Sarcolemmal KATP Channels Research Articles

Abstract 247: Expression and Activity of K ATP Channel in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells of Nondiabetic and Type II Diabetic Patients

Pharmacological preconditioning with volatile anesthetics (APC) protects the myocardium against ischemia/reperfusion injury via multiple pathways that include the cardiac sarcolemmal (sarc) KATP channel. However, APC is altered or absent in metabolic diseases such as diabetes. To investigate the underlying mechanisms we evaluated the expression and activity of cardiac sarcKATP channel in cardiomyocytes (CMs) differentiated from induced pluripotent stem cells of non-diabetic (N-iPSCs) and type II diabetic (DM-iPSCs) patients. Differentiation of human iPSCs into CMs was confirmed by positive immunostaining for cardiac-specific markers and expression of green fluorescent protein under transcriptional control of cardiac promoter myosin light chain-2v that was delivered by lentiviral vector. Immunocytochemical analysis of cardiac sarcKATP channel subunits was carried out with Anti-Kir6.2 and Anti-SUR2A antibodies. Images were captured using a laser-scanning confocal microscope. Single KATP channel activity was monitored from excised inside-out patches at membrane potentials +80 mV to -80 mV, in symmetrical 140 mM K+. The channel was closed by 1 µM glibenclamide and 2 mM ATP. Immunocytochemical analysis showed the presence and colocalization of Kir6.2 and SUR2A proteins in CMs derived from both N-iPSCs and DM-iPSCs. Interestingly, sarcolemmal expression of these subunits appeared lower in DM-iPSCs. Electrophysiological recordings demonstrated that at 5 µM ATP internal single sarcKATP channels were open more frequently in N-iPSCs (7 of 16 cultures) than in DM-iPSCs (3 of 16 cultures). When measured at +40 mV, the single channel current amplitude (2.2 pA) and conductance (55 pS) were typical for the cardiac sarcKATP channel in N-iPSCs. By contrast, the DM-iPSC channels flickered between the open and closed state, resulting in higher noise level and decreased current amplitude (1.6 pA). This is the first report of sarcKATP channel expression and activity in CMs differentiated from human N-iPSCs and DM-iPSCs. The study suggests that compromised APC in human diabetic heart may in part result from altered expression of sarcKATP channel subunits and altered channel kinetics.

A New pROM King for the MitoK ATP Dance

Ischemic heart disease remains the leading cause of death in the developed world, and mechanisms to reduce cardiac ischemic damage are being actively investigated. A potent protective mechanism for the heart is ischemic preconditioning (IPC), a phenomenon in which brief ischemic episodes protect the heart from tissue damage and cell death resulting from subsequent periods of ischemia and reperfusion.1 The precise molecular mechanisms at play during IPC are not known, but the opening of a mitochondrial ATP-sensitive potassium channel, mitoKATP, is believed to be necessary for IPC-induced activation of several prosurvival pathways and processes.2 Article, see p 446 Although the discovery of a putative mitoKATP occurred more than 20 years ago,3 progress on identifying its molecular composition has been limited. The mitoKATP was determined to be both functionally and molecularly distinct from sarcolemmal KATP channels, which have a relatively minimal role in IPC protection and are insensitive to several drugs that affect the mitoKATP.4, 5 Initial studies using immunoreactivity identified the inward rectifying K+-channel subunit Kir6.1 as localizing to mitochondria,6,7 and thus Kir6.1 became an attractive candidate as a possible subunit of the mitoKATP. However, further research by mass spectrometry into the specificity of the Kir6.1 antibody revealed that the antibody does not recognize Kir6.1, and that Kir6.1 is not isolated from more thorough proteomic screens of mitochondria.8 Another investigation led to purification of an inward-rectifying K+-channel component of …

Promoter DNA Methylation Regulates Murine SUR1 (Abcc8) and SUR2 (Abcc9) Expression in HL-1 Cardiomyocytes

Two mammalian genes encode the SURx (SUR1, Abcc8 and SUR2, Abcc9) subunits that combine with Kir6.2 (Kcnj11) subunits to form the ATP-sensitive potassium (KATP) channel in cardiac myocytes. Different isoform combinations endow the channel with distinct physiological and pharmacological properties, and we have recently reported that the molecular composition of sarcolemmal KATP channels is chamber specific in the mouse heart. KATP channel composition is determined by what subunits are expressed in a cell or tissue. In the present study, we explore the role of CpG methylation in regulating SUR1 and SUR2 expression. In HL-1 cardiomyocytes, as in atrial myocytes, SUR1 expression is markedly greater than SUR2. Consistent with CpG methylation-dependent silencing of SUR2 expression, bisulfite sequencing of genomic DNA isolated from HL-1 cells demonstrates that 57.6% of the CpGs in the promoter region of the SUR2 gene are methylated, compared with 0.14% of the the CpG residues in the SUR1 sequence. Moreover, treatment with 10 µM 5-aza-2′-deoxycytidine (Aza-dC) significantly increased both the unmethylated fraction of the SUR2 CpG island and mRNA expression. However, we cannot rule out additional mechanisms of Aza-dC action, as Aza-dC also causes a decrease in SUR1 expression and lower doses of Aza-dC do not alter the unmethylated DNA fraction but do elicit a small increase in SUR2 expression. The conclusion that DNA methylation alone is not the only regulator of SUR subunit expression is also consistent with observations in native myocytes, where the CpG islands of both SUR genes are essentially unmethylated in both atrial and ventricular myocytes. Collectively, these data demonstrate the potential for CpG methylation to regulate SURx subunit expression and raises the possibility that regulated or aberrant CpG methylation might play a role in controlling channel structure and function under different physiological conditions or different species.

Cyp‐epoxygenases mediate adenosine A2A receptor induced vascular relaxation via KATP channels

A2A adenosine receptor (AR) causes aortic relaxation through generation of epoxyeicosatrienoic acids (EETs) formed via Cyp‐epoxygenases (Nayeem et al, AJP 2008). We hypothesize that A2AAR‐mediated aortic relaxation occurs via EETs through opening of KATP channels in smooth muscle cells. Organ bath experiments were done with isolated aortas from A2AKO and wild type (WT) mice. Pinacidil (KATP opener; 10−6M) caused higher relaxation in WT aorta compared to A2AKO (48.09±5.23% vs. 25.41±2.37%; p<0.05). Cyp‐epoxygenase inhibitor MSPPOH (10−5M) caused significant decrease (p<0.05) in pinacidil response in WT (48.09±5.23% vs. 24.93±5.34%) and A2AKO (25.41±2.37% vs. 14.66±3.88%) aorta. Adenosine analog NECA (10−5M) caused significant relaxation in WT aorta (19.65±3.25%) compared to contraction (13.94±6.57%) in A2AKO, that was lowered by KATP blocker glibenclamide in WT (0.17±6.43%) while higher contraction was observed in KO (28.47±5.1%). Relaxation to A2A agonist CGS 21680 (10−6M) in WT (19.02±2.75%) changed to contraction (23.66±6.31%; p<0.05) with glibenclamide. Mitochondrial KATP opener diazoxide had no effect on precontracted tissues, while 5‐HD (mitochondrial KATP blocker, 100uM) had no effect on CGS 21680 response in WT and A2AKO aorta. These data suggest adenosine mediated aortic relaxation occurs via generation of EETs through sarcolemmal KATP channels. (HL027339, HL09444, HL07180)

“Cardiac K ATP ”

Understanding the etiology of and development of appropriate treatment for myocardial ischemia and associated sudden cardiac death is a major goal of cardiovascular research. In a recent review,1 a compelling case is presented that sarcolemmal ATP-sensitive potassium (KATP) channels are an excellent target for antiarrhythmic therapy. Because opening of cardiac KATP channels shortens the action potential and reduces the refractory period, its activation can establish an arrhythmogenic substrate, supporting reentry. Hence, inhibition of KATP could be an ideal way to stop or even prevent arrhythmias: these channels tend to be closed under normal circumstances and to open only when cell metabolism is inhibited; therefore, any agents that inhibit KATP activity should specifically target channels only during ischemia, leaving nonischemic myocardium unaffected. On the other hand, activation of cardiac KATP channels has consistently been shown to protect the heart from damage during ischemia by limiting Ca entry.2 Finding a way to balance these opposing effects on the heart before, during, and after an ischemic event remains an intractable issue. Until recently, efforts to manipulate cardiac KATP have generally been conducted under a single underlying assumption, namely that cardiac KATP channels are structurally and functionally the same throughout the heart. However, recent studies, including the new report of Bao and colleagues3 in this issue, reveal that “cardiac KATP” is not a single entity and is more accurately a collection of structurally distinct channels. The relative density of each one varies regionally in the heart, may differ between species, and may be under dynamic control. Article see p 926 KATP channels are expressed in many different excitable tissues, where they couple cell energetic status with membrane excitability and function.4 In different tissues, KATP channels exhibit different properties. This …

Nicotinamide-rich diet improves physical endurance by up-regulating SUR2A in the heart.

SUR2A is an ATP-binding protein that serves as a regulatory subunit of cardioprotective ATP-sensitive K+ (KATP) channels. Based on signalling pathway regulating SUR2A expression and SUR2A role in regulating numbers of fully assembled KATP channels, we have suggested that nicotinamide-rich diet could improve physical endurance by stimulating SUR2A expression. We have found that mice on nicotinamide-rich diet significantly improved physical endurance, which was associated with significant increase in expression of SUR2A. Transgenic mice with solely overexpressed SUR2A on control diet had increased physical endurance in a similar manner as the wild-type mice on nicotinamide-rich diet. The experiments focused on action membrane potential and intracellular Ca2+ concentration have demonstrated that increased SUR2A expression was associated with the activation of sarcolemmal KATP channels and steady Ca2+ levels in cardiomyocytes in response to β-adrenergic stimulation. In contrast, the same challenge in the wild-type was characterized by a lack of the channel activation and rise in intracellular Ca2+. Nicotinamide-rich diet was ineffective to increase physical endurance in mice lacking KATP channels. This study has shown that nicotinamide-rich diet improves physical endurance by increasing expression of SUR2A and that this is a sole mechanism of the nicotinamide-rich diet effect. The obtained results suggest that oral nicotinamide is a regulator of SUR2A expression and has a potential as a drug that can improve physical endurance in conditions where this effect would be desirable.

The Role of PI3K Isoforms and Sarcolemmal KATP Channel in Epoxyeicosatrienoic Acid Mediated Cardioprotection

The Role of PI3K Isoforms and Sarcolemmal KATP Channel in Epoxyeicosatrienoic Acid Mediated Cardioprotection

Isoflurane‐induced cardioprotection: role of sarcolemmal KATP channels and mitochondria

We investigated the role of sarcKATP channels and mitochondria in anesthetic-induced preconditioning (APC) using a knockout mouse model with deleted Kir6.2 subunit, the pore-forming part of sarcKATP channel (Kir6.2 C57BL/6 KO). Flavoprotein (FP) fluorescence, reactive oxygen species (ROS) production, and mitochondrial membrane potential, were monitored in isolated myocytes by laser scanning confocal microscopy during exposure to 1 MAC isoflurane. In the presence of isoflurane FP fluorescence increased to 180% and 190% of baseline in cardiomyocytes of WT and Kir6.2 KO mice, respectively. ROS increased to 118% and 124% of baseline in WT and Kir6.2 KO myocytes. Mitochondrial membrane potential decreased to 87% and 86% of baseline in WT and Kir6.2 KO. Oxidative stress induced cell death in 31% and 44% of WT and Kir6.2 KO. APC attenuated cell death to 21% and 35% in WT and Kir6.2 KO myocytes. Deletion of pore-forming subunit of sarcKATP channel did not alter characteristic mitochondrial responses to isoflurane in Kir6.2 KO cardiomyocytes. APC is preserved in the Kir6.2 KO cardiomyocytes, but their susceptibility to oxidative stress appears greater under basal conditions. Thus, sarcKATP channels are an important component of a cardioprotective pathway independent of mitochondria-mediated protection. Supported by P01GM066730 (ZJB) from the NIH, Bethesda, MD.

Molecular Mechanisms in Exercise-Induced Cardioprotection

Physical inactivity is increasingly recognized as modifiable behavioral risk factor for cardiovascular diseases. A partial list of proposed mechanisms for exercise-induced cardioprotection include induction of heat shock proteins, increase in cardiac antioxidant capacity, expression of endoplasmic reticulum stress proteins, anatomical and physiological changes in the coronary arteries, changes in nitric oxide production, adaptational changes in cardiac mitochondria, increased autophagy, and improved function of sarcolemmal and/or mitochondrial ATP-sensitive potassium channels. It is currently unclear which of these protective mechanisms are essential for exercise-induced cardioprotection. However, most investigations focus on sarcolemmal KATP channels, NO production, and mitochondrial changes although it is very likely that other mechanisms may also exist. This paper discusses current information about these aforementioned topics and does not consider potentially important adaptations within blood or the autonomic nervous system. A better understanding of the molecular basis of exercise-induced cardioprotection will help to develop better therapeutic strategies.

HMR 1098 is not an SUR isotype specific inhibitor of heterologous or sarcolemmal KATP channels

Murine ventricular and atrial ATP-sensitive potassium (KATP) channels contain different sulfonylurea receptors (ventricular KATP channels are Kir6.2/SUR2A complexes, while atrial KATP channels are Kir6.2/SUR1 complexes). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been considered as a selective sarcolemmal (i.e. SUR2A-dependent) KATP channel inhibitor. However, it is not clear whether HMR 1098 would preferentially inhibit ventricular KATP channels over atrial KATP channels. To test this, we used whole-cell patch clamp techniques on mouse atrial and ventricular myocytes as well as 86Rb+ efflux assays and excised inside-out patch clamp techniques on Kir6.2/SUR1 and Kir6.2/SUR2A channels heterologously expressed in COSm6 cells. In mouse atrial myocytes, both spontaneously activated and diazoxide-activated KATP currents were effectively inhibited by 10μM HMR 1098. By contrast, in ventricular myocytes, pinacidil-activated KATP currents were inhibited by HMR 1098 at a high concentration (100μM) but not at a low concentration (10μM). Consistent with this finding, HMR 1098 inhibits 86Rb+ effluxes through Kir6.2/SUR1 more effectively than Kir6.2/SUR2A channels in COSm6 cells. In excised inside-out patches, HMR 1098 inhibited Kir6.2/SUR1 channels more effectively, particularly in the presence of MgADP and MgATP (mimicking physiological stimulation). Finally, dose-dependent enhancement of insulin secretion from pancreatic islets and decrease of blood glucose level confirm that HMR 1098 is an inhibitor of Kir6.2/SUR1-composed KATP channels.

Differential roles for SUR subunits in KATP channel membrane targeting and regulation

since their discovery over 25 years ago, ATP-sensitive K+ (KATP) channels have been studied extensively in many tissues, including heart, pancreas, brain, skeletal and smooth muscle, pituitary, and kidney ([6][1], [10][2], [18][3], [19][4]). KATP channels are critical sensors that couple cellular

Autophagy And Exercise Cardioprotection: The Role Of Mitochondrial And Sarcolemmal KATP Channels

Exercise, prior to an ischemic event, preconditions the heart against necrotic and apoptotic forms of tissue death. Related to tissue viability, recent understanding of the ischemic heart indicates that autophagic activity is essential to post ischemic survival. From the perspective that low levels of constitutive autophagic control are needed, severe ATP shortages experienced during ischemia-reperfusion (IR) will diminish basal autophagy processes and are thought to be pathologic. The role of autophagy in the exercise preconditioned heart is currently unknown. PURPOSE: This study was undertaken to determine whether exercise preconditioning extends to autophagic activity following acute IR. Due to the ATP-dependent nature of autophagy, the role of the energy sensing mitochondrial and sarcolemmal KATP channels were examined in exercised hearts. METHODS: Young male Sprague Dawley rats performed either short duration cardioprotective exercise (treadmill habituation plus 3 consecutive days of running 30 meters/minute, 50 minutes, ∼70 VO2max) or remained sedentary. Exercised and sedentary animals were exposed to an identical IR challenge (50 minutes I, 120 minutes R), in vivo, known to induce necrotic and apoptotic tissue death. A subset of exercised animals received pharmacologic inhibitors against mitochondrial and sarcolemmal KATP channels (10mg/kg 5HD and 10mg/kg HMR1098, respectively) prior to IR. Evan's blue dye was infused, moments before heart excision, to demark ischemic and perfused cardiac ventricular tissue for subsequent western blot analyses. Ischemic and perfused ventricular samples were assayed for autophagic biomarkers (Atg5, Atg7, Atg12, beclin1, LC3B). RESULTS: As compared to hearts from Sham animals, ventricular tissues exposed to IR from exercised and sedentary treatments were not different for the autophagic markers Atg5, Atg7, or Atg12. Ischemic ventricular tissue from sedentary and exercised hearts with mitochondrial and sarcolemmal KATP channel inhibition, however, expressed a drop in beclin1 and LC3B activity. CONCLUSIONS: These data indicate that constitutive autophagic activity is diminished acutely following an ischemic insult, but that exercise preserves basal autophagy levels. Further, it appears that the mitochondrial and sarcolemmal KATP channels may play a role in preserving autophagy levels in the exercise heart.

Ankyrin-B Regulates Kir6.2 Membrane Expression and Function in Heart

Ankyrin polypeptides are critical for normal membrane protein expression in diverse cell types, including neurons, myocytes, epithelia, and erythrocytes. Ankyrin dysfunction results in defects in membrane expression of ankyrin-binding partners (including ion channels, transporters, and cell adhesion molecules), resulting in aberrant cellular function and disease. Here, we identify a new role for ankyrin-B in cardiac cell biology. We demonstrate that cardiac sarcolemmal K(ATP) channels directly associate with ankyrin-B in heart via the K(ATP) channel alpha-subunit Kir6.2. We demonstrate that primary myocytes lacking ankyrin-B display defects in Kir6.2 protein expression, membrane expression, and function. Moreover, we demonstrate a secondary role for ankyrin-B in regulating K(ATP) channel gating. Finally, we demonstrate that ankyrin-B forms a membrane complex with K(ATP) channels and the cardiac Na/K-ATPase, a second key membrane transporter involved in the cardiac ischemia response. Collectively, our new findings define a new role for cardiac ankyrin polypeptides in regulation of ion channel membrane expression in heart.

Both Mitochondrial KATP Channel Opening and Sarcolemmal KATP Channel Blockage Confer Protection Against Ischemia/Reperfusion-Induced Arrhythmia in Anesthetized Male Rats

this study was performed to assess the effect of selective sarcolemmal adenosine triphosphate (ATP)-sensitive K(+) channel (K(ATP)) inhibition and the mitochondrial K(ATP) channel activation on ischemia and reperfusion (I/R)-induced arrhythmias in different gender of rats. We compared the effect of a selective sarcolemmal K(ATP) channel blocker HMR 1098, a selective mitochondrial K(ATP) channel opener diazoxide, a nonselective K(ATP) channel opener pinacidil, and the combination of pinacidil with HMR 1098 on the incidence and duration of ventricular arrhythmias in 2 groups: anesthetized males (n = 31) and females (n = 31). ischemia and reperfusion was produced by occluding the left main coronary artery of Sprague-Dowley rats for 6 minutes followed by re-opening of the artery for 6 minutes. the arrhythmia score and the duration of arrhythmias were significantly reduced by HMR 1098, diazoxide, and pinacidil in male rats. The combination of the pinacidil with HMR 1098 did not change the antiarrhythmic effect of pinacidil. The duration of arrhythmas was shorter in females than that in the corresponding males. Drug treatments were not effective in decreasing arrhythmias in female groups to the same extent as in the male group. However, the mitochondrial K( ATP) channel activation that is provided by the combination of pinacidil with HMR 1098 significantly decreased the total length of arrhythmias in females. results of the current study indicate that both mitochondrial K(ATP) channel activation and sarcolemmal K(ATP) channel inhibition exert antiarrhythmic action in male rats. The antiarrhythmic effect of pinacidil is not depend on the sarcolemmal K(ATP) channel opening. These results also indicate that K(ATP) channel modulators show no discernable effect in female rats due to the already low incidence of arrhythmias in females.

Thrombin and Its Receptor Enhance ST-Segment Elevation in Acute Myocardial Infarction by Activating the KATP Channel

ST-segment elevation is the major clinical criterion for committing patients with chest pain to have emergent coronary revascularizations; however, the mechanism responsible for ST-segment elevation is unknown. In a guinea pig model of ST-segment elevation acute myocardial infarction (AMI), local application of hirudin, a thrombin antagonist, significantly decreased AMI-induced ST-segment elevation in a dose-dependent manner. Hirudin-induced (5 antithrombin units [ATU]) decrease in ST elevation was reversed by 250 nmol/L thrombin receptor activator peptide (TRAP). TRAP (250 nmol/L [100 microL]) significantly induced ST-segment elevation in hearts without AMI. The TRAP effect was blocked by 4 mg/kg glibenclamide and 4 mg/kg HMR1098 and partially blocked by 3 mg/kg 5HD. Pinacidil (0.45 mg/kg) simulated the effect of TRAP (250 nmol/L [100 microL]) on hearts without AMI. Moreover, single-channel recordings showed that TRAP induced ATP-sensitive K+ channel (KATP channel) activity, and this effect was blocked by HMR1098 but not 5HD. Finally, TRAP significantly shortened the monophasic action potential (MAP) at 90% repolarization (MAP90) and epicardial MAP (EpiMAP) duration. These effects of TRAP were completely reversed by HMR1098 and partially reversed by 5HD. Thrombin and its receptor activation enhanced ST-segment elevation in an AMI model by activating the sarcolemmal KATP channel.

Glycolytic oscillations in single ischemic cardiomyocytes at near anoxia

Previous studies have shown that oscillations of the metabolism can occur in cardiomyocytes under conditions simulating ischemia/reperfusion. It is not known whether they can also occur during real ischemia with near-anoxic oxygen tension. Here, using oxygen clamp in on-chip picochambers, we exposed single resting cardiomyocytes to near anoxia (pO2 < 0.1 mm Hg). We show that at near anoxia, the mitochondrial membrane potential (ΔΨ) was kept by the F1F0-ATPase reversal, using glycolytic adenosine triphosphate (ATP). In many cells, activation of current through sarcolemmal KATP channels (IKATP) started after a delay with one or several oscillations (frequency of 0.044 ± 0.002 Hz). These oscillations were time correlated with oscillations of ΔΨ. Metabolic oscillations at near anoxia are driven by glycolysis because (a) they were inhibited when glycolysis was blocked, (b) they persisted in cells treated with cytoplasmic reactive oxygen species scavengers, and (c) the highest rate of ATP synthesis during an oscillation cycle was associated with the generation of reducing equivalents. Glycolytic oscillations could be initiated upon rapid, but not slow, transition to near anoxia, indicating that the speed of ATP/ADP ratio drop is a determinant of their occurrence. At enhanced oxidative stress, the rate of ATP consumption was increased as indicated by rapid IKATP activation with large-scale oscillations. These results show that metabolic oscillations occur in cardiomyocytes at near anoxia and are driven by glycolysis and modulated by mitochondria through the rate of ATP hydrolysis, which, in turn, can be accelerated by oxidative stress.

Nicotinamide-rich diet protects the heart against ischaemia–reperfusion in mice: A crucial role for cardiac SUR2A

It is a consensus view that a strategy to increase heart resistance to ischaemia–reperfusion is a warranted. Here, based on our previous study, we have hypothesized that a nicotinamide-rich diet could increase myocardial resistance to ischaemia–reperfusion. Therefore, the purpose of this study was to determine whether nicotinamide-rich diet would increase heart resistance to ischaemia–reperfusion and what is the underlying mechanism. Experiments have been done on mice on control and nicotinamide-rich diet (mice were a week on nicotinamide-rich diet) as well as on transgenic mice overexpressing SUR2A (SUR2A mice), a regulatory subunit of cardioprotective ATP-sensitive K+ (KATP) channels and their littermate controls (WT). The levels of mRNA in heart tissue were measured by real-time RT-PCR, whole heart and single cell resistance to ischaemia–reperfusion and severe hypoxia was measured by TTC staining and laser confocal microscopy, respectively. Nicotinamide-rich diet significantly decreased the size of myocardial infarction induced by ischaemia–reperfusion (from 42.5±4.6% of the area at risk zone in mice on control diet to 26.8±1.8% in mice on nicotinamide-rich diet, n=6–12, P=0.031). The cardioprotective effect of nicotinamide-rich diet was associated with 11.46±1.22 times (n=6) increased mRNA levels of SUR2A in the heart. HMR1098, a selective inhibitor of the sarcolemmal KATP channels opening, abolished cardioprotection afforded by nicotinamide-rich diet. Transgenic mice with a sole increase in SUR2A expression had also increased cardiac resistance to ischaemia–reperfusion. We conclude that nicotinamide-rich diet up-regulate SUR2A and increases heart resistance to ischaemia–reperfusion.

Mitochondrial ATP-sensitive K+channels, protectors of the heart

ATP-sensitive K+ (KATP) channels were first identified in the sarcolemma of cardiac myocytes as inwardly rectifying K+ channels that were inhibited by intracellular ATP (Noma, 1983). It was proposed that KATP channels would have a cardioprotective effect during ischaemia by shortening action potential duration and thereby decreasing Ca2+ influx into myocytes. Then it was found that K+ channel opener compounds (KCOs), that are known to activate sarcolemmal KATP channels, exert cardioprotective effects under ischaemia–reperfusion (Grover et al. 1989; Grover & Garlid, 2000). However, several groups of investigators found that KCOs exerted cardioprotection at concentrations below those causing action potential shortening (Yao & Gross, 1994; Grover et al. 1995a,b; Garlid et al. 1997), indicating that KCOs may have targets other than sarcolemmal KATP channels. Alternative targets for KCOs include mitochondrial KATP (mitoKATP) channels (Grover & Garlid, 2000).

Mitochondrial Reactive Oxygen Species Control Metabolic Oscillations in Cardiomyocytes at Near-Anoxia

Metabolic oscillations frequently occur under conditions simulating ischemia. As oxygen tension is a determinant of mitochondrial function and reactive oxygen species (ROS) production, we studied metabolic oscillations in single resting cardiomyocytes at near-anoxia (pO2 < 0.1 mm Hg) using on-chip picochambers. Activation of current through sarcolemmal KATP channels (IKATP), sensing the cytosolic ATP concentration, was measured simultaneously with either the mitochondrial membrane potential, delta Psi (TMRM fluorescence), or the cellular redox state (H2DCF fluorescence). Upon transition to near anoxia, activation of IKATP started with one or several current oscillations, which were time-correlated with oscillations of delta Psi and H2DCF oxidation. Metabolic oscillations persisted in cells treated with either cytoplasmic ROS scavengers or mitochondrial inhibitors of ROS production, and were stimulated when enhancing the mitochondrial ROS production with either antimycin or rotenone. Oscillations of IKATP could be either initiated or potentiated upon rapid, but not slow, transition to near-anoxia and they were closely paralleled by depolarization of delta Psi, indicative of a transient inability of the F1F0-ATPase to keep delta Psi. At elevated oxidative stress, rapid transition to near-anoxia caused a burst of H2DCF oxidation which correlated with an increased rate of IKATP activation. These results show that metabolic oscillations occur in cardiomyocytes at near-anoxia and that these oscillations are controlled by mitochondria through the rate of ATP hydrolysis which in turn depends on ROS production.

HMR 1098 is not an Sur Isotype Specific Inhibitor of Sarcolemmal or Heterologous KATP Channels

Murine ventricular and atrial KATP channels contain different sulfonylurea receptors (ventricular KATP channels are Kir6.2/SUR2A complexes, while atrial KATP channels are Kir6.2/SUR1 complex). HMR 1098, the sodium salt of HMR 1883 {1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea}, has been used as a selective sarcolemmal (i.e. SUR2A-dependent) KATP channel inhibitor. However, specificity for ventricular versus atrial channels has not been examined. We used whole-cell patch-clamp techniques on mouse ventricular and atrial myocytes as well as 86Rb+ efflux assays and excised inside-out patch-clamp techniques on Kir6.2/SUR2A and Kir6.2/SUR1 channels heterologously expressed in COSm6 cells.