Within the aliphatic and aromatic regions (–0.05–9.5 parts per million [ppm]) of the nuclear magnetic resonance (NMR) spectra, resonances were assigned as: (1) 3-trimethylsilyl-2,2,3,3-tetradeuteropropionate; (2) leucine; (3) isoleucine; (4) valine; (5) beta-hydroxybutyrate; (6) lactate; (7) alanine; (8) acetate; (9) glutamate; (10) succinate; (11) glutamine; (12) aspartate; (13) creatine and phosphocreatine; (14) choline; (15) phosphocholine; (16) carnitine; (17) taurine; (18) glycine; (19) creatine; (20) glucose; (21) fumarate; (22) tyrosine; (23) AMP, ADP, and ATP; (24) NAD and NADH; (25) formate; and (26) NAD and NADH. The presence of glycerophosphocholine within the carnitine peak was ruled out by NMR analysis at low pH. ADP = adenosine diphosphate; AF = atrial fibrillation; AMP = adenosine monophosphate; ATP = adenosine triphosphate; NAD = nicotinamide adenine dinucleotide; NADH = reduced form of nicotinamide adenine dinucleotide; SR = sinus rhythm.

Protein extracts were separated on a pH 3 to 10 nonlinear immobilized pH gradient strip, followed by separation on a 12% sodium dodecyl sulfate polyacrylamide gel. Spots were detected by silver staining. The figure represents a direct overlay of average gels from hearts in sinus rhythm (SR) and atrial fibrillation (AF) (blue and orange for SR and AF, respectively) created by using the Proteomweaver software (Definiens AG, Munich, Germany). Differentially expressed spots are numbered. Proteins were identified by mass spectrometry and are listed in Table 2.

(A) Representative areas of 2-dimensional gels from patients in SR and AF highlight quantitative differences in images. Numbers correspond to proteins listed in Table 2. (B) Quantification of protein abundance using 1-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Note the decline of peroxiredoxin 1 levels in persistent AF. SOD = superoxide dismutase; other abbreviations as in Figure 2.

The illustration shows the TCA cycle, oxidative phosphorylation, and protein and metabolite changes observed in patients with persistent AF (red). Notably, the probability of 3 metabolites being associated with 3 enzymes out of the 17 differentially expressed proteins by chance was estimated at <5% after running 1 million simulations on Matlab (Mathworks Limited). Each simulation consisted of randomly selecting 3 metabolites out of the 24 measured, generating 1,500 proteins, randomly associating 300 of them with pairs of metabolites (based on the fact that approximately 20% of spots on 2-dimensional gel maps from human hearts are metabolic enzymes), and randomly selecting 17 of the 1,500 proteins. A success was defined as the 3 selected metabolites being associated with at least 3 proteins among the 17 selected. CoA = coenzyme A; e = electron; TCA = tricarboxylic acid; other abbreviations as in Figures 1 and 2.

¹H-NMR metabolite profiles underwent principal component analysis as described in "Materials and Methods." (A) Plots of the 4 principal components (PC 1, 3, 5, and 6) were used for discrimination. Red triangles and blue circles represent patients who developed post-operative AF and patients, who maintained SR, respectively. Note that more than 80% of patients were correctly classified using either PC1 versus PC5 (B) or PC3 versus PC6 (C). Abbreviations as in Figure 2.

(A) Reduction of the glucose to acetate ratio in patients susceptible to post-operative AF irrespective of the underlying cardiac pathology. (B) Positive correlation between the ratio of glycolytic end products (alanine, lactate) to end products of lipid metabolism (acetate) and the onset of post-operative AF in patients with coronary artery disease. ANOVA = analysis of variance; CABG = coronary artery bypass grafting; other abbreviations as in Figure 2.