Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis

We assessed viable Pax7(-/-) mice in 129Sv/J background and observed reduced growth and marked muscle wasting together with a complete absence of functional satellite cells. Acute injury resulted in an extreme deficit in muscle regeneration. However, a small number of regenerated myofibers were detected, suggesting the presence of residual myogenic cells in Pax7-deficient muscle. Rare Pax3(+)/MyoD+ myoblasts were recovered from Pax7(-/-) muscle homogenates and cultures of myofiber bundles but not from single myofibers free of interstitial tissues. Finally, we identified Pax3+ cells in the muscle interstitial environment and demonstrated that they coexpressed MyoD during regeneration. Sublaminar satellite cells in hind limb muscle did not express detectable levels of Pax3 protein or messenger RNA. Therefore, we conclude that interstitial Pax3+ cells represent a novel myogenic population that is distinct from the sublaminar satellite cell lineage and that Pax7 is essential for the formation of functional myogenic progenitors from sublaminar satellite cells.     

The Transition from Proliferation to Differentiation Is Delayed in Satellite Cells from Mice Lacking MyoD

Satellite cells from adult rat muscle coexpress proliferating cell nuclear antigen and MyoD upon entry into the cell cycle, suggesting that MyoD plays a role during the recruitment of satellite cells. Moreover, the finding that muscle regeneration is compromised in MyoD-/- mice, has provided evidence for the role of MyoD during myogenesis in adult muscle. In order to gain further insight into the role of MyoD during myogenesis in the adult, we compared satellite cells from MyoD-/- and wildtype mice as they progress through myogenesis in single-myofiber cultures and in tissue-dissociated cell cultures (primary cultures). Satellite cells undergoing proliferation and differentiation were traced immunohistochemically using antibodies against various regulatory proteins. In addition, an antibody against the mitogen-activated protein kinases ERK1 and ERK2 was used to localize the cytoplasm of the fiber-associated satellite cells regardless of their ability to express specific myogenic regulatory factor proteins. We show that during the initial days in culture the myofibers isolated from both the MyoD-/- and the wildtype mice contain the same number of proliferating, ERK+ satellite cells. However, the MyoD-/- satellite cells continue to proliferate and only a very small number of cells transit into the myogenin+ state, whereas the wildtype cells exit the proliferative compartment and enter the myogenin+ stage. Analyzing tissue-dissociated cultures of MyoD-/- satellite cells, we identified numerous cells whose nuclei were positive for the Myf5 protein. In contrast, quantification of Myf5+ cells in the wildtype cultures was difficult due to the low level of Myf5 protein present. The Myf5+ cells in the MyoD-/- cultures were often positive for desmin, similar to the MyoD+ cells in the wildtype cultures. Myogenin+ cells were identified in the MyoD-/- primary cultures, but their appearance was delayed compared to the wildtype cells. These "delayed" myogenin+ cells can express other differentiation markers such as MEF2A and cyclin D3 and fuse into myotubes. Taken together, our studies suggest that the presence of MyoD is critical for the normal progression of satellite cells into the myogenin+, differentiative state. It is further proposed that the Myf5+/MyoD- phenotype may represent the myogenic stem cell compartment which is capable of maintaining the myogenic precursor pool in the adult muscle.     

Myf5 and MyoD activation define independent myogenic compartments during embryonic development

Gene targeting has indicated that Myf5 and MyoD are required for myogenic determination because skeletal myoblasts and myofibers are missing in mouse embryos lacking both Myf5 and MyoD. To investigate the fate of Myf5:MyoD-deficient myogenic precursor cells during embryogenesis, we examined the sites of epaxial, hypaxial, and cephalic myogenesis at different developmental stages. In newborn mice, excessive amounts of adipose tissue were found in the place of muscles whose progenitor cells have undergone long-range migrations as mesenchymal cells. Analysis of the expression pattern of Myogenin-lacZ transgene and muscle proteins revealed that myogenic precursor cells were not able to acquire a myogenic fate in the trunk (myotome) nor at sites of MyoD induction in the limb buds. Importantly, the Myf5-dependent precursors, as defined by Myf5(nlacZ)-expression, deficient for both Myf5 and MyoD, were observed early in development to assume nonmuscle fates (e.g., cartilage) and, later in development, to extensively proliferate without cell death. Their fate appeared to significantly differ from the fate of MyoD-dependent precursors, as defined by 258/-2.5lacZ-expression (-20 kb enhancer of MyoD), of which a significant proportion failed to proliferate and underwent apoptosis. Taken together, these data strongly suggest that Myf5 and MyoD regulatory elements respond differentially in different compartments.     

Interaction between satellite cells and skeletal muscle fibers

Single myofibers with attached satellite cells isolated from adult rats were used to study the influence of the mature myofiber on the proliferation of satellite cells. The satellite cells remain quiescent when cultured in serum containing medium but proliferate when exposed to mitogen from an extract of crushed adult muscle. The response of satellite cells to mitogen was measured under three situations with respect to cell contact: (1) in contact with a viable myofiber and its basal lamina, (2) detached from the myofiber by centrifugal force and deposited on the substratum and (3) beneath the basal lamina of a Marcaine killed myofiber. The results show that satellite cells in contact with the plasmalemma of a viable myofiber have reduced mitogenic response. Since inhibiting growth may induce differentiation, I tested whether satellite cells proliferating on the surface of a myofiber would fuse. Although the satellite cell progeny were fusion competent, they did not fuse with the myofiber. To determine whether fusion competence of the myofiber changes with time in culture, embryonic myoblasts were challenged to fuse with myofibers that had been stripped of satellite cells and cultured for several days. The myoblasts fused with pseudopodial sprouts growing from the ends of the myofiber, but did not fuse with the original myofiber surface. These results indicate that contact with the surface of a mature myofiber suppresses proliferation of myogenic cells but the cells do not fuse with the myofiber.     

Bmi1 is expressed in postnatal myogenic satellite cells, controls their maintenance and plays an essential role in repeated muscle regeneration

Satellite cells are the resident stem cell population of the adult mammalian skeletal muscle and they play a crucial role in its homeostasis and in its regenerative capacity after injury. We show here that the Polycomb group (PcG) gene Bmi1 is expressed in both the Pax7 positive (+)/Myf5 negative (-) stem cell population as well as the Pax7+/Myf5+ committed myogenic progenitor population. Depletion of Pax7+/Myf5- satellite cells with reciprocal increase in Pax7+/Myf5+ as well as MyoD positive (+) cells is seen in Bmi1-/- mice leading to reduced postnatal muscle fiber size and impaired regeneration upon injury. Bmi1-/- satellite cells have a reduced proliferative capacity and fail to re-enter the cell cycle when stimulated by high serum conditions in vitro, in keeping with a cell intrinsic defect. Thus, both the in vivo and in vitro results suggest that Bmi1 plays a crucial role in the maintenance of the stem cell pool in postnatal skeletal muscle and is essential for efficient muscle regeneration after injury especially after repeated muscle injury.     

Angiotensin II Inhibits Satellite Cell Proliferation and Prevents Skeletal Muscle Regeneration

Cachexia is a serious complication of many chronic diseases, such as congestive heart failure (CHF) and chronic kidney disease (CKD). Although patients with advanced CHF or CKD often have increased angiotensin II (Ang II) levels and cachexia and Ang II causes skeletal muscle wasting in rodents, the potential effects of Ang II on muscle regeneration are unknown. Muscle regeneration is highly dependent on the ability of a pool of muscle stem cells (satellite cells) to proliferate and to repair damaged myofibers or form new myofibers. Here we show that Ang II reduced skeletal muscle regeneration via inhibition of satellite cell (SC) proliferation. Ang II reduced the number of regenerating myofibers and decreased expression of SC proliferation/differentiation markers (MyoD, myogenin, and active-Notch) after cardiotoxin-induced muscle injury in vivo and in SCs cultured in vitro. Ang II depleted the basal pool of SCs, as detected in Myf5^nLacZ/+ mice and by FACS sorting, and this effect was inhibited by Ang II AT1 receptor (AT1R) blockade and in AT1aR-null mice. AT1R was highly expressed in SCs, and Notch activation abrogated the AT1R-mediated antiproliferative effect of Ang II in cultured SCs. In mice that developed CHF postmyocardial infarction, there was skeletal muscle wasting and reduced SC numbers that were inhibited by AT1R blockade. Ang II inhibition of skeletal muscle regeneration via AT1 receptor-dependent suppression of SC Notch and MyoD signaling and proliferation is likely to play an important role in mechanisms leading to cachexia in chronic disease states such as CHF and CKD.