Postischemic cardiac recovery in heme oxygenase-1 transgenic ischemic/reperfused mouse myocardium

Abstract

Heme oxygenase-1 (HO-1) transgenic mice (Tg) were created using a rat HO-1 genomic transgene. Transgene expression was detected by RT-PCR and Western blots in the left ventricle (LV), right ventricle (RV) and septum (S) in mouse hearts, and its function was demonstrated by the elevated HO enzyme activity. Tg and non-transgenic (NTg) mouse hearts were isolated and subjected to ischemia/reperfusion. Significant post-ischemic recovery in coronary flow (CF), aortic flow (AF), aortic pressure (AOP) and first derivative of AOP (AOPdp/dt) were detected in the HO-1 Tg group compared to the NTg values. In HO-1 Tg hearts treated with 50 μmol/kg of tin protoporphyrin IX (SnPPIX), an HO enzyme inhibitor, abolished the post-ischemic cardiac recovery. HO-1 related carbon monoxide (CO) production was detected in NTg, HO-1 Tg and HO-1 Tg + SnPPIX treated groups, and a substantial increase in CO production was observed in the HO-1 Tg hearts subjected to ischemia/reperfusion. Moreover, in ischemia/reperfusion-induced tissue Na(+) and Ca(2+) gains were reduced in HO-1 Tg group in comparison with the NTg and HO-1 Tg + SnPPIX treated groups; furthermore K(+) loss was reduced in the HO-1 Tg group. The infarct size was markedly reduced from its NTg control value of 37 ± 4% to 20 ± 6% (P < 0.05) in the HO-1 Tg group, and was increased to 47 ± 5% (P < 0.05) in the HO-1 knockout (KO) hearts. Parallel to the infarct size reduction, the incidence of total and sustained ventricular fibrillation were also reduced from their NTg control values of 92% and 83% to 25% (P < 0.05) and 8% (P < 0.05) in the HO-1 Tg group, and were increased to 100% and 100% in HO-1 KO(-/-) hearts. Immunohistochemical staining of HO-1 was intensified in HO-1 Tg compared to the NTg myocardium. Thus, the HO-1 Tg mouse model suggests a valuable therapeutic approach in the treatment of ischemic myocardium.

Fig 1

Schematic presentation of the experimental time course.

Fig 2

The detection of rat HO-1 transgene by PCR (A) and Western blot (B) and HO enzyme activities (C) in mouse hearts. Total RNA obtained from mouse LV, RV and S, respectively, was used to synthesize first strand cDNA. HO-1 originated from rat was amplified by PCR exon 5-specific primer and visualized with ethidium bromide as a 166-bp band by agarose gel electrophoresis. In two clones, the specific band was amplified from RNA samples of RV, LV and S, respectively, in transgene (transgene 1 and transgene 2) mice (A, upper and middle parts), but it was absent in the RNA sample of NTg littermate (A, lower part). Western blots (B) also depict the expression of rat transgene 1, transgene 2 and NTg in the LV, RV and S of mouse hearts. HO enzyme activities are also shown in the LV, RV and S in NTg and Tg myocardium (C). n = 6 in each group, mean ± S.E.M., *P < 0.05, comparisons were made to the NTg values in each group.

Fig 3

Representative GC chromatograms for the demonstration of endogenous CO production in NTg [chromatogram (a)], HO-1 Tg [chromatogram (b)] and HO-1 Tg mouse myocardium treated with SnPPIX [HO-1 Tg + SnPPIX, chromatogram (c)]. Hearts were subjected to 20 min. of normothermic global ischemia followed by 120 min. of reperfusion. The results show, in NTg myocardium [chromatogram (a)], that there is a well-detectable endogenous CO production after 20 min. of ischemia followed by 120 min. of reperfusion. The endogenous CO production was substantially increased in HO-1 Tg myocardium (chromatogram (b)], and a reduction of endogenous CO production was recorded in HO-1 myocardium treated with SnPPIX [chromatogram (c)].

Fig 4

Infarct size and HO-1 staining in isolated NTg, Tg and HO-1 KO mouse hearts subjected to 20 min. of normotermic global ischemia followed by 120 min. of reperfusion. *P < 0.05 compared to the NTg control values. n = 6 in each group. Infarct is represented by white area surrounding by the living red tissues [(A), to the right lower part]. Immunochemical localization of HO-1 is stained by blue in NTg, Tg and KO myocardium [(A), to the left lower part]. (B) shows the incidence (%) of total (open bars) and sustained (hatched bars) reperfusion-induced VF in isolated NTg, Tg and KO mouse hearts. The incidence of reperfusion-induced VF was registered, and comparisons were made to the values of NTg group. n = 12 in each group, *P < 0.05. Because of the non-parametric distribution in the incidence of total and sustained VF, the chi-square non-parametric test was used to compare individual groups. This figure and data were presented at the FEAM Meeting held in Bucharest, Romania, in March, 2010, and published in the current issue of the JCMM, 2011, by Bak et al.