Muscle biology
Abstract
The mechanisms that modulate the kinetics of muscle relaxation are critically important for muscle function. A prime example of the impact of impaired relaxation kinetics is nemaline myopathy caused by mutations in KBTBD13 (NEM6). In addition to weakness, NEM6 patients have slow muscle relaxation, compromising contractility and daily-life activities. The role of KBTBD13 in muscle is unknown, and the pathomechanism underlying NEM6 is undetermined. A combination of transcranial magnetic stimulation-induced muscle relaxation, muscle fiber- and sarcomere-contractility assays, low angle X-ray diffraction and super-resolution microscopy revealed that the impaired muscle relaxation kinetics in NEM6 patients are caused by structural changes in the thin filament, a sarcomeric microstructure. Using homology modeling, binding- and contractility assays with recombinant KBTBD13, novel Kbtbd13-knockout and Kbtbd13R408C-knockin mouse models and a GFP-labeled Kbtbd13- transgenic zebrafish model we discovered that KBTBD13 binds to actin – a major constituent of the thin filament - and that mutations in KBTBD13 cause structural changes impairing muscle relaxation kinetics. We propose that this actin-based impaired relaxation is central to NEM6 pathology.
Authors
Josine M. de Winter, Joery P. Molenaar, Michaela Yuen, Robbert van der Pijl, Shengyi Shen, Stefan Conijn, Martijn van de Locht, Menne Willigenburg, Sylvia J.P. Bogaards, Esmee S.B. van Kleef, Saskia Lassche, Malin Persson, Dilson E. Rassier, Tamar E. Sztal, Avnika A. Ruparelia, Viola Oorschot, Georg Ramm, Thomas E. Hall, Zherui Xiong, Christopher N. Johnson, Frank Li, Balazs Kiss, Noelia Lozano-Vidal, Reinier A. Boon, Manuela Marabita, Leonardo Nogara, Bert Blaauw, Richard J. Rodenburg, Benno Kϋsters, Jonne Doorduin, Alan H. Beggs, Henk Granzier, Ken Campbell, Weikang Ma, Thomas Irving, Edoardo Malfatti, Norma B. Romero, Robert J. Bryson-Richardson, Baziel G.M. van Engelen, Nicol C. Voermans, Coen A.C. Ottenheijm
Abstract
Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated Ca2+-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, we visualized annexin cap formation during injury. We found that annexin cap size positively correlated with increasing Ca2+ concentrations. We also found that annexin overexpression promoted external blebs enriched in Ca2+ and correlated with a reduction of intracellular Ca2+ at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identify annexins as mediators of membrane-associated Ca2+ release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity.
Authors
Alexis R. Demonbreun, Katherine S. Fallon, Claire C. Oosterbaan, Elena Bogdanovic, James L. Warner, Jordan J. Sell, Patrick G. Page, Mattia Quattrocelli, David Y. Barefield, Elizabeth M. McNally
Abstract
Myocardin (MYOCD) is the founding member of a class of transcriptional co-activators that bind serum response factor to activate gene expression programs critical in smooth muscle (SM) and cardiac muscle development. Insights into the molecular functions of MYOCD have been obtained from cell culture studies and, to date, knowledge about in vivo roles of MYOCD comes exclusively from experimental animals. Here, we defined an often lethal congenital human disease associated with inheritance of pathogenic MYOCD variants. This disease manifested as a massively dilated urinary bladder, or megabladder, with disrupted SM in its wall. We provided evidence that monoallelic loss-of-function variants in MYOCD caused congenital megabladder in males only, whereas biallelic variants were associated with disease in both sexes, with a phenotype additionally involving the cardiovascular system. These results were supported by co-segregation of MYOCD variants with the phenotype in four unrelated families, by in vitro transactivation studies where pathogenic variants resulted in abrogated SM gene expression, and finding megabladder in two distinct mouse models with reduced Myocd activity. In conclusion, we have demonstrated that variants in MYOCD result in human disease, and the collective findings highlight a vital role for MYOCD in mammalian organogenesis.
Authors
Arjan C. Houweling, Glenda M. Beaman, Alex V. Postma, T. Blair Gainous, Klaske D. Lichtenbelt, Francesco Brancati, Filipa M. Lopes, Ingeborg van der Made, Abeltje M. Polstra, Michael L. Robinson, Kevin D. Wright, Jamie M. Ellingford, Ashley R. Jackson, Eline Overwater, Rita Genesio, Silvio Romano, Letizia Camerota, Emanuela D'Angelo, Elizabeth J. Meijers-Heijboer, Vincent M. Christoffels, Kirk M. McHugh, Brian L. Black, William G. Newman, Adrian S. Woolf, Esther E. Creemers
Abstract
Receptor activator of Nfkb ligand (RANKL) activates, while osteoprotegerin (OPG) inhibits, osteoclastogenesis. In turn a neutralizing Ab against RANKL, denosumab improves bone strength in osteoporosis. OPG also improves muscle strength in mouse models of Duchenne’s muscular dystrophy (mdx) and denervation-induce atrophy, but its role and mechanisms of action on muscle weakness in other conditions remains to be investigated. We investigated the effects of RANKL inhibitors on muscle in osteoporotic women and mice that either overexpress RANKL (HuRANKL-Tg+), or lack Pparb and concomitantly develop sarcopenia (Pparb-/-). In women, denosumab over 3 years improved appendicular lean mass and handgrip strength compared to no treatment, whereas bisphosphonate did not. HuRANKL-Tg+ mice displayed lower limb force and maximal speed, while their leg muscle mass was diminished, with a lower number of type I and II fibers. Both OPG and denosumab increased limb force proportionally to the increase in muscle mass. They markedly improved muscle insulin sensitivity and glucose uptake, and decrease anti-myogenic and inflammatory gene expression in muscle, such as myostatin and protein tyrosine phosphatase receptor-γ. Similarly, in Pparb-/-, OPG increased muscle volume and force, while also normalizing their insulin signaling and higher expression of inflammatory genes in skeletal muscle. In conclusions, RANKL deteriorates, while its inhibitor improves, muscle strength and insulin sensitivity in osteoporotic mice and humans. Hence denosumab could represent a novel therapeutic approach for sarcopenia.
Authors
Nicolas Bonnet, Lucie Bourgoin, Emmanuel Biver, Eleni Douni, Serge Ferrari
Abstract
Aside from its catalytic function in protein synthesis, leucyl-tRNA synthetase (LRS) has a nontranslational function in regulating cell growth via the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway by sensing amino acid availability. mTOR also regulates skeletal myogenesis, but the signaling mechanism is distinct from that in cell growth regulation. A role of LRS in myogenesis has not been reported. Here we report that LRS negatively regulated myoblast differentiation in vitro. This function of LRS was independent of its regulation of protein synthesis, and it required leucine-binding but not tRNA charging activity of LRS. Local knock down of LRS accelerated muscle regeneration in a mouse injury model, and so did the knock down of Rag or Raptor. Further in vitro studies established a Rag-mTORC1 pathway, which inhibits the IRS1-PI3K-Akt pathway, to be the mediator of the nontranslational function of LRS in myogenesis. BC-LI-0186, an inhibitor reported to disrupt LRS-Rag interaction, promoted robust muscle regeneration with enhanced functional recovery, and this effect was abolished by cotreatment with an Akt inhibitor. Taken together, our findings revealed what we believe is a novel function for LRS in controlling the homeostasis of myogenesis, and suggested a potential therapeutic strategy to target a noncanonical function of a housekeeping protein.
Authors
Kook Son, Jae-Sung You, Mee-Sup Yoon, Chong Dai, Jong Hyun Kim, Nidhi Khanna, Aditi Banerjee, Susan A. Martinis, Gyoonhee Han, Jung Min Han, Sunghoon Kim, Jie Chen
Abstract
Ca2+ channel β-subunit interactions with pore-forming α-subunits are long-thought to be obligatory for channel trafficking to the cell surface and for tuning of basal biophysical properties in many tissues. Unexpectedly, we demonstrate that transgenic expression of mutant cardiac α1C subunits lacking capacity to bind CaVβ because of alanine-substitutions of three conserved residues — Y467, W470, and I471 in the α-interaction domain of rabbit α1C — can traffic to the sarcolemma in adult cardiomyocytes in vivo and sustain normal excitation-contraction coupling. However, these β-less Ca2+ channels cannot be stimulated by β-adrenergic pathway agonists, and thus adrenergic-augmentation of contractility is markedly impaired in isolated cardiomyocytes and in hearts. Similarly, viral-mediated expression of a β-subunit-sequestering-peptide sharply curtailed β-adrenergic stimulation of wild-type Ca2+ channels, identifying an approach to specifically modulate β-adrenergic regulation of cardiac contractility. Our data demonstrate that β subunits are required for β-adrenergic regulation of CaV1.2 channels and positive inotropy in the heart, but are dispensable for CaV1.2 trafficking to the adult cardiomyocyte cell surface, and for basal function and excitation-contraction coupling.
Authors
Lin Yang, Alexander Katchman, Jared S. Kushner, Alexander Kushnir, Sergey I. Zakharov, Bi-xing Chen, Zunaira Shuja, Prakash Subramanyam, Guoxia Liu, Arianne Papa, Daniel D. Roybal, Geoffrey S. Pitt, Henry M. Colecraft, Steven O. Marx
Abstract
Cardiac two pore domain potassium channels (K2P) exist in organisms from Drosophila to humans, however their role in cardiac function is not known. We identified a K2P gene, CG8713 (sandman), in a Drosophila genetic screen and show that sandman is critical to cardiac function. Mice lacking an ortholog of sandman, TWIK related potassium channel (TREK-1 or Kcnk2), exhibit exaggerated pressure overload induced concentric hypertrophy and alterations in fetal gene expression, yet retain preserved systolic and diastolic cardiac function. While cardiomyocyte specific deletion of TREK-1 in response to in vivo pressure overload resulted in cardiac dysfunction, TREK-1 deletion in fibroblasts prevented deterioration in cardiac function. The absence of pressure overload induced dysfunction in TREK-1 KO mice was associated with diminished cardiac fibrosis and reduced activation of c-Jun N-terminal kinase activity (JNK) in cardiomyocytes and fibroblasts. These findings indicate a central role for cardiac fibroblast TREK-1 in the pathogenesis of pressure overload-induced cardiac dysfunction and serve as a conceptual basis for its inhibition for as a potential therapy.
Authors
Dennis M. Abraham, Teresa E. Lee, Lewis J. Watson, Lan Mao, Gurangad S. Chandok, Hong-Gang Wang, Stephan Frangakis, Geoffrey S. Pitt, Svati H. Shah, Matthew J. Wolf, Howard A. Rockman
Abstract
Skeletal muscle has emerged as a critical, disease-relevant target tissue in spinal and bulbar muscular atrophy, a degenerative disorder of the neuromuscular system caused by a CAG/polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. Here, we used RNA-Seq to identify pathways that are disrupted in diseased muscle using AR113Q knock-in mice. This analysis unexpectedly identified significantly diminished expression of numerous ubiquitin-proteasome pathway genes in AR113Q muscle, encoding approximately 30% of proteasome subunits and 20% of E2 ubiquitin conjugases. These changes were age-, hormone- and glutamine length-dependent and arose due to a toxic gain-of-function conferred by the mutation. Moreover, altered gene expression was associated with decreased level of the proteasome transcription factor NRF1 and its activator DDI2 and resulted in diminished proteasome activity. Ubiquitinated ADRM1 was detected in AR113Q muscle, indicating the occurrence of stalled proteasomes in mutant mice. Finally, diminished expression of Drosophila orthologues of NRF1 or ADRM1 promoted the accumulation of polyQ AR protein and increased toxicity. Collectively, these data indicate that AR113Q muscle develops progressive proteasome dysfunction that leads to the impairment of quality control and the accumulation of polyQ AR protein, key features that contribute to the age-dependent onset and progression of this disorder.
Authors
Samir R. Nath, Zhigang Yu, Theresa A. Gipson, Gregory B. Marsh, Eriko Yoshidome, Diane M. Robins, Sokol V. Todi, David E. Housman, Andrew P. Lieberman
Abstract
The remarkable regeneration capability of skeletal muscle depends on coordinated proliferation and differentiation of satellite cells. The self-renewal of satellite cells is critical for long-term maintenance of muscle regeneration potential. Hypoxia profoundly affects the proliferation, differentiation, and self-renewal of cultured myoblasts. However, the physiological relevance of hypoxia and hypoxia signaling in satellite cells in vivo remains largely unknown. Here, we report that satellite cells are in an intrinsic hypoxic state in vivo and express hypoxia-inducible factor 2A (HIF2A). HIF2A promotes the stemness and long-term homeostatic maintenance of satellite cells by maintaining the quiescence, increasing the self-renewal and blocking the myogenic differentiation of satellite cells. HIF2A stabilization in satellite cells cultured under normoxia augmented their engraftment potential in regenerative muscle. Reversely, HIF2A ablation led to the depletion of satellite cells and the consequent regenerative failure in the long-term. In contrast, transient pharmacological inhibition of HIF2A accelerated muscle regeneration by increasing satellite cell proliferation and differentiation. Mechanistically, HIF2A induces the quiescence/self-renewal of satellite cells by binding the promoter of Spry1 gene and activating Spry1 expression. These findings suggest that HIF2A is a pivotal mediator of hypoxia signaling in satellite cells and may be therapeutically targeted to improve muscle regeneration.
Authors
Liwei Xie, Amelia Yin, Anna S. Nichenko, Aaron M. Beedle, Jarrod A. Call, Hang Yin
Abstract
Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget’s disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid–phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S–persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.
Authors
YouJin Lee, Per Harald Jonson, Jaakko Sarparanta, Johanna Palmio, Mohona Sarkar, Anna Vihola, Anni Evilä, Tiina Suominen, Sini Penttilä, Marco Savarese, Mridul Johari, Marie-Christine Minot, David Hilton-Jones, Paul Maddison, Patrick Chinnery, Jens Reimann, Cornelia Kornblum, Torsten Kraya, Stephan Zierz, Carolyn Sue, Hans Goebel, Asim Azfer, Stuart H. Ralston, Peter Hackman, Robert C. Bucelli, J. Paul Taylor, Conrad C. Weihl, Bjarne Udd
Copyright © 2019 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)