Effect of nitrite on Ca2+ movements and fatigue in muscle

The recent study by Bailey and colleagues (Bailey et al. 2019) presents results on the effects of nitrite exposure on fatigue development in isolated skeletal muscle fibres and concluded that “increased exposure to NO2 blunts fatigue development at near-physiological … through enhancing SR Ca2+ pumping rate”. We question this conclusion, noting that the study did not provide any evidence that the blunting of the fatigue was in any way caused by enhancement of SR Ca2+ pumping. We point out that particular aspects of the data presented by Bailey et al. in fact suggest a quite different underlying cause of the blunted fatigue development, which also helps account for the unexplained disparities between their findings and those of previous work in single muscle fibres (Andrade et al. 1998). First, we note that it is difficult for readers to precisely gauge the extent to which the nitrite exposure affected fatigue development in the paper of Bailey et al. because no actual data records are presented, neither the real temporal profiles of the trains of tetani during the fatigue protocol, nor any examples of the force and Ca2+ profiles for typical individual tetani during those trains. Instead, Fig. 1 presents a cartoon representation of the train profiles, with identical panels drawn for both the control and nitrite representations. Consequently, it is unclear exactly how the individual tetani, and the temporal profiles of the trains of tetani, differed with and without nitrite, raising a question about whether the development of fatigue was adequately gauged simply from the time taken for the peak tetanic force to drop to 50% of its initial level. Nevertheless, the summarized data from the force-frequency runs in their Fig. 3B highlights a critical action of the nitrite treatment not recognized by the authors. Specifically, following the nitrite treatment the peak cytoplasmic [Ca2+] to tetanic stimulation was significantly decreased across all stimulation frequencies, including when the fibre was very potently stimulated by simultaneously applying both 120 Hz stimulation and 10 mm caffeine. Such decreased Ca2+ release in response to combined electrical and caffeine stimulation is strongly indicative that the SR Ca2+ content had been decreased to some extent by the nitrite treatment. This, in fact, was to be expected, because NO donors have been shown to increase Ca2+ leak out of the SR in skeletal muscle fibres (Pouvreau et al. 2004), via an action on the ryanodine receptors (Sun et al. 2003) that is augmented under the low conditions used by Bailey et al. Importantly, Bailey et al. applied the nitrite for 60 min before ascertaining the fibre properties, and over such a period even a very small increase in RyR Ca2+ leakage could be expected to cause partial depletion of the SR Ca2+ content, with the cytoplasmic [Ca2+] remaining at close to control levels because it is determined in the longer term by the flux of Ca2+ across the surface membrane not that across the SR. The partial depletion of Ca2+ in the SR accounts for why they observed that SR Ca2+ release was lower at all frequencies following the 60 min nitrite treatment, because decreasing SR Ca2+ content below the normal endogenous level results in decreased Ca2+ release to each action potential (Posterino & Lamb, 2003). In contrast to Bailey et al., the earlier study by Andrade et al. (1998) found that nitrite and other NO donors increased the Ca2+ release at all frequencies; in those experiments the measurements were made after exposing the fibre to the nitrite or other agents for only 3–5 min, which sensitized the RyRs but was not long enough to result in appreciable depletion of SR Ca2+. In the experiments of Bailey et al., the SR Ca2+ depletion was only partial, as evidently there was still sufficient Ca2+ release to initially get maximal force. However, the total amount of Ca2+ released and then taken back up again by the SR on each tetanus was decreased by the nitrite treatment. Consequently, less ATP would have been utilized in recovering the released Ca2+ on each tetanus, and so the onset of fatigue would have been slowed because of the reduced build-up of inorganic phosphate and free Mg2+ and depletion of the ATP levels (see Allen et al. 2008). ATP usage may also have been slowed because the nitrite treatment caused S-nitrosylation of myosin heavy chains, which can slow crossbridge cycling (Nogueira et al. 2009) and increase force (Evangelista et al. 2010). Thus, net Ca2+ uptake by the SR pump would have decreased less quickly in the nitrite-treated fibres compared to the controls, as observed, because the ATP level was better maintained. Finally, the SR Ca2+ depletion and related effects observed by Bailey et al. probably do not occur with nitrate supplementation in humans, where the plasma nitrite concentration reached is orders of magnitude lower than that applied to the isolated fibres. Instead, the enhanced performance might be attributable to sensitization of the RyRs at low NO levels, and possibly also to upregulation of the SR Ca2+ binding protein calsequestrin, as reported to occur with prolonged nitrate supplementation (Hernandez et al. 2012). Further, it is possible that the enhanced performance might also be aided by S-glutathionylation of fast troponin I (TnIf); the single accessible cysteine on TnIf can undergo S-glutathionylation, which increases the Ca2+ sensitivity of the contractile apparatus, or S-nitrosylation or other oxidative changes, which decreases Ca2+ sensitivity (Dutka et al. 2017). Application of nitrite or other NO donors can produce a combination of both S-nitrosylation and S-glutathionylation, by both direct action of cellular GSH on the nitrosylated sites and the production of GSNO (Dutka et al. 2011; Kovacs et al. 2015). As a consequence, following prolonged nitrite treatment there may be little net change in the Ca2+ sensitivity of the contractile apparatus, but the S-glutathionylated sites on TnIf would be resistant to damaging oxidative change, which might account for the contractile properties reported by Bailey et al. None of the authors have any conflicts of interest. No funding has been received in regard to this work.