Gabriela de Castro Magalhães ¹, Ana Carolina Lara Ribeiro¹, Nícia Pedreira Soares¹, Marcos Eliezeck¹, Bruno Sanches¹, Sérgio Ricardo Aluotto Scalzo Júnior¹, André Luis Lima Monteiro¹, Robson Augusto Souza dos Santos ¹, Vladimir Gorshkov², Frank Kjeldsen², Silvia Guatimosim¹, Thiago Verano Braga¹

Gut dysbiosis is a reduction in microorganisms diversity caused, for example, by poor fiber intake, chronical stress and sedentarism. Chronical treatment with antibiotics can lead to dysbiosis, which can lead to cardiac disorders and potentially modify the cardiac proteome. To investigate whether the depletion of gut microbiota by antibiotics can lead to cardiac dysfunctions and a dysregulation of the cardiac proteome. C57BL/6 male mice (8-10 weeks old) were treated (ATB) or not (CTL) with antibiotics for 14 days. After euthanasia, cardiac tissues were collected and prepared for proteomics, phosphoproteomics and N-glycoproteomics analysis. The samples were analyzed by liquid chromatography (EASY-Nlc) coupled with mass spectrometry (Orbitrap Eclipse Tribid). The data was processed with MaxQuant, then analyzed with the programs Perseus and DanteR, it was utilized one-way ANOVA (p < 0.05) and a log2 fold-change of > 0.38 (positively regulated) or < -0.38 (negatively regulated). Also, cardiac tissues were collected and cardiomyocytes isolated for further experiments (CEUA: 99/2020). We identified a total of 3,682 proteins and 5,513 post-translational modifications (PTM) sites, including 3,109 phosphorylation sites and 2,404 former SA-containing N-glycosylation sites in ATB mice hearts. Out of the total proteins found, 98 were differentially regulated, with 36 downregulated and 62 upregulated, also in terms of PTM regulation, 118 sites were differentially modified, 31 downregulated and 87 upregulated, and 34 former SA-containing N-glycosylation sites were altered, 20 downregulated and 14 upregulated. Using the KEGG database, the pathway analysis revealed that the regulated proteins and PTMs in the ATB mice hearts were associated with hypertrophic cardiomyopathy and dilated cardiomyopathy. In addition, we observed a decrease in the contraction-relaxation time (p=0.0032), contraction time (p=0.0019), relaxation time (p=0.0363), contraction speed (p=0.0002), relaxation speed (p=0.005) and shortening area (p<0.0001) in cardiomyocytes derived from ATB mice. Also, a deleterious effect in cardiac Ca2+ handling was observed, where the Ca2+ decay time (50%) was decreased in ATB mice cardiomyocytes, although the Ca2+ peak (F/F0) was not altered. The cardiac tissue’s proteome, phosphoproteome and N-glycoproteome were altered by the antibiotic-induced dysbiosis, in which were found differentially regulated proteins associated to hypertrophic cardiomyopathy and dilated cardiomyopathy, that was corroborated with the negatively impacted cardiomyocytes’ contractility and calcium handling. This study reveals the relationship between gut microbiota modulation and cardiac function.

Agradecimentos: CNPq, Fapemig, and Capes.