HEXIM1 and its partner P-TEFb have been demonstrated to regulate a wide range of physiological and pathological processes, such as embryonic and cardiac development, cardiomyocyte hypertrophic growth, and human immunodeficiency virus 1 infection (28
). Such diverse functions of the HEXIM1/P-TEFb pathway are believed to be a result of its role in the regulation of global gene expression during transcription pausing escape and productive elongation (28
). Recently, several reports showed evidence of gene-specific recruitment of P-TEFb by various transcription factors, indicating that P-TEFb not only permits global transcription elongation but also could promote the expression of specific genes by shortening or bypassing transcription pausing (49
). MyoD, the master transcription factor of myogenic differentiation, has been shown to interact with P-TEFb during the transcription of MyoD target genes, and P-TEFb activity is required for the MyoD-mediated differentiation of satellite cells in vitro (33
). In this study, we further demonstrate that skeletal muscle regeneration, which is achieved by the de novo formation of myofibers from the satellite cell pool, is controlled by the HEXIM1/P-TEFb pathway that regulates satellite cell expansion after injury. Mechanistically, during regeneration satellite cells undergo proliferation and then terminal differentiation, both of which are implicated to be modulated by P-TEFb by in vitro studies based on nonmyogenic cell lines (44
). Here, we show that relieved inhibition of P-TEFb by HEXIM1 promotes proliferation and expansion while repressing early myogenic differentiation of satellite cells during skeletal muscle regeneration, which substantiates the role of HEXIM1/P-TEFb as a general regulator of cell proliferation and differentiation (44
). In light of this, we speculate that other regenerative processes involving a proliferation-to-differentiation switch analogous to that of satellite cells, such as adult neurogenesis and wound healing, may be regulated similarly by HEXIM1/P-TEFb. Considering this promising potential, current knowledge regarding the precise effect of HEXIM1/P-TEFb on context-specific transcription is insufficient and calls for further investigation of downstream targets of HEXIM1/P-TEFb and their physiological significance.
Upregulation of P-TEFb in Hexim1+/–
mice increases muscle mass and myofiber number without hypertrophic growth after injury, which is a potentially beneficial effect. In contrast, intramuscular injection of satellite cells after injury increases the size and mass of regenerated muscles and induces hypertrophic growth of myofibers (16
). This difference could be explained by the fact that intramuscular injection of satellite cells increases satellite cell density only in a specific region near the delivery site due to limited migration capability of transplanted satellite cells (20
), whereas the enrichment of satellite cells by enhancing their proliferation in our model was relatively uniform throughout the muscle. Moreover, due to the large amount of cells delivered during injection, these satellite cells may not be completely consumed by the early regeneration process and thus could serve as an extra myogenic source at the later stages of regeneration. Therefore, enhanced proliferation in Hexim1+/–
muscles is unlikely to result in muscle hypertrophy as induced by injection of satellite cells.
Blockade of myostatin, a powerful inhibitor of skeletal muscle growth, enhances global myogenesis, including both muscle maintenance and regeneration (51
), while haplodeficiency of Hexim1
specifically promotes muscle regeneration after injury. The preserved functional capacity of Hexim1+/–
muscles also indicates a clear mechanistic departure from that of myostatin blockade where there is reduced functional capacity (52
). It has been shown that muscle function is not only proportional to the muscle size but also depends on the myonuclei density in each myofiber, which is maintained by satellite cell differentiation and fusion with myofibers (53
). Others have reported that satellite cells undergo limited self-renewal by asymmetric division during muscle growth and maintenance but proliferate symmetrically with vigorous self-renewal and expansion during regeneration (39
). In fact, the cytokines secreted by infiltrating immune cells during early regeneration, which can activate P-TEFb to promote the rapid expansion of the satellite cell pool, are present at much lower levels under resting conditions (1
), rendering the reduced HEXIM1 level sufficient to maintain normal P-TEFb activity for the maintenance of the satellite cell pool. Therefore, we reasoned that haplodeficiency of Hexim1
does not affect muscle function during maintenance, due to low stimulation of the HEXIM1/P-TEFb pathway under resting conditions, but promotes muscle regeneration and functional recovery by augmenting the activation of P-TEFb, leading to rapid expansion and accumulation of satellite cells after injury. In contrast, recent reports showed that myostatin inhibition–induced hypertrophy was independent of satellite cell activity and possibly resulted from altered metabolism of muscle proteins (55
), and this may explain the additional effect of myostatin inhibition on muscle maintenance.
Transplantation of satellite cells is a promising therapy to cure muscular dystrophies and promote muscle regeneration, while several obstacles, including the large numbers of satellite cells required to regenerate sufficient myofibers and the rapid loss of proliferative and regenerative potential after ex vivo expansion, prevent its wide application (10
). We have shown that cultured satellite cells proliferate at an optimal rate, without losing stem cell characteristics or regenerative potential when the cellular HEXIM1 protein level is reduced by half. Spontaneous cell cycle exit and early myogenic differentiation, a common but undesirable phenomenon in therapeutic satellite cell cultures, were rarely observed in Hexim1+/–
cultures. Furthermore, transplantation of Hexim1+/–
satellite cells leads to enhanced expansion of the satellite cell pool after injury and improves the outcome of muscle regeneration more effectively than transplantation of WT satellite cells. Therefore, lowering the cellular HEXIM1 protein level could be a useful approach to improve the efficacy of therapeutic satellite cell cultures.
It is noteworthy that cardiomyocyte-specific hyperactivation of P-TEFb by overexpression of cyclin T1 leads to hypertrophic growth of cardiomyocytes and accelerates the progression of heart failure (45
). However, because of the relatively low numbers of successfully engrafted satellite cells, as compared with the endogenous satellite cell pool as shown in clinical trials (10
), it is possible that only a small fraction of myonuclei in the regenerated myofibers could be derived from transplanted satellite cells that have reduced HEXIM1 levels. It is also conceivable that WT myonuclei could increase Hexim1
expression to compensate for the Hexim1+/–
myonuclei located in the same myofiber, which therefore maintains a normal level of overall gene expression and protein synthesis. In fact, the injured WT muscles receiving Hexim1+/–
satellite cell transplantation did not develop hypertrophy during regeneration, despite the significant increase in size and myofiber numbers after regeneration. In summary, our findings suggest that transient inhibition of HEXIM1 may improve the ex vivo expansion of satellite cells as well as the therapeutic efficacy of satellite cell transplantation following ex vivo cultures and that controlled inhibition of HEXIM1 in vivo, if it has no deleterious side effect, could possibly benefit patients with muscular disorders.