溶血磷脂膽鹼對於新生大鼠心肌細胞間隙接合的影響

Abstract

溶血磷脂膽鹼(Lysophosphatidylcholine, lysolecithins,簡稱LPC)是phophatidylcholine經由phospholipase A2的氧化作用而產生的。溶血磷脂膽鹼的生理濃度大約介於5–180 μM。目前的研究指出，缺血的心臟會造成溶血磷脂膽鹼的堆積，並造成心律不整(arrhythmogenesis)以及縮收異常(contractile dysfunction)，但目前並沒有太多相關的研究去探討溶血磷脂膽鹼對於心肌細胞間隙接合(gap junction)所可能造成的影響。我們主要的研究目的便是希望探討溶血磷脂膽鹼對於心肌細胞間隙接合的作用以及影響。首先，細胞處理溶血磷脂膽鹼後並不影響細胞存活。雖然在心肌細胞的自發性收縮平均速率上沒有產生變化，但可以觀察到有快慢不一的現象。利用dye transfer技術評估其間隙接合的能力，發現溶血磷脂膽鹼處理後使dye transfer的距離變短，證明加入溶血磷脂膽鹼的處理確實會在短時間內使間隙接合能力(gap junction intercellular communication ; GJIC)下降。我們也發現溶血磷脂膽鹼處理後，會造成PKCalpha的活化，活化的PKCalpha會對Connexin43-ser368 ¬(P0)位置進行磷酸化並導致GJIC功能下降，而PKCalpha抑制劑(ev1-2)的前處理則可以恢復下降的GJIC功能。另外，我們發現溶血磷脂膽鹼處理後引起細胞內鈣離子濃度上升、PKCalpha和CaMKII的活化、以及Cx43在心肌細胞內的分布由細胞膜散落到細胞質。而鈣離子螯合劑(BAPTA-AM)、PKCalpha、CaMKII的抑制劑(Gö6976、KN93)都可有效的恢復經溶血磷脂膽鹼處理而下降的GJIC功能，而Gö6976、KN93還可以恢復溶血磷脂膽鹼引起的Cx43分布位置改變。同時，溶血磷脂膽鹼可以使P2-Cx43及P1-Cx43表現量以及Cx43的mRNA表現量上升，但前項結果並不會受到Gö6976以及KN93的處理而恢復。因此我們認為溶血磷脂膽鹼引起的P2-Cx43及P1-Cx43磷酸化的增加有可能是經由其他的激酶參與，此推論需要進一步的研究證實，待未來有更多的時間可以補足這方面的研究。

Lysophosphatidylcholine (lysolecithins, referred to LPC) is oxidized from phosphatidylcholine by phospholipase A2. The physiological concentration of LPC in the blood is about 5-180 μM. LPC is accumulated during heart ischemia and contributes to arrhythmia and contractile dysfunction. However, the mechanism underlying LPC-induced damage remains unclear. We aimed to study the effects of LPC on cardiomyocyte gap junction. First, LPC did not affect cell survival. Although LPC did not alter the spontaneous contraction rates of cardiomyocytes, but arrhythmic beating was found. In functional study of scrape loading dye transfer assay, the distance of dye transfer decreased after LPC treatment, indicated that LPC impaired the gap junction intercellular communication (GJIC). We also found LPC treatment induced PKCalpha activation. This activation of PKCalpha increased the phosphorylation of connenix43-Ser368(P0-Cx43) and led to the decreased GJIC. Pre-treatment with the PKCalpha inhibitor (eV1-2) could restore the function of GJIC. In addition, we found that LPC increased intracellular calcium, induced activation of PKCalpha and CaMKII, and altered the distribution of Cx43 from the membrane to the cytoplasm. The calcium chelator (BAPTA-AM), PKCalpha inhibitor (Gö6976), and CaMKII inhibitor (KN93) could recover the function of GJIC. Gö6976 and KN93also recover the LPC-induced Cx43 redistribution. The expression of P2-Cx43 and P1-Cx43 also increased 1.3 folds after LPC treatment, but were not prevented by Gö6976 and KN93. Thus, LPC-induced phosphorylation of P2-Cx43 and P1-Cx43 might include other kinase. The hypothesis needs further studies to elucidate.