SARCOPLASMIC-RETICULUM CA2-1 INDUCED HYPERTROPHY OF CULTURED RAT CARDIOMYOCYTES( ATPASE PROMOTER ACTIVITY DURING ENDOTHELIN)

Objectives: Characterization of an in vitro model of endothelin-1 indu ced hypertrophy of cultured neonatal rat ventricular myocytes and subs equent analysis of transcription regulation of the rat promoter of the sarcoplasmic reticulum Ca2+ ATPase gene. Methods: Neonatal rat ventri cular myocytes were cultured in serum free medium and hypertrophy was induced by addition of endothelin-1 to 10(-8) M up to 48 h. Hypertroph y was characterized biochemically, and gene expression regulation was evaluated by Northern blotting. A sarcoplasmic reticulum Ca2+ ATPase p romoter fragment, isolated from a rat library was cloned in a reporter vector. Promoter activity during hypertrophy was assessed after trans fection of the reporter plasmid to cultured cardiomyocytes. Results: S timulation with endothelin-1 resulted in increased cell size, as indic ated by protein/DNA ratio as well as by augmented protein synthesis. W hen compared to angiotensin II or alpha(1)-adrenergic agonist, endothe lin-1 was the strongest inducer of hypertrophy (protein/DNA ratio) aft er 48 h of stimulation. Endothelin-1 induced hypertrophy was accompani ed by a twofold increase in total RNA content per cell as well as to i ncreased glyceraldehydephosphate dehydrogenase mRNA levels. The level of atrial natriuretic factor mRNA was increased more than twofold, rel ative to glyceraldehydephosphate dehydrogenase, while the expression o f the sarcoplasmic reticulum Ca2+ pump and phospholamban genes was dec reased (by 26 and 49%, respectively) after induction of hypertrophy by stimulation with endothelin-l. In the same model, a 1.9 kb sarcoplasm ic reticulum Ca2+ pump gene promoter fragment (including 0.4 kb of the 5' UTR of the mRNA) directed down-regulation of the expression of the reporter gene to the same magnitude as endogenous Ca2+ pump mRNA rela tive to glyceraldehydephosphate dehydrogenase mRNA. However, absolute mRNA level per cell did not change for either the reporter gene or the endogenous Ca2+ pump. Conclusions: Endothelin-1 can induce phenotypic changes in cultured rat ventricular myocytes that are reminiscent of hypertrophy in vivo. In this model, a 1.9 kb sarcoplasmic reticulum Ca 2+ pump promoter fragment directed gene expression of a reporter gene identical to the endogenous regulation of the Ca2+ pump. Furthermore, expression of the Ca2+ pump during hypertrophy was only downregulated when compared to (increased levels of) glyceraldehydephosphate dehydro genase mRNA, but absolute Ca2+ ATPase mRNA amounts remained unchanged. This suggests that the Ca2+ pump promoter is not responding to the in crease in transcriptional activity that accompanies hypertrophy. (C) 1 998 Elsevier Science B.V.