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Kunesidingo sesistimu ye-in vitro ethembekile engaphinda ikhiqize ngokunembile indawo yomzimba yenhliziyo ukuze kuhlolwe izidakamizwa. Ukutholakala okulinganiselwe kwezinhlelo zokukhulisa izicubu zenhliziyo zabantu kuholele ekuchazeni okungalungile kwemiphumela yemithi yenhliziyo. Lapha, sithuthukise imodeli yokukhulisa izicubu zenhliziyo (i-CTCM) evuselela ngogesi izingcezu zenhliziyo futhi idlule ekunwetshweni ngokomzimba ngesikhathi sezigaba ze-systolic kanye ne-diastolic zomjikelezo wenhliziyo. Ngemva kwezinsuku ezingu-12 zokukhulisa, le ndlela yathuthukisa kancane ukusebenza kwezingxenye zenhliziyo, kodwa ayizange ilondoloze ngokugcwele ubuqotho bazo besakhiwo. Ngakho-ke, ngemva kokuhlolwa kwama-molecule amancane, sithole ukuthi ukungezwa kwe-100 nM triiodothyronine (T3) kanye ne-1 μM dexamethasone (Dex) endaweni yethu yokuxhumana kwagcina isakhiwo esincane sezingxenye izinsuku ezingu-12. Ngokuhambisana nokwelashwa kwe-T3/Dex, uhlelo lwe-CTCM lugcine amaphrofayili okubhala, ukusebenza, umsebenzi we-metabolic, kanye nobuqotho besakhiwo ezingeni elifanayo nezicubu zenhliziyo ezintsha izinsuku ezingu-12. Ngaphezu kwalokho, ukwelula kakhulu kwezicubu zenhliziyo emasikweni kubangela izimpawu zenhliziyo ezihambisana ne-hypertrophic, okunikeza ubufakazi bekhono le-CTCM lokulingisa izimo ezihambisana ne-hypertrophic ezibangelwa ukwelula kwenhliziyo. Ekuphetheni, i-CTCM ingalingisa i-physiology kanye ne-pathophysiology yenhliziyo esikweni isikhathi eside, okwenza kube lula ukuhlolwa kwemithi okuthembekile.
Ngaphambi kocwaningo lwezokwelapha, kudingeka izinhlelo ezithembekile ze-in vitro ezingaphinda zikhiqize ngokunembile indawo yomzimba yenhliziyo yomuntu. Izinhlelo ezinjalo kufanele zilingise ukwelula okushintshiwe kwemishini, izinga lokushaya kwenhliziyo, kanye nezakhiwo ze-electrophysiological. Amamodeli ezilwane avame ukusetshenziswa njengeplatifomu yokuhlola i-physiology yenhliziyo enokwethenjelwa okulinganiselwe ekuboniseni imiphumela yemithi enhliziyweni yomuntu1,2. Ekugcineni, i-Ideal Cardiac Tissue Culture Experimental Model (CTCM) iyimodeli ezwela kakhulu futhi eqondile ekungeneleleni okuhlukahlukene kokwelapha kanye nemithi, iphinda ikhiqize ngokunembile i-physiology kanye ne-pathophysiology yenhliziyo yomuntu3. Ukungabikho kwesistimu enjalo kunciphisa ukutholakala kwezindlela ezintsha zokwelapha ukwehluleka kwenhliziyo4,5 futhi kuholele ekubeni nesifo senhliziyo nezidakamizwa njengesizathu esikhulu sokuphuma emakethe6.
Eminyakeni eyishumi edlule, imithi eyisishiyagalombili engeyona eyenhliziyo nemithambo yegazi iye yahoxiswa ekusetshenzisweni kwezokwelapha ngoba ibangela ukwelulwa kwesikhathi se-QT okuholela ekushayweni kwenhliziyo kanye nokufa okuzumayo7. Ngakho-ke, kunesidingo esikhulayo samasu okuhlola angaphambi kokwelashwa athembekile ukuhlola ukusebenza kahle kwenhliziyo kanye nobuthi. Ukusetshenziswa kwamuva nje kwama-cardiomyocytes atholakala kumaseli enhliziyo abangelwa abantu (ama-hiPS-CM) ekuhlolweni kwezidakamizwa nasekuhlolweni kobuthi kunikeza ikhambi eliyingxenye yale nkinga. Kodwa-ke, isimo sokungavuthwa kwama-hiPS-CM kanye nokuntuleka kobunzima bamaseli amaningi ezicubu zenhliziyo kuyimingcele emikhulu yale ndlela. Izifundo zamuva zibonise ukuthi lo mkhawulo unganqotshwa ngokwengxenye ngokusebenzisa ama-hiPS-CM okuqala ukwakha ama-hydrogel ezicubu zenhliziyo ngokushesha nje ngemva kokuqala kokufinyela okuzenzakalelayo nokukhulisa kancane kancane ukukhuthazwa kukagesi ngokuhamba kwesikhathi. Kodwa-ke, lawa ma-microtissue e-hiPS-CM awanazo izakhiwo ze-electrophysiological ezivuthiwe kanye ne-contractile ze-myocardium yabantu abadala. Ngaphezu kwalokho, izicubu zenhliziyo yomuntu zinesakhiwo esiyinkimbinkimbi kakhulu, esakhiwe yingxube ehlukahlukene yezinhlobo ezahlukene zamaseli, okuhlanganisa amaseli e-endothelial, ama-neurons, nama-stromal fibroblast, axhunywe ngamasethi athile amaprotheni e-extracellular matrix. Lokhu kungafani kwezinhlobo ezingezona eze-cardiomyocyte11,12,13 enhliziyweni yabantu abadala abancelisayo kuyisithiyo esikhulu ekubumbeni izicubu zenhliziyo kusetshenziswa izinhlobo zamaseli ngazinye. Lezi mikhawulo ezinkulu zigcizelela ukubaluleka kokuthuthukisa izindlela zokukhulisa izicubu ze-myocardial ezingaguquki ngaphansi kwezimo zomzimba neze-pathological.
Izingxenye ezincane ezikhuliswe kahle (300 µm) zenhliziyo yomuntu ziye zabonakala ziyimodeli ethembisayo ye-myocardium yomuntu engashintshi. Le ndlela inikeza ukufinyelela ohlelweni oluphelele lwe-3D multicellular olufana nezicubu zenhliziyo yomuntu. Kodwa-ke, kuze kube ngu-2019, ukusetshenziswa kwezingxenye zenhliziyo ezikhuliswe kahle kwakunqunyelwe ukusinda okufushane (amahora angu-24) kwesiko. Lokhu kungenxa yezici eziningana ezihlanganisa ukuntuleka kokunwebeka ngokomzimba-kwemishini, isikhombimsebenzisi somoya-uketshezi, kanye nokusetshenziswa kwemidiya elula engasekeli izidingo zezicubu zenhliziyo. Ngo-2019, amaqembu amaningana ocwaningo abonise ukuthi ukufaka izici zemishini ezinhlelweni zesiko lezicubu zenhliziyo kungandisa impilo yesiko, kuthuthukise ukubonakaliswa kwenhliziyo, futhi kulingise isifo senhliziyo. Izifundo ezimbili ezinhle kakhulu 17 no-18 zibonisa ukuthi umthwalo we-uniaxial mechanical unomthelela omuhle ku-phenotype yenhliziyo ngesikhathi sesiko. Kodwa-ke, lezi zifundo azisebenzisanga umthwalo we-physico-mechanical onamandla amathathu womjikelezo wenhliziyo, njengoba izingxenye zenhliziyo zazigcwele amandla okudonsa e-isometric 17 noma umthwalo we-auxotonic oqondile 18. Lezi zindlela zokwelula izicubu zaholela ekucindezelweni kwezakhi zofuzo eziningi zenhliziyo noma ukubonakaliswa ngokweqile kwezakhi zofuzo ezihambisana nezimpendulo ezingavamile zokwelula. Okuphawulekayo ukuthi, uPitoulis et al. 19 bathuthukise i-dynamic heart slice culture bath yokwakhiwa kabusha komjikelezo wenhliziyo besebenzisa impendulo ye-force transducer kanye nokushayela kokucindezeleka. Nakuba lolu hlelo luvumela ukumodelwa komjikelezo wenhliziyo okunembe kakhudlwana, ubunzima kanye nokusetshenziswa okuphansi kwendlela kunciphisa ukusetshenziswa kwalolu hlelo. Ilabhorethri yethu isanda kuthuthukisa uhlelo lokukhulisa olulula olusebenzisa ukuvuselela kukagesi kanye nendawo elungiselelwe ukugcina ukusebenza kwezingxenye zezicubu zenhliziyo yengulube neyabantu kuze kube yizinsuku eziyi-6.
Embhalweni wamanje, sichaza imodeli yokukhuliswa kwezicubu zenhliziyo (i-CTCM) sisebenzisa izingxenye zenhliziyo yengulube ezihlanganisa izinkomba ze-humoral ukuze kuphindwe kabili i-physiology yenhliziyo enezinhlangothi ezintathu kanye nokwanda kwe-pathophysiological ngesikhathi somjikelezo wenhliziyo. Le CTCM ingandisa ukunemba kokubikezela izidakamizwa zangaphambi kokwelashwa kuze kube sezingeni elingakaze lifinyelelwe ngaphambili ngokuhlinzeka ngesistimu yenhliziyo engabizi kakhulu, ephakathi nendawo elingisa i-physiology/pathophysiology yenhliziyo yezilwane ezincelisayo ukuze kuhlolwe izidakamizwa zangaphambi kokwelashwa.
Izimpawu ze-hemodynamic mechanical zidlala indima ebalulekile ekugcineni umsebenzi we-cardiomyocyte ku-vitro 22,23,24. Embhalweni wamanje, sakhe i-CTCM (Isithombe 1a) esingalingisa indawo yenhliziyo yomuntu omdala ngokubangela kokubili ukukhuthazwa kagesi kanye nokwemishini kumaza okuphila (1.2 Hz, ama-beats angu-72 ngomzuzu). Ukuze kugwenywe ukwelula kwezicubu ngokweqile ngesikhathi se-diastole, kwasetshenziswa idivayisi yokuphrinta ye-3D ukwandisa usayizi wezicubu ngo-25% (Isithombe 1b). Ukuhamba kukagesi okubangelwa uhlelo lwe-C-PACE kwanqunywa ukuthi kuqale nge-100 ms ngaphambi kwe-systole kusetshenziswa uhlelo lokuthola idatha ukuze kukhiqizwe ngokugcwele umjikelezo wenhliziyo. Uhlelo lokukhulisa izicubu lusebenzisa i-actuator ye-pneumatic ehlelwe kahle (LB Engineering, Germany) ukuze kwandiswe ulwelwesi lwe-silicone oluguquguqukayo ukuze kubangele ukwanda kwezingcezu zenhliziyo ekamelweni eliphezulu. Uhlelo lwaluxhunywe emgqeni womoya wangaphandle nge-transducer yokucindezela, okwenza kwaba nokwenzeka ukulungisa ngokunembile ingcindezi (± 1 mmHg) nesikhathi (± 1 ms) (Isithombe 1c).
a Namathisela ingxenye yezicubu endandatho yokusekela engu-7 mm, eboniswe ngombala oluhlaza okwesibhakabhaka, ngaphakathi kwegumbi lokukhulisa ledivayisi. Igumbi lokukhulisa lihlukaniswe negumbi lomoya nge-membrane encane ye-silicone eguquguqukayo. Beka i-gasket phakathi kwegumbi ngalinye ukuvimbela ukuvuza. Isembozo sedivayisi siqukethe ama-electrode e-graphite ahlinzeka ngokuvuselela kagesi. b Ukumelwa kwe-schematic kwedivayisi enkulu yezicubu, indandatho yokuqondisa kanye nendandatho yokusekela. Izingxenye zezicubu (ezinsundu) zibekwe kudivayisi enkulu kakhulu ngendandatho yokuqondisa ebekwe emseleni ongaphandle wedivayisi. Usebenzisa umhlahlandlela, beka ngokucophelela indandatho yokusekela eboshwe nge-tissue acrylic adhesive phezu kwengxenye yezicubu zenhliziyo. c Igrafu ekhombisa isikhathi sokuvuselela kagesi njengomsebenzi wokucindezela kwegumbi lomoya okulawulwa yi-actuator ye-pneumatic ehlelwe kahle (PPD). Idivayisi yokuthola idatha yasetshenziswa ukuvumelanisa ukuvuselela kagesi kusetshenziswa izinzwa zokucindezela. Lapho ingcindezi ekamelweni lokukhulisa ifinyelela umkhawulo obekiwe, isignali ye-pulse ithunyelwa ku-C-PACE-EM ukuze iqalise ukuvuselela kagesi. d Isithombe sama-CTCM amane abekwe eshalofini le-incubator. Amadivayisi amane axhunywe ku-PPD eyodwa ngesekethe yomoya, futhi izinzwa zokucindezela zifakwa ku-valve ye-hemostatic ukuze ziqaphe ingcindezi esesekethe yomoya. Idivayisi ngayinye inezingxenye eziyisithupha zezicubu.
Sisebenzisa i-actuator eyodwa yomoya, sikwazile ukulawula amadivayisi angu-4 e-CTCM, ngalinye lawo elingabamba izingxenye zezicubu ezingu-6 (Isithombe 1d). Ku-CTCM, ingcindezi yomoya ekamelweni lomoya iguqulwa ibe yingcindezi ehambisanayo ekamelweni loketshezi futhi ibangela ukwanda ngokomzimba kwengxenye yenhliziyo (Isithombe 2a kanye ne-Supplementary Movie 1). Ukuhlolwa kokwelulwa kwezicubu ku-80 mm Hg. Art. kubonise ukwelulwa kwezingxenye zezicubu ngo-25% (Isithombe 2b). Lokhu kwelulwa kwephesenti kuboniswe ukuthi kuhambisana nobude be-sarcomere bomzimba obungu-2.2–2.3 µm ukuze kube nokugoba kwengxenye yenhliziyo evamile17,19,25. Ukunyakaza kwezicubu kuhlolwe kusetshenziswa izilungiselelo zekhamera ezenziwe ngokwezifiso (Isithombe Esingeziwe 1). Ubukhulu kanye nesivinini sokunyakaza kwezicubu (Isithombe 2c, d) kuhambisana nokwelula ngesikhathi somjikelezo wenhliziyo nesikhathi ngesikhathi se-systole kanye ne-diastole (Isithombe 2b). Ukunwebeka kanye nesivinini sezicubu zenhliziyo ngesikhathi sokugoba kanye nokuphumula kwahlala kungaguquki izinsuku ezingu-12 ekukhuleni (Isithombe 2f). Ukuze sihlole umphumela wokukhuthazwa kukagesi ekuqineni ngesikhathi sokukhula, sakha indlela yokunquma ukukhubazeka okusebenzayo sisebenzisa i-algorithm yokufiphaza (Isithombe Esingeziwe 2a,b) futhi sakwazi ukuhlukanisa phakathi kokukhubazeka okukhona kanye nokungenakho ukukhuthazwa kukagesi. Ingxenye efanayo yenhliziyo (Isithombe 2f). Esifundeni esinyakazayo sokusika (R6-9), i-voltage ngesikhathi sokukhuthazwa kukagesi yayiphezulu ngo-20% kunalapho kungekho ukukhuthazwa kukagesi, okubonisa umnikelo wokukhuthazwa kukagesi emsebenzini wokufiphaza.
Izimpawu ezimele zokucindezela kwegumbi lomoya, ingcindezi yegumbi loketshezi, kanye nokulinganisa ukunyakaza kwezicubu kuqinisekisa ukuthi ingcindezi yegumbi ishintsha ingcindezi yegumbi loketshezi, okubangela ukunyakaza okuhambisanayo kwesilayi sezicubu. b Izimpawu ezimele zokwelula kwamaphesenti (oluhlaza okwesibhakabhaka) kwezingxenye zezicubu ezihambisana nokwelula kwamaphesenti (ophuzi). c Ukunyakaza okulinganisiwe kwesilayi senhliziyo kuhambisana nesivinini esilinganisiwe sokunyakaza. (d) Izindlela ezimele zokunyakaza okujikelezayo (umugqa oluhlaza okwesibhakabhaka) kanye nesivinini (umugqa onamachashazi e-orange) esiqeshini senhliziyo. e Ukulinganiswa kwesikhathi somjikelezo (n = izingcezu ezingu-19 ngeqembu, ezivela ezingulubeni ezahlukene), isikhathi sokufinyela (n = izingcezu ezingu-19 ngeqembu), isikhathi sokuphumula (n = izingcezu ezingu-19 ngeqembu, ezivela ezingulubeni ezahlukene), ukunyakaza kwezicubu (n = 25). izingcezu)/iqembu ezivela ezingulubeni ezahlukene), ijubane le-systolic eliphakeme (n = 24(D0), izingcezu ezingu-25(D12)/iqembu ezivela ezingulubeni ezahlukene) kanye nesilinganiso sokuphumula esiphezulu (n=24(D0), izingcezu ezingu-25(D12)/iqembu ezivela ezingulubeni ezahlukene). Ukuhlolwa kwe-t komfundi onemisila emibili akubonisanga mehluko obalulekile kunoma iyiphi ipharamitha. f Ukuhlaziywa kobunzima obumele kwezingxenye zezicubu ezine-(red) kanye ne-(blue) stimulation kagesi, izindawo eziyishumi zesifunda zezingxenye zezicubu ezivela esigabeni esifanayo. Amaphaneli angezansi abonisa ukulinganiswa komehluko wamaphesenti ekucindezelweni kwezingxenye zezicubu ezine-na-na-na-na-na-stimulation kagesi ezindaweni eziyishumi ezivela ezingxenyeni ezahlukene. (n = izingcezu ezingu-8/iqembu ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-t komfundi onemisila emibili; ****p < 0.0001, **p < 0.01, *p < 0.05). (n = izingcezu ezingu-8/iqembu ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-t komfundi onemisila emibili; ****p < 0.0001, **p < 0.01, *p < 0.05). (n = 8 срезов/группу от разных свиней, проводится двусторонний t-критерий Стьюдента; ****p<0,0001, **p<0,01, *p<0,05). (n = izigaba ezingu-8/iqembu elivela ezingulubeni ezahlukene, ukuhlolwa kwe-t komfundi okunemisila emibili; ****p<0.0001, **p<0.01, *p<0.05). （n = 8 片/组，來自不同的猪，进行双尾学生t 检验；****p <0.0001,**p <0.01,*p <0.05）. （n = 8 片/组，來自不同的猪，进行双尾学生t 检验；****p <0.0001,**p <0.01,*p <0.05）. (n = 8 срезов/группу, от разных свиней, двусторонний критерий Стьюдента; ****p <0,0001, **p <0,01, *p <0,05). (n = izigaba ezingu-8/iqembu, ezivela ezingulubeni ezahlukene, ukuhlolwa kwe-t komfundi okunemisila emibili; ****p<0.0001, **p<0.01, *p<0.05).Amabha amaphutha amelela ukuphambuka okujwayelekile okungu-±.
Kuhlelo lwethu lwangaphambilini lokukhulisa izingcezu zenhliziyo ezimile okwe-static [20, 21], silondoloze ukusebenza, ukusebenza, kanye nobuqotho besakhiwo sezingcezu zenhliziyo izinsuku eziyi-6 ngokusebenzisa ukuvuselela kagesi kanye nokwenza ngcono ukwakheka kwe-medium. Kodwa-ke, ngemva kwezinsuku eziyi-10, lezi zibalo zehle kakhulu. Sizobhekisela ezigabeni ezikhuliswe ohlelweni lwethu lwangaphambilini lokukhulisa izingcezu ze-static biomimetic 20, 21 (Ctrl) futhi sizosebenzisa i-medium yethu elungiselelwe ngaphambilini njengezimo ze-MC kanye nokukhulisa ngaphansi kokuvuselela okwenziwa ngomshini kanye nogesi ngesikhathi esisodwa (CTCM). okubizwa ngokuthi . Okokuqala, sithole ukuthi ukuvuselela okwenziwa ngomshini ngaphandle kokuvuselela kagesi kwakwanele ukugcina ukusebenza kwezicubu izinsuku eziyi-6 (Isifanekiso Esingeziwe 3a,b). Ngokuthakazelisayo, ngokwethulwa kokuvuselela kwe-physio-mechanical kanye nogesi kusetshenziswa i-STCM, ukusebenza kwezingxenye zenhliziyo zezinsuku eziyi-12 kwahlala kufana nasezingxenyeni zenhliziyo ezintsha ngaphansi kwezimo ze-MS, kodwa hhayi ngaphansi kwezimo ze-Ctrl, njengoba kuboniswe ukuhlaziywa kwe-MTT (Isifanekiso 1). 3a). Lokhu kusikisela ukuthi ukuvuselela okwenziwa ngomshini kanye nokulingisa umjikelezo wenhliziyo kungagcina izingxenye zezicubu ziphila isikhathi eside kabili kunalokho okubikwe ohlelweni lwethu lwangaphambilini lokukhulisa izingcezu zenhliziyo. Kodwa-ke, ukuhlolwa kobuqotho besakhiwo sezingxenye zezicubu ngokufaka i-immunolabeling ye-cardiac troponin T kanye ne-connexin 43 kubonise ukuthi ukubonakaliswa kwe-connexin 43 kwakuphakeme kakhulu ezicutshini ze-MC ngosuku lwe-12 kunasezilawulini ngosuku olufanayo. Kodwa-ke, ukubonakaliswa kwe-connexin 43 okufanayo kanye nokwakheka kwe-Z-disc akuzange kugcinwe ngokugcwele (Isithombe 3b). Sisebenzisa uhlaka lwe-artificial intelligence (AI) ukuze silinganise ubuqotho besakhiwo sezicubu26, ipayipi lokufunda okujulile elisekelwe esithombeni elisekelwe ku-troponin-T kanye ne-connexin staining43 ukuze silinganise ngokuzenzakalelayo ubuqotho besakhiwo kanye nokugqama kwezingcezu zenhliziyo ngokuya ngamandla endawo. Le ndlela isebenzisa i-Convolutional Neural Network (CNN) kanye nohlaka lokufunda okujulile ukuze silinganise ngokwethembeka ubuqotho besakhiwo sezicubu zenhliziyo ngendlela ezenzakalelayo nengachemile, njengoba kuchaziwe ku-reference. 26. Izicubu ze-MC zibonise ukufana kwesakhiwo okuthuthukisiwe nosuku lwe-0 uma kuqhathaniswa nezingxenye zokulawula ezimile. Ngaphezu kwalokho, ukudaya kwe-trichrome kukaMasson kwembule iphesenti eliphansi kakhulu le-fibrosis ngaphansi kwezimo ze-MS uma kuqhathaniswa nezimo zokulawula ngosuku lwe-12 lokukhuliswa (Isithombe 3c). Ngenkathi i-CTCM yandisa ukusebenza kwezingxenye zezicubu zenhliziyo ngosuku lwe-12 kuya ezingeni elifana nelezicubu zenhliziyo ezintsha, ayizange ithuthukise kakhulu ubuqotho besakhiwo sezingxenye zenhliziyo.
Igrafu yebha ikhombisa ukulinganiswa kokusebenza kwe-MTT kwezingcezu zenhliziyo ezintsha (D0) noma ukukhuliswa kwezingcezu zenhliziyo izinsuku eziyi-12 kungaba kukhuliswa okungaguquki (D12 Ctrl) noma ku-CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) izingcezu/iqembu elivela ezingulubeni ezahlukene, indlela eyodwa eyenziwa ngayo ukuhlolwa kwe-ANOVA; ####p < 0.0001 uma kuqhathaniswa no-D0 kanye no-**p < 0.01 uma kuqhathaniswa no-D12 Ctrl). Igrafu yebha ikhombisa ukulinganiswa kokusebenza kwe-MTT kwezingcezu zenhliziyo ezintsha (D0) noma ukukhuliswa kwezingcezu zenhliziyo izinsuku eziyi-12 kungaba kukhuliswa okungaguquki (D12 Ctrl) noma ku-CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) izingcezu/iqembu elivela ezingulubeni ezahlukene, indlela eyodwa eyenziwa ngayo ukuhlolwa kwe-ANOVA; ####p < 0.0001 uma kuqhathaniswa no-D0 kanye no-**p < 0.01 uma kuqhathaniswa no-D12 Ctrl).i-histogram ikhombisa ukulinganiswa kokusebenza kwezingxenye zenhliziyo ezintsha ze-MTT (D0) noma isiko lezingxenye zenhliziyo izinsuku eziyi-12 ekukhuleni okungaguquki (ukulawula kwe-D12) noma i-CTCM (D12 MC) (n = 18 (D0), 15 (ukulawula kwe-D12). ) ), izingxenye/iqembu le-12 (D12 MC) ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA okuyindlela eyodwa;####p < 0,0001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 uma kuqhathaniswa no-D0 kanye no-**p < 0.01 uma kuqhathaniswa no-D12 Ctrl). a 条形图显示在静态培养(D12 Ctrl) 或CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 12 或片(D0) 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 1天的MTT 活力的量化)，來自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；与D0 相比，####p <2比比比，####p <2比 10D1 < 0.01). a 条形图显示在静态培养(D12 Ctrl) 或CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 中北切片/组，进行单向ANOVA 测试；与D0 相比，####p <0.0001, 与D12 Ctrl 相比，**p.)i-histogram ekhombisa ukulinganiswa kokusebenza kwe-MTT ezingxenyeni zenhliziyo entsha (D0) noma izingxenye zenhliziyo ezikhuliswe izinsuku eziyi-12 ekukhuleni okungaguquki (ukulawula kwe-D12) noma i-CTCM (D12 MC) (n = 18 (D0), 15 (ukulawula kwe-D12)), 12 (D12 MC) izingxenye/iqembu elivela ezingulubeni ezahlukene, ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa;####p < 0,0001 по сравнению с D0, **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 uma kuqhathaniswa no-D0, **p < 0.01 uma kuqhathaniswa no-D12 Ctrl).b I-Troponin-T (eluhlaza okotshani), i-connexin 43 (ebomvu) kanye ne-DAPI (eluhlaza okwesibhakabhaka) ezingxenyeni zenhliziyo ezisanda kuhlukaniswa (D0) noma ezingxenyeni zenhliziyo ezikhuliswe ngaphansi kwezimo ezimile (Ctrl) noma izimo ze-CTCM (MC) izinsuku ezingu-12) zezithombe ezimele i-immunofluorescence (isikali esingenalutho = 100 µm). Ukulinganisa ubuhlakani bokwenziwa kobuqotho besakhiwo sezicubu zenhliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izingcezu/iqembu ngalinye elivela engulubeni ehlukene, kwenziwa ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ####p < 0.0001 uma kuqhathaniswa no-D0 kanye no-****p < 0.0001 uma kuqhathaniswa no-D12 Ctrl). Ukulinganisa ubuhlakani bokwenziwa kokuqina kwesakhiwo sezicubu zenhliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izingcezu/iqembu ngalinye kusuka ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA kwendlela eyodwa; ####p < 0.0001 uma kuqhathaniswa no-D0 kanye no-****p < 0.0001 uma kuqhathaniswa no-D12 Ctrl). Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) сруправи/ проводится однофакторный тест ANOVA; ####p <0,0001 по сравнению с D0 и ****p < 0,0001 по сравнению с D12 Ctrl). Ukulinganiswa kobuqotho besakhiwo sezicubu zenhliziyo ngobuhlakani bokwenziwa (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izingxenye/amaqembu avela ezingulubeni ezahlukene, kwenziwe ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ####p < 0.0001 vs. no-D0 kanye no-****p < 0.0001 uma kuqhathaniswa no-D12 Ctrl).人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izingcezu/iqembu ngalinye lengulube ehlukile, isivivinyo sendlela eyodwa ye-ANOVA;###00D0 <10.相比,****p <0.0001 与D12 Ctrl 相比）.人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izingcezu/iqembu lengulube ngayinye ehlukene, isivivinyo sendlela eyodwa ye-ANOVA;##0#0D0D **** **** <0.0001 与D12 Ctrl 相比）. I-Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC/грка) разных свиней, односторонний тест ANOVA; ####p <0,0001 vs. D0 Для сравнения ****p < 0,0001 по сравнению с D12 Ctrl). Ubuhlakani bokwenziwa ukuze kulinganiswe ubuqotho besakhiwo sezicubu zenhliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izingxenye/iqembu ngalinye lezingulube ezahlukene, ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ####p<0.0001 vs .D0 Ukuze kuqhathaniswe ****p < 0.0001 uma kuqhathaniswa ne-D12 Ctrl). c Izithombe ezimele (kwesobunxele) kanye nokulinganisa (kwesokudla) kwezingcezu zenhliziyo ezifakwe ibala likaMasson's trichrome (Isikali singenalutho = 500 µm) (n = izingcezu eziyi-10/iqembu ngalinye elivela engulubeni ehlukile, kwenziwa ukuhlolwa kwe-ANOVA kwendlela eyodwa; ####p < 0.0001 uma kuqhathaniswa no-D0 kanye no-***p < 0.001 uma kuqhathaniswa no-D12 Ctrl). c Izithombe ezimele (kwesobunxele) kanye nokulinganisa (kwesokudla) kwezingcezu zenhliziyo ezifakwe ibala likaMasson's trichrome (Isikali singenalutho = 500 µm) (n = izingcezu eziyi-10/iqembu ngalinye elivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA kwendlela eyodwa; #### p < 0.0001 uma kuqhathaniswa no-D0 kanye no-***p < 0.001 uma kuqhathaniswa no-D12 Ctrl). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Массок00 = мкм) (n = 10 срезов/группу от разных свиней, выполняется односторонний тест ANOVA; #### p < 0,0001 по сравнению с D0не ***p. c Izithombe ezimele (kwesobunxele) kanye nokulinganisa (kwesokudla) kwezingxenye zenhliziyo ezifakwe ibala likaMasson's trichrome (isikali esingagqokwanga = 500 µm) (n = izingxenye/iqembu eziyi-10 ezivela ezingulubeni ezahlukene, kwenziwe ukuhlolwa kwe-ANOVA kwendlela eyodwa; #### p < 0 .0001 uma kuqhathaniswa no-D0 kanye no-***p < 0.001 uma kuqhathaniswa no-D12 Ctrl). c 用Masson 三色染料染色的心脏切片的代表性图像（左）和量化(右）（裸尺度= 500 1) = 500 µ个切片/组，每组來自不同的猪，进行单向ANOVA 测试；#### p <0.0001 与D0 相比，***p <0.001 Ctrl ．D12 . C 用 mason 三 色 染料的 心脏 切片 的 代表性 （左 左） 量化 （右） 裸尺庰裸尺庰裸尺庰裸尺庰裸尺庰裸尺庰裸尺庰裸尺= 500 µm） （n = 10 个 切片 组 每 组 來自 不同 猪 , 进行 单向 单向 Anova 测试；#### D0 <0 0.0.0. < 0.001 Faka i-D12 Ctrl. c Репрезентативные изображения (слева) и количественный анализ (справа) срезов сердца, окрашенных трихромным красителем Массом Массом 5 (n = 10 срезов/группа, каждый от другой свиньи, протестировано с помощью однофакторного дисперсионного анализа ;### #p < 0,00,000 cindezela u-D12 Ctrl). c Izithombe ezimele (kwesobunxele) kanye nokulinganisa (kwesokudla) kwezingxenye zenhliziyo ezifakwe ibala likaMasson's trichrome (elingenalutho = 500 µm) (n = izingxenye/iqembu eziyi-10, ngasinye sivela engulubeni ehlukile, sihlolwe ngokuhlaziywa kokuhlukahluka kwendlela eyodwa ;### # p < 0.0001 uma kuqhathaniswa no-D0, ***p < 0.001 uma kuqhathaniswa no-D12 Ctrl).Amabha amaphutha amelela ukuphambuka okujwayelekile okungu-±.
Sicabange ukuthi ngokungeza ama-molecule amancane endaweni yokukhulisa amangqamuzana, ubuqotho be-cardiomyocyte bungathuthukiswa futhi ukuthuthukiswa kwe-fibrosis kunciphe ngesikhathi sokukhulisa i-CTCM. Ngakho-ke sihlole ama-molecule amancane sisebenzisa amasiko ethu okulawula aqinile20,21 ngenxa yenani elincane lezici ezididayo. I-Dexamethasone (Dex), i-triiodothyronine (T3), kanye ne-SB431542 (SB) zakhethwa kulesi sivivinyo. Lawa ma-molecule amancane asetshenziswe ngaphambilini kumasiko e-hiPSC-CM ukuze akhuthaze ukuvuthwa kwama-cardiomyocyte ngokwandisa ubude be-sarcomere, ama-T-tubules, kanye nesivinini sokuqhuba. Ngaphezu kwalokho, kokubili i-Dex (i-glucocorticoid) kanye ne-SB kwaziwa ukuthi kuvimbela ukuvuvukala29,30. Ngakho-ke, sihlole ukuthi ukufakwa kwe-molecule eyodwa noma inhlanganisela yalawa ma-molecule amancane kuzothuthukisa ubuqotho besakhiwo sezingxenye zenhliziyo. Ekuhlolweni kokuqala, umthamo wenhlanganisela ngayinye ukhethwe ngokusekelwe ekugxilweni okusetshenziswa kakhulu kumamodeli okukhulisa amangqamuzana (1 μM Dex27, 100 nM T327, kanye no-2.5 ​​μM SB31). Ngemva kwezinsuku ezingu-12 zokukhula, ukuhlanganiswa kwe-T3 ne-Dex kwaholela ekuqineni kwesakhiwo se-cardiomyocyte kanye nokulungiswa okuncane kwe-fibrous (Izithombe Ezingeziwe 4 no-5). Ngaphezu kwalokho, ukusetshenziswa kwala mazinga amabili noma amabili e-T3 ne-Dex kwaveza imiphumela emibi uma kuqhathaniswa namazinga ajwayelekile (Isithombe Esingeziwe 6a,b).
Ngemva kokuhlolwa kokuqala, senze ukuqhathanisa ngqo kwezimo ezine zokulima (Isithombe 4a): I-Ctrl: izingxenye zenhliziyo ezikhuliswe kusiko lethu elingaguquki esichazwe ngaphambilini sisebenzisa i-medium yethu elungiselelwe; I-20.21 TD: I-T3 kanye ne-Ctrl s Added Dex ngoLwesithathu; I-MC: izingxenye zenhliziyo ezikhuliswe ku-CTCM sisebenzisa i-medium yethu elungiselelwe ngaphambilini; kanye ne-MT: I-CTCM ene-T3 kanye ne-Dex engezwe ku-medium. Ngemva kwezinsuku ezingu-12 zokulima, ukuphila kwezicubu ze-MS kanye ne-MT kwahlala kufana nokwezicubu ezintsha ezihlolwe ngokuhlolwa kwe-MTT (Isithombe 4b). Ngokuthakazelisayo, ukungezwa kwe-T3 kanye ne-Dex ku-transwell cultures (TD) akuzange kuholele ekuthuthukisweni okukhulu kokuphila uma kuqhathaniswa nezimo ze-Ctrl, okubonisa indima ebalulekile yokukhuthaza ngomshini ekugcineni ukuphila kwezingxenye zenhliziyo.
umdwebo womklamo wokuhlola obonisa izimo ezine zokulima ezisetshenziswa ukuhlola imiphumela yokuvuselelwa kwemishini kanye nokwengezwa kwe-T3/Dex endaweni ephakathi izinsuku ezingu-12. b Igrafu yebha ikhombisa ukulinganiswa kokuphila ezinsukwini eziyi-12 ngemuva kokukhuliswa kuzo zonke izimo zokukhuliswa ezine (Ctrl, TD, MC, kanye ne-MT) uma kuqhathaniswa nezingcezu zenhliziyo ezintsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kanye ne-D12 MT), izingcezu/iqembu eziyi-12 (D12 MC) ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ####p < 0.0001, ###p < 0.001 uma kuqhathaniswa ne-D0 kanye **p < 0.01 uma kuqhathaniswa ne-D12 Ctrl). b Igrafu yebha ikhombisa ukulinganiswa kokuphila ezinsukwini eziyi-12 ngemuva kokukhuliswa kuzo zonke izimo zokukhuliswa ezine (Ctrl, TD, MC, kanye ne-MT) uma kuqhathaniswa nezingcezu zenhliziyo ezintsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kanye ne-D12 MT), izingcezu/iqembu eziyi-12 (D12 MC) ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ####p < 0.0001, ###p < 0.001 uma kuqhathaniswa ne-D0 kanye **p < 0.01 uma kuqhathaniswa ne-D12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после культивирования во всех 4 условиях культивирования во всех 4 условиях культивирования, TD сравнению со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD and D12 MT), 12 (D12 MC) срезов/группу от разныхстонстонстенный I-ANOVA; ####p <0,0001, ###p < 0,001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). b Igrafu yebha ikhombisa ukulinganiswa kokuphila ezinsukwini eziyi-12 ngemuva kokukhuliswa kuzo zonke izimo zokukhuliswa ezine (ukulawula, i-TD, i-MC, kanye ne-MT) uma kuqhathaniswa nezingxenye zenhliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, i-D12 TD, kanye ne-D12 MT), izingxenye/iqembu eziyi-12 (D12 MC) ezivela ezingulubeni ezahlukene, ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ####p < 0.0001, ###p < 0.001 vs. D0 kanye no **p < 0.01 by uma kuqhathaniswa ne-D12 Ctrl). b 条形图显示所有4 种培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) (n = 18 (D0)),15 (D12 Ctrl,D12 TD) MT)，來自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；####p < 0.0001,###p < 0.001 与D0 相比，*0p与D12控制）.b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC and MT) по сравнению со свежими срезами сердца (D0) (n = 18 (D12), D12), MT), от разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA b I-Histogram ekhombisa zonke izimo ezine zokukhula (ukulawula, i-TD, i-MC kanye ne-MT) uma kuqhathaniswa nezingxenye zenhliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kanye ne-D12 MT), ezivela ezingulubeni ezahlukene 12 (D12 MC) izingxenye/iqembu, ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ####p<0.0001, ###p<0.001 vs. D0, **p<0.01 vs. ukulawula D12). Igrafu yebha ikhombisa ukulinganiswa kokushintshashintsha kweglucose ezinsukwini eziyi-12 ngemuva kokukhuliswa kuzo zonke izimo zokukhuliswa ezine (Ctrl, TD, MC, kanye ne-MT) uma kuqhathaniswa nezingcezu zenhliziyo ezintsha (D0) (n = izingcezu ezi-6/iqembu ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ###p < 0.001, uma kuqhathaniswa no-D0 kanye no-***p < 0.001 uma kuqhathaniswa no-D12 Ctrl). Igrafu yebha ikhombisa ukulinganiswa kokushintshashintsha kweglucose ezinsukwini eziyi-12 ngemuva kokukhuliswa kuzo zonke izimo zokukhuliswa ezine (Ctrl, TD, MC, kanye ne-MT) uma kuqhathaniswa nezingcezu zenhliziyo ezintsha (D0) (n = izingcezu ezi-6/iqembu ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ###p < 0.001, uma kuqhathaniswa no-D0 kanye no-***p < 0.001 uma kuqhathaniswa no-D12 Ctrl). c Гистограмма показывает количественную оценку потока глюкозы через 12 дней после культивирования во всех 4 условиях культивирования, MCTD, MC. сравнению со свежими срезами сердца (D0) (n = 6 срезов/группу от разных свиней, односторонний Выполняется тест ANOVA; ##не#p <0,00 cindezela inkinobho D12 Ctrl). c I-Histogram ikhombisa ukulinganiswa kokuhamba kweglucose ezinsukwini eziyi-12 ngemuva kokukhuliswa ngaphansi kwazo zonke izimo ezine zokukhuliswa (ukulawula, i-TD, i-MC kanye ne-MT) uma kuqhathaniswa nezingxenye zenhliziyo entsha (D0) (n = izingxenye/iqembu ezi-6 ezivela ezingulubeni ezahlukene, kwenziwe ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ###p < 0.001 uma kuqhathaniswa ne-D0 kanye ***p < 0.001 uma kuqhathaniswa ne-D12 Ctrl). c 条形图显示所有4 种培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) 相比，培养后12天的葡萄糖通量定量（n = 6 片/组，來自不同猪，单向执行ANOVA 测试；###p < 0.001，与D0 相比 1 与D0 <0.001 Ctrl D0 1 2 *.相比）. C 条形图 显示 所有 4 种 条件 (（ctrl 、 td 、 mc 和 mt) 12 天 的 通量 定量 （n = 6 片/组 ， 來自 猪 , , , , , , , , 猪 猪单向执行ANOVA 测试；#0#01 <0.相比,***p < 0.001 ku-D12 Ctrl 相比）. c Гистограмма, количественную оценку потока глюкозы через 12 дней после культивирования для всех 4 условий культивирования сравнению со свежими срезами сердца (D0) (n = 6 срезов/группа, от разных свиней, односторонний Были проведены тесты ANOVA; ###0p1 <0,0, *** 0,001 по сравнению с D12 (контроль). c I-Histogram ekhombisa ukulinganiswa kokuhamba kweglucose ezinsukwini eziyi-12 ngemuva kokukhuliswa kwazo zonke izimo ezine zokukhuliswa (ukulawula, i-TD, i-MC, kanye ne-MT) uma kuqhathaniswa nezingxenye zenhliziyo entsha (D0) (n = izingxenye ezi-6/iqembu, ezivela ezingulubeni ezahlukene, ohlangothini olulodwa. Ingabe kwenziwa ukuhlolwa kwe-ANOVA, ###p < 0.001 uma kuqhathaniswa ne-D0, ***p < 0.001 uma kuqhathaniswa ne-D12 (ukulawula).d Iziqephu zokuhlaziywa kobunzima bezicubu ezintsha (eziluhlaza okwesibhakabhaka), usuku lwe-12 MC (oluhlaza), kanye nosuku lwe-12 MT (obomvu) ezindaweni eziyishumi zesigaba sezicubu zesifunda (n = izingcezu ezi-4/iqembu, ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; bekungekho mehluko obalulekile phakathi kwamaqembu). e Iziqephu zentaba-mlilo ezibonisa izakhi zofuzo ezivezwe ngokuhlukile ezingxenyeni zenhliziyo entsha (D0) uma kuqhathaniswa nezingxenye zenhliziyo ezikhuliswe ngaphansi kwezimo ezimile (Ctrl) noma ngaphansi kwezimo ze-MT (MT) izinsuku eziyi-10-12. f Imephu yokushisa yezakhi zofuzo ze-sarcomere zezingxenye zenhliziyo ezikhuliswe ngaphansi kwezimo ngazinye zezitshalo. Amabha amaphutha amelela isilinganiso ± ukuphambuka okujwayelekile.
Ukuthembela kwe-metabolic ekushintsheni kusuka ku-oxidation ye-fatty acid kuya ku-glycolysis kuyisici esibalulekile sokuhlukana kwe-cardiomyocyte. Ama-cardiomyocyte angakavuthwa asebenzisa i-glucose ngokuyinhloko ekukhiqizweni kwe-ATP futhi ane-mitochondria ye-hypoplastic ene-cristae embalwa5,32. Ukuhlaziywa kokusetshenziswa kwe-glucose kubonise ukuthi ngaphansi kwezimo ze-MC ne-MT, ukusetshenziswa kwe-glucose kwakufana nalokho okwakusezicutshini zosuku 0 (Isithombe 4c). Kodwa-ke, amasampula e-Ctrl abonise ukwanda okukhulu kokusetshenziswa kwe-glucose uma kuqhathaniswa nezicubu ezintsha. Lokhu kubonisa ukuthi inhlanganisela ye-CTCM ne-T3/Dex ithuthukisa ukusebenza kwezicubu futhi igcina i-phenotype ye-metabolic yezingxenye zenhliziyo ezikhuliswe izinsuku ezingu-12. Ngaphezu kwalokho, ukuhlaziywa kokucindezeleka kubonise ukuthi amazinga okucindezeleka ahlala efana nakwasezicutshini zenhliziyo ezintsha izinsuku ezingu-12 ngaphansi kwezimo ze-MT ne-MS (Isithombe 4d).
Ukuze sihlaziye umthelela ophelele we-CTCM kanye ne-T3/Dex endaweni yokubhalwa kwezicubu zenhliziyo, senze i-RNAseq ezicucwini zenhliziyo ezivela kuzo zonke izimo ezine ezahlukene zesiko (Idatha Engeziwe 1). Ngokuthakazelisayo, izingxenye ze-MT zibonise ukufana okuphezulu kokubhalwa kwezicubu zenhliziyo ezintsha, kanti zingu-16 kuphela ezivezwe ngendlela ehlukile kwezakhi zofuzo ezingu-13,642. Kodwa-ke, njengoba sibonisile ngaphambili, izingcezu ze-Ctrl zibonise izakhi zofuzo ezingu-1229 ezivezwe ngendlela ehlukile ngemva kwezinsuku ezingu-10-12 ekukhulisweni (Isithombe 4e). Le datha iqinisekiswe yi-qRT-PCR yezakhi zofuzo zenhliziyo kanye ne-fibroblast (Isithombe Engeziwe 7a-c). Ngokuthakazelisayo, izingxenye ze-Ctrl zibonise ukwehla kwezakhi zofuzo zenhliziyo kanye nomjikelezo weseli kanye nokusebenza kwezinhlelo zezakhi zofuzo ezivuvukalayo. Le datha iphakamisa ukuthi ukwahlukana, okuvame ukwenzeka ngemva kokukhula isikhathi eside, kuncishiswa ngokuphelele ngaphansi kwezimo ze-MT (Isithombe Engeziwe 8a,b). Ucwaningo olucophelelayo lwezakhi zofuzo ze-sarcomere lukhombisile ukuthi ngaphansi kwezimo ze-MT kuphela lapho izakhi zofuzo ezifaka ikhodi ku-sarcomere (Umfanekiso 4f) kanye nesiteshi se-ion (Umfanekiso Ongeziwe 9) zigcinwa khona, zivikele ekucindezelweni ngaphansi kwezimo ze-Ctrl, TD, kanye ne-MC. Le datha ikhombisa ukuthi ngokuhlanganiswa kokukhuthazwa komshini kanye nokuhlasimulisa (T3/Dex), i-transcriptome ye-heart slice ingahlala ifana nezingcezu zenhliziyo ezintsha ngemva kwezinsuku ezingu-12 ekukhuleni.
Lokhu okutholakele kokubhalwa phansi kusekelwa iqiniso lokuthi ubuqotho besakhiwo sama-cardiomyocytes ezingxenyeni zenhliziyo bugcinwa kangcono ngaphansi kwezimo ze-MT izinsuku ezingu-12, njengoba kuboniswe yi-connexin engaguquki neyasendaweni 43 (Isithombe 5a). Ngaphezu kwalokho, i-fibrosis ezingxenyeni zenhliziyo ngaphansi kwezimo ze-MT yehliswe kakhulu uma kuqhathaniswa ne-Ctrl futhi ifana nezingxenye zenhliziyo entsha (Isithombe 5b). Le datha ikhombisa ukuthi inhlanganisela yokuvuselelwa kwemishini kanye nokwelashwa kwe-T3/Dex kulondoloza ngempumelelo isakhiwo senhliziyo ezingxenyeni zenhliziyo emasikweni.
izithombe ezimele i-immunofluorescence ze-troponin-T (eluhlaza okotshani), i-connexin 43 (ebomvu), kanye ne-DAPI (eluhlaza okwesibhakabhaka) ezingxenyeni zenhliziyo ezisanda kuhlukaniswa (D0) noma ezikhuliswe izinsuku eziyi-12 kuzo zonke izimo ezine ze-section yenhliziyo (ibha yesikali = 100 µm). Ukuhlolwa kobuhlakani bokwenziwa kokuqina kwesakhiwo sezicubu zenhliziyo (n = 7 (D0 kanye ne-D12 Ctrl), 5 (D12 TD, D12 MC kanye ne-D12 MT) izingcezu/iqembu elivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; ####p < 0.0001 uma kuqhathaniswa ne-D0 kanye ne-*p < 0.05, noma ****p < 0.0001 uma kuqhathaniswa ne-D12 Ctrl). Ukuhlolwa kobuhlakani bokwenziwa kokuqina kwesakhiwo sezicubu zenhliziyo (n = 7 (D0 kanye ne-D12 Ctrl), 5 (D12 TD, D12 MC kanye ne-D12 MT) izingcezu/iqembu elivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; #### p < 0.0001 uma kuqhathaniswa ne-D0 kanye ne-*p < 0.05, noma ****p < 0.0001 uma kuqhathaniswa ne-D12 Ctrl). I-Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 7 (D0 kanye ne-D12 Ctrl), 5 (D12 TD, D12 MT/D12 MT) kanye от разных свиней, проведен однофакторный тест ANOVA; #### p <0,0001 по сравнению с D0 и *p <0,05 или ****p <0,0001 по сравнению с D12 ). Ukuhlolwa kokuqina kwesakhiwo sezicubu zenhliziyo kusetshenziswa ubuhlakani bokwenziwa (n = 7 (D0 kanye ne-D12 Ctrl), 5 (D12 TD, D12 MC kanye ne-D12 MT) izingxenye/iqembu elivela ezingulubeni ezahlukene, kwenziwe ukuhlolwa kwe-ANOVA okuhamba ngendlela eyodwa; #### p < 0.0001 uma kuqhathaniswa ne-D0 kanye ne-*p < 0.05 noma ****p < 0.0001 uma kuqhathaniswa ne-D12 Ctrl).对不同猪的心脏组织结构完整性（n = 7（D0 和D12 Ctrl）、5（D12 TD、D12 MC 和D12 MT）切片/组）进行人工智能量化，进行单向ANOVA 测试;相比）.对 不同 猪 的 心脏 结构 完整性 (n = 7 （d0 和 d12 ctrl)智能量 化 进行 单向 单向 单向 测试 ； ########## p < 0.0001 与D0 和*p <0.05 相比 <2 Ctrl 1 0.****0p相比）.Ukulinganiswa kobuqotho besakhiwo sezicubu zenhliziyo kusetshenziswa ubuhlakani bokwenziwa ezingulubeni ezahlukene (n = 7 (D0 kanye ne-D12 Ctrl), 5 (D12 TD, D12 MC kanye ne-D12 MT) izingxenye/iqembu) ngokuhlolwa kwe-ANOVA okwenziwa ngendlela eyodwa;#### p < 0,0001 по сравнению с D0 и *p < 0,05 или ****p < 0,0001 по сравнению с D12 Ctrl). #### p < 0.0001 uma kuqhathaniswa no-D0 kanye no-*p < 0.05 noma ****p < 0.0001 uma kuqhathaniswa no-D12 Ctrl). b Izithombe ezimele kanye nokulinganisa izingcezu zenhliziyo ezifakwe ibala likaMasson's trichrome (Ibha yesikali = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, kanye ne-D12 MC), izingcezu/iqembu ezingu-9 (D12 MT) ezivela ezingulubeni ezahlukene, ukuhlolwa kwe-ANOVA kwendlela eyodwa kwenziwa; ####p < 0.0001 uma kuqhathaniswa no-D0 kanye no-***p < 0.001, noma ****p < 0.0001 uma kuqhathaniswa no-D12 Ctrl). b Izithombe ezimele kanye nokulinganisa izingcezu zenhliziyo ezifakwe ibala likaMasson's trichrome (Ibha yesikali = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, kanye ne-D12 MC), izingcezu/iqembu ezingu-9 (D12 MT) ezivela ezingulubeni ezahlukene, ukuhlolwa kwe-ANOVA kwendlela eyodwa kwenziwa; ####p < 0.0001 uma kuqhathaniswa no-D0 kanye no-***p < 0.001, noma ****p < 0.0001 uma kuqhathaniswa no-D12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Массона (масштабная 10) = 50 = 10 (D0, D12 Ctrl, D12 TD и D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполняется односторонний тест ANOVA < ####p0p 0,0 иняется односторонний тест ANOVA; 0,001 noma ****p <0,0001 по cindezela u-D12 Ctrl). b Izithombe ezimele kanye nokulinganisa izingxenye zenhliziyo ezifakwe umbala we-Masson's trichrome stain (isikali sebha = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD kanye ne-D12 MC), izingxenye/iqembu ezingu-9 (D12 MT) ezivela ezingulubeni ezahlukene, ezenziwe ngendlela eyodwa i-ANOVA; ####p < 0.0001 vs. D0 kanye ***p < 0.001 noma ****p < 0.0001 vs. D12 Ctrl). b 用Masson 三色染料染色的心脏切片的代表性图像和量化（比例尺= 500 µm）（n = 10（D2D,D 、 D MC. 0.0001 cindezela u-D12 Ctrl 相比）. b 用 masson 三 色 染料 的 心脏 切片 的 代表性 和 量化 （比例 尺 尺 尺 = 500 µm） （n d 1, d 1, d 1, d 1, d 1, d 1, d 1, d 1, d = 1, d 1, d 2, d 1, d 1 = 1 d 1. d12 td 和 d12 mc） 來自 不同 的 9 个 d12 mt 切片 切片 切片 切片 切片 切片 切片 切片切片 切片 切片 切片 切片/组，进行单因素方差分析；####p <0.0001 与D0 相00p1 0.0001 Susa i-D12 Ctrl 相比）. b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом Массона (масштабная линейка = 500, DSL = 500, D. I-D12 TD ne-D12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA; D12 Ctrl). b Izithombe ezimele kanye nokulinganiswa kwezingxenye zenhliziyo ezifakwe i-trichrome kaMasson (ibha yesikali = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD kanye ne-D12 MC), izingxenye ezingu-9 (D12 MT) ezivela ezingulubeni/eqenjini elihlukene, indlela eyodwa ye-ANOVA; ####p < 0.0001 uma kuqhathaniswa no-D0, ***p < 0.001 noma ****p < 0.0001 uma kuqhathaniswa no-D12 Ctrl).Amabha amaphutha amelela ukuphambuka okujwayelekile okungu-±.
Ekugcineni, ikhono le-CTCM lokulingisa i-hypertrophy yenhliziyo lahlolwa ngokwanda kokunwebeka kwezicubu zenhliziyo. Ku-CTCM, umfutho wegumbi lomoya ophezulu ukhuphuke kusuka ku-80 mmHg kuya ku-80 mmHg. Ubuciko. (ukwelula okuvamile) kwafika ku-140 mmHg Ubuciko. (Umfanekiso 6a). Lokhu kuhambelana nokwenyuka okungu-32% kokunwebeka (Umfanekiso 6b), okwaboniswa ngaphambilini njengokwelulwa kwephesenti elihambisanayo elidingekayo ukuze izingxenye zenhliziyo zifinyelele ubude be-sarcomere obufana nobo obubonakala ku-hypertrophy. Ukunwebeka kanye nesivinini sezicubu zenhliziyo ngesikhathi sokufinyela nokuphumula kwahlala kungaguquki phakathi nezinsuku eziyisithupha zokukhuliswa (Umfanekiso 6c). Izicubu zenhliziyo ezivela ezimweni ze-MT zahlushwa ukunwebeka okuvamile (MT (Normal)) noma izimo zokunwebeka ngokweqile (MT (OS)) izinsuku eziyisithupha. Kakade ngemva kwezinsuku ezine ekukhulisweni, i-biomarker ye-hypertrophic NT-ProBNP yaphakanyiswa kakhulu endaweni ephakathi ngaphansi kwezimo ze-MT (OS) uma kuqhathaniswa nezimo ze-MT (normal) (Umfanekiso 7a). Ngaphezu kwalokho, ngemva kwezinsuku eziyisithupha zokukhulisa, usayizi weseli ku-MT (OS) (Umfanekiso 7b) wanda kakhulu uma kuqhathaniswa nezingxenye zenhliziyo ye-MT (ejwayelekile). Ngaphezu kwalokho, ukudluliselwa kwe-NFATC4 kwenuzi kwanda kakhulu ezicutshini ezinwetshiwe ngokweqile (Umfanekiso 7c). Le miphumela ikhombisa intuthuko eqhubekayo yokuguqulwa kwe-pathological ngemva kokwanda kwe-hyperdistension futhi isekela umqondo wokuthi idivayisi ye-CTCM ingasetshenziswa njengeplatifomu yokufunda i-stretch-induced cardiac hypertrophy signaling.
Izinkomba ezimele zokucindezela kwegumbi lomoya, ukucindezela kwegumbi loketshezi, kanye nokulinganisa ukunyakaza kwezicubu kuqinisekisa ukuthi ukucindezela kwegumbi kushintsha ukucindezela kwegumbi loketshezi, okubangela ukunyakaza okuhambisanayo kwesilayi sezicubu. b Amaphesenti amele ukwelula kanye namajika esilinganiso sokwelula ezingxenyeni zezicubu ezivame ukwelulwa (orenji) kanye nezelulwe kakhulu (oluhlaza okwesibhakabhaka). c Igrafu yebha ekhombisa isikhathi somjikelezo (n = izingcezu ezingu-19 ngeqembu, ezivela ezingulubeni ezahlukene), isikhathi sokufinyela (n = izingcezu ezingu-18-19 ngeqembu, ezivela ezingulubeni ezahlukene), isikhathi sokuphumula (n = izingcezu ezingu-19 ngeqembu, ezivela ezingulubeni ezahlukene) ), ubukhulu bokunyakaza kwezicubu (n = izingcezu ezingu-14/iqembu, ezivela ezingulubeni ezahlukene), ijubane le-systolic eliphakeme (n = izingcezu ezingu-14/iqembu, ezivela ezingulubeni ezahlukene) kanye nesilinganiso sokuphumula esiphezulu (n = 14 (D0), 15 (D6) ) izingxenye/amaqembu) ezivela ezingulubeni ezahlukene), ukuhlolwa kwe-t komfundi okunemisila emibili akubonisanga mehluko obalulekile kunoma iyiphi ipharamitha, okubonisa ukuthi la mapharamitha ahlala engaguquki phakathi nezinsuku ezingu-6 zokukhuliswa nge-overvoltage. Amabha amaphutha amelela ukuphambuka okujwayelekile ± okujwayelekile.
Ukulinganiswa kwegrafu yebha kokuhlushwa kwe-NT-ProBNP kumidiya yokukhulisa kusuka ezinceleni zenhliziyo ezikhuliswe ngaphansi kwezimo ze-MT normal stretch (Norm) noma ukwelula ngokweqile (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kanye ne-D4 MTOS) izingcezu/iqembu ezivela ezingulubeni ezahlukene, kwenziwa i-ANOVA enezindlela ezimbili; **p < 0.01 uma kuqhathaniswa nokulula okuvamile). Ukulinganiswa kwegrafu yebha yokuhlushwa kwe-NT-ProBNP kumidiya yokukhulisa kusuka ezinceleni zenhliziyo ezikhuliswe ngaphansi kwezimo ze-MT normal stretch (Norm) noma ukwelula ngokweqile (OS) (n ​​= 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kanye ne-D4 MTOS) izingcezu/iqembu ezivela ezingulubeni ezahlukene, kwenziwa i-ANOVA enezindlela ezimbili; **p < 0.01 uma kuqhathaniswa nokulula okuvamile).I-histogram yobuningi bokuhlushwa kwe-NT-ProBNP endaweni yokukhulisa kusuka ezinceleni zenhliziyo ezikhuliswe ngaphansi kwezimo zokwelula okuvamile kwe-MT (okujwayelekile) noma ukwelula ngokweqile (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kanye ne-D4). I-MTOS) izingcezu /iqembu ezivela ezingulubeni ezahlukene, kwenziwa ukuhlaziywa kwezici ezimbili kokuhlukahluka;**p < 0,01 по сравнению с нормальным растяжением). **p < 0.01 uma kuqhathaniswa nokwelula okuvamile). a 在MT 正常拉伸(Okuvamile) 或过度拉伸(OS) 条件下培养的心脏切片培养基中NT-ProBNP 浓麦的条彈n 浓麦的条彈2 = MTNorm)、 3（D2 MTOS、D4 MTNorm 和D4 MTOS）來自不同猪的切片/组，进行双向方差分析；**与正常拉伸1 . i-Quantification ye-NT-ProBNP yokugxilisa ingqondo ezingcezuni zenhliziyo ezikhuliswe ngaphansi kwezimo ze-MT ezijwayelekile zokwelula (Okuvamile) noma i-Overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm neD4 MTOS) kusuka ku-猪的切片/组，可以双向双经ngokwelula okuvamile, p <0.01).i-histogram Ukulinganiswa kokuhlushwa kwe-NT-ProBNP ezingcezwini zenhliziyo ezikhuliswe ngaphansi kwezimo zokwelula okujwayelekile kwe-MT (okujwayelekile) noma ukwelula ngokweqile (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) kanye ne-D4 MTOS) izingcezu/iqembu ezivela ezingulubeni ezahlukene, ukuhlaziywa okubili kokuhlukahluka;**p < 0,01 по сравнению с нормальным растяжением). **p < 0.01 uma kuqhathaniswa nokwelula okuvamile). b Izithombe ezimele izingcezu zenhliziyo ezifakwe i-troponin-T kanye ne-WGA (kwesobunxele) kanye nokulinganiswa kosayizi weseli (kwesokudla) (n = 330 (D6 MTOS), 369 (D6 MTNorm) amaseli/iqembu elivela ezingcezu eziyi-10 ezahlukene ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-t-Student okunemisila emibili; ****p < 0.0001 uma kuqhathaniswa nokwelula okuvamile). b Izithombe ezimele izingcezu zenhliziyo ezifakwe i-troponin-T kanye ne-WGA (kwesobunxele) kanye nokulinganiswa kosayizi weseli (kwesokudla) (n = 330 (D6 MTOS), 369 (D6 MTNorm) amaseli/iqembu elivela ezingcezu eziyi-10 ezahlukene ezivela ezingulubeni ezahlukene, Ukuhlolwa kwe-t komfundi okunemisila emibili kwenziwa; ****p < 0.0001 uma kuqhathaniswa nokwelula okuvamile). b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного определения размера клеток30 (Dn = 6) 369 (D6 MTNorm) клеток/группу из 10 разных срезов от разных свиней, два- проводится хвостовой t-критерий Стьюной Стьюномном ****pыдента ****pымномнта ****pымномнта растяжением). b Izithombe ezimele izingxenye zenhliziyo ezifakwe i-troponin-T kanye ne-AZP (kwesobunxele) kanye nokulinganiswa kosayizi weseli (kwesokudla) (n = 330 (D6 MTOS), amaseli/iqembu le-369 (D6 MTNorm) elivela ezigabeni eziyi-10 ezahlukene ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-t komfundi okunemisila emibili; ****p < 0.0001 uma kuqhathaniswa nobunzima obujwayelekile). b 用肌钙蛋白-T 和WGA（左）和细胞大小量化（右）染色的心脏切片的代表性图像（右）染色的心脏切片的代表性图像（n =D60) MTOS) 0.0001). b Izithombe ezimele izingcezu zenhliziyo ezifakwe i-calcarein-T kanye ne-WGA (kwesobunxele) kanye nosayizi weseli (kwesokudla) (n = 330 (D6 MTOS), 369 kusukela kuzingcezu eziyi-10 ezahlukene (D6 MTNorm)) Amaseli/组，两方法有尾学生t test；uma kuqhathaniswa nokwelula okuvamile，****p < 0.0001). b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оценка размера клеток (S69 MT) (S69 MT) (D6 MTNorm) из 10 различных срезов от разных свиней Клетки/группа, двусторонние критерий Стьюдента; b Izithombe ezimele izingxenye zenhliziyo ezifakwe i-troponin-T kanye ne-AZP (kwesobunxele) kanye nokulinganiswa kosayizi weseli (kwesokudla) (n = 330 (D6 MTOS), 369 (D6 MTNorm) ezivela ezigabeni eziyi-10 ezahlukene ezivela ezingulubeni ezahlukene) Amaseli/iqembu, isici sokulinganisa esinemisila emibili t yomfundi; ****p < 0.0001 uma kuqhathaniswa nobunzima obujwayelekile). c Izithombe ezimele izingcezu zenhliziyo ze-MTOS zosuku 0 nosuku 6 ezifakwe i-immunolabeled ye-troponin-T kanye ne-NFATC4 kanye nokulinganiswa kokudluliselwa kwe-NFATC4 kuma-nuclei e-CM (n = 4 (D0), izingcezu/iqembu le-3 (D6 MTOS) ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-t-Student okunemisila emibili; *p < 0.05). c Izithombe ezimele izingcezu zenhliziyo ze-MTOS zosuku 0 nosuku 6 ezifakwe i-immunolabeled ye-troponin-T kanye ne-NFATC4 kanye nokulinganiswa kokudluliselwa kwe-NFATC4 kuma-nuclei e-CM (n = 4 (D0), izingcezu/iqembu le-3 (D6 MTOS) ezivela ezingulubeni ezahlukene, kwenziwa ukuhlolwa kwe-t-Student okunemisila emibili; *p < 0.05). c Репрезентативные изображения для срезов сердца 0 futhi 6 дней MTOS, иммуномеченых для тропонина-Т kanye NFATC4, kanye количественная оцнло NFATCED кавернозных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней , выполняется двусторонний t-критерий Стьюдента, *0; c Izithombe ezimele izingxenye zenhliziyo ezinsukwini ezingu-0 nezingu-6 ze-MTOS, ezifakwe i-immunolabeled ye-troponin-T kanye ne-NFATC4, kanye nokulinganiswa kokudluliselwa kwe-NFATC4 ku-nucleus yamaseli e-cavernous (n = 4 (D0), izingcezu/iqembu le-3 (D6 MTOS) ezivela ezingulubeni ezahlukene) zenze ukuhlolwa kwe-t komfundi okunemisila emibili; *p < 0.05). c 用于肌钙蛋白-T 和NFATC4 免疫标记的第0 天和第6 天MTOS 心脏切片的代表性图像，以及來自萌TC4猪细胞核的量化（n = 4 (D0),3 (D6 MTOS) 切片/组, 进行双尾学生t 检验；*p <0.05). c Izithombe ezimele ze-calcanin-T ne-NFATC4 immunolabeling 第0天和第6天MTOS izingcezu zenhliziyo, kanye ne-NFATC4 evela ku-NFATC4 易位至CM cell nucleus的quantity化 (n = 4 (D0), 3 (D6 MTOS)/时间双尾学生et 电影；*p <0.05). c Репрезентативные изображения срезов сердца MTOS ku-0 futhi 6 день для иммуномаркировки тропонином-Т kanye ne-NFATC4 kanye ne-количественная оценская оценская от разных свиней (n = 4 (D0), 3 (D6 MTOS) срез/группа, два- хвостатый t-критерий Стьюдента; *p <0,05). c Izithombe ezimele izingcezu zenhliziyo ze-MTOS ngosuku 0 no-6 zokufaka ama-immunolabeling e-troponin-T kanye ne-NFATC4 kanye nokulinganisa ukudluliselwa kwe-NFATC4 ku-nucleus ye-CM evela ezingulubeni ezahlukene (n = 4 (D0), 3 (D6 MTOS) izingcezu/iqembu, i-t-criterion enemisila emibili. Abafundi; *p < 0.05).Amabha amaphutha amelela isilinganiso ± ukuphambuka okujwayelekile.
Ucwaningo lwe-cardiovascular oluhunyushiwe ludinga amamodeli eselula akhiqiza ngokunembile indawo yenhliziyo. Kulolu cwaningo, kwasungulwa idivayisi ye-CTCM futhi yachazwa engakhuthaza izingxenye ezincane kakhulu zenhliziyo. Uhlelo lwe-CTCM luhlanganisa ukukhuthazwa kwe-electromechanical okuvumelaniswe ngokomzimba kanye nokucebisa uketshezi lwe-T3 kanye ne-Dex. Lapho izingxenye zenhliziyo yengulube zivezwa kulezi zici, ukusebenza kwazo, ubuqotho besakhiwo, umsebenzi we-metabolic, kanye nokubonakaliswa kokubhalwa kwahlala kufana nokwezicubu zenhliziyo ezintsha ngemva kwezinsuku ezingu-12 zokukhula. Ngaphezu kwalokho, ukwelula ngokweqile kwezicubu zenhliziyo kungabangela i-hypertrophy yenhliziyo ebangelwa yi-hyperextension. Sekukonke, le miphumela isekela indima ebalulekile yezimo ze-physiological culture ekugcineni i-phenotype evamile yenhliziyo futhi inikeze ipulatifomu yokuhlola izidakamizwa.
Izici eziningi zinegalelo ekudaleni indawo efanele yokusebenza nokusinda kwama-cardiomyocyte. Ezisobala kakhulu kulezi zici zihlobene (1) ukusebenzisana phakathi kwamaseli, (2) ukukhuthazwa kwe-electromechanical, (3) izici ze-humoral, kanye (4) ne-metabolic substrates. Ukusebenzisana kwe-physiological cell-to-cell kudinga amanethiwekhi ayinkimbinkimbi anezinhlangothi ezintathu ezinhlobo eziningi zamaseli asekelwa yi-extracellular matrix. Ukusebenzisana okunjalo okuyinkimbinkimbi kwamaseli kunzima ukukwakha kabusha in vitro ngokukhuliswa kwezinhlobo zamaseli ngazinye, kodwa kungatholakala kalula ngokusebenzisa uhlobo lwe-organotypic lwezingxenye zenhliziyo.
Ukwelula komshini kanye nokukhuthaza kagesi kwama-cardiomyocyte kubalulekile ekugcineni i-phenotype yenhliziyo33,34,35. Ngenkathi ukuvuselela komshini kusetshenziswe kabanzi ekulungiseni nasekuvuthweni kwe-hiPSC-CM, izifundo eziningana ezinhle muva nje ziye zazama ukuvuselela komshini izingcezu zenhliziyo ekukhuliseni kusetshenziswa ukulayisha okukodwa. Lezi zifundo zibonisa ukuthi ukulayisha okukodwa kwe-2D kunomthelela omuhle ku-phenotype yenhliziyo ngesikhathi sokukhula. Kulezi zifundo, izingxenye zenhliziyo zazigcwele amandla okudonsa e-isometric17, ukulayisha okukodwa kwe-auxotonic18, noma umjikelezo wenhliziyo wadalwa kabusha kusetshenziswa impendulo ye-force transducer kanye nokushayela kokucindezeleka. Kodwa-ke, lezi zindlela zisebenzisa ukwelula kwezicubu okukodwa ngaphandle kokulungiswa kwemvelo, okuholela ekucindezelweni kwezakhi zofuzo eziningi zenhliziyo noma ukuvezwa ngokweqile kwezakhi zofuzo ezihlotshaniswa nezimpendulo zokwelula ezingavamile. I-CTCM echazwe lapha inikeza isikhuthazo se-electromechanical se-3D esilingisa umjikelezo wenhliziyo wemvelo ngokwesikhathi somjikelezo kanye nokwelula komzimba (ukwelula okungu-25%, i-systole engu-40%, i-diastole engu-60%, kanye nokushaya okungu-72 ngomzuzu). Nakuba lokhu kukhuthaza kwemishini okunezinhlangothi ezintathu kuphela kunganele ukugcina ubuqotho bezicubu, inhlanganisela yokukhuthaza kwe-humoral kanye nokwemishini kusetshenziswa i-T3/Dex iyadingeka ukuze kulondolozwe ukusebenza kahle kwezicubu, ukusebenza, kanye nobuqotho bazo.
Izici ezihlekisayo zidlala indima ebalulekile ekuguquleni i-phenotype yenhliziyo yomuntu omdala. Lokhu kwaqokonyiswa ezifundweni ze-HiPS-CM lapho i-T3 ne-Dex zenezelwa khona kwimidiya yokukhulisa ukuze kusheshiswe ukuvuthwa kwamaseli. I-T3 ingathonya ukuthuthwa kwama-amino acid, ushukela kanye ne-calcium kuwo wonke ama-membranes eseli. Ngaphezu kwalokho, i-T3 ikhuthaza ukuvezwa kwe-MHC-α kanye nokwehla kwe-MHC-β, okukhuthaza ukwakheka kwama-myofibrils asheshayo kuma-cardiomyocytes avuthiwe uma kuqhathaniswa nama-myofibrils asheshayo e-CM yengane. Ukuntuleka kwe-T3 ezigulini ezine-hypothyroid kuholela ekulahlekelweni kwama-myofibrillar bands kanye nesilinganiso esinciphile sokuthuthukiswa kwethoni. I-Dex isebenza kuma-receptor e-glucocorticoid futhi iboniswe ukuthi yandisa ukugoba kwe-myocardial ezinhliziyweni ezihlukanisiwe ezigcwele amanzi; 38 lokhu kuthuthuka kucatshangwa ukuthi kuhlobene nomphumela ekungeneni kwe-calcium deposit-driven (SOCE) 39,40. Ngaphezu kwalokho, i-Dex ibopha kuma-receptor ayo, okubangela impendulo ebanzi yangaphakathi kweseli ecindezela ukusebenza komzimba kanye nokuvuvukala 30.
Imiphumela yethu ikhombisa ukuthi ukukhuthazwa kwemishini yomzimba (i-MS) kuthuthukise ukusebenza kokukhuliswa okuphelele uma kuqhathaniswa ne-Ctrl, kodwa kwehlulekile ukugcina ukuphila, ubuqotho besakhiwo, kanye nokubonakaliswa kwenhliziyo ezinsukwini eziyi-12 ekukhulisweni. Uma kuqhathaniswa ne-Ctrl, ukwengezwa kwe-T3 kanye ne-Dex kuma-CTCM (MT) cultures kuthuthukise ukuphila futhi kwagcina amaphrofayili afanayo okukhuliswa, ubuqotho besakhiwo, kanye nomsebenzi we-metabolic ngezicubu zenhliziyo ezintsha izinsuku eziyi-12. Ngaphezu kwalokho, ngokulawula izinga lokunwebeka kwezicubu, imodeli ye-hypertrophy yenhliziyo ebangelwa yi-hyperextension yadalwa kusetshenziswa i-STCM, ekhombisa ukuguquguquka kohlelo lwe-STCM. Kufanele kuqashelwe ukuthi yize ukuguqulwa kwenhliziyo kanye ne-fibrosis kuvame ukubandakanya izitho eziphelele amaseli azo ajikelezayo anganikeza ama-cytokines afanele kanye ne-phagocytosis nezinye izici zokuguqulwa, izingxenye zenhliziyo zisengalingisa inqubo ye-fibrotic ekuphenduleni ukucindezeleka kanye nokulimala. Lokhu kuye kwahlolwa ngaphambilini kule modeli yesilayidi senhliziyo. Kufanele kuqashelwe ukuthi amapharamitha e-CTCM angaguqulwa ngokushintsha ingcindezi/i-amplitude kagesi kanye nemvamisa ukuze kulingise izimo eziningi ezifana ne-tachycardia, i-bradycardia, kanye nokusekelwa kokujikeleza kwegazi kwemishini (inhliziyo engenawo ugesi). Lokhu kwenza uhlelo lube yindlela ephakathi yokuhlola izidakamizwa. Ikhono le-CTCM lokulingisa i-hypertrophy yenhliziyo ebangelwa ukuzikhandla ngokweqile livula indlela yokuhlola lolu hlelo ukuze kutholakale ukwelashwa komuntu siqu. Ekuphetheni, lolu cwaningo lubonisa ukuthi ukwelula komshini kanye nokukhuthaza i-humoral kubalulekile ekugcineni isiko lezingxenye zezicubu zenhliziyo.
Nakuba idatha evezwe lapha isikisela ukuthi i-CTCM iyipulatifomu ethembisayo kakhulu yokwenza imodeli ye-myocardium engaguquki, le ndlela yokukhulisa inemikhawulo ethile. Umkhawulo oyinhloko wokukhula kwe-CTCM ukuthi ubeka ukucindezeleka okuqhubekayo komshini ezingcezu, okuvimbela ikhono lokuqapha ngenkuthalo ukufinyela kwezingcezu zenhliziyo ngesikhathi somjikelezo ngamunye. Ngaphezu kwalokho, ngenxa yobukhulu obuncane bezingxenye zenhliziyo (7 mm), ikhono lokuhlola umsebenzi we-systolic ngaphandle kwezinhlelo zokukhulisa kusetshenziswa izinzwa zamandla zendabuko lilinganiselwe. Embhalweni wamanje, sinqoba kancane lo mkhawulo ngokuhlola i-voltage ye-optical njengesibonakaliso somsebenzi wokukhulisa. Kodwa-ke, lo mkhawulo uzodinga umsebenzi owengeziwe futhi ungabhekwana nawo esikhathini esizayo ngokwethula izindlela zokuqapha ukusebenza kwezingcezu zenhliziyo ekukhuliseni, njengemephu ye-optical esebenzisa amadayi azwela i-calcium kanye ne-voltage. Omunye umkhawulo we-CTCM ukuthi imodeli yokusebenza ayilawuli ukucindezeleka komzimba (ukulayisha kusengaphambili kanye nokulayisha ngemuva). Ku-CTCM, ingcindezi yabangelwa ngezindlela ezihlukile ukuze ikhiqize ukwelula komzimba okungu-25% ku-diastole (ukwelula okugcwele) kanye ne-systole (ubude bokufinyela ngesikhathi sokuvuselelwa kukagesi) ezicutshini ezinkulu kakhulu. Lo mkhawulo kufanele ususwe eziklanyweni ze-CTCM zesikhathi esizayo ngokucindezela okwanele ezicutshini zenhliziyo ezivela ezinhlangothini zombili nangokusebenzisa ubudlelwano obuqondile bokucindezela nomthamo okwenzeka emakamelweni enhliziyo.
Ukuguqulwa okubangelwa ukwelula kakhulu okubikwe kulo mbhalo wesandla kukhawulelwe ekulingiseni izimpawu ze-hyperstretch ezibangelwa ukwelula. Ngakho-ke, lo modeli ungasiza ekufundweni kokubonakaliswa kwe-hypertrophic okubangelwa ukwelula ngaphandle kwesidingo sezinto ezibangela ukuhlazeka noma ze-neural (ezingekho kulolu hlelo). Kudingeka izifundo ezengeziwe ukuze kwandiswe ukuphindaphindeka kwe-CTCM, isibonelo, ukukhulisa ndawonye amaseli omzimba, ukujikeleza kwe-plasma humoral factors, kanye nokugcinwa lapho kukhula ndawonye namaseli e-neuronal kuzothuthukisa amathuba okwenza imodeli yesifo nge-CTCM.
Kusetshenziswe izingulube eziyishumi nantathu kulolu cwaningo. Zonke izinqubo zezilwane zenziwe ngokuhambisana neziqondiso zezikhungo futhi zavunyelwa yiKomidi Lokunakekelwa Kwezilwane Nezokusetshenziswa Kwe-University of Louisville Institutional. I-aortic arch yaqiniswa futhi inhliziyo yagcotshwa nge-1 L ye-sterile cardioplegia (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U/mL heparin, pH kufika ku-7.4); Izinhliziyo zagcinwa esixazululweni se-cardioplegic esibandayo kuze kube yilapho zithuthelwa elabhorethri eqhweni okuvame ukuba ngaphansi kwemizuzu eyi-10. Izinhliziyo zagcinwa esixazululweni se-cardioplegic esibandayo kuze kube yilapho zithuthelwa elabhorethri eqhweni okuvame ukuba ngaphansi kwemizuzu eyi-10. сердца хранили в ледяном кардиоплегическом растворе до транспортировки в лабораторию на льду, что обычно занимает <10 мин. Izinhliziyo zazigcinwa esixazululweni se-cardioplegic esibandayo kuze kube yilapho zithuthwa ziye elabhorethri eqhweni, okuvame ukuthatha ngaphansi kwemizuzu eyi-10.将心脏保存在冰冷的心脏停搏液中，直到冰上运送到实验室，通常<10分钟。将心脏保存在冰冷的心脏停搏液中，直到冰上运送到实验室，通常<10分钟。 I-Держите сердца in ледяной кардиоплегии до транспортировки in лабораторию льду, обычно <10 мин. Gcina izinhliziyo ziseqhweni le-cardioplegia kuze kube yilapho zithuthwa ziye elabhorethri eqhweni, ngokuvamile ngaphansi kwemizuzu eyi-10.
Idivayisi ye-CTCM yathuthukiswa ngesofthiwe yokuklama okusizwa yikhompyutha ye-SolidWorks (CAD). Amakamelo okukhulisa, abahlukanisi kanye namakamelo omoya enziwe ngepulasitiki ye-acrylic ecacile ye-CNC. Indandatho yokusekela engu-7mm ububanzi yenziwe nge-polyethylene ephezulu (HDPE) phakathi nendawo futhi inomsele we-o-ring ukuze ifake indandatho ye-silicone o-ring esetshenziselwa ukuvala imidiya engaphansi. I-membrane encane ye-silica ihlukanisa igumbi lokukhulisa nepuleti lokuhlukanisa. I-membrane ye-silicone inqunywe nge-laser kusuka ephepheni le-silicone elingu-0.02″ ubukhulu futhi inobunzima obungu-35A. Ama-gasket e-silicone angaphansi naphezulu anqunywe nge-laser kusuka ephepheni le-silicone eliwu-1/16″ ubukhulu futhi anobunzima obungu-50A. Izikulufo zensimbi engagqwali ezingu-316L kanye namakinati ephiko asetshenziselwa ukubopha ibhloko nokudala uphawu olungangeni moya.
Ibhodi lesifunda eliphrintiwe elizinikele (i-PCB) lenzelwe ukuhlanganiswa nohlelo lwe-C-PACE-EM. Amasokhethi okuxhumanisa omshini waseSwitzerland ku-PCB axhunywe kuma-electrode e-graphite ngezintambo zethusi ezifakwe isiliva kanye nezikulufo zethusi ezingu-0-60 ezifakwe kuma-electrode. Ibhodi lesifunda eliphrintiwe lifakwa ekhava yephrinta ye-3D.
Idivayisi ye-CTCM ilawulwa yi-programmable pneumatic actuator (PPD) edala umfutho wokujikeleza kwegazi olawulwayo ofana nomjikelezo wenhliziyo. Njengoba umfutho ngaphakathi kwegumbi lomoya ukhuphuka, ulwelwesi lwe-silicone oluguquguqukayo luyanda phezulu, luphoqa indawo ngaphansi kwendawo yezicubu. Indawo yezicubu izobe seyelulwa yilokhu kukhishwa koketshezi, okulingisa ukwanda ngokomzimba kwenhliziyo ngesikhathi se-diastole. Esicongweni sokuphumula, ukukhuthazwa kukagesi kwasetshenziswa ngama-electrode e-graphite, okunciphisa umfutho ekamelweni lomoya futhi kwabangela ukufinyela kwezingxenye zezicubu. Ngaphakathi kwepayipi kukhona i-valve ye-hemostatic enenzwa yokucindezela ukuthola umfutho ohlelweni lomoya. Umfutho ozwa yinzwa yokucindezela usetshenziswa kumqoqi wedatha oxhunywe kwi-laptop. Lokhu kuvumela ukuqapha okuqhubekayo komfutho ngaphakathi kwegumbi legesi. Lapho umfutho wegumbi ophezulu ufinyelelwe (ojwayelekile ongu-80 mmHg, 140 mmHg OS), idivayisi yokuthola idatha yayalwa ukuba ithumele isignali ohlelweni lwe-C-PACE-EM ukuze ikhiqize isignali ye-voltage ye-biphasic engu-2 ms, esethwe ku-4 V.
Kutholwe izingxenye zenhliziyo futhi izimo zokukhula emithonjeni engu-6 zenziwa kanje: Dlulisa izinhliziyo ezivuniwe zisuka emthonjeni wokudlulisa ziye ethileyini equkethe i-cardioplegia ebandayo (4° C.). I-ventricle yesobunxele yahlukaniswa nge-blade engenasici yasikwa yaba yizicucu ezingu-1-2 cm3. Lawa mabhulokhi ezicubu anamathiselwe ezisekelweni zezicubu nge-adhesive yezicubu futhi afakwa ebhavini lezicubu ezincane ezidlidlizayo eliqukethe isixazululo sikaTyrode futhi afakwa umoya-mpilo njalo (3 g/L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g). ), 6 mM KCl (0.447 g), 10 mM D-glucose (1.86 g), 10 mM HEPES (2.38 g), 1 mM MgCl2 (1 ml 1 M solution), 1.8 mM CaCl2 (1.8 ml 1 M solution), kuze kufike ku-1 L ddH2O). I-microtome edlidlizayo yayimiselwe ukusika izingcezu ezingama-300 µm ubukhulu ngemvamisa engu-80 Hz, ubukhulu bokudlidliza obuvundlile obungu-2 mm, kanye nesivinini esithuthukisiwe esingu-0.03 mm/s. Ibhavu lezicubu lalizungezwe yiqhwa ukuze kugcinwe ikhambi lipholile futhi izinga lokushisa ligcinwe ku-4°C. Dlulisa izingxenye zezicubu kusuka ebhavuni le-microtome uye ebhavuni lokufukula eliqukethe ikhambi le-Tyrode elinomoya-mpilo njalo eqhweni kuze kube yilapho kutholakala izingxenye ezanele zepuleti elilodwa lokufukula. Kuma-transwell cultures, izingxenye zezicubu zinamathiselwe ezisekelweni ze-polyurethane ezihlanzekile ezingama-6 mm ububanzi futhi zafakwa ku-6 ml we-medium elungiselelwe (199 medium, 1x ITS supplement, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-alkaline kanye ne-2X antibiotic-antifungal ). Ukuvuselelwa kukagesi (10 V, imvamisa engu-1.2 Hz) kwafakwa ezingxenyeni zezicubu nge-C-Pace. Ezimweni ze-TD, i-T3 entsha kanye ne-Dex kwanezelwa ku-100 nM kanye ne-1 μM ekushintsheni ngakunye okuphakathi. Indawo igcwele umoya-mpilo ngaphambi kokushintshwa izikhathi ezintathu ngosuku. Izingxenye zezicubu zakhuliswa ku-incubator ku-37°C kanye ne-5% CO2.
Kuma-CTCM cultures, izingxenye zezicubu zabekwa kuphrinta ye-3D eyenziwe ngokwezifiso esitsheni sikaPetri equkethe isixazululo sikaTyrode esiguquliwe. Le divayisi yenzelwe ukwandisa usayizi wesilayi senhliziyo ngo-25% wendawo yendandatho yokusekela. Lokhu kwenziwa ukuze izingxenye zenhliziyo zinganwebi ngemva kokudluliselwa kusuka kusisombululo sikaTyrode kuya endaweni ephakathi nendawo nangesikhathi se-diastole. Usebenzisa iglue ye-histoacrylic, izingxenye ezingama-300 µm ubukhulu zaqiniswa endandatho yokusekela engu-7 mm ububanzi. Ngemva kokuhlanganisa izingxenye zezicubu endandatho yokusekela, nquma izingxenye zezicubu ezengeziwe bese ubeka izingxenye zezicubu ezinamathiselwe emuva ebhavini lesisombululo sikaTyrode eqhweni (4°C) kuze kube yilapho izingxenye ezanele sezilungiselelwe idivayisi eyodwa. Isikhathi sokucubungula sonke samadivayisi akufanele sidlule amahora ama-2. Ngemva kokuthi izingxenye zezicubu ezi-6 zinamathiselwe emasongweni azo okusekela, idivayisi ye-CTCM yahlanganiswa. Igumbi lokukhulisa i-CTCM ligcwaliswa kusengaphambili nge-medium engu-21 ml efakwe umoya-mpilo. Dlulisa izingxenye zezicubu ekamelweni lokukhulisa bese ususa ngokucophelela noma yimaphi amabhamuza omoya nge-pipette. Ingxenye yezicubu ibe isiqondiswa emgodini bese icindezelwa ngobumnene endaweni yayo. Ekugcineni, beka isivalo se-electrode kudivayisi bese udlulisela idivayisi ku-incubator. Bese uxhuma i-CTCM ku-air tube kanye nohlelo lwe-C-PACE-EM. I-pneumatic actuator iyavuleka bese i-air valve ivula i-CTCM. Uhlelo lwe-C-PACE-EM lwalungiselelwa ukuletha ama-V angu-4 ku-1.2 Hz ngesikhathi sokuhamba nge-biphasic isikhathi esingama-ms amabili. I-medium yashintshwa kabili ngosuku kanti ama-electrode ashintshwa kanye ngosuku ukuze kugwenywe ukunqwabelana kwe-graphite kuma-electrode. Uma kudingeka, izingxenye zezicubu zingasuswa emithonjeni yazo yokukhulisa ukuze kukhishwe noma yimaphi amabhamuza omoya okungenzeka ukuthi awele ngaphansi kwawo. Ezimweni zokwelapha ze-MT, i-T3/Dex yengezwa isha ngokushintsha ngakunye okuphakathi nge-100 nM T3 kanye ne-1 μM Dex. Amadivayisi e-CTCM akhuliswa ku-incubator ku-37°C kanye ne-5% CO2.
Ukuze kutholakale izindlela ezinwetshiwe zezingcezu zenhliziyo, kwasungulwa uhlelo lwekhamera olukhethekile. Ikhamera ye-SLR (Canon Rebel T7i, Canon, Tokyo, Japan) yasetshenziswa nelensi ye-Navitar Zoom 7000 18-108mm macro (Navitar, San Francisco, CA). Ukubona ngeso lengqondo kwenziwa ekushiseni kwegumbi ngemuva kokushintsha indawo nge-medium entsha. Ikhamera ibekwe ekhoneni elingu-51° futhi ividiyo iqoshwa kumafreyimu angu-30 ngomzuzwana. Okokuqala, isofthiwe yomthombo ovulekile (MUSCLEMOTION43) yasetshenziswa ne-Image-J ukuze kulinganiswe ukunyakaza kwezingcezu zenhliziyo. Imaski yadalwa kusetshenziswa i-MATLAB (MathWorks, Natick, MA, USA) ukuze kuchazwe izindawo ezithakazelisayo zokushaya izingcezu zenhliziyo ukuze kugwenywe umsindo. Izingcezu zemaski ezihlukaniswe ngesandla zifakwa kuzo zonke izithombe ngokulandelana kohlaka bese zidluliselwa ku-plug-in ye-MUSCLEMOTION. I-Muscle Motion isebenzisa ukuqina okumaphakathi kwamaphikseli kuhlaka ngalunye ukuze kulinganiswe ukunyakaza kwayo maqondana nohlaka lokubhekisela. Idatha yaqoshwa, yahlungwa futhi yasetshenziswa ukulinganisa isikhathi somjikelezo nokuhlola ukwelula kwezicubu ngesikhathi somjikelezo wenhliziyo. Ividiyo eqoshiwe yacutshungulwa ngemuva kokusebenzisa isihlungi sedijithali se-zero-phase yokuqala. Ukuze kulinganiswe ukwelula kwezicubu (i-peak-to-peak), ukuhlaziywa kwe-peak-to-peak kwenziwa ukuze kuhlukaniswe phakathi kweziqongo kanye ne-troughs esignalini eqoshiwe. Ngaphezu kwalokho, ukuwohloka kwenziwa kusetshenziswa i-polynomial ye-6th order ukuze kuqedwe ukuzulazula kwesignali. Ikhodi yohlelo yathuthukiswa ku-MATLAB ukuze kunqunywe ukunyakaza kwezicubu zomhlaba wonke, isikhathi somjikelezo, isikhathi sokuphumula, kanye nesikhathi sokufinyela (Ikhodi Yohlelo Engeziwe 44).
Ukuze kuhlaziywe ukucindezeleka, sisebenzisa amavidiyo afanayo adalelwe ukuhlolwa kokwelula komshini, siqale salandela izithombe ezimbili ezimele iziqongo zokunyakaza (amaphuzu aphezulu kakhulu (aphezulu) naphansi kakhulu (aphansi) okunyakaza) ngokusho kwesofthiwe ye-MUSCLEMOTION. Sabe sesihlukanisa izifunda zezicubu futhi sasebenzisa uhlobo lwe-algorithm yokufiphaza ezicutshini ezihlukanisiwe (Isithombe Esingeziwe 2a). Izicubu ezihlukanisiwe zabe sezihlukaniswa zaba yizindawo ezingaphansi eziyishumi, futhi ukucindezeleka ebusweni ngabunye kwabalwa kusetshenziswa i-equation elandelayo: Ukucindezeleka = (Sup-Sdown)/Sdown, lapho i-Sup ne-Sdown kuyibanga lesimo ukusuka ezithunzini eziphezulu nezaphansi zendwangu, ngokulandelana (Isithombe Esingeziwe .2b).
Izingxenye zenhliziyo zaqiniswa ku-4% we-paraformaldehyde amahora angu-48. Izicubu eziqinile zancishiswa amanzi ku-10% kanye no-20% we-sucrose ihora eli-1, bese kuba ku-30% we-sucrose ubusuku bonke. Izingxenye zabe sezifakwa ku-optimum cutting temperature compound (OCT compound) futhi zaqandiswa kancane kancane ku-isopentane/dry ice bath. Gcina amabhlogo okushumeka e-OCT ku--80 °C kuze kube yilapho kuhlukaniswa. Amaslayidi alungiswa njengezingxenye ezinobukhulu obungu-8 μm.
Ukuze ususe i-OCT ezingxenyeni zenhliziyo, fudumeza amaslayidi ku-heating block ku-95 °C imizuzu emi-5. Faka i-1 ml ye-PBS ku-slide ngayinye bese uyifukamela imizuzu engama-30 ekushiseni kwegumbi, bese ugcwalisa izingxenye ngokusetha i-0.1% Triton-X ku-PBS imizuzu eyi-15 ekushiseni kwegumbi. Ukuze uvimbele ama-antibodies angaqondile ukuthi ahlangane nesampula, engeza i-1 ml yesisombululo se-BSA esingu-3% kumaslayidi bese uyifukamela ihora eli-1 ekushiseni kwegumbi. I-BSA yabe isisuswa futhi amaslayidi agezwa nge-PBS. Maka isampula ngayinye ngepensela. Ama-antibodies ayinhloko (axutshwe ngo-1:200 ku-1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) kanye ne-troponin-T (Thermo Scientific; #MA5-12960) angezwe ngemizuzu engama-90, kwabe sekufakwa ama-antibodies esibili (axutshwe ngo-1:200 ku-1% BSA) ngokumelene negundane i-Alexa Fluor 488 (Thermo Scientific; #A16079), ngokumelene nonogwaja i-Alexa Fluor 594 (Thermo Scientific; #T6391) imizuzu engama-90 eyengeziwe. Ahlanzwe izikhathi ezi-3 nge-PBS. Ukuze sihlukanise ukudaya okuqondiwe kusuka ngemuva, sisebenzise i-antibody yesibili kuphela njengokulawula. Ekugcineni, kwafakwa i-DAPI nuclear stain futhi amaslayidi afakwa ku-vectashield (Vector Laboratories) futhi avalwa nge-nail polish. -x magnification) kanye ne-Keyence microscope enokukhulisa okungu-40x.
I-WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) ku-5 μg/ml ku-PBS yasetshenziselwa ukufaka imibala ku-WGA futhi yafakwa ezigabeni eziqinile imizuzu engama-30 ekushiseni kwegumbi. Amaslayidi abe esegezwa nge-PBS kwathi i-Sudan black yafakwa kuslayidi ngasinye futhi yafakwa esikhumbeni imizuzu engama-30. Amaslayidi abe esegezwa nge-PBS kwathi i-vectashield embedding medium yanezelwa. Amaslayidi aboniswe nge-microscope ye-Keyence ku-40x magnification.
I-OCT isusiwe kumasampula njengoba kuchaziwe ngenhla. Ngemva kokususa i-OCT, cwilisa amaslayidi esisombululo sikaBouin ubusuku bonke. Amaslayidi abese ehlanzwa ngamanzi acwecwe ihora eli-1 bese efakwa esixazululweni se-Bibrich aloe acid fuchsin imizuzu eyi-10. Amaslayidi abese ehlanzwa ngamanzi acwecwe bese efakwa esixazululweni se-5% phosphomolybdenum/5% phosphotungstic acid imizuzu eyi-10. Ngaphandle kokuhlanza, dlulisela amaslayidi ngqo esixazululweni esiluhlaza okwesibhakabhaka se-aniline imizuzu eyi-15. Amaslayidi abese ehlanzwa ngamanzi acwecwe bese efakwa esixazululweni se-1% acetic acid imizuzu eyi-2. Amaslayidi omisiwe ku-200 N ethanol bese edluliselwa ku-xylene. Amaslayidi anombala abonakala kusetshenziswa i-microscope ye-Keyence enenhloso eyi-10x. Iphesenti lendawo ye-Fibrosis lalinganiswa kusetshenziswa isofthiwe ye-Keyence Analyzer.
I-CyQUANT™ MTT Cell Viability Assay (Invitrogen, Carlsbad, CA), inombolo yekhathalogi V13154, ngokwenqubo yomenzi enezinguquko ezithile. Ikakhulukazi, kwasetshenziswa ukuhlinzwa okunobubanzi obungu-6 mm ukuqinisekisa ubukhulu bezicubu obufanayo ngesikhathi sokuhlaziywa kwe-MTT. Izicubu zafakwa ngazinye emigodini yepuleti enezinsika ezingu-12 equkethe i-MTT substrate ngokwenqubo yomenzi. Izingxenye zifakwa ku-37° C. amahora ama-3 futhi izicubu eziphilayo ziguqula i-MTT substrate ukuze zenze i-compound ensomi ye-formazan. Faka esikhundleni isixazululo se-MTT nge-1 ml ye-DMSO bese uyifaka ku-37°C imizuzu eyi-15 ukuze kukhishwe i-formazan ensomi ezingxenyeni zenhliziyo. Amasampula ancishiswe ngo-1:10 ku-DMSO emapuletini aphansi acacile angu-96 kanye nobukhulu bombala onsomi obulinganiswa ku-570 nm kusetshenziswa i-Cytation plate reader (BioTek). Ukufundwa kwalungiswa kwaba yisisindo sengxenye ngayinye yenhliziyo.
I-heart slice media ithathelwe indawo yi-media equkethe i-1 μCi/ml [5-3H]-glucose (Moravek Biochemicals, Brea, CA, USA) ukuze kuhlolwe ukusetshenziswa kwe-glucose njengoba kuchaziwe ngaphambilini. Ngemva kwamahora ama-4 okufukamela, engeza i-100 µl ye-medium ku-microcentrifuge tube evulekile equkethe i-100 µl ye-0.2 N HCl. Ngemuva kwalokho i-tube yafakwa ku-scintillation tube equkethe i-500 μl ye-dH2O ukuze iphele [3H]2O amahora angama-72 ku-37°C. Bese ususa i-microcentrifuge tube ku-scintillation tube bese ufaka i-10 ml ye-scintillation fluid. Izibalo ze-scintillation zenziwa kusetshenziswa i-Tri-Carb 2900TR liquid scintillation analyzer (Packard Bioscience Company, Meriden, CT, USA). Ukusetshenziswa kwe-glucose kwabe sekubalwa kucatshangelwa umsebenzi othize we-[5-3H]-glucose, ukulingana okungaphelele kanye nesizinda, ukuncishiswa kwe-[5-3H]-ku-glucose engabhalwanga, kanye nokusebenza kahle okuphikisana ne-scintillation. Imininingwane ilinganiswa ngokulingana nesisindo sezingxenye zenhliziyo.
Ngemva kokuhlanganiswa kwezicubu eTrizol, i-RNA yahlukaniswa nezingxenye zenhliziyo kusetshenziswa i-Qiagen miRNeasy Micro Kit #210874 ngokwenqubo yomenzi. Ukulungiswa komtapo wolwazi we-RNAsec, ukulandelana kanye nokuhlaziywa kwedatha kwenziwa kanje:
I-1 μg ye-RNA ngesampula ngayinye yasetshenziswa njengezinto zokuqala zokulungiselela umtapo wezincwadi we-RNA. Imitapo yolwazi yokulandelelana yakhiqizwa kusetshenziswa i-NEBHext UltraTM RNA Library Preparation Kit ye-Illumina (NEB, USA) kulandela izincomo zomenzi, futhi amakhodi enkomba angeziwe ku-attribute sequences yesampula ngayinye. Kafushane, i-mRNA yahlanzwa ku-RNA iyonke kusetshenziswa ubuhlalu obunamandla obuxhunywe ngama-poly-T oligonucleotides. Ukuqhekeka kwenziwa kusetshenziswa ama-divalent cations ekushiseni okuphezulu ku-NEBHext First Strand Synthesis Reaction Buffer (5X). I-cDNA yokuqala ye-strand yahlanganiswa kusetshenziswa ama-primer angahleliwe e-hexamer kanye ne-M-MuLV reverse transcriptase (RNase H-). I-cDNA yesibili ye-strand yahlanganiswa kusetshenziswa i-DNA polymerase I kanye ne-RNase H. Ama-overhang asele aguqulwa abe yiziphetho ezibuthuntu ngomsebenzi we-exonuclease/polymerase. Ngemuva kokwehliswa kokuphela kwe-3′ kwengxenye ye-DNA, i-NEBHext Adapter enesakhiwo se-hairpin loop inamathiselwe kuyo ukuze ilungiselele i-hybridization. Ukuze kukhethwe izingcezu ze-cDNA ezinobude obukhethwayo, izingcezu zomtapo wolwazi eziyi-150-200 bp. zahlanzwa kusetshenziswa uhlelo lwe-AMPure XP (Beckman Coulter, Beverly, USA). Kwabe sekusetshenziswa i-3 μl USER Enzyme (NEB, USA) ene-cDNA ekhethiwe ngobukhulu ene-adaptha imizuzu eyi-15 ku-37°C bese kuba imizuzu emi-5 ku-95°C ngaphambi kwe-PCR. I-PCR yabe seyenziwa kusetshenziswa i-Phusion High-Fidelity DNA polymerase, ama-primer e-PCR e-universal, kanye nama-primer e-Index (X). Ekugcineni, imikhiqizo ye-PCR yahlanzwa (uhlelo lwe-AMPure XP) futhi ikhwalithi yomtapo wolwazi yahlolwa ohlelweni lwe-Agilent Bioanalyzer 2100. Umtapo wolwazi we-cDNA wabe usuhlelwa kusetshenziswa i-Novaseq sequencer. Amafayela ezithombe ezingavuthiwe avela ku-Illumina aguqulwa abe ama-reads angavuthiwe kusetshenziswa i-CASAVA Base Calling. Idatha engavuthiwe igcinwa kumafayela efomethi ye-FASTQ(fq) aqukethe ama-read sequences kanye nezimfanelo zesisekelo ezihambisanayo. Khetha i-HISAT2 ukuze ufanise ukufundwa kokulandelana okuhlungiwe ne-genome yereferensi ye-Sscrofa11.1. Ngokuvamile, i-HISAT2 isekela ama-genome anoma yiluphi usayizi, kufaka phakathi ama-genome amakhulu kunezisekelo eziyizigidi eziyizinkulungwane ezine, futhi amanani azenzakalelayo asethelwe amapharamitha amaningi. Ukuhlanganisa ukufundwa kusuka kudatha ye-RNA Seq kungalungiswa kahle kusetshenziswa i-HISAT2, uhlelo olushesha kakhulu olutholakalayo njengamanje, ngokunemba okufanayo noma okungcono kunanoma iyiphi enye indlela.
Ubuningi bemibhalo eqoshiwe bubonisa ngqo izinga lokuvezwa kwezakhi zofuzo. Amazinga okuvezwa kwezakhi zofuzo ahlolwa ngobuningi bemibhalo eqoshiwe (inani lokulandelana) ehlotshaniswa ne-genome noma i-exons. Inani lokufunda lilingana namazinga okuvezwa kwezakhi zofuzo, ubude bezakhi zofuzo, kanye nokujula kokulandelana. I-FPKM (izingcezu ngamabhangqa esisekelo ayinkulungwane wemibhalo eqoshiwe ehlelwe ngamabhangqa esisekelo ayisigidi) zabalwa futhi amanani e-P okuvezwa okuhlukile anqunywa kusetshenziswa iphakheji ye-DESeq2. Sabe sesibala izinga lokutholwa okungamanga (i-FDR) yenani ngalinye le-P sisebenzisa indlela ye-Benjamini-Hochberg9 ngokusekelwe ku-R-function eyakhelwe ngaphakathi ethi “p.adjust”.
I-RNA ehlukaniswe nezingxenye zenhliziyo yaguqulwa yaba yi-cDNA ekugxilweni okungu-200 ng/μl kusetshenziswa ingxube ye-SuperScript IV Vilo Master evela ku-Thermo (Thermo, cat. no. 11756050). I-Quantitative RT-PCR yenziwe kusetshenziswa ipuleti yokusabela ecacile ye-Applied Biosystems Endura Plate Microamp 384-well (Thermo, cat. no. 4483319) kanye ne-microamp optical adhesive (Thermo, cat. no. 4311971). Ingxube yokusabela yayiqukethe ingxube ye-5 µl Taqman Fast Advanced Master (Thermo, cat # 4444557), i-0.5 µl Taqman Primer kanye ne-3.5 µl H2O exutshwe ngomthombo ngamunye. Kwaqhutshwa imijikelezo ejwayelekile ye-qPCR futhi amanani e-CT alinganiswa kusetshenziswa ithuluzi le-PCR le-Applied Biosystems Quantstudio 5 real-time (384-well module; umkhiqizo # A28135). Ama-primer e-Taqman athengwe ku-Thermo (GAPDH (Ss03375629_u1), PARP12 (Ss06908795_m1), PKDCC (Ss06903874_m1), CYGB (Ss06900188_m1), RGL1 (Ss06868890_m1), ACTN1 (Ss01009508_mH), GATA4 (Ss03383805_u1), GJA1 (Ss03374839_u1), COL1A2 (Ss03375009_u1 ), COL3A1 (Ss04323794_m1), ACTA2 (Ss04245588_m1) Amanani e-CT awo wonke amasampula ahlelwe ngendlela evamile ku-housekeeping gene GAPDH.
Ukukhishwa kwe-NT-ProBNP kwabezindaba kuhlolwe kusetshenziswa ikhithi ye-NT-ProBNP (ingulube) (Cat. No. MBS2086979, MyBioSource) ngokwenqubo yomenzi. Kafushane, ama-250 µl esampula ngayinye kanye nezinga elijwayelekile kwanezelwa kabili emthonjeni ngamunye. Ngokushesha ngemva kokufaka isampula, engeza ama-50 µl e-Assay Reagent A emthonjeni ngamunye. Nyikinya ipuleti kancane bese uvala nge-sealant. Amaphilisi abe esefakwa ku-37°C ihora eli-1. Bese uphefumula ikhambi bese ugeza imithombo izikhathi ezine nge-350 µl yesisombululo sokugeza esingu-1X, ufaka ikhambi lokugeza imizuzu eyi-1-2 isikhathi ngasinye. Bese ufaka ama-100 µl e-Assay Reagent B emthonjeni ngamunye bese uvala nge-plate sealant. Ithebhulethi yanyakaziswa kancane futhi yafakwa ku-37°C imizuzu engama-30. Faka isixazululo bese ugeza imithombo izikhathi ezi-5 ngesisombululo sokugeza esingu-350 µl esingu-1X. Engeza isixazululo esingu-90 µl se-substrate emthonjeni ngamunye bese uvala ipuleti. Faka ipuleti ku-37°C imizuzu eyi-10-20. Engeza isixazululo sokumisa esingu-50 µl emthonjeni ngamunye. Ipuleti lalinganiswa ngokushesha kusetshenziswa isethi yesifundi sepuleti se-Cytation (BioTek) ku-450 nm.
Ukuhlaziywa kwamandla kwenziwe ukuze kukhethwe osayizi beqembu abazohlinzeka ngamandla angaphezu kuka-80% ukuthola ushintsho oluphelele oluyi-10% kupharamitha enesilinganiso samaphutha sohlobo lwe-5% Uhlobo I. Ukuhlaziywa kwamandla kwenziwe ukuze kukhethwe osayizi beqembu abazohlinzeka ngamandla angaphezu kuka-80% ukuthola ushintsho oluphelele oluyi-10% kupharamitha enesilinganiso samaphutha sohlobo lwe-5% Uhlobo I. I-Aнализ мощности был выполнен для выбора размеров групп, которые обеспечат >80% мощности для обнаружения 10% абеспечат частотой ошибок типа I. Ukuhlaziywa kwamandla kwenziwe ukuze kukhethwe osayizi beqembu abazohlinzeka ngamandla angaphezu kuka-80% ukuthola ushintsho oluphelele lwamapharamitha angu-10% ngesilinganiso samaphutha sohlobo lwe-5% I.进行功效分析以选择将提供> 80％功效以检测参数中10％绝对变化和5％I型错误率的。进行功效分析以选择将提供> 80％功效以检测参数中10％绝对变化和5％I型错误率的。 Был проведен анализ мощности для выбора размера группы, который обеспечил бы > 80% мощности для обнаружения 10% абеспечил 5% isaphulelo sika-типа I. Kwenziwe ukuhlaziywa kwamandla ukuze kukhethwe usayizi weqembu ozohlinzeka ngamandla angaphezu kuka-80% ukuthola ushintsho oluphelele lwamapharamitha angu-10% kanye nesilinganiso samaphutha sohlobo I esingu-5%.Izingxenye zezicubu zakhethwa ngokungahleliwe ngaphambi kokuhlolwa. Zonke izihlaziyi zazingabonakali esimweni futhi amasampula ahlukaniswa kuphela ngemva kokuba yonke idatha isihlaziyiwe. Isofthiwe ye-GraphPad Prism (eSan Diego, CA) yasetshenziswa ukwenza zonke izihlaziyi zezibalo. Kuzo zonke izibalo, amanani e-p abhekwa njengabalulekile kumanani angaphansi kuka-0.05. Kuzo zonke izibalo, amanani e-p abhekwa njengabalulekile kumanani angaphansi kuka-0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Kuzo zonke izibalo, amanani e-p abhekwa njengabalulekile kumanani angaphansi kuka-0.05.对于所有统计数据，p 值在值<0.05 时被认為是显着的。对于所有统计数据，p 值在值<0.05 时被认為是显着的。 Для всей статистики p-значения считались значимыми при значениях <0,05. Kuzo zonke izibalo, amanani e-p abhekwa njengabalulekile kumanani angaphansi kuka-0.05.Ukuhlolwa kwe-t komfundi onemisila emibili kwenziwe kudatha ngokuqhathanisa okubili kuphela. I-ANOVA enendlela eyodwa noma enezindlela ezimbili yasetshenziswa ukunquma ukubaluleka phakathi kwamaqembu amaningi. Lapho kwenziwa izivivinyo zangemva kwe-hoc, ukulungiswa kukaTukey kwasetshenziswa ekubhekeni ukuqhathanisa okuningi. Idatha ye-RNAsec inokucabangela okukhethekile kwezibalo lapho kubalwa i-FDR kanye ne-p.adjust njengoba kuchaziwe esigabeni sezindlela.
Ukuze uthole ulwazi olwengeziwe mayelana nomklamo wocwaningo, bheka isifinyezo se-Nature Research Report esixhunywe kulesi sihloko.