Myocardial expression of NHE1 was determined at protein level by immunoblot analysis. In order to avoid potential problems with differential recoveries of membranes from unused donor and recipient heart samples, immunoblot analysis was conducted using unfractionated tissue homogenates as described recently (32). Na+/Ca2+ exchange (NCE) expression was also determined as a positive control for the presence of sarcolemmal protein in the samples. Ventricular tissue samples (approximately 0.2 g) obtained from regions without overt signs of fibrosis or damage were rapidly thawed, weighed and homogenized for 3 to 4 min in lysis buffer (sorbitol [5%], histidine [pH 7.4; 25 mmol/L], Na2EDTA [50 mmol/L], KCl [50 mmol/L], leupeptin [1 μg/μL], PMSF [0.5 mmol/L] and benzamidine [1 mmol/L]). For NHE analysis, 0.5% SDS and 0.1% beta-mercaptoethanol were added to 25 μL of sample containing 100 μg of protein. After boiling for 5 min, 55 μL of lysis buffer and 5 μL of polyoxyethylene-8-lauryl ether (Sigma, Poole, United Kingdom) were added to the sample. After incubation at 37°C for 15 h, 50 μL of 3× SDS-sample buffer was added and the sample boiled for 10 min. For NCE analysis, SDS-sample buffer (×1) was added directly to an aliquot of tissue homogenate to obtain a final protein concentration of 2 μg/μL and the sample boiled for 10 min. After centrifugation, all samples (100 μg protein) were subjected to electrophoresis using a 7.5% SDS-polyacrylamide gel, and the separated proteins were transferred to polyvinylidene difluoride membranes. Immunoblot analysis was performed using mouse monoclonal antibody for NHE1 (1:500 dilution; #MAB3140, Chemicon International Inc., Harrow, United Kingdom) or NCE (1:500 dilution; #C2C12, Cambridge BioScience, United Kingdom) in conjunction with antimouse secondary antibody and enhanced chemiluminescence (Amersham Pharmacia Biotech, Little Chalfont, United Kingdom).