Receptor-Independent Cardiac Protein Kinase Cα Activation by Calpain-Mediated Truncation of Regulatory Domains

Rationale: Protein kinase (PK)Cs and calpain cysteine proteases are highly expressed in myocardium. Ischemia produces calcium overload that activates calpains and conventional PKCs. However, calpains can proteolytically process PKCs, and the potential in vivo consequences of this interaction are unknown. Objective: To determine the biochemical and pathophysiological consequences of calpain-mediated cardiac PKC alpha proteolysis. Methods and Results: Isolated mouse hearts subjected to global ischemia/reperfusion demonstrated cleavage of PKC alpha. Calpain 1 overexpression was not sufficient to produce PKC alpha cleavage in normal hearts, but ischemia-induced myocardial PKC alpha cleavage and myocardial injury were greatly increased by cardiac-specific expression of calpain 1. In contrast, calpain 1 gene ablation or inhibition with calpastatin prevented ischemia/reperfusion induced PKC alpha cleavage; infarct size was decreased and ventricular function enhanced in infarcted calpain 1 knockout hearts. To determine consequences of PKC alpha fragmentation on myocardial protein phosphorylation, transgenic mice were created conditionally expressing full-length PKC alpha or its N-terminal and C-terminal calpain 1 cleavage fragments. Two-dimensional mapping of ventricular protein extracts showed a distinct PKC alpha phosphorylation profile that was exaggerated and distorted in hearts expressing the PKC alpha C-terminal fragment. MALDI mass spectroscopy revealed hyperphosphorylation of myosin-binding protein C and phosphorylation of atypical substrates by the PKC alpha C-terminal fragment. Expression of parent PKC alpha produced a mild cardiomyopathy, whereas myocardial expression of the C-terminal PKC alpha fragment induced a disproportionately severe, rapidly lethal cardiomyopathy. Conclusions: Proteolytic processing of PKC alpha by calcium-activated calpain activates pathological cardiac signaling through generation of an unregulated and/or mistargeted kinase. Production of the PKC alpha C-terminal fragment in ischemic hearts occurs via a receptor-independent mechanism. (Circ Res. 2010;107:903-912.)