In Vitro Studies of ET-1 Induced Hypertrophy Figure 1. Cultured cardiac trabeculae continuously electrically stimulated to twitch contract at 1 Hz for 25 hours at a preload on only 1 mN/mm2 in presence (ET-1, n = 17) and absence (Control, n = 14) of 1 nM ET-1. Compared to control, in ET-1-treated muscles an enhanced active developed force was demonstrated (A), whereas time to peak force (B) and 90% of relaxation time (RT90, C) were similar between two groups. doi:10.1371/journal.pone.0043179.g001 Endothelin Induces Cardiac Inotropy It has been argued that Ca2+-signal-dependent cardiac hyper- trophy is due to increased diastolic Ca2+ level or altered Ca2+ cycling [34,35]. To test whether an ET-1 induced long-term inotropic response is associated with enhanced Ca2+ cycling or enhanced MAPK signaling pathway, various signaling inhibitors were applied to the cultured cardiac trabeculae. Within half an hour before ET-1 was applied, no significant effect of inhibitors on developed force was observed except in the muscle treated with 3.0 mM GF109203X, a non-specific PKC inhibitor (Figure 3B). Figure 3D shows the response to inhibitor after 24 hours. At 25 hours in culture; KB-R7943, PD98095, KN-93, INCA6, and 2- APB inhibited the ET-1-induced increase in force of contraction. Although no significant difference in developed force between ET- 1 treated muscles and ET-1+ GF109203X treated muscles was observed after 24 hours, slight increases in developed force from the beginning indicated that the PKC inhibitor potentially attenuates the inotropic effect of ET-1 (Figure 4G). Interestingly, the presence of the inhibitor of NFAT-calcineurin association (INCA) inhibited ET-1 induced increases in developed force in a biphasic fashion. Prior to 10 hours incubation with ET-1 there was no increase in developed force. After 10 hours incubation with 40 mM INCA-6 there was a significant decline in developed force potentially due to a delayed response to downstream inhibition (Figure 4E). The use of cyclosporin A (a direct calcineurin inhibitor) produce a slightly decreased developed force with no blunting of the ET-1 inotropic effect (Figure 4F). This result likely implies that there exists two mechanistic phases underlying an increase in developed force after ET-1 stimulation. In contrast, cyclosporine A, which is proposed to inhibit calcineurin activation [25], could not prevent the inotropic effect of ET-1 suggesting an enhanced response to further downstream targeting of the calcineurin pathway. As expected, Akt inhibition and PDE-5 inhibition did not interfere with the inotropic effect of ET-1. Endothelin Signals Induce Cardiac Hypertrophy Increases in the diameter of a muscle after 24 hours in culture are a direct parameter determining cardiac hypertrophy (Figure 1). To determine the signaling pathway involved in ET-1 activated hypertrophic response, muscle diameters were recorded after 24 hours in culture for all treatment groups (Figure 5). There was no change in muscle diameter before and after culture in ET-1 treated plus KB-R7943, PD98095, KN-93, INCA6, or cyclospor- ine A (paired T-test). In contrast, GF109203X and 2-APB, which inhibit the inotropic effect of ET-1, could not attenuate the hypertrophic component of the response. Also, no effects regarding hypertrophy from Akt inhibition and PDE-5 inhibition on ET-1 induced hypertrophy were observed. Results from each set of experiments reveal a general correlation between increased developed force and muscle hypertrophy (Table 1). Figure 2. Endothelin-1-induced cardiac hypertrophy. A: % changes in muscle diameter showed a significant increase in ET-1 treated muscle (n = 11), but not in control group (n = 5) by pair’s t-test. Significantly increase in myocyte width (B) but not in myocyte length (C) confirmed hypertrophic effect of ET-1. Values are mean 6 SEM from 3 muscles in each group. *P,0.05 vs. control by student t-test. doi:10.1371/journal.pone.0043179.g002 PLOS ONE | www.plosone.org 3 August 2012 | Volume 7 | Issue 8 | e43179