For tissue collection, the fresh hearts were quickly removed and perfused for 1 to 2 min with cold modified Krebs-Henseleit solution (200 mM sucrose, 200 mM Tris-HCL, 0.4 mM CaCl, pH 2
7.0). After removing fat and connective tissues, heart was snap frozen in liquid nitrogen and stored at -80 ?C. To obtain protein extracts, the left ventricle was ground in liquid nitrogen and homogenized for 15 sec twice in lysis buffer (9 M urea, 2% CHAPS, 0.5% DTT, Protease inhibitor cocktail and 0.14% PMSF) at the maximal speed. The resulting homogenate was centrifuged for 10 min at 10000 g, supernatant was collected and treated with 2-D Cleanup Kit (Amersham Biosciences). Protein concentration of sample was measured using 2-D Quant Kit (Amersham Biosciences).