Invasive hemodynamic studies and M-mode echocardiography were performed using standard techniques. prevented ischemia-reperfusion induced PKC cleavage; infarct size was decreased and ventricular function enhanced in infarcted calpain 1 knockout hearts. To determine consequences of PKC fragmentation on myocardial protein phosphorylation, transgenic mice were created conditionally expressing full length PKC or its N-terminal and C-terminal calpain 1 cleavage fragments. Two-dimensional mapping of ventricular protein extracts showed a distinct PKC phosphorylation profile that was exaggerated and distorted in hearts expressing the PKC C-terminal fragment. MALDI mass spectroscopy revealed hyper-phosphorylation of MyBP-C and phosphorylation of atypical substrates by the PKC C-terminal fragment. Expression of parent PKC produced a mild cardiomyopathy, whereas myocardial expression of the C-terminal PKC fragment induced a disproportionately severe, rapidly lethal cardiomyopathy. == Conclusions == Proteolytic processing of PKC by calcium-activated calpain activates pathological cardiac signaling through generation of an unregulated and/or mistargeted kinase. Production of the PKC C-terminal fragment in ischemic hearts occurs via a receptor-independent mechanism. Keywords:Protein kinase C, calpain 1, cardiomyopathy, ischemia-reperfusion injury, myocardial infarction myosin binding protein C == Introduction == Receptor-mediated activation of protein kinase Cs (PKCs) via Gq and phospholipase C signaling is usually implicated in myocardial contractile abnormalities, cardiac hypertrophy, and the progression to heart failure1,2. Of the twelve acknowledged PKC isoenzymes3, myocardium expresses significant amounts of four: two conventional isoenzymes (PKC and PKC) and two novel isoemzymes (PKC and PKC). PKC is the most abundant PKC in mouse myocardium, and is specifically implicated in cardiac hypertrophy, contractile MTC1 dysfunction and cardiomyopathy development48. The canonical mechanism for PKC activation is usually via phospholipase C- mediated increases in cytosolic free calcium and phospholipids. Walsh and co-workers BML-277 have suggested that oxidative stress can stimulate a pathway to PKC activation that is impartial of these receptor-coupled signaling events, although specific mechanisms were not identified9. PKCs consist of an N-terminal regulatory domain name connected to a C-terminal catalytic domain name via an inter-domain V3 hinge region10. In their inactive state, the N-terminal regulatory and C-terminal catalytic domains interact to sequester crucial BML-277 membrane targeting and substrate-binding domains. Conventional PKC isoenzymes, including PKC, are activated when calcium and phospholipid bind to specific domains within the N-terminus and disrupt intra-molecular bonding, thus unfolding the molecule and exposing C-terminal substrate binding domains11. Conventional wisdom holds that concomitant with PKC activation, calcium and phospholipid initiate PKC inactivation through calpain-mediated proteolysis1214. Calpains are ubiquitous calcium-dependent cysteine proteases15. We previously detected low levels of calpain 1, but not calpain 2, proteolytic activity in non-stressed myocardium16. However, myocardial free calcium levels rise dramatically to 6001000 nmol/L (depending upon perfusate calcium concentration) during ischemia-reperfusion17, exceeding levels that induce calpain activation in cardiac myocytes18. For this reason, calpain-mediated degradation of myofilament proteins has been implicated in post-ischemic myocardial injury and stunning1922and in preload-induced proteolysis23. Calpain proteolysis of myocardial PKC has not been examined. Here, we show that calpain 1 cleaves myocardial PKC as a specific response to ischemia-reperfusion. We demonstrate that this C-terminal PKC calpain cleavage fragment is a potent myocardial kinase. Finally, we determine that this PKC C-terminal fragment, but not the N-terminal fragment, induces cardiomyopathic features, impartial of myocardial ischemia or calpain activation. == Methods == == Transgenic mice == Mice conditionally overexpressing calpain 1 with a tetracycline-suppressible (tet-off) -myosin heavy chain-driven conditional transgenic system were described previously16. The current studies used the lower-expressing L2 line with calpain induction at 8 weeks of age. This line of calpain 1 transgenic mice does not exhibit spontaneous proteolysis and does not develop BML-277 a cardiac phenotype after transgene induction in adult hearts. Calpain 1 knockout mice were previously described24. Human PKC and fragments encoding peptides corresponding to the calpain N-terminal cleavage fragment (PKCNT) and C-terminal cleavage fragment (PKCCT) were expressed using the same conditional cardiac-specific expression system. Invasive hemodynamic studies and M-mode echocardiography were performed using standard techniques. Isolated perfused mouse hearts underwentex vivoischemia-reperfusion injury as described25. Mice were housed and studied according to procedures approved by Animal Studies Committee at Washington University School of Medicine. == Immunoblot analysis == SDS-PAGE and immunoblotting used standard techniques. Antibodies used were: anti-PKC C-terminus (Santa Cruz, sc-208; recognizes an epitope at the C-terminus of human PKC), anti-PKC (Santa Cruz, sc-80; recognizes an epitope within the N-terminal hinge region (residues 292-317) of human PKC), anti-MYBPC3.