Role of Endothelin in the Induction of Cardiac Hypertrophy In Vitro Tepmanas Bupha-Intr, Kaylan M. Haizlip, Paul M. L. Janssen* Department of Physiology and Cell Biology and D. Davis Heart Lung Research Institute, College of Medicine, The Ohio State University, Columbus, Ohio, United States of America Abstract Endothelin (ET-1) is a peptide hormone mediating a wide variety of biological processes and is associated with development of cardiac dysfunction. Generally, ET-1 is regarded as a molecular marker released only in correlation with the observation of a hypertrophic response or in conjunction with other hypertrophic stress. Although the cardiac hypertrophic effect of ET-1 is demonstrated, inotropic properties of cardiac muscle during chronic ET-1-induced hypertrophy remain largely unclear. Through the use of a novel in vitro multicellular culture system, changes in contractile force and kinetics of rabbit cardiac trabeculae in response to 1 nM ET-1 for 24 hours can be observed. Compared to the initial force at t = 0 hours, ET-1 treated muscles showed a ,2.5 fold increase in developed force after 24 hours without any effect on time to peak contraction or time to 90% relaxation. ET-1 increased muscle diameter by 12.563.2% from the initial size, due to increased cell width compared to non-ET-1 treated muscles. Using specific signaling antagonists, inhibition of NCX, CaMKII, MAPKK, and IP3 could attenuate the effect of ET-1 on increased developed force. However, among these inhibitions only IP3 receptor blocker could not prevent the increase muscle size by ET-1. Interestingly, though calcineurin-NFAT inhibition could not suppress the effect of ET-1 on force development, it did prevent muscle hypertrophy. These findings suggest that ET-1 provokes both inotropic and hypertrophic activations on myocardium in which both activations share the same signaling pathway through MAPK and CaMKII in associated with NCX activity. Citation: Bupha-Intr T, Haizlip KM, Janssen PML (2012) Role of Endothelin in the Induction of Cardiac Hypertrophy In Vitro. PLoS ONE 7(8): e43179. doi:10.1371/ journal.pone.0043179 Editor: Xiongwen Chen, Temple University, United States of America Received December 22, 2011; Accepted July 18, 2012; Published August 17, 2012 Copyright: 2012 Bupha-Intr et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Work supported by American Heart Association Established Investigator Award. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: janssen.10@osu.edu Introduction Cardiac hypertrophy is a form of myocyte remodeling that can be induced by both physiological and pathological stresses. Numerous studies have highlighted the effects of pressure overload and endogenous substances on the hypertrophic response of the heart. Among these substances, endothelin has been of interest for well over a decade, due to the association in stretch-induced inotropic and hypertrophic responses [1,2]. However, its mech- anism of action remains incompletely understood. Endothelin exists natively in three subtypes (ET-1, ET-2, and ET-3) with ET-1 produced in endothelium and myocytes. Endothelin-1 is a potent vasoconstricting agent and within the heart functions mainly as a positive inotrope, chronotrope, and stimulator of the renin- angiotensin-aldosterone system [3]. Inotropic and hypertrophic effects of ET-1 have been widely investigated on cardiomyocytes [4,5,6,7,8]. The mechanism of action of ET-1 on G-protein coupled receptors mainly activates phospholipase C which hydrolyzes phosphatidylinositol 4,5- biphosphate to diacylglycerol and inositol 1,4,5-trisphosphate (IP3) [9]. IP3 then activates an increased in intracellular Ca2+ levels, while diacylglycerol causes the translocation of protein kinase C (PKC) resulting in activation of the small G-protein Ras and consequently, the extracellular signal regulated kinase 1/2 (ERK1/2) cascade [10]. Along with the effects on the hypertrophic response, these messengers could also mediate the intracellular Ca2+ transients and myofilament Ca2+ sensitivity, subsequently, affecting contractility [9,11,12]. It however still remains unclear whether there is one specific signaling cascade or more than one that orchestrates the modulation of inotropic activity and induction of cardiomyocyte hypertrophy. In the present work, we demonstrate the effects of ET-1 on inotropic and hypertrophic responses using cultured rabbit trabeculae in the absence of systemic regulation and preload. Previous studies from our lab have shown the feasibility to induce hypertrophy via culturing muscles ex vivo at high preloads [13,14]. We use this system to further elucidate the mechanism of the ET-1 induced hypertrophic response and alterations in the inotropic response, with the working hypothesis that ERK1/2 activation is a major contributor to both responses. While the mechanism of action of ET-1 on cardiac hypertrophy still remains elusive, we were able to show that 1) the addition of ET-1 during the culture of intact muscle preparations in the absence of preload leads to an increase in the size and force production of that muscle over time, indicating a hypertrophic response, 2) Na+-Ca2+ exchanger, CaMKII, and MAPK are involved in both inotropic and hypertrophic effects of ET-1, and 3) there is only a weak association between ET-1 induced inotropic and hypertrophic response and ERK 1/2 activation. PLOS ONE | www.plosone.org 1 August 2012 | Volume 7 | Issue 8 | e43179