Histochemical Methods—Visualizing Chemicals and Enzymes in Tissue

Three fundamental muscle fiber properties are identified using histochemical methods. ("Histochemical" [histo = tissue] implies that the chemical reaction is occurring in the tissue itself, rather than in a test tube or other reaction vessel.) These histochemical methods rely on the fact that enzymes located in thin (6-8 µm) frozen sections of muscle fibers can be chemically reacted with certain products in order to visualize the activity of the enzyme.

The basic requirement for a histochemical assay is similar, at least in principle, to the requirement for any biochemical assay. First, a substrate (fuel) is provided for the enzyme to be studied. Second, an energy source is provided that allows the enzyme to utilize the substrate. Finally, a reaction product is linked to another product that forms a precipitate so it can be visualized microscopically.

Let us now proceed to the three histochemical assays typically used to determine muscle fiber types. These three assays are the myosin ATPase (mATPase) assay, the succinate dehydrogenase (SDH) assay, and the a-glycerophosphate dehydrogenase (αGPD) assay.

Myofibrillar ATPase: Identifying Fast and Slow Fibers

The histochemical assay for myofibrillar ATPase activity is used to distinguish between fast- and slow-contracting muscle fibers. Recall that the during the cross-bridge cycle, the myosin molecule itself binds and hydrolyzes ATP during force generation. Because myosin ATPase activity is positively correlated with muscle contraction velocity, measures of ATPase activity can be interpreted in terms of contraction speed. The first step of this assay is a preincubation with either an acid (pH ~4) or basic (pH~10) solution. Acid preincubations inhibit the myosin ATPase activity in fast (mammalian type 2A, 2X, and 2B) fiber types, but not slow (mammalian type 1, see Figure 1A). Conversely, basic preincubations inhibit myosin ATPase in slow fiber types only (Figure 1B).

For this assay, ATP is the reaction energy source and substrate, P_i is the reaction product, and myosin is the enzyme. Since P_i is invisible histochemically, the assay requires that P_i be chemically reacted with calcium (Ca) in order to form the precipitate calcium phosphate (CaPO₄, limestone). Subsequent steps in the process convert CaPO₄ into colbalt sulfide (CoS₂) which is brownish-black, more easily viewable, and less soluble. Thus, effectively, as P_i is released by the myosin molecule's consumption of ATP, a brownish-black product is deposited on the muscle tissue section. Fast-contracting muscle fibers hydrolyze ATP faster than slow-contracting fibers. When given equivalent times, fast-contracting fibers appear dark histochemically, and slow-contracting fibers appear light. When this method is used in conjunction with preincubation steps to accentuate or reverse this pattern, the myosin ATPase assay can be used to distinguish between fast- and slow-contracting muscle fibers.

Figure 1: (A) mATPase reaction following an acid preincubation, demonstrating that only type 1 fibers stain darkly. (B) mATPase following alkaline preincubation, where the opposite pattern is seen.

Succinate Dehydrogenase: Identifying Oxidative Potential

The histochemical assay for SDH is used to distinguish between oxidative and nonoxidative (actually, “less” oxidative) fibers. Recall that fibers with a high oxidative capacity generate ATP via oxidative phosphorylation in the mitochondria. It follows that muscle cells which contain more mitochondria will have a higher oxidative capacity. The SDH enzyme is located in the inner membrane of the mitochondrion, bound to the cristae. SDH is responsible for oxidizing succinate to fumarate in the citric acid cycle. As this reaction proceeds, succinate is oxidized, and the reduced form of NADH is produced. Succinate is therefore the substrate, NADH is the reaction product (actually, a different electron acceptor is used for practical reasons), and SDH is the enzyme. The electron acceptor is chemically reacted with nitro blue tetrazolium (NBT), a purple salt, to visualize the reaction, and this results in a speckled pattern of the mitochondria (Figure 2), proportional to the number of mitochondria and the SDH activity within them. Similar to the ATPase assay, the more SDH (and therefore mitochondria) a fiber contains, the greater the intensity of the stain. Oxidative fibers have a relatively dense, purple speckled appearance, while nonoxidative fibers have only scattered purple speckles. Therefore, this histochemical assay reflects the relative oxidative potential of muscle fibers.

Figure 2: SDH assay demonstrating stronger staining in the more oxidative fibers.

α-Glycerophosphate Dehydrogenase: Identifying Glycolytic Potential

The αGPD enzyme is used to distinguish among fibers based on their relative glycolytic potential. Recall that glycolysis is used to generate ATP in the absence of oxygen (anaerobically). The chemical reactions involved in glycolysis take place in the muscle cell cytoplasm (myoplasm). The role of αGPD in glycolysis is to shuttle the NADH that is produced into the mitochondria where ATP can be produced. It is thus related to glycolytic activity in the sense that the more NADH that can be shuttled into the mitochondrion, the more energy that can be produced. It would be preferable to directly and selectively stain a rate-limiting glycolytic enzyme (such as phosphofructokinase [PFK], an important glycolysis regulatory enzyme), but technically, this is rather difficult to achieve.

As such, αGPD activity is directly related to the energy production of the glycolytic pathway. As with SDH, αGPD is an enzyme that “dehydrogenates” or oxidizes its substrate, glycerol-1-phosphate. The histochemical method is completely analogous to the SDH assay where the reaction product is linked to NBT. Unlike SDH, however, the reaction appears more homogeneous across the entire cell since the αGPD enzyme is not organelle bound. This assay can thus distinguish between glycolytic and nonglycolytic fibers.

Fiber Type Classifications Using Histochemical Methods

In their most basic form, the three histochemical methods described above can classify muscle fibers into fast or slow, oxidative or nonoxidative, and glycolytic or nonglycolytic. Given that there are three parameters with two choices each, we could potentially obtain any of the eight fiber types. In reality (and fortunately), however, over 95% of normal muscle fibers can be classified into one of only three categories.

Figure 3: An example of serial cross-sections of muscle stained for (A) hematoxylin and eosin to visualize morphology, (B) myosin ATPase, (C) SDH and (D) αGPD, to determine fiber type classification.

We have created a classification scheme that fulfills the criteria set forth above: It can classify most muscle fibers, and it can be related to physiologic, biochemical, and morphologic measurements. While classification schemes are, by definition, artificial, this one is less so in that it interleaves well with many different experimental methodologies.