Name: methods for the determination of rates of glucose and fatty acid oxidation in the isolated working rat heart
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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.

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The authors declare that they have no competing financial interests.

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?...

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。

<table><tbody><tr><td>Sodium Chloride (NaCl)</td><td>Fisher Scientific</td><td>BP358</td><td></td></tr><tr><td>Potassium Chloride (KCl)</td><td>Fisher Scientific</td><td>BP366</td><td></td></tr><tr><td>Potassium Phosphate Monobasic (KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>P284</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate (MgSO&lt;sub&gt;4&lt;/sub&gt;*7H&lt;sub&gt;2&lt;/sub&gt;O)</td><td>Fisher Scientific</td><td>M63</td><td></td></tr><tr><td>Sodium Bicarbonate (NaHCO&lt;sub&gt;3&lt;/sub&gt;)</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>Calcium Chloride (CaCl&lt;sub&gt;2&lt;/sub&gt;)</td><td>Sigma-Aldrich</td><td>C5670</td><td></td></tr><tr><td>AG 1-X8 resin, chloride form, 100 - 200 dry mesh size, 500 g</td><td>Bio-Rad</td><td>1401441</td><td>This item can be replaced by purchasing directly the hydoxide form&amp;nbsp; (see reference below), but this will cost almost 8 times more</td></tr><tr><td>AG 1-X8 resin, hydroxide form, 100 - 200 dry mesh size, 100 g</td><td>Bio-Rad</td><td>1432445</td><td>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)</td></tr><tr><td>Glass Microanalysis Vacuum Filter Holder</td><td>Fisher Scientific</td><td>09-753-2</td><td></td></tr><tr><td>Sodium Hydroxide (NaOH)</td><td>Fisher Scientific</td><td>S318</td><td>Corrosive. Consult the product MSDS for appropriate handling and storage.</td></tr><tr><td>Gas Dispersion Tube with Fritted Cylinder</td><td>Fisher Scientific</td><td>11-138B</td><td></td></tr><tr><td>Probumin Bovine Serum Albumin Fatty Acid Free, Powder</td><td>EMD Millipore</td><td>820027</td><td>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.</td></tr><tr><td>Sodium Oleate</td><td>Sigma-Aldrich</td><td>O7501</td><td></td></tr><tr><td>Oleic Acid, [9,10-&lt;sup&gt;3&lt;/sup&gt;H(N)]-</td><td>PerkinElmer</td><td>NET289005MC</td><td>Radioactive material. Follow your Institution&#039;s radiation safety office guidelines for ordering and handling.</td></tr><tr><td>Dialysis Membrane Tubing, 29 mm diameter</td><td>Fisher Scientific</td><td>08-667E</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Sigma-Aldrich</td><td>G7021</td><td></td></tr><tr><td>Glucose, D-[14C(U)]-</td><td>PerkinElmer</td><td>NEC042B005MC</td><td>Radioactive material. Follow your Institution&#039;s radiation safety office guidelines for ordering and handling.</td></tr><tr><td>Humulin R U-100</td><td>Eli Lilly and Company</td><td>NDC 0002-8215-01 (HI-210)</td><td></td></tr><tr><td>Inactin Hydrate</td><td>Sigma-Aldrich</td><td>T133</td><td>Controlled substance on USDEA Schedule III</td></tr><tr><td>3-0 Silk Black Braid</td><td>Roboz Surgical</td><td>SUT-15-3</td><td></td></tr><tr><td>10x Hyamine Hydroxide</td><td>PerkinElmer</td><td>6003005</td><td>Highly toxic and causes severe burns. Consult the product MSDS for appropriate handling and storage</td></tr><tr><td>20 ml Glass Scintillation Vials</td><td>Fisher Scientific</td><td>03-341-25E</td><td>Use glass vials for quantitative recovery of &lt;sup&gt;14&lt;/sup&gt;CO&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>20 ml HDPE Scintillation Vials</td><td>Fisher Scientific</td><td>03-337-23B</td><td>Use HDPE vials for quantitative recovery of &lt;sup&gt;3&lt;/sup&gt;H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Red Rubber Sleeve Stoppers</td><td>Fisher Scientific</td><td>14-126DD</td><td>Fit 20 mL scintillation vials; Reusable</td></tr><tr><td>BD PrecisionGlide Needle 23G x 40 mm</td><td>BD</td><td>305194</td><td>Use to inject perchloric acid through the rubber sleeve stopper of the CO&lt;sub&gt;2&lt;/sub&gt; trap</td></tr><tr><td>Perchloric Acid, 60%</td><td>Fisher Scientific</td><td>A228</td><td>Highly corrosive and may act as an oxidizer and/or cause an explosion hazard. Consult the product MSDS for appropriate handling and storage</td></tr><tr><td>Ultima Gold, Scintillation Cocktail</td><td>PerkinElmer</td><td>6013327</td><td></td></tr><tr><td>Glass Wool</td><td>Fisher Scientific</td><td>AC38606</td><td></td></tr><tr><td>Decon Dri-Clean Detergent Powder</td><td>Fisher Scientific</td><td>04-355</td><td>For cleaning of glassware, plastic parts, and tubing</td></tr><tr><td>Alconox Tergazyme Enzyme-Active Powered Detergent</td><td>Fisher Scientific</td><td>16-000-115</td><td>For cleaning of &quot;hard to reach&quot; surfaces (tubing, glassware) contaminated by fatty acid-BSA residue</td></tr></tbody></table>

methods for the determination of rates of glucose and fatty acid oxidation in the isolated working rat heart

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。

两个代表性实验在下面的图中描述。在这两种情况下，一个16周龄的雄性Sprague Dawley大鼠的心脏中分离，并与根据前述方案制备KH液的工作模式灌注。在每个实验中，将心脏经受应力条件影响心脏的工作。心脏收缩功能是由脉压的连续记录通过主动脉线压力传感器的插入和通过测定心肌力量评估。在提供能量的底物的利用每个应力条件下的后果是同时由葡萄糖和油的氧化速?...

Watch this Scientific Journal Video about Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart at JoVE.com

Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

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.

为葡萄糖和脂肪酸氧化率在大鼠离体的测定方法

The mammalian heart is a major consumer of ATP and requires a constant supply of energy substrates for contraction. Not surprisingly, alterations of ...

methods-for-determination-rates-glucose-fatty-acid-oxidation-isolated

Research

JoVE Journal

Biology

14.6K 次观看.  University of Mississippi Medical Center. 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.

视频: Methods for the Determination of Rates of Glucose and Fatty Acid Oxidation in the Isolated Working Rat Heart

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

科研

发育生物学

NADH Fluorescence Imaging of Isolated Biventricular Working Rabbit Hearts

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited for studies of cardiac metabolism, which require the heart to perform work within the context of physiologic preload and afterload pressures. Neely introduced modifications to the Langendorff technique to establish appropriate left ventricular (LV) preload and afterload pressures3. The model is known as the isolated LV working heart model and has been used extensively to study LV performance and metabolism4-6. This model, however, does not provide a properly loaded right ventricle (RV). Demmy et al. first reported a biventricular model as a modification of the LV working heart model7, 8. They found that stroke volume, cardiac output, and pressure development improved in hearts converted from working LV mode to biventricular working mode8. A properly loaded RV also diminishes abnormal pressure gradients across the septum to improve septal function. Biventricular working hearts have been shown to maintain aortic output, pulmonary flow, mean aortic pressure, heart rate, and myocardial ATP levels for up to 3 hours8.
When studying the metabolic effects of myocardial injury, such as ischemia, it is often necessary to identify the location of the affected tissue. This can be done by imaging the fluorescence of NADH (the reduced form of nicotinamide adenine dinucleotide)9-11, a coenzyme found in large quantities in the mitochondria. NADH fluorescence (fNADH) displays a near linearly inverse relationship with local oxygen concentration12 and provides a measure of mitochondrial redox state13. fNADH imaging during hypoxic and ischemic conditions has been used as a dye-free method to identify hypoxic regions14, 15 and to monitor the progression of hypoxic conditions over time10.
The objective of the method is to monitor the mitochondrial redox state of biventricular working hearts during protocols that alter the rate of myocyte metabolism or induce hypoxia or create a combination of the two. Hearts from New Zealand white rabbits were connected to a biventricular working heart system (Hugo Sachs Elektronik) and perfused with modified Krebs-Henseleit solution16 at 37 &deg;C. Aortic, LV, pulmonary artery, and left &amp; right atrial pressures were recorded. Electrical activity was measured using a monophasic action potential electrode. To image fNADH, light from a mercury lamp was filtered (350&plusmn;25 nm) and used to illuminate the epicardium. Emitted light was filtered (460&plusmn;20 nm) and imaged using a CCD camera. Changes in the epicardial fNADH of biventricular working hearts during different pacing rates are presented. The combination of the heart model and fNADH imaging provides a new and valuable experimental tool for studying acute cardiac pathologies within the context of realistic physiological conditions.

The objective is to monitor the mitochondrial redox state of isolated hearts within the context of physiologic preload and afterload pressures. A biventricular working rabbit heart model is presented. High spatiotemporal resolution fluorescence imaging of NADH is used to monitor the mitochondrial redox state of epicardial tissue.

NADH的荧光成像隔离双心室工作离体兔心

Since its inception by Langendorff1, the isolated perfused heart remains a prominent tool for studying cardiac physiology2. However, it is not well-suited ...

医学

Measurement of Fatty Acid &#946;-Oxidation in a Suspension of Freshly Isolated Mouse Hepatocytes

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional processes, such as ketogenesis and gluconeogenesis, which are essential to maintain whole-body glucose homeostasis and support extra-hepatic organ function in the fasted state. Fatty acid &#946;-oxidation occurs within the mitochondria and peroxisomes and is regulated through multiple mechanisms, including the uptake and activation of fatty acids, enzyme expression levels, and availability of cofactors such as coenzyme A and NAD+. In assays that measure fatty acid &#946;-oxidation in liver homogenates, cell lysis and the common addition of supraphysiological levels of cofactors mask the effects of these regulatory mechanisms. Furthermore, the integrity of the organelles in the homogenates is hard to control and can vary significantly between preparations. The measurement of fatty acid &#946;-oxidation in intact primary hepatocytes overcomes the above pitfalls. This protocol describes a method for the measurement of fatty acid &#946;-oxidation in a suspension of freshly isolated primary mouse hepatocytes incubated with 14C-labeled palmitic acid. By avoiding hours to days of culture, this method has the advantage of better preserving the protein expression levels and metabolic pathway activity of the original liver, including the activation of fatty acid &#946;-oxidation observed in hepatocytes isolated from fasted mice compared to fed mice.

Fatty acid &#946;-oxidation is an essential metabolic pathway responsible for generating energy in many different cell types, including hepatocytes. Here, we describe a method to measure fatty acid &#946;-oxidation in freshly isolated primary hepatocytes using 14C-labeled palmitic acid.

测量新鲜分离的小鼠肝细胞悬浮液中的脂肪酸β氧化

Fatty acid &#946;-oxidation is a key metabolic pathway to meet the energy demands of the liver and provide substrates and cofactors for additional ...

生物化学

Preparation and Respirometric Assessment of Mitochondria Isolated from Skeletal Muscle Tissue Obtained by Percutaneous Needle Biopsy

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining samples of human Vastus lateralis, isolating mitochondria from minimal amounts of skeletal muscle tissue, and plate based respirometric profiling using an extracellular flux (XF) analyzer. Comparison of respirometric profiles obtained using 1.0, 2.5 and 5.0 &#956;g of mitochondria indicate that 1.0 &#956;g is sufficient to measure respiration and that 5.0 &#956;g provides most consistent results based on comparison of standard errors. Western blot analysis of isolated mitochondria for mitochondrial marker COX IV and non-mitochondrial tissue marker GAPDH indicate that there is limited non-mitochondrial contamination using this protocol. The ability to study mitochondrial respirometry in as little as 20 mg of muscle tissue allows users to utilize individual biopsies for multiple study endpoints in clinical research projects.

Methods for biopsy of Vastus lateralis, preparation of purified mitochondria, and respirometric profiling are described. The use of small muscle volume makes this technique suitable for clinical research applications.

线粒体的制备及透气性评估，从骨骼肌组织通过穿刺活检获得的分离

Respirometric profiling of isolated mitochondria is commonly used to investigate electron transport chain function. We describe a method for obtaining ...

An Optimized Protocol to Analyze Glycolysis and Mitochondrial Respiration in Lymphocytes

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration and differentiation, and cytokine production. All of these events are tightly linked to the energy status of the cell, and therefore studying the energy-producing pathways may give clues about the overall functionality of these cells. The extracellular flux analyzer is a commonly used device for evaluating the performance of glycolysis and mitochondrial respiration in many cell types. This system has been used to study immune cells in a few published reports, yet a comprehensive protocol optimized particularly for lymphocytes is lacking. Lymphocytes are fragile cells that survive poorly in ex vivo conditions. Oftentimes lymphocyte subsets are rare, and working with low cell numbers is inevitable. Thus, an experimental strategy that addresses these difficulties is required. Here, we provide a protocol that allows for rapid isolation of viable lymphocytes from lymphoid tissues, and for the analysis of their metabolic states in the extracellular flux analyzer. Furthermore, we provide results of experiments in which the metabolic activities of several lymphocyte subtypes at different cell densities were compared. These observations suggest that our protocol can be used to achieve consistent, well-standardized results even at low cell concentrations, and thus it may have broad applications in future studies focusing on the characterization of metabolic events in immune cells.

The study of metabolism is becoming increasingly relevant to immunological research. Here, we present an optimized method for measuring glycolysis and mitochondrial respiration in mouse splenocytes, and T and B lymphocytes.

一种优化的协议分析和糖酵解线粒体呼吸的淋巴细胞

Lymphocytes respond to a variety of stimuli by activating intracellular signaling pathways, which in turn leads to rapid cellular proliferation, migration ...

免疫与感染

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

生物工程

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

生物学

Analyzing Oxygen Consumption Rate in Primary Cultured Mouse Neonatal Cardiomyocytes Using an Extracellular Flux Analyzer

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an important contributing factor to impaired cardiac function found in heart failure. By contrast, restoring defective mitochondrial function may have beneficial effects to improve cardiac function in the failing heart. Therefore, studying the regulatory mechanisms and identifying novel regulators for mitochondrial function could provide insight which could be used to develop new therapeutic targets for treating heart disease. Here, cardiac myocyte mitochondrial respiration is analyzed using a unique cell culture system. First, a protocol has been optimized to rapidly isolate and culture high viability neonatal mouse cardiomyocytes. Then, a 96-well format extracellular flux analyzer is used to assess the oxygen consumption rate of these cardiomyocytes. For this protocol, we optimized seeding conditions and demonstrated that neonatal mouse cardiomyocytes oxygen consumption rate can be easily assessed in an extracellular flux analyzer. Finally, we note that our protocol can be applied to a larger culture size and other studies, such as intracellular signaling and contractile function analysis.

The goal of this protocol is to illustrate how to use mouse neonatal cardiomyocytes as a model system to examine how various factors can alter oxygen consumption in the heart.

细胞外通量分析仪分析原代培养小鼠新生心肌细胞的耗氧率

Mitochondria and oxidative metabolism are critical for maintaining cardiac muscle function. Research has shown that mitochondrial dysfunction is an ...

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. In addition, photoreceptors also exhibit high aerobic glycolysis like cancer cells. Precise measurements of these metabolic activities can provide valuable insights into cellular homeostasis under physiological conditions and in disease states. High throughput microplate-based assays have been developed to measure mitochondrial respiration and various metabolic activities in live cells. However, a vast majority of these are developed for cultured cells and have not been optimized for intact tissue samples and for application ex vivo. Described here is a detailed step-by-step protocol, using microplate-based fluorescence technology, to directly measure oxygen consumption rate (OCR) as an indicator of mitochondrial respiration, as well as extracellular acidification rate (ECAR) as an indicator of glycolysis, in intact ex vivo retinal tissue. This method has been used to successfully assess metabolic activities in adult mouse retina and demonstrate its application in investigating cellular mechanisms of aging and disease.

Determination of Mitochondrial Respiration and Glycolysis in Ex Vivo Retinal Tissue Samples

Described here is a detailed protocol for performing mitochondrial stress assay and glycolytic rate assay in ex vivo retinal tissue samples using a commercial bioanalyzer.

离体视网膜组织样品中线粒体呼吸和糖酵解的测定

Mitochondrial respiration is a critical energy-generating pathway in all cells, especially retinal photoreceptors that possess a highly active metabolism. ...

神经科学

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

测量外产酸和分析，以确定糖酵解率

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...