LCZ696 Attenuated Doxorubicin-Induced Chronic Cardiomyopathy Through the TLR2-MyD88 Complex Formation

Figures

• 

  FIGURE 1. LCZ696 treatment and TLR2 deficiency attenuated doxorubicin-challenged systolic dysfunction. (A) Representative echocardiogram images of each group. (B) Representative H&E staining of heart tissues showing the effect of LCZ696 and TLR2 deficiency on doxorubicin-induced dilated cardiomyopathy in mice [original magnification 1×].

• 

  FIGURE 2. LCZ696 treatment and TLR2 deficiency attenuated doxorubicin-induced cardiac fibrosis. (A) Representative images of Sirius Red and Masson trichrome staining of longitudinal sections of the hearts (200×). (B,C) Quantification of interstitial fibrotic areas (%) from Sirius Red-stained heart sections (B) and Masson’s Trichome staining (C). (D,E) The mRNA levels of Col1a1 (D) and Tgfb (E) were detected by real-time PCR in the heart tissue from the above transplanted mice (data were normalized to Actb). (F) Heart tissue from each group of mice was homogenized for western blot analysis of collagen I, TGF-β, and GAPDH. (n = 7; *** P < 0.001 as indicated; ###P < 0.001 compared to Ang II; and ns is not significant).

• 

  FIGURE 3. LCZ696 treatment and TLR2 knockdown attenuated doxorubicin-induced cardiac inflammation in mice. (A) Representative images of anti-TNF-α staining in the hearts of each group (200×). (B) TNF-α levels in mouse heart tissue homogenates determined by ELISA. (C) Western blot analysis of IκBα levels in heart tissue. GAPDH was used as a loading control. (D) Nuclei were isolated from mouse heart tissue, and NF-κB in the nucleus was detected by western blot. Lamin B and GAPDH were used as controls. (E–G) Real-time PCR was used to determine the mRNA levels of Tnfa (E), Mcp1 (F), and Il-6 (G) in mouse heart tissue (data were normalized to Actb). (n = 7; *** P < 0.001 vs. WT control group; ### P < 0.001 vs. DOX group).

• 

  FIGURE 4. LCZ696 treatment and TLR2 knockdown attenuated doxorubicin-induced H9C2 cell fibrosis in vitro. (A) H9C2 cells were transfected with control siRNA (NC) or TLR2 siRNA (siTLR2) and treated with LCZ696 (20 μmol/l) for 24 h. Western blot detected TLR2 and GAPDH protein levels. (B) Densitometric quantification of TLR2/GAPDH levels in the immunoblots presented in panel (A). (C) H9C2 cells were transfected with control siRNA or TLR2 siRNA and pre-treated with LCZ696 (20 μmol/l) and then stimulated with DOX (1 μM) for 24 h. Western blot analysis of the protein levels of collagen I and TGF-β in whole cell lysates. (D) Densitometric quantification of collagen I/GAPDH and TGF-β/GAPDH levels in immunoblots presented in panel (C). (E,F) The mRNA levels of Col1a1 (E) and Tgfb (F) were detected by real-time PCR. (n = 5; *** P < 0.001 vs. Ctrl group; ##P < 0.01 compared to Ang II; ### P < 0.001 vs. DOX group).

• 

  FIGURE 5. LCZ696 treatment and TLR2 knockdown attenuated the doxorubicin-induced H9C2 cell inflammatory response in vitro. (A,B) H9C2 cells were transfected with control siRNA, TLR2 siRNA, and LCZ696 (20 μmol/l) and then stimulated with DOX (1 μM) for 8 h. Western blot detected IκBa and GAPDH protein levels in whole cell lysates (A) and detected NF-κB, Lamin B, and GAPDH protein levels in the nuclear extractions (B). (C) Densitometric quantification of IκBa/GAPDH levels in the immunoblots presented in panel (A). (D) Densitometric quantification of NF-κB/Lamin B levels in the immunoblots presented in panel (B). (E–G) The mRNA levels of Tnfa (E), Mcp1 (F), and Il-6 (G) were measured by real-time PCR. (n = 5; *** P < 0.001 vs. Ctrl group; ### P < 0.001 vs. DOX group).

• 

  FIGURE 6. LCZ696 treatment attenuated doxorubicin-induced cardiac injury by inhibiting TLR2-MyD88 complex formation. (A) Time course of the DOX-induced TLR2-MyD88 interaction in H9C2 cells. Cells were treated with 1 μM DOX for up to 1 h. Quantification is shown below. (B) H9C2 cells were pre-treated with LCZ696 (20 μmol/l) for 1 h and then stimulated with DOX (1 μM) for 30 min. The TLR2-MyD88 interaction was analyzed. (C) A co-immunoprecipitation assay determined the TLR2-MyD88 interaction in mice heart tissues. (D) The total of 20 binding conformations (right) are produced by the docking program QVina-W, of which pose 1 (marked yellow) has the best docking score. The details of the binding pose of DOX with the lowest binding energy for TLR2-TIR is shown in the middle, and the details of the binding pose of DOX with TLR2-TIR calculated by MM/GBSA method is shown in the right. (E) The details of the binding pose of LCZ696 (Sacubitril-right and Valsartan-left) with the lowest binding energy is shown for TLR2-TIR. Magenta is the optimal conformation for scoring (unit is kcal/mol). ***P < 0.001 compared to ctrl.

• 

  FIGURE 7. LCZ696 attenuated doxorubicin-induced heart injury through the TLR2-MyD88 pathway. The mechanism by which LCZ696 relieves DOX-induced cardiac inflammation, fibrosis and heart failure lies in reducing the formation of TLR2-MyD88 complexes.