Abstract 13808: Investigating Autophagy in Experimental Autoimmune Myocarditis Using Single Cell RNA Sequencing and Spatial Transcriptomics

Background: Autophagy has emerged as an important process in the context of heart failure. Experimental autoimmune myocarditis (EAM) represents an animal model of acute myocarditis (d19) and post-inflammatory dilated cardiomyopathy (d35). Aim: To explore autophagy dynamics in a mouse model of EAM at the single cell level. Methods: 6-8-week-old BALB/c mice were immunized with αMyHC peptide and complete Freund’s adjuvant. Single-cell RNA (scRNA) libraries were prepared using 10x Genomics technology and sequenced, targeting 50’000 reads/cell. For spatial transcriptomics, snap-frozen hearts embedded in OTC were sectioned into 10 μm-thick fragments and placed on 10x Genomics Visium Gene Expression Slide and processed using 10x Genomics Visium protocols. The data were analyzed using RStudio software. LC3B and Beclin-1 proteins were detected by IHC. Results: After quality control check, we included in the RNAseq analysis 25’058 cells clustering in 11 distinct cell populations (Fig. 1A). Expression of autophagic genes Atg7 and Becn1 was relatively consistent across all cell populations, whereas Atg3 and Map1lc3b (encoding LC3B) showed high expression specifically in monocytes. Analysis of monocytes indicated up-regulation of Atg3, Atg7 and Becn1 during the progression of EAM. Spatial transcriptomics analysis identified 5911 spots under the tissue and results showed an up-regulation of Atg3, Atg7, Becn1 and Map1lc3b in the hearts during the inflammatory phase of EAM. This increased expression was found throughout the entire heart section (Fig. 1B). Moreover, we detected the presence of LC3B and Beclin-1 proteins specifically localized within the inflammatory compartment of inflamed hearts (d19). Conclusions: In the EAM model, acute inflammation in the heart is associated with activated autophagy. Targeting this autophagic response could potentially improve disease outcomes. Financed by the National Science Centre (Poland), grant 2019/35/B/NZ5/00551