Biology subjects

Naim, A.

Publications and source records attributed to Naim, A..

3 recordsLinked to original sources

bioRxiv2025

Direct measurement of mavacamten and deoxyATP perturbation of the SRX/DRX ratio in porcine cardiac myofibrils using a simple, accessible and multiplexed approach

Cardiac muscle adapts to varying physiological demands by modulating the number of active myosin II motors available for contraction. These motors are organized into thick filaments in the sarcomere and generate force through an ATP-dependent interaction with thin filaments that contain actin. To conserve energy, when demand is low myosin can occupy the super-relaxed (SRX) state, which acts as a reserve. Here, we build upon the earlier studies from the Cooke lab to quantify the size of this cardiac reserve using fluorescence imaging of Cy3-ATP directly in myofibrils. This approach employs a pulse-chase method and exploits the high permeability of isolated myofibrils to monitor nucleotide release in situ. By preserving sarcomeric architecture while enabling rapid reagent exchange, this method bridges the gap between complex single-molecule imaging and traditional stopped-flow bulk assays using MANT-ATP. Using this approach we have studied biochemical perturbation of the SRX reserve with deoxyATP and mavacamten. DeoxyATP caused large depletion of the cardiac reserve, and mavacamten increased its size, consistent with its clinical application. Our results demonstrate the utility of this technique and the potential for further enhancement using multiplexing, holding promise for future applications in health and disease.

biophysics↗

bioRxiv2025

Myosin modulator Aficamten inhibits force in cardiac muscle by altering myosin's biochemical activity without changing thick filament structure

BackgroundInhibiting 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. MethodsPermeabilized 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. ResultsIn 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. ConclusionsWe 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.

biophysics↗

bioRxiv2024

Spatially resolving how phosphorylation affects β-cardiac myosin activity in porcine myofibril sarcomeres with single molecule resolution

Cardiac muscle contraction is mediated by myosin binding from the thick filament of the sarcomere 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. Additionally, the number of heads available for participation in contraction is also regulated, resulting in a dynamically variable reserve of heads for controlling contractile force. We aimed to discover the size of this reserve and how it is modulated by phosphorylation. Using single molecule imaging of fluorescently labelled ATP molecules binding and releasing myosins within porcine cardiac sarcomeres, we could determine myosin activity with high spatial resolution. We find three kinetic species when examining the myosin ATPase. The fastest is consistent with non-specific ATP binding to myosins surface, and the slower two species are consistent with the previously identified DRX and SRX states. The former is thought to represent myosins in an ON state, ready to interact with the thin filament and the latter an OFF state with slowed ATPase that constitutes the cardiac reserve. We find that the cardiac reserve is 50% in the sarcomere and this can be sub-divided into the P-, C- and D-zones, with the D-zone having the least population of OFF heads (44%). Treatment with PKA phosphorylates cardiac myosin binding protein-C (cMyBP-C) leading to a 16% reduction in reserve in the C-zone (where cMyBP-C is found), a 10% reduction in the P-zone, and an unexpected 8% increase in the D-zone. By contrast, myosin regulatory light chain (RLC) phosphorylation with myosin light chain kinase (MLCK) resulted in a large 24% decrease in reserve myosins, interestingly the least affected area of the sarcomere was the C-zone. Altogether these data suggest that cMyBP-Cs interaction with RLC governs the degree of activation due to RLC phosphorylation.

biophysics↗