RT-qPCR was used to measure the NHE1 mRNA expression levels. Briefly, the hearts were harvested following reperfusion, total RNA was extracted from the left ventricular heart tissues using TRIzol reagent and the RNA purity was quantified spectrophotometrically at a ratio of 260 to 280 nm. cDNA was obtained from 1 µg total RNA using the AMV First Strand cDNA Synthesis kit according to the manufacturer's instructions (New England Biolabs Inc., Ipswich, MA, USA). Quantitative (real-time) PCR (qPCR) was performed using iQ SYBR-Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The NHE1 and GAPDH (internal control) primer sequences were defined as follows: NHE1 forward, 5′-GCGGCGAGCAGATCAATAA-3′ and reverse, 5′-ACAGTGACGGCATCGTTGAG-3′; and GAPDH forward, 5′-CAAGTTCAACGGCACAGTCAA-3′ and reverse, 5′-CGCCAGTAGACTCCACGACA-3′. The reaction conditions were as follows: 10 sec at 95°C, 40 sec at 60°C and 45 sec at 72°C for 40 cycles. Amplification of the products was followed by melting curve analysis using Applied Biosystems 7500 system software, as previously described (28 (link)) (Life Technologies, Grand Island, NY, USA). The value of NHE1 mRNA was expressed relative to that of GAPDH from the same sample.