Acute myocardial infarction induces sex-specific, time-dependent remodeling of the gut microbiome and intestinal immune compartment in retired breeder C57BL/6N mice

Figure 1. Microbiota composition at baseline. Fecal pellets from retired breeder mice (n=19 females, n=22 males) were collected after 4 weeks of acclimation to the vivarium and prior to surgery to analyze baseline gut microbiota composition. 16S rRNA sequencing of fecal DNA enabled microbiota profiling. Analyses were performed using MicrobiomeAnalyst v2.0. LEfSe-based cladogram was prepared using Galaxy, an open-source platform hosted by George Mason University. (A) Differences in alpha (within-sample) diversity between male and female microbiota. Chao1 and Shannon indices of alpha diversity reflect taxonomic richness (Chao1 and Shannon) and evenness (Shannon) of microbiota. **p < 0.01, ***p < 0.001. (B) Bray-Curtis Index with PERMANOVA was used to calculate beta (between-sample) diversity in microbiota samples collected from males and females at baseline. Results are visualized by spatial clustering on PCoA plots. (C) Relative abundance of the 15 most abundant bacterial families at baseline in males and females. * indicates significant differential abundance (unpaired Student’s t-test with Benjamini-Hochberg FDR correction, q < 0.05). (D) Left: Microbial features differentially enriched…

Figure 2. Microbiota composition at different timepoints after SH and MI in males. Fecal pellets were collected from male mice on the day of surgery (D0) and day 3 (D3) or day 7 (D7) after SH or MI. 16S rRNA sequencing of fecal DNA enabled microbiota profiling. Analyses were performed using MicrobiomeAnalyst v2.0. Each dot indicates a sample from an individual mouse. (A) Differences in alpha (within-sample) diversity after SH and MI at different timepoints. Chao1 and Shannon indices of alpha diversity reflect taxonomic richness (Chao1 and Shannon) and evenness (Shannon) of microbiota. **p < 0.01, ***p < 0.001. (B) Bray-Curtis Index with PERMANOVA was used to calculate beta (between-sample) diversity in microbiota samples collected at different timepoints after SH and MI. Results are visualized by spatial clustering on PCoA plots. (C) Changes in percent relative abundance of the 15 most abundant bacterial phyla and families after SH or MI. * indicates significantly differential abundance (one-way ANOVA with Benjamini-Hochberg FDR correction, q < 0.05). (D) LEfSe showing most differentially abundant taxa in male microbiota at different timepoints after SH and MI (LDA score >2, q < 0.05). LDA…

Figure 3. Microbiota composition at different timepoints after SH and MI in females. Fecal pellets were collected from female mice on the day of surgery (D0) and day 3 (D3) or day 7 (D7) after SH or MI. 16S rRNA sequencing of fecal DNA enabled microbiota profiling. Analyses were performed using MicrobiomeAnalyst v2.0. Each dot indicates a sample from an individual mouse. (A) Differences in alpha (within-sample) diversity after SH and MI at different timepoints. Chao1 and Shannon indices of alpha diversity reflect taxonomic richness (Chao1 and Shannon) and evenness (Shannon) of microbiota. (B) Bray-Curtis Index with PERMANOVA was used to calculate beta (between-sample) diversity in microbiota samples collected at different timepoints after SH and MI. Results are visualized by spatial clustering on PCoA plots. (C) Changes in percent relative abundance of the 15 most abundant bacterial phyla and families after SH or MI. * indicates significantly differential abundance (one-way ANOVA with Benjamini-Hochberg FDR correction, q < 0.05). (D) LEfSe showing most differentially abundant taxa in female microbiota at different timepoints after SH and MI (LDA score >2, q < 0.05). LDA scores represent…

Figure 4. Sex-specific changes in microbiota after SH and MI. Fecal pellets were collected from male and female mice 3 days (D3) or 7 days (D7) after SH or MI. 16S rRNA sequencing of fecal DNA enabled microbiota profiling. Analyses were performed using MicrobiomeAnalyst v2.0. Each dot indicates a sample from an individual mouse. (A) Bray-Curtis Index with PERMANOVA was used to calculate beta (between-sample) diversity in microbiota samples collected at different timepoints after SH and MI from males and females. Results are visualized by spatial clustering on PCoA plots. (B) LEfSe showing most differentially abundant taxa in male and female microbiota at different timepoints after SH and MI (LDA score >2, q < 0.05). LDA scores represent the effect size of each taxon. (C, D) Relative abundance of the most discriminative bacterial families (C) and species (D) from male and female MI D3 samples. Significance was calculated using ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5. Gut metabolite profiles and microbe-metabolite associations after MI. Cecum contents were collected from NoSx or MI mice on D3. Untargeted UPLC-MS in the negative and positive ion modes allowed for metabolite quantification and characterization. 16S rRNA sequencing of cecal DNA enabled microbiota profiling. Analyses were performed using MetaboAnalyst v6.0 and MicrobiomeAnalyst v2.0. Each dot indicates a sample from an individual mouse. (A) Principal Component Analysis (PCA) of both negative and positive ion mode metabolites shows significant differences in metabolite profiles between NoSx and MI D3 cecal metabolomes in males and females (p-values calculated with PERMANOVA). Results are visualized on PCA plots. (B) Volcano plot shows significant changes in male MI D3/female MI D3 metabolite abundance (log2 fold change [log2FC]; significance determined using an empirical Bayes-moderated statistic) in the positive ion mode, revealing 2 upregulated (increased) metabolites and 1 downregulated (decreased) metabolite in male over female samples (adjusted p-value < 0.05). (C) Left: Pearson r correlation analyses of positive ion mode metabolites showing male-female pattern correlation…

Figure 6. Changes to small intestine structure over time after MI in male and female mice. The small intestine was isolated, the contents extruded, external fat and Peyer’s Patches removed, and tissue weighed. Portions of the jejunum were fixed, embedded in paraffin, sectioned, stained with H&E, and digitally scanned. Scanned sections from NoSx, MI D3, and MI D7 cohorts were analyzed using QuPath v6.0. Each dot indicates the average result from the analyses of 3–8 villi from an individual mouse. (A) Whole small intestine weights indexed to body weight in males and females. Significance calculated using ANOVA and Tukey’s post-hoc test. *p < 0.05, ***p < 0.001, ****p < 0.0001. (B) A representative small intestine villus demonstrating the measurement of villi height, crypt thickness, muscularis thickness and identification of endothelial and goblet cells. (C, D) Differences in villi height, crypt length, epithelial and goblet cell numbers/µm villi, and the ratio of goblet:epithelial cell numbers in male (C) and female (D) mice at different timepoints after MI. Significance was calculated using ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001.

Figure 7. Innate and T cell populations in the small intestine. The small intestine was isolated, the contents extruded, external fat and Peyer’s Patches removed, and tissue weighed. A portion of the jejunum was weighed and used to prepare single cell preparations that were incubated with fluorescently labeled antibodies for analysis by flow cytometry. Flow data were analyzed using FlowJo software v10.10. Each dot indicates a sample from an individual mouse. These populations were identified and enumerated using the gating strategy shown in Supplementary Figure 4. Cell numbers normalized to total small intestine weight and are shown as numbers per gram of small intestine. (A, B) Differences in absolute numbers of innate cells (first row) and T cells (second row) cells per gram of intestine in male (A) and female (B) mice between NoSx and SH or MI at different timepoints. Y-axis scales are matched to allow for easier comparisons of cell numbers between males and females. Significance was calculated using ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Significant sex differences are indicated by a dagger symbol in (B).

Figure 8. Innate and T cell populations in the heart. The heart was isolated, blood removed, and tissue weighed. Single cells suspensions were prepared with enzymatic tissue digestion and filtration and subjected to incubation with fluorescently labeled antibodies for flow cytometry analysis. Data were analyzed using FlowJo software v10.10. Each dot indicates a sample from an individual mouse. These populations were identified and enumerated using the gating strategy shown in Supplementary Figure 4. Cell numbers are shown as numbers per whole heart. (A, B) Differences in absolute numbers of innate cells (first row) and T cells (second row) cells per whole heart in male (A) and female (B) mice between NoSx and SH or MI at different timepoints. Y-axis scales are matched to allow for easier comparisons of cell numbers between males and females. Significance was calculated using ANOVA with Tukey’s post-hoc test. *p < 0.05, **p < 0.01. Significant sex differences are indicated by a dagger symbol in (B).