Effects Of GO-sECM Microenvironment On The Development Of Excitation-contraction Coupling Function In Cardiomyocytes

Objective:How to make the cultured cardiomyocytes in vitro approach the state of in vivo development has always been an urgent problem to be solved in the clinical application of cardiac regenerative medicine,and it is also a research hotspot in myocardial tissue engineering.The extracellular matrix(ECM)of the cardiomyocytes is the natural microenvironment for the growth and development of cardiomyocytes,but its source is limited.The previous research of our group suggested that the composition and structure of skeletal muscle ECM(sECM)are similar to myocardial ECM,which is easy to obtain and is more conducive to the survival of myocardial cells.It is reported in the literature that graphene oxide(GO)has good electrical conductivity and good mechanical properties,and has the potential to support cell adhesion,proliferation and differentiation.Can the combination of GO and sECM create a more suitable microenvironment for the development of isolated myocardial function? Therefore,our research group used human induced pluripotent stem cell derived cardiomyocytes(hi PSC-CMs)as the isolated myocardial development model to explore the effects of GO and sECM in vitro microenvironment on their survival and the development of excitation-contraction coupling function.Methods:Porcine skeletal muscle was subjected to a three-step decellularization process to prepare sECM.The 13-day-old hiPSC-CMs induced by the modified Gi Wi method were inoculated on sECM,and they were co-cultured with standard medium containing low and high concentrations of GO for 2 weeks.Live-dead cell staining and lactate dehydrogenase content were used to detect the biocompatibility of the microenvironment and cardiomyocytes.Using real-time PCR to observe the effects of different microenvironments on the expression of genes related to myocardial sarcoplasmic reticulum calcium manipulation.WGA fluorescent staining was used to observe the effects of different microenvironments on the development of myocardial T-tubes.Results:1)The GO-sECM microenvironment of different concentrations has no obvious cytotoxicity to cardiomyocytes,and promotes the orderly growth of cells.2)The GO-sECM microenvironment can promote the expression of genes related to calcium manipulation in cardiomyocytes.3)The low concentration of GO microenvironment is more conducive to the development of myocardial T-tubes.Conclusion:The GO-sECM microenvironment may be beneficial to the development of cardiomyocyte excitation-contraction coupling function.