Akkermansia muciniphila prevents cold-related atrial fibrillation in rats by modulation of TMAO induced cardiac pyroptosis

Abstract

Background: Cold exposure is one of the most important risk factors for atrial fibrillation (AF), and closely related to the poor prognosis of AF patients. However, the mechanisms underlying cold-related AF are poorly understood.

Methods: Various techniques including 16S rRNA gene sequencing, fecal microbiota transplantation, and electrophysiological examination were used to determine whether gut microbiota dysbiosis promotes cold-related AF. Metabonomics were performed to investigate changes in fecal trimethylamine (TMA) and plasma trimethylamine N-oxide (TMAO) during cold exposure. The detailed mechanism underlying cold-related AF were examined in vitro. Transgenic mice were constructed to explore the role of pyroptosis in cold-related AF. The human cohort was used to evaluate the correlation between A. muciniphila and cold-related AF.

Findings: We found that cold exposure caused elevated susceptibility to AF and reduced abundance of Akkermansia muciniphila (A. muciniphila) in rats. Intriguingly, oral supplementation of A. muciniphila ameliorated the pro-AF property induced by cold exposure. Mechanistically, cold exposure disrupted the A. muciniphila, by which elevated the level of trimethylamine N-oxide (TMAO) through modulation of the microbial enzymes involved in trimethylamine (TMA) synthesis. Correspondingly, progressively increased plasma TMAO levels were validated in human subjects during cold weather. Raised TMAO enhanced the infiltration of M1 macrophages in atria and increased the expression of Casp1-p20 and cleaved-GSDMD, ultimately causing atrial structural remodeling. Furthermore, the mice with conditional deletion of caspase1 exhibited resistance to cold-related AF. More importantly, a cross-sectional clinical study revealed that the reduction of A. muciniphila abundance was an independent risk factor for cold-related AF in human subjects.

Interpretation: Our findings revealed a novel causal role of aberrant gut microbiota and metabolites in pathogenesis of cold-related AF, which raises the possibility of selectively targeting microbiota and microbial metabolites as a potential therapeutic strategy for cold-related AF.

Funding: This work was supported by grants from the State Key Program of National Natural Science Foundation of China (No.81830012), and National Natural Science Foundation of China (No.82070336, No.81974024), Youth Program of the National Natural Science Foundation of China (No.81900374, No.81900302), and Excellent Young Medical Talents supporting project in the First Affiliated Hospital of Harbin Medical University (No. HYD2020YQ0001).

Keywords: Akkermansia muciniphila; Atrial fibrillation; Cold; Gut microbiota; TMAO.

Figure 1

Cold exposure induces AF and gut microbiota dysbiosis in rats. (A) Endocardial and surface electrograms recordings in response to burst pacing in RT and Cold rats. (B) Number of rats in which AF could be reproducibly induced by right atrial burst pacing. (C) AF duration in RT and Cold groups (n=13 per group). (D) Representative images of Masson's trichrome staining and the collagen volume fraction in the atria of RT and Cold rats (n = 3 per group). Magnification × 200, scale bar = 50μm. (E) Representative bands and quantification of expressions of TGF-β1 and α-SMA in rats of RT and Cold rats groups (n = 6 per group). (F) Principal coordinate analysis (PCoA) of unweighted UniFrac revealed clustering of the gut microbiota after temperatures. Each dot represents a single sample of feces. (G) The top 10 of relative abundance of gut microbiota at family level in feces of RT and Cold rats (n = 13 per group). GAPDH was used for normalization. Data are expressed as mean ± SEM and compared by Student's t test (D and E) or Wilcoxon test (C). AF inducibility (B) was presented as numbers and compared by Fisher exact test. RT, room temperature. AF, atrial fibrillation. SR, sinus rhythm.

Figure 2

Oral supplementation of A. muciniphila protects Cold rats against AF. (A) Endocardial and surface electrograms recordings in response to burst pacing in Cold and Cold + A. muciniphila group. Supplementation with A. muciniphila reduced AF inducibility (B) and duration (C) in Cold rats (n = 10 per group). (D) Representative images of Masson's trichrome staining and the collagen volume fraction in the atria of Cold and Cold+A. muciniphila rats (n = 4 per group). Magnification × 200, scale bar = 50μm. (E) Representative bands and quantification of expressions of TGF-β1 and α-SMA in rats of Cold and Cold+A. muciniphila groups (n = 6 per group). AF inducibility (F) and AF duration (G) in Cold (n = 10) and Cold + pasteurised A. muciniphila group (n = 9). GAPDH was used for normalization. Data are expressed as mean ± SEM and compared by Student's t test (D and E) or Wilcoxon test (C and G). AF inducibility (B and F) was presented as numbers and compared by Fisher exact test.

Figure 3

A. muciniphila reverses the elevated TMAO induced by cold exposure through restraint of TMA synthesis. (A) Heat map of the nontargeted metabolomics in RT and Cold rats (n = 12 per group). (B) Heat map of relative abundances of TMAO-related metabolites in plasma samples from RT and Cold rats detected by targeted metabolomics (n = 9 per group). Comparison of TMAO (C), Choline (D), and Carnitine (E) levels in plasma between RT and Cold groups (n = 9 per group). (F) The results of plasma TMAO levels in human donors (n=1305) during 12 months. (G) Oral supplementation of A. muciniphila reduced the concentration of TMAO and TMA in Cold rats (n = 10 per group). (H) Statistical results for the expression levels of CntA, CntB, CutC, CutD, YEAX and YEAW in feces from rats of Cold and Cold+ A. muciniphila group (n = 9 per group). Throughout, data are expressed as mean ± SEM and compared by Student's t test. TMAO, trimethylamine N-oxide. TMA, trimethylamine.

Figure 4

TMAO induces pyroptosis of cardiac myocytes and fibroblasts by enhancing M1 macrophages infiltration. BMDMs were cultured in the absence or presence of TMAO (10 μM) for 48 hours. (A) CD86⁺ M1 population were sorted from CD11b/c⁺ macrophages and caspase1-p20 were sorted from CD86⁺ M1 population. (B) CD163⁺ M2 population were sorted from CD11b/c⁺ macrophages. (C) The percentages of CD86⁺ M1 phenotype or CD163⁺ M2 phenotype out of CD11b/c⁺ macrophages were determined (n = 5 per group). (D) Representative bands and quantification of expressions of Casp1-p20 and Cleaved-GSDMD in macrophages in the absence or presence of TMAO (n = 6 per group). CM or CF were respectively co-cultured with macrophages in the absence or presence of TMAO (10 μM). (E) Representative bands of Casp1-p20 and Cleaved-GSDMD in CM and CF (n = 6 per group). Quantification of expressions of Casp1-p20 and Cleaved-GSDMD in CM (F) and CF (G). GAPDH was used for normalization. The data are given as mean ± SEM and compared by Student's t test. BMDMs, Bone marrow derived macrophages; CM, cardiac myocytes; CF, cardiac fibroblasts.

Figure 5

NSA treatment protects against cold-related AF in rats by attenuation of atrial pyroptosis. Representative immunofluorescence staining of DAPI (blue), CD86 (red), CD163 (green) on the atrial tissue from Cold and RT rats (n = 5 per group). Scale bar = 100 μm. (B) and (C) Quantitative analysis of CD86 and CD163 positive cells. (D) Representative bands of Casp1-p20 and Cleaved-GSDMD expressions in atria of RT and Cold rats (n = 6 per group). (E) Representative bands of Casp1-p20 and Cleaved-GSDMD expressions in Cold and Cold + NSA rats (n = 6 per group). (F and G) Quantification of Casp1-p20 and Cleaved-GSDMD in rats of RT and Cold groups, or Cold and Cold + NSA groups. (H) Representative images of Masson staining, and quantification of collagen volume fractions in atria of Cold (n = 3) and Cold+NSA groups rats (n = 4). Magnification ×200, scale bar = 50 μm. AF induction rate (I) and AF duration (J) in Cold (n = 6) and Cold+NSA groups (n = 10). GAPDH was used for normalization. The data are given as mean ± SEM and compared by Student's t test (B, C, F, G and H) or Wilcoxon test (J). AF inducibility (I) compared by Fisher exact test. NSA, necrosulfonamide.

Figure 6

Caspase1 knockout protects against cold-related AF in mice by restraint of atrial pyroptosis. AF induction rate (A) and AF duration (B) in RT and Cold mice (n=7 per group). (C) Representative bands and quantification of Casp1-p20 and Cleaved-GSDMD in atria of RT and Cold mice (n=6 per group). (D) Endocardial and surface electrograms recordings in response to burst pacing in Casp1^flox/flox and Casp1^mef2c/^mef2c mice with a three-week cold exposure (n=7 per group). (E) AF induction rate. (F) AF duration. Representative bands of the protein expressions of Casp1-p20 and Cleaved-GSDMD (G), TGF-β1 and α-SMA (H) in atrial tissue from Casp1^flox/flox and Casp1^mef2c/^mef2c mice (n=6 per group). (I) Quantification of Casp1-p20, Cleaved-GSDMD, TGF-β1 and α-SMA. GAPDH was used for normalization. The data are given as mean ± SEM and compared by Student's t test (C and I) or Wilcoxon test (B and F). AF inducibility (A and E) compared by Fisher exact test.

Figure 7

The decreased A. muciniphila was associated with AF in human subjects during winter. (A) The NMDS of unweighted UniFrac showed that the gut taxonomic composition was significantly different between winter AF and other groups. The closer the spatial distance of the sample, the more similar the species composition of the sample. (B) Chao1, Shannon and Simpson indices were analysed in human. Chao1 indices reflect community richness, and the Shannon and Simpson indices represent community diversity. (C) Heatmap of fecal microbiota from summer SR(n=16), summer AF(n=11), winter SR(n=13) and winter AF(n=12) patients. (D) q-RT PCR validation of the relative abundance of A. muciniphila in feces collected from human subjects during summer (n = 106) or winter (n = 103). (E) The relative abundance of A. muciniphila in feces collected from subjects with SR (n = 178) or AF (n = 31). (F) Prevalence of AF according to A. muciniphila abundance stratified by season. A. muciniphila abundance was categorized into three levels according to the tertiles of A. muciniphila. Seasons were categorized into summer and winter. Throughout, data are expressed as mean ± SEM and compared by one-way ANOVA (B) and Student's t test (D and E). NMDS, non-metric multidimensional scaling; OTUs, operational taxonomic units; Summer SR, patients with sinus rhythm during summer; Summer AF, patients with atrial fibrillation during summer; Winter SR, patients with sinus rhythm during winter; Winter AF, patients with atrial fibrillation during winter; SR, sinus rhythm; AF, atrial fibrillation; A. muciniphila, Akkermansia muciniphila.

Figure 8

Summary scheme outlining AF-promoting mechanisms from cold microbiota dysbiosis. Cold exposure led to an decrease in A. muciniphila, by which promoted intestinal-derived TMA production and augmented the levels of TMAO in plasma, then TMAO enhanced M1 macrophages polarization and promoted atrial pyroptosis, ultimately leading to atrial structural remodeling and atrial fibrillation.