Muscle biology
Abstract
Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy. When human induced pluripotent stem cells (hiPSCs) were differentiated into myoblasts, the myoblasts derived from DMD patients’ hiPSCs (DMD hiPSC-derived myoblasts) exhibited an identifiable DMD relevant phenotype: myogenic fusion deficiency. Based on this model, we developed a DMD hiPSC-derived myoblast screening platform employing a high-content imaging (BD pathway 855) approach to generate parameters describing morphological as well as myogenic marker protein expression. Following treatment of the cells with 1524 compounds from the Johns Hopkins Clinical Compound Library, compounds that enhanced myogenic fusion of DMD hiPSC-derived myoblasts were identified. The final hits were ginsenoside Rd and fenofibrate. Transcriptional profiling revealed that ginsenoside Rd is functionally related to FLT3 signaling, while fenofibrate is linked to TGF-β signaling. Preclinical tests in mdx mice showed that treatment with these two hit compounds can significantly ameliorate some of the skeletal muscle phenotypes caused by dystrophin deficiency, supporting their therapeutic potential. Further study with hiPSC-derived cardiomyocytes revealed that fenofibrate could inhibit mitochondria-induced apoptosis in the DMD hiPSC-derived cardiomyocytes. We have developed a platform based on DMD hiPSC-derived myoblasts for drug screening and identified two promising small molecules with in vivo efficacy.
Authors
Congshan Sun, In Young Choi, Yazmin I. Rovira Gonzalez, Peter Andersen, C. Conover Talbot Jr., Shama R. Iyer, Richard M. Lovering, Kathryn R. Wagner, Gabsang Lee
Abstract
Muscle progenitor cell fusion is required for the formation and regeneration of multinucleated skeletal muscle fibers. Chronic muscle regeneration in Duchenne muscular dystrophy (DMD) is characterized by ongoing fusion of satellite cell (SC) progeny, but the effects of fusion on disease and the mechanisms by which fusion is accomplished in this setting are not fully understood. Using the mdx mouse model of DMD, we deleted the fusogenic protein Myomaker in SCs or myofibers. Following deletion in SCs, mice displayed a complete lack of myocyte fusion, resulting in severe muscle loss, enhanced fibrosis, and significant functional decline. Reduction of Myomaker in mature myofibers in mdx mice, however, led to minimal alterations in fusion dynamics. Unexpectedly, myofiber-specific deletion of myomaker resulted in improvement of disease phenotype, with enhanced function and decreased muscle damage. Our data indicate that Myomaker has divergent effects on dystrophic disease severity depending upon its compartment of expression. These findings show that myocyte fusion is absolutely required for effective regeneration in DMD, but persistent Myomaker expression in myofibers due to ongoing fusion may have unintended deleterious consequences for muscle integrity. Thus, sustained activation of a component of the myogenic program in dystrophic myofibers exacerbates disease.
Authors
Michael J. Petrany, Taejeong Song, Sakthivel Sadayappan, Douglas P. Millay
Abstract
Advanced colorectal cancer (CRC) is often accompanied by development of liver metastases (LMs) and skeletal muscle (SkM) wasting, i.e. cachexia. Despite plaguing the majority of CRC patients, cachexia remains unresolved. By using mice subcutaneously (C26) or intrasplenically injected with C26 tumor cells to mimic hepatic dissemination of cancer cells (mC26), here we aimed to further characterize functional, molecular and metabolic effects on SkM and examine whether LMs exacerbate CRC-induced cachexia. C26-derived LMs were associated with progressive loss of body weight, as well as with significant reductions in SkM size and strength, in line with reduced phosphorylation of markers of protein anabolism and enhanced protein catabolism. mC26 hosts showed prevalence of fibers with glycolytic metabolism and enhanced lipid accumulation, consistent with abnormalities of mitochondrial homeostasis and energy metabolism. In a comparison with mice bearing subcutaneous C26, cachexia appeared exacerbated in the mC26 hosts, as also supported by differentially expressed pathways within SkM. Overall, our model recapitulates the cachectic phenotype of metastatic CRC and reveals that formation of LMs resulting from CRC exacerbate cancer-induced SkM wasting by promoting differential gene expression signatures.
Authors
Joshua R. Huot, Leah J. Novinger, Fabrizio Pin, Ashok Narasimhan, Teresa A. Zimmers, Thomas M. O'Connell, Andrea Bonetto
TGF-β–driven muscle degeneration and failed regeneration underlie disease onset in a DMD mouse model
Abstract
Duchenne muscular dystrophy (DMD) is a chronic muscle disease characterized by poor myogenesis and replacement of muscle by extracellular matrix. Despite the shared genetic basis, severity of these deficits varies among patients. One source of these variations is the genetic modifier that leads to increased TGF-β activity. While anti–TGF-β therapies are being developed to target muscle fibrosis, their effect on the myogenic deficit is underexplored. Our analysis of in vivo myogenesis in mild (C57BL/10ScSn-mdx/J and C57BL/6J-mdxΔ52) and severe DBA/2J-mdx (D2-mdx) dystrophic models reveals no defects in developmental myogenesis in these mice. However, muscle damage at the onset of disease pathology, or by experimental injury, drives up TGF-β activity in the severe, but not in the mild, dystrophic models. Increased TGF-β activity is accompanied by increased accumulation of fibroadipogenic progenitors (FAPs) leading to fibro-calcification of muscle, together with failure of regenerative myogenesis. Inhibition of TGF-β signaling reduces muscle degeneration by blocking FAP accumulation without rescuing regenerative myogenesis. These findings provide in vivo evidence of early-stage deficit in regenerative myogenesis in D2-mdx mice and implicates TGF-β as a major component of a pathogenic positive feedback loop in this model, identifying this feedback loop as a therapeutic target.
Authors
Davi A.G. Mázala, James S. Novak, Marshall W. Hogarth, Marie Nearing, Prabhat Adusumalli, Christopher B. Tully, Nayab F. Habib, Heather Gordish-Dressman, Yi-Wen Chen, Jyoti K. Jaiswal, Terence A. Partridge
Abstract
Most prostate cancers depend on androgens for growth and therefore the mainstay treatment for advanced, recurrent or metastatic prostate cancer is androgen deprivation therapy (ADT). A prominent side effect in patients receiving ADT is an obese frailty syndrome that includes fat gain and sarcopenia, defined as the loss of muscle function accompanied by reduced muscle mass or quality. Mice bearing Pten deficient prostate cancers were examined to gain mechanistic insight into ADT-induced sarcopenic obesity. Castration induced fat gain as well as skeletal muscle mass and strength loss. Catabolic TGFß-family myokine protein levels were increased immediately prior to strength loss and pan-myokine blockade using a soluble receptor (ActRIIB-Fc) completely reversed the castration-induced sarcopenia. The onset of castration-induced strength and muscle mass loss, as well as the increase in catabolic TGFß-family myokine protein levels, were coordinately accelerated in tumor-bearing mice relative to tumor-free mice. Notably, GDF11 increased in muscle after castration only in tumor-bearing mice, but not in tumor free mice. An early surge of GDF11 in prostate tumor tissue and in the circulation suggests that endocrine GDF11 signaling from tumor to muscle is a major driver of the accelerated ADT-induced sarcopenic phenotype. In tumor-bearing mice, GDF11 blockade largely prevented castration-induced strength loss but did not preserve muscle mass, which confirms a primary role for GDF11 in muscle function and suggests an additional role for the other catabolic myokines.
Authors
Chunliu Pan, Neha Jaiswal Agrawal, Yanni Zulia, Shalini Singh, Kai Sha, James L. Mohler, Kevin H. Eng, Joe Chakkalakal, John J. Krolewski, Kent L. Nastiuk
Abstract
The mitochondrial calcium uniporter is widely accepted as the primary route of rapid calcium entry into mitochondria, where increases in matrix calcium contribute to bioenergetics but also mitochondrial permeability and cell death. Hence, regulation of uniporter activity is critical to mitochondrial homeostasis. The uniporter subunit EMRE is known to be an essential regulator of the channel-forming protein MCU in cell culture, but EMRE’s impact on organismal physiology is less understood. Here we characterize a novel mouse model of EMRE deletion and show that EMRE is indeed required for mitochondrial calcium uniporter function in vivo. EMRE–/– mice are born less frequently; however, the mice which are born are viable, healthy, and do not manifest overt metabolic impairment, at rest or with exercise. Finally, to investigate the role of EMRE in disease processes, we examine the effects of EMRE deletion in a muscular dystrophy model associated with mitochondrial calcium overload.
Authors
Julia C. Liu, Nicole C. Syder, Nima S. Ghorashi, Thomas B. Willingham, Randi J. Parks, Junhui Sun, Maria M. Fergusson, Jie Liu, Kira M. Holmström, Sara Menazza, Danielle A. Springer, Chengyu Liu, Brian Glancy, Toren Finkel, Elizabeth Murphy
Abstract
OBJECTIVES: Idiopathic inflammatory myopathies (IIM) are characterized by muscle inflammation and weakness, myositis specific autoantibodies (MSAs) and extramuscular organ damage. The role of neutrophil dysregulation and neutrophil extracellular traps (NETs) in IIM is unclear. We assessed if pathogenic neutrophil subsets (low-density granulocytes, LDGs) and NETs were elevated in IIM, associated with clinical presentation and MSAs, and their effect on skeletal myoblasts and myotubes.METHODS: Circulating NETs and LDGs were quantified and correlated with clinical measures. Specific MSAs were tested for their ability to induce NETs. NETs and neutrophil gene expression were measured in IIM biopsies. Whether NETs damage skeletal myoblasts and myotubes was tested.RESULTS: Circulating LDGs and NETs were increased in IIM. IIM LDGs had enhanced ability to form NETs. LDGs and NETs correlated with IIM disease activity and muscle damage. The serum MSA anti-MDA5 correlated with circulating and tissue NETs and directly enhanced NET formation. An enhanced neutrophil gene signature was present in IIM muscle and associated with muscle injury and tissue interferon gene signatures. IIM NETs decreased the viability of myotubes in a citrullinated histone-dependent manner. CONCLUSION: Dysregulated neutrophil pathways may play pathogenic roles in IIM through their ability to directly injure muscle cells and other affected tissues.
Authors
Nickie L. Seto, Jose Jiram Torres-Ruiz, Carmelo Carmona-Rivera, Iago Pinal-Fernandez, Katherine Pak, Monica M. Purmalek, Yuji Hosono, Catia Fernandes-Cerqueira, Prateek C. Gowda, Nathan Arnett, Alexander Gorbach, Olivier Benveniste, Diana Gómez-Martín, Albert Selva-O'Callaghan, Jose C. Milisenda, Josep M. Grau-Junyent, Lisa Christopher-Stine, Frederick W. Miller, Ingrid E. Lundberg, J. Michelle Kahlenberg, Adam I. Schiffenbauer, Andrew L. Mammen, Lisa G. Rider, Mariana J. Kaplan
Abstract
Dystrophic muscle is characterised by chronic injury, and a steady recruitment of inflammatory Ly6Chi monocytes. Recent studies have identified the spleen as the dominant reservoir of these cells during chronic inflammation. Here we investigated the hitherto unexplored contribution of splenic Ly6Chi monocytes to dystrophic muscle pathology. Using the mdx mouse model of muscular dystrophy, we show that Ly6Chi monocytes accumulate in great numbers in the spleen over the course of the disease. The chemokine receptor CCR2 was upregulated on Ly6Chi monocytes in mdx spleen before disease onset, thereby enabling their recruitment to dystrophic muscle. Splenectomy performed before disease onset significantly reduced the number of Ly6Chi monocytes infiltrating dystrophic limb muscle. Moreover, in the absence of splenic Ly6Chi monocytes there was a significant reduction in dystrophic muscle inflammation and necrosis, along with improved regeneration during early disease. However, during late disease, lack of splenic Ly6Chi monocytes adversely affected muscle fiber repair, due to a delay in the phenotypic shift of pro-inflammatory F4/80+/Ly6Chi/CD206lo to anti-inflammatory F4/80+/Ly6Clo/CD206+ macrophages. Overall, we show that the spleen is an indispensable source of Ly6Chi monocytes in muscular dystrophy, and that splenic monocytes are critical players in both muscle fiber injury and repair.
Authors
Giuseppe Rizzo, Rosanna Di Maggio, Anna Benedetti, Jacopo Morroni, Marina Bouche, Biliana Lozanoska-Ochser
Abstract
Massive tears of the rotator cuff (RC) are associated with chronic muscle degeneration due to fibrosis, fatty infiltration, and muscle atrophy. The microenvironment of diseased muscle often impairs efficient engraftment and regenerative activity of transplanted myogenic precursors. Accumulating myofibroblasts and fat cells disrupt the muscle stem cell niche and myogenic cell signaling and deposit excess disorganized connective tissue. Therefore, restoration of the damaged stromal niche with non–fibro-adipogenic cells is a prerequisite to successful repair of an injured RC. We generated from human embryonic stem cells (hES) a potentially novel subset of PDGFR-β+CD146+CD34–CD56– pericytes that lack expression of the fibro-adipogenic cell marker PDGFR-α. Accordingly, the PDGFR-β+PDGFR-α– phenotype typified non–fibro-adipogenic, non-myogenic, pericyte-like derivatives that maintained non–fibro-adipogenic properties when transplanted into chronically injured murine RCs. Although administered hES pericytes inhibited developing fibrosis at early and late stages of progressive muscle degeneration, transplanted PDGFR-β+PDGFR-α+ human muscle-derived fibro-adipogenic progenitors contributed to adipogenesis and greater fibrosis. Additionally, transplanted hES pericytes substantially attenuated muscle atrophy at all tested injection time points after injury. Coinciding with this observation, conditioned medium from cultured hES pericytes rescued atrophic myotubes in vitro. These findings imply that non–fibro-adipogenic hES pericytes recapitulate the myogenic stromal niche and may be used to improve cell-based treatments for chronic muscle disorders.
Authors
Gina M. Mosich, Regina Husman, Paras Shah, Abhinav Sharma, Kevin Ressadeh, Temidayo Aderibigbe, Vivian J. Hu, Daniel J. McClintick, Genbin Wu, Jonathan D. Gatto, Haibin Xi, April D. Pyle, Bruno Péault, Frank A. Petrigliano, Ayelet Dar
Abstract
In humans, chronic glucocorticoid use is associated with side effects like muscle wasting, obesity, and metabolic syndrome. Intermittent steroid dosing has been proposed in Duchenne Muscular Dystrophy patients to mitigate the side effects seen with daily steroid intake. We evaluated biomarkers from Duchenne Muscular Dystrophy patients, finding that, compared with chronic daily steroid use, weekend steroid use was associated with reduced serum insulin, free fatty acids, and branched chain amino acids, as well as reduction in fat mass despite having similar BMIs. We reasoned that intermittent prednisone administration in dystrophic mice would alter muscle epigenomic signatures, and we identified the coordinated action of the glucocorticoid receptor, KLF15 and MEF2C as mediators of a gene expression program driving metabolic reprogramming and enhanced nutrient utilization. Muscle lacking Klf15 failed to respond to intermittent steroids. Furthermore, coadministration of the histone acetyltransferase inhibitor anacardic acid with steroids in mdx mice eliminated steroid-specific epigenetic marks and abrogated the steroid response. Together, these findings indicate that intermittent, repeated exposure to glucocorticoids promotes performance in dystrophic muscle through an epigenetic program that enhances nutrient utilization.
Authors
Mattia Quattrocelli, Aaron S. Zelikovich, Zhen Jiang, Clara Bien Peek, Alexis R. Demonbreun, Nancy L. Kuntz, Grant D. Barish, Saptarsi M. Haldar, Joseph Bass, Elizabeth M. McNally
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