Fig. 6. Effect of ER stress on mitochondrial integrity and associated signaling molecules.

Effects of in vitro and in vivo thapsigargin challenge on mitochondrial function, activation of Akt and GSK3β were evaluated in cardiomyocytes from adult WT and MyAkt mice. Mitochondrial integrity was assessed by mitochondrial membrane potential (MMP) and mitochondrial permeability transition pore opening (mPTP). For MMP and mPTP, cardiomyocytes from WT and MyAkt mice were exposed to thapsigargin (3 μM) for 4 hrs in vitro in the presence or absence of the NADPH oxidase inhibitor apocynin (5 μM) prior to biochemical assessments. A: Representative JC-1 fluorochrome images depicting MMP in cardiomyocytes from adult WT and MyAkt mice treated with or without TG or apocynin (or the positive control CCCP, 10 μM); B: Pooled data of MMP (ratio of JC-1 red to green fluorescence); The mitochondrial uncoupler CCCP (10 μM) was used as a positive control; C: Mitochondrial permeability transition pore opening evaluated by NAD⁺, a marker for mitochondrial permeability transition pore opening; D-E: Expression of phosphorylated Akt (Ser⁴⁷³) and GSK3β (Ser⁹) shown as pAkt-to-Akt and pGSK3β-to-GSK3β ratios in murine hearts following in vivo thapsigargin challenge (3 mg/kg, i.p. 48 hrs); Insets: Representative gel blots depicting expression of pan and phosphorylated Akt and GSK3β using specific antibodies; Mean ± SEM, n = 5–7 mice per group, * p < 0.05 vs. WT group; # p < 0.05 vs. WT-Thapsigargin (TG) group.