Key Points
- To characterize an in vitro model of endothelin-1-induced hypertrophy in neonatal rat ventricular myocytes and analyze the transcriptional regulation of the sarcoplasmic reticulum Ca2+ ATPase promoter.
- Cultured neonatal rat ventricular myocytes under serum-free conditions and induced hypertrophy with 10⁻⁸ M endothelin-1 for up to 48 hours, comparing outcomes against angiotensin II and α1-adrenergic agonists.
- Assessed gene expression by Northern blotting and evaluated promoter activity using a cloned 1.9 kb rat sarcoplasmic reticulum Ca2+ ATPase promoter reporter construct transfected into cultured cardiomyocytes.
- Endothelin-1 was the strongest inducer of hypertrophy, producing a twofold increase in total cellular RNA and a >twofold increase in atrial natriuretic factor mRNA relative to GAPDH.
- Endothelin-1 reduced relative mRNA expression of the sarcoplasmic reticulum Ca2+ pump by 26% and phospholamban by 49% relative to GAPDH, whereas absolute Ca2+ pump mRNA levels per cell remained unchanged.
- A 1.9 kb promoter fragment drove reporter gene downregulation relative to GAPDH identical to endogenous Ca2+ pump mRNA, confirming that the promoter does not match the general transcriptional upregulation of hypertrophy.
Structured PICO
PPopulationCultured neonatal rat ventricular myocytes in serum free medium
IInterventionEndothelin-1 (10(-8) M) up to 48 h
CComparatorAngiotensin II, alpha 1-adrenergic agonist, or unstimulated state
OOutcomeSarcoplasmic reticulum Ca2+ ATPase promoter activity and gene expression regulationsurrogate
In an in vitro model of endothelin-1 induced hypertrophy, the absolute mRNA levels of the sarcoplasmic reticulum Ca2+ pump remain unchanged, indicating its promoter does not respond to the general increase in transcriptional activity associated with hypertrophy.