The Experimental Study On The Role Of Signal Transduction Of TGF-β₁/Smad3 During Cardiomyocyte Ischemic Preconditioning

Members of the TGF-βsuperfamily,comprising the TGF-β,activin and BMP family, are the classical activators of Smads proteins.They participate in a wide range of processes, from tissue differentiation during development through to regulation of mesenchymal and immune cell functions.In recent years,elevated expression of proteins of the transcription factor family Smads was found under several pathophysiological situations in the heart,i.e. after myocardial infarction or in diverse forms of cardiomyopathy.Smads proteins are described to have different effects on heart development,cell proliferation,cell growth, and apoptosis.These different consequences of Smads activation are dependent on different Smad isoforms,interaction of Smads with other transcription factors in the particular situation,and modulation of Smads activity by various kinases.Some studies showed that BMP/Smad1 Protects Cardiomyocytes from Ischemia-Reperfusion Injury.But the study on TGF-β/Smads in the process of Ischemic preconditioning and Ischemia-Reperfusion Injury was very few.Studies on mechanisms of ischemia-reperfusion injury and myocardial protection are the spotlight in present day.In order to fight for cardiomyocytes ischemic necrosis and protect myocardium,lots of medicine was studied for myocardial protection(for example,β-antagonist,free-radical scavenger,calcium antagonist).The studies on cardioprotection of ischemic preconditioning were always the most fascinating.Ischemic preconditioning may lead to a reduction in infarct size,enhance the heart function,protect the ultrastructure of myocardium and reduce the arrhythmia after Ischemia-Reperfusion Injury.But the mechanisms of these protective effects remain unclear.During recent years,the mechanisms of ischemic preconditioning have focused on mito-KATP channels,PKC and NF-κB,ect.In our experiments cultured neonatal rat cardiomyocytes were performed to prepared ischemia-reperfusion injury and ischemic preconditioning model in vitro,and were interfered with TGF-β₁.TUNEL,LDH detection,fluorescence staining of DAPI and flow cytometry were performed to identify the apoptosis of cardiaomyocytes during ischemia-reperfusion injury,and ELISA was performed to detect the Smad3.The main methods and results are described as follows: 1.Neonatal rat cardiomyocytes culture and ischcmia-reperfusion and ischemic preconditioning model preparation1.1 Neonatal rat cardiomyocytes cultureNeonatal rat cardiomyocytes were digested according the Simpson's method and cultured by a selective attached technique.The purity of cardiomyocytes was over 90%. The synchronous pulsation was seen after 4 days.1.2 ischemia-reperfusion and ischemic preconditioning model preparationCardiomyocytes subjected to hypoxic substrate-free solution containing elevated potassium,acidosis,lactate accumulation and reinstated with normal culture solutions to mimic Ischemia-Reperfusion process.At the same time,TGF-β₁ was added in the culture solution.1.3 To identify the efficacy of the ischemia-reperfusion and ischemic preconditioning modelThe method to identify the efficacy of the ischemia-reperfusion and ischemic preconditioning model is to study the ratio of apoptosis.The ratio of Control group is 5.92±1.88%,the ratio of IR group is 28.99±6.96%,the ratio of IPC group is 13.85±1.40%, and there is significient deference among these groups.2.The signal transduction of TGF-β₁/Smad3 during ischemia-reperfusion injury and ischemic preconditioning.The myocardiocytes of neonatal SD rats were randomized into 6 groups:Group A(Control group):The cells were cultured with modified DMEM with 10% FBS in a modified chamber and mixture gas(95%air.5%CO₂) for 48h,then change the culture solution and continue culturing for 3h;Group B(Ischemia-Reperfusion Injury group):The cells were cultured with low carbohydrates DMEM under the condition of hypoxia(95%N₂ and 5%CO₂;the O₂ partial pressure was lower than 5 mmHg) for 48h,then cultured with modified DMEM with 10% FBS in a modified chamber and mixture gas(95%air,5%CO₂) for 3h;Group C(Ischemia-Reperfusion Injury and TGF-β₁ group):TGF-β₁(5ng/ml) was put in DMEM,the others are the same to B group;Group D(Ischemia-Reperfusion Injury and Smad3 inhibitor group):LY364947 (60nmol/L) were put in DMEM,the others are the same to B group; Group E(Ischemic Preconditioning group):The cells were cultured with low carbohydrates DMEM under the condition of hypoxia(95%N₂ and 5%CO₂;the O₂ partial pressure was lower than 5 mmHg) for 6h,then cultured with modified DMEM with 10% FBS in a modified chamber and mixture gas(95%air,5%CO₂) for 3h,then the same to B group;Group F(ischemic preconditioning and TGF-β₁ group):TGF-β₁(5ng/ml) was put in DMEM,the others are the same to E group.2.1 Detection of cardiac myocyte apoptosis2.1.1 In situ terminal deoxynucleotidy transferase(TdT) labeling(TUNEL)TUNEL positive apoptotic cells could be detected in all groups,and the radio of positive cells in group C(54.60±8.49%) was significantly higher compared to the other groups,group D(20.48±1.94%) is less than group B(31.10±4.21%);group E(12.21±0.92%) and group F(14.16±1.66%) have no significant difference;group A(5.24±0.99%) is least.2.1.2 Cardiac myocyte apoptosis index were determined by flow cytometry with Annexin-V and propidinm iodiode(PI) stainingThe radio of positive cells in group C(54.19±11.86%) was significantly higher compared to the other groups,group D(21.31±3.78%) is less than group B(30.75±4.88%), group E(12.45±1.52%) and group F(14.10±1.78%) have no significant difference; group A(5.50±1.38%) is least.2.1.3 LDH detectionKeep the culture solution in refrigerator of 4℃for detection,and the LDH will identify the ratios of cardiomyocyte apoptosis.Group C(121.94±12.98U/L) was significantly higher compared to the other groups,group D(39.73±3.23 U/L) is less than group B(52.98±4.94 U/L);group E(14.80±2.49 U/L) and group F(15.70±1.29%) have no significant difference;group A(6.59±1.45 U/L) is least.2.2 detection of the Smad3 that induced by TGF-β₁ during Ischemia-Reperfusion Injury and Ischemic PreconditioningKeep the culture solution in refrigerator of 4℃and collect the cell by mechanical method.Add the cell to the collected culture solution and split the cell with ultrasound and detect the Smad3 with ELISA method.Group A:21.77±2.32 ng/ml,Group B:35.7±2.43 ng/ml,Group C:47.51±4.60 ng/ml,Group D:25.66±1.51ng/ml,Group E:29.47±1.36 ng/ml,Group F:34.12±1.90 ng/ml.There are singnal deference between group C and the other groups,but there is no significant deference between group B and group D.There is no significant deference between group E and group F.There is no significant deference between group E and group D.Conclusion:In our experiments cultured neonatal rat cardiomyocytes were performed to prepared ischemia-reperfusion injury and ischemic preconditioning model in vitro,and were interfered with TGF-β₁.The cardiomyocytes apoptosis and Smad3 were detected with several methods,by which we can study the signal transduction of TGF-β₁/Smad3 during ischemic preconditioning.1.The purity of cardiomyocytes that were digested according the Simpson's method and cultured by a selective attached technique is over 90%.2.TGF-β₁ may induce the Smad3 and increase the cardiomyocytes apoptosis during Ischemia-Reperfusion injury.3.Ischemic Preconditioning may prevent the cardiomyocytes apoptosis during Ischemia-Reperfusion injury by inhibit the Smad3.