Manuscript R-00559-2005/R1 Inverse Regulation of Preproendothelin-1 and Endothelin-Converting Enzyme-1β Genes in Cardiac Cells by Mechanical Load

Mechanical stretch and paraand/or autocrine factors, including endothelin-1, induce hypertrophy of cardiac myocytes and proliferation of fibroblasts. To investigate the effect of mechanical load on endothelin-1 production and on endothelin system gene expression in neonatal rat ventricular myocytes and fibroblasts, we exposed cells to cyclic mechanical stretch in vitro (0.5 Hz, 10% to 25% elongation, from 1 min to 24 hours). Endothelin-1 peptide levels were measured from culture media of myocytes and fibroblasts and human umbilical vein endothelial cells (positive control) by specific radioimmunoassay. Preproendothelin-1 promoter activity was measured by using transfection of reporter plasmids and mRNA levels with Northern blot or quantitative RT-PCR. Activity of extracellular signal regulated kinase was quantified with specific kinase assay. We found that stretching of myocytes activated preproendothelin-1 gene expression including promoter activation, transient increase in mRNA levels and augmented endothelin-1 secretion. In contrast, preproendothelin-1 gene expression was inhibited in stretched fibroblasts. Endothelin-converting enzyme-1β mRNA levels elevated in stretched fibroblasts, but decreased in stretched myocytes. Endothelin receptor type-A mRNA levels declined in stretched myocytes, while levels were below detection in fibroblasts. Stretch activated extracellular signal regulated kinase in myocytes, and when the kinase activity was pharmacologically inhibited, the preproendothelin-1 induction was suppressed. Transient overexpression of mitogenactivated ERK activating kinase -1 induced preproendothelin-1 promoter in myocytes. In summary, mechanical stretch distinctly regulates endothelin system gene expression in cardiac myocytes and fibroblasts. The inhibition of endothelin system may affect on cardiac mechanotransduction and therefore provides an approach in treatment of loadinduced cardiac pathology. endothelins; gene regulation; protein kinases; mechanotransduction _________________________________________________________ EMERGING evidence has indicated that a complex pattern of humoral and autocrine/paracrine factors, including endothelin-1 (ET-1) and angiotensin II, participate in the pathophysiology of cardiovascular diseases (1). Plasma levels of ET-1 precursor peptide, big ET-1, have been shown to increase in patients with cardiac hypertrophy and the levels correlate with the clinical severity of the heart failure (2). Beside the potential role as a humoral factor, more than 80% of ET-1 is secreted to basolateral compartment by endothelial cells (3), and ET-1 may act as an important local regulator of vascular tone as well as cardiac growth and function. Pressure overload increases the levels of preproendothelin-1 (ppET-1) mRNA in rat hypertrophied myocardium (4). Moreover, both in rats with chronic heart failure due to myocardial infarction (5) and in dogs with tachycardia-induced heart failure (6), cardiac ET-1 peptide and ppET-1 mRNA levels increase. ET-1 treatment of cultured cardiac myocytes increases myocyte size accompanied by sarcomere organization and activated protein synthesis and transcription of hypertrophy associated genes (7; 8). In the nonmyocyte pool of cardiac cells, ET-1 stimulates the proliferation of cardiac fibroblasts (9). In the deoxycorticosterone acetate-salt fed rats with hypertension and activated ET-1system, inhibition of ET-1 system reduces development of cardiac fibrosis (10). Similarly, chronic treatment with endothelin receptor antagonist bosentan reduces myocardial fibrosis in rats with heart failure due to myocardial infarction (11). Collectively, ET-1 may regulate both myocyte growth and proliferation of fibroblasts in the development of cardiac hypertrophy. The mechanisms that control ET-1 system are of fundamental interest in the pathophysiology of the heart failure. To directly examine the effect of mechanical loading on ET-1 system, we used an in vitro model of mechanical load. We studied which cardiac cells respond to load by increasing ET-1 production. Our results suggest that the effect of mechanical stretch on ET-1 system is cell-type specific: ET-1 generation is activated in myocytes and decreased in fibroblasts. Interestingly, mechanical stretch induces opposite changes in mRNA levels of endothelin-converting enzyme-1β (ECE-1β) and ppET-1. Our results further demonstrate that activation of ppET-1 gene transcription in stretched ventricular myocytes is mediated via extracellular signal regulated kinase (ERK). MATERIALS AND METHODS Plasmids. Plasmid for Rous Sarcoma virus-promoter linked to β-galactosidase gene (RSV-βgal) was purchased from Clontech (Palo Alto, CA). pGL3 basic vector driven by 1.3 kb fragment of rat ppET-1 promoter (ppET-1-luc) has been described previously (12). Plasmid encoding wild type mitogen-activated ERK activating kinase-1 (pCMV-MEK1) was a kind gift from Dr K.L. Guan (University of Michigan, Ann Arbor, USA) and pMT2 plasmid was generously provided by Dr. D.B. Wilson (Boston Children's Hospital, Massachusetts, USA). Cell Culture and Transfection. Cell cultures of neonatal rat ventricular myocytes and fibroblasts and human umbilical vein endothelial cells (HUVEC) were prepared and transfected as previously desribed (7; 13). The Ethical and The Animal Use and Care Committees of the University of Oulu approved the experimental design. Briefly, after digestion of ventricular tissue with collagenase (2 mg/ml), cell suspension was pre-plated for 30 to 45 minutes, and the attached cells were further cultured for two passages (divided at a ratio of 1:5 per passage) in the presence of 10% fetal bovine serum (FBS) to ensure proliferation of fibroblasts over other cell types. The non-attached cells or myocyte-enriched cells (MC), were plated at the density of 2 x 10 /cm on cell culture plates (Falcon) or on flexible bottomed collagen I-coated elastomere plates (Bioflex, Flexcell Int.) for stretch experiments, and cultured overnight with DMEM/F12 medium containing 10% of FBS, and thereafter in complete serum free medium (CSFM). Fibroblasts were propagated in 10% FBS containing DMEM/F12 medium until 24 hours prior experiment when medium was replaced with CSFM. HUVEC culture was prepared as previously described (13). To examine the presence of endothelial cells, cells were stained for an endothelial cell marker with an antibody raised against von Willebrandt factor (vWF) (14). Second passage of fibroblasts and primary MC cultures stained negative for vWF, while more than 95% of HUVECs stained positive (data not shown). Transfection of cells (if designated) was performed on the second day in culture. Cells were exposed to 1,5 μl of FuGENE 6 and 0,75 μg of DNA (0,5 μg of Luc, 0.25 μg of βgal plasmids) per ml of CSFM for 6 hours and cultured thereafter in CSFM. For cotransfection experiments (15), cells were grown on 24-well plates and transfected with 0.5 μg ppET-1-luc plasmid and 0.025 μg pCMV-MEK1 or empty control plasmid (pMT2) with 0.25 μg RSV β-gal and 1.5 μl FuGENE 6 per ml. Reporter gene activities were measured by using luciferase and β-galactosidase (to correct transfection efficiency) assays (Promega) with luminometer (7). Cells on Bioflex plates were exposed to cyclic mechanical stretch. Frequency of cyclic stretch was 0.5 Hz with pulsation of 10% to 25% elongation of cells from 1 min to 24 hours. Cells were stretched by applying a cyclic vacuum suction under Bioflex plates by computer controlled equipment (FX-3000, Flexcell Int.). This in vitro model of mechanical load leads to hypertrophic phenotype of cardiac myocytes within 24 to 48 hours (activation of hypertrophy-associated genes, reorganization of sarcomeric proteins) (16). mRNA Analysis and Radioimmunoassay. RNA was isolated from cells by the guanidine thiocyanate-CsCl method (17). Northern blots with fibroblast RNA were hybridized with specific cDNA probes for rat ET-1 and rat ribosomal 18S labeled with [P]dCTP (Amersham-Pharmacia) with a T7 Quick Prime Kit (Amersham-Pharmacia). ET-1 levels of culture medium were measured with radioimmunoassay for ET-1 (18). In addition, c-Fos, ECE-1β, endothelin receptor type-A (ETA), ET-1 mRNA and 18S RNA levels were measured by real-time quantitative reverse transcription–polymerase chain reaction analysis (see Supplemental Table 1.), using Taqman chemistry on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) as described (19). ERK Activity Assay. Activity of ERK was measured as described previously (20). Briefly, cells were sonicated and supernatant was collected after centrifugation. 15 μl of protein extract was incubated at 30 °C for 15 minutes with 10 μl substrate buffer containing specific ERK-substrate peptide in the presence of 1 μCi [γ-P] ATP. Terminated reaction was blotted on peptide binding paper discs, which were washed with 75 mM orthophosphoric acid repeatedly. Incorporated radioactivity was measured with scintillation counter (Rackbeta II, LKB Wallac). Fibroblasts were lysed and subsequently samples (6.8 μg of protein/lane) were subjected to western blot measurement of sitespecific phosphorylation of ERK (Phospho-p44/42 MAP Kinase (Thr202/Tyr204) Antibody, New England Biolabs) and total levels of ERK (p44/42 MAP Kinase Antibody, New England Biolabs) as described in detail earlier (20). Statistical Analysis. Results are expressed as mean ± SEM. To determine the statistical significance between two groups and for analysis of multiple groups, Student's t-test and one-way ANOVA followed by Bonferroni post hoc test were used, respectively. Differences at the 95 % level were considered statistically significant.