In this study, the difference between the healthy control group and the heart failure group was analyzed by the metabonomics technique of NMR The results of principal component analysis showed that the healthy control group, hfmref group and hfreef group were obviously separated on the integral scatter diagram. The results showed that the biomarkers related to metabolic remodeling could be obtained by analyzing the plasma of heart failure patients with metabonomics. The results showed that the contents of 3-hydroxybutyric acid, alanine, citrulline and acetone in heart failure subgroup were increased. It is difficult to distinguish HFrEF patients and HFmrEF patients by traditional clinical laboratory examination indexes, but the metabonomics method based on NMR can distinguish HFrEF patients and HFmrEF patients directly from the principal component analysis and OPLS-DA score chart, which shows the advantages of metabonomics method in disease diagnosis.
Cardiac metabolism is a tightly regulated and incessant process that can be divided into three stages: substrate utilization, energy production and ATP transport shuttle[7].The substrate utilization phase starts with substrate uptake by the cardiomyocytes and culminates in the generation of reducing equivalents from the tricarboxylic acid cycle (TCA). The heart primarily uses free fatty acids (FFAs) and glucose as its substrates, and 60%-90% of the ATP required for cardiac activities is derived from aerobic oxidation of FFAs[8], 10%-40% from aerobic oxidation of glucose[9], and the remaining 1%-3% from anaerobic glycolysis and the decomposition of amino acids and ketone bodies. In addition, the myocardium can also utilize glycogen and lactic acid[10–11]. The major pathological changes seen in heart failure include myocardial remodeling and myocardial fibrosis. Myocardial remodeling reduces the number of capillaries per unit organ weight and increases the distance for passive oxygen diffusion, thus causing myocardial anoxia. In addition, the activity of myocardial ATPase is reduced by 20%-40% during heart failure, leading to impaired energy utilization and reduced myocardial contractility. In the early stages of heart failure, glucose utilization is increased, and that of FFAs is either unaffected or only slightly increased. In severe heart failure however, FFA usage is significantly reduced.
We found that 3-hydroxybutyrate and acetone levels in the serum were increased in the heart failure patients, along with decreased LVEF. Both are the intermediates of fatty acid catabolism that are produced in hepatocyte mitochondria and later enter the bloodstream. Studies show that oxidation of ketone bodies can inhibit the uptake and oxidation of fatty acids in the myocardium [12–13]. During physiological conditions, the ketones are rapidly oxidized in the extrahepatic tissues, which results in extremely low levels in the blood. During some pathological conditions however, glucose metabolism is inhibited, which results in increased fat utilization and ketone bodies. When the production of ketone bodies in the liver overwhelms the metabolic ability of extrahepatic tissues, the excess amounts are released in the bloodstream. The heart failure patients in our cohort presented decreased glucose and lipid metabolism and ketone body production, which significantly altered cardiac substrate uptake and utilization. In addition, decreased fatty acid utilization in heart failure patients leads to lipid deposition and cardiotoxicity, which further aggravates the metabolic disorder in the myocardium.
We also observed a significant increase in the concentrations of alanine and citrulline in the heart failure patients, indicating weakened amino acid metabolism, which could be due to reduced activity of certain amino acid metabolizing enzymes. Citrulline is involved in the urea cycle, and can be converted to the essential amino acid L-arginine. Excess levels of the latter is related to hypertension, atherosclerosis, diabetes, heart failure and ischemia reperfusion injury. Studies show that increased serum levels of citrulline in patients with heart failure can aggravate metabolic disorder in the cardiomyocytes, while decreased cardiac function also triggers abnormal citrulline metabolism. Alanine is a 4ketogenic amino acid that can be converted into ketone bodies through fatty acid metabolic pathways. In addition, it can also produce pyruvic acid and free ammonia through deamination, as well as glucose through gluconeogenesis. Taken together, our findings suggest that patients with heart failure have abnormal amino acid and glycolipid metabolism.
Since the discovery of brain natriuretic peptide (BNP) as a biomarker of heart failure, studies on cardiovascular diseases have increasingly focused on screening for more biomarkers. However, no single biomarker identified so far is an accurate indicator of myocardial metabolic remodeling[14]. A metabonomics approach can identify significantly altered metabolites through methodologies with advantages like minimal sample processing, high reproducibility, easy quantification and high-throughput analysis. Using this approach, we found significant differences between the healthy controls and heart failure patients in terms of the concentration of key metabolites, which pointed to disordered glucose and fatty acid metabolism in the affected myocardial cells that would lead to abnormalities in myocardial structure and function. In addition, the heart failure patients with lower and mid-range left ventricular ejection fraction differed on the basis of the four metabolites. Therefore, indices of altered myocardial metabolism can be a reliable diagnostic tool for the objective evaluation of cardiac remodeling in heart failure patients, as well as a basis to investigate the underlying mechanisms of heart failure.