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Table of Contents
ORIGINAL ARTICLE
Year : 2022  |  Volume : 65  |  Issue : 6  |  Page : 290-300

Optimization of exercise preconditioning duration in protecting from exhausted exercise-induced cardiac injury in rats


Department of Cardiology and Nephrology, The 82nd Group Army Hospital of PLA (252 Hospital of PLA), Baoding, Hebei, China

Date of Submission16-Apr-2022
Date of Decision18-Aug-2022
Date of Acceptance19-Sep-2022
Date of Web Publication26-Dec-2022

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


DOI: 10.4103/0304-4920.365457

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  Abstract 


The effect of different duration of exercise preconditioning (EP) on protecting from exhaustive exercise-induced cardiac injury (EECI) has been optimized in rats. Male Sprague-Dawley rats were divided into six groups: the control group, exhaustive exercise (EE) group, EP 20-min + EE group, EP 40-min + EE group, EP 60-min + EE group and EP 80-min + EE group. The EP groups were subjected to treadmill running at the intensity of 74.0% V̇O2 max. Changes of exercise capacity, cardiac pathology, myocardial enzymology, electrocardiogram (ECG), cardiac function, and mitochondrial respiratory function were compared. Compared to the C group, the EE group has shown significant decrease of exercise capacity, elevation of serum N-terminal pro B-type natriuretic peptide (NT-proBNP) and cardiac troponin-I (cTn-I) levels, cardiac morphology change, ECG disturbance, cardiac dysfunction and reduction of myocardial mitochondrial respiration function. Compared to the EE group, the EP groups have shown significant elevation of exercise capacity, decrease of serum NT-proBNP and cTn-I, improvement of cardiac function and myocardial mitochondrial electron transfer pathway complex I, II and IV activity. The correlation analyses showed protection of EP was proportional to EP duration from 20-min to 60-min. EE caused cardiac injury. EP could protect from EECI by alleviating myocardial damage, improving cardiac function and mitochondrial ETP complex I, II and IV activity. EP protection was positively correlated to EP duration from 20-min to 60-min with EP intensity fixed at 74.0% V̇O2 max.

Keywords: Exercise preconditioning, exhaustive exercise-induced cardiac injury, mitochondrial respiratory function


How to cite this article:
Ping Z, Li J, Sun Y, Zhang X, Wang Z, Cao X. Optimization of exercise preconditioning duration in protecting from exhausted exercise-induced cardiac injury in rats. Chin J Physiol 2022;65:290-300

How to cite this URL:
Ping Z, Li J, Sun Y, Zhang X, Wang Z, Cao X. Optimization of exercise preconditioning duration in protecting from exhausted exercise-induced cardiac injury in rats. Chin J Physiol [serial online] 2022 [cited 2023 Jan 28];65:290-300. Available from: https://www.cjphysiology.org/text.asp?2022/65/6/290/365457




  Introduction Top


Exhaustive exercise-induced cardiac injury (EECI) refers to the adverse effects on the heart caused by high-intensity or unsuitable exercise, and its clinical manifestations include changes in cardiac morphology, abnormal myocardial injury markers, exercise-induced arrhythmia, syncope, and even sudden cardiac death (SCD).[1] Recent reports suggest that prodigious amounts of exercise may increase markers for, and even the incidence of cardiovascular disease. Klinkenberg et al.[2] discovered that prolonged endurance-type exercise is associated with elevated cardiac troponin (cTn) levels in athletes. Guasch et al.[3] reported strenuous physical activity was a trigger for ventricular arrhythmias and sudden death. An Italian study identified a 2.5-times relative risk for SCD in adolescents engaged in competitive sports versus an age-matched nonathletic population.[4] A French investigation found the relative risk of sports-related SCD was 4.5 times greater in competitive young athletes compared with noncompetitive sports participants.[5] The incidence of EECI is widespread and obscure with great social harm and developing a pragmatic countermeasure to reduce EECI is vital.

Recent studies have well documented that exercise can reproduce the "ischemic preconditioning," which refers to the capacity of short periods of ischemia to render the myocardium more resistant to subsequent ischemic insult and to limit infarct size during prolonged ischemia, thus protect myocardium from EECI and ischemia/re-perfusion (I/R) injury.[6],[7] Exercise preconditioning (EP) could enhance the expression of cardiac protective substances, make the cardiac structure and function undergo adaptive change, reduce myocardial fiber rupture, and increase myocardial tolerance to longtime ischemia and hypoxia.[8] The cardioprotective mechanisms of EP on EECI are multiple. EP can improve myocardial energy metabolism by up-regulating mitochondrial biogenesis pathway PGC-1α-NRF1/NRF2 and the activities of mitochondrial electron transport pathway complex I, II and IV in EECI rats. EP can alleviate myocardial oxidative stress by down-regulating TXNIP/TRX/NF-κBp65/NLRP3 inflammatory signaling pathway and reduce the content of downstream inflammatory factors in exhausted rats.[9],[10]

As a kind of physical stimulus, EP has important practical significance for formulating healthy training scheme and preventing EECI. However, researches on the effects of different EP duration on EECI are rare and inconsistent. Hamilton et al.[11] reported both short-term and long-term exercise training enhanced myocardial recovery after an I/R insult. Sun and Pan[12] found that calcitonin-related gene peptide did not synthesized in short-term EP, but synthetically released in long-term EP. The inhibiting effects of long-term EP on myocardial cell apoptosis were stronger than those of short-term EP.[13] Therefore, exploring an optimal duration of EP protocol is of great significance for basic research model design and clinical implication.

In the previous research, we have discussed EP protection mechanisms on EECI and optimized the EP intensity which turned to be 74.0% V̇O2 max.[14] Based on this, we continued to optimize EP duration in protecting from EECI so as to establish an optimal EP protocol which can be used as an "EP prescription" in basic research. In the study, we have discussed the protection rule of different duration of EP from EECI by observing changes in exercise capacity, cardiac and left ventricular weight (LVW), myocardial morphology, myocardial injury markers, electrocardiogram (ECG), cardiac function, and mitochondrial function with EP intensity fixed at 74.0% V̇O2 max.


  Materials and Methods Top


Materials

Enzyme-linked immunoassay (ELISA) kits for heart injury markers were obtained from R and D Systems (USA). Reagents for measuring mitochondrial respiratory function were purchased from Sigma-Aldrich (St. Louis, MO, USA).

The following main instruments were used in the present study: An animal treadmill (Taimeng, China), an Optical Microscope (Olympus, Japan), a transmission electron microscopy (HT7700, Hitachi, Japan), a MultiscanGO enzyme standard instrument (Thermo, USA), a pressure volume catheter (SPR838, Millar Company, USA), a PowerLab data acquisition and analysis system (AD Instruments, Australia), a bio-electric amplifier (AD Instruments, Australia), a needle electrode (AD Instruments, Australia) and a high-resolution respirometry (Oroboros Instruments, Austria).

We obtained 90 male Sprague-Dawley rats (300 g ± 20 g) from Beijing Zhongke Dasheng Biological Technology Co., Ltd. (Beijing, China), License Number: SCXK (Beijing)-2016-0002. The animals were housed at 25°C–26°C under a 12 h dark and 12 h light cycle. Food and water were provided ad libitum during experimental period. All experiments were conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals of the 82nd Group Army Hospital of PLA, and all procedures were approved by the Ethics Committee for the Use of Experimental Animals at the 82nd Group Army Hospital of PLA (Date 2018-12-01; Decision number 2018012).

Animal model and treatment

A total of 72 rats were randomly divided into six groups (n = 6): the sedentary control group (C), exhaustive exercise group (EE), EP 20-min + EE group (EP20), EP 40-min + EE group (EP40), EP 60-min + EE group (EP60), EP 80-min + EE group (EP80). All rats have adopted an adaptive treadmill running for a week, and the rats that could not complete the training were excluded. In this study, we have adopted the optimal EP intensity training protocol demonstrated in our previous work, which was treadmill running at the speed of 26 m/min with a slope of 5° (74.0% V̇O2 max).[14] The EP20, EP40, EP60 and EP80 groups have undergone 20-min, 40-min, 60-min, and 80-min of EP training, respectively for 5 days a week, and the scheme lasted for 3 weeks. After the EP models were established, except the C group, the other groups underwent treadmill running at the speed of 30–40 m/min for 4 h to 5 h until they were exhausted. The exhaustive judgment principle was as follows: The running of the rats was 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, and there was significant change in the motion state under the various stimulus.[8],[15] At the point of exhaustion, all rats were anesthetized by the intraperitoneal injection of pentobarbital (50 mg/kg). The serum was collected and preserved at −80°C and the hearts were harvested. The body weight (BW), heart weight (HW), and LVW of the rats were measured. The HW/BW ratio and LVW/BW ratio were calculated. The left ventricular tissue was isolated and part was immediately used for mitochondrial function measurements and histomorphometric analysis. Since the pressure-volume guidewire experiment was invasive, it is easy to cause deviation to other relative experiments such as myocardial specimens and serum; therefore, 6 rats of each group were used for cardiac function and ECG testing, and the remaining 6 were used for mitochondrial respiratory function and specimen collection.

Exercise capacity

The distance and duration of EE and EE + EP groups during the exhaustion process were documented and compared.

Electrocardiogram tracing

Adaptive electrocardiography training was performed in all experimental rats. ECGs were recorded from the rats of C group in a quiet state for 5-min. In EE and EP + EE groups, 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.[14]

Cardiac function

Millar pressure volume catheters were applied to record cardiac function. The rats were weighed and anesthetized with pentobarbital sodium (40 mg/kg, intraperitoneal injection), and fixed on the operating table in supine position. After endotracheal intubation, we separated the right carotid artery, and calibrated pressure with MPVS control software. The Millar catheter was inserted into the left ventricle from the right carotid artery. The left ventricular pressure volume waveforms of the anesthetized rats were recorded with Chart 7.0 software in real-time. The basic waveform was recorded for 10-min. A ventral midline incision was performed on the abdomen, and the inferior vena cava was occluded and the waveform was recorded. 30% NaCl solution (30 μL) was injected in the left jugular vein and the pressure-volume waveform was recorded. Then the catheter tips were submerged in the holes of a calibration cuvette which was filled with fresh heparinized warm blood respectively and recorded the conductance changes in the volume channel, finally the volume can be calculated. The parameters: Stroke work, cardiac output (CO), stroke volume (SV), heart rate (HR), end-systolic volume (ESV), end-diastolic volume (EDV), end-systolic pressure (ESP), end diastolic pressure (EDP), ejection fraction (EF), peak rate of pressure rise (dp/dtmax), peak rate of pressure decline (dp/dtmin), ESP volume relationship (ESPVR), end-diastolic pressure volume relationship (EDPVR), relaxation time constant (Tau) were detected.

Histomorphometric analysis

Rat hearts were harvested and rinsed with cold normal saline. Myocardial tissue of the left ventricle was collected for light microscopy. The tissue was fixed with 10% formaldehyde, paraffin-embedded, sectioned, dehydrated with different concentration gradients of alcohol, stained with HE, and observed by an optical microscope.

A small piece (2 mm × 1 mm × 1 mm) of subendocardial myocardium from the root of the left ventricular papillary muscle was harvested and fixed with fresh prepared 4% glutaraldehyde for 5 h, washed using phosphate buffer, then postosmicated in 1% osmium tetroxide for 2 h, dehydrated with gradient acetone, embedded, sectioned, electron stained with uranyl acetate and lead citrate, and then observed by transmission electron microscopy.

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

The serum levels of N-terminal pro B-type natriuretic peptide (NT-proBNP) and cTn-I were tested by ELISA. The serum was removed from the −80°C freezer. ELISA assays were performed according to the instructions of the kits. The 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.[14]

In situ studies of mitochondrial respiratory function

We used permeabilized myocardial fibers, which provides an excellent way to study the mitochondria in situ without isolating them from tissue. Myocardial fibers were isolated by dissecting muscle tissue (left ventricle) in BIOPS solution on ice followed by saponin permeabilization. Cell membrane permeabilization with saponin enables the study of organelle function while maintaining cellular architecture and controlling the intracellular milieu. Mitochondrial function was measured by high-resolution respirometry at 37°C using dual-chamber titration injection respirometers. The respiration medium (MiR05) included 110 mM sucrose, 60 mM K-lactobionate, 0.5 mM EGTA, 1 g/L bovine serum albumin (essentially fatty acid-free), 3 mM MgCl2, 20 mM taurine, 10 mM KH2PO4, and 20 mM HEPES (pH 7.1). DatLab software was used for data acquisition and analysis. A series of respiratory titration protocols were designed to test for multiple mitochondrial defects, including cytochrome c depletion. We used 1 mM adenosine diphosphate (ADP) to stimulate respiration (state 3) and measured it sequentially through complex I (10 mM glutamate and 2 mM malate), complex II (10 mM succinate and 0.5 M rotenone), and complex IV (0.5 mM TMPD, 5 mM ascorbate, and 2.5 M antimycin A). Respiration was measured before and after stimulation by adding cytochrome C (10 M) to test the integrity of the mitochondrial outer membrane. Respiratory rates were expressed per mg wet weight.[1]

Statistical analyses

SPSS statistics 22.0 (SPSS Inc., Chicago, IL, USA) was used for statistical analysis. The results were expressed as means ± standard deviation. One-way analysis of variance was used to compare the data among multiple groups. Dunnett t-test or SNK q-test was used for multiple comparisons between the groups. Levene method was used to test the homogeneity of variance. Difference was considered significant statistically when P < 0.05.

A correlation analysis was performed by calculating Spearman's correlation coefficients between different duration of EP and EECI markers, and P < 0.05 was considered to indicate a significant difference.


  Results Top


Different duration of exercise preconditioning increased cardiac weight and exercise capacity in exhausted rats

As shown in [Table 1], compared with the C group and the EE group, the HW and HW/BW ratio of different duration of EP groups were increased significantly respectively (P < 0.05); compared with the C group and the EE group, the LVW and LVW/BW ratio of the EP40, EP60 and EP80 groups were increased significantly respectively (P < 0.05). As shown in [Figure 1], compared with EE group, the duration of exhausted treadmill running of all the EP groups were increased (P < 0.05) among which the EP80 was the most obvious and the distance of that of all the EP groups were increased (P < 0.05) among which the EP60 group was the most significant.
Figure 1: Exercise capacity of different duration of exercise preconditioning groups. C: Sedentary control group. EE: Exhaustive exercise. EP20, EP40, EP60, and EP80: Different duration of exercise preconditioning which have been performed for 20 min, 40 min, 60 min, and 80 min, respectively. SD: Standard deviation. Data are expressed as means ± SD, n = 6; #P < 0.05 versus the EE group, and ΔP < 0.05 versus the EP20 group, P < 0.05 versus the EP40 group, P < 0.05 versus the EP60 group.

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Table 1: Effects of different duration of exercise preconditioning on cardiac mass indices in exhausted rats

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Different duration of exercise preconditioning attenuated myocardial pathological injury in exhausted rats

Light microscopy

[Figure 2]a showed an optical microscopy analysis of the rat myocardial structure. In the C group, myocardial fibers were arranged neatly as an interstitial substance without edema, the muscle membranes showed no damage, and the muscle fibers had no fractures, degeneration, or necrosis. In the EE group, myocardial fibers were arranged irregularly as interstitial substance with edema, there was muscle membrane damage, and the muscle fibers showed evidence of fracture, degeneration, and necrosis. In the EP20 group, myocardial fibers were arranged disordered, myocardial interstitium was edema, cells swelled and necrotic, and inflammatory cell infiltration was seen. The myocardium structure of EP40 was better than the EP20 group. In the EP60 group, the myocardial fibers were arranged more neatly, and there were different degrees of thickening, and the staining was more uniform. The EP80 group did not improve significantly compared with EP60 group.
Figure 2: Effects of different duration of exercise preconditioning on myocardial morphology. (a) Hematoxylin and eosin (H and E) staining (×400). EP20, EP40, EP60, and EP80: Different duration of exercise preconditioning that have been performed for 20 min, 40 min, 60 min, and 80 min, respectively. (b) Transmission electron microscopy (×20,000). C: Sedentary control group. EE: Exhaustive exercise. EP20, EP40, EP60, and EP80: Different duration of exercise preconditioning that have been performed for 20-min, 40-min, 60 min and 80 min, respectively. Note the mitochondrial vacuolation (red arrows), myofilament dissolution and fracture (yellow arrows).

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Electron microscopy

[Figure 2]b showed the cardiomyocyte ultrastructure. In the C group, sarcomeres were arranged neatly, the density was uniform, the organelles had no edema, and the membrane and crest of the mitochondria were normal. In the EE group, the overall cardiomyocyte structure was abnormal, myofilament lysis and rupture, muscular nodules obscured, mitochondria became oval, reduced, cristae decreased, partially dissolved, and part of mitochondria was vacuolated. In the EP20 group, the overall structure of myocardial cells was normal, the nucleus was normal, the myofilament was broken, the number of mitochondria was reduced, the cristae was reduced, and partly dissolved. In the EP40 group, some myofilaments were lysed and broken, and the mitochondrial cristae was reduced and partially dissolved. In the EP60 group, the myofilament was slightly lysed and broken, the light and dark bands were clearly visible, the musculature was clearly discernible, and the mitochondria were oval, the cristae were arranged neatly. No cristae lysis vacuoles were seen. In the EP80 group, the overall structure of myocardial cells was normal, the myofilament was arranged neatly, the light and dark bands were clearly visible; the musculature was clearly discernible, the mitochondria were oval and more, the cristae were arranged neatly, and some mitochondria were vacuolated.

Different duration of exercise preconditioning decreased the serum level of cardiac enzymes in exhausted rats

As shown in [Figure 3], compared with the C group, the content of cTn-I in the EE group and the EP groups were increased (P < 0.05). Compared with the EE group, the content of cTn-I of the EP40, EP60 and EP80 groups were decreased significantly (P < 0.05). Compared with the EP20 group, the EP40, EP60 and the EP80 groups were reduced (P < 0.05). Compared with the EP40 group, the EP60 group was reduced (P < 0.05).
Figure 3: Comparison of the contents of cTn-I and NT-proBNP in rat serum. C: Sedentary control group. cTn-I: cardiac troponin I. EE: Exhaustive exercise. EP20, EP40, EP60, and EP80: Different duration of exercise preconditioning which have been performed for 20 min, 40 min, 60 min, and 80 min, respectively. NTproBNP: N-terminal pro B-type natriuretic peptide. SD: Standard deviation. Data are expressed as means ± SD, n = 6; *P < 0.05 versus the C group, #P < 0.05 versus the EE group, ΔP < 0.05 versus the EP20 group, P < 0.05 versus the EP40 group.

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Compared with the C group, the serum levels of NT-proBNP in the EE and EP groups were increased significantly (P < 0.05). Compared with the EE group, the level of NT-proBNP in each EP group was reduced (P < 0.05). Compared with the EE group, the EP40, EP60 and EP80 groups were decreased (P < 0.05). Compared with the EP20 group, the EP40, EP60 and EP80 groups were significantly decreased (P < 0.05). Compared with the EP40 group, the EP60 and EP80 groups were decreased significantly (P < 0.05).

Different duration of exercise preconditioning improved electrocardiogram indicators in exhausted rats

As shown in [Table 2] and [Supplementary Figure 1] in the online-only Data Supplement, compared with the C group, the HR of the EE group was significantly elevated (P < 0.05). Compared with the EE group, EP60 and EP80 groups were decreased significantly (P < 0.05).
Table 2: Effects of different duration of exercise preconditioning on electrocardiogram indexes in exhausted rats

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Compared with the C group, the ST segment of rats in EE group was significantly elevated (P < 0.05). Compared with the EE group, the ST segment in EP40, EP60 and EP80 groups were significantly decreased (P < 0.05).

Compared with the C group, the QRS interval in the EE and EP20 group was wider (P < 0.05). Compared with the EE group, the QT interval of the EP40, EP60 and EP80 groups were significantly shortened (P < 0.05).

Different duration of exercise preconditioning improved cardiac function of exhausted rats

As shown in [Table 3] and [Figure 4], the cardiac function was measured by hemodynamic parameters. The EE group showed the decreased width in P-V loops, and the original diagram reflected reduced SV along with increased ESV and EDV. EF, CO, dp/dtmax, and ESPVR were all decreased significantly, these suggested deteriorated systolic performance in rats after EE. Significant decrease in -dp/dtmin and increase in EDPVR and Tau indicated impaired diastolic function. However, these damaging changes could be mitigated by EP as we observed in the EP groups. Compared with the EE group, the EF, CO, SV, ESP, +dp/dtmax, ESPVR, and -dp/dtmin of the EP60 group were all increased significantly (P < 0.05), the EDV, EDP, EDPVR and Tau of the EP60 group were decreased significantly, indicating EP provided cardio-protective function to EE. Compared to the EP20 group, EF, CO, SV, ESP, and ESV of the EP60 group were increased significantly (P < 0.05); EDV, EDPVR, and Tau were decreased significantly (P < 0.05). Compared to the EP40 group, the EF, SV, ESP of the EP60 group were increased significantly (P < 0.05), the ESV and Tau were decreased significantly (P < 0.05). These suggested that the protection of cardiac function of EP60 is better than other EP groups.
Figure 4: Original recording showing the pressure-volume loops. C: Sedentary control group. EE: Exhaustive exercise. EP20, EP40, EP60 and EP80: Different duration of exercise preconditioning performed for 20 min, 40 min, 60 min, and 80 min, respectively. The EE group showed reduced SV along with increased ESV and increased EDV, thus the position of P-V loops shifted to right. The EP groups showed decreased ESV and decreased EDV, thus the position of P-V loops shifted to left, and among them the EP60 group is the most obvious. EDV: End-diastolic volume, ESV: End-systolic volume.

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Table 3: Effects of different duration of exercise preconditioning on hemodynamic parameters and indices of systolic and diastolic function in exhausted rats

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Different duration of exercise preconditioning improved myocardial mitochondrial respiration function of exhausted rats

As shown in [Figure 5], with glutamate and malate as electron donors for complex I, compared to the C group, the state 3 respiration rates of the EE group and the EP groups were reduced significantly (P < 0.05). Compared with the EE group, the state 3 respiration rates of the EP40, EP60 and EP80 groups were increased (P < 0.05). Compared with the EP20 group, the EP60 and EP80 groups were increased (P < 0.05). Compared with the EP40 group, the EP60 and EP80 groups were increased (P < 0.05).
Figure 5: Maximal respiratory capacity in permeabilized myocardial fibers. (a) Original recording. (b) Comparison. C: Sedentary control group. EE: Exhaustive exercise. EP20, EP40, EP60 and EP80: Different duration of exercise preconditioning that have been performed for 20-min, 40-min, 60-min, and 80-min, respectively. MAL: Malate. Cyt C: Cytochrome C. ADP: Adenosine diphosphate. GLU: Glutamate. SUC: Succinate. ROT: Rotenone. AMA: Antimycin A. ASC: Ascorbate. SD: Standard deviation. Data are expressed as means ± SD, n = 6; *P < 0.05 versus the C group, #P < 0.05 versus the EE group, and ΔP < 0.05 versus the EP20 group, P < 0.05 versus the EP40 group, P < 0.05 versus the EP60 group.

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Using succinate as a substrate for complex II, compared to the C group, the state 3 respiration rates of the EE group and the EP groups were reduced significantly (P < 0.05). Compared with the EE group, the state 3 respiratory rates of the EP40, EP60 and EP80 groups were increased (P < 0.05). Compared with the EP20 group, the EP60 and EP80 groups were increased significantly (P < 0.05).

With ascorbate/TMPD being used as substrates for complex IV, compared to the C group, the state 3 respiratory rates of the EE group and the EP groups were reduced significantly (P < 0.05). Compared with the EE group, the state 3 respiration rates of the EP40, EP60 and EP80 groups were increased (P < 0.05). Compared with the EP20 group, the EP60 and EP80 groups were increased (P < 0.05). Compared with the EP40 group, the EP60 and EP80 groups were increased (P < 0.05).

Cardiac protection of exercise preconditioning is correlated to duration in a certain range

According to Spearman's correlation analysis, with regards to exercise capacity, the exhaustive treadmill running duration and distance were both positively correlated with the EP duration (P < 0.05). The serum levels of cTn-I and NT-proBNP were strongly and negatively correlated with the EP intensity (P < 0.05). As for the ECG parameters, the HR, QRS duration, ST height and QT interval were negatively correlated with the EP duration significantly (P < 0.05). Regarding cardiac function parameters, the CO, SV, ESP, EF, dp/dtmin and ESPVR were and positively correlated with the EP duration significantly (P < 0.05), and the ESV, EDV, EDP, Tau and EDPVR were negatively correlated with the EP duration significantly (P < 0.05). Concerning the activities of mitochondrial ET pathway complexes, the state 3 respiration rates of complex I, II and IV were positively correlated with the EP duration significantly (P < 0.05) [Table 4].
Table 4: Analysis of correlation between exercise preconditioning duration and effect parameters

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  Discussion Top


In present study, we have investigated effect of different EP duration on EECI. The main findings are EP can protect from EECI by alleviating myocardial damage, improving cardiac function and myocardial mitochondrial electron transfer pathway complex I, II and IV activity. We also gained new insights that EP protection was positively correlated to EP duration from 20-min to 60-min with EP intensity fixed at 74.0% V̇O2 max among all the setting EP groups.

Exercise capacity was characterized by the running time and distance to exhaustion in present research. Both of the two parameters of the EP groups were prolonged compared to the EE group, indicating EP has a great effect on improving exercise performance. Huang et al.[16] also reported 12-week of motorized treadmill running of rats increased running time to exhaustion (80 ± 5 and 151 ± 13-min, for group exhaustively exercised and trained plus exhaustively exercised, respectively, P = 0.0001). Correlation analyses showed the running distance to exhaustion was proportionally to EP duration in the time range 20 min to 60 min, indicating the optimal EP duration is 60 min. Notably, the EP80 running duration is higher than the EP60, but the running distance is lower than the EP60, and that's because the speed during exhaustion was in the range of 30–40 m/min and the average speed of the EP60 group (≈35 m/min) was higher than that of EP80 (≈31 m/min). The running distance of EP60 is higher than that of EP80 also indirectly indicates the better exercise capacity of EP60.

The cardiac mass data showed that after 3-week EP running, the rats' heart mass and left ventricular mass were increased significantly. Considering other indexes such as improved cardiac systolic function and decreased serum levels of myocardial injury enzymes compared with EE group, we deem the hypertrophy of myocardium after 3-week of EP is adaptive and physiological. Bei et al.[17] also observed that 3-week of swimming exercise for 90-min twice a day was effective to induce physiological cardiac growth, as evidenced by increased HW, HW/BW ratio, and HW/tibia length ratio. Exercise-induced adaptive cardiac growth is a physiological but no pathological process that is largely related to the increase in cardiomyocyte size, accompanied by increased protein synthesis and protective metabolic changes.[18],[19],[20]

cTn-I was often used to diagnose myocardial infarction in clinic as a sensitive biomarker of myocardial injury. In some cases, cTn-I acted as a monitor to evaluate the prolonged exercise induced minor myocardial injury.[21] BNP is a biomarker for cardiac dysfunction. It is secreted by the heart in response to excessive stretching of the heart muscle cells and is released as preproBNP, which comprises the biologically active BNP and the inactive metabolite N-terminal fragment (NT-proBNP).[22] In this research, the serum cTn-I and NT-proBNP levels of rats in the EE group have increased significantly, indicating EE caused myocardial damage and cardiac wall stress or dysfunction. The significant elevation of serum cTn-I and NT-proBNP levels after exhaustive treadmill running at the speed of 30 m/min in rats was also reported by Wang et al.[23] Except the EP20 group, the cTn-I and NT-proBNP of the other EP groups have decreased significantly compared with the EE group, suggesting EP could resist myocardial injury by reducing the myocardial injury induced by ischemia and hypoxia, which was in line with our previous results.[1],[9],[14] Sun and Mao[24] also reported EP significantly lowered serum cTn-I level in EE-induced myocardial ischemia injury rats. The correlation analyses showed the decrease of cTn-I and NT-proBNP were time-dependent in the range of 20-min to 60-min. When the EP duration was 60-min, the effect was most obvious.

The optical and electron microscopy results showed cardiomyocyte structure was damaged as a consequence of EE, which is consistent with Xu's and Li's reports.[25] The EP groups have shown attenuated pathological injury of the myocytes, and as EP duration prolongs, the myocardial structure of the EP groups have shown improvement in cardiomyocytes, among which the 60-min of EP was more close to the sedentary group.

The results of ECG parameters showed that after exhaustion, rats' HR was increased, QRS interval was prolonged, and ST segment was elevated, indicating overload exercise induced myocardial ischemia and hypoxia, decreased ventricular conduction capacity, abnormal ventricular wall motion and sequence of myocardial repolarization and slowed ventricular depolarization. Benito et al.[26] also observed sustained ventricular tachyarrhythmias in 42% rats compared with only 6% of sedentary rats and increased QRS duration after 16-week of intense treadmill running, indicating arrhythmia susceptibility following long-term endurance training. Compared with the EE group, the QRS interval and ST-segment elevation of the EP40, EP60 and EP80 were decreased significantly, indicating 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.[27] The correlation analyses showed effect of EP on cardiac electrophysiological activity was correlated with the increase of EP duration from 20-min to 60-min (HR: r = -0.422, P < 0.05; QRS Duration: r = -0.648, P < 0.05), indicating different EP training duration could improve the ECG disturbances to different extent.

The present study described left ventricular (LV) pressure and volume relations and provided characterization of cardiac function. After EE, impaired inotropic state of the heart resulted in a suboptimal transfer of blood from the left ventricular to the periphery with more excessively decline in SV, CO, and EF, which further influenced ESV and EDV, and led to compensatory increase in HR. The significant drop of dp/dtmax indicated EE has induced heart failure.[9] Oláh et al.[28] also demonstrated that overload exercise could damage the contractile function of the rat's myocardium. The cardiac function of different duration of EP groups were improved to different extent compared with the exhausted group, which is consistent with Li's study.[10] The EF of the EP20 and the EP40 group were significantly lower than 50%, which was in the phase of reduction of EF in acute heart failure. The EF of the EP60 and EP80 groups were higher than 50%, which was in the EF retention phase in acute heart failure. The correlation analyses showed protection of EP was increased as the EP duration prolonged from 20-min to 60-min.

The left ventricular diastolic objective indicators (EDV, EDP, -dp/dtmin, Tau) of the EE group have changed significantly, suggesting the left ventricular diastolic function were damaged by the EE. 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 diastolic functions of the myocardium were improved. Li et al.[9] also reported EP alleviated the EE-induced myocardial dysfunction. According to the correlation analyses, EP protection on the left ventricular diastolic function was increased as the prolong of the EP duration in the range of 20-min to 60-min, which amounted to the the peak at 60-min, while as the duration continued to prolong to 80-min, the effect began to decline. Hamilton et al.[11] also reported 60 min/day and 30 m/min (70% V̇O2 max) treadmill exercise improved ventricular contraction and diastolic function and myocardial tolerance to I/R.

The position of the P-V ring of the EE group has shift to the right, indicating EE led to ischemia and hypoxia, resulting in myocardial necrosis, decreased cardiac work, and cardiac dysfunction. Compared with the EE group, the PV Loop of the EP groups have shift to the left to different degrees, suggesting different training duration of EP have different effect on heart function.

Muscle oxidative capacities have been assessed by measuring respiration of mitochondria in permeabilized fibres in situ with no limitation of substrates, ADP, or oxygen. Results showed that the state 3 of ET pathway complexes I, II, and IV in the EE group were decreased 69.80%, 42.69% and 52.55% respectively, which suggested that EE induced depression of myocardial mitochondrial respiration function. In order to fulfil the energy needs of myocytes, the ET pathway would compensate with producing large amount of super oxide which in turn injures myocardium and causes the respiration apparatus damage and energy production failure.[29] Compared to the EE group, the EP groups have shown improvements of respiration rates of complex I, II, and IV to different extent, indicating EP promoted changes in myocardial mitochondrial energy metabolism which may contribute to the cardiac protection induced by EE, which was in correspondence with our previous studies.[14] The correlation analyses indicated with the prolong of EP duration in the range of 20-min to 60-min, the mitochondrial function has been improved proportionally (Complex I: r = 0.746, P < 0.05; Complex II: r = 0.663, P < 0.05; Complex IV: r = 0.764, P < 0.05), among which the 60-min group was most obvious. Lee et al.[30] demonstrated 5 days of treadmill running for 60 min/d at 30 m/min could protect the heart against IR-induced mitochondrial functional impairment and oxidative damage in rats.

The correlation analysis results showed that EP protection was proportional to EP duration in the range of 20-min to 60-min, including reducing serum levels of cTn-I (r = −0.839, P < 0.05) and NTproBNP (r = −0.730, P < 0.05), improving ECG activity (QRS Duration: r = 0.648, P < 0.05), improving cardiac function (CO: r = 0.541; SV: r = 0.383; EF: r = 0.648; ESV: r = −0.445; EDV: r = −0.563; EDP: r = −0.451; dp/dtmin: r = 0.414; Tau: r = −0.539; ESPVR: r = 0.663; EDPVR: r = −0.474; P < 0.05) and elevating mitochondrial ET pathway complex I (r = 0.746, P < 0.05), II (r = 0.663, P < 0.05) and IV (r = 0.764, P < 0.05) activity. The protection of EP on serum myocardial injury enzymes, heart function and mitochondrial respiration function was most obvious when EP duration was 60-min. Starnes et al.[31] reported rats treadmill training at 55%–60% V̇O2 max and 40 min/d was below the threshold intensity necessary to induce intrinsic cardiac protection against ischemia reperfusion injury. However, in the study by Lennon et al. showed that both moderate (60-min at 55% V̇O2 max) and high-intensity (60-min at 75% V̇O2 max) treadmill running of rats provided equivalent protection against ischemia re-perfusion injury.[32] The difference between the two studies was the exercise duration. It appears that the exercise duration of a single exercise session should last for 60 min and should be performed at about 75% maximum oxygen consumption to achieve exercise-induced cardio-protection, which was in accordance with our results.

Limitations of the study

This research has some limitations. First, the mechanism of EP cardio-protection on EECI was not involved in this study which was mainly focused on exploration of optimization of EP duration and protection rule on EECI, due to the mechanism of EP protection on EECI has been discussed from several different angles in our previous work, energy metabolism, oxidative stress, inflammation and apoptosis, for instance. Second, the notion of EE being injurious to the heart was still controversial. To elucidate whether EECI was transient or pathological, more comprehensive EECI models and experiments need to be designed. For example, repetitive EE models need to be established and the trend of cardiac markers at different time points after exhausted exercise should be observed. Finally, this conclusion is only based on the animal model, but optimal EP protocol should be gradually connected with clinical practice. It should be compiled into a clinical exercise guide as an prescription based on clinical experimental evidence in hope to bringing benefits to more people.


  Conclusion Top


In summary, exhausted exercise caused definite cardiac injury, including myocardial damage, cardiac morphology change, cardiac dysfunction, ECG disturbance, and reduction of myocardial mitochondrial electron transfer pathway complex I, II, and IV; activity in the rat model. Exercise preconditioning can protect from EECI by alleviating myocardial damage, improving cardiac function and myocardial mitochondrial electron transfer pathway complex I, II, and IV; activity. EP protection was correlated to EP duration from 20 min to 60 min, which was most remarkable when EP duration was 60 min with EP intensity fixed at 74.0% V̇O2 max among all the setting EP groups. It appears exercise preconditioning duration and intensity are both important factors in achieving a cardio-protective phenotype and the result can provide pragmatic reference for EP training scheme design in the basic research and human clinical practice.

Financial support and sponsorship

This study was financially supported by Natural Science Foundation of China (no. 32100939) and Medical science research project of Hebei Province (no. 20210744).

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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

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



 

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