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

Abstract 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.


| INTRODUC TI ON
In recent years, with the development of new therapies, the mortality of acute myocardial infarction (MI) is decreasing, but it is still a major cause of disease-related death globally. 1 MI patients may ultimately develop ischaemic cardiomyopathy (ICM), whose 5-year survival rate only 40%-50%. Ventricular remodelling during ICM is a continuous, diverse and complex process, contributes to a deterioration of heart function and poor prognosis. Persistent and chronic inflammation plays an important role in the process. 2 Our past studies have demonstrated that the cholinergic antiinflammatory pathway (CAP) has a protective role against fatal ventricular arrhythmias. 3 The CAP, excited by α7 nicotinic acetylcholine receptors (α7-nAChR), is part of a highly conserved endogenous mechanism regulates the magnitude of inflammatory responses and benefits a wide range of inflammatory diseases. 4 Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation, suppresses the production of proinflammatory cytokines, 5 and natural killer cells are less efficient to trigger dendritic cells (DC) maturation under the activated-CAP. 6 Eliciting α7-nAChR suppresses the inflammatory response in autoimmune encephalomyelitis, 7 autoimmune myocarditis 8 and the inflammation mediated by microglia in global ischaemia rats. 9 Recent years, many researchers suggest that inflammation disrupts energy metabolism, wherein cytokines impede a range of metabolic pathways and cause further damage. [10][11][12] Adenosine monophosphate-activated protein kinase (AMPK) consists of three subunits, α, β and γ, which together form a functional enzyme that works as an energy sensor to provide metabolic adaptations. The activation of AMPK appears to have a protective effect on many heart diseases by adjusting energy metabolism and the immune system, 13 such as attenuating the development of atherosclerosis by reducing Drp1-mediated mitochondrial fission. 14 And Krawczyk et al implicated AMPK as an inhibitor of DC activation that modulated inflammation response. 15 The CAP and AMPK signalling both contribute a protective role in many involve conditions, especially in anti-inflammation. Thus, we hypothesized that the protective effects of the CAP are associated with AMPK signalling in ICM and aimed to investigate this hypothesis. and photoperiod (12-hour light/dark cycle). Food and water were supplied by Wenzhou Medical University Animal Facilities.

| ICM model
We made the model described by Jayasankar et al 16

| Echocardiography
Transthoracic echocardiography with an M-mode transducer (12-MHz phased-array transducer; Sonos 5500, Philips USA, Bothell, WA) was performed by an experienced technician who was uninformed of the research groups. At the papillary muscle level, short-axis views of M-mode tracings were recorded through the anterior and posterior LV walls to measure LV end-diastolic dimension (LVEDd), LV end-systolic dimension (LVESd) and LV fraction shortening (LVFS). LVEF values were obtained according to the Simpson approach.

| Electrocardiograph, left ventricular pressure and ventricular programmed electric stimulation
Rats were anaesthetized under isoflurane and an electrical physiology system (PowerLab 8/36; AD Instruments, Colorado Springs, CO) based on a computer was applied to measure the electrocardiograph (ECG) and pressure. The right carotid artery was isolated and pressure transducers were introduced into the left ventricle to measure heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), LV systolic pressure (LVSP) and LV end-diastolic pressure (LVEDP). Biopotential leads were immobilized subcutaneously in the bottom left portion of the chest wall and the right pectoral muscle.
LabChart 8 software (AD Instruments) was used to record the RR, PR, QRS and QT duration data. The Bazett formula was used in the calibration of the QTc for the heart rate. After ECG measurements, a thoracotomy was carried out on the rats to expose the heart. Two stimulating electrodes (made with silver metal with a diameter under 0.2 mm, except for the two contact points and covered by Teflon for insulation) were gently stuck into the heart and placed in contact with the apex of heart. The programmable-stimulators were allowed to produce the pacing using PowerLab 8/36. The programmed electric stimulation (PES) protocol and scoring table were developed as described by Bélichard et al. 18 Under six beats with a self-terminating ventricular tachyarrhythmia was considered 'non-inducible'. Ventricular tachyarrhythmia lasted >15 beats was 'sustained group', while a ventricular tachyarrhythmia that lasted ≤15 beats was 'non-sustained group'. The modes of PES ( Figure 1E) were: S 0 consisted of 20 pace beats at a basic cycle length of 100 ms; S 1 consisted of a premature stimulus that was applied to identify the effective refractory period after eight paced beats that were uniformly distributed at a basic cycle length of 100 ms; S 2 , S 3 and S 4 consisted of S 1 with 1×, 2× and 3× extra stimuli.

| The extent of fibrosis
The middle 1/3 of the left ventricle (the LV middle ring) was embedded in paraffin, sectioned into 5 μm-thick slices and the Masson's trichrome-stained dyeing method was applied. The degree of fibrosis was expressed as the mean percentage of the total border zone and the protein expression level of collagen I and III.

| Transmission electron microscopy
Fresh tissue pieces (approximately 1 mm × 1 mm × 1 mm) from the mid-myocardium of middle 1/3 of the LV of the infarct border zone were fixed in glutaraldehyde and osmium tetroxide, embedded in epoxy resin. An experienced technician who was ignorant about the groups processed the treated samples into ultrathin sections and made all measurements.

| Immunocytochemistry
Fresh tissue samples embedded in OCT Compound (−30°C) were sectioned into 5 μm-thick slices. Thawed slides were fixed in 4°C acetones for 10 minutes. Cell samples were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.5% Triton X-100 for 10 minutes. Slides were washed in 1× PBS three times after each step.
Sufficient reagent was used to cover the specimen and the reagents were removed after each step using suction. The specimens were first incubated with 10% blocking serum in PBS at room temperature for 30 minutes and then washed off with PBS for 5 minutes each. Next, the specimens were incubated with primary antibodies overnight at 4°C, and then the antibodies were washed off with PBS for 5 minutes each. The secondary antibodies were then added and the specimens were incubated at room temperature for 60 minutes.

| Western blotting
Protein was extracted from the treated primary macrophages and the marginal area around the infarct area. RIPA lysate (ThermoFisher, 89900), protease inhibitors (Beyotime, Jiangsu, China, ST506-2) and phosphatase inhibitors (Applygen Technologies, Beijing, China, P1260) were added in to lyse the cells or tissues. A BCA protein assay kit (ThermoFisher, 23235) was used to measure the protein concentration. Then the protein was mixed with double distilled H 2 O and 5× loading buffer to a final volume ratio of loading buffer to sample of 1:5 to finally get the mixture sample. The mixture samples should have the same protein concentration, and the protein should not higher than 80% volume of homologous mixture sample.
After that, boiled the mixture at 100°C for 10 minutes. Next, each sample was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membranes were blocked in 5% milk (Skim Milk; BD, Difco TM ). 1× mixture of Tris-buffered saline and Polysorbate 20 was applied to wash the membranes (5 mintues, three times). After being incubated in specific primary (12 hours, 4°C) and secondary antibodies (1-hour, room temperature), the amount of protein on the membranes were measured by enhanced

| Suppressing AMPK signalling blunted the improvement of cardiac function and PES-induced ventricular tachyarrhythmia elicited by the CAP
In Figure 1,

| Suppressing AMPK signalling blunted the decrease in fibrosis and proinflammatory cytokine production elicited by the CAP in the infarcted border zone
The area of fibrosis in the infarcted border zone was shown by Masson's trichrome-staining, and the collagen synthesis/release and proinflammatory cytokine production were displayed by western blotting ( Figure 2). Compared to the Sham group, the area of fibrosis in the ICM group was significantly increased (ICM group vs the Sham group, P < 0.05), as well as the synthesis/release of collagen (ICM group vs the Sham group, Collagen I/GAPDH, P < 0.05; Collagen III/ GAPDH, P < 0.05) and the proinflammatory cytokine production (ICM group vs the Sham group, p-NF-κB p65/NF-κB p65, P < 0.05; TNFα, P < 0.05; IL-6, P < 0.05; IL-1β, P < 0.05). PNU treatment improved collagen synthesis/release, decreased proinflammatory cytokine production and decreased the area of fibrous (all compared to the ICM group, P < 0.05). The modified effects of PNU treatment were inhibited by BML (all compared to the ICM + PNU group, P < 0.05).

| Suppressing AMPK signalling blunted the preservation of Connexin-43 (Cx-43) elicited by the CAP
In Figure

| Suppressing AMPK signalling blunted the effect of the CAP on inflammatory and reparative macrophages
Ly-6C high and Ly-6C low macrophages are two subsets of macrophages.
The Ly-6C high macrophages are inflammatory macrophages that ap-

| D ISCUSS I ON
In this study, we chose a specific α7-nAChR agonist to investigate how eliciting the CAP would improve the cardiac function of ICM and to understand the potential mechanism behind it. We found that  un-phosphorylated STAT3 could interfere with the LPS-induced proinflammatory response via binding of NF-κB, prevents the activation of NF-κB. 48,49 The activation of AMPKα have an inhibitory effect on STAT3. 50 Our experimental data support the findings above.
In addition, α7-nAChR agonists can up-regulate the receptor expression, 51 We think this may be a mechanism of feedback. The neurovirulence of oxaliplatin decreased the expression of the alpha7 receptor, and the PNU increased the level of the receptor. In our preliminary experiment the alpha7 receptor expression of ICM rats was increased, and the treatment with PNU restored the normal level of the receptor (see supplements, Figure S3). These two opposite effects are both for regulating the abnormal expression of the alpha7 receptor in the pathological condition. They are protective effects and a mechanism of feedback. Furthermore, evidence indicates that α7-nAChR can desensitize rapidly in response to high agonist concentrations in vitro, which may preclude us from observing the effect of receptor activation. 52,53 In the normal neuromuscular junction, the delivery and removal of acetylcholine is very rapid so that desensitization is usually not considered to be important. But the process of desensitization is complicated and still worth for further researches. Study the desensitization will lead us to the best dosage of the agonist for most beneficial effect.

| CON CLUS IONS
In this study, we demonstrated that eliciting the CAP can exert protective effects in cardiac ischaemia and ICM-induced heart failure and ventricular arrhythmia via activation of AMPK signalling. The CAP could be a potential therapeutic for ICM-induced heart failure and an alternative therapeutic strategy for ICM-induced ventricular arrhythmia.

ACK N OWLED G EM ENTS
LZH and LJF designed and drafted the original research. LZH, WSJ, ZC, LYZ, LH, LWQ performed the experiments. All authors approved it for publication.

D I SCLOS U R E S
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

R E FE R E N C E S
1. Lozano R, Naghavi M, Foreman K, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: