Biology subjects

Granzier, H.

Publications and source records attributed to Granzier, H..

7 recordsLinked to original sources

bioRxiv2025

KLHL41 orchestrates sarcomere assembly and size to drive skeletal muscle hypertrophy in vivo

Sarcomere assembly and growth are fundamental processes essential for the development, function, and repair of skeletal muscle. However, the mechanisms underlying sarcomere formation and assembly in vivo, which are critical for the formation of functional myofibers in vertebrates, remain poorly understood. Defects in sarcomeres contribute to muscle dysfunction in numerous genetic and acquired myopathies, yet the lack of a clear understanding of specific sarcomeric defects has hindered the development of effective therapies. Nemaline myopathy (NM) is caused by mutations in genes that primarily affect sarcomere structure and function. The disease is clinically heterogeneous, with the congenital form being the most severe. Using zebrafish models of congenital forms of NM, we investigated sarcomere assembly in vivo during skeletal muscle development to identify key steps contributing to myofibril formation and muscle growth under both normal and disease conditions. Our findings demonstrate that the gene encoding the sarcomeric protein KLHL41 plays a critical role in the formation and directional organization of new sarcomeres within developing myofibrils, facilitating both radial and longitudinal muscle growth. Dysregulation of sarcomeric proteins and impaired protein turnover in the KLHL41-NM zebrafish model resulted in the development of sarcomeric defects. Moreover, we show that the interaction between the KLHL41-troponin complex is essential for muscle growth and cross-bridge regulation in developing muscle. These studies address a significant gap in the understanding of sarcomeric defects in myopathies and will help guide the development of targeted therapies aimed at rescuing these processes.

genetics↗

bioRxiv2024

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. As the small-molecule Mavacamten is known to promote the myosin super-relaxed state and reduce the ATP demand, here, 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 over-consumption. Following this, we assessed its short-term in vivo effects by using the conditional nebulin knock-out (cNeb KO) mouse model and by subsequently performing global proteomics profiling in dissected soleus myofibres. After a four-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 summaryO_LINo cure exists for nemaline myopathy, a type of genetic skeletal muscle disease mainly derived from mutations in genes encoding myofilament proteins. C_LIO_LIApplying Mavacamten, a small molecule directly targeting the myofilament, to isolated membrane-permeabilized muscle fibres from human patients restored myosin energetic disturbances. C_LIO_LITreating a mouse model of nemaline myopathy in vivo with Mavacamten for four weeks, remodeled the skeletal muscle fibre proteome without any noticeable effects on energetic proteins. C_LIO_LIShort-term Mavacamten treatment may not be sufficient to reverse the muscle phenotype in nemaline myopathy. C_LI

physiology↗

bioRxiv2023

Characterization of NEB mutations 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. Mutations in the nebulin gene (NEB) cause NEB-based nemaline myopathy (NEM2), a genetically heterogeneous disorder characterized by hypotonia and muscle weakness, currently lacking therapies targeting the underlying pathological mechanisms. In this study, we examined a cohort of ten NEM2 patients, each with unique mutations, aiming to understand their impact on mRNA, protein, and functional levels. Results show that truncation mutations 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 splicing mutations which is expected to disrupt the actin-binding sites of nebulin. Determination of protein levels revealed patients with relatively normal nebulin levels and others with markedly reduced nebulin. We observed a positive relation between the reduction in nebulin and a reduction in TFL, and a positive relation between the reduction in nebulin level and the reduction in tension (both maximal and submaximal tension). Interestingly, our study revealed a duplication mutation in nebulin that resulted in a 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 I muscle fibers of NEM2 patients. OM treatment substantially increased submaximal tension across all NEM2 patients ranging from 87-318%, 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 splicing mutations. 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.

genetics↗

bioRxiv2023

Late-life Rapamycin Treatment Enhances Cardiomyocyte Relaxation Kinetics and Reduces Myocardial Stiffness

Diastolic dysfunction is a key feature of the aging heart. We have shown that late-life treatment with mTOR inhibitor, rapamycin, reverses age-related diastolic dysfunction in mice but the molecular mechanisms of the reversal remain unclear. To dissect the mechanisms by which rapamycin improves diastolic function in old mice, we examined the effects of rapamycin treatment at the levels of single cardiomyocyte, myofibril and multicellular cardiac muscle. Compared to young cardiomyocytes, isolated cardiomyocytes from old control mice exhibited prolonged time to 90% relaxation (RT90) and time to 90% Ca2+ transient decay (DT90), indicating slower relaxation kinetics and calcium reuptake with age. Late-life rapamycin treatment for 10 weeks completely normalized RT90 and partially normalized DT90, suggesting improved Ca2+ handling contributes partially to the rapamycin-induced improved cardiomyocyte relaxation. In addition, rapamycin treatment in old mice enhanced the kinetics of sarcomere shortening and Ca2+ transient increase in old control cardiomyocytes. Myofibrils from old rapamycin-treated mice displayed increased rate of the fast, exponential decay phase of relaxation compared to old controls. The improved myofibrillar kinetics were accompanied by an increase in MyBP-C phosphorylation at S282 following rapamycin treatment. We also showed that late-life rapamycin treatment normalized the age-related increase in passive stiffness of demembranated cardiac trabeculae through a mechanism independent of titin isoform shift. In summary, our results showed that rapamycin treatment normalizes the age-related impairments in cardiomyocyte relaxation, which works conjointly with reduced myocardial stiffness to reverse age-related diastolic dysfunction.

physiology↗

bioRxiv2022

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). RBM20 is a splicing factor with two canonical domains, an RNA recognition motif (RRM) and an arginine-serine rich (RS) domain. RRM loss-of-function disrupts the splicing of RBM20 target transcripts and leads to systolic dysfunction without overt DCM, while mutations in the RS domain precipitate DCM. We show that mice lacking the RS domain (Rbm20{Delta}RS) manifest DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 is mis-localized in Rbm20{Delta}RS mice but not in mice lacking the RRM, which are also deficient in RBM20 splicing. We determine that the RS domain, not other domains including the RRM, is critical for RBM20 nuclear import and define the core nuclear localization signal (NLS) within this domain. Mutation analysis of phosphorylation sites within the RS domain indicate that phosphorylation is dispensable for RBM20 nuclear import. Collectively, our findings establish disruption of the NLS in RBM20 as a causative mechanism in DCM through nucleocytoplasmic transport.

cell biology↗

bioRxiv2020

Late-life restoration of mitochondrial function reverses cardiac dysfunction in old mice

Diastolic dysfunction is a prominent feature of cardiac aging in both mice and humans. We show here that 8-week treatment of old mice with the mitochondrial targeted peptide SS-31 (elamipretide) can substantially reverse this deficit. SS-31 normalized the increase in proton leak and reduced mitochondrial ROS in cardiomyocytes from old mice, accompanied by reduced protein oxidation and a shift towards a more reduced protein thiol redox state in old hearts. Improved diastolic function was concordant with increased phosphorylation of cMyBP-C Ser282 but was independent of titin isoform shift. Late-life viral expression of mitochondrial-targeted catalase (mCAT) produced similar functional benefits in old mice and SS-31 did not improve cardiac function of old mCAT mice, implicating normalizing mitochondrial oxidative stress as an overlapping mechanism. These results demonstrate that pre-existing cardiac aging phenotypes can be reversed by targeting mitochondrial dysfunction and implicate mitochondrial energetics and redox signaling as therapeutic targets for cardiac aging.

physiology↗

bioRxiv2019

Impairments in contractility and cytoskeletal organisation cause nuclear defects in nemaline myopathy

Nemaline myopathy (NM) is a genetically heterogeneous skeletal muscle disorder caused by mutations predominately affecting contractile filaments, in particular thin filament structure and/or regulation. The underlying cellular pathophysiology of this disease remains largely unclear. Here, we report novel pathological defects in skeletal muscle fibres of mice and patients with NM, including disrupted nuclear envelope, altered chromatin arrangement, and disorganisation of the cortical cytoskeleton. We demonstrate that such nuclear defects are caused by impairment of muscle fibre contractility, and that cytoskeletal organisation determines nuclear morphology. Our results overlap with findings in diseases caused by mutations in nuclear envelope or cytoskeletal proteins. Given the important role of nuclear shape and envelope in regulating gene expression, and the cytoskeleton in maintaining muscle fibre integrity, our findings are likely to underlie some of the hallmarks of NM, which include broad transcriptional alterations, arrested muscle fibre growth, contractile filament disarray and altered mechanical properties.

cell biology↗