中國醫藥大學機構典藏 China Medical University Repository, Taiwan:Item 310903500/32579

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(1)! !. ύ!!୯!!ᙴ!!ᛰ!!ε!!Ꮲ! ᖏ ġ ‫ ׉‬ġ ᙴ ġ Ꮲ ġ ࣴ ġ ‫ ز‬ġ ‫!܌‬ റ γ Ꮲ Տ ፕ Ў! ! ! CAPON ፓ௓Ј᠌ӆཱུϯǺᖏ‫׉‬᛾Ᏽᆶϩηᐒᙯ CAPON Modulates Cardiac Repolarization: Clinical Evidence and Molecular Mechanisms ! ! !. ࡰ ! Ꮴ ! ௲ ! ௤ Ǻ ጰᇶϘ!!௲!!௤! Ӆ ӕ ࡰ Ꮴ ௲ ௤ Ǻ ‫׵‬म໢!!௲!!௤! Ӆ ӕ ࡰ Ꮴ ௲ ௤ Ǻ ໳ࣿᒸ!!௲!!௤!. ! ࣴ!‫!ز‬ғǺ!!஭!‫!!!҅!ڷ‬. ! ύ๮҇୯ΐΜΐԃΎД!.

(2) ᇞ. ᖴ. Ј᠌ႝ਻ғ౛Ꮲ‫ޑ‬ᙴᕍǵ௲Ꮲᆶࣴ‫ز‬Ǵനಖ‫ޑ‬Ҟ኱ࢂૈ ᑼ཮ೣ೯୷ᘵᆶᖏ‫ޕޑ׉‬᛽Ǵ೭ࢂ‫ঁך‬Γ΋‫ޔ‬ոΚ‫ޑ‬БӛǴ ΟԃٰǴӧύ୯ᙴᛰεᏢᖏ‫׉‬ᙴᏢࣴ‫ޑ܌ز‬ᏢಞǴᡣ‫ך‬ӧ೭ ঁሦୱัԖ຾৖ǴϣЈкᅈགৱǶ २Ӄ‫ך‬ाགᖴǴ௴วᆶ௲Ꮴ‫وך‬Εᖏ‫׉‬ႝғ౛ሦୱ‫ޑ‬Դ ৣǺֆቺਟ௲௤ǵယහࢪ௲௤ᆶഋӹమᙴৣǴдॺ‫ޑ‬௲ᏤǴ ᡣ‫ڀך‬ഢΑᖏ‫׉‬ႝғ౛‫ޕޑ‬᛽ᆶ‫ೌמ‬ǴωૈӧӢጔሞ཮Πၟ ᒿࢫྷ݊௲௤ӣ҆ਠว৖Ǵය໔Ԗ۩ᆶ໳Н‫ڷ‬௲௤Ӆ٣Ǵ٠ ᆾ‫ځ‬௢ᙚ‫ ܭ‬2005 ԃ 4 ДԿऍ୯ Johns Hopkins UniversityǴၟ ᒿ ಒ झ ႝ ғ ౛ ε ৣ Eduardo Marban ௲ ௤ Ꮲ ಞ molecular genetics ᆶ patch clampǴDr. Marban кϩЍ࡭ᆶࡰᏤǴᡣ‫ך‬ ‫ޑ‬Ј᠌ႝ਻ғ౛‫ޕ‬᛽҅ԄၠΕಒझቫԛǶӧ 2007 ԃ 7 Дӣ ୯ࡕǴ‫܍‬ᆾ‫׵‬म໢௲௤ǵጰᇶϘ௲௤ǵ໳ࣿᒸ௲௤ᆶᙔӃϡ ‫ڐߏ܌‬շᆶࡰᏤǴωૈӧᕷԆ‫ޑ‬ᙴᕍ཰୍ΠǴೕჄࣴ‫ز‬Б ӛǴ೴‫؁‬۳߻ᗌ຾Ƕ‫׳ך‬ाགᖴύ୯ᙴᕍᡏ‫س‬ጰဠ٣ߏǵ݅ ҅ϟଣߏǴ΋ၡ‫ޑ‬Ѝ࡭ᆶග‫ܘ‬Ǵᡣ‫ך‬Ԗᐒ཮ॅऍ୯຾অᆶ൩ ᠐ᖏ‫׉‬ᙴᏢࣴ‫܌ز‬Ǵჴ౜ფགྷǶ നࡕǴ‫ך‬ाགᖴР҆‫ޑ‬ਭ୻Ǵ‫ޑך‬ϣΓ஭లЎᆶ‫ޑך‬ζ.

(3) ‫ٽ‬஭◚উ‫ޑ‬Ѝ࡭ᆶх৒Ǵᡣ‫ך‬คࡕ៝ϐኁǴωૈӄΚаॅֹ ԋᏢ཰Ƕ. ஭‫҅ڷ‬ᙣᇞ‫ܭ‬ ύ୯ᙴᛰεᏢᖏ‫׉‬ᙴᏢࣴ‫܌ز‬ ҇୯ΐΜΐԃΎД.

(4) Ҟ. ᒵ. Ҟᒵɡკ. ii. Ҟᒵɡ߄. iii. ύЎᄔा. iv-vii. मЎᄔा. viii-xii. ಃ΋ക ߻‫ق‬. 1. ಃ΋࿯ ࣴ‫ز‬ङඳ. 1-4. ಃΒ࿯ ࣴ‫ز‬Ҟ‫ޑ‬. 4-7. ಃΒക ࣴ‫ز‬Б‫ݤ‬. 8. ಃ΋࿯ ୷ᘵࣴ‫ز‬. 8-18. ಃΒ࿯ ᖏ‫زࣴ׉‬. 18-28. ಃΟ࿯ ಍ीБ‫ݤ‬. 29-30. ಃΟക ࣴ‫่݀ز‬. 31. ಃ΋࿯ ୷ᘵࣴ‫ز‬. 31-39. ಃΒ࿯ ᖏ‫زࣴ׉‬. 40-45. ಃѤക ૸ፕ. 46. ಃ΋࿯ ୷ᘵࣴ‫ز‬. 46-48. ಃΒ࿯ ᖏ‫زࣴ׉‬. 49-50. ಃΟ࿯ ࣴ‫ز‬ज़‫ڋ‬. 50-51. ಃϖക ่ፕ. 52. ୖԵЎ᝘. 53-60. ߕᒵ. 61-74 i.

(5) Ҟ კ1. ᒵ ɡ კ. Arking et al. २ࡋճҔ genome-wide association study ว. 4. ౜ CAPON ‫ ک‬QTc ‫࣬ޑߏۯ‬ᜢ‫܄‬ კ2. rs10494366 homozygote ᆶ QTc ‫ߏۯ‬Ǵ4 ঁεࠠ࣬ᜢ‫ࣴ܄‬. 5. ‫ز‬ϐКၨ კ3. Jaffery et al. २ࡋว౜ CAPON ӧઓ࿶ಒझ‫ת‬ᄽख़ाфૈ. 6. კ4. CAPON ӧЈ᠌߄ၲ‫ޑ‬᛾ᏵȐAǺWestern blotǹBǺխࣝ. 32. ಒझࢉՅȑ კ5. CAPON ፓ௓ЈԼಒझ୏բႝՏᆶ້ᚆηႝࢬ. 33. კ6. CAPON ፓ௓ЈԼಒझႇᚆηႝࢬ. 35. კ7. CAPON ᆶ NOS1 ӧЈ᠌և౜ೈқᆶೈқϐҬϕբҔ. 36. კ8. ӧЈԼಒझ CAPON ၸࡋ߄ၲёᛙ‫ ۓ‬NOS1 ೈқ. 37. კ9. ӧЈ᠌ CAPON ၸࡋ߄ၲёගϲЈԼಒझ‫ ޑ‬NO ౢໆ. 38. კ 10 L-NAME ё࡮‫ל‬Ӣ CAPON ၸࡋ߄ၲЇଆЈԼಒझ୏բ. 39. ႝՏᕭอᆶ້ᚆηႝࢬᡂλ კ 11 methadone Ꮚໆᆶ QTc ӧ‫܄ت‬և౜҅࣬ᜢ. 41. კ 12 methadone Ꮚໆᆶ QTc ӧζ‫܄‬όև҅࣬ᜢ‫܄‬. 41. კ 13 Longitudinal ϩ‫݋‬Ǵ‫ੰ܄ت‬Γӧ methadone ‫ݯ‬ᕍࡕ QTc ᡉ. 43. ๱ᡉ๱‫ߏۯ‬ კ 14 Longitudinal ϩ‫݋‬Ǵζ‫ੰ܄‬Γӧ methadone ‫ݯ‬ᕍࡕ QTc ؒ. 43. Ԗᡉ๱‫ߏۯ‬ კ 15 Ӆ೫ขᡉ༾᜔ᔠಒझխࣝᑻӀࢉՅǴᡉҢ CAPON ᆶ NOS1 և౜ಒझϣ colocalization ‫ޑ‬౜ຝ ii. 47.

(6) Ҟ. ᒵ ɡ ߄. ߄1. നཥӃϺ‫ ܄‬Long QT Syndrome ‫ޑ‬ϩᜪ. ߄2. cross-section ੰΓಔ‫୷ޑ‬ҁၗ਑. 40. ߄3. cross-section ϩ‫݋‬Ǵ‫ੰ܄ت‬Γ‫ ޑ‬methadone Ꮚໆᆶ QTc և. 41. 3. ҅࣬ᜢ‫܄‬ ߄4. cross-section ϩ‫݋‬Ǵζ‫ੰ܄‬Γ‫ ޑ‬methadone Ꮚໆᆶ QTc ό. 42. և҅࣬ᜢ‫܄‬ ߄5. Longitudinal ϩ‫݋‬ಔੰΓ‫ޑ‬ၗ਑. 43. ߄6. CAPON ୷Ӣᡂ౦ǵmethadone ‫ ک‬QTc ‫࣬ޑ‬ᜢ‫܄‬. 45. iii.

(7) ύЎᄔा CAPON ࢂ 1998 ԃ२ࡋӧတಔᙃว౜‫ޑ‬ೈқǴ‫ڀ‬Ԗፓ࿯ nNOS ౢғ NO ‫ޑ‬ख़ाфૈǶ2006 ԃ 4 ДǴ΋໨җऍ୯ऊᑣᓅදߎථεᏢ Ϸቺ୯ኀѭ໵εᏢЬᏤ‫ޑ‬εೕኳΓᜪ୷Ӣᡏ SNP association studyǴว ߄ӧ Nature Genetics ᚇᇞǴԜࣴ‫ز‬२ࡋว౜ CAPON ‫୷ޑ‬Ӣᡂ౦཮ቹ ៜЈ᠌ӆཱུϯǴϸᔈӧЈႝკ QT interval ‫ޑ‬ᡂϯǶҗ‫ ܭ‬QT interval ϼߏ‫܈‬ϼอǴࣣ৒ܰ೷ԋЈӢ‫₽܄‬ԝǴӢԜǴ೭໨ࣴ‫ڀ่݀ز‬Ԗ൳໨ ख़εཀကǴॶள຾΋‫؁‬௖૸Ǻಃ΋ǵCAPON ёૈࢂ΋໨ЇଆЈӢ‫܄‬ ₽ԝ‫ޑ‬჻ཥӒᓀӢηǹಃΒǵCAPON ҁ‫ي‬٠ߚᚆη೯ၰೈқǴՠࠅ ૈቹៜЈ᠌ᚆη೯ၰ‫ޑ‬фૈǹಃΟǵCAPON-nNOS-target protein-NO ϐૻ৲բҔ೼৩ӧЈ᠌ғ౛‫ੰ܈‬౛‫ݩރ‬ΠǴ‫ת‬ᄽख़ा‫فޑ‬ՅǶՠ CAPON ‫ز‬ഖӧЈ᠌‫ת‬ᄽՖᅿ‫ف‬ՅǴѬΞӵՖቹៜЈ᠌ӆཱུϯǴၸѐ ΋ค‫ޕ܌‬Ǵ‫ॺך‬வࣴ‫ ز‬CAPON ࢂցӧЈ᠌߄ၲ๱ЋǴӆ௖૸ CAPON ᆶ NOS1 ‫ޑ‬ೈқҬϕբҔǴ٠ճҔဏੰࢥբၩᡏࣴ‫ ز‬CAPON ೸ၸᆶ NOS1 ‫ޑ‬ҬϕբҔǴ྽୷Ӣၸࡋ߄ၲਔǴ཮‫້ڋ׭‬ᚆη೯ၰႝࢬǴ٠ ຾ԶᕭอЈԼಒझӆཱུϯ‫ޑ‬ਔ໔Ǵ࿶җԜࣴ‫่݀ޑز‬ёаӑ᛾Γᜪ௼ ဂ CAPON ୷Ӣᡂ౦‫ ک‬QT ߏอ‫ڀ‬Ԗ࣬ᜢ‫ޑ܄‬ว౜ǶฅԶǴ‫ॺך‬ᇡࣁ ќԖΟεୢᚒǴ࡚ሡှเǺCAPON ୷Ӣᡂ౦ჹ୯ΓЈႝკ QT interval ‫ޑ‬ቹៜǴ྽Ј᠌ೀӧੰ౛‫ރ‬ᄊΠ CAPON ‫ת‬ᄽՖᅿ‫ف‬ՅǴϷ CAPON. iv.

(8) ӵՖፓ௓Ј᠌ႝ਻ᆶԏᕭфૈϐҬϕբҔǶ ଞჹಃ΋ঁୢᚒǴ‫ॺך‬ଷ೛ CAPON ୷Ӣᡂ౦ዴჴ཮ቹៜ୯ΓЈ ႝკ QT intervalǴ೭ᅿቹៜёаϸᔈӧ଼ந‫ঁޑ‬ΓǴ‫܈‬྽ঁΓௗ‫ڙ‬ё Їଆ QT ‫ޑߏۯ‬ᛰ‫ݯނ‬ᕍਔǴ஥Ԗ CAPON ୷Ӣᡂ౦‫ޣ‬ёૈ຾΋‫ۯ؁‬ ߏ‫ ځ‬QT intervalǴ‫ޑॺך‬฼ౣࢂ: ୷Ӣ᠘‫ۓ‬΋ဂੇࢶӢȐheroinȑԋ ᠅҅ӧௗ‫ ڙ‬methadone ‫ݯ‬ᕍ‫ޑ‬ԋԃ‫ت‬ζǴ߃යෳ၂‫ঁٿ‬ख़ा‫ ޑ‬CAPON SNPǴrs10494366 ᆶ rs1415263Ǵٰ࣮஥Ԗ୷Ӣᡂ౦‫ࢂޣ‬ցӧܺҔ methadone ߻ǵࡕቹៜ‫ځ‬Јႝკ QT intervalǶᜢ‫ܭ‬೭ঁଷ೛Ǵӧᆒઓ ϩ຋஻‫ܺޣ‬Ҕ risperidone ‫ݯ‬ᕍ‫ޑ‬௼ဂȐ58 Γȑ΢Ǵ‫؁߃ॺך‬ว౜஥ Ԗ rs1415263 homozygous T alleles ‫ޣ‬Ǵӧௗ‫ ڙ‬risperidone ‫ݯ‬ᕍࡕ‫ځ‬Ј ႝკ QTc interval җ 396.7±26.7 ms ‫ ࣁߏۯ‬403.3±20.6 ms Ȑ6.7 msȑ Ǵ Զ ஥ Ԗ CC Ȑ 407.7±29.8 ms vs. 397.7±17.4 ms ȑ ᆶ CT alleles Ȑ401.0±17.1 ms vs. 394.5±18.2 msȑ‫ޣ‬ӧܺҔ risperidone ࡕ QTc interval ϸԶΠफ़ǴԶ rs10494366 ೭ঁӧΟεኻऍ௼ဂࣴ‫཮ࣣز‬೷ԋ QTc ‫ ޑߏۯ‬SNPǴ߃‫ٰ࣮؁‬٠όቹៜ೭ঁ੝‫ۓ‬Ѡ᡼ᆒઓϩ຋௼ဂܺҔ risperidone ߻ǵࡕ‫ ޑ‬QTc intervalǶ୷‫ܭ‬೭ঁ߃‫่݀؁‬Ǵߦԋ‫ॺך‬གྷ຾ ΋‫؁‬௖૸ᛰ᠅஻‫ޣ‬ӧௗ‫ ڙ‬methadone ‫ݯ‬ᕍ‫ޑ‬ၸำǴ஥Ԗ CAPON ୷Ӣ ᡂ౦ࢂց཮೷ԋᝄख़ QTc ‫ߏۯ‬ǴЇวठ‫܄ڮ‬Ј࠻ό᏾ેԶᏤठ₽ԝǶ ќѦǴ‫ॺך‬ΨीฝаεႵ࡚‫܈܄‬٥࡚‫܄‬ЈԼఒ༞‫ޑ‬ኳԄѐ௖૸. v.

(9) CAPON ӧЈԼఒ༞೽Տᆶ҅தЈԼ߄ၲࢂցόӕǴ٠Ъ٬Ҕൂ΋Ј Լಒझࣴ‫ ز‬CAPON ߄ၲໆόӕਔǴჹ‫ܭ‬Ј᠌ႝ਻ᆶԏᕭфૈϐҬϕ բҔȐexcitation-contraction couplingȑ‫ޑ‬ቹៜǶ ӧಃ΋കǴ‫ॺך‬ӣ៝ЈԼಒझጢ΢Ӛᅿᚆηႝࢬ،‫ۓ‬Јႝკ QT ߏอ‫ޑ‬ᐒᙯǴᇥܴࣴ‫ ز‬QT ‫ޑ‬ख़ा‫܄‬ǴҞ߻ congenital long QT syndrome ‫ޑ‬ϩᜪǴ௖૸ቹៜ QT ϐεࠠ genome-wide association studyǴᙖҗၸ ѐ‫ ޕ܌‬CAPON ӧઓ࿶‫س‬಍‫ޑ‬բҔǴ٠຾Զࣴ‫ ز‬CAPON ӧЈ᠌‫ת܌‬ ᄽ‫فޑ‬ՅǶ ӧಃΒകύǴӧࣴ‫ ز‬CAPON ‫ޑ‬ϩηᐒᙯБय़Ǵ‫ॺך‬ճҔဏੰࢥ բၩᡏа in vivo gene transfer ‫ޑ‬БԄǴஒ CAPON ၸࡋ߄ၲǴ௖૸ CAPON ‫ޑ‬ғ౛фૈǹӧᖏ‫ زࣴ׉‬CAPON ᡂ౦ᆶЈႝკ QT ࣬ᜢ‫܄‬ Бय़Ǵ‫ॺך‬᠘‫ ۓ‬24 ໨த‫ ޑـ‬CAPON ୷Ӣᡂ౦Ǵϩ‫݋‬Јႝკ‫ ޑ‬QTc ӧൂપ methadone ቹៜΠᆶ methadone у୷Ӣᡂ౦ቹៜΠ‫࣬ޑ‬ᜢ‫܄‬Ƕ ӧಃΟകύǴӧ CAPON ‫ޑ‬ϩηᐒᙯБय़Ǵ‫ॺך‬և౜ CAPON ቹ ៜЈ᠌ӆཱུϯ‫ޑ‬ϩηᐒᙯǴќѦ‫ॺך‬ΨЇҔ‫ځ‬дᐒᄬཥ߈ว߄ϐЈԼ ఒ༞୏‫ނ‬ኳԄа௖૸ CAPON ϐ‫ف‬Յфૈǹӧᖏ‫׉‬᛾ᏵБय़Ǵ‫ॺך‬ග р methadone ‫ ܈‬methadone у CAPON ୷Ӣᡂ౦ჹЈႝკ QTc ‫ޑ‬ቹៜ ‫่݀ޑ‬Ƕ ಃѤകǴ๱ख़‫૸ܭ‬ፕ౜Ԗ CAPON ϩηᐒᙯᆶᖏ‫่݀ޑزࣴ׉‬Ǵ. vi.

(10) ٠‫ک‬ၸѐ࣬ᜢ‫زࣴޑ‬բКၨǴаೕჄ຾΋‫ޑزࣴ؁‬БӛǴ٠ᖐр౜Ԗ ࣴ‫ޑز‬ज़‫ڋ‬Ƕ ಃϖകǴ߾ᇥܴ࿶җ೭٤ࣴ‫ز‬ԋ݀Ǵёаᡣ‫ॺך‬வᖏ‫׉‬᛾Ᏽᆶϩ ηᐒᙯӄय़ᙶమ CAPON ӧЈ᠌ғ౛ᆶੰ౛‫ݩރ‬Π‫ת܌‬ᄽ‫فޑ‬ՅǴዴ ‫ ۓ‬CAPON ୷Ӣᡂ౦ჹ୯Γௗ‫ ڙ‬methadone ‫ݯ‬ᕍϐ੝‫ۓ‬௼ဂЈ᠌ӆཱུ ϯ‫ޑ‬ቹៜǴ੝ձࢂǴࢂց཮೷ԋᝄख़ QTc ‫ߏۯ‬ǴЇวठ‫܄ڮ‬Јࡓό ᏾Ǵ຾ԶԖշ‫ܭ‬ٛ‫ݯ‬ЈӢ‫₽܄‬ԝǶ ᜢᗖຒǺQTcǵЈ᠌ӆཱུϯǵ₽ԝǵᛰ᠅ǵmethadoneǶ. vii.

(11) Abstract Congenital long or short QT syndrome caused by gene mutations is well recognized as capable of predisposing to ventricular tachycardia and sudden cardiac death. Apart from the rare disease-causing mutations, common genetic variants in a neuronal nitric oxide synthase (NOS1) regulator, CAPON gene, have recently been associated with QT interval variations in a human whole-genome SNP association study in 2006; this association has been replicated in two different Caucasian populations in 2007. We have recently identified CAPON expression and interaction with NOS1-NO pathways to modulate cardiac repolarization in the heart, which provides the rationale for the association of CAPON gene variants and extremes of QT interval in Caucasian populations. However, there are at least three important issues remaining to be answered. Firstly, the association of CAPON gene variants and extremes of QT interval has not been documented in non-Caucasian populations; secondly, the role of CAPON in cardiac repolarization is not clear in pathological or diseased model, and lastly, CAPON-mediated modulation of excitation-contraction coupling has not been investigated. To answer the first question, we hypothesize that CAPON gene. viii.

(12) variants affect healthy Taiwan Chinese population and the CAPON gene variants cause further QTc prolongation if the subjects are on QT-prolonging drugs. Our strategies are to genotype two important candidate SNPS, rs10494366 and rs1415263, in a heroin-addicted "healthy" Taiwan Chinese population who are undergoing methadone (a QT-prolonging drug via blocking IKr) treatment to assess the effects of gene variants on the pre- and the post-treatment QTc intervals. This approach is based on our preliminary data from a schizophrenic population (n=58). We found that when the schizophrenic patients carry the TT alleles at rs1415263, they tended to have QTc prolongation after receiving risperidone (another QT-prolonging drug via blocking IKr) treatment (396.7±26.7 ms vs. 403.3±20.6 ms), while those bearing the CC or CT alleles did not. Interestingly, rs1419263, a highly significant SNP associated with QTc prolongation in Caucasians did not affect the QT interval in this Taiwanese population both before and after risperidone treatment. With the findings, we are motivated to investigate the influence of CAPON gene variants on the development of QTc prolongation and potential lethal ventricular tachyarrhythmias in a drug-addicted Taiwan. ix.

(13) Chinese population undergoing methadone treatment. This study might enable us to identify a novel genetic risk factor for life-threatening ventricular tachyarrhythmias occurring in this particular population while undergoing methadone treatment and lead to development of new therapeutic interventions to prevent sudden cardiac death. In chapter 1, we stress the importance of studying QT interval and the underlying ionic mechanisms responsible for QTc prolongation. In addition to the well-known congenital long QT syndrome, Arking et al. has identified a new genetic marker, CAPON (NOS1AP), as a novel QT modifier in a recent genome-wide association study. This finding was soon replicated and confirmed by 3 other studies in different populations. As CAPON was previously unexpected to play a role in the heart, we designed an animal study to explore the molecular functions of CAPON in the heart. Additionally, we wanted to know whether the particular CAPON gene variants are also associated QTc prolongation in a Taiwanese population. In chapter 2, we used in vivo gene transfer technique to overexpress CAPON. in. guinea. pig. heart. then. exploring. the. cellular. electrophysiological remodeling after CAPON overexpression with and. x.

(14) without pharmacological intervention. For the CAPON gene variants and QTc association study, we genotyped 24 CAPON single nucleotide polymorphisms (SNPs) using a high throughput SNP array technique in a heroin addiction population undergoing methadone treatment. We wanted to answer the effects of CAPON gene variants on the QTc with and without methadone. In chapter 3, we found the expression of CAPON protein in the heart and when CAPON is overexpressed, it interacts with NOS1-NO pathway to affect the ICa,L and IKr currents causing acceleration of cardiac repolarization. (shortening. of. action. potential. duration).. The. electrophysiological remodeling of CAPON could be reversed by L-NAME, attesting the mechanistic link of CAPON to NOS1-NO pathway. In clinical part, we found low maintenance dose of methadone is associated with QTc prolongation and methadone-associated QTc prolongation appears to be gender-dependent. In this study, men appear to be more susceptible than women to methadone associated QTc prolongation. For the CAPON SNP association study, we found rs10918594 C>G, CAPON variant but not rs10494366 T>G is associated QTc prolongation in this Taiwanese population and use of methadone in. xi.

(15) patients with rs10918594 homozygous variant might potentiate the QTc prolongation. In chapter 4, we discussed the molecular mechanisms by which CAPON modulated cardiac repolarization and pointed out several potential limitations of the basic study. Indeed, future studies, particularly using a CAPON knock-out model are necessary to further elucidate CAPON function in the heart. For the clinical study, it seems that rs10918594 C>G is also associated QTc prolongation in this Taiwanese population, yet it needs to be confirmed in a larger population study and the underlying mechanisms of gender-dependent susceptibility of methadone associated QTc prolongation needs to be explored. In chapter 5, we conclude that CAPON is a new QT modifier in the heart which exists not only in the western population but likely also in Aisan population. Therefore, people carrying the genetic variants might be at a greater risk of developing serious ventricular tachyarrhythmias and sudden death when superimposed with additional long QT conditions, such as drugs and/or electrolytes imbalance. Keywords: QTc, cardiac repolarization, sudden death, drug-addiction, methadone. xii.

(16) ಃ΋ക ߻‫ق‬ ಃ΋࿯ ࣴ‫ز‬ङඳ Јႝკ‫ ޑ‬QT interval ࢂࡰவЈႝკ‫ ޑ‬QRS ‫ݢ‬ଆᗺ‫ ډ‬T ‫ޑ״่ݢ‬ ਔ໔Ǵа msec ߄ҢǴ೭ࢤය໔೏Ҕٰж߄Ј᠌ϐӆཱུϯਔ໔ǴԶ QT interval Ψ࣬྽‫ܭ‬Ј࠻ಒझֹԋ΋ঁ୏բႝՏ‫ޑ‬ਔ໔Ǵ྽Ј࠻ಒझѐ ཱུϯǴ٬ளಒझጢ΢‫ޑ‬ႝՏৡ΢ϲၲ‫ॶ⸣ډ‬Ǵ஥҅ႝ‫໊ޑ‬ᚆηႝࢬ ȐINaȑεໆ‫ޑ‬෢ΕಒझϣǴ٬ளጢႝՏ΢ϲ‫ډ‬നଯᗺǴௗ๱஥҅ႝ‫ޑ‬ ႇᚆηႝࢬȐItoȑࢬӛಒझѦǴ٬ЈԼಒझِೲӆཱུϯǴԜอኩِೲ ‫ޑ‬ӆཱུϯǴΞӢ஥҅ႝ‫້ޑ‬ᚆηȐICaȑႝࢬ෢ΕಒझϣԶᖿ጗Ǵௗ๱ ஥҅ႝ‫ޑ‬ႇᚆηႝࢬȐIKȑӛಒझѦࢬрǴ٬ಒझֹԋӆཱུϯǴ٠җ IK1 ᆢ࡭ᓉЗጢႝՏ‫ޑ‬ᛙ‫ۓ‬Ƕ 㵝ϙሶाࣴ‫ز‬Јႝკ QT intervalǻவၸѐ‫زࣴޑ‬ᡉҢǴόፕࢂ଼ ந‫܈ޣ‬ᑡ஻੯ੰ௼ဂȐ‫ٯ‬ӵᑗֿੰ‫߷܈‬Јੰȑ ǴQT interval ‫ߏۯ‬೿ࢂ ЇวЈӢ‫₽܄‬ԝ‫ޑ‬΋ঁख़ाࡰ኱Ǵ‫܌‬аॶளుΕࣴ‫ز‬ǶԶ‫ॺך‬ς‫ޕ‬ε ऊ 30ʘ‫ ޑ‬QT interval ࢂҗ୷Ӣ،‫ۓ‬Ǵ‫܌‬аόᆅӧӃϺ‫ࡕ܈‬Ϻ long QT syndromeǴ୷ӢӢન೿‫ת‬ᄽ๱ᜢᗖ‫فޑ‬ՅǴаӃϺ‫ ܄‬long QT syndrome Զ‫ق‬Ǵவ 1957 ԃಃ΋ੰ‫ٯ‬೏᛾ჴࢂҗ୷ӢँᡂЇଆࡕǴԿϞ 50 ԃٰǴ ς࿶ഌុว౜ 12 ࠠϐӃϺ‫ ܄‬long QT syndromeȐ߄ 1ȑǴନΑಃ 4 ȐAnkyrin-Bȑ ǵಃ 11 ȐAKAP9, Yotiaoȑᆶಃ 12ȐSNTA1, -1 Syntrophinȑ 1.

(17) ࠠࢂҗ‫ܭ‬ፓ௓ᚆη೯ၰϐೈқวғँᡂаѦǴ‫ځ‬Ꭹࣣҗᚆη೯ၰೈқ ҁ‫ँي‬ᡂᏤठǶԶᖏ‫׉‬΢ಃ 1 Կಃ 3 ࠠ‫ ޑ‬long QT syndrome ੰ‫ٯ‬э 99ʘа΢ǴӧЈႝკ‫ ޑ‬phenotype Бय़Ǵಃ 1 ࠠ‫ ޑ‬long QT syndrome ‫ڀ‬Ԗቨε‫ ޑ‬T ‫ݢ‬Ǵಃ 2 ࠠ‫ڀ‬ԖλԶϩΰࠠϐ T ‫ݢ‬ǴԶಃ 3 ࠠ‫ڀ‬Ԗၨ ߏϐ ST ‫ࢤݢ‬ϷλԶӾࠠϐ T ‫ݢ‬Ǵջߡ genotype ໚‫ޑ܄‬஻‫ޣ‬Ǵ‫ ځ‬QTc ‫ߏޑ‬อᆶࢂցวғၸཀྵല‫܈‬ЈӢ‫₽܄‬ԝǴϝࢂ،‫ੰۓ‬ΓႣࡕᆶᒧ᏷Ֆ ᅿ‫ݯ‬ᕍ‫ޑ‬ख़ाࡰ኱Ǵၸѐࣴ‫ز‬ᡉҢǴӵ݀ QTc λ‫ ܭ‬500 msec ٠Ъவ ҂วғၸཀྵലǴ‫ ځ‬5 ԃϣวғЈӢ‫₽܄‬ԝ‫ޑ‬ᐒ཮ǴѝԖ 0.5ʘǴՠऩ QTc ε‫ ܭ‬500 msecǴ‫܈‬ၸѐමวғၸ࡚௱Ǵ߾ 5 ԃϣวғЈӢ‫₽܄‬ԝ ‫ޑ‬ᐒ཮ଯၲ 14ʘǴฅԶǴᖏ‫׉‬΢ϝԖଯၲ 30ʘа΢‫ੰޑ‬Γ‫ي‬΢‫פ‬ό ‫ډ‬೭٤ς‫ ޑޑޕ‬long QT syndrome ‫୷ޑ‬ӢँᡂǴӆ‫ޣ‬Ǵ΋٤ᛰ‫܈ނ‬ႝ ှ፦౦த‫ ޑ‬long QT syndrome ੰ‫ٯ‬Ǵ೭٤໺಍‫ ޑ‬long QT ୷ӢँᡂΨ ค‫ֹݤ‬ӄှញ‫ ځ‬long QT phenotypeǴҗ‫ܭ‬೭٤ज़‫ڋ‬Ǵߡ௪ଆΑεࠠ‫ޑ‬ ୷Ӣࣴ‫ز‬Ǵ၂კٰ‫פ‬൨ཥࠠቹៜ QT interval ‫୷ޑ‬Ӣᡂ౦Ƕ. 2.

(18) ߄ 1ǺനཥӃϺ‫ ܄‬Long QT Syndrome ‫ޑ‬ϩᜪǶ. Congenital Long QT Syndrome Genetics of Long QT Syndrome炷LQTS炸 LQT Type. Gene. Chromosome Locus. Autosomal-dorminant 炷Romano-Ward炸 LQT1炷1991炸 KCNQ1炷KVLQT1炸11p15.5 LQT2炷1994炸 KCNH2炷HERG炸 7q35-36 LQT3炷1994炸 SCN5A 3p21-24 LQT4炷1995炸 4q25-27 Ankyrin-B LQT5炷1997炸 KCNE1炷minK炸 21q22.1-22.2 LQT6炷1999炸 KCNE2炷MiRP1炸 21q22.1-22.2 LQT7炷2001炸 KCNJ2 17q23 Autosomal-recessive 炷Jervell and Lange-Nielsen炸 KCNQ1炷KVLQT1炸11p15.5 JLN1 KCNE1炷minK炸 JLN2 21q22.1-22.2. Ion Channel. Effects. Percent of LQTS. - subunit of IKs - subunit of IKr - subunit of INa. IKs IKr INa. - subunit of IKs - subunit of IKr Kir2.1. IKs IKr IK1. 50 炴 45 炴 3-4 炴烋1 炴烋1 炴烋1 炴烋1 炴. - subunit of IKs -subunit of IKs. IKs IKs. 烋1 炴烋1 炴. LQT8:CACNA1C炷Timothy syndrome炸--- Cav1.2 LQT9: Caveolin-3--- late INa LQT10:SCN4B--- late INa LQT11: AKAP9, Yotiao--- I Kr LQT12: SNTA1, -1 Syntrophin--- late INa. ӧ 2006 ԃ 4 ДǴ΋໨җऍ୯ऊᑣᓅදߎථεᏢϷቺ୯ኀѭ໵ε ᏢЬᏤ‫ޑ‬εೕኳΓᜪ୷Ӣᡏ SNP association studyǴว߄ӧ Nature Genetics ᚇᇞǴԜࣴ‫ز‬२ࡋว౜ CAPON ‫୷ޑ‬Ӣᡂ౦཮ቹៜЈ᠌ӆཱུ ϯǴϸᔈӧЈႝკ QT interval ‫ޑ‬ᡂϯǶԜࣴ‫ࢂز‬ӧቺ୯ KORA ‫܌‬຾ ՉǴಃ΋໘ࢤࢂᑔᒧ QTc നதᆶനอ‫ ޑ‬7.5 percentileǴբ genome-wide association studyȐGWAȑ Ǵ‫פ‬рനᡉ๱ৡձ‫୷ޑ‬Ӣᡂ౦ȐSNPȑ Ǵӆ຾ ՉಃΒ໘ࢤ‫ޑ‬ෳ၂ǴQTc ‫ཱུঁٿ‬ᆄӚ 300 ΓǴӆᒧрനԖᡉ๱ৡ౦‫ޑ‬. 3.

(19) SNPsǴ຾ՉಃΟ໘ࢤ 3400 Γ‫ޑ‬ϩ‫݋‬ǴཀѦ‫פ‬р CAPON ࢂ΋ቹៜЈ ႝკ QTc ‫ޑ‬ӄཥ୷ӢǶ 10 9 8. -log (p-value). 7 6 5 4 3 2 1 0 158,718. OLFML2B. 158,768. CAPON. 158,818. 158,868. 158,918. Genomic Position (kb). A whole-genome SNP association study. Arking, Nature Genetics 2006. კ 1ǺArking et al. २ࡋճҔ genome-wide association study ว౜ CAPON ‫ ک‬QTc ‫࣬ޑߏۯ‬ᜢ‫܄‬Ƕ. ಃΒ࿯. ࣴ‫ز‬Ҟ‫ޑ‬. ᆙௗ๱ӧ೭ಃ΋ঁ GWA ว߄ࡕǴഌុԖ൳ঁख़ा‫ ޑ‬replication ࣴ‫ز‬Ǵ२Ӄࢂ 2007 ԃଞჹऍ୯ old order Amish ௼ဂ‫࣬ޑ‬ᜢ‫زࣴ܄‬Ǵ ว౜ CAPON rs10494366(T/G)‫୷ޑ‬Ӣᡂ౦ၟ QTc ‫ߏۯ‬Ԗᡉ๱࣬ᜢǴௗ ๱ӕԃӧ಻ើജ੝Ϗ‫زࣴޑ‬ǴΨᡉҢ CAPON ୷Ӣᡂ౦ rs10494366. 4.

(20) Ϸ rs10918594 ᆶ QTc ‫ߏۯ‬Ԗᡉ๱‫࣬ޑ‬ᜢ‫܄‬Ǵӧ 2008 ԃኻဴऍ୯Γᑗ ֿੰৎ௼‫زࣴޑ‬Ǵ‫׳‬຾΋‫؁‬᛾ჴ CAPON ୷Ӣᡂ౦ rs10494366 Ϸ rs10918594 ᆶ QTc ‫ߏۯ‬Ԗߚதᡉ๱‫࣬ޑ‬ᜢ‫܄‬Ǵҗа΢όӕ௼ဂ‫ࣴޑ‬ ‫ز‬ᡉҢ rs10494366 ᆶ QTc ‫࣬ޑߏۯ‬ᜢ‫܄‬നࣁᡉ๱Ȑკ 2ȑ Ƕ. QTc (ms). 10.0 7.5 9.3. 5.0. 7.2 6.1. 2.5. 5.3. n en ,D ia be te s. ds e, C. Le. ht in. irc ul at io. ity er ed Aa rn ou. ,H st Po. Ar ki ng ,. N. um an. at ur e. H. G. en et ic s. 0.0. კ 2Ǻrs10494366 homozygote ᆶ QTc ‫ߏۯ‬Ǵ4 ঁεࠠ࣬ᜢ‫زࣴ܄‬ϐ КၨǶ. ೭٤ўค߻‫୷ٯ‬Ӣࣴ‫่݀ޑز‬Ǵεε‫ࡷޑ‬Ꮿ‫ॺך‬ၸѐჹ‫ ܭ‬QT ғ ౛‫ޑ‬ᇡ᛽Ƕ CAPON ‫ ܭ‬1998 ԃ२ࡋ‫ܭ‬εႵ‫ޑ‬တઓ࿶ಔᙃ೏ว౜Ǵࢂ΋ঁଯࡋ conserved ‫ޑ‬ೈқǴεႵᆶΓᜪ‫ ޑ‬CAPON ೈқऊԖ 92ʘữ୷ለ‫ׇ‬ӈ ࢂ࣬՟‫ޑ‬ǴCAPON ೈқ‫ڀ‬Ԗ΋ঁ amino-terminus phospho-tyrosine bindingȐPTBȑdomain ‫ک‬΋ঁ carboxyl-terminus ‫ ޑ‬PDZ binding 5.

(21) domainǴӧတಔᙃ CAPON ёа೸ၸ‫ ک‬PDZ domain ‫ޑ‬ᗖ่Ǵ‫ ک‬PSD95 ᝡ‫ݾ‬ᆶ NOS1 ‫่ޑ‬ӝǴ຾Զ٬ NMDA-NOS1-NO ‫ૻޑ‬৲໺ሀၡ৩‫ڙ‬ ‫ڋ׭ډ‬ǴCAPON ёаᇥࢂ NOS1 ‫ ޑ‬regulator ‫ ک‬adaptorǴёа‫ ע‬NOS1 ஥‫ډ‬኱‫ޑ‬ೈқ຾Չ੝‫ޑۓ‬ғ౛բҔȐკ 3ȑ ǴᏃᆅӵԜ CAPON ࢂցΨ ӧЈ᠌‫ת‬ᄽᜪ՟‫فޑ‬ՅǴӧ྽ਔ΋ค‫ޕ܌‬Ƕ CAPON protein in neuron 20. 490. 180. PTB domain. 1) Jaffrey, Neuron 1998 2) Fang, Neuron 2000 3) Jaffrey, PNAS 2002. 503. PDZ binding domain. Ca2+ membrane. Ca2+ NMDA receptor. cytoplasm. membrane. PSD95. NMDA receptor. cytoplasm. PSD95. CA PO N. PDZ-PDZ binding. Dexras1-GDP NOS1 NOS1. NO Ca2+/Calmodulin. Ca2+/Calmodulin. SH Dexras1-GTP NO SNO. კ 3ǺJaffery et al. २ࡋว౜ CAPON ӧઓ࿶ಒझ‫ת‬ᄽख़ाфૈǶ. NOS1 ᆶ NOS3 ‫ޣٿ‬ӧЈԼಒझࣣև౜ಔԋ‫߄܄‬ၲǴNOS1 ё‫ک‬ ಒझጢ΢‫ ޑ‬Na+-K+ ATPase ᆶ Ca2+/calmodulin-dependent Ca2+ ATPase (PMCA)ҬϕբҔǴӧϣ፦ᆛȐsarcoplasmatic reticulumǴSRȑNOS1 ӧ่ᄬ΢ᆶ ryanodine receptor 2ȐRyR2ȑϷ cardiac SR Ca2+ ATPase ȐSERCA2ȑᆙஏ่ӝǴаፓ࿯ಒझϣ‫້ޑ‬ൻᕉϷᑫᏟ-ԏᕭϕ୏. 6.

(22) Ȑexcitation-contraction couplingȑ Ǵӧ٬Ҕ୷Ӣᙯ෗Ⴕஒ NOS1 ၸࡋ߄ ၲ‫ޑ‬ჴᡍᡉҢ NOS1 ཮‫ک‬ಒझጢ΢‫ ޑ‬L-type ້ᚆη೯ၰ่ӝ٠Ъ‫׭‬ ‫ ڋ‬L-type ້ᚆηႝࢬȐICa,Lȑ Ǵ೭٤᛾ᏵᡉҢ NOS1 ӧЈ᠌ғ౛‫ת‬ᄽ ख़ा‫ف‬ՅǴӢԜ‫ॺך‬ଷ೛ CAPON ΨӧЈ᠌߄ၲ٠Ъ೸ၸ NOS1 ຾Չ ғ౛բҔǴӢԜ‫ॺך‬२ӃவӧЈ᠌‫פ‬൨ϣғ‫ ޑ܄‬CAPON ೈқ໒‫ۈ‬Ǵ ٠ЪճҔဏੰࢥၩᡏ຾Չ in vivo ୷ӢᏤΕ‫ޑ‬БԄஒ CAPON ၸࡋ߄ၲ ٰ௖૸ CAPON ‫ת܌‬ᄽ‫ޑ‬ғ౛‫ف‬ՅǴ٠຾Զࣴ‫ ز‬CAPON ᆶ NOS-NO ૻ৲ၡ৩‫ޑ‬ҬϕբҔǶ ӧΑှΑ CAPON ӧЈ᠌‫ޑ‬ϩηᐒᙯࡕǴ‫ॺך‬Ψ຾Չᖏ‫زࣴ׉‬௖ ૸த‫ ޑـ‬CAPON ୷Ӣᡂ౦ȐSNPsȑࢂցᆶ୯Γ QTc ‫ڀߏۯ‬Ԗ࣬ᜢ ‫܄‬Ƕ. 7.

(23) ಃΒക ࣴ‫ز‬Б‫ݤ‬ ಃ΋࿯ ୷ᘵࣴ‫ز‬ Cloning of CAPON cDNA, Plasmid Construction, and Adenovirus Preparation. Total RNA was extracted from the ventricular myocardium of adult guinea pig with an RNEasy mini kit (Qiagen) according to the manufacturer's instructions and then quantified by a UV spectrometer. Total RNA (2 mg) was subjected to a 20.5-ml reverse transcriptase reaction to synthesize cDNA after the SuperScript II first-strand cDNA synthesis protocol (Invitrogen). The full-length CAPON cDNA was cloned and amplified by PCR using the first-strand cDNA as template with a forward primer of 5'-ACCATGCCCAGCAAAACCAAGTAC-3' and a reverse primer of 5'-CTACACGGCGATCTCATCAT-3'. The CAPON cDNA PCR product was inserted into pTOPO (Invitrogen) plasmid to generate pTOPO-CAPON, according to the manufacturer's protocol. The CAPON cDNA was then cut out of pTOPO-CAPON from the NsiI restriction enzyme site and ligated with the compatible PstI site in the multiple cloning sites of AdCIG plasmid to generate the adenoviral plasmid, pAdCMV-CAPON-IRES-GFP, capable of coexpressing GFP as a reporter. The construction of the adenoviral plasmid was verified by 8.

(24) multiple restriction enzymes digestion testing and DNA sequencing. Adenovirus vectors (AdCAPON-GFP) were produced by Cre-lox recombination of purified y5 viral DNA and shuttle vector DNA as described in previously. Overexpression of CAPON protein was confirmed by Western blotting and immunofluorescent staining. The recombinant viral products were plaque-purified, expanded, and then purified to generate the concentration in the order of 1010 pfu/ml. Western Blot Analysis. Fresh ventricular myocardium and brain and lung tissues were harvested from adult guinea pig and rapidly frozen in liquid nitrogen and stored at -80°C for further processing. The preparation and quantification of the frozen protein tissues have been described. Briefly, the tissue samples were homogenized in a 4´ volume of RIPA buffer (Sigma-Aldrich) plus complete protease inhibitors (Roche). The protein concentration was determined by Lowry assay. Each protein sample was heated in SDS sample buffer and loaded on a 4-12% gradient Bistris Nu-PAGE gel (Invitrogen). Proteins were transferred to polyvinylidene difluoride (Bio-Rad) or nitrocellulose membrane (Invitrogen) in transfer buffer without SDS. According to the experimental purposes, blots were probed with primary antibodies, 9.

(25) including a rabbit polyclonal antibody against the C terminus of CAPON (sc-9138, 1:200-1:1,000 dilution; Santa Cruz Biotechnology), a mouse monoclonal anti-NOS1 antibody (sc-5302, 1:200 dilution; Santa Cruz Biotechnology), or a rabbit polyclonal anti-NOS3 antibody (sc-654, 1:200 dilution; Santa Cruz Biotechnology), respectively, and detected by SuperSignal West Femto chemiluminescent reagents (Pierce) with an anti-rabbit (sc-2004; Santa Cruz Biotechnology) or anti-mouse (GE Healthcare) secondary antibody coupled to horseradish peroxidase. To verify CAPON bands, blots were also probed with a goat polyclonal antibody against the N-terminal epitope (2-14 aa) of CAPON (ab3928, 1:200-1:1,000. dilution;. abcam). for. comparison.. For. GAPDH. normalization, blots were incubated in the stripping buffer containing 62.5 mM Tris×HCl (pH 6.7), 2% SDS, and 100 mM b-mercaptoethanol at 50°C for 30 min, followed by reacting with a mouse monoclonal antibody to GAPDH (Fitzgerald Industries International, Inc.) and visualized by using SuperSignal West Pico chemiluminescent agents (Pierce) with an anti-mouse secondary antibody (GE Healthcare). For protein samples from the culture ventricular myocytes, the GAPDH was probed by a goat polyclonal antibody (IMG-3073, 1:15,000 dilution; IMGENEX,) and. 10.

(26) detected by SuperSignal West Femto chemiluminescent substrates (Pierce) with. an. anti-goat. secondary. antibody. (sc-2020;. Santa. Cruz. Biotechnology). Coimmunoprecipitation of CAPON and Nitric Oxide Synthase (NOS). The rabbit anti-CAPON antibody (sc-9138; Santa Cruz Biotechnology) or the rabbit anti-NOS1 antibody (sc-648; Santa Cruz Biotechnology) was bound and immobilized to a protein G support with cross-linking agent (DSS) according to the manufacturer's instructions (Pierce). The tissue homogenates from normal guinea pig ventricular myocytes were immunoprecipitated overnight with the antibody-protein G complex at 4°C. After an extensive wash, the immunoprecipitated antigens were then eluted and became the samples for Western blotting. To prepare the sample for Western blot analysis, 5 ml of sample buffer (Pierce) was added to a 20-ml elution sample and boiled at 100°C for 5 min before SDS/PAGE. To probe NOS1, NOS3, and CAPON, the blots were reacted with the monoclonal mouse anti-NOS1 antibody (sc-5302; Santa Cruz Biotechnology), the rabbit polyclonal anti-NOS3 antibody (sc-654; Santa Cruz Biotechnology), and the rabbit anti-CAPON antibody (sc-9138; Santa Cruz Biotechnology), respectively, and then detected 11.

(27) with. SuperSignal. West. Femto. chemiluminescent. reagents. with. corresponding secondary antibodies. For control experiments, the ventricular myocardial homogenates were incubated overnight with antibody-free protein G complex and then processed in the same ways for Western blot analysis. Cell Culture and Adenovirus Transduction. HEK293 cells (American Type Culture Collection) maintained in DMEM (Invitrogen) with 10% FBS (Invitrogen) and 1% penicillin/streptomycin (Invitrogen) were seeded at ~50% confluence in T25 flasks (Salsdect) and cultured in a 5% CO2 and 37°C environment. Cells were transduced with 10 ml of AdCAPON-GFP at 80% confluence and cultured for 48 h to ensure adequate transgenic expression. Cells were then harvested, lysed, homogenized, and subjected to Western blotting as described above. A T25 flask of HEK293 cells of same confluence without viral transduction was used as a control. For myocytes culture, the freshly isolated ventricular myocytes dispersed in DMEM with 5% FBS and 1% penicillin/streptomycin were plated into 6-well culture dishes precoated with laminin and cultured for 1 h to let cells attach to the bottoms of the culture dishes. Then, the medium was replaced with fresh medium, and 12.

(28) cells were infected with ~108 pfu/ml AdCAPON-GFP or AdGFP or not infected with either virus and cultured for 2 days. After that, cells were harvested for Western blot analysis. To compare the NOS1 level in freshly isolated ventricular myocytes, when the cells became attached to the bottoms of the culture dishes 1 h after plating, the medium was replaced with AdCAPON-GFP-containing or noncontaining DMEM, and then the cells were harvested immediately for protein analysis, which was designated as 0 h of cell culture. Immunofluorescent Staining and Confocal Microscopic Imaging. Freshly isolated guinea pig ventricular myocytes were attached to coverslips precoated with laminin, fixed in 4% paraformaldehyde, and then permeabilized with 0.2% Triton X-200. After blocking with 10% normal goat serum, the myocytes were reacted overnight with the rabbit polyclonal anti-CAPON antibody (sc-9138, 1:50-1:100 dilution; Santa Cruz Biotechnology) at 4°C, followed by a Texas red-conjugated anti-rabbit secondary antibody (sc-2780; Santa Cruz Biotechnology). Background fluorescence caused by unspecific binding of the secondary antibody was estimated by omitting preincubation with primary CAPON antibody in a subset of myocytes. Nuclear counterstaining was carried out 13.

(29) by using a DNA-specific dye, Hoechst 33342 (Molecular Probes). Ventricular myocytes were examined by a laser scanning confocal microscope with multiple track techniques (LSM510; Zeiss). Texas red was excited by helium/neon laser (543 nm), GFP by argon laser (488 nm), and Hoechst by diode UV laser (405 nm). In Vivo Gene Transfer and Myocyte Isolation. Adult guinea pigs (weight, 300-400 g) were anesthetized with 4% isoflurane, intubated, and supported by a ventilator with a maintenance isoflurane dose of 1.5-2% during the operation. The guinea pigs then underwent open-chest intramyocardial injection of the adenoviral vectors, 2×1010 pfu/ml AdCAPON-GFP, or 3×1010 pfu/ml AdGFP into the left ventricular apex with a total volume of 200 ml by using 30-gauge needle as described. The ventricular myocytes were isolated 72 h after in vivo gene transfer, typically within 3-5 days, with standard techniques. The transduced ventricular myocytes were judged by their vivid green fluorescence by using a xenon arc lamp at 488/530 nm (excitation/emission). Electrophysiological and Pharmacological Studies. Experiments were performed by using whole-cell patch clamp technique with an Axopatch 200B amplifier (Axon Instruments) while sampling at 10 kHz 14.

(30) for current recordings or 2 kHz for voltage recordings and filtering at 2 kHz. Pipettes had tip resistances of 2-4 MW when filled with internal solutions. All recordings were performed at 33 ±1°C, but INa was recorded at room temperature (22°C). Cells were superfused with a physiological saline solution containing 140 mmol/liter NaCl, 5 mmol/liter KCl, 2 mmol/liter CaCl2, 1 mmol/liter MgCl2, 10 mmol/liter Hepes, and 10 mmol/liter glucose; the pH was adjusted to 7.4 with NaOH. The pipette solution was comprised of 100 mmol/liter potassium aspartate, 20 mmol/liter KCl, 1 mmol/liter MgCl2, 5 mmol/liter Hepes, 5 mmol/liter EGTA, 5 mmol/liter MgATP, and adjusted to pH 7.3 by KOH. Action potentials were elicited continuously by brief suprathreshold depolarizing current pulses at 1, 2, and 0.05 Hz until steady state was reached. APD was measured as the time from overshoot to 10% (APD10), 25% (APD25), 50% (APD50), 75% (APD75), and 90% repolarization (APD90). For IK1 and total IK recordings, 10 mM nitrendipine (Sigma-Aldrich) was added to the external solution to block calcium currents. For separation of IKr and IKs, after recordings of the total IK, cells were exposed to 30 mM chromanol 293B (Aventis Pharma) to fully suppress the IKs and to obtain the remaining IKr. For measurement of ICa,L, the external solution contained. 15.

(31) 128 mmol/liter NaCl, 10 mmol/liter CsCl, 1 mmol/liter MgCl2, 10 mmol/liter NaHepes, 2 mmol/liter CaCl2, adjusted to pH 7.4 by HCl; and the pipette solution consisted of 110 mmol/liter CsCl, 0.5 mmol/liter MgCl2, 10 mmol/liter Hepes, 20 mmol/liter tetraethylammonium chloride (TEA-Cl), 5 mmol/liter BATPA, 5 mmol/liter MgATP, adjusted by pH 7.2 by CsOH. To measure ICa,L, the INa was steady-state-inactivated by a prepulse from -80 to -40 mV, and the membrane potential was held at -40 mV throughout the recordings. INa was recorded by using symmetrical 10 mmol/liter Na+ with external solution containing 10 mmol/liter NaCl, 2 mmol/liter MgCl2, 5 mmol/liter CsCl, 120 mmol/liter TEA-Cl, 20 mmol/liter Hepes, adjusted to pH 7.4 by NaOH; the internal solution contained 10 mmol/liter NaCl, 140 mmol/liter CsCl, 10 mmol/liter Hepes, adjusted to pH 7.2 by CsOH. Because CAPON is a regulator and adaptor of NOS1, to understand the role of the NOS-NO pathway in CAPON overexpression-mediated electrophysiological changes is mechanistically important. For this purpose, freshly isolated ventricular myocytes from the same animal were divided into two fractions; fraction 1 was pretreated with 1 mM L-NG-nitro-L-arginine methyl ester (L-NAME) (Sigma-Aldrich) for 30 16.

(32) min before the patch clamp study and then continuously exposed to the same concentration of L-NAME in the bath solution throughout the study, whereas fraction 2 was not exposed to L-NAME for comparison. Intracellular NO Imaging with DAR-4M AM. DAR-4M AM (AXXORA) is a membrane-permeable rhodamine-based chromophore, capable of detecting intracellular NO as low as 7 nM. When incubated with cells, it enters the cells because of the presence of the acetoxymethyl ester (AM) moiety. Once inside the cells, the AM moiety is hydrolyzed by cytoplasmic esterase to form the membrane-impermeable DAR-4M. When NO is produced intracellularly, DAR-4M can react with it to form a. triazole. compound,. DAR-4MT.. DAR-4MT. is. a. stable,. membrane-impermeable, and fluorescent compound that can be visualized with a fluorescent microscope with rhodamine filter. For chromophore loading, freshly isolated ventricular myocytes dispersed in DMEM without phenol red (GIBCO) were incubated with 10 mM DAR-4M AM in 35-mm glass-bottom culture dishes (P35G-0-14-C; MatTek Corporation) for 30 min at room temperature or incubated at 37°C with the addition of 2 mM L-arginine (Sigma-Aldrich) for 60 min. After incubation, the extracellular chromophore was carefully washed 17.

(33) away by DMEM without phenol red three times. The cells were then subjected to confocal microscopic imaging by using an LSM510 system with an excitation wavelength of 543 nm and emission wavelength of 560 nm to image the NO fluorescent marker in CAPON-overexpressing ventricular myocytes and control myocytes. The fluorescent intensity was quantified by LSM510 software. L-NAME (2 mM) and sodium nitroprusside (1 mM; Sigma-Aldrich) were used to inhibit or enhance the NO fluorescent marker to verify the specificity of NO detection by DAR-4M AM chromophore.. ಃΒ࿯ ᖏ‫زࣴ׉‬ Study Subjects Evaluation Between September 2008 and May 2010, consecutive heroin dependence patients admitted to outpatient methadone clinics of the Division of Addiction Psychiatry of China Medical University Hospital, Taichung, TAIWAN were recruited for the study. The diagnosis of opioid dependence was according to DSM-IV criteria made by a specialist in psychiatry. Eligible male or female patients who had opioid addiction history of more than 1 year should be aged ɪ18 and ɩ65 years with 18.

(34) good physical health determined by complete physical examination, laboratory tests, and a 12-lead ECG screening for entry of the study. To be eligible, patients or their reliable caregivers are being expected to ensure acceptable compliance and visit attendance throughout the study period. Patients were excluded from the study if they are currently with severe physical and mental disorders including an uncontrolled and/or clinically significant cardiac, hepatic or renal dysfunction as well as premorbid mental retardation or multiple substances dependence that would compromise patient safety or preclude study participation. Women of childbearing potential, not using adequate contraception as adjusted by investigators or not willing to comply with contraception for the duration of the study and females who are pregnant or nursing were also excluded from the study. The Institutional Review Board for the Protection of Human Subjects at China Medical University Hospital approved this study protocol. At baseline (immediately before methadone induction), a complete medical history, physical examination and laboratory tests were obtained. Each individual subject gave a signed informed consent before inclusion. Recent drug and alcohol use was assessed by patient report. Particular attention was given to screen for medications affecting cardiac. 19.

(35) conduction, repolarization, or methadone metabolism. All patients received a 20 mg starting dose of oral methadone followed by subsequent 10 mg incremental increases. Doses were titrated to maintenance levels based on self-reported heroin use, presence of opioid withdrawal symptoms, and urine toxicology evidence of illicit opioid abuse. A standard digital 12-lead electrocardiography (ECG) (GE Healthcare, USA) was performed when the participants underwent stable methadone maintenance treatment program, typically 1 month after initiation of methadone. In laboratory, urine toxicology assays for illicit opioids and amphetamines were performed at regular intervals during the study. The psychiatric. evaluation. tools. consisted. of. Mini. International. Neuropsychiatric Inventory (MINI) and Structured Clinical Interview for DSM-IV, Addiction severity index (ASI) and Tridimensional Personality Questionnaire (TPQ) for a comprehensive patient assessment and care. Electrocardiographic Measurements All 12-lead ECGs were recorded by a GE Marquette’s MAC 5500 ECG recorder (GE Medical Systems, Milwaukee, WI, USA), using a standardized protocol with special attention to precise localization of each electrode. A 10-second resting 12-lead ECG recorded at a sampling. 20.

(36) frequency of 500 Hz and was digitally transmitted and stored to the MUSE system (GE Marquette) at the core ECG laboratory of China Medical University Hospital for subsequent analysis. All the stored ECGs were visually inspected by an experienced cardiologist blinded to the study to exclude those with technical errors and inadequate quality before being processed for automatic measurement by the 2001 version of the Marquette 12SL program (GE Marquette). The machine-read QT interval measurements were based on a "Global Median" beat, a superimposition of 12 representative median beats. The 12SL measures the earliest onset of the Q wave in any lead and the latest offset of the T wave in any lead to derive the QT interval which was then corrected by heart rate (QTc) using Bazett’s formula (QTc=QT/RR1/2). The 12SL algorithm includes the U wave in the QT measurement if the U wave merges with the preceding T wave. Otherwise, the U wave is excluded from the QT measurement. A 12-lead ECG was obtained when the subjects were receiving stable maintenance methadone treatment, typically one month after the initiation of methadone. The ECG recording was performed after observed intake of individual participant’s daily methadone. To study the cross sectional association between oral methadone doses and QTc duration, all eligible. 21.

(37) ECGs from subjects undergoing maintenance methadone therapy were used. In a subset of participants, ECGs were obtained before, 1 month and 3 months after the initiation of methadone therapy to assess the chronological changes of the QTc intervals. ECGs with a wide QRS duration of 120 ms caused by complete right or left bundle-branch block, ventricular preexcitation, or intraventricular conduction delay were excluded from analyses. In addition, we also exclude ECGs with atrial fibrillation and evidence of myocardial infarction to avoid potential non-methadone influence on the QT intervals. Similarly, other confounding factors that might affect the QT duration at the time of ECG recording including the concomitant use of any other QT-altering drugs, electrolyte imbalance with hypokalemia or hypocalcemia are excluded from analyses. In laboratory, urine toxicology assays for illicit opioids and amphetamines will be perform at regular intervals during the study and blood samples will collect to extract DNA before initiation of methadone. 1. Inclusion criteria Patients must meet all of these inclusion criteria to be eligible for enrollment into the study:. 22.

(38) A. Capacity and willingness to give written informed consent B. A diagnosis of opioid dependence according to DSM-IV criteria made by a specialist in psychiatry C. Male or female patient aged ʁ18 and ʀ65 years D. Good. physical. health. determined. by. complete. physical. examination, laboratory tests, and EKG E. Patient or a reliable caregiver can be expected to ensure acceptable compliance and visit attendance for the duration of the study. 2. Exclusion criteria The presence of any of the following will exclude a patient from study enrollment: A. Current evidence of an uncontrolled and/or clinically significant medical condition, e.g., cardiac, hepatic and renal failure that would compromise patient safety or preclude study participation. B. Premorbid mental retardation C. Current Other major Axis-I DSM-IV diagnosis other than opioid dependence such as multiple substance dependence D. Increase in total SGOT, SGPT, gamma-GT, BUN and creatinine by more than 3X ULN (upper limit of normal). 23.

(39) E. Women of childbearing potential, not using adequate contraception as per investigator judgment or not willing to comply with contraception for the duration of the study. F. Females who are pregnant or nursing. 3. Evaluation tools A. Mini International Neuropsychiatric inventory (MINI) and Structured Clinical Interview for DSM-IV: for psychiatric disorder evaluation and diagnosis B. Addiction severity index (ASI): evaluation of substance related family, physical, occupational, legal, economical factors C. Tridimensional Personality Questionnaire (TPQ): Self rating scale, TPQ, will be used as an instrument to investigate the personality trait of the patient with opioid dependence. The Chinese version of the TPQ used in the present study is a 100-item, self-administered, true–false instrument. Consisting of two subscales, the Cronbach’s of the novelty-seeking subscale (NS) was 0.70, and that of the harm-avoidance subscale (HA) was 0.87. Since the reward dependency (RD) dimension has no adequate reliability among Han Chinese in Taiwan, only NS and HA dimensions will be. 24.

(40) analyzed in this study. DNA extraction and genotyping We will obtain DNA from the participants via blood by phlebotomy. DNA will be extracted from blood samples using standard procedures. All samples will be obtain on 9 A.M. before and after 6 weeks of treatment and 10 ml blood will be obtain. After extracting of DNA and mRNA of blood mononuclear cells, the sample will be storage under -80ʚ. Genotyping of the two candidate SNPs of CAPON, rs10494366 and rs1415263, will be performed by using 1 ng genomic DNA extracted from leukocytes, as previously reported. DNA extraction, amplification, and mutation screening: Total genomic DNA was extracted from 10 to 15 ml of venous blood from each participant after informed consent was obtained. DNA was purified from lymphocyte pellets according to standard procedures using a. Puregene. kit. (Gentra. Systems,. Minneapolis,. MN). or. the. phenol-chloroform extraction method. Twenty-four SNPs spanning the non-coding region of CAPON gene were identified using GenBank database. Multiplex PCR and SNP analyses were performed with the GenomeLab SNPstream genotyping platform (Beckman Coulter Inc.. 25.

(41) Fullerton, CA) and its accompanying SNPstream software suite. The primers for the multiplex PCR and single base extension primers were optimally designed by Web-based software provided at Beckman Coulter Inc. PCR primers were designed to amplify a short stretch of DNA (90 bp) that encompasses the SNP of interest. The tagged extension primers used to identify the SNPs were designed in two parts. The 5' portion of the probe is complementary to one of 12 unique single stranded DNA oligonucleotides that are microarrayed at a specific location within each well of a 384-well microplate. The 3' portion of the probe is complementary and precisely adjacent to the SNP, which enables detection of the presence of either or both nucleotides of the SNP through the incorporation of a fluorescent-labeled terminating nucleotide. Twelve-plex PCR reactions were performed in 384-well plates (MJS BioLynx, Brockville, ON) in a 5 l volume using 6 ng of DNA, 75 M dNTPs, 0.5 U of AmpliTaq Gold (Perkin-Elmer, Wellesley, MA), and the 24 PCR primers at a concentration of 50 nM each in 1 X PCR buffer. Thermal cycling was performed in GeneAmp PCR system 9700 thermal cyclers (Applied Biosystems, Foster City, CA) using the following program: initial denaturation at 95 °C for 5 min followed by 40 cycles of. 26.

(42) 95 °C for 30 s, 50-55 °C for 55 s, 72 °C for 30 s. After the last cycle, the reaction was held at 72 °C for 7 min. Following PCR, plates were centrifuged briefly and 3 l of a mixture containing 0.67 U Exonuclease I (Amersham Pharmacia, Buckinghamshire, UK) and 0.33 U shrimp alkaline phosphatase (Amersham Pharmacia) were added to each well. The plates were sealed and incubated for 30 min at 37 °C and at 95 °C for 10 min. The tagged extension primers were extended with single TAMRA- or bodipy-fluorescein-labeled nucleotide terminator reactions and then spatially resolved by hybridization to the complementary oligonucleotides arrayed on the 384-well microplates (SNPware Tag array). The Tag array plates were imaged with a two-laser, two-color charged couple de- vice-based imager (GenomeLab SNPstream array imager). The 12 individual SNPs were identified by their position and fluorescent color in each well according to the position of the tagged oligonucleotides. Sample genotype data were generated on the basis of the relative fluorescent intensities for each SNP and computer processed for graphical review. PCR was performed on 50 ng of genomic DNA with a GeneAmp PCR system 9700 thermocycler (Applied Biosystems). The Touchdown PCR cycling condition was performed as follows: initial. 27.

(43) preheating step for 11 min at 95 °C to achieve a hot start, followed by 12 cycles of 94 °C for 20 s, 68 °C for 20 s, and 72 °C for 45 s, the annealing temperature decreased 0.5 °C per cycle until 62 °C; followed by 30 cycles of 94 °C for 20 s, 62 °C for 20 s, and 72 °C for 45 s; with a final elongation at 72 °C for 10 min. Amplified PCR products were separated by agarose gel electrophoresis and visualized by staining with ethidium bromide. They were then purified in purification columns (QIAquick; Qiagen, Valencia, CA) and sequenced using dye terminator chemistry (BigDye Terminator ver. 3.1 on a model 3100 Genetic Analyzer; Applied Biosystems, Foster City, CA). Sequences were trimmed for quality and aligned using BioEdit software (version 5.0.6). Normal and affected individual DNA sequences were aligned to the known reference genomic sequence (GenBank NT_011362), available via the National Center for Biotechnology Information (NCBI) database and compared for sequence variation.. 28.

(44) ಃΟ࿯ġ ಍ीБ‫ݤ‬ Statistical Analysis All continuous variables are expressed as mean ± SD and all categorical variables are expressed as count (percentage). Two-sample t test, paired t test and chi-square test were used to perform univariate comparison. Pearson’s correlation, simple and multiple regression models were used to assess the association between QTc intervals and methadone doses. All analyses were performed for male and female separately. The variables in the model included age, gender, methadone doses, age at exposure to heroin, duration of heroin addition, duration of methadone treatment and the QTc intervals. The relations between gender and clinical variables such as SGOT, SGPT, -GT, HBsAg, and HCV Ab were assessed with the use of a Fisher’s exact test. The QTc intervals were compared with the two-sample t-test for the normal and abnormal groups according to the classification from SGOT, SGPT, r-Gt, HBsAg, and HCV Ab. All statistical tests were two-sided, and a p value of less than 0.05 was considered to indicate statistical significance. Analyses were performed with the use of SAS software (version 9.1, SAS Institute).. 29.

(45) To examine the association of the SNP, we used the 2 test to compare the alteration of genotypes between patients and controls. The. 2 test evaluates the difference between the observed genotype frequency and the expected frequency under the null hypothesis of no association. However, when the expected frequency is small, the Fisher exact test is conducted instead. Bonferroni correction was applied for the multiple tests. Next, all detected SNPs were assessed for Hardy-Weinberg disequilibrium using the 2 test. We conducted logistic regression analysis with a stepwise approach. The independent variables were values of SNP (homozygote, 2; heterozygote, 3; wild type, 1). The covariates of interactions between SNP were also included in the model. The final optimal model contains the statistically significant variables (SNP) that were selected by the stepwise procedure. These statistical analyses were performed using SPSS software (ver. 10.1; SPSS Science, Chicago, IL).. 30.

(46) ಃΟക ࣴ‫่݀ز‬ ಃ΋࿯ ୷ᘵࣴ‫ز‬ २ࡋว౜ϣྍ‫ ܄‬CAPON ӧЈ᠌߄ၲ ‫ॺך‬٬Ҕ Western Blotting ‫ೌמޑ‬ǴவϺไႵ‫ޑ‬ѰЈ࠻ЈԼ൨‫פ‬ CAPON protein ߄ၲǴЈԼ‫ ޑ‬protein bands ‫ک‬တϷ‫ޤ‬ಔᙃ protein bands բКჹǴ‫ॺך‬ΨճҔନΑ௖‫؃‬ϣྍ‫ ޑ܄‬capon аѦǴ‫ॺך‬Ψ٬Ҕဏੰ ࢥၩᡏȐAdCAPON-GFPȑஒ CAPON ၸࡋ߄ၲ‫ ܭ‬HEK293 ಒझǴ Western Blot ‫่݀ޑ‬ᡉҢЈԼр౜΋ঁ 60-Kda protein bandǴ࣬྽‫ܭ‬ϣ ྍ‫ ޑ܄‬full-length CAPONǴӧ 70-Kda protein bandǴёૈࢂ CAPON ᙯ᝿ࡕ‫ׯ‬ᡂ‫ߚ܈‬੝౦‫܄‬ϸᔈǴॶள‫ݙ‬ཀ‫ࢂޑ‬ǴӧЈԼќѦр౜΋చ 30-Kda bandǴ࣬྽‫ ܭ‬short-form CAPONǴќѦଞჹཥϩᚆϐЈ࠻ಒ झբխࣝϷᑻӀࢉՅΨᡉҢ CAPON protein ӧಒझ፦ϩթ٠ӧಒझ ਡᆶಒझጢ‫ڬ‬ൎԖуம‫ޑ‬౜ຝǴӧ CAPON ၸࡋ߄ၲ‫ޑ‬ЈԼಒझΨև ౜խࣝᑻӀቚம౜ຝǴᡉҢဏੰࢥ୷ӢᏤΕԋфȐAdCAPON-GFPȑ Ȑკ 4ȑǶ. 31.

(47) A. B HEK 293 NT. T. Lung VM. CAPON. Merge. Brain. 1. ˑ. 80. GFP. Guinea Pig. 60. CAPON-L. 30. CAPON-S. 2. GAPDH. 3. კ 4ǺCAPON ӧЈ᠌߄ၲ‫ޑ‬᛾ᏵǶAǺWestern blotǹBǺխࣝಒझ ࢉՅǶ. ဏੰࢥҁ‫ي‬٠όቹៜಒझႝғ౛ ࣁΑ᛾ჴဏੰࢥҁ‫ي‬٠ό཮‫ׯ‬ᡂಒझႝғ౛Ǵ‫ॺך‬Кၨ୏բႝՏ ᆶ ້ ᚆ η ႝ ࢬ ‫ ܭ‬AdGFP Ꮴ Ε Ȑ transduction ȑ ᆶ ҂ Ꮴ Ε Ȑnon-transductionȑ‫ޑ‬ЈԼಒझǴ٠ว౜೭‫ޣٿ‬٠คᡉ๱‫ޑ‬ৡ౦Ȑკ 5 A-Bȑ Ǵӧዴ‫ۓ‬Αဏੰࢥၩᡏ٠όԿᏤठಒझႝғ౛‫ׯޑ‬ᡂǴ‫ॺך‬ω ૈ୼຾΋‫؁‬Кၨႝғ౛੝‫ ܭ܄‬AdCAPO-GFP ᏤΕᆶ҂ᏤΕ‫ޑ‬ЈԼಒ झǶ. CAPON ၸࡋ߄ၲуೲЈ࠻ಒझ୏բႝՏ ӧ 1-Hz ႝ ‫ ڈ‬ᐟ ਔ Ǵ CAPON ၸ ࡋ ߄ ၲ ‫ ޑ‬Ј Լ ಒ झ (n ɨ 9) ‫ځ‬ APD10ǵAPD50ǵAPD75 ᆶ APD90 ϩձफ़Կ 54.0 ± 7.2ǵ198.8 ± 8.1ǵ221.8 32.

(48) ± 7.9 ᆶ 233.9 ± 7.7 msǴԶჹྣϐЈԼಒझ߾ࣁ 96.6 ± 11.5ǵ291.0 ± 16.4ǵ316.1 ± 17.2 ᆶ 327.5 ± 17.9 ms (nɨ13, Pɦ0.05) (კ 5 C-E)Ƕ᏾ ᡏԶ‫ق‬ǴCAPON ၸࡋ߄ၲᏤठ APD90 ᕭอ 28.6ʘǴௗ๱‫ॺך‬຾΋‫؁‬ ௖૸ՖᅿᚆηႝࢬᏤठ୏բႝՏᕭอǶ B control GFP p=NS p=NS. p=NS. 200 100 0. 10. 13. 8. 8. 11. 8. 0 -2 -4. 2 Hz. C. -8 -10. 50 ms p=NS. Peak ICa,L (pA/pF). 20 mV. 20 mV. 100 ms. E. **. 200. *. 100. : p<0.05. 0 0. 25. 50. 500. ** **. *. * ** : p<0.001 75. 100. Repolarization (%). 1 Hz APD90. 1 Hz APD50. 400 300. 0 -2 -4 -6 -8 -10 -12 -14. H CAPON Control. 300 200 100. CAPON. 12. 8 p<0.05. pA/pF. 400. 100. Control. -40-30-20-10 0 10 20 30 40 50 60. 500. 200. Co nt ro CA l PO N. 300. G. 0. APD (ms). Control CAPON. 5 pA/pF 50 ms. 100 ms. 400. 5 pA/pF. CAPON-1 Hz. 1 Hz. 0 pA. 25. -6. Control-1 Hz. D APD (ms). 18. 0.05 Hz. 0. CAPON. 0 pA. -12. 1 Hz. Control. GFP. Co nt ro CA l PO N. 300. F Control. APD (ms). APD90 (ms). 400. Peak ICa,L (pA/pF). A. mV. -2 -4 -6 -8 -10 -12 -14. კ 5ǺCAPON ፓ௓ЈԼಒझ୏բႝՏᆶ້ᚆηႝࢬǶ. L-type ້ᚆηႝࢬ ᆶჹྣϐЈԼಒझբКၨǴCAPON ၸࡋ߄ၲ‫ޑ‬ЈԼಒझ‫ځ‬ஏࡋ ܴᡉ෧ϿȐ-7.2 ± 0.5 pA/pF, at +20 mV, nɨ8 vs. -11.0 ± 1.0 pA/pF, at +10 mV, nɨ12; Pɦ0.05ȑ Ȑკ 5 F- Gȑ᏾ᡏԶ‫ق‬Ǵӧ CAPON ၸࡋ߄ ၲ‫ޑ‬ЈԼಒझ້ᚆηஏࡋऊ෧Ͽ 35ʘǴԶ peak current density-voltage relationship (I-V curve) ϩ‫݋‬ᡉҢӧ CAPON ၸࡋ߄ၲ‫ޑ‬ЈԼಒझǴ‫ځ‬ 33.

(49) ጢႝՏவ-30 mV ‫ډ‬+40 mVǴ‫້ځ‬ᚆηႝࢬ೿ܴᡉ‫ޑ‬෧λȐკ 5 Hȑ Ǵ җ‫ܭ‬຾Εಒझ‫້ޑ‬ᚆηႝࢬ෧ϿǴёᏤठ CAPON ၸࡋ߄ၲϐЈԼಒ झ୏բႝՏᕭอǶ. ໊ᚆηႝࢬ ໊ᚆηႝࢬ٠҂‫ ډڙ‬CAPON ၸࡋ߄ၲ‫ޑ‬ቹៜȐკ 6 A-BȑǶ. ӛѦ᏾ࢬϐႇᚆηႝࢬ (Outward Rectifier Potassium Current) ӧϺ‫ޞ‬Ⴕ‫ޑ‬Ј࠻ԼԺಒझԖ‫ٿ‬ᅿ‫ۯ‬ᒨӛѦ᏾ࢬϐႝࢬǴջِೲࢲ ϯ‫܄‬ႇᚆηႝࢬȐIKrȑϷ጗ᄌ‫ࢲ܄‬ϯႇႝࢬȐIKsȑǴٰॄೢಖЗ୏բ ႝՏ‫ޑ‬ѳচයǴӧ௖૸ CAPON ၸࡋ߄ၲӧ೭‫ٿ‬ᅿႝࢬ‫ޑ‬ቹៜǴ‫ॺך‬ ٬Ҕ΋ᅿ੝౦‫ ޑ܄‬IKs ߔᘐᏊ chromanol 293B ٰϩᚆ IKr Ϸ IKsǴ‫ॺך‬ ว౜ӧ capon ၸࡋ߄ၲ‫ޑ‬ЈԼಒझ IKr ‫ޑ‬നε‫׀‬ႝࢬஏࡋᇸ༾ቚу Ȑ0.92 ± 0.07 pA/pF, nɨ10 vs. 0.61 ± 0.08 pA/pF, nɨ7 ; Pɦ0.05ȑ Ȑკ 6 C-Dȑ೭ঁ่݀ᡉҢନΑ້ᚆηႝࢬ෧ϿѦǴIKr ႝࢬ‫ޑ‬΢ϲΨԖշ‫ܭ‬ CAPON ၸࡋЈԼಒझϐ୏բႝՏ‫ޑ‬ᕭอǶ. ӛϣ᏾ࢬϐႇᚆηႝࢬ (Inward Rectifier Potassium CurrentǴIK1) ӛϣ᏾ࢬϐႇᚆηႝࢬ (IK1)٠҂‫ ډڙ‬CAPON ‫ޑ‬ቹៜȐკ 6 Eȑ Ƕ. 34.

(50) A. B. CAPON. Control. pA/pF -80-70-60-50-40-30-20-10 10 20 mV 0 -10. 0 pA. 0 pA. -20. p=NS. -30. 20 pA/pF. Control CAPON. 20 pA/pF 10 ms. 10 ms 5s. + 40 mV. - 40 mV. + chromanol 293B. 2.5. 0.7 pA/pF. E. 2s. 1.5. p<0.05. 1.0. *. 7. 10. 0.5. 7. IK. 10. IKr pA/pF 10 5. -10 -15 -20 -25 -30. Control CAPON. 2s. 7. 10. p=NS. 20 40 60 80. 0.7 pA/pF. p=NS. IKs. -120-100-80 -60 -40 -20-5. + chromanol 293B. CAPON. Control CAPON. p=NS. 2.0. 0.0. Control. -50. D Peak Tail Current (pA/pF). C. -40. mV. კ 6ǺCAPON ፓ௓ЈԼಒझႇᚆηႝࢬǶ. CAPON ᆶ NOS1 ೈқ-ೈқҬϕբҔ ӧዴ‫ ۓ‬full-length CAPON ᆶ short-form CAPON ዴჴӧЈ᠌߄ ၲǴ٠Ъ CAPON ၸࡋ߄ၲ཮೸ၸ෧Ͽ້ᚆηႝࢬǴϷቚம IKr ႝࢬ ٬ளЈԼಒझϐ୏բႝՏᕭอࡕǴ‫ך‬䜹ௗ๱௖૸ CAPON ၸࡋ߄ၲࡕ ೭٤ႝғ౛౜ຝ‫ׯޑ‬ᡂࢂց࿶җፓ௓ NOS-NO ೼৩Ǵ२Ӄ‫ॺך‬٬Ҕ CO-IP Western blot ٰ௖૸ CAPON-NOS ೈқ-ೈқҬϕբҔǴ҅தϺ ‫ޞ‬ႵЈԼಒझ‫֡ޑ‬Ϭషӝ‫ނ‬ǴӃ‫ ک‬anti-CAPON ‫ ܈‬anti-NOS1 ่ӝ‫ޑ‬ Protein G complex խࣝϸᔈࡕа SDS/PAGE ϩᚆǴӧϩձҔ CAPON NOS1 ‫ל‬ᡏѐϸᔈǴ‫ॺך‬ว౜ CAPON ѝ‫ ک‬NOS1Ȑკ 7 A-BȑԶό‫ک‬ 35.

(51) NOS3Ȑკ 7 C-Dȑ‫׎‬ԋխࣝ؈ᐘǴ೭ঁ่݀ᡉҢ CAPON ‫ ک‬NOS1. NOS1. N Be ad s. -C AP O. VM 220. IP. B Be ad s. VM. A. IP -C AP ON. Զόࢂ‫ ک‬NOS3 ԋࣁғ౛‫܄‬ፄӝᡏǶ. CAPON-L. 80 60. 120. 30. CAPON-S. WB: NOS1. WB: CAPON. VM. -C AP O IP. VM. IP -C AP O. N. D N. C. CAPON. NOS3. WB: CAPON. WB: NOS3. კ 7ǺCAPON ᆶ NOS1 ӧЈ᠌և౜ೈқᆶೈқϐҬϕբҔǶ. CAPON ၸࡋ߄ၲ NOS1 ᛙ‫ ۓ‬NOS1 ࣁΑΑှ CAPON ၸࡋ߄ၲਔ NOS1 ೈқӵՖ‫ׯ‬ᡂǴཥϩᚆ‫ޑ‬Ј ࠻ԼԺಒझௗ‫ ڙ‬AdCAPON-GFP ‫ ܈‬AdGFP ϐ in vitro ୷ӢᏤΕǴ‫܈‬ ҂ௗ‫ڙ‬ҺՖ୷ӢᏤΕǴ୷ӢᏤΕ‫ޑ‬ਏ౗ӧ AdGFP ಔऊࣁ 100ʘȐკ 8 Aȑ Ǵӧ AdCAPON-GFP ಔऊࣁ 50ʘȐკ 8 Aȑ Ǵin vitro CAPON ୷Ӣ ᏤΕёஒ CAPON ೈқ‫߄ޑ‬ၲໆቚуࣁ 1.9 ७Ȑკ 8 Bȑ Ǵӧ҂բಒझ ୻Ꭶϐ߻ǴNOS1 ೈқໆӧ AdCAPON-GFP ᏤΕᆶ҂ௗ‫୷ڙ‬ӢᏤΕ‫ޑ‬. 36.

(52) ЈԼಒझࢂ࣬߈‫ޑ‬Ȑკ 8 Cȑ Ǵёࢂӧ࿶ၸ 40.3± 2.3 λਔ‫ޑ‬ಒझ୻Ꭶ ࡕǴNOS1 ೈқӧ҂ௗ‫୷ڙ‬ӢᏤΕᆶௗ‫ ڙ‬AdGFP ᏤΕ‫ޑ‬ЈԼಒझԖ ܴᡉ‫ޑ‬Πफ़ǴԶӧ AdCAPON-GFP ᏤΕ‫ޑ‬ЈԼಒझ߾և౜ᛙ‫ۓ‬ᆶቚ. BF. A. GFP. B. No virus. AdCAPON-GFP. Normalized CAPON. уǴ Ȑ2.78 ± 1.75ʘ vs. 18.54 ± 5.99ʘ, Pɦ0.05ȑ Ȑკ 8 Dȑ Ƕ. 75 50 25 0. Control. CAPON. CAPON GAPDH. AP O VM. D. Ad C. VM. FP. AdCAPON-GFP. Ad G. No virus. N o. C. vi ru s. N -G. FP. AdGFP. NOS1. NOS1. 10.0 7.5 5.0 2.5 0.0. Culture (Hrs). GAPDH Normalized NOS1. Normalized NOS1. GAPDH 30. 10 0. Control. CAPON 0. *. 20. Culture (Hrs). Control. CAPON. 40.3 r 2.3. კ 8ǺӧЈԼಒझ CAPON ၸࡋ߄ၲёᛙ‫ ۓ‬NOS1 ೈқǶ. CAPON ၸࡋ߄ၲЈԼಒझёߦ٬ಒझϣ NO ౢໆቚу ӧΑှЈԼಒझϣ CAPON ᆶ NOS1 ่ӝǴёᛙ‫ ۓ‬NOS1 ࡕǴ‫ך‬ ॺ຾΋‫؁‬௖૸ CAPON ၸࡋ߄ၲ‫ޑ‬ЈԼಒझ‫ځ‬ಒझϣ NO ‫ޑ‬ౢໆࢂց ‫ׯ‬ᡂǴ‫ॺך‬٬Ҕ DAR-4M AM ٰෳ၂Ӹࢲಒझϣϐ NO ‫֖ޑ‬ໆǴ೭٤. 37.

(53) ЈԼಒझࢂӧ in vivo CAPON ୷ӢᏤΕ 3-5 Ϻࡕϩᚆ‫܌‬ளǴDAR-4M AM ‫ޑ‬ᑻӀ੝౦‫܄‬Ӄ࿶ၸуΕ L-NAME ࡕᑻӀ෧১ǴᆶуΕ sodium nitroprusside ᑻӀቚமԶ᛾ჴȐკ 9 AȑǴ‫ॺך‬ௗ๱ӧ baseline ਔᆶу Ε L-arginine ࡕǴКၨಒझϣ‫ ޑ‬NO ౢໆ٠ว౜྽ЈԼಒझуΑ L-arginine ϐࡕǴCAPON ၸࡋ߄ၲ‫ޑ‬ЈԼಒझᑻӀமࡋε‫ܭ‬ჹྣಔ‫ޑ‬ ЈԼಒझǴȐ1420.9 ± 28.1 a.u. nɨ43 vs. 1329.0 ± 19.1 a.u., nɨ166, P. ɦ0.05ȑȐკ 9 B-DȑǶ. A. L-NAME + DAR-4M AM. B. SNP + DAR-4M AM. DAR-4M AM. Baseline. L-arginine. NO. GFP. NO. BF. C. D. NO. p<0.05 1500. *. 1250 1000 750 500 250 0. C on tr ol C A PO C on N tr ol -L C -a A rg PO N -L -a rg. Fluorescent Intensity (A.U.). GFP. კ 9ǺӧЈ᠌ CAPON ၸࡋ߄ၲёගϲЈԼಒझ‫ ޑ‬NO ౢໆǶ. L-NAME ࡠൺ CAPON ၸࡋ߄ၲЇଆ‫୏ޑ‬բႝՏᕭอᆶ້ᚆηႝࢬ ෧Ͽ ӧ 3-5 ϺࡕǴஔ‫ ڙ‬in vivo CAPON ୷ӢᏤΕϐЈԼಒझϩᚆ 38.

(54) ࡕǴуᆶόу 1mM L-NAME ࡕǴӧϩձෳ၂‫୏ځ‬բႝՏᆶ້ᚆηႝ ࢬ‫ޑ‬ቹៜǴ‫ॺך‬ว౜‫ ܭ‬CAPON ၸࡋ߄ၲ‫ޑ‬ЈԼಒझуΑ L-NAME ࡕǴচҁᕭอ‫ ޑ‬APD90 ࡠൺԿᆶჹྣಔಒझ࣬߈‫ ޑ‬APD90Ȑ1 Hz, 348.3 ± 47.8ms, nɨ7 vs. 221.6 ± 19.9 ms, nɨ6, Pɦ0.05ȑ Ȑკ 10 A-Bȑ ǴԶচ ҁ෧Ͽ‫້ޑ‬ᚆηႝࢬΨࡠൺԿჹྣಔಒझ‫ޑ‬НྗȐ-6.2 ± 0.6 pA/pF, n ɨ14 vs. -4.8 ± 0.3 pA/pF, nɨ14; Pɦ0.05ȑ Ȑკ 10 C-EȑǶ. B. 1 Hz. APD90 (ms). 0 Control-L-NAME. 300 200 7. 50 ms. Control-L-NAME. CAPON-L-NAME. -6 -8. N A. M E. M E -L -. C. CAPON-L-NAME. -4. CAPON-L-NAME. Control-L-NAME. 70. A PO N. tr. ol -L. -N A. A PO N. ol. C. tr. Control-L-NAME. 4 pA/pF 50 ms. 50. 14. 14. 14. 14. mV. E. -2. 0 pA. 4 pA/pF. 30. CAPON. pA/pF 0 pA. 10 0. CAPON. CAPON. 7. Control. 50 ms. Control. 5. 0 C on. Control 50 ms. 4 pA/pF. 6. D -50 -30 -10. 4 pA/pF. **. C on. 100 ms 0 pA. *. 400. CAPON. 0 pA. p<0.05. 100. Control CAPON-L-NAME. C. p<0.01 500. Peak ICa,L (pA/pF). 20 mV. A. 0 -1 -2 -3 -4 -5 -6 -7 -8. *. p<0.001. **. p<0.05. კ 10ǺL-NAME ё࡮‫ל‬Ӣ CAPON ၸࡋ߄ၲЇଆЈԼಒझ୏բႝՏ ᕭอᆶ້ᚆηႝࢬᡂλǶ. 39.

(55) ಃΒ࿯ ᖏ‫زࣴ׉‬ Methadone Ꮚໆ‫ک‬Јႝკ QTcɡCross Section ϩ‫݋‬ ӧ೭΋೽ҽϩ‫္݋‬य़ӅԖ 203 ՏੇࢶӢԋ᠅҅ӧௗ‫ ڙ‬Methadone ‫ݯ‬ᕍ‫ੰޑ‬ΓǴдॺ‫୷ޑ‬ҁၗ਑ӵ߄ 2 ‫܌‬ҢǴ‫܄ت‬Ԗ 164 ΓǴζ‫܄‬Ԗ 39 ΓǴԃइϩձࣁ‫ ܄ت‬38.9 ± 7.1 ྃǴζ‫ ܄‬35.2 ± 6.7 㹺Ǵௗ‫ڙ‬ Methadone ‫ݯ‬ᕍ‫ޑ‬ᏊໆǴ‫ ܄ت‬48.6 ± 25.6 mgǴζ‫ ܄‬44.6 ± 25.7 mgǶ ߄ 2Ǻcross-section ੰΓಔ‫୷ޑ‬ҁၗ਑Ƕ All炷N=203炸 Variables. Male炷N=164炸 Female炷N=39炸. p-value. meanƲSD. meanƲSD. meanƲSD. 38.2 Ʋ 7.2. 38.9 Ʋ 7.1. 35.2 Ʋ 6.7. 0.0029b. 26 Ʋ 7.2. 26 Ʋ 7.4. 26 Ʋ 6.7. 0.9962 b. 430.1 Ʋ 22.0. 429.1 Ʋ 21.7. 434.5 Ʋ 23.0. 0.1621 b. Normal, N炷炴炸. 116炷57.1炸. 86炷52.4炸. 30炷76.9炸. 0.0055 c. Abnormala,N炷炴炸. 87炷42.9炸. 78炷47.6炸. 9炷23.1炸. Methadone dose炷mg炸. 47.8 Ʋ 25.6. 48.6 Ʋ 25.6. 44.6 Ʋ 25.7. 0.3819 b. Heroin addiction炷years炸. 12.2 Ʋ 6.1. 12.9 Ʋ 6.0. 9.2 Ʋ 6.0. 0.0005 b. 246 Ʋ 191.3. 254.3 Ʋ 194.6. 211 Ʋ 174.8. 0.2045 b. Age炷year炸 First use of heroin炷age, years炸 QTc炷ms炸. MMT炷days炸. a烉 Female and QTc烍450 ms烊male and QTc烍430 ms b烉 Two sample t-test c烉 Chi-square test. ӧ‫ ܄ت‬Methadone Ꮚໆ‫ک‬Јႝკ‫ ޑ‬QTC և౜ᡉ๱‫࣬҅ޑ‬ᜢȐკ 11ȑǴԶӧζ‫ੰ܄‬Γ߾ؒԖ࣬ᜢ‫܄‬Ȑკ 12ȑǴӧ‫ੰ܄ت‬ΓǴൂᡂ࣬ᆶ ӭᡂ࣬ϩ‫݋‬೿ᡉҢ Methadone Ꮚໆࢂ‫ ک‬QTc ‫ߏۯ‬നᡉ๱࣬ᜢ‫ޑ‬Ӣન Ȑ߄ 3ȑ Ǵӧζ‫ੰ܄‬ΓǴόᆅҔൂᡂ࣬ᆶӭᡂ࣬ϩ‫݋‬೿ᡉҢ Methadone. 40.

(56) Ꮚໆࢂ‫ ک‬QTc ‫ؒߏۯ‬Ԗᡉ๱‫࣬ޑ‬ᜢ‫܄‬Ȑ߄ 4ȑǶ. Male: Dose-dependent Correlation. Female: Dose-dependent Correlation. mean methadone dose 48.6 mg. mean methadone dose 44.6 mg. კ 11ȐѰȑǺmethadone Ꮚໆᆶ QTc ӧ‫܄ت‬և౜҅࣬ᜢǶ კ 12ȐѓȑǺmethadone Ꮚໆᆶ QTc ӧζ‫܄‬όև҅࣬ᜢ‫܄‬Ƕ. ߄ 3Ǻcross-section ϩ‫݋‬Ǵ‫ੰ܄ت‬Γ‫ ޑ‬methadone Ꮚໆᆶ QTc և҅ ࣬ᜢ‫܄‬Ƕ. Positive Dose-dependent Association Between Methadone and QTc in Male Subjects Male炷N=164炸. Univariate analysis. Univariate analysis. Beta. p-value. Beta. p-value. Methadone dose炷mg炸. 0.165. 0.012. 0.187. 0.005. Age炷years炸. 0.373. 0.119. 0.555. 0.033. First use of heroin炷age, years炸 0.392. 0.090. Heroin addiction炷years炸. -0.076. 0.793. -0.327. 0.282. MMT炷days炸. 0.006. 0.503. 0.004. 0.667. 41.

(57) ߄ 4Ǻcross-section ϩ‫݋‬Ǵζ‫ੰ܄‬Γ‫ ޑ‬methadone Ꮚໆᆶ QTc όև҅ ࣬ᜢ‫܄‬Ƕ. Negative Dose-dependent Association Between Methadone and QTc in Female Subjects Female炷N=39炸. Univariate analysis. Univariate analysis. Beta. p-value. Beta. p-value. Methadone dose炷mg炸. 0.093. 0.529. 0.191. 0.278. Age炷years炸. 0.413. 0.467. 0.935. 0.211. First use of heroin炷age, years炸 0.492. 0.384. Heroin addiction炷years炸. -0.099. 0.875. -0.545. 0.472. MMT炷days炸. -0.007. 0.760. 0.004. 0.853. ! Methadone Ꮚໆ‫ک‬Јႝკ QTcɡLongitudinal ϩ‫݋‬ ӧ೭΋೽ҽϩ‫္݋‬य़ӅԖ 55 ՏੇࢶӢԋ᠅҅ӧௗ‫ ڙ‬Methadone ‫ݯ‬ᕍ‫ੰޑ‬ΓǴдॺ‫୷ޑ‬ҁၗ਑ӵ߄ 5 ‫܌‬ҢǴ‫܄ت‬Ԗ 46 ΓǴζ‫܄‬Ԗ 9 ΓǴԃइϩձࣁ‫ ܄ت‬37.5 ± 7.0 ྃǴζ‫ ܄‬33.1 ± 5.1 㹺Ǵ‫܄ت‬ӧௗ‫ڙ‬ Methadone ‫ݯ‬ᕍ߻‫ ޑ‬QTc ࢂ 427.0 ± 23.4 msǴ‫ݯ‬ᕍ΋ঁДࡕȐ55.3 ± 24.0 mgȑ ǴQTc ᡉ๱‫ ࣁߏۯ‬431.0 ± 21.3 ms ȐPɨ0.026ȑ Ȑკ 13ȑ ǴԶ ζ‫܄‬ӧௗ‫ ڙ‬Methadone ‫ݯ‬ᕍ߻‫ ޑ‬QTc ࢂ 437.6 ± 32.9 msǴ‫ݯ‬ᕍ΋ঁД ࡕȐ52.5 ± 23.1 mgȑ ǴQTc ٠คᡉ๱ᡂϯࣁ 420.5 ± 22.4 msȐPɨ0.297ȑ Ȑკ 14ȑ Ƕ. 42.

(58) ߄ 5ǺLongitudinal ϩ‫݋‬ಔੰΓ‫ޑ‬ၗ਑Ƕ. Baseline Characteristics of Study Subjects Table 1. When time = 0 All炷N=55炸 Male炷N=46炸 Female炷n=9炸 p-valuea. Variables Age炷years炸. 36.8 ± 6.9. 37.5 ± 7.0. 33.2 ± 5.1. 0.096. 25 ± 7.1. 24.8 ± 7.3. 26.4 ± 6.2. 0.32. Heroin addiction炷years炸. 11.7 ± 6.6. 12.7 ± 6.5. 6.8 ± 4.7. 0.007. MMT炷days炸. 25 ± 94.9. 29.9 ± 103.3. 23.2 ± 102.3. 0.316. First use of heroin炷age, years炸. a烉Wilcoxon rank sum test. Methadone dose. 55.3 Ʋ 24.0. 61.6 Ʋ 28.0. Methadone dose. Significant QTc Prolongation After Methadone Treatment in Male. 52.5 Ʋ 23.1. 60.0 Ʋ 28.3. Insignificant QTc Change After Methadone Treatment in Female. კ 13ȐѰȑ ǺLongitudinal ϩ‫݋‬Ǵ‫ੰ܄ت‬Γӧ methadone ‫ݯ‬ᕍࡕ QTc ᡉ๱‫ߏۯ‬Ƕ კ 14Ȑѓȑ ǺLongitudinal ϩ‫݋‬Ǵζ‫ੰ܄‬Γӧ methadone ‫ݯ‬ᕍࡕ QTc ؒԖᡉ๱‫ߏۯ‬Ƕ. 43.

(59) CAPON ୷Ӣᡂ౦ǵMethadone ᏊໆᆶЈႝკ QTc ‫୷؁߃ॺך‬Ӣ᠘‫ ۓ‬24 ঁ CAPON SNPsǴхࡴ rs10494366ǵ rs10918594ǵrs1415263 ฻Ǵٰ࣮஥Ԗ CAPON SNPs ‫ࢂޣ‬ց຾΋‫؁‬у ம Methadone ჹ QTC ‫ޑ‬ቹៜǴ‫ॺך‬а 111 Տੇࢶमԋ᠅Ⴃഢௗ‫ڙ‬ methadone ‫ݯ‬ᕍ‫ ޑ‬101 Տ‫ੰ܄ت‬Γ଺ϩ‫݋‬ǴӧᗋؒԖௗ‫ ڙ‬methadone ‫ݯ‬ᕍ߻Ǵа rs10918594CC ࣁ reference (frequency ࣁ 52.81%)Ǵ‫ ځ‬QTc ࣁ 421.9± 23.8 msǴ஥Ԗ rs10918594CG ‫(ޣ‬frequency ࣁ 16.85%)Ǵ‫ځ‬ QTc ‫ ߏۯ‬7.46 msȐpɨ0.284ȑ ǴԶ஥Ԗ rs10918594GG ‫(ޣ‬frequency ࣁ 30.34%)Ǵ‫ ځ‬QTc ຾΋‫ ߏۯ؁‬11.60 msȐpɨ0.040ȑ ǴᡉҢ rs10918594 ୷Ӣᡂ౦ᆶ QTc ‫ߏۯ‬Ǵ‫ڀ‬Ԗ dose-dependent effectǴԶќ΋๱Ӝ SNPǵ rs10494366 ӧኻऍ௼ဂ‫ ک‬QTc ‫ڀߏۯ‬Ԗߚதᡉ๱࣬ᜢ‫୷ޑ܄‬Ӣᡂ ౦ǴӧҞ߻‫زࣴॺך‬௼ဂǴ߾ό‫ڀ‬Ԗ‫ ک‬QTc ‫࣬ޑߏۯ‬ᜢ‫܄‬ǶӧܺҔ methadoneȐ48.6 ± 10.3 mgȑ 36 ± 10 ϺࡕǴ஥Ԗ rs10918594CG ‫ޣ‬Ǵ ‫ ځ‬QTc ‫ޑ‬ᡂϯόܴᡉȐ-0.88 msǴpɨ0.930ȑ ǴԶ஥Ԗ rs10918594GG ‫ޣ‬Ǵ‫ ځ‬QTc ຾΋‫ ࣁߏۯ؁‬15.41 msȐpɨ0.0486ȑ ǴԜ่݀ᡉҢ CAPON ୷Ӣᡂ౦Ȑrs10918594ȑу΢ methadone ٬ҔǴჹ QTc ‫ڀߏۯ‬Ԗуԋ բҔȐ߄ 6ȑǶ. 44.

(60) ߄ 6ǺCAPON ୷Ӣᡂ౦ǵmethadone ‫ ک‬QTc ‫࣬ޑ‬ᜢ‫܄‬Ƕ. CAPON gene variants, methadone and QTc Variables. Before Methadone 95% CI. After Methadone. n. QTc. p. rs1964052_CC. 63. rs1964052_CT. 26. 14. rs1964052-TT. 1. reference 7.6 炷-3.2 , 18.3炸 NS 20.2 炷-26.3 , 66.8炸 NS. 1. -0.4 炷-15.9 , 15.1炸 NS 8.5 炷-42.2 , 59.2炸 NS. rs10918594_CC rs10918594_CG rs10918594_GG. 47 15 27. reference 7.5 炷-6.3 , 21.2炸 0.284 11.6 炷0.6 , 22.6炸 0.040. 34 7 14. -0.9 炷-20.8 , 19.0炸 NS 15.4 炷0.1 , 30.7炸 0.0486. rs10494366_GG. 48. 26. rs10494366_GT rs10494366_TT. 35 7. reference -6.9 炷-17.2 , 3.4炸 NS -4.9 炷-23.7 , 13.8炸 NS. rs348624_CC rs348624_CT. 61 22. rs348624_TT. 1. reference 8.4 炷-3.4 , 20.2炸 NS 20.7 炷-27.2 , 68.5炸 NS. 45. n. QTc. 95% CI. p. 40. 24 5 41 13 1. -6.0 炷-20.1 , 8.2炸 NS -4.0 炷-28.4 , 20.5炸 NS -0.7 炷-16.4 , 15.0炸 NS 9.2 炷-40.6 , 58.9炸 NS.

(61) ಃѤക ૸ፕ ಃ΋࿯ ୷ᘵࣴ‫ز‬ ‫ॺך‬२ࡋ᛾ჴ CAPON ೭΋ঁӧတઓ࿶ಔᙃ࣬྽ख़ा‫ ޑ‬NOS1 ‫ޑ‬ adaptor/regulator ೈқΨӧЈ᠌߄ၲ٠Ъ‫ ک‬NOS1 ҬϕբҔǴ྽ CAPON ၸࡋ߄ၲਔ཮ࢲϯ NOS1-NO ૻ৲໺ሀၡ৩Ǵ٬ள້ᚆηႝ ࢬ෧λǴIkr ႝࢬቚуǴᏤठ୏բႝՏᕭอǶ. ϣྍ‫ ܄‬CAPON ‫ܭ‬Ј᠌߄ၲ Ծவ 1998 ԃ CAPON ӧεႵတઓ࿶೏ว౜аࡕǴXu. et al. ຾΋ ‫؁‬᛾ჴ CAPON ೈқԖ‫ٿ‬ᅿ isooformsǺfull-length CAPON ᆶ short-form C terminus CAPONǴfull-length CAPON ֖Ԗ 10 ঁ exonsǴ٠‫׎‬ԋ PTBᆶ PDZ-binding dormainsǴ‫ॺך‬Ψ२ࡋ᛾ჴ೭‫ٿ‬ᅿ CAPON isoforms ೿ӧЈ᠌߄ၲǴಒझխࣝᑻӀᡉҢ CAPON ೈқϩ೽‫ܭ‬ಒझ፦Ǵ٠Ъ ӧಒझਡ‫ڬ‬ൎᆶಒझጢԖቚம౜ຝǶ. CAPON ၸࡋ߄ၲࢲϯ NOS1-NO ၡ৩ ࣁΑ᛾ჴ CAPON ၸࡋ߄ၲૈࢲϯ NOS1-NO ၡ৩Ǵ٠Ъ೷ԋಒ झႝғ౛‫ׯޑ‬ᡂǴ‫ॺך‬٬Ҕ Co-IP Western blot ᛾ჴ CAPON NOS1 ‫ڀ‬ Ԗೈқ-ೈқҬϕբҔǴ೭ᅿҬϕբҔΨ࿶җ٬ҔӅ೫ขᡉ༾᜔ᔠಒ झխࣝᑻӀࢉՅǴᡉҢ CAPON ᆶ NOS1 և౜ಒझϣ colocalization ‫ޑ‬. 46.

(62) ౜ຝȐკ 15ȑ ǴԖ፪‫ࢂޑ‬ѝԖ full-length CAPON ωᆶ NOS1 ԖҬϕբ ҔǴ೭‫ک‬ၸѐӧတઓಒझᢀჸ‫ޑډ‬౜ຝ΋ठǴௗ๱‫ॺך‬٬ҔᡏѦ୷Ӣ ᏤΕᆶಒझ୻Ꭶ‫ޑ‬БԄǴ᛾ჴ CAPON ၸࡋ߄ၲёаᛙ‫ ۓ‬NOS1ǴԜ 㵝 CAPON ᆶ NOS1 ӧЈ᠌ϐҬϕբҔග‫ޔࣁ׳ٮ‬ௗϐ᛾ᏵǴനࡕ‫ך‬ ॺ٬Ҕ DAR-4M AM ٰ‫ޔ‬ௗෳ၂ࢲᡏЈԼಒझϣ NO ‫ޑ‬ౢໆǴ٠ว౜ CAPON ၸࡋ߄ၲಒझϣ NO ౢໆ΢ϲǴᗨฅКଆჹྣಔЈԼಒझ CAPON ၸࡋ߄ၲѝࢂ೷ԋϿ೚ NO ౢໆቚуȐ7 ʘȑ Ǵՠҗ‫ ܭ‬NO ‫ޑ‬ ғ౛բҔǴᆶॄೢౢғ NO ‫ ޑ‬NOS ‫܌‬ӧ‫ޑ‬Տ࿼৲৲࣬ᜢǴԶ CAPON ࡐёૈ‫ ע‬NOS ஥‫ډ‬੝‫ޑۓ‬኱‫ޑ‬ೈқǴ٬ NO วච੝‫ޑۓ‬բҔǴќѦ җ‫ ܭ‬CAPON ૈ୼‫ځک‬дೈқᝡ‫ ݾ‬PDZ-bindingǴCAPON ΨԖёૈ࿶ җԜ΋ᐒᙯՉ٬‫ځ‬ғ౛բҔǶ. კ 15ǺӅ೫ขᡉ༾᜔ᔠಒझխࣝᑻӀࢉՅǴᡉҢ CAPON ᆶ NOS1 և౜ಒझϣ colocalization ‫ޑ‬౜ຝǶ. 47.

(63) CAPON ၸࡋ߄ၲ೸ၸ‫້ڋ׭‬ᚆηႝࢬᆶගϲ Ikr ٰᕭอ୏բႝՏ җ‫ܭ‬ϺไႵ‫ޑ‬ЈԼಒझલϿ Ito ႝࢬǴਥᏵҁჴᡍ่݀Ǵ້ᚆη ႝࢬ෧Ͽ 35ʘǴIkr ႝࢬቚу 50ʘǴς‫ى‬аှញ୏բႝՏᕭอ 30ʘӧ CAPON ၸࡋ߄ၲ‫ޑ‬ЈԼಒझǶ. L-NAME ёࡠൺ CAPON ၸࡋ߄ၲ‫܌‬Їଆ‫ޑ‬ႝғ౛ᡂϯ ‫ॺך‬ϩձу L-NAME ၸࡋၲ‫ޑ‬ЈԼಒझᆶჹྣಔ‫ޑ‬ЈԼಒझǴ ่݀ว౜ L-NAME ‫ ܭ‬CAPON ၸࡋ߄ၲ‫ޑ‬ЈԼಒझ‫ ߏۯܭ‬APD90 Ȑ57 ʘ vs. 13ʘȑᆶගϲ້ᚆηႝࢬȐ29ʘ vs.5ʘȑ‫ޑ‬மࡋ֡ၨჹྣಔЈ Լಒझ‫ࣁ׳‬ᡉ๱ǴӵԜё௨ନ L-NAME ‫ ޑ‬background effectǴ٠᛾ჴ CAPON ၸࡋ߄ၲ೷ԋ‫ޑ‬ႝғ౛‫ׯ‬ᡂዴჴࢂ೸ၸ NOS1-NO ೼৩‫܌‬ ठǴԿ‫ ܭ‬NO ӵՖ࿶җ‫້ڋ׭‬ᚆηႝࢬϷගϲ Iks ႝࢬٰᕭอ୏բႝ ՏǴၸѐࣴ‫ز‬ᡉҢ NO ёа೸ၸ cGMP-dependent ᆶ cGMP-independent ‫ޑ‬೼৩ٰ‫ڋ׭‬ಒझጢ້ᚆηႝࢬǴ࣬ӕ‫ ޑ‬NO Ψёૈ೸ၸ࣬ӕ‫ޑ‬ᐒ ‫ڋ‬Ǵቹៜಒझጢ Ikr ႝࢬǴਥᏵ Burkard ‫زࣴޑ‬ᡉҢӧ NOS1 ၸࡋ߄ၲ ‫୷ޑ‬Ӣᙯ෗ႵǴӧ NOS1 ၸࡋ߄ၲ‫ޑ‬ЈԼಒझ‫້ځ‬ᚆηႝࢬ෧Ͽ 39 ʘǴ೭ࢂӢࣁ NOS1 ೏஥‫ډ‬ಒझጢ٠‫ ک‬L-type ້ᚆη೯ၰೈқҬϕ բҔ‫܌‬ठǴӢԜ‫ࡐॺך‬Ԗ౛җ࣬ߞ྽ CAPON ၸࡋ߄ၲਔǴஒ NOS1 ஥‫ډ‬ಒझጢ٠ᆶಒझጢ‫ޑ‬ᚆηೈқҬϕբҔǴ٬ள້ᚆηႝࢬ෧Ͽᆶ Ikr ႝࢬቚуǴԶᏤठ୏բႝՏᕭอǶ 48.

(64) ಃΒ࿯ ᖏ‫زࣴ׉‬ 1ȑ Methadone Ꮚໆᆶ QTc ᜢ߯ ‫ॺך‬ว౜όᆅவ cross-section ‫ ܈‬longitudinal ϩ‫݋‬Ǵ‫ੰ܄ت‬Γ‫ ޑ‬QTc ‫ ک‬methadone Ꮚໆ೿և౜ࡐᡉ๱‫࣬҅ޑ‬ᜢ‫܄‬ǴԶζ‫ੰ܄‬Γ߾คԜ ౜ຝǴԖ፪‫ࢂޑ‬Ҟ߻Ў᝘ൔ֋ methadone Ꮚໆ‫ ک‬QTc ‫ߏۯ‬և౜҅ ࣬ᜢ‫܄‬Ǵ‫܌ځ‬٬Ҕ‫ޑ‬ѳ֡ኧ‫܈‬ύՏኧ methadone Ꮚໆ೿ӧ 100 mg а΢ǴԶӧҁࣴ‫ز‬ύ‫ੰ܄ت‬Γ٬Ҕ methadone ‫ޑ‬ѳ֡ᏊໆѝԖ 48.6 mgǴ೭ࢂಃ΋ጇ‫زࣴޑ‬ᡉҢեᏊໆ‫ ޑ‬methadone ϝฅᆶ QTc ‫ۯޑ‬ ߏԖ҅࣬ᜢ‫܄‬ǴԜѦǴmethadone Ꮚໆᆶ QTc ‫ࢂߏۯ‬ցӸӧ‫܄‬ձ‫ޑ‬ ৡ౦ǴFanoe ӧдॺ‫ ޑ‬cross-section ࣴ‫ز‬ύࡰрǴ‫ੰ܄ت‬Γ‫࣬ޑ‬ᜢ ߯ኧ correlation coefficient ࣁ 0.28ǴԶζ‫܄‬ѝԖ 0.12Ǵ՟Я೸៛р ௗ‫ ڙ‬methadone ‫ݯ‬ᕍ‫ੰ܄تޑ‬Γၨ৒ܰр౜ QTc ‫ߏۯ‬ǴԶ‫ޑॺך‬ ࣴ‫ࢂز‬२ࡋ٬Ҕ cross-section Ϸ longitudinal ‫ޑ‬ϩ‫݋‬Ǵࡰр‫܄ت‬К ζ‫܄‬ӧௗ‫ ڙ‬methadone ‫ݯ‬ᕍࡕ‫ܰ׳‬р౜ QTc ‫ߏۯ‬Ǵ೭‫ک‬໺಍΢ζ ‫ੰ܄‬Γၨ৒ܰЇଆᛰ‫ ܄ނ‬QTc ‫ޑߏۯ‬ᇡ‫ޕ‬Ԗ‫܌‬όӕǶ 2ȑ CAPON ୷Ӣᡂ౦ǵmethadone ᆶ QTc ᜢ߯ ‫ॺך‬ਥᏵЎ᝘ൔ֋ᑔᒧ 24 ঁёૈၟ QTc ‫ߏۯ‬Ԗᜢ‫ ޑ‬CAPON ୷Ӣ ᡂ ౦ Ǵ ߃ ‫ ؁‬ว ౜ rs10918594GG ‫ ୷ ޑ‬Ӣ ᡂ ౦ Ȑ wild type ࣁ rs10918594CC ȑǴ ᆶ QTc ‫ ڀ ߏ ۯ‬Ԗ ᡉ ๱ ‫ ࣬ ޑ‬ᜢ ‫ ܄‬Ǵ ೭ ٤ ஥ Ԗ. 49.