Mitochondrial Isolevuglandins Contribute to Vascular Oxidative Stress and Mitochondria-Targeted Scavenger of Isolevuglandins Reduces Mitochondrial Dysfunction and Hypertension

Abstract

Hypertension remains a major health problem in Western Societies, and blood pressure is poorly controlled in a third of patients despite use of multiple drugs. Mitochondrial dysfunction contributes to hypertension, and mitochondria-targeted agents can potentially improve treatment of hypertension. We have proposed that mitochondrial oxidative stress produces reactive dicarbonyl lipid peroxidation products, isolevuglandins, and that scavenging of mitochondrial isolevuglandins improves vascular function and reduces hypertension. To test this hypothesis, we have studied the accumulation of mitochondrial isolevuglandins-protein adducts in patients with essential hypertension and Ang II (angiotensin II) model of hypertension using mass spectrometry and Western blot analysis. The therapeutic potential of targeting mitochondrial isolevuglandins was tested by the novel mitochondria-targeted isolevuglandin scavenger, mito2HOBA. Mitochondrial isolevuglandins in arterioles from hypertensive patients were 250% greater than in arterioles from normotensive subjects, and ex vivo mito2HOBA treatment of arterioles from hypertensive subjects increased deacetylation of a key mitochondrial antioxidant, SOD2 (superoxide dismutase 2). In human aortic endothelial cells stimulated with Ang II plus TNF (tumor necrosis factor)-α, mito2HOBA reduced mitochondrial superoxide and cardiolipin oxidation, a specific marker of mitochondrial oxidative stress. In Ang II-infused mice, mito2HOBA diminished mitochondrial isolevuglandins-protein adducts, raised Sirt3 (sirtuin 3) mitochondrial deacetylase activity, reduced vascular superoxide, increased endothelial nitric oxide, improved endothelium-dependent relaxation, and attenuated hypertension. Mito2HOBA preserved mitochondrial respiration, protected ATP production, and reduced mitochondrial permeability pore opening in Ang II-infused mice. These data support the role of mitochondrial isolevuglandins in endothelial dysfunction and hypertension. We conclude that scavenging of mitochondrial isolevuglandins may have therapeutic potential in treatment of vascular dysfunction and hypertension.

Keywords: Sirtuin 3; blood pressure; mitochondria; oxidative stress; superoxide dismutase.

Figure 1.

Western blots of mitochondrial isoLGs (A) in human arterioles from normotensive and hypertensive subjects (n=5), (B) development of mitochondria-targeted isoLG scavenger mito2HOBA and (C, D) SOD2 acetylation in human arterioles isolated from normotensive and hypertensive subjects and treated ex vivo with mito2HOBA (0.5 μM, 24 hours, DMEM). Data were normalized by Complex I levels (Sham is 100%). Data are mean ± SEM. *P<0.01 vs Normotensive Sham, **P<0.01 vs Hypertensive (n=5).

Figure 2.

Effect of mito2HOBA on mitochondrial superoxide and cardiolipin oxidation in human aortic endothelial cells induced by angiotensin II plus TNFα. (A) Mitochondrial superoxide was measured by HPLC analysis of mitoSOX-superoxide specific product, mito-2-hydroxyethidium (Mito-2OH-Et⁺).^, Mito2HOBA (50 nM) abolishes stimulation of mitochondrial superoxide while similar dose of untargeted isoLG scavenger 2HOBA (50 nM) or high dose of isoLG-inactive analog 4HOBA (10 μM) are not protective. *P<0.01 vs control, **P<0.001 vs Angiotensin II + TNFα. (B) Cardiolipin oxidation induced by Angiotensin II + TNFα measured my LC/MS. Cardiolipin oxidation is significantly attenuated by mito2HOBA (50 nM) while untargeted 2HOBA (10 μM) is not effective. Supplemental figure S1 shows typical chromatograms. Data are mean ± SEM. *P<0.01 vs Control, **P<0.01 vs Angiotensin II + TNFα (n=4).

Figure 3.

Effect of mito2HOBA on angiotensin II-induced hypertension and accumulation of mitochondrial isoLGs protein adducts. (A) Blood pressure tail-cuff measurements in male Sham or angiotensin II-infused mice supplied with mito2HOBA in drinking water (0.1 g/L) or equimolar amount of untargeted analog 2HOBA (0.17 mmol/L). (B) Telemetry studies of blood pressure in angiotensin II-infused mice supplied with mito2HOBA or plain water as a vehicle. (C) Representative LC/MS/MS chromatograms of isoLG-Lysyl-Lactam adduct; (D) isoLG-Lys-Lactam levels in kidney mitochondria isolated from Sham or angiotensin II-infused mice supplied with mito2HOBA. Results are mean ± SEM. *P<0.01 vs Sham, **P<0.01 vs Ang II (n=8).

Figure 4.

Western blot analysis of mitochondrial acetylation in aortas isolated from Sham and angiotensin II-infused mice treated with mito2HOBA. (A) Typical Western blots isoLG-protein adducts (D11 ab), Sirt3, Acetyl-Lysine, SOD2-K68-Acetylation, CypD Acetylation, isoLG adduct with complex I NDUFS1 75 KDa subunit and mitochondrial complex I; (B) Sirt3 levels; (C) mitochondrial protein lysine acetylation; (D) SOD2-K68-Acetyl levels; and (E) CypD-Acetyl levels. Mice supplied with mito2HOBA (m2H) in drinking water (0.1 g/L) and angiotensin II (osmotic pump, 0.7 mg/kg/day) for 14 days. Data were normalized by Complex I levels (Sham is 100%). Results are mean ± SEM (n=5). *P<0.01 vs Sham, **P<0.01 vs angiotensin II (Ang II).

Figure 5.

Effect of mito2HOBA supplementation on mitochondrial superoxide (A), vascular superoxide (B), endothelial nitric oxide (C) and endothelial-dependent relaxation (D) in angiotensin II-infused mice. Mitochondrial and vascular O₂^• was measured by mitochondria-targeted superoxide probe mitoSOX (1 μM) or untargeted superoxide probe DHE (50 μM) using HPLC. Endothelial nitric oxide was analyzed by NO spin trap Fe(DETC)₂ and ESR. C57Bl/6J mice were infused with Ang II and mito2HOBA was provided in the drinking water (0.1 g/L). Supplemental figure S2 shows typical HPLC chromatograms. Supplemental figure S3 shows representative ESR spectra of nitric oxide measurements. Results are mean ± SEM. *P<0.01 vs Sham, **P<0.01 vs Ang II (n=6).

Figure 6.

Mito2HOBA reduces mPTP opening and prevents mitochondrial dysfunction. C57Bl/6J mice were infused with Ang II (0.7 mg/kg/ml) and mito2HOBA in the drinking water (0.1 g/L). Following 14 days of Ang II infusion the animals were sacrificed and kidneys were isolated for mitochondrial studies. Addition of CaCl₂ to mitochondria above Ca²⁺ retention capacity led to mPTP opening and mitochondria swelling. Mitochondria isolated from Ang II-infused mice had significant reduction in Ca²⁺ capacity due to increased mPTP opening and CypD inhibitor Cyclosporine A (CsA) rescued Ca²⁺ retention capacity (A). Respiratory control ratio (State 3/State 4) was measured in isolated kidney mitochondria with glutamate and malate (B). Control level is 100%. (C) Renal ATP was measured in freshly isolated tissue by luciferase-based luminescent assay. Results are mean ± SEM. *P<0.01 vs Sham, **P<0.01 vs Angiotensin II (n=5).