| Objective:The purpose of this study was to observe the changes of expressions of canonical transient receptor potential (TRPC) in the angiotensin (Ang)Ⅱ-induced hypertrophic cardiomyocytes and to investigate the molecular mechanisms of TRPC expressions and the roles of TRPC in hypoxia/reoxygenation- induced apoptosis.Methods:1. Ventricular myocytes of neonatal Spraque Dawley (SD) rats (2-5 days) were isolated and cultured.2. The hypertrophy of the cardiomyocytes was induced by 0.1μM of AngⅡfor 48hrs.3. The hypertrophy of the cardiomyocytes was identified according to the levels of protein contents determined by methods of bicinchoninic acid (BCA) and [3H]-Leucine incorporation. The surface areas of the cardiomyocytes were measured by using Motic Image 1.3 software package.4. The samples were divided into 7 groups, i.e, the control group, AngⅡgroup, SKF96365+ AngⅡgroup, Losartan+AngⅡgroup, PD123319+ AngⅡgroup, U73122+AngⅡgroup and CsA+AngⅡgroup.5. The cardiomyocytes were cultured under the hypoxic condition (5%CO2+95%N2) for 6 hrs first, then under reoxigenic condition (5%CO2+21%O2) for 4hrs so as to simulate the ischemia/reperfusion of the cardiomyocytes.6. Some samples were cultured for determining TRPC1-C7 mRNA expressions by real time PCR.7. The proteins of TRPC1, C3, C6 and the expressions ofα-fodrin (a structure protein of myocytes) were measured by the method of western blot.8. The apoptosis rates were assessed by the method of annexin V FITC/PI and Hoechst 33258 staining.Results:1. The productions of hypertrophic cardiomyocytes were successfully induced by 0.1μM of AngⅡ. Compared with the control group, the incorporated [3H]-Leucine increased significantly at 48thhrs after AngⅡwas added (1239±154 cpm/well, 2103±203cpm/well respectively, n=6, p<0.05). The total protein contents in AngⅡgroup were higher than those in the control group(1503±146μg/well, 1075±118μg/well, respectively, n=6,p<0.05).The surface areas of the cardiomyocytes in AngⅡgroup were 1.46 times higher than those in the control group obtained by using Motic Images 1.3 software packages.2. After 48hrs of AngⅡstimulation, the mRNA expressions of TRPC1 and TRPC3 were upregulated measured by the method of quantitative RT-PCR. TRPC1 and TRPC3 mRNA were 1.45 and1.24 times those in the control groups, however, there were no significant differences in the TRPC6 mRNA between AngⅡgroup and the control group (p>0.05,n=4). The TRPC2, TRPC4, TRPC5 and TRPC7 mRNA in AngⅡgroup were lower than those in the control group (p<0.05,n=4).3. The expressions of TRPC1 protein measured by western blot increased after 48hrs of AngⅡstimulation, but there were no changes for the expressions of TRPC3 and TRPC6 proteins, because the relative density ratio of TRPC1 to GAPDH in AngⅡgroup was higher than that in the control group, there were no significant differences in relative density ratio of TRPC3 to GAPDH and relative density ratio of TRPC6 to GAPDH between AngⅡgroup and the control group.4. Before adding AngⅡ, AT1 receptor blocker (Lorsartan), AT2 receptor blocker (PD123319), phospholipase C inhibitor (U73122), TRPC blocker (SKF96365) and calcineurin inhibitor (cyclosporin A, CsA) were added, it was found that TRPC1 density ratio of Losartan+AngⅡgroup to the control group obtained by the method of western blot were lower than that of AngⅡgroup to the control group, the same results were also obtained in U73122+AngⅡgroup, SKF96365+AngⅡgroup and CsA+AngⅡgroup except for PD123319+AngⅡgroup.This indicated that Losartan, U73122, SKF96365 and CsA were able to inhibit AngⅡ-induced over-expressions of TRPC1 in the cardiomyocytes, but AT2 receptor blocker PD123319 didn’t show the same effects.5. It was also found that the radioactivity (1452±172 cpm/well) in SKF96365+ AngⅡgroup was lower than that in AngⅡgroup (2103±203 cpm/well) by the method of [3H]-Leucine incorporation. The surface areas of the cardiomyocytes in SKF96365+ AngⅡgroup were less than those in AngⅡgroup, which were 1.29 times those in the control group,while the surface areas of the cardiomyocytes in AngⅡgroup were 1.46 times those in the control group (p<0.05). This indicated that SKF96365 was able to partly inhibit AngⅡ-induced hypertrophy of cardiomyocytes.6. The determination of hypoxia/reoxygenation injuries under 6h-hypoxia and 4h- reoxygenation conditions were as follows:(1)There were no significant differences in LDH and necrosis rates measured by the method of annexin V FITC -PI among the control group, AngⅡgroup and SKF96365+ AngⅡgroup.(p>0.05).(2)Apoptosis rates measured by both annexin V and Hoechst staining in AngⅡgroup were the highest among the control group, AngⅡgroup and SKF96365+ AngⅡgroup, the apoptosis rates in SKF96365+ AngⅡgroup were lower than those in AngⅡgroup (p<0.05),which indicated SKF96365 was able to reduce apoptosis rates.(3)The ratio of cleavedα-fodrin to noncleavedα-fodrin in SKF96365+ AngⅡgroup was higher than that in the control group, but lower than that in AngⅡgroup.Conclusions:1. The productions of hypertrophic cardiomyocytes were successfully induced by 0.1μM of AngⅡ. 2. The expressions of TRPC1 mRNA and protein in hypertrophic cardiomyocytes induced by AngⅡare upregulated. Block of TRPC can partly inhibit AngⅡ-induced cardiomyocyte hypertrophy. TRPC1 plays an important role in the progress of cardiomyocyte hypertrophy.3. Mechanisms of TRPC1 over-expressions induced by AngⅡmay be through AT1 receptor, PLC, calcineurin and nuclear factor of activated T cells in hypertrophic cardiomyocytes and the progression of feed forward of TRPC1.4. Under hypoxia/reoxigenation conditions AngⅡ-induced hypertrophic cardiomyocytes are more likely to cause apoptosis because of over-expression of TRPC1. TRPC1 takes part in apoptosis of hypertrophic cardiomyocytes associated with calpain.5. It is possible that TRPC1 is used as a new treatment site, through the treatment of which we can alleviate cardiomyocyte injuries caused by hypoxia/reoxigenation, thus the prognosis of patients may become better. |
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