Upregulation of endothelial DRP1 promotes a pathological vasodilation mechanism in response to flow-induced dilation

Abstract

Background: Endothelial microvascular dysfunction is a strong and independent risk factor for coronary artery disease (CAD), and improvements in microvascular function improve rates of survivability. Microvascular endothelium of healthy individuals releases nitric oxide (NO) in response to increased blood flow, while individuals with CAD release mitochondria-derived hydrogen peroxide (H2O2), a reactive oxygen species (ROS) molecule, in response to the same stimulus. Mitochondrial fission, a mitochondrial regulatory mechanism that is mediated by DRP1, is associated with increased ROS production as well as a multitude of cardiometabolic disease including obesity, pulmonary arterial hypertension, and diabetes. Aim: We hypothesize that increased endothelial DRP1 mediates the phenotypic shift in the release of NO to H2O2 in response to increased blood flow in individuals with CAD. Methods & Results: Atrial and adipose tissue was obtained from surgical discard tissue from individuals with and without CAD (non-CAD). Western blot analysis of LV tissue shows increased DRP1 expression in individuals with CAD compared to non-CAD (1.97 to 0.99, N=3-7, p<0.05). CD31 magnetic isolation was implored on digested pericardial adipose tissue and on intact blood vessels followed by COXIV immunofluorescent antibody staining and confocal microscopy. Immunofluorescent imaging reveals that individuals with CAD have increased mitochondrial fragmentation counts compared to non-CAD (3.3 to 1.2, N=3-5, p<0.05). This difference is even more stark in intact arterioles from human patients with CAD compared to non-CAD (45.0 to 5.5, p<0.05). Assessment of flow-mediated dilation (FMD) of microvessels is a suitable approach to assess endothelial-dependent vasodilation functionality. Microvessels were obtained from individuals with CAD and treated intraluminally overnight with a DRP1 siRNA (si-DRP1) or siRNA scramble control. Endothelial function was then assessed by FMD in the presence of L-NAME (NOS inhibitor) or PEG-catalase (peg-cat, H2O2 scavenger). FMD was blocked by L-NAME (p<0.05) in the vessels treated with si-DRP1 indicating that blocking DRP1 restores physiological NO-mediated vasodilation. Lastly, microvessels from non-CAD were treated overnight with adeno-associated virus (AAV) inoculated with DPR1 mRNA (or GFP mRNA as control). FMD was blocked by peg-cat in the vessels treated with AAV-DRP1 indicating that upregulating DRP1 promotes a pathological H2O2-mediated vasodilation in humans. Production of H2O2 was confirmed by inoculating non-CAD microvessels with AAV-DRP1 using a H2O2-fluorescent probe in intact blood vessels in response to flow. Production of NO was confirmed by transfection of CAD microvessels with si-DRP1 using a NO-fluorescent probe in intact blood vessels in response to flow. Conclusion: Identifying the role of mitochondrial dynamics and ROS pathways on endothelial function may provide novel avenues of therapeutic potential for the treatment of CAD. American Physiology Society (Porter Fellowship, CG), NIH/NHLBI R01 HL-135901 (Gutterman), NIH/NHLBI R01 HL-133029 (Beyer), AHA SFRN, Medical College of Wisconsin (Beyer) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.