Table 1.
Clinical characteristics of patients with chronic heart failure due to DCM (mean ± SD).
Table 2.
Holter Electrocardiographic Findings (mean ± SD).
Figure 1.
Titers and incidence of β1-AA in 95 healthy subjects and 95 patients with DCM.
A. Titers of β1-AA from 95 healthy subjects (open squares) and 95 patients with DCM (filled squares). Scatter plot represents the titers of β1-AA for each patient in each group. Experiments were repeated twice per sample. B. Percentage of β1-AA-positive sera from two different groups. N, normal group, DCM dilated cardiomyopathy. **p<0.01 versus N group.
Figure 2.
β1-AA from DCM patients bound to β1-ARs on the surface of CD3+T cells. A.
After FACS, the purity of selected rat CD3+T lymphocytes by immunomagnetic separation was 92.2%. B, C. The binding of β1-AA (25 µg/ml) with the β1-ARs on the CD3+T cells was determined by confocal microscopy, and β1-AR was identified using an anti-β1-AR antibody (green). Nuclei were labeled with DAPI (blue). The negative control was performed by omitting primary antibodies during the incubation. Bar, 30 µm.
Figure 3.
β1-AA from DCM patients increased the beat frequency of cultured cardiomyocytes.
The bar graph shows the increase in beat frequency of isolated myocardial cells stimulated by β1-AA (25 µg/ml) or isoproterenol (0.1 µmol/l). **p<0.01 versus vehicle group. Data were presented as means ± SD of three independent experiments. ISO: isoproterenol, MET: metoprolol.
Figure 4.
β1-AA from DCM patients significantly promoted the proliferation of CD3+T cells.
A. β1-AA promoted CD3+T cell proliferation in a concentration-dependent manner. B. CD3+T cells (5×105 cells/ml) were incubated for 48 h at 37°C and 5% CO2 in the presence of β1-AA (25 µg/ml) or isoproterenol (0.1 µmol/l). Cell proliferation was measured at 450 nm by CCK-8 uptake assay. **p<0.01 versus vehicle group; ##p<0.01 versus negative IgG group. n = 9 per group. C. CD3+T cells were labeled with 4 µmol/l CFSE, and cell proliferation was measured by flow cytometry. Data shown here are representative of one of three different experiments with similar results. D. The bar graph shows the percentage of proliferating (CFSElo) T cells among the total CD3+T cell population. n = 3, *p<0.05 versus vehicle group. **p<0.01 versus vehicle group; ##p<0.01 versus Negative IgG group, ISO: isoproterenol.
Figure 5.
β1-AA-mediated T cells proliferation through the β1-AR/cAMP/PKA pathway.
A. T cells were stimulated with the selective β1-AR antagonist metoprolol (1 µmol/l) and the selective PKA inhibitor H89 (1 µmol/l) for 1 h at 37°C in 5% CO2 before the addition of β1-AA (25 µg/ml). *p<0.05, **p<0.01 versus vehicle group; #p<0.05, ##p<0.01 versus β1-AA group. n = 9 per group. B. The effect of β1-AA or isoproterenol on the production of cAMP (expressed as pg/ml) in T lymphocytes was examined by ELISA, **p<0.01 versus vehicle group, ##p<0.01 versus negative IgG group, ΔΔp<0.01 versus β1-AA group. Data were presented as means ± SD of 6 independent experiments. C. Immunoblot detection of phosphorylated VASP (p-VASP) and total VASP from CD3+T cells stimulated with β1-AA for 30 min. Images are representative of 3 independent experiments. D. The bar graph shows the ratio of p-VASP to total VASP. n = 3, **p<0.01 versus vehicle group, ΔΔp<0.01 versus β1-AA group, MET: metoprolol.
Figure 6.
β1-AA-mediated T cell proliferation mediated by activation of p38 MAPK.
A. T cells were pretreated with the selective PKA inhibitor H89 (1 µmol/l) and the selective p38 MAPK inhibitor SB203580 (1 µmol/l) for 1 h at 37°C in 5% CO2 before stimulation by β1-AA (25 µg/ml). *p<0.05, **p<0.01 versus vehicle group; #p<0.05 versus β1-AA group. n = 9 per group. B. Immunoblot detection of phosphorylated p38 MAPK (p-p38 MAPK) and total p38 MAPK from CD3+T cells stimulated with β1-AA for 30 min. Images are representative of three independent experiments. C. The bar graph shows the ratio of p-p38 MAPK to total p38 MAPK. n = 3, **p<0.01 versus Vehicle group, ΔΔp<0.01 versus β1-AA group, SB: SB203580.
Figure 7.
β1-AA inhibited IFN-γ secretion and promoted IL-4 production in T cells.
A. T cells (5 × 105 cells/ml) were pretreated with 1 µmol/l metoprolol in the presence of β1-AA (25 µg/ml) for 48 h, and then IFN-γ levels were analyzed by ELISA. **p<0.01 versus vehicle group, ##p<0.01 versus negative IgG group, n = 9/group B. The effect of β1-AA (25 µg/ml) on IL-4 levels was examined by ELISA, **p<0.01 versus vehicle group, ##p<0.01 versus negative IgG group, ΔΔp<0.01 versus β1-AA group, n = 9 per group. Data are presented as means ± SD of 6 independent experiments.