马骨骼肌的高分辨率氟呼吸测定法
Summary
马具有非凡的有氧运动能力，使马骨骼肌成为研究马运动生理学和哺乳动物线粒体生理学的重要组织。本文介绍了全面评估马骨骼肌线粒体功能的技术。
Abstract
线粒体功能——氧化磷酸化和活性氧的产生——对健康和疾病都至关重要。因此，测量线粒体功能是生物医学研究的基础。骨骼肌是线粒体的强大来源，特别是在具有非常高有氧能力的动物中，例如马，使其成为研究线粒体生理学的理想对象。本文演示了使用高分辨率呼吸测量法与同步荧光测定法，以及新鲜收获的骨骼肌线粒体，以量化不同线粒体状态下氧化底物的能力，并确定线粒体呼吸不同元素的相对能力。四甲基罗丹明甲酯用于证明由底物氧化产生的线粒体膜电位的产生，包括通过计算每单位并发氧通量产生的相对膜电位来计算线粒体的相对效率。由于腺苷酸对镁的亲和力不同，ADP转化为ATP导致反应室中镁浓度的变化。因此，镁绿可用于测量ATP合成速率，从而可以进一步计算氧化磷酸化效率（磷酸化与氧化的比率[P / O]）。最后，使用Amplex UltraRed与过氧化氢结合时产生荧光产物（试卤灵），可以量化线粒体呼吸过程中活性氧的产生，以及ROS产生与同时呼吸之间的关系。这些技术允许在各种不同的模拟条件下对线粒体生理学进行稳健的量化，从而揭示了这种关键细胞成分对健康和疾病的贡献。
Introduction
真核细胞的线粒体产生细胞用于工作和维持的大部分ATP1。ATP线粒体产生的关键步骤是将氧气转化为水，因此线粒体和相关细胞的代谢能力经常通过测量氧气消耗量来量化2。然而，线粒体生理学比简单的耗氧过程更复杂，并且仅依赖该终点提供了对线粒体功能和功能障碍对细胞健康影响的不完整评估。线粒体功能的全面表征不仅需要评估耗氧量，还需要评估ATP的产生以及活性氧（ROS）。
关键线粒体功能的额外测量可以与通过使用特定荧光团测量呼吸同时完成。四甲基罗丹明甲酯（TMRM）是一种阳离子荧光团，其在线粒体基质中与线粒体跨膜电压电位成比例地积累，导致由于这种积累而降低荧光强度3。TMRM可用作线粒体膜电位相对变化的指标，或可用于量化跨膜电压的精确变化，并进行额外的实验以确定允许将荧光信号转换为mV的常数。镁绿（MgG）是一种荧光团，与Mg2+结合时会发出荧光，用于根据ADP和ATP对镁二价阳离子4的差异亲和力测量ATP合成。研究人员必须确定在特定分析条件下ADP和ATP的特定亲和力/解离常数（Kd），以将MgG荧光的变化转化为ATP浓度的变化。Amplex UltraRed （AmR） 是用于测量线粒体呼吸过程中过氧化氢和其他 ROS 产生的荧光团5。H2O2 和AmR（由辣根过氧化物酶催化）之间的反应产生试卤灵，可通过530nM的荧光检测到。这些测定中的每一个都可以单独添加到实时线粒体呼吸测定中，以同时测量线粒体生理学的各个方面，从而提供呼吸和线粒体输出之间的直接联系。
马能够产生非常高的质量比氧消耗率，部分原因是马骨骼肌的线粒体含量非常高，这使得这种组织与研究线粒体生理学高度相关。随着高分辨率呼吸测量法的发展，使用这种新技术的研究有助于确定马骨骼肌线粒体对马显着运动能力和骨骼肌疾病的病理生理学的贡献6，7，8，9，10，11，12，13，14.对马骨骼肌线粒体功能的研究特别有利，因为获得大量的这种组织是非终末性的。因此，马受试者不仅可以为线粒体功能的完整表征提供足够的组织，还可以作为线粒体生理学高质量机械研究的纵向对照。出于这个原因，已经开发了额外的测定来量化线粒体膜电位，ATP合成和ROS的产生，以补充该组织中氧气消耗量的测量，以便提供更可靠的线粒体生理学表征马骨骼肌。
Protocol
Representative Results
Discussion
在高分辨率呼吸计的标准输出中添加荧光信号可提供有关线粒体生理学的宝贵信息，但对荧光信号进行细致校准对于高质量数据至关重要。使用MgG的原始方案表明，计算镁-腺苷酸解离常数时产生的校准曲线可以应用于后续测定4;然而，对于这种方法，来自MgG的荧光信号在测定之间可能没有足够的重现性。因此，每次测定都使用自动 MgCl2 滴定进行校准，以及用于计算该单?…
Declarações
The authors have nothing to disclose.
Acknowledgements
作者要感谢约翰和黛比奥克斯利马运动医学捐赠主席和格雷森赛马会研究基金会的慷慨支持。
Materials
| ADP | Sigma-Aldrich (MilliporeSigma) | A5285 | |
| Amplex UltraRed | Life Technologies | A36006 | |
| ATP | Sigma-Aldrich (MilliporeSigma) | A2383 | |
| BSA | Sigma-Aldrich (MilliporeSigma) | A6003 | |
| Calcium carbonate | Sigma-Aldrich (MilliporeSigma) | C4830 | |
| CCCP | Sigma-Aldrich (MilliporeSigma) | C2759 | |
| DatLab 7.0 | Oroboros Inc | Software to operate O2K fluororespirometer | |
| Dithiothreitol | Sigma-Aldrich (MilliporeSigma) | D0632 | |
| DTPA | Sigma-Aldrich (MilliporeSigma) | D1133 | |
| EGTA | Sigma-Aldrich (MilliporeSigma) | E4378 | |
| Glutamate | Sigma-Aldrich (MilliporeSigma) | G1626 | |
| HEPES | Sigma-Aldrich (MilliporeSigma) | H7523 | |
| Horseradish peroxidase | Sigma-Aldrich (MilliporeSigma) | P8250 | |
| Hydrogen peroxide | Sigma-Aldrich (MilliporeSigma) | 516813 | Must be made fresh daily prior to assay |
| Imidazole | Sigma-Aldrich (MilliporeSigma) | I2399 | |
| K-MES | Sigma-Aldrich (MilliporeSigma) | M8250 | |
| Magnesium chloride hexahydrate | Sigma-Aldrich (MilliporeSigma) | M9272 | |
| Magnesium Green | Thermo Fisher Scientific | M3733 | |
| Malate | Sigma-Aldrich (MilliporeSigma) | M1000 | |
| Mannitol | Sigma-Aldrich (MilliporeSigma) | M9647 | |
| Mitochondrial isolation kit | Sigma-Aldrich (MilliporeSigma) | MITOISO1 | |
| O2K fluororespirometer | Oroboros Inc | Multiple units required to run full spectrum of assays concurrently. | |
| Phosphocreatine | Sigma-Aldrich (MilliporeSigma) | P7936 | |
| Potassium hydroxide | Sigma-Aldrich (MilliporeSigma) | P1767 | |
| Potassium lactobionate | Sigma-Aldrich (MilliporeSigma) | L2398 | |
| Potassium phosphate | Sigma-Aldrich (MilliporeSigma) | P0662 | |
| Pyruvate | Sigma-Aldrich (MilliporeSigma) | P2256 | Must be made fresh daily prior to assay |
| Rotenone | Sigma-Aldrich (MilliporeSigma) | R8875 | |
| Succinate | Sigma-Aldrich (MilliporeSigma) | S2378 | |
| Sucrose | Sigma-Aldrich (MilliporeSigma) | 84097 | |
| Superoxide dismutase | Sigma-Aldrich (MilliporeSigma) | S8160 | |
| Taurine | Sigma-Aldrich (MilliporeSigma) | T0625 | |
| Titration pump | Oroboros Inc | ||
| Titration syringes | Oroboros Inc | ||
| TMRM | Sigma-Aldrich (MilliporeSigma) | T5428 | |
| UCH biopsy needle | Millenium Surgical Corp | 72-238067 | Available in a range of sizes |
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