Recombinant human angiotensin-converting enzyme 2 plays a protective role in mice with sepsis-induced cardiac dysfunction through multiple signaling pathways dependent on converting angiotensin II to angiotensin 1–7

Introduction

Sepsis is a life-threatening syndrome caused by the host’s dysregulated immune response to infection, which consecutively causes organ dysfunction, disability, and even death (1). Evidence accumulated over the past decades shows a high incidence of sepsis, especially in the intensive care unit (ICU) all over the world (2-5). Though the understanding and management of sepsis have been improving during the past years, the reported mortality remains as high as 20–40% (2,4,6,7).

Sepsis-induced cardiac dysfunction (SICD) is a common complication that exacerbates the poor prognosis of patients, most of which is characterized by reduced left ventricular ejection fraction (LVEF) and diminished contractility (8). In patients with SICD, the mortality is increased 2–3 times (9), which is reported as approximately 70–90% (10). Precise mechanisms and effective therapy in patients with SICD remain unclear and need further exploration to improve its management and outcomes.

The renin-angiotensin-aldosterone system (RAAS) includes classic pathway in which angiotensin II (Ang II) is the main effector and non-classic RAAS pathway in which angiotensin 1–7 (Ang 1–7) plays the key role. Both pathways are expressed in various cells in heart including cardiomyocytes, fibroblasts, endothelia, and immune cells (11-14). Ang II is known to promote heart failure through inducing cardiac hypertrophy, fibrosis, and ventricular remodeling depending on its pro-inflammatory effects (15,16). On the contrary, the non-classic RAAS in heart exerts a cardiac protective role opposite to Ang II (17). Angiotensin-converting enzyme 2 (ACE2), which can inactivate Ang II by catalyzing it to Ang 1–7 (18) shows cardiac beneficial effects depending on its cleavage action of Ang II (19,20).

Evidences indicated that Ang II is enhanced in sepsis and involved in the pathophysiology of organ injury (21,22). Ang II leads to an up-regulation of pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-1 (IL-1) and subsequent macrophage infiltration, micro-vascular ischemia, and cardiac dysfunction in sepsis (23). Treatment targeted to balance the disturbed RAAS in septic animal models show anti-inflammatory effects and are beneficial to heart function and survival (24).

ACE2, which is a homolog of ACE1, is a negative regulator of RAAS by converting Ang II to Ang 1–7 (15). A recombinant human ACE2 (rhACE2; APN01, GSK2586881) has been found to have protective effects on cardiac dysfunction and injury in both inflammatory and non-inflammatory cardiac injury models and to be safe, with no negative hemodynamic effects in healthy volunteers and in patients with acute respiratory distress syndrome (ARDS) (25,26).

The nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, which consists of 3 members, namely apoptosis-associated speck-like protein (ASC), NLRP3, and pro-caspase-1 (27), is a vital mediator pathway of the innate immune system and has recently gained close attention in SICD research.

Previous studies have shown that NLRP3 inflammasome triggers the cardiomyopathy of polymicrobial sepsis induced by cecal ligation and puncture (CLP) (28) and Ang II is involved in nuclear factor κB (NF-κB)-mediated NLRP3 inflammasome activation (29). Therefore, we hypothesized that regulating NLRP3 inflammasome through modulating the activation of RAAS may play a protective role in mice with SICD.

Lipopolysaccharide (LPS) is widely accepted to be used in vivo and in vitro to simulate the clinical conditions of sepsis and SICD for years (30,31). By intraperitoneal injection to mouse, we can get a mouse model of SICD easily and economically.

In our study, we built a model of LPS-induced cardiac dysfunction with mouse to investigate the effects of rhACE2 on SICD and the probable mechanism of it. We present the following article in accordance with the ARRIVE reporting checklist (available at https://atm.amegroups.com/article/view/10.21037/atm-22-6016/rc).

Methods

Animals and treatment

Male healthy C57/B6 mice (animal certification No. 110011210104951884) aged 8–10-week, and weighing 20–25 g, were purchased from Vital River Laboratories, Beijing, China. Before initiating the experiment, a week of acclimation was allowed. The animal was housed in standard pathogen-free environment (22 ℃, light/dark cycle of 12/12 h) with enough standard food and water. A protocol was prepared before the study without registration. The investigator (PZ) in charge of group allocation was the only person who know the group allocation in our team. The study was approved by the laboratory animal ethical committee of Animal Experiment Center of the Fourth Hospital of Hebei Medical University (No. 2020002). Hebei Key Laboratory of Critical Disease Mechanism and Intervention was informed and agreed with this study. All procedures were conducted in the Animal Experiment Center of the institute above and Hebei Key Laboratory of Critical Disease Mechanism and Intervention, in accordance with the Guide for the Care and Use of Laboratory Animals of Institutes of Health (Eighth Edition).

The SICD model was established via intraperitoneal LPS injection. According to the previous work of our team, we obtained 60% of mice with significant myocardial dysfunction at 3 hours after intraperitoneal LPS injection and significance of outcome measures among groups, each of which contained 6 mice (32). The experiment was carried out in two parts. In the first part, the control, LPS, and LPS + rhACE2 groups were arranged to investigate the effects of rhACE2 on mice with LPS-induced cardiac dysfunction. In detail, following the table of random digit, 6 mice were distributed to the control group and at least 20 mice were allocated to a model-establish group that would be subsequently randomly assigned to a LPS group and a LPS + rhACE2 group (6 per group). Mice in the model-establish group were injected with LPS (10 mg/kg, Escherichia coli 055:B5, Cat. No. L8880, Solarbio, Beijing, China) intraperitoneally, and mice in the control group were intraperitoneally injected with an isovolumetric saline. Mice that were injected with LPS yet showed no significant decrease of LVEF and left ventricular fractional shortening (LVFS) at 3 hours after LPS administration compared to the control group were excluded and euthanized by cervical dislocation to reduce their suffering. The LPS-injected mice that displayed a significant decrease of LVEF and LVFS at 3 hours after LPS administration were included and allocated to either LPS group or LPS + rhACE2 group according to the random number method. RhACE2 (Cat No. HY-P7442, MedChemExpress, USA) was dissolved in normal saline 0.9% and intraperitoneally injected at a dose of 200 µg/kg (25,33) into mice allocated to the LPS + rhACE2 group immediately after group allocation. The remaining mice were treated with isovolumetric saline. All mice underwent echocardiographic re-evaluation at 6 hours after LPS injection and were sacrificed rapidly by cervical dislocation. Inhaling isoflurane (2%) anesthesia was applied to all mice to reduce their suffering during echocardiographic examination. The blood and myocardial tissues were collected. To minimize the influence of the order of treatments and measurements on the outcome, we applied the treatment, echocardiographic examination, and sample collection in the same order. The serum samples were isolated through centrifugation (3,000 rpm, 15 minutes) at 4 ℃ and frozen at −80 ℃ immediately. The heart samples were rinsed with phosphate-buffered saline (PBS) to remove the residual blood and frozen at −80 ℃ for further investigation.

A779, a selective mas receptor antagonist of Ang 1–7, was used to further elucidate the mechanism of the effects of rhACE2 on LPS-induced cardiac dysfunction in the second part. In detail, four groups (the control group, the LPS group, LPS + rhACE2 group, and LPS + rhACE2 group +A779, each containing 6 mice), were used to investigate the LVEF and LVFS, cardiac structure injury, pyroptosis cell rates, and NLRP3 expression in heart tissue. The establishment of the SICD model, and the criteria of inclusion and exclusion were same to those of the first part. Random numbers were also used for group allocation. Besides the LPS group and LPS + rhACE2 group, mice with LPS-induced cardiac dysfunction were also distributed randomly to the LPS + rhACE2 +A779 group, receiving 10 mg/kg A779, (HY-P0216, MedChemExpress, Monmouth Junction, NJ, USA) by tail vein injection (34) prior to receiving 200 µg/kg rhACE2 by intraperitoneal injection. All the mice were also sacrificed after cardiac function evaluation by echocardiography at 6-hour after LPS treatment under inhaling isoflurane anesthesia. The serum samples and myocardial tissues were collected as described previously.

Echocardiography

LVEF and LVFS were obtained from M-mode detection of left ventricular (LV) to evaluate cardiac function of mice by a special ultrasound instrument (Vevo 2100 imaging system, VisualSonics, Ontario, Canada) for small animals with a 13-MHz linear transducer under isoflurane inhalation anesthesia. The sonographer was blinded to the group allocation.

Indicators of myocardial injury and inflammation

Commercial enzyme-linked immunosorbent assay (ELISA) kits were obtained to examine the serum levels of indicators myocardial injury and inflammation. Cardiac troponin I (cTnI; Cat No. E-EL-M1203c, Elabscience Biotechnology, Wuhan, China) was the indicator of cardiac injury we used. The inflammatory factors included TNF-α (Cat No. KE10002, Proteintech, Wuhan, China), IL-18 (Cat No. CSB-E04609m, CUSABIO, Wuhan, China), and IL-1β (Cat No. KE10003, Proteintech).

Angiotensin in serum

Serum concentrations of Ang II (Cat No. CSB-E04495m, CUSABIO) and Ang 1–7 (Cat No. CSB-E13763m, CUSABIO) were investigated through ELISA kits on the basis of the manufacturer’s instructions.

Hematoxylin and eosin (HE) staining

Myocardial tissues were fixed in 4% paraformaldehyde for over 24 hours, embedded in paraffin, cut into 4 µm-thick sections, and stained with HE at room temperature. Two skilled pathologists who were blind to the interventions examined the extent of myocardial injury with a light microscope (DM3000 LED, Leica, Wetzlar, Germany) at 200× magnification and photographed them with a digital camera. The extent of myocardial injury showed in sections were scored using a semi-quantitative method as previously described: 0 for no damage; 1 for damage involving no more than 25% of the myocardium and 2 for 25–50%, 3 for 50–75%, and 4 for 75%, respectively (35,36). The average score of two sections of each sample were used for analysis.

Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining

TUNEL staining was performed to identify pyroptosis cells in heart tissue sections with a TUNEL assay kit (Servicebio, G1501, Wuhan, China). Myocardial tissue sections were deparaffinized with xylene and rehydrated with ethanol, and then were reacted with proteinase K working solution (Servicebio, G1205) at 37 ℃ for 25 minutes and completely washed with PBS. Sections were further incubated with permeabilize working solution (the membrane breaking fluid was 0.1% triton. Configuration method, triton stock solution:PBS =1:1,000) for 20 minutes and washed. TUNEL staining was performed following the manufacturer’s protocol. Microscopic examination was performed, and images were collected through a fluorescence microscope (Nikon, Tokyo, Japan). Cells that were positively labeled (green) were quantified in 10 different fields per slide by Image J software (1.53a, Wayne Rasband, NIH, USA). The means and standard deviations (SDs) were derived from counting a minimum of three fields from two different coverslips per sample.

Western blot (WB) analysis

According to the instructions, whole proteins were extracted from heart tissues in radioimmunoprecipitation assay (RIPA) buffer (P0013B, Beyotime, Nanjing, China) with phosphatase inhibitor in it. The protein content was measured with a bicinchoninic acid (BCA) kit (Cat No. SW101-02, SEVEN, Beijing, China). Protein samples (40 µg per lane) were separated by 8% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore-Upstate, Beijing, China, 0.45 or 0.22 µm). Nonfat milk (Cat No. 1172GR500, Saiguo, Guangzhou, China) or albumin (Solarbio) resolved in tris-buffered saline solution with 0.1% Tween-20 (TBST) were used to block the membranes (37 ℃ for 2 hours). Membranes were then incubated at 4 ℃ overnight with primary antibodies against {[NLRP3 1:1,000, anti-NLRP3 antibody (EPR23094-1), Cat No. ab263899, Abcam, Cambridge, UK], gasdermin D [GSDMD; 1:1,000, anti-GSDMD (EPR19828), Cat No. ab209845, Abcam], caspase-1 [1:1,000, Cat No. ab179515, anti-pro-caspase-1 + p10 + p12 antibody (EPR 16883), Abcam], NF-κB (1:500, NF-κB p65 Ab, Cat No. AF 5006, Affinity Biosciences, Liyang, China), p-NF-κB [1:500, phospho-NF-κB p65 (Ser536) Ab, Cat No. AF 2006, Affinity Biosciences], AMP-activated protein kinase (AMPK; 1:500 catalog number 10929-2-AP AMPK alpha 1 rabbit polyclonal antibody, Proteintech), p-AMPK [1:500; phospho-AMPK (Thr172) (D4D6D), Cat No. #50081, Cell Signaling Technology, Danvers, MA, USA], p38 mitogen-activated protein kinase (MAPK; 1:500, p38 MAPK rabbit polyclonal antibody, Cat No. 14064-1-AP, Proteintech), p-P38 MAPK [1:500, phosph-p38 MAPK Thr180/Tyr182(D3F9)XP Rabbit mAb, Cat No. #4511, Cell Signaling Technology], and α-tubulin (alpha tubulin rabbit polyclonal antibody 1:5,000, Cat No. 11224-1-AP, Proteintech)}. The membranes were washed in TBST 3 times (15 minutes each time) followed by incubation with HRP-linked secondary antibodies (1:10,000, Cat No. SA00001-2, Proteintech) (room temperature for 2 hours). Enhanced chemiluminescence (ECL) detection kit (Cat No. P10300, NcmBiotech, Suzhou, China) and BLT GelView 6000Plus (Fluorchem E, Santa Clara, CA, USA) were used to visulize the protein bands. Image J software (1.53a, Wayne Rasband) was used to quantify the intensities of the bands. Alpha-tubulin was used as internal control.

Statistical analyses

The results were represented as mean ± SD and analyzed using Graph Prism 8.0 (GraphPad Software, San Diego, CA, USA) and SPSS 26.0 software (IBM Corp., Armonk, NY, USA). The differences between the two groups were compared by Student’s unpaired t-test. Differences among multiple groups were analyzed by one-way analysis of variance (ANOVA) followed by a post hoc analysis by least significance difference (LSD) test or Tamhane’s T2 test, depending on the deference of variances. Correlation between serum levels of Ang II and levels of NLRP3 expression in heart tissue was analyzed by Pearson correlation analysis. Statistical significance was regarded as P<0.05.

Results

RhACE2 improved cardiac function in mice with LPS-induced cardiac dysfunction

In the first section of the study, 18 (6 per group) out of the total 28 mice that were enrolled completed the experiment. Due to absence of significant decline in LVEF and LVFS, 10 mice were excluded. Manifestation of the animal received LPS injection included lethargy, mild closure of the eyes, ruffled fur, and diarrhea consistent to those of endotoxemia. All mice among groups weighed comparably and survived at 6 hours after intervention. LVEF and LVFS of mice at 3 hours after LPS injection in the LPS group and LPS + rhACE2 group were significantly lower than those in control group. Compared with the LPS group, LVEF and LVFS improved significantly in the LPS + rhACE2 group at 3 hours after rhACE2 treatment (Figure 1A-1C).

Figure 1 RhACE2 administration improves cardiac function in mice with LPS-induced cardiac dysfunction. (A) Transthoracic echocardiography (M-mode) of mice among three groups (6 per group). (B,C) Comparison of LVEF and LVFS of mice among three groups (6 per group). Data are expressed as mean ± SD. LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; SD, standard deviation.

RhACE2 administration relieved myocardial injury in mice with LPS-induced cardiac dysfunction

The HE staining of myocardial sections indicated widespread myocardial structure disorder, interstitial hemorrhage and edema, and infiltration with neutrophil granulocytes in LPS-induced cardiac dysfunction mice, which were relieved significantly by rhACE2 treatment in the LPS + rhACE2 group (Figure 2A,2B). Compared with the control group, cTnI concentration in serum increased significantly at 6 hours in the LPS group. Compared with the LPS group, cTnI concentration in serum in the LPS + rhACE2 group decreased significantly (Figure 2C).

Figure 2 RhACE2 administration relieved myocardial injury in mice with LPS-induced cardiac dysfunction. (A) Representative pictures of HE-stained heart tissue of mice in three groups (n=6). Magnification, ×200. (B) Myocardial injury score in three groups (n=6). (C) Evaluation of myocardial injury by cTnI in serum from mice in three groups (n=6). Data are expressed as mean ± SD. LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; HE, hematoxylin and eosin, cTnI, cardiac troponin I; SD, standard deviation.

RhACE2 significantly inhibited activation of NLRP3 inflammasome followed by significant decrease of NLRP3 inflammasome-related inflammatory factors in serum and cell pyroptosis in heart tissues induced by LPS

According to WB analysis of heart tissue, expression of NLRP3 in the LPS group were significantly higher than those in control group, and expression of NLRP3 in the LPS + rhACE2 group was significantly lower than that in the LPS group. Expressions of the other two classic pyroptosis-associated proteins, N-terminal cleavage product of GSDMD (GSDMD-NT) and caspase-1 p12 (cleavage product of caspase-1 p46) in heart tissues were also increased in the LPS group and then decreased after rhACE2 treatment (Figure 3A-3D). Serum concentrations of IL-1β and IL-18, the inflammatory factors matured through NLRP3 inflammasome activation, increased significantly in the LPS group and decreased significantly under the treatment of rhACE2 (Figure 3E,3F). And the significant increase in serum of TNF-α, the representative inflammatory factor in sepsis, was also suppressed by rhACE2 (Figure 3G). TUNEL staining of heart tissue sections showed that pyroptosis cell rates in the LPS group significantly increased compared with those in control group, and decreased significantly in the LPS + rhACE2 group compared with those in LPS group (Figure 3H,3I).

Figure 3 RhACE2 significantly inhibited activation of NLRP3 inflammasome followed by significant decrease of NLRP3 inflammasome related inflammatory factors in serum and cell pyroptosis in heart tissues in mice with LPS-induced cardiac dysfunction. (A-D) Representative bands of WB and quantitative analysis of pyroptosis-related proteins in three groups (n=6). α-tubulin was used as an internal control. (E) The serum levels of IL-1β in three groups (n=6). (F) The serum levels of IL-18 in three groups (n=6). (G) The serum levels of TNF-α in three groups (n=6). (H) Representative images of cell pyroptosis (indicated by TUNEL positive cells) in heart tissue of mice in three groups (6 per group). Nuclei were depicted by DAPI (blue). The pyroptotic cells were depicted by TUNEL (green). Magnification, ×200. (I) Comparison of pyroptosis cell rates in heart tissue from mice in three groups (6 per group). Data are expressed as mean ± SD. GSDMD, gasdermin D; GSDMD-NT, N-terminal cleavage product of GSDMD; NLRP3, nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3; LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; IL, interleukin; TNF-α, tumor necrosis factor-α; DAPI, 4',6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; WB, western blot; SD, standard deviation.

RhACE2 regulated changes of angiotensin in LPS-induced cardiac dysfunction mice, and the levels of Ang II in serum had a significantly positive correlation with levels of NLRP3 expression in heart tissue

In the LPS group, Ang II and Ang 1–7 levels in serum were significantly higher than those in the control group. Compared with the LPS group, levels of Ang II in serum decreased significantly in the LPS + rhACE2 group, and levels of Ang 1–7 further increased in the LPS + rhACE2 group (Figure 4A,4B). As indicated by WB analysis, in mice with LPS-induced cardiac dysfunction, NLRP3 expression in heart tissue increased significantly compared with the control group, but decreased significantly after rhACE2 treatment (Figure 3A,3B). The levels of Ang II in serum had a significantly positive correlation with levels of NLRP3 expression in heart tissue (Figure 4C).

Figure 4 RhACE2 regulated the changes of angiotensin in mice with LPS-induced cardiac dysfunction, and the levels of Ang II in serum correlated with the levels of NLRP3 expression in heart tissue positively. (A) Ang II levels in serum of mice in three groups (n=6). (B) Ang 1–7 serum levels in serum of mice in three groups (n=6). (C) Ang II levels in serum were positive correlated with levels of NLRP3 expression in heart tissue. Data are expressed as mean ± SD. Ang II, angiotensin II; LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; Ang 1–7, angiotensin 1–7; NLRP3, nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3; SD, standard deviation.

RhACE2 regulated activation of the NF-κB, p38 MAPK, and AMPK pathways in heart tissue

In order to determine the possible mechanisms and relationship between LPS-induced cardiac dysfunction and rhACE2, we further evaluated the activation levels of the NF-κB, p38 MAPK, and AMPK-α1 signaling pathways-associated proteins in heart tissue, which were involved in cardiac injury due to either Ang II/Ang 1–7 imbalance or sepsis, by WB analysis. We found that the increases of levels of p65 (a subunit of NF-κB) and p38 MAPK phosphorylation in the LPS group were diminished by rhACE2 treatment. On the contrary, levels of AMPK-α1 signaling pathway phosphorylation decreased in the LPS group and recovered partly after rhACE2 treatment (Figure 5A-5D).

Figure 5 RhACE2 significantly inhibited phosphorylation of NF-κB, p38 MAPK signaling pathway, and partly recovered activation of AMPK signaling pathway changed in mice with LPS-induced cardiac dysfunction. (A) Representative bands of WB and quantitative analysis of NF-κB, p38 MAPK, and AMPK-α1 signaling pathway related proteins expression in three groups. (B) Quantitative analysis phosphorylation levels of phosphorylation levels of NF-κB in three groups (n=6). (C) Quantitative analysis phosphorylation levels of p38 MAPK in three groups (n=6). (D) Quantitative analysis phosphorylation levels of AMPK-α1 in three groups (n=6). α-tubulin was used as an internal control. Data are expressed as mean ± SD. p-, phosphorylated-; NF-κB, nuclear factor κB; AMPK, AMP-activated protein kinase; MAPK, mitogen-activated protein kinase; LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; WB, western blot; SD, standard deviation.

A779 administration offset the inhibitive effects of rhACE2 on NLRP3 expression and cardiac-protective effects on mice with LPS-induced cardiac dysfunction

In the second section, 12 of the total 36 mice were excluded for absence of cardiac dysfunction. Finally, 24 mice were allocated to four groups (6 per group) as described in methods and completed all the investigations. Symptoms following LPS injection were similar with those described previously. Also, all mice among groups weighed comparably and survived at 6 hours after interventions. The LVEF and LVFS of mice with LPS-induced cardiac dysfunction that were treated with both rhACE2 and A779 were lower than those of the mice were treated with rhACE2 only. The NLRP3 expression in heart tissue of mice with LPS-induced cardiac dysfunction that were treated with both rhACE2 and A779 were higher than in those that were treated with rhACE2 only. Myocardial injury and pyroptosis cell rates in mice with LPS-induced cardiac dysfunction who were treated with both rhACE2 and A779 were also higher than that who were treated with rhACE2 only. Therefore, A779 administration offset the inhibitive effects of rhACE2 on NLRP3 expression and cardiac-protective effects on LPS-induced cardiac dysfunction (Figure 6A-6J).

Figure 6 A779 administration offset the improve effect of rhACE2 in mice with LPS-induced cardiac dysfunction. (A) Transthoracic echocardiography (M-mode) of mice in each group (6 per group). (B) Comparison of LVEF of mice among four groups (6 per group). (C) Comparison of LVFS of mice among four groups (6 per group). (D) Representative pictures of HE-stained heart tissue of mice in four groups (n=6). Magnification, ×200. (E) Myocardial injury score in four groups (n=6). (F) Evaluation of myocardial injury by cTnI in serum from mice in four groups (n=6). (G) Representative images of cell pyroptosis (indicated by TUNEL labeled cells) in heart tissue of mice in four groups (6 per group). Nuclei were depicted by DAPI (blue). The pyroptotic cells were depicted by TUNEL (green). Magnification, ×200. (H) Comparison of pyroptosis cell rates in heart tissue from mice in four groups (6 per group). (I) WB for NLRP3 expression in heart tissue of mice from four groups (n=6), α-tubulin was used as an internal control. (J) Quantitative analysis of NLRP3 expression in heart tissue of mice from four groups (n=6). Data are expressed as mean ± SD. LPS, lipopolysaccharide; rhACE2, recombinant human angiotensin-converting enzyme 2; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; DAPI, 4',6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase dUTP nick-end labeling; NLRP3, nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3; HE, hematoxylin and eosin; cTnI, cardiac troponin I; WB, western blot; SD, standard deviation.

Discussion

Recently, SICD has been attracting much attention because of its contribution to mortality and complexity of management in patients with sepsis (37). However, the mechanism and treatment of SICD are still full of uncertainties. Since the hypothesis that SICM develop at the cause of gross coronary hypoperfusion was denied by the landmark study which showed the increased coronary blood flow in patients with septic shock (38), increasing numbers of studies showed disturbance of microcirculatory and microvascular thrombosis in septic heart (39,40). The notion that myocardial inhibitors, such as TNF-α, IL-1β, IL-6, ROS, and RNS (41-45), derived form either inflammation or mitochondrial dysfunction, or the crosstalk between them suppress the cardiac function is consistent with the findings that the systolic dysfunction is global (46). Additionally, influence of Ca²⁺ dysregulation and β-adrenergic alteration on myocardial contractility were also discussed in detail (47-49). Disturbance of energy metabolism due to either mitochondrial dysfunction or substrate utilization disorder is hot topic in researches (50,51). Though cell death is not the significant character in septic heart, different forms of programmed myocardial cell death including apoptosis, pyroptosis, and ferroptosis have been being investigated because of their influence on inflammation and mitochondrial function (52-56). The RAAS has been a focus of research for the complex crosstalk among the components and the robust association with heart disease (19,57,58). How it is involved in sepsis had been investigated for decades (59) and attracted more attention again because of the global pandemic of COVID-19 recently for that ACE2 is known to be the receptor of SARS-CoV-2 (18). Besides the drug aimed to increase myocardial contractility and mechanical device aimed to support the cardiac pump function, various treatments targeted to regulate inflammation, mitochondrial dynamics and function, energy metabolism to improve cardiac conditions have been investigated in animal or cell experiments based on the mechanisms (60-62). However, the conclusions are variable and the data of clinical study is absence.

In this study, we demonstrated that rhACE2 regulated Ang II and Ang 1–7 serum levels followed by relief of myocardial injury and improvement of LVEF in a mouse model of LPS-induced cardiac dysfunction. The effects of rhACE2 on activations of the NF-κB, p38 MAPK, and AMPK-α1 signaling pathways that were demonstrated in the present study indicated that rhACE2 ameliorated cardiac injury and dysfunction in mice with LPS-induced cardiac dysfunction through the NF-κB, p38 MAPK, and AMPK-α/NLRP3 inflammasome pathways depending on conversion of Ang II to Ang 1–7 during sepsis. To the best of our knowledge, the present study is the first to report the protective role of rhACE2 in SICD, in spite of a large body of research demonstrating the protective effects of rhACE2 on heart function in conditions with pressure overload, diabetic cardiomyopathy, myocardial infarction, or doxorubicin-induced cardiomyopathy.

LPS, which is a cell wall component of Gram-negative bacteria, is known to be effective to trigger an intense inflammatory response and induce sepsis (63). LPS intraperitoneal injection has been used to build an SICD model successfully for years (30,31). In the present study, LVEF declined significantly at 3 hours and reached to the bottom at 6 hours after LPS injection in about 60% of LPS treated mice, which was consistent with the data reported by a previous study (64). Changes of cTnI in serum and what were observed in HE staining heart tissue sections showed obvious cardiac injury in mice with LPS-induced cardiac dysfunction. Using the mouse model of SICD, we elucidated the actions of rhACE2 on SICD successfully.

Various mechanisms participate in myocardial dysfunction induced by sepsis. Pyroptosis, a form of inflammation programmed cell death, is involved in either inflammatory or non-inflammatory heart disease (65,66). NLRP3 inflammasome activation plays a significant role in initiating inflammatory response and pyroptosis, contributing to cardiac injury and dysfunction in both ischemic and inflammatory heart disease (67,68).

In the present study, we observed that NLRP3 expression, caspase-1 and GSDMD activation in heart tissue increased significantly in mice with LPS-induced cardiac dysfunction, and dropped significantly under rhACE2 treatment. Up-regulation of pyroptotic cell rates in heart tissue indicated by TUNEL staining, and increase of IL-1β and IL-18 in serum of mice with SICD were also diminished by rhACE2 treatment in our study. According to previous report, increase of NLRP3 protein expression is the priming step of NLRP3 inflammasome activation (27). Caspase-1, the key effector of NLRP3 inflammasome, self-cleaves to active caspase-1 on the platform NLRP3 assembled during activation. Active caspase-1 then promotes pro-IL-1β and pro-IL-18 maturate to IL-1β and IL-18 and cleaves GSDMD to free its N-terminal domain and induce the formation of pores at the membrane, leading to pyroptosis (69). TNF-α is a main inflammatory cytokine in sepsis. Maturation and release of IL-1β and IL-18 amplify the inflammatory response as indicated by the changes of TNF-α among groups in the study. TNF-α and IL-1β are regarded as the main myocardial inhibitors in septic shock contribute to cardiac depression. Pyroptosis procedure mediated by GSDMD cleavage has a detrimental effect on cardiac function (70). Inhibitors of NLRP3 inflammasome-related pro-inflammatory factors and depletion of pyroptosis by knocking out NLRP3 or other components of NLRP3 inflammasome (ASC, GSDMD, or caspase-1) attenuate cardiac injury and improve cardiac function (67,68,70-72). Our observation in the present study also indicated the involvement of NLRP3 inflammasome activation in LPS-induced cardiac dysfunction. Further, the inhibitive role of rhACE2 on NLRP3 inflammasome activation as well as the following inflammatory response and cell pyroptosis demonstrated in our study is the probable mechanism of the protective role of rhACE2 in SICD. This is also consistent with previous researches reporting that rhACE2 shows a protective role in sepsis-induced organ injury (26,33), and cardiac dysfunction caused by doxorubicin (73) through inhibition of NLRP3 inflammasome.

As a negative regulator of RAAS, ACE2 catalyzes the vasoconstrictive, pro-inflammatory, and fibrogenic Ang II peptide to the vasodilatory, anti-inflammatory, and anti-fibrogenic Ang 1–7 peptide, which plays a protective role in heart disease (74,75). The catalyzing action from Ang II to Ang 1–7 of rhACE2 was also revealed in our study. Ang II in serum increased significantly in the LPS group, and dropped significantly in the LPS + rhACE2 group. In contrast, Ang 1–7 concentrations went up significantly in the LPS group and further increased in the LPS + rhACE2 group. Increase of Ang II and Ang 1–7 in LPS group just the results of activation of RAAS during sepsis and SICD. Decrease of Ang II and Increase of Ang 1–7 indicated the converting action of rhACE2 on Ang II. Additionally, our study showed that changes of NLRP3 expression in heart tissue were significantly positively correlated with the changes of Ang II in serum. Therefore, it could be Ang II that influenced the NLRP3 expression in heart tissue and caused cardiac injury. In fact, Ang II has been shown by previous reports to induce NLRP3 and reactive oxygen species (ROS) production which participates the disturbed redox cycle in sepsis and leads to cardiac injury and dysfunction (76,77).

Ang 1–7, the effects of which are opposite to those of Ang II, exerts its effects of inhibiting NLRP3 inflammasome activation through the Mas receptor and is implicated in physiological and pathophysiological processes, including leukocyte recruitment and inflammation, cardiac remodeling, and vascular alteration (78,79). Mice deficient for Mas receptor show cardiovascular-related phenotypes, including myocardial contractile dysfunction and endothelial dysfunction (74,80,81). On the contrary, increase of Ang 1–7 in the heart by either infusion or overexpression from transgenes elicited cardioprotective actions in cardiac damage models (82,83). When A779, a selective mas receptor antagonist of Ang 1–7, was administrated to mice with SICD, the protective role of rhACE2 on cardiac injury and function as well as the inhibitive role of it on NLRP3 expression were offset according to our study.

Not only A779 offset LVEF improve due to rhACE2 treatment in mice with SICD, but it also offset the effects of rhACE2 treatment on changes of cTnI concentration in serum, cell pyroptosis in heart tissue and myocardial injury induced by LPS insult. Furthermore, administration of A779 offset the increase of NLRP3 induced by LPS insult. What were observed in the A779 part of the experiment verified that the protective effects of rhACE2 on SICD depended on its conversion action of Ang II and the resulting increase of Ang 1–7. The results are similar to the report of Liu et al., in which A779 offset the beneficial effects on acute lung injury resulted from sepsis (84).

As shown in this study, rhACE2 treatment decreased Ang II and further enhanced Ang 1-7 in serum of mice with SICD. In line with the activation of NLRP3 inflammasome, the NF-κB and p38 MAPK signaling pathways were activated in mice with LPS induced cardiac dysfunction. In line with the suppression of NLRP3 inflammasome activation by rhACE2 treatment, activation of the NF-κB and p38 MAPK signaling pathways were also suppressed in mice with LPS-induced cardiac dysfunction. Therefore, we inferred that the NF-κB and p38 MAPK signaling pathways were involved in the protective role of rhACE2 in SICD. The activation of toll-like receptors (TLRs)/NF-κB has been extensively accepted as pathways contributing to development of sepsis and following MODS including SICD (85). In response to microbial molecules, NF-κB stimulates the expression of NLRP3, which is the priming step of canonical NLRP3 inflammasome activation (69,86,87). Also, previous researches demonstrated that p38 MAPK plays a role in inflammasome priming signals (88) and regulation of downstream p38 MAPK activation is critical for NLRP3 expression and caspase-1 activation (89). It has been reported that the p38 MAPK pathway is closely related to LPS-induced septic shock (90). A recent study demonstrated that activation of the p38 MAPK pathway promotes NLRP3 inflammasome activation in septic shock (91). Ang II has been shown to induce NLRP3, in various cardiac cells in a non-septic cardiac damage model, depending on the AT1R/NF-κB or p38 MAPK pathway or crosstalk among them (76,77,92). By contrast, Ang 1–7 has inhibitory effects on activated NF-κB and p38 MAPK pathways in these cells (93) and is beneficial for improved contractility (94).

Additionally, in the present study, the AMPK-α1 signaling pathway was inhibited in mice with LPS-induced cardiac dysfunction and was partly recovered by rhACE2 administration. Previous reports have shown that AMPK activation inhibited NLRP3 inflammasome-induced pyroptosis in microglia (95) and relieved myocardial dysfunction through the AMPK-α/NLRP3 inflammasome dependent pathway (96). Deficiency of AMPK-α1 influences systemic production of cytokines and results in lung injury, liver damage after sepsis, and exacerbates sepsis-induced mortality in male mice (97). Evidence showed that ACE2 deficiency impaired cardiac function and contributed to suppressing the phosphorylation of AMPK, aggravating cardiac lipotoxicity, and myocardial insulin resistance (98). In contrast, increasing activation of AMPK-α and the PI3K/AKT pathway by an ACE2 activator improved cardiac function in diabetic cardiomyopathy (99). Therefore, enhancement of AMPK-α1 activation could represent another signaling pathway with a protective role of rhACE2 to SICD.

Thus, multiple mechanisms, including the NF-κB, p38 MAPK, and AMPK-α1 signaling pathways, were involved in the cardiac protective role of rhACE2 in SICD depending on converting Ang II to Ang 1–7 followed by the regulation of activation of NLRP3 inflammasome. By modulating NLRP3 inflammasome activation, rhACE2 is supposed to regulating the followed inflammatory process and redox imbalance in septic heart, which might protect the heart from injury.

In summary, we established a model of SICD in vivo with mouse and detected the beneficial role of rhACE2 on SICD; the results might provide a new direction of management for patients with SICD.

Additionally, as the receptor of the novel SARS-CoV-2, ACE2 mediates the entry of virus into cells, contributing to the subsequent infection and pathological process such as inflammation and organ injury (18). There are two forms of ACE2 existing in plasma (which is named soluble ACE2, sACE2) and anchoring on cell membrane respectively. It is the membrane-located ACE2 that can be bound to the viral surface spike protein to facilitate the virus internalization and subsequent replication (100). Additionally, the entry of virus mediated by transmembrane ACE2 led to down-regulation of the trans-membrane ACE2 followed by increase of local Ang II which aggravated the inflammation in tissue (18). As a competitive interceptor, sACE2 can neutralize the viruses and block their entry into target cells. It is verified by the study in which soluble rhACE2 inhibited SARS-CoV-2 infection in engineered human tissues (101). Therefore, supplementing soluble rhACE2 to patients with SARS-CoV-2 infection is expected to benefit the population through both infection control and RAAS negative modulation and deserves further investigation in preclinical and clinical settings.

Limitations

Compared with the CLP model, the SICD model in the present study, which was established via LPS intraperitoneal injection, does not simulate the common pattern of sepsis well in clinical conditions, since sepsis often derives from infection caused by either Gram-negative, or Gram-positive bacterial, or both of them. However, management of cardiac dysfunction induced by endotoxemia, which excludes the influence of unsatisfactory infection control on progression of sepsis and organ injury, can clarify the effects of the intervention aiming to more adequately modulate the dysregulated host response to infection.

In the absence of hemodynamic monitoring, we could not provide accuracy data on preload and could not clarify the influence of fluid resuscitation on LVEF. However, the left ventricular end diastolic volume (LVEDV) of mice among groups were comparable (Figure S1), which indicated the comparable preload among groups in a certain degree.

In the absence of a cell experiment, we could not present the cell-specific effects of rhACE2 on SICD, especially effects on cardiomyocytes. Indeed, in our study, the main pathologic discovery of heart section was not cardiomyocyte necrosis but infiltration, interstitial edema, and cardiomyocyte disorders. Consistently, cardiomyocyte necrosis is probably of less importance as it has been found only in a small proportion of human autopsy specimens, and its occurrence during experimental sepsis has not been universally confirmed (102). In fact, pyroptosis occurred not only in cradiomyocytes, but also in immune cells, cardiac fibroblast cells, and endothelial cells that exist in heart tissue and amplified inflammatory, which contributes to the reversible functional damage of the septic heart (30,103-106).

Only function of the left heart was evaluated in our study. Since it is common (107) and associates with higher short-term and long-term mortality during sepsis (108), right ventricular (RV) function in sepsis is worthy of further investigation to compensate our understanding of SICD, which will be taken into consideration in our subsequent studies.

Additionally, cardiac diastolic dysfunction was also detected in patients with SICD (9). However, we did not detect a significant difference among groups when we examined the diastolic function of left ventricle by E/A (Figure S2). A better animal model is needed to evaluate the diastolic function of the septic heart and further study is needed to explore the management of diastolic dysfunction of SICD.

Conclusions

In conclusion, rhACE2 played a protective role in the SICD mice model, ameliorating cardiac injury and contractile dysfunction in mice with LPS-induced cardiac dysfunction through the NF-κB, p38 MAPK, and AMPK-α1/NLRP3 inflammasome pathway dependent on converting Ang II to Ang 1–7. These findings provide a new idea about research and management of SICD.

Well-designed preclinical and clinical studies are needed to investigate the exact influence and mechanism of rhACE2 on SICD in depth, which are expected to enable a precise understanding and treatment of SICD.

Acknowledgments

Funding: This study was supported by the Department of Science and Technology of Hebei Province of China (No. 20277707D).

Footnote

Reporting Checklist: The authors have completed the ARRIVE reporting checklist. Available at https://atm.amegroups.com/article/view/10.21037/atm-22-6016/rc

Data Sharing Statement: Available at https://atm.amegroups.com/article/view/10.21037/atm-22-6016/dss

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://atm.amegroups.com/article/view/10.21037/atm-22-6016/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Animal experiments were performed under a project license (No. 2020002) granted by the laboratory animal ethical committee of Animal Experiment Center of the Fourth Hospital of Hebei Medical University, complying with the Guide for the Care and Use of Laboratory Animals of Institutes of Health (Eighth Edition). Hebei Key Laboratory of Critical Disease Mechanism and Intervention was informed and agreed with this study.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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(English Language Editor: J. Jones)