Titin-based mechanosensing modulates muscle hypertrophy

Background: Titin is an elastic sarcomeric filament that has been proposed to play a key role in mechanosensing and trophicity of muscle. However, evidence for this proposal is scarce due to the lack of appropriate experimental models to directly test the role of titin in mechanosensing. Methods: We used unilateral diaphragm denervation (UDD) in mice, an in vivo model in which the denervated hemidiaphragm is passively stretched by the contralateral, innervated hemidiaphragm and hypertrophy rapidly occurs. Results: In wildtype mice, the denervated hemidiaphragm mass increased 48 ± 3% after 6 days of UDD, due to the addition of both sarcomeres in series and in parallel. To test whether titin stiffness modulates the hypertrophy response, RBM20ΔRRM and TtnΔIAjxn mouse models were used, with decreased and increased titin stiffness, respectively. RBM20ΔRRM mice (reduced stiffness) showed a 20 ± 6% attenuated hypertrophy response, whereas the TtnΔIAjxn mice (increased stiffness) showed an 18 ± 8% exaggerated response after UDD. Thus, muscle hypertrophy scales with titin stiffness. Protein expression analysis revealed that titin-binding proteins implicated previously in muscle trophicity were induced during UDD, MARP1 & 2, FHL1, and MuRF1. Conclusions: Titin functions as a mechanosensor that regulates muscle trophicity

COVID-19 is associated with distinct myopathic features in the diaphragm of critically ill patients

Introduction The diaphragm is the main muscle of inspiration, and its dysfunction contributes to adverse clinical outcomes in critically ill patients. We recently reported the infiltration of SARS-CoV-2, and the development of fibrosis, in the diaphragm of critically ill patients with COVID-19. In the current study, we aimed to characterise myofiber structure in the diaphragm of critically ill patients with COVID-19. Methods Diaphragm muscle specimens were collected during autopsy from patients who died of COVID-19 in three academic medical centres in the Netherlands in April and May 2020 (n=27). We studied diaphragm myofiber gene expression and structure and compared the findings obtained to those of deceased critically ill patients without COVID-19 (n=10). Results Myofibers of critically ill patients with COVID-19 showed on average larger cross-sectional area (slow-twitch myofibers: 2441±229 vs 1571±309 μm 2; fast-twitch myofibers: 1966±209 vs 1225±222 μm 2). Four critically ill patients with COVID-19 showed extremely large myofibers, which were splitting and contained many centralised nuclei. RNA-sequencing data revealed differentially expressed genes involved in muscle regeneration. Conclusion Diaphragm of critically ill patients with COVID-19 has distinct myopathic features compared with critically ill patients without COVID-19, which may contribute to the ongoing dyspnoea and fatigue in the patients surviving COVID-19 infection

Cardiac troponin T N-domain variant destabilizes the actin interface resulting in disturbed myofilament function

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 Ca2+ 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 Ca2+ 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 Ca2+ activation. Additionally, in the low Ca2+-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 Ca2+, 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

Dysfunctional sarcomere contractility contributes to muscle weakness in ACTA1-related nemaline myopathy (NEM3)

Objective: Nemaline myopathy (NM) is one of the most common congenital nondystrophic myopathies and is characterized by muscle weakness, often from birth. Mutations in ACTA1 are a frequent cause of NM (ie, NEM3). ACTA1 encodes alpha-actin 1, the main constituent of the sarcomeric thin filament. The mechanisms by which mutations in ACTA1 contribute to muscle weakness in NEM3 are incompletely understood. We hypothesized that sarcomeric dysfunction contributes to muscle weakness in NEM3 patients. Methods: To test this hypothesis, we performed contractility measurements in individual muscle fibers and myofibrils obtained from muscle biopsies of 14 NEM3 patients with different ACTA1 mutations. To identify the structural basis for impaired contractility, low angle X-ray diffraction and stimulated emission-depletion microscopy were applied. Results: Our findings reveal that muscle fibers of NEM3 patients display a reduced maximal force-generating capacity, which is caused by dysfunctional sarcomere contractility in the majority of patients, as revealed by contractility measurements in myofibrils. Low angle X-ray diffraction and stimulated emission-depletion microscopy indicate that dysfunctional sarcomere contractility in NEM3 patients involves a lower number of myosin heads binding to actin during muscle activation. This lower number is not the result of reduced thin filament length. Interestingly, the calcium sensitivity of force is unaffected in some patients, but decreased in others. Interpretation: Dysfunctional sarcomere contractility is an important contributor to muscle weakness in the majority of NEM3 patients. This information is crucial for patient stratification in future clinical trials. Ann Neurol 2018;83:269–282

Pathogenic variants in TNNC2 cause congenital myopathy due to an impaired force response to calcium

Troponin C (TnC) is a critical regulator of skeletal muscle contraction; it binds Ca2+ to activate muscle contraction. Surprisingly, the gene encoding fast skeletal TnC (TNNC2) has not yet been implicated in muscle disease. Here, we report 2 families with pathogenic variants in TNNC2. Patients present with a distinct, dominantly inherited congenital muscle disease. Molecular dynamics simulations suggested that the pathomechanisms by which the variants cause muscle disease include disruption of the binding sites for Ca2+ and for troponin I. In line with these findings, physiological studies in myofibers isolated from patients’ biopsies revealed a markedly reduced force response of the sarcomeres to [Ca2+]. This pathomechanism was further confirmed in experiments in which contractile dysfunction was evoked by replacing TnC in myofibers from healthy control subjects with recombinant, mutant TnC. Conversely, the contractile dysfunction of myofibers from patients was repaired by replacing endogenous, mutant TnC with recombinant, wild-type TnC. Finally, we tested the therapeutic potential of the fast skeletal muscle troponin activator tirasemtiv in patients’ myofibers and showed that the contractile dysfunction was repaired. Thus, our data reveal that pathogenic variants in TNNC2 cause congenital muscle disease, and they provide therapeutic angles to repair muscle contractility

KBTBD13 is an actin-binding protein that modulates muscle kinetics

The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low-angle x-ray diffraction, and superresolution microscopy revealed that the impaired muscle-relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling and binding and contractility assays with recombinant KBTBD13, Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models, and a GFP-labeled Kbtbd13-transgenic zebrafish model, we discovered that KBTBD13 binds to actin - a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle-relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology