Cardiac myosin-binding protein-C phosphorylation and cardiac function.

Division of Molecular Cardiovascular Biology, Department of Pediatrics, Cincinnati Children's Hospital Medical Center, OH, USA.
Circulation Research (Impact Factor: 11.86). 12/2005; 97(11):1156-63. DOI: 10.1161/01.RES.0000190605.79013.4d
Source: PubMed

ABSTRACT The role of cardiac myosin binding protein-C (cMyBP-C) phosphorylation in cardiac physiology or pathophysiology is unclear. To investigate the status of cMyBP-C phosphorylation in vivo, we determined its phosphorylation state in stressed and unstressed mouse hearts. cMyBP-C phosphorylation is significantly decreased during the development of heart failure or pathologic hypertrophy. We then generated transgenic (TG) mice in which the phosphorylation sites of cMyBP-C were changed to nonphosphorylatable alanines (MyBP-C(AllP-)). A TG line showing &40% replacement with MyBP-C(AllP-) showed no changes in morbidity or mortality but displayed depressed cardiac contractility, altered sarcomeric structure and upregulation of transcripts associated with a hypertrophic response. To explore the effect of complete replacement of endogenous cMyBP-C with MyBP-C(AllP-), the mice were bred into the MyBP-C(t/t) background, in which less than 10% of normal levels of a truncated MyBP-C are present. Although MyBP-C(AllP-) was incorporated into the sarcomere and expressed at normal levels, the mutant protein could not rescue the MyBP-C(t/t) phenotype. The mice developed significant cardiac hypertrophy with myofibrillar disarray and fibrosis, similar to what was observed in the MyBP-C(t/t) animals. In contrast, when the MyBP-C(t/t) mice were bred to a TG line expressing normal MyBP-C (MyBP-CWT), the MyBP-C(t/t) phenotype was rescued. These data suggest that cMyBP-C phosphorylation is essential for normal cardiac function.

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    ABSTRACT: Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin binding protein-C (cMyBP-C), a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca2+ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0-C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2.0 um) and long (2.3 um) sarcomere length (SL). Our data demonstrate that the presence of hC0-C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca2+ sensitivity. Moreover, hC0-C1f led to increased cooperative activation, cross-bridge cycling kinetics and tension cost, with greater effects at short SL. We further established that the effects of hC0-C1f occur through direct interaction with actin and α-tropomyosin (α-TM). Our data demonstrate that the presence of hC0-C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0-C1f to bind actin and α-TM, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cMyBP-C with the thin filament.
    Journal of Biological Chemistry 02/2014; · 4.65 Impact Factor
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    ABSTRACT: Cardiac myosin binding protein-C (cMyBP-C) is a cardiac-specific, thick-filament regulatory protein that is differentially phosphorylated at Ser(273), Ser(282), and Ser(302) by various kinases and modulates contraction. In this study, phosphorylation-site-specific effects of cMyBP-C on myocardial contractility and cross-bridge kinetics were studied by sinusoidal analysis in papillary and trabecular muscle fibers isolated from t/t (cMyBP-C-null) mice and in their counterparts in which cMyBP-C contains the ADA (Ala(273)-Asp(282)-Ala(302)), DAD (Asp(273)-Ala(282)-Asp(302)), and SAS (Ser(273)-Ala(282)-Ser(302)) mutations; the results were compared to those from mice expressing the wild-type (WT) transgene on the t/t background. Under standard activating conditions, DAD fibers showed significant decreases in tension (∼50%), stiffness, the fast apparent rate constant 2πc, and its magnitude C, as well as its magnitude H, but an increase in the medium rate constant 2πb, with respect to WT. The t/t fibers showed a smaller drop in stiffness and a significant decrease in 2πc that can be explained by isoform shift of myosin heavy chain. In the pCa-tension study using the 8 mM phosphate (Pi) solution, there was hardly any difference in Ca(2+) sensitivity (pCa50) and cooperativity (nH) between the mutant and WT samples. However, in the solutions without Pi, DAD showed increased nH and slightly decreased pCa50. We infer from these observations that the nonphosphorylatable residue 282 combined with phosphomimetic residues Asp(273) and/or Asp(302) (in DAD) is detrimental to cardiomyocytes by lowering isometric tension and altering cross-bridge kinetics with decreased 2πc and increased 2πb. In contrast, a single change of residue 282 to nonphosphorylatable Ala (SAS), or to phosphomimetic Asps together with the changes of residues 273 and 302 to nonphosphorylatable Ala (ADA) causes minute changes in fiber mechanics.
    Biophysical Journal 03/2014; 106(5):1112-1122. · 3.67 Impact Factor
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    ABSTRACT: Diastolic dysfunction prominently contributes to heart failure with preserved ejection fraction (HFpEF). Owing partly to inadequate understanding, HFpEF does not have any effective treatments. Cardiac myosin-binding protein-C (cMyBP-C), a component of the thick filament of heart muscle that can modulate cross-bridge attachment/detachment cycling process by its phosphorylation status, appears to be involved in the diastolic dysfunction associated with HFpEF. In patients, cMyBP-C mutations are associated with diastolic dysfunction even in the absence of hypertrophy. cMyBP-C deletion mouse models recapitulate diastolic dysfunction despite in vitro evidence of uninhibited cross-bridge cycling. Reduced phosphorylation of cMyBP-C is also associated with diastolic dysfunction in patients. Mouse models of reduced cMyBP-C phosphorylation exhibit diastolic dysfunction while cMyBP-C phosphorylation mimetic mouse models show enhanced diastolic function. Thus, cMyBP-C phosphorylation mediates diastolic function. Experimental results of both cMyBP-C deletion and reduced cMyBP-C phosphorylation causing diastolic dysfunction suggest that cMyBP-C phosphorylation level modulates cross-bridge detachment rate in relation to ongoing attachment rate to mediate relaxation. Consequently, alteration in cMyBP-C regulation of cross-bridge detachment is a key mechanism that causes diastolic dysfunction. Regardless of the exact molecular mechanism, ample clinical and experimental data show that cMyBP-C is a critical mediator of diastolic function. Furthermore, targeting cMyBP-C phosphorylation holds potential as a future treatment for diastolic dysfunction.
    Pflügers Archiv - European Journal of Physiology 01/2014; · 4.87 Impact Factor


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