Restoration of normal blood flow in atherosclerotic arteries promotes plaque stabilization.

Blood flow is a key regulator of atherosclerosis. Disturbed blood flow promotes atherosclerotic plaque development, whereas normal blood flow protects against plaque development. We hypothesized that normal blood flow is also therapeutic, if it were able to be restored within atherosclerotic arteries. Apolipoprotein E-deficient (ApoE-/-) mice were initially instrumented with a blood flow-modifying cuff to induce plaque development and then five weeks later the cuff was removed to allow restoration of normal blood flow. Plaques in decuffed mice exhibited compositional changes that indicated increased stability compared to plaques in mice with the cuff maintained. The therapeutic benefit of decuffing was comparable to atorvastatin and the combination had an additive effect. In addition, decuffing allowed restoration of lumen area, blood velocity, and wall shear stress to near baseline values, indicating restoration of normal blood flow. Our findings demonstrate that the mechanical effects of normal blood flow on atherosclerotic plaques promote stabilization.

A Link Between Methylglyoxal and Heart Failure During HIV-1 Infection.

Early-onset heart failure (HF) continues to be a major cause of morbidity and mortality in people living with human immunodeficiency virus type one (HIV-1) infection (PLWH), yet the molecular causes for this remain poorly understood. Herein NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ humanized mice (Hu-mice), plasma from PLWH, and autopsied cardiac tissues from deceased HIV seropositive individuals were used to assess if there is a link between the glycolysis byproduct methylglyoxal (MG) and HF in the setting of HIV-1 infection. At five weeks post HIV infection, Hu-mice developed grade III-IV diastolic dysfunction (DD) with an associated two-fold increase in plasma MG. At sixteen-seventeen weeks post infection, cardiac ejection fraction and fractional shortening also declined by 26 and 35%, and plasma MG increased to four-fold higher than uninfected controls. Histopathological and biochemical analyses of cardiac tissues from Hu-mice 17 weeks post-infection affirmed MG increase with a concomitant decrease in expression of the MG-degrading enzyme glyoxalase-1 (Glo1). The endothelial cell marker CD31 was found to be lower, and coronary microvascular leakage and myocardial fibrosis were prominent. Increasing expression of Glo1 in Hu-mice five weeks post-infection using a single dose of an engineered AAV2/9 (1.7 × 1012 virion particles/kg), attenuated the increases in plasma and cardiac MG levels. Increasing Glo1 also blunted microvascular leakage, fibrosis, and HF seen at sixteen weeks post-infection, without changes in plasma viral loads. In plasma from virally suppressed PLWH, MG was also 3.7-fold higher. In autopsied cardiac tissues from seropositive, HIV individuals with low viral log, MG was 4.2-fold higher and Glo1 was 50% lower compared to uninfected controls. These data show for the first time a causal link between accumulation of MG and HF in the setting of HIV infection.

Macrophage depletion in stellate ganglia alleviates cardiac sympathetic overactivation and ventricular arrhythmogenesis by attenuating neuroinflammation in heart failure.

Cardiac sympathetic overactivation is involved in arrhythmogenesis in patients with chronic heart failure (CHF). Inflammatory infiltration in the stellate ganglion (SG) is a critical factor for cardiac sympathoexcitation in patients with ventricular arrhythmias. This study aims to investigate if macrophage depletion in SGs decreases cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. Surgical ligation of the coronary artery was used for induction of CHF. Clodronate liposomes were microinjected into bilateral SGs of CHF rats for macrophage depletion. Using cytokine array, immunofluorescence staining, and Western blot analysis, we found that macrophage expansion and expression of TNFα and IL-1ß in SGs were markedly increased in CHF rats. Flow cytometry data confirmed that the percentage of macrophages in SGs was higher in CHF rats than that in sham rats. Clodronate liposomes significantly reduced CHF-elevated proinflammatory cytokine levels and macrophage expansion in SGs. Clodronate liposomes also reduced CHF-increased N-type Ca2+ currents and excitability of cardiac sympathetic postganglionic neurons and inhibited CHF-enhanced cardiac sympathetic nerve activity. ECG data from 24-h, continuous telemetry recording in conscious rats demonstrated that clodronate liposomes not only restored CHF-induced heterogeneity of ventricular electrical activities, but also decreased the incidence and duration of ventricular tachycardia/fibrillation in CHF. Macrophage depletion with clodronate liposomes attenuated CHF-induced cardiac sympathetic overactivation and ventricular arrhythmias through reduction of macrophage expansion and neuroinflammation in SGs.

Inhibition of N-type calcium channels in cardiac sympathetic neurons attenuates ventricular arrhythmogenesis in heart failure.

AIMS: Cardiac sympathetic overactivation is an important trigger of ventricular arrhythmias in patients with chronic heart failure (CHF). Our previous study demonstrated that N-type calcium (Cav2.2) currents in cardiac sympathetic post-ganglionic (CSP) neurons were increased in CHF. This study investigated the contribution of Cav2.2 channels in cardiac sympathetic overactivation and ventricular arrhythmogenesis in CHF. METHODS AND RESULTS: Rat CHF was induced by surgical ligation of the left coronary artery. Lentiviral Cav2.2-α shRNA or scrambled shRNA was transfected in vivo into stellate ganglia (SG) in CHF rats. Final experiments were performed at 14 weeks after coronary artery ligation. Real-time polymerase chain reaction and western blot data showed that in vivo transfection of Cav2.2-α shRNA reduced the expression of Cav2.2-α mRNA and protein in the SG in CHF rats. Cav2.2-α shRNA also reduced Cav2.2 currents and cell excitability of CSP neurons and attenuated cardiac sympathetic nerve activities (CSNA) in CHF rats. The power spectral analysis of heart rate variability (HRV) further revealed that transfection of Cav2.2-α shRNA in the SG normalized CHF-caused cardiac sympathetic overactivation in conscious rats. Twenty-four-hour continuous telemetry electrocardiogram recording revealed that this Cav2.2-α shRNA not only decreased incidence and duration of ventricular tachycardia/ventricular fibrillation but also improved CHF-induced heterogeneity of ventricular electrical activity in conscious CHF rats. Cav2.2-α shRNA also decreased susceptibility to ventricular arrhythmias in anaesthetized CHF rats. However, Cav2.2-α shRNA failed to improve CHF-induced cardiac contractile dysfunction. Scrambled shRNA did not affect Cav2.2 currents and cell excitability of CSP neurons, CSNA, HRV, and ventricular arrhythmogenesis in CHF rats. CONCLUSIONS: Overactivation of Cav2.2 channels in CSP neurons contributes to cardiac sympathetic hyperactivation and ventricular arrhythmogenesis in CHF. This suggests that discovering purely selective and potent small-molecule Cav2.2 channel blockers could be a potential therapeutic strategy to decrease fatal ventricular arrhythmias in CHF.

Sympathoinhibition and Vasodilation Contribute to the Acute Hypotensive Response of the Superoxide Dismutase Mimic, MnTnBuOE-2-PyP5+, in Hypertensive Animals.

The pathogenesis of hypertension has been linked to excessive levels of reactive oxygen species (ROS), particularly superoxide (O2•-), in multiple tissues and organ systems. Overexpression of superoxide dismutase (SOD) to scavenge O2•- has been shown to decrease blood pressure in hypertensive animals. We have previously shown that MnTnBuOE-2-PyP5+ (BuOE), a manganese porphyrin SOD mimic currently in clinical trials as a normal tissue protector for cancer patients undergoing radiation therapy, can scavenge O2•- and acutely decrease normotensive blood pressures. Herein, we hypothesized that BuOE decreases hypertensive blood pressures. Using angiotensin II (AngII)-hypertensive mice, we demonstrate that BuOE administered both intraperitoneally and intravenously (IV) acutely decreases elevated blood pressure. Further investigation using renal sympathetic nerve recordings in spontaneously hypertensive rats (SHRs) reveals that immediately following IV injection of BuOE, blood pressure and renal sympathetic nerve activity (RSNA) decrease. BuOE also induces dose-dependent vasodilation of femoral arteries from AngII-hypertensive mice, a response that is mediated, at least in part, by nitric oxide, as demonstrated by ex vivo video myography. We confirmed this vasodilation in vivo using doppler imaging of the superior mesenteric artery in AngII-hypertensive mice. Together, these data demonstrate that BuOE acutely decreases RSNA and induces vasodilation, which likely contribute to its ability to rapidly decrease hypertensive blood pressure.

HIV-1-Associated Left Ventricular Cardiac Dysfunction in Humanized Mice.

The molecular cause(s) for early onset heart failure in people living with HIV-1 infection (PLWH) remains poorly defined. Herein, longitudinal echocardiography was used to assess whether NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice reconstituted with human hematopoietic stem cells (Hu-NSG mice) and infected with HIV-1ADA can recapitulate the salient features of this progressive human disease. Four weeks post infection, Hu-NSG mice of both sexes developed left ventricular (LV) diastolic dysfunction (DD), with 25% exhibiting grade III/IV restrictive DD with mitral regurgitation. Increases in global longitudinal and circumferential strains and declines in LV ejection fraction and fractional shortening were observed eight weeks post infection. After twelve weeks of infection, 33% of Hu-NSG mice exhibited LV dyskinesia and dyssynchrony. Histopathological analyses of hearts seventeen weeks post infection revealed coronary microvascular leakage, fibrosis and immune cell infiltration into the myocardium. These data show for the first time that HIV-1ADA-infected Hu-NSG mice can recapitulate key left ventricular cardiac deficits and pathophysiological changes reported in humans with progressive HIV-1 infection. The results also suggest that HIV-1 infected Hu-NSG mice may be a useful model to screen for pharmacological agents to blunt LV dysfunction and associated pathophysiologic causes reported in PLWH.

Extracellular vesicular MicroRNA-27a* contributes to cardiac hypertrophy in chronic heart failure.

Under stress, the heart undergoes extensive remodeling resulting in cardiac fibrosis and hypertrophy, ultimately contributing to chronic heart failure (CHF). Alterations in microRNA levels are associated with dysfunctional gene expression profiles involved in the pathogenesis of heart failure. We previously showed that myocardial infarction-induced microRNA-enriched extracellular vesicles (EVs) contribute to the reduction in antioxidant enzymes by targeting Nrf2 signaling in CHF. MicroRNA-27a (miRNA-27a) is the predominant microRNA contained in cardiac fibroblast-derived EVs contributing to oxidative stress along with hypertrophic gene expression in cardiomyocytes. In the present study, we observed that miRNA-27a passenger strand (miRNA-27a*) was markedly upregulated in the non-infarcted area of the left ventricle of rats with CHF and encapsulated into EVs and secreted into the circulation. Bioinformatic analysis revealed that PDZ and LIM domain 5 (PDLIM5) is one of the major targets of miRNA-27a*, playing a major role in cardiac structure and function, and potentially contributing to the progression of cardiac hypertrophy. Our in vivo data demonstrate that PDLIM5 is down-regulated in the progression of heart failure, accompanied with the upregulation of hypertrophic genes and consistent with alterations in miRNA-27a*. Moreover, exogenous administration of miRNA27a* mimics inhibit PDLIM5 translation in cardiomyocytes whereas a miRNA27a* inhibitor enhanced PDLIM5 expression. Importantly, we confirmed that infarcted hearts have higher abundance of miRNA-27a* in EVs compared to normal hearts and further demonstrated that cultured cardiac fibroblasts secrete miRNA27a*-enriched EVs into the extracellular space in response to Angiotensin II stimulation, which inhibited PDLIM5 translation, leading to cardiomyocyte hypertrophic gene expression. In vivo studies suggest that the administration of a miRNA-27a* inhibitor in CHF rats partially blocks endogenous miR-27a* expression, prevents hypertrophic gene expression and improves myocardial contractility. These findings suggest that cardiac fibroblast-secretion of miRNA27a*-enriched EVs may act as a paracrine signaling mediator of cardiac hypertrophy that has potential as a novel therapeutic target.

Exercise Training Promotes Cardiac Hydrogen Sulfide Biosynthesis and Mitigates Pyroptosis to Prevent High-Fat Diet-Induced Diabetic Cardiomyopathy.

Obesity increases the risk of developing diabetes and subsequently, diabetic cardiomyopathy (DMCM). Reduced cardioprotective antioxidant hydrogen sulfide (H2S) and increased inflammatory cell death via pyroptosis contribute to adverse cardiac remodeling and DMCM. Although exercise training (EX) has cardioprotective effects, it is unclear whether EX mitigates obesity-induced DMCM by increasing H2S biosynthesis and mitigating pyroptosis in the heart. C57BL6 mice were fed a high-fat diet (HFD) while undergoing treadmill EX for 20 weeks. HFD mice developed obesity, hyperglycemia, and insulin resistance, which were reduced by EX. Left ventricle pressure-volume measurement revealed that obese mice developed reduced diastolic function with preserved ejection fraction, which was improved by EX. Cardiac dysfunction was accompanied by increased cardiac pyroptosis signaling, structural remodeling, and metabolic remodeling, indicated by accumulation of lipid droplets in the heart. Notably, EX increased cardiac H2S concentration and expression of H2S biosynthesis enzymes. HFD-induced obesity led to features of type 2 diabetes (T2DM), and subsequently DMCM. EX during the HFD regimen prevented the development of DMCM, possibly by promoting H2S-mediated cardioprotection and alleviating pyroptosis. This is the first report of EX modulating H2S and pyroptotic signaling in the heart.

Therapeutic Effects of Nrf2 Activation by Bardoxolone Methyl in Chronic Heart Failure.

Oxidative stress plays an important role in the pathogenesis of chronic heart failure (CHF) in many tissues. Increasing evidence suggests that systemic activation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) signaling can protect against postinfarct cardiac remodeling by reducing oxidative stress. However, it remains to be elucidated if Nrf2 activation exerts therapeutic effects in the CHF state. Here, we investigated the beneficial hemodynamic effects of bardoxolone methyl (2-Cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid methyl ester, CDDO-Me), a pharmacological activator of Nrf2, in a rodent model of CHF. Based on echocardiographic analysis, rats at 12 weeks post-myocardial infarction (MI) were randomly split into four groups. CDDO-Me (5 mg/kg, i.p.) was administered daily for another 2 weeks in sham and CHF rats and compared with vehicle treatment. Echocardiographic and hemodynamic analysis suggest that short-term CDDO-Me administration increased stroke volume and cardiac output in CHF rats and decreased left ventricle end-diastolic pressure. Molecular studies revealed that CDDO-Me-induced cardiac functional improvement was attributed to an increase of both Nrf2 transcription and translation, and a decrease of oxidative stress in the noninfarcted areas of the heart. Furthermore, CDDO-Me reduced NF-κB binding and increased Nrf2 binding to the CREB-binding protein, which may contribute to the selective increase of Nrf2 downstream targets, including NADPH Oxidase Quinone 1, Heme Oxygenase 1, Catalase, and Glutamate-Cysteine Ligase Catalytic Subunit, and the attenuation of myocardial inflammation in CHF rats. Our findings suggest that Nrf2 activation may provide beneficial cardiac effects in MI-mediated CHF. SIGNIFICANCE STATEMENT: Chronic heart failure (CHF) is the leading cause of death among the aged worldwide. The imbalance between pro- and antioxidant pathways is a determinant in the pathogenesis of CHF. Systemic activation of Nrf2 and antioxidant protein signaling by bardoxolone methyl may have beneficial effects on cardiac function and result in improvements by enhancing antioxidant enzyme expression and attenuating myocardial inflammation.

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.

Chronic Heart Failure Abolishes Circadian Rhythms in Resting and Chemoreflex Breathing.

Physiological systems often display 24 h rhythms that vary with the light/dark cycle. Disruption of circadian physiological rhythms have been linked to the progression of various cardiovascular diseases, and advances in the understanding of these rhythms have led to novel interventions and improved clinical outcomes. Although respiratory function has been known to vary between the light and dark periods, circadian rhythms in breathing have been understudied in clinical conditions. In the current study, we have begun to assess light/dark variations in respiration in chronic heart failure (CHF), a condition associated with abnormal resting and chemoreflex breathing as well as exercise intolerance. CHF was induced using coronary artery ligation and verified using echocardiography. Sham animals underwent a thoracotomy without coronary artery ligation. Tidal volume, respiratory frequency, and minute ventilation were all determined by whole body plethysmography under resting conditions and in response to chemoreflex challenges during the light and dark periods. Light/dark differences in voluntary exercise were assessed using a running wheel. The sham control group showed light/dark differences in resting and chemoreflex breathing, as well as arterial pressure, and these effects were eliminated in the CHF group. Both groups completed more rotations on the running wheel during the dark period compared to during the light period. The data suggest that CHF disrupts cardiovascular and respiratory circadian rhythms.

Emerging role of hydrogen sulfide-microRNA crosstalk in cardiovascular diseases.

Despite an obnoxious smell and toxicity at a high dose, hydrogen sulfide (H2S) is emerging as a cardioprotective gasotransmitter. H2S mitigates pathological cardiac remodeling by regulating several cellular processes including fibrosis, hypertrophy, apoptosis, and inflammation. These encouraging findings in rodents led to initiation of a clinical trial using a H2S donor in heart failure patients. However, the underlying molecular mechanisms by which H2S mitigates cardiac remodeling are not completely understood. Empirical evidence suggest that H2S may regulate signaling pathways either by directly influencing a gene in the cascade or interacting with nitric oxide (another cardioprotective gasotransmitter) or both. Recent studies revealed that H2S may ameliorate cardiac dysfunction by up- or downregulating specific microRNAs. MicroRNAs are noncoding, conserved, regulatory RNAs that modulate gene expression mostly by translational inhibition and are emerging as a therapeutic target for cardiovascular disease (CVD). Few microRNAs also regulate H2S biosynthesis. The inter-regulation of microRNAs and H2S opens a new avenue for exploring the H2S-microRNA crosstalk in CVD. This review embodies regulatory mechanisms that maintain the physiological level of H2S, exogenous H2S donors used for increasing the tissue levels of H2S, H2S-mediated regulation of CVD, H2S-microRNAs crosstalk in relation to the pathophysiology of heart disease, clinical trials on H2S, and future perspectives for H2S as a therapeutic agent for heart failure.

Exercise ameliorates high fat diet induced cardiac dysfunction by increasing interleukin 10.

Increasing evidence suggests that a sedentary lifestyle and a high fat diet (HFD) leads to cardiomyopathy. Moderate exercise ameliorates cardiac dysfunction, however underlying molecular mechanisms are poorly understood. Increased inflammation due to induction of pro-inflammatory cytokine such as tumor necrosis factor-alpha (TNF-α) and attenuation of anti-inflammatory cytokine such as interleukin 10 (IL-10) contributes to cardiac dysfunction in obese and diabetics. We hypothesized that exercise training ameliorates HFD- induced cardiac dysfunction by mitigating obesity and inflammation through upregulation of IL-10 and downregulation of TNF-α. To test this hypothesis, 8 week old, female C57BL/6J mice were fed with HFD and exercised (swimming 1 h/day for 5 days/week for 8 weeks). The four treatment groups: normal diet (ND), HFD, HFD + exercise (HFD + Ex) and ND + Ex were analyzed for mean body weight, blood glucose level, TNF-α, IL-10, cardiac fibrosis by Masson Trichrome, and cardiac dysfunction by echocardiography. Mean body weights were increased in HFD but comparatively less in HFD + Ex. The level of TNF-α was elevated and IL-10 was downregulated in HFD but ameliorated in HFD + Ex. Cardiac fibrosis increased in HFD and was attenuated by exercise in the HFD + Ex group. The percentage ejection fraction and fractional shortening were decreased in HFD but comparatively increased in HFD + Ex. There was no difference between ND and ND + Ex for the above parameters except an increase in IL-10 level following exercise. Based on these results, we conclude that exercise mitigates HFD- induced cardiomyopathy by decreasing obesity, inducing IL-10, and reducing TNF-α in mice.

Cigarette smoke-induced effects on bone marrow B-cell subsets and CD4+:CD8+ T-cell ratios are reversed by smoking cessation: influence of bone mass on immune cell response to and recovery from smoke exposure.

Cigarette smoking adversely affects the immune system, and is a risk factor for developing osteoporosis. How smoking contributes to osteoporosis is unclear, but since lymphocytes help maintain bone homeostasis and lymphocyte depletion results in bone loss, one potential mechanism for how smoke exposure promotes osteoporosis is by reducing bone marrow lymphocytes. Since the risk for developing osteoporosis is reportedly greater in smokers with polymorphisms in LRP5, a gene involved in canonical Wnt signaling that regulates bone metabolism, smoking-induced effects on lymphocytes may be influenced by Lrp5 functionality. To test these possibilities, we examined how the duration and cessation of cigarette smoke exposure affects lymphocyte distribution and function in normal mice and mice predisposed to low or high bone mass due to disruption or mutation of Lrp5. We find that, independent of genotype, mice exposed to cigarette smoke for 3-12 weeks showed a significant reduction in bone marrow B220(+)CD43(-) B cells and splenic transitional T1 B cells, and exhibited a splenic CD4(+):CD8(+) T-cell ratio that was skewed toward CD8(+) T cells. Smoke exposure had little or no effect on other lymphocyte subsets or on lymphocyte function ex vivo. Interestingly, these differences were no longer apparent after 6 weeks without smoke exposure, except in mice with high bone mass where bone marrow B220(+)CD43(-) B cells failed to fully recover. These data provide the first evidence that smoke exposure reduces bone marrow B cells, providing a plausible mechanism for how smoking contributes to osteoporosis.