This final study examined the cellular localisation of the six NKA isoforms expressed in human skeletal muscle, α1 – α3 and β1 – β3, by quantitatively analysing and comparing
fluorescence from the plasma membrane and intracellular regions, which encompassed the t-tubules, sarcoplasm and cytoskeleton, as well as fluorescence between type I and type II fibres. Both the NKA α2 and β1 isoforms were expressed with greater density in
the plasma membrane, regardless of fibre type (Table 6.1). Previous immunofluorescence research has been of a qualitative nature on rat skeletal muscle (Williams et al., 2001; Zhang et al., 2006), so the relative distribution between regions cannot be accurately compared between studies. Analysis of NKA isoform expression in fibre types revealed NKA α1 and α3 were expressed with greater density in type II and
type I fibres, respectively (Tables 6.1 - 6.2). This has possible functional relevance for NKA activity levels, with NKA α3 having a lower affinity for [Na+]i compared to α1 and
α2 (Munzer et al., 1994) and potentially resulting in a lower NKA activity in type I
Table 6.1 Summary of immunofluorescence localisation in human vastus lateralis muscle results from Study 3.
NKA isoform Location Fibre type difference
α1 PM ≈ IC I < II α2 PM 2x > IC I ≈ II α3 PM ≈ IC I 2x > II β1 PM 47% > IC I ≈ II β2 PM 44% > IC I ≈ II β3 PM ≈ IC NM
PM plasma membrane; IC intracellular; NM not measured.
The fibre type specific expression of the NKA α1 and α3 also has important implications
for studies investigating changes in NKA isoforms following exercise interventions. Most human muscle analyses are performed on biopsies obtained from the vastus lateralis muscle, which is of a mixed fibre type (Staron et al., 2000). If adaptations occur in specific fibre types specific to the exercise mode utilised, then results from mixed whole homogenates may not provide an accurate representation of changes in different fibre types. This could contribute to the lack of change seen in protein abundance in both Study 1 and Study 2. As both of these utilised high intensity exercise, suggesting NKA changes would most likely be seen in type II fibres, which constitute approximately only half of the fibres present in the vastus lateralis. Analysis of dissected human vastus lateralis muscle segments demonstrated that following acute high intensity exercise, phosphorylation of FXYD1 (PLM) was increased only in type II fibres and unchanged in type I fibres (Thomassen et al., 2013). If the protein analysis involved had examined type I and type II fibres separately, it would likely have been more sensitive to detect any changes that occurred.
Table 6.2 Summary of NKA immunofluorescence density in human vastus lateralis muscle results from Study 3.
PM Density (a.u.) NKA isoform MHC I MHC II α1 7.2 9.0 α2 7.9 7.5 α3 9.2 3.9 β1 14.4 13.9 β2 4.6 4.3 β3 NM NM
a.u. arbitrary units; NM not measured.
All three α isoforms, as well as the β1 isoform demonstrated strong intracellular
fluorescence, which appeared likely to be localisation of the isoforms within the t- tubular system. T-tubular localisation of the NKA is consistent with previous studies on NKA α2 in developing mouse skeletal muscle (Cougnon et al., 2002) and
immunofluorescence labelling of NKA α1 and α2 in rat EDL (Williams et al., 2001). It
has been previously shown that during strenuous exercise the plasma [K+] in venous blood draining contracting muscle can double from resting values (Sjøgaard et al., 1985; Bangsbo et al., 1996) and reach even higher values in the muscle interstitium (Nordsborg et al., 2003). Electrical stimulation of surface fibres of the EDL of rats showed that [K+] in the innermost t-tubules, estimated via modelling, reached approximately 13 mmol.l-1 when stimulated at 30 Hz (Fraser et al., 2011). Therefore, the change in K+ homeostasis in the t-tubules may be particularly pronounced following activation, and K+ released in to the t-tubule can likely only be rapidly removed by reuptake mediated by transport proteins located within the t-tubule membrane (Kristensen & Juel, 2010). This suggests that the localisation of NKA α1 and α2 within
6.2
Conclusions
The major conclusions from this thesis are:
Study 1
1. An acute oral glucose load elevated arterial plasma [insulin], decreased arterial and venous plasma [K+] and also increased the plasma [K+]a-v diff across the
relatively inactive forearm musculature.
2. Acute high-intensity intermittent exercise did not change skeletal muscle NKA α1-3, β1 or β2 isoform protein abundance.
3. Acute high-intensity intermittent exercise increased the skeletal muscle NKA β3
isoform protein abundance.
Study 2
4. Acute RS exercise did not change the skeletal muscle NKA α1-3 or β1-3 isoform
protein abundance.
5. Four weeks of RSE training with chronic NaHCO3 supplementation did not
change the skeletal muscle NKA α1-3, β2 or β3 isoform protein abundances, but
decreased the NKA β1 protein abundance.
Study 3
Immunofluorescence analysis of localisation in human vastus lateralis muscle showed that:
6. NKA α1 is expressed with a 24% greater density in type II fibres, while NKA α3
is expressed with a 90% greater density in type I fibres.
7. NKA α2 and β1 isoforms were expressed with greater density in the plasma
membrane than in the intracellular region which encompassed the t-tubules, sarcoplasm and cytoskeleton, but with a strong t-tubular localisation suggested
8. NKA β2 co-localised with the cell nuclei.
9. NKA β demonstrated a possible co-localisation with vascular tissue.