Mean data are presented throughout the text as stimulated Irel (current amplitude, normalized to the maximum KATP current per patch). Data analysis Data were analyzed using ClampFit and Microsoft Excel software. SUR1 suppress KATP channel HAS2 activity in cultured neonatal myocytes18 and atrial myocytes are more sensitive to the SUR1-specific KCO diazoxide19;20 than ventricular. SUR1?/? hearts show improved functional recovery after ischemia21, whereas extensive arrhythmias and sudden death are observed in mice overexpressing SUR1, but not SUR2A, in the heart22. In this study we have examined the regional expression of SUR1 and the functional properties of KATP channels, in wild type and SUR1?/? hearts. The striking results demonstrate that sarcolemmal KATP is markedly different in structure in the atria and ventricles, SUR1 being an essential component of the atrial channel but having no obvious role in the ventricle. These findings have significant implications for the pharmacology and patho-physiology of atrial versus ventricular KATP channels. Materials And Methods The generation of SUR1?/? and SUR1 TG mice are described elsewhere23;24. All procedures complied with the standards for the care and use of animal subjects as stated in the (NIH publication No. 85-23, revised 1996) and protocols were approved by the Animal Studies Committee at Washington University School of Medicine. Quantitative RT-PCR Expression of SUR1 and SUR2A mRNA was examined using real time RT-PCR23. Total RNA was isolated from cardiac atrial or ventricular tissue using TRIzol (Invitrogen) following manufacturers protocols. Isolated RNA was then treated with DNAseI to digest residual genomic DNA and further purified using a silica-based column protocol (Rneasy, Qiagen). [RNA] was determined spectrophotometrically (Nanodrop Technologies, Inc). Reverse transcription and PCR were carried out in a single tube in an ABI Prism 7000 sequence detection system (Applied Biosystems, Inc.). 75 ng of template RNA were used in all reactions. Following the RT reaction (45 min, 48C), 40 cycles of PCR were carried out. Double stranded DNA was fluorescently labeled with SYBR green (Applied Biosystems, Inc.). Gene-specific primers for SUR1, SUR2A and -actin Crovatin were designed using the PrimerExpress software (ABI) and purchased from Integrated DNA Technologies. Reactions with each primer pair and template were performed in duplicate. Following baseline correction, a fluorescence threshold was established and the cycle when this threshold was crossed (Ct) was determined for each reaction. To control for variability in RNA quantity, the normalized value, Ct, for each sample was calculated using the formula Ct = Ct(SUR) C Ct(actin). Relative mRNA expression is reported as 2?Ct * 1000. Generation of anti-SUR1 antibody Antibodies to SUR1 were raised and purified using standard protocols. Briefly, a peptide (CKDSVFASFVRADK) corresponding to the final 13 amino acids of the mouse SUR1 Crovatin subunit and containing an amino-terminal cysteine for downstream purification reactions was synthesized, and antibodies to the COOH-terminal peptide were raised in rabbits (Strategic Biosolutions) and affinity-purified against the peptide (Sulfolink, Pierce) following manufacturer protocols. Protein isolation and analysis Recombinant channel protein expressed in COSm6 cells was analyzed as described15. Hearts were excised and washed in ice cold PBS, and atrial appendages/ventricular tissue (or ventricular myocytes, isolated as below) were separated and placed in cardiac homogenization buffer (300 mM sucrose, Crovatin 10 mM Tris, pH 7.4) supplemented with protease inhibitors. Tissue Crovatin was disrupted using a Polytron homogenizer. Crude membrane extract was obtained by ultracentrifugation at 65,000 rpm for 60 minutes and pellet was resuspended in COS cell lysis solution (150 mM NaCl, 20 mM HEPES, 5 mM EDTA, 1% NP-40, pH 7.4) supplemented with protease inhibitors. Protein concentration was determined by the bicinchoninic method (Pierce). For Western analysis, proteins (typically 50 g) were separated by 7.5% PAGE and transferred to PVDF membrane. Antibodies were used at 1:1000 dilution, and signal was visualized with either the SuperSignal West Femto or Supersignal West Pico ECL substrates (Pierce). For immunohistochemistry, freshly isolated cardiomyocytes were attached to laminin-coated glass cover slips and fixed with 10% formalin for 15 minutes, followed by 100% methanol overnight at 4 C. Following fixation, cells were permeabilized with PBS.
