The maintenance of skeletal muscle mass is critical for sustaining health; however, the mechanisms responsible for muscle loss with aging and chronic diseases, such as diabetes and obesity, are poorly understood. We found that expression of a member of the AMPK-related kinase family, the SNF1-AMPK-related kinase (SNARK, also known as NUAK2), increased with muscle cell differentiation. SNARK expression increased in skeletal muscles from young mice exposed to metabolic stress and in muscles from healthy older human subjects. The regulation of SNARK expression in muscle with differentiation and physiological stress suggests that SNARK may function in the maintenance of muscle mass. Consistent with this hypothesis, decreased endogenous SNARK expression (using siRNA) in cultured muscle cells resulted in increased apoptosis and decreased cell survival under conditions of metabolic stress. Likewise, muscle-specific transgenic animals expressing a SNARK dominant-negative inactive mutant (SDN) had increased myonuclear apoptosis and activation of apoptotic mediators in muscle. Moreover, animals expressing SDN had severe, age-accelerated muscle atrophy and increased adiposity, consistent with sarcopenic obesity. Reduced SNARK activity, in vivo and in vitro, caused downregulation of the Rho kinase signaling pathway, a key mediator of cell survival. These findings reveal a critical role for SNARK in myocyte survival and the maintenance of muscle mass with age.
Sarah J. Lessard, Donato A. Rivas, Kawai So, Ho-Jin Koh, André Lima Queiroz, Michael F. Hirshman, Roger A. Fielding, Laurie J. Goodyear
Here, we have described and validated a strategy for monitoring skeletal muscle protein synthesis rates in rodents and humans over days or weeks from blood samples. We based this approach on label incorporation into proteins that are synthesized specifically in skeletal muscle and escape into the circulation. Heavy water labeling combined with sensitive tandem mass spectrometric analysis allowed integrated synthesis rates of proteins in muscle tissue across the proteome to be measured over several weeks. Fractional synthesis rate (FSR) of plasma creatine kinase M-type (CK-M) and carbonic anhydrase 3 (CA-3) in the blood, more than 90% of which is derived from skeletal muscle, correlated closely with FSR of CK-M, CA-3, and other proteins of various ontologies in skeletal muscle tissue in both rodents and humans. Protein synthesis rates across the muscle proteome generally changed in a coordinate manner in response to a sprint interval exercise training regimen in humans and to denervation or clenbuterol treatment in rodents. FSR of plasma CK-M and CA-3 revealed changes and interindividual differences in muscle tissue proteome dynamics. In human subjects, sprint interval training primarily stimulated synthesis of structural and glycolytic proteins. Together, our results indicate that this approach provides a virtual biopsy, sensitively revealing individualized changes in proteome-wide synthesis rates in skeletal muscle without a muscle biopsy. Accordingly, this approach has potential applications for the diagnosis, management, and treatment of muscle disorders.
Mahalakshmi Shankaran, Chelsea L. King, Thomas E. Angel, William E. Holmes, Kelvin W. Li, Marc Colangelo, John C. Price, Scott M. Turner, Christopher Bell, Karyn L. Hamilton, Benjamin F. Miller, Marc K. Hellerstein
The Popeye domain–containing 1 (
Roland F.R. Schindler, Chiara Scotton, Jianguo Zhang, Chiara Passarelli, Beatriz Ortiz-Bonnin, Subreena Simrick, Thorsten Schwerte, Kar-Lai Poon, Mingyan Fang, Susanne Rinné, Alexander Froese, Viacheslav O. Nikolaev, Christiane Grunert, Thomas Müller, Giorgio Tasca, Padmini Sarathchandra, Fabrizio Drago, Bruno Dallapiccola, Claudio Rapezzi, Eloisa Arbustini, Francesca Romana Di Raimo, Marcella Neri, Rita Selvatici, Francesca Gualandi, Fabiana Fattori, Antonello Pietrangelo, Wenyan Li, Hui Jiang, Xun Xu, Enrico Bertini, Niels Decher, Jun Wang, Thomas Brand, Alessandra Ferlini
Satellite cells are a stem cell population within adult muscle and are responsible for myofiber regeneration upon injury. Satellite cell dysfunction has been shown to underlie the loss of skeletal muscle mass in many acquired and genetic muscle disorders. The transcription factor paired box-protein-7 (PAX7) is indispensable for supplementing the reservoir of satellite cells and driving regeneration in normal and diseased muscle. TNF receptor–associated factor 6 (TRAF6) is an adaptor protein and an E3 ubiquitin ligase that mediates the activation of multiple cell signaling pathways in a context-dependent manner. Here, we demonstrated that TRAF6-mediated signaling is critical for homeostasis of satellite cells and their function during regenerative myogenesis. Selective deletion of
Sajedah M. Hindi, Ashok Kumar
Conditions such as muscular dystrophies (MDs) that affect both cardiac and skeletal muscles would benefit from therapeutic strategies that enable regeneration of both of these striated muscle types. Protocols have been developed to promote induced pluripotent stem cells (iPSCs) to differentiate toward cardiac or skeletal muscle; however, there are currently no strategies to simultaneously target both muscle types. Tissues exhibit specific epigenetic alterations; therefore, source-related lineage biases have the potential to improve iPSC-driven multilineage differentiation. Here, we determined that differential myogenic propensity influences the commitment of isogenic iPSCs and a specifically isolated pool of mesodermal iPSC-derived progenitors (MiPs) toward the striated muscle lineages. Differential myogenic propensity did not influence pluripotency, but did selectively enhance chimerism of MiP-derived tissue in both fetal and adult skeletal muscle. When injected into dystrophic mice, MiPs engrafted and repaired both skeletal and cardiac muscle, reducing functional defects. Similarly, engraftment into dystrophic mice of canine MiPs from dystrophic dogs that had undergone TALEN-mediated correction of the MD-associated mutation also resulted in functional striatal muscle regeneration. Moreover, human MiPs exhibited the same capacity for the dual differentiation observed in murine and canine MiPs. The findings of this study suggest that MiPs should be further explored for combined therapy of cardiac and skeletal muscles.
Mattia Quattrocelli, Melissa Swinnen, Giorgia Giacomazzi, Jordi Camps, Ines Barthélemy, Gabriele Ceccarelli, Ellen Caluwé, Hanne Grosemans, Lieven Thorrez, Gloria Pelizzo, Manja Muijtjens, Catherine M. Verfaillie, Stephane Blot, Stefan Janssens, Maurilio Sampaolesi
Maintenance of skeletal muscle structure and function requires a precise stoichiometry of sarcomeric proteins for proper assembly of the contractile apparatus. Absence of components of the sarcomeric thin filaments causes nemaline myopathy, a lethal congenital muscle disorder associated with aberrant myofiber structure and contractility. Previously, we reported that deficiency of the kelch-like family member 40 (KLHL40) in mice results in nemaline myopathy and destabilization of leiomodin-3 (LMOD3). LMOD3 belongs to a family of tropomodulin-related proteins that promote actin nucleation. Here, we show that deficiency of LMOD3 in mice causes nemaline myopathy. In skeletal muscle, transcription of
Bercin K. Cenik, Ankit Garg, John R. McAnally, John M. Shelton, James A. Richardson, Rhonda Bassel-Duby, Eric N. Olson, Ning Liu
Muscle satellite cells promote regeneration and could potentially improve gene delivery for treating muscular dystrophies. Human satellite cells are scarce; therefore, clinical investigation has been limited. We obtained muscle fiber fragments from skeletal muscle biopsy specimens from adult donors aged 20 to 80 years. Fiber fragments were manually dissected, cultured, and evaluated for expression of myogenesis regulator PAX7. PAX7+ satellite cells were activated and proliferated efficiently in culture. Independent of donor age, as few as 2 to 4 PAX7+ satellite cells gave rise to several thousand myoblasts. Transplantation of human muscle fiber fragments into irradiated muscle of immunodeficient mice resulted in robust engraftment, muscle regeneration, and proper homing of human PAX7+ satellite cells to the stem cell niche. Further, we determined that subjecting the human muscle fiber fragments to hypothermic treatment successfully enriches the cultures for PAX7+ cells and improves the efficacy of the transplantation and muscle regeneration. Finally, we successfully altered gene expression in cultured human PAX7+ satellite cells with Sleeping Beauty transposon–mediated nonviral gene transfer, highlighting the potential of this system for use in gene therapy. Together, these results demonstrate the ability to culture and manipulate a rare population of human tissue-specific stem cells and suggest that these PAX7+ satellite cells have potential to restore gene function in muscular dystrophies.
Andreas Marg, Helena Escobar, Sina Gloy, Markus Kufeld, Joseph Zacher, Andreas Spuler, Carmen Birchmeier, Zsuzsanna Izsvák, Simone Spuler
Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (
Matthew S. Alexander, Juan Carlos Casar, Norio Motohashi, Natássia M. Vieira, Iris Eisenberg, Jamie L. Marshall, Molly J. Gasperini, Angela Lek, Jennifer A. Myers, Elicia A. Estrella, Peter B. Kang, Frederic Shapiro, Fedik Rahimov, Genri Kawahara, Jeffrey J. Widrick, Louis M. Kunkel
The ubiquitously expressed multifunctional cytolinker protein plectin is essential for muscle fiber integrity and myofiber cytoarchitecture. Patients suffering from plectinopathy-associated epidermolysis bullosa simplex with muscular dystrophy (EBS-MD) and mice lacking plectin in skeletal muscle display pathological desmin-positive protein aggregation and misalignment of Z-disks, which are hallmarks of myofibrillar myopathies (MFMs). Here, we developed immortalized murine myoblast cell lines to examine the pathogenesis of plectinopathies at the molecular and single cell level. Plectin-deficient myotubes, derived from myoblasts, were fully functional and mirrored the pathological features of EBS-MD myofibers, including the presence of desmin-positive protein aggregates and a concurrent disarrangement of the myofibrillar apparatus. Using this cell model, we demonstrated that plectin deficiency leads to increased intermediate filament network and sarcomere dynamics, marked upregulation of HSPs, and reduced myotube resilience following mechanical stretch. Currently, no specific therapy or treatment is available to improve plectin-related or other forms of MFMs; therefore, we assessed the therapeutic potential of chemical chaperones to relieve plectinopathies. Treatment with 4-phenylbutyrate resulted in remarkable amelioration of the pathological phenotypes in plectin-deficient myotubes as well as in plectin-deficient mice. Together, these data demonstrate the biological relevance of the MFM cell model and suggest that this model has potential use for the development of therapeutic approaches for EBS-MD.
Lilli Winter, Ilona Staszewska, Eva Mihailovska, Irmgard Fischer, Wolfgang H. Goldmann, Rolf Schröder, Gerhard Wiche
α-Actinin-3 deficiency occurs in approximately 16% of the global population due to homozygosity for a common nonsense polymorphism in the
Jane T. Seto, Kate G.R. Quinlan, Monkol Lek, Xi Fiona Zheng, Fleur Garton, Daniel G. MacArthur, Marshall W. Hogarth, Peter J. Houweling, Paul Gregorevic, Nigel Turner, Gregory J. Cooney, Nan Yang, Kathryn N. North