Angiotensin-(1-7) ameliorates sepsis-induced cardiomyopathy by alleviating inflammatory response and mitochondrial damage through the NF-κB and MAPK pathways

Abstract

Background: There is no available viable treatment for Sepsis-Induced Cardiomyopathy (SIC), a common sepsis complication with a higher fatality risk. The septic patients showed an abnormal activation of the renin angiotensin (Ang) aldosterone system (RAAS). However, it is not known how the Ang II and Ang-(1-7) affect SIC.

Methods: Peripheral plasma was collected from the Healthy Control (HC) and septic patients and Ang II and Ang-(1-7) protein concentrations were measured. The in vitro and in vivo models of SIC were developed using Lipopolysaccharide (LPS) to preliminarily explore the relationship between the SIC state, Ang II, and Ang-(1-7) levels, along with the protective function of exogenous Ang-(1-7) on SIC.

Results: Peripheral plasma Ang II and the Ang II/Ang-(1-7) levels in SIC-affected patients were elevated compared to the levels in HC and non-SIC patients, however, the HC showed higher Ang-(1-7) levels. Furthermore, peripheral plasma Ang II, Ang II/Ang-(1-7), and Ang-(1-7) levels in SIC patients were significantly correlated with the degree of myocardial injury. Additionally, exogenous Ang-(1-7) can attenuate inflammatory response, reduce oxidative stress, maintain mitochondrial dynamics homeostasis, and alleviate mitochondrial structural and functional damage by inhibiting nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways, thus alleviating SIC.

Conclusions: Plasma Ang-(1-7), Ang II, and Ang II/Ang-(1-7) levels were regarded as significant SIC biomarkers. In SIC, therapeutic targeting of RAAS, for example with Ang-(1-7), may exert protective roles against myocardial damage.

Keywords: Angiotensin (1–7); Angiotensin II; Apoptosis; Mitochondria; Oxidative stress; Sepsis-induced cardiomyopathy.

Fig. 1

Clinical correlation between plasma Ang II, Ang II/Ang-(1–7), and Ang-(1–7) levels and SIC patients. A–C Ang-(1–7), Ang II, Ang II/Ang-(1–7), expression levels in the peripheral plasma samples of HC (n = 12), non-SIC (n = 18), and SIC (n = 16) patients. D Correlation matrix of plasma Ang-(1–7), Ang II, and Ang II/Ang-(1–7) levels with biochemical indices and clinical scores in the SIC patients (n = 16). The matrix values included the Spearman correlation coefficient (p < 0.05). NS: no significant difference, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 2

Ang-(1–7) reduces systemic inflammatory response, alleviates myocardial injury, and increases survival in SIC mice. A Schematic representation of the animal experiments. Mice were injected with Ang-(1–7) (or same saline volume), IP, daily for 3 days. 30 min after administering the compounds (or saline) for 3 days of the administration, the animals in the corresponding groups were again IP injected by LPS (10 mg/kg) or saline. Echocardiography images were captured 12 h later and mice were sacrificed for further experiments. B–F Serum Ang II, IL-6, cTnT, IL-1β, and TNF-α levels (n = 6). G M-mode echocardiography representative images. H Quantitative assessment of LVEF, LVEDV, LVESV, LVID; s, LVID; d, LVFS in every mouse group (n = 4). I Representative images of HE staining. J Histopathological scores of the heart (n = 6). K mRNA levels of BNP in the myocardial samples (n = 4). L Influence of Ang-(1–7) pretreatment on the 3^rd day of survival of septic mice (n = 12). NS: no significant difference, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 3

Ang-(1–7) attenuates inflammatory response, macrophage infiltration, and oxidative stress in myocardial tissues of the SIC mice. A Representative immunofluorescence images of macrophage infiltration in myocardial tissue of each group (20 μm; × 400, scale bar). B Number of F4/80⁺ macrophages (n = 4). C–G The IL-6, Ang II, TNF-α, Ang-(1–7), and IL-1β protein concentrations in the myocardial specimens (n = 6). H–J TNF-α, IL-1β, and IL-6 mRNA levels in myocardial tissue samples (n = 4). K Images showing the DHE staining in myocardial tissues (50 μm; × 200, scale bar). L Relative fluorescence intensity of the DHE procedure (n = 4). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 4

Ang-(1–7) maintains the mitochondrial dynamic equilibrium of cardiomyocytes in SIC mice, reverses their structural dysfunction and damage, and alleviates cardiomyocyte apoptosis. A Representative TEM image of myocardial mitochondria. B Flameng score of TEM images of mitochondria of cardiomyocytes (n = 4). C MMP in myocardial tissue specimens of mice was detected using JC-1 staining (n = 4). D Mitochondrial ATP concentration in myocardial tissues (n = 4). E TUNEL staining images of the myocardial cellular apoptosis (× 200, scale bar: 50 μm). F No. of TUNEL positive cells (n = 4). G Western blot images of Bcl-2, cleaved-caspase-3, Bax, Drp1, Mfn2, and caspase-9 in myocardial tissues. (H–M) Quantification of the caspase-9, Drp1, cleaved-caspase-3, Bcl -2, Mfn2, and Bax proteins concentrations in myocardial specimens (n = 4). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 5

Ang-(1–7) suppresses the upregulation of the NF-κB and MAPK pathways in the myocardial tissues of SIC mice. A Western blot images of p-IκBα, IκBα, p-P65, and P65 in the myocardial specimens. B Western blot images of p-P38, p-JNK, JNK, p-ERK, P38, ERK in the myocardial tissues. C–G Quantifying the p-P38/P38, p-IκBα/IκBα, p-ERK/ERK, p-P65/P65, and p-JNK/JNK concentrations in myocardial tissues (n = 4). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 6

Ang-(1–7) inhibits inflammatory response, mitochondrial dysfunctioning, and oxidative stress, in LPS-induced H9c2 cells. A The H9c2 cell viability, after being pretreated with varying concentrations of Ang-(1–7) (10^–6, 10^–7, 10^–8 mol/L) for 12 h, was measured using the CCK8 assay (n = 6). B The H9c2 cell viability after Ang-(1–7) (10^–6, 10^–7, 10^–8 mol/L) pretreatment for 1 h before LPS stimulation for 12 h was measured using the CCK8 assay (n = 6). C IL-1β protein concentration in the H9c2 cells (n = 6). D–F The IL-1β, TNF-α, and IL-6 mRNA expression levels in H9c2 cells (n = 4). G, H TNF-α and IL-6 protein concentrations in H9c2 cells (n = 6). I, J Quantitative analysis and flow cytometry histograms depicting the ROS production in H9c2 cells (n = 6). K, L Quantitative analysis and flow cytometry plots describing the ratio of monomer JC-1 cells (representing mitochondrial damage) in every group (n = 4). Cells with monomeric JC-1 were green in the P2 region, representing damaged mitochondria. B ^*p < 0.05 than control, ^#p < 0.05 than LPS mice. D–L Ang-(1–7) intervention using the pretreatment concentration of 10^–6 mol/L for 1 h, and LPS activation of 1 ug/mL for 12 h. NS: no significant difference, ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 7

Ang-(1–7) maintained mitochondrial dynamic equilibrium in H9c2 cardiomyocytes and alleviated LPS-induced apoptosis. A Western blot images of Drp1, Mfn2, caspase-9, cleaved caspase-3, Bcl-2, and Bax in H9c2 cells. B–G Quantification of the concentrations of cleaved caspase-3, Drp1, Mfn2, Bcl-2, caspase-9, and Bax proteins in H9c2 cells (n = 4). H, I Flow cytometry plots and quantitative analysis depicting the proportion of H9c2 apoptotic cells (n = 4). J Before LPS stimulation of H9c2 cells for 12 h, cells in each group were pretreated using differing concentrations of A-779 (10^–5,10^–6,10^–7 mol/L) for 2 h, using Ang-(1–7) (10^–6 mol/L) for 1 h and then with CCK8 assay for cell viability (n = 6). ^*p < 0.05 than the control group, ^#p < 0.05 than LPS group, ^&p < 0.05 in comparison to LPS + Ang-(1–7) group. K, L) The IL-6 and TNF-α protein concentrations in H9c2 cells (n = 6). M–P IL-6, Bcl-2, TNF-α, and the Bax mRNA levels in the H9c2 cells (n = 4). K–P The A-779 intervention was pretreatment with 10^–5 mol/L for 2 h, the Ang-(1–7) intervention included 10^–6 mol/L pretreatment for 1 h, and LPS activation of 1 µg/mL for 12 h. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001

Fig. 8

Ang-(1–7) inhibits LPS-induced activation of NF-κb and MAPK pathways in H9c2 cardiomyocytes. A Western blotting images of the p-P65, p-IκBα, P65, and IκBα in H9c2 cells. B Western blots of p-JNK, p-P38, p-ERK, P38, ERK, and JNK in H9c2 cells. C–G Quantifying the concentrations of p-IκBα/IκBα, p-ERK/ERK, p-JNK/JNK, p-P38/P38, and p-P65/P65 proteins in H9c2 cells (n = 4). ^***p < 0.001, ^**p < 0.01, ^*p < 0.05

Fig. 9

Schematic diagram showing the protective effect of Ang-(1–7) on SIC mice and its underlying mechanism. LPS can induce RAAS activation and increase Ang II secretion in blood, tissues, and cells. LPS and Ang II can activate NF-κB and MAPK pathways and generate a large number of inflammatory mediators. Excessive production of proinflammatory factors aggravates oxidative stress, mitochondrial damage, and dynamic imbalance in cardiomyocytes, and further leads to increased apoptosis of cardiomyocytes. Ang-(1–7) may regulate inflammation, reduce oxidative stress, maintain mitochondrial homeostasis, and alleviate mitochondrial structural and functional damage through NF-κB and MAPK pathways