Heart muscle has the unique property that it can never rest; all cardiomyocytes contract with each heartbeat which requires a complex control mechanism to regulate cardiac output to physiological requirements. Changes in calcium concentration regulate the thin filament activation. A separate but linked mechanism regulates the thick filament activation, which frees sufficient myosin heads to bind the thin filament, thereby producing the required force. Thick filaments contain additional nonmyosin proteins, myosin-binding protein C and titin, the latter being the protein that transmits applied tension to the thick filament. How these three proteins interact to control thick filament activation is poorly understood. Here, we show using 3-D image reconstruction of frozen-hydrated human cardiac muscle myofibrils lacking exogenous drugs that the thick filament is structured to provide three levels of myosin activation corresponding to the three crowns of myosin heads in each 429Å repeat. In one crown, the myosin heads are almost completely activated and disordered. In another crown, many myosin heads are inactive, ordered into a structure called the interacting heads motif. At the third crown, the myosin heads are ordered into the interacting heads motif, but the stability of that motif is affected by myosin-binding protein C. We think that this hierarchy of control explains many of the effects of length-dependent activation as well as stretch activation in cardiac muscle control.

Missense variant Ile79Asn in human cardiac troponin T (cTnT-I79N) has been associated with hypertrophic cardiomyopathy and sudden cardiac arrest in juveniles. cTnT-I79N is located in the cTnT N-terminal (TnT1) loop region and is known for its pathological and prognostic relevance. A recent structural study revealed that I79 is part of a hydrophobic interface between the TnT1 loop and actin, which stabilizes the relaxed (OFF) state of the cardiac thin filament. Given the importance of understanding the role of TnT1 loop region in Ca ²⁺ regulation of the cardiac thin filament along with the underlying mechanisms of cTnT-I79N-linked pathogenesis, we investigated the effects of cTnT-I79N on cardiac myofilament function. Transgenic I79N (Tg-I79N) muscle bundles displayed increased myofilament Ca ²⁺ sensitivity, smaller myofilament lattice spacing, and slower crossbridge kinetics. These findings can be attributed to destabilization of the cardiac thin filament’s relaxed state resulting in an increased number of crossbridges during Ca ²⁺ activation. Additionally, in the low Ca ²⁺ -relaxed state (pCa8), we showed that more myosin heads are in the disordered-relaxed state (DRX) that are more likely to interact with actin in cTnT-I79N muscle bundles. Dysregulation of the myosin super-relaxed state (SRX) and the SRX/DRX equilibrium in cTnT-I79N muscle bundles likely result in increased mobility of myosin heads at pCa8, enhanced actomyosin interactions as evidenced by increased active force at low Ca ²⁺ , and increased sinusoidal stiffness. These findings point to a mechanism whereby cTnT-I79N weakens the interaction of the TnT1 loop with the actin filament, which in turn destabilizes the relaxed state of the cardiac thin filament.

Cardiac muscle contraction is distinct from the contraction of other muscle types. The heart continuously undergoes contraction–relaxation cycles throughout an animal’s lifespan. It must respond to constantly varying physical and energetic burdens over the short term on a beat-to-beat basis and relies on different mechanisms over the long term. Muscle contractility is based on actin and myosin interactions that are regulated by cytoplasmic calcium ions. Genetic variants of sarcomeric proteins can lead to the pathophysiological development of cardiac dysfunction. The sarcomere is physically connected to other cytoskeletal components. Actin filaments, microtubules and desmin proteins are responsible for these interactions. Therefore, mechanical as well as biochemical signals from sarcomeric contractions are transmitted to and sensed by other parts of the cardiomyocyte, particularly the nucleus which can respond to these stimuli. Proteins anchored to the nuclear envelope display a broad response which remodels the structure of the nucleus. In this review, we examine the central aspects of mechanotransduction in the cardiomyocyte where the transmission of mechanical signals to the nucleus can result in changes in gene expression and nucleus morphology. The correlation of nucleus sensing and dysfunction of sarcomeric proteins may assist the understanding of a wide range of functional responses in the progress of cardiomyopathic diseases.

Four insect orders have flight muscles that are both asynchronous and indirect; they are asynchronous in that the wingbeat frequency is decoupled from the frequency of nervous stimulation and indirect in that the muscles attach to the thoracic exoskeleton instead of directly to the wing. Flight muscle thick filaments from two orders, Hemiptera and Diptera, have been imaged at a subnanometer resolution, both of which revealed a myosin tail arrangement referred to as “curved molecular crystalline layers”. Here, we report a thick filament structure from the indirect flight muscles of a third insect order, Hymenoptera, the Asian bumble bee Bombus ignitus. The myosin tails are in general agreement with previous determinations from Lethocerus indicus and Drosophila melanogaster. The Skip 2 region has the same unusual structure as found in Lethocerus indicus thick filaments, an α-helix discontinuity is also seen at Skip 4, but the orientation of the Skip 1 region on the surface of the backbone is less angled with respect to the filament axis than in the other two species. The heads are disordered as in Drosophila, but we observe no non-myosin proteins on the backbone surface that might prohibit the ordering of myosin heads onto the thick filament backbone. There are strong structural similarities among the three species in their non-myosin proteins within the backbone that suggest how one previously unassigned density in Lethocerus might be assigned. Overall, the structure conforms to the previously observed pattern of high similarity in the myosin tail arrangement, but differences in the non-myosin proteins.

Striated muscle thick filaments are composed of myosin II and several non-myosin proteins which define the filament length and modify its function. Myosin II has a globular N-terminal motor domain comprising its catalytic and actin-binding activities and a long $\alpha$-helical, coiled tail that forms the dense filament backbone. Myosin alone polymerizes into filaments of irregular length, but striated muscle thick filaments have defined lengths that, with thin filaments, define the sarcomere structure. The motor domain structure and function are well understood, but the myosin filament backbone is not. Here we report on the structure of the flight muscle thick filaments from Drosophila melanogaster at 4.7 Å resolution, which eliminates previous ambiguities in non-myosin densities. The full proximal S2 region is resolved, as are the connecting densities between the Ig domains of stretchin-klp. The proteins, flightin, and myofilin are resolved in sufficient detail to build an atomic model based on an AlphaFold prediction. Our results suggest a method by which flightin and myofilin cooperate to define the structure of the thick filament and explains a key myosin mutation that affects flightin incorporation. Drosophila is a genetic model organism for which our results can define strategies for functional testing.

Invertebrate striated muscle myosin filaments are highly variable in structure. The best characterized myosin filaments are those found in insect indirect flight muscle (IFM) in which the flight-powering muscles are not attached directly to the wings. Four insect orders, Hemiptera, Diptera, Hymenoptera, and Coleoptera, have evolved IFM. IFM thick filaments from the first three orders have highly similar myosin arrangements but differ significantly among their non-myosin proteins. The cryo-electron microscopy of isolated IFM myosin filaments from the Dipteran Drosophila melanogaster described here revealed the coexistence of two distinct filament types, one presenting a tubular backbone like in previous work and the other a solid backbone. Inside an annulus of myosin tails, tubular filaments show no noticeable densities; solid filaments show four paired paramyosin densities. Both myosin heads of the tubular filaments are disordered; solid filaments have one completely and one partially immobilized head. Tubular filaments have the protein stretchin-klp on their surface; solid filaments do not. Two proteins, flightin and myofilin, are identifiable in all the IFM filaments previously determined. In Drosophila, flightin assumes two conformations, being compact in solid filaments and extended in tubular filaments. Nearly identical solid filaments occur in the large water bug Lethocerus indicus, which flies infrequently. The Drosophila tubular filaments occur in younger flies, and the solid filaments appear in older flies, which fly less frequently if at all, suggesting that the solid filament form is correlated with infrequent muscle use. We suggest that the solid form is designed to conserve ATP when the muscle is not in active use.