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Table of Contents
ORIGINAL ARTICLE
Year : 2021  |  Volume : 64  |  Issue : 2  |  Page : 97-105

Study on the time-effectiveness of exercise preconditioning on heart protection in exhausted rats


1 Department of Cardiology, The Hospital of the 82nd Group Army, Baoding, Hebei, China
2 Department of Clinical Pharmacy, The Hospital of the 82nd Group Army, Baoding, Hebei, China
3 Department of Central Laboratory, The Hospital of the 82nd Group Army, Baoding, Hebei, China
4 Department of Gynaecology, The Hospital of the 82nd Group Army, Baoding, Hebei, China

Date of Submission07-Aug-2020
Date of Decision15-Mar-2021
Date of Acceptance16-Mar-2021
Date of Web Publication28-Apr-2021

Correspondence Address:
Dr. Xuebin Cao
Department of Cardiology, The Hospital of the 82nd Group Army, No. 991 Baihua East Road, Lianchi District, Baoding, Hebei
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/CJP.CJP_65_20

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  Abstract 


To investigate the persistence time and the effectiveness of exercise preconditioning (EP) on myocardial protection in exhausted rats from myocardial enzymes, electrocardiogram (ECG), cardiac function, and mitochondrial respiratory function after cessation of exercise training. One hundred and twelve healthy male Sprague–Dawley rats were randomly divided into seven groups (n = 16): control group (CON), exhaustive exercise (EE) group, EP group, and EE after EP (EP + EE); furthermore, EP + EE group was randomly divided into 1D, 3D, 9D, and 18D groups (1D, 3D, 9D, and 18D) and performed exhaustive treadmill exercise at a speed of 30 m/min on the 1st, 3rd, 9th, and 18th days separately after EP exercise stopped. We detected the serum contents of N-terminal pro B type natriuretic peptide (NT-proBNP) and cardiac troponin I (cTnI) by the enzyme-linked immunosorbent assays method, recorded ECG, detected heart function by pressure volume catheter, measured the respiratory rates of rat myocardial mitochondria state 3 and 4 of complex I, complex II, and IV by high-resolution breathing apparatus. EP could decrease the serum content of NT-proBNP and cTnI, improved the electrical derangement and the left ventricular function in exhausted rats. Moreover, the protective effect was more obvious in the 9th day after EP stopped, whereas it would disappear when EP stopped for more than 18 days. Compared with EE group, the respiratory rate value of myocardial mitochondrial complex increased in 1D, 3D, and 9D groups. Therefore, the protective effect of EP on the heart of exhausted rats decreased with the prolongation of stopping training time, and the effect was significant within 3 days of discontinuing training, then decreased gradually, and completely disappeared in the 18th day. EP enhanced the cardiac function in exhausted rats through raising the nicotinamide adenine diphosphate hydride (NADH) electron transport chain and increased the respiration rates of mitochondrial respiratory complex I and IV state 3, thereby improved myocardial mitochondrial respiratory function and energy metabolism.

Keywords: Cardiac function, exercise preconditioning, exhaustive exercise, mitochondrial respiratory chain, rats


How to cite this article:
Su Y, Wang Y, Xu P, Sun Y, Ping Z, Huang H, Cao X. Study on the time-effectiveness of exercise preconditioning on heart protection in exhausted rats. Chin J Physiol 2021;64:97-105

How to cite this URL:
Su Y, Wang Y, Xu P, Sun Y, Ping Z, Huang H, Cao X. Study on the time-effectiveness of exercise preconditioning on heart protection in exhausted rats. Chin J Physiol [serial online] 2021 [cited 2021 Jun 22];64:97-105. Available from: https://www.cjphysiology.org/text.asp?2021/64/2/97/315092

The authors Ye Su and Yang Wang contributed equally to this work.





  Introduction Top


As a source of stress, exercise has “double-sided nature.” Suitable exercise can protect the body, while unsuitable strenuous exercise can cause damage to the body. Exhaustive exercise (EE) means that the body continues to move in a state of fatigue which is beyond the limit of the body and leads to relative or absolute ischemia and hypoxia to the heart[1] and generate myocardial damage. It is commonly seen in athletes, soldiers, and other special groups that often engage in intense training. Exercise preconditioning (EP) can cause repeated and transient myocardial ischemia and hypoxia through sustained or intermittent intensive exercise in a short time, thus the tolerance of myocardial tissue is improved, and the myocardial injury that under the long time ischemia and hypoxia is reduced.[2] EP can enhance the expression of cardiac protective substances, makes the heart structure and function undergo adaptive change,[3] reduces myocardial fiber rupture, and increases myocardial tolerance to long-time ischemia and hypoxia.[1] EP promotes the expression of mitochondrial biogenic expression pathway PGC-1α-NRF1/NRF2 protein, so the activities of mitochondrial respiratory complex I, II, and IV are enhanced, and the energy metabolism level of myocardial cell is improved. In addition, EP can weaken oxidative stress and TXNIP/TRX/NF-κBp65/NLRP3 inflammatory signaling pathway and reduce the content of downstream inflammatory factors.[4] Moreover, EP also can activate PI3K-Akt signaling pathway, inhibit mitochondrial pathway, reduce myocardial cell apoptosis, and fight against heart injury caused by EE.[3] EP has the characteristics of simple, feasible, and controllable intensity, and it can be used as a preventive and therapeutic method for athletic heart injury, which is of great significance to improve the training level of athletes and combat effectiveness of the army.

The protective effect of EP on the heart has two time windows. The first time window is generated immediately after the exercise is completed, and the protective effect gradually decays after 3 h, this is known as the early protective effect. The second time window is defined as that the protective effect begins at 24 h after the exercise is completed and lasts for several days, this is known as the late protective effect. Moreover, the action intensity of the late protective effect is obviously weaker than the early protective effect, but it has more practical and effective value due to its longer action period.[5] Lennon et al. found that preconditioning with different exercise intensity all could improve the recovery degree of myocardial injury caused by ischemic/reperfusion (I/R) and alleviate cardiac suppression, meanwhile the cardiac function of moderate and high-intensity EP was improved significantly because of the late preconditioning protective effect.[6]

The cardiac protective effect induced by EP will decay gradually and return to the preexercise state slowly. It shows that the athletic heart has the adjustability and remodeling. At present, there are few studies on the timeliness of the protective effect after stopping exercise. The duration of EP protection has not been agreed. Lennon et al. found that the protective effect of EP on I/R injury could be maintained for about 9 days and recovered to the level of the control group within 9-18 days.[7] Another study found that participants were given a continuous quantitative exercise to measure their heart function, respectively, before and 21 days after they stopped training, the heart rate (HR) increased significantly, the stroke volume (SV) decreased, the cardiac output decreased and the oxygen uptake decreased when they stopped training after 21 days; these might be the results of a combined decrease in cardiac volume, blood flow, total plasma volume, and vasoconstriction capacity. Calvert et al.[8] found that exercise could reduce myocardial injury in mice after a 4-week training period, and these protective effects could last for at least 1 week after the cessation of training, but the effects would disappear completely when the exercise was stopped for 4 weeks.

In accordance with the Lennon experiment, we established an EP model and selected an EP exercise program with the best protective effect of intermittent long-term medium and high intensity load training, so as to explore the time-effectiveness of the late protective effect about EP.[7],[9] We observed the protective effect of EP on cardiac injury caused by exhausting exercise at different time after EP suspension from different perspectives of cardiac enzymes, electrocardiogram (ECG), cardiac function and mitochondrial respiratory function, and discussed the time-effectiveness of EP-induced myocardial protective effect.


  Materials and Methods Top


Experimental animal

The male Sprague-Dawley rats (200 ± 20 g) were provided by Beijing Zhongkeda Biotechnology Co., LTD., License Number: SCXK (Beijing)-2016-0002. National standard rodent dry feed was provided ad libitum, the indoor temperature was maintained at 18°C-22°C, and the relative humidity was maintained at 40%-55%. All experiments were conducted in compliance with the guide for the Care and Use of Laboratory Animals and reviewed and approved by the Ethics Committee for the Use of Experimental Animals at the PLA 82nd Group Military Hospital (CA19-02).

Drugs and main instruments

The main reagents used in the present study are listed below. The cardiac troponin I (cTnI) and N-terminal pro B type natriuretic peptide (NT-proBNP) enzyme-linked immunoassay kits were obtained from Shanghai Kmaels Biologic Technology Co., Ltd., ADP potassium salt, cytochrome c, lactobionate, ATP-Na2, Na2Phosphocreatine, EGTA, ascorbic acid, taurine, imidazole, DTT, HEPES, MES, glutamate, malate, succinate, TMPD, antimycin A and rotenone were purchased from Sigma (USA). The following main instruments were used in the present study: an animal treadmill (Taimeng, China), a PowerLab signal acquisition and analysis system, a MultiscanGO enzyme standard instrument (Thermo, USA), a pressure volume catheter (SPR-838, Millar Company, USA), a PowerLab data acquisition and analysis system (AD Instruments, Australia), a bioelectric amplifier (AD Instruments, Australia), a needle electrode (AD Instruments, Australia) and a high-resolution respirometry (Oroboros Instruments, Austria).

Establishment and grouping of animal models

One hundred and twelve healthy male Sprague–Dawley rats were randomly divided into seven groups (n = 16): control group (CON), EE group, EP group, EE after EP group (EP + EE), and EP + EE group was randomly divided into 1D group, 3D group, 9D group, and 18D group. Half of the 16 animals in each group were used for the pressure volume catheter detection of cardiac function, which was an invasive experiment. These animals were euthanized after the experiment. ECG and serum were collected from the remaining animals (n = 8 animals per group). We established the animal model of EP by referring the studies of Domenech and Lennon. [6,10] EP + EE group (1D, 3D, 9D, and 18D) was performed EE at a speed of 30 m/min at the 1st, 3rd, 9th, and 18th days separately after EP exercise was stopped. EP animal model was established by referring to the studies of Lennon et al. and Domenech et al.[6],[10] Intermittent treadmill exercise was performed at a speed of 26 m/min with the slope of 5° (75% VO2 max) for 60 min. Exercise for 15 min, then rest for 5 min, this was regarded as a set, three sets were repeated in the 60 min exercise process, and the intermittent treadmill exercise was carried out for 3 weeks, and exhaustive treadmill exercise was carried out at a speed of 30 m/min at the 1st, 3rd, 9th and 18th days after the exercise was stopped. Rat exhaustion criteria: the running of the rats was significantly more difficult in the late stages of exercise than in the early stages, unable to bear the original running speed, speed gradually decreased, movement posture also changed from the ground running to half supine position running, even stayed on the back third of the runway as many as 10 times, under the various stimulus, there was significant change in the motion state of the rats. After the exercise, the rats were obviously characterized by shortness of breath, fatigue, lying on the spot, slow reaction, and failure to avoid being caught in time.

Collection and preparation of serum

Rats were subjected to abdominal anesthesia with pentobarbital sodium (40 mg/kg), the chest was opened, and blood was collected from the inferior thoracic vena cava. The blood was centrifuged at 3000 r/min for 20 min, and the supernatant was collected and stored in a -80°C freezer until the detection of serum indicators.

Enzyme-linked immunoassays for N-terminal pro B type natriuretic peptide and cardiac troponin I levels in the serum

The serum was removed from the −80°C freezer. Enzyme-linked immunosorbent assays were performed according to the instructions of the kits. The optical density (OD) value of each sample was measured at 450 nm. The OD value for the standard was measured, and a standard curve was constructed with the OD value on the y-axis and the concentration on the x-axis. The concentration of the indicated marker in each sample was obtained from the standard curve.

Electrocardiography

Adaptive electrocardiography training was performed in all experimental rats. ECGs were recorded from the rats of CON and EP groups in a quiet state for 5 min. In EP + EE group, ECGs were recorded for 5 min immediately after EE. Anesthetic rats were placed in the rat cage, and all the sets of limbs and the right forearm were routinely disinfected. Subcutaneous punctures in extremities were created to insert the electrodes (the left hind leg was used as the positive electrode, the right foreleg was used as the negative electrode, and the left foreleg was used as the grounding electrode), and the electrode needle was fixed. The dynamic ECG results were recorded by a PowerLab data acquisition and analysis system. The HR, PR interval, QRS interval, ST height, and T amplitude were obtained.

Determination of cardiac function parameters with a pressure volume catheter

Rats were anesthetized with pentobarbital sodium (40 mg/kg, i.p.), and the closed-chest approach was chosen for catheter insertion. The animal was fixed in the supine position on the operating table. The skin of the neck was disinfected before a midline neck incision, and the trachea was separated and intubated. The right carotid artery was separated from the common carotid artery. Two 4-0 silk threads were sewn through the common carotid artery, and one of the silk threads was used to ligate the proximal end of the carotid artery. A cut was made at the end of the heart to complete the knot. The pressure volume catheter was inserted through the incision into the left chamber along the inverse blood flow of the carotid artery and calibrated with MPVS control software (Millar Company, USA). The left ventricular pressure volume waveforms of the anesthetized rats were recorded with Chart7 software (AD Instruments, Australia) in real-time. Vessels and catheters were fixed with another silk thread. The baseline data were recorded for 15 min. Moreover, the abdominal skin was disinfected, a median incision was made, the inferior vena cava was occluded, and changes in the waveform were recorded. A 20 μl solution of 30% NaCl was rapidly injected into the anterior jugular vein, and pressure-volume waveform changes were recorded. The first 4 holes of a calibration cuvette with known diameters (provided by the manufacturer) were quickly filled, and the catheter tip was submerged in fresh heparinized warm blood. The conductance changes in the volume channel were recorded, and the volume was calculated.[11]

The HR, end-systolic pressure (Pes), end-diastolic pressure (Ped), end-systolic volume (Ves), end-diastolic volume (Ved), SV, ejection fraction (EF), peak rate of the increase in pressure (dP/dtmax), peak rate of the decrease in pressure (−dP/dtmin), slope of the end-systolic pressure volume relationship (ESPVR), relaxation time constant (Tau), and slope of the end-diastolic pressure volume relationship (EDPVR) were detected. The pressure volume loop (PV Loop) was drawn with the pressure on the Y-axis and the volume on the X-axis.

The tests of myocardial mitochondrial respiratory function

The rat hearts were removed in phosphate-buffered saline buffer to clean the blood. Then a part of the left ventricular apex was extracted for mitochondrial respiratory function tests by a sharp blade. The myocardium was separated along the edge into small muscle bundles and each consisted of six to eight muscle fibers. The separated muscle bundles were quickly placed into a tube containing 2 ml BIOPS. Then oscillated it slightly for 30 min by a microoscillator, and the permeation took place on the ice. The elution of osmotic fluid should be repeated three times on the ice. The wet weight of the specimen was weighed by an electronic balance. Muscle fibers were put into 2 ml MIR06 solution, meanwhile added 2 ml MIR06 into each of the reaction compartments of Oxygraph-2k and put the cardiac muscle tissue into the reaction compartments, then added oxygen to the reaction compartments and the oxygen concentration did not exceed 400 μM. The test operation was carried out after the pure oxygen was injected into the reaction chamber for 10 - 15 min in order that the state was stable.

The respiration rate of mitochondrial complex I state 3 was obtained by sampling 10 μL glutamate acid (Glu) and 5 μL malate acid (Mal). The respiration rate of complex I state 4 was obtained by adding 20 μL ADP. The respiratory control ratio (OR respiratory control index RCR) of mitochondrial complex I was the ratio of respiratory rate of state 3 and state 4, also known as respiratory regulation ratio.

The respiration rate of complex I and II was acquired by adding 20 μL succinic acid (Suc), and the respiration rate of complex II was got by adding 5 μL rotenone (Rot) to inhibit the respiration rate of complex I.

The respiration rate of antimycin III was obtained by adding 1 μL antimycin A (ANT-A) which was an inhibitor of complex III in mitochondria, and the respiration rate of complex IV was got by adding 5 μL TMPD and 5 μL ascorbic (ASC) when the curves stabilized.

Washing the reaction compartments repeatedly with 70% ethanol and pure water at least three times, respectively, and soaking them with 100% ethanol for 30 min. Finally, cleaning them with pure water and add a small amount of pure water into the reaction compartments.

Statistical analysis

The data were presented as means ± standard deviation. SPSS17.0 statistical software (SPSS Company, USA) was used to analyze all experimental data. A single factor analysis of variance was used for the comparisons of multiple means after a one-way ANOVA and a homogeneity test was first performed. Comparisons of mean values between the two groups were performed by LSD test when the variance was equal or Dunnett's T3 method when the variance was unequal. A correlation analysis was performed by calculating Pearson's correlation coefficients. A single factor regression analysis was performed. P < 0.05 was considered to indicate a significant difference.


  Results Top


Effects of exercise preconditioning on serum N-terminal pro B type natriuretic peptide and cardiac troponin I levels in exhausted rats

Compared with CON group, the serum contents of NT-proBNP and cTnI in each exhaustive group were all significantly increased (P < 0.05). Compared with EE group, the contents of NT-proBNP and cTnI in 1D, 3D, and 9D groups were all significantly reduced (P < 0.05). For the content of serum NT-proBNP, 9D and 18D groups both increased significantly compared with 1D group (P < 0.05), and compared with 9D group, 3D group was significantly reduced and 18D group was significantly increased (P < 0.05). With regard to the content of serum cTnI, compared with 9D group, 18D group increased significantly (P < 0.05). The results are shown in [Figure 1] and [Figure 2].
Figure 1: Effects of EP on serum NT-proBNP levels in exhausted rats. The data are presented as means ± SD, n = 8 animals per group. *P< 0.05 compared with the Con group, #P < 0.05 compared with the EE group, P < 0.05 compared with the EP group, P < 0.05 compared with the 1D group, P < 0.05 compared with the 9D group. NT-proBNP: N-terminal pro B type natriuretic peptide, CON: Control group, EE: Exhaustive exercise group, EP: Exercise preconditioning group, 1D, 3D, 9D, and 18D: Exhaustive exercise, respectively, performed on 1st, 3rd, 9th, and 18th days after EP was stopped, SD: Standard deviation.

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Figure 2: Effects of EP on serum and cTn-I levels in exhausted rats. The data are presented as means ± SD, n = 8 animals per group. For groups, see the footnote to Figure 1. *P < 0.05 compared with the CON group, #P < 0.05 compared with the EE group, P < 0.05 compared with the EP group, P < 0.05 compared with the 1D group, P < 0.05 compared with the 9D group. cTn-I: Cardiac troponin I, CON: Control group, EE: Exhaustive exercise group, EP: Exercise preconditioning group, 1D, 3D, 9D and 18D: Exhaustive exercise, respectively, performed on 1st, 3rd, 9th, and 18th days after EP was stopped, SD: Standard deviation.

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Effects of exercise preconditioning on electrocardiogram indexes in exhausted rats

Compared with CON group, there were significant differences in the increase of HR and ST segment elevation in each exhaustive group (P < 0.05). Compared with EE group, the shortening of QRS interval and the elevation of ST segment all decreased in 1D, 3D, and 9D groups (P < 0.05). Compared to 3D group, HR increased and ST segment elevated significantly in 9D and 18D groups (P < 0.05). Compared with 9D group, the QRS interval of 18D group was significantly prolonged (P < 0.05). The results are shown in [Figure 3] and [Table 1].
Figure 3: Original recording showing the electrokardiogram.

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Table 1: Effects of exercise preconditioning on electrocardiogram indexes in exhausted rats

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Effects of exercise preconditioning on cardiac function indexes in exhausted rats

Compared with CON group, the HR and the absolute values of dP/dtmax and −dP/dtmin in each exhaustive group were decreased, SV and EF decreased and Ves increased in EE group and 18D group (P < 0.05). Compared with EE group, HR, Ped and Ves decreased significantly in 1D and 3D groups, whereas the absolute values of Pes, dP/dtmax and −dP/dtmin increased prominently (P < 0.05). Compared with 1D group, the absolute values of dP/dtmax and −dP/dtmin in 9D and 18D groups reduced observably, showing statistical difference (P < 0.05). Compared with 9D group, the absolute values of dP/dtmax and −dP/dtmin increased in 3D group (P < 0.05), whereas 18D group decreased significantly (P < 0.05). Compared to 1D group, CO, SV and EF decreased and Ves increased prominently in 18D group. Compared with 9D group, SV decreased, Ves increased and EF decreased significantly in 18D group (P < 0.05). Compared with CON group, ESPVR value of EE, 9D and 18D groups all decreased notably, while EDPVR and Tau value increased prominently (P < 0.05). Compared with EE group, ESPVR increased and EDPVR decreased in 1D, 3D, and 9D groups (P < 0.05). Compared with group 9D, ESPVR of 3D group and EDPVR of 18D group both increased, while EDPVR of 3D group and ESPVR of 18D group both decreased significantly (P < 0.05). The results are shown in [Figure 4] and [Table 2], [Table 3], [Table 4].
Figure 4: Original recording showing the pressure-volume loops. For groups, see the footnote to Figure 1. CON: Control group, EE: Exhaustive exercise group, EP: Exercise preconditioning group, 1D, 3D, 9D, and 18D: Exhaustive exercise, respectively, performed on the 1st, 3rd, 9th, and 18th days after EP was stopped.

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Table 2: Effects of exercise preconditioning on cardiac function parameters in exhausted rats

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Table 3: Effects of exercise preconditioning on systolic indices in exhausted rats

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Table 4: Effects of exercise preconditioning on diastolic indices in exhausted rats

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Comparison of myocardial mitochondrial complex between each group

Comparison of state 4, state 3 respiration rate, and respiratory control ratio of myocardial mitochondrial complex I between each group

Compared with CON group, the state 4 respiration rates of mitochondrial complex I increased significantly in EE, 3D, 9D, and 18D groups (P < 0.05). The state 4 respiration rates of complex I in 18D group increased prominently (P < 0.05). Compared with CON group, the state 3 respiration rates of complex I in every EE group reduced prominently (P < 0.05). Compared with EE group, the state 3 in 1D, 3D, and 9D groups all increased notable (P < 0.05), whereas 18D group decreased prominently (P < 0.05); the state 3 respiration rates of complex I in 8D group reduced prominently compared with 9D group (P < 0.05). The results are shown in [Figure 5].
Figure 5: Comparison of respiration rate of myocardial mitochondrial complex I between each group. The data are presented as means ± SD, n = 8 animals per group. For groups, see the footnote to Figure 1. *P < 0.05 compared with the CON group, #P < 0.05 compared with the EE group, P < 0.05 compared with the EP group, P < 0.05 compared with the 1D group, P < 0.05 compared with the 9D group. CON: Control group, EE: Exhaustive exercise group, EP: Exercise preconditioning group, 1D, 3D, 9D, and 18D: Exhaustive exercise, respectively, performed on the 1st, 3rd, 9th, and 18th days after EP was stopped, SD: Standard deviation.

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Compared with CON group, the RCR of complex I in every EE group reduced prominently (P < 0.05); compared with EE group, the complex I RCR in 1D, 3D, and 9D groups increased obviously (P < 0.05). The complex I RCR in 18D group reduced significantly compared with 9D group (P < 0.05). The results are shown in [Figure 6].
Figure 6: Comparison of myocardial mitochondrial respiratory control ratio between each group. The data are presented as means ± SD, n = 8 animals per group. For groups, see the footnote to Figure 1. *P < 0.05 compared with the CON group; #P < 0.05 compared with the EE group. P < 0.05 compared with the EP group; P < 0.05, compared with the 1D group, P < 0.05, compared with the 9D group. CON: Control group, EE: Exhaustive exercise group, EP: Exercise preconditioning group, 1D, 3D, 9D, and 18D: Exhaustive exercise, respectively, performed on the 1st, 3rd, 9th, and 18th days after EP was stopped, SD: Standard deviation.

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Comparison of state 3 respiration rate of myocardial mitochondrial complex II and complex IV between each group

Compared with CON group, the state 3 respiration rate of complex II in every EP group decreased prominently (P < 0.05). Compared with CON group, the state 3 respiration rate of complex IV in EE, 3D, 9D, and 18D groups all decreased obviously (P < 0.05). Compared with EE group, the state 3 respiration rate of complex IV in 1D, 3D, and 9D groups rose prominently (P < 0.05). Compared with 1D group, the state 3 respiration rate of complex IV in 18D group decreased significantly (P < 0.05). The results are shown in [Figure 7].
Figure 7: Comparison of respiration rate of myocardial mitochondrial complex II, IV between each group. The data are presented as means ± SD, n = 8 animals per group. For groups, see the footnote to Figure 1. *P < 0.05 compared with the CON group, #P < 0.05 compared with the EE group, P < 0.05 compared with the EP group, P < 0.05 compared with the 1D group, P < 0.05 compared with the 9D group. CON: Control group, EE: Exhaustive exercise group, EP: Exercise preconditioning group, 1D, 3D, 9D, and 18D: Exhaustive exercise, respectively, performed on the 1st, 3rd, 9th, and 18th days after EP was stopped, SD: Standard deviation.

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Analysis of the correlations between cardiac function parameters and myocardial mitochondrial complex respiration rate

According to Pearson linear correlation analysis, the respiration rate of complex II was strongly and positively correlated with CO formation in CON group (P < 0.01), negatively correlated with HR, and positively correlated with dP/dtmax (P < 0.05). In EE group, the state 4 respiration rate of complex I was strongly positively correlated with EDPVR, and negatively correlated with dP/dtmax (P < 0.05). The state 3 respiration rate of complex I was positively correlated with CO and positively correlated with EF (P < 0.05). The respiration rate of complex II was positively correlated with Pes (P < 0.05). In EP group, the respiration rate of complex II and complex IV was negatively correlated with HR value. In 1D group, the state 3 respiration rate of complex I and the respiratory rate of complex IV were strongly positive correlation with CO. In the 3D group, the respiration rate of complex IV was positively correlated with that of CO (P < 0.05). In 18D group, the state 4 respiration rate of complex I was positively correlated with Ped (P < 0.05). The state 3 respiration rate of complex I was positively correlated with CO (P < 0.05). The results are shown in [Table 5].
Table 5: Analysis of the correlations between cardiac function parameters and myocardial mitochondrial complex respiration rate (r)

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  Discussion Top


During exhausting exercise, myocardial oxygen consumption increased sharply, and with the rapid enhancement of heart pumping activity and the redistribution of heart blood flow, myocardial ischemia and hypoxic injury occurred. In addition, the cardiomyocyte was pulled by the overloaded pressure and capacity of the heart chamber, which resulted in serum levels of cTnI and NT-proBNP increased after EE.[12],[13],[14],[15] The protective effect of EP was gradually weakened after the EP training was stopped. At last, the effect of EP on myocardial injury of exhausted rats disappeared and the myocardial injury was serious.

EE induced abnormal electrocardiographic activity in rats. EP was shown to improve electrical disturbances in the heart caused by EE within 9 days after stopping EP training. This might be related to EP could enhance the tolerance level of myocardium to ischemia and hypoxia and reduce the influence of myocardial injury caused by EE on electrical activity.[3] The protective effect of EP on cardiac activity in exhausted rats disappeared after 18 days of EP training stopped.

During exercise, myocardial oxygen consumption increased, and stronger contractility of the myocardium was required to deliver more oxygen to the muscles. Increasing the ability of CO was still one of the key factors in determining exercise capacity.[16] Through EP training, the heart underwent adaptive changes, and the systolic and pumping functions of the heart were enhanced, which were conducive to the empties and filling of the left ventricle, the compliance of the myocardium was enhanced, and the systolic and diastolic functions of the myocardium were improved.

EP can improve the damage of EE to the rats' cardiac function, enhance the elasticity of the ventricular wall, and increase myocardial systolic and diastolic ability. The cardioprotective effect induced by EP weakened with time prolongation after stopping the training, and the effect was strongest within 3 days after stopping training and disappeared on the 18th day. Studies had found that 60% of the physiological myocardial hypertrophy could be reversed within 7 days after the athletes stopped training.[17] The thickness of the posterior wall of the left ventricle and the interventricular septum both decreased proportionally, and the end-diastolic diameter of left ventricle was significantly shrunk in 7 days after exercise stopped. Pelliccia evaluated 40 superior athletes in the recovery period, he found that the maximum oxygen consumption dropped by 50% in the first 3 weeks after stopped training, and dropped to 16% at the 12th week. The maximum cardiac output dropped by 80% within 3 weeks after stopped training.[18]

The mitochondrial respiratory chain played an important role in oxidative phosphorylation and energy conversion etc.[19] There were two main ways in which the energy generation of mitochondrial respiratory chain, one was the NADH respiratory chain pathway which was composed of complexes I, III, and IV, the other was the FADH2 respiratory chain pathway which was composed of complexes II, III, and IV. The decreased activity or structure damage of the complex enzymes would affect the function of the respiratory chain and ultimately caused a decrease of ATP and an increase of free radicals, thus caused mitochondrial respiratory dysfunction. Different load exercises had different effects on the activity of the complex enzymes in the mitochondrial respiratory chain. The FADH2 respiratory chain was more sensitive to high-intensity training. Moreover, the NADH respiratory chain was mainly affected by moderate-intensity training. Studies have found that the activities of various myocardial complex enzymes were increased during moderate-intensity training, while low-intensity training modes had no significant effect on the activities of these complex enzymes.[20]

Mitochondrial respiration could be divided into state 3 and state 4 respirations. When ADP was present, the substrate was sufficient, the proton gradient was consumed, the electron transfer was smooth, and the oxygen consumption was large, this respiration state was called state 3 respiration; when ADP was exhausted, the proton gradient could not be consumed, the process of electron transfer was blocked and the oxygen consumption decreased, this respiration state was called state 4 respiration.[21] The ratio of the respiration rate of State 3 (ADP added) to state 4 (ADP depleted) was called RCR, it was also called respiratory regulation ratio. The decrease of RCR would result in the dysfunction of mitochondrial ATP synthetic and the impairment of the respiratory function. Conversely, the increase of RCR indicated that the mitochondria had complete structure, normal function, active cellular respiration, and accelerated metabolism.[22] However, EP activated mitochondrial respiration and accelerated metabolism. EP could enhance the integrity of the myocardial mitochondria in exhausted rats within 9 days after training and protect the normal function of the mitochondrial respiration. The decrease of state 3 respiration rates of mitochondrial respiratory complexes I, II, and IV showed that EE damaged the mitochondrial respiratory chain. The activity of respiratory chain complex enzyme was significantly reduced, and the production of ATP was decreased. EP could improve the activities of myocardial mitochondrial respiratory complex I and IV enzymes in exhausted rats by affecting NADH electron transport chain and increased the composition of ATP. EP had a high protective effect on rats' myocardial mitochondrial function within 9 days after stopping training, but the protective effect disappeared on the 18th day and returned to pretraining level.

The correlation results showed that the respiratory rate index of mitochondrial complex was correlated with cardiac function parameters. The mitochondrial NADH and FADH2 electron transport chains were both related to cardiac function during EE. Reducing the respiratory rates of mitochondrial respiratory complexes I, II and increasing the state 4 respiratory rate of complex I could lead to myocardial energy metabolism disorders and cardiac function reduced. After EP, EP increased the respiratory rates of mitochondrial respiratory complex I and IV by affecting the mitochondrial NADH electron transport chain, improved the myocardial mitochondrial respiratory function, improved the myocardial energy metabolism and enhanced cardiac function. The effect of the mitochondrial respiratory electron transport chain on the cardiac function was more obvious within 3 days after the training. But after 18 days, the state 4 and state 3 respiratory rates of complex I were related to cardiac function. These indicated that the cardiac function increased with the improvement of mitochondrial respiratory function, if the function of myocardial mitochondrial respiratory chain improved, the myocardial energy metabolism would increase, and the cardiac function would be strengthened.

In this study, the effects of EP on the mitochondrial respiratory chain of exhausted rats were studied, and the timeliness of the protective effect on the heart of exhausted rats was analyzed by combining cardiac function with energy metabolism. This study reminds us that we should pay attention to the adaptive deterioration of the cardiac structure and function after stopping exercise. People must make reasonable arrangements for their recovery training after stopping exercise. We only conducted research on rats in this experiment, but EP should be gradually connected with clinical practice. It should be compiled into a clinical exercise guide based on clinical experimental evidence in hope to bringing good news to more people.


  Conclusion Top


The protective effect of EP on the heart of exhausted rats decreased with the prolongation of stopping training time, and the effect was significant within 3 days of discontinuing training, then decreased gradually, and completely disappeared in the 18th day. EP enhanced the cardiac function in exhausted rats through raising the NADH electron transport chain and increased the respiration rates of mitochondrial respiratory complex I and IV state 3, thereby improved myocardial mitochondrial respiratory function and energy metabolism.

Data availability statement

All datasets analyzed to support the findings of the current study are available from the corresponding author upon reasonable request.

Financial support and sponsorship

This study was supported by grants from the Medical Science Research Program of the Chinese Army (CWS12J064), the Logistical Science Research Project of the Chinese Army (CBJ13J002), the Natural Science Foundation of Hebei Province (H2019104017), and the Heart Injury Prevention Team Foundation Caused By Exercise For Military Personnel.

Authors' Contributions

Ye Su and Yang Wang contributed equally to this work. They are co-first authors.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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