Electrical Pulse Stimulation Protects C2C12 Myotubes against Hydrogen Peroxide-Induced Cytotoxicity via Nrf2/Antioxidant Pathway.

Skeletal muscle contraction evokes numerous biochemical alterations that underpin exercise benefits. This present study aimed to elucidate the mechanism for electrical pulse stimulation (EPS)-induced antioxidant adaptation in C2C12 myotubes. We found that EPS significantly upregulated Nrf2 and a broad array of downstream antioxidant enzymes involved in multiple antioxidant systems. These effects were completely abolished by pretreatment with a ROS scavenger, N-acetylcysteine. MitoSOX-Red, CM-H2DCFDA, and EPR spectroscopy revealed a significantly higher ROS level in mitochondria and cytosol in EPS cells compared to non-stimulated cells. Seahorse and Oroboros revealed that EPS significantly increased the maximal mitochondrial oxygen consumption rate, along with an upregulated protein expression of mitochondrial complexes I/V, mitofusin-1, and mitochondrial fission factor. A post-stimulation time-course experiment demonstrated that upregulated NQO1 and GSTA2 last at least 24 h following the cessation of EPS, whereas elevated ROS declines immediately. These findings suggest an antioxidant preconditioning effect in the EPS cells. A cell viability study suggested that the EPS cells displayed 11- and 36-fold higher survival rates compared to the control cells in response to 2 and 4 mM H2O2 treatment, respectively. In summary, we found that EPS upregulated a large group of antioxidant enzymes in C2C12 myotubes via a contraction-mitochondrial-ROS-Nrf2 pathway. This antioxidant adaptation protects cells against oxidative stress-associated cytotoxicity.

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.

Systemic mapping of organ plasma extravasation at multiple stages of chronic heart failure.

Introduction: Chronic Heart failure (CHF) is a highly prevalent disease that leads to significant morbidity and mortality. Diffuse vasculopathy is a commonmorbidity associated with CHF. Increased vascular permeability leading to plasma extravasation (PEx) occurs in surrounding tissues following endothelial dysfunction. Such micro- and macrovascular complications develop over time and lead to edema, inflammation, and multi-organ dysfunction in CHF. However, a systemic examination of PEx in vital organs among different time windows of CHF has never been performed. In the present study, we investigated time-dependent PEx in several major visceral organs including heart, lung, liver, spleen, kidney, duodenum, ileum, cecum, and pancreas between sham-operated and CHF rats induced by myocardial infarction (MI). Methods: Plasma extravasation was determined by colorimetric evaluation of Evans Blue (EB) concentrations at 3 days, ∼10 weeks and 4 months following MI. Results: Data show that cardiac PEx was initially high at day 3 post MI and then gradually decreased but remained at a moderately high level at ∼10 weeks and 4 months post MI. Lung PEx began at day 3 and remained significantly elevated at both ∼10 weeks and 4 months post MI. Spleen PExwas significantly increased at ∼10 weeks and 4 months but not on day 3 post MI. Liver PEx occurred early at day 3 and remain significantly increased at ∼10 weeks and 4 months post MI. For the gastrointestinal (GI) organs including duodenum, ileum and cecum, there was a general trend that PEx level gradually increased following MI and reached statistical significance at either 10 weeks or 4 months post MI. Similar to GI PEx, renal PEx was significantly elevated at 4 months post MI. Discussion: In summary, we found that MI generally incites a timedependent PEx of multiple visceral organs. However, the PEx time window for individual organs in response to the MI challenge was different, suggesting that different mechanisms are involved in the pathogenesis of PEx in these vital organs during the development of CHF.

Extracellular Vesicle MicroRNAs in Heart Failure: Pathophysiological Mediators and Therapeutic Targets.

Extracellular vesicles (EVs) are emerging mediators of intracellular and inter-organ communications in cardiovascular diseases (CVDs), especially in the pathogenesis of heart failure through the transference of EV-containing bioactive substances. microRNAs (miRNAs) are contained in EV cargo and are involved in the progression of heart failure. Over the past several years, a growing body of evidence has suggested that the biogenesis of miRNAs and EVs is tightly regulated, and the sorting of miRNAs into EVs is highly selective and tightly controlled. Extracellular miRNAs, particularly circulating EV-miRNAs, have shown promising potential as prognostic and diagnostic biomarkers for heart failure and as therapeutic targets. In this review, we summarize the latest progress concerning the role of EV-miRNAs in HF and their application in a therapeutic strategy development for heart failure.

The superior cervical ganglion is involved in chronic chemoreflex sensitization during recovery from acute lung injury.

Introduction: Acute lung injury (ALI) initiates an inflammatory cascade that impairs gas exchange, induces hypoxemia, and causes an increase in respiratory rate (fR). This stimulates the carotid body (CB) chemoreflex, a fundamental protective reflex that maintains oxygen homeostasis. Our previous study indicated that the chemoreflex is sensitized during the recovery from ALI. The superior cervical ganglion (SCG) is known to innervate the CB, and its electrical stimulation has been shown to significantly sensitize the chemoreflex in hypertensive and normotensive rats. We hypothesized that the SCG is involved in the chemoreflex sensitization post-ALI. Methods: We performed a bilateral SCG ganglionectomy (SCGx) or sham-SCGx (Sx) in male Sprague Dawley rats 2 weeks before inducing ALI (Week -2 i.e., W-2). ALI was induced using a single intra-tracheal instillation of bleomycin (bleo) (day 1). Resting-fR, Vt (Tidal Volume), and V̇ E (Minute Ventilation) were measured. The chemoreflex response to hypoxia (10% O2, 0% CO2) and normoxic-hypercapnia (21% O2, 5% CO2) were measured before surgery on W (-3), before bleo administration on W0 and on W4 post-bleo using whole-body plethysmography (WBP). Results: SCGx did not affect resting fR, Vt and V̇E as well as the chemoreflex responses to hypoxia and normoxic hypercapnia in either group prior to bleo. There was no significant difference in ALI-induced increase in resting fR between Sx and SCGx rats at W1 post-bleo. At W4 post-bleo, there were no significant differences in resting fR, Vt, and V̇E between Sx and SCGx rats. Consistent with our previous study, we observed a sensitized chemoreflex (delta fR) in response to hypoxia and normoxic hypercapnia in Sx rats at W4 post-bleo. However, at the same time, compared to Sx rats, the chemoreflex sensitivity was significantly less in SCGx rats in response to either hypoxia or normoxic hypercapnia. Discussion: These data suggest that SCG is involved in the chemoreflex sensitization during ALI recovery. Further understanding of the underlying mechanism will provide important information for the long-term goal of developing novel targeted therapeutic approaches to pulmonary diseases to improve clinical outcomes.

Potential Neuromodulation of the Cardio-Renal Syndrome.

The cardio-renal syndrome (CRS) type 2 is defined as a progressive loss of renal function following a primary insult to the myocardium that may be either acute or chronic but is accompanied by a decline in myocardial pump performance. The treatment of patients with CRS is difficult, and the disease often progresses to end-stage renal disease that is refractory to conventional therapy. While a good deal of information is known concerning renal injury in the CRS, less is understood about how reflex control of renal sympathetic nerve activity affects this syndrome. In this review, we provide insight into the role of the renal nerves, both from the afferent or sensory side and from the efferent side, in mediating renal dysfunction in CRS. We discuss how interventions such as renal denervation and abrogation of systemic reflexes may be used to alleviate renal dysfunction in the setting of chronic heart failure. We specifically focus on a novel cardiac sensory reflex that is sensitized in heart failure and activates the sympathetic nervous system, especially outflow to the kidney. This so-called Cardiac Sympathetic Afferent Reflex (CSAR) can be ablated using the potent neurotoxin resinferitoxin due to the high expression of Transient Receptor Potential Vanilloid 1 (TRPV1) receptors. Following ablation of the CSAR, several markers of renal dysfunction are reversed in the post-myocardial infarction heart failure state. This review puts forth the novel idea of neuromodulation at the cardiac level in the treatment of CRS Type 2.

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.

Time-dependent alteration in the chemoreflex post-acute lung injury.

Acute lung injury (ALI) induces inflammation that disrupts the normal alveolar-capillary endothelial barrier which impairs gas exchange to induce hypoxemia that reflexively increases respiration. The neural mechanisms underlying the respiratory dysfunction during ALI are not fully understood. The purpose of this study was to investigate the role of the chemoreflex in mediating abnormal ventilation during acute (early) and recovery (late) stages of ALI. We hypothesized that the increase in respiratory rate (fR) during post-ALI is mediated by a sensitized chemoreflex. ALI was induced in male Sprague-Dawley rats using a single intra-tracheal injection of bleomycin (Bleo: low-dose = 1.25 mg/Kg or high-dose = 2.5 mg/Kg) (day 1) and respiratory variables- fR, Vt (Tidal Volume), and VE (Minute Ventilation) in response to 10% hypoxia (10% O2, 0% CO2) and 5% hypercapnia/21% normoxia (21% O2, 5% CO2) were measured weekly from W0-W4 using whole-body plethysmography (WBP). Our data indicate sensitization (∆fR = 93 ± 31 bpm, p < 0.0001) of the chemoreflex at W1 post-ALI in response to hypoxic/hypercapnic gas challenge in the low-dose bleo (moderate ALI) group and a blunted chemoreflex (∆fR = -0.97 ± 42 bpm, p < 0.0001) at W1 post-ALI in the high-dose bleo (severe ALI) group. During recovery from ALI, at W3-W4, both low-dose and high-dose groups exhibited a sensitized chemoreflex in response to hypoxia and normoxic-hypercapnia. We then hypothesized that the blunted chemoreflex at W1 post-ALI in the high-dose bleo group could be due to near maximal tonic activation of chemoreceptors, called the "ceiling effect". To test this possibility, 90% hyperoxia (90% O2, 0% CO2) was given to bleo treated rats to inhibit the chemoreflex. Our results showed no changes in fR, suggesting absence of the tonic chemoreflex activation in response to hypoxia at W1 post-ALI. These data suggest that during the acute stage of moderate (low-dose bleo) and severe (high-dose bleo) ALI, chemoreflex activity trends to be slightly sensitized and blunted, respectively while it becomes significantly sensitized during the recovery stage. Future studies are required to examine the molecular/cellular mechanisms underlying the time-course changes in chemoreflex sensitivity post-ALI.

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.

Cardiac Spinal Afferent Denervation Attenuates Renal Dysfunction in Rats With Cardiorenal Syndrome Type 2.

Cardiorenal syndrome type 2 (CRS2) is defined as a chronic cardiovascular disease, usually chronic heart failure (CHF), resulting in chronic kidney disease. We hypothesized that the cardiac spinal afferent reflex (CSAR) plays a critical role in the development of CRS2. Our data suggest that cardiac afferent ablation by resiniferatoxin not only improves cardiac function but also benefits the kidneys and increases long-term survival in the myocardial infarction model of CHF. We also found that renal denervation has a similar reno-protective effect in CHF rats. We believe this novel work contributes to the development of a unique neuromodulation therapy to treat CHF patients.

Research Opportunities in Autonomic Neural Mechanisms of Cardiopulmonary Regulation: A Report From the National Heart, Lung, and Blood Institute and the National Institutes of Health Office of the Director Workshop.

This virtual workshop was convened by the National Heart, Lung, and Blood Institute, in partnership with the Office of Strategic Coordination of the Office of the National Institutes of Health Director, and held September 2 to 3, 2020. The intent was to assemble a multidisciplinary group of experts in basic, translational, and clinical research in neuroscience and cardiopulmonary disorders to identify knowledge gaps, guide future research efforts, and foster multidisciplinary collaborations pertaining to autonomic neural mechanisms of cardiopulmonary regulation. The group critically evaluated the current state of knowledge of the roles that the autonomic nervous system plays in regulation of cardiopulmonary function in health and in pathophysiology of arrhythmias, heart failure, sleep and circadian dysfunction, and breathing disorders. Opportunities to leverage the Common Fund's SPARC (Stimulating Peripheral Activity to Relieve Conditions) program were characterized as related to nonpharmacologic neuromodulation and device-based therapies. Common themes discussed include knowledge gaps, research priorities, and approaches to develop novel predictive markers of autonomic dysfunction. Approaches to precisely target neural pathophysiological mechanisms to herald new therapies for arrhythmias, heart failure, sleep and circadian rhythm physiology, and breathing disorders were also detailed.

Safety and efficacy of renal denervation in patients with heart failure with reduced ejection fraction (HFrEF): A systematic review and meta-analysis.

INTRODUCTION: A systematic analysis of clinical trials was performed in order to assess the effectiveness and risks of bilateral renal denervation (RDN) in patients with chronic heart failure with reduced ejection fraction (HFrEF). METHODS: A systematic review was conducted of all clinical trials exploring the effectiveness of RDN in patients with HF who had reduced (<50%) EF. Primary outcomes were NYHA class, 6-min walk test, N-terminal pro-B-type natriuretic peptide (NT-proBNP) levels, left ventricular ejection fraction (LVEF) and other cardiac parameters including left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), and left atrium diameter (LAD). Secondary outcomes were systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR), glomerular filtration rate (GFR), and creatinine. RESULTS: Seven studies were included in this analysis. From baseline to 6 months after RDN, the pooled mean NYHA class was decreased (mean difference [MD], -0.9; 95% confidence interval [CI], -1.6 to -0.2; P = 0.018), the mean 6-min walk test was increased (MD, 79.5 m; 95% CI, 26.9 to 132.1; P = 0.003), and the average NT-proBNP level was decreased (MD, -520.6 pg/mL; 95% CI, -1128.4 to 87.2; P = 0.093). Bilateral RDN increased the LVEF (MD, 5.7%; 95% CI, 1.6 to 9.6; P = 0.004), decreased the LVESD (MD, -0.4 cm; 95% CI, -0.5 to -0.2; P < 0.001), decreased the LVEDD (MD, -0.5 cm; 95% CI, -0.6 to -0.3; P < 0.001), and decreased the LAD (MD, -0.4 cm; 95% CI, -0.8 to 0; P = 0.045). In addition, RDN significantly decreased systolic BP (MD, -9.4 mmHg; 95% CI, -16.3 to -2.4; P = 0.008) and diastolic BP (MD, -4.9 mmHg; 95% CI, -9.5 to -0.4; P = 0.033), and decreased HR (MD, -4.5 bpm; 95% CI, -8.2to -0.9; P = 0.015). RDN did not significantly change GFR (MD, 7.9; 95% CI, -5.0 to 20.8; P = 0.230), or serum creatinine levels (MD, -7.2; 95% CI, -23.7 to 9.4; P = 0.397). CONCLUSION: Bilateral RDN appears safe and well-tolerated in patients with HF. RDN improved the signs and symptoms of HF and slightly decreased systolic and diastolic BP without affecting renal function in the clinical trials performed to date.

Timeline of Multi-Organ Plasma Extravasation After Bleomycin-Induced Acute Lung Injury.

Acute lung injury (ALI) is characterized by the abrupt onset of clinically significant hypoxemia in the context of non-hydrostatic pulmonary edema. Acute lung injury is associated with cytokine release and plasma extravasation (PEx) that can cause pulmonary edema and subsequently acute respiratory distress syndrome (ARDS). Therefore, it is critical we understand the relationship between ALI and lung PEx. In addition, it is also important to assess PEx in the lungs and other organs post-ALI since ALI/ARDS often causes multi-organ failure. We hypothesized that ALI induces time-dependent lung PEx, which promotes extravasation in the heart, liver, kidney, spleen, pancreas, and gastrointestinal (GI) tract, in a time-dependent manner. To test our hypothesis, we administered bleomycin or saline via tracheal intubation in 8-week-old Sprague Dawley rats. At the terminal experiments, Evans Blue was injected (IV) through the femoral vein to allow for the visualization of PEx. Plasma extravasation of desired organs was evaluated at 3-, 7-, 14-, 21-, and 28-days after bleomycin or saline treatment by evaluating Evans Blue concentrations calorimetrically at fluorescence excitation wavelength of 620 nm (bandwidth 10 nm) and an emission wavelength of 680 nm (bandwidth 40 nm). Data show that ALI induces lung PEx beginning at day 3 and peaking between 7 and 21 days. Extravasation was also seen in all organs at varying degrees beginning at day 3 and peaking between days 7 and 14. Resolution appears to start after day 21 and continues past day 28. We conclude that ALI caused by bleomycin incites a time-dependent PEx of the lungs and multiple other organs.

Macrophage activation in stellate ganglia contributes to lung injury-induced arrhythmogenesis in male rats.

AIM: Patients suffering from acute lung injury (ALI) are at high risk of developing cardiac arrhythmias. We hypothesized that stellate ganglia (SG) neural inflammation contributes to ALI-induced arrhythmia. METHODS: We created an ALI rat model using a single tracheal instillation of bleomycin (2.5 mg/kg), with saline as a sham control. We recorded ECGs by implanted radiotelemetry in male bleomycin and sham rats treated with and without oral minocycline (20 mg/kg/d), an anti-inflammatory drug that inhibits microglia/macrophage activation. The SG neuronal excitability was assessed by electrophysiology experiments. RESULTS: ECG data showed that bleomycin-exposed rats exhibited significantly more spontaneous premature ventricular contractions (PVCs) from 1- to 3-week post-induction compared with sham rats, which was mitigated by chronic oral administration of minocycline. The bleomycin-exposed rats displayed a robust increase in both the number of Iba1-positive macrophages and protein expression of interferon regulatory factor 8 in the SG starting as early at 1-week post-exposure and lasted for at least 4 weeks, which was largely attenuated by minocycline. Heart rate variability analysis indicated autonomic imbalance during the first 2-week post-bleomycin, which was significantly attenuated by minocycline. Electrical stimulation of the decentralized SG triggered more PVCs in bleomycin-exposed rats than sham and bleomycin + minocycline rats. Patch-clamp data demonstrated enhanced SG neuronal excitability in the bleomycin-exposed rats, which was attenuated by minocycline. Co-culture of lipopolysaccharide (LPS)-pretreated macrophages with normal SG neurons enhanced SG neuronal excitability. CONCLUSION: Macrophage activation in the SG contributes to arrhythmogenesis in bleomycin-induced ALI in male rats.

Skeletal Muscle Nrf2 Contributes to Exercise-Evoked Systemic Antioxidant Defense Via Extracellular Vesicular Communication.

This review explores the hypothesis that the repetitive contraction-relaxation that occurs during chronic exercise activates skeletal myocyte nuclear factor erythroid-derived 2-like 2 (Nrf2) to upregulate antioxidant enzymes. These proteins are secreted into the circulation within extracellular vesicles and taken up by remote cells, thus providing remote organs with cytoprotection against subsequent oxidative stress.

Regulation of Nrf2 signaling pathway in heart failure: Role of extracellular vesicles and non-coding RNAs.

The balance between pro- and antioxidant molecules has been established as an important driving force in the pathogenesis of cardiovascular disease. Chronic heart failure is associated with oxidative stress in the myocardium and globally. Redox balance in the heart and brain is controlled, in part, by antioxidant proteins regulated by the transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2), which is reduced in the heart failure state. Nrf2 can, in turn, be regulated by a variety of mechanisms including circulating microRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) derived from multiple cell types in the heart. Here, we review the role of the Nrf2 and antioxidant enzyme signaling pathway in mediating redox balance in the myocardium and the brain in the heart failure state. This review focuses on Nrf2 and antioxidant protein regulation in the heart and brain by miRNA-enriched EVs in the setting of heart failure. We discuss EV-mediated intra- and inter-organ communications especially, communication between the heart and brain via an EV pathway that mediates cardiac function and sympatho-excitation in heart failure. Importantly, we speculate how engineered EVs with specific miRNAs or antagomirs may be used in a therapeutic manner in heart failure.