Hypertrophic Cardiomyopathy in Cardiac Myosin Binding Protein-C Knockout Mice

Abstract

Abstract-Familial hypertrophic cardiomyopathy (FHC) is an inherited autosomal dominant disease caused by mutations in sarcomeric proteins. Among these, mutations that affect myosin binding protein-C (MyBP-C), an abundant component of the thick filaments, account for 20% to 30% of all mutations linked to FHC. However, the mechanisms by which MyBP-C mutations cause disease and the function of MyBP-C are not well understood. Therefore, to assess deficits due to elimination of MyBP-C, we used gene targeting to produce a knockout mouse that lacks MyBP-C in the heart. Knockout mice were produced by deletion of exons 3 to 10 from the endogenous cardiac (c) MyBP-C gene in murine embryonic stem (ES) cells and subsequent breeding of chimeric founder mice to obtain mice heterozygous (ϩ/Ϫ) and homozygous (Ϫ/Ϫ) for the knockout allele. Wild-type (ϩ/ϩ), cMyBP-C ϩ/Ϫ , and cMyBP-C Ϫ/Ϫ mice were born in accordance with Mendelian inheritance ratios, survived into adulthood, and were fertile. Western blot analyses confirmed that cMyBP-C was absent in hearts of homozygous knockout mice. Whereas cMyBP-C ϩ/Ϫ mice were indistinguishable from wild-type littermates, cMyBP-C Ϫ/Ϫ mice exhibited significant cardiac hypertrophy. Cardiac function, assessed using 2-dimensionally guided M-mode echocardiography, showed significantly depressed indices of diastolic and systolic function only in cMyBP-C Ϫ/Ϫ mice. Ca 2ϩ sensitivity of tension, measured in single skinned myocytes, was reduced in cMyBP-C Ϫ/Ϫ but not cMyBP-C ϩ/Ϫ mice. These results establish that cMyBP-C is not essential for cardiac development but that the absence of cMyBP-C results in profound cardiac hypertrophy and impaired contractile function. Key Words: myosin binding protein-C Ⅲ heart Ⅲ myocardium Ⅲ gene knockout Ⅲ sarcomeric proteins M yosin binding protein-C (MyBP-C), also known as C-protein, 1 is a thick filament accessory protein that is present in nearly all vertebrate striated muscles but whose function is unknown. Nonetheless, there is compelling evidence to suggest that MyBP-C is a significant determinant of muscle contractile properties. In particular, cardiac MyBP-C (cMyBP-C) is a target for phosphorylation in response to various inotropic stimuli, including sympathetic stimuli that effect trisphosphorylation of cMyBP-C via cAMP-dependent protein kinase (PKA). 2 In addition, mutations of the cMyBP-C gene are a leading cause of familial hypertrophic cardiomyopathy (FHC), 3 an inherited disorder linked to mutations in cardiac contractile proteins (for review, see Bonne et al 4 and Seidman and Seidman 5 ).However, despite clues suggesting the importance of cMyBP-C to cardiac health, the function of cMyBP-C has remained enigmatic. For instance, although numerous studies have investigated effects of PKA on cardiac contractility (eg, Strang et al 6 and Patel et al 7 ), the role, if any, of cMyBP-C in mediating contractile responses to PKA has been difficult to discern. 8 -10 Similarly, the mechanisms by which cMyBP-C mutations affect cardiac function are not well understo...

Cardiac myosin binding protein-C modulates actomyosin binding and kinetics in the in vitro motility assay

“…Several investigators have proposed that the reduced unloaded shortening velocity in fiber studies is the result of cMyBP-C functioning either as a tether restricting myosin's interactions with actin [15,29,[43][44][45] and/or imparting a viscous load to thick-thin filament sliding [46]. Our data would indicate that neither of these mechanisms can account for the functional effects observed with cMyBP-C in this study.…”

“…For example, the effect of cMyBP-C on half maximal calcium activated tension (i.e. calcium sensitivity) has been reported to be decreased, unchanged or increased in fibers depending on the experimental approach [12][13][14][15][16][17].…”

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

“…Perhaps Myk461 counters this effect by altering myosin head conformation in a manner that increases XB stability and/or slows XB turnover in KO myocardium, partly explaining the reduced force deficits induced by Myk461 in KO myocardium—a hypothesis that requires further validation using molecular studies specifically geared towards elucidating the contributions of cMyBPC on Myk461‐mediated modulation of XB behavior. Nevertheless, the Myk461‐induced slowing in k rel in the KO myocardium would be expected to reduce overall ATP turnover rate and tension cost as XB detachment was shown to correlate well with tension cost,56, 57 which may improve systolic function in vivo and reduce pathological hypertrophy and remodeling in models of hypertrophy induced by reductions in cMyBPC 30, 32. Notably, a slowed k rel and increased XB “ on ” time following Myk461 incubation may underlie our observation that Myk461‐induced force generation reductions were not as pronounced in the KO myocardium (Table 2; Figure 3).…”

“…Wild‐type (WT) mice expressing full‐length cMyBPC were used as controls. Mice lacking cMyBPC (knockout (KO)) in their myocardium used in this study were previously generated and well‐characterized 32. Male and female mice (SV/129 strain) aged 3 to 6 months were used in this study.…”

Increased transport of acetyl‐CoA into the endoplasmic reticulum causes a progeria‐like phenotype

“…Transthoracic echocardiography was performed as described previously (Harris et al, 2002). Further details are in Supporting Information.…”