Study On Regulation Of Signal Pathways In Motor Motility By MicroRNAs

The term "athlete’s heart" has been widely used to characterize the changes that occur in the heart due to long-term physical exercise in athletes. Left and right ventricle can be hypertrophic in physiological cardiac hypertrophy, but Left ventricular hypertrophy induced by exercise training is a major change that compensates for chronic increases in hemodynamic overload. The hypertrophic level is associated with the style, intensity and duration of exercise. Physical exercise results in brachycardia, increasing stroke volume and cardiac reserve. Increasing number and function of organells, including Golgi complex, rough endoplasmic reticulum, specific atrial granule and organelle, are happen in the process of cardiac remolding. At the same time, the physical exercise increases the number of myofibril, and improves the function of transverse tubular system and sarcoplasmic reticulum. All of these will induce the enhancement in endocrine, aerobic metabolism, and myocardium contractibility. However, the molecular mechanism of physiological cardiac hypertrophy is still unknown, and the correlated study is still on the way. miRNAs have recently emerged as a large group of short (18-25nucleotides), non-coding, small RNA molecules that negatively regulate gene expression. Although little is yet known about the specific biological functions of most miRNAs, these small molecules are believed to constitute a large gene regulatory network that can impact the expression of up to30%of total cellular proteins. There is increasing data supporting the roles of miRNAs in the regulation of a range of physiological responses, including development, cellular apoptosis, differentiation, proliferation, and cancer. Moreover, recent studies have shown the roles played by miRNAs in different forms of cardiovascular disease and pathological LVH. However, which do microRNAs play important roles in the physiological LVH? Are these microRNAs associated with signaling pathways which induce the physiological LVH? All answers of these are still on the way. The present study hypothesized that swimming exercise alters specific miRNAs that regulate their cardiac signaling genes. It will be help for the development of molecular mechanism of exercise-induced physiological LVH.Purpose:In our research, we determined the molecular mechanism of physiological cardiac hypertrophy via analyzing the expression of miRNAs in physiological LVH rats. The results from this study will provide theoretic foundation for optimal sports program, athlete’s heart health promotion, physical exercise prescription preparation and cardiovascular diseases prevention.Methods:1. For8weeks, from Monday to Friday, the rats in the SE group completed a1-hour swimming exercise in order to build a cardiac hypertrophy model in rats.24hours after last exercise session Blood Pressure (BP) and Heart Rate (HR) were recorded. The hemodynamic parameters of the rats were measured by the tail-cuff method. The rats’heart weights (HW) and left ventricle (LV) were weighed. Left cardiac hypertrophy was assessed by the measurement of the ratio of LV weight to HW in milligrams and HW in milligrams to animal body weight (BW) in grams. The LV was cut in8μm and subsequently stained with hematoxylin and eosin (HE) in order to capture an image of the heart structures;2. The different expression of miRNAs between SC and SE group was analyzed by miRNA array;3. The functions of signaling pathways were analyzed via determining the mRNA and protein expression of the correlated signal molecule that participate in the signaling pathway associated with cell igrowth and proliferation (ERK and PI3K/AKT/mTOR signaling pathway);4. TargetScan software was used to search the miRNAs’target. The expression of miRNA was precisely determined by real time PCR via TaqMan microRNA assay;5. The mRNA expression levels of microRNAs’ targets were determined by RT-PCR, and protein levels were tested by Western-blot.Results:1. After8weeks, the rats’body weight was lighter in the SE group when compared with the SC group, but there was no significant difference (274.8±17.9g vs.267.2±13.4g, P>0.05). The mean blood pressure was decreased in the SE group when compared with the SC group, but there was no significant difference (114.7±6.8mmHg vs.110.4±6.1mmHg, P>0.05). On the other hand, the resting heart rate was significantly decreased in the SE group when compared with the SC group (340.8±11.5bpm vs.298.6±13.2bpm, P<0.05). The HW of the SE group increased after the swimming exercise by38.7%(4.3±0.45mg/g; P<0.05); by comparison, the SC group was lower (3.1±0.16mg/g). Likewise, the LV hypertrophy demonstrated in the SE group was28.6%(2.7±0.13mg/g; P<0.01), again higher than the SC group (2.1±0.09mg/g). The increase in the diameter of the LV myocyte in the SE group (13.6±1.3μm), when compared with the SC group (11.3±1.2μm; P<0.05). All the results demonstrate that physiological LVH models have been built by exercise training;2. After8weeks, the mRNA expression of ANP (1.461±0.271vs.1.350±0.179, P>0.05) and a-actin (1.474±0.358vs.1.317±0.305, P>0.05) was no significant difference between the SC and SE group. Similarly, the ratio of α/β-MHC was also no difference between two groups (P>0.05). All the results show that no pathological changes occur in the LVH rats.3. After8weeks, according to the microRNA array analysis, miR-21, miR-144, miR-145, miR-130a, miR-130b*, miR-1, miR-653*, miR-3120, miR-103-1*, miR-200b, miR-377*, miR-224*, miR-183*, miR-331*, miR-3568, miR-742, miR-758*and let-7a-2*were significantly up-regulated, whereas miR-124, miR-3597, miR-352, miR-181a, miR-152, miR-26b, miR-126*, miR-598-3p, miR-743a, miR-376b-5p, miR-129-2*, miR-204, miR-219-5p, miR-497, miR-142-3p, miR-153*, miR-30c-1*, miR-451, miR-199a-5p, miR-223, miR-181b, miR-664-1*, miR-674-3p, miR-338, miR-146a, miR-322, miR-29b-2*, let-7d, let-7f, let-7a, let-7a-l*/7c-2were down-regulated in the SE group when compared with the SC group;4. After8weeks, the protein expression levels of ERK1/2increased by9%in the SE group when compared with the SC group (0.793±0.095vs.0.864±0.063, P>0.05). phosphoThr202/Tyr204-ERKl/2decreased by5%in the SE group when compared with the SC group (0.812±0.016vs.0.771±0.046, P>0.05). Both AKT1and mTOR expression increased2%in the SE group when compared with the SC group (1.209±0.094vs.1.185±0.114;0.466±0.048vs.0.476±0.023, P>0.05). However, the phosphoser473-AKT and phosphoSer2448-mTOR were respectively increased by46%(0.698±0.037vs.1.019±0.115, P<0.01) and38%(0.362±0.034vs.0.450±0.034, P<0.05) in the SE group when compared with SC group;5. The microarray analysis of miRNA was restricted to those miRNAs that underwent a significant change from the baseline (the ratio of SC/SE was≥2or≤0.5fold). Fig.4A shows the miRNA targeting phosphatase and tensin homolog (PTEN):In the SC group, the relative expression value of miRNA-21was381±21arbitrary units (AU), and the value of miRNA-144was2250±178AU. In the SE group, the values were1150±32(202%increase; P<0.01) and4820±194(114%increase; P<0.01) AU, respectively, in comparison with the SC group. In addition, in the SE group, the swimming exercise increased the relative expression value of miRNA-124, targeting PIK3a. The value was decreased by54%(1015±119AU; P<0.01), when compared with SC (2205±48AU). Finally, the swimming exercise increased miRNA-145, targeting tuberous sclerosis complex2(TSC2, tuberin). This is a116%increase (4555±128AU; P<0.01) over the SC group (2105±161AU);6. To confirm the microRNAs that targeted associated genes in physiological LVH, the miRNAs-21,124,144, and145were quantified by RT-PCR. miRNAs-21,144, and145were respectively up-regulated in the SE group (152%,128%, and101%relative increases over the SC group; P<0.01), whereas miRNA-124was down-regulated in the SE group (38%decrease compared with the SC group; P<0.05). The miRNA expression in the SE group, when compared with the SC group demonstrated the microarray results;7. By the Targetscan software, the target of miRNA-124is PI3K(pll0a), the target of miRNA-21and144is PTEN, and the target of miRNA-145is TSC2;8. After8weeks, the mRNA and protein expression of PI3K(p110α) respectively increased by213%(1.184±0.165vs.3.710±0.780, P<0.01) and36%(0.832±0.057vs.1.132±0.128, P<0.05) in the SE group when compared with the SC group;9. After8weeks, the mRNA and protein expression of PTEN respectively decreased by50%(1.043±0.065vs.0.524±0.118,P<0.05) and37%(1.211±0.109vs.0.763±0.092, P<0.05) in the SE group when compared with the SC group;10. After8weeks, the mRNA and protein expression of TSC2respectively decreased by55%(0.977±0.068vs.0.446±0.099, P<0.05) and22%(0.817±0.042vs.0.637±0.048, P<0.05), but the phosphoThr1462-TSC2increased by48%(0.582±0.017vs.0.861±0.059, P<0.01) in the SE group when compared with the SC group.Conclusion:1. According to previous studies from Femandes et al. and Oliveira et al, the exercise-induced LVH SD rat models were reproduced in this experiment; 2. The results of the swimming exercise of trained rats indicate explicitly that LVH is physiological and is associated with the activation of the PI3K/AKT/mTOR signaling pathway. By contrast, the ERKl/2signaling is unavailable in exercise-induced LVH;3. The expression of microRNAs were different between the SC and SE group;4. These results increase our understanding of the mechanisms of physiological LVH, and imply that a basis for treatment to prevent the development of pathological LVH might be to regulate specific miRNAs, probably through antisense or small interfering RNA, so as to activate PI3K/AKT/mTOR signaling via PI3Kα, PTEN, and TSC2;5. Exercise mediates physiological LVH via PI3K/AKT/mTOR pathway.