Cardiac pressure overload hypertrophy is differentially regulated by β-adrenergic receptor subtypes

Abstract

In isolated myocytes, hypertrophy induced by norepinephrine is mediated via α(1)-adrenergic receptors (ARs) and not β-ARs. However, mice with deletions of both major cardiac α(1)-ARs still develop hypertrophy in response to pressure overload. Our purpose was to better define the role of β-AR subtypes in regulating cardiac hypertrophy in vivo, important given the widespread clinical use of β-AR antagonists and the likelihood that patients treated with these agents could develop conditions of further afterload stress. Mice with deletions of β(1), β(2), or both β(1)- and β(2)-ARs were subjected to transverse aortic constriction (TAC). After 3 wk, β(1)(-/-) showed a 21% increase in heart to body weight vs. sham controls, similar to wild type, whereas β(2)(-/-) developed exaggerated (49% increase) hypertrophy. Only when both β-ARs were ablated (β(1)β(2)(-/-)) was hypertrophy totally abolished. Cardiac function was preserved in all genotypes. Several known inhibitors of cardiac hypertrophy (FK506 binding protein 5, thioredoxin interacting protein, and S100A9) were upregulated in β(1)β(2)(-/-) compared with the other genotypes, whereas transforming growth factor-β(2), a positive mediator of hypertrophy was upregulated in all genotypes except the β(1)β(2)(-/-). In contrast to recent reports suggesting that angiogenesis plays a critical role in regulating cardiac hypertrophy-induced heart failure, we found no evidence that angiogenesis or its regulators (VEGF, Hif1α, and p53) play a role in compensated cardiac hypertrophy. Pressure overload hypertrophy in vivo is dependent on a coordination of signaling through both β(1)- and β(2)-ARs, mediated through several key cardiac remodeling pathways. Angiogenesis is not a prerequisite for compensated cardiac hypertrophy.

Fig. 1.

Heart weight-to-body weight ratios (HW/BW) comparing transverse aortic constriction (TAC) with sham-operated controls for each genotype (n = 10 in each group). The β₁^−/− mice showed no significant difference in the degree of hypertrophy [NS vs. wild type (WT)]; in contrast, β₂^−/− mice showed exaggerated hypertrophy (P < 0.05 vs. WT). Only in the absence of both β-adrenergic receptor (AR) subtypes was hypertrophy ablated. *P < 0.05 vs. sham. †P < 0.05 vs. WT TAC.

Fig. 2.

A: myocyte cross-sectional area in TAC vs. sham-operated controls for WT, β₂^−/−, and β₁β₂^−/− mice (n = 10 in each group). *P < 0.05 vs. sham. †P < 0.01 vs. WT TAC. B: micrographs comparing WT sham with TAC in WT, β₂^−/−, and β₁β₂^−/− mice.

Fig. 3.

A: Doppler-derived pressure gradient was not different between genotypes (n = 10 in each group). B: validation of echo Doppler measurement of transaortic gradient compared with the double cannulation method in a separate group of mice with a wide variation in TAC gradients (see materials and methods for details, n = 12). C: left ventricular fractional shortening was also not different between genotypes and was unchanged between TAC and sham-operated controls, demonstrating the absence of heart failure in this model. D: left ventricular dimensions (d, diastole; s, systole) comparing sham vs. TAC for each genotype. *P < 0.05, TAC vs. sham.

Fig. 4.

A: VEGF expression in sham-operated controls compared with TAC after 1, 2, and 3 wk (n = 7 in each group). There were no differences in VEGF expression in any of the genotypes at any time point after TAC compared with sham controls. Baseline, i.e., sham-operated, VEGF expression was increased in the β₂^−/− (†P < 0.005) and decreased in the β₁β₂^−/− (†P < 0.005) compared with WT. VEGF was did not change in WT or β1β2^−/− after TAC but fell in the β₂^−/− (*P < 0.05). B: p53 expression was unchanged with TAC in each genotype (n = 7 in each group). However, similar to VEGF, p53 expression was increased in the sham-operated β₂^−/− (†P < 0.005) and decreased in the sham-operated β₁β₂^−/− (†P < 0.005) compared with WT. C: sham operation significantly increased VEGF expression at 2 wk compared with nonoperated controls (n = 4 in each group) demonstrating the importance of using sham controls at all time points after TAC (*P < 0.05). D: Hif-1α expression was not increased after TAC at any time point (n = 4 in each group). +Con is positive control (nuclear extract from cobalt chloride-treated COS-7 cells).

Fig. 5.

Vessel-to-myocyte ratios, measured by 2 independent observers blinded to experimental condition, were unchanged at both 2 and 3 wk after TAC in WT, β₂^−/−, and β₁β₂^−/− mice (n = 4 in each group).

Fig. 6.

Expression of known regulators of cardiac hypertrophy after TAC in each genotype. Each candidate gene was initially identified as significantly differentially regulated by gene microarray analysis. Data shown are results of confirmatory quantitative (Q)RT-PCR (n = 9 in each group). Transcripts which were differentially increased with TAC in β₁β₂^−/− vs. all other genotypes include FK506 binding protein 5 (Fkbp5; A), thioredoxin interacting protein (Txnip; B), and S100A9 (C). In contrast, transforming growth factor-β₂ (TGF-β₂; D) expression was increased in all genotypes after TAC except for the β₁β₂^−/−. *P < 0.05 vs. sham.

Fig. 7.

Expression of hypertrophy genes after TAC in each genotype by QRT-PCR (n = 9 in each group). A: expression of α-skeletal actin was significantly increased in all TAC groups except for the β₁/β₂^−/−. B: atrial natriuretic peptide (ANP) expression was increased in all TAC groups, although the greatest increase was in the excessively hypertrophic β₂^−/− (13-fold increase over sham). C: sarcoplasmic reticulum calcium ATPase (SERCA) decreased in both WT and β₂^−/− but was unchanged in the β₁^−/− and β₁β₂^−/−. D: calcium/calmodulin-dependent serine protein kinase (CaM kinase) was increased significantly only in the β₁^−/−. *P < 0.05 vs. sham.