Characterization of Mitochondria from Primary Isolated Mouse Aortic Smooth Muscle Cells

Sexual dimorphism is demonstrated in cardiovascular disease (CVD). Vascular dysfunction is a critical player in CVD involving mitochondrial respiration in aortic smooth muscle cells. In this study, we characterized contractile and synthetic gene expression, and mitochondrial respiration by sex in aortic smooth muscle cells (ASMC). We hypothesize that female than male ASMC have increased contractile genes and better mitochondrial energetics. Adult male and female C57BL6J mice were euthanized, and thoracic aorta excised. Aortic tissue sections were subject to enzymatic digestion and cultured for subsequent experiments. The 2-delta delta CT method using beta actin as housekeeping gene was used for analysis. For mitochondrial respiration, confluent (70%) cells were trypsinized, enumerated, and seeded in Seahorse mini-culture plates (20,000 cells/well). After overnight incubation mitochondrial stress test was performed to measure basal respiration, maximal respiration, spare capacity, ATP production, proton leak, and non-mitochondrial respiration. All mitochondrial respiratory parameters were expressed as oxygen consumption rate normalized with protein levels (OCR, picomoles/L of O2 per microgram protein, n=8-10 wells/group). Unpaired t-test was used for statistical analysis and P<0.05 was considered significant. To characterize the sex of isolated cells (n=6-8/group), we used sex specific primers that showed significantly higher Sry (P<0.001) and Xist (P<0.001) gene expression of male and female ASMC, respectively. Next, we assessed contractile and synthetic genes including myocardin, alpha actin, and myosin heavy chain 11 that were higher in female than male ASMC (P<0.05). However, myosin heavy chain 10 was higher (P=0.002) in male than female cells. The basal respiration and ATP production were significantly high in female ASMC by 79% and 77%, respectively, when compared with male ASMC (2.48 ± 0.13 vs. 1.38 ± 0.15, P= 0.0001; 2.11 ± 0.11 vs. 1.19 ± 0.11, P= 0.0001). Female cells indicated increased spare respiratory capacity compared to male ASMC (1.67 ± 0.31 vs. 1.1 ± 0.23, p= 0.01). The maximal respiration and proton leak were significantly higher in female than male cells by 69% and 109% (4.21 ± 0.33 vs. 2.49 ± 0.31, P= 0.001: 0.42 ± 0.02 vs. 0.2 ± 0.04, P= 0.001). Female and male cells did not show any significant difference in non-mitochondrial respiration. Based on these results, female than male ASMC exhibit increased contractile gene expression and better mitochondrial energetics at basal and stressed conditions. The sex-specific differences in mitochondrial function may attribute to sex bias in CVD. R00HL155841 and R25HL105400 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.