The mean serum digoxin concentration (SDC) was 0.8 nM, or ~0.62 ng.ml-1 following 14 days of digoxin administration at 0.25mg.day-1. Similar SDC of 0.9 nM was reported following the same dose in healthy males (Schenck-Gustafsson et al 1987). Higher SDC have been reported (~1 nM) following 14 days of digoxin at 0.5 mg.day-1 in healthy humans (Sundqvist et al 1983). The resultant SDC observed in the current study satisfies the preferred therapeutic range of ~0.65-1.15 nM or ~0.5-0.9 ng.ml-1 which has previously demonstrated reduced hospitalisation and mortality rates, and alleviation of numerous clinical symptoms in heart failure patients (Ahmed et al 2006; Wang & Song 2005). Whilst higher SDC can be attained however, SDC greater than 1.2 nM in heart failure patients has been associated with increased mortality (Rathore et al 2003). Therefore the dosage administered within the present study, and subsequent SDC, is clinically relevant.
4.4.2 Chronic digoxin therapy does not impair K+ homeostasis during exercise
Muscle contractions increase muscle interstitial [K+] and this can potentially impair force development, but this effect is countered primarily by NKA activation (Sejersted & Sjøgaard, 2000). Digoxin binds to and partially inhibits skeletal muscle NKA. Furthermore this binding in skeletal muscle increases with exercise (Joretag & Jogestrand, 1983), consistent with increased ouabain binding found during muscle stimulation in rat muscle (McKenna et al, 2003). On the basis of expected DIG binding in resting muscle, an increase in digoxin binding to skeletal muscle NKA during
exercise was also expected. Subsequently an exacerbation of plasma [K+] elevation during exercise was hypothesised with DIG in healthy humans.
The elevated SDC indicates that some inhibition of NKA in skeletal muscle should have occurred. Contrary to our hypothesis, and surprisingly, 14 days of chronic digoxin therapy did not increase any of the [K+]a, [K+]v, [K+]a-v or the calculated muscle K+ efflux,
whether at rest, during exercise or in recovery. During muscle contractions, the rise in plasma [K+] is dependent on the rates of K+ entry and removal from plasma (Sejersted and Sjøgaard, 2000), with entry via contracting muscle K+ release. Numerous studies have found K+ uptake into red blood cells during high intensity cycling exercise (Lindinger et al, 1992; McKelvie et al, 1991, 1992), therefore it is possible that some K+ entered plasma during contractions via K+ release from red blood cells. However these findings are in contrast to others, whereby red blood cells did not take up K+ during intense forearm exercise (Maassen et al, 1998) or during submaximal knee extension exercise (Juel et al, 1999). Therefore increases in red blood cell [K+] during exercise might be due to a decrease in red cell volume (Juel et al, 1999; Maassen et al, 1998). Thus the contribution of red cell K+ release to the increase in plasma [K+] during intense contractions in the present study is likely to be negligible, though inconclusive. Clearance of K+ accumulation in plasma is subsequently facilitated via NKA in contracting (Verberg et al, 1999; Sahlin and Broberg, 1989; Chapter 3) and non- contracting muscle (Kowalchuk et al, 1988; Lindinger et al, 1990; Chapter 5). K+ uptake by contracting muscle was not impaired by digoxin during intermittent exercise or recovery in the present study. Although [K+]a-v across inactive muscle was not
assessed, there was no DIG effect on [K+]a-v found across inactive muscle in the same
participants during cycling exercise (Chapter 5), therefore a DIG effect on K+ uptake across inactive muscle would be unlikely during forearm contractions. There was only a very small rise in arterial [K+] during small muscle mass contractions, which also highlights that the predominantly large volume of inactive muscle has a substantial capacity for K+ clearance. In fact there was remarkable consistency of K+ data between the DIG and CON trials. The lack of change in [K+]a-v or K+ fluxes were not due to
differences in forearm blood flow, which was not affected by digoxin. This is consistent with Glover et al (1967), who found no effects on forearm or hand blood flow and venous tone following intravenous ouabain infusion (0.05 mg.min-1, 10 min) in healthy males. Furthermore, altered fluid shifts cannot explain the unchanged [K+] with digoxin, as there were no digoxin effects on ∆PV or ∆BV during exercise.
The effects of digoxin-induced NKA inhibition and subsequent K+ homeostasis in healthy humans undertaking whole body exercise is largely unknown, and was
investigated in detail in Chapter 5. During three submaximal bouts of incremental
cycling exercise (10 min each at 33%, 67%
V
•O2peak, and the final bout of 90%V
•O2peak
to fatigue), K+ homeostasis was not impaired by digoxin, nor was there an effect on muscle fatigue, which is consistent with the present forearm exercise study. Digoxin occupancy of NKA was not analysed for the forearm muscle, therefore the fraction of digoxin binding specific to the contracting musculature is unknown. However, digoxin binding to vastus lateralis muscle NKA was measured in the same volunteers on the same days (appendix 7). These muscle analyses revealed no change in NKA content (measured by [3H]ouabain binding site content) or in NKA activity (measured by 3-O- MFPase activity) at rest after digoxin. Importantly, they indicated 7% digoxin occupancy following Digibind® antibody removal of bound digoxin (appendix 7). It is highly likely that similar digoxin occupancy occurred in forearm muscle. Thus the most likely interpretation is that an upregulation of NKA occurred in skeletal muscle after 14 d DIG in healthy humans. The net result was that NKA content and maximal NKA activity was therefore unchanged, and this fits with the highly consistent [K+] data between DIG and CON trials. The small exercising muscle mass in the present study does not provide a rationale to explain the lack of DIG effect on K+ homeostasis. Whilst [K+]a changes are
small in absolute terms, marked changes in [K+]v during exercise were observed, and
the [K+]a-v demonstrates a large net influx of K+ into plasma during exercise as blood
traversed the exercising forearm muscle. Blood flow was high across the forearm, consequently K+ efflux was pronounced, but there was no DIG effect observed. During cycling exercise in the companion study (Chapter 5) where exercising muscle mass was considerably greater than the forearm, and [K+]a was substantially higher, there
was still no digoxin effect observed. The rationale for insufficient forearm muscle NKA activation to explain a lack of DIG effect is not valid, as there was a complete reversal of K+ release into plasma to K+ from plasma during the recovery transition between each intermittent exercise bout, therefore in-vivo NKA activity was high.
No studies have thoroughly investigated K+ homeostasis during arm exercise in digitalized humans. Janssen et al (2009) recently reported that venous [K+] during handgrip exercise was not different between digoxin and control in healthy individuals. However, the rise in [K+]v was surprisingly small, suggesting either post-exercise blood
sampling, with a consequent lower [K+]v, or an inappropriate exercise model.
Furthermore the digoxin uptake by muscle was not quantified, nor was blood flow evaluated, thus making their results difficult to interpret.
It is possible that the elevated plasma [K+] found during cycling exercise with DIG in cardiac patients (Norgaard et al, 1991; Schmidt et al, 1995) might be greater due to the
reduced skeletal muscle NKA content in these patients due to the concomitant effects of heart disease, inactivity, as well as the specific inhibitory digoxin effects on NKA. Whilst digoxin occupancy in skeletal muscle NKA was ~9% after 3 d treatment (Schmidt et al, 1995) and ~13% in muscle post-mortem (Schmidt et al, 1993), there was no evidence of compensatory upregulation of NKA content. The absence of upregulated NKA content has also been confirmed by Green et al (2001) in digitalised chronic heart failure patients.
Serum digoxin concentration was within the therapeutic range; therefore it appears likely that the unchanged K+ at rest, during exercise and in recovery with DIG was associated with a compensatory up-regulation of NKA in healthy human skeletal muscle. This finding is consistent with K+ being a tightly regulated physiological variable. However, the lack of change in [K+] with digoxin makes it not possible to conclude on the importance of K+ regulation to muscle fatigue.