Abstract 21401: Mitofusin 2 Gene Therapy Increases PGC-1α Expression in Pulmonary Artery Smooth Muscle Cells but Does Not Alter Mitochondrial Oxygen Consumption
Introduction: Vasoconstriction and proliferation of pulmonary artery smooth muscle cells (PASMCs) is important in the development of pulmonary arterial hypertension. Mitochondrial morphology is very dynamic in these cells and is regulated by several proteins including mitofusin 2 (Mfn2), an outer mitochondrial membrane protein which promotes mitochondrial accumulation. We tested whether changes in mitochondrial morphology influence mitochondrial activity and energy metabolism.
Methods: PASMCs were cultured from Sprague Dawley rats and transfected with Mfn2 or LacZ overexpressing adenoviruses. Mitochondrial fragmentation was calculated after transfection of mitochondria-targeted DsRed (number of mitochondria divided by total DsRed pixel count). Oxygen consumption was measured in a Mitocell MT200 (Strathkelvin). Mitochondrial mass (1μM MitoTracker Green for 20′) and proliferation (10μM BrdU for 1h) were determined by flow cytometry.
Results: Adenoviral transduction upregulated Mfn2 expression 317±64 fold and the mitochondrial fragmentation factor was decreased (LacZ 2.3±0.2 vs Mfn2 1.6±0.2, n=20 cells/group, P<0.05). Mfn2 gene therapy increased Glucose Transporter 1 (Glut1) expression 3.7±0.6 fold (n=6, P<0.01) and Peroxisome Proliferator-activated Receptor gamma Coactivator 1 alpha (PGC1a) 65.2±26.7 fold (P<0.05). Mitochondrial mass was slightly increased (LacZ 48.4±5.5 vs. Mfn2 60.82±0.9 arbitrary MitoTracker Green fluorescence, P=0.12). Both Mfn2 and LacZ treated PASMCs had identical baseline oxygen consumption (LacZ 3.9±0.4 vs. Mfn2 4.0±0.5 nmol/min*106 cells, n=6, P=0.91) and also maximal oxygen consumption in response to the uncoupler FCCP was very similar (LacZ 6.4±0.4 vs. Mfn2 5.4±0.6 nmol/min*106 cells, P=0.41). Proliferation was decreased approximately with 20% after Mfn2 gene therapy.
Conclusions: Mfn2 gene therapy increases mitochondrial fusion and Glut1 and PGC1a expression, suggesting increased glucose uptake and mitochondrial biogenesis respectively. Unexpectedly, this did not increase mitochondrial activity as measured with oxygen consumption. This suggests increased glycolysis but further research is necessary to establish whether increased PGC1a reflects a homeostatic mechanism.
- © 2010 by American Heart Association, Inc.