Deacetylation mimetic mutation of mitochondrial SOD2 attenuates ANG II-induced hypertension by protecting against oxidative stress and inflammation

Abstract

Almost one-half of adults have hypertension, and blood pressure is poorly controlled in a third of patients despite the use of multiple drugs, likely because of mechanisms that are not affected by current treatments. Hypertension is linked to oxidative stress; however, common antioxidants are ineffective. Hypertension is associated with inactivation of key intrinsic mitochondrial antioxidant, superoxide dismutase 2 (SOD2), due to hyperacetylation, but the role of specific SOD2 lysine residues has not been defined. Hypertension is associated with SOD2 acetylation at lysine 68, and we suggested that deacetylation mimetic mutation of K68 to arginine in SOD2 inhibits vascular oxidative stress and attenuates hypertension. To test this hypothesis, we have developed a new deacetylation mimetic SOD2-K68R mice. We performed in vivo studies in SOD2-K68R mice using angiotensin II (ANG II) model of vascular dysfunction and hypertension. ANG II infusion in wild-type mice induced vascular inflammation and oxidative stress and increased blood pressure to 160 mmHg. SOD2-K68R mutation completely prevented increase in mitochondrial superoxide, abrogated vascular oxidative stress, preserved endothelial nitric oxide production, protected vasorelaxation, and attenuated ANG II-induced hypertension. ANG II and cytokines contribute to vascular oxidative stress and hypertension. Treatment of wild-type aortas with ANG II and cytokines in organoid culture increased mitochondrial superoxide twofold, which was completely prevented in aortas isolated from SOD2-K68R mice. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and pathogenesis of hypertension. We conclude that strategies to reduce SOD2 acetylation may have therapeutic potential in the treatment of vascular dysfunction and hypertension.

NEW & NOTEWORTHY Essential hypertension is associated with hyperacetylation of key mitochondrial antioxidant SOD2; however, the pathophysiological role of SOD2 acetylation has not been defined. Our animal study of angiotensin II hypertension model shows that deacetylation mimetic SOD2-K68R mutation prevents pathogenic increase in vascular mitochondrial superoxide, abrogates vascular oxidative stress, preserves endothelial nitric oxide, protects endothelial-dependent vasorelaxation, and attenuates hypertension. These data support the important role of SOD2-K68 acetylation in vascular oxidative stress and the pathogenesis of hypertension.

INTRODUCTION

Hypertension is a major health problem in Western societies and a risk factor for stroke, myocardial infarction, and heart failure (1). Hypertension is a multifactorial disorder involving perturbations of the vasculature, the kidney, and the central nervous system (2). Despite treatment with multiple drugs, 37% of patients with hypertension remain hypertensive (3), likely because of mechanisms contributing to blood pressure elevation that is not affected by current treatments. Recent studies showed that mitochondria become dysfunctional in hypertension and overproduction of mitochondrial superoxide (O₂^•−) promotes hypertension (4, 5). Superoxide dismutase 2 (SOD2) is a key mitochondrial antioxidant enzyme, and SOD2 overexpression attenuates hypertension (4), while SOD2 depletion increases mitochondrial oxidative stress and augments hypertension (6, 7). Interestingly, analysis of human cells and vascular tissue did not show SOD2 depletion in essential hypertension; however, SOD2 activity was substantially reduced because of acetylation of lysine residues (8). Meanwhile, the role of specific SOD2 lysine acetylation is not defined.

Protein acetylation is the major mitochondrial posttranslational modification driven by enzymatic (9) and nonenzymatic (10) processes. SOD2 catalytic center has 10 lysine residues altering enzyme stability and substrate recognition, and SOD2 acetylation plays a key role in the regulation of SOD2 activity, particularly because of acetylation of highly conserved catalytic center SOD2 lysines 68 (K68) and 122 (K122) inhibiting enzymatic activity (11). Mass spectrometry study showed that SOD2-K68 was the most abundant SOD2 acetylation site detected both in sham and angiotensin II (ANG II) mice (6). Several studies have shown the key role of sirtuin 3 (Sirt3) in deacetylation of K68 and K122, which activates SOD2 likely because of electrostatic and steric hindrance effects (8, 12–14). Our previous studies in animals and human subjects with essential hypertension have shown SOD2 inactivation associated with SOD2-K68 acetylation, which was linked to reduced expression and activity of Sirt3 (6). We proposed that SOD2 inactivation in hypertension is linked to acetylation of lysine 68 (K68), and mutation of lysine 68 to arginine (K68R) mimics SOD2 deacetylation, inhibits vascular oxidative stress, and attenuates ANG II-induced hypertension.

Mitochondrial dysfunction contributes to the pathogenesis of hypertension and cardiovascular disease (15, 16); however, there are no approved drugs that directly target mitochondria (17). Mitochondrial dysfunction is associated with increased mitochondrial oxidative stress; however, common antioxidants, like ascorbate and vitamin E, are ineffective in preventing cardiovascular diseases and hypertension (18). These agents are unlikely to reach key sites of oxidant production such as the mitochondria. We suggest that inactivation of intrinsic antioxidant SOD2 by acetylation promotes vascular oxidative stress, while deacetylation mimetic SOD2-K68R protects from mitochondrial oxidative stress. In the current study, we used deacetylation mimetic SOD2-K68R mice to test if SOD2-K68R protects from ANG II-induced superoxide overproduction 1) preserves endothelial nitric oxide levels and vascular relaxation and 2) attenuates ANG II-induced hypertension. Understanding the molecular mechanisms of mitochondrial oxidative stress may provide a new target to improve the treatment of endothelial dysfunction and hypertension.

MATERIALS AND METHODS

The authors declare that all supporting data are available within the article. All methods have corresponding literature references. Additional protocol information is available from the corresponding author upon reasonable request.

Reagents

Mitochondria-targeted superoxide probe MitoSOX and untargeted cellular superoxide probe DHE were supplied by Invitrogen (Grand Island, NY). Acetyl-K68-SOD2 (ab137037), p65 (ab97726), Complex IV (ab22094), and GAPDH (ab8245) antibodies were from Abcam. SOD2 (sc-30080), VCAM (sc-13160), ICAM (sc-18908) were from Santa Cruz. D11, an isoLG-lysyl adducts-specific scFv antibody, has been previously characterized (19). Secondary antibodies conjugated with horseradish peroxidase were purchased from Amersham (anti-rabbit IgG NA934V or anti-mouse IgG NA931V). All other reagents were obtained from Sigma (St. Louis, MO).

Animal Experiments

Deacetylation mimetic SOD2-K68R mutant mice were developed in collaboration with Vanderbilt University Transgenic Mouse Core (TMESCSR) by CRISPR/Cas9 technology. Homozygous SOD2-K68R mice genotype was confirmed by PCR, restriction digest, and Sanger sequencing. The lack of K68 acetylation was confirmed by Western blot analysis using highly specific anti-acetyl-K68-SOD2 antibodies (ab137037, Abcam) validated in sirtuin 3 knockout (Sirt3^−/−) mice and site-directed mutagenesis (13). No difference in body weights, food, or water intake between wild-type and SOD2-K68R mice was noted. Hypertension was induced by ANG II infusion (0.7 mg/kg/day, 14 days) using osmotic pumps (Alzet, model 1002) as previously described (20). To test the potential protective effect of deacetylation mimic SOD2-K68R mutation, 4–5-mo-old wild-type C57Bl/6J (Jackson Laboratories) and SOD2-K68R male and female mice received saline or ANG II minipump placement. Blood pressure was monitored by the telemetry and tail-cuff measurements as previously described (21, 22). Mice were anesthetized with an intraperitoneal injection of ketamine-xylazine. After isolation of the left common carotid artery, the catheter connected to the transducer was introduced into the carotid and advanced until the tip was just inside the thoracic aorta. The DSI transmitter was positioned along the right flank, close to the hindlimb. After telemetry implantation, mice were allowed to recover for 10 days before the osmotic minipumps were placed. Following 14 days of ANG II infusion, the animals were euthanized by CO₂ inhalation, and aortas were extracted for the vascular studies. The Vanderbilt Institutional Animal Care and Use Committee approved the procedures (Protocol M1700207), and the mice were housed and cared for in accordance with the Guide for the Care and Use of Laboratory Animals, US Department of Health and Human Services. Simple randomization was used to select animals for sham or ANG II groups for equal chance of being allocated to treatment groups.

Superoxide Measurements Using HPLC

Mouse aortic segments were loaded with mitochondria-targeted MitoSOX (1 µM) or cellular untargeted superoxide probe DHE (50 µM) in Krebs-HEPES buffer for 30 min incubation in a tissue culture incubator at 37°C. The tissue was then placed in methanol (300 µL) and homogenized with a glass pestle. The homogenate was passed through a 0.22-μm filter and filtrates were analyzed by HPLC (23). The superoxide-specific product mito-2-hydroxyethidium was detected using a C-18 reverse-phase column (Nucleosil 250 to 4.5 mm) and a mobile phase containing 0.1% trifluoroacetic acid and an acetonitrile gradient (from 37% to 47%) at a flow rate of 0.5 mL/min. Mito-2-hydroxyethidium was quantified by fluorescence detector using an emission wavelength of 580 nm and an excitation of 480 nm as previously described (4). DHE-superoxide-specific product 2-hydroxyethidium was detected using a C-18 reverse-phase column (Nucleosil 250 to 4.5 mm) as previously described (4).

Electron Spin Resonance Analysis of Mitochondrial Superoxide

Five aortic sections (2 mm) were incubated with 25 µM mitoTEMPO-H for 40 min at 37°C in Krebs-HEPES buffer containing 10 µM DTPA and then placed in a 1-mL syringe and frozen in liquid nitrogen as previously described (24). Electron spin resonance (ESR) spectra were recorded in the quartz finger Dewar flask. The amplitude of the ESR signal was measured, and the amount of detected superoxide was determined by accumulation of stable nitroxide radical (25) using TEMPOL calibration curve.

Nitric Oxide Measurements by ESR

Endothelial nitric oxide was quantified by ESR and colloid spin trap Fe(DETC)₂ (26). All ESR samples were placed in quartz Dewar (Corning, New York, NY) filled with liquid nitrogen. ESR spectra were recorded using a EMX ESR spectrometer (Bruker Biospin, Billerica, MA) and a super high Q microwave cavity. The ESR settings were as follows: field sweep, 160 Gauss; microwave frequency, 9.42 GHz; microwave power, 10 mW; modulation amplitude, 3 Gauss; scan time, 150 ms; time constant, 5.2 s; and receiver gain, 60 dB (n = 4 scans).

Aortic Mitochondria Isolation and Analysis of SOD2 Activity

Mice were euthanized by CO₂ inhalation and aortas were collected with fat tissue removed and placed in ice-slurry cold-isolation medium. Aortas were minced, washed with the isolation medium, and homogenized using a Polytron disintegrator with six pulses of 2 s each. Homogenate was diluted sevenfold (wt/vol) and homogenized with 10 strokes by glass-Teflon homogenizer. Mitochondria were isolated by differential centrifugation with final centrifugation at 8,000 g (27). Isolation medium contained (in mM) 75 mannitol, 175 sucrose, 20 MOPS (pH 7.2), and 1 EGTA. Mitochondria were washed in isolation medium, resuspended in buffer containing 0.25 M sucrose and (in mM) 1 EDTA, 10 MOPS, 20 NaCl, 10 MgCl₂, and 5 KH₂PO₄ (pH 7.2), and homogenized by sonication for 5 s, three times with medium power (Fisher Cl18). Mitochondrial protein concentration was measured by Bradford method. SOD2 activity was measured by xanthine oxidase assay and electron spin resonance (ESR) using spin probe CAT1H (25). Mitochondrial homogenate (10–40 µg/mL) was mixed with xanthine oxidase (2 mU/mL), hypoxanthine (200 µM), and 0.5 mM CAT1H then placed in 1,000-µL capillary tube for ESR analysis. Xanthine oxidase superoxide production was followed by CAT1 accumulation in xanthine oxidase plus hypoxanthine samples measured after 1, 5, and 10 min using the following ESR setting: field sweep, 60 Gauss; microwave frequency, 9.85 GHz; microwave power, 20 milliwatts; modulation amplitude, 2 Gauss; scan time, 100 s; time constant, 655 s; and receiver gain, 60 dB. SOD2 activity was calculated by reduction in CAT1 levels compared with SOD2 calibration curve (0.2–1.6 U/mL). SOD2 activity was normalized by protein concentration.

Western Blot Analysis

Human arterioles and mouse aortas were homogenized in RIPA lysis buffer (Sigma, R0278) with 2.0 mmol/L sodium orthovanadate (Na₃VO₄), 1.0 mM fluoride phenylmethylsulfonyl (PMSF) containing inhibitors of proteolytic enzymes: 10 μg/mL aprotinin, 10 μg/mL leupeptin, 10 μg/mL pepstatin (Sigma-Aldrich). The concentration of protein in lysates was determined using the DC Assay Protein kit (Bio-Rad). Protein (30 μg) separation was carried out in polyacrylamide gels (4–12%) and transferred to PVDF membrane (Bio-Rad) at 4°C. Nonspecific binding sites on the membrane were blocked with 5% skim milk or 3% BSA in Tris-buffered saline solution with Tween (TBS-T) for 1 h at room temperature and then membranes were incubated with anti-acetyl-K68-SOD2 antibodies (Abcam, ab137037, 1:1,000), anti-SOD2 (Santa Cruz, sc30080, 1:1,000), anti-VCAM (Santa Cruz, sc-13160, 1:1,000), ICAM (Santa Cruz, sc-18908, 1:1,000), anti-p65 (Abcam, ab97726, 1:1,000), anti-GAPDH (Abcam, ab8245, 1:1,000), and complex IV (ab22094), overnight at 4°C. The next day, membranes were incubated with secondary antibodies conjugated with horseradish peroxidase (HRP). Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific) was used for chemiluminescence based. Final scans were performed using X-ray films or Azure c500 Western Blot Imaging System (Azure Biosystem), Odyssey FC Imaging System (LI-COR). Densitometric analyses were performed using ImageJ software or ImageStudio software (LI-COR). Data were normalized by GAPDH levels.

Vasodilatation Study

Isometric tension studies were performed on 2-mm mouse mesenteric arteries and aortic rings dissected free of perivascular fat. Studies were performed in a horizontal wire myograph (DMT, Aarhus, Denmark, Models 610 M and 620 M) containing physiological salt solution with the composition of (in mM) 118 NaCl, 4.7 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 25 NaHCO₃, 11 glucose, and 1.8 CaCl₂. The isometric tone of each vessel was recorded using LabChart Pro v7.3.7 (AD Instruments, Australia). The aortic rings and mesenteric arteries were equilibrated over 2 h by heating and stretching the vessels to an optimal baseline tension before contracting them with 60 mM KCl physiological saline solution. Endothelial-dependent and independent vascular relaxation were tested after preconstriction with 1 µM phenylephrine. Once the vessels reached a steady state contraction, increasing concentrations of acetylcholine or sodium nitroprusside were administered, and the response to each concentration of drug was recorded.

Statistics

The normality of continuous variable distribution was examined with the Shapiro–Wilk test. Comparisons of the normally distributed continuous variables were assessed by the two-way analysis of variance (ANOVA) followed by a Tukey post hoc test. For telemetry blood pressure measurements over time and aortic relaxation, two-way ANOVA with repeated measures was employed. All statistical analyses were done using GraphPad Prizm 10. P values < 0.05 were considered significant.

RESULTS

Deacetylation Mimetic SOD2-K68R Attenuates SOD2 Acetylation and Prevents ANG II-Induced Superoxide Overproduction

We have previously reported SOD2-K68 acetylation in vascular tissue from patients with essential hypertension (28), and ANG II mouse model of hypertension associated with SOD2-K68 hyperacetylation (6). To test the pathophysiological role of SOD2-K68 acetylation, we have developed deacetylation mimetic SOD2-K68R mice using CRISPR/Cas9 technology. We suggested that the replacement of lysine 68 to arginine should prevent the ANG II-induced SOD2-K68 hyperacetylation. Indeed, Western blot analysis of aortic lysates showed a robust SOD2 acetylation in wild-type mice infused with ANG II, which was prevented in deacetylation mimetic SOD2-K68R mice (Fig. 1, A and B). It has been previously reported that SOD2 acetylation at K68 reduces SOD2 activity because the entry of the superoxide anion into the coordinated core of SOD2 was inhibited (Fig. 1B) (29). We hypothesized that SOD2-K68R mice would retain SOD2 activity because of the lack of K68R acetylation. To test this hypothesis, we measured SOD2 activity in aortic mitochondria isolated from sham and ANG II-infused wild-type and SOD2-K68R mice. As expected, SOD2 activity in ANG II-infused wild-type mice was significantly reduced to 45% compared with sham wild-type mice. Interestingly, the SOD2 activity in sham SOD2-K68R mice trended to be slightly higher than in wild-type and it did not reduce after ANG II infusion (Fig. 1, C and D). These data support critical role of SOD2-K68 acetylation in regulation of SOD2 activity.

Figure 1.

Analysis of SOD2 acetylation and SOD2 activity in sham and ANG II-infused (ANG II) wild-type and SOD2-K68R mice and schematic presentation of SOD2 inactivation by K68 acetylation. A: typical Western blots of SOD2 acetylation (SOD2-K68-Ac) measured by anti-SOD2-K68-acetyl antibodies. Total SOD2 levels were not different between wild-type and SOD2-K68R mice and were not significantly changed by ANG II infusion. B: densitometry of SOD2 acetylation in aortic samples. Data were normalized by GAPDH. SOD2-K68R mice are protected from ANG II-induced SOD2-K68 acetylation. *P = 1.0 × 10⁻¹⁰ vs. WT, **P = 2.0 × 10⁻¹⁰ vs. WT + ANG II (n = 6). C: schematic presentation of SOD2 inactivation by acetylation of highly conserved catalytic center SOD2-K68 inhibiting enzymatic activity in ANG II-infused wild-type mice (6). D: ESR analysis of SOD2 activity in aortic mitochondria. The SOD2 inactivation is prevented in acetylation-resistant SOD2-K68R mutant. *P = 9.2 × 10⁻⁵ vs. WT, **P = 3.3 × 10⁻⁶ vs. WT + ANG II (n = 6). Data were analyzed using two-way ANOVA and Tukey post hoc multiple comparisons. Results are means ± SD. ANG II, angiotensin II; ANOVA, analysis of variance; SOD2, superoxide dismutase 2.

We have suggested that preserved SOD2 activity in SOD2-K68R mice protects from ANG II-induced superoxide overproduction. To test this hypothesis, we analyzed mitochondrial and cytoplasmic superoxide in sham and ANG II-infused wild-type and SOD2-K68R mice. Following 2 wk of ANG II treatment (0.7 mg/kg/day) mice were euthanized, and aortas were isolated for superoxide analysis. Mitochondrial superoxide was measured by HPLC analysis of the superoxide-specific product, mito-2-hydroxyethidium (30), in tissue incubated with mitochondria-targeted superoxide probe MitoSOX (1 µM) (28). It is important to note that 1 μM MitoSOX, rather than the commonly used 2–5 μM, is the optimal concentration of MitoSOX for detecting mitochondrial superoxide (31) due to potential mitochondria overload by high concentrations of MitoSOX, leak of MitoSOX into cytoplasm, and contamination with cytoplasmic superoxide at 5 μM MitoSOX (32). MitoSOX/HPLC analysis of mitochondrial superoxide in intact aortas showed a robust increase in wild-type ANG II-infused mice (Supplemental Fig. S1; all Supplemental material is available at https://dx.doi.org/10.6084/m9.figshare.26053141 and Fig. 2A). As expected, deacetylation mimetic SOD2-K68R mutation abolished the ANG II-induced overproduction of mitochondrial superoxide in SOD2-K68R mice (Fig. 2A), supporting an important role of K68 site in the regulation of SOD2 activity in vivo.

Figure 2.

HPLC analysis of mitochondrial superoxide (A) and cellular superoxide (B) in aortas isolated from sham and ANG II-infused wild-type and SOD2-K68R mice. Mice were infused with vehicle (saline) or ANG II (0.7 mg/kg/day) for 14 days before superoxide analysis. A: mitochondrial superoxide (O₂^•−) was measured by MitoSOX and HPLC following accumulation of superoxide-specific product of MitoSOX, mito-2-hydroxyethidium (4). Inset: typical HPLC diagram of MitoSOX samples with superoxide-specific product mito-2-hydroxyethidium (2-OH-Mito-E⁺) and nonspecific oxidation product mito-ethidium (Mito-E⁺). *P = 1.0 × 10⁻⁹ vs. WT, **P = 4.3 × 10⁻⁹ vs. WT + ANG II. B: cellular O₂^•− was measured by DHE and HPLC following accumulation of superoxide-specific product of DHE, 2-hydroxyethidium (4). Inset: typical HPLC diagram of DHE samples with superoxide-specific product 2-hydroxyethidium (2-OH-E⁺) and nonspecific oxidation product ethidium (E⁺). *P = 4.5 × 10⁻⁷ vs. WT, **P = 3.2 × 10⁻⁷ vs. WT + ANG II. Data were analyzed using two-way ANOVA and Tukey post hoc multiple comparisons. Results are means ± SD (n = 7). ANG II, angiotensin II; ANOVA, analysis of variance; SOD2, superoxide dismutase 2.

Cellular superoxide was measured by untargeted superoxide probe DHE (50 μM) and HPLC detection of superoxide-specific product, 2-hydroxyethidium (30). DHE probe is insensitive to antimycin A-induced mitochondrial superoxide, and it reflects cytoplasmic superoxide production by NADPH oxidases (5). DHE/HPLC analysis of cellular superoxide exhibited a twofold increase in aortas from ANG II-infused wild-type mice (Fig. 2B and Supplemental Fig. S2) in line with previously reported data (28). Interestingly, deacetylation mimetic SOD2-K68R mutation abolished the ANG II-induced superoxide overproduction of cellular superoxide (Fig. 2B), suggesting that blocking the ANG II-induced mitochondrial superoxide (Fig. 2A) downregulates superoxide production in the cytoplasm as well. This can be due to ROS-induced ROS release (33) and the cross talk between mitochondria and NADPH oxidases (34); therefore, blocking the mitochondrial superoxide inhibits the overproduction of cytoplasmic superoxide by NADPH oxidases (35).

Deacetylation Mimetic SOD2-K68R Attenuates Hypertension and Protects Endothelial Function

We have previously reported that SOD2 overexpression attenuates hypertension (4). Meanwhile, essential hypertension did not alter the SOD2 expression in human subjects, while both human hypertension and animal models of hypertension are associated with SOD2 hyperacetylation (6, 28). We have hypothesized that deacetylation mimetic SOD2-K68R mutation attenuates hypertension and protects endothelial function due to inhibition of oxidative stress. To test this hypothesis, we used sham or ANG II-infused wild-type and SOD2-K68R mice. Analysis of systolic blood pressure by telemetry at the end of the 14 days of ANG II infusion (0.7 mg/kg/day) in wild-type mice showed increased blood pressure from 110 (sham) to 158 (ANG II) mmHg. Meanwhile, ANG II-induced hypertension in SOD2-K68R mice was significantly attenuated, and systolic blood pressure was changed from 105 to 135 mmHg (Fig. 3A).

Figure 3.

Systolic blood pressure (A), endothelial nitric oxide (B), endothelial-dependent relaxation (C), and accumulation of mitochondrial lipid oxidation product isolevuglandins (C) in wild-type and SOD2-K68R mice. A: systolic blood pressure after 14 days of ANG II infusion was measured by telemetry. *P = 4.3 × 10⁻¹⁰ vs. WT, **P = 1.4 × 10⁻⁵ vs. WT + ANG II (n = 7). B: endothelial NO was analyzed by NO spin trap FeDETC₂ and EPR (inset) (26). *P = 9.5 × 10⁻¹² vs. WT, **P = 6.9 × 10⁻¹⁰ vs. WT + ANG II (n = 7). C: endothelial-dependent relaxation was measured by dose response to acetylcholine (ACh). Aortic relaxation data were analyzed using two-way ANOVA with repeated measurements. Data were analyzed using two-way ANOVA and Tukey post hoc multiple comparisons. *P = 2.1 × 10⁻⁹ vs. WT, **P = 5.9 × 10⁻⁸ vs. WT + ANG II (n = 8). D: typical Western blot of protein-isolevuglandins adducts in mitochondria isolated from aortas from wild-type and SOD2-K68R mice using D11 antibodies normalized by complex IV (36). Results are means ± SD (n = 6). ANG II, angiotensin II; ANOVA, analysis of variance; SOD2, superoxide dismutase 2.

Endothelial nitric oxide production plays an important role in blood pressure regulation; however, vascular oxidative stress reduces nitric oxide levels. SOD2-K68R mice are protected from oxidative stress (Fig. 2); therefore, we tested if SOD2-K68R mutation preserves nitric oxide production. Endothelial nitric oxide was measured by spin trap Fe(DETC)₂ and electron spin resonance (ESR) as we have previously described (26). ESR analysis showed strong decrease in endothelial nitric oxide by 56% in aortas from ANG II-infused wild-type mice (Fig. 3B and Supplemental Fig. S3). Meanwhile, ANG II infusion in SOD2-K68R mice reduced NO only by 18%, indicating that deacetylation mimetic SOD2-K68R mutation protects endothelial nitric oxide production (Fig. 3B).

Inactivation of vascular nitric oxide impairs vascular relaxation in hypertension, and we tested if deacetylation mimetic SOD2-K68R mutation preserves endothelial-dependent relaxation because of lack of oxidative stress and protection of nitric oxide. Indeed, analysis of acetylcholine-induced endothelium-dependent relaxation in aortas and mesenteric-resistant arteries was substantially impaired in ANG II-infused wild-type mice (Fig. 3C and Supplemental Fig. S4); however, it was completely preserved in ANG II-infused SOD2-K68R mice (Fig. 3C and Supplemental Fig. S4). These data demonstrate that deacetylation mimetic SOD2-K68R protects endothelial function.

Recently, our group and others have shown that highly reactive and cytotoxic lipid peroxidation products, isolevuglandins, promote hypertension (36, 37). We tested if deacetylation mimetic SOD2-K68R mice are protected from isolevuglandins formation because of lack of oxidative stress. To test this hypothesis, we performed Western blot analysis of isolevuglandin-protein adducts using D11 antibodies. Isolevuglandins vividly react with protein lysine residues yielding very stable cytotoxic adducts (38). Western blot analysis showed substantial accumulation of isolevuglandin-protein adducts in aortas from ANG II-infused wild type (Fig. 3D and Supplemental Fig. S3). Interestingly, SOD2-K68R mice were protected from ANG II-induced isolevuglandins accumulation (Fig. 3D). These data support the pathophysiological role of mitochondrial superoxide and SOD2 acetylation in the formation of cytotoxic isolevuglandins.

SOD2-K68R Mutant Mice Are Protected from Cytokine and ANG II-Induced Inflammation

We have previously reported that ANG II and inflammatory cytokines cooperatively induce vascular oxidative stress, vascular inflammation, and hypertension (39, 40). We tested if SOD2-K68R deacetylation mutation attenuates vascular inflammation and inhibits cytokine-induced vascular oxidative stress. To test this hypothesis, we performed Western blot studies of inflammation markers p65 (NF-κB subunit), VCAM, and ICAM in aortas isolated from sham and ANG II-infused mice. It was found that ANG II-induced hypertension significantly increased vascular expression of inflammation markers in ANG II-infused wild-type mice (Fig. 4A), which is in line with the Western blot studies of arterioles from human subjects with essential hypertension (28). Interestingly, deacetylation mimetic SOD2-K68R mutation abolished the ANG II-induced increase in vascular inflammation markers (Fig. 4A and Supplemental Fig. S5). This can be associated with reduced vascular oxidative stress and diminished production of isolevuglandins in ANG II-infused SOD2-K68R mice compared with wild-type mice (Figs. 2 and 3). We have tested directly if deacetylation mimetic SOD2-K68R mutation attenuates superoxide overproduction (MitoSOX/HPLC) in response to proinflammatory cytokines (TNFα and IL17A) and ANG II using organoid culture model as we previously described (39). It was found that ex vivo treatment of aortas from wild-type mice with a mixture of ANG II, IL17A, and TNFα increased mitochondrial superoxide by 2.5-fold, while deacetylation mimetic SOD2-K68R mutation completely inhibited the cytokine-induced mitochondrial superoxide overproduction (Fig. 4B and Supplemental Fig. S6). These data support the pathophysiological role of SOD2-K68 acetylation in vascular inflammation.

Figure 4.

Western blot of vascular inflammation markers (A) and cytokine-induced mitochondrial superoxide (B) in aortas from wild-type and SOD2-K68R mice. A: typical Western blot of p65, component of NF-κB transcription factor, VCAM, vascular cell adhesion molecule-1, and ICAM, intercellular adhesion molecule-1. Aortas were isolated from sham and ANG II-infused (0.7 mg/kg/day, 14 days) mice. Data were normalized by GAPDH. Presented aortic samples are the same as in Fig. 1A. B: analysis of mitochondrial O₂^•− by superoxide-specific product of MitoSOX, mito-2-hydroxyethidium (4). Aortas were incubated ex vivo in DMEM for 24 h before analysis with vehicle or combination of ANG II (200 nM), IL17A (100 ng/mL) plus TNFα (10 ng/mL) (39). Data were analyzed using two-way ANOVA and Tukey post hoc multiple comparisons. *P = 6.4 × 10⁻⁸ vs. WT, **P = 3.9 × 10⁻⁸ vs. WT + ANG II + IL17A + TNFα. Results are means ± SD (n = 7). ANG II, angiotensin II; ANOVA, analysis of variance; SOD2, superoxide dismutase 2.

Resveratrol Reduces SOD2 Acetylation, Inhibits Mitochondrial O₂^•−, and Rescues Endothelial NO

Human hypertension and mouse models of hypertension showed vascular deficiency in mitochondrial deacetylase Sirt3, which leads to SOD2 hyperacetylation. We have tested if ex vivo treatment of aortas isolated from hypertensive mice with sirtuin activator resveratrol reduces SOD2 acetylation, inhibits mitochondrial O₂^•−, and rescues endothelial NO. To test this hypothesis, we used organoid culture of aortas isolated from sham and ANG II-infused wild-type mice supplemented with resveratrol (10 µM), Sirt3-inactive analog dihydroresveratrol (10 µM) (41), or saline (vehicle) for 24 h in DMEM. Western blot analysis showed that resveratrol decreased SOD2 acetylation in aortas isolated from hypertensive mice, while dihydroresveratrol was not effective (Fig. 5A and Supplemental Fig. S7). ESR analysis of mitochondrial superoxide using spin probe mitoTEMPO-H (25) showed reduction of mitochondrial O₂^•− by resveratrol, while Sirt3-inactive analog with a similar chemical structure dihydroresveratrol did not affect the superoxide levels (Fig. 5B and Supplemental Fig. S8). ESR analysis of endothelial nitric oxide showed twofold decline in aortas isolated from hypertensive mice, which was restored to a normal level by resveratrol, while dihydroresveratrol was not effective (Fig. 5C and Supplemental Fig. S9). These data suggest that strategies to reduce SOD2 acetylation may have therapeutic potential.

Figure 5.

Inhibition of SOD2 acetylation (A), mitochondrial O₂^•− (B), and rescue of endothelial nitric oxide production (C) in aortas isolated from hypertensive mice and treated ex vivo with resveratrol (Resv, 10 µM) or Sirt3-inactive analog dihydroresveratrol (DHResv, 10 µM) for 24 h in DMEM tissue culture. Mice were infused with saline (sham) or ANG II (0.7 mg/kg/day) for 14 days before isolation of aortas. A: representative Western blots of K68-acetyl-SOD2 and total SOD2 levels (n = 4). B: ESR analysis of mitochondrial O₂^•− by mitochondria-targeted spin probe mitoTEMPO-H (25). Inset: typical EPR spectra of aortic tissue incubated with mitoTEMPO-H. *P = 1.0 × 10⁻⁷ vs. sham, **P = 2.1 × 10⁻⁹ vs. ANG II. #P = 1.8 × 10⁻⁷ vs. ANG II + Resv. C: EPR analysis of endothelial nitric oxide using specific NO spin trap Fe(DETC)₂. Inset: typical EPR spectra of NO-Fe(DETC)₂ (26). *P = 3.0 × 10⁻⁷ vs. sham, **P = 1.1 × 10⁻⁵ vs. ANG II. #P = 5.2 × 10⁻⁵ vs. ANG II + Resv. Data were analyzed using two-way ANOVA and Tukey post hoc multiple comparisons. Results are means ± SD (n = 4–6). ANG II, angiotensin II; ANOVA, analysis of variance; SOD2, superoxide dismutase 2.

DISCUSSION

We have previously reported that patients with essential hypertension have hyperacetylation of key antioxidant, mitochondrial superoxide dismutase (SOD2) (6, 28). There are multiple SOD2 lysine residues that can be acetylated leading to inhibition of SOD2 activity, particularly, catalytic center SOD2 lysines 68 (K68) and 122 (K122) (11). SOD2-K68 was remarkably acetylated in the animal models of hypertension (6). In the current study, we tested the hypothesis that deacetylation mimetic SOD2-K68R mice are protected from vascular oxidative stress and hypertension. This study provides the first evidence that deacetylation mimetic SOD2-K68R mutation prevents ANG II-induced superoxide increase in the vascular mitochondria (Fig. 2A). It was found that deacetylation mimetic SOD2-K68R mutation abolishes the ANG II-mediated stimulation of mitochondrial superoxide in SOD2-K68R mice because of the lack of K68 acetylation (Figs. 1 and 2). Interestingly, deacetylation mimetic SOD2-K68R mice are also protected from ANG II-induced overproduction of cellular superoxide, suggesting that blocking of the ANG II-induced mitochondrial superoxide downregulates superoxide production in the cytoplasm (Fig. 2, A and B). Indeed, we have previously reported that SOD2 overexpression or treatment with mitochondria-targeted SOD2 mimetic, mitoTEMPO, reduces not only mitochondrial superoxide but also downregulates NADPH oxidase activity leading to diminished superoxide production in the cellular cytoplasm (4). It is important to note that decreased superoxide production in the cytoplasm is not a result of “off-target” effects of SOD2 or mitoTEMPO, but consequence of physiological cross talk between mitochondrial superoxide and cytoplasmic NADPH oxidases (34). Indeed, SOD2 overexpression and mitoTEMPO did not affect the basal cytoplasmic superoxide levels but only reduced the feed-forward stimulation of NADPH oxidases by mitochondrial oxidative stress (4, 35). Meanwhile, SOD2 expression is not significantly affected in essential hypertension and mouse models of hypertension but SOD2-K68 acetylation was linked to reduced SOD2 activity (6). In this work, we found that deacetylation mimetic SOD2-K68R mice are protected from ANG II-induced endothelial dysfunction and hypertension (Fig. 3), supporting the specific pathophysiological role of SOD2-K68 acetylation in these pathological conditions.

Hypertension is a multifactorial disorder involving perturbations of the vasculature, the kidney, and the central nervous system; however, oxidative stress may contribute to all of these factors (42). Overproduction of superoxide in the endothelium, smooth muscle, inflammatory, and neural cells promotes vasoconstriction, vascular remodeling, inflammation, and increased sympathetic outflow leading to hypertension (43). One of the limitations of this study is that we used global SOD2-K68R mice with mutation affecting superoxide production in multiple cells and tissues. In this work, we found that endothelial function is protected in SOD2-K68R mice; however, this may result from both direct and indirect vascular effects. Western blot studies showed reduced expression of inflammation markers in aortas from ANG II-infused SOD2-K68R mice compared with wild-type mice (Fig. 4A). This can be due to reduced cytokine production or increased vascular cytokine resistance. We have tested directly if deacetylation mimetic SOD2-K68R mutation attenuates cytokine-induced superoxide overproduction in ex vivo organoid culture studies. It was found that deacetylation mimetic SOD2-K68R mutation completely inhibited the cytokine-induced vascular mitochondrial superoxide overproduction (Fig. 4B). These data support the pathophysiological role of SOD2-K68 acetylation in vascular inflammation; however, further studies are required to define the cell-specific effects of SOD2-K68 acetylation.

The prevalence of hypertension in early adulthood among women is three times lower than in men (44), and animals studies showed attenuated ANG II-induced hypertension in female mice compared with male littermates, which was associated with male and female hormones (45). In our studies, we did not notice significant differences between male and female SOD2-K68R mice (Supplemental Fig. S10). Meanwhile, deacetylation mimetic SOD2-K68R mutation in male mice reduced ANG II-induced hypertension by 24 mmHg, whereas in SOD2-K68R female mice, hypertension was decreased only by 10 mmHg. The sex difference in K68R-mediated antihypertensive effects can be attributed to several factors including 1) the role of sex in ANG type 2 receptor/type 1 receptor balance (46) and 2) increased SOD2 activity in females compared with males (47). The increased antihypertensive response to deacetylation mimetic SOD2-K68R mutation in male mice can be associated with diminished ANG II type 1 receptor-mediated activation of redox-dependent NADPH oxidases (5) shifting the balance to protective ANG II type 2 receptor signaling. On the other hand, females have been reported to have increased antioxidant activity (47, 48); therefore, the potential benefits of increased SOD2 activity can be smaller in females compared with males. Meanwhile, sex-specific aspects of hypertension are poorly understood, and women or female-specific risk factors are understudied in basic, clinical, and population research. Therefore, further studies are required to define the sex-specific role of SOD2-K68 acetylation.

Acetylation is a major posttranslational modification of mitochondrial proteins modulating antioxidant, oxidative phosphorylation, fatty acid β-oxidation, and mitochondrial dynamics pathways (49). We have reported hyperacetylation of SOD2, CypD, and many other mitochondrial proteins in hypertension (6). Enzymatic and nonenzymatic acetylation of highly reactive lysine residues in the catalytic center normally affects the enzymatic activity of mitochondrial proteins; however, the pathophysiological role of acetylation of specific proteins is not clear. Several in vitro cellular studies identified specific acetylation sites in mitochondrial cyclophilin D (50), very-long chain acyl-CoA dehydrogenase (51), long-chain acyl-CoA dehydrogenase (52), medium-chain acyl-CoA dehydrogenase (52), dynamin-related protein 1, succinate dehydrogenase A (53), and SOD2 (29); however, in vivo studies are lacking, and functional role of acetylation of specific enzymes in pathological conditions is not defined. Our study provides the first in vivo evidence for antioxidant protective effect of deacetylation mimetic SOD2-K68R in hypertension. Future in vivo studies are warranted to define the pathophysiological role of acetylation of other mitochondrial proteins, which can identify new pathophysiological pathways and identify novel therapeutic targets.

Hypertension is linked to diminished production of endothelial nitric oxide and inactivation of vascular nitric oxide by free radicals such as superoxide. Previous studies showed impairment of endothelial NO synthase activity because of uncoupling, phosphorylation, and inhibition by dimethylarginine (54–56), and these pathways are also associated with oxidative stress. Our data showed that deacetylation mimetic SOD2-K68R mutation preserves endothelial nitric oxide levels (Figs. 3 and 5). Additional studies are needed to define specific effects of SOD2 acetylation on endothelial NO synthase function and nitric oxide inactivation.

We have previously reported that hypertension is linked to Sirt3 deficiency, which leads to SOD2 acetylation, and recombinant Sirt3 rescues activity of SOD2 isolated from aortas of hypertensive mice (6). In this work, we showed that ex vivo treatment of aortas from hypertensive mice with pan-specific sirtuin activator resveratrol improves SOD2 deacetylation, reduces mitochondrial superoxide, and rescues endothelial nitric oxide production (Fig. 5). These data support the therapeutic potential of targeting SOD2 acetylation; however, there are several limitations of this experimental approach. First, resveratrol has a very broad pharmacological activity (57) not limited to sirtuins, despite the fact that resveratrol activates Sirt3 (58). Second, resveratrol-mediated Sirt3 activation leads to deacetylation/activation of multiple mitochondrial proteins and not specific to SOD2 (59). Third, potential adverse effects of resveratrol must be considered (60). Additional studies are warranted to define optimal strategies for targeting SOD2 acetylation. Finally, future studies must test the potential detrimental effects of SOD2 deacetylation. SOD2 acetylation represents physiological modulation of SOD2 activity and there may be situations where high SOD2 activity is detrimental. For example, blocking mitochondrial superoxide attenuates EGF redox signaling in endothelial cells (5), which can impair angiogenic responses.

Conclusions

Our work demonstrates that SOD2-K68 deacetylation plays an important role in antioxidant protection from mitochondrial superoxide overproduction. Furthermore, SOD2-K68 deacetylation attenuates cytoplasmic superoxide production due to mitochondria-NADPH oxidase cross talk; therefore, SOD2-K68 deacetylation provides global antioxidant protection normalizing superoxide levels and inhibiting the formation of harmful reactive lipid oxidation products, isolevuglandins. Human hypertension is associated with SOD2-K68 acetylation (6, 28), and this work demonstrates that deacetylation mimetic SOD2-K68R mutation protects from ANG II-induced endothelial dysfunction and hypertension. Furthermore, deacetylation mimetic SOD2-K68R inhibits vascular inflammation and attenuates cytokine-induced vascular oxidative stress. Multiple risk factors such as a sedentary lifestyle, smoking, aging, metabolic conditions, and inflammation can promote SOD2 acetylation (40, 61–63). Mitochondrial oxidative stress has been implicated in cardiovascular disease, inflammation, and neurodegeneration; therefore, strategies directed to reduce SOD2 acetylation may have therapeutic potential in these conditions.

DATA AVAILABILITY

Data will be made available upon reasonable request.

SUPPLEMENTAL MATERIAL

Supplemental Figs. S1–S10 can be accessed at https://dx.doi.org/10.6084/m9.figshare.26053141.

GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants R01HL144943 and R01HL157583. Dr. Dikalova was supported by American Heart Association Transformational Project Awards 23TPA1077648 and 19TPA34910157.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.D. and S.D. conceived and designed research; A.D., M.A., L.T., and S.D. performed experiments; A.D., M.A., L.T., and S.D. analyzed data; A.D., M.A., and S.D. interpreted results of experiments; A.D., M.A., and S.D. prepared figures; S.D. drafted manuscript; A.D. and S.D. edited and revised manuscript; A.D., M.A., L.T., and S.D. approved final version of manuscript.