CARDIOVASCULAR JOURNAL OF AFRICA • Volume 34, No 2, May/June 2023 78 AFRICA for cardioprotection,16,33 others focus on either ischaemic or pharmacological late preconditioning. Late preconditioning is a delayed adaptive response that renders the heart relatively resistant to IRI 12 to 24 hours after the initial preconditioning treatment. In some of these studies inhibition of NFκB activation abolished cardioprotection induced by adenosine A3 receptor activation19 and late ischaemic preconditioning,18 while in others, translocation of NFκB to the nucleus and DNA binding was increased by late ischaemic preconditioning35 and late pharmacological preconditioning with erythropoietin.33 The fact that these studies are in agreement with ours is interesting since the mechanism of early and late preconditioning is suggested to be different, with early preconditioning eliciting changes in proteins that have already been synthesised, and late preconditioning relying on the synthesis of new proteins.36 One might think that this is due to NFκB remaining activated until just prior to the ischaemic insult, however Xuan et al.18 demonstrated that any increase in NFκB activation or nuclear translocation is resolved within four hours of the late preconditioning stimulus (long before the ischaemic insult). To our knowledge, only two studies assessed the relationship between TNF and NFκB in cardioprotection. The first is in agreement with our study and demonstrated that NFκB activation was increased within 30 minutes after the late ischaemic preconditioning stimulus. Although, they did not measure earlier time points, these data indicate a rapid increase in NFκB. Furthermore, they found that this increase was abolished in TNF-/- mice, suggesting that this rapid activation of NFκB by endogenous TNF is required for late ischaemic preconditioning.35 The second study demonstrated that late preconditioning with erythropoietin was also mediated by rapid activation of NFκB shortly after the stimulus, however they did not see any increase in TNF in response to erythropoietin treatment, suggesting that TNF was not involved in this protection.33 Together, these results suggest that while rapid activation of NFκB seems to be a key mediator of late preconditioning, it may or may not be activated by TNF. While the relevance of these findings to early preconditioning stimuli are unclear, the findings of our study suggest that TNF does lead to cytoprotection via rapid activation of NFκB. The timing of this activation is important. To date, drug candidates for minimising the damage caused by ischaemia– reperfusion have not translated well into the clinical setting.2 A better understanding of the temporal nature of signalling mechanisms downstream of TNF-induced cytoprotection may help us identify not only novel targets for drug development but also treatment protocols with optimal timing for prevention of IRI in ischaemic heart disease. Interestingly, the activation of TNF and STAT-3, key components of the SAFE pathway, has been described during both the conditioning stimulus and at the onset of reperfusion.2 During the conditioning stimulus, binding of TNF to its receptor TNFR2 leads to the activation of STAT-3, protein kinase C, reactive oxygen species and the mitochondrial potassium ATP-dependent (mKATP) channel.14,37 At the onset of reperfusion, activation of the SAFE pathway by TNF leads to the closure of the mitochondrial permeability transition pore and the modulation of miR-34b and miR-337.25,38 It would be of interest to explore how NFκB interacts with other key components of the SAFE pathway that have already been identified during the conditioning stimulus. On this note, our data highlight the fact that TNF-induced changes in respiratory parameters were inhibited in the presence of the NFκB inhibitor. This suggests that TNF induces protection via NFκB-induced effects on mitochondria. Although, we have previously shown that TNF-induced cytoprotection is mediated by mitochondrial effects,13 the fact that this is mediated by NFκB has not previously been shown. This is in line with studies using shRNA to demonstrate that activation of TNFR2 leads to mitochondrial effects that are mediated by activation of NFκB.26 In those studies, the mitochondrial effects involved transcription of OPA1 and mitochondrial fusion. Since assessment of mitochondrial function in our study took place seven hours after TNF exposure and NFκB activation, this is a possibility. However, it is also possible that these effects were mediated by faster mechanisms. Although remote ischaemic preconditioning confers cardioprotection via activation of NFκB and downstream opening of mitochondrial KATP channels, 20 further studies are required to assess which of these downstream mechanisms might be involved in the TNF–NFκB-induced changes in mitochondrial respiration. Limitations There are three main limitations of this study. Firstly, the C2C12 cell culture model used in our study is derived from the murine skeletal muscle. In a serum-deprived medium, the myoblasts differentiate into myotubes, which exhibit behavioural characteristics of cardiomyocytes. The advantage of C2C12 cells is that they propagate rapidly and it is easy to obtain large quantities of cells to work with. However, any cell culture model does not reflect the complexity of cellular interactions found in vivo, and specific to TNF, our investigation does not consider the intracoronary activation of leukocytes, which may affect the levels of TNF and produce negative effects in the setting of myocardial ischaemia. Therefore, this study should be confirmed in an in vivo animal model. Secondly, this study was only carried out in one cell line and so the conclusion of the findings needs to be taken with caution. Further studies will need to be carried out to confirm these findings in other cell lines and in animal models of IRI. Thirdly, we measured IkB phosphorylation to examine the activation of NFκB. This is an indirect technique that may not reflect the reality as it is possible that there may be some protein deterioration. During this study, we also attempted to assess the p65 and p50 subunits of NFκB using Western blot analyses, however this method was not sensitive enough to study the activity of NFκB in our model. A more direct measurement of NFκB by means of an electrophoretic mobility shift assay would be required to confirm the data. Conclusions In this study, we demonstrated that preconditioning of cells with the cytokine TNF induced cytoprotection against simulated ischaemia–reperfusion and that this protection was mediated by activation of NFκB prior to ischaemia. We also demonstrated that TNF improved mitochondrial respiration and that this improvement was abolished by administration of an NFκB
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