Abstract 15684: A Novel Approach to Quantifying the Fragmentation of Mitochondria Induced by Hypoxia in Pulmonary Artery Smooth Muscle Cells
Introduction and hypothesis: Pulmonary arterial hypertension (PAH) is characterized by an excessive proliferation of pulmonary artery smooth muscle cells (PASMC) and is in part mediated by activation of HIF-1α (hypoxia inducible factor-1 alpha). Mitochondrial fragmentation is a necessary prerequisite for cell division and may thus contribute to PASMC proliferation which is believed to contribute to the development of PAH. In this study we developed a novel imaging approach to quantify mitochondrial fragmentation and then tested the hypothesis that hypoxia leads to mitochondrial fragmentation by activating HIF-1α.
Methods: The mitochondrially targeted constructs Mito-DS-Red (red fluorescent protein) and Mito-PA-GFP (photo-activatable green fluorescent protein) were co-expressed in PASMCs. We performed automated particle counting of the number of Mito-DS-Red-expressing mitochondrial fragments. This number was normalized to the total number of mitochondria population and expressed as the mitochondrial fragmentation factor (MFF). We compared this approach to the established approach of evaluating the mitochondrial network with photo-activated Mito-PA-GFP.
Results: Our novel approach confirmed a fragmented mitochondrial state after overnight hypoxia with 1%O2 (Normoxia: 1.30 ± 0.20, N=11; Hypoxia: 2.62 ± 0.23, N=10; P<0.001). The HIF-1α activator cobalt chloride (500 μM) resulted in an even greater MFF (CoCl2: 4.80 ± 0.56 N=11; P<0.001). These results mirrored those obtained with the use of Mito-PA-GFP.
Conclusions: Hypoxia and direct HIF-1α activation induce significant mitochondrial fragmentation in PASMC, as assessed with automated particle counting. Our automated approach allows for high through-put evaluation of therapies that modify the mitochondrial network. Since higher level of mitochondrial fragmentation is observed under conditions that support proliferation, modifying the mitochondrial network may inhibit smooth muscle proliferation in PAH.
- © 2010 by American Heart Association, Inc.