Transforming growth factor β₁ oppositely regulates the hypertrophic and contractile response to β-adrenergic stimulation in the heart

Abstract

Background: Neuroendocrine activation and local mediators such as transforming growth factor-β₁ (TGF-β₁) contribute to the pathobiology of cardiac hypertrophy and failure, but the underlying mechanisms are incompletely understood. We aimed to characterize the functional network involving TGF-β₁, the renin-angiotensin system, and the β-adrenergic system in the heart.

Methods: Transgenic mice overexpressing TGF-β₁ (TGF-β₁-Tg) were treated with a β-blocker, an AT₁-receptor antagonist, or a TGF-β-antagonist (sTGFβR-Fc), were morphologically characterized. Contractile function was assessed by dobutamine stress echocardiography in vivo and isolated myocytes in vitro. Functional alterations were related to regulators of cardiac energy metabolism.

Results: Compared to wild-type controls, TGF-β₁-Tg mice displayed an increased heart-to-body-weight ratio involving both fibrosis and myocyte hypertrophy. TGF-β₁ overexpression increased the hypertrophic responsiveness to β-adrenergic stimulation. In contrast, the inotropic response to β-adrenergic stimulation was diminished in TGF-β₁-Tg mice, albeit unchanged basal contractility. Treatment with sTGF-βR-Fc completely prevented the cardiac phenotype in transgenic mice. Chronic β-blocker treatment also prevented hypertrophy and ANF induction by isoprenaline, and restored the inotropic response to β-adrenergic stimulation without affecting TGF-β₁ levels, whereas AT₁-receptor blockade had no effect. The impaired contractile reserve in TGF-β₁-Tg mice was accompanied by an upregulation of mitochondrial uncoupling proteins (UCPs) which was reversed by β-adrenoceptor blockade. UCP-inhibition restored the contractile response to β-adrenoceptor stimulation in vitro and in vivo. Finally, cardiac TGF-β₁ and UCP expression were elevated in heart failure in humans, and UCP--but not TGF-β₁--was downregulated by β-blocker treatment.

Conclusions: Our data support the concept that TGF-β₁ acts downstream of angiotensin II in cardiomyocytes, and furthermore, highlight the critical role of the β-adrenergic system in TGF-β₁-induced cardiac phenotype. Our data indicate for the first time, that TGF-β₁ directly influences mitochondrial energy metabolism by regulating UCP3 expression. β-blockers may act beneficially by normalizing regulatory mechanisms of cellular hypertrophy and energy metabolism.

Figure 1. Characterization of myocardial tissue in wild type (WT) and TGF-β₁ transgenic mice (TGF-β₁) that have been treated with either metoprolol (METO), telmisartan (TELMI), or soluble TGF-βR-Fc (sR-Fc).

(A) Myocardial TGF-β₁ protein expression as determined by Western blotting in heart homogenates from the various groups as indicated. RasGAP served as lysate control. (B) Body weight, heart weight, and heart/body weight ratio (n = 30–57 animals in each group). (C–F) Morphometric analysis of myocardial tissue (n = 5–9 animals in each group). Shown are the fractional areas of connective tissue (C), cardiac fibroblasts (D), cardiac myocytes (E), and cardiomyocyte diameter (F). *p<0.05 vs. WT, ^# p<0.05 vs. untreated TGF-β₁ mice.

Figure 2. Induction of (A) atrial natriuretic factor (ANF) and (B) ornithine decarboxylase (ODC) mRNA by isoprenaline in hearts from TGF-β₁ transgenic mice (TGF-β₁) that have been treated with either metoprolol (METO), telmisartan (TELMI), or soluble TGF-βR-Fc (sR-Fc).

Data are expressed as fold-increase relative to saline-perfused hearts. *p<0.05 vs. WT mice.

Figure 3. Echocardiographic evaluation of wild type (WT) and TGF-β₁ transgenic mice (TGF-β₁) that have been treated with either metoprolol (METO), telmisartan (TELMI), or soluble TGF-βR-Fc (sR-Fc).

(A) Representative short axis views during diastole and systole in WT and TGF-β₁ mice. (B) Left ventricular mass. (C) Left ventricular ejection fraction at rest. (D) Resistive Index. Data in B–D represent means ± SEM from 5–7 animals in each group. *p<0.05 vs. WT; ^# p<0.05 vs. untreated TGF-β₁ mice.

Figure 4. Dobutamine stress echocardiography (DSE).

(A) DSE protocol as applied in mice. (B) Representative m-mode registrations in a WT mouse at rest, and at various concentrations of dobutamine (Dobu). (C) Contractile reserve in response to cumulative concentrations of dobutamine in wild type (WT) and TGF-β₁ transgenic mice (TGF-β₁) that have been treated with either metoprolol (METO), telmisartan (TELMI), or soluble TGF-βR-Fc (sR-Fc), n = 5–6 in each group. *p<0.05 vs. rest; ^# p<0.05 vs. WT.

Figure 5. Contractility of isolated cardiac myocytes from wild type (WT) and TGF-β₁ transgenic (TGF-β₁) mice.

(A) Representative original registrations of cell shortening (dL/L) under basal conditions and upon isoprenaline stimulation (10 µM). (B) Contractile response of isolated cardiomyocytes from WT and TGF-β₁ mice to increasing concentrations of isoprenaline (n = 100 in each group). (C) Contractile response of isolated cardiomyocytes to 10 µM isoprenaline in the various treatment groups (n = 97–135 in each group). *p<0.05 vs. WT.

Figure 6. A role for uncoupling proteins (UCPs) for the diminished contractile reserve in TGF-β₁ transgenic mice.

(A) Stimulation of rat cardiac myocytes with TGF-β₁ (10 ng/ml) leads to upregulation of UCP2 and UCP3 mRNA (n = 4 in each group). (B) Western blot analysis of UCP3 expression in mitochondria isolated from myocardial tissue of WT and TGF-β₁ mice. COX-I served as a loading control, and UCP3 knockout mice served as a negative control. The bar graph represents means ± SEM from 7 animals in each group. (C and D) Expression of UCP2 and UCP3 mRNA in the various treatment groups. (E) Functional role of UCPs in the heart. Inhibition of UCPs by genipin (100 mg/kgBW) restored the contractile response to dobutamine (40 µg/kg/min) in TGF-β₁ mice. (F) Genipin (5 µM) restored the contractile response to isoprenaline (10 µM) in isolated cardiac myocytes (n = 26–30 in each group).

Figure 7. TGF-β₁ (A) and UCP3 (B) expression in human heart.

Myocardial samples were obtained from non-failing myocardium (NF; n = 3), and from DCM hearts of patients who had not received β-blocker treatment (DCM; n = 5) or patients who were treated with metoprolol (DCM-METO; n = 3). *p<0.05 vs. DCM.