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系統識別號 U0026-1208201320390300
論文名稱(中文) 活化雌激素貝他增加心臟L型鈣離子通道及誘導新生鼠心臟生理性肥大外表型
論文名稱(英文) Activated ERbeta increases the expression of L-type Ca2+ channel and enhances the hypertrophic phenotype of neonatal rat hearts
校院名稱 成功大學
系所名稱(中) 生理學研究所
系所名稱(英) Department of Physiology
學年度 101
學期 2
出版年 102
研究生(中文) 王忻慈
研究生(英文) Hsin-Tzu Wang
學號 s36004038
學位類別 碩士
語文別 英文
論文頁數 60頁
口試委員 指導教授-蔡美玲
口試委員-洪麗滿
口試委員-甘宗旦
中文關鍵字 17貝它雌激素  雌激素接受器貝它  L-型鈣離子通道  鈣調控蛋白  心臟細胞骨架蛋白  QT間隔 
英文關鍵字 17beta-estradiol  ERbeta  L-type Ca2+ channel  calcium-handling protein  cardiac cytoskeleton proteins  QT interval 
學科別分類
中文摘要 自著床後的懷孕期間,雌激素(E2)的濃度不斷的上升直到分娩。同時心臟發育從胚胎期到剛出生持續改變它的生長型態,由分裂(增加細胞數目)至分化(加大細胞面積),但不增加心臟與身體之重量比。因此,本研究假設:雌激素具有誘導生理性心臟肥大的能力。目前研究顯示E2可作用至兩種接受器,分別是ERα和ERβ。在成熟小鼠中,剃除體內ERβ增加L型鈣離子通道(L-type Ca2+ channel)電流,延長心臟週期,並誘導心臟肥大但剃除體內ERβ對L型鈣離子通道(L-type Ca2+ channel)電流與心臟形態並無影響。然而在實驗室先前的實驗結果顯示,的ERβ蛋白表現量隨著心臟的成熟而下降,所以推測新生鼠ERβ表現量高的情況下會影響L型鈣離子通道,進而誘導出生理性心臟肥大。因此本研究的目的是利用DPN,探討活化ERβ是否增加L-type Ca2+ channel,引發新生鼠生理性心臟肥大表現型。新生大鼠皮下注射DPN (20 µg/ kg/ day) 一連七天當作是本研究的動物實驗模式。另外,由大鼠胚胎心臟分離出的H9c2 (心臟前驅細胞)當作細胞實驗模式,進一步去證實動物實驗模式。維他命A酸(retinoic acid), 白血病抑制因子(leukemia inhibitory factor)以及異丙腎上腺素(isoproterenol)改變細胞型態且增加L-type Ca2+ channel以及細胞骨架蛋白( α-sarcomeric actinin,β-tubulin)蛋白表現量,顯示出心臟肥大的早期外表型。以上這些作為細胞模式的正向控制組。實驗結果顯示,雌激素和DPN並不會影響新生鼠的心跳,心臟和體重。但是相較於E2,DPN會影響心電圖之心臟週期(QT interval) ,使其與心跳呈逆相關,增加在Lead I肢導之R+S波。另外也增加鈣處置蛋白(calcium-handling protein)及細胞骨架蛋白(cytoskeletal proteins)的表現。Nifedipine (L-type Ca2+ channel blocker) 不讓細胞外鈣離子流入,DPN促進nifedipine抑制的Lead I肢導S 波以及R+S 波以及抑制nifedipine促進的心跳速率。H9c2培養在低血清濃度下七天,生長型態像是retinoic acid, leukemia inhibitory factor以及isoproterenol的組別,長度增加但寬度不變的非同心肥大,並且增加ER蛋白表現,促進L-type Ca2+ channel和 α-sarcomeric actinin,β-tubulin的蛋白表現量。在tamoxifen (ER αand ERβ 拮抗劑, 10-8 M)或PHTPP (ERβ 拮抗劑, 10-7 M)存在下會降低DPN的促進L-type Ca2+ channel的作用。另外ERβshRNA 抑制L-type Ca2+ channel的蛋白表現。利用全細胞膜片箝制技術(whole-cell patch clamp method)偵測DPN所促進的L-type Ca2+ channel電流。本研究之細胞及動物實驗的結果顯示,在長時間活化ERβ,促使鈣處置蛋白以及細胞骨架蛋白表現量增加,推論雌激素貝他扮演著啟動初期生理性心臟肥大的能力。
英文摘要 With the elevation of 17 β-estradiol (E2) from implantation to parturition during pregnancy, cardiac development from the embryonic to neonatal stages changes its growth pattern from hyperplasia to hypertrophy without increasing the ratio of heart to body weight. It is possible that E2 may induce cardiac hypertrophy in neonatal hearts. Now, two estrogen receptors are discovered. Deletion of ERβ increases the current of the L-type calcium channel, prolongs the duration of the cardiac cycle, and induces cardiac hypertrophy in adult mice but depletion of ERβ does not Our preliminary data showed higher levels of ERβ protein in pre-pubertal hearts than those in adult hearts. Therefore, the purpose of the study was to determine whether activation of ERβ by DPN increased the expression of L-type Ca2+ channels and enhanced the hypertrophic phenotype of neonatal hearts. Neonatal SD rats were injected with DPN (20 µg/kg/day) subcutaneously for 7 days. H9c2 derived from cardiac tissue-derived cardiomyoblasts were used to confirm our study in neonatal rats. A 7-day treatment with retinoic acid (RA), LIF, or isoproterenol which changed cell morphology and increased the expression of α-actinin and L-type calcium channel showed early sign of hypertrophic growth. Those were used as our positive controls in vitro. Our results indicated that both E2 and DPN did not affect heart rate, heart weight, and body weight. Compared with E2, DPN improved the inverse correlation between heart rate and the duration of an electrical cycle of the heart (QTc), enlarged the amplitude of the QRS complex in Lead I, and increased the relative abundance of calcium handling proteins and cytoskeletal proteins. Co-treatment with nifedipine increased the amplitude of the S wave in Lead I and heart rate. A 7-day culture with DPN in vitro, changed cell morphology as RA, LIF, or ISO without altering cell size and increased the protein expressions of ER β,L-type Ca2+ channel, α-actinin, and β-tubulin. Co-treatment with tamoxifen (ER αand ER β antagonist, 10-8M) or PHTPP (an ER βantagonist, 10-8M) lowered the positive effect of DPN on the expression of L-type Ca2+ channel protein expression. Knockdown of ER βinhibited L-type Ca2+ channel protein expression as well. Whole-cell patch clamp showed the increased current of L-type Ca2+ channel by DPN enhanced. These in vivo and in vitro results showed the increases of calcium-handling proteins, cytoskeletal proteins, calcium current, and the amplitude of the QRS complex in Lead I with the increased expression of ERβ suggesting the role of ER β in initiating early development of cardiac hypertrophy.
論文目次 中文摘要 I
ABSTRACT III
誌 謝 V
INDEX VI
LIST OF TABLES IX
LIST OF FIGURES X
LIST OF SUPPLEMENTAL FIGURES XII
Part 1. General literature review 1
Ca2+-signaling pathway in hypertrophy cardiomyocytes 1
Cardiac cytoskeletons and microtubules both involved in cardiac hypertrophy 1
Leukemia inhibitory factor (LIF) elicits a potent hypertrophic factor in cardiomyocytes. 2
Isoproterenol-induced cardiac hypertrophy via -adrenergic receptor 3
Concentric and eccentric cardiac hypertrophy growth 3
Measurement of electrical profile of Lead I electrocardiogram (ECG) on cardiac hypertrophy 4
Part 2. Introduction 5
2-1 Estrogen (E2) is positively associated with hypertrophic growth of the hearts during pregnancy 5
2-2 Physiological remodeling of maternal heart from pregnancy to parturition 5
2-3 ER and ER knockout on hypertrophic phenotype 6
2-4 The purpose and working model of this study 6
Part 3. Materials and Methods 8
3-1 Reagents and antibodies 8
3-2 Animal care 8
3-3 Electrocardiogram 8
3-4 Heart-derived myoblasts-H9c2 cells and cell culture 9
3-5 Whole cell lysate 9
3-6 Western blot analysis 9
3-7 Immunofluorescences staining 10
3-8 Measurement of cell length-to-width ratio and size 11
3-9 Transfection 11
3-10 Electrophysiology 11
3-11 Data analysis and statistical evaluation 11
Part 4. Results 12
4-1 Postnatal cardiac development involve the functional and morphological change for cardiac hypertrophy phenotypes 12
4-2 Long-term expose estrogen and ER agonist on estrogen receptors protein expression 13
4-3 Estrogen-mediated downregulation of cardiac Kv4.3 protein expression 13
4-4 Assessment of cardiac structure and electrical profile of lead I ECG in ER activated neonatal rat hearts 13
4-5 Activated-ER induced cardiac hypertrophy is related to calcium-handling protein and cytoskeleton protein abundance 14
4-6 Relation between L-type Ca2+ channel and electrical profile of lead I ECG 15
4-7 ER-induced cardiac hypertrophy through result from change the growth pattern in cardiomyocytes and H9c2 15
4-8 Activated-ER increase L-type Ca2+ current in electrophysiology 16
Part 5. Discussion 17
5-1 Summary of this study 17
5-2 Interpret findings & key literature support 17
5-2-1 Exposure to E2 regulates L-type Ca2+ channel via ER 17
5-2-2 Contribution of E2 to L-type Ca2+ channel affect ECG profiles of Lead I in neonatal hearts 18
5-2-3 Ca2+ regulates -sarcomeric actinin abundance and leads to cardiac hypertrophy via ER 19
5-2-4 -sarcomeric actinin regulates cardiac cell growth pattern 19
5-3 Significant of this study 20
Part 6. References 20
Part 7. Tables 24


List of tables
TABLE 1. LIST OF PRIMARY AND SECONDARY ANTIBODIES USED IN THIS STUDY 24
TABLE 2. LIST OF DRUGS USED IN THIS STUDY 25

List of figures
FIGURE 1. CARDIAC DEVELOPMENT ON HEART WEIGHT/ BODY WEIGHT AND HEART RATE CHANGE 26
FIGURE 2. CARDIAC DEVELOPMENT ON ELECTRICAL PROFILE OF LEAD I ECG 27
FIGURE 3. CARDIAC DEVELOPMENT ON ELECTRICAL PROFILE OF LEAD III ECG 28
FIGURE 4. EFFECT OF ACTIVATED ER BY DPN ON ER AND ER PROTEIN EXPRESSION 29
FIGURE 5. EFFECT OF ACTIVATED ER ON KV4.3 PROTEIN EXPRESSION 30
FIGURE 6. EFFECT OF ACTIVATED ER ON HEART RATE, HEART WEIGHT AND BODY WEIGHT 31
FIGURE 7. EFFECT OF ACTIVATED ER ON LEAD I QTC EXPRESSION 32
FIGURE 8. EFFECT OF ACTIVATED ER ON CALCIUM APPARATUS PROTEIN EXPRESSION 33
FIGURE 9. EFFECT OF ACTIVATED ER ON CARDIAC PROTEIN EXPRESSION 34
FIGURE 10. EFFECT OF NIFEDIPINE ON ELECTRICAL PROFILE OF LEAD I ECG 35
FIGURE 11. EFFECT OF NIFEDIPINE ON ELECTRICAL PROFILE OF QTC AND HEART RATE 36
FIGURE 12. EFFECT OF ACTIVATED ER ON THE PRIMARY CULTURE CARDIOMYOCYTES 37
FIGURE 13. EFFECT OF ACTIVATED ER ON THE CARDIOMYOCYTES GROWTH PATTERN 38
FIGURE 14. EFFECT OF ACTIVATED ER ON ER PROTEIN EXPRESSION 39
FIGURE 15. EFFECT OF ACTIVATED ER ON H9C2 GROWTH PATTERN 40
FIGURE 16. EFFECT OF ACTIVATED ER ON ER PROTEIN EXPRESSION 41
FIGURE 17. EFFECT OF ACTIVATED ER ON THE SUBCELLULAR LOCALIZATION OF F-ACTIN 42
FIGURE 18. EFFECT OF ACTIVATED ER ON L-TYPE CA2+ CHANNEL PROTEIN EXPRESSION 43
FIGURE 19. EFFECT OF KNOCKDOWN ER ON L-TYPE CA2+ CHANNEL PROTEIN EXPRESSION 44
FIGURE 20. EFFECT OF ACTIVATED ER ON -SARCOMERIC ACTININ PROTEIN EXPRESSION 45
FIGURE 21. EFFECT OF ACTIVATED ER ON L-TYPE CA2+ CHANNEL OPENING 46

List of supplemental figures
SUPPLEMENTAL 1. CORRELATION 47
SUPPLEMENTAL 2. EFFECT OF ACTIVATED ER ON KV4.3 48
SUPPLEMENTAL 3. EFFECT OF ACTIVATED ER ON CARDIAC PROTEIN EXPRESSION 49
SUPPLEMENTAL 4. EFFECT OF ACTIVATED ER ON CARDIAC PROTEIN EXPRESSION 50
SUPPLEMENTAL 5. EFFECT OF LIF AND ISO ON H9C2 CELL LENGTH 51
SUPPLEMENTAL 6. EFFECT OF LIF AND ISO ON H9C2 CELL WIDTH 52
SUPPLEMENTAL 7. EFFECT OF LIF AND ISO ON H9C2 CELL LENGTH/ WIDTH 53
SUPPLEMENTAL 8. EFFECT OF LIF AND ISO ON H9C2 AREA 54
SUPPLEMENTAL 9. EFFECT OF LIF AND ISO ON H9C2 AREA 55
SUPPLEMENTAL 10. EFFECT OF LIF AND ISO ON L-TYPE CA2+ CHANNEL OPENING AS SLIDE 21 56
SUPPLEMENTAL 11. EFFECT OF LIF AND ISO ON L-TYPE CA2+ CHANNEL OPENING 57
SUPPLEMENTAL 12. CARDIAC DEVELOPMENT ON ER, LIF, AND -SARCOMERIC ACTININ PROTEIN EXPRESSION 58
SUPPLEMENTAL 13. CARDIAC DEVELOPMENT ON L-TYPE CA2+ CHANNEL PROTEIN EXPRESSION 59
SUPPLEMENTAL 14. EFFECT OF LIF AND ISO ON THE PRIMARY CULTURE CARDIOMYOCYTES 60
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