Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.

Troponin Variants in Congenital Myopathies: How They Affect Skeletal Muscle Mechanics.

The troponin complex is a key regulator of muscle contraction. Multiple variants in skeletal troponin encoding genes result in congenital myopathies. TNNC2 has been implicated in a novel congenital myopathy, TNNI2 and TNNT3 in distal arthrogryposis (DA), and TNNT1 and TNNT3 in nemaline myopathy (NEM). Variants in skeletal troponin encoding genes compromise sarcomere function, e.g., by altering the Ca2+ sensitivity of force or by inducing atrophy. Several potential therapeutic strategies are available to counter the effects of variants, such as troponin activators, introduction of wild-type protein through AAV gene therapy, and myosin modulation to improve muscle contraction. The mechanisms underlying the pathophysiological effects of the variants in skeletal troponin encoding genes are incompletely understood. Furthermore, limited knowledge is available on the structure of skeletal troponin. This review focusses on the physiology of slow and fast skeletal troponin and the pathophysiology of reported variants in skeletal troponin encoding genes. A better understanding of the pathophysiological effects of these variants, together with enhanced knowledge regarding the structure of slow and fast skeletal troponin, will direct the development of treatment strategies.

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 µm2; fast-twitch myofibers: 1966±209 vs 1225±222 µm2). 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.

Muscle ankyrin repeat protein 1 (MARP1) locks titin to the sarcomeric thin filament and is a passive force regulator.

Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin-N2A element, found in titin's molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin-N2A. Mechanics and super-resolution microscopy revealed that MARP1 "locks" titin-N2A to the sarcomeric thin filament, causing increased extension of titin's elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.

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.

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer.

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of serially linked sarcomeres, the smallest contractile units in muscle. Sarcomeric dysfunction contributes to muscle weakness in patients with mutations in genes encoding for sarcomeric proteins. The study of myofibril mechanics allows for the assessment of actin-myosin interactions without potential confounding effects of damaged, adjacent myofibrils when measuring the contractility of single muscle fibers. Ultrastructural damage and misalignment of myofibrils might contribute to impaired contractility. If structural damage is present in the myofibrils, they likely break during the isolation procedure or during the experiment. Furthermore, studies in myofibrils provide the assessment of actin-myosin interactions in the presence of the geometrical constraints of the sarcomeres. For instance, measurements in myofibrils can elucidate whether myofibrillar dysfunction is the primary effect of a mutation in a sarcomeric protein. In addition, perfusion with calcium solutions or compounds is almost instant due to the small diameter of the myofibril. This makes myofibrils eminently suitable to measure the rates of activation and relaxation during force production. The protocol described in this paper employs an optical force probe based on the principle of a Fabry-Pérot interferometer capable of measuring forces in the nano-Newton range, coupled to a piezo length motor and a fast-step perfusion system. This setup enables the study of myofibril mechanics with high resolution force measurements.

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.

End-diastolic force pre-activates cardiomyocytes and determines contractile force: role of titin and calcium.

Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca2+ ], titin-based stretch pre-activates cardiomyocytes during diastole and is a major determinant of force production in the subsequent contraction. Through this mechanism titin would play an important role in active force development and length-dependent activation. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) result in expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work loops that mimic the cardiac cycle in wild-type (WT) and heterozygous (HET) RBM20-deficient rats. In addition, we studied the role of diastolic [Ca2+ ] in membrane-permeabilized WT and HET cardiomyocytes. Intact cardiomyocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT) at low pacing frequencies, but this difference disappeared at high pacing frequencies. Length-dependent activation (force-sarcomere length relationship) was blunted in HET cardiomyocytes, but the force-end-diastolic force relationship was not different between HET and WT cardiomyocytes. To delineate the effects of diastolic [Ca2+ ] and titin pre-activation on force generation, measurements were performed in detergent-permeabilized cardiomyocytes. Cardiac twitches were simulated by transiently exposing permeabilized cardiomyocytes to 2 µm Ca2+ . Increasing diastolic [Ca2+ ] from 1 to 80 nm increased force development twofold in WT. Higher diastolic [Ca2+ ] was needed in HET. These findings are consistent with our hypothesis that pre-activation increases active force development. Highly compliant titin allows cells to function at higher diastolic [Ca2+ ].

Recessive MYH7-related myopathy in two families.

Myopathies due to recessive MYH7 mutations are exceedingly rare, reported in only two families to date. We describe three patients from two families (from Australia and the UK) with a myopathy caused by recessive mutations in MYH7. The Australian family was homozygous for a c.5134C > T, p.Arg1712Trp mutation, whilst the UK patient was compound heterozygous for a truncating (c.4699C > T; p.Gln1567*) and a missense variant (c.4664A > G; p.Glu1555Gly). All three patients shared key clinical features, including infancy/childhood onset, pronounced axial/proximal weakness, spinal rigidity, severe scoliosis, and normal cardiac function. There was progressive respiratory impairment necessitating non-invasive ventilation despite preserved ambulation, a combination of features often seen in SEPN1- or NEB-related myopathies. On biopsy, the Australian proband showed classical myosin storage myopathy features, while the UK patient showed multi-minicore like areas. To establish pathogenicity of the Arg1712Trp mutation, we expressed mutant MYH7 protein in COS-7 cells, observing abnormal mutant myosin aggregation compared to wild-type. We describe skinned myofiber studies of patient muscle and hypertrophy of type II myofibers, which may be a compensatory mechanism. In summary, we have expanded the phenotype of ultra-rare recessive MYH7 disease, and provide novel insights into associated changes in muscle physiology.