Sympathetic nerve hyperactivity is a primary reason for fatal ventricular arrhythmias (VAs) following myocardial infarction (MI). Pro-inflammatory cytokines produced in the paraventricular nucleus (PVN) post-MI are associated with sympathetic overexcitation; however, the precise mechanism needs further investigation. Our aim was to explore the mechanism of toll-like receptor 4 (TLR4) and its downstream molecular pathway in mediating sympathetic activity post-MI within the PVN. A rat MI model was developed via left anterior descending coronary artery ligation. TLR4 was primarily localized in microglia and increased markedly within the PVN at 3 days in MI rats. Sympathoexcitation also increased, as indicated by high levels of renal sympathetic nerve activity (RSNA) and norepinephrine (NE) concentration. TLR4 knockdown via shRNA microinjection to the PVN resulted in decreased activation of Fos protein (+) neurons in the PVN and peripheral sympathetic nerve activity. TLR4 knockdown also exhibited a lower arrhythmia score following programmed electrical stimulation than those treated with MI surgery only, indicating that the knockdown of TLR4 decreased the incidence of malignant ventricular arrhythmias following MI. LPS-induced inflammatory response was analyzed to explore the underlying mechanism of TLR4 in sympathetic hyperactivity. High levels of NF-κB protein, the pro-inflammatory cytokines IL-1β and TNF-α, and ROS production were observed in the LPS group. PVN-targeted injection of the NF-κB inhibitor PDTC attenuated NF-κB expression and sympathetic activity. Taken together, the results suggested that knockdown of microglial TLR4 within the PVN decreased sympathetic hyperactivity and subsequent VAs post-MI. The downstream NF-κB pathway and ROS production participated in the process. Interventions targeting TLR4 signaling in the PVN may be a novel approach to ameliorate the incidence of VAs post-MI.

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.

Malignant ventricular arrhythmias (VAs) following myocardial infarction (MI) is a lethal complication resulting from sympathetic nerve hyperactivity. Numerous evidence have shown that inflammation within the paraventricular nucleus (PVN) participates in sympathetic hyperactivity. Our aim was to explore the role of Macrophage‐inducible C‐type lectin (Mincle) within the PVN in augmenting sympathetic activity following MI,and whether NOD‐like receptor family pyrin domain‐containing 3 (NLRP3) inflammasome/IL‐1β axis is involved in this activity. MI was induced by coronary artery ligation. Mincle expression localized in microglia within the PVN was markedly increased at 24 hours post‐MI together with sympathetic hyperactivity, as indicated by measurement of the renal sympathetic nerve activity (RSNA) and norepinephrine (NE) concentration. Mincle‐specific siRNA was administrated locally to the PVN, which consequently decreased microglial activation and sympathetic nerve activity. The MI rats exhibited a higher arrhythmia score after programmed electric stimulation than that treated with Mincle siRNA, suggesting that the inhibition of Mincle attenuated foetal ventricular arrhythmias post‐MI. The underlying mechanism of Mincle in sympathetic hyperactivity was investigated in lipopolysaccharide (LPS)‐primed naïve rats. Recombinant Sin3A‐associated protein 130kD (rSAP130), an endogenous ligand for Mincle, induced high levels of NLRP3 and mature IL‐1β protein. PVN‐targeted injection of NLRP3 siRNA or IL‐1β antagonist gevokizumab attenuated sympathetic hyperactivity. Together, the data indicated that the knockdown of Mincle in microglia within the PVN prevents VAs by attenuating sympathetic hyperactivity and ventricular susceptibility, in part by inhibiting its downstream NLRP3/IL‐1β axis following MI. Therapeutic interventions targeting Mincle signalling pathway could constitute a novel approach for preventing infarction injury.

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.

Serine and thiol proteases react with peptide substrates to form an acyl-enzyme. We have synthesized inhibitors which are pseudo-substrates and react with the proteases to generate acyl-enzymes which hydrolize slowly. This is achieved by incorporating an electron-donating group near the carbonyl group of inhibitors I [Ac-Phe--C(O)NH--NH--C(O)X] and II [benzyl-O-C(O)-psiAla-Leu-ArgOMe]. The acyl-enzymes derived from the reaction of I with papain and II with chymotrypsin hydrolyze with t1/2 of 12 and 1 h, respectively. The increased electron density on the carbonyl group of the inhibitor also reduces the rate of acyl-enzyme formation. Components were incorporated into the inhibitor which interact with the leaving group binding site (S' subsite) and which accelerate the rate of reaction of inhibitor with enzyme. For inhibitor I, X = NH(CH3), k(on) < 0.13 M(-1) s(-1) for the reaction papain, but if X = psiLeu(CH3)2,k(on) =10(5) M(-1) s(-1). Similar results were obtained with II and chymotrypsin. Concomitant with acyl-enzyme formation, X is released and a slowly hydrolyzing acyl-enzyme remains.

BackgroundPulmonary arterial hypertension (PAH) is a devastating disease that lacks sufficient treatment. Studies have shown that the Nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome contributes to PAH pathogenesis, but the role of the upstream molecular P2X7 receptor (P2X7R) has remained unexplored. We investigated the role of P2X7R in the pathogenesis of PAH.Methods and resultsPH was induced by a single subcutaneous injection of monocrotaline (MCT) (60 mg/kg) on left pneumonectomised Sprague-Dawley rats, as validated by significant increases in pulmonary artery pressure and vessel wall thickness. Marked P2X7R was detected by predominant PA immunostaining in lungs from PH rats. Western blot revealed a significant increase in the protein levels of P2X7R as well as NLRP3 and caspase-1 in the diseased lung tissue compared with normal tissue. The rats received A-740003 (a selective P2X7 receptor antagonist, 30 mg/kg) daily starting from 1 week before or 2 weeks after MCT injection. Consequently, A-740003 reversed the NLRP3 inflammasome upregulation, significantly decreased the mean right ventricular (RV) pressure and RV hypertrophy, and reversed pulmonary arterial remodelling 4 weeks after MCT injection, as both a pretreatment and rescue intervention. Notably, A-740003 significantly reduced macrophage and pro-inflammatory cytokine levels, as measured via bronchoalveolar lavage. The recruitment of macrophages as well as collagen fibre deposition in the perivascular areas were also reduced, as confirmed by histological staining.ConclusionsP2X7R contributes to the pathogenesis of PH, probably in association with activation of the NLRP3 inflammasome. Blockade of P2X7R might be applied as a novel therapeutic approach for the treatment of PAH.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-017-0603-0) contains supplementary material, which is available to authorized users.

Background Pulsed electromagnetic fields (PEMF) have been shown to accelerate proliferation and regeneration to the damaged tissue, but its mechanism remains unclear. Here, we examine whether PEMF therapy improves postnatal neovascularization using murine model of hindlimb ischemia, and investigate the underlying cellular/molecular mechanisms. Methods and Results Hindlimb ischemia was induced by unilateral femoral artery resection using 6–8 week-old male C57BL6 mice. PEMF therapy (8 min/cycle, 30 ± 3 Hz, 5 mT) was then performed 4 cycles/day. At postoperative day 21, accelerated wound healing, decreased prevalence of gangrene and increased capillary density were observed among the PEMF-exposed mice. Moreover, the levels of vascular endothelial growth factor (VEGF) expression, endothelial nitric oxide synthase (eNOS) and Akt phosphorylation contained in the ischaemic muscles were increased in response to PEMF therapy. In vitro , PEMF interrupted the process of hypoxia-induced apoptosis and contributed to the tube-formative and proliferative capacities of human umbilical vein endothelial cells. PEMF exposure increased the level of VEGF in the conditioned media, as well as the eNOS and Akt phosphorylation in the cell lysate, and these PEMF-mediated angiogenic benefits could be blocked after PI3K inhibitor was administrated. Conclusions In conclusion, Our data indicated that PEMF enhanced ischemia-mediated angiogenesis, through up-regulating VEGF expression and activating the PI3K-Akt-eNOS pathway. Therefore, PEMF may be valuable for the treatment of the patients with critical limb ischemia.

Aim:Overactivation of the sympathetic nerve may lead to severe ventricular arrhythmias (VAs) after myocardial infarction (MI). Thus, targeting sympathetic nerve activity is an effective strategy to prevent VAs clinically. The superior cervical ganglion (SCG), the extracardiac sympathetic ganglion innervating cardiac muscles, has been found to have a GABAergic signalling system, the physiological significance of which is obscure. We aimed to explore the functional significance of SCG post MI and whether the GABAergic signal system is involved in the process. Methods: Adult male Sprague-Dawley rats were divided into seven different groups. Rats in the MI groups underwent ligation of the left anterior descending coronary artery. All animals were used for electrophysiological testing, renal sympathetic nerve activity (RSNA) testing, and ELISA. Primary SCG sympathetic neurons were used for the in vitro study. Results: The GABA A receptor agonist muscimol significantly decreased the ATPinduced increase in intracellular Ca 2+ (P < 0.05). GABA treatment in MI rats significantly attenuated the level of serum and cardiac norepinephrine (NE; P < 0.05).Sympathetic activity and inducible VAs were also lower in MI + GABA rats than in MI rats (P < 0.05). Knockdown of the GABA A Rs β 2 subunit (GABA A Rβ 2 ) in the SCG of MI rats increased the NE levels in serum and cardiac tissue, RSNA and inducible VAs compared with vehicle shRNA (P < 0.05). Conclusion: The GABAergic signalling system is functionally expressed in SCG sympathetic neurons, and activation of this system suppresses sympathetic activity, thereby facilitating cardiac protection and making it a potential target to alleviate VAs. K E Y W O R D Sarrhythmias, GABA, myocardial infarction, superior cervical ganglia, sympathetic nerve activity 2 of 14 | SHI et al.