Nrf2-Keap1 in Cardiovascular Disease: Which Is the Cart and Which the Horse?

High levels of oxidant stress in the form of reactive oxidant species are prevalent in the circulation and tissues in various types of cardiovascular disease including heart failure, hypertension, peripheral arterial disease, and stroke. Here we review the role of nuclear factor erythroid 2-related factor 2 (Nrf2), an important and widespread antioxidant and anti-inflammatory transcription factor that may contribute to the pathogenesis and maintenance of cardiovascular diseases. We review studies showing that downregulation of Nrf2 exacerbates heart failure, hypertension, and autonomic function. Finally, we discuss the potential for using Nrf2 modulation as a therapeutic strategy for cardiovascular diseases and autonomic dysfunction.

Extracellular Vesicles Regulate Sympatho-Excitation by Nrf2 in Heart Failure.

BACKGROUND: Chronic heart failure (CHF) is associated with redox imbalance. Downregulation of Nrf2 (nuclear factor [erythroid-derived 2]-like 2) plays important roles in disrupting myocardial redox homeostasis and mediating sympathetic nerve activity in the setting of CHF. However, it is unclear if circulating extracellular vesicles (EVs) elicit sympathetic excitation in CHF by disrupting central redox homeostasis. We tested the hypothesis that cardiac-derived EVs circulate to the presympathetic rostral ventrolateral medulla and contribute to oxidative stress and sympathetic excitation via EV-enriched microRNA-mediated Nrf2 downregulation. METHODS: Data were collected on rats with CHF post-myocardial infarction (MI) and on human subjects with ischemic CHF. EVs were isolated from tissue and plasma, and we determined the miRNAs cargo that related to targeting Nrf2 translation. We tracked the distribution of cardiac-derived EVs using in vitro labeled circulating EVs and cardiac-specific membrane GFP+ transgenic mice. Finally, we tested the impact of exogenously loading of antagomirs to specific Nrf2-related miRNAs on CHF-EV-induced pathophysiological phenotypes in normal rats (eg, sympathetic and cardiac function). RESULTS: Nrf2 downregulation in CHF rats was associated with an upregulation of Nrf2-targeting miRNAs, which were abundant in cardiac-derived and circulating EVs from rats and humans. EVs isolated from the brain of CHF rats were also enriched with Nrf2-targeting miRNAs and cardiac-specific miRNAs. Cardiac-derived EVs were taken up by neurons in the rostral ventrolateral medulla. The administration of cardiac-derived and circulating EVs from CHF rats into the rostral ventrolateral medulla of normal rats evoked an increase in renal sympathetic nerve activity and plasma norepinephrine compared with Sham-operated rats, which were attenuated by exogenously preloading CHF-EVs with antagomirs to Nrf2-targeting miRNAs. CONCLUSIONS: Cardiac microRNA-enriched EVs from animals with CHF can mediate crosstalk between the heart and the brain in the regulation of sympathetic outflow by targeting the Nrf2/antioxidant signaling pathway. This new endocrine signaling pathway regulating sympathetic outflow in CHF may be exploited for novel therapeutics.

Functional, proteomic and bioinformatic analyses of Nrf2- and Keap1- null skeletal muscle.

KEY POINTS: Nrf2 is a master regulator of endogenous cellular defences, governing the expression of more than 200 cytoprotective proteins, including a panel of antioxidant enzymes. Nrf2 plays an important role in redox haemostasis of skeletal muscle in response to the increased generation of reactive oxygen species during contraction. Employing skeletal muscle-specific transgenic mouse models with unbiased-omic approaches, we uncovered new target proteins, downstream pathways and molecular networks of Nrf2 in skeletal muscle following Nrf2 or Keap1 deletion. Based on the findings, we proposed a two-way model to understand Nrf2 function: a tonic effect through a Keap1-independent mechanism under basal conditions and an induced effect through a Keap1-dependent mechanism in response to oxidative and other stresses. ABSTRACT: Although Nrf2 has been recognized as a master regulator of cytoprotection, its functional significance remains to be completely defined. We hypothesized that proteomic/bioinformatic analyses from Nrf2-deficient or overexpressed skeletal muscle tissues will provide a broader spectrum of Nrf2 targets and downstream pathways than are currently known. To this end, we created two transgenic mouse models; the iMS-Nrf2flox/flox and iMS-Keap1flox/flox , employing which we demonstrated that selective deletion of skeletal muscle Nrf2 or Keap1 separately impaired or improved skeletal muscle function. Mass spectrometry revealed that Nrf2-KO changed expression of 114 proteins while Keap1-KO changed expression of 117 proteins with 10 proteins in common between the groups. Gene ontology analysis suggested that Nrf2 KO-changed proteins are involved in metabolism of oxidoreduction coenzymes, purine ribonucleoside triphosphate, ATP and propanoate, which are considered as the basal function of Nrf2, while Keap1 KO-changed proteins are involved in cellular detoxification, NADP metabolism, glutathione metabolism and the electron transport chain, which belong to the induced effect of Nrf2. Canonical pathway analysis suggested that Keap1-KO activated four pathways, whereas Nrf2-KO did not. Ingenuity pathway analysis further revealed that Nrf2-KO and Keap1-KO impacted different signal proteins and functions. Finally, we validated the proteomic and bioinformatics data by analysing glutathione metabolism and mitochondrial function. In conclusion, we found that Nrf2-targeted proteins are assigned to two groups: one mediates the tonic effects evoked by a low level of Nrf2 at basal condition; the other is responsible for the inducible effects evoked by a surge of Nrf2 that is dependent on a Keap1 mechanism.

Quantitative Proteomics Identifies Novel Nrf2-Mediated Adaptative Signaling Pathways in Skeletal Muscle Following Exercise Training.

Exercise training (ExT) improves skeletal muscle health via multiple adaptative pathways. Nrf2 is a principal antioxidant transcription factor responsible for maintaining intracellular redox homeostasis. In this study, we hypothesized that Nrf2 is essential for adaptative responses to ExT and thus beneficial for muscle. Experiments were carried out on male wild type (WT) and iMS-Nrf2flox/flox inducible muscle-specific Nrf2 (KO) mice, which were randomly assigned to serve as sedentary controls (Sed) or underwent 3 weeks of treadmill ExT thus generating four groups: WT-Sed, WT-ExT, KO-Sed, and KO-ExT groups. Mice were examined for exercise performance and in situ tibialis anterior (TA) contractility, followed by mass spectrometry-based proteomics and bioinformatics to identify differentially expressed proteins and signaling pathways. We found that maximal running distance was significantly longer in the WT-ExT group compared to the WT-Sed group, whereas this capacity was impaired in KO-ExT mice. Force generation and fatigue tolerance of the TA were enhanced in WT-ExT, but reduced in KO-ExT, compared to Sed controls. Proteomic analysis further revealed that ExT upregulated 576 proteins in WT but downregulated 207 proteins in KO mice. These proteins represent pathways in redox homeostasis, mitochondrial respiration, and proteomic adaptation of muscle to ExT. In summary, our data suggest a critical role of Nrf2 in the beneficial effects of SkM and adaptation to ExT.

Identification of Cardiac Expression Pattern of Transient Receptor Potential Vanilloid Type 1 (TRPV1) Receptor using a Transgenic Reporter Mouse Model.

Transient receptor potential vanilloid type 1 (TRPV1) channels are structurally related, non-selective cation channels that exhibit a high permeability to calcium. Sensory nerve endings expressing TRPV1 channels play a prominent role in regulating the cardiac sympathetic afferent reflex and contribute to cardiac remodeling and dysfunction in chronic heart failure. However, the precise expression of TRPV1 channels in cardiomyocytes vs. non-cardiomyocytes remains debated. Here we utilized a tdTomato-GFP reporter mouse crossed with a mouse line expressing Cre recombinase under the control of the TRPV1 promoter to map the TRPV1 expression pattern in heart. In this model, TRPV1-negative cells express tdTomato protein (red), whereas TRPV1-positive cells express GFP protein (green). As we expected, substantial GFP expression was found in many small and medium diameter dorsal root ganglia neurons in heterozygous TRPV1-Cre +/-, tdTomato flox/flox +/- male mice, suggesting that this heterozygous model is sufficient for labeling TRPV1-positive cells. Furthermore, these results showed that GFP green staining was not detectable in cardiomyocytes. Instead, we found strong GFP green staining in cardiac blood vessels-thought to be arterioles-in the heart. We also observed strong GFP signals on PGP9.5-positive cardiac nerve endings in the epicardium. In summary, this study does not support the concept that TRPV1 channels are strongly expressed in mouse cardiomyocytes. We conclude that TRPV1 channels in mouse heart are mostly expressed on non-cardiomyocyte cells including cardiac nerve endings and vessels. These data have important implications for the modulations of cardiogenic reflexes.

Overexpression of Central ACE2 (Angiotensin-Converting Enzyme 2) Attenuates the Pressor Response to Chronic Central Infusion of Ang II (Angiotensin II): A Potential Role for Nrf2 (Nuclear Factor [Erythroid-Derived 2]-Like 2).

We investigated the mechanism by which ACE2 (angiotensin-converting enzyme 2) overexpression alters neurohumoral outflow and central oxidative stress. Nrf2 (nuclear factor [erythroid-derived 2]-like 2) is a master antioxidant transcription factor that regulates cytoprotective and antioxidant genes. We hypothesized that upregulation of central ACE2 inhibits the pressor response to Ang II (angiotensin II) by reducing reactive oxygen species through a Nrf2/antioxidant enzyme-mediated mechanism in the rostral ventrolateral medulla. Synapsin human Angiotensin Converting Enzyme 2 positive (SynhACE2+/+) mice and their littermate controls synhACE2-/- were used to evaluate the consequence of intracerebroventricular infusion of Ang II. In control mice, Ang II infusion evoked a significant increase in blood pressure and norepinephrine excretion, along with polydipsia and polyuria. The pressor effect of central Ang II was completely blocked in synhACE2+/+ mice. Polydipsia, norepinephrine excretion, and markers of oxidative stress in response to central Ang II were also reduced in synhACE2+/+ mice. The MasR (Mas receptor) agonist Ang 1-7 and blocker A779 had no effects on blood pressure. synhACE2+/+ mice showed enhanced expression of Nrf2 in the rostral ventrolateral medulla which was blunted following Ang II infusion. Ang II evoked nuclear translocation of Nrf2 in cultured Neuro 2A (N2A) cells. In synhACE2-/- mice, the central Ang II pressor response was attenuated by simultaneous intracerebroventricular infusion of the Nrf2 activator sulforaphane; blood pressure was enhanced by knockdown of Nrf2 in the rostral ventrolateral medulla in Nrf2 floxed (Nrf2f/f) mice. These data suggest that the hypertensive effects of intracerebroventricular Ang II are attenuated by selective overexpression of brain synhACE2 and may be mediated by Nrf2-upregulated antioxidant enzymes in the rostral ventrolateral medulla.

Upregulating Nrf2 in the RVLM ameliorates sympatho-excitation in mice with chronic heart failure.

Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor that maintains redox homeostasis by governing a broad array of antioxidant genes in response to oxidant stress. We hypothesized that overexpression of Nrf2 in the rostral ventrolateral medulla (RVLM) ameliorates sympatho-excitation in mice with coronary artery ligation-induced chronic heart failure (CHF). To address this, we overexpressed Nrf2 in the RVLM using an HIV-CamKIIa-Nrf2 lenti virus in C57BL/6 mice. In addition, we used a Lenti-Cre virus in Keap1flox/flox mice to upregulate Nrf2 non-selectively in the RVLM. Arterial blood pressure (AP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded under conscious and anesthetized conditions, respectively. Protein expression was assayed using western blotting and immunofluorescence staining. We found that (1) Nrf2 and two target proteins, NQO1 and HO-1 in the RVLM were significantly lower in CHF compared to Sham mice. Nrf2 viral transfection of the RVLM upregulated Nrf2 protein. (2) Urinary NE excretion in CHF mice was markedly attenuated following Nrf2 upregulation (812 ±â€¯133 vs 1120 ±â€¯271 ng/24hr mean. ±SE, *p < 0.05, n = 8/group). (3) In the conscious state, CHF mice overexpressing Nrf2 exhibited an enhancement in spontaneous baroreflex gain and in phenylephrine-induced baroreflex control of HR. (4) Acute experiments under anesthetisa revealed a significant decrease in basal RSNA (44.0 ± 6.5 vs 64.7 ± 8.3% of Max. *P < 0.05 n = 8/group) and enhancement in baroreflex sensitivity (Maximal gain -1.8 ± 0.3 vs 1.1 ± 0.2 of mmHg. **p < 0.01. n = 6/group) in CHF mice that were virally transfected with Nrf2 compared with CHF mice transfected with Lenti-GFP. Finally, Lenti-Cre viral overexpression of Nrf2 in Keap1flox/flox mice reduced Keap1 protein and increased Nrf2, NQO1, and HO-1 in the RVLM of Sham and CHF mice. CHF-Cre mice exhibited a significant decrease in baseline RSNA and plasma NE concentration (8.9 ± 1.1 vs 12.7 ± 0.9 ng/mL *P < 0.05 n = 6/group) as compared with CHF-GFP mice. Based on the above data, we conclude that upregulating Nrf2 selectively in the RVLM attenuates sympatho-excitation in CHF mice. Nrf2 may be an important central target for autonomic modulation in cardiovascular disease and during stress.

Curcumin improves exercise performance of mice with coronary artery ligation-induced HFrEF: Nrf2 and antioxidant mechanisms in skeletal muscle.

A hallmark of chronic heart failure (HF) with low ejection fraction (HFrEF) is exercise intolerance. We hypothesized that reduced expression of nuclear factor E2-related factor 2 (Nrf2) in skeletal muscle contributes to impaired exercise performance. We further hypothesized that curcumin, a Nrf2 activator, would preserve or increase exercise capacity in HF. Experiments were carried out in mice with coronary artery ligation-induced HFrEF. Curcumin was deliveried by a subcutaneous osmotic minipump at a dose of 50 mg·kg-1·day-1 for 8 weeks. In vivo, in situ, and in vitro experiments were employed to evaluate exercise capacity, muscle function, and molecular mechanisms. We found that: 1) the maximal speed, running distance to exhaustion, and limb grip force were significantly lower in HFrEF mice compared with sham. Curcumin-treated HF mice displayed enhanced exercise performance compared with vehicle-treated HF mice; 2) both soleus (Sol) and extensor digitorum longus (EDL) muscles of HFrEF mice exhibited reduced force and rapid fatigue, which were ameliorated by curcumin; and 3) protein expression of Nrf2, hemeoxygenase-1, SOD2, myogenin, and MyoD were significantly lower, but total ubiquitinated proteins, MURF1, and atrogen-1 were higher in Sol and EDL of HFrEF compared with sham mice, whereas these alterations in Nrf2 signaling and antioxidant defenses in HFrEF were attenuated by curcumin, which had no effect on cardiac function per se in mice with severe HFrEF. These data suggest that impaired Nrf2 signaling intrinsic to skeletal muscle contributes to exercise intolerance in HFrEF. Skeletal muscle Nrf2 should be considered as a novel therapeutic target in severe HF. NEW & NOTEWORTHY These studies suggest that impaired nuclear factor E2-related factor 2 (Nrf2) signaling is a critical mechanism underlying the enhanced oxidative stress in skeletal muscle in heart failure with low ejection fraction (HFrEF). Curcumin prevents the decline in running performance in HFrEF mice by upregulating antioxidant defenses in skeletal muscle, likely mediated by activating Nrf2 signaling. These findings suggest a novel therapeutic target for the improvement of exercise capacity and quality of life in HFrEF patients.

Prostanoid Receptor Antagonist Effects on Intraocular Pressure, Supported by Ocular Biodisposition Experiments.

PURPOSE: Since all prostanoid receptors affect intraocular pressure (IOP) and endogenous prostanoids are found in ocular tissues, the pressor effects of prostanoid antagonists were comprehensively evaluated. The absence of effects of most of these antagonists was not entirely anticipated. To ensure no false-negative results, ocular biodisposition studies were conducted. METHODS: Monkeys with laser-induced ocular hypertension were used to study antagonist effects on IOP. Ocular biodisposition of each antagonist was assessed in rabbits, with LC/MS/MS analyses of tissue extracts and blood. RESULTS: EP1, EP2, EP3, EP4, FP, IP, and TP prostanoid receptor antagonists did not affect IOP, even at a high 1% dose. These studies were followed by ocular biodisposition studies. Striking differences in ocular tissue bioavailability were observed, which were independent of solubility. Only the EP1 antagonist SC-51322 failed to penetrate sufficiently to be bioavailable in the aqueous humor and ciliary body/iris. This obliged testing an alternative EP1 antagonist, namely ONO-8713, to reliably conclude that an EP1 antagonist does not alter IOP. CONCLUSIONS: These antagonist studies provided no evidence for individual endogenous prostanoids exerting a meaningful role in regulating IOP. They do reaffirm the critical importance of studying ocular bioavailability for confirming negative data. Large differences among the antagonists in anterior segment and even ocular surface tissue biodisposition were observed in rabbits. It appears from these monkey studies, supported by rabbit ocular bioavailability data, that an absence of drug effect in the eye cannot be adequately substantiated without determination of ocular pharmacokinetics.