All three α isoforms, as well as β1 each demonstrated strong IC fluorescence, which from
the higher magnification images, appears highly likely to be due to localisation within the t-tubular system. The t-tubules are an extensive network of invaginations on the surface membrane with a very high surface-to-volume ratio, constituting ~80% of the total fibre surface area but only ~1% of the total fibre volume (Dutka & Lamb, 2007). Early analysis of t-tubules isolated from rabbit skeletal muscle found they were able to accumulate Na+ via an ATP-dependent, K+-sensitive and digitoxin-suppressible process, thus suggesting NKA presence (Lau et al., 1979). Analysis of mechanically skinned EDL muscle fibres from the rat demonstrated that NKA subunits located within the t- tubules played an important role in restoring and maintaining excitability (Nielsen et al., 2004b). Immunofluorescence labelling of NKA α2 in rat EDL muscle demonstrated that
the α2 isoform displayed a distinctive reticular pattern in the intracellular region, that co-
labelled with the DHPR (Williams et al., 2001), consistent with the presence of the α2
muscle. As a result of K+ efflux into the t-tubule with each action potential, the [K+] in the t-tubules may increase substantially above resting levels (Fraser et al., 2011), which could depolarise the membrane and interfere with action potential generation and Ca2+ release (Sejersted & Sjøgaard, 2000; Clausen, 2003; Dutka & Lamb, 2007). Therefore, a high abundance of α2 in both the PM and t-tubules in human muscle indicates that
localisation of the NKA in the t-tubules is particularly important for maintaining Na+/K+ gradients and membrane potential necessary for continued excitability of the interior of the cell.
5.4.4
Limitations
The immunostaining methods used in this study are well established and widely used and published by experts in this field (Trenerry et al., 2007; Murphy et al., 2009; Radzyukevich et al., 2013). There were a number of limitations to this study, including the apparent cross-reactivity of antibodies to the α-isoforms to other proteins as seen in the western blot results. However, the proteins are in a denatured state for western blotting compared to in their natural folded form for immunofluorescence analysis (Murphy & Lamb, 2013). This has the potential to alter the antibody binding properties and therefore limit the relevance of western blot results. The western blot results did show that the strongest band was located at the expected molecular weight for each NKA isoform, suggesting that the majority of the antibody is binding to the NKA, and any non-specific fluorescence likely to be small.
The use of a detergent is a common step in immunostaining procedures performed in order to unmask antigens and to restore immunoreactivity which can be reduced during the fixation process. While detergents are harsh chemicals that generally disrupt proteins when used at high concentrations for long periods of time (Boenisch, 2001), the
concentration and time used in this study (5% for 5 min) should not result in any significant protein disruption or damage. Therefore, the fluorescence results presented should provide an indication of NKA distribution in human skeletal muscle. Interestingly it was noted that the western blot for α2 displayed some non-specific
binding at approximately 10-15kD, which could possibly be PLM, and future studies should include localisation analysis of PLM.
It should also be noted that the possibility of any hybrid fibres with a mixed myosin type weren’t identified and separated for individual analysis. However, previous analysis has shown that the vastus lateralis muscle in humans has very few hybrid fibres (~8%) (Staron et al., 2000), so this is unlikely to have had a major influence on these results. Possible labelling of blood vessels with β3 suggests that future analysis be repeated with
a different protein used to label the PM which is not expressed in blood vessels and also using a specific marker for smooth muscle tissue.
This technique also has limitations due to the inability to separate the intracellular region into the t-tubules and sarcoplasm. The results suggest that the intracellular localisation is largely based in the t-tubules, however because this result also includes the sarcoplasm, the NKA density in the t-tubules is likely to be greater than that calculated here. It is also not sensitive enough around the PM/IC interface and more detailed analysis using confocal microscopy is recommended, along with examination of NKA isoform location in relation to other membrane transport proteins.
5.4.5
Conclusions
This study took a novel approach in quantifying the cellular location of the six NKA isoforms expressed in human skeletal muscle. By utilising antibodies to label cellular proteins known to be expressed in the plasma membrane, individual fibres were able to
be separated into PM and IC regions and the fluorescence quantified for analysis and comparison. This methodology demonstrated firstly that each of the NKA α2, β1 and β2
isoforms were expressed with greater density in the PM than IC, and secondly, analysis of isoform expression in fibre types revealed NKA α1 and α3 were expressed with greater
density in type II and type I fibres, respectively. These results reveal new information regarding isoform-specific expression of the NKA isoforms in human skeletal muscle, and provide a basis for future research into previously suggested isoform specific functions. These results also demonstrate that the NKA isoform distribution is distinctly different in human skeletal muscle compared to previous reports in mice and rats, hence the relevance and transferability of results from one species to human should be carefully considered.