2.2.2.1 [3H]ouabain binding
In skeletal muscle the most widely accepted method for quantification of the total number of functional NKA is through measurement of the [3H]ouabain binding site content in muscle (Clausen, 2003a). The procedure is performed on small pieces of whole muscle samples (typically between 5-20 mg) and is based on the affinity binding of cardiac glycosides to the α subunit of the NKA, with a stoichiometry of 1:1 (Hansen, 1984). By incubation of muscle samples in tritiated ouabain and counting of β particles via liquid scintillation, it is possible to quantify the NKA in molar units, typically expressed as NKA content in pmol.g wet weight-
1(Hansen & Clausen, 1988). This can be contrasted to western blotting, which typically only
gives measurement of relative abundance using an arbitrary unit.
In rat muscle the α1 isoform makes up approximately 20% of the NKA α subunits; 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 detected by [3H]ouabain binding (Wang et al., 2001; Clausen, 2013b). The [3H]ouabain binding site content reported in human skeletal muscle is shown in Table 2. 1. However, the [3H]ouabain binding assay does not identify which of the α isoforms may have been detected and furthermore no information can be obtained regarding
The use of rat skeletal muscle for quantifying NKA content can be used to gain a representation of NKA fibre type specificity, as the rat EDL and soleus muscles are comprised of predominately type II and type I fibres, respectively. Given the [3H]ouabain binding procedure can only be performed in whole muscle pieces, measures conducted in human skeletal muscle are therefore unable to determine any possible fibre type differences due to the heterogeneous nature of human skeletal muscle from the vastus lateralis muscle. In rat skeletal muscle it is unclear whether [3H]ouabain binding site content is greater in a particular fibre-type. The [3H]ouabain binding site content was higher in rat SOL and EDL by ~50% and in red vastus lateralis by ~70%, when compared to white vastus lateralis muscle, with no difference reported between SOL, EDL and red vastus lateralis (Chin & Green, 1993). The [3H]ouabain binding site was 4 times greater in SOL compared with EDL in mouse muscle (Bray et al., 1977). Whilst conversely there was [3H]ouabain binding is between 23-50% higher in EDL compared to SOL (Clausen et al., 1982; Kjeldsen et al., 1984a; Everts & Clausen, 1992). The collation of data from numerous rat studies, all of which measure different muscles and use different aged rats makes a direct comparison between studies difficult.
2.2.2.2 Western blotting
Typically, western blotting is used to determine the relative abundance of an individual isoform compared to a housekeeping protein or the total protein per lane and is expressed in arbitrary units (a.u.). Additionally western blotting can be used to measure the
phosphorylation state of proteins (Murphy et al., 2006b) and specifically for the regulation of NKA, this is mainly FXYD1 phosphorlyation (Thomassen et al., 2013). Many researchers have performed western blotting utilising separated fractions of homogenised skeletal
muscle, to purify the sample analysed (Nielsen et al., 2004; Mohr et al., 2007; Bangsbo et al., 2009). However, there are important adverse implications of this approach in analysing the
supernatant of a muscle homogenate, with the recovery of NKA was reported to only be between 0.2-8.9% (Hansen & Clausen, 1988). Therefore when determining NKA protein relative abundance, the use of a whole muscle homogenate (i.e. with no centrifugation) is a preferred method to recover all NKA molecules and gain the best representation of NKA isoforms in the muscle. However, the use of a whole muscle homogenate derived from human muscle biopsy samples doesn’t allow for any fibre-type differences to be determined, since human skeletal muscle is heterogeneous with a relatively equal proportion of type I and II fibres.
Recently a new method has been developed which allows for the quantification of proteins within a single fibre segment (Murphy, 2011). This approach avoids two common problems with western blotting. Firstly, this allows a “whole muscle” sample to be analysed as the intact fibre segment encases the plasma membrane and all intracellular compartments of the cell, ensuring there is no loss of the NKA as would occur during any centrifugation
procedures. Secondly, this method overcomes the problem of saturating a gel with a large amount of protein, which may potentially mask a research outcome following an intervention (Mollica et al., 2009). Additionally, this technique allows for the detection of all NKA
isoforms in different fibre-types. To date western blotting using single fibres has been used to explore the fibre-type expression of three of the six NKA isoforms (α1-2and β1) expressed in
human skeletal muscle (Thomassen et al., 2013). The determination of possible fibre-type specific expression of all six NKA isoforms expressed in human single skeletal muscle fibres and their adaptability to three interventions and ageing is a key focus of this thesis.
Table 2.1 Vastus lateralis muscle [3H]ouabain binding site content from muscle biopsy samples in healthy young adults aged between 18-35 years.
Study [3H]ouabain (pmol.g wet weight-1)
Norgaard et al., (1984) 278 ± 15 Dorup et al., (1988) 258 ± 16 Kiltgaard et al., (1989) 276 ± 19 Kjeldsen et al., (1990) 308 ± 13 Benders et al. (1992) 360 ± 31 McKenna et al., (1993) 333 ± 19 Green et al., (1993) 339 ± 16 Madsen et al., (1994) 307 ± 43 Schmidt et al. (1994) 223 ± 13 Gullestad et al. (1995) 258 ± 13 Ravn et al. (1997) 276 ± 11 Evertsen et al., (1997) 343 ± 11 Haller et al. (1998) 281 ± 20 Green et al., (1999) 289 ± 22
Green et al.(2000a) 348 ± 12
Medbo et al., (2001) 356 ± 6
Fraser et al., (2002) 311 ± 41
Leppik et al., (2004) 317 ± 17
Nordsborg et al. (2005a) 312 ± 17
Aughey et al., (2005)* 307 ± 41
Aughey et al. (2006)* 318 ± 37
Aughey et al., (2007)* 355 ± 80
McKenna et al., (2012)* 350 ± 108
Data is presented as mean ± SE unless otherwise stated. *SD. Range (Minimum-Maximum) 223- 355 pmol.g wet weight-1. Mean from 23 studies presented is 308 pmol.g wet weight-1.