| Background:Myocardial infarction(MI)causes hypoxia in myocardium,leads to numbers of cardiomyocytes apoptosis or necrosis and cardiac fibrosis,which would induce heart dysfunction and heart failure(HF).Recombined human granulocyte cell-stimulating factor(rhG-CSF)is an effective bone marrow cell(BMCs)mobilization reagent.BMCs could be mobilized into blood circulation by rhG-CSF,and home to infarction region,differentiate into functional cardiomyocytes to repair the injured heart.Interval exercise is an effective method of cardiac rehabilitation,but its mechanism is still not well known.It has no report about whether interval exercise can mobilize endogenous BMCs into blood and induce them homing to the infarcted heart,improve cardiac regeneration.Fibroblast growth factor 1(FGF1)-PI3K-AKT plays a very important role in cell proliferation.However,whether interval exercise with or without rhG-CSF treatment could activate theFGF1-PI3K-AKT signaling and promote cardiomyocyte proliferation after MI are still not clear.In this study,rats were used to establish the MI model and subjected to interval exercise with or without rhG-CSF injection,or CXCR4 antagonist AMD3100 or exogenous recombinant rat FGF1 injection.The heart function,cardiac morphology,cardiomyocyte calcium transients,proliferation and apoptosis,and related molecule mechanism were detected.The aim of this study was to study the effect and possible mechanism of interval exercise with or without rhG-CSF treatment on improving cardiomyocyte proliferation in rats with MI.Methods:3-month old Sprague-Dawley rats were used to establish MI model by ligation of the left anterior descending coronary artery,and randomly divided into 7 groups in part 2 and part3:Sham group(Sham),MI group(MI),MI with interval exercise group(ME),MI with rhG-CSF treatment group(MG),MI with rhG-CSF treatment and interval exercise group(MGE),MI with AMD3100 group(MA)and MI with AMD3100 and interval exercise group(MAE),12 rats in each group.In part 4,we have two experimental model:First,rats were divided into Sham with solvent group(SV),Sham with recombinant rat FGF1 group(SF),MI with solvent group(MV)and MI with recombinant rat FGF1 group(MF);The other,rats were divided into Sham,MI,ME,MG and MGE,12 rats in each group.The rats in MG and MGE groups were injected with rhG-CSF(physiological saline solution diluted)by subcutaneous injection,10 ?g/kg/d×5d.The rats in MA and MAE groups were injected with AMD3100 intraperitoneally,200 ?g/kg/d×5d.The rats in Sham,MI and ME groups were injected the same volume of physiological saline solution as control.The rats in SF and MF groups were administered with recombinant rat FGF1,2ml/kg/d×7d,and the rats in SV and MV groups were injected with solvent as control.Rats in ME,MGE and MAE groups were subjected to interval exercise.The training started at the 8th day after surgery.First,rats were warmed up at 10m/min×10min on the treadmill.Then,25m/min×8min and 15m/min×2min.Repeat it for four times.Finally,25m/min×8min and 10m/min×2min and terminated the training.1h/d,5d/wk×3wk.Peripheral blood mononuclear cells were isolated by reagent kit.Flow cytometry were used to detect the level of c-kit+,CD29+ and CD34+ cells;Cardiac function was detected by color doppler ultrasonic diagnostic instrument and hemodynamic measurement;Individual cardiomyocytes were isolated and the calcium transients and systolic function were detected by single-celled detection system(IonOptix);TTC staining were used to determine the infarcted area;PCNA+ and cTnT+ cardiomyocytes were counted by immunofluorescence staining to analyze cardiomyocyte proliferation;Cardiac transcription factors GATA4 and NKX2-5 gene expression was measured by reverse transcription polymerase chain reaction(RT-PCR),and HIF-1,SDF-1,c-kit,CD29,CD34,Bcl-2,BAX,Caspase 9,FGF1,PI3K,P-PI3K,AKT,P-AKT,P70 S6K,P-P70 S6K,cyclin B1,and cyclinD1 were detected by Western Blot.Results:1.Interval exercise with or without rhG-CSF treatment could stimulate BMCs mobilization in MI rats.(1)MI and rhG-CSF could enhance the level of c-kit+,CD29+ and CD34+mononuclear cells in peripheral blood,which could be reduced by AMD3100.Interval exercise after MI could increase the number of c-kit+,CD29+ and CD34+ mononuclear cells in peripheral blood,and combined with rhG-CSF gave a better effect.These results indicated that interval exercise with or without rhG-CSF treatment can significantly mobilize BMCs into the circulation system.(2)MI and rhG-CSF could up-regulate the expression of HIF-la and SDF-1 in the peri-infarcted area of rats,which could be reduced by AMD3100.Interval exercise after MI could increase the expression of HIF-1? and SDF-1,and combined with rhG-CSF gave a better effect.These results indicated that interval exercise with or without rhG-CSF treatment could improve the chemotaxis capability of BMSCs after MI.(3)MI and rhG-CSF could up-regulate the protein expression of c-kit,CD34 and CD29 in the peri-infarcted area of rats,which could be inhibit by AMD3100.Interval exercise after MI could increase the expression of c-kit,CD34 and CD29,and combined with rhG-CSF gave a better effect.These results indicated that interval exercise with or without rhG-CSF treatment could stimulate BMSCs homing to infarted heart.2.Interval exercise with or without rhG-CSF treatment could stimulate cardiomyocyte proliferation and improve cardiac function in MI rats.(4)Interval exercise and rhG-CSF treatment could increase PCNA+cTnT+ cells significantly in MI rats.The effect was weakened by AMD3100.Combined use of interval exercise and rhG-CSF had a better effect.(5)Interval exercise could reduce infarcted size in MI rats.The effect was weakened by AMD3100.Combined use of interval exercise and rhG-CSF had a better effect.(6)Interval exercise could increase the amplitude of calcium transient and intracellular Ca2+ concentration,decrease the time from peak value to 50%baseline and the time from the baseline to 50%peak;the velocity of monocardiomyocyte contracting increased in ME and MGE group rats significantly contrast to that of MI group rats.It also increased the systolic and diastolic function of cardiomyocytes in the peri-infarct region in MI rats.The effect was weakened by AMD3100.Combined use of interval exercise and rhG-CSF had a better effect.3.Interval exercise with or without rhG-CSF treatment could activateFGFl-PI3K-AKT signaling pathway,improve cardiomyocyte prolifer-ation and cardiac function in MI rats.(7)rhG-CSF could increase the level of FGF1 in peripheral blood and myocardium,and increase the number of PCNA+ cTnT+ cells,reduce the activation of caspase3 in the peri-infarcted area.(8)Interval exercise with or without rhG-CSF treatment could down-regulate the protein expression of p-caspase 9 and BAX,up-regulate the Bcl-2 expression and the ratio of Bcl-2/BAX,reduced cell apoptosis.(9)Interval exercise with or without rhG-CSF treatment could up-regulate the protein expression of FGF1,FGFR,mTOR,p-AKT,p-PI3K P85,p-P70S6K,cyclin B1 and Cyclin D1 and the mRNA expression of GATA4 and NKX2-5 in the peti-infarcted area of MI rats,and combined use of interval exercise and rhG-CSF had a better effect.Interval exercise could activate theFGF1-PI3K-AKT signal pathway,improve cardiomyocyte proliferation and combined use of interval exercise and rhG-CSF had a better effect.(10)Interval exercise had a similar effect as rhG-CSF on promoting the mRNA expression of NKX2-5 and GATA4,regenerating the infarcted heart,decreasing cardiomyocyte apoptosis,activatingFGF1-PI3K-AKT signal pathway and promoting cardiomyocyte proliferation in MI rats.Conclusions:1.Interval exercise with or without rhG-CSF treatment could promote BMCs mobilization to the circulation system and home to the infarct zone and peri-infarct region under the function of SDF-1 in MI rats.Combined use of interval exercise and G-CSF had a better effect;2.Interval exercise with or without rhG-CSF treatment could promote cardiomyocyte proliferation and decrease cardiomyocyte apoptosis,improve cardiomyocyte systolic and diastolic function and calcium transients,and promote cardiac function in MI rats;3.Exogenous injection of recombinant rat FGF1 could increase the FGF1 level in circulation system and myocardium,improve cardiomyocyte proliferation and inhibit cell apoptosis;4.FGF1-PI3K-AKT signal pathway was compensatory activated by MI;Interval exercise could activateFGF1-PI3K-AKT signal pathway and promote cardiomyocyte proliferation.Combined use of interval exercise and G-CSF had a better effect.These results indicated thatFGFl-PI3K-AKT signal pathway plays an important role in the improvement of cardiomyocyte proliferation and cardiac function by interval exercise and rhG-CSF treatment. |
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