Reply from T. L. Dutka, J. P. Mollica, G. S. Posterino and G. D. Lamb

We thank Dr Tsikas for his interest in our work (Dutka et al. 2011). We agree with points Dr Tsikas makes about the chemistry of GSNO, indeed specifically having made some of them ourselves. We do nevertheless wish to make some particular points about our experiments. Firstly, although not explicitly stated in the work in question, we in no way meant to imply that the concentrations of agents applied to the skinned fibre preparation were at all comparable with those pertaining within muscle fibres or other cells. Just as in virtually all biochemical studies of isolated proteins and systems, and as in our previous skinned fibre studies using oxidants such as H2O2 and dithiodipyridine (DTDP) (Lamb & Posterino, 2003; Murphy et al. 2008), the experiments involved applying saturating concentrations of the agents sufficient to induce maximal activation of the particular processes under examination. Furthermore, we need to point out that in these skinned fibre experiments all the diffusible cytoplasmic constituents have been entirely lost from the preparation, and so the additional pathways to which Dr Tsikas refers were absent in these experiments. Indeed, this is one of the very strengths of this type of skinned fibre examination, as it makes it possible to sequentially add back in various normal constituents (e.g. myoglobin, GSH, etc.) in order to determine the effects of each individually, which is not possible in intact cells where the relevant reactions, and identities and concentrations of the myriad reactive entities and intermediates, are unknown and effectively not determinable. In regard to the above points, we note that we wrote the following in our paper in the section Consideration of experimental limitations: ‘Firstly, the results here were all obtained in skinned fibres, where various diffusible intracellular constituents present in vivo would have been lost from the fibre, possibly including redox-sensitive kinases or phosphatases or other co-factors. Consequently, it is possible that ROS and RNS generated within, or applied to, an intact muscle fibre might exert different or additional effects to those found here. We point out, however, that the findings here and in our earlier skinned fibre studies (Lamb & Posterino, 2003; Posterino et al., 2003; Murphy et al., 2008) do seem in generally good accord with those found when applying ROS and RNS in intact fibres (Andrade et al., 1998a,b), such as in regard to the increases or decreases in contractile sensitivity occurring without change in maximum force, and the lack of major changes in fibre excitability and Ca2+ release. This suggests that the major acute effects reported here probably do generally reflect those happening in intact fibres …’ In our study we used various agents including GSNO to produce both S-nitrosylation and S-glutathionylation of proteins in the skinned fibres. We fully agree that the S-nitrosylation of the cysteine residues was probably brought about by higher oxides of •NO (such as N2O3) rather by the •NO itself, or even more likely that it occurred via transnitrosation (Foster et al., 2003; Halliwell & Gutteridge, 2007). Our aim was to induce the final state and not to try to emulate the unknown reactions actually occurring in intact fibres in vivo. We further note that application of similar high concentrations of GSNO to skinned fibres has been previously demonstrated to indeed produce S-nitrosylation of key contractile proteins (including fast troponin I, TnIf), shown both by using antibodies to CysNO and by the biotin switch method (Nogueira et al. 2009). Additionally, we described the conditions under which GSNO caused S-glutathionylation of cysteine residues, namely immediately following its addition to the solution (referred to as GSNOimm), and specifically attributed the effect to generation of other products of the GSNO. In particular we wrote: ‘The propensity of the GSNOimm treatment to cause S-gluthathionylation of proteins is quite possibly explained by the finding that GSNO readily undergoes spontaneous chemical transformation in aqueous solution, generating several glutathione derivatives including glutathione thiosulfinate (GS(O)SG), which is very reactive to any protein thiol and produces S-gluthathiolyation (Huang & Huang, 2002). The overall action of the GSNO solution changed over time from glutathionylation to nitrosylation, which presumably resulted from a change in proportion of GSNO and its various derivatives over time. It is not possible to say, however, whether the same progressive change also occurs when GSNO is generated in the cytoplasm of a cell, nor even whether the GSNO species itself will cause glutathionylation or nitrosylation (or both) of a given target protein. ’ Furthermore, in our paper we noted that the GSNOimm treatment, and not the other GSNO application method (GSNOdel), had been verified using Western blotting to elicit S-glutationylation of contractile proteins, in particular TnIf, and such evidence has since been published in Fig. 6 of Mollica et al. (2012). That work also provides direct evidence that the same S-glutathionylation reaction can be brought about both by exposure to oxidized glutathione (GSSG) alone and by the other specific treatment used in our study (sequential application of DTDP and GSH). Finally, and very importantly, that study (Mollica et al. 2012) also showed that S-glutathionylation of TnIf actually occurs in the skeletal muscle of humans during normal exercise, which highlights the value and relevance of the findings obtained from our skinned fibre experiments under discussion (Dutka et al. 2011).