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Introduction: Microvascular endothelial dysfunction is a strong, independent, and modifiable risk factor for the development of coronary artery disease (CAD). Under physiological conditions, an increase in blood flow stimulates endothelial cells (EC) to release nitric oxide (NO) which mediates microvascular dilation. Under pathological conditions, such as CAD, NO-mediated dilation is lost but dilator capacity is preserved by mitochondria-derived hydrogen peroxide (H 2 O 2 ), a reactive oxygen species (ROS). Recent work in rodent models suggests that ATP is required for NO-mediated and increasing evidence suggests that the balance between mitochondrial ATP vs. ROS production contributes to this phenotypic switch. Mitochondrial fission, an autoregulatory process primarily mediated by DRP1, is associated with various cardiometabolic diseases and is a direct contributor to increased reactive oxygen species (ROS) formation and decreased production of ATP. However, the role of DRP1 in the endothelium and it impacts on vascular function is unknown. Hypothesis: We hypothesize that mitochondrial fission, mediated by DRP1, is a critical regulator of microvascular redox balance. Methods: Microvessels (~50-200μm) were obtained from discarded human adipose surgical tissue from individuals with and without CAD or from rat mesentery and subjected to pressure myography (flow-mediated dilation; FMD). A fluorescent mitochondrial-specific ATP probe was measured in vessels after 10 minutes at maximal shear stress. DRP1 overexpression was achieved by adeno-associated viral (AAV) gene transfer and downregulated via DRP1 targeted siRNA (si-DRP1). To show physiologic and translational relevance, a transgenic rat model with a cre-inducible endothelial-specific overexpression of DRP1 (DRP Tg) was created to test the in vivo effects of EC-DRP1 overexpression. To test whether reduction of ROS improved the observed phenotype, vessels were acutely treated ex vivo in the organ chamber with TEMPOL, a cytosolic antioxidant, and mito-tempo, a mitochondrial antioxidant. Two-way ANOVA, post-hoc analysis was performed using PRISM to best understand the effects of the treatment and pressure-gradient. T-test was performed for comparison analyses between patients with/without CAD. Results:Fluorescent analysis of showed that ATP is increased in vessels obtained from patients without CAD compared to those with CAD (6.3 vs 1.1, mean fluorescent intensity, N=3, p<0.05). In vessels obtained from patients without CAD, AAV-mediated DRP1 overexpression switches the mediator of FMD from NO to H 2 O 2 (inhibitable by PEG-Catalase, max dilation 83.2% vs. 20.2%, N=6, p<0.05). Conversely, arterioles obtained from patients with CAD treated with si-DRP1 restores NO-mediated dilation (inhibitable by L-NAME, max dilation 94.6% vs. 41.7%, N=5, p<0.05). In the rat microcirculation, FMD is significantly impaired in EC-DRP1 rats compared to littermate controls (max dilation 41.2% vs. 80.3%, N=9, p<0.05). Scavenging of ROS (TEMPOL and mito-Tempo) does not restore the phenotype in both human and rat microcirculation suggesting DRP1 mediates dysfunction independent of ROS, which is in line with preliminary observations of reduced flow-induced ATP levels in vessels from patients with CAD. Significance: These data indicate that ROS may be secondary to mitochondrial fission rather than causal to the microvascular phenotype observed in patients with CAD. Therefore, targeting endothelial mitochondrial fission may be of therapeutic benefit to treat cardiovascular disease. AHA Diversity Research Pre-Doctoral Award (CGH), 5P30ES030283-04 (AJL), 5R01HL133029-5 (AB), 5R21OD018306-2 (AB). This is the full abstract presented at the American Physiology Summit 2024 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.
Huerta et al. (Wed,) studied this question.