3. Drug Treatment of CardiomyocytesStock solutions of dobutamine were prepared with normal media. Cells were treated with varying concentrations of dobutamine (0.01�C10��mol/L) for 4 hours, inhibitor Tubacin washed twice with PBS, and removed by trypsinization. The treated cells were then collected and subjected to a gene expression assay. In addition, pretreatment with various inhibitory agents (��1-adenocepotor antagonist (10��mol/L atenolol) [19, 20], ��2-adenocepotor antagonist (10��mol/L butoxamine) [21], calcium chelator (25mmol/L BAPTA-AM), calcineurin inhibitor (1��mol/L cyclosporine A) [22], or CaMK inhibitor (1��mol/L KN-93) [22]) was applied for 30 minutes before the addition of dobutamine. 2.4.

Western Blotting AnalysisProtein was extracted from tissue homogenates and cell lysates using ice-cold radio-immunoprecipitation assay (RIPA) buffer supplemented with phosphatase and protease inhibitors (50mmol/L sodium vanadate, 0.5mmol/L phenylmethylsulphonyl fluoride, 2mg/mL aprotinin, and 0.5mg/mL leupeptin). Protein concentrations were determined with the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Total protein (30��g) was separated by SDS/polyacrylamide gel electrophoresis (10% acrylamide gel) using the Bio-Rad Mini-Protein II system. Protein was transferred to expanded polyvinylidene difluoride membranes (Pierce, Rockford, IL, USA) with a Bio-Rad Trans-Blot system. After the transfer, the membranes were washed with PBS and blocked for 1 hour at room temperature with 5% (w/v) nonfat dry milk (NFDM) in PBS.

Blots were incubated overnight at 4��C with an immunoglobulin-G polyclonal rabbit anti-mouse antibody (Affinity BioReagents, Inc., Golden, CO, USA) diluted 1:500 in 5% (w/v) NFDM dissolved in PBS/Tween 20 (0.5% by volume). The blots were also incubated with goat polyclonal antibody (1:1000) targeted to actin, which served as an internal control. After the removal of the primary antibody, the blots were extensively washed with PBS/Tween 20. The blots were then incubated for 2hours at room temperature with the appropriate peroxidase-conjugated secondary antibody diluted in 5% (w/v) NFDM dissolved in PBS/Tween 20. The blots were developed by autoradiography using the ECL-Western blotting system (Amersham International, Buckinghamshire, UK). The immunoblots were quantified with a laser densitometer.2.5.

Measurement of Intracellular AV-951 Calcium ConcentrationThe changes in intracellular calcium were detected using the fluorescent probe Fura2-AM [23]. Primary cultured cardiomyocytes were placed in a buffered physiological saline solution containing 140mmol/L NaCl, 5.9mmol/L KCl, 1.2mmol/L CaCl2, 1.4mmol/L MgCl2, 11.5mmol/L glucose, 1.8mmol/L Na2HPO4, and 10mmol/L HEPES-Tris. A final concentration of 5��mol/L Fura-2AM was added to the cells which were incubated for 1 hour in humidified 5% CO2 and 95% air at 37��C.