Figure 7.

Effects of losartan (Los), metformin (Met), Nω-nitro-L-arginine methyl ester (L-NAME), splitomicin (SP) or pifithrin-α (Pf) on mitochondrial membrane potential (ΔΨ_m) and permeability transition pores (PTP) in AngII-treated cardiomyocytes. (A) Representative images of JC-1 fluorescence. The images were obtained using a Zeiss LSM510 META (Carl Zeiss) microscope from cells after incubation with JC-1 (10 μg/ml). The cells were treated with Los, Met, SP, L-NAME or Pf in the presence or absence of 200 nM AngII. In addition, to demonstrate specificity of the fluorescence signal, the cells were treated with FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone), a chemical uncoupler of electron transport and oxidative phosphorylation that induces depolarization of the mitochondrial inner membrane. Red fluorescent images of dye aggregates (‘J-aggregates’) indicate high ΔΨ_m, whereas green images of monomeric dye (‘JC-1 monomers’) show low ΔΨ_m. (B) Quantitative results of JC-1 fluorescence intensity, measured using a Spectramax M3 microplate reader (Molecular Devices). Data were normalized to cells and expressed as a percentage of the control group. *P < 0.05 versus control. n = 6–8 per group. (C) Representative images of calcein fluorescence. Cells treated with Los, Met, SP, L-NAME or Pf in the presence or absence of 200 nM AngII for 24 hrs were co-loaded with cobalt chloride (5 mM) and calcein-AM (5 μM) and, then, imaged using an Olympus IX73 (Center Valley, PA, USA) inverted fluorescence microscope. (D) Quantitative results of calcein fluorescence, normalized to individual cells and expressed as a percentage of the control group. *P < 0.05 versus control; n = 4 per group.