Ameliorating Endothelial Mitochondrial Dysfunction Restores Coronary Function via Transient Receptor Potential Vanilloid 1–Mediated Protein Kinase A/Uncoupling Protein 2 PathwayNovelty and Significance by Shiqiang Xiong, Peijian Wang, Liqun Ma, Peng Gao, Liuping Gong, Li Li, Qiang Li, Fang Sun, Xunmei Zhou, Hongbo He, Jing Chen, Zhencheng Yan, Daoyan Liu, and Zhiming Zhu Hypertension Volume 67(2):451-460 January 13, 2016 Copyright © American Heart Association, Inc. All rights reserved.

Effect of transient receptor potential vanilloid 1 (TRPV1) or uncoupling protein 2 (UCP2) deficiency on coronary vasodilatation in ApoE−/− mice. Effect of transient receptor potential vanilloid 1 (TRPV1) or uncoupling protein 2 (UCP2) deficiency on coronary vasodilatation in ApoE−/− mice. A–C, Endothelium-dependent relaxations of the coronary artery rings from ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with normal diet (ND) or high-fat diet (HFD). *P<0.05, **P<0.01 vs ND; ##P<0.01 vs HFD. D–F, Endothelium-independent relaxations of the coronary artery rings from ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with ND or HFD. *P<0.05 vs ND. Data are expressed as mean±SEM. n=6 for each mouse genotype. Concentration–response curves were analyzed via nonlinear regression followed by Student t test for comparisons between 2 groups. 5-HT indicates 5-hydroxytryptamine; and L-NAME, NG-nitro-l-arginine methyl ester. Shiqiang Xiong et al. Hypertension. 2016;67:451-460 Copyright © American Heart Association, Inc. All rights reserved.

Effect of transient receptor potential vanilloid 1 (TRPV1) or uncoupling protein 2 (UCP2) deficiency on coronary blood flow and myocardial perfusion. Effect of transient receptor potential vanilloid 1 (TRPV1) or uncoupling protein 2 (UCP2) deficiency on coronary blood flow and myocardial perfusion. Representative images (A) and summarized data (B) for the left coronary artery flow spectrum of ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with normal diet (ND) or high-fat diet (HFD); these results were acquired using a high-frequency ultrasound system; n=8 to 10 per group. Representative images (C) and summarized data (D) for electrocardiographic recordings and measurements of ST-segment depression obtained from ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with ND or HFD; n=6 per group. **P<0.01 vs ND, #P<0.05 vs ApoE−/− (HFD) group. Data are expressed as mean±SEM. Data were assessed using 2-tailed Student t tests or ANOVA followed by Bonferroni multiple comparisons post hoc tests as appropriate. Shiqiang Xiong et al. Hypertension. 2016;67:451-460 Copyright © American Heart Association, Inc. All rights reserved.

Effect of transient receptor potential vanilloid 1 (TRPV1) or uncoupling protein 2 (UCP2) deficiency on endothelial reactive oxygen species (ROS) generation and NO production. Effect of transient receptor potential vanilloid 1 (TRPV1) or uncoupling protein 2 (UCP2) deficiency on endothelial reactive oxygen species (ROS) generation and NO production. Representative images of 10 mice each group (A) and summarized data (B), ROS generation measured by DHE in the en face endothelium of aortae from ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with normal diet (ND) or high-fat diet (HFD). Red: DHE fluorescence (excitation, 515 nm) in the nucleus of endothelial cells in the aorta. Green: autofluorescence of elastin underneath the endothelium (excitation, 488 nm). Bottom, Merged. Bar, 100 µm. Representative images of 10 mice each group (C) and summarized data (D), nitric oxide (NO) levels measured by DAF-2 DA in the endothelium of aortae rings from ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with ND or HFD. Bar, 200 µm. **P<0.01 vs ND; #P<0.05, ##P<0.01 vs the ApoE−/− (HFD) group. Data are expressed as mean±SEM. Data were compared using 2-tailed Student t tests or ANOVA followed by Bonferroni multiple comparisons post hoc tests as appropriate. DAF-2DA indicates 4,5-diaminofluorescein diacetate; DHE, dihydroethidium; and KO, knockout. Shiqiang Xiong et al. Hypertension. 2016;67:451-460 Copyright © American Heart Association, Inc. All rights reserved.

Roles of transient receptor potential vanilloid 1 (TRPV1) and uncoupling protein 2 (UCP2) in endothelial mitochondrial dysfunction. Roles of transient receptor potential vanilloid 1 (TRPV1) and uncoupling protein 2 (UCP2) in endothelial mitochondrial dysfunction. Levels of cytosolic reactive oxygen species (ROS; A) and mitochondrial ROS (B) induced by oxidized low-density lipoprotein (ox-LDL) in human umbilical vein endothelial cells (HUVECs). **P<0.01 vs control; n=6 to 8 per group. C, Representative profiles of 6 different experiments in each group. D, Summarized data for oxygen consumption in mitochondria of HUVECs exposed to ox-LDL, ox-LDL+capsaicin, or ox-LDL+capsaicin+genipin (inhibitor of UCP2), as measured via Oxygraph-2k high-resolution respirometry. Blue line, decreases in oxygen concentration in the oxygraphy chamber containing living endothelial cells (ECs). Red line, oxygen consumption in response to the application of substrates for complex I (CI) and II (CII). The values are expressed in pmol/s/106 cells. E, After treatment, cells were stained with JC-1 staining solution for mitochondrial membrane potential (MMP) measurement. MMP is expressed as the ratio of red/green fluorescence intensity; n=3 per group. F, Representative immunoblots and quantitative analysis of NADH dehydrogenase [ubiquinone] 1 α subcomplex subunit 9 (NDUFA9; 39 kDa) expression in ECs treated with ox-LDL, ox-LDL+Cap, or ox-LDL+Cap+Gen. n=3. **P<0.01 vs control; #P<0.05, ##P<0.01 vs ox-LDL; &P<0.05, &&P<0.01 vs ox-LDL+Cap. Data are expressed as mean±SEM. Data were compared using 2-tailed Student t tests or ANOVA followed by Bonferroni multiple comparisons post hoc tests as appropriate. Cap indicates capsaicin; CI Leak, leak respiration of CI; CI OXPHOS, CI + CII OXPHOS, CI and CI plus CII oxidative phosphorylation capacity; CI ETS, CI + CII ETS, CI and CI plus CII electron transfer system capacity; Control, vehicle treatment; and Gen, genipin. Shiqiang Xiong et al. Hypertension. 2016;67:451-460 Copyright © American Heart Association, Inc. All rights reserved.

Effect of transient receptor potential vanilloid 1 (TRPV1) activation on endothelial reactive oxygen species (ROS) generation and the protein kinase A (PKA)/uncoupling protein 2 (UCP2) pathway. Effect of transient receptor potential vanilloid 1 (TRPV1) activation on endothelial reactive oxygen species (ROS) generation and the protein kinase A (PKA)/uncoupling protein 2 (UCP2) pathway. Representative immunoblots of phosphorylated-PKA (p-PKA, 40 kDa), PKA (40 kDa), UCP2 (35 kDa), phosphorylated eNOS (p-eNOS, 140 kDa), and eNOS (140 kDa; A). The effect of chronic TRPV1 activation on the levels of p-PKA (B), UCP2 levels (C), and p-eNOS (D) in the aortae of ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed on high-fat diet (HFD) or HFD plus capsaicin (HC). **P<0.01 vs HFD. Data are expressed as mean±SEM; n=3. E, The effect of TRPV1 activation on PKA phosphorylation in cultured endothelial cells (ECs). iRTX, 5′-iodo-RTX. **P<0.01 vs control; ##P<0.01 vs oxidized low-density lipoprotein (ox-LDL); &P<0.05, &&P<0.01 vs ox-LDL+Cap. Data are expressed as mean±SEM; n=6. F, The effect of TRPV1 activation–mediated PKA phosphorylation on UCP2 levels in cultured ECs. **P<0.01 vs control; ##P<0.01 vs ox-LDL; &&P<0.01 vs ox-LDL+Cap1 µmol/L. Data are expressed as mean±SEM, n=6. ECs stained with MitoSOX Red (G) or DHE (H) after culturing in the presence of ox-LDL, ox-LDL+Cap, ox-LDL+Cap+iRTX, ox-LDL+Cap+KT5720, or ox-LDL+Cap+Gen. **P<0.01 vs ox-LDL; #P<0.05, ##P<0.01 vs ox-LDL+Cap. Data are expressed as mean±SEM; n=6 to 8. Data were assessed using 2-tailed Student t tests or ANOVA followed by Bonferroni multiple comparisons post hoc tests as appropriate. Cap indicates capsaicin; Control, vehicle treatment; DHE, dihydroethidium; and Gen, genipin. Shiqiang Xiong et al. Hypertension. 2016;67:451-460 Copyright © American Heart Association, Inc. All rights reserved.

Effect of transient receptor potential vanilloid 1 (TRPV1) activation on coronary function and the prognosis of ApoE−/− mice. Effect of transient receptor potential vanilloid 1 (TRPV1) activation on coronary function and the prognosis of ApoE−/− mice. A–C, The changes in coronary flow spectrum, reactive oxygen species (ROS) generation and nitric oxide (NO) levels in the endothelium of aortae from ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with high-fat diet (HFD) or HFD plus capsaicin (HC) relative to that in mice fed with normal diet (ND; Figure 1). **P<0.01 vs the HFD; n=8 to 10 mice per group. Endothelium-dependent relaxation in coronary artery rings from ApoE−/− (D), ApoE−/−/TRPV1−/− (E), and ApoE−/−/UCP2−/− (F) mice fed with HC when compared with mice fed with ND or HFD. *P<0.05 vs ND; #P<0.05 vs HFD; n=6. G, The changes in ST-segment depression in the ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with HFD or HC relative to that in mice fed with ND (Figure 2). *P<0.05 vs the HFD group. n=6. Survival curves (H) and summarized data (I) from ApoE−/−, ApoE−/−/TRPV1−/−, and ApoE−/−/UCP2−/− mice fed with HFD or HC. **P<0.01 vs HFD; ##P<0.01 vs the HC-fed ApoE−/− group. n=15. All data are expressed as mean±SEM. Data were assessed using 2-tailed Student t tests. Concentration–response curves were analyzed via nonlinear regression followed by Student t tests for comparisons between 2 groups. Survival curves were calculated by the Kaplan–Meier method and compared using the log-rank test. UCP2 indicates uncoupling protein 2. Shiqiang Xiong et al. Hypertension. 2016;67:451-460 Copyright © American Heart Association, Inc. All rights reserved.