Murine double minute-2 expression is required for capillary maintenance and exercise-induced angiogenesis in skeletal muscle.

Exercise-induced angiogenesis is a key determinant of skeletal muscle function. Here, we investigated whether the E3 ubiquitin ligase murine double minute-2 (Mdm2) exerts a proangiogenic function in exercised skeletal muscle. Mdm2 hypomorphic (Mdm2(Puro/Δ7-9)) mice have a 60% reduction in Mdm2 expression compared with that in wild-type animals. Capillary staining on muscle sections from Mdm2(Puro/Δ7-9) sedentary mice with a wild-type or knockout background for p53 revealed that deficiency in Mdm2 resulted in 20% capillary regression independently of p53 status. In response to one bout of exercise, protein expression of the proangiogenic vascular endothelial growth factor-A (VEGF-A) was increased by 64% in muscle from wild-type animals, and endothelial cell outgrowth from exercised muscle biopsy samples cultured in a 3-dimensional collagen gel was enhanced by 37%. These proangiogenic responses to exercise were impaired in exercised Mdm2(Puro/Δ7-9) mice. Prolonged exercise training resulted in increased Mdm2 protein expression (+49%) and capillarization (+24%) in wild-type muscles. However, exercise training-induced angiogenesis was abolished in Mdm2(Puro/Δ7-9) mice. Finally, exercise training restored Mdm2, VEGF-A, and capillarization levels in skeletal muscles from obese Zucker diabetic fatty rats compared with those in healthy animals. Our results define Mdm2 as a crucial regulator of capillary maintenance and exercise-induced angiogenesis in skeletal muscle.

Striated muscle angio-adaptation requires changes in Vasohibin-1 expression pattern.

Vasohibin-1 (VASH-1) was recently identified as a negative feedback regulator of angiogenesis. Here, we analyzed how the expression of the two active anti-angiogenic VASH-1 isoforms p36 and p42 was altered during physiological and pathological muscle angio-adaptation. Our results showed that VASH-1 protein expression was muscle-type specific, with higher levels detected in less vascularized muscles. In rat plantaris and heart muscles, the expression of VASH-1 protein was decreased in response to exercise training, a physiological pro-angiogenic stimulus leading to muscle capillary growth. Interestingly, expression patterns for p36 and p42 were different between plantaris and heart muscles. Next, we analyzed the time-course expression of VASH-1 isoforms in rat soleus muscles subjected to hindlimb unloading, a model that induces muscle capillary regression. Both p36 and p42 isoforms were increased, a signal in favor of some vessel destabilization and regression. Finally, we investigated VASH-1 expression in plantaris muscles from Zucker Diabetic Fatty rats (ZDF) that develop obesity and type-2 diabetes associated with a loss of capillaries in skeletal muscle. VASH-1 expression was higher in sedentary ZDF rats when compared to lean animals, suggesting its potential role during capillary regression. Interestingly, a physiological VASH-1 level was efficiently restored in spontaneously active ZDF animals where muscle capillarization was preserved. In conclusion, our results bring evidence that endogenous VASH-1 isoforms p36 and p42 are key actors of physiological and pathological muscle angio-adaptation.