Doctor of Philosophy, The Ohio State University, 2016, Biomedical Sciences

Cardiovascular disease is the leading cause of death in the United States, claiming nearly 800,000 lives each year. Regardless of the underlying cardiovascular dysfunction, nearly 50% of these patients die of sudden cardiac arrest caused by arrhythmia. Development and sustainment of cardiac arrhythmia begins with dysfunction of excitability and structure at the cellular level. Therefore, in order to improve therapeutic options for these patients, a basic understanding of the molecular mechanisms regulating cardiac cellular excitability and structure is required. Decades of research have demonstrated that intracellular scaffolding polypeptides known as ankyrins are critical for the regulation of cellular excitability and structure in multiple cell types. Ankyrin-G (ANK3) is critical for regulation of action potentials in neurons and lateral membrane development in epithelial cells. Given its central importance for cellular physiology in excitable and non-excitable cell types, we hypothesized that functional ankyrin-G expression is critical for proper cardiac function. To test this hypothesis in vivo, we generated cardiac-specific ankyrin-G knockout (cKO) mice. In the absence of ankyrin-G, mice display significant reductions in membrane targeting of the voltage-gated sodium channel Nav1.5. This disruption in turn causes severely reduced whole cell sodium current, leading to significant conduction abnormalities, bradycardia, and ventricular arrhythmia and atrioventricular nodal block following infusion of NaV channel antagonists. In addition to regulating cardiac excitability, we also demonstrate a critical role for ankyrin-G in the regulation of the cardiomyocyte cytoarchitecture. Specifically, ankyrin-G cKO mice show disrupted cellular distribution of the desmosomal protein plakophilin-2 (PKP2) at baseline. In a setting of pressure overload-induced heart failure we observed severe disruptions to the cellular localization of PKP2. Further, as desmosomes mediate the in (open full item for complete abstract)

Committee: Peter Mohler (Advisor); Noah Weisleder (Committee Chair); Thomas Hund (Committee Member); Philip Binkley (Committee Member) Subjects: Cellular Biology; Physiology

Doctor of Philosophy (PhD), Ohio University, 2010, Biological Sciences (Arts and Sciences)

Mammalian hippocampal circuitry contains copious vesicular Zn2+ and glutamate in the mossy fiber axon terminals which originate from the granule cells of the dentate gyrus. Temporal lobe epilepsy in humans and rodent epilepsy models reveal a peculiar feature in the brain, branches referred to as “recurrent mossy fibers” which sprouts off from mossy fibers. The recurrent mossy fiber terminals of pilocarpine induced epileptic rats were examined for Zn2+ and confirmed with Timm's staining and intracellular fluorescent zinc indicators. These zinc-rich terminals were investigated for release of Zn2+ into the extracellular space detected by a low affinity fluorescent indicator for Zn2+, namely Newport Green. This study provides evidence to support that high frequency electrical stimulation of the mossy fiber axons causes Zn2+ release from not only mossy fibers but also the Zn2+ rich recurrent mossy fiber terminals. This release is frequency dependent; the fluorescent response is attenuated in the presence of Zn2+ specific chelators, low calcium medium and a vesicular uptake inhibitor. Also, the concentration of Zn2+ released from the terminals was estimated to be in the low micromolar range, with significantly higher release observed in epileptic animals when compared to sham treated ones. Evidence for a Zn2+ sensitive voltage gated sodium channel, Nav1.5/ SCN5A expression in the dentate gyrus was obtained by western blotting and real time quantitative PCR and using immunostaining they were seen localized to regions around the soma of granule cells and CA3 pyramidal cells in the dentate gyrus. Interestingly, when epileptic rats were analyzed it was seen that animals that were 16 weeks epileptic had the maximal amount of releasable zinc as well as Nav1.5 protein expression. This was followed by a gradual decline with age. Since these channels could be blocked by Zn2+ and their localization was found near where synaptic Zn2+ is released, it could be postulated that the p (open full item for complete abstract)

Committee: Yang Li PhD (Advisor); Robert Colvin PhD (Committee Member); Mark Berryman PhD (Committee Member); Gary Cordingley PhD (Committee Member) Subjects: Biomedical Research; Molecular Biology

Doctor of Philosophy in Clinical-Bioanalytical Chemistry, Cleveland State University, 2012, College of Sciences and Health Professions

Type-3 long QT syndrome, which is related to type 5 voltage-gated sodium channel alpha subunit (SCN5A) mutation, has been identified since 1995. LQTS mutation in SCN5A is a gain-of-function mutation producing late sodium current, INa,L. Brugada mutation in SCN5A is a loss-of-function causing INa decrease. Whereas, the mechanism for Dilated Cardiomyopathy mutations in SCN5A is still not fully understood. N1325S is one of the first series of mutations identified for type-3 LQTS. Our lab created a mouse model for LQTS by expressing SCN5A mutation N1325S in the mouse hearts (TG-NS) and a matched experimental control line with overexpression of wild- type SCN5A (TG-WT). There are some interesting findings in TG-NS mice: (i) Intracellular sodium (Na+) level is higher in TG-NS myocytes compared with TG-WT myocytes. (ii) Ca2+ handling is abnormal in TG-NS myocytes, but not in TG-WT myocytes. (iii) Apoptosis was also found in TG-NS mouse heart tissue, but not in TG-WT hearts. These results provoke the hypothesis that gain-of-function mutation N1325S in SCN5A leads to LQTS through abnormal cytosolic Ca2+ homeostasis. Another LQTS mutation in SCN5A R1193Q was identified in 2004 and the electrophysiological property is similar to other gain-of-function SCN5A mutations. The transgenic mouse model for this mutation was also established and the surface Electrocardiogram (ECG) results indicate longer corrected QT interval also present in transgenic mice carrying R1193Q mutation. Besides, quinidine, an anti-arrhythmic medication, can cause arrhythmic symptoms such as premature ventricular contraction (PVC), premature atrial contraction (PAC) and atrioventricular (AV) block in R1193Q transgenic mice. In order to further study the relationship between abnormal Ca2+ handling and the type of SCN5A mutation, either gain-of-function or loss-of-function, we have chosen HL-1 cells, a cell line with indefinite passages in culture with all the adult cardiac phenotypes. The similar abnormal (open full item for complete abstract)

Committee: Qing Wang PhD, MBA (Advisor); Yan Xu PhD (Advisor); Xue-long Sun PhD (Committee Member); Robert Wei PhD (Committee Member); Wu Yuping PhD (Other) Subjects: Biology; Chemistry; Genetics; Health Sciences

Doctor of Philosophy in Regulatory Biology, Cleveland State University, 2008, College of Science

FUNCTIONAL CHARACTERIZATION OF SCN5A, THE CARDIAC SODIUMCHANNEL GENE ASSOCIATED WITH CARDIAC ARRHYTHMIAS AND SUDDEN DEATH LING WU ABSTRACT The cardiac sodium channel α subunit Nav1.5 (encoded by the SCN5A gene) plays an important role in the generation and propagation of electrical signals in the heart, and can cause cardiac arrhythmias, heart failure and sudden death when mutated or dysregulated. However, the precise composition of the multi-protein complex for the sodium channel has not been completely defined. The molecular mechanisms by which Nav1.5 mutations cause cardiac arrhythmias have not yet been well-studied in vivo. It remains to be explored whether Nav1.5 is expressed in other tissues and plays novel roles in other tissues or organs. This dissertation addresses these aspects of Nav1.5 regulation. I found that MOG1, a small protein that is highly conserved from yeast to humans, is a central component of the channel complex and distinctly modulates the physiological function of Nav1.5. A yeast two-hybrid screen identified MOG1 as a new protein that interacts with the cytoplasmic loop II (between transmembrane domain DII and III) of Nav1.5. The interaction was further demonstrated by both in vitro GST pull-down and in vivo co-immunoprecipitation assays. Co-expression of MOG1 with Nav1.5 in HEK293 cells increased sodium current densities, whereas two siRNAs that knocked down expression of MOG1 decreased current densities. In neonatal myocytes, over-expression of MOG1 increased current densities nearly two-fold, and MOG1 siRNAs down-regulated the sodium currents. Immunostaining revealed that in the heart, MOG1 was expressed in both atrial and ventricular tissues and was highly localized in the intercalated discs. These results suggest that MOG1 may be a critical regulator of sodium channel function in the heart and reveal a new function for MOG1. Furthermore, I showed that MOG1 increased sodium current density by increasing cell membrane localization of Nav (open full item for complete abstract)

Committee: Dr. Qing Wang (Advisor) Subjects: Biochemistry; Biology; Biomedical Research; Biophysics; Biostatistics