PLD2 deletion ameliorates sepsis-induced cardiomyopathy by suppressing cardiomyocyte pyroptosis via the NLRP3/caspase 1/GSDMD pathway

Abstract

Objective: Sepsis-induced cardiomyopathy (SICM) is a life-threatening complication. Phospholipase D2 (PLD2) is crucial in mediating inflammatory reactions and is associated with the prognosis of patients with sepsis. Whether PLD2 is involved in the pathophysiology of SICM remains unknown. This study aimed to investigate the effect of PLD2 knockout on SICM and to explore potential mechanisms.

Methods: The SICM model was established using cecal ligation and puncture in wild-type and PLD2-knockout mice and lipopolysaccharide (LPS)-induced H9C2 cardiomyocytes. Transfection with PLD2-shRNA lentivirus and a PLD2 overexpression plasmid were used to interfere with PLD2 expression in H9C2 cells. Cardiac pathological alterations, cardiac function, markers of myocardial injury, and inflammatory factors were used to evaluate the SICM model. The expression of pyroptosis-related proteins (NLRP3, cleaved caspase 1, and GSDMD-N) was assessed using western blotting, immunofluorescence, and immunohistochemistry.

Results: SICM mice had myocardial tissue damage, increased inflammatory response, and impaired heart function, accompanied by elevated PLD2 expression. PLD2 deletion improved cardiac histological changes, mitigated cTNI production, and enhanced the survival of the SICM mice. Compared with controls, PLD2-knockdown H9C2 exhibits a decrease in inflammatory markers and lactate dehydrogenase production, and scanning electron microscopy results suggest that pyroptosis may be involved. The overexpression of PLD2 increased the expression of NLRP3 in cardiomyocytes. In addition, PLD2 deletion decreased the expression of pyroptosis-related proteins in SICM mice and LPS-induced H9C2 cells.

Conclusion: PLD2 deletion is involved in SICM pathogenesis and is associated with the inhibition of the myocardial inflammatory response and pyroptosis through the NLRP3/caspase 1/GSDMD pathway.

Keywords: Caspase 1; GSDMD; NLRP3; Phospholipase D2; Pyroptosis; Sepsis-induced cardiomyopathy.

Fig. 1

Increased expression of PLD2 in cardiac tissue was correlated with decreased heart function in SICM mice. SICM was induced in mice by utilizing cecal ligation and puncture (CLP). A The experimental strategy and timetable for the SICM model are shown in (A) (n = 6 mice/group). Histopathological analysis via HE staining of mouse heart section at different points (CLP-12 h, CLP-24 h and CLP-48 h) after CLP is also shown (B). CLP induction in SICM mice showed a more disorganized arrangement of cardiomyocytes, along with interstitial edema (black arrows) and some infiltration of inflammatory cells (red arrows). C Representative left ventricular echocardiographic M-mode images in mice with control and CLP-induced SICM groups. D–F Sequential measurements of left ventricular ejection fraction (LVEF), left ventricular shortening fraction (LVFS), and cardiac output (CO) of mice in each group after sham or CLP. G Serum levels of lactate dehydrogenase (LDH) in mice among groups. mRNA expression levels of various groups of inflammatory factors, including Il-6 (H), Il-1β (I), and Tnf-α (J), in mouse cardiac tissue. Western blotting evaluates the expression of PLD2 protein in cardiac tissues of control mice and SICM mice induced by CLP-48 h (K, L). Magnification, × 100; scale bar, 100 μm. Magnification, × 200; scale bar, 50 μm. Data represent the mean ± SD of three independent experiments. **P < 0.001 compared to the control group

Fig. 2

PLD2 knockdown protected cardiomyocytes from LPS-induced cardiac damage and inflammation in vitro. The PLD2 gene was targeted for knockdown using shRNA to establish the H9C2 stable cell line. The H9C2 cells were transfected with control (sh-NC) or PLD2 shRNA (sh-PLD2) for 48 h and then exposed to LPS (10 μg/ml) for 12 h. The cells were divided into four groups: sh-NC, sh-NC + LPS, sh-PLD2, and sh-PLD2 + LPS. The efficiency of the knockdown was assessed using western blotting analysis (A, B). The impact of varying concentrations of LPS on the viability of H9C2 cardiomyocytes was assessed using the CCK8 assay (C). The asterisk indicates that when the concentration of LPS was 10 μg/ml, cell viability dropped dramatically to 76.5%. LDH level in cell supernatants (D) and the mRNA expression levels of inflammatory markers Tnf-α (E), Il-1β (F), and Il-18 (G) in H9C2 cells. **P < 0.001 compared to the sh-NC group; *P < 0.05, **P < 0.001 compared to the sh-PLD2 + LPS group

Fig. 3

PLD2 deficiency alleviated cardiomyocytes pyroptosis induced by LPS in vitro. Scanning electron micrograph of H9C2 cells after 12 h of treatment with 10 μg/ml LPS. There were four distinct cell groups: sh-NC, sh-NC + LPS, sh-PLD2, and sh-PLD2 + LPS. As shown in Figure A, at a magnification of 2000 × , control cells are characterized by a rounded cellular mass with extended cellular processes. The cells treated with LPS exhibit irregular margins and lack processes. Plasma membranes appear ruptured. At a magnification of 10,000 × (B), the surface of control cells exhibits projections from the plasma membrane and extracellular vesicles. The stimulation of LPS induces the creation of pits and pores of varying sizes, and a pronounced loss of extracellular vesicles (red arrow). The degree of holes and pits in the membrane was reduced in the sh-PLD2 + LPS group as compared to the sh-NC + LPS group. Scale = 5 μm (A) and 1 μm (B)

Fig. 4

PLD2 deficiency alleviated NLRP3/caspase 1/ GSDMD-mediated pyroptosis in cardiomyocytes induced by LPS in vitro. The H9C2 cells were treated with 10 μg/ml LPS for 12 h and were divided into four distinct groups, namely sh-NC, sh-NC + LPS, sh-PLD2, and sh-PLD2 + LPS. The graphical depiction of the immunofluorescence findings for NLRP3, cleaved caspase 1, and GSDMD-N of the cells in different groups is shown by (A, red), (D, green), and (G, green), respectively. The related bar graphs of fluorescence intensity analysis are represented by J, K, and L, respectively. Western blotting analysis revealed the protein expression levels of NLRP3 (B, C), cleaved caspase 1 (E, F), and GSDMD-N (H, I), in the order mentioned. Magnification, × 200; scale bar, 50 μm. Magnification, × 400; scale bar, 20 μm. **P < 0.001 compared to the sh-NC group; *P < 0.05, **P < 0.001 compared to the sh-PLD2 + LPS group

Fig. 5

PLD2 overexpression exacerbates cardiomyocyte injury through NLRP3-mediated inflammatory response in vitro. A plasmid vector was used for the purpose of overexpressing PLD2 and confirming the effective completion of the constructed plasmid (A, B). To investigate the role of PLD2 gene overexpression in LPS-induced myocardial injury, cells were divided into four distinct groups, namely OE-NC, OE-NC + LPS, OE-PLD2, and OE-PLD2 + LPS. Western blotting shows the different groups' levels of NLRP3 expression after 10 μg/ml LPS for 12 h stimulation (C, D). **P < 0.001 compared to the OE-NC group; **P < 0.001 compared to the OE-NC + LPS group

Fig. 6

PLD2 deletion reduced CLP-induced cardiac tissue damage and mortality in SICM mice. The SICM model was constructed 48 h after CLP. Efficient creation and validation of PLD2 gene knockout in mouse heart tissue by western blotting (A, B). The mice were allocated into four distinct groups, namely PLD2^+/+, PLD2^+/+  + CLP, PLD2^−/−, PLD2^−/− + CLP (n = 6 mice/group). Histopathological alterations in the cardiac tissues of four distinct groups of mice were evaluated using the HE staining technique (C). CLP-induced SICM caused cardiac tissue edema (blue arrows), inflammatory cell infiltration (red arrows), and blood congestion (black arrows). Cardiac function was evaluated by representative left ventricular echocardiographic M-mode pictures in mice (D), specifically by measuring parameters such as LVEF (H), LVFS (I), and CO (J). Changes in the expression level of cTNI, a marker of myocardial injury, in mouse serum by ELISA (E) and LDH (F) by LDH cytotoxicity assay reagent. The survival of sham mice and LPS-induced SICM mice was recorded over 72 h (G). Magnification, × 200; scale bar, 50 μm. Magnification, × 400; scale bar, 20 μm. **P < 0.001 compared to the PLD2^+/+ group; *P < 0.05, **P < 0.001 compared to the PLD2^+/+  + CLP group

Fig. 7

PLD2 deletion alleviated cardiac tissues inflammatory response and NLRP3/caspase 1/GSDMD-mediated pyroptosis in CLP-induced SICM mice. The SICM mice were created 48 h after the CLP. The mice were allocated into four distinct groups, namely PLD2^+/+, PLD2^+/+  + CLP, PLD2^−/−, PLD2^−/− + CLP (n = 6 mice/group). The effect of PLD2 deletion on the levels of serum inflammatory factors (Tnf-α, Il-1β, and Il-18) in SICM mice was investigated using ELISA (A–C). D Immunohistochemistry assay shows NLRP3 distributions in the heart tissue of mice. Distributions of pyroptosis-associated proteins cleaved caspase 1 (E, green), GSDMD-N (G, red) and associated fluorescence intensity analysis of cleaved caspase 1 (F), and and GSDMD-N (H) in the heart of mice among the groups. I, K, M Western blotting analysis to determine NLRP3, cleaved caspase 1, and GSDMD-N levels in mice from each group. Quantifications of NLRP3, cleaved caspase 1, and GSDMD-N are shown in (J), (L), and (N) respectively. Magnification × 200, scale bar, 50 μm. **P < 0.001 compared to the PLD2^+/+ group; *P < 0.05, **P < 0.001 compared to the PLD2^−/− + CLP group

Fig. 8

Mechanistic map of PLD2 deletion protects against SICM by suppressing pyroptosis via the NLRP3/caspase 1/ GSDMD pathway