Study In The Electorphysiological Changes Mediated By P2x7Receptor Of Cervical Sympathetic Ganglia In Myocardial Ischemic Rats

Backround and Objective: Cardiac sympathetic postganglion neurons locate insuperior cervical ganglion and stellate ganglion. Clinical studies found that removalof superior cervical ganglion （SCG）, stellate ganglion （SG） and other sympatheticganglia make50-60%of coronary heart disease patients angina symptoms disappear.It’s indicated that cardiac sympathetic nerve involved in the signal transmission ofmyocardial ischemic injury. Myocardial ischemia activates cardiac sympatheticafferent nerve and transmits the information to the brain, which results in sympatheticexcitatory reflex by exciting sympathetic efferent. It causes an increase in heart rateand blood pressure, leading to the development of myocardial ischemia. Theinflammatory mediators, such as adenosine triphosphate （ATP） and bradykinin, canexcit cardiac afferent nerves. Preliminary experiments in our laboratory confirmed thesuperior cervical ganglion and stellate ganglion P2X2,P2X3and P2X2/3receptor isinvolved in signal transduction of myocardial ischemia. There is no report about therelationship between P2X₇receptor and sympathetic excitatory reflex in myocardialischemia, and the mechanism remains unclear. This study was observing P2X₇receptor-mediated electrophysiological changes in the rat superior cervical ganglionneurons, the changes of P2X₇mRNA in the rat superior cervical, stellate sympatheticganglion and myocardium after myocardial ischemia, P2X₇receptor-mediated [Ca²⁺]irise on PC12cells, learning the role and possible mechanism of P2X₇receptor-mediated sympathetic excitatory reflex in myocardial ischemia model and findingnew drug targets for the prevention and treatment of myocardial ischemic injury.Methods: Rats were randomly divided into the control group, myocardialischemic group, sham group and myocardial ischemic rats treated with P2X₇receptorantagonist （oxATP） group.⑴A rat model of myocardial ischemia injury wasestablished by ligating the left anterior descending coronary artery. The expression ofP2X₇receptors mRNA in rat SCG, SG and myocardium were analyzed by Real-timePCR in control group, myocardial ischemic group, sham group and oxATP-treatedmyocardial injury group.⑵P2X₇receptor agonist-activated currents and the effects of P2X₇receptor antagonist on P2X₇agonist-activated currents were studied bywhole-cell patch clamp technique in the cervical sympathetic ganglion neuronsisolated from control and myocardal ischemic rats.⑶With siRNA technology toknockdown the P2X₇receptor of PC12cells cultured by hypoxic-ischemia, a group ofsiRNA fragment was transfected into cultured cells. Verify the knockdown effect byreal-time PCR.⑷M easured thechanges of [Ca²⁺]iin hypoxic-ischemic culturedPC12cells and the cells treated with oxATP, BzATP, siRNA-P2X₇and ERK inhibitorU0126through the method of the Ca²⁺-specific fluorescent probe （fura-2/AM） andfluorescence spectroph-otometry.Results:⑴The levels of P2X₇mRNA were studied by Real-time PCR. Theaverage RQ value of P2X₇mRNA in the SCG, SG and myocardial tissues inmyocardial ischemic group was significantly higher than those in control group, shamgroup and oxATP-treated myocardial injury group（n=10）（p<0.05）. No difference wasfound in control group, sham group and oxATP-treated myocardial injury group（n=10）（p>0.05）.⑵A mplitudes ofthe currents activated by P2X₇receptor agonists inthe same concentration were much larger in the cervical sympathetic ganglionneurons of myocardial ischemic rats than those obtained in control rats; P2X₇receptorantagonist inhibited P2X₇receptor agonist-activated currents in the cervicalsympathetic ganglion neurons; The inhibition of P2X₇receptor antagonist wasconcentration-dependent, and the inhibition was stronger in myocardial ischemic ratsthan that in control rats at the same concentration of P2X₇receptor antagonist;Current-voltage （I-V） relationships for P2X₇receptor agonist-activated currentsshowed that there were no changes at the reversal potentials （0mV） of the currentsin both myocardial ischemic rats and control rats.⑶Small interfering RNA（siRNA）knockdown experiment results showed that P2X₇receptors mRNA expression of P12cells in oxygen and glucose deprivation （OGD） group and OGD+NC group were nosignificant difference （p>0.05）, indicating that Lipofectamine2000agent could’taffect the expression of P2X₇receptors. Compared with OGD group, P2X₇receptorsmRNA expression of PC12cells in OGD+siRNA-P2X₇group were significantlylower （p<0.05）. Interference efficiency of OGD+siRNA-P2X₇group is56.4%.⑷Thechanges of [Ca²⁺]iin each group detected by fluorescence spectrophotometric determinator.100μM ATP can caused a [Ca²⁺]irise in PC12cells. There is a highlevel of original concertraton of [Ca2+]iin OGD+BzATP group. The peak [Ca²⁺]iinOGD+oxATP group and OGD+siRNA-P2X₇group was partially inhibited by oxATP（300mM） and after transfecting with siRNA-P2X₇by about28.8%and37.4%,respectively （p<0.05, n=5）. It was partially inhibited after transfecting with ERKinhibitor U0126（10μM） by about49.9%（P<0.05, n=5）.100μM BzATP can caused a[Ca2+]irise in PC12cells. There is a high level of original concertraton of [Ca2+]iinOGD+BzATP group. The peak [Ca²⁺]iin OGD+oxATP group andOGD+siRNA-P2X₇group was significantly inhibited by oxATP（300mM） and aftertransfecting with siRNA-P2X₇by about68.9%and70.7%, respctively （p<0.05, n=5）.It was partially inhibited after transfecting with ERK inhibitor U0126（10μM） byabout55.1%（P<0.05, n=5）.Conclusions: The myocardial ischemic injury increased the expression of P2X₇receptor mRNA in superior cervical ganglion, stellate ganglion and myocardium.Amplitudes of the currents activated by P2X₇receptor agonists in the sameconcentration were much larger in the cervical sympathetic ganglion neurons ofmyocardial ischemic rats than those obtained in control rats. P2X₇receptor antagonistsignificantly inhibited P2X₇receptor agonist-activated currents in the cervicalsympathetic ganglion neurons. The peak [Ca2+]iin OGD+oxATP group andOGD+siRNA-P2X₇group was partially inhibited by oxATP （300mM） and aftertransfecting with siRNA-P2X₇and ERK inhibitor U0126（10μM） respectively.Therefore, P2X₇receptors in SCG, SG neurons and cardiac tissue were involved inthe transmission of sympathetic excitatory reflex induced by myocardial ischemicnociceptive signal. P2X₇receptor antagonist can inhibit the transmission of thesympathetic excitatory reflex, and produce the protective effect in myocardialischemic injury.