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Transmission Electron Microscopy for Analysis of Mitochondria in Mouse Skeletal Muscle   

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Original research article

A brief version of this protocol appeared in:
The Journal of Cell Biology
Dec 2010

Abstract

Skeletal muscle is the most abundant tissue in the human body and regulates a variety of functions including locomotion and whole-body metabolism. Skeletal muscle has a plethora of mitochondria, the organelles that are essential for aerobic generation of ATP which provides the chemical energy to fuel vital functions such as contraction. The number of mitochondria in skeletal muscle and their function decline with normal aging and in various neuromuscular diseases and in catabolic conditions such as cancer, starvation, denervation, and immobilization. Moreover, compromised mitochondrial function is also associated with metabolic disorders including type 2 diabetes mellitus. It is now clear that maintaining mitochondrial content and function in skeletal muscle is vital for sustained health throughout the lifespan. While a number of staining methods are available to study mitochondria, transmission electron microscopy (TEM) is still the most important method to study mitochondrial structure and health in skeletal muscle. It provides critical information about mitochondrial content, cristae density, organization, formation of autophagosomes, and any other abnormalities commonly observed in various disease conditions. In this article, we describe a detailed protocol for sample preparation and analysis of mouse skeletal muscle mitochondria by TEM.

Keywords: Transmission electron microscopy, Skeletal muscle, Mitochondria, Autophagy, Myopathy, Atrophy, Oxidative metabolism

Background

Skeletal muscle is a highly plastic tissue that undergoes morphological and metabolic adaptations in response to a number of extracellular cues. A number of perturbations including resistance or endurance exercise stimulates mitochondrial biogenesis leading to increased metabolic capacity and resistance to fatigue (Li et al., 2008; Sandri, 2008). By contrast, during aging, inactivity, and in many catabolic disease states, skeletal muscle mitochondrial number and function decline, leading to increased fatigability and insulin resistance (Sandri, 2008). An accumulation of dysfunctional mitochondria may also result in progressive reactive oxygen species-induced damage, producing a further impairment of oxidative capacity in skeletal muscle (Bonnard et al., 2008).

Mitochondria exist as a reticular membrane network that is located in different subcellular compartments in skeletal muscle. The subsarcolemmal (SS) mitochondria, account for 10-15% of the mitochondrial volume and lie directly beneath the sarcolemmal membrane, whereas the intermyofibrillar (IMF) mitochondria are located in close contact with the myofibril (Takahashi and Hood, 1996). Mitochondria are double membrane structures containing an intermembrane space between the outer and inner membranes as well as the inner matrix compartment, where most of the metabolic processes take place. The inner membrane is highly folded, forming so-called cristae, to accommodate its large surface area. The five complexes that make up the respiratory chain where oxidative phosphorylation takes place are embedded within the inner mitochondrial membrane. In this process, a proton gradient across the inner membrane is coupled to ATP synthesis at complex V (Peterson et al., 2012). In addition to producing ATP for cross-bridge cycling between actin and myosin, mitochondria are a source of free radicals which regulate skeletal muscle physiology (Peterson et al., 2012).

Transmission electron microscopy (TEM) is a powerful technique for ultrastructural studies (Watson, 1958). TEM has been very useful in studying mitochondrial structure in skeletal muscle in both physiological and pathological conditions (Picard et al., 2013). For example, TEM can provide information about mitochondrial content, organization, cristae structure, and vacuolization as observed in some neuromuscular disorders such as Amyotrophic lateral sclerosis (Picard et al., 2013). In many muscle wasting conditions, mitochondrial content is reduced through autophagy, also known as mitophagy. In this regard, TEM has been found to be an important approach to study autophagosome formation (Sandri, 2008). We have developed an efficient protocol that can be easily adapted in any laboratory to study the ultrastructure of mouse mitochondria in skeletal muscle by TEM (Paul et al., 2010; Hindi et al., 2014 and 2018). In the following sections, we provide a step-wise protocol for sample preparation and analysis of SS and IMF mitochondria in skeletal muscle. A similar protocol can be used for studying other organelles in skeletal muscle by TEM as well.

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. McMillan, J. D. and Eisenback, M. A. (2018). Transmission Electron Microscopy for Analysis of Mitochondria in Mouse Skeletal Muscle. Bio-protocol 8(10): e2455. DOI: 10.21769/BioProtoc.2455.
  2. Paul, P. K., Gupta, S. K., Bhatnagar, S., Panguluri, S. K., Darnay, B. G., Choi, Y. and Kumar, A. (2010). Targeted ablation of TRAF6 inhibits skeletal muscle wasting in mice. J Cell Biol 191(7): 1395-1411.
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