为葡萄糖和脂肪酸氧化率在大鼠离体的测定方法
Summary
The following protocol describes the preparation and utilization of buffers for the quantitative measurement of rates of glucose and fatty acid oxidation in the isolated working rat heart. The methods used for sample analysis and data interpretation are also discussed.
Abstract
哺乳动物的心脏是ATP的主要消费和需要能量底物进行收缩的稳定供应。不足为奇的是，心肌代谢的改变已被链接到的收缩功能障碍和心脏衰竭的发展。因此，解开新陈代谢和收缩之间的联系应该在某些领导在疾病状态下心脏适应或适应不良的机制线索。分离的工作大鼠心脏制剂可用于跟踪，同时和实时，心脏收缩功能和能量提供基板到氧化代谢途径的通量。本协议的目的是提供在缓冲器的氧化的葡萄糖和脂肪酸，主要能量提供所述心脏的基板用率的定量测量的制备和利用所采用的方法的详细描述。还讨论了用于样品分析和数据解释的方法。简言之，该技术是基于通过常温晶体灌注的14供给的C放射性标记的葡萄糖和3 H-放射性标记长链脂肪酸对离体心脏跳动。14 CO 2和3 H 2 0，结束副产物参与这些提供能量的基板的利用酶反应，然后定量地从冠状动脉流出物中回收。与所使用的放射性标记的底物的比活性的知识，那么有可能单独定量葡萄糖和脂肪酸的通量在氧化途径。离体心脏的收缩功能可以并行确定与适当的记录设备和直接相关代谢通量的值。该技术是研究响应于各种胁迫条件的代谢/收缩关系极为有用，如在前和负荷和缺血，药物或circula之后改变廷因子，或者在基因产物的表达的改变如下。
Introduction
临床相关性
在哺乳动物心脏，有基材的通量之间通过氧化代谢途径，ATP的生成和心脏工作1较强的正相关关系。在过去的二十年中，心脏代谢和功能之间的错综复杂的链路的调查已经导致认识到，在心脏代谢的改变是用于在不同类型的心脏疾病2-4的设置收缩功能障碍和可能的病理结构重建的原因。因此，可以预期，我们的管辖强调心脏的代谢重构机制的理解会导致心脏衰竭5-7的预防或治疗的治疗靶的鉴定。从美国心脏协会科学语句的“评估心脏代谢”的最近的出版物强调了科学界的T的兴趣与日俱增他的研究领域8。不过，虽然在心脏成像的技术进步现在允许心脏形态和功能的一个快速，准确的评估，心脏代谢的体内研究仍然有限和繁重：核磁共振（NMR）谱和正电子发射断层扫描（PET）成像可以用来跟随心脏高能磷酸代谢和克雷布斯循环的活性，但这些技术都受到高的操作费用和他们无法困扰，以确定各种基材在稳态条件9的贡献氧化代谢。对此日期体外工作心脏制备代表鞋底和独特的技术可用来研究，同时，实时，收缩功能和底物的助熔剂装入氧化代谢途径7,9。以下方案的目的在于提供在用于确定大鼠试剂的制备和使用的指南在大鼠离体心脏基板利用率上课。
隔离工作鼠类心脏仪器
虽然技术是近半个世纪之久，隔离工作大鼠心脏的准备仍然首选心血管研究的方法。由于与心脏的Langendorff编制，工作啮齿动物心脏提供了一个相对简单，可靠和廉价的方式从其他器官，神经内分泌等循环因子的混杂效应独立测量范围广泛的心脏参数。但与此相反的的Langendorff灌注心脏，工作心脏继续执行近生理心脏工作，为氧化代谢通量的产生是相关的体内条件级的先决条件。这是通过经由连接到左心房的插管递送灌注缓冲液至左心室（LV）的实现，并作为低压填充和合同，该缓冲器是通过对一个确定的负荷静水压力主动脉线喷射。最初由尼利和他的同事描述的10后来被Taegtmeyer，折边和克雷布斯11好转，但灌注设备的设计至今变化不大。如在原始设备描述的，收缩功能可以通过测定心输出量的评估，使用不超过量筒和秒表来测量主动脉和冠状动脉流10,11。一些厂商现在提供完整的工作啮齿动物心脏灌注系统。这些可商购的装置可与flowprobes，压力传感器，一个压力 – 体积导管和必需的所有心功能数据采集和分析的设备来获得。该供应商提供大量的文档和培训课程，熟悉他们的设备新用户。一些评论文章还详细协议的工作心脏仪器和设备行进，而对使用导管来测量在啮齿类动物12-15心脏功能。因为这个原因，我们将只简要地提及灌注装置和记录设备的设置。本协议，而旨在用的，可以被实现同时测量葡萄糖和长链脂肪酸的氧化的速率，在正常心脏两大能量提供衬底的方法的描述，以补充已经可用的信息。我们在这里描述的所有参与利用放射性标记的能量底物的心肌氧化代谢的评估的步骤，从试剂和缓冲液，以样品的回收和处理的制备中，对数据的分析。
该方法的原理
心肌细胞产生它们的能量的对收缩从脂肪酸（主要的长链脂肪酸）和碳水化合物的氧化磷酸化的体（glucose和乳酸）。心脏有非常有限的精力充沛的储量依赖于从循环这些能源提供基板的稳定供应。葡萄糖通过糖酵解途径分解代谢产生丙酮酸，然后通过线粒体内膜的丙酮酸脱氢酶复合脱羧。长链脂肪酸，从循环白蛋白或脂蛋白甘油三酯萃取，首先活化成酰基-CoA分子在细胞质中，并随后输送的线粒体基质内通过肉碱穿梭进入β-氧化途径。由葡萄糖和脂肪酸分解代谢产生的乙酰-CoA分子燃料三羧酸循环来产生其用于通过电子传递链来构建跨线粒体内膜的质子动力的还原当量（NADH和FADH 2）和通过ATP合酶的活性产生的ATP。水和二氧化碳是的端部的副产物酶反应发生的克雷布斯循环回路内。的14 C和3 H-放射性标记的底物的供给（如14 C-放射性标记的葡萄糖和3 H-放射性标记油酸）的分离工作心脏将因此导致生产14 CO 2和3 的 H 2的O其可以定量地从冠脉流出液回收。 14 CO 2的集合由保持离体灌注心脏成密封室以及由冠状动脉流出物在离开心脏立即恢复进行。一个小的阴离子交换柱用于分离和从冠状动脉流出物中回收3 H 2 O操作。从经处理的样品的放射活性用液体闪烁计数器测定，并与所使用的放射性标记的底物的比活性的知识，那么有可能单独定量葡萄糖和脂肪酸中的磁通氧化通路16,17。
Protocol
Representative Results
Discussion
前述协议的细节的方法，通过在隔离工作大鼠心脏葡萄糖氧化和脂肪酸氧化同时量化底物的通量。然后测量可以叠加到所记录的心功能参数，以确定基线和压力条件下，底物的代谢和心脏工作的关系（在工作量的变化，缺血再灌注等 ）。它也可以评估如何新陈代谢/收缩关系由先前存在的条件，如心脏衰竭和糖尿病的影响。此外，转基因啮齿动物的心脏可用于询问特定蛋白质对心脏代谢和?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This work was supported by National Institutes of Health Grants R00 HL112952 (to R. H.), R01 HL108618 (to J.P.G.), P01 HL051971, and P20 GM104357. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Materials
| Sodium Chloride (NaCl) | Fisher Scientific | BP358 | |
| Potassium Chloride (KCl) | Fisher Scientific | BP366 | |
| Potassium Phosphate Monobasic (KH2PO4) | Fisher Scientific | P284 | |
| Magnesium Sulfate Heptahydrate (MgSO4*7H2O) | Fisher Scientific | M63 | |
| Sodium Bicarbonate (NaHCO3) | Fisher Scientific | S233 | |
| Calcium Chloride (CaCl2) | Sigma-Aldrich | C5670 | |
| AG 1-X8 resin, chloride form, 100-200 dry mesh size, 500 g | Bio-Rad | 1401441 | This item can be replaced by purchasing directly the hydoxide form (see reference below), but this will cost almost 8 times more |
| AG 1-X8 resin, hydroxide form, 100-200 dry mesh size, 100 g | Bio-Rad | 1432445 | Purchasing this item allows to bypass the conversion of the anion exchange resin from the chloride form to the hydroxide form (See section 1.2 of protocol) |
| Glass Microanalysis Vacuum Filter Holder | Fisher Scientific | 09-753-2 | |
| Sodium Hydroxide (NaOH) | Fisher Scientific | S318 | Corrosive. Consult the product MSDS for appropriate handling and storage. |
| Gas Dispersion Tube with Fritted Cylinder | Fisher Scientific | 11-138B | |
| Probumin Bovine Serum Albumin Fatty Acid Free, Powder | EMD Millipore | 820027 | We recommend the use of a charcoal-defatted BSA, as other purification process such as cold ethanol fractionation may leave residues toxic for the heart. |
| Sodium Oleate | Sigma-Aldrich | O7501 | |
| Oleic Acid, [9,10-3H(N)]- | PerkinElmer | NET289005MC | Radioactive material. Follow your Institution's radiation safety office guidelines for ordering and handling. |
| Dialysis Membrane Tubing, 29 mm diameter | Fisher Scientific | 08-667E | |
| D-(+)-Glucose | Sigma-Aldrich | G7021 | |
| Glucose, D-[14C(U)]- | PerkinElmer | NEC042B005MC | Radioactive material. Follow your Institution's radiation safety office guidelines for ordering and handling. |
| Humulin R U-100 | Eli Lilly and Company | NDC 0002-8215-01 (HI-210) | |
| Inactin Hydrate | Sigma-Aldrich | T133 | Controlled substance on USDEA Schedule III |
| 3-0 Silk Black Braid | Roboz Surgical | SUT-15-3 | |
| 10X Hyamine Hydroxide | PerkinElmer | 6003005 | Highly toxic and causes severe burns. Consult the product MSDS for appropriate handling and storage |
| 20 mL Glass Scintillation Vials | Fisher Scientific | 03-341-25E | Use glass vials for quantitative recovery of 14CO2 |
| 20 mL HDPE Scintillation Vials | Fisher Scientific | 03-337-23B | Use HDPE vials for quantitative recovery of 3H2O |
| Red Rubber Sleeve Stoppers | Fisher Scientific | 14-126DD | Fit 20 mL scintillation vials; Reusable |
| BD PrecisionGlide Needle 23G x 40 mm | BD | 305194 | Use to inject perchloric acid through the rubber sleeve stopper of the CO2 trap |
| Perchloric Acid, 60% | Fisher Scientific | A228 | Highly corrosive and may act as an oxidizer and/or cause an explosion hazard. Consult the product MSDS for appropriate handling and storage |
| Ultima Gold, Scintillation Cocktail | PerkinElmer | 6013327 | |
| Glass Wool | Fisher Scientific | AC38606 | |
| Decon Dri-Clean Detergent Powder | Fisher Scientific | 04-355 | For cleaning of glassware, plastic parts, and tubing |
| Alconox Tergazyme Enzyme-Active Powered Detergent | Fisher Scientific | 16-000-115 | For cleaning of "hard to reach" surfaces (tubing, glassware) contaminated by fatty acid-BSA residue |
Referencias
- Neely, J. R., Morgan, H. E. Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Annu Rev Physiol. 36, 413-459 (1974).
- Sen, S., et al. Glucose regulation of load-induced mTOR signaling and ER stress in mammalian heart. J Am Heart Assoc. 2, e004796 (2013).
- Young, M. E., McNulty, P., Taegtmeyer, H. Adaptation and maladaptation of the heart in diabetes: Part II: potential mechanisms. Circulation. 105, 1861-1870 (2002).
- Stanley, W. C., Recchia, F. A., Lopaschuk, G. D. Myocardial substrate metabolism in the normal and failing heart. Physiol Rev. 85, 1093-1129 (2005).
- Fillmore, N., Lopaschuk, G. D. Targeting mitochondrial oxidative metabolism as an approach to treat heart failure. Biochim Biophys Acta. 1833, 857-865 (2013).
- Jaswal, J. S., Keung, W., Wang, W., Ussher, J. R., Lopaschuk, G. D. Targeting fatty acid and carbohydrate oxidation–a novel therapeutic intervention in the ischemic and failing heart. Biochim Biophys Acta. 1813, 1333-1350 (2011).
- Taegtmeyer, H. Cardiac metabolism as a target for the treatment of heart failure. Circulation. 110, 894-896 (2004).
- Taegtmeyer, H., et al. Assessing Cardiac Metabolism: A Scientific Statement From the American Heart Association. Circ Res. , (2016).
- Barr, R. L., Lopaschuk, G. D. Methodology for measuring in vitro/ex vivo cardiac energy metabolism. J Pharmacol Toxicol Methods. 43, 141-152 (2000).
- Neely, J. R., Liebermeister, H., Battersby, E. J., Morgan, H. E. Effect of pressure development on oxygen consumption by isolated rat heart. Am J Physiol. 212, 804-814 (1967).
- Taegtmeyer, H., Hems, R., Krebs, H. A. Utilization of energy-providing substrates in the isolated working rat heart. Biochem J. 186, 701-711 (1980).
- Liao, R., Podesser, B. K., Lim, C. C. The continuing evolution of the Langendorff and ejecting murine heart: new advances in cardiac phenotyping. Am J Physiol Heart Circ Physiol. 303, H156-H167 (2012).
- Cingolani, O. H., Kass, D. A. Pressure-volume relation analysis of mouse ventricular function. Am J Physiol Heart Circ Physiol. 301, H2198-H2206 (2011).
- Pacher, P., Nagayama, T., Mukhopadhyay, P., Batkai, S., Kass, D. A. Measurement of cardiac function using pressure-volume conductance catheter technique in mice and rats. Nat Protoc. 3, 1422-1434 (2008).
- Abraham, D., Mao, L. Cardiac Pressure-Volume Loop Analysis Using Conductance Catheters in Mice. J Vis Exp. , (2015).
- Harmancey, R., et al. Insulin resistance improves metabolic and contractile efficiency in stressed rat heart. FASEB J. 26, 3118-3126 (2012).
- Harmancey, R., Vasquez, H. G., Guthrie, P. H., Taegtmeyer, H. Decreased long-chain fatty acid oxidation impairs postischemic recovery of the insulin-resistant rat heart. FASEB J. 27, 3966-3978 (2013).
- Goodwin, G. W., Taylor, C. S., Taegtmeyer, H. Regulation of energy metabolism of the heart during acute increase in heart work. J Biol Chem. 273, 29530-29539 (1998).
- Lopaschuk, G. D., Ussher, J. R., Folmes, C. D., Jaswal, J. S., Stanley, W. C. Myocardial fatty acid metabolism in health and disease. Physiol Rev. 90, 207-258 (2010).
- Neely, J. R., Denton, R. M., England, P. J., Randle, P. J. The effects of increased heart work on the tricarboxylate cycle and its interactions with glycolysis in the perfused rat heart. Biochem J. 128, 147-159 (1972).
- Katz, J., Dunn, A. Glucose-2-t as a tracer for glucose metabolism. Bioquímica. 6, 1-5 (1967).
- Gillis, A. M., Kulisz, E., Mathison, H. J. Cardiac electrophysiological variables in blood-perfused and buffer-perfused, isolated, working rabbit heart. Am J Physiol. 271, H784-H789 (1996).
- Qiu, Y., Hearse, D. J. Comparison of ischemic vulnerability and responsiveness to cardioplegic protection in crystalloid-perfused versus blood-perfused hearts. J Thorac Cardiovasc Surg. 103, 960-968 (1992).
- Cotter, D. G., Schugar, R. C., Crawford, P. A. Ketone body metabolism and cardiovascular disease. Am J Physiol Heart Circ Physiol. 304, H1060-H1076 (2013).
- Huang, Y., Zhou, M., Sun, H., Wang, Y. Branched-chain amino acid metabolism in heart disease: an epiphenomenon or a real culprit?. Cardiovasc Res. 90, 220-223 (2011).
- Buse, M. G., Biggers, J. F., Friderici, K. H., Buse, J. F. Oxidation of branched chain amino acids by isolated hearts and diaphragms of the rat. The effect of fatty acids, glucose, and pyruvate respiration. J Biol Chem. 247, 8085-8096 (1972).
- Liepinsh, E., et al. The heart is better protected against myocardial infarction in the fed state compared to the fasted state. Metabolism. 63, 127-136 (2014).
- Niu, Y. G., Hauton, D., Evans, R. D. Utilization of triacylglycerol-rich lipoproteins by the working rat heart: routes of uptake and metabolic fates. J Physiol. 558, 225-237 (2004).
- Goodwin, G. W., Arteaga, J. R., Taegtmeyer, H. Glycogen turnover in the isolated working rat heart. J Biol Chem. 270, 9234-9240 (1995).
- Sender, P. M., Garlick, P. J. Synthesis rates of protein in the Langendorff-perfused rat heart in the presence and absence of insulin, and in the working heart. Biochem J. 132, 603-608 (1973).
- Hindlycke, M., Jansson, L. Glucose tolerance and pancreatic islet blood flow in rats after intraperitoneal administration of different anesthetic drugs. Ups J Med Sci. 97, 27-35 (1992).
- Zuurbier, C. J., Keijzers, P. J., Koeman, A., Van Wezel, H. B., Hollmann, M. W. Anesthesia’s effects on plasma glucose and insulin and cardiac hexokinase at similar hemodynamics and without major surgical stress in fed rats. Anesth Analg. 106, 135-142 (2008).
- Oguchi, T., Kashimoto, S., Yamaguchi, T., Nakamura, T., Kumazawa, T. Is pentobarbital appropriate for basal anesthesia in the working rat heart model?. J Pharmacol Toxicol Methods. 29, 37-43 (1993).
- Segal, J., Schwalb, H., Shmorak, V., Uretzky, G. Effect of anesthesia on cardiac function and response in the perfused rat heart. J Mol Cell Cardiol. 22, 1317-1324 (1990).
- Webster, I., Smith, A., Lochner, A., Huisamen, B. Sanguinarine non- versus re-circulation during isolated heart perfusion–a Jekyll and Hyde effect?. Cardiovasc Drugs Ther. 28, 489-491 (2014).
- Belke, D. D., Larsen, T. S., Lopaschuk, G. D., Severson, D. L. Glucose and fatty acid metabolism in the isolated working mouse heart. Am J Physiol. 277, R1210-R1217 (1999).
- Iannaccone, P. M., Jacob, H. J. Rats! . Dis Model Mech. 2, 206-210 (2009).
