Biology subjects

Amrute-Nayak, M.

Publications and source records attributed to Amrute-Nayak, M..

6 recordsLinked to original sources

bioRxiv2026

Myotropes unveil two myosin cycles with distinct kinetics and stroke size

Cardiac contraction relies on cyclic interactions between myosin II motors and actin filaments, powered by the free energy of ATP turnover. Targeting of this fundamental process has emerged as a promising therapeutic strategy; Mavacamten, a cardiac myosin inhibitor was recently approved for treating obstructive hypertrophic cardiomyopathy whereas omecamtiv mecarbil was developed as a myosin activator for heart failure. Both myotropes bind to the same myosin pocket but their mechanisms of action remain incompletely understood. Here, we investigate these using optical tweezers mechanoenzymology and single-molecule fluorescence-based actomyosin kinetics applied to {beta}-myosin from donor hearts and recombinant human {beta}-myosin subfragment 1. Our findings reveal unexpected layers of complexity. Both mavacamten and omecamtiv mecarbil partially slow the ATP turnover, giving one fast (similar to physiological) and one drug-induced slow ATP turnover cycle at saturating drug concentrations with different rate-limiting steps for the two compounds. In one of the parallel cycles, mavacamten increases stroke size and appreciably delays the power-stroke, allowing direct visualization of a strongly bound pre-power-stroke state. No power-stroke delay is seen in the other cycle, but a reduced stroke size is observed. Our findings uncover a new paradigm in cardiac regulation at the single-molecule level, demonstrating partial inhibition of myosin as a powerful therapeutic strategy.

biophysics↗

bioRxiv2026

Single-molecule analysis sheds light on cardiac myosin dysfunction due to hypertrophic cardiomyopathy mutation A57D in ventricular myosin light chain-1 (MLC1v)

Ventricular myosin light chain-1 (MLC1v) is a key structural and function-modulating component of the {beta}-cardiac myosin ({beta}M-II) motor complex. Single-point mutations in MLC1v are linked to severe forms of hypertrophic cardiomyopathy (HCM) and sudden cardiac death (SCD) at a young age. However, the molecular mechanisms underlying the motor dysfunction responsible for HCM phenotype development are not fully understood. Here, we investigated native {beta}M-II motors isolated from septal myectomy sample of an HCM patient, harboring a rare homozygous mutation in MLC1v (A57D). Using a pure population of mutant motors (MUT), and sensitive single-molecule functional analysis approach, we directly assessed the primary functional alterations in {beta}M-II bearing A57D MLC1v mutation. In optical trap single-molecules measurements, the mutant motors displayed increased actomyosin (AM) interaction duration in strongly bound state (ton), corresponding to 3-fold reduced AM detachment rate than wild type myosin (WT). The MUT myosin also generated a shorter powerstroke size ({delta}). Ensemble average analysis of AM interaction events demonstrated that both the first powerstroke ({delta}1) associated with Pi release and the second powerstroke ({delta}2) linked to ADP release were reduced in MUT myosin. Moreover, the increased actomyosin cross-bridge stiffness in the AM.ADP state was observed for MUT compared to WT motors. Consistent with slower AM detachment rate and shorter stroke size, reconstituted human mutant {beta}M-II displayed slower actin filament gliding speed. Alterations in sarcomere-level mechanics included increased Ca2+ sensitivity of force generation and prolonged relaxation time, as predicted by FiberSim modelling. Molecular dynamics simulations indicated that the substitution of alanine by aspartate altered MLC1v interactions with myosin heavy chain (MyHC) and light chain 2 (MLC2v), affecting the curvature and flexibility of the lever arm. Overall, these studies establish the molecular mechanism underlying the primary myosin dysfunction due to A57D MLC1v mutation and further highlight the crucial role of MLC1v-mediated regulation of myosin function. Understanding the precise changes in the mutant myosins biomechanical properties is directly relevant to comprehending the initial triggers for pathological cardiac remodeling in HCM patients and designing tailored therapeutic interventions.

biophysics↗

bioRxiv2024

Sorafenib induces muscle wasting by disrupting the activity of distinct chromatin regulators

Adverse effects of chemotherapies can outweigh the benefits in cancer patients. Various chemotherapeutics are linked to muscle wasting or cachexia, drastically reducing the chance of survivability of cancer patients. Insights into the molecular basis of chemotherapy-induced cachexia is an unmet need to improve the treatment strategies. Here, we investigated the tyrosine kinase inhibitor class of chemotherapeutic agents for their effects on muscle function. Sorafenib, but not Nilotinib and Imatinib, triggered cachexia. System-wide transcriptome and proteome analyses revealed that Sorafenib alters the global transcriptional program and proteostasis in muscle cells. Mechanistically, Sorafenib treatment reduced active epigenetic mark H3K4 methylation on distinct muscle-specific genes due to the defective chromatin association of SET1/A, a catalytic component of the SET1/MLL complex. It favored transcriptionally incompetent chromatin, characterized by diminished association with RNA polymerase II. The transcriptional reorientation led to disrupted sarcomere organization, calcium homeostasis, and mitochondrial respiration. Consequently, the contractile ability of muscle cells was severely compromised. Collectively, we identified an unanticipated transcriptional mechanism underlying Sorafenib-induced cachexia. Our findings hold the potential to strategize therapy regimens to minimize chemotherapy-induced cachexia and improve treatment outcomes.

cell biology↗

bioRxiv2022

Cardiac ventricular myosin and slow skeletal myosin exhibit dissimilar chemo-mechanical properties despite the same myosin heavy chain isoform.

The myosin II motors are ATP-powered, force-generating machines driving cardiac and muscle contraction. Myosin II heavy chain isoform-beta ({beta}-MyHC) is primarily expressed in the ventricular myocardium and slow-twitch muscle fibers, such as in M. soleus. M. soleus-derived myosin II (SolM-II) is often used as an alternative to the ventricular {beta}-cardiac myosin ({beta}M-II); however, the direct assessment of detailed biochemical and mechanical features of the native myosins is limited. By employing the optical trapping method, we examined the mechanochemical properties of the native myosins isolated from rabbit heart ventricle and M. soleus muscles at the single-molecule level. Contrary to previous reports, the purified motors from the two tissue sources, despite the same MyHC isoform, displayed distinct motile and ATPase kinetic properties. {beta}M-II was [~]threefold faster in the actin filament-gliding assay than SolM-II. The maximum acto-myosin (AM) detachment rate derived in single-molecule assays was [~]threefold higher in {beta}M-II. The stroke size for both myosins was comparable. The stiffness of the AM rigor cross-bridge was also similar for both the motor forms. The stiffness of {beta}M-II was found to be determined by the nucleotide state of the actin-bound myosin. Our analysis revealed distinct kinetic differences, i.e., a higher AM detachment rate for the {beta}M-II, corresponding to the ADP release rates from the cross-bridge, thus elucidating the observed differences in the motility driven by {beta}M-II and SolM-II. These studies have important implications for the future choice of tissue sources to gain insights into cardiomyopathies

biophysics↗

bioRxiv2022

Myosin essential light chain 1sa decelerates actin and thin filament gliding on β-myosin molecules

The {beta}-myosin heavy chain expressed in ventricular myocardium and the myosin heavy chain (MyHC) in slow-twitch skeletal soleus muscle type-I fibers are both encoded by MYH7. Thus, these myosin molecules are deemed equivalent. However, some reports suggested variations in the light chain composition between soleus and ventricular myosin, which could influence functional parameters such as maximum velocity of shortening. To test for functional differences of the actin gliding velocity on immobilized myosin molecules we made use of the in vitro motility assay. We found that ventricular myosin moved actin filaments with approx. 0.9 m/s significantly faster than soleus myosin (0.3 m/s). Unregulated actin filaments are not the native interaction partner of myosin and are believed to slow down movement. Yet, using native thin filaments purified from soleus or ventricular tissue, the gliding velocity of soleus and ventricular myosin remained significantly different. When comparing the light chain composition of ventricular and soleus {beta}-myosin a difference became evident. Soleus myosin contains not only the "ventricular" essential light chain (ELC) MLC1sb/v, but also an additional longer and more positively charged MLC1sa. Moreover, we revealed that on a single muscle fiber level, a higher relative content of MLC1sa was associated with significantly slower actin gliding. We conclude that the ELC MLC1sa decelerates gliding velocity presumably by a decreased dissociation rate from actin associated with a higher actin affinity compared to MLC1sb/v. Such ELC/actin interactions might also be relevant in vivo as differences between soleus and ventricular myosin persisted when native thin filaments were used. SummaryCompared to the "ventricular" essential myosin light chain MLC1sb/v, the longer and more positively charged MLC1sa present in slow-twitch soleus muscle fibers decelerates actin filament gliding on {beta}-myosin molecules presumably by a decreased dissociation rate from actin filaments.

physiology↗

bioRxiv2020

Single-molecule analysis reveals the role of regulatory light chains in fine-tuning skeletal myosin-II function

Myosin II is the main force generating motor during muscle contraction. Myosin II exists as different isoforms, involved in diverse physiological functions. The outstanding question is whether the myosin heavy chain (MHC) isoforms alone account for the distinct physiological properties. Unique sets of essential and regulatory light chains (RLCs) assembled with specific MHCs raises an interesting possibility of specialization of myosin functions via light chains (LCs). Here, we ask whether different RLCs contribute to the functional diversification. To investigate this, we generated chimeric motors by reconstituting MHC fast isoform (MyHC-IId) and slow isoform (MHC-I) with different light chain variants. As a result of RLCs swapping, actin filament sliding velocity increased by [~] 10 fold for the slow myosin and decreased by >3 fold for the fast myosin. Ensemble molecule solution kinetics and single molecule optical trapping measurements provided in-depth insights into altered chemo mechanical properties of the myosin motors, thereby affecting the sliding speed. We find that both slow and fast myosins mechanical output is sensitive to the RLC isoform and propose that RLCs are crucial in fine-tuning of the myosin function.

biophysics↗