This work was supported by the Swedish Olympic Committee.

The present protocol using human biopsied skeletal muscle samples was approved by The Regional Committees on Health Research Ethics for Southern Denmark (S-20170198). Muscle biopsies were obtained through an incision in the skin from the vastus lateralis muscle using a Bergstr&#246;m needle with suction after local anesthesia was given subcutaneously (1-3 mL of Lidocaine 2% per incision). If isolated whole rat muscles were used, the animals were sacrificed by cervical dislocation before the muscle biopsies were ...

<ol>
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The authors declare no competing interests.

The critical step of the method is the use of reduced osmium by potassium ferrocyanide during post-fixation. The selectivity of this modified fixative for glycogen detection cannot be fully explained by chemistry, but also includes experimental findings demonstrating no detection of such particles in tissues known to be free of glycogen or in the extracellular space11.
Critical parameters are the precision of the estimates and the fiber-to-fiber variation. By following ...

Glycogen particles are composed of branched polymers of glucose and various associated proteins1 and constitute an important fuel during high metabolic demands2. Although not widely recognized, glycogen particles also constitute a local fuel, where some subcellular processes preferentially utilize glycogen despite the availability of other and more long-lasting fuels as plasma glucose and fatty acids3,4.
The importance of storing glycogen as a subcellular specific localized fuel has been discussed in several reviews5,6 mainly based on some of the earliest documentations of the subcellular distribution of glycogen by transmission electron microscopy (TEM)7,8. The first studies used different protocols to increase the contrast of glycogen from histochemical staining techniques to negative and positive stainings9,10. An important methodological development was the refined post-fixation protocol with the potassium ferrocyanide-reduced osmium11,12,13,14, which significantly improved the contrast of glycogen particles. This refined protocol was not used in some of the pioneering work on exercise-induced glycogen depletion15 but was re-introduced by Graham and colleagues16,17.
Based on the 2-dimensional images, the subcellular distribution of glycogen is most often described as glycogen particles located in three pools: subsarcolemmal (just beneath the surface membrane), intermyofibrillar (between the myofibrils), or intramyofibrillar (within the myofibrils). However, glycogen particles could also be described as associated with, for example, sarcoplasmic reticulum7 or nuclei18. In addition to the subcellular distribution, the advantage of TEM-estimated glycogen content is also that quantification can be conducted at the single fiber level. This allows investigation of fiber-to-fiber variability and correlative analyses with fiber types and cellular components as mitochondria and lipid droplets.
Here, the protocol for the TEM-estimated fiber type-specific volumetric content of the three common subcellular pools of glycogen (subsarcolemmal, intermyofibrillar, and intramyofibrillar) in skeletal muscle fibers is described. The method has been applied to skeletal muscles from humans19, rats20, and mice21; as well as birds and fish22; and cardiomyocytes from rats23.

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			<td>Merck</td>
			<td>75-56-9</td>
			<td></td>
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			<td>Embedding 812 resin medium kit</td>
			<td>Taab</td>
			<td>T031</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde solution 25%</td>
			<td>Merck</td>
			<td>1.04239.0250</td>
			<td></td>
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		<tr>
			<td>ITEM</td>
			<td>Olympus</td>
			<td></td>
			<td>Imaging software</td>
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			<td>Leica EM AC20</td>
			<td>Leica</td>
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			<td>Automatic contrasting system</td>
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			<td>OSIS Veleta digital camera</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
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			<td>Osmium tetroxide 4% solution</td>
			<td>Polysciences</td>
			<td>0972A</td>
			<td></td>
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		<tr>
			<td>Philips CM 100 Transmission EM</td>
			<td>Philips</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium hexacyanoferrate (II) trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>455989-245G</td>
			<td></td>
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			<td>Sodium cacodylatbuffer 0,2 M ph 7.4</td>
			<td>Ampliqon.com</td>
			<td>AMPQ40989.0500</td>
			<td></td>
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			<td>Ultra-microtome Leica UC7</td>
			<td>Leica</td>
			<td></td>
			<td></td>
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			<td>Ultrostain lead citrate 3%, stabilised solution</td>
			<td>Leica</td>
			<td>16707235</td>
			<td></td>
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			<td>Uranyl acetate dihydrate</td>
			<td>Polysciences</td>
			<td>6159-44-0</td>
			<td></td>
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quantification of subcellular glycogen distribution in skeletal muscle fibers using transmission electron microscopy

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This enables quantifications of ultrastructural aspects such as volume fractions, surface area to volume ratios, morphometry, and physical contact sites of different subcellular structures. In the 1970s, a protocol for enhanced staining of glycogen in cells was developed and paved the way for a string of studies on the subcellular localization of glycogen and glycogen particle size using transmission electron microscopy. While most analyses interpret glycogen as if it is homogeneously distributed within the muscle fibers, providing only a single value (e.g., an average concentration), transmission electron microscopy has revealed that glycogen is stored as discrete glycogen particles located in distinct subcellular compartments. Here, the step-by-step protocol from tissue collection to the quantitative determination of the volume fraction and particle diameter of glycogen in the distinct subcellular compartments of individual skeletal muscle fibers is described. Considerations on how to 1) collect and stain tissue specimens, 2) perform image analyses and data handling, 3) evaluate the precision of estimates, 4) discriminate between muscle fiber types, and 5) methodological pitfalls and limitations are included.

Using this protocol, glycogen particles appear black and distinct (Figures 1 and Figure 2). The normal values of glycogen are depicted in Figure 3. These data are based on a total of 362 fibers from 41 healthy young men as collected in different previous studies19,24,29,30,31</...

Watch this Scientific Journal Video about Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy at JoVE.com

Quantification of Subcellular Glycogen Distribution in Skeletal Muscle Fibers using Transmission Electron Microscopy

A modified post-fixation procedure increases the contrast of glycogen particles in tissue. This paper provides a step-by-step protocol describing how to handle the tissue, conduct the imaging, and use stereological methods to obtain unbiased and quantitative data on fiber type-specific subcellular glycogen distribution in skeletal muscle.

With the use of transmission electron microscopy, high-resolution images of fixed samples containing individual muscle fibers can be obtained. This ...

The measure of glycogen content in single cells and subcellular organizations is important because in most cell types, single cells adapt differently to changing physiological conditions and diseases. So, the main advantage of this technique is the very high resolution in the transmission electron microscope. This improves the possibility to have localized subcellular structures and also in this content in this technique here, it's important that the glycogen staining is antibody free.So, we don't have any specificity to worry about. In order to get a better contrast on glycogen, just use potassium ferrocyanide 1.5%instead of potassium ferrocyanide. Prepare 1.6 milliliters of the primary fixative solution by adding 2.5%glutaraldehyde in 0.1 molar sodium cacodylate buffer in a two milliliter micro-centrification tube.Store it at five degrees Celsius for a maximum of 14 days. Isolate a small specimen from the muscle biopsy or whole muscle which has a maximum diameter of one millimeter in any direction and is longer longitudinally than cross-sectionally. Place the specimen in the tube containing the cold primary fixation solution.Store it at five degrees Celsius for 24 hours. Then wash the specimen four times in 0.1 molar sodium cacodylate buffer. Using transfer pipettes, remove the used buffer from the tube leaving the specimen untouched and add the fresh buffer.Post-fix the specimen with 1%osmium tetroxide and 1.5%potassium ferrocyanide in 0.1 molar sodium cacodylate buffer for 120 minutes at four degrees Celsius. Rinse the specimen twice in double distilled water at room temperature. Dehydrate by submerging in a graded series of ethanol at room temperature.Infiltrate the specimen with propylene oxide and epossidic resin graded mixtures at room temperature using the mentioned volume ratios. The following day, embed specimens in 100%fresh epossidic resin in molds and polymerize at 60 degrees Celsius for 48 hours. Mount the block of a specimen on the ultramicrotome holder.Trim the block on the surface with a razor blade to reach the tissue level. Mount a diamond knife in front of the sample and align the sample surface parallel to the knife. Produce a semi-thin section of one micrometer thickness with the diamond knife to check the orientation of the sample.Stain the semi-thin section with toluidine blue for observation with light microscopy. Then trim the block further to reduce the area of interest to get proper ultrathin sections. Cut 60 to 70 nanometer thickness ultrathin sections with a second diamond knife.Collect one to two sections on one whole copper grids using a perfect loop. For contrasting the sections, immerse the grids in uranyl acetate solution for 20 minutes and wash the grids in double distilled water and then immerse the grids in lead citrate for 15 minutes and re-wash the grids in double distilled water. Turn on the transmission electron microscope computer and image recording software, record digital images with a digital Slow Scan CCD Camera and the associated imaging software.After inserting the grid with multiple sections in the microscope stage, screen the grid initially at low magnification to choose the best quality sections and determine the direction of the muscle fibers. Next focus, the image at magnification above 30, 000 with the beam centered on a peripheral fiber in the section and record images with one second exposure time at the desired magnification. Ensure that the images are distributed across the length and width of the fiber in a randomized bot systematic order to obtain unbiased results and repeat the imaging until six to 10 fibers are imaged.Import images to image J by clicking on file followed by open. Set global scale to match the original size of the image by clicking on analyze and then set scale. To zoom in 100%click on image, go to zoom and click on in.Measure the thickness of one Z-disk per image of the myofibrillar space using the straight line tool from the tools menu and calculate the average Z-disk thickness of each of the six to 10 fibers. Use the segmented line tool to measure the length of the outermost myofibril visible just below the subsarcolemmal region. To insert a grid, click on analyze, select tools and then select grid.Now set the area per point at 32, 400 square nanometers. Count the number of hits within the available length in the 12 subsarcolemmal images where a cross hits the subsarcolemmal glycogen. Insert a grid by clicking on analyze, then select tools, followed by grid and set area per point at 160, 000 square nanometers.Count the number of hits in the 12th myofibrillar images where a cross hits the intramyofibrillar space. Then again, to insert a grid, click on analyze, select tools and then select grid. Now set the area per point at 3, 600 square nanometers.Count the number of hits in the 12th myofibrillar images where a cross hits the intramyofibrillar glycogen. Insert a grid by clicking on analyze, then select tools, followed by grid and set area per point at 32, 400 square nanometers. Count the number of hits in the 12th myofibrillar images where a cross hits the intermyofibrillar glycogen.Using the 32, 400 square nanometers grid to randomly choose the glycogen particles, start with the upper left square and move left if necessary. Using straight line tool, measure the diameter of five randomly chosen glycogen particles of each pool for each of the 12 images to obtain an average of 60 particles per pool per fiber. This figure shows the normal values of the three glycogen pools.It can be observed that intermyofibrillar glycogen values are distributed close to normal, whereas both intramyofibrillar and subsarcolemmal glycogen show skewed distribution where fibers sometimes have an excessive amount of glycogen. Amongst all the steps it's very important to use potassium ferrocyanide. The analysis of glycogen can be combined with analysis of mitochondrion and lipid droplets which can improve our understanding of how other key metabolic components correlate with glycogen and muscle health.

quantification-subcellular-glycogen-distribution-skeletal-muscle

Research

JoVE Journal

Biochemistry

3.8K visualizações.  University College South Denmark. A medida do teor de glicogênio em células únicas e organizações subcelulares é importante porque, na maioria dos tipos de células, as células únicas se adaptam de forma diferente às mudanças nas condições fisiológicas e doenças. Então, a principal vantagem desta técnica é a resolução muito alta no microscópio eletrônico de transmissão. Isso melhora a possibilidade de ter estruturas subcelulares localizadas e também neste conteúdo nesta técnica aqui, é importante que...