Effects Of PSEN1in Cardiovascular Development And In Situ Visualizion Of SR Ca²⁺ Channels With Electron Microscopy

Membrane-integral proteins, acting as enzymes, receptors, and channel proteins,play essential roles in cell communication and signal transduction, material transportion,cell connection and so on. It is important for drug development to explore the structure,function, as well as relationship between membrane-integral proteins and their embeddedlipid membrane. Here, genetic operation was used to study the effects of PSEN1, an important membrane-integral protein for Althemia disease, on cardiovascular developmentand diseases. In vivo PSEN1knockout causes abnormal embryonic development, includingsevere head bleeding, defects of coronary artery development, as well as retardation ofheart growth. Using HE staining, we showed that compared with control mice, PSEN1knockout embryos display several defects in cardiovascular system, such as ventricularchamber dilatation and sepal defect, valvular thickening, messy arrangement of aortic wall.Using electron microscopy analysis, PSEN1knockout embryos were showed to have morenaive cardiomyocytes which had less muscle fibers than control. In the cardiomyocytes,PSEN1knockout induces significant defects of cellular connections, increases the numberof mitochondrias which are accompanied with irregular shape and dissolve phenomenon,and causes more glycogen accumulation in the cytoplasm. Echocardiography showed lessleft ventricular cavity at the end of systolic or diastolic stage and more left ventricleejection fraction and fractional shortening in Smo22-PSEN1knockout mice than that of thecontrol mice. These results indicate that membrane-integral PSEN1is essential forcardiovascular development and diseases. Using molecular analysis, we found thatSmo22-PSEN1knockout causes dynamical expression of genes involved in regulatingcalcium storage and releasing, such as DHPR and RyR, in cell connection, such asClaudins and collagens. Although gene knockout technique can reveal the function ofmembrane-integral proteins in pathophysiology, it is still lack of technology to in situobserve the structure of membrane-integral proteins, as well as the relationship with theirsurrounding membrane. It is one of the main technical bottlenecks in current structurestudying of membrane-integral proteins. Here, we established Quick freezing fixation-Electron microscope-Three dimensional reconstruction and surface replacementtechnology to visualize the dilicate structure of membrane-integral proteins, RyR1andDHPR, as well as their arrangement with surrounding bilayer-enviroments at T-Tubule/SRjunctions. The results confirm that it is an effeciant method to illustrate the in situ structureof natural membrane-integral proteins, as well as to study the protein structure of calciumstorage and releasing machinery of skeletal muscle in relaxed and immediately changedphysiological processes. The architecture of T-Tubule/SR junction also provides directarchitectural information for Ca2+storage and releasing model. Advance of technolgy, itcould be expected to study the in situ structure of other membrane-integral proteinscomplexes.