Graduation Year

2008

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Physiology and Biophysics

Major Professor

Eric S. Bennett Ph.D.

Keywords

Glycosylation, Cardiac, Development, Excitability, Sodium channel

Abstract

Cell surfaces are replete with complex, biologically important glycans responsible for multiple cellular functions including cell adhesion and cellular communication. Proper protein glycosylation is essential for normal development and often pathologies are marked by altered glycosylation. Here, data showed that the auxillary subunit, ß1, modified voltage-gated Na? channel (Na[subscript]v) gating in an isoform-specific, sialic acid dependent, and saturating manner. The regulated activity of the hundreds of glycogenes (glycosylation-associated genes) is responsible for protein glycosylation; this could result in a glycome of thousands of glycan structures. Microarray analyses indicated that glycogene expression was highly regulated throughout the heart during development. Specifically, >59% of glycogenes were significantly differentially expressed among neonatal and adult atrial and ventricular myocytes.

Quantitative-PCR of individual genes confirmed the microarray analyses. Such substantial regulation of glycogene expression likely results in changes in glycan structures attached to cell surface proteins. To confirm this, myocyte glycan profiles were determined and compared among neonatal and adult atria and ventricles using mass spectrometry. The data predicted marked differences in glycan structures among myocyte types, indicating that the glycome is remodeled throughout the heart during development. To address the question of whether the remodeled glycome can impact cardiac function, action potentials and Na[subscript]v activity were measured and compared under conditions in which glycogene expression was regulated. That is, atrial and ventricular myocytes were isolated from control mice and from mice in which the polysialyltransferase, STX, was knocked out. STX is expressed in the neonatal atria, and is essentially absent in neonatal ventricle.

Action potential waveforms and Na[subscript]v activity measured in atrial myocytes were impacted by STX expression. No changes in ventricular action potential waveform or in Na[subscript]v activity were observed; as expected since STX is not expressed in the ventricle. The magnitude of the atrial action potential and the rate of depolarization were decreased in the absence of STX. Further, Na[subscript]v gating was shifted consistently in the depolarized direction in STX knockout atrial myocytes. Together, these data indicate that the glycome is tightly controlled and regulated in the heart, and proper glycosylation is essential for normal myocyte function.

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