張 力，周成斌

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心肌細(xì)胞鈣離子代謝與胎兒心肌保護(hù)

張 力，周成斌

［關(guān)鍵詞］：鈣離子代謝；心肌保護(hù)；胎兒心功能

作者單位：510080廣州,南方醫(yī)科大學(xué),廣東省心血管病研究所,廣東省人民醫(yī)院(張 力)；510080廣州,廣東省心血管病研究所,廣東省人民醫(yī)院(周成斌)

隨著胎兒心臟超聲診斷技術(shù)的提高和推廣,大量先天性心臟病(先心病)能夠產(chǎn)前診斷[1]。宮內(nèi)矯治胎兒心臟結(jié)構(gòu)畸形,避免心臟大血管繼發(fā)性改變是提高復(fù)雜先心病救治成功率的重要選擇。在胎羊心臟轉(zhuǎn)流模型的研究中,國(guó)內(nèi)外學(xué)者認(rèn)識(shí)到胎兒心功能的損害可能與體外循環(huán)過程中胎兒心肌細(xì)胞的鈣離子(Ca2＋)代謝有關(guān)。本文探討胎兒心肌細(xì)胞Ca2＋代謝與心功能受損的關(guān)系,為胎兒心肌保護(hù)提供新思路。

1 胎兒心血管系統(tǒng)的生理特點(diǎn)

胎兒在宮內(nèi)處于低氧環(huán)境,經(jīng)胎盤氧合后的臍靜脈血氧分壓僅為20～30 mm Hg,是成人動(dòng)脈血氧分壓的1/4～1/5。胎兒為了適應(yīng)在宮內(nèi)低氧環(huán)境的生長(zhǎng)需要,其營(yíng)養(yǎng)攝取、廢物排泄及氣體交換都由臍帶血管經(jīng)胎盤循環(huán)來完成[2]。胎兒的心臟發(fā)育貫穿整個(gè)孕期,胎兒的心肌細(xì)胞成分以非收縮成分為主(約占60%),在生長(zhǎng)發(fā)育過程中,由于其心臟的儲(chǔ)備能力低下,心排量的維持依賴較高的心率。研究表明增加胎兒心率可以延長(zhǎng)單位時(shí)間內(nèi)心肌細(xì)胞去極化的累計(jì)時(shí)間,增加鈣內(nèi)流以及肌質(zhì)網(wǎng)(sarcoplasmic reticulum,SR)鈣的儲(chǔ)備；同時(shí),高頻率去極化過程增加心肌細(xì)胞膜下的鈉離子(Na＋)濃度,促進(jìn)鈉鈣交換蛋白(Na＋/Ca2＋exchanger,NCX)的Ca2＋內(nèi)流,提高細(xì)胞內(nèi)Ca2＋濃度[3]。

2 心肌細(xì)胞鈣循環(huán)

研究表明,Ca2＋在心臟收縮和舒張過程中發(fā)揮了關(guān)鍵的作用。心肌鈣循環(huán)主要是在心肌細(xì)胞SR內(nèi)進(jìn)行,包括鈣釋放、鈣回?cái)z和鈣存儲(chǔ)三個(gè)部分,任何一個(gè)過程出現(xiàn)異常,都將導(dǎo)致心功能的損害。在正常心肌細(xì)胞鈣循環(huán)過程中,SR為引起興奮-收縮的鈣瞬變提供了約92%的Ca2＋[4],為心肌興奮-收縮偶聯(lián)鈣循環(huán)的主要場(chǎng)所。心肌細(xì)胞去極化是少量Ca2＋通過心肌細(xì)胞膜上的L型鈣離子通道(LCa)迅速進(jìn)入細(xì)胞質(zhì)使Ca2＋濃度增加,Ca2＋與SR上的蘭尼堿受體2(ryanodine receptor 2,RyR2)結(jié)合,使SR上的通道開放,導(dǎo)致大量的Ca2＋從SR中釋放出來,進(jìn)入胞漿,這一過程被稱為鈣誘導(dǎo)鈣釋放(calcium induced calcium release,CICR)[5]。心肌細(xì)胞收縮后SR鈣泵將細(xì)胞質(zhì)內(nèi)的Ca2＋回?cái)z至SR內(nèi)儲(chǔ)存,同時(shí)細(xì)胞膜上的鈉鈣交換體將少量Ca2＋運(yùn)至細(xì)胞膜外,Ca2＋與肌鈣蛋白C解離,心肌細(xì)胞舒張。在心肌鈣代謝過程中,目前認(rèn)為以下受體或蛋白發(fā)揮重要作用。

2.1 Ryanodine受體 Ryanodine受體是一種Ca2＋釋放通道,可分為RyR1、RyR2、RyR3三類。在心肌細(xì)胞中鈣釋放通道的主要類型是RyR2,參與了心肌的興奮收縮耦聯(lián)、心臟起搏和心率失常的過程,CICR是心肌中基礎(chǔ)Ca2＋釋放(鈣火花)的主要方式。有研究顯示,在體內(nèi)剔除RyR2將減少Ca2＋、ATP和氧化代謝,從而導(dǎo)致代謝重排和細(xì)胞死亡,因此RyR2維持了心肌細(xì)胞ATP的生產(chǎn)和生存[6],對(duì)心肌功能起到重要作用。在心衰個(gè)體中,蛋白激酶A信號(hào)通路誘導(dǎo)的RyR2過度磷酸化被認(rèn)為是導(dǎo)致鈣泄漏及心衰的重要原因之一[7]。然而有很多其他實(shí)驗(yàn)數(shù)據(jù)表明鈣/鈣調(diào)素依賴性蛋白激酶Ⅱ(Ca2＋/calmodulin-dependent protein kinaseⅡ,CaMK Ⅱ)依賴的RyR磷酸化是鈣泄漏的重要原因[8-9]。此外,交換蛋白的識(shí)別被cAMP直接激活更進(jìn)一步證實(shí)CaMKⅡ依賴磷酸化通過β受體刺激心臟的重要性[10-12]。最近的報(bào)道提出CaMKⅡ和蛋白激酶A(protein kinase A,PKA)對(duì)RyR均有功能性的調(diào)節(jié)[13]。

2.2 CaMKⅡ CaMKⅡ參與LCa的調(diào)節(jié),應(yīng)用CaM和CaMKⅡ抑制劑觀察對(duì)LCa的鈣依賴性失活(CDI)和鈣依賴性易化(CDF)的影響,發(fā)現(xiàn)在應(yīng)用了CaMKⅡ阻斷劑后,RyRs的磷酸化被抑制,繼而Ca2＋釋放減少,導(dǎo)致細(xì)胞內(nèi)Ca2＋濃度降低,影響了CDI和CDF的時(shí)程[14]。CaMKⅡ還可以通過下游調(diào)控元件拮抗調(diào)節(jié)子(downstream regulatory element antagonist modulator,DREAM)的易位來抑制心肌LCa的α1亞單位基因的轉(zhuǎn)錄[15]。Ronkainen等人進(jìn)行深入研究得出結(jié)論,CaMKⅡ能夠使Ca2＋介導(dǎo)的轉(zhuǎn)錄抑制劑DREAM易位進(jìn)入細(xì)胞核中,從而促進(jìn)DREAM介導(dǎo)的轉(zhuǎn)錄抑制[15]。研究發(fā)現(xiàn),CaMKⅡ與RyR2上的FK506結(jié)合蛋白共同調(diào)節(jié)RyR2的功能[16-17],直接改變?nèi)毖陂g或再灌注期間CaMKⅡ活性,顯示出更顯著的心肌保護(hù)作用。在酸性環(huán)境、缺氧/復(fù)氧等情況下,主要是通過CaMKⅡ途徑來發(fā)揮心肌保護(hù)作用的[18]。

2.3 SR Ca2＋-ATP酶 心肌細(xì)胞收縮后,Ca2＋的清除主要經(jīng)SR上的鈣回?cái)z,即肌質(zhì)網(wǎng)Ca2＋-ATP酶(sarco-endoplasmic reticulum Ca2＋-ATP protein,SERCA2a)將Ca2＋重新攝取回SR,或由細(xì)胞膜上的鈉-鈣交換泵出細(xì)胞外。SERCA2a基因在鈣回?cái)z中有重要作用,是心臟收縮和舒張的主要調(diào)節(jié)之一,改變SERCA2a的活性對(duì)心臟功能尤其有害。有相關(guān)報(bào)道,在各種形式的缺血性心臟病以及早期低壓缺氧反應(yīng)中,SERCA2a表達(dá)受抑制,表明SERCA2a的表達(dá)可能受氧分壓的調(diào)節(jié)。胚胎的SERCA2a調(diào)控提供了一個(gè)模型來研究低氧依賴性SERCA2a的轉(zhuǎn)錄,胚胎在低氧(1%～5%O2)的子宮環(huán)境下發(fā)育[19],最初,胚胎的增大導(dǎo)致缺氧,但在發(fā)育的心臟中,四腔心的形成尤其是冠狀動(dòng)脈的形成,缺氧局限在較小的區(qū)域[20]。而從胎兒到成年小鼠心臟組織的氧合逐步改善與SERCA2a表達(dá)的數(shù)倍增長(zhǎng)是一致的[21]。研究發(fā)現(xiàn),在SERCA2a基因剔除的心臟中,可引起鈣循環(huán)重要場(chǎng)所SR的凋亡[22]。新近研究發(fā)現(xiàn)選擇性調(diào)整SERCA的活性對(duì)心臟興奮收縮偶聯(lián)產(chǎn)生深遠(yuǎn)的影響,這歸因于SR鈣攝取的改變,但對(duì)SR鈣泄漏沒有顯著改變[23]。

2.4 鈣離子通道 心肌細(xì)胞膜上主要鈣離子通道有電壓依賴性LCa、電壓依賴性T型鈣離子通道(TCa)和NCX。通過LCa的慢內(nèi)向鈣內(nèi)流是構(gòu)成心室肌細(xì)胞動(dòng)作電位平臺(tái)期的基礎(chǔ),在心肌細(xì)胞正常生理興奮-收縮偶聯(lián)中有重要作用。TCa雖然也能觸發(fā)鈣內(nèi)流誘導(dǎo)SR釋放Ca2＋,但作用較小[24-25]。NCX在心肌細(xì)胞膜上是一個(gè)非ATP依賴的雙向鈣離子轉(zhuǎn)運(yùn)蛋白,依賴于細(xì)胞膜內(nèi)外鈉、鈣離子濃度的變化,參與調(diào)節(jié)Ca2＋在心肌細(xì)胞內(nèi)外的進(jìn)出。在生理?xiàng)l件下,NCX主要通過正向交換模式將細(xì)胞興奮時(shí)由LCa進(jìn)入細(xì)胞內(nèi)的Ca2＋排出細(xì)胞外,對(duì)維持心肌細(xì)胞鈣穩(wěn)態(tài)和收縮偶聯(lián)起著重要作用。在缺氧狀態(tài)下,胞內(nèi)酸性代謝產(chǎn)物的蓄積,激活膜上的Na＋/H＋交換體,通過Na＋/H＋交換造成細(xì)胞內(nèi)Na＋濃度升高,后者又可以通過增強(qiáng)反向NCX使胞內(nèi)Ca2＋濃度升高,引起Ca2＋超載。細(xì)胞內(nèi)Ca2＋超載是缺血心肌在再灌注過程中細(xì)胞凋亡、壞死、以及心功能降低的重要原因。盡管NCX不依賴ATP,但是當(dāng)ATP充足時(shí),NCX更有利于將Ca2＋排出細(xì)胞外[26]。NCX功能的改變可導(dǎo)致SR內(nèi)Ca2＋的異常釋放以及心肌電活動(dòng)和收縮運(yùn)動(dòng)的異常。有研究顯示在腦細(xì)胞NCX編碼基因的啟動(dòng)子上有缺氧誘導(dǎo)因子1(hypoxia-inducing factor 1,HIF-1)結(jié)合的缺氧反應(yīng)元件,HIF-1促進(jìn)腦細(xì)胞NCX的表達(dá),參與腦缺血預(yù)處理的保護(hù)作用[27]。鈣預(yù)處理能夠有效對(duì)抗心肌損傷,其機(jī)制涉及鈣預(yù)處理時(shí)NCX反向交換活性的上調(diào)[28]。

2.5 線粒體和SR鈣循環(huán)的相互作用 線粒體和SR鈣循環(huán)是相互影響的,有研究發(fā)現(xiàn)心衰大鼠心肌細(xì)胞質(zhì)Na＋濃度的增加引起興奮-收縮耦聯(lián)過程中線粒體鈣回?cái)z減少,破壞了心肌細(xì)胞的能量供應(yīng)[29]。有報(bào)道線粒體-內(nèi)質(zhì)網(wǎng)結(jié)構(gòu)偶聯(lián)中的蛋白分子很多都是與Ca2＋相關(guān)的[30],它們形成有效的細(xì)胞器叫Ca2＋轉(zhuǎn)運(yùn)機(jī)制。有學(xué)者指出心衰時(shí)胞漿Na＋的增加導(dǎo)致心肌細(xì)胞活性氧的增加[31],在缺氧/復(fù)氧過程中,鈣敏感受體激活通過肌醇1,4,5-三磷酸受體途徑,是SR Ca2＋釋放入線粒體,引起心肌細(xì)胞的凋亡。心肌細(xì)胞線粒體的鈣釋放主要通過線粒體NCX,研究發(fā)現(xiàn),灌流線粒體NCX特異性阻斷劑CGP37157使成年大鼠心肌組織中環(huán)磷酸腺苷水平升高,這可能與環(huán)磷酸腺苷活化蛋白激酶A,而蛋白激酶A又使RyR2的特異磷酸化位點(diǎn)ser2808、ser2814磷酸化,FKBP12.6結(jié)合蛋白結(jié)合RyR2的能力降低,導(dǎo)致SR在靜息期的鈣泄漏有關(guān)[32]。Neumann認(rèn)為,線粒體NCX特異性阻斷劑CGP37157促進(jìn)了RyR2的活化且抑制了SERCA2a通道[33]。

3 HIF-1對(duì)鈣離子代謝的影響

筆者在已建立的胎羊心臟轉(zhuǎn)流模型中,利用自身胎盤作為氧合器,進(jìn)行氣體交換,提供胎羊生理性低氧(20～30 mm Hg)[34-38]。低氧是胎兒心臟發(fā)育過程中不可或缺的生長(zhǎng)條件,研究顯示低氧激活HIF-1,參與早期胎兒心臟的形成、冠脈血管的生長(zhǎng)、流出道的成形和中后期的胎兒心臟發(fā)育等[39]。HIF-1是一個(gè)由氧敏感的α亞基和結(jié)構(gòu)性表達(dá)的β亞基組成的異源二聚體。HIF-1β在細(xì)胞內(nèi)的表達(dá)水平相對(duì)穩(wěn)定,而HIF-1α的活性和表達(dá)水平受氧濃度的影響[40-41]。常氧下HIF-1α被脯氨酸羥化酶(prolyl hydroxylases,PHD)作用后,經(jīng)泛素-蛋白酶體途徑迅速被降解；低氧下PHD受到抑制,HIF-1α穩(wěn)定性增強(qiáng),轉(zhuǎn)移到細(xì)胞核與HIF-1β形成HIF-1,作用于靶基因的缺氧反應(yīng)元件,激活轉(zhuǎn)錄過程,其調(diào)控基因涉及胎兒心臟發(fā)育的多個(gè)環(huán)節(jié)[42]。胎羊體外循環(huán)誘導(dǎo)全身炎癥反應(yīng)、手術(shù)應(yīng)激,促進(jìn)肝臟內(nèi)核因子(NF-kappa B,NF-KB)的激活[43]。研究表明NF-KB與HIF-1具有相互促進(jìn)作用,進(jìn)一步誘導(dǎo)凋亡、抗凋亡、胎兒糖酵解等相關(guān)基因的表達(dá)[44]。因此,推測(cè)胎羊體外循環(huán)過度增強(qiáng)HIF-1活性,以適應(yīng)體外循環(huán)狀態(tài)下的低氧環(huán)境。為節(jié)約用氧,低氧下合成ATP的氧化磷酸化過程被抑制,HIF-1促進(jìn)無氧糖酵解增強(qiáng),胎盤代謝的再調(diào)整使得供給胎兒的能量底物減少[45]。胎羊體外循環(huán)的研究也顯示胎羊體內(nèi)儲(chǔ)存的肝糖原被大量消耗,乳酸值顯著升高,孕羊提供的血糖減少,使胎羊的血糖水平下降[46]。在過度激活的HIF-1作用下胎羊能量匱乏,對(duì)胎羊各臟器功能產(chǎn)生不利影響。

國(guó)內(nèi)外胎羊體外循環(huán)的研究已證實(shí)心臟不停跳、生理性低氧的體外循環(huán)影響胎羊心室功能,降低心肌細(xì)胞SERCA2a蛋白含量,促進(jìn)心肌肌鈣蛋白的降解[36,38]。相關(guān)研究證實(shí)胎羊體外循環(huán)后心肌細(xì)胞出現(xiàn)明顯Ca2＋超載現(xiàn)象。胎兒心肌細(xì)胞SR發(fā)育不成熟,鈣離子儲(chǔ)備少,但是SR仍然是CICR機(jī)制中鈣離子釋放和回收的重要細(xì)胞器。通過上述Ca2＋通道進(jìn)入細(xì)胞內(nèi)的Ca2＋作用于SR膜上的RyR2,啟動(dòng)CICR機(jī)制,SR釋放Ca2＋,與細(xì)胞外進(jìn)入的Ca2＋一起產(chǎn)生鈣瞬變,促進(jìn)肌絲的滑動(dòng),發(fā)生心肌收縮現(xiàn)象；達(dá)到鈣瞬變的峰值后,SERCA2a、NCX等轉(zhuǎn)運(yùn)蛋白將胞漿內(nèi)增多的鈣離子回收到SR內(nèi)或泵出細(xì)胞外,使胞漿內(nèi)Ca2＋濃度迅速下降,肌絲滑動(dòng)復(fù)員,心肌舒張[47]。在SERCA2a編碼基因的啟動(dòng)子上也有HIF-1結(jié)合的缺氧反應(yīng)元件,研究顯示低氧和HIF-1抑制胚胎心肌細(xì)胞SERCA2a的表達(dá)和活性[48]。

4 結(jié) 語(yǔ)

綜上所述,心肌細(xì)胞Ca2＋代謝受很多因素的影響,在心肌保護(hù)中起到重要作用。胎兒心肌細(xì)胞興奮收縮耦聯(lián)中的Ca2＋代謝依賴細(xì)胞膜Ca2＋通道和CICR機(jī)制[49-51]。胎羊生理性低氧體外循環(huán)存在過度激活HIF-1的風(fēng)險(xiǎn),一方面通過無氧糖酵解迅速消耗胎羊有限的糖原儲(chǔ)備和母體來源的能量,使胎羊心肌細(xì)胞處于能量匱乏狀態(tài)；另一方面,HIF-1抑制SERCA2a的表達(dá),降低SR攝取鈣離子的能力,同時(shí)HIF-1上調(diào)胎羊心肌細(xì)胞NCX的表達(dá),在能量供應(yīng)不足的情況下,NCX促進(jìn)Ca2＋內(nèi)流,減少鈣離子排出,從而產(chǎn)生Ca2＋超載,激活鈣離子依賴的蛋白酶,影響心肌收縮蛋白的功能,產(chǎn)生心功能不良。因此,HIF-1可能是胎羊體外循環(huán)心功能不良發(fā)生機(jī)制中的關(guān)鍵環(huán)節(jié)。通過降低HIF-1的活性,提供能量底物促進(jìn)Ca2＋的轉(zhuǎn)運(yùn),增強(qiáng)心肌細(xì)胞調(diào)節(jié)Ca2＋的能力,為胎羊體外循環(huán)的心肌保護(hù)提供新方法。

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(修訂日期:2015-11-09)

(收稿日期:2015-09-17)

通訊作者：周成斌,E-mail:zcbwwww@ 163.com

基金項(xiàng)目：國(guó)家自然科學(xué)基金項(xiàng)目(81370274)

DOI:10.13498/j.cnki.chin.j.ecc.2016.01.15