# The Metabolically Resistant Apelin-17 Analogue LIT01-196 Reduces Cardiac Dysfunction and Remodelling in Heart Failure After Myocardial Infarction

Pierre-Emmanuel Girault-Sotias, Robin Deloux, Nadia de Mota, Stéphanie Riché, François Daubeuf, Xavier Iturrioz, Ara Parlakian, Alain Berdeaux, Onnik Agbulut, Dominique Bonnet, et al.

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## ► To cite this version:

Pierre-Emmanuel Girault-Sotias, Robin Deloux, Nadia de Mota, Stéphanie Riché, François Daubeuf, et al.. The Metabolically Resistant Apelin-17 Analogue LIT01-196 Reduces Cardiac Dysfunction and Remodelling in Heart Failure After Myocardial Infarction. Canadian Journal of Cardiology, 2025, 41 (5), pp.911-924. 10.1016/j.cjca.2024.11.034. hal-05217715

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Basic Research

# The Metabolically Resistant Apelin-17 Analogue LIT01-196 Reduces Cardiac Dysfunction and Remodelling in Heart Failure After Myocardial Infarction

Pierre-Emmanuel Girault-Sotias, PhD,ª Robin Deloux, PhD,ᵇ Nadia De Mota, MSc,ª

Stephanie Riché, MSc,ᶜ François Daubeuf, MSc,ᶜ Xavier Iturrioz, PhD,ᵈ Ara Parlakian, PhD,ᵇ

Alain Berdeaux, MD, PhD,ᶜ Onnik Agbulut, PhD,ᵇ Dominique Bonnet, PhD,ᶜ

Solène Emmanuelle Boitard, PhD,ª and Catherine Llorens-Cortes, PhDª,ᵈ

See editorial by Chen et al., pages 925-927 of this issue.

## ABSTRACT

**Background:** To protect patients after myocardial infarction (MI) and preserve cardiac function, the development of new therapeutics remains an important issue. Apelin, a neuro-vasoactive peptide, increases aqueous diuresis and cardiac contractility while reducing vascular resistance. However, its *in vivo* half-life is very short. We therefore developed a metabolically resistant apelin-17 analogue, LIT01-196 and investigated its effects on cardiac function and remodelling in a murine MI model.

**Methods:** The selectivity of LIT01-196 toward the apelin receptor was checked *in vitro*. Its *in vivo* half-life was assessed in male Swiss mice using radioimmunoassay. After permanent coronary artery ligation to induce MI, mice received subcutaneous administration of LIT01-196 (MI + LIT01-196, 9 mg/kg/d) or saline (MI + vehicle) for 4 weeks. Left ventricular (LV) function was assessed using echocardiography and Millar (Houston, TX) catheter, vascular density using immunofluorescence, and cardiac fibrosis using Sirius red staining. Real-time quantitative PCR was used to measure mRNA expression of heart

## RÉSUMÉ

**Contexte** : Pour protéger les patients après un infarctus du myocarde (IM) et préserver la fonction cardiaque, le développement de nouvelles thérapeutiques reste une question importante. L'apéline, un peptide neuro-vasoactif, augmente la diurèse aqueuse et la contractilité cardiaque tout en réduisant la résistance vasculaire. Cependant, sa demi-vie *in vivo* est très courte. Nous avons donc développé un analogue de l'apéline-17 résistant au métabolisme, le LIT01-196, et nous avons étudié ses effets sur la fonction et le remodelage cardiaques dans un modèle murin d'IM.

**Méthodes** : La sélectivité du LIT01-196 vis-à-vis du récepteur de l'apéline a été vérifiée *in vitro*. Sa demi-vie *in vivo* a été évaluée chez des souris mâles de type Swiss à l'aide d'un dosage radioimmunologique. Après ligature permanente de l'artère coronaire pour induire un IM, les souris ont reçu une administration sous-cutanée de LIT01-196 (IM + LIT01-196, 9 mg/kg/j) ou de solution saline (IM + véhicule) pendant 4 semaines. La fonction ventriculaire gauche (VG) a été évaluée par échocardiographie et cathéter Millar (Houston, TX), la

Besides activation of the adrenergic system, activation of the renin-angiotensin system is well identified as a major factor that contributes to cardiac remodelling of the heart after acute myocardial infarction (MI).¹,² Through the increased production of angiotensin II, cardiac fibrosis develops, a progressive increase in cardiac volume and diameter occurs as well as ultimately a reduction in ejection fraction,¹ a hallmark of left ventricular (LV) heart failure (HF). Treatments with

<footnote>ª Laboratory of Central Neuropeptides in the Regulation of Water Balance and Cardiovascular Functions, College de France, CIRB, INSERM U1050/CNRS UMR7241, 75005 Paris, France</footnote>

<footnote>ᵇ Sorbonne Université, Institut de Biologie Paris-Seine (IBPS), CNRS UMR 8263, Inserm U1345, Development, Adaptation and Ageing, 75005, Paris, France</footnote>

<footnote>ᶜ Laboratoire d'Innovation Thérapeutique, UMR7200 CNRS/Université de Strasbourg, Institut du Médicament de Strasbourg, Faculté de Pharmacie, 67401 Illkirch, France</footnote>

<footnote>ᵈ Université Paris Saclay, CEA, Département Médicaments et Technologies pour la Santé (DMTS), SIMoS, 91191 Gif-sur-Yvette, France</footnote>

<footnote>ᵉ INSERM U955-IMRB Equipe 03 Université Paris Est Créteil, 94010 Créteil, France</footnote>

<footnote>Received for publication April 20, 2024. Accepted November 24, 2024.</footnote>

<footnote>Corresponding author: Dr Catherine Llorens-Cortes, CEA centre de Saclay, Bâtiment 152, Point Courrier 24, 91191 Gif Sur Yvette cedex, France. Tel.: +33 6 25 91 48 80.</footnote>

<footnote>E-mail: c.llorens-cortes@college-de-france.fr</footnote>

<footnote>X @PiGirault, @cortes_llorens</footnote>

<footnote>See page 923 for disclosure information.</footnote>
failure (HF) fibrosis biomarkers and sarco/endoplasmic reticulum Ca²⁺-ATPase-2.

**Results:** The *in vivo* half-life of LIT01-196, a specific and selective apelin receptor agonist, was 2.5 hours. MI + LIT01-196 showed significantly improved LV function, reduced HF biomarkers, and enhanced cardiac contractility and sarco/endoplasmic reticulum Ca²⁺-ATPase-2 expression compared with MI + vehicle. LIT01-196 treatment almost doubled cardiac vascular density and maintained LV wall thickness post MI. It also significantly reduced cardiac fibrosis and fibrosis biomarkers, without decreasing arterial blood pressure.

**Conclusions:** Chronic LIT01-196 treatment post MI improves LV function without decreasing blood pressure, increases cardiac vascular density, and reduces cardiac remodelling. This suggests that apelin receptor activation by LIT01-196 might constitute an original pharmacological approach for HF treatment after MI.

densité vasculaire par immunofluorescence et la fibrose cardiaque par coloration au rouge Sirius. La PCR quantitative en temps réel a été utilisée pour mesurer l'expression de l'ARNm des biomarqueurs de la fibrose liée à l'insuffisance cardiaque (IC) et de la Ca²⁺-ATPase-2 du réticulum sarco-endoplasmique.

**Résultats:** La demi-vie *in vivo* du LIT01-196, un agoniste spécifique et sélectif du récepteur de l'apéline, était de 2,5 heures. Le groupe expérimental IM + LIT01-196 a montré une amélioration significative de la fonction du VG, une réduction des biomarqueurs de l'IC et une amélioration de la contractilité cardiaque et de l'expression de la Ca²⁺-ATPase-2 du réticulum sarco/endoplasmique, par rapport au groupe IM + véhicule. Le traitement par LIT01-196 a presque doublé la densité vasculaire cardiaque et maintenu l'épaisseur de la paroi du VG après l'IM. Il a également réduit de manière significative la fibrose cardiaque et les biomarqueurs de la fibrose, sans diminuer la pression artérielle.

**Conclusions :** Le traitement chronique par le LIT01-196 après un IM améliore la fonction VG sans diminuer la pression artérielle, augmente la densité vasculaire cardiaque et réduit le remodelage cardiaque. Cela suggère que l'activation du récepteur de l'apéline par le LIT01-196 pourrait constituer une approche pharmacologique originale pour le traitement de l'IC après un IM.

renin-angiotensin system blockers<sup>3,4</sup> such as angiotensin I converting enzyme inhibitors or antagonists of type 1 angiotensin II receptors are known to reduce cardiac fibrosis and subsequent remodelling of the myocardium post MI. However, despite the current treatments of HF<sup>5</sup> including renin-angiotensin system blockers, neprilysin inhibitors, β-blockers, mineralocorticoid receptor antagonists, and sodium-glucose co-transporter 2 inhibitors, there is always a need for novel drugs that act on new targets for preventing cardiac remodelling, hypotension, renal dysfunction, hypokalemia, and avoiding premature mortality.

In addition to the renin-angiotensin system, other counter-regulatory hormonal systems, such as the natriuretic and apelinergic systems, are also involved in HF.<sup>6,7</sup> Apelin, a neuro-vasoactive peptide, is the endogenous ligand of the human orphan G protein-coupled receptor, APJ, now known as the apelin receptor (ApelinR).<sup>8,9</sup> Apelin is derived from a 77-amino acid precursor, preproapelin.<sup>8</sup> It is expressed, together with its receptor, in the brain, heart, kidneys, and vessels.<sup>10</sup>

Apelin is involved in the regulation of body fluid homeostasis. It increases aqueous diuresis via a central mechanism involving the inhibition of arginine-vasopressin release into the bloodstream<sup>11</sup> and a direct renal mechanism counteracting the antidiuretic effect of arginine-vasopressin mediated by type 2 vasopressin receptors.<sup>12</sup> Apelin is also involved in several cardiovascular functions through a nitric oxide-mediated vasodilatory effect<sup>10,13</sup> and positive inotropic activity.<sup>13,14</sup> Systemic apelin administration in mice increases myocardial contractility, and decreases preload and afterload. Conversely, cardiac contractility is impaired in ApelinR or preproapelin knockout models, spontaneously<sup>15,16</sup> and after coronary artery ligation.<sup>17</sup> Human plasma apelin levels increase in early HF, subsequently decrease, together with LV ApelinR mRNA levels, in later stages of the disease.<sup>18,19</sup> This suggests that the apelinergic system is upregulated early in HF (probably as a compensatory mechanism), but

downregulated at the end stage of the disease. Consistent with this conclusion, intravenous apelin infusion increases cardiac index in healthy humans and HF patients,<sup>20</sup> and apelin administration after aortic banding in rodents prevents myocardial remodelling and cardiac dysfunction.<sup>21,22</sup> Apelin was also shown to reduce infarct size.<sup>23</sup>

However, therapeutic use of the natural peptide apelin is limited by its short half-life *in vivo* (< 1 minute).<sup>24</sup> This led us to generate LIT01-196, a metabolically resistant apelin-17 analogue, by adding a fluorocarbon chain to the N-terminal part of K17F.<sup>13</sup> In this study, we investigated the effects of repeated subcutaneous administrations of LIT01-196 over 4 weeks on cardiac function and remodelling, in an experimental model of post MI induced HF in mice.

## Methods

A detailed description of methods is available in *Supplemental Appendix S1*.

## Animals protocols

Adult male Swiss mice (25-30 g, 4-6 weeks old) were purchased from Janvier Laboratories (Le Genest-Saint-Isle, France). Animals were kept under a 12-hour artificial light/ 12-hour darkness cycle, with *ad libitum* access to normal food and water. Temperature (18°C-22°C) and humidity (40%-60%) were controlled. All procedures conformed to the guidelines of Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the current US National Institutes of Health guidelines. The experimental protocols were approved by the French Committee on Ethics in Animal Experimentation (CEEA no. 59, Ref. APAFIS#22274-2019080111288819). The mouse model of HF used was a model of ischemic MI obtained by permanent surgical ligation of the descending left coronary artery (LAD) as described in *Supplemental*
**Appendix S1.** The validation of this model has already been published.²⁵

For acute treatment, mice received either vehicle (saline, 1 mL/kg) or various doses of LIT01-196 (1, 3, or 9 mg/kg) subcutaneously. For chronic treatment after MI, considering the difficulties posed by anaesthesia after surgery and because at this stage, the fibrous scarring after the infarction has not yet fully set in, no echocardiography or MI size evaluation was performed 48 hours after MI before to start chronic vehicle or LIT01-196 treatment. To avoid as far as possible the differences of MI size between MI + vehicle and MI+ LIT01-196 mice, mice were randomly assigned to 3 groups: (1) mice subjected to sham surgery and to receive vehicle (sham); (2) MI mice to receive vehicle (MI + vehicle);and (3) MI mice to receive LIT01-196 (MI + LIT01-196) at a dose of 9 mg/kg (2700 nmol/kg), once daily subcutaneously for 4 weeks (at different injection sites). The drugs were always administered in the morning using an insulin syringe fitted with a 28-gauge needle, with a volume of 300 µL (the dose was adapted to the animal's weight). The investigators were blind to animal treatment group during the experiments. We performed 3 different series of animals to minimize the differences, a total of 123 mice were studied. Considering the large number of animals studied, the blind MI repartition and treatment, and the same researcher performing the surgery it could be considered that the variability of the MI size among MI + vehicle and MI + LIT01-196 mice was similar. The MI sizes were assessed at the end of the study on histological cryosections after red Sirius staining. MI mice with infarct sizes < 15% at the end of the study were excluded from the analysis (4 MI + vehicle mice and 3 MI + LIT01-196 mice). Cardiac function was assessed before surgery, and then at 2 and 4 weeks post MI, using transthoracic echocardiography as detailed in **Supplemental Appendix S1**.

## Invasive hemodynamic studies

LV hemodynamic parameters (systolic arterial [SA] blood pressure [BP]; [SABP]), diastolic arterial BP (DABP), cardiac contractility (derivative of pressure overtime [dP/dt] maximal [max; dP/dt max] and dP/dt minimal [min; dP/dt min]), LV peak systolic pressure (LVPSP) and LV end-diastolic pressure (LVEDP)] were measured, as previously described,²⁵ with a Millar catheter (Mikro-Tip Catheter Transducers SPR-671). For this purpose, LIT01-196 was subcutaneously administered at day 31. Then at 2 hours 30 minutes after treatment, mice were anaesthetized as described in **Supplemental Appendix S1**.

## Histological quantification of cardiac fibrosis and histopathology examinations of the liver and kidney

Heart, liver, and kidney sections were stained and analyzed as described in **Supplemental Appendix S1**.

## Mini protein G activation monitored using bioluminescence resonance energy transfer

The selectivity of LIT01-196 toward the ApelinR was evaluated by measuring the ability of the compound to block the activation by isoprenaline or angiotensin II of mini protein

G (miniG) recruitment to the plasma membrane in HEK293T cells expressing the β-adrenergic receptors (β1, β2, or β3) or the type 1a angiotensin II receptor (AT1a receptor). In addition, in these cells, we checked the ability of LIT01-196 alone to trigger miniG recruitment to the plasma membrane. The protocol used is described in detail in **Supplemental Appendix S1**.

## Real-time quantification of gene expression

Total RNA was extracted from the cardiac apex using TRIzol reagent (Sigma T9424; Sigma-Aldrich, St Louis, MO) and submitted to real-time quantification of gene expression, as described in **Supplemental Appendix S1**.

## Sodium dodecyl-sulfate polyacrylamide gel electrophoresis and Western blot analysis

Mice hearts were snap frozen on liquid nitrogen and proteins extracted and homogenized in RIPA buffer (ThermoFisher Scientific, Waltham, MA) containing protease and phosphatase inhibitors with Precellys Evolution homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Lysates were then quantified using bicinchoninic acid and 50 µg of protein was subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis and Western blot analysis as described in **Supplemental Appendix S1**.

## Quantification of plasma N-terminal pro hormone B-type natriuretic peptide

Four weeks after MI, MI + vehicle or MI + LIT01-196 mice were euthanized by decapitation and trunk blood (approximately 1 mL) was collected, on ice in chilled tubes containing 0.05 mL of 0.3 mol/m³ ethylenediaminetetraacetic acid (pH 7.4) and centrifuged at 1800g at 4°C for 15 minutes. Plasma N-terminal pro hormone B-type natriuretic peptide (NT-proBNP) levels were then determined using the Mouse Pro Brain Natriuretic Peptide ELISA kit (Clinisciences, Nanterre, France), according to the manufacturer's instructions.

## Simultaneous detection of CD31 and vinculin using double immunofluorescence staining

Hearts were harvested after cervical dislocation, frozen in liquid nitrogen-cooled isopentane, and sectioned into 8-µm thick cryosections using a cryomicrotome. Double immunofluorescence staining of frozen sections was performed as described in **Supplemental Appendix S1**.

## Detection of LIT01-196 using apelin radioimmunoassay

Adult male Swiss mice (25-30 g, 4-6 weeks old) were injected with (9 mg/kg) of LIT01-196 or vehicle subcutaneously (3-7 animals per point). Mice were euthanized by decapitation at different time points (0.5, 1.5, 4, 6, 8, and 12 hours) after treatment and plasma samples were prepared as described in the section, "Quantification of plasma N-terminal pro hormone B-type natriuretic peptide." Plasma (0.5 mL) was mixed with 0.05% bovine serum albumin and acidified with 0.175 mL of 3 mol/m³ HCl and stored at -80°C. Plasma LIT01-196 levels were determined using radioimmunoassay
with the K17F antiserum¹¹ as described in Supplemental Appendix S1.

## Statistical analysis

Data are expressed as mean ± standard error of the mean. Differences were analyzed using 1-way or 2-way analysis of variance for repeated and nonrepeated measures followed by Sidak or Tukey post hoc test for multiple comparisons, as appropriate in experiments including 3 groups. Data normality was assessed using the Shapiro-Wilk test. We used the Student t test or Mann-Whitney test for comparisons between 2 groups. Survival was analyzed using the log-rank test. Values of *P* < 0.05 were considered statistically significant. Statistical analyses were performed with GraphPad Prism software 8.0 (GraphPad Software, La Jolla, CA).

## Results

### In vitro characterization of LIT01-196

LIT01-196 was produced by the original chemical addition of a fluorocarbon group onto K17F (Fig. 1A). LIT01-196, like K17F, bound to rat ApelinR with an affinity in the subnanomolar range (Ki values for K17F and LIT01-196: 0.14 ± 0.02 and 0.34 ± 0.05 nM, respectively). In Chinese hamster ovary cells stably expressing the ApelinR-enhanced green fluorescent protein, LIT01-196 like K17F behaves as a full agonist for cyclic adenosine monophosphate production, extracellular signal-regulated kinase 1/2 phosphorylation, β-arrestin recruitment, and ApelinR internalization.¹³ In our study we showed that preincubation of cells with LIT01-196 (10 µM) for 15 minutes was not able to block isoprenaline (10 µM) or angiotensin II (0.1 µM) activation of miniG recruitment to the plasma membrane in HEK293T cells expressing the β-adrenergic receptors (β1, β2, or β3) or the AT1a receptor (Supplemental Fig. S1). In addition, in these cells, LIT01-196 alone was not able to trigger miniG recruitment to the plasma membrane.

### In vivo half-life of LIT01-196

The antibody directed against K17F used to measure plasma LIT01-196 levels recognized K17F and LIT01-196 with similar affinity (half-maximal inhibitory concentration values: 0.11 ± 0.01 nmol/L and 0.56 ± 0.04 nmol/L, respectively). Plasma apelin levels at baseline corresponded to endogenous apelin levels in the plasma, and those after subcutaneous LIT01-196 (9 mg/kg) administration corresponded with the levels of endogenous apelin plus LIT01-196 in the plasma.

Basal plasma apelin levels in conscious mice were 0.6 ± 0.1 pmol/mL. LIT01-196 treatment led to a sharp increase in plasma apelin levels, with the peak concentration (164 ± 18 pmol/mL) reached 90 minutes after administration (Fig. 1B). These levels then rapidly decreased, reaching basal values after 12 hours (1.2 ± 0.1 pmol/mL). We investigated the kinetics of apelin disappearance after the subcutaneous administration of LIT01-196 with an exponential 1-phase decay equation (GraphPad Prism) corresponding to a first-order elimination

model (*r* = 0.96). This gave an *in vivo* half-life in the mouse bloodstream of 142 minutes for LIT01-196 (Fig. 1B).

## Effects of acute LIT01-196 administration on LV ejection fraction and heart rate

Absolute values for LV ejection fraction (LVEF) and heart rate (HR) after acute LIT01-196 administration in control mice are shown in Supplemental Table S1. Acute subcutaneous LIT01-196 administration (1 or 3 mg/kg) did not significantly modify LVEF relative to vehicle-treated mice (Fig. 1C). In contrast, subcutaneous LIT01-196 administration (9 mg/kg) induced a progressive increase in LVEF. The increase relative to that in control mice receiving vehicle was significant after 2 hours (66.3 ± 1.0%; n = 6), maximal after 3 hours (67.8 ± 1.4%; n = 6), and persisted after 5 hours. LVEF values in the various groups had returned to baseline 24 hours after treatment (Fig. 1C). Acute LIT01-196 treatment had no effect on HR, regardless of dose (Fig. 1D). We therefore used 9 mg/kg LIT01-196 administered subcutaneously to study the effects of chronic LIT01-196 administration, for 4 weeks, on cardiac function in mice after MI.

## Effects of chronic LIT01-196 treatment on cardiac function

In total, 123 mice were randomized to MI or sham surgery groups; 34 mice died during the surgical procedure (28% mortality in the MI groups) within 60 minutes after LAD ligation. Eighty-nine mice were randomized into 3 groups: sham (25 mice), MI + vehicle (36 mice), and MI + LIT01-196 (28 mice). One death was observed in the sham group at later stages. Mortality was 25% in the MI + vehicle group (9 mice) and 18% in the MI + LIT01-196 group (5 mice) during subsequent stages of the study. The effects of chronic LIT01-196 or vehicle treatment on survival rate in mice were reported after different times post MI (Supplemental Fig. S2). The 4-week survival rate after myocardial infarction (MI) was 82% in the MI + LIT01-196 group vs 75% in the MI + vehicle group. The difference in survival rate between both groups using the log-rank test was not significant. The final group sizes were 24 mice for sham, 23 for MI + vehicle mice, and 20 for MI + LIT01-196 mice.

MI was induced by LAD ligation on day 0. Two days after surgery, subcutaneous treatment began, with vehicle or LIT01-196 (9 mg/kg), once daily for 4 weeks (Fig. 2A). As expected, MI decreased LVEF relative to the sham group, 2 and 4 weeks after MI (Fig. 2B-C and E, Supplemental Table S2). At these time points, LVEF was significantly higher in MI + LIT01-196 mice than in MI + vehicle mice, 3 hours after the last LIT01-196 administration on day 16 or 30 (Fig. 2A), but remained significantly lower than those for sham mice (Fig. 2D and E, Supplemental Table S2). Moreover, the improvement of LVEF after chronic treatment with LIT01-196 during 4 weeks was significantly maintained compared with MI + vehicle mice at 6 and 24 hours after the last administration (Supplemental Fig. S3A, Supplemental Table S3)

## Effects of chronic LIT01-196 treatment on LV dilation and wall thickness

Four weeks after MI, mean body weight was identical in all 3 groups (sham: 41.0 ± 0.7 g, n = 24; MI + vehicle: 39.6 ±
A

LIT01-196

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B

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C

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D

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<figure><img src="image_11.png" /></figure>

LIT01-196 (1 mg/kg)

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LIT01-196 (3 mg/kg)

<figure><img src="image_13.png" /></figure>

LIT01-196 (9 mg/kg)

**Figure 1.** *In vivo* half-life (t₁/₂) and acute effects of LIT01-196 on left ventricular ejection fraction and heart rate in control mice. (A) Chemical structure of LIT01-196. (B) *In vivo* half-life of LIT01-196 after acute subcutaneous administration (2700 nmol/kg = 9 mg/kg) in conscious mice (n = 3-7 for each treatment). (C) Left ventricular ejection fraction (LVEF, %); and (D) heart rate (beats per minute; bpm) were measured using echo-cardiography in mice, at different times after the acute subcutaneous administration of LIT01-196 at various doses (green line, 1 mg/kg, n = 6; red line, 3 mg/kg, n = 6; blue line, 9 mg/kg, n = 6), or vehicle (black line, 1 mL/kg, n = 6). Results are mean ± standard error of the mean. Multiple comparison tests were performed using repeated measures 2-way analysis of variance followed by Tukey tests. ** P < 0.01.*

1.0 g, n = 23; and MI + LIT01-196: 38.9 ± 1.0 g, n = 20). MI resulted in significant LV dilation 2 and 4 weeks after the intervention, as shown by the significantly higher LV end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD), LV end-systolic volume (LVESV), and LV end-diastolic volume (LVEDV), and significantly lower fractional shortening (FS) in the MI + vehicle group than in the sham group (Fig. 2F-I, Supplemental Tables S2 and S4).

LIT01-196 treatment significantly decreased LVESD and LVESV relative to MI + vehicle mice, at 2 and 4 weeks post MI (Fig. 2F and H, Supplemental Table S2), but these values nevertheless remained significantly higher than those for sham mice (Fig. 2F and H, Supplemental Table S2). MI + LIT01-196 mice had slightly lower LVEDD and LVEDV values than MI + vehicle mice 2 and 4 weeks post MI, but this difference was not significant (Fig. 2G and I, Supplemental Table S2). LVEDD and LVEDV values were also not significantly different from the corresponding values in sham mice at 2

weeks post MI, whereas this difference became significant after 4 weeks (Fig. 2G and I, Supplemental Table S2).

FS at 2 and 4 weeks post MI in MI + LIT01-196 mice was significantly higher than that in MI + vehicle mice 3 hours after the last LIT01-196 administration on day 16 or day 30 (Fig. 2A), but significantly lower than that in sham mice (Supplemental Table S4). Moreover, the improvement of FS after chronic treatment with LIT01-196 during 4 weeks was significantly maintained compared with MI + vehicle mice 6 and 24 hours after the last LIT01-196 administration (Supplemental Fig. S3B, Supplemental Table S4).

LV posterior wall thickness in systole (LVPWs) and LV posterior wall thickness in diastole is shown in Fig. 2J and K and Supplemental Table S4. Only LVPWs was significantly lower in MI + vehicle mice than sham mice 4 weeks after MI. Four weeks of LIT01-196 treatment significantly increased LVPWs relative to MI + vehicle, with values reaching levels similar to those in the control sham group. Four weeks of
<figure><img src="image_14.png" /><figcaption>**Figure 2.** Effects on cardiac function of repeated subcutaneous administrations of LIT01-196 post myocardial infarction (MI) in mice. (A) Schematic diagram of the experimental design of the study: chronic subcutaneous LIT01-196 treatment (9 mg/kg) was compared with vehicle in mice over a 31-day period, beginning 2 days after (D2) MI. (B-D) Representative echocardiography in 2-dimensional guided M-mode, from parasternal short-axis views outside the MI region, for the various experimental groups. (E) Left ventricular (LV) ejection fraction (LVEF); (F) LV end-systolic diameter (LVESD); (G) LV end-diastolic diameter (LVEDD); (H) LV end-systolic volume (LVESV); (I) LV end-diastolic volume (LVEDV); and (J and K) LV posterior wall thickness (LVPW) in systole (LVPWs) and diastole (LVPWd) measured using echocardiography before surgery and 2 and 4 weeks post MI in sham (grey bars and circles, n = 24), MI + vehicle (blue bars and squares, n = 23), and MI + LIT01-196 mice (orange bars and triangles, n = 20). Results are mean ± standard error of the mean. Multiple comparisons were performed using repeated measures 2-way analysis of variance with Tukey adjustment for multiple comparisons. D0, 16, 30, and 31, day 0, 16, 30, and 31, respectively; LAD, descending left coronary artery; ns, nonsignificant. n corresponds to the number of animals included in the messurer values shown in Fig. 2. \* P < 0.05, \*\* P < 0.01, \*\*\* P < 0.001, \*\*\*\* P < 0.0001.</figcaption></figure>
LIT01-196 treatment did not modify HR relative to MI + vehicle or sham mice (sham: 496 ± 17 beats per minute [bpm], n = 24; MI + vehicle: 516 ± 18 bpm, n = 23; and MI + LIT01-196: 445 ± 26 bpm, n = 20).

## Effects of chronic LIT01-196 treatment on hemodynamic parameters

Thirty-one days post MI, SABP and DABP were significantly lower in MI + vehicle mice than in sham mice (Fig. 3A and B, Supplemental Table S2). SABP and DABP were significantly higher in MI + LIT01-196 mice than in MI + vehicle mice, but were not significantly different from control values measured in sham mice (Fig. 3A and B, Supplemental Table S2).

Thirty-one days after MI, MI + vehicle mice had a lower dP/dt max and a higher dP/dt min than sham mice (Fig. 3C and D, Supplemental Table S2). Chronic LIT01-196 treatment improved cardiac contractility (dP/dt max and dP/dt min) relative to MI + vehicle treatment, with values similar to those in the sham group (Fig. 3C and D, Supplemental Table S2). LVPSP and LVEDP were significantly higher and lower, respectively, in the MI + LIT01-196 mice than in the MI + vehicle mice, reaching values similar to those for the sham group (Fig. 3E and F, Supplemental Table S2). Chronic LIT01-196 treatment did not modify HR relative to MI + vehicle or sham treatments (Supplemental Table S2). Sarco/endoplasmic reticulum Ca²⁺-ATPase-2 (SERCA2) mRNA levels in the cardiac apex were significantly lower, by 67%, in MI + vehicle mice than in sham mice (Fig. 4A). Chronic LIT01-196 treatment significantly increased SERCA2 mRNA levels relative to MI + vehicle treatment, for which values nevertheless remained lower than those for sham treatment (Fig. 4A). We also evaluated using Western blot analysis the cardiac levels of phosphorylated phospholamban (PLN) in mice post MI treated with LIT01-196 or vehicle (Supplemental Fig. S4). The ratio of phosphorylated PLN levels to nonphosphorylated PLN levels in MI + LIT01-196 mice were significantly higher than those measured in MI + vehicle mice (Supplemental Fig. S4).

## Effects of chronic LIT01-196 treatment on HF biomarkers

HF biomarkers were assessed using real-time quantitative polymerase chain reaction and radioimmunoassay on the cardiac apex (Fig. 3G-I, Supplemental Table S5). MI significantly increased atrial natriuretic factor (Anf) and brain natriuretic peptide (Fig. 3G and H) mRNA levels and plasma NT-proBNP levels. LIT01-196 treatment for 31 days post MI in mice significantly reduced Anf and brain natriuretic peptide mRNA levels and plasma NT-proBNP levels relative to MI + vehicle treatment (Fig. 3G-I).

## Effects of chronic LIT01-196 treatment on cardiac vascular density

Our results (Fig. 5) showed that the vascular density in the border zone of the infarcted area, identified by the immunofluorescence labelling of the endothelial cell marker CD31 relative to that of vinculin, was significantly higher in LIT01-196-treated mice than in vehicle-treated mice (6.95 ± 0.61%

in MI + vehicle mice (n = 8) vs 11.27 ± 1.92% in MI + LIT01-196 mice (n = 9); $P \le 0.05$).

## Effects of chronic LIT01-196 treatment on cardiac fibrosis, hypoxia, and mRNA expression of proapelin and apelin receptor

We investigated the effects of 31 days of LIT01-196 treatment post MI on cardiac fibrosis in mice, by determining the percentage of collagen type I (Col I) and III (Col III) fibres using Sirius red staining on 10 µm-thick heart sections (Fig. 6A). Cardiac fibrosis levels, as a percentage of the total heart, were significantly higher in MI + vehicle than in sham mice. Chronic treatment with LIT01-196 markedly reduced cardiac fibrosis relative to MI + vehicle treatment (Fig. 6B, Supplemental Table S6). Transcript levels for proteins involved in fibrosis, such as connective tissue growth factor (Ctgf; Fig. 6C), Col I (Fig. 6D), Col III (Fig. 6A), transforming growth factor-β (TGF-β1; Fig. 6F), α-smooth muscle actin (α-Sma; Fig. 6G), and sphingosine kinase 1 (Sphk1; Fig. 6H), were significantly higher in MI + vehicle mice than in sham mice (Supplemental Table S6). Chronic LIT01-196 treatment significantly decreased mRNA levels for cardiac fibrosis biomarkers relative to MI + vehicle treatment, but the mRNA levels for these biomarkers remained significantly higher than in sham mice, except for α-smooth muscle actin levels, which were not significantly different from those in the sham group (Supplemental Table S6). Transcript levels for hypoxia-inducible factor 1-alpha (Hif1-α), a hypoxia biomarker and for its target gene, angiopoietin 2 (Angpt2), were significantly higher in the MI + vehicle group than in the sham group. Chronic LIT01-196 treatment significantly decreased Hif1-α and reduced Angpt2 mRNA levels relative to MI + vehicle mice, but whereas the Hif1-α levels remained significantly higher than those for the sham group, the Angpt2 levels were not significantly different from sham (Fig. 4B and C, Supplemental Table S6).

Proapelin (Apln) mRNA levels were drastically reduced 4 weeks post MI whereas the slight decrease for apelin receptor (Aplnr) mRNA expression was not significant. Chronic LIT01-196 treatment post MI during 4 weeks significantly increased Apln and Aplnr mRNA expression relative to MI + vehicle mice. Apln mRNA expression after LIT01-196 treatment remained nevertheless significantly inferior to that in the sham group, whereas Aplnr mRNA expression reached levels slightly but significantly above control values measured in the sham group (Fig. 4D and E, Supplemental Table S6).

## Innocuity to the liver and kidneys of chronic LIT01-196 treatment

Histopathology analyses of the livers and kidneys of mice treated with LIT01-196 or vehicle during 4 weeks revealed no tissue damage. This finding was supported by body weight monitoring, which showed no weight loss in MI + LIT01-196 mice relative to MI + vehicle mice (Supplemental Fig. S5).

## Discussion

Our results show that the chronic subcutaneous administration of LIT01-196 for 4 weeks post MI in mice improves
<figure><img src="image_15.png" /><figcaption>**Figure 3.** Hemodynamic parameters and heart failure biomarkers in mice chronically treated with LIT01-196 or vehicle after myocardial infarction (MI). Hemodynamic parameters were measured in anaesthetized mice that received subcutaneous vehicle for 31 days after surgery for sham and MI + vehicle mice, or LIT01-196 (9 mg/kg) for MI + LIT01-196 mice with the last administration 3 hours before measurement. Measurement, using Millar catheter (Houston, TX), of (A) systolic arterial blood pressure (SABP (B) diastolic arterial blood pressure (DABP); (C) dP/dt max; (D) dP/dt min in sham indicated by **grey bars and circles** (n = 6), MI + vehicle (**blue bars and squares**, n = 6), and MI + LIT01-196 mice (**orange bars and triangles**, n = 6); (E) left ventricular peak systolic pressure (LVPSP); and (F) left ventricular end-diastolic pressure (LVEDP) in sham (n = 5), MI + vehicle (n = 5) or MI + LIT01-196 mice (n = 5). (G and H) mRNA levels for atrial natriuretic factor (Anf) and brain natriuretic peptide (Bnp); and (I) plasma N terminal (NT)-pro Bnp levels in sham (n = 10), MI + vehicle (n = 10) and MI + LIT01-196 mice (n = 10). Results are mean ± standard error of the mean. Multiple comparisons were performed using nonrepeated measures 1-way analysis of variance with Tukey adjustment for multiple comparisons. dP/dt, derivative of pressure overtime; ns, non-significant. \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001, \*\*\*\* *P* < 0.0001.</figcaption></figure>
<figure><img src="image_16.png" /><figcaption>**Figure 4.** Effects of chronic post myocardial infarction (MI) treatment with LIT01-196 during 31 days in mice on mRNA expression of sarco/endoplasmic reticulum Ca²⁺-ATPase-2 (SERCA2), hypoxia-inducible factor 1-alpha (Hif1-α), angiopoietin 2 (Angpt2), proapelin (Apln), and apelin receptor (Apln R) in the cardiac apex in mice post MI. Relative mRNA levels for (A) SERCA2; (B) Hif1-α; (C) Angpt2; (D) Apln, and (E) Aplnr. The gene expressions were normalized against sham with 2 housekeeping genes (hydroxymethyl-bilane synthase [Hmbs] and hypoxanthine phosphoribosyltransferase [Hprt]) used as references. Data are presented as mean ± standard error of the mean. Nonrepeated measures 1-way analysis of variance followed by Tukey tests for multiple comparisons. A.U, arbitrary units. \* P < 0.05, \*\* P < 0.01, \*\*\* P < 0.001, \*\*\*\* P < 0.0001.</figcaption></figure>

systolic and diastolic function by increasing LVEF and maintaining hemodynamic parameters, such as dP/dt max and dP/dt min, LVEDP, and LVPSP, at normal values without decreasing arterial BP. It also reduces cardiac remodelling by limiting ventricular dilation, LV wall thinning, and cardiac fibrosis, and increasing vascular density.

LIT01-196 binds ApelinR with subnanomolar affinity, acts as a full agonist for cyclic adenosine monophosphate production, extracellular signal-regulated kinase 1/2 phosphorylation, β-arrestin recruitment, and ApelinR internalization, and is much more stable when added to plasma (half-life > 24

hours) than K17F (4.6 minutes).¹³ Its selectivity for the ApelinR was shown by its lack of agonist or antagonist activity on the β1, β2, and β3-adrenergic receptors and AT1a receptor as well as on the vasopressin type 2 receptor as previously shown.²⁴ In our study LIT01-196 had a half-life *in vivo* of 2 hours 22 minutes in the bloodstream after subcutaneous administration in alert control mice. The maximal plasma concentration reached, 90 minutes after LIT01-196 administration, was 164 nM, 2050 times higher than its affinity (Kᵢ: 0.08 nM) for the ApelinR,¹³ and remained 125 times above the Kᵢ value 8 hours after administration. The longer half-life
<figure><img src="image_17.png" /><figcaption>**Figure 5.** Effects of chronic LIT01-196 treatment on cardiac vascular density. (A-F) Representative images of CD31 and vinculin-stained heart tissue sections in the border zone of the infarcted area in myocardial infarction (MI) + vehicle mice (n = 8) or MI + LIT01-196 mice (n = 9); scale bars = 75 µm. (G) Diagram of the percentage of vascular density in MI + vehicle vs MI + LIT01-196 mice relative to the total surface obtained from the CD31/vinculin staining. For each group, quantifications were performed on n = 8-9 samples. **Bars** indicate mean ± standard error of the mean and *P* values vs each group using Mann-Whitney test.</figcaption></figure>

of LIT01-196 in the bloodstream probably reflects its plasma protein-binding rate of 69%,²⁴ because such binding protects against enzymatic degradation and decreases renal clearance. Acute subcutaneous LIT01-196 administration (9 mg/kg) in control mice significantly improved cardiac performance independently of HR, with kinetics consistent with its *in vivo* half-life.

Daily LIT01-196 (9 mg/kg) administration for 4 weeks after MI in mice slightly but not significantly reduced mortality compared with vehicle-treated mice. Moreover, no tissue damage appeared in the liver and kidneys, in agreement with the observation that repeated administrations of LIT01-196 (3 mg/kg) for 4 days in control rats were not associated with renal failure,²⁴ suggesting that LIT01-196 appears to be safe, which remains to be confirmed in a larger number of animals. Moreover, chronic LIT01-196 treatment for 4 weeks after MI in mice improved cardiac function, as shown by the significant increase in LVEF and FS, LVPSP preservation, and improvement in LVEDP. The increase in LVEF and FS was observed not only 3 hours after the last LIT01-196 administration at 4 weeks post MI but was also maintained after 6 and 24 hours after dosing, showing that the improvement of cardiac function by LIT01-196 was not due to an acute response to drug treatment but was the result of chronic treatment.

The improvement in LVEDP is consistent with the effect of the nonpeptidic ApelinR agonist AM-8123, which improves diastolic function in rats post MI.²⁶ LVEDP improvement might reflect better volume regulation, because LIT01-196 is a potent aquaretic that inhibits the antidiuretic action of arginine-vasopressin in the collecting duct, thereby improving preload.¹³,²⁴ All of these effects, resulting from a

greater stimulation of the ApelinR by chronic LIT01-196 treatment, were strengthened by the LIT01-196 treatment-induced increases in proapelin and ApelinR mRNA expressions. In fact, proapelin mRNA levels were considerably reduced 4 weeks post MI, whereas ApelinR mRNA levels were slightly decreased. After chronic LIT01-196 treatment, proapelin mRNA expression reached values close to those measured in the sham group whereas those of ApelinR were significantly higher.

Chronic LIT01-196 administration also improves cardiac contractility, as shown by the improvement in dP/dt max and dP/dt min relative to the MI + vehicle group. SERCA2 is the primary cardiac isoform that regulates intracellular Ca²⁺ homeostasis.²⁷ An increase in SERCA2 activity results in enhanced calcium import into the sarcoplasmic reticulum resulting in higher amounts of calcium available for discharge through the ryanodine receptor into the cytoplasm during the next contraction cycle,²⁸ improving cardiac contractility. At the opposite, its downregulation in HF leads to slowed removal of cytosolic Ca²⁺ and incomplete relaxation, leading to a loss of cardiac contractility.²⁹ We observed that SERCA2 mRNA levels in LV tissues decreased after MI but were twice as high in LIT01-196-treated mice as in MI + vehicle mice. To strengthen these data and knowing that the phosphorylation of PLN reduces its inhibitory effect on SERCA2 function by reducing its Ca²⁺ affinity,³⁰ we have shown after MI that chronic LIT01-196 treatment significantly increases cardiac phosphorylated PLN protein levels compared with vehicle-treated mice. This suggests that the favourable effect of LIT01-196 on cardiac contractility post MI might partly be due to the increase in SERCA2 expression and phosphorylation of PLN, requested for a correct cardiac contractility.
<figure><img src="image_18.png" /><figcaption>**Figure 6.** Chronic treatment with LIT01-196 reduces the fibrotic remodelling induced by myocardial infarction (MI) in mice. (A) Representative Sirius red staining of heart sections (10 µm-thick) from mice subjected to MI or sham surgery and treated subcutaneously with 1 mL/kg/d vehicle solution or 9 mg/kg/d LIT01-196 for 31 days. (B) Quantification of fibrosis in the total heart for sham (grey bars and circles, n = 13), MI + vehicle (blue bars and squares, n = 14) and MI + LIT01-196 (orange bars and triangles, n = 12); relative mRNA levels for fibrosis biomarkers are shown in: (C) connective tissue growth factor (Ctgf); (D) collagen type I (Col I); (E) Col III; (F) transforming growth factor-β (Tgf-β1); (G) α-smooth muscle actin (α-Sma); and (H) sphingosine kinase 1 (SphK1) in the different groups, 31 days post MI, in the cardiac apex. Results are mean ± standard error of the mean. Nonrepeated measures 1-way analysis of variance followed by Tukey tests for multiple comparisons. AU, arbitrary units; ns, nonsignificant. \* P < 0.05, \*\* P < 0.01, \*\*\* P < 0.001, \*\*\*\* P < 0.0001.</figcaption></figure>
<figure><img src="image_19.png" /><figcaption>**Figure 7.** Proposed model of the mechanism of action of LIT01-196 for decreasing fibrosis post MI in mice. AMPK, AMP-activated protein kinase; APELIN-R, apelin receptor; Ctgf, connective tissue growth factor; α-Sma, α-smooth muscle actin; Sphk, sphingosine kinase; TGFβ, transforming growth factor β.</figcaption></figure>

Interestingly, LIT01-196 treatment prevented the decrease in SABP and DABP induced by MI at 4 weeks. Indeed, SABP and DABP values after LIT01-196 treatment were similar to those for the sham group. This finding is consistent with the effect of LIT01-196 on LVPSP, the value of which did not differ significantly from that in the sham group. This might reflect a decreased vascular tone induced by LIT01-196,¹³ even in the absence of a decrease in BP, decreasing cardiac afterload and improving stroke volume as previously reported for apelin¹³⁻¹⁵ or this might be due to endothelium dysfunction in favour of a vasoconstrictive effect of LIT01-196 mediated by ApelinRs expressed in vascular smooth muscle cells as previously shown for apelin³¹ which remains to be assessed. The lack of change in BP in an MI rat model and in an hypertension-induced cardiac dysfunction rat model, was also observed after treatment with 2 nonpeptidic ApelinR agonists, the AM-8123 and the BMS-986224 compounds.²⁶,³² Angiotensin I-converting enzyme inhibitor treatment is known to reduce cardiac fibrosis and subsequent LV remodelling after MI, but hypotension might occur as an adverse effect. Careful dose titration is, therefore, recommended when initiating angiotensin I-converting enzyme inhibitor treatment in patients with acute MI.⁴ Recently, we showed in the same mouse MI model that chronic oral treatment with the angiotensin I-converting enzyme inhibitor, ramipril (1.25 mg/kg) during 4 weeks significantly decreased SABP and DABP values relative to those of MI + vehicle mice.⁶ Our data suggest that LIT01-196 might improve

cardiac function without lowering BP. This improvement is supported by the significant decrease in mRNA levels for HF biomarkers⁶,³³ in the apex, such as brain natriuretic peptide and Anf or in plasma NT-proBNP levels. Similarly, a significant reduction in the mRNA expression of Hif1-α and a Hif1-α target gene, *Angpt2*, was also observed, suggesting that chronic LIT01-196 treatment after MI could be beneficial to decrease cardiomyocyte hypoxia.³⁴

Chronic LIT01-196 treatment significantly attenuated increases in LV diameter and volume, thereby decreasing LV dilation. It also prevented the decrease in the thickness of the LV posterior wall induced by MI. These changes demonstrate a cardioprotective role for LIT01-196 in cardiac remodelling and are consistent with previous studies, that have shown that chronic apelin-13 treatment post MI in mice decreases infarct size, improves cell survival, and promotes angiogenesis.³⁵,³⁶ In line with this hypothesis, chronic treatment with LIT01-196 almost doubled cardiac vascular density, thereby improving oxygen and nutrient delivery to the cardiomyocytes located in the border zone of the infarcted area, favouring their survival.

Adverse cardiac remodelling is also associated with increases in interstitial collagen deposition, fibrosis, and fibrosis-related gene expression. The most striking effect of LIT01-196 is its ability to decrease cardiac fibrosis strongly throughout the heart; this effect was also observed after the intramyocardial injection of a lentivector-expressing apelin in a mouse MI model.²⁹ TGF-β1 plays a key role in cardiac fibroblast activation after MI,³⁷ increasing the expression of sphingosine kinase (Sphk1), which
mediates TGF-β1-stimulated collagen production.³⁸ In our study mRNA levels for the fibrosis biomarkers Ctgf, Col I, Col III, TGF-β1, α-Sma, and Sphk1 increased considerably in the heart 4 weeks post MI, but these levels were significantly decreased by LIT01-196. Apelin inhibits the TGF-β1-stimulated activation of cardiac fibroblasts by inhibiting α-Sma activation, Sphk1 upregulation, and collagen production, but also by directly inhibiting SphK1 activity through an AMP-activated protein kinase-dependent mechanism.²¹ We therefore propose the following mechanism of action for the LIT01-196-induced decrease in fibrosis (Fig. 7). LIT01-196 activates ApelinR, thereby reducing cardiac fibrosis post MI by inhibiting cardiac SphK1 and TGF-β1 mRNA production, inducing a decrease in α-Sma and Ctgf mRNA levels, with Ctgf expression being correlated with disease severity.³⁹

The advantage of our compound compared with non-
peptidic ApelinR agonists lies in the addition of the fluoro-
carbon chain on the N-terminal part of K17F. This
modification does not change the properties of the endoge-
nous peptide to bind or to activate the different signalling
pathways of ApelinR, ensuring its specificity and selectivity
toward this receptor. In a post MI-induced HF model,
chronic LIT01-196 treatment improved systolic and diastolic
function without decreasing arterial BP, reduced cardiac
fibrosis, increased vascular density, and attenuated cardiac
hypertrophy. The last 2 effects were not reported with the
nonpeptidic ApelinR agonists.²⁶,³² Furthermore, chronic
LIT01-196 treatment corrected Apln and Aplnr mRNA def-
icits induced by severe HF. Thus, the synergy between the
endogenous peptide apelin and LIT01-196 on the ApelinR
ensures effective cardiac protection post MI. Notably, no
tolerance to the beneficial effects of LIT01-196 on cardiac
function was observed after chronic treatment. Additionally,
like the potent type 2 vasopressin receptor antagonist, tol-
vaptan, LIT01-196 increased aqueous diuresis,²⁴ helping to
reduce preload and improving diastolic function.

Limitations

Our study has several limitations. It should be noted that only
male mice were used in the experiments. Another limitation is
the 2.5-hour half-life of LIT01-196 and the fact that we only
administered it once a day. Although we showed that LIT01-196
blood levels remained 125 times higher than its affinity for
ApelinR, even 8 hours after administration, it would be worth
investigating whether cardiac protection induced by LIT01-196
could be better if giving it twice a day during 4 weeks in the MI
model. In this regard, it would be also important to generate
potent metabolically resistant apelin analogues with improved
*in vivo* Absorption, Distribution, Metabolism, and Excretion
properties. In addition, because mortality is fairly high in the MI
model, there might be a survival bias despite that we analyzed a
large number of animals for each group. Finally, evaluation of
LIT01-196 in another HF model such as a hypertension-
induced HF model, would be useful.

Conclusions and Perspectives

Our study demonstrates the high efficacy of chronic sub-
cutaneous administration of LIT01-196, a potent and selective
full ApelinR agonist, in mitigating post-MI cardiac dysfunc-
tion. The sustained elevation of LIT01-196 concentrations in

the bloodstream, well above its affinity for ApelinR, highlights
its pharmacokinetic advantage, providing a prolonged thera-
peutic window for improving LVEF, cardiac contractility, and
intracardiac pressure. Notably, LIT01-196 treatment yielded a
triple benefit: it simultaneously reduced fibrosis, increased
vascular density, and prevented LV wall thinning, thereby fa-
voring cardiomyocyte survival and attenuating adverse
myocardial remodelling post MI. The robust preclinical out-
comes presented herein warrant further exploration of the safety
and efficacy of ApelinR activation in clinical settings, especially
in patients recovering from acute MI.

Acknowledgements

Special thanks to the animal facility and the Orion imaging
facility of the Center for Interdisciplinary Research in Biology,
Collège de France and its staff for their help and guidance. The
English text was edited by J. Sappa from Alex Edelman &
Associates.

Ethics Statement

All procedures conformed to the guidelines of Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes or the current US National Institutes of Health guidelines. The experimental protocols were approved by the French Committee on Ethics in Animal Experimentation (CEEA no 59, Ref APAFIS#22274-2019080111288819).

Patient Consent

The authors confirm that this is a research article involving
animal experiments, with the ethical protocol reviewed and
approved by the relevant committee; therefore, patient con-
sent is not applicable.

Funding Sources

This work was supported by the Institut National de la
Santé et de la Recherche Médicale, the Centre National de la
Recherche Scientifique, the Collège de France, the Fédération
Française de Cardiologie, La Fondation pour la Recherche
Médicale, and the Sorbonne Université. P.-E.G.-S. was sup-
ported by a fellowship from the Fondation pour la Recherche
Médicale (grant number PBR201810007643).

Disclosures

The authors have no conflicts of interest to disclose.

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## Supplementary Material

To access the supplementary material accompanying this article, visit the online version of the *Canadian Journal of Cardiology* at www.onlinecjc.ca and at https://doi.org/10.1016/j.cjca.2024.11.034.