Histochemical analysis of damage and regeneration in skeletal muscle wasting

Cachexia is a severe form of muscle wasting, triggered by elevated levels of cytokines in chronic diseases,which interferes with the management of the primary disease and accounts for the death of a significant percentage of patients. In order to identify mechanisms underlying the loss of muscle homeostasis in cachexia, we analyzed the effects of cytokines on skeletal muscle damage and regenerative capacity. We exploited the properties of Evans Blue Dye to detect sarcolemmal damage in a murine model of cachexia. Fiber damage-associated inflammation was also revealed by immuno-histochemistry for activated macrophages invading muscle fibers. Muscles responded to damage and necrosis by activating a satellite cell -mediated response. The cachectic musculature is enriched in satellite cells expressing high levels of Pax7 and MyoD.However, a morphometric evaluation of the muscle regenerative potential indicated that cachectic muscles lack the ability to fully regenerate following satellite cell activation. The uncoupling between increased fiber necrosis and impaired regeneration resulted in a decreased muscle fiber number per cross-section in the cachectic musculature. Following experimentally-induced damage, intramuscular TNF injection significantly decreased the number and size of the regenerating fibers, an effect abolished by treatment with a caspase inhibitor. Active caspases were highlighted in interstitial cells expressing stem cell markers in the absence of apoptotic phenomena. TNF-dependent impairment of regeneration was rescued by muscle gene delivery of the dominant negative form of PW1 (a TNF effector) or the V1a receptor (to sensitize muscle to the myogenic factor Vasopressin). We conclude that both increased damage and decreased regeneration contribute to muscle wasting in cachexia, suggesting to counteract cytokine effects on muscle regeneration by pharmacological and gene therapy approaches.