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Muscle fiber – a bidirectional approach

Abstract: Molecular investigation of muscle fiber reveals the versatility of its contractile units reflected into conversion within the specific spectrum of muscle fiber phenotype bordered by type I and II. In this regard there have been found three main type I, type IIa and type IIb fibers even though the existence of myosin heavy chain (MHC) isomers enlarges this spectrum. The main factor that triggers and controls this transformation is accounted by the motoneurons units and their different pattern of firing rate. There have also been established different signaling pathways involved in expression of gene in proteins synthesis that make up MHC, sarcoplasmic or endoplasmic reticulum calcium ATPase (SERCA), aerobic and anaerobic enzymatic apparatus specific to each main muscle fiber type. Specificity of contraction in muscle fibers can also be investigated during exercise by measuring the energy turnover which has dynamic kinetic determined by the predominant fraction and type of the muscle fibers recruited. It comes out that the recruitment pattern determined by the exercise duration and intensity conditions the expression of fibers phenotype within a muscle.

Key words: myosin heavy chain, sarcoplasmic or endoplasmic reticulum calcium ATPase,


  How fast, for how long and how powerful the contraction of the muscles is, are the main criteria depending on which, the act of movement in sport is investigated. But these psycho-physical qualities determine a wide range of body movement’s spectrum, made up by the different weight of contribution for each of them. This interpretation is simple and very easy to be understood. The existence of more parameters in quantification of the body movements in sport, in terms of their speed or duration, reveals that skeletal musculature is able to contract and sustain different physical efforts determined by the specific sport requirements. Therefore the muscle fibers must be regarded as versatile muscle units which adapt their molecular structure and function in response to the manner in which the muscle is solicited. In this regard it has been established the existence of three main I, IIA and IIB skeletal muscle fiber types (4) based on the myosin heavy chain (MHC) isoforms MHC I, MHC IIa, MHC IIb identified by ATPase staining. Yet, the human skeletal muscle fibers almost always contain myosin heavy chains that are hybrids (26). Identification of these multiple fiber types has been accomplished by biochemical, histochemical, physiological and morphological approach, but the findings do not always agree (32).
  The aim of this paper is not debating upon these techniques. The histochemical method yet is
discussed here because by this technique the most intimate site of the contraction machinery is explored. Then the in vitro molecular structure and function investigations of the muscle fiber will be paralleled by findings obtained based on the morphological, structural and functional assessments prior, during and after exercise.

Molecular structure and function of the muscle fiber contraction machinery

 Inside the “chinese box” an interesting comparison of the gross structure of the muscle in which the external sheaths epimysium, perimysium and endomisyum are compared with the smaller boxes encompassed inside of a large box, the most important components that dictate the behavior of a muscle are its contractile units, the sarcomeres. More about the gross structure of the muscle can be found elsewhere in literature.
  Detailing the topic, the actin and myosin filaments which by coming repeatedly in contact within cross-bridge cycle represent the core of contraction in each single muscle fiber.
  The thin filament, is a protein whose backbone is a double-stranded α-helical polymer made up by actin molecules. This structure is also called F-actin. Two identical α- helices that wrap around each other and reside along the groove formed by the helical actin strands, represent tropomyosin.
Further, troponin a heterotrimer (troponin T, troponin C and troponin I) is the third component of the complex inside which, the coordinated interaction among actin, tropomyosin and troponin permit and influence actin-myosin reactions triggered by Ca2+ concentration (Fig 1).

Fig 1. Troponin complex and the exposed myosin binding sites caused by Ca2+ interaction with troponin C subunit of the complex.
from Elsevier Ltd. Boron & Boulpaep:Medical Physiology, Updated Edition

The thick filament, myosin structure reveals as the actin filament, a polymeral structure. One myosin molecule is a hexamer formed by the heavy chains represented by two α-helices left handed supercoiled structures which end each other with a head, consisting of 850 amino acids (3).

Fig 2. Molecular structure of the thick filament. In the figure are presented the myosin heavy chain that ends up with the heads displaying the regulatory and essential subunits.
from Elsevier Ltd. Boron & Boulpaep:Medical Physiology, Updated Edition

A α-helix that extends from this domain represents the link for the essential and regulatory light chains (Fig 2).
Within the cross-bridge cycle, binding of one ATP molecule detaches the head of the myosin heavy chain. Further hydrolysis of ATP allows the myosin head to attach again to the binding site along the actin filament and concomitant with the Pi release the power stroke (the shortening of the muscle) occurs (fig 3). One cycle in the actine-myosin interaction is ended up when myosin heavy chain nucleotide binding site is occupied only by ADP. The release of ADP lives the actin-myosin complex into an attached state.

Fig 3. The action of Ca2+ on troponin C subunit resulting in  exposing the myosin binding site and coupling of actin and myosin.
from Elsevier Ltd. Boron & Boulpaep:Medical Physiology, Updated Edition ATPase-based identified muscle fiber types and myosin heavy chain isoforms

 Histochemical staining for myosin ATPase activity has been the most used technique in classifying the muscle fibers in type I, IIA, IIB. This technique is based on exposing the myosin heavy chain head that displays the ATPase activity, to different pH levels and thereafter the muscle tissue preparation is incubated in the presence of Ca2+ and ATP (4).
Depending on how high the acidity level affected activity of the ATP binding site (conformational structure) the ATPase activity will be differently expressed among the fibers of the muscle preparation. Therefore it was noted that the ATPase system in type I fibers is not affected by pH ranging between 3.9 and 10.4. Type IIA fibers displayed an ATPase activity which was inhibited by a pH below 4.9 and above 10.8 whereas in type IIB fibers ATP hydrolysis is not affected by acidity levels from 4.5 to 10.8 pH. However, it can be noted that this technique does not evaluate myosin ATPase hydrolysis rate which has been found to be 2 to 3 time higher in type II than I fibers (35). Based on this method, more recently muscle fibers of type IC, IIC, IIAC and IIAB which posses intermediate myosin ATPase activity have been identified (32).
 Studying the expression of the MHC isoforms in the muscle fibers also allows for fiber type classification. Even though the myosin heavy chain isoforms are identified by imunohistochemical analysis in which are used antimyosin antibodies or sodium dodecyl sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) separation (25) these correspond to the ATPase-based identified muscle fiber types. It is normal because the myosin heavy chains contain the site for ATPase. Except the correspondence between type IIB and MHCx the notation for myosin heavy chains related to fiber type is the same (table 1). The human skeletal muscle does not express the fast myosin heavy chain isoform MHCIIb (15).
 There is also evidence that the light chains of the myosin filament can exist in different isoforms which affects the contractile properties of the fiber (34).

 Dynamic of the muscle fiber type transformation
A very important characteristic of the skeletal muscle is its capability for adaptation to the different daily functional demands. This capability is reflected in the muscle fiber plasticity which presumes conversions among fiber types. The most common conversion has found to be between type IIB and IIA (1, 26) but also an increased expression of MHC isoform IIa related with a bidirectional transformation from both MHC isoform I and IIx towards MHCIIa can be possible (1). A los in type I fibers by a strong conversion into type II was found to be caused by severe deconditioning or spinal cord injury (29).

Table 1. The correspondence between ATPase stained fiber types and myosin heavy chain isoforms
ATPase - stainedMyosin heavy chain
Fiber typeIsoforms

Promoters of the different muscle fiber phenotypes

  The plastic nature of the muscle fiber phenotype has been proved to be accounted by the physiological control exerted by the variations in motor neuron activity (26, 27, 37). The main consequence of the neural stimulation is the increase of intracellular Ca2+ concentration but the mechanism by which this can be the link between motoneurons activity and gene expression is still a debated issue. However some of the gene transcription inducing promoters involved specifically in expression of the two main type I and II fibers have been suggested. Therefore a calcineurin-dependent transcriptional pathway was found to control the expression of gene in slow twitch fibers (5). The proposed mechanism consists in the direct control of calcineurin on the phosphorylation state of nuclear factor of activated protein (NFAT) and its translocation to the nucleus which further causes the activation of the target slow-type muscle proteins in cooperation with myogenic enhancing factor (MEF2) which responds to multiple calcium-regulated signals (39) and others regulatory proteins. Signaling pathway involving Ras and mitogen-activated protein kinase (MAPK) has been also found to influence the generation of slow fibers (24). Calcium-dependent calmodulin kinase activity is also regulated by a low firing rate of the motor units (39). Peroxisome-proliferator-activated receptor-gamma co-activator-1 alpha (PGC-1α) activates transcription in cooperation with MEF2 proteins and serves as a target for calcineurin signaling, which as mentioned before is implicated in slow fiber gene expression (21).
  The shift in the predominant metabolic nature of the effort from aerobic to anaerobic production of the energy is accompanied by a corresponding response in the transcriptional control. In the study by Grifon et al (2004), supplying the predominant slow twitch fibers containing muscle soleus, with Six 1 and its cofactor Eya 1 caused the proliferation of endogenous glycolytic enzymes and fast twitch sarcomeric proteins concomitantly with down regulation of slow oxidative transcription controlling factors. Another important regulator in the type II fiber gene expression has been found to be the hypoxia inducible factor-1α (HIF-1α). Its proliferation was found to correspond with essential hypoxic responses in maintaining a constant ATP level in the cells (23). Therefore the higher recruitment of type II fibers in performing maximal and supramaximal exercise intensities can not be only accidentally associated with the more pronounced hypoxia caused by such efforts.
  It can be concluded that the main transitional factor affecting expression of the gene in muscle contractile proteins synthesis is the dynamic of intracellular Ca2+ concentration. As cross-bridge cycle occurs only following the reaction of calcium ions with troponin in exposing the myosin binding sites of actin filament it results that the pattern of calcium release in response to neural excitation must be mediated by a different activity in Ca2+ pumps. Therefore ATP-dependent Ca2+ pump SERCA, have been found to exist in three isoforms (22). From these SERCA 1 has been found to be more expressed in type II whereas SERCA 2 in type I muscle fibers.
  In summary, it can be stated that the different pattern in muscle contraction caused by motoneurons activity triggers corresponding transcriptional control which targets not only the expression of MHC isoforms, but also glycolytic and/or oxidative enzymatic apparatus as well as SERCA isoforms.

Macromolecular assessment of the muscle fiber phenotype

  It consists in evidencing the findings about molecular structure and functioning of the muscle fibers using assessment of the skeletal muscles behavior during exercise by different methods and procedures which are less invasive and provide faster results.
  The most common approach in identifying the predominant type of muscle fiber bundles recruited during contraction is investigation of muscle contraction intensity and velocity and the corresponding energy turn over. Muscle energy turnover can be addressed either by metabolic or calorimetrically approach (38). Metabolic approach assumes summing the muscle aerobic and anaerobic energy turnover while the calorimetric way consists in measuring the muscle heat production and power output. By in vitro studies based on the energetic cost of short tetani (6), the content of ATP and CP that was found to be proportional with contraction velocity (7) and the heat release during contraction (2) it has been established that in type II contraction velocity is 3-5 times higher then type I fibers.
  In accordance to the findings of these thorough investigations it has been noted that the physiological adjustments occurring in the human body during exercising at different exercise intensities were different. In this regard the oxygen uptake kinetic has been found to present a third slow component during higher exercise intensities indicating higher oxygen consumption due to a higher recruitment of fast twitch fibers (19, 28). This can be due to the lover mitochondrial content and in general the reduced aerobic enzymatic equipment which causes them to have lower efficiency in rephosphorylation of the nucleotides based on oxygen use (16, 18). Moreover type II fibers have been found to express the alpha glycerolphosphate shuttle involved in NADH (nicotine adenine dinucleotide-dependent H+) transport to higher degree than type I (31) which also explains their lower efficiency in obtaining ATP within mitochondrial respiratory chain.
  Besides these, the expression of uncoupling protein 3 (UCP3) was found to correlate with the fraction of FT fibers in the human skeletal muscle (14). It has been suggested that due to this protein, the leakage of the protons across the mitochondrial membrane decreases P/O ratio during exercise. Thus it can be suggested that the shift in the recruitment pattern towards more FT fibers contracting during exercise could increase the metabolic energy cost and thus decreases the mechanical efficiency.
  Estimation of the glycogen stores depletion during exercise revealed that the higher exercise intensities were well correlated with the higher consumption of the glycogen predominantly from type II fibers (9, 36).
  Production of ATP by the immediate phosphagen system namely CP (creatine phosphate) dephosphorylation has been found to be significant higher in type II fibers within intense exercises (30, 33). These are explainable because CP as well as glycogen levels have been found to be higher in type II fibers. Lactate release has been found to increase in parallel with exercise intensity. An interesting finding is that two of the major monocarboxylate transporters (MCTs) residing in the muscle membrane cell and that are involved in lactate transport have different properties in terms of their activity rate. The MCT4 isoform which was predominantly found in fast twitch glycolytic fibers has a relatively high dissociation constant (binds easily but releases hardly the lactate molecule) Km of 20-35 mM whereas MCT1 isoform more expressed in slow twitch oxidative fibers has Km of 3-5 mM (11, 20). This also shows the ability but also the predisposition of type II fibers to produce and use the energy anaerobicaly and this as it has been mentioned above supports the higher velocity of skeletal muscle contraction.

Fig 4. Recruitment of the three main fiber types expressed depending on firing rate and contraction intensity: type I (open squares), type IIb (filled squares) and type IIb (filled triangles); based on data from Hannerz 1974.

  By using muscle biopsy technique it is possible to asses the changes in percentage expression of fiber types within a muscle as well as cross sectional area and capillarization that have been influenced by the specificity of designed protocol.
All together indicate the existence of recruitment pattern according to which low threshold type I fibers are recruited at low intensity and high threshold type II fibers are recruited as exercise intensity increases (13, 17) (Fig 4)
  It becomes interesting to make a parallel between laboratory work findings and eyes free observations in terms of the correspondence between velocity or/and intensity of contractions and the level of oxygen consumption or heart rate ,when measuring of oxygen consumption is not possible, being known that there is a close relationship between heart rate and oxygen uptake (8). Surely, completion of these with measurements of blood and muscle metabolites comes and improves the accuracy in measuring the involvement degree in terms of recruitment and activation level of the three main skeletal muscle fibers.

Mihai Relu Ursta Anghel  
August Krogh Institute, Department of Human Physiology, University of Copenhagen, Denmark  


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August 25,  2007

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