AKT controls protein synthesis and oxidative metabolism via combined mTORC1 and FOXO1 signalling to govern muscle physiology.

BACKGROUND: Skeletomuscular diseases result in significant muscle loss and decreased performance, paralleled by a loss in mitochondrial and oxidative capacity. Insulin and insulin-like growth factor-1 (IGF-1) are two potent anabolic hormones that activate a host of signalling intermediates including the serine/threonine kinase AKT to influence skeletal muscle physiology. Defective AKT signalling is associated with muscle pathology, including cachexia, sarcopenia, and disuse; however, the mechanistic underpinnings remain unresolved. METHODS: To elucidate the role of AKT signalling in muscle mass and physiology, we generated both congenital and inducible mouse models of skeletal muscle-specific AKT deficiency. To understand the downstream mechanisms mediating AKT's effects on muscle biology, we generated mice lacking AKT1/2 and FOXO1 (M-AKTFOXO1TKO and M-indAKTFOXO1TKO) to inhibit downstream FOXO1 signalling, AKT1/2 and TSC1 (M-AKTTSCTKO and M-indAKTTSCTKO) to activate mTORC1, and AKT1/2, FOXO1, and TSC1 (M-QKO and M-indQKO) to simultaneously activate mTORC1 and inhibit FOXO1 in AKT-deficient skeletal muscle. Muscle proteostasis and physiology were assessed using multiple assays including metabolic labelling, mitochondrial function, fibre typing, ex vivo physiology, and exercise performance. RESULTS: Here, we show that genetic ablation of skeletal muscle AKT signalling resulted in decreased muscle mass and a loss of oxidative metabolism and muscle performance. Specifically, deletion of muscle AKT activity during development or in adult mice resulted in a significant reduction in muscle growth by 30-40% (P  < 0.0001; n = 12-20) and 15% (P < 0.01 and P < 0.0001; n = 20-30), respectively. Interestingly, this reduction in muscle mass was primarily due to an ~40% reduction in protein synthesis in both M-AKTDKO and M-indAKTDKO muscles (P < 0.05 and P < 0.01; n = 12-20) without significant changes in proteolysis or autophagy. Moreover, a significant reduction in oxidative capacity was observed in both M-AKTDKO (P < 0.05, P < 0.01 and P < 0.001; n = 5-12) and M-indAKTDKO (P < 0.05 and P < 0.01; n = 4). Mechanistically, activation and inhibition of mTORC1/FOXO1, respectively, but neither alone, were sufficient to restore protein synthesis, muscle oxidative capacity, and muscle function in the absence of AKT in vivo. In a mouse model of disuse-induced muscle loss, simultaneous activation of mTORC1 and inhibition of FOXO1 preserved muscle mass following immobilization (~5-10% reduction in casted M-indFOXO1TSCDKO muscles vs. ~30-40% casted M-indControl muscles, P < 0.05 and P < 0.0001; n = 8-16). CONCLUSIONS: Collectively, this study provides novel insights into the AKT-dependent mechanisms that underlie muscle protein homeostasis, function, and metabolism in both normal physiology and disuse-induced muscle wasting.

Persistent NF-κB activation in muscle stem cells induces proliferation-independent telomere shortening.

During the repeated cycles of damage and repair in many muscle disorders, including Duchenne muscular dystrophy (DMD), the muscle stem cell (MuSC) pool becomes less efficient at responding to and repairing damage. The underlying mechanism of such stem cell dysfunction is not fully known. Here, we demonstrate that the distinct early telomere shortening of diseased MuSCs in both mice and young DMD patients is associated with aberrant NF-κB activation. We find that prolonged NF-κB activation in MuSCs in chronic injuries leads to shortened telomeres and Ku80 dysregulation and results in severe skeletal muscle defects. Our studies provide evidence of a role for NF-κB in regulating stem-cell-specific telomere length, independently of cell replication, and could be a congruent mechanism that is applicable to additional tissues and/or diseases characterized by systemic chronic inflammation.

PMO-based let-7c site blocking oligonucleotide (SBO) mediated utrophin upregulation in mdx mice, a therapeutic approach for Duchenne muscular dystrophy (DMD).

Upregulation of utrophin, a dystrophin related protein, is considered a promising therapeutic approach for Duchenne muscular dystrophy (DMD). Utrophin expression is repressed at the post-transcriptional level by a set of miRNAs, among which let-7c is evolutionarily highly conserved. We designed PMO-based SBOs complementary to the let-7c binding site in UTRN 3'UTR, with the goal of inhibiting let-7c interaction with UTRN mRNA and thus upregulating utrophin. We used the C2C12UTRN5'luc3' reporter cell line in which the 5'- and 3'-UTRs of human UTRN sequences flank luciferase, for reporter assays and the C2C12 cell line for utrophin western blots, to independently evaluate the site blocking efficiency of a series of let-7c PMOs in vitro. Treatment of one-month old mdx mice with the most effective let-7c PMO (i.e. S56) resulted in ca. two-fold higher utrophin protein expression in skeletal muscles and the improvement in dystrophic pathophysiology in mdx mice, in vivo. In summary, we show that PMO-based let-7c SBO has potential applicability for upregulating utrophin expression as a therapeutic approach for DMD.

Genome Editing-Mediated Utrophin Upregulation in Duchenne Muscular Dystrophy Stem Cells.

Utrophin upregulation is considered a promising therapeutic strategy for Duchenne muscular dystrophy (DMD). A number of microRNAs (miRNAs) post-transcriptionally regulate utrophin expression by binding their cognate sites in the 3' UTR. Previously we have shown that miRNA: UTRN repression can be alleviated using miRNA let-7c site blocking oligonucleotides (SBOs) to achieve utrophin upregulation and functional improvement in mdx mice. Here, we used CRISPR/Cas9-mediated genome editing to delete five miRNA binding sites (miR-150, miR-296-5p, miR-133b, let-7c, miR-196b) clustered in a 500 bp inhibitory miRNA target region (IMTR) within the UTRN 3' UTR, for achieving higher expression of endogenous utrophin. Deleting the UTRN IMTR in DMD patient-derived human induced pluripotent stem cells (DMD-hiPSCs) resulted in ca. 2-fold higher levels of utrophin protein. Differentiation of the UTRN edited DMD-hiPSCs (UTRNΔIMTR) by MyoD overexpression resulted in increased sarcolemmal α-sarcoglycan staining consistent with improved dystrophin glycoprotein complex (DGC) restoration. These results demonstrate that CRISPR/Cas9-based UTRN genome editing offers a novel utrophin upregulation therapeutic strategy applicable to all DMD patients, irrespective of the dystrophin mutation status.

Tibetan PHD2, an allele with loss-of-function properties.

Tibetans have adapted to the chronic hypoxia of high altitude and display a distinctive suite of physiologic adaptations, including augmented hypoxic ventilatory response and resistance to pulmonary hypertension. Genome-wide studies have consistently identified compelling genetic signatures of natural selection in two genes of the Hypoxia Inducible Factor pathway, PHD2 and HIF2A The product of the former induces the degradation of the product of the latter. Key issues regarding Tibetan PHD2 are whether it is a gain-of-function or loss-of-function allele, and how it might contribute to high-altitude adaptation. Tibetan PHD2 possesses two amino acid changes, D4E and C127S. We previously showed that in vitro, Tibetan PHD2 is defective in its interaction with p23, a cochaperone of the HSP90 pathway, and we proposed that Tibetan PHD2 is a loss-of-function allele. Here, we report that additional PHD2 mutations at or near Asp-4 or Cys-127 impair interaction with p23 in vitro. We find that mice with the Tibetan Phd2 allele display augmented hypoxic ventilatory response, supporting this loss-of-function proposal. This is phenocopied by mice with a mutation in p23 that abrogates the PHD2:p23 interaction. Hif2a haploinsufficiency, but not the Tibetan Phd2 allele, ameliorates hypoxia-induced increases in right ventricular systolic pressure. The Tibetan Phd2 allele is not associated with hemoglobin levels in mice. We propose that Tibetans possess genetic alterations that both activate and inhibit selective outputs of the HIF pathway to facilitate successful adaptation to the chronic hypoxia of high altitude.

Functional effects of muscle PGC-1alpha in aged animals.

PGC-1 (peroxisome-proliferator-activated receptor-γ coactivator-1) alpha is a potent transcriptional coactivator that coordinates the activation of numerous metabolic processes. Exercise strongly induces PGC-1alpha expression in muscle, and overexpression of PGC-1alpha in skeletal muscle activates mitochondrial oxidative metabolism and neovascularization, leading to markedly increased endurance. In light of these findings, PGC-1alpha has been proposed to protect from age-associated sarcopenia, bone loss, and whole-body metabolic dysfunction, although these findings have been controversial. We therefore comprehensively evaluated muscle and whole-body function and metabolism in 24-month-old transgenic mice that over-express PGC-1alpha in skeletal muscle. We find that the powerful effects of PGC-1alpha on promoting muscle oxidative capacity and protection from muscle fatigability persist in aged animals, although at the expense of muscle strength. However, skeletal muscle PGC-1alpha does not prevent bone loss and in fact accentuates it, nor does it have long-term benefit on whole-body metabolic composition or insulin sensitivity. Protection from sarcopenia is seen in male animals with overexpression of PGC-1alpha in skeletal muscle but not in female animals. In summary, muscle-specific expression of PGC-1alpha into old age has beneficial effects on muscle fatigability and may protect from sarcopenia in males, but does not improve whole-body metabolism and appears to worsen age-related trabecular bone loss.

Author Correction: High-throughput identification of post-transcriptional utrophin up-regulators for Duchenne muscle dystrophy (DMD) therapy.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

High-throughput identification of post-transcriptional utrophin up-regulators for Duchenne muscle dystrophy (DMD) therapy.

Upregulation of endogenous utrophin offers great promise for treating DMD, as it can functionally compensate for the lack of dystrophin caused by DMD gene mutations, without the immunogenic concerns associated with delivering dystrophin. However, post-transcriptional repression mechanisms targeting the 5' and 3' untranslated regions (UTRs) of utrophin mRNA significantly limit the magnitude of utrophin upregulation achievable by promoter activation. Using a utrophin 5'3'UTR reporter assay, we performed a high-throughput screen (HTS) for small molecules capable of relieving utrophin post-transcriptional repression. We identified 27 hits that were ranked using a using an algorithm that we designed for hit prioritization that we call Hit to Lead Prioritization Score (H2LPS). The top 10 hits were validated using an orthogonal assay for endogenous utrophin expression. Evaluation of the top scoring hit, Trichostatin A (TSA), demonstrated utrophin upregulation and functional improvement in the mdx mouse model of DMD. TSA and the other small molecules identified here represent potential starting points for DMD drug discovery efforts.

New Insights into the Lactate Shuttle: Role of MCT4 in the Modulation of the Exercise Capacity.

Lactate produced by muscle during high-intensity activity is an important end product of glycolysis that supports whole body metabolism. The lactate shuttle model suggested that lactate produced by glycolytic muscle fibers is utilized by oxidative fibers. MCT4 is a proton coupled monocarboxylate transporter preferentially expressed in glycolytic muscle fibers and facilitates the lactate efflux. Here we investigated the exercise capacity of mice with disrupted lactate shuttle due to global deletion of MCT4 (MCT4-/-) or muscle-specific deletion of the accessory protein Basigin (iMSBsg-/-). Although MCT4-/- and iMSBsg-/- mice have normal muscle morphology and contractility, only MCT4-/- mice exhibit an exercise intolerant phenotype. In vivo measurements of compound muscle action potentials showed a decrement in the evoked response in the MCT4-/- mice. This was accompanied by a significant structural degeneration of the neuromuscular junctions (NMJs). We propose that disruption of the lactate shuttle impacts motor function and destabilizes the motor unit.

Effect of Interleukin-15 Receptor Alpha Ablation on the Metabolic Responses to Moderate Exercise Simulated by in vivo Isometric Muscle Contractions.

Lack of interleukin 15 receptor alpha (IL15RA) increases spontaneous activity, exercise capacity and protects from diet-induced obesity by enhancing muscle energy metabolism, suggesting a role as exercise mimetic for IL15RA antagonists. Using controlled in vivo muscle stimulation mimicking moderate exercise in normal and Il15ra-/- mice, we mapped and contrasted the metabolic pathways activated upon stimulation or deletion of IL15RA. Stimulation caused the differential regulation of 123 out of the 321 detected metabolites (FDR ≤ 0.05 and fold change ≥ ±1.5). The main energy pathways activated were fatty acid oxidation, nucleotide metabolism, and anaplerotic reactions. Notably, resting Il15ra-/- muscles were primed in a semi-exercised state, characterized by higher pool sizes of fatty acids oxidized to support muscle activity. These studies identify the role of IL15RA in the system-wide metabolic response to exercise and should enable translational studies to harness the potential of IL15RA blockade as a novel exercise mimetic strategy.

Non-immunogenic utrophin gene therapy for the treatment of muscular dystrophy animal models.

The essential product of the Duchenne muscular dystrophy (DMD) gene is dystrophin1, a rod-like protein2 that protects striated myocytes from contraction-induced injury3,4. Dystrophin-related protein (or utrophin) retains most of the structural and protein binding elements of dystrophin5. Importantly, normal thymic expression in DMD patients6 should protect utrophin by central immunologic tolerance. We designed a codon-optimized, synthetic transgene encoding a miniaturized utrophin (µUtro), deliverable by adeno-associated virus (AAV) vectors. Here, we show that µUtro is a highly functional, non-immunogenic substitute for dystrophin, preventing the most deleterious histological and physiological aspects of muscular dystrophy in small and large animal models. Following systemic administration of an AAV-µUtro to neonatal dystrophin-deficient mdx mice, histological and biochemical markers of myonecrosis and regeneration are completely suppressed throughout growth to adult weight. In the dystrophin-deficient golden retriever model, µUtro non-toxically prevented myonecrosis, even in the most powerful muscles. In a stringent test of immunogenicity, focal expression of µUtro in the deletional-null German shorthaired pointer model produced no evidence of cell-mediated immunity, in contrast to the robust T cell response against similarly constructed µDystrophin (µDystro). These findings support a model in which utrophin-derived therapies might be used to treat clinical dystrophin deficiency, with a favorable immunologic profile and preserved function in the face of extreme miniaturization.

The HDAC3 enzymatic activity regulates skeletal muscle fuel metabolism.

Histone deacetylase 3 (HDAC3) is a major HDAC, whose enzymatic activity is targeted by small molecule inhibitors for treating a variety of conditions. However, its enzymatic activity is largely dispensable for its function in embryonic development and hepatic lipid metabolism. HDAC3 plays a pivotal role in regulating muscle fuel metabolism and contractile function. Here, we address whether these muscular functions of HDAC3 require its enzymatic activity. By mutating the NCoR/SMRT corepressors in a knock-in mouse model named NS-DADm, we ablated the enzymatic activity of HDAC3 without affecting its protein levels. Compared to the control mice, skeletal muscles from NS-DADm mice showed lower force generation, enhanced fatigue resistance, enhanced fatty acid oxidation, reduced glucose uptake during exercise, upregulated expression of metabolic genes involved in branched-chain amino acids catabolism, and reduced muscle mass during aging, without changes in the muscle fiber-type composition or mitochondrial protein content. These muscular phenotypes are similar to those observed in the HDAC3-depleted skeletal muscles, which demonstrates that, unlike that in the liver or embryonic development, the metabolic function of HDAC3 in skeletal muscles requires its enzymatic activity. These results suggest that drugs specifically targeting HDAC3 enzyme activity could be developed and tested to modulate muscle energy metabolism and exercise performance.

Elongated mitochondrial constrictions and fission in muscle fatigue.

Mitochondria respond to stress and undergo fusion and fission at variable rates, depending on cell status. To understand mitochondrial behavior during muscle fatigue, we investigated mitochondrial ultrastructure and expression levels of a fission- and stress-related protein in fast-twitch muscle fibers of mice subjected to fatigue testing. Mice were subjected to running at increasing speed until exhaustion at 45 min-1 h. In further experiments, high-intensity muscle stimulation through the sciatic nerve simulated the forced treadmill exercise. We detected a rare phenotype characterized by elongated mitochondrial constrictions (EMCs) connecting two separate segments of the original organelles. EMCs are rare in resting muscles and their frequency increases, albeit still at low levels, in stimulated muscles. The constrictions are accompanied by elevated phosphorylation of Drp1 (Dnm1l) at Ser 616, indicating an increased translocation of Drp1 to the mitochondrial membrane. This is indicative of a mitochondrial stress response, perhaps leading to or facilitating a long-lasting fission event. A close apposition of sarcoplasmic reticulum (SR) to the constricted areas, detected using both transmission and scanning electron microscopy, is highly suggestive of SR involvement in inducing mitochondrial constrictions.

Mitochondrial ultrastructural adaptations in fast muscles of mice lacking IL15RA.

The pro-inflammatory cytokine interleukin-15 (IL15) and its receptor α (IL15RA) participate in the regulation of musculoskeletal function and metabolism. Deletion of the Il15ra gene in mice increases spontaneous activity, improves fatigue resistance in the glycolytic extensor digitorum longus (EDL) and protects from diet-induced obesity. In humans, IL15RA single-nucleotide polymorphisms (SNPs) have been linked to muscle strength, metabolism and performance in elite endurance athletes. Taken together, these features suggest a possible role for IL15RA in muscle mitochondrial structure and function. Here, we have investigated the consequences of loss of IL15RA on skeletal muscle fiber-type properties and mitochondrial ultrastructure. Immunostaining of the EDL for myosin heavy chain (MyHC) isoforms revealed no significant changes in fiber type. Electron microscopy (EM) analysis of the EDL indicated an overall higher mitochondria content, and increased cristae density in subsarcolemmal and A-band mitochondrial subpopulations. The higher cristae density in Il15ra-/- mitochondria was associated with higher OPA1 and cardiolipin levels. Overall, these data extend our understanding of the role of IL15RA signaling in muscle oxidative metabolism and adaptation to exercise.

Functional improvement of dystrophic muscle by repression of utrophin: let-7c interaction.

Duchenne muscular dystrophy (DMD) is a fatal genetic disease caused by an absence of the 427kD muscle-specific dystrophin isoform. Utrophin is the autosomal homolog of dystrophin and when overexpressed, can compensate for the absence of dystrophin and rescue the dystrophic phenotype of the mdx mouse model of DMD. Utrophin is subject to miRNA mediated repression by several miRNAs including let-7c. Inhibition of utrophin: let-7c interaction is predicted to 'repress the repression' and increase utrophin expression. We developed and tested the ability of an oligonucleotide, composed of 2'-O-methyl modified bases on a phosphorothioate backbone, to anneal to the utrophin 3'UTR and prevent let-7c miRNA binding, thereby upregulating utrophin expression and improving the dystrophic phenotype in vivo. Suppression of utrophin: let-7c interaction using bi-weekly intraperitoneal injections of let7 site blocking oligonucleotides (SBOs) for 1 month in the mdx mouse model for DMD, led to increased utrophin expression along with improved muscle histology, decreased fibrosis and increased specific force. The functional improvement of dystrophic muscle achieved using let7-SBOs suggests a novel utrophin upregulation-based therapeutic strategy for DMD.

In Vivo Evaluation of the Mechanical and Viscoelastic Properties of the Rat Tongue.

The tongue is a highly innervated and vascularized muscle hydrostat on the floor of the mouth of most vertebrates. Its primary functions include supporting mastication and deglutition, as well as taste-sensing and phonetics. Accordingly, the strength and volume of the tongue can impact the ability of vertebrates to accomplish basic activities such as feeding, communicating, and breathing. Human patients with sleep apnea have enlarged tongues, characterized by reduced muscle tone and increased intramuscular fat that can be visualized and quantified by magnetic resonance imaging (MRI). The abilities to measure force generation and viscoelastic properties of the tongue constitute important tools for obtaining functional information to correlate with imaging data. Here, we present techniques for measuring tongue force production in anesthetized Zucker rats via electrical stimulation of the hypoglossal nerves and for determining the viscoelastic properties of the tongue by applying passive Lissajous force/deformation curves.

IL15RA is required for osteoblast function and bone mineralization.

Interleukin-15 receptor alpha (IL15RA) is an important component of interleukin-15 (IL15) pro-inflammatory signaling. In addition, IL15 and IL15RA are present in the circulation and are detected in a variety of tissues where they influence physiological functions such as muscle contractility and overall metabolism. In the skeletal system, IL15RA was previously shown to be important for osteoclastogenesis. Little is known, however, about its role in osteoblast function and bone mineralization. In this study, we evaluated bone structural and mechanical properties of an Il15ra whole-body knockout mouse (Il15ra-/-) and used in vitro and bioinformatic analyses to understand the role IL15/IL15RA signaling on osteoblast function. We show that lack of IL15RA decreased bone mineralization in vivo and in isolated primary osteogenic cultures, suggesting a cell-autonomous effect. Il15ra-/- osteogenic cultures also had reduced Rankl/Opg mRNA ratio, indicating defective osteoblast/osteoclast coupling. We analyzed the transcriptome of primary pre-osteoblasts from normal and Il15ra-/- mice and identified 1150 genes that were differentially expressed at a FDR of 5%. Of these, 844 transcripts were upregulated and 306 were downregulated in Il15ra-/- cells. The largest functional clusters, highlighted using DAVID analysis, were related to metabolism, immune response, bone mineralization and morphogenesis. The transcriptome analysis was validated by qPCR of some of the most significant hits. Using bioinformatic approaches, we identified candidate genes, including Cd200 and Enpp1, that could contribute to the reduced mineralization. Silencing Il15ra using shRNA in the calvarial osteoblast MC3T3-E1 cell line decreased ENPP1 activity. Taken together, these data support that IL15RA plays a cell-autonomous role in osteoblast function and bone mineralization.

Cyclin D1 Restrains Oncogene-Induced Autophagy by Regulating the AMPK-LKB1 Signaling Axis.

Autophagy activated after DNA damage or other stresses mitigates cellular damage by removing damaged proteins, lipids, and organelles. Activation of the master metabolic kinase AMPK enhances autophagy. Here we report that cyclin D1 restrains autophagy by modulating the activation of AMPK. In cell models of human breast cancer or in a cyclin D1-deficient model, we observed a cyclin D1-mediated reduction in AMPK activation. Mechanistic investigations showed that cyclin D1 inhibited mitochondrial function, promoted glycolysis, and reduced activation of AMPK (pT172), possibly through a mechanism that involves cyclin D1-Cdk4/Cdk6 phosphorylation of LKB1. Our findings suggest how AMPK activation by cyclin D1 may couple cell proliferation to energy homeostasis. Cancer Res; 77(13); 3391-405. ©2017 AACR.

Dissociation of muscle insulin sensitivity from exercise endurance in mice by HDAC3 depletion.

Type 2 diabetes and insulin resistance are associated with reduced glucose utilization in the muscle and poor exercise performance. Here we find that depletion of the epigenome modifier histone deacetylase 3 (HDAC3) specifically in skeletal muscle causes severe systemic insulin resistance in mice but markedly enhances endurance and resistance to muscle fatigue, despite reducing muscle force. This seemingly paradoxical phenotype is due to lower glucose utilization and greater lipid oxidation in HDAC3-depleted muscles, a fuel switch caused by the activation of anaplerotic reactions driven by AMP deaminase 3 (Ampd3) and catabolism of branched-chain amino acids. These findings highlight the pivotal role of amino acid catabolism in muscle fatigue and type 2 diabetes pathogenesis. Further, as genome occupancy of HDAC3 in skeletal muscle is controlled by the circadian clock, these results delineate an epigenomic regulatory mechanism through which the circadian clock governs skeletal muscle bioenergetics. These findings suggest that physical exercise at certain times of the day or pharmacological targeting of HDAC3 could potentially be harnessed to alter systemic fuel metabolism and exercise performance.