Interpretación de Resultados

As described above, the early studies of rat enolase showed that expression of P- enolase (muscle-specific enolase, MSE) was restricted to skeletal muscle and heart (Rider and Taylor, 1974). In the former, almost all enolase activity was due to the pp- enolase isoenzyme whilst in the latter, where the NNE and MSE subunits were coexpressed, the ap-enolase heterodimer was shown to predominate. In adult rat, mouse and human skeletal muscle, immunohistochemical and in situ hybridisation studies have shown that MSE is preferentially expressed in fast twitch (type II) muscle fibres (Ibi et al., 1983; Kato et al., 1985; Keller et al., 1992) whilst residual NNE is evenly distributed between fast and slow twitch fibres (Keller et al., 1992). Studies in rat and mouse (Fletcher et al., 1978; Kato et al., 1985) showed that moderate levels of MSE were also found in cartilagenous tissue (again with the heterodimer predominating) and low levels of the protein could be detected in organs such as stomach and bladder which contain smooth muscle. Insignificant levels of MSE were found in other tissues.

In a number of mammals, the level o f MSE protein in adult skeletal muscle has been shown to depend upon the functional state of the muscle and its innervation (Kato et al., 1985; Matsushita et al., 1986; 1991; Satoh et al., 1991; Keller et al., 1992a). Thus, MSE levels have been shown to decline following denervation, and this reflects a general decrease in the levels of enolase and other glycolytic enzymes (Prewitt and Salafsky, 1970; Shackelford and Lebherz, 1981). A moderate increase in NNE protein expression also accompanies denervation, and this may help to explain the

Introduction: Chapter l

foetal enolase isoenzyme profiles observed in some neuromuscular disorders (Edwards et al., 1982).

1.4.2 Developmental regulation in vivo and ex vivo

In rats, only the postnatal ontogeny of MSE has been considered in any detail (Rider and Taylor, 1974; Kato et al., 1985; Sakimura et al., 1989). By studying the relative amounts of the three muscle isoenzymes, a clear difference between heart and skeletal muscle ontogeny was demonstrated (Rider and Taylor, 1974; 1975a). In the heart, MSE was first detected postnatally and its contribution to total enolase activity rose from nothing to 30% by postnatal day 80. The profile o f the three isoenzymes conformed to a best fit binomial distribution based on the abundance of the NNE and MSE subunits, indicating that the subunits were coexpressed, the isoenzymes being generated by random dimerisation. In contrast, MSE was already detectable in foetal skeletal muscle, where it contributed 20-40% of total enolase activity. The prevalence of MSE increased until postnatal day 30 when none of the NNE subunit could be detected. During this switchover, the profile of the three isoenzymes failed to fit a best fit binomial distribution, revealing a deficiency for the heterodimer. These data suggested that a rapid switch from NNE to MSE expression occurred during development and that the heterodimer formed only during the transient stage when both subunits were expressed.

In the mouse, isoenzyme analysis showed that postnatal accumulation of MSE protein was similar to the profile observed in rats (Fletcher et al., 1978). The mouse studies have been more informative, however, because the investigators have considered earlier stages of development and have looked at the relative levels of MSE and NNE gene expression at both the protein and mRNA levels. Fletcher and colleagues found that MSE protein was already expressed in skeletal muscle, heart and tongue of the earliest stage mouse embryos they examined (E l3) although in hind limb skeletal muscle, significant accumulation of the MSE protein did not occur until El 7. Northern analysis showed that MSE mRNA could not be detected prior to El 5 in hindlimb skeletal muscle (Barbieri et al., 1990; Lucas et al., 1992). The study by Lucas and colleagues showed that from E l7, MSE became the predominant message; the accumulation was shown to be biphasic, the first steep rise occurring prenatally, and coinciding with the formation of secondary myofibres, the second beginning at P5, and coinciding with their definitive specialisation. The levels of NNE mRNA decreased over the entire developmental period studied, but the greatest decrease

Introduction: Chapter I

occurred postnatally. Quantitative western analysis carried out in parallel showed that the transition from NNE to MSE in the mouse was controlled primarily at the level of transcription. The biphasic accumulation of MSE mRNA was shown to match that of another muscle-specific protein, a-skeletal actin, and the second phase of MSE mRNA accumulation coincided with the accretion of fast type IIB myosin heavy chain transcripts. No correlation between the increase in MSE message and the decline of NNE message could be detected, indicating that the two processes, whilst physiologically related, were regulated independently.

In vivo experiments, showing the onset of MSE gene expression in the hindlimb buds to be coincident with the formation of secondary myofibres, suggested that the gene could be used as a marker of late myogenesis. However, analysis of myoblast differentiation ex vivo indicated that gene expression commenced at a much earlier stage. As a model for myogenesis, Lamande et al., (1989) investigated the modulation of NNE and MSE mRNAs in premyogenic C3H10T'/2 cells and their myogenic derivatives and also in permissive and inducible C2.7 myoblasts. The MSE message was already detectable in both proliferating and quiescent myoblasts and accumulated further during terminal differentiation. The ratio o f MSE to NNE mRNA increased about threefold and a recent study has shown that the increase in the abundance of MSE mRNA was due to upregulation of transcription and not to an increase in RNA stability (Lamande et al., 1995). Other muscle-specific messages, such as the mRNA for a-skeletal actin, were not detected in myoblasts (Lamande et al., 1989). C3H10T‘/2 cells can be induced to differentiate into myoblasts or other cell types (e.g. adipocytes) by different chemical treatments. MSE was absent from undifferentiated C3H10T‘/2 cells, but present in myogenic derivatives generated by treatment with hypomethylation agents or transfection with MyoDI cDNA. Taking the results from the in vivo and ex vivo experiments together, it was suggested that MSE could be expressed in particular subsets of myoblasts, specifically the adult myoblasts (also called satellite cells) which accumulate during late foetal

development and give rise to secondary myofibres, but not in embryonic myoblasts which give rise to primary myofibres (Barbieri et al., 1990). It is possible to discriminate between the two types of myoblast in culture on the basis of their response to various differentiating agents and their accumulation of myosin isoforms. This theory was therefore tested by assaying cultured embryonic and foetal myoblasts for the MSE message; this has been done in mouse (Barbieri et al., 1990) and human cells (Peterson et al., 1992). In the case of the mouse, MSE message was detected in foetal but not embryonic myoblasts as expected. Furthermore, when the myoblasts were differentiated in vitro, the expression of MSE mRNA in the secondary myotubes

Introduction: Chapter 1

was tenfold higher than that observed in the primary myotubes. In humans, undifferentiated embryonic and foetal myoblasts showed levels o f MSE expression similar to the murine myoblasts, however, upon differentiation, both primary and secondary myotubes accumulated comparable levels of the MSE transcript. The authors argued that the apparent discrepancy between these results was attributable to differences in the developmental stage from which the embryonic myoblasts were obtained (Peterson et al., 1992), the mouse embryonic myoblasts being derived from an earlier stage than the human cells. However, the more sensitive in situ

hybridisation technique has been used to investigate MSE expression during very early myogenesis (Keller et al., 1992a) and this analysis showed that a more limited accumulation of the message occurred in primary myofibres, starting as early as E7.8. MSE mRNA was first detected in the cardiac tube, the earliest myogenic structure to form, and expression of the transcript was also observed in the myotomes of rostral somites from E8.75. By El 1.5, most developing skeletal muscles expressed detectable levels of MSE mRNA and MSE protein, but there was still no detectable expression in the limb buds. MSE transcripts were shown to start accumulating in the forelimb buds at E12.5, and in the hindlimb buds at E13.5, much earlier than results from previous northern analyses had suggested (Barbieri et al., 1990; Lucas et al.,

1992). These experiments showed that the ontogeny of MSE was triphasic, the first (embryonic) stage marked by accumulation of the message and protein in primary myofibres at levels detectable by in situ hybridisation and in situ immunohistological assay but not by biochemical analysis, the second stage marked by a more pronounced accumulation in secondary (foetal) myofibres at levels detectable by northern and western procedures and the third (postnatal) stage marked by rapid replacement o f embryonic enolase isoenzymes with MSE, coincident with final differentiation o f the myofibres.