Sarcolemmal and mitochondrial K(ATP) channels and myocardial ischemic preconditioning

Ischemic preconditioning (IPC) is the phenomenon whereby brief periods of ischemia have been shown to protect the myocardium against a sustained ischemic insult. The result of IPC may be manifest as a marked reduction in infarct size, myocardial stunning, or incidence of arrhythmias. While many substances and pathways have been proposed to play a role in the signal transduction mediating the cardioprotective effect of IPC, overwhelming evidence indicates an intimate involvement of the ATP-sensitive potassium channel (K_ATP channel) in this process. Initial hypotheses suggested that the surface or sarcolemmal K_ATP (sarcK_ATP) channel mediated the cardioprotective effects of IPC. However, much research has subsequently supported a major role for the mitochondrial K_ATP channel (mitoK_ATP) as the one involved in IPC-mediated cardioprotection. This review presents evidence to support a role for the sarcK_ATP or the mitoK_ATP channel as either triggers and/or downstream mediators in the phenomenon of IPC.     

Activation of mitochondrial ATP-sensitive potassium channels increases cell viability against rotenone-induced cell death

We recently showed that activation of ATP-sensitive potassium (KATP) channels in PC12 cells induces protection against the neurotoxic effect of rotenone, a mitochondrial complex I inhibitor. In this study, we sought to determine the locus of the KATP channels that mediate this protection in PC12 cells. We found that pretreatment of PC12 cells with diazoxide, a mitochondrial KATP channel selective opener, dose-dependently increases cell viability against rotenone-induced cell death as indicated in trypan blue exclusion assays. The protective effect of this preconditioning is attenuated by 5-hydroxydecanoic acid (5-HD), a selective mitochondrial KATP channel antagonist but not in the presence of HMR-1098, a selective plasma membrane KATP potassium channel antagonist. In contrast, P-1075, a selective plasma membrane KATP channel opener, does not induce protection. Using specific antibodies against SUR1 and Kir6.1, we detected immunoreactive proteins of apparent molecular masses 155 and 50 kDa, corresponding to those previously reported for SUR1 and Kir6.1, respectively, in the mitochondria-enriched fraction of PC12 cells. In addition, whole cell patch-clamp studies revealed that inward currents in PC12 cells are insensitive to P-1075, HMR-1098, glibenclamide and diazoxide, indicating that functional plasma membrane KATP channels are negligible. Taken together, our results demonstrate for the first time that activation of mitochondrial KATP channels elicits protection against rotenone-induced cell death.     

Minoxidil prevents 3,4-methylenedioxymethamphetamine-induced serotonin depletions: role of mitochondrial ATP-sensitive potassium channels, Akt and ERK

Preconditioning has emerged as a valid strategy against different neurotoxic insults. Although the mechanisms underlying preconditioning are not fully understood, the activation of ATP-sensitive potassium (KATP) channels has been proposed to play a pivotal role in neuronal preconditioning. In the present work we examine whether minoxidil a KATP channel activator protects against the long-term toxicity caused by the amphetamine derivative 3,4-methylenedioxymethamphetamine (MDMA) in rats. Our data show that intrastriatal administration of minoxidil prevents MDMA-induced long-term indole depletions in the rat striatum. This effect was not related to an effect on core temperature, as pre-treatment with minoxidil did not significantly alter MDMA-induced hyperthermia. Taking into account that minoxidil opens both sarcolemmal and mitochondrial KATP channels, we examined the role of each type of channels in the protective effects of minoxidil using specific inhibitors. The administration of HMR-1098, a blocker of the sarcolemmal KATP channels, along with minoxidil did not affect the protection afforded by the latter. On the contrary the selective mitochondrial KATP channel blocker 5-hydroxydecanoic acid completely reversed the protection afforded by minoxidil, thereby implicating the involvement of mitochondrial (but not sarcolemmal) KATP channels. Furthermore our data show the participation of Akt and extracellular signal-regulated kinases in minoxidil-afforded protection. Intrastriatal administration of wortmannin or PD98059 (inhibitors of phosphatidylinositol-3-kinase and mitogen-activated protein kinase/extracellular regulated protein kinase, respectively), along with minoxidil abolished the protective effect of minoxidil against the serotonergic toxicity caused by MDMA. These results demonstrate that minoxidil by opening mitochondrial KATP channels completely prevents MDMA toxicity and that Akt and MAP kinases are involved in minoxidil-afforded neuroprotection.     

Diabetes and insulin secretion: whither KATP?

The critical involvement of ATP-sensitive potassium (KATP) channels in insulin secretion is confirmed both by the demonstration that mutations that reduce KATP channel activity underlie many if not most cases of persistent hyperinsulinemia, and by the ability of sulfonylureas, which inhibit KATP channels, to enhance insulin secretion in type II diabetics. By extrapolation, we contend that mutations that increase beta-cell KATP channel activity should inhibit glucose-dependent insulin secretion and underlie, or at least predispose to, a diabetic phenotype. In transgenic animal models, this prediction seems to be borne out. Although earlier genetic studies failed to demonstrate a linkage between KATP mutations and diabetes in humans, recent studies indicate significant association of KATP channel gene mutations or polymorphisms and type II diabetes. We suggest that further efforts to understand the involvement of KATP channels in diabetes are warranted.     

The C. elegans mitochondrial K+(ATP) channel: a potential target for preconditioning

Ischemic preconditioning (IPC) is an evolutionarily conserved endogenous mechanism whereby short periods of non-lethal exposure to hypoxia alleviate damage caused by subsequent ischemia reperfusion (IR). Pharmacologic targeting has suggested that the mitochondrial ATP-sensitive potassium channel (mK_ATP) is central to IPC signaling, despite its lack of molecular identity. Here, we report that isolated Caenorhabditis elegans mitochondria have a K_ATP channel with the same physiologic and pharmacologic characteristics as the vertebrate channel. Since C. elegans also exhibit IPC, our observations provide a framework to study the role of mK_ATP in IR injury in a genetic model organism.     

Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction

We previously demonstrated in the pig that instigation of three cycles of 10 min of occlusion and reperfusion in a hindlimb by tourniquet application (approximately 300 mmHg) elicited protection against ischemia-reperfusion injury (infarction) in multiple distant skeletal muscles subsequently subjected to 4 h of ischemia and 48 h of reperfusion, but the mechanism was not studied. The aim of this project was to test our hypothesis that mitochondrial ATP-sensitive potassium (KATP) (mKATP) channels play a central role in the trigger and mediator mechanisms of hindlimb remote ischemic preconditioning (IPC) of skeletal muscle against infarction in the pig. We observed in the pig that hindlimb remote IPC reduced the infarct size of latissimus dorsi (LD) muscle flaps (8 x 13 cm) from 45 +/- 2% to 22 +/- 3% (n = 10; P < 0.05). The nonselective KATP channel inhibitor glibenclamide (0.3 mg/kg) or the selective mKATP channel inhibitor 5-hydroxydecanoate (5-HD, 5 mg/kg), but not the selective sarcolemmal KATP (sKATP) channel inhibitor HMR-1098 (3 mg/kg), abolished the infarct-protective effect of hindlimb remote IPC in LD muscle flaps (n = 10, P < 0.05) when these drugs were injected intravenously at 10 min before remote IPC. In addition, intravenous bolus injection of glibenclamide (1 mg/kg) or 5-HD (10 mg/kg) at the end of hindlimb remote IPC also abolished the infarct protection in LD muscle flaps (n = 10; P < 0.05). Furthermore, intravenous injection of the specific mKATPchannel opener BMS-191095 (2 mg/kg) at 10 min before 4 h of ischemia protected the LD muscle flap against infarction to a similar extent as hindlimb remote IPC, and this infarct-protective effect of BMS-191095 was abolished by intravenous bolus injection of 5-HD (5 mg/kg) at 10 min before or after intravenous injection of BMS-191095 (n = 10; P < 0.05). The infarct protective effect of BMS-191095 was associated with a higher muscle content of ATP at the end of 4 h of ischemia and a decrease in muscle neutrophilic myeloperoxidase activity at the end of 1.5 h of reperfusion compared with the time-matched control (n = 10, P < 0.05). These observations led us to conclude that mKATP channels play a central role in the trigger and mediator mechanisms of hindlimb remote IPC of skeletal muscle against infarction in the pig, and the opening of mKATP channels in ischemic skeletal muscle is associated with an ATP-sparing effect during sustained ischemia and attenuation of neutrophil accumulation during reperfusion.     

Effects of 5-hydroxydecanoate and ischemic preconditioning on the ischemic-reperfused heart of fed and fasted rats

This investigation aimed to assess whether the mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate (5-HD) could abolish the protection conferred by fasting and ischemic preconditioning (IPC) and to ascertain whether these effects are associated with glycogen breakdown and glycolytic activity. Langendorff perfused hearts of fed and 24-h fasted rats were exposed to 25 min ischemia plus 30 min reperfusion. IPC was achieved by a 3 min ischemia plus a 5 min reperfusion cycle. 5-HD (100 microM) perfusion begun 5 min before IPC or 13 min before sustained ischemia in the non preconditioned groups. Fasting improved the reperfusion recovery of contraction, decreased the contracture and the lactate production, increased glycogenolysis and did not affect the percentage of viable tissue. 5-HD abolished the effects of fasting on the contractile recovery but did not affect the contracture. 5-HD decreased the lactate production in the fed group, increased the preischemic glycogen content in both nutritional groups and did not affect the ischemic glycogen fall. IPC improved the contractile function but prevented the contracture only in the fed group, reduced lactate accumulation and glycogenolysis and evoked an increase of the viable tissue. 5-HD abolished the effects of IPC on the contractile recovery and did not affect its effect on the contracture, lactate production, glycogenolysis and viable tissue. These data suggest that the mitocondrial ATP-sensitive potassium channel is involved in the effects of fasting and IPC on the contractile function but the other cardioprotective and metabolic effects appear evoked through other mechanisms. Also suggest that besides the inhibition of the mitochondrial potassium channel, other mechanisms mediate the effects of 5-HD.     

Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygen-glucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of diazoxide while the non-selective KATP channel blocker glibenclamide did not. The non-selective KATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxide-pre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.     

Effects of hypoxia, anoxia, and metabolic inhibitors on KATP channels in rat femoral artery myocytes

Vascular ATP-sensitive potassium (KATP) channels have an important role in hypoxic vasodilation. Because KATP channel activity depends on intracellular nucleotide concentration, one hypothesis is that hypoxia activates channels by reducing cellular ATP production. However, this has not been rigorously tested. In this study we measured KATP current in response to hypoxia and modulators of cellular metabolism in single smooth muscle cells from the rat femoral artery by using the whole cell patch-clamp technique. KATP current was not activated by exposure of cells to hypoxic solutions (Po2 approximately 35 mmHg). In contrast, voltage-dependent calcium current and the depolarization-induced rise in intracellular calcium concentration ([Ca2+]i) was inhibited by hypoxia. Blocking mitochondrial ATP production by using the ATP synthase inhibitor oligomycin B (3 microM) did not activate current. Blocking glycolytic ATP production by using 2-deoxy-D-glucose (5 mM) also did not activate current. The protonophore carbonyl cyanide m-chlorophenylhydrazone (1 microM) depolarized the mitochondrial membrane potential and activated KATP current. This activation was reversed by oligomycin B, suggesting it occurred as a consequence of mitochondrial ATP consumption by ATP synthase working in reverse mode. Finally, anoxia induced by dithionite (0.5 mM) also depolarized the mitochondrial membrane potential and activated KATP current. Our data show that: 1) anoxia but not hypoxia activates KATP current in femoral artery myocytes; and 2) inhibition of cellular energy production is insufficient to activate KATP current and that energy consumption is required for current activation. These results suggest that vascular KATP channels are not activated during hypoxia via changes in cell metabolism. Furthermore, part of the relaxant effect of hypoxia on rat femoral artery may be mediated by changes in [Ca2+]i through modulation of calcium channel activity.     

Preconditioning involves selective mitophagy mediated by Parkin and p62/SQSTM1

Autophagy-dependent mitochondrial turnover in response to cellular stress is necessary for maintaining cellular homeostasis. However, the mechanisms that govern the selective targeting of damaged mitochondria are poorly understood. Parkin, an E3 ubiquitin ligase, has been shown to be essential for the selective clearance of damaged mitochondria. Parkin is expressed in the heart, yet its function has not been investigated in the context of cardioprotection. We previously reported that autophagy is required for cardioprotection by ischemic preconditioning (IPC). In the present study, we used simulated ischemia (sI) in vitro and IPC of hearts to investigate the role of Parkin in mediating cardioprotection ex vivo and in vivo. In HL-1 cells, sI induced Parkin translocation to mitochondria and mitochondrial elimination. IPC induced Parkin translocation to mitochondria in Langendorff-perfused rat hearts and in vivo in mice subjected to regional IPC. Mitochondrial depolarization with an uncoupling agent similarly induced Parkin translocation to mitochondria in cells and Langendorff-perfused rat hearts. Mitochondrial loss was blunted in Atg5-deficient cells, revealing the requirement for autophagy in mitochondrial elimination. Consistent with previous reports indicating a role for p62/SQSTM1 in mitophagy, we found that depletion of p62 attenuated mitophagy and exacerbated cell death in HL-1 cardiomyocytes subjected to sI. While wild type mice showed p62 translocation to mitochondria and an increase in ubiquitination, Parkin knockout mice exhibited attenuated IPC-induced p62 translocation to the mitochondria. Importantly, ablation of Parkin in mice abolished the cardioprotective effects of IPC. These results reveal for the first time the crucial role of Parkin and mitophagy in cardioprotection.     

Warm ischemic preconditioning improves mitochondrial redox balance during and after mild hypothermic ischemia in guinea pig isolated hearts

Ischemic preconditioning (IPC) induces distinctive changes in mitochondrial bioenergetics during warm (37 degrees C) ischemia and improves function and tissue viability on reperfusion. We examined whether IPC before 2 h of hypothermic (27 degrees C) ischemia affords additive cardioprotection and improves mitochondrial redox balance assessed by mitochondrial NADH and flavin adenine dinucleotide (FAD) autofluorescence in intact hearts. A mediating role of ATP-sensitive K(+) (K(ATP)) channel opening was investigated. NADH and FAD fluorescence was measured in the left ventricular wall of guinea pig isolated hearts assigned to five groups of eight animals each: hypothermia alone, hypothermia with ischemia, IPC with cold ischemia, 5-hydroxydecanoic acid (5-HD) alone, and 5-HD with IPC and cold ischemia. IPC consisted of two 5-min periods of warm global ischemia spaced 5 min apart and 15 min of reperfusion before 2 h of ischemia at 27 degrees C and 2 h of warm reperfusion. The K(ATP) channel inhibitor 5-HD was perfused from 5 min before until 5 min after IPC. IPC before 2 h of ischemia at 27 degrees C led to better recovery of function and less tissue damage on reperfusion than did 27 degrees C ischemia alone. These improvements were preceded by attenuated increases in NADH and decreases in FAD during cold ischemia and the reverse changes during warm reperfusion. 5-HD blocked each of these changes induced by IPC. This study indicates that IPC induces additive cardioprotection with mild hypothermic ischemia by improving mitochondrial bioenergetics during and after ischemia. Because effects of IPC on subsequent changes in NADH and FAD were inhibited by 5-HD, this suggests that mitochondrial K(ATP) channel opening plays a substantial role in improving mitochondrial bioenergetics throughout mild hypothermic ischemia and reperfusion.     

Cardioselective sulfonylthiourea HMR 1098 blocks mitochondrial uncoupling induced by a KATP channel opener,P-1075, in beating rat hearts

We investigated effects of blockade of cardiac ATP-sensitive potassium channels (KATP) with a novel cardioselective sulfonylthiourea, HMR 1098, on metabolic uncoupling caused by a potent KATP opener, P-1075, in Langendorff-perfused rat hearts. We used (1) 87Rb-NMR to detect activation-deactivation of sarcolemmal KATP, (2) 31P-NMR to monitor high-energy phosphates, (3) oxygen uptake measurements to monitor cellular respiration, and (4) myocardial optical absorbance measurements at 603 nm to follow changes in cytochrome c oxidase redox state. Activation of sarcolemmal KATP by P-1075 (5 microM) and a mitochondrial uncoupler 2,4-dinitrophenol (DNP) (50 microM) stimulated Rb+ efflux from the hearts by 130% and 60%, respectively. HMR 1098 (5 and 30 microM) blocked activation of sarcolemmal KATP in situ. HMR 1098 also prevented cardiac arrest and mitochondrial uncoupling induced by P-1075, such as (a) depletion of phosphocreatine and ATP by 40%, (b) two-fold decrease in venous oxygen, and (c) reduction of cytochrome c oxidase (demonstrated by an increase in 603 nm optical absorbance). The metabolic effects of P-1075 can be readily explained by activation of putative mitochondrial KATP. We concluded that blockade of mitochondrial uncoupling by HMR 1098 included an inhibiting effect of HMR 1098 on sarcolemmal and mitochondrial KATP in beating rat hearts.     

The human mitochondrial KATP channel is modulated by calcium and nitric oxide: a patch-clamp approach

ATP-sensitive potassium channels of the inner mitochondrial membrane (mtKATP) are blocked by ATP. They are suggested to be involved in protective mechanisms such as ischemic preconditioning (IPC). Here we identify this channel type for the first time in a human cell line (Jurkat cells). Vesicles of the inner mitochondrial membrane (mitoplasts) were prepared by hypoosmotic shock. Single-channel currents were measured by means of the patch-clamp technique. We identified an outward-rectifying channel with a slope conductance of 15 and 82 pS at negative and positive potentials, respectively. The block by 5-hydroxydecanoic acid and inhibition by ATP characterize this channel as the mtKATP channel. ATP also increased the frequency of events within the burst. This effect was modulated by the Ca2+-bath concentration. We also show that the human mtKATP channel is a direct target for nitric oxide that blocked the channel activity. Although the molecular structure of this channel type is still unknown, its characterization as an outward-rectifying channel and modulation by calcium ions and nitric oxide may help to elucidate its functional significance, which possibly implicates a role in cell survival after IPC.     

内皮素和ATP敏感性钾通道介导缺氧所致家兔窦房结起搏细胞的电生理效应

用细胞内微电极技术研究了ＡＴＰ－敏感性钾（Ｋ_（ＡＴＰ））通道和内皮素（ｅｎｄｏｔｈｅｌｉｎ,ＥＴ）在缺氧所致窦房结起搏细胞负性频率中的作用,主要结果如下：（１）缺氧引起窦房结起搏细胞的ＲＰＦ降低和ＡＰＤ缩短,这一效应随时间延长而加重。（２）Ｋ_（ＡＴＰ）通道开放剂ｃｒｏｍａｋａｌｉｍ浓度依赖性地对窦房结起搏细胞有负性频率作用,且明显缩短ＡＰＤ_（５０）。该通道的阻断剂格列苯脲能部分阻断缺氧对起搏细胞的上述效应,表明缺氧效应中有Ｋ_（ＡＴＰ）通道的参与。（３）ＥＴ－１可显著加重缺氧所致的ＲＰＦ降低,使起搏细胞停跳时间前移;而以ＥＴ_Ａ受体阻断剂ＢＱ－１２３预处理窦房结标本后,则能有效地缓解缺氧对起搏细胞的效应,提示内源性ＥＴ－１的释放在缺氧效应中的作用。上述结果表明,缺氧所致起搏细胞的负性频率作用和ＡＰＤ缩短,与Ｋ_（ＡＴＰ）通道的激活和内源性ＥＴ－１的释放有关。     

Mitochondria are targets for geranylgeranylacetone-induced cardioprotection against ischemia-reperfusion in the rat heart

It has been shown that orally administered geranylgeranylacetone (GGA), an anti-ulcer drug, induces expression of heat shock protein 72 (HSP72) and provides protection against ischemia-reperfusion in rat hearts. The underlying protective mechanisms, however, remain unknown. Mitochondria have been shown to be a selective target for heat stress-induced cardioprotection. Therefore, we hypothesized that preservation of mitochondrial function, owing to an opening of a putative channel in the inner mitochondrial membrane, the mitochondrial ATP-sensitive potassium (mitoK(ATP)) channel, could be involved in GGA- or heat stress-induced cardioprotection against ischemia-reperfusion. Rats were treated with oral GGA or vehicle. Twenty-four hours later, each heart was isolated and perfused with a Langendorff apparatus. GGA-treated hearts showed better functional recovery, and less creatine kinase was released during a 30-min reperfusion period, after 20 min of no-flow ischemia. Concomitant perfusion with 5-hydroxydecanoate (5-HD, 100 microM) or glibenclamide (10 microM) abolished the GGA-induced cardioprotective effect. GGA also showed preserved mitochondrial respiratory function, isolated at the end of the reperfusion period, which was abolished with 5-HD treatment. GGA prevented destruction of the mitochondrial structure by ischemia-reperfusion, as shown by electron microscopy. In cultured cardiomyocytes, GGA induced HSP72 expression and resulted in less damage to cells, including less apoptosis in response to hypoxia-reoxygenation. Treatment with 5-HD abolished the GGA-induced cardioprotective effects but did not affect HSP72 expression. Our results indicate that preserved mitochondrial respiratory function, owing to GGA-induced HSP72 expression, may, at least in part, have a role in cardioprotection against ischemia-reperfusion. These processes may involve opening of the mitoK(ATP) channel.     

Nitric oxide-cGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of ATP-sensitive K+ channels in rat hearts

Nitric oxide (NO) plays an important role in anoxic preconditioning to protect the heart against ischemia-reperfusion injuries. The present work was performed to study better the NO-cGMP-protein kinase G (PKG) signaling pathway in the activation of both sarcolemmal and mitochondrial ATP-sensitive K+ (KATP) channels during anoxic preconditioning (APC) and final influence on reducing anoxia-reperfusion (A/R)-induced cardiac damage in rat hearts. The upstream regulating elements controlling NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection were investigated. The involvement of both inducible and endothelial NO synthases (iNOS and eNOS) in the progression of this signaling pathway was followed. Final cellular outcomes of ischemia-induced injury after different preconditioning in the form of lactate dehydrogenase release, DNA strand breaks, and malondialdehyde formation as indexes of cell injury and lipid peroxidation, respectively, were investigated. The lactate dehydrogenase and malondialdehyde values decreased in the groups that underwent preconditioning periods with specific mitochondrial KATP channels opener diazoxide (100 microM), nonspecific mitochondrial KATP channels opener pinacidil (50 microM), S-nitroso-N-acetylpenicillamine (SNAP, 300 microM), or beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclicmonophosphorothioate, Sp-isomer (10 microM) before the A/R period. Preconditioning with SNAP significantly reduced the DNA damage. The effect was blocked by glibenclamide (50 microM), 5-hydroxydecanoate (100 microM), NG-nitro-L-arginine methyl ester (200 microM), and beta-phenyl-1,N2-etheno-8-bromoguanosine-3',5'-cyclic monophosphorothioate, Rp-isomer (1 microM). The results suggest iNOS, rather than eNOS, as the major contributing NO synthase during APC treatment. Moreover, the PKG shows priority over NO as the upstream regulator of NO-cGMP-PKG signal-induced KATP channel opening that leads to cardioprotection during APC treatment.     

Mitochondrial Src tyrosine kinase plays a role in the cardioprotective effect of ischemic preconditioning by modulating complex I activity and mitochondrial ROS generation

Abstract

While ischemic preconditioning (IPC) and other cardioprotective interventions have been proposed to protect the heart from ischemia/reperfusion (I/R) injury by inhibiting mitochondrial complex I activity upon reperfusion, the exact mechanism underlying the modulation of complex I activity remains elusive. This study was aimed to test the hypothesis that IPC modulates complex I activity at reperfusion by activating mitochondrial Src tyrosine kinase, and induces cardioprotection against I/R injury. Isolated rat hearts were preconditioned by three cycles of 5-min ischemia and 5-min reperfusion prior to 30-min index ischemia followed by 2 h of reperfusion. Mitochondrial Src phosphorylation (Tyr⁴¹⁶) was dramatically decreased during I/R, implying inactivation of Src tyrosine kinase by I/R. IPC increased mitochondrial Src phosphorylation upon reperfusion and this was inhibited by the selective Src tyrosine kinase inhibitor PP2. IPC's anti-infarct effect was inhibited by the selective Src tyrosine kinase inhibitor PP2. Complex I activity was significantly increased upon reperfusion, an effect that was prevented by IPC in a Src tyrosine kinase-dependent manner. In support, Src and phospho-Src were found in complex I. Furthermore, IPC prevented hypoxia/reoxygenation-induced mitochondrial reactive oxygen species (ROS) generation and cellular injury in rat cardiomyocytes, which was revoked by PP2. Finally, IPC reduced LDH release induced by both hypoxia/reoxygenation and simulated ischemia/reperfusion, an effect that was reversed by PP2 and Src siRNA. These data suggest that mitochondrial Src tyrosine kinase accounts for the inhibitory action of IPC on complex I and mitochondrial ROS generation, and thereby plays a role in the cardioprotective effect of IPC.     

Gene delivery of Kir6.2/SUR2A in conjunction with pinacidil handles intracellular Ca2+ homeostasis under metabolic stress.

Metabolic injury is a complex process affecting various tissues, with intracellular Ca2+ loading recognized as a common precipitating event leading to cell death. We have recently observed that cells overexpressing recombinant ATP-sensitive K+ (KATP) channel subunits may acquire resistance against metabolic stress. To examine whether, under metabolic challenge, intracellular Ca2+ homeostasis can be maintained by an activator of channel proteins, we delivered Kir6.2 and SUR2A genes, which encode KATP channel subunits, into a somatic cell line lacking native KATP channels. Hypoxia-reoxygenation was simulated by application and removal of the mitochondrial poison 2,4 dinitrophenol. Under such metabolic stress, Ca2+ loading was induced by Ca2+ influx during hypoxia and release of Ca2+ from intracellular stores during reoxygenation. Delivery of Kir6.2/SUR2A genes, in conjunction with the KATP channel activator pinacidil, prevented intracellular Ca2+ loading irrespective of whether the channel opener was applied throughout the duration of hypoxia-reoxygenation or transiently during the hypoxic or reoxygenation stage. In all stages of injury, the effect of pinacidil was inhibited by the selective antagonist of KATP channel, 5-hydroxydecanoate. The present study provides evidence that combined use of gene delivery and pharmacological targeting of recombinant proteins can handle intracellular Ca2+ homeostasis under hypoxia-reoxygenation irrespective of the stage of the metabolic insult.     

Molecular mechanisms of cardiac protection by adaptation to chronic hypoxia

Effective protection of the heart against ischemia/reperfusion injury is one of the most important goals of experimental and clinical research in cardiology. Besides ischemic preconditioning as a powerful temporal protective phenomenon, adaptation to chronic hypoxia also increases cardiac tolerance to all major deleterious consequences of acute oxygen deprivation such as myocardial infarction, contractile dysfunction and ventricular arrhythmias. Although many factors have been proposed to play a potential role, the detailed mechanism of this long-term protection remains poorly understood. This review summarizes current limited evidence for the involvement of ATP-sensitive potassium channels, reactive oxygen species, nitric oxide and various protein kinases in cardioprotective effects of chronic hypoxia.