目的 分析L型钙电流(IcaL)在犬三层心室肌细胞中的特点,探讨其在LQTl发病机制中的作用.方法 成年杂种犬14只,体重13～15 kg,雌雄不拘.分离犬三层心室肌细胞,采用全细胞膜片钳技术记录动作电位(AP)和ICaL,依次用Chromanol 293B(50ìμmoL/L)阻断慢激活延迟整流性钾电流(IKs)模拟LQTl,用异丙肾上腺素(100 nmo/L)激活13肾上腺素受体(β-AR),观察AP和,ICaL的变化.分三层取少量心室肌组织,采用实时定量逆转录聚合酶链反应(RT-PCR)技术,检测各层L型钙通道a1C亚单位的mRNA含量.结果 正常情况下,犬三层心室肌细胞ICaL电流密度差异无统计学意义[外层(4.253±0.782)pA/pF,中层(4.392±0.714)pA/pF,内层(4.182±0.665)pA/pF,P＞0.05],而中层心室肌细胞动作电位时限(APD)较内层和外层的长[外层(721.48±26.59)ms,中层(911.80±31.24)ms,内层(783.52±25.27)ms,P＜0.05];阻断IKs后ICaL电流密度没有变化,而APD均明显延长[(外层(835.21±27.34)ms,中层(1089.21±30.55)ms,内层(830.64±27.12)ms,与阻断IKs前相比,P＜0.05)];β-AR兴奋使三层心室肌细胞ICaL显著增加,且三者变化差异无统计学意义[(外层(5.654±0.756)pA/pF,中层(5.458±0.702)pA/pF,内层(5.600±0.819)pAZpF,P＞0.05].但β-AR兴奋使外层和内层心室肌细胞APD缩短,中层心室肌细胞APD延长,三者变化差异有统计学意义[外层(792.63±26.71)ms,中层(1127.85±32.10)ms,内层(811.32±27.52)ms,P＜0.05].实时定量RT-PCR结果显示,三层心室肌细胞中alC亚单位的mRNA含量差异无统计学意义(外层0.112±0.019,中层0.077±0.018,内层0.109±0.012,P＞0.05).结论 L型钙通道在犬三层心室肌中的分布没有差异,在LQTl模型中,Iso使三层心室肌细胞,ICaL均匀增加,推测ICsL本身没有引起LQTl复极不稳定.
目的 分析L型鈣電流(IcaL)在犬三層心室肌細胞中的特點,探討其在LQTl髮病機製中的作用.方法 成年雜種犬14隻,體重13～15 kg,雌雄不拘.分離犬三層心室肌細胞,採用全細胞膜片鉗技術記錄動作電位(AP)和ICaL,依次用Chromanol 293B(50ìμmoL/L)阻斷慢激活延遲整流性鉀電流(IKs)模擬LQTl,用異丙腎上腺素(100 nmo/L)激活13腎上腺素受體(β-AR),觀察AP和,ICaL的變化.分三層取少量心室肌組織,採用實時定量逆轉錄聚閤酶鏈反應(RT-PCR)技術,檢測各層L型鈣通道a1C亞單位的mRNA含量.結果 正常情況下,犬三層心室肌細胞ICaL電流密度差異無統計學意義[外層(4.253±0.782)pA/pF,中層(4.392±0.714)pA/pF,內層(4.182±0.665)pA/pF,P＞0.05],而中層心室肌細胞動作電位時限(APD)較內層和外層的長[外層(721.48±26.59)ms,中層(911.80±31.24)ms,內層(783.52±25.27)ms,P＜0.05];阻斷IKs後ICaL電流密度沒有變化,而APD均明顯延長[(外層(835.21±27.34)ms,中層(1089.21±30.55)ms,內層(830.64±27.12)ms,與阻斷IKs前相比,P＜0.05)];β-AR興奮使三層心室肌細胞ICaL顯著增加,且三者變化差異無統計學意義[(外層(5.654±0.756)pA/pF,中層(5.458±0.702)pA/pF,內層(5.600±0.819)pAZpF,P＞0.05].但β-AR興奮使外層和內層心室肌細胞APD縮短,中層心室肌細胞APD延長,三者變化差異有統計學意義[外層(792.63±26.71)ms,中層(1127.85±32.10)ms,內層(811.32±27.52)ms,P＜0.05].實時定量RT-PCR結果顯示,三層心室肌細胞中alC亞單位的mRNA含量差異無統計學意義(外層0.112±0.019,中層0.077±0.018,內層0.109±0.012,P＞0.05).結論 L型鈣通道在犬三層心室肌中的分佈沒有差異,在LQTl模型中,Iso使三層心室肌細胞,ICaL均勻增加,推測ICsL本身沒有引起LQTl複極不穩定.
목적 분석L형개전류(IcaL)재견삼층심실기세포중적특점,탐토기재LQTl발병궤제중적작용.방법 성년잡충견14지,체중13～15 kg,자웅불구.분리견삼층심실기세포,채용전세포막편겸기술기록동작전위(AP)화ICaL,의차용Chromanol 293B(50ìμmoL/L)조단만격활연지정류성갑전류(IKs)모의LQTl,용이병신상선소(100 nmo/L)격활13신상선소수체(β-AR),관찰AP화,ICaL적변화.분삼층취소량심실기조직,채용실시정량역전록취합매련반응(RT-PCR)기술,검측각층L형개통도a1C아단위적mRNA함량.결과 정상정황하,견삼층심실기세포ICaL전류밀도차이무통계학의의[외층(4.253±0.782)pA/pF,중층(4.392±0.714)pA/pF,내층(4.182±0.665)pA/pF,P＞0.05],이중층심실기세포동작전위시한(APD)교내층화외층적장[외층(721.48±26.59)ms,중층(911.80±31.24)ms,내층(783.52±25.27)ms,P＜0.05];조단IKs후ICaL전류밀도몰유변화,이APD균명현연장[(외층(835.21±27.34)ms,중층(1089.21±30.55)ms,내층(830.64±27.12)ms,여조단IKs전상비,P＜0.05)];β-AR흥강사삼층심실기세포ICaL현저증가,차삼자변화차이무통계학의의[(외층(5.654±0.756)pA/pF,중층(5.458±0.702)pA/pF,내층(5.600±0.819)pAZpF,P＞0.05].단β-AR흥강사외층화내층심실기세포APD축단,중층심실기세포APD연장,삼자변화차이유통계학의의[외층(792.63±26.71)ms,중층(1127.85±32.10)ms,내층(811.32±27.52)ms,P＜0.05].실시정량RT-PCR결과현시,삼층심실기세포중alC아단위적mRNA함량차이무통계학의의(외층0.112±0.019,중층0.077±0.018,내층0.109±0.012,P＞0.05).결론 L형개통도재견삼층심실기중적분포몰유차이,재LQTl모형중,Iso사삼층심실기세포,ICaL균균증가,추측ICsL본신몰유인기LQTl복겁불은정.
Objective To explore the characteristics of L-type calcium current in canine ventricular myocytes of three different layers and its effect on LQTl.Methods Whole-cell patch clamp technique was used to record action potential(AP)and ICaL in canine epicardial,midmyocardial,and endocardial ventricular myocytes,when the cells were under baseline conditions;or perfused with chromanol 293 B(50μmol/L)-IKs blocker alone;or isoproterenol(100 nmol/L)-β-adrenergic receptor with the present of chromanol 293B.Realtime RT-PCR was used to detect the mRNA levels of ICaL in three layers of canine ventricular muscle.Results The peak ICaL density showed no significant difference among the epicardial,midmyoeardial and endocardial ventricular myocytes[Epi(4.253±0.782)pA/pF;M(4.392±0.714)pA/pF,Endo(4.182±0.665)pA/pF,P＞0.05],and did not change after the cells were perfused with chromanol 293 B.But chromanol 293 B significantly prolonged the action potential duration(APD)of three types of cells(Epi(721.48±26.59)ms vs(835.21±27.34)ms;M(911.80±31.24)ms vs(1089.21±30.55)ms;Endo(783.52±25.27)ms vs(830.64±27.12)ms,compared with the baseline conditions,P＜0.05]without increasing transmural dispersion of repolarization(TDR).Iso activated ICaL markedly in all the cells,but the increase in the peak ICaL density was identical throughout three layers(Epi(5.654±0.756)pA/pF,M(5.458±0.702)pA/pF,Endo(5.600±0.819)pA/pF,P＞0.05].In contrast,the APDs were prolonged in midmyocardial myocytes,but abbreviated in epicardial and endocardial myocytes when perfused with Iso[Epi(792.63±26.71)ms;M(1127.85±32.10)ms;Endo(81 1.32±27.52)ms,P＜0.05].The changes of APDs resulted in TDR increasing.The mRNA levels of ion conducting subunit of ICaL(alC)was similar among three layers of canine ventricular muscle(Epi:0.112±0.019,M:0.077±0.018,Endo:0.109±0.012,P＞0.05).Conclusion These results indicate that the distribution of ICaL is not different among three layers of canine ventricular muscle with regard to genetic and electrophysiological expression.In the model of LQTl ICaL increased homogeneously by β-adrenergic receptor stimulmion.It may suggest that ICaL is not involved in the repolarization instability to cause LQTl.
