Project description:In cardiomyocytes, Ca2+ influx through L-type voltage-gated calcium channels (LTCCs) following membrane depolarization regulates crucial Ca2+-dependent processes including duration and amplitude of the action potentials and excitation-contraction coupling. LTCCs are heteromultimeric proteins composed of the Cav1, Cav, Cav2 and Cav subunits. Here, using ascorbate peroxidase (APEX)-mediated proximity labeling and quantitative proteomics, we identified 61 proteins in the nano-environments of Cav2 in cardiomyocytes. These proteins are involved in diverse cellular functions such as cellular trafficking, muscular contraction, sarcomere organization and excitation-contraction coupling. Moreover, pull-down assays, co-immunoprecipitation analyses and super-resolution imaging using direct stochastic optical reconstruction microscopy (dSTORM) revealed that Cav2 interacts with the ryanodine receptor 2 (RyR2) in adult cardiomyocytes, probably coupling LTCCs and the RyR2 into a supramolecular complex at the dyads. This interaction is mediated by the Src-homology 3 domain of Cav2 and is necessary for an effective pacing frequency‐dependent increase of the Ca2+-induced Ca2+ release mechanism in cardiomyocytes.
Project description:Here, we characterize the transcriptome of the mouse embryonic stem cell line CM7-1 during differentiation into beating cardiomyocytes and compared the gene expression profiles with those from primary adult murine cardiomyocytes and left ventricular myocardium.
Project description:Over 200 point mutations in ryanodine receptor (RyR2) of cardiac sarcoplasmic reticulum (SR) are known to associate with cardiac arrhythmia. We have already reported on the calcium signaling phenotype of a point mutation in RyR2 Ca2+ binding site Q3925E expressed in human stem-cell derived cardiomyocytes (hiPSC-CMs) that was found to be lethal in a 9 year old girl. CRISPR/Cas9 gene edited mutant cardiomyocytes carrying RyR2-Q3925E mutation exhibited loss of calcium-induced calcium release (CICR) and caffeine-triggered calcium release, but continued to beat arrhythmically without generating significant SR Ca2+ release, consistent with remodeling of calcium signaling pathway. RNAseq heat-map confirmed significant changes of calcium associated genes, supporting the remodeling possibility. To determine the in-situ cardiac phenotype in animal model of this mutation, we generated a knock-in mouse model of RyR2-Q3924E+/– using CRISPR/Cas9 technique. We obtained 3 homozygous and one chimera mice, but they all died before 3 weeks of age preventing the establishment of germline mutation transmission in the offsprings. Histo-pathological analysis of the heart showed significant cardiac hypertrophy suggesting Q3924E-RyR2 mutation was lethal in the mouse.
Project description:To analyse the transcriptome landscape of differentiating cardiomyocytes, GFP labeled cardiac cells were purified by fluorescent activated cell sorting using the TinC*>GFP transgenic line, and expression profile analysed by deep sequencing.
Project description:A novel ppp1r13l sequence variation causes dilated cardiomyopathy and cardiac inflammation. In this experiment we knocked down IASPP (protein product of ppp1r13l) in cardiomyocytes and exposed them to lipopolysaccharide for time interval of 2 and 4 hours. Transcriptome was examined using rna-seq high-throughput sequencing.
