Functional Antagonism\nof Protein Kinase C and A Phosphorylation\nof Cardiac Myosin Binding Protein‑C

Heart\nmuscle systolic and diastolic function is controlled on a\nbeat-to-beat basis by the calcium-dependent activation of the contractile\nmyofilaments but modulated by neurohumoral signaling pathways coupled\nto the activation of intracellular effector molecules such as protein\nkinases. Phosphorylation of myofilament regulatory proteins such as\ncardiac troponin I (cTnI) and cardiac myosin binding protein-C (cMyBP-C)\nhas important regulatory function for the heart by controlling both\ncardiac inotropy and lusitropy. Sympathetic signaling activates both\nα- and β-adrenergic receptors on the surface of cardiomyocytes,\nwhich leads to an increase in cMyBP-C phosphorylation via protein\nkinase C (PKC)/D (PKD) and protein kinase A (PKA) signaling, respectively.\nHowever, the functional interactions between the PKC/PKD and PKA phosphorylation\nsites on cMyBP-C have remained uncharacterized. Here, using a combination\nof site-specific phosphorylation of recombinant N-terminal domains\nof cMyBP-C and in situ functional assays, we show that the PKC/PKD\nand PKA phosphorylation sites have antagonistic effects on myofilament\nactivation. PKA phosphorylation on multiple sites in the N-terminal\ndomains of cMyBP-C reduces both its activating and inhibiting effect\non myofilament activation in the absence and presence of activator\nCa<sup>2+</sup>, respectively. In contrast, PKC phosphorylation increases\nmyofilament activation and blunts the inhibitory effect of PKA phosphorylation.\nOur results lead to a new model of phosphoregulation of cMyBP-C with\nimportant implications for both health and disease states of the heart.