Transforming Growth Factor β1 Oppositely Regulates the Hypertrophic and Contractile Response to β-Adrenergic Stimulation in the Heart

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

Oligonucleotide sequences for primer and probe sets for rat cardiomyocytes.

<p>Oligonucleotide sequences for primer and probe sets for rat cardiomyocytes.</p

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).

<p>(<b>A</b>) 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>B</b>) Body weight, heart weight, and heart/body weight ratio (n = 30–57 animals in each group). (<b>C–F</b>) Morphometric analysis of myocardial tissue (n = 5–9 animals in each group). Shown are the fractional areas of connective tissue (<b>C</b>), cardiac fibroblasts (<b>D</b>), cardiac myocytes (<b>E</b>), and cardiomyocyte diameter (<b>F</b>). *<i>p</i><0.05 vs. WT, ^#<i>p</i><0.05 vs. untreated TGF-β₁ mice.</p

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

<p>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). *<i>p</i><0.05 vs. DCM.</p

Dobutamine stress echocardiography (DSE).

<p>(<b>A</b>) DSE protocol as applied in mice. (<b>B</b>) Representative m-mode registrations in a WT mouse at rest, and at various concentrations of dobutamine (Dobu). (<b>C</b>) 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. *<i>p</i><0.05 vs. rest; ^#<i>p</i><0.05 vs. WT.</p

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).

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

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).

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

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

<p>(<b>A</b>) 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>B</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. (<b>C</b> and <b>D</b>) Expression of UCP2 and UCP3 mRNA in the various treatment groups. (<b>E</b>) 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. (<b>F</b>) Genipin (5 µM) restored the contractile response to isoprenaline (10 µM) in isolated cardiac myocytes (n = 26–30 in each group).</p