Figure 6. Effect of ET-1 on ERK1/2 phosphorylation. A:Western blot analysis of ERK K phosphorylation (pERK) as compared to total ERK K (tERK) is increased upon treatment with 1 nM ET-1 in cultured trabeculae. B: Increases in the phospho-ERK1/2 expression in ET-1 treated muscles are augmented by PD98095, Akt inhibitor and 2-APB. In Vitro Studies of ET-1 Induced Hypertrophy Values are mean 6 SEM from 4–7 muscles in each group. *P,0.05 vs. control by student t-test. doi:10.1371/journal.pone.0043179.g006 response is dependent on NCX, CaMKII, IP3, and MAPK activity. Previous studies have determined NCX alone to regulate the stretch induced positive inotropic activity of the heart [40]. Additional studies have revealed that inhibition of MAPK activity accelerated the stretch-induced slow inotropic response and stretch lead to the phosphorylation of p38 and ERK [42]. In the absences of stretch, under ET-1 activation, the inhibition of MAPK depresses the inotropic response. In contrast, it has been suggested that p38 MAPK induces a negative inotropic effect via decreased myofilament Ca2+ sensitivity [43]. However, a direct effect of mechanical stretch on calcium related signaling cascades, CaMKII and IP3 activation, regarding inotropic activity of the heart has not been established. A previous study shows that direct application of IP3 increased the Ca2+ spark frequency of isolated ventricular myocyte in which IP3 inhibitor could abolish the acute effect of ET-1 on increasing the amplitude of intracellular Ca2+ transients [44]. CaMKII inhibition could also attenuate ET-1 increasing cardiac L-type Ca2+ current [45]. Therefore, if the mechanical stretch-activated inotropic response occurs mainly through ET-1, both CaMKII and IP3 activation should also be involved. The mechanism underlying ET-1 induced cardiac hypertrophy has been demonstrated for many years; however, studies generally reported only one specific signal at the time. Due to many potential ET-1 signaling cascades being activated, multiple pathways may be responsible for differing effects on the hypertrophic response. Inhibition of NCX, MAPK, CaMKII, and calcineurin-NFAT signals suggested a possible involvement of all these pathways together. Based on the ERK1/2 results, one could speculate that ET-1 might initially activate ERK1/2 phosphorylation. As previously reported, phosphorylated ERK1/ 2 increased Na+-H+ exchange activity [46], potential increased intracellular Na+ might induce reverse NCX activation [47]. Consequently, increased NCX leads to increased intracellular Ca2+ which then activates both CaMKII and calcineurin-NFAT signaling cascades [48]. Between the two signaling pathways, only CaMKII activation can possibly induce an increase in intracellular Ca2+ transients [45] and cardiac hypertrophy [49]. However, previous reports on the negative effect of CaMKII on calcineurin dependent NFAT-nuclear translocation [50] suggest that the physiological role of CaMKII likely regulates calcineurin-NFAT signaling activity. Though the entire complex mechanism still remains unclear, we have furthered our knowledge of potential ET-1 pathways through the use of multiple calcium and MAPK inhibitors, but due to limitations of specificity of antagonists readily available we are currently unable to fully determine the effects of each individual step that eventually lead to hypertrophy. Our data does provide critical novel information in that presence of ET-1 alone (without stretch) leads to an increase in developed force produced in our in vitro multicellular model system, where these observations were made in absence of systemic regulation while under controlled mechanical loading conditions. In order to pinpoint the key components in the endothelin-MAPK pathway, future directions would likely need to be focused the effects of inhibition at different points in the pathway. In addition, quantification of the magnitude of responsibility for the different calcium-dependent pathways involved, via dose-dependent test and the inhibition of multiple pathways at a time, would further elucidate the endothelin- induced hypertrophic response. PLOS ONE | www.plosone.org 7 August 2012 | Volume 7 | Issue 8 | e43179