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Abstract The dystrophin protein has well‐characterized roles in force transmission and maintaining membrane integrity during muscle contraction. Studies have reported decreased expression of dystrophin in atrophying muscles during wasting conditions, and that restoration of dystrophin can attenuate atrophy, suggesting a role in maintaining muscle mass. Phosphorylation of S3059 within the cysteine‐rich region of dystrophin enhances binding between dystrophin and β‐dystroglycan, and mimicking phosphorylation at this site by site‐directed mutagenesis attenuates myotube atrophy in vitro. To determine whether dystrophin phosphorylation can attenuate muscle wasting in vivo, CRISPR‐Cas9 was used to generate mice with whole body mutations of S3059 to either alanine (DmdS3059A) or glutamate (DmdS3059E), to mimic a loss of, or constitutive phosphorylation of S3059, on all endogenous dystrophin isoforms, respectively. Sciatic nerve transection was performed on these mice to determine whether phosphorylation of dystrophin S3059 could attenuate denervation atrophy. At 14 days post denervation, atrophy of tibialis anterior (TA) but not gastrocnemius or soleus muscles, was partially attenuated in DmdS3059E mice relative to WT mice. Attenuation of atrophy was associated with increased expression of β‐dystroglycan in TA muscles of DmdS3059E mice. Dystrophin S3059 phosphorylation can partially attenuate denervation‐induced atrophy, but may have more significant impact in less severe modes of muscle wasting.

Abstract 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, resulting in increased non-oxidative glucose metabolism. This metabolic reprogramming was also associated with impaired apoptosis and ferroptosis responses, and 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. Redox drivers of ferroptosis derived from oxidative metabolism were also reduced. The relevance of this pathway was demonstrated by overexpression of loss-of-function HDAC4 and HDAC5 mutants in skeletal muscle of obese db/db mice, which enhanced oxidative metabolic capacity, increased apoptosis and ferroptosis and reduced muscle mass. This study identifies HDAC4 and HDAC5 as repressors of skeletal muscle oxidative metabolism, which is linked to inhibition of cell death pathways and preservation of muscle integrity in response to lipotoxicity.

High baseline clearance of immune checkpoint inhibitors (ICIs), independent of dose or systemic exposure, is associated with cachexia and poor outcomes in cancer patients. Mechanisms linking ICI clearance, cachexia and ICI therapy failure are unknown. Here, we evaluate in four murine models and across multiple antibodies whether altered baseline catabolic clearance of administered antibody requires a tumor and/or cachexia and whether medical reversal of cachexia phenotype can alleviate altered clearance. Key findings include mild cachexia phenotype and lack of elevated pembrolizumab clearance in the MC38 tumor-bearing model. We also observed severe cachexia and decreased, instead of increased, baseline pembrolizumab clearance in the tumor-free cisplatin-induced cachexia model. Liver Fcgrt expression correlated with altered baseline catabolic clearance, though elevated clearance was still observed with antibodies having no (human IgA) or reduced (human H310Q IgG1) FcRn binding. We conclude cachexia phenotype coincides with altered antibody clearance, though tumor presence is neither sufficient nor necessary for altered clearance in immunocompetent mice. Magnitude and direction of clearance alteration correlated with hepatic Fcgrt, suggesting changes in FcRn expression and/or recycling function may be partially responsible, though factors beyond FcRn also contribute to altered clearance in cachexia.

Introduction Acute clinical deterioration in hospital inpatients can be caused by a range of factors including dementia, delirium, substance withdrawal and psychiatric disturbance, creating challenges in diagnosis, often requiring a management plan with input from multiple disciplines. Staff forums and broader literature have confirmed that healthcare staff working in non-mental health settings, may not be as skilled in recognising and managing early signs of emerging and/or escalating clinical agitation. The BoC RRT is a consultation service within the Division of Medicine and CL Psychiatry. Staffed by Medical Registrars and Mental Health Nurses, the collaboration provides a unique healthcare response to acute general wards. The BoC RRT has been implemented to address the rising number of incidences whereby staff and patient safety are compromised. Using evidence-based skills the team aimed to: respond to episodes of clinical agitation that require an internal security response, assist ward referrals by exploring biopsychosocial contributants to behaviour, develop individual patient support plans and review and reduce restrictive intervention practices. Objectives To determine if the rapid response model has influenced: - The impact on staff/patient safety - Frequency of emergency responses for aggression - Frequency of restrictive intervention use Methods This project was approved as a quality assurance project (QA2022018). The patients within scope of the BoC RRT include inpatients in medical and surgical wards. It excludes patients in Emergency Departments, mental health units, outpatient clinics, and visitors. The evaluation of the pilot has used a PDSA (Plan, Do, Study, Act) cycle when implementing new improvements. A mixed methods approach explored the impact of the BoC RRT. Staff consultation will identify challenges in responding to scenarios whereby there is risk of harm to staff and patients. Staff feedback and the emergency response data was monitored. Results In 2021, there was approx. 720 code greys per month, requiring a security response. Since the implementation of BoC RRT, these numbers have reduced to 527. Reviewing restrictive intrvention practices has identified areas for policy review and need for education. Staff consultation found that nurses were confident caring for those patients exhibiting clinical agitation associated with delirium and dementia. However, caring for people with mental health or substance use disorders were more challenging. Conclusions These interim results indicate that BoC RRT has been generally well received by clinical staff. The decline in code grey responses indicates that it is likely having a positive impact in early identification and management of clinical agitation for hospital inpatients. There is support for this response model to continue beyond the pilot phase and further area for research. Disclosure of Interest None Declared

Abstract Background Cachexia is a debilitating complication of cancer characterized by progressive wasting and weakness of skeletal muscles that reduces quality of life and can compromise survival. Many anticancer treatments, such as chemotherapy, also cause muscle wasting, which impairs the response to treatment. Given that many cancer patients present with cachexia at the initiation of treatment, we investigated whether cachectic mice were susceptible to chemotherapy‐induced muscle wasting and to investigate contributing mechanisms, including the dysregulation of microRNAs (miRs). Methods Cachectic colon‐26 (C‐26) tumour‐bearing mice were given 5‐fluourouracil (5‐FU) chemotherapy or vehicle treatment and analysed for muscle mass, fibre size and composition, and miR expression. Mechanisms were validated in vitro using C2C12 cell culture and miR mimics and inhibitors and were confirmed in vivo by injecting muscles of 5‐FU‐treated cachectic mice with recombinant adeno‐associated viral (rAAV) vectors encoding a miR sponge. Results In cachectic tumour‐bearing mice, 5‐FU chemotherapy exacerbated the loss of skeletal muscle mass compared with vehicle treatment (by −12% to −20%, P

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

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.

Abstract Background Skeletal 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. Methods To elucidate if TGF‐β superfamily ligands regulate insulin action, we used an adeno‐associated virus gene editing approach to overexpress an 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). Results Fst288 muscle overexpression markedly increased in vivo insulin‐stimulated (but not basal) glucose uptake (+75%, P < 0.05) and increased protein expression and intracellular insulin signalling of AKT, TBC1D4, PAK1, pyruvate dehydrogenase‐E1α, and p70S6K, while decreasing TBC1D1 signaling (P < 0.05). Fst288 increased both basal and insulin‐stimulated protein synthesis, but no correlation was observed between the Fst288‐driven hypertrophy and the increase in insulin‐stimulated glucose uptake. Importantly, Fst288 completely normalized muscle glucose uptake in insulin‐resistant diet‐induced obese mice. RYGB surgery doubled circulating Fst and reduced activin A (−24%, P < 0.05) concentration 1 week after surgery before any significant weight loss in morbidly obese normoglycemic patients, while major weight loss after 1 year did not further change the concentrations. Conclusions We 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, pyruvate dehydrogenase‐E1α, and PAK1 as Fst targets. Circulating Fst more than doubled post‐RYGB surgery, a treatment that markedly improved insulin sensitivity, suggesting a role for Fst in regulating glycaemic control. 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.

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.

Diabetes is a major contributor to the increasing burden of heart failure prevalence globally, at least in part due to a disease process termed diabetic cardiomyopathy. Diabetic cardiomyopathy is characterised by cardiac structural changes that are caused by chronic exposure to the diabetic milieu. These structural changes are a major cause of left ventricular (LV) wall stiffness and the development of LV dysfunction. In the current study, we investigated the therapeutic potential of a cardiac-targeted bone morphogenetic protein 7 (BMP7) gene therapy, administered once diastolic dysfunction was present, mimicking the timeframe in which clinical management of the cardiomyopathy would likely be desired. Following 18 weeks of untreated diabetes, mice were administered with a single tail-vein injection of recombinant adeno-associated viral vector (AAV), containing the BMP7 gene, or null vector. Our data demonstrated, after 8 weeks of treatment, that rAAV6-BMP7 treatment exerted beneficial effects on LV functional and structural changes. Importantly, diabetes-induced LV dysfunction was significantly attenuated by a single administration of rAAV6-BMP7. This was associated with a reduction in cardiac fibrosis, cardiomyocyte hypertrophy and cardiomyocyte apoptosis. In conclusion, BMP7 gene therapy limited pathological remodelling in the diabetic heart, conferring an improvement in cardiac function. These findings provide insight for the potential development of treatment strategies urgently needed to delay or reverse LV pathological remodelling in the diabetic heart.

Summary: Long non-coding RNAs (lncRNAs) have been demonstrated to influence numerous biological processes, being strongly implicated in the maintenance and physiological function of various tissues including the heart. The lncRNA OIP5-AS1 (1700020I14Rik/Cyrano) has been studied in several settings; however its role in cardiac pathologies remains mostly uncharacterized. Using a series of in vitro and ex vivo methods, we demonstrate that OIP5-AS1 is regulated during cardiac development in rodent and human models and in disease settings in mice. Using CRISPR, we engineered a global OIP5-AS1 knockout (KO) mouse and demonstrated that female KO mice develop exacerbated heart failure following cardiac pressure overload (transverse aortic constriction [TAC]) but male mice do not. RNA-sequencing of wild-type and KO hearts suggest that OIP5-AS1 regulates pathways that impact mitochondrial function. Thus, these findings highlight OIP5-AS1 as a gene of interest in sex-specific differences in mitochondrial function and development of heart failure.

ABSTRACT 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.

Inhibition of myostatin- and activin-mediated SMAD2/3 signaling using ligand traps, such as soluble receptors, ligand-targeting propeptides and antibodies, or follistatin can increase skeletal muscle mass in healthy mice and ameliorate wasting in models of cancer cachexia and muscular dystrophy. However, clinical translation of these extracellular approaches targeting myostatin and activin has been hindered by the challenges of achieving efficacy without potential effects in other tissues. Toward the goal of developing tissue-specific myostatin/activin interventions, we explored the ability of transmembrane prostate androgen-induced (TMEPAI), an inhibitor of transforming growth factor-β (TGF-β1)-mediated SMAD2/3 signaling, to promote growth, and counter atrophy, in skeletal muscle. In this study, we show that TMEPAI can block activin A, activin B, myostatin and GDF-11 activity in vitro. To determine the physiological significance of TMEPAI, we employed Adeno-associated viral vector (AAV) delivery of a TMEPAI expression cassette to the muscles of healthy mice, which increased mass by as much as 30%, due to hypertrophy of muscle fibers. To demonstrate that TMEPAI mediates its effects via inhibition of the SMAD2/3 pathway, tibialis anterior (TA) muscles of mice were co-injected with AAV vectors expressing activin A and TMEPAI. In this setting, TMEPAI blocked skeletal muscle wasting driven by activin-induced phosphorylation of SMAD3. In a model of cancer cachexia associated with elevated circulating activin A, delivery of AAV:TMEPAI into TA muscles of mice bearing C26 colon tumors ameliorated the muscle atrophy normally associated with cancer progression. Collectively, the findings indicate that muscle-directed TMEPAI gene delivery can inactivate the activin/myostatin-SMAD3 pathway to positively regulate muscle mass in healthy settings and models 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. Author Video: Author Video Watch what authors say about their articles Keywords: Glucose metabolism, Enzyme activity, Insulin sensitivity

Cardiac fibrosis is associated with most forms of cardiovascular disease. No reliable therapies targeting cardiac fibrosis are available, thus identifying novel drugs that can resolve or prevent fibrosis is needed. Tilorone, an antiviral agent, can prevent fibrosis in a mouse model of lung disease. We investigated the anti-fibrotic effects of tilorone in human cardiac fibroblasts in vitro by performing a radioisotopic assay for [3H]-proline incorporation as a proxy for collagen synthesis. Exploratory studies in human cardiac fibroblasts treated with tilorone (10 µM) showed a significant reduction in transforming growth factor-β induced collagen synthesis compared to untreated fibroblasts. To determine if this finding could be recapitulated in vivo, mice with established pathological remodelling due to four weeks of transverse aortic constriction (TAC) were administered tilorone (50 mg/kg, i.p) or saline every third day for eight weeks. Treatment with tilorone was associated with attenuation of fibrosis (assessed by Masson’s trichrome stain), a favourable cardiac gene expression profile and no further deterioration of cardiac systolic function determined by echocardiography compared to saline treated TAC mice. These data demonstrate that tilorone has anti-fibrotic actions in human cardiac fibroblasts and the adult mouse heart, and represents a potential novel therapy to treat fibrosis associated with heart failure.

Drugs 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.

Heart failure (HF) is a debilitating and deadly chronic disease, with almost 50% of patients with HF dying within 5 years of diagnosis. With limited effective therapies to treat or cure HF, new therapies are greatly needed. microRNAs (miRNAs) are small non-coding RNA molecules that are powerful regulators of gene expression and play a key role in almost every biological process. Disruptions in miRNA gene expression has been functionally linked to numerous diseases, including cardiovascular disease. Molecular tools for manipulating miRNA activity have been developed, and there is evidence from preclinical studies demonstrating the potential of miRNAs to be therapeutic targets for cardiovascular disease. For clinical application, miRNA sponges and tough decoys have been developed for more stable suppression and targeted delivery of the miRNA of choice. The aim of this study was to generate miRNA sponges and tough decoys to target miR-34 in the mouse heart. We present data to show that using both approaches we were unable to get significant knockdown of miR-34 or regulate miR-34 target genes in the heart in vivo. We also review recent applications of this method in the heart and discuss further considerations for optimisation in construct design and testing, and the obstacles to be overcome before they enter the clinic.

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

Therapeutic inhibition of the miR-34 family (miR-34a,-b,-c), or miR-34a alone, have emerged as promising strategies for the treatment of cardiac pathology. However, before advancing these approaches further for potential entry into the clinic, a more comprehensive assessment of the therapeutic potential of inhibiting miR-34a is required for two key reasons. First, miR-34a has ∼40% fewer predicted targets than the miR-34 family. Hence, in cardiac stress settings in which inhibition of miR-34a provides adequate protection, this approach is likely to result in less potential off-target effects. Secondly, silencing of miR-34a alone may be insufficient in settings of established cardiac pathology. We recently demonstrated that inhibition of the miR-34 family, but not miR-34a alone, provided benefit in a chronic model of myocardial infarction. Inhibition of miR-34 also attenuated cardiac remodeling and improved heart function following pressure overload, however, silencing of miR-34a alone was not examined. The aim of this study was to assess whether inhibition of miR-34a could attenuate cardiac remodeling in a mouse model with pre-existing pathological hypertrophy. Mice were subjected to pressure overload via constriction of the transverse aorta for four weeks and echocardiography was performed to confirm left ventricular hypertrophy and systolic dysfunction. After four weeks of pressure overload (before treatment), two distinct groups of animals became apparent: (1) mice with moderate pathology (fractional shortening decreased ∼20%) and (2) mice with severe pathology (fractional shortening decreased ∼37%). Mice were administered locked nucleic acid (LNA)-antimiR-34a or LNA-control with an eight week follow-up. Inhibition of miR-34a in mice with moderate cardiac pathology attenuated atrial enlargement and maintained cardiac function, but had no significant effect on fetal gene expression or cardiac fibrosis. Inhibition of miR-34a in mice with severe pathology provided no therapeutic benefit. Thus, therapies that inhibit miR-34a alone may have limited potential in settings of established cardiac pathology.

BackgroundImpaired mitochondrial function is fundamental feature of heart failure (HF) and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO) metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury.Methods and resultsIn mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model) we demonstrated a marked reduction in L-arginine uptake (pConclusionThese data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury.

Muscles can be injured in different ways and the trauma and subsequent loss of function and physical capacity can impact significantly on the lives of patients through physical impairments and compromised quality of life. The relative success of muscle repair after injury will largely determine the extent of functional recovery. Unfortunately, regenerative processes are often slow and incomplete, and so developing novel strategies to enhance muscle regeneration is important. While the capacity to enhance muscle repair by stimulating β2-adrenoceptors (β-ARs) using β2-AR agonists (β2-agonists) has been demonstrated previously, the exact role β-ARs play in regulating the regenerative process remains unclear. To investigate β-AR-mediated signaling in muscle regeneration after myotoxic damage, we examined the regenerative capacity of tibialis anterior and extensor digitorum longus muscles from mice lacking either β1-AR (β1-KO) and/or β2-ARs (β2-KO), testing the hypothesis that muscles from mice lacking the β2-AR would exhibit impaired functional regeneration after damage compared with muscles from β1-KO or β1/β2-AR null (β1/β2-KO) KO mice. At 7 days post-injury, regenerating muscles from β1/β2-KO mice produced less force than those of controls but muscles from β1-KO or β2-KO mice did not exhibit any delay in functional restoration. Compared with controls, β1/β2-KO mice exhibited an enhanced inflammatory response to injury, which delayed early muscle regeneration, but an enhanced myoblast proliferation later during regeneration ensured a similar functional recovery (to controls) by 14 days post-injury. This apparent redundancy in the β-AR signaling pathway was unexpected and may have important implications for manipulating β-AR signaling to improve the rate, extent and efficacy of muscle regeneration to enhance functional recovery after injury.

microRNAs regulate the development of myogenic progenitors, and the formation of skeletal muscle fibers. However, the role miRNAs play in controlling the growth and adaptation of post-mitotic musculature is less clear. Here, we show that inhibition of the established pro-myogenic regulator miR-206 can promote hypertrophy and increased protein synthesis in post-mitotic cells of the myogenic lineage. We have previously demonstrated that histone deacetylase 4 (HDAC4) is a target of miR-206 in the regulation of myogenic differentiation. We confirmed that inhibition of miR-206 de-repressed HDAC4 accumulation in cultured myotubes. Importantly, inhibition of HDAC4 activity by valproic acid or sodium butyrate prevented hypertrophy of myogenic cells otherwise induced by inhibition of miR-206. To test the significance of miRNA-206 as a regulator of skeletal muscle mass in vivo, we designed recombinant adeno-associated viral vectors (rAAV6 vectors) expressing miR-206, or a miR-206 "sponge," featuring repeats of a validated miR-206 target sequence. We observed that over-expression or inhibition of miR-206 in the muscles of mice decreased or increased endogenous HDAC4 levels respectively, but did not alter muscle mass or myofiber size. We subsequently manipulated miR-206 levels in muscles undergoing follistatin-induced hypertrophy or denervation-induced atrophy (models of muscle adaptation where endogenous miR-206 expression is altered). Vector-mediated manipulation of miR-206 activity in these models of cell growth and wasting did not alter gain or loss of muscle mass respectively. Our data demonstrate that although the miR-206/HDAC4 axis operates in skeletal muscle, the post-natal expression of miR-206 is not a key regulator of basal skeletal muscle mass or specific modes of muscle growth and wasting. These studies support a context-dependent role of miR-206 in regulating hypertrophy that may be dispensable for maintaining or modifying the adult skeletal muscle phenotype--an important consideration in relation to the development of therapeutics designed to manipulate microRNA activity in musculature.

Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo.

Skeletal muscle nNOSmu (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSmu; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSmu. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSmu signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSmu expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention.

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. 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. 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 FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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 4 week 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 8 weeks 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 8 weeks, 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. [ABSTRACT FROM AUTHOR]

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 Ca2+ handling 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. [Display omitted] • Comparative muscle phosphoproteome of endurance, sprint, and resistance exercise • Identification of the canonical muscle exercise phosphoproteome • Identification of C18ORF25 as an AMPK substrate • Phosphorylation of C18ORF25 regulates skeletal muscle contractile function Blazev et al. characterized the signaling pathways regulated during three different types of exercise in human skeletal muscle. They identified the uncharacterized protein C18ORF25 as a target of AMPK and a new regulator of exercise capacity and skeletal muscle function. [ABSTRACT FROM AUTHOR]

The article discusses a study to identify a key regulator of exercise-induced protection and assess if it can reverse pathological remodeling. It builds a protocol where chronic exercise training protected mouse heart against subsequent cardiac insult. It suggests that phosphoinositide 3-kinase (P13K) is indispensable for exercise-induced cardiac protection and rAAV6-caP13K can improve cardiac function in mice with preexisting pressure overload-induced remodeling and cardiac dysfunction.

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. [ABSTRACT FROM AUTHOR]

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. 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. 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. 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. • 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. [ABSTRACT FROM AUTHOR]