MOLECULAR BIOLOGY OF THE CONTRACTILE SYSTEM 
Bernardo Nadal-Ginard, M.D., Ph.D., Investigator 
During the past year, Dr. Nadal-Ginard's labora- 
tory has continued to focus on three main areas of 
research: 1) developmental biology of the contrac- 
tile system, with emphasis on skeletal and cardiac 
myogenesis; 2) the mechanisms involved in the 
production of multiple protein isoforms from a sin- 
gle gene by alternative splicing; and 3) structural 
and functional characterization of genes involved in 
the regulation of the contractile phenotype. 
I. Developmental Regulation of the Contractile 
System. 
One characteristic of the terminally differentiated 
phenotype of striated muscle cells is its apparent ir- 
reversible withdrawal from the cell cycle. Although 
these cells have functional receptors for a variety of 
growth factors, as demonstrated by their metabolic 
response, they are not induced to re-initiate DNA 
synthesis. It is not known whether these cells have 
lost their ability to replicate their DNA or whether 
their lack of response is due to the presence of a re- 
pressor of cell growth that is induced during differ- 
entiation. The previous isolation of cells that are 
temperature sensitive for the commitment step and 
cannot withdraw irreversibly from the cell cycle, to- 
gether with the demonstration that the BC3H1 cell 
line is a mutant for this phenotype, strongly sug- 
gests that an inhibitor of cell growth is induced 
during terminal differentiation. To develop an ex- 
perimental approach to test this hypothesis and 
identify the putative molecule (s) involved, Dr. 
Nadal-Ginard and his colleagues have explored the 
ability of the SV40 large T antigen to inhibit 
myogenesis. The temperature-sensitive mutant 
tsA58, driven by the metallothioneine promoter, 
has been used to show that expression of this 
oncogene for as little as 5 h in terminally differenti- 
ated muscle cells is sufficient to make the cells re- 
spond to growth factors and reenter the cell cycle. 
The multiple nuclei in these myotubes undergo 
synchronous mitosis and, in some cases, cytokine- 
sis. A similar phenomenon is induced by the adeno- 
ElA protein as well as the phyloma T antigen. 
These results strongly suggest that the terminally 
differentiated phenotype is maintained by a gene 
product whose function is neutralized by the 
oncogene tested. Preliminary results indicate that 
the retinoblastoma gene product is not responsible 
for this phenotype. The involvement of p53 in this 
process is being investigated. In addition, a large va- 
riety of SV40 T antigen mutants that are defective in 
their interaction with different cellular components 
are being studied, in order to map the domain of 
this protein responsible for the reversibility of the 
terminally differentiated phenotype. Experiments 
designed to identify the molecule that putatively in- 
teracts with SV40 T antigen through immunopre- 
cipitation of T antigen under different conditions 
are also under way. 
One of the main obstacles in cardiac biology has 
been the unavailability of cardiocyte cell lines. To 
test the generality of the effect of SV40 T antigen 
on terminally differentiated phenotypes, Dr. Nadal- 
Ginard and his colleagues have transfected primary 
cardiocytes with the tsA58 mutant under the con- 
trol of cardiac-specific promoters. A large number 
of established cell lines have been produced. These 
cells have many of the characteristics of very early 
cardiocytes and depend on the expression of T anti- 
gen for growth. At permissive temperatures these 
cells respond with rapid cell growth to serum 
growth factors and remain undifferentiated. At non- 
permissive temperatures the cells become unre- 
sponsive to growth factors and induce a large array 
of genes characteristic of the differentiated state. 
Approaches similar to those described for the skele- 
tal muscle cells are in progress to identify the mole- 
cule(s) interacting with T antigen and responsible 
for the withdrawal from the cell cycle. 
The transcriptional induction of contractile pro- 
tein genes continues to be studied, using the myo- 
sin heavy-chain gene as a model system. Several cis- 
acting elements on these promoters have been 
identified. None of them is tissue specific. Recently 
the transacting factor that binds to the light-chain 
enhancer has been identified and characterized. Ex- 
periments are in progress for the purification and 
eventual cloning of this molecule. At the same time 
the role of different isoforms of the thyroid hor- 
mone receptor on the induction and repression of 
different myosin heavy-chain genes continues to be 
pursued. 
II. Alternative Splicing of Contractile Protein Genes. 
The laboratory has continued to analyze the reg- 
ulation of alternative splicing, with emphasis on 
mutually exclusive exons, which seem the most 
likely to reveal the underlying molecular mecha- 
nisms. Two main questions have been addressed: 
What is the basis of this mutual exclusivity? How is 
Continued 
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