Skeletal muscle is capable of extensive regeneration following injury. Injury induces an early inflammatory response followed by the activation of resident muscle precursors (satellite cells), which subsequently differentiate to replace and repair muscle fibers . A subpopulation of satellite cells re-enters a quiescent state, providing a reserve myogenic population . These phenomena rely on asymmetric self-renewal and commitment of satellite cells . Multipotent stem cells are also present in adult skeletal muscle, although their anatomical localization has not been clearly resolved and the extent of their contribution to regenerating muscle is unclear . Although satellite cells are located underneath the basal lamina, non-lineage-restricted precursor cells with myogenic potential, such as mesoangioblasts, bone marrow-derived cells, and side population cells, display various localizations in the interstitial space [5, –7] Mesoangioblasts have successfully been exploited in the treatment of muscular dystrophy in mice and dogs . Transplant experiments showed that bone marrow-derived cell incorporation into myofibers occurs at a low frequency and increases following muscle injury . Striking changes in the number of nuclei per fiber over time were reported, negatively affected by atrogenic stimuli such as denervation [10, 11]. The occurrence of fibers with centrally located nuclei associated with muscle hypertrophy in the absence of damage has been reported [12, 13]. These reports highlight the importance of precursor cell recruitment and incorporation into fibers during muscle growth and homeostasis. How cytokines influence muscle precursor cell behavior and muscle regeneration is of relevance in several pathological conditions characterized by elevated levels of cytokines associated with muscle wasting . In particular, cachexia is a devastating fat and skeletal muscle wasting syndrome displayed by patients with chronic diseases, including cancer, AIDS, chronic heart, and kidney failure. Cachexia interferes with therapies and increases morbidity and mortality [15, 16]. Inflammatory cytokines promote cachexia and are targets of therapeutic approaches . Several cytokines, including IL-1, IL-6, and tumor necrosis factor-α (TNF; abbreviation is in accordance with ), as well as other factors of tumor (proteolysis inducing factor) or host (interferon-γ, leukemia inhibitory factor, transforming growth factor-β) origin, have been identified as promoters of cachexia [14, 18]. We have demonstrated that TNF is sufficient to induce cachexia, as well as inhibition of muscle regeneration . TNF has also been shown to downregulate the myogenic factor MyoD in vivo . Muscle atrophy is coupled with an impairment in myogenic potential of muscle precursor cells [21, 22], confirming that these cells are required for muscle recovery. Multiple pathways are involved in the TNF-mediated inhibition of myogenesis, including the downregulation of MyoD and myogenin , decrease in MyoD protein stability , and induction of proliferation through cyclin D1 . We showed that TNF-mediated inhibition of muscle differentiation requires the p53 cell-death effectors PW1 and Bax [26, 27]. Furthermore, we reported that muscle stem cells show constitutively activated p53 and that loss of p53 function alters muscle stem cell number . In addition, tumor-bearing p53-null mice are resistant to cachexia. We have also shown that the p53 effector PW1 cooperates with p53 in regulating stem cell number and muscle atrophy in vivo . In cultured myogenic cells, PW1 recruits p53-dependent apoptotic pathways, including downstream caspase activation, which participates in the regulation of myogenesis . Whereas apoptosis is a most likely cellular outcome of caspase activation, several studies demonstrate that caspases also play a role in mediating cell differentiation in specific lineages [29, –31].
TNF levels are barely detectable in uninjured skeletal muscle and increase following muscle injury [32, 33]. TNF localizes to the infiltrating inflammatory cells during the first 3 days following injury and is subsequently detected in regenerating myofibers [32, 34]. Although in vivo studies reveal a role for TNF during muscle regeneration [20, 32, 34, –36], the molecular and cellular pathways triggered by TNF in this process remain poorly understood.
A direct role for myogenic stem cells during cachexia remains to be clearly demonstrated, although deregulation of stem cell number or behavior leads to decreased muscle mass [37, 38]. We recently reported that cachexia is associated with more hematopoietic stem cells in skeletal muscle, coupled with a compromised regenerative capacity of the musculature [19, 39]. In this context, it is important to address the mechanisms underlying such impaired regenerative potential of the muscle. We propose that TNF abrogates stem cell function, which in turn delays or inhibits muscle regeneration.
In this study, we demonstrated that TNF inhibits muscle regeneration by upregulating caspase activity in a subpopulation of interstitial cells that can be identified by specific stem cell markers, such as Sca-1+, CD34+, and PW1+. Caspase activation occurs in the absence of apoptosis and in a PW1-dependent manner. These results provide new insights in the molecular mechanisms underlying TNF-mediated effects on muscle regeneration.