Temporal and gefitinib-sensitive regulation of cardiac cytokine expression via chronic β-adrenergic receptor stimulation

Abstract

Chronic stimulation of β-adrenergic receptors (βAR) can promote survival signaling via transactivation of epidermal growth factor receptor (EGFR) but ultimately alters cardiac structure and contractility over time, in part via enhanced cytokine signaling. We hypothesized that chronic catecholamine signaling will have a temporal impact on cardiac transcript expression in vivo, in particular cytokines, and that EGFR transactivation plays a role in this process. C57BL/6 mice underwent infusion with vehicle or isoproterenol (Iso)±gefitinib (Gef) for 1 or 2 wk. Cardiac contractility decreased following 2 wk of Iso treatment, while cardiac hypertrophy, fibrosis, and apoptosis were enhanced at both timepoints. Inclusion of Gef preserved contractility, blocked Iso-induced apoptosis, and prevented hypertrophy at the 2-wk timepoint, but caused fibrosis on its own. RNAseq analysis revealed hundreds of cardiac transcripts altered by Iso at each timepoint with subsequent RT-quantitative PCR validation confirming distinct temporal patterns of transcript regulation, including those involved in cardiac remodeling and survival signaling, as well as numerous cytokines. Although Gef infusion alone did not significantly alter cytokine expression, it abrogated the Iso-mediated changes in a majority of the βAR-sensitive cytokines, including CCL2 and TNF-α. Additionally, the impact of βAR-dependent EGFR transactivation on the acute regulation of cytokine transcript expression was assessed in isolated cardiomyocytes and in cardiac fibroblasts, where the majority of Iso-dependent, and EGFR-sensitive, changes in cytokines occurred. Overall, coincident with changes in cardiac structure and contractility, βAR stimulation dynamically alters cardiac transcript expression over time, including numerous cytokines that are regulated via EGFR-dependent signaling.

Keywords: EGFR; cytokines; heart; remodeling; βAR.

Fig. 1.

Effects of chronic isoproterenol (Iso) ± gefitinib (Gef) stimulation on cardiac contractility. A: representative M-mode echocardiography from C57BL/6 mice before treatment and following 1 or 2 wk. Vehicle (Veh) ± Gef or Iso ± Gef. Left ventricular fractional shortening (FS; B) and ejection fraction (EF; C) were measured at the short axis from M mode using VisualSonic Analysis Software. *P ≤ 0.05 vs. Veh; n = 8–16 each, 2-way ANOVA.

Fig. 2.

Effect of chronic Iso ± Gef stimulation on cardiac hypertrophy. Representative hematoxylin-eosin (H&E) staining (A), and gravimetric analysis of heart weight to tibia length (HW/TL; B), of hearts from mice treated with Veh ± Gef or Iso ± Gef for 1 or 2 wk is shown. *P ≤ 0.05, ‡P ≤ 0.001; n = 8–16 each; 1-way ANOVA. ns, Not significant.

Fig. 3.

Effect of chronic Iso ± Gef stimulation on cardiac fibrosis. Representative Masson trichrome staining (A), and quantification of Masson trichrome staining (B), of hearts from mice treated with Veh ± Gef or Iso ± Gef for 1 or 2 wk is shown. *P ≤ 0.05, †P ≤ 0.01, ‡P ≤ 0.001; n = 8–16 each; 1-way ANOVA.

Fig. 4.

Effect of chronic Iso ± Gef stimulation on cardiac survival. Representative transferase-mediated dUTP nick-end labeling (TUNEL) staining (A), and percentage of TUNEL-positive (TUNEL +ve) cells (B), in hearts from mice treated with Veh ± Gef or Iso ± Gef for 1 or 2 wk is shown. *P ≤ 0.05, †P ≤ 0.01, ‡P ≤ 0.001; n = 8–16 each; 1-way ANOVA.

Fig. 5.

Differential temporal regulation of the cardiac transcriptome by chronic β-adrenergic receptor (βAR) stimulation. A: heatmap depicting significant (P ≤ 0.05) changes in cardiac transcript expression in mouse hearts treated with Iso compared with Veh for 1 vs. 2 wk as detected by transcriptome analysis. Each RNAseq sample (Veh 1 wk, Iso 1 wk, Veh 2 wk, and Iso 2 wk) contained total RNA from 4 individual hearts per condition. B: Venn diagram depicting the number of cardiac transcripts significantly (P ≤ 0.05) increased or decreased following 1 or 2 wk Iso treatment. C: ingenuity analysis revealed the 10 most significantly associated cellular function categories of the cardiac transcripts regulated by 1 or 2 wk of Iso infusion.

Fig. 6.

Temporal regulation of transcripts involved in cardiac remodeling and survival signaling by chronic βAR stimulation. RT-quantitative PCR (RT-qPCR) validation of changes in expression of transcripts predicted by transcriptome analysis to be involved in cardiac remodeling and survival following Iso infusion for either 1 wk (A) or 2 wk (B) is shown. *P ≤ 0.05, †P ≤ 0.01 vs. Veh of same transcript; n = 4–6 each; 2-tailed t-test. RQ, relative quantity.

Fig. 7.

Impact of chronic βAR (ADRB) stimulation on cardiac cytokine transcript expression. A: network analysis between ADRB and the 48 cytokines predicted to be regulated by Iso. RT-qPCR analysis of 17 cytokine transcripts were predicted to become altered following Iso infusion for 1 wk (B) or 2 wk (C). *P ≤ 0.05, †P ≤ 0.01, ‡P ≤ 0.001 vs. Veh of same transcript; n = 3–6 each; 2-tailed t-test.

Fig. 8.

Chronic βAR-mediated regulation of cardiac cytokine expression is sensitive to epidermal growth factor receptor (EGFR) inhibition. A: network analysis between EGFR and the 48 cytokines predicted to be regulated by Iso. ●, Cytokines validated to be regulated by chronic Iso infusion via RT-qPCR (in Fig. 7, B and C). The effects of Gef on changes in Iso-mediated transcript expression were assessed by RT-qPCR at 1 and 2 wk timepoints for Thbs1 (B), Ccl2 (C), and Tnfa (D). *P ≤ 0.05, †P ≤ 0.01, ‡P ≤ 0.001; n = 4–6 each; 1-way ANOVA. ELISAs for CCL2 (E) or TNF-α (F) were performed on serum from mice treated for 1 or 2 wk with Veh ± Gef or Iso ± Gef. *P ≤ 0.05; n = 4 each; 1-way ANOVA.

Fig. 9.

Acute βAR-mediated and EGFR-sensitive regulation of cytokine expression occurs primarily in cardiac fibroblasts. RT-qPCR analysis of changes in cytokine expression following acute Iso stimulation (3 h) in primary isolated rat neonatal cardiomyocytes (A) or rat neonatal cardiac fibroblasts (B) is shown. *P ≤ 0.05, †P ≤ 0.01, ‡P ≤ 0.001 vs. Veh of same transcript; n = 4–8 each; 2-tailed t-test. ND, not detected.

Fig. 10.

Temporal- and Gef-sensitive changes in cardiac transcript expression in response to chronic βAR stimulation. βAR stimulation induces differential changes in cardiac transcript expression following Iso infusion for 1 wk (left) vs. 2 wk (right). Transcripts are regulated via EGFR-dependent and -independent mechanisms. *Transcript (Il17a), whose expression is dominantly repressed by βAR-mediated EGFR transactivation but is significantly enhanced in the presence of Gef via EGFR-independent βAR signaling.