Src tyrosine kinase promotes cardiac remodeling induced by chronic sympathetic activation

Abstract

Cardiac remodeling serves as the underlying pathological basis for numerous cardiovascular diseases and represents a pivotal stage for intervention. The excessive activation of β-adrenergic receptors (β-ARs) assumes a crucial role in cardiac remodeling. Nonetheless, the underlying molecular mechanisms governing β-AR-induced cardiac remodeling remain largely unresolved. In the present study, we identified Src tyrosine kinase as a key player in the cardiac remodeling triggered by excessive β-AR activation. Our findings demonstrated that Src mediates isoproterenol (ISO)-induced cardiac hypertrophy, fibrosis, and inflammation in vivo. Furthermore, Src facilitates β-AR-mediated proliferation and transdifferentiation of cardiac fibroblasts, and hypertrophy and cardiomyocytes in vitro. Subsequent investigations have substantiated that Src mediates β-AR induced the extracellular signal-regulated protein kinase (ERK1/2) signaling pathway activated by β-AR. Our research presents compelling evidence that Src promotes β-AR-induced cardiac remodeling in both in vivo and in vitro settings. It establishes the promoting effect of the β-AR/Src/ERK signaling pathway on overall cardiac remodeling in cardiac fibroblasts and underscores the potential of Src as a therapeutic target for cardiac remodeling.

Keywords: Src; cardiac remodeling; cardiomyocyte; β-adrenergic receptor.

Figure 1. Inhibition of Src attenuates ISO-induced cardiac hypertrophy

(A) Representative images of heart size. (B) Representative M-mode echocardiography images were taken to show left ventricular wall thickness. (C) Representative micrographs of myocyte cross-sectional area. Quantitative analysis of myocyte cross-sectional area; bar = 100 μm. F = 70.11, P<0.001. (D) Quantitative analysis of diastolic left ventricular posterior wall thickness; F = 50.20, P<0.001. (E) Quantitative analysis of HW/BW ratio; F = 32.23, P<0.001. (F) Quantitative analysis of HW/TL ratio; F = 65.61, P<0.001. (G) Quantitative analysis of ANP mRNA expression; F = 23.06, P<0.001. (H) Quantitative analysis of BNP mRNA expression; F = 27.61, P<0.001. (I) Left ventricular EF and (J) FS were measured to reflect cardiac contraction function; F(I) = 0.35, P(I)>0.05, F(J) = 0.17, P(J)>0.05. Data represent mean ± SEM. Analyzed by one-way analysis of variance (ANOVA) with Tukey’s post-hoc multiple comparison tests; N=7, *P<0.05, **P<0.01, ***P<0.001; ns, P>0.05.

Figure 2. Inhibition of Src prevents ISO-induced cardiac fibrosis

(A) Representative micrographs of picrosirius red-stained sections of the ventricle. Red parts represent collagen. The scale bars of 10× images are 2 mm, of 200× images are 100 μm. Quantification of cardiac interstitial collagen content from picrosirius red-stained sections with results expressed as the ratio of collagen area to heart area. There is no significance among all the groups; F = 35.76, P<0.001. (B) The mRNA expression of Collagen I in the heart tissue; F = 27.52, P<0.001. (C) The mRNA expression of Collagen III in the heart tissue; F = 42.56, P<0.001. Data represent mean ± SEM. Analyzed by one-way ANOVA with Tukey’s post-hoc multiple comparison tests; N=7, *P<0.05, ***P<0.001.

Figure 3. Inhibition of Src attenuates ISO-induced cardiac inflammation

(A) Representative images showing H&E staining of heart sections. The arrows refer to the infiltrated inflammatory cells. The scale bars of 10× images are 2 mm, of 200× images are 100 μm. (B) The mRNA expression of IL-1β in the heart tissue; F = 36.03, P<0.001. (C) The mRNA expression of IL-6 in the heart tissue; F = 73.46, P<0.001. Data represent mean ± SEM. Analyzed by one-way ANOVA with Tukey’s post-hoc multiple comparison tests; N=7, *P<0.05, **P<0.01, ***P<0.001.

Figure 4. Inhibition of Src reduces ISO-induced cardiomyocyte hypertrophy

(A) Src could be activated by isoproterenol. Cardiomyocytes were starved for 12 h, then stimulated with isoproterenol (10 μM) for 5 or 15 min, the phosphorylation of Src was analyzed using Western blotting. Activation of Src was determined by anti-Tyrosine 416 phospho-specific Src antibody. Experiments were performed in four times; F = 41.11, P<0.001. (B) The mRNA expression of ANP in the cardiomyocyte; F = 79.08, P<0.001. (C) The mRNA expression of BNP in the cardiomyocyte; F = 49.45, P<0.001. (D) Inhibition of Src reduces ISO-induced cardiomyocyte hypertrophy indicator F-actin. Cardiomyocytes were starved for 12 h, treated with or without PP1 (10 μM) for 1 h and then stimulated with isoproterenol (10 μM) in the presence or absence of PP1 for an additional 72 h; F = 67.44, P<0.001; bar = 50 μm. Data represent mean ± SEM. Analyzed by one-way ANOVA with Tukey’s post-hoc multiple comparison tests. N=4, number of NRCM>100 cells; *P<0.05, **P<0.01, ***P<0.001.

Figure 5. Src inhibition prevents ISO-induced cardiac fibroblast proliferation and transdifferentiation

(A) Src could be activated by isoproterenol. Cardiac fibroblasts were starved for 12 h, then stimulated with isoproterenol (10 μM) for 5 or 15 min, the phosphorylation of Src was analyzed using Western blotting. Activation of Src was determined by anti-Tyrosine 416 phospho-specific Src antibody. Experiments were performed in four times; F = 5.12, P<0.05. (B) Inhibition of Src prevents ISO-induced cardiac fibroblasts cell viability. The cell viability was detected by CCK-8 assay; F = 10.58, P<0.001. (C) Inhibition of Src reduces ISO-induced cardiac fibroblasts proliferation indicator PCNA; F = 10.48, P<0.01. (D) Inhibition of Src reduces ISO-induced cardiac fibroblasts transdifferentiation indicator α-SMA; F = 30.81, P<0.001. Cardiac fibroblasts were starved for 12 h, treated with or without PP1 (10 μM) for 1 h and then stimulated with isoproterenol (10 μM) in the presence or absence of PP1 for an additional 24 h. Data represent mean ± SEM. Analyzed by one-way ANOVA with Tukey’s post-hoc multiple comparison tests; N=4, *P<0.05, **P<0.01, ***P<0.001.

Figure 6. Src mediates β-AR-induced ERK1/2 signal pathway

(A) Inhibition of Src decreases ISO-induced ERK1/2 phosphorylation. HEK-293A was starved for 12 h, treated with or without PP1 (10 μM) for 1 h, then stimulated with isoproterenol (10 μM) for 5 or 15 min; F(interaction) = 9.06, P<0.01. (B) Overexpression of Src increases ISO-induced ERK1/2 phosphorylation. HEK-293A was transfected with flag-Src plasmid, starved for 12 h, then stimulated with isoproterenol (10 μM) for 5 or 15 min; F(interaction) = 8.59, P<0.01. Data represent mean ± SEM. Analyzed by two-way ANOVA with Tukey’s post-hoc multiple comparison tests; N=4, *P<0.05, **P<0.01, #P<0.05, ##P<0.01.