• Bishopric, Nanette (PI)

Project: Research project

Project Details


Beta-adrenergic agonists are strongly implicated in myocardial hypertrophy
in vivo, and beta-adrenergic and cyclic AMP (cAMP)-dependent mechanisms
are a frequent target of drugs used in ischemic cardiovascular disease and
heart failure. However, the mechanisms transducing beta-adrenoceptor-
mediated myocardial cell growth and gene expression are poorly understood.
With few exceptions, beta-adrenergic effects in the heart have been
thought to be mediated via modulation of cAMP. We have recently identified
a novel pathway for beta-adrenergic gene regulation in cardiac myocytes
that appears to be coupled to the storage or flux of calcium from
physically or biochemically defined compartments and is independent of
cAMP and cAMP-dependent protein kinase. The skeletal alpha-actin (sACT)
gene, encoding a developmentally regulated actin-actin isoform, is
selectively regulated by this pathway during beta-adrenoceptor-mediated
hypertrophy. In order to identify essential components of this signal
transduction mechanism, we propose to identify pharmacologic mechanisms
for beta-adrenergic regulation of the sACT gene and to examine
interactions between the sACT promoter and DNA-binding transcriptional
activating factors that may be required for beta-adrenergic induction.
Experiments described in part (1) will evaluate the role of beta-
adrenergic, protein kinase A-independent, and calcium-dependent mechanisms
in transcriptional and posttranscriptional regulation of the sACT gene,
employing nuclear run-on and messenger RNA half-life assays. Because we
have determined that transcriptional activating factors Fos and Jun (AP-1)
positively regulates sACT in cardiac myocytes, we will determine the
effects of protein kinase A-dependent, and calcium-dependent mechanisms on
expression of c-fos, c-jun, and related genes as well as on AP-1 binding
activity and immunoreactivity. Pl9 teratocarcinoma cells will be stably
transfected with an sACT/lacZ chimeric gene containing full-length
promoter sequences, most coding sequences and 3' flanking sequences in
order to create a cell line in which the signal-mediated regulation of
this gene can be studied directly. In part (2), beta-adrenergic and AP- 1-
responsive bases in the proximal sACT promoter will be determined by a
combination of in situ mutagenesis, gel mobility retardation assay, and
methylation interference. Cardiac myocyte nuclear proteins will be
evaluated for interaction with the sACT promoter, and a potential
interaction between serum response factor and AP-1 on this promoter will
be assessed. Finally, lambda-gt11 cardiac myocyte cDNA expression
libraries will be constructed and screened with relevant oligonucleotide
sequences from the sACT promoter.
Effective start/end date1/1/9412/31/98


  • National Institutes of Health
  • National Institutes of Health
  • National Institutes of Health: $216,999.00
  • National Institutes of Health: $192,281.00
  • National Institutes of Health: $231,200.00


  • Medicine(all)


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