The ventricular conduction system (VCS) is a multicellular network comprised of highly specialized cardiomyocytes that orchestrate the near synchronous excitation and contraction of the ventricular myocardium. Purkinje cells comprise the most distal portion of the VCS and they have been implicated as arrhythmic triggers in acquired and inherited forms of arrhythmia. The immunoglobulin superfamily cell adhesion molecule (IgSF-CAM) Contactin-2 (CNTN2), was previously identified as a marker of the VCS but its biologic function in this context remains to be elucidated. Through differential transcriptional profiling of FACS-sorted ventricular cardiomyocytes and Purkinje cells expressing Cntn2-EGFP, we found that two additional IgSF-CAMs, NCAM-1 and ALCAM were also highly enriched in Purkinje cells. Immunofluorescence staining showed highly dynamic expression patterns for each IgSF-CAM during embryonic and early post-natal stages, but expression of all three proteins then converged and became restricted to mature Purkinje cells. Targeted loss-of-function mutations revealed that mice deficient in NCAM-1 exhibited QRS prolongation and defects in VCS patterning exhibiting a dysmorphic VCS morphology with reduced levels of Cntn2-EGFP+ cells. Transcriptional profiling of Ncam-1 KO and WT Purkinje cells revealed NCAM-1 to be an important modulator of Purkinje cellular programming and function. Additionally, NCAM-1 has been identified as the major carrier of polysialic acid (PSA), a large cell-surface glycan that regulates embryonic development by attenuating cell adhesion properties between cells. We show that post-translational modification of NCAM-1 by addition of polysialic acid is restricted in time and space to a small population of late fetal and early perinatal subendocardial cardiomyocytes that ultimately comprise the specialized VCS. With the use of CRISPR/Cas9 technology, we generated a PSA-deficient mouse through targeted mutations of the two polysialyltransferases responsible for adding PSA on NCAM-1, ST8sia2 and ST8sia4. While loss-of-function of NCAM-1 itself interferes with proper VCS patterning, targeted disruption of post-translational polysialylation disrupts Purkinje cell-cell contacts and trafficking of sarcolemmal intercalated disc proteins to the junctional membrane. Taken together, our data establish novel functions for IgSF-CAMS in the developing heart and their particular relevance to formation and function of Purkinje cells within the VCS.
|Advisor:||Fishman, Glenn I.|
|Commitee:||Coetzee, William, Delmar, Mario, Cronstein, Bruce, Spray, David|
|School:||New York University|
|Department:||Basic Medical Science|
|School Location:||United States -- New York|
|Source:||DAI-B 81/5(E), Dissertation Abstracts International|
|Subjects:||Molecular biology, Developmental biology, Cellular biology|
|Keywords:||Cardiac conduction system, Cell adhesion, IgSF-CAM, Purkinje cell|
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