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Journal of Molecular and Cellular Cardiology Plus logoLink to Journal of Molecular and Cellular Cardiology Plus
. 2025 Sep 19;14:100485. doi: 10.1016/j.jmccpl.2025.100485

Ac-SDKP and eplerenone confer additive cardioprotection against angiotensin II-induced cardiac injury in C57BL/6J mice

Suhail Hamid a,b, Sarah Sarkar a,b, Hongmei Peng a,b, Khalid Matrougui c, Pablo A Ortiz a,b, Jiang Xu a,b, Tang-Dong Liao a,b, Robert A Knight d, Nour-Eddine Rhaleb a,b,⁎
PMCID: PMC12495342  PMID: 41048927

Abstract

Eplerenone, a mineralocorticoid receptor antagonist, is an anti-hypertensive and cardioprotective drug. We showed that N-Acetyl-Seryl-Aspartyl-Proline (Ac-SDKP) exerts beneficial effects on the heart. Whether Ac-SDKP provides additional cardioprotective effects if combined with eplerenone in angiotensin II (Ang II)-induced hypertension remains unknown. Male C57BL/6J mice were treated with either sham, Ang II (2.9 mg/kg/day, s.c.), Ang II + Ac-SDKP (1.6 mg/kg/day, s.c.), Ang II + Eplerenone (150 mg/kg/day in mouse chow), or Ang II + Ac-SDKP + Eplerenone. Treatment lasted for eight weeks. Systolic blood pressure (SBP) measurements were taken weekly. Echocardiography (Echo) and magnetic resonance imaging (MRI) were performed at the end of the experiment. SBP was increased in all mice with Ang II and was not affected by any treatment. Posterior wall thickness (PWT) and left ventricular (LV) mass were increased in Ang II–treated groups. LV mass was not significantly affected by treatment, but PWT was reduced by both monotherapies and showed the greatest reduction with combined Ac-SDKP and eplerenone. Ejection fraction (EF) decreased in the Ang II group compared to the sham group. EF increased with all treatments (MRI), and there was a further significant increase in EF for mice treated with Ac-SDKP + Eplerenone compared to those receiving a single treatment (Echo). Our data indicate that treatment with Ac-SDKP or Eplerenone improves cardiac function in Ang II-induced hypertension, and supplying Ac-SDKP to Eplerenone provides additive, not synergistic, cardioprotective effects. These beneficial effects were associated with decreased myocardial collagen accumulation, CD68-positive macrophage infiltration, and the expression of CHOP, an endoplasmic reticulum stress mediator. Ac-SDKP could be an effective supplementary treatment alongside Eplerenone for managing hypertension-associated cardiac damage and dysfunction.

Keywords: Ac-SDKP, Eplerenone, Cardiac dysfunction, Angiotensin II, Hypertension

Graphical abstract

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Highlights

  • •

    First preclinical evidence that combining Ac-SDKP with eplerenone provides additive cardioprotection in Ang II–induced hypertensive cardiac injury.

  • •

    Cardioprotective effects occurred independently of blood pressure lowering, emphasizing direct myocardial actions.

  • •

    Combination therapy restored ejection fraction and improved cardiac remodeling, confirmed by both echocardiography and MRI.

  • •

    Treatments reduced fibrosis, inflammation (CD68+ macrophages), ER stress (CHOP), and apoptosis, while enhancing Akt pro-survival signaling.

  • •

    Findings support Ac-SDKP as a safe adjunct to eplerenone for potential therapeutic use in hypertensive heart disease and HFpEF.

1. Introduction

Hypertension is a major risk factor for cardiovascular disease and a leading cause of heart failure (HF), a condition with high morbidity and a five-year mortality rate approaching 50 % [1]. The progression from hypertension to HF involves maladaptive cardiac remodeling characterized by inflammation, cardiomyocyte disarray, fibrosis, and reduced vascularization [2,3].

While both agents are cardioprotective through distinct mechanisms, Ac-SDKP through antifibrotic and anti-inflammatory actions, and eplerenone through mineralocorticoid receptor blockade, their combined use may yield additive or synergistic effects by targeting multiple pathways within cardiac remodeling.

Aldosterone, a key effector of the renin-angiotensin-aldosterone system (RAAS), is produced in the adrenal cortex and cardiovascular tissues, including the heart and vasculature. It contributes to HF pathophysiology by promoting inflammation, myocardial fibrosis, hypertrophy, and arrhythmias [[4], [5], [6]]. Elevated aldosterone levels are associated with increased mortality in HF patients, as demonstrated in the CONSENSUS trial [7]. While spironolactone, a non-selective mineralocorticoid receptor antagonist, reduces cardiac fibrosis, its off-target effects—including gynecomastia and menstrual irregularities—limit clinical utility. Eplerenone (Eple), a selective aldosterone blocker with fewer endocrine side effects, has emerged as a better-tolerated alternative [9]. However, its role in attenuating myocardial fibrosis and dysfunction in the setting of hypertension-induced injury and remodeling, or in chronic heart failure, remains incompletely defined.

Although ACE inhibitors (ACEi) can reduce aldosterone synthesis, this effect is often incomplete. Approximately 20 % of patients with chronic HF demonstrate elevated aldosterone levels despite ACEi therapy, a phenomenon known as “aldosterone escape” [7,8]. In addition to angiotensin II (Ang II), other factors—such as corticotropin, potassium, endothelin, catecholamines, and nitric oxide—can also stimulate aldosterone secretion [[8], [9], [10], [11], [12], [13]], underscoring the need for therapeutic strategies that target multiple nodes within the RAAS.

Several groups, including ours, have previously reported the cardioprotective effects of N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP), an endogenous tetrapeptide derived from thymosin-β4 (Tβ4) by sequential cleavage via meprin-α and prolyl oligopeptidase [[14], [15], [16], [17], [18], [19]]. Ac-SDKP is rapidly degraded by ACE, and its circulating levels increase significantly with ACEi treatment [20]. Using rodent models of hypertension and MI, we and others have shown that Ac-SDKP exerts anti-inflammatory, anti-fibrotic, and pro-angiogenic effects in the heart [[21], [22], [23], [24], [25]]. These actions reduce cardiac rupture, ER stress, inflammatory infiltration, and interstitial collagen accumulation. Importantly, myocardial levels of Tβ4 and Ac-SDKP are significantly reduced in patients and animals with advanced HF, suggesting a pathological deficiency in this protective pathway.

To comprehensively assess cardiac remodeling, we employed both echocardiography and cardiac MRI. This combined approach allowed for the evaluation of both functional and structural parameters, enhancing the rigor and reliability of our findings. [[26], [27], [28]]

Given its favorable safety profile, even at high doses, and absence of known adverse effects, Ac-SDKP represents a promising adjunct to existing therapies for HF. We hypothesize that Ac-SDKP, when combined with eplerenone, will enhance cardioprotection by modulating multiple aspects of RAAS signaling. In this study, using an Ang II-induced mouse model of hypertension and cardiac remodeling, we investigated (1) whether eplerenone alone attenuates myocardial fibrosis and improves cardiac function, and (2) whether co-treatment with Ac-SDKP provides additive or synergistic benefit beyond either agent alone. These investigations are conceptually aligned with prior findings of dynamic LV remodeling after cardiac injury [29] and aim to inform future therapeutic strategies targeting HF progression at multiple mechanistic levels.

2. Materials and methods

2.1. Animals and experimental design

Twelve-week-old male or female C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME) were divided into five groups: 1) Sham, 2) Ang II, 3) Ang II + Ac-SDKP, 4) Ang II + eplerenone, and 5) Ang II + eplerenone + Ac-SDKP. Mice were anesthetized with 1.5–3 % isoflurane. An ALZET osmotic minipump (Model 2004, Durect, Cuppertino, CA) was implanted under aseptic conditions to deliver Ang II (2.9 mg · kg−1 · day−1, Bachem, Torrance, CA), Ac-SDKP (1.6 mg · kg−1 · d−1, Genescript, Piscataway, NJ) or vehicle (saline plus 0.01 N acetic acid). Eplerenone (150 mg · kg−1 · d−1, Breckenridge Pharmaceutical, Inc., Berlin, CT) was given in rodent diet (TestDiet, Richmond, IN) [[30], [31], [32]]. The treatment lasted for 8 weeks. In the present study, we selected the dose of Ac-SDKP at 1.6 mg/kg/day based on our previous studies and reported equivalent surface area dosing conversion factors [24,[33], [34], [35], [36], [37], [38]]. This study was approved by the Henry Ford Hospital Institutional Animal Care and Use Committee (IACUC).

2.2. Delivery of vehicle, Ang II, Ac-SDKP, or eplerenone

Mice were anesthetized with 1.5–3.0 % isoflurane, then prepared for surgery by shaving the back area of the neck and disinfecting with povidone‑iodine (betadine®). A small incision (< 1 cm) was made in the interscapular region to create a subcutaneous pocket, where an osmotic pump was inserted to deliver the vehicle, Ang II, and/or Ac-SDKP. The incision was closed with a 4–0 resorbable suture, and the skin incision site was closed with wound clips (MikRon Precision, Inc., Gardena, CA). Mice were then housed in a temperature and humidity-controlled environment with ad libitum access to food and water. Eplerenone was administered at a dose of 150 mg. kg-1.day-1, mixed into the standard chow. This dose was selected based on body surface area normalization, as described by Freireich et al. [33], and is consistent with previous murine studies demonstrating efficacy without toxicity [31,32,39]. Daily food intake was monitored and found to be consistent across treatment groups. No signs of toxicity, weight loss, or distress were observed in the treated mice.

2.3. Measurement of systolic blood pressure (SBP)

SBP was measured weekly in conscious mice using a noninvasive computerized tail-cuff system (BP-2000, Visitech, Apex, NC) as described previously [40,41].

2.4. Cardiac remodeling and function measurements by Echo and MRI

Cardiac geometry and function were determined in non-anesthetized mice. Diastolic left ventricular dimensions (LVDd) and areas (LVAd), diastolic posterior wall thickness (PWTd), LV ejection fraction (EF), and shortening fraction (SF) were measured using a Doppler Echo with a 15-MHz linear transducer (Acuson c256, Mountain View, CA), as described previously [42].

Cardiac magnetic resonance imaging (MRI) was performed using a 7-Tesla Varian magnet (Varian, Palo Alto, CA) interfaced with a Bruker console running Paravision 6.0 (Bruker BioSpin, Billerica, MA). Mice were anesthetized with 1.0–2.0 % isoflurane in a 3:1 N2O:O2 gas mixture and maintained on a heated imaging platform [28]. Respiratory and ECG signals were continuously monitored.

All animals underwent a baseline scan starting with a three-plane localizer sequence to align the heart in standard orientations. A prospective intragate CINE MRI sequence (Bruker BioSpin, Billeria, MA) was then acquired to evaluate left ventricular (LV) function. CINE imaging captured a series of static images across multiple phases of the cardiac cycle and reconstructed them into dynamic cine loops for functional assessment. A black-blood gradient echo technique was employed to suppress intravascular signal and enhance myocardial contrast. Imaging parameters were as follows: repetition time (TR) = 20 ms, echo time (TE) = 2.6 ms, field of view (FOV) = 25 mm, matrix size = 256 × 256, slice thickness = 1 mm, and acquisition time ≈10 min per scan.

Images were sorted and reconstructed using Bruker's software suite. Quantitative analysis was performed using Segment software (Medviso AB, Lund, Sweden), which enabled automated detection of end-diastolic and end-systolic frames. The following parameters were calculated: EF (EF), end-diastolic volume (EDV), end-systolic volume (ESV), and stroke volume (SV). All MRI analyses were performed by an investigator blinded to group allocation. Animals were randomly assigned to treatment or control groups using a coded numeric scheme prior to imaging. Physiological parameters (e.g., body temperature) were maintained within normal limits throughout the procedure.

2.5. Organ harvest

At the end of the experiment, animals were weighed and anesthetized with pentobarbital sodium (100 mg. kg−1, i.p.). The heart was stopped at diastole by intraventricular injection of 15 % KCL, and rapidly excised. Left ventricle (LV, including the septum) was weighed and sectioned transversely into 3 slices from apex to base. The base slide of the LV was fixed in 10 % formaldehyde solution for morphological studies. The apex slice was rapidly frozen in isopentane precooled in liquid nitrogen and then stored at −80 °C for histology studies. Another slice was snap-frozen and stored at −80 °C for molecular biology studies. The right leg was excised, the tibia muscles removed, and tibia length (TL) measured.

2.6. Plasma Ac-SDKP levels

At the end of each experimental protocol, mice were euthanized by an overdose of sodium pentobarbital (100 mg−1. kg−1, i.p.). Blood was withdrawn via the vena cava with a 1-ml syringe coated with heparin containing captopril (final concentration 10−5 M) and centrifuged at 1600 xg for 20 min at 4 °C. Plasma was collected and stored at −20 °C. Plasma Ac-SDKP concentrations were quantified using an Ac-SDKP enzyme immunoassay kit (SPI-BIO, Massey Cedex, France) and expressed in nM, as previously described [20,23,24,36].

2.7. Determination of interstitial collagen

The 6-μm LV sections were deparaffinized, rehydrated and stained with picrosirius red using a modification of Sweat and Puchtler's method [36,43]. The images of collagen morphometry were captured using a microscope (IX81; Center Valley, PA) equipped with a digital camera (DP70; Olympus American). Interstitial collagen fraction (ICF) was analyzed with Microsuite Biological Imaging software (Olympus) and expressed as percentage of interstitial collagen area in the total area of myocardium.

2.8. Immunohistochemical staining

The 6-μm LV cryosections were fixed in cold acetone and then incubated in 0.3 % hydrogen peroxide to quench endogenous peroxidase activity. They were then preincubated in blocking solution (5 % BSA PBS) for 30 min at room temperature and then incubated with a rat anti-mouse CD68 antibody (1:300, #MCA1957, Bio-Rad Laboratories, Hercules, CA) at 4 °C overnight. Sections were incubated with biotinylated rabbit anti-rat IgG antibody (Vector Laboratories, Burlingame, CA), and then with a VECTASTAIN ABC reagent (Vector Laboratories). Sections were developed with 3-amino-9-ethylcarbazole and counterstained with hematoxylin. The images of the sections were captured using a microscope (IX81; Olympus American, Melville, NY) equipped with a digital camera (DP70; Olympus American) and quantified with Microsuite Biological imaging software (Olympus). Positive cells showed red chromogens staining around blue nuclei. Sections were examined by two investigators in a blinded manner. The numbers of CD68+ cells were counted and expressed as numbers of cells/mm2.

2.9. Western blot to detect CHOP, phospho-Akt (p-Akt), caspase-3 and transforming growth factor-beta1,2,3 (TGF-β1,2,3)

LV lysate samples were prepared as we previously described [44]. 60 μg LV lysates were subjected to 10 % sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing (2-mercaptoethanol) condition for detections of CHOP, p-AKT, AKT and caspase-3, or non-reducing condition for detection of TGF-β1,2,3. The nitrocellulose membranes were immunoblotted overnight at 4 °C with the following primary antibodies: A rabbit antibody against caspase-3 (1:1000, #9662, Cell Signaling Technology, Danvers, MA), p-Akt (ser473) (D9E, 1:1000, #4060, Cell Signaling Technology), or a mouse monoclonal antibody against CHOP (L63F7, 1:1000, #2895, Cell Signaling Technology), TGF-β1,2,3 (1D11, 1 μg/mL, #MAB1835, R & D Systems, Minneapolis, MN). Bound antibodies were visualized using a secondary antibody conjugated to horseradish peroxidase (Cell Signaling Technology) and SuperSignalTM West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Rocklford, IL) according to the manufacturers' instructions. After detection of p-Akt, the membrane was striped with RestoreTM Western Blot Stripping Buffer (#21059, Thermo Scientific), and re-blotted with a rabbit monoclonal antibody against Akt (pan) (C67E7, 1:1000, #4691, Cell Signaling Technology). All the other membranes were re-blotted with a rabbit antibody against GAPDH (#2118, Cell Signaling Technology). Band intensity was quantified with ImageJ. P-Akt was normalized to Akt, and all detected other parameters to GAPDH. The results expressed as fold change compared with sham.

2.10. Histochemistry

LV sections were stained with fluorescein-labeled peanut agglutinin and rhodamine-labeled Griffonia simplicifolia lectin I (GSL I) to assess interstitial collagen fraction (ICF) and capillary density, as previously described in our published studies [25,[44], [45], [46]]. Briefly, three slices per frozen section were pretreated with neuraminidase (3.3 U/mL) for 50 min at room temperature, washed in PBS, then incubated with a mixture of fluorescein-labeled peanut agglutinin (0.12 mg/mL) and rhodamine-labeled GSL I (0.02 mg/mL). Total area, interstitial space, and capillary area were quantified using Microsuite Biological Imaging software. ICF was calculated as the interstitial area minus capillary area, expressed as a percentage of total myocardial area. Capillary density was determined as the number of capillaries per mm2. Data were averaged from an average of eight randomly selected fields across total area.

2.11. Statistical analysis

Data are presented as mean ± SEM unless otherwise specified. Normality of data distribution was assessed using the Shapiro–Wilk test, and homogeneity of variance was evaluated with Levene's test. For systolic blood pressure (SBP), comparisons between groups were performed using the two-sample Wilcoxon test, with Hochberg's correction applied for multiple comparisons. For all other outcomes, group differences were analyzed using one-way Analysis of Variance (ANOVA) followed by Šídák's post hoc test for multiple comparisons, as indicated in the Figure Legends. Statistical significance was defined as p < 0.05 (two-tailed). Analyses were conducted using GraphPad Prism (version 10), except for SBP analyses, which were performed separately.

3. Results

3.1. Ac-SDKP and eplerenone do not lower blood pressure but attenuate cardiac remodeling in Ang II-induced hypertension

SBP significantly increased in all Ang II-treated groups compared to control, with no significant reduction by Ac-SDKP, eplerenone, or their combination (Fig. 1A). Plasma Ac-SDKP levels were markedly elevated in the Ang II + Ac-SDKP and Ang II + Ac-SDKP + eplerenone groups, confirming effective systemic delivery (Fig. 1B).

Fig. 1.

Fig. 1

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Systolic blood pressure, plasma Ac-SDKP (Ac-S), and echocardiographic/structural parameters in mice with Ang II–induced hypertension. (A) Systolic blood pressure (SBP; n = 9–10/group) was significantly increased in all Ang II–treated groups and was not affected by Ac-SDKP, eplerenone (Epl), or their combination. (B) Plasma Ac-SDKP levels (n = 9–10/group) were significantly elevated in groups receiving Ac-SDKP, confirming effective delivery. Echocardiographic indices of cardiac function and remodeling (n = 14–15/group): ejection fraction (EF; C), shortening fraction (SF; D), and cardiac index (CI; E) were significantly reduced in Ang II–treated mice and partially restored by treatment, most effectively with combination therapy. Diastolic left ventricular dimension (LVDd; F) remained unchanged, whereas increased posterior wall thickness (PWTd; G) in Ang II–treated mice was attenuated by treatment. (H) LV weight/tibia length ratio (LVW/TL; n = 9–10/group) was significantly increased in Ang II–treated mice and was not affected by Ac-SDKP, eplerenone, or their combination. (I) LV collagen content (n = 5–7/group) was elevated in Ang II–infused mice and reduced by both Ac-SDKP and eplerenone. All data are shown as mean ± SEM. SBP Data in A was analyzed by two-sample Wilcoxon test with Hochberg's corrections. All data were examined using one-way ANOVA with Šídák's test for multiple comparisons except in A.

Echo assessment revealed that Ang II significantly reduced EF (Fig. 1C) and SF (Fig. 1D), both of which were partially restored by either Ac-SDKP or eplerenone and fully normalized with combination therapy. CI was also significantly decreased in Ang II-treated mice and improved with combined treatment (Fig. 1E). Structural remodeling was evident by increased diastolic posterior wall thickness (PWTd; Fig. 1G) and left ventricular (LV) collagen content (Fig. 1I), both of which were attenuated by Ac-SDKP, eplerenone, or their combination. While the LV weight-to-tibia length ratio (LVW/TL; Fig. 1H) was elevated in the Ang II group, it was not significantly altered by any treatment. Left ventricular diastolic diameter (LVDd; Fig. 1F) remained unchanged across all groups, indicating preserved chamber dimensions.

MRI-based measurements further confirmed these functional and structural changes. End-systolic mass (ESM) was not significantly different among groups (Fig. 2A), but end-systolic volume (ESV) was significantly elevated in Ang II-treated mice and reduced by all treatments, particularly with the combination (Fig. 2B). Stroke volume (SV) was modestly increased only in the combination group (Fig. 2C). End-diastolic volume (EDV; Fig. 2D) and end-diastolic mass (EDM; Fig. 2E) exhibited trends toward reduction with treatment, though not all reached statistical significance. Importantly, EF measured by MRI mirrored the Echo findings: Ang II significantly reduced EF, while all treatments improved it, with the combination restoring EF to control levels (Fig. 2F).

Fig. 2.

Fig. 2

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MRI assessment of cardiac structure and function in Ang II-induced hypertensive mice treated with Ac-SDKP (Ac-S) and/or eplerenone (Epl). Left ventricular parameters were assessed using cardiac MRI after 8 weeks of treatment. (A) End-systolic mass (ESM) was unchanged across groups. (B) End-systolic volume (ESV) was significantly increased in Ang II-treated mice and reduced by Ac-SDKP, eplerenone, and their combination. (C) Stroke volume (SV) was preserved only in the combination treatment group. (D) End-diastolic volume (EDV) showed a non-significant trend toward reduction in treated groups. (E) End-diastolic mass (EDM) was elevated in Ang II mice and reduced by Ac-SDKP. (F) Ejection fraction (EF) was significantly reduced by Ang II and improved by all treatments, with the combination therapy restoring EF to control levels. All data are shown as mean ± SEM, n = 3–10/group. Data were examined using one-way ANOVA with Šídák's test for multiple comparisons.

Together, these data demonstrate that Ac-SDKP and eplerenone, especially when combined, significantly attenuate Ang II-induced cardiac dysfunction and remodeling, with consistent results across both Echo and MRI modalities.

3.2. Ac-SDKP and eplerenone reduce cardiac fibrosis

Picrosirius Red staining revealed extensive interstitial collagen deposition in Ang II-treated mice, which was significantly reduced by Ac-SDKP, Eplerenone, and especially their combination therapy. Quantification of the interstitial collagen fraction (ICF) confirmed the antifibrotic effects of all treatments, with the combination therapy providing the most pronounced reduction (Fig. 3A–B).

Fig. 3.

Fig. 3

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Cardiac fibrotic remodeling in the hearts of C57 BL/6 J mice treated with Ac-SDKP (Ac-S), eplerenone (Epl) or Ac-S + Epl in Ang II-induced hypertension. (A) Representative images of interstitial fibrillar collagen (red) in the left ventricle of C57 BL/6 J mice after 8-week treatment. (B) Quantification of interstitial collagen fraction (ICF) of the study animals. All data are shown as mean ± SEM, n = 9–10/group. Data were examined using one-way ANOVA with Šídák's test for multiple comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.3. Treatment reduces cardiac infiltration of CD68+ macrophages

Immunohistochemical analysis demonstrated marked accumulation of CD68+ macrophages in the myocardium following Ang II infusion. All treatment groups showed a significant decrease in macrophage infiltration (a marker of inflammation), with the combined Ac-SDKP + Eplerenone group showing the lowest levels (Fig. 4A–B), suggesting potent anti-inflammatory effects.

Fig. 4.

Fig. 4

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The appearance of CD68+ macrophages in the hearts of C57 BL/6 J mice treated with Ac-SDKP (Ac-S), eplerenone (Epl) or Ac-S + Epl in Ang II-induced hypertension. Representative images of immunohistochemical staining for CD68+ macrophages (red brown; A) in the myocardium of mice with the treatment for 8 weeks. (B) Quantitative analysis of CD68+ macrophages. All data are shown as mean ± SEM, n = 9/group. Data were examined using one-way ANOVA with Šídák's test for multiple comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.4. Capillary density and reduced fibrosis with combined therapy

Imaging of GSL I-stained capillaries and peanut agglutinin-stained collagen showed that Ang II reduced capillary density and increased fibrosis. Both Ac-SDKP and Eplerenone partially restored capillary density and reduced collagen accumulation. However, the combination therapy failed to provide additional improvement of the microvascular architecture and reducing interstitial fibrosis (Fig. 5A–B).

Fig. 5.

Fig. 5

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Capillary density and interstitial collagen in the hearts of C57 BL/6 J mice treated with Ac-SDKP (Ac-S), eplerenone (Epl) or Ac-S + Epl in Ang II-induced hypertension. (A) Representative images of capillaries (red) and interstitial collagen (green). (B) Quantitative analysis of capillary density (Left panel) and interstitial collagen fraction (ICF, right panel). All data are shown as mean ± SEM, n = 5–7/group. Data were examined using one-way ANOVA with Šídák's test for multiple comparisons. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.5. Combination therapy modulates stress, fibrosis, and survival signaling pathways

Western blot analysis revealed that CHOP, TGF-β, and caspase-3 protein levels were elevated in Ang II-treated hearts and significantly reduced by Ac-SDKP, Eplerenone, and their combination. In parallel, phospho-AKT (P-AKT), a marker of pro-survival signaling, was increased in all treated groups, with the highest levels seen in the combination group (Fig. 6A–D). These findings suggest that treatment modulates ER stress, apoptosis, and survival pathways, independent of blood pressure reduction.

Fig. 6.

Fig. 6

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CHOP, TGF-β1,2,3, caspase 3, and phospho-AKT (P-AKT) expression in the hearts of C57 BL/6 J mice treated with vehicle (V), Ac-SDKP (Ac-S), eplerenone (Epl) or Ac-S + Epl in Ang II-induced hypertension. CHOP (A), TGF-β1,2,3(B), caspase 3 (C), and P-AKT (D) in the heart were analyzed by Western blot, the left panels of A, B, C, or D exhibit representative blots, and the quantitative data of corresponding blot show in the right panels. All data are shown as mean ± SEM, n = 5–7/group. Data were examined using one-way ANOVA with Šídák's test for multiple comparisons.

4. Discussion

This study demonstrates that co-administration of Ac-SDKP and eplerenone yields additive cardioprotective effects in a mouse model of Ang II-induced hypertension, independent of blood pressure lowering. While both agents individually improved cardiac remodeling and function, their combination resulted in superior outcomes across structural, functional, and molecular domains.

Echocardiographic and MRI analyses revealed that the combination therapy provided the most robust improvements in ejection fraction (EF), stroke volume (SV), and cardiac index (CI), along with significant reductions in posterior wall thickness, left ventricular (LV) mass, and collagen content (Fig. 1, Fig. 3). These enhancements were paralleled by decreased CD68+ macrophage infiltration (Fig. 4), restoration of capillary density (Fig. 5), and reduced expression of key stress and fibrotic markers such as CHOP, caspase-3, and TGF-β1,2,3 (Fig. 6). The highest levels of phosphorylated Akt in the combination group further highlight enhanced activation of pro-survival signaling pathways.

Although echocardiography and MRI offer complementary insight into cardiac structure and function, differences in resolution, assumptions, and anesthesia effects can yield modest discrepancies in parameters such as EF, mass, and volume [[26], [27], [28]]. Nonetheless, both imaging modalities consistently supported the cardioprotective benefit of the combination therapy, underscoring the robustness of the findings.

A key methodological difference between MRI and echocardiography lies in accuracy and assumptions. MRI offers superior spatial resolution and directly measures volumes from anatomical slices, making it more precise for assessing end-diastolic volume (EDV), end-systolic volume (ESV), and EF. In contrast, Echo often relies on geometric assumptions and can overestimate EF, especially in small animals like mice, due to limited resolution and angle dependency.

Using both cardiac MRI and echocardiography allows for a complementary assessment of cardiac structure and function, enhancing the rigor of our findings. Echo offers practical, high-throughput evaluation of cardiac performance in conscious or lightly anesthetized mice, providing dynamic measures such as EF and CI. However, its reliance on geometric assumptions and operator variability may limit the accuracy of volume and mass assessments. Cardiac MRI, with its superior spatial resolution and direct volumetric measurements, serves as the gold standard for assessing ventricular volumes and mass. It provides anatomically precise and reproducible data, particularly useful for validating changes observed by echocardiography [[26], [27], [28]]. Anesthesia also impacts measurements: MRI requires anesthesia, which lowers preload and heart rate, potentially underestimating EDV and EF. Echo performed in awake or lightly anesthetized animals better reflects physiological function but is more prone to motion artifacts and stress variability.

Biologically, stroke volume (SV) and related metrics may diverge between modalities. MRI-derived SV may appear lower due to reduced contractility under anesthesia, while Echo-derived CI may be higher under more natural loading conditions. Echo tends to overestimate EF based on wall motion, whereas MRI provides volumetric accuracy. Additionally, MRI captures LV mass more reliably in fibrotic or edematous tissue, while Echo may miss regional hypertrophy due to limitations in wall thickness assessment.

Despite these differences, both techniques consistently demonstrated improved function and structure with Ac-SDKP and eplerenone, validating the robustness of the findings through complementary approaches.

Importantly, neither treatment lowered systolic blood pressure, affirming that the observed benefits stemmed from direct myocardial actions rather than hemodynamic changes. This observation is consistent with previous reports showing that Ac-SDKP exerts antifibrotic and anti-inflammatory effects without affecting blood pressure [16,24]. Nevertheless, more studies are needed to directly determine whether Ac-SDKP exerts its effects by regulating some or all components of the RAAS system, and whether combining Ac-SDKP or eplerenone with other established cardiac therapies, such as thiazide diuretics or β-adrenergic receptor antagonists, provides additive or synergistic benefits. Both Ac-SDKP and eplerenone exhibit antifibrotic effects. However, they appear to do so through distinct but complementary mechanisms. Ac-SDKP, via its putative receptor(s) [[47], [48], [49]], is recognized for its anti-inflammatory, antifibrotic, and pro-angiogenic properties [[21], [22], [23], [24], [25]], while eplerenone, a selective mineralocorticoid receptor antagonist [[4], [5], [6],31,32,39], primarily acts by blocking mineralocorticoid receptor–mediated profibrotic and proinflammatory signaling. Their combination therefore likely provides a broader inhibition of maladaptive remodeling, including fibrosis and apoptosis, than either agent alone. We acknowledge, however, that further mechanistic studies are warranted to comprehensively document and dissect the downstream pathways through which each agent contributes to these beneficial effects.

Mechanistically, this additive effect may reflect dual targeting of RAAS-dependent and -independent pathways. Ac-SDKP modulates fibroblast activation, immune cell recruitment, and endothelial function [3,21,24,[50], [51], [52], [53], [54]], while eplerenone inhibits aldosterone-mediated injury [31,32,55]. The marked suppression of CHOP, caspase-3, and TGF-β, along with increased Akt phosphorylation, indicates convergence on ER stress and survival signaling pathways [24,32]. Furthermore, the combination may more effectively restore perfusion by enhancing angiogenesis and reducing interstitial stiffness [22,39].

These additive effects may also stem from modulation of distinct cardiac cell types at different times. Ac-SDKP predominantly affects endothelial cells and fibroblasts, whereas eplerenone may act more directly on myocytes and inflammatory cells. Such multi-cellular engagement could underlie the superior histological and functional outcomes observed. Importantly, treated mice exhibited no adverse effects, supporting the safety and tolerability of this regimen in our experimental model. Monotherapies may reach a therapeutic plateau due to compensatory mechanisms; by contrast, the combination appears to overcome these limitations.

Despite the exciting data presented in our study, our manuscript is not without limitations that could be addressed in future work. One important limitation is the absence of Doppler-derived diastolic function indices, specifically the E/e' ratio, which serves as a surrogate for left ventricular filling pressures. Although several structural and functional parameters were assessed, the echocardiography platform used in this study (Acuson c256, Mountain View, CA) did not support the acquisition of tissue Doppler imaging (TDI) required to determine E/e. This technical constraint prevented a more comprehensive evaluation of diastolic dysfunction. Future studies employing advanced echocardiography systems with TDI capabilities are warranted to better characterize the effects of Ac-SDKP, eplerenone or other combined pharmacotherapies on diastolic performance.

Another limitation is the use of a relatively high dose of eplerenone (150 mg. kg−1. Day−1) in mice, which exceeds typical human therapeutic doses on a direct mg/kg basis. However, when adjusted for interspecies differences using body surface area normalization, this dose falls within a translationally relevant range and is consistent with previous preclinical studies [33]. Importantly, no signs of toxicity were observed, including weight loss, behavioral changes, or mortality. In addition, more detailed mechanistic studies are warranted to clarify the potential involvement of other immune cells, apoptosis, and angiogenesis in the beneficial effects of monotherapy versus combination therapy. Finally, future studies should examine dose–response effects and evaluate lower doses to further refine translational applicability.

In conclusion, this study provides compelling evidence that combining Ac-SDKP with eplerenone enhances myocardial protection against Ang II-induced injury. The combination therapy reduces fibrosis, inflammation, ER stress, and apoptosis while restoring vascular integrity and cardiac function. Given Ac-SDKP's favorable safety profile and lack of toxicity even at high doses [16,18,20], its use alongside eplerenone represents a promising therapeutic strategy for patients with hypertensive heart disease or heart failure with preserved ejection fraction.

CRediT authorship contribution statement

Suhail Hamid: Writing – review & editing, Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Sarah Sarkar: Writing – review & editing, Investigation, Data curation. Hongmei Peng: Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation. Matrougui Khalid: Writing – review & editing, Writing – original draft, Validation. Pablo A. Ortiz: Writing – review & editing. Jiang Xu: Writing – review & editing, Methodology, Formal analysis, Data curation. Tang-Dong Liao: Writing – review & editing, Methodology, Data curation. Robert A. Knight: Writing – review & editing, Investigation, Formal analysis, Data curation. Nour-Eddine Rhaleb: Writing – review & editing, Writing – original draft, Validation, Supervision, Resources, Project administration, Methodology, Investigation, Funding acquisition, Formal analysis, Data curation, Conceptualization.

Funding sources

This study was supported by the NIH grants HL136456-01 and in part by the Henry Ford Health System institutional funds A10163 (NER); the NIH-HL150014 (KM), NIH-HL151616 (KM), and NIDDK R01DK131114A1 (PAO).

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Nour-Eddine Rhaleb reports financial support was provided by National Institutes of Health, and in part by the Henry Ford Health System. Pablo A. Ortiz reports financial support was provided by National Institute of Diabetes and Digestive and Kidney Diseases. Dr. Khalid Matrougui reports financial support was provided by National Institutes of Health. Reports a relationship with that includes:. Has patent pending to. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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