細胞外間質與整合素交互作用於心臟纖維化過程中對於心房及肺靜脈心肌細胞電生理重塑機轉之研究

Role of collagen-integrin interaction in electrical remodeling of atrial and pulmonary vein cardiomyocytes during cardiac fibrosis

盧彥佑(Yen-Yu Lu)

指導教授 ： 陳亦仁教授

臺北醫學大學/醫學院/臨床醫學研究所/博士(2014年)

摘要

背景：心肌纖維化在心臟衰竭中扮演著至關重要的角色。心肌纖維化造成心臟結構及電生理的重塑，而導致誘發心房性心律不整。心房顫動是最常見的持續性心律不整。然而肺靜脈則是AF發生的重要起源。細胞外膠原蛋白的增加所導致之心臟纖維化透過心臟結構和電生理的重塑與心房顫動的病理生理中起著至關重要的作用。然而，目前尚不清楚是否膠原蛋白可以直接調節鈣離子恆定和心肌細胞的電生理特性。本研究的目的是確定膠原蛋白是否影響心房和肺靜脈心肌細胞的鈣離子恆定以及電生理特性。
方法：在研究I，HL-1心肌細胞分別培養於有無含膠原蛋白以及有無加入Losartan之培養液中。利用全細胞箝制，Indo-1螢光攝影和西方墨點法用來評估動作電位和離子流，細胞內鈣離子恆定和鈣離子調節蛋白之表現。在研究II，分離之雄性新西蘭兔之肺靜脈心肌細胞培養於有無含膠原蛋白之培養液中。利用全細胞箝制來評估動作電位和離子流的變化。
結果:與對照組相比，膠原蛋白處理之HL-1心肌細胞表現出較大的細胞內鈣離子濃度和較大的肌漿網內鈣含量，膠原蛋白處理之HL-1心肌亦有較高SERCA2a蛋白和Thr17磷酸化phospholamban的表現，但Na+/Ca2+exchanger and ryanodine receptor的表現並無差異。膠原蛋白處理HL-1心肌細胞有較大的動作電位及較短的90%動作電位的持續時間。此外，膠原蛋白處理之HL -1細胞有較大的Ito和IKsus值。Losartan則會減少膠原蛋白所造成細胞內鈣離子， 動作電位形態，離子電流， SERCA2a以及Thr17磷酸化phospholamban表現的變化。在研究II中，膠原蛋白處理的肺靜脈心肌細胞跳動較快及較大的delayed afterdepolarization。此外，膠原蛋白處理的肺靜脈心肌細胞表現出較大的Ito，small-conductance Ca2 +-activated K+ current，inward rectifier potassium current，pacemaker current，late sodium current，但sodium current，IKsus和L-type calcium current是相似的。膠原蛋白增加肺靜脈心肌細胞p38 MAPK磷酸化， SB203580 （ p38 MAPK的催化活性抑製劑）則明顯減少膠原蛋白對肺靜脈心肌細胞自動性和動作電位的變化。
結論：這兩項研究表明，膠原蛋白可以透過腎素-血管收縮素系統來直接調節心房心肌細胞的鈣離子動力學和影響心電生理。這些研究結果表明膠原蛋白在纖維化過程中對心臟電生理的重塑起著重要的作用。此外，膠原蛋白可以通過活化p38 MAPK直接增加肺靜脈心肌細胞心律失常，這可能有助於了解心房顫動的發病機制。

關鍵字

纖維化，細胞外間質，電生理，鈣離子恆定，膠原蛋白，肺靜脈，心房顫動 ； p38 MAPK

並列摘要

Background: Myocardial fibrosis plays a critical role in heart failure, resulting in cardiac structural and electrical remodeling which can induce atrial arrhythmias. Atrial fibrillation (AF) is the most common sustained arrhythmia. Pulmonary veins (PVs) are important foci for AF genesis. Cardiac fibrosis with enhanced extracellular collagen plays a critical role in the pathophysiology of AF through structural and electrical remodeling. However, it is not clear whether collagen can directly regulate the calcium homeostasis and the electrophysiologic characteristics of cardiomyocytes. The aim of our study was to determine the effects of collagen on calcium homeostasis and the electrical properties of atrial and pulmonary cardiomyocytes.
Methods: In study I, HL-1 cardiomyocytes were cultured with and without collagen type I (1 or 10 μg/mL) or losartan (10 μmol/L). Whole-cell clamp, indo-1 fluorescence, and Western blotting were used to evaluate the action potential (AP) and ionic currents, intracellular calcium homeostasis, and calcium regulatory proteins. In study II, APs and ionic currents were investigated in isolated male New Zealand rabbit PV cardiomyocytes with and without collagen incubation (10 μg/ml, 5–7 h) using the whole-cell patch-clamp technique.
Results In study I, collagen (10 μg/mL)–treated HL-1 cardiomyocytes exhibited larger intracellular calcium ([Ca2+]i) transients and a larger sarcoplasmic reticulum (SR) calcium content compared with the control samples,. Collagen–treated HL-1 cardiomyocytes had higher expression of SR ATPase (SERCA2a) and Thr17-phosphorylated phospholamban but similar protein expressions of the Na+/Ca2+exchanger and ryanodine receptor. Collagen–treated HL-1 cardiomyocytes had larger AP amplitude and shorter 90% of AP duration than control cells. Moreover, collagen–treated HL-1 cells had larger Ito and IKsus values than control cells. The administration of losartan (10 μmol/L) attenuated collagen-induced changes in [Ca2+]i transients, [Ca2+]i stores, AP morphology, ionic currents, SERCA2a, and Thr17-phosphorylated phospholamban expressions.
In study II, collagen-treated PV cardiomyocytes had a faster beating rate and a larger amplitude of delayed afterdepolarization as compared to control PV cardiomyocytes. Moreover, collagen-treated PV cardiomyocytes showed a larger transient outward potassium current, small-conductance Ca2 +-activated K+ current, inward rectifier potassium current, pacemaker current, and late sodium current than control PV cardiomyocytes, but amplitudes of the sodium current, sustained outward potassium current, and L-type calcium current were similar. Collagen increased the p38 MAPK phosphorylation in PV cardiomyocytes as compared to control. The change of the spontaneous activity and action potential morphology were ameliorated by SB203580 (the p38 MAPK catalytic activity inhibitor).
Conclusions: These two studies demonstrate that collagen can directly modulate the calcium dynamics and electrical activities of atrial cardiomyocytes, which are associated with the renin-angiotensin system. These findings suggest a critical role of collagen in electrical remodeling during fibrosis. Besides, collagen can directly increase PV cardiomyocyte arrhythmogenesis through p38 MAPK activation, which may contribute to the pathogenesis of AF.

並列關鍵字

Fibrosis, extracellular matrix, electrophysiology, calcium homeostasis Collagen ； Pulmonary veins ； Atrial fibrillation ； p38 MAPK

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