Angiotensin II-induced apoptosis in rat cardiomyocyte culture: A possible role of AT₁ and AT₂ receptors

Objectives: To investigate the mechanism of angiotensin II-induced apoptosis in cultured cardiomyocytes by determining which receptor subtype is involved, and what is the relationship between intracellular Ca²⁺ changes and apoptosis. Design and methods: Neonatal rat cardiomyocytes were pretreated with either the AT₁ antagonist irbesartan or the AT₂ antagonist PD123319 before exposure to angiotensin II. Apoptotis was evaluated using morphological technique, staining nuclei by Feulgen and Hoechst methods followed by image analysis and by in situ terminal deoxynucleotidyl transferase nick-end (TUNEL) labelling. TUNEL-positive cardiocytes were distinguished from other cells by double staining with α-sarcomeric actin. Intracellular Ca²⁺ changes were assessed by indo-1 fluorescence microscopy, and the effect of Ca²⁺ on angiotensin II-induced apoptosis was tested using the calcium channel blocker verapamil. Results: Exposure to angiotensin II (10 nmol/l) resulted in cell replication and a three-fold increase in programmed cell death (P < 0.05). Pretreatment with either irbesartan (an AT₁ receptor antagonist, 100 nmol/l) or PD123319 (an AT₂ receptor antagonist, 1 μmol/l) prevented the angiotensin II-induced apoptosis, indicating the presence of both AT₁ and AT₂ receptors on cardiomyocytes. Exposure of myocytes to angiotensin II caused an immediate and dose-dependent increase in the concentration of intracellular free Ca²⁺ that lasted 40-60 s. The effect was sustained in a Ca²⁺ free medium. Pretreatment of cells with irbesartan (100 nmol/l) and PD123319 (10 μmol/l) blocked Ca²⁺ elevation. Pretreatment with verapamil (10 μmol/l) prevented angiotensin II-induced apoptosis. Conclusions: Angiotensin II-induced apoptosis in rat cardiomyocytes is mediated through activation of both AT₁ and AT₂ receptors. The apoptotic mechanism is not related to the immediate angiotensin II-induced Ca²⁺ rise from intracellular stores. However, it is accompanied by cardiomyocyte proliferation and requires Ca²⁺ influx through L-type channel activity.