TAK1 regulates skeletal muscle mass and mitochondrial function

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Abstract

Skeletal muscle mass is regulated by a complex array of signaling pathways. TGF-β–activated kinase 1 (TAK1) is an important signaling protein, which regulates context-dependent activation of multiple intracellular pathways. However, the role of TAK1 in the regulation of skeletal muscle mass remains unknown. Here, we report that inducible inactivation of TAK1 causes severe muscle wasting, leading to kyphosis, in both young and adult mice.. Inactivation of TAK1 inhibits protein synthesis and induces proteolysis, potentially through upregulating the activity of the ubiquitin-proteasome system and autophagy. Phosphorylation and enzymatic activity of AMPK are increased, whereas levels of phosphorylated mTOR and p38 MAPK are diminished upon inducible inactivation of TAK1 in skeletal muscle. In addition, targeted inactivation of TAK1 leads to the accumulation of dysfunctional mitochondria and oxidative stress in skeletal muscle of adult mice. Inhibition of TAK1 does not attenuate denervation-induced muscle wasting in adult mice. Finally, TAK1 activity is highly upregulated during overload-induced skeletal muscle growth, and inactivation of TAK1 prevents myofiber hypertrophy in response to functional overload. Overall, our study demonstrates that TAK1 is a key regulator of skeletal muscle mass and oxidative metabolism.

Authors

Sajedah M. Hindi, Shuichi Sato, Guangyan Xiong, Kyle R. Bohnert, Andrew A. Gibb, Yann S. Gallot, Joseph D. McMillan, Bradford G. Hill, Shizuka Uchida, Ashok Kumar

Mutations causing congenital myasthenia reveal principal coupling pathway in the acetylcholine receptor ε-subunit

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Abstract

We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the β8-β9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between εR218 and εE184, and between εR218 and εE45 from the β1-β2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling between conserved residues within the principal coupling pathway of the α-subunit, but also between corresponding residues in the ε-subunit.

Authors

Xin-Ming Shen, Joan M. Brengman, Shelley Shen, Hacer Durmus, Veeramani Preethish-Kumar, Nur Yuceyar, Seena Vengalil, Atchayaram Nalini, Feza Deymeer, Steven M. Sine, Andrew G. Engel

Functional correction of dystrophin actin binding domain mutations by genome editing

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Abstract

Dystrophin maintains the integrity of striated muscles by linking the actin cytoskeleton with the cell membrane. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene (DMD) that result in progressive, debilitating muscle weakness, cardiomyopathy, and a shortened lifespan. Mutations of dystrophin that disrupt the amino-terminal actin-binding domain 1 (ABD-1), encoded by exons 2–8, represent the second-most common cause of DMD. In the present study, we compared three different strategies for CRISPR/Cas9 genome editing to correct mutations in the ABD-1 region of the DMD gene by deleting exons 3–9, 6–9, or 7–11 in human induced pluripotent stem cells (iPSCs) and by assessing the function of iPSC-derived cardiomyocytes. All three exon deletion strategies enabled the expression of truncated dystrophin protein and restoration of cardiomyocyte contractility and calcium transients to varying degrees. We show that deletion of exons 3–9 by genomic editing provides an especially effective means of correcting disease-causing ABD-1 mutations. These findings represent an important step toward eventual correction of common DMD mutations and provide a means of rapidly assessing the expression and function of internally truncated forms of dystrophin-lacking portions of ABD-1.

Authors

Viktoriia Kyrychenko, Sergii Kyrychenko, Malte Tiburcy, John M. Shelton, Chengzu Long, Jay W. Schneider, Wolfram-Hubertus Zimmermann, Rhonda Bassel-Duby, Eric N. Olson

Physiological adaptations to resistance exercise as a function of age

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Abstract

BACKGROUND. The impact of resistance exercise training (RE-T) across the life span is poorly defined. METHODS. To resolve this, we recruited three distinct age cohorts of young (18–28 years; n = 11), middle-aged (45–55 years; n = 20), and older (nonsarcopenic; 65–75 years; n = 17) individuals to a cross-sectional intervention study. All subjects participated in 20 weeks of fully supervised whole-body progressive RE-T, undergoing assessment of body composition, muscle and vascular function, and metabolic health biomarkers before and after RE-T. Individuals also received stable isotope tracer infusions to ascertain muscle protein synthesis (MPS). RESULTS. There was an age-related increase in adiposity, but only young and middle-age groups demonstrated reductions following RE-T. Increases in blood pressure with age were attenuated by RE-T in middle-aged, but not older, individuals, while age-related increases in leg vascular conductance were unaffected by RE-T. The index of insulin sensitivity was reduced by RE-T in older age. Despite being matched at baseline, only younger individuals increased muscle mass in response to RE-T, and there existed a negative correlation between age and muscle growth; in contrast, increases in mechanical quality were preserved across ages. Acute increases in MPS (upon feeding plus acute RE-T) were enhanced only in younger individuals, perhaps explaining greater hypertrophy. CONCLUSION. Our data indicate that RE-T offsets some, but not all, negative characteristics of ageing — some of which are apparent in midlife. FUNDING. Biotechnology and Biological Sciences Research Council (BB/C516779/1).

Authors

Bethan E. Phillips, John P. Williams, Paul L. Greenhaff, Kenneth Smith, Philip J. Atherton

MicroRNA-29 overexpression by adeno-associated virus suppresses fibrosis and restores muscle function in combination with micro-dystrophin

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Abstract

Duchenne muscular dystrophy (DMD) is caused by dystrophin deficiency resulting in progressive muscle weakness and fibrotic scarring. Muscle fibrosis impairs blood flow, hampering muscle repair and regeneration. Irrespective of the success of gene restoration, functional improvement is limited without reducing fibrosis. The levels of miR-29c, a known regulator of collagen, are reduced in DMD. Our goal is to develop translational, antifibrotic therapy by overexpressing miR-29c. We injected the gastrocnemius muscle with either self-complementary AAV.CMV.miR-29c or single-stranded AAV.MCK.micro-dystrophin alone or in combination in the mdx/utrn^+/– mouse, a DMD mouse model. Treatment of 3-month-old mdx/utrn^+/– mice with AAV.miR-29c showed a reduction in collagen and increased absolute and specific force compared with untreated animals, but neither parameter reached WT levels. Combinatorial gene delivery in 3-month-old mdx/utrn^+/– mice further decreased fibrosis, and showed a reduction of transcript levels for Col1A, Col3A, fibronectin, and Tgfb1. In addition, absolute and specific force was normalized and equivalent to WT. However, protection against eccentric contraction fell short of WT levels at this time point. When this same mouse model was treated with miR-29c/micro-dystrophin combinatorial therapy at 1 month of age, there was complete normalization of specific and absolute force and protection against eccentric contraction–induced injury was comparable to WT. These findings highlight the potential for miR-29c as an important addition to the armamentarium for translational gene therapy, especially when used in combination with micro-dystrophin in DMD.

Authors

Kristin N. Heller, Joshua T. Mendell, Jerry R. Mendell, Louise R. Rodino-Klapac

Muscular dystrophy in PTFR/cavin-1 null mice

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Abstract

ice and humans lacking the caveolae component polymerase I transcription release factor (PTRF, also known as cavin-1) exhibit lipo- and muscular dystrophy. Here we describe the molecular features underlying the muscle phenotype for PTRF/cavin-1 null mice. These animals had a decreased ability to exercise, and exhibited muscle hypertrophy with increased muscle fiber size and muscle mass due, in part, to constitutive activation of the Akt pathway. Their muscles were fibrotic and exhibited impaired membrane integrity accompanied by an apparent compensatory activation of the dystrophin-glycoprotein complex along with elevated expression of proteins involved in muscle repair function. Ptrf deletion also caused decreased mitochondrial function, oxygen consumption, and altered myofiber composition. Thus, in addition to compromised adipocyte-related physiology, the absence of PTRF/cavin-1 in mice caused a unique form of muscular dystrophy with a phenotype similar or identical to that seen in humans lacking this protein. Further understanding of this muscular dystrophy model will provide information relevant to the human situation and guidance for potential therapies.

Authors

Shi-Ying Ding, Libin Liu, Paul F. Pilch

Deletion of neuropilin 2 enhances detrusor contractility following bladder outlet obstruction

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Abstract

Chronic urethral obstruction and the ensuing bladder wall remodeling can lead to diminished bladder smooth muscle (BSM) contractility and debilitating lower urinary tract symptoms. No effective pharmacotherapy exists to restore BSM contractile function. Neuropilin 2 (Nrp2) is a transmembrane protein that is highly expressed in BSM. Nrp2 deletion in mice leads to increased BSM contraction. We determined whether genetic ablation of Nrp2 could restore BSM contractility following obstruction. Partial bladder outlet obstruction (pBOO) was created by urethral occlusion in mice with either constitutive and ubiquitous, or inducible smooth muscle–specific deletion of Nrp2, and Nrp2-intact littermates. Mice without obstruction served as additional controls. Contractility was measured by isometric tension testing. Nrp2 deletion prior to pBOO increased force generation in BSM 4 weeks following surgery. Deletion of Nrp2 in mice already subjected to pBOO for 4 weeks showed increased contractility of tissues tested 6 weeks after surgery compared with nondeleted controls. Assessment of tissues from patients with urodynamically defined bladder outlet obstruction revealed reduced NRP2 levels in obstructed bladders with compensated compared with decompensated function, relative to asymptomatic controls. We conclude that downregulation of Nrp2 promotes BSM force generation. Neuropilin 2 may represent a novel target to restore contractility following obstruction.

Authors

Evalynn Vasquez, Vivian Cristofaro, Stefan Lukianov, Fiona C. Burkhard, Ali Hashemi Gheinani, Katia Monastyrskaya, Diane R. Bielenberg, Maryrose P. Sullivan, Rosalyn M. Adam

Characterization of miRNA-regulated networks, hubs of signaling, and biomarkers in obstruction-induced bladder dysfunction

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Abstract

Bladder outlet obstruction (BOO) induces significant organ remodeling, leading to lower urinary tract symptoms accompanied by urodynamic changes in bladder function. Here, we report mRNA and miRNA transcriptome sequencing of bladder samples from human patients with different urodynamically defined states of BOO. Patients’ miRNA and mRNA expression profiles correlated with urodynamic findings. Validation of RNA sequencing results in an independent patient cohort identified combinations of 3 mRNAs (NRXN3, BMP7, UPK1A) and 3 miRNAs (miR-103a-3p, miR-10a-5p, miR-199a-3p) sufficient to discriminate between bladder functional states. All BOO patients shared cytokine and immune response pathways, TGF-β and NO signaling pathways, and hypertrophic PI3K/AKT signaling pathways. AP-1 and NFkB were dominant transcription factors, and TNF-α was the top upstream regulator. Integrated miRNA-mRNA expression analysis identified pathways and molecules targeted by differentially expressed miRNAs. Molecular changes in BOO suggest an increasing involvement of miRNAs in the control of bladder function from the overactive to underactive/acontractile states.

Authors

Ali Hashemi Gheinani, Bernhard Kiss, Felix Moltzahn, Irene Keller, Rémy Bruggmann, Hubert Rehrauer, Catharine Aquino Fournier, Fiona C. Burkhard, Katia Monastyrskaya

Disease-modifying effects of orally bioavailable NF-κB inhibitors in dystrophin-deficient muscle

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Abstract

Duchenne muscular dystrophy (DMD) is a devastating muscle disease characterized by progressive muscle deterioration and replacement with an aberrant fatty, fibrous matrix. Chronic upregulation of nuclear factor κB (NF-κB) is implicated as a driver of the dystrophic pathogenesis. Herein, 2 members of a novel class of NF-κB inhibitors, edasalonexent (formerly CAT-1004) and CAT-1041, were evaluated in both mdx mouse and golden retriever muscular dystrophy (GRMD) dog models of DMD. These orally bioavailable compounds consist of a polyunsaturated fatty acid conjugated to salicylic acid and potently suppress the pathogenic NF-κB subunit p65/RelA in vitro. In vivo, CAT-1041 effectively improved the phenotype of mdx mice undergoing voluntary wheel running, in terms of activity, muscle mass and function, damage, inflammation, fibrosis, and cardiac pathology. We identified significant increases in dysferlin as a possible contributor to the protective effect of CAT-1041 to sarcolemmal damage. Furthermore, CAT-1041 improved the more severe GRMD phenotype in a canine case study, where muscle mass and diaphragm function were maintained in a treated GRMD dog. These results demonstrate that NF-κB modulation by edasalonexent and CAT-1041 is effective in ameliorating the dystrophic process and these compounds are candidates for new treatments for DMD patients.

Authors

David W. Hammers, Margaret M. Sleeper, Sean C. Forbes, Cora C. Coker, Michael R. Jirousek, Michael Zimmer, Glenn A. Walter, H. Lee Sweeney

Maternal obesity reduces oxidative capacity in fetal skeletal muscle of Japanese macaques

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Abstract

Maternal obesity is proposed to alter the programming of metabolic systems in the offspring, increasing the risk for developing metabolic diseases; however, the cellular mechanisms remain poorly understood. Here, we used a nonhuman primate model to examine the impact of a maternal Western-style diet (WSD) alone, or in combination with obesity (Ob/WSD), on fetal skeletal muscle metabolism studied in the early third trimester. We find that fetal muscle responds to Ob/WSD by upregulating fatty acid metabolism, mitochondrial complex activity, and metabolic switches (CPT-1, PDK4) that promote lipid utilization over glucose oxidation. Ob/WSD fetuses also had reduced mitochondrial content, diminished oxidative capacity, and lower mitochondrial efficiency in muscle. The decrease in oxidative capacity and glucose metabolism was persistent in primary myotubes from Ob/WSD fetuses despite no additional lipid-induced stress. Switching obese mothers to a healthy diet prior to pregnancy did not improve fetal muscle mitochondrial function. Lastly, while maternal WSD alone led only to intermediary changes in fetal muscle metabolism, it was sufficient to increase oxidative damage and cellular stress. Our findings suggest that maternal obesity or WSD, alone or in combination, leads to programmed decreases in oxidative metabolism in offspring muscle. These alterations may have important implications for future health.

Authors

Carrie E. McCurdy, Simon Schenk, Byron Hetrick, Julie Houck, Brian G. Drew, Spencer Kaye, Melanie Lashbrook, Bryan C. Bergman, Diana L. Takahashi, Tyler A. Dean, Travis Nemkov, Ilya Gertsman, Kirk C. Hansen, Andrew Philp, Andrea L. Hevener, Adam J. Chicco, Kjersti M. Aagaard, Kevin L. Grove, Jacob E. Friedman