Circulating protein synthesis rates reveal skeletal muscle proteome dynamics

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Abstract

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.

Authors

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

POPDC1^S201F causes muscular dystrophy and arrhythmia by affecting protein trafficking

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Abstract

The Popeye domain–containing 1 (POPDC1) gene encodes a plasma membrane–localized cAMP-binding protein that is abundantly expressed in striated muscle. In animal models, POPDC1 is an essential regulator of structure and function of cardiac and skeletal muscle; however, POPDC1 mutations have not been associated with human cardiac and muscular diseases. Here, we have described a homozygous missense variant (c.602C>T, p.S201F) in POPDC1, identified by whole-exome sequencing, in a family of 4 with cardiac arrhythmia and limb-girdle muscular dystrophy (LGMD). This allele was absent in known databases and segregated with the pathological phenotype in this family. We did not find the allele in a further screen of 104 patients with a similar phenotype, suggesting this mutation to be family specific. Compared with WT protein, POPDC1^S201F displayed a 50% reduction in cAMP affinity, and in skeletal muscle from patients, both POPDC1^S201F and WT POPDC2 displayed impaired membrane trafficking. Forced expression of POPDC1^S201F in a murine cardiac muscle cell line (HL-1) increased hyperpolarization and upstroke velocity of the action potential. In zebrafish, expression of the homologous mutation (popdc1^S191F) caused heart and skeletal muscle phenotypes that resembled those observed in patients. Our study therefore identifies POPDC1 as a disease gene causing a very rare autosomal recessive cardiac arrhythmia and LGMD, expanding the genetic causes of this heterogeneous group of inherited rare diseases.

Authors

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

TRAF6 regulates satellite stem cell self-renewal and function during regenerative myogenesis

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Abstract

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 Traf6 in satellite cells of adult mice led to profound muscle regeneration defects and dramatically reduced levels of PAX7 and late myogenesis markers. TRAF6 was required for the activation of MAPKs ERK1/2 and JNK1/2, which in turn activated the transcription factor c-JUN, which binds the Pax7 promoter and augments Pax7 expression. Moreover, TRAF6/c-JUN signaling repressed the levels of the microRNAs miR-1 and miR-206, which promote differentiation, to maintain PAX7 levels in satellite cells. We also determined that satellite cell–specific deletion of Traf6 exaggerates the dystrophic phenotype in the mdx (a mouse model of Duchenne muscular dystrophy) mouse by blunting the regeneration of injured myofibers. Collectively, our study reveals an essential role for TRAF6 in satellite stem cell function.

Authors

Sajedah M. Hindi, Ashok Kumar

Mesodermal iPSC–derived progenitor cells functionally regenerate cardiac and skeletal muscle

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Abstract

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.

Authors

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

Severe myopathy in mice lacking the MEF2/SRF-dependent gene leiomodin-3

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Abstract

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 Lmod3 was controlled by the transcription factors SRF and MEF2. Myocardin-related transcription factors (MRTFs), which function as SRF coactivators, serve as sensors of actin polymerization and are sequestered in the cytoplasm by actin monomers. Conversely, conditions that favor actin polymerization de-repress MRTFs and activate SRF-dependent genes. We demonstrated that the actin nucleator LMOD3, together with its stabilizing partner KLHL40, enhances MRTF-SRF activity. In turn, SRF cooperated with MEF2 to sustain the expression of LMOD3 and other components of the contractile apparatus, thereby establishing a regulatory circuit to maintain skeletal muscle function. These findings provide insight into the molecular basis of the sarcomere assembly and muscle dysfunction associated with nemaline myopathy.

Authors

Bercin K. Cenik, Ankit Garg, John R. McAnally, John M. Shelton, James A. Richardson, Rhonda Bassel-Duby, Eric N. Olson, Ning Liu

Human satellite cells have regenerative capacity and are genetically manipulable

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Abstract

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.

Authors

Andreas Marg, Helena Escobar, Sina Gloy, Markus Kufeld, Joseph Zacher, Andreas Spuler, Carmen Birchmeier, Zsuzsanna Izsvák, Simone Spuler

MicroRNA-486–dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy–associated symptoms

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Abstract

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 (Dmd^mdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmd^mdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle–specific miR-486 overexpression in Dmd^mdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle.

Authors

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

Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle

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Abstract

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.

Authors

Lilli Winter, Ilona Staszewska, Eva Mihailovska, Irmgard Fischer, Wolfgang H. Goldmann, Rolf Schröder, Gerhard Wiche

ACTN3 genotype influences muscle performance through the regulation of calcineurin signaling

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Abstract

α-Actinin-3 deficiency occurs in approximately 16% of the global population due to homozygosity for a common nonsense polymorphism in the ACTN3 gene. Loss of α-actinin-3 is associated with reduced power and enhanced endurance capacity in elite athletes and nonathletes due to “slowing” of the metabolic and physiological properties of fast fibers. Here, we have shown that α-actinin-3 deficiency results in increased calcineurin activity in mouse and human skeletal muscle and enhanced adaptive response to endurance training. α-Actinin-2, which is differentially expressed in α-actinin-3–deficient muscle, has higher binding affinity for calsarcin-2, a key inhibitor of calcineurin activation. We have further demonstrated that α-actinin-2 competes with calcineurin for binding to calsarcin-2, resulting in enhanced calcineurin signaling and reprogramming of the metabolic phenotype of fast muscle fibers. Our data provide a mechanistic explanation for the effects of the ACTN3 genotype on skeletal muscle performance in elite athletes and on adaptation to changing physical demands in the general population. In addition, we have demonstrated that the sarcomeric α-actinins play a role in the regulation of calcineurin signaling.

Authors

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

Transcription factor EGR1 directs tendon differentiation and promotes tendon repair

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Abstract

Tendon formation and repair rely on specific combinations of transcription factors, growth factors, and mechanical parameters that regulate the production and spatial organization of type I collagen. Here, we investigated the function of the zinc finger transcription factor EGR1 in tendon formation, healing, and repair using rodent animal models and mesenchymal stem cells (MSCs). Adult tendons of Egr1^–/– mice displayed a deficiency in the expression of tendon genes, including Scx, Col1a1, and Col1a2, and were mechanically weaker compared with their WT littermates. EGR1 was recruited to the Col1a1 and Col2a1 promoters in postnatal mouse tendons in vivo. Egr1 was required for the normal gene response following tendon injury in a mouse model of Achilles tendon healing. Forced Egr1 expression programmed MSCs toward the tendon lineage and promoted the formation of in vitro–engineered tendons from MSCs. The application of EGR1-producing MSCs increased the formation of tendon-like tissues in a rat model of Achilles tendon injury. We provide evidence that the ability of EGR1 to promote tendon differentiation is partially mediated by TGF-β2. This study demonstrates EGR1 involvement in adult tendon formation, healing, and repair and identifies Egr1 as a putative target in tendon repair strategies.

Authors

Marie-Justine Guerquin, Benjamin Charvet, Geoffroy Nourissat, Emmanuelle Havis, Olivier Ronsin, Marie-Ange Bonnin, Mathilde Ruggiu, Isabel Olivera-Martinez, Nicolas Robert, Yinhui Lu, Karl E. Kadler, Tristan Baumberger, Levon Doursounian, Francis Berenbaum, Delphine Duprez