The contribution of cardiac myosin binding protein‐c Ser282 phosphorylation to the rate of force generation and in vivo cardiac contractility

Abstract

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...

Impact of the Myosin Modulator Mavacamten on Force Generation and Cross‐Bridge Behavior in a Murine Model of Hypercontractility

“…Pro‐Q Diamond phosphoprotein stain (Life Technologies) was used to assess myofilament protein phosphorylation in the cardiac samples as described previously29, 33 (Figure 1A). Cardiac myofibrils were prepared by homogenizing frozen ventricular tissue chunks for ≈15 s using a homogenizer (PowerGen 500; Thermo Fischer Scientific) in a relaxing solution containing protease and phosphatase inhibitors (PhosSTOP and cOmplete ULTRA tablets; Roche Applied Science).…”

“…Phosphorylation levels of myofilament proteins were normalized to protein expression levels, which were determined by counterstaining the gels with Coomassie blue. Densitometric scanning of stained gels was performed using Image J software available from the US National Institutes of Health, Bethesda, MD 33, 34…”

“…Frozen ventricular tissue chunks were homogenized in a relaxing solution followed by detergent‐skinning using 1% Triton‐X 100 for 1 hour 33. Multicellular ventricular preparations measuring ≈100 μm in width and ≈400 μm in length were selected for the experiments (Figures 2 and 3).…”

“…Changes in the motor arm position and force transducer signals were sampled at 2000 Hz using a custom‐built sarcomere length control software 41. For all mechanical measurements, the sarcomere length of the ventricular preparations was set to 2.1 μm 33, 37, 38, 40. Force‐pCa relationships were determined by measuring the forces generated by the skinned myocardial preparations in a range of pCa solutions that yield submaximal to maximal forces.…”

“…The stretch activation protocol used here was described in detail in previous studies 33, 39, 42. Skinned myocardial preparations were activated with pCa that generated a range of submaximal forces (eg, ≈40%–45% of maximal force at pCa 6.1 and ≈52%–57% of maximal force at pCa 6.0).…”