Calpains and proteasomes mediate degradation of ryanodine receptors in a model of cardiac ischemic reperfusion |
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Authors: | Zully Pedrozo Gina Sánchez Natalia Torrealba Rodrigo Valenzuela Carolina Fernández Cecilia Hidalgo Sergio Lavandero Paulina Donoso |
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Institution: | 1. Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile;2. Centro de Estudios Moleculares de la Célula, Facultad de Medicina, Universidad de Chile, Santiago, Chile;3. Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile |
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Abstract: | Type-2 ryanodine receptors (RyR2) – the calcium release channels of cardiac sarcoplasmic reticulum – have a central role in cardiac excitation–contraction coupling. In the heart, ischemia/reperfusion causes a rapid and significant decrease in RyR2 content but the mechanisms responsible for this effect are not fully understood. We have studied the involvement of three proteolytic systems – calpains, the proteasome and autophagy – on the degradation of RyR2 in rat neonatal cardiomyocyte cultures subjected to simulated ischemia/reperfusion (sI/R). We found that 8 h of ischemia followed by 16 h of reperfusion decreased RyR2 content by 50% without any changes in RyR2 mRNA. Specific inhibitors of calpains and the proteasome prevented the decrease of RyR2 caused by sI/R, implicating both pathways in its degradation. Proteasome inhibitors also prevented the degradation of calpastatin, the endogenous calpain inhibitor, hindering the activation of calpain induced by calpastatin degradation. Autophagy was activated during sI/R as evidenced by the increase in LC3-II and beclin-1, two proteins involved in autophagosome generation, and in the emergence of GFP-LC3 containing vacuoles in adenovirus GFP-LC3 transduced cardiomyocytes. Selective autophagy inhibition, however, induced even further RyR2 degradation, making unlikely the participation of autophagy in sI/R-induced RyR2 degradation. Our results suggest that calpain activation as a result of proteasome-induced degradation of calpastatin initiates RyR2 proteolysis, which is followed by proteasome-dependent degradation of the resulting RyR2 fragments. The decrease in RyR2 content during ischemia/reperfusion may be relevant to the decrease of heart contractility after ischemia. |
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