Abstract 3391: A Constitutively Active Kinase Produced During Ischemia by Proteolytic Processing of PKCα is Cardiomyopathic
Myocardial ischemia causes calcium overload, activating protein kinase C (PKC) and calcium-dependent proteases (calpains). Roles for calpains in myocardial stunning, and PKCα in contractile dysfunction, are known; a functional interaction in cardiac signaling has not been described. PKCs are “hinged” molecules in which N-terminal regulatory/RACK binding and C-terminal catalytic domains interact to prevent spontaneous PKC substrate phosphorylation. Calcium and phospholipid reduce these intra-molecular interactions, translocating PKCs to subcellular domains and permitting phosphorylation of local substrates.
In vitro studies: Purified calpain 1 cleaved PKCα at the hinge region, releasing two ~40 kDa fragments. Relieved of normal intra-molecular inhibition, the C-terminal catalytic domain can be a promiscuous, constitutively active kinase. Alanine mutagenesis within the hinge region abolished calpain-mediated PKCα proteolysis.
In vivo studies: Transgenic mouse hearts conditionally overexpressing calpain 1 in the heart exhibited proteolytic PKCα cleavage after global ischemia, whereas hearts expressing calpastatin (endogenous calpain inhibitory protein) did not. Calpain 1 transgenic mice had increased mortality (13/19 dead) within 24 hours of experimental myocardial infarction, increased remodeling, and larger infarcts (28±4% vs 17±3% control, P=0.048). To determine if ischemic sensitivity was related to proteolytic processing of PKCα, full-length PKCα, the N-terminal (PKCαNT), or the C-terminal (PKCαCT) fragments were conditionally expressed in mouse hearts. Overexpressed full-length PKCα caused an asymptomatic dilated cardiomyopathy (%FS 35±2 vs 63±2 control, P<0.001) and insensitivity to beta-adrenergic stimulation. Similar levels of PKCα NT did not alter heart structure or function. PKCαCT for the same period, at a fraction of those levels, caused massive bi-ventricular dilation, severe systolic dysfunction (%FS=16±1 vs 70±1 in controls, p<0.001), and premature mortality due to heart failure.
Conclusion: This is an entirely new mechanism for post-ischemic cardiomyopathy, wherein myocardial calpain activation by calcium overload cleaves PKCα and produces a damaging, constitutively active rogue kinase.