Introduction: 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 signals. Effects of cMyBP-C on contraction are complex and attributable to dynamic interactions with both actin and myosin. For instance, cMyBP-C binding to actin can exert either activating or inhibitory effects on contraction by shifting tropomyosin on the thin filament or by blocking myosin heads, respectively. Recently, calmodulin (Ca2+/CaM) was also shown to bind cMyBP-C at a unique sequence within the regulatory M-domain of cMyBP-C (Lu et al, JBC 287:31596, 2012). Importantly, the binding site for CaM overlaps with a conserved actin binding site that we found was important for mediating the activating and inhibitory effects of cMyBP-C on the thin filament (Bezold et al. JBC 288: 21496, 2013).
Hypothesis: Here we hypothesized that CaM competes with actin for cMyBP-C binding and thus influences the functional effects of cMyBP-C that are mediated through the thin filament.
Methods: Recombinant N’-terminal domains of cMyBP-C were used in co-sedimentation binding assays, pull-down assays, and actomyosin ATPase assays to determine effects of CaM binding on cMyBP-C function.
Results: We found that CaM binds to N’-terminal domains of cMyBP-C in the presence of Ca2+ (Ca2+/CaM) with an affinity comparable to cMyBP-C binding to actin (3-10 UM). We further show that Ca2+/CaM reduces apparent binding affinity of cMyBP-C for actin, consistent with Ca2+/CaM competing with actin for binding to cMyBP-C. Also consistent with reduced binding of cMyBP-C to actin, Ca2+/CaM reversed the inhibitory effects of cMyBP-C N’-terminal domains on actin activated myosin ATPase rates. However, apo-CaM showed reduced binding affinity for cMyBP-C, did not compete with actin, and did not reverse the activating effects of cMyBP-C on the thin filament at low Ca2+.
Conclusions: These data suggest that Ca2+/CaM is a novel modulator of cMyBP-C function that can temporally tune the complex effects of cMyBP-C on contraction, potentially as Ca2+ rises and falls during a single heartbeat.