Phosphorylation kinetics of cardiac gap junction regulation during stress
| dc.contributor.author | Stanley, Kari Elizabeth | en |
| dc.contributor.committeechair | Smyth, James | en |
| dc.contributor.committeechair | Lamouille, Samy | en |
| dc.contributor.committeemember | Weger, James David | en |
| dc.contributor.committeemember | Karch, Jessica Mary | en |
| dc.contributor.committeemember | Sassi, Yassine | en |
| dc.contributor.department | Graduate School | en |
| dc.date.accessioned | 2025-01-16T09:01:22Z | en |
| dc.date.available | 2025-01-16T09:01:22Z | en |
| dc.date.issued | 2025-01-15 | en |
| dc.description.abstract | The coordinated contraction of the heart occurs because of the propagation of action potential between the cardiomyocytes. Gap junctions consisting primarily of connexin43 (Cx43) connect cardiomyocytes at regions of contact between cells known as the intercalated disc to facilitate cellular coupling. Cardiac pathologies frequently manifest with disrupted gap junctional intercellular communication which can generate potentially fatal arrhythmias, thus, it is essential to elucidate mechanisms underlying Cx43 regulation and altered intercellular communication. Phosphorylation of residues in the Cx43 carboxyl terminus can alter the subcellular localization, channel gating, and internalization of Cx43. The channel open probability of gap junctions is regulated, in part, by the phosphorylation of S368. Phosphorylation of S365 and S373 have been reported to exert gatekeeper effects on the phosphorylation of S368 and these phosphorylation events further affect protein interactions with 14-3-3 and zonula occludens-1 (ZO-1). While it is established that pS365 creates a conformational change preventing pS368, it is currently unclear precisely how pS373 regulates pS368. Further, it is unclear if alterations to these residues might impact protein binding and pathological cardiac remodeling during stress. Utilizing an ex vivo ischemia model, we find by immunofluorescent confocal microscopy that wildtype Cx43 hearts exhibit significantly decreased Cx43/N-cadherin colocalization during ischemia, while phospho-null mutant hearts retain Cx43/N-cadherin colocalization. Triton X-100 solubility assay indicates S365A/S373A mice have increased junctional Cx43 during ischemia. Additionally, we show that pS368 decay is more rapid in S373A mutants than wildtype suggesting, for the first time, that pS373 may prevent dephosphorylation at Cx43-S368 rather than promote Cx43-pS368. This knowledge could highlight potential therapies for prevention of cardiac remodeling and arrhythmogenesis. | en |
| dc.description.abstractgeneral | A healthy heartbeat is achieved by the passage of electrical signals from cell-to-cell throughout the heart to cause a coordinated contraction. The muscle cells of the heart, known as cardiomyocytes, pass this electrical impulse through channels connecting neighboring cells, known as gap junctions, which are made from the protein connexin43 (Cx43). Gap junction channels allow for the passage of ions and small molecules from one cell to the next and these ions help initiate contraction in the neighboring cell. To regulate the location and function of Cx43, the protein can be modified by the addition of chemical groups which may alter the shape of the protein, affecting its function or ability to interact with other proteins. The addition of a phosphate group is known as phosphorylation. When the heart is under stress, such as low oxygen (hypoxia) or low blood flow (ischemia), Cx43 is phosphorylated at specific sites along the protein which can alter the function of gap junction channels in a harmful way. If the heart can no longer pass the electrical signal necessary for contraction, this can cause disrupted heart rhythms known as arrhythmias and can lead to death. Phosphorylation at S368 has been shown to decrease gap junction channel function. We know that phosphorylation at S365 changes the shape of the connexin protein and prevents phosphorylation of S368. However, it is unknown how phosphorylation of S373 affects phosphorylation of S368. We show that S373 may prevent phosphorylation of S368, and that mutation of these residues prevents cardiac remodeling. These discoveries may lead to therapeutic targets to prevent arrhythmia and sudden cardiac death. | en |
| dc.description.degree | Master of Science | en |
| dc.format.medium | ETD | en |
| dc.identifier.other | vt_gsexam:42360 | en |
| dc.identifier.uri | https://hdl.handle.net/10919/124221 | en |
| dc.language.iso | en | en |
| dc.publisher | Virginia Tech | en |
| dc.rights | In Copyright | en |
| dc.rights.uri | http://rightsstatements.org/vocab/InC/1.0/ | en |
| dc.subject | connexin43 | en |
| dc.subject | arrhythmia | en |
| dc.subject | ischemia | en |
| dc.subject | gap junction | en |
| dc.subject | gatekeeper phosphorylation cascade | en |
| dc.title | Phosphorylation kinetics of cardiac gap junction regulation during stress | en |
| dc.type | Thesis | en |
| thesis.degree.discipline | Translational Biology, Medicine and Health | en |
| thesis.degree.grantor | Virginia Polytechnic Institute and State University | en |
| thesis.degree.level | masters | en |
| thesis.degree.name | Master of Science | en |
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