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.
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
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.
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
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.
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
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.
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
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.
Liwei Xie, Amelia Yin, Anna S. Nichenko, Aaron M. Beedle, Jarrod A. Call, Hang Yin
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.
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
Regulation of skeletal muscle development and organization is a complex process that is not fully understood. Here, we focused on amphiphysin 2 (BIN1, also known as bridging integrator-1) and dynamin 2 (DNM2), two ubiquitous proteins implicated in membrane remodeling and mutated in centronuclear myopathies (CNMs). We generated Bin1–/– Dnm2+/– mice to decipher the physiological interplay between BIN1 and DNM2. While Bin1–/– mice die perinatally from a skeletal muscle defect, Bin1–/– Dnm2+/– mice survived at least 18 months, and had normal muscle force and intracellular organization of muscle fibers, supporting BIN1 as a negative regulator of DNM2. We next characterized muscle-specific isoforms of BIN1 and DNM2. While BIN1 colocalized with and partially inhibited DNM2 activity during muscle maturation, BIN1 had no effect on the isoform of DNM2 found in adult muscle. Together, these results indicate that BIN1 and DNM2 regulate muscle development and organization, function through a common pathway, and define BIN1 as a negative regulator of DNM2 in vitro and in vivo during muscle maturation. Our data suggest that DNM2 modulation has potential as a therapeutic approach for patients with CNM and BIN1 defects. As BIN1 is implicated in cancers, arrhythmia, and late-onset Alzheimer disease, these findings may trigger research directions and therapeutic development for these common diseases.
Belinda S. Cowling, Ivana Prokic, Hichem Tasfaout, Aymen Rabai, Frédéric Humbert, Bruno Rinaldi, Anne-Sophie Nicot, Christine Kretz, Sylvie Friant, Aurélien Roux, Jocelyn Laporte
Skeletal muscle is a key organ in energy homeostasis owing to its high requirement for nutrients. Heterotrimeric G proteins converge signals from cell-surface receptors to potentiate or blunt responses against environmental changes. Here, we show that muscle-specific ablation of Gα13 in mice promotes reprogramming of myofibers to the oxidative type, with resultant increases in mitochondrial biogenesis and cellular respiration. Mechanistically, Gα13 and its downstream effector RhoA suppressed nuclear factor of activated T cells 1 (NFATc1), a chief regulator of myofiber conversion, by increasing Rho-associated kinase 2–mediated (Rock2-mediated) phosphorylation at Ser243. Ser243 phosphorylation of NFATc1 was reduced after exercise, but was higher in obese animals. Consequently, Gα13 ablation in muscles enhanced whole-body energy metabolism and increased insulin sensitivity, thus affording protection from diet-induced obesity and hepatic steatosis. Our results define Gα13 as a switch regulator of myofiber reprogramming, implying that modulations of Gα13 and its downstream effectors in skeletal muscle are a potential therapeutic approach to treating metabolic diseases.
Ja Hyun Koo, Tae Hyun Kim, Shi-Young Park, Min Sung Joo, Chang Yeob Han, Cheol Soo Choi, Sang Geon Kim
Maintenance of muscle structure and function depends on the precise organization of contractile proteins into sarcomeres and coupling of the contractile apparatus to the sarcoplasmic reticulum (SR), which serves as the reservoir for calcium required for contraction. Several members of the Kelch superfamily of proteins, which modulate protein stability as substrate-specific adaptors for ubiquitination, have been implicated in sarcomere formation. The Kelch protein Klhl31 is expressed in a muscle-specific manner under control of the transcription factor MEF2. To explore its functions in vivo, we created a mouse model of Klhl31 loss of function using the CRISPR-Cas9 system. Mice lacking Klhl31 exhibited stunted postnatal skeletal muscle growth, centronuclear myopathy, central cores, Z-disc streaming, and SR dilation. We used proteomics to identify several candidate Klhl31 substrates, including Filamin-C (FlnC). In the Klhl31-knockout mice, FlnC protein levels were highly upregulated with no change in transcription, and we further demonstrated that Klhl31 targets FlnC for ubiquitination and degradation. These findings highlight a role for Klhl31 in the maintenance of skeletal muscle structure and provide insight into the mechanisms underlying congenital myopathies.
James B. Papizan, Glynnis A. Garry, Svetlana Brezprozvannaya, John R. McAnally, Rhonda Bassel-Duby, Ning Liu, Eric N. Olson
Glucocorticoid steroids such as prednisone are prescribed for chronic muscle conditions
such as Duchenne muscular dystrophy, where their use is associated with prolonged
ambulation. The positive effects of chronic steroid treatment in muscular dystrophy are
paradoxical because these steroids are also known to trigger muscle atrophy. Chronic
steroid use usually involves once-daily dosing, although weekly dosing in children has
been suggested for its reduced side effects on behavior. In this work, we tested steroid
dosing in mice and found that a single pulse of glucocorticoid steroids improved
sarcolemmal repair through increased expression of annexins A1 and A6, which mediate
myofiber repair. This increased expression was dependent on glucocorticoid response
elements upstream of annexins and was reinforced by the expression of forkhead box O1
(FOXO1). We compared weekly versus daily steroid treatment in mouse models of acute muscle
injury and in muscular dystrophy and determined that both regimens provided comparable
benefits in terms of annexin gene expression and muscle repair. However, daily dosing
activated atrophic pathways, including F-box protein 32 (
Mattia Quattrocelli, David Y. Barefield, James L. Warner, Andy H. Vo, Michele Hadhazy, Judy U. Earley, Alexis R. Demonbreun, Elizabeth M. McNally