Jin-Jun Liu

Acetylcholine Mediates AMPK-Dependent Autophagic Cytoprotection in H9c2 Cells During Hypoxia/Reoxygenation Injury

Background: Acetylcholine (ACh), a neurotransmitter of vagal nerve, offers tolerance to ischemia/reperfusion injury. Given the regulation of autophagy in cardioprotection, this study was to examine the role of autophagy in ACh-elicited protection against hypoxia/reoxygenation (HR) injury. Methods: H9c2 cells were subjected to HR injury. Autophagy was determined by transmission electron microscopy, MDC staining and western blot. MTT kit, LDH and CK release, ATP content and TUNEL assay were used to evaluate cardiomyocytes injury. Atg7 and AMPK knockdown was performed with siRNA transfection. Results: Following 4, 8, 12 and 16 h reoxygenation, autophagosomes were decreased along with reduced cell viability. ACh during 4 h reoxygenation facilitated autophagy as evidence by increased autophagosomes and MDC labeling autophagic vacuoles. H9c2 cells treated with ACh also underwent a biochemical changes by increased ratio of LC3-II/LC3-I and autophagy flux (decreased p62), while muscarinic receptor antagonist atropine suppressed these effects. Induction of autophagy was correlated with enhanced cell survival and decreased apoptosis. Autophagy inhibition with chloroquine and Atg7 siRNA significantly attenuated ACh-induced cytoprotection. ACh-elicited autophagy activation could be related to increased AMPK phosphorylation and decreased mTOR phosphorylation. AMPK siRNA exhibited an elevation in mTOR phosphorylation and reduced the ratio of LC3-II/LC3-I. Importantly, AMPK knockdown desensitized H9c2 cells to ACh-mediated protection. Conclusions: These data provided first evidence that ACh-induced autophagy elicited cytoprotective effects through muscarinic receptor activated-AMPK-mTOR pathway, and suggested a novel mechanism of ACh-induced tolerance against HR injury.

Optimizing the Parameters of Vagus Nerve Stimulation by Uniform Design in Rats with Acute Myocardial Infarction

Vagus nerve stimulation (VNS) has been shown to improve left ventricular function and survival in rats with acute myocardial infarction (AMI), and this maneuver has also been adopted clinically for the treatment of patients with chronic heart failure (CHF). Recent in vitro and in vivo studies have suggested that VNS can modulate the level of pro-inflammatory factors. Despite the beneficial effects of VNS, the stimulation parameters for obtaining favorable outcomes appear highly variable. To optimize VNS parameters, we set up different stimulation protocols with different pulse width (1–2 ms), frequency (1–6 Hz), voltage (1–6 V) and duration (40–240 min) of VNS by uniform design (UD). Rats were divided into seven groups with (Group1–Group6) or without VNS (MI group). Our results demonstrate that (1) the parameter sets in Group1, Group2 and Group3 yield the best post-MI protection by VNS, while the protective role were not observed in Group4, Group5 and Group6; (2) baroreflex sensitivity and the α7 nicotinic acetylcholine receptor level were also increased in Group1, Group2 and Group3. (3) the parameter set in Group1 (G1:1 ms, 2 Hz, 3 V, 240 min) is judged the most optimal parameter in this study as rats in this group not only showed a reduced myocardial injury with better-preserved cardiac function compared with other groups, more important, but also exhibited minimal heart rate (HR) reduction. (4) the duration of VNS plays an important role in determining the protection effect of VNS. In conclusion, VNS displays a beneficial role in Group1, Group2 and Group3. Of note, the parameter set in Group1 provides the most optimal cardioprotective effect. These results may provide insight into development of novel treatment for ischemic heart diseases.

Exercise improves the dilatation function of mesenteric arteries in postmyocardial infarction rats via a PI3K/Akt/eNOS pathway-mediated mechanism

Myocardial infarction (MI) has been shown to induce endothelial dysfunction in peripheral resistance arteries and thus increase peripheral resistance. This study was designed to investigate the underlying mechanisms of post-MI-related dysfunctional dilatation of peripheral resistance arteries and, furthermore, to examine whether exercise may restore dysfunctional dilatation of peripheral resistance arteries. Adult male Sprague-Dawley rats were divided into three groups: sham-operated, MI, and MI + exercise. Ultrastructure and relaxation function of the mesenteric arteries, as well as phosphatidylinositol-3 kinase (PI3K), Akt kinases (Akt), endothelial nitric oxide synthase (eNOS) activity, and phosphorylation of PI3K, Akt, and eNOS by ACh were determined. Post-MI rats exhibited pronounced ultrastructural changes in mesenteric artery endothelial cells and endothelial dysfunction. In addition, the activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh were significantly attenuated in mesenteric arteries (P < 0.05-0.01). After 8 wk of exercise, not only did endothelial cells appeared more normal in structure, but also ameliorated post-MI-associated mesenteric arterial dysfunction, which were accompanied by elevated activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh (P < 0.05-0.01). Importantly, inhibition of either PI3K or eNOS attenuated exercise-induced restoration of the dilatation function and blocked PI3K, Akt, and eNOS phosphorylation by ACh in the mesenteric arteries. These data demonstrate that MI induces dysfunctional dilation of peripheral resistance arteries by degradation of endothelial structural integrity and attenuating PI3K-Akt-eNOS signaling. Exercise may restore dilatation function of peripheral resistance arteries by protecting endothelial structural integrity and increasing PI3K-Akt-eNOS signaling cascades.

Protection against Ischemia-Induced Oxidative Stress Conferred by Vagal Stimulation in the Rat Heart: Involvement of the AMPK-PKC Pathway

Reactive oxygen species (ROS) production is an important mechanism in myocardial ischemia and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is one of major sources of ROS in the heart. Previous studies showed that vagus nerve stimulation (VNS) is beneficial in treating ischemic heart diseases. However, the effect of VNS on ROS production remains elusive. In this study, we investigated the role of VNS onischemia-induced ROS production. Our results demonstrated that VNS alleviated the myocardial injury, attenuated the cardiac dysfunction, reserved the antioxidant enzyme activity and inhibited the formation of ROS as evidenced by the decreased NADPH oxidase (Nox) activity and superoxide fluorescence intensity as well as the expression of p67phox, Rac1 and nitrotyrosine. Furthermore, VNS resulted in the phosphorylation and activation of adenosine monophosphate activated protein kinase (AMPK), which in turn led to an inactivation of Nox by protein kinase C (PKC); however, the phenomena were repressed by the administration of a muscarinic antagonist atropine. Taken together, these data indicate that VNS decreases ROS via AMPK-PKC-Nox pathway; this may have potential importance for the treatment of ischemic heart diseases.

Acetylcholine Attenuates Hypoxia/ Reoxygenation-Induced Mitochondrial and Cytosolic ROS Formation in H9c2 Cells via M2 Acetylcholine Receptor

Background: The anti-infammatory and cardioprotective effect of acetylcholine (ACh) has been reported; nevertheless, whether and how ACh exhibits an antioxidant property against ischemia/reperfusion (I/R)-induced oxidative stress remains obscure. Methods: In the present study, H9c2 rat cardiomyocytes were exposed to hypoxia/reoxygenation (H/R) to mimic I/R injury. We estimated intracellular different sources of reactive oxygen species (ROS) by measuring mitochondrial ROS (mtROS), mitochondrial DNA (mtDNA) copy number, xanthine oxidase (XO) and NADPH oxidase (NOX) activity and expression of rac 1. Cell injury was determined by lactate dehydrogenase (LDH) release and cleaved caspase-3 expression. The siRNA transfection was performed to knockdown of M2 acetylcholine receptor (M2 AChR) expression. Results: 12-h hypoxia followed by 2-h reoxygenation resulted in an abrupt burst of ROS in H9c2 cells. Administration of ACh reduced the levels of ROS in a concentration-dependent manner. Compared to the H/R group, ACh decreased mtROS, recovered mtDNA copy number, diminished XO and NOX activity, rac 1 expression as well as cell injury. Co- treatment with atropine rather than hexamethonium abolished the antioxidant and cardioprotective effect of ACh. Moreover, knockdown of M2 AChR by siRNA showed the similar trends as atropine co-treatment group. Conclusions: ACh inhibits mitochondria-, XO- and NOX-derived ROS production thus protecting H9c2 cells against H/R-induced oxidative stress, and these benefcial effects are mainly mediated by M2 AChR. Our findings suggested that increasing ACh release could be a potential therapeutic strategy for treatment and prevention of I/R injury.

Tumour necrosis factor‐α and its receptors in the beneficial effects of vagal stimulation after myocardial infarction in rats

1. Acute myocardial infarction (AMI) often activates the sympathetic system and inhibits the vagal system. Long‐term vagal nerve stimulation (VNS) exerts several beneficial effects on the ischaemic heart, including an anti‐inflammatory effect. The aim of the present study was to investigate whether short‐term VNS during AMI could inhibit tumour necrosis factor (TNF)‐α expression and the effect of TNF receptor (TNFR), key components in inflammatory responses to AMI, in a rodent model.

Acetylcholine inhibits hypoxia‐induced tumor necrosis factor‐α production via regulation of MAPKs phosphorylation in cardiomyocytes

Recent findings have reported that up‐regulation of tumor necrosis factor‐alpha (TNF‐α) induced by myocardial hypoxia aggravates cardiomyocyte injury. Acetylcholine (ACh), the principle vagal neurotransmitter, protects cardiomyocytes against hypoxia by inhibiting apoptosis. However, it is still unclear whether ACh regulates TNF‐α production in cardiomyocytes after hypoxia. The concentration of extracellular TNF‐α was increased in a time‐dependent manner during hypoxia. Furthermore, ACh treatment also inhibited hypoxia‐induced TNF‐α mRNA and protein expression, caspase‐3 activation, cell death and the production of reactive oxygen species (ROS) in cardiomyocytes. ACh treatment prevented the hypoxia‐induced increase in p38 mitogen‐activated protein kinase (MAPK) and c‐Jun N‐terminal kinase (JNK) phosphorylation, and increased extracellular signal‐regulated kinase (ERK) phosphorylation. Co‐treatment with atropine, a non‐selective muscarinic acetylcholine receptor antagonist, or methoctramine, a selective type‐2 muscarinic acetylcholine (M2) receptor antagonist, abrogated the effects of ACh treatment in hypoxic cardiomyocytes. Co‐treatment with hexamethonium, a non‐selective nicotinic receptor antagonist, and methyllycaconitine, a selective alpha7‐nicotinic acetylcholine receptor antagonist, had no effect on ACh‐treated hypoxic cardiomyocytes. In conclusion, these results demonstrate that ACh activates the M2 receptor, leading to regulation of MAPKs phosphorylation and, subsequently, down‐regulation of TNF‐α production. We have identified a novel pathway by which ACh mediates cardioprotection against hypoxic injury in cardiomyocytes. J. Cell. Physiol. 226: 1052–1059, 2011.

Vagal nerve modulation: a promising new therapeutic approach for cardiovascular diseases.

1. The physiological activities of the mammalian heart are regulated by the autonomic nervous system. An imbalanced autonomic nervous system with increased sympathetic tone and reduced vagal tone has been implicated in cardiovascular diseases.

Acetylcholine Inhibits Tumor Necrosis Factor α Activated Endoplasmic Reticulum Apoptotic Pathway via EGFR‐PI3K Signaling in Cardiomyocytes

Previous findings have shown that acetylcholine (ACh) decreased hypoxia‐induced tumor necrosis factor alpha (TNF α) production, thus protected against cardiomyocyte injury. However, whether and how ACh affects TNF α‐induced endoplasmic reticulum (ER) stress and cell apoptosis remain poorly defined. This study was aimed at determining the effect of ACh in H9c2 cells after TNF α stimulation. Presence of ER stress was verified using the ER stress protein markers glucose regulatory protein 78 (GRP78) and C/EBP homologous protein (CHOP). Cell apoptosis was shown by caspase‐3 activation and terminal deoxynucleotidyl transferase mediated dUTP‐biotin nick end labeling. Exogenously administered ACh significantly decreased these TNF α‐induced changes. Moreover, when the cells were exposed to nonspecific muscarinic receptor (M AChR) inhibitor atropine, methoctramine (M2 AChR inhibitor) or the epidermal growth factor receptor (EGFR) inhibitor AG1478, the cardioprotection elicited by ACh was diminished. Furthermore, the above effects were also blocked by M2 AChR or EGFR siRNA, indicating that EGFR transactivation by M2 AChR may be the major pathway responsible for the benefits of ACh. In addition, LY294002, a phosphatidylinositol‐3‐kinase (PI3K) inhibitor, displayed the similar trends as AG1478, suggesting that PI3K/Akt signaling may be the downstream of EGFR in ACh‐elicited anti‐apoptotic property. Together, these data indicate that EGFR‐PI3K/Akt signaling is involved in M2 AChR‐mediated ER apoptotic pathway suppression and the subsequent survival of H9c2 cardiomyocytes. We have identified a novel pathway underlying the cardioprotection afforded by ACh. J. Cell. Physiol. 230: 767–774, 2015.

Improving vagal activity ameliorates cardiac fibrosis induced by angiotensin ii: in vivo and in vitro

Cardiac remodeling is characterized by overactivity of the renin–angiotensin system (RAS) and withdrawal of vagal activity. We hypothesized that improving vagal activity could attenuate cardiac fibrosis induced by angiotensin II (Ang II) in vivo and in vitro. Rats were subjected to abdominal aorta constriction (AAC) with or without pyridostigmine (PYR) (31 mg/kg/d). After 8 weeks, PYR significantly decreased Ang II level, AT1 protein expression, and collagen deposition in cardiac tissue and improved heart rate variability, baroreflex sensitivity and cardiac function, which were abolished by atropine. In vitro, treatment of cardiac fibroblasts (CFs) with Ang II (10−7 M) increased cell proliferation, migration, transformation, and secretory properties, which were significantly diminished by acetylcholine (ACh, 10−6 M). Subsequently, Ang II significantly increased collagen type I expression as well as metalloproteinase (MMP)-2 expression and activity. Transforming growth factor (TGF)-β1 expression and Smad3 phosphorylation presented a similar trend. Notably, the knockdown of the acetylcholine M2 receptor by siRNA could abolish ACh anti-fibrotic action. These data implicated cholinesterase inhibitor can increase vagal activity and reduce local Ang II level, and ACh inhibit Ang II pro-fibrotic effects. Our findings suggested that the parasympathetic nervous system can serve as a promising target for cardiac remodeling treatment.