PM2.5-Induced Cardiac Structural Modifications and Declined Pro-Survival Signalling Pathways Are Responsible for the Inefficiency of GSK-3β Inhibitor in Attenuating Myocardial Ischemia-Reperfusion Injury in Rats
, Sheikh F. Ahmad 2, Ahmed Nadeem 2, Mohammad Waseem 3 and Gino A. Kurian 1,*
Round 1
Reviewer 1 Report
The manuscript entitled “GSK-3β may not be a promising therapeutic target in attenuating myocardial ischemia reperfusion injury in rats exposed to PM2.5” explain the therapeutic efficiency of GSK3β as a target in the treatment of myocardial ischemia reperfusion injury. The major finding of this manuscript explains the inefficiency GSK3β as a therapeutic target against ischemia reperfusion injury, if the animals were subjected to prolonged exposure to PM2.5. Further they explain that PM2.5 exposure mediate an inactivation of pro-survival signalling pathway, that leads to poor recovery of rat heart from ischemia reperfusion. I find this article very interesting and relevant to the unmet need of the population.
According to the present study result GSK3β inhibitor is effective in attenuating IR induced changes in heart physiology and tissue injury, but failed to replicate the same function in heart exposed to PM2.5 for 21 days. How will authors explain the cardiotoxicity with single and multiple exposure of Pm2.5? Do the changes are transient or cardiac tissue is having robust adaptive response ability that prevent or reduce the single exposed PM2.5 effect?
The reduced cardiac tolerance with prolonged Pm2.5 exposure is the direct Pm2.5 effect or associated/secondary effect on the basal tissues.
What could be the probable reason for the ineffectiveness of GSK3β inhibitor in IR challenged rat heart from 21 days exposed animal.
In the methodology section, Group A and group B mentioned are little confusing. It may be recommended to include a sentence that state the whole study comprises of 2 sets of experimental animals, where one set was exposed to 21 days and other set was exposed to 1 day.
Inclusion of diagrammatic representations of different experimental groups are recommended.
Consider rearranging the section where IR induction and hemodynamics assessment comes before Experimental protocol as latter contains the group named IR, which was explained later.
The authors need to remove the RT PCR procedure from the methodology section as it is not carried out
Author Response
- According to the present study result GSK3β inhibitor is effective in attenuating IR induced changes in heart physiology and tissue injury, but failed to replicate the same function in heart exposed to PM2.5 for 21 days. How will authors explain the cardiotoxicity with single and multiple exposure of Pm2.5? Do the changes are transient or cardiac tissue is having robust adaptive response ability that prevent or reduce the single exposed PM2.5 effect?
Response: We thank the reviewer for the comment.
How will authors explain the cardiotoxicity with single and multiple exposure of PM2.5?
When the animals were exposed to PM2.5 for a single exposure duration, we had observed changes in the cardiac electrocardiogram, however the significant changes were not reflected in the ECG indices. Further when the animals were exposed to PM2.5 for multiple durations like 7, 14 and 21 days, after the day-14 of exposure, a significant decline was observed in the normal heart rate. In support to the reduced heart rate, the RR interval showed a significant increase. The QT interval was also significantly elevated from the normal control (figure 6f). Exposure to PM2.5 for 21 days resulted in a decreased heart rate and increased RR interval. The PR interval, which represents the time between atrial depolarization and ventricular depolarization was prolonged, similarly the time of ventricular depolarization (QRS) and QT interval also displayed a significant prolongation after 21 days of PM2.5 exposure. The ST segment, a marker of myocardial infarction was elevated significantly after 21 days.
Further we analysed the cardiac function, where we found that the cardiac function was deteriorated only in 21 days PM2.5 exposed group as explained by the hemodynamic data mentioned below:
Do the changes are transient or cardiac tissue is having robust adaptive response ability that prevent or reduce the single exposed PM2.5 effect?
To determine whether the cardiotoxic effects induced by PM2.5 exposure were temporary or permanent, we incorporated a washout period of 24 hours after each exposure duration, which spanned 1, 7, 14, and 21 days. Our results pointed out that, when we introduced the washout period following the 1-day and 7-day exposure durations, the cardiotoxicity that had been caused by PM2.5 exposure was found to be transient. The functional activities of the myocardium, which had been adversely affected by the pollutant, were successfully restored back to normal levels. However, as the exposure duration increased to 14 and 21 days, we observed a significant shift in the outcomes. The introduction of the washout period at this stage lost its efficacy in restoring the functional activities of the myocardium. This suggests that the cardiotoxic effects of PM2.5 exposure, once endured for a more extended period, become less responsive to the washout intervention (results yet to be published).
- The reduced cardiac tolerance with prolonged Pm2.5 exposure is the direct Pm2.5 effect or associated/secondary effect on the basal tissues.
Response: We thank the reviewer for the comment.
The heart's reduced tolerance to PM2.5, resulting from prolonged exposure, is influenced by two main factors: direct and secondary effects on the underlying tissues. When we focus on the direct effect, we have observed that prolonged exposure to PM2.5 leads to the degradation of the mitochondrial membrane potential. This, in turn, causes an accumulation of PM2.5 particles within the mitochondria. Supporting this observation is an increase in the expression of the metallothione-1 gene and a decrease in the expression of mitochondrial detoxification genes. These changes, which arise due to the direct impact of PM2.5 on mitochondria, can initiate secondary events such as oxidative stress and inflammation, consequently disrupting the normal functioning of the heart. Furthermore, PM2.5 exposure can also trigger secondary effects through other organs, with the lungs playing a significant role. Inhaling PM2.5 particles can initiate a cascade of systemic inflammation and oxidative stress responses that eventually affect the health of the heart.
In conclusion, prolonged exposure to PM2.5 can reduce the heart's tolerance through direct effects on mitochondria, leading to oxidative stress and inflammation. Additionally, secondary effects originating from other organs, particularly the lungs, contribute to systemic inflammation, collectively disrupting the heart's normal functioning and potentially posing significant health risks.
- What could be the probable reason for the ineffectiveness of GSK3β inhibitor in IR challenged rat heart from 21 days exposed animal.
Response: We thank the reviewer for the comment. The present study outcome suggest that PM2.5 exposure depleted the cardiac tolerance to withstand additional surgical stress, that makes the cardiac patients highly sensitive to develop co-morbidity or even inflict mortality with surgery. One of the probable reasons for the ineffectiveness of GSK3β inhibitor in IR challenged rat heart exposed to PM2.5 maybe because: PM2.5 exposure instigate mitochondrial damage via considerable uptake of PM2.5 by the organelle that leads depleted mitochondrial quality control, eventually induced cardiac injury and deteriorated contractile function of the heart. In low level of healthy mitochondria in the cardiac tissue, GSK 3β inhibitor will be unable to provide the desirable function that protect the heart from IR injury. In support to this our results demonstrated that:
- GSK3β inhibitor was unable to improve the expression levels of mitochondrial quality control genes in 21days PM5 exposed rat hearts subjected to IR
Figure: mRNA expression levels of A) Mitophay genes (PINK, PARKIN, OPTN), B) Mitofission genes (MFF, DNM1, FIS1), C) Mitofusion genes (MFN1, MFN2), (*p<0.05 vs Normal). D) Protein expression levels of Parkin, MFF and MFN1 via western blot. Groups include: 1: Normal Perfusion, 2: IR control, 3: PM2.5 exposure for 21 days followed by normal perfusion, 4: PM2.5 exposure for 21 days followed by IR induction, 5: GSK3β inhibitor administration followed by IR induction, 6: PM2.5 exposure followed by GSK3β inhibitor administration and normal perfusion, 7: PM2.5 exposure followed by GSK3β inhibitor administration and IR induction)
- Protein expression level of p-GSK3β, GSK3β in cardiac tissue and mitochondria from rat exposed to PM5 for 21 days in presence and absence of GSK3β inhibitor
Figure: Protein expression of GSK3β and p- GSK3β in A) Cytosol, B) Mitochondria, mRNA level expression of C) MT-1 (*p<0.05 vs Normal), D) ICPMS data showing metal deposition in isolated mitochondria. Groups include: 1: Normal Perfusion, 2: IR control, 3: PM2.5 exposure for 21 days followed by normal perfusion, 4: PM2.5 exposure for 21 days followed by IR induction, 5: GSK3β inhibitor administration followed by IR induction, 6: PM2.5 exposure followed by GSK3β inhibitor administration and normal perfusion, 7: PM2.5 exposure followed by GSK3β inhibitor administration and IR induction)
- In the methodology section, Group A and group B mentioned are little confusing. It may be recommended to include a sentence that state the whole study comprises of 2 sets of experimental animals, where one set was exposed to 21 days and other set was exposed to 1 day.
Response: We thank the reviewer for the comment and have accordingly made modifications in the methodology section, which is displayed below for your reference:
Revised version:
Experimental protocol
The animal exposure to PM2.5 was conducted using a modified whole-body animal exposure model, as detailed in our previous publication [15]. The temperature, pressure, and flow rate within the chamber were maintained at optimal levels. To monitor the concentrations of PM, oxygen (O2), carbon monoxide (CO), as well as the temperature and humidity inside the chamber, a PRANA air-sourced CAIR air quality monitor was employed. The experimental animals used in the present study were grouped into two major groups: Group A: exposed to PM2.5 for 21 days and Group B: exposed to PM2.5 for 1 day.
- Inclusion of diagrammatic representations of different experimental groups are recommended.
Response: We thank the reviewer for the comment and have added a diagrammatic representation of the experimental groups, which is also mentioned below for your reference
- Consider rearranging the section where IR induction and hemodynamics assessment comes before Experimental protocol as latter contains the group named IR, which was explained later.
Response: We thank the reviewer for the comment and have accordingly made modifications in the methodology section, which is displayed below for your reference:
Revised version:
IR induction and hemodynamics assessment
To induce IR injury, the hearts were subjected to 30 minutes of ischemia by halting the flow of KH buffer, followed by 60 minutes of reperfusion by resuming the buffer perfusion. Throughout the entire experiment, hemodynamic parameters were continuously monitored using AD instruments' Lab chart software from Australia. At the end of the experiment, the hearts were collected and preserved for subsequent analysis [16].
Experimental protocol
The animal exposure to PM2.5 was conducted using a modified whole-body animal exposure model, as detailed in our previous publication [15]. The temperature, pressure, and flow rate within the chamber were maintained at optimal levels. To monitor the concentrations of PM, oxygen (O2), carbon monoxide (CO), as well as the temperature and humidity inside the chamber, a PRANA air-sourced CAIR air quality monitor was employed. The experimental animals used in the present study were grouped into two major groups: Group A: exposed to PM2.5 for 21 days and Group B: exposed to PM2.5 for 1 day.
Group A: (21 days exposure study): 1: Normal, 2: IR control, 3: PM2.5 exposure control (PM_C): Exposure to PM2.5 at a concentration of 250 µg/m3 for 3hrs daily for 21 days , 4: PM2.5 exposure followed by IR induction (PM_IR): Exposure to PM2.5 followed by IR induction, 5: GSK3β_IR: 0.7mg/kg of GSK3β inhibitor: SB216763 was administered intraperitoneally followed by IR induction, 6: PM+ GSK3β_C: Exposure to PM2.5 at a concentration of 250 µg/m3 for 3hrs daily for 21 days followed by 0.7mg/kg of SB216763 administration and normal perfusion for 120min, PM+ GSK3β_IR: Exposure to PM2.5 at a concentration of 250 µg/m3 for 3hrs daily for 21 days followed by SB216763 administration and IR induction.
Group B: (1 day exposure study): : Normal, 2: IR control, 3: PM2.5 exposure control (PM 1day_C): Exposure to PM2.5 at a concentration of 250 µg/m3 for 1hr, 4: PM2.5 exposure followed by IR induction (PM 1day_IR): Exposure to PM2.5 followed by IR induction, 5: GSK3β_IR: 0.7mg/kg of GSK3β inhibitor: SB216763 was administered intraperitoneally followed by IR induction, 6: PM 1day+ GSK3β_C: Exposure to PM2.5 at a concentration of 250 µg/m3 for 1hr followed by 0.7mg/kg of SB216763 administration and normal perfusion for 120min, PM 1day + GSK3β_IR: Exposure to PM2.5 at a concentration of 250 µg/m3 for 1hr followed by SB216763 administration and IR induction.
- The authors need to remove the RT PCR procedure from the methodology section as it is not carried out
Response: We thank the reviewer for the comment and have accordingly removed RT PCR procedure from the methodology
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Regarding the manuscript titled “ GSK-3β may not be a promising therapeutic target in attenuating myocardial ischemia reperfusion injury in rats exposed to PM2.5” the authors investigated the protective effect of GSK-3β inhibitors against cardiac IR injury and PM2.5 exposure either for 1 or 21 days. The protective effect was revealed in case of 1 day PM2.5 exposure but not with 21 days of exposure. It is a good point of research with a good experimental design except for some points should be considered.
* I think the title need to be more descriptive.
* mention the total number of animals used in this study and the number of animal per each group.
* mention the F statistics if the number of the animals per each group is than 15.
* check the numbering of the titles and subtitles as recommended by the journal author guides.
The quality of English language is good, though minor revision is required.
Author Response
myocardial ischemia reperfusion injury in rats exposed to PM2.5” the authors investigated the protective effect of GSK-3β inhibitors against cardiac IR injury and PM2.5 exposure either for 1 or 21 days. The protective effect was revealed in case of 1 day PM2.5 exposure but not with 21 days of exposure. It is a good point of research with a good experimental design except for some points should be considered.
* I think the title need to be more descriptive.
Response: As per the reviewer’s suggestion we have modified the title as ” PM2.5 induced cardiac structural modifications and declined pro-survival signalling pathways are responsible for the in-efficiency of GSK-3β inhibitor in attenuating myocardial ischemia-reperfusion injury in rats”
* mention the total number of animals used in this study and the number of animal per each group.
Response: We thank the reviewer for the comment and have accordingly added the animal details in the methodology section which is highlighted in the manuscript file and also displayed below for your reference:
Revised version:
Experimental protocol
The animal exposure to PM2.5 was conducted using a modified whole-body animal exposure model, as detailed in our previous publication [15]. The temperature, pressure, and flow rate within the chamber were maintained at optimal levels. To monitor the concentrations of PM, oxygen (O2), carbon monoxide (CO), as well as the temperature and humidity inside the chamber, a PRANA air-sourced CAIR air quality monitor was employed. The experimental animals used in the present study were divided into two major groups: Group A (comprises of 42 rats): exposed to PM2.5 for 21 days and Group B (comprises of 42 rats): exposed to PM2.5 for 1 day. Group A and B are further sub divided into 7 groups comprising of 6 animals per group
* mention the F statistics if the number of the animals per each group is than 15.
Response: We would like to bring to the kind attention of the reviewer that, the number of animals per group were 6 and hence F statistics is not mentioned in the statistical analysis.
* check the numbering of the titles and subtitles as recommended by the journal author guides.
Response: We thank the reviewer for the comment and have accordingly made changes in the manuscript file
Author Response File: Author Response.pdf
Round 2
Reviewer 1 Report
it can be accepted in present form
Reviewer 2 Report
All the corrections have been done properly.
