High-intensity interval training protects the heart against acute myocardial infarction through SDF-1a, CXCR4 receptors, and c-kit levels

One of the best and most effective applied and tolerable approaches for cardioprotection is the regular exercise. In the situation of exercise activity and even cardiac ischemic injury, the activity of the myocardial stem cells and their recruiting factors are changed so that contribute to the adaptation and repairment of the myocardium. The aim of this study was to investigate the effect of myocardial preconditioning with HIIT on SDF-1a myocardial levels, CXCR4 receptors, and c-kit after acute myocardial infarction in male rats. Twenty male Wistar were randomly divided into 4 groups: control (C), training (T), myocardial infarction (MI), and training + myocardial infarction (T + MI). The training groups performed 2 weeks of high-intensity interval training in four sections. Protein expression of SDF-1, C-Kit, and CXCR4 receptors was measured by the western blot method in the myocardial tissue, and myocardial injury enzymes were measured in serum. The results showed that SDF-1, CXCR4 receptors, and C-Kit had a significant increase after two weeks of HIIT and myocardial infarction. Also, serum enzyme measurements showed a positive effect of exercise, so that in the myocardium injury, enzymes significantly increased in the MI group compared with the other three groups (p < 0.001), and there was a significant difference between the groups of T + MI in all of the enzymes of the myocardium injury compared to the C and T groups. Even short terms of HIIT can increase the levels of proteins SDF1-a, C-Kit, and CXCR4 receptors in order to cardioprotection against myocardial injury through the recruitment of stem cells.


Background
Ischemic heart disease is caused by a change in coronary and myocardial arteries by myocardial oxygen deprivation in result decrease in blood flow to the myocardium (Tashakori-Miyanroudi et al. 2020).Heart attack or acute myocardial infarction (AMI) is in fact the same ischemia caused by coronary artery atherosclerosis associated with arterial thrombosis, coronary embolism, spasm, and reduction in the diameter of the coronary arteries, leading to permanent and irreversible cell death in the part of the heart muscle (Baba and Wohlschlaeger 2008).Myocardial ischemia is associated with numerous morphological, molecular, and functional changes in the myocardium.The levels of myocardial damage depend on the ischemic duration and severity that can lead to a structural remodeling and functional changes in the ventricular.Myocardial ischemia causes injuries to the myocardium, eventually leading to terminal pump failure, interstitial fibrosis, cardiac dilation, arrhythmias, and cardiac death (Baba and Wohlschlaeger 2008).MI results in decreased cellular ATP, increased ROS, accumulation of H + , elevated cytosolic calcium levels, and calpain activation; these are essential mechanisms of MI-induced injury (Ramez et al. 2019).Furthermore, studies have shown that, following an acute myocardial infarction, inflammation occurs in the affected areas of the heart, and cytokines play a role in the inducement of this inflammation (Hassanshahi et al. 2008).Among them, a group of cytokines with local presence helps to reduce the inflammation process and prevent the injury development, and in this case, chemokines play an important role (Hassanshahi et al. 2008).Part of this process, through the release of heart tissue cardiokines and chemotactic of stem cells, stimulates endogenous pathways in injured locations (DeWood et al. 1980;Wen et al. 2012a).More studies have shown that the stem cell recruitmenter factors (such as G-CSF, SDF, SCF, and Flt3) are chemokines that recruitment stem cells from the bone marrow and move them to the myocardial tissue.Among the recognized chemokines that are released from the myocyte into the bloodstream (Stromal Drive Cell Factor), the SDF of the CXC family has numerous isoforms that play a very important role in post-myocardial regeneration and the most important isoform is α-1 (Colter et al. 2000).SDF-1α has a variety of functions, including proangiogenic, improved endothelial function, cell regeneration, and stimulating cell proliferation is antiapoptotic.In normal conditions, the level of this chemokine and the recruitment of the stem cells in the body are insignificant, but it has been observed that there is an upregulation immediately after myocardial infarction that reduces the myocyte injury and improves cardiac function by the movement of stem cells from the bone marrow (Yu et al. 2006).Possible mechanisms of this incident can be due to myocardial tissue regeneration by the process of myogenesis and vasculogenesis (new myocyte), damaged tissue remodeling and anti-apoptotic signals, and anti-fibrotic myositis, reducing the structural disorder of the myocardial after acute myocardial infarction, the setting up of paracrine and endocrine mechanisms, regeneration of injured myocytes, as a result, reduced infarct area (Goodman and Hodgson 1962;Harada et al. 2005a;Beltrami et al. 2003).On the other hand, SDF indirectly protects the cells from apoptosis through several signal pathways, the most important of which is the AKT / PKB pathway, and directly affects the BAX / BCL2 ratio, which is the cell death index (Orlic et al. 2001).It can be seen from the evidence that increased SDF and other factors involved in the recruitment of stem cells and as a result, the increase in stem cells can be used to cardioprotection and regeneration of the myocardial tissue in many ways (Brocheriou et al. 2000;Kang and Izumo 2000).
There is increasing evidence that the SDF / CXCR4 axis plays a key role in regulating and regeneration of injurious myocytes (Matsui et al. 1999;Askari et al. 2003;Deglurkar et al. 2006;Elmadbouh et al. 2007;Zhang et al. 2007a).In this regard, Tang et al. (2009) reported that the axis of the SDF / CXCR4 plays an important role in the recruitment of endogenous and exogenous stem cells (Zhang et al. 2007b).Also well known that SDF-1a / CXCR4 axis is directly involved in the movement of stem cells to the area of injury in various tissues (Tang et al. 2009;Ji et al. 2004;Liu et al. 2013).It has been shown that the expression of both SDF-1a and CXCR4 increases following acute hypoxic conditions (Kitaori et al. 2009;Liu et al. 2010).In recent decades, many studies have been done on different types of stem cells that are involved in the regeneration and production of new cardiac cells (Liu et al. 2010;Zhang et al. 2010), among them can mention stem cell CSC (Ramez et al. 2019;Bin et al. 2014).CSC includes several types of cells, that most theme in the regeneration and repair of injured areas in the heart of the c-kit (Roccio et al. 2008).Human and animal studies have shown that c-kit cells are stimulated and activated under hypoxic conditions in response to chemotactic signals such as SDF and SCF to reduce fibrosis of the myocytes (Wen et al. 2012b).Several other studies have reported that CXCR4 expression and activity, in response to the stress of hypoxia, result in the proliferation and migration of endogenous cardiac stem cells (C-kit), and it has been shown that activity and expression of HIF-1a is the main factor in expression of CXCR4, and the suppression of HIF-1a expression reduces the expression of CXCR4 (Bearzi et al. 2007).Also, the hypoxic conditions is led to threefold increase serum and tissue levels of SDF-1a (Matsui et al. 1999;Askari et al. 2003).Studies have shown that stem cell factor (SCF) and its receptor play an important role in the migration of C-kit endogenous stem cells to infarcted area after myocardial infarction and concluded that the role of SCF in the accumulation of CSC (C-kit) stem cells is dependent on the expression of the C-kit receptor.It has also been shown that the expression of the C-kit receptor for the movement of exogenous C-kit cells to the injurious cardiac tissue after myocardial infarction is necessary (Penn 2009).Accordingly, the crosstalk between the signal of the axes mentioned by the chemokine SDF-1a/CXCR4 and the SCF/C-kit stem cell factor plays an important role in stem cell recruitment (Fazel et al. 2008) and cardio protection through intracellular signaling pathways (Ding et al. 2013;Liang et al. 2007;Singh et al. 2010).
Epidemiologic evidence shows that there is a strong relationship between people who are training regularly and are saved from cardiac infarction (Suzuki et al. 2001).Regular exercise seems to be one of the best and most effective practical and tolerable approaches to heart protection (Frasier et al. 2011).Studies in this field showed that in various physiological conditions such as exercise and altituderelated ischemia, and also pathological conditions (such as diseases), the concentration of SDF and other stem factors such as CSF-G, SCF, C-KIT, and SCa-1 are changed (Powers et al. 2008;Ellison et al. 2011). Lu et al. showed that 1 to 2 weeks of incremental training with suitable intensity after stroke ischemia, increased levels of SDF and CXCR4.They showed that the expression of SDF and its receptor CXCR4 following exercise training plays a key role in the proliferation and migration of brain stem cells (Ellison et al. 2011).Another study by Sen et al. (2015) showed that following aerobic exercise training, CD34 cells increased by stimulating SDF-1a and its receptor, CXCR4, and the presence of this chemokine was necessary to move stem cells to injury and inflammatory areas (Luo et al. 2014).In this field, a study also showed that even one session of high-intensity exercise (HIE) above the lactate threshold increased serum levels of FLT3, G-CSF, SDF-1a, and CD34 in adult and immature adults (Sen et al. 2015).And they concluded that the maximal and submaximal exercises increase the levels of these chemokines in circulation and tissue (Zaldivar et al. 2007;Tirakitsoontorn et al. 2001).
However above reasons, although high-intensity interval training is known to be a strong stimulant for the release of recruitmener chemokine of various stem cells from different tissues (Rehman et al. 2004), no research has been found to study the effect of a short high-intensity interval training on the stimulation and release of chemokines that recruitment stem cell factors endogenous with ischemic.Accordingly, the aim of this study was to investigate the effect of highintensity interval training on tissue level of SDF, receptor, and C-Kit proteins in myocardial following acute myocardial infarction in male rats as a cardioprotection mechanism.

Animals and experimental design
In this study, twenty male Wistar rats with a weight of 234.8 + 5.7 g (8 weeks old) were purchased from the Animal Science Laboratory of Pasteur Institute and After transfer to the Center for Experimental Studies, kept at a temperature of 22 ± 2 °C, relative humidity of 50-55%, and a dark-light cycle of 12:12.Rats were randomly divided into four groups of control (C: n = 5), high-intensity interval training (T: n = 5), training with myocardial infarcted (T + MI: n = 5), and myocardial infarcted group (MI: n = 5).In addition, animals were freely available to drinking water and compressed foods for rats during the study.In order to create an accord with the environment, all interventions began at least 2 weeks after the animals were located in the lab.Forty-eight hours after the last training session and 1 week after induction of infarction, the rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg).After confirmation of complete anesthesia, the cardiac tissue extraction was frozen quickly in liquid nitrogen and stored at -80 °C in the freezer.After the end of the study, the animals were delivered to the animal care center to be destroyed in accordance with the ethical principles of the animal ethics committee of the University of Medical Sciences.
All methods including anesthesia and sacrifice procedure were conducted in accordance with the guide for the care and use of laboratory animals, institutes for laboratory animal research, National Institutes of Health (NIH Publication no.85-23, revised 1996), and approved by the Animal Ethics Committee of University of Medical Sciences (28895-1302-1395 IR.IUM S.REC).

Exercise protocols
The reasons for the use of this training method in this study are because of the beneficial effects of high-intensity interval training on cardiovascular disease, angiogenic, mitochondrial biogenesis, extra enhance metabolic needs of the body's muscles, increase stress hormones, and active signal that research variously approves (Sen et al. 2015;Brenner et al. 1998;Rahimi et al. 2015).
Before starting the training period, exercise groups practiced 3 sessions at a speed of 20 m/min for 10 to 15 min (approximately 50% of VO2max) on treadmill in order to familiarize with treadmills (Brenner et al. 1998;Astorino et al. 2012;Ghanimati et al. 2020).After 1 day of rest, the 2-week protocol containing four parts was performed as follows (Table 1).
The first section consisted 3 days of training, two sessions each day, and each session consisting of 4 × 2 min with a speed of 35-40 m/min (~ 85-85% of vo2max) and 3 × 2 min with slow speeds of 25 to 30 m/min (~ 60-70% vo2max) between two high interval trainings.The second part included 2 days of training, 4 × 2 min with 40 to 45 m/min (~ 95-100% vo2max) and 3 × 2 min slow intervals with 28 to 32 m /min (~ 65-75% vo2max).The third part included 3 days of training, including 5 high intervals and 4 slow intervals with the intensity of the second part.The fourth section consisted of 2 days of training, as in the third part, but with 6 high-intensity intervals and 5 slow-intensity intervals (Astorino et al. 2012;Kemi et al. 2005).
In order to ensure the physiological effect of training during two weeks, the functional test of maximum endurance performance capacity at the beginning and end of the training was measured (Fig. 1).The time to exhaust was determined by a mild shock.Whenever the rats contact with the shock set at the end of the barrel twice in 30 s, or the reflection of the return and standing straight on the leg was seen as exhaustion (Aboutaleb et al. 2015).The test protocol includes gradual warming with the intensity of 15 to 25 m/ min for 5 min.Then, in the second stage, the speed and activity time, such as Fig. 1, continued time to exhaustion (Judge et al. 2005).

Induction of myocardial ischemia
Forty-eight hours after the last training session, myocardial infarction was induced by subcutaneous injection of Isoprenaline (150 mg/kg) solution in normal saline for two consecutive days.The use of this substance in animal models, especially in rats, is one of the common ways to cause infarction (Sen et al. 2015;Astorino et al. 2012).One week after induction of infarction, the rats were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/Kg), and their hearts were removed.

Histological methods and confirmation of infarction
Blood enzymes creatine kinase (CK), lactate dehydrogenase (LDH), and troponin T were measured to confirm the injury of the heart tissue.Hematoxylin and Eosin (H&E) staining was also used to investigate the necrosis and Mason's trichrome staining was used to determine the fibrous tissue.After staining, samples were analyzed using an optical microscope of 40 × zoom.The digital quantification of fibrosis areas (stained green) was perfomed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).Fig. 1 The steps of the exhaustion exercise test in order to evaluate of endurance capacity

Proteins measurement
Protein concentrations of SDF-1α, CXCR4 receptors, and c-kit were measured using the western blot method so that 100 mg of left ventricular tissue was located in a RIPA buffer for 30 min.Then, the ventricular tissue was lysed using a homogenizer.The solution was stored in ice for half an hour.And then for 20 min, using a centrifuge set at a temperature of 4 °C and a speed of 12,000 rpm, the supernatant was separated and stored in a freezer at 80 °C until it was used.Nano-Drop evaluations were used to determine the protein concentration.The western blot test was performed based on the determined protocol.In summary, the prepared samples were prepared with 10 ml Tris (pH 6.8), 12.5 ml Glycerol, 2.5 ml β-mercapto ethanol, 0.01 g Bromophenol Blue, and 25 ml SDS (10%)) which are equally combined and boiled in a temperature of 100° C for 7 min.Then, the solution was exposed to SDS-page 12.5%, and proteins were transferred to the PVDF membrane.The concentration of these proteins was identified by specific antibodies to SDF-1α (England, biorbyt, orb227817), CXCR4 receptors (England, biorbyt, orb10305), and c-kit (England, biorbyt, orb374707).Finally, the membrane was incubated with an ECL Western blotting system and exposed to X-ray film.Band density analysis was performed by ImageJ software, whereas β-actin is a component of proteins with stable expression in the cell.The antibody of this protein was used to remove the error of loading equal amounts of protein in the wells.

Statistical analysis
In the present study, the Shapiro-Wilk test was used to determine the normal distribution of data, and one-way ANOVA and Tukey's tests were used for data analysis at the level of 0.05.Statistical analysis was performed using SPSS software.

Endurance capacity
The results of this study showed that 2 weeks of high-intensity interval training significantly increased (p = 0.000) endurance capacity of male rats in the two training groups, that indicates the physiological effect of this duration and type of exercise training on the endurance capacity of the rats.The running distance in the C group at the beginning of the training was 735 m, and the time was 41.5 min, which after 2 weeks was 945 m, and the time was 48.95 min.Also, the running distance in the MI group changed from 774 to 976 m, and its time ranged from 42.75 to 52.85 min, but the running distance in the T group at the beginning of the training was 760 m, and the time was 41.86 min, and after 2 weeks of high intensity interval training, the distance was 3400 m, and the time was 182.75 min.In the T + MI group, the distance changed from 723 to 3324 m and the time from 40.2 to 170.25 min (Table 2).

Cellular injury markers
The results of the one-way analysis showed that there was a significant increase in LDH, CKMB, CK total, and troponin T factors after myocardial infarction (p = 0.000).Tukey's post hoc test showed significant difference between the MI group and the other groups (C (p = 0.000), T (p = 0.000), and T + MI (p = 0.000)).This test also showed that in all four factors, there was a significant difference between the T + MI group with T and C groups.These results mean that myocardial infarction significantly increases LDH, CKMB, and troponin T (Fig. 2).As shown in figure, the results showed that the increase of injury indices in the exercise group (T + MI) is less than the MI group.

Cardiac pathology and tissue injury
Hematoxylin and Eosin (H&E) staining results (Fig. 3) and Mason's trichrome staining (Fig. 4) was shown the amount of fibrosis and necrosis in the heart tissue and can confirm the protective effect of exercise training against infarction.Pathological results showed that injection of isoprenaline in two consecutive days causes severe tissue injury to the heart.As shown in Fig. 3, in the noninfarcted groups, the cardiomyocytes were orderly arranged and dense, and the cardiac muscle fascicles are complete in shape, but in the MI In the Masson staining of myocardial tissue, collagen fibers are stained green (Fig. 4).Compared with the MI group, the T + MI group shows a looser arrangement of the myocardium with a lower amount of blue collagen between the myocardium.The MI group showed a severely disordered arrangement of the myocardium with a widened myocardial gap that a large area of blue fibrotic tissue replaced the normal myocardial tissue.Calculating the percentage of fibrosis area also shows that fibrosis in the T + MI group is less than in the MI group (10.76 and 19.21%, respectively).Therefore, HIIT attenuated ISO-induced cardiac fibrosis (43.98%).

Protein expression
Antibody SDF-1α was used for its expression in the cardiac tissue of four groups of rats.Analysis of western blot results using one-way ANOVA showed that there is a significant difference between the mean of the four groups.The results showed that the SDF-1α protein concentration of the MI group was statistically significant in comparison with the T group, the C group, and the T + MI group (p = 0.000) (Fig. 5A).Also, SDF-1α protein concentration in T + MI group was significant in comparison with the T group (p = 0.002) and C group (p = 0.000).Also, C-Kit tissue protein concentration was studied in four groups using the western blot technique.The result shows that the concentration of this protein in the MI group was significantly higher than in the T group, T + MI, and C group (p = 0.000).As well, the concentration of C-kit protein in the T group and T + MI group was significantly higher than in the C group (p = 0.000) (Fig. 5B).
The results of western blot analysis with one-way ANOVA and Tukey's post hoc test showed that CXCR4 receptors concentration after 2 weeks of high-intensity interval training in the MI group was significantly higher than T group (p = 0.000), T + MI group (p = 0.009), and C group (p = 0.000).Moreover, the results showed that the concentration of this protein in the T + MI group was significantly increased compared to the C group (p = 0.000) and T group (p = 0.017), and the expression of this protein in the T group was significantly increased compared to the C group (p = 0.000) (Fig. 5C).
Protein beta-actin as a control has been studied, and the results of actin expression in groups of T, T + MI, MI, and C, are seen in Fig. 5D.

Discussion
The results of the present study showed a significant increase in endurance capacity, duration, and running distance of male Wistar rats following 2 weeks of high-intensity interval training, which indicated the physiological effects of selected exercise training on performance and physiological adaptations of rat (Dolinsky et al. 2012).
The results showed that levels of c-kit receptor protein tissue were statistically significant increase in T, MI, and T + MI groups compared with the C group.In this regard, studies have shown that stem cell factor (SCF) and its receptor C-kit play an important role in the migration of endogenous stem cells to infarct areas after MI.Kuang et al. (2008) showed that the expression of SCF/C-kit significantly increased immediately after MI in the rats.They concluded that the overexpression of SCF and its receptor in the infarcted area was associated with the accumulation of stem cells (Waring et al. 2015).And they also claimed that the presence and expression of SCF, which is a factor in the accumulation of stem cells, depends on the content of the expression of the receptor of the C-kit.On the other hand, Hang et al. (2011), in a study, showed that the adult heart has a small number of cells expressing stem cell markers such as C-kit, SCa-1, and MDR-1 (Kuang et al. 2008).
Furthermore, studies have shown that following exercise training, the stem cells' recruitment of C-kit and also the expression of NKX2/5 and GATA4 (the key factors transcription of the cardiac stem cells) are increased (Huang et al. 2011;Armiñán et al. 2009).In addition, exercise training activates the PI3K/AKT signal pathways in the myocardial infarcted area, and beneficial effects are induced through the recruitment of C-kit + cells by increasing the synthesis of DNA in the heart infarcted area (Bruneau 2013) The findings of this research, which show a significant increase in the concentration of SDF-1a protein and CXCR4 receptor compared to the C group, state that during the ischemia and hypoxia, key family factors (HIFs) directly activate and increase the growth factors (SCF, HGF, VEGF, PDGF), chemokines (SDF-1a), and cytokines (Ellison et al. 2008;Ceradini et al. 2004;Ockaili et al. 2005).The expression of SDF-1a is regulated through the HIF pathway in endothelial A SDF-1/β actin, *p < 0.001 compared to the control group; # p < 0.01 compared to the T group; + p < 0.001 compared to 3 other groups.B C-kit/β actin, * p < 0.001 compared to 3 other groups; # p < 0.001compared to 3 other groups.C CXCR4/β actin, *p < 0.001 compared to the control group; # p < 0.05 compared to the T group; + p < 0.001 compared to C and T groups; ++ p < 0.01 compared to T + MI group.D Western blot images of the protein bands of SDF-1a, C-kit, and CXCR4 and β actin (as a loading control) in the heart tissue.The ratio of these proteins to β-actin expression in experimental groups was shown and described in A-C.All results are presented as means ± SD cells and its expression in ischemic tissue is proportional to the amount of oxygen depletion (Ellison et al. 2008;Schioppa et al. 2004).Accordingly, the SDF/CXCR4 axis is associated with cardiac survival, new angiogenesis, and cardiac function after cardiac infarction (Zaruba and Franz 2010).Recent studies have shown that the SDF/CXCR4 signal axis plays a role in reducing infarcted size and improving left ventricular function in ischemic injury-redundant models (Kucia et al. 2004).On the other hand, following the ischemia in tissue with the activation of the SDF-1a/CXCR4 signal axis, the migration of CPC/C-kit + stem cells to the infarction area increases (Chen et al. 2010;Rodrigues et al. 2011).In this relation, Tang et al. found that hypoxia increased the expression of CXCR4 and HIF-1a increased CLK/C-kit + migration through SDF-1a (Zhang et al. 2007b).It is necessary to express that CPC/Ckit + cells are endogenous stem cells in the adult heart that are responsible for the regeneration of cardiac myocyte in physiologic and pathological conditions (Zhang et al. 2007b).Studies have shown that stem cell migration is enhanced by the pathways of SDF-1a/CXCR4 and SCF/C-kit, which plays its role by blocking the pathway of signal p38-MAPK (a factor in reducing the migration of stem cells) (Kuang et al. 2008).The research that was done by Ding et al. (2013) showed that crosstalk between the SDF-1α/CXCR4 receptors and SCF/C-kit signaling pathways plays an important role in the profibrotic by recruitment of endogenous C-kit stem cells (Fazel et al. 2008).The research also showed that the expression of the receptor of the C-kit induces MMP-9 activity, which is necessary for the proliferation and migration of the progressive C-kit stem cells to the cardiac after the MI (Penn 2009).On the other hand, TNF-α binding to the C-kit receptor is overexpressed by the C-kit receptor, which increases the regeneration of injured cardiac cells (Penn 2009).
Also, other signaling pathways are activated by axis SDF-1a/CXCR4 receptors, which results in cell survival and stem cell proliferation.So, the expression of CXCR4 is PI3K/ AKT phosphorylation mediator, which results in upregulated vascular growth factor (VEGF) regulation (Ding et al. 2013).AKT and ERK1/2 are other signal pathways that are activated by the SDF-1a/CXCR4 axis, which inactivates the BAD protein expression (associated with cell death) (Liang et al. 2007;Singh et al. 2010).It also increases the expression of cell protection and survival proteins such as BCL2, BCL-XL, Notch-1, β-catenin, and NK-KB.The CAMP/PKA signal path is another path that is activated by the SDF-1a/ CXCR4 axis, which is necessary for the migration of stem cells to injured areas (Tang et al. 2011).
A research showed that tissue ischemia increased the expression of SDF-1, which activates the STST3 signal pathway, improves cell growth, and inhibits cell apoptosis (Kuang et al. 2008).STAT3 is a protective pathway in the heart that, by activating several downstream signal pathways, expresses BCL2, BCL-XL, HSP, and angiogenic factors and decreases the secretion of inflammatory cytokines (Sadana and Dessauer 2009).
Pathologic results of the present study showed that subcutaneous injection of isoproterenol in two continues days caused severe pathological changes such as edema, neutrophilic accumulation, tissue disruption and hemorrhage in rat's cardiac tissue.It also significantly increases indexes injuries cardiac such as LDH, CK total, CKMB enzymes and troponin T enzyme compared to the control and training groups, which was consistent with the results of the study by Farvin et al. (2010) and Tofighi et al. (2017) (Xiao et al. 2014;Tofighi et al. 2017;Farvin et al. 2010).On the other hand, the results of this study showed that cardiac injury factors in T + MI group were less than that in the MI group, which may be the result of the protective effect of exercise training against ischemic attacks and cardiac infarction.The results of this study were similar to the results of Lobo et al. ( 2011) and Nounou et al. (2012) (Tofighi et al. 2017;Nounou et al. 2012).Several studies have reported that exercise training protects against cardiac MI in animal models Induces (Lobo Filho et al. 2011;Brown et al. 2003Brown et al. , 2005;;French et al. 2006;Ramez et al. 2020).Regular training periods seem to be one of the best, most effective, and tolerant approaches that cause cardiac protection (Frasier et al. 2011).Anatomical and physiological changes in the coronary arteries, head shock protein (HSPS), increased activity of cyclooxygenase-2 (COX-2), increased endoplasmic endothelial stress (ER), enhanced potassium function of ATPdependent sarcolemma (sarcoKATP), increased levels of ATPdependent potassium channels in mitochondria (mitoKATP), nitric oxide (NO), and increased the antioxidant capacity of the myocardium are among the cellular-molecular mechanisms involved in cardiac protection from cardiovascular injuries (Ramez et al. 2019;French et al. 2006).In this field, this study showed that the increase in the chemokine SDF and its receptor, as well as the C-kit receptor, would result in cardiac protection and regeneration by high intensity interval training.In the present study and other studies, it has been shown that under normal conditions, the levels of SDF-1α and its receptor CXCR4 in the heart tissue are insignificant (Goodman and Hodgson 1962;Ellison et al. 2011), but in different physiological conditions such as intense training, hypoxia and ischemia, as well as pathological conditions (Such as acute myocardial infarction), the concentration of these factors changes (Borges and Lessa 2015;Sarto et al. 2007).In a similar study, researchers showed that serum levels of chemokines such as SDF-1α and stem cells increased after one-session endurance training, and they increased this as a result of increased levels of cytokines in the trained people (Beltrami et al. 2003;Morici et al. 2005).These results were similar to the results of the present study, which showed that the high-intensity interval training of SDF-1α tissue concentration increased.It seems that exercise training through induce ischemia and low hypoxia in various tissues of the body, including the cardiac, increases the level of SDF-1α and other stem cell promoters, and this increase can recruit the stem cell to the ischemic and hypoxia tissue (Luo et al. 2014).In this field, various conclusions have been report from the effect of exercise training on the levels and effects of chemokines by researchers, as Sen et al. (2015) and Sarto et al. (2007) showed that following the performance of various exercises training, the levels of SDF-1a increased significant (Luo et al. 2014;Sarto et al. 2007).They report that the probable reasons for the significant increase in the SDF-1a/CXCR4 axis were in cardiac increased cortisol levels due to exercise stress, which plays a key role in the onset of the AKT / PKB signal cascade, which improves cardiac function, improves ejection fraction, Left ventricular fractional shortening, increased left ventricular wall thickness and recruitment of stem cells (Sen et al. 2015;Morici et al. 2005).Another study by Mendes-Fereer et al. (2008) found that acute activity exercise and longterm exercise training led to the release of catecholamine's that increase the movement of the progressive hematopoietic stem cells and also the acute and exercise training of the receptor expression Beta-adrenergic α1,2 and β2 increased, via increasing the expression of MMP-9, which is essential for the migration of progressive hematopoietic stem cells (Harada et al. 2005b;Méndez-Ferrer et al. 2008).In addition, the release of epinephrine by the exercise activity induces the expression of the receptor of GSK3β, which increases the sensitivity of the chemotactic signaling pathways of SDF-1 and induces the remodeling of cytoskeletons (Lapid et al. 2008).
Although there is a need for confirmation based on future studies, it can be reasoned that this is probably due to the fact that the tissue levels of SDF, CXCR4 receptors, and C-kit in the training group and training-myocardial infarction group compared to the myocardial infarction group, despite of increasing the values, were not statistically significant.The results of this study in tissue injuries showed that exercise training has a pathway of cardiac protection through inducing multiple signal pathways and other unknown mechanisms that actually injuries resistance to acute ischemia and reduce the injuries caused by ischemia Acute and decreased cellular damage in the cardiac tissue.Therefore, the expected accumulation of chemokine tissue levels in the training-myocardial infarction group may be statistically insignificant.

Conclusion
Despite the need for further research in this field, the general results of the present study suggest that high-intensity interval training can be a type of preconditioning of the cardiac by increasing the levels of recruitment factors and the cardiac tissue regeneration factors itself and, in acute ischemia, reduce the amount of tissue injuries significantly.

Fig. 2
Fig. 2 Cellular injury markers of CK total, LDH, CK MB and (cTI) in experimental groups (n = 5).A Blood levels of creatine kinase total (CKtotal), B blood levels of lactate dehydrogenase (LDH), C blood levels of creatine kinase (CK MB ), and D blood levels of troponin T

Fig. 4 A
Fig. 4 A Masson's trichrome staining, representative histopathological and amount of fibrosis in the heart tissue, (a) C group, (b) T group, (c) T + MI group, and (d) MI group.The green color represents the amount of tissue fibrosis (× 40 magnification).The MI group showed a severely disordered arrangement of myocardium with . In this relation, Ellison et al. also showed that the exercise training increases the ratio of cardiac-to-body weight, volume of myocytes, and also the number of cardiac stem cells in the training group by 5 folds in the ventricular wall.They also showed that exercise training in the first, second, and third weeks resulted in C-kit expression in the left ventricle of rat, but this increase was observed in the right ventricle in the third week (D'Uva et al. 2015).Exercise training with controlled intensity increases the expression of growth factors, initiates the regeneration of myocytes, and then activates the differentiation of the C-Kit, which results in the production of new heart cells.This finding suggests that cardiac physiological adaptation depends on the intensity and duration of the exercise (D'Uva et al. 2015).

Table 1
High-intensity interval training protocol The changes of these indicators (CK total, CKMB, troponin T, and LDH) in the T + MI group have decreased by respectively 44.65, 20.33, 37.06, and 21.5%, compared to the MI group, which indicates the cardioprotection effect of the exercise training.

Table 2
The results of the functional test before and after 2 weeks of HIIT in research groups groups, severe pathological changes were created, including abnormal myocardial architecture, edema, neutrophil accumulation, and separation of fibers in the heart tissue.However, these changes in the T + MI group are less than MI group, which can indicate the positive effect of exercise training on cardioprotection.