Decreased expression of cardiac myosin binding protein-C (cMyBP-C) in the myocardium is thought to be a contributing factor to hypertrophic cardiomyopathy in humans, and the initial molecular defect is likely abnormal cross-bridge (XB) function which leads to impaired force generation, decreased contractile performance, and hypertrophy in vivo. The myosin activator omecamtiv mecarbil (OM) is a pharmacological drug that specifically targets the myosin XB and recent evidence suggests that OM induces a significant decrease in vitro motility velocity and an increase in the XB duty cycle. Thus, the molecular effects of OM maybe beneficial in improving contractile function in skinned myocardium lacking cMyBP-C because absence of cMyBP-C in the sarcomere accelerates XB kinetics and enhances XB turnover rate, which presumably reduces contractile efficiency. Therefore, parameters of XB function were measured in skinned myocardium lacking cMyBP-C prior to and following OM incubation. We measured ktr, the rate of force redevelopment as an index of XB transition from both the weakly- to strongly-bound state and from the strongly- to weakly-bound states and performed stretch activation experiments to measure the rates of XB detachment (krel) and XB recruitment (kdf) in detergent-skinned ventricular preparations isolated from hearts of wild-type (WT) and cMyBP-C knockout (KO) mice. Samples from donor human hearts were also used to assess the effects of OM in cardiac muscle expressing a slow β-myosin heavy chain (β-MHC). Incubation of skinned myocardium with OM produced large enhancements in steady-state force generation which were most pronounced at low levels of [Ca2+] activations, suggesting that OM cooperatively recruits additional XB’s into force generating states. Despite a large increase in steady-state force generation following OM incubation, parallel accelerations in XB kinetics as measured by ktr were not observed, and there was a significant OM-induced decrease in krel which was more pronounced in the KO skinned myocardium compared to WT skinned myocardium (58% in WT vs. 76% in KO at pCa 6.1), such that baseline differences in krel between KO and WT skinned myocardium were no longer apparent following OM-incubation. A significant decrease in the kdf was also observed following OM incubation in all groups, which may be related to the increase in the number of cooperatively recruited XB’s at low Ca2+-activations which slows the overall rate of force generation. Our results indicate that OM may be a useful pharmacological approach to normalize hypercontractile XB kinetics in myocardium with decreased cMyBP-C expression due to its molecular effects on XB behavior.

Key pointsr Phosphorylation of cardiac myosin binding protein-C Ser282 has been proposed to modulate the phosphorylation of Ser273 and Ser302, and thereby the contractile response to increased β-adrenergic stimulation, yet the precise functional role of Ser282 is unknown.r Protein kinase A phosphorylation of Ser273 and Ser302 was unaffected by Ser282 phospho-ablation, suggesting that Ser282 phosphorylation is not required for full phosphorylation of neighbouring residues.r Mice with Ser282 phospho-ablation (TG S282A ) displayed normal basal in vivo cardiac function but impaired rates of pressure development in response to β-adrenergic stimulation.r Basal rates of cross-bridge kinetics were unaffected by Ser282 phospho-ablation; however, the protein kinase A-mediated acceleration of cross-bridge recruitment was blunted in TG S282A myocardium.r Collectively, our data suggests that Ser282 phosphorylation is critical to achieve complete acceleration of cardiac contractile function in response to increased β-adrenergic stimulation, but also implicates Ser273 and Ser302 phosphorylation as important modulators of the cardiac myosin binding protein-C-mediated contractile response.Abstract Cardiac myosin binding protein-C phosphorylation plays an important role in modulating cardiac muscle function and accelerating contraction. It has been proposed that Ser282 phosphorylation may serve as a critical molecular switch that regulates the phosphorylation of neighbouring Ser273 and Ser302 residues, and thereby govern myofilament contractile acceleration in response to protein kinase A (PKA). Therefore, to determine the regulatory roles of Ser282 we generated a transgenic (TG) mouse model expressing cardiac myosin binding protein-C with a non-phosphorylatable Ser282 (i.e. serine to alanine substitution, TG S282A ). Myofibrils isolated from TG S282A hearts displayed robust PKA-mediated phosphorylation of Ser273 and Ser302, and the increase in phosphorylation was identical to TG wild-type (TG WT ) controls. No signs of pathological cardiac hypertrophy were detected in TG S282A hearts by either histological examination of cardiac sections or echocardiography. Baseline fractional shortening, ejection fraction, isovolumic relaxation time, rate of pressure development and rate of relaxation (τ) were unaltered in TG S282A mice. However, the increase in cardiac contractility as well as the acceleration of pressure development observed in response to β-adrenergic stimulation was attenuated in TG S282A mice. In agreement with our in vivo data, in vitro force measurements revealed that PKA-mediated acceleration of cross-bridge kinetics in TG S282A myocardium was significantly attenuated compared to TG WT myocardium. Taken together, our data suggest that Abbreviations cMyBP-C, cardiac myosin binding protein-C; dP/dt, rate of LV pressure development; dP/dt max , peak rate of left ventricular pressure development; EDP, end-diastolic pressure; F max , maximum Ca 2+ activated force; F min , Ca 2+ independent force; H&E, haematoxylin and eosin...

Mutations in cardiac myosin binding protein C (MyBP-C) are a common cause of familial hypertrophic cardiomyopathy (FHC). The majority of MyBP-C mutations are expected to reduce MyBP-C expression; however, the consequences of MyBP-C deficiency on the regulation of myofilament function, Ca²⁺ homeostasis, and in vivo cardiac function are unknown. To elucidate the effects of decreased MyBP-C expression on cardiac function, we employed MyBP-C heterozygous null (MyBP-C+/-) mice presenting decreases in MyBP-C expression (32%) similar to those of FHC patients carrying MyBP-C mutations. The levels of MyBP-C phosphorylation were reduced 53% in MyBP-C+/- hearts compared with wild-type hearts. Skinned myocardium isolated from MyBP-C+/- hearts displayed decreased cross-bridge stiffness at half-maximal Ca²⁺ activations, increased steady-state force generation, and accelerated rates of cross-bridge recruitment at low Ca²⁺ activations (<15% and <25% of maximum, respectively). Protein kinase A treatment abolished basal differences in rates of cross-bridge recruitment between MyBP-C+/- and wild-type myocardium. Intact ventricular myocytes from MyBP-C+/- hearts displayed abnormal sarcomere shortening but unchanged Ca²⁺ transient kinetics. Despite a lack of left ventricular hypertrophy, MyBP-C+/- hearts exhibited elevated end-diastolic pressure and decreased peak rate of LV pressure rise, which was normalized following dobutamine infusion. Furthermore, electrocardiogram recordings in conscious MyBP-C+/- mice revealed prolonged QRS and QT intervals, which are known risk factors for cardiac arrhythmia. Collectively, our data show that reduced MyBP-C expression and phosphorylation in the sarcomere result in myofilament dysfunction, contributing to contractile dysfunction that precedes compensatory adaptations in Ca²⁺ handling, and chamber remodeling. Perturbations in mechanical and electrical activity in MyBP-C+/- mice could increase their susceptibility to cardiac dysfunction and arrhythmia.

Key pointsr β-adrenergic stimulation increases cardiac myosin binding protein C (MyBP-C) and troponin I phosphorylation to accelerate pressure development and relaxation in vivo, although their relative contributions remain unknown.r Using a novel mouse model lacking protein kinase A-phosphorylatable troponin I (TnI) and MyBP-C, we examined in vivo haemodynamic function before and after infusion of the β-agonist dobutamine.r Mice expressing phospho-ablated MyBP-C displayed cardiac hypertrophy and prevented full acceleration of pressure development and relaxation in response to dobutamine, whereas expression of phosphor-ablated TnI alone had little effect on the acceleration of contractile function in response to dobutamine.r Our data demonstrate that MyBP-C phosphorylation is the principal mediator of the contractile response to increased β-agonist stimulation in vivo.r These results help us understand why MyBP-C dephosphorylation in the failing heart contributes to contractile dysfunction and decreased adrenergic reserve in response to acute stress.Abstract β-adrenergic stimulation plays a critical role in accelerating ventricular contraction and speeding relaxation to match cardiac output to changing circulatory demands. Two key myofilaments proteins, troponin I (TnI) and myosin binding protein-C (MyBP-C), are phosphorylated following β-adrenergic stimulation; however, their relative contributions to the enhancement of in vivo cardiac contractility are unknown. To examine the roles of TnI and MyBP-C phosphorylation in β-adrenergic-mediated enhancement of cardiac function, transgenic (TG) mice expressing non-phosphorylatable TnI protein kinase A (PKA) residues (i.e. serine to alanine substitution at Ser23/24; TnI PKA− ) were bred with mice expressing non-phosphorylatable MyBP-C PKA residues (i.e. serine to alanine substitution at Ser273, Ser282 and Ser302; MyBPC PKA− ) to generate a novel mouse model expressing non-phosphorylatable PKA residues in TnI and MyBP-C (DBL PKA− ). MyBP-C dephosphorylation produced cardiac hypertrophy and increased wall thickness in MyBPC PKA− and DBL PKA− mice, and in vivo echocardiography and pressure-volume catheterization studies revealed impaired systolic function and prolonged diastolic relaxation compared to wild-type and TnI PKA-mice. Infusion of the β-agonist dobutamine resulted in accelerated rates of pressure development and relaxation in all mice; however, MyBPC PKA− and DBL PKA− mice displayed a blunted contractile response compared to wild-type and TnI PKA-mice. Furthermore, unanaesthesized MyBPC PKA− and DBL PKA− mice displayed depressed maximum systolic pressure in response to dobutamine as measured using implantable telemetry devices. Taken together, our data show that MyBP-C phosphorylation is a critical modulator of the in vivo acceleration of pressure development and relaxation as a result of enhanced β-adrenergic stimulation, and reduced MyBP-C phosphorylation may underlie depressed adrenergic reserve in heart failure. Abbreviations dp/dt, rate of LV pressure ...

Cardiac myosin binding protein-C (cMyBP-C) phosphorylation is an important regulator of contractile function, however, its contributions to length-dependent changes in cross-bridge (XB) kinetics is unknown. Therefore, we performed mechanical experiments to quantify contractile function in detergent-skinned ventricular preparations isolated from wild-type (WT) hearts, and hearts expressing non-phosphorylatable cMyBP-C [Ser to Ala substitutions at residues Ser273, Ser282, and Ser302 (i.e., 3SA)], at sarcomere length (SL) 1.9 μm or 2.1μm, prior and following protein kinase A (PKA) treatment. Steady-state force generation measurements revealed a blunting in the length-dependent increase in myofilament Ca2+-sensitivity of force generation (pCa50) following an increase in SL in 3SA skinned myocardium compared to WT skinned myocardium. Dynamic XB behavior was assessed at submaximal Ca2+-activations by imposing an acute rapid stretch of 2% of initial muscle length, and measuring both the magnitudes and rates of resultant phases of force decay due to strain-induced XB detachment and delayed force rise due to recruitment of additional XBs with increased SL (i.e., stretch activation). The magnitude (P2) and rate of XB detachment (krel) following stretch was significantly reduced in 3SA skinned myocardium compared to WT skinned myocardium at short and long SL, and prior to and following PKA treatment. Furthermore, the length-dependent acceleration of krel due to decreased SL that was observed in WT skinned myocardium was abolished in 3SA skinned myocardium. PKA treatment accelerated the rate of XB recruitment (kdf) following stretch at both SL's in WT but not in 3SA skinned myocardium. The amplitude of the enhancement in force generation above initial pre-stretch steady-state levels (P3) was not different between WT and 3SA skinned myocardium at any condition measured. However, the magnitude of the entire delayed force phase which can dip below initial pre-stretch steady-state levels (Pdf) was significantly lower in 3SA skinned myocardium under all conditions, in part due to a reduced magnitude of XB detachment (P2) in 3SA skinned myocardium compared to WT skinned myocardium. These findings demonstrate that cMyBP-C phospho-ablation regulates SL- and PKA-mediated effects on XB kinetics in the myocardium, which would be expected to contribute to the regulation of the Frank-Starling mechanism.