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: 11.09). 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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We tested whether cardiac myosin binding protein-C (cMyBP-C) affects myosin cross-bridge kinetics in the two cardiac myosin heavy chain (MyHC) isoforms. Mice lacking cMyBP-C (t/t) and transgenic controls [Formula: see text] were fed L-thyroxine (T4) to induce 90/10 % expression of α/β-MyHC. Non-transgenic (NTG) and t/t mice were fed 6-n-propyl-2-thiouracil (PTU) to induce 100 % expression of β-MyHC. Ca(2+)-activated, chemically-skinned myocardium underwent length perturbation analysis with varying [MgATP] to estimate the MgADP release rate [Formula: see text] and MgATP binding rate [Formula: see text]. Values for [Formula: see text] were not significantly different between [Formula: see text] (102.2 ± 7.0 s(-1)) and [Formula: see text] (91.3 ± 8.9 s(-1)), but [Formula: see text] was lower in [Formula: see text] (165.9 ± 12.5 mM(-1) s(-1)) compared to [Formula: see text] (298.6 ± 15.7 mM(-1) s(-1), P < 0.01). In myocardium expressing β-MyHC, values for [Formula: see text] were higher in [Formula: see text] (24.8 ± 1.0 s(-1)) compared to [Formula: see text] (15.6 ± 1.3 s(-1), P < 0.01), and [Formula: see text] was not different. At saturating [MgATP], myosin detachment rate approximates [Formula: see text], and detachment rate decreased as sarcomere length (SL) was increased in both [Formula: see text] and [Formula: see text] with similar sensitivities to SL. In myocardium expressing β-MyHC, detachment rate decreased more as SL increased in [Formula: see text] (21.5 ± 1.3 s(-1) at 2.2 μm and 13.3 ± 0.9 s(-1) at 3.3 μm) compared to [Formula: see text] (15.8 ± 0.3 s(-1) at 2.2 μm and 10.9 ± 0.3 s(-1) at 3.3 μm) as detected by repeated-measures ANOVA (P < 0.01). These findings suggest that cMyBP-C reduces MgADP release rate for β-MyHC, but not for α-MyHC, even as the number of cMyBP-C that overlap with the thin filament is reduced to zero. Therefore, cMyBP-C appears to affect β-MyHC kinetics independent of its interaction with the thin filament.
    Journal of Muscle Research and Cell Motility 10/2014; DOI:10.1007/s10974-014-9390-0 · 1.93 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Cardiac myosin binding protein-C (cMyBP-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 neighboring 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 cMyBP-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 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 (FS), ejection fraction (EF), isovolumic relaxation time (IVRT), the rate of pressure development, and the 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 suggests that while Ser282 phosphorylation does not regulate the phosphorylation of neighboring Ser residues and basal cardiac function, but full acceleration of cross-bridge kinetics and left ventricular pressure development cannot be achieved in its absence. This article is protected by copyright. All rights reserved.
    The Journal of Physiology 06/2014; 592(17). DOI:10.1113/jphysiol.2014.276022 · 4.54 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Exon skipping mediated by antisense oligoribonucleotides (AON) is a promising therapeutic approach for genetic disorders, but has not yet been evaluated for cardiac diseases. We investigated the feasibility and efficacy of viral-mediated AON transfer in a Mybpc3-targeted knock-in (KI) mouse model of hypertrophic cardiomyopathy (HCM). KI mice carry a homozygous G>A transition in exon 6, which results in three different aberrant mRNAs. We identified an alternative variant (Var-4) deleted of exons 5-6 in wild-type and KI mice. To enhance its expression and suppress aberrant mRNAs we designed AON-5 and AON-6 that mask splicing enhancer motifs in exons 5 and 6. AONs were inserted into modified U7 small nuclear RNA and packaged in adeno-associated virus (AAV-U7-AON-5+6). Transduction of cardiac myocytes or systemic administration of AAV-U7-AON-5+6 increased Var-4 mRNA/protein levels and reduced aberrant mRNAs. Injection of newborn KI mice abolished cardiac dysfunction and prevented left ventricular hypertrophy. Although the therapeutic effect was transient and therefore requires optimization to be maintained over an extended period, this proof-of-concept study paves the way towards a causal therapy of HCM.
    EMBO Molecular Medicine 07/2013; 5(7). DOI:10.1002/emmm.201202168 · 8.25 Impact Factor


Available from