Controlled reoxygenation cardiopulmonary bypass is associated with reduced transcriptomic changes in cyanotic tetralogy of Fallot patients undergoing surgery

Abstract

In cyanotic patients undergoing repair of heart defects, high level of oxygen during cardiopulmonary bypass (CPB) leads to greater susceptibility to myocardial ischemia and reoxygenation injury. This study investigates the effects of controlled reoxygenation CPB on gene expression changes in cyanotic hearts of patients undergoing surgical correction of tetralogy of Fallot (TOF). We randomized 49 cyanotic TOF patients undergoing corrective cardiac surgery to receive either controlled reoxygenation or hyperoxic/standard CPB. Ventricular myocardium biopsies were obtained immediately after starting and before discontinuing CPB. Microarray analyses were performed on samples, and array results validated with real-time PCR. Gene expression profiles before and after hyperoxic/standard CPB revealed 35 differentially expressed genes with three upregulated and 32 downregulated. Upregulated genes included two E3 Ubiquitin ligases. The products of downregulated genes included intracellular signaling kinases, metabolic process proteins, and transport factors. In contrast, gene expression profiles before and after controlled reoxygenation CPB revealed only 11 differentially expressed genes with 10 upregulated including extracellular matrix proteins, transport factors, and one downregulated. The comparison of gene expression following hyperoxic/standard vs. controlled reoxygenation CPB revealed 59 differentially expressed genes, with six upregulated and 53 downregulated. Upregulated genes included PDE1A, MOSC1, and CRIP3. Downregulated genes functionally clustered into four major classes: extracellular matrix/cell adhesion, transcription, transport, and cellular metabolic process. This study provides direct evidence that hyperoxic CPB decreases the adaptation and remodeling capacity in cyanotic patients undergoing TOF repair. This simple CPB strategy of controlled reoxygenation reduced the number of genes whose expression was altered following hyperoxic/standard CPB.

Fig. 1.

Oxygen pressure (A) and 8-isoprostane level (B) changes up to 24 h postoperatively in the hyperoxic/standard compared with the controlled reoxygenation cardiopulmonary bypass (CPB) groups. Time 1, preoperatively; time 2, X-clamp off; time 3, 30 min X-clamp off; time 4, 2 h X-clamp off; time 5, 6 h X-clamp off; time 6, 24 h X-clamp off. **P < 0.01 and *P < 0.05 vs. time 1 in the Hyperoxic/Standard group.

Fig. 2.

A: summary of the regulated genes (≥1.6-fold) in post-op vs. pre-op controlled reoxygenation, post-op vs. pre-op hyperoxic procedure, and post-op hyperoxic vs. post-op controlled reoxygenation. B: regulated transcripts in the 3 comparisons. The histogram shows the proportion of upregulated vs. downregulated genes.

Fig. 3.

Confirmation of the microarray results of changed genes in hyperoxic vs. controlled reoxygenation procedures. Changes in mRNA expression of COL1A2, VCAN, MAPK8, NLK, SLC6A6, NRAS1, PDE1, MOSC1, and CRIP3 were verified in 10 patients (5 using hyperoxic and 5 using controlled reoxygenation procedure) by quantitative real-time-PCR. Results are shown as means (±SE) fold-change compared with hyperoxia. *P < 0.05, **P < 0.01.

Fig. 4.

Protein levels of MOSC1, TAUT, and COL1A2 in ventricular biopsies of controlled reoxygenation vs. hyperoxic patients. Tissues were lysed to isolate protein content and Western blotting analysis performed probing for MOSC1 (A), TAUT (B), COL1A2 (C), and GAPDH. No significant changes were observed in controlled reoxygenation (C) compared with hyperoxic (H) samples. MOSC1, CRIP3, and COL1A2 bands were normalized to GAPDH levels. Data are means ± SE.