Physical exercise prevents age-related heart dysfunction induced by high-salt intake and heart salt-specific overexpression in Drosophila

High-salt intake(HSI) promoted age-related heart dysfunction, dFOXO/PGC-1α decline, and oxidative stress/ dTOR increase

In mammals, both HSI and aging lead to the myocyte hypertrophy, myocardial fibrosis, and mitochondrial dysfunction, which are more likely to end up with the deterioration of the cardiac function and heart failure [3, 13]. However, it is still unclear whether HSI can promote cardiac senescence and cardiac CG2196(salt) gene expression.

In here, our results displayed that in one-week old and five-week old flies, a HSI dramatically reduced heart diastolic interval, heart period, and fractional shortening (P<0.05, P<0.05, P<0.01) (Figure 1A, 1C, 1F), and it dramatically increased arrhythmia index (P<0.05) (Figure 1G), but it did not dramatically change the heart systolic interval, diastolic diameter, and systolic diameter (P>0.05) (Figure 1B, 1D, 1E). In five-week old flies, a HSI dramatically reduced fractional shortening (P<0.05) (Figure 1F), and it dramatically increased heart systolic interval, heart period, arrhythmia index (P<0.05 or P<0.01) (Figure 1B, 1C, 1G, 1H), but it did not dramatically change the heart diastolic interval and diastolic diameter effectively (P>0.05) (Figure 1A, 1D).

Besides, in one-week old flies and five-week old flies, a HSI dramatically up regulated the heart CG2196(salt) gene expression (P<0.01) (Figure 1I). Moreover, a HSI dramatically up regulated the heart dTOR gene expression in both one-week old flies and five-week old flies (P<0.01, P<0.01) (Figure 1J), and it down regulated the heart dFOXO and PGC-1 gene expression in both one-week old flies and five-week old flies (P<0.01, P<0.01) (Figure 1K, 1L). Since the myofibrils and mitochondria of cardiomyocytes are critical to the contractile function of the heart, the ultrastructure of cardiomyocytes was observed by electron microscopy in 5-week old flies. The images showed that a HSI reduced the number of myofibrils and mitochondria, and it destroyed the arrangement of myofibrils (Figure 1O).

In both w¹¹¹⁸ and w¹¹¹⁸+HSI flies, aging dramatically increased diastolic interval, systolic interval, heart period, and arrhythmia index(P<0.01) (Figure 1A–1C, 1G), and it dramatically decreased diastolic diameter and fractional shortening (P<0.05 or P<0.01) (Figure 1D, 1F), but aging did not dramatically change the heart systolic diameter and (P>0.05) (Figure 1A). Aging dramatically up regulated the heart CG2196 (salt) gene expression, dTOR gene expression, and MDA level (P<0.01) (Figure 1I, 1J, 1N). Aging down regulated the heart SOD level, dFOXO expression, and PGC-1α gene expression (P<0.01, P<0.01) (Figure 1K–1M).

Thus, the results suggested that both long-term HSI and aging contributed to age-related accelerated decline of cardiac contractility and age-related accelerated increase of arrhythmias, and the mechanism may be related to up-regulation of heart salt gene expression and oxidative stress/dTOR pathway, and down-regulation of heart dFOXO/PGC-1α pathway. However, the effect of the heart salt gene on heart aging was unknown.

Heart salt specific overexpression(HSSO) promoted age-related heart dysfunction, dFOXO/PGC-1α decline, and oxidative stress/ dTOR increase

HSI up regulates the CG2196(salt) gene expression, and it reduces the lifespan of flies. CG2196(salt) gene overexpression also reduces the survival of flies, and it seems to accelerate aging of flies [12]. However, it remains unknown the relationship between heart salt gene and heart aging.

In this experiment, the HSSO was produced by using UAS/hand-gal4 system, and the HSSO flies were raised up to five weeks of age. Our results displayed that the heart salt gene expression of salt-OE flies was higher than that of salt-control group flies (P<0.01) (Figure 2I), which indicated that HSSO was built successfully. Besides, HSSO dramatically increased the systolic interval, heart period, and systolic diameter in five-week old flies (P<0.05 or P<0.01) (Figure 2B, 2C, 2E). In three-week old flies and five-week old flies, HSSO dramatically decreased the fractional shortening in 5-week old flies (P<0.01) (Figure 2F), and it dramatically increased arrhythmia index (P<0.05 or P<0.01) (Figure 2G, 2H).

In addition, our results showed that HSSO dramatically up regulated heart dTOR expression (P<0.01) (Figure 2J), and it dramatically increased MDA level in five-week old flies (P<0.01) (Figure 2N). However, HSSO dramatically down regulated heart dFOXO and PGC-1α gene expression (P<0.01, P<0.01) (Figure 2K, 2L), and it decreased SOD activity level (P<0.01, P<0.01) (Figure 2M). Finally, the electron microscopy images showed that HSSO decreased the number of myofibrils and mitochondria, and it disrupted the arrangement of myofibrils (Figure 2O).

In both Salt-Control and Salt-OE flies, aging dramatically increased diastolic interval, systolic interval, heart period, diastolic diameter, and arrhythmia index(P<0.01) (Figure 2A–2D, 2G), and it dramatically decreased fractional shortening (P<0.05 or P<0.01) (Figure 2F), but aging did not dramatically change the heart systolic diameter (P>0.05) (Figure 2E).

Therefore, these results suggested that HSSO also promoted age-related impairment of heart contractility and an increase in age-related arrhythmias, and the mechanism of that may be related to up-regulation of heart oxidative stress/dTOR pathway and down-regulation of heart dFOXO/PGC-1α pathway.

Moreover, to explore the relationship between HSSO and flies body aging, the climbing ability and lifespan were also measured. Our results showed that HSSO did not dramatically change the climbing index in aging flies (P>0.05) (Figure 3A), but it dramatically reduced the climbing endurance in 1-week old, 3-week old, and 5-week old flies (P<0.05 or P<0.01) (Figure 3D, 3E, 3F). Moreover, HSSO dramatically reduced the lifespan of flies (P<0.05) (Figure 3B, 3C). Aging dramatically reduced the climbing index and climbing endurance (P<0.01) (Figure 3A, 3G, 3H, 3I). These results indicated that HSSO promoted aging-related decline in exercise capacity and hastened aging-related death.

Long-term exercise (LTE) protected the heart from age-related dysfunction caused by HIS

Numerous studies have shown that LTE can enhance the heart's ability to contract, reduce cardiac dysfunction, and delay cardiac aging [21, 28–30].

Increasing evidence confirms that LTE can improve the heart defects induced by high sugar diet, high fat diet (HFD), or high-salt diet [25, 31–33]. However, it remains unknown whether LTE can prevent heart presenility induced by HSI when LTE and HSI are present throughout lifetime of flies. The relationship between LTE and the heart salt gene is also unclear.

In this experiment, our results showed that in one-week old HSI flies, LTE dramatically increased the heart fractional shortening (P<0.05) (Figure 1F), and it dramatically decreased arrhythmia index (P<0.05) (Figure 1G), but LTE did not dramatically change the heart systolic diameter, diastolic diameter, systolic interval, diastolic interval, and heart period (P>0.05) (Figure 1A–1E). In 3-week old HSI flies, LTE dramatically increased the heart fractional shortening (P<0.05) (Figure 1F), but LTE did not dramatically change the heart systolic diameter, diastolic diameter, systolic interval, diastolic interval, heart period, and arrhythmia index (P>0.05) (Figure 1A–1E, 1G). In five-week old HSI flies, LTE dramatically decreased the heart systolic interval, heart period, and arrhythmia index (P<0.01) (Figure 1B, 1C, 1G), and it dramatically increased fractional shortening (P<0.01) (Figure 1F, 1H), but it did not effectively change the heart systolic diameter, diastolic diameter, and diastolic interval (P>0.05) (Figure 1A, 1D, 1E).

Besides, our results showed that in 1-week old HSI flies, LTE dramatically down regulated the heart CG2196(salt) gene expression (P<0.01) (Figure 1I) and dTOR gene expression(P<0.05) (Figure 1J), and it dramatically decreased MDA level (P<0.05) (Figure 1N). LTE dramatically up regulated the heart dFOXO and PGC-1α gene expression (P<0.01, P<0.01) (Figure 1K, 1L), and LTE increased SOD activity level(P<0.01) (Figure 1M). In 5-week old HSI flies, LTE also dramatically down regulated the heart CG2196(salt) gene expression (P<0.01) (Figure 1I) and dTOR gene expression(P<0.01) (Figure 1J), and it dramatically decreased MDA level (P<0.05) (Figure 1N), but LTE up regulated the heart dFOXO and PGC-1α gene expression (P<0.01, P<0.01) (Figure 1K, 1L), and it increased SOD activity level(P<0.01) (Figure 1M). The electron microscopy images showed that LTE increased the number of myofibrils and mitochondria, and it enhanced the arrangement of myofibrils (Figure 1O).

In w¹¹¹⁸+HSI+E flies, aging dramatically increased diastolic interval, systolic interval, heart period, and arrhythmia index(P<0.01) (Figure 1A–1C, 1G), and it dramatically decreased diastolic diameter and fractional shortening (P<0.05 or P<0.01) (Figure 1D, 1F), but aging did not dramatically change the heart systolic diameter and (P>0.05) (Figure 1A). Aging dramatically up regulated the heart CG2196 (salt) gene expression, dTOR gene expression, and MDA level (P<0.01) (Figure 1I, 1J, 1N). Aging down regulated the heart dFOXO expression, PGC-1α gene expression, and SOD level (P<0.01, P<0.01) (Figure 1K–1M).

Thus, these results suggested that LTE prevented age-related accelerated decline of cardiac contractility and age-related accelerated increase of arrhythmias induced by a HSI, and the mechanism of that may be related to dow-regulation of heart salt gene expression and oxidative stress/dTOR pathway, and up-regulation of heart dFOXO/PGC-1α pathway.

Long-term exercise (LTE) improved age-relate heart dysfunction induced by HSSO

To explore whether LTE could resist the damages induced by HSSO in flies, the HSSO flies were taken exercise from 1-week old to 5-week old. Previous studies have confirmed that exercise training can prevent heart defects, mobility decline, and lifespan reduction induced by CG9940 gene or dSir2 gene mutation [23–25]. Therefore, as a mild intervention, exercise training has potential therapeutic significance for genetic diseases.

In this experiment, our results showed that in one-week old HSSO flies, LTE dramatically increased the diastolic diameter and heart fractional shortening (P<0.05) (Figure 2D, 2F), but LTE did not dramatically change the heart systolic diameter, diastolic diameter, systolic interval, diastolic interval, heart period, and arrhythmia index (P>0.05) (Figure 2A–2C, 2E, 2G). In three-week old HSSO flies, the diastolic diameter and heart fractional shortening (P<0.05) (Figure 2D, 2F), and it dramatically decreased arrhythmia index (P<0.05) (Figure 2G), but LTE did not dramatically change the heart systolic diameter, systolic interval, diastolic interval, and heart period (P>0.05) (Figure 2A–2E, 2G). In 5-week old HSSO flies, LTE dramatically decreased the heart systolic interval, heart period, systolic diameter, and arrhythmia index (P<0.01) (Figure 2B, 2C, 2E, 2G, 2H), and it dramatically increased diastolic diameter and fractional shortening (P<0.05) (Figure 2D, 2F), but LTE did not effectively change the heart diastolic diameter (P>0.05) (Figure 2A).

In 5-week old HSSO flies, LTE also dramatically down regulated the heart dTOR gene expression (P<0.01) (Figure 2J), and it dramatically decreased MDA level (P<0.05) (Figure 2N), but LTE up regulated the heart dFOXO and PGC-1α gene expression (P<0.01) (Figure 2K, 2L), and it increased SOD activity level(P<0.01) (Figure 2M). The electron microscopy images showed that LTE increased the number of myofibrils and mitochondria, and it enhanced the arrangement of myofibrils (Figure 2O). LTE did not effectively change the heart salt expression level(P>0.05) (Figure 2I).

In Salt-OE+E flies, aging dramatically increased diastolic interval, systolic interval, heart period, and arrhythmia index (P<0.01) (Figure 2A–2C, 2G), and it dramatically decreased diastolic diameter and fractional shortening (P<0.05 or P<0.01) (Figure 2D, 2F), but aging did not dramatically change the heart systolic diameter and (P>0.05) (Figure 2A).

Therefore, LTE prevented age-related accelerated decline of cardiac contractility and age-related accelerated increase of arrhythmias induced by HSSO, and the mechanism of that may be related to down-regulation of oxidative stress/dTOR pathway and up-regulation of heart dFOXO/PGC-1α pathway.

Our results showed that LTE dramatically increased the climbing index, climbing endurance, and survival in HSSO flies (P<0.05 or P<0.01) (Figure 3A–3F), and aging dramatically reduced the climbing index and climbing endurance(P<0.01) (Figure 3A, 3D, 3F, 3F). These indicated that LTE resisted the body aging induced by HSSO.

Heart salt specific RNAi(HSSR) prevented heart age-related abnormalities induced by HSI

It has been reported that salt gene knockdown in flies can prevent lifespan decrease from high salt diet, but it remains unknown whether knock-down of heart salt gene can resist heart presenility induced by HSI. In this experiment, our results displayed that heart salt expression of salt-RNAi flies was dramatically lower than that of salt-control flies (P<0.01) (Figure 4I), which indicated that HSSR was built successfully.

Our results showed that in five-week old flies, HSSR didn't dramatically change heart diastolic diameter, systolic diameter, fractional shortening, diastolic interval, systolic interval, heart period, arrhythmia index, SOD activity level, MDA level, dTOR gene, dFOXO gene, and PGC-1α gene expression (P>0.05) (Figure 4A–4H, 4J–4N). These indicated that HSSR did not dramatically delay the natural aging of the heart. Besides, in 5-week old HSSR flies, HSI didn't dramatically change heart diastolic diameter, systolic diameter, fractional shortening, diastolic interval, systolic interval, heart period, arrhythmia index, SOD activity level, MDA level, dTOR gene, dFOXO gene, and PGC-1α gene expression (P>0.05) (Figure 4A–4H, 4J–4N).

In five-week old HSI flies, HSSR dramatically decreased systolic interval, heart period, and arrhythmia index (P<0.05 or P<0.01) (Figure 4B, 4C, 4G, 4H), and it dramatically increased fractional shortening (P<0.01) (Figure 4F). Moreover, HSSR dramatically decreased salt expression, dTOR expression, and MDA level(P<0.01) (Figure 4I, 4J, 4N), and it dramatically increased up regulated the heart dFOXO gene expression, PGC-1α gene expression, and SOD activity level(P<0.01) (Figure 4K, 4L, 4M).

HSSR didn't dramatically change climbing index, climbing endurance, survival, and average lifespan in aging flies (P>0.05) (Figure 4A–4H). HSI dramatically reduced the climbing index, climbing endurance, survival, and average lifespan in 5-week old HSSR flies (P<0.05 or P<0.01) (Figure 5A–5H).

These indicated that HSSR did not delay the body aging induced by HSSO, but HSSO did resist heart presenility induced by HSI.

Long-term exercise(LTE) delayed heart aging and body aging induced by HSI in HSSR Drosophila

To explore whether LTE combined with HSSR did better combat the harm induced by HSI, the HSSR flies were taken exercise training and fed a HSI from young to old.

Our results showed that LTE dramatically reduced diastolic interval, heart period heart, diastolic diameter, and fractional shortening in 5-week old HSSR-HSI flies (P<0.05 or P<0.01) (Figure 4A, 4C, 4D, 4F), and it dramatically decreased arrhythmia index (P<0.01) (Figure 4G, 4H), but it did not effectively change the heart systolic interval and systolic diameter (P>0.05) (Figure 4B, 4E). Next, in 5-week old HSSR-HSI flies, LTE dramatically down regulated the heart dTOR gene expression and decreased MDA level (P<0.01) (Figure 4J, 4N), and it increased SOD activity level and up regulated the heart dFOXO and PGC-1α gene expression (P<0.01) (Figure 4K, 4L, 4M), but it did not effectively change the heart salt gene expression (P>0.05) (Figure 4I).

In 5-week old HSI+E flies, HSSR dramatically increased heart period, diastolic diameter, and fractional shortening(P<0.05) (Figure 4C, 4D, 4F), but it dramatically decreased arrhythmia index (P<0.01) (Figure 4G), and HSSR didn't dramatically change diastolic interval, systolic interval, systolic diameter (P>0.05) (Figure 4A, 4B, 4E). Besides, in 5-week old HSI+E flies, HSSR dramatically decreased salt expression, dTOR expression, and MDA level(P<0.01) (Figure 4I, 4J, 4N), and it dramatically increased up regulated the heart dFOXO expression, PGC-1α gene expression, and SOD activity level(P<0.01) (Figure 4K, 4L, 4M).

Finally, In 5-week old HSI+E flies, LTE dramatically increased the climbing index, climbing endurance, and lifespan (P<0.05 or P<0.01) (Figure 5A–5H). In 5-week old HSI+E flies, HSSR did not dramatically change the climbing index, climbing endurance, and lifespan (P<0.05 or P<0.01) (Figure 5A–5H).

Therefore, these results indicated that LTE combined with HSSR further enhanced heart contractility and reduced arrhythmia in HSI flies, and the mechanism was related to down-regulation of heart dTOR expression and oxidative stress decrease, and up-regulation of heart dFOXO and PGC-1α gene expression. Besides, LTE could also resist whole body aging induced by a HSI in HSSR flies.

Drosophila stock and diet

The salt-UAS-RNAi fly(Genotype:w¹¹¹⁸; P{GD12732}v28349/TM3; stock ID: v28349) was obtained from the Vienna Drosophila RNAi Center. The salt-UAS-overexpression fly (Genotype: w^*; TI{TI}mir-1014^KO salt^mir-1014-KO; stock ID: 58888) was obtained from the Bloomington Stock Center. The w¹¹¹⁸ fly and hand-Gal4 fly were gifts from Xiu-shan Wu (Heart Development Center of Hunan Normal University).

The w¹¹¹⁸ virgin female flies were divided into 3 groups: w¹¹¹⁸, w¹¹¹⁸+HSI(high-salt intake), and w¹¹¹⁸+HSI+E(exercise). For the transgenic groups, maternal origin was selected as the genetic control group to avoid the influence of genetic background differences on the results. The “UAS-salt-overexpression virgin female flies”, “hand-gal4>UAS-salt-overexpression virgin female flies”, and “hand-gal4>UAS-salt-overexpression exercised virgin female flies” were respectively represented as “salt-control”, “salt-OE”, and “salt-OE+E”. Besides, The “UAS-salt-RNAi virgin female flies”, “hand-gal4>UAS-salt-RNAi virgin female flies”, “hand-gal4>UAS-salt-RNAi HSI virgin female flies”, and “hand-gal4>UAS-salt-RNAi HSI exercised virgin female flies” were respectively represented as “salt-control”, “salt-RNAi”, “salt-RNAi +HSI”, and “salt-RNAi+HSI+E”.

Normal food contained 2% agar, 10% sucrose, and10% yeast [54]. During the experiment, all experimental fruit flies were housed in a 12-h light/dark cycle and a 25° C incubator with 50% humidity. All experimental fruit flies were fed fresh food every other day during the experiment. High-salt foods were prepared by adding 2% of salt (NaCl) to normal foods [18]. All experimental flies would be provided with 1ml of pure water to drink, which was injected into a sponge plug. Compared with Stergiopoulos’study, our experimental conditions could be regarded as a mild high-salt diet since they did not provide pure water for Drosophila on the high-salt diet [12].

Exercise training protocols

Using the characteristics of Drosophila negative geotactic, Drosophila in exercise group underwent climbing training for 5 weeks. The exercise group began to exercise at 1 week of age and ended at the end of 5 weeks of age. The flies in the exercise group were trained for 1.5 hours a day for five days a week and then rested for two days. The flies were trained to climb in a 10cm vials. After each reversal of the vials, the flies had 12 seconds to climb. The vials rotated at a rate of the 0.16 rev/s. Drosophila exercise training device was independently developed according to Power Tower and Tread Wheel principles (Figure 7) [65–67].

Analysis of cardiac function

The head, ventral thorax, and ventral abdominal cuticle of anesthetized flies were removed, and the hearts were exposed. Oxygenated artificial hemolymph could maintain the normal function of the heart in Drosophila. High-speed cameras captured video of a fruit fly’s heart beating, and the video was shot at 120 FPS and took 30 seconds. The video of the heart beating was analyzed using SOHA software. The heart period, systolic period, diastolic period, fractional shortening, diastolic diameter, systolic diameter, and arrhythmia index could be measured by this method [68].

The qRT-PCR assay

The flies’ hearts were placed in Trizol, and hearts were homogenized. The total RNA was purified from the Trizol by organic solvent extraction. The RNA was used to generate oligo dT-primed cDNAs after being treated with DNase I. In here, internal reference was Rp49 gene, and the quantity of total RNAs was normalized by Rp49. The SYBR green was used to measured Real-time PCR. Gene expression was calculated based on CT values. Primer sequences of salt: F: 5′-TTAATCGCAGGCGCGTCAGTG-3′; R: 5′-GGACGAGACCACCGTGTTAATCAG-3′. Primer sequences of dFOXO: F: 5’-AACAACAGCAGCATCAGCAG-3’; R: 5’-CTGAACCCGAGCATTCAGAT-3’. Primer sequences of dTOR: F: 5’- GAATTGTGGGCAGATGACCT-3’; R: 5’- CCTGCCTGTTGCA CTGATTA-3’. Primer sequences of PGC-1: F: 5’-TGTTGCTGCTACTGCTGCTT-3’; R: 5’-GCCTCTG CATCACCTACACA-3’. Primer sequences of Rp49: F: 5′-CTAAGCTGTCGCACAAATGG-3’; R: 5’- AACT TCTTGAATCCGGTGGG-3’.

The SOD and MDA assay

The flies’ hearts were placed in PBS (pH 7.2–7.4), and these hearts were homogenized by freezing repeatedly with liquid nitrogen. The homogenate maintained at 2° C -8° C after melting. Next, the homogenate was centrifuged for 20 minutes at a speed of 2000-300rmp, and then the supernatant was removed. The methods and steps of SOD and MDA detection were strictly according to the operation requirements of the kits, and the insect SOD activity ELISA Kits and insect MDA ELISA Kits were provided by MLBIO(Shanghai, China) [18].

Fatigue assay

The fatigue assay mainly based on the research of Tinkerhess MJ et al [65]. In a nutshell, 10 vials containing 20 flies each were were placed on the training device, and these flies were trained to be tired. When five flies or fewer than five flies were climbing in a vial, we considered the flies of this vial to be exercise-trained to fatigue, and we recorded the duration of exercise training as fatigue time of this vial. The training fatigue of Drosophila was observed and recorded every 30 minutes. The log-rank analysis was used to analyze and compare the differences in fatigue time.

Climbing index assay

The climbing index assay mainly based on the research of Tinkerhess MJ et al [65]. In short, twenty fruit flies were placed in an 18-cm-long vial with an inner diameter of 2.8 cm. Before assessing negative geotaxis, flies were allowed to adapt to the vial for 10 minutes. A light box was placed behind the vials. Once the flies have been flipped back to the bottom of the vials, a picture was snapped by a timed digital camera after 8 seconds. 5 pictures were taken of each test vial. The climbing index was calculated based on the average climb height of the flies in the pictures.

Longevity assays

The number of flies that died was recorded daily by the experimenters. The average lifespan of each group was calculated based on the survival time of each fruit fly. The survival curve of each group was made according to the survival time of each fruit fly. The log-rank analysis was used to analyze and compare the differences in longevity. Between 200 and 210 flies in each group were tested for longevity [69].

Statistical analyses

Using a one-way analysis of variance (ANOVA) followed by an LSD test among different groups of the same age flies. The differences between 1-week-old flies and 5-week-old flies were verified by using an independent-sample t tests. P-values for lifespan curves and climbing endurance curves were calculated by the log-rank test. The GraphPad Prism and Social Sciences (SPSS) version 16.0 were used for statistical analysis. The statistical significance was set at P<0.05. Data are represented as means ± SEM.