Ca2+/CaM Modulates the Functional Effects of cMyBP-C on the Thin Filament
PublisherThe University of Arizona.
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AbstractBackground. Cardiac myosin binding protein-C (cMyBP-C) is an essential regulator of heart muscle function that is necessary for both normal contraction and for increased contractility in response to inotropic stimuli(1-7). Effects of cMyBP-C on contraction are due to dynamic interactions of cMyBP-C with both actin and myosin, but the mechanism(s) by which cMyBP-C binding to these ligands is modulated are only partly understood. Recently, calmodulin (CaM) was shown to bind to cMyBP-C in the regulatory M-domain(8, 9) near a conserved actin binding site(10). Here we investigated whether CaM competes with actin for binding to cMyBP-C and thus whether CaM affects cMyBP-C function. Methods. Recombinant N’-terminal domains of cMyBP-C were used in pull-down assays, co-sedimentation binding assays, and actin activated myosin ATPase assays to determine effects of CaM binding on cMyBP-C. Results. In accordance with previous reports, we found that CaM binds to N’-terminal domains of cMyBP-C in the presence of Ca2+ (Ca2+/CaM) with a binding affinity comparable to cMyBP-C binding to actin (3-10 μM). We further show that Ca2+/CaM reduces cMyBP-C apparent binding affinity for actin, consistent with the competition between Ca2+/CaM and actin for binding to cMyBP-C. Ca2+/CaM also reversed the inhibitory effects of cMyBP-C N’-terminal domains on actin activated myosin ATPase rates, consistent with reduced cMyBP-C interactions with actin. However, apo-CaM (calcium-free calmodulin) did not influence the ability of cMyBP-C to activate actomyosin ATPase rates at low Ca2+. Phosphorylation of cMyBP-C by PKA significantly increased its binding to both Ca2+/CaM and apo-CaM. Phosphorylated cMyBP-C was also a less potent regulator of cross-bridge cycling in ATPase assays. Conclusions. These data demonstrate that Ca2+/CaM competes with actin for binding to cMyBP-C and selectively reverses the inhibitory effects of cMyBP-C on actomyosin interactions. However, apo-CaM does not compete with cMyBP-C binding to actin and does not affect the ability of cMyBP-C to activate the thin filament at low Ca2+. Ca2+/CaM may serve as additional regulatory mechanism in addition to phosphorylation of cMyBP-C to regulate its function. These data suggest that Ca2+/CaM is a novel modulator of cMyBP-C function that can dynamically tune cMyBP-C effects on contraction potentially as [Ca2+]i rises and falls during the time course of a single heart beat.
Degree ProgramGraduate College
Cellular and Molecular Medicine