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Titin (TTN) is one of the largest and most complex proteins expressed in humans, and truncation variants are the most prevalent genetic lesion identified in individuals with dilated cardiomyopathy (DCM) or other disorders of impaired cardiac contractility. Two reports in this issue of the JCI shed light on a potential mechanism involving truncated TTN sarcomere integration and the potential for disruption of sarcomere structural integrity. Kellermayer, Tordai, and colleagues confirmed the presence of truncated TTN protein in human DCM samples. McAfee and authors developed a patient-specific TTN antibody to study truncated TTN subcellular localization and to explore its functional consequences. A "poison peptide" mechanism emerges that inspires alternative therapeutic approaches while opening new lines for inquiry, such as the role of haploinsufficiency of full-length TTN protein, mechanisms explaining sarcomere dysfunction, and explanations for variable penetrance.

Myofilaments and their associated proteins, which together constitute the sarcomeres, provide the molecular-level basis for contractile function in all muscle types. In intact muscle, sarcomere-level contraction is strongly coupled to other cellular subsystems, in particular the sarcolemmal membrane. Skinned muscle preparations (where the sarcolemma has been removed or permeabilized) are an experimental system designed to probe contractile mechanisms independently of the sarcolemma. Over the last few decades, experiments performed using permeabilized preparations have been invaluable for clarifying the understanding of contractile mechanisms in both skeletal and cardiac muscle. Today, the technique is increasingly harnessed for preclinical and/or pharmacological studies that seek to understand how interventions will impact intact muscle contraction. In this context, intrinsic functional and structural differences between skinned and intact muscle pose a major interpretational challenge. This review first surveys measurements that highlight these differences in terms of the sarcomere structure, passive and active tension generation, and calcium dependence. We then highlight the main practical challenges and caveats faced by experimentalists seeking to emulate the physiological conditions of intact muscle. Gaining an awareness of these complexities is essential for putting experiments in due perspective., (© 2022 Lewalle et al.)

We have created a novel in-vitro platform to study reverse remodeling of engineered heart tissue (EHT) after mechanical unloading. EHTs were created by seeding decellularized porcine myocardial sections with a mixture of primary neonatal rat ventricular myocytes and cardiac fibroblasts. Each end of the ribbon-like constructs was fixed to a plastic clip, allowing the tissues to be statically stretched or slackened. Inelastic deformation was introduced by stretching tissues by 20% of their original length. EHTs were subsequently unloaded by returning tissues to their original, shorter length. Mechanical characterization of EHTs immediately after unloading and at subsequent time points confirmed the presence of a reverse-remodeling process, through which stress-free tissue length was increased after chronic stretch but gradually decreased back to its original value within 9 days. When a cardiac myosin inhibitor was applied to tissues after unloading, EHTs failed to completely recover their passive and active mechanical properties, suggesting a role for actomyosin contraction in reverse remodeling. Selectively inhibiting cardiomyocyte contraction or fibroblast activity after mechanical unloading showed that contractile activity of both cell types was required to achieve full remodeling. Similar tests with EHTs formed from human induced pluripotent stem cell-derived cardiomyocytes also showed reverse remodeling that was enhanced when treated with omecamtiv mecarbil, a myosin activator. These experiments suggest essential roles for active sarcomeric contraction and fibroblast activity in reverse remodeling of myocardium after mechanical unloading. Our findings provide a mechanistic rationale for designing potential therapies to encourage reverse remodeling in patient hearts., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

KBTBD13 is a protein expressed in striated muscle whose precise function is unknown. Work by de Winter et al. in this issue of the JCI provides evidence that KBTBD13 localizes to the sarcomere and can directly bind actin. A mutation in KBTBD13 that is associated with nemaline myopathy alters the protein's effects on actin, apparently increasing thin-filament stiffness and ultimately depressing contractile force and relaxation rate. We discuss here the implications of this new sarcomeric protein, some alternate explanations for the effects of KBTBD13R408C, and the advantages of using computational models to interpret functional data from muscle.

A skeletal muscle fiber that is stimulated to contract and then stretched from L₁ to L₂ produces more force after the initial transient decays than if it is stimulated at L₂. This behavior has been well studied experimentally, and is known as residual force enhancement. The underlying mechanism remains controversial. We hypothesized that residual force enhancement could reflect mechanical interactions between heterogeneous half-sarcomeres. To test this hypothesis, we subjected a computational model of interacting heterogeneous half-sarcomeres to the same activation and stretch protocols that produce residual force enhancement in real preparations. Following a transient period of elevated force associated with active stretching, the model predicted a slowly decaying force enhancement lasting >30 seconds after stretch. Enhancement was on the order of 13% above isometric tension at the post-stretch muscle length, which agrees well with experimental measurements. Force enhancement in the model was proportional to stretch magnitude but did not depend strongly on the velocity of stretch, also in agreement with experiments. Even small variability in the strength of half-sarcomeres (2.1% standard deviation, normally distributed) was sufficient to produce a 5% force enhancement over isometric tension. Analysis of the model suggests that heterogeneity in half-sarcomeres leads to residual force enhancement by storing strain energy introduced during active stretch in distributions of bound cross-bridges. Complex interactions between the heterogeneous half-sarcomeres then dissipate this stored energy at a rate much slower than isolated cross-bridges would cycle. Given the variations in half-sarcomere length that have been observed in real muscle preparations and the stochastic variability inherent in all biological systems, half-sarcomere heterogeneity cannot be excluded as a contributing source of residual force enhancement.

We developed a Markov model of cardiac thin filament activation that accounts for interactions among nearest-neighbor regulatory units (RUs) in a spatially explicit manner. Interactions were assumed to arise from structural coupling of adjacent tropomyosins (Tms), such that Tm shifting within each RU was influenced by the Tm status of its neighbors. Simulations using the model demonstrate that this coupling is sufficient to produce observed cooperativity in both steady-state and dynamic force-Ca(2+) relationships. The model was further validated by comparison with reported responses under various conditions including inhibition of myosin binding and the addition of strong-binding, non-force-producing myosin fragments. The model also reproduced the effects of 2.5 mM added P(i) on Ca(2+)-activated force and the rate of force redevelopment measured in skinned rat myocardial preparations. Model analysis suggests that Tm-Tm coupling potentiates the activating effects of strongly-bound cross-bridges and contributes to force-Ca(2+) dynamics of intact cardiac muscle. The model further predicts that activation at low Ca(2+) concentrations is cooperatively inhibited by nearest neighbors, requiring Ca(2+) binding to >25% of RUs to produce appreciable levels of force. Without excluding other putative cooperative mechanisms, these findings suggest that structural coupling of adjacent Tm molecules contributes to several properties of cardiac myofilament activation., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)