Periostin‐CCL3 Feedforward Signaling Loop Promotes Cardiac Fibrosis and Cardiomyocyte Necroptosis in Arrhythmogenic Cardiomyopathy

Figures

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  FIGURE 1. POSTN is upregulated in the myocardium of DSP‐ACM mice and predominantly derived from cardiac fibroblasts. (A,B) The mRNA (A) (n = 12) and protein (B) levels of POSTN in the myocardium of WT (DSPf/+ ) and DSP‐ACM (CreDSPf/+ ) mice. (C–E) Representative images of immunohistochemical (C,D) and immunofluorescence (E) staining of POSTN from WT and DSP‐ACM mouse cardiac sections. Scale bars, 500 µm (C) and 50 µm (D,E). (F) Masson's Trichrome staining and immunohistochemical staining of POSTN in cardiac continuous sections from DSP‐ACM mice. Scale bars, 50 µm. (G,H) The mRNA (G) (n = 12) and protein (H) levels of POSTN in CMs and NCMs isolated from the hearts of DSP‐ACM mice. (I) Representative immunofluorescence co‐staining of POSTN with cardiac myofibroblasts (Vimentin), adipocytes (Perilipin), macrophage (CD68), and endothelial cells (CD31) in cardiac sections of DSP‐ACM mice. Scale bars, 50 µm. (J) RNA‐FISH staining of cardiac myofibroblasts (Vimentin) and Postn in cardiac sections of DSP‐ACM mice. Scale bars, 50 µm. Statistical analyses were performed by unpaired two‐tailed Student's t‐test (A,G). Data are mean ± SEM. **** p < 0.0001.

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  FIGURE 2. POSTN deficiency attenuates cardiac dysfunction and increases the survival rate of DSP‐ACM mice. (A) H&E staining in cardiac sections from WT and DSP‐ACM mice with or without POSTN deficiency. Scale bars, 500 µm. (B) mRNA levels of Nppa and Nppb in the myocardium of WT and DSP‐ACM mice with or without POSTN deficiency (n = 5). (C–E) Representative images of echocardiographs (C) and statistical data (D,E) on EF%, FS% in each group (n = 4). Scale bars, 2 mm. (F) Running time and HW/TL ratio in WT and DSP‐ACM mice with or without POSTN deficiency (n = 6). (G) Kaplan‐Meier survival curve analysis of WT and DSP‐ACM mice with or without POSTN deficiency (n = 20). (H) H&E staining in cardiac sections from WT and DSP‐ACM mice with or without AAV‐mediated cardiac fibroblast‐targeted deletion of Postn. Scale bars, 500 µm. (I) mRNA levels of Nppa and Nppb in the myocardium of WT and DSP‐ACM mice with or without AAV‐mediated cardiac fibroblast‐targeted deletion of Postn (n = 8). (J–L) Representative images of echocardiographs (J) and statistical data (K,L) on EF%, FS% in each group (n = 4). Scale bars, 2 mm. (M) Running time and HW/TL ratio in WT and DSP‐ACM mice with or without AAV‐mediated…

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  FIGURE 3. POSTN deficiency alleviates cardiac fibrosis in DSP‐ACM mice. (A,B) Masson's Trichrome staining (A) and immunohistochemical staining of α‐SMA (B) in cardiac sections from WT and DSP‐ACM mice with or without POSTN deficiency. Scale bars, 50 µm. (C) Western blot analysis of α‐SMA in the cardiac tissue lysates of WT and DSP‐ACM mice with or without POSTN deficiency. (D–G) mRNA levels of Acta2 (D), Col1a1 (E), Col3a1 (F), and Tgf‐β3 (G) in the myocardium of WT and DSP‐ACM mice with or without POSTN deficiency (n = 6). (H,I) Masson's Trichrome staining (H) and immunohistochemical staining of α‐SMA (I) in cardiac sections from WT and DSP‐ACM mice with or without AAV‐mediated cardiac fibroblast‐targeted deletion of Postn. Scale bars, 50 µm. (J) Western blot analysis of α‐SMA in the cardiac tissue lysates of WT and DSP‐ACM mice with or without AAV‐mediated cardiac fibroblast‐targeted deletion of Postn. (K–N) mRNA levels of Acta2 (K), Col1a1 (L), Col3a1 (M), and Tgf‐β3 (N) in the myocardium of WT and DSP‐ACM mice with or without AAV‐mediated cardiac fibroblast‐targeted deletion of Postn (n = 8). Statistical analyses were performed by one‐way ANOVA analysis followed by Tukey's multiple…

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  FIGURE 4. POSTN promotes cardiomyocyte necroptosis via the integrin‐JNK‐RIP3‐MLKL axis. (A,C) The activities of lactate dehydrogenase (LDH) in the serum isolated from WT and DSP‐ACM mice with or without either global (A) (n = 5) or cardiac fibroblast‐specific (C) (n = 8) POSTN deficiency. (B,D) Western blot analysis of the indicated proteins associated with necroptosis in the myocardium of WT and DSP‐ACM mice with or without either global (B) or cardiac fibroblast‐specific (D) POSTN deficiency. (E,F) Western blot analysis of the indicated proteins associated with necroptosis in NMCMs (E) and AC16 cells (F) transfected with siDSP in the presence or absence of TSZ, rmPOSTN or rhPOSTN. (G) Western blot analysis of p‐JNK and JNK in the myocardium of WT and DSP‐ACM mice with or without POSTN deficiency. (H,I) The protein levels of p‐JNK and JNK in NMCMs (H) and AC16 cells (I) transfected with siDSP in the presence or absence of rmPOSTN or rhPOSTN. (J–O) Western blot analysis of p‐JNK, JNK, p‐RIP3, RIP3, p‐MLKL and MLKL in NMCMs and AC16 cells transfected with siDSP in the presence or absence of rmPOSTN or rhPOSTN, as well as αvβ3 or αvβ5 integrin neutralizing antibody (J,M), PD98059 or SP600125…

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  FIGURE 5. Integrin‐JNK‐ETS2 signaling mediates POSTN‐induced CCL3 upregulation in cardiomyocytes. (A) Differential gene expression from RNA‐seq analysis in the myocardium of CreDSPf/+Postn−/− mice compared to CreDSPf/+Postn+/+ mice (n = 3). (B,C) Relative mRNA (B) (n = 8) and protein (C) levels of CCL3 in the myocardium of WT and DSP‐ACM mice with or without POSTN deficiency. (D,E) Relative mRNA (D) (n = 8) and protein (E) levels of CCL3 in CMs derived from WT and DSP‐ACM mice with or without POSTN deficiency. (F,G) Relative mRNA (F) (n = 8) and protein (G) levels of CCL3 in CMs isolated from CreDSPf/+Postn+/+ mice, which were cultured alone or co‐cultured indirectly with CFs isolated from CreDSPf/+Postn+/+ mice and CreDSPf/+Postn−/− mice. (H,I) Relative mRNA (H) (n = 8) and protein levels (I) of CCL3 in NMCMs transfected with siDSP in the presence or absence of rmPOSTN. (J–M) Relative mRNA (n = 8) and protein levels of CCL3 in NMCMs transfected with siDSP in the presence or absence of rmPOSTN, as well as αvβ3 or αvβ5 integrin neutralizing antibody (J,K), PD98059 or SP600125 (L,M). (N) The protein levels of ETS2 in the myocardium of WT and DSP‐ACM mice with or without POSTN deficiency.…

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  FIGURE 6. CCL3‐CCR5‐NF‐κB axis mediates cardiac fibroblast activation and POSTN expression. (A–C) Relative mRNA (A,B) (n = 6) and protein (C) levels of α‐SMA and POSTN in CFs derived from DSPf/+Postn+/+ mice, which were cultured alone or co‐cultured indirectly with CMs derived from DSPf/+Postn+/+ mice or CreDSPf/+Postn+/+ mice. (D) Relative mRNA levels of Ccr5 in the myocardium of WT and DSP‐ACM mice with or without POSTN deficiency (n = 8). (E–H) Relative mRNA expression of Acta2 (E,G) and Postn (F,H) in NMCFs treated with rmCCL3 for the indicated time or indicated doses for 24 h (n = 6). (I) The protein levels of α‐SMA and POSTN in NMCFs treated with PBS or rmCCL3. (J–L) Relative mRNA (J,K) (n = 6) and protein (L) levels of α‐SMA and POSTN in NMCFs treated with or without rmCCL3 in the presence or absence of Maraviroc. (M) The protein levels of ILK, p‐P65, and P65 in the myocardium of WT and DSP‐ACM mice with or without POSTN deficiency. (N) Western blot analysis of ILK, p‐P65, and P65 in CFs derived from DSPf/+Postn+/+ mice, which were cultured alone or co‐cultured indirectly with CMs derived from DSPf/+Postn+/+ mice or CreDSPf/+Postn+/+ mice. (O,P) The protein levels of ILK, p‐P65, and…

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  FIGURE 7. POSTN expression positively correlates with cardiomyocyte necroptosis in clinical and murine ACM hearts. (A) Masson's Trichrome staining, immunohistochemical staining of POSTN, Perilipin, and p‐MLKL in cardiac sections from normal controls and patients with ACM. Scale bars, 50 µm. (B) Immunofluorescence co‐staining of CCL3 (red) and POSTN (green) in cardiac sections from normal controls and patients with ACM. Scale bars, 50 µm. (C) Immunofluorescence co‐staining of CCL3 (green) and cTNT (red) in cardiac sections from patients with ACM. Scale bars, 50 µm. (D) RNA‐FISH staining of Ccl3 and cardiomyocytes (cTNT) in cardiac sections of patients with ACM. Scale bars, 50 µm. (E,F) Immunofluorescence co‐staining of p‐RIP3 (E) or CCL3 (F) (red) and POSTN (green) in cardiac sections from WT and DSP‐ACM mice with or without POSTN deficiency. Scale bars, 50 µm.

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  FIGURE 8. Pharmacological inhibition of the JNK‐CCL3‐CCR5 axis attenuates cardiac remodeling and necroptosis in ACM mice. (A) H&E staining in cardiac sections from Vehicle‐ and Maraviroc‐treated ACM mice. Scale bars, 50 µm. (B) mRNA levels of Nppa and Nppb in the myocardium of Vehicle‐ and Maraviroc‐treated ACM mice (n = 8). (C,D) Masson's Trichrome staining (C) and immunohistochemical staining of α‐SMA (D) in cardiac sections from Vehicle‐ and Maraviroc‐treated ACM mice. Scale bars, 50 µm. (E) Western blot analysis of POSTN and α‐SMA in the cardiac tissue lysates of Vehicle‐ and Maraviroc‐treated ACM mice. (F) mRNA levels of Postn, Acta2, Col1a1, Col3a1, and Tgfβ3 in the myocardium of Vehicle‐ and Maraviroc‐treated ACM mice (n = 8). (G) The activities of LDH in the serum isolated from Vehicle‐ and Maraviroc‐treated ACM mice (n = 8). (H) Western blot analysis of the indicated proteins associated with necroptosis in the myocardium derived from Vehicle‐ and Maraviroc‐treated ACM mice. (I) The protein levels of p‐JNK, JNK, ETS2, and CCL3 in the myocardium derived from Vehicle‐ and Maraviroc‐treated ACM mice. (J) H&E staining in cardiac sections from Vehicle‐ and SP600125‐treated ACM mice.…