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Phosphorylation and acetylation of sarcomeric proteins are important for fine-tuning myocardial contractility. Here, we used bottom-up proteomics and label-free quantification to identify novel post-translational modifications (PTMs) on beta-myosin heavy chain (β-MHC) in normal and failing human heart tissues. We report six acetylated lysines and two phosphorylated residues: K34-Ac, K58-Ac, S210-P, K213-Ac, T215-P, K429-Ac, K951-Ac, and K1195-Ac. K951-Ac was significantly reduced in both ischemic and non-ischemic failing hearts compared to non-diseased hearts. Molecular dynamics simulations show that K951-Ac may impact stability of thick filament tail interactions and ultimately myosin head positioning. K58-Ac altered the solvent exposed SH3 domain surface – known for protein-protein interactions – but did not appreciably change motor domain conformation or dynamics under conditions studied. Together, K213-Ac/T215-P altered loop 1’s structure and dynamics – known to regulate ADP-release, ATPase activity, and sliding velocity. Our study suggests that β-MHC acetylation levels may be influenced more by the PTM location than the type of heart disease since less protected acetylation sites are reduced in both heart failure groups. Additionally, these PTMs have potential to modulate interactions between β-MHC and other regulatory sarcomeric proteins, ADP-release rate of myosin, flexibility of the S2 region, and cardiac myofilament contractility in normal and heart failure hearts.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca(2+)-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca(2+) activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC–cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.

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.

The cardiac troponin C (TnC)-A8V mutation is associated with hypertrophic and restrictive cardiomyopathy (HCM and RCM) in human and mice. The residue affected lies in the N-helix, a region known to affect Ca 2+ -binding affinity to the N-terminal domain. Here we report on the functional effects of this mutation in skinned papillary muscle fibers from homozygous knock-in TnC-A8V mice. Muscle fibers from left ventricle were activated at 25°C under the ionic conditions of working cardiomyocytes. The pCa-tension relationship showed a 3× increase in Ca 2+ -sensitivity and a decrease (0.8×) in cooperativity ( n H ) in mutant fibers. The elementary steps of the cross-bridge (CB) cycle were investigated by sinusoidal analysis. The ATP study revealed that there is no significant change in the affinity of ATP ( K 1 ) for the myosin head. In TnC-A8V mutant fibers, the CB detachment rate ( k 2 ) and its equilibrium constant ( K 2 ) increased (1.5×). The phosphate study revealed that rate constant of the force-generation step ( k 4 ) decreased (0.5×), reversal step ( k −4 ) increased (2×), and the phosphate-release step (1/ K 5 ) increased (2×). Pro-Q Diamond staining of the skinned fibers samples revealed no significant changes in total phosphorylation of multiple sarcomeric proteins. Further investigation using liquid chromatography-tandem mass spectrometry revealed hypophosphorylation of the rod domain of myosin heavy chain in TnC-A8V mutant fibers compared to wild-type. Immunoblotting confirmed the results observed in the mass spectrometry analysis. The results suggest perturbed CB kinetics—possibly caused by changes in the α -myosin heavy chain phosphorylation profile—as a novel mechanism, to our knowledge, by which a mutation in TnC can have rippling effects in the myofilament and contribute to the pathogenesis of HCM/RCM.

Recent genetic and functional data with the Ala8Val missense mutation in cardiac troponin C (cTnC-A8V) suggest its pathogenicity for the development of hypertrophic cardiomyopathy (HCM) in humans and mice. Previous functional studies performed using cTnC-A8V knock-in mice revealed increased Ca2+-sensitivity of contraction and impaired kinetics of cardiac muscle relaxation of skinned and intact cardiomyocytes. This study aims to further understand the molecular mechanisms that underlie the development of HCM caused by the cTnC-A8V mutation by measuring the kinetics of tension redevelopment (kTR) and sinusoidal stiffness at two distinct sarcomere lengths (SL). Papillary muscles were isolated from the left ventricle of cTnC-A8V knock-in homozygote and WT mice. SL of skinned preparations was set to 1.9µm or 2.1µm using HeNe laser diffraction after the ends were chemically fixed with glutaraldehyde to minimize end-compliance. SL dependent changes in mechanical properties were similar in WT and cTnC-A8V preparations: increased pCa50, with increased stiffness and decreased kTR at any given activation level. However, regardless of SL, cTnC-A8V preparations exhibited an increased pCa50, kTR and sinusoidal stiffness at submaximum Ca2+ levels relative to WT. Maximum sinusoidal stiffness increased 2x in WT preparations stretched from SL 1.9µm to 2.1µm, while for the cTnC-A8V preparations the SL-dependent increase in maximum sinusoidal stiffness was blunted. To determine whether the difference in cross-bridge kinetics at SL 2.1µm between cTnC-A8V and WT were due to the increased Ca2+-sensitivity of cTnC, WT preparations were treated with 300μM Bepridil, a Ca2+-sensitizer. As seen with the cTnC-A8V, sensitizing the thin filament with Bepridil resulted in an increased pCa50 and kTR. These data suggest an important role of the thin filament controlling not only the number of cross-bridges during Ca2+-activation, but may also influence cross-bridge kinetics.