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Sarcolemmal Versus Mitochondrial ATP-Sensitive K+ Channels and Myocardial Preconditioning
Abstract
Ischemic preconditioning (IPC) is a phenomenon in which single or multiple brief periods of ischemia have been shown to protect the heart against a more prolonged ischemic insult, the result of which is a marked reduction in myocardial infarct size, severity of stunning, or incidence of cardiac arrhythmias. Although a number of substances and signaling pathways have been proposed to be involved in mediating the cardioprotective effect of IPC, the overwhelming majority of evidence suggests that the ATP-sensitive potassium channel (KATP channel) is an important component of this phenomenon and may serve as the end effector in this process. Initially, it was hypothesized that the surface or sarcolemmal KATP (sarc KATP) channel mediated protection observed after IPC; however, subsequent evidence suggested that the recently identified mitochondrial KATP channel (mito KATP) may be the potassium channel mediating IPC-induced cardioprotection. In this review, evidence will be presented supporting a role for either the sarc KATP or the mito KATP in IPC and potential mechanisms by which opening these channels may produce cardioprotection; additionally, we will address important questions that still need to be investigated to define the role of the sarc or mito KATP channel, or both, in cardiac pathophysiology.
... The exact mechanism behind the opening o f the mKATp and the observed protection is not fully understood. u i K a t p opening causes depolarisation of the inner mitochondrial membrane, matrix swelling, enhanced respiration and reduced calcium overload (Gross et al, 1999). ...
... In relation to IPC, the K a t p channel opening appears to play a central role, either as a distal mediator (Grover et al, 2000) or, as has been suggested more recently, as a proximal (trigger) event . Opening o f both the sarcolemmal and mitochondrial K a t p ( s K a t p and mKATp) channels has been shown to Chapter 5 be enhanced following activation o f intracellular mediators or kinases (Gross et al, 1999). While s K a t p channel opening is responsible for shortening o f the myocyte action potential, membrane hyperpolarisation, decreased calcium entry and preservation o f ATP, mKATP channel opening causes membrane depolarisation, matrix swelling, enhanced respiration and reduced calcium overload (Gross et al, 1999). ...
... Opening o f both the sarcolemmal and mitochondrial K a t p ( s K a t p and mKATp) channels has been shown to Chapter 5 be enhanced following activation o f intracellular mediators or kinases (Gross et al, 1999). While s K a t p channel opening is responsible for shortening o f the myocyte action potential, membrane hyperpolarisation, decreased calcium entry and preservation o f ATP, mKATP channel opening causes membrane depolarisation, matrix swelling, enhanced respiration and reduced calcium overload (Gross et al, 1999). ...
Type-B natriuretic peptide (BNP) is an important hormone abundantly present as a pro-peptide in cardiomyocytes. Its release is triggered by exercise, hypoxia and myocardial ischaemia, in addition to chronic haemodynamic cardiac overloading states. BNP's main endocrine actions of vasodilation and natriuresis are mediated by a particulate receptor guanylyl cyclase, natriuretic peptide receptor-A (NPR-A), with subsequent elevation of intracellular cGMP. The role and mechanisms of action of BNP in cardiac ischaemia are not known. We hypothesised that BNP mediates cardioprotection during acute ischaemia-reperfusion, via guanylyl cyclase and cGMP elevation, and examined the role of KATP channel opening in the protective mechanism. The role of nitric oxide (NO) in BNP's signal transduction was also evaluated. Pharmacological studies were carried out in the Langendorff perfused rat heart. Endogenous BNP release was assessed by radio-immunoassay of coronary effluent samples following global normothermic ischemia. Peak concentrations in the first min of reperfusion were markedly elevated following 2 min, 5 min and 20 min of ischaemia. In rat hearts subjected to 35 min regional ischaemia, exogenous BNP limited infarct size in a concentration-dependent manner. The protective action of BNP was sensitive to inhibition by glibenclamide and 5-hydroxydecanoate, blockers of the mitochondrial KATP channel, but not by HMR1098, a blocker of the sarcolemmal KATP channel. Radio-immunoassayed cGMP in cardiac tissue showed a proportionate rise when hearts were subjected to graded durations of ischaemia and when perfused with BNP. Hearts perfused with varying concentrations of 8-bromo-cGMP, a cell-permeable cGMP analogue, were protected against infarction at the lower concentration. L-NAME a blocker of nitric oxide synthase (NOS), and ODQ a blocker of soluble guanylyl cyclase (sGC), both abolished cardioprotection when co-perfused with BNP, suggesting that NO and its activation of sGC play key roles in the protective effect of BNP. To evaluate the source of NOS, studies were undertaken using Western Blot techniques to probe the involvement of endothelial isoform of NOS (eNOS) known to be partially responsible for BNP's vasodilation. However, we found no evidence for acute phosphorylation of eNOS at serine 1177, following BNP or acute ischaemia. Finally, together, our findings indicate a previously-unrecognised cardioprotective action of exogenous BNP via opening of the putative mitochondrial KATP channel with the involvement of basal nitric oxide in the NO/sGC, and cGMP release in the signal transduction. The work contained in this thesis thus confirms a cytoprotective role for BNP in myocardial ischaemia-reperfusion injury and requires further studies in other species and in transgenic models.
... В настоящее время преобладает мнение, что транзиторные ишемические атаки являются прежде всего фактором риска мозгового инсульта. Упоминания об их саногенном эффекте в публикациях последних лет встречаются довольно редко [55][56][57][58][59]. ...
... Katp-каналы идентифицированы в сарколемме и в митохондриальных мембранах. Первоначально ответственными за ИП рассматривались сарколеммные каналы Katp, активация которых сокращает продолжительность потенциала действия, ингибирует поток кальция через каналы L-типа, в результате чего уменьшается внутриклеточная перегрузка кальцием в процессе ишемии [56,59]. Позднее было установлено, что укорочение потенциала действия активацией сарколемных Katp-каналов не существенно для индуцированной противоишемической защиты миокарда, а при активации митохондриальных Katp-каналов воспроизводится эффект ИП [56,57]. ...
... Первоначально ответственными за ИП рассматривались сарколеммные каналы Katp, активация которых сокращает продолжительность потенциала действия, ингибирует поток кальция через каналы L-типа, в результате чего уменьшается внутриклеточная перегрузка кальцием в процессе ишемии [56,59]. Позднее было установлено, что укорочение потенциала действия активацией сарколемных Katp-каналов не существенно для индуцированной противоишемической защиты миокарда, а при активации митохондриальных Katp-каналов воспроизводится эффект ИП [56,57]. В последнее время показано, что активация Katp-каналов внутренней митохондриальной мембраны является не только необходимым, но и достаточным условием для реализации защитного эффекта гипоксического и ишемического прекондиционирования [35]. ...
The phenomenon of ischemic preconditioning based on the S.P. Botkin’s idea about defense effect of disturbing factors acting in small intensities is observed in the review. The modern literature data about main types of preconditioning exposure, triggers and mechanisms of ischemic preconditioning are reviewed. This phenomenon was supported in many experiments in vivo and in vitro on animals of different spices as well as in humans in clinical conditions. Ischemic preconditioning is qualified as transient positive changes in the organs and tissues produced by activation of rapid endogenous adoptive processes in them during the short period of subletal ischemia and reperfusion and which defend them from subsequent ischemic episodes. There are early and late ischemic preconditioning (the second window of defense). The first type of ischemic preconditioning belongs to classic type of preconditioning and is produced by the short ischemic episodes (3-5 min) and similar intervals of reperfusion. Ischemic preconditioning observed in a day or more after preconditioning stimuli is named as late preconditioning with genes expression, synthesis of heat shock proteins (HSP 72 in particular) and NO synthase as the basis mechanisms underlying of it. Administration of triggers like adenosine, forbol ether, bradykinine or glycerol derivatives into the blood or ischemic tissues produces defense action similar to ischemic preconditioning and qualified as pharmacological preconditioning. Preconditioning induced by pharmacological agents are more preference than short ischemic episodes. Antihypoxic effects of benzimidazol derivatives in both an acute hypoxia and hypoxic preconditioning are described in the article. Other perspectives of pharmacological preconditioning in practical use are also discussed.
... Cardiac myocytes contain two K ATP channel entities: one located in the sarcolemmal membrane (sK ATP ) and a proposed mitochondrial channel (mitoK ATP ). The structure and function of sK ATP and mitoK ATP appear to be related but with important differences [34][35][36] . The well-characterized sK ATP channel has been cloned and is present at a very high density in the heart (2,000-3,000/myocyte) [37] . ...
... Multiple K ATP channel openers have been studied for cardioprotection, and each has a different pharmacology. Pinacidil was used in many early studies evaluating K ATP channel openers and cardioprotection, but many researchers have since focused on diazoxide due to its proposed specificity for mitoK ATP channels, unlike most other K ATP channel openers [34] . Garlid found that diazoxide has a 2,000-fold greater affinity for mitoK ATP compared to the sarcolemmal K ATP channel [95] . ...
The many ways in which ATP-sensitive potassium (KATP) channels can be exploited for human benefit have expanded over recent decades. Especially since the early 2000s, research has improved our understanding of the components and mechanisms of KATP channels. They have the potential to have a prominent role in cardiac surgery. Pharmacologic and non-pharmacologic activation of KATP channels has been shown to be both cardioprotective and neuroprotective in early basic science and clinical studies. However, many questions remain unanswered and require further study, necessitating further basic science work and large human clinical trials. This review discusses the history and recent progress in the research relating to the use of KATP channels for cardiac surgery.
... Localized transient elevation of ROS in mitochondria has been shown to be protective in myocardium subjected to ischaemic injury. ATP-sensitive potassium channels (K ATP ; K ir 6.2) have received major attention as a molecular mediator of myocardial protection by ischaemic preconditioning via generating ROS (Gross and Fryer, 1999;Oldenburg et al., 2002;Lacza et al., 2003). Transient K + influx via activated mitochondrial ATP-sensitive potassium channel (mitoK ATP ) channels stimulate respiration and augment electron flux through q cycle, both of which stimulate ROS generation (Becker et al., 1999). ...
... Diazoxide is 2000-fold more selective for mitochondrial than for plasmalemmal channels (Garlid et al., 1996), which indeed supports the critical role of mitoK ATP channels in the mitochondrial signalling in different experimental settings (Bajgar et al., 2001;Farkas et al., 2004;Katakam et al., 2009Katakam et al., , 2013Katakam et al., , 2016. However, it is important to note that the structure of the mitoK ATP channel is not yet known with certainty but appears to be pharmacologically distinct basing on the selectivity of the agonists (Garlid et al., 1996 andGross and Fryer, 1999). ...
Background and Purpose
CD34⁺ haematopoietic stem/progenitor cells have revascularization potential and are now being tested for the treatment of ischaemic vascular diseases in clinical trials. We tested the hypothesis that mitochondrial depolarization stimulates the reparative functions of CD34⁺ cells.
Experimental Approach
Peripheral blood was obtained from healthy individuals (n = 63), and mononuclear cells (MNCs) were separated. MNCs were enriched for lineage negative cells, followed by isolation of CD34⁺ cells. Vascular repair‐relevant functions of CD34⁺ cells, proliferation and migration, were evaluated in the presence and absence of diazoxide. Mitochondrial membrane potential, ROS and NO levels were evaluated by flow cytometry by using JC‐1, mitoSOX and DAF‐FM respectively.
Key Results
Diazoxide stimulated the proliferation and migration of CD34⁺ cells that were comparable to the responses induced by stromal‐derived factor‐1α (SDF) or VEGF. Effects of diazoxide were blocked by either 5‐hydroxydecanoate (5HD), a selective mitochondrial ATP‐sensitive potassium channel (mitoKATP) inhibitor, or by L‐NAME. Diazoxide induced mitochondrial depolarization, and NO and cGMP generation that were 5HD‐sensitive. The generation of NO and cGMP by diazoxide was blocked by an endothelial NOS (eNOS)‐selective inhibitor, NIO, but not by a neuronal (n)NOS‐selective inhibitor, Nω‐propyl‐L‐arginine (NPA). A Ca²⁺ chelator, BAPTA, Akt inhibitor, triciribine, or PI3K inhibitor, LY294002, inhibited the NO release induced by diazoxide. Phosphorylation of eNOS at Ser¹¹⁷⁷ and dephosphorylation at Thr⁴⁹⁵ were increased. Diazoxide‐induced ROS generation and phosphorylation of eNOS at Ser¹¹⁷⁷ were reduced by NPA.
Conclusion and Implications
Diazoxide stimulates vascular repair‐relevant functions of CD34⁺ cells via the mitoKATP‐dependent release of NO and ROS.
Linked Articles
This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc
... K ATP channels mediate insulin secretion in pancreatic islet beta cells, and controlling vascular tone [77] . Under pathophysiological conditions, K ATP channels play cytoprotective role in cardiac myocytes and neurons during ischemia and/or hypoxia [78][79][80][81] . ...
... Ischemic preconditioning is when one or several intermittent periods of ischemia disconcertingly results in protection against tissue damage by a subsequent and sustained ischemic injury [79] . K ATP channel activation prior to ischemic event mimics the effects of ischemic preconditioning [78,80,92] . Similarly, in the brain K ATP channels play a role in ischemic tolerance in stroke, conferring neuroprotection [81] . ...
ATP-sensitive potassium (KATP) channels are ubiquitously expressed on the plasma membrane of cells in multiple organs, including the heart, pancreas and brain. KATP channels play important roles in controlling and regulating cellular functions in response to metabolic state, which are inhibited by ATP and activated by Mg-ADP, allowing the cell to couple cellular metabolic state (ATP/ADP ratio) to electrical activity of the cell membrane. KATP channels mediate insulin secretion in pancreatic islet beta cells, and controlling vascular tone. Under pathophysiological conditions, KATP channels play cytoprotective role in cardiac myocytes and neurons during ischemia and/or hypoxia. KATP channel is a hetero-octameric complex, consisting of four pore-forming Kir6.x and four regulatory sulfonylurea receptor SURx subunits. These subunits are differentially expressed in various cell types, thus determining the sensitivity of the cells to specific channel modifiers. Sulfonylurea class of antidiabetic drugs blocks KATP channels, which are neuroprotective in stroke, can be one of the high stoke risk factors for diabetic patients. In this review, we discussed the potential effects of KATP channel blockers when used under pathological conditions related to diabetics and cerebral ischemic stroke.
... Specifically, the repolarization phase involves potassium (K + ) channels [1]. Several studies have focused on the modulation of K + channels from a cardioprotective perspective in order to reproduce the extensively documented positive anti-ischemic effects due to the ischemic pre-conditioning (IPC) mechanism, a self-defense phenomenon characterized by brief periods of I/R able to increase the resistance to the injury due to a subsequent, more prolonged ischemic episode [2]. Later, another preventive phenomenon was described and named as ischemic post-conditioning (IPostC). ...
Modulation of mitochondrial K channels represents a pharmacological strategy to promote cardioprotective effects. Isothiocyanates emerge as molecules capable of releasing hydrogen sulfide (H2S), an endogenous pleiotropic gasotransmitter responsible for anti-ischemic cardioprotective effects also through the involvement of mitoK channels. Erucin (ERU) is a natural isothiocyanate resulting from the enzymatic hydrolysis of glucosinolates (GSLs) present in Eruca sativa Mill. seeds, an edible plant of the Brassicaceae family. In this experimental work, the specific involvement of mitoKATP channels in the cardioprotective effect induced by ERU was evaluated in detail. An in vivo preclinical model of acute myocardial infarction was reproduced in rats to evaluate the cardioprotective effect of ERU. Diazoxide was used as a reference compound for the modulation of potassium fluxes and 5-hydroxydecanoic acid (5HD) as a selective blocker of KATP channels. Specific investigations on isolated cardiac mitochondria were carried out to evaluate the involvement of mitoKATP channels. The results obtained showed ERU cardioprotective effects against ischemia/reperfusion (I/R) damage through the involvement of mitoKATP channels and the consequent depolarizing effect, which in turn reduced calcium entry and preserved mitochondrial integrity.
... Vasodilatation is attributed to the opening of K ATP channels in the sarcolemmal membrane of vascular smooth muscle. This effect produces both coronary and systemic vasodilatation and coronary blood flow is enhanced (Gross and Fryer, 2000;Lilleberg et al., 1998;Pataricza et al., 2003). ...
... Although it is now generally believed that the mitochondrial K + -ATP channels play a more significant role in the beneficial effects of IP, the SL K + -ATP channels have increased in importance for inducing the harmful effects of oxidative stress [139]. Earlier observations have revealed that the beneficial actions of IP may involve the SL K + -ATP channels because they are in an open state when exposed to ROS and this correlated well with the cytoprotective properties [140][141][142][143]. Subsequent studies demonstrated that the SL K + -ATP channels appear to act as an effector of IP and facilitated in improving functional recovery. ...
It is now well established that ischemia/reperfusion (I/R) injury is associated with the compromised recovery of cardiac contractile function. Such an adverse effect of I/R injury in the heart is attributed to the development of oxidative stress and intracellular Ca2+-overload, which are known to induce remodeling of subcellular organelles such as sarcolemma, sarcoplasmic reticulum, mitochondria and myofibrils. However, repeated episodes of brief periods of ischemia followed by reperfusion or ischemic preconditioning (IP) have been shown to improve cardiac function and exert cardioprotective actions against the adverse effects of prolonged I/R injury. This protective action of IP in attenuating myocardial damage and subcellular remodeling is likely to be due to marked reductions in the occurrence of oxidative stress and intracellular Ca2+-overload in cardiomyocytes. In addition, the beneficial actions of IP have been attributed to the depression of proteolytic activities and inflammatory levels of cytokines as well as the activation of the nuclear factor erythroid factor 2-mediated signal transduction pathway. Accordingly, this review is intended to describe some of the changes in subcellular organelles, which are induced in cardiomyocytes by I/R for the occurrence of oxidative stress and intracellular Ca2+-overload and highlight some of the mechanisms for explaining the cardioprotective effects of IP.
... However, it is still unclear, which role the mitoK ATP channel has. Is it a trigger or a distal effector or maybe both, which some studies have shown (Gross and Fryer 1999;Schulz et al. 2001). ...
KATP channels are ubiquitously expressed and link metabolic state to electrical excitability. In heart, in response to ischaemic stress, they play a protective role and in vascular smooth muscle regulation of vascular tone (vasorelaxation). Functional KATP channels are hetero-octamers composed of two subunits, a pore forming Kir6, which is a member of the inwardly rectifying potassium channels family and a regulatory sulphonylurea receptor (SUR). In response to nucleotides and pharmacological agents, SUR allosterically regulate KATP channel gating. Multidisciplinary techniques (molecular biology, biochemistry, electrophysiology, pharmacology) were used to study the allosteric regulation between these two heterologous subunits in KATP channels.
This project was divided into three major sub-projects: 1) Application of site directed mutagenesis and biochemical techniques to identify the cognate interaction domain on Kir6.2 for SUR2A-NBD2 (nucleotide binding domain 2). 2) Electrophysiological techniques to investigate the allosteric information transfer between heterologous subunits Kir6 and SUR2A. 3) Recombinant fusion protein to express and purify the cytoplasmic domains of Kir6.2 for structural analysis of the interaction between the two subunits.
This study reports on the identification of three cytoplasmic electrostatic interfaces between Kir6 and SUR2A involved in determining the sensitivity of KATP channel agonist, pinacidil, and antagonist, glibenclamide, from SUR2A to the Kir6 channel pore.
For structural study of cytoplasmic domains of Kir6.2, bacterial TM1070 was used as fusion partner with Kir6.2. A TM1070-Kir6.2 NC (CT-His6 tag) fusion construct expressed in Arctic Express competent cells permitted successful expression of folded cytoplasmic domains of Kir6.2 in near native form. Immobilized metal ion affinity chromatography, IMAC (Ni2+), and gel filtration chromatography (GFC) column as second purification step were performed to purify this recombinant protein. The purification was confirmed by CBS and Western blot analysis.
Possibly, this new information on channel structure-function relationships may contribute to the design of novel and more effective drugs.
... Additionally, they showed that treatment with aprikalim a K atp channel opener, mimicked the protective effects of IPC . This finding was subsequently found in a number of different species and experimental models including, humans (Gross & Fryer, 1999). The question is, how would the opening of K atp channels lead to a protective effect? ...
Very few therapeutic modalities are beneficial in the treatment of acute myocardial infarction. However, the phenomenon of ischaemic preconditioning (IPC) reduces cell necrosis and therefore, may offer protection against ischaemia-reperfusion injury. Bradykinin has been implicated in IPC as a trigger of this protective phenomenon. The protective effects of both IPC and bradykinin are largely under-investigated in models of chronic myocardial hypertrophy. Furthermore, the kallikrein-kinin system is thought to be implicated in hypertension, indeed studies have demonstrated that levels of bradykinin are attenuated in hypertension. Therefore, the aim of this thesis was to further elucidate the cardioprotective actions of bradykinin in both the normal and hypertrophied myocardium. In preliminary experiments, the deoxycorticosterone acetate (DOCA)-salt rat was used to represent a mild model of left ventricular hypertrophy (LVH) associated with short term hypertension. Although IPC was found to reduce infarct size in the DOCA-salt rat hearts subjected to ischaemic-reperfusion injury, bradykinin induced cardioprotection was impaired in these hearts. Drugs that inhibit bradykinin degradation, namely, angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) inhibitors can be used therapeutically to augment bradykinin levels. Previous studies have demonstrated that ACE inhibitors can potentiate a subthreshold preconditioning stimulus, however, it is not known whether dual ACE and NEP inhibition also potentiates IPC. It was found that the dual ACE and NEP inhibitor, omapatrilat, analogous to captopril, augmented a subthreshold IPC stimulus via activation of the bradykinin B2 receptor. In contrast to captopril, omapatrilat also evoked protection when administered directly (ie, in the absence of preconditioning ischaemia), an effect also dependent upon 82 receptor activation. The effects of IPC in chronic myocardial hypertrophy associated with long term hypertension were investigated. IPC evoked protection in hearts isolated from young and middle aged SHR and normotensive age matched, WKY rats. However, IPC did not protect the ageing SHR/WKY rat hearts. Therefore, the combination of ageing and long standing hypertrophy interfere with the occurrence of IPC. In an attempt to raise bradykinin levels, captopril was used in conjunction with the IPC protocol. However, no protection was observed in hearts isolated from the ageing SHR. In contrast, modest protection was seen in age matched WKY rat hearts. In the normal myocardium, it was found that bradykinin administered just prior to reperfusion also induced cardioprotection possibly via activation of the PIS kinase pathway. However, the protective effect of bradykinin at reperfusion could not be duplicated in the ageing SHR myocardium. Even though numerous studies have demonstrated that bradykinin elicits classical preconditioning, its role in delayed preconditioning remains elusive. The final set of experiments in this thesis investigated whether bradykinin triggers delayed preconditioning. It was found that a bradykinin bolus given 24 hours prior to infarction triggered protection, an effect dependent upon the generation of nitric oxide. The work contained in this thesis confirms the cardioprotective potential of bradykinin and bradykinin modifying drugs in normal myocardium. However, the impairment of cardioprotective pathways in hypertensive myocardium was a consistent finding of these studies and therefore requires further investigation.
... The inner membrane anion channel (IMAC) and chloride intracellular channel proteins 4 and 5 (CLIC4 and CLIC5) represent the anion channel family of the IMM [82,86]. Since the discovery in 1997 that the activation of the mitochondrial ATP-sensitive K + channel (mitoK ATP ) protects hearts against ischemia/reperfusion (I/R) injury [87,88], an important role of IMM K + permeability in cytoprotection has been described in numerous cellular models and various insults [89]. It all started with the discovery that the activation of the mitoK ATP channel mimics ischemic preconditioning (IPC)-a well-known phenomenon in which brief periods of ischemia protect cells against the subsequent injury resulting from sustained ischemia [90,91]. ...
A large number of diverse mechanisms that lead to cytoprotection have been described to date. Perhaps, not surprisingly, the role of mitochondria in these phenomena is notable. In addition to being metabolic centers, due to their role in cell catabolism, ATP synthesis, and biosynthesis these organelles are triggers and/or end-effectors of a large number of signaling pathways. Their role in the regulation of the intrinsic apoptotic pathway, calcium homeostasis, and reactive oxygen species signaling is well documented. In this review, we aim to characterize the prospects of influencing cytoprotective mitochondrial signaling routes by natural substances of plant origin, namely, flavonoids (e.g., flavanones, flavones, flavonols, flavan-3-ols, anthocyanidins, and isoflavones). Flavonoids are a family of widely distributed plant secondary metabolites known for their beneficial effects on human health and are widely applied in traditional medicine. Their pharmacological characteristics include antioxidative, anticarcinogenic, anti-inflammatory, antibacterial, and antidiabetic properties. Here, we focus on presenting mitochondria-mediated cytoprotection against various insults. Thus, the role of flavonoids as antioxidants and modulators of antioxidant cellular response, apoptosis, mitochondrial biogenesis, autophagy, and fission and fusion is reported. Finally, an emerging field of flavonoid-mediated changes in the activity of mitochondrial ion channels and their role in cytoprotection is outlined.
... The evidence supporting a role for K^yp channels in ischaemic preconditioning comes from studies in rats (129), rabbits (262), dogs (263), pigs (264) and man (265) where the blockade of these channels with sulphonylurea receptor antagonists was shown to abolish the protective effects of classic preconditioning. Conversely, pharmacological activation of K^yp channels with bimakalim, cromokalin, or pinacidil produced cardioprotective effects comparable to that of ischaemic preconditioning (266). Furthermore, PKC has been shown to activate K^yp channels in human and rabbit ventricular myocytes (267) and a synergistic action of adenosine and PKC on K^yp channels and shortening of action potential duration has been reported (268). ...
Introduction: Ischaemic heart disease is the leading cause of mortality and morbidity in the Western world. The possibility of exploiting an innate adaptive mechanism to protect ischaemic myocardium has generated considerable excitement and enthusiastic research. Brief periods of ischaemia induce subacute myocardial protection against subsequent ischaemic injury, a phenomenon termed delayed preconditioning which appears to be mediated by adenosine. The temporal profile and the cellular mechanisms underlying this delayed adaptation in experimental animals and in patients with coronary artery disease were the subject of this thesis. Methods and Results: In experimental studies, delayed preconditioning was induced in in vivo rabbit and in vitro rat models of regional myocardial ischaemia-reperfusion with the selective adenosine A1 receptor (A1R) agonist 2-chloro-N6-cyclopentyladenosine (CCPA). Infarct size expressed as a percentage of risk zone was nearly halved by prior intermittent A1R activation with CCPA (5 doses at 48h intervals) implying that animals had been maintained in a preconditioned state over 10 days. Using selective inhibitors, it was demonstrated that A1R induced delayed preconditioning is dependent on a signalling mechanism involving protein kinase C (PKC) and tyrosine kinases (TKs). Downstream of these kinases, A1R activation induced subacute activation of p38 MAPK, phosphorylation of Hsp27 and enhanced expression of Mn-SOD, implying involvement of these cytoprotective proteins in mediating delayed protection. No role was demonstrated for a nitric oxide-dependent pathway in induction of protection. Delayed myocardial protection, in terms of enhanced tolerance to ischaemia during exercise was demonstrated in patients with coronary artery disease. This adaptation was independent of adenosine. Conclusions: These results suggest that the cellular events downstream of A1R involve both PKC and TKs which in turn, results in phosphorylation/activation of Hsp27, and enhanced activity of Mn-SOD, two potential end-effectors of delayed cardioprotection. The exercise study is the first to demonstrate delayed preconditioning in man. The ability to maintain myocardium in a protected state over several days suggests that A1R activation may hold promise as a new approach to long-term cardioprotection in patients at increased risk of myocardial infarction.
... [63][64][65] However, due to its role in cardioprotection most of the attention has been focused on the mitochondrial ATP-sensitive (ie, inhibited by physiological levels of ATP) K + channel (mitoK ATP ). [66][67][68][69][70] The maintenance of matrix volume depends on the net balance between K + uptake and release. 64,71 A K + cycle across the IMM exists whereby the electrophoretic influx driven by Δѱ m is balanced by the electroneutral K + /H + antiporter. ...
Acute myocardial infarction (AMI) and the heart failure (HF) that often result remain the leading causes of death and disability worldwide. As such, new therapeutic targets need to be discovered to protect the myocardium against acute ischaemia/reperfusion (I/R) injury in order to reduce myocardial infarct (MI) size, preserve left ventricular function and prevent the onset of HF. Mitochondrial dysfunction during acute I/R injury is a critical determinant of cell death following AMI, and therefore, ion channels in the inner mitochondrial membrane, which are known to influence cell death and survival, provide potential therapeutic targets for cardioprotection. In this article, we review the role of mitochondrial ion channels, which are known to modulate susceptibility to acute myocardial I/R injury, and we explore their potential roles as therapeutic targets for reducing MI size and preventing HF following AMI.
... These energysensing channels, present in the sarcolemma (sarcoK ATP ) and in mitochondria (mitoK ATP ) of cardiac myocytes, are important regulators of cardiac myocyte function. SarcoK ATP or mitoK ATP channel opening before an IR insult confers cardioprotection [116]. The activation of sar-coK ATP channels by a conditioning strategy can shorten the cardiac action potential duration, and then protect the myocardium. ...
Ischemic heart disease is one of the main causes of morbidity and mortality worldwide. Physical exercise is an effective lifestyle intervention to reduce the risk factors for cardiovascular disease and also to improve cardiac function and survival in patients with ischemic heart disease. Among the strategies that contribute to reduce heart damages during ischemia and reperfusion, regular physical exercise is efficient both in rodent experimental models and in humans. However, the cellular and molecular mechanisms of the cardioprotective effects of exercise remain unclear. During ischemia and reperfusion, mitochondria are crucial players in cell death, but also in cell survival. Although exercise training can influence mitochondrial function, the consequences on heart sensitivity to ischemic insults remain elusive. In this review, we describe the effects of physical activity on cardiac mitochondria and their potential key role in exercise-induced cardioprotection against ischemia-reperfusion damage. Based on recent scientific data, we discuss the role of different pathways that might help to explain why mitochondria are a key target of exercise-induced cardioprotection.
... Certainly both sarcK ATP and mitoK ATP channels are responsible for the cardio-protection, forgeting about way that several differentiations in their qualities among animal species and exploratory conditions. It has been questionable that sarc-K ATP or mitoK ATP match some regular highlights in IPC [19]. The present examination has demonstrated that sarcK ATP channel work is crucial for IPC, which is a high-heart-rate creature assortment in any way in mouse. ...
A study of potassium channels correlates the fundamentals of mechanistic pathways and various physiological functions. The knowledge of these pathways provides the background, how to determine unit cell functions and to affect cardio protection. ATP sensitive potassium channels adjust excitability of membrane and functions as per metabolic status of cell. A lot of energy consumption primarily occurred in skeletal muscles which also express high number of potassium channels. The increase in calcium release and high heat production is occurred due to loss of potassium channels. Such type of mediations determines metabolic changes and energy required in the dissipation. IPC reduces infarct size in anesthetized mice. In ischemic-reperfusion, pressure in left ventricle was watched while contractile power recovery did not happen. It was seen that elements of intact potassium channel are fundamental for Ischemic preconditioning (IPC). If more prominent is enactment of potassium channels and their cardiologic effects create high heart rate. All the more as of late, it has been suggested that mitochondrial ATP sensitive potassium channels are critical as closing stage effectors which trigger IPC as opposed to sarcolemmal potassium channels. Nevertheless, the importance of the potassium channels reconsidered in cardio-protection in present findings. These discoveries recommend that potassium channels in the adjusting ischemic-reperfusion damage in mice. The heart rate of the mouse occurred during ischemia; enhance watchful extrapolation applied to larger warm blooded animals.
... H 2 S-induced hyperpolarization of SMC membrane is also abolished by glibenclamide. The opening of K ATP channels in myocardium has been seen to play a pivotal role in cardioprotection during I/R injury, which is specifically seen in cardiac ischemic preconditioning [108]. It was observed that in the perfused rat heart preparation, NaHS concentration-dependently limited the size of infarction induced by left coronary artery ligation, and this protective effect was abolished by K ATP channel blockers glibenclamide and 5-hydroxydecanoate [106]. ...
In addition to nitric oxide and carbon monoxide, hydrogen sulfde (H2S) is the third gasotransmitter in mammals.
It is synthesized from L-cysteine by cystathionine β-synthase, cystathionine γ-lyase or by sequential action of alanine
aminotransferase and 3-mercaptopyruvate sulfur transferase. Although initially it was suggested that in the vascular
wall H
2S is synthesized only by smooth muscle cells and relaxes them by activating ATP-sensitive potassium channels,
more recent studies indicate that H
2S is synthesized in endothelial cells as well. The physiological functions of H2S
are mediated by different molecular targets, such as different ion channels and signaling proteins. Endogenous H2S is involved in the regulation of many physiological processes in the cardiovascular system including the regulation of vascular tone, blood pressure and inhibits atherogenesis. Many new technologies have been developed to detect endogenous H2S production, and novel H2S-delivery compounds have been invented to aid therapeutic intervention of diseases related to abnormal H2S metabolism. The primary purpose of this review was to provide an overview of the role of H2S in the blood vessel, methods of endogenous production detections and common ion channels used to produce its biological effect describe its benefcial effects.
... These include, but not limited to, the following: firstly, opening of K ATP channels-this channel opens as ATP levels fall and is inhibited when levels are high. The K ATP channel, which exists in both mitochondria and sarcolemmal membrane has been recognized by Gross et al. [122] to be a likely end effector of ischaemic preconditioning, and earlier work [123] suggests that the sarcolemmal channel surface might be an important effector of the cardioprotective effects of ischemic/hypoxic preconditioning. Secondly, restrict mitochondria permeability transition pore (MPTP) from opening-this pore has been identified by Bains et al. [124] and others [125] as an effector of preconditioning. ...
Heart failure (HF) is a physiological state in which cardiac output is insufficient to meet the needs of the body. It is a clinical syndrome characterized by impaired ability of the left ventricle to either fill or eject blood efficiently. HF is a disease of multiple aetiologies leading to progressive cardiac dysfunction and it is the leading cause of deaths in both developed and developing countries. HF is responsible for about 73,000 deaths in the UK each year. In the USA, HF affects 5.8 million people and 550,000 new cases are diagnosed annually. Cardiac remodelling (CD), which plays an important role in pathogenesis of HF, is viewed as stress response to an index event such as myocardial ischaemia or imposition of mechanical load leading to a series of structural and functional changes in the viable myocardium. Protein kinase C (PKC) isozymes are a family of serine/threonine kinases. PKC is a central enzyme in the regulation of growth, hypertrophy, and mediators of signal transduction pathways. In response to circulating hormones, activation of PKC triggers a multitude of intracellular events influencing multiple physiological processes in the heart, including heart rate, contraction, and relaxation. Recent research implicates PKC activation in the pathophysiology of a number of cardiovascular disease states. Few reports are available that examine PKC in normal and diseased human hearts. This review describes the structure, functions, and distribution of PKCs in the healthy and diseased heart with emphasis on the human heart and, also importantly, their regulation in heart failure.
... Initial observations suggested a role for surface K ATP channels in IPC [108][109][110], with depressed contractility thought to be the mechanism of cardioprotection [107,111,112]. However, the discovery that K ATP activators [diazoxide (DZX) [113], cromakalim [114], and aprikalim [115]] were capable of protecting noncontracting myocytes, in a manner blocked by K ATP inhibitors [5-hydroxydecanoate (5-HD), glyburide, and HMR1098 [116][117][118]], suggested a protective mechanism independent of depressed contractility. The discovery of a mitochondrial K ATP channel with sensitivity to DZX and cromakalim [79,83,119] provided a candidate mechanism, with further support provided by evidence that cardiac surface K ATP channels are insensitive to DZX [120][121][122][123]. Subsequently, a channel with pharmacologic properties ascribed to mitoK ATP was recognized as a key player in IPC signaling, despite an ongoing debate regarding the molecular identity of this channel. ...
Mitochondria play an important role in tissue ischemia and reperfusion (IR) injury, with energetic failure and the opening of the mitochondrial permeability transition pore being the major causes of IR-induced cell death.Thus, mitochondria are an appropriate focus for strategies to protect against IR injury.Two widely studied paradigms of IR protection, particularly in the field of cardiac IR, are ischemic preconditioning (IPC) and volatile anesthetic preconditioning (APC).While the molecular mechanisms recruited by these protective paradigms are not fully elucidated, a commonality is the involvement of mitochondrial K+ channel opening.In the case of IPC, research has focused on a mitochondrial ATP-sensitive K+ channel (mitoKATP), but, despite recent progress, the molecular identity of this channel remains a subject of contention.In the case of APC, early research suggested the existence of a mitochondrial large-conductance K+ (BK, big conductance of potassium) channel encoded by the Kcnma1 gene, although more recent work has shown that the channel that underlies APC is in fact encoded by Kcnt2.In this review, we discuss both the pharmacologic and genetic evidence for the existence and identity of mitochondrial K+ channels, and the role of these channels both in IR protection and in regulating normal mitochondrial function.
... Pharmacological agents that selectively open the K ATP channels have been demonstrated to have infarct-limiting effects as potent as IPC. However, there is still a controversy as to whether the sarcK ATP or mitoK ATP channels play a more important role in the mechanisms of cardioprotection (57). ...
Hydrogen sulfide (H2S) was identified as the third gasotransmitter in 1996 following the discoveries of the biological importance of nitric oxide and carbon monoxide. Although H2S has long been considered a highly toxic gas, the discovery of its presence and enzymatic production in mammalian tissues supports a critical role for this physiological signaling molecule. H2S is synthesized endogenously by three enzymes: cystathionine β-synthase, cystathionine-γ-lyase, and 3-mercaptopyruvate sulfurtransferase. H2S plays a pivotal role in the regulation of cardiovascular function as H2S has been shown to modulate: vasodilation, angiogenesis, inflammation, oxidative stress, and apoptosis. Perturbation of endogenous production of H2S has been associated with many pathological conditions of the cardiovascular system such as diabetes, heart failure, and hypertension. As such, modulation of the endogenous H2S signaling pathway or administration of exogenous H2S has been shown to be cytoprotective. This review article will provide a summary of the current body of evidence on the role of H2S signaling in the setting of myocardial ischemia and heart failure.
... Current studies have shown that sarcolemmal K + ATP channels are a potential mechanism in the protection against I/R resulting from exercise. Thus, the opening of sarcolemmal K + ATP channels accelerates cardiomyocyte depolarization through increasing the rate of K + exit and decreasing the action potential duration (36). Considering the value of this study, the duration of the training period (16 weeks) and the performance of regular exercise can justify these mentioned adaptations. ...
Background: Cardiovascular diseases are among the major causes of mortality in industrialized countries. Prevention of cardio- vascular diseases and increasing stress tolerance are two of the main goals of physical training.
Objectives: This study was designed to compare the effects of two exercise programs of different intensities on rat hearts with isoproterenol-induced myocardial injuries.
Methods: Animals were randomly divided into four groups (n = 8 per group): control group (CTL); ISO group, administered iso- proterenol (85 mg/kg subcutaneously) for two consecutive days; low-intensity interval training + isoproterenol group (LIIT+ ISO: 5 minutes warm up at 40% VO2 max,5 x 10 minutes at50% -60% VO2 max [about 20-24 m/min]); and high-intensity interval training + isoproterenol group (HIIT+ ISO: 5 minutes warm-up at 40% VO2 max, 5 x 5 min at 95% -105% VO2 max [45 -50 m/min]). The train- ing groups performed high- and low-intensity interval training programs (5 days/week) on a motor-driven treadmill for 16 weeks. Seventy-two hours after the last training session, isoproterenol (85 mg/kg) was injected on two consecutive days. On the third day, hemodynamic parameters were recorded, blood samples were taken, and hearts were removed for laboratory analysis.
Results: ISO-induced heart injury raised cardiac troponin I levels, significantly decreased + dp/dt max(P< 0.05) and-dp/dt max(P < 0.05), and significantly increased serum CTnI and tissue TNF α levels (P < 0.05). Exercise training had no significant effects on HR, LVSP, and LVEDP. Impairments of + dp/dt max and-dp/dt max were significantly improved in the HIIT+ ISO and LIIT+ISO groups (P < 0.05 for both groups versus ISO). In addition, exercise training groups especially HIIT + ISO to some reduce exacerbated the myocardial lesions induced by ISO (P < 0.05).
Conclusions: These biochemical and histopathological findings suggest there is a protective role provided by both high- and low- intensity interval training protocols on ischemic hearts.
... Binding of fentanyl to the μ-opioid receptor induces activation of heterotrimeric G-protein complex, leading to activation of protein kinase C (PKC). Activation of PKC enhances opening of the mitoK ATP channels (as proposed also by others 86,87 ) and reduces mitochondrial respiration rates, an effect that is prevented by preincubation with naloxone or 5-hydroxydecanoate (5-HD). Reduced cellular respiration leads to a decrease in cellular adenosine triphosphate (ATP) content. ...
Background:
Remifentanil interferes with hepatic mitochondrial function. The aim of the present study was to evaluate whether hepatic mitochondrial function is affected by fentanyl, a more widely used opioid than remifentanil.
Methods:
Human hepatoma HepG2 cells were exposed to fentanyl or pretreated with naloxone (an opioid receptor antagonist) or 5-hydroxydecanoate (5-HD, an inhibitor of mitochondrial adenosine triphosphate (ATP)-sensitive potassium [mitoKATP] channels), followed by incubation with fentanyl. Mitochondrial function and metabolism were then analyzed.
Results:
Fentanyl marginally reduced maximal mitochondrial complex-specific respiration rates using exogenous substrates (decrease in medians: 11%-18%; P = 0.003-0.001) but did not affect basal cellular respiration rates (P = 0.834). The effect on stimulated respiration was prevented by preincubation with naloxone or 5-HD. Fentanyl reduced cellular ATP content in a dose-dependent manner (P < 0.001), an effect that was not significantly prevented by 5-HD and not explained by increased total ATPase concentration. However, in vitro ATPase activity of recombinant human permeability glycoprotein (an ATP-dependent drug efflux transporter) was significantly stimulated by fentanyl (P = 0.004).
Conclusions:
Our data suggest that fentanyl reduces stimulated mitochondrial respiration of cultured human hepatocytes by a mechanism that is blocked by a mitoKATP channel antagonist. Increased energy requirements for fentanyl efflux transport may offer an explanation for the substantial decrease in cellular ATP concentration.
... Similar to Brown's study (Brown et al. 2005a), no change was observed in the expression of mitochondrial type — both Kir 6.2 and SUR 2 . In spite of several clear reports which indicate the mitochondrial type of K ATP channels is an important component for cardioprotective effects of ischemic preconditioning (Bolli 2000; Gross and Fryer 1999; Murata et al. 2001), there is no agreement in the findings of published studies. Some basic experimental studies are required to be conducted to elucidate the interaction of androgen analogs, exercise and cardiac K ATP channels to clarify the underlying mechanisms involved. ...
... Similar to Brown's study (Brown et al. 2005a), no change was observed in the expression of mitochondrial typeboth Kir 6.2 and SUR 2 . In spite of several clear reports which indicate the mitochondrial type of K ATP channels is an important component for cardioprotective effects of ischemic preconditioning (Bolli 2000;Gross and Fryer 1999;Murata et al. 2001), there is no agreement in the findings of published studies. ...
ATP-sensitive potassium channels are supposed to have a substantial role in improvement of cardiac performance. This study was performed to evaluate whether nandrolone decanoate (ND) and (or) exercise training could affect the expression of cardiac K ATP channel subunits. Thirty-five male albino Wistar rats were randomly divided into 5 groups, including sedentary control (SC), sedentary vehicle (SV), sedentary ND (SND), exercise control (EC), and exercise and ND (E+ND). Exercise training was performed on a treadmill 5 times per week. ND was injected (10 mg/kg/week, i.m.) to the rats in the SND and E+ND groups. Following cardiac isolation, the expression of both sarcolemmal and mitochondrial subunits of K ATP channel was measured using Western blot method. The expression of sarcolemmal, but not mitochondrial, subunits of K ATP channel (Kir 6.2 and SUR 2) of EC group was significantly higher compared with SC group while ND administration (SND group) did not show any change in their expression. In the E+ND group, ND administration led to decrease of the over-expression of sarcolemmal Kir 6.2 and SUR 2 which was previously induced by exercise. There was no significant association between the mitochondrial expression of either Kir 6.2 or SUR 2 proteins and administration of ND or exercise. Supra-physiological dosage of ND negatively reverses the effects of exercise on the cardiac muscle expression of sarcolemmal, but not mitochondrial, K ATP channel subunits.
... Similar to Brown's study (Brown et al. 2005a), no change was observed in the expression of mitochondrial typeboth Kir 6.2 and SUR 2 . In spite of several clear reports which indicate the mitochondrial type of K ATP channels is an important component for cardioprotective effects of ischemic preconditioning (Bolli 2000;Gross and Fryer 1999;Murata et al. 2001), there is no agreement in the findings of published studies. ...
Can J Physiol Pharmacol. 2016 Mar;94(3):324-31. doi: 10.1139/cjpp-2015-0040. Epub 2015 Sep 18.
Nandrolone decanoate negatively reverses the beneficial effects of exercise on cardiac muscle via sarcolemmal, but not mitochondrial KATP channel.
Bayat G1, Javan M2, Safari F3, Khalili A4, Shokri S5, Goudarzvand M1, Salimi M2, Hajizadeh S2.
Author information
Abstract
ATP-sensitive potassium channels are supposed to have a substantial role in improvement of cardiac performance. This study was performed to evaluate whether nandrolone decanoate (ND) and (or) exercise training could affect the expression of cardiac KATP channel subunits. Thirty-five male albino Wistar rats were randomly divided into 5 groups, including sedentary control (SC), sedentary vehicle (SV), sedentary ND (SND), exercise control (EC), and exercise and ND (E+ND). Exercise training was performed on a treadmill 5 times per week. ND was injected (10 mg/kg/week, i.m.) to the rats in the SND and E+ND groups. Following cardiac isolation, the expression of both sarcolemmal and mitochondrial subunits of KATP channel was measured using Western blot method. The expression of sarcolemmal, but not mitochondrial, subunits of KATP channel (Kir6.2 and SUR2) of EC group was significantly higher compared with SC group while ND administration (SND group) did not show any change in their expression. In the E+ND group, ND administration led to decrease of the over-expression of sarcolemmal Kir6.2 and SUR2 which was previously induced by exercise. There was no significant association between the mitochondrial expression of either Kir6.2 or SUR2 proteins and administration of ND or exercise. Supra-physiological dosage of ND negatively reverses the effects of exercise on the cardiac muscle expression of sarcolemmal, but not mitochondrial, KATP channel subunits.
KEYWORDS:
KATP channel; Kir6.2; SUR2; canal KATP; décanoate de nandrolone; entraînement physique; exercise; mitochondria; mitochondrie; nandrolone decanoate; sarcolemma; sarcolemme
PMID: 26909616 [PubMed - in process]
... Bunların sonucu olarak da iskemi süresince ve olasılıkla da erken reperfüzyonda aşırı Ca ++ yükü engellenir ve eş zamanlı olarak hücre yaşamsal aktivitesinde artma gözlenebilir. MitoK ATP kanallarının açılması ile de membran depolarizasyonu, mitokondriyal matrikste şişme, solunum fonksiyonlarında artış ve aşırı Ca ++ yükünün düşmesi izlenir 28 . ...
ÖZET
Önce 1986’da Murray ve ark tarafından, “iskemik ön
koşullanma”, sonrasında da 2003’te Vinten-Johansen
grubu tarafından “sonradan koşullanma” tanımlanmış ve
bu güçlü endojen mekanizmaların dokuyu iskemi-reperfüzyon
hasarına karşı koruyucu etkileri, bugüne dek
birçok organda, birçok çalışma ile gösterilmiştir.
Her iki fenomenle ilgili birçok mekanizma ileri sürülse de
iskemik ön koşullanma yoluyla olan adaptasyonun; adenozin
etkisi ile oluşan, potasyum-ATP (KATP) kanal aktivasyonu
tarafından oluşturulduğu bildirilmiştir. Sonradan
koşullanmanın ise; ekstraselüler sinyal regüle kaskad ½,
fosfatidil inozitol 3-kinaz, guanilil siklaz ile mitokondriyal
potasyum-ATP kanallarının aktivasyonuna ve nitrik oksit
üretimine bağlı olduğu ileri sürülmüştür.
İÖ ve SK’nın koruyucu mekanizmaları henüz kesin olarak
bilinmemekle birlikte, potasyum-ATP kanallarının bu
mekanizmalar içinde çok önemli bir yer tuttuğu dikkati
çekmektedir.
Anahtar Kelimeler: İskemi; reperfüzyon; iskemik ön
koşullanma; sonradan koşullanma; KATP kanalları
ABSTRACT
Potassium-ATP channels (mitochondrial and
sarcolemmal): One of the mechanisms of ischemic
preconditioning and postconditioning
Firstly in 1986 “ischemic preconditioning” was determined
by Murray et al, then in 2003 “postconditioning” by the
group of Vinten-Johansen and the preventive effects on
the tissue of these strong endogeneous mechanisms
against ischemia-reperfusion has been shown with lots
of study on many organs up to date.
Although lots of mechanisms were propounded for both
phenomenon, the adaptation via ischemic preconditioning
was created by potassium-ATP channels which was
occurred by the effect of adenosine was expressed. In
the aspect of ischemic postconditioning; it is asserted
that, it depends on the activation of extracellular signalregulated
kinase cascade ½, phosphatidyl inositol 3-
kinase, guanylyl cyclase and mitochondrial potassium-
ATP channels and production of nitric oxide.
In spite of the protective mechanisms of ischemic
preconditioning and postconditioning were not clearly
known until now, considerable importance of potassium-
ATP channels among those mechanisms is remarkable.
Key Words: Ischemia; reperfusion; ischemic preconditioning;
postconditioning; K-ATP channels
... adenosine, bradykinin, noradrenaline and endorphins acting via their receptors) or intervening reperfusion (reactive oxygen species) have been strongly implicated (Vanden Hoek et al. 1998;Baines et al. 1999;Cohen et al. 2001). It has also been proposed that sulphonylureasensitive ATP-sensitive potassium (K ATP ) channels may be involved since K ATP channel openers and blockers can mimic and block IPC, respectively (Gross & Fryer, 1999;Szewczyk & Marban, 1999;D'Hahan et al. 1999;Ghosh et al. 2000). More recent pharmacological evidence has led to the proposal that it is K ATP channels in the mitochondrial inner membrane (mitoK ATP channels) rather than those in the plasma membrane that are important for this effect. ...
... Similar to Brown's study (Brown et al. 2005a), no change was observed in the expression of mitochondrial typeboth Kir 6.2 and SUR 2 . In spite of several clear reports which indicate the mitochondrial type of K ATP channels is an important component for cardioprotective effects of ischemic preconditioning (Bolli 2000;Gross and Fryer 1999;Murata et al. 2001), there is no agreement in the findings of published studies. ...
ATP-sensitive potassium channels are supposed to have a substantial role in improvement of cardiac performance. This study was performed to evaluate whether nandrolone decanoate (ND) and (or) exercise training could affect the expression of cardiac KATP channel subunits. Thirty-five male albino Wistar rats were randomly divided into 5 groups, including sedentary control (SC), sedentary vehicle (SV), sedentary ND (SND), exercise control (EC), and exercise and ND (E+ND). Exercise training was performed on a treadmill 5 times per week. ND was injected (10 mg/kg/week, i.m.) to the rats in the SND and E+ND groups. Following cardiac isolation, the expression of both sarcolemmal and mitochondrial subunits of KATP channel was measured using Western blot method. The expression of sarcolemmal, but not mitochondrial, subunits of KATP channel (Kir6.2 and SUR2) of EC group was significantly higher compared with SC group while ND administration (SND group) did not show any change in their expression. In the E+ND group, ND administration led to decrease of the over-expression of sarcolemmal Kir6.2 and SUR2 which was previously induced by exercise. There was no significant association between the mitochondrial expression of either Kir6.2 or SUR2 proteins and administration of ND or exercise. Supra-physiological dosage of ND negatively reverses the effects of exercise on the cardiac muscle expression of sarcolemmal, but not mitochondrial, KATP channel subunits. © 2015, National Research Council of Canada. All Right Reserved.
The heart is an organ that requires a significant energy input for its contractile function. Energy reserves being low, it is dependent on blood supply. Myocardial ischemia results from an imbalance between intake and myocardial oxygen requirements.
This chapter describes the molecular and cellular mechanisms that enable the heart of some fish species to maintain function during acute changes in physiological and environmental conditions, as well as to remodel in response to chronic stressors. Specifically, we cover hypoxic preconditioning in rainbow trout, as well as the remodeling response of the heart from a number of fish species to hypoxia, anoxia, and thermal acclimation. This remodeling response represents a highly integrated and regulated process across multiple levels of biological organization. In addition, we examine the capacity of the zebrafish heart to regenerate following cardiac injury, the mechanisms involved in this response, and how this ability differs from that seen in mammalian species.
Diabetic patients present high co-morbidities of neuropathy and severer consequences of coronary heart disease. But the pathological mechanism is still unclear. Here we investigated a potential association of diabetic impairment of sensory nerves with increase of vulnerability of myocardium in acute myocardial ischemia/reperfusion. A rat model of diabetes mellitus was induced by high fat and sugar diet plus a small dose of streptozotocin. Impairment of sensory nerves was evaluated by measurement of changes in tail flick latency to noxious thermal stimulation and calcitonin gene-related peptide (CGRP) and substance P (SP) in the dorsal root ganglia (DRG) and the myocardium of the heart were examined. The myocardial injury was examined by infarct size, apoptosis ratio of cardiomyocytes and cardiac troponin I in the animals underwent acute myocardial ischemia (for 30min) and reperfusion (for 120min). The effects of CGRP and SP on cardiomyocyte injury induced by high glucose and hypoxia/reoxygenation were tested in cultured myocytes. The diabetic animals presented significant elevation of noxious thermal threshold with obvious reduction of the contents of CGRP and SP in the DRG and the myocardium. Importantly, the diabetic animals showed significant increases of infarct size, myocyte apoptosis and serum cardiac troponin I after acute myocardial ischemia/reperfusion, compared to the non-diabetic control. Furthermore, exogenously administered CGRP and SP attenuated the myocyte injury induced by the high concentration of glucose and hypoxia/reoxygenation. These findings suggested that impairment of sensory nerves with significant reduction of CGRP and SP in DRG, ventricular myocardium and serum may be associated with increase of myocardial vulnerability in acute myocardial ischemia/reperfusion in streptozotocin-induced diabetic rats.
The authors’ analysis of literature sources provided evidence that the main candidates for the role of end effector of ischemic preconditioning of the heart are: MPT pores (mitochondrial permeability transition pores), nexuses, and the cytoskeleton. Almost all known intracellular signal cascades ultimately converge on MPT pores, cytoskeletal components, and nexuses.
Sulfur-containing gaseous signal molecules including hydrogen sulphide and sulfur dioxide were previously recognized as toxic gases. However, extensive studies have revealed that they can be generated in the cardiovascular system via a sulfur-containing amino acid metabolic pathway, and have an important role in cardiovascular physiology and pathophysiology. Ion channels are pore-forming membrane proteins present in the membrane of all biological cells; their functions include the establishment of a resting membrane potential and the control of action potentials and other electrical signals by conducting ions across the cell membrane. Evidence has now accumulated suggesting that the sulfur-containing gaseous signal molecules are important regulators of ion channels and transporters. The aims of this review are (1) to discuss the recent experimental evidences in the cardiovascular system regarding the regulatory effects of sulfur-containing gaseous signal molecules on a variety of ion channels, including ATP-sensitive potassium, calcium-activated potassium, voltage-gated potassium, L- and T-type calcium, transient receptor potential and chloride and sodium channels, and (2) to understand how the gaseous signal molecules affect ion channels and cardiovascular diseases.
Linked articles:
This article is part of a themed section on Spotlight on Small Molecules in Cardiovascular Diseases. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.8/issuetoc.
Introduction:
Nicorandil, a vasodilatory drug used to treat angina, was reported to protect against myocardial ischemia-reperfusion injury in various animal models. However, its cardioprotective action following cardiac arrest is unknown. We examined the cardioprotective effects of nicorandil in a porcine model of cardiac arrest and resuscitation.
Methods:
Ventricular fibrillation was induced electrically for 4min in anesthetized domestic swine, followed by cardiopulmonary resuscitation. Sixteen successfully resuscitated animals were randomized to saline control (n=8) or nicorandil (n=8) groups. Nicorandil (150μg/kg) was administered by central intravenous injection at onset of restoration of spontaneous circulation (ROSC), followed by 3μg/kg/min infusion until reperfusion end. Sham-operated animals received surgery only (n=4). Hemodynamic parameters were monitored continuously. Blood samples were taken at baseline, 5, 30, 180, and 360min after ROSC. Left ventricular ejection fraction was assessed by echocardiography at baseline and 6h after ROSC. The animals were euthanized 6h after ROSC, and the cardiac tissue was removed for analysis.
Results:
6 h after ROSC, nicorandil had significantly improved all hemodynamic variables (all P<0.05) except the maximum rate of left ventricular pressure decline and heart rate (P>0.05) compared with the control group. Control animals showed elevated cardiac troponin I and lactate levels compared with sham animals, which were significantly decreased following nicorandil treatment (P<0.05). In the saline control group, the adenosine triphosphate (ATP) content was largely reduced but subsequently rescued by nicorandil (P<0.05). Histopathologic injury was reduced with nicorandil treatment. Nicorandil reduced cardiomyocyte apoptosis as evidenced by reduced terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, decreased Bax and caspase-3 expression, and increased Bcl-2 expression in the myocardium (all P<0.05).
Conclusion:
Nicorandil exhibited cardioprotective effects on myocardial injury following cardiac arrest via improvement in post-resuscitation myocardial dysfunction and energy metabolism, reduction in myocardial histopathologic injury, and antiapoptotic effects.
ATP-sensitive potassium (KATP) channels are known as the potassium-conducting channels coupling cellular metabolic status to membrane electrical activity. Either an increase in ADP or decrease in ATP levels opens KATP channels and hyperpolarizes the membrane potential. Knocking out the inward rectifier K+ channel (Kir6.2) subunit of the KATP channels or pharmacologically blocking KATP channels increases brain injury. Overexpression of the Kir6.2 subunit or pharmacologically opening KATP channel reduces neuronal injury from ischemic insults. Hypoxic preconditioning (HPC) provides neuroprotection against subsequent ischemic brain injury. Similar to its effects in heart, KATP channels contribute to the hypoxic preconditioning-induced neuroprotection. KATP channels may therefore serve as therapeutic targets in ischemic or hypoxic-ischemic brain injury.
Our previous study has indicated that diazoxide protected myocardium against ischemia-reperfusion injury. This study tests the hypothesis that the maintenance of mitochondrial membrane potential (ΔΨm) in myocytes is responsible for cell protection against ischemia. This was specifically tested in myocytes after activation of the mitoKATP channel. Myocyte damage by 3 hrs anoxia and 2 hrs reoxygenation (A-R) was evaluated by cell viability, membrane permeability and apoptosis. Mitochondrial function was indicated by the concentration of ATP. Mitochondrial morphology was observed by staining myocytes with Mito Tracker Orange CMTMRos and by electron microscopy. Immunostaining was used to determine the distribution of cytochrome c. ΔΨm was assayed by staining with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine iodide (JC-1) and observed by con-focal microscopy. Results show that 1) An extensive damage was observed in cultured myocytes as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis and decreased ATP concentration after A-R. 2) Mitochondria in A-R myocytes were swollen and exhibited a collapsed ΔΨm. Cytochrome c was released from mitochondria into the cytosol. 3) Diazoxide (100μmol/L) significantly prevented myocyte and mitochondrial damage, cytochrome c loss, and stabilized ΔΨm. 4) This protection was blocked by 5-hydroxydecanoate (5-HD, 500μmol/L), a mitoKATP channel selective inhibitor but not by HMR-1098 (30μmol/L), a putative sarcolemmal KATP channel selective inhibitor. 5) Diazoxide reduced ΔΨm in normal cultured myocytes in a concentration- and time-independent pattern. It is concluded that activation of mitoKATP channel with diazoxide prevented disruption of ΔΨm resulting in protection against A-R induced injury.
Myocardial protection by ischemic preconditioning is effective in experimental studies, and ischemic preconditioning can also prevent cellular damage in many tissues and organs. This has encouraged investigators in various fields to study ischemic preconditioning intensively. In search of the essential cardioprotective factors, they have begun to clarify the major events during brief periods of ischemia. Ca2+ overload, free radicals, catecholamines, cytokines, and hormones have been proposed as candidate causes of ischemic damage but have also been identified as triggers for ischemic preconditioningderived cardioprotection. Ischemic preconditioning leads to the activation of intracellular messengers, including nitric oxide (NO) and KATP channels, and other enzymes to produce a cardioprotective effect. These two agents have essentially different properties. However, they appear to use the analogous pathways to reduce the severity of both myocardial infarction and myocardial dysfunction such as stunning, hibernating myocardium, and remodeling. Because preconditioning ischemia must precede lethal ischemia for these effects to occur, the underlying mechanisms such as NO and the opening of KATP channel should be effectively applied to strategies for protection after ischemic insults. Here, we summarize previous and current investigations related to the interaction of NO and KATp channels, especially in cardioprotection including controversial issues, and discuss the future directions of investigation, including some successfully Proceeding clinical trials.
Le myocarde est un organe qui requiert un apport énergétique important pour assurer sa fonction contractile. Ses réserves énergétiques étant faibles, il est dépendant des apports sanguins. L’ischémie myocardique résulte d’un déséquilibre entre les apports et les besoins en oxygène du myocarde.
Preconditioning refers to a phenomenon whereby tissues are rendered resistant to the deleterious effects of prolonged ischemia and reperfusion (I/R) by prior exposure to oxidants, brief periods of vascular occlusion, endotoxin derivatives, heat shock, a variety of pharmacologic agents (chemical or pharmacologic preconditioning) or chronic ethanol consumption (6, 11, 16, 26, 29, 35, 40, 42, 48, 55, 71). Of these perturbations, our best understanding of the mechanisms involved in conferring protection is related to ischemic preconditioning (IPC). The beneficial effects of IPC are not related to improved collateral flow but instead represent an enhanced intrinsic ability of tissues to tolerate ischemia. However, recent work indicates that in addition to its anti-ischemic effects, IPC also completely prevents the reperfusion component of tissue injury by abrogating the inflammatory changes that are initiated in the microcirculation upon restitution of blood flow (1, 7, 14, 29, 36, 40, 41, 75). Additional support for this concept is provided by the demonstration that endothelium-dependent vasoregulatory mechanisms remain intact in preconditioned tissues (42, 71). Taken together, these observations indicate that adaptive changes in the microvasculature play a key role in the development of the preconditioned state. Thus, understanding the mechanisms whereby both arterial and venular endothelium enter a protective or defensive phenotype as a result of preconditioning may lead to the development of novel therapeutic strategies to manage I/R injury.
Neurons are strongly dependent on continuous substrate delivery; accordingly physiologic resistance of neuronal tissues against ischemia is very low. Even short periods of cerebral ischemia can, therefore, cause severe neuronal damage. Certain surgical procedures (for example, during cardiovascular- or neuro-surgery) may involve periods of decreased or diminished cerebral perfusion, putting the patient at risk for neuronal damage and subsequent neurological deficit. Physicians caring for these patients will try to prevent or reduce damage using different neuroprotective strategies (aimed mainly at the reduction of cerebral metabolism). In addition to this, a modulation of neuronal ischemic tolerance (‘preconditioning’) prior to respective operative interventions may add substantially to the further improvement in patient outcome.
It has been over 20 years since Hearse described the “oxygen paradox” and the “calcium paradox” in hearts undergoing ischaemia-reperfusion (I-R) [1]. The concept that it is reperfusion itself that increases and potentiates ischaemia-induced damage has progressively gained ground, and it is now one of the main factors taken into account in the treatment of various pathological states, from crush injury to transplantation [2, 3] . Even when reperfusion itself is the goal of therapy, such as during thrombolysis for infarcted myocardium, reperfusion-associated dysfunctions, which range from arrhythmias to stunning, must be considered as they significantly affect morbidity and mortality rates [4, 5]. Regardless of the cause and the modality that have provoked it, the sequence of ischaemia and reperfusion actually induces a typical inflammatory response which is not restricted to the injured tissue but frequently has a systemic recoil [6].
The activation of cardiac cell membrane ATP-sensitive potassium channels during myocardial ischemia promotes potassium efflux, reductions in action potential duration, and inhomogeneities in repolarization, thereby creating a substrate for reentrant arrhythmias. Antiarrhythmic drugs inhibit ATP-sensitive potassium channels at therapeutic concentrations. This article discusses the relationship between ischemically induced alterations in extracellular potassium and arrhythmia formation and evaluates the antiarrhythmic potential of ATP-sensitive potassium channel antagonists, drugs that may act selectively on the ischemic myocardium.
Keywords:
potassium channels;
antiarrhythmic drugs;
ischemic myocardium;
arrhythmia;
extracellular potassium formation
Antiarrhythmic agents play an important role in the termination and suppression of both atrial and ventricular arrhythmias as primary or adjunctive therapy. The use of hybrid treatment, combining drugs with radiofrequency ablation or ICD implantation, is expected to rise as the number of patients with complex arrhythmias continues to increase (1,2). In this evolving management scenario, selection of an effective yet safe pharmacologic agent is challenging. The challenge arises from factors intrinsic to the patient, disease condition or the drug itself. These factors primarily include variability in the pathophysiologic substrate, diverse arrhythmia mechanisms, multiple clinical presentation with differing prognostic implications, along with variability in drug disposition and/or response in a highly heterogeneous patient population. Moreover, the availability of multiple therapeutic options and the narrow therapeutic index with limited ability to determine satisfactory endpoints further emphasize the need for better understanding of interactions between drug, end target, and disease condition.
ATP-sensitive K+ (KATP) channels are recognized by their biophysical fingerprint, unique heteromultimeric structure, and distinct nucleotide-dependent regulation (Noma, 1983; Aguilar-Bryan and Bryan, 1999; Seino, 1999). These weakly inwardly rectifying, high-conductance, potassium-selective channels are kept closed by intracellular ATP and activated by intracellular ADP. Thereby, KATP channels set the membrane potential according to changes in the cellular metabolic state (Weiss and Venkatesh, 1993; O’Rourke et al., 1994; Dzeja and Terzic, 1998). KATP channels are distributed in the plasmalemma of various metabolically active tissues, including the heart (Noma, 1983), pancreatic β-cells (Ashcroft, 1996), skeletal (Vivaudou et al., 1991) and smooth (Quayle et al., 1997) muscle, and the brain (Spanswick et al., 1997). A related channel has been recognized in the inner membrane of mitochondria (Inoue et al., 1991; Paucek et al., 1992), underscoring the role of KATP channels in signaling networks that transduce intracellular metabolic events.
Through gene duplication and natural selection, the ancestral cytochrome P450 gene evolved to perform many essential functions in higher organisms. The role of P450 enzymes in metabolizing steroids, hormones, and xenobiotic chemicals is well established. More recently, members of several P450 subfamilies were found to be expressed in cardiovascular tissues and active in the metabolism endogenous fatty acids to oxidized lipids. These fatty acids, such as arachidonic acid, are released from cell membranes by phospholipases and mono-oxidized to epoxides called epoxyeicosatrienoic acids (EETs) or hydroxyls called hydroxyeicosatetraenoic acids (HETEs) by CYP2 and CYP4 family members. After generation of prostaglandin H2 by cyclooxygenases, CYP5 and CYP8 enzymes perform isomerase reactions to act as terminal synthases for thromboxane and prostacyclin production. The biologically active eicosanoids and related compounds play important roles in cardiovascular homeostasis and pathology. This chapter discusses the reactions, regulation, localization, and physiological consequences of this extrahepatic cytochrome P450 expression and activity.
Ischemic conditioning is an intrinsic protective mechanism in which repeated short episodes of reversible ischemia protects the tissue and increases its tolerance against a subsequent longer period of ischemia (index ischemia). Bradykinin is a physiologically and pharmacologically active peptide of the kallikrein-kinin system. Besides the involvement of bradykinin in a variety of physiological and pathological responses such as pain, inflammation and in cardiovascular system as a potent vasodilator, it also acts as an endogenous cytoprotective mediator in the ischemic tissue. Pretreatment with various pharmacological modulators of bradykinin has confirmed the involvement of bradykinin in ischemic conditioning-induced protection. The protective actions of bradykinin in three major paradigms of ischemic conditioning i.e ischemic preconditioning, ischemic postconditioning and remote ischemic preconditioning involves activation and regulation of various endogenous signaling cascades to render the heart resistant to infarction. In ischemic preconditioning, bradykinin exerts cardioprotective effect via activation of PI3K/Akt/eNOS signaling pathway and regulation of redox state via NO release. The role of bradykinin and its B2 receptors in ischemic-postconditioning induced neuroprotection has been described mainly due to its increased redox sigaling cascade and activation of mitochondrial anti-apoptotic pathway. Furthermore, its cardioprotective role during remote ischemic preconditioning has been associated with activation of B2 receptors mediated neurogenic pathway and internalization of B2 receptors along with the formation of signalosomes that activates intracellular cytoprotective transduction pathways. The present review focuses on the potential role of bradykinin in mediating different forms of ischemic conditioning (pre/post/remote)-induced cardioprotection and neuroprotection along with the possible mechanisms.
In experiments on the anaesthetized dogs the influence of a new fluorine-containing opener of ATP-sensitive potassium (K(ATP)) channels flocalin on the cardiohemodynamic of great animals in vivo was studied. Flocalin introduced intravenously in doses 0.01 - 1.5 mgs/kg. It is shown that it reduces in dose-dependent manner a system arterial pressure, perfusion pressure in coronary artery and general peripheral resistance of vessels with maximal effects on 56.8 +/- 2.7, 22.4 +/- 4.7 and 47.2% +/- 6.5% accordingly at most dose 1.5 mgs/kg. Flocalin causes development of cardiodepressive reactions in heart, that is exhibited in dose-dependent the decrease of pressure in the left ventricle, speed of growth (dP/dt(max)) and reduction (dP/dt(min)) in it's of pressure with maximal effects on 37.1 +/- 5.1, 51.2 +/- 9.4 and 55.6% +/- 6.9% accordingly at introduction of most dose of flocalin. Diminish of the cardiac out put and heart rate with a maximal effects on 23.1% +/-12.7% and 19.2% +/- 1.7% accordingly at a dose 1.0 mgs/kg was shown. It should be noted that considerable reduction of heart rate and general peripheral resistance of vessels takes place only at the large doses of flocalin - 1 and 1.5 mgs/kg. Thus, it is shown that activation of K(ATP) channels by flocalin causes the dose-dependent decrease of pressure in the system of circulation of blood and contraction activity of myocardium.
Theinfarct-limiting effect ofischemic preconditioning isbelieved tobe a transient phenomenon. We examined thedelayed effects ofrepetitive brief ischemia on limiting infarct sizeinan open-chest dog modelbyan occlusion (90minutes) oftheleft anterior descending coronaryartery (LAD)followed by reperfusion (5hours). Thedogswere preconditioned withfourbrief repeated ischemic episodes induced by5-minute LAD occlusions withsubsequent reperfusion. Thesizeofinfarcts initiated bya sustained occlusion immediately or 24hoursafter preconditioning was significantly smaller whencompared with infarcts insham-operated dogs(for theimmediate occlusion, 14.4±2.0o versus39.0+3.7%, respectively (p
The transport properties of mitochondria are such that net potassium flux across the inner membrane determines mitochondrial volume. It has been known that K+ uptake is mediated by diffusive leak driven by the high electrical membrane potential maintained by redox-driven, electrogenic proton ejection and that regulated K+ efflux is mediated by an 82-kDa inner membrane K+/H+ antiporter. There is also long-standing suggestive evidence for the existence of an inner membrane protein designed to catalyze electrophoretic K+ uptake into mitochondria. We report reconstitution of a highly purified inner membrane protein fraction from rat liver and beef heart mitochondria that catalyzes electrophoretic K+ flux in liposomes and channel activity in planar lipid bilayers. The unit conductance of the channel at saturating [K+] is about 30 pS. Reconstituted K+ flux is inhibited with high affinity by ATP and ADP in the presence of divalent cations and by glibenclamide in the absence of divalent cations. The mitochondrial ATP-dependent K+ channel is selective for K+, with a Km of 32 mM, and does not transport Na+. K+ transport depends on voltage in a manner consistent with a channel activity that is not voltage-regulated. Thus, the mitochondrial ATP-dependent K+ channel exhibits properties that are remarkably similar to those of the ATP-dependent K+ channels of plasma membranes.
The biochemical properties of the mitochondrial K channel are very similar to those of plasma membrane K channels, including inhibition by low concentrations of ATP and glyburide (Paucek, P., Mironova, G., Mahdi, F., Beavis, A.
D., Woldegiorgis, G., and Garlid, K. D.(1992) J. Biol. Chem. 267, 26062-26069). Plasma membrane K channels are highly sensitive to the family of drugs known as K channel openers, raising the question whether mitochondrial K channels are similarly sensitive to these agents. We addressed this question by measuring K flux in intact rat liver mitochondria and in liposomes containing K channels purified from rat liver and beef heart mitochondria. K channel openers completely reversed ATP inhibition of K flux in both systems. In liposomes, ATP-inhibited K flux was restored by diazoxide (K = 0.4 μM), cromakalim (K = 1 μM), and two developmental cromakalim analogues, EMD60480 and EMD57970 (K = 6 nM). Similar K values were observed in intact mitochondria. These potencies are well within the range observed with plasma membrane K channels. We also compared the potencies of these K channel openers on the plasma membrane K channel purified from beef heart myocytes. The K channel from cardiac mitochondria is 2000-fold more sensitive to diazoxide than the channel from cardiac sarcolemma, indicating
that two distinct receptor subtypes coexist within the myocyte. We suggest that the mitochondrial K channel is an important intracellular receptor that should be taken into account in considering the pharmacology of K channel openers.
ATP-sensitive potassium (KATP) channels link cellular metabolism to electrical activity in nerve, muscle, and endocrine tissues. They are formed as a functional complex of two unrelated subunits-a member of the Kir inward rectifier potassium channel family, and a sulfonylurea receptor (SUR), a member of the ATP-binding cassette transporter family, which includes cystic fibrosis transmembrane conductance regulators and multidrug resistance protein, regulators of chloride channel activity. This recent discovery has brought together proteins from two very distinct superfamilies in a novel functional complex. The pancreatic KATP channel is probably formed specifically of Kir6.2 and SUR1 isoforms. The relationship between SUR1 and Kir6.2 must be determined to understand how SUR1 and Kir6.2 interact to form this unique channel. We have used mutant Kir6.2 subunits and dimeric (SUR1-Kir6.2) constructs to examine the functional stoichiometry of the KATP channel. The data indicate that the KATP channel pore is lined by four Kir6.2 subunits, and that each Kir6.2 subunit requires one SUR1 subunit to generate a functional channel in an octameric or tetradimeric structure.
Heat stress is known to confer protection against ischemia, but the mechanisms involved are yet to be elucidated. Opening of ATP-sensitive potassium (K(ATP)) channels has been demonstrated to be involved in other endogenous forms of cardioprotection, in particular 'classic' ischemic preconditioning and delayed preconditioning following treatment with the endotoxin derivative, monophosphoryl lipid A. We therefore speculated that there may be a role for K(ATP) channels in delayed heat stress-induced cardioprotection. This hypothesis was investigated in an in vivo rabbit model of acute myocardial infarction using two structurally dissimilar K channel blockers, glibenclamide and sodium 5-hydroxydecanoate. Sodium pentobarbitone-anesthetized rabbits were subjected to either transient heat stress at 42 ± 0.2°C for 15 minutes or sham anesthesia. Twenty-four hours later, animals were reanesthetized ('Hypnorm' and sodium pentobarbitone) and a midline sternotomy and pericardiotomy were performed. An anterolateral branch of the circumflex coronary artery was occluded for 30 minutes and reperfused for 2 hours. The infarct-to-risk ratio was significantly limited in vehicle-treated rabbits from 41.3 ± 4.0% in controls (n = 10) to 24.1 ± 5.0% (n = 9; P = 0.014 by one-factor ANOVA) in heat-stressed hearts. This limitation in infarct size was abolished by 0.3 mg/kg iv glibenclamide or 5 mg/kg iv 5-hydroxydecanoate when administered 10 minutes prior to coronary occlusion (45.2 ± 6.4%; n = 9 and 41.5 ± 5.0%; n = 5, respectively.) The same doses of glibenclamide and 5-hydroxydecanoate in sham-anesthetized hearts had no effect (42.3 ± 5.1%; n = 10 and 51.9 ± 2.2%; n = 6, respectively). The adequacy of the heat stress protocol was confirmed by Western blot analysis of the inducible 72-kD heat stress protein. It is concluded, therefore, that K(ATP) channels appear to play a role in the heat stress response. The underlying mechanisms involved are, however, unclear.
The mitochondrial KATP channel (mitoKATP) is hypothesized to be the receptor for the cardioprotective effects of K+ channel openers (KCO) and for the blocking of cardioprotection by glyburide and 5-hydroxydecanoate (5-HD). Studies on glyburide have indicated that this drug is inactive in isolated mitochondria. No studies of the effects of 5-HD on isolated mitochondria have been reported. This paper examines the effects of glyburide and 5-HD on K+ flux in isolated, respiring mitochondria. We show that mitoKATP is completely insensitive to glyburide and 5-HD under the experimental conditions in which the open state of the channel is induced by the absence of ATP and Mg2+. On the other hand, mitoKATP became highly sensitive to glyburide and 5-HD when the open state was induced by Mg2+, ATP, and a physiological opener, such as GTP, or a pharmacological opener, such as diazoxide. In these open states, glyburide (K1/2 values 1-6 microM) and 5-HD (K1/2 values 45-75 microM) inhibited specific, mitoKATP-mediated K+ flux in both heart and liver mitochondria from rat. These results are consistent with a role for mitoKATP in cardioprotection and show that different open states of mitoKATP, although catalyzing identical K+ fluxes, exhibit very different susceptibilities to channel inhibitors.
Reactive oxygen species (ROS) have been proposed to participate in the induction of cardiac preconditioning. However, their
source and mechanism of induction are unclear. We tested whether brief hypoxia induces preconditioning by augmenting mitochondrial
generation of ROS in chick cardiomyocytes. Cells were preconditioned with 10 min of hypoxia, followed by 1 h of simulated
ischemia and 3 h of reperfusion. Preconditioning decreased cell death from 47 ± 3% to 14 ± 2%. Return of contraction was observed
in 3/3 preconditioned versus 0/6 non-preconditioned experiments. During induction, ROS oxidation of the probe dichlorofluorescin (sensitive to H2O2) increased ∼2.5-fold. As a substitute for hypoxia, the addition of H2O2 (15 μmol/liter) during normoxia also induced preconditioning-like protection. Conversely, the ROS signal during hypoxia was
attenuated with the thiol reductant 2-mercaptopropionyl glycine, the cytosolic Cu,Zn-superoxide dismutase inhibitor diethyldithiocarbamic
acid, and the anion channel inhibitor 4,4′-diisothiocyanato-stilbene-2,2′-disulfonate, all of which also abrogated protection.
ROS generation during hypoxia was attenuated by myxothiazol, but not by diphenyleneiodonium or the nitric-oxide synthase inhibitor
l-nitroarginine. We conclude that hypoxia increases mitochondrial superoxide generation which initiates preconditioning protection.
Furthermore, mitochondrial anion channels and cytosolic dismutation to H2O2 may be important steps for oxidant induction of hypoxic preconditioning.
Discovered in the cardiac sarcolemma, ATP-sensitive K+ (KATP) channels have more recently also been identified within the inner mitochondrial membrane. Yet the consequences of mitochondrial KATP channel activation on mitochondrial function remain partially documented. Therefore, we isolated mitochondria from rat hearts and used K+ channel openers to examine the effect of mitochondrial KATP channel opening on mitochondrial membrane potential, respiration, ATP generation, Ca2+ transport, and matrix volume. From a mitochondrial membrane potential of -180 ± 15 mV, K+ channel openers, pinacidil (100 μM), cromakalim (25 μM), and levcromakalim (20 μM), induced membrane depolarization by 10 ± 7, 25 ± 9, and 24 ± 10 mV, respectively. This effect was abolished by removal of extramitochondrial K+ or application of a KATP channel blocker. K+ channel opener-induced membrane depolarization was associated with an increase in the rate of mitochondrial respiration and a decrease in the rate of mitochondrial ATP synthesis. Furthermore, treatment with a K+ channel opener released Ca2+ from mitochondria preloaded with Ca2+, an effect also dependent on extramitochondrial K+ concentration and sensitive to KATP channel blockade. In addition, K+ channel openers, cromakalim and pinacidil, increased matrix volume and released mitochondrial proteins, cytochrome c and adenylate kinase. Thus, in isolated cardiac mitochondria, KATP channel openers depolarized the membrane, accelerated respiration, slowed ATP production, released accumulated Ca2+, produced swelling, and stimulated efflux of intermembrane proteins. These observations provide direct evidence for a role of mitochondrial KATP channels in regulating functions vital for the cardiac mitochondria.
We tested 5-hydroxydecanoate (5-HD), a specific blocker of ATP-sensitive potassium channels (IK.ATP), to determine if it mitigates electrophysiologic changes produced by regional myocardial ischemiain vivo. A sequence of 5-minute occlusion of the distal LAD and 30-minute reperfusion was repeated while recording the monophasic action potential (MAP) and bipolar electrogram (EG) from the epicardial center of the ischemic myocardium in anesthetized dogs. 5-HD (30 mg/kg, IV) or glibenclamide (0.15 or 0.3 mg/kg, IV) was administered before the third occlusion, and the data were compared to the second occlusion data. 5-HD did not affect baseline MAP duration at 90% and 50% repolarization (APD90, APD50) before LAD occlusion but suppressed occlusion-induced shortening of APD90 (16 2% during the second occlusion vs. 5 3% during the third occlusion, n=8, p
The present study was performed to test whether the ischemic preconditioning could reduce mitochondrial O2.- production and prevent mitochondrial respiratory impairment upon reperfusion of ischemic hearts. The isolated perfused rat hearts were subjected to 30 min of global ischemia and 20 min of reperfusion. Ischemic preconditioning was performed, involving three 5-min periods of ischemia, each followed by a 5-min reperfusion just before a sustained ischemia. Ischemic preconditioning improved the post-ischemic cardiac function and reduced LDH release and malondialdehyde production upon reperfusion. O2.- generation of mitochondria isolated from the preconditioned hearts was significantly lower than that of mitochondria from the non-preconditioned hearts, and none of the activities of mitochondrial antioxidant enzymes(SOD, catalase, glutathione peroxidase) was altered as a consequence of the ischemic preconditioning alone. The impairment of mitochondrial state 3 respiration induced by ischemia and reperfusion was prevented by ischémie preconditioning. Amytal, a reversible respiratory chain blocker suppressing O2.- production in mitochondria, prevented the ischemia/reperfusion injury. The cardioprotective effect of Amytal could not be distinguished from that of ischemic preconditioning. These results suggest that the cardioprotective effect of ischemic preconditioning against the ischemia/reperfusion injury is attributed partly to the reduction of mitochondrial oxygen radical generation and prevention of the respiratory impairment during ischemia and reperfusion.
The direct effects of diazoxide on mitochondrial membrane potential, Ca ²⁺ transport, oxygen consumption and ATP generation were investigated in mouse pancreatic B‐cells and rat liver mitochondria.
Diazoxide, at concentrations commonly used to open adenosine 5′‐triphosphate (ATP)‐dependent K ⁺ ‐channels (K ATP channels) in pancreatic B‐cells (100 to 1000 μ M ), decreased mitochondrial membrane potential in mouse intact perifused B‐cells, as evidenced by an increase of rhodamine 123 fluorescence. This reversible decrease of membrane potential occurred at non‐stimulating (5 m M ) and stimulating (20 m M ) glucose concentrations.
A decrease of mitochondrial membrane potential in perifused B‐cells was also caused by pinacidil, but no effect could be seen with levcromakalim (500 μ M each).
Measurements by a tetraphenylphosphonium‐sensitive electrode of the membrane potential of rat isolated liver mitochondria confirmed that diazoxide decreased mitochondrial membrane potential by a direct action. Pretreatment with glibenclamide (2 μ M ) did not antagonize the effects of diazoxide.
In Fura 2‐loaded B‐cells perifused with the Ca ²⁺ channel blocker, D 600, a moderate, reversible increase of intracellular Ca ²⁺ concentration could be seen in response to 500 μ M diazoxide. This intracellular Ca ²⁺ mobilization may be due to mitochondrial Ca ²⁺ release, since the reduction of membrane potential of isolated liver mitochondria by diazoxide was accompanied by an accelerated release of Ca ²⁺ stored in the mitochondria.
In the presence of 500 μ M diazoxide, ATP content of pancreatic islets incubated in 20 m M glucose for 30 min was significantly decreased by 29%. However, insulin secretion from mouse perifused islets induced by 40 m M K ⁺ in the presence of 10 m M glucose was not inhibited by 500 μ M diazoxide, suggesting that the energy‐dependent processes of insulin secretion distal to Ca ²⁺ influx were not affected by diazoxide at this concentration.
The effects of diazoxide on oxygen consumption and ATP production of liver mitochondria varied depending on the respiratory substrates (5 m M succinate, 10 m M α‐ketoisocaproic acid, 2 m M tetramethyl phenylenediamine plus 5 m M ascorbic acid), indicating an inhibition of respiratory chain complex II. Pinacidil, but not levcromakalim, inhibited α‐ketoisocaproic acid‐fuelled ATP production.
In conclusion, diazoxide directly affects mitochondrial energy metabolism, which may be of relevance for stimulus‐secretion coupling in pancreatic B‐cells.
British Journal of Pharmacology (1998) 123 , 781–788; doi: 10.1038/sj.bjp.0701663
The aims were: (1) to determine if a new ischaemia selective ATP dependent potassium (KATP) channel antagonist, sodium 5-hydroxydecanoate (5-HD), blocks ischaemic preconditioning in dogs; (2) to determine whether a small intracoronary dose of glibenclamide, a classical sulphonylurea KATP channel antagonist, could block ischaemic preconditioning independent of systemic metabolic effects.
Barbitone anaesthetised dogs were subjected to 60 min of left circumflex coronary artery occlusion followed by 5 h of reperfusion. Preconditioning was produced by a single 5 min left circumflex occlusion followed by 10 min of reperfusion prior to the 60 min occlusion period. 5-HD (150 micrograms.kg-1 x min-1) or vehicle was given by intracoronary infusion into the ischaemic region over 20 min, beginning 15 min prior to the 60 min occlusion period in the presence or absence of preconditioning. Glibenclamide (3 micrograms.kg-1 x min-1) was given by intracoronary infusion into the left circumflex artery during the 5 min preconditioning period or during the first 5 min of occlusion in preconditioned or non-preconditioned dogs. Transmural myocardial blood flow was measured by radioactive microspheres and infarct size determined by triphenyltetrazolium staining and expressed as a percent of the area at risk.
There were no differences in haemodynamic variables, myocardial blood flow, area at risk, or blood glucose between groups. Infarct size was markedly reduced in preconditioned dogs compared to control animals, at 7(SEM 2)% v 29(4)%, p < 0.05 The reduction in infarct size by preconditioning was blocked completely by intracoronary 5-HD, or by intracoronary glibenclamide given during preconditioning or during the first 5 min of the prolonged occlusion period. Neither 5-HD nor glibenclamide affected infarct size in the absence of preconditioning at the doses studied.
These results further strengthen the hypothesis that activation of myocardial KATP channels is involved in the mechanism of ischaemic preconditioning in dogs.
Single or multiple brief periods of ischemia (preconditioning) have been shown to protect the myocardium from infarction after a subsequent more prolonged ischemic insult. To test the hypothesis that preconditioning is the result of opening ATP-sensitive potassium (KATP) channels, a selective KATP channel antagonist, glibenclamide, was administered before or immediately after preconditioning in barbital-anesthetized open-chest dogs subjected to 60 minutes of left circumflex coronary artery (LCX) occlusion followed by 5 hours of reperfusion. Preconditioning was elicited by 5 minutes of LCX occlusion followed by 10 minutes of reperfusion before the 60-minute occlusion period. Glibenclamide (0.3 mg/kg i.v.) or vehicle was given 10 minutes before the initial ischemic insult in each of four groups. In a fifth group, glibenclamide was administered immediately after preconditioning. In a final series (group 6), a selective potassium channel opener, RP 52891 (10 micrograms/kg bolus and 0.1 micrograms/mg/min i.v.) was started 10 minutes before occlusion and continued throughout reperfusion. Transmural myocardial blood flow was measured at 30 minutes of occlusion, and infarct size was determined by triphenyltetrazolium staining and expressed as a percent of the area at risk. There were no significant differences in hemodynamics, collateral blood flow, or area at risk between groups. The ratio of infarct size to area at risk in the control group (28 +/- 6%) was not different from the group pretreated with glibenclamide in the absence of preconditioning (31 +/- 6%). Preconditioning produced a marked reduction (p less than 0.002) in infarct size (28 +/- 6% to 6 +/- 2%), whereas glibenclamide administered before or immediately after preconditioning completely abolished the protective effect (28 +/- 6% and 30 +/- 8%, respectively). RP 52891 also produced a significant (p less than 0.03) reduction (28 +/- 6% to 13 +/- 3%) in infarct size. These results suggest that myocardial preconditioning in the canine heart is mediated by activation of KATP channels and that these channels may serve an endogenous myocardial protective role.
Mitochondria take up and extrude various inorganic and organic ions, as well as larger substances such as proteins. The technique of patch clamping should provide real-time information on such transport and on energy transduction in oxidative phosphorylation. It has been applied to detect microscopic currents from mitochondrial membranes and conductances of ion channels in the 5-1,000 pS range in the outer and inner membranes. These pores are not, however, selective for particular ions. Here we use fused giant mitoplasts prepared from rat liver mitochondria to identify a small conductance channel highly selective for K+ in the inner mitochondrial membrane. This channel can be reversibly inactivated by ATP applied to the matrix side under inside-out patch configuration; it is also inhibited by 4-aminopyridine and by glybenclamide. The slope conductance of the unitary currents measured at negative membrane potentials was 9.7 +/- 1.0 pS (mean +/- s.d., n = 6) when the pipette solution contained 100 mM K+ and the bathing solution 33.3 mM K+. Our results indicate that mitochondria depolarize by generating a K+ conductance when ATP in the matrix is deficient.
The role of ATP-regulated K+ channels in protecting the myocardium against ischemia/reperfusion damage was explored using glibenclamide and pinacidil to block and activate the channels, respectively. Electrical and mechanical activity of arterially perfused guinea pig right ventricular walls was recorded simultaneously via an intracellular microelectrode and a force transducer. The preparations were subjected to either 1) 20 minutes of no-flow ischemia with or without glibenclamide (1 and 10 microM) followed by reperfusion, or 2) 30 minutes of no-flow ischemia with or without pinacidil (1 and 10 microM) followed by reperfusion. No-flow ischemia for 20 minutes produced changes in electrical and mechanical activity that were completely reversed on reperfusion; resting membrane potential declined by 13 +/- 1.2 mV, action potential duration at 90% repolarization (APD90) decreased by 62%, and developed tension fell by greater than 95%, but resting tension did not change significantly. Glibenclamide (10 microM) had no effect on activity during normal perfusion, but during ischemia, resting membrane potential fell slightly further (17 +/- 1.8 mV) and APD90 declined by only 24%. Developed tension declined more slowly and to a lesser extent, but resting tension rose significantly between 10 and 20 minutes of ischemia. Reperfusion of glibenclamide-treated tissues elicited arrhythmias (extrasystoles and tachycardia), and the preparations failed to recover mechanical function. Glibenclamide at 1 microM produced qualitatively similar effects, albeit less severe. After 30 minutes of no-flow ischemia in untreated tissues, resting tension increased by approximately 130% during the no-flow period. Reperfusion caused arrhythmias (extrasystoles, tachyarrhythmias, and fibrillation) and failed to restore resting or developed tension to preischemic levels. Pinacidil at 1 microM did not affect electrical or contractile function, but at 10 microM it had a negative inotropic effect, decreasing APD90 and developed tension by 5% and 18%, respectively. Both concentrations of the drug caused a faster and greater decline in APD90 during the no-flow period. Resting tension did not change during 30 minutes of no-flow ischemia in the presence of pinacidil, and reperfusion led to 85% and complete recovery of electrical and mechanical activity at 1 and 10 microM, respectively. The data indicate that glibenclamide enhances whereas pinacidil reduces myocardial damage caused by ischemia/reperfusion. The results are consistent with the hypothesis that activation of ATP-regulated K+ channels during ischemia is an important adaptive mechanism for protecting the myocardium when blood flow to the tissue is compromised.
An outward current of unknown nature increases significantly when cardiac cells are treated with cyanide or subjected to hypoxia, and decreases on intracellular injection of ATP. We report here that application of the patch-clamp technique to CN-treated mammalian heart cells reveals specific K+ channels which are depressed by intracellular ATP (ATPi) at levels greater than 1 mM. For these channels, conductance in the outward direction is much larger than the inward rectifier K+ channel which is insensitive to ATP. AMP had no effect on the ATP-sensitive K+ channel, and ADP was less effective than ATP. Thus, the ATP-sensitive K+ channel seems to be important for regulation of cellular energy metabolism in the control of membrane excitability.
We endeavored to determine if the enhanced shortening of the myocardial action potential duration (APD) during ischemia can be dissociated from the cardioprotective effects of the adenosine triphosphate (ATP) sensitive potassium channel (KATP) opener cromakalim. To establish if there is a relationship between APD shortening and the cardioprotective effect of cromakalim, we determined the effect of a dose of the delayed rectifier (IKr) blocker dofetilide (which abolishes the APD shortening effect of cromakalim) on the cardioprotective activity of cromakalim. Cromakalim was infused at a previously determined cardioprotective dose (10 micrograms/kg + 0.3 micrograms/kg/min infusion i.c.), and we determined the effect of 1 mg/kg (followed by a 0.01 mg/kg/min i.v. infusion) dofetilide alone and in combination with cromakalim on APD shortening and infarct size (90-min coronary occlusion and 5-h reperfusion) in anesthetized dogs. Dofetilide completely abolished the APD shortening effects of cromakalim during ischemia such that APD was similar to preischemic values. Cromakalim only shortened the APD during ischemia, although this effect was attenuated late into ischemia. Cromakalim significantly reduced infarct size (40% reduction from vehicle group value), whereas dofetilide alone had no effect. Dofetilide, at a dose that prevented the cromakalim-induced shortening of APD in ischemic tissue, did not attenuate the cardioprotective effects of cromakalim. No differences in collateral blood flow for any of the groups were observed. Dofetilide did cause a slight bradycardia, but this effect is unlikely to affect the interpretation of the results. These data suggest that APD shortening observed with the KATP opener cromakalim is not correlated with its cardioprotective effects.
The ATP-sensitive potassium channel (KATP) has been shown to serve an endogenous cardioprotective role in a number of experimental models of myocardial stunning and infarction. More importantly, a majority of evidence has also been obtained which suggests that the KATP channel may be intimately involved in both triggering and maintaining the cardioprotection afforded by the phenomenon of ischemic preconditioning particularly in large animal models such as dogs and pigs. Although the evidence for an involvement of KATP in ischemic pre-conditioning is equivocal in smaller animal species such as rabbits and rats, activation of this channel by KATP channel openers produces cardioprotection in all species studied. Whether this channel is an important mediator of ischemic preconditioning in all animal species including man and the mechanism by which this cardioprotective effect is obtained await further experimental studies. Nevertheless, the use of selective potassium channel openers to mimic preconditioning in selected clinical settings may be a desirable future therapeutic goal.
This study evaluated the importance of ATP-dependent potassium channels (KATP) for ischemic preconditioning (IP) in swine. Swine were studied because due to the sparsity of their innate collateral circulation infarct size (IS) development closely resembles that observed in humans. Ninety minutes of ischemia at a blood flow reduction sufficient to reduce regional myocardial work by 90% caused 13.2 +/- 8.9% (SD) IS of the area at risk. A single cycle of 10-min preconditioning ischemia followed by 15-min reperfusion reduced IS after 90 min of ischemia to 2.8 +/- 2.7%. The epicardial monophasic action potential duration at 50% repolarization (MAP50) was decreased more markedly during the initial 10 min of the prolonged ischemia than during the first 10 min of the preconditioning ischemic period (84 +/- 4 vs. 89 +/- 2%). Transmural myocardial adenosine (ADO) uptake was reversed to net release during both ischemic periods and during the initial phase of reperfusion. Glibenclamide (0.5 mg/kg, followed by 50 micrograms/min i.v.) abolished the reduction in MAP50 without altering ADO release. Glibenclamide did not alter IS per se (13.0 +/- 7.6%) but abolished the beneficial effect of IP (IS: 13.6 +/- 6.2%). Thus blockade of KATP with glibenclamide abolishes the IS-reducing effect of IP in swine but does not reduce ADO release.
The purpose of the present study was to determine whether enhanced activation of myocardial ATP-dependent potassium channels (KATP) with a potassium channel opener, bimakalim, can reduce the time necessary to produce the protective effect of ischemic preconditioning and to determine whether this effect is mediated via accelerating the rate of action potential shortening during preconditioning. Barbital-anesthetized dogs were subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 4 h of reperfusion. Ten minutes of preconditioning was found to markedly reduce myocardial infarct size from 30.6 +/- 4.7 to 7.1 +/- 2.6%. Subsequently, it was observed that either 3 min of LAD occlusion or a 3-min intracoronary infusion with 0.3 micrograms/min of bimakalim did not reduce myocardial infarct size. However, intracoronary infusion with bimakalim during the 3-min preconditioning period markedly reduced myocardial infarct size to a similar extent as that of ischemic preconditioning (12.2 +/- 1.9%). In addition, it was observed that bimakalim markedly accelerated the ischemia-induced shortening of the action potential during preconditioning. These results are the first to demonstrate that activation of KATP channels with a potassium channel opener reduces the threshold of time necessary to produce preconditioning in anesthetized dogs. These data also suggest that KATP channel activation may produce this effect by enhancing the rate of ischemic myocardial action potential shortening during preconditioning.
We previously reported a delayed phase of protection against infarction 24 hours after ischemic preconditioning in the rabbit. In the present study, we investigated the possibility that this "second window of protection," like the well-described early phase of protection in the rabbit, might be associated with adenosine receptor activation.
In the first series of experiments, we examined whether adenosine receptor blockade with 8-(p-sulfophenyl)-theophylline (SPT) during preconditioning could abolish the delayed protection against infarction 24 hours later. Open-chest rabbits were subjected to myocardial preconditioning (PC) with the four 5-minute coronary occlusions or they were sham operated on (SHAM). During these procedures, animals received either SPT (PC + SPT, n = 6; and SHAM + SPT, n = 6) or vehicle (PC + VEH, n = 12; and SHAM + VEH, n = 11). Twenty-four hours later, infarct development after a 30-minute coronary occlusion/120-minute reperfusion insult was assessed with triphenyltetrazolium staining. In vehicle-treated rabbits, the infarct-to-risk ratio (I/R) was reduced from 53.6 +/- 5.7% (SHAM + VEH) to 32.9 +/- 4.6% (PC + VEH) (P < .05), clearly indicating a delayed phase of protection. Although I/R was not significantly different between SHAM + VEH (53.6 +/- 5.7%) and SHAM + SPT (61.7 +/- 5.4%), in PC + SPT the delayed protection was abolished (I/R = 56.8 +/- 3.8%). In the second series of experiments, we examined if pharmacological adenosine A1 receptor stimulation could evoke a delayed phase of protection. Conscious rabbits were pretreated with intravenous boluses of saline or the A1 receptor-selective agonist 2-chloro-N6-cyclopentyladenosine (CCPA), and infarct size in response to 30-minute ischemia/120-minute reperfusion was assessed 24 hours later. I/R was 54.5 +/- 2.7% in saline-pretreated controls (n = 12). Pretreatment with 25 micrograms/kg CCPA (n = 6), 50 micrograms/kg CCPA (n = 6), or 100 micrograms/kg CCPA (n = 6) resulted in I/R ratios of 37.1 +/- 4.2% (P < .01), 37.7 +/- 2.2% (P < .01), and 26.3 +/- 5.7% (P < .01), respectively. In both series of experiments, there were no differences in systemic hemodynamics during the infarct protocol, assessed as rate-pressure product, between the different experimental groups.
Twenty-four hours after repetitive brief coronary occlusions, susceptibility to infarction in rabbit myocardium is reduced, an effect that may have clinical relevance. Results of the present study suggest that this second window of protection following preconditioning may, like the early phase of protection, be initiated by an adenosine-related mechanism.
We used three interventions to test critically the theory that ischemic preconditioning is the result of translocation of cytosolic protein kinase C (PKC) into the membranes where it can be activated. If that theory were true then kinase activity should not be necessary during the preconditioning ischemia and thus blocking kinase activity at this time should not block protection. Secondly, since most translocation processes in the cell are accomplished by cytoskeletal microtubules, disrupting them with colchicine should also block protection from preconditioning. Finally, translocating PKC by transient exposure to PMA, should still require adenosine receptor activation to reactivate the PKC pathway during the subsequent ischemia. Blocking kinase activity with staurosporine during a 30 min insult completely blocks protection in preconditioned hearts but when staurosporine treatment was confined to the preconditioning episode protection was not blocked in five of the eight hearts studied. Microtubule disruption with colchincine did block the protective effect of preconditioning (38.3 +/- 1.9% infarction v 40.6 +/- 4.1% in non-preconditioned). Colchicine had no effect on infarct size in the non-preconditioned group. Five min PMA treatment plus 10 min washout significantly limited infarct size in isolated rabbit hearts subjected to 30 min regional ischemia (5.9 +/- 1.1% v 31 +/- 3.5% infarction in control). PMA's protection was blocked by adding the adenosine receptor blocker, SPT, during the sustained ischemia (38.1 +/- 6.1% infarction). All three of these experiments strongly support the translocation theory of ischemic preconditioning.
Adenosine has been proposed to be an important mediator of ischemic preconditioning. Intracoronary administration of adenosine has recently been shown to mimic the effects of preconditioning in isolated rabbit hearts. However, it is not known whether this agent can duplicate the effects of preconditioning in vivo or in other species. Thus, the first objective of the present study was to determine whether adenosine can limit myocardial necrosis to the same extent as preconditioning in anesthetized dogs. A second objective was to determine whether the duration of the adenosine-induced cardioprotection persisted as long as that of ischemic preconditioning. Finally, a third aim was to determine whether adenosine mediates its cardioprotection via the KATP channel, which has been shown to be an important mediator of preconditioning in several animal species, including dogs.
Barbital-anesthetized open-chest dogs were subjected to 60 minutes of left anterior descending coronary artery (LAD) occlusion followed by 4 hours of reperfusion. Preconditioning was elicited by 10 minutes of LAD occlusion followed by 10 or 60 minutes of reperfusion before the 60-minute occlusion period. Adenosine (400 micrograms/min) or an equivalent volume of saline was infused into the LAD for 10 minutes, followed by a 10- or 60-minute drug-free period before the 60-minute ischemic insult. Glibenclamide (0.3 mg/kg i.v.), a selective KATP channel blocker, was given 15 minutes before adenosine administration, and another selective KATP channel blocker, 5-hydroxydecanoate (5-HD, 3 mg/min IC) was infused concomitantly with adenosine into the LAD for 10 minutes. Transmural myocardial blood flow was measured at 5 minutes of occlusion, and infarct size was determined by triphenyltetrazolium staining and expressed as a percent of the area at risk (AAR). There were no significant differences in hemodynamics, collateral blood flow, or AAR between groups. Preconditioning with either 10 or 60 minutes of reperfusion produced a marked reduction (P < .05) in infarct size (6.7 +/- 2.5% and 8.7 +/- 2.6%, respectively, versus 26.9 +/- 4.3% in controls). Administration of adenosine with a 10-minute drug-free period before 60 minutes of occlusion resulted in a marked decrease in infarct size similar to that seen with preconditioning (9.6 +/- 1.7% versus 26.9 +/- 4.3% in controls); however, the protection disappeared when a 60-minute drug-free period was allowed after adenosine administration (23.0 +/- 2.4% versus 26.9 +/- 4.3% in controls). In addition, treatment with either glibenclamide or 5-HD completely abolished the protective effects of adenosine (26.4 +/- 6.8 and 25.0 +/- 4.4%, respectively, versus 26.9 +/- 4.3% in controls).
These results clearly reveal that (1) a 10-minute intracoronary infusion of adenosine exhibits the same efficacy as ischemic preconditioning in reducing myocardial necrosis in dogs; (2) similar to preconditioning, adenosine mediates its cardioprotection via a cardiac KATP channel-linked mechanism; and (3) adenosine-induced cardioprotection is transient (disappearing within 60 minutes), whereas ischemic preconditioning persists for at least 60 minutes. These data support the hypothesis that endogenous adenosine released during ischemia is an important mediator of ischemic preconditioning; however, important differences exist between the time course of effects of exogenously administered adenosine and preconditioning, which suggests that other factors may also be involved.
The major purpose of the present study was to determine the effect of the potassium channel opener bimakalim, administered intracoronary only during the initial 10 minutes of ischemia, on myocardial infarct size in anesthetized dogs. A second aim was to test the possibility that bimakalim mediates its cardioprotective effects by accelerating the rate of myocyte action potential shortening during early ischemia. A third aim was to determine the relative potency of bimakalim to open coronary vascular ATP-regulated potassium (KATP) channels versus myocyte KATP channels.
Barbital-anesthetized open-chest dogs were used. In the initial studies, bimakalim (0.1 to 10 micrograms/min) was infused into the left anterior descending coronary artery (LAD), and changes in coronary blood flow and monophasic action potential duration (MAPD) were used as indexes of coronary vascular KATP channel and myocyte KATP channel activity, respectively. In subsequent infarct studies, dogs were subjected to 60 minutes of LAD occlusion followed by 4 hours of reperfusion. Based on preliminary studies, two doses of bimakalim that did not shorten MAPD during nonischemic conditions (0.1 and 0.3 microgram/min) and one that markedly shortened MAPD during nonischemic conditions (3.0 micrograms/min) or an equal volume of vehicle were infused into the LAD during the initial 10 minutes of coronary artery occlusion. Transmural myocardial blood flow was measured at 5 and 30 minutes of occlusion by the radioactive microsphere technique, and infarct size was determined at the end of 4 hours of reperfusion by triphenyltetrazolium staining. The monophasic action potential duration at 50% repolarization (MAPD50) was measured by an epicardial probe placed in the center of the ischemic area. Bimakalim had an approximately 10-fold greater affinity for the coronary vascular than the myocardial KATP channel (ED50 coronary, approximately 0.3 microgram/min; ED50 myocyte, approximately 3.0 micrograms/min). Three doses of bimakalim (0.1, 0.3, and 3.0 micrograms/min) all markedly reduced infarct size expressed as percent of the area at risk (12.6 +/- 3.3%, 14.5 +/- 2.2%, and 14.2 +/- 5.3%, respectively, versus 27.2 +/- 5.7% in controls) to nearly equal extents. Subsequently, we found that the two higher doses of bimakalim (0.3 and 3.0 micrograms/min) markedly accelerated yet the 0.1-microgram/min dose of bimakalim did not significantly affect the ischemia-related shortening of the action potential during the initial 5 minutes of occlusion. In addition, 0.1 and 0.3 microgram/min bimakalim did not increase the incidence of ventricular fibrillation during the 60 minutes of occlusion (0 of 7 and 0 of 8 dogs, respectively), whereas 3.0 micrograms/min bimakalim had a profibrillatory effect (6 of 6) compared with the control group (1 of 8). There were no significant differences between groups in systemic hemodynamics, myocardial oxygen demand, ischemic bed size, or collateral blood flow to the ischemic region.
The results of the present study clearly reveal that a small dose (0.1 or 0.3 microgram/min) of the KATP channel opener bimakalim administered only during the initial 10-minute period of ischemia markedly reduced myocardial infarct size to an extent equal to that of a higher profibrillatory dose in barbital-anesthetized dogs. These data also suggest that bimakalim and other potassium channel openers may partially exert their cardioprotective effects by accelerating KATP channel activation during early ischemia as evidenced by an enhanced rate of ischemic myocyte action potential shortening. However, the results also suggest that other cellular mechanisms may be involved in mediating the cardioprotection produced by a low dose of bimakalim (0.1 microgram/min) because it did not significantly accelerate the shortening of the action potential duration, yet it had an efficacy to redu
The major purpose of the present study was to de
The major objectives of the present study were to examine the ability of acetylcholine (ACh) to mimic ischemic preconditioning in dogs and to determine the role of cardiac ATP-sensitive potassium (KATP) channels in mediating its effects. Barbital-anesthetized open-chest dogs were subjected to 60 min of left anterior descending coronary artery (LAD) occlusion followed by 4 h of reperfusion. Preconditioning was elicited by 10 min of LAD occlusion followed by 10 min of reperfusion before the 60-min occlusion period. ACh (10 micrograms/min) or an equivalent volume of saline were infused into the LAD for 10 min followed by a 10-min drug-free period before the 60-min ischemic insult. In another group, the specific KATP channel blocker glibenclamide (0.3 mg/kg iv) was given 15 min before ACh administration. Transmural myocardial blood flow was measured at 30 min of occlusion, and infarct size (IS) was determined by triphenyltetrazolium staining and expressed as a percentage of the anatomic area at risk (AAR). There were no significant differences in hemodynamics, collateral blood flow, or AAR between groups. Preconditioning produced a marked reduction (P < 0.05) in IS (5.3 +/- 3.0 vs. 23.7 +/- 5.9% in the controls). ACh, similar to preconditioning, resulted in a dramatic decrease in IS (10.0 +/- 2.9%), whereas glibenclamide completely abolished its protective effects (20.9 +/- 4.8%). These results are the first to indicate that ACh mimics ischemic preconditioning via a cardiac KATP channel-sensitive mechanism in dogs.
The aim was to examine whether ischaemic preconditioning delays the onset of cellular electrical uncoupling during ischaemia, and whether the effect of preconditioning is mediated by the activation of ATP sensitive K+ channels (IK-ATP).
Onset of uncoupling, action potential duration (APD80), and conduction velocity were measured in an isolated perfused rabbit papillary muscle. Preconditioning consisted of 10 min occlusion and 10 min reperfusion prior to 40 min sustained ischaemia. Five groups were studied: (1) control (sustained ischaemia only); (2) preconditioning; (3) preconditioning with 20 microM glibenclamide, a blocker of IK-ATP, added for 10 min during the reperfusion period; (4) sustained ischaemia after 15 min perfusion with 20 microM cromakalim (BRL 34915), an opener of IK-ATP; (5) sustained ischaemia after 10 min perfusion with 20 microM glibenclamide without preconditioning.
Uncoupling started at 15.0(SEM 0.7) min of ischaemia in the control group and at 22.8(1.5) min after preconditioning (p < 0.001 v control group). Blocking IK-ATP during the preconditioning protocol with glibenclamide abolished the delay of uncoupling: onset was at 14.7(1.2) min. Activation of IK-ATP with cromakalim resulted in uncoupling at 23.3(1.9) min (p < 0.002 v control). Glibenclamide without preconditioning had no effect on uncoupling: onset was at 15.6(1.0) min. APD80 during ischaemia was significantly shorter in the preconditioning and cromakalim groups than in the control group from 5 min of ischaemia onward. In the preconditioning+glibenclamide group and the glibenclamide group APD80 was at no point significantly different from the control group. Conduction velocity during ischaemia decreased to about 70% of baseline after 10 min and was not different between the five groups.
(1) Preconditioning delays the onset of electrical uncoupling; (2) the protective effect of preconditioning may be caused by activation of the IK-ATP channel; (3) the protective effect is associated with reduction of action potential duration, but not with changes of conduction velocity.
To test the hypothesis that the heat shock response is associated with myocardial salvage, the heat stress protein (HSP) content of cardiac tissue was increased by either ischemic or thermal stress.
Rabbits were divided into four groups. Ischemic pretreatment (n = 15) comprised four 5-minute episodes of coronary ligation separated by 10 minutes of reperfusion. The corresponding control group (n = 21) underwent surgical preparation without coronary ligation. Thermal pretreatment (n = 16) involved whole-body temperature elevation to 42 degrees C for 15 minutes; corresponding controls (n = 15) were treated with anesthetic alone. Twenty-four hours later, hearts were removed for HSP estimation or infarct size assessment after a 30-minute coronary ligation. Myocardial HSP72 content assessed by Western blotting was elevated by both ischemic and thermal pretreatments (2.5 +/- 0.2 units, n = 4, and 2.8 +/- 0.3 units, n = 4, mean +/- SEM; P = NS, respectively) compared with the corresponding control groups (1.0 +/- 0.3, n = 4, P < or = .01 and 0.3 +/- 0.1, n = 4, P < or = .01, respectively). HSP60 was preferentially elevated by ischemic pretreatment. After a 30-minute coronary occlusion and 120 minutes of reperfusion, ischemic and thermal pretreatments limited infarct size as a percentage of the volume at risk by 28.8 +/- 5.2% vs 52.0 +/- 5.2%, P < or = .01 and 32.8 +/- 3.8% vs 56.9 +/- 6.5%, P < or = .01, respectively.
Myocardial stress protein induced by either sublethal thermal or ischemic injury is associated with myocardial salvage. Our findings suggest that stress protein elevation, rather than the nonspecific effects of thermal or ischemic stress, may be responsible for the myocardial protection seen in this model. Our observations may have important implications regarding myocardial adaptation to brief periods of ischemia.
The infarct-limiting effect of ischemic preconditioning is believed to be a transient phenomenon. We examined the delayed effects of repetitive brief ischemia on limiting infarct size in an open-chest dog model by an occlusion (90 minutes) of the left anterior descending coronary artery (LAD) followed by reperfusion (5 hours). The dogs were preconditioned with four brief repeated ischemic episodes induced by 5-minute LAD occlusions with subsequent reperfusion. The size of infarcts initiated by a sustained occlusion immediately or 24 hours after preconditioning was significantly smaller when compared with infarcts in sham-operated dogs (for the immediate occlusion, 14.4 +/- 2.0% versus 39.0 +/- 3.7%, respectively [p < 0.01]; and for the delayed occlusion, 18.8 +/- 3.4% versus 35.1 +/- 4.6%, respectively [p < 0.05]); however, when the infarction was induced 3 hours (31.2 +/- 3.7% versus 37.5 +/- 4.2%, respectively) or 12 hours (25.4 +/- 4.8% versus 35.0 +/- 5.3%, respectively) after repetitive ischemia, the infarct size did not differ. No differences were seen in regional myocardial blood flow or rate-pressure products between the two groups. These results indicate that an infarct-limiting effect of brief repeated ischemia can be observed 24 hours after sublethal preconditioning.
Calcium tolerant rabbit cardiomyocytes, isolated by collagenase perfusion, were preincubated for varying periods of time followed by resuspension in fresh media and centrifugation into an ischaemic pellet with restricted extracellular fluid. Pellets were incubated for 240 min under oil at 37 degrees C to mimic severe ischaemia. Time to onset of ischaemic contracture (rod to square transformation) and trypan blue permeability following resuspension in 85 mOSM media were monitored at sequential times. The protocol of Series 1 was a 5-10 min pre-incubation, immediately followed by ischaemic pelleting. Preincubation with pinacidil (50 microM) protected cells from ischaemic insult, but pinacidil added only into the ischaemic pellet did not protect. Protection was abolished by the protein kinase (PKC) inhibitors chelerythrine (10 microM) added with pinacidil and calphostin C (200nM) added only into the ischaemic pellet. Neither PKC inhibitor had an effect on injury of untreated ischaemic myocytes (data not shown). Series 2-5 were preconditioning protocols with a 10 min intervention period, followed by a 30 min oxygenated drug-free period, prior to ischaemic pelleting. In series 2 pinacidil protected cells from ischaemic insult and this protection was abolished when glyburide (10 microM) was present during preincubation, or during post-incubation and ischaemia. Glyburide only partially inhibited the protection when glyburide was added only into the ischaemic pellet. In Series 3, 8-sulfophenyltheophyline (SPT)(100 microM) or adenosine deaminase during preincubation, or SPT only added into the ischaemic pellet abolished pinacidil's protection. In Series 4, cardiomyocytes were ischaemically preconditioned by pelleting for 10 min followed by 30 min reoxygenation. Glyburide during initial ischaemic blocked protection, but when added during post incubation and into the final pellet protection was not reduced. In Series 5 8-cyclopentyl-1,3,dipropylxanthine (DPCPX) (10 microM) added into the final pellet abolished protection by pinacidil, but not protection following ischaemic preconditioning. In contrast to pinacidil, ischaemically preconditioned cells maintain protection in the presence of glyburide, indicating that: (1) pinacidil does not exactly mimic preconditioning and (2) ischaemically preconditioned cells do not require opened K+ATP channels for protection, although they appear to be important during initiation of the preconditioned state. It is hypothesized that pinacidil opening of K+ channels may facilitate induction of preconditioning.
We determine if action potential duration (APD) shortening and cardioprotection are separable phenomena in ATP-sensitive potassium channel (KATP) openers which protect ischemic myocardium via a glyburide-reversible mechanism.
We determined the effect of the weakly vasodilating KATP opener, BMS-180448, and the less cardiac-selective cromakalim, on APD in normal, hypoxic or ischemic guinea pig papillary muscles or isolated hearts and compared their APD effects with their cardioprotective activity in isolated guinea pig hearts.
In isolated ischemic guinea pig hearts, cromakalim and BMS-180448 had similar cardioprotective potencies (EC25 of 3.2 and 3.3 microM, respectively, for increasing time to the onset of contracture). At 10 microM, BMS-180448 produced no APD shortening, yet was equally protective at this concentration compared to cromakalim, which produced profound APD shortening under either hypoxic or ischemic conditions. The cardioprotective effects of both compounds at 10 microM were abolished by 0.3 microM glyburide.
APD shortening is not correlated with cardioprotective activity for BMS-180448 and cromakalim while their cardioprotective effects are abolished by glyburide. These results suggest the possibility of reduced proarrhythmic activity in some KATP openers and that their cardioprotective activity is not associated with sarcolemmal KATP opening.
We tested the hypothesis that late preconditioning is associated with increased antioxidant enzyme activity induced by initial oxidative stress.
Isolated rat myocytes were preconditioned either with two cycles of 5 minutes of anoxia and 5 minutes of reoxygenation or with exogenous superoxide anion (O2-) generated by reaction of xanthine oxidase with xanthine. Myocytes were allowed to recover for 60 minutes or 24 hours, after which they were subjected to 60 minutes of anoxia and 60 minutes of reoxygenation. After 60 minutes or 24 hours, the protection was evidenced by decreased O2- production, increased Mn superoxide dismutase (Mn-SOD) activity, increased call viability, decreased LDH release, reduced malondialdehyde formation, high-energy phosphate preservation, and improved call morphology in preconditioned and O2(-)-treated myocytes. Immediately after treatment with O2- or repetitive, brief anoxia, O2- production was increased in myocytes. Longer anoxia resulted in loss of Mn-SOD activity in anoxic controls 24 hours later, whereas it was significantly increased in preconditioned and O2- -treated myocytes. O2- production was inhibited in preconditioned and O2(-)-myocytes. Myocytes treated with Mn-SOD during short, intermittent anoxia exhibited decreased activity of Mn-SOD and increased O2- production 24 hours later. Mn-SOD activity in late preconditioning was considerably higher than that in classic preconditioning.
These results suggest that a burst of oxygen free radicals generated during the initial periods of brief, repetitive anoxia increases myocardial antioxidant activity 24 hours later and that it contributes to the late cardioprotective effect of preconditioning.
We examined the effects of oxygen free radicals (OFRs) on action potentials and membrane currents of guinea pig ventricular myocytes. OFRs produced biphasic changes in the action potential duration, initial lengthening (30 s after exposure to OFRs) and subsequent shortening (within 5 min). In voltage-clamp experiments, OFRs suppressed the L-type calcium current, the delayed rectifier K+ current, and the inward rectifier K+ current. In addition, OFRs increased the time-independent outward current (I(term)) at potentials greater than -30 mV. The increases in I(term) reflected activation of the ATP-sensitive K+ (KATP) channels, as glibenclamide (1 microM) blocked this current. In inside-out patches, OFRs significantly increased the open probability of the channel at a relatively narrow range of ATP concentrations (0.2-2 mM), and this effect was enhanced in the presence of ADP (0.1 mM) and abolished in the presence of either free radical scavengers or gliben-clamide. These findings are compatible with the notion that OFRs activate KATP channels by modulating ATP binding sites of the KATP channels, without affecting ADP binding or glibenclamide binding sites.
Conductive chloride transport in the small intestine is an important factor controlling fluid movement from the blood to the lumen of the gut. Several proteins with potential conductive chloride ion channel activity are expressed in the enterocyte cell population. However, it is not clear whether one or more than one protein species is normally responsible for mediating conductive chloride transport. We have raised monoclonal antibodies that inhibit conductive chloride transport in apical membrane vesicles prepared from porcine ileal enterocytes. These monoclonal antibodies have been used to identify a unique protein involved with this conductive chloride transport. Here, we report that anti-chloride conductance monoclonal antibodies did not detect any antigen in Western blots of enterocyte apical membrane protein. Dot blotting and immunoprecipitation experiments indicated that the antigen recognized by these monoclonal antibodies was not the cystic fibrosis transmembrane conductance regulator. The antigen was localized to both villus and crypt regions of ileum on immunohistochemistry. A 90-kDa protein species was immunoprecipitated from a primary enterocyte cell line by these monoclonal antibodies. This 90-kDa protein may be a chloride ion channel or may play some regulatory role in conductive chloride transport in enterocyte apical membrane vesicles.
ATP-sensitive potassium channels are thought to play an important role in preconditioning. possibly due to shortening of the action potential duration (APD). The purpose of this study was to determine the effect of the class III antiarrhythmic agent dofetilide on preconditioning at a dose that abolishes APD shortening during ischemia A pilot study was performed to find a dose of dofetilide that would abolish the APD shortening effect of preconditioning Anesthetized dogs were subjected to 5-min coronary occlusion (or sham) and 10-min reperfusion followed by 60-min coronary occlusion. Monophasic action potentials were recorded periodically throughout the experiment. Significant APD shortening was observed during the 5- and 60-min ischemic periods, although preconditioning did not further enhance APD shortening during the prolonged ischemia. Dofetilide (1 mg/kg + 0.01 mg.kg-1.h-1 iv) abolished the APD shortening effect of ischemia. The effect of this dose of dofetilide on the protective action of preconditioning was then determined. Preconditioning significantly reduced infarct size expressed as a percentage of the area at risk compared with nonpreconditioned hearts. Dofetilide had no effect on infarct size when given to nonpreconditioned hearts. In addition, dofetilide did not alter the protective effect of preconditioning. No differences in collateral blood flow during ischemia were observed for any group. This study shows that the class III antiarrhythmic agent dofetilide does not abolish preconditioning and that the cardioprotective effect of preconditioning is independent of APD shortening below baseline values.
The effects of hydrogen peroxide (H2O2) on the activity of adenosine 5'-triphosphate-sensitive potassium (K+ ATP) channels in ventricular cells isolated from guinea-pig hearts were investigated in inside-out membrane patches using the patch-clamp technique. H2O2 at concentrations of 5 mM but not 1 mM increased K+ ATP channel activity in the presence of 0.3 mM ATP. The presence of 10 microM ADP together with 0.3 mM ATP led to activation of the K+ ATP channel by 1 mM H2O2. This potentiation of the H2O2-induced activation of the K+ ATP channel by ADP depended on the presence of Mg-ATP. Channel activation was due to an increase in the open-state probability and was not associated with a change in the single-channel conductance or the mean open and closed times during burst-like openings. The relationship between channel activity and ATP concentration could be fitted by the Hill equation with a Hill coefficient of 2 and the half maximal inhibition at 85 microM ATP in the presence of 10 microM ADP. The curve was shifted to higher ATP concentrations in a non-parallel manner by 1 mM H2O2. Analysis of open-state probability for membrane patches containing several channels indicated that H2O2 activated the individual channels to a differing extent. It is concluded that H2O2 activates the K+ ATP channel directly by decreasing the sensitivity to ATP. This effect was potentiated by the presence of ADP.
The cardioprotective effect of myocardial preconditioning (PC) to reduce infarct size has been shown to last approximately 90 min (early PC), and then a second window of protection (SWOP or late PC) appears 24 h later. Although much work has been done to characterize early PC, little has been done to investigate potential mediators of SWOP. To that end, we have used monophosphoryl lipid A (MLA), a nontoxic endotoxin derivative, to produce SWOP and have examined the role of ATP-sensitive potassium (KATP) channels in mediating its cardioprotection. Adult mongrel dogs were given MLA (3, 10, or 35 micrograms/kg i.v.) 24 h before a 60-min left anterior descending coronary artery occlusion and 3 h of reperfusion. After reperfusion, the hearts were stained for myocardial infarction with triphenyltetrazolium. MLA produced a dose-dependent reduction in infarct size that was associated with an enhanced shortening of the monophasic action potential duration during early ischemia. To further examine the role of KATP channels, animals were treated with MLA (35 micrograms/kg) and 24 h later were administered either glibenclamide (0.3 mg/kg i.v.) or 5-hydroxydecanoate (7.5 mg/kg intracoronary over 20 min), two structurally distinct KATP-channel antagonists. Both glibenclamide and 5-hydroxydecanoate abolished the cardioprotection produced by MLA. These results demonstrate that the cardioprotective effect of late PC produced by MLA is dependent on functional KATP channels and is the first study to suggest that late PC may be the result of an increased KATP current during ischemia.
Monophosphoryl lipid A (MLA), a detoxified derivative of the lipid A portion of the endotoxin molecule, given as a pretreatment 24 h prior to cardiac ischemia/reperfusion reduces myocardial stunning and infarction in dogs. This study was undertaken to evaluate the ability of MLA pretreatment to reduce infarct size in a rabbit model of in situ regional myocardial ischemia and reperfusion. Secondly, the potential role of modulation of ATP-sensitive potassium (KATP) channel in MLA's cardioprotection was evaluated using in vivo pharmacologic antagonism with a KATP channel blocker, as was the role of tumor necrosis factor using an enzyme-linked immunosorbent assay method of serum cytokine analysis.
Rabbits were pretreated intravenously with MLA or vehicle injection 24 h prior to initiation of 30 min in situ left anterior descending coronary artery occlusion followed by 3 h reperfusion. In animals receiving glibenclamide, the potassium channel antagonist was administered 30 min prior to inducing ischemia. Animals receiving glibenclamide, which possesses hypoglycemic effects, underwent serial blood glucose evaluation prior to drug and throughout the ischemia and reperfusion periods. Hemodynamics were monitored; infarct size and area at risk were assessed by contrast dye staining (triphenyltetrazolium chloride). Serum tumor necrosis factor was measured by enzyme-linked immunosorbent method in animals administered cardioprotective doses of MLA as well as pyrogenic doses of MLA and endotoxin (positive control) to determine if elaboration of this cytokine could be associated with the cardioprotective effect of MLA.
MLA administered as a single intravenous dose 24 h prior to ischemia reduced infarct size, expressed as a percent of the area at risk, 64 and 71% at doses of 35 and 10 micrograms/kg, respectively. Lower doses of MLA (2.5 and 5 micrograms/kg) did not significantly reduce infarct size. Administration of glibenclamide (300 micrograms/kg) 30 min prior to ischemia completely blocked the ability of MLA pretreatment to limit infarct size, while MLA vehicle-glibenclamide-treated control rabbits displayed infarcts not significantly different from MLA-vehicle-treated control rabbits. A cardioprotective dose of MLA (35 micrograms/kg) did not induce the elaboration of tumor necrosis factor into rabbit serum (within the limits of assay sensitivity).
Single-dose pretreatment with MLA administered intravenously to rabbits substantially reduces infarct size when administered 24 h prior to ischemia. Pharmacologic preconditioning with MLA appears to be mediated through KATP channels as the channel blocker, glibenclamide, reversed the cardioprotective activity of MLA when administered 1 day following MLA pretreatment, yet 30 min prior to ischemia. In this model the cardioprotective does not appear to be associated with increases in serum tumor necrosis factor.
Results from numerous studies have shown that there is an important link between adenosine A1 receptors and ATP-sensitive potassium (KATP) channels in mediating the cardioprotective effects of ischemic preconditioning (PC). The major aim of the present study was to determine whether occupation of A1 receptors and/or the opening of KATP channels is involved in the time delay between the PC stimulus and the prolonged ischemic insult or the "memory" of PC to reduce infarct size. Barbital sodium-anesthetized dogs were subjected to 1 h of left anterior descending coronary artery (LAD) occlusion followed by 4 h of reperfusion. Ischemic PC was elicited by 10 min of LAD occlusion followed by 1 h of reperfusion (1-h memory) before the 1-h occlusion period. Either adenosine (800 g/min), bimakalim (3 g/min), a combination of two lower doses of each agent (400 g/min of adenosine and 0.3 g/min of bimakalim), or an equivalent volume of saline was infused into the LAD for 10 min followed by a 1-h drug-free period before the 1-h ischemic insult. In another series, glibenclamide, 8-cyclopentyl-1,3-dipropylxanthine (a selective A1-receptor blocker), or PD-115199 (a nonselective adenosine-receptor antagonist) was administered 50 min after ischemic PC (10 min before the 1-h occlusion period). Infarct size (IS) was expressed as a percentage of the area at risk. PC with 1 h of reperfusion resulted in a marked reduction in IS (8.1 +/- 6.5 vs. 29.8 +/- 5.8% in control dogs). Administration of adenosine or bimakalim followed by a 1-h drug-free period had no effect on IS; however, the simultaneous administration of adenosine and bimakalim resulted in a marked decrease in IS (11.5 +/- 2.7%). One hour after ischemic PC, administration of glibenclamide blocked the protective effect of ischemic PC, whereas 8-cyclopentyl-1,3-dipropylxanthine or PD-115199 did not affect it. These results provide evidence that the opening of myocardial KATP channels may play an important role in the memory of ischemic PC in the canine heart and also suggest that adenosine and the KATP channel may have a synergistic interaction that is important for the memory phase of PC.
Recently, much interest has been focused on the pharmacology of ATP-sensitive potassium channels (KATP) in myocardial ischemia. KATP are thought to be involved with the mechanism of myocardial preconditioning, therefore further increasing the level of interest in these channels. Pharmacologic KATP openers have been shown by numerous investigators to protect ischemic-reperfused myocardium. These agents reduce necrosis, improve postischemic functional recovery, and inhibit contracture formation. These protective effects are abolished by KATP blockers. The cardioprotective effects of KATP openers are independent of vasodilator and cardiodepressant effects, but seem to be mediated by energy conservation (reduced ATP hydrolysis). Action potential shortening is also not correlated with cardioprotection, suggesting a role for intracellular (mitochondrial) KATP. Agents have been developed that retain the glyburide-reversible cardioprotective effects of cromakalim but are devoid of vasodilator and action potential shortening activity. Currently, studies are underway to determine the mechanism of cardioprotection. The potential role of mitochondrial KATP in the pathogenesis of ischemia is discussed in this review article.
Heat shock protects against myocardial ischemia-reperfusion injury possibly via increased expression of heat shock proteins. The direct evidence of heat shock protein protection in vivo remains circumstantial, and no other new mechanism of protection has been proposed. Recent studies suggest that opening of ATP-sensitive K+ channels (KATP channels) plays an important role in ischemic preconditioning; however, it is not known whether this channel is also important in delayed protection conferred by heat shock. Anesthetized rabbits underwent heat shock treatment by raising core temperature to 42 degrees C for 15 min. Twenty-four hours later, the animals were reanesthetized and subjected to regional ischemia-reperfusion. The specific KATP channel blockers glibenclamide (0.3 mg/kg i.p.) and sodium 5-hydroxydecanoate (5HD; 5 mg/kg i.v.) were used to block the channel function. The drugs were administered at two different times, either pre-heat stress or preischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The 72-kDa heat shock protein (HSP 72) was measured by Western blots. Our results show that heat shock produced a marked reduction in infarct size (39.4 +/- 8.1 to 14.3 +/- 2.5% of risk area, P < 0.05). Glibenclamide and 5HD completely abolished heat shock-induced reduction in infarct size (42.3 +/- 0.32 and 33.7 +/- 4.8%) when given before ischemia-reperfusion; however, these antagonists failed to block protection when administered before the onset of heat shock. Furthermore, the enhanced expression of HSP 72 in heat shock groups was not diminished by glibenclamide or 5HD, suggesting a lack of a direct role of this protein in conferring cardiac protection by heat shock. The complete blockade of cardiac protection by glibenclamide and 5HD strongly suggests that opening of this channel is a very important component of heat shock-induced ischemic protection in rabbit hearts.
Previous studies showed a poor correlation between sarcolemmal K+ currents and cardioprotection for ATP-sensitive K+ channel (KATP) openers. Diazoxide is a weak cardiac sarcolemmal KATP opener, but it is a potent opener of mitochondrial KATP, making it a useful tool for determining the importance of this mitochondrial site. In reconstituted bovine heart KATP, diazoxide opened mitochondrial KATP with a K1/2 of 0.8 mumol/L while being 1000-fold less potent at opening sarcolemmal KATP. To compare cardioprotective potency, diazoxide or cromakalim was given to isolated rat hearts subjected to 25 minutes of global ischemia and 30 minutes of reperfusion. Diazoxide and cromakalim increased the time to onset of contracture with a similar potency (EC25, 11.0 and 8.8 mumol/L, respectively) and improved postischemic functional recovery in a glibenclamide (glyburide)-reversible manner. In addition, sodium 5-hydroxydecanoic acid completely abolished the protective effect of diazoxide. While-myocyte studies showed that diazoxide was significantly less potent than cromakalim in increasing sarcolemmal K+ currents. Diazoxide shortened ischemic action potential duration significantly less than cromakalim at equicardioprotective concentrations. We also determined the effects of cromakalim and diazoxide on reconstituted rat mitochondrial cardiac KATP activity. Cromakalim and diazoxide were both potent activators of K+ flux in this preparation (K1/2 values, 1.1 +/- 0.1 and 0.49 +/- 0.05 mumol/L, respectively). Both glibenclamide and sodium 5-hydroxydecanoic acid inhibited K+ flux through the diazoxide-opened mitochondrial KATP. The profile of activity of diazoxide (and perhaps KATP openers in general) suggests that they protect ischemic hearts in a manner that is consistent with an interaction with mitochondrial KATP.
The effects of potassium channel opening drugs and intracellular nucleotides on the ATP-sensitive K+ (KATP) channel composed of SUR2A and Kir6.2 in HEK293T cells were examined using the patch-clamp technique. The SUR2A/Kir6.2 channel was activated effectively by pinacidil, marginally by nicorandil but not by diazoxide. The pinacidil-activated channel currents were inhibited by glibenclamide with a K
i value of 160 nM. Upon formation of inside-out (I-O) patches, spontaneous openings of the channels appeared, which were inhibited by intracellular ATP (ATPi) equipotently in the presence and in the absence of intracellular Mg2+ (Mg2+i). The channel activity ran-down gradually in I-O patches. The run-down channels could be reactivated by ATPi only in the presence of Mg2+i. Uridine 5’-diphosphate (UDP) antagonized the ATPi-mediated inhibition of the channel activity before run-down. After run-down, UDP activated the channel without antagonizing ATPi-mediated channel inhibition. Thus, the SUR2A/Kir6.2 reproduced the major properties of the native cardiac KATP channel well in terms of nucleotide regulation and pharmacology, and therefore can be a useful tool with which to elucidate the molecular mechanisms characterizing the KATP channel.
Heat stress (HS) is known to protect against myocardial ischaemia‐reperfusion injury by improving mechanical dysfunction and decreasing necrosis. However, the mechanisms responsible for this form of cardioprotection remain to be elucidated. ATP‐sensitive potassium (K ATP ) channels have been shown to be involved in the delayed phase of protection following ischaemic preconditioning, a phenomenon closely resembling the HS‐induced cardioprotection. The aim of this study was thus to investigate the role of K ATP channels in HS‐induced protection of the isolated rat heart.
Twenty four hours after whole body heat stress (at 42°C for 15 min) or sham anaesthesia, isolated perfused hearts were subjected to a 15 min stabilization period followed by a 15 min infusion of either 10 μ M glibenclamide (Glib), 100 μ M sodium 5‐hydroxydecanoate (5HD) or vehicle (0.04% DMSO). Regional ischaemia (35 min) and reperfusion (120 min) were then performed.
Prior heat stress significantly reduced infarct‐to‐risk ratio (from 42.4±2.4% to 19.4±2.9, P <0.001). This resistance to myocardial infarction was abolished in both Glib‐treated (40.1±1.8% vs 42.3±1.8%) and 5HD‐treated (41.2±1.8% vs 41.8±1.2%) groups.
The results of this study suggest that K ATP channel activation contributes to the cytoprotective response induced by heat stress.
British Journal of Pharmacology (1998) 123 , 1085–1088; doi: 10.1038/sj.bjp.0701710
Brief interruptions of coronary blood flow paradoxically protect the heart from subsequent prolonged ischemia. The basis of such endogenous cardioprotection, known as "ischemic preconditioning," remains uncertain. Pharmacological evidence has implicated ATP-dependent potassium (KATP) channels in the mechanism of preconditioning; however, the effects of sarcolemmal KATP channels on excitability cannot account for the protection.
We simultaneously measured flavoprotein fluorescence, an index of mitochondrial redox state, and sarcolemmal KATP currents in intact rabbit ventricular myocytes. Our results show that diazoxide, a KATP channel opener, selectively activates mitochondrial KATP channels. Diazoxide induced reversible oxidation of flavoproteins with an EC50 of 27 micromol/L but did not activate sarcolemmal KATP channels. The subcellular site of diazoxide action is further localized to mitochondria by confocal imaging of fluorescence arising from flavoproteins and tetramethylrhodamine ethyl ester. In a cellular model of simulated ischemia, inclusion of diazoxide decreased the rate of cell death to about half of that in controls. Both the redox changes and protection are inhibited by the KATP channel blocker 5-hydroxydecanoic acid.
Our results demonstrate that diazoxide targets mitochondrial but not sarcolemmal KATP channels and imply that mitochondrial KATP channels may mediate the protection from KATP channel openers.
Brief transient episodes of nonlethal myocardial ischemia protect or "precondition" the heart and render the myocardium resistant to a subsequent more sustained ischemic insult. The hallmark of this phenomenon--documented in virtually all species and experimental models evaluated to date in countless laboratories worldwide--is the profound reduction in infarct size seen in preconditioned groups versus time-matched controls. Efforts to identify the cellular mechanisms responsible for this paradoxical ischemia-induced cardioprotection, to expand the definition of ischemic preconditioning beyond infarct size reduction, and, perhaps most importantly, to evaluate the efficacy of preconditioning in disease models and in the clinical setting, are all topics of intensive ongoing investigation.
The aim of the present study was to determine the effect of 5-hydroxydecanoate (5-HD) on extracellular K+ levels during global ischemia for 30 min employing K+-sensitive electrodes in isolated guinea-pig hearts. 5-HD (100 microM) reduced the K+ accumulation during the early period of ischemia, but did not inhibit the elevation of extracellular K+ in the latter half of the ischemic period which was selectively enhanced by ouabain (3 microM). Thus, 5-HD appears to exert a similar mode of action as glibenclamide on extracellular K+ accumulation in the ischemic guinea-pig hearts. The present study also strengthens the previous conclusion that an ATP-sensitive K+ channel contributes only to the initial increasing phase of extracellular K+ accumulation during ischemia in guinea-pig hearts.
Pharmacological openers of mitochondrial ATP-dependent K+ (mitoKATP) channels mimic ischemic preconditioning, and such cardioprotection can be prevented by mitoKATP channel blockers. It is also known that protein kinase C (PKC) plays a key role in the induction and maintenance of preconditioning. To look for possible mechanistic links between these 2 sets of observations, we measured mitochondrial matrix redox potential as an index of mitoKATP channel activity in rabbit ventricular myocytes. The mitoKATP channel opener diazoxide (100 micromol/L) partially oxidized the matrix redox potential. Exposure to phorbol 12-myristate 13-acetate (PMA, 100 nmol/L) potentiated and accelerated the effect of diazoxide. These effects of PMA were blocked by the mitoKATP channel blocker 5-hydroxydecanoate, which we verified to be a selective blocker of the mitoKATP channel in simultaneous recordings of membrane current and flavoprotein fluorescence. The inactive control compound 4alpha-phorbol (100 nmol/L) did not alter the effects of diazoxide. We conclude that the activity of mitoKATP channels can be regulated by PKC in intact heart cells. Potentiation of mitoKATP channel opening by PKC provides a direct mechanistic link between the signal transduction of ischemic preconditioning and pharmacological cardioprotection targeted at ATP-dependent K+ channels.
The activation of the ATP-sensitive potassium channel (KATP) during myocardial ischemia leads to potassium efflux, reductions in action potential duration and the formation of ventricular fibrillation (VF). Drugs that inactivate KATP should prevent these changes and thereby prevent VF. However, most KATP antagonists also alter pancreatic channels, which promote insulin release and hypoglycemia. Recently, a cardioselective KATP antagonist, HMR 1883, has been developed that may offer cardioprotection without the untoward side effects of existing compounds. Therefore, VF was induced in 13 mongrel dogs with healed myocardial infarctions by a 2-min coronary artery occlusion during the last minute of a submaximal exercise test. On subsequent days, the exercise-plus-ischemia test was repeated after pretreatment with HMR 1883 (3.0 mg/kg i.v., n = 13) or glibenclamide (1.0 mg/kg i.v., n = 7). HMR 1883 (P < .001) and glibenclamide (P < .01) prevented VF in 11 of 13 and 6 of 7 animals, respectively. Glibenclamide, but not HMR 1883, elicited increases in plasma insulin and reductions in blood glucose. Glibenclamide also reduced (P < .01) both mean coronary blood flow and left ventricular dP/dt maximum as well as the reactive hyperemia induced by 15-sec coronary occlusions (-30.3 +/- 11%), whereas HMR 1883 did not alter this increase in coronary flow (-3.0 +/- 4.7%). Finally, myocardial ischemia (n = 10) significantly (P < .01) reduced refractory period (control, 121 +/- 2 msec; occlusion, 115 +/- 2 msec), which was prevented by either glibenclamide or HMR 1883. Thus, the cardioselective KATP antagonist HMR 1883 can prevent ischemically induced reductions in refractory period and VF without major hemodynamic effects or alterations in blood glucose levels. These data further suggest that the activation of KATPs may play a particularly important role in both the reductions in refractory period and lethal arrhythmia formation associated with myocardial ischemia.
The novel sulfonylthiourea HMR 1883 was investigated in in vitro systems. The rilmakalim-induced shortening of the APD90 in guinea pig right papillary muscle at pHo = 6.0 was antagonized half-maximally by glibenclamide and HMR 1883 with 0.14 microM and 0. 6 microM, respectively. Hypoxia-induced shortening of the APD90 was significantly attenuated by the sulfonylureas when applied 60 min after induction of hypoxia. In isolated guinea pig ventricular myocytes the APD90 as well as the whole-cell current was measured with the patch-clamp technique. The rilmakalim-induced shortening of the APD90 was half-maximally antagonized by glibenclamide and HMR 1883 with 10 nM and 0.4 microM, respectively (pHo = 6.5). The rilmakalim-induced whole-cell current (at 0 mV clamp-potential) was inhibited by glibenclamide and HMR 1883 half-maximally with 20 nM and 0.8 microM, respectively (pHo = 7.4). In isolated perfused guinea pig hearts, the coronary flow (CF) was increased by perfusion with hypoxic solution (20% O2). Whereas 1 microM glibenclamide completely inhibited the hypoxia-induced increase in CF, 10 microM HMR 1883 reduced it by only 18%. Pancreatic effects were investigated in rat insulinoma cells (RINm5F), which were hyperpolarized with 100 microM diazoxide. Addition of glibenclamide or HMR 1883 depolarized the cell potential half-maximally with concentrations of 9 nM and approximately 20 microM, respectively. In conclusion, the sulfonylthiourea HMR 1883 blocks KATPs in cardiac muscle cells with 10-50 fold higher potency than in pancreatic beta-cells and has little effect on the coronary vascular system. Therefore, HMR 1883 has pharmacological selectivity for cardiac myocytes and thereby may be a promising substance for the prevention of ischemia-induced ventricular fibrillation.