Biology subjects

Vilchez, J. J.

Publications and source records attributed to Vilchez, J. J..

3 recordsLinked to original sources

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

Deletion of exons 45 to 55 in the DMD gene: from the therapeutic perspective to the in vitro model

Gene editing therapies in development for correcting out-of-frame DMD mutations in Duchenne muscular dystrophy aim to replicate benign spontaneous deletions. Deletion of 45-55 DMD exons (del45-55) was described in asymptomatic subjects, but recently serious skeletal and cardiac complications have been reported. Uncovering why a single mutation like del45-55 is able to induce diverse phenotypes and grades of severity may impact the strategies of emerging therapies. Cellular models are essential for this purpose, but their availability is compromised by scarce muscle biopsies. Here, we have introduced through CRISPR-Cas9 edition, a del45-55 mimicking the intronic breakpoints harboured by a subset of patients of this form of dystrophinopathy, into a Duchenne patients cell line. Dystrophin expression was restored in edited myoblasts and the myogenic defects were ameliorated. Besides confirming the potential of CRISPR-Cas9 to create tailored mutations as a useful approach to generate in vitro models, we also generated an immortalized myoblast line derived from a patient with a specific del45-55. Overall, we provide helpful resources to deepen into unknown factors responsible for DMD-pathophysiology. SUMMARY STATEMENTWe restored dystrophin expression in a DMD culture by replicating the exact deletion in exons 45-55 harboured by mild patients, testing this therapeutic approach, and creating a new cell model.

cell biology↗

bioRxiv2023

Human light meromyosin mutations linked to skeletal myopathies disrupt the coiled coil structure and myosin head sequestration

Myosin heavy chains encoded by MYH7 and MYH2 are among the most abundant proteins in human skeletal muscle. After decades of intense research using a wide range of biophysical and biological approaches, their functions have begun to be elucidated. Despite this, it remains unclear how mutations in these genes and resultant proteins disrupt myosin structure and function, inducing pathological states and skeletal myopathies termed myosinopathies. Here, we have analysed the effects of several common MYH7 and MYH2 mutations located in light meromyosin (LMM) using a broad range of approaches. We determined the secondary structure and filament forming capabilities of expressed and purified LMM constructs in vitro, performed in-silico modelling of LMM constructs, and evaluated the incorporation of eGFP-myosin heavy chain constructs into sarcomeres in cultured myotubes. Using muscle biopsies from patients, we applied Mant-ATP chase protocols to estimate the proportion of myosin heads that were super-relaxed, X-ray diffraction measurements to estimate myosin head order and myofibre mechanics to investigate contractile function. We found that human MYH7 and MYH2 LMM mutations commonly disrupt myosin coiled-coil structure and packing of filaments in vitro; decrease the myosin super-relaxed state in vivo and increase the basal myosin ATP consumption; but are not associated with myofibre contractile deficits. Altogether, these findings indicate that the structural remodelling resulting from LMM mutations induces a pathogenic state in which formation of shutdown heads is impaired, thus increasing myosin head ATP demand in the filaments, rather than affecting contractility. These key findings will help in the design of future therapies for myosinopathies.

physiology↗