Effects Of Exhaustive Exercise On The Pacemaker Ionic Channel Of Rat Sinoatrial Node

The effect of long term and high-intensity exercise may result in many types ofcardiac sinoatrial node dysfunction, including sinus bradycardia, sinus arrhythmia,sick sinus syndrome and so on, sinus node disease may increase the risk of suddencardiac death during the competition.The pacemaker activity is generated by the ionicchannels on the sinoatrial node cells, such as: HCN、L-Ca²⁺、T-Ca²⁺、I_Kr、I_Ks、I_KATPand so on. A larger number of studies reveal that the sinoatrial node dysfunctionassociated with the changes of ionic channels current density and subunit geneexpression.This paper proposed that changes of ionic channels electrophysiology （PartI） and molecular structure （Part II） of sinoatrial node cells can be as the entry point tostudy the exercise-induced sinus node dysfunction. In order to discover therelationship between ionic channels remolding of SAN cells andexercise-induced sinus node disease, providing experimental evidence for clarifyingthe mechanism of exercise-induced sinus node dysfunction.Part I Effects of Exhaustive Exercise on the changes of Ionic Channel currents ofRat Sinoatrial NodeObjectiveIn order to analysis the effects of two weeks exhaustive exercise on the currentdensity changes of ionic channel of rat sinoatrial node, including: I_f, I_CaL, I_CaT, I_Kr, I_Ksand I_KATP.MethodGroup:180healthy adult male SD rats were grouped into9groups （20rats for eachgroup） as1control groups（C group）,4groups of one-time exhaustive swimminggroup（O group） and4groups of2-week repeated exhaustive swimming group （Rgroup）. In order to discover the relationship between the current density changes andrecovery time, rats were sacrificed at0,4,12, and24hours after exhaustiveswimming, The rats of single exhaustive swimming group were named O-0h、O-4h、O-12h、O-24h, respectively, the rats of repeated exhaustive swimming group werenamed R-0h、R-4h、R-12h、R-24h, respectively.Exercise Programme: Rats underwent swimming with loading（3%of bodyweight）on their tails.2-hour exhaustive swimming was carried out for the repeatedswimming groups6days per week for a total of2weeks. After2-week normalfeeding,2-hour exhaustive swimming was carried out for the single exhaustiveswimming group.Experiment Method:1. SAN cells isolation: the heart was excised and perfusion byLangendroff device, the collection of SAN cells by enzymatically isolation.2. The ionic channel current density were analyzed by whole-cell patch clamp. Result1. The effects of exhaustive exercise on If: single exhaustive swimming have noaffect on the Ifcurrent density（P>0.05）, the Ifcurrent density of the repeatedexhaustive swimming group at4time phrases were significantly lower than that ofcontrol group and one-time exhaustive swimming group （P<0.01）.2. The effects of exhaustive exercise on ICa,L: the current density of C group is-7.78±0.76pA/pF, single exhaustive swimming have no affect on the ICa,Lcurrentdensity（P>0.05）, the ICa,Lcurrent density of the repeated exhaustive swimming groupat0h,4h,12h,24h were-6.19±0.38pA/pF,-6.26±0.06pA/pF,-6.22±0.95pA/pFand-6.33±0.68pA/pF, respectively, significantly lower than that of control group（P<0.05, P<0.05, P<0.05）.3. The effects of exhaustive exercise on ICa,T: the current density of C group is-11.23±0.79pA/pF, single exhaustive swimming have no affect on the ICa,Tcurrentdensity（P>0.05）, the ICa,Tcurrent density of the repeated exhaustive swimming groupat0h,4h,12h,24h were-6.89±1.42pA/pF,-6.62±0.42pA/pF,-7.02±0.84pA/pFand-6.58±0.56pA/pF, respectively, significantly lower than that of controlgroup（P<0.01, P<0.01, P<0.01）.4. The effects of exhaustive exercise on IKr: the current density of C group is1.73±0.04pA/pF, single exhaustive swimming have no affect on the IKrcurrentdensity（P>0.05）, the IKrcurrent density of the repeated exhaustive swimming group at0h,4h,12h,24h were0.93±0.05pA/pF,0.89±0.17pA/pF,0.96±0.03pA/pF and0.92±0.29pA/pF, respectively, significantly lower than that of control group（P<0.01,P<0.01, P<0.01）.5. The effects of exhaustive exercise on I_KATP: the current density of C group is1.02±0.07pA/pF（P<0.01）, single exhaustive swimming have no affect on the I_KATPcurrent density（P>0.05）, the I_KATPcurrent density of the repeated exhaustiveswimming group at0h was3.77±0.05pA/pF（P<0.01）, were significantly higher thanthat of C group and O-0h group.Conclusion1. Two weeks exhaustive exercise reduce the I_f, I_Ca,L, I_Ca,T, I_Krcurrent, increase theI_KATPcurrent, the total effect of5ionic channels may slow the rate of sinoatrialnode pacemaker activiey, these may result in sinus bradycardia, sinus arrhythmia,sick sinus syndrome and so on.2. The effects of high intensity and repeat exercise training in long term on cardiacsinoatrial node pacemaker activity may lead to ectopic pacemaker appeared, suchas atrial tachycardia, atrial fibrillation, atrial flutter and ventricular arrhythmia andheart electric excitement generated anomaly, and may also lead to atrioventricularblock, atrioventricular dissociation and conduction disorders. Therefore, the changes of pacemaker ionic channels electrophysiology may be one of the mostimportant mechenism of exercise-induced SAN dysfunction and arrhythmias. Part II Effects of Exhaustive Exercise on the gene expression changes of IonicChannel Subunit of Rat Sinoatrial NodeObjectiveIn order to analysis the effects of two weeks exhaustive exercise on the mRNA andprotein expression changes of ionic channel subunits of rat sinoatrial node, including:HCN1、HCN2、HCN4、Cav1.2、Cav1.3、Cav3.1、Cav3.2、ERG、KvLQT1、Kir6.2.MethodGroup and Exercise programme: As part I.Experiment Method:1. Ionic channel subunit mRNA expression were analyzed byreal-time fluorescent quantitative PCR.2. Ionic channel subunit protein expression were analyzed by Western-blot.Results1. The effects of exhaustive exercise on HCN subunit:（1） mRNA level:the HCN1/4mRNA expression of the single exhaustive swimming group O-0h were significantlyhigher than that of control group, respectively（P<0.05, P<0.01）, O-0h weresignificantly higher than that of control group, respectively（P<0.01, P<0.05）；theHCN2/4mRNA expression of the repeated exhaustive swimming group at4timephrases were significantly lower than that of control group（P<0.01）.（2） Protein level:the normalized image density of HCN2/4subunit of the repeated exhaustiveswimming group at4time phrases were significantly lower than that of controlgroup（P<0.01）.2. The effects of exhaustive exercise on L-Ca²⁺subunit:（1） mRNA level:there wereno significient difference between group on Cav1.2mRNA expression；the Cav1.3mRNA expression of the repeated exhaustive swimming group at4time phrases weresignificantly lower than that of control group and O-0h group, respectively（P<0.01,P<0.01）.（2） Protein level: the normalized image density of Cav1.3subunit of therepeated exhaustive swimming group at4time phrases were significantly lower thanthat of control group and O-0h group, respectively（P<0.01, P<0.01）.3. The effects of exhaustive exercise on T-Ca²⁺subunit:（1） mRNA level: theCav3.1mRNA expression of the repeated exhaustive swimming group at4timephrases were significantly lower than that of control group and O-0h group,respectively（P<0.01, P<0.05）, there were no significient difference between group on Cav3.2mRNA expression.（2） Protein level: the normalized image density of Cav3.1subunit of the repeated exhaustive swimming group at4time phrases weresignificantly lower than that of control group and O-0h group, respectively（P<0.01,P<0.01）.4. The effects of exhaustive exercise on rapidly activated delayed rectifierpotassium channel subunit:（1） mRNA level: the ERG mRNA expression of therepeated exhaustive swimming group at4time phrases were lower than that of controlgroup, respectively（P<0.05, P<0.05, P<0.05, P<0.05）.（2） Protein level: thenormalized image density of ERG subunit of the repeated exhaustive swimminggroup at4time phrases were significantly lower than that of control group and O-0hgroup, respectively（P<0.01, P<0.01）.5. The effects of exhaustive exercise on ATP-sensitive potassium channel subunit:（1） mRNA level: the Kir6.2mRNA expression of the repeated exhaustive swimminggroup at4time phrases were significantly higher than that of control group （P<0.01）.（2） Protein level: the normalized image density of Kir6.2subunit of the repeatedexhaustive swimming group at4time phrases were significantly higher than that ofcontrol group（P<0.01）.ConclusionTwo weeks exhaustive and repeated exercise changed the sinoatrial node pacemakerionic channels subunit mRNA and protein expression, including HCN、L-Ca²⁺、T-Ca²⁺、rapidly activated delayed rectifier K⁺channel and ATP-sensitive K⁺channels,along with the corresponding current density. Therefore, ion channel subunit maybeone of the most important structural factors and molecular mechanism for changes ofchannel electrophysiology.