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

CD Ileal RNA-seq Study

Our IBDGC post-op ileal study integrates bulk RNA-seq data from the terminal ileum, single cell RNA-seq data from ileal resection samples, and GSA genotype data

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

Increased angiogenesis in scar area after myocardial injection of myoblasts.

<p>(A) Representative images of heart sections 1 week after cell injection. X-gal positive cells in the scar area indicate the progeny of injected cells. Green fluorescence indicates CD31⁺ endothelial cells in the vasculature. Nuclei were counter-stained with DAPI (blue). Scale bar = 50 µm. (B) Vascular density was measured using anti-CD31 antibody staining of scar tissue cross sections 1 week after injection of wild-type myoblasts, <i>MyoD^−/−</i> myoblasts, or medium (n = 3). (C) FACS analysis showed an increase in the number of CD45⁺ blood cells (arrows) and CD45^- CD31⁻ Sca-1⁺ cardiac progenitor cells (black boxes) and a decrease in the number of CD45⁻ CD31⁺ endothelial cells (red boxes) in the hearts of mice receiving wild-type myoblasts, <i>MyoD^−/−</i> myoblasts, or medium alone. (D) The total number of CD45⁺ blood cells, CD45^- CD31⁻ Sca-1⁺ cardiac progenitors, and CD45⁻ CD31⁺ endothelial cells was estimated using FACS data (n = 3).</p

Engraftment of wild-type and <i>MyoD^−/−</i> myoblasts after myocardial injection into infarcted heart.

<p>(A) These panels show MI induced by left coronary artery ligation. Wild-type and <i>MyoD^−/−</i> myoblasts were directly injected into the peri-infarct regions of LV. After 1 week, X-gal staining of whole heart indicated that more <i>MyoD^−/−</i> myoblasts engrafted than wild-type myoblasts (arrows). Arrowheads indicate left coronary artery ligation points. X-gal staining of cross sections indicated that more <i>MyoD^−/−</i> myoblasts than wild-type myoblasts engrafted in both injured and uninjured areas of the heart. Arrows indicate engrafted lacZ⁺ wild-type and <i>MyoD^−/−</i> myoblasts. Scale bars = 1 mm. (B) Engraftment of injected cells in the infarcted heart. More <i>MyoD^−/−</i> myoblasts than wild-type myoblasts engraft in the infarcted heart 3 day to 4 weeks post-transplantation. Y-axis indicates the survival rates of engrafted cells after an injection of 1×10⁶ wild-type or <i>MyoD^−/−</i> myoblasts. (C) By day 3, anti-activated caspase-3 (Casp-3) and anti-phospho-histone H3 (p-His-H3) antibody staining demonstrated that lacZ⁺ wild-type and <i>MyoD^−/−</i> myoblasts proliferate or undergo apoptotic cell death (n = 4). Nuclei were counter-stained with DAPI (blue). Scale bar = 50 µm. (D) Comparison of the relative numbers of lacZ⁺/activated caspase-3⁺ or lacZ⁺/phospho-histone H3⁺ cells for wild-type and <i>MyoD^−/−</i> myoblasts 3 days after transplantation.</p

Expression of myogenic markers in wild-type and <i>MyoD^−/−</i> myoblast cultures.

<p>(A) Fluorescence views of myoblasts isolated from wild-type and <i>MyoD^−/−</i> mice. Immunostaining for Pax7 (green) and MyoD (red) showed that nearly all wild-type myoblasts expressed Pax7 and MyoD while <i>MyoD^−/−</i> cells expressed only Pax7 (n = 3). Scale bar = 25 µm. (B) After 3 days of culture in low serum conditions, sarcomeric myosin heavy chain (MHC) immunostaining clearly demonstrated that wild-type myoblasts differentiated into MHC⁺ myocytes and myotubes while myogenic differentiation was delayed for <i>MyoD^−/−</i> myoblasts (n = 3). Scale bar = 50 µm. (C) More than 90% of stable wild-type and <i>MyoD^−/−</i> myoblast transformants expressed nuclear lacZ after co-transfection of PGK-nlacZ-MAR/PGK-puro followed by puromycin selection. Scale bar = 50 µm. Nuclei were counter-stained with DAPI (blue).</p

Skeletal muscle fiber differentiation of engrafted wild-type and <i>MyoD^−/−</i> myoblasts following transplantation.

<p>(A) Two weeks post-transplantation, immunofluorescence staining of heart cross sections showed that the progeny of lacZ⁺ wild-type and <i>MyoD^−/−</i> myoblasts formed nestin⁺ multinucleated skeletal myotubes. Laminin (red) indicates cardiomyocytes and skeletal myotubes. Arrows indicate lacZ⁺ donor cell-derived nuclei in nestin⁺ myotubes. (B) Comparison of the relative numbers of lacZ⁺/nestin⁺ myotubes for wild-type and <i>MyoD^−/−</i> myoblasts 2 weeks after injection (n = 3). (C) Two weeks post-transplantation, immunofluorescence staining of heart cross sections showed lacZ⁺<i>MyoD^−/−</i> myoblast-derived cardiomyocyte-like cells express cardiac troponin T (cTnT, red) in their cytoplasm and myocyte enhancer factor 2 (MEF2, green) in their nucleus (arrows). (D) Secondary antibodies alone did not show any background staining. Nuclei were counter-stained with DAPI (blue). Scale bars = 50 µm.</p