Thesis (Ph. D.)--Ohio State University, 2008.

Thesis (M. Phil.)--University of Hong Kong, 2008. / Includes bibliographical references (leaves 75-100) Also available online.

Thesis (Ph. D.)--University of California, San Diego and San Diego State University, 2000. / Includes bibliographical references (leaves 118-135).

Thesis (M.S.)--University of Alabama at Birmingham, 2006. / Thesis not released until Summer 2007. Description based on contents viewed Oct. 5, 2007; title from title screen. Includes bibliographical references.

Thesis (Ph. D.)--University of Nevada, Reno, 2008. / "May, 2008." Includes bibliographical references. Online version available on the World Wide Web.

Physiologic β-adrenergic activation of PKA during the sympathetic “fight-or-flight” response increases calcium influx through CaV1.2 in cardiomyocytes, leading to increased cardiac contractility. The molecular mechanisms of β-adrenergic regulation of CaV1.2 in cardiomyocytes are incompletely known, but activation of PKA is required for this process. The second chapter of this dissertation describes our investigation of the functional PKA phosphorylation target for β-adrenergic regulation of CaV1.2. Recent data confirm that β-adrenergic regulation of CaV1.2 does not require any combination of PKA phosphorylation sites on α1C or β2B subunits. Proximity proteomic labeling methods led us to other potential PKA targets near the CaV1.2 complex, including Rad, a calcium channel inhibitor that changes its position within the calcium channel neighborhood after β-adrenergic stimulation. With expression of α1C, β2B, and Rad in a heterologous expression system, we reconstituted forskolin-PKA regulation of CaV1.2. By mutating potential PKA phosphorylation sites on Rad, we identified specific residues that are critical for this mechanism to occur and validated Rad as the functional PKA target for regulation of CaV1.2. In the third chapter, we probe the contribution of both CaV1.2 α1C and β subunits in β-adrenergic regulation. Previous results have shown that binding between α1C and β subunits is required for adrenergic stimulation of the calcium channel in the heart. Using transgenic mouse models, we demonstrate that this phenomenon requires a rigid IS6-AID linker in the α1C subunit, as introduction of glycines in this region increased flexibility of the linker and abolished a response to adrenergic agonists even though α1C was able to bind to β. The fourth chapter examines the role of the auxiliary β subunit in β-adrenergic regulation of CaV1.2. Binding of Rad to the β subunit is also necessary for this mechanism to occur. Although the β2B isoform is the predominant subunit in the heart, we show that transgenic mice with β3 or β4 replacing β2B in the heart are viable and still have normal β-adrenergic regulation of CaV1.2, indicating that this mechanism is universal to other voltage gated calcium channels that bind to β subunits and RGK proteins. The fifth chapter verifies that Rad is the PKA target in the heart. Using a knock-in mouse model with four PKA phosphorylation sites mutated to alanine, we definitively show that phosphorylation of Rad is necessary for β-adrenergic regulation of CaV1.2 in the heart. We investigate the importance of Rad phosphorylation on many levels. First, we study Rad’s role in regulating the calcium channel. Second, we observe the effect phosphorylation of Rad has on the calcium transient using isolated cardiomyocytes. Third, we examine cardiovascular function in vivo using radiotelemetry and echocardiograms. Finally, we assess the “fight-or-flight” response in an animal model with exercise capacity testing. Together, these findings conclusively show that in the heart, phosphorylation of Rad is the essential mechanism for the sympathetic nervous system control of calcium influx in both atrial and ventricular cardiomyocytes. Additionally, Rad modulates both heart rate and contractility in vivo. In the sixth chapter, we explore the mechanism of Rad modulation of CaV1.2 in depth using a flow-cytometry Förster resonance energy transfer (FRET) two-hybrid assay. We closely examine the roles of phosphorylation sites on both Rad’s N-terminus and C-terminus. By creating phosphomimetic mutations on Rad, we uncover the importance of phosphorylating the C-terminus for release of Rad from both the membrane and the β subunit. Taken together, these findings elucidate the mechanism behind β-adrenergic regulation of CaV1.2 in the heart – a longstanding query for over forty years in the cardiovascular ion channel field. At baseline, Rad “tunes” the amount of calcium influx into the cell by inhibiting a population of channels as a functional reserve. Upon adrenergic stimulation, Rad is phosphorylated, lessening its interaction with the membrane, and releasing inhibition of the calcium channel. The enhanced local calcium influx allows for additional calcium release into the cytoplasm through ryanodine receptors leading to increased contractility of the heart, a notable characteristic of the evolutionary survival mechanism— “fight-or-flight.”

Cardiovascular disease remains the leading cause of death worldwide. A lack of curative treatments and a shortage of transplant hearts necessitate new approaches to cardiac repair. Recent advances, including the advent of pluripotent stem cell-derived cardiomyocytes and the development of tissue engineering techniques, represent promising new directions to remuscularize the heart or induce endogenous regeneration. However, these approaches are currently limited by the immaturity of differentiated cardiomyocytes and the inability of cardiomyocytes to functionally integrate with the damaged myocardium. Mimicking the cardiomyocyte niche, the myriad signals surrounding the cardiomyocyte, may enhance the utility of these cells. In this dissertation, each of the three aspects of the cardiomyocyte niche: physical signals, the extracellular matrix, and soluble factors, are examined for their ability to guide cardiomyocyte growth and function. We first explore the effect of electrical stimulation, a physical signal pervasive in the heart, on pluripotent stem cell-derived cardiomyocyte development and function. Stimulated cardiomyocytes are more mature, show greater cell-cell connectivity, and are more resistant to tachycardic stress. Cardiomyocytes adapt their beating rate to the stimulation frequency, an effect mediated by the emergence of a rapidly depolarizing cell type and ion channel expression. We next engineer cardiovascular tissue architecture, critical components of the extracellular matrix, using a micromolding approach and determine geometric parameters necessary for the induction of cardiomyocyte alignment and tissue synchrony. We finally test pluripotent stem cell-derived cardiomyocyte exosomes, soluble nanovesicles specifically packaged and secreted by the cell, in vitro and in vivo, demonstrating functional improvement and reduction of arrhythmia in the heart. Therefore, the use of the cardiomyocyte niche supports the interrogation of cellular function to enable new cell-based approaches for the reduction of arrhythmia or induction of repair in the heart.

Thesis (MSc)--Stellenbosch University, 2005. / ENGLISH ABSTRACT: Introduction: Exercise increases skeletal muscle glucose uptake via AMP-activated protein kinase (AMPK) activation and GLUT4 translocation from cytosol to cell membrane. It also promotes glucose utilisation in type 2 diabetic patients via increased insulin sensitivity. Insulin stimulates GLUT4 translocation by activating P13- kinase and protein kinase B (PKB/Akt). We therefore postulated that a connection exists between these two pathways upstream of GLUT4 translocation. Understanding this connection is important in the development of treatment strategies for type 2 diabetes. This exercise-induced increase in AMP-activated protein kinase (AMPK) activation can be mimicked by a pharmacological agent, 5'-aminoimidazole-4- carboxamide ribonucleoside (AlGAR), which is converted intracellularly into 5'- aminoimidazole-4-carboxamide-ribonucleosidephosphate (ZMP), an AMP analogue. Aim: To investigate the effect of two pharmacological AMPK-activating compounds, ZMP and AlGAR, on the phosphorylation of AMPK, the phosphorylation of PKB/Akt as well as possible feedback on insulin-stimulated glucose uptake and GLUT4 translocation. Materials and Methods: Adult ventricular cardiomyocytes were isolated from male Wistar rats by collagenase perfusion and treated with 1 mM AlGAR or 1 mM ZMP in the presence or absence of 100 nM insulin or 100 nM wortmannin, an inhibitor of P13- kinase. Glucose uptake was measured via eH]-2-deoxyglucose (2DG) accumulation. PKB/Akt and AMPK phosphorylation and GLUT4 translocation was detected by Western blotting. Purinergic receptors were blocked with 8-cyclopentyl-1,3- dipropylxanthine (8CPT) and the effect on AMPK phosphorylation noted. Certain results were confinned or refuted by repeating experiments using the isolated rat heart model. Results: AICAR and ZMP promoted AMPK phosphorylation. Neither drug increased glucose uptake but in fact inhibited basal glucose uptake, although GLUT4 translocation from cytosol to membrane occurred. Both compounds also attenuated insulin stimulated glucose uptake. Wortmann in abolished glucose uptake and PKB/Akt phosphorylation elicited by insulin while, in the presence of wortmannin, AICAR and ZMP increased levels of PKB/Akt phosphorylation. Although AICAR and ZMP increased glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. However both compounds increased GLUT4 translocation, clearly demonstrating that translocation and activation of GLUT4 are separate processes. 8CPT had no effect on the phosphorylation of AMPK by either AICAR or ZMP indicating that there was no involvement of the purinergic receptors. Conclusion: Although AICAR and ZMP increase glucose uptake in skeletal muscle, this was not seen in cardiomyocytes. Conversely, both compounds inhibited both basal and insulin stimulated glucose uptake despite increasing GLUT4 translocation. Inhibition of PI3-kinase in presence or absence of insulin unmasked hitherto unknown effects of AICAR and ZMP on PKB phosphorylation. / AFRIKAANSE OPSOMMING: Agtergrond: Oefening verhoog skeletspier glukose opname via AMP-geaktiveerde protein kinase (AMPK) aktivering en GLUT4 translokering vanaf die sitosol na die selmembraan. Dit verbeter ook glukose verbruik in tipe 2 diabetes pasiënte via verhoogde insulien sensitiwiteit. Insulien stimuleer GLUT4 translokering deur P13- kinase en protein kinase B (PKB/Akt) te aktiveer. Dit word dus gepostuleer dat daar 'n verbinding tussen hierdie twee paaie, wat beide betrokke is by GLUT4 translokering, bestaan. Dit is belangrik om hierdie verbinding te verstaan aangesien dit in behandelingstrategieë van tipe 2 diabetes geteiken kan word. Die oefening geïnduseerde verhoging in AMPK aktivering, kan deur 'n farmakologiese middel 5'- aminoimidasool-4-karboksamied ribonukleosied (AICAR), wat intrasellulêr omgesit word na 5'-aminoimidasool-4-karboksamied-ribonukleosiedfosfaat (ZMP), 'n AMP analoog, nageboots word. Doel: Om die effek van twee farmakologiese AMPK-aktiveringsmiddels, AICAR en ZMP, op die fosforilering van AMPK en PKB/Akt, sowel as moontlike effekte daarvan op insulien-gestimuleerde glukose opname en GLUT4 translokering, te ondersoek. Materiale en Metodes: Volwasse ventrikulêre kardiomiosiete is uit manlike Wistar rotharte geïsoleer d.m.v kollagenase perfusies en behandel met 1 mM AICAR of 1 mM ZMP in die teenwoordigheid of afwesigheid van 100 nM insulien of 100 nM wortmannin. Glukose opname is gemeet via intrasellulêre [3H]-2-deoksiglukose akkumulasie; PKB/Akt en AMPK fosforilering sowel as GLUT4 translokering is bepaal deur Western blot analises. Purinergiese reseptore is geblokkeer met 8-siklopentiel- 1,3-dipropielxanthien (8CPT) en die effek daarvan op AMPK fosforilering genoteer. Ten einde resultate wat in die geïsoleerde kardiomiosiet-model verkry is, te bevestig, is sekere eksperimente in die geïsoleerde rothart herhaal. Resultate: Beide AIGAR en ZMP stimuleer AMPK fosforilering. Die middels kan nie glukose opname verhoog nie, inteendeel, basale glukose opname is onderdruk alhoewel GLUT4 translokering vanaf die sitosol na die selmembraan wel plaasgevind het. Wortmannin kon insulien gemedieerde glukose opname en PKB/Akt fosforilering onderdruk. In die teenwoordigheid van wortmannin het beide AIGAR en ZMP PKB/Akt fosforilering verhoog. Alhoewel beide AIGAR en ZMP glukose opname in skeletspier verhoog, was dit nie die geval in kardiomiosiete nie. Beide middels het wel GLUT 4 translokering verhoog, wat duidelik demonstreer dat die translokering en aktivering van GLUT4, verskillende prosesse is. 8GPT het geen effek gehad op die fosforilering van AMPK deur AIGAR of ZMP nie, wat bewys dat daar geen betrokkenheid van die purinergiese reseptore was nie. Gevolgtrekking: Alhoewel AIGAR en ZMP glukose opname in skeletspier verhoog is dit nie die geval in kardiomiosiete nie. Beide middels inhibeer basale en insuliengestimuleerde glukose opname maar stimuleer GLUT4 translokeering. Inhibisie van PI3-kinase in die teenwoordigheid of afwesigheid van insulien, ontmasker voorheen onbekende effekte van AIGAR en ZMP op PKB/Akt fosforilering.

published_or_final_version / abstract / Physiology / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy