| Part 1 Expressions of TRPCs in pressure overload-inducedmyocardial fibrosis in ratsObjective: To investigate the expressions of TRPCs in pressure overload-inducedmyocardial fibrosis in rats.Methods: Myocardial fibrosis was induced by coarctaion of abdominal aorta (CAA) inSprague-Dawley rats. The rats were divided into 2 groups: The sham-oprerated group andCAA group.Blood pressure, Left ventricular mass index (LVMI), cardiac collegen volumefraction (CVF) and the expression of TRPC channels were examined after 4 weeks ofoperation.Results: Compared with sham-operated group, blood pressure, LVMI, CVF andexpressions of TRPC1 mRNA and protein were increased remarkably in CAA group.However, TRPC3, TRPC6 mRNA and protein expressions did not changed obviously intwo groups.Conclusion: The expression of TRPC1 was significantly upregulated in pressureoverload-induced myoardial fibrosis in rats, which possible played important role inpressure overload-induced myocardial fibrosis in rats. Part 2 The role of TRPC1 in cardiac fibroblasts proliferationand the expression of TGF-β1 induced by AngⅡObjective To investigate the role of TRPC1 in cardiac fibroblasts proliferation and theexpression of transforming growth factor beta 1(TGF-β1) induced by AngⅡ.Methods Cardiac fibroblasts were prepared from the ventricles of 1-3-day-oldSprague-Dawley rats. The cells were treated with various concentration of angiotensinⅡ(10-7,10-6, 10-5mol·L-1) for 24h, or treated with angiotensinⅡ(10-5mol·L-1) for 0, 12 and 24h,respectively. The expressions of TRPC1 and TGF-β1 were determined by real-time quantitativepolymerase chain reaction and western blot. Under high concentration of angiotensinⅡ(10-5mol·L-1), cardiac fibroblasts were treated with the inhibitors of the calcium for 24 hours,including SKF96365 (an inhibitor of TRPC1), thapsigargin (an inhibitor of Ca2+-ATP) andverapamil (an inhibitor of L-Ca2+ channel). TGF-β1 was determined by real-time quantitativepolymerase chain reaction and western blot. In addition, cells were pretreated with SKF96365or thapsigargin for 30 mins, and then treated with angiotensinⅡ(10-5mol·L-1), the intracellularCa2+ was analyzed by confocal laser microscopy.Results The expressions of TRPC1 mRNA and protein increased when treated withangiotensinⅡin a concentration- and time-dependent manner. SKF96365 and thapsigarginsignificantly inhibited fibroblasts proliferation and the TGF-β1 expression in cardiacfibroblasts induced by AngⅡ(10-5mol·L-1) for 24 hours. Verapamil also inhibitedfibroblasts proliferation (P＜0.01), but has no effect on TGF-β1 expression (P＞0.05).AngiotensinⅡ(10-5mol·L-1) induced an increase in intracellular Ca2+. And pretreatmentwith thapsigargin for 30 minutes, the intracellular Ca2+ sustained increasing by AngⅡinthe presence of external Ca2+ and only a mild and transient rise initiated by AngⅡin theabsence of external Ca2+ in cardiac fibroblasts. And pretreatment with SKF96365 for 30mins, the intracellular Ca2+ concentration did not increased by AngⅡin cardiac fibroblasts.Conclusion The expression of TRPC1 increased in cardiac fibroblasts when treated with angiotensinⅡin a concentration- and time-dependent manner. AngⅡcan increaseintracellular Ca2+ depending on the Ca2+ entry through SOC. And TRPC1 may be thepotential molecular entity for SOC, which can promote the cardiac fibrosis and TGF-β1expression in cardiac fibroblasts induced by AngⅡ. SKF96365 can prevent the cardiacfibrosis by inhibiting the expression and function of TRPC1 in cardiac fibroblasts inducedby AngⅡ. Part 3 The effects of valsartan on the expression of TRPC1 incardiac fibroblastsObjective To examine the effects of valsartan on the expression of TRPC1 in culturedcardiac fibroblasts under high AngⅡlevel.Methods Cardiac fibroblasts were prepared from the ventricles of 1-3-day-oldSprague-Dawley rats. They were treated with 10-5mol·L-1 angiotensinⅡin the presence ofvarious concentration valsartan (100umol·L-1, 10umol·L-1or 5umol·L-1) for 24 hours. Thefibroblasts proliferation was measured by MTT. The TRPC1 and TGF-β1 mRNA andprotein expression were assayed by real time PCR and Western blot.Results AngiotensinⅡincreased the expressions of TRPC1 and TGF-β1. Comparedwith the angiotensinⅡalone group, valsartan inhibited the proliferation of cardiacfibroblasts and expression of TGF-β1. In addition, the expression of TRPC1 wasobviously inhibited by valsartan in a dose dependent manner.Conclusion Valsartan may decrease the expression of TRPC1 to inhibit proliferationof cardiac fibroblasts and the expression of TGF-β1 induced by angiotensinⅡ. |
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