Syfte: Syftet med den här studien var att utvärdera ögontorrhetsparametrar och synskärpa hos patienter som fått Visian ICL implanterat och sedan jämföra med en tidigare studie gjord om LASIK, för att se vilken metod som gav minst påverkan på patienternas tårfilm och gav minst biverkningar gällande torra ögon Metod: En grupp på sex stycken som implanterades med en ICL-lins undersöktes på operationsdagen och sedan cirka tre veckor efter operation. Undersökningarna som gjordes före och efter operation var en OSDI-enkät innehållande 12 frågor gällande torrhetsproblem, lipidskikt, NITBUT och tårmenisken utvärderades med hjälp av ett Tearscope, och sist kontrollerades visus. Resultat: 4 personer upplevde att de hade färre torrhetsproblem postoperativt. 6 ögon visar på ett tunnare lipidskikt postoperativt, 2 ögon visar på ett tjockare lipidskikt postoperativt och 4 ögon hade ett oförändrat lipidskikt. 5 ögon visar på en tunnare tårmenisk postoperativt, 2 ögon visar på en tjockare tårmenisk postoperativt och 5 ögon visar på ett oförändrat resultat. Medelvärdet på NITBUT ökade från 16,8±6,7 till 20,3±8,9 sekunder postoperativt, alltså en ökning på 3,2 sekunder. 2 ögon visar på ett försämrat visus postoperativt, 4 ögon visar på ett förbättrat visus postoperativt och 6 ögon visar på ett oförändrat resultat. Slutsats: Den här studien visar på att Visian ICL är en bra och säker metod för synkorrigering och att metoden ger färre biverkningar gällande torra ögon än LASIK. / The Visian Implantable Collamer Lens (ICL) is a lens implanted into the posterior chamber as an intraocular lens. It is effective for moderate to high ametropia correction. The ICL increases contrast sensetivity and the method is reversible (Iagarashi et al., 2014). The aim of this study was to evaluate dry eye parameters and visual acuity in Visian ICL patiens and then compare with a previous study about LASIK to see which method that gave the least impact on the patients tear film, and to examine with which method patients experienced least dry eye problems. Method: A group of six patients who had ICL lenses implanted were examined on the day of the surgery and then about three weeks after surgery. The same examination was conducted before and after surgery, an OSDI questionnaire containing 12 questions about dry eye symptoms, lipid layer, NITBUT and tear meniscus were evaluated using a Tearscope, and visual acuity measured and compared with their preoperative fully corrected visual acuity. Results: 5 people felt that they had less dry eye symptoms postoperatively. 6 eyes showed a thinner lipid layer postoperatively, 2 eyes had a thicker lipid layer postoperatively and 4 eyes had an unchanged lipid layer. 5 eyes showed a thinner tear meniscus postoperatively, 2 eyes showed a thicker tear meniscus postoperatively and 5 eyes showed an unchanged result. The mean NITBUT significantly increased from 16.8±6,7 to 20.3±8,9 seconds postoperatively, an increase of 3.2 seconds. 2 eyes had a poorer visual acuity postoperatively, 4 eyes showed an improved visual acuity postoperative and 6 eyes shows an unchanged result. Conclusion: This study indicates that the Visian ICL is a good and safe method of vision correction and that the method provides fewer side effects regarding dry eye symptoms than LASIK.

<p>The protein Snm1B binds to the telomere binding protein TRF2 to help protect telomeres from DNA damage during S phase. In addition, Snm1B protects DNA from agents that induce interstrand crosslinks (ICLs), lethal lesions that covalently attach the opposite strands of DNA together. To elucidate how Snm1B performs these functions I performed a yeast two-hybrid screen to identify proteins binding to the C-terminus of Snm1B. From this screen I identified PSF2 (GINS2), a member of the tetrameric protein complex GINS, to bind Snm1B. The GINS complex is required for replication initiation and elongation. Of interest, the knockdown of PSF2 sensitizes cells to ICL inducing agents. I therefore tested the interaction of PSF2 and Snm1B by co-immunoprecipitation from human cells and discovered that PSF2 binds to two regions of Snm1B. Deletion of the first of these regions inhibited the ability of Snm1B to co-immunoprecipitate with the protein Mus81, a structure specific endonuclease that is required to form double strand breaks (DSBs) as an intermediate in ICL repair. Deletion of the second binding region reduced the ability of PSF2 to localize Snm1B onto chromatin. These data support a role for an interaction of Snm1B with PSF2 in ICL repair.</p> / Dissertation

DNA crosslinking agents exhibit a variety of DNA lesions, such as monoadducts, DNA-DNA interstrand or intrastrand crosslinks or DNA-protein crosslinks. Agents that produce interstrand crosslinks (ICLs) exist naturally and are widely used in chemotherapy. Therefore, it is important to understand how the lesions induced by these agents are repaired. In bacteria, the repair is mainly dependent on nucleotide excision repair (NER) together with homologous recombination (HR) or translesion synthesis (TLS). In human cells, it is not clear how these lesions are repaired, and it is believed to be a more complicated process in which NER does not play as important a role as in prokaryotes. Here, we investigated the repair mechanisms mainly after treatment with psoralen but also with acetaldehyde, cisplatin and mitomycin C in some studies. As expected from studies on plasmids and in bacteria, we used new techniques to confirm that various ICL-inducing agents block replication fork elongation in mammalian cells. We also found that the replication fork was unable to bypass these lesions. We confirmed that ERCC1/XPF and the HR proteins BRCA2 and XRCC2/3 are vital for protection against ICL treatments. These proteins were also found to be equally important for the repair of monoadducts. To better understand ICL repair in mammalian cells, we developed a method to study the induction and unhooking of ICL in human fibroblasts. We found that ICLs were repaired and that 50% of the induced ICLs were unhooked within 3 hours following exposure. Additionally, we determined that XPA, but not XPE, is involved in ICL unhooking, although not affecting lethality. A step in ICL repair is the formation of double-strand breaks (DSBs), and we identified a replication-dependent formation of DSBs following ICL treatment. Furthermore, ERCC1/XPF was not necessary for DSB formation. The repair of these DSBs was performed by HR and involved ERCC1/XPF. Additionally, we were able to quantify the ICL unhooking in human fibroblasts and found that they can unhook ~2500 ICL/h. We also determined that a dose of approximately 400 ICL/cell is lethal to 50% of the cells, indicating that ICL unhooking is not the most critical step during the repair process. / DNA-skadande ämnen är vanligt i cancerbehandling, då snabbt växande celler, såsom cancerceller är betydligt känsligare än normala celler för DNA skador. En grupp av ämnen som vanligen används i cancerbehandling är korsbindare av DNA. Dessa ämnen kommer reagera två gånger med DNA och skapa två bindningar mitt emot varandra. DNA strängen, som består av två delar, måste kunna separeras och kopieras (replikation) på ett tillförlitligt sätt för att cellerna ska kunna dela sig och bli flera. DNA strängen måste också kunna dela sig och bli avläst rätt för att nya proteiner ska kunna bildas (transkription). När korsbindarna har bundit till DNA strängarna, hindrar detta deras separation och därigenom förhindras även avläsningen och kopieringen.  För att göra undersökningarna av DNA korsbindande ämnen ännu lite svårare, så ger korsbindare flera olika typer av skador. Dels kan det bli flera olika typer av korsbindningar, både mellan två DNA-strängar (ICL) vilket är den farligaste och mest svårreparerade typen, men det kan också ske inom samma DNA-sträng (intrastrand crosslink) eller mellan en DNA-sträng och ett protein (DNA-protein crosslink). Korsbindare kan även bilda enbindningsskador (monoaddukt), vilket innebär den bara binder en gång till DNA. För att cellen ska kunna överleva, så måste den reparera skadorna och ta bort korsbindningen eller monoaddukten. Hur detta sker i människor är inte helt klarlagt men det verkar som det sker i flera steg. Till att börja med klipps DNA sönder i ena strängen på båda sidorna om korsbindningen, detta gör att den kvarvarande delen av korsbindningen kan böjas bort. Därefter kommer cellen att skapa nytt DNA för att fylla mellanrummet som bildats. Cellen använder sig av den andra DNA strängen som mall för att sätta in rätt DNA baser, men i fallet med korsbindande ämnen så är även den strängen skadad och därför finns det en stor risk för att fel DNA baser sätts in och då uppstår mutationer. Nästa steg är att klippa den kvarvarande delen av korsbindningen, även denna gång skapas ett mellanrum som måste fyllas med nya baser. Den första artikeln i avhandlingen handlar om att försöka reda ut om det är ICLen eller monoaddukten som är orsak till olika effekter som påträffas efter behandling med korsbindande ämnen. Det vi fann var att även om det bara var från ICLs som vi kunde mäta en effekt på replikationen, så fick vi nästan lika stark effekt från monoaddukterna, som från ICL, för en av de vanligast använda markörerna (kännetecknen) för båda DNA strängarna var brutna på samma ställe (dubbelstränsbrott). Detta berodde dock inte på att även monoaddukterna skapade dubbelsträngsbrott, utan på att markören vi använde var ospecifik. Vi fann även att även om ICLs har mycket större effekt än monoaddukten på cellens överlevnad m.m., så kan man inte bortse ifrån effekten av monoaddukten och att den troligen har en betydande roll för de korsbindande ämnen som endast ger en liten del ICLs. I artikel två har vi utvecklat en ny metod, som gör det möjligt att mäta hur många ICLs som bildas vid en viss dos av de korsbindande ämnen vi undersöker. Vi kan även mäta hur fort ICLerna kan repareras i mänskliga celler med hjälp av metoden. Tack vare en kombination av våra mätningar och med hjälp av datorsimuleringar, kunde vi räkna ut hur många ICLs som bildades per dos för tre vanliga korsbindare. Vi kunde även visa att 50 % av ICLen har påbörjat reparationen och kommit så långt att de var bortklippta från ena stängen inom 3 timmar efter behandlingen. I artikel tre undersöker vi vilka proteiner som är inblandade i den tidiga delen av ICL reparationen, alltså fram till och med att celler klipper ut korsbindningen på båda sidorna om skadan i ena strängen. Här visar vi att celler som är defekta i reparationsprotein kallat XPA, har en betydligt långsammare borttagning av ICLer än vad båda normala celler och celler defekta i reparationsprotein XPE har. Vi visar även att detta inte påverkar cellens replikationshastighet, eller har någon effekt på cellens överlevnad. Den fjärde artikeln handlar om acetaldehyd, som bildas när alkohol förbränns i kroppen. Acetaldehyd har föreslagits bilda ICL och därför undersökte vi vilka effekter den har på cellerna. Vi visar i den här artikeln att det krävs nysyntes av DNA för att acetaldehyd ska leda till dubbelsträngsbrott. Celler kan reparera dessa dubbelsträngsbrott med hjälp av reparationssystem, som kallas homolog rekombination, men att reparationen ibland blir felaktig. I den femte och sista artikeln i avhandligen undersöker vi ett av de vanligast föreslagna proteinen för att sköta klippningen av DNA (ERCC1/XPF) och hur den är inblandad i reparationen av korsbindningar. Vi kan här visa att även det krosbindande ämnet mitomycin C bromsar replikationshastigheter och att ERCC1/XPF är nödvändigt för att kunna fullfölja homolog rekombination av ICLs. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 1: Submitted. Paper 2: Manuscript. Paper 3: Manuscript. Paper 4: Submitted.</p>

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20668号 / 医博第4278号 / 新制||医||1024(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 武田 俊一, 教授 岩井 一宏, 教授 清水 章 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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A quimiorresistência de tumores constitui um dos maiores obstáculos que levam comumente ao fracasso da terapia. Os mecanismos relevantes que contribuem para a resistência celular incluem: bombas de efluxo; alterações na interação entre a droga e o seu alvo e mudanças nas respostas celulares, em particular uma habilidade aumentada de reparar os danos induzidos no DNA e defeitos nas vias apoptóticas. A capacidade de reparar os danos no DNA e a evasão da apoptose são de grande importância, uma vez que a maioria dos quimioterápicos tem sua ação baseada na indução de citotoxicidade pela capacidade de gerar lesões no DNA. Desta forma, uma importante estratégia para melhorar a quimioterapia é o desenvolvimento de abordagens mais seletivas e mecanismos que contornem a resistência tumoral. Neste trabalho, através de um estudo sobre os genes e suas respectivas vias envolvidas no reparo, capacidade de sobrevivência e sinalização de danos induzidos pela nimustina (ACNU) - um agente cloroetilante comumente utilizado em tratamentos quimioterápicos de tumores sólidos - identificamos genes potencialmente alvos para uma terapia adjuvante. Demonstramos que células de glioma p53mt tem menor capacidade de reparo de ICLs induzidos por esta droga do que células p53wt. Também, que a via de NHEJ (\'\'Non Homologous End Joining\'\') não é uma via preferencial de reparo dessas lesões, mas que a via de NER (\'\'Nucleotide Excision Repair\'\') (ou especificamente os produtos gênicos XPA, XPC e XPF) é bastante importante. Curiosamente, na ausência de XPA, NHEJ assume uma participação no reparo dessas lesões, provavelmente devido a um aumento no número de DSBs e saturação das outras vias de reparo. Da mesma forma, verificamos que a DNA polimerase POLH (XPV), envolvida em TLS (\'\'Translesion Synthesis\'\'), também participa na tolerância dessas lesões. Neste contexto, encontramos evidências de que a polimerase TLS (especificamente POLH e POLK) apresentam-se superexpressas em amostras de gliomas, podendo desta forma concorrerem tanto para a tumorigênese quanto para a resistência observada nestes tipos tumorais. Por fim, realizamos o silenciamento gênico através da teconologia de RNAi, que reprimem os genes pela eliminação do transcrito mRNA correspondente, prevenindo a síntese protéica. Os genes-alvo escolhidos para o silenciamento foram, desta forma, XPC, XPF, POLH e POLK. O silenciamento gênico de XPC, XPF e POLH demonstraram-se capazes de sensibilizar significativamente células de glioma, permitindo-nos sugerir estas proteínas como elementos importantes na quimioresistência de gliomas ao ACNU e colocando a inibição dessas moléculas como uma estratégia importante na sensibilização de gliomas ao ACNU e potencialmente a outros agentes quimioterápicos com o mesmo mecanismo de ação. / The chemoresistance of tumors is one of the most important obstacles that commonly lead to the failure of therapy. The main mechanisms that contribute to cellular resistance include efflux pumps; changes in the interaction between the drug and its target and changes in cellular responses, in particular an increased ability to repair induced DNA damages and defects in apoptotic pathways. The ability to repair DNA damage and evasion of apoptosis are of great importance, since most chemotherapy has its action based on the induction of cytotoxicity by the ability to generate DNA lesions. Thus, an important strategy for improving chemotherapy is the development of more selective mechanisms that circumvent tumor resistance approaches. In this work, through a study of genes and pathways involved in the repair, survival and damage signaling induced by nimustine (ACNU) - a cloroethylating agent commonly used in treatments of solid tumors - we aimed to identify target genes for a potentially adjuvant therapy. We demonstrated that glioma cells p53mt have less ability to repair ICLs induced by this drug then p53wt cells. Also, that the NHEJ (\'\'Non Homologous End Joining\'\') pathway is not the main route of repair of these lesions, but that the NER (\'\'Nucleotide Excision Repair\'\') pathway (or specifically the gene products XPA, XPC and XPF) is very important. Interestingly, in the absence of XPA, NHEJ takes place in the repair of those lesions, probably due to an increase in the number of DSBs and saturation of other repair pathways. Likewise, we found that DNA polimerase involved in TLS (\'\'Translesion Synthesis\'\') POLH (XPV) also participates in tolerance of such lesions. We also found evidence that TLS polimerases (specifically POLH and POLK) are overexpressed in gliomas samples and could play a role in the tumorigenesis and in the resistance observed in these tumor types. Finally, we performed gene silencing through RNAi teconology, which repress genes by eliminating the corresponding mRNA transcript, preventing protein synthesis. The target genes selected for silencing were XPC, XPF, POLH and POLK. The knockdown of XPC, XPF and POLH proved to significantly sensitize glioma cells, suggesting these proteins as important elements in the chemoresistance of gliomas and highlighting the inhibition of these molecules as an important strategy in the sensitization of gliomas to ACNU and probably to other chemotherapeutic agents with the same mechanisms of action.

A quimiorresistência de tumores constitui um dos maiores obstáculos que levam comumente ao fracasso da terapia. Os mecanismos relevantes que contribuem para a resistência celular incluem: bombas de efluxo; alterações na interação entre a droga e o seu alvo e mudanças nas respostas celulares, em particular uma habilidade aumentada de reparar os danos induzidos no DNA e defeitos nas vias apoptóticas. A capacidade de reparar os danos no DNA e a evasão da apoptose são de grande importância, uma vez que a maioria dos quimioterápicos tem sua ação baseada na indução de citotoxicidade pela capacidade de gerar lesões no DNA. Desta forma, uma importante estratégia para melhorar a quimioterapia é o desenvolvimento de abordagens mais seletivas e mecanismos que contornem a resistência tumoral. Neste trabalho, através de um estudo sobre os genes e suas respectivas vias envolvidas no reparo, capacidade de sobrevivência e sinalização de danos induzidos pela nimustina (ACNU) - um agente cloroetilante comumente utilizado em tratamentos quimioterápicos de tumores sólidos - identificamos genes potencialmente alvos para uma terapia adjuvante. Demonstramos que células de glioma p53mt tem menor capacidade de reparo de ICLs induzidos por esta droga do que células p53wt. Também, que a via de NHEJ (\'\'Non Homologous End Joining\'\') não é uma via preferencial de reparo dessas lesões, mas que a via de NER (\'\'Nucleotide Excision Repair\'\') (ou especificamente os produtos gênicos XPA, XPC e XPF) é bastante importante. Curiosamente, na ausência de XPA, NHEJ assume uma participação no reparo dessas lesões, provavelmente devido a um aumento no número de DSBs e saturação das outras vias de reparo. Da mesma forma, verificamos que a DNA polimerase POLH (XPV), envolvida em TLS (\'\'Translesion Synthesis\'\'), também participa na tolerância dessas lesões. Neste contexto, encontramos evidências de que a polimerase TLS (especificamente POLH e POLK) apresentam-se superexpressas em amostras de gliomas, podendo desta forma concorrerem tanto para a tumorigênese quanto para a resistência observada nestes tipos tumorais. Por fim, realizamos o silenciamento gênico através da teconologia de RNAi, que reprimem os genes pela eliminação do transcrito mRNA correspondente, prevenindo a síntese protéica. Os genes-alvo escolhidos para o silenciamento foram, desta forma, XPC, XPF, POLH e POLK. O silenciamento gênico de XPC, XPF e POLH demonstraram-se capazes de sensibilizar significativamente células de glioma, permitindo-nos sugerir estas proteínas como elementos importantes na quimioresistência de gliomas ao ACNU e colocando a inibição dessas moléculas como uma estratégia importante na sensibilização de gliomas ao ACNU e potencialmente a outros agentes quimioterápicos com o mesmo mecanismo de ação. / The chemoresistance of tumors is one of the most important obstacles that commonly lead to the failure of therapy. The main mechanisms that contribute to cellular resistance include efflux pumps; changes in the interaction between the drug and its target and changes in cellular responses, in particular an increased ability to repair induced DNA damages and defects in apoptotic pathways. The ability to repair DNA damage and evasion of apoptosis are of great importance, since most chemotherapy has its action based on the induction of cytotoxicity by the ability to generate DNA lesions. Thus, an important strategy for improving chemotherapy is the development of more selective mechanisms that circumvent tumor resistance approaches. In this work, through a study of genes and pathways involved in the repair, survival and damage signaling induced by nimustine (ACNU) - a cloroethylating agent commonly used in treatments of solid tumors - we aimed to identify target genes for a potentially adjuvant therapy. We demonstrated that glioma cells p53mt have less ability to repair ICLs induced by this drug then p53wt cells. Also, that the NHEJ (\'\'Non Homologous End Joining\'\') pathway is not the main route of repair of these lesions, but that the NER (\'\'Nucleotide Excision Repair\'\') pathway (or specifically the gene products XPA, XPC and XPF) is very important. Interestingly, in the absence of XPA, NHEJ takes place in the repair of those lesions, probably due to an increase in the number of DSBs and saturation of other repair pathways. Likewise, we found that DNA polimerase involved in TLS (\'\'Translesion Synthesis\'\') POLH (XPV) also participates in tolerance of such lesions. We also found evidence that TLS polimerases (specifically POLH and POLK) are overexpressed in gliomas samples and could play a role in the tumorigenesis and in the resistance observed in these tumor types. Finally, we performed gene silencing through RNAi teconology, which repress genes by eliminating the corresponding mRNA transcript, preventing protein synthesis. The target genes selected for silencing were XPC, XPF, POLH and POLK. The knockdown of XPC, XPF and POLH proved to significantly sensitize glioma cells, suggesting these proteins as important elements in the chemoresistance of gliomas and highlighting the inhibition of these molecules as an important strategy in the sensitization of gliomas to ACNU and probably to other chemotherapeutic agents with the same mechanisms of action.

With the start of the Large Hadron Collider(LHC) at CERN, we will obtain a new understanding of the physics beyond our current limits. New discoveries will be made; but we will require a deeper understanding, which the LHC machine, being a hadron collider, will not be able to elucidate. Instead, we will need an e+e- collider to make precision measurements of the newly discovered phenomena. Electroweak symmetry breaking and the origin of fermion and boson masses are fundamental issues in our understanding of particle physics. The essential piece of electroweak symmetry breaking - the Higgs boson - will probably be discovered at the LHC. If there are one, or more, Higgs boson(s) precise measurements of all properties of the Higgs will be very important. In this thesis I present two measurements of Standard Model Higgs boson properties in the context of the International Linear Collider (ILC) at √s = 500 GeV, using the proposed International Linear Detector (ILD). First a performance study of ILD to measure the branching ratios of the Higgs boson with mH = 120 GeV, where the Higgs boson is produced with a Z-boson via the Higgsstralung process, and the Z decays into e+e- or μ+μ-. It will also be essential to study the Higgs Yukawa coupling. Therefore, in the second part of this thesis, I present a study of e+e- → tt¯H with the aim of making a direct measurement of the the top-Higgs coupling, using the semi-leptonic nal state and mH of 120 GeV. I show that the top-Higgs coupling can be measured with an accuracy of better than 28%.

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in models of cardiovascular disease. We previously suggested sphingosine-1-phosphate (S1P) activates volume-sensitive Cl- current (ICl,swell) by ROS-dependent signaling. S1P and its analog, FTY720 (fingolimod), primarily act via G-protein coupled receptors (S1PR; S1PR1-3 in heart), but several intracellular S1P ligands are known. We investigated how these agents regulate ICl,swell. ICl,swell was elicited by bath S1P (500 nM), FTY720 (S1PR1,3 agonist; 10 μM), and SEW2871 (S1PR1 agonist; 10 μM) and was fully inhibited by DCPIB, a specific blocker. These data suggested role of S1PR in activation of ICl,swell. Surprisingly, neither CAY10444 (S1PR3 antagonist; 10 μM) nor VPC23019 (S1PR1,3 antagonist; 13 μM) blocked FTY720-induced ICl,swell. Also, gallein a pan Gbeta-gamma inhibitor, failed to block the S1P-induced current. Moreover, 100 nM FTY720 applied via the pipette evoked a larger, faster activating current than 10 μM bath FTY720. Similarly, 500 nM S1P gave larger, faster activating ICl,swell when added to the pipette than when added in the bath. In contrast to FTY720, bath S1P-induced ICl,swell was blocked by CAY10444, but a 3-fold higher concentration failed to eliminate the response to pipette S1P, and VPC23019 failed to suppress bath and pipette S1P-induced currents. Taken together, inconsistencies in the responses to S1PR agents and the greater sensitivity to pipette than bath S1P and FTY720 support the notion that intracellular ligands rather than sarcolemmal S1PR activated ICl,swell. Next we tested if S1P and FTY720, like osmotic swelling, require both NADPH oxidase and mitochondrial ROS production to evoke ICl,swell. S1P- and FTY720-induced ICl,swell were blocked by rotenone but were insensitive to gp91ds-tat, suggesting only mitochondrial ROS production was needed. One possibility is that S1P and FTY720 elicit ICl,swell by binding to mitochondrial prohibitin-2, an S1P ligand whose knockdown augments mitochondrial ROS productions. These data suggest ICl,swell may be activated by S1P accumulation in ischemia-reperfusion and CHF. Understanding S1P-signaling that elicits ICl,swell may provide insight into electrophysiological mechanisms of cardiac pathology and help identify novel targets for therapy.

Swelling-activated Cl− current (ICl,swell) is an outwardly-rectifying current that plays an important role in cardiac electrical activity, cellular volume regulation, apoptosis, and acts as a potential effector of mechanoelectrical feedback. Persistent activation of ICl,swell has been observed in a number of models of cardiovascular disease. Previously we showed that angiotensin II (Ang II), endothelin-1 (ET-1), endothelial growth factor receptor (EGFR), and reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and mitochondria are involved in the activation of ICl,swell by both osmotic swelling and Beta1 integrin stretch. Sphingolipid metabolism is modulated in several cardiopathologies and because sphingolipids are bioactive lipids involved in signaling cascades that overlap significantly with these modulators of ICl,swell, we investigated the role of sphingolipids in the regulation of ICl,swell in cardiac ventricular myocytes. Under isoosmotic conditions that isolate anions currents, addition of exogenous, cell permeant C2-ceramide (C2-Cer) elicited an outwardly-rectifying Cl− current that reversed near the Cl− equilibrium potential (ECl) in both physiological and symmetrical Cl− gradients. This current was inhibited by the ICl,swell-specific blockers DCPIB. Dihydro-C2-ceramide (C2-H2Cer), the inactive analogue of C2-Cer, failed to elicit current. These data strongly suggest that the identity of C2-Cer-induced Cl− current is ICl,swell and indicate that sphingolipid signaling pathways may be involved. Bacterial sphingomyelinase (SMase), which converts endogenous sphingomyelin in the outer leaflet of the sarcolemmal membrane to native chain-length ceramides, elicited a DCPIB-sensitive Cl− current. SMase-induced current is also suppressed by tamoxifen, which under conditions that isolate anion currents is a specific inhibitor of ICl,swell. SMase-induced ICl,swell was abrogated by ebselen, a membrane permeant glutathione peroxidase mimetic that dismutates H2O2 to H2O. This suggests that ROS are required mediators of SMase-induced activation of ICl,swell. Both NOX and mitochondria are important sources of ROS in cardiomyocytes and both have been implicated in modulating ICl,swell. Blocking NOX with apocynin or the NOX fusion peptide inhibitor gp91ds-tat had no effect on SMase-induced current. However, pretreatment of cardiomyocytes with gp91ds-tat reduced the maximum current amplitude of SMaseinduced ICl,swell, indicating that NOX may play a time-dependent role in this mechanism. By contrast, the mitochondrial Complex I blocker rotenone, which suppresses extramitochondrial ROS release by Complex III, completely suppresses SMase-induced ICl,swell. Additionally, SMase-induced ICl,swell is partially inhibited by blockade of mitochondrial KATP (mitoKATP) channels with 5-hydroxy-decanoic acid (5-HD). MitoKATP channels have been implicated as modulators of mitochondrial ROS release. Thus these data suggest that mitochondrial ROS generation is required for SMaseinduced activation of ICl,swell. Ceramides are metabolized to form several sphingolipids, including sphingosine-1-phosphate (S1P). We tested whether ceramide metabolites are responsible for eliciting ICl,swell. Under isosmotic conditions that isolate anion currents, SMase-induced ICl,swell was abrogated by blockade of ceramidase, which converts ceramide to sphingosine, with Derythro-MAPP. SMase-induced ICl,swell also was suppressed by inhibition of sphingosine kinase with DL-threo-dihydrosphingosine. These data suggested that the ceramide metabolite S1P is likely to stimulate ICl,swell. As expected, exogenous S1P elicited an outwardly rectifying Cl− current that was fully inhibited by DCPIB. As seen with SMaseinduced ICl,swell, S1P-induced ICl,swell was fully inhibited by rotenone. In contrast to results with SMase, S1P-induced current was partially inhibited by blockade of NOX with apocynin. These data indicate that S1P is a necessary component of SMase-induced ICl,swell activation and that the action of exogenous S1P involves ROS from both mitochondria and NOX. Importantly, the fact that exogenous C2-ceramide also activates ICl,swell even though C2-ceramide may not metabolized to S1P in native cells. Thus, it seems likely that ceramides can elicit ICl,swell via S1P and also by a distinct pathway and that both pathways converge at mitochondrial ROS. In order to determine the role of ERK in the proposed signaling pathway that regulates ICl,swell, we examined the effect of ERK inhibitors PD98059 and U0126 on the activation of ICl,swell. Both of these agents partially inhibited SMase-induced activation of ICl,swell, indicating SMase acts through both ERK-dependent and ERK-independent signaling pathways. HL-1 cells are derived from a murine atrial cell line that retains phenotypic characteristics of adult cardiomyocytes. Recently, ICl,swell has been observed in HL-1 cells with similar regulatory mechanisms to those seen in native cells. We showed that SMase elicits an outwardly-rectifying, DCPIB-sensitive Cl− current that reverses near ECl in HL-1 cells. Finally, we confirmed the production of ROS by SMase-induced signaling by flow cytometry in HL-1 cells using the nominally H2O2-selective fluorescent probe CH2DCFDA-AM. Exposure to SMase increased ROS production, as did the positive control H2O2. SMase-induced ROS generation was suppressed by pretreatment with rotenone but was unaffected by pretreatment with gp91ds-tat. These data indicate that exogenous and endogenous sphingolipids elicit ICl,swell in cardiomyocytes by stimulating mitochondrial ROS production. NOX may contribute to the ROS generation, but is not a required step in this mechanism. Sphingolipid signaling is likely to play an important role in stimulating ROS production and activating ICl,swell in a number of cardiovascular diseases.