Abstract 19465: 3-D Super-Resolution Microscopy of Mitochondrial Internalization in Cardiac Cells
Ischemia and reperfusion of the heart negatively impacts the function of mitochondria, which results in energy deficits attributable to the impairment of oxidative phosphorylation through loss of high energy phosphate reserves, accumulation of calcium, and excessive reactive oxygen species generation. While these events are initiated during ischemia, they extend throughout reperfusion to compromise functional recovery and cell viability. To preserve cardiac viability and functional performance, we developed a unique approach to support the heart by injecting ischemic regions with exogenous mitochondria isolated from a remote, autologous, non-ischemic tissue at the onset of reperfusion. We discovered treated hearts have increased adenosine triphosphate (ATP) stores, reduced infarct sizes, and improved contractile function. Intriguingly, we observed some transplanted mitochondria were internalized by cardiomyocytes; however, little was known about their intracellular fate. Here, we used a baculovirus-expression system to target fluorescent proteins to specific organelles and cell structures in iPS-derived human cardiomyocytes and human cardiac fibroblasts. Cells were incubated with isolated mitochondria labeled with a different colored fluorophore for 0, 1, 2, 4, 8, and 24 hours. Following incubation, samples were prepared for 3-D super-resolution microscopy. Using structured illumination, we found internalized exogenous mitochondria fused with the endogenous mitochondrial network in both cell types. Although some internalized mitochondria were associated with early endosomes, there was little or no co-localization with late endosomes, lysosomes, the Golgi complex, or sarcoplasmic reticulum. Mitochondrial fusion was obvious by 4 hours and persisted for 24 hours. Measurements of ATP production and oxygen consumption in parallel experiments confirmed the addition of exogenous mitochondria functionally benefited treated cells. Further investigation of this biological phenomenon may guide the development of treatments directed at replacing or augmenting impaired mitochondria in the ischemic heart as well as in a wide range of mitochondrial diseases.
Author Disclosures: D.B. Cowan: None. R. Yao: None. J.K. Thedsanamoorthy: None. P.J. del Nido: None. J.D. McCully: None.
- © 2016 by American Heart Association, Inc.