Date of Award

1-1-2012

Document Type

Campus Access Dissertation

Department

Biological Sciences

First Advisor

Lydia E Matesic

Abstract

Sudden cardiac death (SCD) is the most common cause of mortality worldwide, accounting for more than 3 million deaths annually with estimates in the United States ranging between 300,000 to 350,000 deaths each year. Because SCD is the common endpoint of a variety of heart pathologies that predispose to ventricular arrhythmogenesis (e.g., coronary artery disease, cardiomyopatheis, or channelopathies), therapies that prevent SCD would have a major impact in medical practice as there are few options that currently exist. In the heart, gap junctions function as specialized channels that localize to the boundary between neighboring cardiomyocytes in a structure known as the intercalated disc (ID). Each gap junction is composed of protein subunits called connexins (Cx) that work in concert to maintain a coordinated wave of electrical excitation among cardiomyocytes in order to ensure a proper and synchronized contraction of the myocardium. The precise localization of gap junctions to the ID is established during postnatal cardiac development in a progressive and highly orchestrated manner. Once complete, this process results in the transition of the cardiac electrical conduction properties towards a more uniform rate and electrical anisotropy. Therefore, altering the organization or expression pattern of these Cx in the adult (a pathology called gap junction remodeling) causes abnormal impulse propagation throughout the myocardium, resulting in ventricular arrhythmia and SCD.

Our lab is interested in examining the role of the ubiquitin ligase Wwp1 in regulating Cx expression during normal postnatal cardiac development as well as in gap junction remodeling associated with cardiac dysfunction. Thus, we created a mouse model which completely lacks Wwp1 activity (Wwp1-/-) as well as an inducible transgenic mouse model that enables us to activate overexpression of Wwp1 in a tissue-and temporal- specific manner upon exposure to the cre recombinase. Using both of these systems, we found that while the Wwp1-/- mice were viable and fertile, all of the transgenic mice overexpressing Wwp1 (either ubiquitously throughout the body or in a cardiomyocyte-specific fashion) died suddenly in early adulthood (approximately 8 weeks of age in mice) due to ventricular arrhythmias. In addition to the SCD phenotype, the Wwp1 overexpressers exhibited structural remodeling of the heart specifically characterized by the development of left ventricular hypertrophy as detected by echocardiography. Furthermore, multi-immunofluorescence staining of cardiac tissue derived from the Wwp1 overexpressers at different stages of postnatal heart development showed mislocalization of the gap junction proteins Cx43 and Cx45. However, further analysis of these Cx by western blotting revealed a decrease in the amount of Cx43 protein while the protein levels of Cx45 were unaltered. We hypothesized that Wwp1 specifically affected Cx43 because the expression, localization and structure of other ID-associated intercellular junction proteins (i.e., components of the fascia adherens and desmosomes) were not altered as assessed by immunofluorescence staining in conjunction with confocal microscopy, by transmission electron microscopy and by western blot analysis. Consistent with the loss of myocyte electrocoupling due to dysregulated Cx expression, electrocardiogram abnormalities were noted in the Wwp1 overexpressers at different stages of heart development and these electrical disturbances resulted in extreme sensitivity to induced and spontaneous arrhythmias. In contrast to the transgenic mice, the Wwp1-/- animals showed increased junctional Cx43 without a statistically significant increase in the amount of total Cx43 protein. However, this was not associated with any overt phenotype. Taken together, these data suggest that Wwp1 contributes to the homeostasis of Cx43 pools within cardiomyocytes, and changes in the expression level of Wwp1 influence the electroconduction properties of the myocardium, contributing to SCD. This newly identified role of Wwp1 in cardiac arrhythmogenesis is emerging as a powerful portal in the development of innovative and more effective therapeutic interventions for SCD.

Rights

© 2012, Wassim Antoine Basheer

Download

Share

COinS