α-Melanocyte-stimulating hormone alleviates pathological cardiac remodeling via melanocortin 5 receptor

α-Melanocyte-stimulating hormone (α-MSH) regulates diverse physiological functions by activating melanocortin receptors (MC-R). However, the role of α-MSH and its possible target receptors in the heart remain completely unknown. Here we investigate whether α-MSH could be involved in pathological cardiac remodeling. We found that α-MSH was highly expressed in the mouse heart with reduced ventricular levels after transverse aortic constriction (TAC). Administration of a stable α-MSH analog protected mice against TAC-induced cardiac hypertrophy and systolic dysfunction. In vitro experiments revealed that MC5-R in cardiomyocytes mediates the anti-hypertrophic signaling of α-MSH. Silencing of MC5-R in cardiomyocytes induced hypertrophy and fibrosis markers in vitro and aggravated TAC-induced cardiac hypertrophy and fibrosis in vivo. Conversely, pharmacological activation of MC5-R improved systolic function and reduced cardiac fibrosis in TAC-operated mice. In conclusion, α-MSH is expressed in the heart and protects against pathological cardiac remodeling by activating MC5-R in cardiomyocytes. These results suggest that analogs of naturally occurring α-MSH, that have been recently approved for clinical use and have agonistic activity at MC5-R, may be of benefit in treating heart failure.


Response:
We thank the reviewer for these comments and acknowledge that further mechanistic experiments to investigate the dependency of the observed mouse phenotypes on the JNK pathway would be valuable.Unfortunately, we have discontinued the maintenance of Mc5r-cKO mouse line due to breeding problems (-> only cryopreserved sperm available) and performing new experiments with this mouse model would not be possible within a reasonable time frame.
Instead, we performed a wider screen for phosphoproteins in PG-901-treated H9c2 cells.We selected targets that are known to be modulated by melanocortin 5 receptor signaling in other cells/models (Xu Y et al., Cell Mol Life Sci. 2020 Oct;77(19):3831-3840), and quantified these by Western blotting.As shown below (Figure R1), PG-901 did not affect the phosphorylation status of CREB (B), ERK (C), p38 (D), Akt (E) or AMPK (F).We also did the same screening for samples from Mc5r-silenced H9c2 cells (Extended Figure 4A in the revision).The only significant change was observed in JNK phosphorylation, which was increased in Mc5r-silenced cells (Extended Figure 4B).We also performed a new experiment with primary cardiomyocytes (NMCMs) to investigate whether the induction of Nppb expression is similarly dependent on JNK signaling as observed in H9c2 cells (Figure 4M).Indeed, JNK inhibition with SP600125 completely reversed the upregulation of Nppb in Mc5r-silenced NMCMs (Extended Figure 4C), further consolidating the link between MC5-R signaling, JNK modulation and cardiomyocyte hypertrophy.
However, based on the existing and conflicting literature on the role of JNK in cardiac hypertrophy (reviewed in Physiol Rev. 2010 Oct;90(4):10.1152/physrev.00054.2009), it would be highly challenging to prove the contribution of the JNK pathway to the observed phenotype in vivo.Earlier findings indicate that JNK activation is a dynamic and transient signaling event (peaking at ~30 min) after applying pressure overload and that different JNK isoforms might have separate and non-redundant roles in the process.Although in vitro studies strongly argue for a prohypertrophic role of JNKs, loss-of-function approaches to silence JNK or its upstream regulators in vivo have resulted in promotion as well as attenuation of cardiac growth response (as discussed on manuscript page 15, lines 531-539).Furthermore, JNK activation in vivo (in transgenic animal models) does not induce cardiac hypertrophy.
Therefore, we have been careful not to claim that the observed in vivo phenotypes would be dependent on the JNK pathway, and disclose the uncertainty related to the involvement of JNK signaling in the observed in vivo phenotypes (manuscript page 15, lines 535-539): "In vitro experiments in the current study demonstrate that MC5-R regulates hypertrophic growth of cardiomyocytes in a JNK-dependent manner.However, further studies are warranted to determine whether MC5-R-induced JNK reduction has a direct effect on cardiomyocyte growth in vivo".
Regarding the mechanistic insight of the manuscript, we think that the most important finding is the demonstration that α-MSH evokes an anti-hypertrophic effect by activating MC5-R in cardiomyocytes.From a drug development perspective, this is the key mechanistic finding revealing that cardiomyocytes are the main effector cells (among other cell types of the heart) and that MC5-R is the target receptor (among MC-R subtypes) for the anti-hypertrophic and -fibrotic effect of α-MSH.
2) Figure 1A: instead of hypothalamus, the levels in the pituitary should be shown, where α-MSH is mainly produced.
Response: As requested by the reviewer, we measured α-MSH concentration in the pituitary gland and present this new data in Figure 1A (instead of hypothalamic α-MSH level).

3) Figure 1H: Fibrosis should be measured in hearts by histology.
Response: We fully agree with the reviewer and quantified the level of LV fibrosis in α-MSH-treated mice.Consistent with the phenotype observed in Mc5r-cKO mice and PG-901-treated mice, TAC-induced fibrosis was significantly attenuated by chronic α-MSH treatment (Figure 1H & 1N).

4) How is fibrosis regulated by α-MSH?
Could there be a cross talk of myocytes to fibroblasts?More mechanistic insight is clearly needed.
Response: We thank the reviewer for this comment.We found that α-MSH downregulated fibrotic markers such as TGFb and CTGF in cultured cardiomyocytes, which are known to promote cardiac fibrosis though humoral and growth factormediated pathways.
We are aware that in the context of cardiac hypertrophy, cardiac myocyte-to-cardiac fibroblast (CM-to-CF) and CF-to-CM crosstalk significantly contribute to pathological cardiac remodeling.Mice with CM-specific gene manipulation might have a significant effect on cardiac fibrosis without any clear signs of cardiac hypertrophy.For instance, mice with CM-specific overexpression of CTGF show enhanced cardiac fibrosis after TAC-induced pressure overload but are not sensitized to the development of cardiac hypertrophy (Yoon PO et al., J Mol Cell Cardiol. 2010 Aug;49(2):294-303).This might have also relevance to our findings of downregulated CTGF expression in PG-901 treated cells and in TAC-operated mice as well as upregulated CTGF expression in Mc5r-silenced cardiomyocytes.
Against this background, it is very likely that α-MSH regulates fibrosis, at least in part, through CM-to-CF cross talk.However, since we identified cardiac myocytes as the main effector cells for α-MSH-mediated anti-hypertrophic and -fibrotic regulation, we feel that it is beyond the scope of this manuscript to investigate cross talk between CM and CF in this regard.Particularly, taking into account that the mechanisms of CM-to-CF cross talk are not fully understood and involve e.g. release of paracrine factors, direct cell-cell interactions and cell interaction with the extracellular matrix (Zhang P & U Mende JS, Am J Physiol Heart Circ Physiol. 2012 Dec 15;303(12):H1385-96), it would be challenging and time-consuming to start addressing these possibilities.

5) P-JNK Western blots need to be analyzed in the myocardium in the mouse models shown (Figures 1, 6, 7, 8).
Response: As requested by the reviewer, we analyzed cardiac p-JNK levels in our mouse models.Second, we analyzed the LV samples from PG-901-treated C57Bl/6N mice for p-JNK, p-ERK and p-p38 levels (Figure R3).PG-901-treatmeant did not significantly change the expression of these phosphoproteins in TAC-operated C57Bl/6N mice.There was however a tendence for lower p-JNK level (P=0.09by 1-way ANOVA and Dunnett's post hoc test) in TAC mice treated with 0.5 mg/kg of PG-901.Overall, it is difficult to draw any valid conclusion from these results.First, the samples from these experiments were already relatively old, which caused challenges in detecting clear bands for the phosphorylated forms of these proteins.Second, the signal for e.g.p-JNK in these Western blots is derived from both cardiomyocytes and non-cardiomyocytes such as fibroblasts, endothelial cells and leukocytes, thus possibly diluting the effect that might be occurring in cardiomyocytes.
These results imply that PG-901 or MC5-R deficiency does not affect the expression p-JNK at the investigated time points after TAC operation, but taking into account the uncertainties related to these analyses, we would rather omit these data from the manuscript.Since the expression levels of the other phosphoproteins were also unaffected, the results do not help in delineating the signaling mechanisms behind the observed in vivo phenotypes.

6) Figure 4: H9c2 cells are not a good model for cardiomyocyte hypertrophy at all.
Response: We agree with the reviewer and have therefore used neonatal mouse ventricular cardiac myocytes (NMCM) in parallel with H9c2 cells.To comply with the 3R principles, we first aimed to identify α-MSH-mediated responses in H9c2 cells and then validated the key findings in NMCMs.Since the main signaling responses appeared similarly in H9c2 cells and NMCMs, we relied more on the H9c2 model when performing subsequent mechanistic experiments to reduce the number of mice.
Based on the reviewer's comment, we have performed new experiments with NMCMs to further demonstrate that the MC5-R mediated effects are appearing similarly in both in vitro models (Extended Figure 3 & Figure R5).

7) Figure 5: α-MSH should be measured in failing human hearts. The effects of PG-901 on cardiac gene-expression are extremely small. Cell size should be measured.
Response: Measurement of α-MSH level in failing human hearts would be indeed interesting and relevant.Based on the increased POMC expression in the human cardiomyopathy patient samples, it is expected that α-MSH expression is similarly increased.This hypothesis is also supported by the clinical finding of increased α-MSH concentration in the plasma of cardiomyopathy patients (Yamaoka-Tojo M et al, Intern Med. 2006;45:429-434).However, due to the lack of respective protein samples from the LV, this hypothesis cannot be tested.
We agree that the effects of PG-901 on gene expression in hiPSC-CMs are extremely small and therefore measurement of cell size could provide further evidence on the role of MC5-R in modulating hypertrophic responses in these cells.However, based on our previous experiments, cell size, as measured by cross-sectional area, does not significantly change in response to ET-1 treatment, although Nppa and Nppb are robustly upregulated.There might be an increase in cell volume in ET1-treated hiPSC-CMs (Johansson M et al., Biol Open. 2020 Sep 21;9(9):bio052381), but we do not have a validated protocol for this purpose.Therefore, instead of cell size or volume, we quantified NT-proBNP protein expression, which could be used a molecular marker of cell hypertrophy (Pohjolainen L et al., Front Pharmacol. 2021 Jan 20:11:553852) and is considered as the gold standard biomarker in determining the diagnosis and prognosis of human heart failure.We observed that PG-901 treatment attenuated the hypertrophic effect of ET-1 in hiPSC-CMs (Figure 5H, manuscript page 11, lines 355-357).

8) Figure 8: Why is there no anti-hypertrophic effect of PG-901 in vivo?
Response: The lack of effect of PG-901 on hypertrophic response in TAC-operated C57Bl/6N might relate to the controversial role of JNK signaling in pathological cardiac hypertrophy in vivo (as discussed above; response to comment 1 and on manuscript page 15, lines 531-535).Therefore, the impact of JNK inhibition on the hypertrophic response could be sensitive to nuances in the experimental settings (e.g.C57Bl/6J vs C57Bl/6N, time point and timing of dosing).
However, we consider that the most likely explanation is that PG-901 evokes offtarget effect(s) in cardiomyocytes and/or non-myocytes that are counteracting/masking the possible anti-hypertrophic effect that is mediated via MC5-R.This issue is more thoroughly discussed in response to comment 11 by Referee #3 (page 17 of this response letter) and on manuscript page 14/lines 507-514.

Referee #2
In As suggested by the reviewer, we also measured the level of α-MSH in the heart/LV and plasma of Ang II-infused mice.Consistent with the earlier findings of unchanged cardiac MC5-R expression, Ang II-infusion for 4 weeks did not affect circulating or cardiac α-MSH levels (Extended Figure 1H and I).Therefore, it appears that the α-MSH/MC5-R axis responds in a cohesive way to different hypertrophic stimuli, i.e. reduced α-MSH and MC5-R expression in TAC-operated mice, while no effect is observed in Ang II-infused mice.
2. Across the entire study, most if not all differences are small, yet significant.While I do not doubt the significance of the difference and the implication of the work, it is striking to me regarding the small differences.Does this mean that a-MSH signaling is not major?I would recommend some explanation/discussion from the authors.Even better, the authors could compare this phenotype with findings from other similar hormones.

Response:
The reviewer raises an important point, which should be discussed in the manuscript.It might well be that MC5-R signaling does not play a major role in cardiac remodeling.However, although we have not done head-to-head comparison in our experiments, it appears that the effect of α-MSH is stronger compared to the MC5-R agonist in both in vitro and in vivo models.This raises a possibility that PG-901 is a partial or biased agonist of MC5-R and therefore, does not fully recapitulate the actions of α-MSH.PG-901 might also evoke off-target effects that mask the primary effect occurring through MC5-R activation (more detailed discussion below/response to comment 3).
It is also well-established that melanocortin receptors, like other G protein-coupled receptors, undergo agonist-mediated desensitization and internalization, which becomes particularly dominant after repeated, long-term treatment with an MC-R agonist.In any case, we want to emphasize that PG-901 was only used as pharmacological tool to further dissect the role of MC5-R in cardiac remodeling.
Further experiments are clearly warranted to design new MC5-R agonists and test their effectiveness in similar disease models.This would better allow addressing the question whether MC5-R signaling is major in cardiac remodeling or not, and whether targeting MC5-R holds promise as a therapeutic strategy in managing heart failure.

Figure 8, PG-091 did not affect cardiac hypertrophy but improve cardiac function under pressure overload. This finding is not consistent with the in vitro results in figure 4E&H. Any explanations?
Response: We have to acknowledge that there is clearly something with PG-901 that we have not figured out.For example, in hiPSC-CMs, PG-901 evokes a significant and dose-dependent effect on NPPB expression, but the direction of effect varies from experiment to experiment (Figure R4).Basal MC5R expression, which varies between different batches of hiPSC-CM differentiation, might explain this confusing finding.PG-901 downregulated NPPB in cells that have a higher MC5R expression (Exp 1 and Exp 3), while an opposite effect was observed in cells that have a lower MC5R expression (Exp 2 and Exp 4).

Figure R4. Quantitative PCR analysis of NPPB mRNA expression in hiPSC-CMs treated with different concentrations of PG-901 for 24 hours in the absence (A) or presence (B) of ET-1 (100 nM). (C) Relative MC5R mRNA expression in unstimulated control cells in different experiments.
This could indicate that there is an off-target effect that becomes particularly dominant, when the signaling through MC5-R is weaker (i.e. in cells with lower MC5R expression).Therefore, in a model of advanced heart failure that was shown to be associated with reduced MC5-R dimerization (Figure 3L), this off-target effect might also become dominant and mask the potential anti-hypertrophic effect of MC5-R activation.This notion is further supported by the new in vivo experiment, which was performed to address the comment 3 of Referee #3.In sham-operated mice that   6G).This early time point (4 weeks after TAC) was selected to phenotype Mc5r-cKO mice, since advanced cardiac hypertrophy and heart failure was associated with reduced MC5-R expression in the heart (Figure 3L), which could limit the incremental effect of genetically-induced MC5-R deficiency on the hypertrophic response as discussed on manuscript page 15/lines 548-551.

Figure 8, the treatment of PG-901 was initiated since TAC. This is to test the prevention effect of a-MSH signaling. Could the authors try to test the rescue effect? For example, treat mice with PG-901 after the establishment of cardiac dysfunction (8 weeks after TAC).
Response: We thank the reviewer for this suggestion.It would be indeed valuable to test the rescue effect of PG-901 after the establishment of cardiac dysfunction.
However, PG-901 treatment was applied to TAC-operated C57Bl/6N mice that develop marked heart failure compared to C57Bl/6J (background strain for MYH6-MCM, Mc5r fl/fl and Mc5r-cKO mice) and according to our experience, a significant number of C57Bl/6N mice would not survive beyond the 8-week time point.Furthermore, based on our data, it appears that PG-901 is not an optimal compound for selective targeting of MC5-R, because it does not precisely mimic the actions of α-MSH (i.e.no anti-hypertrophic effect in vivo) and might even evoke off-target effects (as discussed in response to comment 3).We would therefore prefer not to perform new in vivo experiments with PG-901considering that the effects on EF and other functional parameters were only subtle at 5 weeks post-TAC.More detailed information on the pharmacokinetic and -dynamic characteristics of PG-901 would aid in designing future experiment with refined dosing regimens.Ideally, other selective MC5-R agonist could be tested and compared with PG-901 for their antihypertrophic and -fibrotic effects in TAC-operated mice.

This work presents a novel target involved in cardiac remodeling development. The authors show for the first time that α-MSH and the MC5R are expressed in mouse and human hearts. Most importantly, they report that MC5R is functionally active in ventricular myocytes. They also report that administration of a stable α-MSH analogue and an agonist for MC5R protects mice against TAC-induced cardiac remodeling. Furthermore, targeting specifically M5CR seems to improve cardiac function after TAC. The paper is interesting
and the novelty of the results is high, nonetheless, there are some major points that need to be addressed.

Response:
We thank the reviewer for the positive feedback as well as for the constructive comments.

Major concerns: 1. A major criticism is the organization of the results which makes difficult to the reader to understand what the authors want to show. The reviewer suggests to the authors to gather the in vitro parts.
Response: Thank you for pointing out the lack of clarity.Unfortunately, we weren't able to change the order of figures without strong disruptions to the logical flow of the manuscript, and dropping out significant amount of data.However, we have gone through each result section heading and updated the text where necessary to more carefully introduce the next topic.

Regarding the in vivo experiments, the number of animals is not the same in the different echocardiography parameters. For instance, LVPW (CTRL n= 11) and ejection fraction (CTRL n=9). If some animals are erased as outliers in any part of the work, they should be mentioned and considered for all the parameters. The number of animals used for each experiment and each condition should be detailed in the figure legends.
Response: We thank the reviewer for picking up this mistake.The graph for LVPW (Fig 1M) contained accidentally duplication of two data points.This has been corrected (->CTRL n=9) in the revised manuscript.This does not however affect the results/conclusion in any way as the difference between CTRL and TAC groups remains highly significant.We have also specified in more detail the number of animals used for each experiment in the figure legends.S2).Low-or high-dose treatment with PG-901 did not significantly affect systolic or diastolic performance of the heart as measured by echocardiography (Appendix Table S2).

All along the paper one of the most consistent results is the presence of fibrosis, therefore this should be measured for alpha-MSH treated mice (Figure 1H-N).
Response Response: We thank the reviewer for these remarks.We agree that the wording in that particular sentence was misleading and thus revised the text as follows: "..PG-901 was the only compound that showed similar responses to α-MSH".We have also included additional details on the pharmacological properties of PG-901 in the discussion (page 14/lines 507-514).The binding and antagonism of MC3-R and MC4-R might indeed explain some of the observed discrepancies related to PG-901.
The rationale for choosing the PG-901 compound was based on previous publications as this appears to be the most widely used MC5-R selective agonist and also the only compound that has been previously used in in vivo studies (Trotta MC et al, Front Physiol. 2018 Oct 26;9:1475. & Rossi S et al, Mediators Inflamm. 2016:2016:7368389).A few other MC5-R agonists have been developed and characterized but none of these are commercially available.3E-H).For instance, in the case of TGFB1 it is difficult to mention that MSH treated samples are different when treated with PG-20N.Authors should comment on this.

Upon treatment with PG-20N there seems to be a decrease in p-JNK and gene levels (Figures
Response: Admittedly, this is something that has also puzzled us, since we expected that PG-20N alone would increase p-JNK and Nppb expression if anything.
Nevertheless, the effect of PG-20N did not reach statistical significance in post hoc comparisons.Furthremore, in post hoc comparisons, there is a significant difference between Control/α-MSH and PG-20N/α-MSH treatments, further indicating that the effect of α-MSH is reversed by PG-20N.We have revised Figure 3E-H accordingly and displayed all the significant post hoc comparisons in the graphs.

Regarding inhibition of Gi with PTX, the results and description are both confusing. Authors state: " Further mechanistic experiments using H9c2 cells revealed that inhibition of Gi signaling with pertussis toxin (PTX) induced a further reduction in the amount of phosphorylated JNK and downregulation of Nppb and Acta2. Although PTX appeared to block the effect of PG-901 on JNK phosphorylation, it did not abrogate the gene expression changes evoked by MC5-R activation"
The comments are contradictory between the first and the second sentence.Following what was said before one would have expected an increase of the pJNK levels with PTX.The results show a reduction similar to that obtained with PG-901.Regarding gene expression, it seems there is a further decrease in some of them.It is rather difficult to draw a clear conclusion.
Response: We acknowledge the reviewer's concern and agree that the text in this regard is confusing.We have therefore revised the text as follows: "Further mechanistic experiments using H9c2 cells revealed that inhibition of Gi signaling with pertussis toxin (PTX) induced a further reduction in the amount of phosphorylated JNK and downregulation of Nppb and Acta2.Importantly, it did not abrogate the gene expression changes evoked by MC5-R activation As elegantly pointed out by the reviewer, if Gi signaling mediates the reduction in p-JNK, then PTX would increase (rather than decrease) JNK phosphorylation.It might well be that the sensitivity of that particular ELISA assay was not sufficient to detect a further reduction in p-JNK level of PTX and PG-901-treated samples.Therefore, we focus on gene expression changes and particularly on Nppb expression, which serves as a molecular marker of cardiomyocyte hypertrophy.PTX did not reverse the downregulation of Nppb in PG-901 treated cells (Appendix Figure S4), indicating that Gi signaling does not mediate this effect.We also confirmed this result in primary cardiomyocytes (NMCMs), which showed downregulation of Nppb in response to PTX treatment (Figure R5).Importantly, the effect of PG-901 on Nppb expression was retained in PTX-treated NMCMs (Figure R5).

Figure 4J. There seems to be no increase in leucine incorporation after angiotensin II treatment in this experiment. Is this increase significant?
Response: This change is non-significant by 2-way ANOVA.According to our experience, Opti-MEM medium, which is used in combination with Lipofectamine, blunts the hypertrophic response to angiotensin II and other similar agonists.

Results in human samples are interesting but difficult to integrate with the data obtained in animals. The authors state that there is an increase in the MC5-R in the DCM/ICM patients when one would expect a reduction given the supposed beneficial effect of the receptor. Nonetheless, when they measured monomer and dimer of MC5R in mice they observed an increase in at least one of the forms. In line with this, the expression of POMC was also increased, when in the mouseTAC model the levels of alpha-MSH were clearly reduced. Yet, in the IPSC, contrary to what the authors observed in human samples, MC5R was reduced after stretching stress and ET-1 treatment. In the introduction, it is mentioned that a-MSH plasma levels are higher in heart failure patients than in control and in the discussion that plasma a-MSH levels are inversely correlated with NYHA functional class in heart failure. Controversially, after TAC in mice the levels of a-MSH were lower than in the control. The elevation or decrease of the a-MSH and MC5R levels in the heart and plasma in the context of a potential treatment should be further clarified and discussed.
Response: We acknowledge that these results might be confusing.However, the finding of differential expression of MC5-R in human cardiomyopathy samples is not necessarily contradictory, because there are multiple factors that might affect the outcome: e.g.mouse vs. human, protein vs. mRNA, age of subjects, etiology of disease and interfering signal from non-myocytes (in case of heart lysates).For example, it is not uncommon that mRNA level and corresponding protein product of a gene are showing opposite effects, i.e. reduced protein level (due to enhanced degradation) could lead to a compensatory increase in mRNA level.Therefore, the direction of effect in the MC5R expression in human cardiomyopathy samples cannot be directly compared with MC5-R protein expression in the mouse heart after TACinduced cardiac hypertrophy.Furthermore, the levels of α-MSH in the plasma/heart cannot be directly compared between human cardiomyopathy patients and TACchallenged mice.We observed that there was a biphasic response to TAC in terms of cardiac Pomc expression, i.e. increased expression during the early phase after TAC and then declining expression towards advanced heart failure (Figure 1E and F).This fits with the clinical finding of higher plasma α-MSH concentration in patients with hypertrophic cardiomyopathy (vs dilated cardiomyopathy) and with the negative association between plasma α-MSH concentration and NYHA functional class, i.e. declining α-MSH level in more advanced heart failure (Yamaoka-Tojo M et al., Intern Med. 2006;45:429-434).
However, as pointed out by the reviewer, the downregulation of MC5R in stretched and ET-1-treated hiPSC-CMs is conflicting with the increased MC5R in DCM and ICM patient samples.This discrepancy might relate to the handling of the control and patient samples: LV samples from DCM and ICM patients were collected immediately upon heart cardiac transplantation, while control LV samples were collected at autopsy.Although control and patient samples showed stable and consistent Ct values for the housekeeping gene, the difference in sample handling (i.e.delay in freezing of control LV samples) might introduce a technical error in quantification of certain target genes.Therefore, a more reliable and important finding in human heart samples is the highly significant correlation between POMC and MC5R expression in both DCM and ICM groups.
If the reviewers and editors feel that these results are conflicting and confusing for the readers, we could exclude the data from human cardiomyopathy samples (Figure 5A-C) and related text parts completely from the manuscript.6&8), which was included in the echocardiography protocol based on the in vitro findings that α-MSH and PG-901 down-regulated fibrotic markers, which might have an influence on myocardial stiffness and diastolic function in vivo.Our ultrasound system was also upgraded with tools for pulsed-wave and tissue Doppler imaging after we had already completed the in vivo experiment for Figure 1.

For the TAC model in the cardiac-specific mice there are several points to review. Statistical differences between CTRL and CTRL TAC should be shown in all the parameters, to see if the model works, main effect is not sufficient when one of the conditions is the treated one, post-hoc test should be displayed. In addition, the differences between the KO and the 2 controls are not in all cases significant, making the result difficult to interpret, please comment on this. On the other hand, the TAC model (4 weeks) seems to have no effect on cardiac function, taking into consideration the TAC model used in Figure one (8 weeks) where the functional effect is obvious, The reviewer does not understand why this experiment was performed at 4 weeks and not 8 weeks. Also, the authors add endocardial FAC and IVRT analysis, which they do not analyze in the other case, and is only different from one of the control groups. Finally, the authors discuss the tamoxifen in the discussion section, this should be described in the results and the low ejection fraction in the SHAM
As requested by the reviewer, we moved the discussion of the tamoxifen-effect to the results section (page 11/lines 369-375).Ejection fraction was consistently low in sham-operated mice between different experiments (Figure 1,6 & 8).The EF values are also comparable to our previous publications (Perjés Á et al., Basic Res Cardiol. 2016 Jan;111(1):2 & Szabó Z et al., Hypertension. 2014 Jun;63(6):1235-40) and thereby more dependent on the anesthesia protocol than on tamoxifen-related cardiotoxicity.
11.In the last experiment with PG-901 there seems to be some relevant functional data.Nonetheless, cardiac hypertrophy is not modified by the treatment.This might be due to the off-target effect of the drug which could explain the absence of any effect at higher doses.This should be discussed in light of a potential treatment and further clarified.
Response: This is an important remark that was also brought up by Referee #2.Admittedly, there is something with PG-901 that we have not figured out.For example, in hiPSC-CMs, PG-901 evokes a significant and dose-dependent effect on NPPB expression, but the direction of effect varies from experiment to experiment (Figure R4).Basal MC5R expression, which varies between different batches of hiPSC-CM differentiation, might explain this confusing finding.PG-901 downregulated NPPB in cells that have a higher MC5R expression (Exp 1 and Exp 3), while an opposite effect was observed in cells that have a lower MC5R expression (Exp 2 and Exp 4).

Figure R4. Quantitative PCR analysis of NPPB mRNA expression in hiPSC-CMs treated with different concentrations of PG-901 for 24 hours in the absence (A) or presence (B) of ET-1 (100 nM). (C) Relative MC5R mRNA expression in unstimulated control cells in different experiments
This could indicate that there is an off-target effect that becomes particularly dominant, when the signaling through MC5-R is weaker (i.e. in cells with lower MC5R expression).Therefore, in a model of advanced heart failure that was shown to be associated with reduced MC5-R dimerization (Figure 3L), this off-target effect might also become dominant and mask the potential anti-hypertrophic effect of MC5-R activation.This notion is further supported by the new in vivo experiment, which was performed to address the comment 3.In sham-operated mice that have a 'normal' cardiac MC5-R expression level, PG-901 induced a subtle reduction in ventricular weight at the higher dose (0.5 mg/kg) (P=0.03vs Vehicle by Student's t test, P=0.08 by 1-way ANOVA and Dunnett post hoc test).

MC5R expression in unstimulated control cells
Furthermore, as a part of more thorough phenotyping, we quantified total leukocytes and their different subsets in the blood of PG-901-treated TAC mice (Figure R6), since MC5-R is known to regulate immune responses in leukocytes.This immunophenotyping revealed that PG-901 inhibits the TAC-induced increase in circulating neutrophils (Figure R6B) and decrease in B cells (Figure R6D) in a dosedependent manner, i.e. the higher dose of PG-901 was more effective than the lower dose.Consequently, the possible off-target effect appears to be heart-specific, since PG-901 showed classical dose-responsiveness in other cells.The issue related to offtarget effect of PG-901 is discussed on manuscript page 14/lines 507-514.

The work is lacking a mechanism analysis that could explains the obtained results. Though the JNK pathway seems plausible, there are no measurements of JNK in any of the animal models used. Even the authors discard GS and GI pathways, for cAMP there are different effectors from PKA that could be playing a role and other JNK partners should be evaluated. Also, given the role of JNK in apoptosis, this should be evaluated both in vitro and in vivo.
Response: Our in vitro data show that the MC5-R-mediated effects on hypertrophyand fibrosis-associated markers are dependent on JNK signaling (Figure 3L-N).In the revision, this dependency was also confirmed to exist in primary cardiomyocytes (Extended Figure 4C).We also did a further screening of possible targets that are known to be modulated by MC5-R and other MC-R subtypes.However, none of the other intracellular targets were affected by MC5-R signaling (Figure R1 and Extended Figure 4A and B).For further details, please see also response to comment 1 of Referee #1 (page 2 and 3 of this response letter).
We also analyzed p-JNK level in the heart of Mc5-cKO and PG-901-treated mice, but we did not find any significant changes in this regard (for further details and the results, please see response to comment 2 by Referee #1).Based on the existing and conflicting literature on the role of JNK in cardiac hypertrophy (reviewed in Physiol Rev. 2010 Oct;90(4):10.1152/physrev.00054.2009), it would be highly challenging to prove the contribution of the JNK pathway to the observed phenotypes in vivo.Earlier findings indicate that JNK activation is a dynamic and transient signaling event (peaking at ~30 min) after applying pressure overload and that different JNK isoforms might have separate and non-redundant roles in the process.Although in vitro studies strongly argue for a prohypertrophic role of JNKs, loss-of-function approaches to silence JNK or its upstream regulators in vivo have resulted in promotion as well as attenuation of cardiac growth response (as discussed on manuscript page 15, lines 531-535).Furthermore, JNK activation in vivo (in transgenic animal models) does not induce cardiac hypertrophy.
Therefore, we have been careful not to claim that the observed in vivo phenotypes would be dependent on the JNK pathway, and disclose the uncertainty related to the involvement of JNK signaling (manuscript page 15, lines 535-539): "In vitro experiments in the current study demonstrate that MC5-R regulates hypertrophic growth of cardiomyocytes in a JNK-dependent manner.However, further studies are warranted to determine whether MC5-R-induced JNK reduction has a direct effect on cardiomyocyte growth in vivo".
As requested by the reviewer, we analyzed markers of apoptosis in Mc5r-silenced NMCMs.MC5-R deficiency was associated with increased expression of proapoptotic markers such as Casp3, p53, Bax and Noxa (Extended Figure 4D-H).In good agreement with this finding, TAC-operated Mc5r-cKO mice showed increased number of apoptotic TUNEL + cells in the heart (Appendix Figure S9C).Conversely, long-term treatment with PG-901 reduced the number of TUNEL + cells in the heart (Extended Figure 5).Given the established role of JNK in apoptosis, it was surprising that the induction of apoptotic markers in Mc5r-silenced NMCMs occurred in a JNK-independent manner (Extended Figure 4D-H).In the screening of other targets, there was a tendency that p38 phosphorylation (P=0.14 vs Control siRNA) could be also affected in Mc5r-silenced cells (Extended Figure 4A and B).Therefore, we also investigated whether p38 inhibition with TAK-715 could reverse the upregulation of the pro-apoptotic markers in Mc5r-silenced NMCMs.TAK-715 alone increased the expression of Nppb (Figure R7A), Casp 3 (B) and Noxa (C), and did not reverse the effect of Mc5r knockdown on these genes, thus excluding the possibility that the increased apoptosis is mediated by p38 signaling.

Response:
We do not fully understand the point raised by the reviewer.We speculate that the difference (in vitro vs in vivo) might be caused by the dominant influence of noncardiomyocyte cells such as fibroblasts as mediators of Ang II-induced hypertrophic response in vivo.Vasoconstriction and consequent increase in blood pressure also contribute to cardiac hypertrophy in Ang II-infused mice.Accordingly, the mechanisms of Ang II-induced cardiomyocyte hypertrophy differ between in vitro and in vivo settings.This could explain why MC5-R activation/inactivation evokes a clear phenotype (in the context of Ang II stimulation) only in cultured cardiomyocytes, when the cells are isolated from the influence of other cell types such as fibroblasts.(Figure 2K-M) like in 4F-G, as the gene expression changes occurred quickly after applying the treatment in these cells.

Throughout the paper the loading control for the western blot changes arbitrarily. This should be homogenized or explained.
Response: For revision, we have limited the use of loading controls to b-actin and vinculin, and excluded GAPDH that was originally used in Extended Figure 1F.For an unknown reason, our protocol for stripping and re-probing of WB membranes has led to inconsistent results in terms of wiping out the primary antibody.Therefore, in few cases, we needed to use vinculin (as a loading control) that is a higher MW protein, which signal did not overlap with the signal from the primary protein target.

Protein levels of MC5R in the cardiac-specific KO are not convincing given the unconvincing signal of the Vinculin.
Response: Based on the reviewer's concern, we rerun the samples and replaced the the representative blots with these new images to improve the quality of the bands (Appendix Figure S7C).4 for not showing a response to MC5R agonist and JNK pathway.

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Figure R1 .
Figure R1.The effects of the MC5-selective agonist PG-901 on intracellular signaling pathways in H9c2 cells.(A through F) Representative Western blots and quantification of p-CREB (normalized to total CREB), p-ERK (normalized to total ERK), p-p38 (normalized to total p38), p-Akt (normalized to total Akt) and p-AMPK (normalized to total AMPK) in H9c2 cells treated with PG-901 (0.1 nM) for 5-60 minutes.Data are mean ± SEM, n=4-6 per group.
First, we analyzed the LV samples from cardiomyocyte-specific Mc5r-cKO mice for p-JNK levels as well as for the phosphorylation level of other MAP kinases (ERK and p38) and CREB.The results of the WB analysis are shown below (FigureR2).Sham-or TAC-operated Mc5r-cKO mice did not show any alteration in p-JNK (B), p-ERK (C), p-p38 (D) or p-CREB (E) level compared to control mice.
normal' cardiac MC5-R expression level, PG-901 induced a subtle reduction in ventricular weight at the higher dose (0.5 mg/kg) (

:
We fully agree with the reviewer and quantified the level of LV fibrosis in α-MSH-treated mice.Consistent with the phenotype observed in Mc5r-cKO mice and PG-901-treated mice, TAC-induced cardiac fibrosis was significantly attenuated by chronic α-MSH treatment (Figure 1H & 1N).5.The effect of the MC5-R agonist looks different from the rest (Figure 3A-D), but there is no statistical difference as there is for the MSH in vitro.It is difficult to observe the "clearly mimicked" stated by the authors.Also, it is important to consider that according to the paper cited by the authors (Grieco et al. 2002, Biochem.Biophys.Res.Commu, DOi:10.1006/bbrc.2002.6739),PG-901 behaves as a full agonist at the human MC5R but also as a full antagonist at MC3R and MC4R.Thefore, the authors should explain and discuss the rationale for choosing the PG-901 compound.

Figure R5 .
Figure R5.Quantitative real-time PCR (qPCR) analysis of Nppb mRNA expression in NMCMs treated with or without PTX for 18 hours followed by PG-901 treatment for 3 hours.Data are mean ± SEM, n=5-6 per group in each graph.* P<0.05 for the indicated post hoc comparisons.#### P<0.0001 for the main effect of PTX by 2-way ANOVA.
group should also be discussed.Response:We have carefully checked the figures (Fig1 and Fig 8)and included missing statistical differences between CTRL and CTRL TAC by 1-way ANOVA and post hoc tests.In Figure6and 7, we argue for displaying the main effect of TAC by 2-way ANOVA, because GraphPad Prism 9 does not allow analyzing pre-defined comparison in post hoc tests of 2-way ANOVA.If we choose to analyze and display all the possible post hoc comparisons, we will end up with 15 different comparisons making the graphs extremely busy and the interpretation whether the TAC model works or not even more obscure for the reader.Two-way ANOVA is a valid statistical test to analyze the effect of two independent variables (i.e.TAC and genotype).We have modified the presentation of statistical significances in Fig 6 and 7 to make it clearer whether TAC has an effect on the analyzed parameters.It is true that the differences between the Mc5r cKO and the 2 controls (Mc5r

Figure R6 .
Figure R6. .Quantification of total leukocytes (A), neutrophils (B), monocytes (C) and B cells (D) in the peripheral blood of sham-and TAC-operated mice treated with either vehicle or PG-901.Data are mean ± SEM, each dot represents individual mouse.* P<0.05 for the indicated comparisons by 1-way ANOVA and Dunnett post hoc tests.

this study, Suominen et al. focused on alpha-melanocyte-stimulating hormone (a-MSH) in cardiac remodeling under pressure overload. The authors found that a-MSH was actually highly expressed in the heart, compared to its conventional expression location, the hypothalamus. They moved to showed that cardiomyocytes showed high expression of a-MSH. Importantly, a-MSH treatment ameliorated cardiac remodeling under pressure overload. At the molecular level, the authors found that a-MSH suppressed JNK signaling, which was accompanied by downregulation of a number of hypertrophy markers. In addition, they showed that MC5-R is the receptor for a-MSH, suggesting a-MSH exerts a paracrine function in the heart. Finally, MC5-R deletion mice displayed exacerbated pathological remodeling in response to pressure overload. Overall, the experiments were thoughtfully designed and nicely executed. I have a few comments for the authors to consider. Response:
We thank the reviewer for the constructive and insightful comments.

lines 507-514) 4. MC5-R deletion from cardiomyocytes elevated cardiac hypertrophy under TAC. However, cardiac function was not affected. Is it due to the fact that control mice have not developed cardiac dysfunction yet? The TAC was too mild? Response:
P=0.03 vs Vehicle by Student's t test, P=0.08 by 1-way ANOVA and Dunnett post hoc test).We have discussed this issue also in the manuscript (page 14, We did not observe any change in LV ejection fraction (EF%) but the other functional indexes (FAC and IVRT) were reduced in response to TAC challenge, and Mc5-cKO showed further reduction of FAC and IVRT, which indicates deterioration of systolic and diastolic performance.Fractional area change (FAC) measures the change in circumferential area of the LV and is thus analogous to LV ejection fraction (EF).Considering that EF is based upon (extrapolated) volume, FAC could be more sensitive than EF and detect subtle changes in LV systolic performance that are not necessarily captured by measuring EF.IVRT, on the other hand, is typically increased during the development of diastolic dysfunction, but in the case of restrictive filling (as in TAC-operated mice), IVRT declines as reported also in other mouse studies (e.g.Richards DA et al, Sci Rep. 2019 Apr 10;9(1):5844 &  Sung MM et al, Circ Heart Fail.2015Jan;8(1):128-37).Admittedly, as pointed out by the reviewer, control MYH6-MCM and Mc5r fl/fl mice had not developed considerable cardiac dysfunction 4 weeks after the TAC operation.This mild phenotype might be caused by the genetic background (C57Bl/6J) of the (MYH6-MCM, Mc5r fl/fl and Mc5r-cKO) mice.It has been systematically shown that the C57Bl/6J substrain is more resilient to TAC-induced heart failure and develop variable cardiac phenotypes compared to C57Bl/6N substrain(Garcia-Melendez L et  al., Am J Physiol Heart Circ Physiol.2013Aug 1; 305(3): H397-H402 & Zi M et al.,  Curr Res Physiol.2019Dec;1:1-10).Four weeks after TAC, LV ejection fraction is marginally decreased and only a small percentage (~20%) of C57Bl/6J mice will eventually develop heart failure.These earlier observations are consistent with our data from MYH6-MCM, Mc5r fl/fl and Mc5r-cKO mice (Figure

with PG-901 or alpha-MSH should include SHAM mice treated with the drugs in order to show basal effects. Response:
As requested by the reviewer, we performed new in vivo experiments to investigate the effects of α-MSH and PG-901 in sham-operated mice.We observed that long-term treatment with α-MSH induced a subtle but significant reduction in ventricular weight and left ventricular posterior wall thickness in sham-operated mice without affecting functional parameters (Extended Figure2).Likewise, high-dose of PG-901 tended (P=0.03vs Vehicle by Student's t test, P=0.08 by 1-way ANOVA and Dunnett post hoc test) to reduce ventricular weight compared to vehicle-treated shamoperated mice (Appendix Table fl/fl and MYH6-MCM) are not in all cases significant.However, the main findings, which are increased ventricular size and cardiac fibrosis, are significant in comparisons against the 2 controls.Four weeks after TAC, LV ejection fraction is marginally decreased and only a small percentage (~20%) of C57Bl/6J mice will eventually develop heart failure.These earlier observations are consistent with our data from MYH6-MCM, Mc5r fl/fl and Mc5r-cKO mice.Since LV EF, as a primary functional parameter, was not changed in Mc5r-cKO at 4-weeks post-TAC, we did a more extensive analysis by echocardiography to detect more subtle changes in LV systolic performance.Fractional area change (FAC) measures the change in circumferential area of the LV and is thus analogous to LV ejection fraction (EF).Considering that EF is based upon (extrapolated) volume, FAC could be more sensitive than EF and detect changes in LV systolic function that are not necessarily captured by measuring EF.IVRT was analyzed as a part of evaluation of diastolic function (Figure In Fig 6and 7, we chose to phenotype Mc5r-cKO mice already at 4 weeks post-TAC, because advanced cardiac hypertrophy and heart failure was associated with reduced MC5-R expression in the heart (Figure3L), which could limit the incremental effect of genetically-induced MC5-R deficiency on the hypertrophic response (as discussed on manuscript page 15/lines 548-551).It has been systematically shown that the C57Bl/6J substrain (background strain of Mc5r-cKO mice) is more resilient to TACinduced heart failure and develop variable cardiac phenotypes compared to C57Bl/6N substrain(Garcia-Melendez L et al., Am J Physiol Heart Circ Physiol.2013Aug 1;  305(3): H397-H402 & Zi M et al., Curr Res Physiol.2019Dec;1:1-10).

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