The Effect Of "Telomere-p53-PGC1α" Axis On Intracellular Calcium In Aged Mice With Atrial Fibrillation And Its Mechanism

Background:Atrial fibrillation is one of the most common arrhythmias.China is the leading disease of atrial fibrillation.Although aging is considered to be an independent risk factor for atrial fibrillation,the specific mechanism of atrial fibrillation caused by aging is unknown.Most scholars believe that there is a correlation between aging-related molecule telomere length and atrial fibrillation.Some scholars even proposed that telomere length can be used as a new early warning factor for atrial fibrillation or telomere length shortening,and decreased telomerase activity can be used as a predictor of arrhythmia in patients with ischemic cardiomyopathy.Telomere length decreases with age.Telomerase replication of telomere repeat fragments is the main mechanism of telomere length maintenance.Telomerase includes reverse transcriptase（telomere reverse transcriptase,TERT）and RNA element（telomere RNA component,TERC）.Previous studies have found that the abnormal telomere function of mouse cardiomyocytes activates p53 and then binds and inhibits the promoter of PGC-1α,which leads to mitochondrial dysfunction and the increase of reactive oxygen species,namely"Telomere-p53-PGC1αregulatory axis".The main molecules of this regulatory axis,p53 and PGC1α,are considered to be related to the regulation of intracellular calcium,while the decrease of L-type calcium current and intracellular calcium overload in elderly atrial cells are widely considered to be one of the important mechanisms of electrical remodeling in atrial fibrillation.Objective:To study the mechanism of atrial fibrillation in the elderly and the role of"Telomere-p53-PGC1α"axis in it.Methods:Firstly,the mice were divided into four groups:Young group,Young+Stimulation group（Young+TEAP group）,Old group and Old Stimulation group（Old+TEAP group）.Atrial fibrillation was induced by one-month periodic transesophageal atrial pacing（TEAP）in the two stimulation groups,and the incidence of atrial fibrillation was compared between the two groups.The atrial myocytes of each group were isolated,and the levels of action potential（AP）and I_Ca,L were detected by patch clamp technique.The occurrence of DAD and TA was observed.Calcium release and calcium wave were observed by confocal microscope and Ion Optix single cell contraction and ion detection microscope.Protein and DNA in trial tissue were extracted and the expression of calcium transport related protein was detected by Western Blotting.The relative length of telomere was measured by RT-PCR.The changes of atrial structure and fibrosis were observed by HE staining and Masson staining.Then TERT^-/-mice were constructed and compared with wild-type（WT）mice.Results:（1）Telomere length in the Old group was shorter than that in the Young group,while the expression of p53 increased and the expression of PGC-1αdecreased.Compared with WT,the telomere of TERT^-/-group was shorter,the expression of p53 was higher,and the expression of PGC-1αwas lower（P<0.05）.（2）The incidence of atrial fibrillation in old mice was significantly higher than that in young mice（26.4%vs 47.1%,P<0.05）.The incidence of atrial fibrillation in TERT^-/-group was higher than that in wild type group（25.9%vs 76.1%,P<0.05）.The incidence of spontaneous atrial fibrillation in Old+TEAP group and TERT^-/-group was 19.6%and 17.8%respectively,while spontaneous AF was not observed in other groups.There was no significant difference of the duration of atrial fibrillation or mortality rate in each group.（3）The APD₅₀ of the Old group was significantly shorter than that of the Young group,and the APD₅₀ was further shortened after administration of TEAP.There was no significant difference of the action potential amplitude（APA）,maximal velocity of action potential(V_max)and rest potential（RP）in each group.The incidence of delayed afterdepolarization（DAD）and triggered activity（TA）in the Old group was higher than that in the Young group（P<0.05）.Among them,the rate of DAD and TA in Old+TEAP group was the highest（15.33%and 11.67%,respectively）.The APD₅₀ of TERT^-/-group was shorter than that of WT group,with no significant difference of APA,V_max and RP observed between two groups.The incidence of DAD and TA was higher than that of WT group（2.0%vs 13.4%,2.3%vs 12.0%,respectively）.（4）From non-atrial fibrillation to atrial fibrillation,from young to old,the current density and amplitude of I_Ca,L decreased,the SSA curve of L-type calcium channel shifted to the right,and the SSI curve shifted to the left,and the recovery curve didn’t show obvious change.Compared with WT group,I_Ca,L in TERT^-/-group decreased,SSA moved to the right and the recovery after inactivation curve moved to the direction of depolarization,with no obvious difference of SSI observed within two groups.（5）The amplitude of calcium release in the Old group was higher than that in the Young mice,and the time to baseline 50%（t to bl 50%）and elimination time constant（sin exp tau）was prolonged,and it showed no difference of time to peak 50%（t to peak 50%）in four groups.These differences were further enhanced after the administration of TEAP（P<0.05）.The amplitude of calcium release in TERT^-/-group was higher than that in WT group,and t to bl 50%and sin exp tau was longer than that in WT group（P<0.05）.The frequency of calcium sparks and spontaneous calcium waves in the Old group was higher than that in the Young group,and that in the TERT^-/-group was higher than the WT group.（6）The expression of p-Ry R2increased,while the expression of SERCA2a decreased in Old group（P<0.05）.Compared with WT,the expression p-Ry R2 was strengthened,and the expression of SERCA2a was supressed（P<0.05）.（7）The degree of myocardial fibrosis in the Old group and the Old+TEAP group was higher than that in the Young group and the Young+TEAP group,but there was no significant difference between the TERT^-/-group and the WT group.Conclusion:（1）The telomere length of Old mice was smaller than that of Young mice,and the expression of p53 increased,while the expression of PGC-1αdecreased.The telomere length of TERT^-/-mice was smaller than that of WT mice,the expression of p53 rose,while the expression of PGC-1αwas reduced.（2）The incidence of atrial fibrillation in Old mice was higher than that in Young mice,and that in TERT^-/-mice was higher than that in WT group,and spontaneous atrial fibrillation occurred in Old+TEAP group and TERT^-/-group.（3）The APD of atrial myocytes in aged mice was shortened,the incidence of DAD and TA was significantly increased,and I_Ca,L decreased,especially after induction of atrial fibrillation.Compared with WT,the APD of atrial myocytes in TERT^-/-mice was shortened,the incidence of DAD and TA was significantly increased,and I_Ca,L was reduced.（4）Intracellular calcium disorders were found in Old mice:the amplitude of calcium release increased,calcium recovery slowed down,and the incidence of calcium sparks and spontaneous calcium waves increased.These disorders of calcium regulation were further aggravated after induction of atrial fibrillation.Similar calcium disorders were also observed in TERT^-/-mice.The expression of p-Ry R2 increased,while the expression of SERCA2a decreased in Old mice and TERT^-/-mice.