Exercise Training Upregulates Cardiac mtp Expression in Drosophila melanogaster with HFD to Improve Cardiac Dysfunction and Abnormal Lipid Metabolism

Simple Summary It is well-established that the heart regulates systemic lipid metabolism, but the exact molecular mechanisms remain largely unclear. A high-fat diet (HFD) can lead to systemic lipid overload and a range of cardiac dysfunctions such as arrhythmias, fibrillation and reduced contractility. Although pharmacological treatment is often effective, there are always side effects. Accordingly, exercise training represents an effective alternative intervention to medication. In this experiment, we established a high-fat Drosophila model by HFD feeding and conducted exercise training intervention to investigate whether the exercise intervention altered the expression of the target gene (mtp), thus affecting systemic lipid metabolism and cardiac function. Our results suggest that specific knockdown of mtp mitigates HFD-induced elevation of systemic triglycerides and protects cardiac contractility to some extent. Further analysis showed that exercise training could upregulate the expression of mtp to restore dysregulated systemic lipid metabolism and cardiac function induced by HFD. Overall, we explored the important role of mtp in systemic lipid metabolism and cardiac function, providing new insights for future clinical studies related to lipotoxic cardiomyopathy and the potential use of exercise training to treat abnormal lipid metabolism and cardiac dysfunction due to HFD. Abstract Current evidence suggests that the heart plays an important role in regulating systemic lipid homeostasis, and high-fat diet (HFD)-induced obesity is a major cause of cardiovascular disease, although little is known about the specific mechanisms involved. Exercise training can reportedly improve abnormal lipid metabolism and cardiac dysfunction induced by high-fat diets; however, the molecular mechanisms are not yet understood. In the present study, to explore the relationship between exercise training and cardiac mtp in HFD flies and potential mechanisms by which exercise training affects HFD flies, Drosophila was selected as a model organism, and the GAL4/UAS system was used to specifically knock down the target gene. Experiments revealed that HFD-fed Drosophila exhibited changes in body weight, increased triglycerides (TG) and dysregulated cardiac contractility, consistent with observations in mammals. Interestingly, inhibition of cardiac mtp expression reduced HFD-induced cardiac damage and mitigated the increase in triglycerides. Further studies showed that in HFD +w1118, HFD + Hand > w1118, and HFD+ Hand > mtpRNAi, cardiac mtp expression downregulation induced by HFD was treated by exercise training and mitochondrial β-oxidation capacity in cardiomyocytes was reversed. Overall, knocking down mtp in the heart prevented an increase in systemic TG levels and protected cardiac contractility from damage caused by HFD, similar to the findings observed after exercise training. Moreover, exercise training upregulated the decrease in cardiac mtp expression induced by HFD. Increased Had1 and Acox3 expression were observed, consistent with changes in cardiac mtp expression.


Introduction
Unhealthy eating habits cause an excessive accumulation of lipids in the body (i.e., "obesity"), which represents a high-risk factor for harmful effects on metabolism and heart function and can lead to the development of a range of diseases such as heart disease, type 2 diabetes and cancer. After hydrolysis into glycerol and fatty acids, fats are packaged in the endoplasmic reticulum of small intestinal cells as chylomicrons (CMs) [1,2] and the liver cells as very low-density lipoproteins (VLDLs) [3]. Microsomal triglyceride transfer protein (mtp) is involved in the assembly of lipoproteins, transferring lipids to lipoprotein B (apoB) [4], which allows apoB to fold correctly and assemble primitive lipoprotein particles involved in systemic lipid metabolism [5]. An increasing body of evidence suggests that mice lacking mtp in the liver or intestine exhibit systemic or specific reductions in plasma triglyceride and cholesterol levels [6,7]. Studies on humans have shown that mtp is associated with lipid droplets in brown and white fat and plays an important role in lipid droplet formation and/or turnover [8]. In addition, mtp is composed of α and β subunits, and mitochondrial β-oxidation is the main metabolic system for the catabolism of fatty acids, generating the main energy source for various cellular processes. β-oxidation catalyzes fatty acids through four reactions (dehydration, oxidation, hydration, and thiolysis) to produce acetyl coenzyme A. Importantly, mtp catalyzes the last three steps of β-oxidation of long-chain fatty acids. The heart can influence mitochondrial β-oxidation by regulating the expression of mtp, which affects systemic lipid metabolism.
Exercise training is recognized as one of the most effective treatments for obesity and cardiovascular disease. In mouse studies, exercise training improved HFD-induced glucose intolerance and insulin resistance while increasing acetylcholine levels, ChAT activity and PKC activity [9]. It could also inhibit inflammation in the adipose tissue of obese mice induced by a high-fat diet [9,10]. Several human metabolic diseases, including obesity, impaired glucose tolerance and type 2 diabetes, have been advocated for prevention and treatment with exercise training [11][12][13][14].
Unlike other animal models such as Mice and Zebrafish, Drosophila has a short life cycle (from egg to adult in about 10 days), produces many offspring (it can produce 30 generations a year, is convenient to feed and can be used for genome editing. More importantly, sequencing and annotation of the Drosophila genome have shown that genes involved in the normal development of several organs, including the heart, are highly conserved and less redundant than in vertebrates. Moreover, it has been shown that a large number of human disease genes have Drosophila counterparts and that their hearts exhibit developmental and functional similarities to the vertebrate heart [15][16][17]. In addition, several models of HFD-induced obesity have been successfully developed in Drosophila, encapsulating the distinctive features of human obesity and diabetes [18].
An increasing body of evidence from recently published studies has confirmed that the heart plays an important role in regulating systemic lipid metabolism [19]. Animal experiments showed that cardiac MED13 expression regulation could control systemic lipid metabolism [20]. However, the specific mechanisms warrant further exploration. By establishing a Drosophila model of HFD and conducting cardiac function measurements [21][22][23], we investigated how exercise training affects HFD-induced cardiac disorders and abnormalities in lipid metabolism. Our findings suggest that specific knockdown of mtp in the heart reduces the elevation of systemic TG and alleviates HFD-induced cardiac dysfunction. In addition, in HFD + w 1118 , HFD + Hand > w 1118 , and HFD + Hand > mtp RNAi , exercise training could restore decreased cardiac mtp expression induced by HFD and improve mitochondrial β-oxidation capacity in cardiomyocytes. Our findings improve current understanding of the role of mtp on systemic lipid metabolism and cardiac function and provide new insights for future clinical studies related to lipotoxic cardiomyopathy and the potential use of exercise training to treat abnormal lipid metabolism and cardiac dysfunction induced by HFD.

Drosophila Strains and Husbandry
w 1118 Drosophila were obtained from the Bloomington Drosophila Stock Center, and UAS-mtp RNAi Drosophila from the Drosophila Center, Tsinghua University. To reduce mtp expression in the heart, we used Hand-Gal4 (cardiomyocyte-specific driver BL48396). The normal food diet (NFD) consisted of a combination of yeast, maize and starch; the high-fat food diet (HFD) was made from 30% coconut oil mixed with the 70% volume of NFD [24,25]. All fruit flies were fed with NFD for five days, then high-fat group fruit flies were fed HFD for five days.
Hand-Gal4 male flies were crossed with w 1118 female flies and female flies of the F1 generation were collected within 12 h of eclosion. These flies and virgin flies of w 1118 underwent different interventions (NFD +w 1118 , HFD + w 1118 , HFD + E + w 1118 , NFD + Hand > w 1118 , HFD + Hand > w 1118 , and HFD + E + Hand > w 1118 (E is for exercise)). Hand-Gal4 male flies were crossed with UAS-mtp RNAi female flies, and female flies from the F1 generation were eclosion was collected within 12 h of eclosion. These flies were defined as cardiomyocyte-specific knockdown of mtp groups and were subjected to different interventions (NFD + Hand > mtp RNAi , HFD + Hand > mtp RNAi and HFD + E + Hand > mtp RNAi ). A total of 1800 virgin flies that were 10 days old were obtained (200 in each group). All flies were placed in transparent glass tubes (20-30 per tube). NFD-fed flies were placed in a constant temperature and humidity incubator (25 • C, 50% humidity, 12 h day-night cycle). HFD-fed flies were reared in an environment of 21-22 • C to prevent part of the high-fat food from melting and causing the flies to stick to the food and die.

Heart Function Tests
Based on previous studies, flies were anesthetized with FlyNap (Triangle Biotechnology, Shanghai, China), and the dissected fly hearts were preserved in oxygenated saline. The heartbeats were captured using an EM-CCD high-speed camera (130 fps, 30-s video), and heart rate, cardiac period, arrhythmia, contractility, shortening fraction, etc., were analyzed using semi-automatic optical heartbeat analysis software (SOHA) [26,27].

Body Weight and Triglyceride (TG) Measurement
An electronic microbalance (Shimadzu, AUW220D, Kyoto Japan) was used to weigh the fruit flies and record the weight of each fly for analysis.
TG concentration was measured using a triglyceride (TG) assay kit (Nanjing Jiancheng Bioengineering Institution, Nanjing, China) according to the manufacturer's instructions [28].

Oil Red O Dye Staining
Flies were anesthetized and dissected in ice-cold PBS. The ventral cuticle was removed from the abdomen using microscissors, and the viscera and genitalia were excised, leaving intact abdominal fat bodies attached to the dorsal cuticle. The plates were fixed with 4% paraformaldehyde for 20 min and washed three times with PBS for 10 min each time. The oil Red O solution (3:2 for staining solution and distilled water) was filtered, then incubated at room temperature for 30 min and washed three times with PBS. The photographs were taken under a Leica stereomicroscope, and after processing the images using Adobe Photoshop, the staining area of oil red was calculated using ImageJ software (ImageJ2,LOCI, Bethesda, MD, USA) [29].

Sports Training Devices and Programs
Consistent with previously established protocols, all fruit flies were fed under NFD conditions for 5 days, and those in the locomotor training group were trained from day 6 for 5 days, ending with training on day 10. Drosophila exercised for 1.5 h per day for 5 days [30][31][32]. During the locomotor training intervention, Drosophila spent 1.5 h in a glass tube without food. The fruit flies in the locomotor training control group were also placed in the glass tubes without food for 1.5 h, thus ensuring that all flies were in the same environment to avoid affecting their feeding rate [33].

Statistical Analysis
Figures were generated using GraphPad Prism6 software. Analyses were performed using the Statistical Package for the Social Sciences for Windows (SPSS) version 21.0 (SPSS-Inc., Chicago, IL, USA) [30][31][32]. All data are expressed as SEM ± mean. One-way ANOVA was used to identify differences between NFD, HFD and HFD + E groups of Drosophila with the same genetic background. Independent sample t-tests were used to identify differences between the two groups. A two-tailed p-value < 0.05 was statistically significant.

HFD Causes Abnormal Lipid Metabolism and Cardiac Dysfunction in Drosophila
First, we established a Drosophila high-fat model. Female Drosophila were fed on NFD for 5 days and then on HFD for 5 days [24], and morphological photographs of Drosophila were taken ( Figure 1A). Compared to the NFD group, the HFD-fed Drosophila exhibited a larger body size and a bulging abdomen with significantly higher body weight than the NFD group ( Figure 2A). Moreover, the wing and ovary sizes of NFD and HFD flies were comparable. These findings suggested lipid uptake was responsible for the apparent abdominal bulge in HFD flies ( Figure 1B-D). In addition, the oil red O staining area ( Figure 2B,C) and TG content ( Figure 2D) of abdominal fat were significantly greater in the HFD group of Drosophila than in the NFD group, demonstrating that our high fat protocol successfully induced a model of diet-induced obesity in Drosophila.
To investigate whether cardiac mtp is involved in obesity-induced cardiac dysfunction, we measured various cardiac function parameters and mtp mRNA expression in Drosophila NFD and Drosophila HFD, including heart rate (HR), cardiac cycle or heart period (HP), arrhythmia (AI), diastolic interval (DI) and systolic interval (SI), fibrillation (FL), diastolic diameter (DD), systolic diameter (SD), and fractional shortening (FS). We found varying degrees of increase in HR, AI and FL ( Figure 3A,C,F) under HFD compared to NFD. It is well-established that fibrillation is the most common sustained arrhythmia and a high-risk factor for morbidity and mortality due to obesity [34]. Significant decreases in HP,SI, DD and FS (Figure 3B,E,G,I) and diastolic diameter and fractional shortening indicated a decrease in myocardial contractility in Drosophila. No significant differences were observed in DI and SD ( Figure 3D,H). This finding suggests that HFD can lead to an increased heart rate in Drosophila and a concomitant increase in the frequency of arrhythmias and fibrillations, shorter cardiac periods and decreased pumping capacity of the heart (shortened fractions and diastolic diameters). Indeed, increased levels of fat and circulating blood lipids lead to cardiac dysfunction. For example, increased expression of lipid transport proteins in the heart leads to elevated lipids in cardiac myocytes with cardiac dysfunction [35]; cardiac pathological remodeling (i.e., hypertrophy and fibrosis) occurs during chronic HFD feeding, which affects systolic and diastolic function in the hearts of obese mice [36], consistent with findings of previous studies of lipotoxic cardiomyopathy [37]. Moreover, we found a significant decrease in mtp expression in the hearts of Drosophila in the HFD group compared to the NFD group ( Figure 4A). These results suggest that increased dietary fat leads to obesity and cardiac dysfunction in Drosophila, which may be related to reduced mtp in cardiac myocytes ( Figure 4A,B).  Whole-body TG levels were significantly higher in the HFD group than in the NFD group. N = 10, repeated three times. One-way ANOVA was used for (A,D). An independent sample t-test was used for (C).

Specific Knockdown of mtp in the Heart Alleviates HFD-Induced Cardiac Dysfunction, Similar to the Effect of Exercise Training
We subjected high-fat Drosophila to five days of exercise and then measured mtp mRNA expression levels, cardiac function and whole-body triglyceride content. The results showed that exercise training significantly increased cardiac mtp expression in all groups of HFD Drosophila ( Figure 5A). After exercise training, compared to HFD + w 1118 and HFD + Hand > w 1118 , HFD + E+w 1118 and HFD + E+Hand > w 1118 Drosophila showed slower heart rate, increased cardiac period, decreased arrhythmia index, increased diastolic diameter, decreased systolic diameter, reduced fibrillation and increased shortening fraction ( Figure 6A-C,F-I), with significant improvements in both cardiac rhythm and systolic function, effectively treating HFD-induced cardiac dysfunction. Exercise training also significantly reduced whole-body TG levels in HFD + w 1118 and HFD + Hand > w 1118 Drosophila, demonstrating that exercise training could upregulate mtp expression in cardiac myocytes while modulating whole-body lipid levels and reducing whole-body TG, thereby protecting the heart from HFD-induced dysfunction.  HFD deteriorated cardiac function in Drosophila and caused a significant decrease in mRNA levels of mtp in the heart. Next, to investigate the relationship between HFD-induced cardiomyopathy and cardiomyocyte mtp, we targeted KD against mtp in cardiomyocytes and assessed whole-body TG levels in Drosophila with HFD. We found no significant difference in mRNA levels of mtp between NFD + Hand > mtp RNAi and HFD + Hand > mtp RNAi ( Figure 5A). Moreover, the knockdown of mtp in the heart prevented an increase in systemic TG levels ( Figure 6J), suggesting that mtp in cardiomyocytes plays an important role in regulating systemic lipid metabolism. Interestingly, no significant differences were found between the NFD + Hand > mtp RNAi group and the HFD + Hand > mtp RNAi group in DD, SD, FS and FL ( Figure 6F-I); and differences were found in HR, HP, AI, DI and SI ( Figure 6A-E) during M-mode ECG. These results suggest that NFD + Hand > mtp RNAi and HFD + Hand > mtp RNAi differed significantly, indicating that HFD mainly affected the cardiac rhythmicity of Hand > mtp RNAi . Compared to the control HFD + w 1118 and HFD + Hand > w 1118 groups, knocking down the cardiac mtp of HFD + Hand > mtp RNAi could reduce the damage of myocardial contractile function by HFD to some extent ( Figure 5B). Knockdown of the cardiac mtp alone yielded effects similar to exercise training in treating cardiomyopathy in Drosophila with HFD, whereas the HFD + Hand > mtp RNAi group of Drosophila showed a significant increase in cardiac mtp expression ( Figure 5A) and systemic TG levels ( Figure 6J) after exercise training. We also found a decrease in HR ( Figure 6A) and AI ( Figure 6C) and an increase in HP, DI and SI ( Figure 6B,D,E), indicating that the abnormal heart rhythm caused by HFD was significantly improved and the cardiac function of Drosophila was restored.  All samples were virgin flies of 10 days, N = 10, and measurements were repeated three times. Whole-body TG levels were significantly higher in Drosophila in the HFD + E group compared to the HFD group. One-way ANOVA was used to identify differences between the strains of Drosophila NFD, HFD and HFD + E. LSD was used for post hoc testing.

Exercise Training Upregulates Cardiomyocyte mtp Expression and Reverses the HFD-Induced Decrease in Cardiac Beta-Oxidation Capacity
It is well-established that β-oxidation catalyzes the degradation of long-chain fatty acids through four steps (dehydration, oxidation, hydration, and thiolysis) to produce acetyl coenzyme A. mtp consists of a hetero-octamer of four mtpα and four mtpβ subunits. The mtpα subunit has long-chain 3-enoyl coenzyme A hydratase and long-chain 3-hydroxy coenzyme A dehydrogenase activities, catalyzing the second and third steps, respectively; the mtpβ subunit has long-chain 3-ketoacyl coenzyme A Thiolase activity, catalyzing the fourth step. This study was based on previous approaches to β-oxidation function as assessed by acyl-coenzyme A dehydrogenase (β Hydroxy acid dehydrogenase 1 abbreviated as Had1) and 3-hydroxyacyl coenzyme A dehydrogenase (Acyl-CoA oxidase 3 abbreviated as Acox3) [38][39][40]. As seen in Figure 7A, HFD caused a decrease in Had1 expression in w 1118 and Hand > w 1118 Drosophila cardiomyocytes, as well as a decrease in Acox3 expression in w 1118 , Hand > w 1118 and Hand > mtp RNAi Drosophila cardiomyocytes, indicating that HFD can cause a decrease in cardiac β-oxidation and a reduction in the ability to catalyze long-chain fatty acids, predisposing to excessive accumulation of fat in the heart. However, after exercise training, there was a general increase in Had1 and Acox3 in w 1118 , Hand > w 1118, and Hand > mtp RNAi Drosophila cardiomyocytes, suggesting that exercise training could reverse the decrease in cardiac β-oxidation induced by HFD ( Figure 7B), consistent with changes in cardiac mtp expression ( Figure 5A).

Discussion
Unhealthy lifestyle habits, especially excessive intake of high-fat foods, represent a risk factor that predisposes to cardiovascular disease. Drosophila is widely acknowledged as an excellent model for studying cardiovascular function. More importantly, 77% of human disease genes have Drosophila counterparts, 26 of which have been associated with cardiovascular disease [15][16][17]. Exercise training is an effective means of preventing and treating obesity and associated cardiovascular disease [11][12][13][14]. Accordingly, we performed exercise training on high-fat Drosophila to explore how exercise training might impact high-fat diet-induced cardiac dysfunction.
As an important target in regulating systemic lipid metabolism, mtp, when expressed in the liver and adipose, is involved in the correct folding and assembly of downstream apoB, affecting the secretion of chylomicrons (CMs) and the synthesis of very low-density lipoproteins (VLDL) [3]. In addition, mtp in cardiac myocytes is involved in regulating systemic lipid metabolism, and knocking down mtp in the heart alone can prevent the increase in systemic TG levels caused by HFD. Furthermore, knocking down mtp in the heart can protect systolic cardiac function from HFD to a certain extent, similar to treating Drosophila with HFD by exercise training. Although it is well-recognized that the heart plays an important role in regulating systemic lipid metabolism, and previous studies have found that mtp in cardiac myocytes can regulate systemic lipid metabolism under HFD conditions, the exact mechanism remains unclear [41].
Current evidence suggests that HFD induces myocardial hypertrophy and fibrosis, reduced coronary reserve and cardiac function. Importantly, exercise limits lipid metabolism disorders, cardiac hypertrophy and fibrosis and helps prevent lipotoxic cardiomyopathy. It is well-established that exercise lowers the heart rate [42] and alleviates cardiac oxidative stress by inhibiting LOX-1 receptor expression [43]. In addition, exercise-based cardiac rehabilitation reduces hospitalization rates and cardiovascular mortality and improves the quality of life [44]. A study found that exercise-based cardiac rehabilitation reduced hospitalization rates, cardiovascular mortality and improved quality of life. Interestingly, exercise was found to mediate cardioprotection through upregulation of miR-344g-5p, which targets Hmgcs2 mRNA, and inhibits HMGCS2 upregulation, thereby suppressing lipotoxicity [45]. It has also been suggested that aerobic exercise reverses cardiac remodeling by reducing inflammation, fibrosis and apoptosis in HFD rats, to a certain extent by inhibiting P2X7R expression in cardiomyocytes [46]. Herein, we showed that exercise training had a therapeutic effect on the decrease in cardiac mtp expression caused by HFD, both in w 1118 , Hand > w 1118 and Hand > mtp RNAi Drosophila; Had1 and Acox3mRNA expression in the heart of HFD flies was elevated to some extent after exercise training, suggesting that the ability of cardiomyocytes to catalyze long-chain fatty acid catabolism may have increased.

Conclusions
Overall, we provided compelling evidence that knocking down mtp in the heart could prevent an increase in systemic TG levels and protect cardiac contractility from HFDinduced damage, which is similar to performing exercise training to some extent. Moreover, exercise training upregulated the decrease in cardiac mtp expression induced by HFD. An increase in the expression of Had1 and Acox3, which are downstream of mtp, was observed, which was highly consistent with the changes in cardiac mtp. This finding suggests that the upregulation of cardiac mtp by exercise training may be an important pathway to treat HFD-induced cardiac dysfunction and abnormal lipid metabolism. It is conceivable that elevated cardiac mtp expression indirectly drives up downstream β oxidase, thereby perhaps increasing the ability of cardiomyocyte mitochondria to break down triglycerides. Given the important role of cardiomyocyte mtp in regulating systemic lipid metabolism and protecting the heart from lipotoxicity, the ameliorative effects of exercise training on cardiac mtp provide new insights for future clinical studies related to lipotoxic cardiomyopathy, especially on the potential use of exercise training to treat abnormalities in lipid metabolism and cardiac dysfunction due to HFD.
Author Contributions: T.P. and M.D. conceived and designed the experiments and wrote the manuscript. T.P., Q.L., H.Y. and P.Z. collected the samples. T.P., M.D., R.T. and P.Z. performed the experiments. T.P. and L.Z. analyzed the data. All authors have read and agreed to the published version of the manuscript.