GLA-modified RNA treatment lowers GB3 levels in iPSC-derived cardiomyocytes from Fabry-affected individuals.

Recent studies in non-human model systems have shown therapeutic potential of nucleoside-modified messenger RNA (modRNA) treatments for lysosomal storage diseases. Here, we assessed the efficacy of a modRNA treatment to restore the expression of the galactosidase alpha (GLA), which codes for α-Galactosidase A (α-GAL) enzyme, in a human cardiac model generated from induced pluripotent stem cells (iPSCs) derived from two individuals with Fabry disease. Consistent with the clinical phenotype, cardiomyocytes from iPSCs derived from Fabry-affected individuals showed accumulation of the glycosphingolipid Globotriaosylceramide (GB3), which is an α-galactosidase substrate. Furthermore, the Fabry cardiomyocytes displayed significant upregulation of lysosomal-associated proteins. Upon GLA modRNA treatment, a subset of lysosomal proteins were partially restored to wild-type levels, implying the rescue of the molecular phenotype associated with the Fabry genotype. Importantly, a significant reduction of GB3 levels was observed in GLA modRNA-treated cardiomyocytes, demonstrating that α-GAL enzymatic activity was restored. Together, our results validate the utility of iPSC-derived cardiomyocytes from affected individuals as a model to study disease processes in Fabry disease and the therapeutic potential of GLA modRNA treatment to reduce GB3 accumulation in the heart.

Characterization of the skeletal muscle arginine methylome in health and disease reveals remodeling in amyotrophic lateral sclerosis.

Arginine methylation is a protein posttranslational modification important for the development of skeletal muscle mass and function. Despite this, our understanding of the regulation of arginine methylation under settings of health and disease remains largely undefined. Here, we investigated the regulation of arginine methylation in skeletal muscles in response to exercise and hypertrophic growth, and in diseases involving metabolic dysfunction and atrophy. We report a limited regulation of arginine methylation under physiological settings that promote muscle health, such as during growth and acute exercise, nor in disease models of insulin resistance. In contrast, we saw a significant remodeling of asymmetric dimethylation in models of atrophy characterized by the loss of innervation, including in muscle biopsies from patients with myotrophic lateral sclerosis (ALS). Mass spectrometry-based quantification of the proteome and asymmetric arginine dimethylome of skeletal muscle from individuals with ALS revealed the largest compendium of protein changes with the identification of 793 regulated proteins, and novel site-specific changes in asymmetric dimethyl arginine (aDMA) of key sarcomeric and cytoskeletal proteins. Finally, we show that in vivo overexpression of PRMT1 and aDMA resulted in increased fatigue resistance and functional recovery in mice. Our study provides evidence for asymmetric dimethylation as a regulator of muscle pathophysiology and presents a valuable proteomics resource and rationale for numerous methylated and nonmethylated proteins, including PRMT1, to be pursued for therapeutic development in ALS.

Treatment of type 2 diabetes with the designer cytokine IC7Fc.

The gp130 receptor cytokines IL-6 and CNTF improve metabolic homeostasis but have limited therapeutic use for the treatment of type 2 diabetes. Accordingly, we engineered the gp130 ligand IC7Fc, in which one gp130-binding site is removed from IL-6 and replaced with the LIF-receptor-binding site from CNTF, fused with the Fc domain of immunoglobulin G, creating a cytokine with CNTF-like, but IL-6-receptor-dependent, signalling. Here we show that IC7Fc improves glucose tolerance and hyperglycaemia and prevents weight gain and liver steatosis in mice. In addition, IC7Fc either increases, or prevents the loss of, skeletal muscle mass by activation of the transcriptional regulator YAP1. In human-cell-based assays, and in non-human primates, IC7Fc treatment results in no signs of inflammation or immunogenicity. Thus, IC7Fc is a realistic next-generation biological agent for the treatment of type 2 diabetes and muscle atrophy, disorders that are currently pandemic.

Integrated Glycoproteomics Identifies a Role of N-Glycosylation and Galectin-1 on Myogenesis and Muscle Development.

Many cell surface and secreted proteins are modified by the covalent addition of glycans that play an important role in the development of multicellular organisms. These glycan modifications enable communication between cells and the extracellular matrix via interactions with specific glycan-binding lectins and the regulation of receptor-mediated signaling. Aberrant protein glycosylation has been associated with the development of several muscular diseases, suggesting essential glycan- and lectin-mediated functions in myogenesis and muscle development, but our molecular understanding of the precise glycans, catalytic enzymes, and lectins involved remains only partially understood. Here, we quantified dynamic remodeling of the membrane-associated proteome during a time-course of myogenesis in cell culture. We observed wide-spread changes in the abundance of several important lectins and enzymes facilitating glycan biosynthesis. Glycomics-based quantification of released N-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown digalactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labeling and analysis of enriched glycopeptides with multiple fragmentation approaches identified glycoproteins modified by these regulated glycans including several integrins and growth factor receptors. Myogenesis was also associated with the regulation of several lectins, most notably the upregulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion of Lgals1 inhibited differentiation and myotube formation, suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes in N-glycosylation and the upregulation of galectin-1 during postnatal skeletal muscle development were observed in mice. Treatment of new-born mice with recombinant adeno-associated viruses to overexpress galectin-1 in the musculature resulted in enhanced muscle mass. Our data form a valuable resource to further understand the glycobiology of myogenesis and will aid the development of intervention strategies to promote healthy muscle development or regeneration.

Sex-Specific Control of Human Heart Maturation by the Progesterone Receptor.

BACKGROUND: Despite in-depth knowledge of the molecular mechanisms controlling embryonic heart development, little is known about the signals governing postnatal maturation of the human heart. METHODS: Single-nucleus RNA sequencing of 54 140 nuclei from 9 human donors was used to profile transcriptional changes in diverse cardiac cell types during maturation from fetal stages to adulthood. Bulk RNA sequencing and the Assay for Transposase-Accessible Chromatin using sequencing were used to further validate transcriptional changes and to profile alterations in the chromatin accessibility landscape in purified cardiomyocyte nuclei from 21 human donors. Functional validation studies of sex steroids implicated in cardiac maturation were performed in human pluripotent stem cell-derived cardiac organoids and mice. RESULTS: Our data identify the progesterone receptor as a key mediator of sex-dependent transcriptional programs during cardiomyocyte maturation. Functional validation studies in human cardiac organoids and mice demonstrate that the progesterone receptor drives sex-specific metabolic programs and maturation of cardiac contractile properties. CONCLUSIONS: These data provide a blueprint for understanding human heart maturation in both sexes and reveal an important role for the progesterone receptor in human heart development.

Intravascular Follistatin gene delivery improves glycemic control in a mouse model of type 2 diabetes.

Type 2 diabetes (T2D) manifests from inadequate glucose control due to insulin resistance, hypoinsulinemia, and deteriorating pancreatic ß-cell function. The pro-inflammatory factor Activin has been implicated as a positive correlate of severity in T2D patients, and as a negative regulator of glucose uptake by skeletal muscle, and of pancreatic ß-cell phenotype in mice. Accordingly, we sought to determine whether intervention with the Activin antagonist Follistatin can ameliorate the diabetic pathology. Here, we report that an intravenous Follistatin gene delivery intervention with tropism for striated muscle reduced the serum concentrations of Activin B and improved glycemic control in the db/db mouse model of T2D. Treatment reversed the hyperglycemic progression with a corresponding reduction in the percentage of glycated-hemoglobin to levels similar to lean, healthy mice. Follistatin gene delivery promoted insulinemia and abundance of insulin-positive pancreatic ß-cells, even when treatment was administered to mice with advanced diabetes, supporting a mechanism for improved glycemic control associated with maintenance of functional ß-cells. Our data demonstrate that single-dose intravascular Follistatin gene delivery can ameliorate the diabetic progression and improve prognostic markers of disease. These findings are consistent with other observations of Activin-mediated mechanisms exerting deleterious effects in models of obesity and diabetes, and suggest that interventions that attenuate Activin signaling could help further understanding of T2D and the development of novel T2D therapeutics.

The Hippo Signaling Pathway in the Regulation of Skeletal Muscle Mass and Function.

The Hippo signaling pathway regulates the activity of the proteins Yes-associated protein (Yap) and transcriptional co-activator with PDZ-binding motif (Taz) to control tissue growth in many different cell types. Previously, we demonstrated that Yap is a critical regulator of skeletal muscle mass. We hypothesize that alterations in Yap and Taz activity modulate the anabolic adaptations of skeletal muscle to resistance exercise.

Disparities in spatially variable gene calling highlight the need for benchmarking spatial transcriptomics methods.

Identifying spatially variable genes (SVGs) is a key step in the analysis of spatially resolved transcriptomics data. SVGs provide biological insights by defining transcriptomic differences within tissues, which was previously unachievable using RNA-sequencing technologies. However, the increasing number of published tools designed to define SVG sets currently lack benchmarking methods to accurately assess performance. This study compares results of 6 purpose-built packages for SVG identification across 9 public and 5 simulated datasets and highlights discrepancies between results. Additional tools for generation of simulated data and development of benchmarking methods are required to improve methods for identifying SVGs.

Liver-Secreted Hexosaminidase A Regulates Insulin-Like Growth Factor Signaling and Glucose Transport in Skeletal Muscle.

Nonalcoholic fatty liver disease (NAFLD) and impaired glycemic control are closely linked; however, the pathophysiological mechanisms underpinning this bidirectional relationship remain unresolved. The high secretory capacity of the liver and impairments in protein secretion in NAFLD suggest that endocrine changes in the liver are likely to contribute to glycemic defects. We identify hexosaminidase A (HEXA) as an NAFLD-induced hepatokine in both mice and humans. HEXA regulates sphingolipid metabolism, converting GM2 to GM3 gangliosides-sphingolipids that are primarily localized to cell-surface lipid rafts. Using recombinant murine HEXA protein, an enzymatically inactive HEXA(R178H) mutant, or adeno-associated virus vectors to induce hepatocyte-specific overexpression of HEXA, we show that HEXA improves blood glucose control by increasing skeletal muscle glucose uptake in mouse models of insulin resistance and type 2 diabetes, with these effects being dependent on HEXA's enzymatic action. Mechanistically, HEXA remodels muscle lipid raft ganglioside composition, thereby increasing IGF-1 signaling and GLUT4 localization to the cell surface. Disrupting lipid rafts reverses these HEXA-mediated effects. In this study, we identify a pathway for intertissue communication between liver and skeletal muscle in the regulation of systemic glycemic control.

Proteome-wide systems genetics identifies UFMylation as a regulator of skeletal muscle function.

Improving muscle function has great potential to improve the quality of life. To identify novel regulators of skeletal muscle metabolism and function, we performed a proteomic analysis of gastrocnemius muscle from 73 genetically distinct inbred mouse strains, and integrated the data with previously acquired genomics and >300 molecular/phenotypic traits via quantitative trait loci mapping and correlation network analysis. These data identified thousands of associations between protein abundance and phenotypes and can be accessed online (https://muscle.coffeeprot.com/) to identify regulators of muscle function. We used this resource to prioritize targets for a functional genomic screen in human bioengineered skeletal muscle. This identified several negative regulators of muscle function including UFC1, an E2 ligase for protein UFMylation. We show UFMylation is up-regulated in a mouse model of amyotrophic lateral sclerosis, a disease that involves muscle atrophy. Furthermore, in vivo knockdown of UFMylation increased contraction force, implicating its role as a negative regulator of skeletal muscle function.

Tissue-specific expression of Cas9 has no impact on whole-body metabolism in four transgenic mouse lines.

OBJECTIVE: CRISPR/Cas9 technology has revolutionized gene editing and fast tracked our capacity to manipulate genes of interest for the benefit of both research and therapeutic applications. Whilst many advances have, and continue to be made in this area, perhaps the most utilized technology to date has been the generation of knockout cells, tissues and animals. The advantages of this technology are many fold, however some questions still remain regarding the effects that long term expression of foreign proteins such as Cas9, have on mammalian cell function. Several studies have proposed that chronic overexpression of Cas9, with or without its accompanying guide RNAs, may have deleterious effects on cell function and health. This is of particular concern when applying this technology in vivo, where chronic expression of Cas9 in tissues of interest may promote disease-like phenotypes and thus confound the investigation of the effects of the gene of interest. Although these concerns remain valid, no study to our knowledge has yet to demonstrate this directly. METHODS: In this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models that express Cas9 in the heart, liver, skeletal muscle or adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. RESULTS: We demonstrate that Cas9 expression in these tissues had no detrimental effect on whole body health of the animals, nor did it induce any tissue-specific effects on whole body energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. CONCLUSIONS: Our data suggests that these models are suitable for studying the tissue specific effects of gene deletion using the LSL-Cas9-Tg model, and that phenotypes observed utilizing these models can be confidently interpreted as being gene specific, and not confounded by the chronic overexpression of Cas9.

Perturbed BMP signaling and denervation promote muscle wasting in cancer cachexia.

Most patients with advanced solid cancers exhibit features of cachexia, a debilitating syndrome characterized by progressive loss of skeletal muscle mass and strength. Because the underlying mechanisms of this multifactorial syndrome are incompletely defined, effective therapeutics have yet to be developed. Here, we show that diminished bone morphogenetic protein (BMP) signaling is observed early in the onset of skeletal muscle wasting associated with cancer cachexia in mouse models and in patients with cancer. Cancer-mediated factors including Activin A and IL-6 trigger the expression of the BMP inhibitor Noggin in muscle, which blocks the actions of BMPs on muscle fibers and motor nerves, subsequently causing disruption of the neuromuscular junction (NMJ), denervation, and muscle wasting. Increasing BMP signaling in the muscles of tumor-bearing mice by gene delivery or pharmacological means can prevent muscle wasting and preserve measures of NMJ function. The data identify perturbed BMP signaling and denervation of muscle fibers as important pathogenic mechanisms of muscle wasting associated with tumor growth. Collectively, these findings present interventions that promote BMP-mediated signaling as an attractive strategy to counteract the loss of functional musculature in patients with cancer.

Yap is a novel regulator of C2C12 myogenesis.

The expression, regulation and function of mammalian Hippo pathway members in skeletal muscle is largely unknown. The aim of this study was thus to test the hypothesis that core members of the mammalian Hippo pathway are expressed in skeletal muscle and that the transcriptional co-factor Yap, a core member of the Hippo pathway, regulates C2C12 myogenesis. We found that the major components of the mammalian Hippo pathway including Yap are all expressed in skeletal muscles, C2C12 myoblasts and myotubes. In C2C12 myoblasts, Yap Ser127 phosphorylation is low and Yap localises to nuclei. Upon differentiation, Yap Ser127 phosphorylation increases approximately 20-fold and Yap translocates from the nucleus to the cytosol. To test whether the observed increase of Yap Ser127 phosphorylation is required for differentiation we overexpressed hYAP1 S127A, a mutant that can not be phosphorylated at Ser127, in C2C12 myoblasts. We found that overexpression of hYAP S127A prevented myotube formation, whereas the overexpression of wildtype hYAP1 or empty vector had no effect. In addition, more hYAP1 S127A overexpressing cells progressed through the S phase of the cell cycle and the expression of MRFs (myogenin, Myf5), Mef2c and cell cycle regulators (p21, cyclin D1) was significantly changed when compared to wildtype hYAP1 and empty vector overexpressing cells. This data suggests that the phosphorylation of Yap at Ser127 leads to a changed expression of MRFs and cell cycle regulators and is required for C2C12 myoblasts to differentiate into myotubes.

SB431542 treatment promotes the hypertrophy of skeletal muscle fibers but decreases specific force.

The small molecule inhibitor SB431542 inhibits activin type I receptors. The muscle growth-inhibitor myostatin binds to and signals via these receptors. The aim of this study was to test the hypothesis that SB431542 can inhibit myostatin-related Smad signaling and induce muscle growth in cultured C2C12 myotubes and increase growth and specific force in cultured Xenopus muscle fibers. The effect of SB431542 was assessed in vitro on C2C12 myotubes and ex vivo using mature Xenopus muscle fibers. SB431542 treatment reduced myostatin-induced C-terminal Smad2 phosphorylation and resulted in the formation of enlarged myotubes. However myogenin expression was unchanged, while p70 S6k phosphorylation at Thr389, total myosin heavy chain, and the rate of protein synthesis were all reduced. Mature Xenopus muscle fibers that were treated with SB431542 had a higher fiber cross-sectional area but decreased specific force production than control. SB431542 can initially antagonize myostatin signaling, but long-term unexpected signaling effects occur. Muscle fibers hypertrophy, but their specific force decreases compared to control.

Extracellular Vesicles Provide a Means for Tissue Crosstalk during Exercise.

Exercise stimulates the release of molecules into the circulation, supporting the concept that inter-tissue signaling proteins are important mediators of adaptations to exercise. Recognizing that many circulating proteins are packaged in extracellular vesicles (EVs), we employed quantitative proteomic techniques to characterize the exercise-induced secretion of EV-contained proteins. Following a 1-hr bout of cycling exercise in healthy humans, we observed an increase in the circulation of over 300 proteins, with a notable enrichment of several classes of proteins that compose exosomes and small vesicles. Pulse-chase and intravital imaging experiments suggested EVs liberated by exercise have a propensity to localize in the liver and can transfer their protein cargo. Moreover, by employing arteriovenous balance studies across the contracting human limb, we identified several novel candidate myokines, released into circulation independently of classical secretion. These data identify a new paradigm by which tissue crosstalk during exercise can exert systemic biological effects.

Regulation of Tissue Growth by the Mammalian Hippo Signaling Pathway.

The integrative control of diverse biological processes such as proliferation, differentiation, apoptosis and metabolism is essential to maintain cellular and tissue homeostasis. Disruption of these underlie the development of many disease states including cancer and diabetes, as well as many of the complications that arise as a consequence of aging. These biological outputs are governed by many cellular signaling networks that function independently, and in concert, to convert changes in hormonal, mechanical and metabolic stimuli into alterations in gene expression. First identified in Drosophila melanogaster as a powerful mediator of cell division and apoptosis, the Hippo signaling pathway is a highly conserved regulator of mammalian organ size and functional capacity in both healthy and diseased tissues. Recent studies have implicated the pathway as an effector of diverse physiological cues demonstrating an essential role for the Hippo pathway as an integrative component of cellular homeostasis. In this review, we will: (a) outline the critical signaling elements that constitute the mammalian Hippo pathway, and how they function to regulate Hippo pathway-dependent gene expression and tissue growth, (b) discuss evidence that shows this pathway functions as an effector of diverse physiological stimuli and

Integrated expression analysis of muscle hypertrophy identifies Asb2 as a negative regulator of muscle mass.

The transforming growth factor-ß (TGF-ß) signaling network is a critical regulator of skeletal muscle mass and function and, thus, is an attractive therapeutic target for combating muscle disease, but the underlying mechanisms of action remain undetermined. We report that follistatin-based interventions (which modulate TGF-ß network activity) can promote muscle hypertrophy that ameliorates aging-associated muscle wasting. However, the muscles of old sarcopenic mice demonstrate reduced response to follistatin compared with healthy young-adult musculature. Quantitative proteomic and transcriptomic analyses of young-adult muscles identified a transcription/translation signature elicited by follistatin exposure, which included repression of ankyrin repeat and SOCS box protein 2 (Asb2). Increasing expression of ASB2 reduced muscle mass, thereby demonstrating that Asb2 is a TGF-ß network-responsive negative regulator of muscle mass. In contrast to young-adult muscles, sarcopenic muscles do not exhibit reduced ASB2 abundance with follistatin exposure. Moreover, preventing repression of ASB2 in young-adult muscles diminished follistatin-induced muscle hypertrophy. These findings provide insight into the program of transcription and translation events governing follistatin-mediated adaptation of skeletal muscle attributes and identify Asb2 as a regulator of muscle mass implicated in the potential mechanistic dysfunction between follistatin-mediated muscle growth in young and old muscles.

The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass.

Although the canonical transforming growth factor ß signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin-dependent process, whereas increased BMP-Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders.