Modulation of DRP1 and MFN1/2 alters mitochondrial morphology and dynamics in endothelial cells

Endothelial microvascular dysfunction is a modifiable risk factor that predicts major adverse cardiac events. Mitochondrial fission and fusion are important regulators of cellular and mitochondrial homeostasis and regulate and we hypothesize that altering mitochondrial fission and fusion may play a critical role in modulating endothelial function. Mitochondrial fission is primarily mediated by Dynamin-related protein 1 (DRP1). Levels of DRP1 are known to be elevated in individuals with pulmonary arterial hypertension, obesity, and diabetes. In contrast, mitochondrial fusion enhances mitochondrial function by allowing for the exchange of important metabolites, enzymes, and mitochondrial DNA, overall promoting cellular health and ATP production. Fusion is mediated by OPA1 and OMA1 which conjoin the mitochondrial internal membrane while MFN1 and MFN2 conjoin the mitochondrial outer membrane. To investigate this, we modulated DRP1 and MFN1/2 using an adeno-associated viral (AAV) vector and silencing RNA (siRNA) to HMEC-1 cells and in a proof-of-principle experiment, subsequently measured the amount of protein expressed for each by western blot analysis. We then used immunofluorescence to visualize mitochondrial networks and hypothesized that increasing DRP1 would lead to increased fragmentation. To assess mitochondrial function, we conducted a series of Seahorse experiments to assess oxygen consumption rate (OCR). Western blot analysis revealed that levels of DRP1 increased 30% with overnight AAV treatment and decreased by 38% when treating overnight with siRNA. IF analysis presented that AAV-DRP1-treated cells had increased mitochondrial fragmentation (MFC = 3.2 to 0.83, p<0.05); this phenotype was also observed when HMEC1 cells treated with AAV-MFN1/2 (MFC = 2.5 to 0.83, p<0.05). Seahorse analysis showed that treating overnight with AAV-MFN1 increased mitochondria OCR, but interestingly treatment with AAV-MFN2 did not. Treatment with AAV-DRP1 did not alter HMEC1 mitochondria OCR. In contrast, treatment with siMFN1, siMFN2, and siDRP1 all increased mitochondria OCR in this cell type. Our results show that altering DRP1 and MFN1/2 lead to distinct phenotypes but overall show that causing a disbalance of mitochondria fission and fusion is linked to morphologic changes coupled with changes in mitochondrial ATP production efficiency. Therefore, it is likely that mitochondrial dynamics play an important role in modulating endothelial microvascular function. Supported by the NIH/NHLBI Grant nos. HL173549 and HL157025 (to A.M.B.). C.G.H. was supported by funding through the NIH/NHLBI (1F31HL176057). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.