Histochemical Fiber Types

Basic Concept - Cell Differentiation

The formation of muscle fibers from mesodermal cells through a series of transitional cell types (premyoblast, myoblast and myotube or secondary fiber) is a classical example of cellular differentiation. Cellular differentiation leads to an efficient and mutually advantageous division of labor among the tissues and organs of the body.

 In skeletal muscles, differentiation continues after the fibers have been formed and have reached a functional state.

 Physiological differentiation follows cellular differentiation, and creates populations of fast and slow fibers with appropriate sources of energy for contraction,

 either aerobic (using blood-borne oxygen for complete oxidation of substrates)

 or anaerobic (incomplete oxidation of carbohydrates without need for oxygen).

Red and White Muscle

Certain muscles of the carcass are particularly dark or red. This color difference is caused by a red pigment, myoglobin, in the sarcoplasm (cytoplasm) of muscle fibers.

 Hemoglobin, the pigment of red blood cells, brings oxygen to capillaries on the muscle fiber surface.

From here, the transport of oxygen to the interior of the fiber is facilitated by myoglobin. Thus, fibers specialized for aerobic metabolism develop a high myoglobin concentration.

The dominant work of some muscles is to maintain a standing posture or to contract slowly during locomotion, chewing or breathing. Such muscles tend to contain a high proportion of slow-contracting and fatigue-resistant fibers with a high myoglobin concentration. The capillary bed of red muscles is more dense than in white muscles.
 
 

Way back in 1873, the great French histologist Ranvier had already found that dark red muscles

Don't get longitudinal striations mixed up with In transverse sections of muscle fibers, differences in myofibrillar size, in the regularity of myofibrillar arrangement, and in the degree of myofibrillar separation may create two distinct patterns named by German histologists, felderstruktur in slow fibers and fibrillenstruktur in fast fibers.
 
  For every generalization, we can expect an underlying complexity of exceptions! A detailed explanation is available elsewhere.
 
 

Fast and Slow Fibers

At first sight, historically speaking, it appeared that the relationship between fast and slow fibers in meat animals was quite simple. From the time of Ranvier onwards, it had been known that fast fibers were usually white, while slow fibers were usually red. When redness was found to be due to myoglobin, and myoglobin was found to be correlated with aerobic metabolism, this explained the relationship between redness and speed of contraction. The pale or white fibers with a low aerobic potential were found to be well endowed with glycolytic enzymes that enabled them to obtain energy rapidly by the incomplete oxidation of glycogen. This explained why white fibers soon became fatigued once their glycogen stores were depleted and why they had to wait for the removal of lactate by the circulatory system.

 At the extremes of the range in physiological differentiation (fast white fibers versus slow red fibers) these discoveries are still valid. The problem, as we see it now, is that there are also fibers with a fast contraction speed and a dual energy supply.

In other words, some fast fibers have both aerobic and anaerobic capabilities.

The discovery of these fibers coincided in a most confusing way with a growing awarenesss that slow red fibers in meat animals and poultry were rather different from those of frogs and other creepy animals so frequently used in biomedical research. It is difficult to write a research report on muscle fiber types without giving them names. Unfortunately, everybody seemed to use different names, and the numbers of fiber types that were recognized tended to be a function of the number of histochemical techniques used to identify them. What a bummer.

Cutting a long story short, we may generalize as follows.

Here is an example of an ATPase reaction.

A frozen section of muscle is exposed to ATP solution and the ATPase obligingly cleaves off the phosphate. But the phosphate is invisible and tries to move around. First we stop it moving by precipitating the phosphate with cobalt, then we make the cobalt salt go black so we can see where it is by converting it to a sulfide. If that is all we do, all the fibers may go black, because they have all got ATPase. So first off all, before we start the reactions described above, we expose the frozen sections of meat to solutions (acetic acid, formaldehyde, etc) that will knock out the isoenzyme in either the fast or slow fibers. Then we can see differences between fibers, as above. It's a lot more complicated than this in reality, but hopefully this will help you understand this image!

Here is an example of an SDH reaction.

 SDH = succinate dehydrogenase, an enzyme specific to mitochondria. Each little granule of diformazan (the reaction product of nitroblue tetrazolium) indicates where there are mitochondria.
 
 

Here is an example of a phosphorylase reaction.

Phosphorylase is the first enzyme involved in glycogenolysis. It normally breaks down glycogen, but we can trick it into running backwards so that it makes new glycogen (amylose) that we can stain with iodine. The catch is, that the reaction works best if there is some natural glycogen present in the muscle fiber to start the reaction. Thus, absence of a phosphorylase reaction does not automatically mean that there is no phosphorylase present!
 
 

Here is an example of a stain for triglyceride - Sudan Black B.

Sudan black has stained the lipid droplets inside red muscle fibers in this slice of pork, and it has also stained a large triangle of intramuscular (marbling) adipose cells.

Many of the cellular features associated with aerobic and anaerobic metabolism in muscle fibers are fairly straightforward. Aerobic fibers are

Quantitatively, however, the range from aerobic to anaerobic metabolism is usually a continuous variable and is seldom broken into discontinuous steps.

From which we may deduce two points: Thus, to some researchers, the histochemical categorization of muscle fibers by any method, including myofibrillar ATPase, is merely a useful, but artificial subdivision of a continuously variable range. We (because this is the view I support) conclude that
 
 

muscle fibers undergo a continual alteration throughout life as an adaptation to changing functional demands, and that "fiber type" merely reflects the consitution of a fiber at any particular time.

However, click on to another researcher's home page, and you might read the opposite! From an agricultural viewpoint, this is particularly interesting since it suggests the existance of some degree of genetic or developmental plasticity in the fiber type continuum. In meat animals, this might be a vital link in relating muscle growth to meat quality.

Intracellular differentiation

Physiological differentiation may vary intracellularly along and across individual muscle fibers, at least as far as aerobic metabolism is concerned. But as far as is known at present, factors relating to contraction speed are fairly uniform within individual fibers. Aerobic metabolism, as indicated by the distribution of mitochondria, may be graduated radially so that the subsarcolemmal region has a high level of aerobic metabolism while the central axis has a low level. Mitochondria from peripheral and axial regions of the muscle fiber may differ in their biochemical characteristics, and. proportional mitochondrial volume and maximal rate of oxygen consumption are linearly related among different muscle regions.

The subsarcolemmal concentration of mitochondria in some types of muscle fibers may be related to the fact that the supply of oxygen to individual muscle fibers arrives in capillaries that wind over the surface of the muscle fiber. Mitochondria are larger in red fibers than in intermediate or white fibers and, in red fibers, they may form thick longitudinal columns between the myofibrils. The arterial and venous elements of muscle capillaries tend to occur in an alternating manner along the length of the fiber, with longer arterial segments of capillaries in white muscle relative to red muscle.

This image taken off my research computer shows the results of automatic mapping of the SDH deposits in a muscle fiber. Dark blue shows high SDH, and light blue shows low SDH (and cyan is medium). Once on the computer, these data can be used for studying the radial gradients of SDH activity in different types of meat animals. Gradients have been measured for pigs,geese,ducks, and turkeys.

TROPHIC EFFECT OF NERVES

Motor neurons exert a long term regulation over the physiological and metabolic properties of the fibers in their motor unit. This is often called the trophic effect of nerve on muscle. The word trophic implies something of a nutritive effect, as if the nerve was feeding the muscle, but its current usage sometimes includes possible non-nutritive effects such as the frequency patterns of nerve impulses to the muscle. The idea that nerves might have a trophic function is far from new, and probably originates from ancient observations on the degenerative fate that overtakes many organs once they have been denervated.

Trophic effects may be bi-directional since there are some retrograde trophic effects that travel from the muscle to the nerve. For example, presynaptic terminal boutons on motor neuron perikarya are lost when axons are cut, and they are restored when neuromuscular contact is re-established. Similarly, there are soluble fractions from skeletal muscle that may promote growth and differentiation in the embryonic spinal cord.

FIBER TYPE CHANGES DURING GROWTH

Histochemical fiber types are important in meat animals because they influence meat quality. Histochemical fiber types also react differently during the conversion of muscles to meat, because they contain different levels of glycogen and anaerobic enzymes. Before it became known that fibers could change from one type to another, growth-related changes in fiber types were not adequately controlled in agricultural experiments on muscle fiber histochemistry.

 This three dimensional plot shows the sorts of changes that may occur as fiber type clusters are transformed during muscle growth.