Interactions of muscle regulatory factors and isoforms of insulin growth factor-1 in the muscle regenerative process

Skeletal muscle regeneration following damage is mainly attributed to activation, proliferation and myogenic differentiation of muscle stem cells known as satellite cells. Satellite cells lie beneath the basal lamina that surrounds muscle fibres. These pluripotent cells respond to muscle injury to initiate the process of satellite cell myogenesis, regulated by several transcription factors known as myogenic regulatory factors (MRFs): MyoD, Myf5, myogenin and MRF4. Pax-7-expressing quiescent satellite cells, upon activation, up-regulate MyoD and Myf5 expression to generate myogenic lineage cells, or myoblasts. Myoblasts proliferate and subsequently up-regulate myogenin and MRF4 levels leading to terminal differentiation into muscle fibres (Charge & Rudnicki, 2004). On the other hand, insulin-like growth factor−1 (IGF-1) signalling was implicated in the process of myogenesis. In vitro studies demonstrated that IGF-1 positively regulated myogenesis in a two-step process, first by cell proliferation of stem cells and later by enhancing differentiation process (Rosenthal & Cheng, 1995; Engert et al. 1996). Transgenic mice with muscle-specific IGF-1 isoform supplementation have muscle regenerative capacity in advanced ages, suggesting muscle-specific IGF-1 in the regenerative process (Musaro et al. 2001). To date, three isoforms of IGF-1 have been detected in skeletal muscle: IGF-1Ea, IGF-1Eb and IGF-1Ec (IGF-1Ec is referred to as mechano-growth factor, MGF) (Rotwein, 1986; Yang et al. 1996). The roles of these isoforms in muscle repair, particularly their regulation of MRFs, was not elucidated in detail. A recent report by McKay et al. (2008) in The Journal of Physiology investigated the temporal gene expression of MRFs and IGF-1 isoforms in human quadriceps femoris upon exercise-induced muscle damage. Eight male subjects were recruited to perform intensive muscle-lengthening exercise. Muscle biopsies were collected before (0 h), and after exercise at 4, 24, 72 and 120 h. Immunofluorescence assays were done to determine localization of IGF-1 and Pax-7 proteins after the exercise-induced damage. The authors speculate about the interaction of IGF-1 signalling and transcription of MRFs based on gene expression and immunoflourescence data. In summary, MyoD and Myf5 gene expressions were significantly up-regulated at 4 h and 24 h post-exercise, respectively. Up-regulation of myogenin gene expression was observed from 4 h onwards, and MRF4 expression was significantly up-regulated at 72 and 120 h. These observations are partly inconsistent with accepted cascade of myogenic differentiation, as the authors observed up-regulation of myogenin prior to Myf5. This clearly suggests limitations of interpretations based on crude muscle samples and gene expression analyses to acutely delineate a cascade in response to damage. The authors also measured transcript levels of IGF-1Ea, IGF-1Eb and MGF. MGF and IGF-1Ea were significantly up-regulated at 24 h and 72 h, respectively, whereas IGF-1Eb was up-regulated at 72 and 120 h. These data were then used to determine correlations between IGF-1 isoforms and MRF gene expressions. The authors report a correlation of MGF with Myf5, while IGF-1Ea and IGF-1Eb correlated with MRF4 expression. The observed correlations are limited in their capacity to provide biological information, because IGF-1Ea and IGF-1Eb correlated with only one of the differentiation markers (MRF4), and MGF was correlated with only one of the myogenic precursor markers (Myf5). Adding to this conundrum as noted above is the paradoxical up-regulation of myogenin before Myf5. In essence, the gene expression analyses along with statistical analyses are limited to provide insight into signalling pathways connecting IGF-1 isoforms and MRFs. The observations from similar studies were contradictory to the current report; for example, Psilander et al. (2003) reported that MyoD was up-regulated immediately after exercise and reached baseline after 1 h onwards, whereas MRF4 and myogenin were significantly up-regulated only at 2 h and 6 h, respectively. It has to be noted that the changes in gene expression reported by Psilander et al. were rapid i.e. within 6 h of exercise. At later time-points the levels were not different from baseline. They report data suggesting a decline in IGF-1a levels at 1 h and 6 h post-exercise. These gene expression patterns are difficult to interpret and the apparent decline of IGF-1a in the muscle regeneration process is unclear. These apparent discrepancies in the timeline of gene expression changes and relationship of IGF-1 and MRFs may be attributed to limitations of interpretations solely based upon gene expression changes, different muscle injury protocols and biological variation in subjects chosen for the two studies. Interpretations based on gene expression analyses are highly influenced by temporal effects depending on kinetics of promoter activation (pulsatile association of transcription factors on promoter), pre-mRNA to mature mRNA formation and half-life of mature mRNA. Due to intrinsic differences in these regulatory processes among transcripts, several minutes to days may be necessary to reliably quantify gene expression changes in response to injury. Thus, interpretations supported by associated protein changes will provide strength to delineate muscle regenerative process. On the other hand, McKay et al. claim that IGF-1 protein was localized only in satellite cells at 24 h, but present both in satellite cells and myofibres at 72 h and 120 h post-exercise. Immunofluorescence data were difficult to interpret due to colocalization of DAPI with IGF-1 but, nonetheless, suggest that IGF-1 is localized to the nucleus of satellite cells. This is unexpected of IGF-1, a secreted protein that is localized in secretory vesicles. Thus, it is difficult to decipher biological information that links different isoforms of IGF and MRFs based on the immunoflourescence data. Although in vitro/rodent models laid foundations for fundamental understanding of muscle regeneration process, understanding details of myogenic regulatory mechanisms in human beings is needed for realization of potential for therapeutic interventions. In this regard, use of human subjects by authors is highly appreciated, as it has the potential for discovering physiologically relevant molecular mechanisms. Future studies should include an examination as to whether the regenerative process is influenced by the extent of muscle damage and associated inflammatory response upon injury. This can be accomplished by determining the levels of serum creatine kinase, an indicator of muscle damage, and also by quantifying the inflammatory response by histological measurement of invading neutrophils and macrophages in injured muscle. This could then be followed by further analyses to determine the relationship between serum creatine kinase, inflammatory response and expression profiles of MRFs/IGF-1 isoforms. The extent of the regenerative process may also depend on basal satellite cell population, their activation and proliferation. In this context, measuring transcript levels of satellite cell marker Pax-7 will provide in indirect measure of relative satellite cell population/activity at different stages of the repair process. The presence of three isoforms of IGF-1 in muscle and their putative roles in repair underlines the importance of understanding their complicated regulatory and signalling mechanisms. Toward understanding IGF-1 biology in the muscle regenerative process, it is important to understand if one or more of the MRFs directly/indirectly regulate transcription or splicing of IGF-1 genes. There is a greater need for distinguishing the three isoforms of IGF-1 at the protein level and quantifying each of these isoforms, and also to determine their signalling. This becomes complex, as IGF proteins upon secretion bind to IGF-binding proteins (IGFBPs). There are several IGFBPs, and specific interactions of the IGF isoforms and IGFBPs may lead to different results, such as inhibitition or stimulation of IGF-1 signalling. This complexity can be partially addressed by conducting studies using transgenic rodent models with over-expression of each of the splice variants in muscle and determine IGF signalling and the myogenic process after injury. Further research in vitro may also be used to determine isoform-specific IGF signalling to gene expression of different MRFs, which involves determining the activated transcription factors and their responsive cis-elements on different MRFs. In addition, recent genetic models such as zebrafish can also be used to better understand the fundamentals of molecular regulation of the myogenic process, as the mechanisms of embryonic muscle development are similar to muscle regeneration. In conclusion, there is a clear need for complementary approaches using different models to understand regulatory and signalling mechanisms of each of the IGFs and MRFs. In the long term, studies using these approaches will help us to better understand and treat muscle degeneration.