Abstract
BackgroundChanges in cardiac gene expression due to myocardial injury are usually assessed in whole heart tissue. However, as the heart is a heterogeneous system, spatial and temporal heterogeneity is expected in gene expression.ResultsIn an ischemia/reperfusion (I/R) rat model we evaluated gene expression of mitochondrial and cytoplasmatic superoxide dismutase (MnSod, Cu-ZnSod) and thioredoxin reductase (trxr1) upon short (4 h) and long (72 h) reperfusion times in the right ventricle (RV), and in the ischemic/reperfused (IRR) and the remote region (RR) of the left ventricle. Gene expression was assessed by Real-time reverse-transcription quantitative PCR (RT-qPCR). In order to select most stable reference genes suitable for normalization purposes, in each myocardial region we tested nine putative reference genes by geNorm analysis. The genes investigated were: Actin beta (actb), Glyceraldehyde-3-P-dehydrogenase (gapdh), Ribosomal protein L13A (rpl13a), Tyrosine 3-monooxygenase (ywhaz), Beta-glucuronidase (gusb), Hypoxanthine guanine Phosphoribosyltransferase 1 (hprt), TATA binding box protein (tbp), Hydroxymethylbilane synthase (hmbs), Polyadenylate-binding protein 1 (papbn1). According to our findings, most stable reference genes in the RV and RR were hmbs/hprt and hmbs/tbp/hprt respectively. In the IRR, six reference genes were recommended for normalization purposes; however, in view of experimental feasibility limitations, target gene expression could be normalized against the three most stable reference genes (ywhaz/pabp/hmbs) without loss of sensitivity. In all cases MnSod and Cu-ZnSod expression decreased upon long reperfusion, the former in all myocardial regions and the latter in IRR alone. trxr1 expression did not vary.ConclusionsThis study provides a validation of reference genes in the RV and in the anterior and posterior wall of the LV of cardiac ischemia/reperfusion model and shows that gene expression should be assessed separately in each region.
Highlights
Changes in cardiac gene expression due to myocardial injury are usually assessed in whole heart tissue
As far as the sham group is concerned, we found a significant heterogeneity in the expression level within the two left ventricle (LV) areas for both mitochondrial and cytosolic SOD, with the higher levels expressed in IRR (p < 0.05 for both). trxr1 expression did not vary among the three ventricular regions
GeNorm analysis has shown that reference gene stability varies among the three myocardial regions analyzed: hmbs, hprt and hmbs, tbp, hprt are suitable reference genes in the right ventricle and in the Remote region respectively
Summary
Changes in cardiac gene expression due to myocardial injury are usually assessed in whole heart tissue. Restoration of blood flow to the ischemic myocardium modulates the size of myocardial infarct and the chance of cell survival. This process, termed reperfusion, per se can induce injury. Activity or gene expression of antioxidant enzymes has been reported to either increase or decrease upon ischemia/reperfusion (I/R) [17,18,19,20,21,22,23]. This may be due to different experimental conditions and/or to variation of cardiac endogenous antioxidant expression at different times of reperfusion [20]
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