Fig. 6.

Transplantation of autologous mitochondria in rat neonatal cardiomyocytes and high-energy synthesis. A: neonatal rat cardiomyocytes incubated for 24 h with Lewis rat liver mitochondria labeled with MTRed. Nuclei (DAPI, blue; left), F-actin (green; left middle), Lewis rat liver mitochondria (red; right middle), and the merged image (right). Scale bars, 25 μm. B: cardiomyocytes stained for total mitochondria with MitoFluor Red and DNA with DAPI (blue). Control cardiomyocytes (left) and cardiomyocytes cocultured with HeLa cell mitochondria detected with anti-human MTC02 and Alexa 488 anti-mouse antibody (green) for 2 (left middle), 8 (right middle), and 24 h (right). The 2-h time period shows that many mitochondria are extracellular, whereas the 8-h time period shows that most mitochondria are internalized in the cardiomyocytes. The 24-h time period shows a mixture of intra- and extracellular mitochondria. Scale bars, 25 μm. C: cardiomyocytes cocultured with HeLa mitochondria detected using MTC02 and Alexa 488 anti-mouse antibody (green) and CM-DiI for 4 h to reveal the cell membranes. Nuclei are stained blue. Scale bars are 25 μm. D: transmission electron microscopy of syngeneic rat liver mitochondria inside cardiomyocytes in plastic section is shown at left. To detect xenogenic HeLa mitochondria in cardiomyocytes, frozen sections were incubated with anti-human MTC02, which was detected with an anti-mouse secondary antibody and a protein A-gold conjugate (shown as black dots, indicated by white arrows at middle and right). Transplanted mitochondria range in size from 500 to 1,200 nm in diameter. Scale bars, 500 nm. m, Native mitochondria. Arrows represent transplanted mitochondria. E: oxygen consumption rate (OCR) and rate of acid efflux (ECAR) in day 2 neonatal rat cardiomyocytes in control, cocultured with respiration media only (Con), and in cardiomyocytes cocultured with respiration media containing 1.26 × 10⁶ rat liver mitochondria at 2, 4, or 8 h following coculture. Results are means ± SE; n = 4–8 each. *P < 0.05 vs. control. F: costaining with the acidotropic probe LysoTracker (shown in red). G: costaining with the lysosomal protein LAMP-1 (shown in red). H and I (higher magnification): costaining with cadaverine (autophagosome; shown in red). Scale bars, 500 nm. There was no colocalization of mitochondria with any of the lysosomal or autophagosomal markers.