The Role Of TGF-β1/Smad Signaling Pathway In The Development Of Exercise-Induced Myocardial Fibrosis

Objective:Exercise-induced arrhythmia has always been the highlight in sports science and medicine field. It is partly due to pathological injury of the heart caused by long-term repeated intensive exercise training. It often has adverse effects on athelteˊs health, systematic training and cometition. In recent years, some studies found that repeated high intensity exercise myocardial fibrosis damage mybe related to the occurrence of exercise-induced cardiac arrhythmia. While it remains unclear the relationship between extent of cardiac fibrosis and exercise duration or cardia chamber. TGF-β1/Smad signaling pathway is the key to fibrosis, whether it participates in the regulation of exercise-induced myocardial fibrosis, has not been reported. Therefore, through establishment of long-term extensive exercise training animal model, our reserch will explore the role of TGF-β1/Smad pathway and its downsteam factor in the process.The first part: The effects of long-term exercise on cardiac damage and fibrosis biomarker of rats.Methods:72 SD rats were divided into 3 groups, the control group(C), modrate intensity(M) and high intensity group(H). Each group is divided into 8-week, 12-week and 16-week group, running for 8 weeks, 12 weeks and 16 weeks respectively. M Group runed at speed of 15m/min, slope of 5°(equivalent to 58.4 +/-1.7%VO2max), H group runned at speed of 28 m/min, slope of 10°(the equivalent of 81.0%+/-3.5% VO2max) for 1 hour every day, 5 days per week. Myocardial structure and function were detected by small animal ultrasound cariograph, c Tn I were analysed by Elisa. Cell ultrastructure histomorpholoy were observed by transmission electron microscopy and HE staining. Collagen content were detected by hydrolysis. Sirius red staining were used to observe the distribution of collagen in heart, then CVF were calculated. Immunofluorescence histochemical and RT-PCR to detect the distribution and content of type I and III collagen. Degree of Cardiac damage, fibrosis and collagen content were observed along with exercise duration and intensity. α-SMA distribution and expression were also detcteted by Immunofluorescence histochemical and Western Blot.Results:(1) Body weight decreased, while HWI increased gradully with the time of exercise in Both M and H group.(2) Left venricle srtucture change is not obvious after 8-weeks exercise. LVEF of M group increased significantly, but not in H group. RVDs and RVDs of right ventricle have marked increase but not RVFS and RVEF. LVDd and LVDs was augmented remarkably in both the group, while H group had a greater range after 16-week exercise. Both LVEF and LVFS reduced in H group. RVDs and RVDd had varying increase. RVEF reduced significantly in H group but not M group.(3) c Tn I increased significantly in H group but not M group after 8/12/16 weeks exercise.(4) Transmission electron microscope indicated increased mitochondria in M and H group at all weeks. Muscle fiber fracture and discontinuous intercalated disc was found in H group but M group. HE staining indicated cardiac damage in H group, and the right ventricle damage seriously than left ventricle.(5) Hydroxyproline and CVF in atrium and right ventricle increased with entensive exercise duration, but not left ventricle. Hydroxyproline and CVF of M group had also mild increase, but no significance.(6) Col-I had a remarkbable increase in myocardium, while the degree of increase in atrium and right ventricle was great than left ventricle.(7) Col-III m RNA and protein in both group increased with exercise duration. There was significant increase in atium and both ventricle.(8) Ratio of Col-I/Col-III in atium and right ventricle of H group had markedly increase at 16 weeks, but not in left ventricle. There was mild increase in M group without significant difference.(9) α-SMA has not been found in all group at different exercise duration.Conclusions: Long-term moderate exercise promoted biventricle dilatation and assisted cardiac ejection, which can be contrubuted to modest increase of collagen. While long-term intensive exercise induced cardiac dysfunction, myocardial cellula damage, major in atrium and right ventricle. Myocardial fibrosis mainly occurring in atrium and right ventricle due to disproportionality of Col-I/Col-III maybe mechnism of cardial dysfuntion and arrhythmia. But this could remain in early stage of myocardial fibrosis, because of no evident of cardiac fibroblasts.The second part: Regulation by TGF-beta1/Smad signaling pathway in exercise-induced myocardial fibrosisMethods: RT-PCR and Western Blot to measure fibrosis key factor(TGF-beta1), pathway factors(Smd-2, Smad-3, Smad-4, Smad-7), downstream collagen degradation factor(MMP-1, TIMP-1, MMP-2, TIMP-2) and factors of collagen synthesis(CTGF and micro RNA-21) in C and H group.Results:(1) TGF-beta1 of Group H in atrial, right ventricular and left ventricle significantly increased after 8 weeks. TGF-beta1 of Group H in atrial and right ventricle significantly increased after 12 weeks and 16 weeks but not left ventricle. TGF-beta1 of Group H reduced gradully with time.(2) Extensive exercise had no effects on Smad-2 and Smad-3.(3) Smad-4 increased while Smad-7 declined gradully with time. Both Smad-4 and Smad-7 had remarkable change at 16 week. Degree of change atrium and right ventricle is great than left ventricle.(4) There was notable alteration of MMP-1. TIMP-1 of Group H in atrial and right ventricle significantly increased after 12 weeks and 16 weeks but not left ventricle. TIMP increased while MMP-1 declined gradully with time in H group.(5) MMP-2 of group H increased gradully with exercise duration. MMP-2 but TIMP-2, ratio of MMP-2/TIMP-2, increased significantly after 16 exercise. The degree of increase of atrium and right ventricle is great than left venticle.(6) CTGF increased with exercise duration but no significance at 8/12 week. CTGF of group H increased significantly in atrium but biventricle.(7) Mir-21 of group H increased significantly after 8/12/16 weeks. The degree of increase of atrium and right ventricle is great than left venticle.Conclusions:(1) Incease of TGF-beta1 caused by long-term intensive exercise, induced imbalance of MMP-1/TIMP-1 and MMP-2/TIMP-2, increase of CTGF and mi R-21. This may caused disorder of collagen distribution as well as increase of callagen synthesis, inducing accumulation in myocardium, thus maybe the molecular mechanism of cardiac funtion and arrhythmia. Degree of change of TGF-beta1/Smad signaling pathway and downstream factors in atrium and right ventricle is greater than left ventricle, this may be mechanism that atrium and right ventricle had more serious damage and more susceptible to myocardial fibrosis.