The activation of cardiac dSir2-related pathways mediates physical exercise resistance to heart aging in old Drosophila

Abstract

Cardiac aging is majorly characterized by increased diastolic dysfunction, lipid accumulation, oxidative stress, and contractility debility. The Sir2/Sirt1 gene overexpression delays cell aging and reduces obesity and oxidative stress. Exercise improves heart function and delays heart aging. However, it remains unclear whether exercise delaying heart aging is related to cardiac Sir2/Sirt1-related pathways. In this study, cardiac dSir2 overexpression or knockdown was regulated using the UAS/hand-Gal4 system in Drosophila. Flies underwent exercise interventions from 4 weeks to 5 weeks old. Results showed that either cardiac dSir2 overexpression or exercise remarkably increased the cardiac period, systolic interval, diastolic interval, fractional shortening, SOD activity, dSIR2 protein, Foxo, dSir2, Nmnat, and bmm expression levels in the aging flies; they also notably reduced the cardiac triacylglycerol level, malonaldehyde level, and the diastolic dysfunction index. Either cardiac dSir2 knockdown or aging had almost opposite effects on the heart as those of cardiac dSir2 overexpression. Therefore, we claim that cardiac dSir2 overexpression or knockdown delayed or promoted heart aging by reducing or increasing age-related oxidative stress, lipid accumulation, diastolic dysfunction, and contractility debility. The activation of cardiac dSir2/Foxo/SOD and dSir2/Foxo/bmm pathways may be two important molecular mechanisms through which exercise works against heart aging in Drosophila.

Keywords: dSir2; exercise; heart aging; lipid accumulation; oxidative stress.

Figure 1

Influence of cardiac dSir2 knockdown on heart lipid accumulation and oxidative stress. (A) Cardiac dSir2 expression level. (B) Cardiac Foxo expression. (C) Cardiac SOD activity level. (D) Cardiac MDA level. (E) Cardiac bmm expression. (F) Cardiac TAG level. (G) The normal line of TAG, the normal line of SOD, and the normal line of MDA. Independent-sample t tests were used to assess differences between the 1-week-old and 7-week-old flies to explore the effects of aging on the heart. Independent-sample t tests were used to assess differences between hand-Gal4>w¹¹¹¹⁸ and hand-Gal4>UAS-dSir2-RNAi flies to explore the effects of cardiac dSir2 knockdown on the heart. Data are represented as means ± SEM. *P<0.05; **P <0.01. Sample size was 80 hearts for each group, and measurements were taken 3 times.

Figure 2

Heart function was measured by M-mode trace. (A) Heart period. (B) Heart systolic period. (C) Heart diastolic period. (D) Heart diastolic dysfunction index. The diastolic dysfunction index is diastolic interval standard deviation/diastolic interval median). (E) Fractional shortening. (F) Diastolic diameter. (G) Systolic diameter. (H) Microscopic image of cardiac function from M-mode trace in 5-week-old and 7-week-old Drosophila. 1: 5-week-old hand-Gal4>w¹¹¹¹⁸ flies; 2: 5-week-old hand-Gal4>UAS-dSir2-RNAi flies; 3: 7-week-old hand-Gal4>w¹¹¹¹⁸ flies; 4: 7-week-old hand-Gal4>UAS-dSir2-RNAi flies. It can be observed from 1, 2, 3, and 5 that the cardiac dSir2 knockdown could reduce heart period and fractional shortening, and increase diastolic dysfunction. Independent-sample t tests were used to assess differences between the 1-week-old” and 7-week-old flies to explore the effects of aging on the heart. Independent-sample t tests were used to assess differences between the hand-Gal4>w¹¹¹¹⁸ and hand-Gal4>UAS-dSir2-RNAi flies to explore the effects of cardiac dSir2 knockdown on the heart. Data are represented as means ± SEM. *P<0.05; **P <0.01. Sample size was 30 hearts for each group.

Figure 3

Effect of cardiac dSir2 knockdown on young hearts. (A) Cardiac dSir2 expression level. (B) Cardiac Foxo expression. (C) Cardiac SOD activity level. (D) Cardiac MDA level. (E) Cardiac TAG level. (F) Cardiac bmm expression. (G) Heart period. (H) Heart systolic period. (I) Heart diastolic period. (J) Heart diastolic dysfunction index. The diastolic dysfunction index is diastolic interval standard deviation/diastolic interval median). (K) Fractional shortening. (L) Diastolic diameter. (M) Systolic diameter. (N) Microscopic image of cardiac function from M-mode trace. 1: hand-Gal4>w¹¹¹¹⁸ flies; 2: hand-Gal4>UAS-dSir2^RNAi flies; 3: 7-week-old hand-Gal4>w¹¹¹¹⁸ flies; 4: 7-week-old hand-Gal4>UAS-dSir2-RNAi flies. It can be observed from 1 and 2 that the cardiac dSir2 knockdown could reduce heart period and fractional shortening, and increase diastolic dysfunction. Independent-sample t tests were used to assess differences between the hand-Gal4>w¹¹¹¹⁸ and hand-Gal4>UAS-dSir2^RNAi flies to explore the effects of cardiac dSir2 knockdown on the heart. Data are represented as means ± SEM. *P<0.05; **P <0.01. Sample size was the same as in our previous experiments.

Figure 4

The influence of cardiac dSir2 overexpression on the heart in 7-weeek-old flies. (A) Cardiac dSir2 expression. (B) Cardiac Foxo expression. (C) Cardiac SOD activity level. (D) Cardiac MDA level. (E) Cardiac TAG level. (F) Cardiac bmm expression. (G) Heart period. (H) Heart systolic period. (I) Heart diastolic period. (J) Heart diastolic dysfunction index. The diastolic dysfunction index is diastolic interval standard deviation/diastolic interval median). (K) Fractional shortening. (L) Diastolic diameter. (M) Systolic diameter. (N) Microscopic image of cardiac function from M-mode trace. It can be observed that the cardiac dSir2 overexpression could increase heart period and fractional shortening, and decrease diastolic dysfunction. (O) Cardiac dnaJ-H expression level. One-way analysis of variance (ANOVA) with least significant difference (LSD) tests were used to identify differences among the hand-Gal4>w¹¹¹¹⁸, UAS-dSir2-overexpression>w¹¹¹¹⁸, and hand-Gal4>UAS-dSir2-overexpression flies to explore the effects of cardiac dSir2 overexpression on aging hearts. Data are represented as means ± SEM. *P<0.05; **P <0.01. The sample size was the same as in our previous experiments.

Figure 5

Effect of exercise training on cardiac functions in cardiac Sir2 differential-expression and 5-week-old flies. (A) Heart period. (B) Systolic period. (C) Diastolic period. (D) Diastolic dysfunction index. (E) Fractional shortening. (F) Heart SOD activity level. (G) Heart MDA level. (H) Heart Foxo expression level. (I) Heart TAG level. (J) Heart bmm expression level. (K) Heart Nmnat expression level. (L) Heart SIR2 protein level. “1” indicates hand-Gal4>w1118, “2” indicates hand-Gal4>w1118+Exercise, “3” indicates hand-Gal4>UAS-dSir2-RNAi, “4” indicates hand-Gal4>UAS-dSir2-RNAi+E, “5” indicates hand-Gal4>UAS-dSir2-overexpression, and “6” indicates hand-Gal4>UAS-dSir2-overexpression+E. Independent-sample t tests were used to assess differences between the “Control group” and “Exercise group” in cardiac dSir2 differential-expression flies to explore the effects of exercise training on the heart. Data are represented as means ± SEM. *P<0.05; **P <0.01. Sample size and repetitions were the same as before. (M) Hand-Gal4>w1118 group. (N) hand-Gal4>UAS-dSir2-RNAi, and (O) hand-Gal4>UAS-dSir2-overexpression. In m, n, and o, 1: ultrastructure image of myocardium in the non-exercise group; 2: ultrastructure image of myocardium in the exercise group; 3: microscopic image of cardiac function in the non-exercise group; and 4: microscopic image of cardiac function in the exercise group. It can be observed from m1, n1, and o1 that the cardiac dSir2 knockdown could reduce the number of mitochondria and make the arrangement of myofibrils irregular, but cardiac dSir2 overexpression can increase the number of mitochondria and make the myofibrils more orderly. It can be observed from m3, n3, and o3 that the cardiac dSir2 knockdown can reduce heart period and increase diastolic dysfunction, and cardiac dSir2 overexpression can extend heart period and decrease diastolic dysfunction. Moreover, it can be observed from m, n, and o that exercise training can increase the number of heart mitochondria, make the myofibrils more orderly, extend heart period, and reduce diastolic dysfunction in cardiac dSir2 differential-expression flies.

Figure 6

Effect of cardiac dSir2 differential-expression on the climbing index and average lifespan in Drosophila. (A) The climbing index change curves with aging of cardiac dSir2 knockdown flies. (B) The climbing index of cardiac dSir2 knockdown flies. The sample size was about 100 flies for each group. (C) The climbing index change curves with aging in cardiac dSir2 overexpression flies. (D) The climbing index of cardiac dSir2 overexpression flies. The sample size was about 100 flies for each group. (E) Fly population survival (%) curve of cardiac dSir2 knockdown flies. The leftmost curve represents the cardiac dSir2 knockdown group, of which flies had the shortest lifespan. (F) The average lifespan of cardiac dSir2 knockdown flies. The sample size was 200–220 flies for each group. (G) Fly population survival (%) curve of cardiac dSir2 overexpression flies. The leftmost curve represents the cardiac dSir2 overexpression group, of which flies had the longest lifespan. (H) The average lifespan of cardiac dSir2 overexpression flies. The sample size was 200–220 flies for each group. P-values for lifespan curves were calculated by the log-rank test. Data are represented as means ± SEM. *P<0.05; **P <0.01.

Figure 7

Effect of exercise and cardiac Sir2 on the climbing index and average lifespan in Drosophila. (A) The climbing index. The sample size was about 100 flies for each group. (B) The average lifespan. The sample size was 200–220 flies for each group. (C) Percents of survival curve. The rightmost curve represents the cardiac dSir2 overexpression combined with exercise group, of which flies had the longest lifespan. P-values for lifespan curves were calculated by the log-rank test. Data are represented as means ± SEM. *P<0.05; **P <0.01.