| 1 ObjectiveThe study stemmed from a hypothesis to a potential mechanism that changes of gene expression in the Calcium signal pathway may play a part in the age-related decline of cardiac function as well as in protective effects of exercise. At first, we based on a method of UAS-RNAi system to screening calcium signaling genes, which played an important role in cardiac function of aging Drosophila. The second, we investigated Ca MKII’s function in the protective effects of exercise on aging flies based on screening results by four aspects of experimental tests which including heart function, activity index, climbing ability and life-cycle.2 MethodsScreening of Calcium signaling gene: Hand-Cal4 and UAS-RNAi male lines were crossed to wild-type strain w1118 female flies respectively, then collected F1 virgin flies that carry both Transgenic element and wild-type genetic background as the control group. Hand-Cal4 male and UAS-RNAi female lines were crossed, and virgin progeny was collected as Cardiac-specific Calcium signaling gene KD group. Heart function of young flies(1-week-old) or late-middle-old flies(5-week-old) were examined by M-mode methods, which genes KD in cardiomyocyte can lead to heart dysfunction would be considered as candidategene.Study the function of Ca MKII related protective effects of exercise: UAS-RNAi male lines were crossed to wild-type strain w1118 female flies, then collected F1 virgin flies as control group. Hand-Cal4 and Arm-Cal4 male lines were crossed to UAS-Ca MKII-RNAi male female flies respectively, then collected F1 virgin flies as Cardiac-specific Calcium signaling gene KD group or Calcium signaling gene KD group. Heart function of flies with different ages were examined by M-mode methods, circadian rhythm and climbing ability were tested after the period of exercise training, and survival statistics of different groups were recorded.3 Results 3.1 The conservative analysis of Ca MKII between Drosophila and HumanWe found 12 kinds of splice variant isoforms of Ca MKII in Drosophila highly conserved with isoform1 or isoform2 expressed by Ca MKIIδ in Human. Blast results showed more than 74% of sequence identity and 82% of sequence similarity in these comparisons.3.2 Results of q RT-PCRReal-time PCR showed: the expression level of myocardial Ca MKII in Hand> Ca MKII RNAi-C had a markedly decline with 79.70% compared to Ca MKII RNAi/+-C; the expression level of Ca MKII in Arm> Ca MKII RNAi-C had a markedly decline with 64.54% compared to Ca MKII RNAi/+-C.3.3 Results of Cardiac function testHP, AI, FL increased and FS decreased with aging. Group with Cardiac-specific Ca MKII knockdown have a significantly decreased in FS compared to normal Drosophila flies at 2-weeks-old and 4-weeks-old. Group with Cardiac-specific Ca MKII knockdown have a significantly increased in AI compared to normal Drosophila flies at 4-weeks-old and 6-weeks-old. Group with Cardiac-specific Ca MKII knockdown have a significantly increased in FS compared to normal Drosophila flies at all ages. In addition, the FS of Ca MKII RNAi / +-E was significantly higher than the flies of Ca MKII RNAi / +-C, the AI of Hand> Ca MKII RNAi-E compared Hand> Ca MKII RNAi-C decreased significantly.3.4 Results of circadian activity rhythm monitoringUnder LD or DD conditions, the exercise group compared with Drosophila genotype night sleep time was significantly longer in the control group of Drosophila.3.5 Results of climbing ability testFlies with cardiac-specific Ca MKII gene knockdown showed a significantly decreased than the normal group in climbing ability at 6-weeks-old and 7-weeks-old(P <0.001); Flies of Ca MKII RNAi / +-E have a lower climbing ability compared to Ca MKII RNAi / +-C at 6-weeks-old; Flies of Hand> Ca MKII RNAi –E have a lower climbing ability compared to Hand> Ca MKII RNAi –C in different ages(P <0.001).3.6 results of Life Cycle testThe results showed a lower survival of Ca MKII gene KD flies(Median survival=55 days) than that of control group(Median survival=67 days, P<0.0001). In addition, Hand> Ca MKII RNAi –E(Median survival=64 days) have a higher survival compared to Hand> Ca MKII RNAi –C(P<0.0001).4 Conclusion4.1 Ca MKII played an important role in maintenance of Drosophila heart function, exercise capacity and life cycle. long-term low expression level of cardiac Ca MKII may lead to a acceleration of heart dysfunction and the decline of exercise capacity with aging.4.2 Three weeks of regular exercise have significantly delayed the Drosophila heart pumping capacity which declined with aging, and extended the sleep at night of flies, suggesting the protective effects of exercise to aging heart of drosophila worked some similarities with human.4.3 The protective effects to drosophila’s heart of three weeks of regular exercise may work on Ca MKII mediated pathway in drosophila’s heart. |
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