Mas receptor activation regulates the functional phenotype of myocardial macrophages through the Akt/Nrf2 signaling pathway to alleviate sepsis-induced cardiomyopathy

Abstract

Background

Sepsis-induced cardiomyopathy (SIC) has high mortality due to uncontrolled cardiac inflammation and macrophage-mediated mitochondrial damage. The Ang-(1–7)/Mas receptor (MasR) axis exhibits anti-inflammatory effects, but its role in modulating macrophage phenotypes in SIC remains unclear. This study aimed to determine if MasR activation confers cardioprotective effects in SIC via the macrophage polarization-regulating Akt/Nrf2 pathway.

Methods

Blood samples from healthy controls and patients with SIC were analyzed for MasR expression on monocytes via flow cytometry. SIC was induced in mice using the cecal ligation and puncture model, with pretreatment using the MasR agonist AVE0991. Cardiac macrophage-specific MasR overexpression was achieved using an AAV9 vector. Cardiac function, myocardial injury, macrophage phenotypes, mitochondrial morphology, and apoptosis were assessed by echocardiography, histopathology, transmission electron microscopy, TUNEL staining, and multiplex flow cytometry. Bone marrow-derived macrophages (BMDM) were exposed to LPS and treated with AVE0991 and the Nrf2 inhibitor ML385 in vitro. The role of the Akt/Nrf2 pathway in macrophage polarization and cardiomyocyte protection was confirmed through Western blot, flow cytometry, and a BMDM-H9c2 cardiomyocyte co-culture system.

Results

MasR expression on monocytes was significantly elevated in patients with SIC and correlated positively with IL-6, lactate, and TnI levels. In SIC mice, pretreatment with AVE0991 or cardiac macrophage-specific MasR overexpression significantly improved survival, enhanced cardiac function, and reduced myocardial injury and apoptosis. Mechanistically, MasR activation enhanced Akt phosphorylation and Nrf2 nuclear translocation in both myocardial tissue and BMDM, promoting a shift in cardiac macrophages from an inflammatory M1 state toward a protective M2 state, thereby reducing inflammatory infiltration. Additionally, MasR activation preserved mitochondrial homeostasis, enhancing mitochondrial membrane potential and ATP production. The Nrf2 inhibitor ML385 completely abolished the protective effects induced by AVE0991. In the co-culture system, conditioned medium from AVE0991-treated BMDM alleviated oxidative stress, mitochondrial damage, and apoptosis in H9c2 cells.

Conclusions

MasR activation alleviates SIC by initiating the Akt/Nrf2 signaling axis, reprogramming cardiac macrophages towards a protective M2 phenotype, thereby reducing myocardial inflammation, enhancing mitochondrial function, and inhibiting cardiomyocyte apoptosis. This study suggests that targeting the MasR-Akt/Nrf2-macrophage axis may offer a novel therapeutic strategy for SIC.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-026-08171-5.

Introduction

Sepsis-induced cardiomyopathy (SIC) is a reversible cardiac injury resulting from sepsis, typically presenting as acute systolic and/or diastolic dysfunction of one or both ventricles, independent of coronary artery disease [1, 2]. The precise pathogenesis of SIC remains unclear, but it may involve excessive inflammation, autonomic nervous system dysfunction, calcium imbalance, oxidative stress, and mitochondrial dysfunction [1, 2]. SIC is associated with high mortality, and currently, effective prevention or treatment options are lacking [3, 4]. Therefore, further investigation into its pathogenesis and the development of novel therapeutic strategies is critical.

Recent work by Wu et al. demonstrated that CD5 ablation in T cells enhances their persistence and antitumor efficacy by mitigating exhaustion and promoting cytotoxic function, highlighting the broad therapeutic potential of targeting immunoregulatory receptors [5]. Macrophages also serve as key immunoregulatory cells and exhibit remarkable plasticity, polarizing into distinct phenotypes that regulate immune responses, and targeted manipulation of this polarization presents a promising strategy for restoring immune balance and treating sepsis [6]. In SIC, cardiac macrophages are fulfilling dual roles as both protectors and disruptors within the cardiac immune microenvironment [7, 8]. Under normal conditions, resident cardiac macrophages maintain tissue homeostasis [7]. However, during the early stages of SIC, these macrophages, along with numerous recruited monocytes, polarize into the pro-inflammatory M1 state, releasing high levels of inflammatory mediators [9–12]. These factors not only impair myocardial contractility but also generate excessive reactive oxygen species (ROS), which damage myocardial mitochondria. This mitochondrial dysfunction leads to uncoupling of oxidative phosphorylation, ATP depletion, and activation of the mitochondria-dependent intrinsic apoptosis pathway [13, 14]. Conversely, alternatively activated M2 macrophages exert anti-inflammatory effects and promote tissue repair [15, 16]. Therefore, promoting the shift of macrophages from the M1 to the M2 phenotype represents a promising immune regulatory strategy to mitigate SIC.

The renin-angiotensin-aldosterone system (RAAS) plays a pivotal role in regulating cardiovascular homeostasis, comprising several interconnected axes where the bioactive molecules exert mutual influence [17–19]. In sepsis, the expression of key RAAS components—such as Angiotensinogen, renin, angiotensin II (Ang II), and Ang-(1–7)—is closely linked to the severity and prognosis of septic patients [20–24]. Ang II binds to the angiotensin II type 1 receptor (AT1R), triggering vasoconstriction, enhanced sympathetic nervous activity, increased oxidative stress, and excessive immune cell infiltration [25]. Evidence suggests that early intervention targeting the Ang II/AT1R axis can reduce immune cell infiltration and regulate macrophage polarization, thus alleviating SIC [26].

Mas, a G protein-coupled receptor encoded by the proto-oncogene Mas1, is widely expressed across various tissues and immune cells in both humans and mice [27]. Activation of the Ang-(1–7)/Mas axis has shown significant cardioprotective effects, including anti-inflammatory, antifibrotic, and antioxidant properties in animal models of hypertension, heart failure, and myocardial infarction [28–30]. Studies have indicated that exogenous Ang-(1–7) can mitigate inflammatory responses and organ dysfunction in sepsis models [31–34]. However, the peptide nature of Ang-(1–7) results in a short half-life, limiting its clinical utility [35, 36]. AVE0991, an analog of Ang-(1–7) that is non-peptide, has a longer half-life and binds specifically to the Mas receptor [37, 38]. It reproduces the physiological actions of Ang-(1–7) within vascular, cerebral, and renal systems and demonstrates protective capabilities such as combating oxidative stress, preventing apoptosis, and reducing inflammation [36, 39, 40]. Recent research has shown that the MasR agonist AVE0991 can modulate microglial/macrophage function and phenotype, effectively alleviating end-organ damage in conditions such as salt-sensitive hypertension and reducing neuroinflammation and neuronal apoptosis in intracerebral hemorrhage (ICH) models [41, 42]. However, it remains unclear whether MasR activation can specifically improve cardiac dysfunction in sepsis and whether this effect is mediated by modulation of the local cardiac immune microenvironment, particularly through macrophage phenotype regulation.

MasR activation may stimulate nitric oxide synthesis and initiate the activity of phospholipase A2 (PLA2), along with key signaling pathways such as phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) and protein kinase A (PKA) [43, 44]. Similar to the engineered T cell receptors discussed by Cheng et al., which require high signaling sensitivity to target low-density intracellular antigens, the efficiency of MasR-mediated signaling in macrophages may also depend on the integrity of downstream signal amplification cascades [45]. Akt plays a central role in regulating cell survival, proliferation, and metabolism. Concurrently, nuclear factor erythroid 2-related factor 2 (Nrf2), a key transcription factor, is essential for cellular defense against oxidative damage by activating the expression of antioxidant and cytoprotective genes [46]. Evidence indicates that phosphorylation of Akt enhances Nrf2 expression, which in turn mediates antioxidant defense, cellular protection, anti-inflammatory responses, and regulation of microglial phenotypes [47, 48]. In the context of SIC, however, it remains unclear whether MasR activation may protect the injured myocardium by reprogramming the functional phenotype of cardiac macrophages via the Akt/Nrf2 signaling axis.

In this study, a positive correlation between MasR expression in monocytes and disease severity was identified in patients with SIC. Next, in an animal model of SIC, the MasR agonist AVE0991 was administered, and a cardiac macrophage-specific MasR overexpression mouse model was created to target the relevant cell type. Lastly, this study employed an in vitro co-culture system of bone marrow-derived macrophages (BMDM) and cardiomyocytes (H9c2), combined with specific inhibitors, to further explore the role of the Akt/Nrf2 axis. This study aims to systematically elucidate the molecular mechanisms by which MasR activation alleviates SIC, providing theoretical support and new targets for the development of drugs aimed at preventing and treating SIC.

Materials and methods

Subjects

Healthy controls (HCs) and patients with SIC were enrolled between 2023 and 2025. Patients with SIC met the following inclusion criteria: (1) diagnosis of sepsis; (2) BNP > 100 pg/mL or Troponin I > 0.08 ng/mL (wherein normal concentrations at the hospital ranged between 0 and 0.08 ng/mL) within 24 h of admission; (3) LVEF < 50% within 24 h of admission; (4) age between 18 and 80 years. The diagnostic criteria for SIC were established based on previously published studies and current clinical guidelines [2, 26, 32, 49, 50]. While there is no universally accepted definition for SIC, the combination of elevated cardiac biomarkers (BNP or Troponin I) and reduced LVEF (< 50%) within 24 h of admission has been widely adopted in clinical research to identify sepsis-associated myocardial dysfunction [26, 32, 49, 50]. Exclusion criteria included prior diagnoses of organ failure, cardiomyopathy, chronic cardiac insufficiency, autoimmune disorders, or malignancy, as well as age < 18 or > 80 years. Blood samples were collected, and clinical data were recorded within 2 h of admission. All participants, or their legal guardians, provided written informed consent, and the research protocol was approved by the Institutional Ethics Committee.

Study design and animal experiment

Male C57BL/6J mice were obtained from Hunan Silek Jingda Experimental Animal Co., Ltd. Mice were randomly assigned to experimental groups prior to intervention. While the operating investigator was unblinded during cecal ligation and puncture (CLP) and drug administration for safety reasons, all outcome assessments—including echocardiography, tissue collection, and histological analyses—were conducted by investigators blinded to group allocation. The study consisted of two parts. In the first part, to examine the effects of MasR activation, mice received intraperitoneal injections of AVE0991 (3 mg/kg) or an equal volume of DMSO vehicle for three consecutive days prior to CLP-induced SIC. The animals were divided into Sham, CLP + Vehicle, and CLP + AVE0991 groups. In the second part, 6-week-old male mice received either Mas-overexpressing AAV9 or a control viral vector (8 × 10¹¹ v.g.) via tail vein injection. After four weeks, transduction efficiency was confirmed by immunofluorescence, followed by CLP to induce SIC. The groups included Sham + NC, Sham + OV, CLP + NC, and CLP + OV.

BMDM culture and intervention

Bone marrow was harvested from the femurs and tibias of C57BL/6J mice, passed through a 40-µm filter, and centrifuged at 300–400 × g. The resulting pellet was resuspended in DMEM containing 10% fetal bovine serum (CellMax, Beijing, China; cat. no. SA211.02), 1% penicillin/streptomycin, and 20 ng/mL M-CSF, and incubated at 37 °C with 5% CO₂. Cells were differentiated into mature macrophages over 7 days with continuous M-CSF induction, and maturation was confirmed by F4/80 and CD11b immunostaining. BMDM were pretreated for 2 h with the MasR agonist AVE0991 (10 µM), its vehicle control (Vehicle1), the NRF2 inhibitor ML385 (5 µM), the Akt inhibitor MK-2206 (2 µM), or their respective vehicle controls (Vehicle2 for ML385, Vehicle3 for MK-2206), either alone or in combination. Subsequently, cells were stimulated with LPS (1 µg/mL) for 24 h, followed by the corresponding analyses. One experimental group was divided into: Control, LPS+Vehicle1, LPS + AVE0991, LPS + AVE0991 + ML385, and LPS + AVE0991 + Vehicle2. The other experimental group was divided into: Control, LPS+Vehicle1 + Vehicle3, LPS+AVE0991 + Vehicle3, and LPS+AVE0991 + MK-2206.

Conditioned medium and cardiomyocyte indirect coculture model

H9c2 cardiomyocytes were indirectly co-cultured with conditioned medium (CM) from BMDM as described previously. BMDM were pretreated for 2 h with AVE0991 (10 µM) or the NRF2 inhibitor ML385 (5 µM), then exposed to LPS (1 µg/mL) for 24 h. The CM was collected, centrifuged, and filtered (0.22 μm) before being applied to H9c2 cells for 24 h. After the co-culture period, apoptosis, ROS levels, and MMP in H9c2 cells were assessed by flow cytometry (FCM).

Laser speckle contrast imaging

Mice were anesthetized with isoflurane and surgically prepared under iodophor disinfection. A ventral or dorsal incision was made to expose target organs (e.g., liver, kidneys, mesentery), which were isolated with gauze. Using a laser speckle contrast imaging (LSCI) system (RWD, China), the field was centered on the tissue with the indicator light set to minimal magnification. Magnification was adjusted, the light was turned off, and perfusion images along with quantitative data were acquired.

Histologic analysis

Myocardial tissue was fixed in 4% PFA, dehydrated, embedded, and sectioned. Following dewaxing, HE staining was performed for light microscopy. Histopathological injury was scored (0–3) based on myocardial structure, fiber alignment, nuclear changes, and interstitial inflammation/edema. Two pathologists conducted blinded evaluations.

Immunofluorescence analysis

Heart sections were dewaxed, blocked with 10% BSA, and incubated overnight at 4 °C with primary antibodies against CD206, Mas, CD86, and F4/80 (all diluted 1:100). After PBS washes, sections were incubated with Cy3- and Alexa Fluor 488-conjugated secondary antibodies for 1 h at 37 °C in the dark. Images were captured using fluorescence microscopy. Heart, liver, spleen, and lung sections from CLP + NC and CLP + OV mice were stained for MasR together with cell-specific markers: cTnI, α-SMA, CD31, or F4/80. Using ImageJ, MasR mean fluorescence intensity (MFI) was measured within regions defined by each marker. Cardiac macrophage transduction efficiency was calculated as the percentage of F4/80 + cells with MasR MFI > mean of NC group. Off-target expression in liver, spleen, and lung was assessed by comparing MasR MFI in F4/80 + cells between groups. All analyses were performed blinded.

Western blotting

Proteins from both cells and mouse myocardial tissues were extracted using RIPA buffer (Solarbio) and homogenized on ice. Protein concentrations were determined using a BCA assay (Beyotime). Samples (40 µg protein) were denatured in loading buffer, separated by SDS–PAGE, and transferred to methanol-activated PVDF membranes (Millipore). After blocking with 5% non-fat milk, membranes were incubated with primary antibodies at 4 °C overnight, followed by secondary antibody incubation. Protein bands were visualized with ECL reagent (Millipore) using a Bio-Rad imaging system and quantified using ImageJ (NIH). Antibody details are provided in the Supplementary Materials. Western blot antibodies included: anti-β-actin (#3700), anti-Bax (#14796), anti-caspase-9 (#9508), anti-cleaved caspase-3 (#9664), anti-Bcl-2 (#3498), anti-iNOS (#13120), anti-Nrf2 (#12721), and anti-Arg-1 (#93668) from Cell Signaling Technology; anti-AKT (#60203-2-Ig), anti-IL-1β (#26048-4-AP), anti-TNF-α (#17590-1-AP), anti-Mas (#20080-1-AP), and anti-p-AKT (#66444-1-Ig) from Proteintech; anti-Mfn2 (#sc-515647) and anti-Drp1 (#sc-271583) from Santa Cruz Biotechnology. All antibodies were used at a 1:1000 dilution unless specified.

Flow cytometry

Apoptosis in H9c2 cells was assessed with an Annexin V-FITC kit (Servicebio), while BMDM polarization was evaluated using fluorescent antibodies: APC-anti-CD86, FITC-anti-CD11b, PE-anti-F4/80, and PB450-anti-CD206. Single-cell suspensions from mouse hearts were adjusted to 1 × 10⁶ cells/mL, blocked with anti-CD16/32 (4 °C, 30 min), and stained in the dark (4 °C, 30 min) with antibodies against CD11b-FITC, CD45-APC-A750, CD86-APC and F4/80-PE. After washing, samples were analyzed on a Beckman flow cytometer. Flow cytometry data were acquired using standardized templates, and gating strategies were predefined based on fluorescence-minus-one (FMO) controls. All analyses were performed by an operator blinded to sample identity to ensure unbiased gating and quantification.

Echocardiography

Twenty-four hours post-modeling, cardiac function was assessed by transthoracic echocardiography on anesthetized mice (1% isoflurane) using a Vinno v6vet system with a 30 MHz transducer. Mice were positioned supine on a heated pad at 37 °C under 1% isoflurane anesthesia. The left ventricular (LV) long-axis view was obtained from the LV outflow tract, followed by adjustment to capture the short-axis view at the papillary muscle level. Two-dimensional and M-mode images were analyzed to determine LV end-systolic volume (LVESV), LV end-diastolic volume (LVEDV), LV internal diameters at end-systole (LVIDs) and end-diastole (LVIDd), as well as LV ejection fraction (LVEF) and LV fractional shortening (LVFS). Echocardiography was performed by a trained technician who was blinded to the experimental groups. All acquired images were analyzed offline by a separate blinded investigator to ensure unbiased measurement of cardiac function parameters.

Transmission electron microscopy (TEM)

The LV myocardium was excised using a sterile scalpel and immediately immersed in 2.5% glutaraldehyde. The tissue was cut into 1 mm³ blocks and fixed in fresh glutaraldehyde for 24 h. Following phosphate buffer rinses, samples were post-fixed in osmium tetroxide, washed, dehydrated, and embedded in resin. Sections were stained and examined by TEM, and mitochondrial injury was semi-quantitatively assessed using Flameng criteria.

Dihydroethidium (DHE) staining

ROS levels in H9c2 cells were assessed using DHE staining according to the manufacturer’s protocol (Beyotime, Shanghai, China). Cells were incubated in a 10 µM DHE solution at 37 °C without serum for 30 min in darkness. FCM was used to measure MFI.

Mitochondrial membrane potential (MMP) assay

MMP was evaluated by Solarbio assay kit in both H9c2 cells and myocardial tissue. Mitochondria were isolated from myocardial samples, and protein concentration was determined using the Bradford method. Isolated mitochondria were incubated with JC-1, and fluorescence intensities of aggregates (Ex/Em: 525/590 nm) and monomers (Ex/Em: 490/530 nm) were measured using a microplate reader (BioTek). MMP was expressed as the aggregate-to-monomer ratio. For H9c2 cells, MMP was assessed by FCM: cells were stained with JC-1, incubated at 37 °C for 20 min, washed, and analyzed.

Statistical analysis

Data normality was evaluated with the Shapiro–Wilk test. Normally distributed data are shown as mean ± SD and analyzed by t-test or one-way ANOVA (Bonferroni correction). Non-normal data are reported as median (interquartile range) and compared using Mann–Whitney or Kruskal–Wallis tests (Dunn’s post-hoc). Survival was analyzed via Kaplan–Meier curves and log-rank test. Spearman’s correlation was applied for association analysis. All experiments in vitro were repeated at least three times. The figure legends specify the value of n, representing the number of independent biological replicates. Data from the 2 × 2 factorial design (Sham/CLP and NC/OV) were analyzed using two-way ANOVA to assess main effects and interactions, followed by Sidak’s post-hoc test for multiple comparisons when appropriate. All statistical analyses were performed in GraphPad Prism v8.0, with P < 0.05 considered significant.

Results

Clinical association of monocytic MasR expression in patients with SIC

This study involved 16 HCs and 15 patients with SIC who met the inclusion criteria. Attached File 1: Table S1 presents the clinical data of the subjects included in the study. MasR expression on CD14 monocytes in peripheral blood was measured by FCM across the study groups. The gating strategy for FCM is shown in Fig. 1A and B. A significant increase in MasR expression on CD14 monocytes was observed in patients with SIC compared to HCs (Fig. 1A–D).

Fig. 1.

Expression and clinical significance of MasR on peripheral blood monocytes in patients with SIC. (A, B) Flow cytometric analysis of monocytes and MasR expression on their surface in patients with SIC. (C, D) MasR expression levels on monocytes in HCs (n = 16) and patients with SIC (n = 15). (E) Correlation matrix between MasR expression (MFI) on monocytes and clinical parameters in patients with SIC. Values in the matrix represent Spearman correlation coefficients (p < 0.05). NS: no significant difference, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

To account for potential interpatient heterogeneity in SIC, the correlations between MasR expression on monocytes and clinical/biochemical parameters in all patients with SIC were analyzed (Fig. 1E). Our analysis revealed significant positive correlations between the MFI of MasR on peripheral blood CD14 monocytes and the following parameters: TnI levels (r = 0.654, P = 0.008), APACHE II score (r = 0.560, P = 0.030), lactate levels (r = 0.581, P = 0.023), IL-6 levels (r = 0.573, P = 0.026), and SOFA score (r = 0.664, P = 0.007). These results suggest that MasR expression on monocytes is associated with sepsis complicated by myocardial dysfunction. Targeting MasR regulation may therefore represent a viable therapeutic approach for SIC.

To explore potential confounding, we performed exploratory multivariate linear regression. Given the small sample size, these analyses are underpowered and warrant cautious interpretation. Lactate and IL-6, both positively correlated with MasR expression and TnI, were identified as potential confounders. After adjusting for IL-6 and lactate, the association between MasR MFI and TnI was attenuated and no longer significant (P = 0.212). Likewise, adjustment for TnI and IL-6 attenuated the MasR MFI–SOFA association (P = 0.111), and adjustment for IL-6 and lactate attenuated the MasR MFI–APACHE II association (P = 0.340). Although not statistically significant in this small cohort, the consistent trends observed suggest that the relationship between monocyte MasR expression and disease severity warrants further investigation in larger, adequately powered studies.

AVE0991 pretreatment decreased systemic inflammation, alleviated cardiac damage, and enhanced survival rates in SIC mice

AVE0991 was used to activate MasR and assess its effects in SIC mice. Mice were intraperitoneally injected with either AVE0991 or the DMSO solvent for three consecutive days, followed by cecal ligation and puncture (CLP) to induce the SIC model (Fig. 2A). Survival rates were monitored for 72 h across the experimental groups. The survival rate was 100% in the Sham group, 25% in the CLP + Vehicle group, and 50% in the CLP + AVE0991 group. Notably, AVE0991 pretreatment significantly improved survival in SIC mice compared to the CLP + Vehicle group (Fig. 2B). HE staining of myocardial tissue revealed that Sham group mice had well-preserved myocardial structure, with neatly arranged myocardial cells, no nuclear swelling, and minimal edema or immune cell infiltration in the interstitium (Fig. 2C, D). In contrast, the CLP + Vehicle group showed extensive myocardial damage, with disorganized myocardial fibers, nuclear fragmentation, myolysis, and significant immune cell infiltration in the interstitium (Fig. 2C, D). In comparison, the CLP + AVE0991 group exhibited a marked reduction in myocardial damage, with more orderly myocardial fiber arrangement and significantly reduced immune cell infiltration (Fig. 2C, D).

Fig. 2.

AVE0991 pretreatment decreased systemic inflammation, mitigated heart injury, and enhanced survival rates in SIC mice. (A) Experimental schematic: Mice received AVE0991 (3 mg/kg, i.p.) or vehicle (DMSO) daily for three days, followed by CLP to induce SIC and subsequent analyses. (B) Effect of AVE0991 pretreatment on 72-hour survival rates in SIC mice (n = 16). (C, D) Representative HE-stained heart sections and corresponding pathological injury scores for each group (n = 4). (E, F) Serum levels of cTnT and IL-6 in mice from each group (n = 5). (G–M) Representative echocardiograms and quantitative analysis of cardiac function parameters, including LVFS, LVEDV, LVIDd, LVESV, LVEF, and LVIDs, in all experimental groups (n = 5). NS: no significant difference, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

ELISA quantification of serum levels of cTnT and IL-6 showed that these biomarkers were significantly higher in the CLP + Vehicle group compared to the Sham group (Fig. 2E, F). Importantly, AVE0991 pretreatment effectively attenuated the CLP-induced increases in these biomarkers (Fig. 2E, F). Echocardiography was used to evaluate cardiac function in mice from each group (Fig. 2G–M). The results indicated that 24 h post-model induction, the CLP + Vehicle group had reduced LVFS and LVEF, along with increased LVIDs and LVESV compared to the Sham group (Fig. 2G–M). However, the CLP + AVE0991 group exhibited significantly higher LVEF and LVFS, and lower LVESV and LVIDs compared to the CLP + Vehicle group (Fig. 2G–M). No significant differences were observed in LVIDd or LVEDV among the three groups (Fig. 2G–M). These results demonstrate that AVE0991 pretreatment effectively improves myocardial dysfunction in SIC mice.

AVE0991 preserves mitochondrial dynamics homeostasis, attenuates mitochondrial damage, and reduces cardiomyocyte apoptosis in myocardial cells

Mitochondrial dysfunction is a critical pathological mechanism in the progression of SIC. TEM was used to assess ultrastructural changes in myocardial mitochondria across experimental groups. Compared to Sham controls, CLP + Vehicle mice exhibited disorganized myocardial fiber arrangement, with dissolution, along with characteristic mitochondrial abnormalities such as swelling, cristae reduction/rupture, and partial vacuolar degeneration (Fig. 3A, B). Notably, AVE0991 pretreatment significantly mitigated these CLP-induced mitochondrial ultrastructural damages (Fig. 3A, B). Mitochondrial membrane damage leads to reduced MMP, impaired oxidative phosphorylation, and diminished ATP generation. Our data revealed that, compared to the Sham group, both MMP and ATP levels in myocardial tissue from the CLP + Vehicle group were significantly decreased (Fig. 3C, D). However, AVE0991 pretreatment prior to CLP administration reversed the decline in MMP and ATP levels induced by CLP in myocardial tissue (Fig. 3C, D).

Fig. 3.

AVE0991 preserves mitochondrial dynamics, attenuates mitochondrial damage, and reduces cardiomyocyte apoptosis. (A, B) Representative transmission electron micrographs of myocardial mitochondria and corresponding mitochondrial injury scores in each group (n = 4). (C, D) ATP content and MMP levels in myocardial mitochondria across groups (n = 4). (E-K) Expression levels of Mfn2, Drp1, Bcl-2, cleaved-caspase-3, caspase-9, and Bax in cardiac tissues of each group (n = 3). (L, M) Representative TUNEL staining and quantification of TUNEL-positive cells in myocardial tissue (n = 4). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

An imbalance in mitochondrial fusion and fission dynamics contributes significantly to increased cardiomyocyte apoptosis. Our results showed that, relative to the Sham group, protein levels of Drp1, Bax, caspase-9, and cleaved caspase-3 were significantly elevated, while Mfn2 and Bcl-2 expression were notably reduced in myocardial tissue from the CLP + Vehicle group (Fig. 3E-K). AVE0991 pretreatment effectively reversed these CLP-induced protein expression changes (Fig. 3E–K). Additionally, myocardial cell apoptosis was assessed in mice from each group using the TUNEL assay. The results showed a significant increase in apoptotic cardiomyocytes in the CLP + Vehicle group compared to the Sham group (Fig. 3L, M). However, AVE0991 pretreatment prior to CLP significantly alleviated the exacerbation of myocardial cell apoptosis induced by CLP (Fig. 3L, M).

AVE0991 attenuated macrophage infiltration, regulated macrophage polarization, and decreased the release of pro-inflammatory cytokines in the hearts of SIC mice

Immune cell infiltration and functional changes in cardiac tissue are critical factors influencing SIC. FCM was used to assess changes in myeloid cells and macrophages in cardiac tissue across experimental groups. Results indicated that, compared to the Sham group, the CLP + Vehicle group exhibited significantly higher counts of myeloid cells (CD45⁺CD11b⁺), total macrophages (CD45⁺CD11b⁺F4/80⁺), and M1 macrophages (CD45⁺CD11b⁺F4/80⁺CD86⁺) in cardiac tissue (Fig. 4A–F). In contrast, compared to the CLP + Vehicle group, the CLP + AVE0991 group showed significantly reduced numbers of these cell populations in the myocardium (Fig. 4A-F).

Fig. 4.

AVE0991 attenuates macrophage infiltration, regulates macrophage polarization, and decreases pro-inflammatory cytokine release in the hearts of SIC mice. (A-F) Representative flow cytometry plots and percentages of myeloid cells (CD45⁺CD11b⁺), total macrophages (CD45⁺CD11b⁺F4/80⁺), and M1 macrophages (CD45⁺CD11b⁺F4/80⁺CD86⁺) in cardiac tissue for each group (n = 4). (G-K) Protein expression levels of TNF-α, iNOS, IL-1β, and Arg-1 in myocardial tissues of mice across groups (n = 3)

Macrophage-associated protein expression in cardiac tissue was also evaluated by Western blot. Compared to the Sham group, the CLP + Vehicle group showed increased expression of IL-1β, TNF-α, and iNOS proteins in cardiac tissue, with no change in arginase-1 (Arg-1) expression between the two groups (Fig. 4G-K). In contrast, the CLP + AVE0991 group exhibited decreased levels of IL-1β, TNF-α, and iNOS proteins, along with increased Arg-1 expression in cardiac tissue (Fig. 4G–K). These results suggest that AVE0991 helps maintain the homeostasis of the myocardial immune microenvironment by reducing macrophage infiltration and modulating macrophage polarization in SIC mice.

Targeting myocardial macrophage MasR can alleviate myocardial tissue pathological damage and cardiac dysfunction in SIC mice, thereby improving survival rate

To further investigate the effect of MasR expression on myocardial macrophages in SIC mice, a myocardial macrophage-specific MasR overexpression model was generated using AAV9 (AAV9-F4/80-Mas). The experimental procedure and groupings are detailed in Fig. 5A. To assess cell-type specificity, we examined MasR expression in non-macrophage cardiac cells. No significant differences in MasR MFI were observed in cTnI⁺ cardiomyocytes, α-SMA⁺ fibroblasts, or CD31⁺ endothelial cells between CLP + NC and CLP + OV mice (Additional File 2: Fig S2.A–F), confirming macrophage-specific expression. Using a stringent threshold (MasR MFI > mean of NC group), approximately 43.44 ± 4.27% of F4/80⁺ cardiac macrophages in CLP + OV mice exhibited high MasR expression (Fig. 5B), indicating efficient transduction. No intergroup differences in MasR MFI were detected in spleen or lung macrophages (Additional File 2: Fig S3.A–D). In the liver, MasR MFI was modestly increased in CLP + OV mice (1.76 ± 0.26 vs. 1.14 ± 0.22, P = 0.035; Additional File 2: Fig S3.E–F), but this increase was far lower than in cardiac macrophages, likely reflecting lower transduction efficiency in Kupffer cells due to rapid viral degradation.

Fig. 5.

Targeting myocardial macrophage MasR alleviates myocardial tissue pathological damage and cardiac dysfunction in SIC mice, thereby improving the survival rates. (A) Schematic of the animal experiment: AAV9 (AAV9-f4/80-Mas) was used to construct a mouse model with cardiac macrophage-specific for subsequent experiments. (B) Validation of MasR overexpression in myocardial macrophages within the SIC mouse model: Representative immunofluorescence images showing co-localization of MasR and F4/80 in myocardial tissues from CLP + NC and CLP + OV mice. (C–I) Representative echocardiograms from each group of mice, along with quantitative analysis of cardiac functional parameters, including LVFS, LVEDV, LVIDd, LVEF, LVIDs, and LVESV (n = 4). (J) Serum levels of cTnT in each group of mice (n = 4). (K–L) Representative HE-stained images of cardiac tissues and corresponding pathological injury scores for each group (n = 4). (M) Effect of myocardial macrophage-specific MasR overexpression on the 72-hour survival rate of SIC mice (n = 16)

To assess whether MasR overexpression modifies CLP effects, two-way ANOVA was performed. Significant CLP × OV interactions were observed for LVEF, LVFS, LVESV, and LVIDs (all P < 0.05), indicating modulation by MasR overexpression. Post-hoc comparisons showed that, relative to Sham + NC, the CLP + NC group had decreased LVEF and LVFS and increased LVESV and LVIDs, whereas the CLP + OV group showed opposite changes (Fig. 5C–I). No significant differences or interactions were observed for LVEDV or LVIDd (P > 0.05). For myocardial injury, two-way ANOVA revealed a significant CLP × OV interaction for serum cTnT (P < 0.05). Serum cTnT levels were significantly elevated in the CLP + NC group relative to the Sham + NC group (Fig. 5J). However, cTnT levels were markedly reduced in the CLP + OV group compared to the CLP + NC group (Fig. 5J), further confirming that macrophage-specific MasR overexpression improves myocardial dysfunction in SIC mice.

HE staining revealed that myocardial cells in both the Sham + NC and Sham + OV groups were well-organized, with no significant interstitial edema (Fig. 5K, L). In contrast, the CLP + NC group exhibited extensive myocardial damage, including disorganized fibers, nuclear fragmentation, and myolysis, accompanied by significant immune cell infiltration into the myocardial interstitium (Fig. 5K, L). Notably, myocardial tissue damage and immune cell infiltration were significantly reduced in the CLP + OV group compared to the CLP + NC group (Fig. 5K, L). The 72-hour survival rates across experimental groups were monitored, with 100% survival in both Sham + NC and Sham + OV groups, 18.75% survival in the CLP + NC group, and 50.00% survival in the CLP + OV group (Fig. 5M). Notably, CLP + OV treatment significantly improved survival rates compared to the CLP + NC group (Fig. 5M).

Activation of myocardial macrophage MasR can alleviate tissue and organ blood flow perfusion in SIC mice, improve mitochondrial dynamics imbalance in myocardial tissue, and reduce myocardial cell apoptosis

LSCI was used to assess blood flow perfusion in the kidney, mesentery, and liver of mice from each group. Two-way ANOVA demonstrated a significant CLP × OV interaction for blood perfusion in the mesentery, liver, and kidney (all P < 0.05), indicating that MasR overexpression alleviated CLP-induced hypoperfusion. The results showed no significant differences in blood flow between the Sham + NC and Sham + OV groups in mesenteric, hepatic, and renal tissues (Fig. 6A–D). However, compared to the Sham + NC group, the CLP + NC group exhibited significantly reduced blood perfusion in these tissues (Fig. 6A–D). In contrast, perfusion in the mesentery, liver, and kidney was notably improved in the CLP + OV group compared to the CLP + NC group (Fig. 6A–D).

Fig. 6.

Activation of myocardial macrophage MasR alleviates tissue and organ blood flow perfusion in SIC mice, improves mitochondrial dynamics imbalance in myocardial tissue, and reduces myocardial cell apoptosis. (A-D) Representative LSCI images and quantitative perfusion analysis of mesenteric, hepatic, and renal tissues in all experimental groups (n = 4). (E-K) Myocardial protein expression levels of Mfn2, Bcl-2, cleaved-caspase-3, Drp1, caspase-9, and Bax in each group (n = 4)

Two-way ANOVA revealed significant CLP × OV interactions for Mfn2, Drp1, Bcl-2, Bax, caspase-9, and cleaved caspase-3 (all P < 0.05), confirming that MasR overexpression counteracted CLP-induced mitochondrial imbalance and apoptosis. The results indicated that, compared to the Sham + NC group, the CLP + NC group showed significantly increased expression of Drp1, Bax, caspase-9, and cleaved-caspase-3, along with decreased expression of Mfn2 and Bcl-2 (Fig. 6E–K). In contrast, the CLP + OV group exhibited significantly lower levels of cleaved-caspase-3, Drp1, Bax, and caspase-9, and higher levels of Mfn2 and Bcl-2 compared to the CLP + NC group (Fig. 6E–K). These results suggest that MasR activation in cardiac macrophages can alleviate mitochondrial dynamic imbalance and reduce cardiomyocyte apoptosis in SIC mice.

Activation of MasR in cardiac macrophages modulates their polarization via the Akt/Nrf2 axis in SIC mice, thereby preserving myocardial cytokine homeostasis

Following the onset of SIC, cardiac macrophage phenotypes undergo dynamic changes. Therefore, the phenotypic alterations of cardiac macrophages in SIC mice and the effects of MasR activation on macrophage polarization were examined. Two-way ANOVA revealed significant CLP × OV interactions for total macrophage count, M1 proportion, and M2 proportion in cardiac tissue (all P < 0.05), indicating that MasR overexpression modulates macrophage polarization in SIC. As shown in Fig. 7A–D, total macrophage numbers and M1/M2 ratios were comparable between Sham + NC and Sham + OV groups. Relative to Sham + NC, the CLP + NC group showed increased total macrophages and M1 proportion, and decreased M2 proportion. MasR activation reversed these changes, reducing total macrophages and M1 fraction while increasing M2 fraction (Fig. 7A–D). For cytokine levels, two-way ANOVA demonstrated significant CLP × OV interactions for IL-6 and IL-10 (P < 0.05). Compared to the Sham + NC group, the CLP + NC group exhibited elevated IL-6 levels with unchanged IL-10 expression. In contrast, the CLP + OV group displayed reduced IL-6 and increased IL-10 levels compared to the CLP + NC group (Fig. 7E, F).

Fig. 7.

Activation of MasR in cardiac macrophages modulates their polarization via the Akt/Nrf2 axis in SIC mice, preserving myocardial cytokine homeostasis. (A-D) Representative immunofluorescence images and quantitative analysis of total macrophages, M1/M2 macrophage subtypes in cardiac tissue from each group (× 200, scale bar: 50 μm) (n = 3). (E, F) IL-6 and IL-10 levels in myocardial tissues from each group (n = 5). (G-L) Expression levels of p-Akt, Akt, Nrf2, iNOS, Arg-1, and TNF-α proteins in cardiac tissues across groups (n = 4)

To explore the cardioprotective mechanisms of MasR activation, Western blotting was performed on cardiac tissues to evaluate alterations in the Akt/Nrf2 pathway and macrophage-related markers. Two-way ANOVA revealed significant CLP × OV interactions for p-Akt, Nrf2, iNOS, TNF-α, and Arg-1 (all P < 0.05). Compared to the Sham + NC group, the CLP + NC group showed unchanged levels of p-Akt, Nrf2, and Arg-1, with elevated expression of iNOS and TNF-α (Fig. 7G–L). In contrast, the CLP + OV group exhibited increased myocardial expression of p-Akt, Nrf2, and Arg-1, and decreased levels of iNOS and TNF-α (Fig. 7G–L). These results demonstrate that cardiac MasR activation maintains cytokine balance in SIC via Akt/Nrf2-mediated macrophage polarization.

MasR activation regulates LPS-induced BMDM polarization via the Akt/Nrf2 pathway and attenuates H9c2 cell apoptosis mediated by the pro-inflammatory response of BMDM

To further confirm the role of Akt/Nrf2 signaling in MasR activation and its regulation of LPS-induced BMDM polarization, BMDM were pretreated with AVE0991 and ML385. FCM analysis revealed that LPS + AVE0991 treatment significantly reduced the proportion of M1 macrophages and increased the proportion of M2 macrophages compared to the LPS + Vehicle1 group (Fig. 8A–C). Notably, co-treatment with ML385 (LPS + AVE0991 + ML385 group) reversed these effects, significantly increasing the M1 macrophage proportion and reducing the M2 macrophage proportion compared to the LPS + AVE0991 + Vehicle2 group (Fig. 8A–C). Furthermore, compared to the Control, LPS + Vehicle1 treatment notably elevated IL-1β, iNOS, and TNF-α protein levels in BMDM. In contrast, the LPS + AVE0991 group showed reduced IL-1β, iNOS, and TNF-α levels, alongside increased Arg-1, p-Akt, and Nrf2 expression compared to the LPS + Vehicle1 group (Fig. 8D–J). However, treatment with ML385 (LPS + AVE0991 + ML385) reversed these changes, increasing IL-1β, iNOS, and TNF-α levels, while decreasing Arg-1, p-Akt, and Nrf2 expression compared to the LPS + AVE0991 + Vehicle2 group (Fig. 8D–J). To determine whether Akt is required for MasR-mediated Nrf2 activation and M2 polarization, we used the Akt-specific inhibitor MK-2206. AVE0991 treatment (LPS+AVE0991 + Vehicle3) markedly upregulated Nrf2 and Arg-1, an effect completely abolished by MK-2206 co-treatment (LPS+AVE0991 + MK-2206), demonstrating that Akt activation is essential for MasR-induced Nrf2 expression and subsequent M2 polarization (Additional File 2: Fig S1.A-C). These results further confirm that MasR activation regulates LPS-induced BMDM polarization via the Akt/Nrf2 pathway, thereby reducing pro-inflammatory cytokine levels.

Fig. 8.

MasR activation regulates LPS-induced BMDM polarization via the Akt/Nrf2 pathway and attenuates H9c2 cell apoptosis mediated by the pro-inflammatory response of BMDM. (A–J) BMDM were pretreated for 2 h with the MasR agonist AVE0991 (10 µM), its vehicle control (Vehicle1), the NRF2 inhibitor ML385 (5 µM), its vehicle control (Vehicle2), either alone or in combination. Subsequently, the cells were exposed to LPS (1 µg/mL) for 24 h, and the relevant analyses were conducted. Data are from four independent experiments (n = 4). (A–C) FCM plots and percentages of M1 and M2 macrophages in BMDM per group. (D) Representative western blot of Arg-1, iNOS, p-AKT, TNF-α, AKT, NRF2, and IL-1β in BMDM from each group. (E-J) Quantitative analysis of the relative expression of Arg-1, iNOS, p-AKT/AKT, NRF2, IL-1β, and TNF-α in each group of BMDM. (K) BMDM were pretreated for 2 h with the MasR agonist AVE0991 (10 µM), its vehicle control (Vehicle1), the NRF2 inhibitor ML385 (5 µM), or its vehicle control (Vehicle2), either alone or in combination. Subsequently, the cells were stimulated with LPS (1 µg/mL) for 24 h. The culture supernatants from each group were then collected and used as conditioned media to co-culture with H9c2 cells for 24 h, after which FCM analysis was performed. (L–Q) FCM plots and quantitative analysis graphs of ROS levels, JC-1 monomer ratio, and apoptosis rate in H9c2 cells across different groups. Data are from three independent experiments (n = 3). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

To investigate whether MasR activation alleviates the pro-inflammatory impact of BMDM on H9c2 cells via the Akt/Nrf2 pathway, BMDM from different treatment groups were used to collect conditioned media, which were then co-cultured with H9c2 cells for 24 h. The experimental procedure and groupings are illustrated in Fig. 8K. The results showed that the LPS + Vehicle1 group exhibited significantly increased intracellular ROS levels, a higher JC-1 monomer ratio (indicative of mitochondrial damage), and an elevated apoptosis rate in H9c2 cells compared to the control (Fig. 8L–Q). In contrast, these markers were significantly lower in the LPS + AVE0991 group relative to the LPS + Vehicle1 group (Fig. 8L–Q). However, compared to the LPS + AVE0991 + Vehicle2 group, treatment with LPS + AVE0991 + ML385 significantly increased intracellular ROS levels, the JC-1 monomer ratio, and the apoptosis rate in H9c2 cells (Fig. 8L–Q). These results indicate that MasR activation regulates LPS-induced BMDM polarization via the Akt/Nrf2 pathway, reducing H9c2 cell apoptosis by modulating the pro-inflammatory response of BMDM.

Discussion

This study revealed elevated MasR expression on peripheral blood monocytes in patients with SIC, which correlated with myocardial injury severity, suggesting its involvement in SIC pathogenesis and potential as a therapeutic target. To validate this hypothesis, various intervention strategies were employed to activate MasR and evaluate its role and molecular mechanisms in both in vivo and in vitro SIC models. An overview of the study design is provided in Additional File 3: Fig S1. This study provides initial systematic evidence that MasR activation reprograms cardiac macrophage phenotypes via the Akt/Nrf2 axis, modulating inflammatory cytokine release and cardiomyocyte apoptosis, thereby offering protection against sepsis-induced cardiac injury. This discovery establishes a connection between the RAAS protective axis, immune metabolic regulation, and mitochondrial homeostasis, providing new insights into the complex pathological network of SIC and identifying novel targets for its prevention and treatment.

Key components of the RAAS—such as intact angiotensinogen, active renin, and angiotensin-converting enzyme 2 (ACE2)—are significantly correlated with the prognosis of patients with sepsis [21, 51–53]. Moreover, in patients with SIC, plasma levels of Ang II and the Ang II/Ang-(1–7) ratio are elevated, while Ang-(1–7) is reduced, and AT1R expression on peripheral monocytes is heightened [26, 32]. These changes correlate positively with the extent of myocardial injury. Our clinical observations indicate that MasR expression on CD14⁺ monocytes in the peripheral blood of patients with SIC is significantly increased, which may be related to the severity of the disease (assessed by APACHE II and SOFA scores) as well as the levels of IL-6, lactate and TnI. Similarly, studies have shown that MasR expression on macrophages is significantly upregulated under inflammatory stimulation [54]. The reason for this may be that in SIC patients, under severe inflammatory responses, the level of Ang-(1–7) is reduced, and the body may initiate a compensatory response represented by the upregulation of MasR. These results reinforce the critical role of the Ang-(1–7)/MasR axis in SIC, highlighting that pharmacological or genetic modulation of MasR could be a promising therapeutic strategy for preventing and managing SIC. A universally accepted definition for SIC is currently lacking, and diagnostic thresholds vary across studies. While our criteria were based on prior literature and clinical relevance [26, 32, 49, 50], this heterogeneity may limit comparability across cohorts and the generalizability of our findings. Standardized criteria would facilitate cross-study comparisons and meta-analyses.

In this study, both prophylactic AVE0991 treatment and cardiac macrophage-specific MasR overexpression significantly improved survival, alleviated cardiac dysfunction, and reduced myocardial damage in CLP mice. Current literature further supports the cardioprotective roles of ACE2, Ang-(1–7), Ang-(1–9), alamandine, and its MrgD receptor in sepsis [55–57]. Research also indicates that pharmacological enhancement of the ACE2/Ang-(1–7)/MasR axis mitigates myocardial inflammation and apoptosis, improving SIC outcomes [11, 58, 59]. Notably, this study introduces a novel cardiac macrophage-specific MasR overexpression model, providing direct evidence that MasR’s cardioprotective effects are mediated through macrophages—key regulators in the cardiac immune microenvironment. This approach shifts the understanding of MasR’s function from a traditional “direct cardiomyocyte protection” model to a new paradigm of “indirect myocardial protection via immune cell regulation.”

Myeloid ACE2 mitigates sepsis-induced hypotension and vascular dysfunction by suppressing oxidative stress, regulating nitric oxide production, and modulating macrophage polarization through the Ang-(1–7)/MasR axis [60]. Additionally, activation of the Ang-(1–7)/MasR axis during infection not only regulates macrophage polarization but also enhances macrophage migration and phagocytosis, aiding in pathogen clearance [61]. Previous research has demonstrated that Ang-(1–7) elevates p-eNOS/eNOS and suppresses iNOS via the Mas/Akt pathway, thereby reducing oxidative stress [62]. Akt signaling also modulates the Nrf2 transcription factor, contributing to the reduction of oxidative stress and downregulation of inflammatory responses [63–66]. Recent studies have highlighted that MasR activation with AVE0991 regulates microglial polarization through the Akt/Nrf2 pathway, promoting neural recovery following ICH [42]. In this study, animal experiments demonstrated that activating MasR in cardiac macrophages alleviated myocardial injury in SIC mice by modulating macrophage polarization via the Akt/Nrf2 axis, thereby maintaining cytokine balance in myocardial tissue. Of note, the M1/M2 framework used here is an operational simplification that does not fully capture the heterogeneity of cardiac macrophages. Recent single-cell studies have identified more refined subsets in sepsis, including TREM2-high resident macrophages, iNOS⁺Arg1⁺ macrophages, and Mertk⁺ or Ccr2⁺ macrophages, each playing distinct roles in homeostasis, inflammation, and tissue repair during SIC [9, 67, 68]. Whether MasR activation protects the heart by targeting specific subsets warrants further investigation at the single-cell level. Furthermore, the precise molecular mechanisms through which MasR controls macrophage polarization and the functional impact of macrophages on cardiomyocytes remain poorly understood. To address these gaps, we used ML385 and MK-2206 to validate the roles of the Nrf2 and Akt pathways in AVE0991-mediated macrophage polarization. In a macrophage–cardiomyocyte co-culture system, AVE0991 pretreatment attenuated cardiomyocyte oxidative stress, mitochondrial dysfunction, and apoptosis driven by the macrophage pro-inflammatory response; these effects were abolished by ML385. Thus, MasR activation regulates LPS-induced BMDM polarization via the Akt/Nrf2 pathway, thereby reducing H9c2 cell apoptosis driven by BMDM pro-inflammatory responses. The MasR-mediated Akt/Nrf2 pathway does not act in isolation but networks with other RAAS protective mechanisms. Losartan attenuates SIC by blocking TLR4‑dependent NF‑κB/MAPK signaling [26]; ACE2 activation mitigates SIC by inhibiting NF‑κB/STAT1 and promoting M2 polarization [11], and also boosts mitochondrial biogenesis via Sirt1 [59]. These pathways may exhibit crosstalk with the MasR-Akt/Nrf2 axis. Mechanistically, Nrf2 activation inhibits NF‑κB transcriptional activity and synergistically enhances anti‑inflammatory effects via the NF‑κB/MAPK and NF‑κB/STAT1 pathways [69]. In parallel, Sirt1 activates Nrf2 through deacetylation and may also modulate signaling via Akt [70, 71]. Thus, the RAAS protective axis likely counteracts SIC through coordinated interplay of multiple signaling pathways. Elucidating the precise interactions among these pathways represents an important direction for future research.

SIC can progress to cardiogenic shock, exacerbating hypoperfusion in the intestines, liver, and kidneys [1, 72]. Intestinal dysbiosis and accumulation of harmful metabolites further aggravate myocardial injury, creating a vicious cycle [73]. LSCI effectively monitors tissue perfusion and predicts outcomes in sepsis [74, 75], and prior studies have shown significantly reduced perfusion in septic mice across multiple organs [76, 77]. In our study, MasR activation in cardiac macrophages improved mesenteric, hepatic, and renal perfusion in SIC mice, suggesting systemic hemodynamic benefits. As the central driver of the circulatory system, improved cardiac function significantly enhances perfusion to organs throughout the body. Furthermore, attenuation of local cardiac inflammation may positively influence systemic vascular tone, potentially by reducing circulating inflammatory cytokines or improving autonomic nervous system balance [78, 79]. We observed a modest increase in MasR expression in Kupffer cells of CLP + OV mice, suggesting improved hepatic perfusion reflects local MasR effects on liver macrophages, not solely secondary to cardiac function. This likely reflects F4/80-driven expression, but efficiency was lower than in cardiac macrophages, possibly due to rapid viral degradation in the liver [80]. Importantly, this mild elevation does not alter our conclusions, as: (i) its magnitude was far lower than the robust upregulation in cardiac macrophages; (ii) no off-target expression was detected in spleen or lung; and (iii) multi-organ perfusion improvement, consistent with systemic hemodynamic effects. These findings indicate that MasR activation in myocardial macrophages has a significant therapeutic effect on SIC.

Several limitations exist. First, lack of septic patients without myocardial dysfunction precludes determining whether MasR association is SIC-specific or reflects sepsis severity. Second, small sample size reduced statistical power; multivariate results were non-significant after adjustment, likely due to Type II error. Thus, our findings indicate association, not independent prediction, warranting validation in larger studies. Third, both AVE0991 and AAV9 were administered prophylactically—appropriate for mechanistic proof-of-concept but not reflective of clinical practice. Future studies with delayed intervention are needed to define the therapeutic window. Fourth, MasR’s role in other cardiac cell types and their crosstalk with macrophages remains unexplored. Finally, other signaling pathways downstream of MasR warrant further investigation.

Conclusions

In conclusion, this study identified a significant clinical correlation between MasR expression on peripheral blood monocytes and SIC, which could inform the optimization of clinical diagnosis, treatment, and prognosis assessment for SIC. Additionally, it was confirmed that activation of MasR in cardiac macrophages reprograms their phenotype through the Akt/Nrf2 axis, modulating inflammatory cytokine release and cardiomyocyte apoptosis, ultimately alleviating SIC (Fig. 9). These findings present a promising therapeutic approach for patients with SIC.

Fig. 9.

Schematic diagram showing that MasR activation in cardiac macrophages alleviates SIC by modulating macrophage polarization through the Akt/Nrf2 axis

Supplementary Information

Below is the link to the electronic supplementary material.

Author contribution

Xin-sen Chen: Conceptualization, Data curation, Funding acquisition, Visualization, Writing – original draft. Meng Shao, Hua-jun Ge: Formal analysis, Methodology, Project administration, Writing – original draft. Ting Jiang, Shuo Fan: Investigation, Methodology, Data Curation, Visualization. Juan Zhou, Na Li: Validation, Formal analysis. Min Huang, Lu Zhang: Supervision, Funding acquisition, Writing – Review & Editing. All authors approved the final manuscript.

Funding

This research received financial support through grants from the National Natural Science Foundation of China (No. 82402570), the Hubei Provincial Natural Science Foundation (No. 2024AFB529), Xiangyang Central Hospital hospital-level scientific research project (No.2024YJ01A, No.2022YB12), Xiangyang Central hospital personnel training project (2025RCYC-038), Xiangyang Central Hospital doctoral starting fund (No. 2023BS02).

Data availability

Any data involved in this study can be requested from the corresponding author.

Declarations

Ethics approval and consent to participate

All human participants or their legal guardians provided written informed consent. This study was conducted in accordance with the Declaration of Helsinki. The study protocol was reviewed and approved by the Ethics Committee of Xiangyang Central Hospital, Hubei University of Arts and Sciences. All animal experiments complied with NIH guidelines and were approved by the same institution’s Animal Ethics Committee.

Consent for publication

Not applicable.

Competing interests

All authors have no competing interests.