Gene and protein expression changes in response to normoxic perfusion in mouse hearts

Abstract

Introduction: Although crystalloid-perfused isolated heart models are widely used in cardiovascular research, there are several limitations of these techniques. Changes in cardiac gene expression pattern due to normoxic perfusion itself have not been studied, despite its potential importance to provide useful information on limitations of this model. Therefore, here we investigated the time-dependent effect of normoxic, normothermic perfusion on global gene expression at mRNA and protein levels. Methods: Hearts from male CFLP mice were perfused according to the Langendorff technique. We assessed relative gene expression changes by DNA microarray analysis of 8000 genes after 0, 60 and 120 min perfusion. Results: Twelve genes exhibited significant up-regulation and 27 showed repression in hearts perfused for 60 or 120 min as compared to 0 min controls. Expression changes of 17 selected genes were verified and an additional 19 genes were examined by real-time quantitative PCR. Genes with altered expression included those coding for Creatin kinase, Lactate dehydrogenase, Voltage-dependent anion channel 1, a Disintegrin and Metalloprotease domain 3, Integrin alpha 7, Long-chain acyl-CoA dehydrogenase, Casein kinase II, Ketohexokinase, Chloride ion current inducer protein, Matrix metalloproteinase 2 and 9, Superoxide dismutases and Nitric oxide synthases, etc. Discussion: Our results show that normoxic crystalloid perfusion itself results in time-dependent changes in cardiac gene expression which should be considered when designing ex vivo perfusion protocols in the mouse heart to mimic cardiac pathologies as many of these genes have been suspected to influence several cardiovascular diseases.

MicroRNAs associated with ischemia-reperfusion injury and cardioprotection by ischemic pre- and postconditioning: protectomiRs

“…MicroRNAs were labeled using the miRNA Complete Labeling and Hyb kit system (Agilent Technologies, Palo Alto, CA). Labeled samples were completely vacuum dried on the medium-high (45°C) heat setting and hybridized onto the surface of a 8 ϫ 15k Rat miRNA Microarray (Agilent Technologies), which was done in microarray hybridization chambers (Agilent Technologies), as previously described (13). Each array was scanned as previously described (16).…”

Functional Genomics of Cardioprotection by Ischemic Conditioning and the Influence of Comorbid Conditions: Implications in Target Identification

“…These early studies have been confirmed by several papers showing that both ischemic preconditioning and postconditioning trigger a cardioprotective gene expression profile in the heart at the transcript level [14][15][16][17]. In line with these findings, changes in the expression of the posttranscriptional regulators of gene expression, i.e.…”

“…cathepsin G protease [11] PDE9A1 phosphodiesterase [12] peroxredoxin 4 antioxidant defence [18] PDGFR signaling [18] ZAC1 transcription factor [20] microRNA-139-5p unknown [17] microRNA-125b* unknown [17] let-7b unknown [17] microRNA-487b unknown [17] Hyperlipidemia and obesity ATF3 transcription factor [25] NADH-ubiquinone oxidoreductase energy metabolism [27] Hsp86 stress response [27] procollagen structural proteins [27] argininosuccinate synthetase Arg metabolism [28] Hsp70 stress response [28] cytochrome C oxidase energy metabolism [30] MMP-9 remodeling [30] microRNA-25 oxidative stress [33] microRNA-27b hypertrophy [34][35][36] Diabetes vanilloid receptor 1 signaling [64] histone deacetylase 2 gene regulation [64] apolipoprotein B lipid metabolism [64] MMP-13 remodeling [64] microRNA-208a hypertrophy [54,55] microRNA-24 vascularization [54,55] microRNA-21 hypertrophy [54,55] …”

Purification of high-quality micro RNA from the heart tissue

“…These mixes were used for the hybridization, which was done as previously described [5,10]. After hybridization the slides were washed in Gene Expression Wash buffer 1 containing Triton X-102 from Agilent Technologies at room temperature for 1 minute than in Gene Expression Wash buffer 2 containing Triton X-102 at 37 °C for another 1 minutes before scanning.…”