File(s) under embargo
until file(s) become available
Determine the Function of a Specific Immunoglobulin Domain of Titin in Zebrafish
thesisposted on 01.12.2020, 00:00 by Xinghang Jiang
Mutations in genes encoding cardiac ion channels, transcription factors, and myocardial structural proteins have been identified in patients with early-onset atrial fibrillation (EOAF). However, the underlying mechanisms by which structural proteins give rise to atrial fibrillation (AF) remain unclear. Titin (TTN) is a multi-domain protein essential for sarcomere assembly during heart development and the restoration of normal sarcomere length after cardiomyocyte contraction. Previous work on a truncated TTN suggested that disruption of the sarcomeric organization may lead to EOAF. However, it remains unknown what role a specific TTN domain might play in abnormal sarcomere development and/or maturation. Our collaborator identified a TTN missense mutation (T31115I) in a patient with EOAF and showed that it co-segregates in the patient’s family for three generations. To elucidate whether the T31115I mutant causes AF and what the underlying mechanisms might be, I adopted two CRISPR-Cas9-mediated approaches: 1) homology-directed repair to introduce this mutation into the zebrafish ortholog, ttna; and 2) generation of two mutant ttna alleles, ttnaΔ6 and ttnaΔ9, with 6 or 9 amino acids deleted, respectively, within a highly conserved region containing the site of T31115I mutation. ttnaΔ9/Δ9 zebrafish embryos demonstrated inflow tract edema, pericardial edema, blood clots, atrial enlargement, and reduced cardiac function, as indicated by lower ventricular contraction and cardiac output. These data suggest that the specific region deleted in ttnaΔ9 is important for proper cardiac function. As the highly conserved region contains the EOAF-associated TTN point mutation, further investigation of this region may not only reveal more biological pathways that involve titin, but also provide more insights into the pathogenesis of EOAF.