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Sarcomeric proteins have been shown to be a target of post-translational modifications (PTMs). Phosphorylation and acetylation of several sarcomeric proteins have been reported to be important for fine-tuning of myocardial contractility. Given the emerging importance of understanding the potential role of PTMs on cardiac muscle performance in healthy and diseased states, we sought to identify novel PTMs on human cardiac beta-myosin heavy chain (beta-MHC). We found several high confidence beta-MHC peptides modified by K-acetylation and S- and T-phosphorylation found in non-diseased, ischemic, and non-ischemic human heart samples. Using bottom-up proteomics and label-free quantification, we identified seven high-confidence peptides (K34, K58, S210, T215, K429, K951, K1195) with K951 displaying significant reduction in acetylation levels in both ischemic and non-ischemic failing hearts compared to donor hearts. Molecular dynamics simulations were performed to better understand the functional significance of the beta-MHC PTMs. Focus was placed on modifications in the regions with greatest potential functional significance as well as modified residues with significantly altered abundance in diseased states (K951-Ac at the myosin tail nearby a binding site for myosin heads in the super-relaxed state). K951 is located in the myosin tail (S2) at the C-terminal end of simulated structure. In both unmodified and modified simulations, the tail fragment showed significant flexibility and partial unfolding at the C-terminus. In the unmodified simulations, the inter- and intra-helical contacts were maintained. However, when beta-MHC is acetylated at residue 951, these helical contacts were altered as the uncharged acetylated residue no longer formed strong hydrogen bonds with a residue of the opposite chain. This facilitated changes increase in inter-helical contacts, an increase in inter-helical distance, and disruption of the coiled-coil tail domain structure. Our study suggests that there are distinct differences in beta-MHC acetylation levels that appear to be influenced more by location of the modified residues than the type of heart disease (ischemic- and non-ischemic heart failure). Additionally, we speculate that these PTMs have the potential to modulate the interactions between beta-MHC and other regulatory sarcomeric proteins, as well as ADP-release rate of myosin, flexibility of S2 fragment, and cardiac myofilament contractility under normal and heart failure condition.

Considerable effort has gone into investigating mechanisms that underlie the developmental transition in which mammalian cardiomyocytes (CMs) switch from being able to proliferate during development, to essentially having lost that ability at maturity. This problem is interesting not only for scientific curiosity, but also for its clinical relevance because controlling the ability of mature CMs to replicate would provide a much-needed approach for restoring cardiac function in damaged hearts. In this review, we focus on the propensity of mature mammalian CMs to be multinucleated and polyploid, and the extent to which this may be necessary for normal physiology yet possibly disadvantageous in some circumstances. In this context, we explore whether the concept of the myonuclear domain (MND) in multinucleated skeletal muscle fibers might apply to cardiomyocytes, and whether cardio-MND size might be related to the transition of CMs to become multinuclear. Nuclei in CMs are almost certainly integrators of not only biochemical, but also—because of their central location within the myofibrils—mechanical information, and this multimodal, integrative function in adult CMs—involving molecules that have been extensively studied along with newly identified possibilities—could influence both gene expression as well as replication of the genome and the nuclei themselves.

Acto-myosin cross-bridge kinetics are important for beat-to-beat regulation of cardiac contractility; however, physiological and pathophysiological mechanisms for regulation of contractile kinetics are incompletely understood. Here we explored whether thin filament-mediated Ca(2+) sensitization influences cross-bridge kinetics in permeabilized, osmotically compressed cardiac muscle preparations. We used a murine model of hypertrophic cardiomyopathy (HCM) harboring a cardiac troponin C (cTnC) Ca(2+)-sensitizing mutation, Ala8Val in the regulatory N-domain. We also treated wild-type murine muscle with bepridil, a cTnC-targeting Ca(2+) sensitizer. Our findings suggest that both methods of increasing myofilament Ca(2+) sensitivity increase cross-bridge cycling rate measured by the rate of tension redevelopment (k(TR)); force per cross-bridge was also enhanced as measured by sinusoidal stiffness and I(1,1)/I(1,0) ratio from X-ray diffraction. Computational modeling suggests that Ca(2+) sensitization through this cTnC mutation or bepridil accelerates k(TR) primarily by promoting faster cross-bridge detachment. To elucidate if myofilament structural rearrangements are associated with changes in k(TR), we used small angle X-ray diffraction to simultaneously measure myofilament lattice spacing and isometric force during steady-state Ca(2+) activations. Within in vivo lattice dimensions, lattice spacing and steady-state isometric force increased significantly at submaximal activation. We conclude that the cTnC N-domain controls force by modulating both the number and rate of cycling cross-bridges, and that the both methods of Ca(2+) sensitization may act through stabilization of cTnC’s D-helix. Furthermore, we propose that the transient expansion of the myofilament lattice during Ca(2+) activation may be an additional factor that could increase the rate of cross-bridge cycling in cardiac muscle. These findings may have implications for the pathophysiology of HCM.