Enzyme Histochemistry: Identification of Fast vs Slow Twitch Muscle fibers in Rat Tissue

Skeletal muscle is made up of bundles of individual muscle fibers called myocytes. Each myocyte contains many myofibrils, which are strands of proteins (actin and myosin) that can grab on to each other and pull. This shortens the muscle and causes muscle contraction. It is generally accepted that muscle fiber types can be broken down into two main types slow twitch (Type I) muscle fibers and fast twitch (Type II) muscle fibers. !ast twitch fibers can be further categori"ed into Type IIa and Type IIb fibers. Slow twitch muscles are more efficient at using o#ygen to generate more $T% for continuous, e#tended muscle contractions over a long time. They fire more slowly than fast twitch fibers and can go for a long time before they fatigue. Therefore, slow twitch fibers are great at helping athletes run marathons and bicycle for hours. Since fast twitch muscle fibers use anaerobic metabolism to create fuel, they are much better at generating short bursts of strength or speed than slow muscles. &owever, they fatigue more 'uickly. !ast twitch fibers generally produce the same amount of force per contraction as slow muscles, but they get their name because they are able to fire more rapidly. &aving more fast twitch fibers can be an asset to a sprinter since s(he needs to 'uickly generate a lot of force. &istochemical methods can be used to classify muscle fibers into fast or slow, o#idative or non)o#idative, and glycolytic or non)glycolytic. *&istochemical* +histo , tissue- implies that the chemical reaction is occurring in the tissue itself, rather than in a test tube or other reaction vessel. &istochemical methods rely on the fact that en"ymes located in thin (.)/ 0m) fro"en sections of muscle fibers can be chemically reacted with certain products in order to visuali"e the activity of the en"yme. The basic re'uirement for a histochemical assay is similar, at least in principle, to the re'uirement for any biochemical assay. !irst, a substrate is provided for the en"yme to be studied. Second, an energy source is provided that allows the en"yme to utili"e the substrate. !inally, a reaction product is linked to another product that forms a precipitate so it can be visuali"ed microscopically. There are three histochemical assays typically used to determine muscle fiber types. These three assays are the myosin $T%ase (m$T%ase) assay, the succinate dehydrogenase (S1&) assay, and the )glycerophosphate dehydrogenase (23%1) assay. The m$T%ase assay is used to distinguish between fast) and slow)twitch muscle fibers. Since $T% is hydroly"ed during force generation, the level of $T%ase activity can be correlated to contraction speed. The a)31% assay is used to identify glycolytic potential. The function of )31% is to shuttle 4$1& into mitochondria where the electrons it carries can be used to make $T%. 5ytoplasmic 4$1& is produced during glycolysis and therefore is an indirect measure of glycolytic activity. The histochemical assay for S1& is used to distinguish between o#idative and non) o#idative (actually, 6less7 o#idative) fibers. !ibers with a high o#idative capacity generate $T% via o#idative phosphorylation in the mitochondria. It follows that muscle cells that contain more mitochondria will have a higher o#idative capacity and therefore will have a higher contraction speed (a fast fiber). Therefore S1& levels can be an indicator if a muscle fiber is a fast twitch or a slow twitch fiber. In this lab, you will dissect muscle tissue from a rat. This muscle tissue will be fro"en in isopentane cooled to )89::5 followed by sectioning with a cryostat. The fro"en sections will then be stained for S1& activity. 8

S1& is located in the inner membrane of the mitochondrion, bound to the cristae. S1& is responsible for o#idi"ing succinate to fumarate in the ;reb<s 5ycle. $s this reaction proceeds, succinate is o#idi"ed, and the reduced form of 4$1& is produced. Succinate is therefore the substrate, 4$1& is the reaction product (actually, a different electron acceptor is used for practical reasons), and S1& is the en"yme. The electron acceptor is chemically reacted with nitro blue tetra"olium (4=T), a purple salt, to visuali"e the reaction, and this results in a speckled pattern of the mitochondria (!igure 8), proportional to the number of mitochondria and the S1& activity within them. Similar to the $T%ase assay, the more S1& (and therefore mitochondria) a fiber contains, the greater the intensity of the stain. >#idative fibers have a relatively dense, purple speckled appearance, while non)o#idative fibers have only scattered purple speckles. Therefore, this histochemical assay reflects the relative o#idative potential of muscle fibers. Purpose: The purpose of the this lab is to use a histochemical stain for a SDH to identify fast and slow muscle fibers in rat thigh muscle, determine the percentage of these fibers in the tissue and the average diameter of each fiber type.

!ig. 8 !ro"en section of skeletal muscle stained for S1& showing fast fibers (dark) and slow fibers (light). ?etrieved from the @orld @ide @eb at http ((muscle.ucsd.edu(musintro(histochem.shtml on Aanuary B, C::/ Procedure: 8. !ree"ing muscle specimens a. 1issect out muscle specimen. Suggestions would be abdominal or leg muscle. b. ?inse tissue in ice cold %=S to remove blood. Deave in %=S until ready to free"e. c. %lace the plastic container into a larger plastic container. !ill the smaller container E full with isopentane (C)methylbutane). Slowly add small chips of dry ice to the isopentane. @atch out for boiling over. ;eep adding dry ice until the isopentane stops boiling. d. Fsing forceps drop your tissue into the isopentane and allow to remain for about 8G sections. Then remove the specimen and place on the Saran wrap on dry ice. 1on<t let the sample thaw. C. Hounting specimen for sectioning a. Hake sure the cryostat is set to )C:o5. b. Transport the specimen to the cryostat on dry ice. c. Hount the specimen on a disk using free"ing medium. C

d. e. f. g.

$llow the specimen to sit in the cryostat for 8: minutes to warm up. Section specimen at / m. Sections should be collected toward the bottom of the slide. Slides can be kept in a slide bo# until ready for staining.

I. Staining slides for S1& activity. a. Incubate sections for I: minutes at IJo5 in incubation medium placed in a 5oplin Kar. b. ?inse section in physiological saline. This can be done by dipping the slides several times in a 5oplin Kar containing the saline. c. !i# sections in 8:L formalin)saline solution (5oplin Kar) for I)G minutes. d. ?inse in 8GL alcohol for G minutes (5oplin Kar). e. Hount with an a'ueous mounting medium and let sit for C)I minutes. f. Seal edges of cover slip with clear nail polish and let dry. g. Miew with a microscope. 0.2 M. Sodium phosphate monobasic C.J/ g(8:: ml 0.2 M Sodium phosphate dibasic G.IJg(8::ml 0.2 M Phosphate Buffer, pH 7.6 8I ml :.C H sodium phosphate monobasic /J ml :.C H sodium phosphate dibasic 5heck p& and adKust to J.. using 84 4a>& or 84 &5D. Store in the refrigerator. 0.2 M Sodium succinate solution C.J: g Sodium succinate (4a>5>5&C5&C5>>4aN.&C: G: ml d&C: %repare fresh NB solution :.8 g 4=T (Sigma O 4)./J.) G: mls d&C: Hake up only G: ml at a time and store in the refrigerator. !ncubation medium Aust before use, mi# G ml :.C H phosphate buffer G ml sodium succinate solution G ml 4=T solution G ml d&C>

Ph"siolo#ical Saline /.G g 4a5l 8 D d&C> Store at room temperature I

$0% &ormalin'saline solution 9G ml physiological saline G ml 9:L formaldehyde %repare fresh each time $(% alcohol 8G ml ET>& /G ml d&C> Buffered )l"cerol mountin# medium Either :.8H %hosphate buffer (p& J.9) 8: ml or :.8H T?IS buffer (p& B.:) 8: ml $nti)fading agent Either p)%henylenediamine hydrochloride 8:: mg or n)propyl gallate G:: mg 3lycerol B: ml ;eeps for at least I months, probably much longer, in darkness (which protects the anti)fade agent) at )C:5. The working bottle is kept at 95, for a week or two. *ata anal"sis: 8. 1etermine the percentage of fast vs slow fibers. C. 1etermine the average diameter of fast and slow fibers. I. Take a picture of your preparation. +ab report: 8. @rite an abstract and a results section of your research including all necessary data properly presented. C. Include a picture of your slide that includes a si"e marker. I. $nswer the following 'uestionsP a. !or each of the following indicate its purpose. 4=T Succinate %hosphate buffer !ormalin)saline b. @hy are some muscle fibers stained darker than othersQ c. E#plain how this stain works to label mitochondria. d. @hat is the difference between fast and slow fibersQ e. @hy do muscle cells have these two different types of fibersQ f. If you are an endurance runner, is it better have more fast fibers or slow fibersQ E#plain.

This protocol was obtained at http ((www.ihcworld.com(Rprotocols(histology(a'ueousRmountingRmedium.htm. ?etrieved from the @orld @ide @eb on Aanuary 8:, C::/ This lab was adapted from &istotechnology $ Self)Instructional Te#t (8BBJ) by !reida D 5arson, $S5% %ress, p. C.G)C.J 9