ORIGEN Y DESARROLLO DEL DERECHO REGULATIVO

The fibres which express fast and/or slow myosin heavy chains can be characterized metabohcaUy (ie for oxidative capacity) by measuring the activities of key enzymes of the aerobic and anaerobic respiratory pathways, and also physiologically by determining their speed of contraction. However, the staining intensity of myosin ATPase at different pH remains one of the most widely used histochemical methods of isoform classification. The latter method, although rapid, is unsatisfactory for several reasons (Sant'Ana Periera).

Sahin (1977) combined these methods to classify human fast and slow fibre types; the fibres expressing type I MyHCs were found to be highly oxidative due to high activities of enzymes within the TCA cycle. The rate at which ATP is used and supplied determines the fatigue resistance of that fibre and thus muscle. The type I fibres also possess low glycolytic capacity but do not stain for mATPase activity at either set of pre-incubations (Table 5.0 shows the staining intensities of different fibre types under alkaline and acidic conditions). The blood supply to these fibres is dense and there is also a high density of mitochondria associated with the type I fibres both ofwiiich contribute to the highly fatigue-resistant properties of the type I fibres. The

postural muscles such as the soleus for example are largely composed of these fibre types since they are in constant use. In contrast, the properties of fast-twitch type HB fibres are opposite to those described for the type I fibres. They were shown to have a high glycolytic:oxidative ratio and stained intensely for mATPase after both pHlO.3 and pH4.6-4.8 pre-incubations. They have a poor blood supply, contain very few mitochondria and obtain their fuel from endogenous glycogen stores which supply ATP via the anaerobic pathway. The fast type HA were identified by their positive mATPase staining at pHlO.3 but not with the double pre-incubations. These fibre- types form the intermediate between the type I and IIB isoforms in terms of enzymatic activity and speed of contraction.

Property Type I Type HA

(oxidative)

Type nB*

(glycolytic)

ContractUe Speed slow fast fast

ATPase (pH 10.3) - +++ +++

ATPase (pH 4.6-4.S + 10.3) - - +++

Glycolytic Capacity low moderate high

Oxidative Capacity high moderate low

Blood Supply good moderate poor

Table 5 .0 Properties o f hum an m yosin heavy chain isoform s (N e w sh o lm e 1986).

*Ennion e t a l (1 9 9 5 ) sh ow ed that the IIB isoform as characterized in the rat is not exp ressed in hum an m u s c le . In fact the com b in ed results o f im m unochem istry, h istology and c lo n in g techniques dem onstrated that the so called hum an IIB fibres express a m y o sin heavy chain isoform equivalent to the rat IIX , n ot the IIB.

In addition to the metaboHc differences between isoforms, different fibre-types are also subject to different patterns of neuronal stimulation. Each fibre is supphed by only one branch of motomeurone Wiich also stimulates many others within the motor unit. The fast and slow fibres can be distinguished by their abihty to produce force either rapidly or over a longer period of time due to their pattern of innervation. The rate at v\frich force is developed as well as the maximum rate of shortening (Vmax), correlates weU with the mATPase activity of the myosin heavy chain expressed. Type

II fibres are innervated by large motor neurones which fire in short intense bursts while the type I innervating neurones supply a more steady repetitive transmission of activity. Other 6 ctors influence force and power production; these include the number

of muscle fibres and the size of each fibre. In addition different isoforms of the troponin/tropontyosin complex may exist which have different sensitivities to calcium levels in the local environment. This would alter the speed at which cross-bridges are formed regardless of myosin heavy chain isoform properties.

5.2.2 Myosin Heavy Chain Genes

Each of the skeletal, cardiac and developmental MyHC Class II isoforms are encoded by separate genes which are highly homologous in nucleotide and amino acid sequence. This is in contrast to the single myosin heavy chain gene identified in

Drosophila which is alternatively sphced to produce the functionally diverse myosin proteins expressed in flight and leg muscles. In mammalian systems there exist type I (slow), type DA, DB, EX (fast), a- and P-cardiac, embryonic and developmental isoforms as well as some other uncharacterized genes. Although there is a developmental and tissue-specific expression of these genes, the myosin heavy chain corqposition of a muscle can change according to functional demands made upon it (Goldspink 1992).

The skeletal and cardiac genes are encoded on separate chromosomes. In humans the embryonic, neonatal, fast skeletal and extraoccular muscles have been mapped to chromosome 17 (Yoon 1992). The same genes are encoded on chromosome 11 in the mouse (Weydert 1985). The a- and P- cardiac genes are both found arranged tandemly on chromosome 14 in mouse and human and separated by 4-5kb.

The Class II myosin heavy chain genes have a genomic length of approximately 23kb which includes either 40 or 41 exons. Alternative splicing of exon 37 in some isoforms (such as the rat embryonic) is responsible for the 'additional' exon. Exons 1 and 2 are untranslated and do not contribute to any coding regions of the resulting protein. The ATG start codon is located in the 5' region of exon 3 which encodes an mRNA size of approximately 6kb. The resulting myosin heavy chain protein has a high molecular weight of approximately 200kDa. There is an untranslated region between 100 and 120 nucleotides after the stop codon which has been useful in distinguishing different myosin heavy chain transcripts. Despite functional diversity there is considerable conservation of nucleotide and amino acid sequence across all isoforms. Two regions however are significantly divergent between genes encoding seemingly random amino acids which form flexible loops that protrude from the surface of the myosin protein. Because of their unconserved nature it has been thought that they serve no function. However according to other studies (Kelley 1993; Spudich 1994; Uyeda 1994) it is suggested that in fact they play significant roles in determining the contractile properties of each isofoim These hypervariable regions are described in detail below (see Chapter 10), with particular emphasis upon the loop that forms part of the nucleotide binding pocket where ATP is hydrolyzed.