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Non-alcoholic fatty liver disease (NAFLD) and impaired glycaemic 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 glycaemic defects. We identify hexosaminidase A (HEXA) as a 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 viral 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 insulin-like growth factor 1 signalling and glucose transporter 4 localization to the cell surface. Disrupting lipid rafts reverses these HEXA-mediated effects. Together, this study identifies a novel pathway for inter-tissue communication between liver and skeletal muscle in the regulation of systemic glycaemic control.

High blood cholesterol affects ~30% of adult Canadians and doubles the risk of cardiac events. Statins are widely used for lowering cholesterol levels, but statins have pleiotropic effects that can be mediated by lowering protein prenylation. The most prevalent side effect of statin therapy is myopathy. Overt myotoxicity and rhabdomyolysis are rare (S127A) in C2C12 mitigated statin-mediated increases in atrogin-1 and lower myotube diameter compared to vector/vehicle controls in LPS primed myotubes. Statins also promoted the nuclear accumulation of FOXO1 and decreased levels of phosphorylated FOXO1/3a. Restoring protein prenylation and blocking the NLRP3 inflammasome prevented statin-induced nuclear accumulation of FOXO1 and mitigated decreased levels of p-FOXO1/3a. These results showed that prenylation and activation of NLRP3 inflammasome are both upstream of the FOXO activation. Our results demonstrated that activation of YAP in the presence of statins mitigated measures of statin-induced myopathy and that NLRP3 activation is upstream of FOXO activation, which is a known regulator of Atrogin-1. Further investigation to characterize the mechanism of statin-induced myopathy can lead to an enhanced understanding of statin-induced myopathy and the establishment of next-generation statin therapies without muscular side effects. Natural Sciences and Engineering Research Council (NSERC) of Canada This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

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Cachexia is a life-threatening wasting syndrome lacking effective treatment, which arises in many cancer patients. Although ostensibly induced by multiple tumor-produced cytokines (tumorkines), their functional contribution to initiation and progression of this syndrome has proven difficult to determine. In this study, we used adeno-associated viral vectors to elevate circulating levels of the tumorkines IL6 and/or activin A in animals in the absence of tumors as a tactic to evaluate hypothesized roles in cachexia development. Mice with elevated levels of IL6 exhibited 8.1% weight loss after 9 weeks, whereas mice with elevated levels of activin A lost 11% of their body weight. Co-elevation of both tumorkines to levels approximating those observed in cancer cachexia models induced a more rapid and profound body weight loss of 15.4%. Analysis of body composition revealed that activin A primarily triggered loss of lean mass, whereas IL6 was a major mediator of fat loss. Histologic and transcriptional analysis of affected organs/tissues (skeletal muscle, fat, and liver) identified interactions between the activin A and IL6 signaling pathways. For example, IL6 exacerbated the detrimental effects of activin A in skeletal muscle, whereas activin A curbed the IL6-induced acute-phase response in liver. This study presents a useful model to deconstruct cachexia, opening a pathway to determining which tumorkines are best targeted to slow/reverse this devastating condition in cancer patients. Cancer Res; 76(18); 5372–82. ©2016 AACR.

Titrating rAAV6:IL-6 doses in wild type mice to elevate circulating levels of IL-6. High doses of rAAV6:IL-6 result in cachexia.

TA muscles of mice injected with rAAV6:TNF-α and rAAV6:Tweak exhibited muscle loss and increases in the phosphorylation of p65, but did not induce cachexia.

Comparing the rate of lean and fat mass loss in mice bearing colon-26 tumors and mice injected with rAAV6:activin A and/or rAAV6:IL-6.

In mice injected with AAV:activin A and/or AAV:IL6, the liver was examined for expression of extracellular matrix genes and collagen deposition.

In mice injected with rAAV6:activin A and/or rAAV6:IL-6, the mRNA expression of E3 ubiquitin ligases was determined in quadriceps muscles. In addition, skeletal muscle, epididymal WAT and BAT depots were probed for IL-6 target genes or genes involved in lipolysis and browning.

Mice bearing colon-26 tumors rapidly lose lean and fat mass, together with significant wasting of muscle groups. These losses correlate with increases in the phosphorylation of the transcription factors, Stat3 and Smad3, which are activated by IL-6 and activin A, respectively. Smad3 target genes are also altered in cachectic mice.

mRNA expression of activin A and Il-6 in the TA muscle, heart, liver and spleen of mice bearing colon-26 tumors.

Lipotoxicity, the accumulation of lipids in non-adipose tissues, alters the metabolic transcriptome and mitochondrial metabolism in skeletal muscle. The mechanisms involved remain poorly understood. Here we show that lipotoxicity increased histone deacetylase 4 (HDAC4) and histone deacetylase 5 (HDAC5), which reduced the expression of metabolic genes and oxidative metabolism in skeletal muscle. This metabolic reprogramming was linked with reduced expression of p53 pathway genes that mediate apoptosis and ferroptosis, as well as preserved muscle cell viability in response to lipotoxicity. Mechanistically, increased HDAC4 and 5 decreased acetylation of p53 at K120, a modification required for transcriptional activation of apoptosis, while metabolically sensitive redox drivers of ferroptosis were also reduced. Overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obesedb/dbmice enhanced oxidative capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of the oxidative state of skeletal muscle, which is linked to inhibition of cell death pathways and preservation of muscle integrity in response to lipotoxicity.

Type 2 diabetes (T2D) manifests from inadequate glucose control due to insulin resistance, hypoinsulinemia, and deteriorating pancreatic β-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 β-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 β-cells, even when treatment was administered to mice with advanced diabetes, supporting a mechanism for improved glycemic control associated with maintenance of functional β-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.

BackgroundSkeletal muscle wasting is often associated with insulin resistance. A major regulator of muscle mass is the transforming growth factor β (TGF-β) superfamily, including activin A, which causes atrophy. TGF-β superfamily ligands also negatively regulate insulin-sensitive proteins, but whether this pathway contributes to insulin action remains to be determined.MethodsTo elucidate if TGF-β superfamily ligands regulate insulin action we used an adeno-associated virus gene editing approach to overexpress the activin A inhibitor, follistatin (Fst288) in mouse muscle of lean and diet-induced obese mice. We determined basal and insulin-stimulated 2 deoxy-glucose uptake using isotopic tracers in vivo. Furthermore, to evaluate whether circulating Fst and activin A concentrations are associated with obesity, insulin resistance, and weight loss in humans we analysed serum from morbidly obese subjects before, 1 week, and 1 year after Roux-en-Y gastric bypass (RYGB).ResultsFst288 muscle overexpression markedly increased in vivo insulin-stimulated (but not basal) glucose uptake (+75%, pConclusionsWe here present evidence that Fst is a potent regulator of insulin action in muscle and in addition to AKT and p70S6K, we identify TBC1D1, TBC1D4 and PAK1 as Fst targets. A possible role for Fst in regulating glycemic control is suggested because circulating Fst more than doubled post RYGB surgery, a treatment that markedly improved insulin sensitivity. These findings demonstrate the therapeutic potential of inhibiting TGF-β superfamily ligands to improve insulin action and Fst’s relevance to muscle wasting associated insulin resistant conditions in mice and humans.

Recombinant adeno-associated viruses (rAAV) have proven to be a safe and successful vector for transferring genetic material to treat various health conditions in both the laboratory and the clinic. However, pre-existing neutralizing antibodies (NAbs) against AAV capsids pose an ongoing challenge for the successful administration of gene therapies in both large animal experimental models and human populations. Preliminary screening for host immunity against AAV is necessary to ensure the efficacy of AAV-based gene therapies as both a research tool and as a clinically viable therapeutic agent. This protocol describes a colorimetric in vitro assay to detect neutralizing factors against AAV serotype 6 (AAV6). The assay utilizes the reaction between an AAV encoding an alkaline phosphatase (AP) reporter gene and its substrate NBT/BCIP, which generates an insoluble quantifiable purple stain upon combination. In this protocol, serum samples are combined with an AAV expressing AP and incubated to permit potential neutralizing activity to occur. Virus serum mixture is subsequently added to cells to allow for viral transduction of any AAVs that have not been neutralized. The NBT/BCIP substrate is added and undergoes a chromogenic reaction, corresponding to viral transduction and neutralizing activity. The proportion of area colored is quantitated using a free software tool to generate neutralizing titers. This assay displays a strong positive correlation between coloration and viral concentration. Assessment of serum samples from sheep before and after administration of a recombinant AAV6 led to a dramatic increase in neutralizing activity (125 to10,000-fold increase). The assay displayed adequate sensitivity to detect neutralizing activity in1:32,000 serum dilutions. This assay provides a simple, rapid, and cost-effective method to detect NAbs against AAVs.

MuRF1 (TRIM63) is a muscle-specific E3 ubiquitin ligase and component of the ubiquitin proteasome system. MuRF1 is transcriptionally upregulated under conditions that cause muscle loss, in both rodents and humans, and is a recognized marker of muscle atrophy. In this study, we used in vivo electroporation to determine whether MuRF1 overexpression alone can cause muscle atrophy and, in combination with ubiquitin proteomics, identify the endogenous MuRF1 substrates in skeletal muscle. Overexpression of MuRF1 in adult mice increases ubiquitination of myofibrillar and sarcoplasmic proteins, increases expression of genes associated with neuromuscular junction instability, and causes muscle atrophy. A total of 169 ubiquitination sites on 56 proteins were found to be regulated by MuRF1. MuRF1-mediated ubiquitination targeted both thick and thin filament contractile proteins, as well as, glycolytic enzymes, deubiquitinases, p62, and VCP. These data reveal a potential role for MuRF1 in not only the breakdown of the sarcomere but also the regulation of metabolism and other proteolytic pathways in skeletal muscle.

Exercise induces signaling networks to improve muscle function and confer health benefits. To identify divergent and common signaling networks during and after different exercise modalities, we performed a phosphoproteomic analysis of human skeletal muscle from a cross-over intervention of endurance, sprint, and resistance exercise. This identified 5,486 phosphosites regulated during or after at least one type of exercise modality and only 420 core phosphosites common to all exercise. One of these core phosphosites was S67 on the uncharacterized protein C18ORF25, which we validated as an AMPK substrate. Mice lacking C18ORF25 have reduced skeletal muscle fiber size, exercise capacity, and muscle contractile function, and this was associated with reduced phosphorylation of contractile and Casup2+/suphandling proteins. Expression of C18ORF25 S66/67D phospho-mimetic reversed the decreased muscle force production. This work defines the divergent and canonical exercise phosphoproteome across different modalities and identifies C18ORF25 as a regulator of exercise signaling and muscle function.

Homozygosity for the common ACTN3 null polymorphism (ACTN3 577X) results in α-actinin-3 deficiency in ~20% of humans worldwide and is linked to reduced sprint and power performance in both elite athletes and the general population. α-Actinin-3 deficiency is also associated with reduced muscle mass and strength, increased risk of sarcopenia in the elderly, and altered response to muscle wasting induced by denervation and immobilisation. ACTN3 genotype is also a disease modifier for Duchenne muscular dystrophy (DMD), with α-actinin-3 deficiency associated with slower disease progression. Here we show that α-actinin-3 plays a key role in the regulation of protein synthesis and breakdown signalling in skeletal muscle, and its influence on muscle mass begins during early postnatal muscle development. Actn3 genotype also influences the skeletal muscle response to the glucocorticoid dexamethasone. Following acute dexamethasone exposure, transcriptomic analyses by RT-qPCR and RNA-sequencing show reduced atrophy signalling (Mstn, Tmem100, mRas, Fbxo32, Trim63) and anti-inflammatory response in α-actinin-3 deficient mice compared to wild-type. α-Actinin-3 deficiency also protects against muscle wasting following prolonged daily treatment with dexamethasone in female, but not male mice. In combination, these data suggest that ACTN3 R577X is a pharmacogenetic variant influencing the anti-inflammatory and muscle wasting response to glucocorticoid therapy.

Mitochondrial dynamics refers to the constant remodeling of mitochondrial populations by multiple cellular pathways that help maintain mitochondrial health and function. Disruptions in mitochondrial dynamics often lead to mitochondrial dysfunction, which is frequently associated with disease in rodents and humans. Consistent with this, obesity is associated with reduced mitochondrial function in white adipose tissue, partly via alterations in mitochondrial dynamics. Several proteins, including the E3 ubiquitin ligase membrane-associated RING-CH-type finger 5 (MARCH5), are known to regulate mitochondrial dynamics; however, the role of these proteins in adipocytes has been poorly studied. Here, we show that MARCH5 is regulated by peroxisome proliferator-activated receptor-γ (PPARγ) during adipogenesis and is correlated with fat mass across a panel of genetically diverse mouse strains, in ob/ob mice, and in humans. Furthermore, manipulation of MARCH5 expression in vitro and in vivo alters mitochondrial function, affects cellular metabolism, and leads to differential regulation of several metabolic genes. Thus our data demonstrate an association between mitochondrial dynamics and metabolism that defines MARCH5 as a critical link between these interconnected pathways.

Obesity is a major risk factor underlying the development of metabolic disease and a growing public health concern globally. Strategies to promote skeletal muscle metabolism can be effective to limit the progression of metabolic disease. Here, we demonstrate that the levels of the Hippo pathway transcriptional co-activator YAP are decreased in muscle biopsies from obese, insulin-resistant humans and mice. Targeted disruption of Yap in adult skeletal muscle resulted in incomplete oxidation of fatty acids and lipotoxicity. Integrated ‘omics analysis from isolated adult muscle nuclei revealed that Yap regulates a transcriptional profile associated with metabolic substrate utilisation. In line with these findings, increasing Yap abundance in the striated muscle of obese (db/db) mice enhanced energy expenditure and attenuated adiposity. Our results demonstrate a vital role for Yap as a mediator of skeletal muscle metabolism. Strategies to enhance Yap activity in skeletal muscle warrant consideration as part of comprehensive approaches to treat metabolic disease., The mechanisms driving metabolic dysfunction in obesity remain incompletely understood. Here, the authors show that the levels of Hippo pathway effector YAP are reduced in muscle from individuals with insulin resistance and obese-diabetic mice, and that YAP promotes skeletal muscle lipid metabolism and limits adiposity in obese mice.

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., Highlights • Detailed characterization of 4 tissue specific Cas9 TG mice in relevant metabolic tissues. • Demonstration that these models express robust Cas9 in a tissue specific manner. • Detailed phenotyping demonstrates that chronic Cas9 expression has no impact on tissue weight, body composition or body weight. • Metabolic phenotyping demonstrates that Cas9 expression does not impact whole body glucose tolerance, or heart function. • Tissue specific characterization confirms that there is no discernible effect on tissue health or function.

Polyamines have been shown to be absolutely required for protein synthesis and cell growth. The serine/threonine kinase, the mechanistic target of rapamycin complex 1 (mTORC1), also plays a fundamental role in the regulation of protein turnover and cell size, including in skeletal muscle, where mTORC1 is sufficient to increase protein synthesis and muscle fiber size, and is necessary for mechanical overload-induced muscle hypertrophy. Recent evidence suggests that mTORC1 may regulate the polyamine metabolic pathway, however, there is currently no evidence in skeletal muscle. This study examined changes in polyamine pathway proteins during muscle hypertrophy induced by mechanical overload (7 days), with and without the mTORC1 inhibitor, rapamycin, and during muscle atrophy induced by food deprivation (48 h) and denervation (7 days) in mice. Mechanical overload induced an increase in mTORC1 signaling, protein synthesis and muscle mass, and these were associated with rapamycin-sensitive increases in adenosylmethione decarboxylase 1 (Amd1), spermidine synthase (SpdSyn), and c-Myc. Food deprivation decreased mTORC1 signaling, protein synthesis, and muscle mass, accompanied by a decrease in spermidine/spermine acetyltransferase 1 (Sat1). Denervation, resulted increased mTORC1 signaling and protein synthesis, and decreased muscle mass, which was associated with an increase in SpdSyn, spermine synthase (SpmSyn), and c-Myc. Combined, these data show that polyamine pathway enzymes are differentially regulated in models of altered mechanical and metabolic stress, and that Amd1 and SpdSyn are, in part, regulated in a mTORC1-dependent manner. Furthermore, these data suggest that polyamines may play a role in the adaptive response to stressors in skeletal muscle.

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 by taking advantage of Cas9 function to promote indels in precise locations in the genome. Whilst the advantages of this technology are many fold, 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. Thus, in this study we used the lox-stop-lox (LSL) spCas9 ROSA26 transgenic (Tg) mouse line to generate four tissue-specific Cas9-Tg models with expression in the heart, liver, skeletal muscle and adipose tissue. We performed comprehensive phenotyping of these mice up to 20-weeks of age and subsequently performed molecular analysis of their organs. We demonstrated 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 energy metabolism, liver health, inflammation, fibrosis, heart function or muscle mass. Thus, 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.

The development of the musculoskeletal system and its maintenance depends on the reciprocal relationship between muscle and bone. The size of skeletal muscles and the forces generated during muscle contraction are potent sources of mechanical stress on the developing skeleton, and they shape bone structure during growth. This is particularly evident in hypermuscular global myostatin (Mstn)‐null mice, where larger muscles during development increase bone mass and alter bone shape. However, whether muscle hypertrophy can similarly influence the shape of bones after the embryonic and prepubertal period is unknown. To address this issue, bone structure was assessed after inducing muscle hypertrophy in the lower hindlimbs of young‐adult C57BL/6J male mice by administering intramuscular injections of recombinant adeno‐associated viral vectors expressing follistatin (FST), a potent antagonist of Mstn. Two FST isoforms were used: the full‐length 315 amino acid isoform (FST‐315) and a truncated 288 amino acid isoform (FST‐288). In both FST‐treated cohorts, muscle hypertrophy was observed, and the anterior crest of the tibia, adjacent to the tibialis anterior muscle, was lengthened. Hypertrophy of the muscles surrounding the tibia caused the adjacent cortical shell to recede inward toward the central axis: an event driven by bone resorption adjacent to the hypertrophic muscle. The findings reveal that inducing muscle hypertrophy in mice can confer changes in bone shape in early adulthood. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

BackgroundLong ncRNAs (lncRNAs) are known to influence numerous biological processes including cellular differentiation and tissue development. They are also implicated in the maintenance, health and physiological function of many tissues including the heart. Indeed, manipulating the expression of specific lncRNAs has been shown to improve pathological cardiac phenotypes such as heart failure. One lncRNA studied in various settings is OIP5-AS1 (also known as 1700020I14Rik and Cyrano), however its role in cardiac pathologies remains mostly uncharacterised.MethodsWe used data generated from FACS sorted murine cardiomyocytes, human iPSC derived cardiomyocytes, as well as heart tissue from various animal models to investigate OIP5-AS1 expression in health and disease. Using CRISPR we engineered a global OIP5-AS1 knock out (KO) mouse model and performed cardiac pressure overload experiments to study heart failure in these animals. RNA-sequencing of left ventricles provided mechanistic insight between WT and KO mice.ResultsWe demonstrate that OIP5-AS1 expression is regulated during cardiac development and cardiac specific pathologies in both rodent and human models. Moreover, we demonstrate that global female OIP5-AS1 KO mice develop exacerbated heart failure, but male mice do not. Transcriptomics and gene set enrichment analysis suggests that OIP5-AS1 may regulate pathways that impact mitochondrial function.ConclusionsOIP5-AS1 is regulated in cardiac tissue and its deletion leads to worsening heart function under pressure overload in female mice. This may be due to impairments in mitochondrial function, highlighting OIP5-AS1 as a gene of interest in sex-specific differences in heart failure.

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.

Ubiquitination is a posttranslational protein modification that has been shown to have a range of effects, including regulation of protein function, interaction, localization, and degradation. We have previously shown that the muscle-specific ubiquitin E3 ligase, ASB2β, is downregulated in models of muscle growth and that overexpression ASB2β is sufficient to induce muscle atrophy. To gain insight into the effects of increased ASB2β expression on skeletal muscle mass and function, we used liquid chromatography coupled to tandem mass spectrometry to investigate ASB2β-mediated changes to the skeletal muscle proteome and ubiquitinome, via a parallel analysis of remnant diGly-modified peptides. The results show that viral vector-mediated ASB2β overexpression in murine muscles causes progressive muscle atrophy and impairment of force-producing capacity, while ASB2β knockdown induces mild muscle hypertrophy. ASB2β-induced muscle atrophy and dysfunction were associated with the early downregulation of mitochondrial and contractile protein abundance and the upregulation of proteins involved in proteasome-mediated protein degradation (including other E3 ligases), protein synthesis, and the cytoskeleton/sarcomere. The overexpression ASB2β also resulted in marked changes in protein ubiquitination; however, there was no simple relationship between changes in ubiquitination status and protein abundance. To investigate proteins that interact with ASB2β and, therefore, potential ASB2β targets, Flag-tagged wild-type ASB2β, and a mutant ASB2β lacking the C-terminal SOCS box domain (dSOCS) were immunoprecipitated from C2C12 myotubes and subjected to label-free proteomic analysis to determine the ASB2β interactome. ASB2β was found to interact with a range of cytoskeletal and nuclear proteins. When combined with the in vivo ubiquitinomic data, our studies have identified novel putative ASB2β target substrates that warrant further investigation. These findings provide novel insight into the complexity of proteome and ubiquitinome changes that occur during E3 ligase-mediated skeletal muscle atrophy and dysfunction., Graphical abstract, Highlights • Proteomic and ubiquitinomic analysis of increased ASB2β in skeletal muscle • ASB2β increased the abundance of proteome and cytoskeletal proteins • ASB2β reduced the abundance of mitochondrial and contractile proteins • No simple relationship between changes in protein abundance and ubiquitination, In Brief The E3 ubiquitin ligase ASB2β has been identified as a regulator of skeletal muscle mass. To gain insights into potential mechanisms of action, mouse muscles expressing a Flag-tagged ASB2β were investigated using quantitative proteomic methods. The results identified ASB2β-induced changes in the abundance and ubiquitination of proteins associated with mitochondria, the sarcomere, and the cytoskeleton. Additional in vitro studies identified novel putative ASB2β target substrates. The results highlight the complex relationship between protein abundance and ubiquitination in ASB2β-mediated muscle adaptation.

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 remain 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 releasedN-linked glycans confirmed remodeling of the glycome consistent with the regulation of glycosyltransferases and glycosidases responsible for their formation including a previously unknown di-galactose-to-sialic acid switch supporting a functional role of these glycoepitopes in myogenesis. Furthermore, dynamic quantitative glycoproteomic analysis with multiplexed stable isotope labelling 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 up-regulation of galectin-1 (LGALS1). CRISPR/Cas9-mediated deletion ofLgals1inhibited differentiation and myotube formation suggesting an early functional role of galectin-1 in the myogenic program. Importantly, similar changes inN-glycosylation and the up-regulation 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.

Aims: The glucose-driven enzymatic modification of myocardial proteins by the sugar moiety, β-N-acetylglucosamine (O-GlcNAc), is increased in pre-clinical models of diabetes, implicating protein O-GlcNAc modification in diabetes-induced heart failure. Our aim was to specifically examine cardiac manipulation of the two regulatory enzymes of this process on the cardiac phenotype, in the presence and absence of diabetes, utilising cardiac-targeted recombinant-adeno-associated viral-vector-6 (rAAV6)-mediated gene delivery. Methods and results: In human myocardium, total protein O-GlcNAc modification was elevated in diabetic relative to non-diabetic patients, and correlated with left ventricular (LV) dysfunction. The impact of rAAV6-delivered O-GlcNAc transferase (rAAV6-OGT, facilitating protein O-GlcNAcylation), O-GlcNAcase (rAAV6-OGA, facilitating de-O-GlcNAcylation) and empty vector (null) were determined in non-diabetic and diabetic mice. In non-diabetic mice, rAAV6-OGT was sufficient to impair LV diastolic function and induce maladaptive cardiac remodelling, including cardiac fibrosis and increased Myh-7 and Nppa pro-hypertrophic gene expression, recapitulating characteristics of diabetic cardiomyopathy. In contrast, rAAV6-OGA (but not rAAV6-OGT) rescued LV diastolic function and adverse cardiac remodelling in diabetic mice. Molecular insights implicated impaired cardiac PI3K(p110α)-Akt signalling as a potential contributing mechanism to the detrimental consequences of rAAV6-OGT in vivo. In contrast, rAAV6-OGA preserved PI3K(p110α)-Akt signalling in diabetic mouse myocardium in vivo and prevented high glucose-induced impairments in mitochondrial respiration in human cardiomyocytes in vitro. Conclusion: Maladaptive protein O-GlcNAc modification is evident in human diabetic myocardium, and is a critical regulator of the diabetic heart phenotype. Selective targeting of cardiac protein O-GlcNAcylation to restore physiological O-GlcNAc balance may represent a novel therapeutic approach for diabetes-induced heart failure. Translational perspective: There remains a lack of effective clinical management of diabetes-induced cardiac dysfunction, even via conventional intensive glucose-lowering approaches. Here we reveal that the modification of myocardial proteins by O-GlcNAc, a glucose-driven process, is not only increased in human diabetic myocardium, but correlates with reduced cardiac function in affected patients. Moreover, manipulation of the two regulatory enzymes of this process exert opposing influences on the heart, whereby increasing O-GlcNAc transferase (OGT) is sufficient to replicate the cardiac phenotype of diabetes (in the absence of this disease), while increasing O-GlcNAc-ase (OGA) rescues diabetes-induced impairments in both cardiac dysfunction and remodelling. Cardiac O-GlcNAcylation thus represents a novel therapeutic target for diabetes-induced heart failure.

Diabetic cardiomyopathy is a distinct form of heart disease that represents a major cause of death and disability in diabetic patients, particularly, the more prevalent type 2 diabetes patient population. In the current study, we investigated whether administration of recombinant adeno-associated viral vectors carrying a constitutively active phosphoinositide 3-kinase (PI3K)(p110α) construct (rAAV6-caPI3K) at a clinically relevant time point attenuates diabetic cardiomyopathy in a preclinical type 2 diabetes (T2D) model. T2D was induced by a combination of a high-fat diet (42% energy intake from lipid) and low-dose streptozotocin (three consecutive intraperitoneal injections of 55 mg/kg body wt), and confirmed by increased body weight, mild hyperglycemia, and impaired glucose tolerance (all P < 0.05 vs. nondiabetic mice). After 18 wk of untreated diabetes, impaired left ventricular (LV) systolic dysfunction was evident, as confirmed by reduced fractional shortening and velocity of circumferential fiber shortening (Vcfc, all P < 0.01 vs. baseline measurement). A single tail vein injection of rAAV6-caPI3K gene therapy (2×1011vector genomes) was then administered. Mice were followed for an additional 8 wk before end point. A single injection of cardiac targeted rAAV6-caPI3K attenuated diabetes-induced cardiac remodeling by limiting cardiac fibrosis (reduced interstitial and perivascular collagen deposition, P < 0.01 vs. T2D mice) and cardiomyocyte hypertrophy (reduced cardiomyocyte size and Nppa gene expression, P < 0.001 and P < 0.05 vs. T2D mice, respectively). The diabetes-induced LV systolic dysfunction was reversed with rAAV6-caPI3K, as demonstrated by improved fractional shortening and velocity of circumferential fiber shortening (all P < 0.05 vs pre-AAV measurement). This cardioprotection occurred in combination with reduced LV reactive oxygen species ( P < 0.05 vs. T2D mice) and an associated decrease in markers of endoplasmic reticulum stress (reduced Grp94 and Chop, all P < 0.05 vs. T2D mice). Together, our findings demonstrate that a cardiac-selective increase in PI3K(p110α), via rAAV6-caPI3K, attenuates T2D-induced diabetic cardiomyopathy, providing proof of concept for potential translation to the clinic. NEW & NOTEWORTHY Diabetes remains a major cause of death and disability worldwide (and its resultant heart failure burden), despite current care. The lack of existing management of heart failure in the context of the poorer prognosis of concomitant diabetes represents an unmet clinical need. In the present study, we now demonstrate that delayed intervention with PI3K gene therapy (rAAV6-caPI3K), administered as a single dose in mice with preexisting type 2 diabetes, attenuates several characteristics of diabetic cardiomyopathy, including diabetes-induced impairments in cardiac remodeling, oxidative stress, and function. Our discovery here contributes to the previous body of work, suggesting the cardioprotective effects of PI3K(p110α) could be a novel therapeutic approach to reduce the progression to heart failure and death in diabetes-affected patients.