CXCR4-overexpressed exosomes from cardiosphere-derived cells attenuate myocardial ischemia/reperfusion injury by transferring miRNA to macrophages and regulating macrophage polarization.

Cardiosphere-derived cells (CDCs) are emerging as ideal candidates for managing cardiac inflammation, albeit with some limitations. Recent literatures have indicated that exosomes secreted by CDCs with C-X-C motif chemokine receptor 4 (CXCR4) overexpression can promote cardiac function after myocardial infarction and there have been some reports of miRNAs involved in ischemia/reperfusion (I/R) therapy. Therefore, we are interested in the role of CXCR4-overexpressed CDC-derived exosomes in delivering specific miRNA after myocardial I/R injury. In this research, we first constructed CDC-derived exosomes that overexpressed CXCR4 and miR-27a-5p, miR-182, or miR-101a. Then, we co-cultured the engineered exosomes with RAW264.7 cells and injected them intravenously into myocardial I/R model mice. In vitro, results showed that proinflammatory cytokines levels in the culture supernatant were decreased and the expression of M2 phenotypic markers were increased. Administration of engineered exosomes improved cardiac function, reduced infarct size, alleviated macrophage infiltration, and regulated M2 macrophage polarization after myocardial I/R, suggesting their implications in cardiac injury repair.

Inhibition of Notch signaling pathway attenuates sympathetic hyperinnervation together with the augmentation of M2 macrophages in rats post-myocardial infarction.

Inflammation-dominated sympathetic sprouting adjacent to the necrotic region following myocardial infarction (MI) has been implicated in the etiology of arrhythmias resulting in sudden cardiac death; however, the mechanisms responsible remain to be elucidated. Although being a key immune mediator, the role of Notch has yet to be explored. We investigated whether Notch regulates macrophage responses to inflammation and affects cardiac sympathetic reinnervation in rats undergoing MI. MI was induced by coronary artery ligation. A high level of Notch intracellular domain was observed in the macrophages that infiltrated the infarct area at 3 days post-MI. The administration of the Notch inhibitor N-N-(3,5-difluorophenacetyl-L-alanyl)-S-phenylglycine-t-butyl ester (DAPT) (intravenously 30 min before MI and then daily until death) decreased the number of macrophages and significantly increased the M2 macrophage activation profile in the early stages and attenuated the expression of nerve growth factor (NGF). Eventually, NGF-induced sympathetic hyperinnervation was blunted, as assessed by the immunofluorescence of tyrosine hydroxylase. At 7 days post-MI, the arrhythmia score of programmed electric stimulation in the vehicle-treated infarcted rats was higher than that in rats treated with DAPT. Further deterioration in cardiac function and decreases in the plasma levels of TNF-α and IL-1ß were also detected. In vitro studies revealed that LPS/IFN-γ upregulated the surface expression of NGF in M1 macrophages in a Notch-dependent manner. We concluded that Notch inhibition during the acute inflammatory response phase is associated with the downregulation of NGF, probably through a macrophage-dependent pathway, thus preventing the process of sympathetic hyperinnervation.

Valsartan Attenuates KIR2.1 by Downregulating the Th1 Immune Response in Rats Following Myocardial Infarction.

BACKGROUND: Myocardial infarction (MI) results in decreased inward-rectifier K⁺ current (IK1), which is mediated primarily by the Kir2.1 protein and is accompanied by upregulated T cells. Interferon γ (IFN-γ), secreted predominantly by Th1 cells, causes a decrease in IK1 in microglia. Whether Th1 cells can induce IK1/Kir2.1 remodeling following MI and whether valsartan can ameliorate this phenomenon remain unclear. METHODS: Rats experiencing MI received either valsartan or saline for 7 days. Th1-enriched lymphocytes and myocytes were cocultured with or without valsartan treatment. Th1 cells were monitored by flow cytometry. The protein levels of Kir2.1 were detected by Western blot analyses. IK1 was recorded through whole-cell patch clamping. The plasma levels of IFN-γ, interleukin 2, and tumor necrosis factor α were detected by enzyme-linked immunosorbent assay. RESULTS: Th1 cell number and cytokine expression levels were higher following MI, and the Kir2.1 protein level was decreased. In MI rats, valsartan reduced Th1 cell number and cytokine expression levels and increased the Kir2.1 expression and the IK1 current compared with the rats that received saline treatment; these results are consistent with the effect of valsartan in cocultured lymphocytes and myocytes. In vitro, IFN-γ overexpression suppressed the IK1 current, whereas interleukin 2 and tumor necrosis factor α had no significant effect on the current, establishing that Th1 cell regulation of IK1/Kir2.1 expression is mainly dependent on IFN-γ. CONCLUSIONS: Valsartan ameliorates IK1/Kir2.1 remodeling by downregulating the Th1 immune response following MI.

Semaphorin 3A attenuates cardiac autonomic disorders and reduces inducible ventricular arrhythmias in rats with experimental myocardial infarction.

BACKGROUND: To investigate the effects of semaphorin 3A (sema 3A) on cardiac autonomic regulation and subsequent ventricular arrhythmias (VAs) in post-infarcted hearts. METHOD AND RESULTS: In order to explore the functions of sema 3A in post-infarcted hearts, lentivirus-Sema 3A-shRNA and negative control vectors were delivered to the peri-infarcted myocardium rats respectively. Meanwhile, recombinant sema 3A and control (0.9% NaCl solution) were injected intravenously into infarcted rats to test the therapeutic potential of sema 3A. Results indicated that levels of sema 3A were higher in post-infarcted hearts compared with sham rats. However, sema 3A silencing leaded to sympathetic hyperinnervation, increased myocardial norepinephrine (NE) content and inducible VAs. Conversely, the intravenous administration of sema 3A to infarcted rats reduced sympathetic nerve sprouting, improved cardiac autonomic regulation, and decreased the incidence of inducible VAs. However, both infarct size and cardiac function were similar among infarcted hearts. CONCLUSIONS: The upregulation and administration of sema 3A exerted beneficial effects on infarction-induced cardiac autonomic disorders by increasing cardiac electrical stability and reducing VAs. Sema 3A might be a potential therapeutic agent for cardiac autonomic abnormalities induced arrhythmias.

Valsartan Upregulates Kir2.1 in Rats Suffering from Myocardial Infarction via Casein Kinase 2.

PURPOSE: Myocardial infarction (MI) results in an increased susceptibility to ventricular arrhythmias, due in part to decreased inward-rectifier K+ current (IK1), which is mediated primarily by the Kir2.1 protein. The use of renin-angiotensin-aldosterone system antagonists is associated with a reduced incidence of ventricular arrhythmias. Casein kinase 2 (CK2) binds and phosphorylates SP1, a transcription factor of KCNJ2 that encodes Kir2.1. Whether valsartan represses CK2 activation to ameliorate IK1 remodeling following MI remains unclear. METHODS: Wistar rats suffering from MI received either valsartan or saline for 7 days. The protein levels of CK2 and Kir2.1 were each detected via a Western blot analysis. The mRNA levels of CK2 and Kir2.1 were each examined via quantitative real-time PCR. RESULTS: CK2 expression was higher at the infarct border; and was accompanied by a depressed IK1/Kir2.1 protein level. Additionally, CK2 overexpression suppressed KCNJ2/Kir2.1 expression. By contrast, CK2 inhibition enhanced KCNJ2/Kir2.1 expression, establishing that CK2 regulates KCNJ2 expression. Among the rats suffering from MI, valsartan reduced CK2 expression and increased Kir2.1 expression compared with the rats that received saline treatment. In vitro, hypoxia increased CK2 expression and valsartan inhibited CK2 expression. The over-expression of CK2 in cells treated with valsartan abrogated its beneficial effect on KCNJ2/Kir2.1. CONCLUSIONS: AT1 receptor antagonist valsartan reduces CK2 activation, increases Kir2.1 expression and thereby ameliorates IK1 remodeling after MI in the rat model.

Exogenous nerve growth factor promotes the repair of cardiac sympathetic heterogeneity and electrophysiological instability in diabetic rats.

OBJECTIVES: Diabetic cardiac autonomic neuropathy can lead to an increased incidence of ventricular arrhythmias (VAs). However, few data are available regarding the pathogenesis and therapy of the VAs accompanying diabetic cardiac autonomic neuropathy. We aimed to explore whether or not exogenous nerve growth factor (NGF) can reduce the sympathetic heterogeneity and the incidence of VAs in diabetes mellitus (DM). METHODS: Male Wistar rats were randomly divided into 3 groups: controls, rats with DM with saline infused into the left stellate ganglion (LSG), i.e. the DS group and rats with DM with NGF infused into the LSG, i.e. the DN group. After 28 weeks, all rats were subjected to electrophysiological experiments. Sympathetic innervations and NGF were studied by immunostaining, RT-PCR or Western blot analysis. RESULTS: The incidence of inducible VAs was significantly higher in the DS group than in the control group, but was markedly decreased in the DN group. In the DS group, the tyrosine hydroxylase (TH) and NGF expression were significantly lower than in the other groups, and significant proximal-distal heterogeneities existed regarding the TH and NGF expression in the left ventricle, but were markedly repaired in the DN group. CONCLUSIONS: NGF intervention in the LSG can reduce the heterogeneity of cardiac sympathetic innervations and the incidence of VAs in diabetic rats.

Metoprolol-mediated amelioration of sympathetic nerve sprouting after myocardial infarction.

OBJECTIVES: Systemic or local inflammation causes cardiac nerve sprouting and consequent arrhythmia. Metoprolol can prevent sympathetic nerve remodeling after myocardial infarction (MI), but the underlying mechanism is unclear. In this study, we evaluated the role of metoprolol in ameliorating sympathetic sprouting. METHODS: Rabbits underwent ligation of the coronary artery for MI. MI rabbits received metoprolol or saline for 7 days. Immunohistochemistry was used to measure cardiac nerve sprouting and sympathetic innervations. Nuclear factor-κB (NF-κB) DNA binding activity was analyzed by electrophoretic mobility shift assay. The protein levels of NF-κB p65, inhibitor κBα (IκBα) and nerve growth factor (NGF) were detected by Western blot analysis. The mRNA levels of NGF, interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) were examined by quantitative real-time PCR. RESULTS: MI rabbits showed nerve sprouting and sympathetic hyperinnervation. In MI rabbits, as compared with saline treatment, metoprolol reduced NF-κB DNA binding activity and NF-κB p65 level, and increased IκBα level. Moreover, metoprolol downregulated IL-1ß, TNF-α and NGF levels, and reduced the density of sympathetic nerve fibers. CONCLUSIONS: Metoprolol ameliorates sympathetic nerve sprouting in rabbits after MI and is associated in part with inhibiting NF-κB activity.

Mesenchymal stem cell therapy improves diabetic cardiac autonomic neuropathy and decreases the inducibility of ventricular arrhythmias.

BACKGROUND: Diabetic cardiac autonomic neuropathy (DCAN) may cause fatal ventricular arrhythmias and increase mortality in diabetics. Mesenchymal stem cells (MSCs) can secrete various cytokines and growth factors exerting neurosupportive effects. In this study, we investigated the effect of MSC on DCAN in diabetic rats. METHODS: Forty rats were divided into normal control, diabetes mellitus (DM) control, MSC treatment (6 × 10(6) MSCs via direct myocardial injection) and MSC-conditioned medium group (100 µl via direct myocardial injection). Immunohistochemistry was used to measure choline acetyltransferase (ChAT, a marker for parasympathetic nerves) and tyrosine hydroxylase (TH, a marker for sympathetic nerves) positive nerve fibres in the ventricular myocardium. Heart rate variability and programmed electrical stimulation was used to assess the inducibility of ventricular arrhythmias in the animals. RESULTS: Two weeks after MSC treatment, the density of ChAT- and TH-positive nerve fibres in MSCs and MSC-conditioned medium group was higher than in DM control group (P < 0.05 or P < 0.01). The ChAT/TH ratio in MSC group was higher than in DM control group (0.37 ± 0.014 vs. 0.27 ± 0.020, P < 0.01). The standard deviation of normal-to-normal R-R intervals in MSCs (5.13 ± 0.69) and MSC-conditioned medium group (4.30 ± 0.56) was higher than in DM control group (3.45 ± 0.60, P < 0.05). The inducibility of VAs in the MSC group was lower than in the DM control group. CONCLUSIONS: MSC therapy may promote cardiac nerve sprouting and increase the ratio of parasympathetic to sympathetic nerve fibres. It may also suppress the inducibility of ventricular arrhythmias in the diabetic rats.

Risk of ventricular arrhythmias after myocardial infarction with diabetes associated with sympathetic neural remodeling in rabbits.

BACKGROUND: Abnormal sympathetic innervation underlies both long-term hyperglycemia and myocardial infarction (MI). The incidence of ventricular arrhythmias (VAs) after MI is higher in diabetic than in nondiabetic patients. However, the exact mechanism remains unclear. In this study, we aimed to explore sympathetic neural remodeling after MI in diabetic rabbits and its relationship with VAs. METHODS: Rabbits were randomly assigned to 4 groups: control, diabetes mellitus (DM), MI and diabetic myocardial infarction (DI). After electrophysiological experiments in vivo, immunohistochemistry and real-time RT-PCR were used to measure sympathetic innervations. To test the function of sympathetic nerve fibers, norepinephrine levels were measured by high-performance liquid chromatography. RESULTS: The corrected QT interval and QT dispersion were significantly more prolonged with DI than other conditions. The density of tyrosine hydroxylase-positive fibers and corresponding mRNA abundance was significantly higher with DI than with DM and under control conditions, but was lower than with the MI group. Moreover, the distribution and structure of regenerated nerve was heterogeneous in DI rabbits. Norepinephrine content was higher in the DI group, and accompanied by an increased quantity of tyrosine hydroxylase-positive fibers. CONCLUSION: MI results in sympathetic neural remodeling in diabetic rabbits, which may be responsible in part for the increased occurrence of VAs.

Astaxanthin attenuates contrast-induced acute kidney injury in rats via ROS/NLRP3 inflammasome.

OBJECTIVE: To explore the protective effect and mechanism of astaxanthin on the kidney of rats with contrast-induced acute kidney injury. METHODS: Forty SD rats were randomly divided into five groups: Control group (CON); Astaxanthin control group (AST); Contrast media group (CM); Astaxanthin pre-treatment group (AST + CM); N-acetylcysteine pre-treatment group (NAC + CM), each group with eight rats. The rats were killed 72 h after the modeling, the blood supernatant and kidneys were collected, and then the serum creatinine and blood urea nitrogen levels were measured; HE staining was used to observe the pathological changes in kidney tissue; TUNEL was used to detect apoptosis level in renal tubular epithelial cells; frozen section was used to observe the expression of ROS in renal tissue by reactive oxygen staining; the expression of NLRP3, ASC, caspase-1, IL-1ß, IL-18 were detected by immunohistochemistry and western blot. RESULTS: The CI-AKI rat model was induced by iohexol. Then the elevated level of ROS activated the inflammatory response mediated by NLRP3 inflammasome (NLRP3, ASC, caspase-1). Subsequently, the increase in renal tubular epithelial cell apoptosis caused the destruction of the pathological structure of the kidney and finally led to renal impairment. While after the pretreatment of astaxanthin, the level of ROS was decreased. The activation level of NLRP3 inflammasome and its mediated inflammatory response were alleviated significantly. Eventually, the level of renal tubular epithelial cell apoptosis and renal damage were significantly mitigated. CONCLUSION: Astaxanthin can protect the kidney in CI-AKI by inhibiting the activation of NLRP3 inflammasome-IL-1ß/IL-18 through inhibition of the production of ROS.

Development and Validation of a Risk Nomogram Model for Predicting Contrast-Induced Acute Kidney Injury in Patients with Non-ST-Elevation Acute Coronary Syndrome Undergoing Primary Percutaneous Coronary Intervention.

OBJECTIVE: To establish a nomogram model to predict the risk of contrast-induced acute kidney injury (CI-AKI) by analyzing the risk factors of CI-AKI and to evaluate its effectiveness. METHODS: Retrospectively analyze the clinical data of non-ST-elevation acute coronary syndrome (NSTE-ACS) patients who underwent percutaneous coronary intervention (PCI) in our cardiology department from September 2018 to June 2021. Of these, patients who underwent PCI in an earlier period formed the training cohort (70%; n = 809) for nomogram development, and those who underwent PCI thereafter formed the validation cohort (30%; n = 347) to confirm the model's performance. The independent risk factors of CI-AKI were determined by LASSO regression and multivariable logistic regression analysis. By using R software from which nomogram models were subsequently generated. The nomogram was developed and evaluated based on discrimination, calibration, and clinical efficacy using the concordance statistic (C-statistic), calibration plot, and decision curve analysis (DCA), respectively. RESULTS: The nomogram consisted of six variables: age >75, left ventricular ejection fraction, diabetes mellitus, fibrinogen-to-albumin ratio, high-sensitive C-reactive protein, and lymphocyte count. The C-index of the nomogram is 0.835 (95% CI: 0.800-0.871) in the training cohort and 0.767 (95% CI: 0.711-0.824) in the validation cohort, respectively. The calibration plots exhibited that the nomogram was in good agreement between prediction and observation in the training and validation cohorts. Decision curve analysis and clinical impact curve suggested that the predictive nomogram had clinical utility. CONCLUSION: The nomogram model established has a good degree of differentiation and accuracy, which is intuitively and individually to screen high-risk groups and has a certain predictive value for the occurrence of CI-AKI in NSTE-ACS patients after PCI.

Resveratrol Pretreatment Improved Heart Recovery Ability of Hyperglycemic Bone Marrow Stem Cells Transplantation in Diabetic Myocardial Infarction by Down-Regulating MicroRNA-34a.

AIM: To examine the effect of resveratrol (RSV) on bone marrow mesenchymal stem cells (BMSCs) under hyperglycemic conditions and on BMSCs transplantation in diabetic rats with myocardial infarction (MI). METHODS: In vitro, BMSCs were isolated from 3-week-old male Sprague Dawley (SD) rats and cultured under hyperglycemic conditions for up to 28 days. Cell viability was analyzed by cell counting kit-8 (CCK-8) assays. The expression of miR-34a was measured by RT-qPCR. Western blotting was used to examine the protein expression of SIRT1, P21, P16, VEGF and HIF-1α. A senescence-associated ß-galactosidase assay was used to examine the senescence level of each group. In vivo, a diabetes model was established by feeding rats a high-sugar and high-fat diet for 8 weeks, injecting the animals with streptozotocin (STZ) and continuing high-sugar and high-fat feeding for 4 additional weeks. Then, left anterior descending coronary artery (LAD) cessation was used to established the myocardial infarction (MI) models. Each group of rats was transplanted with differentially preconditioned BMSCs after myocardial infarction. Ultrasound was used to analyze cardiac function 1 and 3 weeks after the operation, and frozen heart sections were used for immunohistochemical analysis, Masson staining and CD31 measurement. In addition, ELISA analysis of serum cytokine levels was performed. RESULTS: This study showed that the viability of BMSCs cultured under hyperglycemic conditions was decreased, the cells became senescent. Besides, an obviously increased in the expression of miR-34a was detected. Moreover, RSV preconditioning reduced the expression of miR-34a in BMSCs after high glucose stimulation and rejuvenated BMSCs under hyperglycemic conditions. Further analysis showed that the transplantation of RSV-BMSCs were benefit to heart recovery following infarction in diabetic rats, promoted proangiogenic factor release and increased arteriole and capillary densities. CONCLUSION: RSV rejuvenated BMSCs after chronic hyperglycemia-induced senescence by interacting with miR-34a and optimized the therapeutic effect of BMSCs on diabetes with myocardial infarction.

Impact of small and dense low-density lipoprotein (sd-LDL)on contrast-induced acute kidney injury in patients with acute ST-segment elevation myocardial infarction undergoing primary percutaneous coronary intervention.

OBJECTIVE: To investigate the impact of serum small and dense low-density lipoprotein (sd-LDL) on contrast-induced acute kidney injury (CI-AKI) after emergency percutaneous coronary intervention (PCI) in patients with acute ST-segment elevation myocardial infarction (STEMI). METHOD: From November 2019 to August 2020, 352 patients with STEMI who underwent primary PCI were recruited consecutively. Patients were divided into CI-AKI group (n = 71) and non-CI-AKI group (n = 281). CI-AKI was defined as an increase in serum creatinine (≥ 25% or ≥ 0.5 mg/dL) from baseline occurring 72 h after PCI. All subjects were tested for sd-LDL. RESULTS: In the 352 eligible patients with STEMI receiving emergency PCI, 71 patients (20.2%) developed CI-AKI. The levels of sd-LDL in CI-AKI group was higher than those in the non-CI-AKI group, and the difference was statistically significant (P < 0.05). The area under the curve (AUC) of the sd-LDL was 0.741 [95% confidence interval (CI) 0.538-0.636] in the STEMI patients receiving emergency PCI. CI-AKI model included the following five predictors: sd-LDL, NLR, Diabetes, Pre-PCI eGFR, and Log NT-proBNP. The AUC of forecast probability was 0.835 [95% confidence interval (CI) 0.786-0.883].The Hosmer-Lemeshow test has a P value of 0.519, which confirms the model's goodness of fit. CONCLUSION: Increased sd-LDL is independently associated with risk of CI-AKI in STEMI patients treated by primary PCI.

EPO protects mesenchymal stem cells from hyperglycaemic injury via activation of the Akt/FoxO3a pathway.

INTRODUCTION: Mesenchymal stem cell (MSC)-based therapies have demonstrated positive outcomes for treating cardiovascular disease. However, the proliferative ability of MSCs decreases during chronic exposure to hyperglycaemia; their ability to contribute to endogenous injury repair is thus reduced. Erythropoietin (EPO) was recently reported to protect against hyperglycaemia-related injury in various cells and may be a good candidate for enhancing MSC functions under hyperglycaemic conditions. METHODS: Bone marrow-derived MSCs were isolated from male donor rats weighing 60-80 g. The roles of EPO in regulating cell viability, senescence, angiogenesis and inflammation were investigated using the Cell Counting Kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assays; senescence-associated ß-galactosidase (SA-ß-gal) staining; VEGF, HGF, IGF, bFGF ELISAs and TNF-α ELISA, respectively. ROS production was measured by flow cytometry. The expression levels of Akt, forkhead box class O3a (FoxO3a) and VEGF proteins in MSCs were analysed by western blotting. Matrigel was used for tube formation assays. RESULTS: The results of the current study showed that EPO has beneficial effects on MSCs exposed to hyperglycaemia by promoting proliferation, inhibiting senescence and the release of pro-inflammatory factors, increasing the secretion of proangiogenic cytokines, and enhancing the ability of MSCs to stimulate tube formation among human umbilical vein endothelial cells (HUVECs). In addition, the beneficial effects of EPO may result from the activation of the Akt/FoxO3a signalling pathway. CONCLUSIONS: Our study demonstrates for the first time that EPO protects MSCs from hyperglycaemia-induced damage by targeting the Akt/FoxO3a signalling pathway.

Nicorandil modulated macrophages activation and polarization via NF-κb signaling pathway.

Nicorandil, a drug with both nitrate-like and ATP-sensitive potassium (KATP) channel-activating properties, has been well demonstrated in various aspects of myocardial infarction (MI), especially in inhibiting cell apoptosis and increasing coronary flow. However, the role of nicorandil in regulating inflammation and angiogenesis following myocardial infarction is still unrevealed. In the present study, we explored the effect of nicorandil on macrophage phenotype transition and inflammation regulation and the potential underlying mechanisms. For the phenotype transition and phagocytosis ability of macrophages detection, flow cytometry analysis was used. The inflammation factors were measured with ELISA and qRT-PCR. Western blot was used to assess the levels of NF-κb and its target genes and VEGF expression. The tube formation ability of endothelial cells was examined on matrigel. We discovered that nicorandil can obviously inhibit the differentiation of monocytes into mature macrophages and decrease M1 phenotype transition both in peritoneal macrophages and cultured macrophage cell line in normal or hypoxia and serum deprivation (H/SD) conditions. Meanwhile, nicorandil can induce an anti-inflammatory M2 phenotype. Thereby, nicorandil regulated macrophages switching to M1/M2 status. Our data further showed that NF-κb and the expression of its target genes were pivotal players in the regulation of macrophages phenotype. Besides, we also showed that nicorandil can promote the tube formation and VEGF expression in endothelial cells. We concluded that nicorandil may serve as an effective modulator of NF-κb signaling pathway during the pathogenesis of MI via regulating M1/M2 status and promoting angiogenesis.

Valsartan ameliorates KIR2.1 in rats with myocardial infarction via the NF-κB-miR-16 pathway.

BACKGROUND: MicroRNAs have an important role in regulating arrhythmogenesis. MicroRNA-16 (miR-16) is predicted to target KCNJ2. The regulation of miR-16 is primarily due to NF-κB. Whether valsartan could downregulate miR-16 via the inhibition of NF-κB after MI and whether miR-16 targets KCNJ2 remain unclear. METHODS: MI rats received valsartan or saline for 7days. The protein levels of NF-κB p65, inhibitor κBα (IκBα), and Kir2.1 were detected by Western blot analysis. The mRNA levels of Kir2.1 and miR-16 were examined by quantitative real-time PCR. Whole cell patch-clamp techniques were applied to record IK1. RESULTS: MiR-16 expression was higher in the infarct border, and was accompanied by a depressed IK1/KIR2.1 level. Additionally, miR-16 overexpression suppressed KCNJ2/KIR2.1 expression. In contrast, miR-16 inhibition or binding-site mutation enhanced KCNJ2/KIR2.1 expression, establishing KCNJ2 as a miR-16 target. In the MI rats, compared to saline treatment, valsartan reduced NF-κB p65 and miR-16 expression and increased IκBα and Kir2.1 expression. In vitro, angiotensin II increased miR-16 expression and valsartan inhibited it. Overexpressing miR-16 in cells treated with valsartan abrogated its beneficial effect on KCNJ2/Kir2.1. NF-κB activation directly upregulates miR-16 expression. CONCLUSIONS: miR-16 controls KCNJ2 expression, and valsartan ameliorates Kir2.1 after MI partly depending on the NF-κB-miR-16 pathway.

Atorvastatin attenuates sympathetic hyperinnervation together with the augmentation of M2 macrophages in rats postmyocardial infarction.

OBJECTIVES: Inflammation after myocardial infarction (MI) causes cardiac nerve sprouting and consequent ventricular arrhythmias (VAs). Macrophages, as major immune cells, are involved in the entire inflammation response process and serve as a link between inflammation and sympathetic hyperinnervation by regulating nerve growth factor (NGF) expression. Accumulating evidence shows that statins possess antiarrhythmogenic properties, and the aim of this study was to explore the mechanism by which atorvastatin ameliorates cardiac sympathetic nerve sprouting via regulating macrophage polarization. METHODS: Rat models of MI were created by ligating the left coronary artery. MI-operated rats received either atorvastatin or phosphate-buffered saline for 7 days. Immunohistochemical analyses and immunofluorescence staining were used to analyze macrophage infiltration after MI and to detect the distribution and density of growth-associated protein-43 (GAP-43) and tyrosine hydroxylase (TH) in nerve fibers in peri-infarct zones after MI. The polarity of the macrophages that were obtained from the rat peritoneal cavity was examined via flow cytometry. The protein levels of NGF were detected via Western blot analysis, and the concentrations of NGF in the supernatants were determined via enzyme-linked immunosorbent assay. The mRNA levels of NGF, inducible nitric oxide synthase (iNOS), Arginase-1 (Arg1), interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) were examined by quantitative real-time polymerase chain reaction. The association between VAs and MI was evaluated using programmed electrophysiological stimulation. RESULTS: Macrophages were successfully induced to the M1 (classically activated) and M2 (alternatively activated) phenotypes by stimulation with lipopolysaccharide and interferon-γ (LPS + IFN-γ) and interleukin-4 (IL-4), respectively. Atorvastatin markedly downregulated IL-1ß, TNF-α, iNOS, and NGF and upregulated Arg1, shifting the macrophage phenotype from M1 to M2. Moreover, atorvastatin significantly reduced TH levels and the density of GAP-43-positive nerve fibers and decreased inducible VAs. CONCLUSION: Atorvastatin effectively ameliorated cardiac sympathetic nerve remodeling and prevented VAs after MI by significantly downregulating the expression of NGF, IL-1ß, and TNF-α via together with the augmentation of M2 macrophage.

In rats the duration of diabetes influences its impact on cardiac autonomic innervations and electrophysiology.

Diabetic cardiac autonomic neuropathy (DCAN) may cause fatal ventricular arrhythmias and increase mortality in diabetics. However, limited data are available with regard to the precise changes in cardiac autonomic denervation after diabetes onset. In this study, we dynamically observed the progression of DCAN and its relationship with the inducibility of ventricular arrhythmias in diabetic rats. Rats were randomly divided into normal control and diabetes mellitus (DM) groups. The rats were sacrificed at 3 or 6 months post-treatment. Heart rate variability and programmed electrical stimulation were used to assess the electrophysiological characteristics and the inducibility of ventricular arrhythmias in the animals. Immunohistochemistry and real-time RT-PCR were used to measure choline acetyltransferase and tyrosine hydroxylase-positive nerve fibers and the corresponding mRNA expression levels in the proximal and distal regions of the left ventricle. Short-term diabetes resulted in distal myocardial parasympathetic denervation with sparing of the proximal myocardium. By 6 months, both parasympathetic and sympathetic denervation were further aggravated. Moreover, electrophysiological experiments demonstrated a sympatho-parasympathetic imbalance and an increase in ventricular arrhythmia inducibility in the diabetic rats. These results suggest that DM causes cardiac nerve denervation, relative sympathetic hyperinnervation and inhomogeneous neural innervations, which may be associated with an increase in the induction of ventricular arrhythmia in diabetic rats.