Na+, k+ ATPase in single skeletal muscle fibres and the effects of ageing, training and inactivity

Na+, K+-ATPase in single skeletal muscle fibres and the effects of ageing, training and inactivity Submitted by Victoria L Wyckelsma B Appl Sci (Hons) April 2014 A thesis submitted in fulfilment of the requirements of the degree Doctor of Philosophy Supervisor: Professor Michael J McKenna Co-supervisors: Dr Aaron Petersen Dr Itamar Levinger Dr Cedric Lamboley Muscle Ions and Exercise Research Group Institute of Sport, Exercise and Active Living College of Sport and Exercise Science Victoria University, Melbourne, Australia I ABSTRACT The Na+,K+-ATPase (NKA) is a key protein involved in the maintenance of skeletal muscle excitability and comprises subunits (α and β), each of which express multiple isoforms at a protein level in skeletal muscle (α1-3 and β1-3) The fibre-specific expression, adaptability and roles of each isoform in human skeletal muscle are explored in this thesis Research utilising muscle biopsies typically uses samples obtained from the vastus lateralis muscle, which in healthy young people comprises similar proportions of type I and II fibres Analyses using whole muscle pieces don’t allow the detection of fibre-type specific differences and changes occurring at a cellular level Hence analysis of skeletal muscle samples at the single fibre level offers important advantages in understanding NKA regulation This thesis therefore investigated the isoform abundance of the NKA in human skeletal muscle single fibres and their adaptability following intense repeated-sprint exercise (RSE) training in healthy young adults (Study 1); with ageing (Study 2) and after high-intensity interval training (HIT) in the elderly (Study 3) and after voluntary inactivity and resistance training in healthy young adults (Study 4) Study The NKA plays a key role in muscle excitability, but little is known in human skeletal muscle about possible fibre-type specific differences in NKA isoform expression or adaptability Hence a vastus lateralis muscle biopsy was taken in 17 healthy young adults and the NKA isoform protein relative abundance contrasted between type I and II fibres The muscle fibre-type specific NKA adaptability in eight of these adults was then investigated following four weeks of repeated-sprint exercise (RSE) training, comprising three sets of 5x4-s sprints, three days/week Single fibres were separated and myosin heavy chain (MHC I, MHC II) and NKA (α1-3 and β1-3) isoform abundance were determined via western blotting All six NKA isoforms were found to be expressed in both type I and II muscle fibres; II however, only the β2 NKA isoform exhibited fibre-type specific differences in abundance RSE training increased the β1 in type I fibres only (Pre-Train 0.58±0.28, Post-Train 0.76±0.38 a.u., 30%, p[...]... characteristics and performance measures of healthy older adults before and after 12 weeks of high-intensity interval training or control………………………… 125 Table 5.2 Na+, K+- ATPase isoform abundance in type I and II fibres before and after 12 weeks of high-intensity interval training in the elderly…………………………………….126 Table 6.1 Number of fibres and participants in type I fibres for each Na+, K+- ATPase isoform before,... β1-3 isoforms to intermittent training in human single skeletal muscle fibres Poster Presentation International Union Physiological Society (IuPS) Meeting Birmingham, United Kingdom, July 2013 XIII 4 Wyckelsma VL, Murphy RM, Levinger I, Petersen AC, McKenna MJ The effects of high-intensity interval training on aerobic power and skeletal muscle Na+, K+- ATPase in single fibres of healthy older adults Oral... investigated whether 12 weeks of high-intensity interval training (HIT) in aged individuals induced adaptations in NKA isoforms in a fibretype specific manner, in a whole muscle homogenate, further [3H]ouabain binding site content in whole muscle could be detected 29 Physical training increases the [3H]ouabain binding site content in rat human and rat skeletal muscle, with high levels of physical activity... lateralis muscle, which comprises a similar proportion of type I and II fibres Western blotting approaches use typically whole or fractioned muscle homogenate, which therefore contain a mixture of both type I and II fibres The use of whole muscle has been effective in detecting general adaptability of the [3H]ouabain binding site content and NKA isoforms following training, inactivity induced by injury and. .. plausible that other NKA isoforms may also be expressed or adapt in a fibretype specific manner following various interventions It therefore would be advisable to undertake bio-molecular measurements of NKA isoforms in single skeletal muscle fibres to have a more complete understanding of the underlying molecular properties and adaptations of the NKA following training, inactivity and in ageing In animals,... Implications of changes of NKA isoform abundance in single fibres of older adults 110 XVIII 4.4.2 No decreases in [3H]ouabain binding in ageing 112 4.4.3 Methodological considerations - Single fibre and whole muscle homogenate western blotting in ageing 113 4.5 CONCLUSIONS 115 CHAPTER 5 The effect of high-intensity interval training in older adults on Na+, K +ATPase. .. investigated possible fibre-type specific expression of the other three isoforms (α3, β2, β3) The expression of all NKA isoforms in skeletal muscle single fibres was investigated in Chapter 3 Several of the NKA isoforms are adaptable to exercise training, with various high-intensity training protocols inducing upregulation of the key NKA α1, α2 and β1 isoforms (Mohr et al., 2007; Green et al., 2008; Iaia... blots of Na+, K+- ATPase at baseline, following ULLS and following four weeks of resistance training …………………………………………… 141 Figure 6.3 Na+, K+- ATPase isoforms in type I and II fibres before, following 23 days of ULLS and 4 weeks of resistance training ……………………………………………… 143 Figure 7.1 Representative blot of skeletal muscle fibres for fibre-typing using myosin heavy chain I and II antibodies…………………………………………………………………….166... isoforms in single fibres 80 3.3.2 Fibre-type specific adaptability to RSE training 83 3.3.3 Antibody testing 83 3.4 DISCUSSION 86 3.4.1 Homogenous expression of the α isoforms in type I and II fibres in human skeletal muscle 86 3.4.2 Fibre-specific expression of the beta isoforms in type I and II fibres 88 3.4.3 Effects of RSE training on NKA isoforms... however its lower affinity to cardiac glycosides doesn’t allow for α1 to be detected using the standard [3H]ouabain binding sites measurement (Hansen, 2001) Hence, in rat skeletal muscle, the α2 is the only NKA α isoform which is measured in the [3H]ouabain binding analysis In contrast in humans, in skeletal muscle and other tissues, the three α isoforms have a similar ouabain affinity and so can be readily ... declare that the PhD thesis entitled Na+, K+- ATPase in single skeletal muscle fibres and the effects of ageing, training and inactivity is no more than 100,000 words in length, exclusive of tables,... isoforms in single skeletal muscle fibres to have a more complete understanding of the underlying molecular properties and adaptations of the NKA following training, inactivity and in ageing In. .. and II fibres The use of whole muscle has been effective in detecting general adaptability of the [3H]ouabain binding site content and NKA isoforms following training, inactivity induced by injury