Wenjuan Cheng

Targeted NGF siRNA Delivery Attenuates Sympathetic Nerve Sprouting and Deteriorates Cardiac Dysfunction in Rats with Myocardial Infarction

Nerve growth factor (NGF) is involved in nerve sprouting, hyper-innervation, angiogenesis, anti-apoptosis, and preservation of cardiac function after myocardial infarction (MI). Positively modulating NGF expression may represent a novel pharmacological strategy to improve post-infarction prognosis. In this study, lentivirus encoding NGF short interfering RNA (siRNA) was prepared, and MI was modeled in the rat using left anterior descending coronary artery ligation. Rats were randomly grouped to receive intramyocardial injection of lentiviral solution containing NGF-siRNA (n = 19, MI-SiNGF group), lentiviral solution containing empty vector (n = 18, MI-GFP group) or 0.9% NaCl solution (n = 18, MI-control group), or to receive thoracotomy and pericardiotomy (n = 17, sham-operated group). At 1, 2, 4, and 8 wk after transduction, rats in the MI-control group had higher levels of NGF mRNA and protein than those in the sham-operated group, rats in the MI-GFP group showed similar levels as the MI-control group, and rats in the MI-SiNGF group had lower levels compared to the MI-GFP group, indicating that MI model was successfully established and NGF siRNA effectively inhibited the expression of NGF. At 8 wk, echocardiographic and hemodynamic studies revealed a more severe cardiac dysfunction in the MI-siRNA group compared to the MI-GFP group. Moreover, rats in the MI-siRNA group had lower mRNA and protein expression levels of tyrosine hydroxylase (TH) and growth-associated protein 43-positive nerve fibers (GAP-43) at both the infarcted border and within the non-infarcted left ventricles (LV). NGF silencing also reduced the vascular endothelial growth factor (VEGF) expression and decreased the arteriolar and capillary densities at the infarcted border compared to the MI-GFP group. Histological analysis indicated a large infarcted size in the MI-SiNGF group. These findings suggested that endogenous NGF silencing attenuated sympathetic nerve sprouting and angiogenesis, enlarged the infarct size, aggravated cardiac dysfunction, and potentially contributed to an unfavorable prognosis after MI.

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.

Effects of Atrioventricular Nodal Ablation on Permanent Atrial Fibrillation Patients With Cardiac Resynchronization Therapy: A Systematic Review and Meta-analysis

Cardiac resynchronization therapy (CRT) is a well‐established therapy for patients with heart failure (HF) and wide QRS configuration, especially for those in sinus rhythm. However, for those with permanent AF, atrioventricular nodal (AVN) ablation use remains under debate. Our objective was to evaluate clinical outcomes and mortality of AVN ablation in HF patients with permanent AF receiving CRT. Electronic publication database and reference lists through October 1, 2013 were searched. Observational cohort studies comparing CRT patients with AF who received either AVN ablation or medical therapy were selected. Outcomes included mortality, CRT nonresponse, changes in left ventricular remodeling, and functional outcomes, such as New York Heart Association (NYHA) functional class, quality of life, and 6‐minute hall walk distance. Of 1641 reports identified, 13 studies with 1256 patients were included. Among patients with permanent AF and insufficient biventricular pacing (<90%), those who had undergone AVN ablation compared to those who did not had numerically lower all‐cause mortality (risk ratio [RR]: 0.63, 95% confidence interval [CI]: 0.42 to 0.96, P = 0.03) and significantly lower nonresponse to CRT (RR: 0.41, 95% CI: 0.31 to 0.54, P < 0.00001). Furthermore, AVN ablation was not associated with additional improvements on left ventricular ejection fraction, NYHA functional class, 6‐minute hall walking distance, and quality of life. In patients with permanent AF undergoing CRT, AVN ablation tended to reduce mortality potentially and improved clinical response when it was applied to patients with inadequate biventricular pacing (<90%). Randomized controlled trials are needed to further address the efficacy of AVN ablation among this population.

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.

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-1β 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.

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 κBa (IκBa) and nerve growth factor (NGF) were detected by Western blot analysis. The mRNA levels of NGF, interleukin-1ß (IL-1ß) and tumor necrosis factor-a (TNF-a) 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 IκBa level. Moreover, metoprolol downregulated IL-1ß, TNF-a 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.

Myocardial infarction induces sympathetic hyperinnervation via a nuclear factor-κB-dependent pathway in rabbit hearts.

Cardiac sympathetic hyperinnervation after myocardial infarction (MI) is associated with a high incidence of lethal arrhythmia. However, the mechanisms of nerve sprouting induced by MI are unclear. In this study, we showed a nuclear factor-κB (NF-κB) signaling pathway involved in cardiac sympathetic hyperinnervation after MI in rabbit hearts. An MI model was induced by ligation of the coronary artery in rabbits, which were then euthanized after 7 days. Rabbits with MI showed sympathetic hyperinnervation, as revealed by immunohistochemical analysis of the density of nerve fibers positive for growth-associated protein 43 (GAP43) and tyrosine hydroxylase (TH). Using western blot and real-time RT-PCR techniques, we found that MI was associated with activation of NF-κB signaling and consequent upregulation of nerve growth factor. Intravenous administration with the NF-κB inhibitor pyrrolidine dithiocarbamate (100mg/kg/day) inhibited NF-κB activation and ameliorated sympathetic hyperinnervation after MI. These results suggest that cardiac nerve sprouting after MI is associated in part with NF-κB activation and may be one of the mechanisms responsible for sympathetic hyperinnervation induced by MI.

Mechanisms of atypical flutter wave morphology in patients with isthmus-dependent atrial flutter

Sixty-three episodes of isthmus-dependent atrial flutter (AFL) in 55 patients were studied to characterize variations in flutter wave morphology and to investigate the mechanisms of the atypical flutter waves on surface ECG. The activation patterns of coronary sinus (CS) and their relationship with flutter wave morphology on the ECG were analyzed. In 46 episodes of counterclockwise AFL (CCW-AFL), there were four types of flutter waves on ECG. Typical and atypical flutter waves were found in 47.8% and 13.0% of the episodes, respectively. Atypical flutter waves had broad positive terminal portion or entirely positive wave in the inferior leads and in V1, with a distal-to-proximal or fused activation pattern in the CS, and an average activation time of 21.3 ± 11.4 ms. In 17 episodes of clockwise AFL (CW-AFL), typical and atypical flutter waves were identified in 41.2% and 41.2%, respectively. Atypical flutter waves had negative waves in the inferior ECG leads and in V1, a proximal-to-distal activation pattern in the CS, and an average activation time of 42.4 ± 14.4 ms. We conclude that atypical flutter waves are common in the isthmus-dependent AFL. The clockwise or counterclockwise conduction in the right atrium, and the activation patterns or conduction sequences between the right and the left atrium, are associated with the variations in the flutter wave morphology on body surface ECG.

P2X7 receptor inhibition attenuated sympathetic nerve sprouting after myocardial infarction via the NLRP3/IL‐1β pathway

Mounting evidence supports the hypothesis that inflammation modulates sympathetic sprouting after myocardial infarction (MI). The myeloid P2X7 signal has been shown to activate the nucleotide‐binding and oligomerization domain‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome, a master regulator of inflammation. We investigated whether P2X7 signal participated in the pathogenesis of sympathetic reinnervation after MI, and whether NLRP3/interleukin‐1β (IL‐1β) axis is involved in the process. We explored the relationship between P2X7 receptor (P2X7R) and IL‐1β in the heart tissue of lipopolysaccharide (LPS)‐primed naive rats. 3′‐O‐(4‐benzoyl) benzoyl adenosine 5′‐triphosphate (BzATP), a P2X7R agonist, induced caspase‐1 activation and mature IL‐1β release, which was further neutralized by a NLRP3 inhibitor (16673‐34‐0). MI was induced by coronary artery ligation. Following infarction, a marked increase in P2X7R was localized within infiltrated macrophages and observed in parallel with an up‐regulation of NLRP3 inflammasome levels and the release of IL‐1β in the left ventricle. The administration of A‐740003 (a P2X7R antagonist) significantly prevented the NLRP3/IL‐1β increase. A‐740003 and/or Anakinra (an IL‐1 receptor antagonist) significantly reduced macrophage infiltration as well as macrophage‐based IL‐1β and NGF (nerve growth factor) production and eventually blunted sympathetic hyperinnervation, as assessed by the immunofluorescence of tyrosine hydroxylase (TH) and growth‐associated protein 43 (GAP 43). Moreover, the use of Anakinra partly attenuated sympathetic sprouting. This indicated that the effect of P2X7 on neural remodelling was mediated at least partially by IL‐1β. The arrhythmia score of programmed electric stimulation was in accordance with the degree of sympathetic hyperinnervation. In vitro studies showed that BzATP up‐regulated secretion of nerve growth factor (NGF) in M1 macrophages via IL‐1β. Together, these data indicate that P2X7R contributes to neural and cardiac remodelling, at least partly mediated by NLRP3/IL‐1β axis. Therapeutic interventions targeting P2X7 signal may be a novel approach to ameliorate arrhythmia following MI.

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.