MG53 protects against septic cardiac dysfunction by ubiquitinating ATF2

Figures

• 

  Fig. 1. MG53 deficiency exaggerates the LPS-induced septic cardiac dysfunction. (A) Representative images of M mode echocardiographic analysis performed 12 h post intraperitoneal injection of saline or LPS in WT and MG53-KO mice (n = 6). (B) Quantification of left ventricular ejection fraction (EF%), fraction shortening (FS%), left ventricular internal diastolic diameter (LVIDd), and left ventricular internal systolic diameter (LVIDs) (n = 6). (C) The serum expression of ANP and BNP in LPS-induced WT and MG53-KO mice, analyzed by ELISA (n = 6). (D) Hematoxylin-eosin (HE) staining showing the anatomical structure of WT and MG53-KO mice hearts at 12 h after LPS injection (scale bar = 50 μm; n = 6). (E) Representative images of TUNEL staining (E1) and quantification (E2) of TUNEL+ cells in WT and MG53-KO mice heart sections (scale bar = 50 μm; n = 6). Data shown are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

• 

  Fig. 2. MG53 overexpression improves heart function and survival rate in SCD mice. (A) Representative images of M mode echocardiographic analysis performed 12 h post intraperitoneal injection of saline or LPS in AAV9-cTnT-Control and AAV9-cTnT-MG53 mice (n = 6). (B) Quantification of left ventricular ejection fraction (EF%), fraction shortening (FS%), left ventricular internal diastolic diameter (LVIDd), and left ventricular internal systolic diameter (LVIDs) (n = 6). (C) The serum expression of ANP and BNP in LPS-induced AAV9-cTnT-Control mice and AAV9-cTnT-MG53 mice, analyzed by ELISA (n = 6). (D) Hematoxylin-eosin (HE) staining showing the anatomical structure of AAV9-cTnT-Control and AAV9-cTnT-MG53 mice hearts at 12 h after LPS injection (scale bar = 50 μm; n = 6). (E) Representative images of TUNEL staining (E1) and quantification (E2) of TUNEL+ cells in AAV9-cTnT-Control and AAV9-cTnT-MG53 mice heart sections (scale bar = 50 μm; n = 6). (F) Survival rate of AAV9-cTnT-Control mice and AAV9-cTnT-MG53 mice that received intraperitoneal injection of saline or LPS, monitored every 1 h for a 30-h period (n = 10 mice per group). Data shown are mean ± SEM. *P < 0.05, **P < 0.01, ***P <…

• 

  Fig. 3. rhMG53 administration improves the impaired cardiac function in SCD mice. (A) Representative images of M mode echocardiographic analysis performed 12 h post intraperitoneal injection of saline or LPS, with or without rhMG53 treatment. (B) Quantification of left ventricular ejection fraction (EF%), fraction shortening (FS%), left ventricular internal diastolic diameter (LVIDd), and left ventricular internal systolic diameter (LVIDs) (n = 8). (C) The serum expression of ANP and BNP in mice, analyzed by ELISA (n = 3). (D) Hematoxylin–eosin (HE) staining showing the anatomical structure of mice hearts 12 h after LPS with or without rhMG53 injection (scale bar = 50 μm; n = 3). (E) Representative images of TUNEL staining (E1) and quantification of TUNEL+ cells in heart tissue (E2) (scale bar = 50 μm; n = 3). (F) Survival rate of mouse intraperitoneally injected with PBS, LPS with or without rhMG53, monitored every 1 h for a 30 h period. (Control, n = 10; LPS + saline, n = 15; LPS + rhMG53, n = 15). Data shown are mean ± SEM. **P < 0.01, ***P < 0.001, ****P < 0.0001.

• 

  Fig. 4. rhMG53 protects against CLP-induced septic cardiac dysfunction. (A) Schematic diagram of the administration of rhMG53 in the CLP model. (B) Quantification of left ventricular ejection fraction (EF%), fraction shortening (FS%), left ventricular internal diastolic diameter (LVIDd), and left ventricular internal systolic diameter (LVIDs) (n = 6). (C) Representative images of M mode echocardiographic analysis performed 12 h post CLP, with or without rhMG53 treatment. (D) The serum expression of ANP and BNP in mice heart, analyzed by ELISA (n = 6). (E) Hematoxylin–eosin (HE) staining showing the anatomical structure of mice hearts 12 h after CLP with or without rhMG53 injection (scale bar = 50 μm; n = 6). (F) Representative images of TUNEL staining (F1) and quantification of TUNEL+ cardiomyocytes in heart tissue (F2) (scale bar = 50 μm; n = 6). Data shown are mean ± SEM. *P < 0.05. **P < 0.01, ***P < 0.001, ****P < 0.0001.

• 

  Fig. 5. MG53 protects cardiomyocytes by down-regulating TLR4 expression. (A) Quantification of TLR4 in LPS-induced AAV9-cTnT-Control and AAV9-cTnT-MG53 mice heart, analyzed by qPCR (n = 6). GAPDH mRNA levels were used for normalization. (B) Representative image of TLR4 protein expression in LPS-induced AAV9-cTnT-Control and AAV9-cTnT-MG53 mice heart, analyzed by Western blot (n = 4). (C) Quantification of TLR4 in mice heart injected with PBS, LPS with or without rhMG53, analyzed by qPCR (n = 4). GAPDH mRNA levels were used for normalization. (D) Representative image of TLR4 protein expression in mice heart injected with PBS, LPS with or without rhMG53, analyzed by Western blot (n = 4). (E) The serum expression of IL‐1β, IL‐6 and TNF‐α by ELISA in serum of LPS-induced AAV9-cTnT-Control and AAV9-cTnT-MG53 mice heart at 12 h after LPS injection (n = 6). (F) The serum expression of IL‐1β, IL‐6 and TNF‐α measured by ELISA in serum of rhMG53 treated septic cardiac dysfunction mice (n = 3). (G) Representative image of IL‐1β, IL‐6 and TNF‐α protein expression of LPS-induced AAV9-cTnT-control and AAV9-cTnT-MG53 mice heart, analyzed by Western blot (n = 3). (H) Representative image of IL‐1β, IL‐6…

• 

  Fig. 6. MG53 inhibits TLR4 transcriptional activity by regulation of ATF2 ubiquitination. (A) Representative immunofluorescence image of exogenous rhMG53 wrapped in Rhodamine in NRVMs. (B) The Venn diagram showing the overlap between transcription factors predicted by FactorBook (n = 887), Animal TFDB (n = 577), MotifMap (n = 530) and GTRD (n = 288). (C) Predicted ATF2-binding sites in the promoter region of TLR4. (D) ChIP-qPCR was performed in NRVMs to identify the enrichment of ATF2 on the TLR4 promoter region. The input represents 1 % of the whole lysate used for each immunoprecipitation (n = 3). (E) Representative Western blot images of ATF2 in NRVMs treated with or without Ad-MG53, stimulated by LPS. (F) The mRNA expression of ATF2 in MG53 overexpressed NRVMs treated with LPS (n = 3). GAPDH mRNA levels were used for normalization. (G) Co-immunoprecipitation of MG53 with ATF2 in NRVMs treated only with or without Ad-MG53. (H) Ubiquitination of ATF2 in NRVMs transfected only with or without Ad-MG53. (I) Ubiquitination of ATF2 in NRVMs transfected with full length MG53, C14A or ΔRING, analyzed by Western blot. Data shown are mean ± SEM. *P < 0.05.

• 

  Fig. 7. MG53 protects against septic cardiac dysfunction by ubiquitinating ATF2. MG53 inhibits the transcriptional activity of TLR4 by regulating the ubiquitination of ATF2, which a transcription factor of TLR4.