Exploring The Role Of SPEG In Dilated Cardiomyopathy Based On A Human Cardiomyocyte Mode

BackgroundStriated muscle preferentially expressed protein kinase(SPEG)is a member of the myosin light streptokinase family.SPEG plays a crucial role in maintaining regular cardiac calcium activity by preserving phosphorylation of proteins associated with transverse tubule formation and membrane-coupled complexes.Previous studies have found that SPEG can interact with various proteins such as Sarco-endoplasmic reticulum ATPase-2a(SERCA2a),Ryanodine receptor 2(RyR2),and Junctophilin 2(JPH2),and regulate calcium activity.Mutations and down-regulation of SPEG expression are closely related to various cardiac diseases such as human dilated cardiomyopathy,atrial fibrillation,heart failure,but the definitive pathogenic mechanism remains to be studied in detail.PurposeIn this study,we used SPEG knockout human embryonic stem cell-derived cardiomyocytes as a model to detect the abnormal phenotype of SPEG-deficient cardiomyocytes and to investigate the potential pathological mechanisms of SPEG abnormalities leading to cardiac diseases.MethodIn this study,we used CRISPR/Cas9 gene editing technology to knock out SPEG in a human embryonic stem cell line and differentiated SPEG knockout human embryonic stem cells into cardiomyocyte-like cells in vitro using a small molecule differentiation method with a well-defined chemical composition.We detected the normal phenotype by flow cytometry,calcium fluorescence assay,contractility assay,electrophysiological assay ang etc.,and analyzed the pathogenic mechanism using transcriptome sequencing technology.In addition,in this study,wild-type JPH2 was overexpressed in Speg-deficient cardiomyocytes by lectin transfection to detect the recovery of abnormal phenotype,and the expression of JPH2 and its phosphorylation in cardiomyocytes after SPEG knockout was detected by Western Blot assay.ResultBased on the SPEG knockout cell model,we found that the deletion of SPEG did not affect the multipotentiality of H9-hESC,and that the efficiency of myocardial differentiation and the ratio of atrial to ventricular subtype differentiation were similar in SPEG-deficient cardiomyocytes and wild-type cardiomyocytes.After SPEG deletion,cardiomyocytes showed hypertrophy phenotype with disorganized intracellular myofilament myonodular structure,reduced cell contractility and uneven pulsation.Transcriptomic analysis indicated that the expression of calcium pathway related genes was significantly downregulated after SPEG knockout,and calcium fluorescence imaging also showed abnormal calcium activity in cardiomyocytes.MEA test results demonstrated that SPEG deficient cardiomyocytes showed abnormal excitatory contraction coupling phenotype.JPH2 expression and phosphorylated JPH2 expression were significantly reduced after SPEG deletion.RT-PCR results showed that the downregulation of JPH2 expression lagged behind the up-regulation of heart failure markers.In addition,overexpression of JPH2 can partially restore the abnormal phenotype of cardiomyocytes after SPEG deletion.ConclusionIn this study,we successfully constructed a cardiomyocyte-like cell model of SPEG knockout human embryonic stem cell origin,and found that abnormal calcium signaling pathway may be an important reason for the cardiomyocyte-like phenotype of amplified heart disease after SPEG deletion through transcriptome sequencing analysis.The significant reduction of phosphorylated JPH2 in cells after SPEG deletion may be an important reason for the cellular defects.Finally,the study overexpressed JPH2 in SPEG-deficient cardiomyocytes and found that the abnormal phenotype of SPEG-deficient cardiomyocytes could be partially restored,providing a potential target for the treatment of cardiac diseases related to SPEG abnormalities.