Mayank Verma

MyoD regulates apoptosis of myoblasts through microRNA-mediated down-regulation of Pax3

Suppression of the myogenic transcription factor MyoD is required for maintenance of muscle stem cells.

Increased survival of muscle stem cells lacking the MyoD gene after transplantation into regenerating skeletal muscle

MyoD is a myogenic master transcription factor that plays an essential role in muscle satellite cell (muscle stem cell) differentiation. To further investigate the function of MyoD in satellite cells, we examined the transplantation of satellite cell-derived myoblasts lacking the MyoD gene into regenerating skeletal muscle. After injection into injured muscle, MyoD−/− myoblasts engrafted with significantly higher efficiency compared with wild-type myoblasts. In addition, MyoD−/− myoblast-derived satellite cells were detected underneath the basal lamina of muscle fibers, indicating the self-renewal property of MyoD−/− myoblasts. To gain insights into MyoD gene deficiency in muscle stem cells, we investigated the pathways regulated by MyoD by GeneChip microarray analysis of gene expression in wild-type and MyoD−/− myoblasts. MyoD deficiency led to down-regulation of many muscle-specific genes and up-regulation of some stem cell markers. Importantly, in MyoD−/− myoblasts, many antiapoptotic genes were up-regulated, whereas genes known to execute apoptosis were down-regulated. Consistent with these gene expression profiles, MyoD−/− myoblasts were revealed to possess remarkable resistance to apoptosis and increased survival compared with wild-type myoblasts. Forced expression of MyoD or the proapoptotic protein Puma increased cell death in MyoD−/− myoblasts. Therefore, MyoD−/− myoblasts may preserve stem cell characteristics, including their resistance to apoptosis, expression of stem cell markers, and efficient engraftment and contribution to satellite cells after transplantation. Furthermore, our data offer evidence for improved therapeutic stem cell transplantation for muscular dystrophy, in which suppression of MyoD in myogenic progenitors would be beneficial to therapy by providing a selective advantage for the expansion of stem cells.

MyoD Gene Suppression by Oct4 Is Required for Reprogramming in Myoblasts to Produce Induced Pluripotent Stem Cells

Expression of the four transcription factors, that is, Oct4, Sox2, cMyc, and Klf4 has been shown to generate induced pluripotent stem cells (iPSCs) from many types of specialized differentiated somatic cells. It remains unclear, however, whether fully committed skeletal muscle progenitor cells (myoblasts) have the potency to undergo reprogramming to develop iPSCs in line with previously reported cases. To test this, we have isolated genetically marked myoblasts derived from satellite cell of adult mouse muscles using the Cre‐loxP system (Pax7‐CreER:R26R and Myf5‐Cre:R26R). On infection with retroviral vectors expressing the four factors, these myoblasts gave rise to myogenic lineage tracer lacZ‐positive embryonic stem cell (ESC)‐like colonies. These cells expressed ESC‐specific genes and were competent to differentiate into all three germ layers and germ cells, indicating the successful generation of myoblast‐derived iPSCs. Continuous expression of the MyoD gene, a master transcription factor for skeletal muscle specification, inhibited this reprogramming process in myoblasts. In contrast, reprogramming myoblasts isolated from mice lacking the MyoD gene led to an increase in reprogramming efficiency. Our data also indicated that Oct4 acts as a transcriptional suppressor of MyoD gene expression through its interaction with the upstream enhancer region. Taken together, these results indicate that suppression of MyoD gene expression by Oct4 is required for the initial reprogramming step in the development of iPSCs from myoblasts. This data suggests that the skeletal muscle system provides a well‐defined differentiation model to further elaborate on the effects of iPSC reprogramming in somatic cells. STEM CELLS 2011;505–516

Flt-1 haploinsufficiency ameliorates muscular dystrophy phenotype by developmentally increased vasculature in mdx mice.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disease caused by mutations in the gene coding for the protein dystrophin. Recent work demonstrates that dystrophin is also found in the vasculature and its absence results in vascular deficiency and abnormal blood flow. This induces a state of ischemia further aggravating the muscular dystrophy pathogenesis. For an effective form of therapy of DMD, both the muscle and the vasculature need to be addressed. To reveal the developmental relationship between muscular dystrophy and vasculature, mdx mice, an animal model for DMD, were crossed with Flt-1 gene knockout mice to create a model with increased vasculature. Flt-1 is a decoy receptor for vascular endothelial growth factor, and therefore both homozygous (Flt-1(-/-)) and heterozygous (Flt-1(+/-)) Flt-1 gene knockout mice display increased endothelial cell proliferation and vascular density during embryogenesis. Here, we show that Flt-1(+/-) and mdx:Flt-1(+/-) adult mice also display a developmentally increased vascular density in skeletal muscle compared with the wild-type and mdx mice, respectively. The mdx:Flt-1(+/-) mice show improved muscle histology compared with the mdx mice with decreased fibrosis, calcification and membrane permeability. Functionally, the mdx:Flt-1(+/-) mice have an increase in muscle blood flow and force production, compared with the mdx mice. Consequently, the mdx:utrophin(-/-):Flt-1(+/-) mice display improved muscle histology and significantly higher survival rates compared with the mdx:utrophin(-/-) mice, which show more severe muscle phenotypes than the mdx mice. These data suggest that increasing the vasculature in DMD may ameliorate the histological and functional phenotypes associated with this disease.

Vascular-targeted therapies for Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is the most common muscular dystrophy and an X-linked recessive, progressive muscle wasting disease caused by the absence of a functional dystrophin protein. Dystrophin has a structural role as a cytoskeletal stabilization protein and protects cells against contraction-induced damage. Dystrophin also serves a signaling role through mechanotransduction of forces and localization of neuronal nitric oxide synthase (nNOS), which produces nitric oxide (NO) to facilitate vasorelaxation. In DMD, the signaling defects produce inadequate tissue perfusion caused by functional ischemia due to a diminished ability to respond to shear stress induced endothelium-dependent dilation. Additionally, the structural defects seen in DMD render myocytes with an increased susceptibility to mechanical stress. The combination of both defects is necessary to generate myocyte damage, which induces successive rounds of myofiber degeneration and regeneration, loss of calcium homeostasis, chronic inflammatory response, fibrosis, and myonecrosis. In individuals with DMD, these processes inevitably cause loss of ambulation shortly after the first decade and an abbreviated life with death in the third or fourth decade due to cardio-respiratory anomalies. There is no known cure for DMD, and although the culpable gene has been identified for more than twenty years, research on treatments has produced few clinically relevant results. Several recent studies on novel DMD therapeutics are vascular targeted and focused on attenuating the inherent functional ischemia. One approach improves vasorelaxation capacity through pharmaceutical inhibition of either phosphodiesterase 5 (PDE5) or angiotensin-converting enzyme (ACE). Another approach increases the density of the underlying vascular network by inducing angiogenesis, and this has been accomplished through either direct delivery of vascular endothelial growth factor (VEGF) or by downregulating the VEGF decoy-receptor type 1 (VEGFR-1 or Flt-1). The pro-angiogenic approaches also seem to be pro-myogenic and could resolve the age-related decline in satellite cell (SC) quantity seen in mdx models through expansion of the SC juxtavascular niche. Here we review these four vascular targeted treatment strategies for DMD and discuss mechanisms, proof of concept, and the potential for clinical relevance associated with each therapy.

Improved regenerative myogenesis and muscular dystrophy in mice lacking Mkp5

Duchenne muscular dystrophy (DMD) is a degenerative skeletal muscle disease caused by mutations in dystrophin. The degree of functional deterioration in muscle stem cells determines the severity of DMD. The mitogen-activated protein kinases (MAPKs), which are inactivated by MAPK phosphatases (MKPs), represent a central signaling node in the regulation of muscle stem cell function. Here we show that the dual-specificity protein phosphatase DUSP10/MKP-5 negatively regulates muscle stem cell function in mice. MKP-5 controlled JNK to coordinate muscle stem cell proliferation and p38 MAPK to control differentiation. Genetic loss of Mkp5 in mice improved regenerative myogenesis and dystrophin-deficient mdx mice lacking Mkp5 exhibited an attenuated dystrophic muscle phenotype. Hence, enhanced promyogenic MAPK activity preserved muscle stem cell function even in the absence of dystrophin and ultimately curtailed the pathogenesis associated with DMD. These results identify MKP-5 as an essential negative regulator of the promyogenic actions of the MAPKs and suggest that MKP-5 may serve as a target to promote muscle stem cell function in the treatment of degenerative skeletal muscle diseases.

Acute failure of action potential conduction in mdx muscle reveals new mechanism of contraction‐induced force loss

•  A primary feature of skeletal muscle lacking the protein dystrophin, as occurring in Duchenne muscular dystrophy, is a hypersensitivity to eccentric contraction‐induced injury. Dystrophin is associated with the plasmalemma which in healthy muscle has the physiological function of maintaining resting membrane potential to facilitate action potential generation and conduction during muscle activation. •  We tested the hypothesis that the physiological function of the plasmalemma is impaired as a result of eccentric contractions in dystrophic skeletal muscles. •  Electromyographic analysis of dystrophic muscle from mdx mice, the murine model of Duchenne muscular dystrophy, during and immediately after eccentric contractions revealed impairment in the muscles ability to generate and conduct action potentials. •  In agreement with our electromyographic analysis, assessment of resting membrane potentials showed that dystrophic muscle cells are depolarized immediately after an injurious bout of eccentric contractions. •  Our results suggest a major plasmalemma‐based mechanism of strength loss underlying eccentric contraction‐induced injury in dystrophic muscle.

Increased angiogenesis and improved left ventricular function after transplantation of myoblasts lacking the MyoD gene into infarcted myocardium.

Skeletal myoblast transplantation has therapeutic potential for repairing damaged heart. However, the optimal conditions for this transplantation are still unclear. Recently, we demonstrated that satellite cell-derived myoblasts lacking the MyoD gene (MyoD−/−), a master transcription factor for skeletal muscle myogenesis, display increased survival and engraftment compared to wild-type controls following transplantation into murine skeletal muscle. In this study, we compare cell survival between wild-type and MyoD−/− myoblasts after transplantation into infarcted heart. We demonstrate that MyoD−/− myoblasts display greater resistance to hypoxia, engraft with higher efficacy, and show a larger improvement in ejection fraction than wild-type controls. Following transplantation, the majority of MyoD−/− and wild-type myoblasts form skeletal muscle fibers while cardiomyocytes do not. Importantly, the transplantation of MyoD−/− myoblasts induces a high degree of angiogenesis in the area of injury. DNA microarray data demonstrate that paracrine angiogenic factors, such as stromal cell-derived factor-1 (SDF-1) and placental growth factor (PlGF), are up-regulated in MyoD−/− myoblasts. In addition, over-expression and gene knockdown experiments demonstrate that MyoD negatively regulates gene expression of these angiogenic factors. These results indicate that MyoD−/− myoblasts impart beneficial effects after transplantation into an infarcted heart, potentially due to the secretion of paracrine angiogenic factors and enhanced angiogenesis in the area of injury. Therefore, our data provide evidence that a genetically engineered myoblast cell type with suppressed MyoD function is useful for therapeutic stem cell transplantation.

Post-mitotic role of nucleostemin as a promoter of skeletal muscle cell differentiation.

Nucleostemin (NS) is a nucleolar protein abundantly expressed in a variety of proliferating cells and undifferentiated cells. Its known functions include cell cycle regulation and the control of pre-rRNA processing. It also has been proposed that NS has an additional role in undifferentiated cells due to its downregulation during stem cell differentiation and its upregulation during tissue regeneration. Here, however, we demonstrate that skeletal muscle cell differentiation has a unique expression profile of NS in that it is continuously expressed during differentiation. NS was expressed at similar levels in non-proliferating muscle stem cells (satellite cells), rapidly proliferating precursor cells (myoblasts) and post-mitotic terminally differentiated cells (myotubes and myofibers). The sustained expression of NS during terminal differentiation is necessary to support increased protein synthesis during this process. Downregulation of NS inhibited differentiation of myoblasts to myotubes, accompanied by striking downregulation of key myogenic transcription factors, such as myogenin and MyoD. In contrast, upregulation of NS inhibited proliferation and promoted muscle differentiation in a p53-dependent manner. Our findings provide evidence that NS has an unexpected role in post-mitotic terminal differentiation. Importantly, these findings also indicate that, contrary to suggestions in the literature, the expression of NS cannot always be used as a reliable indicator for undifferentiated cells or proliferating cells.

Efficient Single Muscle Fiber Isolation from Alcohol-Fixed Adult Muscle following β-Galactosidase Staining for Satellite Cell Detection

Staining for β-galactosidase activity for whole tissues, sections, and cells is a common method to detect expression of β-galactosidase reporter transgene as well as senescence-dependent β-galactosidase activity. Choice of fixatives is a critical step for detection of β-galactosidase activity, subsequent immunostaining, and enzymatic digestion of tissue to dissociate cells. In this report, the authors examined several aldehyde and alcohol fixatives in mouse skeletal muscle tissues for their efficiency at improving detection of β-galactosidase activity as well as detection by immunostaining. In addition, fixatives were also analyzed for their efficiency for collagenase digestion to isolate single muscle fibers on postfixed β-galactosidase-stained whole skeletal muscle tissues. The results show that fixing cells with isopropanol yields the greatest reliability and intensity in both β-galactosidase staining as well as double staining for β-galactosidase activity and antibodies. In addition, isopropanol and ethanol, but not glutaraldehyde or paraformaldehyde, allow for the isolation of single muscle fibers from the diaphragm and tibialis anterior muscles following postfixed β-galactosidase staining. Using this method, it is possible to identify the amount of cells that occupy the satellite cell compartment in single muscle fibers prepared from any muscle tissues, including tibialis anterior muscle and diaphragm.