FIGURE 3.

SERCA2a-dependent [Ca²⁺]c overload activated mitofusin 2 (Mfn2)-mediated [Ca²⁺]m overload and was accompanied by increased Parkin/PINK-dependent mitophagy (n = 3/group). (A,B) Western blot analysis was performed to detect the expression of Mfn2. Hypoxia and pretreatment with tBHQ increased the expression of Mfn2, while overexpression of SERCA2a, pretreatment with BAPTA and knockdown of the Mfn2 gene reversed the Mfn2 expression level. (C,D) Coimmunofluorescence of SERCA2a and Mfn2 in cardiac microvascular endothelial cells (CMECs). The overlap of fluorescence intensity changed in the different groups and was consistent with the changes observed in WB. (E,F) The [Ca²⁺]c probe (Fluo-3) and [Ca²⁺]m probe (Rhod2) were used to observe changes in Ca²⁺ concentration via fluorescence. (G,H) Western blot analysis was performed to detect Parkin/PINK-dependent mitophagy- and mito-apoptosis-related protein expression in the different groups. (I) Electron microscopy was used to observe autophagosome formation. *p < 0.05 compared with the Ad-Ctrl; ^#p < 0.05 compared with the Hypo(Ad-Ctrl).