| Objective:Acute myocardial ischemia induced severe ventricular arrhythmias,leading to poor prognosis and even sudden cardiac death.Many factors are involved in the pathological process of ischemic arrhythmias.The over-activation of sympathetic nerves and cardiac sensory afferent nerves play an important role in the occurrence and development of ischemic arrhythmias.Our previous study found that antagonizing excessive rise of endogenous nociceptin/orphanin FQ in the progress of acute myocardial ischemia can inhibit the occurrence of ischemic arrhythmias,but its exact mechanism keeps unclear.The aim of this study was to investigate the interaction between endogenous nociceptin/orphanin FQ and?1-adrenergic receptor??1-AR?during ischemic arrhythmias,and to further explore the relationship between sympathetic nerves and cardiac sensory afferent nerves in the acute phase of myocardial ischemia.Methods:SPF grade healthy male SD rats with a body mass of?260±20?g were used in all experiments.The model of acute myocardial ischemia was prepared by ligating the left anterior descending branch of the coronary artery of rats.The experiment was divided into three parts:Part one:The effect of endogenous nociceptin/orphanin FQ on ventricular arrhythmias and cardiac function during early stage of acute myocardial ischemia in ratsThe animals were divided into three groups:sham-operated group?Sham group?,coronary artery occlusion group?CAO group?and pretreatment of nociceptin/orphanin FQ receptor antagonist?UFP-101?group?U+CAO group?.In the Sham group,only the chest was threaded but the left anterior descending coronary artery was not ligated;In the CAO group,the left anterior descending coronary artery was ligated after opening the chest;In the U+CAO group,the specific nociceptin/orphanin FQ receptor antagonist UFP-101?1×10-99 mol/L?was administrated in a volume of 1 mL·kg-1,i.v.,at 10 min before CAO.The other two groups were given an equal volume of normal saline.The changes of ECG and LVSP,LVEDP,HR,+dp/dtmax,-dp/dtmax and other indicators were analyzed from 10 min before CAO to 1 h after CAO.Part two:The effect of endogenous nociceptin/orphanin FQ on the expression of?1-AR during early stage of acute myocardial ischemia in ratsAccording to the results of the first part of the experiment,the myocardium at the risk of ischemia was collected at 15 minutes?massive arrhythmias?and 1 hour?few arrhythmias?after coronary artery occlusion.?1-AR of plasma membrane and whole cell?total protein?and?1-AR mRNA level was determined.Part three:Acute isolation of rat cardiomyocytes simulating acute myocardial ischemia in vivo to observe the expression and distribution of?1-AR in cardiomyocytesAfter anaesthesia,the rat heart was removed quickly and left ventricular cardiomyocytes were separated.The isolated cell suspension was inoculated into 6-well plate with prepared cell sheets and allowed to stand at room temperature for 3 hours for climbing films.According to the way of treatment,negative control?NC?group,norepinephrine?NE?group,nociceptin/orphanin FQ?N/OFQ?group and NE+N/OFQ group.NC group was not treated;NE group was added with norepinephrine to a concentration of 10-55 mol·L-1;N/OFQ group was added with N/OFQ to a concentration of 10-66 mol·L-1;NE+N/OFQ group was added NE and N/OFQ to a concentration of 10-5mol/L and 10-66 mol·L-1,respectively.After 15 min incubation,4%paraformaldehyde was added to fix dehydration,and then immunofluorescence staining was performed.Results:1.The occurrence of ventricular arrhythmia after myocardial ischemia in each group:Single-ventricular ectopic beat?VEB?can occur in the Sham group after myocardial threading,and no ventricular tachycardia?VT?or ventricular fibrillation?VF?occurs.Rats were suffered to induction of electrical disorders after coronary artery occlusion?CAO?.High incidences of ventricular arrhythmias were observed in the rats of CAO group compared with those in Sham group,which appeared within 30 min and peaked at15 min following coronary artery occlusion.Pretreatment with UFP-101 led to reduction of VEB,duration of VT+VF,and arrhythmogenic scores?all P<0.05?,while the episodes of VT+VF displayed no statistical difference,compared with the CAO group.2.The effect of endogenous nociceptin/orphanin FQ on cardiac function in rats:Slight changes were observed in the indicators of cardiac functions?less than 15%of baseline?after coronary artery occlusion.After injection of UFP-101,LVSP and left ventricular end diastolic pressure?LVEDP?from 0-15 min after coronary artery occlusion were significantly decreased and increased,compared with CAO group?P<0.05?.However,no significant difference was found in LVSP and LVEDP between the two groups at 16-30 min,31-45 min and 46-60 min after coronary artery occlusion.No significant change in HR,LV+dp/dtmax and LV-dp/dtmax was observed during the whole periods of coronary artery occlusion.3.Expression of?1-AR protein and its mRNA in rats of each group:The dynamic change of?1-AR and its mRNA was detected during 60 min period after coronary artery occlusion?CAO?.At 15 min after CAO:compared with Sham group,the?1-AR of total protein and its mRNA in CAO group presents significant reductions?all P<0.05?,however,the?1-AR of plasma membrane presenting a marked up-regulation?P<0.05?;Pretreatment with UFP-101 led to reduction of?1-AR of plasma membrane and increases of?1-AR of total protein and its?1-AR mRNA?all P<0.05?,compared with CAO group.At 60 min after CAO:compared with Sham group,the total protein of?1-AR and its mRNA presents significant up-regulations?all P<0.05?,while the?1-AR of plasma membrane presenting a marked reduction?P<0.05?.4.Immunofluorescence showed the optical density and distribution of?1-AR in acutely isolated rat cardiomyocytes:Acutely isolated rat cardiomyocytes display rod-shaped,?1-AR was labeled as red fluorescence,and nuclei were labeled as blue fluorescence by DAPI.Compared with the NC group:the average optical density of?1-AR in the N/OFQ+NE treatment group was decreased?P<0.05?,but the fluorescence intensity of the cell membrane was significantly stronger than that of the cytoplasm;No significant changes in optical density and changes in cell membrane fluorescence intensity were observed in the NE and N/OFQ alone treatment groups.The results suggested that N/OFQ+NE co-treatment can lead to the decrease of?1-AR expression in cardiomyocytes and cause significant externalization of?1-AR,while N/OFQ and NE treatment alone have no significant effect on?1-AR of cardiomyocytes.Conclusion:Significant externalization of?1-AR in acute phase of myocardial ischemia can cause ischemic arrhythmias,and endogenous nociceptin/orphanin FQ is one of the causes of?1-AR externalization in this process.Namely endogenous nociceptin/orphanin FQ can mediate ischemic arrhythmias by regulating the externalization of?1-AR in cardiomyocytes. |
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