Influence Of Aging And Clk Gene Down-regulation On Sleep-activity Rhythm And Cardiac Rhythm In Drosophila And Effects Of Exercise Intervention

1 ObjectiveWe use Drosophila as the model organism and use Gal4-UAS system to establish the brain Clk gene knockdown strain and the peripheral cardiac Clk gene knockdown strain respectively based on the transgenic technology and give regular exercise.Clk gene knockdown on the main clock or on the peripheral clock of the heart affects sleep/activity rhythm and heartbeat rhythm,and observes whether exercise can improve pathological or physiological rhythm decline and is intended for exercise in rhythm provide a theoretical basis for the treatment of disorders in the movement.2 MethodsThe virgin female flies of UAS-Clk RNAi strain were crossed with male W1118,Pdf-Gal4 and Hand-Gal4 respectively.The F1 generation of female flies in each cross were the gene normal expression control group(Clk RNAi/+),brain Clk knockdown group(Pdf>Clk RNAi)and cardiac Clk knockdown group(Hand>Clk RNAi),each group collected 1200 female flies and randomly divided into 3 weeks old control group(3W_C),6 weeks of control group(6W_C)and 6 weeks of exercise group(6W_E).The training device adopts the self developed Drosophila motion rotary device developed by our laboratory.The movement mode is a 180-degree turning movement starting from the vertical ground.The rotation speed of the motor is 240 s/r,and the residence time after turning is 15 s.In the exercise group,the fruit fly started its regular exercise for 3 weeks starting from the 22nd day of age.It was exercised for 2.5 hours per day for 5 days and rested for 2 days.The exercise time was controlled from 9:00 to 12:00 in the morning.The relative expression of Clk mRNA in brain or heart of Drosophila is detected by real-time PCR.The relative expression of CLK protein in brain or heart of 3W Drosophila was detected by Western blotting.The collection time is from 9:00 to 12:00.The Drosophila Behaviour Monitoring System(DAMS)monitored the sleep-activity status of the fruit flies.Among them,3 W fruit flies are placed in DAMS by 18:00 on the 20d,6 W flies are placed before 18:00 on the 41d,and analyze the data for second days.EM-CCD camera and HC-Image software were used to record and process 30s microscopic imaging of half-exposed heart beats in each group.Heart beat rhythm was analyzed by semi-automatic optical heart beat analysis software(SOHA).Combined with M-mode cardiogram,the cardiac rhythm and arrhythmia indexes of Drosophila melanogaster were analyzed.3 Results3.1 real-time PCR resultsWith aging,the relative expression of each group Clk mRNA in the heart or brain significantly decreased.Clk gene knockdown in 3 W_C and 6W_C Drosophila brain Clk mRNA decreased to varying degrees;cardiac Clk knockdown 3W_C and 6W_C Drosophila heart Clk mRNA levels decreased to varying degrees.Exercise to make the gene normal expression Drosophila brain and heart Clk mRNA relative expression decreased,so that the Clk gene knockdown Drosophila brain Clk mRNA relative expression increased,the same cardiac Clk knockdown Drosophila Clk mRNA relative expression also increased.3.2 Western blot test resultsThe CLK protein was detected in the brain or heart of all the crosses.The protein expression of Drosophila in the brain and heart was decreased by 17%and 13%,respectively.3.3 Drosophila sleep-activity rhythm monitoring resultsCompared with the 3W_C group,the normal expression of 6W_C Drosophila at night and daytime significantly increased the number of sleep times and activities significantly reduced,and the number of activities per hour was significantly lower than 3W_C Drosophila,night and daytime increased the number of second deep sleep,Sleep latency decreased significantly;Clk knockdown of brain Drosophila total sleep time and the total number of activities,hourly activity rhythm and sleep quality indicators did not change significantly,but the number of activities per minute during night and day awakening increased;heart Drosophila Clk knockdown was similar to Drosophila with normal expression of genes,that is,the total sleep time during nighttime and daytime increased,the total number of activities decreased and the number of activities per hour also increased.The sleep quality indicators,the number of night sleep segments Increased,the first and second days of deep sleep during the day increased the number of sleep latency decreased.Dysfunction of brain Clk knockdown was similar to that of 6W Drosophila with normal expression of genes,that is,the total sleep time during night and day increased,the total number of activities decreased,the number of activities per hour also decreased significantly,and the quality of sleep showed the first daytime The number of times of deep sleep increased,the number of deep sleep at night decreased for the second time,because of its activity weakened,sleep latency also decreased.Cardiac Clk knockdown 3W_C Drosophila total sleep time decreased during the day,the total number of activities increased during the night,the number of sleep segments increased during the day,6W_C Drosophila during the day the number of sleep segments,the first increase in the number of deep sleep,night and day Second,the number of deep sleep decreased.However,exercise intervention had no significant effect on normal gene expression and knockdown of Clk gene knockout in Drosophila.However,in the cardiac Clk knockdown group,the total nocturnal and daytime sleeptime increase caused by aging increased,the total number of activities and hourly Decreased the number of activities,and also improved the quality of sleep indicators of aging or Clk mRNA expression decreased caused by the first and second days of deep sleep increase.3.4 The cardiac rhythm analysis resultsCompared with 3W_C group,the gene normal expression and Clk gene knockdown in 6W_C Drosophila melanogaster significantly increased the incidence of arrhythmia,while in the heart of Clk knockdown Drosophila,did not show age-related heart rhythm disorders.Clk knockdown of the brain and heart increased the occurrence of irregular heartbeat in 3W_C Drosophila melanogaster and that of the knockout 6W_C Drosophila melanogaster in the brain showed an increase in the rate of cardiac cycle and bradycardia only whereas the knockdown of cardiac Clk gene by a 6W_C systolic interval.And the incidence of bradycardia decreased and no significant changes in other rhythm indicators.Regular exercise can reduce the Clk gene knockdown in the brain Drosophila caused by the aging of the heart rhythm disorder,but make the gene expression and cardiac Clk knockdown Drosophila arrhythmia increase.4 Conclusions4.1 With the increase of age,the gene normal expression Drosophila of Clk in the brain(gene normal expression group and cardiac Clk knockdown group)with an increased sleep volume and reduced activities;the brain Clk gene is involved in the regulation of sleep-activity rhythm and cardiac rhythm,and its low expression can make the fruit fly enter the aging state in advance.4.2 Cardiac rhythm of gene normal expression Drosophila of Clk in the heart(gene normal expression group and brain Clk knockdown group)showed an age-related decline;The cardiac Clk gene is mainly involved in the regulation of the rhythm of the heartbeat,to some extent affect the quality of sleep in fruit flies,but does not participate in the regulation of sleep-activity.4.3 The 3-week regular exercise can ameliorate the activity decline caused by aging and increase the number of deep sleep during the day in cardiac Clk gene knocked-out Drosophila.4.4 The 3-week regular exercise reduced the onset of arrhythmia caused by the aging in the brain Clk gene knockout Drosophila,but increased the incidence of arrhythmia in gene normal expression Drosophila and cardiac Clk gene knockdown flies.