Cardiac muscle-restricted expression of the alpha-myosin heavy-chain (alpha-MHC) 
gene is regulated by multiple elements in the proximal enhancer/promoter. Within 
this region, an M-CAT site and an A-rich site were identified as potential 
regulatory elements. Site-specific mutations in each site, individually, reduced 
activity from the wild-type promoter by approximately 85% in the adult rat 
heart, demonstrating that these sites were positive regulatory elements. 
alpha-MHC, beta-MHC, and chicken cardiac troponin T (cTnT) M-CAT sites 
interacted with an M-CAT-binding factor (MCBF) from rat heart nuclear extracts 
that was immunologically related to transcriptional enhancer factor 1, a factor 
that binds within the simian virus 40 enhancer. The factor that bound the A-rich 
region (ARF) was antigenically related to the RSRF family of proteins, ARF was 
distinct from myocyte-specific enhancer factor 2 (MEF-2) on the basis of 
DNA-binding specificity and developmental expression. Like MEF-2, ARF 
DNA-binding activity was present in the heart and brain; however, no ARF 
activity was detected in extracts from skeletal muscle or C2C12 myotubes. MCBF 
and ARF DNA-binding activities were developmentally regulated with peak levels 
in the 1- to 2-day neonatal heart. The activity of both factors increased nearly 
fivefold in adult rat hearts subjected to a pressure overload. By comparison, 
the levels of alpha-MHC binding factor 2 did not change during hypertrophy. 
Binding sites for MCBF and ARF are present in several genes that are upregulated 
during cardiac hypertrophy. Our results suggest that these factors participate 
in the alterations in gene expression that occur during cardiac development and 
hypertrophy.