Atrial Natriuretic Peptide Regulates Calcium Approach In Mouse ES Derived Cardiomyocytes

Objective:Voltage dependent L-type calcium channels(VDCC) are responsible for the transmembrane Ca2+ influx of cardiomyocytes during the plateau phase of action potentials(AP), they are critical in the excitation-contraction coupling of the heart.atrial natriuritic peptide(ANP) is polypeptide that secreted by heart from the development of early phase heart,How do it decrease calcium currents in cardiomyocytes is debated. Therefore, My project is about ANP regulates the calcium approach in the embryonic stem cell derived cardiomyocytes.Methods:We induced ES cells differentiate into cardiomyocytes by hanging-drop method and isolate those ESMC by collagenase B at different stages; The whole-cell configuration of Patch-Clamp technique was used to record ANP and relative factor including agonists and inhibitors for different signal ways regulate calcium current in ESMC.α- MHC is special protein of cardiomyocyte and ANP expression were measured by immunofluorescence technique, Moreover , The level of ANP expression in embryonic cardiomyocytes was measured by RT-PCR method.Results: We gained beating EB by hanging-drop method and single beating cardiomyocytes was isolated by collagenase B.α- MHC, calcium current and AP were generated in VEDS cardiomyocytes.ANP has a pronounced negative chronotropic effect on EDS and LDS cardiomyocytes,Moreover it decreases L type of calcium current which basal and Iso stimulated in early (7+2-4) and late(7+9-12) ESMC. The decrease of calcium current by ANP is through a pathway that independent no NO-producing enzymes,PTX sensitive G proteins(Go,Gi) and soluble GC. The ANP induced inhibition of calcium current was mediated through activation of the particulate GC dependent PDEⅡand the resulting increased cAMP breakdown .ANP levels rise continuously during embryonic and postnatal development while high ANP levels tend to decrease rapidly after birth. By RT-PCR and immunofluorescence method, ANP transcripts are first detectable in mouse cardiomyocytes at embryonic day 8(E8).After E12, stronge expression is detected in heart after birth, ANP levels absolutely decrease during adult stage.it was located by immunofluorescene,which symmetrically expresses in cytoplasm at embryonic stages,however, Its expression emerges polarity at ealy development of embryo.