α₁A-adrenergic receptors regulate cardiac hypertrophy in vivo through interleukin-6 secretion

Abstract

The role of α₁-adrenergic receptors (ARs) in the regulation of cardiac hypertrophy is still unclear, because transgenic mice demonstrated hypertrophy or the lack of it despite high receptor overexpression. To further address the role of the α₁-ARs in cardiac hypertrophy, we analyzed unique transgenic mice that overexpress constitutively active mutation (CAM) α₁A-ARs or CAM α₁B-ARs under the regulation of large fragments of their native promoters. These constitutively active receptors are expressed in all tissues that endogenously express their wild-type counterparts as opposed to only myocyte-targeted transgenic mice. In this study, we discovered that CAM α₁A-AR mice in vivo have cardiac hypertrophy independent of changes in blood pressure, corroborating earlier studies, but in contrast to myocyte-targeted α₁A-AR mice. We also found cardiac hypertrophy in CAM α₁B-AR mice, in agreement with previous studies, but hypertrophy only developed in older mice. We also discovered unique α₁-AR-mediated hypertrophic signaling that was AR subtype-specific with CAM α₁A-AR mice secreting atrial naturietic factor and interleukin-6 (IL-6), whereas CAM α₁B-AR mice expressed activated nuclear factor-κB (NF-κB). These particular hypertrophic signals were blocked when the other AR subtype was coactivated. We also discovered that crossbreeding the two CAM models (double CAM α₁A/B-AR) inhibited the development of hypertrophy and was reversible with single receptor activation, suggesting that coactivation of the receptors can lead to novel antagonistic signal transduction. This was confirmed by demonstrating antagonistic signals that were even lower than normal controls in the double CAM α₁A/B-AR mice for p38, NF-κB, and the IL-6/glycoprotein 130/signal transducer and activator of transcription 3 pathway. Because α₁A/B double knockout mice fail to develop hypertrophy in response to IL-6, our results suggest that IL-6 is a major mediator of α₁A-AR cardiac hypertrophy.

Fig. 1.

Southern blot analysis of CAM α_1A-AR and CAM α_1B-AR crossbreeding to produce double CAM α_1A/B-AR transgenic mice. Pups from CAM α_1A-AR × CAM α_1B-AR breeding were genotyped from tail DNA and subjected to Southern blot analysis. Each pup DNA was screened against an α_1A-AR–specific probe, designated as “A” on the blot (21) or an α_1B-AR specific probe, designated as “B” on the blot (14). Pup DNA that demonstrated positive results for both probes (A⁺/B⁺) were used as founders for the CAM α_1A/B-AR mouse line and verified for homozygosity by back-breeding to wild-type mice.

Fig. 2.

Expression and constitutive activity of CAM α_1A/B-AR. (A) Saturation binding was performed using [¹²⁵I]-HEAT to determine the density of α₁-ARs in hearts of transgenic and normal mice. *P < 0.01; ^#P < 0.05 (significant difference compared with normal hearts). (B) IP₃ concentrations were measured in heart tissue from transgenic and normal mice and normalized to wet tissue weight. ⁺Significant activation of IP3 over nonstimulated tissue in normal hearts. ^†Significance of basal IP₃ over nonstimulated tissue. Data represent the mean ± S.E.M. of 4–8 mice of equal sexes.

Fig. 3.

HW/BW ratios (A), fibrosis (B), ANF/BNP levels (C), and BP (D). (A) The HW/BW ratio was determined in mice aged 6–8-months. (B) Hearts were subjected to Masson Trichrome staining and the amount of fibrosis determined through Image J analysis. (C) Total RNA from hearts were subjected to Northern blot analysis and probed for ANF and BNP mRNA. (D) Measurement of the mean carotid artery BP in conscious mice. BP studies in CAM α_1B-AR are published (Zuscik et al., 2001). Data represent the mean ± S.E.M. of 4–8 mice of equal sexes. *P < 0.05 (significant difference compared with nontransgenic hearts).

Fig. 4.

Protein levels of phosphorylated p38 and IKBα. Hearts were homogenized from normal, CAM α_1A-AR (CAM A), CAM α_1B-AR (CAM B), and CAM α_1A/B-AR (CAM A/B) mice and subjected to Western analysis. Phosphorylated proteins were normalized to total protein and glyceraldehyde 3-phosphate dehydrogenase. Data represent the mean ± S.E.M. of 4–6 mice of equal sexes. *P < 0.05 (significant difference compared with control).

Fig. 5.

Echocardiographic analysis of posterior wall dimensions and chamber size at ages 4–6 months and 11–12 months. Mice were subjected to echocardiographic analysis and anesthetized with isoflurane (0.2% v/v). M-mode echocardiograms (G) obtained from 9 to 10 beats per mouse allowed direct measurement (mean ± S.E.M.) of posterior wall thickness (A and B) and ventricular dimensions end systolic dimensions (C and D), and left ventricular end diastolic dimensions (E and F). *P < 0.05 (significance compared with age-matched normal controls). n = 6–8 mice of equal sexes.

Fig. 6.

α₁-AR subtype induced cardiac hypertrophy and suppression by coactivation. Normal or CAM mice were subjected to i.p. injections of various α₁-AR agonists and antagonists. α_1A-ARs were stimulated using cirazoline (0.3 mg/kg i.p.). α_1B-ARs were stimulated using NE (1 mg/kg i.p.) and the α_1A-AR antagonist, 5-methyurapidil (10 μg/kg i.p.). Control mice were injected with saline (0.9% NaCl). All mice were injected twice daily for 2 weeks and HW/BW ratios determined. Data represent the mean ± S.E.M. of 6–8 mice of equal sexes. *P < 0.05 (significant difference compared with control).

Fig. 7.

IL-6/gp130/STAT3 levels and mediated hypertrophy in CAM mice. (A) Serum IL-6 was determined using the Quantikine mouse kit following the manufacturer’s instructions. (B) Levels of gp130, phosphorylated, and total STAT3 as assessed by Western blot. (C) Mice were injected daily for 2 weeks i.p. with IL-6 (0.1 ml, 40 ng) and HW/BW ratios determined. Data represent the mean ± S.E.M. of 4–6 mice of equal sexes. *P < 0.05 (significant difference compared with nontransgenic mice).

Fig. 8.

Schematic of α_1A-AR–mediated cardiac hypertrophy and antagonistic hypertrophic signaling initiated with coactivation with the α_1B-AR. α_1A-ARs mediate the secretion of IL-6 into the bloodstream from various cell types such as myocytes, vascular smooth muscle cells, fibroblasts, lymphocytes, and endothelial cells. The secreted IL-6 acts on the myocyte to mediate cardiac hypertrophy through STAT3 nuclear signaling. α_1A-ARs also phosphorylate STAT3 independent of IL-6 secretion. α_1B-ARs mediate hypertrophic NF-κB signaling. When α_1A- and α_1B-ARs are coexpressed and coactivated, hypertrophic signals through p38, NK-κB, and STAT3 are inhibited. Inhibition of both p38 and NF-κB downregulate the expression and secretion of IL-6 from the myocyte.