Molecular Exercise Physiology Fibre Phenotype Regulation Presentation 4 Henning Wackerhage

1. Molecular Exercise PhysiologyFibre Phenotype RegulationPresentation 4Henning Wackerhage

2. Learning outcomes • At the end of this presentation you should be able to: • Explain how fibre types can be visualised and describe the features of different fibre types. Also be able to critically comment on the fibre type concept. • Explain how fibre types and proteins therein respond to changes in contractile activity; • Explain how the calcineurin and ERK1/2 pathway may regulate proteins within muscle fibres.

3. Fibre Phenotype RegulationPart 1Fibre types

4. Fibre types Muscle consist of thousands of muscle fibres. For example there are about half a million muscle fibres in the vastus lateralis of a young male. Muscle fibres are thin but can be very long; i.e. a fibre can probably span the whole length of a muscle from tendon to tendon. Muscle fibres are thus the largest cells of the human body. Muscle fibres are heterogenous: At the end of the 19th century, “translucent” and “opaque” fibres were distinguished by microscopy. Fast muscle(s) (fibres) have a higher ATPase activity Barany et al. (1965, 1967) then found that fast and slow muscles had different ATPase activities which correlated with the speed of contraction. Thus, muscles that can split ATP quickly can contract quicker. ATPase activity is thus a good measure to distinguish between muscle fibres. Barany et al.’s data are shown on the next slide. The figure shows that there is a close correlation between contraction speed (v) and myosin ATPase activity.

5. Fibre types Later, Guth and Samaha (1969) and Brooke and Kaiser (1970) developed histological stains for ATPase which allowed them to distinguish between muscle fibres with different ATPase activity: I (slow), IIa (intermediate) and IIb (fast).

6. ATPase histochemistry How does the ATPase fibre stain work? ATPase is an enzyme that splits ATP into ADP and Pi. Thus, muscle sections are incubated with an ATP solution and the ATPase in the section will split ATP into ADP and Pi. The more ATPase, the more ADP and Pi will be produced during incubation. The invisible Pi is then visualised as a dark stain with calcium chloride, cobalt chloride and ammonium sulphate. The result of an ATPase stain is shown above. Slow (type I) fibres are white and fast IIa and IIb fibres are stained black.

7. ATPase histochemistry The ATPase activity also depends strongly on the pH of a so-called preincubation solution as is shown in the table below. The table also features IIx fibres which are another fibre type that was discovered later. It was also found that the fast muscles fibres in human muscle are IIx rather than IIb fibres.

8. NBT stain for a mitochondrial enzyme Fibres also differ in their mitochondrial content. Mitochondria can be stained with a so-called nitro blue tetrazolium (NBT) stain. Enzymes in the mitochondria convert the substrate in a brown blue dye. The more mitochondria, the darker the slider after the incubation period. In this weeks practical, you will both perform an ATPase and a NBT stain.

9. Myosin heavy chain Myosin is the key motor that converts the chemical energy from the ATPase reaction into mechanical energy (i.e. muscle contraction) and heat. The ATPase function of myosin was discovered by Engelhardt and Lyubimova in 1939. Later it was discovered that myosin consisted of light and heavy chains and that there were different myosin heavy chains. The following slide shows an experiment that visualises the different MHCs in fast and slow muscles.

10. SDS gel electrophoresis for myosin heavy chain isoforms Myosin heavy chains were separated by an electrical field in a gel and then stained with a protein stain. The I (slow), IIa (intermediated) and IIx and IIb (fast) myosin heavy chain isoforms can be distinguished. M. soleus Diaphragm M. gastrocnemius

11. Task What is the standard technique to measure the concentration of a protein?

12. Summary Muscle fibres can be distinguished into type I, IIa, IIx and IIb fibres based on the predominant MHC isoform, mitochondrial content, colour and contraction speed among other. The table below gives an overview. Fibre type I IIa IIb (IIx)1) Functional description slow intermediate fast Predominant MHC isoform I IIa IIb (IIx)1) Mitochondrial content high high low Contraction speed slow intermediate high Endurance high intermediate low 1)Human fast fibres are usually not reactive to mammalian MHC IIb antibodies. Fast human fibres predominantly express MHC IIx. MHC Myosin heavy chain.

13. Fibre types: an oversimplification It is now generally recognised that fibre types occur not as three (or four) distinct forms but that they present a continuum based on combinations of MHC isoform content, oxidative and glycolytic enzymes, myoglobin, and calcium-handling proteins. Thus, the differentiation of three different fibre types is an oversimplification.

14. Fibre Phenotype RegulationPart 2Fibre type and training

15. Fibre type variations Soon the ATPase stain developed around 1970 and other stains were used for human muscle samples in order to detect variations in fibre types between individuals and to see whether there is a link between fibre type percentages and performance and to see whether training can affect the percentages of fibre types. Landmark studies by Gollnick and Saltin Landmark studies were performed by Gollnick and Saltin’s groups in the early 1970s. In the first study, Gollnick et al. (1972) found that endurance athletes had more slow twitch (type I) fibres than untrained subjects in their vastus lateralis. In the second study, Gollnick et al. (1973) reported that endurance training for 1 h at 4 days per week for 5 months had no significant effect on the percentages of fast and slow fibres. However, they had only investigated 6 subjects and thus large differences could not be detected. Later studies did investigate the variability of fibres in a muscle and the effect of training on fibre types in more detail.

16. Fibre percentages differ between athletes This study shows the % of type I fibres in track athletes. The data suggest a relationship between the length of the event and the percentage of slow fibres. 70 60 50 40 30 20 10 0 % type I fibres Middle distance Long distance Untrained Sprint Costill et al. (1976)

17. Slow fibres increase with time The Costill group also investigated whether the percentage of fibres changes over a 20-year period. The authors found a significant increase in type I fibres in fit and untrained subjects but not highly endurance-trained subjects were the type I fibre percentage was high already. The increase maybe partially due to a loss of type II muscle fibres (Lexell et al. 1988). 80 70 60 50 40 * * * 1973 1993 % type I fibres Fit Untrained Total Highly trained Trappe et al. (1995)

18. Fibre type variations Type IIx-to-IIa shifts by endurance training The Gollnick et al. (1973) paper suggested that training did not stimulate changes from type I to type II fibres. However, later studies suggested that there was a decrease in IIx and an increase in IIa fibres (Jansson & Kaijser, 1977;Ingjer, 1979). In addition, most training studies are not long enough to allow a conclusive answer regarding whether type II fibres can be turned into type I fibres by endurance training over years. Promotion of type II fibres in most denervation models No muscle activity usually results in an increase of type II fibres and a decrease of type I fibres. In rat soleus and adductor longus muscles, MHC I is gradually exchanged by MHC IIx mRNA and protein starting at day 15-30 up to day 90 (Huey et al., 2001). A longitudinal study in human beings with spinal cord injury suggest that slow MHC isoform fibres drop between 1 and 20 months after trauma and that by 70 months fibres almost exclusively express type II MHC isoforms (Burnham et al., 1997).

19. Chronic electrical low-frequency stimulation Chronic electrical stimulation can convert type II into type I fibres Stimulating a motor nerve continuously transforms the a type II to type I fibre type change in the innervated muscle. The extent of transformation depends on the stimulation dose. Sutherland et al. (1998) stimulated rabbit tibialis anterior muscle with 2.5, 5 or 10 Hz for 10 months. In the control muscle, there are nearly no type I fibres (tibialis anterior is a fast muscle) and 10 Hz stimulation > 95% of fibres are type I fibres. Stimulation with 2.5 or 5% stimulates a limited increase of type I fibres. Nuhr et al. (2003) stimulated human muscle with 15 Hz via surface electrodes (less effective than nerve stimulation). The found an approximately 20% decrease in the relative concentration of MHCIId/x (from 28% to 22%) and an approximately 10% increase in the relative concentration of MHCI (from 30% to 34%). Although they measured MHC isoform content and did not count fibres, these data suggest that a type II to type I expression change may be possible in human muscle if the dose is high enough.

20. Metabolic enzymes respond as well Chronic electrical stimulation also affects the expression of oxidative and glycolytic enzymes in rabbit tibialis anterior muscle. Mitochondrial, oxidative enzymes Glycolytic enzymes Henriksson et al. (1986)

21. Summary (1) Inactivation (paraplegia, denervation) partially transforms type I fibres into fast type II. (2) Chronic electrical stimulation turns fast type IIb/x fibres into intermediate IIa or slow type I fibres  complete fast-to-slow transformation can be achieved. (3) Endurance and strength training mainly turn very fast type IIb/x into intermediate type IIa fibres  incomplete fast-to-slow transformation. (1) (3) (2) Electrode (10 Hz stimulation) Motor nerve

22. Fibre Phenotype RegulationPart 3Signal transduction

23. Calcineurin hypothesis Before 1998, virtually nothing was known about the regulation of the adaptation to endurance exercise. In 1998, Eva Chin published a landmark paper where her research group linked the exercise signal calcium (calcium makes muscles contract) to the induction of “slow” genes via the calcineurin pathway. The paper is: Chin et al. A calcineurin-dependent transcriptional pathway controls skeletal muscle fibre type. Genes & Dev. 2499-2509, 1998. Eva Chin and co-workers demonstrated that a blockade of calcineurin with cyclosporin A led to an increased in the percentage of fast fibres. This suggested that the non-blocked, exercise/calcium activated pathway would promote a fast-to-slow fibre phenotype conversion. The cyclosporin A blockade data are shown on the next slide.

24. Mean Mean Cyclosporin A treated Control Calcineurin hypothesis 40 30 20 10 0 Blockade of the calcineurin pathway with cyclosporin A increased the type II fibre percentage in rat soleus muscle. Fast fibres (%) Chin et al. (1998)

25. Calcineurin hypothesis Chin et al. hypothesised that the calcineurin pathway would be a “molecular mechanism by which different patterns of motor nerve activity promote the specialised characteristics of slow and fast myofibres”. A schematical overview over the hypothesized function of the calcineurin pathway is shown on the next slide.

26. Slow type I genes [Ca2+] Calmodulin NFAT NFAT CnB CnA NFAT P Calcineurin hypothesis Muscle fibre Sarcoplasmic reticulum Nucleus Whenever we exercise, calcium is released from the sarcoplasmic reticulum. Calcium binds to calmodulin which in turn activates calcineurin (Cn). Calcineurin consists of a regulatory (CnB) and catalytic subunit (CnA). Calcineurin is a protein phosphatase and activated calcineurin dephosphorylates the nuclear factor of activated T-cells (NFAT). Dephosphorylation of NFAT exposes a nuclear localisation signal (NLS) which leads to the import of NFAT into the nucleus. NFAT then binds to its transcription factor binding site and increases the expression of “slow” genes that respond e.g. to endurance training.

27. Task Carry out a literature search for other functions of the calcineurin pathway.

28. ERK1/2 pathway Various studies showed that the calcineurin story was more difficult that previously imagined. For example, Swoap et al. (2001) reported that the calcineurin pathway also upregulated some fast genes, contradicting Chin et al.’s hypothesis. In 2000 Murgia reported that the activation of the ERK signal transduction pathway by its upstream activator Ras could induce slow fibres in regenerating muscle. Murgia et al.: Ras is involved in nerve-activity-dependent regulation of muscle genes. Nature Cell Biology. 2 142-147, 2000. Some of their results are shown on the next slide.

29. ERK1/2 pathway Figure. Regenerating soleus (a) without and (b) activated RasV12-ERK1/2. Light stains indicate the expression of slow myosin heavy chain I. Figure: Murgia et al. (2000) activated with transgenic methods RasV12, an upstream activator of the extracellular-signal regulated kinase (ERK1/2) pathway in denervated, regenerating soleus muscle. The muscle normally regenerates as a fast muscle that does not express slow myosin heavy chain I, which is shown in figure a. RasV12 activation, however, let to an increase in the expression of myosin heavy chain I in many fibres which is shown on the right (light grey and white fibres).

30. ERK1/2 pathway Several researchers have shown that the ERK1/2 pathway is activated by exercise in isolated and intact rat and human skeletal muscle. However, the signals that lead to an activation of ERK1/2 are unknown. The following slide shows data from the study of Yu et al. (2003), who reported an increased ERK1/2 phosphorylation in response to exercise.

31. Exercise and ERK1/2 phosphorylation Western blot with phospho-specific ERK1/2 antibody Figure. The signal transduction protein ERK1/2 is more phosphorylated in response to exercise in both untrained and trained subjects. ERK1/2 is an example for the phosphorylation and activation of a signal transduction protein by exercise. Yu et al. (2003)

32. ERK1/2 pathway What is the ERK1/2 pathway? The ERK1/2 pathway belongs to the mitogen activated protein kinase (MAPK) signal transduction pathways that sense signals such as mitogens and oxidative stress. MAPK pathways are protein kinase cascades where one kinase phosphorylates the next one downstream until the last kinase (such as ERK1/2) usually enters the nucleus. The major three MAPK pathways are ERK1/2, p38 and JNK. Researchers at the University of Dundee, such as Sir Philip Cohen, have contributed landmark papers to analyse these pathways. The following slide shows a schematical overview over the ERK1/2 pathway.

33. ? Ras Exercise signal or stress Raf P P P P MEK1/2 ERK1/2 ERK1/2 P P ERK1/2 pathway Muscle fibre Slow type I genes ? Nucleus The ERK1/2 pathway is a cascade of kinases that each phosphorylate each other. Ras phosphorylates Raf which phosphorylates MEK1/2 which phosphorylates ERK1/2. ERK1/2 phosphorylation leads to the import of ERK1/2 into the nucleus. Inside the nucleus, ERK1/2 is believed to activate transcription factors but the targets in muscle are unknown. This leads to the expression of slow genes.

34. Summary The three major human muscle fibre types are I (slow) IIa (intermediate) and IIx (fast). Rodents have also a IIb isoform. However, the fibre type complex is problematic because many transition forms exist. Fibres are a mix of 1000nds of proteins. Very high amounts of contractile activity (chronic electrical stimulation) can achieve II-to-I fibre type transformations. Training studies suggest that IIx fibres decrease and IIa fibres increase. Denervation studies usually cause a I-to-II fibre type transformation. Calcineurin and ERK1/2 are both activated in contracting muscle and promote the expression of at least some “slow” muscle genes. It was shown last week that the induction of the transcriptional co-factor PGC-1 by AMPK can also promote the formation of slow muscle fibres. To conclude, fibre type transformations are probably mediated by several pathways including the calcineurin, ERK1/2 and AMPK-PGC-1 pathways. Remember, there are hundreds of slow proteins that need to be up-regulated and equally hundreds of fast proteins that need to be down-regulated. It ain’t simple!

35. The End