Sudden cardiac death occurs due to aberrations in the multifactorial process that is cardiac conduction. Conduction velocity (CV) and its modulation by several determinants, like cellular excitability, tissue structure and electrical coupling by gap junctions (GJ), have been extensively studied. However, there are several discrepancies in cardiac electrophysiology research that have extended over decades, suggesting elements that are still not completely understood about this complex phenomenon. This dissertation will focus on one such mechanism, ephaptic coupling (EpC). The purpose of this work is twofold, 1) to identify ionic determinants of EpC, and its interactions with gap junctional coupling (GJC) and, 2) to investigate the possible role of serum ion modulation in cardiac arrhythmia therapy. First, the effects of altering extracellular ion concentration – sodium, potassium and calcium at varying GJ protein expression were studied. Briefly, reducing sodium was related to CV slowing under conditions of reduced EpC (wide intercalated disc nanodomains – perinexi) and GJC (reduced GJ protein – Connexin43). On the other hand, increasing potassium slowed CV in hearts with wide perinexi independent of GJC. Elevating calcium, reduced perinexal width and was associated with fast CV during physiologic sodium concentration. However, under conditions associated with disease, like hyponatremia, elevating calcium still reduced perinexal width but slowed CV. These findings are the first to suggest that ionic modulators of EpC could modulate CV during health and disease. Next, the potential of perfusate ion modulation in cardiac arrhythmia therapy was investigated. Briefly, in a model of myocardial inflammation, TNFα, a pro-inflammatory cytokine, slowed CV relative to control conditions and this was associated with widening of the perinexus (reduced EpC). Increasing extracellular calcium restored CV to control values by improving not only EpC but also GJC. Finally, in a model of metabolic ischemia in the heart, CV response due to solutions with varying sodium and calcium concentrations were tested. The solutions that were associated with wider perinexi and elevated sodium performed best during ischemia by attenuating CV slowing, reducing arrhythmias and increasing time to asystole. Taken together, these findings provide evidence for the possibility of ionic determinants of EpC in cardiac arrhythmia therapy. / Ph. D.

Connexins are transmembrane proteins that form hemichannels allowing the exchange of molecules between the extracellular space and the cell interior. Two hemichannels from adjacent cells dock and form a continuous gap junction pore, thereby permitting direct intercellular communication. Connexin 36 (Cx36), expressed primarily in neurons, is involved in the synchronous activity of neurons and may play a role in aberrant synchronous firing, as seen in seizures. To understand the reciprocal interactions between Cx36 and seizure-like neural activity, we examined three questions: a) does Cx36 deficiency affect seizure susceptibility, b) does seizure-like activity affect Cx36 expression patterns, and c) does acute blockade of Cx36 conductance increase seizure susceptibility. We utilize the zebrafish pentylenetetrazol (PTZ; a GABA(A) receptor antagonist) induced seizure model, taking advantage of the compact size and optical translucency of the larval zebrafish brain to assess how PTZ affects brain-wide neuronal activity and Cx36 protein expression. We exposed wild-type and genetic Cx36-deficient (cx35.5-/-) zebrafish larvae to PTZ and subsequently mapped neuronal activity across the whole brain, using phosphorylated extracellular-signal-regulated kinase (pERK) as a proxy for neuronal activity. We found that cx35.5-/- fish exhibited region-specific susceptibility and resistance to PTZ-induced hyperactivity compared to wild-type controls, suggesting that genetic Cx36 deficiency may affect seizure susceptibility in a region-specific manner. Regions that showed increased PTZ sensitivity include the dorsal telencephalon, which is implicated in human epilepsy, and the lateral hypothalamus, which has been underexplored. We also found that PTZ-induced neuronal hyperactivity resulted in a rapid reduction of Cx36 protein levels within. 30 minutes and one-hour exposure to 20 mM PTZ significantly reduced the expression of Cx36. This Cx36 reduction persists after one-hour of recovery but recovered after 3-6 hours. This acute downregulation of Cx36 by PTZ is likely maladaptive, as acute pharmacological blockade of Cx36 by mefloquine results in increased susceptibility to PTZ-induced neuronal hyperactivity. Together, these results demonstrate a reciprocal relationship between Cx36 and seizure-associated neuronal hyperactivity: Cx36 deficiency contributes region-specific susceptibility to neuronal hyperactivity, while neuronal hyperactivity-induced downregulation of Cx36 may increase the risk of future epileptic events. / Doctor of Philosophy / Within the brain, cells (neurons) communicate with each other to pass along information. This communication is important for normal functions of the brain such as learning and memory, muscle movement, etc. Epilepsy is a disease of the brain that is caused by rapid over synchronized communication between cells. This leads to seizures which can include convulsions, loss of attention, and much more. Currently, 30% of patients suffering from epilepsy do not have a treatment option that works for them, it is, therefore, imperative to investigate new targets for treatment in this disease. Connexin36 is a protein in the brain that directly connects cells so they can pass information quickly between them. Connexin36, therefore, might make a good target for treatment. Previous work has aimed to understand this relationship but has been limited in their ability to look at the entire brain at any one time. The goal of this study was to understand the relationship between connexin 36 and brain hyperactivity across the whole brain simultaneously. To understand this relationship, we first determined what happened to brain activity if the protein was missing entirely after exposure to a seizure causing drug. We were asking: How does connexin 36 affect hyperactivity. We found that different regions of the brain responded differently without the connexin 36 protein. This suggests that one size does not fit all, and one must look at the whole brain to understand the effects of the connexin 36 protein. Next, we asked a similar question, but in the opposite direction, how does hyperactivity affect connexin 36? We found, in the short-term, hyperactivity reduced the amount of connexin 36 present in certain regions of the brain. This continued until 3 hours following exposure to the seizure causing drug Pentylenetetrazol (PTZ). Lastly, to determine if this short-term reduction in connexin 36 meant that an individual was more likely to experience hyperactivity. To do this, we used a connexin 36 blocking drug, then introduced the seizure causing drug at different concentrations. We found, at all concentrations, the connexin 36 blocking drug caused significant changes in neuronal activity, depending on the brain regions. Overall, our results showed that connexin 36 plays an important role in hyperactivity and that a short-term reduction in connexin 36 is detrimental, and may contribute to an increase in the possibility of subsequent hyperactivity.

Hintergrund: Connexine (Cx) spielen eine wichtige Rolle bei Wachstum und Differenzierung. Die Angiogenese ist an vielen physiologischen und pathologischen Prozessen beteiligt. Jedoch ist noch unklar, ob Connexine einen Einfluss auf die Angiogenese haben. Fragestellung; In dieser Arbeit sollte die Rolle endothelialer Connexine an der Angiogenese untersucht werden. Methoden: Venöse Zellen der menschlichen Nabelschnur (HUVEC) wurden kultiviert bis sie subkonfluent waren. Vor der 3D Kultur im Matrigel wurden die Zellen entweder mit Nicotin, dem Gap Junction Inhibitor Palmitoleinsäure (PA), einem siRNA-Knockdown von entweder Cx37, Cx40 oder Cx43 oder mit Isoprenalin behandelt. Die Zellen wurden dann auf ein in vitro Angiogenese-Assay (3D-Kultur, Matrigel) gegeben und nach 18h wurden unterschiedliche Angiogeneseparameter erhoben, um die Komplexität der Angiogenese zu beurteilen. Änderungen der Expression der Cx mRNA- und Proteinexpression sowie der Kommunikation über Gap Junctions wurden mittels PCR, Immunfluoreszenzmikroskopie, Western Blot und Lucifer Yellow Dye Transfer untersucht. Bei Gewebeproben der A. mammaria von Rauchern und Nichtrauchern wurden mittels Immunofluoreszenzmikroskopie die Expression der Cx beurteilt. Ergebnisse: Die Behandlung mit spezifischer siRNA führte zu einer signifikanten Abnahme der Expression des jeweiligen Connexins in den HUVECs. Sowohl der Knockdown als auch die Behandlung mit PA verringerte die Kommunikation über Gap Junctions signifikant und reduzierte die Anzahl der Abzweigungen im Angiogenese-Assay. Der Knockdown des Cx43 und Cx40 sowie die Behandlung mit PA reduzierten ebenfalls die Komplexität des Musters im Matrigel-Assay. Nicotin führte zu einer Reduktion der Cx43- und Cx37-Proteinexpression, wohingegen Cx40 durch transskriptionelle Gegenregulation konstant gehalten wurde, sowie zu einer Abnahme der Länge der Kapillar-ähnlichen-Strukturen, der Anzahl der Abzweigungen und des Musters im Matrigelassay, während die Anzahl der Zellen zunahm. Die mRNA-Expression der Connexine war hingegen erhöht. In Gewebeproben von Rauchern konnte analog eine verminderte Expression von Cx43 und Cx37, aber nicht von Cx40, in der Intima gezeigt werden. Isoprenalin erhöhte die Proteinexpression der endothelialen Connexine und verbesserte sowohl die interzelluläre Kommunikation als auch das Muster im Matrigel-Assay. Ebenso waren die Kapillar-ähnlichen Strukturen im Vergleich zur Kontrolle länger. Schlussfolgerung: Aus den erhobenen Ergebnissen lässt sich der Schluss ziehen, dass Connexine bei der Angiogenese involviert sind, vor allem beim Vorgang der Verzweigung. Dies kann teilweise die Veränderung erklären, die eine Nicotinbehandlung auf die Angiogenese hat.

Gap Junction Kanäle sind wesentlich für die interzelluläre Kommunikation im Herzen. Die wichtigsten kardialen Gap Junction Proteine sind Connexin 43, Connexin 40 und in frühen Entwicklungsstadien Connexin 45. Da Katecholamine eine wichtige Rolle in der kardialen Physiologie bzw. Pathophysiologie spielen, sollte geklärt werden, ob Katecholamine die Expression von Connexin 40 und Connexin 43 beeinflussen. Es wurden neonatale Rattenkardiomyozyten in Zellkultur 24 Stunden mit aufsteigenden Noradrenalinkonzentrationen (1-10000 nM) (physiologischer Agonist für α-und β-Adrenozeptoren) inkubiert. Hier zeigte sich ein signifikanter Anstieg der Connexin 43 Expression, während die Connexin 40 Expression nicht beeinflußt wurde. Um zu zeigen über welchen Adrenozeptor (α oder β) die Hochregulation von Cx 43 durch Noradrenalin erfolgt, wurden die Zellkulturen in zwei weiteren Versuchsreihen zusätzlich mit einem β-Blocker (Propranolol 100µM) bzw. einem α- Blocker (Prazosin 10 µM) über 24 Std. inkubiert. Hier zeigte sich eine deutliche Inhibierung des Noradrenalineffektes durch die Blockade der α-Rezeptoren durch Prazosin. Eine Blockade der β-Rezeptoren hatte keinen inhibitorischen Effekt auf die Hochregulation der Expression von Cx 43 durch Noradrenalin. Es kam hier sogar zu einer verstärkten Expression verglichen mit der Cx 43 Expression unter Noradrenalin ohne Rezeptorblockade. Desweiteren wurde überprüft, ob es auch unter der Behandlung von Sprague– Dawley-Ratten mit einem β-Mimetikum (Isoprenalin) bzw. einem α-Mimetikum (Phenylephrin) zu einer Veränderung der Connexinexpression kommt. Die Western Blot Analysen ergaben hierbei wieder einen α-Rezeptoren vermittelten Effekt, indem es zu einem wesentlichen Konzentrationsanstieg von Connexin 43 unter der Behandlung mit Phenylephrin kam.

As a highly versatile signal, Ca2+ operates over a wide temporal range to regulate many different cellular processes, impacting nearly every aspect of cellular life including excitability, exocytosis, motility, apoptosis, and transcription. While it has been well recognized that Ca2+ acts as both a second messenger to regulate cell-cell communication upon external stimuli and as a first messenger to integrate extracellular with intracellular signaling in various cell types. Molecular bases for such regulation and related human diseases are largely hampered by the challenges related to key membrane proteins. In the present study, we first investigated the regulatory role of intracellular Ca2+ ([Ca2+]i) on Connexin45 (Cx45) gap junction through a ubiquitous Ca2+ sensor protein-Calmodulin (CaM). Using bioluminescence resonance energy transfer assay, this study provides the first evidence of direct association of Cx45 and CaM in a Ca2+-dependent manner in cells. Complementary approaches including bioinformatics analysis and various biophysical methods identified a putative CaM-binding site in the intracellular loop of Cx45 with high Ca2+/CaM-binding affinity and Ca2+-dependent binding mode that is different from alpha family of connexins. To understand the role of extracellular calcium in regulation of gap junction hemichannels, we would like to prove a possible Ca2+-binding site predicted by our computational algorithm MUGSR in Connexin 26 (Cx26) through mutagenesis study, metal binding affinity measurement, conformational changes examination of purified Cx26 protein from Sf9; however, we failed to achieve this goal due to either the limitation of available methods or lethal effect of mutating the predicted Ca2+-binding ligand. Additionally, in this study, we identified a putative Ca2+-binding site in metabotropic glutamate receptor 5 (mGluR5) and demonstrated the importance of this Ca2+-binding site in activation of mGluR5 and modulating the actions of other orthosteric ligands on mGluR5. In addition, we successfully solved the first crystal structure of the extracellular domain of Ca2+-sensing receptor (CaSR) bound with Mg2+ and an unexpected Trp derivative. The extensive study of mechanism of CaSR function specifically through Mg2+-binding site and the unexpected ligand-binding site was done using several cell-based assays in wild type CaSR and mutants. Studies in this dissertation provides more information on how Ca2+ regulates gap junction channels, modulates mGluR5 activities and structural basis for regulation of CaSR by Mg2+ and an unexpected Trp derivative co-agonist.

Gap junctions are intercellular channels that permit passage of electrical and chemical signals between neighbouring cells. Vascular endothelium typically co-expresses Cx37 and Cx40, but may downregulate its expression of Cx37 (and upregulate Cx43) in response to changes in flow. The specific regulatory roles mediated by vascular endothelial connexins, and the consequences of altered connexin expression, remain unclear. In this study, we hypothesized that Cx37 and Cx40 regulate distinct vascular responses. We further hypothesize that Cx37 is predominantly involved in vascular growth control, whereas vascular growth is not affected by ablation of Cx40 expression. We show herein that Cx37, but not Cx40 or Cx43, suppresses growth of a highly-proliferative cancer cell line by inducing G1 cell cycle accumulation. We further show that Cx37-deficient mice, lacking Cx37's putative growth inhibitory effect on the vasculature, exhibit a more extensive native and post-ischemic collateral circulation, and greater ischemia-induced microvascular density. In addition, Cx37-/- mice demonstrate a functional improvement in recovery over wild-type animals in two models of hindlimb ischemia. By contrast, Cx40-/- mice fail to recover distal limb flow following unilateral hindlimb ischemia, resulting in necrosis. Long-term angiotensin II antagonism normalized post-ischemic hindlimb bloodflow, reduced macrophage infiltration, and delayed (but did not reverse) the necrotic phenotype of these animals. In summary, we show a distinct role for each of the endothelial connexins, Cx37 and Cx40, in regulating post-ischemic vascular responses.

Lymphatic valves (LVs) are cusped luminal structures that permit the movement of lymph in only one direction and are therefore critical for proper lymphatic vessel function. Congenital valve aplasia or agenesis can, in some cases, be a direct cause of lymphatic disease. Knowledge about the molecular mechanisms operating during the development and maintenance of LVs may thus aid in the establishment of novel therapeutic approaches to treat lymphatic disorders. In this study, we examined the role of Connexin43 (Cx43), a gap junction protein expressed in lymphatic endothelial cells (LECs), during valve development. Mouse embryos with a null mutation in Cx43 (Gja1) were previously shown to completely lack mesenteric LVs at embryonic day 18. However, interpreting the phenotype of Cx43(-/-) mice was complicated by the fact that global deletion of Cx43 causes perinatal death due to heart defects during embryogenesis. We have now generated a mouse model (Cx43(∆LEC)) with a lymphatic-specific ablation of Cx43 and show that the absence of Cx43 in LECs causes a delay (rather than a complete block) in LV initiation, an increase in immature valves with incomplete leaflet elongation, a reduction in the total number of valves, and altered lymphatic capillary patterning. The physiological consequences of these lymphatic changes were leaky valves, insufficient lymph transport and reflux, and a high incidence of lethal chylothorax. These results demonstrate that the expression of Cx43 is specifically required in LECs for normal development of LVs.

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Thu Annelise Nguyen / Preclinical animal models of mammary carcinoma formation are vital for the advancement of cancer research, specifically in drug development. Two different types of animal models were utilized to determine the efficacy of combinational treatment of common antineoplastic drugs and the new class of primaquines that act as gap junction enhancers (PQs) at attenuating mammary tumor growth. The classic xenograft mouse model was used to show that PQs could increase the efficacy of cisplatin and paclitaxel. Combinational treatment induced an upregulation of connexin and caspase expression in the isolated tumor. Next the transgenic PyVT mouse model was characterized by multiple factors, including hormone receptor status, molecular markers for survival and proliferation, tissue histopathology, and secondary metastases during multiple stages of tumor development. This model showed limited therapeutic response to the antineoplastic drugs tested. PQ1 effectively attenuated tumor growth at all stages of tumorigenesis in the PyVT model, while PQ7 was determined to be an effective chemopreventive compound rather than chemotherapeutic. The PQs altered the expression profiles of connexins during tumorigenesis. Together the results indicate that PQs have an anticancer effect that is more efficient at attenuating tumor growth than the common antineoplastic compounds. Lastly the PyVT mouse model was used to determine the efficacy of antineoplastic compounds on male mammary carcinoma development. Interestingly, the antineoplastic compound that attenuated female mammary carcinoma growth did not produce a therapeutic response in the males and vice versa, suggesting a need for further studies into the male response to therapy.

Previous work has shown D1/D2 requisite synergism can still occur in the striatum in the absence of action potentials. Some nonclassical communication such as gap junctions may be allowing the segregated dopamine (DA) receptors to interact to produce stereotyped motor activity. Connexin-32 (Cx32) and connexin-36 (Cx36) were targeted for study due to their abundance in neural tissues and presence in the striatum. Mice lacking either the Cx32 or Cx36 gene and their respective wildtype littermates were compared on a climbing behavior task used to gauge their dopaminergic activity after receiving either saline, D1 agonist, D2 agonist, or both D1 and D2 agonists. The results showed that D1/D2 requisite synergism was still intact in both strains of mice. The Cx32 WT mice displayed significantly greater scores than the KO mice in the D1/D2 treatment. The Cx36 mice did not display a significant genotype difference, but a trend was observed with the KO females having larger scores relative to WT females or to males of either genotype.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system leads to the rewarding properties of alcohol. The mesolimbic DA system, which plays an important role in regulating reward and addiction, consists of DA neurons in the midbrain ventral tegmental area (VTA) that innervate the nucleus accumbens (NAc). It is believed that VTA DA neurons are inhibited by local gamma-aminobutyric acid (GABA) interneurons that express connexin-36 (Cx36) gap junctions (GJs). We have previously demonstrated that blocking Cx36 GJs suppresses electrical coupling between VTA GABA neurons and reduces ethanol intoxication and consumption suggesting that electrical coupling between mature VTA GABA neurons underlies the rewarding properties of ethanol. The aim of this study was to further investigate the role of VTA GABA neurons expressing Cx36 GJs in regulating DA neuron activity and release and mediating ethanol effects on VTA GABA neurons. To this end, we customized a Designer Receptor Exclusively Activated by Designer Drugs (DREADDs) viral vector to target VTA GABA neurons expressing Cx36 GJs in order to chemogenetically modulate their activity. In order to more conclusively demonstrate the role of this sub population of VTA GABA neurons in regulating DA neural activity and release we used electrophysiology to characterize the electrical changes that occur in VTA DA and GABA neurons when Cx36-expressing VTA GABA cells were selectively activated. In addition, we evaluated the effects of activation of VTA GABA neurons on brain stimulation reward and alcohol consumption in ethanol naive and dependent mice. Results indicate that there are two populations of GABA neurons in the VTA, one that is GAD65+/Cx36+ and one that is GAD67+/Cx36-. Activation of Cx36+ VTA GABA neurons by clozapine-n-oxide (CNO) in mice injected with Gq DREADD activated VTA DA neurons and subsequent DA release in the NAc, suggesting that Cx36-containing GABA neurons are inhibiting non-Cx36 GABA neurons to disinhibit DA neurons. In hM3Dq animals, CNO administration provided a rewarding stimulus in the conditioned pace preference paradigm, and reduced consumption in the drink-in-the-dark ethanol consumption paradigm in dependent and naïve mice. A better understanding of the circuitry of the mesolimbic DA system is key to understanding the mechanisms that lead to addiction and may ultimately lead to improved therapies for substance abuse.