Oxidation of Protein Kinase A Regulatory Subunit PKARI? Protects Against Myocardial Ischemia-Reperfusion Injury by Inhibiting Lysosomal-Triggered Calcium Release.

ABSTRACT:

Background

Kinase oxidation is a critical signaling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, PKARI? (type-1 protein kinase A) can be reversibly oxidized, forming interprotein disulfide bonds in the holoenzyme complex. However, the effect of PKARI? disulfide formation on downstream signaling in the heart, particularly under states of oxidative stress such as ischemia and reperfusion (I/R), remains unexplored.

Methods

Atrial tissue obtained from patients before and after cardiopulmonary bypass and reperfusion and left ventricular (LV) tissue from mice subjected to I/R or sham surgery were used to assess PKARI? disulfide formation by immunoblot. To determine the effect of disulfide formation on PKARI? catalytic activity and subcellular localization, live-cell fluorescence imaging and stimulated emission depletion super-resolution microscopy were performed in prkar1 knock-out mouse embryonic fibroblasts, neonatal myocytes, or adult LV myocytes isolated from "redox dead" (Cys17Ser) PKARI? knock-in mice and their wild-type littermates. Comparison of intracellular calcium dynamics between genotypes was assessed in fura2-loaded LV myocytes, whereas I/R-injury was assessed ex vivo.

Results

In both humans and mice, myocardial PKARI? disulfide formation was found to be significantly increased (2-fold in humans, P=0.023; 2.4-fold in mice, P<0.001) in response to I/R in vivo. In mouse LV cardiomyocytes, disulfide-containing PKARI? was not found to impact catalytic activity, but instead led to enhanced AKAP (A-kinase anchoring protein) binding with preferential localization of the holoenzyme to the lysosome. Redox-dependent regulation of lysosomal two-pore channels by PKARI? was sufficient to prevent global calcium release from the sarcoplasmic reticulum in LV myocytes, without affecting intrinsic ryanodine receptor leak or phosphorylation. Absence of I/R-induced PKARI? disulfide formation in "redox dead" knock-in mouse hearts resulted in larger infarcts (2-fold, P<0.001) and a concomitant reduction in LV contractile recovery (1.6-fold, P<0.001), which was prevented by administering the lysosomal two-pore channel inhibitor Ned-19 at the time of reperfusion.

Conclusions

Disulfide modification targets PKARI? to the lysosome, where it acts as a gatekeeper for two-pore channel-mediated triggering of global calcium release. In the postischemic heart, this regulatory mechanism is critical for protection from extensive injury and offers a novel target for the design of cardioprotective therapeutics.

SUBMITTER: Simon JN 

PROVIDER: S-EPMC7846288 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature