Analysis Of The Functional Relevance Of Human Beta-myosin Heavy Chain Post-translational Modifications

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.