Dilated cardiomyopathy (DCM) is associated with mutations in cardiomyocyte sarcomeric proteins, including α-tropomyosin. In conjunction with troponin, tropomyosin shifts to regulate actomyosin interactions. Tropomyosin molecules overlap via tropomyosin-tropomyosin head-to-tail associations, forming a continuous strand along the thin filament. These associations are critical for propagation of tropomyosin’s reconfiguration along the thin filament and key for the cooperative switching between heart muscle contraction and relaxation. Here, we tested perturbations in tropomyosin structure, biochemistry and function caused by DCM-linked mutation, M8R, which is located at the overlap junction. Localized and non-localized structural effects of the mutation were found in tropomyosin that ultimately perturb its thin-filament regulatory function. Comparison of mutant and wild-type α-tropomyosin was carried out using in-vitro motility assays, circular dichroism, actin co-sedimentation, and molecular dynamics simulations. Regulated thin filament velocity measurements showed the presence of M8R tropomyosin decreased calcium sensitivity and thin filament cooperativity. The co-sedimentation of actin and tropomyosin showed weakening of actin-mutant tropomyosin binding. Troponin-T’s N-terminus’ binding to the actin-mutant tropomyosin complex was also weakened. Circular dichroism and molecular dynamics indicate that the M8R mutation disrupts the 4-helix bundle at the head-to-tail junction, leading to weaker tropomyosin-tropomyosin binding and weaker tropomyosin-actin binding. Molecular dynamics revealed that altered end-to-end bond formation has affects extending towards the central region of the tropomyosin molecule, which alter the azimuthal position of tropomyosin, likely disrupting the mutant thin filament response to calcium. These results demonstrate that mutation-induced alterations in tropomyosin-thin filament interactions underlie the altered regulatory phenotype and ultimately the pathogenesis of DCM.
Muscle is highly hierarchically organized, with functions shaped by genetically controlled expression of protein ensembles with different isoform profiles at the sarcomere scale. However, it remains unclear how isoform profiles shape whole muscle performance. We compared two mouse hind limb muscles, the slow, relatively parallel-fibered soleus (SOL) and the faster, more pennate-fibered tibialis anterior (TA), across scales: from gene regulation, isoform expression and translation speed, to force-length-velocity-power for intact muscles. Expression of myosin heavy-chain (MHC) isoforms directly corresponded with contraction velocity. The fast-twitch TA with fast MHC isoforms had faster unloaded velocities (actin sliding velocity, VACTIN; peak fiber velocity, VMAX) than slow-twitch SOL. For SOL, VACTIN was biased towards VACTIN for purely slow MHC I, despite this muscle's even fast and slow MHC isoform composition. Our multi-scale results clearly identified a consistent and significant dampening in fiber shortening velocities for both muscles, underscoring an indirect correlation between VACTIN and fiber VMAX that may be influenced by differences in fiber architecture, along with internal loading due to both passive and active effects. These influences correlate with the increased peak force and power in the slightly more pennate TA, leading to a broader length range of near-optimal force production. Conversely, a greater force-velocity curvature in the near-parallel fibered SOL highlights the fine-tuning by molecular-scale influences including myosin heavy and light chain expression along with whole muscle characteristics. Our results demonstrate that the individual gene, protein, and whole fiber characteristics do not directly reflect overall muscle performance but that intricate fine-tuning across scales shapes specialized muscle function.
with the most frequent events (pCa: 6.6, 6.2, 5.8, and 5.4) by unbiased observers. Whereas, at low calcium (pCa 6.6), the distribution appears indistinguishable from exponential, short run times are increasingly suppressed as pCa is decreased in the assay. The pCa 5.4 histogram most resembles gamma rather than exponential distribution. Goodness of fit of the expected and observed frequencies using chi square statistic confirms the calciumdependent trend from exponential to gamma distribution. Contrary to our expectations, adding tropomyosin-troponin complex to same flow cell decreases rather than increases the proportion of short run times, thereby shifting the distributions away from exponential and toward gamma at all pCa. We can explain these observations mathematically if events during the run delay the rate that pause events arrive by a thermodynamic process. Given the constraint of constant free energy, we propose that the delay events arise by changes in mechanical force acting on the position of tropomyosin during the run. These results motivate further investigation of motion-dependent fluctuations and run-lengthening agents.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.