Grant R. Budas

Deficiency of PDK1 in cardiac muscle results in heart failure and increased sensitivity to hypoxia.

We employed Cre/loxP technology to generate mPDK1−/− mice, which lack PDK1 in cardiac muscle. Insulin did not activate PKB and S6K, nor did it stimulate 6‐phosphofructo‐2‐kinase and production of fructose 2,6‐bisphosphate, in the hearts of mPDK1−/− mice, consistent with PDK1 mediating these processes. All mPDK1−/− mice died suddenly between 5 and 11 weeks of age. The mPDK1−/− animals had thinner ventricular walls, enlarged atria and right ventricles. Moreover, mPDK1−/− muscle mass was markedly reduced due to a reduction in cardiomyocyte volume rather than cardiomyocyte cell number, and markers of heart failure were elevated. These results suggested mPDK1−/− mice died of heart failure, a conclusion supported by echocardiographic analysis. By employing a single‐cell assay we found that cardiomyocytes from mPDK1−/− mice are markedly more sensitive to hypoxia. These results establish that the PDK1 signalling network plays an important role in regulating cardiac viability and preventing heart failure. They also suggest that a deficiency of the PDK1 pathway might contribute to development of cardiac disease in humans.

Creatine kinase is physically associated with the cardiac ATP-sensitive k+ channel in vivo

Cardiac sarcolemmal ATP‐sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, couple the metabolic status of cells with the membrane excitability. Based on previous functional studies, we have hypothesized that creatine kinase (CK) may be a part of the sarcolemmal KATP channel protein complex. The inside‐out and whole cell patch clamp electrophysiology applied on guinea pig cardiomyocytes showed that substrates of CK regulate KATP channels activity. Following immunoprecipitation of guinea‐pig cardiac membrane fraction with the anti‐SUR2 antibody, Coomassie blue staining revealed, besides Kir6.2 and SUR2A, a polypeptide at ~48 kDa. Western blotting analysis confirmed the nature of putative Kir6.2 and SUR2A, whereas matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analysis identified p48 kDa as a muscle form of CK. In addition, the CK activity was found in the anti‐SUR2A immunoprecipitate and the cross reactivity between an anti‐CK antibody and the anti‐SUR2A immunoprecipitate was observed as well as vice verse. Further results obtained at the level of recombinant channel subunits demonstrated that CK is directly physically associated with the SUR2A, but not the Kir6.2, subunit. All together, these results suggest that the CK is associated with SUR2A subunit in vivo, which is an integral part of the sarcolemmal KATP channel protein complex.

AMP-activated protein kinase mediates preconditioning in cardiomyocytes by regulating activity and trafficking of sarcolemmal ATP-sensitive K+ channels

Brief periods of ischemia and reperfusion that precede sustained ischemia lead to a reduction in myocardial infarct size. This phenomenon, known as ischemic preconditioning, is mediated by signaling pathway(s) that is complex and yet to be fully defined. AMP‐activated kinase (AMPK) is activated in cells under conditions associated with ATP depletion and increased AMP/ATP ratio. In the present study, we have taken advantage of a cardiac phenotype overexpressing a dominant negative form of the α2 subunit of AMPK to analyze the role, if any, that AMPK plays in preconditioning the heart. We have found that myocardial preconditioning activates AMPK in wild type, but not transgenic mice. Cardiac cells from transgenic mice could not be preconditioned, as opposed to cells from the wild type. The cytoprotective effect of AMPK was not related to the effect that preconditioning has on mitochondrial membrane potential as revealed by JC‐1, a mitochondrial membrane potential‐sensitive dye, and laser confocal microscopy. In contrast, experiments with di‐8‐ANEPPS, a sarcolemmal‐potential sensitive dye, has demonstrated that intact AMPK activity is required for preconditioning‐induced shortening of the action membrane potential. The preconditioning‐induced activation of sarcolemmal KATP channels was observed in wild type, but not in transgenic mice. HMR 1098, a selective inhibitor of sarcolemmal KATP channels opening, inhibited preconditioning‐induced shortening of action membrane potential as well as cardioprotection afforded by AMPK. Immunoprecipitation followed by Western blotting has shown that AMPK is essential for preconditioning‐induced recruitment of sarcolemmal KATP channels. Based on the obtained results, we conclude that AMPK mediates preconditioning in cardiac cells by regulating the activity and recruitment of sarcolemmal KATP channels without being a part of signaling pathway that regulates mitochondrial membrane potential. J. Cell. Physiol. 210: 224–236, 2007.

Gender-specific difference in cardiac ATP-sensitive K+channels ☆

OBJECTIVES The main objective of this study was to establish whether gender regulates expression and/or properties of cardiac ATP-sensitive K(+) (K(ATP)) channels. BACKGROUND Recently, evidence has been provided that differing cardiac responses in males and females to metabolic stress may result from gender-specific difference(s) in the efficiency of endogenous cardioprotective mechanism(s) such as K(ATP) channels. METHODS A reverse transcription polymerase chain reaction (RT-PCR) using primers specific for Kir6.2, Kir6.1 and SUR2A subunits was performed on total RNA from guinea pig ventricular tissue. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Whole-cell, single-channel electrophysiology and digital epifluorescent Ca(2+) imaging were performed on isolated guinea pig ventricular cardiomyocytes. RESULTS The RT-PCR revealed higher levels of SUR2A, but not Kir6.1 and Kir6.2, messenger RNA in female tissue relative to male tissue, while much higher levels of both Kir6.2 and SUR2A proteins in cardiac membrane fraction in female tissue compared with male tissue were found. In both male and female tissue, pinacidil (100 microM), a K(ATP) channel opener, induced outward whole-cell currents. The current density of the pinacidil-sensitive component was significantly higher in female tissue than it was in male tissue, while no differences in single K(ATP) channel properties between genders were observed. Ischemia-reperfusion challenge induced significant intracellular Ca(2+) loading in male, but not female, cardiomyocytes. To test the hypothesis that SUR2A expression is the limiting factor in K(ATP) channel formation, we took different volumes of Kir6.2 and SUR2A complementary DNA (cDNA) from the same cDNA pool and subjected them to PCR. In order to obtain a band having 50% of the maximal intensity, a volume of SUR2a cDNA approximately 20 times the volume of Kir6.2 cDNA was required. CONCLUSIONS This study has demonstrated that female tissue expresses higher levels of functional cardiac K(ATP) channels than male tissue due to the higher expression of the SUR2A subunit, which has an impact on cardiac response to ischemia-reperfusion challenge.

17β-Estradiol regulates expression of KATPchannels in heart-derived H9c2 cells ☆

Abstract Objectives The main objective of the present study was to establish whether 17β-estradiol (E2) regulates expression of cardiac adenosine triphosphate-sensitive potassium (K ATP ) channel. Background Based on our previous studies that demonstrate gender-specific differences in sarcolemmal K ATP channels, we have hypothesized that the main estrogen, E2, may regulate expression of cardiac K ATP channels. Methods Reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for Kir6.2 and sulfonylurea receptor 2A (SUR2A) subunits was performed on total ribonucleic acid (RNA) from rat embryonic heart-derived H9c2 cells. Immunoprecipitation and Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was done on membrane fraction of H9c2 cells. Whole cell electrophysiology and digital epifluorescent Ca 2+ imaging were performed on living H9c2 cells. All experiments were done in cells incubated 24 h with or without 100 nM E2. Results The RT-PCR revealed higher levels of SUR2A, but not Kir6.2, messenger RNA (mRNA) in E2-treated, relative to untreated, cells. Increase of the level of only the SUR2A subunit could change the number of sarcolemmal K ATP channels only if the Kir6.2 is in excess over SUR2A. Indeed, RT-PCR analysis demonstrated considerably lower levels of SUR2A mRNA compared with Kir6.2 mRNA. Significantly higher levels of both Kir6.2 and SUR2A protein subunits were found in the membrane fraction of E2-treated cells compared with untreated ones, and the density of current evoked by pinacidil (100 μM), a K ATP channel opener, was significantly higher in E2-treated compared with untreated cells. To test the effect of E2 on cellular response to hypoxia-reoxygenation, we have measured on-line, intracellular concentration of Ca 2+ in H9c2 cells exposed to hypoxia-reoxygenation. Intracellular Ca 2+ loading induced by hypoxia-reoxygenation was significantly decreased by treatment with E2. This E2-mediated protection was inhibited by HMR 1098 (30 μM), but not by 5-hydroxydecanoate (50 μM). Conclusions In conclusion, this study has demonstrated that E2 increases levels of SUR2A subunit, stimulates K ATP channel formation and protects cardiac cells from hypoxiareoxygenation.

M-LDH serves as a sarcolemmal KATP channel subunit essential for cell protection against ischemia

ATP‐sensitive K+ (KATP) channels in the heart are normally closed by high intracellular ATP, but are activated during ischemia to promote cellular survival. These channels are heteromultimers composed of Kir6.2 subunit, an inwardly rectifying K+ channel core, and SUR2A, a regulatory subunit implicated in ligand‐dependent regulation of channel gating. Here, we have shown that the muscle form (M‐LDH), but not heart form (H‐LDH), of lactate dehydrogenase is directly physically associated with the sarcolemmal KATP channel by interacting with the Kir6.2 subunit via its N‐terminus and with the SUR2A subunit via its C‐terminus. The species of LDH bound to the channel regulated the channel activity despite millimolar concentration of intracellular ATP. The presence of M‐LDH in the channel protein complex was required for opening of KATP channels during ischemia and ischemia‐resistant cellular phenotype. We conclude that M‐LDH is an integral part of the sarcolemmal KATP channel protein complex in vivo, where, by virtue of its catalytic activity, it couples the metabolic status of the cell with the KATP channels activity that is essential for cell protection against ischemia.

Hypoxia-induced preconditioning in adult stimulated cardiomyocytes is mediated by the opening and trafficking of sarcolemmal KATP channels.

The opening of sarcolemmal and mitochondrial ATP‐sensitive K+ (KATP) channels in the heart is believed to mediate ischemic preconditioning, a phenomenon whereby brief periods of ischemia/reperfusion protect the heart against myocardial infarction. Here, we have applied digital epifluorescent microscopy, immunoprecipitation and Western blotting, perforated patch clamp electrophysiology, and immunofluorescence/laser confocal microscopy to examine the involvement of KATP channels in cardioprotection afforded by preconditioning. We have shown that adult, stimulated‐to‐beat, guinea‐pig cardiomyocytes survived in sustained hypoxia for ~17 min. An episode of 5‐min‐long hypoxia/5‐min‐long reoxygenation before sustained hypoxia dramatically increased the duration of cellular survival. Experiments with different antagonists of KATP channels, applied at different times during the experimental protocol, suggested that the opening of sarcolemmal KATP channels at the beginning of sustained hypoxia mediate preconditioning. This conclusion was supported by perforated patch clamp experiments that revealed activation of sarcolemmal KATP channels by preconditioning. Immunoprecipitation and Western blotting as well as immunofluorescence and laser confocal microscopy showed that the preconditioning is associated with the increase in KATP channel proteins in sarcolemma. Inhibition of trafficking of KATP channel subunits prevented preconditioning without affecting sensitivity of cardiomyocytes to hypoxia in the absence of preconditioning. We conclude that the preconditioning is mediated by the activation and trafficking of sarcolemmal KATP channels.

Overexpression of SUR2A generates a cardiac phenotype resistant to ischemia

ATP‐sensitive K+ (KATP) channels are present in the sarcolemma of cardiac myocytes where they link membrane excitability with the cellular bioen‐ergetic state. These channels are in vivo composed of Kir6.2, a pore‐forming subunit, SUR2A, a regulatory subunit, and at least four accessory proteins. In the present study, real‐time RT‐PCR has demonstrated that of all six sarcolemmal KATP channel‐forming proteins, SUR2A was probably the least expressed protein. We have generated mice where the SUR2A was under the control of a cytomegalovirus promoter, a promoter that is more efficient than the native promoter. These mice had an increase in SUR2A mRNA/protein levels in the heart whereas levels of mRNAs of other channel‐forming proteins were not affected at all. Imunopre‐cipitation/Western blot and patch clamp electrophysi‐ology has shown an increase in KATP channel numbers in the sarcolemma of transgenic mice. Cardiomyocytes from transgenic mice responded to hypoxia with shortening of action membrane potential and were significantly more resistant to this insult than cardiomyocytes from the wild‐type. The size of myocardial infarction in response to ischemia‐reperfusion was much smaller in hearts from transgenic mice compared to those in wild‐type. We conclude that overexpression of SUR2A generates cardiac phenotype resistant to hypoxia/isch‐emia/reperfusion injury due at least in part to increase in levels of sarcolemmal KATP channels.—Du, Q., Jovanovic, S., Clelland, A., Sukhodub, A., Budas, G., Phelan, K., Murray‐Tait, V., Malone, L., Jovanovic, A. Overexpression of sur2a generates cardiac phenotype resistant to ischemia. FASEBJ. 20, 1131–1141 (2006)

Glyceraldehyde 3‐phosphate dehydrogenase serves as an accessory protein of the cardiac sarcolemmal KATP channel

Cardiac sarcolemmal ATP‐sensitive K+ (KATP) channels, composed of Kir6.2 and SUR2A subunits, are regulated by intracellular ATP and they couple the metabolic status of the cell with the membrane excitability. On the basis of previous studies, we have suggested that glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) may be a part of the sarcolemmal KATP‐channel protein complex. A polypeptide of ∼42 kDa was immunoprecipitated with an anti‐SUR2A antibody from guinea‐pig cardiac membrane fraction and identified as GAPDH. Immunoprecipitation/western blotting analysis with anti‐Kir6.2, anti‐SUR2A and anti‐GAPDH antibodies showed that GAPDH is a part of the sarcolemmal KATP‐channel protein complex in vivo. Further studies with immunoprecipitation/western blotting and the membrane yeast two‐hybrid system showed that GAPDH associates physically with the Kir6.2 but not the SUR2A subunit. Patch‐clamp electrophysiology showed that GAPDH regulates KATP‐channel activity irrespective of high intracellular ATP, by producing 1,3‐bisphosphoglycerate, a KATP‐channel opener. These results suggest that GAPDH is an integral part of the sarcolemmal KATP‐channel protein complex, where it couples glycolysis with the KATP‐channel activity.

Ageing is associated with a decrease in the number of sarcolemmal ATP-sensitive K+ channels in a gender-dependent manner

The opening of sarcolemmal K(ATP) channels is considered to be an important endogenous cardioprotective mechanism. On the other hand, age-dependent changes in the myocardial susceptibility to ischemia and hypoxia have been observed in different species, including humans. Here, we have hypothesized that aging might be associated with the changes in sarcolemmal K(ATP) channels. Therefore, the main objective of the present study was to establish whether aging changes expression of cardiac sarcolemmal ATP-sensitive K+ (K(ATP)) channels. RT-PCR using primers specific for K(ATP) channel subunits, Kir6.2, Kir6.1 and SUR2A subunits was performed using total RNA from guinea-pig ventricular tissue. Whole cell electrophysiology was done on isolated guinea-pig ventricular cardiomyocytes. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Tissue and cells were harvested from young and old, male and female guinea-pigs. RT-PCR analysis did not reveal significant age-related changes in levels of Kir6.1 or Kir6.2 mRNAs. However, levels of SUR2A were significantly lower in old than in young females. Such age-differences were not observed with cardiac tissue from male animals. In both old and young males, pinacidil (100 microM) induced outward currents. The difference between current density of pinacidil-sensitive component in females, but not males, was statistically significant. Western blotting analysis revealed higher levels of Kir6.2 and SUR2A proteins in cardiac membrane fraction from young than old females. The present study demonstrates that in females, but not males, aging is associated with decrease in number of cardiac K(ATP) channels which is due to decrease in levels of the SUR2A subunit.