Abstract 15558: Characterization of Sarcolemmal K Atp Channels in Human Ipsc-derived Cardiomyocytes

Background: The ATP-sensitive potassium channel (KATP) plays a key role in protecting heart muscle during metabolic challenges such as ischemia. KATP activation causes action potential shortening that reduces calcium entry and contraction thus reducing calcium overload induced damage and preserving energy reserves. Cardiomyocytes derived from human inducible pluripotent stem cell (hiPSC) have emerged as a model to study cardiac function, however there are few studies that have focused on KATP. Methods: In the present study, cardiomyocytes were either generated from hiPSC using heparin- based chemically defined media or purchased from Cellular Dynamics (iCells2). Expression of the pore-forming (Kir6.2) and regulatory (SUR1 & SUR2) subunits of the KATP channel during differentiation were assessed using western blot. KATP function was assessed by measuring the field potential duration (FPD) and spontaneous beat rate in a confluent monolayer using the Axion Maestro multielectrode array system. Cells were probed using the KATP activators P1075 and diazoxide, specific for SUR2 and SUR1, respectively. Results: We found that the pore-forming subunit of the sarcolemmal KATP channel (Kir6.2) was expressed in iPSC and maintained throughout the course of differentiation. Consistent with the typical composition of sarcolemmal KATP, we observe a significant increase of SUR2 but little SUR1 protein following Wnt inhibition. Functionally, the FPD is markedly reduced by P1075 in a concentration-dependent manner, with 24% reduction at 100 nM and 92 % reduction at 100 μM. Moreover, glibenclamide 10μM reduces FPD shortening confirming a role for KATP. Finally, we observe little change in FPD when cells are exposed to diazoxide (100 μM) consistent with reduced SUR1 protein levels. Conclusion: These results indicate that cardiomyocytes derived from human iPSC express the KATP channel composed of primarily the SUR2 isoform and suggest that iPSC derived cardiomyocytes would be an effective model for studying the role of KATP during metabolic challenges.

Ischemic Postconditioning Reduces Reperfusion Arrhythmias by Adenosine Receptors and Protein Kinase C Activation but Is Independent of KATP Channels or Connexin 43

Ischemic postconditioning (IPoC) reduces reperfusion arrhythmias but the antiarrhythmic mechanisms remain unknown. The aim of this study was to analyze IPoC electrophysiological effects and the role played by adenosine A1, A2A and A3 receptors, protein kinase C, ATP-dependent potassium (KATP) channels, and connexin 43. IPoC reduced reperfusion arrhythmias (mainly sustained ventricular fibrillation) in isolated rat hearts, an effect associated with a transient delay in epicardial electrical activation, and with action potential shortening. Electrical impedance measurements and Lucifer-Yellow diffusion assays agreed with such activation delay. However, this delay persisted during IPoC in isolated mouse hearts in which connexin 43 was replaced by connexin 32 and in mice with conditional deletion of connexin 43. Adenosine A1, A2A and A3 receptor blockade antagonized the antiarrhythmic effect of IPoC and the associated action potential shortening, whereas exogenous adenosine reduced reperfusion arrhythmias and shortened action potential duration. Protein kinase C inhibition by chelerythrine abolished the protective effect of IPoC but did not modify the effects on action potential duration. On the other hand, glibenclamide, a KATP inhibitor, antagonized the action potential shortening but did not interfere with the antiarrhythmic effect. The antiarrhythmic mechanisms of IPoC involve adenosine receptor activation and are associated with action potential shortening. However, this action potential shortening is not essential for protection, as it persisted during protein kinase C inhibition, a maneuver that abolished IPoC protection. Furthermore, glibenclamide induced the opposite effects. In addition, IPoC delays electrical activation and electrical impedance recovery during reperfusion, but these effects are independent of connexin 43.

Altered K+ current profiles underlie cardiac action potential shortening in hyperkalemia and β-adrenergic stimulation

Hyperkalemia is known to develop in various conditions including vigorous physical exercise. In the heart, hyperkalemia is associated with action potential (AP) shortening that was attributed to altered gating of K+ channels. However, it remains unknown how hyperkalemia changes the profiles of each K+ current under a cardiac AP. Therefore, we recorded the major K+ currents (inward rectifier K+ current, IK1; rapid and slow delayed rectifier K+ currents, IKr and IKs, respectively) using AP-clamp in rabbit ventricular myocytes. As K+ may accumulate at rapid heart rates during sympathetic stimulation, we also examined the effect of isoproterenol on these K+ currents. We found that IK1 was significantly increased in hyperkalemia, whereas the reduction of driving force for K+ efflux dominated over the altered channel gating in case of IKr and IKs. Overall, the markedly increased IK1 in hyperkalemia overcame the relative decreases of IKr and IKs during AP, resulting in an increased net repolarizing current during AP phase 3. β-Adrenergic stimulation of IKs also provided further repolarizing power during sympathetic activation, although hyperkalemia limited IKs upregulation. These results indicate that facilitation of IK1 in hyperkalemia and β-adrenergic stimulation of IKs represent important compensatory mechanisms against AP prolongation and arrhythmia susceptibility.