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, 1842 (5), 701-11

Loss of duplexmiR-223 (5p and 3p) Aggravates Myocardial Depression and Mortality in Polymicrobial Sepsis

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Loss of duplexmiR-223 (5p and 3p) Aggravates Myocardial Depression and Mortality in Polymicrobial Sepsis

Xiaohong Wang et al. Biochim Biophys Acta.

Abstract

Sepsis is the leading cause of death in critically ill patients. While myocardial dysfunction has been recognized as a major manifestation in severe sepsis, the underlying molecular mechanisms associated with septic cardiomyopathy remain unclear. In this study, we performed a miRNA array analysis in hearts collected from a severe septic mouse model induced by cecal ligation and puncture (CLP). Among the 19 miRNAs that were dys-regulated in CLP-mouse hearts, miR-223(3p) and miR-223*(5p) were most significantly downregulated, compared with sham-operated mouse hearts. To test whether a drop of miR-223 duplex plays any roles in sepsis-induced cardiac dysfunction and inflammation, a knockout (KO) mouse model with a deletion of the miR-223 gene locus and wild-type (WT) mice were subjected to CLP or sham surgery. We observed that sepsis-induced cardiac dysfunction, inflammatory response and mortality were remarkably aggravated in CLP-treated KO mice, compared with control WTs. Using Western-blotting and luciferase reporter assays, we identified Sema3A, an activator of cytokine storm and a neural chemorepellent for sympathetic axons, as an authentic target of miR-223* in the myocardium. In addition, we validated that miR-223 negatively regulated the expression of STAT-3 and IL-6 in mouse hearts. Furthermore, injection of Sema3A protein into WT mice revealed an exacerbation of sepsis-triggered inflammatory response and myocardial depression, compared with control IgG1 protein-treated WT mice following CLP surgery. Taken together, these data indicate that loss of miR-223/-223* causes an aggravation of sepsis-induced inflammation, myocardial dysfunction and mortality. Our study uncovers a previously unrecognized mechanism underlying septic cardiomyopathy and thereby, may provide a new strategy to treat sepsis.

Keywords: Inflammation; MicroRNA; Myocardial depression; Sema3A; Sepsis.

Conflict of interest statement

Conflict of Interest Disclosures:

None

Figures

Figure 1
Figure 1
miRNA expression profile in mouse hearts after severe cecal ligation and puncture [CLP(s)] surgery. (A) The mortality curves of mice underwent sham operation, moderate (m) and severe (s) CLP –induced septic surgery. The survival rate was determined every 12 h until 5 days after surgery (n=10 animals per group, p<0.001). (B) A partial heat-map of the upregulated and downregulated miRNAs (Red means upregulation and green represents downregulation) in mouse hearts 6 h after severe CLP (n=4). (C) Microarray data are summarized by volcano plot graph, which displays both fold-change and t-test criteria (log odds). (D/E) Alterations of miR-223 and miR-223* expression were validated in CLP(s)-treated mouse hearts by qRT-PCR (normalized to control U6) (n=4, *, p<0.05 vs. shams). (F/G) Both miR-223 and miR-223* were dramatically upregulated in mouse hearts at 6 h post-CLP (m). (n=4, *, p<0.05 vs. shams).
Figure 1
Figure 1
miRNA expression profile in mouse hearts after severe cecal ligation and puncture [CLP(s)] surgery. (A) The mortality curves of mice underwent sham operation, moderate (m) and severe (s) CLP –induced septic surgery. The survival rate was determined every 12 h until 5 days after surgery (n=10 animals per group, p<0.001). (B) A partial heat-map of the upregulated and downregulated miRNAs (Red means upregulation and green represents downregulation) in mouse hearts 6 h after severe CLP (n=4). (C) Microarray data are summarized by volcano plot graph, which displays both fold-change and t-test criteria (log odds). (D/E) Alterations of miR-223 and miR-223* expression were validated in CLP(s)-treated mouse hearts by qRT-PCR (normalized to control U6) (n=4, *, p<0.05 vs. shams). (F/G) Both miR-223 and miR-223* were dramatically upregulated in mouse hearts at 6 h post-CLP (m). (n=4, *, p<0.05 vs. shams).
Figure 1
Figure 1
miRNA expression profile in mouse hearts after severe cecal ligation and puncture [CLP(s)] surgery. (A) The mortality curves of mice underwent sham operation, moderate (m) and severe (s) CLP –induced septic surgery. The survival rate was determined every 12 h until 5 days after surgery (n=10 animals per group, p<0.001). (B) A partial heat-map of the upregulated and downregulated miRNAs (Red means upregulation and green represents downregulation) in mouse hearts 6 h after severe CLP (n=4). (C) Microarray data are summarized by volcano plot graph, which displays both fold-change and t-test criteria (log odds). (D/E) Alterations of miR-223 and miR-223* expression were validated in CLP(s)-treated mouse hearts by qRT-PCR (normalized to control U6) (n=4, *, p<0.05 vs. shams). (F/G) Both miR-223 and miR-223* were dramatically upregulated in mouse hearts at 6 h post-CLP (m). (n=4, *, p<0.05 vs. shams).
Figure 2
Figure 2
MiR-223-deficient mouse hearts display reduced cardiac contractile function to a greater degree compared with shams upon severe CLP, determined by echocardiography (A-C, *P < 0.05, n=8) and ex vivo langendorff setting (D-F, *P < 0.05, n=5). (G) The mortality curves of WT (miR-223+/y) and KO (miR-223−/y) mice subjected to severe CLP-induced septic surgery. The survival rate was determined every 2 h until 24h after surgery (n=12 animals per group, *P<0.001).
Figure 2
Figure 2
MiR-223-deficient mouse hearts display reduced cardiac contractile function to a greater degree compared with shams upon severe CLP, determined by echocardiography (A-C, *P < 0.05, n=8) and ex vivo langendorff setting (D-F, *P < 0.05, n=5). (G) The mortality curves of WT (miR-223+/y) and KO (miR-223−/y) mice subjected to severe CLP-induced septic surgery. The survival rate was determined every 2 h until 24h after surgery (n=12 animals per group, *P<0.001).
Figure 3
Figure 3
Absence of miR-223 duplex aggravates sepsis-induced myocardial inflammation and neutrophil infiltration. The levels of TNF-α (A) , IL-1β (B) and IL-6 (C) were measured in heart homogenates using ELISA kits (*P<0.05 vs. miR-223+/y mice, n=6). (D) Activity of myeloperoxidase (MPO) which represents the amount of neutrophils was determined in the heart tissue of mice 6 h after severe CLP, using a fluorometric assay kit (*P<0.05 vs. miR-223+/y mice, n=5).
Figure 4
Figure 4
Knockout of miR-223 exacerbates the peritoneal and the systemic inflammation in mice following severe CLP surgery. The levels of TNF-α (A), IL-6 (C) and IL-1β (E) were assessed in peritoneal fluid of mice 6 h after CLP. The serum levels of TNF-α (B), IL-6 (D) and IL-1β (F) were measured in mice at 6 h post-CLP (*P<0.05 vs. miR-223+/y mice, n=5-7). Bacterial load was counted in blood (G) and peritoneal lavage fluid (H) at 12 h after severe CLP. (*P<0.05 vs. miR-223+/y mice, n=8-10 mice/group).
Figure 5
Figure 5
Sema3A is a target of miR-223* and STAT3 is a target of miR-223 in the mouse heart. (A) A scheme of pre-miR-223 structure and miR-223-5p (miR-223*) binding to the 3’-UTR of Sema3A as well as miR-223-3p (miR-223) binding to 3’-UTRs of STAT3 and IL-6. (B) Reciprocal expression of miR-223/STAT3 and miR-223*/Sema3A was determined in (B) WT and KO mouse hearts (*P<0.05 vs. miR-223+/y mice, n=6), (C) sham-operated and severe CLP-treated mouse hearts (*P<0.05 vs. shams, n=5), and (D) sham-treated and moderated CLP-treated mouse hearts(*P<0.05 vs. shams, n=5). α-Actin was used as a loading control.
Figure 5
Figure 5
Sema3A is a target of miR-223* and STAT3 is a target of miR-223 in the mouse heart. (A) A scheme of pre-miR-223 structure and miR-223-5p (miR-223*) binding to the 3’-UTR of Sema3A as well as miR-223-3p (miR-223) binding to 3’-UTRs of STAT3 and IL-6. (B) Reciprocal expression of miR-223/STAT3 and miR-223*/Sema3A was determined in (B) WT and KO mouse hearts (*P<0.05 vs. miR-223+/y mice, n=6), (C) sham-operated and severe CLP-treated mouse hearts (*P<0.05 vs. shams, n=5), and (D) sham-treated and moderated CLP-treated mouse hearts(*P<0.05 vs. shams, n=5). α-Actin was used as a loading control.
Figure 6
Figure 6
A luciferase-reporter assay for determining the interaction of miR-223* with the Sema3A 3’-UTR. Diagram of plasmid construction indicating four regions of Sema3A 3’ UTR inserted downstream of the luciferase-encoding sequence. Dual luciferase activity was measured in HEK-293 cells co-transfected with the plasmid containing the segment of Sema3A 3’ UTR and either miR-223* or a mimic miRNA control. β–gal vector was also co-transfected as an internal control. (* P<0.05 relative to miR mimic controls). Similar results were observed in three additional, independent experiments.
Figure 7
Figure 7
Injection of Sema3A protein re-captures the pro-inflammatory consequence of miR-223*-KO in mice following severe CLP surgery. WT male mice were pre-treated with either mouse Sema3A protein or IgG1 control protein at a dosage of 25 µg/kg body weight 1h before severe CLP surgery. Inflammatory cytokines TNF-α (A/C) and IL-6 (B/D) in peritoneal fluid (A/B) and heart homogenates (C/D) collected 6 h after CLP were determined by ELISA. (*P<0.05 vs. IgG1, n=6/group). (E/F) Cardiac function was measured in Sema3A or IgG1 protein-treated mice at 6h post-CLP by the ex vivo Langendorff system. (n=5 mice for each sham group, n=7 mice for each CLP group, *P<0.05 vs. IgG1 control).
Figure 8
Figure 8
Proposed scheme for aggravation of sepsis-induced inflammation and myocardial depression in miR-223-KO mice. Severe sepsis or septic shock causes a remarkable downregulation of miR-223 duplex (5p&3p) in the heart, leading to upregulation of Sema3A and STAT3/IL-6. As a result, multiple levels of inflammatory response might be activated which induces myocardial depression and lethality.

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