Misoprostol treatment prevents hypoxia-induced cardiac dysfunction through a 14-3-3 and PKA regulatory motif on Bnip3
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Abstract
Systemic hypoxia is a common element in most perinatal emergencies and is a known driver of Bnip3 expression in the neonatal heart. Bnip3 plays a prominent role in the evolution of necrotic cell death, disrupting ER calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests a cardioprotective role for the prostaglandin E1 analog misoprostol during periods of hypoxia, but the mechanisms for this protection are not completely understood. Using a combination of mouse and cell models, we tested if misoprostol is cardioprotective during neonatal hypoxic injury by altering Bnip3 function. Here we report that hypoxia elicits mitochondrial-fragmentation, MPT, reduced ejection fraction, and evidence of necroinflammation, which were abrogated with misoprostol treatment or Bnip3 knockout. Through molecular studies we show that misoprostol leads to PKA-dependent Bnip3 phosphorylation at threonine-181, and subsequent redistribution of Bnip3 from mitochondrial Opa1 and the ER through an interaction with 14-3-3 proteins. Taken together, our results demonstrate a role for Bnip3 phosphorylation in the regulation of cardiomyocyte contractile/metabolic dysfunction, and necroinflammation. Furthermore, we identify a potential pharmacological mechanism to prevent neonatal hypoxic injury.
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Reply to the reviewers
We wish to thank the reviewers for their detailed and constructive comments on our manuscript. This valuable feedback has resulted in substantial improvements to our paper. A detailed list addressing the reviewers’ comments and the changes to our manuscript since the first submission is outlined below:
Reviewer #1:
__The manuscript by Martens et al investigates the mechanisms of Bnip3-mediated cell damage during hypoxia. The Authors show that modulation of prostaglandin (PG) E1 signaling with misoprostol prevents cardiac dysfunction, mitochondrial impairment and cell death induced by hypoxia. In addition, they show that the effect of misoprostol is …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
We wish to thank the reviewers for their detailed and constructive comments on our manuscript. This valuable feedback has resulted in substantial improvements to our paper. A detailed list addressing the reviewers’ comments and the changes to our manuscript since the first submission is outlined below:
Reviewer #1:
__The manuscript by Martens et al investigates the mechanisms of Bnip3-mediated cell damage during hypoxia. The Authors show that modulation of prostaglandin (PG) E1 signaling with misoprostol prevents cardiac dysfunction, mitochondrial impairment and cell death induced by hypoxia. In addition, they show that the effect of misoprostol is dependent on PKA-mediated Thr181 phosphorylation. The Authors also suggest that there is a possible interaction between Bnip3 and 14-3-3beta that prevents ER Ca2+ release and mitochondrial Ca2+ overload. The Authors conclude that Bnip3 phosphorylation plays a key role in the regulation of cardiac and metabolic dysfunction and identify misoprostol treatment as a therapeutic intervention to prevent hypoxia-induced cardiac injury. __
__ **Major Comments:** __
__ 1.Results presented in Figs. 1, 2 and 3 pertaining to hypoxia-induced changes in Bnip3 expression, changes in mitochondrial function, cell death, Bnip3-dependent Ca2+ transfer from ER to mitochondria and the effect of misoprostol have partially been demonstrated in a previous publication from the same group (PMID: 30275982). __
Thank you for noting our previous work using predominantly the HCT-116 cell line and a rat model of neonatal hypoxia published in the journal Cell Death Discovery. The work in the current manuscript builds on our previous papers, and extends these findings utilizing a neonatal mouse model, primary neonatal ventricular myocytes, human iPSC-derived cardiomyocytes, and H9c2 cells. These models not only demonstrate the robust nature of the effects of misoprostol treatment on the hypoxic neonatal cardiomyocytes, but they also allowed us to utilize powerful genetic models, such as the Bnip3 knockout mouse, and knockout mouse embryonic fibroblasts (MEFs), which phenocopy many of the effects of misoprostol treatment. These findings strongly implicate Bnip3 as a primary target of misoprostol treatment in the hypoxic neonatal heart in rodents.
In addition, Figure 1 contains very important in vivo endpoints that we have not previously utilized, including echocardiography to assess neonatal cardiac function, transmission electron microscopy to assess mitochondrial ultrastructure, cardiac ATP and lactate levels, an array of gene expression, and HMGB1 immunofluorescence (Fig.1 I in the current version of the manuscript) implicating a necro-inflammatory phenotype that is modulated by misoprostol treatment. Moreover, in Figures 2 and 3 we confirm previous observations related to hypoxia- and Bnip3-mediated mitochondrial function, and calcium signaling, but also extend these observations to include the impact of hypoxia, Bnip3 and misoprostol treatment on mitochondrial morphology and necro-inflammatory markers, in additional to utilizing human cardiomyocytes and knockout MEFs. Finally, Figures 4-7 of our manuscript describes a novel mechanism by which misoprostol treatment can therapeutically target Bnip3 function both in vivo and in cell models (see below).
Although based on our previous observations, we feel the work in the present manuscript is highly novel and original, and adds substantially to our knowledge of both Bnip3 function, and neonatal hypoxic injury, which currently represents a world-wide health crisis that is underrepresented in the biomedical literature.
__2.The very same publication from 2018 shows that misoprostol treatment of pups exposed to hypoxia for 7 days is able to prevent the increase in Bnip3 protein levels. Yet, in the present manuscript misoprostol treatment had no effect on Bnip3 protein levels in the same model (Fig. 1E). This raises some concerns regarding the solidity and soundness of the results presented. __ Thank you for noting this in our previous work. In our 2018 paper, we treated hypoxic neonatal rats with misoprostol and observed a complete repression of Bnip3 expression in the gut and hippocampus, but only a partial repression in the heart. This observation prompted us to explore other mechanisms by which misoprostol could inhibit Bnip3 function. We have increased our sample size for the data in Figure 1 J and K to be more statistically conclusive. The evidence is now stronger that misoprostol only the partial represses Bnip3 expression in the neonatal mouse heart. In addition, we provided a representative western blot (Fig. 1 J), which is consistent with the result in Figure 3C in MEF cells.
__ 3.The validation of the custom antibody against p-Thr181 needs to be shown. Fig. 4E shows that p-Bnip3 band is quite strong in H9c2 cells, despite total endogenous Bnip3 levels are barely detectable. In addition, phosphorylation of the Bnip3 Thr181 residue in cells and/or in vivo should be confirmed by mass spectrometry. __
Our plan in the next revision, we will be to provide additional validation of the p-Thr181 antibody, as we have done previously (PMID: 33044904). In addition, we will re-run the western blots noted above to improve their quality, as the difference between total Bnip3 and p-Bnip3 in H9c2 cells is likely due to different exposures of the two blots. Confirmation of Bnip3 phosphorylation using mass spectrometry in extracts from intact cells was previously published (PMID: 26102349), however, the nature of the signaling pathways leading to phosphorylation was not determine, nor was the mechanism of Bnip3 inhibition previously determined.
__ 4.Fig. 4L shows that misoprostol treatment of H9c2 cells leads to an increase in Bnip3 phosphorylation, but this does not seem to be the case in normoxic conditions in vivo (Fig. 4N). Moreover, shouldn't this presumable increase in phosphorylation induced by misoprostol in normoxic conditions lead to Bnip3 accumulation in the cytosol thereby reducing its colocalization with mitochondria (Fig. 6B)? The results obtained with the colocalization method should be corroborated using different methods, such as cell fractionation.__
In the previous version of the manuscript, we reported that misoprostol treatment increases Bnip3 phosphorylation in H9c2 cells following acute exposure (Supplement 5 B). In the updated version of the manuscript, we confirmed this *in vitro *observation in PVNC’s, demonstrating that in culture, acute misoprostol drug treatments during normoxia result in Bnip3 phosphorylation (Supplement 5 C). However, when we increased our N in the revised manuscript this difference remained not statistically sustained after 7 days of misoprostol treatment in vivo (Fig. 4 M, N). Importantly though, our observation that hypoxia exposure resulted in reduced Bnip3 phosphorylation, and that misoprostol drug treatment was sufficient to restore it is particularly novel, and ties together with our new colocalization data (Fig. 4 M, N). What is very intriguing about the data in Figure 6B is that myc-Bnip3 did not colocalize with the mitochondrial matrix-targeted mito-Emerald under normoxic conditions, and thus was not impacted by misoprostol treatment in normoxia. However, in hypoxic cells the colocalization coefficient between myc-Bnip3 and mito-Emerald increased and was abrogated by misoprostol treatment. This observation suggests that Bnip3 is actively translocated deeper into the mitochondria ultrastructure during hypoxic stress, and that misoprostol treatment can prevent this phenomenon. This observation is consistent with the pBnip3 data shown in Figure 4M. In the most recent version of the manuscript, we have performed additional confocal experiments to substantiate this novel observation. New data clearly demonstrates that hypoxia exposure in vivo increases the colocalization of Bnip3 with the inner mitochondrial membrane protein Opa1 (Fig. 6 C). However, when mice are treated with misoprostol, the colocalization with Opa1 is reduced and the colocalization of Bnip3 with 14-3-3b increases (Fig. 6 M). We have also shown in H9c2 cells that expression of Opa1 prevents Bnip3-induced mitochondrial fission (Fig. 3 J), and that when both Bnip3 and 14-3-3b are ectopically expressed, misoprostol treatment can increase their colocalization (Fig. 6 N, O), suggesting that this is regulated by post-translational modification and not alterations in Bnip3 expression due to hypoxia. Our plan is to include additional fractionation experiments in the next revision of the manuscript; however, this approach may not be as sensitive as confocal microscopy.
__ 5.In relation to Fig. 4 M, N (page 19), the Authors concluded that the reduction in Bnip3 phosphorylation suggests an increase in Bnip3 activity in the hypoxic neonatal hearts. Nevertheless, this has not been demonstrated. __
Thank you for pointing this out. At this time, we do not have data to suggest that a reduction in Bnip3 phosphorylation increases its activity in vivo. In the revised manuscript, we have new confocal-based colocalization experiments using fixed sections from hypoxic and misoprostol treated hearts that provide insightful information into the subcellular localization of Bnip3 (Please see above; Fig. 6 C, M). Importantly, based on our data in figure 5, particularly Fig.5F using Bnip3-null MEFs, the protective effect of misoprostol is completely prevented by reconstitution of the T181A mutant, but not wild-type Bnip3, suggesting phosphorylation at T181 is an important mechanism by which misoprostol inhibits Bnip3-induced mitochondrial depolarization. Finally, we have also been careful not to overstate our conclusions is the most recent version of the manuscript, have been more specific with our language, and have avoided vague terms like ‘activity’.
__ 6.Along that line, the Authors concluded that misoprostol-induced cytoprotection is dependent on PKA Thr181 phosphorylation. Nevertheless, this dependence has not been convincingly demonstrated in hypoxic cells and in vivo.__
The new data described outline above, in point #4 and #5, have provided assurances regarding the role of Bnip3 phosphorylation on its subcellular location in vivo and in cultured cells. To further address the dependency of PKA on T181 phosphorylation, we have performed experiments using the PKA inhibitor, H89, in cellular experiments and evaluate whether the protective effect of misoprostol is lost in the presence of this inhibitor. This new data has been added to the most recent version of the manuscript (Fig. 4 G).
__ 7.Previous studies showed that Bnip3 induces mitochondrial fragmentation and mitophagy (PMID: 16645637, 20436456). What is the hypothesis for the inhibition of mitochondrial fragmentation induced by misoprostol in the present study? Does it prevent Bnip3 interaction with Opa1 or is this event downstream of ER Ca2+ release and mitochondrial Ca2+ overload? Does misoprostol affect mitophagy? __
We have added new experimental data to address the hypothesis that Bnip3 colocalizes with Opa1 to induce mitochondrial fragmentation (as noted by the reviewer, they were previously shown to physically interact), and that this is inhibited by misoprostol treatment (Fig. 6 C). We have also added new data to the supplemental material demonstrating that misoprostol inhibits hypoxia- and Bnip3-induced mitophagy. Based on our data, we proposal that misoprostol inhibits both Bnip3-induced ER-calcium release and Opa1-dependent mitochondrial fusion. This is based on our data that misoprostol prevents Bnip3 accumulation at both the ER and mitochondria, respectively.
__ 8.The link between Bnip3 interaction with 14-3-3 and Bnip3 Thr181 phosphorylation, if there is any, is not clear. The Authors mention that Thr181 lies within the 14-3-3 binding domain. Is Thr181 phosphorylation required for 14-3-3 binding or are these events unrelated? What is the significance of these events in hypoxia, does 14-3-3 binding to Bnip3 occur in vivo? Is Bnip3 localization affected by hypoxia, 14-3-3 binding and/or misoprostol treatment in vivo? __
Previously, we described the role of phosphorylation of Bnip3L (Nix) at Ser-212 how this regulates the interaction with 14-3-3 (PMID: 33044904). This phosphorylation site is conserved in Bnip3 as T181. Interestingly, phosphorylation of Nix by PKA was not required for interested with 14-3-3b, but the interaction between Nix and 14-3-3b was enhanced by phosphorylation. Our plan is to perform similar experiments with Bnip3 and 14-3-3b to determine if this mechanism is conserved. However, as noted above we have new in vivo data showing that misoprostol increased the colocalization of Bnip3 and 14-3-3b in the hypoxic heart (Fig. 6 M).
__ 9.Fig. 6P shows the presence of myc-tag after IP for HA-tag, even when HA-14-3-3 was not expressed (middle lane). How is this possible? __
This appears to be a small amount of non-specific interaction between the HA antibody and myc-Bnip3. This is relatively small compared to the band in lane 3, which demonstrates specificity, and the importance of including this control condition. Our plan is to re-run this CO-IP to improve the western blot quality.
__ **Minor Comments:**__
__ 1.Please co-stain with the cardiomyocyte marker in Fig. 2A (such as alpha-actinin).__
Yes, good suggestion.
__ 2.The Methods are not sufficiently detailed. For instance, it is not clear what is the Ca2+ concentration used for Ca2+ pulses in the CRC experiment. The fact that cardiac mitochondria are able to uptake only two Ca2+ pulses raises some concerns regarding the quality of mitochondrial preparation. What is the reason for isolating mitoplasts instead of intact mitochondria? __
We have provided more detail in the revised manuscript.
__ 3.TMRM fluorescence should be measured before and after FCCP administration, to account for the difference in plasma membrane potential (the results should be expressed as F/FFCCP).__
We can provide some additional control experiments in the revised manuscript, if necessary.
__ 4.Measurement of extracellular acidification is mentioned in the methods, but the relative results are not shown. __
Thank you, this has been removed.
__ 5.RNAi experiments targeting Bnip3 are also mentioned in the methods, but the results are not described. __
Thank you. This has been fixed.
__ Reviewer #1 (Significance (Required)): __
__ Previous studies have demonstrated that Bnip3 is upregulated by hypoxia and plays a key role in inducing mitochondrial dysfunction and PTP opening that eventually results in cell death (PMID: 12169648, 10922063). Along that line, misoprostol has been shown to prevent damaging effects of hypoxia by repressing Bnip3 and promoting the expression of pro-survival alternative splicing isoforms (PMID: 30275982). Indeed, the same study showed that misoprostol treatment prevents loss of mitochondrial membrane potential, ROS formation and impairment in mitochondrial oxygen consumption caused by hypoxia in primary neonatal cardiomyocytes. The present manuscript recapitulates these previously published findings. The truly novel findings concern the identification of Bnip3 residue Thr181 as target for PKA phosphorylation and the possible interaction of Bnip3 with 14-3-3. However, the role and/or involvement of these events has not been thoroughly investigated in relation to hypoxia and misoprostol treatment in cells or in vivo.__
Thank you for noting our previous work and identifying the novelty in our present work. As stated above, for Reviewer #1 comments 4, 6, and 8. We have provided additional mechanistic and in vivo data to more fully describe the role of T181 phosphorylation and the interaction with 14-3-3 chaperones in the revised manuscript.
Reviewer #2:
Systemic hypoxia, a major complication associated with reduced gestational time, affects more 60% of preterm infants and is a known driver of hypoxia-induced Bcl-2-like 19kDa-interacting protein 3 (Bnip3) expression in neonatal heart. At the level of the cardiomyocyte, Bnip3 activity plays a prominent role in the evolution of necrotic cell death, disrupting subcellular calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests both a cardioprotective role for protein kinase A (PKA) through stimulatory prostaglandin (PG) E1 signalling during prolonged periods of hypoxia, and a cytoprotective role for Bnip3 phosphorylation, indicating that post-translational modifications of Bnip3 may be a point of convergence for these two protective pathways. Using a combination of in vivo and multiple cell models, including human iPSC-derived cardiomyocytes, the authors tested if the PGE1 analogue misoprostol is cardioprotective during neonatal hypoxic injury by altering the phosphorylation status of Bnip3. Here we report that hypoxia exposure significantly increases Bnip3 expression, mitochondrial-fragmentation, -ROS, -calcium accumulation and -permeability transition, while reducing mitochondrial membrane potential, all of which were restored to control levels with addition of misoprostol, despite elevated Bnip3 protein expression. Through both gain- and loss-of function genetic studies, the authors show that misoprostol-induced protection directly affects Bnip3, preventing mitochondrial perturbations. They demonstrate that this is a result of PG EP4 receptor signalling, PKA activation, and direct Bnip3 phosphorylation at threonine-181. Furthermore, when this PKA phosphorylation site within Bnip3 is neutralized, the protective misoprostol effect is lost. They also provide evidence that misoprostol traffics Bnip3 away from the ER through a physical interaction with 14-3-3β, thereby preventing aberrant ER calcium release and MPT. In vivo studies further demonstrate that misoprostol treatment increases Bnip3 phosphorylation at threonine-181 in the mouse heart, while both misoprostol treatment and genetic ablation of Bnip3 prevented hypoxia-induced reductions in contractile function. Taken together, these results demonstrate a foundational role for Bnip3 phosphorylation in the molecular regulation of cardiomyocyte contractile and metabolic dysfunction and identifies EP4 signaling as a potential pharmacological mechanism to prevent hypoxia-induced neonatal cardiac injury. While this work is interesting, a number of issues remain.
__ 1.English expression needs some attention. For example, the first sentence of the abstract - "more than 60%...."; Page 20, line 9 "We observed that misoprostol's ability to to". Many sections should be broken into 2 or 3 sentences.__
Thank you, we have made these changes and have fully proof-read our manuscript.
__ 2.Evidence from In vivo studies such as those described in section 3.7 is minimal. Much more in vivo evidence is needed. It is unclear the authors established this in vivo model of hypoxia - supposedly gestational hypoxia should be considered. Consider citing these reviews on maternal over- and under-nutrition for postnatal heath (PMID 33181042; 22982026).__
Thank you, we will cite these papers and have clarified our in vivo model, related to comparable human gestation time, in the Methods section. We have also revised the Introduction and Discussion to be more consistent with our in vivo model. In addition, and noted above, we have preformed addition in vivo experiments in both the hypoxia/misoprostol model, and in Bnip3 KO mice to more fully support our conclusions. Additional HMGB1 immunofluorescence has already been added to Figures 1 and 7, and we have include additional confocal-based colocalization experiments from fixed tissues (described in more detail above; Fig. 7 D).
__ 3.Which one does misoprostol exactly execute its action? Phosphorylation through PKA or trafficking Bnip3 away from the ER through a physical interaction with 14-3-3β?__
This is a very good question. Based on our previous work on Bnip3L (Nix; PMID: 33044904,) PKA-induced phosphorylation of the transmembrane domain increased the physical interaction with 14-3-3b, which acts as a chaperone to translocate Nix away from the mitochondria and ER/SR. We will preform similar experiments with Bnip3 and 14-3-3b for the next revision to provide additional support for this conclusion.
__ 4.More in vivo proof of concept studies are needed to validate the signaling mechanism - this is an invitro-based study (hypoxia challenge occurs in vitro).__
We have already included additional in vivo immunofluorescence to Figure 1 and 7, and have performed additional colocalization experiments to validate the signaling pathway in this revision. Many of these experiments are described above.
__ 5.Quality of figures is somewhat poor.__
Our images are the highest possible resolution within the confines of the figure size limit. Perhaps the reviewer received a web-optimized version of the figures for review.
__ Reviewer #2 (Significance (Required)):__
__ Relatively high - although in vivo evidence is needed. __
Thank you. This is provided in the revised manuscript.
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Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
Systemic hypoxia, a major complication associated with reduced gestational time, affects more 60% of preterm infants and is a known driver of hypoxia-induced Bcl-2-like 19kDa-interacting protein 3 (Bnip3) expression in neonatal heart. At the level of the cardiomyocyte, Bnip3 activity plays a prominent role in the evolution of necrotic cell death, disrupting subcellular calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests both a cardioprotective role for protein kinase A (PKA) through stimulatory prostaglandin (PG) E1 signalling during prolonged periods of hypoxia, and a …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
Systemic hypoxia, a major complication associated with reduced gestational time, affects more 60% of preterm infants and is a known driver of hypoxia-induced Bcl-2-like 19kDa-interacting protein 3 (Bnip3) expression in neonatal heart. At the level of the cardiomyocyte, Bnip3 activity plays a prominent role in the evolution of necrotic cell death, disrupting subcellular calcium homeostasis and initiating mitochondrial permeability transition (MPT). Emerging evidence suggests both a cardioprotective role for protein kinase A (PKA) through stimulatory prostaglandin (PG) E1 signalling during prolonged periods of hypoxia, and a cytoprotective role for Bnip3 phosphorylation, indicating that post-translational modifications of Bnip3 may be a point of convergence for these two protective pathways. Using a combination of in vivo and multiple cell models, including human iPSC-derived cardiomyocytes, the authors tested if the PGE1 analogue misoprostol is cardioprotective during neonatal hypoxic injury by altering the phosphorylation status of Bnip3. Here we report that hypoxia exposure significantly increases Bnip3 expression, mitochondrial-fragmentation, -ROS, -calcium accumulation and -permeability transition, while reducing mitochondrial membrane potential, all of which were restored to control levels with addition of misoprostol, despite elevated Bnip3 protein expression. Through both gain- and loss-of function genetic studies, the authors show that misoprostol-induced protection directly affects Bnip3, preventing mitochondrial perturbations. They demonstrate that this is a result of PG EP4 receptor signalling, PKA activation, and direct Bnip3 phosphorylation at threonine-181. Furthermore, when this PKA phosphorylation site within Bnip3 is neutralized, the protective misoprostol effect is lost. They also provide evidence that misoprostol traffics Bnip3 away from the ER through a physical interaction with 14-3-3β, thereby preventing aberrant ER calcium release and MPT. In vivo studies further demonstrate that misoprostol treatment increases Bnip3 phosphorylation at threonine-181 in the mouse heart, while both misoprostol treatment and genetic ablation of Bnip3 prevented hypoxia-induced reductions in contractile function. Taken together, these results demonstrate a foundational role for Bnip3 phosphorylation in the molecular regulation of cardiomyocyte contractile and metabolic dysfunction and identifies EP4 signaling as a potential pharmacological mechanism to prevent hypoxia-induced neonatal cardiac injury. While this work is interesting, a number of issues remain.
1.English expression needs some attention. For example, the first sentence of the abstract - "more than 60%...."; Page 20, line 9 "We observed that misoprostol's ability to to". Many sections should be broken into 2 or 3 sentences.
2.Evidence from In vivo studies such as those described in section 3.7 is minimal. Much more in vivo evidence is needed. It is unclear the authors established this in vivo model of hypoxia - supposedly gestational hypoxia should be considered. Consider citing these reviews on maternal over- and under-nutrition for postnatal heath (PMID 33181042; 22982026) .
3.Which one does misoprostol exactly execute its action? Phosphorylation through PKA or trafficking Bnip3 away from the ER through a physical interaction with 14-3-3β?
4.More in vivo proof of concept studies are needed to validate the signaling mechanism - this is an invitro-based study (hypoxia challenge occurs in vitro).
5.Quality of figures is somewhat poor.
Significance
relatively high - although in vivo evidence is needed
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript by Martens et al investigates the mechanisms of Bnip3-mediated cell damage during hypoxia. The Authors show that modulation of prostaglandin (PG) E1 signaling with misoprostol prevents cardiac dysfunction, mitochondrial impairment and cell death induced by hypoxia. In addition, they show that the effect of misoprostol is dependent on PKA-mediated Thr181 phosphorylation. The Authors also suggest that there is a possible interaction between Bnip3 and 14-3-3beta that prevents ER Ca2+ release and mitochondrial Ca2+ overload. The Authors conclude that Bnip3 phosphorylation plays a key role in the regulation of cardiac and …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript by Martens et al investigates the mechanisms of Bnip3-mediated cell damage during hypoxia. The Authors show that modulation of prostaglandin (PG) E1 signaling with misoprostol prevents cardiac dysfunction, mitochondrial impairment and cell death induced by hypoxia. In addition, they show that the effect of misoprostol is dependent on PKA-mediated Thr181 phosphorylation. The Authors also suggest that there is a possible interaction between Bnip3 and 14-3-3beta that prevents ER Ca2+ release and mitochondrial Ca2+ overload. The Authors conclude that Bnip3 phosphorylation plays a key role in the regulation of cardiac and metabolic dysfunction and identify misoprostol treatment as a therapeutic intervention to prevent hypoxia-induced cardiac injury.
Major Comments:
1.Results presented in Figs. 1, 2 and 3 pertaining to hypoxia-induced changes in Bnip3 expression, changes in mitochondrial function, cell death, Bnip3-dependent Ca2+ transfer from ER to mitochondria and the effect of misoprostol have partially been demonstrated in a previous publication from the same group (PMID: 30275982).
2.The very same publication from 2018 shows that misoprostol treatment of pups exposed to hypoxia for 7 days is able to prevent the increase in Bnip3 protein levels. Yet, in the present manuscript misoprostol treatment had no effect on Bnip3 protein levels in the same model (Fig. 1E). This raises some concerns regarding the solidity and soundness of the results presented.
3.The validation of the custom antibody against p-Thr181 needs to be shown. Fig. 4E shows that p-Bnip3 band is quite strong in H9c2 cells, despite total endogenous Bnip3 levels are barely detectable. In addition, phosphorylation of the Bnip3 Thr181 residue in cells and/or in vivo should be confirmed by mass spectrometry.
4.Fig. 4L shows that misoprostol treatment of H9c2 cells leads to an increase in Bnip3 phosphorylation, but this does not seem to be the case in normoxic conditions in vivo (Fig. 4N). Moreover, shouldn't this presumable increase in phosphorylation induced by misoprostol in normoxic conditions lead to Bnip3 accumulation in the cytosol thereby reducing its colocalization with mitochondria (Fig. 6B)? The results obtained with the colocalization method should be corroborated using different methods, such as cell fractionation.
5.In relation to Fig. 4 M, N (page 19), the Authors concluded that the reduction in Bnip3 phosphorylation suggests an increase in Bnip3 activity in the hypoxic neonatal hearts. Nevertheless, this has not been demonstrated.
6.Along that line, the Authors concluded that misoprostol-induced cytoprotection is dependent on PKA Thr181 phosphorylation. Nevertheless, this dependence has not been convincingly demonstrated in hypoxic cells and in vivo.
7.Previous studies showed that Bnip3 induces mitochondrial fragmentation and mitophagy (PMID: 16645637, 20436456). What is the hypothesis for the inhibition of mitochondrial fragmentation induced by misoprostol in the present study? Does it prevent Bnip3 interaction with Opa1 or is this event downstream of ER Ca2+ release and mitochondrial Ca2+ overload? Does misoprostol affect mitophagy?
8.The link between Bnip3 interaction with 14-3-3 and Bnip3 Thr181 phosphorylation, if there is any, is not clear. The Authors mention that Thr181 lies within the 14-3-3 binding domain. Is Thr181 phosphorylation required for 14-3-3 binding or are these events unrelated? What is the significance of these events in hypoxia, does 14-3-3 binding to Bnip3 occur in vivo? Is Bnip3 localization affected by hypoxia, 14-3-3 binding and/or misoprostol treatment in vivo?
9.Fig. 6P shows the presence of myc-tag after IP for HA-tag, even when HA-14-3-3 was not expressed (middle lane). How is this possible?
Minor Comments:
1.Please co-stain with the cardiomyocyte marker in Fig. 2A (such as alpha-actinin).
2.The Methods are not sufficiently detailed. For instance, it is not clear what is the Ca2+ concentration used for Ca2+ pulses in the CRC experiment. The fact that cardiac mitochondria are able to uptake only two Ca2+ pulses raises some concerns regarding the quality of mitochondrial preparation. What is the reason for isolating mitoplasts instead of intact mitochondria?
3.TMRM fluorescence should be measured before and after FCCP administration, to account for the difference in plasma membrane potential (the results should be expressed as F/FFCCP).
4.Measurement of extracellular acidification is mentioned in the methods, but the relative results are not shown.
5.RNAi experiments targeting Bnip3 are also mentioned in the methods, but the results are not described.
Significance
Previous studies have demonstrated that Bnip3 is upregulated by hypoxia and plays a key role in inducing mitochondrial dysfunction and PTP opening that eventually results in cell death (PMID: 12169648, 10922063). Along that line, misoprostol has been shown to prevent damaging effects of hypoxia by repressing Bnip3 and promoting the expression of pro-survival alternative splicing isoforms (PMID: 30275982). Indeed, the same study showed that misoprostol treatment prevents loss of mitochondrial membrane potential, ROS formation and impairment in mitochondrial oxygen consumption caused by hypoxia in primary neonatal cardiomyocytes. The present manuscript recapitulates these previously published findings. The truly novel findings concern the identification of Bnip3 residue Thr181 as target for PKA phosphorylation and the possible interaction of Bnip3 with 14-3-3. However, the role and/or involvement of these events has not been thoroughly investigated in relation to hypoxia and misoprostol treatment in cells or in vivo.
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