Figure 4.

Fusion of exogenous mitochondria with the cardiomyocyte mitochondrial network. (a) iPS-CMs with RFP-labelled mitochondria (red) treated for 4 h with isolated GFP-labelled HCF mitochondria (green). After fixation, the nuclei were stained with DAPI (blue) and slides were imaged using 3-D SR-SIM. Individual red, green, and blue channels are shown along with the combined color rendering. Fusion of endocytosed mitochondria with the endogenous mitochondrial network is readily apparent. Mitochondrial fusion was apparent at all times examined (0.5, 1, 2, and 4 h) and four separate experiments were performed. Scale bars, 0.5 µm. (b) Immunoblot analysis of whole cell and isolated mitochondrial lysates (25 µg per lane) from iPS-CMs and HCFs using antibodies directed against MFN1, MFN2, OPA1, and mitochondria (MTC02). Lanes 1 to 4 contained the following lysates: iPS-CM cell proteins, HCF cell proteins, iPS-CM mitochondrial proteins, and HCF mitochondrial proteins. Arrows indicate the specifically detected protein(s) for each antibody and immunoblot experiments were repeated six times. (c) 3-D SR-SIM of human cardiomyocytes containing RFP-labelled mitochondria (red) incubated with isolated GFP-labelled exogenous mitochondria (green) for 30 min. Coverslips were fixed and stained with a mitofusin-1 (MFN1) antibody (white). The top panels show the three separate color channels and the combined image (left to right). The boxed regions have been magnified in successive images (top right to lower panels, left to right) to show the reactivity of the MFN1 antibody with fusing mitochondria and the presence of this antibody throughout the cytosol. Parallel experiments were performed at 1, 2, and 4 h and the presented images are representative of four separate experiments. Scale bars, 0.5 µm.