Effect Of CG9940 Gene Expression Changes And Exercise On Age-related Changes Of Cardiac Function In Drosophila

1 ObjectiveTo study the effects of overexpression and RNAi of CG9940 gene combine with endurance exercise on age-related changes of cardiac function in Drosophila melanogaster, This experiment was adopted the UAS-Gal4 system for regulating the NAD synthase CG9940 gene expression of Drosophila melanogaster, used the 3rd motion device for Drosophila endurance exercise, then detected heart functions as well as measured exercise capacity and lifespan. For further comprehensive and science studying in age-related changes of cardiac function in Drosophila melanogaste.2 Materials and methodsDrosophila gene lines: CG9940 over-expression line is y[1]w[67c23]P{y[+m8]=Mae-UAS.6.11}CG9940[GG01267](Stock No.:14158),CG9940 RNAi line is PBac{WH}CG9940f04110(Stock No.: 18730), wild line is W1118 and regulation line is arm-Gal4, all lines were bought from Bloomington at Indiana University in USA. For regulating gene expression, virgin females drosophila of y[1]w[67c23]P{y[+m8]=Mae-UAS. 6.11}CG9940[GG01267], PBac{WH}CG9940f04110and W1118 were were separately crossed to male drosophila of arm-Gal4, then collected virgin females of F1 generations from different lines. We grouped 9 based on inequable conditions,there were 1W-C-W1118/arm-Gal4, 6W-C-W1118/arm-Gal4, 6W-E-W1118/arm-Gal4, 1W-C-Opr-CG9940/arm- Gal4, 6W-C-Op-CG9940/armGal4, 6W-E-Op-CG9940/arm-Gal4, 1W-C-RNAi-CG9940/arm-Gal4, 6W-C-RNAi-CG9940/arm-Gal4, 6W-E-RNAi-CG9940/arm-Gal4. each group were 400 drosophilas, 20/tube, four groups were put in the HWS intelligent constant temperature and humidity flies incubator, the temperature is 25 degrees and keeps 50% humidity,12 hours a day/night cycle.By use of the third generation of Drosophila movement device,6W-Exercise-Overexpression-CG9940/arm-Gal4,6W-Exerci se-W1118/arm-Gal4 and 6W-Exercise-RNAi-CG9940/arm-Gal4 were set in motion from eighth days of age, exercise load was gradient increase or decrease according to the aging in drosophila,motor speed from second to sixth weeks respectively were control for 0.18r/s, 0.20r/s, 0.18r/s, 0.16r/s and 0.14r/s,1.5h per day, 5 days in a row per week, continuous training for5 weeks, weekly rest were 2 days. we did next stepped experiments after 24 hours, when exercise program ended.Detection: from 1W-W1118/arm-Gal4, 1W-Op-CG9940/arm-Gal4 and 1W-RNAi-CG9940/arm-Gal4 were randomly selected 10 flies for RT-PCR test to make sure whether or not Drosophila melanogaster strains had constructed, about 30 drosophilas were randomly selected for M-mold cardiac functions detection and 100 flies do climbing ability test at seventh days age.Another 6 groups were also randomly selected 30 fruit flies for M-mold cardiac functions detection, 100 flies do climbing ability test at forty-second days age, we counted dead drosophilas from the same days age to make lifespan statistics.3 Results3.1 The result of RT-PCR1118Compared with C-W1118/arm-Gal4 group, C-Op-CG9940/arm-Gal4 group expression increased(P<0.01), rate of overexpression was127.27%; C-RNAi-CG9940/arm-Gal4 group expression decreased(P<0.01), rate of overexpression was-72.72%. Suggesting that CG9940 gene of overexpression, RNAi lines were constructed successfully in F1 generation of drosophila.3.2 The result of M-mold cardiac functions in drosophilaCompared with the 6W-C-W1118/arm-Gal4 group, there were no significant difference in heart rate(P=0.87), fraction shortening increased(P<0.05), arrhythmia index decreased(P<0.05) in 6W-C- Op-CG9940/arm-Gal4 group; Heart rate decreased(P<0.05), fraction shortening had no obvious change(P=0.43), no differences in arrhythmia index(P=0.58) in6W-C-RNAi-CG9940/arm-Gal4 group. It suggested that heart function would enhance and the risk of arrhythmia would reduce in aging Drosophila of CG9940 gene expression is up-regulated,While down regulation of CG9940 gene expression in aging in Drosophila led to decreased heart rate.Compared with 6W-C-W1118/arm-Gal4 group, the heart rate decreased(P<0.05), fraction shortening increased(P<0.01),arrhythmia index decreased(P<0.05) in 6W-E-W1118/arm-Gal4 group.Sugg esting that exercise training could improve heart function,and reduce the risk of arrhythmia occurrence in aging wild-type Drosophila.Compared with 6W-C-Op-CG9940/arm-Gal4 group, heart rate decreased(P<0.01), fraction shortening increased(P<0.01),arrhythmia index decreased(P<0.01) in 6W-E-Op-CG9940/arm-Gal4 group. Suggesting that exercise training could improve heart function, and reduce the risk of arrhythmia occurrence in aging Drosophila of CG9940 gene overexpression.Compared with 6W-C-RNAi-CG9940/arm-Gal4 group, 6W-ERNAi-CG9940/arm-Gal4 group heart rate and fraction shortening had no obvious change, arrhythmia index reduced(P<0.01).Suggesting that there were no effect on heart contractile function after endurance training in aged Drosophila of reduced CG9940 gene expression, but could reduce the risk of arrhythmia occurrence.3.3 climbing ability test resultsCompared with 6W-C-W1118/arm-Gal4 group, 6W-C-Op-CG9940/arm-Gal4 group’s climbing index increased(P<0.05), 6W-CRNAi-CG9940/arm-Gal4 group ’ s climbing index decreased(P<0.01), suggesting that CG9940 gene expression was upregulated in aged Drosophila could improve exercise capacity,and down-regulation of CG9940 gene expression could lead to weakening of movement ability in aging Drosophila.Compared with 6W-C-W1118/arm-Gal4 group, 6W-E-W1118/arm- Gal4group’s climbing index increased(P<0.05); compared with6W-C-Op-CG9940/arm-Gal4 group, 6W-E-Op-CG9940/arm-Gal4group’s climbing index increased(P<0.05); compared with that in 6W-C-RNAi-CG9940/arm-Gal4 group, 6W-E-RNAi-CG9940/armGal4 group ’ s the climbing index had no obvious change. Suggesting that exercise training can enhance movement ability in aging Drosophila of the wild line and the overexpression of CG9940 gene line, but no significant influences on movement ability in aging flies of down-regulation of CG9940 gene expression.3.4 Results of the lifespan statisticsCompared with the C-W1118/arm-Gal4 group, E-W1118/arm-Gal4group’s lifespan was prolonged(P<0.05); compared with C-OpCG9940/arm-Gal4 group, E-Op-CG9940/arm-Gal4 group’s lifespan was extended(P<0.05); There was no significant difference between the C-RNAi-CG99940/arm-Gal4 group and E-RNAi-CG9940/arm-Gal4 group in lifespan(P>0.05). Tipping at the genetic background of normal expression of CG9940 gene and up-regulated the expression of CG9940 gene, exercise training helps to prolong the lifespan in drosophila, but down-regulation of CG9940 gene, exercise training has no good affect on lifespan prolonged.4 Conclusion4.1 CG9940 gene expression was up-regulated and down regulated in Drosophila melanogaster strains constructed successfully by using UAS-Gal4 system, we found that the up-regulation of NAD synthase gene CG9940 expression can improve heart function and exercise capacity in young fruit flies, delay heart function and exercise capacity decline in aging drosophila, reduce the risk of arrhythmia occurrence and prolong life; The down-regulation of NAD synthase gene CG9940 expression can improve exercise capacity, but has no obvious influence on cardiac contracted function in young drosophila, and can accelerate heart rate and athletic ability degradation in aging drosophila, and shorten the lifespan.4.2 At different genetic background, there are different influences on heart function and exercise capacity with age-related changes after exercise training. For the wild line and up-regulation of CG9940 gene expression line, exercise training contribute heart function and exercise capacity to delaying aging decline, improve heart automatic rhythmicity,prolongs the service lifespan, however exercise training shows the best contributions to heart function, exercise capacity and lifespan at genetic background of up-regulation of CG9940 gene expression; down- regulation of G9940 gene expression line,exercise intervention can reduce arrhythmia occurrence, but can not delay heart function and exercise capacity decline in elder drosophila, not help toxinz expectancy life extended.4.3 Drosophila NAD synthase gene CG9940 is closely related with the heart function, exercise capacity and aging. When Exercise training combines with up-regulation of NAD synthase gene CG9940, it will generate superposition beneficial effects in aging drosophila, which will show the strongest heart function and exercise capacity, the best quality of life and the longest lifespan.