Background and Rationale: Connexin-43 (Cx43) is an integral membrane phosphoprotein and the major constituent of cardiac gap junctions, intercellular channels mediating metabolic and electrical coupling. In addition, Cx43 is involved in the regulation of cell proliferation, acting as an inhibitor of DNA synthesis, and is suggested to be essential for the ability of the heart to become resistant to injury in response to ischemic or pharmacological preconditioning. Cx43 is the target of several different signal transduction cascades culminating in its phosphorylation at several sites located at its C-terminal tail. Previous work in my mentor’s laboratory provided evidence that: cardioprotection of isolated perfused rat hearts by ischemic preconditioning or fibroblast growth factor 2 (FGF-2) administration was closely correlated with above-physiological Cx43 phosphorylation at protein kinase C (PKC) target sites such as serine S262; (ii), the FGF-2-induced cardiac Cx43 phosphorylation at S262 was mediated by protein kinase Cε (PKCε), and this cancelled the ability of Cx43 to inhibit DNA synthesis in neonatal cardiomyocytes. The overall purpose of this study was to further investigate the relationship between Cx43, its phosphorylation at PKCε-target sites such as S262, and PKCε-mediated end-points such as cytoprotection/cardioprotection, and stimulation of DNA synthesis. The main hypotheses were that (a) Cx43 phosphorylation at S262 mediates cardiomyocyte protection from ischemic injury by FGF-2, PKC and ischemic preconditioning, and (b) Cx43-associated inhibition of DNA synthesis is caused by activation of the transforming growth factor beta (TGFβ) pathway. Materials and Methods: Primary cultures of neonatal rat ventricular cardiomyocytes as well as the Cx43-deficient cell line HEK293 were used as in vitro models. Simulated ischemia was achieved in the presence of a low pH medium and incubation in a hypoxia chamber. Cell injury or cell death was assessed using the lactate dehydrogenase or TUNEL assay kits, respectively. Protein expression and localization were examined by western blotting and immunofluorescence using well characterized antibodies. The fraction of cells synthesizing DNA was obtained by determining the BrdU labeling index. Gene transfer in cardiomyocytes was achieved by infection with various adenoviruses carrying: wild type Cx43 or the Cx43 phosphorylation mutants such as S262A-Cx43, or a Cx43 mutant containing a serine to aspartate (S262D-) substitution; dominant negative Smad2 or TGFRII; wild type PKCε. Gene transfer in HEK293 cells was achieved using the TransIT reagent. Results: In the cytoprotection experiments, a modest overexpression of wild type Cx43, which localized predominantly to cell-cell contact sites, increased the resistance of cardiomyocytes to ischemic injury. In contrast, expression of a C-terminal-HA tagged Cx43, which showed localization to perinuclear sites, or of the channel domain-deficient C-terminal fragment of Cx43 (Cx43CT), which localized to cytosol and nucleus, rendered cardiomyocytes more vulnerable to ischemic injury. Preventing Cx43 phosphorylation at S262 by overexpressing S262A-Cx43 also rendered cardiomyocytes more vulnerable to ischemic injury, and fully abolished the ability of either FGF2, or PKC to confer cytoprotection. Conversely, the protective effect of ischemic preconditioning was only partially prevented by S262A-Cx43 expression. In addressing the mechanism by which Cx43 inhibits DNA synthesis in cardiomyocytes we found that the inhibition of several components of TGF-associated signal transduction including TGFβRI (with SB431542), or TGFβRII (by overexpressing dominant negative TGFβRII), or Smad2 (by overexpressing dominant negative Smad2) had no effect on Cx43-mediated inhibition of DNA synthesis. Treatment of cardiomyocytes with TGFβ prevented the FGF-2-induced Cx43 phosphorylation at S262, detected by phospho-specific antibodies. In HEK293 cells, we found that expression of Cx43CT, lacking the channel-forming domain, retained the ability to inhibit DNA synthesis, and that this event was dependent on S262: expression of S262A-Cx43CT elicited maximal inhibition of DNA synthesis, while S262D-Cx43CT (simulating constitutive phosphorylation) had no inhibitory effect. Conclusions: Phosphorylation of Cx43 at S262 is likely a mediator of FGF-2-induced and PKC-mediated cardiomyocyte resistance to ischemic injury, while protection by ischemic preconditioning may be only partially dependent on Cx43 and its phosphorylation at S262. The mechanism of cytoprotection by Cx43 phosphorylation at S262 is linked to preservation of membrane targeting and intercellular channel formation, as it was abolished in the absence of channel-forming ability (Cx43CT) or aberrant Cx43 localization (Cx43-HA). The mechanism by which Cx43 inhibits DNA synthesis is not dependent on downstream activation of TGF signal transduction. It is possible, however, that a component of TGF-triggered inhibition of DNA synthesis includes prevention of mitogen-induced Cx43 phosphorylation at S262. Furthermore, inhibition of DNA synthesis by Cx43, and its regulation by S262 phosphorylation are independent of channel-forming ability or subcellular localization. Targeting Cx43 and its phosphorylation at S262 may provide a novel strategy to improve cardiac response to injury, by decreasing tissue loss through increased resistance as well as improving a regenerative response by disinhibiting cardiomyocyte proliferation.

Background and Rationale: Connexin-43 (Cx43) is an integral membrane phosphoprotein and the major constituent of cardiac gap junctions, intercellular channels mediating metabolic and electrical coupling. In addition, Cx43 is involved in the regulation of cell proliferation, acting as an inhibitor of DNA synthesis, and is suggested to be essential for the ability of the heart to become resistant to injury in response to ischemic or pharmacological preconditioning. Cx43 is the target of several different signal transduction cascades culminating in its phosphorylation at several sites located at its C-terminal tail. Previous work in my mentor’s laboratory provided evidence that: cardioprotection of isolated perfused rat hearts by ischemic preconditioning or fibroblast growth factor 2 (FGF-2) administration was closely correlated with above-physiological Cx43 phosphorylation at protein kinase C (PKC) target sites such as serine S262; (ii), the FGF-2-induced cardiac Cx43 phosphorylation at S262 was mediated by protein kinase Cε (PKCε), and this cancelled the ability of Cx43 to inhibit DNA synthesis in neonatal cardiomyocytes. The overall purpose of this study was to further investigate the relationship between Cx43, its phosphorylation at PKCε-target sites such as S262, and PKCε-mediated end-points such as cytoprotection/cardioprotection, and stimulation of DNA synthesis. The main hypotheses were that (a) Cx43 phosphorylation at S262 mediates cardiomyocyte protection from ischemic injury by FGF-2, PKC and ischemic preconditioning, and (b) Cx43-associated inhibition of DNA synthesis is caused by activation of the transforming growth factor beta (TGFβ) pathway. Materials and Methods: Primary cultures of neonatal rat ventricular cardiomyocytes as well as the Cx43-deficient cell line HEK293 were used as in vitro models. Simulated ischemia was achieved in the presence of a low pH medium and incubation in a hypoxia chamber. Cell injury or cell death was assessed using the lactate dehydrogenase or TUNEL assay kits, respectively. Protein expression and localization were examined by western blotting and immunofluorescence using well characterized antibodies. The fraction of cells synthesizing DNA was obtained by determining the BrdU labeling index. Gene transfer in cardiomyocytes was achieved by infection with various adenoviruses carrying: wild type Cx43 or the Cx43 phosphorylation mutants such as S262A-Cx43, or a Cx43 mutant containing a serine to aspartate (S262D-) substitution; dominant negative Smad2 or TGFRII; wild type PKCε. Gene transfer in HEK293 cells was achieved using the TransIT reagent. Results: In the cytoprotection experiments, a modest overexpression of wild type Cx43, which localized predominantly to cell-cell contact sites, increased the resistance of cardiomyocytes to ischemic injury. In contrast, expression of a C-terminal-HA tagged Cx43, which showed localization to perinuclear sites, or of the channel domain-deficient C-terminal fragment of Cx43 (Cx43CT), which localized to cytosol and nucleus, rendered cardiomyocytes more vulnerable to ischemic injury. Preventing Cx43 phosphorylation at S262 by overexpressing S262A-Cx43 also rendered cardiomyocytes more vulnerable to ischemic injury, and fully abolished the ability of either FGF2, or PKC to confer cytoprotection. Conversely, the protective effect of ischemic preconditioning was only partially prevented by S262A-Cx43 expression. In addressing the mechanism by which Cx43 inhibits DNA synthesis in cardiomyocytes we found that the inhibition of several components of TGF-associated signal transduction including TGFβRI (with SB431542), or TGFβRII (by overexpressing dominant negative TGFβRII), or Smad2 (by overexpressing dominant negative Smad2) had no effect on Cx43-mediated inhibition of DNA synthesis. Treatment of cardiomyocytes with TGFβ prevented the FGF-2-induced Cx43 phosphorylation at S262, detected by phospho-specific antibodies. In HEK293 cells, we found that expression of Cx43CT, lacking the channel-forming domain, retained the ability to inhibit DNA synthesis, and that this event was dependent on S262: expression of S262A-Cx43CT elicited maximal inhibition of DNA synthesis, while S262D-Cx43CT (simulating constitutive phosphorylation) had no inhibitory effect. Conclusions: Phosphorylation of Cx43 at S262 is likely a mediator of FGF-2-induced and PKC-mediated cardiomyocyte resistance to ischemic injury, while protection by ischemic preconditioning may be only partially dependent on Cx43 and its phosphorylation at S262. The mechanism of cytoprotection by Cx43 phosphorylation at S262 is linked to preservation of membrane targeting and intercellular channel formation, as it was abolished in the absence of channel-forming ability (Cx43CT) or aberrant Cx43 localization (Cx43-HA). The mechanism by which Cx43 inhibits DNA synthesis is not dependent on downstream activation of TGF signal transduction. It is possible, however, that a component of TGF-triggered inhibition of DNA synthesis includes prevention of mitogen-induced Cx43 phosphorylation at S262. Furthermore, inhibition of DNA synthesis by Cx43, and its regulation by S262 phosphorylation are independent of channel-forming ability or subcellular localization. Targeting Cx43 and its phosphorylation at S262 may provide a novel strategy to improve cardiac response to injury, by decreasing tissue loss through increased resistance as well as improving a regenerative response by disinhibiting cardiomyocyte proliferation.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21255号 / 医博第4373号 / 新制||医||1029(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 長船 健二, 教授 柳田 素子, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Cardiovascular disease is the leading cause of death world-wide, with 42% of sudden cardiac death in young adults caused by myocarditis. Viruses represent the main cause of myocarditis, with adenovirus being a leading pathogen. However, it is not understood how adenoviruses cause sudden cardiac arrest. Myocarditis is defined by two phases, acute and chronic. The acute phase involves viral-mediated remodeling of subcellular structures in the myocardium, which is thought to contribute to arrhythmogenesis. The chronic phase is immune response-mediated, where the host immune system causes damage that induces gross remodeling of the heart, which can result in cardiac arrest or heart failure. Electrical impulses of the heart are propagated by cardiomyocytes, via gap junctions, ion channels, and intracellular junctions, creating the healthy heartbeat. Cx43, the primary gap junction protein in the myocardium, not only propagates electrical signals, but also anti-viral molecules. Viral targeting of gap junction function leads to reduced anti-viral responses in neighboring cells. However, reduced cellular communication would dangerously alter cardiac conduction. Using a cardiotropic adenovirus, MAdV-3, we find that viral genomes are significantly enriched in the heart, with a decrease of gap junction and ion channel mRNA in infected hearts, however, their protein levels were unchanged. Phosphorylation of Cx43 at serine 368, known to reduce gap junction open probability, was increased in infected hearts. Ex vivo optical mapping illustrated decreased conduction velocity in the infected heart and patch clamping of isolated cardiomyocytes revealed prolonged action potential duration, along with decreased potassium current density during infection. Pairing mouse work with human induced pluripotent stem cell-derived cardiomyocytes, we found that human adenovirus type-5 infection increased pCx43-Ser368 and perturbation of intercellular coupling, as we observed with in vivo MAdV-3 infection. Allowing adenovirus infection to progress in vivo, we find myocardium remodeling and immune cell infiltration. Together, these data demonstrate the complexity of cardiac infection from viral-infection induced subcellular alterations in electrophysiology to immune-mediated cardiomyopathy of cardiac adenoviral infection. Our data describe virally induced mechanisms of arrhythmogenesis, which could lead to the development of new diagnostic tools and therapies, to help protect patients from arrhythmia following infection. / Doctor of Philosophy / Viral infection has long thought to be a cause of unexplained sudden cardiac death, especially in young adults. Viruses have been identified to cause many cases of deleterious remodeling of the heart, which can result in heart failure. The heart relies on electrical signaling that moves in a coordinated fashion to contract and pump blood throughout the body. The cells within the heart that do this are called cardiomyocytes, and they join end-to-end to communicate with each other via gap junctions. Gap junctions are tunnels that allow for ions that create electrical impulses to pass, and molecules, such as ones that are important in immune responses to infection. In addition to gap junctions in the heart, ion channels, which are highly selective to allow only one ion flow, unlike gap junctions, create the healthy heartbeat. The most common gap junction in the heart comprises Cx43 proteins. If a virus were to alter how Cx43 connects to a neighboring cell, this would cause a better environment for the virus, as this would keep anti-viral surveillance low, however, this would change how the electrical signal moves throughout the heart, creating arrhythmias. Adenoviruses are a common cold virus, but have been found in the hearts of many cardiac arrest patients. However, little is known on how adenoviruses may cause cardiac arrest, because human adenoviruses are only successful in humans, and mouse adenoviruses are only successful in mice. This creates a challenge when studying the dynamic heart, which does not translate well to cells in a dish. A mouse adenovirus, called Mouse Adenovirus Type-3 (MAdV-3) was reported to favor infecting the heart in mice, but no research has been published on if this virus can answer how adenoviruses change the heart. Because of this virus, and our prior research that adenoviruses can decrease Cx43 within skin cells in a dish, we used MAdV-3 to understand if, how adenoviruses could cause sudden cardiac arrest, and if longer infection could change the overall structure of the heart. We find that MAdV-3 infection prefers the heart to other organs, and that early stages, reduce both the speed of the electrical signal moves through heart and, looking within a cardiomyocyte, how it creates that electrical signal. These changes are arrhythmogenic and accompany modification of Cx43 that would close the gap junction between two cells, changing how ions and molecules move between cells. Using a human adenovirus infection in human cardiomyocytes created from stem cells, this result is also observed. If infection is allowed to continue in the mouse to cause chronic infection, the heart itself changes shape and is diseased. Together, this work shows that adenoviruses create a diseased heart, first the virus changes how the electrical signal moves and then later, causes thinning of the heart muscle. These data illustrate the role viruses play in causing cardiac arrest and could lead to diagnostic or drug targets.

No description available.

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.

Die Ergebnisse dieser Arbeit lassen vermuten, dass die Connexin43 Anordnung an der der Zellmembran humaner Herzmuskelzellen pharmakologisch beeinflussbar ist. Es ist bekannt, dass sich Connexin43 an der polaren und lateralen Zellmembran beim Vorhofflimmern und Sinusrhythmus unterschiedlich anordnet. Während beim Sinusrhythmuspatienten Connexin43 kaum an der lateralen Zellmembran zu finden ist, zeigt sich beim Vorhofflimmern vor allem an der lateralen Zellmembran eine verstärkte Connexin43 Anhäufung. Neben dem Rhythmustyp hat auch β-Adrenozeptorstimulation Einfluss auf die Connexin43 Expression. Aus diesem Grund untersucht die vorliegende Arbeit den Einfluss einer pharmakologischen Blockade der β-Adrenozeptoren durch Betablocker. Dafür wurden 38 die untersuchten Patienten anhand ihres Rhythmustyps, ihrer kardialen Begleiterkrankung und ihrer Pharmakotherapie (Betablocker: ja/nein) unterteilt und neben deren klinischen Daten ihre intraoperativ gewonnenen Herzohrbiopsien immunhistochemisch gefärbt und anschließend ausgewertet. Dabei zeigte sich, dass es zum einen zu einer unterschiedlichen Anordnung von Connexin43 bei den beiden Rhythmustypen kommt. Während beim Sinusrhythmus Connexin43 vor allem polar an der Zellmembran zu finden ist, ist es beim Vorhofflimmern vor allem an den lateralen Zellgrenzen zu finden. Betablockade geht hierbei vor allem beim Patienten mit Vorhofflimmern und Mitralklappenvitium mit einer Reduktion der Lateralisierung und einem positiven Effekt auf die Polarisierung einher.

Connexin43 (Cx43) is one of the most abundant gap junction proteins in the central nervous system. Abnormal opening of Cx43 hemichannels after ischemic insults causes apoptotic cell death. In this study, we found persistently increased expression of Cx43 8 h to 7 d after hypoxia/ischemia (HI) injury in neonatal rats. Pre-treatment with Gap26 and Gap27, two Cx43 mimetic peptides, significantly reduced cerebral infarct volume. Gap26 treatment at 24 h after ischemia improved functional recovery on muscle strength, motor coordination, and spatial memory abilities. Further, Gap26 inhibited Cx43 expression and reduced active astrogliosis. Gap26 interacted and co-localized with Cx43 together in brain tissues and cultured astrocytes. After oxygen glucose deprivation, Gap26 treatment reduced the total Cx43 level in cultured astrocytes; but Cx43 level in the plasma membrane was increased. Degradation of Cx43 in the cytoplasm was mainly via the ubiquitin proteasome pathway. Concurrently, phosphorylated Akt, which phosphorylates Cx43 on Serine373 and facilitates the forward transport of Cx43 to the plasma membrane, was increased by Gap26 treatment. Microdialysis showed that increased membranous Cx43 causes glutamate release by opening Cx43 hemichannels. Extracellular glutamate concentration was significantly decreased by Gap26 treatment in vivo. Finally, we found that cleaved caspase-3, an apoptosis marker, was attenuated after HI injury by Gap26 treatment. Effects of Gap27 were analogous to those of Gap26. In summary, our findings demonstrate that modulation of Cx43 expression and astroglial function is a potential therapeutic strategy for ischemic brain injury.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24493号 / 医博第4935号 / 新制||医||1063(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 浅野 雅秀, 教授 万代 昌紀, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM