Cardioprotection via Metabolism for Rat Heart Preservation Using the High-Pressure Gaseous Mixture of Carbon Monoxide and Oxygen

Abstract

The high-pressure gas (HPG) method with carbon monoxide (CO) and oxygen (O₂) mixture maintains the preserved rat heart function. The metabolites of rat hearts preserved using the HPG method (HPG group) and cold storage (CS) method (CS group) by immersion in a stock solution for 24 h were assessed to confirm CO and O₂ effects. Lactic acid was significantly lower and citric acid was significantly higher in the HPG group than in the CS group. Moreover, adenosine triphosphate (ATP) levels as well as some pentose phosphate pathway (PPP) metabolites and reduced nicotinamide adenine dinucleotide phosphate (NADPH) were significantly higher in the HPG group than in the CS group. Additionally, reduced glutathione (GSH), which protects cells from oxidative stress, was also significantly higher in the HPG group than in the CS group. These results indicated that each gas, CO and O₂, induced the shift from anaerobic to aerobic metabolism, maintaining the energy of ischemic preserved organs, shifting the glucose utilization from glycolysis toward PPP, and reducing oxidative stress. Both CO and O₂ in the HPG method have important effects on the ATP supply and decrease oxidative stress for preventing ischemic injury. The HPG method may be useful for clinical application.

Keywords: carbon monoxide; cardioprotection; high-pressure gas; ischemic injury; metabolomics; oxygen; rat; transplantation.

Figure 1

Assessment of transplanted hearts after 24-h preservation in three groups: control group (CT), nonpreservation; cold storage (CS) group, preservation in the University of Wisconsin (UW) solution for 24 h; high-pressure gas (HPG) group, preservation using the mixture of high-pressure carbon monoxide (CO) and oxygen (O₂) for 24 h. (a) Heart rate at 60 min after transplantation. (b) Hematoxylin and eosin staining in transplanted hearts after 24-h preservation. Black bar represents 40 μm. (c) Representative midmyocardial cross-sections of 2,3,5-triphenyltetrazolium chloride-stained hearts. Red-stained areas indicate viable tissues, and white areas indicate infarct tissues. White bar represents 500 μm. Myocardial infarction areas in the hearts of each group were quantified. Data in each bar are presented as mean ± standard error of the mean. N.S., not significant. * p < 0.05.

Figure 2

Assessment of cardiac morphology and mitochondrial activities after 24-h preservation. (a) Hematoxylin and eosin staining in hearts immediately after 24-h preservation. Black bar represents 40 μm. (b) Adenosine triphosphate (ATP) content. (c) Alterations in reduced nicotinamide adenine dinucleotide phosphate (NADPH), NADP⁺, and NADPH/NADP⁺ ratio. Bar graphs indicate fold changes relative to the CT group. (d) Alterations in reduced glutathione (GSH), oxidized glutathione (GSSG), and the GSH/GSSG ratio. Data in each bar are presented as mean ± standard error of the mean. N.S., not significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Metabolite profiling of hearts preserved using the cold storage (CS) method and high-pressure gas (HPG) method. (a) Principal components analysis (PCA) score plot. Blue and red circles represent 95% confidence interval of CS and HPG, respectively. (b) Factor loading plot for PC1. A total of 70 statistically significant metabolites were selected with an absolute value of 0.8 of PC1 factor loading. Blue and red dotted lines show the significantly negative and positive levels, respectively. (c) A heat map of 70 statistically significant metabolites in the CS and HPG groups. Red, green, and blue letters show metabolites of the pentose phosphate pathway and nucleic acid, tricarboxylic acid (TCA) cycle, and glycolysis, respectively. The color scale from blue to red indicates low to high amount of metabolites between CS and HPG.

Figure 4

Metabolome data map after 24-h heart preservation in glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway (PPP), glutaminolysis, and polyol pathway. Bar graphs indicate fold changes relative to the control (CT) group. The lack of a bar graph representation for a given metabolite means that the metabolite was not detected. Data in each bar are presented as mean ± standard error of the mean. N.S., not significant. * p < 0.05, ** p < 0.01, *** p < 0.001. CS, cold storage; HPG, high-pressure gas; G6P, glucose 6-phosphate; F6P, fructose-6-phosphate; F1,6P, fructose 1,6-bisphosphate; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; BPG, bisphosphoglycerate; 3-PG, 3-phosphoglycerate; 2-PG, 2-phosphoglycerate; PEP, phosphoenolpyruvate; α-KG, α-ketoglutarate; 6PG, 6-phosphogluconate; Ru5P, ribulose-5-phosphate; R5P, ribose-5-phosphate; Glu, glutamic acid; Gln, glutamine.

Figure 5

Schematic representation of proposed metabolic pathways for the preservation in comparison between the cold storage (CS) method and high-pressure gas (HPG) method. The CS preservation method is characterized by lactic acid production, a by-product of anaerobic metabolism, the polyol pathway that causes oxidative stress, and anaplerotic utilization of glutaminolysis. Conversely, the HPG preservation method is characterized by adenosine triphosphate (ATP) production and a shift in glucose metabolism toward the pentose phosphate pathway (PPP). PPP acceleration increases cellular reduced nicotinamide adenine dinucleotide phosphate (NADPH) amounts, providing reduced glutathione (GSH) that benefits reducing oxidative stress.

Figure 6

Schematic representation of the preservation method and experimental design. (a) The pressure tight chamber was filled with CO [partial pressure of carbon monoxide (PCO) = 1500 hPa] and O₂ [partial pressure of oxygen (PO₂) = 2000 hPa]. During heart preservation, a flask with 50 mL distilled water was placed in the chamber to maintain humidity. (b) Three groups were examined: nonpreserved hearts (CT group), hearts preserved in University of Wisconsin (UW) solution for 24 h (CS group), and hearts preserved with the high-pressure gas preservation containing CO and O₂ for 24 h (HPG group). To assess mitochondrial activities and metabolomic analysis, the hearts were sampled immediately after heart extraction in the CT group and after preservation in the CS and HPG groups (Sampling 1: white arrow). For morphological and functional assessment, the hearts were sampled after 90 min from transplantation in the CS and HPG groups (Sampling 2: black arrow).