Mechanoelectrical Coupling and Reorganisation of Cardiomyocytes and Fibroblasts under Shear Stress

Abstract

In this work, I studied the influence of changes such as the abundance of myofibroblasts in fibrotic conditions and the increased shear stress on cellular mechanical properties, morphology, contractility and connectivity. By using co-cultures of cardiomyocytes and myofibroblasts as an in vitro model system for fibrosis I observed that their communication occurs via both electrical and mechanical junctions. Co-cultures with low ratios of cardiomyocytes overexpressed electrical and mechanical junction proteins, Connexin43 (Cx43) and N-Cadherin (N-Cad), respectively, inducing alterations in the electrophysiology of cardiomyocytes: decrease of beating frequency and outbreak of spiral waves. The enhanced expression of N-Cad shows that myofibroblasts may influence the function of cardiomyocytes by applying contractile forces via mechanical junctions. N-Cad in turn is responsible for the transmission of contractile forces between myofibroblasts throughout the fibrotic scar. AFM-based microrheological measurements revealed that viscoelastic properties change under fibrotic conditions. Co-culture was observed to be stiffer than both cardiomyocytes and fibroblasts monocultures and was characterised by a solid-like behaviour at almost all frequencies. Another physiological change that occurs in LVR is the increase of mechanical load on cardiomyocytes, in particular shear stress, which is often neglected in vitro and in silico models. By combining impedance spectroscopy and optical microscopy I have shown that high values of shear stress stimulation lead to an immediate decrease of cell-substrate distances at the flow onset, cell spreading up to 48 hours, and a gradual reorientation of the actin fibers along the direction of the flow that took upto 108 hours. Additionally, beating frequency and cell-cell connectivity of cardiomyocytes increased under shear stress. Furthermore, contraction of cardiomyocytes synchronised in the presence of shear stress. The application of shear stress leads to the increase of beating frequency of co-cultures and induces contractility of fibrotic co-cultures with 9:1 ratio of myofibroblasts and cardiomyocytes, which do not present any activity without flow stimulation. Flow stimulation increased the beating frequency of the cardiomyocytes and myofibroblasts co-cultures similar to the monocultures, and for the first time, I observed contractility in co-cultures with 9:1 ratio of myofibroblasts after shear stimulation, whereas the non-sheared co-cultures did not show any activity. In conclusion this work proves that stimulating cardiomyocytes with high shear stress is a reliable in vitro pathological model to reproduce conditions similar to the in vivo situation. Furthermore, experimental and modelling studies used to understand pathophysiology during LVR should take into account the presence of high shear stress and its influence on mechanoelectrical coupling and cellular morphology.