Significance: Heartbeats rely on cyclical interactions between myosin thick and actin thin filaments orchestrated by rising and falling Ca ²⁺ . During systole, Ca ²⁺ binds to the thin filament and allows its interaction with the thick filament to produce force required for contraction. The structure of the thin filament at physiological Ca ²⁺ levels is unknown, which limits our understanding of the thin filament regulation by Ca ²⁺ . Here, we directly observe thin filament structural states along individual filaments at systolic Ca ²⁺ levels to show that the thin filament is activated stochastically with short-range cooperativity evident only on one strand of the thin filament. We suggest how cardiac muscle can be regulated by a narrow range of Ca ²⁺ levels upon transition from diastole to systole.

Fast skeletal myosin-binding protein-C (fMyBP-C) is one of three MyBP-C paralogs and is predominantly expressed in fast skeletal muscle. Mutations in the gene that encodes fMyBP-C, MYBPC2, are associated with distal arthrogryposis, while loss of fMyBP-C protein is associated with diseased muscle. However, the functional and structural roles of fMyBP-C in skeletal muscle remain unclear. To address this gap, we generated a homozygous fMyBP-C knockout mouse (C2^−/−) and characterized it both in vivo and in vitro compared to wild-type mice. Ablation of fMyBP-C was benign in terms of muscle weight, fiber type, cross-sectional area, and sarcomere ultrastructure. However, grip strength and plantar flexor muscle strength were significantly decreased in C2^−/− mice. Peak isometric tetanic force and isotonic speed of contraction were significantly reduced in isolated extensor digitorum longus (EDL) from C2^−/− mice. Small-angle X-ray diffraction of C2^−/− EDL muscle showed significantly increased equatorial intensity ratio during contraction, indicating a greater shift of myosin heads toward actin, while MLL4 layer line intensity was decreased at rest, indicating less ordered myosin heads. Interfilament lattice spacing increased significantly in C2^−/− EDL muscle. Consistent with these findings, we observed a significant reduction of steady-state isometric force during Ca^2+-activation, decreased myofilament calcium sensitivity, and sinusoidal stiffness in skinned EDL muscle fibers from C2^−/− mice. Finally, C2^−/− muscles displayed disruption of inflammatory and regenerative pathways, along with increased muscle damage upon mechanical overload. Together, our data suggest that fMyBP-C is essential for maximal speed and force of contraction, sarcomere integrity, and calcium sensitivity in fast-twitch muscle. Significance Myosin-binding protein-C (MyBP-C) is a thick filament regulatory protein found exclusively in the C-zone of the A-band in the sarcomeres of vertebrate striated muscle. Cardiac, slow skeletal, and fast skeletal MyBP-C (fMyBP-C) paralogs perform different functions. All three paralogs share similar protein structures but likely differ substantially in terms of expression and function, which may serve the distinct physiologies of fast and slow muscle fibers. However, the functional role of fMyBP-C in fast skeletal muscle is completely unknown. Genetic mutations in human fMyBP-C lead to skeletal myopathies. In this study, we used knockout mice to define the molecular basis for fMyBP-C paralog diversity. We demonstrate that fMyBP-C modulates the speed and force of fast skeletal muscle contraction.

Myocyte disarray is a hallmark of many cardiac disorders. However, the relationship between alterations in the orientation of individual myofibrils and myofilaments to disease progression has been largely underexplored. This oversight has predominantly been because of a paucity of methods for objective and quantitative analysis. Here, we introduce a novel, less-biased approach to quantify myofibrillar and myofilament orientation in cardiac muscle under near-physiological conditions and demonstrate its superiority as compared with conventional histological assessments. Using small-angle x-ray diffraction, we first investigated changes in myofibrillar orientation at increasing sarcomere lengths in permeabilized, relaxed, wild-type mouse myocardium from the left ventricle by assessing the angular spread of the 1,0 equatorial reflection (angle σ). At a sarcomere length of 1.9 μm, the angle σ was 0.23 ± 0.01 rad, decreased to 0.19 ± 0.01 rad at a sarcomere length of 2.1 μm, and further decreased to 0.15 ± 0.01 rad at a sarcomere length of 2.3 μm (p < 0.0001). Angle σ was significantly larger in R403Q, a MYH7 hypertrophic cardiomyopathy model, porcine myocardium (0.24 ± 0.01 rad) compared with wild-type myocardium (0.14 ± 0.005 rad; p < 0.0001), as well as in human heart failure tissue (0.19 ± 0.006 rad) when compared with nonfailing samples (0.17 ± 0.007 rad; p = 0.01). These data indicate that diseased myocardium suffers from greater myofibrillar disorientation compared with healthy controls. Finally, we showed that conventional, histology-based analysis of disarray can be subject to user bias and/or sampling error and lead to false positives. Our method for directly assessing myofibrillar orientation avoids the artifacts introduced by conventional histological approaches that assess myocyte orientation and only indirectly evaluate myofibrillar orientation, and provides a precise and objective metric for phenotypically characterizing myocardium. The ability to obtain excellent x-ray diffraction patterns from frozen human myocardium provides a new tool for investigating structural anomalies associated with cardiac diseases.

Nuclear factor κ-B (NFκB) is activated in iPSC-cardiac myocytes from patients with arrhythmogenic cardiomyopathy (ACM) under basal conditions, and inhibition of NFκB signaling prevents disease in Dsg2 ^mut/mut mice, a robust mouse model of ACM. Here, we used genetic approaches and single-cell RNA-Seq to define the contributions of immune signaling in cardiac myocytes and macrophages in the natural progression of ACM using Dsg2 ^mut/mut mice. We found that NFκB signaling in cardiac myocytes drives myocardial injury, contractile dysfunction, and arrhythmias in Dsg2 ^mut/mut mice. NFκB signaling in cardiac myocytes mobilizes macrophages expressing C-C motif chemokine receptor-2 (CCR2 ⁺ cells) to affected areas within the heart, where they mediate myocardial injury and arrhythmias. Contractile dysfunction in Dsg2 ^mut/mut mice is caused both by loss of heart muscle and negative inotropic effects of inflammation in viable muscle. Single nucleus RNA-Seq and cellular indexing of transcriptomes and epitomes (CITE-Seq) studies revealed marked proinflammatory changes in gene expression and the cellular landscape in hearts of Dsg2 ^mut/mut mice involving cardiac myocytes, fibroblasts, and CCR2 ⁺ macrophages. Changes in gene expression in cardiac myocytes and fibroblasts in Dsg2 ^mut/mut mice were dependent on CCR2 ⁺ macrophage recruitment to the heart. These results highlight complex mechanisms of immune injury and regulatory crosstalk between cardiac myocytes, inflammatory cells, and fibroblasts in the pathogenesis of ACM.

Aberrant regulation of myocardial force production represents an early biomechanical defect associated with sarcomeric cardiomyopathies, but the molecular mechanisms remain poorly defined. Here, we evaluated the pathogenicity of a previously unreported sarcomeric gene variant identified in a pediatric patient with sporadic dilated cardiomyopathy, and we determined a molecular mechanism. Trio whole-exome sequencing revealed a de novo missense variant in TNNC1 that encodes a p.I4M substitution in the N-terminal helix of cardiac troponin C (cTnC). Reconstitution of this human cTnC variant into permeabilized porcine cardiac muscle preparations significantly decreases the magnitude and rate of isometric force generation at physiological Ca²⁺-activation levels. Computational modeling suggests that this inhibitory effect can be explained by a decrease in the rates of cross-bridge attachment and detachment. For the first time, we show that cardiac troponin T (cTnT), in part through its intrinsically disordered C terminus, directly binds to WT cTnC, and we find that this cardiomyopathic variant displays tighter binding to cTnT. Steady-state fluorescence and NMR spectroscopy studies suggest that this variant propagates perturbations in cTnC structural dynamics to distal regions of the molecule. We propose that the intrinsically disordered C terminus of cTnT directly interacts with the regulatory N-domain of cTnC to allosterically modulate Ca²⁺ activation of force, perhaps by controlling the troponin I switching mechanism of striated muscle contraction. Alterations in cTnC–cTnT binding may compromise contractile performance and trigger pathological remodeling of the myocardium.