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Kunesidingo sesistimu ye-in vitro ethembekile engakhiqiza kabusha ngokunembile indawo ephilayo yenhliziyo ukuze kuhlolwe izidakamizwa.Ukutholakala okulinganiselwe kwezinhlelo zesiko lezicubu zenhliziyo yomuntu kuye kwaholela ekuchazeni okungalungile kwemiphumela yezidakamizwa zenhliziyo.Lapha, senze imodeli yesiko lezicubu zenhliziyo (CTCM) eyenza i-electromechanical ivuse izingcezu zenhliziyo futhi idlulele ukunwebeka komzimba phakathi nezigaba ze-systolic ne-diastolic zomjikelezo wenhliziyo.Ngemva kwezinsuku ezingu-12 zesiko, le ndlela yathuthukisa kancane ukusebenza kwezingxenye zenhliziyo, kodwa ayizange ilondoloze ngokugcwele ubuqotho bazo besakhiwo.Ngakho-ke, ngemva kokuhlolwa kwe-molecule encane, sithole ukuthi ukungezwa kwe-100 nM triiodothyronine (T3) kanye ne-1 μM i-dexamethasone (i-Dex) ku-medium yethu kugcina i-microstructure yezigaba izinsuku ezingu-12.Ngokuhambisana nokwelashwa kwe-T3/Dex, uhlelo lwe-CTCM lugcine amaphrofayili okubhalwa phansi, ukusebenza kahle, umsebenzi we-metabolic, nobuqotho besakhiwo ezingeni elifanayo nezicubu zenhliziyo ezintsha izinsuku eziyi-12.Ukwengeza, ukwelula ngokweqile kwezicubu zenhliziyo emasikweni kubangela ukubonakaliswa kwenhliziyo ye-hypertrophic, okunikeza ubufakazi bekhono le-CTCM ukulingisa izimo ze-hypertrophic ezibangelwa ukwelula kwenhliziyo.Sengiphetha, i-CTCM ingamodela i-physiology kanye ne-pathophysiology yenhliziyo ngesiko phakathi nesikhathi eside, ivumele ukuhlolwa kwezidakamizwa okuthembekile.
Ngaphambi kocwaningo lomtholampilo, izinhlelo ze-in vitro ezithembekile ziyadingeka ezingakhiqiza kabusha ngokunembile indawo yokuphila yenhliziyo yomuntu.Amasistimu anjalo kufanele alingise ukunwebeka kwemishini okushintshile, izinga lokushaya kwenhliziyo, kanye nezakhiwo ze-electrophysiological.Amamodeli ezilwane avame ukusetshenziswa njengenkundla yokuhlola ye-cardiac physiology enokwethenjelwa okulinganiselwe ekuboniseni imiphumela yezidakamizwa enhliziyweni yomuntu1,2.Ekugcineni, i-Ideal Cardiac Tissue Culture Experimental Model (CTCM) iyimodeli ezwela kakhulu futhi eqondile yokungenelela okuhlukahlukene kwezokwelapha kanye nemithi, ikhiqiza kabusha ngokunembile i-physiology kanye ne-pathophysiology yenhliziyo yomuntu3.Ukungabikho kohlelo olunjalo kunciphisa ukutholakala kwemithi emisha yokwelapha isifo senhliziyo4,5 futhi kuholele ekubeni nezidakamizwa ze-cardiotoxicity njengesizathu esikhulu sokuphuma emakethe6.
Kule minyaka eyishumi edlule, izidakamizwa eziyisishiyagalombili ezingezona ezenhliziyo ziye zahoxiswa ekusetshenzisweni komtholampilo ngoba zibangela ukwelulwa kwesikhawu se-QT okuholela ku-ventricular arrhythmias nokufa kungazelelwe7.Ngakho-ke, kunesidingo esikhulayo samasu okuhlola angaphambi komtholampilo ukuze kuhlolwe ukusebenza kahle kwenhliziyo kanye nobuthi.Ukusetshenziswa kwakamuva kwe-pluripotent stem cell-derived cardiomyocytes (i-hiPS-CM) eyenziwe ngumuntu ekuhlolweni kwezidakamizwa nokuhlola ubuthi kunikeza isisombululo esiyingxenye kule nkinga.Kodwa-ke, isimo esingavuthiwe se-hiPS-CMs kanye nokuntuleka kobunzima be-multicellular yezicubu zenhliziyo yimikhawulo emikhulu yale ndlela.Ucwaningo lwakamuva lubonise ukuthi lo mkhawulo unganqotshwa ngokwengxenye ngokusebenzisa i-hiPS-CM yokuqala ukwakha ama-hydrogel ezicubu zenhliziyo ngokushesha ngemva kokuqala kokufinyela okuzenzakalelayo futhi kancane kancane ukwandisa ukukhuthazwa kagesi ngokuhamba kwesikhathi.Kodwa-ke, lezi zicubu ezincane ze-hiPS-CM azinazo izici ezivuthiwe ze-electrophysiological kanye nenkontileka ye-myocardium yabantu abadala.Ngaphezu kwalokho, izicubu zenhliziyo yomuntu zinesakhiwo esiyinkimbinkimbi, esihlanganisa ingxube ehlukahlukene yezinhlobo zamaseli ahlukene, okuhlanganisa amangqamuzana e-endothelial, ama-neurons, nama-stromal fibroblasts, axhunywe amasethi athile amaprotheni e-extracellular matrix.Lokhu kuhlukahluka kwezibalo ezingezona ze-cardiomyocyte11,12,13 enhliziyweni yesilwane esincelisayo esikhulile kuyisithiyo esikhulu ekufanekiseni izicubu zenhliziyo kusetshenziswa izinhlobo zamaseli ngamanye.Le mikhawulo emikhulu igcizelela ukubaluleka kokuthuthukisa izindlela zokuhlakulela izicubu ze-myocardial ezingaguquki ngaphansi kwezimo ze-physiological and pathological.
Izingxenye zenhliziyo yomuntu ezacile ezikhulisiwe (300 µm) zifakazele ukuthi ziyimodeli ethembisayo ye-myocardium yomuntu eqinile.Le ndlela inikeza ukufinyelela ohlelweni oluphelele lwe-3D multicellular efana nezicubu zenhliziyo yomuntu.Kodwa-ke, kuze kube ngu-2019, ukusetshenziswa kwezingxenye zenhliziyo ezithuthukisiwe kwakunqunyelwe ukusinda kwesiko elifushane (24 h).Lokhu kungenxa yezinto eziningi ezihlanganisa ukuntuleka kokunwebeka komzimba, isixhumi esibonakalayo soketshezi lomoya, 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 zikanokusho 17 kanye ne-18 zibonisa ukuthi ukulayisha uniaxial ngomshini kunomphumela omuhle ku-phenotype yenhliziyo phakathi nesiko.Kodwa-ke, lezi zifundo azizange zisebenzise ukulayishwa okunamandla kwe-physico-mechanical okushukumisayo komjikelezo wenhliziyo, njengoba izigaba zenhliziyo zilayishwe noma ngabe i-isometric tensile force 17 noma i-auxotonic eqondile yokulayisha i-18.Lezi zindlela zokwelula izicubu zibangele ukucindezelwa kwezakhi zofuzo eziningi zenhliziyo noma ukuvezwa ngokweqile kwezakhi zofuzo ezihlobene nezimpendulo ezinwebekayo ezingavamile.Ngokuphawulekayo, uPitoulis et al.I-19 yakha indawo yokugeza yesiko locezu lwenhliziyo eguqukayo ukuze kwakhiwe kabusha umjikelezo wenhliziyo kusetshenziswa impendulo ye-transducer enamandla kanye nokushayela kwengcindezi.Nakuba lolu hlelo luvumela ukulinganisa okunembe kakhudlwana kwe-in vitro cardiac cycle modeling, inkimbinkimbi kanye nokuphuma okuphansi kwendlela kunciphisa ukusetshenziswa kwalolu hlelo.Ilabhorethri yethu isanda kuthuthukisa uhlelo lwesiko olwenziwe lula olusebenzisa amandla kagesi kanye nendawo ethuthukisiwe ukuze kugcinwe ukusebenza kwezingxenye zengulube nezicubu zenhliziyo yomuntu kuze kube yizinsuku ezingu-620,21.
Embhalweni wesandla wamanje, sichaza imodeli yesiko lezicubu zenhliziyo (CTCM) sisebenzisa izingxenye zenhliziyo yengulube ehlanganisa ama-humoral cues ukuze kubuyiselwe i-physiology yenhliziyo enezinhlangothi ezintathu kanye ne-pathophysiological distension ngesikhathi somjikelezo wenhliziyo.Le CTCM ingakhuphula ukunemba kokubikezela izidakamizwa zangaphambi komtholampilo kuze kube sezingeni elingakaze lifinyelelwe ngokuhlinzeka ngohlelo lwenhliziyo olungabizi, oluphakathi nolwedlule olulingisa i-physiology/pathophysiology yenhliziyo yesilwane esincelisayo ukuze kuhlolwe izidakamizwa zangaphambi komtholampilo.
Izimpawu zemishini ye-Hemodynamic zidlala indima ebalulekile ekugcineni umsebenzi we-cardiomyocyte ku-vitro 22,23,24.Embhalweni wesandla wamanje, senze i-CTCM (Umfanekiso 1a) engalingisa indawo yenhliziyo yabantu abadala ngokuyenga kokubili ukukhuthazwa kagesi nokomshini kumafrikhwensi okuphila (1.2 Hz, 72 beats per minute).Ukuze ugweme ukwelula kwezicubu ngokweqile ngesikhathi se-diastole, idivayisi yokunyathelisa ye-3D yasetshenziselwa ukwandisa usayizi wezicubu ngo-25% (Fig. 1b).Ukuhamba kukagesi okudalwe uhlelo lwe-C-PACE kwanqunywa ukuthi kuqale ngo-100 ms ngaphambi kwe-systole kusetshenziswa isistimu yokutholwa kwedatha ukuze kuphinde kukhiqizwe ngokugcwele umjikelezo wenhliziyo.Uhlelo lwe-tissue Culture actuator (LB Engineering, Germany) ukuze lunwebe ngomjikelezo ulwelwesi lwe-silicone oluguquguqukayo ukuze lubangele ukunwebeka kwezingcezu zenhliziyo ekamelweni eliphezulu.Uhlelo luxhunywe kumugqa womoya wangaphandle ngokusebenzisa i-transducer yokucindezela, okwenza kube lula ukulungisa ngokunembile ukucindezela (± 1 mmHg) nesikhathi (± 1 ms) (Fig. 1c).
a Namathisela ingxenye yesicubu kwendandatho yokusekela engu-7 mm, ekhonjiswe ngokuluhlaza okwesibhakabhaka, ngaphakathi kwegumbi lamasiko ledivayisi.Igumbi lamasiko lihlukaniswe negumbi lomoya ngolwelwesi oluncane lwe-silicone oluguquguqukayo.Faka i-gasket phakathi kwegumbi ngalinye ukuvimbela ukuvuza.Isivalo socingo siqukethe ama-electrode e-graphite ahlinzeka ngokuvuselela kagesi.b Ukumelwa okuhleliwe kocingo olukhulu lwesicubu, iringi yomhlahlandlela kanye neringi yokusekela.Izingxenye zethishu (ezinsundu) zibekwe emshinini omkhulukazi ngendandatho yomhlahlandlela ebekwe emseleni onqenqemeni olungaphandle lwedivayisi.Usebenzisa umhlahlandlela, beka ngokucophelela indandatho yokusekela eboshwe nge-adhesive ye-acrylic yethishu phezu kwengxenye yezicubu zenhliziyo.c Igrafu ebonisa isikhathi sokuvuselela ugesi njengomsebenzi wokucindezela kwegumbi lomoya olawulwa yi-programmable pneumatic actuator (PPD).Idivayisi yokutholwa kwedatha isetshenziswe ukuvumelanisa isivuseleli sikagesi kusetshenziswa izinzwa zokucindezela.Lapho ingcindezi egumbini lamasiko ifinyelela embundwini omisiwe, isignali ye-pulse ithunyelwa ku-C-PACE-EM ukuze kuqalise ukukhuthazwa 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 kuqashwe ingcindezi kumjikelezo womoya.Ithuluzi ngalinye linezingxenye eziyisithupha zamathishu.
Ukusebenzisa i-actuator eyodwa ye-pneumatic, sakwazi ukulawula amadivaysi e-4 CTCM, ngayinye engakwazi ukubamba izingxenye zezicubu ze-6 (Fig. 1d).Ku-CTCM, ingcindezi yomoya egumbini lomoya iguqulelwa ekucindezelweni okuvumelanayo ekamelweni eliwuketshezi futhi ibangele ukunwetshwa komzimba kocezu lwenhliziyo (Umfanekiso 2a kanye ne-Supplementary Movie 1).Ukuhlolwa kokunwebeka kwezicubu ku-80 mm Hg.Art.wabonisa ukwelula kwezingxenye zezicubu ngama-25% (Fig. 2b).Lokhu kunwetshwa kwephesenti kuboniswe ukuthi kuhambisana nobude be-physiological sarcomere obungu-2.2–2.3 µm ngenkontileka yesigaba senhliziyo esivamile17,19,25.Ukunyakaza kwezicubu kwahlolwa kusetshenziswa izilungiselelo zekhamera yangokwezifiso (Umfanekiso Owengeziwe 1).I-amplitude kanye nesivinini sokunyakaza kwezicubu (Fig. 2c, d) kuhambelana nokwelula phakathi nomjikelezo wenhliziyo kanye nesikhathi ngesikhathi se-systole ne-diastole (Fig. 2b).Ukwelula kanye nesivinini sezicubu zenhliziyo phakathi nokufinyela nokuphumula kwahlala kungashintshile izinsuku eziyi-12 kusiko (Fig. 2f).Ukuhlola umthelela wokugqugquzelwa kukagesi kunkontileka phakathi nesiko, sithuthukise indlela yokunquma ukukhubazeka okusebenzayo sisebenzisa i-algorithm ye-shading (I-Supplementary Fig. 2a,b) futhi sakwazi ukuhlukanisa phakathi kokukhubazeka ngokushukumisa ugesi nangaphandle.Ingxenye efanayo yenhliziyo (Fig. 2f).Esifundeni esinyakazayo sokusikwa (i-R6-9), i-voltage ngesikhathi sokuvuselela ugesi yayiphezulu ngo-20% kunokungabikho kokuvuselela kagesi, okubonisa umnikelo wokuvuselela ugesi emsebenzini wenkontileka.
Iminonjana emele yokucindezela kwegumbi lomoya, ingcindezi yegumbi loketshezi, nezilinganiso zokunyakaza kwezicubu ziqinisekisa ukuthi umfutho wegumbi ushintsha umfutho wegumbi loketshezi, okubangela ukunyakaza okuhambisanayo kocezu lwethishu.b Izimpawu zokulandelana kwephesenti lokunwebeka (okuluhlaza okwesibhakabhaka) kwezingxenye zethishu ezihambisana nokwelukwa kwephesenti (owolintshi).c Ukunyakaza okukaliwe kocezu lwenhliziyo kuhambisana nesivinini esilinganisiwe sokunyakaza.(d) Ama-trajectories amelela ukunyakaza kwe-cyclic (umugqa oluhlaza okwesibhakabhaka) kanye nesivinini (umugqa wamachashazi awolintshi) ocezwini lwenhliziyo.e Ukulinganisa isikhathi somjikelezo (n = izingcezu ezingu-19 eqenjini ngalinye, ezingulubeni ezahlukene), isikhathi sokufinyela (n = izingcezu ezingu-19 iqembu ngalinye), isikhathi sokuphumula (n = izingcezu ezingu-19 iqembu ngalinye, ezingulubeni ezahlukene), ukunyakaza kwezicubu (n = 25).izingcezu)/iqembu elivela ezingulubeni ezahlukene), isivinini esikhulu se-systolic (n = 24(D0), 25(D12) izingcezu/iqembu elivela ezingulubeni ezahlukene) kanye nezinga lokuphumula eliphakeme (n=24(D0), 25(D12) izingcezu/iqembu elivela ezingulubeni ezahlukene).Ukuhlola kuka-t koMfundi onemisila emibili akubonisi mehluko omkhulu kunoma iyiphi ipharamitha.f Ukuhlaziywa kohlobo olumele ukulandelwa kwezingxenye zethishu ezinogesi (obomvu) nangaphandle (oluhlaza okwesibhakabhaka), izindawo eziyishumi zesifunda zezingxenye zethishu ezivela esigabeni esifanayo.Amaphaneli angezansi abonisa ukulinganisa komehluko wamaphesenti obunzima ezigabeni zethishu ngokushukumisa ugesi nangaphandle kwezindawo eziyishumi ezivela ezigabeni ezihlukene. (n = izingcezu ezingu-8/iqembu elivela ezingulubeni ezihlukene, Ukuhlolwa kuka-t koMfundi onemisila emibili kuyenziwa; ****p <0.0001, **p <0.01, *p <0.05). (n = izingcezu ezingu-8/iqembu elivela ezingulubeni ezihlukene, Ukuhlolwa kuka-t koMfundi onemisila emibili kuyenziwa; ****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 = 8 izigaba/iqembu elivela ezingulubeni ezihlukene, ukuhlolwa kuka-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 = 8 izigaba/iqembu, elivela ezingulubeni ezihlukene, ukuhlolwa kuka-t koMfundi okunemisila emibili; ****p<0.0001, **p<0.01, *p<0.05).Amabha amaphutha amelela isilinganiso ± ukuchezuka okujwayelekile.
Kusistimu yethu yangaphambilini ye-static biomimetic heart slice [20, 21], sigcine ukusebenza, ukusebenza, nobuqotho besakhiwo sezingcezu zenhliziyo izinsuku ezingu-6 ngokusebenzisa ukuvuselela ugesi nokuthuthukisa ukwakheka okuphakathi nendawo.Nokho, ngemva kwezinsuku ezingu-10, lezi zibalo zehla kakhulu.Sizobhekisela ezingxenyeni ezithuthukiswe ohlelweni lwethu lwangaphambilini lwe-static biomimetic culture 20, izimo zokulawula ezingama-21 (Ctrl) futhi sizosebenzisa indlela yethu ethuthukiswe ngaphambilini njengezimo ze-MC kanye nesiko ngaphansi kokuvuselela kanyekanye kokusebenza kukagesi (CTCM).ebizwa .Okokuqala, sinqume ukuthi ukugqugquzelwa kwemishini ngaphandle kokuvuselela kagesi kwakungenele ukugcina ukusebenza kwezicubu izinsuku ezingu-6 (I-Supplementary Fig. 3a,b).Kuyathakazelisa ukuthi ngokusungulwa kokuvuselela kwe-physio-mechanical kanye nogesi kusetshenziswa i-STCM, ukusebenza kwezingxenye zenhliziyo zezinsuku ezingu-12 kwahlala kufana nezigaba zenhliziyo entsha ngaphansi kwezimo ze-MS, kodwa hhayi ngaphansi kwezimo ze-Ctrl, njengoba kuboniswe ukuhlaziywa kwe-MTT (Fig. 1).3a).Lokhu kusikisela ukuthi ukugqugquzelwa kwemishini kanye nokulingisa umjikelezo wenhliziyo kungagcina izingxenye zezicubu zisebenza ngokuphindwe kabili njengoba kubikwe ohlelweni lwethu lwangaphambili lwesiko elimile.Kodwa-ke, ukuhlolwa kobuqotho besakhiwo sezingxenye zezicubu ngokufakwa kwe-immunolabeling ye-cardiac troponin T kanye ne-connexin 43 kubonise ukuthi inkulumo ye-connexin 43 yayiphezulu kakhulu kumathishu e-MC ngosuku lwe-12 kunezilawuli ngosuku olufanayo.Kodwa-ke, i-uniform connexin 43 expression kanye ne-Z-disc formation ayizange igcinwe ngokugcwele (Fig. 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 ne-fluorescence yezingcezu zenhliziyo ngokwamandla okwenziwa kwasendaweni.Le ndlela isebenzisa i-Convolutional Neural Network (CNN) kanye nohlaka lokufunda olujulile ukuze kulinganiswe ubuningi bobuqotho besakhiwo sezicubu zenhliziyo ngendlela ezenzakalelayo nengachemile, njengoba kuchazwe kusithenjwa.I-26. Izicubu ze-MC zibonise ukufana kwesakhiwo okuthuthukisiwe ngosuku lwe-0 uma kuqhathaniswa nezigaba zokulawula ezimile.Ngaphezu kwalokho, ukungcola kwe-Masson kwe-trichrome kwembula iphesenti eliphansi kakhulu le-fibrosis ngaphansi kwezimo ze-MS uma kuqhathaniswa nezimo zokulawula ngosuku lwe-12 lwesiko (Fig. 3c).Nakuba i-CTCM yandisa ukusebenza kwezigaba zezicubu zenhliziyo ngosuku lwe-12 kuya ezingeni elifana nelezicubu zenhliziyo entsha, ayizange ithuthukise kakhulu ubuqotho besakhiwo sezingxenye zenhliziyo.
igrafu yebha ibonisa ubuningi bokusebenza kwe-MTT kwezingcezu zenhliziyo entsha (D0) noma isiko lezingcezu zenhliziyo izinsuku ezingu-12 kungaba ngesiko elimile (D12 Ctrl) noma nge-CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) izingcezu/iqembu le-1 kuqhathaniswa nokuhlolwa kwe-1 ##0 eyodwa kusuka ku-1#0VA ukuhlolwa okukodwa; 0 kanye no-**p <0.01 uma kuqhathaniswa ne-D12 Ctrl). igrafu yebha ibonisa ubuningi bokusebenza kwe-MTT kwezingcezu zenhliziyo entsha (D0) noma isiko lezingcezu zenhliziyo izinsuku ezingu-12 kungaba ngesiko elimile (D12 Ctrl) noma nge-CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl ), 12 (D12 MC) izingcezu/iqembu indlela eyodwa ye-1 ##0 kuqhathaniswa ne-ANOVA ehlukile I-D0 kanye ne-**p <0.01 uma kuqhathaniswa ne-D12 Ctrl).i-histogram ibonisa ubuningi bokusebenza kwezingxenye zenhliziyo entsha ye-MTT (D0) noma isiko lezigaba zenhliziyo izinsuku ezingu-12 kunoma iyiphi isiko elimile (D12 control) noma i-CTCM (D12 MC) (n = 18 (D0), 15 (D12 control). )), 12 (D12 MC) izigaba/iqembu le-VA elihlukile-iqembu le-ANO lenziwa ngendlela ehlukile;####p < 0,0001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). ####p <0.0001 uma kuqhathaniswa ne-D0 kanye ne-**p <0.01 uma kuqhathaniswa ne-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) 或片片(D0) 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 1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 <0.0001} . . . . 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) 中文 12 片(D0)1) (D12 MC)组，进行单向ANOVA 测试；与D0 相比，####p <0.0001，与D12 Ctrl 相比，**p.)i-histogram ebonisa ubuningi bokusebenza kwe-MTT ezigabeni zenhliziyo entsha (D0) noma izigaba zenhliziyo ezikhuliswe izinsuku ezingu-12 kusiko lwe-static (D12 control) noma i-CTCM (D12 MC) (n = 18 (D0), 15 (D12 control)) , 12 (D12 MC) izigaba/iqembu elivela ezingulubeni ezahlukene ze-ANOVA, isivivinyo esisodwa;####p < 0,0001 по сравнению с D0, **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 uma kuqhathaniswa ne-D0, **p <0.01 uma kuqhathaniswa ne-D12 Ctrl).b I-Troponin-T (eluhlaza), i-connexin 43 (ebomvu) ne-DAPI (eluhlaza okwesibhakabhaka) ezigabeni zenhliziyo ezisanda kuhlukaniswa (D0) noma ezigabeni zenhliziyo ezikhuliswe ngaphansi kwezimo ezimile (Ctrl) noma izimo ze-CTCM (MC) izinsuku eziyi-12) zezithombe ezimele i-immunofluorescence (isikali esingenalutho = 100 µm). Ukulinganisa kobuhlakani bokwenziwa kobuqotho besakhiwo sezicubu zenhliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izingcezu/iqembu ngalinye elivela engulubeni ehlukile, ukuhlolwa kwe-ANOVA yendlela eyodwa kwenziwa; ####p <0.0001 uma kuqhathaniswa ne-D0 kanye ne-****p <0.000 Ctrl1 uma kuqhathaniswa ne-D010). Ukulinganisa kobuhlakani bokwenziwa kobuqotho besakhiwo sezicubu zenhliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izingcezu/iqembu ngalinye elivela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kuyenziwa; ####p <0.0001 uma kuqhathaniswa ne-D0 kanye ne-****p <0.000 Ctrl1 uma kuqhathaniswa ne-D010). Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) срезов/ ся однофакторный тест ANOVA; ####p < 0,0001 по сравнению с D0 и ****p < 0,0001 по сравнению с D12 Ctrl). Ukulinganisa ubuqotho besakhiwo sezicubu zenhliziyo ngobuhlakani bokwenziwa (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izigaba/amaqembu avela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa okwenziwe; ####p <0.0001 vs. nge-D0 kanye ne-****p <10 10 kuqhathaniswa ne-D0 kanye no-****p <10 10).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 ehlukile, isivivinyo se-ANOVA yendlela eyodwa;##0#00D **** 盯節0001 kusetshenziswa i-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#00D010p. 001 ku-D12 Ctrl 相比. I-Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC/грузей) сзрка ней, односторонний тест ANOVA; ####p <0,0001 vs. D0 Для сравнения ****p < 0,0001 по сравнению с D12 Ctrl). Ubuhlakani bokwenziwa bokukala ubuqotho besakhiwo bezicubu zenhliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izigaba/iqembu lengulube ngayinye ehlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa; ####p<0.0001 vs .D0 Ukuze uqhathanise ****p10 <10 10 . c Izithombe ezimele (kwesokunxele) nomthamo (kwesokudla) wezingcezu zenhliziyo ezingcoliswe ibala le-trichrome lika-Masson (Isikali esingenalutho = 500 µm) (n = izingcezu ezingu-10/iqembu ngalinye elisuka engulubeni ehlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kuyenziwa; ####p <0.0001 uma kuqhathaniswa ne-D00 kanye ne-Dtpl 01 kanye ne-D*20). c Izithombe ezimele (kwesokunxele) nomthamo (kwesokudla) wezingcezu zenhliziyo ezingcoliswe ibala le-trichrome lika-Masson (Isikali esingenalutho = 500 µm) (n = izingcezu ezingu-10/iqembu ngalinye elivela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kuyenziwa; #### p <0.0001 uma kuqhathaniswa ne-D01 kanye ne-Dtpl 01 kanye no-D010 ***p). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Масзкбо Массом 5 (n = 10 срезов/группу от разных свиней, выполняется односторонний тест ANOVA; #### p < 0,0001 по сравнению с D0 nase ***p <0,002ю по с Dравнению с D0 kanye ***p <0,002ю по с Dравнению с D0 kanye ***p <0,002ю по с Dравнению). c Izithombe ezimele (kwesokunxele) nomthamo (kwesokudla) wezingxenye zenhliziyo ezingcoliswe ibala le-trichrome lika-Masson (isikali esingagcotshiwe = 500 µm) (n = izigaba ezingu-10/iqembu elivela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kwenziwe; #### p <0 .0001 uma kuqhathaniswa ne-D0 kanye ne-D00 ***1 kuqhathaniswa ne-D0.1 ***21 <). 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)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 与D12 Ctrl 相比。 C 用 mason 三 色 染料 的 心脏 切片 的 代表性 （左 左） 量化 （右） 裸尺庰 裸尺度 裸尺度 裸尺度 裸尺度 裸尺度 裸尺庰 裸尺庰 裸尺庰裸尺尺度 左 左） 量化µm） （n = 10 个 切片 组 每 组 來自 不同 猪 , 进行 单向 单向 Anova 测试；#### p <0.00001 Faka i-D12 Ctrl. c Репрезентативные изображения (слева) и количественный анализ (справа) срезов сердца, окрашенных трихромным красителем Массом 10 = Массоя 1 = 10 протестировано с помощью однофакторного дисперсионного анализа Ctrl). c Izithombe ezimele (kwesokunxele) nenani (kwesokudla) lezingxenye zenhliziyo ezingcoliswe ibala le-trichrome lika-Masson (elingenalutho = 500 µm) (n = 10 izigaba/iqembu, ngasinye sivela engulubeni ehlukile, sihlolwe ngokuhlaziywa kwendlela eyodwa yokuhluka ;### # p <0.0001 < 0.0001 uma kuqhathaniswa no-D010, ***p).Amabha amaphutha amelela isilinganiso ± ukuchezuka okujwayelekile.
Sacabanga ukuthi ngokungeza ama-molecule amancane endaweni yesiko, ubuqotho be-cardiomyocyte bungathuthukiswa futhi ukuthuthukiswa kwe-fibrosis kuncishiswe phakathi nesiko le-CTCM.Ngakho-ke sihlole ama-molecule amancane sisebenzisa amasiko ethu okulawula okumile20,21 ngenxa yenani elincane lezici ezididayo.I-Dexamethasone (Dex), triiodothyronine (T3), ne-SB431542 (SB) zikhethelwe lesi sikrini.Lawa ma-molecule amancane ake asetshenziswa emasikweni e-hiPSC-CM ukuze enze ukuvuthwa kwama-cardiomyocyte ngokwandisa ubude be-sarcomere, ama-T-tubules, kanye nesivinini sokuqhuba.Ngaphezu kwalokho, kokubili i-Dex (i-glucocorticoid) ne-SB zaziwa ngokucindezela ukuvuvukala29,30.Ngakho-ke, sihlole ukuthi ukufakwa kweyodwa noma inhlanganisela yala ma-molecule amancane kungathuthukisa ubuqotho besakhiwo sezingxenye zenhliziyo.Ukuze kuhlolwe okokuqala, umthamo wenhlanganisela ngayinye ukhethiwe ngokusekelwe ekugxiliseni okuvame ukusetshenziswa kumamodeli we-cell culture (1 μM Dex27, 100 nM T327, kanye no-2.5 ​​μM SB31).Ngemva kwezinsuku ezingu-12 zesiko, inhlanganisela ye-T3 ne-Dex ibangele ubuqotho besakhiwo se-cardiomyocyte kanye nokulungiswa okuncane kwe-fibrous (Izibalo Ezingeziwe 4 kanye ne-5).Ukwengeza, ukusetshenziswa kokuphindwe kabili noma kabili kwalokhu kugxilwa kwe-T3 ne-Dex kukhiqize imiphumela esusayo uma kuqhathaniswa nokugxila okuvamile (I-Supplementary Fig. 6a,b).
Ngemva kokuhlola kokuqala, senze ukuqhathanisa kwekhanda kuya kwekhanda kwezimo zempucuko ezi-4 (Umfanekiso 4a): Ctrl: izigaba zenhliziyo ezikhuliswe ngesiko lethu elimile elichazwe ngaphambilini kusetshenziswa indlela yethu ethuthukisiwe;20.21 TD: T3 kanye no-Ctrl s Added Dex ngoLwesithathu;I-MC: izigaba zenhliziyo ezikhuliswe ku-CTCM kusetshenziswa indawo yethu elungiselelwe ngaphambilini;kanye ne-MT: I-CTCM ene-T3 kanye ne-Dex yengezwe phakathi nendawo.Ngemuva kwezinsuku ze-12 zokutshala, ukusebenza kwezicubu ze-MS ne-MT kwahlala kufana nezicubu ezintsha ezihlolwe ukuhlolwa kwe-MTT (Fig. 4b).Kuyathakazelisa ukuthi ukungezwa kwe-T3 ne-Dex kumasiko e-transwell (TD) akuzange kubangele ukuthuthukiswa okuphawulekayo kokusebenza uma kuqhathaniswa nezimo ze-Ctrl, okubonisa indima ebalulekile yokuvuselela kwemishini ekugcineni ukusebenza kwezigaba zenhliziyo.
umdwebo womklamo wokuhlola obonisa izimo ezine zesiko elisetshenziselwa ukuhlola imiphumela yokukhuthazwa komshini kanye nokwengeza kwe-T3/Dex kokumaphakathi izinsuku eziyi-12. b Igrafu yebha ikhombisa ubuningi bokusebenza kwesiko lokuthunyelwe kwezinsuku eziyi-12 kuzo zonke izimo zempucuko ezi-4 (Ctrl, TD, MC, kanye ne-MT) uma kuqhathaniswa nezingcezu zenhliziyo ezintsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kanye ne-D12 MT), 12 (D12 MC) izingcezu/iqembu le-indlela eyodwa 0 #0, #0 isivivinyo senziwe nge-pigs ehlukile #0; ###p <0.001 uma kuqhathaniswa ne-D0 kanye ne-**p <0.01 uma kuqhathaniswa ne-D12 Ctrl). b Igrafu yebha ikhombisa ubuningi bokusebenza kwesiko lokuthunyelwe kwezinsuku eziyi-12 kuzo zonke izimo zempucuko ezi-4 (Ctrl, TD, MC, kanye ne-MT) uma kuqhathaniswa nezingcezu zenhliziyo ezintsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kanye ne-D12 MT), 12 (D12 MC) izingcezu/iqembu le-indlela eyodwa 0 #0, #0 isivivinyo senziwe nge-pigs ehlukile #0; ###p <0.001 uma kuqhathaniswa ne-D0 kanye ne-**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 ю со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD and D12 MT), 12 (D12 MC) срезов/группу от разных свиней #0тей свиней, пронсто#дисто#дисто#дисто#дисто#дисто#дисто#дисто#дистонсто#дисто#дися ; ,0001, ###p < 0,001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). b Igrafu yebha ibonisa ubuningi bokusebenza kusiko lokuthunyelwe kwezinsuku eziyi-12 kuzo zonke izimo zamasiko ezingu-4 (control, TD, MC, and MT) uma kuqhathaniswa nezingxenye zenhliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, and D12 MT), 12 (D12 MC ezahlukene) #00p; #0pgroup ehlukile #0p; 1, ###p < 0.001 vs. D0 kanye ne-**p <0.01 ngokuqhathaniswa 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 CT MT1,D12 自和D12 MT1,D12 自和2 MC) 切片/组，进行单向ANOVA 测试；####p < 0.0001,###p < 0.001 与D0 相比，**p < 0.01 与D12）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), Ctrl (D12) разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA; ####p <0,0001, ###p <0,001 по сравнению с D0, **p <0,01 по компение 2). b I-Histogram ebonisa zonke izimo zesiko ezingu-4 (ukulawula, i-TD, MC kanye ne-MT) uma kuqhathaniswa nezigaba zenhliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kanye ne-D12 MT), kusukela ezingulubeni ezihlukene 12 (D12 MC) izigaba/iqembu, ukuhlolwa kwe-ANOVA yendlela eyodwa; ##0#0 vs. 0.01 vs. control D12). c Igrafu yebha ikhombisa inani le-glucose flux ezinsukwini eziyi-12 zesiko lokuthunyelwe kuzo zonke izimo zempucuko ezi-4 (Ctrl, TD, MC, kanye ne-MT) uma kuqhathaniswa nezingcezu zenhliziyo entsha (D0) (n = izingcezu ezingu-6/iqembu elivela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kuyenziwa; ###p <0.001, uma kuqhathaniswa ne-D0 kanye ne-**** 1 uma kuqhathaniswa ne-D1 kanye no-****1  kuqhathaniswa ne-D0). c Igrafu yebha ikhombisa inani le-glucose flux ezinsukwini eziyi-12 zesiko lokuthunyelwe kuzo zonke izimo zempucuko ezi-4 (Ctrl, TD, MC, kanye ne-MT) uma kuqhathaniswa nezingcezu zenhliziyo entsha (D0) (n = izingcezu ezingu-6/iqembu elivela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kuyenziwa; ###p <0.001, uma kuqhathaniswa ne-D0 kanye ne-**** 1 uma kuqhathaniswa ne-D1 kanye no-****1  kuqhathaniswa ne-D0). Вь голе поле после - 12 (TD, MC и Mt) Со Совнении пов С ыпппеяеееееееееееso; ### p <0,001 по Сравнению с D12 по Сравнению с D12 По c I-Histogram ibonisa ubuningi be-glucose flux ezinsukwini ezingu-12 emva kwesiko ngaphansi kwazo zonke izimo ze-4 zamasiko (control, TD, MC kanye ne-MT) uma kuqhathaniswa nezigaba zenhliziyo entsha (D0) (n = 6 izigaba/iqembu elivela ezingulubeni ezahlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa okwenziwe ; ###p <0.001 uma kuqhathaniswa ne-D0 kanye ne-****p <0.00 <0.00 Ctrl 1). c 条形图显示所有4 种培养条件（CT、TD、MC 和MT）与新鲜心脏切片(D0) 相比，培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) 相比，培养养宇后12 6 片/组，來自不同猪，单向执行ANOVA 测试；###p < 0.001，与D0 相比，***p < 0.001 与D12 Ctrl 相比）. C 条形图 显示 所有 4 种 条件 （(ctrl 、 td 、 mc 和 mt） 新鲜 心脏 切片 切片 切剹光 切特 切片 1的 通量 定量 （n = 6 片/组 ， 來自 猪 , , , , , , , 猪 猪单行向执ANOVA 测试；###p <0.00D10. Faka i-D12 Ctrl. c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после культивирования для всех 4 условий культивирования ю со свежими срезами сердца (D0) (n = 6 срезов/группа, от разных свиней, односторонний Были проведены тесты ANOVA; ###p < 0,000 12 (контроль). c I-Histogram ebonisa ubuningi be-glucose flux ezinsukwini ezingu-12 emva kwesiko kuzo zonke izimo ze-4 zamasiko (control, TD, MC, and MT) uma kuqhathaniswa nezigaba zenhliziyo entsha (D0) (n = 6 izigaba/iqembu, elivela ezingulubeni ezahlukene, unilateral Ingabe ukuhlolwa kwe-ANOVA kwenziwa, ###p <0.001 uma kuqhathaniswa no-D01, ***1 kuqhathaniswa no-D0. ***1).d Iziqephu zokuhlaziywa kobunzima zezicubu ezintsha (eziluhlaza okwesibhakabhaka), usuku lwe-12 MC (okuluhlaza okotshani), nosuku lwe-12 MT (obomvu) ezindaweni eziyishumi zesigaba sethishu zesifunda (n = izingcezu ezi-4/iqembu, ukuhlolwa kwe-ANOVA yendlela eyodwa; kwakungekho umehluko obalulekile phakathi kwamaqembu).e Isakhiwo sentaba-mlilo esibonisa izakhi zofuzo ezivezwe ngokuhlukile ezigabeni zenhliziyo entsha (D0) uma kuqhathaniswa nezingxenye zenhliziyo ezikhuliswe ngaphansi kwezimo ezimile (Ctrl) noma ngaphansi kwezimo ze-MT (MT) izinsuku ezingu-10-12.f Imephu yokushisa yezakhi zofuzo ze-sarcomere zezingxenye zenhliziyo ezikhuliswe ngaphansi kwesimo ngasinye samasiko.Amabha amaphutha amelela isilinganiso ± ukuchezuka okujwayelekile.
Ukuncika kwe-metabolism ekushintsheni kusuka ku-fatty acid oxidation kuya ku-glycolysis kuwuphawu lwe-cardiomyocyte dedifferentiation.Ama-cardiomyocyte angavuthiwe asebenzisa ngokuyinhloko i-glucose ekukhiqizeni i-ATP futhi ane-hypoplastic mitochondria nama-cristae5,32 ambalwa.Ukuhlaziywa kokusetshenziswa kwe-glucose kubonise ukuthi ngaphansi kwezimo ze-MC ne-MT, ukusetshenziswa kwe-glucose kwakufana nalokho osukwini lwezicubu ze-0 (Umfanekiso 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 ilondoloze i-phenotype ye-metabolic yezigaba zenhliziyo ezikhulisiwe zezinsuku eziyi-12.Ukwengeza, ukuhlaziywa kwezinkinga kubonise ukuthi amazinga okucindezeleka ahlala efana nezicubu zenhliziyo entsha izinsuku ezingu-12 ngaphansi kwezimo ze-MT ne-MS (Fig. 4d).
Ukuze sihlaziye wonke umthelela we-CTCM kanye ne-T3/Dex kukwakheka kwezwe okulotshiweyo komhlaba wonke kwezicubu zocezu lwenhliziyo, senze i-RNAseq ezingcezu zenhliziyo ezivela kuzo zonke izimo ezine zamasiko ezihlukene (Idatha Eyengeziwe 1).Kuyathakazelisa ukuthi izigaba ze-MT zibonise ukufana okuphezulu kokubhaliwe kwezicubu zenhliziyo entsha, ezine-16 kuphela ezivezwe ngokuhlukile kuzakhi zofuzo ze-13,642.Kodwa-ke, njengoba sibonisile ngaphambili, izingcezu ze-Ctrl zibonise izakhi zofuzo eziyi-1229 ezivezwe ngokuhlukile ngemuva kwezinsuku eziyi-10-12 kusiko (Fig. 4e).Le datha iqinisekiswe yi-qRT-PCR yezakhi zofuzo zenhliziyo ne-fibroblast (I-Supplementary Fig. 7a-c).Kuyathakazelisa ukuthi izigaba ze-Ctrl zibonise ukwehliswa kwezakhi zofuzo zomjikelezo wenhliziyo kanye namaseli kanye nokusebenza kwezinhlelo zofuzo ezivuthayo.Le datha iphakamisa ukuthi ukuhlukaniswa, okuvame ukwenzeka ngemva kokutshala isikhathi eside, kuncishisiwe ngokuphelele ngaphansi kwezimo ze-MT (I-Supplementary Fig. 8a,b).Ukucwaninga ngokucophelela kwezakhi zofuzo ze-sarcomere kubonise ukuthi ngaphansi kwezimo ze-MT kuphela izakhi zofuzo ezifaka ikhodi ye-sarcomere (Fig. 4f) kanye nesiteshi se-ion (Supplementary Fig. 9) zigcinwe, ziwavikela ekucindezelweni ngaphansi kwezimo ze-Ctrl, TD, ne-MC.Le datha ibonisa ukuthi ngenhlanganisela yokukhuthazwa komshini nokuhlekisayo (T3/Dex), i-transcriptome yocezu lwenhliziyo ingahlala ifana nezingcezu zenhliziyo ezintsha ngemva kwezinsuku eziyi-12 zesiko.
Lokhu okutholakele okubhalwe phansi kusekelwa ukuthi ubuqotho besakhiwo se-cardiomyocytes ezigabeni zenhliziyo kugcinwa kangcono ngaphansi kwezimo ze-MT izinsuku ezingu-12, njengoba kuboniswe yi-connexin engaphelele futhi yendawo ye-43 (Fig. 5a).Ngaphezu kwalokho, i-fibrosis ezigabeni zenhliziyo ngaphansi kwezimo ze-MT yancishiswa kakhulu uma kuqhathaniswa ne-Ctrl futhi ifana nezingxenye zenhliziyo entsha (Fig. 5b).Le datha ibonisa ukuthi inhlanganisela yokuvuselela kwemishini kanye nokwelashwa kwe-T3/Dex kugcina kahle ukwakheka kwenhliziyo ezigabeni zenhliziyo kusiko.
izithombe ezimele i-immunofluorescence ze-troponin-T (eluhlaza), i-connexin 43 (ebomvu), ne-DAPI (eluhlaza okwesibhakabhaka) ezigabeni zenhliziyo ezisanda kuhlukaniswa (D0) noma ezikhuliswe izinsuku eziyi-12 kuzo zonke izimo zesiko lesigaba senhliziyo ezine (isikali sebha = 100 µm).). Ukulinganisa kobuhlakani bokwenziwa kobuqotho besakhiwo sezicubu zenhliziyo (n = 7 (D0 kanye no-D12 Ctrl), izingcezu/iqembu le-5 (D12 TD, D12 MC kanye ne-D12 MT) elivela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kuyenziwa; <###p <0.0001 uma kuqhathaniswa ne-D000 kanye ne-D002 <0.002 noma i-D002. Ctrl). Ukulinganisa kobuhlakani bokwenziwa kobuqotho besakhiwo sezicubu zenhliziyo (n = 7 (D0 kanye no-D12 Ctrl), izingcezu/iqembu le-5 (D12 TD, D12 MC kanye ne-D12 MT) elivela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kuyenziwa; <### p <0.0001 uma kuqhathaniswa ne-D000 kanye ne-D002 <0.0001 noma D002. Ctrl). I-Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 7 (D0 kanye ne-D12 Ctrl), 5 (D12 TD, D12 зно MT) kanye ых свиней, проведен однофакторный тест ANOVA; #### p <0,0001 по сравнению с D0 и *p < 0,05 или ****p < 0,0001 по сравнению с D12 Ctrl). Ukulinganisa ubuqotho besakhiwo sezicubu zenhliziyo kusetshenziswa ubuhlakani bokwenziwa (n = 7 (D0 no-D12 Ctrl), 5 (D12 TD, D12 MC kanye ne-D12 MT) izigaba/iqembu elivela ezingulubeni ezihlukene, ukuhlolwa kwe-ANOVA yendlela eyodwa kwenziwe; #### p <0.0001 <0.0001 <0.0001 Ctrl                                                      </</em>          </</em>                 kwenziwa ukuhlolwa kwe-ANOVA ).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/滄人）切片/组行单向ANOVA 测试；#### p <0.0001 与D0 和*p < 0.05 相比，或****p < 0.0001 与D12 Ctrl 相比）.对 不同 猪 的 心脏 结构 完整性 (n = 7 （d0 和 d12 ctrl） （5 （d12 td,d12 mc 和 d12 mc 和 d12 mc)化 进行 单向 单向 单向 测试 ; ########### p <0.0001 与D0 和*p <0.05 相比，或****p <0.00012 .Ukulinganisa ubuqotho besakhiwo sezicubu zenhliziyo kusetshenziswa ubuhlakani bokwenziwa ezingulubeni ezahlukene (n = 7 (D0 kanye ne-D12 Ctrl), izigaba/iqembu le-5 (D12 TD, D12 MC kanye ne-D12 MT) ngokuhlolwa kwe-ANOVA kwendlela 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 ne-D0 kanye ne-*p <0.05 noma ****p <0.0001 uma kuqhathaniswa ne-D12 Ctrl). b Izithombe ezimele kanye nomthamo wezingcezu zenhliziyo ezingcoliswe ibala le-trichrome lika-Masson (Ibha yesikali = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, ne-D12 MC), 9 (D12 MT) izingcezu/iqembu elisuka ezingulubeni ezihlukene, indlela eyodwa 0#0 iqhathaniswa ne-ANOVA #0#p *** iqhathaniswa 0#0 ne-D#p 0.001, noma ****p <0.0001 uma kuqhathaniswa ne-D12 Ctrl). b Izithombe ezimele kanye nomthamo wezingcezu zenhliziyo ezingcoliswe ibala le-trichrome lika-Masson (Ibha yesikali = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, ne-D12 MC), 9 (D12 MT) izingcezu/iqembu elisuka ezingulubeni ezihlukene, indlela eyodwa 0#0 iqhathaniswa ne-ANOVA #0#p *** iqhathaniswa 0#0 ne-D#p 0.001, noma ****p <0.0001 uma kuqhathaniswa ne-D12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Массона (масштабная 10Dке млине) = D10 (10D = D10) U-Ctrl, D12 TD ne-D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполняется односторонний тест ANOVA; ####p < 0,0001 по D00 и0 и0 , *** 1 kuya ku-D12 Ctrl). b Izithombe ezimelelayo nobukhulu bezingxenye zenhliziyo ezingcoliswe ibala le-trichrome lika-Masson (ibha yesikali = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD ne-D12 MC), 9 (D12 MT) izigaba/iqembu elivela ezingulubeni ezihlukene, lenze indlela eyodwa <#001 kanye ne-D#001. ##001 kanye ne-DANOVA. 1 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（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D（D）同猪的9 个（D12 MT）切片/组，进行单因素方差分析；####p < 0.0001 与D0 相比,***p <0.00D爸比,***p <0.00D縥 1 2 1 1 1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0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 2, d 1, d 1, d 1, d 2, d 1, d 2, d 1 d 2, d 1 d 2, d 2, d 1 d 2, d 1 d 2,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 相毅 <0.0001 与D0 相毅 <0 与D0 <0 10 D0 <0. 2 Ctrl 相比）. b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом Массона (масштабная линейка линейка = 500, D1 = 500m, D1 = CTRL) 12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA; ####p < 0,0001 по сравнению с D0, ***p < 0,001 или 0****p 1  Ctrl, 0 ****p 1  Ctrl, 0 ****p 1). b Izithombe ezimelelayo nomthamo wezingxenye zenhliziyo ezingcoliswe i-trichrome ka-Masson (isikali sebha = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD kanye ne-D12 MC), 9 (D12 MT) izigaba ezisuka ezingulube/iqembu, indlela eyodwa ye-ANOVA; #0.0. p <0.0001 uma kuqhathaniswa ne-D12 Ctrl).Amabha amaphutha amelela isilinganiso ± ukuchezuka okujwayelekile.
Ekugcineni, ikhono le-CTCM lokulingisa i-hypertrophy yenhliziyo yahlolwa ngokwandisa ukunwebeka kwezicubu zenhliziyo.Ku-CTCM, ingcindezi ephezulu yegumbi lomoya yenyukile isuka ku-80 mmHg yaya ku-80 mmHg.Art.(ukunwebeka okujwayelekile) kufika ku-140 mmHg Art.(Umdwebo 6a).Lokhu kuhambisana nokwenyuka kwe-32% kokunwetshwa (Fig. 6b), okwakuboniswe ngaphambili njengephesenti elihambisanayo elidingekayo ukuze izingxenye zenhliziyo zifinyelele ubude be-sarcomere obufana nalokhu okubonwa ku-hypertrophy.Ukwelula kanye nesivinini sezicubu zenhliziyo ngesikhathi sokufinyela nokuphumula kwahlala kungashintshi phakathi nezinsuku eziyisithupha zesiko (Fig. 6c).Izicubu zenhliziyo ezisuka ezimeni ze-MT zazingaphansi kokwelula okuvamile (MT (Okuvamile)) noma izimo zokunwebeka ngokweqile (MT (OS)) izinsuku eziyisithupha.Kakade ngemva kwezinsuku ezine kusiko, i-hypertrophic biomarker NT-ProBNP yayiphakanyiswe kakhulu phakathi kwezimo ngaphansi kwezimo ze-MT (OS) uma kuqhathaniswa nezimo ze-MT (ezivamile) (Fig. 7a).Ngaphezu kwalokho, ngemva kwezinsuku eziyisithupha zokulima, usayizi weseli ku-MT (OS) (Fig. 7b) wanda kakhulu uma kuqhathaniswa nezigaba zenhliziyo ye-MT (evamile).Ukwengeza, i-NFATC4 i-nuclear translocation yanda kakhulu ezicutshini ezinwetshiwe (Fig. 7c).Le miphumela ibonisa ukuthuthukiswa okuqhubekayo kokulungiswa kabusha kwe-pathological ngemva kwe-hyperdistension futhi isekela umqondo wokuthi idivayisi ye-CTCM ingasetshenziswa njengenkundla yokutadisha ukusayinda kwe-hypertrophy yenhliziyo enwetshiwe.
Iminonjana emele yokucindezela kwegumbi lomoya, ingcindezi yegumbi loketshezi, nezilinganiso zokunyakaza kwezicubu ziqinisekisa ukuthi umfutho wegumbi ushintsha umfutho wegumbi loketshezi, okubangela ukunyakaza okuhambisanayo kocezu lwethishu.b Iphesenti lokunwebeka elimele kanye namajika esilinganiso sokunwebeka ezingxenyeni zethishu ezinwetshiwe ezivamile (eziwolintshi) nezinwetshiwe (eziluhlaza okwesibhakabhaka).c Igrafu yebha ebonisa isikhathi somjikelezo (n = 19 izingcezu iqembu ngalinye, kusukela ezingulubeni ezahlukene), isikhathi contraction (n = 18-19 tincetu iqembu ngalinye, kusukela ezingulubeni ezahlukene), isikhathi sokuphumula (n = 19 tincetu iqembu ngalinye, kusukela ezingulubeni ezahlukene)), amplitude ukunyakaza kwezicubu (n = 14 tincetu/iqembu, kusukela ezingulubeni ezahlukene), isiqongo 4 izingcezu ze-pigs / i-peak ye-peak (i-pigs ye-peak) kanye ne-1 n = 14 (D0), 15 (D6) ) izigaba/amaqembu) avela ezingulubeni ezahlukene), Ukuhlolwa kwe-t koMfundi enemisila emibili akuzange kubonise umehluko obalulekile kunoma iyiphi ipharamitha, okubonisa ukuthi le mingcele yahlala injalo phakathi nezinsuku ezingu-6 zesiko ngokugcwala ngokweqile.Amabha amaphutha amelela isilinganiso ± ukuchezuka okujwayelekile.
ukulinganisa kwegrafu yebha yokugxiliswa kwe-NT-ProBNP kumidiya yesiko kusuka ezincekeni zenhliziyo ezikhuliswe ngaphansi kwezimo ze-MT ezelulekile ezijwayelekile (Norm) noma ezinwebeka ngokweqile (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kanye ne-D4 MTOS) izingcezu/iqembu elisuka ezingulubeni ezihlukene, i-ANOVA yezindlela ezimbili uma iqhathaniswa ne-0 evamile yenziwa. **1 yenziwa i-ANOVA evamile). ukulinganisa kwegrafu yebha yokugxilisa ingqondo ye-NT-ProBNP kumidiya yesiko kusuka ezincekwini zenhliziyo ezikhuliswe ngaphansi kwezimo ze-MT ezelulekile ezijwayelekile (Norm) noma ezinwebeka ngokweqile (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kanye ne-D4 MTOS) izingcezu/iqembu elisuka ezingulubeni ezihlukene, i-ANOVA yezindlela ezimbili uma iqhathaniswa ne-ANOVA0 yenziwa ngezindlela ezimbili.I-histogram yobuningi bokugxiliswa kwe-NT-ProBNP kusiko eliphakathi nendawo kusuka ezincekeni zenhliziyo ezikhuliswe ngaphansi kwezimo zokwelula kwe-MT evamile (okujwayelekile) noma i-overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, ne-D4).MTOS) izingcezu / iqembu elivela ezingulubeni ezahlukene, ukuhlaziywa kwezinto ezimbili ezihlukene kwenziwa;**p < 0,01 по сравнению с нормальным растяжением). **p <0.01 uma kuqhathaniswa nokwelula okuvamile). a 在MT 正常拉伸(Okujwayelekile) 或过度拉伸(OS) 条件下培养的心脏切片培养基中NT-ProBNP 浓麦的条彈彈3, 浓度的条彈3 （D2 MTOS、D4 MTNorm 和D4 MTOS）来自不同猪的切片/组，进行双向方差分析；**与正常拉伸相比； i-Quantification ye-NT-ProBNP yokugxilisa ingqondo ezincekwini zenhliziyo ezikhuliswe ngaphansi kwezimo ze-MT ezijwayelekile zokwelula (Okujwayelekile) noma ze-Overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm neD4 MTOS) kusuka ku-猪的切片/组，可以双双向 eyahlukene p <0.01).i-histogram Ukulinganisa kwe-NT-ProBNP okugxilile ezincekwini zenhliziyo ezikhuliswe ngaphansi kwezimo ze-MT evamile elula (evamile) noma i-overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) kanye ne-D4 MTOS) izingcezu/iqembu elivela ezingulubeni ezahlukene, ukuhlaziywa okubili kokuhluka;**p < 0,01 по сравнению с нормальным растяжением). **p <0.01 uma kuqhathaniswa nokwelula okuvamile). b Izithombe ezimele izingcezu zenhliziyo ezingcoliswe i-troponin-T ne-WGA (kwesobunxele) kanye nokulinganisa usayizi weseli (kwesokudla) (n = 330 (D6 MTOS), amaseli/iqembu angu-369 (D6 MTNorm) asuka kuzingcezu eziyi-10 ezihlukene ezingulubeni, Ukuhlolwa kwe-t-umfundi onemisila emibili kuyenziwa. b Izithombe ezimele izingcezu zenhliziyo ezingcoliswe i-troponin-T ne-WGA (kwesobunxele) kanye nokulinganisa usayizi weseli (kwesokudla) (n = 330 (D6 MTOS), amaseli/iqembu angu-369 (D6 MTNorm) asuka ezingcezu ezingu-10 ezihlukene ezingulubeni, Ukuhlolwa kwe-t-T yoMfundi onemisila emibili kuyenziwa. 0 ****001 p. b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного определения размера клетокток (6) (6 = 6D = 6) I-MTNorm) клеток/группу из 10 разных срезов от разных свиней, два- проводится хвостовой t-критерий Стьюдента; ****p <0,00001 проводится хвостовой t-критерий Стьюдента; b Izithombe ezimele izigaba zenhliziyo ezingcoliswe i-troponin-T ne-AZP (kwesobunxele) kanye nokulinganisa usayizi weseli (kwesokudla) (n = 330 (D6 MTOS), amaseli/iqembu le-369 (D6 MTNorm) elisuka ezigabeni eziyi-10 ezihlukene ezingulubeni, Ukuhlolwa kwe-t-tailed yoMfundi enemisila emibili <i-0 kwenziwa isitimela esivamile esingu-1 ku-0. ****p . 30个不同切片的369（D6 MTNorm）细胞/组，两进行有尾学生t 检验；与正常拉伸相比，****p <1）0. b Izithombe ezimelela izingcezu zenhliziyo ezingcoliswe i-calcarein-T ne-WGA (kwesobunxele) nosayizi weseli (kwesokudla) (n = 330 (D6 MTOS), 369 kusuka ezingcezu ezihlukene eziyi-10 (D6 MTNorm)) Amaseli/组，两方法有尾学，pap0 ukwelula okujwayelekile <**** com 0 nokuhlolwa okujwayelekile . b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оценка размера клеток (S69MT3) (D3 Nomt3) rm) из 10 различных срезов от разных свиней Клетки/группа, двусторонние критерий Стьюдента; ****p < 0,0001 по сравнению с номнистенные). b Izithombe ezimele izigaba zenhliziyo ezingcoliswe i-troponin-T ne-AZP (kwesokunxele) kanye nokulinganisa kosayizi weseli (kwesokudla) (n = 330 (D6 MTOS), 369 (D6 MTNorm) ezigabeni ezingu-10 ezihlukene ezingulubeni) Amaseli/iqembu, umbandela onemisila emibili t;****p <0.0001 uma kuqhathaniswa nobunzima obujwayelekile). c Izithombe ezimele usuku olungu-0 nosuku 6 izingcezu zenhliziyo ze-MTOS ezifakwe amasosha omzimba ku-troponin-T ne-NFATC4 kanye nomthamo wokudluliselwa kwe-NFATC4 ku-nuclei yama-CMs (n = 4 (D0), 3 (D6 MTOS) izingcezu/iqembu elivela ezingulubeni ezihlukene, I-Two-tailed 0 Student . c Izithombe ezimele usuku olungu-0 nosuku 6 izingcezu zenhliziyo ze-MTOS ezifakwe amasosha omzimba ku-troponin-T ne-NFATC4 kanye nomthamo wokudluliselwa kwe-NFATC4 ku-nuclei yama-CMs (n = 4 (D0), 3 (D6 MTOS) izingcezu/iqembu elivela ezingulubeni ezahlukene , Ukuhlolwa kwe-Two-tailed 0 Umfundi. c Репрезентативные изображения для срезов сердца 0 futhi 6 дней MTOS, иммуномеченых для тропонина-Т kanye ne-NFATC4, kanye ne-количественная цинсанка зных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней , выполняется двусторонний t-критерий Стьюдента, *5 <p. c Izithombe ezimele izigaba zenhliziyo ngezinsuku ezingu-0 neziyi-6 ze-MTOS, ezifakwe amasosha omzimba e-troponin-T ne-NFATC4, kanye nomthamo we-NFATC4 translocation ku-nucleus yamaseli e-cavernous (n = 4 (D0), 3 (D6 MTOS) izingcezu/iqembu elivela ezingulubeni ezahlukene) lenze umfundi onemisila 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，以及來自第TC-CM4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 kanye ne-NFATC4 immunolabeling 第0天和第6天MTOS izingcezu zenhliziyo, kanye ne-NFATC4 evela ku-NFATC4 ehlukile 易位至CM cell nucleus的quantity化 (n = 4 (D0), 3 (D6 MTOS) 其公物生et 电影；*p <0.05). c I-Репрезентативные изображения срезов сердца MTOS ku-0 futhi 6 день для иммуномаркировки тропонином-Т kanye ne-NFATC4 kanye ne-количественная оценспенка TCFAD зных 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 ze-troponin-T kanye ne-NFATC4 immunolabeling kanye nomthamo we-NFATC4 translocation ku-nucleus ye-CM kusuka ezingulubeni ezahlukene (n = 4 (D0), 3 (D6 MTOS) izingcezu/iqembu, t-tailed two-t -criterion <5p.'Amabha amaphutha amelela ukuchezuka okujwayelekile okungu-±.
Ucwaningo lokuhumusha lwenhliziyo nemithambo yegazi ludinga amamodeli amaselula akhiqiza ngokunembile indawo yenhliziyo.Kulolu cwaningo, kwasungulwa idivayisi ye-CTCM futhi yabonakala engakwazi ukuvuselela izingxenye zenhliziyo ze-ultrathin.Uhlelo lwe-CTCM luhlanganisa ukugqugquzelwa kwe-electromechanical okuvunyelaniswe ngokomzimba kanye nokunothisa uketshezi lwe-T3 kanye ne-Dex.Lapho izingxenye zenhliziyo yengulube zivezwa kulezi zici, ukusebenza kwazo, ubuqotho besakhiwo, umsebenzi we-metabolic, nokusho okubhaliwe kwahlala kufana nezicubu zenhliziyo ezintsha ngemva kwezinsuku eziyi-12 zesiko.Ngaphezu kwalokho, ukwelula ngokweqile kwezicubu zenhliziyo kungabangela i-hypertrophy yenhliziyo ebangelwa i-hyperextension.Sekukonke, le miphumela isekela indima ebalulekile yezimo zesiko lempilo ekugcineni i-phenotype evamile yenhliziyo futhi inikeze inkundla yokuhlolwa kwezidakamizwa.
Ziningi izici ezinomthelela ekudaleni indawo ekahle yokusebenza nokuphila kwama-cardiomyocyte.Okusobala kakhulu kwalezi zici kuhlobene (1) nokusebenzisana kwe-intercellular, (2) ukukhuthazwa kwe-electromechanical, (3) izici ezihlekisayo, kanye (4) nama-substrates e-metabolic.Ukusebenzisana komzimba kweseli kuya kumaseli kudinga amanethiwekhi ayinkimbinkimbi anamacala amathathu ezinhlobo zamaseli amaningi asekelwa i-matrix engaphandle kwamaseli.Ukusebenzisana okunjalo okuyinkimbinkimbi kwamaselula kunzima ukuphinda kwakhiwe kabusha ku-vitro ngokuhlanganyela kwesiko lezinhlobo zamaseli ngamanye, kodwa kungafezwa kalula kusetshenziswa imvelo ye-organotypic yezingxenye zenhliziyo.
Ukwelula kwemishini kanye nokuvuselela kagesi kwe-cardiomyocyte kubalulekile ekugcineni i-phenotype yenhliziyo33,34,35.Ngenkathi ukuvuselela komshini sekusetshenziswe kabanzi ukulungisa nokuvuthwa kwe-hiPSC-CM, izifundo eziningi ezinhle zisanda kuzama ukuvuselela izingcezu zenhliziyo kusiko kusetshenziswa ukulayisha okukodwa.Lezi zifundo zibonisa ukuthi ukulayisha kwe-2D uniaxial mechanical kunomphumela omuhle ku-phenotype yenhliziyo phakathi nesiko.Kulezi zifundo, izingxenye zenhliziyo zazilayishwe amandla e-isometric tensile17, i-linear auxotonic loading18, noma umjikelezo wenhliziyo wenziwa kabusha kusetshenziswa impendulo ye-transducer yamandla kanye nokushayela kwengcindezi.Kodwa-ke, lezi zindlela zisebenzisa i-uniaxial tissue stretch ngaphandle kokulungiswa kwemvelo, okuholela ekucindezelweni kwezakhi zofuzo eziningi zenhliziyo noma ukucindezeleka ngokweqile kwezakhi zofuzo ezihambisana nezimpendulo ezilula ezingavamile.I-CTCM echazwe lapha inikeza i-3D electromechanical stimulus elingisa umjikelezo wenhliziyo wemvelo ngokwesikhathi somjikelezo kanye nokwelula komzimba (ukunwebeka okungu-25%, i-systole engu-40%, i-diastole engu-60%, namabhithi angu-72 ngomzuzu).Nakuba lokhu kukhuthazwa komshini wezinhlangothi ezintathu kuphela akwanele ukugcina ubuqotho bezicubu, inhlanganisela yokuvuselela okuhlekisayo nokusetshenziswa komshini usebenzisa i-T3/Dex iyadingeka ukuze kugcinwe ngokwanele ukusebenza kwezicubu, ukusebenza, nobuqotho.
Izici ezihlekisayo zidlala indima ebalulekile ekuguquleni i-phenotype yenhliziyo yabantu abadala.Lokhu kugqanyiswe ezifundweni ze-HiPS-CM lapho i-T3 ne-Dex zengezwe kumidiya yezamasiko ukuze kusheshiswe ukuvuthwa kwamaseli.I-T3 ingaba nomthelela ekuthuthweni kwama-amino acid, ushukela kanye ne-calcium kuwo wonke ulwelwesi lwamaseli36.Ukwengeza, i-T3 ikhuthaza ukubonakaliswa kwe-MHC-α kanye nokwehliswa kwe-MHC-β, ikhuthaza ukwakheka kwe-myofibrils esheshayo ku-cardiomyocyte evuthiwe uma kuqhathaniswa ne-slow twitch myofibrils ku-CM yengane.Ukuntuleka kwe-T3 ezigulini ze-hypothyroid kuholela ekulahlekelweni kwamabhande e-myofibrillar kanye nezinga elincishisiwe lokuthuthukiswa kwethoni37.I-Dex isebenza kuma-receptors e-glucocorticoid futhi iboniswe ukwandisa ukufinyela kwe-myocardial ezinhliziyweni ezixubile ezihlukene;38 lokhu kuthuthukiswa kucatshangwa ukuthi kuhlobene nomthelela ekungeneni kwe-calcium deposit-driven (SOCE) 39,40.Ngaphezu kwalokho, i-Dex ibophezela kuma-receptors ayo, okubangela impendulo ebanzi ye-intracellular ecindezela ukusebenza komzimba nokuvuvukala30.
Imiphumela yethu ibonisa ukuthi ukukhuthazwa kokusebenza ngokomzimba (MS) kuthuthukise ukusebenza kwesiko kukonke uma kuqhathaniswa ne-Ctrl, kodwa kwehlulekile ukugcina ukusebenza kahle, ubuqotho besakhiwo, nokubonakaliswa kwenhliziyo phakathi kwezinsuku eziyi-12 kusiko.Uma kuqhathaniswa ne-Ctrl, ukungezwa kwe-T3 ne-Dex kumasiko e-CTCM (MT) kuthuthukise ukusebenza futhi kwagcina amaphrofayili afanayo alotshiweyo, ubuqotho besakhiwo, nomsebenzi we-metabolic ngezicubu zenhliziyo ezintsha izinsuku eziyi-12.Ukwengeza, ngokulawula izinga lokwelula kwezicubu, imodeli ye-hyperextension-induced cardiac hypertrophy yadalwa kusetshenziswa i-STCM, ebonisa ukuguquguquka kwesistimu ye-STCM.Kumele kuqashelwe ukuthi nakuba ukuguqulwa kwenhliziyo kanye ne-fibrosis ngokuvamile kuhilela izitho eziqinile amangqamuzana ajikelezayo anganikeza ama-cytokines afanelekile kanye ne-phagocytosis nezinye izici zokuvuselela, izingxenye zenhliziyo zisengakwazi ukulingisa inqubo ye-fibrotic ekuphenduleni ukucindezeleka nokuhlukumezeka.ku-myofibroblasts.Lokhu kuye kwahlolwa ngaphambilini kule modeli yocezu lwenhliziyo.Kumele kuqashelwe ukuthi imingcele ye-CTCM ingashintshwa ngokushintsha ingcindezi / i-amplitude kagesi kanye nemvamisa ukuze ilingise izimo eziningi ezifana ne-tachycardia, i-bradycardia, kanye nokusekelwa kokujikeleza kwemishini (inhliziyo engalayishiwe).Lokhu kwenza isistimu ibe i-output emaphakathi yokuhlolwa kwezidakamizwa.Ikhono le-CTCM lokumodela i-hypertrophy yenhliziyo eyenziwe ngokuzikhandla ngokweqile livula indlela yokuhlola lolu hlelo ukuze kutholakale ukwelashwa komuntu siqu.Sengiphetha, ucwaningo lwamanje lubonisa ukuthi ukunwebeka kwemishini kanye nokugqugquzela ama-humoral kubalulekile ekugcineni isiko lezigaba zezicubu zenhliziyo.
Nakuba idatha evezwe lapha iphakamisa ukuthi i-CTCM iyinkundla ethembisayo kakhulu yokumodela i-myocardium eqinile, le ndlela yesiko inokulinganiselwa okuthile.Umkhawulo oyinhloko wesiko le-CTCM ukuthi ibeka ukucindezeleka okuqhubekayo okuguquguqukayo ezincekwini, okuvimbela ikhono lokuqapha ngenkuthalo ukufinyela kocezu lwenhliziyo phakathi nomjikelezo ngamunye.Ngaphezu kwalokho, ngenxa yobukhulu obuncane bezingxenye zenhliziyo (7 mm), ikhono lokuhlola umsebenzi we-systolic ngaphandle kwezinhlelo zesiko kusetshenziswa izinzwa zamandla endabuko lilinganiselwe.Embhalweni wesandla wamanje, sinqoba kancane lo mkhawulo ngokuhlola i-optical voltage njengenkomba yomsebenzi wenkontileka.Kodwa-ke, lo mkhawulo uzodinga umsebenzi owengeziwe futhi ungase uxazululwe ngokuzayo ngokwethula izindlela zokuqapha ngokubona umsebenzi wezingcezu zenhliziyo esikweni, njengemephu yokubona kusetshenziswa i-calcium nodayi abazwela amandla kagesi.Omunye umkhawulo we-CTCM ukuthi imodeli yokusebenza ayisebenzisi ukucindezeleka ngokomzimba (ukulayisha kuqala kanye nokulayisha ngemuva).Ku-CTCM, ingcindezi yenziwa ngezindlela ezihlukile ukuze ikhiqize kabusha i-25% ye-physiological stretch ku-diastole (ukwelula okugcwele) kanye ne-systole (ubude bokufinyela ngesikhathi sokuvuselela ugesi) ezicutshini ezinkulu kakhulu.Lo mkhawulo kufanele ususwe emiklamo ye-CTCM yesikhathi esizayo ngokucindezela okwanele kwezicubu zenhliziyo ezivela ezinhlangothini zombili kanye nokusebenzisa ubudlelwano obuqondile bokucindezela kwevolumu okwenzeka emakamelweni enhliziyo.
Ukulungiswa kabusha okudalwe ukwelula okubikwe kulo mbhalo wesandla kukhawulelwe ekulingiseni amasignali e-hypertrophic hyperstretch.Ngakho-ke, lo modeli ungasiza ekutadisheni i-stretch-induced hypertrophic signaling ngaphandle kwesidingo sezinto ezihlekisayo noma ze-neural (ezingekho kulolu hlelo).Ucwaningo olwengeziwe luyadingeka ukuze kwandiswe ukuphindaphindeka kwe-CTCM, isibonelo, ukuhlanganisana namaseli omzimba, ukujikeleza kwezici ze-plasma humoral, kanye nokungabi nandawo lapho ukulima ngokubambisana namaseli e-neuronal kuzothuthukisa amathuba okuba nesifo imodeli nge-CTCM.
Kusetshenziswe izingulube eziyishumi nantathu kulolu cwaningo.Zonke izinqubo zezilwane zenziwe ngokuhambisana neziqondiso zesikhungo futhi zagunyazwa yi-University of Louisville Institutional Animal Care and Use Committee.I-aorta arch yayiboshiwe futhi inhliziyo yagcotshwa ngo-1 L we-cardioplegia eyinyumba (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 emakhazeni e-ice-cold cardioplegic kuze kube yilapho zithuthelwa elebhu eqhweni elivame ukuba <10 min. izinhliziyo zagcinwa emakhazeni e-ice-cold cardioplegic kuze kube yilapho zithuthelwa elebhu eqhweni elivame ukuba <10 min. сердца хранили в ледяном кардиоплегическом растворе до транспортировки в лабораторию на льду, что обычно занимает <10 мин. izinhliziyo zazigcinwe kusixazululo senhliziyo esibandayo kuze kube yilapho zithuthwa endaweni yokucwaninga ngeqhwa, ngokuvamile okuthatha <10 min.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 ku-ice cardioplegia kuze kube yilapho uthuthwa endaweni yokucwaninga ngeqhwa, ngokuvamile u-<10 min.
Idivayisi ye-CTCM yakhiwe ngesofthiwe ye-SolidWorks yomklamo osizwa ngekhompiyutha (CAD).Amakamelo amasiko, abahlukanisi kanye namagumbi omoya enziwe ngepulasitiki ye-acrylic ecacile ye-CNC.Indandatho yesipele engu-7mm yenziwe nge-polyethylene (i-HDPE) ephakeme kakhulu phakathi nendawo futhi ine-o-ring groove ukuze ifake i-silicone o-ring esetshenziselwa ukuvala imidiya ngaphansi.Ulwelwesi oluncane lwe-silica luhlukanisa igumbi lamasiko nepuleti lokuhlukanisa.Ulwelwesi lwe-silicone lusikwe nge-laser ukusuka ku-0.02″ ishidi eliwugqinsi le-silicone futhi linobunzima obungu-35A.Ama-silicone gaskets aphansi nangaphezulu ayi-laser asikwa ukusuka ku-1/16″ ishidi eliwugqinsi le-silicone futhi anobunzima obungu-50A.Izikulufu zensimbi engagqwali ezingu-316L namantongomane aphiko asetshenziselwa ukubopha ibhulokhi nokudala isivalo esingangenisi umoya.
Ibhodi lesekethe eliphrintiwe elizinikele (PCB) lenzelwe ukuhlanganiswa nohlelo lwe-C-PACE-EM.Amasokhethi okuxhuma umshini we-swiss ku-PCB axhunywe kuma-electrode e-graphite ngezintambo zethusi ezicwecwe ngesiliva nezikulufu zethusi ezingu-0-60 ezifakwe kuma-electrode.Ibhodi lesekethe eliphrintiwe libekwe kukhava yephrinta ye-3D.
Idivayisi ye-CTCM ilawulwa i-programmable pneumatic actuator (PPD) eyenza ukucindezela okujikelezayo okulawulwayo okufana nomjikelezo wenhliziyo.Njengoba ukucindezela ngaphakathi kwekamelo lomoya kwanda, ulwelwesi lwe-silicone oluguquguqukayo lunwebeka phezulu, luphoqelele okuphakathi ngaphansi kwesizinda sezicubu.Indawo yezicubu izobe inwetshwa yilokhu kuxoshwa koketshezi, kulingisa ukwanda komzimba wenhliziyo ngesikhathi se-diastole.Esiqongweni sokuphumula, ukuvuselela kagesi kwasetshenziswa ngama-electrode e-graphite, okunciphisa ukucindezela ekamelweni lomoya futhi kubangele ukugoqa kwezingxenye zezicubu.Ngaphakathi kwepayipi kune-valve ye-hemostatic enenzwa yokucindezela ukuze ibone ukucindezela ohlelweni lomoya.Ingcindezi ezwa inzwa yokucindezela isetshenziswa kumqoqi wedatha oxhunywe kukhompuyutha ephathekayo.Lokhu kuvumela ukuqapha okuqhubekayo kokucindezela ngaphakathi kwegumbi legesi.Lapho ukucindezela kwegumbi okukhulu kufinyelelwa (okujwayelekile okungu-80 mmHg, 140 mmHg OS), idivayisi yokutholwa kwedatha yayalelwa ukuthi ithumele isignali ohlelweni lwe-C-PACE-EM ukuze kukhiqizwe isignali kagesi ye-biphasic engu-2 ms, isethelwe ku-4 V.
Izingxenye zenhliziyo zatholwa futhi izimo zamasiko emithonjeni engu-6 zenziwa kanje: Dlulisa izinhliziyo ezivuniwe kusuka emkhunjini wokudlulisa kuya ethreyini equkethe amakhaza (4° C.) i-cardioplegia.I-ventricle yesokunxele yahlukaniswa nge-blade oyinyumba futhi yasikwa izingcezu ze-1-2 cm3.Lawa mabhuloki ezicubu anamathiselwe ezisekelweni zethishu ngokunamathela kwezicubu futhi afakwa kubhavu wethishu ye-microtome edlidlizayo equkethe isixazululo se-Tyrode futhi efakwe umoya-mpilo ngokuqhubekayo (3 g/L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g) . ), 6 mM KCl (0.1 mPE) D-Glucose (0.401M) DG (0.1 mPE) D. (2.38 g), 1 mM MgCl2 (1 ml 1 M isixazululo), 1.8 mM CaCl2 ( 1.8 ml 1 M isixazululo), kufika ku-1 L ddH2O).I-microtome edlidlizayo yayisethelwe ukusika izingcezu eziwugqinsi ezingu-300 µm ngemvamisa engu-80 Hz, i-amplitude yokudlidliza evundlile engu-2 mm, kanye nesilinganiso sangaphambili esingu-0.03 mm/s.Indawo yokugeza izicubu yayizungezwe yiqhwa ukuze kugcinwe ikhambi lipholile futhi izinga lokushisa laligcinwe ku-4°C.Dlulisa izingxenye zezicubu ukusuka kubhavu we-microtome uye kubhavu ekufukanyelwayo equkethe isixazululo seTyrode esigcwele umoya-mpilo eqhweni kuze kube yilapho kutholwa izingxenye ezanele zepuleti elilodwa lempucuko.Emasikweni e-transwell, izigaba zezicubu zazinanyathiselwe kuzisekelo ze-polyurethane eziyinyumba eziyi-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).Ukukhuthazwa kukagesi (i-10 V, imvamisa engu-1.2 Hz) kusetshenziswe ezigabeni zethishu nge-C-Pace.Ezimweni ze-TD, i-T3 entsha ne-Dex zengezwe ku-100 nM no-1 μM ekushintsheni ngakunye ngakunye.I-medium igcwele umoya-mpilo ngaphambi kokushintshwa izikhathi ezi-3 ngosuku.Izingxenye zezicubu zakhuliswa ku-incubator ku-37°C no-5% CO2.
Kwamasiko e-CTCM, izigaba zethishu zabekwa ephrinta ye-3D eyenziwe ngokwezifiso esitsheni se-Petri esiqukethe isisombululo se-Tyrode esilungisiwe.Idivayisi yakhelwe ukwandisa usayizi wocezu lwenhliziyo ngo-25% wendawo yeringi yokusekela.Lokhu kwenziwa ukuze izingxenye zenhliziyo zinganwebeki ngemva kokususwa kwesisombululo seTyrode ziye endaweni ephakathi nendawo nangesikhathi se-diastole.Kusetshenziswa iglue ye-histoacrylic, izigaba eziwugqinsi ongu-300 µm zalungiswa eringini yosekelo engu-7 mm ububanzi.Ngemva kokunamathisela izingxenye zezicubu eringini yokusekela, nqamula izingxenye zezicubu ezingaphezulu bese ubuyisela izingxenye zezicubu ezinamathiselwe kubhavu wengxube ye-Tyrode eqhweni (4°C) kuze kube yilapho sekulungiselelwe izingxenye ezanele zomshini owodwa.Isikhathi esiphelele sokucubungula sawo wonke amadivayisi akumele sidlule amahora ama-2.Ngemuva kokuthi izingxenye zezicubu ze-6 zifakwe emasongweni azo okusekela, idivayisi ye-CTCM yahlanganiswa.Igumbi lesikompilo le-CTCM ligcwaliswa ngaphambilini nge-pre-oxygenated medium engu-21 ml.Dlulisa izingxenye zezicubu ekamelweni lamasiko futhi ususe ngokucophelela noma yimaphi ama-bubbles emoyeni nge-pipette.Ingxenye yethishu ibe isiqondiswa emgodini bese icindezelwa kahle endaweni yayo.Ekugcineni, faka isivalo se-electrode kudivayisi bese udlulisela idivayisi ku-incubator.Bese uxhuma i-CTCM kushubhu lomoya kanye nohlelo lwe-C-PACE-EM.I-actuator yomoya iyavuleka bese ivalvu yomoya ivula i-CTCM.Isistimu ye-C-PACE-EM yalungiselelwa ukuthi ilethe i-4 V ku-1.2 Hz phakathi nokuhamba kwe-biphasic ku-2 ms.I-medium yayishintshwa kabili ngosuku futhi ama-electrode ayeshintshwa kanye ngosuku ukuze kugwenywe ukunqwabelana kwegraphite kuma-electrode.Uma kunesidingo, izingxenye zezicubu zingasuswa emithonjeni yazo yesiko ukuze kukhishwe noma yimaphi amabhamuza omoya okungenzeka awele ngaphansi kwawo.Ezimweni zokwelashwa kwe-MT, i-T3/Dex yengezwe entsha ngoshintsho olumaphakathi ngalunye nge-100 nM T3 kanye ne-1 μM Dex.Amadivaysi e-CTCM akhuliswe ku-incubator ku-37°C naku-5% CO2.
Ukuze kutholwe izindlela ezinwetshiwe zezingcezu zenhliziyo, kwasungulwa isimiso sekhamera esikhethekile.Ikhamera ye-SLR (Canon Rebel T7i, Canon, Tokyo, Japan) isetshenziswe ne-Navitar Zoom 7000 18-108mm macro lens (Navitar, San Francisco, CA).Ukubona ngeso lengqondo kwenziwa ekamelweni lokushisa ngemva kokufaka okuphakathi nendawo entsha.Ikhamera ibekwe ku-engeli engu-51° futhi ividiyo irekhodwa kumafreyimu angu-30 ngomzuzwana.Okokuqala, isofthiwe yomthombo ovulekile (MUSCLEMOTION43) yasetshenziswa ne-Image-J ukuze kulinganise ukunyakaza kwezingcezu zenhliziyo.Imaski idalwe kusetshenziswa i-MATLAB (MathWorks, Natick, MA, USA) ukuze ichaze izifunda ezinentshisekelo kuzo zokushaya izingcezu zenhliziyo ukugwema umsindo.Amamaski ahlukaniswe mathupha asetshenziswa kuzo zonke izithombe ngokulandelana kozimele bese edluliselwa ku-plug-in ye-MUSCLEMOTION.I-Muscle Motion isebenzisa ukuqina okumaphakathi kwamaphikseli kufreyimu ngayinye ukuze kulinganise ukunyakaza kwayo ngokuhlobene nohlaka lwesithenjwa.Idatha yarekhodwa, yahlungwa futhi yasetshenziselwa ukulinganisa isikhathi somjikelezo nokuhlola ukunwetshwa kwezicubu phakathi nomjikelezo wenhliziyo.Ividiyo erekhodiwe yacutshungulwa ngemuva kusetshenziswa isihlungi sedijithali se-oda lokuqala elinguziro.Ukuze kulinganise ukunwebeka kwezicubu (ukuphakama-kuya-kuphakameni), ukuhlaziya kwe-peak-to-peak kwenziwa ukuze kuhlukaniswe phakathi kweziqongo nama-troughs kusiginali erekhodiwe.Ngaphezu kwalokho, ukuhoxiswa kwenziwa kusetshenziswa i-polynomial ye-6th ukuze kuqedwe ukukhukhuleka kwesignali.Ikhodi yohlelo yathuthukiswa ku-MATLAB ukuze kunqunywe ukunyakaza kwezicubu zomhlaba wonke, isikhathi somjikelezo, isikhathi sokuphumula, nesikhathi sokufinyela (Ikhodi Yohlelo Eyengeziwe 44).
Ukuze kuhlaziywe ubunzima, kusetshenziswa amavidiyo afanayo adalelwe ukuhlola ukunwebeka komshini, siqale salandelela izithombe ezimbili ezimele iziqongo zokunyakaza (amaphoyinti okunyakaza aphezulu kakhulu (aphezulu) naphansi (aphansi) ngokuya ngesoftware ye-MUSCLEMOTION.Sabe sesihlukanisa izifunda zezicubu futhi sasebenzisa uhlobo lwe-algorithm ye-shading kuzicubu ezihlukene (I-Supplementary Fig. 2a).Izicubu ezihlukene zabe sezihlukaniswa zaba izindawo ezingaphansi eziyishumi, futhi ukucindezeleka endaweni ngayinye kubalwa kusetshenziswa i-equation elandelayo: I-Strain = (I-Sup-Sdown)/Sdown, lapho i-Sup ne-Sdown ingamabanga wesimo kusukela phezulu nangaphansi kwethunzi lendwangu, ngokulandelanayo (I-Supplementary Fig. .2b).
Izingxenye zenhliziyo zalungiswa ku-4% paraformaldehyde amahora angama-48.Izicubu ezingaguquki zaphelelwa amanzi ngo-10% kanye no-20% we-sucrose ihora elingu-1, kwase kuba ngama-30% e-sucrose ngobusuku obubodwa.Izingxenye zabe sezishunyekwa ku-optimum cutting temperatures compound (OCT compound) futhi kancane kancane ziqandiswe endaweni yokugeza ye-isopentane/eyomile yeqhwa.Gcina amabhulokhi wokushumeka we-OCT ku- -80 °C kuze kube ukuhlukana.Amaslayidi alungiswa njengezingxenye ezinogqinsi luka-8 μm.
Ukuze ususe i-OCT ezigabeni zenhliziyo, shisisa amaslayidi endaweni yokushisa engu-95 °C imizuzu emi-5.Engeza i-1 ml ye-PBS kusilayidi ngasinye bese ufukamela imizuzu engu-30 ekamelweni lokushisa, bese ungena ezingxenyeni ngokubeka u-0.1% we-Triton-X ku-PBS imizuzu engu-15 ekamelweni lokushisa.Ukuze uvimbele amasosha omzimba angaqondile ekubopheni isampula, engeza u-1 ml wesisombululo se-BSA esingu-3% kumaslayidi futhi ufukamele ihora elingu-1 ekamelweni lokushisa.I-BSA yabe isikhishwa futhi amaslayidi ahlanzwa nge-PBS.Maka isampula ngayinye ngepensela.Amasosha omzimba ayinhloko (ahlanjululwe ngu-1:200 ku-1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) kanye ne-troponin-T (Thermo Fcientific; #MA5-12960) angezwe ngaphezu kwemizuzu engu-90, kwase kuba amasosha omzimba angu-4% e-Alexaluted ku-12% esibili: 8 (Thermo Scientific; #A16079), ngokumelene nogwaja u-Alexa Fluor 594 (Thermo Scientific; #T6391) amaminithi angu-90 engeziwe Sigeze izikhathi ezingu-3 nge-PBS Ukuze sihlukanise ukungcola okuqondisiwe kusukela kungemuva, sasebenzisa kuphela isivikela-mzimba sesibili njengesilawuli. Ekugcineni, i-DAPI nuclear stain yafakwa futhi kwafakwa i-nail ye-vectarato ye-nail e-Vectato -x magnification) kanye nesibonakhulu sikaKeyence esinokukhulisa okungu-40x.
I-WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) ku-5 μg/ml ku-PBS yasetshenziselwa ukungcolisa i-WGA futhi yasetshenziswa ezigabeni ezigxilile imizuzu engu-30 ekamelweni lokushisa.Ama-slides abe esewashwa nge-PBS futhi i-Sudan emnyama yafakwa esilayidini ngasinye futhi ifakwe imizuzu engu-30.Amaslayidi abe esegezwa nge-PBS futhi kwafakwa i-vectashield medium.Amaslayidi aboniswe ngesibonakhulu se-Keyence ekukhuliseni okungu-40x.
I-OCT ikhishiwe kumasampuli njengoba kuchazwe ngenhla.Ngemva kokukhipha i-OCT, gxilisa amaslayidi esixazululweni sika-Bouin ngobusuku bonke.Amaslayidi abe esehlanzwa ngamanzi acwengekile ihora elingu-1 abese efakwa kusixazululo se-Bibrich aloe acid fuchsin imizuzu eyi-10.Khona-ke ama-slides ahlanjululwa ngamanzi a-distilled futhi afakwa kwisisombululo se-5% phosphomolybdenum/5% phosphotungstic acid imizuzu engu-10.Ngaphandle kokuwageza, dlulisela amaslayidi ngqo kusixazululo esiluhlaza okwesibhakabhaka se-aniline imizuzu eyi-15.Khona-ke ama-slides ahlanjululwa ngamanzi acwengekile futhi afakwa ku-1% isixazululo se-acetic acid imizuzu engu-2.Amaslayidi omisiwe ku-200 N ethanol futhi adluliselwa ku-xylene.Amaslayidi anamabala abonwa ngeso lengqondo kusetshenziswa isibonakhulu se-Keyence esinenjongo engu-10x.Iphesenti lendawo ye-Fibrosis yalinganiswa kusetshenziswa isofthiwe ye-Keyence Analyzer.
I-CyQUANT™ MTT Cell Viability Assay (Invitrogen, Carlsbad, CA), inombolo yekhathalogi V13154, ngokuya ngephrothokholi yomkhiqizi enezinguquko ezithile.Ikakhulukazi, i-punch yokuhlinzwa enobubanzi be-6 mm yasetshenziselwa ukuqinisekisa usayizi wezicubu ezifanayo ngesikhathi sokuhlaziywa kwe-MTT.Ama-tissue afakwe ngawodwana emithonjeni yepuleti elinemithombo engu-12 eliqukethe i-MTT substrate ngokuvumelana nephrothokholi yomkhiqizi.Izingxenye zifukanyelwa ku-37° C. amahora angu-3 futhi izicubu eziphilayo zigaya i-substrate ye-MTT ukuze yakhe inhlanganisela ye-formazan ensomi.Faka esikhundleni sesixazululo se-MTT nge-1 ml DMSO bese ufukamela ku-37 °C imizuzu engu-15 ukuze ukhiphe i-formazan ensomi ezingxenyeni zenhliziyo.Amasampuli ahlanjululwe ngo-1:10 ku-DMSO kumapuleti aphansi acace kahle angu-96 kanye nokuqina kombala onsomi kukalwa ngo-570 nm kusetshenziswa i-Cytation plate reader (BioTek).Ukufundwa kwenziwa ngendlela evamile ngokwesisindo socezu ngalunye lwenhliziyo.
Imidiya yocezu lwenhliziyo yathathelwa indawo imidiya equkethe i-1 μCi/ml [5-3H] -glucose (Moravek Biochemicals, Brea, CA, USA) yokuhlola ukusetshenziswa kweglucose njengoba kuchazwe ngaphambilini.Emuva kwamahora angu-4 okufukamela, engeza u-100 µl wokuphakathi neshubhu le-microcentrifuge elivulekile eliqukethe u-100 µl we-0.2 N HCl.Khona-ke leli shubhu lalifakwa eshubhuni yokugcoba equkethe u-500 μl we-dH2O ukuze ihwamuke [3H]2O amahora angu-72 ku-37°C.Bese ususa ithubhu ye-microcentrifuge kusuka kushubhu ye-scintillation bese wengeza u-10 ml we-scintillation fluid.Izibalo ze-scintillation zenziwe kusetshenziswa i-Tri-Carb 2900TR liquid scintillation analyzer (Packard Bioscience Company, Meriden, CT, USA).Ukusetshenziswa kweglucose kwabe sekubalwa kucatshangelwa umsebenzi othize we-glucose engu- [5-3H], ukulingana okungaphelele nesizinda, ukuhlanjululwa kuka- [5-3H] -kuya ku-glucose engenamalebula, kanye nokusebenza kahle kwe-scintillation counter.Idatha ijwayele ubuningi bezingxenye zenhliziyo.
Ngemuva kokuhlanganiswa kwezicubu ku-Trizol, i-RNA yahlukaniswa nezingxenye zenhliziyo kusetshenziswa i-Qiagen miRNeasy Micro Kit #210874 ngokuya ngephrothokholi yomkhiqizi.Ukulungiswa komtapo wezincwadi we-RNAsec, ukulandelana nokuhlaziywa kwedatha kwenziwe kanje:
I-1 μg ye-RNA isampula ngayinye isetshenziswe njengesiqalo sokulungiswa komtapo wezincwadi we-RNA.Amalabhulali alandelanayo akhiqizwa kusetshenziswa i-NEBNext UltraTM RNA Library Preparation Kit ye-Illumina (NEB, USA) ngokulandela izincomo zomkhiqizi, namakhodi ezinkomba engeziwe ekulandeleni kwezibaluli zesampula ngayinye.Kafushane, i-mRNA yahlanzwa ku-RNA isiyonke kusetshenziswa ubuhlalu kazibuthe obunamathiselwe nge-poly-T oligonucleotides.Ukuhlukaniswa kwenziwa kusetshenziswa ama-divalent cations ezingeni lokushisa eliphezulu ku-NEBNext First Strand Synthesis Reaction Buffer (5X).I-cDNA yomucu wokuqala yahlanganiswa kusetshenziswa iziqalo ze-hexamer ezingahleliwe kanye ne-M-MuLV reverse transcriptase (RNase H-).I-cDNA yomucu wesibili bese ihlanganiswa kusetshenziswa i-DNA polymerase I kanye ne-RNase H. Ama-overhang asele aguqulwa abe iziphetho ezibuthuntu ngomsebenzi we-exonuclease/polymerase.Ngemva kokwahlukaniswa kokuphela kwe-3′ yesiqeshana se-DNA, i-adaptha ye-NEBNext ene-hairpin loop structure ixhunywe kuyo ukuze ilungiselele ukuxutshwa.Ukuze kukhethwe izingcezu ze-cDNA zobude obuncanyelwayo obungu-150-200 bp.izingcezwana zomtapo wolwazi zahlanzwa kusetshenziswa uhlelo lwe-AMPure XP (Beckman Coulter, Beverly, USA).Bese, 3 μl USER Enzyme (NEB, USA) ene-cDNA ekhethiwe usayizi exhunywe nge-adaptha yasetshenziswa imizuzu engu-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, i-PCR primers, kanye neziqalo ze-Index (X).Ekugcineni, imikhiqizo ye-PCR yahlanzwa (uhlelo lwe-AMPure XP) kanye nekhwalithi yelabhulali yahlolwa kuhlelo lwe-Agilent Bioanalyzer 2100.Umtapo wezincwadi we-cDNA wabe usulandelwa kusetshenziswa isilandeleli se-Novaseq.Amafayela esithombe angahlungiwe asuka ku-Illumina aguqulelwe ekufundweni okungaphekiwe kusetshenziswa i-CASAVA Base Calling.Idatha eluhlaza igcinwa ngamafayela efomethi ye-FASTQ(fq) aqukethe ukulandelana kokufundiwe kanye nezimfanelo eziyisisekelo ezihambisanayo.Khetha i-HISAT2 ukuze ufanise ukulandelana okuhlungiwe okufundwe ku-Sscrofa11.1 reference genome.Ngokuvamile, i-HISAT2 isekela ama-genomes anoma yimuphi usayizi, okuhlanganisa ama-genomes amakhulu kunezisekelo eziyizigidi eziyizinkulungwane ezingu-4, futhi amanani azenzakalelayo asethelwe amapharamitha amaningi.I-Splicing ifundeka kudatha ye-RNA Seq ingaqondaniswa kahle kusetshenziswa i-HISAT2, isistimu eshesha kakhulu etholakalayo njengamanje, enembayo efanayo noma engcono kunanoma iyiphi enye indlela.
Ubuningi bemibhalo elotshiweyo bubonisa ngokuqondile izinga lenkulumo yofuzo.Amaleveli esisho sofuzo ahlolwa ngobuningi bemibhalo ebhaliwe (isibalo sokulandelana) esihlotshaniswa ne-genome noma ama-exons.Inombolo yokufundwayo ilingana namazinga wokuchazwa kofuzo, ubude bofuzo, nokujula kokulandelana.I-FPKM (izingcezu eziyinkulungwane zamapheya esisekelo sombhalo olandelanayo ngamapheya esisekelo ayisigidi) abaliwe futhi amanani e-P wenkulumo ehlukile anqunywa kusetshenziswa iphakheji ye-DESeq2.Sibe sesibala izinga lokutholwa okungamanga (FDR) ivelu ngayinye engu-P sisebenzisa indlela ye-Benjamin-Hochberg9 ngokusekelwe kumsebenzi owakhelwe ngaphakathi we-R othi “p.adjust”.
I-RNA ehlukanisiwe nezingxenye zenhliziyo yaguqulelwa ku-cDNA ekugxilweni kuka-200 ng/μl kusetshenziswa i-SuperScript IV Vilo Master mix kusukela ku-Thermo (Thermo, cat. no. 11756050).I-quantitative RT-PCR yenziwe kusetshenziswa i-Applied Biosystems Endura Plate Microamp 384-well transparent reaction plate (Thermo, cat. no. 4483319) kanye ne-microamp optical adhesive (Thermo, cat. no. 4311971).Ingxube yokusabela yayihlanganisa 5 µl Taqman Fast Advanced Master mix (Thermo, cat # 4444557), 0.5 µl Taqman Primer kanye no-3.5 µl H2O okuxutshwe emthonjeni ngamunye.Imijikelezo evamile ye-qPCR yasetshenziswa futhi amanani e-CT alinganiswa kusetshenziswa ithuluzi le-PCR le-Applied Biosystems Quantstudio 5 lesikhathi sangempela (imojula ye-384-well; umkhiqizo # A28135).I-Taqman Primers yathengwa kusuka ku-Thermo (GAPDH (SS033756299_U1), Parp12 (SS06908795_M1), i-PKDCC (SS06903874_M1), i-CLGB1 (SS06868890_M1), ACTN1 (SS01009508 _mh), Gata4 (SS03383805_U1), GJA1 (SS03374839_U1 (SS03374839_U1 (SS0337500399 Dh.
Ukukhishwa kwemidiya kwe-NT-ProBNP kwahlolwa kusetshenziswa ikhithi ye-NT-ProBNP (ingulube) (Cat. No. MBS2086979, MyBioSource) ngokuya ngephrothokholi yomkhiqizi.Kafushane, u-250 µl wesampula ngalinye nezinga lingeziwe ngokuphindwe kabili emthonjeni ngamunye.Ngokushesha ngemva kokwengeza isampula, engeza u-50 µl we-Assay Reagent A emthonjeni ngamunye.Gcoba ipuleti ngobumnene bese uvala nge-sealant.Khona-ke amaphilisi afakwa ku-37 ° C ihora elingu-1.Bese ufisa isixazululo bese ugeza imithombo izikhathi ezingu-4 ngo-350 µl wesisombululo sokugeza esingu-1X, ufukamele isixazululo sokugeza imizuzu engu-1-2 isikhathi ngasinye.Bese wengeza u-100 µl we-Assay Reagent B emthonjeni ngamunye bese uvala nge-plate sealant.Ithebhulethi yanyakaziswa kahle futhi yafakwa ku-37 ° C imizuzu engu-30.Funa isixazululo bese ugeza imithombo izikhathi ezi-5 ngo-350 µl wesisombululo sokugeza esingu-1X.Engeza u-90 µl wesixazululo se-substrate emthonjeni ngamunye bese uvala ipuleti.Faka ipuleti ku-37 ° C imizuzu engu-10-20.Engeza i-50 µl Stop Solution emthonjeni ngamunye.Ipuleti likalwa ngokushesha kusetshenziswa isifundi sepuleti se-Cytation (BioTek) esisethwe ku-450 nm.
Ukuhlaziywa kwamandla kwenziwe ukuze kukhethwe osayizi beqembu abazonikeza >80% amandla okuthola uguquko oluphelele elingu-10% kupharamitha enezinga lephutha elingu-5%. Ukuhlaziywa kwamandla kwenziwe ukuze kukhethwe osayizi beqembu abazonikeza >80% amandla okuthola uguquko oluphelele elingu-10% kupharamitha enezinga lephutha elingu-5%. Ukushintshanisa okungcono kakhulu kwe- 80% kufinyelela ku-10% yemali engenayo той ошибок типа I. Ukuhlaziywa kwamandla kwenziwe ukuze kukhethwe osayizi beqembu ababezonikeza >80% amandla okuthola ushintsho oluphelele lwepharamitha engu-10% ngesilinganiso sephutha esingu-5%.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 обнаружения 10% абеспечил бы aстоты ошибок типа I. Ukuhlaziywa kwamandla kwenziwe ukuze kukhethwe usayizi weqembu onganikeza >80% amandla okuthola u-10% woshintsho oluphelele lwepharamitha kanye nesilinganiso sephutha esingu-5%.Izigaba zezicubu zikhethwe ngokungahleliwe ngaphambi kokuhlolwa.Konke ukuhlaziya bekungaboni kahle futhi amasampula aqoshwa kuphela ngemuva kokuthi yonke idatha isihlaziywe.Isofthiwe ye-GraphPad Prism (i-San Diego, CA) yasetshenziswa ukwenza konke ukuhlaziya kwezibalo. Kuzo zonke izibalo, amanani we-p athathwe njengabalulekile kumanani angu-<0.05. Kuzo zonke izibalo, amanani we-p athathwa njengokubalulekile kumanani <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Kuzo zonke izibalo, amanani we-p athathwa njengokubalulekile kumanani <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 we-p athathwa njengokubalulekile kumanani <0.05.Ukuhlolwa kuka-t Komfundi onemisila emibili kwenziwa kudatha ngokuqhathaniswa okungu-2 kuphela.I-ANOVA yendlela eyodwa noma emibili isetshenziswe ukuze kutholwe ukubaluleka phakathi kwamaqembu amaningi.Lapho kuhlolwa i-post hoc, ukulungiswa kuka-Tukey kwasetshenziswa ku-akhawunti ukuze kuqhathaniswe kaningi.Idatha ye-RNAsec inokucatshangelwa kwezibalo okukhethekile lapho kubalwa i-FDR kanye ne-p.lungisa njengoba kuchazwe esigabeni Sezindlela.
Ukuze uthole ulwazi olwengeziwe mayelana nesakhiwo socwaningo, bheka i-Nature Research Report abstract exhunywe kulesi sihloko.