Enkosi ngokutyelela iNature.com. Inguqulelo yesikhangeli oyisebenzisayo inenkxaso encinci yeCSS. Ukuze ufumane amava angcono, sicebisa ukuba usebenzise isikhangeli esihlaziyiweyo (okanye ukhubaze iMowudi yokuHlangana kwi-Internet Explorer). Okwangoku, ukuqinisekisa inkxaso eqhubekayo, siza kuyinika le ndawo ngaphandle kwezitayile kunye neJavaScript.
Kukho imfuneko yenkqubo ethembekileyo ye-in vitro enokuphinda ivelise ngokuchanekileyo imeko-bume yempilo yentliziyo ukuze kuvavanywe amayeza. Ukunqongophala kweenkqubo zokukhulisa izicubu zentliziyo yomntu kukhokelele ekutolikweni okungachanekanga kwemiphumo yeziyobisi zentliziyo. Apha, siphuhlise imodeli yokukhulisa izicubu zentliziyo (CTCM) evuselela ngombane izilayi zentliziyo kwaye idlule ekunwetshweni kwempilo ngexesha lezigaba ze-systolic kunye ne-diastolic zomjikelo wentliziyo. Emva kweentsuku ezili-12 zokukhulisa, le ndlela yaphucula kancinci ukusebenza kwamacandelo entliziyo, kodwa ayizange igcine ngokupheleleyo ukuthembeka kwawo kwesakhiwo. Ke ngoko, emva kokuhlolwa kweemolekyuli ezincinci, sifumanise ukuba ukongezwa kwe-100 nM triiodothyronine (T3) kunye ne-1 μM dexamethasone (Dex) kwindawo yethu yokugcina ulwakhiwo lwecandelo kangangeentsuku ezili-12. Ngokudibeneyo nonyango lwe-T3/Dex, inkqubo ye-CTCM igcine iiprofayili zokubhala, ukusebenza, umsebenzi we-metabolic, kunye nokuthembeka kwesakhiwo kwinqanaba elifanayo nezicubu zentliziyo ezintsha kangangeentsuku ezili-12. Ukongeza, ukolula kakhulu kwezicubu zentliziyo kwinkcubeko kubangela ukuhanjiswa kwemiqondiso yentliziyo e-hypertrophic, nto leyo enika ubungqina bokuba i-CTCM iyakwazi ukulinganisa iimeko ze-hypertrophic ezibangelwa kukolula kwentliziyo. Ukuqukumbela, i-CTCM inokwenza umzekelo we-physiology kunye ne-pathophysiology yentliziyo kwinkcubeko ixesha elide, nto leyo evumela ukuba kuhlolwe amayeza ngendlela ethembekileyo.
Ngaphambi kophando lwezonyango, kufuneka iinkqubo ezithembekileyo ze-in vitro ezinokuphinda zivelise ngokuchanekileyo imeko-bume yempilo yentliziyo yomntu. Ezi nkqubo kufuneka zifane nokutshintsha kwendlela yokwelula, ukubetha kwentliziyo, kunye neempawu ze-electrophysiological. Iimodeli zezilwanyana zihlala zisetyenziswa njengeqonga lokuhlola i-physiology yentliziyo kunye nokuthembeka okuncinci ekuboniseni imiphumo yamayeza entliziyweni yomntu1,2. Ekugqibeleni, iModeli yoVavanyo lweSifo seNtliziyo seNtliziyo yeNtliziyo yeNtliziyo (i-CTCM) yimodeli enovakalelo olukhulu kwaye ikhethekileyo kwiindlela ezahlukeneyo zonyango kunye neyeza, iphinda ivelise ngokuchanekileyo i-physiology kunye ne-pathophysiology yentliziyo yomntu3. Ukungabikho kwenkqubo enjalo kuthintela ukufunyanwa konyango olutsha lokusilela kwentliziyo4,5 kwaye kukhokelele ekubeni ne-cardiotoxicity yamayeza njengesizathu esiphambili sokuphuma kwimarike6.
Kwiminyaka elishumi edlulileyo, amayeza asibhozo angengowentliziyo aye arhoxiswa ekusetyenzisweni kweklinikhi kuba abangela ukwandiswa kwexesha le-QT okukhokelela kwi-ventricular arrhythmias kunye nokufa ngequbuliso7. Ke ngoko, kukho isidingo esikhulayo seendlela zokuhlola ezithembekileyo ngaphambi konyango lokuvavanya ukusebenza kakuhle kwentliziyo kunye nobuthi. Ukusetyenziswa kwakutshanje kwe-cardiomyocytes ezithathwe kwi-human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) ekuhlolweni kwamayeza kunye novavanyo lobuthi kubonelela ngesisombululo esincinci kule ngxaki. Nangona kunjalo, imeko yokungavuthwa kwe-hiPS-CMs kunye nokungabikho kobunzima beeseli ezininzi zezicubu zentliziyo yimida ephambili yale ndlela. Izifundo zakutshanje zibonise ukuba lo mda unokoyiswa ngokuyinxenye ngokusebenzisa i-hiPS-CM yokuqala ukwenza ii-hydrogels zezicubu zentliziyo emva nje kokuqala kokungqubana okuzenzekelayo kunye nokunyusa kancinci ukuvuselelwa kombane ngokuhamba kwexesha. Nangona kunjalo, ezi microtissues ze-hiPS-CM azinazo iipropati ze-electrophysiological kunye ne-contractile ezivuthiweyo ze-myocardium yabantu abadala. Ukongeza, izicubu zentliziyo yomntu zinesakhiwo esintsonkothileyo, esiqulathe umxube owahlukeneyo weentlobo ezahlukeneyo zeeseli, kuquka iiseli ze-endothelial, ii-neurons, kunye ne-stromal fibroblasts, ezidityaniswe ziiseti ezithile zeeproteni ze-extracellular matrix. Olu mahluko phakathi kwe-non-cardiomyocyte populations11,12,13 entliziyweni yabantu abadala abancancisayo ngumqobo omkhulu ekubumbeni izicubu zentliziyo kusetyenziswa iintlobo zeeseli nganye. Le mida iphambili igxininisa ukubaluleka kokuphuhlisa iindlela zokukhulisa izicubu zentliziyo ezingaguqukiyo phantsi kweemeko zomzimba neze-pathological.
Amacandelo amancinci akhuliswe kakuhle (300 µm) entliziyo yomntu abonakalise ukuba ngumzekelo othembisayo we-myocardium yomntu engaguqukiyo. Le ndlela ibonelela ngokufikelela kwinkqubo epheleleyo ye-3D multicellular efana nezicubu zentliziyo yomntu. Nangona kunjalo, kude kube ngu-2019, ukusetyenziswa kwamacandelo entliziyo akhuliswe kakuhle kwakunqunyelwe kukusinda kwexesha elifutshane (iiyure ezingama-24) lokukhula. Oku kungenxa yezinto ezininzi eziquka ukungabikho kolulo lwe-physical-mechanical, ujongano lomoya-lwe-liquid, kunye nokusetyenziswa kweendaba ezilula ezingaxhasi iimfuno zezicubu zentliziyo. Ngo-2019, amaqela ophando aliqela abonise ukuba ukufaka izinto ze-mechanical kwiinkqubo zenkcubeko yezicubu zentliziyo kunokwandisa ubomi benkcubeko, kuphucule ukubonakaliswa kwentliziyo, kwaye kufane ne-cardiac pathology. Izifundo ezimbini ezintle 17 kunye 18 zibonisa ukuba umthwalo we-uniaxial mechanical unefuthe elihle kwi-phenotype yentliziyo ngexesha lokukhula. Nangona kunjalo, ezi zifundo azisebenzisanga umthwalo we-physico-mechanical onemilinganiselo emithathu womjikelo wentliziyo, kuba amacandelo entliziyo ayene-isometric tensile forces 17 okanye i-linear auxotonic loading 18. Ezi ndlela zokolula izicubu zibangele ukucinezelwa kwezakhi zofuzo ezininzi zentliziyo okanye ukubonakaliswa kakhulu kwezakhi zofuzo ezinxulumene neempendulo ezingaqhelekanga zokolula. Okuphawulekayo kukuba, uPitoulis et al. 19 baphuhlise i-dynamic heart slice culture bath yokwakhiwa kwakhona komjikelo wentliziyo besebenzisa impendulo ye-force transducer kunye ne-tension drives. Nangona le nkqubo ivumela ukumodelwa komjikelo wentliziyo we-in vitro ngokuchanekileyo, ubunzima kunye nomthamo ophantsi wendlela kunciphisa ukusetyenziswa kwale nkqubo. Ilabhoratri yethu isandula ukuphuhlisa inkqubo yokukhulisa eyenziwe lula esebenzisa ukuvuselela ngombane kunye nendlela ephuculweyo yokugcina ukusebenza kwamacandelo eenyamakazi kunye nezicubu zentliziyo yomntu ukuya kuthi ga kwiintsuku ezi-6.
Kwincwadi yangoku, sichaza imodeli yokukhulisa izicubu zentliziyo (i-CTCM) sisebenzisa amacandelo entliziyo yehagu aquka iimpawu ze-humoral ukuze aphinde achaze i-physiology yentliziyo enemilinganiselo emithathu kunye nokukhula kwe-pathophysiological ngexesha lomjikelo wentliziyo. Le CTCM inokunyusa ukuchaneka kokubikezela amayeza ngaphambi konyango ukuya kwinqanaba elingazange lifikelelwe ngaphambili ngokubonelela ngenkqubo yentliziyo engabizi kakhulu, ephakathi elinganisa i-physiology/pathophysiology yentliziyo yezilwanyana ezincancisayo ukuze kuvavanywe amayeza ngaphambi konyango.
Imiqondiso ye-Hemodynamic mechanical idlala indima ebalulekileyo ekugcineni umsebenzi we-cardiomyocyte kwi-vitro 22,23,24. Kwincwadi yangoku, siphuhlise i-CTCM (Umfanekiso 1a) enokulinganisa imeko-bume yentliziyo yomntu omdala ngokukhuthaza zombini ukuvuselelwa kombane kunye noomatshini kwi-physiological frequency (1.2 Hz, 72 beats ngomzuzu). Ukuthintela ukolulwa kwezicubu ngokugqithisileyo ngexesha le-diastole, isixhobo sokuprinta se-3D sisetyenzisiwe ukwandisa ubungakanani bezicubu nge-25% (Umfanekiso 1b). Isantya sombane esibangelwa yinkqubo ye-C-PACE sabekwa ixesha lokuqala nge-100 ms ngaphambi kokuba i-systole isebenzise inkqubo yokufumana idatha ukuvelisa ngokupheleleyo umjikelo wentliziyo. Inkqubo yokukhulisa izicubu isebenzisa i-actuator ye-pneumatic ecwangcisiweyo (LB Engineering, Germany) ukwandisa i-membrane ye-silicone eguquguqukayo ngokujikelezayo ukuze kubangele ukwanda kwezilayi zentliziyo kwigumbi eliphezulu. Inkqubo yayiqhagamshelwe kumgca womoya wangaphandle nge-transducer yoxinzelelo, okwenza kube nokwenzeka ukulungisa ngokuchanekileyo uxinzelelo (± 1 mmHg) kunye nexesha (± 1 ms) (Umfanekiso 1c).
a Ncamathisela icandelo lezicubu kwiringi yokuxhasa eyi-7 mm, eboniswe ngombala oluhlaza okwesibhakabhaka, ngaphakathi kwegumbi lokukhulisa lesixhobo. Igumbi lokukhulisa lahlulwe kwigumbi lomoya yi-membrane encinci ye-silicone eguquguqukayo. Beka i-gasket phakathi kwegumbi ngalinye ukuthintela ukuvuza. Isiciko sesixhobo siqulathe ii-electrode ze-graphite ezibonelela ngokuvuselela ngombane. b Ukubonakaliswa kwesicwangciso sesixhobo esikhulu sezicubu, iringi yesikhokelo kunye neringi yokuxhasa. Amacandelo ezicubu (amdaka) abekwa kwisixhobo esikhulu kunye neringi yesikhokelo ebekwe kwi-groove kumda wangaphandle wesixhobo. Usebenzisa isikhokelo, beka ngononophelo iringi yokuxhasa egqunywe nge-tissue acrylic adhesive phezu kwecandelo lezicubu zentliziyo. c Igrafu ebonisa ixesha lokuvuselela ngombane njengomsebenzi woxinzelelo lwegumbi lomoya olulawulwa yi-programmable pneumatic actuator (PPD). Isixhobo sokufumana idatha sisetyenziselwe ukuvumelanisa ukuvuselela ngombane kusetyenziswa izinzwa zoxinzelelo. Xa uxinzelelo kwigumbi lokukhulisa lufikelela kumda omiselweyo, isignali ye-pulse ithunyelwa kwi-C-PACE-EM ukuze iqalise ukuvuselela ngombane. d Umfanekiso wee-CTCM ezine ezibekwe kwishelufu ye-incubator. Izixhobo ezine ziqhagamshelwe kwi-PPD enye nge-pneumatic circuit, kwaye ii-pressure sensors zifakwa kwi-hemostatic valve ukuze zijonge uxinzelelo kwi-pneumatic circuit. Isixhobo ngasinye sinamacandelo amathandathu ezicubu.
Sisebenzisa i-actuator enye ye-pneumatic, sikwazile ukulawula izixhobo ezi-4 ze-CTCM, ngasinye sazo esinokubamba amacandelo ama-6 ezicubu (Umzobo 1d). Kwi-CTCM, uxinzelelo lomoya kwigumbi lomoya luguqulwa lube luxinzelelo oluhambelanayo kwigumbi lolwelo kwaye lubangela ukwanda kwe-physiological kwesilayi sentliziyo (Umfanekiso 2a kunye ne-Supplementary Movie 1). Uvavanyo lokwelulwa kwezicubu kwi-80 mm Hg. Art. lubonise ukolulwa kwamacandelo ezicubu nge-25% (Umzobo 2b). Olu kolulwa kwepesenti luboniswe ukuba luhambelana nobude be-physiological sarcomere obuyi-2.2–2.3 µm kwi-contractility eqhelekileyo yecandelo lentliziyo17,19,25. Intshukumo yezicubu ihlolwe kusetyenziswa useto lwekhamera oluqhelekileyo (Umfanekiso ongezelelweyo 1). Ubukhulu kunye nesantya sokuhamba kwezicubu (Umzobo 2c, d) zihambelana nokolulwa ngexesha lomjikelo wentliziyo kunye nexesha ngexesha le-systole kunye ne-diastole (Umzobo 2b). Ukolulwa kunye nesantya sezicubu zentliziyo ngexesha lokukothuka kunye nokuphumla kwahlala kungaguquki iintsuku ezili-12 kwinkcubeko (Umzobo 2f). Ukuvavanya impembelelo yokuvuselelwa kombane kwi-contractility ngexesha lokukhulisa, siphuhlise indlela yokufumanisa ukukhubazeka okusebenzayo sisebenzisa i-shading algorithm (Umzobo oNcedisayo 2a,b) kwaye sikwazile ukwahlula phakathi kokukhubazeka okunokuvuselelwa kombane kunye nokungashukunyiswa kombane. Icandelo elifanayo lentliziyo (Umzobo 2f). Kwindawo eshukumayo ye-cut (R6-9), i-voltage ngexesha lokuvuselelwa kombane yayiphezulu ngama-20% kunaxa kungekho kuvuselelwa kombane, nto leyo ebonisa igalelo lokuvuselelwa kombane kumsebenzi wokukhupha.
Iimpawu ezimele uxinzelelo lwegumbi lomoya, uxinzelelo lwegumbi lolwelo, kunye nokulinganisa ukuhamba kwezicubu ziqinisekisa ukuba uxinzelelo lwegumbi lutshintsha uxinzelelo lwegumbi lolwelo, nto leyo ebangela intshukumo ehambelanayo yesilayi sezicubu. b Iimpawu ezimele ipesenti yokwelula (oluhlaza okwesibhakabhaka) kwamacandelo ezicubu ahambelana nepesenti yokwelula (orenji). c Intshukumo elinganisiweyo yesilayi sentliziyo ihambelana nesantya esilinganisiweyo sentshukumo. (d) Iindlela ezimele intshukumo yokujikeleza (umgca oluhlaza okwesibhakabhaka) kunye nesantya (umgca onamachaphaza eorenji) kwisilayi sentliziyo. e Ubungakanani bexesha lomjikelo (n = izilayi ezili-19 kwiqela ngalinye, kwiihagu ezahlukeneyo), ixesha lokuqina (n = izilayi ezili-19 kwiqela ngalinye), ixesha lokuphumla (n = izilayi ezili-19 kwiqela ngalinye, kwiihagu ezahlukeneyo), intshukumo yezicubu (n = 25). izilayi)/iqela kwiihagu ezahlukeneyo), isantya se-systolic esiphezulu (n = 24(D0), izilayi ezingama-25(D12)/iqela kwiihagu ezahlukeneyo) kunye nesantya sokuphumla esiphezulu (n=24(D0), izilayi ezingama-25(D12)/iqela kwiihagu ezahlukeneyo). Uvavanyo lwe-t lomfundi olunemisila emibini alubonisanga mahluko abalulekileyo kuyo nayiphi na iparameter. f Uhlalutyo olumeleyo lweengxaki zamacandelo ezicubu anokukhuthazwa kombane (obomvu) kwaye ngaphandle kokukhuthazwa kombane (oluhlaza okwesibhakabhaka), iindawo ezilishumi zengingqi zamacandelo ezicubu ezivela kwicandelo elinye. Iiphaneli ezisezantsi zibonisa ubungakanani bomahluko wepesenti kuxinzelelo kumacandelo ezicubu anokukhuthazwa kombane nangaphandle kokukhuthazwa kombane kwiindawo ezilishumi ezivela kumacandelo ahlukeneyo. (n = izilayi ezi-8/iqela elivela kwiihagu ezahlukeneyo, uvavanyo lwe-t lomfundi olunemisila emibini lwenziwa; ****p < 0.0001, **p < 0.01, *p < 0.05). (n = izilayi ezi-8/iqela elivela kwiihagu ezahlukeneyo, uvavanyo lwe-t lomfundi olunemisila emibini lwenziwa; ****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 = amacandelo asi-8/iqela elivela kwiihagu ezahlukeneyo, uvavanyo lwe-t lomfundi olunemisila emibini; ****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 = amacandelo asi-8/iqela, avela kwiihagu ezahlukeneyo, uvavanyo lwe-t lomfundi olunemisila emibini; ****p<0.0001, **p<0.01, *p<0.05).Iibha zempazamo zimele i-mean ± standard deviation.
Kwinkqubo yethu yangaphambili ye-static biomimetic heart slice culture [20, 21], sigcine ukusebenza, ukusebenza, kunye nokuqina kwesakhiwo see-heart slices kangangeentsuku ezi-6 ngokusebenzisa ukuvuselela ngombane kunye nokwenza ngcono ukwakheka kwe-medium. Nangona kunjalo, emva kweentsuku ezili-10, la manani ehle kakhulu. Siza kubhekisa kumacandelo akhuliswe kwinkqubo yethu yangaphambili ye-static biomimetic culture 20, 21 control conditions (Ctrl) kwaye siza kusebenzisa i-medium yethu eyayilungisiwe ngaphambili njengeemeko ze-MC kunye ne-culture phantsi kokuvuselela ngoomatshini kunye nombane ngaxeshanye (CTCM). ebizwa ngokuba yi-. Okokuqala, sigqibe kwelokuba ukuvuselela ngoomatshini ngaphandle kokuvuselela ngombane kwakunganelanga ukugcina ukusebenza kwezicubu kangangeentsuku ezi-6 (Umzobo ongezelelweyo 3a,b). Okunomdla kukuba, ngokungeniswa kokuvuselela nge-physio-mechanical kunye nombane kusetyenziswa i-STCM, ukusebenza kwe-12-day heart sections kwahlala kufana nakwi-fresh heart sections phantsi kwe-MS, kodwa kungekhona phantsi kwe-Ctrl, njengoko kuboniswe luhlalutyo lwe-MTT (Umzobo 1). 3a). Oku kuthetha ukuba ukuvuselela ngoomatshini kunye nokulinganisa umjikelo wentliziyo kunokugcina amacandelo ezicubu ephila ixesha elide kabini njengoko kuxeliwe kwinkqubo yethu yangaphambili ye-static culture. Nangona kunjalo, uvavanyo lokuqina kwesakhiwo seenxalenye zezicubu nge-immunolabeling ye-cardiac troponin T kunye ne-connexin 43 lubonise ukuba ukubonakaliswa kwe-connexin 43 kwakuphezulu kakhulu kwiithishu ze-MC ngomhla we-12 kunakwiilawuli ngosuku olunye. Nangona kunjalo, ukubonakaliswa kwe-connexin 43 efanayo kunye nokwakheka kwe-Z-disc akuzange kugcinwe ngokupheleleyo (Umzobo 3b). Sisebenzisa isakhelo se-artificial intelligence (AI) ukulinganisa ukuqina kwesakhiwo sezicubu26, umbhobho wokufunda okunzulu osekelwe kumfanekiso osekelwe kwi-troponin-T kunye ne-connexin staining43 ukulinganisa ngokuzenzekelayo ukuqina kwesakhiwo kunye ne-fluorescence yezilayi zentliziyo ngokwendlela yamandla eendawo. Le ndlela isebenzisa i-Convolutional Neural Network (CNN) kunye nesakhelo sokufunda okunzulu ukulinganisa ngokuthembekileyo ukuqina kwesakhiwo sezicubu zentliziyo ngendlela ezenzekelayo nengakhethi cala, njengoko kuchaziwe kwisalathiso. 26. Ithishu ye-MC ibonise ukufana okuphuculweyo kwesakhiwo nomhla we-0 xa kuthelekiswa namacandelo olawulo oluzinzileyo. Ukongeza, ukudaya kwe-trichrome kaMasson kutyhile ipesenti ephantsi kakhulu ye-fibrosis phantsi kweemeko ze-MS xa kuthelekiswa neemeko zolawulo ngosuku lwe-12 lokukhula (Umzobo 3c). Nangona i-CTCM yonyuse ukusebenza kwamacandelo ezicubu zentliziyo ngosuku lwe-12 ukuya kwinqanaba elifanayo nelezicubu zentliziyo ezintsha, ayizange iphucule kakhulu ukuqina kwesakhiwo samacandelo entliziyo.
Igrafu yeBar ibonisa ubungakanani bokuphila kwe-MTT kwezilayi zentliziyo ezintsha (D0) okanye inkcubeko yezilayi zentliziyo iintsuku ezili-12 nokuba kukukhula okungaguqukiyo (D12 Ctrl) okanye kwi-CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) izilayi/iqela elivela kwiihagu ezahlukeneyo, indlela enye eyenziwa ngayo uvavanyo lwe-ANOVA; ####p < 0.0001 xa kuthelekiswa ne-D0 kunye ne **p < 0.01 xa kuthelekiswa ne-D12 Ctrl). Igrafu yeBar ibonisa ubungakanani bokuphila kwe-MTT kwezilayi zentliziyo ezintsha (D0) okanye inkcubeko yezilayi zentliziyo iintsuku ezili-12 nokuba kukukhula okungaguqukiyo (D12 Ctrl) okanye kwi-CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) izilayi/iqela elivela kwiihagu ezahlukeneyo, indlela enye eyenziwa ngayo uvavanyo lwe-ANOVA; ####p < 0.0001 xa kuthelekiswa ne-D0 kunye ne **p < 0.01 xa kuthelekiswa ne-D12 Ctrl).i-histogram ibonisa ubungakanani bokuphila kwe-MTT fresh heart sections (D0) okanye i-culture of heart sections kangangeentsuku ezili-12 kwi-static culture (D12 control) okanye kwi-CTCM (D12 MC) (n = 18 (D0), 15 (D12 control). ) ), 12 (D12 MC) sections/group ezivela kwiingulube ezahlukeneyo, kwenziwa uvavanyo lwe-ANOVA oluya kwicala elinye;####p < 0,0001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 xa kuthelekiswa no-D0 kunye no-**p < 0.01 xa kuthelekiswa 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) 或片(D0) 或叶光 (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*10D0 CTRL 1,000 < 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 ebonisa ubungakanani bokuphila kwe-MTT kwiindawo zentliziyo entsha (D0) okanye kwiindawo zentliziyo ezikhuliswe iintsuku ezili-12 kwinkcubeko engashukumiyo (ulawulo lwe-D12) okanye kwi-CTCM (D12 MC) (n = 18 (D0), 15 (ulawulo lwe-D12)), 12 (D12 MC) iindawo/iqela elivela kwiingulube ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye;####p < 0,0001 по сравнению с D0, **p < 0,01 по сравнению с D12 Ctrl). ####p < 0.0001 xa kuthelekiswa no-D0, **p < 0.01 xa kuthelekiswa no-D12 Ctrl).b I-Troponin-T (eluhlaza), i-connexin 43 (ebomvu) kunye ne-DAPI (eluhlaza okwesibhakabhaka) kwiindawo zentliziyo ezisandula ukwahlulwa (D0) okanye kwiindawo zentliziyo ezikhuliswe phantsi kweemeko ezingashukumiyo (Ctrl) okanye iimeko ze-CTCM (MC) kangangeentsuku ezili-12) zemifanekiso emele i-immunofluorescence (isikali esingenanto = 100 µm). Ukulinganiswa kobukrelekrele bokwenziwa kobume bentliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izilayi/iqela ngalinye elivela kwihagu eyahlukileyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ####p < 0.0001 xa kuthelekiswa ne-D0 kunye ****p < 0.0001 xa kuthelekiswa ne-D12 Ctrl). Ukulinganiswa kobukrelekrele bokwenziwa kobume besakhiwo sezicubu zentliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) izilayi/iqela ngalinye kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ####p < 0.0001 xa kuthelekiswa ne-D0 kunye ****p < 0.0001 xa kuthelekiswa ne-D12 Ctrl). Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) сруправи/ проводится однофакторный тест ANOVA ; Ukulinganiswa kobume besakhiwo sezicubu zentliziyo ngokusebenzisa ubukrelekrele bokwenziwa (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) amacandelo/amaqela avela kwiihagu ezahlukeneyo, kwenziwe uvavanyo lwe-ANOVA oluhamba ngendlela enye; ####p < 0.0001 vs. no-D0 kunye no-****p < 0.0001 xa kuthelekiswa 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) izilayi/iqela nganye yehagu eyahlukileyo, indlela enye yovavanyo lwe-ANOVA <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) izilayi/iqela nganye yeehagu ezahlukeneyo, indlela enye yovavanyo lwe-ANOVA; <0.0001 与D12 Ctrl 相比）. Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC/MC) разных свиней, односторонний тест ANOVA; Ubukrelekrele bokwenziwa ukuze kulinganiswe ukuthembeka kwesakhiwo sezicubu zentliziyo (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) amacandelo/iqela ngalinye leengulube ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye; ####p<0.0001 vs .D0 Ukuthelekisa ****p < 0.0001 xa kuthelekiswa ne-D12 Ctrl). c Imifanekiso emeleyo (ekhohlo) kunye nokulinganiswa (ekunene) kwezilayi zentliziyo ezidaywe ngebala likaMasson le-trichrome (Isikali singenanto = 500 µm) (n = izilayi ezili-10/iqela ngalinye elivela kwihagu eyahlukileyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ####p < 0.0001 xa kuthelekiswa no-D0 kunye ***p < 0.001 xa kuthelekiswa no-D12 Ctrl). c Imifanekiso emeleyo (ekhohlo) kunye nokulinganiswa (ekunene) kwezilayi zentliziyo ezidaywe ngebala likaMasson le-trichrome (Isikali singenanto = 500 µm) (n = izilayi ezili-10/iqela ngalinye kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; #### p < 0.0001 xa kuthelekiswa no-D0 kunye no-***p < 0.001 xa kuthelekiswa no-D12 Ctrl). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Масязк00 мкм) (n = 10 срезов/группу от разных свиней, выполняется односторонний тест ANOVA; c Imifanekiso emeleyo (ekhohlo) kunye nokulinganiswa (ekunene) kwamacandelo entliziyo anombala webala likaMasson le-trichrome (isikali esingagqunywanga = 500 µm) (n = amacandelo ali-10/iqela elivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwe; #### p < 0 .0001 xa kuthelekiswa ne-D0 kunye ***p < 0.001 xa kuthelekiswa ne-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)个切片/组，每组來自不同的猪，进行单向ANOVA 测试； C 用 mason 三 色 染料的 心脏 切片 的 代表性 （左 左） 量化= 500 µm） （n = 10 个 切片 组 每 來自 不同 猪 , 进行 单向 单向 Anova 测试；#### D0 <0.0.0. < 0.001 Xhuma i-D12 Ctrl 相比）. c Репрезентативные изображения (слева) и количественный анализ (справа) срезов сердца, окрашенных трихромным красителем Массом 5 (n = 10 срезов/группа, каждый от другой свиньи, протестировано с помощью однофакторного дисперсионного анализа ;### #p <0,00,000 сравнению с D12 Ctrl). c Imifanekiso emele (ekhohlo) kunye nobungakanani (ekunene) beenxalenye zentliziyo ezidaywe ngebala likaMasson le-trichrome (elingenanto = 500 µm) (n = iinxalenye ezili-10/iqela, ngalinye livela kwihagu eyahlukileyo, livavanywe ngohlalutyo lwendlela enye lokwahluka ;### # p < 0.0001 xa kuthelekiswa no-D0, ***p < 0.001 xa kuthelekiswa no-D12 Ctrl).Iibha zempazamo zimele i-mean ± standard deviation.
Sicinge ukuba ngokongeza iimolekyuli ezincinci kwindawo yokukhulisa iiseli, ukuthembeka kwentliziyo kunokuphuculwa kwaye uphuhliso lwe-fibrosis lunciphe ngexesha lokukhulisa iiseli ze-CTCM. Ngoko ke sihlolisise iimolekyuli ezincinci sisebenzisa iinkcubeko zethu zokulawula ezizinzileyo20,21 ngenxa yenani elincinci lezinto ezididayo. I-Dexamethasone (Dex), i-triiodothyronine (T3), kunye ne-SB431542 (SB) zikhethwe kolu vavanyo. Ezi molekyuli zincinci bezisetyenziswa ngaphambili kwiinkcubeko ze-hiPSC-CM ukukhuthaza ukuvuthwa kweeseli zentliziyo ngokwandisa ubude be-sarcomere, ii-T-tubules, kunye nesantya sokuqhuba. Ukongeza, zombini i-Dex (i-glucocorticoid) kunye ne-SB zaziwa ngokucinezela ukuvuvukala29,30. Ke ngoko, sivavanye ukuba ukufakwa kwenye okanye indibaniselwano yezi molekyuli zincinci kuya kuphucula ukuthembeka kwesakhiwo seenxalenye zentliziyo. Ukuhlolwa kokuqala, idosi yekhompawundi nganye ikhethwe ngokusekelwe kumanqanaba asetyenziswa rhoqo kwiimodeli zenkcubeko yeseli (1 μM Dex27, 100 nM T327, kunye ne-2.5 μM SB31). Emva kweentsuku ezili-12 zokukhula, ukudityaniswa kwe-T3 kunye ne-Dex kwaphumela ekubeni kubekho ukuqina kwesakhiwo se-cardiomyocyte kunye nokulungiswa okuncinci kwe-fibrous (Imifanekiso eyongezelelweyo 4 kunye no-5). Ukongeza, ukusetyenziswa kokuphindwe kabini okanye kabini kwezi ngxinano ze-T3 kunye ne-Dex kuye kwavelisa iziphumo ezinobungozi xa kuthelekiswa nokuqhelekileyo (Umzobo owongezelelweyo 6a,b).
Emva kovavanyo lokuqala, senze uthelekiso lweemeko ezine zenkcubeko (Umfanekiso 4a): I-Ctrl: amacandelo entliziyo akhuliswe kwinkcubeko yethu engaguqukiyo echazwe ngaphambili kusetyenziswa indlela yethu ephuculweyo; I-20.21 TD: I-T3 kunye ne-Ctrl s Added Dex ngoLwesithathu; I-MC: amacandelo entliziyo akhuliswe kwi-CTCM kusetyenziswa indlela yethu ephuculweyo ngaphambili; kunye ne-MT: I-CTCM ene-T3 kunye ne-Dex yongezwe kwisixhobo. Emva kweentsuku ezili-12 zokulima, ukuphila kwezicubu ze-MS kunye ne-MT kwahlala kufana nakwizicubu ezintsha ezihlolwe ngovavanyo lwe-MTT (Umzobo 4b). Okunomdla kukuba, ukongezwa kwe-T3 kunye ne-Dex kwiinkcubeko ze-transwell (TD) akuzange kubangele ukuphucuka okukhulu kokuphila xa kuthelekiswa neemeko ze-Ctrl, nto leyo ebonisa indima ebalulekileyo yokukhuthaza umatshini ekugcineni ukuphila kwecandelo lentliziyo.
umzobo woyilo lovavanyo obonisa iimeko ezine zenkcubeko ezisetyenziselwa ukuvavanya imiphumo yokuvuselelwa koomatshini kunye nokongezwa kwe-T3/Dex kwindawo ephakathi kangangeentsuku ezili-12. b Igrafu yebha ibonisa ubungakanani bokuphila emva kweentsuku ezili-12 emva kokukhuliswa kuzo zonke iimeko zokukhuliswa ezine (Ctrl, TD, MC, kunye neMT) xa kuthelekiswa nezilayi zentliziyo ezintsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kunye neD12 MT), izilayi/iqela ezili-12 (D12 MC) ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ####p < 0.0001, ###p < 0.001 xa kuthelekiswa ne-D0 kunye **p < 0.01 xa kuthelekiswa ne-D12 Ctrl). b Igrafu yebha ibonisa ubungakanani bokuphila emva kweentsuku ezili-12 emva kokukhuliswa kuzo zonke iimeko zokukhuliswa ezine (Ctrl, TD, MC, kunye neMT) xa kuthelekiswa nezilayi zentliziyo ezintsha (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kunye neD12 MT), izilayi/iqela ezili-12 (D12 MC) ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ####p < 0.0001, ###p < 0.001 xa kuthelekiswa ne-D0 kunye **p < 0.01 xa kuthelekiswa ne-D12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после культивирования во всех 4 сравнению со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD kunye D12 MT), 12 (D12 MC) срезов/группу от разных програзией, ANOVA; ####p <0,0001, ###p < 0,001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). b Igrafu yebha ibonisa ubungakanani bokuphila kwiintsuku ezili-12 emva kokukhuliswa kuzo zonke iimeko zokukhuliswa ezine (ulawulo, i-TD, i-MC, kunye ne-MT) xa kuthelekiswa namacandelo entliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, i-D12 TD, kunye ne-D12 MT), amacandelo/iqela eli-12 (D12 MC) elivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye; ####p < 0.0001, ###p < 0.001 vs. D0 kunye **p < 0.01 by xa kuthelekiswa 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 ne MT) по сравнению со свежими срезами сердца (D0) (n = 15,D12), CTRL 18 (D12) MT), от разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA; b I-Histogram ebonisa zonke iimeko ezine zenkcubeko (ulawulo, i-TD, i-MC kunye ne-MT) xa kuthelekiswa namacandelo entliziyo entsha (D0) (n = 18 (D0), 15 (D12 Ctrl, i-D12 TD kunye ne-D12 MT), ezivela kwiihagu ezahlukeneyo 12 (D12 MC) amacandelo/iqela, uvavanyo lwe-ANOVA oluya kwicala elinye; ####p<0.0001, ###p<0.001 vs. D0, **p<0.01 vs. ulawulo D12). c Igrafu yebha ibonisa ubungakanani bokuhamba kweglucose kwiintsuku ezili-12 emva kokukhula kuzo zonke iimeko zokukhula ezine (iCtrl, iTD, iMC, kunye neMT) xa kuthelekiswa nezilayi zentliziyo entsha (D0) (n = izilayi ezi-6/iqela ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ###p < 0.001, xa kuthelekiswa ne-D0 kunye ne ***p < 0.001 xa kuthelekiswa ne-D12 Ctrl). c Igrafu yebha ibonisa ubungakanani bokuhamba kweglucose kwiintsuku ezili-12 emva kokukhula kuzo zonke iimeko zokukhula ezine (iCtrl, iTD, iMC, kunye neMT) xa kuthelekiswa nezilayi zentliziyo entsha (D0) (n = izilayi ezi-6/iqela ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ###p < 0.001, xa kuthelekiswa ne-D0 kunye ne ***p < 0.001 xa kuthelekiswa ne-D12 Ctrl). c Гистограмма показывает количественную оценку потока глюкозы через 12 дней после культивирования во всех 4 условиях культивирования, которые сравнению со свежими срезами сердца (D0) (n = 6 срезов/группу от разных свиней, односторонний Выполняется тест ANOVA; ##не#p 0,00 по сравнению с D12 Ctrl). c I-Histogram ibonisa ubungakanani bokuhamba kweglucose kwiintsuku ezili-12 emva kokukhuliswa phantsi kwazo zonke iimeko zokukhula ezine (ulawulo, i-TD, i-MC kunye ne-MT) xa kuthelekiswa namacandelo entliziyo entsha (D0) (n = amacandelo ama-6/iqela elivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwe; ###p < 0.001 xa kuthelekiswa ne-D0 kunye ***p < 0.001 xa kuthelekiswa 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 *D0 CTRL12 ,*.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 切片 切片 相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 与D12 Ctrl 相比）. c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после культивирования для всех 4 сравнению со свежими срезами сердца (D0) (n = 6 срезов/группа, от разных свиней, односторонний Были проведены тесты ANOVA; 0,001 по сравнению с D12 (контроль). c I-Histogram ebonisa ubungakanani bokuhamba kweglucose kwiintsuku ezili-12 emva kokukhuliswa kwazo zonke iimeko zokukhuliswa ezine (ulawulo, i-TD, i-MC, kunye ne-MT) xa kuthelekiswa namacandelo entliziyo entsha (D0) (n = amacandelo ama-6/iqela, kwiihagu ezahlukeneyo, kwicala elinye Ngaba kwenziwe uvavanyo lwe-ANOVA, ###p < 0.001 xa kuthelekiswa ne-D0, ***p < 0.001 xa kuthelekiswa ne-D12 (ulawulo).d Iiploti zohlalutyo loxinzelelo lwezicubu ezintsha (eziluhlaza okwesibhakabhaka), usuku lwe-12 MC (eziluhlaza), kunye nosuku lwe-12 MT (ezibomvu) kwiindawo ezilishumi zecandelo lezicubu zengingqi (n = izilayi ezi-4/iqela, uvavanyo lwe-ANOVA oluhamba ngendlela enye; bekungekho mahluko ubalulekileyo phakathi kwamaqela). e Iploti yentaba-mlilo ebonisa iijini ezivezwe ngokwahlukileyo kwiindawo zentliziyo entsha (D0) xa kuthelekiswa neendawo zentliziyo ezikhuliswe phantsi kweemeko ezimileyo (Ctrl) okanye phantsi kweemeko ze-MT (MT) kangangeentsuku ezili-10-12. f Imephu yobushushu yeejini ze-sarcomere kwiindawo zentliziyo ezikhuliswe phantsi kweemeko zenkcubeko nganye. Iibha zempazamo zimele i-avareji ± ukuphambuka okuqhelekileyo.
Ukuxhomekeka kwi-metabolism kwi-switch ukusuka kwi-fatty acid oxidation ukuya kwi-glycolysis luphawu lokwahlukana kwe-cardiomyocyte. Ii-cardiomyocytes ezingekavuthwa zisebenzisa i-glucose kwimveliso ye-ATP kwaye zine-hypoplastic mitochondria ene-cristae encinci5,32. Uhlalutyo lokusetyenziswa kwe-glucose lubonise ukuba phantsi kweemeko ze-MC kunye ne-MT, ukusetyenziswa kwe-glucose kwakufana noko kwizicubu zosuku lwe-0 (Umfanekiso 4c). Nangona kunjalo, iisampulu ze-Ctrl zibonise ukwanda okukhulu kokusetyenziswa kwe-glucose xa kuthelekiswa nezicubu ezintsha. Oku kubonisa ukuba indibaniselwano ye-CTCM kunye ne-T3/Dex iphucula ukusebenza kwezicubu kwaye igcina i-metabolism phenotype yeentsuku ezili-12 zentliziyo ezikhuliswe. Ukongeza, uhlalutyo loxinzelelo lubonise ukuba amanqanaba oxinzelelo ahlala efana nakwizicubu zentliziyo ezintsha kangangeentsuku ezili-12 phantsi kweemeko ze-MT kunye ne-MS (Umfanekiso 4d).
Ukuze sihlalutye impembelelo iyonke ye-CTCM kunye ne-T3/Dex kwi-transcriptional landscape yehlabathi yezicubu zesilayi sentliziyo, senze i-RNAseq kwizilayi zentliziyo ezivela kuzo zonke iimeko ezine ezahlukeneyo zenkcubeko (Idatha eyongezelelweyo 1). Okunomdla kukuba, amacandelo e-MT abonise ukufana okuphezulu kwe-transcriptional nezicubu zentliziyo ezintsha, kunye ne-16 kuphela ezivezwe ngokwahlukileyo kwi-13,642 yezakhi zofuzo. Nangona kunjalo, njengoko sibonisile ngaphambili, izilayi ze-Ctrl zibonise ii-genes ezili-1229 ezivezwe ngokwahlukileyo emva kweentsuku ezili-10-12 kwinkcubeko (Umzobo 4e). Ezi datha ziqinisekiswe yi-qRT-PCR yezakhi zofuzo zentliziyo kunye ne-fibroblast (Umzobo ongezelelweyo 7a-c). Okunomdla kukuba, amacandelo e-Ctrl abonise ukwehla kwezakhi zofuzo zentliziyo kunye nomjikelo weseli kunye nokusebenza kweenkqubo zezakhi zofuzo ezivuthayo. Ezi datha zibonisa ukuba ukwahlukana, okuqhele ukwenzeka emva kokukhulisa ixesha elide, kuncitshiswa ngokupheleleyo phantsi kweemeko ze-MT (Umzobo ongezelelweyo 8a,b). Uphononongo olucokisekileyo lwee-genes ze-sarcomere lubonise ukuba phantsi kweemeko ze-MT kuphela apho ii-genes ezibhala i-sarcomere (Umzobo 4f) kunye ne-ion channel (Umzobo oNcedisayo 9) zigcinwa khona, zikhusela ekucinezelweni phantsi kweemeko ze-Ctrl, TD, kunye ne-MC. Ezi datha zibonisa ukuba ngokudibanisa ukukhuthazwa koomatshini kunye nokuhlazisa (T3/Dex), i-transcriptome ye-heart slice inokuhlala ifana nee-heart slices ezintsha emva kweentsuku ezili-12 ekukhuleni.
Ezi ziphumo zokubhala zixhaswa yinto yokuba ukuqina kwesakhiwo se-cardiomyocytes kumacandelo entliziyo kugcinwa ngcono phantsi kweemeko ze-MT kangangeentsuku ezili-12, njengoko kuboniswe yi-connexin engaguqukiyo neyasekuhlaleni 43 (Umzobo 5a). Ukongeza, i-fibrosis kumacandelo entliziyo phantsi kweemeko ze-MT incitshiswe kakhulu xa kuthelekiswa ne-Ctrl kwaye ifana namacandelo entliziyo entsha (Umzobo 5b). Ezi datha zibonisa ukuba indibaniselwano yokuvuselela ngoomatshini kunye nonyango lwe-T3/Dex igcina ngokufanelekileyo isakhiwo sentliziyo kumacandelo entliziyo kwinkcubeko.
Imifanekiso emele i-immunofluorescence ye-troponin-T (eluhlaza), i-connexin 43 (ebomvu), kunye ne-DAPI (eluhlaza okwesibhakabhaka) kwiindawo zentliziyo ezisandula ukwahlulwa (D0) okanye ezikhuliswe iintsuku ezili-12 kuzo zonke iimeko ezine zenkcubeko yesigaba sentliziyo (isikali sebha = 100 µm). Ukulinganiswa kobukrelekrele bokwenziwa kobume besakhiwo sezicubu zentliziyo (n = 7 (D0 kunye ne-D12 Ctrl), 5 (D12 TD, D12 MC kunye ne-D12 MT) izilayi/iqela ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ####p < 0.0001 xa kuthelekiswa ne-D0 kunye ne-*p < 0.05, okanye ****p < 0.0001 xa kuthelekiswa ne-D12 Ctrl). Ukulinganiswa kobukrelekrele bokwenziwa kobume besakhiwo sezicubu zentliziyo (n = 7 (D0 kunye ne-D12 Ctrl), 5 (D12 TD, D12 MC kunye ne-D12 MT) izilayi/iqela ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; #### p < 0.0001 xa kuthelekiswa ne-D0 kunye ne-*p < 0.05, okanye ****p < 0.0001 xa kuthelekiswa ne-D12 Ctrl). Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 7 (D0 kunye D12 Ctrl), 5 (D12 TD, D12 MT) kunye neD12 MT от разных свиней, проведен однофакторный тест ANOVA; Ukulinganiswa kobume besakhiwo sezicubu zentliziyo kusetyenziswa ubukrelekrele bokwenziwa (n = 7 (D0 kunye ne-D12 Ctrl), 5 (D12 TD, D12 MC kunye ne-D12 MT) amacandelo/iqela elivela kwiingulube ezahlukeneyo, kwenziwe uvavanyo lwe-ANOVA oluya kwicala elinye; #### p < 0.0001 xa kuthelekiswa ne-D0 kunye ne-*p < 0.05 okanye ****p < 0.0001 xa kuthelekiswa 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 ;相比）.Ukulinganiswa kobume besakhiwo sezicubu zentliziyo kusetyenziswa ubuchule bokwenziwa kwiihagu ezahlukeneyo (n = 7 (D0 kunye neD12 Ctrl), 5 (D12 TD, D12 MC kunye neD12 MT) amacandelo/iqela) ngovavanyo lwe-ANOVA oluya kwicala elinye;#### p < 0,0001 по сравнению с D0 и *p < 0,05 okanye ****p < 0,0001 по сравнению с D12 Ctrl). #### p < 0.0001 xa kuthelekiswa no-D0 kunye no-*p < 0.05 okanye ****p < 0.0001 xa kuthelekiswa no-D12 Ctrl). b Imifanekiso emeleyo kunye nokulinganiswa kwezilayi zentliziyo ezidaywe ngebala likaMasson le-trichrome (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, kunye ne-D12 MC), izilayi/iqela ezili-9 (D12 MT) ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ####p < 0.0001 xa kuthelekiswa ne-D0 kunye ne-***p < 0.001, okanye ****p < 0.0001 xa kuthelekiswa ne-D12 Ctrl). b Imifanekiso emeleyo kunye nokulinganiswa kwezilayi zentliziyo ezidaywe ngebala likaMasson le-trichrome (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, kunye ne-D12 MC), izilayi/iqela ezili-9 (D12 MT) ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-ANOVA oluhamba ngendlela enye lwenziwa; ####p < 0.0001 xa kuthelekiswa ne-D0 kunye ne-***p < 0.001, okanye ****p < 0.0001 xa kuthelekiswa ne-D12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Массока (масштабная 10) = 50 = 10 (D0, D12 Ctrl, D12 TD kunye ne-D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполняется односторонний тест ANOVA <0,***#p 0,001 okanye ****p < 0,0001 по сравнению с D12 Ctrl). b Imifanekiso emeleyo kunye nokulinganiswa kwamacandelo entliziyo anombala we-Masson's trichrome stain (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD kunye ne-D12 MC), amacandelo/iqela eli-9 (D12 MT) avela kwiihagu ezahlukeneyo, ezenziwe ngendlela enye i-ANOVA; ####p < 0.0001 vs. D0 kunye ***p < 0.001 okanye ****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（D2,D2、D2（D3（D（D（D（D（D（D3（D（D（D（D（D（D3（D（D（D（D（D（D（D（D（D（2（D（D0 MC. 0.0001 与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 d0（n d1（n（（（（（（（ （（（（（（（（（（（（（（（（（（（（（（（0（0（0（0（0（0（0（0（（0（0（0（0（0（0（ d0 d 1 d 用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 Imveliso 0.0001 与D12 Ctrl 相比）. b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом Массона (масштабная линейка = 500, D1 = 500, D. I-D12 TD kunye ne-D12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA; D12 Ctrl). b Imifanekiso emeleyo kunye nokulinganiswa kwamacandelo entliziyo anombala we-Masson's trichrome (isikali sebha = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD kunye ne-D12 MC), amacandelo ayi-9 (D12 MT) avela kwiihagu/iqela ezahlukeneyo, indlela enye ye-ANOVA; ####p < 0.0001 xa kuthelekiswa ne-D0, ***p < 0.001 okanye ****p < 0.0001 xa kuthelekiswa ne-D12 Ctrl).Iibha zempazamo zimele i-mean ± standard deviation.
Ekugqibeleni, amandla e-CTCM okulinganisa i-hypertrophy yentliziyo ahlolwe ngokwandisa ukolula kwezicubu zentliziyo. Kwi-CTCM, uxinzelelo lwegumbi lomoya oluphezulu lunyuke ukusuka kwi-80 mmHg ukuya kwi-80 mmHg. Ubugcisa. (ukolula okuqhelekileyo) ukuya kuthi ga kwi-140 mmHg Ubugcisa. (Umfanekiso 6a). Oku kuhambelana nokunyuka kwe-32% kokolula (Umfanekiso 6b), okwaboniswa ngaphambili njengokolula kwepesenti ehambelanayo efunekayo kumacandelo entliziyo ukuze kufezekiswe ubude be-sarcomere obufana nobo bubonwa kwi-hypertrophy. Ukolula kunye nesantya sezicubu zentliziyo ngexesha lokuqiniswa kunye nokuphumla kwahlala kungaguquki kwiintsuku ezintandathu zokukhuliswa (Umfanekiso 6c). Izicubu zentliziyo ezivela kwiimeko ze-MT zafakwa kwi-stretch eqhelekileyo (MT (Normal)) okanye kwiimeko zokunwebeka kakhulu (MT (OS)) kangangeentsuku ezintandathu. Emva kweentsuku ezine kwi-culture, i-hypertrophic biomarker NT-ProBNP yaphakanyiswa kakhulu kwindawo ephakathi phantsi kweemeko ze-MT (OS) xa kuthelekiswa neemeko ze-MT (normal) (Umfanekiso 7a). Ukongeza, emva kweentsuku ezintandathu zokukhulisa, ubungakanani beeseli kwi-MT (OS) (Umzobo 7b) banda kakhulu xa kuthelekiswa namacandelo entliziyo ye-MT (eqhelekileyo). Ukongeza, ukudluliselwa kwenyukliya kwe-NFATC4 kwanda kakhulu kwizicubu ezoluliweyo kakhulu (Umzobo 7c). Ezi ziphumo zibonisa uphuhliso oluqhubekayo lokuguqulwa kwezifo emva kokwanda kwe-hyperdistension kwaye zixhasa ingcamango yokuba isixhobo se-CTCM singasetyenziswa njengeqonga lokufunda i-stretch-induced cardiac hypertrophy signaling.
Iimpawu ezimele uxinzelelo lwegumbi lomoya, uxinzelelo lwegumbi lolwelo, kunye nokulinganisa ukuhamba kwezicubu ziqinisekisa ukuba uxinzelelo lwegumbi lutshintsha uxinzelelo lwegumbi lolwelo, nto leyo ebangela intshukumo ehambelanayo yesilayi sezicubu. b Iigrafu ezimele ipesenti yokwelula kunye nesantya sokwelula kwiindawo zezicubu ezinwetshiweyo (orenji) kunye nezoluliweyo kakhulu (oluhlaza okwesibhakabhaka). c Igrafu yebha ebonisa ixesha lomjikelo (n = izilayi ezili-19 kwiqela ngalinye, kwiihagu ezahlukeneyo), ixesha lokucutheka (n = izilayi ezili-18-19 kwiqela ngalinye, kwiihagu ezahlukeneyo), ixesha lokuphumla (n = izilayi ezili-19 kwiqela ngalinye, kwiihagu ezahlukeneyo) ), ubukhulu bokuhamba kwezicubu (n = izilayi ezili-14/iqela, kwiihagu ezahlukeneyo), isantya se-systolic esiphezulu (n = izilayi ezili-14/iqela, kwiihagu ezahlukeneyo) kunye nesantya sokuphumla esiphezulu (n = 14 (D0), 15 (D6) ) kwiindawo/amaqela) kwiihagu ezahlukeneyo), uvavanyo lwe-t lomfundi olunemisila emibini alubonisanga mahluko abalulekileyo kuyo nayiphi na iparameter, nto leyo ebonisa ukuba ezi parameters zahlala zihleli ngexesha leentsuku ezi-6 zokukhula nge-overvoltage. Iibha zempazamo zimele i-mean ± standard deviation.
Ukulinganiswa kwegrafu yebha yoxinzelelo lwe-NT-ProBNP kwimidiya yenkcubeko evela kwizilayi zentliziyo ezikhuliswe phantsi kweemeko ze-MT normal stretch (Norm) okanye ezolula kakhulu (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kunye ne-D4 MTOS) izilayi/iqela ezivela kwiihagu ezahlukeneyo, i-ANOVA yeendlela ezimbini iyenziwa; **p < 0.01 xa kuthelekiswa nokolula okuqhelekileyo). Ukulinganiswa kwegrafu yebha yoxinzelelo lwe-NT-ProBNP kwimidiya yenkcubeko evela kwizilayi zentliziyo ezikhuliswe phantsi kweemeko ze-MT normal stretch (Norm) okanye ezolula kakhulu (OS) (n ​​= 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kunye ne-D4 MTOS) izilayi/iqela ezivela kwiingulube ezahlukeneyo, kwenziwa i-ANOVA eneendlela ezimbini; **p < 0.01 xa kuthelekiswa nokolula okuqhelekileyo).I-histogram yobungakanani boxinzelelo lwe-NT-ProBNP kwi-culture medium evela kwii-heart slices ezikhuliswe phantsi kweemeko ze-MT stretch eqhelekileyo (norm) okanye i-overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, kunye ne-D4). I-MTOS) izilayi /iqela ezivela kwiingulube ezahlukeneyo, kwenziwa uhlalutyo lwezinto ezimbini zokwahluka;**p < 0,01 по сравнению с нормальным растяжением). **p < 0.01 xa kuthelekiswa nolusu oluqhelekileyo). a 在MT 正常拉伸(Eqhelekileyo) 或过度拉伸(OS) 条件下培养的心脏切片培养基中NT-ProBNP 浓度的条彈 = D 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 yoxinaniso kwizilayi zentliziyo ezikhuliswe phantsi kwe-MT eqhelekileyo yokwelula (Eqhelekileyo) okanye i-OS) yeemeko (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm neD4 MTOS) ukusuka ezahlukeneyo 猪的切片/组，可以双向双向kunye nokwelula okuqhelekileyo, p <0.01).ihistogram Ukulinganiswa koxinzelelo lwe-NT-ProBNP kwizilayi zentliziyo ezikhuliswe phantsi kweemeko zokolula okuqhelekileyo kwe-MT (isiqhelo) okanye ukolula kakhulu (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) kunye ne-D4 MTOS) izilayi/iqela ezivela kwiingulube ezahlukeneyo, uhlalutyo oluvela kwiindlela ezimbini zokwahluka;**p < 0,01 по сравнению с нормальным растяжением). **p < 0.01 xa kuthelekiswa nolusu oluqhelekileyo). b Imifanekiso emele izilayi zentliziyo ezidaywe nge-troponin-T kunye ne-WGA (ekhohlo) kunye nokulinganiswa kobukhulu beseli (ekunene) (n = 330 (D6 MTOS), 369 (D6 MTNorm) iiseli/iqela elivela kwizilayi ezili-10 ezahlukeneyo ezivela kwiihagu ezahlukeneyo, Uvavanyo lwe-t-Student olunemisila emibini luyenziwa; ****p < 0.0001 xa kuthelekiswa nolwandiso oluqhelekileyo). b Imifanekiso emele izilayi zentliziyo ezidaywe nge-troponin-T kunye ne-WGA (ekhohlo) kunye nokulinganiswa kobukhulu beseli (ekunene) (n = 330 (D6 MTOS), 369 (D6 MTNorm) iiseli/iqela elivela kwizilayi ezili-10 ezahlukeneyo ezivela kwiihagu ezahlukeneyo, Uvavanyo lwe-t lomfundi olunemisila emibini luyenziwa; ****p < 0.0001 xa kuthelekiswa nolwandiso oluqhelekileyo). b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного определения размера клеток (с) 369 (D6 MTNorm) клеток/группу из 10 разных срезов от разных свиней, два- проводится хвостовой t-критерий Стьюной Стьюном ****pыдента ****pымномнта

b Imifanekiso emele amacandelo entliziyo anombala we-troponin-T kunye ne-AZP (ekhohlo) kunye nokulinganiswa kobukhulu beseli (ekunene) (n = 330 (D6 MTOS), 369 (D6 MTNorm) iiseli/iqela elivela kumacandelo ali-10 ahlukeneyo kwiihagu ezahlukeneyo, uvavanyo lwe-t lomfundi olunemisila emibini lwenziwe; ****p < 0.0001 xa kuthelekiswa noxinzelelo oluqhelekileyo). 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（D330（D330). MTOS. 0.0001. b Imifanekiso emele izilayi zentliziyo ezidaywe nge-calcarein-T kunye ne-WGA (ekhohlo) kunye nobukhulu beseli (ekunene) (n = 330 (D6 MTOS), 369 ukusuka kwizilayi ezili-10 ezahlukeneyo (D6 MTNorm)) Iiseli/组，两方法有尾学生t test； xa kuthelekiswa nokwelula okuqhelekileyo，****p < 0.0001). b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оценка размера клеток (0 = MT63) (D6 MTNorm) из 10 различных срезов от разных свиней Клетки/группа, двусторонние критерий Стьюдента ****p < 0,0001 по сравнильмы . b Imifanekiso emele amacandelo entliziyo anombala we-troponin-T kunye ne-AZP (ekhohlo) kunye nokulinganiswa kobukhulu beseli (ekunene) (n = 330 (D6 MTOS), 369 (D6 MTNorm) ukusuka kumacandelo ali-10 ahlukeneyo kwiihagu ezahlukeneyo) Iiseli/iqela, ikhrayitheriya yomsila emibini t yomfundi; ****p < 0.0001 xa kuthelekiswa noxinzelelo oluqhelekileyo). c Imifanekiso emele usuku lwe-0 kunye nosuku lwe-6 lwe-MTOS heart slices ezifakwe i-immunolabeled ye-troponin-T kunye ne-NFATC4 kunye nokulinganiswa kokudluliselwa kwe-NFATC4 kwi-nuclei ye-CMs (n = 4 (D0), 3 (D6 MTOS) slices/group ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-t-olunemisila emibini lwenziwa; *p < 0.05). c Imifanekiso emele usuku lwe-0 kunye nosuku lwe-6 lwe-MTOS heart slices ezifakwe i-immunolabeled ye-troponin-T kunye ne-NFATC4 kunye nokulinganiswa kokudluliselwa kwe-NFATC4 kwi-nuclei ye-CMs (n = 4 (D0), 3 (D6 MTOS) slices/group ezivela kwiihagu ezahlukeneyo, uvavanyo lwe-t-Student olunemisila emibini lwenziwa; *p < 0.05). c Репрезентативные изображения для срезов сердца 0 и 6 дней MTOS , иммуномеченых для тропонина-Т kunye NFATC4 кавернозных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней , выполняется двусторонний t-критерий Стьюдента, *5. c Imifanekiso emele amacandelo entliziyo kwiintsuku ezi-0 nezi-6 ze-MTOS, efakwe i-immunolabeled ye-troponin-T kunye ne-NFATC4, kunye nokulinganiswa kokudluliselwa kwe-NFATC4 kwi-nucleus yeeseli ze-cavernous (n = 4 (D0), izilayi ezi-3 (D6 MTOS)/iqela ezivela kwiihagu ezahlukeneyo) zenze uvavanyo lwe-t-olunemisila emibini yoMfundi; *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，以及來自肉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 Ummeli wemifanekiso ye-calcanin-T kunye ne-NFATC4 immunolabeling 第0天和第6天MTOS izilayi zentliziyo, kunye ne-NFATC4 evela kwi-NFATC4 eyahlukileyo 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 на 0 и 6 день для иммуномаркировки тропонином-Т kunye NFATC4 kunye количественная оценценка от разных свиней (n = 4 (D0), 3 (D6 MTOS) срез/группа, два- хвостатый t-критерий Стьюдента; *p <0,05). c Imifanekiso emele izilayi zentliziyo ze-MTOS ngomhla we-0 nowe-6 ze-troponin-T kunye ne-NFATC4 immunolabeling kunye nokulinganisa ukudluliselwa kwe-NFATC4 kwi-nucleus ye-CM evela kwiihagu ezahlukeneyo (n = 4 (D0), 3 (D6 MTOS) izilayi/iqela, i-t-criterion enemisila emibini. Abafundi; *p < 0.05).Iibha zempazamo zimele i-mean ± ukuphambuka okuqhelekileyo.
Uphando oluguqulelweyo lwentliziyo lufuna iimodeli zeseli ezivelisa ngokuchanekileyo imeko-bume yentliziyo. Kolu phononongo, kwaveliswa isixhobo se-CTCM saza sachazwa esinokuvuselela iindawo ezincinci kakhulu zentliziyo. Inkqubo ye-CTCM ibandakanya ukuvuselelwa kwe-electromechanical okuvumelaniswe physiologically kunye nokutyetyiswa kolwelo lwe-T3 kunye ne-Dex. Xa iindawo zentliziyo yeengulube zazichatshazelwa zezi zinto, ukusebenza kwazo, ukuqina kwesakhiwo, umsebenzi we-metabolic, kunye nokubonakaliswa kwe-transcriptional kwahlala kufana nakwizicubu zentliziyo ezintsha emva kweentsuku ezili-12 zokukhula. Ukongeza, ukolula kakhulu kwezicubu zentliziyo kunokubangela i-hypertrophy yentliziyo ebangelwa yi-hyperextension. Ngokubanzi, ezi ziphumo zixhasa indima ebalulekileyo yeemeko zenkcubeko ye-physiological ekugcineni i-phenotype eqhelekileyo yentliziyo kwaye zibonelela ngeqonga lokuhlolwa kweziyobisi.
Izinto ezininzi zinegalelo ekudaleni imeko-bume efanelekileyo yokusebenza nokusinda kwe-cardiomyocytes. Ezona zinto zicacileyo kwezi zinxulumene (1) nokusebenzisana phakathi kweeseli, (2) ukuvuselela nge-electromechanical, (3) izinto ezi-humoral, kunye (4) ne-metabolic substrates. Ukusebenzisana kwe-physiological cell-to-cell kufuna iinethiwekhi ezintsonkothileyo zeentlobo ezininzi zeeseli ezixhaswa yi-extracellular matrix. Ukunxibelelana okunjalo okuntsonkothileyo kweeseli kunzima ukukwakha kwakhona kwi-vitro ngokukhuliswa kweentlobo zeeseli nganye, kodwa kunokufezekiswa ngokulula ngokusebenzisa uhlobo lwe-organotypic lwamacandelo entliziyo.
Ukolulwa kwemithambo kunye nokuvuselelwa kombane kwee-cardiomyocytes kubalulekile ekugcineni i-phenotype yentliziyo33,34,35. Nangona ukuvuselelwa kwemithambo kuye kwasetyenziswa kakhulu kwi-hiPSC-CM conditioning kunye nokuvuthwa, izifundo ezininzi ezintle kutshanje ziye zazama ukuvuselelwa kwemithambo yeentliziyo kwinkcubeko kusetyenziswa ukulayisha kwe-uniaxial. Ezi zifundo zibonisa ukuba ukulayisha kwe-uniaxial mechanical kwe-2D kunefuthe elihle kwi-phenotype yentliziyo ngexesha lenkcubeko. Kwezi zifundo, amacandelo entliziyo ayene-isometric tensile forces17, ukulayisha kwe-auxotonic okuthe ngqo18, okanye umjikelo wentliziyo waphinda wadalwa kusetyenziswa impendulo ye-force transducer kunye ne-tension drives. Nangona kunjalo, ezi ndlela zisebenzisa ukolulwa kwezicubu ze-uniaxial ngaphandle kokulungiswa kwendalo, okubangela ukucinezelwa kwezakhi zofuzo ezininzi zentliziyo okanye ukubonakaliswa kakhulu kwezakhi zofuzo ezinxulumene neempendulo ze-stretch ezingaqhelekanga. I-CTCM echazwe apha inika isikhuthazo se-3D electromechanical esilinganisa umjikelo wentliziyo wendalo ngokwexesha lomjikelo kunye nokolulwa kwe-physiological (ukolulwa kwe-25%, i-40% systole, i-60% diastole, kunye ne-72 beats ngomzuzu). Nangona olu vuselelo loomatshini olunemilinganiselo emithathu lulodwa lunganelanga ukugcina ukuthembeka kwezicubu, indibaniselwano yovuselelo oluhlazo nolusebenzisa i-T3/Dex iyadingeka ukugcina ukuqina kwezicubu, ukusebenza, kunye nokuthembeka kwazo ngokwaneleyo.
Izinto ezihlekisayo zidlala indima ebalulekileyo ekuguquleni i-phenotype yentliziyo yomntu omdala. Oku kugqanyisiwe kwizifundo ze-HiPS-CM apho i-T3 kunye ne-Dex zongezwa kwi-culture media ukuze kukhawuleziswe ukuvuthwa kweeseli. I-T3 inokuchaphazela ukuthuthwa kwee-amino acids, iiswekile kunye ne-calcium kwii-membranes zeseli36. Ukongeza, i-T3 ikhuthaza ukubonakaliswa kwe-MHC-α kunye nokwehla kwe-MHC-β, ikhuthaza ukwakheka kwe-fast twitch myofibrils kwi-cardiomyocytes evuthiweyo xa kuthelekiswa ne-slow twitch myofibrils kwi-CM ye-fetal. Ukungabikho kwe-T3 kwizigulane ezine-hypothyroid kubangela ukulahleka kwee-myofibrillar bands kunye nesantya esinciphileyo sophuhliso lwethoni37. I-Dex isebenza kwi-glucocorticoid receptors kwaye ibonakalisiwe ukuba yonyusa i-myocardial contractility kwiintliziyo ezixutyiweyo;38 olu phuculo lucingelwa ukuba lunxulumene nesiphumo sokungena kwe-calcium deposit-driven (SOCE) 39,40. Ukongeza, i-Dex ibopha kwi-receptors zayo, ibangela impendulo ebanzi yangaphakathi kweseli ecinezela ukusebenza komzimba kunye nokuvuvukala30.
Iziphumo zethu zibonisa ukuba ukukhuthazwa koomatshini bomzimba (i-MS) kuphucule ukusebenza kwenkcubeko iyonke xa kuthelekiswa ne-Ctrl, kodwa kungaphumeleli ukugcina amandla okuphila, ukuthembeka kwesakhiwo, kunye nokubonakaliswa kwentliziyo kwiintsuku ezili-12 kwinkcubeko. Xa kuthelekiswa ne-Ctrl, ukongezwa kweenkcubeko ze-T3 kunye ne-Dex kwi-CTCM (MT) kuphucule amandla okuphila kwaye kwagcina iiprofayili ezifanayo zokubhala, ukuthembeka kwesakhiwo, kunye nomsebenzi we-metabolic ngezicubu zentliziyo ezintsha kangangeentsuku ezili-12. Ukongeza, ngokulawula izinga lokunwebeka kwezicubu, imodeli ye-hypertrophy yentliziyo ebangelwa yi-hyperextension yenziwe kusetyenziswa i-STCM, ebonisa ukuguquguquka kwenkqubo ye-STCM. Kufuneka kuqatshelwe ukuba nangona ukulungiswa kwentliziyo kunye ne-fibrosis kudla ngokubandakanya amalungu angasebenziyo aneeseli ezijikelezayo ezinokubonelela ngee-cytokines ezifanelekileyo kunye ne-phagocytosis kunye nezinye izinto zokulungisa, amacandelo entliziyo asenokulinganisa inkqubo ye-fibrotic ekuphenduleni uxinzelelo kunye nokulimala. kwi-myofibroblasts. Oku kuye kwavavanywa ngaphambili kule modeli yesilayi sentliziyo. Kufuneka kuqatshelwe ukuba iiparameter ze-CTCM zinokuguqulwa ngokutshintsha uxinzelelo/i-amplitude yombane kunye ne-frequency ukulinganisa iimeko ezininzi ezifana ne-tachycardia, i-bradycardia, kunye nenkxaso ye-mechanical circulatory (intliziyo engenazingxobo). Oku kwenza le nkqubo ibe yindlela ephakathi yokuvavanya amayeza. Amandla e-CTCM okubonisa i-hypertrophy yentliziyo ebangelwa kukudinwa kakhulu avula indlela yokuvavanya le nkqubo ukuze kufumaneke unyango lomntu ngamnye. Ukuqukumbela, olu phononongo lubonisa ukuba ukolula koomatshini kunye nokukhuthaza ukudumba kubalulekile ekugcineni inkcubeko yamacandelo ezicubu zentliziyo.
Nangona idatha evezwe apha ibonisa ukuba i-CTCM liqonga elithembisayo kakhulu lokwenza imodeli ye-myocardium engaguqukiyo, le ndlela yokukhulisa inemida ethile. Umda ophambili wenkcubeko ye-CTCM kukuba ibeka uxinzelelo oluqhubekayo lwe-mechanical kwiislices, nto leyo ethintela amandla okujonga ngokukhutheleyo ukucotha kwe-cardiac slice ngexesha lomjikelo ngamnye. Ukongeza, ngenxa yobukhulu obuncinci beenxalenye zentliziyo (7 mm), amandla okuvavanya umsebenzi we-systolic ngaphandle kweenkqubo zokukhulisa kusetyenziswa izinzwa zamandla zendabuko anqongophele. Kwincwadi yangoku, siyawoyisa ngokuyinxenye lo mda ngokuvavanya i-optical voltage njengesalathiso somsebenzi we-contractile. Nangona kunjalo, lo mda uya kufuna umsebenzi ongaphezulu kwaye unokujongwa kwixesha elizayo ngokwazisa iindlela zokujonga ukusebenza kweeslices zentliziyo kwinkcubeko, ezifana ne-optical mapping kusetyenziswa i-calcium kunye ne-voltage-sensitive dyes. Omnye umda we-CTCM kukuba imodeli yokusebenza ayilawuli uxinzelelo lwe-physiological (preload kunye ne-afterload). Kwi-CTCM, uxinzelelo lwenziwa kwicala elichaseneyo ukuze luvelise i-25% physiological stretch kwi-diastole (full stretch) kunye ne-systole (ubude bokucotha ngexesha lokuvuselelwa kombane) kwizicubu ezinkulu kakhulu. Lo mda kufuneka ususwe kwiindlela ze-CTCM ezizayo ngokucinezela ngokwaneleyo izicwili zentliziyo kumacala omabini nangokusebenzisa ubudlelwane obuchanekileyo boxinzelelo nomthamo owenzeka kumagumbi entliziyo.
Ukulungiswa okubangelwa kukwanda kakhulu okuxelwe kulo mbhalo-ngqangi kunqunyelwe ekulinganiseni imiqondiso ye-hyperstretch egqithisileyo. Ngoko ke, le modeli inokunceda ekufundweni kwe-stretch-induced hypertrophic signaling ngaphandle kwesidingo se-humoral okanye i-neural factors (engekhoyo kule nkqubo). Izifundo ezongezelelweyo ziyafuneka ukwandisa ubuninzi be-CTCM, umzekelo, ukukhulisa kunye neeseli zomzimba, ukujikeleza kwe-plasma humoral factors, kunye nokugcinwa xa kukhula kunye neeseli ze-neuronal kuya kuphucula amathuba okubonisa isifo nge-CTCM.
Kusetyenziswe iihagu ezilishumi elinesithathu kolu phononongo. Zonke iinkqubo zezilwanyana zenziwe ngokwemigaqo yesikhungo kwaye zavunywa yiKomiti yoLondolozo lweZilwanyana yeYunivesithi yaseLouisville. I-aortic arch yaxinwa kwaye intliziyo yaxutywa 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 ukuya kuthi ga kwi-7.4); Iintliziyo zigcinwe kwisisombululo se-cardioplegic esibandayo de zathunyelwa kwilebhu enomkhenkceni odla ngokuba ngaphantsi kwemizuzu eli-10. Iintliziyo zigcinwe kwisisombululo se-cardioplegic esibandayo de zathunyelwa kwilebhu enomkhenkceni odla ngokuba ngaphantsi kwemizuzu eli-10. сердца хранили в ледяном кардиоплегическом растворе до транспортировки в лабораторию на льду, что обычно занимает <10 мин. Iintliziyo zazigcinwa kwisisombululo sentliziyo esibandayo de zithuthwe ziye kwilebhu enomkhenkce, nto leyo edla ngokuthatha imizuzu engaphantsi kwe-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. Держите сердца в ледяной кардиоплегии до транспортировки в лабораторию на льду, обычно <10 мин. Gcina iintliziyo zikwi-ayisikhilimu ye-cardioplegia de zithuthwe ziye kwilebhu kwi-ayisikhilimu, ngokuqhelekileyo zingaphantsi kwemizuzu eli-10.
Isixhobo se-CTCM senziwe kwisoftware yoyilo oluncediswa yikhompyutha yeSolidWorks (CAD). Amagumbi okukhulisa, izahluli kunye namagumbi omoya enziwe ngeplastiki ye-acrylic ecacileyo ye-CNC. Isangqa sokuxhasa esinobubanzi obuyi-7mm senziwe nge-polyethylene enoxinano oluphezulu (HDPE) embindini kwaye sinomngxuma we-o-ring ukuze singene kwi-silicone o-ring esetyenziselwa ukutywina i-media engaphantsi. I-membrane encinci ye-silica yahlula igumbi lokukhulisa kwipleyiti yokwahlula. I-membrane ye-silicone inqunyulwe nge-laser kwi-silicone sheet eyi-0.02″ ubukhulu kwaye inobunzima obuyi-35A. Ii-gasket ze-silicone ezisezantsi neziphezulu zinqunyulwe nge-laser kwi-silicone sheet eyi-1/16″ ubukhulu kwaye zinobunzima obuyi-50A. Izikrufu zentsimbi engagqwaliyo ezingama-316L kunye neenati zamaphiko zisetyenziselwa ukubopha ibhloko kunye nokwenza isitywina esingangeni moya.
Ibhodi yesekethe eprintiweyo (i-PCB) yenzelwe ukudityaniswa nenkqubo ye-C-PACE-EM. Izikhonkwane zomatshini waseSwitzerland kwi-PCB ziqhagamshelwe kwii-electrode ze-graphite ngeentambo zobhedu ezifakwe isilivere kunye nezikrufu zebronze 0-60 ezifakwe kwii-electrodes. Ibhodi yesekethe eprintiweyo ibekwe kwisigqubuthelo seprinta ye-3D.
Isixhobo se-CTCM silawulwa yi-programmable pneumatic actuator (PPD) edala uxinzelelo olulawulwayo lokujikeleza kwegazi olufana nomjikelo wentliziyo. Njengoko uxinzelelo olungaphakathi kwigumbi lomoya lusanda, i-membrane ye-silicone eguquguqukayo iyakhula iye phezulu, inyanzela i-medium phantsi kwendawo yezicubu. Indawo yezicubu iya kolulwa ngolu hlobo lokukhupha ulwelo, oluxelisa ukwanda kwentliziyo ngexesha le-diastole. Kwincopho yokuphumla, ukuvuselelwa kombane kusetyenziswa ii-electrodes ze-graphite, ezinciphise uxinzelelo kwigumbi lomoya kwaye zabangela ukuxinana kwamacandelo ezicubu. Ngaphakathi kombhobho kukho ivalvu ye-hemostatic ene-sensor yoxinzelelo yokufumanisa uxinzelelo kwinkqubo yomoya. Uxinzelelo oluviwa yi-sensor yoxinzelelo lusetyenziswa kumqokeleli wedatha oqhagamshelwe kwi-laptop. Oku kuvumela ukujonga okuqhubekayo uxinzelelo olungaphakathi kwigumbi legesi. Xa uxinzelelo oluphezulu lwegumbi lufikelelwe (umgangatho we-80 mmHg, i-140 mmHg OS), isixhobo sokufumana idatha sayalelwa ukuba sithumele isignali kwinkqubo ye-C-PACE-EM ukuvelisa isignali ye-voltage ye-biphasic ye-2 ms, emiselwe kwi-4 V.
Kufunyenwe amacandelo entliziyo kwaye iimeko zokukhula kwimithombo emi-6 zenziwe ngolu hlobo lulandelayo: Ukuhambisa iintliziyo ezivuniweyo ukusuka kwisitya sokudlulisa ukuya kwitreyi equlethe i-cardioplegia ebandayo (4° C.). I-ventricle yasekhohlo yahlulwa nge-blade engenazintsholongwane yaza yanqunyulwa yaba ziingceba ze-1-2 cm3. Ezi bhloko zezicubu zancamathiselwa kwizixhasi zezicubu nge-tissue adhesive zaza zafakwa kwi-vibrating microtome tissue bath equlethe isisombululo sikaTyrode kwaye i-oxygenated rhoqo (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 isisombululo), 1.8 mM CaCl2 (1.8 ml 1 M isisombululo), ukuya kuthi ga kwi-1 L ddH2O). I-microtome engcangcazelayo yayimiselwe ukusika izilayi ezingama-300 µm ubukhulu kwi-frequency ye-80 Hz, i-amplitude yokungcangcazela ethe tye ye-2 mm, kunye nesantya esiphezulu se-0.03 mm/s. I-tissue bath yayijikelezwe ngumkhenkce ukugcina isisombululo sipholile kwaye ubushushu bugcinwe kwi-4°C. Dlulisa amacandelo ezicubu ukusuka kwi-microtome bath ukuya kwi-incubation bath equlethe isisombululo seTyrode esineoksijini rhoqo kwiqhwa de kufumaneke amacandelo aneleyo kwipleyiti enye yokukhulisa. Kwi-transwell cultures, amacandelo ezicubu aqhotyoshelwe kwi-polyurethane supports ecocekileyo eyi-6 mm ububanzi kwaye afakwa kwi-6 ml ye-optimized medium (199 medium, 1x ITS supplement, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-alkaline kunye ne-2X antibiotic-antifungal). Ukuvuselelwa kombane (10 V, frequency 1.2 Hz) kufakwe kumacandelo ezicubu nge-C-Pace. Kwiimeko ze-TD, i-T3 entsha kunye ne-Dex zongezwa kwi-100 nM kunye ne-1 μM kutshintsho ngalunye oluphakathi. Olu hlobo lugcwele ioksijini ngaphambi kokutshintshwa kathathu ngemini. Amacandelo ezicubu atyalwe kwi-incubator kumaqondo obushushu angama-37°C kunye ne-5% CO2.
Kwi-CTCM cultures, amacandelo ezicubu abekwe kwiprinta ye-3D eyenziwe ngokwezifiso kwisitya sePetri esinesisombululo seTyrode esilungisiweyo. Esi sixhobo senzelwe ukwandisa ubungakanani besilayi sentliziyo nge-25% yendawo yeringi yokuxhasa. Oku kwenziwa ukuze amacandelo entliziyo angasoluli emva kokudluliselwa kwisisombululo seTyrode ukuya kwindawo ephakathi nangexesha le-diastole. Usebenzisa iglu ye-histoacrylic, amacandelo angama-300 µm ubukhulu aqiniswe kwiringi yokuxhasa enobubanzi obuyi-7 mm. Emva kokuncamathisela amacandelo ezicubu kwiringi yokuxhasa, nqumla amacandelo ezicubu angaphezulu kwaye ubeke amacandelo ezicubu aqhotyoshelweyo umva kwibhafu yesisombululo seTyrode kwiqhwa (4°C) de kube kulungiselelwe amacandelo aneleyo kwisixhobo esinye. Ixesha elipheleleyo lokucubungula zonke izixhobo akufuneki lidlule iiyure ezi-2. Emva kokuba amacandelo ezicubu ama-6 enamathele kwiringi yazo yokuxhasa, isixhobo seCTCM sahlanganiswa. Igumbi lokukhulisa i-CTCM lizaliswa kwangaphambili nge-21 ml ye-pre-oxygenated medium. Dlulisa amacandelo ezicubu kwigumbi lokukhulisa kwaye ususe ngononophelo naziphi na iibhamuza zomoya nge-pipette. Icandelo lezicubu emva koko likhokelwa lingene emngxunyeni lize licinezelwe ngobunono endaweni yalo. Okokugqibela, beka isiciko se-electrode kwisixhobo uze udlulisele isixhobo kwi-incubator. Emva koko qhagamshela i-CTCM kwityhubhu yomoya kunye nenkqubo ye-C-PACE-EM. I-actuator ye-pneumatic iyavuleka kwaye i-valve yomoya ivula i-CTCM. Inkqubo ye-C-PACE-EM yenzelwe ukuhambisa i-4 V kwi-1.2 Hz ngexesha le-biphasic pacing kangange-2 ms. I-medium yatshintshwa kabini ngemini kwaye ii-electrode zatshintshwa kanye ngemini ukuze kuthintelwe ukuqokelelana kwe-graphite kwii-electrodes. Ukuba kuyimfuneko, amacandelo ezicubu anokususwa kwimithombo yawo yokukhulisa ukuze kukhutshwe naziphi na iibhamuza zomoya ezinokuba ziwele phantsi kwazo. Kwiimeko zonyango lwe-MT, i-T3/Dex yongezwa ngokutsha ngotshintsho ngalunye oluphakathi nge-100 nM T3 kunye ne-1 μM Dex. Izixhobo ze-CTCM zakhuliswa kwi-incubator kuma-37°C kunye ne-5% CO2.
Ukuze kufunyanwe iindlela ezoluliweyo zezilayi zentliziyo, kwaveliswa inkqubo yekhamera ekhethekileyo. Ikhamera ye-SLR (Canon Rebel T7i, Canon, Tokyo, Japan) yasetyenziswa nelensi ye-Navitar Zoom 7000 18-108mm macro (Navitar, San Francisco, CA). Ukubona ngeliso lengqondo kwenziwe kubushushu begumbi emva kokutshintsha isilayi ngesixhobo esitsha. Ikhamera ibekwe kwi-engile ye-51° kwaye ividiyo irekhodwa kwiifreyimu ezingama-30 ngomzuzwana. Okokuqala, isoftware yomthombo ovulekileyo (MUSCLEMOTION43) yasetyenziswa ne-Image-J ukulinganisa intshukumo yezilayi zentliziyo. Imaski yenziwe kusetyenziswa i-MATLAB (MathWorks, Natick, MA, USA) ukuchaza iindawo ezinomdla zokubetha izilayi zentliziyo ukuze kuthintelwe ingxolo. Iimaski ezihlukaniswe ngesandla zifakwa kuyo yonke imifanekiso kulandelelwano lwesakhelo kwaye emva koko zidluliselwe kwi-plug-in ye-MUSCLEMOTION. I-Muscle Motion isebenzisa amandla aphakathi ee-pixels kwisakhelo ngasinye ukulinganisa intshukumo yayo ngokumalunga nesakhelo sesalathiso. Idatha yarekhodwa, yahluzwa yaza yasetyenziswa ukulinganisa ixesha lomjikelo kunye nokuvavanya ukolula kwezicubu ngexesha lomjikelo wentliziyo. Ividiyo erekhodiweyo yacutshungulwa emva kokusetyenziswa kwesihluzo sedijithali se-zero-phase yokuqala. Ukulinganisa ukolula kwezicubu (i-peak-to-peak), uhlalutyo lwe-peak-to-peak lwenziwa ukwahlula phakathi kweencochoyi kunye nee-troughs kwisignali erekhodiweyo. Ukongeza, ukuchithwa kwe-trending kwenziwa kusetyenziswa i-polynomial ye-6th order ukuphelisa ukushukuma kwesignali. Ikhowudi yenkqubo yaphuhliswa kwi-MATLAB ukumisela intshukumo yezicubu zehlabathi, ixesha lomjikelo, ixesha lokuphumla, kunye nexesha lokuqunjelwa (Ikhowudi yeNkqubo eyongezelelweyo 44).
Uhlalutyo loxinzelelo, sisebenzisa iividiyo ezifanayo ezenzelwe uvavanyo lokwelula koomatshini, siqale salandela imifanekiso emibini emele iincopho zentshukumo (amanqaku aphezulu (aphezulu) nasezantsi (asezantsi) okunyakaza) ngokwesoftware ye-MUSCLEMOTION. Emva koko sahlulahlula iindawo zezicubu saza sasebenzisa uhlobo lwe-algorithm yomthunzi kwizicubu ezihluliweyo (Umzobo oNcedisayo 2a). Izicubu ezihluliweyo zahlulwahlulwa zaba ziindawo ezilishumi ezingaphantsi komhlaba, kwaye uxinzelelo kumphezulu ngamnye lwabalwa kusetyenziswa i-equation elandelayo: Uxinzelelo = (Sup-Sdown)/Sdown, apho i-Sup kunye ne-Sdown ziyimigama yesimo ukusuka kwizithunzi eziphezulu nezisezantsi zelaphu, ngokulandelelana (Umzobo oNcedisayo .2b).
Inxalenye yentliziyo iqiniswe kwi-4% ye-paraformaldehyde kangangeeyure ezingama-48. Izicubu ezizinzileyo zancitshiswa kwi-10% kunye ne-20% ye-sucrose iyure e-1, emva koko kwi-30% ye-sucrose ubusuku bonke. Inxalenye emva koko zafakwa kwi-optimum cutting temperature compound (i-OCT compound) zaza zaqandiswa kancinci kancinci kwi-isopentane/dry ice bath. Gcina iibhloko ze-OCT embedding kwi--80 °C de zahlulwe. Iislayidi zalungiswa njengeenxalenye ezinobukhulu obuyi-8 μm.
Ukuze ususe i-OCT kwiindawo zentliziyo, fudumeza iislayidi kwibhloko yokufudumeza kwi-95 °C imizuzu emi-5. Yongeza i-1 ml ye-PBS kwisilayidi ngasinye uze uyifukame imizuzu engama-30 kubushushu begumbi, uze uzalise iindawo ngokuseta i-0.1% yeTriton-X kwi-PBS imizuzu eli-15 kubushushu begumbi. Ukuthintela ii-antibodies ezingezizo ezithile ukuba zingabotshelelwa kwisampuli, yongeza i-1 ml yesisombululo se-3% se-BSA kwiislayidi uze uyifukame iyure e-1 kubushushu begumbi. Emva koko i-BSA yasuswa kwaye iislayidi zahlanjwa nge-PBS. Phawula isampuli nganye ngepensile. Ii-antibodies eziphambili (ezixutywe yi-1:200 kwi-1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) kunye ne-troponin-T (Thermo Scientific; #MA5-12960) zongezwa kwimizuzu engama-90, emva koko ii-antibodies zesibini (ezixutywe yi-1:200 kwi-1% BSA) ngokuchasene negundane i-Alexa Fluor 488 (Thermo Scientific; #A16079), ngokuchasene nomvundla i-Alexa Fluor 594 (Thermo Scientific; #T6391) kangangemizuzu engama-90 eyongezelelweyo. Zihlanjwe amaxesha ama-3 nge-PBS Ukwahlula idayi ekujoliswe kuyo ukusuka kwimvelaphi, sisebenzise kuphela i-antibody yesibini njengolawulo. Ekugqibeleni, kongezwa idayi yenyukliya ye-DAPI kwaye iislayidi zafakwa kwi-vectashield (Vector Laboratories) zaza zavalwa nge-nail polish. -x magnification) kunye ne-Keyence microscope enokwandiswa kwe-40x.
I-WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) kwi-5 μg/ml kwi-PBS yasetyenziselwa ukufaka idayi kwi-WGA yaza yasetyenziswa kwiindawo ezizinzileyo kangangemizuzu engama-30 kubushushu begumbi. Emva koko iislayidi zahlanjwa nge-PBS kwaye i-Sudan black yongezwa kwislayidi nganye yaza yafakwa kwi-incubator imizuzu engama-30. Emva koko iislayidi zahlanjwa nge-PBS kwaye kongezwa i-vectashield embedding medium. Iislayidi zabonwa kwi-Keyence microscope kwi-40x magnification.
I-OCT isusiwe kwiisampuli njengoko kuchaziwe apha ngasentla. Emva kokususa i-OCT, faka iislayidi kwisisombululo sikaBouin ubusuku bonke. Emva koko iislayidi zahlanjwa ngamanzi acocekileyo kangangeyure e-1 zaze zafakwa kwisisombululo seBibrich aloe acid fuchsin imizuzu eli-10. Emva koko iislayidi zahlanjwa ngamanzi acocekileyo zaza zafakwa kwisisombululo se-5% phosphomolybdenum/5% phosphotungstic acid imizuzu eli-10. Ngaphandle kokuhlanjwa, dlulisela iislayidi ngqo kwisisombululo esiluhlaza okwesibhakabhaka se-aniline imizuzu eli-15. Emva koko iislayidi zahlanjwa ngamanzi acocekileyo zaza zafakwa kwisisombululo se-1% acetic acid imizuzu emi-2. Iislayidi zomiswa kwi-200 N ethanol zaze zadluliselwa kwi-xylene. Iislayidi ezinemibala zabonwa kusetyenziswa i-Keyence microscope ene-10x objective. Ipesenti yendawo ye-Fibrosis yalinganiswa kusetyenziswa isoftware ye-Keyence Analyzer.
Uvavanyo lweCyQUANT™ MTT Cell Viability Assay (Invitrogen, Carlsbad, CA), inombolo yekhathalogu V13154, ngokwemigaqo yomenzi kunye notshintsho oluthile. Ngokukodwa, ukubethelwa ngotyando okunobubanzi obuyi-6 mm kwasetyenziswa ukuqinisekisa ubungakanani obufanayo bezicubu ngexesha lohlalutyo lwe-MTT. Izicubu zafakwa ngokwahlukeneyo kwimithombo yepleyiti ene-12-well equlethe i-MTT substrate ngokwemigaqo yomenzi. Amacandelo afakwa kwi-37° C. iiyure ezi-3 kwaye izicubu eziphilayo ziguqula i-MTT substrate ukuze zenze i-purple formazan compound. Buyisela isisombululo se-MTT nge-1 ml ye-DMSO kwaye ufakwe kwi-37°C imizuzu eli-15 ukuze kukhutshwe i-purple formazan kwiindawo zentliziyo. Iisampulu zancitshiswa nge-1:10 kwi-DMSO kwiipleyiti ezisezantsi ezicacileyo ezingama-96-well kunye nobunzima bombala omfusa obulinganiswa kwi-570 nm kusetyenziswa i-Cytation plate reader (BioTek). Ukufundwa kwalungiswa ngokobunzima besilayi ngasinye sentliziyo.
I-heart slice media ithathelwe indawo yi-media equlethe i-1 μCi/ml [5-3H]-glucose (Moravek Biochemicals, Brea, CA, USA) yokuvavanya ukusetyenziswa kweglucose njengoko kuchaziwe ngaphambili. Emva kweeyure ezi-4 zokufunxa, yongeza i-100 µl ye-medium kwityhubhu ye-microcentrifuge evulekileyo equlethe i-100 µl ye-0.2 N HCl. Emva koko ityhubhu ibekwe kwityhubhu ye-scintillation equlethe i-500 μl ye-dH2O ukuze iphele [3H]2O kangangeeyure ezingama-72 kwi-37°C. Emva koko susa ityhubhu ye-microcentrifuge kwityhubhu ye-scintillation kwaye wongeze i-10 ml ye-scintillation fluid. Ubalo lwe-scintillation lwenziwe kusetyenziswa i-Tri-Carb 2900TR liquid scintillation analyzer (Packard Bioscience Company, Meriden, CT, USA). Ukusetyenziswa kweglucose emva koko kwabalwa kuthathelwa ingqalelo umsebenzi othile weglucose [5-3H], ukulingana okungaphelelanga kunye nemvelaphi, ukuxutywa kweglucose [5-3H] ukuya kwiglucose engabhalwanga, kunye nokusebenza kakuhle kwe-scintillation. Idatha ilungelelaniswa nobunzima bamacandelo entliziyo.
Emva kokuba izicubu zikwazi ukuhlangana kwi-Trizol, i-RNA yahlulwa kwiindawo zentliziyo kusetyenziswa i-Qiagen miRNeasy Micro Kit #210874 ngokwemigaqo yomenzi. Ukulungiswa kwethala leencwadi le-RNAsec, ukulandelelana kunye nohlalutyo lwedatha kwenziwe ngolu hlobo lulandelayo:
I-1 μg ye-RNA kwisampulu nganye isetyenzisiwe njengezinto zokuqala ukulungiselela ilayibrari ye-RNA. Iilayibrari zokulandelelanisa zenziwe kusetyenziswa i-NEBNext UltraTM RNA Library Preparation Kit ye-Illumina (NEB, e-USA) emva kweengcebiso zomenzi, kwaye iikhowudi ze-index zongezwa kwi-attribute sequences kwisampulu nganye. Ngamafutshane, i-mRNA yacocwa kwi-RNA iyonke kusetyenziswa ii-magnetic beads eziqhotyoshelwe kwi-poly-T oligonucleotides. Ukuqhekeka kwenziwa kusetyenziswa ii-divalent cations kubushushu obuphezulu kwi-NEBNext First Strand Synthesis Reaction Buffer (5X). I-cDNA yokuqala ye-strand yenziwe kusetyenziswa ii-random hexamer primers kunye ne-M-MuLV reverse transcriptase (RNase H-). I-cDNA yesibini ye-strand emva koko yenziwe kusetyenziswa i-DNA polymerase I kunye ne-RNase H. Ii-overhangs eziseleyo ziguqulwa zibe ziziphelo ezi-blunt ngumsebenzi we-exonuclease/polymerase. Emva kwe-adenylation yesiphelo se-3′ se-DNA fragment, i-NEBNext Adapter enesakhiwo se-hairpin loop iqhotyoshelwe kuyo ukuze ilungiselele i-hybridization. Ukukhetha iziqwenga ze-cDNA ezinobude obukhethwayo, iziqwenga zelayibrari eziyi-150-200 bp, zacocwa kusetyenziswa inkqubo ye-AMPure XP (Beckman Coulter, eBeverly, e-USA). Emva koko, i-3 μl USER Enzyme (NEB, e-USA) ene-cDNA ekhethiweyo ngobukhulu efakwe i-adaptha yasetyenziswa imizuzu eli-15 kwi-37°C yaze emva koko yasetyenziswa imizuzu emi-5 kwi-95°C ngaphambi kwe-PCR. I-PCR emva koko yenziwa kusetyenziswa i-Phusion High-Fidelity DNA polymerase, ii-universal PCR primers, kunye nee-Index (X) primers. Ekugqibeleni, iimveliso ze-PCR zacocwa (inkqubo ye-AMPure XP) kwaye umgangatho welayibrari wavavanywa kwinkqubo ye-Agilent Bioanalyzer 2100. Ilayibrari ye-cDNA yacwangciswa kusetyenziswa i-Novaseq sequencer. Iifayile zemifanekiso eluhlaza ezivela kwi-Illumina zaguqulwa zaba zi-raw reads kusetyenziswa i-CASAVA Base Calling. Idatha eluhlaza igcinwa kwiifayile zefomathi ye-FASTQ(fq) eziqulathe ulandelelwano lokufunda kunye neempawu zesiseko ezihambelanayo. Khetha i-HISAT2 ukuze ihambelane nokufundwa kokulandelelana okuhluziweyo kwi-genome yesalathiso se-Sscrofa11.1. Ngokubanzi, i-HISAT2 ixhasa ii-genome zalo naluphi na ubungakanani, kuquka ii-genome ezinkulu kunee-bases ezi-4 zeebhiliyoni, kwaye amaxabiso amiselweyo amiselwe uninzi lweeparameter. Ukudibanisa ukufundwa kwidatha ye-RNA Seq kunokulungelelaniswa ngokufanelekileyo kusetyenziswa i-HISAT2, inkqubo ekhawulezayo ekhoyo ngoku, ngokuchanekileyo okufanayo okanye okungcono kunayo nayiphi na enye indlela.
Ubuninzi bee-transcript bubonisa ngokuthe ngqo inqanaba lokubonakaliswa kwe-gene. Amanqanaba okubonakaliswa kwe-gene avavanywa ngobuninzi bee-transcript (inani lokulandelelana) ezinxulunyaniswa ne-genome okanye ii-exons. Inani lee-reads lihambelana namanqanaba okubonakaliswa kwe-gene, ubude be-gene, kunye nobunzulu bokulandelelana. I-FPKM (iziqwenga ngee-pairs zesiseko eziliwaka ze-transcript ezilandelelaniswa ngee-pairs zesiseko ezizizigidi) zibaliwe kwaye amaxabiso e-P e-differential expression amiselwa kusetyenziswa iphakheji ye-DESeq2. Emva koko sibale i-false discovery rate (FDR) yexabiso ngalinye le-P sisebenzisa indlela ye-Benjamini-Hochberg9 esekelwe kwi-R-function eyakhelwe ngaphakathi ethi "p.adjust".
I-RNA ehlukanisiweyo kwiindawo zentliziyo yaguqulwa yaba yi-cDNA kuxinzelelo lwe-200 ng/μl kusetyenziswa umxube weSuperScript IV Vilo Master ovela kwiThermo (Thermo, cat. no. 11756050). I-Quantitative RT-PCR yenziwe kusetyenziswa ipleyiti ye-reaction ecacileyo ye-Applied Biosystems Endura Plate Microamp 384-well (Thermo, cat. no. 4483319) kunye ne-microamp optical adhesive (Thermo, cat. no. 4311971). Umxube we-reaction wawuqulathe umxube we-5 µl Taqman Fast Advanced Master (Thermo, cat # 4444557), i-0.5 µl Taqman Primer kunye ne-3.5 µl H2O exutywe ngomthombo ngamnye. Imijikelo ye-qPCR eqhelekileyo yaqhutywa kwaye amaxabiso e-CT alinganiswa kusetyenziswa isixhobo se-Applied Biosystems Quantstudio 5 real-time PCR (imodyuli ye-384-well; imveliso # A28135). Iiprimer zeTaqman zithengwe kwiThermo (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) Amaxabiso e-CT azo zonke iisampuli ahlengahlengiswe kwi-housekeeping gene GAPDH.
Ukukhutshwa kwe-NT-ProBNP kwimidiya kuhlolwe kusetyenziswa i-NT-ProBNP kit (ihagu) (Cat. No. MBS2086979, MyBioSource) ngokwemigaqo yomenzi. Ngamafutshane, i-250 µl yesampulu nganye kunye nomgangatho wongezwe kabini kwiqula ngalinye. Emva nje kokongeza isampulu, yongeza i-50 µl ye-Assay Reagent A kwiqula ngalinye. Shukumisa ipleyiti ngobunono kwaye uyivale nge-sealant. Emva koko iipilisi zafakwa kwi-incubation kwi-37°C iyure e-1. Emva koko phefumla isisombululo kwaye uhlambe imigodi izihlandlo ezi-4 nge-350 µl yesisombululo sokuhlamba se-1X, ufukame isisombululo sokuhlamba imizuzu e-1-2 ngexesha ngalinye. Emva koko yongeza i-100 µl ye-Assay Reagent B kwiqula ngalinye kwaye uyivale nge-plate sealant. Ithebhulethi yashukunyiswa kancinci kwaye yafakwa kwi-incubation kwi-37°C imizuzu engama-30. Bilisa isisombululo uze uhlambe imingxuma izihlandlo ezi-5 nge-350 µl yesisombululo sokuhlamba esingu-1X. Yongeza i-90 µl yesisombululo se-substrate emthonjeni ngamnye uze uvale ipleyiti. Faka ipleyiti kwi-37°C imizuzu eli-10-20. Yongeza i-50 µl yesisombululo sokumisa emthonjeni ngamnye. Ipleyiti yalinganiswa ngoko nangoko kusetyenziswa iseti ye-Cytation (BioTek) plate reader kwi-450 nm.
Uhlalutyo lwamandla lwenziwe ukuze kukhethwe ubungakanani beqela obuya kubonelela ngamandla angaphezu kwama-80% ukufumanisa utshintsho olupheleleyo lwe-10% kwiparameter enezinga lempazamo yohlobo lwe-5% Uhlobo I. Uhlalutyo lwamandla lwenziwe ukuze kukhethwe ubungakanani beqela obuya kubonelela ngamandla angaphezu kwama-80% ukufumanisa utshintsho olupheleleyo lwe-10% kwiparameter enezinga lempazamo yohlobo lwe-5% Uhlobo I. Анализ мощности был выполнен для выбора размеров групп, которые обеспечат >80% мощности для обнаружения 10% абеспечат частотой ошибок типа I. Uhlalutyo lwamandla lwenziwe ukuze kukhethwe ubungakanani beqela eliza kubonelela ngamandla angaphezu kwama-80% ukufumanisa utshintsho olupheleleyo lwe-10% kwiparameter kunye ne-5% Type I error rate.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% обеспечил 5% частоты ошибок типа I. Uhlalutyo lwamandla lwenziwe ukuze kukhethwe ubungakanani beqela eliza kubonelela ngamandla angaphezu kwama-80% okufumanisa utshintsho olupheleleyo lwe-10% kwiparameter kunye nezinga lempazamo yohlobo lwe-1 le-5%.Amacandelo ezicubu akhethwe ngokungacwangciswanga ngaphambi kovavanyo. Zonke iinkcazo azizange zijongwe kwimeko kwaye iisampulu zahlulwahlulwa kuphela emva kokuba yonke idatha ihlalutyiwe. Isoftware yeGraphPad Prism (eSan Diego, CA) yasetyenziswa ukwenza zonke iinkcazo zezibalo. Kuzo zonke izibalo, ixabiso le-p lithathwe njengelibalulekileyo kumaxabiso angaphantsi kwe-0.05. Kuzo zonke izibalo, ixabiso le-p lithathwe njengelibalulekileyo kumaxabiso angaphantsi kwe-0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Kuzo zonke izibalo, ixabiso le-p lithathwe njengelibalulekileyo kumaxabiso angaphantsi kwe-0.05.对于所有统计数据，p 在值<0.05 时被认為是显着的.对于所有统计数据，p 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, ixabiso le-p lithathwe njengelibalulekileyo kumaxabiso angaphantsi kwe-0.05.Uvavanyo lwe-t lomfundi olunemisila emibini lwenziwe kwidatha ngokuthelekiswa oku-2 kuphela. I-ANOVA yendlela enye okanye ezimbini yasetyenziswa ukufumanisa ukubaluleka phakathi kwamaqela amaninzi. Xa kwenziwa uvavanyo lwe-post hoc, ukulungiswa kukaTukey kwasetyenziswa ukuqwalasela ukuthelekiswa okuninzi. Idatha ye-RNAsec ineengqalelo ezikhethekileyo zezibalo xa kubalwa i-FDR kunye ne-p.adjust njengoko kuchaziwe kwicandelo leeNdlela.
Ukuze ufumane ulwazi oluthe kratya malunga noyilo lwesifundo, jonga isishwankathelo seNgxelo yoPhando lweNdalo esidityaniswe neli nqaku.