Materials and Methods The present study conforms to the NIH Guide for the care and Use of Laboratory Animals (NIH publication No.85-23, revised 1996). All of the animals handled and experiments conducted according to a protocol (2009A0174) approved by the review board of the animal care and use committee of The Ohio State University. Multicellular Myocardial Culture The cardiac trabeculae culture procedure has been detailed previously. Our lab and those of others have shown that these cultured multicellular preparations muscles remain stable in their protein expression/generation [15] and contractile function for up to 5 days [16]. Multicellular preparations can be used from various species [14,17], including human [16], and this system allows for functional protein product expression of virus-mediated gene transfer [18,19] and the observation of slow process such as load- induced changes in protein expression [13,20] or apoptosis [21,22]. Briefly, New Zealand White rabbits (1.5–2.0 kg) were heparinized and anesthetized by infusion of pentobarbital sodium (50 mg/kg) into the ear vein. After foot-pinch and eye-touch reflexes were absent, hearts were rapidly excised and retrogradely perfused in a modified Langendorff perfusion system with a BDM- containing low calcium Krebs-Henseleit solution. Non-branched, linear trabeculae from the free wall of the right ventricle were dissected and then mounted between a force transducer and a micromanipulator screw a semi-closed circuit culture system. The solution was exchanged with a normal Krebs-Henseleit solution (1.5 mM Ca2+) and the muscles were stimulated at 1 Hz at 37uC as previously described [14]. The muscles were then subjected to isometric contractions by stretching to a low passive tension of 2 around 1 mN/mm . Force and kinetics of contraction were continuously monitored for 25 hours. Endothelin-1 was added into the medium to a concentration of 1 nM at 60 minutes after contractions were initiated, whereas inhibitors or 0.1% DMSO (solvent control) were added 30 minutes prior to endothelin administration. Pharmacological Antagonists Several pharmacological agonists and antagonists were used to evaluate the signaling mechanism of endothelin-induced cardiac hypertrophy. For the Ca2+-dependent hypertrophic pathway, KB- R7943 (0.5 mM) and KN-93 (1.0 mM) were applied for the inhibition of reverse-mode Na2+-Ca2+ exchange and CAMKII, respectively [23,24]. Cyclosporine A (1.0 mM) was used to attenuate calcineurin activity [25], while INCA-6 (40 mM) prevented NFAT-calcineurin association [26]. GF109203X (3.0 mM) is a non-specific PKC inhibitor [27], and 2-APB (4.0 mM) is an IP3 receptor blocker [28]. For the MAPK pathway, PD98095 (10 mM) was utilized in order to inhibit MAPKK activation [29]. An Akt inhibitor (10 mM) was used to attenuate possible Akt-dependent cardiac hypertrophy [30]. Based on previous report that phosphodiesterase type 5 inhibition could attenuate cardiac hypertrophy through cGMP degradation, T- 0156 (0.1 mM), a potent phosphodiesterase type-5 inhibitor was applied [31]. Concentrations of these compounds used were at 10 to 100 times their Ki value. Since a single experiment takes up to 3 full days to set-up and collect and an additional 3–6 days to analyze, a dose-response curve for each inhibitor used would take 4–5 months per drug to conduct, and was deemed beyond the current capability and scope. In Vitro Studies of ET-1 Induced Hypertrophy Protein Electrophoresis of ERK1/2 Phosphorylation Using only the middle 2/3 of the trabeculae (to prevent including tissue from potentially damaged-ends), Immunoblot- analysis was performed using a standard protocol [14]. Anti- phosphorylated ERK 1/2 (1:1000) and anti-total ERK (1:2000) antibodies were obtained from Cell Signaling Technology. By randomized distribution of all samples over multiple gels, blot-to- blot variation impact on statistical analysis was minimized. Data analysis and Statistics Force and kinetics of contraction were recorded and calculated off-line using custom designed (LabView-based) program. Immu- noblot densitometry was calculated using the ImageJ 1.37 v program (NIH). Multiple group comparisons were performed using ANOVA followed by Bonferroni post-hoc analysis. Muscle hypertrophy was determined by a comparison of muscle diameters before and after 25 hours of culture using Student paired T-test as described previously [14]. Values are given as mean 6 SEM. A two-tailed P value of ,0.05 was considered to be statistically significant. Results Endothelin Affects Myocardial Contractile Performance and Hyperotrophy To evaluate whether ET-1 could induce a hypertrophic response, and/or functional change in contractile function in mammalian myocardium, cardiac trabeculae under low preload, were subjected to isometric contractions at 1-Hz electrical stimulation (37uC, pH 7.4) in the presence or absence of 1 nM ET-1 for up to 24 hours. Muscle diameters were compared before and after experiments to assess overall cardiac hypertrophy. We found that ET-1 induced a gradual increase in developed force throughout the 24-hour culture period (Figure 1A). Developed forces were significantly higher in ET-1 treated muscles than in control muscles (no ET-1) after three hours of incubation with ET- 1. After 24 hours, ET-1 treated muscles showed a 4.760.6 (P,0.05) fold increase in developed force (based on average individual change, overall the group-averaged increase was ,2.5 fold), while developed force increased only slightly (1.660.3 fold, P,0.05) in the control group. No change in time to peak force or in time to 90% relaxation was detected in either control muscles or ET-1 treated muscles (Figure 1B and C). Since the various contractile timing kinetics are linked [32,33], we did not analyze additional contractile parameters. Hypertrophic effects of ET-1 were revealed by a significant increase in muscle diameter and cell width (Figure 2). While control muscles demonstrated a minimal (3.562.1%) increase in muscle diameter compared to their initial size, the presence of ET- 1 significantly increased diameter by 12.563.2% (P,0.05). To confirm the hypertrophic effect of endothelin-1 at the myocyte level, and to rule out potential edema, cultured cardiac trabeculae were fixed and myocytes were isolated as previously described [14]. Compared to controls, myocytes from ET-1 treated muscles showed an increase in cell width by 16.8% compared to controls (Figure 2B). No significant difference in the length of myocytes between the two groups was observed (Figure 2C). This result indicates that increased muscle diameter by ET-1 was a result of myocyte hypertrophy, and not tissue edema. Hence, this first series of experiments demonstrated that ET-1 directly exerts inotropic effects on the myocardium in which gradual increases in force could be at least partially due to increased number of functional sarcomeres added in parallel in cardiomyocytes. PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43179