Ablation of cardiac myosin–binding protein-C accelerates contractile kinetics in engineered cardiac tissue

Department of Pediatrics, University of Wisconsin School of Medicine and Public Health, Madison, WI 53792.
The Journal of General Physiology (Impact Factor: 4.79). 01/2013; 141(1):73-84. DOI: 10.1085/jgp.201210837
Source: PubMed


Hypertrophic cardiomyopathy (HCM) caused by mutations in cardiac myosin-binding protein-C (cMyBP-C) is a heterogenous disease in which the phenotypic presentation is influenced by genetic, environmental, and developmental factors. Though mouse models have been used extensively to study the contractile effects of cMyBP-C ablation, early postnatal hypertrophic and dilatory remodeling may overshadow primary contractile defects. The use of a murine engineered cardiac tissue (mECT) model of cMyBP-C ablation in the present study permits delineation of the primary contractile kinetic abnormalities in an intact tissue model under mechanical loading conditions in the absence of confounding remodeling events. We generated mechanically integrated mECT using isolated postnatal day 1 mouse cardiac cells from both wild-type (WT) and cMyBP-C-null hearts. After culturing for 1 wk to establish coordinated spontaneous contraction, we measured twitch force and Ca(2+) transients at 37°C during pacing at 6 and 9 Hz, with and without dobutamine. Compared with WT, the cMyBP-C-null mECT demonstrated faster late contraction kinetics and significantly faster early relaxation kinetics with no difference in Ca(2+) transient kinetics. Strikingly, the ability of cMyBP-C-null mECT to increase contractile kinetics in response to adrenergic stimulation and increased pacing frequency were severely impaired. We conclude that cMyBP-C ablation results in constitutively accelerated contractile kinetics with preserved peak force with minimal contractile kinetic reserve. These functional abnormalities precede the development of the hypertrophic phenotype and do not result from alterations in Ca(2+) transient kinetics, suggesting that alterations in contractile velocity may serve as the primary functional trigger for the development of hypertrophy in this model of HCM. Our findings strongly support a mechanism in which cMyBP-C functions as a physiological brake on contraction by positioning myosin heads away from the thin filament, a constraint which is removed upon adrenergic stimulation or cMyBP-C ablation.

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Available from: Adrian Grimes, Feb 28, 2014
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    • "cMyBP-C appears to repress cross bridge cycling. Cardiomyocytes from cMyBP-C knock out mice display faster cross bridge cycling as evident from faster twitch kinetics [15], faster force development following a stretch [16] [17] [18] and increased shortening velocity [17] [19]. "
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    ABSTRACT: Cardiac myosin binding protein C (cMyBP-C) is an essential regulator of cross bridge cycling. Through mechanisms that are incompletely understood the N-terminal domains (NTDs) of cMyBP-C can activate contraction even in the absence of calcium and can also inhibit cross bridge kinetics in the presence of calcium. In vitro studies indicated that the proline-alanine rich (p/a) region and C1 domain are involved in these processes, although effects were greater using human proteins compared to murine proteins (Shaffer et al. J Biomed Biotechnol 2010, 2010: 789798). We hypothesized that the p/a and C1 region are critical for the timing of contraction. In this study we tested this hypothesis using a mouse model lacking the p/a and C1 region (p/a-C1(-/-) mice) to investigate the in vivo relevance of these regions on cardiac performance. Surprisingly, hearts of adult p/a-C1(-/-) mice functioned normally both on a cellular and whole organ level. Force measurements in permeabilized cardiomyocytes from adult p/a-C1(-/-) mice and wild type (Wt) littermate controls demonstrated similar rates of force redevelopment both at submaximal and maximal activation. Maximal and passive force and calcium sensitivity of force were comparable between groups as well. Echocardiograms showed normal isovolumetric contraction times, fractional shortening and ejection fraction, indicating proper systolic function in p/a-C1(-/-) mouse hearts. p/a-C1(-/-) mice showed a slight but significant reduction in isovolumetric relaxation time compared to Wt littermates, yet this difference disappeared in older mice (7-8months of age). Moreover, stroke volume was preserved in p/a-C1(-/-) mice, corroborating sufficient time for normal filling of the heart. Overall, the hearts of p/a-C1(-/-) mice showed no signs of dysfunction even after chronic stress with an adrenergic agonist. Together, these results indicate that in mice the p/a region and the C1 domain of cMyBP-C are not critical for normal cardiac contraction and that these domains have little if any impact on cross bridge kinetics at least not in mice. These results thus contrast with in vitro studies utilizing proteins encoding the human p/a region and C1 domain. More detailed insight in how individual domains of cMyBP-C function and interact, across species and over the wide spectrum of conditions in which the heart has to function, will be essential to a better understanding of how cMyBP-C tunes cardiac contraction.
    Journal of Molecular and Cellular Cardiology 10/2015; 88. DOI:10.1016/j.yjmcc.2015.09.006 · 4.66 Impact Factor
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    • "Although KO adWT and KO ad258 mECT remodeled similarly and appeared indistinguishable by gross inspection , KO ad258 mECT were marginally but significantly narrower than KO adWT mECT (974 ± 24 nm vs. 1,097 ± 41 nm; P = 0.018). The absence of hypertrophy in our model may reflect that we use neonatal cardiomyocytes isolated from cMyBP-C–null newborn mice that have yet to develop the hypertrophy that develops later (de Lange et al., 2013). The acute expression of the E258K mutation on this background provides the opportunity to evaluate the contractile phenotype, free of remodeling. "
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    ABSTRACT: Mutations in cardiac myosin binding protein C (cMyBP-C) are prevalent causes of hypertrophic cardiomyopathy (HCM). Although HCM-causing truncation mutations in cMyBP-C are well studied, the growing number of disease-related cMyBP-C missense mutations remain poorly understood. Our objective was to define the primary contractile effect and molecular disease mechanisms of the prevalent cMyBP-C E258K HCM-causing mutation in nonremodeled murine engineered cardiac tissue (mECT). Wild-type and human E258K cMyBP-C were expressed in mECT lacking endogenous mouse cMyBP-C through adenoviral-mediated gene transfer. Expression of E258K cMyBP-C did not affect cardiac cell survival and was appropriately incorporated into the cardiac sarcomere. Functionally, expression of E258K cMyBP-C caused accelerated contractile kinetics and severely compromised twitch force amplitude in mECT. Yeast two-hybrid analysis revealed that E258K cMyBP-C abolished interaction between the N terminal of cMyBP-C and myosin heavy chain sub-fragment 2 (S2). Furthermore, this mutation increased the affinity between the N terminal of cMyBP-C and actin. Assessment of phosphorylation of three serine residues in cMyBP-C showed that aberrant phosphorylation of cMyBP-C is unlikely to be responsible for altering these interactions. We show that the E258K mutation in cMyBP-C abolishes interaction between N-terminal cMyBP-C and myosin S2 by directly disrupting the cMyBP-C-S2 interface, independent of cMyBP-C phosphorylation. Similar to cMyBP-C ablation or phosphorylation, abolition of this inhibitory interaction accelerates contractile kinetics. Additionally, the E258K mutation impaired force production of mECT, which suggests that in addition to the loss of physiological function, this mutation disrupts contractility possibly by tethering the thick and thin filament or acting as an internal load.
    The Journal of General Physiology 09/2013; 142(3):241-55. DOI:10.1085/jgp.201311018 · 4.79 Impact Factor
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    • "Determining and understanding the post-translational modifications of contractile proteins are critical to our understanding of muscle physiology and pathology. Post-translational modification of cMyBP-C is known to affect the Ca 2+ -sensitivity of force development, the cross-bridge cycling rates, the length-dependent activation, force development and protein stability [11] [12] [13]. "
    Journal of Molecular and Cellular Cardiology 05/2013; 62. DOI:10.1016/j.yjmcc.2013.05.015 · 4.66 Impact Factor
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