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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 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.

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.

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.

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.

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.

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.

Aims: The induction of heat shock protein 72 (Hsp72) via heating, genetic manipulation or pharmacological activation is metabolically protective in the setting of obesity‐induced insulin resistance across mammalian species. In this study, we set out to determine whether the overexpression of Hsp72, specifically in skeletal muscle, can protect against high‐fat diet (HFD)‐induced obesity and insulin resistance. Materials and methods: An Adeno‐Associated Viral vector (AAV), designed to overexpress Hsp72 in skeletal muscle only, was used to study the effects of increasing Hsp72 levels on various metabolic parameters. Two studies were conducted, the first with direct intramuscular (IM) injection of the AAV:Hsp72 into the tibialis anterior hind‐limb muscle and the second with a systemic injection to enable body‐wide skeletal muscle transduction. Results: IM injection of the AAV:Hsp72 significantly improved skeletal muscle insulin‐stimulated glucose clearance in treated hind‐limb muscles, as compared with untreated muscles of the contralateral leg when mice were fed an HFD. Despite this finding, systemic administration of AAV:Hsp72 did not improve body composition parameters such as body weight, fat mass or percentage body fat, nor did it lead to an improvement in fasting glucose levels or glucose tolerance. Furthermore, no differences were observed for other metabolic parameters such as whole‐body oxygen consumption, energy expenditure or physical activity levels. Conclusions: At the levels of Hsp72 over‐expression reported herein, skeletal muscle‐specific Hsp72 overexpression via IM injection has the capacity to increase insulin‐stimulated glucose clearance in this muscle. However, upon systemic injection, which results in lower muscle Hsp72 overexpression, no beneficial effects on whole‐body metabolism are observed.

Loss of expression of ACTN3, due to homozygosity of the common null polymorphism (p.Arg577X), is underrepresented in elite sprint/power athletes and has been associated with reduced muscle mass and strength in humans and mice. To investigate ACTN3 gene dosage in performance and whether expression could enhance muscle force, we performed meta-analysis and expression studies. Our general meta-analysis using a Bayesian random effects model in elite sprint/power athlete cohorts demonstrated a consistent homozygous-group effect across studies (per allele OR = 1.4, 95% CI 1.3-1.6) but substantial heterogeneity in heterozygotes. In mouse muscle, rAAV-mediated gene transfer overexpressed and rescued α-actinin-3 expression. Contrary to expectation, in vivo "doping" of ACTN3 at low to moderate doses demonstrated an absence of any change in function. At high doses, ACTN3 is toxic and detrimental to force generation, to demonstrate gene doping with supposedly performance-enhancing isoforms of sarcomeric proteins can be detrimental for muscle function. Restoration of α-actinin-3 did not enhance muscle mass but highlighted the primary role of α-actinin-3 in modulating muscle metabolism with altered fatiguability. This is the first study to express a Z-disk protein in healthy skeletal muscle and measure the in vivo effect. The sensitive balance of the sarcomeric proteins and muscle function has relevant implications in areas of gene doping in performance and therapy for neuromuscular disease.

Experimental autoimmune orchitis (EAO) is a rodent model of chronic testicular inflammation that mimics the pathology observed in some types of human infertility. In a previous study, testicular expression of the inflammatory/immunoregulatory cytokine, activin A, was elevated in adult mice during the onset of EAO, indicating a potential role in the regulation of the disease. Consequently, we examined the development of EAO in mice with elevated levels of follistatin, an endogenous activin antagonist, as a potential therapeutic approach to testicular inflammation. Prior to EAO induction, mice received a single intramuscular injection of a non-replicative recombinant adeno-associated viral vector carrying a gene cassette of the circulating form of follistatin, FST315 (FST group). Serum follistatin levels were increased 5-fold in the FST group compared with the control empty vector (EV) group at 30 and 50 days of EAO, but intra-testicular levels of follistatin or activin A were not significantly altered. Induction of EAO was reduced, but not prevented, with mild-to-severe damage in 75% of the EV group and 40% of the FST group, at 50 days following immunisation with testicular homogenate. However, the EAO damage score (based on disruption of the blood–testis barrier, apoptosis, testicular damage and fibrosis) and extent of intratesticular inflammation (expression of inflammatory mediators) were directly proportional to the levels of activin A measured in the testis at 50 days. These data implicate activin A in the progression of EAO, thereby providing a potential therapeutic target; however, elevating circulating follistatin levels were not sufficient to prevent EAO development.

Phosphoinositide 3-kinase [PI3K (p110α)] is able to negatively regulate the diabetes-induced increase in NADPH oxidase in the heart. Patients affected by diabetes exhibit significant cardiovascular morbidity and mortality, at least in part due to a cardiomyopathy characterized by oxidative stress and left ventricular (LV) dysfunction. Thus, PI3K (p110α) may represent a novel approach to protect the heart from diabetes-induced cardiac oxidative stress and dysfunction. In the present study, we investigated the therapeutic potential of a delayed intervention with cardiac-targeted PI3K gene therapy, administered to mice with established diabetes-induced LV diastolic dysfunction. Diabetes was induced in 6-week-old male mice by streptozotocin (STZ). After 8 weeks of untreated diabetes, LV diastolic dysfunction was confirmed by a reduction in echocardiography-derived transmitral E/A ratio. Diabetic and non-diabetic mice were randomly allocated to receive either recombinant adeno-associated viral vector-6 carrying a constitutively-active PI3K construct (recombinant adeno-associated-virus 6-constitutively active PI3K (p110α) (caPI3K) (rAAV6-caPI3K), single i.v. injection, 2 × 1011 vector genomes) or null vector, and were followed for a further 6 or 8 weeks. At study endpoint, diabetes-induced LV dysfunction was significantly attenuated by a single administration of rAAV6-caPI3K, administered 8 weeks after the induction of diabetes. Diabetes-induced impairments in each of LV NADPH oxidase, endoplasmic reticulum (ER) stress, apoptosis, cardiac fibrosis and cardiomyocyte hypertrophy, in addition to LV systolic dysfunction, were attenuated by delayed intervention with rAAV6-caPI3K. Hence, our demonstration that cardiac-targeted PI3K (p110α) gene therapy limits diabetes-induced up-regulation of NADPH oxidase and cardiac remodelling suggests new insights into promising approaches for the treatment of diabetic cardiomyopathy, at a clinically relevant time point (after diastolic dysfunction is manifested).

In cancer, elevated activin levels promote cachectic wasting of muscle, irrespective of tumor progression. In excess, activins A and B use the myostatin signaling pathway in muscle, triggering a decrease in protein synthesis and an increase in protein degradation, which ultimately leads to atrophy. Recently, we demonstrated that local delivery of engineered activin and myostatin propeptides (natural inhibitors of these growth factors) could induce profound muscle hypertrophy in healthy mice. Additionally, the expression of these propeptides effectively attenuated localized muscle wasting in models of dystrophy and cancer cachexia. In this study, we examined whether a systemically administered recombinant propeptide could reverse activin A–induced cachectic wasting in mice. Chinese hamster ovary cells stably expressing activin A were transplanted into the quadriceps of nude mice and caused an 85-fold increase in circulating activin A levels within 12 days. Elevated activin A induced a rapid reduction in body mass (−16%) and lean mass (−10%). In agreement with previous findings, we demonstrated that adeno-associated virus–mediated delivery of activin propeptide to the tibialis anterior muscle blocked activin-induced wasting. In addition, despite massively elevated levels of activin A in this model, systemic delivery of the propeptide significantly reduced activin-induced changes in lean and body mass. Specifically, recombinant propeptide reversed activin-induced wasting of skeletal muscle, heart, liver, and kidneys. This is the first study to demonstrate that systemic administration of recombinant propeptide therapy effectively attenuates tumor-derived activin A insult in multiple tissues.

KEY POINTS: Adeno-associated viral vector was used to elevate the expression of muscle specific kinase (MuSK) and rapsyn (a cytoplasmic MuSK effector protein) in the tibialis anterior muscle of wild-type and dystrophic (mdx) mice. In mdx mice, enhanced expression of either MuSK or rapsyn ameliorated the acute loss of muscle force associated with strain injury. Increases in sarcolemmal immunolabelling for utrophin and β-dystroglycan suggest a mechanism for the protective effect of MuSK in mdx muscles. MuSK also caused subtle changes to the structure and function of the neuromuscular junction, suggesting novel roles for MuSK in muscle physiology and pathophysiology. ABSTRACT: Muscle specific kinase (MuSK) has a well-defined role in stabilizing the developing mammalian neuromuscular junction, but MuSK might also be protective in some neuromuscular diseases. In the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy, limb muscles are especially fragile. We injected the tibialis anterior muscle of 8-week-old mdx and wild-type (C57BL10) mice with adeno-associated viral vectors encoding either MuSK or rapsyn (a cytoplasmic MuSK effector protein) fused to green fluorescent protein (MuSK-GFP and rapsyn-GFP, respectively). Contralateral muscles injected with empty vector served as controls. One month later mice were anaesthetized with isoflurane and isometric force-producing capacity was recorded from the distal tendon. MuSK-GFP caused an unexpected decay in nerve-evoked tetanic force, both in wild-type and mdx muscles, without affecting contraction elicited by direct electrical stimulation of the muscle. Muscle fragility was probed by challenging muscles with a strain injury protocol consisting of a series of four strain-producing eccentric contractions in vivo. When applied to muscles of mdx mice, eccentric contraction produced an acute 27% reduction in directly evoked muscle force output, affirming the susceptibility of mdx muscles to strain injury. mdx muscles overexpressing MuSK-GFP or rapsyn-GFP exhibited significantly milder force deficits after the eccentric contraction challenge (15% and 14%, respectively). The protective effect of MuSK-GFP in muscles of mdx mice was associated with increased immunolabelling for utrophin and β-dystroglycan in the sarcolemma. Elevating the expression of MuSK or rapsyn revealed several distinct synaptic and extrasynaptic effects, suggesting novel roles for MuSK signalling in muscle physiology and pathophysiology.

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., Graphical Abstract, Highlights • Proteomic, glycomic and glycoproteomic analysis of myogenesis. • Mechanistic insights into site-specific glycoproteome regulation via discrete glycosidases and glycosyltransferases. • Quantification and validation of glycan-binding proteins. • Functional analysis of LGALS1 reveals a role in myogenesis and muscle development., In Brief The proteome, N-glycome, and N-glycoproteome have been determined during myotube formation. This revealed a complex regulation of N-glycosylation including a switch in sialylation linkages, digalactosylation, and paucimannosylation on receptors and adhesion molecules. The data also revealed regulation of several glycan-binding proteins including LGALS1, and functional validation revealed a role of this protein in myogenesis and skeletal muscle development.

Objective The insulin-like growth factor binding proteins (IGFBPs) are thought to modulate cell size and homeostasis via IGF-I-dependent and -independent pathways. There is a considerable dearth of information regarding the function of IGFBPs in skeletal muscle, particularly their role in the pathophysiology of Duchenne muscular dystrophy (DMD). In this study we tested the hypothesis that intramuscular IGFBP-2 overexpression would ameliorate the pathology in mdx dystrophic mice. Design 4week old male C57Bl/10 and mdx mice received a single intramuscular injection of AAV6-empty or AAV6-IGFBP-2 vector into the tibialis anterior muscle. At 8weeks post-injection the effect of IGFBP-2 overexpression on the structure and function of the injected muscle was assessed. Results AAV6-mediated IGFBP-2 overexpression in the tibialis anterior (TA) muscles of 4-week-old C57BL/10 and mdx mice reduced the mass of injected muscle after 8weeks, inducing a slower muscle phenotype in C57BL/10 but not mdx mice. Analysis of inflammatory and fibrotic gene expression revealed no changes between control and IGFBP-2 injected muscles in dystrophic ( mdx ) mice. Conclusions Together these results indicate that the IGFBP-2-induced promotion of a slower muscle phenotype is impaired in muscles of dystrophin-deficient mdx mice, which contributes to the inability of IGFBP-2 to ameliorate the dystrophic pathology. The findings implicate the dystrophin-glycoprotein complex (DGC) in the signaling required for this adaptation.

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.

SummaryDrugs that recapitulate aspects of the exercise adaptive response have the potential to provide better treatment for diseases associated with physical inactivity. We previously observed reduced skeletal muscle class IIa HDAC (histone deacetylase) transcriptional repressive activity during exercise. Here, we find that exercise-like adaptations are induced by skeletal muscle expression of class IIa HDAC mutants that cannot form a corepressor complex. Adaptations include increased metabolic gene expression, mitochondrial capacity, and lipid oxidation. An existing HDAC inhibitor, Scriptaid, had similar phenotypic effects through disruption of the class IIa HDAC corepressor complex. Acute Scriptaid administration to mice increased the expression of metabolic genes, which required an intact class IIa HDAC corepressor complex. Chronic Scriptaid administration increased exercise capacity, whole-body energy expenditure and lipid oxidation, and reduced fasting blood lipids and glucose. Therefore, compounds that disrupt class IIa HDAC function could be used to enhance metabolic health in chronic diseases driven by physical inactivity.

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.

Growth/differentiation factor 8 (GDF8), or myostatin, negatively regulates muscle mass. GDF8 is held in a latent state through interactions with its N-terminal prodomain, much like TGF-β. Using a combination of small-angle X-ray scattering and mutagenesis, we characterized the interactions of GDF8 with its prodomain. Our results show that the prodomain:GDF8 complex can exist in a fully latent state and an activated or "triggered" state where the prodomain remains in complex with the mature domain. However, these states are not reversible, indicating the latent GDF8 is "spring-loaded." Structural analysis shows that the prodomain:GDF8 complex adopts an "open" configuration, distinct from the latency state of TGF-β and more similar to the open state of Activin A and BMP9 (nonlatent complexes). We determined that GDF8 maintains similar features for latency, including the alpha-1 helix and fastener elements, and identified a series of mutations in the prodomain of GDF8 that alleviate latency, including I56E, which does not require activation by the protease Tolloid. In vivo, active GDF8 variants were potent negative regulators of muscle mass, compared with WT GDF8. Collectively, these results help characterize the latency and activation mechanisms of GDF8.

The mammalian heart undergoes maturation during postnatal life to meet the increased functional requirements of an adult. However, the key drivers of this process remain poorly defined. We are currently unable to recapitulate postnatal maturation in human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), limiting their potential as a model system to discover regenerative therapeutics. Here, we provide a summary of our studies, where we developed a 96-well device for functional screening in human pluripotent stem cell-derived cardiac organoids (hCOs). Through interrogation of >10,000 organoids, we systematically optimize parameters, including extracellular matrix (ECM), metabolic substrate, and growth factor conditions, that enhance cardiac tissue viability, function, and maturation. Under optimized maturation conditions, functional and molecular characterization revealed that a switch to fatty acid metabolism was a central driver of cardiac maturation. Under these conditions, hPSC-CMs were refractory to mitogenic stimuli, and we found that key proliferation pathways including β-catenin and Yes-associated protein 1 (YAP1) were repressed. This proliferative barrier imposed by fatty acid metabolism in hCOs could be rescued by simultaneous activation of both β-catenin and YAP1 using genetic approaches or a small molecule activating both pathways. These studies highlight that human organoids coupled with higher-throughput screening platforms have the potential to rapidly expand our knowledge of human biology and potentially unlock therapeutic strategies.

We previously showed that medium chain acyl-coenzyme A dehydrogenase (MCAD, key regulator of fatty acid oxidation) is positively modulated in the heart by the cardioprotective kinase, phosphoinositide 3-kinase (PI3K(p110α)). Disturbances in cardiac metabolism are a feature of heart failure (HF) patients and targeting metabolic defects is considered a potential therapeutic approach. The specific role of MCAD in the adult heart is unknown. To examine the role of MCAD in the heart and to assess the therapeutic potential of increasing MCAD in the failing heart, we developed a gene therapy tool using recombinant adeno-associated viral vectors (rAAV) encoding MCAD. We hypothesised that increasing MCAD expression may recapitulate the cardioprotective properties of PI3K(p110α). rAAV6:MCAD or rAAV6:control was delivered to healthy adult mice and to mice with pre-existing pathological hypertrophy and cardiac dysfunction due to transverse aortic constriction (TAC). In healthy mice, rAAV6:MCAD induced physiological hypertrophy (increase in heart size, normal systolic function and increased capillary density). In response to TAC (~15 weeks), heart weight/tibia length increased by ~60% in control mice and ~45% in rAAV6:MCAD mice compared with sham. This was associated with an increase in cardiomyocyte cross-sectional area in both TAC groups which was similar. However, hypertrophy in TAC rAAV6:MCAD mice was associated with less fibrosis, a trend for increased capillary density and a more favourable molecular profile compared with TAC rAAV6:control mice. In summary, MCAD induced physiological cardiac hypertrophy in healthy adult mice and attenuated features of pathological remodelling in a cardiac disease model.

The transforming growth factor-β (TGF-β) network of ligands and intracellular signaling proteins is a subject of intense interest within the field of skeletal muscle biology. To define the relative contribution of endogenous TGF-β proteins to the negative regulation of muscle mass via their activation of the Smad2/3 signaling axis, we used local injection of adeno-associated viral vectors (AAVs) encoding ligand-specific antagonists into the tibialis anterior (TA) muscles of C57BL/6 mice. Eight weeks after AAV injection, inhibition of activin A and activin B signaling produced moderate (∼20%), but significant, increases in TA mass, indicating that endogenous activins repress muscle growth. Inhibiting myostatin induced a more profound increase in muscle mass (∼45%), demonstrating a more prominent role for this ligand as a negative regulator of adult muscle mass. Remarkably, codelivery of activin and myostatin inhibitors induced a synergistic response, resulting in muscle mass increasing by as much as 150%. Transcription and protein analysis indicated that this substantial hypertrophy was associated with both the complete inhibition of the Smad2/3 pathway and activation of the parallel bone morphogenetic protein (BMP)/Smad1/5 axis (recently identified as a positive regulator of muscle mass). Analyses indicated that hypertrophy was primarily driven by an increase in protein synthesis, but a reduction in ubiquitin-dependent protein degradation pathways was also observed. In models of muscular dystrophy and cancer cachexia, combined inhibition of activins and myostatin increased mass or prevented muscle wasting, respectively, highlighting the potential therapeutic advantages of specifically targeting multiple Smad2/3-activating ligands in skeletal muscle.

The transforming growth factor beta (TGFβ) superfamily comprises a large number of secreted proteins that regulate various fundamental biological processes underlying embryonic development and the postnatal regulation of many cell types and organs. Sequence similarities define two ligand subfamilies: the TGFβ/activin subfamily and the bone morphogenetic protein (BMP) subfamily. The discovery that myostatin, a member of the TGFβ/activin subfamily, negatively controls muscle mass attracted attention to this pathway. However, recent findings of a positive role for BMP-mediated signaling in muscle have challenged the model of how the TGFβ network regulates skeletal muscle phenotype. This review illustrates how this complex network integrates crosstalk among members of the TGFβ superfamily and downstream signaling elements to regulate muscle in health and disease.

α-Actinin-3 deficiency occurs in approximately 16% of the global population due to homozygosity for a common nonsense polymorphism in the ACTN3 gene. Loss of α-actinin-3 is associated with reduced power and enhanced endurance capacity in elite athletes and nonathletes due to “slowing” of the metabolic and physiological properties of fast fibers. Here, we have shown that α-actinin-3 deficiency results in increased calcineurin activity in mouse and human skeletal muscle and enhanced adaptive response to endurance training. α-Actinin-2, which is differentially expressed in α-actinin-3–deficient muscle, has higher binding affinity for calsarcin-2, a key inhibitor of calcineurin activation. We have further demonstrated that α-actinin-2 competes with calcineurin for binding to calsarcin-2, resulting in enhanced calcineurin signaling and reprogramming of the metabolic phenotype of fast muscle fibers. Our data provide a mechanistic explanation for the effects of the ACTN3 genotype on skeletal muscle performance in elite athletes and on adaptation to changing physical demands in the general population. In addition, we have demonstrated that the sarcomeric α-actinins play a role in the regulation of calcineurin signaling.

Patients with advanced cancer often succumb to complications arising from striated muscle wasting associated with cachexia. Excessive activation of the type IIB activin receptor (ActRIIB) is considered an important mechanism underlying this wasting, where circulating procachectic factors bind ActRIIB and ultimately lead to the phosphorylation of SMAD2/3. Therapeutics that antagonize the binding of ActRIIB ligands are in clinical development, but concerns exist about achieving efficacy without off-target effects. To protect striated muscle from harmful ActRIIB signaling, and to reduce the risk of off-target effects, we developed an intervention using recombinant adeno-associated viral vectors (rAAV vectors) that increase expression of Smad7 in skeletal and cardiac muscles. SMAD7 acts as an intracellular negative regulator that prevents SMAD2/3 activation and promotes degradation of ActRIIB complexes. In mouse models of cachexia, rAAV:Smad7 prevented wasting of skeletal muscles and the heart independent of tumor burden and serum levels of procachectic ligands. Mechanistically, rAAV:Smad7 administration abolished SMAD2/3 signaling downstream of ActRIIB and inhibited expression of the atrophy-related ubiquitin ligases MuRF1 and MAFbx. These findings identify muscle-directed Smad7 gene delivery as a potential approach for preventing muscle wasting under conditions where excessive ActRIIB signaling occurs, such as cancer cachexia.

Skeletal muscle possesses remarkable ability to change its size and force-producing capacity in response to physiological stimuli. Impairment of the cellular processes that govern these attributes also affects muscle mass and function in pathological conditions. Myostatin, a member of the TGF-β family, has been identified as a key regulator of muscle development, and adaptation in adulthood. In muscle, myostatin binds to its type I (ALK4/5) and type II (ActRIIA/B) receptors to initiate Smad2/3 signalling and the regulation of target genes that co-ordinate the balance between protein synthesis and degradation. Interestingly, evidence is emerging that other TGF-β proteins act in concert with myostatin to regulate the growth and remodelling of skeletal muscle. Consequently, dysregulation of TGF-β proteins and their associated signalling components is increasingly being implicated in muscle wasting associated with chronic illness, ageing, and inactivity. The growing understanding of TGF-β biology in muscle, and its potential to advance the development of therapeutics for muscle-related conditions is reviewed here.

Anabolic β2-adrenoceptor (β2-AR) agonists have been proposed as therapeutics for treating muscle wasting but concerns regarding possible off-target effects have hampered their use. We investigated whether β2-AR-mediated signalling could be modulated in skeletal muscle via gene delivery to the target tissue, thereby avoiding the risks of β2-AR agonists. In mice, intramuscular administration of a recombinant adeno-associated virus-based vector (rAAV vector) expressing the β2-AR increased muscle mass by >20% within 4 weeks. This hypertrophic response was comparable to that of 4 weeks’ treatment with the β2-AR agonist formoterol and was not ablated by mTOR inhibition. Increasing expression of inhibitory (Gαi2) and stimulatory (GαsL) G-protein subunits produced minor atrophic and hypertrophic changes in muscle mass, respectively. Furthermore, Gαi2 over-expression prevented AAV:β2-AR mediated hypertrophy. Introduction of the non-muscle Gαs isoform, GαsXL elicited hypertrophy comparable to that achieved by AAV:β2-AR. Moreover, GαsXL gene delivery was found to be capable of inducing hypertrophy in the muscles of mice lacking functional β1- and β2-ARs. These findings demonstrate that gene therapy-based interventions targeting the β2-AR pathway can promote skeletal muscle hypertrophy independent of ligand administration and highlight novel methods for potentially modulating muscle mass in settings of disease.

Objective The development of skeletal muscle insulin resistance is an early physiological defect, yet the intracellular mechanisms accounting for this metabolic defect remained unresolved. Here, we have examined the role of glucose-6-phosphate dehydrogenase (G6PDH) activity in the pathogenesis of insulin resistance in skeletal muscle. Methods Multiple mouse disease states exhibiting insulin resistance and glucose intolerance, as well as obese humans defined as insulin-sensitive, insulin-resistant, or pre-diabetic, were examined. Results We identified increased glucose-6-phosphate dehydrogenase (G6PDH) activity as a common intracellular adaptation that occurs in parallel with the induction of insulin resistance in skeletal muscle and is present across animal and human disease states with an underlying pathology of insulin resistance and glucose intolerance. We observed an inverse association between G6PDH activity and nitric oxide synthase (NOS) activity and show that increasing NOS activity via the skeletal muscle specific neuronal (n)NOSμ partially suppresses G6PDH activity in skeletal muscle cells. Furthermore, attenuation of G6PDH activity in skeletal muscle cells via (a) increased nNOSμ/NOS activity, (b) pharmacological G6PDH inhibition, or (c) genetic G6PDH inhibition increases insulin-independent glucose uptake. Conclusions We have identified a novel, previously unrecognized role for G6PDH in the regulation of skeletal muscle glucose metabolism., Highlights • Defective skeletal muscle G6PDH activity in multiple insulin resistant animal models. • Demonstration of defective skeletal muscle G6PDH activity in pre-diabetic individuals. • Identification of nNOSμ as a regulator of G6PDH activity in skeletal muscle. • G6PDH activity modulates insulin-independent glucose uptake in skeletal muscle.

Background— Numerous molecular and biochemical changes have been linked with the cardioprotective effects of exercise, including increases in antioxidant enzymes, heat shock proteins, and regulators of cardiac myocyte proliferation. However, a master regulator of exercise-induced protection has yet to be identified. Here, we assess whether phosphoinositide 3-kinase (PI3K) p110α is essential for mediating exercise-induced cardioprotection, and if so, whether its activation independent of exercise can restore function of the failing heart. Methods and Results— Cardiac-specific transgenic (Tg) mice with elevated or reduced PI3K(p110α) activity (constitutively active PI3K [caPI3K] and dominant negative PI3K, respectively) and non-Tg controls were subjected to 4 weeks of exercise training followed by 1 week of pressure overload (aortic-banding) to induce pathological remodeling. Aortic-banding in untrained non-Tg controls led to pathological cardiac hypertrophy, depressed systolic function, and lung congestion. This phenotype was attenuated in non-Tg controls that had undergone exercise before aortic-banding. Banded caPI3K mice were protected from pathological remodeling independent of exercise status, whereas exercise provided no protection in banded dominant negative PI3K mice, suggesting that PI3K is necessary for exercise-induced cardioprotection. Tg overexpression of heat shock protein 70 could not rescue the phenotype of banded dominant negative PI3K mice, and deletion of heat shock protein 70 from banded caPI3K mice had no effect. Next, we used a gene therapy approach (recombinant adeno-associated viral vector 6) to deliver caPI3K expression cassettes to hearts of mice with established cardiac dysfunction caused by aortic-banding. Mice treated with recombinant adeno-associated viral 6-caPI3K vectors had improved heart function after 10 weeks. Conclusions— PI3K(p110α) is essential for exercise-induced cardioprotection and delivery of caPI3K vector can improve function of the failing heart.

Smad3/Akt/mTOR/S6K/S6RP signaling plays a critical role in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms., Follistatin is essential for skeletal muscle development and growth, but the intracellular signaling networks that regulate follistatin-mediated effects are not well defined. We show here that the administration of an adeno-associated viral vector expressing follistatin-288aa (rAAV6:Fst-288) markedly increased muscle mass and force-producing capacity concomitant with increased protein synthesis and mammalian target of rapamycin (mTOR) activation. These effects were attenuated by inhibition of mTOR or deletion of S6K1/2. Furthermore, we identify Smad3 as the critical intracellular link that mediates the effects of follistatin on mTOR signaling. Expression of constitutively active Smad3 not only markedly prevented skeletal muscle growth induced by follistatin but also potently suppressed follistatin-induced Akt/mTOR/S6K signaling. Importantly, the regulation of Smad3- and mTOR-dependent events by follistatin occurred independently of overexpression or knockout of myostatin, a key repressor of muscle development that can regulate Smad3 and mTOR signaling and that is itself inhibited by follistatin. These findings identify a critical role of Smad3/Akt/mTOR/S6K/S6RP signaling in follistatin-mediated muscle growth that operates independently of myostatin-driven mechanisms.

MicroRNAs (miRs) are emerging as prominent players in the regulation of many biological processes, including myogenic commitment and skeletal muscle formation. Members of the TGF-β family can influence the proliferation and myogenic differentiation of cells, although it is presently not clear what role miRNAs play in the TGF-β-mediated control of myogenic differentiation. Here, we demonstrate in the myogenic C2C12 cell line, and in primary muscle cells, that miR-206 and miR-29-two miRs that act on transcriptional events implicated in muscle differentiation are down-regulated by TGF-β. We further demonstrate that TGF-β treatment of myogenic cells is associated with increased expression of histone deacetylase 4 (HDAC4), a key inhibitor of muscle differentiation that has been identified as a target for regulation by miR-206 and miR-29. We confirmed that increased expression of miR-206 and miR-29 resulted in the translational repression of HDAC4 in the presence or absence of TGF-β via interaction with the HDAC4 3′-untranslated region. Importantly, we found that miR-206 and miR-29 can attenuate the inhibitory actions of TGF-β on myogenic differentiation. Furthermore, we present evidence that the mechanism by which miR-206 and miR-29 can inhibit the TGF-β-mediated up-regulation of HDAC4 is via the inhibition of Smad3 expression, a transducer of TGF-β signaling. These findings identify a novel mechanism of interaction between TGF-β and miR-206 and -29 in the regulation of myogenic differentiation through HDAC4.

Recombinant adeno-associated viral (rAAV) vector-mediated gene transfer represents a promising approach for many diseases. However, the applicability of rAAV vectors has long been hindered by the small (~4.8 kb) DNA packaging capacity. This limitation can hamper the packaging and delivery of critical regulatory elements and/or larger coding sequences, such as the ~14-kb dystrophin complementary DNA (cDNA) that is of interest for gene therapy of Duchenne muscular dystrophy (DMD). Here, we have demonstrated reconstitution of an expression cassette (7.3 kb) encoding a highly functional "minidystrophin" protein (ΔH2-R19, 222 kd) in vivo following intravascular co-delivery of two independent rAAV6 vectors sharing a central homologous recombinogenic region of 372 nucleotides. Similar to previously reported trans-splicing approaches, one rAAV vector provides the promoter with the ~1/2 initial portion of minidystrophin, while the second vector provides the remaining minidystrophin cDNA followed by the polyadenylation signal. Significantly, administering a modest dose [2 × 10(12) vector genomes (vg)] of the two minidystrophin-encoding rAAV vectors to dystrophic mice elicited an improvement of physiological performance indicative of prevention or amelioration of the disease state. These studies provide evidence that functional dystrophin transgenes larger than that typically carried by a single rAAV genome can be reconstituted in vivo by homologous recombination (HR) following intravascular co-delivery with rAAV6.

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.

A growing body of research supports the development of recombinant adeno-associated viral (rAAV) vectors for delivery of gene expression cassettes to striated musculature as a method of treating severe neuromuscular conditions. However, it is unclear whether delivery protocols that achieve extensive gene transfer in mice can be adapted to produce similarly extensive gene transfer in larger mammals and ultimately patients. Consequently, we sought to investigate methodological modifications that would facilitate rAAV-mediated gene transfer to the striated musculature of canines. A simple procedure incorporating acute (i) occlusion of limb blood flow, (ii) exsanguination via compression bandage, and (iii) vector “dwell” time of

Angiotensin-converting enzyme (ACE) 2 is a recently identified homologue of ACE. There is great interest in the therapeutic benefit for ACE2 overexpression in the heart. However, the role of ACE2 in the regulation of cardiac structure and function, as well as maintenance of systemic blood pressure, remains poorly understood. In cell culture, ACE2 overexpression led to markedly increased myocyte volume, assessed in primary rabbit myocytes. To assess ACE2 function in vivo, we used a recombinant adeno-associated virus 6 delivery system to provide 11-week overexpression of ACE2 in the myocardium of stroke-prone spontaneously hypertensive rats. ACE2, as well as the ACE inhibitor enalapril, significantly reduced systolic blood pressure. However, in the heart, ACE2 overexpression resulted in cardiac fibrosis, as assessed by histological analysis with concomitant deficits in ejection fraction and fractional shortening measured by echocardiography. Furthermore, global gene expression profiling demonstrated the activation of profibrotic pathways in the heart mediated by ACE2 gene delivery. This study demonstrates that sustained overexpression of ACE2 in the heart in vivo leads to the onset of severe fibrosis.

Duchenne muscular dystrophy (DMD), the most prevalent lethal genetic disorder in children, is caused by mutations in the 2.2-MB dystrophin gene. Absence of dystrophin and the dystrophin-glycoprotein complex (DGC) from the sarcolemma leads to severe muscle wasting and eventual respiratory and/or cardiac failure. There is presently no effective therapy for DMD. Several lines of evidence have suggested that methods to increase expression of utrophin, a dystrophin paralog, show promise as a treatment for DMD. Adeno-associated viral (AAV) vectors are a promising vehicle for gene transfer to muscle, but microutrophin transgenes small enough to be carried by AAV have not been tested for function. In this study, we intravenously administered recombinant AAV (rAAV2/6) harboring a murine codon-optimized microutrophin (DeltaR4-R21/DeltaCT) transgene to adult dystrophin(-/-)/utrophin(-/-) (mdx:utrn(-/-)) double-knockout mice. Five-month-old mice demonstrated localization of microutrophin to the sarcolemma in all the muscles tested. These muscles displayed restoration of the DGC, increased myofiber size, and a considerable improvement in physiological performance when compared with untreated mdx:utrn(-/-) mice. Overall, microutrophin delivery alleviated most of the pathophysiological abnormalities associated with muscular dystrophy in the mdx:utrn(-/-) mouse model. This approach may hold promise as a treatment option for DMD because it avoids the potential immune responses that are associated with the delivery of exogenous dystrophin.

Restoring dystrophin expression in the muscles of patients with Duchenne muscular dystrophy (DMD) may halt or reverse the degenerative wasting and weakness that causes premature death. However, the therapeutic efficacy of an intervention may be limited by the extent of disease progression prior to treatment. In this study, we considered the potential for ameliorating the pathology in a mouse model of advanced-stage muscular dystrophy by systemic administration of recombinant adeno-associated viral (rAAV6) vectors encoding a microdystrophin expression construct. The treatment of 20-month-old mdx mice restored body-wide expression of a dystrophin-based protein in striated musculature. In aged mice that received treatment, the resultant dystrophin expression was associated with improved hindlimb and respiratory muscle morphology and function, concomitant with reduced muscle fiber degeneration. The findings demonstrate that an established dystrophic state remains amenable to improvement with appropriate intervention and, by some measures, may even achieve benefits similar to those observed with intervention early in disease progression. The capacity to ameliorate the pathology in an animal model of advanced-stage muscular dystrophy suggests that interventions ultimately proven to exert a therapeutic effect in young patients may offer benefits to older patients or those with advanced conditions of progressive muscular dystrophy.

Muscular dystrophies are a diverse group of severe degenerative muscle diseases. Recent interest in the role of the Golgi complex (GC) in muscle disease has been piqued by findings that several dystrophies result from mutations in putative Golgi-resident glycosyltransferases. Given this new role of the Golgi in sarcolemmal stability, we hypothesized that abnormal Golgi distribution, regulation and/or function may constitute part of the pathology of other dystrophies, where the primary defect is independent of Golgi function. Thus, we investigated GC organization in the dystrophin-deficient muscles of mdx mice, a mouse model for Duchenne muscular dystrophy. We report aberrant organization of the synaptic and extrasynaptic GC in skeletal muscles of mdx mice. The GC is mislocalized and improperly concentrated at the surface and core of mdx myofibers. Golgi complex localization is disrupted after the onset of necrosis and normal redistribution is impaired during regeneration of mdx muscle fibers. Disruption of the microtubule cytoskeleton may account in part for aberrant GC localization in mdx myofibers. Golgi complex distribution is restored to wild type and microtubule cytoskeleton organization is significantly improved by recombinant adeno-associated virus 6-mediated expression of DeltaR4-R23/DeltaCT microdystrophin showing a novel mode of microdystrophin functionality. In summary, GC distribution abnormalities are a novel component of mdx skeletal muscle pathology rescued by microdystrophin expression.

Adeno-associated virus-based vector (AAV)-mediated gene delivery has been successful in some animal models of human disease such as the mdx mouse model of human Duchenne muscular dystrophy (DMD). However, recent evidence of immune-mediated loss of vector persistence in dogs and humans suggests that immune modulation might be necessary to achieve successful long-term transgene expression in these species. We have previously demonstrated that direct intramuscular injection of AAV2 or AAV6 in wild-type random-bred dogs resulted in a robust immune response to capsid or capsid-associated proteins. We now demonstrate that a brief course of immunosuppression with a combination of anti-thymocyte globulin (ATG), cyclosporine (CSP), and mycophenolate mofetil (MMF) is sufficient to permit long-term and robust expression of a canine micro-dystrophin (c-micro-dys) transgene in the skeletal muscle of a dog model for DMD (canine X-linked muscular dystrophy, or cxmd dog) and that its expression restored localization of components of the dystrophin-associated protein complex at the muscle membrane. This protocol has potential applications to human clinical trials to enhance AAV-mediated therapies.

Much progress has been made over the past decade elucidating the molecular basis for a variety of muscular dystrophies (MDs). Accordingly, there are examples of mouse models of MD whose disease progression has been halted in large part with the use of viral vector technology. Even so, we must acknowledge significant limitations of present vector systems that must be overcome prior to successful treatment of humans with such approaches. This review will present a variety of viral-mediated therapeutic strategies aimed at counteracting the muscle-wasting symptoms associated with muscular dystrophy. We include viral vector systems used for muscle gene transfer, with a particular emphasis on adeno-associated virus. Findings of several encouraging studies focusing on repair of the mutant dystrophin gene are also included. Lastly, we present a discussion of muscle compensatory therapeutics being considered that include pathways involved in the up-regulation of utrophin, promotion of cellular adhesion, enhancement of muscle mass, and antagonism of the inflammatory response. Considering the complexity of the muscular dystrophies, it appears likely that a multilayered approach tailored to a patient sub-group may be warranted in order to effectively contest the progression of this devastating disease.

Recombinant adeno-associated virus (rAAV)-mediated gene transfer has shown promise for treating diseases in various animal models including the mdx mouse model of Duchenne muscular dystrophy (DMD). In many cases, however, preclinical studies in inbred mice have not successfully predicted human clinical responses. To assess the potential clinical utility of treating human DMD patients by AAV-mediated gene delivery, we performed a series of direct intramuscular injections in random-bred wild-type dogs. AAV serotypes 2 and 6 carrying different promoter-transgene cassettes were produced as previously described for murine studies and administered intramuscularly. The injection sites were biopsied at various time points and analyzed for transgene expression and immunohistochemical analysis. In contrast to the generally nonimmunogenic nature of these vectors in murine studies, both AAV2 and AAV6 vectors elicited robust cellular immune responses regardless of the transgene expressed, the cellular specificity of the promoter, and the muscle type injected. Viral purification by various methods did not diminish T cell-mediated infiltration. Our data indicate that AAV2 and AAV6 capsid proteins can elicit primary cellular immune responses when injected into the skeletal muscle of random-bred dogs, and suggest the possibility of cellular immunity to AAV vectors in humans.

SummaryThe cytokine TWEAK and its cognate receptor Fn14 are members of the TNF/TNFR superfamily and are upregulated in tumors. We found that Fn14, when expressed in tumors, causes cachexia and that antibodies against Fn14 dramatically extended lifespan by inhibiting tumor-induced weight loss although having only moderate inhibitory effects on tumor growth. Anti-Fn14 antibodies prevented tumor-induced inflammation and loss of fat and muscle mass. Fn14 signaling in the tumor, rather than host, is responsible for inducing this cachexia because tumors in Fn14- and TWEAK-deficient hosts developed cachexia that was comparable to that of wild-type mice. These results extend the role of Fn14 in wound repair and muscle development to involvement in the etiology of cachexia and indicate that Fn14 antibodies may be a promising approach to treat cachexia, thereby extending lifespan and improving quality of life for cancer patients.

Follistatin is an inhibitor of TGF-β superfamily ligands that repress skeletal muscle growth and promote muscle wasting. Accordingly, follistatin has emerged as a potential therapeutic to ameliorate the deleterious effects of muscle atrophy. However, it remains unclear whether the anabolic effects of follistatin are conserved across different modes of non-degenerative muscle wasting. In this study, the delivery of a recombinant adeno-associated viral vector expressing follistatin (rAAV:Fst) to the hind-limb musculature of mice two weeks prior to denervation or tenotomy promoted muscle hypertrophy that was sufficient to preserve muscle mass comparable to that of untreated sham-operated muscles. However, administration of rAAV:Fst to muscles at the time of denervation or tenotomy did not prevent subsequent muscle wasting. Administration of rAAV:Fst to innervated or denervated muscles increased protein synthesis, but markedly reduced protein degradation only in innervated muscles. Phosphorylation of the signalling proteins mTOR and S6RP, which are associated with protein synthesis, was increased in innervated muscles administered rAAV:Fst, but not in treated denervated muscles. These results demonstrate that the anabolic effects of follistatin are influenced by the interaction between muscle fibres and motor nerves. These findings have important implications for understanding the potential efficacy of follistatin-based therapies for non-degenerative muscle wasting.

Apoptosis-inducing factor (AIF) is a mitochondrial flavoprotein with dual roles in redox signaling and programmed cell death. Deficiency in AIF is known to result in defective oxidative phosphorylation (OXPHOS), via loss of complex I activity and assembly in other tissues. Because the kidney relies on OXPHOS for metabolic homeostasis, we hypothesized that a decrease in AIF would result in chronic kidney disease (CKD). Here, we report that partial knockdown of Aif in mice recapitulates many features of CKD, in association with a compensatory increase in the mitochondrial ATP pool via a shift toward mitochondrial fusion, excess mitochondrial reactive oxygen species production, and Nox4 upregulation. However, despite a 50% lower AIF protein content in the kidney cortex, there was no loss of complex I activity or assembly. When diabetes was superimposed onto Aif knockdown, there were extensive changes in mitochondrial function and networking, which augmented the renal lesion. Studies in patients with diabetic nephropathy showed a decrease in AIF within the renal tubular compartment and lower AIFM1 renal cortical gene expression, which correlated with declining glomerular filtration rate. Lentiviral overexpression of Aif1m rescued glucose-induced disruption of mitochondrial respiration in human primary proximal tubule cells. These studies demonstrate that AIF deficiency is a risk factor for the development of diabetic kidney disease.