Expression of lactate dehydrogenase, myosin heavy chain and myogenic regulatory factor genes in rabbit embryonic muscle cell cultures.

The expression of myogenic regulatory factors (MRFs), lactate dehydrogenase (LDH) and myosin heavy chains (MyHC), as markers of myogenesis, metabolism and contractility respectively, were investigated during differentiation of rabbit embryonic muscle cells in primary culture. Myf5, MyoD and myogenin mRNAs were abundantly expressed at day 1 of culture. The expression of Myf5 and MyoD mRNA transcripts decreased sharply as myoblasts fused and differentiated into myotubes, whilst myogenin mRNA was maintained throughout the duration of the culture. In contrast, MRF4 mRNA was weakly expressed on day 1 of culture, its expression increased slightly as myoblasts fused and reached a maximum level in 7-day-old cultures containing striated myofibres. The specific activity of LDH increased linearly during myoblast proliferation and fusion. In 7-day-old cultures, LDH-M mRNA (dominant in glycolytic muscles) and LDH-H mRNA (predominant in perinatal and oxidative muscles) represented 38% and 62% of total LDH mRNA respectively. At this stage, immunocytochemical staining with perinatal and adult-type MyHC antibodies showed that embryonic and perinatal MyHC isoforms were expressed in all myotubes, while few of them were stained by type I MyHC antibody. However, none of them expressed adult type II MyHC. The latter results were further supported by RT-PCR analysis of adult-type MyHC mRNA which showed that only the type I MyHC mRNA transcript was expressed. These data were in agreement with those reported in vivo on perinatal rabbit muscles. They differed from those obtained on cultured satellite cells isolated from adult rabbit fast-twitch or slow-twitch muscles which did not express embryonic MyHC, and instead expressed fast- or slow-type MyHC according to their muscle origin. Taken together, these results further suggest that myogenic mononucleated cells express different properties in vitro according to their developmental origin as well as properties related to those of the muscles from which they were isolated.

Transformation of slow- or fast-twitch rabbit muscles after cross-reinnervation or low frequency stimulation does not alter the in vitro properties of their satellite cells.

We previously showed that satellite cells isolated from rabbit fast-twitch and slow-twitch muscles presented different behaviours in culture; cells from slow muscle differentiated more quickly and fused into more numerous myotubes than those from fast muscle. Moreover, only slow-muscle derived satellite cells expressed in vitro the slow type I myosin heavy chain isoform (MyHC). We wanted to investigate whether the properties of satellite cells originating from different muscles were under the influence of the adult fibre type on which they were located. For this purpose, we transformed the properties of the adult rabbit fast-twitch semimembranosus accessorius (SMa; approximately 100% type II fibres) and the slow-twitch semimembranosus proprius (SMp; 100% type I fibre) muscles by (1) cross-reinnervating the SMp with the main branch of the fast SMa nerve; or (2) electrical stimulation at 10 Hz of the SMa muscle. We studied their satellite cells in vitro. Five-month cross-reinnervation of the SMp induced a large shift of its MyHC type characteristics towards those of a fast muscle, and three-month electrical stimulation at low frequency transformed the fast-twitch SMa into a slow-twitch muscle, as shown by SDS-PAGE of MyHC. In spite of the transformation of their muscle characteristics, satellite cells in culture kept their original properties. Indeed, as shown by MyoD and myogenin gene expression as markers of fusion, satellite cells isolated from cross-reinnervated and from control SMp began to fuse by eight days of culture, and expressed MyoD and myogenin at that stage. Later they differentiated into numerous myotubes. Satellite cells isolated from electrically stimulated and control SMa presented a similar behaviour in culture: they did not express MyoD and myogenin at eight days, and fused by ten days into only a few myotubes. Moreover, MyHC gene expression showed that, in contrast with slow-muscle derived satellite cells, the type I MyHC gene was not expressed by satellite cells isolated from the stimulated SMa in spite of its homogeneous type I fibre composition. Taken together, these data support the idea that once constituted, muscle fibre types per se do not influence the properties of their associated satellite cells.

Rabbit slow and fast skeletal muscle-derived satellite myoblast phenotypes do not involve constitutive differences in the components of the insulin-like growth factor system.

The insulin-like growth factor (IGF) system is actively involved in the control of proliferation and differentiation of several myogenic cell lines, and phenotypic differences between myoblasts are associated with modifications of the equilibrium of the components of the IGF system. To determine whether this observation is a physiologic feature that also concerns the phenotypes of ex vivo adult satellite myoblasts in primary cell culture, we investigated the IGF system in rabbit slow-twitch muscle-derived satellite myoblasts (SSM), which differ phenotypically from fast-twitch muscle-derived satellite myoblasts (FSM) by their proliferation and differentiation kinetics in vitro. The expression of IGF-I and IGF-II were similar in SSM and FSM as well as their concentrations measured in cell-conditioned media. Ligand blotting of conditioned media samples indicated the presence of five IGF binding protein (IGFBP) species of Mr 37-40, 32, 30-31, 28, and 24 kDa. The 30-31 kDa doublet was visible in SSM-conditioned medium only and associated with the presence of a 22-kDa protein, which may represent a proteolytic fragment. In contrast, the 32-kDa band was observed in FSM conditioned medium only. The other IGFBP moieties were present in both SSM- and FSM-conditioned media. Cross-linking experiments revealed the presence of the M6P/IGF-II receptor on both SSM and FSM membranes. We also observed an IGF-I receptor form bearing unusual high affinity for IGF-II: the binding of [125I]IGF-I on this receptor was preferentially displaced by IGF-I but that of [125I]IGF-II was mostly inhibited by IGF-II, suggesting that the two tracers did not bind on the same epitopes. [125I]IGF-II binding to this receptor was greater on SSM than on FSM membranes. Autophosphorylation of WGA-purified receptors revealed an approximately 400-kDa band after SDS-PAGE under nonreducing conditions, which corresponded to the alpha 2 beta 2 form of the IGF-I receptor, and two beta subunit moieties of Mr 101 and 105 kDa under reducing conditions in both SSM and FSM extracts. Phosphorylation of the 105-kDa moiety was more intensively increased than that of the 101-kDa protein after growth factor stimulation. Basal phosphorylation state of the two beta subunits was similarly stimulated by IGF-I and IGF-II and less by insulin. Since both insulin and IGF-I receptors were expressed in FSM and SSM, one of the two beta subunits may actually correspond to that of the insulin receptor. We conclude that the IGF system is not considerably affected by the phenotypes of SSM and FSM. The differences observed, which mostly concern IGFBP species, more likely appear as regulatory adaptations than as phenotypic changes targeting the components of the IGF system.

Expression of myosin isoforms in denervated, cross-reinnervated, and electrically stimulated rabbit muscles.

The expression of myosin heavy (MyHC) and light (MyLC) chain isoforms was analyzed after denervation and cross-reinnervation by a fast nerve of the slow-twitch Semimembranosus proprius (SMp) muscle, and after denervation and electrical stimulation at low frequency of the fast-twitch Semimembranous accessorius (SMa) muscle of the rabbit. The control SMp (100% type I fibers) expressed 100% type I MyHC and 100% slow-type (1S', 1S and 2S) MyLC isoforms. Five month denervation did not alter significantly the MyHC expression of the muscle, but induced the expression of a new type 1 MyLC corresponding most probably to an embryonic MyLC. Five-month cross-reinnervation of the SMp by the fast SMa nerve induced a large change of its fiber type properties. As shown by immunocytochemistry, almost all fibers were stained by fast myosin antibody, but a high proportion of them co-expressed slow myosin. This result was in agreement with biochemical data showing that fast MyHC and MyLC isoforms became predominant. The control SMa (nearly 100% type II fibers) expressed almost 100% type II MyHC (70% type IIb and 22% IIx/d) and 100% fast-type (1F, 2F and 3F) MyLC isoforms. Five month denervation of the SMa induced a shift in its MyHC, with 98% type IIx/d and 2% type IIb isoforms, and no change in the proportions of its MyLC. Three month electrical stimulation at 10 Hz of the SMa transformed its fiber type composition. All fibers reacted with the slow myosin antibody and a minor proportion of them were stained by the fast myosin antibody. These observations were in agreement with the biochemical analysis showing a large predominance of the slow-type MyHC and MyLC isoforms. Taken together, these results obtained from rabbit muscles which are normally homogeneous in either fast-twitch or slow-twitch fiber types, further support the idea that the different myosin isoforms, particularly the MyHC, are differentially regulated by motor innervation. Type I MyHC is maintained in denervated SMp muscle, but is not expressed in denervated SMa. Type IIb isoform is the most sensitive to neural influence, as it disappears rapidly in denervated and electrically stimulated fast-twitch SMa muscle, and is barely expressed in cross-reinnervated slow-twitch SMp muscle. In contrast, type IIa and type IIx/d are less dependent upon motor innervation. In addition to the previous studies of d'Albis et al. analysis of these results leads us to conclude that, in the rabbit, sensitivity to motor innervation increases from the glycolytic to the oxydative types of fibers, in the order IIB > IIX/IID > IIA > I.

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures.

We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.

Expression of acetylcholinesterase gene during in vitro differentiation of rabbit muscle satellite cells.

We investigated the myogenic properties and the expression of acetylcholinesterase (AChE) in culture of satellite cells (SCs) isolated from slow and fast rabbit muscles. Slow SCs form myotubes more rapidly (day 9 vs day 11) than fast SCs, and differentiate further into striated and contractile fibers. AChE activity and mRNA expression are higher in SCs cultured from slow than from fast muscles, as also observed in the muscles themselves. However, the two types of SC cultures do not show obvious difference in their patterns of AChE molecular forms. Taken together, these preliminary data support the view that there might be more than one SC population in skeletal muscles.

Expression of acetylcholinesterase molecular forms during in vitro differentiation of rabbit embryonic muscle cells.

We investigated the myogenic properties of rabbit myoblasts in culture, and examined particularly the expression of acetylcholinesterase (AChE). In the presence of horse serum, myoblasts proliferate quickly and begin to fuse by day 3 of culture. Differentiated myotubes presenting some spontaneous contractions are clearly visible from days 6 to 9. The cultures degenerate from day 10 onwards. The specific activity of AChE is low during the proliferation phase of myogenic cells, increases sharply as myotubes differentiated, then decreases as cultures degenerate. In 7-day-old cultures, globular forms are largely predominant and asymmetric forms, detected as A12, represent only 1% of the total activity.