Effect of Acute High-Intensity Exercise on Rat Myocardium metabolic Proles. An LC-MS Based Metabolomics Study

Acute high-intensity exercise is a harmful manner associated with a series of myocardial injuries. Metabolism disorder of myocardium is one of the most serious conditions. However, few metabolomics-based studies provide data on the effect of exercise along with myocardial metabolism. Our study aimed to identify metabolic signatures in rat myocardium during acute high-intensity exercise and evaluate their diagnostic potential to sports injuries. SD rats were divided into control group and acute high-intensity exercise group and their myocardium samples were analyzed by LC-MS to explore metabolic alterations of rats’ myocardium. This study showed myocardium metabolism clearly differed between the two groups. there were 6 target metabolic pathways and 12 potential metabolic markers for acute high-intensity exercise. Our ndings provide an insight that myocardium metabolism during acute high-intensity exercise have distinct disorders in complex lipids and fatty acids. Moreover, an increase of purine degradation products as well as signs of impaired glucose metabolism were observed. However, the amino acid was enhanced, which had a certain protective effect on the myocardium.

Added 100ul acetonitrile/water (1:1/V: V) mixture and blended thoroughly to reconstitute sample. After centrifugation at 4°C and 14000rcf for 15 minutes, the supernatant was taken for mass spectrometry injection analysis.
The samples were prepared as quality control samples (QC) for testing the instrument and system status. The QC sample was analyzed 4 times at random.

Data processing
Collected data from LC-MS. Used SPSS 24.0 for univariate statistical analysis and SIMCA 14.1 for multivariate statistical analysis based on peak areas data of the detected metabolites. Statistically signi cant compounds were evaluated by using ROC curve analysis. Made the volcano map and cluster map and Searched Met-PA databases to screen out target metabolic pathways and potential metabolic markers.

QC analysis
The UHPLC-Q-TOF MS ion chromatograms of the QC sample were overlapped and compared (Figure 1, a). the QC sample chromatograms overlapped well, indicating the instrument was in good condition and the experimental data was reliable.
Overall sample Hotellings T2 Overall sample Hotellings T2 analysis was used to detect outliers in this experiment. Here, all samples were within the 99% con dence interval and there were no outliers (Figure 1, b).
Typical metabolic pro le Myocardial samples were detected by LC-MS to obtain typical metabolic spectrums ( Figure 2). The contours of the myocardial metabolites between the two groups had changed to different degrees. The differences can be found by further analysis.

PLS-DA analysis results
PLS-DA uses to identify experimental data and predict differences between the two groups. This experiment supervised the data of the two groups after myocardial preconditioning and established a regression model. The model parameters (7 cycles of veri cation) showed the model establishment was stable and reliable (Table 1). Constructed a PSL-DA model score chart (Figure 3). The positive and negative ion points between group C and group E had a signi cant separation trend but within the group were more concentrated, indicating the metabolites of the two groups had differences. Table1 PLS-DA analysis model parameter table   Grouping Negative ion mode Positive ion mode Note: A is the number of main components; R 2 is the explanatory rate of model variables to X or Y; Q 2 is the predictive ability of the model; the closer R 2 Y and Q 2 are to 1, the more stable and reliable the model is; the model is stable and reliable when Q 2 is greater than 0.5

OPLS-DA analysis results
OPLS-DA statistical analysis revises the PLS-DA experimental data and enhances the signi cance of the differences between groups. The model parameters were shown in table 2. In this study, R 2 Y and Q 2 were both greater than 0.5, indicating the two groups had signi cant differences and the OPLS-DA model is stable and reliable. Constructed the OPLS-DA model score chart (Figure 4). The separation trend of positive and negative ions between the two groups was obvious but the tendency of aggregation within the group was obvious, indicating the metabolites of the two groups had signi cant differences.
Created 200 models on the basis of OPLS-DA to perform permutation tests on the random sorting of categorical variables Y and determined the R 2 and Q 2 values of the random model ( Figure 5). On the same abscissa, the R 2 value was greater than the Q 2 value and can be well separated. The rightmost points of R 2 and Q 2 were both larger than the other points, the leftmost value of Q 2 was less than 0, indicating the model veri cation of this research had passed, the metabolites of the two groups existed differences and the analysis of PLS-DA, OPLS-DA results was meaningful. Note: A is the number of main components; R 2 is the explanatory rate of model variables to X or Y; Q 2 is the predictive ability of the model; the closer R 2 Y and Q 2 are to 1, the more stable and reliable the model is; the model is stable and reliable when Q 2 is greater than 0.5

Univariate statistical analysis results
Combined T test and FC analysis to make Volcano Plot ( Figure 6). Visually displayed the signi cantly changed metabolites between the two groups and speed up the screening of potential metabolic markers involved in the pathway. On the basis of p<0.05, FC>1.5 or FC<0.67, the substances represented by the red dots in the upper left and upper right corners of the coordinates were the difference metabolites.

Comparison results of different metabolites
The VIP value obtained from OPLS-DA analysis screened the difference metabolites. The T test and FC analysis judged the signi cance of difference metabolites between the two groups and whether the difference metabolites increased or decreased. VIP>1.0, p<0.05, FC>1.5 represented the differential metabolites were signi cantly increased; VIP>1.0, p<0.05, FC<0.67 represented the differential metabolites were signi cantly decreased. The metabolites of the two groups were screened. It was found there were 32 different metabolites between group C and group E in the positive and negative ion mode (Table 3). Note: * means p<0.05 for comparison between the two groups; ** means p<0.01 for the comparison between the two groups; ↑ means that the change of the difference is an upward trend; ↓ means that the change of the difference is an upward trend Hierarchical clustering analysis of differential metabolites The difference metabolites of myocardial samples between the two groups were analyzed by hierarchical cluster analysis ( Figure 7). The red was a signi cant increase in metabolites, The blue was a signi cant decrease in metabolites. Here, the color changes of the samples in the same group were relatively concentrated and that of different groups were sharply contrasted, indicating the differences of the myocardial metabolites within the group were small but the differences between the groups were obvious. The selected different metabolites were reliable.
Pathway analysis of target metabolites MetaboAnalyst 4.0 was used to analyze the differential metabolites between the two groups by Met-PA approach. Imported the data of 32 different metabolites into Pathway Analysis to explore the weight of the metabolic pathways involved ( Figure 8). There were 26 metabolic pathways involved during highintensity exercise (Table 4). Here, Raw p<0.05 and Pathway Impact>0.05 were used as the critical point to screen the above-mentioned metabolic pathways. We found there were 6 potential target metabolic pathways that affect the myocardial metabolism of rats during acute high-intensity exercise, namely fructose and mannose metabolism, Linoleic acid metabolism, pyrimidine metabolism, niacin and nicotinamide metabolism, arginine metabolism, amino sugar and nucleotide sugar metabolism ( Figure  9). Note: Total is the total number of compounds in the pathway; Expected is the expected value; His is the number of accurate matches in the uploaded marker data; Raw P is the original P value obtained through the analysis of the pathway score map; FDR is the error trigger rate; Impact is obtained through topological analysis Out-of-path in uence value

Metabolic markers of rat myocardium
The receiver operating characteristic curve (ROC) evaluated the diagnostic ability of differential metabolites during acute high-intensity exercise. Combined the area value (AUC) and P value (P<0.05) under the ROC curve to screen out the potential metabolism of the above 6 acute high-intensity exercise metabolic pathways. It was Thymine ( In this study, these 12 metabolites were regarded as potential markers affecting fatigue metabolism.

Discussion
Metabolomics studies the metabolic mechanism from the overall metabolite pro le. This study used LC-MS to explore the in uence of acute high-intensity exercise on rat myocardial metabolism. We found there were 32 different metabolites, participating in 26 metabolic pathways during acute high-intensity exercise. Among them, fructose and mannose metabolism, linoleic acid metabolism, pyrimidine metabolism, niacin and nicotinamide metabolism, arginine metabolism, amino sugar and nucleotide sugar metabolism were the 6 target metabolic pathways during acute high-intensity exercise involved in 12 potential metabolic markers.
Phosphatidyl choline (PC), 1-stearoyl-2-oleoyl lecithin (SOPC) and linoleic acid (LA) participated in the metabolic pathway of linoleic acid (Figure 10.a). After acute exercise, the content of PC in subjects' plasma increased, leading to impaired utilization of cardiac fatty acids and in ammation-mediated metabolic disorders, which induced heart failure [7] . The reason may be that under the high oxidative stress conditions, PCs were easily transformed into lysophosphatidylcholine (LPC) catalyzed by phospholipase A2 (PLA2). LPC induces the production of in ammatory factors such as TGF-β1, IL-1β, accelerates the apoptosis of cardiomyocytes and promotes the development of coronary heart disease [8] . Lu's study showed the myocardium produced a large amount of free radicals during high-intensity exercise and leaded to the overexpression of PLA2, furthermore, accelerated the production of LPC, which nally induced heart damage [9] . The metabolic process and biological effects of SOPC are consistent with those of PC. Research showed after excessive consumption of red meat, the content of SOPC was high and trimethylamine oxide (TMAO) was produced, which was a risk factor to induce coronary atherosclerosis and cardiovascular diseases [10] . In our study, SOPC and PC in group E increased signi cantly, indicating acute high-intensity exercise increased the cardiac oxidative stress sharply, possibly producing the lipid peroxidation and toxic substances, damaging the health of myocardium. LA were essential nutrients for organisms, which has the functions of lowering blood pressure and promoting microcirculation [11] . However, patients with diastolic dysfunction were found a signi cant increase of LA in the neointimal part of the myocardium and atherosclerotic plaques which improved lipid metabolism to provide energy for the heart [12] . Study also showed LA increased when the myocardium is in a pathological state, inducing myocardial hypertrophy through the calcineurin-activated T cell nuclear factor signaling pathway. In addition, the oxidation products of LA can be easily produced because of oxidative stress during acute high-intensity exercise, which causes macrophage apoptosis and induced coronary plaque rupture, thrombus formation or myocardial infarction [13] . In this study, LA in group E was signi cantly increased, indicating the organism accordingly improved the utilization of myocardium fatty acids during acute high-intensity exercise. However, myocardial ischemia and hypoxia is prone to generate linoleic acid oxidation products or its derivatives, causing heart damage. D-mannose and D-mannose-1-phosphate participated in the metabolic pathway of fructose and mannose (Figure10.c). D-Mannose exists as a component of mannan in the body, which will be phosphorylated into D-mannose-6-phosphate by hexokinase. Later, a small part is isomerized to form D-mannose-1phosphate [14] . D-mannose is structurally similar to glucose. When its content in organism is high, glucose transporter will be snatched by D-mannose to produce high levels of D-mannose-6-Phosphoric acid, which disrupts the aerobic oxidation of glucose, accelerates glycolysis and causes abnormal energy supply to the myocardium. These changes will hinder succinate-mediated activation of hypoxia-inducible factors, thereby inhibiting the expression of vascular endothelial growth factor and heme oxygenase 1, reducing angiogenesis and cardiac antioxidant capacity and ultimately leading to heart disease [15] . Study showed D-mannose in type 2 diabetic rats was signi cantly increased with the risk of myocardial infarction [16] . Ultramarathon runners also been detected high levels of D-mannose in their urine [17] . Dmannose-1-phosphate easily reacts with proteins or lipids to participates in the glycosylation process and generate glycosylation end products (AGEs) [14] . AGEs cause myocardial lipid metabolism disorders, induce atherosclerosis and mediate myocardial chronic in ammation or cell apoptosis through the myeloid differentiation receptor 2/toll-like receptor 4 pathway, which leads to chronic heart failure [18] .
Myocardial pressure, corresponding to heart failure, overloaded during high-intensity exercise, which will made material metabolism abnormal and produced more AGEs [19] . In this study, D-mannose and Dmannose 1-phosphate (p<0.05) in group E were signi cantly increased, indicating the aerobic oxidation of glucose in the rat myocardium during acute high-intensity exercise was blocked and the glucose metabolism was disturbed. At this time, cardiovascular regeneration and antioxidant capacity decreased, promoting the production of AGEs and inducing heart failure.
Cytidine-phosphate-N-acetylneuraminic acid (CMP-Neu5Ac), D-mannose-1-phosphate and Dmannose participated in the metabolic pathway of amino sugar and nucleotide sugar (Figure 10.e). CMP-Neu5Ac is the activated form of Neu5Ac, existing as the component of glycolipids and glycoproteins. The two is positively correlated. Neu5Ac is synthesized in the cytoplasm of eukaryotic cells and transferred to the nucleus. It is activated by CMP-Neu5Ac synthase to transfer cytidine monophosphate (CMP) residues from cytidine triphosphate (CTP) and generate CMP-Neu5Ac [20] . Study showed the increase of CMP-Neu5Ac caused cardiomyocyte apoptosis and in ammatory response through Rho/ROCK-JNK/ERK signaling pathway, interfered with lipid metabolism and accelerated the occurrence of atherosclerosis, resulting in myocardial injury [21] . Neu5Ac can also cause myosin light chain phosphorylation and integrin aggregation, increase the permeability of endothelial cells and promote the release of oxidized lowdensity lipoproteins and in ammatory factors, which destroied the intravascular microenvironment and apoptosis of arterial smooth muscles, nally causing a cardiovascular disease [22] . During high-intensity exercise, myocardium ischemic necrosis increased, which promoted the movement of Neu5Ac in serum to the conjugate in plasma, inducing myocardial injury [23] , so the concentration of Neu5AC can represent the level of in ammatory response and be served as a marker for heart diseases. In this study, CMP-Neu5Ac (p<0.01) increased signi cantly in group E, indicating the heart damage during acute highintensity exercise may be related to the in ammatory reaction of the heart. Niacinamide and L-aspartic acid (Asp) participated in the metabolic pathway of niacin and niacinamide ( Figure 10.b). Niacinamide is the precursor of Coenzyme I (NAD+) and has a signi cant antioxidant effect [24] . Nicotinamide can increase the bioavailable NO content and up-regulate the expression of forkhead box protein 1 (Foxo1) by activating Silent Information Regulator 1 (SIRT1), thereby enhancing angiogenesis activity, inhibiting cardiomyocyte apoptosis and maintaining cardiovascular health [25] .
After excessive exercise, more nutrients were consumed, resulting in myocardial ischemia or hypoxia and a signi cant decrease in Niacinamide [26] . However, supplementing nicotinamide during exercise will increase the antioxidant enzyme activity of cardiomyocytes and mitochondrial protein, activate autophagy to degrade damaged cell components in a timely manner, maintain the homeostasis of cardiomyocytes and improve the exercise endurance of rats [27] . In this study, nicotinamide in group E (p<0.05) decreased signi cantly, showing acute high-intensity exercise reduced myocardial niacinamide, which easily induces myocardial pathological damage. Asp is an important substrate of gluconeogenesis with the effects of protecting cardiovascular health and promoting fatigue recovery [28] . Study showed Asp can reduce hyperammonemia caused by high-intensity exercise and prolong exercise exhaustion time by promoting muscle glycogen retention, free fatty acid oxidation and gluconeogenesis [29] .
Supplementing Asp in repeated cycling sprints can increase the concentrations of glutamic acid, alanine, phenylalanine and total amino acid in blood, reduce the body's latic acid production by generating carbonates and maintain the normal PH value of the blood, thereby alleviating exercise fatigue and enhancing the output power in bicycle sprinting [30] . In this study, Asp (p<0.05) in group E increased signi cantly, indicating during acute high-intensity exercise, the rat myocardium will produce Asp to resist the damage of myocardium by improving the oxidative stress, glucose metabolism and lipid metabolism, thereby protecting heart health to a certain extent.
L-arginine (L-Arg) and L-aspartic acid participated in the metabolic pathway of arginine (Figure 10.f). L-Arg is an essential amino acid in human's body with the functions of detoxi cation and alleviating fatigue [31] . Research showed L-Arg can produced NO, which dilated blood vessels and increased cardiac blood ow to enhance lung ventilation and maximum oxygen uptake, thereby improving coronary perfusion or cardiomyocyte death during strenuous exercise and maintaining the normal cardiopulmonary function [32] . L-Arg can also promote the phosphorylation of PI3K/Akt and then accelerated the secretion of insulin to regulate the glucose transport process, improved the utilization of glucose and alleviated secondary heart damage in diabetic patients [33] . Exogenous supplementation of L-Arg can reduce the MDA of myocardium, lipid peroxides and free radicals, enhance the activity of antioxidant enzymes and ATPase, thereby reducing heart oxidative stress damage after a one-time continuous downhill running [34] . In this study, L-Arg and Asp (p<0.05) in group E increased signi cantly, indicating during acute high-intensity exercise, the metabolism of amino acid was enhanced, which provided energy for the myocardium and protected the heart.
Uracil, thymidine and thymine participated in the metabolic pathway of pyrimidine (Figure 10.d). The three play an important role in the regulatory functions and energy metabolism. Uracil nucleotides in the venous plasma of patients with myocardial ischemia increased signi cantly to improve cardiac output, protect the ischemic heart and reduce TNF-a-mediated myocardial apoptosis, which were the important positive inotropic factors and alleviated chronic heart failure [35] . Laitano's research showed during the recovery period after high-intensity running wheel training, the level of uracil in the myocardium of mice was signi cantly increased, which was essential for myocardial repair after strenuous exercise [36] . Study showed the plasma thymine in mice with acute myocardial ischemia increased signi cantly and the expansion of thymine was closely related to the risk of heart disease [37] . Peng's research showed a signi cant decrease in thymidine in rats' serum can be used as a biomarker of early acute myocardial infarction [38] . After strenuous exercise, the increase of thymine in rats' serum had been con rmed and it will induce cardiac ischemia damage. Thymidine supplementation can promote the regeneration of rat myocardial cells, provide energy for the heart and improve anti-fatigue ability [39] . In this study, thymine (p<0.01) in group E increased signi cantly, indicating during acute high-intensity exercise, the myocardium was in an ischemic state and the heart may have pathological changes. At this time, uracil and thymidine (p<0.01) in myocardium increased which can improve cardiac function.

Conclusion
In this study, we found compared with the control, rat myocardium acute high-intensity exercise had 32 different metabolites and 12 potential metabolic markers which participated in 6 target metabolic pathways by LC-MS and metabolic pathway analysis. Cardiac dysfunction caused by acute high-intensity exercise may be related to myocardial lipid peroxidation, lipid and glucose metabolism disorders. At this moment, the increase of amino acid and nucleotide metabolism in organism can speed up the repair of damaged myocardium and provide energy for myocardium to maintain the normal physiological function of heart. Therefore, improving myocardial material metabolism may be an important target for the treatment of heart disease.   Acute high-intensity exercise metabolic pathways constructed by MetPA database.

Declarations
Note: The abscissa pathway impact is the importance value of the metabolic pathway obtained by topological analysis, and the ordinate -logP is the signi cance level of the metabolic pathway enrichment analysis; the greater the pathway impact and -logP value are, the higher the correlation of the metabolic differences between different groups is, the bigger the circle is.

Figure 9
Metabolic pathways involved in differential metabolites. Red is the potential marker of the pathway involved in this study; Blue is not in the metabolites of this study