A NOVEL CARDIAC-RESTRICTED TARGET FOR DOXORUBICIN - CARP, A NUCLEAR MODULATOR OF GENE-EXPRESSION IN CARDIAC PROGENITOR CELLS AND CARDIOMYOCYTES

Doxorubicin (Dox), a cardiotoxic antineoplastic drug, disrupts the car diac-specific program of gene expression (Kurabayashi, M., Dutta, S., Jeyaseelan, R., and Kedes, L. (1995) Mel. Cell. Biol. 15, 6386-6397; J eyaseelan, R., Poizat, C., Wu, H. Y., and Kedes, L. (1997) J. Biol. Ch em. 272, 5828-5832). To determine whether this drug might interfere wi th the function of cardiac-specific regulatory pathways, we used a dif ferential display strategy to clone from neonatal rat cardiomyocyte ca ndidate mRNAs that were rapidly sensitive to Box. We report here the i dentification of a constitutively expressed, cardiac-restricted, nucle ar protein whose mRNA level is exquisitely sensitive to Box Hence we h ave named this protein cardiac adriamycin-responsive protein (CARP). C ARP mRNA is present at the earliest stages of cardiac morphogenesis. I t was detected by in situ hybridization within the cardiogenic plate o f 7.5-day post coitum (p.c.) embryos, and in 8.5-day p.c. embryos CARP transcripts are present in uniformly high levels in the myocardium. T hroughout cardiac development, GARP expression is specific for the myo cardium; endocardial cushions and valves exhibit only background level s of signal. Transcript levels persist but gradually decrease in neona tal, 2-week-old, and adult hearts. There were no stages when CARP mRNA could not be detected. The pattern and timing of CARP mRNA expression , including transient expression in the tongue at 14.5 days p.c., coin cides with that of Nkx2.5/Csx (a putative homolog of tinman, the Droso phila melanogaster gene responsible for cardiac development). The clon ed full-length 1749 nucleotide CARP cDNA encodes a 319-amino acid 40-k Da polypeptide containing five tandem ankyrin repeats. CARP appears to be the rat homolog of a previously reported human single-copy gene (C -193; Chu, W., Burns, D. K., Swerlick, R. A., and Presky, D. H. (1995) J. Biol. Chem. 270, 10236-10245), whose mRNA is inducible by cytokine s only in human endothelial cells. CARP appears to function as a negat ive regulator of cardiac-specific gene expression. Over-expression of CARP in cardiomyocytes suppresses cardiac troponin C and atrial natriu retic factor transcription. Cotransfection experiments in HeLa cells i ndicate that CARP inhibits Nkx2.5 transactivation of atrial natriureti c factor promoter. When fused to a GAL4 DNA-binding domain, CARP has t ranscriptional inhibitory properties in noncardiac cells. CARP thus re presents the first example of a cardiac-restricted transcriptional reg ulatory protein that is sensitive to Dox.