Graduation Year

2012

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Medical Sciences

Major Professor

Eric S. Bennett

Keywords

Action Potential, Arrhythmia, Ion Channel, Sialic Acid, Voltage-Clamp

Abstract

In the heart, electrical signaling is responsible for its rhythmicity and is necessary to initiate muscle contraction. The net electrical activity in a cardiac myocyte during a contraction cycle is observed as the action potential (AP), which describes a change in membrane potential as a function of time. In ventricular cardiac myocytes, voltage-gated sodium channels (Nav) and voltage-gated potassium channels (Kv) play antagonistic roles in shaping the AP with the former initiating membrane depolarization and the latter repolarizing it. Functional changes in the primary cardiac Nav isoform, Nav 1.5, or any one of the many Kv isoforms expressed in the ventricle, as evidenced by those characterized in various congenital and/or acquired etiologies, can lead to severe cardiac pathologies. Nav and Kv are large transmembrane proteins that can be extensively post-translationally modified through processes that include glycosylation. The sequential glycosylation process typically ends with negatively charged sialic acid residues added through trans-Golgi sialyltransferase activity. Sialyltransferases belong to a much larger group of glycogene products that number in the hundreds and are responsible for creating a complex and variable glycan profile (glycome) unique to different cell types and tissues. Sialic acids impact Nav and Kv function likely by contributing to the extracellular surface potential and thereby causing channels to gate following smaller depolarizations. Additionally, developmentally regulated sialylation contributes to cardiac myocyte excitability in the neonatal mouse atria. However, little is understood concerning how the glycosylation machinery (glycogene products) influences cell and tissue electrical signaling. The sialytransferase Β-galactoside α-2,3-sialyltransferase 4 (ST3Gal4) adds sialic acids to galactose residues of N- and O-linked glycans through α-2,3-linkgages. ST3Gal4 is uniformly expressed throughout the chambers and developmental stages of the heart and therefore is likely a useful target to question whether and how glycosylation impacts these events. Additionally, diseases of glycosylation often cause symptoms that are consistent with changes in excitability that include arrhythmias and seizures. Congenital disorders of glycosylation lead to variably reduced glycoprotein and glycolipid glycosylation. However, because sialic acids are typically the terminal residues added to glycan structures, disease-related reduced glycosylation often leads to fewer sialic acids being attached. In addition, Chagas disease, which results in pathological changes in cardiac electrical function, may reduce sialic acids directly. Because of this, the ST3Gal4-/- strain was also used to investigate the role of glycosylation in the pathological cardiac electrical remodeling often associated with these diseases. The methodologies included cellular, tissue and whole-animal electrophysiology as well as biochemical assays. The data indicate that deletion of ST3Gal4 significantly affects Nav sialylation and gating with no change in maximum current density or protein expression. ST3Gal4 deletion also depolarizes the activation gating of both voltage-dependent kinetic components of repolarization found in the mouse ventricle: Ito and IKslow; however unlike the effect on INa, ST3Gal4 gene deletion causes a reduction in the peak IK density. Protein expression of the putative Kv isoforms responsible for Ito and IKslow was variably affected by ST3Gal4 gene deletion with Kv1.5 and Kv4.2 demonstrating no differences in protein densities. Contrastingly, a small but significant reduction in Kv2.1 protein from ST3Gal4-/- ventricular tissue was observed. In addition to effects on Nav and Kv activity, ST3Gal4 expression is necessary for normal cellular electrical signaling as demonstrated by a reduction in cellular refractory period and alterations in AP waveforms that include a slowing of cellular conduction and an extension of AP duration in ventricular myocytes from ST3Gal4-/- mice. Concurrent with aberrant excitability at the cellular level, the ST3Gal4-/- left ventricular epicardium demonstrated a reduced refractory period and was more susceptible to arrhythmias as observed through optical mapping studies. Additionally, ECGs of ambulatory ST3Gal4-/- mice demonstrated that deletion of the gene causes modest aberrant conduction under basal conditions and, in preliminary studies, appears to increase susceptibility to arrhythmias following a cardiac challenge, in the form of a low dosage of the Β-adrenergic agonist isoproterenol, suggesting a reduction in repolarization reserve in ST3Gal4 hearts. Based on the data reported here, it is apparent that relatively minor perturbations in the cardiac glycome cause significant changes in cardiac electrical signaling. These data highlight the role of glycosylation in normal physiology and underscore it as an important mediator in diseases where it may be altered.

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