Background/Objective: Reperfusion after ischemia can result in cytosolic and mitochondrial Ca overload in cardiomyocytes leading to contractile dysfunction, arrhythmias and cell death. Recently, the protein kinase RNA-like endoplasmic reticulum kinase (PERK) has been shown to increase cytoplasmic Ca levels after activation by endoplasmic reticulum stress in cardiomyocytes of rats with streptozotocin-induced diabetic cardiomyopathy. PERK is also known to be activated by ischemia/reperfusion. We tested the hypothesis that PERK inhibition may reduce cellular Ca overload in a cardiomyocyte model of reperfusion injury by hypoxia/reoxygenation (H/R).

Methods: Ventricular cardiomyocytes were isolated from adult Sprague Dawley rats (n=7). Ischemia was simulated by hypoxia (0.5% O₂/5% CO₂/94.5% N₂ at 37°C) and starvation (buffer containing 20mM 2-Deoxyglucose) for a total of 2 hours. Reoxygenation was accomplished at 95% air/5% CO₂ at 37°C for 15 min in Media 199. Controls were incubated in 95% air/5% CO₂ at 37°C in Media 199. Mitochondrial and cytosolic Ca was simultaneously measured in myocytes (Rhod-2-AM, 5µM, 120 min at 4°C cold loading, Fluo-4-AM, 10µM, 12.5 min at 25°C) by confocal laser scanning microscopy using dual excitation (488nm/555nm). Emission light was split into two beams using a variable secondary dichroic beamsplitter (set to 555nm) and recorded using two photomultipliers, each with an optical filter (PMT1: SP 555 d10, PMT2: LP 560 d10). For some experiments, mitochondrial-specific loading of Rhod-2-AM was confirmed by co-loading with Mitotracker Green FM (fig. 1A, 200nM, 30min at 37°C). Statistical analysis was conducted with a one-way ANOVA test for repeated measures with Sidak’s post-hoc correction. 

Results: Co-loading with Mitotracker Green revealed a high enrichment of Rhod-2 fluorescence in mitochondria (fig. 1A, original frame scans). Expectedly, exposure of myocytes to H/R significantly increased both cytosolic and mitochondrial Ca (original frame scans and mean data in fig. 1B&C, n=7 rats, p<0.05 each). Interestingly, pre-incubation with the selective PERK inhibitor GSK 2656157 (2 µM) completely abolished the H/R-dependent increase in cytosolic Ca (fig. 1B&C, p<0.05 vs. H/R vehicle) but did not inhibit the increase in mitochondrial Ca (fig. 1B&C, p=0.97 vs. H/R vehicle).

Conclusion: PERK inhibition reduces H/R-dependent cytoplasmic Ca overload, which may have therapeutic implications.