The Gut Microbiota and Seleno-Compound as Emerging Risk Factors for Acute Myocardial Infarction

Background Methods The feces of the 44 subjects (AMI: 19; control: 25) were collected for fecal genomic DNAs extraction. The variable region V3–V4 of the 16S rRNA gene was sequencing by the platform of Illumina Miseq. The abundances of metabolites were analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways.


Introduction
Acute myocardial infarction (AMI) is a growing epidemic in the developing countries, and the leading cause of death in the industrialized societies. The episodes of AMI are related to transmural myocardial ischemia, and result in myocardial injury or necrosis. 1 The pathophysiology of AMI is initiated by the erosion, ssuring, dissection or rupture of the atherosclerotic plaques of the coronary arteries. The onset of the plaque rupture results in the cascade of platelet adhesion, activation, aggregation, and atherothrombosis formation. 1 Therefore, the blood ow of the coronary artery is disrupted abruptly, and occluded by the thrombus formation. The traditional risk factors of AMI have been known as hypertension, diabetes mellitus, dyslipidemia, family history, smoking, age, and post-menopause. 1 Nowadays, the microbiota, which is a dynamic ecosystem shaped by a few factors such as microbiome, genetics, diet, and environment has been investigated for its crucial function for the human health. There are more than 2000 species of bacterial organisms in the human body, and the gut microbiome is by far the greatest mass of microbiota. 2 These bacterial organisms have developed complicated connection with the human bodies. Many studies have shown that intestinal ora disorders are closely related to the occurrence of in ammation, metabolic, and systemic diseases (e.g., obesity, diabetes, dyslipidemia, malignancies, psychiatric problems, autoimmune disorders, and cardiovascular diseases. 3 Therefore, gut microbiota which contributes to the in ammatory metabolism, might possibly be associated with the AMI episodes. Based on this point, and given the need for effective prevention or treatment of AMI, we conduct this comparative study to investigate the composition, and functional differences between the patients with /without AMI by 16S ribosomal RNA (rRNA) microbiome analysis.
We attempt to clarify the speci c pro les of gut microbiota in AMI patients, and their potentiality for the treatment of AMI.

Methods
The Patient and Public Involvement statement This study was approved by the ethics committee and institutional review board (IRB) on human research of the Medical Research Department of National Taiwan University Hospital, Taipei, Taiwan. All subjects provided informed consent before participating in the study. The participants provided their written informed consent to participate in this study. All research was performed in accordance with relevant guidelines/regulations, and include in their manuscript a statement con rming that informed consent was obtained from all participants and/or their legal guardians. Research involving human research participants must have been performed in accordance with the Declaration of Helsinki.

Study Setting and Participants
In this single-center and case-control study, 44 patients (AMI: 19 vs control: 25) were enrolled and collected their stool for microbiome analysis between June 2020 and Oct 2020. AMI was de ned by the criteria based on a combination of two of three characteristics (e.g., typical symptoms, the electrocardiographic pattern, and cardiac enzyme raise). The control subjects were without coronary artery disease (CAD) based on clinical history, noninvasive stress testing, and coronary imaging studies including coronary computed tomography and/or coronary angiography. Subjects with prior gastrointestinal surgery (e.g., colectomy, ileectomy, and gastrectomy), current administration of antibiotics, in ammatory bowel disease, auto-immune diseases, and malignancy were excluded. The gastrointestinal function of these subjects was normal without vomiting, diarrhea, and/or constipation to the day of stool collection.

Fecal Collection and Processing
The rst defecation sample in the morning was collected from each subject. The samples were collected and frozen in liquid nitrogen for future isolation of bacterial genomic DNA. The inner part of the samples was used for sequencing to avoid environmental contamination. Total bacterial DNA was extracted from fecal samples within 2 weeks by the stool DNA Kit (Omega Biotek, Norcross, GA, USA) according to the manufacturer's protocol.

16S rRNA Gene Sequencing
To investigate the compositional change of microbiome associated with AMI, PCR ampli cation was performed on the V3− V4 region of the 16S rRNA gene with TransStart Fastpfu DNA Polymerase (Takara) following by sequencing on the Illumina MiSeq v3 chemistry (Illumina Inc., San Diego, CA, United States) in multiple runs and pooling all 44 samples together according to the manufacturer's instructions.

Comparison of Gut Microbiome Composition
The raw 16S data were analyzed by Pandaseq, processed through the QIIME (version 1.8.0), clustered into operational taxonomic units (OTUs) with a 97% identity cutoff and taxonomically. The alphadiversity measures were calculated based on the OTUs counts. Number of observed OTUs indicates microbial richness, which measures the number of taxa in each sample. The linear discriminant analysis effect size (LEfSe) was calculated using the online version of Galaxy3. The linear discriminate analysis (LDA) was performed using a one-against-all strategy, and OTUs showing a score higher than 2 are selected. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways database was used to predict differences in bacterial biochemical pathways between the patients with /without AMI.

STATISTICS
We applied the metagenomic sequencing for intestinal ora analysis (e.g., ora composition, diversity, functional, and metabolite analyses). Descriptive statistical analyses were used to summarize the clinical features. Other analyses were performed by SPSS 20.0 (Chicago, IL, USA), including unpaired t-test and continuous variables are expressed as the mean ± standard deviation (SD). p < 0.05 was de ned as statistically signi cant.

Analysis of Diversity between Groups
The alpha-diversity (richness, uniformity, and Shannon index of diversity) was an indicator for describing species diversity. After analyzing intestinal microbial metagenomics, there was a difference in the relative richness of bacteria between the groups ( Fig. 1 and Fig. 2). The abundance of bacteria was more enriched in the AMI group (p = 0.01, 95% CI). The phylogenetic analogue of taxon analyses also revealed more richness of bacteria in the AMI group (Fig. 3, p < 0.001, 95% CI). There was no difference of evenness and uniformity between the groups (Fig. 4, p = 0.4). The Shannon index was an information statistic index, which assumed all species were represented in a sample and that they were randomly sampled. The Shannon index between the groups was similar (Fig. 5, p = 0.23, 95% CI).
For further exploring these ndings, we performed a LEfSe analysis to identify differences in abundant taxa between the samples of patients with/without AMI. There were over expression of several bacterial genera between the groups ( Because the gut ora could produce and consume many metabolites, we assumed certain metabolites were associated with the abundance of the ora. The KEGG metabolic pathways was assisted to estimate the difference in metabolic potential between the groups. There was a difference of the seleno-compound distribution between the groups. The seleno-compound was more abundant in the AMI group at the Family, Genus, and Species levels (Fig. 8, 11, and 14; all LDA scores > 2).

Discussion
Trillions of microbial cells harbor at the human intestine as one part of our physiological ecosystem. These communities of bacteria, fungi, archaea, and viruses are collectively referred as "microbiota," and their genome as the "microbiome." 4 The microbial colonization of the gastrointestinal tract initiates at birth and the composition of the species-level phylotypes differs individually. 5 Approximately 99% of human gut microbiota are composed by the four phyla of bacterial species, including Firmicutes, Bacteroides, Actinobacteria, and Proteobacteria. 6 Among them, Firmicutes and Bacteroidetes phyla are the two major species accounting for almost 90% of bacterial species in the human intestines. 7 The gut microbiota plays an important role in the immune support and host metabolism. Abnormal changes of the gut ora might affect host health by inducing immune response. 7 From the previous studies, the gut microbiota could code various enzymes, synthesize vitamins, produce amino acids, and digest various nondigestible dietary components (e.g., large polysaccharides, resistant starch, pectin, cellulose, hemicellulose, alcohols, and sugars, etc. ). 5 The interindividual variabilities of the microbial communities are interfered by host-microbial interactions, host genetics, host diets, host life-style, and environmental conditions (e.g., PH gradient, gastric motility, oxygen content, and nutrition, etc.). 4 The diverse, and various metabolites from gut microbiota also affect the host physiology vice versa. 6 The Main Findings of This Study In this study, the DNAs of the gut microbiota were extracted from the fecal samples of the subjects, and the DNA libraries were constructed by 16S rRNA gene sequencing on the MiSeq (Illumina) platform. The highly similar sequences were grouped into the OTUs. After alpha diversity analysis, the OTUs and faith phylogenetic diversity (PD) were noted to be different between AMI and control groups (p-value = 0.01 and < 0.001, individually). Therefore, the abundance of gut microbiota was different between the AMI and control groups.
The taxonomic cladogram reported all clades of the gut microbiota, and there was less abundance of Selenomonadales in the AMI group relative to the control group at the Family, Genus, and Species levels. The KEGG database resource was comparing for evaluating the possibly metabolic pathways involved with the AMI episodes. After the metagenome functional analysis, the seleno-compound wa noted to be more abundant in the AMI group.
From the current studies, there was relatively increasing abundance of Ruminococcus gnavus in the patients with CAD. 8 In addition to coronary atherosclerosis, the Ruminococcus gnavus was also linked to speci c in ammatory process, production of in ammatory polysaccharide, and in ammatory bowel disease. 8 On the contrary, the abundances of Lachnospiraceae and Ruminococcus gauvreauii were decreasing in the subjects with CAD. 8 This is the rst study to demonstrate "another distinct" gut microbiome-Selenomonadales and seleno-compound to be associated with the occurrence of AMI. The possible mechanisms for altering microbiota, and the potential metabolome in association with the AMI episodes are discussed in the following paragraphs.
The Possibly Metabolic Pathways of Selenomonadales in Correlation with AMI Episodes

Selenomonadales and short-chain free fatty acids (SCFAs)
Generally, the primary products of anaerobic fermentation of undigested nutrients (e.g., resistant starch, dietary ber, and various complex polysaccharides) after bacterial hydrolysis are monosaccharides. Those monosaccharides are further fermented to various fatty acids ranging from 1 to 6 carbon chains, commonly referred as short-chain fatty acids (SCFAs) such as acetate, butyrate, and propionate. 4 Acetate is utilized by butyrate producers to produce butyrate while the butyryl-CoA: acetate-CoAtransferase pathway is the main process for the biosynthesis of butyrate. 9 Butyrate acts as histone deacetylases inhibitor, and involves in epigenetic regulation of T-cell development and maintenance. 10 Those SCFAs (e.g., acetate, butyrate, and propionate) are transported by the monocarboxylate transporters on the mucosal epithelium of the intestines, and into the systemic circulation. Although 5-10% SCFAs, particularly butyrate serves as energy substrates for epithelial cells of the intestines, SCFAs also serve as signaling molecules. SCFAs interact with G-protein receptor (e.g., GPR41 and GPR43), and olfactory receptor 78 (Olfr78), which exhibit various physiological functions (e.g., histone deacetylases inhibition, chemotaxis, phagocytosis modulation, reactive oxygen species induction, regulating immune cell proliferation, lipid, and glucose metabolism, etc. Selenomonadales are the members of Firmicutes, and the class of Negativicutes which are Gramnegative. Selenomonadales have been reported to ferment carbohydrates into acetate and lactate. 9 Therefore, those SCFAs metabolites generated by the Selenomonadales might lead to the downstream metabolic alterations, and affect coronary atherosclerosis in correlation with AMI episodes.

Dysbiosis of gut microbiota in AMI subjects
An abnormal imbalance of the gut microbiota is called "dysbiosis". Foods are important to maintain the integrity of the diversity, and keep balance of microbiota for producing SCFAs e ciently. However, high fat, and modern diets tend to hamper the gut microbiota ecosystem, which may explain the increasing incidence of metabolic diseases recently. 10 Shifts from the animal-based diets to be plant-based diets could alter, and modify the production of SCFAs. 4 An increase in Firmicutes has been related to increase enzymes for disintegrating polysaccharides from food, and produce SCFAs as well. 7 Those SCFAs are critical for repairing the cardiac structure after AMI episodes from the animal models. 4 The intestinal microbiota, and their metabolites might stimulate the immune system via intestinal lymphoid tissues. 4 The relative less abundance of Selenomonadales in the AMI subjects might be associated with deprivation of SCFAs in the intestine, and resulting in the loss of bene ts from SCFAs mentioned above. Therefore, it might be speculated that propagation or sterilization of bacterial species speci c for augmenting SCFAs generation, might prevent in ammatory process including coronary atherosclerosis, and could be bene cial for the treatment of AMI subjects.

Intervention of Selenomonadales with probiotic
In addition to changing dietary habits, probiotic is another possible method to modulate gut microbiota pro les. In a rat myocardial infarction model, administration of either Lactobacillus plantarum or Lactobacillus rhamnosus GR-1 is associated with attenuation of cardiac remodeling after AMI. 4 As Selenomonadales are considered, Clostridium butyricum, which is an oral diet-added probiotic, has been demonstrated to promote the abundance of Selenomonadales dramatically. Clostridium. butyricum is an anaerobic, Gram-positive, butyric acid-producing bacillus, and has a protective role after intestinal injury by modulating gut microbial metabolites, such as SCFAs. 9 After using Clostridium butyricum probiotic for two weeks, Selenomonadales replace Clostridiales to be the major and dominant bacteria in the intestines. 9 Seleno-compound Metabolism with the Association of AMI We are the rst to nd the relationship between seleno-compound and AMI episodes by metabolomic analysis. Selenium (Se) is a naturally occurring, and essential trace element necessary for activation of speci c enzymes (e.g. glutathione peroxidases and thioredoxin reductase) after oxidative stress. 11 Secontaining enzymes, especially glutathione peroxidase, are involved in regulating the redox balance in almost all tissues 12 and very important for the detoxi cation of reactive oxygen species (e.g., peroxides and hydroperoxides). 11 While the human body is under stress, for instanced, oxidative stress caused by the intense growth activity of the fetus during pregnancy, the rst line of defense against the oxidative stress are the endogenous antioxidants, such as the Se containing compounds. During pregnancy, the placenta also plays an important role for activating seleno-compounds such as glutathione-peroxidase and thioredoxinreductase. 12 Therefore, it is reasonable that the increasing abundance of selenocompound in the AMI subjects might be correlated with the reaction of oxidative stress after AMI episodes.
The Interaction of Selenomonadales, Seleno-compound, and Dietary Se At present, the Selenomonadales have not been reported to be associated with the bioavailable Se-protein compound for supplying selenium to the organisms. 13 The relationship between the Selenomonadales, and the seleno-compound needs to be clari ed in the future. However, the dietary Se has been proved to be cardioprotective for reducing oxidative stress, lowering connexin-43 dephosphorylation, and decreasing TNF-α expression from the rat models. 14 For the patients with cardiac dysfunction, the Se intake might be helpful for improving cardiac remodeling even when the provided Se is within the normal range of physiological values. 14 For the dietary Se, the Se-enriched plants (e.g., onion, broccoli, wild leek, and garlic) possess protective effects on the anti-in ammation, ant-cancer, and anti-oxidant activities via Se-methyl selenocysteine or gamma-glutamyl-Se-methyl selenocysteine. 15,16 Additionally, the combination treatment with Vit E, Se, and anthocyanin from purple carrots shows greater antioxidant activities against D-galactose-induced oxidative damage in rats than those of individual treatments, suggesting the synergistic antioxidant effects of these antioxidants. 17 The protection from Vit E against the adverse effects of nitrites/nitrates is attributed to its ability to reduce ONOO-formation, while Se exerts its protective effects via selenoenzymes/compounds, which reduce ONOO-formed. 18

DATA SHARING
Whether data collected for the study, including participant data and a data dictionary de ning each eld in the set, will be made available to others after publication. These data will be made available after approval of a proposal and a signed data access agreement by e-mail request (e-mail: jeremysnc1000@gmail.com).

Conclusion
The research of gut microbiota has garnered much interest because of its importance in nutrition, disease, and health. This study demonstrates that there is a decreasing abundance of Selenomonadales gut microbiome in the AMI subjects, and the seleno-compound is also increasing after AMI episodes. Our ndings raise the potential role of microbial composition to be a valuable target for preventing, and treatment of AMI. The relationships between the immune microenvironment, intestinal ora, and AMI warrant further investigation. By doing so, gut microbiota-targeted therapy for the AMI could be carried out in the future.

DECLARATION OF INTERESTS
All authors disclose no nancial and personal relationships with other people or organizations that could inappropriately in uence (bias) their work. Analyzed the data and prepared for the data interpretation.
Analyzed the data and prepared for the data interpretation.

Figure 9
Cladogram showing different abundant taxa at Genus level. Alphabets correspond to those in parentheses (CTL: control; ME: AMI).
Page 19/21   Taxonomy differential abundance analysis for the seleno-compound metabolism at Genus level (CTL: control; ME: AMI).

Figure 12
Cladogram showing different abundant taxa at Species level. Alphabets correspond to those in parentheses (CTL: control; ME: AMI).  Taxonomy differential abundance analysis for the seleno-compound metabolism at Species level (CTL: control; ME: AMI).