Figure 6.

FUNDC1 mediates mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) formation. A, Co-localization of FUNDC1 with mitochondria and ER. Mitochondria was labeled with MitoTracker Red; ER was labeled with pDsRed2-ER; FUNDC1 was indicated by anti-FUNDC1; Nucleus was stained by DAPI. Representative confocal microscopy image is shown. The images of each single channel were given in Supplemental Figure 15. Scale bar, 10 μm. B, Traces of fluorescence intensity spatial profiles through the red line shown in A for MitoTracker Red, DsRed2-ER, and FUNDC1 fluorescence. C, Western blot analysis of the protein levels of FUNDC1 in homogenates (H), as well as crude mitochondria (Mc), pure mitochondria (Mp), MAMs, ER, and cytosolic (C) fractions from mouse hearts. Calreticulin was used as an ER marker, voltage dependent anion channel 1 (VDAC1) was used as a mitochondrial marker, and inositol 1,4,5-trisphosphate type 2 receptor (IP₃R2) was used as a MAMs component (n = 5 mice per group). D, Representative confocal images showing the association between ER (DsRed2-ER) and mitochondria (MitoTracker Red) in Fundc1^wt/Y/Cre^αMyHC+/− and Fundc1^f/Y/Cre^αMyHC+/− neonatal cardiomyocytes (Scale bars, 10 μm; n = 5 mice per group). The images of each single channel were given in Supplemental Figure 16. E, Pearson’s coefficient, calculated by ZEN software, indicates the contact of ER and mitochondria (mean ± SD, n = 5 independent experiments; ^*P < 0.05 versus Fundc1^wt/Y/Cre^αMyHC+/−). F, Representative transmission electron microscopy (TEM) images indicating the ER and mitochondrial contacts in Fundc1^wt/Y/Cre^αMyHC+/− and Fundc1^f/Y/Cre^αMyHC+/− hearts. Asterisks indicate mitochondria; Arrows indicate ER. Scale bars, 500 nm. G, Quantification of the ER adjacent to mitochondria in the heart was normalized by total ER length (mean ± SD, n = 5 independent experiments; ^*P < 0.05 versus Fundc1^wt/Y/Cre^αMyHC+/−). H, Cardiac MAMs fractions were prepared from Fundc1^wt/Y/Cre^αMyHC+/− and Fundc1^f/Y/Cre^αMyHC+/− mice. Protein levels of IP₃R2, phosphofurin acidic cluster sorting protein 2 (PACS-2), protein disulfide isomerase (PDI), and sigma-1 receptor (Sig1R) in the homogenates were assayed by western blot. I, Densitometric analysis of the protein levels in H (mean ± SD, n = 5 independent experiments; ^**P < 0.01 versus Fundc1^wt/Y/Cre^αMyHC+/−). J, The interaction between FUNDC1 and IP₃R2 in Fundc1^wt/Y/Cre^αMyHC+/− and Fundc1^f/Y/Cre^αMyHC+/− hearts was determined by immunoprecipitation (IP) and western blot (WB) (n = 5 mice per group).