Elsevier

Methods in Enzymology

Volume 457, 2009, Pages 349-372
Methods in Enzymology

Chapter 20: Functional Assessment of Isolated Mitochondria In Vitro

https://doi.org/10.1016/S0076-6879(09)05020-4Get rights and content

Abstract

Mitochondria play a pivotal role in cellular function, not only as a major site of ATP production, but also by regulating energy expenditure, apoptosis signaling, and production of reactive oxygen species. Altered mitochondrial function is reported to be a key underlying mechanism of many pathological states and in the aging process. Functional measurements of intact mitochondria isolated from fresh tissue provides distinct information regarding the function of these organelles that complements conventional mitochondrial assays using previously frozen tissue as well as in vivo assessment using techniques such as magnetic resonance and near‐infrared spectroscopy. This chapter describes the process by which mitochondria are isolated from small amounts of human skeletal muscle obtained by needle biopsy and two approaches used to assess mitochondrial oxidative capacity and other key components of mitochondrial physiology. We first describe a bioluminescent approach for measuring the rates of mitochondrial ATP production. Firefly luciferase catalyzes a light‐emitting reaction whereby the substrate luciferin is oxidized in an ATP‐dependent manner. A luminometer is used to quantify the light signal, which is proportional to ATP concentration. We also review a method involving polarographic measurement of oxygen consumption. Measurements of oxygen consumption, which previously required large amounts of tissue, are now feasible with very small amounts of sample obtained by needle biopsy due to recent advances in the field of high‐resolution respirometry. We illustrate how careful attention to substrate combinations and inhibitors allows an abundance of unique functional information to be obtained from isolated mitochondria, including function at various energetic states, oxidative capacity with electron flow through distinct complexes, coupling of oxygen consumption to ATP production, and membrane integrity. These measurements, together with studies of mitochondrial DNA abundance, mRNA levels, protein expression, and synthesis rates of mitochondrial proteins provide insightful mechanistic information about mitochondria in a variety of tissue types.

Introduction

Mitochondria are believed to have originated in eukaryotic cells by endosymbiosis∼2 billion years ago (Gray et al., 1999). Although most mitochondrial proteins are encoded by nuclear DNA and imported into the organelle, mitochondria contain ribosomes and between 2 and 10 copies of circular DNA containing 13 protein‐encoding regions and 22 tRNA‐encoding genes. Mitochondria are an essential part of normal cellular function, particularly in their role in oxidizing carbon substrates to satisfy cellular energy requirements. Hydrolysis of ATP to ADP or AMP releases free energy to drive energy‐requiring processes such as cross‐bridge cycling, maintenance of membrane potentials, sarcoplasmic reticulum calcium uptake, signal transduction, membrane transport, and protein synthesis. Nearly 90% of cellular ATP demand is satisfied by mitochondrial ATP synthesis, which, as illustrated in (Fig. 20.1), is driven by a proton gradient across the inner mitochondrial membrane as a result of oxidation of carbon substrates in the tricarboxylic acid (TCA) cycle and electron flow through various enzymes and proteins at the inner mitochondrial membrane. These organelles not only produce chemical energy in the form of ATP, but also liberate energy through thermogenic uncoupling, regulate apoptosis, and are a major source of reactive oxygen species (ROS). While ROS production plays an important role in cell signaling, increased ROS levels can result in oxidative damage to mitochondrial DNA, particularly when ROS production exceeds the capacity of antioxidant defense systems (glutathione peroxidase, catalase, and superoxide dismutase) and DNA repair. Indeed, accumulated oxidative damage to various molecules has been proposed as a mechanism by which the aging process is accelerated (Harman, 1956). The importance of mitochondria in cellular function and energy balance has spawned much interest in their role in the aging process and metabolic diseases such as type 2 diabetes. Accurate, precise assessment of tissue mitochondrial function is necessary to understand the aging process and the underlying mechanisms of many diseases. Various tools, each with distinct advantages and caveats, have been used to probe mitochondrial properties in vitro and in vivo. For example, magnetic resonance spectroscopy permits noninvasive in vivo assessment of muscle oxidative capacity (Argov et al., 1987, Kent‐Braun and Ng, 2000, Lanza et al., 2007), steady‐state mitochondrial ATP synthesis rate (Befroy et al., 2008, Lebon et al., 2001, Petersen et al., 2003), and TCA cycle flux (Befroy et al., 2008, Lebon et al., 2001, Petersen et al., 2004). The ability to assess mitochondrial function under conditions where circulatory and regulatory systems are intact is a strength of these in vivo approaches. Experiments performed ex vivo permit a reductionist approach to probe specific molecular levels within the complex pathway of mitochondrial ATP synthesis; information which is crucial to understanding mechanisms by which mitochondrial function is altered with aging and disease and pathways by which exercise and pharmacological interventions exert beneficial effects. Moreover, the ability to quantify mitochondrial DNA copy numbers (Asmann et al., 2006, Barazzoni et al., 2000, Chow et al., 2007, Short et al., 2005), expression (mRNA and protein) of various mitochondrial proteins (Lanza et al., 2008, Short et al., 2005), mitochondrial enzyme activities (Rooyackers et al., 1996, Short et al., 2003), and stable isotope‐based synthesis of mitochondrial proteins (Rooyackers et al., 1996) allows an enormous amount of complementary data to be obtained from the same biopsy sample that is used for measurements of mitochondrial function. In addition, a recent approach to measure synthesis rates of individual skeletal muscle mitochondrial proteins has been reported (Jaleel et al., 2008), which offers an opportunity to determine the translational efficiency of gene transcripts, especially when combined with large scale gene transcript measurements (Asmann et al., 2006). Together, these methods permit comprehensive investigation of mitochondrial function and its regulation at various molecular and cellular levels to understand the underlying mechanisms of mitochondrial changes that occur in conditions related to aging, disease, and physical activity.

Historically, the maximal activities of key mitochondrial enzymes have been widely used as indices of mitochondrial oxidative capacity (Houmard et al., 1998, Rooyackers et al., 1996, Wicks and Hood, 1991). Citrate synthase is a common matrix enzyme marker, while succinate dehydrogenase and cytochrome c oxidase are frequently measured as representative enzymes from the inner mitochondrial membrane. Since only small amounts of previously frozen tissue are required, spectrophotometric‐based enzyme activity assays are well‐suited for human studies where tissue quantities are limited. Although it is not unreasonable to relate maximal activities of these marker enzymes to mitochondrial oxidative capacity, it is unlikely that a single enzyme can accurately reflect the collective function of an organelle as complex as the mitochondrion. Thus, there is a critical need to implement more direct functional measurements of mitochondrial function and oxidative capacity. Measurement of oxygen consumption in isolated mitochondria, pioneered by Britton Chance over 50 years ago (Chance and Williams, 1956), has long been used to assess function of freshly isolated mitochondria. Accurate respiration measurements using conventional respirometers necessitated large quantities of tissue, limiting its practicality for routine human studies. However, recent technological advances in the field of high‐resolution respirometry allow these types of measurements using very small amounts of sample (Gnaiger, 2001, Haller et al., 1994), permitting investigators to obtain a wealth of information regarding mitochondrial function from small amounts of human biopsy tissue. In addition to respiration‐based measurements, it is also possible to assess mitochondrial function by measuring synthesis of ATP, the ultimate end‐product of oxidative phosphorylation. The ability to measure ATP production in freshly isolated intact mitochondria offers an alternative approach for assessing mitochondrial function that provides distinct, complementary information to traditional measurements of oxygen consumption. By exploiting a photon‐emitting reaction involving ATP, luciferin, and firefly luciferase, it is possible to quantify the rates of ATP production under various conditions.

For many years, luciferase‐based measurements of skeletal muscle mitochondrial ATP production rates (MAPR) (Wibom and Hultman, 1990) have become an integral part of studies in our laboratory. We have applied this method in various ways, including to demonstrate an age‐related decline in mitochondrial oxidative capacity (Short et al., 2005) and the absence of this trend in individuals who engage in chronic endurance exercise (Lanza et al., 2008). Using this technique, we have observed paradoxically elevated MAPR in Asian Indian individuals in spite of stark insulin resistance compared to people of northern European descent (Nair et al., 2008). To help elucidate the controversial relationship between insulin resistance and mitochondrial function, we have demonstrated that insulin stimulates MAPR in healthy controls but not in insulin resistant individuals (Stump et al., 2003). These studies point to insulin as a key regulator of mitochondrial function and impaired insulin signaling as a potential mechanism by which mitochondrial dysfunction manifests in insulin resistant people (Asmann et al., 2006). Combining measurements of maximal ATP production with measures of mitochondrial respiration using various substrates offer an opportunity to asses defects at specific levels within the process of mitochondrial energy metabolism.

This chapter describes methodology for assessing the function of mitochondria isolated from skeletal muscle, with particular attention to the procedures for isolating mitochondria from skeletal muscle samples, a luciferase‐based bioluminescent measurement of mitochondrial ATP production, and mitochondrial oxygen consumption measurements using high‐resolution respirometry. Although we describe the detailed procedures for functional measurements in isolated mitochondria from skeletal muscle, similar approaches with relatively minor modifications could be applied to investigate other tissues such as liver, adipose, kidney, heart, brain, permeabilized fibers, and cell culture.

Section snippets

Mitochondrial Isolation Procedures

Skeletal muscle contains two distinct populations of mitochondria (Cogswell et al., 1993). Subsarcolemmal mitochondria (∼20% of total mitochondrial content) are defined as those within 2 μm from the sarcolemma, often densely clustered in the proximity of nuclei and easily liberated by gentle, mechanical homogenization (Elander et al., 1985). Intermyofibrillar mitochondria (∼80% of total mitochondria) are imbedded amongst the contractile machinery and are best liberated by softening the tissue

Principles of the bioluminescent approach

Luciferase from the firefly Photinus pyralis catalyzes a two‐step reaction that oxidizes luciferin in an ATP‐dependent reaction that generates a light signal in proportion to ATP concentration (DeLuca and McElroy, 1974). Luciferyl adenylate and inorganic pyrophosphate are formed when ATP is added to luciferin in the presence of luciferase. Oxyluciferin and AMP are subsequently formed when luciferyl adenylate reacts with oxygen, and light is generated when the products are released from the

Overview

Oxygen in solution can be measured polarographically with a Clark‐type oxygen electrode. Clark electrodes have gold or platinum cathodes and silver or silver/silver chloride anodes, which are connected by a salt bridge and covered by an oxygen‐permeable membrane. As oxygen diffuses across the membrane, it is reduced by a fixed voltage between the cathode and anode which generates current in proportion to the concentration of oxygen in solution. By calibrating the voltage with known oxygen

Summary

The function and capacity of intact mitochondria isolated from small amounts of tissue can be objectively determined using polarographic‐based measurements of oxygen consumption and luciferase‐based bioluminescent measurements of ATP synthesis. Careful selection of substrate combinations and inhibitors enable quantitative measurement of mitochondrial function in various energetic states, oxidative capacity with electron flow through distinct complexes, coupling of oxygen consumption to ATP

References (51)

  • I.A. Trounce et al.

    Assessment of mitochondrial oxidative phosphorylation in patient muscle biopsies, lymphoblasts, and transmitochondrial cell lines

    Methods Enzymol.

    (1996)
  • Z. Argov et al.

    Bioenergetic heterogeneity of human mitochondrial myopathies: Phosphorus magnetic resonance spectroscopy study

    Neurology

    (1987)
  • Y.W. Asmann et al.

    Skeletal muscle mitochondrial functions, mitochondrial DNA copy numbers, and gene transcript profiles in type 2 diabetic and nondiabetic subjects at equal levels of low or high insulin and euglycemia

    Diabetes

    (2006)
  • D.E. Befroy et al.

    Increased substrate oxidation and mitochondrial uncoupling in skeletal muscle of endurance‐trained individuals

    Proc. Natl. Acad. Sci. USA

    (2008)
  • R. Boushel et al.

    Patients with type 2 diabetes have normal mitochondrial function in skeletal muscle

    Diabetologia

    (2007)
  • L.S. Chow et al.

    Impact of endurance training on murine spontaneous activity, muscle mitochondrial DNA abundance, gene transcripts, and function

    J. Appl. Physiol.

    (2007)
  • A.M. Cogswell et al.

    Properties of skeletal muscle mitochondria isolated from subsarcolemmal and intermyofibrillar regions

    Am. J. Physiol.

    (1993)
  • M. DeLuca et al.

    Kinetics of the firefly luciferase catalyzed reactions

    Biochemistry

    (1974)
  • B. Drew et al.

    Method for measuring ATP production in isolated mitochondria: ATP production in brain and liver mitochondria of Fischer‐344 rats with age and caloric restriction

    Am. J. Physiol. Regul. Integr. Comp. Physiol.

    (2003)
  • R. Edwards et al.

    Needle biopsy of skeletal muscle in the diagnosis of myopathy and the clinical study of muscle function and repair

    N. Engl. J. Med.

    (1980)
  • A. Elander et al.

    Biochemical and morphometric properties of mitochondrial populations in human muscle fibres

    Clin. Sci. (Lond.)

    (1985)
  • E. Gnaiger et al.

    Mitochondrial respiration at low levels of oxygen and cytochrome c

    Biochem. Soc. Trans.

    (2002)
  • E. Gnaiger et al.

    High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia

    Proc. Natl. Acad. Sci. USA

    (2000)
  • M.W. Gray et al.

    Mitochondrial evolution

    Science

    (1999)
  • D. Harman

    Aging: A theory based on free radical and radiation chemistry

    J. Gerontol.

    (1956)
  • Cited by (185)

    • Mitochondrial techniques for physiologists

      2024, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular Biology
    • Ultrasensitive sensors reveal the spatiotemporal landscape of lactate metabolism in physiology and disease

      2023, Cell Metabolism
      Citation Excerpt :

      Seahorse XFe96 analyzer (Agilent, XFe96, US) was used to measure NADH oxidation-based OCR of freshly isolated mitochondria in the absence or presence of 1 mM NADH (Lanza and Nair, 2009; Puchowicz et al., 2004). Citrate synthase activity is measured spectrophotometrically from the mitochondria samples using previously described methods (Lanza and Nair, 2009). Briefly, 5 μL mitochondria samples (4.6∼6.1 μg protein per well) were loaded into the 96-well clear flat-bottom plate and absorbance at 412 nm was measured every 13 seconds for 10 minutes at 30 °C immediately after the addition of 100 μL 0.1 M Tris-HCl assay buffer (pH 8.0) containing 0.1 mM dithionitrobenzoic acid (DNTB), 0.05 mM acetyl-CoA and 0.25 mM oxaloacetate.

    View all citing articles on Scopus
    View full text