Abstract 9506: Calpain Activation in Early Reperfusion Triggers Pyroptosis Resulting in Myocardial Ischemia/Reperfusion Injury

Abstract

Sterile inflammation contributes to reperfusion injury. During myocardial ischemia/reperfusion (I/R), inflammasomes proteolytically activate caspase-1 (cas-1) which then kills cardiomyocytes by liberating N-terminal fragments of gasdermin D which form large pores in the sarcolemma (pyroptosis). We and others find hearts from cas-1 knockout (KO) mice have less myocardial infarction (MI) after I/R than those of wild type (WT) hearts. Pharmacologic cas-1 blockade when administered prior to ischemia or at the onset of reperfusion provides similar protection against MI but protection is lost when blockade is started 10min after reperfusion revealing that cas-1 does its killing in the first 10min of reperfusion. We wondered what could trigger cas-1 activation at reperfusion. Ca ++ floods myocytes at reperfusion making Ca ++ a suspect. We tested if adenyl cyclase isoform 10 (AC10), which is stimulated by Ca ++ and in one study promoted I/R injury in cardiomyocytes, could be responsible. However, there were no differences in infarct size among AC10 -/- KO, AC10 -/+ heterozygous, and WT buffer-perfused mouse hearts after 50min global ischemia/2h reperfusion. Ca ++ also activates calpain which releases membrane-bound inactive procas-1 which is required for infammasome assembly. Adding calpain antagonist calpeptin (CPT) to the perfusate of isolated AC10 -/+ hearts reduced MI from 90% of risk zone to 55% (p<0.001). Cas-1 KO mouse hearts were similarly protected after I/R (40% infarction) and treating KO hearts with CPT resulted in no added protection suggesting both cas-1 and calpain are steps in the same mechanism of cell killing. This result was amplified by lack of added protection when mice received combined CPT and emricasan (EMRI), a pan-cas blocker. In contrast, EMRI plus preconditioning does give additive protection. Hence calpain activity in the initial minutes of reperfusion is also involved in injurious pyroptosis and may be a viable target in treatment of acute MI.