In Vitro Studies of ET-1 Induced Hypertrophy Figure 5. Potential signaling mechanism of ET-1 induced muscle hypertrophy. Muscle diameter before and after culture was analyzed the percent change. Hypertrophy effect of ET-1 was inhibited by the inhibitors of NCX, MEK-1, CaMKII, NFAT-calcineurin association, and calcineurin. Values are mean 6 SEM from 5–8 muscles. *P,0.05 compared between before and after culture using paired t-test. doi:10.1371/journal.pone.0043179.g005 Therefore, ET-1 might not be the main signal activating the inotropic response following acute or chronic stretch, and the exact role of ET-1 alone would remain elusive. Because ET-1 secretion is often correlated with mechanical stretch, the effects of ET-1 alone, in absence of increased mechanical load, would for a large part explain the mechanistic signaling of endothelin on the heart. To determine if ET-1 by itself can produce an inotropic and/or hypertrophic response we studied the effects of ET-1 incubation in absence of changes in stretch. Although our study was not designed to fully elucidate the entire signaling pathway(s) involved, we briefly will discuss a few observations and our interpretation, and will leave the final interpretation of our results to the readership. Based on the specific inhibitors, our results suggest that the ET-1 mediated inotropic Table 1. Effect of specific antagonists on inotropic response, muscle hypertrophy, and ERK1/2 phosphorylation in 24-hr endothelin-1 treated trabeculae. Inotropic effect Hypertrophy phospho-ERK1/2 *GF109203X induced a significant increase in developed force within 1 hour of incubation. doi:10.1371/journal.pone.0043179.t001 PLOS ONE | www.plosone.org 6 August 2012 | Volume 7 | Issue 8 | e43179 Specific antagonists KB-R7943 NCX inhibitor KN-93 CaMKII inhibitor PD98095 MAPKK inhibitor Cyclosporin A Calcineurin inhibitor INCA6 Inhibiton of NFAT-calcineurin association 2-APB IP3 receptor blocker GF109203X Non-specific PKC inhibitor Akt inhibitor T-0156 Phosphodiesterase type-5 inhibitor Inhibit Inhibit Inhibit No Inhibit Inhibit Inhibit * No No Inhibit Inhibit Inhibit Inhibit Inhibit No No No No No No No No Inhibit Inhibit No Inhibit No