PLASTICITY OF SKELETAL MUSCLE STUDIED BY STEREOLOGY

The present contribution provides an overview of stereological methods applied in the skeletal muscle research at the Institute of Anatomy of the Medical Faculty in Ljubljana. Interested in skeletal muscle plasticity we studied three different topics: (i) expression of myosin heavy chain isoforms in slow and fast muscles under experimental conditions, (ii) frequency of satellite cells in young and old human and rat muscles and (iii) capillary supply of rat fast and slow muscles. We analysed the expression of myosin heavy chain isoforms within slow rat soleus and fast extensor digitorum longus muscles after (i) homotopic and heterotopic transplantation of both muscles, (ii) low frequency electrical stimulation of the fast muscle and (iii) transposition of the fast nerve to the slow muscle. The models applied were able to turn the fast muscle into a completely slow muscle, but not vice versa. One of the indicators for the regenerative potential of skeletal muscles is its satellite cell pool. The estimated parameters, number of satellite cells per unit fibre length, corrected to the reference sarcomere length (Nsc/Lfib) and number of satellite cells per number of nuclei (myonuclei and satellite cell nuclei) (Nsc/Nnucl) indicated that the frequency of M-cadherin stained satellite cells declines in healthy old human and rat muscles compared to young muscles. To access differences in capillary densities among slow and fast muscles and slow and fast muscle fibres, we have introduced Slicer and Fakir methods, and tested them on predominantly slow and fast rat muscles. Discussing three different topics that require different approach, the present paper reflects the three decades of the development of stereological methods: 2D analysis by simple point counting in the 70's, the disector in the 80's and virtual spatial probes in the 90's. In all methods the interactive computer assisted approach was utilised.

CAPILLARY NETWORK IN SLOW AND FAST MUSCLES AND IN OXIDATIVE AND GLYCOLYTIC MUSCLE FIBRES

The aim of this study was to compare capillary network in slow and fast muscles and also in oxidative and glycolytic muscle fibres. Soleus (SOL) and extensor digitorum longus (EDL) muscles were excised from five female rats. Capillaries and muscle fibres were demonstrated on thick tissue sections by a triple immunofluorescent method. Stacks of perfectly registered optical images were captured by a confocal microscope and further analysed. Applying stereological methods (POINTGRID, FAKIR and SLICER plugin- modules of the Ellipse programme), we estimated the mean length of capillaries, adjacent to individual muscle fibre, per unit fibre length (Lcap/Lfib), per unit surface area of the fibre (Lcap/Sfib) and per unit fibre volume (Lcap/Vfib) in the slow SOL and in predominantly fast EDL muscle, and separately in oxidative and glycolytic fibres of EDL muscle. The length of capillaries per unit fibre length was larger in SOL than in EDL muscle, however, capillary length per unit fibre volume was larger in EDL muscle. There was no difference in the length of capillaries per unit fibre surface area between the two muscles. Oxidative and glycolytic fibres differ in the length of capillaries per unit fibre surface area (Lcap/Sfib). This parameter probably reflects the oxidative capacity of muscle fibres. In conclusion, capillary supply is evidently well adapted to different muscle fibre types; consequently, an average capillary supply of a heterogeneous muscle depends on the muscle composition. The estimated mean values blur some intrinsic differences.

Extracellular signal-regulated kinase pathway is differentially involved in β-agonist-induced hypertrophy in slow and fast muscles

The molecular mechanisms controlling β-adrenergic receptor agonist (BA)-induced skeletal muscle hypertrophy are not well known. We presently report that BA exerts a distinct muscle- and muscle fiber type-specific hypertrophy. Moreover, we have shown that pharmacologically or genetically attenuating extracellular signal-regulated kinase (ERK) signaling in muscle fibers resulted in decreases ( P < 0.05) in fast but not slow fiber type-specific reporter gene expressions in response to BA exposure in vitro and in vivo. Consistent with these data, forced expression of MAPK phosphatase 1, a nuclear protein that dephosphorylates ERK1/2, in fast-twitch skeletal muscle ablated ( P < 0.05) the hypertrophic effects of BA feeding (clenbuterol, 20 parts per million in water) in vivo. Further analysis has shown that BA-induced phosphorylation and activation of ERK occurred to a greater ( P < 0.05) extent in fast myofibers than in slow myofibers. Analysis of the basal level of ERK activity in slow and fast muscles revealed that ERK1/2 is activated to a greater extent in fast- than in slow-twitch muscles. These data indicate that ERK signaling is differentially involved in BA-induced hypertrophy in slow and fast skeletal muscles, suggesting that the increased abundance of phospho-ERK1/2 and ERK activity found in fast-twitch myofibers, compared with their slow-twitch counterparts, may account, at least in part, for the fiber type-specific hypertrophy induced by BA stimulation. These data suggest that fast myofibers are pivotal in the adaptation of muscle to environmental cues and that the mechanism underlying this change is partially mediated by the MAPK signaling cascade.

Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat

To investigate the plasticity of slow and fast muscles undergoing slow-to-fast transition, rat soleus (SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscles were exposed for 14 days to 1) unweighting by hindlimb suspension (HU), or 2) treatment with the β2-adrenergic agonist clenbuterol (CB), or 3) a combination of both (HU-CB). In general, HU elicited atrophy, CB induced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoforms revealed HU- and CB-induced slow-to-fast transitions in SOL (increases of MHCIIa with small amounts of MHCIId and MHCIIb) and the upregulation of the slow MHCIa isoform. The HU- and CB-induced changes in GAS consisted of increases in MHCIId and MHCIIb (“fast-to-faster transitions”). Changes in the MLC composition of SOL and GAS consisted of slow-to-fast transitions and mainly encompassed an exchange of MLC1s with MLC1f. In addition, MLC3f was elevated whenever MHCIId and MHCIIb isoforms were increased. Because the EDL is predominantly composed of type IID and IIB fibers, HU, CB, and HU-CB had no significant effect on the MHC and MLC patterns.

Myotubes originating from single fast and slow satellite cells display similar patterns of AChE expression

Slow- and fast-contracting skeletal muscles of both rats and mice display significant differences in their patterns of acetylcholinesterase (AChE) expression. Although neural influences are known to account for a large proportion of these differences, intrinsic variations between fast and slow myogenic precursor cells have been implicated. In the present study, we have capitalized on the use of Immorto transgenic mice to obtain single myogenic precursor cells isolated from either slow or fast muscle fibers and determined whether these cells generated myotubes that produced distinct patterns of AChE expression as observed in vivo between slow and fast muscles. These two myotube populations displayed similar cell-associated and secreted AChE enzyme activity as well as comparable levels of AChE transcripts. Both myotube populations also expressed nearly identical molecular form profiles. By contrast, AChE activity and transcript levels were approximately two- and fivefold greater in fast skeletal muscles compared with slow ones. Together, these findings indicate that differences in AChE expression between fast and slow muscles are not due to inherent differences in myogenic precursor cells, thereby suggesting that other factors, such as innervation, play a predominant role in establishing the distinct patterns of AChE expression in these muscle types.

Effect of thyroid hormone on the myosin heavy chain isoforms in slow and fast muscles of the rat.

The myosin heavy chain (MHC) was studied by biochemical methods in the slow-twitch (soleus) and two fast-twitch leg muscles of the triiodothyronine treated (hyperthyroid), thyroidectomized (hypothyroid) and euthyroid (control) rats. The changes in the contents of individual MHC isoforms(MHC-1, MHC-2A, MHC-2B and MHC-2X) were evaluated in relation to the muscle mass and the total MHC content. The MHC-1 content decreased in hyperthyreosis, while it increased in hypothyreosis in the soleus and in the fast muscles. The MHC-2A content increased in hyperthyreosis and it decreased in hypothyreosis in the soleus muscle. In the fast muscles hyperthyreosis did not affect the MHC-2A content, whereas hypothyreosis caused an increase in this MHC isoform content. The MHC-2X, present only in traces or undetected in the control soleus muscle, was synthesised in considerable amount in hyperthyreosis; in hypothyreosis the MHC-2X was not detected in the soleus. In the fast muscles the content of MHC-2X was not affected by any changes in the thyroid hormone level. The MHC-2B seemed to be not influenced by hyperthyreosis in the fast muscles, whereas the hypothyreosis caused a decrease of its content. In the soleus muscle the MHC-2B was not detected in any groups of rats. The results suggest that the amount of each of the four MHC isoforms expressed in the mature rat leg muscles is influenced by the thyroid hormone in a different way. The MHC-2A and the MHC-2X are differently regulated in the soleus and in the fast muscles; thyroid hormone seems to be necessary for expression of those isoforms in the soleus muscle.