Early moderate exercise benefits myocardial infarction healing via improvement of inflammation and ventricular remodelling in rats

Abstract Thus far, the cellular and molecular mechanisms related to early (especially within 24 hours after acute myocardial infarct (MI)) exercise‐mediated beneficial effects on MI have not yet been thoroughly established. In the present study, we demonstrated that acute MI rats that underwent early moderate exercise training beginning one day after MI showed no increase in mortality and displayed significant improvements in MI healing and ventricular remodelling, including an improvement in cardiac function, a decrease in infarct size, cardiomyocyte apoptosis, cardiac fibrosis and cardiomyocyte hypertrophy, and an increase in myocardial angiogenesis, left ventricular wall thickness and the number of cardiac telocytes in the border zone. Integrated miRNA‐mRNA profiling analysis performed by the ingenuity pathway analysis system revealed that the inhibition of the TGFB1 regulatory network, activation of leucocytes and migration of leucocytes into the infarct zone comprise the molecular mechanism underlying early moderate exercise‐mediated improvements in cardiac fibrosis and the pathological inflammatory response. The findings of the present study demonstrate that early moderate exercise training beginning one day after MI is safe and leads to significantly enhanced MI healing and ventricular remodelling. Understanding the mechanism behind the positive effects of this early training protocol will help us to further tailor suitable cardiac rehabilitation programmes for humans.


| INTRODUC TI ON
Myocardial infarction (MI) is a major cause of human morbidity and mortality worldwide. 1 Patients who survive MI frequently develop systolic heart failure because of the infarct-induced loss of functional myocardium per se and the remodelling of the left ventricle (LV), which involves cardiomyocyte necrosis, cardiomyocyte hypertrophy, LV wall thinning, infarct expansion and collagen accumulation.
Although several therapeutic approaches have been proven to ameliorate post-infarction cardiac remodelling, the prognosis remains poor, and LV dysfunction often progresses to heart failure. 2 For many years, avoiding physical activity after MI was recommended; however, the current view proposes that moderate exercise training should be a part of cardiac rehabilitation programmes. There is increasing evidence that exercise training, including exercise training before and after infarction, provides promising effects on the repair of the infarcted heart, regardless of the decrease in infarct size and cardiac fibrosis, the attenuation of apoptosis in the myocardium, improvements in ventricular remodelling and inflammation. [3][4][5][6][7][8][9][10] However, various studies in animals and humans have also shown conflicting results concerning the effects of exercise training, including neutral [11][12][13][14] and adverse [15][16][17] effects, on LV remodelling after MI.
The variation of the effects might be due to differences in exercise intensity after MI. High-intensity exercise can overload the infarcted heart, whereas low-intensity exercise exerts little if any physiological impact. The timing at which exercise training starts after MI might also be a critical factor in its effect on regeneration after MI.
Studies in rats have indicated that exercise that is started late (more than three weeks after MI) does not aggravate 13,18 or blunts 4,6,19 LV dilation and hypertrophy, whereas exercise started less than a week after MI results in beneficial, 20 no 11,21 or detrimental 15,16 effects on LV remodelling. In a more recent human randomized controlled trial study, it was documented that early exercise-based rehabilitation in which patients underwent a supervised early mobilization exercise programme twice a day beginning 12 hours after acute MI (the inpatient phase) improved health-related quality of life and functional capacity in patients with low cardiovascular risk who experienced acute MI. 22 In rats that experienced a large induced MI, early voluntary exercise training (started within 24 hours after MI) had no impact on survival or LV remodelling but attenuated global LV dysfunction 23 and restored cardiomyocyte contraction via improving the myofilament Ca 2+ response and diastolic Ca 2+ handling. 24 In this study, a voluntary exercise programme in which rats ran approximately 5 km a day on average was utilized. It is not yet clear whether and how early exercise of moderate intensity (600 m/d) 25 beginning one day after MI, in contrast to voluntary exercise (approximately 5 km/d on average), benefits the repair of the infracted heart.
In addition, it has been documented that the number of cardiac telocytes increases significantly in the heart following a 4-week ramp swimming exercise programme in mice. 26 These findings suggest that cardiac interstitial cells, such as cardiac telocytes, might be involved in exercise-mediated beneficial effects in the myocardium and might promote the healing and regeneration of the damaged myocardium.
The present study was therefore designed to evaluate the underlying mechanism of the beneficial effects mediated by moderate exercise conducted one day after MI and mainly focused on the effects of exercise on the inflammatory response, ventricular remodelling and cardiac telocyte density in MI.

| Myocardial infarction induction
Myocardial infarct was generated through left anterior descending coronary artery (LAD) ligation in three-month-old female Sprague Dawley rats, as previously described. 27,28 For details, see Appendix S1.

| Treadmill exercise
All animals were habituated to a motorized treadmill by running following a gradual acceleration protocol (0° grade; 10 m/min for 5 minutes, 15 m/min for 5 minutes and 20 m/min for 20 min) each day for 2 weeks. Following 2 days of rest, the rats were randomly F I G U R E 1 Early moderate exercise reduces infarct size and cardiomyocyte apoptosis and improves cardiac function in myocardial infarct (MI). (I) Schematics of the training programme. (II) Masson's trichrome staining showed that early moderate exercise reduced infarct size. n = 6-8. (III) Echocardiography revealed that early moderate exercise improved the ejection fraction (a) and fractional shortening (b). n = 5-8. and a moderate exercise group (n = 23); set-2, a sedentary control group (n = 7) and a moderate exercise group (n = 10). Both sets underwent LAD ligation to induce MI as described above. The rats in the moderate exercise group ran on the treadmill beginning one day after MI for 2 weeks using the moderate exercise protocol, which was set at 20 m/min for 30 min/d (hereafter called the exercise group). 25 The rats in the sedentary control group were allowed to be sedentary in their cages for 2 weeks after MI ( Figure 1I). The set-1 animals were used for all observations except the cardiac function analysis, whereas the set-2 animals were used to analyse of cardiac function. Both sets of animals were included to analyse mortality rate.

| Echocardiography
Cardiac function was evaluated by echocardiography as described in Appendix S1.

| Histological staining
Masson's trichrome staining, the TUNEL assay and immunohistochemistry were performed as described in Appendix S1. mRNAs and miRNAs with differential expression equal to or greater than a two-fold change were considered to be differentially expressed and were selected for ingenuity pathway analysis (IPA).

| mRNA and miRNA sequencing
For details, see Appendix S1.

| mRNA-miRNA integrative IPA
The selected mRNAs and miRNAs with a log 2 expression ratio greater than 1 or less than −1 were used for further mRNA-miRNA integrative analysis to identify regulatory networks and disease function analysis using IPA (http://www.ingen uity.com). The analysis results were used for an additional core analysis in the IPA system. In the present study, only the analysed data that were used to predict "decrease" or "increase", which was indicated by a P value <.001 and a z-score larger than 2 or less than −2, were selected as positive predictors. For details, see Appendix S1.

| Real-time quantitative PCR
Gene and miRNA expression levels were analysed using SYBR greenbased quantitative PCR (qPCR). Relative expression was determined using the 2 −ΔΔCt comparative threshold method. For details, see Appendix S1. The primer list is shown in Appendix S2.

| Statistics
All measured data are presented as the means ± standard errors.
Two-tailed Student's t test was used to calculate the statistical significance between two groups. P values <.05 were considered significant.

| Early moderate exercise does not affect mortality
After LAD ligation to induce MI, 43 LAD-ligated rats were randomly divided into the early moderate exercise group (n = 23) and the F I G U R E 2 Early moderate exercise improves angiogenesis, fibrosis and ventricular remodelling in myocardial infarct (MI). (I) Immunohistochemical staining for vWF showed that blood vessel density in the infarct zone and border zone in early moderate exercise hearts was significantly higher than those in sedentary hearts. (n = 17), 10 rats from the early moderate exercise group and 7 rats from the sedentary group, were used to compare cardiac function and further confirm the difference in mortality rate between animals F I G U R E 3 Early moderate exercise increases the number of cardiac telocytes in the border zone but not in the infarct zone. (I) Immunofluorescence staining for PDGFRα + /CD34 + revealed that the density of CTs in the early moderate exercise group was slightly increased compared with that in the sedentary group; however, the difference was not statistically significant. (II) Immunofluorescence staining for PDGFRα + /CD34 + revealed that, in the border zone, the density of CTs in the early moderate exercise group was significantly increased compared with that in the sedentary group. (a1-4) Sedentary heart. (b1-4) Early moderate exercise heart. (c) Semiquantification of (I) and (II). n = 5 per group. The animals were trained on early moderate exercise for two weeks beginning one day after MI that underwent exercise and those that did not. The mortality rate of the early moderate exercise group (2/10; 20%) was even lower than that of the sedentary control group (2/7; 28.57%). The total mortality rates of the two sets were 21.21% (7/33) and 22.22% (6/27) in the early moderate exercise group and the sedentary control group, respectively.

| Early moderate exercise reduces infarct size and apoptosis of cardiomyocytes and improves cardiac function in acute MI
The infarct size in the exercise group was significantly smaller than that in the sedentary group ( Figure 1II;

| Early moderate exercise improves angiogenesis, fibrosis and ventricular remodelling after MI
In the present study, the effect of early moderate exercise on post-MI regeneration was evaluated in terms of angiogenesis, fibrosis and the remodelling of the infracted heart. The vWF immunohistochemistry results showed that the blood vessel density in the infarct and border zone in the exercise group was significantly higher than that in the sedentary group ( Figure

| Early moderate exercise increases the number of cardiac telocytes in the border zone but not in the infarct zone
As interstitial cells, cardiac telocytes (CTs) have been recently identified as a distinct interstitial cell type that exists in heart and other tissues and organs. [29][30][31][32][33] We previously reported that CTs are distributed longitudinally and within the cross-network of the myocardium, which experiences serious cell death and is significantly decreased in the infarct zone after acute MI in rats. 34,35 In addition, the number of CTs in the heart has been reported to be increased significantly following a 4-week ramp swimming exercise programme in mice. 26 These data suggest that CTs might be involved in exercise-mediated beneficial effects on MI. To reveal the CT networks in the MI heart, cells positive for PDGFRα and CD34, two makers generally used to identify CTs, 26,31 were identified by using immunofluorescence staining. In the infarct zone, the density of CTs was slightly increased in the exercise group compared with the sedentary group ( Figure 3I; P > .05). However, in the border zone, the density of CTs was significantly increased in the exercise group compared with the sedentary group ( Figure 3II; P < .05).

| Early moderate exercise inhibits the inflammatory response in the infracted myocardium
To determine whether early moderate exercise has beneficial effects on the inflammatory response in MI, we assessed the characteris-

| miRNA-mRNA integrative IPA reveals that the inhibition of the TGFB1 regulatory network is a major underlying mechanism of the early moderate exercise-mediated improvement of cardiac fibrosis and ventricular architecture remodelling in MI
To reveal the underlying molecular mechanism of the significant pro-  36 Therefore, we also applied IPA to analyse the differential expression of 87 miRNAs in the infarct zone between early moderate exercise MI hearts and sedentary MI hearts. IPA showed that, at the miRNA level, no predicted activation or inhibition effects regarding the canonical pathway, upstream signalling, or molecular and physiological function were identified (z-score < 2). Conversely, IPA of disease toxicity function identified some miRNAs that are related to cardiotoxicity (z-score < 2), such as miR-150-5p and miR-133a-3p, which are involved in the regulation of cardiac fibrosis and cardiac hypertrophy, as shown in Appendix S4.
It is well established that the functional role of miRNAs is mainly to modulate gene expression through both mRNA degradation and translational repression mechanisms 37,38 ; therefore, the integrated analysis of differential expression profiles of miRNAs-mRNAs be- and FN1 tended to be down-regulated ( Figure 5II). IPA also revealed that 74 genes (63 down-regulated genes and 11 up-regulated genes) are included in the regulatory network of TGFB1 inhibition, as shown in Appendix S5.

| miRNA-mRNA integrative IPA reveals that the inhibition of leucocyte activation and migration is the other major underlying mechanism by which early moderate exercise improves the inflammatory response in MI
The integrated IPA of miRNA and mRNA expression data also pre- tion. In addition, miR-125b-5p was also able to down-regulate ITGA1, and FCGR2A was able to down-regulate CCL2, and these both inhibited leucocyte activation. Moreover, KLF2 was also able to inhibit CCL2 and led to the inhibition of leucocyte activation ( Figure 6I). and the up-regulation of CR2, KLF2 and miR-125b-5p led to the inhibition of leucocyte migration. In addition, miR-125b-5p was able to down-regulate VTCN1 and VDR, whereas KLF2 was able to downregulate THBS1 and CCL2, and these were revealed to contribute to leucocyte migration inhibition (Figure 7).

F I G U R E 4
Early moderate exercise inhibits the inflammatory response in infarcted myocardium. (I) Immunofluorescence staining for CD45 + leucocytes revealed that the density of CD45 + leucocyte infiltration in the infarct zone was significantly decreased in early moderate exercise hearts compared with sedentary hearts. (II) Immunofluorescence staining for CD68 + macrophages (M1 macrophages) demonstrated that the density of M1 macrophages in the infarct zone was significantly decreased in early moderate exercise hearts compared with sedentary hearts. (III) Immunofluorescence staining for CD206 + macrophages (M2) showed that the density of CD206 + macrophages in the infarct zone was increased significantly in early moderate exercise hearts compared with sedentary hearts. (IV) Immunofluorescence staining for CD163 + macrophages (M2) showed that the density of CD163 + macrophages in the infarct zone was increased significantly in early moderate exercise hearts compared with sedentary hearts.

| D ISCUSS I ON
Recent research has revealed that post-MI exercise training is associated with reductions in mortality and reinfarction; therefore, exercise training rather than the traditional instructions to avoid physical activity should be a part of cardiac rehabilitation programmes for MI. 39,40 However, the timing of the exercise training after MI has not yet been optimized. It has been revealed that moderate exercise beginning 5-7 days after MI is safe. 8,41 Much earlier exercise (beginning within 24 hours after MI) has no negative impact on the survival of rats with severe MI. 23,24 Similar to this observation, our results revealed that an exercise programme that began one day after MI was also safe; the mortality of the exercise group was similar to that of the sedentary group. Our findings strengthen the view that post-MI moderate exercise training can start very early, even within a day after infarct without causing higher mortality.
Beside the timing, the intensity of the exercise training is also a critical factor for a successful training protocol for MI patients.
In our study and studies performed by de Warrd et al 23  When MI occurs, LV wall thinning in the infarcted area, the dilation of the ventricular cavity and cardiomyocyte hypertrophy are mortality remodelling phenomena that induce heart failure as the infarct expands. 42,43 Similar to other reported exercise training protocols, 9,44 the early moderate exercise protocol that was applied in the present study decreased the collagen content in the infarct zone, increased the thickness of the ventricular wall and reduced F I G U R E 5 Inhibition of the TGFB1 regulatory network in the infarct zone of the early moderate exercise-trained heart. (I) The upstream analysis of the integrated ingenuity pathway analysis between miRNAs and mRNAs in the infarct zone predicted that compared with the sedentary MI heart, the primary upstream molecule was TGFB1, which was inhibited in the infarct zone of the early moderate exercise MI heart (P-value = 1.78E-08; zscore = −2.266). The inhibition of 10 genes (TGFB1, FN1, MAPK14, SP1, SP3, ESR1, SMAD4, EGR1, CREBBP and SMAD3) and the activation of 3 genes (HDAC2, TP73 and SMAD7) played a role in the inhibition of the TGFB1 regulatory network. The network displays the regulatory relationship of these 13 genes in which SP1, SMAD4 and SMAD3, the direct downstream genes that are regulated by TGFB1, were inhibited, whereas SMAD7, the inhibitor of the TGFB1 pathway, was activated. In addition, the indirect downstream genes, FN1, MAPK14, SP3, ESR1, EGR1 and CREBBP, were predicted to be inhibited, whereas TP73 was activated. (II) The qPCR quantifications of the expression levels of selected genes included in the TGFB1 network. The animals were trained on early moderate exercise for two weeks beginning one day after MI. n = 3 per group. *P < .05 vs the sedentary group cardiomyocyte hypertrophy. Our results suggest that early moderate exercise for two weeks beginning one day after MI provides the same benefits to the MI heart as other reported exercise training protocols in different timeframes via attenuating ventricular remodelling.
Whether cardiac telocytes are involved in exercise-mediated beneficial effects in MI is an intriguing issue. In the present study, we demonstrated that early moderate exercise significantly increases the number of cardiac telocytes in the border zone. This is not unpredictable, as it has been reported that exercise is able to increase the number of cardiac telocytes in the heart under physiological conditions. 26 The findings of the present study clearly reveal that an increase in cardiac telocytes in the noninfarcted area is a novel beneficial effect of early moderate exercise on MI. The early moderate exercise-mediated increase in cardiac telocytes observed in the border zone of MI might have a beneficial effect on the survival of cardiomyocytes in the border zone. The underlying mechanism related to this effect needs to be investigated more deeply in the future. However, our results also documented that early moderate exercise fails to significantly increase the number of cardiac telocytes in the infarct zone. This suggests that the protective effect derived from early moderate exercise is still not enough to protect cardiac telocytes in the infarct zone from cell death. Indeed, our previous study has demonstrated that cardiac telocytes experience serious cell death and are significantly decreased in number in the border zone and especially in the infarct zone during acute MI in rats. 34,35 They have also suggested that exercise rehabilitation alone is not enough to protect cells in the F I G U R E 6 Inhibition of leucocyte activation played a role in the early moderate exercise-mediated improvement of inflammation. (I) The integrated ingenuity pathway analysis between miRNAs and mRNAs predicted that the activation of leucocytes in the infarct zone was decreased (P-value = 3.51E-04; z-score = −2.209) in the early moderate exercise MI heart compared with the sedentary MI heart. The regulatory network demonstrates that 27 molecules (20 down-regulated genes, 3 up-regulated gene and 4 up-regulated miRNAs) were included in the regulatory network for the decreased activation of leucocytes. The directive inhibition relationship, which included miR-125b-5p for ITGA1,   56,59 were found to be increased in exercise MI hearts compared with sedentary hearts (data not shown). This suggests that maintaining fatty acid metabolism as well as glucose uptake and utilization is an important adaptive response mediated by early moderate exercise in MI hearts and might play an important role as an upstream effector to initiate changes in related gene expression, such as that of the genes and miRNAs identified in the present study, as well as others. Therefore, the detailed molecular mechanism of energy metabolism as an upstream target of the adaptive response mediated by early moderate exercise in MI hearts needs to be investigated in the future.
In the present study, MI rats were trained on treadmills, and this is highly similar to aerobic cardiac rehabilitation programmes for humans in which training is predominantly performed in the clinic. Recently, an early mobilization exercise programme beginning 12 hours after MI was shown to improve health-related quality of life in humans. 22 Therefore, the early moderate exercise protocol that was applied in the present study might also be applicable for designing a clinical trial protocol for improving cardiac inflammation and ventricular remodelling after MI in humans. However, the difference in many aspects between rodent and human in cardiovascular physiology should be in mind before transposing the protocol to humans. Furthermore, the early moderate exercise training window identified protective effects for MI healing, cardiac remodelling, the maintenance of cardiac telocytes and gene regulatory networks for the inhibition of the TGFB1 pathway, and leucocyte activation and migration; these findings might help us further tailor precise cardiac rehabilitation programmes to humans.

ACK N OWLED G EM ENTS
This work was supported by grants from the National Natural Science

CO N FLI C T O F I NTE R E S T
The authors declare that they have no competing interests.

AUTH O R CO NTR I B UTI O N S
ZL DL YC YL KZ HC and XZ performed most of the experiments and analysed data; RH ZY HZ QP and XQ contributed to discussion and manuscript writing; and DC conceived and designed this work and wrote the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available on request from the corresponding author.