Autoantibodies Against β ₁ ‐Adrenoceptor Exaggerated Ventricular Remodeling by Inhibiting CTRP9 Expression

Figures

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  Figure 1. The correlation between CTRP9 (C1q tumor necrosis factor‐related protein 9) levels and autoantibodies against the second extracellular loop of β1‐adrenergic receptor (β1‐AA) levels. A, Serum titers of β1‐AA in healthy subjects and patients with coronary heart disease (CAD). B, The positive rate of β1‐AA in healthy subjects and patients with CAD. C, Circulating CTRP9 levels in healthy subjects and patients with CAD. D, Circulating CTRP9 levels in β1‐AA–positive group (n=86) and β1‐AA–negative group (n=45). E, Serum CTRP9 concentrations were inversely correlated with β1‐AA (n=131, r=−0.31). Data are presented as mean±SD or median±interquartile range (25th–75th percentile). OD indicates optical density. **P<0.01, ***P<0.001.

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  Figure 2. Autoantibodies against the second extracellular loop of β1‐adrenergic receptor monoclonal antibodies (β1‐AAmAbs) decreased the levels of CTRP9 (C1q tumor necrosis factor‐related protein 9) in vitro and in vivo. A, Serum CTRP9 levels. B, The mRNA expression levels CTRP9 (normalized to GAPDH). Vehicle is for 8 weeks. C, Western blot analysis for CTRP9 expression. Vehicle is for 8 weeks. D, Western blot analysis for CTRP9 expression in neonatal rat cardiomyocytes (NRVMs) in the presence of β1‐AAmAbs (10−7 mol/L, 48 hours). n=8. 4w indicates fourth week after immunization; 8w, eighth week after immunization; gCTRP9, globular CTRP9. **P<0.01 vs before immunization (0 week) or vehicle group.

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  Figure 3. CTRP9 (C1q tumor necrosis factor‐related protein 9) overexpression restored cardiac function. A, Western blot analysis for CTRP9 expression in vehicle, autoantibodies against the second extracellular loop of β1‐adrenergic receptor monoclonal antibody (β1‐AAmAb), and β1‐AAmAb+CTRP9 groups. B, Echocardiographic left ventricular ejection fraction (EF) of mice. C, Echocardiographic left ventricular fractional shortening (FS) of mice. D, Echocardiographic left ventricular inner dimension of diastolic (LVID[d]). E, Echocardiographic left ventricular inner dimension of systolic (LVID[s]). n=8 in each group. *P<0.05, **P<0.01, ***P<0.001 vs vehicle; # P<0.05, ## P<0.01, ### P<0.001 vs β1‐AAmAb group.

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  Figure 4. CTRP9 (C1q tumor necrosis factor‐related protein 9) overexpression attenuated autoantibodies against the second extracellular loop of β1‐adrenergic receptor monoclonal antibody (β1‐AAmAb)–induced ventricular remodeling. A, Hematoxylin‐eosin staining. B, Masson's trichrome staining. C, Western blot analysis for matrix metalloproteinase‐2 (MMP2) expression. D, Western blot analysis for MMP9 expression. n=8 in each group. *P<0.05 vs vehicle; # P<0.05 vs β1‐AAmAb group.

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  Figure 5. CTRP9 (C1q tumor necrosis factor‐related protein 9) overexpression attenuated autoantibodies against the second extracellular loop of β1‐adrenergic receptor monoclonal antibody (β1‐AAmAb)–induced apoptosis and necrosis. A, Western blot analysis for cleaved caspase‐3 expression. B, Western blot analysis for full‐length caspase‐3 expression. C, Terminal deoxynucleotidyl transferase‐mediated dUTP nick‐end labeling (TUNEL) staining. D, Lactate dehydrogenase (LDH) level. n=8 per group. DAPI indicates 4′,6‐diamidino‐2‐phenylindole; FITC, fluorescein isothiocyanate. *P<0.05, **P<0.01 vs vehicle; # P<0.05, ## P<0.01 vs β1‐AAmAb group.

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  Figure 6. Both cAMP/protein kinase A (PKA) and G‐protein–coupled receptor kinase (GRK)/adiponectin receptor 1 (AdipoR1)/AMP‐activated protein kinase (AMPK) pathways were activated in autoantibodies against the second extracellular loop of β1‐adrenergic receptor monoclonal antibody (β1‐AAmAb) group. A, cAMP levels. B, The phosphorylation levels of PKA (P‐PKA). C, PKA activity. D, Western blot analysis for GRK expression. E, Coimmunoprecipitation for AdipoR1 phosphorylation. F, Western blot analysis for the expression and phosphorylation of AMPK (P‐AMPK). n=8. 4w indicates fourth week after immunization; 8w, eighth week after immunization; CTRP9, C1q tumor necrosis factor‐related protein 9; IB, immunoblot; IP, immunoprecipitation; Neg, negative; Non‐pA1, nonphosphorylated PepTag A1 peptide; p‐A1, phosphorylated PepTag A1 peptide; Pos, positive. *P<0.05, **P<0.01, ***P<0.001.

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  Figure 7. CTRP9 (C1q tumor necrosis factor‐related protein 9) overexpression regulated G‐protein–coupled receptor kinase 2 (GRK2)/adiponectin receptor 1/AMP‐activated protein kinase pathway. A, Protein kinase A activity. B and C, The phosphorylation and expression levels of GRK (p‐GRK). D, The phosphorylation levels of AMPK. n=8 in each group. β1‐AAmAb indicates autoantibodies against the second extracellular loop of β1‐adrenergic receptor monoclonal antibody; Neg, negative; Non‐pA1, nonphosphorylated PepTag A1 peptide; p‐A1, phosphorylated PepTag A1 peptide; Pos, positive. *P<0.05, **P<0.01, ***P<0.001 vs vehicle; # P<0.05 vs β1‐AAmAb group.