2161Redox-mediated PKA-RIalpha localisation to the lysosome inhibits myocardial calcium release and robustly reduces myocardial injury

Abstract

Abstract Background Kinase oxidation is a critical signalling mechanism through which changes in the intracellular redox state alter cardiac function. In the myocardium, type-1 protein kinase A (PKARIα) can be reversibly oxidised, forming interprotein disulphide bonds within the holoenzyme complex. However, the effect of PKARIα oxidation on downstream signalling in the heart, particularly under states of oxidative stress, remains unexplored. Purpose To determine the direct functional consequences of PKARIα oxidation in the heart and investigate their impact on ischaemia/reperfusion (I/R) injury. Methods and results Experiments using the AKAR3ev FRET biosensor in murine left ventricular (LV) myocytes and Fluorescence Recovery After Photobleaching (FRAP) of GFP-tagged wild-type (WT) and mutant RIα proteins expressed in RIα-null fibroblasts showed that PKARIα oxidation does not increase the kinases' catalytic activity, but enhances its binding to A-kinase anchoring proteins (AKAP; n=30–39/N=3, p<0.01). Super-resolution microscopy revealed localisation of oxidised PKARIα to lysosomes in WT myocytes, which was completely absent in “redox dead” Cys17Ser PKARIα knock-in mice (KI; panel A; n=38–41/N=3, p<0.01) and reduced when AKAP binding was prevented using the RIAD disruptor peptide (30.6±5.1% reduction; n=35–37/N=3, p<0.01). Displacement of PKARIα from lysosomes resulted in spontaneous sarcoplasmic reticulum calcium release and dramatic calcium oscillations in KI LV myocytes (panel B), which were preventable by ryanodine receptor blockade (1 mM tetracaine; n=14, p<0.01), acute depletion of endolysosomal calcium stores (100 nM bafilomycin; n=7; p<0.01), or lysosomal two-pore channel inhibition (5 μM Ned-19; n=9; p<0.05). I/R (secondary to cardiopulmonary bypass) was found to induce PKARIα oxidation in the myocardium of patients undergoing cardiac surgery (panel C; n=18, p<0.05). Absence of this response in KI mouse hearts resulted in 2-fold larger infarcts (p<0.01) and a concomitant reduction in LV contractile recovery (final LVDP of 55.9±8.6 vs 82.5±7.1 mmHg in WT; n=7–8, p<0.05), both which were prevented by addition of Ned-19 at the time of reperfusion (panel D; n=4, p<0.01). Conclusions Oxidised PKARIα acts as a potent inhibitor of intracellular calcium release in the heart through its redox-dependent interaction with the lysosome. In the setting of I/R, where PKARIα oxidation is induced, this regulatory mechanism is critical for protecting the heart from injury and offers a novel target for the design of cardioprotective therapeutics. Acknowledgement/Funding British Heart Foundation CH/12/3/29609, RG/16/12/32451; Garfield-Weston Foundation MPS/IVIMS-11/12-4032; Wellcome Trust Fellowship 0998981Z/12/Z