The mechanical environment regulates the extent of aortic valve calcification and thickness: an ex vivo whole mouse heart study

Abstract Background Aortic stenosis (AS) is a narrowing of the aortic valve opening due to calcification and thickening of the leaflets. Calcifications are mostly located at regions with high mechanical stress and disturbed flow suggesting that mechanical environment is important in the regulation of calcification. However, direct evidence linking valve calcification and mechanical stress is still lacking. Recently, we established an ex vivo calcification model for whole mouse hearts (Miniature Tissue Culture System MTCS) which allows the study of the mechanical environment underlying aortic valve calcification. Purpose Aim of this study was to investigate the role of mechanical stress on aortic valve calcification and thickening using the MTCS. Methods Whole mouse hearts were cultured in the MTCS with 2 distinct configurations: 1) medium flowing from the aorta towards the aortic valve to create a continuous closed aortic valve (n=28) and 2) medium flowing from the left ventricle through the aortic valve towards the aorta to create a continuous open aortic valve (n=17) (Figure1). The hearts were subjected to low (300 ul/min) or high (3000 ul/min) flow speed in the presence of calcifying medium (3mM phosphate buffer). After culture for 1 week, the hearts were isolated and fixed overnight with 4% PFA/PBS. Alizarin red staining was performed to determine calcification presence. Quantifications were performed using Caseviewer software. Results Aortic valves in closed configuration exposed to a high flow speed, exhibited significant increase in calcification compared to aortic valves in the closed configuration exposed to low flow speed or aortic valve cultured in the open position (p<0.01, Figure2A). Of interest, the extent of calcification in the aortic valve in closed position exposed to high flow speed strongly correlated with the diameter of the aortic annulus (R²=0.86, p<0.0001, Figure2B). Conversely, leaflet thickness was significantly lower in the closed aortic valve configuration as compared with the open aortic valves configuration (p<0.05, p<0.001, Figure2C). Conclusion Our study demonstrates the influence of mechanical environment on aortic valve calcification and thickening. The presence of calcification in the closed but not the open aortic valve configuration exposed to high flow suggests that increased pressure promotes calcification. The correlation of the extent of aortic valve calcification with the diameter of the aortic annulus implies that configuration of the valve is an important determinator of calcification, with potential implications for the results of aortic valve repair. The presence of a minimal amount of mechanical stress as experienced by aortic valves in the open position, however, resulted in thicker aortic valves, indicating that the right balance of mechanical stresses is required to maintain normal valve morphology. These findings provide valuable insights into AS pathogenesis, guiding future therapeutic approaches.ex vivo cultured aortic valvesCalcification in ex vivo aortic valves