Exposure to Serum From Exclusive Heated Tobacco Product Smokers Induces mTOR Activation and Fibrotic Features in Human Cardiac Fibroblasts

Introduction: Smokers are at higher risk of fibrosis-related atrial fibrillation. The use of heated tobacco products (HTPs) is rising worldwide with unclear effects on health. The SUR-VAPES chronic trial showed that exclusive HTP smokers have increased serum markers of oxidative/endothelial stress versus non-smokers (NS), comparable to cigarette smokers. Hypothesis: We hypothesized that the serological profile of HTP smokers can activate atrial fibroblasts towards a pro-fibrotic phenotype. Methods: CFs were isolated from discarded atrial tissue from elective cardiac surgery procedures, and exposed to serum lots (20 healthy young subjects each) from HTP smokers (mean exclusive use=1.5 years) or clinically matched NSs from the SUR-VAPES Chronic trial. ANOVA testing was applied. Results: CFs treated with HTP serum increased expression of fibrotic genes, including PDGFA (1.18-fold, p<0.01) and THY1 (1.22-fold, p<0.01), and increased aSMA protein levels (1.87-fold, p<0.01), compared to NS serum. Connexin 43 was downregulated at both mRNA (1.17-fold, p<0.05) and protein levels (1.56-fold, p<0.01) in CFs after exposure to HTP serum versus NS. COL1A1 expression increased (1.46-fold, p<0.01), with higher soluble collagen release (1.56-fold, p<0.05). CFs pre-treated with HTP serum released lower levels of cardioprotective proteins (e.g. Adiponectin, Chi3L1, VEGF) by protein arrays/ELISA (p<0.05 vs NS). Paracrine support of CF- conditioned media to endothelial tube formation (63.3±4.7 vs 37.3±5.7 mesh #/well, p<0.05) and to cardiomyocyte viability under starvation (0.60±0.04 vs 0.47±0.01 normOD vs t0, p<0.001) were reduced when pre- treated with HTP versus NS serum. Array screening of 37 phospho-proteins in CFs exposed to HTP serum for 1h found several beneficial pathways blunted, with many inhibiting mTOR, including decreased P- PRAS40 (-2.43-fold) and P-p53 (-2.32-fold, p<0.05). Western blot analysis on CFs after 48h of HTP serum treatment confirmed increased P-mTOR (1.40-fold), and its targets P-S6K (1.39-fold) and P-4EBP1 (1.83-fold, all p<0.05). Conclusions: The serological profile of chronic exclusive HTP smokers induces CF activation and fibrotic features pointing to a potential risk for atrial fibrosis.