Regular Exercise Rescues Heart Function Defects and Shortens the Lifespan of Drosophila Caused by dMnM Downregulation

Although studies have shown that myomesin 2 (MYOM2) mutations can lead to hypertrophic cardiomyopathy (HCM), a common cardiovascular disease that has a serious impact on human life, the effect of MYOM2 on cardiac function and lifespan in humans is unknown. In this study, dMnM (MYOM2 homologs) knockdown in cardiomyocytes resulted in diastolic cardiac defects (diastolic dysfunction and arrhythmias) and increased cardiac oxidative stress. Furthermore, the knockdown of dMnM in indirect flight muscle (IFM) reduced climbing ability and shortened lifespan. However, regular exercise significantly ameliorated diastolic cardiac dysfunction, arrhythmias, and oxidative stress triggered by dMnM knockdown in cardiac myocytes and also reversed the reduction in climbing ability and shortening of lifespan caused by dMnM knockdown in Drosophila IFM. In conclusion, these results suggest that Drosophila cardiomyocyte dMnM knockdown leads to cardiac functional defects, while dMnM knockdown in IFM affects climbing ability and lifespan. Furthermore, regular exercise effectively upregulates cardiomyocyte dMnM expression levels and ameliorates cardiac functional defects caused by Drosophila cardiomyocyte dMnM knockdown by increasing cardiac antioxidant capacity. Importantly, regular exercise ameliorates the shortened lifespan caused by dMnM knockdown in IFM.


Introduction
Cardiovascular disease (CVD) is the leading cause of death worldwide, with more than four million deaths recorded in Europe yearly (46% of all deaths) [1,2]. Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease. HCM affects 1 in 500 people and is the leading cause of heart failure and sudden death [3][4][5]. Although recent studies have identified mutations in the myojunction gene MYOM2 in HCM patients [6], these patients have no mutations in the 12 most common pathogenic genes, such as MYH7, MYBPC3, etc. Therefore, MYOM2 may serve as a new candidate gene for HCM pathogenesis.
Myomesin 2 (MYOM2), also known as M-protein, is located in the M-band of the transverse muscle [7] and belongs to the myomesin gene family. To date, only one myomesin-2 isoform has been identified, with major differences at the protein terminus. This isoform has a much shorter N-terminal than myomesin 1, and its C-terminal contains a unique, strongly basic multi-proline sequence with no similarity to myomesin 1 [8,9]. The myomesin gene family contains three members (MYOM1, MYOM2, and MYOM3) and encodes myosin proteins that play crucial roles in heart and skeletal muscle development [10,11]. Myomesin is a molecular spring whose elasticity protects the stability of myosins, similar to actin [12]. Although the three proteins encoded by the Myomesin gene family have a similar structure, their expression in muscle tissue is significantly different [13]. Myomesin isoforms are associated with the contractile properties of different fiber types. For example, myomesin 1 2 of 16 is expressed in all transverse muscles, myomesin 2 is expressed in adult heart and fast fibers and myomesin 3 is expressed only in skeletal muscle intermediate fiber types [14].
Currently, there is no mammalian model of MYOM2, and only myomesin 3 has been studied in detail using zebrafish [15]. Drosophila is a classical model organism with a short life cycle and a relatively simple genetic structure characterized by a smaller number of genomic repeats than vertebrates [16]. About 75% of human disease genes have homologs in Drosophila [17,18]. Drosophila is the only experimental invertebrate model with a heart that is homologous to vertebrates in terms of cardiac development and function [19]. In addition, it has highly conserved biochemical and genetic pathways with mammals, and its cardiac decline is highly similar to that of the human heart. Furthermore, the mature genetic toolkit of Drosophila makes it suitable for studying MYOM2. dMnM is the homolog of MYOM2 in Drosophila and is expressed in Drosophila heart and skeletal muscle [6]. Drosophila can also be constructed to overexpress or knockout any gene in tissues in a time-specific manner, making it an excellent model for ageing studies. Muscle function can be easily determined by measuring the ability of Drosophila to fly and climb [20]. Drosophila has been widely used to study muscle development and disease. Skeletal muscle accounts for 75% of body mass in flying organisms, such as Drosophila, and flight muscles alone account for 65% of total body mass [21]. The IFM of the Drosophila thorax is the largest and the only fibrous muscle present in Drosophila [22]. Furthermore, the constituent proteins of the IFM show a high degree of homology with their vertebrate counterparts at the sequence, structural, and functional levels [23]. IFM mainly provides power for flight. Although studies have shown that MYOM2 serves as a major component of the myofibrillar myogenic fiber M-band and as a pivotal gene in myofibrillar gene interactions [6], the role of MYOM2 in cardiac function and IFM is unknown.
Oxidative stress is closely related to cardiomyopathy. Furthermore, oxidative damage to the myocardium may be due to low levels of antioxidant enzymes [24]. In addition, the effects of oxidative stress injury on myocardial toxicity are characterized by decreased antioxidant stress products, including glutathione peroxidase (GPX), catalase (CAT), and superoxide dismutase (SOD) [24]. Glycolipotoxicity increases the production of reactive oxygen species (ROS) by stimulating cardiomyocytes, thus enhancing the development of diabetic cardiomyopathy (DCM) [25][26][27][28]. Oxidative stress also promotes the development of alcoholic cardiomyopathy (ACM) [29]. Oxidative stress may also play a key role in hypertrophic cardiomyopathy (HCM) [30], where the development and progression of HCM depend on primary damage to myofascicles caused by mutations. However, the development and progression of HCM may also be associated with secondary alterations in the heart due to increased oxidative stress [31], leading to the exacerbation of myofascicular protein mutations. However, the relationship between dMnM knockdown in Drosophila cardiomyocytes and oxidative stress in the heart is unknown. Regular exercise can reduce the incidence of cardiovascular disease [32,33]. Exercise may even lead to favorable cardiac remodeling [34,35] in mammals or Drosophila. In addition, exercise is a cost-effective way of preventing or improving certain heart diseases. For example, exercise attenuates HFD-induced cardiac fibrosis, reductions in the shortening fraction, and arrhythmias in rats and Drosophila [36][37][38]; improves cardiac dysfunction and lipid accumulation induced by Nmnat knockdown in Drosophila [39]; improves cardiac function and myocardial fibrosis in diabetic cardiomyopathy mice [40]; and prevents or ameliorates thioacetamide (TAA)-induced cardiac-dysfunctional pathological cardiac structural remodeling [41]. However, functional studies of MYOM2 in the heart and IFM are lacking, and the relationship between regular exercise and MYOM2 is unclear. Furthermore, the biological functions of regular exercise and MYOM2 and their role in cardiomyopathy are unknown. In this study, a Drosophila model was used to demonstrate the role of dMnM in cardiac function. Results showed that regular exercise could reverse the cardiac function defects caused by low dMnM expression. Regular exercise also reversed the reduction in climbing ability and shortening of lifespan caused by the low expression of dMnM in the IFM of Drosophila.

Exercise Programs
A locomotor device was designed to induce Drosophila to climb upward based on the natural negative ground-tending behavior of Drosophila [42]. Vials were loaded horizontally into a steel tube with 20 Drosophila per tube, then rotated around the horizontal axis at a gear-controlled axial speed, with each vial rotating along its long axis. Most Drosophila actively climbed upward during the rotation, and the few that could not climb actively walked on the inner walls of the vials. The vials were rotated at 24 s/revolution, and the exercise was conducted for 2.5 h per day [42] for two weeks with two days of rest.

Semi-Intact Drosophila Heart Preparation and Cardiac Function
First, 30 flies were fixed on Petri dishes after being anesthetized with FlyNap (Sangon Biotech, Shanghai, China) for 2-3 min. The head, thorax, cuticle, and all internal organs (except the heart) were quickly removed. Dissection was then performed using haemolymph containing 108 mM NaCl 2 , 5 mM KCl, 2 mM CaCl 2 , 8 mM MgCl 2 , 1 mM NaH 2 PO 4 , 4 mM NaHCO 3 , 15 mM 4-(2-hydroxyethyl)-1-piperazinebisulfonic acid, 10 mM sucrose, and 5 mM alginate at a pH of 7.1 and room temperature (24 • C) to expose the heart tube (to visualize the beating heart of the Drosophila) [43]. Oxygen was pumped at room temperature for 15 min. An EM-CCD 9300 high-speed camera (Hamamatsu; Shizuoka; Japan 100-140 fps/sec) taking high-speed digital videos of the beating heart at 130 fps/sec was used to record Drosophila heartbeats, while HCImage software (Hamamatsu; Shizuoka; Japan) was used to record recording ECG data. A semi-automatic optical heartbeat analysis (SOHA, provided by Ocorr and Bodmer) was used to analyze Drosophila cardiac function parameters. SOHA allows accurate quantification of heart rate (HR), cardiac cycle (HP), diastolic diameter (DD), systolic diameter (SD), systolic interval (SI), diastolic interval (DI), arrhythmia index (AI), fibrillation (FL), and shortening fraction (FS) for the assessment of Drosophila heart function parameters. In addition, cardiac M-patterns, such as qualitative recordings showing the temporal motion of the heart edges, were generated using an optical heartbeat analysis for further analysis of abnormal cardiac contractions [44].

Phalloidine
Semi-intact Drosophila hearts were prepared and confirmed to show rhythmic beating in oxygenated ADH [44]. ADH was quickly replaced with a relaxation buffer (ADH containing 10 mM EGTA). The hearts were fixed with 4% formaldehyde at room temperature for 20 min, then washed thrice using PBS at room temperature (10 min for each wash). The hearts were stained with ghost pen cyclic peptide (Phalloidin-iFluor 594) for 40 min and washed thrice with PBS at room temperature (10 min for each wash). Fluorescence staining images were obtained with a confocal laser scanning microscope (Carl Zeiss; Oberkochen; Germany).

ROS Staining
The flies were dissected to expose the cardiac canal as described by Alexander Lam et al. [45] and then stained for reactive oxygen ROS. The Drosophila were fixed after continuous exposure to CO 2 for 1 min before dissection. The adult flies were dissected to maintain a semi-intact beating heart before staining. The fly was immersed in 1:1000 dihydroethidium (DHE, HY-D0079). PBS for 30 min after exposing the heart tube. The flies were rinsed thrice with PBS at room temperature (10 min for each wash). A Leica stereomicroscope (Leica; Wetzlar; Germany) was used to obtain images, which were then processed with Adobe Photoshop (Adobe, CA, USA).

Climbing and Lifespan Statistics
The Drosophila climbing apparatus consisted of an 18 cm long clear glass tube (Canghzhou four stars glass; Canghzhou; China) with a diameter of 2.8 cm and two 2 cm plugs (Canghzhou four stars glass; Canghzhou; China) at the ends of the glass to prevent Drosophila from climbing out. The Drosophila were gently shaken at the bottom and aggressively climbed upwards according to their own negative tropism [20]. The climbing was recorded using a video camera (Sony; Tokyo; Japan), and the fourth, fifth, and sixth images were intercepted at the end of the sixth second. The glass tube was divided into nine equal regions, which were scored as 1, 2, 3, 4, 5, 6, 7, 8, and 9 from bottom to top, and the number of Drosophila in each region was counted. The climbing index was calculated as follows: climbing index = total score/total number of flies.
Lifespan statistics of Drosophila were counted daily from the day of plumage until the last Drosophila died. The medium was changed every 24 h. The average lifespan and survival curve were used to characterize the lifespan. The sample contained about 200 individuals per group [46].

Statistical Analyses
Data analysis was conducted using SPSS version 22.0 software(SPSS, Chicago, IL, USA). An independent-samples t-test was used to assess the differences between groups. However, nonparametric tests were used when the variances were not equal. Data are expressed as means ± the standard error of the mean (SEM), α = 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001.
The UAS-Hand-Gal4 system was used to determine the effect of dMnM knockdown in Drosophila cardiomyocytes on cardiac function. Compared with the W1118 > Hand-Gal4 group, the heart rate, inter-systolic interval, and shortening fraction significantly decreased in the dMnMRNAi > Hand-Gal4 group ( Figure 1). In addition, cardiac cycle, diastolic interval, arrhythmia index, and fibrillation significantly increased in the dMnMRNAi > Hand-Gal4 group. Meng Ding et al. [47] indicated that arrhythmias include heart rate, arrhythmia index, and fibrillation. In this study, dMnM knockdown decreased heart rate, increased the arrhythmia index, and increased fibrillation, indicating an increase in arrhythmias. Furthermore, dMnM knockdown increased diastolic time, decreased shortening fraction, and decreased cardiac pumping capacity. However, dMnM knockdown did not significantly change the diastolic diameter or systolic diameter, suggesting that the cardiomyocyte dMnM knockdown cannot induce changes in Drosophila heart diameter and systolic diameter, indicating diminished cardiac contractile performance. In conclusion, these results suggest that cardiomyocyte dMnM knockdown causes defects in cardiac function. Long and short black lines represent diastolic diameter and systolic diameter, respectively. The red square shows fibrillation. All M-mode ECGs were intercepted at 10 s. An independent-samples t-test was used to evaluate the differences between the two groups, * p < 0.05, ** p < 0.01, *** p < 0.001, ns is not statistically significant.

dMnM Knockdown in Drosophila Cardiomyocytes Causes Myofibril Destruction
The adult Drosophila melanogaster heart consists of a single layer of contractible monolayer cardiomyocytes that forms a simple linear tube.
The changes in myogenic fibers in the myocardium were examined to explore the Long and short black lines represent diastolic diameter and systolic diameter, respectively. The red square shows fibrillation. All M-mode ECGs were intercepted at 10 s. An independent-samples t-test was used to evaluate the differences between the two groups, * p < 0.05, ** p < 0.01, *** p < 0.001, ns is not statistically significant.

dMnM Knockdown in Drosophila Cardiomyocytes Causes Myofibril Destruction
The adult Drosophila melanogaster heart consists of a single layer of contractible monolayer cardiomyocytes that forms a simple linear tube.
The changes in myogenic fibers in the myocardium were examined to explore the changes in myogenic fibers associated with genetic mutations and regular motility. The F-actin was labeled with a ghost pencil loop peptide. The changes in the Drosophila heart tube were observed using confocal microscopy. Compared with the W1118 > Hand-Gal4 group (Figure 2A), the dMnM RNAi > Hand-Gal4 group showed disruption of myogenic fibers( Figure 2B), suggesting that dMnM knockdown in cardiac myocytes severely damages myogenic fiber structure in Drosophila hearts.

dMnM Knockdown in Drosophila Cardiomyocytes Increases Cardiac ROS Production and Decreases mRNA Expression of dMnM, Upheld, sod2, Cat, and phgpx
Reactive oxygen species (ROS) production was assessed using fluorescent dye dihydroethidium (DHE). qRT-PCR was used to detect dMnM knockdown efficiency. Compared with the W1118 > Hand-Gal4 group, mRNA expression of dMnM, upheld (cardiac troponin, an important marker of myocardial injury), and the antioxidant enzymes sod2, cat, and phgpx (homologs of SOD2, CAT, and GPX4) significantly decreased in the dMnM RNAi > Hand-Gal4 group.
DHE staining of Drosophila hearts was used to determine the relationship between low dMnM expression in Drosophila cardiomyocytes and oxidative stress in the heart in the W1118 > Hand-Gal4 group and the dMnM RNAi > Hand-Gal4 group. Cardiomyocyte dMnM knockdown resulted in brighter cardiac DHE staining fluorescence, indicating that knockdown increases cardiac ROS production and oxidative stress ( Figure 3A-C). In addition, qRT-PCR was used to assess isolated Drosophila hearts, including dMnM, upheld, sod2, cat, and phgpx. Compared with the W1118 > Hand-Gal4 group, the mRNA expression levels of dMnM, upheld, sod2,cat and phgpx were significantly decreased in the dMnM RNAi > Hand-Gal4 group ( Figure 3D-H). dMnM mRNA expression decreased by about 80%, indicating that the cardiomyocyte dMnM knockdown strain upheld is a key marker of myocardial injury. The decreased expression of upheld indicated myocardial injury, while the decreased expression of phgpx, sod2, and cat indicated that Drosophila cardiomyocyte dMnM knockdown reduced cardiac antioxidant capacity.

dMnM Knockdown in Drosophila Cardiomyocytes Increases Cardiac ROS Production and
Decreases mRNA Expression of dMnM, Upheld, sod2, Cat, and phgpx Reactive oxygen species (ROS) production was assessed using fluorescent dye dihydroethidium (DHE). qRT-PCR was used to detect dMnM knockdown efficiency. Compared with the W1118 > Hand-Gal4 group, mRNA expression of dMnM, upheld (cardiac troponin, an important marker of myocardial injury), and the antioxidant enzymes sod2, cat, and phgpx (homologs of SOD2, CAT, and GPX4) significantly decreased in the dMnM RNAi > Hand-Gal4 group.
DHE staining of Drosophila hearts was used to determine the relationship between low dMnM expression in Drosophila cardiomyocytes and oxidative stress in the heart in the W1118 > Hand-Gal4 group and the dMnM RNAi > Hand-Gal4 group. Cardiomyocyte dMnM knockdown resulted in brighter cardiac DHE staining fluorescence, indicating that knockdown increases cardiac ROS production and oxidative stress ( Figure 3A-C). In addition, qRT-PCR was used to assess isolated Drosophila hearts, including dMnM, upheld, sod2, cat, and phgpx. Compared with the W1118 > Hand-Gal4 group, the mRNA expression levels of dMnM, upheld, sod2,cat and phgpx were significantly decreased in the dMnM RNAi > Hand-Gal4 group ( Figure 3D-H). dMnM mRNA expression decreased by about 80%, indicating that the cardiomyocyte dMnM knockdown strain upheld is a key marker of myocardial injury. The decreased expression of upheld indicated myocardial injury, while the decreased expression of phgpx, sod2, and cat indicated that Drosophila cardiomyocyte dMnM knockdown reduced cardiac antioxidant capacity. . T-test for differences between the two groups using independent samples, * p < 0.05, ** p < 0.01, *** p < 0.001.

Effects of dMnM Knockdown in Cardiomyocytes and IFM on Climbing and Lifespan of Drosophila
dMnM was knocked down in Drosophila cardiomyocytes and indirect flight muscle to explore the effects of dMnM knockdown in cardiomyocytes and IFM on the climbing and lifespan of Drosophila. Compared with the W1118 > Hand-Gal4 group, cardiomyocyte dMnM knockdown reduced the climbing ability of the dMnM RNAi > Hand-Gal4 group (Figure 4A). However, cardiomyocyte dMnM knockdown did not affect lifespan ( Figure  4B,C). Compared with the W1118 > Hand-Gal4 group, IFM dMnM knockdown significantly decreased the climbing ability and lifespan of the dMnM RNAi > Act88F-Gal4 group ( Figure 4D-F). These results suggest that dMnM knockdown in cardiac myocytes affects the climbing ability of Drosophila and has less effect on lifespan, while IFM dMnM knockdown significantly affects the climbing ability and lifespan of Drosophila. . T-test for differences between the two groups using independent samples, * p < 0.05, ** p < 0.01, *** p < 0.001.

Effects of dMnM Knockdown in Cardiomyocytes and IFM on Climbing and Lifespan of Drosophila
dMnM was knocked down in Drosophila cardiomyocytes and indirect flight muscle to explore the effects of dMnM knockdown in cardiomyocytes and IFM on the climbing and lifespan of Drosophila. Compared with the W1118 > Hand-Gal4 group, cardiomyocyte dMnM knockdown reduced the climbing ability of the dMnM RNAi > Hand-Gal4 group ( Figure 4A). However, cardiomyocyte dMnM knockdown did not affect lifespan ( Figure 4B,C). Compared with the W1118 > Hand-Gal4 group, IFM dMnM knockdown significantly decreased the climbing ability and lifespan of the dMnM RNAi > Act88F-Gal4 group ( Figure 4D-F). These results suggest that dMnM knockdown in cardiac myocytes affects the climbing ability of Drosophila and has less effect on lifespan, while IFM dMnM knockdown significantly affects the climbing ability and lifespan of Drosophila. climbing results for the W1118 > Act88F-Gal4 and dMnM RNAi > Act88F-Gal4 group; (E,F) lifespan and survival curves for dMnM RNAi > Hand-Gal4 group, respectively. Climbing sample n = 100; lifespan sample n = 200. The difference between the two groups was tested using an independen samples t-test, ** p < 0.01, *** p < 0.001, ns is not statistically significant.

Regular Exercise Ameliorates Cardiac Function Defects Caused by dMnM Knockdown in Drosophila Cardiomyocytes
The flies underwent regular exercise for two weeks after cardiomyocyte dMn knockdown. Compared with the dMnM RNAi > Hand-Gal4 group, cardiac cycle, arrhythm index, fibrillation, and diastolic interval were significantly reduced in the dMnM RNA Hand-Gal4 + E group( Figure 5B,F,H,I). However, heart reat and shortening fraction we significantly increased( Figure 5A,C). These results indicate that regular exercise can s nificantly improve and reverse the arrhythmia and diastolic dysfunction caused dMnM knockdown in Drosophila cardiomyocytes. Regular exercise improved the shorte ing fraction of the heart and increased the pumping function of the heart based on t decreased systolic diameter and increased shortening fraction( Figure 5D). However, re ular exercise did not significantly change diastolic diameter and systolic intervals (Figu 5E,G), suggesting that regular exercise does not alter the changes in cardiac diame caused by dMnM knockdown in cardiomyocytes. In addition, regular exercise reduc fibrillation in the dMnM RNAi > Hand-Gal4 + E group. In conclusion, these results sugg that regular exercise reverses dMnM knockdown-induced arrhythmias and diastolic dy function in Drosophila cardiomyocytes. The difference between the two groups was tested using an independent-samples t-test, ** p < 0.01, *** p < 0.001, ns is not statistically significant.

Regular Exercise Ameliorates Cardiac Function Defects Caused by dMnM Knockdown in Drosophila Cardiomyocytes
The flies underwent regular exercise for two weeks after cardiomyocyte dMnM knockdown. Compared with the dMnM RNAi > Hand-Gal4 group, cardiac cycle, arrhythmia index, fibrillation, and diastolic interval were significantly reduced in the dMnM RNAi > Hand-Gal4 + E group ( Figure 5B,F,H,I). However, heart reat and shortening fraction were significantly increased( Figure 5A,C). These results indicate that regular exercise can significantly improve and reverse the arrhythmia and diastolic dysfunction caused by dMnM knockdown in Drosophila cardiomyocytes. Regular exercise improved the shortening fraction of the heart and increased the pumping function of the heart based on the decreased systolic diameter and increased shortening fraction( Figure 5D). However, regular exercise did not significantly change diastolic diameter and systolic intervals ( Figure 5E,G), suggesting that regular exercise does not alter the changes in cardiac diameter caused by dMnM knockdown in cardiomyocytes. In addition, regular exercise reduced fibrillation in the dMnM RNAi > Hand-Gal4 + E group. In conclusion, these results suggest that regular exercise reverses dMnM knockdown-induced arrhythmias and diastolic dysfunction in Drosophila cardiomyocytes. respectively. The red square shows fibrillation. Rectangular area represents fibrillation. All M-mo ECGs were intercepted for 10 s. An independent-samples t-test and nonparametric test were u to assess the differences between the two groups, * p < 0.05, ** p < 0.01, *** p < 0.001, ns is not sta tically significant.

Regular Exercise Improves Myocardial Myogenic Fiber Destruction Caused by Cardiomyocyte dMnM Knockdown
The myocardial F-actins of the dMnM RNAi > Hand-Gal4 group and dMnM RNAi > Hand-G + E group were subjected to fluorescence image observation to investigate the effect of regu exercise on cardiac myogenic fiber destruction caused by dMnM-specific knockdown of c diomyocytes. The dMnM RNAi > Hand-Gal4 + E group was also subjected to regular exercise two weeks. Compared with the dMnM RNAi > Hand-Gal4 group (Figure 6A), the dMnM RN Hand-Gal4 + E group showed a tighter myofilament arrangement ( Figure 6B), indicating t regular exercise protects against the myofibril destruction caused by dMnM knockdown cardiac myocytes. (J,K) Drosophila M-mode ECG pattern. Long and short black lines represent diastolic diameter and systolic diameter, respectively. The red square shows fibrillation. Rectangular area represents fibrillation. All M-mode ECGs were intercepted for 10 s. An independent-samples t-test and nonparametric test were used to assess the differences between the two groups, * p < 0.05, ** p < 0.01, *** p < 0.001, ns is not statistically significant.

Regular Exercise Improves Myocardial Myogenic Fiber Destruction Caused by Cardiomyocyte dMnM Knockdown
The myocardial F-actins of the dMnM RNAi > Hand-Gal4 group and dMnM RNAi > Hand-Gal4 + E group were subjected to fluorescence image observation to investigate the effect of regular exercise on cardiac myogenic fiber destruction caused by dMnM-specific knockdown of cardiomyocytes. The dMnM RNAi > Hand-Gal4 + E group was also subjected to regular exercise for two weeks. Compared with the dMnM RNAi > Hand-Gal4 group( Figure 6A), the dMnM RNAi > Hand-Gal4 + E group showed a tighter myofilament arrangement ( Figure 6B), indicating that regular exercise protects against the myofibril destruction caused by dMnM knockdown in cardiac myocytes.

Regular Exercise Reduces ROS Production and Upregulates mRNA Expression of dM Upheld, phgpx, sod2, and Cat in Cardiomyocytes after dMnM Knockdown
DHE staining of Drosophila hearts was conducted in the dMnM RNAi > Hand-Gal4 and the dMnM RNAi > Hand-Gal4 + E group to determine the effect of regular exer cardiac oxidative stress caused by dMnM knockdown in cardiomyocytes. Compar the dMnM RNAi > Hand-Gal4 group, the DHE staining of hearts in the dMnM RNAi > Ha + E group had darker fluorescence intensity, indicating that regular exercise reduc diac ROS production and cardiac oxidative stress ( Figure 7A,B).
Proper exercise facilitates cardiac remodeling and improves the antioxidant c of the heart. However, the relationship between regular exercise and cardiac dMn cific knockdown resulting in reduced expression of dMnM, upheld, sod2, cat and is unknown. qRT-PCR assays showed that the mRNA levels of dMnM, upheld, so and phgpx were significantly upregulated in dMnM RNAi > Hand-Gal4 + E group com with the dMnM RNAi > Hand-Gal4 group ( Figure 7D-H). Regular exercise significan viated the effect of dMnM knockdown in Drosophila, indicating that exercise resc ameliorated the cardiac knockdown damage to the heart. The upregulation of so and phgpx after regular exercise increased the antioxidant level of the heart. 3.7. Regular Exercise Reduces ROS Production and Upregulates mRNA Expression of dMnM, Upheld, phgpx, sod2, and Cat in Cardiomyocytes after dMnM Knockdown DHE staining of Drosophila hearts was conducted in the dMnM RNAi > Hand-Gal4 group and the dMnM RNAi > Hand-Gal4 + E group to determine the effect of regular exercise on cardiac oxidative stress caused by dMnM knockdown in cardiomyocytes. Compared with the dMnM RNAi > Hand-Gal4 group, the DHE staining of hearts in the dMnM RNAi > Hand-Gal4 + E group had darker fluorescence intensity, indicating that regular exercise reduced cardiac ROS production and cardiac oxidative stress ( Figure 7A,B).
Proper exercise facilitates cardiac remodeling and improves the antioxidant capacity of the heart. However, the relationship between regular exercise and cardiac dMnM-specific knockdown resulting in reduced expression of dMnM, upheld, sod2, cat and phgpx is unknown. qRT-PCR assays showed that the mRNA levels of dMnM, upheld, sod2, cat and phgpx were significantly upregulated in dMnM RNAi > Hand-Gal4 + E group compared with the dMnM RNAi > Hand-Gal4 group ( Figure 7D-H). Regular exercise significantly alleviated the effect of dMnM knockdown in Drosophila, indicating that exercise rescued or ameliorated the cardiac knockdown damage to the heart. The upregulation of sod2, cat and phgpx after regular exercise increased the antioxidant level of the heart.
Proper exercise facilitates cardiac remodeling and improves the antioxidant capacity of the heart. However, the relationship between regular exercise and cardiac dMnM-specific knockdown resulting in reduced expression of dMnM, upheld, phgpx, sod2, and cat is unknown. qRT-PCR assays showed that the mRNA levels of dMnM, upheld, phgpx, sod2, and cat were significantly upregulated in the dMnM RNAi > Hand-Gal4 + E group compared to the dMnM RNAi > Hand-Gal4 group ( Figure 7D-H). Regular exercise significantly alleviated the effect of dMnM knockdown in Drosophila, indicating that exercise reversed or ameliorated the cardiac knockdown damage to the heart. The upregulation of phgpx, sod2, and cat after regular exercise increased the antioxidant level in the heart. An independent-samples t-test was used to assess differences between the two groups, * p < 0.05, ** p < 0.01, *** p < 0.001.
Proper exercise facilitates cardiac remodeling and improves the antioxidant capacity of the heart. However, the relationship between regular exercise and cardiac dMnM-specific knockdown resulting in reduced expression of dMnM, upheld, phgpx, sod2, and cat is unknown. qRT-PCR assays showed that the mRNA levels of dMnM, upheld, phgpx, sod2, and cat were significantly upregulated in the dMnM RNAi > Hand-Gal4 + E group compared to the dMnM RNAi > Hand-Gal4 group ( Figure 7D-H). Regular exercise significantly alleviated the effect of dMnM knockdown in Drosophila, indicating that exercise reversed or ameliorated the cardiac knockdown damage to the heart. The upregulation of phgpx, sod2, and cat after regular exercise increased the antioxidant level in the heart. An independent-samples t-test was used to assess differences between the two groups, * p < 0.05, ** p < 0.01, *** p < 0.001.

Effects of Regular Exercise on Climbing and Lifespan after dMnM Knockdown in Cardiac Myocytes and IFM of Drosophila
Regular exercise improved the climbing ability in the dMnM RNAi > Hand-Gal4 + E group more significantly than in the dMnM RNAi > Hand-Gal4 group after dMnM knockdown in cardiac myocytes ( Figure 8A). However, regular exercise did not significantly affect lifespan in the dMnM RNAi > Hand-Gal4 + E group compared to the dMnM RNAi > Hand-Gal4 group ( Figure 8B,C). In contrast, regular exercise significantly increased the climbing ability and lifespan of Drosophila after dMnM knockdown in cardiomyocytes and IFM compared to the dMnM RNAi > Act88F-Gal4 group ( Figure 8D-F). group more significantly than in the dMnM RNAi > Hand-Gal4 group after dMnM knockdown in cardiac myocytes ( Figure 8A). However, regular exercise did not significantly affect lifespan in the dMnM RNAi > Hand-Gal4 + E group compared to the dMnM RNAi > Hand-Gal4 group (Figure 8B,C). In contrast, regular exercise significantly increased the climbing ability and lifespan of Drosophila after dMnM knockdown in cardiomyocytes and IFM compared to the dMnM RNAi > Act88F-Gal4 group ( Figure 8D-F). An independent-samples t-test was used to assess the difference between the two groups, ** p < 0.01, ns is not statistically significant.

Discussion
The effect of dMnM knockdown on cardiac functional defects, such as cardiac arrhythmias and diastolic dysfunction, is unknown. Furthermore, the relationship between exercise, dMnM expression, and cardiac function is unclear. This study aimed to illustrate the effects of low dMnM expression on cardiac function and of regular exercise on dMnM in cardiomyocytes using a Drosophila model.
Cardiac pumping function is mainly determined by systolic and diastolic functions, the percentage reduction in wall diameter during the systole, and the shortening fraction (change in diameter); thus, it can be used to estimate the contractility of the Drosophila heart [48]. In this study, dMnM knockdown in Drosophila cardiomyocytes increased the systolic diameter of the heart. This decreased the shortening fraction, thus reducing the heart pumping capacity. A previous study showed that a moderate reduction in Drosophila dMnM results in cardiac dilation, while a complete loss or strong knockdown causes restriction [6]. In this study, cardiomyocyte dMnM knockdown did not significantly change diastolic diameter, indicating that cardiomyocyte dMnM knockdown cannot An independent-samples t-test was used to assess the difference between the two groups, ** p < 0.01, ns is not statistically significant.

Discussion
The effect of dMnM knockdown on cardiac functional defects, such as cardiac arrhythmias and diastolic dysfunction, is unknown. Furthermore, the relationship between exercise, dMnM expression, and cardiac function is unclear. This study aimed to illustrate the effects of low dMnM expression on cardiac function and of regular exercise on dMnM in cardiomyocytes using a Drosophila model.
Cardiac pumping function is mainly determined by systolic and diastolic functions, the percentage reduction in wall diameter during the systole, and the shortening fraction (change in diameter); thus, it can be used to estimate the contractility of the Drosophila heart [48]. In this study, dMnM knockdown in Drosophila cardiomyocytes increased the systolic diameter of the heart. This decreased the shortening fraction, thus reducing the heart pumping capacity. A previous study showed that a moderate reduction in Drosophila dMnM results in cardiac dilation, while a complete loss or strong knockdown causes restriction [6]. In this study, cardiomyocyte dMnM knockdown did not significantly change diastolic diameter, indicating that cardiomyocyte dMnM knockdown cannot cause myocardial dilation, possibly due to the Drosophila genetic background and varying diets. Interestingly, dMnM knockdown prolonged diastolic interval and increased arrhythmias. The increased diastolic interval indicates diastolic dysfunction [49], characterized by impaired relaxation, decreased dilatability, and increased myocardial stiffness, which may be caused by excessive actin interactions [50,51]. However, this suggests that the role of dMnM is not limited to cardiac dilation. Meng Ding et al. [47] suggested that arrhythmias include heart rate, the arrhythmia index, and fibrillation. In this study, dMnM knockdown decreased heart rate, increased the arrhythmia index, and increased fibrillation, thus increasing arrhythmias. In addition, prolongation of the diastolic interval decreases cardiac contractility. These results suggest that dMnM plays an essential role in the heart. Cardiac function defects caused by dMnM knockdown in cardiomyocytes may be due to decreased cardiac antioxidant capacity. In addition, cardiac troponin T (cTnT) is a sensitive indicator of myocardial injury [52]. In this study, Drosophila cTnT may have been sensitive to low dMnM expression. Therefore, cTnT mutations can reduce the contractile performance of cardiomyocytes or even affect the whole heart when dMnM under-expression causes flocculation of myocardial contractile mechanisms [53]. Upheld is a cTnT homolog in Drosophila. In this study, dMnM knockdown in cardiomyocytes decreased mRNA expression of upheld, suggesting that cardiomyocyte dMnM knockdown may impair the contractile function of the myocardium.
Regular physical activity can reduce the risk of cardiovascular diseases [54][55][56]. In this study, regular exercise reduced the diastolic interval, heart rate, arrhythmia index, and fibrillation, suggesting that regular exercise can significantly protect against the development of cardiac diastolic defects and arrhythmias triggered by dMnM knockdown. In addition, regular exercise reduced the systolic diameter and increased the shortening fraction, suggesting that exercise improves cardiac contractility and pumping capacity.
In conclusion, these results show that two-week regular exercise can protect Drosophila cardiomyocytes against diastolic defects and arrhythmias caused by dMnM knockdown.
Oxidative stress in the excessive production of ROS is relative to antioxidant levels. Reactive oxygen species are oxy-chemicals with high reactivity [57]. ROS have deleterious effects on cardiovascular diseases, such as heart failure, cardiomyopathy, and coronary artery disease [58]. Excess ROS cause cellular dysfunction, protein and lipid peroxidation, and DNA damage and can lead to irreversible cellular damage and death. Intracellular ROS are mainly scavenged by antioxidant enzymatic substances, including superoxide dismutase (SOD2), catalase (CAT), and glutathione peroxidase (GPX). Moreover, antioxidant enzymatic substances are also the most important intracellular antioxidants in humans [59]. The three antioxidant enzymatic substances (SOD2, CAT, and GPX) are homologs of sod2, cat, and phgpx in Drosophila, respectively. Exercise promotes health through the maintenance of the biological adaptation of mitochondrial function to oxidative stress. Furthermore, exercise intervention has effects on antioxidants, such as SOD2, CAT, and GPX. For example, a four-week exercise intervention can promote SOD2 expression in a healthy population (SOD2 is positively correlated with exercise habit) [60]. Furthermore, glutathione peroxidase 4 (GPX4) activated by nuclear factor erythroid 2-related factor 2 (Nrf2) has a protective effect on myocardial injury in mice on a high-fat diet during aerobic exercise [61]. In this study, cardiomyocyte dMnM knockdown decreased sod2, cat, and phgpx expressions, suggesting that cardiomyocyte dMnM knockdown reduces cardiac antioxidant capacity. However, regular exercise significantly upregulated the mRNA expression of sod2, cat, and phgpx compared to the knockdown group, suggesting that regular exercise improves the antioxidant capacity of the heart by upregulating the expression of antioxidant enzymes.
French et al. indicated that exercise-induced changes in key antioxidant enzymes play an essential role in exercise-induced cardioprotection [62]. Herein, the DHE cardiac ROS fluorometry showed that the knockdown group had higher brightness after dMnM knockdown in cardiomyocytes than the exercise group. The exercise group also had reduced ROS production compared to the knockdown group. These results confirm that regular exercise can reduce oxidative stress caused by dMnM knockdown in cardiomyocytes.
Furthermore, the knockdown group showed disrupted F-actin myofilament arrangement and altered cardiac morphology compared to the paired group after staining cardiomyocyte myogenic fibers with ghost pencil cyclic peptide. However, regular exercise made the F-actin myofilament arrangement tighter and more complete, suggesting that regular exercise can reverse the structural disruption of myofibrils caused by dMnM knockdown. Importantly, dMnM knockdown in cardiac myocytes only reduced the climbing ability of Drosophila, while dMnM knockdown in IFM decreased both the climbing ability and lifespan of Drosophila. However, regular exercise significantly improved the climbing ability and lifespan decreased by dMnM knockdown in cardiac myocytes and IFM.
In summary, dMnM knockdown in Drosophila cardiomyocytes increases cardiac arrhythmias, increases diastolic dysfunction, and reduces the shortening fraction, possibly due to increased cardiac oxidative stress. In addition, dMnM knockdown in IFM reduces the climbing ability and lifespan of Drosophila. However, regular exercise can upregulate cardiomyocyte dMnM expression levels and alleviate the cardiac dysfunction caused by cardiomyocyte dMnM knockdown by increasing cardiac antioxidant capacity. Regular exercise can also improve the reduced climbing ability and shortened lifespan of Drosophila caused by IFM dMnM knockdown. In conclusion, this study demonstrates the protective effect of regular exercise on cardiac function in cardiac myocytes.

Conclusions
Drosophila cardiomyocyte dMnM knockdown leads to cardiac functional defects, while IFM dMnM knockdown affects climbing and lifespan. Regular exercise can upregulate cardiomyocyte dMnM expression levels and ameliorate cardiac functional defects caused by Drosophila cardiomyocyte dMnM knockdown by increasing cardiac antioxidant capacity. In addition, regular exercise can reverse the shortening of lifespan caused by IFM dMnM knockdown.

Data Availability Statement:
The raw data supporting the conclusions of this article will be made available by the authors without undue reservation.