Molecular Screen Identifies Cardiac Myosin-Binding Protein-C as a Protein Kinase G-Iα Substrate

Background-Pharmacological activation of cGMP-dependent protein kinase G I (PKGI) has emerged as a therapeutic strategy for humans with heart failure. However, PKG-activating drugs have been limited by hypotension arising from PKG-induced vasodilation. PKGI alpha antiremodeling substrates specific to the myocardium might provide targets to circumvent this limitation, but currently remain poorly understood. Methods and Results-We performed a screen for myocardial proteins interacting with the PKGI alpha leucine zipper (LZ)-binding domain to identify myocardial-specific PKGI alpha antiremodeling substrates. Our screen identified cardiac myosin-binding protein-C (cMyBP-C), a cardiac myocyte-specific protein, which has been demonstrated to inhibit cardiac remodeling in the phosphorylated state, and when mutated leads to hypertrophic cardiomyopathy in humans. GST pulldowns and precipitations with cGMP-conjugated beads confirmed the PKGI alpha-cMyBP-C interaction in myocardial lysates. In vitro studies demonstrated that purified PKGI alpha phosphorylates the cMyBP-C M-domain at Ser-273, Ser-282, and Ser-302. cGMP induced cMyBP-C phosphorylation at these residues in COS cells transfected with PKGI alpha, but not in cells transfected with LZ mutant PKGI alpha, containing mutations to disrupt LZ substrate binding. In mice subjected to left ventricular pressure overload, PKGI alpha activation with sildenafil increased cMyBP-C phosphorylation at Ser-273 compared with untreated mice. cGMP also induced cMyBP-C phosphorylation in isolated cardiac myocytes. Conclusions-Taken together, these data support that PKGI alpha and cMyBP-C interact in the heart and that cMyBP-C is an anti remodeling PKGI alpha kinase substrate. This study provides the first identification of a myocardial-specific PKGI alpha LZ-dependent antiremodeling substrate and supports further exploration of PKGI alpha myocardial LZ substrates as potential therapeutic targets for heart failure.