Study Of The Cardiomyocytes Hypertrophy Induced By TGF-β₁ And Effects Of Puerarin Intervention In Rat

Left ventricular hypertrophy (LVH)was one of the most important functional lesion of target organ(TO)about hypertension. 30% hypertension patients were complicated by LVH and 17% patients died of coronary heart disease(CHD)or heart failure (HF) coherented with hypertension. LVH was the independent risk factor for cardiovascular events, and for this reason retroconversion of LVH may became one of the end-all targets for hypertension therapy. The main pathological change of LVH was cardiomyocytes hypertrophy. Although the characters and mechanisms were thinked highly of people there was still lack of deeply learning about cardiomyocytes hypertrophy development. The study of the mechanisms about signal transduction intra-cellular may be helpful to investigate pathogenesy of cardiomyocytes hypertrophy to move forward a signle step.Transtorming growth factor beta 1(TGF-β1) was one of the significant cell factors who regulated growth with various functions. TGF-β1 has been predicted to contribute to the pathologies of LVH,CHD and HF after myocardial infarction(MI). Angiotensin II receptor (ATR II) was reported to stimulate paracrine and autocrine about TGF-β1 that a wide array of regulater for proliferation and hypertrophy of cardiac fibroblasts( CFs ),hyperplasy of extracellular matrix ( ECM ) and cardiomyocytes hypertrophy.The specific mechanisms of the regulation about TGF-β1 and TGF/Smad (Drosophlia mothers against dpp and Celegans Sma) for cardiomyocytes were still unknown. In our study cardiomyocytes hypertrophy were induced by TGF-β1 in rat to research mechanisms about cardiomyocytes hypertrophy though detecting cardiomyocytes hypertrophy and apoptosis, expressions of Smads, changes in p15 and c-myc. Intervention of puerarin was also observated to supply exprement data for drug prevention and cure. The experiments contained there parts as below.Part One: Primary culture and identify of cardiomyocytes in rat ObjectiveTo construct the cardiomyocytes primary culture model in rat.MethodsCardiomyocytes were isolated by trypsin digestion and differential attachment methods from neonatal Sprague Dawley (SD) rats, identified by inverted microscope and transmission electron microscope (TEM) observation.ResultsObserved with inverted microscope, primary cardiomyocytes appeared irregularity triangle and polygonm, and the most part of cardiomyocytes spontaneous beated rhythmically. Observed with TEM, cardiomyocytes were plentiful with mitochondria, and actin filaments were lined up in order.ConclusionIt was succeed to primary culture neonatal SD rat cardiomyocytes by trypsin digestion and differential attachment methods.Part Two: Cardiomyocytes hypertrophy and apoptosis induced by TGF-β1 to observe Smads pathway and oncogene protein expressionsObjectivesTo observe the hypertrophy and apoptosis of cardiomyocytes induced by TGF-β1 and determine the expression of Smads pathway in neonatal SD rat catdiomyocytes, and the expressions of oncogene protein p15 and c-myc were also detected. MethodsCardiomyocytes were stimulated by TGF-β1 with different concentrations (0.1μg/L,1μg/L,3μg/L) , and TGF-β1(3μg/L) stimulated cardiomyocytes for different periods of 15min,30min,1h,2h and 24h. Velocity of cardiomyocytes protein were observed by 3H-Leu incorporation, and Propidium iodide (PI)staining was used to assayed RNA contents. Apoptosis were assayed by flow cytometry (FCM) and FragEL staining. The hypertrophic response was assayed by measuring the expressions of ANF,α-MHC andβ-MHCmRNA detected by real-time PCR. Expressions of hypertrophy and apoptosis were detected by TEM. The Western-blot was performed to detected protein expressions of Smad2,pSmad2,Smad2/3,p15 and c-myc. RNAi was used to be a specific inhibitor to Smad2 as a control. Cardiomyocytes in Smad2siRNA control group were essayed by 3H-Leu incorporation,real-time PCR and western-blot.Results1. The effects of TGF-β1 on cardiomyocytes3H-Leu incorporation in cardiomyocytes Stimulated with TGF-β1 for 24h, to compare with the black control, the rate of protein synthesis in cardiomyocytes was elevated significantly in concentration-dependent manner (P﹤0.01). While in the Smad2siRNA control group the rate of protein synthesis was decreased than TGF-β1group(P﹤0.01).RNA contents detected by PI staining Fluorescence intensity of PI in TGF-β1 group was obviously higher than control group (P﹤0.01).Embryon gene detected by real-time PCR Expression Levels of ANFmRNA andβ-MHCmRNA were higher in TGF-β1 group and elevated significantly in concentration-dependent manner (P﹤0.01).Apoptosis detected by TdT-FragEL staining Rates of apoptosis in cardiomyocytes induced by TGF-β1were improved(P<0.01).FCM assaied apoptosis Changes of cardiomyocytes apoptosis induced by TGF-β1 was associated with upregulation of Caspase-3(P<0.01).TEM observed cardiomyocytes hypertrophy and apoptosis Cardiomyocytes in control group were plenty of cytomicrosome. Cardiomyocytes in TGF-β1 group showed hypertrophy with increase of actin filament and tumefaction of cytomicrosome, and a part of cardiomyocytes in TGF-β1 group also expressed apoptosis by concentrate of caryon and vacuoled of cytomicrosome. Hypertrophy and apoptosis apperanced in TGF-β1 group cardiomyocytes at the same time.2. TGF-β1 activates Smads signalSmad2 mRNA levels Smad2 mRNA level was higher than black control group in cardiomyocytes induced by TGF-β1 and the Smad2 mRNA level was also efficiently downregulated by RNAi to Smad2(P<0.01).Smads proteins TGF-β1 induced early activation of pSmad2 and Smad2/3 at 30 minutes and max expression at 2h, and TGF-β1 induced significant pSmad2 expressions until 24 hours(P<0.01). Expressions of Smad2 were not as obviously as pSmad2 ( P<0.05 ) . Smad2 phosphorylation was dowenregulated efficiently in Smad2siRNA control group(P<0.01)3. Oncogene p15 and c-myc TGF-β1 induced the early activation of c-myc and late activation of p15 in cardiomyocytes compared with control group (P<0.01). In Smad2siRNA control group p15 was inhibited alone with pSmad2(P<0.01).Conclusions1. TGF-β1 induced cardiomyocytes hypertrophy and apoptosis at the same time.2. TGF-β1 activatied early and late Smad2 activation and upregulation to induced cardiomyocytes hypertrophy.3. TGF-β1 also activated ealy upregulation of c-myc and late upregulation of p15. C-myc and p15 expressions maybe assiocatied with cardiomyocytes hypertrophy. p15 could be the downstream signaling molecule of Smads pathway.Part Three: Study of puerarin intervetion on hypertrophic cardiomyocytes induced by TGF-β1ObjectiveTo investigate the effects of puerain intervention on hypertrophic cardiomyocytes induced by TGF-β1 and obveried the inhibition of puerarin to phosphorylation of Smad2 that was compared with Smad2siRNA intervention. MethodsPuerarin(0.1g/L,1g/L,5g/L) with TGF-β1(3μg/L ) co-stimulated or Smad2siRNA with TGF-β1(3μg/L) co-stimulated for 2h and 24h for assessing protein synthesis rate, ANF,β-MHC andα-MHCmRNA expression, pSmad2, Smad2, p15 and c-myc proteins changes in cardiomyocyte.The methods of 3H-Leu incorporation, real time PCR, FCM and Western-blot were used.Results1. Hypertrophy induced by TGF-β1 were obviousily changed by intervention of puerain and Smad2siRNA. Compared with TGF-β1 group, the protein synthesis rate in cardiomyocytes was downregulated significantly in puerain group than in siRNA group (P<0.01).2. It was shown that levels of ANFmRNA andβ-MHCmRNA were downregulated by puerain and Smad2siRNA especialiy by puerain (P<0.01).3. Puerarin(1g/L) with TGF-β1(3μg/L ) co-stimulated cardiomyocytes were able to decrease apoptosis rate compared with TGF-β1(3μg/L) group (P<0.01). Level of Caspase-3 was also downregulated (P<0.01).4. PSmad2 protein was significantly downregulated by puerarin. p15 protein was inhibited by puerarin intervention. And Smad2siRNA intervention could also inhibited pSmad2 and p15 protein expression(P<0.01).Conclusions1. Puerarin was able to amendment cardiomyocytes hypertrophy and apoptosis induced by TGF-β1 in rat.2. Hypertrophy of cardiomyocytes was depressed by puerain with marked downregulation of Smad2 phosphorylation. Intervention of puerarin could drcreased p15 protein expressions in cardiomyocytes.3. Compared with Smad2siRNA intervention, puerarin intervention had preferable effect.