用超极化[1-13C]丙酮酸和 13C / 31P NMR波谱研究分离灌注小鼠心脏中的心脏代谢
Summary
我们描述了一种实验装置，用于在连续灌注模式下向分离的灌注小鼠心脏施用超极化 13种C标记代谢物。专用的 13C-NMR采集方法能够实时定量代谢酶活性，多参数 31P-NMR分析可以测定组织ATP含量和pH值。
Abstract
新陈代谢是细胞生命重要过程的基础。表征代谢网络如何在活组织中发挥作用，为理解疾病的机制和设计治疗方法提供了关键信息。在这项工作中，我们描述了实时研究逆行灌注小鼠心脏中细胞内代谢活动的程序和方法。将心脏原位分离，结合心脏骤停以尽量减少心肌缺血，并在核磁共振（NMR）波谱仪内灌注。在光谱仪和连续灌注下，将超极化[1-13 C]丙酮酸施用于心脏，随后的超极化[1-13C]乳酸和[13C]碳酸氢盐产生速率用于实时确定乳酸脱氢酶和丙酮酸脱氢酶的速率。超极化[1-13C]丙酮酸的这种代谢活性使用产物选择性饱和激发采集方法以模型自由方式定量。31在超极化采集之间应用P光谱来监测心脏能量和pH值。该系统对于研究健康和患病小鼠心脏中的代谢活动具有独特的用途。
Introduction
心脏代谢的改变与多种心肌病有关，并且通常构成潜在病理生理机制的基础1。然而，研究活组织中的代谢存在许多障碍，因为大多数生化测定需要组织和细胞裂解和/或放射性示踪的均质化。因此，迫切需要新的工具来研究活组织中的心肌代谢。超极化 13个 C 标记底物的磁共振 （MR） 允许实时测量活组织2 中的代谢，而无需使用电离辐射，方法是将标记位点的 MR 信噪比提高几个数量级3。在这里，我们描述了一种实验装置，一种采集方法和分析方法，用于研究分离的小鼠心脏中的快速代谢，并同时呈现一般组织能量和酸度的指标。心脏pH值是一个有价值的指标，因为在心脏疾病和心肌缺血、适应不良肥大和心力衰竭等疾病的早期阶段，酸碱平衡被破坏了6。
超极化[1-13C]乳酸和[13 C]碳酸氢盐从超极化[1-13C]丙酮酸产生有助于确定乳酸脱氢酶（LDH）和丙酮酸脱氢酶（PDH）的产生速率。以前在分离的啮齿动物心脏中使用超极化底物进行的大多数研究要么使用复杂的动力学模型来推导出LDH和PDH的酶活性，要么报告超极化产物与底物的信号强度比，而不计算实际的酶活性率2，4，5，6，7，8，9，10，11，12，13，14.在这里，我们使用了产物选择性饱和激发方法15，该方法允许以无模型的方式监测酶活性15，16。通过这种方式，确定了绝对酶促速率（即每单位时间内产生的产物摩尔数）。31采用磷谱观察无机磷酸盐（Pi）、磷酸肌酸（PCr）和三磷酸腺苷（ATP）的信号。使用多参数分析来表征心脏的pH分布，如组织Pi信号中的异质化学变化所证明的那样。
逆行灌注的小鼠心脏（Langendorff心脏）17，18，19是完整跳动心脏的离体模型。在该模型中，心脏活力和pH值至少保留80分钟20，并且在长期缺血性损伤后显示出恢复的潜力21，22。然而，显微手术过程中的无意变异可能导致心脏组织活力的变异。以前的研究报告了这颗心脏随着时间的推移而恶化19;例如，已经观察到每小时收缩功能降低5%-10%18。三磷酸腺苷（ATP）信号先前已被证明可以报告心肌能量状态和活力23。在这里，我们注意到灌注的心脏可能偶尔会显示出活力水平的无意变化，正如ATP含量所证明的那样，尽管我们有不间断的灌注和氧气供应。我们在这里证明，将LDH和PDH率标准化为心脏的ATP含量可以减少这些速率的心间变异性。
在下面的协议中，我们描述了用于在NMR波谱仪中进行心脏插管，分离和随后灌注的外科手术。值得注意的是，其他旨在分离和灌注小鼠心脏的手术方法已在24，25之前描述。
还描述了用于获取与跳动心脏中的酶速率（使用13 C波谱和超极化[1-13C]丙酮酸）以及心脏活力和酸度（使用31P NMR波谱）相关的数据的方法。最后，解释了测定代谢酶活性以及组织活力和酸度的分析方法。
Protocol
希伯来大学和哈达萨医学中心的联合伦理委员会（IACUC）批准了动物福利研究方案（MD-19-15827-1）。 1. 克雷布斯-亨赛莱特缓冲液制备 实验前一天，准备克雷布斯-亨塞莱特缓冲液（KHB）26的修改版本。最初，将 118 mM NaCl、4.7 mM KCl、0.5 mM 丙酮酸、1.2 mM MgSO 4、25 mM NaHCO3 和 1.2 mM KH 2 PO4 溶解在双蒸 H2O 中。</s…
Representative Results
从注入KHB的小鼠心脏和仅从缓冲液记录的 31P谱图如图 1A所示。在心脏中观察到α，β和γ-ATP，PCr和Pi的信号。Pi信号由两个主要成分组成：在较高的场（信号的左侧），Pi信号主要是由于pH值为7.4时的KHB;在较低的场（信号的右侧），由于酸性环境较强，Pi信号更宽且更不均匀。后一种模式起源于心脏组织。通过减去KHB的Pi信号（图1B）提取心脏组织…
Discussion
我们展示了一种实验装置，旨在研究分离的小鼠心脏模型中的超极化[1-13C]丙酮酸代谢，组织能量和pH值。
方案中的关键步骤如下：1）确保缓冲液的pH值为7.4;2）确保包括缓冲液的所有成分;3）通过注射肝素避免心脏血管中的血液凝固;4）通过降低代谢活动（KCl注射和冰冷缓冲液）避免对心脏的缺血性损害;5）避免在手术的任何时候将气泡引入心脏;6）通过组织的颜色验?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
该项目根据第1379/18号赠款协议获得以色列科学基金会的资助;以色列科学技术部应用和工程科学雅博廷斯基奖学金，直接博士生编号3-15892;以及欧盟根据第 858149 号赠款协议（AlternativesToGd）实施的地平线 2020 研究和创新计划。
Materials
| Equipment | |||
| HyperSense DNP Polariser | Oxford Instruments | 52-ZNP91000 | HyperSense, 3.35 T, preclinical dissolution-DNP hyperpolarizer |
| NMR spectrometer | RS2D | NMR Cube, 5.8 T, equiped with a 10 mm broad-band probe | |
| Peristaltic pump | Cole-Parmer | 07554-95 | |
| Temperature probe | Osensa | FTX-100-LUX+ | NMR compatible temprature probe |
| Somnosuite low-flow anesthesia system | Kent Scientific | ||
| Lines, tubings, suture | |||
| Platinum cured silicone tubes | Cole-Parmer | HV-96119-16 | L/S 16 I.D. 3.1 mm |
| Thin polyether ether ketone (PEEK) lines | Upchurch Scientific | id. 0.040” | |
| Intravenous catheter | BD Medical | 381323 | 22 G |
| Silk suture | Ethicon | W577H | Wire diameter of 3-0 |
| Chemicals and pharmaceuticals | |||
| [1-13C]pyruvic acid | Cambridge Isotope Laboratories | CLM-8077-1 | |
| Calcium chloride | Sigma-Aldrich | 21074 | CAS: 10043-52-4 |
| D-(+)-Glucose | Sigma-Aldrich | G7528 | CAS: 50-99-77 |
| Heparin sodium | Rotexmedica | HEP5A0130C0160 | |
| Hydrochloric acid 37% | Sigma-Aldrich | 258148 | CAS: 7647-01-0 |
| Insulin aspart (NovoLog) | Novo Nordisk | ||
| Isoflurane | Terrel | ||
| Magnesium Sulfate | Sigma-Aldrich | 793612 | CAS: 7487-88-9 |
| Potassium chloride | Sigma-Aldrich | P4504 | CAS: 7447-40-7 |
| Potassium phosphate monobasic | Sigma-Aldrich | P9791 | CAS: 7778-77-0 |
| Sodium bicarbonate | Gadot Group | CAS: 144-55-8 | |
| Sodium chloride | Sigma-Aldrich | S9625 | CAS: 7647-14-5 |
| Sodium hydroxide | Sigma-Aldrich | 655104 | CAS: 1310-73-2 |
| Sodium phosphate dibasic | Sigma Aldrich | S7907 | CAS: 7558-79-4 |
| Sodium phosphate monbasic dihydrate | Merck | 6345 | CAS: 13472-35-0 |
| TRIS (biotechnology grade) | Amresco | 0826 | CAS: 77-86-1 |
| Trityl radical OX063 | GE Healthcare AS | NC100136 | OX063 |
| NMR standards | |||
| 13C standard sample | Cambridge Isotope Laboratories | DLM-72A | 40% p-dioxane in benzene-D6 |
| 31P standard sample | Made in house | 105 mM ATP and 120 mM phenylphosphonic acid in D2O | |
| Software | |||
| Excel 2016 | Microsoft | ||
| MNova | Mestrelab Research |
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Citer Cet Article
Shaul, D., Sapir, G., Lev-Cohain, N., Sosna, J., Gomori, J. M., Katz-Brull, R. Investigating Cardiac Metabolism in the Isolated Perfused Mouse Heart with Hyperpolarized [1-13C]Pyruvate and 13C/31P NMR Spectroscopy. J. Vis. Exp. (194), e63188, doi:10.3791/63188 (2023).