Elevated Expression Of VEGFA-AKT-MafG Signaling Promotes Myocardial Angiogenesis In Mice By Exercise

Purposes:To establish a mouse model of exercise-induced cardiac hypertrophy and a VEGFA-stimulated mouse cardiac microvascular endothelial cell（MCMEC）model to analyze the role and molecular mechanism of VEGFA-ERK-MafG signaling in promoting angiogenesis in exercise-induced cardiac hypertrophy.Methods:Forty healthy 7-week-old male C57/BL6 mice were randomly divided into control group（CON group,n=16）and swimming exercise group（EX group,n=24）.Swimming exercise training.Through two-dimensional ultrasound detection,left ventricular coefficient（left ventricular weight/body weight,LW/BW）,cardiac coefficient（full heart weight/body weight,HW/BW）,hematoxylin and eosin（HE staining）,combined with mouse myocardial pathological markers The expression of these substances was used to comprehensively evaluate the establishment of an animal model of exercise-induced cardiac hypertrophy.By using CD31 immunohistochemical staining on left ventricular tissue,the number of new microvessels in mouse myocardial tissue was counted,and the angiogenesis in mice was evaluated.The m RNA expressions of VEGFA and VEGFR2 in the left ventricle of mice were detected by real-time quantitative PCR（RT-q PCR）,and the proteins of VEGFA,VEGFR2,and P-VEGFR2（Tyr1175）in the left ventricle of mice were detected by Western Blot.To determine whether the VEGFA/VEGFR2 signaling pathway was activated in the EX group.MCMECs were stimulated with VEGFA at a concentration of 50ng/ml,and the cells were collected at 0,4,and 12 hours,respectively,and the stimulation effect of VEGFA was verified by cell scratch experiments.Tyr1175),AKT,ERK,P-AKT,P-ERK,Nrf2 and MafG protein expression were detected.Results:（1）8-week swimming endurance training successfully induced exercise-induced myocardial hypertrophy in mice.After 8 weeks of swimming endurance training,the cardiac coefficient（full heart weight/body weight）in the EX group was significantly increased compared with the CON group（P<0.05）;the left ventricular coefficient（left ventricular weight/body weight）was significantly increased（P<0.01）.The myocardial volume of the cardiomyocytes in the EX group was increased,the structure was normal,the myocardial fibers were obviously thickened,and the diameter of the cardiomyocytes was significantly increased（P<0.01）.The indexes of ventricular septal thickness（VS）and ejection fraction（EF）in EX group were significantly higher than those in CON group（P<0.05）,indicating that the cardiac function of mice in EX group was significantly enhanced.The m RNA expressions of ANP,BNP,α-MHC,β-MHC and α-actin in left ventricular tissue had no significant difference between the two groups（p>0.05）,indicating that the mice did not have pathological cardiac hypertrophy.The above results indicate that the 8-week swimming exercise training program implemented in this study successfully induced exercise-induced myocardial hypertrophy in mice.The left ventricle of exercise-induced myocardial hypertrophy mice showed angiogenesis and the VEGFA/VEGFR2 pathway was activated.After 8 weeks of swimming exercise training,the number of left ventricular microvessels in the EX group was significantly higher than that in the CON group（P<0.001）,indicating angiogenesis in the exercise-induced myocardial hypertrophy mice.The m RNA expressions of VEGFA and VEGFR2 in EX group were significantly higher than those in CON group（P<0.001,P<0.01）.The levels of VEGFA and P-VEGFR2（Tyr1175）/VEGFR2 in the left ventricle of the EX group were significantly higher than those of the CON group（P<0.01,P<0.01）,indicating that the VEGFA/VEGFR2 signaling pathway was activated.（2）VEGFA/VEGFR2/P-AKT signaling pathway was successfully activated in MCMEC.Under the stimulation of VEGFA,P-VEGFR2（Tyr1175）/VEGFR2 and P-AKT/AKT in MCMEC were significantly increased at 4h and 12h（P<0.01）,indicating that the VEGFA/VEGFR2/P-ERK signaling pathway was successfully activated.However,the expression of P-ERK/ERK did not increase under the stimulation of VEGFA（P>0.05）,indicating that this signaling pathway was not activated.Under the stimulation of VEGFA,the protein expression levels of Nrf2 and MafG were significantly increased at 4h（P<0.05）,and the protein expression level of Nrf2 was significantly increased after 12h（P<0.01）,indicating that VEGFA has a significant effect on Nrf2,MafG protein has obvious activation effect.Conclusions:（1）8-week swimming exercise training can induce exercise-induced myocardial hypertrophy in mice,increase the expression of VEGFA in the mouse myocardium,and cause angiogenesis in the left ventricle of mice.（2）The results of MCMEC in vitro experiments showed that the VEGFA/VEGFR2 signaling pathway can increase the expression of Nrf2 and MafG through the activation of VEGFR2 phosphorylation site Tyr1175,thereby inducing exercise-induced cardiac hypertrophy.