Selective Ablation Of The Ligament Of Marshall Attenuates Myocardial Ischemia Reperfusion-Induced Ventricular Arrhythmias And Injury And Its Underlying Mechanisms

Background:Reperfusion therapy is the most effective treatment for acute myocardial infarction（AMI）,such as thrombolysis,percutaneous coronary intervention（PCI）,and coronary artery bypass grafting.However,reperfusion arrhythmias and reperfusion injury（RI）,including myocardial apoptosis and necrosis,are important factors limiting the efficacy of reperfusion therapy or even lead to death.Therefore,how to prevent and treat ischemia and reperfusion（IR）arrhythmias and injury has become one of the important tasks in the treatment of patients with acute myocardial infarction..Cardiac sympathetic overactivation is a main cause for ventricular arrhythmia.Cardiac sympathetic denervation,such as left stellate ganglion（LSG）ablation and renal sympathetic nerve ablation,can significantly reduce the prevalence of myocardial infarction-induced ventricular arrhythmia（VAs）and the risk of sudden cardiac death.At the same time,a large number of studies have confirmed that sympathetic nerves can regulate tissue inflammatory response and oxidative stress levels,and inhibition of sympathetic overactivation can significantly reduce the inflammatory response and oxidative stress levels in patients with myocardial infarction and hypertension.Inflammation and oxidative stress are the classic mechanisms of myocardial IR injury.Activation of them further induce cascades of damage mechanisms such as excessive autophagy,myocardial apoptosis,and calcium overload in cardiomyocytes.Therefore,the present study intends to explore the effects of cardiac sympathetic denervation on ventricular arrhythmias and myocardial injury in myocardial IR,and explore whether inhibition of sympathetic overactivation can regulate IR inflammatory response,oxidative stress,autophagy and calcium overload.This would be helpful to clarify the mechanism of myocardial ischemia reperfusion injury,and will give a new sight to prevent the myocardial ischemia reperfusion injury.Methods:IR induced ventricular arrhythmia in canines:34 adult male beagle dogs（8-13 kg）were randomly divided into IR group（n=11）,LOM distal portion denervation group(LOM_LSPV denervation,LOM_LSPVD,n=9)and left stellate ganglion denervation group（LSGD,n=13）.LOM_LSPV was stained with hematoxylin-eosin（HE）,Tyrosine hydroxylase（TH）staining and Acetylcholine transferase（CHAT）.The basic state of each group and the heart rate variability（HRV）after ablation were recorded using LabChart（ADinstruments,Shanghai,China）,including low frequency（LF）（0.04-0.15）,high frequency（HF）（0.15-0.5）and LF/LF.Lead 7000（Sichuan,Jinjiang）was used to record VAs,including Ventricular Premature Beat（VPB）,paired room early（salvo of VPB,s-VPB）,ventricular tachycardia（Ventricular Tachycardia,VT）,ventricular Tachycardia Duration（VTD）and Ventricular Fibrillation（VF）.Elisa assay was used to detect serum levels of epinephrine（E）and noradrenaline（NE）.IR injury in canines:part I:Thirty-four male beagle canines were randomly divided into three groups:Sham operated group（SO,n=6）,Ischemia and reperfusion group（IR,n=15）,and LOM_LSPVD+IR group（n=13）.Sympathetic tone（heart rate variability（HRV）,interstitial noradrenaline（iNE）levels）,cardiac injury markers including serum creatine kinase（CK-MB）,lactate dehydrogenase（LDH）,and cardiac Troponin I（cTnI）levels,myocardial infarct size,cardiac inflammatory cytokines including high mobility group box 1（HMGB1）,interleukin-17A（IL-17A）,and IL-1β,cardiac autophagy level（beclin-1 levels and LC3-II/I）]were detected.Part II:twenty-three male hybrid canines were randomly divided into three groups:Sham operated group（SO,n=6）,Ischemia and reperfusion group（IR,n=9）,and LOM_LSPVD+IR group（n=8）,cardiac injury markers（cTnI and CK-MB）,iNE,myocardial oxidative stress levels[superoxide dismutase（SOD）,malondialdehyde（MDA）],calcium transporter[Ryanodine receptor 2（RyR2）,calcium Phosphorylated L-type-dependent protein kinase II（P-CaMKII）and sodium/calcium exchanger（NCX）],myocardial apoptosis[cleaved caspase-3,B cell lymphoma 2（Bcl-2）,Bcl-2 associated X protein（Bax）and TUNEL]were detected.Results:1.IR induced ventricular arrhythmia in canines:Hematoxylin-Eosin（HE）and Immunohistochemistry staining revealed that LOM_LSPV contained bundles of sympathetic but not parasympathetic nerves.IR increased the cardiac sympathetic tone[serum concentrations of noradrenaline（NE）and epinephrine（E）]and induced the prevalence of VAs[ventricular premature beat（VPB）,salvo of VPB,ventricular tachycardia（VT）,VT duration（VTD）and ventricular fibrillation（VF）].Both LOM_LSPVSPV denervation and LSG denervation could reduce the cardiac sympathetic tone in Baseline（BS）[heart rate variability（HRV）].Compared with IR group,LOM_LSPV denervation and LSG denervation similarly reduced sympathetic tone[NE(1.39±0.068 ng/ml in LOM_LSPVD+IR group,1.29±0.081 ng/ml in LSGD+IR group vs 2.32±0.17 ng/ml in group 1,P<0.05)and E(114.64±9.22 pg/ml in LOM_LSPVD+IR group,112.60±9.69pg/ml in LSGD+IR group vs 166.18±15.78 pg/ml in group 1,P<0.05),^]and VAs[VT(0±3.00 in LOM_LSPVD+IR group,0±1.75 in LSGD+IR group vs 8.00±11.00 in group 1,P<0.05)and VTD(0±4 s in LOM_LSPVD+IR group,0±0.88s in LSGD+IR group vs 10.0±22.00s in group 1,P<0.05)]after 2h reperfusion.2.IR injury:Part I:Compared with the SO group,the sympathetic activity,the expression of myocardial inflammatory factors significantly increased in the IR group,myocardial autophagy,apoptosis significantly increased.Compared with the IR group,LOM_LSPV significantly inhibited sympathetic activity before（LF:0.98±0.83 ms2 vs3.20±1.43 ms2,P<0.05 and LF/HF:0.32±0.10 vs 1.71±1.13,P<0.05）and after（iNE:2.56±0.95 ng/ml vs 4.46±1.21 ng/ml,P<0.05）IR,inhibiting inflammatory cytokine expression（HMGB1:16.17±2.13 ng/ml vs 20.75±3.37 ng/ml,IL-17A:445.10±67.63 ng/ml vs 700.27±132.95 ng/ml;IL-1β:157.26±20.53 ng/ml vs 269.74±53.32ng/ml,all P<0.05）,decreased myocardial autophagy（Beclin-1:0.16±0.015 vs 0.235±0.014;LC3II/I:1.04±0.20 vs 2.26±0.25,P<0.05）and myocardial apoptosis（apoptosis index:15.73±3.62 vs 22.20±4.58,P<0.05）.Part II:Compared with the SO group,the sympathetic activity,the oxidative stress level,the calcium transporter activation and expression and myocardial apoptosis significantly increased in IR group.Compared with the IR group,LOM_LSPV significantly inhibited sympathetic activity（iNE:2.54±0.45 vs 4.48±0.69,P<0.05）,decreased oxidative stress level（MDA expression:5.32±1.50 nmol/mgprot vs 10.82±1.52 nmol/mgprot,P<0.05）,increased antioxidant capacity(SOD:26.73±4.96 U/mgprot Vs 16.04±1.68 U/mgprot;T-Aoc（42.88±5.80 U/mgprot vs 16.54±3.75 U/mgprot）,reduced calcium transporter expression and activation（RyR2:0.38±0.044 vs 0.60±0.54;p-CaMKII:0.65±0.14 vs 0.95±0.20;NCX:0.16±0.021 vs 0.25±0.033;P<0.05）,decreased myocardial apoptosis（cleaved-caspase-3:0.34±0.027 vs 0.42±0.027,Bcl-2/Bax:1.81±0.41 vs 0.56±0.24;myocardial apoptosis index:24%±5%vs 33%±6%,P<0.05）Conclusions:1.In vitro experiments we confirmed that HMGB1 and IL-17A can induce autophagy in myocardial hypoxia reoxygenation.HMGB1 could up regulate the expression level of IL-17A,then exacerbate of H/R on myocardial cells injury2.LOM_LSPVD,an effective approach of cardiac sympathetic denervation,significantly reduced myocardial IR injury by suppressing inflammatory cytokines,oxidative stress,Ca2+overload,excessive autophagy and apoptosis.