Date of Award

2003

Embargo Period

8-1-2024

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

First Advisor

Robert G. Gourdie

Second Advisor

Paul J. McDermott

Third Advisor

Terrance X. O'Brien

Fourth Advisor

Tim C. McQuinn

Fifth Advisor

Edward L. Krug

Abstract

The remodeling of connexin43-containing gap junctions (GJs), as defined by changes in size, distribution, or function, is a prominent feature of both the developing and diseased myocardium. During postnatal development of the mammalian ventricle, there is a progressive remodeling of GJs into intercalated disks. This change in GJ quantity and distribution is correlated with alterations in the velocity and anisotropy of impulse spread within differentiating myocardium. Orderly distribution of GJs is perturbed in many cardiac diseases, and reemergence of immature patterns of myocyte coupling may be a factor in the arrhythmogenic potential attending such pathologies. Although mechanisms responsible for this remodeling process remain to be determined, previous reports have suggested involvement of the actin-binding MAGUK protein Z01. Here we report that in the rat myocardium, connexin43 and Z01 show low to moderate levels of association, as determined by confocal microscopy, immunoelectron microscopy, and co-immunoprecipitation. However, connexin43-Z01 association is increased following dispersion of myocytes from intact myocardium, a process known to induce GJ remodeling. To further probe roles for Z01 in GJ remodeling, connexin43-Z01 interactions were inhibited using approaches that targeted pertinent binding sites on either connexin43 or Z01. In the first approach, infection of myocytes in vitro and in vivo with adenovirus expressing a putative dominant negative inhibitor of Z01-connexin43 interaction resulted in increases in connexin43 particle size and alterations in connexin43 distribution from the membrane to the cytoplasm. However, N-cadherin-containing adherens junctions were also disrupted by this treatment, suggesting that this inhibitor may not be wholly specific for the connexin43-Z01 interaction. In a second approach, the use of a novel, rationally designed inhibitor peptide, based on the PDZ binding domain of connexin43, resulted in striking increases in connexin43 GJ size and quantity between cultured neonatal myocytes. Unlike the Z01 truncation mutant, the inhibitory peptide did not induce GJ redistribution to the cytoplasm or cause disruption to mechanical junctions. Finally, increased GJ size was correlated with decreased connexin43-Z01 interaction over normal postnatal growth in vivo. Based on the data reported herein, it was concluded that Z01 interaction is a key regulator of the size and level of GJs containing connexin43.

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