Pathomechanisms of Monoallelic variants in TTN causing skeletal muscle disease.

Pathogenic variants in the titin gene (TTN) are known to cause a wide range of cardiac and musculoskeletal disorders, with skeletal myopathy mostly attributed to biallelic variants. We identified monoallelic truncating variants (TTNtv), splice site or internal deletions in TTN in probands with mild, progressive axial and proximal weakness, with dilated cardiomyopathy frequently developing with age. These variants segregated in an autosomal dominant pattern in 7 out of 8 studied families. We investigated the impact of these variants on mRNA, protein levels, and skeletal muscle structure and function. Results reveal that nonsense-mediated decay likely prevents accumulation of harmful truncated protein in skeletal muscle in patients with TTNtvs. Splice variants and an out-of-frame deletion induce aberrant exon skipping, while an in-frame deletion produces shortened titin with intact N- and C-termini, resulting in disrupted sarcomeric structure. All variant types were associated with genome-wide changes in splicing patterns, which represent a hallmark of disease progression. Lastly, RNA-seq studies revealed that GDF11, a member of the TGF-ß superfamily, is upregulated in diseased tissue, indicating that it might be a useful therapeutic target in skeletal muscle titinopathies.

Myosin-binding protein H-like regulates myosin-binding protein distribution and function in atrial cardiomyocytes.

Mutations in atrial-enriched genes can cause a primary atrial myopathy that can contribute to overall cardiovascular dysfunction. MYBPHL encodes myosin-binding protein H-like (MyBP-HL), an atrial sarcomere protein that shares domain homology with the carboxy-terminus of cardiac myosin-binding protein-C (cMyBP-C). The function of MyBP-HL and the relationship between MyBP-HL and cMyBP-C is unknown. To decipher the roles of MyBP-HL, we used structured illumination microscopy, immuno-electron microscopy, and mass spectrometry to establish the localization and stoichiometry of MyBP-HL. We found levels of cMyBP-C, a major regulator of myosin function, were half as abundant compared to levels in the ventricle. In genetic mouse models, loss of MyBP-HL doubled cMyBP-C abundance in the atria, and loss of cMyBP-C doubled MyBP-HL abundance in the atria. Structured illumination microscopy showed that both proteins colocalize in the C-zone of the A-band, with MyBP-HL enriched closer to the M-line. Immuno-electron microscopy of mouse atria showed MyBP-HL strongly localized 161 nm from the M-line, consistent with localization to the third 43 nm repeat of myosin heads. Both cMyBP-C and MyBP-HL had less-defined sarcomere localization in the atria compared to ventricle, yet areas with the expected 43 nm repeat distance were observed for both proteins. Isometric force measurements taken from control and Mybphl null single atrial myofibrils revealed that loss of Mybphl accelerated the linear phase of relaxation. These findings support a mechanism where MyBP-HL regulates cMyBP-C abundance to alter the kinetics of sarcomere relaxation in atrial sarcomeres.

NRAP reduction rescues sarcomere defects in nebulin-related nemaline myopathy.

Nemaline myopathy (NM) is a rare neuromuscular disorder associated with congenital or childhood-onset of skeletal muscle weakness and hypotonia, which results in limited motor function. NM is a genetic disorder and mutations in 12 genes are known to contribute to autosomal dominant or recessive forms of the disease. Recessive mutations in nebulin (NEB) are the most common cause of NM affecting about 50% of patients. Because of the large size of the NEB gene and lack of mutational hot spots, developing therapies that can benefit a wide group of patients is challenging. Although there are several promising therapies under investigation, there is no cure for NM. Therefore, targeting disease modifiers that can stabilize or improve skeletal muscle function may represent alternative therapeutic strategies. Our studies have identified Nrap upregulation in nebulin deficiency that contributes to structural and functional deficits in NM. We show that genetic ablation of nrap in nebulin deficiency restored sarcomeric disorganization, reduced protein aggregates and improved skeletal muscle function in zebrafish. Our findings suggest that Nrap is a disease modifier that affects skeletal muscle structure and function in NM; thus, therapeutic targeting of Nrap in nebulin-related NM and related diseases may be beneficial for patients.

Titin's cardiac-specific N2B element is critical to mechanotransduction during volume overload of the heart.

The heart has the ability to detect and respond to changes in mechanical load through a process called mechanotransduction. In this study, we focused on investigating the role of the cardiac-specific N2B element within the spring region of titin, which has been proposed to function as a mechanosensor. To assess its significance, we conducted experiments using N2B knockout (KO) mice and wildtype (WT) mice, subjecting them to three different conditions: 1) cardiac pressure overload induced by transverse aortic constriction (TAC), 2) volume overload caused by aortocaval fistula (ACF), and 3) exercise-induced hypertrophy through swimming. Under conditions of pressure overload (TAC), both genotypes exhibited similar hypertrophic responses. In contrast, WT mice displayed robust left ventricular hypertrophy after one week of volume overload (ACF), while the KO mice failed to undergo hypertrophy and experienced a high mortality rate. Similarly, swim exercise-induced hypertrophy was significantly reduced in the KO mice. RNA-Seq analysis revealed an abnormal ß-adrenergic response to volume overload in the KO mice, as well as a diminished response to isoproterenol-induced hypertrophy. Because it is known that the N2B element interacts with the four-and-a-half LIM domains 1 and 2 (FHL1 and FHL2) proteins, both of which have been associated with mechanotransduction, we evaluated these proteins. Interestingly, while volume-overload resulted in FHL1 protein expression levels that were comparable between KO and WT mice, FHL2 protein levels were reduced by over 90% in the KO mice compared to WT. This suggests that in response to volume overload, FHL2 might act as a signaling mediator between the N2B element and downstream signaling pathways. Overall, our study highlights the importance of the N2B element in mechanosensing during volume overload, both in physiological and pathological settings.

Myosin ATPase inhibition fails to rescue the metabolically dysregulated proteome of nebulin-deficient muscle.

Nemaline myopathy (NM) is a genetic muscle disease, primarily caused by mutations in the NEB gene (NEB-NM) and with muscle myosin dysfunction as a major molecular pathogenic mechanism. Recently, we have observed that the myosin biochemical super-relaxed state was significantly impaired in NEB-NM, inducing an aberrant increase in ATP consumption and remodelling of the energy proteome in diseased muscle fibres. Because the small-molecule Mavacamten is known to promote the myosin super-relaxed state and reduce the ATP demand, we tested its potency in the context of NEB-NM. We first conducted in vitro experiments in isolated single myofibres from patients and found that Mavacamten successfully reversed the myosin ATP overconsumption. Following this, we assessed its short-term in vivo effects using the conditional nebulin knockout (cNeb KO) mouse model and subsequently performing global proteomics profiling in dissected soleus myofibres. After a 4 week treatment period, we observed a remodelling of a large number of proteins in both cNeb KO mice and their wild-type siblings. Nevertheless, these changes were not related to the energy proteome, indicating that short-term Mavacamten treatment is not sufficient to properly counterbalance the metabolically dysregulated proteome of cNeb KO mice. Taken together, our findings emphasize Mavacamten potency in vitro but challenge its short-term efficacy in vivo. KEY POINTS: No cure exists for nemaline myopathy, a type of genetic skeletal muscle disease mainly derived from mutations in genes encoding myofilament proteins. Applying Mavacamten, a small molecule directly targeting the myofilaments, to isolated membrane-permeabilized muscle fibres from human patients restored myosin energetic disturbances. Treating a mouse model of nemaline myopathy in vivo with Mavacamten for 4 weeks, remodelled the skeletal muscle fibre proteome without any noticeable effects on energetic proteins. Short-term Mavacamten treatment may not be sufficient to reverse the muscle phenotype in nemaline myopathy.

Aberrations in Energetic Metabolism and Stress-Related Pathways Contribute to Pathophysiology in the Neb Conditional Knockout Mouse Model of Nemaline Myopathy.

Nemaline myopathy (NM) is a genetically and clinically heterogeneous disease that is diagnosed on the basis of the presence of nemaline rods on skeletal muscle biopsy. Although NM has typically been classified by causative genes, disease severity or prognosis cannot be predicted. The common pathologic end point of nemaline rods (despite diverse genetic causes) and an unexplained range of muscle weakness suggest that shared secondary processes contribute to the pathogenesis of NM. We speculated that these processes could be identified through a proteome-wide interrogation using a mouse model of severe NM in combination with pathway validation and structural/functional analyses. A proteomic analysis was performed using skeletal muscle tissue from the Neb conditional knockout mouse model compared with its wild-type counterpart to identify pathophysiologically relevant biological processes that might impact disease severity or provide new treatment targets. A differential expression analysis and Ingenuity Pathway Core Analysis predicted perturbations in several cellular processes, including mitochondrial dysfunction and changes in energetic metabolism and stress-related pathways. Subsequent structural and functional studies demonstrated abnormal mitochondrial distribution, decreased mitochondrial respiratory function, an increase in mitochondrial transmembrane potential, and extremely low ATP content in Neb conditional knockout muscles relative to wild type. Overall, the findings of these studies support a role for severe mitochondrial dysfunction as a novel contributor to muscle weakness in NM.

Characterization of NEB pathogenic variants in patients reveals novel nemaline myopathy disease mechanisms and omecamtiv mecarbil force effects.

Nebulin, a critical protein of the skeletal muscle thin filament, plays important roles in physiological processes such as regulating thin filament length (TFL), cross-bridge cycling, and myofibril alignment. Pathogenic variants in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking curative therapies. In this study, we examined a cohort of ten NEM2 patients, each with unique pathogenic variants, aiming to understand their impact on mRNA, protein, and functional levels. Results show that pathogenic truncation variants affect NEB mRNA stability and lead to nonsense-mediated decay of the mutated transcript. Moreover, a high incidence of cryptic splice site activation was found in patients with pathogenic splicing variants that are expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with either relatively normal or markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, or reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a pathogenic duplication variant in nebulin that resulted in a four-copy gain in the triplicate region of NEB and a much larger nebulin protein and longer TFL. Additionally, we investigated the effect of Omecamtiv mecarbil (OM), a small-molecule activator of cardiac myosin, on force production of type 1 muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87 to 318%, with the largest effects in patients with the lowest level of nebulin. In summary, this study indicates that post-transcriptional or post-translational mechanisms regulate nebulin expression. Moreover, we propose that the pathomechanism of NEM2 involves not only shortened but also elongated thin filaments, along with the disruption of actin-binding sites resulting from pathogenic splicing variants. Significantly, our findings highlight the potential of OM treatment to improve skeletal muscle function in NEM2 patients, especially those with large reductions in nebulin levels.

Titin M-line insertion sequence 7 is required for proper cardiac function in mice.

Titin is a giant sarcomeric protein that is involved in a large number of functions, with a primary role in skeletal and cardiac sarcomere organization and stiffness. The titin gene (TTN) is subject to various alternative splicing events, but in the region that is present at the M-line, the only exon that can be spliced out is Mex5, which encodes for the insertion sequence 7 (is7). Interestingly, in the heart, the majority of titin isoforms are Mex5+, suggesting a cardiac role for is7. Here, we performed comprehensive functional, histological, transcriptomic, microscopic and molecular analyses of a mouse model lacking the Ttn Mex5 exon (ΔMex5), and revealed that the absence of the is7 is causative for dilated cardiomyopathy. ΔMex5 mice showed altered cardiac function accompanied by increased fibrosis and ultrastructural alterations. Abnormal expression of excitation-contraction coupling proteins was also observed. The results reported here confirm the importance of the C-terminal region of titin in cardiac function and are the first to suggest a possible relationship between the is7 and excitation-contraction coupling. Finally, these findings give important insights for the identification of new targets in the treatment of titinopathies.

RBM20 phosphorylation and its role in nucleocytoplasmic transport and cardiac pathogenesis.

Arginine-serine (RS) domain(s) in splicing factors are critical for protein-protein interaction in pre-mRNA splicing. Phosphorylation of RS domain is important for splicing control and nucleocytoplasmic transport in the cell. RNA-binding motif 20 (RBM20) is a splicing factor primarily expressed in the heart. A previous study using phospho-antibody against RS domain showed that RS domain can be phosphorylated. However, its actual phosphorylation sites and function have not been characterized. Using middle-down mass spectrometry, we identified 16 phosphorylation sites, two of which (S638 and S640 in rats, or S637 and S639 in mice) were located in the RSRSP stretch in the RS domain. Mutations on S638 and S640 regulated splicing, promoted nucleocytoplasmic transport and protein-RNA condensates. Phosphomimetic mutations on S638 and S640 indicated that phosphorylation was not the major cause for RBM20 nucleocytoplasmic transport and condensation in vitro. We generated a S637A knock-in (KI) mouse model (Rbm20S637A ) and observed the reduced RBM20 phosphorylation. The KI mice exhibited aberrant gene splicing, protein condensates, and a dilated cardiomyopathy (DCM)-like phenotype. Transcriptomic profiling demonstrated that KI mice had altered expression and splicing of genes involving cardiac dysfunction, protein localization, and condensation. Our in vitro data showed that phosphorylation was not a direct cause for nucleocytoplasmic transport and protein condensation. Subsequently, the in vivo results reveal that RBM20 mutations led to cardiac pathogenesis. However, the role of phosphorylation in vivo needs further investigation.

Pharmacological Inhibition of Myostatin in a Mouse Model of Typical Nemaline Myopathy Increases Muscle Size and Force.

Nemaline myopathy is one of the most common non-dystrophic congenital myopathies. Individuals affected by this condition experience muscle weakness and muscle smallness, often requiring supportive measures like wheelchairs or respiratory support. A significant proportion of patients, approximately one-third, exhibit compound heterozygous nebulin mutations, which usually give rise to the typical form of the disease. Currently, there are no approved treatments available for nemaline myopathy. Our research explored the modulation of myostatin, a negative regulator of muscle mass, in combating the muscle smallness associated with the disease. To investigate the effect of myostatin inhibition, we employed a mouse model with compound heterozygous nebulin mutations that mimic the typical form of the disease. The mice were treated with mRK35, a myostatin antibody, through weekly intraperitoneal injections of 10 mg/kg mRK35, commencing at two weeks of age and continuing until the mice reached four months of age. The treatment resulted in an increase in body weight and an approximate 20% muscle weight gain across most skeletal muscles, without affecting the heart. The minimum Feret diameter of type IIA and IIB fibers exhibited an increase in compound heterozygous mice, while only type IIB fibers demonstrated an increase in wild-type mice. In vitro mechanical experiments conducted on intact extensor digitorum longus muscle revealed that mRK35 augmented the physiological cross-sectional area of muscle fibers and enhanced absolute tetanic force in both wild-type and compound heterozygous mice. Furthermore, mRK35 administration improved grip strength in treated mice. Collectively, these findings indicate that inhibiting myostatin can mitigate the muscle deficits in nebulin-based typical nemaline myopathy, potentially serving as a much-needed therapeutic option.