Ophiopogonin D maintains Ca2+ homeostasis in rat cardiomyocytes in vitro by upregulating CYP2J3/EETs and suppressing ER stress

Aim: CYP2J3 in myocardium metabolizes arachidonic acid to 4 regioisomeric epoxyeicosatrienoic acids (EETs), which have diverse biological activities in rat heart. In this study we examined whether CYP2J3 was involved in cardioprotective effects of ophiopogonin D (OPD), a steroidal glycoside isolated from Chinese herb Radix ophiopogonis. Methods: Rat cardiomyoblast cell line (H9c2 cells) was tested. Intracellular Ca2+ concentrations ([Ca2+](i)) were measured using Fluo-4/AM. The expression of calcium-regulating molecules and ER stress signaling molecules was measured with qRT-PCR and Western blot analyses. Cell apoptosis was quantified with Hoechst 33258 staining and TUNEL assay. The level of 14,15-DHET, a stable metabolite of 14,15-EET, was assessed with ELISA. Results: Angiotensin II (10(-6) mol/L) significantly decreased the expression of calcium-regulating molecules (SERCA2a, PLB, RyR2 and FKBP12.6), and elevated [Ca2+](i) in H9c2 cells. Furthermore, angiotensin II markedly increased the expression of ER stress signaling molecules (GRP78, CHOP, p-JNK and cleaved caspase-12) and ER stress-mediated apoptosis. OPD (100, 250 and 500 nmol/L) dose-dependently increased CYP2J3 expression and 14,15-DHET levels in normal H9c2 cells. Pretreatment of H9c2 cells with OPD suppressed angiotensin II-induced abnormalities in Ca2+ homeostasis, ER stress responses and apoptosis. Overexpression of CYP2J3 or addition of exogenous 14,15-EET also prevented angiotensin II-induced abnormalities in Ca2+ homeostasis, whereas transfection with CYP2J3 siRNA diminished the effects of OPD on Ca2+ homeostasis. Furthermore, the intracellular Ca2+ chelator BAPTA suppressed angiotensin II-induced ER stress responses and apoptosis in H9c2 cells. Conclusion: OPD is a novel CYP2J3 inducer that may offer a therapeutic benefit in treatment of cardiovascular diseases related to disturbance of Ca2+ homeostasis and ER stress.