TRPV1 Activation Is Associated with Improved Mitochondrial Function and Cardioprotection in Experimental Hypertension

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript presents interesting findings on TRPV1 activation and mitochondrial protection in hypertension. However, several methodological, statistical, and interpretative issues need to be addressed:

Figure2: Clarify if all treated groups significantly differ from H.

Figure 3: The authors state they used Student's t-test for infarct size data (line 133: "Statistical analysis was performed using Student's t test"), but there are 5 experimental groups. A t-test is inappropriate for multiple comparisons. Use one-way ANOVA with post-hoc test (Tukey) for all group comparisons.

Table1: The RC value for H+CS+CZ is reported as 5.8 ± 0.82 this is even higher than the control group (4.96 ± 0.12), which is surprising. ¿Did the combination treatment truly improve coupling beyond controls? Verify the raw data and statistical análisis and discuss why CS+CZ shows better RC than CS alone.

Figure 6. The authors state that catalase activity increased in hypertensive rats and decreased with CS treatment. However, catalase is usually decreased in chronic oxidative stress due to inactivation. Increased catalase may indicate compensatory upregulation.

Line 357-359: Capsaicin was given at 5 mg/kg/day for 4 days (total 20 mg/kg). This is a relatively high cumulative dose. Systemic effects (e.g., hypothermia, pain, autonomic activation) could confound results. Discuss why a short 4-day treatment was chosen versus longer protocols.

Author Response

Reviewer1

Comments and Suggestions for Authors:

The manuscript presents interesting findings on TRPV1 activation and mitochondrial protection in hypertension. However, several methodological, statistical, and interpretative issues need to be addressed:

Response to Reviewer 1

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the valuable comments and suggestions, which have significantly improved the quality and clarity of the work. All comments were carefully considered and addressed as detailed below.

Comment 1

Figure 2: Clarify if all treated groups significantly differ from H.

Response:

We thank the reviewer for this observation. We have revised the figure legend and the Results section to explicitly indicate the statistical differences among groups. All treated groups (H+CS, H+CZ, and H+CS+CZ) showed significantly lower coronary vascular resistance compared with the hypertensive group (H), as determined by one-way ANOVA followed by Tukey’s post hoc test. The figure legend has been modified accordingly to improve clarity.

Comment 2

Figure 3: The authors state they used Student's t-test for infarct size data (line 133: "Statistical analysis was performed using Student's t test"), but there are 5 experimental groups. A t-test is inappropriate for multiple comparisons. Use one-way ANOVA with post-hoc test (Tukey) for all group comparisons.

Response:

We appreciate the reviewer for identifying this issue. We agree that the use of Student’s t-test was not appropriate for comparisons involving more than two groups. The infarct size data have now been reanalyzed using one-way ANOVA followed by Tukey’s post hoc test. The statistical analysis section, Results section, and Figure 3 legend have been revised accordingly. Importantly, the overall conclusions of the study remain unchanged after reanalysis.

Comment 3

Table 1: The RC value for H+CS+CZ is reported as 5.8 ± 0.82; this is even higher than the control group (4.96 ± 0.12), which is surprising. Did the combination treatment truly improve coupling beyond controls? Verify the raw data and statistical analysis and discuss why CS+CZ shows better RC than CS alone.

Response:

We thank the reviewer for this important observation. We carefully reexamined the raw data and statistical analyses and confirmed that the RC value reported for the H+CS+CZ group is correct. Although the mean RC value was numerically higher than that observed in control animals, the difference was not statistically significant due to the variability within the group. We agree that this finding deserves further discussion. Accordingly, a paragraph has been added to the Discussion section addressing possible explanations. One possibility is that the combined administration of capsaicin and capsazepine may provide additive protection against oxidative damage through mechanisms that are not exclusively dependent on TRPV1 modulation. Both compounds contain phenolic moieties that have been associated with antioxidant properties, and capsazepine has also been reported to exert anti-inflammatory effects in experimental models. These actions could contribute to improved preservation of mitochondrial coupling. Additionally, incomplete TRPV1 blockade at the dose of capsazepine used cannot be excluded. Nevertheless, the precise mechanism remains uncertain and warrants further investigation.

Comment 4

Figure 6. The authors state that catalase activity increased in hypertensive rats and decreased with CS treatment. However, catalase is usually decreased in chronic oxidative stress due to inactivation. Increased catalase may indicate compensatory upregulation.

Response:

We thank the reviewer for this insightful comment. We agree that elevated catalase activity under chronic oxidative stress conditions may represent a compensatory adaptive response rather than a direct indicator of reduced oxidative damage. Accordingly, we have revised the interpretation throughout the manuscript. We now discuss that the increased catalase activity observed in hypertensive rats likely reflects an adaptive response to enhanced reactive oxygen species production associated with nitric oxide deficiency and mitochondrial dysfunction. Furthermore, we clarify that the reduction in catalase activity following capsaicin treatment may indicate attenuation of oxidative stress and a diminished need for compensatory antioxidant enzyme upregulation, rather than suppression of antioxidant defenses. The Results and Discussion sections have been modified accordingly.

Comment 5

Line 357-359: Capsaicin was given at 5 mg/kg/day for 4 days (total 20 mg/kg). This is a relatively high cumulative dose. Systemic effects (e.g., hypothermia, pain, autonomic activation) could confound results. Discuss why a short 4-day treatment was chosen versus longer protocols.

Response:

We thank the reviewer for this valuable comment. The capsaicin treatment protocol used in the present study was selected based on our previous work employing the same hypertension model and dosing regimen, in which significant cardiovascular and endothelial effects were observed without evidence of overt toxicity or adverse systemic effects. Importantly, the treatment was initiated after hypertension had already been established and was intentionally limited to four days in order to evaluate the acute therapeutic effects of TRPV1 activation on cardiac and mitochondrial function, rather than its potential role in preventing the development of hypertension. We acknowledge that the cumulative capsaicin dose used in this study may induce systemic physiological responses, including thermoregulatory changes, sensory neuron activation, and autonomic effects, which could potentially influence cardiovascular outcomes. However, no overt signs of toxicity or behavioral abnormalities were observed during the treatment period. Nevertheless, we recognize that indirect systemic effects cannot be completely excluded and may have contributed, at least in part, to the observed cardioprotective responses. To address this concern, we have expanded the Discussion section to clarify the rationale for the treatment protocol and to acknowledge the potential contribution of systemic TRPV1-mediated effects. Future studies will be necessary to distinguish direct myocardial actions from systemic mechanisms and to compare the effects of short-term versus long-term capsaicin administration. A justification for the capsaicin dose and treatment duration has been added to the Materials and Methods and Discussion sections. In addition, the potential influence of systemic TRPV1-mediated responses has been acknowledged as a limitation of the study.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

In their manuscript, Angélica Ruiz-Ramírez and colleagues aimed to show the cardioprotective role of TRPV1 receptor activation by capsaicin in a hypertensive rat model. The topic of the manuscript is interesting, however, there are serious concerns about the animal model and its basic characterization as well as with the study design. Moreover, there is a serious mistake in the concept of the study, i.e. it is not clear, why the authors suppose that infarction develops in an intact heart of a rat with systemic hypertension but without ischemic event or other accepted models of making myocardial infarction.

 

A protocol figure is necessary to include

 

Abstract: The model is missing from the abstract, namely coronary occlusion type (in vivo/isolated heart?)/duration is not mentioned, only infarct size suggests that myocardial infarction was induced.

 

Results section:

• it is not clear, what the point is of measuring infarct size without inducing myocardial ischemia by using any accepted method.
• If ischemia has not been induced, talking about area at risk does not also make any sense, as the entire myocardium could be the subject of myocardial injury.
• In addition, the sample is size is less than acceptable, n=3 sample size in infarction studies is not sufficient to draw any conclusion from the results.
• The measuring unit of infarct size is also not provided – is it expressed in percentage of the area of the left ventricle? please clarify.
• 3-mm thick slices from the heart of a rat weighing 300 to 350g result in only 3 to 4 slices, which are too thick, and thereby unsuitable to accurately represent and estimate myocardial infarct size
• The representative image for the H group shows much larger infarct size than shown in the diagram – this discrepancy also highlights the necessity to specify a method for normalizing infarct size

 

Except Figure 1 and 2, the dimensions /units of the measured parameters are missing from the graphs. Please add measuring units accordingly.

 

In the discussion, a reference should be made to previous relevant findings indicating that rats treated with capsaicin to induce selective sensory desensitization exerted an increased left ventricular end-diastolic pressure measured in an ex vivo Neely system (“working heart” preparation), which contributed to impaired myocardial relaxation (PMID: 18037908). In addition, a comprehensive review about the cardioprotective role of TRPV1 channels in cardiac pathologies should also be included in the discussion section and cited (PMID: 32586044).

 

Finally, the authors are advised to ask help for English editing from a native English speaker to improve the readability of the manuscript.

Author Response

Reviewer 2

 

Comments and Suggestions for Authors

 

Response to Reviewer 2

 

We sincerely thank the reviewer for the detailed evaluation of our manuscript and for the constructive comments and suggestions. We carefully considered all observations and revised the manuscript accordingly. Our detailed responses are provided below.

 

Comment 1

 

In their manuscript, Angélica Ruiz-Ramírez and colleagues aimed to show the cardioprotective role of TRPV1 receptor activation by capsaicin in a hypertensive rat model. The topic of the manuscript is interesting, however, there are serious concerns about the animal model and its basic characterization as well as with the study design. Moreover, there is a serious mistake in the concept of the study, i.e. it is not clear why the authors suppose that infarction develops in an intact heart of a rat with systemic hypertension but without ischemic event or other accepted models of making myocardial infarction.

 

Response:

 

We thank the reviewer for this important observation and agree that the term “infarct size” may be misleading in the absence of an experimentally induced ischemic event. In the present study, hearts were not subjected to coronary occlusion or ischemia-reperfusion protocols. Rather, TTC staining was used as an indicator of myocardial tissue viability and to identify areas of irreversible tissue injury associated with chronic hypertension. To avoid confusion, we have revised the manuscript and replaced the term “infarct size” with “TTC-negative myocardial area” throughout the text. Likewise, references to “area at risk” have been removed, since no regional ischemic insult was induced and the entire myocardium was analyzed. We acknowledge this limitation and have clarified the interpretation of the TTC data accordingly in the Methods, Results, Discussion, and Conclusions sections of the revised manuscript.

 

Comment 2

 

A protocol figure is necessary to include.

 

Response:

 

We thank the reviewer for this valuable suggestion. To improve the clarity of the experimental design and facilitate understanding of the study workflow, a schematic figure illustrating the complete experimental protocol has been incorporated into the revised manuscript. The figure summarizes the induction of hypertension with L-NAME, the treatment schedules for capsaicin and capsazepine, the study timeline, and the subsequent cardiac, histological, and mitochondrial assessments performed at the end of the experimental period. A new figure entitled “Experimental Design and Study Workflow” has been added to the manuscript as Figure 7.

 

Comment 3

 

Abstract: The model is missing from the abstract, namely coronary occlusion type (in vivo/isolated heart?)/duration is not mentioned, only infarct size suggests that myocardial infarction was induced.

 

Response:

 

We thank the reviewer for this observation. The Abstract has been revised to provide a clearer description of the experimental model, including the induction of hypertension with L-NAME and the methodology used for cardiac assessment. Additional details regarding the experimental procedures have been incorporated to avoid ambiguity concerning myocardial injury assessment and TTC staining analysis. The Abstract was revised to clarify the experimental model and study design.

 

Comment 4

 

It is not clear what the point is of measuring infarct size without inducing myocardial ischemia by using any accepted method. If ischemia has not been induced, talking about area at risk does not also make any sense, as the entire myocardium could be the subject of myocardial injury. In addition, the sample size is less than acceptable, n = 3 sample size in infarction studies is not sufficient to draw any conclusion from the results. The measuring unit of infarct size is also not provided. The representative image for the H group shows a much larger infarct size than shown in the diagram, highlighting the need to specify the normalization method.

 

Response:

 

We thank the reviewer for these important observations and agree that the terminology used in the original manuscript may have led to confusion. In the present study, no coronary occlusion or ischemia-reperfusion protocol was performed. Therefore, TTC staining was not intended to quantify classical myocardial infarct size, but rather to assess myocardial tissue viability and identify TTC-negative areas associated with chronic hypertension-induced cardiac injury. Accordingly, we have revised the manuscript and replaced the term “infarct size” with “TTC-negative myocardial injury area” throughout the text. References to “area at risk” have been removed, since no regional ischemic insult was induced and the entire myocardium was analyzed. We have also clarified the quantification method. TTC-negative areas were measured using ImageJ software and expressed as a percentage of the total ventricular tissue area analyzed. This information has been incorporated into the Methods and Results sections. Regarding the sample size, we acknowledge that the number of hearts analyzed by TTC staining (n = 3 per group) is limited and represents a study limitation. Therefore, TTC data were interpreted as supportive evidence and considered together with the functional, histological, and mitochondrial findings rather than as a standalone endpoint. This limitation has now been acknowledged in the Discussion section. Finally, we carefully reexamined the representative images and quantitative analysis. The figure legend and image processing methodology have been clarified to improve consistency between representative images and quantitative measurements. The terms “infarct size” were removed throughout the manuscript and replaced by terminology reflecting myocardial tissue viability and injury assessment. The TTC quantification method was clarified, measurement units were added, and study limitations regarding sample size were acknowledged in the Discussion section.

 

Comment 5

 

Except Figure 1 and 2, the dimensions/units of the measured parameters are missing from the graphs. Please add measuring units accordingly.

 

Response:

 

We thank the reviewer for identifying this oversight. The corresponding units have now been added to all figure axes and graph labels where appropriate in the revised manuscript. Measurement units were incorporated into all relevant figures and figure legends.

 

Comment 6

 

In the discussion, a reference should be made to previous relevant findings indicating that rats treated with capsaicin to induce selective sensory desensitization exerted an increased left ventricular end-diastolic pressure measured in an ex vivo Neely system (“working heart” preparation), which contributed to impaired myocardial relaxation (PMID: 18037908). In addition, a comprehensive review about the cardioprotective role of TRPV1 channels in cardiac pathologies should also be included in the discussion section and cited (PMID: 32586044).

 

Response:

 

We thank the reviewer for these valuable suggestions. Both references were incorporated into the revised Discussion section. The study associated with PMID: 18037908 was included to discuss the complexity of TRPV1 modulation and the potential effects of capsaicin-induced sensory desensitization on cardiac function. Likewise, the comprehensive review corresponding to PMID: 32586044 was incorporated to strengthen the discussion regarding the cardioprotective role of TRPV1 channels in cardiovascular diseases and myocardial injury.

 

Comment 7

 

Finally, the authors are advised to ask help for English editing from a native English speaker to improve the readability of the manuscript.

 

Response:

 

We thank the reviewer for this recommendation. The manuscript has been thoroughly revised for English language usage, grammar, and readability. In addition, professional language editing was performed to improve clarity and overall presentation. The entire manuscript was carefully revised to improve English language quality and readab

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

The research is correctly and precisely presented. I believe that the authors just need to explain how they chose the doses of capsaicin and capsazepine and whether there is any data on the dose dependence of their effect in relation to TRPV1 receptors.

Author Response

Reviewer 3:

 

Comments and Suggestions for Authors

 

Response to Reviewer 3

 

We sincerely thank the reviewer for the positive evaluation of our work and for the valuable suggestion.

 

Comment

 

The research is correctly and precisely presented. I believe that the authors just need to explain how they chose the doses of capsaicin and capsazepine and whether there is any data on the dose dependence of their effect in relation to TRPV1 receptors.

 

Response:

 

We appreciate the reviewer’s important observation. The doses of capsaicin and capsazepine used in this study were selected based on our previous experimental studies, as well as reports from the literature demonstrating effective modulation of TRPV1 receptor activity in cardiovascular and oxidative stress-related models. Specifically, the capsaicin dose employed in the present study has previously been shown to activate TRPV1 and exert cardioprotective effects, whereas the selected capsazepine dose effectively antagonizes TRPV1-mediated responses under in vivo experimental conditions. In our previous studies, these doses were successfully used to investigate the role of TRPV1 activation in cardiovascular and metabolic disorders, showing significant effects on oxidative stress, inflammation, vascular function, and cardiac protection. Therefore, the selected doses were not arbitrary but were based on prior pharmacological validation and experimental evidence supporting their effectiveness in modulating TRPV1-related responses. Regarding dose dependence, previous studies have demonstrated that both capsaicin and capsazepine exhibit dose-dependent effects on TRPV1 activation or inhibition. However, the primary objective of the present study was not to establish a dose–response relationship, but rather to evaluate the effects of TRPV1 modulation using doses previously validated in experimental cardiovascular models. We acknowledge that a comprehensive dose–response analysis could provide additional mechanistic information and represents an interesting area for future investigation. To address the reviewer’s suggestion, we have added a brief justification of the selected doses in the Materials and Methods section and included the corresponding references supporting their use.

 

1. Castrejón-Téllez V, Del Valle-Mondragón L, Pérez-Torres I, Guarner-Lans V, Pastelín-Hernández G, Ruiz-Ramírez A, Díaz-Juárez JA, Varela-López E, Oidor-Chan VH, Vargas-González A, Martínez-Memije R, Flores-Chávez P, León-Ruíz B, Arriaga-Carrillo S, Torres-Narváez JC. TRPV1 Contributes to Modulate the Nitric Oxide Pathway and Oxidative Stress in the Isolated and Perfused Rat Heart during Ischemia and Reperfusion. Molecules. 2022 Feb 3;27(3):1031. doi: 10.3390/molecules27031031.
2. Varela-López E, Del Valle-Mondragón L, Castrejón-Téllez V, Pérez-Torres I, Arenas AP, Rojas FM, Guarner-Lans V, Vargas-González A, Pastelín-Hernández G, Torres-Narváez JC. Role of the Transient Receptor Potential Vanilloid Type 1 (TRPV1) in the Regulation of Nitric Oxide Release in Wistar Rat Aorta. Oxid Med Cell Longev. 2021 Aug 2;2021:8531975. doi: 10.1155/2021/8531975.
3. Torres-Narváez JC, Pérez-Torres I, Castrejón-Téllez V, Varela-López E, Oidor-Chan VH, Guarner-Lans V, Vargas-González Á, Martínez-Memije R, Flores-Chávez P, Cervantes-Yañez EZ, Soto-Peredo CA, Pastelín-Hernández G, Del Valle-Mondragón L. The Role of the Activation of the TRPV1 Receptor and of Nitric Oxide in Changes in Endothelial and Cardiac Function and Biomarker Levels in Hypertensive Rats. Int J Environ Res Public Health. 2019 Sep 25;16(19):3576. doi: 10.3390/ijerph16193576.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

I have reviewed "Activation of the TRPV1 Receptor Confers Myocardial Protection in Hypertensive Rats Via Modulation of Mitochondrial Function". However, in order to meet the quality requirements of the journal, the author still needs to make major revision to the manuscript.

 

Q1：Section 2.1: Following capsaicin (CS) treatment, the mean arterial pressure of rats decreased to 144 ± 6 mmHg. However, following the antagonist capsazepine (CZ) treatment, the mean arterial pressure unexpectedly showed a more pronounced reduction to 128 ± 7 mmHg. Conceptually, this finding appears to conflict with the conclusion proposed in the manuscript that TRPV1 activation mediates the regulation of hypertension. Perhaps the authors should provide a more convincing explanation regarding this point.

 

Q2: Section 2.2: As a CS antagonist, CZ failed to disrupt the CS-induced decrease in Coronary Vascular Resistance. Additionally, CZ alone also demonstrated the ability to alleviate Coronary Vascular Resistance, showing the same trend described in Section 2.3. Does this suggest that the observed improvement in Coronary Vascular Resistance might not be mediated by TRPV1-specific regulation? The authors' explanation regarding this point is not entirely convincing.

 

Q3: Sections 2.3 & 2.4: Regarding the experimental design, CS and CZ interventions were administered for only the final 4 days of a 40-day study. Remarkably, this brief 4-day treatment significantly reversed organic cardiac lesions in hypertensive rats. Perhaps the authors should provide an explanation for this phenomenon.

 

Q4: Table 1: Please use the standard three-line table format.

 

Q5: Figure 5: The figure caption states that the data underwent statistical analysis, yet no statistical symbols (* or **) are visible in the graph itself.

 

Q6: The authors state in the manuscript that TRPV1 regulates mitochondrial Ca²⁺ flux to maintain mitochondrial homeostasis. However, no data related to mitochondrial Ca²⁺ is included in the manuscript. It is suggested to supplement relevant experiments.

 

Q7: Conclusion: The current findings indicate that TRPV1 may not be the sole or primary protective pathway. It is suggested that the authors revise the conclusion or supplement relevant experiments to provide robust data support.

 

Q9: Typographical Errors: Line 195: "(figure %B)", Line 204: please clarify "(C vs CS?)".

Author Response

Reviewer 4

 

Comments and Suggestions for Authors

 

I have reviewed "Activation of the TRPV1 Receptor Confers Myocardial Protection in Hypertensive Rats Via Modulation of Mitochondrial Function". However, in order to meet the quality requirements of the journal, the author still needs to make major revision to the manuscript.

 

Response to Reviewer 4

 

We sincerely thank the reviewer for the detailed evaluation of our manuscript and for the valuable comments and suggestions. We carefully revised the manuscript accordingly. Our detailed responses are presented below.

 

Comment Q1

 

Section 2.1: Following capsaicin (CS) treatment, the mean arterial pressure of rats decreased to 144 ± 6 mmHg. However, following the antagonist capsazepine (CZ) treatment, the mean arterial pressure unexpectedly showed a more pronounced reduction to 128 ± 7 mmHg. Conceptually, this finding appears to conflict with the conclusion proposed in the manuscript that TRPV1 activation mediates the regulation of hypertension. Perhaps the authors should provide a more convincing explanation regarding this point.

 

Response:

 

We thank the reviewer for this important observation. We agree that the antihypertensive effect observed in the capsazepine-treated group suggests that mechanisms beyond direct TRPV1 activation may contribute to the cardiovascular responses observed in this study. Capsazepine is known to exert biological effects beyond TRPV1 antagonism, including modulation of oxidative stress, inflammatory signaling, calcium homeostasis, and vascular reactivity. As the reviewer comment, we made a mistake in the value in the blood pressure observed in the H+CZ group the correct value was 165 ± 5. To address this concern, we revised the Discussion and Conclusions sections to avoid overstating the exclusive role of TRPV1 activation in mediating the antihypertensive effects observed. We now indicate that TRPV1 modulation appears to participate in the cardioprotective and mitochondrial effects observed, although additional mechanisms may also contribute to the overall response.

 

Comment Q2

 

Section 2.2: As a CS antagonist, CZ failed to disrupt the CS-induced decrease in Coronary Vascular Resistance. Additionally, CZ alone also demonstrated the ability to alleviate Coronary Vascular Resistance. Does this suggest that the observed improvement in Coronary Vascular Resistance might not be mediated by TRPV1-specific regulation? The authors' explanation regarding this point is not entirely convincing.

 

Response:

 

We thank the reviewer for this insightful observation. We agree that the coronary vascular resistance (CVR) results do not allow us to attribute the observed vascular protection exclusively to TRPV1 activation. Although capsaicin significantly reduced CVR in hypertensive rats, capsazepine alone also produced a similar effect, and co-administration of capsaicin and capsazepine did not abolish the reduction in CVR. Therefore, we acknowledge that mechanisms independent of TRPV1 activation may contribute to the observed vascular response. Previous studies have reported that capsazepine possesses biological activities beyond TRPV1 antagonism, including antioxidant and anti-inflammatory properties, which may improve vascular function under conditions of oxidative stress. Since hypertension induced by L-NAME is characterized by endothelial dysfunction and increased reactive oxygen species production, it is plausible that capsazepine attenuated vascular damage through mechanisms unrelated to TRPV1 blockade. Nevertheless, the observation that capsaicin, capsazepine, and their combination all improved coronary vascular resistance suggests that preservation of vascular function is a robust finding of the study, although the precise contribution of TRPV1 signaling cannot be established from the present experimental design. Accordingly, we have revised the Discussion section to clarify that the reduction in coronary vascular resistance cannot be interpreted as exclusively mediated by TRPV1 activation and that TRPV1-independent effects of capsazepine may have contributed to the observed response. We have also included this aspect as a limitation of the study and indicated that future investigations using genetic approaches or more selective pharmacological tools will be required to establish the specific contribution of TRPV1 to coronary vascular regulation under hypertensive conditions.

 

Comment Q3.

 

Sections 2.3 & 2.4: Regarding the experimental design, CS and CZ interventions were administered for only the final 4 days of a 40-day study. Remarkably, this brief 4-day treatment significantly reversed organic cardiac lesions in hypertensive rats. Perhaps the authors should provide an explanation for this phenomenon.

 

Response:

 

We thank the reviewer for this valuable observation. We agree that the relatively short duration of capsaicin (CS) and capsazepine (CZ) treatment may appear insufficient to completely reverse structural cardiac lesions developed during the 40-day hypertensive period. However, we would like to clarify that our findings should not be interpreted as complete reversal of established cardiac remodeling. Rather, the observed histological and functional improvements likely reflect attenuation of ongoing myocardial injury and preservation of tissue viability during the late stage of hypertension. Importantly, several of the endpoints evaluated in this study, including TTC-negative myocardial areas, mitochondrial dysfunction, oxidative stress, and apoptotic signaling, reflect ongoing cellular processes rather than fixed structural lesions and may therefore respond relatively rapidly to pharmacological interventions. Previous evidence indicates that TRPV1 activation improves nitric oxide bioavailability, coronary perfusion, mitochondrial function, and redox balance, thereby reducing cellular injury and apoptosis. Consequently, even a short treatment period may be sufficient to produce measurable improvements in myocardial function and tissue integrity without necessarily reversing all pre-existing structural alterations induced by chronic hypertension. To avoid overinterpretation, we have revised the Discussion section to clarify that the beneficial effects observed after CS treatment should be interpreted as attenuation of active myocardial damage and preservation of myocardial structure rather than complete regression of hypertension-induced cardiac remodeling. We have also acknowledged that longer treatment protocols will be required to determine the extent to which sustained TRPV1 activation may promote structural recovery.

 

Comment Q4

 

Table 1: Please use the standard three-line table format.

 

Response:

 

We thank the reviewer for this suggestion. Table 1 was reformatted according to the journal style requirements using the standard three-line table format.

 

Comment Q5

 

Figure 5: The figure caption states that the data underwent statistical analysis, yet no statistical symbols ( or ) are visible in the graph itself.

 

Response:

 

We appreciate the reviewer’s observation. Statistical significance symbols were inadvertently omitted from the figure during manuscript preparation. Figure 5 was corrected in the revised manuscript, and the corresponding statistical annotations were properly included.

 

Comment Q6

 

The authors state in the manuscript that TRPV1 regulates mitochondrial Ca2+ flux to maintain mitochondrial homeostasis. However, no data related to mitochondrial Ca2+ is included in the manuscript. It is suggested to supplement relevant experiments.

 

Response:

 

We thank the reviewer for this important observation. We agree that the present study does not include direct measurements of mitochondrial Ca²⁺ flux and, therefore, does not provide experimental evidence demonstrating that TRPV1 regulates mitochondrial Ca²⁺ handling in our hypertensive model. Our discussion of mitochondrial Ca²⁺ regulation was based on previous studies reporting a functional relationship between TRPV1 activation and mitochondrial Ca²⁺ homeostasis in cardiac and non-cardiac cells. However, we acknowledge that our data do not directly address this mechanism. Therefore, we have revised the manuscript to avoid implying a direct causal demonstration and now present mitochondrial Ca²⁺ regulation as a potential mechanism that may contribute to the observed cardioprotective effects of TRPV1 activation. We have also included this limitation in the Discussion and Conclusions sections, emphasizing that future studies should directly evaluate mitochondrial Ca²⁺ flux using appropriate experimental approaches to determine whether the mitochondrial protection observed in the present study is mediated through modulation of calcium homeostasis.

 

Comment Q7

 

Conclusion: The current findings indicate that TRPV1 may not be the sole or primary protective pathway. It is suggested that the authors revise the conclusion or supplement relevant experiments to provide robust data support.

 

Response:

 

We thank the reviewer for this thoughtful comment. We agree that the present findings do not demonstrate that TRPV1 is the sole mechanism responsible for the cardioprotective effects observed in this study. Although capsaicin treatment produced significant improvements in cardiac function, mitochondrial parameters, and myocardial structure, some protective effects were also observed in the capsazepine-treated groups, suggesting that additional TRPV1-independent mechanisms may contribute to the overall response. Therefore, we have revised the Conclusions section to avoid overstating the role of TRPV1 and to more accurately reflect the scope of our findings. The revised text emphasizes that TRPV1 activation was associated with cardioprotective effects in our experimental model and may represent an important contributor to mitochondrial preservation under hypertensive conditions; however, other pathways cannot be excluded based on the current data. We have also strengthened the Discussion by acknowledging the potential contribution of TRPV1-independent mechanisms and by identifying the need for future studies employing direct measurements of mitochondrial Ca²⁺ handling, genetic approaches, and more selective pharmacological tools to establish the specific contribution of TRPV1 to myocardial protection.

 

Comment Q8

 

Typographical Errors: Line 195: "(figure %B)", Line 204: please clarify "(C vs CS?)".

 

Response:

 

We thank the reviewer for carefully identifying these typographical errors. The indicated errors were corrected throughout the revised manuscript, and the text and figure annotations were clarified accordingly.

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

1. Title and scope: The topic is interesting and relevant; however, the title is long and should be made more concise. Additionally, the manuscript needs to explain the main theme of the research: is it focusing on TRPV1 activation, pharmacology of capsaicin, or mitochondrial protection in hypertension?
2. Mechanistic interpretation is not fully supported: The authors propose that TRPV1 activation has protective effects on the myocardium through mitochondrial regulation, however direct evidence for mitochondrial Ca2+ flux or receptor-specific mitochondrial signaling is not provided. Conclusions should be toned down or backed up by further experiments.
3. The effects of capsazepine on TRPV1-specific conclusions: Capsazepine was unexpectedly found to enhance some parameters such as coronary vascular resistance and infarct size. It makes it hard to say how much the protective effects are due to TRPV1 activation. It is a limitation that should be discussed with more critical consideration by the authors and not overinterpreted as solely TRPV1-mediated actions of capsaicin.
4. Experimental Controls are insufficient: Including a normotensive capsaicin-only and capsazepine-only group would enhance the interpretation of drug-specific effects. The lack of these controls makes it difficult to differentiate the effect of hypertension from the isolated pharmacological effects of capsaicin and capsazepine.
There is a small sample size for some endpoints: Several of the key analyses (such as infarct size and mitochondrial respiration) seem to have small group sizes. The authors should explain the choice of sample size, and indicate if there was a power analysis performed, and that they recognize the limitations of statistical robustness.
6. Statistical reporting should be improved: In the manuscript, exact p-values should be reported (when possible) and all statistical comparisons should be clearly defined in each figure legend. Some figures do use different statistical tests and the reasons for using Student's t-test rather than ANOVA should be given.
The presentation of figures and tables needs revision: Figures are informative but would benefit from clearer labels, consistent abbreviations and better resolution. The units/statistical annotations in the Table 1 are not as clear as the caption is repeated.
8. There is a need for better quantification of the histological analysis: The histological findings are mostly descriptive. The authors should offer detailed quantitative examination of fibrosis, necrosis, inflammatory infiltration and collagen deposition – preferably blind-scored or by standard image analysis.
The conversation is long and repetitive: There is some repetition of points about oxidative stress, mitochondrial dysfunction and apoptosis. It needs to be shortened and restructured to highlight the key results, gaps in understanding, and weaknesses of the study.
Language and formatting need editing: There are typographical and grammatical errors, such as inconsistent spacing, punctuation and terminology. These include: “Hypetensive,” “evaluated TRPV1-mediated effects,” “marked of myocardial injury,” and varying usage of CS/CZ abbreviations.
11. Translational relevance is over-exaggerated: Findings are encouraging, but the study is conducted in a short-term model of pharmacologic intervention in an experimental rat model. Statements suggesting a therapeutic relevance in humans should be more cautious.
12. The conclusion should be balanced: Although the cardioprotective effects of capsaicin described in this model are suggestive of TRPV1 involvement, however, it remains unclear if it is the direct effect of capsaicin or the effect of capsazepine that is responsible for the protection.

Comments on the Quality of English Language

requied

Author Response

Reviewer 5

 

Comments and Suggestions for Authors

 

Response to Reviewer 5

 

We sincerely thank the reviewer for the careful evaluation of our manuscript and for the constructive comments and suggestions. We have carefully considered all observations and substantially revised the manuscript accordingly. Several concerns raised by the reviewer overlapped with comments from other reviewers and were addressed through extensive modifications to the title, Introduction, Discussion, Conclusions, figures, tables, statistical reporting, and acknowledgment of study limitations. Our detailed responses are provided below.

 

Comment 1.

 

Title and scope. The topic is interesting and relevant; however, the title is long and should be made more concise. Additionally, the manuscript needs to explain the main theme of the research: is it focusing on TRPV1 activation, pharmacology of capsaicin, or mitochondrial protection in hypertension?

 

Response:

 

We appreciate this observation and agree that the original title could be improved. Accordingly, the title has been revised to better reflect the primary objective of the study while improving conciseness. We have also clarified throughout the Introduction and Discussion that the main focus of the work is to investigate the contribution of TRPV1 activation to myocardial protection in hypertension and its association with preservation of mitochondrial function. Capsaicin and capsazepine were employed as pharmacological tools to modulate TRPV1 activity rather than as the primary focus of the study.

 

Comment 2.

 

Mechanistic interpretation is not fully supported: The authors propose that TRPV1 activation has protective effects on the myocardium through mitochondrial regulation, however direct evidence for mitochondrial Ca2+ flux or receptor-specific mitochondrial signaling is not provided. Conclusions should be toned down or backed up by further experiments.

 

Response:

 

We agree that direct measurements of mitochondrial Ca²⁺ flux and receptor-specific mitochondrial signaling were not performed in the present study. Accordingly, we have revised the manuscript to avoid overinterpretation of the mechanistic findings. The Discussion and Conclusions sections now explicitly acknowledge that the proposed involvement of mitochondrial calcium homeostasis is based on previous literature and remains speculative in the context of the present study. We have also incorporated this limitation and emphasized the need for future studies directly assessing mitochondrial Ca²⁺ handling and TRPV1-dependent signaling pathways. While direct measurements of mitochondrial Ca²⁺ flux was not performed, the observed changes in cytochrome c release and BAX translocation are consistent with improved mitochondrial integrity and reduced activation of apoptotic pathways. Nevertheless, these findings do not constitute direct evidence of mitochondrial calcium regulation and have been interpreted accordingly in the revised manuscript.

 

Comment 3.

 

The effects of capsazepine on TRPV1-specific conclusions: Capsazepine was unexpectedly found to enhance some parameters such as coronary vascular resistance and infarct size. It makes it hard to say how much the protective effects are due to TRPV1 activation. It is a limitation that should be discussed with more critical consideration by the authors and not overinterpreted as solely TRPV1-mediated actions of capsaicin.

 

Response:

 

We appreciate this important observation. We agree that some protective effects observed in the capsazepine-treated groups complicate the interpretation of an exclusively TRPV1-mediated mechanism. Therefore, we have revised the Discussion and Conclusions sections to acknowledge that TRPV1-independent effects may contribute to the observed responses. In particular, we discuss the possibility that capsazepine exerts antioxidant and anti-inflammatory actions independent of TRPV1 antagonism. The conclusions have been moderated accordingly to avoid attributing all protective effects exclusively to TRPV1 activation.

 

Comment 4.

 

Experimental Controls are insufficient: Including a normotensive capsaicin-only and capsazepine-only group would enhance the interpretation of drug-specific effects. The lack of these controls makes it difficult to differentiate the effect of hypertension from the isolated pharmacological effects of capsaicin and capsazepine.

 

Response:

 

We acknowledge that inclusion of normotensive groups treated with capsaicin or capsazepine would provide additional information regarding the isolated pharmacological effects of these compounds. However, the primary objective of this study was to evaluate whether modulation of TRPV1 activity could influence myocardial injury and mitochondrial function under hypertensive conditions. For this reason, the experimental design focused on comparisons between normotensive controls and hypertensive animals receiving TRPV1 modulation. We recognize the absence of normotensive drug-treated groups as a limitation and have included this consideration in the revised manuscript.

 

Comment 5.

 

There is a small sample size for some endpoints: Several of the key analyses (such as infarct size and mitochondrial respiration) seem to have small group sizes. The authors should explain the choice of sample size, and indicate if there was a power analysis performed, and that they recognize the limitations of statistical robustness.

 

Response:

 

We appreciate this observation. Sample sizes were selected based on our previous experience using this experimental model and are consistent with those commonly reported in cardiovascular and mitochondrial physiology studies. We acknowledge that some endpoints were obtained from relatively small groups, which may limit statistical power for detecting subtle differences. This limitation has been acknowledged in the revised manuscript. Despite this consideration, statistically significant differences were detected for several key functional, histological, and mitochondrial parameters, supporting the robustness of the principal findings. Nevertheless, we recognize that larger sample sizes would further strengthen the statistical power of future studies.

 

Comment 6.

 

Statistical reporting should be improved: In the manuscript, exact p-values should be reported (when possible) and all statistical comparisons should be clearly defined in each figure legend. Some figures do use different statistical tests and the reasons for using Student's t-test rather than ANOVA should be given.

 

Response:

 

We thank the reviewer for this recommendation. The statistical analysis section, figure legends, and tables have been revised to improve clarity regarding the statistical tests employed and the comparisons performed. In addition, statistical analyses have been reviewed and corrected where necessary. Appropriate explanations for the statistical tests used have been incorporated, and figure legends were revised to clearly indicate the comparisons performed.

 

Comment 7.

 

The presentation of figures and tables needs revision: Figures are informative but would benefit from clearer labels, consistent abbreviations and better resolution. The units/statistical annotations in the Table 1 are not as clear as the caption is repeated.

 

Response:

 

We agree with the reviewer and have revised the figures and tables to improve clarity, consistency of abbreviations, labeling, statistical annotations, and overall presentation. The table legends and figure captions were also reviewed and corrected where necessary, and image quality was improved throughout the manuscript.

 

Comment 8.

 

There is a need for better quantification of the histological analysis: The histological findings are mostly descriptive. The authors should offer detailed quantitative examination of fibrosis, necrosis, inflammatory infiltration and collagen deposition – preferably blind-scored or by standard image analysis.

 

Response:

 

We appreciate this comment. We agree that quantitative histological assessment would strengthen the study. However, the present investigation primarily focused on functional, biochemical, and mitochondrial endpoints, with histological evaluation serving as complementary evidence of myocardial injury. We acknowledge that more extensive quantitative histological analyses, including fibrosis assessment, collagen quantification, and inflammatory scoring, would provide additional mechanistic insight and represent an important direction for future studies. This limitation has been acknowledged in the revised Discussion section.

 

Comment 9.

 

The conversation is long and repetitive: There is some repetition of points about oxidative stress, mitochondrial dysfunction and apoptosis. It needs to be shortened and restructured to highlight the key results, gaps in understanding, and weaknesses of the study.

 

Response:

 

We thank the reviewer for this suggestion. The Discussion section has been carefully revised and condensed to reduce redundancy and improve readability. Repetitive descriptions regarding oxidative stress, mitochondrial dysfunction, and apoptosis have been minimized, while greater emphasis has been placed on integrating the principal findings, study limitations, unresolved mechanistic questions, and future research directions.

 

Comment 10.

 

Language and formatting need editing: There are typographical and grammatical errors, such as inconsistent spacing, punctuation and terminology. These include: “Hypetensive,” “evaluated TRPV1-mediated effects,” “marked of myocardial injury,” and varying usage of CS/CZ abbreviations.

 

Response:

 

We appreciate this observation. The manuscript has undergone thorough revision to correct typographical errors, grammatical inconsistencies, terminology usage, abbreviations, punctuation, and formatting throughout the text. Special attention was given to consistency of abbreviations, scientific terminology, and overall readability.

 

Comment 11.

 

Translational relevance is over-exaggerated: Findings are encouraging, but the study is conducted in a short-term model of pharmacologic intervention in an experimental rat model. Statements suggesting a therapeutic relevance in humans should be more cautious.

 

Response:

 

We agree with the reviewer that caution is warranted when extrapolating findings from an experimental animal model to human disease. Accordingly, statements regarding potential therapeutic applications have been revised and moderated throughout the Discussion and Conclusions. The manuscript now emphasizes that the findings provide experimental evidence supporting a potential role of TRPV1 signaling in myocardial protection during hypertension, while recognizing that further mechanistic, preclinical, and translational studies are required before clinical implications can be established.

 

Comment 12.

 

The conclusion should be balanced: Although the cardioprotective effects of capsaicin described in this model are suggestive of TRPV1 involvement, however, it remains unclear if it is the direct effect of capsaicin or the effect of capsazepine that is responsible for the protection.

 

Response:

 

We appreciate this important comment. The Conclusions section has been revised to avoid overstating the role of TRPV1. The revised text now indicates that TRPV1 activation was associated with cardioprotective effects and preservation of mitochondrial function in our experimental model; however, the current findings do not allow definitive exclusion of additional TRPV1-independent mechanisms. This limitation is now explicitly acknowledged in both the Discussion and Conclusions sections and is highlighted as an important area for future investigation. Once again, we sincerely thank the reviewer for the constructive comments, which have substantially improved the quality, rigor, clarity, and scientific balance of the manuscript.

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have satisfactorily addressed all the comments raised in the previous review. The revised manuscript is substantially improved and provides valuable information regarding the role of TRPV1 in myocardial protection and mitochondrial function under hypertensive conditions. I have no additional suggestions and consider the manuscript suitable for publication in its current form.

Author Response

Reviewer1

 

Comments and Suggestions for Authors

 

The authors have satisfactorily addressed all the comments raised in the previous review. The revised manuscript is substantially improved and provides valuable information regarding the role of TRPV1 in myocardial protection and mitochondrial function under hypertensive conditions. I have no additional suggestions and consider the manuscript suitable for publication in its current form.

 

Response:

 

We thank the reviewer for the positive evaluation of our revised manuscript. We appreciate the time and effort dedicated to reviewing our work and are pleased that the changes made have satisfactorily addressed the previous concerns.

 

No further changes were required.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Thank you for providing a flow chart about the experimental design. A timeline scale figure would be much more appropriate for this protocol, allowing the authors to plot the various treatments (i.e., L-NAME model development and CS/CZ or CS+CZ) along the timeline. The current figure does not adequately illustrate the chronological progression of the treatments relative to one another

Thank you for citing PMID 18037908 paper in the discussion section. Although the authors stated in their response that they had cited the other article (PMID: 32586044), which is published in a D1-ranked widely acknowledged journal, and which I suggested for inclusion in the discussion section, it is not actually included in the revised manuscript. Instead, another publication is included (see ref. No.33), even though the content of the paper I suggested focuses more on the link between TRPV1 and cardiovascular diseases than the article actually cited, which, moreover, is not even available in the PubMed database, and the journal in which it published is not registered in either Scimago or the Journal Citation Report system.

It is not clear what the O.D. label stands for in the labels of the Y axis in the bar charts of Figures 5 and 6. The name of the parameter should be given as the mean label (e.g. Catalase activity) and in the brackets, the authors should include the dimensions of the measured parameter (e.g. arbitrary units or A.U. or % etc.) as it is in Figure 1-4. Please, do not use abbreviations without providing their explanation.

The “Conclusions” section is excessively long and redundant. It should be limited to the final paragraph of the actual conclusions. The remaining sections should be moved to the beginning of the “Discussion” section, and a section entitled “Limitations” should be created in place of lines 544–554.

The quality and the resolution of the figures must be significantly improved in the final/published version

Comments on the Quality of English Language

In the revised manuscript, English editing has significantly been improved.

Author Response

Reviewer 2

 

Comments and Suggestions for Authors

 

Comment 1:

 

Thank you for providing a flow chart about the experimental design. A timeline scale figure would be much more appropriate for this protocol, allowing the authors to plot the various treatments (i.e., L-NAME model development and CS/CZ or CS+CZ) along the timeline. The current figure does not adequately illustrate the chronological progression of the treatments relative to one another.

 

Response:

 

We thank the reviewer for this valuable suggestion. We agree that a timeline representation better illustrates the chronological sequence of the experimental protocol. Therefore, Figure 7 has been redesigned as a timeline showing the development of hypertension induced by L-NAME and the administration periods of capsaicin, capsazepine, and combined treatment.

 

Comment 2:

 

Thank you for citing PMID 18037908 paper in the discussion section. Although the authors stated in their response that they had cited the other article (PMID: 32586044), which I suggested for inclusion in the discussion section, it is not actually included in the revised manuscript. Instead, another publication is included (see ref. No.33), even though the content of the paper I suggested focuses more on the link between TRPV1 and cardiovascular diseases than the article actually cited, which, moreover, is not even available in the PubMed database, and the journal in which it published is not registered in either Scimago or the Journal Citation Report system.

 

Response:

 

We thank the reviewer for noting this omission. We apologize for the oversight. The suggested article (PMID: 32586044) has now been included in the Discussion section and added to the reference list, as it provides relevant information regarding the role of TRPV1 in cardiovascular diseases and strengthens the context of our findings.

 

Comment 3:

 

It is not clear what the O.D. label stands for in the labels of the Y axis in the bar charts of Figures 5 and 6. The name of the parameter should be given as the mean label (e.g. Catalase activity) and in the brackets, the authors should include the dimensions of the measured parameter (e.g. arbitrary units or A.U. or % etc.) as it is in Figure 1-4. Please, do not use abbreviations without providing their explanation.

 

Response:

 

We thank the reviewer for this observation. The figure labels have been revised to replace the abbreviation "O.D." with more descriptive parameter names, and the corresponding units (arbitrary units, A.U.) have been included in the Y-axis labels. This modification improves figure clarity and avoids the use of unexplained abbreviations.

 

Comment 4:

 

The “Conclusions” section is excessively long and redundant. It should be limited to the final paragraph of the actual conclusions. The remaining sections should be moved to the beginning of the “Discussion” section, and a section entitled “Limitations” should be created in place of lines 544–554.

 

Response:

 

We thank the reviewer for this constructive suggestion. The Conclusions section has been shortened and focused on the main findings of the study. The explanatory and interpretative paragraphs previously included in the Conclusions have been moved to the Discussion section. In addition, a separate subsection entitled “Limitations” has been incorporated to clearly present the limitations of the study.

 

Comment 5:

 

The quality and the resolution of the figures must be significantly improved in the final/published version.

 

Response:

 

We agree with the reviewer. All figures have been regenerated and will be provided in high-resolution format according to the language quality and readability.

Author Response File: Author Response.pdf

Reviewer 4 Report

Comments and Suggestions for Authors

The authors have addressed all my comments.

Author Response

Reviewer 4

 

Comments and Suggestions for Authors

 

The authors have addressed all my comments.

 

Response:

 

We thank the reviewer for the positive evaluation of our revised manuscript. We appreciate the time and effort devoted to reviewing our work and are pleased that all concerns have been satisfactorily addressed.

 

No further changes were required.

 

Author Response File: Author Response.pdf

Reviewer 5 Report

Comments and Suggestions for Authors

acceptable 

Author Response

Reviewer 5

 

Comments and Suggestions for Authors

 

Acceptable

 

Response:

 

We thank the reviewer for the positive evaluation of our manuscript and for the time devoted to the review process. We appreciate the reviewer’s assessment and support for publication.

 

No further changes were required.

Author Response File: Author Response.pdf