NBCe1 Na+-HCO3- cotransporter ablation causes reduced apoptosis following cardiac ischemia-reperfusion injury in vivo

Figure 1 Targeting strategy for generation of Slc4a4 cardiac myocyte conditional KO mice. The targeting construct contained the neomycin resistance gene (neo) to allow selection of ES cells after homologous recombination. The neo gene was flanked with flippase recognition target (FRT) sites, which allowed removal of neo when mice carrying the targeted allele were bred with transgenic mice expressing Flip recombinase. After confirming the deletion of neo, mice carrying the floxed allele were bred with mice expressing Cre recombinase controlled by the β-MHC promoter, which mediates the deletion of exon 12 of Slc4a4 in cardiac myocytes.

Figure 2 Polymerase chain reaction genotyping and quantitative reverse transcriptase-polymerase chain reaction of NBCe1 mRNA. A: PCR analysis of DNA from tail biopsies, using primers flanking the proximal LoxP site, showing bands for the alleles in Slc4a4^+/+, Slc4a4^flx/+ and Slc4a4^flx/flx mice. The larger size of the band for the floxed allele is due to the presence of the LoxP site; B: Quantitative RT-PCR analysis of cDNA prepared from whole heart RNA (n = 6 for each genotype) showing a reduction of Slc4a4 mRNA in hearts of KO (Slc4a4^flx/flx(Cre)) relative to WT (Slc4a4^flx/flx without Cre) mice. PCR: Polymerase chain reaction.

Figure 3 Cardiovascular performance in WT and KO mice. Intraventricular and intra-arterial pressure measurements were recorded using transducers in the left ventricle and right femoral artery of anesthetized 2-3 mo old WT (Slc4a4^flx/flx) and KO (Slc4a4^flx/flx(Cre)) mice under both basal conditions and in response to β-adrenergic stimulation (intravenous infusion of increasing doses of dobutamine). A: Heart rate; B: Systolic left ventricular pressure; C: Mean arterial pressure; D: Positive dP/dt in mm Hg/sec; E: Positive dP/dt at 40 mm Hg; F: Negative dP/dt in mm Hg/sec is shown for WT and KO mice. n = 8 WT (4 female, 4 male) and 8 KO (4 female, 4 male) mice.

Figure 4 TUNEL staining to quantify apoptosis in WT and KO heart sections after ischemia and reperfusion. Mice were subjected to 30 min of ischemia by occlusion of the LAD, followed by 3 h of reperfusion, and heart sections were processed to analyze apoptosis. A: Representative images from WT and KO heart sections, taken from a region approximately 1 mm distal to the occlusion site, showing nuclei stained with DAPI (blue), TUNEL (red) and sarcomeric actin (green). Yellow arrows point to apoptotic nuclei (pink); B: Quantification of apoptotic nuclei. n = 4 mice of each genotype, P ≤ 0.05.