Molecular Analysis of Mtiscle Development 
and Function 
Bernardo Nadal-Ginard, M.D., Ph.D. — Investigator 
Dr. Nadal-Ginard is also the Alexander S. Nadas Professor of Pediatrics and Professor of Cellular and 
Molecular Physiology at Harvard Medical School and Cardiologist- in- Chief at the Children's Hospital, 
Boston. He received his M.D. degree from the University of Barcelona, Spain, and his Ph.D. degree in 
biology from Yale University. After training in internal medicine and cardiology, he was a student and 
postdoctoral fellow with Clement Markert at Yale. He was a professor of cell biology at Albert Einstein 
College of Medicine before assuming his present position. 
OUR laboratory continues to be interested in 
elucidating the molecular mechanisms that 
regulate the production and function of the con- 
tractile system in muscle cells. This apparatus 
converts the chemical energy generated by food- 
stuffs and stored in the form of ATP, and is thus 
the molecular motor for locomotion and for the 
heartbeat. One of its fundamental roles is in 
maintaining cell shape. There are variations of 
the contractile system in every cell of the 
organism. 
The functional unit of the contractile system is 
the sarcomere, which is composed of a precisely 
arranged set of proteins. These are produced by a 
small family of genes, each making a different 
isoform. In addition, a single gene can in many 
cases produce several kinds of proteins by a pro- 
cess called alternative splicing. Combinations of 
the different isoforms arising from these two 
mechanisms can lead to millions of different 
types of sarcomeres. Thus significant functional 
differences among sarcomeres are produced by 
changing their components, either through gene 
switching at the transcriptional level or alterna- 
tive splicing from the same gene. In addition, the 
performance of a given sarcomere can be affected 
by changing the availability of ions in the muscle 
cells. These three aspects of muscle cell biology 
continue to be the focus of our research. 
Transcriptional Regulation of Contractile 
Protein Genes 
Which sarcomeres assemble in a cell depends 
on which member of the multigene family of 
contractile protein genes is expressed at that par- 
ticular time. To analyze the mechanisms involved 
in switching from one gene to another in the same 
gene family, we have concentrated on those cod- 
ing for the myosin heavy chain (MHC) . This is the 
most important protein of the sarcomere, since it 
contains the enzymatic activity responsible for 
generating force. We are currently exploring two 
main questions in the regulation of the MHC 
genes: What determines which one is expressed 
in a given cell type at a particular developmental 
stage? What determines the level of expression? 
To date, the best-characterized muscle-specific 
regulatory factors are the myogenic basic helix- 
loop-helix (bHLH) proteins of the MyoD family. 
Muscle-specific induction by these proteins de- 
pends on specific DNA binding to a particular se- 
quence, called an E box, present in many muscle 
enhancers and promoters. The MyoDs interact 
with the DNA in conjunction with other HLH pro- 
teins that are distributed more widely and are 
present in limiting amounts. 
During the past year we have cloned and char- 
acterized two new proteins that function as he- 
terodimeric partners of MyoD. These are alterna- 
tively spliced products of gene £2.2 and are 
particularly abundant in skeletal muscle, heart, 
and brain. Several lines of evidence indicate that 
in vivo these proteins are the partners of MyoD in 
muscle and that they also play an important role 
in other tissues. The fact that these proteins inter- 
act with the product of the retinoblastoma gene 
suggests that they are involved in the regulation 
of cell proliferation. 
One of the paradoxes of the MyoD paradigm for 
muscle-specific gene regulation is that not all 
muscle genes contain E boxes, although they are 
uniformly required for efficient muscle-specific 
expression. In addition, many of the genes in- 
duced by MyoD in skeletal muscle are also ex- 
pressed in cardiac and, in some cases, smooth 
muscle, where myogenic bHLH proteins have not 
been found. It was expected, therefore, that other 
transcription factors, which in skeletal muscle 
might be regulated directly or indirectly by the 
MyoD family, mediate activation and high-level 
expression of these genes. A candidate for such a 
factor is the myocyte-specific enhancer-binding 
factor 2 (MEF2), which binds to a specific DNA 
sequence known to be important for high-level 
expression in skeletal and cardiac muscle. 
During the past year we have cloned and char- 
acterized a family of MEF2 factors. In humans 
these factors are encoded by a family of at least 
four genes, which through alternative splicing 
generate a larger number of proteins. All these 
proteins share domains that are strikingly similar 
to the DNA-binding and dimerization domains of 
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