Effects of periodic and sustained stretching on cardiac culture

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姓名

劉閔禎(Ming-Chen Liu) 查詢紙本館藏

畢業系所

生物物理研究所

論文名稱

(Effects of periodic and sustained stretching on cardiac culture)

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摘要(中)

一般來說我們認為心臟跳動是藉由電訊號傳遞來控制的。但是研究發現除了電訊號之外，機械力也能夠誘導心肌細胞上的機械力敏感離子通道開啟並且產生電流進而影響心臟的電活動。本實驗主要是探討機械力對心臟細胞跳動週期的影響，另外我們也探討纖維母細胞的數量多寡是否影響跳動週期。

心臟細胞是由大鼠胚胎（懷孕17天）的心臟取得，並且培養在可變形的腔室中，我們以單軸方向拉腔室。本實驗使用了兩種形式來拉伸細胞：(1) 週期性拉伸：1 Hz下持續二到四個小時，並以鈣離子螢光染色來觀察細胞跳動，(2) 持續性拉伸：將腔室維持在拉的情況下五分鐘再放掉腔室，並在明視野顯微鏡下觀測。本實驗使用了拉伸激活離子通道阻斷劑：釓(Gd3+)，這是一個應用在機械力敏感通道上常見的阻斷劑。

心臟細胞在週期性拉伸過後，我們發現細胞的跳動週期會接近拉伸的週期。
在持續性拉伸下，細胞的跳動週期變長。加入釓之後我們發現，拉伸對細胞跳動週期的影響小很多。除此之外，本實驗抑制了纖維母細胞的增長，發現在低密度纖維母細胞的情況下，跳動週期以及心律變異度明顯的高於高密度纖維母細胞的心臟細胞。

摘要(英)

Traditionally, it is believed that cardiac dynamics is controlled only by electrical activities. However, it is known that mechanical forces can also activate mechanical sensitive channels in the cell which can bring changes in electrical activity. The purpose of our experiment is to understand whether mechanical forces affect the cardiac beating dynamics. We also want to understand whether the number of fibroblasts which is mechanical sensitive affect the beating dynamics. Cardiomyocytes were dissected from rat embryos and seeded on a deformable silicon chamber in which stretching can be realized by elongation. Two kinds of stretching were used: (1) Periodic stretching at 1 Hz for 2 to 4 hours and the dynamics are observed by calcium imaging, (2) Sustained stretching for 5 minutes and the dynamics are observed by bright field microscopy. The stretch-activated ion channels (SACs) blocker, Gadolinium (Gd3+), was used in our experiment to investigate its effect on the beating dynamics. Under periodic stretching, we found that the frequency of cardiomyocytes can almost be synchronized by the stretching frequency. As for sustained stretching, we found that the heart rate decreases during stretching. After treating with $Gd^{3+}$, the difference of inter-beat interval (IBI) between stretched and non-stretched samples significantly decreases. In addition, we also investigated the beating of cardiac culture in the presence of high and low density fibroblasts. As for low density of fibroblasts, the IBI and heart rate variability is higher than those in the high density one.

關鍵字(中)

★ 心臟
★ 心肌細胞
★ 拉伸
★ 周期拉伸
★ 持續拉伸
★ 拉伸激活通道
★ 機械力敏感通道
★ 纖維母細胞

關鍵字(英)

★ cardiac
★ cardiomyocyte
★ stretch
★ fibroblast
★ periodic stretching
★ sustained stretching
★ stretch-activated ion channel
★ SACs
★ Gadolinium

論文目次

摘要 i
Abstract iii
致謝 v
List of Figures xi
List of Tables xvii
Symbols xix
Chapter 1 Introduction 1
1.1 Objective 1
1.2 The function and structure of the heart 2
1.3 Conduction system 3
1.3.1 SA node 3
1.3.2 Conduction 4
1.3.3 The action potential of cardiomyocyte cell 6
1.3.4 Excitation-contraction(EC) coupling 8
1.3.5 The membrane potential of fibroblast 9
1.3.6 Gap junction 11
1.4 Calcium imaging 11
1.5 Heart rate variability 12
1.6 Previous work on effect of stretching 13
1.6.1 Inward current through stretch-activated channels 14
1.6.2 Morphology changes 15
1.6.3 Increase in Ca2+ spark rate 16
1.6.4 Gap junction conductance 17
1.7 Goal of this work 17
Chapter 2 Method and Experimental Setup 19
2.1 Overview 19
2.2 Sample preparation 20
2.3 Optical system 21
2.3.1 Fluorescent optics system 21
2.3.2 Phase contrast optics system 23
2.4 Stretching system 24
2.4.1 Setup 24
2.4.2 Periodic stretching 27
2.4.3 Sustained stretching 28
2.4.4 Our works 28
2.5 Image Analysis 29
2.5.1 Calcium imaging 29
2.5.2 Bright field image 30
Chapter 3 Results 33
3.1 Overview 33
3.2 Result of periodic stretching (E1) 34
3.2.1 Periodic stretching increase the beating 34
3.2.2 Increasing of reentrant propagation frequency after stretching 34
3.3 Result of sustained stretching 35
3.3.1 Stretching effects on IBI and HRV (E2) 37
3.3.2 Effect of Gadolinium (E3) 38
3.4 Effects on different densities of fibroblasts (E4,E5) 42
Chapter 4 Discussions 49
4.1 Effects of stretching on cardiac dynamics 49
4.1.1 The variation of IBI and HRV on cardiac culture 49
4.1.2 Synchronization between every cluster 50
4.1.3 Effects of different density of fibroblast 50
4.1.4 Effects of SACs blocker Gadolinium(Gd3+) 51
4.2 Mechanism of stretching on cardiac culture 51
4.2.1 Activities of ion channels 51
4.2.2 Activities of fibroblasts 52
4.3 Future work and improvement 53
4.3.1 Stretchable microelectrode array 53
4.3.2 Immunostaining 53
4.3.3 Compressing on cardiac culture 53
Appendix A 55
A.1 Culture medium preparation 55
A.2 Coating preparation 55
A.2.1 Fibronectin 55
A.2.2 Coating procedure 55
A.3 Dissection 55
A.3.1 Preparation 55
A.3.2 Tissue fragments preparation 56
A.3.3 Digestion 56
A.3.4 Cell suspension preparation 57
A.3.5 Pre-plating (removing the fibroblast) 57
A.3.6 Cell counting / sample plating 57
Appendix B 59
B.1 Tyrode solution (400 mL) 59
B.1.1 Stock solution 59
B.1.2 Working solution 60
B.2 Fluo-8 AM loading stock solution 60
B.3 Staining protocol for cardiac culture is seeded on chamber 60
Appendix C 61
References 63

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指導教授

陳志強(Chi-Keung Chan)

審核日期

2016-1-5

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