Muscle contraction is controlled at two levels: the thin and the thick filaments. The latter level of control involves three states of myosin heads: active, disordered relaxed (DRX), and super-relaxed (SRX), the distribution of which controls the number of myosins available to interact with actin. How these are controlled is still uncertain. Using fluorescently labeled ATP, we were able to spatially assign the activity of individual myosins within the sarcomere. We observed that SRX comprises 53% of all heads in the C-zone compared with 35% and 44% in the P- and D-zones, respectively. The recently FDA-approved hypertrophic cardiomyopathy drug, mavacamten (mava), significantly decreased DRX, favoring SRX in both the C- and D-zones at 60% and 63%, respectively. Since thick filament regulation is in part regulated by the myosin-binding protein-C (MyBP-C), we also studied PKA phosphorylation. This had the opposite effect as mava, specifically in the C-zone where it decreased SRX to 34%, favoring DRX. These results directly show that excess concentrations of mava do increase SRX, but the effect is limited across the sarcomere, suggesting mava is less effective on skeletal muscle. In addition, we show that PKA directly affects the contractile machinery of skeletal muscle leading to the liberation of repressed heads. Since the effect is focused on the C-zone, this suggests it is likely through MyBP-C phosphorylation, although our data suggest that a further reserve of myosins remain that are not accessible to PKA treatment.

β-cardiac myosin mediates cardiac muscle contraction within the sarcomere by binding to the thin filament in an ATP-powered reaction. This process is highly regulated on a beat-to-beat basis by calcium interactions with the thin filament, but also contractile force is highly regulated by controlling the number of myosins available, resulting in a dynamic reserve. Our goal was to examine the size of this reserve and how it is modulated by cardiac myosin binding protein-C (cMyBP-C). We used single-molecule imaging to determine myosin activity with high spatial resolution by measuring fluorescently tagged ATP molecules binding to and releasing from myosins within the cardiac sarcomere. Three myosin ATPase states were detected: the fastest species was consistent with nonspecific ATP binding to myosin’s surface, and the slower two species were consistent with the previously identified DRX and SRX states. The former represents myosins in a state ready to interact with the thin filament, and the latter in a cardiac reserve state with slowed ATPase. We found the cardiac reserve was 46% across the whole sarcomere in porcine myofibrils. Subdividing into the P-, C-, and D-zones revealed the D-zone has the smallest population of reserve heads (44%). Treatment with PKA that phosphorylates cMyBP-C led to a 16% reduction of reserve in the C-zone (where cMyBP-C is found) and a 10% reduction in the P-zone, with an unexpected 15% increase in the D-zone. Interestingly, the changes in SRX myosin head distribution by PKA phosphorylation of cMyBP-C across each subsarcomeric zone mirror the changes we identified in human cardiac myofibrils isolated from a hypertrophic cardiomyopathy patient mutation (MYBPC3-c.772G>A) that exhibits cMyBP-C haploinsufficiency. These results provide novel insights into how the C-zone functions in both porcine and human β-cardiac myosin–containing thick filaments, revealing a possible compensatory change in the D-zone upon altered cMyBP-C phosphorylation and/or haploinsufficiency.

I. The R403Q mutation in the sarcomere protein beta-myosin heavy chain (β-MHC) is a known genetic cause of hypertrophic cardiomyopathy (HCM), associated with ventricular hypercontractility, impaired relaxation, and cardiac arrhythmias. Despite extensive research, the mutations impact on myosin contractile properties remains unclear, likely due, at least in part, to discrepancies cross different model systems. In this study, we used a multidisciplinary approach to explore mutational effects using two distinct heterozygous R403Q systems: a Yucatan minipig model and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). X-ray diffraction of R403Q minipig ventricular muscle demonstrated reduced order of the thick filament, suggesting destabilization of the inhibited OFF (vs. ON) state of myosin in relaxed muscle, which correlated with elevated force at submaximal calcium. Super-resolution, single-molecule fluorescence microscopy indicated elevated ATPase activity in thick filament zones lacking cMyBP-C. Furthermore, R403Q myofibrils exhibited slower activation and relaxation kinetics, with reduced sensitivity to ADP. Molecular dynamics simulations suggested that altered interactions at the actomyosin interface contribute to these effects, rather than changes at the nucleotide binding pocket, typically associated with ADP release. Human engineered heterozygous R403Q hiPSC-CMs exhibited reduced maximal myofibril force, slowed contractile kinetics, and hypercontraction in engineered heart tissue constructs−consistent with HCM phenotypes observed in the heterozygous porcine model. Our results demonstrate that the R403Q mutation induces early and persistent contractile dysfunction, and that hypercontractility and slower contractile kinetics may result from a combination of an increased population of activated (ON) myosin heads and delayed detachment during cross-bridge cycling, respectively.

Background: Inhibiting contractility by targeting cardiac myosin is an effective treatment for patients with hypertrophic cardiomyopathy (HCM). Aficamten is a second in class myosin inhibitor with promising clinical data showing improvements in hemodynamics and symptoms in patients with HCM. While it is known that Aficamten inhibits force and cardiomyocyte contractility by stabilizing the weak pre-powerstroke conformation, effects on myosin structure and kinetics during loaded contraction are lacking. Methods: Permeabilized porcine cardiac tissue and myofibrils were used for single-molecule imaging of ATP turn over, X-ray diffraction, and mechanical measurements. Engineered heart tissues from human induced pluripotent stem cell cardiomyocytes were used to evaluate effects on force and contraction kinetics. Results: In contrast to Mavacamten, Aficamten does not structurally sequester myosin heads along the thick filament. Aficamten inhibits ATPase activity by shifting myosin heads from higher to slower ATPase state, with the emergence of a super slow biochemical nucleotide turnover state. This results in decreased force and calcium sensitivity without altering cross-bridge cycling. These contractile mechanical changes are comparable to Mavacamten. Our myofibril mechanical assay showed inhibition of force with accelerated relaxation. In EHTs, while Mavacamten and Aficamten inhibit cardiac twitch forces, Mavacamten reduces the activation kinetics while both result in faster relaxation. Conclusions: We used a combination of biochemical and biomechanical assays to show that Aficamten inhibits myosin ATPase without appreciably altering myosin structure. This is different from Mavacamten that strongly affects both. While both compounds inhibit contractility, differences in mechanisms of action and kinetics of force activation and relaxation could allow use in different patient populations.

Coagulation factor VIII (FVIII) is a key therapeutic protein for hemophilia A, an inherited bleeding disorder caused by FVIII deficiency or dysfunction. The development and production of FVIII-based therapeutics remains challenging due to the intrinsic complexity of FVIII biosynthesis, folding, secretion, and functional stability. Advances in the understanding of the FVIII life cycle, as well as studies of its natural homologs, have created opportunities to develop improved FVIII variants. In parallel, therapeutic strategies to restore FVIII activity have evolved from replacement therapy with exogenous FVIII to gene-based delivery of a functional F8 copy. Among these approaches, AAV-mediated gene therapy has emerged as a clinically viable platform with a generally favorable safety profile, prompting the development of multiple therapeutic candidates for hemophilia A. This review summarizes established and current knowledge of FVIII structure, biosynthesis, and immunology in the context of developing effective and clinically applicable AAV-mediated therapies for hemophilia A. Particular attention is given to engineered FVIII variants and the latest clinical findings in AAV gene therapy, as well as to long-term efficacy and safety.