Nitric oxide regulates vascular adaptive mitochondrial dynamics

Abstract

Cardiovascular disease risk factors, such as diabetes, hypertension, dyslipidemia, obesity, and physical inactivity, are all correlated with impaired endothelial nitric oxide synthase (eNOS) function and decreased nitric oxide (NO) production. NO-mediated regulation of mitochondrial biogenesis has been established in many tissues, yet the role of eNOS in vascular mitochondrial biogenesis and dynamics is unclear. We hypothesized that genetic eNOS deletion and 3-day nitric oxide synthase (NOS) inhibition in rodents would result in impaired mitochondrial biogenesis and defunct fission/fusion and autophagy profiles within the aorta. We observed a significant, eNOS expression-dependent decrease in mitochondrial electron transport chain (ETC) protein subunits from complexes I, II, III, and V in eNOS heterozygotes and eNOS null mice compared with age-matched controls. In response to NOS inhibition with NG-nitro-L-arginine methyl ester (L-NAME) treatment in Sprague Dawley rats, significant decreases were observed in ETC protein subunits from complexes I, III, and IV as well as voltage-dependent anion channel 1. Decreased protein content of upstream regulators of mitochondrial biogenesis, cAMP response element-binding protein and peroxisome proliferator-activated receptor-γ coactivator-1α, were observed in response to 3-day L-NAME treatment. Both genetic eNOS deletion and NOS inhibition resulted in decreased manganese superoxide dismutase protein. L-NAME treatment resulted in significant changes to mitochondrial dynamic protein profiles with decreased fusion, increased fission, and minimally perturbed autophagy. In addition, L-NAME treatment blocked mitochondrial adaptation to an exercise intervention in the aorta. These results suggest that eNOS/NO play a role in basal and adaptive mitochondrial biogenesis in the vasculature and regulation of mitochondrial turnover.

Keywords: endothelial nitric oxide synthase; vascular mitochondria.

Fig. 1.

Endothelial nitric oxide synthase (eNOS) null aortas have reduced mitochondrial electron transport chain (ETC). Aortas were taken from 18-wk-old eNOS partial null heterozygotes and eNOS null mice and submitted to SDS-PAGE. Western blotting was performed for mitochondrial ETC subunits representing complexes I–V as follows: I, complex I subunit NDUFA9; II, complex II subunit succinate dehydrogenase complex, subunit A (SDHA); III, complex III core protein; IV, complex IV subunit 4; V, complex V α-subunit. A and B: representative blots (A) and compiled data (B) from Western analysis. Graph shown is the mean ± SE from 6–7 animals/group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 2.

N^G-nitro-l-arginine methyl ester (l-NAME) injection decreases mitochondrial content. Twelve-week-old male Sprague Dawley rats were injected with 50 mg·kg⁻¹·day⁻¹ l-NAME or vehicle (PBS) for 3 days. Aortas were harvested 24 h following the last injection and subjected to SDS-PAGE. Western blotting was performed for mitochondrial ETC subunits representing complexes I–V. A: representative blots (A1) and compiled data (A2) from Western analysis of ETC complexes. B: representative blots and compiled data for voltage-dependent acnion channel 1 (VDAC1). Graphs shown are means ± SE from 10 animals/group. *P < 0.05 and **P < 0.01.

Fig. 3.

The effect of 3-day l-NAME injection on mitochondrial biogenesis signaling. Twelve-week-old male Sprague Dawley rats were injected with 50 mg·kg⁻¹·day⁻¹ l-NAME or vehicle (PBS) for 3 days. Aortas were harvested 24 h following the last injection and subjected to SDS-PAGE. Western blotting was done for cAMP response element-binding protein (CREB) content and activity, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) content, and silent mating type information regulation 2 homolog 1 (SIRT1) content. A: representative blots for CREB and pCREB. B and C: compiled data from Western analysis of CREB and pCREB. D: representative blots and compiled data from Western analysis of PGC-1α. E: representative blots and compiled data from Western analysis of SIRT1. Graphs shown are means ± SE from 10 animals/group. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4.

Three-day l-NAME injection decreases markers of mitochondrial fusion and increases markers of mitochondrial fission. Twelve-week-old male Sprague Dawley rats were injected with 50 mg·kg⁻¹·day⁻¹ l-NAME or vehicle (PBS) for 3 days. Aortas were harvested 24 h following the last injection and subjected to SDS-PAGE. Western blotting was done for mitofusin 1, mitofusin 2, optic atrophy 1 (OPA1), fission 1 (Fis1), and dynamin-related protein 1 (DRP-1). A: representative blots and compiled data for mitofusin 1. B: representative blots and compiled data for mitofusin 2. C: representative blots and compiled data for OPA1. D: representative blots and compiled data for Fis1. E: representative blots and compiled data for DRP-1. Graphs shown are means ± SE from 10 animals/group. *P < 0.05, **P < 0.01, and ***P < 0.001

Fig. 5.

Three-day l-NAME injection has minimal effect on autophagy. Twelve-week-old male Sprague Dawley rats were injected with 50 mg·kg⁻¹·day⁻¹ l-NAME or vehicle (PBS) for 3 days. Aortas were harvested 24 h following the last injection and subjected to SDS-PAGE. Western blotting was done for LC3b, Beclin1, p62, and BCL2/adenovirus E1B 19-kDa protein-interacting protein 3 (BNIP3). A: representative blots of LC3b. LC3b is expressed as a ratio of the 17-kDa fragment to the 19-kDa fragment. B: representative blots and compiled data for Beclin1. C: representative blots and compiled data for p62. D: representative blots and compiled data for BNIP3. Graphs shown are means ± SE from 10 animals/group. *P < 0.05.

Fig. 6.

l-NAME treatment perturbs the mitochondrial adaptive response to exercise. Twelve-week-old male Sprague Dawley rats were injected with 50 mg·kg⁻¹·day⁻¹ l-NAME or vehicle (PBS) for 8 days. Cohorts were separated into either exercised or sedentary groups. Exercised animals were run on the treadmill (0% grade) at 15 m/min for 30 min on days 1 and 2 and for 45 min on days 3–8. Injections began on the 1st day of exercise, and injections and exercise continued for 8 days. After the last exercise bout and injection (24 h), animals were killed, and aorta tissue was removed and subjected to SDS-PAGE. A1: compiled data from Western blotting performed for mitochondrial ETC subunits representing complexes I–V. A2: compiled data for mitofusin 2. A3: compiled data for Fis1. B1: compiled data from Western analysis for ETC complex subunits from animals receiving l-NAME injection. B2: compiled data for mitofusin 2. B3: compiled data for Fis1. Graphs shown are means ± SE from 5 animals/group. *P < 0.05.