ACE2 alleviates sepsis-induced cardiomyopathy through inhibiting M1 macrophage via NF-κB/STAT1 signals

Abstract

Angiotensin-converting enzyme 2 (ACE2), a crucial element of the renin-angiotensin system (RAS), metabolizes angiotensin II into Ang (1-7), which then combines with the Mas receptor (MasR) to fulfill its protective role in various diseases. Nevertheless, the involvement of ACE2 in sepsis-induced cardiomyopathy (SIC) is still unexplored. In this study, our results revealed that CLP surgery dramatically impaired cardiac function accompanied with disruption of the balance between ACE2-Ang (1-7) and ACE-Ang II axis in septic heart tissues. Moreover, ACE2 knockin markedly alleviated sepsis induced RAS disorder, cardiac dysfunction and improved survival rate in mice, while ACE2 knockout significantly exacerbates these outcomes. Adoptive transfer of bone marrow cells and in vitro experiments showed the positive role of myeloid ACE2 by mitigating oxidative stress, inflammatory response, macrophage polarization and cardiomyocyte apoptosis by blocking NF-κB and STAT1 signals. However, the beneficial impacts were nullified by MasR antagonist A779. Collectively, these findings showed that ACE2 alleviated SIC by inhibiting M1 macrophage via activating the Ang (1-7)-MasR axis, highlight that ACE2 might be a promising target for the management of sepsis and SIC patients.

Keywords: ACE2; Bone marrow transplantation; Macrophage polarization; Sepsis-induced cardiomyopathy.

Fig. 1

CLP disturbs the RAS balance in heart. A Experimental schematic diagram of WT mice subjected to CLP and observed for 24–72 h. B Effect of sham or CLP surgery on survival rate (n = 30). C Activity of ACE and level of Ang II in heart after CLP in WT mice (n = 6). D Activity of ACE2 and level of Ang (1–7) in the heart determined by ELISA (n = 4). E Western blot analysis of ACE, AT1R, ACE2, and MasR proteins in the hearts of mice after CLP. F Quantification of relative protein bands (n = 4). Data are presented as the mean ± SEM, and n represents the number of animals in each group

Fig. 2

Overexpression of ACE2 attenuated myocardial injury, oxidative stress and M1 macrophage polarization induced by CLP. A ELISA analysis of activity of ACE2 and levels of Ang II and Ang (1–7) in the heart in WT or ACE2-KI mice after sham or CLP and Ang II to Ang (1–7) ratio (n = 4–6). B Survival rate of each group from 24 to 74 h (n = 20). C Measurement of systolic blood pressure (SBP) in each group by noninvasive tail-cuff method (n = 6). D Analysis of LDH levels in plasma and heart tissues in each group (n = 6). E M-mode echocardiography of the LV chamber (left) and measurement of the EF% and FS% (right, n = 6). F Dihydroethidium (DHE) staining of heart sections with quantification of the fluorescence intensity (left and middle) and qPCR analysis of the mRNA levels of NOX2 (right) in each group (n = 6). G Immunohistochemical staining of heart sections using Mac-2 antibody (left) with the percentage of Mac-2-positive areas (right, n = 6). H-I qPCR analysis of the mRNA levels of macrophage markers (CD80, IL-1β, IL-6, TNF-α, CD206, IL-10 and ARG-1) (n = 6). Data are presented as the mean ± SEM, and n represents the number of animals in each group

Fig. 3

Deficiency of ACE2 aggravated myocardial injury, oxidative stress and M1 macrophage polarization induced by CLP. A ELISA analysis of ACE2 activity, levels of Ang II and Ang (1–7) and Ang II to Ang (1–7) ratio in the heart of WT or ACE2-KO mice following sham or CLP (n = 4–6). B Survival rate of each group from 24 to 74 h (n = 20). C Measurement of systolic blood pressure (SBP) in each group during 24–48 h by noninvasive tail-cuff method (n = 6). D Detection of LDH levels in plasma and heart tissues in each group (n = 6). E M-mode echocardiography of the LV chamber (left) and detection of the EF% and FS% (right, n = 6). F DHE staining of heart sections (left) and quantification of the fluorescence intensity (middle) and qPCR analysis of NOX2 mRNA levels (right, n = 6). G Immunohistochemical staining of heart sections with anti-Mac-2 antibody (left) and quantification of Mac-2-positive macrophages (right, n = 6). H-I qPCR analysis of the mRNA levels of CD80, IL-1β, IL-6, TNF-α, CD206, IL-10 and ARG-1 (n = 6). Data are presented as the mean ± SEM, and n represents the number of animals in each group

Fig. 4

BM-derived cells overexpressing ACE2 prevent SIC. A Western blot experiment suggests increased expression of ACE2 in BM cells. B Experimental schematic diagram of BM transplantation. C Activity of ACE2 in the heart after CLP and levels of Ang II and Ang (1–7) and the ratio of the two determined by ELISA (n = 6). D Plasma (left) and heart tissue (right) LDH levels in each group (n = 6). E M-mode echocardiography of the LV chamber (left) and measurement of the EF% and FS% (right, n = 6). F DHE staining of heart sections with quantification of the fluorescence intensity (left and middle) and qPCR analysis of the NOX2 mRNA levels (right) (n = 6). G Immunohistochemical staining of heart sections using Mac-2 antibody (left) with the percentage of Mac-2-positive areas (right, n = 6). H-I qPCR analysis of the mRNA levels of CD80, IL-1β, IL-6, TNF-α, CD206, IL-10 and ARG-1 (n = 6). Data are presented as the mean ± SEM, and n represents the number of animals in each group

Fig. 5

ACE2 overexpression mitigates macrophages polarization and hyperinflammatory response by inhibiting NF-κB and STAT1 pathways through activating the Ang (1–7)-MasR axis. BMDMs from WT or ACE2-KI mice were pretreated with the MasR antagonist A779 (10^–5 Mol/L) for 1 h and then stimulated by saline or LPS (1 ng/μL) for another 24 h. A DHE staining of BMDMs (left) with quantification of the ROS fluorescence intensity (right, n = 3). B-C qPCR analysis of the mRNA levels of CD80, IL-1β, IL-6, TNF-α, CD206, IL-10 and ARG-1 in BMDMs of each group (n = 4). D Western blot analysis of P-p65 and P-STAT1 (left) in BMDMs with quantification of the relative protein bands (right, n = 3). Data are presented as the mean ± SEM, and n represents the number of independent experiments in each group

Fig. 6

ACE2 overexpression on macrophages mitigates cardiomyocyte injury through activating the Ang (1–7)-MasR axis. A Experimental schematic diagram of co-culture BMDMs and NRCMs. B Measurement of LDH activity in the supernatant of NRCMs in each group (n = 3). C The ratio of Bax to Bcl-2 at mRNA level in NRCMs (n = 3). D Immunostaining of heart sections with TUNEL (red), α-actinin (green) and DAPI (blue) (left), and quantification of TUNEL-positive nuclei (right, n = 3). E DHE staining of NRCMs (left) with quantification of the ROS fluorescence intensity (right, n = 3). Data are presented as the mean ± SEM, and n represents the number of independent experiments in each group

Fig. 7

Mechanistic diagram illustrating the role of ACE2 in SIC. Sepsis reduces ACE2 expression in myeloid cells, leading to enhanced activation of the ACE-Ang II-AT1R axis and subsequent triggering of the NF-κB and STAT1 signaling pathways. This activation prompts macrophage polarization toward M1 phenotype, triggering the production of pro-inflammatory cytokines and ROS, promoting cardiomyocyte apoptosis, ultimately causing cardiac injury. Conversely, ACE2 overexpression in myeloid cells counteracts these effects by converting Ang II to Ang (1-7), which binds to MasR, inhibiting inflammation and ROS production. Consequently, this restoration of balance in the renin-angiotensin system promotes cardiac recovery. These findings underscore the potential of enhancing ACE2 activity or expression as a promising therapeutic strategy for the prevention and treatment of sepsis-induced cardiac dysfunction