segments of myocardium that appear dysfunctional distal to coronary stenoses and that can improve after coronary revascularization is of considerable clinical importance. Although the diagnostic and therapeutic aspects of this problem are clearly defined, the pathophysiological mechanisms underlying the dysfunctional myocardium are controversial.
It was demonstrated more than 20 years ago1 2 that resting wall-motion abnormalities in patients with CAD can improve after administration of an inotropic agent or after coronary bypass. An article published in 1978 by Diamond et al3 presaged the concept of hibernating myocardium: “Reports of sometimes dramatic improvement in segmental left ventricular function following coronary bypass surgery, although not universal, leaves the clear implication that ischemic non-infarcted myocardium can exist in a state of function hibernation.” Rahimtoola, in an article published in 1985,4 popularized this concept and later suggested that “hibernating myocardium is a state of persistently impaired myocardial and left ventricular function at rest due to reduced coronary blood flow that can be partially or completely restored to normal either by improving blood flow or by reducing oxygen demand.”5
Since the introduction of the term “hibernation,”3 4 5 6 the clinical importance of reversible left ventricular dysfunction has been widely accepted. The concept of an adaptive process that decreases myocardial oxygen consumption in the presence of either chronically or intermittently reduced oxygen delivery has generated considerable clinical and experimental interest.
Accordingly, our aims were to (1) review the current criteria of the definition of hibernating myocardium, (2) summarize recent clinical as well as experimental data pertaining to this subject, …
Cardiac myocytes are electrically coupled by gap junctions, clusters of low-resistance intercellular channels composed of connexins. Variations in the quantities and spatial distribution of different connexin types have been implicated in regional differentiation of electrophysiological properties in the heart. Although independent studies have demonstrated that connexin43 is abundant in working ventricular myocardium and that connexin40 is preferentially expressed in the atrioventricular conduction system of a number of species, information on the spatial distribution of connexin45 in the heart is limited to data obtained using an antibody raised to a single peptide sequence. In the present study, we report on the production and characterization of a new anti-connexin45 antibody and its application to the investigation of connexin45 expression in mouse and rat myocardium. The affinity-purified antiserum, raised in guinea pig to residues 354 to 367 of human connexin45, recognized a single 45-kD band on Western blots of HeLa cells transfected to express connexin45 and gave punctate immunolabeling at the cell borders, demonstrated by freeze-fracture cytochemistry to represent gap junctions. Only low levels of connexin45 mRNA were detected on Northern blots of mouse and rat cardiac tissues, and connexin45 protein levels were below the limit of detection on Western blots. Confocal microscopy of immunolabeled ventricular tissue revealed that the major part of the working myocardium was immunonegative for connexin45. A clearly defined zone containing connexin45-expressing cells was, however, localized to the endocardial surface, overlapping with connexin40-expressing myocytes of the conduction system. As these results contrast with the prevailing view that connexin45 is widely distributed in working ventricular myocytes, we compared the immunolabeling pattern obtained with a commercially supplied anti-connexin45 antiserum raised against the same peptide that was used in previous studies. The commercial connexin45 antiserum gave widespread labeling throughout the ventricular myocardium, but this labeling was inhibited by a six-amino acid peptide matching part of the connexin43 sequence, indicating cross-reaction of the commercial connexin45 antiserum with connexin43 in the tissue. Further evidence for such cross-reactivity came from observations on connexin43-transfected cells, which gave positive immunolabeling with the commercial anti-connexin45 antiserum. Our demonstration of a specific association of connexin45 with connexin40-expressing myocytes in rat and mouse ventricle raises the possibility that connexin45 contributes to the modulation of electrophysiological properties in the ventricular conduction system and highlights the need for reappraisal of the distribution and role of connexin45 in other species.
The regional wall motion impairment and predisposition to arrhythmias in human ventricular hibernation may plausibly result from abnormal intercellular propagation of the depolarizing wave front. This study investigated the hypothesis that altered patterns of expression of connexin43, the principal gap junctional protein responsible for passive conduction of the cardiac action potential, contribute to the pathogenesis of hibernation.
Patients with poor ventricular function and severe coronary artery disease underwent thallium scanning and MRI to predict regions of normally perfused, reversibly ischemic, or hibernating myocardium. Twenty-one patients went on to coronary artery bypass graft surgery, during which biopsies representative of each of the above classes were taken. Hibernation was confirmed by improvement in segmental wall motion at reassessment 6 months after surgery. Connexin43 was studied by quantitative immunoconfocal laser scanning microscopy and PC image software. Analysis of en face projection views of intercalated disks revealed a significant reduction in relative connexin43 content per unit area in reversibly ischemic (76.7+/-34.6%, P<.001) and hibernating (67.4+/-24.3%, P<.001) tissue compared with normal (100+/-30.3%); ANOVA P<.001. The hibernating regions were further characterized by loss of the larger gap junctions normally seen at the disk periphery, reflected by a significant reduction in mean junctional plaque size in the hibernating tissues (69.5+/-20.8%) compared with reversibly ischemic (87.4+/-31.2%, P=.012) and normal (100+/-31.5%, P<.001) segments; ANOVA P<.001.
These results indicate progressive reduction and disruption of connexin43 gap junctions in reversible ischemia and hibernation. Abnormal impulse propagation resulting from such changes may contribute to the electromechanical dysfunction associated with hibernation.
Since altered expression of gap junction proteins (connexins) in diseased myocardial tissue may lead to abnormal electrical coupling between cardiomyocytes and hence contribute to arrhythmogenesis, the expression of connexin(Cx)40 and Cx43 was studied in atrial appendage from goats in sinus rhythm (SR) and persistent atrial fibrillation (AF).
Biopsies were taken from the left and right atrial appendages from goats in SR or after pacing-induced persistent AF. Analyses of Cx40 and Cx43 mRNA and protein levels, using quantitative (competitive) polymerase chain reaction and western blotting, respectively, revealed no significant changes in the overall expression of Cx40 and Cx43 as a result of persistent AF. At the cellular level, immunohistochemistry and confocal laser scanning microscopy showed a homogeneous distribution of either connexin in atrial sections taken during SR. After induction of AF, the distribution of Cx43 gap junctions was unchanged whereas the Cx40 pattern showed marked inhomogeneities with small areas (0.15 to 0.6 mm in diameter, 25% of section surface area) of low-density Cx40 located between larger areas of normal (unchanged) Cx40 density. Activation mapping (244 electrodes, spatial resolution 2.25 mm) of the right atrial wall did not reveal changes in atrial conduction velocity.
Pacing-induced persistent AF in the goat gave rise to changes in the spatial organization of Cx40 gap junctions. Although the overall conduction velocity appeared not to have changed, microheterogeneities in conduction due to the local redistribution of Cx40 gap junctions might have contributed to the initiation and maintenance of AF.
Atrial fibrillation, the most common sustained cardiac arrhythmia and a major cause of stroke, results from simultaneous reentrant wavelets. Its spontaneous initiation has not been studied.
We studied 45 patients with frequent episodes of atrial fibrillation (mean [+/-SD] duration, 344+/-326 minutes per 24 hours) refractory to drug therapy. The spontaneous initiation of atrial fibrillation was mapped with the use of multielectrode catheters designed to record the earliest electrical activity preceding the onset of atrial fibrillation and associated atrial ectopic beats. The accuracy of the mapping was confirmed by the abrupt disappearance of triggering atrial ectopic beats after ablation with local radio-frequency energy.
A single point of origin of atrial ectopic beats was identified in 29 patients, two points of origin were identified in 9 patients, and three or four points of origin were identified in 7 patients, for a total of 69 ectopic foci. Three foci were in the right atrium, 1 in the posterior left atrium, and 65 (94 percent) in the pulmonary veins (31 in the left superior, 17 in the right superior, 11 in the left inferior, and 6 in the right inferior pulmonary vein). The earliest activation was found to have occurred 2 to 4 cm inside the veins, marked by a local depolarization preceding the atrial ectopic beats on the surface electrocardiogram by 106+/-24 msec. Atrial fibrillation was initiated by a sudden burst of rapid depolarizations (340 per minute). A local depolarization could also be recognized during sinus rhythm and abolished by radiofrequency ablation. During a follow-up period of 8+/-6 months after ablation, 28 patients (62 percent) had no recurrence of atrial fibrillation.
The pulmonary veins are an important source of ectopic beats, initiating frequent paroxysms of atrial fibrillation. These foci respond to treatment with radio-frequency ablation.