Effects of Drosophila melanogaster regular exercise and apolipoprotein B knockdown on abnormal heart rhythm induced by a high-fat diet.

Abnormal heart rhythm is a common cardiac dysfunction in obese patients, and its pathogenesis is related to systemic lipid accumulation. The cardiomyocyte-derived apoLpp (homologous gene in Drosophila of the human apolipoprotein B) plays an important role in whole-body lipid metabolism of Drosophila under a high-fat diet (HFD). Knockdown of apoLpp derived from cardiomyocytes can reduce HFD-induced weight gain and abdominal lipid accumulation. In addition, exercise can reduce the total amount of apoLpp in circulation. However, the relationship between regular exercise, cardiomyocyte-derived apoLpp and abnormal heart rhythm is unclear. We found that an HFD increased the level of triglyceride (TG) in the whole-body, lipid accumulation and obesity in Drosophila. Moreover, the expression of apoLpp in the heart increased sharply, the heart rate and arrhythmia index increased and fibrillation occurred. Conversely, regular exercise or cardiomyocyte-derived apoLpp knockdown reduced the TG level in the whole-body of Drosophila. This significantly reduced the arrhythmia induced by obesity, including the reduction of heart rate, arrhythmia index, and fibrillation. Under HFD conditions, flies with apoLpp knockdown in the heart could resist the abnormal cardiac rhythm caused by obesity after receiving regular exercise. HFD-induced obesity and abnormal cardiac rhythm may be related to the acute increase of cardiomyocyte-derived apoLpp. Regular exercise and inhibition of cardiomyocyte-derived apoLpp can reduce the HFD-induced abnormal cardiac rhythm.

Introduction

Obesity increases the risk of cardiovascular disease, increases blood lipids and impairs exercise capacity [1]. Drosophila is a good model for metabolism and diet-related diseases and its core pathway for regulating energy has been highly conserved in evolution [2]. It is similar to mammals in that excessive chemical energy in the form of lipids and glycogen are stored in the fat body [3]. Fat bodies are similar to human adipose tissue and have a liver function [3]. They store lipids in lipid droplets (an organelle). Drosophila females have more triglyceride storage than males and triglycerides respond more slowly to lipolysis stimuli. In addition, the female Drosophila body is larger and easier to observe and dissect. When the flies are maintained on a high-fat diet (HFD) the obesity phenotype appears. This reduces heart contractility, blocks conduction, increases structural disease, and causes an abnormal heart rhythm [4]. Therefore, an important way to manage HFD-induced abnormal cardiac rhythm is by controlling lipid metabolism.

Atrial fibrillation (AF) in humans is a clinically severe arrhythmia, similar to the fibrillation of the Drosophila myocardium. A HFD in Drosophila can increase the heart rate and arrhythmia index and cause fibrillation [5]. Fibrillation of the Drosophila heart may result from lipotoxic damage related to the insulin-TOR signal, which is moderate reduction in insulin-TOR signaling prevents HFD-induced obesity and cardiac dysfunction [4]. Apolipoprotein B (apoB) is related to lipid metabolism. It is the carrier of very-low-density lipoprotein (VLDL) and chylomicrons (CM) [6]. In Drosophila, HFD feeding leads to overexpression of apoLpp (apolipoprotein B homologous gene) in the heart and lipid accumulation while inhibiting the expression of cardiomyocyte-derived apoLpp can reduce the effects of an HFD [7]. Some studies have reported the role of Drosophila apoLpp in lipid metabolism but the effects of cardiomyocyte-derived apoLpp on abnormal heart rhythm under an HFD are unclear.

Exercise is an effective means for treating obesity and improving cardiovascular health [8, 9]. Obesity increases the incidence of atrial fibrillation, but this can be reduced by exercise [10]. In Drosophila, moderate exercise can reduce HFD-induced lipid toxicity damage to the heart, reduce the occurrence of fibrillation, and improve heart rhythm and contractile function [5, 11, 12]. However, the effects of regular exercise combined with cardiomyocyte apoLpp knockdown on abnormal heart rhythm under HFD are unclear. This study documented the effects of regular exercise and apoLpp knockdown in cardiomyocytes on abnormal heart rhythms in Drosophila melanogaster.

Materials and methods

Fly stocks and diet

We obtained wild-type W and Hand-Gal4 (W; P{y w = GMR88D05-GAL4}attP2) Drosophila melanogaster from the fruit fly breeding room of Hunan Provincial Key Laboratory of Physical Fitness and Sports Rehabilitation of Hunan Normal University; UAS-apoLpp RNAi (y sc v sev; P{TRiP.HMS00265}attP2/TM3, Sb) were purchased from the Bloomington Drosophila Stock Center, Bloomington, IN, USA. Females of the UAS-apoLppRNAi strain were crossed with males of W and F1 generation virgin flies were collected as a control group. Females of the UAS-apoLppRNAi strain were crossed with males of Hand-Gal4, and the F1 generation virgin flies were collected for the intervention group (excluding the female flies with short bristles). All of the flies are placed in an HWS intelligent incubator and maintained at 25°C, 50% relative humidity (RH), and a 12:12 h (L:D) photoperiod. Unless otherwise stated, all flies used in the experiment were females. Their larger size made dissection easier and they were used in previous imaging work in our lab [13]. Normal food (NF) was a combination of yeast, corn starch and molasses. An HFD was made with 30% coconut oil mixed with 70% NF [4].

HFD feeding regime

Virgin flies were collected and placed on NF medium for five days of aging. They were then transferred to fresh HFD medium for five days [4].

Regular exercise protocols

Based on the negative geotaxis behavior of flies [14], we developed a Drosophila exercise device, which rotates a glass tube to induce the flies to climb upwards [11]. The experiment used HFD fed flies that also exercised. The exercise device rotated 180° every 24 s, and the exercise duration was 1.5 h daily for five days. This exercise intensity was sufficient to reduce the body lipid level of the flies [11]. The flies are placed in an incubator at 25°C with 50% RH and a 12:12 h (L:D) photoperiod. Exercise was conducted regularly in a temperature-controlled room at 25°C. A sponge plug was adjusted before exercise so that all flies had the same movement distance in the glass tube. In the exercise training intervention, flies spent 1.5 h in a glass tube without food. Flies in the exercise-trained control group were also placed in a glass tube without food for 1.5 h (they were in the same environment as the control group but had no exercise training). We ensured that all flies were in the same environment to avoid influencing their feeding rate [15].

qRT-PCR

A total of 60 flies were dissected in PBS to open the abdominal cuticle and expose the heart. A vacuum pump was used to suck up the excess impurities around the heart (fat and arterial muscle attached to the heart, as well as the surrounding kidney cells). We put 60 isolated hearts into 1 mL of TRIzol reagent lysis solution to extract RNA, and added 10 μL of RNase Free dH2O to dissolve them. We used a Takara reverse transcription kit for reverse transcription. The cDNA was diluted to 40 μL with RNase Free dH2O and stored at −20°C. We used a Takara qRT-PCR kit to perform real-time quantification on a Bio-Rad 96-well fluorescent quantitative RT-PCR instrument (ABI7300, Applied Biosystems, USA). We determined the relative gene expression level by comparing the CT method. apoLpp primer: F = 5’-AATTCGCGGATGGTCTGTGT-3’; R = 5’-GCCCCTTAGGGATAGCCTTT-3’. Gapdh primer: F = 5’-GCGTCACCTGAAGATCCCAT-3’; R = 5’-GAAGTGGTTCGCCTGGAAGA-3’.

Semi-intact Drosophila preparation and image analysis

We measured cardiac function parameters using previously described methods [16, 17]. FlyNap (Sangon Biotech, Shanghai, China) was used to anesthetize the flies and then we performed semi-exposed heart dissection. We used an EM-CCD high-speed camera to capture the fly heartbeat (130 fps, 30-s video), semi-automated optical heartbeat analysis software (SOHA) to quantify the heart rate (HR), arrhythmia index (Arrhythmia Index, AI), and fibrillations (FL).

Body weight and triglycerides assay

We used an electronic microbalance (Uni bloc, AUW220D, Japan) to weigh the flies and we recorded the weight of each fly for analysis (S1 Table).

We used the Insect TG ELISA Kit (mlbio, China) to measure the TG concentration according to manufacturer instructions. For quantification of TGs in whole flies, flies (15 per genotype) were weighed and homogenized in PBS containing 0.1% Triton-X100 in an amount (μl) that was 8 X the total weight of the flies (μg). Then, centrifugation was used and the supernatant was obtained. Fifty microliters of standard or sample were added to the appropriate wells. Blank wells had nothing added. One hundred microliters of enzyme conjugate were added to standard wells and sample wells except for the blank well; they were covered with an adhesive strip and incubated for 60 minutes at 37°C. The microtiter plate was washed four times then, Substrate A (50 μl) and Substrate B (50 μl) were both added to each well, mixed gently, and incubated for 15 minutes at 37°C (while being protected from light). Following this, 50 μl stop solution was added to each well. The color in the wells should change from blue to yellow. If the well color was green or the color change does not appear uniform, the plate was gently tapped to ensure thorough mixing. The optical density (OD) was read at 450 nm using a microtiter plate reader within 15 minutes. Using the OD value of the measured standard product as the abscissa and the standard product’s concentration value as the ordinate, the standard curve was drawn to obtain the linear regression equation. The OD value of the sample was substituted into the equation to calculate the concentration of the sample. The triglyceride standard curve obtained is presented in the (S1 Fig).

Negative geotaxis assay

Negative geotaxis is an innate escape response of Drosophila [18]. To test the climbing ability of flies, we transferred the flies to a 20-cm-long glass tube divided into nine areas and sealed with a sponge plug to leave 18 cm as the fly climbing area. Each area had a fixed score. The tube was shaken every 30 s to make the flies fall to the bottom of the tube and this was repeated three times. Before shooting, we allowed the flies to acclimate in the glass tube for 10 min. We then used a digital camera to take a video of flies climbing, capture the climbing image after 8 s, and calculate the total score. Climbing index = total score/total number (S2 Table).

Quantification of ORO staining

Photoshop software was used to quantify the intensity of the ORO staining [19]. The all of the eggs and intact intestines of flies were removed in PBS, fixed with 4% paraformaldehyde for 20 min, and washed twice with PBS. They were dyed with fresh ORO dye solution (6 ml of 0.1% ORO in isopropanol and 4 ml of distilled water) for 20 min and rinsed with distilled water [20]. It should be noted that the heart and other internal organs of the fruit flies must be removed gently to avoid entrainment of fat and reduce the staining effect. A Leica stereomicroscope was used to collect bright-field images. We used Adobe Photoshop to adjust brightness and contrast to clarify the images. And then invert these color photos into black and white images in Photoshop.These color images were inverted (image > adj. > invert) in Photoshop to obtain black/white images. Intensity was measured using histogram analysis (mean pixels). Five individual sections (using a constant square area) were measured and averaged within each flies. The photos used for analysis are in the (S1 File).

Statistical analyses

We used SPSS (version 22.0 for Windows; SPSS Inc., Chicago, IL, USA) for statistical analysis, and comparisons between the NF group and other experimental groups used an independent t-test. We used two-way analysis of variance (ANOVA) and a least significant difference (LSD) test for analysis between groups (HFD, HE, HFD+KD, and HFD+E+KD). When the variance was not uniform, we used non-parametric tests such as Kruskal–Wallis one-way ANOVA (K sample) for post-adjustment. All of the data are expressed as mean±standard error (X±SEM), α = 0.05. *p<0.05, **p<0.01, ***p<0.001*.

Ethical statement

This study was approved by the Ethics Committee of Hunan Normal University.

Results

Increased dietary fat causes obesity and abnormal heart rhythms in Drosophila

To study the effect of lipid accumulation on cardiac function in Drosophila, we first determined the effect of an HFD on the whole-body lipid metabolism in Drosophila. In mammals, elevated TG levels are the main risk factor for obesity and metabolic syndrome [21, 22]. Obesity also increases the risk of cardiovascular disease and weakens physical activity [23]. After five days of feeding on an HFD, the flies gained weight (Fig 1A) and the intensity of ORO staining increased (Fig 1B). Furthermore, the whole-body TG level increased (Fig 1H), and the climbing index decreased (Fig 1I). These results demonstrated that HFD feeding can induce obesity in Drosophila.

Fig 1

UAS-apoLpp RNAi>W1118 group was exposed to HFD for 5 days, resulting in obesity and abnormal heart rhythm.

(A) Photographs and body weights of 10-day-old flies. The body weight was obtained by weighing flies on an electronic microbalance, N = 15. The flies in the HFD group were heavier compared to the NF group. (B) ORO staining of the abdomen of flies. Quantification of ORO intensity, N = 5. (B) ORO staining of fly abdomens. Quantification of ORO intensity, N = 5. The intensity of ORO staining in the abdomen of flies in the HFD group was higher, compared to the HF group. (C) Drosophila M-mode cardiogram, the red rectangle represents fibrillation, and the interception length is 10 s (This refers to the length of the electrocardiogram of 0–10 s). (D-F) M-Mode analysis. Quantification of the fly heart rate, arrhythmia index, and fibrillation, N = 30. The heart rate, arrhythmia index, and fibrillation of flies in the HFD group were higher than those in the NF group. (G) The relative expression level of apoLpp in cardiomyocytes of flies. The apoLpp in the cardiomyocytes of flies in the HFD group was significantly greater than in the NF group. The samples included at least 60 isolated hearts. (H) Whole-body TG levels in flies. The whole-body TG level of the HFD group was significantly higher than that of the NF group. N = 5, repeated three times. (I) Drosophila climbing index. The climbing index of the HFD group was significantly less than that of the NF group. The climbing index = number of flies at the top/total number of flies, N = 50, repeated three times. The detailed method is given in the (S2 Table).

Obesity is an important risk factor for atrial fibrillation. Atrial fibrillation is the most common persistent arrhythmia and can cause morbidity and mortality [24]. To detect whether the hearts of obese Drosophila were abnormal, we performed semi-intact preparations and M-mode analysis. Compared with the NF group, the heart rate of flies in the HFD group increased (Fig 1D), and the arrhythmia index and increased fibrillation increased (Fig 1E and 1F). These results indicate that HFD flies have abnormal heart rhythms and fibrillation (Fig 1C). In addition, apolipoprotein B is closely correlated to the occurrence of cardiovascular disease. When the level of apoB increases, individuals have an increased risk of cardiovascular disease, while reducing the level of apoB can reduce cardiovascular disease [25, 26]. In Drosophila, apoB plays an important role in controlling systemic lipid metabolism [7]. To determine whether HFD increased the expression of apoLpp, we detected apoLpp levels in the hearts of HFD Drosophila. The expression level of apoLpp in the heart of HFD Drosophila significantly increased, compared with the level in the NF group (Fig 1G). These results indicate that an increase in dietary fat leads to obesity and abnormal heart rhythm in Drosophila. The mechanism may be related to the rise in cardiomyocyte apoLpp.

Cardiomyocyte-specific apoLpp knockdown can resist HFD-induced obesity and abnormal heart rhythm

It is unclear how obesity induces abnormal heart rhythms. The apoLpp is related to systemic lipid metabolism and heart function in Drosophila [7]. Therefore, we tested whether cardiomyocyte-derived apoLpp is involved in the abnormal heart rhythm caused by obesity. To determine whether knocking down apoLpp can resist abnormal heart rhythm caused by obesity, we used Hand-Gal4 to specifically inhibit the expression of cardiomyocyte apoLpp (Fig 2G). We first tested whether the increase in whole-body TG levels due to HFD was suppressed in the HFD+KD group of flies. The knockdown of cardiomyocyte apoLpp significantly reduced the whole-body TG level of flies (Fig 2H). Compared with the HFD group, the body weight of flies in the HFD+KD group was reduced, and the intensity of ORO staining was reduced (Fig 2A and 2B). These results indicate that reducing the expression of apoLpp in cardiomyocytes helps to weaken the obesity caused by HFD. Since it has been demonstrated that HFD can induce an abnormal heart rhythm in Drosophila, and the inhibition of apoLpp can reduce the obesity caused by HFD, we speculated that the inhibition of apoLpp in the cardiomyocytes of flies can reduce the abnormal heart rhythm induced by HFD. We tested whether the inhibition of apoLpp in cardiomyocytes can reduce abnormal heart rhythms under HFD conditions. As expected, compared with the HFD group, the heart rate of flies in the HFD+KD group decreased (Fig 2D), and the arrhythmia index (Fig 2E) and fibrillation (Fig 2F) were reduced. The abnormal heart rhythm of flies was improved (Fig 2C). These findings indicate that the inhibition of cardiomyocyte apoLpp in Drosophila can reduce HFD-induced obesity, which may help reduce the adverse effects of obesity on the heart.

Fig 2

Under HFD feeding, inhibition of apoLpp in cardiomyocytes can reduce obesity and abnormal heart rhythm.

(A) Photographs and body weights of 10-day-old flies. The body weight was obtained with an electronic microbalance, N = 15. The flies in the HFD+KD group were lighter compared to the HFD group. (B) ORO staining of the fly abdomens. Quantification of ORO intensity, N = 5. The intensity of ORO staining in the fly abdomens in the HFD+KD group was reduced, compared with the HFD group. (C) Drosophila M-mode cardiogram, the red rectangle represents fibrillation, and the interception length was 10 s (This refers to the length of the electrocardiogram of 0–10 s). (D-F) M-Mode analysis. Quantification of fly heart rate, arrhythmia index, and fibrillation, N = 30. The heart rate, arrhythmia index, and fibrillation of flies in the HFD+KD group were lower than in the HFD group. (G) The relative expression level of apoLpp in the cardiomyocytes of flies. The apoLpp in cardiomyocytes of flies in the HFD+KD group was significantly lower than that in the HFD group. The test samples included at least 60 isolated hearts. (H) Whole-body TG levels in flies. The whole-body TG level of the HFD+KD group was significantly lower than that of the HFD group. N = 5, repeated three times.

Regular exercise reduced the expression level of apoLpp mRNA in cardiomyocytes and reversed the abnormal heart rhythm caused by the HFD diet

Appropriate exercise can aid in weight reduction and can reduce the risk of cardiovascular events [9, 27]. Exercise training can increase cardiac lipid metabolism and prevent cardiovascular disease [28]. However, it is unclear if regular exercise can improve abnormal heart rhythm by changing apoLpp levels in cardiomyocytes. To test this, we made flies exposed to an HFD exercise regularly. The expression of apoLpp in cardiomyocytes of flies in the HE group was significantly lower than that in the HFD group (Fig 3H). In addition, after regular exercise, flies lost body weight, the intensity of ORO staining decreased and the level of TG in the whole-body decreased (Fig 3A, 3B and 3G). To further determine whether regular exercise can reduce the abnormal heart rhythm caused by obesity, we used M-mode to analyze the heart function of flies exposed to HFD. Compared with the HFD group, the heart rate of flies in the HE group decreased (Fig 3D) and the arrhythmia index (Fig 3E) and fibrillation (Fig 3F) also decreased. The abnormal heart rhythm of the flies was improved (Fig 2C). These findings show that regular exercise can downregulate the expression level of apoLpp in cardiomyocytes, thereby reducing the abnormal heart rhythm caused by obesity.

Fig 3

Regular exercise can reduce the expression level of apoLpp mRNA in cardiomyocytes and reverse the abnormal heart rhythm caused by the HFD diet.

(A) Photographs and body weights of 10-day-old flies. The body weight was obtained using an electronic microbalance, N = 15. The body weight of flies in the HE group was lower than the weight of the HFD group. (B) ORO staining of the abdomen of flies. Quantification of ORO intensity, N = 5. The intensity of ORO staining in the abdomen of flies in the HFD+E group was reduced compared to the HFD group. (C) Drosophila M-mode cardiogram, the red rectangle represents fibrillation, and the interception length is 10 s (This refers to the length of the electrocardiogram of 0–10 s). (D-F) M-Mode analysis. Quantification of the fly heart rate, arrhythmia index and fibrillation, N = 30 and 20. The heart rate, arrhythmia index, and fibrillation of flies in the HFD+E group were lower than those in the HFD group. (G) Whole-body TG levels in flies. The whole-body TG level of the HFD+E group was significantly lower than that of the HFD group. N = 5, repeated three times. (H) Relative expression level of apoLpp in cardiomyocytes of flies. The apoLpp in cardiomyocytes of flies in the HFD+E group was significantly lower than that in the HFD group. The samples included at least 60 isolated hearts.

Regular exercise combined with the knockdown effect of cardiomyocytes apoLpp has a significant effect on obesity-induced abnormal heart rhythm

The question the above findings raise is whether regular exercise and the knockdown effect of cardiomyocytes apoLpp have a significant effect on abnormal heart rhythm caused by obesity. To make it easier to observe the difference, we compare the data of the HFD control group with the other three groups side by side. We found that the body weight of the HFD group was higher than the other three groups. In addition, compared with the NF and HFD+KD groups, the HFD+E+KD group has no significant difference in the body weight of the flies. (Fig 4A). For ORO staining, the ORO staining intensity of HFD+KD and HFD+E+KD groups was significantly lower than that of HFD group, but they were higher than NF group. (Fig 4B). These data indicate that the knockdown of apoLpp in cardiomyocytes or the intervention of knockdown, combined with exercise, can resist the increase in body weight of flies due to HFD. In addition, although the above-mentioned intervention method can control body weight, it is less effective in controlling abdominal fat, because the intensity of ORO staining was higher than that of the NF group. M-mode data showed that the heart rate, arrhythmia index and fibrillation of the HFD group were significantly higher than those of the other three groups. In addition, compared with the NF group, there were no significant differences in heart rate, arrhythmia index, and fibrillation in the HFD+E+KD group (Fig 4C–4F). Compared to the NFD+KD group, the HFD+E+KD group was not different in heart rate and fibrillation (Fig 4C, 4D and 4F), while the arrhythmia index was significantly lower (Fig 4E). These results indicate that regular exercise, combined with the knockdown of cardiomyocytes apoLpp, significantly impacts the arrhythmia index. In addition, the expression level of apoLpp mRNA in flies cardiomyocytes in the HFD group was significantly higher than that of the other three groups. And compared with flies in the NF and HFD+KD groups, the expression level of apoLpp in the heart of the HFD+E+KD group was significantly reduced (Fig 4G), and the level of TG in the whole-body was reduced (Fig 4H). These results indicate that regular exercise, in combination with apoLpp knockdown in cardiomyocytes, can further reduce the expression of apoLpp in cardiomyocytes. This reduces the increase in TG levels of flies under HFD conditions, even lower than normal. In summary, these data indicate that in addition to regular exercise, cardiomyocyte apoLpp is required to improve HFD-inducted abnormal heart rhythm requires, because apoLpp is more flexible during HFD feeding.

Fig 4

Combined effect of regular exercise and apoLpp knockdown in cardiomyocytes.

(A) Photographs and body weights of 10-day-old flies. The body weight was obtained using an electronic microbalance, N = 15. The body weight of flies in the HFD group was significantly higher than the other three groups. (B) ORO staining of the abdomen of flies. Quantification of ORO intensity, N = 5. The intensity of ORO staining of flies in the HFD group was significantly higher than that of the other three groups. (C) Drosophila M-mode cardiogram and the interception length was 10 s (This refers to the length of the electrocardiogram of 0–10 s). (D-F) M-Mode analysis. Quantification of fly heart rate, arrhythmia index and fibrillation, N = 30. The heart rate, arrhythmia index and fibrillation of the three groups of NF, HFD+KD and HFD+E+KD were significantly lower than those of the HFD group. Compared with the HFD+KD group, the HFD+E+KD group had no significant difference in heart rate and fibrillation, but the arrhythmia index was significantly lower. (G) The relative expression level of apoLpp in cardiomyocytes of flies. The expression level of apoLpp mRNA in cardiomyocytes of HFD group was significantly higher than that of the other three groups. In addition, compared with HFD+KD, the expression of apoLpp mRNA in cardiomyocytes of HFD+E+KD group was significantly reduced. The samples included at least 60 isolated hearts. (H) Whole-body TG levels in flies. The whole-body TG level in the HFD group was significantly higher than the other three groups. In addition, compared with the NF and HFD+KD groups, the HFD+E+KD group’s whole-body TG levels were significantly lower. N = 5, repeated three times.

Discussion

Effects of high-fat diet on the expression of apoLpp in the cardiomyocytes of Drosophila

The imbalance between energy intake and energy expenditure leads to obesity and obesity can cause abnormalities in cardiovascular hemodynamics, heart shape and ventricular function [29]. In mice, an HFD period can cause abnormal lipid metabolism, which is mainly manifested as abnormal blood lipid/glycemia, insulin resistance, and damage to heart function [30]. In Drosophila, the damage to heart function caused by obesity induced by an HFD is evolutionarily conserved [31]. Other studies have found that an HFD can cause increased expression of apoLpp in Drosophila myocardial cells, weakened myocardial contractility, abnormal heart rhythm and remodeling of the heart structure [4]. After exposure to HFD for five days, the fly whole-body TG level increased, body weight increased, ORO staining intensity increased and cardiac function became abnormal (rapid heart rate, arrhythmia, fibrillation). We also found that an HFD-induced abnormal heart rhythm and fibrillation are related to the increased expression of apoLpp in cardiomyocytes.

Elevated levels of apolipoprotein B are a sign of metabolic syndrome, including obesity, diabetes, and heart disease [32]. The function of apoLpp in Drosophila is similar to that of apolipoprotein B in mammals (its receptor is homologous to mammals), and they can transport lipids and sterols to surrounding tissues to control the storage and mobilization of neutral lipids in cells [33, 34]. Drosophila cardiomyocyte apoLpp plays an essential role in controlling systemic lipid metabolism and actively responds to HFDs [7]. Therefore, the effect of an HFD on the apoLpp of cardiomyocytes is highly correlated, but the relationship between apoLpp and abnormal heart rhythm induced by an HFD is still unclear.

The effects of cardiomyocytes apoLpp knockdown in HFD flies on abnormal heart rhythm

An HFD can cause cardiac lipid toxicity and induce cardiac dysfunction, mainly including arrhythmia, fibrillation, and weakened contractility [5, 11, 12, 31]. However, it is unclear whether abnormal heart rhythm induced by an HFD is affected by apoLpp. To further explore the abnormal heart rhythm induced by an HFD, we knocked down the apoLpp gene in the cardiomyocytes of flies. The apoLpp expression level of fly cardiomyocytes in the HFD+KD group was reduced by 87.7%, compared with the HFD group, which indicated the successful construction of the knockdown strain. The M-type results showed that the heart rate was slowed, the arrhythmia index was reduced, and fibrillation did not occur, reflecting the improvement of heart function. These data indicate that the knockdown of apoLpp in cardiomyocytes can resist the abnormal heart rhythm caused by HFD. Moreover, in the flies exposed to HFD, after apoLpp knockdown in cardiomyocytes, whole-body triglyceride levels were significantly reduced, body weight decreased, and ORO staining intensity decreased. These data indicate that the knockdown of cardiac apoLpp can significantly improve lipid accumulation caused by a HFD, which is consistent with the results of other studies [7]. Knockdown of apoLpp in cardiomyocytes can improve abnormal heart rhythm, which may be caused by changes in the lipid overload environment [35].

Effects of apoLpp knockdown in fly cardiomyocytes, combined with regular exercise, on abnormal heart rhythm

Physical exercise can enhance heart function and reduce diabetic cardiomyopathy, coronary heart disease, and heart failure [36, 37]. Both rats and mice can improve heart disease induced by high glucose or high fat through regular exercise [38, 39]. Drosophila has shown similar results to mammals in terms of heart damage caused by high fat or high sugar [40, 41]. In addition, exercise also has many benefits for flies. Examples include lipotoxic cardiomyopathy and improvement of heart rhythm [11, 12], as well as resistance to age-related degenerative changes in the heart. Previous studies showed that exercise in aged flies can effectively reduce the occurrence of fibrillation [11]. Regular exercise of flies provides partial resistance to the heart function damage induced by an HFD but it is still unclear how regular exercise combined with knockdown of apoLpp gene in cardiomyocytes affects arrhythmia. To clarify this issue, we performed regular exercises on the flies exposed to the HFD. Compared to the respective control group, the expression of apoLpp in the cardiomyocytes of flies in the exercise group was significantly reduced. In addition, whole-body triglyceride levels, body weight, and intensity of ORO staining were reduced. These data demonstrate that regular exercise can reduce the expression of apoLpp in cardiomyocytes caused by an HFD, thereby resisting obesity. M-mode results also showed that regular exercise can reduce heart rate, reduce arrhythmias and reduce fibrillation. In addition, the Drosophila cardiogram indicated that the HFD caused more serious arrhythmia in flies such as fibrillation. When flies exposed to HFD performed regular exercise, there was no fibrillation, and arrhythmia improved. When flies exposed to HFD had apoLpp knocked down in the cardiomyocytes, fibrillation also did not occur. In addition, regular exercise combined with the knockdown of cardiomyocytes apoLpp can further improve the arrhythmia of flies under HFD. This was manifested as a decrease in heart rate, an increase in the cardiac cycle and a decrease in arrhythmia index. Therefore, not only the knockdown effect of apoLpp, but also regular exercise can drive the expression of apoLpp, which in turn affects the arrhythmia induced by an HFD. This study also found that, compared with flies exposed to an HFD and knocked down apoLpp in cardiomyocytes, regular exercise resulted in lower levels of apoLpp in cardiomyocytes, and a slight decrease in heart rate, arrhythmia index, and fibrillation. These results indicate that the benefit of regular exercise on abnormal arrhythmia requires cardiomyocyte apoLpp, because cardiomyocyte apoLpp RNAi is more flexible during HFD. It is worth noting that the feeding rate is an important reference for studying metabolic regulation [42]. Although HFD itself has a profound impact on feeding rate, it does not affect the establishment of the HFD model in this study. In addition, exercise can also affect food intake, such as overeating after exercise [43]. In the future, it will be interesting to explore the effect of Drosophila exercise-regulated feeding rate on lipid metabolism.

Other studies have reported that ovarian triglyceride levels represent only a small part of whole-body triglyceride levels [44, 45] in females. The accumulation of lipids is also not negligible in reproduction, because the ovary is an important area of fat accumulation in female Drosophila. The relationship between lipid metabolism in the ovary of female Drosophila and heart function remains unclear. Considering the shuttle mechanism of lipoproteins, when cardiomyocytes apoLpp is specifically knocked down, it will not lead to a sudden decrease of apolipoprotein B. This may be because apolipoprotein B from the fat body enters the heart through hemolymph circulation. Although this subtle difference is interesting, it does not affect the contribution of inhibiting apoLpp in cardiomyocytes to the total amount of apoLpp in the whole-body. In summary, this study shows that apoLpp reduction may be involved in exercise-induced protection against HFD.

Standard curve diagram of D. melanogaster whole-body triglyceride detection.

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Data from all weight panels in this study.

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Drosophila climbing index.

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ORO stained photo of flies abdomen.

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5 Aug 2021

PONE-D-21-21284

The effect of regular exercise and apolipoprotein B knockdown on abnormal cardiac rhythm induced by HFD

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Reviewer #1: Partly

Reviewer #2: Partly

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Reviewer #1: No

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #2: Yes

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Reviewer #1: Manuscript compares the effect of heart-specific apoLpp knockdown and the effect of exercise on consequences of a high fat diet. Although the effects of HFD, apoLpp and exercise on heart performance have all been examined previously, it is of some interest to look at the combination. There are several significant problems with the study that reduce enthusiasm, including genetic background issues, lack of controls for female-specific dietary responses to egg laying, and inappropriate use of Oil Red O as a lipid quantitation assay.

Major:

1. Not enough attention has been paid to rigor in the control of genetic background. Comparisons are made between outcrossed animals and isogenic control animals without recognizing possible effects of hybrid vigor on phenotypes.

2. Rescue experiments are compared to each other (e.g. exercise to apoLpp RNAi) without including comparisons to wild type. This makes it impossible to quantitate the extent of rescue.

3. No attention has been given to possible effects of any of the treatments on feeding rate.

4. Although experiments are done in females, no attention has been given to effects of reproduction on lipid accumulation. This is an essential point to address, because eggs are a substantial subset of the lipid accumulation in any female.

5. No rationale is given for limiting experiments to females.

6. Figures showing representative flies are clearly of different lengths and overall sizes, which surely must complicate assessment of whether they have "flatter abdomens". Abdominal bulging may result from reduced egg deposition and must be controlled for.

7. Oil Red O staining is not a quantitative assay for lipid content.

8. It is a major stretch to say that apoLpp RNAi phenotypes prove that effects of HFD are because of abnormal lipoprotein concentration.

9. Discussion section does not effectively place the work in context, and basically restates the Introduction. Also, it is labelled "Discuss".

Minor:

1. ApoLpp should be spelled out and defined at first mention

2. Manuscript should be heavily edited for grammar.

3. methods descriptions should be in past tense, and some of the statistical methods do not appear quite right. For example, pairwise comparisons after a 2-way ANOVA should use post-hoc adjustment, such as a Tukey test. All the comparisons in the paper appear to be pairwise, so it is not clear why the stats methods describe something different?

4. What is interception length?

5. does "half-exposure surgery" mean semi-intact preparation?

6. Description of Figure 1 refers incorrectly to figure 2 in several places, which is very confusing

7. meaning of "superimposing effect" is not clear

Reviewer #2: Ding et al present an interesting group of studies showing the role of exercise and the apoLpp protein in high-fat-induced cardiac dysfunction. This is a nice short paper with high quality data and will be of interest to those studying the roles of diet and lipid transport on cardiac function. However, a few changes need to be made before I can recommend it for publication in PLoS One.

The triglyceride (TG) assay method is not described correctly- there are no reagents listed and an antibody/substrate approach makes no sense in this protocol because triglycerides are not typical protein antigens. A standard curve should also be used in this assay so the ug TG/mg body weight can be reported. Without the proper methods, the data on TG cannot be evaluated.

It would also be helpful if you described briefly how you isolated hearts; on line 210, it says that qPCR was used to quantify mRNA in cardiomyocytes, but the heart has nephrocytes and aliary muscles that are stuck to it and are sometimes included in the prep.

The heart rate of ~ 2 sec is surprising to me because most research papers have a rate of 2-3 beats per second. Perhaps the units should be Hz instead of seconds? Please double check this.

For Fig 2B’s legend, “there is no accumulation of fat in the abdomen of flies in the HC-KD group” is not supported by the image. Reduced accumulation even looks like a stretch, but seems to be corroborated by the quantitative data in 2H provided that a legitimate TG assay was done.

Figure 3’s title text “Regular exercise improves abnormal heart rhythm by reducing the expression of apoLpp mRNA in cardiomyocytes” is not supported by the data. The authors have only shown that apoLpp is reduced by exercise, not that this is the mechanism by which heart function is improved. For that, I think you’d have to overexpress apoLpp in the heart during exercise and show that heart function differs from the exercised control genotype. This experiment is one I would request if this were a different journal, but isn’t necessary to publish in PLoS One.

Fig 3G is TG and Fig 3H is apoLpp mRNA, but the legend has it the other way around.

With respect to the figure legends for panel A in Figs 1-4, I think it would be more clear if the text read simply “the abdomen is larger” (or smaller) rather than more prominent or flatter. It’s harder to think about how prominence and flatness are being measured and these might mean different things to different people. Large/small is straightforward- it would be even better if you graphed their wet or dry weights, data you might already have from the triglyceride assays. For 4A, remove significant; significant is reserved for statistical significance in my mind. You might say “no dramatic difference” instead. Again in 4A, I would focus on the size (smaller or larger), rather than “morphology,” which is not as easily ascertained from the images provided. There is only one view shown (perhaps the dorsal abdomen is spotted!) and the size is the most apparent difference in all of these panels.

It would be of interest to directly compare control and apoLpp RNAi responses to exercise. This is difficult considering the way the figures are currently set up; perhaps it could be helpful to add a table or expand the Results or Discussion to mention these. I found the term “superimposed” difficult to understand. It could be rephrased as “Cardiac apoLpp is required for (some of) the benefits of exercise” – then you might consider why there is no longer a benefit from exercise. It may be because apoLpp RNAi hearts are more resilient during HF feeding. So cardiac apoLpp knockdown could be metabolically equivalent to exercising… this would be great, like an exercise mimetic. When I compare 3D and 4D, it looks like the heart rate is already an “exercised” value in the RNAi flies without exercise; the same seems to be true for fibrillation rates in 3F vs 4 F.

There are several small mistakes in writing that should be addressed- I will only mention a few here. Genes and mRNAs should be italicized. Line 110 should say reagent and line 111 and elsewhere should say H2O with a subscript #2. The use of female flies appears four times in the Fig 1 legend and three times in Figure 4’s legend and should be compressed to be as concise as possible. You can mention all flies are female early in the methods and results- then maybe in the Discussion (not Discuss, as on line 336) when comparing your findings to other high fat diet studies.

Finally, I am sleep deprived but I found it hard to keep it in my mind that HFD=HC. Why not use HFD throughout all of the text and figures? It would be easier for some people.

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Reviewer #2: No

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21 Sep 2021

List of Responses

Dear Editors and Reviewers:

Thank you for your letter and for the reviewers’ comments concerning our manuscript entitled “The effect of regular exercise and apolipoprotein B knockdown on abnormal cardiac rhythm induced by HFD” (ID: PONE-D-21-21284). Those comments are all valuable and very helpful for revising and improving our paper, as well as the important guiding significance to our researches. We have studied comments carefully and have made correction which we hope meet with approval. Revised portion are marked in red in the paper. The main corrections in the paper and the responds to the reviewer’s comments are as flowing:

Responds to the editor's comments:

1.We carefully modify the format of the manuscript to ensure that the manuscript meets the style requirements of PLOS ONE.

2.After reviewers’ suggestions, some experiments were added, and qualitative analysis was transformed into quantitative analysis, making the article more objective.

3.This research was funded by the following: National Natural Science Foundation of China (project number: 32071175); the Hunan Province Graduate Education Innovation Project and Professional Ability Enhancement Project Fund (Project Number: CX20200533); China Postdoctoral Science Foundation funded project（Project Number: 2017M622580). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

4.The data in this study are shared. We have generated supporting information files and will upload them together with the manuscript.

5.We have added a complete ethics statement in the Methods section of the manuscript.

6.Supporting information has been added to the end of the manuscript, and update any in-text citations to match accordingly.

Responds to the reviewer’s comments:

Reviewer #1:

Major:

Q1. Not enough attention has been paid to rigor in the control of genetic background. Comparisons are made between outcrossed animals and isogenic control animals without recognizing possible effects of hybrid vigor on phenotypes.

A1. We acknowledge that we did not use multiple GAL4 drivers to generate tissue-specific knockdowns. But before the study, we did some related work on the control of genetic background. In this study, we used the F1 generation crossed between W1118 and UAS-RNAi as a control. These flies are the offspring of a single cross. Secondly, we observed that the traits of the F1 generation of GAL4>UAS-RNAi are stable.

Q2. Rescue experiments are compared to each other (e.g. exercise to apoLpp RNAi) without including comparisons to wild type. This makes it impossible to quantitate the extent of rescue.

A2. In the rescue experiment, we supplemented the control group and quantified the degree of rescue. The specific content is in the revised manuscript.

Q3. No attention has been given to possible effects of any of the treatments on feeding rate.

A3. We carefully considered this issue and referred to the related article (DOI: 10.1371/journal.pone.0006063). The feeding rate of fruit flies is related to circadian rhythm, group size, and diet composition. In this experiment, we strictly follow the formula to make food for raising fruit flies. In addition, the fruit flies are reared in a constant incubator (25°C, 12 hours day and night). For the collected virgin flies, each bottle contained only 30 fruit flies to avoid the influence of group size on the feeding rate. In addition, in the exercise training intervention, fruit flies were allocated to spend 1.5 hours in a glass tube without food. At the same time, the fruit flies in the exercise-trained control group will also be allocated to a glass tube without food for 1.5 hours (they are in the same environment, the control group just has no exercise training). In short, the control of these conditions can overcome the influence of different treatments on the feeding rate of fruit flies to a certain extent.

Q4. Although experiments are done in females, no attention has been given to effects of reproduction on lipid accumulation. This is an essential point to address, because eggs are a substantial subset of the lipid accumulation in any female.

A4. At present, the study of heart-derived apoLpp for systemic lipid regulation is not comprehensive, and its mechanism is likely to be related to ovarian lipid synthesis. This is an excellent suggestion. Although the data of this experiment does not involve the effect of reproduction on lipid accumulation, we will further study the relationship between exercise-regulated heart-derived apoLpp and eggs in the future.

Q5. No rationale is given for limiting experiments to females.

A5. This is omitted in the introduction. There are two reasons why the subjects are restricted to females. Objective reason: In Drosophila, females have more triglyceride storage than males, and the triglyceride decomposition speed in response to lipolysis stimuli is slower. Subjective reasons: females are large and easy to observe and dissect. We have added relevant explanations in the Introduction.

Q6. Figures showing representative flies are clearly of different lengths and overall sizes, which surely must complicate assessment of whether they have "flatter abdomens". Abdominal bulging may result from reduced egg deposition and must be controlled for.

A6. We made some changes to this section. We added a chart of the wet weight of fruit flies on the basis of representative photos and used it to analyze the effects of different treatments on the bodyweight of fruit flies. The detailed revision is in the uploaded manuscript.

Q7. Oil Red O staining is not a quantitative assay for lipid content.

A7. We recognized this error and revised the description in the full text. We refer to a reasonable plan to use ORO to quantify abdominal lipids. (doi:10.1016/j.cub.2017.06.004.)

Q8. It is a major stretch to say that apoLpp RNAi phenotypes prove that effects of HFD are because of abnormal lipoprotein concentration.

A8. Indeed, this statement is wrong without the support of western blot experiments, and we have changed it in the article.

Q9. Discussion section does not effectively place the work in context, and basically restates the Introduction. Also, it is labelled "Discuss".

A9. For the discussion part, we reorganized the logic and revised it.

Minor:

Q1. apoLpp should be spelled out and defined at first mention

A1. The spelling and definition of apoLpp have been added in the corresponding place.

Q2. Manuscript should be heavily edited for grammar.

A2. Due to the limitation of the native language, we handed over the manuscript to a professional polishing company for grammar revision.

Q3. Methods descriptions should be in past tense, and some of the statistical methods do not appear quite right. For example, pairwise comparisons after a 2-way ANOVA should use post-hoc adjustment, such as a Tukey test. All the comparisons in the paper appear to be pairwise, so it is not clear why the stats methods describe something different?

A3. This is a descriptive error. In the case of two factors, we used a two-way analysis of variance and used the LSD test for post-hoc adjustment.

Q4. What is interception length?

A4. We took a video of the heartbeat of fruit flies and output an electrocardiogram. "Interception length is 5s." This refers to the length of the electrocardiogram of 0-5s. During the re-production process, we increased the 5s to 10s and modified the corresponding description.

Q5. Does "half-exposure surgery" mean semi-intact preparation?

A5. Yes, we have changed to semi-intact preparation.

Q6. Description of Figure 1 refers incorrectly to figure 2 in several places, which is very confusing

A6. We have corrected this error and carefully compared the picture and text again.

Q7. Meaning of "superimposing effect" is not clear

A7. The superimposing effect means that regular exercise and apoLpp mRNA knockdown have a cumulative effect on the effect of systemic lipids. This statement was changed in the revised manuscript.

Reviewer #2:

Q1. The triglyceride (TG) assay method is not described correctly- there are no reagents listed and an antibody/substrate approach makes no sense in this protocol because triglycerides are not typical protein antigens. A standard curve should also be used in this assay so the ug TG/mg body weight can be reported. Without the proper methods, the data on TG cannot be evaluated.

A1. We re-described the determination method of triglycerides and added a standard curve in the supplementary information.

Q2. It would also be helpful if you described briefly how you isolated hearts; on line 210, it says that qPCR was used to quantify mRNA in cardiomyocytes, but the heart has nephrocytes and aliary muscles that are stuck to it and are sometimes included in the prep.

A2. We have added some details of separating the heart in the method section.

Q3. The heart rate of ~ 2 sec is surprising to me because most research papers have a rate of 2-3 beats per second. Perhaps the units should be Hz instead of seconds? Please double check this.

A3. I'm very sorry, this is a basic and serious mistake. The unit of heart rate is Hz. In addition, we checked the full text and corrected it.

Q4. For Fig 2B’s legend, “there is no accumulation of fat in the abdomen of flies in the HC-KD group” is not supported by the image. Reduced accumulation even looks like a stretch, but seems to be corroborated by the quantitative data in 2H provided that a legitimate TG assay was done.

A4. Indeed, ORO stained photos cannot directly quantify fat. We found a way to quantify the intensity of ORO staining using Photoshop (DOI: 10.1016/j.cub.2017.06.004). The specific modification is shown in the uploaded manuscript.

Q5. Figure 3’s title text “Regular exercise improves abnormal heart rhythm by reducing the expression of apoLpp mRNA in cardiomyocytes” is not supported by the data. The authors have only shown that apoLpp is reduced by exercise, not that this is the mechanism by which heart function is improved. For that, I think you’d have to overexpress apoLpp in the heart during exercise and show that heart function differs from the exercised control genotype. This experiment is one I would request if this were a different journal, but isn’t necessary to publish in PLoS One.

A5. We made some changes to the title of Figure 3 to make the description more accurate. “Regular exercise can reduce the expression level of apoLpp mRNA in cardiomyocytes and reverse the abnormal heart rhythm caused by the HFD diet”.

Q6. Fig 3G is TG and Fig 3H is apoLpp mRNA, but the legend has it the other way around.

A6. We have corrected this error.

Q7. With respect to the figure legends for panel A in Figs 1-4, I think it would be more clear if the text read simply “the abdomen is larger” (or smaller) rather than more prominent or flatter. It’s harder to think about how prominence and flatness are being measured and these might mean different things to different people. Large/small is straightforward- it would be even better if you graphed their wet or dry weights, data you might already have from the triglyceride assays. For 4A, remove significant; significant is reserved for statistical significance in my mind. You might say “no dramatic difference” instead. Again in 4A, I would focus on the size (smaller or larger), rather than “morphology,” which is not as easily ascertained from the images provided. There is only one view shown (perhaps the dorsal abdomen is spotted!) and the size is the most apparent difference in all of these panels.

A7. This is an excellent suggestion. We added a wet weight chart. This can be a good description of the information in Figure 1-4 panel A, and analyze the significance.

Q8. It would be of interest to directly compare control and apoLpp RNAi responses to exercise. This is difficult considering the way the figures are currently set up; perhaps it could be helpful to add a table or expand the Results or Discussion to mention these. I found the term “superimposed” difficult to understand. It could be rephrased as “Cardiac apoLpp is required for (some of) the benefits of exercise” – then you might consider why there is no longer a benefit from exercise. It may be because apoLpp RNAi hearts are more resilient during HF feeding. So cardiac apoLpp knockdown could be metabolically equivalent to exercising… this would be great, like an exercise mimetic. When I compare 3D and 4D, it looks like the heart rate is already an “exercised” value in the RNAi flies without exercise; the same seems to be true for fibrillation rates in 3F vs 4 F.

A8. We accept the reviewer's suggestion and delete the word "superimposed" and replace it with what is suggested in the comments. In addition, we also mentioned the response of apoLpp RNAi to exercise in the discussion section.

Q9. There are several small mistakes in writing that should be addressed- I will only mention a few here. Genes and mRNAs should be italicized. Line 110 should say reagent and line 111 and elsewhere should say H2O with a subscript #2. The use of female flies appears four times in the Fig 1 legend and three times in Figure 4’s legend and should be compressed to be as concise as possible. You can mention all flies are female early in the methods and results- then maybe in the Discussion (not Discuss, as on line 336) when comparing your findings to other high fat diet studies.

A9. We put "all fruit flies are female" in the method section. In addition, we also checked the full text and corrected minor flaws.

Q10. Finally, I am sleep deprived but I found it hard to keep it in my mind that HFD=HC. Why not use HFD throughout all of the text and figures? It would be easier for some people.

A10. We replaced HC in the full text with HFD.

9 Nov 2021