| Objective:Long QT Syndromes (LQTs) is a cardiac disorder characterized by a prolongation of the QT interval on ECG. LQTs predispose the hearts to develop the potentially lethal ventricular tachyarrhythmia, called torsades de pointes (TdP), which cause episodic syncope and sudden death. Women are known to be at higher risk to congenital and acquired form of TdP, but in adolescents (human< 14 years old and rabbit<42 days old) before the surge of sex steroids, the risk of TdP is reserved with male being more susceptible to TdP. Some evidences suggest that estrogen may be a contributory factor for the greater prevalence of drug-induced TdP in women. Sex-differences in the propensity to early afterdepolarizations (EADs) and TdP are reversed in adolescence and correlate with higher levels of L type Ca2+ channels (Ca-L) and current, Ica-L, and sodium calcium exchangers (NCX) also play a role in sex difference of arrhythmia phenotype. In this study, we analyzed the sex, age and regional difference in NCX and studied the regulation and mechanism of estrogen on Ca-L and NCX in rabbit cardiomyocytes.Method:(1) Single left ventricular myocardial cells of rabbits were isolated by enzymic method. According to the aim of experiment, myocytes were incubated with DMSO,17-β-Estradiol (E2) or other reagents. Ica-L was recorded by the whole-cell configuration of voltage clamp thechnique. The level of Cav1.2 (alC) protein was measured by immunocytochemistry and the level of Cavl.2 (alC) mRNA was measured by single-cell quantitative real time RT-PCR. To evaluate the effect of estrogen on dofetilide-induced EAD, action potential (AP) was recorded under current-clamp mode, and then dofetilide was added to prolong APD and induced EADs. (2) Single left ventricular myocardial cells were isolated from prepubetal (<42-days old) and adult (-3-months old) rabbits. INCXwas recorded by the whole-cell configuration of voltage clamp thechnique. The level of NCX1 protein was measured by immunocytochemistry. The role of NCX in prevalence to arrhythmia was evaluated by simultaneous optical mapping of voltage and Cai. (3) Single left ventricular myocardial cells of rabbits were isolated by enzymic method. According to the aim of experiment, myocytes were incubated with DMSO,17-β-Estradiol (E2) or other reagents. INCX was recorded by the whole-cell configuration of voltage clamp thechnique. The level of NCX1 protein was measured by immunocytochemistry. To evaluate the role of estrogen-induced INCX up-regulation, action potential (AP) was recorded under current-clamp mode, and then dofetilide was added to prolong APD and induced EADs, KB-R7943, NCX inhibitor, was added to suppress EADs.Result:(1) estrogen (E2) incubation caused an increase of ICa-L density, whereas progesterone was ineffective. And E2 effect on ICa-L was dose and region dependent. In addition, the enhancement of ICa-L was blocked by estrogen receptor (ER) antagonist ICI182,780 (10μM) and mimicked by ERa agonist PPT (5 nM). Moreover, E2 did not change ion channel gating, instead it increased Cav1.2a expression. And increased Ica-L by E2 was prevented by transcriptional inhibitor actinomycin D (4μM) and protein synthesis inhibitor cycloheximide (71μM). Finally, dofetilide (IKr-blocker) induced EADs in female base-myocytes cultured for 1-day if incubated with E2, but not without E2 or with E2+fulvestrant. (2) In adult rabbits, INCX was similar for male base (1.76±0.27 pA/pF), male apex (1.70±0.30 pA/pF) and female apex myocyte (1.30±0.50 pA/pF) but was significantly higher in female base (2.59±0.32 pA/pF, p<0.05). In prepubetal rabbits, INCX was similar for female base (1.21±0.25 pA/pF) and female apex (1.28±0.28 pA/pF) (p=0.84); INCX in male base (3.96±0.35 pA/pF) was significantly higher than INCX in male base (3.96±0.35 pA/pF) and in female base and apex. In prepubetal rabbits, NCX1 protein level in male base was significantly higher than that in male apex, female apex and female base (p<0.05); but after sexual maturity, NCX1 protein level at the base of female heart was 38% higher than male base and the apex of both sexes, In adult female rabbits and prepubetal male rabbits, EADs and TdP could be induced after perfusion with E4031 (0.5μM), Ikr inhibitor. The co-perfusion of KB-R7943(1μM) suppressed EADs after 10 min. (3) Myocytes were isolated from the base and apex, incubated in serum free of sex steroids and INCX was measured from freshly isolated myocytes (2-5 hours) then at days 1,2 and 3. In freshly prepared myocytes, INCX density was consistently greater in myocytes from the base compared to the apex. At the base, INCX density decreased between day 0 and day 2 and leveled off by day 3.In contrast, INCX at apex-myocytes from the same hearts did not significantly change. After 24 h-72 h of incubation, estrogen caused a marked up-regulation of INCX in base myocytes but had no effect on myocytes from the apex. Estrogen also caused no significant up-regulation in female base endocardial or male base epicardial cells. Female base-myocytes treated with E2 for 24 h also expressed a significantly greater density of NC1 than female apex-myocytes. INCX up-regulation by estrogen was blunted by an estrogen-receptor antagonist (ICI182,780, 1μM), inhibition of transcription (actinomycin-D=5μg/ml) or translation (cycloheximide=20μg/ml). After incubated with estrogen for 24 h, the myocytes were vulnerable to dofetilide-induced EAD. And KB-R7943 could suppress the EADs.Conclusion:INCX density and NCX1 protein level vary with sex, age and apex-base, and the difference is similar to the variation in Ca-L. Estrogen upregulates the function of Ca-L and NCX in a sexual and regional dependent manner which contributes to the enhanced propensity to EADs and TdP in female hearts. Estrogen upregulates NCX and Ca-L by a genomic mechanism mediated by estrogen receptors. |
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