Dobutamine is the widely used cardiac agent for patients with heart failure. However, the theory is that the action trichostatin a mechanism of action of dobutamine occurs via the activation of PPAR�� remained obscure. In this study, we used the neonatal rat cardiomyocytes to investigate the role of PPAR�� in dobutamine-induced action. Moreover, we determined the possible signaling pathways for increase of PPAR�� induced by dobutamine.2. Methods2.1. MaterialsDobutamine, atenolol, butoxamine, and cyclosporine A were purchased from Sigma-Aldrich (St Louis, MO, USA). BAPTA-AM and KN93 were purchased from Calbiochem-Novabiochem Corp (La Jolla, CA, USA). The fluorescent probe, Fura2-AM, was obtained from Molecular Probes (Eugene, OR, USA). The Opti-MEM I Reduced Serum Medium, Stealth Select RNAi (siRNA-PPAR��), scramble siRNA (siRNA-control), and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA, USA).

Antibodies to PPAR�� and actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies to cardiac TnI and phospho-TnI (Ser 23/24) were purchased from Cell Signaling Technology (Beverly, MA, USA).2.2. Cell CulturePrimary cultures of neonatal rat cardiomyocytes were prepared by the modification of a previously described method [14]. Briefly, under anesthesia with 2% isoflurane, hearts of 1-to-2-day-old Wistar rats were excised, cut into 1-2mm pieces, and predigested with trypsin to remove red blood cells. The heart tissue was then digested with 0.25% trypsin and 0.05% collagenase. The dissociated cells were placed in uncoated 100mm dishes and incubated at 37��C in a 5% CO2 incubator for at least 1 hour to remove the nonmyocytic cells.

This procedure caused fibroblasts to predominantly attach to the dishes while most of the cardiomyocytes remained in suspension. The cardiomyocyte-enriched population was collected and counted. The cells were cultured in Dulbecco/Vogt modified Eagle’s minimal essential medium (DMEM) with 1mmol/L pyruvate, 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 units/mL streptomycin. Over 95% of the collected cells were characterized cardiomyocytes on the basis of the sarcomeric myosin content. On the second day, the medium was Entinostat replaced. After 3 to 4 days in culture, the cells were exposed to hyperglycemic conditions. The high glucose-treated cardiomyocytes were generated by applying 30mmol/L glucose to the cells for 24 hours [14]. This animal experiment was approved and conducted in accordance with local institutional guidelines for the care and use of laboratory animals in the Chi-Mei Medical Center (number 100052307) and followed the Guide for the Care and Use of Laboratory Animals published by the U.S.