Original article
Endothelin-1 upregulation mediates aging-related cardiac fibrosis

https://doi.org/10.1016/j.yjmcc.2015.01.001Get rights and content

Highlights

  • Endothelin-1 (ET-1) is upregulated in senescent cardiac fibroblasts and aged hearts.

  • Fibrotic signals were increased in senescent cardiac fibroblasts and aged hearts.

  • Inhibition of ET-1 suppresses expression of fibrotic signals in the senescent fibroblasts.

Abstract

Endothelin-1 (ET-1) plays a major role in regulating myocardial fibrosis in several pathological conditions, such as hypertension and diabetes. Aging is an independent risk factor for myocardial fibrosis. We hypothesized that ET-1 upregulation may be a basis of enhanced collagen synthesis in the senescent fibroblasts resulting in cardiac fibrosis with aging. To examine this hypothesis, we cultured mouse cardiac fibroblasts to passage-30 (P30). β–Galactosidase activity and several other aging markers were markedly increased in P30 (vs. P3) fibroblasts, indicating that these cells were indeed undergoing senescence. Importantly, ET-1 expression was markedly upregulated in P30 (vs. P3) fibroblasts. Of note, estrogen receptor-α (ER-α), an important negative regulator of ET-1, was downregulated in P30 fibroblasts. We also studied aged (130-weeks old, female) mice hearts, and observed that ET-1 was upregulated and ER-α was downregulated in these hearts (vs. 6-week old mice hearts, female). Similar observations were made in the fibroblasts isolated from aged mice hearts. ET-1 upregulation with aging was also seen in ≈ 70-year old (vs. ≈ 30-year old) human heart sections. In concert with ET-1 upregulation, the expression of fibronectin and collagens was found to be markedly increased in P30 cardiac fibroblasts in culture, fibroblasts isolated from the aged mice hearts, and in aged human hearts. Interestingly, inhibition of ET-1 in the senescent P30 fibroblasts by 2 different strategies (the use of siRNA and the use of endothelin converting enzyme inhibitors) markedly suppressed expression of fibrosis signals. Further, treatment with synthetic ET-1 enhanced fibronectin and collagen expression in P3 cardiac fibroblasts. These observations in mice and human hearts suggest that aging-related cardiac fibrosis is, at least partially, dependent on the upregulation of ET-1.

Introduction

Heart failure is a major problem affecting a large number of patients around the world [1], [2]. Excessive fibrosis, a hallmark of late stage heart failure, occurs in longstanding hypertension and myocardial ischemia. Prevalence of heart failure increases with age, and aging itself is an independent risk factor for cardiac fibrosis [3], [4]. Many strides have been made in understanding etiologic basis of fibrosis. The aging-related changes in the aged human heart include myocardial hypertrophy, fibrosis and diastolic dysfunction [5]. Aging-dependent collagen accumulation in heart would be expected to lead to a progressive increase in ventricular stiffness and impaired diastolic function [1].

Endothelin-1 (ET-1) is a potent vasoconstrictor peptide that is mainly produced by vascular endothelial cells in response to hypoxia, oxidized LDL, Ang II, pro-inflammatory cytokines, and bacterial toxins [6]. Activation of estrogen receptor-α (ER-α), a nuclear receptor for estrogen, has been shown to negatively regulate ET-1 expression [7], [8]. ET-1 is also produced by other cell types, such as smooth muscle cells, epithelial cells, cardiomyocytes, fibroblasts and certain cancer cells [6], [9], [10]. Mature ET-1 is formed from pre-pro-ET-1 via a 39-amino acid intermediate, big ET-1 [6], [11]; this process requires several enzymes, including the endothelin converting enzymes (ECEs) [6]. Fibroblasts from different tissues can generate significant amounts of ET-1 [12], [13], [14], [15], and the exposure of fibroblasts to ET-1 in turn stimulates collagen synthesis [16]. Excessive production of ET-1 has been incriminated in the development and progression of pulmonary and systemic hypertension and other cardiovascular disorders such as atherosclerosis [17], [18]. It is of note that ET-1 secretion has been shown to promote fibroblast proliferation and cardiac fibrosis in pathological states, such as diabetes and hypertension [18], [19]; however, the role of ET-1 in aging-related heart fibrosis is still not clear. In our preliminary study, we observed ET-1 expression to be hugely increased in senescent cardiac fibroblasts and aged mice hearts. So, we hypothesized that excess ET-1 expression is a major component of the pathogenesis of cardiac fibrosis with aging. This study was designed to address hypothesis.

To address our hypothesis, we established senescent cardiac fibroblasts by serial passages. We assessed ET-1 expression and fibrosis signals in young (passage-3, P3) and senescent (passage-30, P30) fibroblasts, and thereafter in the aged (130-week old) mice hearts and in the fibroblasts isolated from aged mice hearts. To further confirm the observation of upregulated ET-1 expression in the mice hearts and senescent fibroblasts, we evaluated the expression of ET-1 and collagens in the aged human hearts. Lastly, we studied the role of ET-1 upregulation in fibrogenesis with the use of ET-1 inhibition and over-expression strategies.

Section snippets

Cardiac fibroblast culture

Ten-week-old C57BL/6 female mice were anesthetized with sodium pentobarbital (80 mg/kg, i.p), and their hearts quickly removed and used for isolation of cardiac fibroblasts. Isolation and culture of mice cardiac fibroblasts were performed as described [2]. The cells were cultured for up to passage-30 (P30). Experiments described subsequently in cultured fibroblasts were mainly performed in passage-3 (P3) and P30 cardiac fibroblasts.

Collection of heart tissue from mice and humans

Hearts were collected from young (6-week-old) and old

SA-β-Gal activity in P3 and P30 cardiac fibroblasts

Senescence-associated beta-galactosidase (SA-β-Gal) activity is a well-known indicator of aging [20]. As shown in Fig. 1A, the SA-β-Gal activity was markedly increased in P30 cardiac fibroblasts as compared with that in P3 cells. SA-β-Gal activity was similarly increased in the old mice hearts (Fig. 1B). The increased SA-β-Gal activity and other aging maker such as FOXO1A, Klotho, β-catenin and AT1R (shown in Suppl Fig. 1) indicated that P30 fibroblasts were indeed undergoing senescence.

Expression of ET-1 and ER-α in senescent cardiac fibroblasts

Discussion

The precise basis of aging-related cardiac fibrosis largely remains undefined. We designed this study to determine the role of ET-1 in this malady. We studied this phenomenon in senescent fibroblasts in culture and in aged hearts. We observed that the expression of fibrosis signals, such as fibronectin, increased several-fold as cardiac fibroblasts went through senescence. Most importantly, expression of ET-1 was markedly increased in senescent cardiac fibroblasts in culture as well as in the

Disclosures

No conflicts to disclose.

Acknowledgments

This study was supported by funds from the Biomedical Laboratory Research and Development, VA Office of Research and Development, (BX-000282-05), Washington, DC. Additional support was provided by the National Natural Science Foundation of China (No. 81370428). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.

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