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Abstract
Voltage-dependent Ca2+ channels and store-operated Ca2+ channels (SOCs) are the major routes of Ca2+ entry into mammalian cells. Previously, we reported that pharmacological preconditioning (PPC) leads to a decrease in the amplitude of L-type calcium channel current in the heart. In this study, we examined PPC-associated changes in SOC function. We measured adult cardiomyocyte membrane currents using the whole-cell patch-clamp technique, and we evaluated reactive oxygen species (ROS) production and intracellular Ca2+ levels in cardiomyocytes using fluorescent probes. Diazoxide (Dzx) and thapsigargin (Tg) were used to induce PPC and to deplete internal stores of Ca2+, respectively. Ca2+ store depletion generated inward currents with strong rectification, which were suppressed by the SOC blocker GSK-7975-A. These currents were completely abolished by PPC, an effect that could be countered with 5-hydroxydecanoate (5-HD; a selective mitochondrial ATP-sensitive K+ channel blocker), an intracellular mitochondrial energizing solution, or Ni2+ [a blocker of sodium–calcium exchanger (NCX)]. Buffering of ROS and intracellular Ca2+ also prevented PPC effects on SOC currents. Refilling of intracellular stores was largely suppressed by PPC, as determined by measuring intracellular Ca2+ with a fluorescent Ca2+ indicator. These results indicate that influx of Ca2+ through SOCs is inhibited by their ROS and Ca2+-dependent inactivation during PPC and that NCX is a likely source of PPC-inactivating Ca2+. We further showed that NCX associates with Orai1. Down-regulation of SOCs by PPC may play a role in cardioprotection following ischemia–reperfusion.
Highlights
Severe damage to heart muscle caused by ischemia and reperfusion (I/R) has been shown to involve Ca2+ overload during reperfusion (Murphy and Steenbergen, 2008)
In experiments testing whether pharmacological preconditioning (PPC)-related elevation of [Ca2+]i plays a role in SOC inactivation, we found that Tginduced inward currents were not inhibited by PPC when myoplasmic Ca2+ was buffered with BAPTA-acetoxymethyl ester (AM) (Figure 1F)
The main findings of this work are that PPC results in a large inhibition of SOC currents upon depletion of sarcoplasmic reticulum (SR), and that this effect is due to reactive oxygen species (ROS) and Ca2+-dependent channel inactivation
Summary
Severe damage to heart muscle caused by ischemia and reperfusion (I/R) has been shown to involve Ca2+ overload during reperfusion (Murphy and Steenbergen, 2008). Mitochondria are key participants and regulators of myocardial injury during I/R. I-induced mitochondrial stress leads to the onset of mitochondrial permeability transition pore (MPTP) opening, release of cytochrome c through MPTPs, and, eventually, consequent cell death during reperfusion (Lesnefsky et al, 2017). Ischemia and reperfusion injury can be mitigated by preconditioning with brief periods of ischemia (Murry et al, 1986) or by pharmacological preconditioning (PPC) with agents, such as diazoxide (Dzx), that open mitochondrial ATP-sensitive K+ channels (mitoKATPs). Diazoxide and SOCs in Heart (Garlid et al, 1997; Pain et al, 2000). Blockade of mitoKATP channels prevents both ischemic preconditioning and PPC (Ardehali and O’Rourke, 2005; Halestrap et al, 2007). Pharmacological activation of mitoKATPs depends on reactive oxygen species (ROS) generated in mitochondria during preconditioning, which act to prevent mitochondrial Ca2+
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