Activating α7nAChR helps post-myocardial infarction healing by regulating macrophage polarization via the STAT3 signaling pathway.

BACKGROUND: Monocytes/macrophages play critical roles in inflammation and cardiac remodeling following myocardial infarction (MI). The cholinergic anti-inflammatory pathway (CAP) modulates local and systemic inflammatory responses by activating α7 nicotinic acetylcholine receptors (α7nAChR) in monocytes/macrophages. We investigated the effect of α7nAChR on MI-induced monocyte/macrophage recruitment and polarization and its contribution to cardiac remodeling and dysfunction. METHODS: Adult male Sprague Dawley rats underwent coronary ligation and were intraperitoneally injected with the α7nAChR-selective agonist PNU282987 or the antagonist methyllycaconitine (MLA). RAW264.7 cells were stimulated with lipopolysaccharide (LPS) + interferon-gamma (IFN-γ) and treated with PNU282987, MLA, and S3I-201 (a STAT3 inhibitor). Cardiac function was evaluated by echocardiography. Masson's trichrome and immunofluorescence were used to detect cardiac fibrosis, myocardial capillary density, and M1/M2 macrophages. Western blotting was used to detect protein expression, and the proportion of monocytes was measured using flow cytometry. RESULTS: Activating the CAP with PNU282987 significantly improved cardiac function and reduced cardiac fibrosis and 28-day mortality after MI. On days 3 and 7 post-MI, PNU282987 reduced the percentage of peripheral CD172a + CD43low monocytes and the infiltration of M1 macrophages in the infarcted hearts, whereas it increased the recruitment of peripheral CD172a + CD43high monocytes and M2 macrophages. Conversely, MLA exerted the opposite effects. In vitro, PNU282987 inhibited M1 macrophage polarization and promoted M2 macrophage polarization in LPS + IFN-γ-stimulated RAW264.7 cells. These PNU282987-induced changes in LPS + IFN-γ-stimulated RAW264.7 cells were reversed by administering S3I-201. CONCLUSION: Activating α7nAChR inhibits the early recruitment of pro-inflammatory monocytes/macrophages during MI and improves cardiac function and remodeling. Our findings suggest a promising therapeutic target for regulating monocyte/macrophage phenotypes and promoting healing after MI.

CB1 receptor antagonist rimonabant protects against chronic intermittent hypoxia-induced bone metabolism disorder and destruction in rats.

OBJECTIVE: The endocannabinoid system (ECS) regulates bone turn-over and remodeling. Chronic intermittent hypoxia (CIH) occurring during obstructive sleep apnea (OSA) may lead to disorders of the ECS and bone metabolism abnormalities. This study aimed to investigate whether or not the cannabinoid receptor 1 (CB1R) antagonist rimonabant (Ri) alleviates bone metabolism abnormalities and bone destruction induced by chronic intermittent hypoxia (CIH). METHODS: Healthy male Sprague Dawley (SD) rats (n=48) were randomly divided into 6 groups of 8 rats: 2 normal control (NC) groups, 2 intermittent hypoxia (IH) groups, and 2 IH + Ri groups. Rats in NC groups breathed room air for 4 weeks (4w NC group) and 6 weeks (6w NC group). Rats in IH groups experienced IH environment for 4 weeks (4w IH group) and 6 weeks (6w IH group). In addition to the same IH exposure, rats in IH + Ri group were given daily intraperitoneal injection of Ri at the dosage of 1.5 mg/kg/d for 4 weeks (4w IH + Ri group) and 6 weeks (6w IH + Ri group). Levels of serum tartrate-resistant acid phosphatase (TRAP, a marker of bone resorption) were determined by ELISA. Hematoxylin and eosin (HE) staining was performed on bone sections to observe the changes in bone microstructure. Expression of CB1R in bone tissue was determined by immunohistochemistry. RESULTS: TRAP levels were higher in the 4w IH and 6w IH groups than in the 4w NC and 6w NC groups; TRAP levels were lower in the 4w IH + Ri and 6w IH + Ri groups than in the 4w IH and 6w IH groups. HE staining showed that the morphology of bone cells in the NC group was normal, but the 4w IH group had mild edema of bone cells, reduction in trabecular bone, and destruction of bone microstructure. Changes were more severe in the 6w IH group than 4w IH. The 4w IH + Ri group was slightly improved compared with the 4w IH group. The 6w IH + Ri group was improved compared with the 4w IH + Ri group. The results of immunohistochemistry showed that the expression of CB1R in IH group was significantly higher than that in NC group. The expression of CB1R in the IH + Ri group was lower than that in the IH group. With the prolongation of hypoxia, the expression of CB1R in bone cells of IH group increased. The expression level of CB1R in IH + Ri group decreased with the prolongation of intervention time. Correlation analysis showed that the expression rate of CB1R in bone cells was positively correlated with the level of TRAP in serum. CONCLUSION: CIH increases serum TRAP levels and triggers metabolic bone disorder by activating bone CB1R. Intervention with CB1R antagonist (rimonabant) reduces the bone dysmetabolism in the CIH rat model.

Eliciting α7-nAChR exerts cardioprotective effects on ischemic cardiomyopathy via activation of AMPK signalling.

Our previous studies have reported that agonist of α7 nicotinic acetylcholine receptors prevented electrophysiological dysfunction of rats with ischaemic cardiomyopathy (ICM) by eliciting the cholinergic anti-inflammatory pathway (CAP). Adenosine monophosphate-activated protein kinase (AMPK) signalling is widely recognized exerting cardioprotective effect in various cardiomyopathy. Here, we aimed to investigate whether the protective effects of the CAP are associated with AMPK signalling in ICM. In vivo, coronary artery of rats was ligated for 4 weeks to induce the ICM and then treated with PNU-282987 (CAP agonist) and BML-275 dihydrochloride (AMPK antagonist) for 4 weeks. In vitro, primary macrophages harvested from rats were induced inflammation by Lipopolysaccharide (LPS) treatment and then treated with PNU-282987 and BML-275 dihydrochloride. In vivo, exciting CAP by PUN-282987 elicited an activation of AMPK signalling, alleviated ventricular remodeling, modified the cardiac electrophysiological function, reduced the cardiac expression of collagens and inflammatory cytokines and maintained the integrity of ultrastructure in the ischemic heart. However, the benefits of CAP excitation were blunted by AMPK signaling antagonization. In vitro, excitation of the CAP was observed inhibiting the nuclear transfer of NF-κB p65 of macrophages and promoting the transformation of Ly-6Chigh macrophages into Ly-6Clow macrophages. However, inhibiting AMPK signalling by BML-275 dihydrochloride reversed the CAP effect on LPS-treated macrophages. Finally, our findings suggest that eliciting the CAP modulates the inflammatory response in ICM through regulating AMPK signalling.

Origins location of the outflow tract ventricular arrhythmias exhibiting qrS pattern or QS pattern with a notch on the descending limb in lead V1.

BACKGROUND: Ventricular outflow tract(VOT) ventricular arrhythmias(VAs) presenting qrS pattern or QS pattern with a notch on the descending limb in lead V1 were consistently thought of arising from the commissure between left and right coronary cusp (L-RCC) by previous studies. However, we found they could originate from other anatomic structures in VOT. This study aimed to investigate the exact origin of this kind VAs. METHODS: Forty-nine patients of VOT premature ventricular contrations/ventricular tachycardia(PVCs/VT) with lead V1 presenting qrS pattern or QS pattern with a notch on the descending limb undergoing successful radiofrequency catheter ablation(RFCA) in our center were analyzed. RESULTS: 12-lead electrocardiogram(ECG) of these PVCs/VT were summarized. Among these PVCs/VT, 37 cases exhibited qrS morphology in lead V1, 12 cases presented QS pattern with a notch on the descending limb in the same lead. Based on the successful ablation sites, these PVCs/VT were divided into 2 groups: (1)Right ventricular outflow tract(RVOT) group (26 cases), and (2) Left ventricular outflow tract (LVOT) group(23 cases, 4 cases originating from the left coronary cusp(LCC), 2 from the right coronary cusp(RCC), 16 from the L-RCC, 1 from the area inferior to LCC(ILCC)). The ECG characteristics of each PVCs/VT were analyzed. Among these PVCs/VT, applying the precordial transitional zone index(TZ index) < 0 to predict LVOT origin was demonstrated with sensitivity of 95.65%, specificity of 96.15%, positive predicting value(PPV) of 95.65% and negative predicting value(NPV) of 96.15%. In LVOT group, further applying the r, R, m,or Rs morphology in lead I to predict L-RCC and RCC origin was demonstrated with sensitivity of 94.44%, specificity of 60.00%, PPV of 89.47% and NPV of 75.00%. CONCLUSIONS: Ventricular outflow tract PVCs/VT with lead V1 presenting qrS pattern or QS pattern with a notch on descending limb not only arising from L-RCC, but also RVOT, LCC, RCC and ILCC. Combining TZ index and QRS morphology in lead I to predict origin site of these kind VAs is a convenient, simple and reliable method and facilitates the RFCA procedure.

Approach selection of radiofrequency catheter ablation for ventricular arrhythmias originating from the left ventricular summit: potential relevance of Pseudo Delta wave, Intrinsicoid deflection time, maximal deflection index.

BACKGROUND: Ventricular arrhythmias (VAs) originating from the left ventricular summit is a challenge for radiofrequency catheter ablation (RFCA). The present study aimed to investigate the appropriate RFCA strategy for VAs originating from the left ventricular summit. METHODS: Forty-five consecutive patients with VAs arising from the left ventricular summit were successfully ablated at our cardiac electrophysiology center and reviewed in the study. RESULTS: Thirty-two cases of VAs were eliminated in the left ventricular endocardium by retrograde transaortic (n = 22, 22/45, 48.9%) or antegrade transseptal (n = 10, 10/45, 22.2%) approaches, the other 13 cases were eliminated in the left ventricular epicardium by distal great cardiac vein (DGCV) approach (n = 13, 13/45, 28.9%). Though these VAs were similar in electrocardiographic (ECG) morphology, the pseudo delta waves (PDW), intrinsicoid deflection time (IDT), maximal deflection index (MDI) differed among them, PDW >53 ms, IDT > 74 ms, MDI > 0.45 strongly indicated that ablating left ventricular summit VAs by DGCV approach. During mean follow-up of 19.5 ± 13.2 (range, 3-60) months, 2 (4.4%) patients experienced VAs recurrence. CONCLUSION: This retrospective study showed that VAs of left ventricular summit origin can be effectively cured with RFCA. For these VAs, prolonged PdW, IDT, MDI indicating RFCA by DGCV approach can be attempted firstly.

Aerobic Exercise Attenuates Pressure Overload-Induced Myocardial Remodeling and Myocardial Inflammation via Upregulating miR-574-3p in Mice.

BACKGROUND: Exercise training can promote cardiac rehabilitation, thereby reducing cardiovascular disease mortality and hospitalization rates. MicroRNAs (miRs) are closely related to heart disease, among which miR-574-3p plays an important role in myocardial remodeling, but its role in exercise-mediated cardioprotection is still unclear. METHODS: A mouse myocardial hypertrophy model was established by transverse aortic coarctation, and a 4-week swimming exercise training was performed 1 week after the operation. After swimming training, echocardiography was used to evaluate cardiac function in mice, and histopathologic staining was used to detect cardiac hypertrophy, myocardial fibrosis, and cardiac inflammation. Quantitative real-time polymerase chain reaction was used to detect the expression levels of miR-574-3p and cardiac hypertrophy markers. Western blotting detected the IL-6 (interleukin-6)/JAK/STAT inflammatory signaling pathway. RESULTS: Echocardiography and histochemical staining found that aerobic exercise significantly improved pressure overload-induced myocardial hypertrophy (n=6), myocardial interstitial fibrosis (n=6), and cardiac inflammation (n=6). Quantitative real-time polymerase chain reaction detection showed that aerobic exercise upregulated the expression level of miR-574-3p (n=6). After specific knockdown of miR-574-3p in mouse hearts with adeno-associated virus 9 using cardiac troponin T promoter, we found that the protective effect of exercise training on the heart was significantly reversed. Echocardiography and histopathologic staining showed that inhibiting the expression of miR-574-3p could partially block the effects of aerobic exercise on cardiac function (n=6), cardiomyocyte cross-sectional area (n=6), and myocardial fibrosis (n=6). Western blotting and immunohistochemical staining showed that the inhibitory effects of aerobic exercise on the IL-6/JAK/STAT pathway and cardiac inflammation were partially abolished after miR-574-3p knockdown. Furthermore, we also found that miR-574-3p exerts cardioprotective effects in cardiomyocytes by targeting IL-6 (n=3). CONCLUSIONS: Aerobic exercise protects cardiac hypertrophy and inflammation induced by pressure overload by upregulating miR-574-3p and inhibiting the IL-6/JAK/STAT pathway.

Cardiac-Specific Overexpression of Caveolin-1 in Rats With Ischemic Cardiomyopathy Improves Arrhythmogenicity and Cardiac Remodelling.

BACKGROUND: Ischemic cardiomyopathy (ICM) is associated with electrical and structural remodelling, leading to arrhythmias. Caveolin-1 (Cav1) is a membrane protein involved in the pathogenesis of ischemic injury. Cav1 deficiency has been associated with arrhythmogenicity. The current study aimed to determine how Cav1 overexpression inhibits arrhythmias and cardiac remodelling in ICM. METHODS: ICM was modelled using left anterior descending (LAD) artery ligation for 4 weeks. Cardiac-specific Cav1 overexpression in ICM on arrhythmias, excitation-contraction coupling, and cardiac remodelling were investigated using the intramyocardial injection of an adeno-associated virus serotype 9 (AAV-9) system, carrying a specific sequence expressing Cav1 (AAVCav1) under the cardiac troponin T (cTnT) promoter. RESULTS: Cav1 overexpression decreased susceptibility to arrhythmias by upregulating gap junction connexin 43 (CX43) and reducing spontaneous irregular proarrhythmogenic Ca2+ waves in ventricular cardiomyocytes. It also alleviated ischemic injury-induced contractility weakness by improving Ca2+ cycling through normalizing Ca2+-handling protein levels and improving Ca2+ homeostasis. Masson stain and immunoblotting revealed that the deposition of excessive fibrosis was attenuated by Cav1 overexpression, inhibiting the transforming growth factor-ß (TGF-ß)/Smad2 signalling pathway. Coimmunoprecipitation assays demonstrated that the interaction between Cav1 and cSrc modulated CX43 expression and Ca2+-handling protein levels. CONCLUSIONS: Cardiac-specific overexpression of Cav1 attenuated ventricular arrhythmia, improved Ca2+ cycling, and attenuated cardiac remodelling. These effects were attributed to modulation of CX43, normalized Ca2+-handling protein levels, improved Ca2+ homeostasis, and attenuated cardiac fibrosis.

Loss of USP22 alleviates cardiac hypertrophy induced by pressure overload through HiF1-α-TAK1 signaling pathway.

Ubiquitin-specific protease 22 (USP22) is a member of the ubiquitin specific protease family (ubiquitin-specific protease, USPs), the largest subfamily of deubiquitinating enzymes, and plays an important role in the treatment of tumors. USP22 is also expressed in the heart. However, the role of USP22 in heart disease remains unclear. In this study, we found that USP22 was elevated in hypertrophic mouse hearts and in angiotensin II (Ang II)-induced cardiomyocytes. The inhibition of USP22 expression with adenovirus significantly rescued hypertrophic phenotype and cardiac dysfunction induced by pressure overloaded. Consistent with in vivo study, silencing by USP22 shRNA expression in vitro had similar results. Molecular analysis revealed that transforming growth factor-ß-activating protein 1 (TAK1)-(JNK1/2)/P38 signaling pathway and HIF-1α was activated in the Ang II-induced hypertrophic cardiomyocytes, whereas HIF-1α expression was decreased after the inhibition of USP22. Inhibition of HIF-1α expression reduces TAK1 expression. Co-immunoprecipitation and ubiquitination studies revealed the regulatory mechanism between USP22 and HIF1α.Under hypertrophic stress conditions, USP22 enhances the stability of HIF-1α through its deubiquitination activity, which further activates the TAK1-(JNK1/2)/P38 signaling pathway to lead to cardiac hypertrophy. Inhibition of HIF-1α expression further potentiates the in vivo pathological effects caused by USP22 deficiency. In summary, this study suggests that USP22, through HIF-1α-TAK1-(JNK1/2)/P38 signaling pathway, may be potential targets for inhibiting pathological cardiac hypertrophy induced by pressure overload.

Activation of the Cholinergic Anti-inflammatory Pathway Attenuated Angiotension II-Dependent Hypertension and Renal Injury.

Background: Angiotensin II (AngII) induces renal fibrosis, characterized by fibroblast proliferation, inflammatory cell infiltration and excessive extracellular matrix deposition, all of which was relevant closely to hypertension. The vagus nerve-related cholinergic anti-inflammatory pathway (CAP) modulates local and systemic inflammatory responses. The aim of present study was to determine the effect of CAP on renal inflammation and fibrosis. Methods and Results: AngII-induced hypertension was induced in vivo by 14-days low-dose AngII infusion from osmotic minipumps. We used GTS-21 dihydrochloride, a selective nicotinic acetylcholine receptor agonist. Daily intraperitoneal GTS-21 injection and/or vagotomy started after hypertension was confirmed and continued for 4 weeks. The elevated blood pressure caused by AngII was significantly attenuated by GTS-21. Improved baroreflex sensitivity was observed after GTS-21 administration. Masson stain and immunoblotting revealed that deposition of excessive fibrosis and overexpression of inflammatory cytokines induced by AngII was reduced by GTS-21. To determine the role of autonomic control in CAP, unilateral vagotomy was performed. Vagotomy weakened the effect of CAP on AngII-induced hypertension. In vitro, GTS-21 suppressed NF-κB activation, attenuated AngII-induced epithelial-mesenchymal transition and reduced inflammation and fibrosis in NRK-52E cells; α-bungarotoxin (α-Bgt, an α7-nAChR selective antagonist) partly inhibited these effects. Conclusion: CAP protected against AngII-induced hypertension via improvement in autonomic control, suppression of NF-κB activation, and reduction of renal fibrosis and inflammatory response.

Dexmedetomidine Exerted Anti-arrhythmic Effects in Rat With Ischemic Cardiomyopathy via Upregulation of Connexin 43 and Reduction of Fibrosis and Inflammation.

BACKGROUND: Persistent myocardial ischemia post-myocardial infarction can lead to fatal ventricular arrhythmias such as ventricular tachycardia and fibrillation, both of which carry high mortality rates. Dexmedetomidine (Dex) is a highly selective α2-agonist used in surgery for congenital cardiac disease because of its antiarrhythmic properties. Dex has previously been reported to prevent or terminate various arrhythmias. The purpose of the present study was to determine the anti-arrhythmic properties of Dex in the context of ischemic cardiomyopathy (ICM) after myocardial infarction. METHODS AND RESULTS: We randomly allocated 48 rats with ICM, created by persistent ligation of the left anterior descending artery for 4 weeks, into six groups: Sham (n = 8), Sham + BML (n = 8), ICM (n = 8), ICM + BML (n = 8), ICM + Dex (n = 8), and ICM + Dex + BML (n = 8). Treatments started after ICM was confirmed (the day after echocardiographic measurement) and continued for 4 weeks (inject intraperitoneally, daily). Dex inhibited the generation of collagens, cytokines, and other inflammatory mediators in rats with ICM via the suppression of NF-κB activation and increased the distribution of connexin 43 (Cx43) via phosphorylation of adenosine 5'-monophosphate-activated protein kinase (AMPK). Dex reduced the occurrence of spontaneous ventricular arrhythmias (ventricular premature beat or ventricular tachycardia), decreased the inducibility quotient of ventricular arrhythmias induced by PES, and partly improved cardiac contraction. The AMPK antagonist BML-275 dihydrochloride (BML) partly weakened the cardioprotective effect of Dex. CONCLUSION: Dex conferred anti-arrhythmic effects in the context of ICM via upregulation of Cx43 and suppression of inflammation and fibrosis. The anti-arrhythmic and anti-inflammatory properties of Dex may be mediated by phosphorylation of AMPK and subsequent suppression of NF-κB activation.

Alteration of Cholinergic Anti-Inflammatory Pathway in Rat With Ischemic Cardiomyopathy-Modified Electrophysiological Function of Heart.

BACKGROUND: With chronic ischemia after myocardial infarction, the resulting scar tissue result in electrical and structural remodeling vulnerable to an arrhythmogenic substrate. The cholinergic anti-inflammatory pathway elicited by vagal nerve via α7 nicotinic acetylcholine receptors (α7-nAChR) can modulate local and systemic inflammatory responses. Here, we aimed to clarify a novel mechanism for the antiarrhythmogenic properties of vagal nerve during the ischemic cardiomyopathy (ICM). METHODS AND RESULTS: Left anterior descending artery of adult male Sprague-Dawley rats was ligated for 4 weeks to develop ICM. Western blot revealed that eliciting the cholinergic anti-inflammatory pathway by nicotine treatment showed a significant reduction in the amounts of collagens, cytokines, and other inflammatory mediators in the left ventricular infarcted border zone via inhibited NF-κB activation, whereas it increased the phosphorylated connexin 43. Vagotomy inhibited the anti-inflammatory, anti-fibrosis, and anti-arrhythmogenic effect of nicotine administration. And immunohistochemistry confirmed that the nicotine administration-induced increase of connexin 43 was located in intercellular junctions. Furthermore nicotine treatment suppressed NF-κB activation in lipopolysaccharide-stimulated RAW264.7 cells, and α-bungarotoxin (an α7-nAChR selective antagonist) partly inhibited the nicotine-treatment effect. In addition, 4-week nicotine administration slightly improved the cardiac function, increased cardiac parasympathetic tone, decreased the prolonged QTc, and decreased the arrhythmia score of programmed electric stimulation-induced ventricular arrhythmia. CONCLUSIONS: Eliciting the cholinergic anti-inflammatory pathway exerts anti-arrhythmogenic effects against ICM-induced ventricular arrhythmia accompanied by downregulation of cytokines, downgenerating of collagens, decrease in sympathetic/parasympathetic ratio, and prevention of the loss of phosphorylated connexin 43 during ICM. Our findings may suggest a promising therapy for the generation of ICM-induced ventricular arrhythmia by eliciting the cholinergic anti-inflammatory pathway.