UA was negatively correlated to cardiac function and positively correlated to circulating fatty acid level in HFrEF patients.
To investigate the correlation between UA and cardiac function, we extracted clinical data of 4772 patients diagnosed with metabolic syndrome from First Affiliated Hospital of Xi’an Jiaotong University from Biobank, including 190 heart failure with reduced ejection fraction(HFrEF) patients, 259 heart failure with mid-range ejection fraction(HFmrEF) patients and 4323 normal ejection fraction(EF) patients. Baseline information were shown in Table.S1. UA value was significantly higher in HFrEF and HFmrEF groups compared to normal EF group (Fig. 1A-B).Linear regression was performedand it is identified that UA negatively correlated to cardiac function, as quantified by cardiac ventricular size (Fig. 1C left ventricular end-diastolic dimension,LVEDD; left ventricular end-systolic dimension, LVESD) and EF value (Fig. 1D). In addition, UA also significantly correlated to blood lipid profiles, with negative correlation to high density lipoprotein(HDL-C, Fig. 1E) and positively to TG(triglycerides, Fig. 1F).
Since the data from clinical cohorts with metabolic syndrome revealed significant higher UA levels in patients with impaired cardia function, and possible correlation between UA and circulating lipids levels, we then investigated the relationship between UA and specific circulating lipid components. To this end, targeted metabolomics analysis of FFAs were applied in participants with normal EF and reduced EF (Fig. 2A); the basic characters for this cohort were shown in Table.S2. In this age and gender matched cohort, UA was around 20% higher in HFrEF patients as compared to controls (Fig. 2B 276.72 vs. 366.90 umol/L p < 0.001). Both non-essential and essential FA profiles were increased in heart failure patients (Fig. 2C). Almost all non-essential FFA and C18:2 showed positive correlation with UA concentration and reverse association with cardiac function in the whole cohort. Besides, in network correlation analysis, UA also showed “stronger positive connection” with FFA alteration as compared to other clinical biochemical parameters, including but not limited to: HbA1c, CRE, TSH and HGB (Fig. 3A-C, Fig.S1).
Ua Aggravates Iso Induced Heart Failure And Caused Energy Metabolism In Zebrafish
To further understand the specific mechanism how UA regulated FFA levels and heart fuction, zebrafish experiments were performed, as zebrafish embryo can tolerate the absence of blood flow for few days because its oxygen is delivered by diffusion rather than by the cardiovascular system, making it an excellent model for studying heart failure35. 1mM UA was used for the study to mimick the hyperuricemia conditions in patients. At first, 1mM UA treatment can slightly decrease heart rate and increase natriuretic peptide B (nppb/pro-BNP) level, but no alteration in cardiovascular size compared to control larvae (Fig. 4A-D) occured; Then, isoproterenol (ISO), a β-adrenergic receptor agonist, commonly used to trigger cardiac hypertrophy in animal models and cardiomyocytes36, was used to construct heart failure model in zebrafish. According to the previous study, 1mM ISO can lead to severe heart failure in zebrafish larvae37. To understand the effect of UA on cardiac function in the present study, 0.5mM Iso was used in the following experiments to mimic a moderate heart failure. 1mM UA treatment aggravated 0.5mM ISO induced moderate heart failure in Tg(fli1:EGFP) zebrafish larvae at 72, 96 and 120 hpf(Fig. 4A), quantified by around 100 percent increased cardiovascular cavity size (B), decreased heart beats per minute(C) and up to 20-fold increased nppb expression(D).
To investigate the exact cardiovascular structure alteration after UA treatment, Tg(myl7:egfp) zebrafish larvae were applied, in which cardiomyocytes were marked with green fluorescence protein38. 0.5mM ISO induced zebrafish heart failure caused pericardial edema and long-stretched heart with increased angle between atrium and ventricle (looping failure) at 3 dpf (Fig. 4E). 1mM UA treatment aggravated ISO induced heart failure by increasing “no loop” phenotype, from 39.1–57.1% (Fig. 3E). Furthermore, in metabolomics analysis, UA aggravated ISO induced heart failure and lead to impaired energy metabolism in zebrafish. (Fig.S2)
UA aggravated heart failure is most likely driven by FFA accumulation through SREBP1/FASN pathway.
To identify fatty acid accymulation after UA aggravated heart failure, expression analyses of lipid metabolism related genes including fatty acid synthase (Fasn), fatty acid desaturase 2(fads2), fatty acid elongase 2(elov2), and stearoyl-CoA desaturase(scd) were performed (Fig.S3), and Fasn was increased in UA treated zebrafish heart failure model (Fig. 5A), indicating the regulatory role of UA in heart failure via dysfunction of FFA synthesis. Lipid profile further identified increased fatty acids in both UA treated and ISO induced heart failure zebrafish larvae at 96 hpf (Fig. 6A and C). Through MetaboAnalyst analysis, enrichment of lipid metabolism pathways were identified in both groups as compared to controls, such as linoleic acid metabolism, biosynthesis of unsaturated fatty acids and arachidonic acid metabolism (Fig. 6B). At last, to further clarify the causal relationship between UA, fatty acid and heart failure, fatty aicd incubation experiments was performed in zebrafish larvae. Saturated fatty acids, including 50 uM palmitic acid and stearic acid, results in partially heart failure, quantified by increased cardiovascular cavity size (Fig. 5B-D) and around 100 percent increased nppb expression (Fig. 5F). Besides, fasn was not altered (Fig. 5E). This implied that external fatty acids can directly contribute to heart failure; whereas, UA may induce heart failure possibly through pathological FFA regulation.
To validate the above findings revealed by zebrafish work and sought for potential signalling pathway in UA related heart failure, we performed UA incubation experiments in human hepatoellular carcinomas (HepG2), and transfected it with sterol regulatory element binding protein 1 (SREBP1) siRNA to check the rescue effects(Fig. 7A). First, UA increased around 50% mRNA expression of FASN and SREBP1, which were restored with siRNA induced SERBP1 knockdown (Fig. 7B). Also, we observed similar trend in protein level, with both increased FASN and full length SREBP1(FL-SREBP1) and splicing SREBP1 (S-SREBP1) after UA treatment and rescued after SREBP1 silence (Fig. 7C). Finally, through FFA profile analysis, UA incubation was found to increase C20:0, C20:2, C22:6, etc, which was reversed after SREBP1 knock down (Fig. 7D), in accordance with our cell culture and clinical data(Fig. 7E), through our zebrafish, cell culture and clinical collection work, our findings suggest that UA-SREBP1-FASN signaling exacerbates cardiac dysfunction during FFA accumulation (Fig. 8).