In Vitro Studies of ET-1 Induced Hypertrophy Figure 3. Effect of signaling inhibitors on developed force before and after ET-1 treatment. No significant difference in developed force at start among group as well as 30 min later before inhibitors was added (A, B). After 30 min incubation with GF109203X, significant increase in developed force was observed (C). Significant increases in developed force by ET-1 after 24 hours treatment was observed by the inhibitors of NCX, MEK-1, CaMKII, NFAT-calcineurin association, PKC, and IP3 receptors (D). Values are mean 6 SEM from 5–8 muscles in each group. *P,0.05 vs. control by ANOVA. doi:10.1371/journal.pone.0043179.g003 Endothelin Phosphorylates ERK1/2 The MAPK pathway has been established as being involved in the inotropic effects of ET-1 as well as in the induction of cardiac hypertrophy. Previously, it has been suggested that ET-1 stimulated MAPK activation occurred through ERK1/2 signaling [25]. To investigate whether this was occurring in our model, and if it played a role in the hypertrophic response induced by endothelin-1, we first measured phosphorylation of ERK1/2 at 24 hours in culture after ET-1 treatment. We observed that indeed an increase in phosphorylated ERK1/2 levels after exposure with ET-1 occurred (Figure 6A). We then measured the phosphoryla- tion level of ERK1/2, using specific MAPK signaling and calcium signaling antagonists, to determine whether hypertrophy was stimulated through the MAPK pathway. With no clear target of interest, PD98095, a MEK-1 inhibitor, led to the inhibition of the ET-1 induced increase in phospho-ERK1/2. Interestingly, 2APB (an non-specific IP3 inhibitor) and Akt also prevented the ERK1/ 2 phosphorylation, while the remaining inhibitors tested had no effect. This data, taken in combination with the observed increases in muscle diameter, suggests that ET-1-stimulated cardiac hypertrophy is not specifically related to ERK1/2 activation. Discussion The results of the present study show the effects of endothelin-1 on intact isolated muscle preparations in an in vitro culture system using near-physiologically conditions. This study is the first to show an inotropic effect of ET-1, in absence of stretch (preload), on intact muscles developing over time, distinctly different from the acute inotropic effect that is well known and arises in mere minutes [36]. Furthermore, in addition we showed that ET-1 caused an increase in myocyte width, and resulted in an overall increase in muscle mass, indicative of hypertrophy. These observations were made in a multicellular preparation, containing cardiomyocytes, fibroblasts, and endothelial cells. In this system, muscles have the ability to react to biochemical and mechanical stimuli and can remodel and adapt contractile function in a 24–48 hour time span [13,15,16,17,18,20,21,37]. Thus, results from this model may differ from other in vitro models in which isolated PLOS ONE | www.plosone.org 4 August 2012 | Volume 7 | Issue 8 | e43179