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 downregulated. 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.apoptosis ͉ cell therapy ͉ microarrays ͉ muscular dystrophy ͉ satellite cell

Background. Activation of the adenosine A2B receptor (A2BR) can reduce myocardial ischemia/reperfusion (IR) injury. However, the mechanism underlying the A2BR-mediated cardioprotection is less clear. The present study was designed to investigate the potential mechanisms of cardioprotection mediated by A2BR. Methods and Results. C57BL/6 mice underwent 40-minute ischemia and 60-minute reperfusion. ATL-801, a potent selective A2BR antagonist, could not block ischemic preconditioning induced protection. BAY 60-6583, a highly selective A2BR agonist, significantly reduced myocardial infarct size, and its protective effect could be blocked by either ATL-801 or wortmannin. BAY 60-6583 increased phosphorylated Akt (p-Akt) levels in the heart at 10 min of reperfusion, and this phosphorylation could also be blocked by ATL-801 or wortmannin. Furthermore, BAY 60-6583 significantly increased M2 macrophages and decreased M1 macrophage and neutrophils infiltration in reperfused hearts, which also could be blocked by wortmannin. Meanwhile, confocal imaging studies showed that the majority of Akt phosphorylation in the heart was colocalized to CD206+ cells in both control and BAY 60-6583 pretreated hearts. Conclusion. Our results indicated that pretreatment with BAY 60-6583 protects the heart against myocardial IR injury by its anti-inflammatory effects, probably by modulating macrophages phenotype switching via a PI3K/Akt pathway.

Targeting therapeutic gene expression to skeletal muscle following intravenous administration is an attractive strategy for treating peripheral arterial disease (PAD), except that vector access to the ischemic limb could be a limiting factor. Since AAV serotype 9 transduces skeletal muscle at high efficiency following systemic delivery, we employed AAV-9 vectors bearing luciferase or enhanced green fluorescent protein (eGFP) reporter genes to test the hypothesis that increased desialylation of cell surface glycans secondary to hindlimb ischemia (HLI) might help offset the reduction in tissue perfusion that occurs in mouse models of PAD. The utility of the creatine kinase-based (CK6) promoter for restricting gene expression to skeletal muscle was also examined by comparing it to the cytomegalovirus (CMV) promoter after systemic administration following surgically-induced HLI. Despite reduced blood flow to ischemic limbs, CK6 promoter-driven luciferase activities in ischemic gastrocnemius (GA) muscles were ~34-, ~28-, and ~150-fold higher than in fully-perfused contralateral GA, heart, and liver, respectively, 10 days after intravenous administration. Furthermore, luciferase activity from the CK6 promoter in ischemic GA muscles was ~2-fold higher than with CMV, while in the liver CK6-driven activity was ~42-fold lower than with CMV, demonstrating that the specificity of ischemic skeletal muscle transduction can be further improved with muscle-specific promoters. Studies with Evans blue dye and fluorescently-labeled lectins revealed that vascular permeability and desialylation of cell surface glycans were increased in ischemic hindlimbs. Furthermore, AAV9/CK6/Luc vector genome copy numbers were ~6-fold higher in ischemic muscle compared to non-ischemic muscle in the HLI model, whereas this trend was reversed when the same genome was packaged in the AAV-1 capsid (which binds sialylated, as opposed to desialylated glycans), further underscoring the importance of desialylation in the ischemic enhancement of transduction displayed by AAV-9. Taken together, these findings suggest two complementary mechanisms contributing to the preferential transduction of ischemic muscle by AAV-9: increased vascular permeability and desialylation. In conclusion, ischemic muscle is preferentially targeted following systemic administration of AAV-9 in a mouse model of HLI. Unmasking of the primary AAV-9 receptor as a result of ischemia may contribute importantly to this effect.