Abstract-Normal cardiac function requires dynamic modulation of contraction. ␤1-Adrenergic-induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non-PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, k rel (s-1), and delayed force development, k df (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline k rel and k df similar to WT myocardium, but, unlike WT, k rel and k df were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction. Key Words: cMyBP-C Ⅲ phosphorylation Ⅲ contraction kinetics T he strength and kinetics of cardiac contraction vary on a beat-to-beat basis as a way to match cardiac output to the circulatory demands of the body. Reduced capacity to modulate contraction has long been recognized as a key feature of dysfunction in heart failure 1 and more recently in hypertrophic cardiomyopathy (HCM). 2 This study explores the possibility that phosphorylation of cardiac myosin binding protein (cMyBP)-C modulates contraction in skinned and living myocardium.MyBP-C is a component of the thick filament in striated muscle 3 and is evident as 7 to 9 bands at 43-nm intervals 4 within the center of each half-thick filament in the A-band. Its location at every third crossbridge crown, ie, every 42.9 nm 5 suggests that cMyBP-C has a regulatory role with respect to thick filament activity. Unlike the skeletal muscle isoform, cMyBP-C is readily phosphorylated by protein kinase (PK)A, 6,7 calcium calmodulin kinase (CaMK) II, 6,7 and PKC. 8,9 Phosphorylation of cMyBP-C may promote actin-myosin interactions by either relieving a structural constraint on myosin to allow closer proximity with actin 10,11 or reducing the binding of cMyBP-C to the S2 domain of myosin to allow greater flexibility...

A normal heart increases its contractile force with increasing heart rate. Although calcium handling and myofibrillar proteins have been implicated in maintaining this positive force-frequency relationship (FFR), the exact mechanisms by which it occurs have not been addressed. In this study, we have developed an analytical method to define the calcium-force loop data, which characterizes the function of the contractile proteins in response to calcium that is independent of the calcium handling proteins. Results demonstrate that increasing the stimulation frequency causes increased force production per unit calcium concentration and decreased frequency-dependent calcium sensitivity during the relaxation phase. We hypothesize that phosphorylation of myosin binding protein-C (MyBP-C) and troponin I (TnI) acts coordinately to change the rates of force generation and relaxation, respectively. To test this hypothesis, we performed simultaneous calcium and force measurements on stimulated intact mouse papillary bundles before and after inhibition of MyBP-C and TnI phosphorylation using the calcium/calmodulin kinase II (CaMK2)

Background Heart failure with preserved ejection fraction (HFpEF) accounts for approximately 50% of all cases of heart failure and currently has no effective treatment. Diastolic dysfunction underlies HFpEF; therefore, elucidation of the mechanisms that mediate relaxation can provide new potential targets for treatment. Cardiac myosin binding protein-C (cMyBP-C) is a thick filament protein that modulates cross-bridge cycling rates via alterations in its phosphorylation status. Thus, we hypothesize that phosphorylated cMyBP-C accelerates rate of cross-bridge detachment, thereby enhancing relaxation to mediate diastolic function. Methods and Results We compared mouse models expressing phosphorylation deficient cMyBP-C(S273A/S282A/S302A)-cMyBP-C(t3SA), phosphomimetic cMyBP-C(S273D/S282D/S302D)-cMyBP-C(t3SD), and WT-control cMyBP-C(tWT) to elucidate the functional effects of cMyBP-C phosphorylation. Decreased voluntary running distances, increased lung/body weight ratios, and increased brain natriuretic peptide (BNP) levels in cMyBP-C(t3SA) mice demonstrate that phosphorylation deficiency is associated with signs of heart failure. Echocardiography (ejection fraction, myocardial relaxation velocity) and pressure/volume measurements (−dP/dtmin, pressure decay time constant Tau-Glantz, passive filling stiffness) show that cMyBP-C phosphorylation enhances myocardial relaxation in cMyBP-C(t3SD) mice while deficient cMyBP-C phosphorylation causes diastolic dysfunction with preserved ejection fraction in cMyBP-C(t3SA) mice. Simultaneous force and [Ca2+]i measurements on intact papillary muscles show that enhancement of relaxation in cMyBP-C(t3SD) mice and impairment of relaxation in cMyBP-C(t3SA) mice are not due to altered [Ca2+]i handling, implicating that altered cross-bridge detachment rates mediate these changes in relaxation rates. Conclusions cMyBP-C phosphorylation enhances relaxation while deficient phosphorylation causes diastolic dysfunction and phenotypes resembling HFpEF. Thus, cMyBP-C is a potential target for treatment of HFpEF.

Contraction and relaxation are fundamental aspects of cardiomyocyte functional biology. They reflect the response of the contractile machinery to the systolic increase and diastolic decrease of the cytoplasmic Ca(2+) concentration. The analysis of contractile function and Ca(2+) transients is therefore important to discriminate between myofilament responsiveness and changes in Ca(2+) homeostasis. This article describes an automated technology to perform sequential analysis of contractile force and Ca(2+) transients in up to 11 strip-format, fibrin-based rat, mouse, and human fura-2-loaded engineered heart tissues (EHTs) under perfusion and electrical stimulation. Measurements in EHTs under increasing concentrations of extracellular Ca(2+) and responses to isoprenaline and carbachol demonstrate that EHTs recapitulate basic principles of heart tissue functional biology. Ca(2+) concentration-response curves in rat, mouse, and human EHTs indicated different maximal twitch forces (0.22, 0.05, and 0.08 mN in rat, mouse, and human, respectively; P < 0.001) and different sensitivity to external Ca(2+) (EC50: 0.15, 0.39, and 1.05 mM Ca(2+) in rat, mouse, and human, respectively; P < 0.001) in the three groups. In contrast, no difference in myofilament Ca(2+) sensitivity was detected between skinned rat and human EHTs, suggesting that the difference in sensitivity to external Ca(2+) concentration is due to changes in Ca(2+) handling proteins. Finally, this study confirms that fura-2 has Ca(2+) buffering effects and is thereby changing the force response to extracellular Ca(2+).