Distinct myofibre domains of the human myotendinous junction revealed by single-nucleus RNA sequencing.

The myotendinous junction (MTJ) is a specialized domain of the multinucleated myofibre that is faced with the challenge of maintaining robust cell-matrix contact with the tendon under high mechanical stress and strain. Here, we profiled 24,124 nuclei in semitendinosus muscle-tendon samples from three healthy males by using single-nucleus RNA sequencing (snRNA-seq), alongside spatial transcriptomics, to gain insight into the genes characterizing this specialization in humans. We identified a cluster of MTJ myonuclei represented by 47 enriched transcripts, of which the presence of ABI3BP, ABLIM1, ADAMTSL1, BICD1, CPM, FHOD3, FRAS1 and FREM2 was confirmed at the MTJ at the protein level in immunofluorescence assays. Four distinct subclusters of MTJ myonuclei were apparent, comprising two COL22A1-expressing subclusters and two subclusters lacking COL22A1 expression but with differing fibre type profiles characterized by expression of either MYH7 or MYH1 and/or MYH2. Our findings reveal distinct myonuclei profiles of the human MTJ, which represents a weak link in the musculoskeletal system that is selectively affected in pathological conditions ranging from muscle strains to muscular dystrophies.

MYH13, a superfast myosin expressed in extraocular, laryngeal and syringeal muscles.

MYH13 is a unique type of sarcomeric myosin heavy chain (MYH) first detected in mammalian extraocular (EO) muscles and later also in vocal muscles, including laryngeal muscles of some mammals and syringeal muscles of songbirds. All these muscles are specialized in generating very fast contractions while producing relatively low force, a design appropriate for muscles acting against a much lower load than most skeletal muscles inserting into the skeleton. The definition of the physiological properties of muscle fibres containing MYH13 has been complicated by the mixed fibre type composition of EO muscles and the coexistence of different MYH types within the same fibre. A major advance in this area came from studies on isolated recombinant myosin motors and the demonstration that the affinity of actin-bound human MYH13 for ADP is much weaker than those of fast-type MYH1 (type 2X) and MYH2 (type 2A). This property is consistent with a very fast detachment of myosin from actin, a major determinant of shortening velocity. The MYH13 gene arose early during vertebrate evolution but was characterized only in mammals and birds and appears to have been lost in some teleost fish. The MYH13 gene is located at the 3' end of the mammalian fast/developmental gene cluster and in a similar position to the orthologous cluster in syntenic regions of the songbird genome. MYH13 gene regulation is controlled by a super-enhancer in the mammalian locus and deletion of the neighbouring fast MYH1 and MYH4 genes leads to abnormal MYH13 expression in mouse leg muscles.

Transcriptional programming of lipid and amino acid metabolism by the skeletal muscle circadian clock.

Circadian clocks are fundamental physiological regulators of energy homeostasis, but direct transcriptional targets of the muscle clock machinery are unknown. To understand how the muscle clock directs rhythmic metabolism, we determined genome-wide binding of the master clock regulators brain and muscle ARNT-like protein 1 (BMAL1) and REV-ERBα in murine muscles. Integrating occupancy with 24-hr gene expression and metabolomics after muscle-specific loss of BMAL1 and REV-ERBα, here we unravel novel molecular mechanisms connecting muscle clock function to daily cycles of lipid and protein metabolism. Validating BMAL1 and REV-ERBα targets using luciferase assays and in vivo rescue, we demonstrate how a major role of the muscle clock is to promote diurnal cycles of neutral lipid storage while coordinately inhibiting lipid and protein catabolism prior to awakening. This occurs by BMAL1-dependent activation of Dgat2 and REV-ERBα-dependent repression of major targets involved in lipid metabolism and protein turnover (MuRF-1, Atrogin-1). Accordingly, muscle-specific loss of BMAL1 is associated with metabolic inefficiency, impaired muscle triglyceride biosynthesis, and accumulation of bioactive lipids and amino acids. Taken together, our data provide a comprehensive overview of how genomic binding of BMAL1 and REV-ERBα is related to temporal changes in gene expression and metabolite fluctuations.

Fiber types in mammalian skeletal muscles.

Mammalian skeletal muscle comprises different fiber types, whose identity is first established during embryonic development by intrinsic myogenic control mechanisms and is later modulated by neural and hormonal factors. The relative proportion of the different fiber types varies strikingly between species, and in humans shows significant variability between individuals. Myosin heavy chain isoforms, whose complete inventory and expression pattern are now available, provide a useful marker for fiber types, both for the four major forms present in trunk and limb muscles and the minor forms present in head and neck muscles. However, muscle fiber diversity involves all functional muscle cell compartments, including membrane excitation, excitation-contraction coupling, contractile machinery, cytoskeleton scaffold, and energy supply systems. Variations within each compartment are limited by the need of matching fiber type properties between different compartments. Nerve activity is a major control mechanism of the fiber type profile, and multiple signaling pathways are implicated in activity-dependent changes of muscle fibers. The characterization of these pathways is raising increasing interest in clinical medicine, given the potentially beneficial effects of muscle fiber type switching in the prevention and treatment of metabolic diseases.

Knockout of human muscle genes revealed by large scale whole-exome studies.

Large scale whole-exome sequence studies have revealed that a number of individuals from different populations have predicted loss-of-function of different genes due to nonsense, frameshift, or canonical splice-site mutations. Surprisingly, many of these mutations do not apparently show the deleterious phenotypic consequences expected from gene knockout. These homozygous null mutations, when confirmed, can provide insight into human gene function and suggest novel approaches to correct gene dysfunction, as the lack of the expected disease phenotype may reflect the existence of modifier genes that reveal potential therapeutic targets. Human knockouts complement the information derived from mouse knockouts, which are not always good models of human disease. We have examined human knockout datasets searching for genes expressed exclusively or predominantly in striated muscle. A number of well-known muscle genes was found in one or more datasets, including genes coding for sarcomeric myosins, components of the sarcomeric cytoskeleton, sarcoplasmic reticulum and plasma membrane, and enzymes involved in muscle metabolism. The surprising absence of phenotype in some of these human knockouts is critically discussed, focusing on the comparison with the corresponding mouse knockouts.

Skeletal muscle mass is controlled by the MRF4-MEF2 axis.

PURPOSE OF REVIEW: The review is focused on the unexpected role of myogenic regulatory factor 4 (MRF4) in controlling muscle mass by repressing myocyte enhancer binding factor 2 (MEF2) activity in adult skeletal muscle, and on the emerging role of MEF2 in skeletal muscle growth. RECENT FINDINGS: The MRF4s of the MyoD family (MyoD, MYF5, MRF4, myogenin) and the MEF2 factors are known to play a major role in embryonic myogenesis. However, their function in adult muscle tissue is not known. A recent study shows that MRF4 loss in adult skeletal muscle causes muscle hypertrophy and prevents denervation atrophy. This effect is mediated by MEF2 factors that promote muscle growth, with MRF4 acting as a repressor of MEF2 activity. The role of MEF2 in skeletal muscle growth is supported by the finding that muscle regeneration is impaired by muscle-specific triple knockout of Mef2a, c, and d genes. SUMMARY: The finding that the MRF4-MEF2 axis controls muscle growth opens a new perspective for preventing muscle wasting. A unique feature of this pathway is that MRF4 is exclusively expressed in skeletal muscle, thus reducing the risk that interventions aimed at down-regulating MRF4 or interfering with the interaction between MRF4 and MEF2 may have off-target effects in other tissues.

Developing a toolkit for the assessment and monitoring of musculoskeletal ageing.

The complexities and heterogeneity of the ageing process have slowed the development of consensus on appropriate biomarkers of healthy ageing. The Medical Research Council-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) is a collaboration between researchers and clinicians at the Universities of Liverpool, Sheffield and Newcastle. One of CIMA's objectives is to 'Identify and share optimal techniques and approaches to monitor age-related changes in all musculoskeletal tissues, and to provide an integrated assessment of musculoskeletal function'-in other words to develop a toolkit for assessing musculoskeletal ageing. This toolkit is envisaged as an instrument that can be used to characterise and quantify musculoskeletal function during 'normal' ageing, lend itself to use in large-scale, internationally important cohorts, and provide a set of biomarker outcome measures for epidemiological and intervention studies designed to enhance healthy musculoskeletal ageing. Such potential biomarkers include: biochemical measurements in biofluids or tissue samples, in vivo measurements of body composition, imaging of structural and physical properties, and functional tests. This review assesses candidate biomarkers of musculoskeletal ageing under these four headings, details their biological bases, strengths and limitations, and makes practical recommendations for their use. In addition, we identify gaps in the evidence base and priorities for further research on biomarkers of musculoskeletal ageing.

Single muscle fiber proteomics reveals unexpected mitochondrial specialization.

Mammalian skeletal muscles are composed of multinucleated cells termed slow or fast fibers according to their contractile and metabolic properties. Here, we developed a high-sensitivity workflow to characterize the proteome of single fibers. Analysis of segments of the same fiber by traditional and unbiased proteomics methods yielded the same subtype assignment. We discovered novel subtype-specific features, most prominently mitochondrial specialization of fiber types in substrate utilization. The fiber type-resolved proteomes can be applied to a variety of physiological and pathological conditions and illustrate the utility of single cell type analysis for dissecting proteomic heterogeneity.

Spaceflight on the ISS changed the skeletal muscle proteome of two astronauts.

Skeletal muscle undergoes atrophy and loss of force during long space missions, when astronauts are persistently exposed to altered gravity and increased ionizing radiation. We previously carried out mass spectrometry-based proteomics from skeletal muscle biopsies of two astronauts, taken before and after a mission on the International Space Station. The experiments were part of an effort to find similarities between spaceflight and bed rest, a ground-based model of unloading, focused on proteins located at the costameres. We here extend the data analysis of the astronaut dataset and show compartment-resolved changes in the mitochondrial proteome, remodeling of the extracellular matrix and of the antioxidant response. The astronauts differed in their level of onboard physical exercise, which correlated with their respective preservation of muscle mass and force at landing in previous analyses. We show that the mitochondrial proteome downregulation during spaceflight, particularly the inner membrane and matrix, was dramatic for both astronauts. The expression of autophagy regulators and reactive oxygen species scavengers, however, showed partially opposite expression trends in the two subjects, possibly correlating with their level of onboard exercise. As mitochondria are primarily affected in many different tissues during spaceflight, we hypothesize that reactive oxygen species (ROS) rather than mechanical unloading per se could be the primary cause of skeletal muscle mitochondrial damage in space. Onboard physical exercise might have a strong direct effect on the prevention of muscle atrophy through mechanotransduction and a subsidiary effect on mitochondrial quality control, possibly through upregulation of autophagy and anti-oxidant responses.

FoxO3 coordinately activates protein degradation by the autophagic/lysosomal and proteasomal pathways in atrophying muscle cells.

Muscle atrophy occurs in many pathological states and results primarily from accelerated protein degradation and activation of the ubiquitin-proteasome pathway. However, the importance of lysosomes in muscle atrophy has received little attention. Activation of FoxO transcription factors is essential for the atrophy induced by denervation or fasting, and activated FoxO3 by itself causes marked atrophy of muscles and myotubes. Here, we report that FoxO3 does so by stimulating overall protein degradation and coordinately activating both lysosomal and proteasomal pathways. Surprisingly, in C2C12 myotubes, most of this increased proteolysis is mediated by lysosomes. Activated FoxO3 stimulates lysosomal proteolysis in muscle (and other cell types) by activating autophagy. FoxO3 also induces the expression of many autophagy-related genes, which are induced similarly in mouse muscles atrophying due to denervation or fasting. These studies indicate that decreased IGF-1-PI3K-Akt signaling activates autophagy not only through mTOR but also more slowly by a transcription-dependent mechanism involving FoxO3.

FoxO3 controls autophagy in skeletal muscle in vivo.

Autophagy allows cell survival during starvation through the bulk degradation of proteins and organelles by lysosomal enzymes. However, the mechanisms responsible for the induction and regulation of the autophagy program are poorly understood. Here we show that the FoxO3 transcription factor, which plays a critical role in muscle atrophy, is necessary and sufficient for the induction of autophagy in skeletal muscle in vivo. Akt/PKB activation blocks FoxO3 activation and autophagy, and this effect is not prevented by rapamycin. FoxO3 controls the transcription of autophagy-related genes, including LC3 and Bnip3, and Bnip3 appears to mediate the effect of FoxO3 on autophagy. This effect is not prevented by proteasome inhibitors. Thus, FoxO3 controls the two major systems of protein breakdown in skeletal muscle, the ubiquitin-proteasomal and autophagic/lysosomal pathways, independently. These findings point to FoxO3 and Bnip3 as potential therapeutic targets in muscle wasting disorders and other degenerative and neoplastic diseases in which autophagy is involved.

Translational suppression of atrophic regulators by microRNA-23a integrates resistance to skeletal muscle atrophy.

Muscle atrophy is caused by accelerated protein degradation and occurs in many pathological states. Two muscle-specific ubiquitin ligases, MAFbx/atrogin-1 and muscle RING-finger 1 (MuRF1), are prominently induced during muscle atrophy and mediate atrophy-associated protein degradation. Blocking the expression of these two ubiquitin ligases provides protection against muscle atrophy. Here we report that miR-23a suppresses the translation of both MAFbx/atrogin-1 and MuRF1 in a 3'-UTR-dependent manner. Ectopic expression of miR-23a is sufficient to protect muscles from atrophy in vitro and in vivo. Furthermore, miR-23a transgenic mice showed resistance against glucocorticoid-induced skeletal muscle atrophy. These data suggest that suppression of multiple regulators by a single miRNA can have significant consequences in adult tissues.

NFAT isoforms control activity-dependent muscle fiber type specification.

The intracellular signals that convert fast and slow motor neuron activity into muscle fiber type specific transcriptional programs have only been partially defined. The calcium/calmodulin-dependent phosphatase calcineurin (Cn) has been shown to mediate the transcriptional effects of motor neuron activity, but precisely how 4 distinct muscle fiber types are composed and maintained in response to activity is largely unknown. Here, we show that 4 nuclear factor of activated T cell (NFAT) family members act coordinately downstream of Cn in the specification of muscle fiber types. We analyzed the role of NFAT family members in vivo by transient transfection in skeletal muscle using a loss-of-function approach by RNAi. Our results show that, depending on the applied activity pattern, different combinations of NFAT family members translocate to the nucleus contributing to the transcription of fiber type specific genes. We provide evidence that the transcription of slow and fast myosin heavy chain (MyHC) genes uses different combinations of NFAT family members, ranging from MyHC-slow, which uses all 4 NFAT isoforms, to MyHC-2B, which only uses NFATc4. Our data contribute to the elucidation of the mechanisms whereby activity can modulate the phenotype and performance of skeletal muscle.

Long Covid: where we stand and challenges ahead.

Post-acute sequelae of SARS-CoV-2 (PASC), also known as Post-Covid Syndrome, and colloquially as Long Covid, has been defined as a constellation of signs and symptoms which persist for weeks or months after the initial SARS-CoV-2 infection. PASC affects a wide range of diverse organs and systems, with manifestations involving lungs, brain, the cardiovascular system and other organs such as kidney and the neuromuscular system. The pathogenesis of PASC is complex and multifactorial. Evidence suggests that seeding and persistence of SARS-CoV-2 in different organs, reactivation, and response to unrelated viruses such as EBV, autoimmunity, and uncontrolled inflammation are major drivers of PASC. The relative importance of pathogenetic pathways may differ in different tissue and organ contexts. Evidence suggests that vaccination, in addition to protecting against disease, reduces PASC after breakthrough infection although its actual impact remains to be defined. PASC represents a formidable challenge for health care systems and dissecting pathogenetic mechanisms may pave the way to targeted preventive and therapeutic approaches.

Drugs for the prevention and treatment of COVID-19 and its complications: An update on what we learned in the past 2 years.

The COVID-19 Committee of the Lincei Academy has reviewed the scientific evidence supporting the efficacy and safety of existing and new drugs/biologics for the preventing and treating of COVID-19 and its complications. This position paper reports what we have learned in the field in the past 2 years. The focus was on, but not limited to, drugs and neutralizing monoclonal antibodies, anti-SARS-CoV-2 agents, anti-inflammatory and immunomodulatory drugs, complement inhibitors and anticoagulant agents. We also discuss the risks/benefit of using cell therapies on COVID-19 patients. The report summarizes the available evidence, which supports recommendations from health authorities and panels of experts regarding some drugs and biologics, and highlights drugs that are not recommended, or drugs for which there is insufficient evidence to recommend for or against their use. We also address the issue of the safety of drugs used to treat underlying concomitant conditions in COVID-19 patients. The investigators did an enormous amount of work very quickly to understand better the nature and pathophysiology of COVID-19. This expedited the development and repurposing of safe and effective therapeutic interventions, saving an impressive number of lives in the community as well as in hospitals.

The proteomic profile of the human myotendinous junction.

Proteomics analysis of skeletal muscle has recently progressed from whole muscle tissue to single myofibers. Here, we further focus on a specific myofiber domain crucial for force transmission from muscle to tendon, the myotendinous junction (MTJ). To overcome the anatomical constraints preventing the isolation of pure MTJs, we performed in-depth analysis of the MTJ by progressive removal of the muscle component in semitendinosus muscle-tendon samples. Using detergents with increasing stringency, we quantified >3000 proteins across all samples, and identified 112 significantly enriched MTJ proteins, including 24 known MTJ-enriched proteins. Of the 88 novel MTJ markers, immunofluorescence analysis confirmed the presence of tetraspanin-24 (CD151), kindlin-2 (FERMT2), cartilage intermediate layer protein 1 (CILP), and integrin-alpha10 (ITGA10), at the human MTJ. Together, these human data constitute the first detailed MTJ proteomics resource that will contribute to advance understanding of the biology of the MTJ and its failure in pathological conditions.

Signatures of muscle disuse in spaceflight and bed rest revealed by single muscle fiber proteomics.

Astronauts experience dramatic loss of muscle mass, decreased strength, and insulin resistance, despite performing daily intense physical exercise that would lead to muscle growth on Earth. Partially mimicking spaceflight, prolonged bed rest causes muscle atrophy, loss of force, and glucose intolerance. To unravel the underlying mechanisms, we employed highly sensitive single fiber proteomics to detail the molecular remodeling caused by unloading and inactivity during bed rest and changes of the muscle proteome of astronauts before and after a mission on the International Space Station. Muscle focal adhesions, involved in fiber-matrix interaction and insulin receptor stabilization, are prominently downregulated in both bed rest and spaceflight and restored upon reloading. Pathways of antioxidant response increased strongly in slow but not in fast muscle fibers. Unloading alone upregulated markers of neuromuscular damage and the pathway controlling EIF5A hypusination. These proteomic signatures of mechanical unloading in muscle fiber subtypes contribute to disentangle the effect of microgravity from the pleiotropic challenges of spaceflight.

Molecular Mechanisms of Skeletal Muscle Hypertrophy.

Skeletal muscle hypertrophy can be induced by hormones and growth factors acting directly as positive regulators of muscle growth or indirectly by neutralizing negative regulators, and by mechanical signals mediating the effect of resistance exercise. Muscle growth during hypertrophy is controlled at the translational level, through the stimulation of protein synthesis, and at the transcriptional level, through the activation of ribosomal RNAs and muscle-specific genes. mTORC1 has a central role in the regulation of both protein synthesis and ribosomal biogenesis. Several transcription factors and co-activators, including MEF2, SRF, PGC-1α4, and YAP promote the growth of the myofibers. Satellite cell proliferation and fusion is involved in some but not all muscle hypertrophy models.