In Vitro Studies of ET-1 Induced Hypertrophy Figure 4. Temporal resolution during 24 hours of impact of signaling inhibitors on developed force. Values are mean 6 SEM from 5–8 muscles. The control muscles and the ET-1 treated muscles are included for each inhibitor for direct comparison. Concentration of inhibitor (all in presence of ET-1) and number of experiments are indicated in each panel. Data was analyzed in one ANOVA test. doi:10.1371/journal.pone.0043179.g004 cardiomyocytes alone are investigated, or from whole-animal models where systemic regulation is present. Lastly, in an initial attempt to start unraveling the underlying molecular mechanism, we studied potentially involved pathways. Using specific inhibitors, ET-1 activation of the cardiac inotropic response via the activation of MAPK, CAMKII, NCX, and IP3 was determined. In addition, ET-1-induced myocardial hypertrophy is involved in the activa- tion of MAPK, CAMKII, NCX, and calcineurin-NFAT pathway. Although some signaling pathways cross-talk and are involved in both inotropic and hypertrophic responses, we showed for the first time that the effects of ET-1 are independent of each other, yet governed, in part, by similar mechanisms with no obvious dependence on the phosphorylation state of ERK1/2. Previous studies have shown a correlation between mechanical stress and endothelin release, however, the isolated effects of endothelin on induction of hypertrophy have, to our knowledge, never been observed in a non load-bearing, contracting prepara- tion. There are many endogenous regulators of the hypertrophic response and we have devised a system that allows us to remove these contributors, and look more closely at the mechanism involved with endothelin-induced ventricular hypertrophy. Studies conducted on neonatal cardiomyocytes and rat myocytes have shown that stretch leads to the release of endothelin and hypertrophy [38,39]. However, the mechanism of action of endothelin release in adult myocardium and in a more physiolog- ical model remains unclear. The stretch activated slow inotropic (second phase) response mediated by increased intracellular Ca2+ is still being debated as to whether it occurs dependent or independent of ET-1 [40,41]. Moreover, our previous study showed that neither an ETA receptor antagonist nor a non- selective ET receptor inhibited the increase in developed force after 24-hour of high-load in cultured rabbit trabeculae [13]. PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43179