Respirometric皂甙通透心脏纤维的氧化磷酸化的评估
Summary
皂甙与respirometric氧化磷酸化的分析，结合通透的纤维制备提供了线粒体功能的综合评估。线粒体呼吸在生理和病理状态下，可以反映不同监管规定的影响，包括线粒体相互作用，形态和生物化学。
Abstract
线粒体参与能量代谢，氧化应激，细胞凋亡，延缓衰老，线粒体encephalomyopathies和药物毒性，线粒体功能的调查代表心脏生理学的一个重要参数。鉴于此，技术来衡量心脏线粒体功能的需求。一项技术，它采用一种综合的方法来衡量线粒体的功能是respirometric氧化磷酸化（OXPHOS）分析。
respirometric OXPHOS评估的原则是围绕利用克拉克电极测量氧气浓度。通透的纤维束消耗氧气，氧气浓度在封闭腔下降。使用选择底物抑制剂 – 解偶联剂滴定协议，电子提供电子传递链的特定网站，使线粒体功能的评价。之前线粒体功能的respirometric分析，机械和化学的筹备技术是利用通透肌纤维肌膜。化学通透采用皂素，在保持细胞结构的同时，有选择性的细胞膜穿孔。
本文深入介绍了在准备测量耗氧量的皂素皮肤的心脏纤维，以评估线粒体OXPHOS所涉及的步骤。此外，故障排除建议以及具体的基板，可用于确定在特定网站的电子传递链的线粒体功能抑制剂和uncouplers提供。重要的是，所描述的协议可能很容易地应用到各种动物模型和人体样本的心脏和骨骼组织。
Protocol
1。试剂准备 1稍作修改，准备放松和保存液（RP解决方案） 。简言之，RP解决方案包括2.77mM CAK 2 EGTA，7.23mM K 2 EGTA，20MM咪唑，0.5mm的二硫苏糖醇，牛磺酸20MM，50MM的K – MES，6.56氯化镁2，5.7mM三磷酸腺苷，磷酸肌酸14.3mM，pH值7.1调整，在室温（RT）。通过0.45微米的过滤器进行消毒过滤的解决方案。分成15毫升的部分（猎鹰聚丙烯管）保存在-20 ° C配方的讨论。 </l…
Discussion
皂甙通透的心脏纤维技术，提供了一个独特的妥协之间 ， 在体外和体内线粒体OXPHOS耗氧量评估。这种技术的优点包括增加生理的相关性比较孤立的细胞结构线粒体被保留下来。虽然质膜降解，细胞内的膜结构，包括线粒体12，14，14肌质网， 肌丝和17的骨架1，保持不变。此外，线粒体和细胞骨架，17之间的相互作用也保持不变。透纤维线粒体增加稳定…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究是由加拿大卫生研究院和加拿大基因组研究所的支持。的JS从阿尔伯塔省遗产基金会加拿大的医学研究，心脏及中风基金会和加拿大糖尿病协会举行的薪水支持奖。实验室要感谢Oroboros仪器的技术援助，在通透的皂素纤维技术收购。
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| 100% Ethanol | Fisher Scientific | HC600 | ||
| 70% Ethanol | Fisher Scientific | HC-1000 | ||
| Adenosine 5′-diphosphate monopotassium salt dihydrate (ADP) | Sigma | A5285 | ||
| Albumin from bovine serum essentially fatty acid–free | Sigma | A-6003 | ||
| Antimycin A | Sigma | A8674 | ||
| Ascobic acid | Sigma | A4403 | ||
| Adenosine 5′-triphosphate disodium salt hydrate (ATP) | Sigma | A2383 | ||
| Atractyloside | Sigma | A6882 | ||
| Calcium carbonate | Sigma | C4830 | ||
| Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) | Sigma | C2920 | ||
| Cytochrome c | Sigma | C7752 | ||
| Digitonin | Sigma | D141 | ||
| Dithiothreitol | Sigma | D9779 | ||
| Ethylene glycol-bis-(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) | Sigma | E4378 | ||
| Glutamic acid | Sigma | 27647 | ||
| HEPES | Sigma | H4034 | ||
| Imidazole | Sigma | I5513 | ||
| Ketamine | Pfizer | Ketaset | ||
| Lactobionic acid | Sigma | 153516 | ||
| Magnesium chloride (MgCl2) | Sigma | M9272 | ||
| Magnesium chloride hexahydrate (MgCl2∙6H2O) | Sigma | M9272 | ||
| Malic acid | Sigma | M1000 | ||
| MES | Sigma | M3671 | ||
| N,N,N’,N’-Tetramethyl- pphenylenediamine Dihydrochloride (TMPD) | Sigma | T3134 | ||
| Oligomycin | Sigma | O4876 | ||
| Phosphocreatine | Sigma | P7936 | ||
| Potassium Chloride | Sigma | P9541 | ||
| Potassium Hydroxide | Sigma | P5958 | ||
| Potassium cyanide | Fluka | 60178 | ||
| Potassium phosphate monobasic | Sigma | P5655 | ||
| Rotenone | Sigma | R8875 | ||
| Saponin | Sigma | 47036 | ||
| Sodium Pentobarbital | Ceva Sante Animale | 1715 138 | Conc. 54.7 mg/ml | |
| Sodium pyruvate | Sigma | P2256 | ||
| Succinic acid | Sigma | S3674 | ||
| Sucrose | Sigma | S7903 | ||
| Taurine | Sigma | T8691 | ||
| Xylazine | Bayer | Rompun | ||
| ddH2O | ||||
| Ice | ||||
| Oroboros Oxygraph-2k | Oroboros Instruments | |||
| Kimwipes | VWR | 21905-026 | ||
| 15ml polypropylene centrifuge tubes | VWR | 89004-368 | ||
| 50ml polypropylene centrifuge tubes | VWR | 89004-364 | ||
| Straight Jewelers Forceps | George Tiemann & Co. | 160-50B | ||
| Curved Jewelers Forceps | George Tiemann & Co. | 160-57B | ||
| Straight Surgery Scissors | George Tiemann & Co. | 105-402 | ||
| Sterile Surgical Blade | VWR | BD371610 | ||
| 0.45-μm Syringe filters | VWR | CA28145-485 | ||
| pH meter | VWR | CA11388-308 | ||
| Glass Petri dishes | VWR | 89000-300 | ||
| 12-well Polystyrene Tissue Culture Plates | VWR | 82050-926 | ||
| Plate Stirrer | VWR | 97042-594 | ||
| Fisherbrand Microbars | Fisher Scientific | 14-511-67 | ||
| Weigh Scale | VWR | CA11278-162 | ||
| 10μl Hamilton Micro Syringe | Fisher Scientific | 14-815-1 | ||
| 25μl Hamilton Micro Syringe | Fisher Scientific | 14-824-7 | ||
| 50μl Hamilton Micro Syringe | Fisher Scientific | 14-824-5 | ||
| Nalgene Squeeze Bottles | Wilkem Scientific | LNA2407-1000 | ||
| Polystyrene Weighing Dishes | VWR | 89106-750 | ||
| Dissecting Microscope | Olympus |
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Tags
Respirometric Oxidative PhosphorylationSaponin-permeabilized Cardiac FibersMitochondrial FunctionEnergy MetabolismOxidative StressApoptosisAgingMitochondrial EncephalomyopathiesDrug ToxicityClark ElectrodeSubstrate-inhibitor-uncoupler Titration ProtocolsElectron Transport ChainSarcolemmaMuscle FibersSaponin-skinned Cardiac FibersOxygen Consumption Measurements
Cite This Article
Hughey, C. C., Hittel, D. S., Johnsen, V. L., Shearer, J. Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers. J. Vis. Exp. (48), e2431, doi:10.3791/2431 (2011).