Despite its frequent use and clinical efficiency, the anticancer anthracycline doxorubicin (Dox) shows strong side effects including cardiotoxicity (McGowan et al., 2017; Tacar et al., 2013). Dox cardiotoxicity can be acute with immediate cardiac disorders (arrhythmias) or chronic with remodeling cardiomyopathies (dilated cardiomyopathy [DCM], heart failure [HF]) years after treatment (Lipshultz et al., 2010).

Mechanistically, Dox, a DNA topoisomerase II inhibitor, induces DNA double strand break by direct DNA interaction (L’Ecuyer et al., 2006), oxidative stress (Cappetta et al., 2017; Rochette et al., 2015), and decreased ATP production (Tokarska-Schlattner et al., 2006). This induces apoptosis and necrosis through p53 cascade and ROS production/ATP depletion, respectively (Liu et al., 2008) and is proposed to be a primary mechanism of Dox-induced cardiomyopathy (Zhang et al., 2009; Casey et al., 2007). Dox also induces mitochondrial biogenesis alteration and energy defects (Goormaghtigh et al., 1990; Guo et al., 2014; Kavazis et al., 2017; Dorn et al., 2015). DNA damage and HF are prevented in cardiac-specific topoisomerase IIβ (TopIIβ) knock-out (KO) mice, leading to the conclusion that TopIIβ is fundamental in Dox-induced cardiotoxicity (Zhang et al., 2012). However, alternative mechanisms have been proposed to explain Dox-induced cardiotoxicity (Sawicki et al., 2021). Currently, the only approved medication against anthracyclines cardiotoxicity is dexrazoxane (Lipshultz et al., 2010). However, the drug is not devoid of adverse effects, such as potential second malignancies incidence and reduced cancer treatment efficacy (Swain et al., 1997; Reichardt et al., 2018; Tebbi et al., 2007). Thus, there is a need to search for new therapeutics which would provide long-term cardioprotection against Dox-induced cardiotoxicity without compromising its antitumoral efficacy.

The exchange factor EPAC (exchange protein directly activated by cAMP) contributes to the hypertrophic effect of β-adrenergic receptor chronic activation in a cAMP-dependent but PKA-independent manner (Métrich et al., 2008; Morel et al., 2005). Two EPAC genes are present in vertebrates, EPAC1 and EPAC2 (Robichaux and Cheng, 2018). EPAC1, encoded by the Rapgef3 gene, is the main isoform expressed in human hearts and its expression is increased in HF (Métrich et al., 2008). EPAC is a guanine nucleotide exchange factor for the small G-protein Rap (de Rooij et al., 1998; Kawasaki et al., 1998) and in pathological conditions, promotes cardiac remodeling through pro-hypertrophic signaling pathways, which involve nuclear Ca²⁺/H-Ras/CaMKII/MEF2 and cytosolic Ca²⁺/Rac/H-Ras/calcineurin/NFAT (Morel et al., 2005; de Rooij et al., 1998; Pereira et al., 2012; Cazorla et al., 2009; Oestreich et al., 2009). Although the role of EPAC in cardiomyocyte apoptosis remains controversial (Mangmool et al., 2015; Okumura et al., 2014; Suzuki et al., 2010), several molecular intermediates in the EPAC1 signalosome (e.g. Ca²⁺ homeostasis, RhoA, Rac) have been shown to be involved in Dox-induced cardiotoxicity (Sag et al., 2011; Riganti et al., 2008; Huelsenbeck et al., 2012). There is also evidence that EPAC inhibition may be cardioprotective in ischemia/reperfusion injury (Fazal et al., 2017). Moreover, EPAC was shown to contribute to cardiac hypertrophy and amyloidosis induced by radiotherapy (Monceau et al., 2014). Finally, EPAC was shown to play a role in various cancers (Huk et al., 2018; Kumar et al., 2018) and tumoral processes (Jansen et al., 2016; Almahariq et al., 2015; Almahariq et al., 2016), suggesting EPAC as a potential therapeutic target for cancer treatments (Kumar et al., 2018; Wang et al., 2017).

All this suggests that EPAC may play a role in Dox-induced cardiotoxicity. Thus, the aim of this study was to explore this hypothesis by evaluating whether inhibition of EPAC1, either pharmacologically (using the specific EPAC1 inhibitor, CE3F4; Courilleau et al., 2012) or genetically (EPAC1 KO mice), is cardioprotective against Dox-induced cardiotoxicity.

Dox is a potent chemotherapeutic agent used in clinic to treat a wide range of cancers but this anthracycline is also well known for its cardiotoxic side effects. Since decades, Dox-induced toxicity is considered to occur mostly through DNA damage, ROS generation, energetic distress, and cell death (apoptosis, necrosis, etc.). However, non-canonical/alternative pathways are nowadays emerging and Dox-induced cardiotoxicity was recently proposed to occur through mechanisms other than those mediating its anticancer activity (Zhang et al., 2012; Li et al., 2018; Zhang et al., 2019). Moreover, no widely accepted therapeutic strategies to minimize Dox-induced cardiac injury have been established. The iron chelator dexrazoxane is the only FDA- and EMEA-approved cardioprotector despite limited and still controversial application (Tebbi et al., 2007). Here, we demonstrated that pharmacological inhibition (CE3F4) and the use of EPAC1 KO mice prevented Dox-induced cardiotoxicity in vitro and in vivo. We report an up-regulation of expression and activity of EPAC1 following Dox treatment. Moreover, specific EPAC1 inhibition with CE3F4, but not EPAC2 inhibition with ESI-05, reduced DNA damage, mitochondrial alterations, and apoptosis elicited by Dox in cardiomyocytes, indicating that EPAC1, but not EPAC2, inhibition protects the heart against Dox-induced cardiotoxicity. This was confirmed in vivo in EPAC1 KO mice. Moreover, EPAC1 inhibition by CE3F4 not only protected cardiac cells, but also increased the toxicity of Dox against human cancer cell lines (MCF-7 human breast cancer and HeLa human cervical cancer). Therefore, these results strongly suggest that inhibition of EPAC1 could be a promising strategy to prevent heart damage in anthracyclines-treated cancer patients.

At the molecular level, there is evidence that Dox and EPAC signaling pathways share common features and downstream effectors (Sag et al., 2011; Riganti et al., 2008; Huelsenbeck et al., 2012). However, no direct link between these two pathways has been reported so far. Our in vitro kinetic analysis revealed that Dox induces an increase of EPAC1 expression in the first hours after treatment (3–6 hr), followed by a down-regulation (16–24 hr). Despite this drop, EPAC activity remained elevated during the 24 hr of Dox treatment. This was confirmed by the progressive increase of the active form Rap-GTP, the direct EPAC effector, and was most likely due to the observed elevation of cAMP concentration in cardiomyocytes during the first 24 hr following Dox treatment. A subsequent decrease of EPAC1 protein level was observed which likely represents a protective cellular response, since we found that chemical or genetic EPAC1 inhibition was cardioprotective.

We show that Dox induces a mitochondrial caspase-dependent apoptosis characterized by DNA damage, Ca²⁺-dependent MPTP opening and mitochondrial membrane permeabilization, caspase activation, nuclear fragmentation, and cell size reduction which is consistent with previous report (Zhang et al., 2009). However, by contrast to other studies (Lim et al., 2004; Lebrecht and Walker, 2007), 1 µM Dox did not induce necrosis in NRVM (measured by PI staining), which was only observed when higher doses of Dox (up to 10 µM) were used (data not shown), indicating that induction of cardiac necrosis by Dox may be dose-dependent.

The role of EPAC1 in cardiomyocyte apoptosis is still unclear owing to controversial reports (Mangmool et al., 2015; Okumura et al., 2014; Suzuki et al., 2010; Fazal et al., 2017). Indeed, EPAC1 has anti- or pro-apoptotic effect in the heart, depending on the type of cardiomyopathy and its induction mode. For instance, EPAC1 was reported to be cardioprotective and to participate in antioxidant and anti-apoptotic effects of exendin-4, an agonist of the glucagon-like peptide-1 receptor (Mangmool et al., 2015). By contrast, EPAC1 KO mice showed no cardiac damage induced by pressure overload, chronic catecholamine injection, or ischemia-reperfusion (Okumura et al., 2014; Fazal et al., 2017), suggesting a deleterious effect of EPAC1. This deleterious role of EPAC1 is also found in other tissues as Rapgef3 deletion or its inhibition by ESI-09 reduces nerve injury and inflammation, leading to allodynia upon anticancer drug paclitaxel treatment (Singhmar et al., 2018). The protective/detrimental action of EPAC1 is thus not completely understood and might depend on the tissues and the pathophysiological mechanisms of the disease. Here, using pharmacological (CE3F4) and genetic approaches, we found that EPAC1 inhibition is cardioprotective in the context of Dox-induced apoptosis and cardiotoxicity.

Mechanistically, we demonstrated that EPAC1 inhibition prevents MPTP opening induced by Dox. Consistent with our observation, MPTP opening is known to result in mitochondrial membrane permeabilization, pro-apoptotic factors release, activation of caspases, and cell dismantling (Childs et al., 2002; Deniaud et al., 2008). Recently, it has been demonstrated that EPAC1 KO mice are protected against myocardial ischemia/reperfusion injury (Fazal et al., 2017). The EPAC1 activation leads to increased mitochondrial Ca²⁺ overload, which in turn promotes MPTP opening, pro-apoptotic factor release, caspase activation, and cardiomyocyte death. The demonstration that EPAC1 inhibition prevents Dox-induced MPTP opening and cardiomyocyte death suggests that EPAC1 may play a similar deleterious role in Dox-associated cardiotoxicity and ischemia/reperfusion injury and that MPTP is a major downstream effector in the cardiac cell death signaling cascade regulated by EPAC1.

We found that Dox elicited mitochondrial dysfunction in cardiomyocytes, in agreement with previous report (Carvalho et al., 2014). Indeed, ΔΨm was up-regulated together with a strong decrease of the activity of the mitochondrial respiratory complex I and IV in cardiomyocytes exposed to Dox. Importantly, inhibition of EPAC1 by CE3F4 counteracted the mitochondrial alterations induced by Dox. These results could be correlated with a previous study showing that during vascular injury, mitochondrial fission and cell proliferation were suppressed by inhibition of EPAC1 in vascular smooth muscle cells (Wang et al., 2016). Therefore, inhibiting EPAC1 can help prevent Dox cardiotoxicity as observed in vascular proliferative diseases.

One of the major mechanisms of Dox-induced cardiomyocyte death and mitochondria reprogramming involves TopIIβ. This gyrase is an enzyme that regulates DNA winding by the formation of DNA/TopII cleavable complexes and generation of DNA double strand breaks (Corbett and Berger, 2004). Thus, by its important role in replication and transcription, TopIIβ is of particular interest in cancer therapy (Nitiss, 2009). Recently, on the cardiac side, a new paradigm in Dox-induced cardiotoxicity has been put forward, conferring a central role to TopIIβ. Indeed, TopIIβ is required for ROS generation, DNA damage, and cell death induction, the three canonical mechanisms involved in Dox side effects (Zhang et al., 2012). Our results showed that EPAC1 inhibition prevents Dox-induced formation of DNA/TopIIβ cleavable complexes, as evidenced by higher protein level of free TopIIβ, suggesting that EPAC1 inhibition blocks deleterious Dox effects in part through the TopIIβ pathway.

Recent data have reported that EPAC1 could be essential to modulate cancer development and metastasis formation (Kumar et al., 2018). In addition, inhibition of EPAC1 was proposed as a therapeutic strategy for the treatment of cancers such as melanoma, pancreatic, or ovarian cancers (Almahariq et al., 2016; Baljinnyam et al., 2014; Baljinnyam et al., 2011; Gao et al., 2016). For instance, the genetic deletion of EPAC1 or the in vivo EPAC1 inhibition by ESI-09 decreases migration and metastasis of pancreatic cancer cells (Almahariq et al., 2015). Of note, in breast cancer cells, which are generally sensitive to Dox therapy, EPAC1 inhibition was shown to inhibit cell migration and induce apoptosis (Kumar et al., 2017). Here, we found that inhibition of EPAC1 by CE3F4 increased Dox-induced cell death in two human cancer cell lines, including breast cancer cells. Therefore, our data show that EPAC1 inhibition not only protects cardiac cells from Dox-induced toxicity but also enhances the sensitivity of cancer cells to Dox. Similarly, PI3Kγ blockade (Li et al., 2018) or the novel agent named biotin-conjugated ADTM analog (BAA) (Zhang et al., 2019) were shown to prevent Dox-induced cardiotoxicity and to synergize with its antitumor activity against breast cancer. Therefore, the identification of new promising chemical compound, such as BAA or CE3F4, could be the starting point to the next therapeutic treatments that would protect patients from acute and chronic cardiotoxicity of Dox without altering its cytotoxicity toward cancer cells.

In conclusion, we propose EPAC1 as a new regulator of Dox-induced toxicity in cardiac cells. In response to Dox, the pharmacological inhibition of EPAC1 by CE3F4 recapitulated EPAC1 KO phenotype, suggesting the potential therapeutic efficacy of this EPAC1 inhibitor to alleviate Dox-associated side effects in the heart, while maintaining or enhancing anticancer effect. Thus, EPAC1 inhibition represents a promising therapeutic strategy both to prevent Dox cardiotoxicity and to enhance its antitumoral activity.

All data generated or analysed during this study are included in the manuscript and supporting file; Source Data files have been provided for Figures 1 to 7 and Figure 5-figure supplement 1.

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Decision letter after peer review:

Thank you for submitting your article "EPAC1 inhibition protects the heart from doxorubicin-induced toxicity" for consideration by eLife. Your article has been reviewed by 2 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and a Senior Editor. The following individual involved in the review of your submission has agreed to reveal their identity: Cecilia Mundiña-Weilenmann (Reviewer #2).

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this letter to help you prepare a revised submission.

Essential revisions:

1) Extended Figure 2 – why do the WT and KO mice weights vary at the start … 25 vs >27.5g? This can make a difference in the way the weights changed over time as is evident in the way both Saline groups performed across the weeks.

2) Request raw uncropped blot image for Figure 2a.

3) Figure 4a: Some of the bar references are missing.

4) Figure 4i – What happens if you use a higher dose of CE3F4?

5) Figure 4i – Not 'most'. Figure disagrees with this assertion. Perhaps show a magnified image too for better clarity.

6) Figure 4k: Please specify the time of Dox-treatment in adult cardiac myocytes.

7) There is no reference to Figure 5a in the text.

8) Figure 5d: The level of EPAC1 expression in the representative blot of 15 weeks after the last i.v. injection of saline solution in WT mice is considerably higher than that of 2 and 6 weeks after the last injection of saline solution. Is there any change in the basal expression of EPAC1 with age? Request authors to send in uncropped data for Figure 5d. A full Western blot is requested.

9) Figure 5e – can authors explain why there is an apparent 4-fold difference between mice in Actin levels? As a further comment, the 2 WT mice Actin EPAC1 levels are quite different too. Was there technical difficulty in collecting protein?

10) Figure 6 a-c: The authors claimed that: "while cardiomyocytes isolated from Dox-treated WT mice had a reduced unloaded cell contraction (cell shortening, Figure 6a), a reduced [ca²⁺]i transient amplitude (peak F/F0, Figure 6b), and a decreased sarcoplasmic reticulum ca²⁺ load (Figure 6c), all these alterations were absent in myocytes isolated from Dox-treated EPAC1 KO mice". In fact, in cardiomyocytes from EPAC1 KO mice, Dox-treatment enhanced Ca transient amplitude and sarcoplasmic reticulum Ca load. Can the authors comment on that?

11) Figure 6d – unacceptable, as actin bands are heavily pixelated. Provide a more resolved image. Did the authors observe a slowdown in the kinetics of Ca transient decay as a consequence of the decrease in SERCA2a expression in myocytes from Dox-treated WT mice?

12) Prevention of Dox-induced cardiac dysfunction in WT mice by pharmacological inhibition of EPAC1, would strengthen the putative clinical application of the current findings.

13) Recommend including https://academic.oup.com/jpp/article/65/2/157/6132943 citation as it is a comprehensive review of the toxic effects of Dox, including its effects on the heart.

14) 'genetic invalidation' – change this terminology.

15) 'human cancer' – Be specific here. Which types of cancer cell lines were studied exactly?

16) 'EPAC1 inhibition was shown to inhibit cell migration and induce apoptosis' … So, are you able to show the combined effect of EPAC1i and Dox on breast cancer cells in your study? Or maybe the cervical line you were using?

17) You did not use females due to hormonal influence – provide a citation as evidence of this phenomenon.

18) Dissociation of Neonatal (NRVM) and Adult (ARVM) Rat Ventricular Myocytes – How did you ensure you got a high purity yield of your target cell type?

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

Thank you for resubmitting your work entitled "EPAC1 inhibition protects the heart from doxorubicin-induced toxicity" for further consideration by eLife. Your revised article has been evaluated by a Senior Editor and a Reviewing Editor.

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

– The version of the manuscript uploaded does not indicate which figure is which at the end where all figures are presented. On page 31, the figure 'D', can the authors explain why in the EPAC KO Dox lane, the bands for the two markers are not aligned, ie. one drops L-R, while the other rises L-R. Are they from the same experiment, as the lane to the immediate left bands look quite okay. This needs to be confirmed by having a look at the whole blot.

– Figure 6A: for the KO condition, why aren't the upper bands shown? The cropping is too close (compare with the WT condition where a wider region is shown above band of interest).

– Also Figure 6D graph y-axis should be 1-100%, and higher, not only 0-1 and higher. Compare 6E.

– Regarding the in vivo weight data there should be an explanation along the lines of slopes of weight curves as supplementary data (ie. Supp Figure 2A and B). A 3.5g difference is > 10% of the weight of animals.

Essential revisions:

1) Extended Figure 2 – why do the WT and KO mice weights vary at the start … 25 vs >27.5g? This can make a difference in the way the weights changed over time as is evident in the way both Saline groups performed across the weeks.

The figure name has been modified according to eLife instructions as follow: Figure 5—figure supplement 1 (line 176, 760 and 766).

The animals were raised in our animal core facility at the Faculty of Pharmacy with a limited number of transgenic animals. All animals (transgenic and associated littermate) used for experimentation were 12 weeks +/- 3 days of age, which could partly explain the observed variations in weight. However, following your comment, we compared the slope of weight gain between WT and Epac1 KO Ctl mice, and between Dox treated WT and Dox Treated Epac1 KO mice. As you can see in appendix of revisions (Figure 5—figure supplement 1a/b), there was no difference between the slopes (P=0.0708 and P=0.6487, respectively).

2) Request raw uncropped blot image for Figure 2a.

We have replaced the previous blot with a new one. The uncropped raw data are provided.

3) Figure 4a: Some of the bar references are missing.

The missing bar references have been added.

4) Figure 4i – What happens if you use a higher dose of CE3F4?

The dose of CE3F4 was chosen according to the previous study performed by Courilleau et al. (Biophysical Research Communications, 2013)¹. We used the R-CE3F4 enantiomer at 10 M based on an IC50 value of R-CE3F4 on recombinant Epac1 of 5.8 M. From our data, this concentration of CE3F4 was sufficient to produce a biological response, namely a decrease in DNA damage, cleaved caspase 3 and 9, and cell death. We preferred not to increase the dose of CE3F4 to remain selective on EPAC1 and to avoid potential side effects such as protein denaturation, e.g. as documented with ESI-09 when used at >25 M (Rehmann et al., 2013², Zhu et al., 2015³).

5) Figure 4i – Not 'most'. Figure disagrees with this assertion. Perhaps show a magnified image too for better clarity.

We apologize if the old Figure 4i was not clear enough. We now provide new micrographs which we hope are more convincing. We would like to keep the description as proposed in the text (“…most CE3F4 supplemented-cells exhibited normal shape.”) because this is really what reflects the results consistently obtained in cell culture.

6) Figure 4k: Please specify the time of Dox-treatment in adult cardiac myocytes.

The time was 24h. This information has been added in the legend to Figure 4k (line 703).

7) There is no reference to Figure 5a in the text.

The reference to Figure 5a has been added in the text (line 176).

8) Figure 5d: The level of EPAC1 expression in the representative blot of 15 weeks after the last i.v. injection of saline solution in WT mice is considerably higher than that of 2 and 6 weeks after the last injection of saline solution. Is there any change in the basal expression of EPAC1 with age? Request authors to send in uncropped data for Figure 5d. A full Western blot is requested.

The original membranes have been reconstructed since they were originally cut out for separated antibodies incubation/revelation and time saving. They were again incubated with EPAC1 and actin antibodies in order to show the full membrane as requested. In addition, the “15 weeks” membrane coming from the mice after 15 weeks of treatment (saline or Dox) has been incubated/revealed with GAPDH as reference protein. These raw data are available in Figure 5—figure supplement 1.

Concerning the interesting question on the increase in basal expression of EPAC1 with age, we have no clear answer. We found one report showing that EPAC1 expression in heart is increasing during development, with a 140% increase measured at 3 weeks and 12 weeks in comparison to the foetal stage and neonatal stage (Ulucan et al. 2007⁴). In any case, EPAC1 expression was strongly inhibited by doxorubicin as indicated in the text.

9) Figure 5e – can authors explain why there is an apparent 4-fold difference between mice in Actin levels? As a further comment, the 2 WT mice Actin EPAC1 levels are quite different too. Was there technical difficulty in collecting protein?

These experiments have been repeated using heart tissues from the same mice and the resulting western blots are shown in new Figure 5e.

10) Figure 6 a-c: The authors claimed that: "while cardiomyocytes isolated from Dox-treated WT mice had a reduced unloaded cell contraction (cell shortening, Figure 6a), a reduced [ca²⁺]i transient amplitude (peak F/F0, Figure 6b), and a decreased sarcoplasmic reticulum ca²⁺ load (Figure 6c), all these alterations were absent in myocytes isolated from Dox-treated EPAC1 KO mice". In fact, in cardiomyocytes from EPAC1 KO mice, Dox-treatment enhanced Ca transient amplitude and sarcoplasmic reticulum Ca load. Can the authors comment on that?

A similar increase in Ca transient amplitude and sarcoplasmic reticulum Ca load was observed in our previous study (Llach et al. 2019⁵) in WT mice after 6 weeks of Dox treatment, as compared to a 2-week treatment, while both parameters were depressed again after 15 weeks of treatment (as seen in this study). This intermediate phase, which was accompanied by an increase in the expression of SERCA 2A and a decrease in the expression of phospholamban, was considered as a transient compensatory cardiac response to Dox, followed by a decompensation at 15 weeks. If, as proposed in our current study, the absence of EPAC1 might prevent the long-term depression of cardiac function induced by Dox, it may not affect the compensatory phase which in this case would last much longer that in WT mice.

We have added a small discussion in the revised manuscript (line 189-194).

11) Figure 6d – unacceptable, as actin bands are heavily pixelated. Provide a more resolved image. Did the authors observe a slowdown in the kinetics of Ca transient decay as a consequence of the decrease in SERCA2a expression in myocytes from Dox-treated WT mice?

As in the comment 8, original membranes have been reconstructed since they were originally cut out for separated antibodies incubation/revelation and time saving. They were again incubated with SERCA 2A and calsequestrin antibody in order to have a better protein reference and to show the full membrane as requested. Actin images have been replaced by calsequestrin one and the uncropped raw data are provided in the source data files.

Concerning the kinetics of Ca transient decay, we indeed observed a 20% slowdown in the kinetics of the Ca transient decay as shown in a new panel e in Figure 6 (see additions in line 195-196, Figure 6e and line 748-750). A similar observation was reported in our previous study (Llach et al. 2019⁵).

12) Prevention of Dox-induced cardiac dysfunction in WT mice by pharmacological inhibition of EPAC1, would strengthen the putative clinical application of the current findings.

This is obviously our current goal and experiments are already undertaken to test this hypothesis. Our plan is actually to use mice subcutaneously injected with tumor cells and treat the mice with saline, Dox, CE3F4 or Dox+CE3F4 (see also our response to comment 16 below). However, as shown in two recent studies, the use of CE3F4 in in vivo studies is challenging due to its poor plasmatic stability^{6, 7}. Therefore, it will take a substantial amount of time and effort to complete these experiments. We hope the reviewers will understand that this is not achievable within the revision of this manuscript, and will rather constitute a whole and separate study.

13) Recommend including https://academic.oup.com/jpp/article/65/2/157/6132943 citation as it is a comprehensive review of the toxic effects of Dox, including its effects on the heart.

This excellent review has been added in the introduction (ref 2 in line 53 and line 456-457 for reference).

14) 'genetic invalidation' – change this terminology.

Corrected: the original terminology has been replaced by the following: “Here, we demonstrated that pharmacological inhibition (CE3F4) and the use of EPAC1 knock-out mice prevented…” (line 224).

15) 'human cancer' – Be specific here. Which types of cancer cell lines were studied exactly?

The cancer cell lines were already indicated in the Methods section. We have added the details in the Discussion (line 231): “(MCF-7 human breast cancer and HeLa human cervical cancer)”.

16) 'EPAC1 inhibition was shown to inhibit cell migration and induce apoptosis' … So, are you able to show the combined effect of EPAC1i and Dox on breast cancer cells in your study? Or maybe the cervical line you were using?

Actually, we do have preliminary experiments which shows this. As shown in Author response image 1, addition of 10 mM of CE3F4 potentiates the effect of doxorubicin on two parameters measured in HeLa cancer cell line: SubG1 cells (Author response image 1A) and healing velocity (Author response image 1B). However, as indicated in the response to comment 12 above, these results are part of another study comparing the effect of Dox, CE3F4 or Dox+CE3F4 in mice subcutaneously injected with tumor cells. We hope the reviewer will accept that we prefer to hold these results for this new and separate study.

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17) You did not use females due to hormonal influence – provide a citation as evidence of this phenomenon.

A previous study from our lab showed a differential response to doxorubicin in male and female adult rats⁸. The authors showed that “After 7 weeks of doxorubicin (2 mg/kg per week), males developed major signs of cardiomyopathy with cardiac atrophy, reduced left ventricular ejection fraction and 50% mortality. In contrast, no females died and their left ventricular ejection fraction was only moderately affected.” We have added a brief statement on this in the revised manuscript (line 325-326 and reference added in line 610-611).

18) Dissociation of Neonatal (NRVM) and Adult (ARVM) Rat Ventricular Myocytes – How did you ensure you got a high purity yield of your target cell type?

First, atria were removed before the dissociation of NRVM, and after the Langendorff perfusion for ARVM isolation. Then, NRVM go through a percoll gradient, which separates in different layers the fibroblasts and the cardiomyocytes⁹, while ARVM passed by two BSA phases in order to remove the maximal amount of non cardiomyocytes cells and ensure a high purity yield¹⁰.

References

1. Courilleau, D., Bouyssou, P., Fischmeister, R., Lezoualc'h, F. and Blondeau, J.P. The (R)-enantiomer of CE3F4 is a preferential inhibitor of human exchange protein directly activated by cyclic AMP isoform 1 (Epac1). Biochemical and biophysical research communications 440, 443-448 (2013).

2. Rehmann, H. Epac-inhibitors: facts and artefacts. Scientific reports 3, 3032 (2013).

3. Zhu, Y. et al. Biochemical and pharmacological characterizations of ESI-09 based EPAC inhibitors: defining the ESI-09 "therapeutic window". Scientific reports 5, 9344 (2015).

4. Ulucan, C. et al. Developmental changes in gene expression of Epac and its upregulation in myocardial hypertrophy. American journal of physiology. Heart and circulatory physiology 293, H1662-1672 (2007).

5. Llach, A. et al. Progression of excitation-contraction coupling defects in doxorubicin cardiotoxicity. Journal of molecular and cellular cardiology 126, 129-139 (2019).

6. Toussaint, B., Hillaireau, H., Cailleau, C., Ambroise, Y. and Fattal, E. Stability, pharmacokinetics, and biodistribution in mice of the EPAC1 inhibitor (R)-CE3F4 entrapped in liposomes and lipid nanocapsules. Int J Pharm 610, 121213 (2021).

7. Toussaint, B. et al. Interspecies comparison of plasma metabolism and sample stabilization for quantitative bioanalyses: Application to (R)-CE3F4 in preclinical development, including metabolite identification by high-resolution mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 1183, 122943 (2021).

8. Moulin, M. et al. Sexual dimorphism of doxorubicin-mediated cardiotoxicity: potential role of energy metabolism remodeling. Circ Heart Fail 8, 98-108 (2015).

9. Wollert, K.C. et al. Cardiotrophin-1 activates a distinct form of cardiac muscle cell hypertrophy. Assembly of sarcomeric units in series VIA gp130/leukemia inhibitory factor receptor-dependent pathways. The Journal of biological chemistry 271, 9535-9545 (1996).

10. Verde, I., Vandecasteele, G., Lezoualc'h, F. and Fischmeister, R. Characterization of the cyclic nucleotide phosphodiesterase subtypes involved in the regulation of the L-type ca²⁺ current in rat ventricular myocytes. British journal of pharmacology 127, 65-74 (1999).

[Editors' note: further revisions were suggested prior to acceptance, as described below.]

The manuscript has been improved but there are some remaining issues that need to be addressed, as outlined below:

– The version of the manuscript uploaded does not indicate which figure is which at the end where all figures are presented. On page 31, the figure 'D', can the authors explain why in the EPAC KO Dox lane, the bands for the two markers are not aligned, ie. one drops L-R, while the other rises L-R. Are they from the same experiment, as the lane to the immediate left bands look quite okay. This needs to be confirmed by having a look at the whole blot.

We added the missing figure numbers on each figure page.

The blots in Figure 6D are from the same experiment. However, as explained in our “Essential revision comment 11”, the original membranes have been reconstructed since they were originally cut out for separated antibodies incubation/revelation and time saving. They were again incubated with SERCA 2A and calsequestrin antibody in order to have a better protein reference and to show the full membrane as requested. Actin images have been replaced by calsequestrin ones and the uncropped raw data are provided in the source data files.

The non-alignment of the bands is likely due to the fact that this is the last well of the blot and the migration might have been perturbed. This is confirmed by a LI-COR staining (all protein staining) made on this blot which is now added the source data files.

– Figure 6A: for the KO condition, why aren't the upper bands shown? The cropping is too close (compare with the WT condition where a wider region is shown above band of interest).

We are sorry, but we are not sure to understand your question. The only blots with EPAC1 KO condition are in Figure 5E and 6D. In both cases the width of the regions shown are the same between WT and KO condition. In case you are referring to differences in the EPAC1 blots of Figure 5D and E, i.e. to the presence of two bands in Figure 5E and one band in Figure 5D for EPAC1, the reason is the following: the two blots were obtained using different types of gels. The one in Figure 5D was obtained with a 10% gel, and the one in Figure 5E was obtained with a gradient gel, which separates differently the proteins. EPAC1 was thus revealed as two bands with the gradient gel while the 10% gel was giving only one band. Please note that the gel in Figure 5E was repeated to reply to “Essential revision comment 9”.

– Also Figure 6D graph y-axis should be 1-100%, and higher, not only 0-1 and higher. Compare 6E.

Again, we do not understand the point. The graph in Figure 6D shows results from western blots which are presented in each figure of the manuscript as a relative level between the treated versus control, the control value being 1 (see e.g. Figure 1F, 1G, 1J, 2A, 2B, 3A, 3B, 3C, 3D, 4E, 4F, 5D). Figure 6E, which represents the kinetics of calcium transients, are presented as the other calcium data shown in Figure 6A, B and C, i.e. as percentage of control, the control value being 100%, and thus cannot be compared with western blot data.

– Regarding the in vivo weight data there should be an explanation along the lines of slopes of weight curves as supplementary data (ie. Supp Figure 2A and B). A 3.5g difference is > 10% of the weight of animals.

We added an explanation of the slopes of the weight curves in the legend to Figure 5—figure supplement 1:

“The slopes were 0.41 and 0.34 g/week in a, and 0.21 and 0.18 g/week in b for WT and EPAC1 KO mice, respectively, and were not statistically different (p=0.0708 and 0.6487, respectively in a and b). The number of mice was 5 and 6 for Sal and 7 and 5 for Dox, in WT and EPAC1 KO, respectively.”

Author details

1. Marianne Mazevet

   Université Paris-Saclay, Orsay, France

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Writing – original draft

   Competing interests

   No competing interests declared

2. Anissa Belhadef

   Université Paris-Saclay, Orsay, France

   Contribution

   Formal analysis, Investigation, Visualization, Writing – original draft

   Competing interests

   No competing interests declared

3. Maxance Ribeiro

   Université Paris-Saclay, Orsay, France

   Contribution

   Conceptualization, Formal analysis, Investigation, Visualization, Writing – original draft

   Competing interests

   No competing interests declared

4. Delphine Dayde

   Université Paris-Saclay, Orsay, France

   Contribution

   Validation, Visualization, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8543-2352

5. Anna Llach

   Université Paris-Saclay, Orsay, France

   Contribution

   Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

6. Marion Laudette

   Institut des Maladies Metaboliques et Cardiovasculaires - I2MC, INSERM, Université de Toulouse, Toulouse, France

   Contribution

   Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

7. Tiphaine Belleville

   Innovations Thérapeutiques en Hémostase - UMR-S 1140, INSERM, Faculté de Pharmacie, Université Paris Descartes, Sorbonne Paris Cité, Paris, France

   Contribution

   Formal analysis, Investigation, Visualization

   Competing interests

   No competing interests declared

8. Philippe Mateo

   Université Paris-Saclay, Orsay, France

   Contribution

   Resources, Formal analysis, Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

9. Mélanie Gressette

   Université Paris-Saclay, Orsay, France

   Contribution

   Formal analysis, Supervision, Investigation, Visualization

   Competing interests

   No competing interests declared

10. Florence Lefebvre

    Université Paris-Saclay, Orsay, France

    Contribution

    Supervision, Investigation

    Competing interests

    No competing interests declared

11. Ju Chen

    Basic Cardiac Research UCSD School of Medicine La Jolla, San Diego, United States

    Contribution

    Resources

    Competing interests

    No competing interests declared

12. Christilla Bachelot-Loza

    Innovations Thérapeutiques en Hémostase - UMR-S 1140, INSERM, Faculté de Pharmacie, Université Paris Descartes, Sorbonne Paris Cité, Paris, France

    Contribution

    Resources

    Competing interests

    No competing interests declared

13. Catherine Rucker-Martin (deceased)

    1. Faculté de Médecine, Université Paris-Saclay, Le Kremlin Bicêtre, France
    2. Inserm UMR_S 999, Hôpital Marie Lannelongue, Le Plessis Robinson, France

    Contribution

    Resources

    Competing interests

    No competing interests declared

14. Frank Lezoualch

    Institut des Maladies Metaboliques et Cardiovasculaires - I2MC, INSERM, Université de Toulouse, Toulouse, France

    Contribution

    Resources

    Competing interests

    No competing interests declared

15. Bertrand Crozatier

    Université Paris-Saclay, Orsay, France

    Contribution

    Writing – review and editing

    Competing interests

    No competing interests declared

16. Jean-Pierre Benitah

    Université Paris-Saclay, Orsay, France

    Contribution

    Writing – review and editing

    Competing interests

    No competing interests declared

17. Marie-Catherine Vozenin

    Laboratoire de Radio-Oncologie, CHUV, Lausanne, Switzerland

    Contribution

    Resources

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0002-2109-8073

18. Rodolphe Fischmeister

    Université Paris-Saclay, Orsay, France

    Contribution

    Resources, Writing – review and editing

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-2086-9865

19. Ana-Maria Gomez

    Université Paris-Saclay, Orsay, France

    Contribution

    Resources, Writing – review and editing

    Competing interests

    No competing interests declared

20. Christophe Lemaire

    1. Université Paris-Saclay, Orsay, France
    2. Université Paris-Saclay, UVSQ, Inserm, Orsay, France

    Contribution

    Conceptualization, Supervision, Methodology, Writing – original draft

    Contributed equally with

    Eric Morel

    Competing interests

    No competing interests declared

21. Eric Morel

    Université Paris-Saclay, Orsay, France

    Contribution

    Conceptualization, Supervision, Funding acquisition, Writing – original draft, Project administration, Writing – review and editing

    Contributed equally with

    Christophe Lemaire

    For correspondence

    eric.morel@universite-paris-saclay.fr

    Competing interests

    No competing interests declared

    "This ORCID iD identifies the author of this article:" 0000-0003-1960-0121

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