心臟毒素（CTX）是眼鏡蛇毒夜中主要成分，在組織表面心臟毒素會引發發炎反應。根據先前研究顯示，台灣西部與東部眼鏡蛇的細胞與肌肉毒性不盡相同，且來自西部的CTX A3會誘導粒線體分裂使型態變碎，但目前心臟毒素如何影響粒線體動態平衡尚未釐清。因此，本論文透過人類骨肉瘤細胞來研究心臟毒素對粒線體之間的關係。本實驗結果顯示加入來自東邊和西邊的心臟毒素 (CTX A3)及東邊的心臟毒素(CTX A6)使粒線體胞器網路形態分別呈現碎裂及網絡狀。進一步的實驗證明CTX A3誘導粒線體變碎必須依靠粒線體的分裂蛋白Drp1。此外，我們的研究指出相較於CTX A6，CTX A3會使粒線體功能變差。在本研究的實驗成果提供更多資訊了解心臟毒素對粒線體功能與動態平衡的影響，也有助於了解粒線體動態平衡機制以及台灣東部及西部眼鏡蛇心臟毒素的差異。

Cardiotoxin (CTX) is the major component of cobra Naja atra snake venom that causes tissue inflammation. The interactions among CTXs and biological molecules on cellular membrane are suggested to be the main cause for CTXs toxicity. CTX A6 from east coast causes more severe tissue necrosis than CTX A3 from west coast of Taiwan according to the reported hospital cases. However, what mechanisms led CTX A3 and A6 to trigger such differences remain obscure. Previous studies had evidences that CTX may target to mitochondria. We hypothesize that regulation of mitochondrial dynamics balance is one of the signaling pathways that CTXs affect. The specific aim of this research is to clarify the effects of CTX A3 and A6 on mitochondrial dynamics and activities. Our results indicated that when cells encountered CTX A3 and CTX A6, mitochondrial network turned into fragmentation and hyperfused respectively. In addition, mitochondrial fragmentation induced by CTX A3 was fission protein Drp1-dependent. Furthermore, our results found mitochondrial membrane potential and reserve respiration capacity decreased after cells were treated with CTX A3 but not CTX A6. These data suggested that the toxicity of CTXs would disrupt mitochondrial dynamic balance. The results also provide us the functional differences of cardiotoxins from eastern and western coast of Taiwan.

致謝.............................................................I
中文摘要................................................ III
ABSTRACT....................................................... IV
CONTENTS................................................... V
LIST OF FIGURES....................................... VII
Chapter 1 Introduction................................................ 1
1.1 Mitochondria are double-membrane organelles within the eukaryotic cells............................................... 1
1.2 Cellular energy production depends on mitochondrial oxidative phosphorylation................................................ 1
1.3 Mitochondrial dynamics regulate the mitochondrial quality ................................................................2
1.4 Important factors involved in mitochondrial dynamics... 3
1.4.1 Mitochondrial dynamics - fusion........................ 4
1.4.2 Mitochondrial dynamics – fission....................... 5
1.5 The actin filaments serve in mitochondrial fission..... 6
1.6 Dysregulation of mitochondrial dynamics leads to neurodegenerative diseases......................................6
1.7 Mitochondria are required for apoptosis and necrosis... 7
1.8 Cardiotoxin............................................ 8
1.9 Specific aim........................................... 9
Chapter 2 Material and Method............................ 10
2.1 Cell culture........................................... 10
2.2 Plasmid transfection................................... 10
2.3 Immunofluorescence staining............................ 11
2.4 Mdivi-1 treatment...................................... 11
2.5 Cell viability assay................................... 12
2.6 Protein extraction............................. .12
2.7 SDS-PAGE and western blot.............................. 12
2.8 Antibodies............................................. 13
2.9 Measurement of mitochondrial membrane potential........ 14
2.10 Measurement of mitochondrial and cytosol reactive oxygen species........................................................ 14
2.11 Measurement of mitochondrial oxygen consumption rate... 15
2.12 Statistical analysis................................... 15
Chapter 3 Results........................................ 16
3.1 Cardiotoxins affects mitochondrial dynamics in U-2OS cell ................................................................16
3.2 The effects of cardiotoxin A3 treatment can be attenuated by Drp1 inhibitor Mdivi-1...................................... 17
3.3 Cardiotoxin A3 treatment can be attenuated by Drp1 siRNA ................................................................17
3.4 Mitochondrial related proteins were not affected by CTX A3 and CTX A6..................................................... 18
3.5 CTX A3 affects mitochondrial membrane potential but not CTX A6......................................................... 18
3.6 Cellular superoxide and mitochondrial superoxide levels were not altered by CTX A3 and A6.............................. 19
3.7 Mitochondrial reserved respiration capacity decreased after treating with CTX A3 but not A6.......................... 19
3.8 Mdivi-1 has minor effects on cell viability of CTX A3 treated U-2OS cells............................................ 21
3.9 Rhodamine-labeled CTX A3 did not target to mitochondrial membrane as expected............................................21
Chapter 4 Conclusion and discussion...................... 22
4.1 Cardiotoxins affect mitochondrial dynamics in U-2OS cells ................................................................22
4.2 Cardiotoxins have no effects on mitochondrial dynamics related proteins............................................... 22
4.3 Mitochondrial activity was altered after CTX A3 but not CTX A6 treatment............................................... 23
4.4 Cardiotoxin A3 did not target to mitochondria as expected ................................................................24
Chapter 5 Perspective.................................... 25
REFERENCE...................................................... 26
LIST OF FIGURES
Figure 1. Mitochondria are highly dynamic organelles in mammalian cells.......................................................... 34
Figure 2. Mitochondrial fission and fusion machinery in mammalian cells.......................................................... 35
Figure 3. Sequences and 3D structures of CTX A3 and A6......... 37
Figure 4. To clarify the effects of CTX A3 and A6 on mitochondrial dynamic........................................................ 38
Figure 5. Cardiotoxins have effects on mitochondrial dynamics in U-2OS cell..................................................... 40
Figure 6. CTX A3 treatment can be partially blocked by Drp1 inhibitor Mdivi-1.............................................. 42
Figure 7. CTX A3 treatment can be partially rescued by Drp1 siRNA ................................................................44
Figure 8. Mitochondrial related proteins were not altered by CTX ................................................................46
Figure 9. Mitochondrial membrane potential was decreased under CTX A3 treatment................................................... 48
Figure 10. Cellular superoxide and mitochondrial superoxide levels were not affected by CTX A3 and A6............................. 51
Figure 11. Mitochondrial respiration after CTX A3 and A6 treatments..................................................... 56
Figure 12. Reserve capacity and proton leak after CTXs treatments ................................................................58
Figure 13. The effects of CTX A3 and Mdivi-1 on cell viability. 60
Figure 14. CTX A3 did not target to mitochondrial membrane..... 62


 

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