Control of In Vivo Contraction/Relaxation Kinetics by Myosin Binding Protein C

Abstract

Background-Cardiac myosin binding protein-C (cMyBP-C) is a thick-filament protein whose presence and phosphorylation by protein kinase A (PKA) regulates cross-bridge formation and kinetics in isolated myocardium. We tested the influence of cMyBP-C and its PKA-phosphorylation on contraction/relaxation kinetics in intact hearts and revealed its essential role in several classic properties of cardiac function. Methods and Results-Comprehensive in situ cardiac pressure-volume analysis was performed in mice harboring a truncation mutation of cMyBP-C (cMyBP-C (t/t) ) that resulted in nondetectable protein versus hearts re-expressing solely wild-type (cMyBP-C WT:(t/t) ) or mutated protein in which known PKA-phosphorylation sites were constitutively suppressed (cMyBP-C AllP-:(t/t) ). Hearts lacking cMyBP-C had faster early systolic activation, which then terminated prematurely, limiting ejection. Systole remained short at faster heart rates; thus, cMyBP-C (t/t) hearts displayed minimal rate-dependent decline in diastolic time and cardiac preload. Furthermore, prolongation of pressure relaxation by afterload was markedly blunted in cMyBP-C (t/t) hearts. All 3 properties were similarly restored to normal in cMyBP-C WT:(t/t) and cMyBP-C AllP-:(t/t) hearts, which supports independence of PKA-phosphorylation. However, the dependence of peak rate of pressure rise on preload was specifically depressed in cMyBP-C AllP-:(t/t) hearts, whereas cMyBP-C (t/t) and cMyBP-C AllP-:(t/t) hearts had similar blunted adrenergic and rate-dependent contractile reserve, which supports linkage of these behaviors to PKA-cMyBP-C modification. Conclusions-cMyBP-C is essential for major properties of cardiac function, including sustaining systole during ejection, the heart-rate dependence of the diastolic time period, and relaxation delay from increased arterial afterload. These are independent of its phosphorylation by PKA, which more specifically modulates early pressure rise rate and adrenergic/heart rate reserve.

Cardiac Myosin-binding Protein C and Troponin-I Phosphorylation Independently Modulate Myofilament Length-dependent Activation

“…cMyBP-C Non-transgenic (NTG), AAA, and DDD Mouse Models-The phospho-mimetic and phospho-null mouse models have been previously described (31)(32)(33). For the phospho-null mouse model, the PKA sites (Ser-273, Ser-282, and Ser-302) were mutated to alanine, whereas for the phosphomimetic model these sites were mutated to aspartic residues.…”

“…For these studies, hearts from 10 NTG wild-type, 11 AAA, and 10 DDD mice were used. The AAA mouse model is associated with the development of hypertrophy, increased expression ␤-myosin, and potentially altered PKA activity (31)(32)(33). To minimize the impact of this potentially confounding factor, relatively young mice were used for this study (ϳ8 weeks of age).…”

“…Here, to resolve whether cMyBP-C phosphorylation affects LDA and to determine whether the impact of cMyBP-C and cTnI phosphorylation are synergistic or merely additive, we employed two transgenic mouse models in which cMyBP-C is replaced by either PKA phospho-null (AAA) or phosphomimic (DDD) versions of the protein (31)(32)(33). Isolated permeabilized single myocyte fragments were either treated with PKA or exchanged for troponin containing PKA phospho-null or phospho-mimic cTnI.…”

The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner

“…Phosphorylation of cMyBP-C is essential for its regulatory effects because elimination of phosphorylation sites (serine to alanine substitutions) abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge cycling kinetics and blunts cardiac responses to inotropic stimuli (6). The substitutions further impair cardiac function, reduce contractile reserve, and cause cardiac hypertrophy in transgenic mice (6,7). By contrast, substitution of aspartic acids at these sites to mimic constitutive phosphorylation is benign or cardioprotective (8).…”

“…Although a role for cMyBP-C in modulating cross-bridge kinetics is supported by several transgenic and knock-out mouse models (6,7,9,10), the precise mechanisms by which cMyBP-C exerts these effects are not completely understood. For instance, the unique regulatory motif or "m-domain" of cMyBP-C binds to the S2 subfragment of myosin in vitro (11) and binding is abolished by PKA-mediated phosphorylation of the m-domain (12).…”