Since all vertebrate species studied to date have been shown to possess multiple isoforms o f the MyoHC which are encoded by a multigene family it is difl&cult to avoid the question o f why so many isoforms should exist. A number o f theories have been proposed for the reasons why animals have so many different MyoHC isoforms and the evidence available to date is not sufficient to conq)letely rule any o f them out.
According to the so called "fimctional" hypothesis (Gros and Buckingham 1987), the differential expression o f various MyoHC isoforms throughout development and in the different fibre types o f the adult is a consequence o f various fimctional demands on the molecule dictating evolution by selective pressure. Such fimctional demands may possibly reside in the SI region o f the molecule where differences in primary structure could effect the actin binding properties and ATPase activity o f the molecule. The tail beat frequency o f fish changes dramatically with development and is far higher in fiy when conq)ared to adult fish. Therefore, developmental changes o f MyoHC gene expression observed in this study may confer the necessary ATPase activities or actin binding properties necessary for these differing modes o f swimming. Alternatively the fimctional demands which dictate evolutionary pressure for multiple MyoHC isoforms may reside in the LMM region where differences in structure may be required for the correct construction of the thick filament in particular cellular environments (Taylor and Bandman, 1989). This theory is supported by the fact that during situations o f muscle regeneration in aduh mammals, the embryonic and neonatal isoforms are expressed (see Chapter 1). Also, if the expression o f the carp type 7 MyoHC isoform in the small diameter newly developing white muscle fibres is driven by fimctional demands then one would hypothesise that properties in the LMM which facihtated the correct formation of thick filaments could be inq)ortant since it is difficult to envisage changes in ATPase in such a population o f fibres affecting the locomotory ability o f the fiish.
An alternative to the "fimctional" hypothesis is that isoform polymorphism confers regulatory advantages at the level o f gene expression rather than changes in protein function. Thus the MyoHC isoforms themselves would be very similar in their functional properties but differ significantly in the regulatory regions o f their genes.
It has also been proposed that isoforms are a consequence o f evolutionary dictated specialisation based on chromosomal geography (Gros and Buckingham, 1987). Hence two identical genes which were originally generated by a gene duphcation event may have diverged independently depending on their chromosomal location. However, due to the location o f the MyoHC genes in the mammaliati genome such an argument could only be used to explain the divergence between the cardiac and skeletal isoforms.
6.6 F uture studies
The isolation o f isoform specific carp MyoHC gene probes in this study has provided the molecular tools for many possible future lines o f investigation. One interesting finding firom this work which warrants further investigation is the hybridisation pattern o f the type 7 MyoHC probe. The MyoHC gene corresponding to this probe was shown, by in situ hybridisation, to be expressed exclusively in the small diameter white muscle fibres o f adult carp which had been acclimated to a warm environmental temperature. As discussed previously (Chapter 5) such small diameter muscle fibres have been implicated to be involved in the fibre hyperplasia process. A fundamental question regarding fibre hyperplasia in fish however, remains unanswered and this is the source of the new fibres Le. do they arise from fibre sphtting or from myosatellite cells. One approach to this question is to culture carp white muscle satelhte cehs (Koumans et al., 1990) and using the probes isolated in this current study, investigate which MyoHC isoforms are expressed when the cehs start to differentiate. If expression o f the type 7 MyoHC isofbrm is observed in such differentiating satelhte cehs then this can be taken to suggest that the smah diameter fibres have arose from satelhte cehs rather than fibre sphtting. Such studies are currently being performed.
Whilst the work described in Chapter 4 o f this thesis showed that the post hatching development o f carp fry involves the expression o f at least frve MyoHC isoforms, fiuther investigations which more accurately map the sequential expression of MyoHC isoforms during fiy and postlarval development are required. Such a study could easily be performed by Northem blot and in situ hybridisation analysis with the isoform specific probes isolated in this study and wih be undertaken on fiy from the next
seasons (1995) spawning.
With regards to the role o f MyoHC isoforms in the tenq)erature acclimation process in carp, the results presented in this study demonstrate that expression certain MyoHC isoforms can be upregulated in response to an increase in terr^erature. Whilst it seems likely that the type 7 MyoHC gene is involved in hyperplasic growth rather than an acclimatory response, the type 6 MyoHC still remains a candidate for a MyoHC gene involved in acclimation to warm tençerature and the expression o f this isoform requires fiirther investigation. None o f the seven MyoHC isoforms isolated in this study were shown to be e?qpressed specifically in response to cold tenq)erature acclimation. Isolation o f such a MyoHC gene in which expression is upregulated in response to cold tenq)eratures would provide more conclusive evidence that MyoHC isoforms facihtate tenqperature acclimation since the expression cannot be confiised with a growth response. The use o f degenerate ohgonucleotides in RACE PCR on cDNA prepared from cold acclimated carp may be more suited to isolation o f such an isoform than the FG2EXN40 oligonucleotide employed in this study.