Muscle contraction is governed by tropomyosin (Tpm) shifting azimuthally between three states on F-actin (B-, C-, and M-states) in response to calcium binding to troponin and actomyosin cross-bridge formation. The Tpm coiled coil polymerizes head to tail along the long-pitch helix of F-actin to form continuous superhelical cables that wrap around the actin filaments. The end-to-end bonds formed between the N-and C-terminus of adjacent Tpm molecules define Tpm continuity and play a critical role in the ability of Tpm to cooperatively bind to actin, thus facilitating Tpm conformational switching to cooperatively propagate along F-actin. We expect that a missense mutation in this critical overlap region associated with dilated cardiomyopathy, A277V, will alter Tpm binding and thin filament activation by altering the overlap structure. Here, we used cosedimentation assays and in vitro motility assays to determine how the mutation alters Tpm binding to actin and its ability to regulate actomyosin interactions. Analytical viscometry coupled with molecular dynamics simulations showed that the A277V mutation results in enhanced Tpm end-to-end bond strength and a reduced curvature of the Tpm overlap domain. The mutant Tpm exhibited enhanced actin-Tpm binding affinity, consistent with overlap stabilization. The observed A277V-induced decrease in cooperative activation observed with regulated thin filament motility indicates that increased overlap stabilization is not correlated with Tpm-Tpm overlap binding strength or mechanical rigidity as is often assumed. Instead, A277V-induced structural changes result in local and delocalized increases in Tpm flexibility and prominent coiled-coil twisting in pseudorepeat 4. An A277V-induced decrease in Ca 2þ sensitivity, consistent with a mutationinduced bolstering of the B-state Tpm-actin electrostatic contacts and an increased Tpm troponin T1 binding affinity, was also observed.

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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.

The contractility of cardiac muscle is regulated by thick-filament based mechanisms in addition to the classical calcium/thin-filament mediated mechanisms. Here we studied the role of structural changes of the thick filament in length-dependent activation in cardiac muscle. We used fluorescence polarization from bifunctional sulphorhodamine probes on the N-and C-lobes of the myosin regulatory light chain (RLC) to monitor changes in the orientation of the myosin motors induced by increasing sarcomere length in relaxed and partially calcium-activated demembranated trabeculae.