Chronic Heart Failure (CHF) is a major health problem worldwide and is the end result of almost the entire forms of cardiovascular disease (1–3). The pathological mechanisms of CHF include hemodynamic disturbances, dysregulation of neurosecretory activation, and the relationship between the inflammatory response and the effects of the overexpression of oxidative stress (4–6). Despite recent developments in modern combination therapy strategies, the high readmission rate and poor prognosis of CHF are not promising (2, 7). Therefore, researchers have started to explore new pathogenesis and treatment pathways for heart failure, focusing on the intestinal flora to explore new methods to prevent and treat CHF. The intestinal flora is composed of trillions with bacteria within the gastrointestinal tract. Its role in maintaining good health, absorbing nutrients, creating hormones and vitamins, building the mucosal barrier, regulating the immune system and inhibiting the colonization of pathogenic bacteria is crucial (8–11). Studies have shown that reduced cardiac output and systemic organ system congestion can lead to edema and localized ischemia in the intestine, resulting in raised intestinal bacterial translocation and circulating endotoxin levels. This induces inflammation-related cytokine production, which in turn can promote inflammatory responses and trigger cardiac fibrosis and microvascular and cardiac dysfunctions, thereby aggravating heart failure (12–15). For the past few years, several researches have shown that the component and function of the gut flora are different in heart failure patients and healthy persons. Phenylacetylglutamine (PAGln), one of the common chronic kidney disease toxins, was previously thought to only be produced by the combination of phenylacetate/phenylbutyrate (phenylacetate precursor) and glutamine, which acts as an ammonia-lowering agent via the urea cycle bypass pathway (16). In contrast, recent studies have found that PAGln, which can also be generated through the metabolism of the essential amino acid phenylalanine by intestinal microorganisms, has high plasma concentrations in patients with Major Adverse Cardiovascular Events (MACE) and is considered to be an independent predictor of MACE risk (17, 18). However, there are few studies on the changes in the PAGln concentration in the plasma of CHF patients. Thus, in this research, we selected patients of different grades in CHF and healthy controls as the study subjects. By sequencing their fecal flora genes and detecting the PAGln levels in their plasma, we investigated (1) the relationship between intestinal flora diversity in patients with varying grades in CHF and that in the healthy controls, (2) the changes in the PAGln levels in patients with varying grades in CHF and the healthy controls, and (3) the effects of intestinal flora diversity and PAGln on CHF.

Tang et al. (14) put the “gut hypothesis of heart failure” forward for the first time. The hypothesis indicates that decreased cardiac production owning to heart failure can bring about reduced intestinal perfusion, mucosal ischemia, and intestinal mucosal destruction. These alterations in the intestinal barrier function, in turn, can result in raised intestinal permeability, intestinal malnutrition, bacterial translocation, and lifted circulating endotoxins, leading to potential inflammation that is connected with CHF. To minimize confounding factors, we recruited patients with age, sex, and cardiovascular disease similar to that of CHF patients as control group. In this study, we found that although there was no substantially difference in the CHF risk factors (coronary heart disease and hypertension) among the three groups, the three groups had significantly different intestinal flora structures as determined by the 16S rRNA amplification sequence assay. A comparison of the distribution of the OTUs in all three groups at the phylum level disclosed that the amount of OTUs specific to the control group was 20.7%, the number of OTUs specific to NYHA class III was 3.4%, and the number of OTUs specific to NYHA class IV was 6.7%. At the genus level, the number of control-specific OTUs was 27.2%, the number of NYHA class III-specific OTUs was 1.7%, and the number of NYHA class IV-specific OTUs was 2.9%. Therefore, the number of NYHA class III and NYHA class IV unique OTUs was considerably less than that of the control group, indicating that the microbial diversity degree in the CHF group was lower than that in the control group. By comparing the microbial diversity of the three groups, we found that the Chao 1 index was lower in both NYHA class III and NYHA class IV than that in the control group (p < 0.05). According to the above results, the more severe the level of heart failure, the lower the number of flora species in heart failure patients. The β-diversity analysis revealed significant separation between the three groups of sample points and clustering within a certain range, indicating differences in the gut flora composition between the three groups. This indicates that there is a connection in the severity of heart failure and the distribution and components of the gut flora. The research demonstrated that the intestinal perfusion of CHF patients was insufficient, and the intestinal structure and function were changed, causing a clear reduction in the diversity of the intestinal flora (19). Additionally, in terms of the species composition and difference analysis, we identified statistically marked differences in the Firmicutes abundance among the three groups at the phylum level (60.5:48.8:51.1%, p < 0.05), with a marked reduction in the Firmicutes abundance in the CHF group. It was also discovered that Bacteroidete’s abundance was raised and Firmicute’s abundance was reduced in the CHF patients, and the abundance of Firmicutes and intestinal microbial diversity were positively correlated with the intake of dietary fiber (20). However, CHF patients generally had digestive symptoms such as nausea due to long-term gastrointestinal stasis, which led to insufficient dietary fiber intake and led to a reduction of the abundance of thick-walled bacteria phyla. Thus, we consider that the decrease in the abundance of Firmicutes is one of dysbiosis’ typical manifestations in CHF patients. In terms of the species composition and analysis of variance, we found that the control, NYHA class III, and NYHA class IV Escherichia-Shigella (1.7:7.2:6.6), Bifidobacterium (0.8:4.2:1.6), Klebsiella (0.5:1.34:4.7), and Lactobacillus (0.6:1.17:4.28) had significantly different abundance shares at the genus level (p < 0.05). The LEfSe analysis demonstrated that at the genus level, Agathobacter was significantly higher in the control, and Escherichia and Bifidobacterium were significantly higher in NYHA class III. Then, Klebsiella and Lactobacillus were significantly higher in NYHA class IV. The genera that were statistically different in both test analyses were Bifidobacterium, Klebsiella, and Lactobacillus. Moreover, Bifidobacterium was higher in NYHA class III than that in NYHA class IV, and Klebsiella in NYHA class III was lower than that in NYHA class IV. This suggests that Bifidobacterium and Klebsiella may be the key organisms that change under different degrees of CHF. Studies have shown that Klebsiella is elevated in cardiovascular disease and that it regulates the trimethylamine synthesis pathway, which can be metabolized by intestinal microbes to produce trimethylamine N-oxide (TMAO). Additionally, researches have found that elevated plasma TMAO levels in CHF patients predict a higher risk of death (21). The Choline-TMA-TMAO production pathway can directly contribute to the development of adverse ventricular remodeling and heart failure. It was found that mice fed with a high choline diet had higher TMAO levels and were prone to ventricular remodeling. The degree of ventricular diastole, ventricular wall thinning, and myocardial fibrosis were significantly increased compared to the control group, and ventricular wall contractility was significantly decreased (22). Furthermore, animal models have shown that Lactobacillus plantarum Lp299v-containing beverages can reduce myocardial hypertrophy and ventricular remodeling, and Lactobacillus rhamnosus GR-1 could significantly improve the prognosis of rats with ischemic heart failure (23). Wang et al. found that Bifidobacterium can extensively reduced plasma TMAO levels in choline-fed mice (24). Meanwhile, Bifidobacterium exerts various physiological effects as a beneficial bacterium, including reducing the number of harmful bacteria (such as E. coli), inhibiting the generation of pro-inflammatory cell factors, regulating host organism immunity, and enhancing the gut environment by depressing ammonia concentration in feces and intestinal pH. Therefore, the increased abundance of Bifidobacterium with heart failure could be the consequence of a feedback mechanism of the organism, acting against pathogenic bacteria and toxic mediators. For instance, some studies have shown an increased abundance of Bifidobacterium in heart failure patients (25). This is consistent with our findings and indicates that alterations in the structural composition of the gut flora might be a potential factor in the progression of CHF.

Phenylacetylglutamine is a byproduct of the catabolism of the essential amino acid phenylalanine by intestinal microorganisms. Phenylalanine is catabolized by the gut flora to form phenylpyruvate and phenylacetic acid, and liver enzymes catalyze the formation of PAGln from phenylacetic acid and glutamine (26, 27). The PAGln plasma concentration was found to independently predict the risk of MACE (18). Then, BNP is the most widely used bioactive hormone for the diagnosis in heart failure. Also, LVEDD and LVESD are common clinical echocardiographic indices that can effectively reflect structural changes in the left ventricle ejection fraction. The Spearman correlation analysis in this study showed that BNP (r = 0.924, p < 0.001), LVESD (r = 0.529, p < 0.001), LVEDD (r = 0.477, p < 0.001) were positively correlated with PAGln, and LVEF (r = -0.0.587, p < 0.001) was negatively correlated with PAGln. The CCA analysis showed that five significant clinical phenotypic data explained 72.7% of the species distribution. The arrows representing BNP and PAGln had the longest lengths, indicating that BNP and PAGln had the greatest effect on the change in the flora; the BNP and PAGln arrows had similar lengths, indicating that both have similar effect sizes on the flora of the CHF group. Hyperactivation of the sympathetic nervous system is a distinctive characteristic of CHF, and prolonged activation can lead to the worsening of CHF. Thus, PAGln can act on the β2-adrenergic receptors in the heart, resulting in hyperexcitation of the sympathetic nervous system, which can exacerbate heart failure (28). PAGln also was substantially different among patients who did and did not experience heart failure-related events (29). Therefore, the level of PAGln can be considered an indicator for screening for heart failure and predicting prognosis.

In our study, the plasma PAGln concentrations were substantially higher in the CHF group compared to the control group(p < 0.001), and the plasma PAGln concentrations were substantially higher in the patients in NYHA class IV than those in NYHA class III (p < 0.001), indicating that the more severe the degree of heart failure, the higher the PAGln concentration. The correlation analysis revealed that Parabacteroides and Bacteroides were negatively correlated with BNP and PAGln, and Romboutsia and Blautia were negatively correlated with PAGln. Klebsiella was positively correlated with BNP, and Escherichia-Shigella was positively correlated with BNP and PAGln. Furthermore, Alistipes was negatively correlated with BNP, and Parabacteroides was negatively correlated with LVEDD. Inflammation and myocardial fibrosis are the basic pathological mechanisms of heart failure. With heart failure, there is a lack of intestinal motility, impaired absorption in the mucosal villi, and damage to the mucosal barrier of the intestinal epithelium, which subsequently leads to increased intestinal permeability and failure to remove bacteria and food residues from the intestinal lumen promptly. This promotes the proliferation of Gram-negative bacteria such as Klebsiella, resulting in the easy entry of toxic mediators such as lipopolysaccharides (LPS) into the circulation, further stimulating the activation of the inflammation and immune response and exacerbating the progression of heart failure. Therefore, we suggest that Klebsiella exacerbates the progression of heart failure by releasing LPS. Also, we speculate that PAGln is a microbial metabolite that is present in Klebsiella, and the role and mechanism of Klebsiella in the development of heart failure should be investigated more thoroughly in the future. In addition, there were significant differences in the LVEF, LVESD, LVEDD, and BNP indices across the three groups. These indices are often involved in the diagnosis and differential diagnosis of heart failure, assessing the extent of the disease, and evaluating the prognosis, and are widely used in clinical practice. In mice given an ob/ob and high-fat diet, Wang et al. (30) reported that Parabacteroides distasonis had positive metabolic effect by decreasing weight gain, insulin resistance, and hepatic steatosis. Interventional treatment of mice fed a high-fat diet using P. distasonis revealed that P. distasonis dramatically altered the bile acid profile, elevating the content of lithocholic acid and ursodeoxycholic acid and increasing the level of succinic acid in the intestine. Additionally, Romboutsia species, such as Romboutsia sedimentorum (31) and Romboutsia ilealis (32), were capable to apply carbohydrates and glucose to create short-chain fatty acids(SCFAs). In a mouse model, SCFAs inhibited hypertension-induced cardiac inflammatory responses, myocardial hypertrophy, and fibrosis (33–35). Previous studies have found that corticosterone can combine with and activate mineralocorticoid receptors, leading to sodium retention, which can lead to increased blood pressure (36, 37). A study (38)showed that the intestinal corticosterone levels were increased 4.4-fold in Wistar rats on a high salt diet. This study also found that corticosterone is significantly and negatively associated with some high abundance bacteria such as Bacteroides and Alistipes. Bacteroides can create arachidonic acid to prevent intestinal corticosterone synthesis and cut down intestinal corticosterone’s elevation owning to a high-salt diet, therefore, exerting a hypotensive effect. Previous studies have found a reduced abundance of Blautia in patients with CHF (19). Blautia is one of the important SCFA-producing bacteria, with anti-inflammatory effects, which contributes to intestinal mucosal injury’s recovery and is correlated with the visceral fat content inversely (39). Then, Empagliflozin improved cardiovascular disease risk factors, increased levels of SCFA-producing bacteria, and reduced levels of severely harmful bacterial species, including Escherichia-Shigella (40). This leads us to suggest that the above bacteria may affect cardiac function through different pathways.

We found out that the presence of disturbed intestinal flora outstandingly elevated the levels of PAGln, and the correlation among the florae was materially high. Therefore, the metabolites and cardiac function indicators in patients with heart failure suggested that the normal function of the intestinal flora is impaired with heart failure and that pathophysiological changes in the intestine in turn aggravate the development of heart failure. Our study showed a positive correlation between BNP and Pagln. And also found that the more severe the heart failure, the higher the level of Pagln. Consequently, it can be used as an indicator for early screening, diagnosis, and treatment for CHF. In addition, colony transplantation therapy has been used in patients with irritable bowel syndrome, obesity, diabetes, and Alzheimer’s disease, and it may become a new direction for heart failure treatment in the future as this treatment technique matures.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: NCBI repository, accession number PRJNA909972.

The studies involving human participants were reviewed and approved by Ethics Committee of Shanghai Fengxian District Central Hospital. The patients/participants provided their written informed consent to participate in this study.

BC and ZZ conducted the investigation and wrote the original draft. BC, ZZ, YS, and HD reviewed and edited the manuscript. HW and ZQ provided the conceptualization and funding. ZQ was the supervisor and conducted project administration. All authors contributed to the article and approved the submitted version.