Dynamic Monitoring Of Mechanical Properties Of Myocardial Cells And Evaluation Of Cardiovascular Drugs Using QCM

The cardiomyocytes cultured in vitro can maintain the ability of spontaneous beating, so the primary cardiomyocytes are often used as the main model for cardiovascular diseases and drug testing. However, complicated extraction steps and a large number of animals are needed to obtain enough and higly purified primary cardiomyocytes. In recent years, measuring of cardiomyocytes’s mechanical properties accompanied with their contraction is becoming a hotspot of cardiomyocytes’s research, but there is currently no suitable technique which can dynamically measure the cardiomyocytes’mechanical properties in a sustained manner, greatly impeding its application in clinical diagnosis and treatment evaluation research.In this thesis, "double resonator" QCM (quartz crystal microbalance) technique,9 MHz AT and BT-cut crystals with naked gold electrodes and KRGD modified gold electrodes formed through self-assembly, were used for the dynamic measuring of the magnitude and direction of cell generated force of H9C2 cardiomyocytes during their adhesion and under the treatments of two cardiovascular drugs isoprenaline (ISO) and verapamil (VRP). We used cell viscoelastic index (CVI) to characterize the cells’softness.The main results are as follows:1) Under the effect of either hypertonic stress or ISO, cells-QCM surface adhesion became stronger and H9C2 cells became stiffer, and the higher the osmotic stress or the ISO concentration, the stiffer the cells; cells-QCM surface adhesion became weaker and H9C2 cells became softer under the effect of either hypotonic stress or VRP, and the lower the osmotic stress or the higher the VRP concentration, the softer the cells.2) Compared to bare gold electrode, the MPA/RGD-modified gold electrode yielded enhanced frequency shift (△f) and motional resistance change (△R) with the adhesion of H9C2 cells. In the tested H9C2 concentration range (5x10~4-4×10~5 cells/mL), Af was linearly related to the cell concentration, whereas △R was a power function of the cell concentration.3) The"double resonator" QCM results indicated that the H9C2 cells adhered to the naked gold electrode were mainly in contractile or slightly spreading status; the cell generated contractile force increased under the treatment of positive inotropic drug ISO and the cells became stiffer, and the cell generated relaxation force increased under the treatment of negative inotropic drug VRP and the cells became softer. Whereas on the KRGD modified gold electrode, the H9C2 cells were well spread as indicated by the measured cell generated force.In summary, our results as shown in this thesis demonstrated that although H9C2 cardiomyocytes do not have the beating function, they still have the ability to contract and expand. Moreover, they are homogeneous and immortal, making them a potentially suitable cell model for the study of contraction and relaxation characteristics of cardiomyocytes, in particular for pre-assessment and toxicity testing of cardiovascular drugs where high-throughput and repetitions are required, breaking the bottleneck of the major cell model of primary cardiomyocytes. In addition, our results demonstrated for the first time that the "double resonator" QCM technique can be used for measuring the dynamic changes of the cell generated contractile or relaxation force and viscoelasticity during adhesion of cardiomyocytes and under the treatments of contratile or relaxation agents, making it an ideal tool for assessing the contractility of populations of cardiomyocytes and evaluating of cardiovascular drugs and toxicity.