Early opening of sarcolemmal ATP-sensitive potassium channels is not a key step in PKC-mediated cardioprotection

Abstract

ATP-sensitive potassium (K ATP ) channels are abundantly expressed in the myocardium.Although a definitive role for the channel remains elusive they have been implicated in the phenomenon of cardioprotection, but the precise mechanism is unclear. We set out to test the hypothesis that the channel protects by opening early during ischemia to shorten action potential duration and reduce electrical excitability thus sparing intracellular ATP. This could reduce reperfusion injury by improving calcium homeostasis.Using a combination of contractile function analysis, calcium fluorescence imaging and patch clamp electrophysiology in cardiomyocytes isolated from adult male Wistar rats, we demonstrated that the opening of sarcolemmal K ATP channels was markedly delayed after cardioprotective treatments; ischemic preconditioning, adenosine and PMA. This was due to the preservation of intracellular ATP for longer during simulated ischemia therefore maintaining sarcolemmal K ATP channels in the closed state for longer. As the simulated ischemia progressed, K ATP channels opened to cause contractile, calcium transient and action potential failure, however there was no indication of any channel activity early during simulated ischemia to impart an energy sparing hyperpolarization or action potential shortening.We present compelling evidence to demonstrate that early opening of sarcolemmal K ATP channels during simulated ischemia is not part of the protective mechanism imparted by ischemic preconditioning or other PKC-dependent cardioprotective stimuli. On the contrary, channel opening was actually delayed. We conclude that sarcolemmal K ATP channel opening is a consequence of ATP depletion, not a primary mechanism of ATP preservation in these cells. 3Highlights:• Opening of the SarcoK ATP channel was proposed to be cardioprotective• Channel opening was delayed after cardioprotective stimuli• Ca 2+ & ATP levels were maintained during ischemia independent of SarcoK ATP opening• Mitochondrial function preserved during ischemia, independent of SarcoK ATP opening• Early opening of SarcoK ATP is not involved in PKC-dependent cardioprotection

ATP-Sensitive Potassium Channels Mediate the Cardioprotective Effect of Panax notoginseng Saponins against Myocardial Ischaemia–Reperfusion Injury and Inflammatory Reaction

“…MitoK ATP is located in the inner membrane of mitochondria, whereas sarcK ATP is located in the inner membrane of cells. Although some studies have reported mitoK ATP to play the predominant role in this process [ 31 ], in our experiment, sarcK ATP may play this role. Of course, this means that Gly could inhibit the K ATP channel on both the mitochondrial and cell membrane, thus inhibiting cardioprotection from PNS.…”

A novel form of glycolytic metabolism‐dependent cardioprotection revealed by PKCα and β inhibition

“…The protocol for isolation of ventricular cardiomyoyctes was as described previously Rainbow et al 2005;Sims et al 2014;Brennan et al 2015). For rat and guinea pig cardiomyocytes isolations, the heart was rapidly excised, temporarily placed into cold, nominally Ca 2+ -free NT solution to stop contraction and quickly cannulated via the aorta on a Langendorff type apparatus.…”

“…Typically this method yielded 70-90% rod-shaped cardiomyocytes, which were stored in NT solution at room temperature and used within 8 h of isolating. Ischaemic preconditioning (IPC) was imparted to cardiomyocytes using a protocol involving three cycles of halted perfusion followed by re-perfusion (Rodrigo & Samani, 2008;Sims et al 2014;Brennan et al 2015). IPC cardiomyocytes were used up to 6 h after isolation.…”

“…Cardiomyocyte contraction and cell death was investigated using two different metabolic poisons. (i) Substrate-free metabolic inhibition Tyrode's solution (SFT-MI; containing 2 mM cyanide to inhibit oxidative phosphorylation and 1 mM iodoacetic acid to inhibit GAPDH) Rainbow et al 2004;Sims et al 2014;Brennan et al 2015). (ii) Ischaemic buffer containing (in mmol L -1 ): 108 NaCl, 16 KCl, 0.33 NaH 2 PO 4 , 10 PIPES, 20 deoxyglucose, 20 Na-lactate, 1 MgCl 2 , 2 CaCl 2 and 0.2 2,4-dinitrophenol (pH 6.8 with NaOH) (Brennan et al 2015).…”

Ischemic postconditioning and pinacidil suppress calcium overload in anoxia-reoxygenation cardiomyocytes via down-regulation of the calcium-sensing receptor

“…Many studies indicated that KATP was the end effector of many cardiac protective strategies, such as the ischemic preconditioning (Brennan et al, 2015), remote preconditioning (Hu et al, 2014b) and exercise (Kraljevic et al, 2015). KATP also contributed to ischemic myocardium protection effects of IPC.…”