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Pakufunika dongosolo lodalirika la in vitro lomwe lingathe kubereka molondola malo okhudza thupi la mtima pofuna kuyesa mankhwala. Kupezeka kochepa kwa machitidwe a chikhalidwe cha minofu ya mtima waumunthu kwachititsa kuti kutanthauzira kolakwika kwa zotsatira za mankhwala a mtima. Pano, tapanga chitsanzo cha chikhalidwe cha mtima (CTCM) chomwe electromechanically chimayambitsa magawo a mtima ndikukhala ndi thupi lotambasula panthawi ya systolic ndi diastolic ya mtima wamtima. Pambuyo pa masiku 12 a chikhalidwe, njirayi inathandiza kuti magawo a mtima azitha kugwira ntchito, koma sanasunge umphumphu wawo. Chifukwa chake, titayang'ana mamolekyu ang'onoang'ono, tidapeza kuti kuwonjezera kwa 100 nM triiodothyronine (T3) ndi 1 μM dexamethasone (Dex) kwa sing'anga yathu kunasunga microstructure ya magawo kwa masiku 12. Kuphatikizana ndi chithandizo cha T3 / Dex, dongosolo la CTCM linasunga mbiri yolembera, kutheka, ntchito ya kagayidwe kachakudya, ndi kukhulupirika kwapangidwe pamlingo wofanana ndi minofu yatsopano ya mtima kwa masiku 12. Kuonjezera apo, kutambasula kwakukulu kwa minofu yamtima mu chikhalidwe kumapangitsa kuti zizindikiro za hypertrophic mtima ziwonetsedwe, zomwe zimapereka umboni wa luso la CTCM kutsanzira mikhalidwe ya hypertrophic yomwe imayambitsidwa ndi kutambasula kwa mtima. Pomaliza, CTCM ikhoza kutsanzira physiology ndi pathophysiology ya mtima pachikhalidwe kwa nthawi yayitali, ndikupangitsa kuyezetsa kodalirika kwa mankhwala.
Asanafufuze zachipatala, machitidwe odalirika a in vitro amafunikira omwe amatha kuberekanso bwino chilengedwe cha mtima wamunthu. Makina oterowo amayenera kutsanzira kusinthasintha kwamakina, kugunda kwamtima, komanso mphamvu zamagetsi. Zitsanzo za zinyama nthawi zambiri zimagwiritsidwa ntchito ngati malo owonetsera zamoyo wamtima ndi kudalirika kochepa powonetsera zotsatira za mankhwala mu mtima wa munthu1,2. Pamapeto pake, Ideal Cardiac Tissue Culture Experimental Model (CTCM) ndi chitsanzo chomwe chimakhala chodziwika bwino komanso chodziwika bwino pazithandizo zosiyanasiyana zamankhwala ndi zamankhwala, kutulutsa molondola zakuthupi ndi matenda a mtima wamunthu3. Kusapezeka kwa dongosolo loterolo kumachepetsa kupezeka kwa mankhwala atsopano a mtima kulephera4,5 ndipo kwadzetsa ku cardiotoxicity yamankhwala ngati chifukwa chachikulu chotulutsira msika6.
Pazaka khumi zapitazi, mankhwala asanu ndi atatu omwe sali amtima adachotsedwa pakugwiritsa ntchito kuchipatala chifukwa amayambitsa kutalika kwa nthawi ya QT kumabweretsa kugunda kwamtima komanso kufa mwadzidzidzi7. Chifukwa chake, pali kufunikira kowonjezereka kwa njira zodalirika zowunikira zowunikira kuti awone momwe mtima ulili komanso kawopsedwe. Kugwiritsidwa ntchito kwaposachedwa kwa pluripotent stem cell-derived cardiomyocytes (hiPS-CM) poyang'ana mankhwala osokoneza bongo komanso kuyesa poizoni kumapereka njira yothetsera vutoli. Komabe, chikhalidwe chachinyamata cha hiPS-CMs ndi kusowa kwa zovuta zambiri zamagulu a mtima wamtima ndizolephereka zazikulu za njirayi. Kafukufuku waposachedwa wawonetsa kuti izi zitha kugonjetsedwera pang'ono pogwiritsa ntchito hiPS-CM yoyambirira kupanga ma hydrogel amtundu wamtima patangopita nthawi pang'onopang'ono kugunda kwapang'onopang'ono ndikuwonjezera kukondoweza kwamagetsi pakapita nthawi. Komabe, ma microtissues a hiPS-CM awa alibe mphamvu yokhwima ya electrophysiological ndi contractile ya myocardium wamkulu. Kuphatikiza apo, minofu yapamtima yamunthu imakhala ndi mawonekedwe ovuta kwambiri, omwe amakhala ndi kusakanikirana kosiyanasiyana kwamitundu yosiyanasiyana ya maselo, kuphatikiza ma endothelial cell, neurons, ndi stromal fibroblasts, olumikizidwa ndi ma seti apadera a mapuloteni a extracellular matrix. Kusiyanasiyana kumeneku kwa anthu omwe si a cardiomyocyte11,12,13 mu mtima wa mammalian akuluakulu ndi chotchinga chachikulu cha kutsanzira minofu ya mtima pogwiritsa ntchito maselo amodzi. Zolepheretsa zazikuluzikuluzi zikugogomezera kufunikira kopanga njira zokulitsa minofu ya myocardial yokhazikika pansi pa zochitika zakuthupi ndi zakuthambo.
Zigawo zoonda (300 µm) za mtima wa munthu zatsimikizira kukhala chitsanzo chabwino cha myocardium yamunthu. Njirayi imapereka mwayi wopeza dongosolo lathunthu la 3D multicellular lofanana ndi minofu yamtima wamunthu. Komabe, mpaka chaka cha 2019, kugwiritsa ntchito magawo amtima otukuka kudachepetsedwa ndi kupulumuka kwakanthawi kochepa (24 h). Izi zimachitika chifukwa cha zinthu zingapo kuphatikizapo kusowa kwa kutambasula kwa thupi-makina, mawonekedwe a mpweya wamadzimadzi, komanso kugwiritsa ntchito njira zosavuta zomwe sizikugwirizana ndi zosowa za minofu ya mtima. Mu 2019, magulu ofufuza angapo adawonetsa kuti kuphatikiza zinthu zamakina mumayendedwe amtundu wamtima kumatha kukulitsa moyo wachikhalidwe, kusintha mawonekedwe amtima, komanso kutsanzira matenda amtima. Maphunziro awiri okongola 17 ndi 18 amasonyeza kuti uniaxial mechanical loading imakhala ndi zotsatira zabwino pamtima phenotype panthawi ya chikhalidwe. Komabe, maphunzirowa sanagwiritse ntchito mphamvu zitatu za physico-mechanical loading ya mtima wamtima, popeza zigawo za mtima zinali zodzaza ndi mphamvu za isometric tensile 17 kapena linear auxotonic loading 18. Njira izi zotambasulira minofu zinapangitsa kuti ma jini ambiri amtima atsekedwe kapena kuchulukirachulukira kwa majini okhudzana ndi mayankho otambasulira osadziwika bwino. Makamaka, Pitoulis et al. 19 adapanga malo osambira osinthika amtundu wamtima kuti amangenso mtima wozungulira pogwiritsa ntchito mphamvu ya transducer ndi ma drive amanjenje. Ngakhale kuti dongosololi limalola kuti pakhale ndondomeko yolondola ya in vitro cardiac cycle modeling, zovuta komanso zochepa zomwe zimapangidwira zimalepheretsa kugwiritsa ntchito dongosololi. Laborator yathu posachedwapa yapanga dongosolo losavuta lachikhalidwe pogwiritsa ntchito kukondoweza kwamagetsi komanso sing'anga yabwino kuti isunge magwiridwe antchito a magawo a nkhumba ndi minofu yamtima wamunthu mpaka masiku 620,21.
M'mipukutu yamakono, tikufotokoza za chikhalidwe cha chikhalidwe cha mtima (CTCM) pogwiritsa ntchito zigawo za mtima wa nkhumba zomwe zimaphatikizira zizindikiro za humoral kuti zibwereze physiology yamtima wapamtima ndi matenda a pathophysiological panthawi ya mtima wamtima. CTCM iyi ikhoza kuonjezera kulondola kwa kuneneratu kwa mankhwala osokoneza bongo mpaka kufika pamlingo womwe sunakwaniritsidwepo popereka ndondomeko ya mtima yamtengo wapatali, yapakati-pakatikati yomwe imatsanzira physiology / pathphysiology ya mammalian heart of pre-clinical pre-clinical test drug.
Zizindikiro zamakina a hemodynamic zimagwira ntchito yofunika kwambiri pakusunga ntchito ya cardiomyocyte mu vitro 22,23,24. M'mipukutu yamakono, tapanga CTCM (Chithunzi 1a) chomwe chingatsanzire chikhalidwe cha mtima wachikulire mwa kukakamiza mphamvu zamagetsi ndi makina pamayendedwe a thupi (1.2 Hz, 72 kugunda pamphindi). Pofuna kupewa kutambasula minofu yambiri pa diastole, chipangizo chosindikizira cha 3D chinagwiritsidwa ntchito kuonjezera kukula kwa minofu ndi 25% (mkuyu 1b). Mayendedwe amagetsi opangidwa ndi dongosolo la C-PACE adayikidwa nthawi kuti ayambe 100 ms isanakwane systole pogwiritsa ntchito njira yopezera deta kuti ibereke bwino kuzungulira kwa mtima. Dongosolo la chikhalidwe cha minyewa limagwiritsa ntchito makina opangira pneumatic actuator (LB Engineering, Germany) kukulitsa kaphatikizidwe ka membrane wosinthika wa silikoni kuti apangitse kukula kwa magawo amtima kuchipinda chapamwamba. Dongosololi lidalumikizidwa ndi mzere wakunja wa mpweya kudzera pa transducer yokakamiza, zomwe zidapangitsa kuti zitheke kuwongolera kupanikizika (± 1 mmHg) ndi nthawi (± 1 ms) (mkuyu 1c).
a Gwirizanitsani gawo la minofu ku mphete yothandizira ya 7 mm, yowonetsedwa mubuluu, mkati mwa chipinda cha chikhalidwe cha chipangizocho. Chipinda cha chikhalidwe chimasiyanitsidwa ndi chipinda cha mpweya ndi membrane yopyapyala ya silicone. Ikani gasket pakati pa chipinda chilichonse kuti musatayike. Chivundikiro cha chipangizocho chimakhala ndi ma electrode a graphite omwe amapereka mphamvu zamagetsi. b Kuyimira kwadongosolo kwa chipangizo chachikulu cha minofu, mphete yolondolera ndi mphete yothandizira. Magawo a minofu (yabulauni) amayikidwa pa chipangizo chokulirapo ndi mphete yolondolera yomwe imayikidwa poyambira pamphepete mwakunja kwa chipangizocho. Pogwiritsa ntchito bukhuli, ikani mosamala mphete yothandizirayo yokutidwa ndi zomatira za acrylic pagawo la minofu yamtima. c Graph yomwe ikuwonetsa nthawi yakukondoweza kwamagetsi ngati ntchito yamphamvu yachipinda cha mpweya yomwe imayendetsedwa ndi pulogalamu ya pneumatic actuator (PPD). Chipangizo chopezera deta chinagwiritsidwa ntchito kugwirizanitsa kukondoweza kwa magetsi pogwiritsa ntchito masensa amphamvu. Kupanikizika mu chipinda cha chikhalidwe kukafika pachimake chokhazikitsidwa, chizindikiro cha pulse chimatumizidwa ku C-PACE-EM kuti chiyambe kuyambitsa magetsi. d Chithunzi cha ma CTCM anayi atayikidwa pa shelefu ya chofungatira. Zipangizo zinayi zimalumikizidwa ndi PPD imodzi kudzera pagawo la pneumatic, ndipo masensa opanikizika amalowetsedwa mu valavu ya hemostatic kuti ayang'anire kuthamanga kwa mpweya. Chida chilichonse chimakhala ndi zigawo zisanu ndi chimodzi za minofu.
Pogwiritsa ntchito makina amodzi a pneumatic actuator, tinatha kulamulira zipangizo za 4 CTCM, zomwe aliyense amatha kugwira zigawo za 6 (mkuyu 1d). Mu CTCM, kuthamanga kwa mpweya mu chipinda cha mpweya kumasinthidwa kukhala kukakamiza kofanana mu chipinda chamadzimadzi ndipo kumapangitsa kukula kwa thupi la kagawo wa mtima (Chithunzi 2a ndi Supplementary Movie 1). Kuyeza kwa minofu kumatambasula pa 80 mm Hg. Art. adawonetsa kutambasula kwa zigawo za minofu ndi 25% (mkuyu 2b). Kutambasula uku kwawonetsedwa kuti kukugwirizana ndi kutalika kwa thupi la sarcomere la 2.2-2.3 µm kwa mgwirizano wapakati wapamtima17,19,25. Kusuntha kwa minofu kunayesedwa pogwiritsa ntchito makonda a kamera (Chithunzi Chowonjezera 1). The matalikidwe ndi liwiro la minofu kayendedwe (mkuyu 2c, d) lolingana kutambasula pa mtima mkombero ndi nthawi pa systole ndi diastole (mkuyu. 2b). Kutambasula ndi kuthamanga kwa minofu yamtima panthawi yopumula ndi kupumula kunakhalabe kosalekeza kwa masiku a 12 mu chikhalidwe (mkuyu 2f). Kuti tione zotsatira za kukondoweza magetsi pa mgwirizano pa chikhalidwe, tinapanga njira yodziwira kupunduka kogwira ntchito pogwiritsa ntchito shading algorithm (Supplementary Fig. 2a, b) ndipo tinatha kusiyanitsa pakati pa zowonongeka ndi zopanda mphamvu zamagetsi. Gawo lomwelo la mtima (mkuyu 2f). M'dera losasunthika la odulidwa (R6-9), voteji panthawi ya kukondoweza kwa magetsi inali 20% kuposa pamene palibe mphamvu yamagetsi, zomwe zimasonyeza kuthandizira kwamagetsi ku ntchito ya contractile.
Zomwe zimayimira kupanikizika kwa chipinda cha mpweya, kuthamanga kwa chipinda chamadzimadzi, ndi miyeso ya kayendedwe ka minyewa zimatsimikizira kuti kupanikizika kwa chipinda kumasintha kupanikizika kwa m'chipinda chamadzi, zomwe zimapangitsa kuti kagawo kakang'ono kake kakhale kofanana. b Zoyimira zoyimira za kutambasuka (buluu) za zigawo zofananira ndi kuchuluka kwa kutambasula (lalanje). c Kuyenda koyezedwa kwa kagawo ka mtima kumagwirizana ndi liwiro loyezedwa la kuyenda. (d) Mayendedwe oyimira a cyclic motion (mzere wabuluu) ndi liwiro (mzere wa madontho alalanje) pagawo la mtima. e Kuwerengera kwa nthawi yozungulira (n = 19 magawo pa gulu, kuchokera ku nkhumba zosiyana), nthawi yochepetsera (n = 19 magawo pa gulu), nthawi yopuma (n = 19 magawo pa gulu, kuchokera ku nkhumba zosiyana), kayendedwe ka minofu (n = 25). magawo)/gulu lochokera ku nkhumba zosiyanasiyana), nsonga ya systolic velocity (n = 24(D0), 25(D12) magawo/gulu lochokera ku nkhumba zosiyanasiyana) ndi kumasuka kwambiri (n=24(D0), 25(D12) magawo/gulu la nkhumba zosiyanasiyana). Mayeso a t a Wophunzira a michira iwiri sanawonetse kusiyana kwakukulu pazigawo zilizonse. f Kuwunika koyimilira kwapang'onopang'ono kumayang'ana zigawo za minofu ndi (zofiira) komanso zopanda (buluu) zokondoweza zamagetsi, madera khumi a zigawo za zigawo za minofu kuchokera ku gawo lomwelo. Pansi mapanelo akuwonetsa kuchuluka kwa kusiyana kwa kuchuluka kwa zovuta m'magawo a minofu ndi popanda kukondoweza kwamagetsi m'madera khumi kuchokera kumadera osiyanasiyana. (n = 8 magawo / gulu lochokera ku nkhumba zosiyanasiyana, Mayeso a t-tailed Wophunzira awiri amachitidwa; ****p <0.0001, **p <0.01, *p <0.05). (n = 8 magawo / gulu lochokera ku nkhumba zosiyanasiyana, Mayeso a t-tailed Wophunzira awiri amachitidwa; ****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 magawo/gulu lochokera ku nkhumba zosiyanasiyana, t-test ya Ophunzira a michira iwiri; ****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 zigawo/gulu, kuchokera ku nkhumba zosiyana, t-mayeso a Wophunzira a michira iwiri; ****p<0.0001, **p<0.01, *p<0.05).Mipiringidzo yolakwika imayimira ± kupatuka kokhazikika.
Mu dongosolo lathu lakale la static biomimetic heart slice culture system [20, 21], tidasungabe kutheka, kugwira ntchito, komanso kukhulupirika kwa magawo amtima kwa masiku 6 pogwiritsa ntchito mphamvu zamagetsi ndikuwongolera kapangidwe kake. Komabe, patatha masiku 10, ziwerengerozi zidatsika kwambiri. Tidzanena za magawo omwe adakhazikitsidwa mumayendedwe athu am'mbuyomu a static biomimetic culture system 20, 21 control condition (Ctrl) ndipo tidzagwiritsa ntchito sing'anga yathu yomwe idakonzedwa kale ngati mikhalidwe ya MC ndi chikhalidwe munthawi imodzi ndi kukondoweza kwamagetsi (CTCM). kuyitanidwa . Choyamba, tinatsimikiza kuti kusonkhezera kwamakina popanda kukakamiza magetsi kunali kosakwanira kusunga minofu kwa masiku a 6 (Supplementary Fig. 3a, b). Chochititsa chidwi n'chakuti, poyambitsa physio-mechanical ndi stimulation magetsi pogwiritsa ntchito STCM, kuthekera kwa magawo a mtima wa masiku a 12 kunakhalabe mofanana ndi magawo a mtima watsopano pansi pa MS, koma osati pansi pa Ctrl mikhalidwe, monga momwe MTT ikuwonetsera (mkuyu 1). 3a). Izi zikusonyeza kuti kusonkhezera kwa makina ndi kuyerekezera kwa kayendedwe ka mtima kungathe kusunga zigawo za minofu kukhala zogwira ntchito kawiri monga momwe tafotokozera m'dongosolo lathu lakale la chikhalidwe cha static. Komabe, kuwunika kwa kukhulupirika kwa magawo a minofu mwa kuyika chitetezo cha mtima wa troponin T ndi connexin 43 kunawonetsa kuti mawu a connexin 43 anali apamwamba kwambiri m'matenda a MC pa tsiku la 12 kuposa momwe amawongolera tsiku lomwelo. Komabe, yunifolomu ya connexin 43 mawu ndi mapangidwe a Z-disc sanasamalidwe mokwanira (mkuyu 3b). Timagwiritsa ntchito dongosolo la Artificial Intelligence (AI) kuti tiyese kukhulupirika kwa minofu26, njira yophunzirira mozama yozikidwa pazithunzi yozikidwa pa troponin-T ndi connexin staining43 kuti tizingodziwiratu kuchuluka kwa magawo amtima komanso kulimba kwa magawo amtima. Njirayi imagwiritsa ntchito Convolutional Neural Network (CNN) ndi ndondomeko yozama yophunzirira kuti iwonetsetse modalirika kukhulupirika kwa minofu ya mtima mwadongosolo komanso mopanda tsankho, monga momwe tafotokozera muzofotokozera. 26. Minofu ya MC inasonyeza kufanana kwapangidwe kofanana ndi tsiku la 0 poyerekeza ndi magawo olamulira osasunthika. Komanso, Masson a trichrome madontho anavumbula kwambiri m'munsi kwambiri kuchuluka kwa fibrosis pansi MS mikhalidwe poyerekeza ndi kulamulira zinthu pa tsiku 12 chikhalidwe (mkuyu. 3c). Ngakhale kuti CTCM inawonjezera mphamvu ya zigawo za minofu ya mtima pa tsiku la 12 kufika pamtunda wofanana ndi wa minofu yatsopano ya mtima, sizinasinthe kwambiri kukhulupirika kwa zigawo za mtima.
Chithunzi cha Bar chikuwonetsa kuchuluka kwa magwiridwe antchito a MTT a magawo atsopano amtima (D0) kapena chikhalidwe cha magawo amtima kwa masiku 12 mwina muchikhalidwe chokhazikika (D12 Ctrl) kapena mu CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) magawo / gulu lochokera ku ###p #ANOVA; 0.0001 poyerekeza ndi D0 ndi **p <0.01 poyerekeza ndi D12 Ctrl). Chithunzi cha Bar chikuwonetsa kuchuluka kwa magwiridwe antchito a MTT a magawo atsopano amtima (D0) kapena chikhalidwe cha magawo amtima kwa masiku 12 mwina muchikhalidwe chokhazikika (D12 Ctrl) kapena mu CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) magawo / gulu limodzi kuchokera ku ### p. 0.0001 poyerekeza ndi D0 ndi **p <0.01 poyerekeza ndi D12 Ctrl).histogram imasonyeza kuchuluka kwa magawo a mtima wa MTT (D0) kapena chikhalidwe cha magawo a mtima kwa masiku a 12 mu chikhalidwe chokhazikika (D12 control) kapena CTCM (D12 MC) (n = 18 (D0), 15 (D12 control).####p < 0,0001 по сравнению с D0 ndi **p < 0,01 по сравнению с D12 Ctrl). ####p <0.0001 poyerekeza ndi D0 ndi **p <0.01 poyerekeza ndi D12 Ctrl). a 条形图显示在静态培养(D12 Ctrl) 或CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 12 或片(D0) 2天的MTT 活力的量化)，來自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；与D0 相比，####p <2比比比，####p <2.00D 10.00D <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) 中文 (D0) 中文切片/组，进行单向ANOVA 测试；与D0 相比，####p <0.0001，与D12 Ctrl 相比，**p.)histogram kusonyeza quantification wa MTT viability mu magawo atsopano a mtima (D0) kapena magawo a mtima otukuka kwa masiku a 12 mu chikhalidwe chosasunthika (D12 control) kapena CTCM (D12 MC) (n = 18 (D0), 15 (D12 control)), 12 (D12 MC) magawo / gulu kuchokera ku nkhumba zosiyana siyana, kuyesa kwa ANOVA;####p < 0,0001 по сравнению с D0, **p <0,01 по сравнению с D12 Ctrl). ####p <0.0001 poyerekeza ndi D0, **p <0.01 poyerekeza ndi D12 Ctrl).b Troponin-T (wobiriwira), connexin 43 (wofiira) ndi DAPI (buluu) m'magawo a mtima omwe angodzipatula (D0) kapena magawo a mtima omwe amakula pansi pazikhalidwe zosasunthika (Ctrl) kapena CTCM (MC) kwa masiku 12) a zithunzi zoimira immunofluorescence (sikelo yopanda kanthu = 100 µm). Kuwerengera kwanzeru kwapakatikati kwa minofu yamtima (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) magawo / gulu lililonse kuchokera ku nkhumba yosiyana, kuyesa kwa njira imodzi ya ANOVA kumachitika; ####p <0.0001 poyerekeza ndi D0 ndi ****p <0.000 kuyerekeza ndi D010). Kuwerengera kwanzeru kwapakatikati kwa minofu yamtima (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) magawo / gulu lililonse kuchokera ku nkhumba zosiyanasiyana, kuyesa kwa njira imodzi ya ANOVA kumachitika; ####p <0.0001 poyerekeza ndi D0 ndi ****p <0.000 kuyerekeza ndi D010). Kuwongolera kwapakatikati (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) kukopera, 5 (D12 MC) проводится однофакторный тест ANOVA; ####p <0,0001 по сравнению с D0 ndi ****p <0,0001 по сравнению с D12 Ctrl). Kuchulukitsidwa kwa kapangidwe ka minofu yamtima ndi luntha lochita kupanga (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) magawo / magulu ochokera ku nkhumba zosiyanasiyana, kuyesa kwa njira imodzi ya ANOVA kunachitika; ####p <0.0001 vs. ndi D0 ndi ****0p <0 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) magawo/gulu lililonse la nkhumba yosiyana, kuyesa njira imodzi ya ANOVA;##0#0D010. Sinthani, ****p <0.0001 pa 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) magawo/gulu lililonse la nkhumba zosiyanasiyana, kuyesa njira imodzi ya ANOVA;##0#0D0D **** 10. <0.0001 Ctrl ndi D12. Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC/pang'onopang'ono) p. разных свиней, односторонний тест ANOVA; ####p <0,0001 vs. D0 Для сравнения ****p <0,0001 по сравнению с D12 Ctrl). Luntha lochita kupanga kuti liwerengere kuchuluka kwa minofu yamtima (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) magawo/gulu lililonse la nkhumba zosiyanasiyana, kuyesa kwa ANOVA kwa njira imodzi; ####p<0.0001 vs .D0 Poyerekeza 0. c Zithunzi zoyimira (kumanzere) ndi kuchuluka (kumanja) kwa magawo amtima odetsedwa ndi banga la Masson's trichrome (Scale bare = 500 µm) (n = 10 magawo / gulu lililonse kuchokera ku nkhumba yosiyana, kuyesa kwa ANOVA kwa njira imodzi kumachitidwa; ####p <0.0001 poyerekeza ndi D00 ndi D*20). c Zithunzi zoyimira (kumanzere) ndi kuchuluka (kumanja) kwa magawo amtima odetsedwa ndi banga la Masson's trichrome (Scale bare = 500 µm) (n = 10 magawo / gulu lililonse kuchokera ku nkhumba zosiyanasiyana, kuyesa kwa ANOVA kwa njira imodzi kumachitidwa; #### p <0.0001 poyerekeza ndi D00 ndi D*20). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Масязкен Масязкон м mbing) (n = 10 сров / оты1 Провний < раВнию С D12 CTRL). c Zithunzi zoyimira (kumanzere) ndi kuchuluka (kumanja) kwa zigawo zamtima zodetsedwa ndi banga la Masson's trichrome (sikelo yosatsekedwa = 500 µm) (n = 10 magawo / gulu lochokera ku nkhumba zosiyanasiyana, kuyesa kwa njira imodzi ya ANOVA kuchitidwa; #### p <0 .0001 poyerekeza ndi D0. 1 ***p
Tidaganiza kuti powonjezera mamolekyu ang'onoang'ono ku chikhalidwe cha chikhalidwe, umphumphu wa cardiomyocyte ukhoza kukhala wabwino komanso kukula kwa fibrosis kuchepetsedwa pa chikhalidwe cha CTCM. Chifukwa chake tidayesa mamolekyu ang'onoang'ono pogwiritsa ntchito zikhalidwe zathu za static control20,21 chifukwa cha kuchepa kwazinthu zosokoneza. Dexamethasone (Dex), triiodothyronine (T3), ndi SB431542 (SB) adasankhidwa pachithunzichi. Mamolekyu ang'onoang'onowa akhala akugwiritsidwa ntchito m'zikhalidwe za hiPSC-CM kuti apangitse kusasitsa kwa cardiomyocytes powonjezera kutalika kwa sarcomere, T-tubules, ndi kuthamanga kwa conduction. Kuphatikiza apo, onse Dex (glucocorticoid) ndi SB amadziwika kuti amaletsa kutupa29,30. Chifukwa chake, tidayesa ngati kuphatikizika kwa mamolekyu ang'onoang'ono kapena kuphatikiza kwa mamolekyu ang'onoang'ono kungathandize kuti magawo a mtima akhale ogwirizana. Pakuwunika koyambirira, mlingo wa pawiri uliwonse udasankhidwa kutengera kuchuluka komwe kumagwiritsidwa ntchito mumitundu yama cell (1 μM Dex27, 100 nM T327, ndi 2.5 μM SB31). Pambuyo pa masiku 12 a chikhalidwe, kuphatikiza kwa T3 ndi Dex kunapangitsa kuti cardiomyocyte structural integrity ikhale yabwino komanso kukonzanso kochepa kwa fibrous (Supplementary Figures 4 ndi 5). Kuphatikiza apo, kugwiritsa ntchito kawiri kapena kuwirikiza kawiri izi za T3 ndi Dex kumabweretsa zotsatira zoyipa poyerekeza ndi zomwe zili bwino (Supplementary Fig. 6a,b).
Pambuyo poyang'ana koyamba, tidafanizira mutu ndi mutu wa chikhalidwe cha 4 (Chithunzi 4a): Ctrl: magawo a mtima omwe amakula mu chikhalidwe chathu chomwe tafotokoza kale pogwiritsa ntchito sing'anga yathu yabwino; 20.21 TD: T3 ndi Ctrl s Added Dex Lachitatu; MC: magawo amtima otukuka mu CTCM pogwiritsa ntchito sing'anga yomwe idakonzedwa kale; ndi MT: CTCM yokhala ndi T3 ndi Dex yowonjezeredwa pakati. Pambuyo pa masiku a 12 a kulima, kutheka kwa minofu ya MS ndi MT kunakhalabe mofanana ndi minofu yatsopano yomwe imayesedwa ndi MTT assay (Fig. 4b). Chochititsa chidwi n'chakuti, kuwonjezera kwa T3 ndi Dex ku transwell zikhalidwe (TD) sikunapangitse kusintha kwakukulu kochita bwino poyerekeza ndi mikhalidwe ya Ctrl, kusonyeza ntchito yofunikira ya makina otsitsimula kuti apitirizebe kugwira ntchito kwa zigawo za mtima.
Chithunzi chojambula choyesera chosonyeza zikhalidwe zinayi zomwe zimagwiritsidwa ntchito poyesa kukopa kwamakina ndi T3/Dex supplementation pa sing'anga kwa masiku 12. b Bar graph ikuwonetsa kuchuluka kwa chikhalidwe chamasiku a 12 m'mikhalidwe yonse ya 4 (Ctrl, TD, MC, ndi MT) poyerekeza ndi magawo atsopano amtima (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD ndi D12 MT), 12 (D12 MC) magawo / gulu limodzi # # ANOVA yoyesedwa ndi ##p; 0.0001, ###p <0.001 poyerekeza ndi D0 ndi **p <0.01 poyerekeza ndi D12 Ctrl). b Bar graph ikuwonetsa kuchuluka kwa chikhalidwe chamasiku a 12 m'mikhalidwe yonse ya 4 (Ctrl, TD, MC, ndi MT) poyerekeza ndi magawo atsopano amtima (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD ndi D12 MT), 12 (D12 MC) magawo / gulu limodzi # # ANOVA yoyesedwa ndi ##p; 0.0001, ###p <0.001 poyerekeza ndi D0 ndi **p <0.01 poyerekeza ndi D12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после культивирования во всех 4 условиях культивирования во всех 4 условиях культивирования сравнению свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD ndi D12 MT), 12 (D12 MC) срезов/группу от разных пронстонстенный, ANOVA b Grafu ya bar ikuwonetsa kuchuluka kwa zochitika pamasiku a 12 positi chikhalidwe pazikhalidwe zonse za 4 (kuwongolera, TD, MC, ndi MT) poyerekeza ndi zigawo zamtima zatsopano (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, ndi D12 MT), 12 (D12 MC) # ANOp # gulu losiyana # VA / gulu limodzi # VA / gulu limodzi; 0.0001, ###p <0.001 vs. D0 ndi **p <0.01 poyerekezera ndi 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和D12 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 Chithunzi cha D12控制.b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC ndi MT) по сравнению свежими TD срезами сердца (D0) (n = 15,D122), D0) (n = 15,D12) MT), от разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA; b Histogram yosonyeza zikhalidwe zonse za 4 (kulamulira, TD, MC ndi MT) poyerekeza ndi magawo atsopano a mtima (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD ndi D12 MT), kuchokera ku nkhumba zosiyana 12 (D12 MC) zigawo / gulu, njira imodzi ANOVA kuyesa; ##0#0 #0 D0, **p<0.01 vs. control D12). c Bar graph ikuwonetsa kuchuluka kwa glucose flux masiku 12 positi chikhalidwe m'mikhalidwe yonse ya 4 (Ctrl, TD, MC, ndi MT) poyerekeza ndi magawo atsopano amtima (D0) (n = 6 magawo / gulu kuchokera ku nkhumba zosiyanasiyana, kuyesa kwa njira imodzi ANOVA kumachitika; ###p <0.001, poyerekeza ndi D0 ndi ***p <0). c Bar graph ikuwonetsa kuchuluka kwa glucose flux masiku 12 positi chikhalidwe m'mikhalidwe yonse ya 4 (Ctrl, TD, MC, ndi MT) poyerekeza ndi magawo atsopano amtima (D0) (n = 6 magawo / gulu kuchokera ku nkhumba zosiyanasiyana, kuyesa kwa njira imodzi ANOVA kumachitika; ###p <0.001, poyerekeza ndi D0 ndi ***p <0). c Гистограмма показывает количественную оценку потока глюкозы через 12 дней после культивирования во всех 4 условиях культивирования, MCTD, MCTD сравнению со свежими срезами сердца (D0) (n = 6 срезов/группу от разных свиней, односторонний Выполняется тест ANOVA; ##не#p <0,00 0,00 kulumikiza ndi D12 Ctrl). c Histogram ikuwonetsa kuchuluka kwa shuga m'masiku a 12 pambuyo pa chikhalidwe pansi pazikhalidwe zonse za 4 (kuwongolera, TD, MC ndi MT) poyerekeza ndi magawo atsopano amtima (D0) (n = 6 magawo / gulu lochokera ku nkhumba zosiyanasiyana, kuyesa kwa njira imodzi ya ANOVA kuchitidwa; ###p <0.001 poyerekeza ndi D0 ndi ***p <0.00 Ctrl <0.00 Ctrl 1). 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0p1 与0D0 Ctrl 1 t*.相比）. C 条形图 显示 所有 4 种 条件 （(ctrl 、 td 、 mc 和 mt) 12 天 的 通量 定量 （n = 6 片/组 ， 來自 猪 , , , , , , , , 猪 猪单向执行ANOVA 测试；#0#01 <0. fufuzani, ***p <0.001 pa D12 Ctrl 相比） c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после культивирования для всех 4 условий кулькозы, TD сравнению со свежими срезами сердца (D0) (n = 6 срезов/группа, от разных свиней, односторонний Были проведены тесты ANOVA; ###0p <0,0,0 0,001 по сравнению с D12 (контроль). c Histogram yosonyeza kuchuluka kwa shuga wamagazi pamasiku a 12 pambuyo pa chikhalidwe cha chikhalidwe chonse cha 4 (control, TD, MC, ndi MT) poyerekeza ndi magawo atsopano a mtima (D0) (n = 6 zigawo / gulu, kuchokera ku nkhumba zosiyana, unilateral Were ANOVA mayesero anachitidwa, ###p <0.001 poyerekeza ndi D01. ***1 poyerekeza ndi D01.d Kusanthula kwazovuta zamagulu atsopano (abuluu), tsiku la 12 MC (wobiriwira), ndi tsiku la 12 MT (ofiira) pamagulu khumi a zigawo zamagulu (n = 4 magawo / gulu, kuyesa kwa njira imodzi ya ANOVA; panalibe kusiyana kwakukulu pakati pa magulu). e Chiwembu cha Volcano chomwe chikuwonetsa majini owonetsedwa m'magawo atsopano amtima (D0) poyerekeza ndi magawo amtima omwe amakula mokhazikika (Ctrl) kapena pansi pa MT mikhalidwe (MT) kwa masiku 10-12. f Kutentha kwa majini a sarcomere a magawo a mtima otukuka pansi pa chikhalidwe chilichonse. Mipiringidzo yolakwika imayimira ± kupatuka kokhazikika.
Kudalira kwa metabolic pakusintha kuchoka ku mafuta acid oxidation kupita ku glycolysis ndichizindikiro cha cardiomyocyte dedifferentiation. Ma cardiomyocyte osakhwima amagwiritsa ntchito shuga popanga ATP ndipo amakhala ndi mitochondria ya hypoplastic yokhala ndi ma cristae5,32 ochepa. Kuwunika kwa kugwiritsidwa ntchito kwa glucose kunawonetsa kuti pansi pamikhalidwe ya MC ndi MT, kugwiritsa ntchito shuga kunali kofanana ndi m'matenda a tsiku la 0 (Chithunzi 4c). Komabe, zitsanzo za Ctrl zikuwonetsa kuwonjezeka kwakukulu kwa kugwiritsidwa ntchito kwa glucose poyerekeza ndi minofu yatsopano. Izi zikuwonetsa kuti kuphatikiza kwa CTCM ndi T3 / Dex kumapangitsa kuti minyewa ikhale yogwira ntchito komanso imasunga metabolic phenotype ya magawo amtima amasiku a 12. Kuonjezera apo, kuwunika kwa zovuta kunasonyeza kuti zovuta zowonongeka zimakhala zofanana ndi minofu yatsopano ya mtima kwa masiku a 12 pansi pa MT ndi MS mikhalidwe (Fig. 4d).
Kuti tiwone momwe CTCM ndi T3/Dex zimakhudzira mawonekedwe amtundu wapadziko lonse wa minofu yamtima, tidachita RNAseq pamagawo amtima kuchokera kumitundu yonse inayi (Supplementary Data 1). Chochititsa chidwi n'chakuti, zigawo za MT zimasonyeza kufanana kwakukulu kwa zilembo zamtundu watsopano wamtima, ndi 16 yokha yowonetsedwa mosiyana ndi majini a 13,642. Komabe, monga taonera kale, Ctrl magawo anasonyeza 1229 differentially anasonyeza majini pambuyo 10-12 masiku chikhalidwe (mkuyu. 4e). Deta iyi idatsimikiziridwa ndi qRT-PCR yamtundu wa mtima ndi fibroblast (Supplementary Fig. 7a-c). Chochititsa chidwi n'chakuti, zigawo za Ctrl zimasonyeza kuchepa kwa majini a mtima ndi ma cell ndi kuyambitsa mapulogalamu a jini otupa. Deta iyi imasonyeza kuti kusiyanitsa, komwe kumachitika kawirikawiri pambuyo pa kulima kwa nthawi yaitali, kumachepetsedwa kwathunthu pansi pa MT (Supplementary Fig. 8a, b). Kufufuza mosamala za majini a sarcomere kunasonyeza kuti pokhapokha pazikhalidwe za MT ndi majini omwe amasungira sarcomere (mkuyu 4f) ndi njira ya ion (Supplementary Fig. 9) yosungidwa, kuwateteza ku kuponderezedwa pansi pa Ctrl, TD, ndi MC. Deta iyi ikuwonetsa kuti kuphatikiza kwamakina ndi kukopa chidwi (T3 / Dex), gawo la mtima la transcriptome limatha kukhala lofanana ndi magawo atsopano amtima pakatha masiku 12 pachikhalidwe.
Zotsatira za transcriptal izi zimathandizidwa ndi mfundo yakuti mapangidwe a cardiomyocytes m'magulu a mtima amasungidwa bwino pansi pa MT kwa masiku a 12, monga momwe akuwonetsera ndi connexin 43 yosasunthika (Fig. 5a). Kuonjezera apo, fibrosis m'magawo a mtima pansi pa MT mikhalidwe inachepetsedwa kwambiri poyerekeza ndi Ctrl ndi zofanana ndi magawo a mtima watsopano (mkuyu 5b). Deta iyi ikuwonetsa kuti kuphatikiza koyambitsa makina ndi chithandizo cha T3 / Dex kumateteza bwino dongosolo la mtima m'magawo amtima mu chikhalidwe.
a Representative immunofluorescence zithunzi za troponin-T (zobiriwira), connexin 43 (red), ndi DAPI (buluu) m'zigawo zapamtima zapayekha (D0) kapena zotukuka kwa masiku 12 m'mikhalidwe yonse inayi ya chikhalidwe cha mtima (scale bar = 100 µm). ). Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0 ndi D12 Ctrl), 5 (D12 TD, D12 MC ndi D12 MT) magawo/gulu kuchokera ku nkhumba zosiyanasiyana, kuyesa kwa njira imodzi ya ANOVA kumachitika; <###p <0.0001 poyerekeza ndi D00 ndi 0*0 5 ku D12 Ctrl). Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0 ndi D12 Ctrl), 5 (D12 TD, D12 MC ndi D12 MT) magawo/gulu kuchokera ku nkhumba zosiyanasiyana, kuyesa kwa njira imodzi ya ANOVA kumachitidwa; <### p <0.0001 poyerekeza ndi D00 ndi 0*0 5 ku D12 Ctrl). Njira zopangira zida zopangira zida zopangira zida zopangira zida zamagetsi (n = 7 (D0 ndi D12 Ctrl), 5 (D12 TD, D12 MT/D12 MT) ndi p. от разных свиней, проведен однофакторный тест ANOVA; #### p <0,0001 по сравнению с D0 и *p <0,05 or ****p <0,0001 по сравнению с D12 ). Kuchul D12 Ctrl).对不同猪的心脏组织结构完整性（n = 7（D0 和D12 Ctrl）、5（D12 TD、D12 MC 和D12 MT）切片/组）进行人工智能量化，进行单向ANOVA 测试;相比）.对 不同 猪 的 心脏 结构 完整性 (n = 7 （d0 和 d12 ctrl)智能量 化 进行 单向 单向 单向 测试 ; ########### p < 0.0001 与D0 和*p <0.05 相比 <0.05 相比 1 Ctrl 1 20D1 0.****0p相比）.Kuwerengera kwa mapangidwe a minofu ya mtima pogwiritsa ntchito luntha lochita kupanga mu nkhumba zosiyanasiyana (n = 7 (D0 ndi D12 Ctrl), 5 (D12 TD, D12 MC ndi D12 MT) magawo / gulu) ndi njira imodzi yoyesera ANOVA;#### p <0,0001 по сравнению с D0 и *p <0,05 kapena ****p <0,0001 по сравнению с D12 Ctrl). #### p <0.0001 poyerekeza ndi D0 ndi *p <0.05 kapena ****p <0.0001 poyerekeza ndi D12 Ctrl). b Zithunzi zoyimira ndi kuchuluka kwa magawo amtima opaka utoto wa Masson's trichrome (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, ndi D12 MC), 9 (D12 MT) magawo/gulu kuchokera ku nkhumba zosiyanasiyana, #0 kuyerekeza ndi D00; #0 kuyerekeza ANOVA #0 njira imodzi. ndi ***p <0.001, kapena ****p <0.0001 poyerekeza ndi D12 Ctrl). b Zithunzi zoyimira ndi kuchuluka kwa magawo amtima opaka utoto wa Masson's trichrome (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, ndi D12 MC), 9 (D12 MT) magawo/gulu kuchokera ku nkhumba zosiyanasiyana, #0 kuyerekeza ndi D00; #0 kuyerekeza ANOVA #0 njira imodzi. ndi ***p <0.001, kapena ****p <0.0001 poyerekeza ndi D12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Массона (масштабная 10) = 50 = 10 = (D0, D12 Ctrl, D12 TD ndi D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполняется односторонний тест ANOVA <0 0,001 kapena ****p <0,0001 podina pa D12 Ctrl). b Zithunzi zoyimira ndi kuchuluka kwa magawo amtima odetsedwa ndi banga la Masson's trichrome (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD ndi D12 MC), 9 (D12 MT) magawo/gulu kuchokera ku nkhumba zosiyanasiyana, anachita njira imodzi.#00 ndi 01. 0.001 kapena ****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(D11D TD), D10（D2D0 MC. 0.0001 ndi D12 Ctrl fungulo. b 用 Massan 三 的 的 和 切片 量化 量化 尺 尺 尺 = 500, 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; 0.001, 或****p <0.0001 pa D12 Ctrl + . b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом Массона (масштабная линейка = 500, D3, Ctrl = 500, D1 = 500, D. D12 TD ndi D12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA; D12 Ctrl). b Zithunzi zoyimira ndi kuchuluka kwa magawo amtima odetsedwa ndi Masson's trichrome (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD ndi D12 MC), 9 (D12 MT) magawo osiyanasiyana a nkhumba/gulu, njira imodzi ya ANOVA; #0. 0.001 kapena ****p <0.0001 poyerekeza ndi D12 Ctrl).Mipiringidzo yolakwika imayimira ± kupatuka kokhazikika.
Potsirizira pake, kuthekera kwa CTCM kutsanzira hypertrophy ya mtima kunayesedwa ndi kuwonjezeka kwa minofu ya mtima. Mu CTCM, kuthamanga kwapamwamba kwa chipinda cha mpweya kunakwera kuchoka pa 80 mmHg kufika ku 80 mmHg. Art. (kutambasula kwabwinobwino) mpaka 140 mmHg Art. (Mku. 6a). Izi zikufanana ndi kuwonjezeka kwa 32% mu kutambasula (mkuyu 6b), yomwe idawonetsedwa kale ngati gawo lofanana la kutambasula kofunikira kuti magawo a mtima akwaniritse kutalika kwa sarcomere mofanana ndi momwe amawonekera mu hypertrophy. Kutambasula ndi kuthamanga kwa minofu ya mtima panthawi yopumula ndi kupumula kunakhalabe kosalekeza masiku asanu ndi limodzi a chikhalidwe (mkuyu 6c). Minofu yamtima yochokera ku MT inakhala yotambasula bwino (MT (Normal)) kapena mikhalidwe yowonjezereka (MT (OS)) kwa masiku asanu ndi limodzi. Kale patatha masiku anayi mu chikhalidwe, hypertrophic biomarker NT-ProBNP inali yokwezeka kwambiri pakati pa MT (OS) mikhalidwe poyerekeza ndi MT (yachibadwa) mikhalidwe (Fig. 7a). Kuonjezera apo, patatha masiku asanu ndi limodzi a culturing, kukula kwa selo mu MT (OS) (mkuyu 7b) kunakula kwambiri poyerekeza ndi zigawo za MT mtima (zachibadwa). Kuonjezera apo, kutumizidwa kwa nyukiliya ya NFATC4 kunawonjezeka kwambiri m'magulu ochulukitsitsa (Mkuyu 7c). Zotsatirazi zikuwonetsa kukula kwapang'onopang'ono kwa kukonzanso kwa pathological pambuyo pa hyperdistension ndikuthandizira lingaliro lakuti chipangizo cha CTCM chingagwiritsidwe ntchito ngati nsanja yophunzirira kutambasula-induced cardiac hypertrophy signing.
Zomwe zimayimira kupanikizika kwa chipinda cha mpweya, kuthamanga kwa chipinda chamadzimadzi, ndi miyeso ya kayendedwe ka minyewa zimatsimikizira kuti kupanikizika kwa chipinda kumasintha kupanikizika kwa m'chipinda chamadzi, zomwe zimapangitsa kuti kagawo kakang'ono kake kakhale kofanana. b Machulukidwe oyimira ndi ma curve otambasulira omwe amakhala otambasulidwa (lalanje) ndi magawo otambasuka (abuluu). c Bar graph yosonyeza nthawi yozungulira (n = 19 magawo pa gulu, kuchokera ku nkhumba zosiyana), nthawi yochepetsera (n = 18-19 magawo pa gulu, kuchokera ku nkhumba zosiyana), nthawi yopuma (n = 19 magawo pa gulu, kuchokera ku nkhumba zosiyanasiyana)), matalikidwe a kayendedwe ka minofu (n = 14 magawo / gulu, kuchokera ku nkhumba zosiyana), nsonga za 4 magawo osiyanasiyana (pigs / gulu) , nsonga za syricity 4 ndi 1 nsonga yopumula (n = 14 (D0), 15 (D6) ) magawo / magulu) ochokera ku nkhumba zosiyana), t-mayeso a Wophunzira amichira iwiri sanasonyeze kusiyana kwakukulu pazigawo zilizonse, kusonyeza kuti magawowa anakhalabe osasintha m'masiku a 6 a chikhalidwe ndi overvoltage. Mipiringidzo yolakwika imayimira ± kupatuka kokhazikika.
a Bar graph quantification of NT-ProBNP concentration mu chikhalidwe media kuchokera ku magawo amtima otukuka pansi pa MT normal stretch (Norm) kapena overstretching (OS) mikhalidwe (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, ndi D4 MTOS) magawo / gulu kuchokera ku nkhumba zosiyanasiyana, njira ziwiri
Kutanthauzira kwamtima kwamtima kumafuna zitsanzo zama cell zomwe zimaberekanso bwino malo amtima. Mu phunziro ili, chipangizo cha CTCM chinapangidwa ndikudziwika chomwe chingathe kulimbikitsa zigawo za ultrathin za mtima. Dongosolo la CTCM limaphatikizapo kukondoweza kwa ma electromechanical ndi physiologically synchronized electromechanical stimulation ndi T3 ndi Dex fluid kulemeretsa. Pamene magawo a mtima wa nkhumba adadziwika ndi zinthu izi, mphamvu zawo, kukhulupirika kwawo, machitidwe a kagayidwe kachakudya, ndi mawu olembedwa amakhalabe ofanana ndi minofu yamtima yatsopano pambuyo pa masiku 12 a chikhalidwe. Kuonjezera apo, kutambasula kwambiri kwa minofu ya mtima kungayambitse hypertrophy ya mtima chifukwa cha hyperextension. Pazonse, zotsatirazi zimathandizira gawo lofunikira la chikhalidwe cha chikhalidwe cha thupi posunga mtima wabwinobwino wa phenotype ndikupereka nsanja yowunikira mankhwala.
Zinthu zambiri zimathandizira kuti pakhale malo abwino kwambiri ogwirira ntchito komanso kukhala ndi moyo kwa ma cardiomyocytes. Zomwe zikuwonekera kwambiri pazifukwa izi zimagwirizana ndi (1) kuyanjana kwapakati, (2) kukondoweza kwa electromechanical, (3) humoral factor, ndi (4) magawo a metabolic. Kuyanjana kwa ma cell-to-cell kumafunikira maukonde amitundu itatu amitundu ingapo omwe amathandizidwa ndi matrix a extracellular. Kulumikizana kwa ma cell kovuta kotereku kumakhala kovuta kukonzanso mu vitro ndi chikhalidwe chamagulu amtundu uliwonse, koma zitha kutheka mosavuta pogwiritsa ntchito gawo la mtima la organotypic.
Kutambasula kwamakina ndi kukondoweza kwamagetsi kwa cardiomyocytes ndikofunikira kuti mtima ukhalebe wa phenotype33,34,35. Ngakhale kukondoweza kwamakina kumagwiritsidwa ntchito kwambiri pakusintha kwa hiPSC-CM ndi kusasitsa, maphunziro angapo owoneka bwino ayesa posachedwapa kusonkhezera magawo amtima pachikhalidwe pogwiritsa ntchito uniaxial loading. Kafukufukuyu akuwonetsa kuti 2D uniaxial mechanical loading imakhala ndi zotsatira zabwino pa phenotype ya mtima pa chikhalidwe. M'maphunzirowa, magawo a mtima anali odzaza ndi mphamvu za isometric tensile17, linear auxotonic loading18, kapena kuzungulira kwa mtima kunapangidwanso pogwiritsa ntchito mphamvu ya transducer ndi mphamvu zoyendetsa. Komabe, njirazi zimagwiritsa ntchito kutambasula kwa minofu ya uniaxial popanda kukhathamiritsa kwa chilengedwe, zomwe zimapangitsa kuponderezedwa kwa majini ambiri a mtima kapena kuwonjezereka kwa majini okhudzana ndi mayankho otambasula osadziwika bwino. CTCM yomwe yafotokozedwa apa imapereka 3D electromechanical stimulus yomwe imatsanzira kayendedwe ka mtima wachilengedwe malinga ndi nthawi yozungulira komanso kutambasula kwa thupi (25% kutambasula, 40% systole, 60% diastole, ndi 72 kugunda pamphindi). Ngakhale kuti kusonkhezera kwamakina atatuwa kokha sikukwanira kusunga umphumphu wa minofu, kusakaniza kochititsa chidwi ndi makina ogwiritsira ntchito T3 / Dex kumafunika kuti asunge bwino minofu, ntchito, ndi kukhulupirika.
Zinthu zoseketsa zimathandizira kwambiri pakuwongolera mtima wamunthu wamkulu. Izi zidawonetsedwa mu maphunziro a HiPS-CM momwe T3 ndi Dex adawonjezedwa ku media media kuti apititse patsogolo kukula kwa ma cell. T3 ikhoza kukhudza kayendedwe ka amino acid, shuga ndi calcium kudutsa ma cell membranes36. Kuonjezera apo, T3 imalimbikitsa MHC-α kufotokozera ndi MHC-β kuchepetsa, kulimbikitsa mapangidwe a myofibrils ofulumira mu cardiomyocytes okhwima poyerekeza ndi pang'onopang'ono myofibrils mu fetal CM. Kuperewera kwa T3 kwa odwala omwe ali ndi hypothyroidism kumabweretsa kutayika kwa magulu a myofibrillar komanso kuchepa kwa kukula kwa mawu37. Dex amachita pa glucocorticoid zolandilira ndipo zasonyezedwa kuonjezera contractility myocardial mu akutali perfused mitima; 38 kuwongolera uku akuganiziridwa kuti kukugwirizana ndi zotsatira za calcium deposit-driven entry (SOCE) 39,40. Kuphatikiza apo, Dex imamangiriza ku zolandilira zake, zomwe zimapangitsa kuyankha kwakukulu kwa intracellular komwe kumachepetsa chitetezo chamthupi ndi kutupa30.
Zotsatira zathu zikuwonetsa kuti kukondoweza kwa thupi (MS) kunapangitsa kuti zikhalidwe zonse ziziyenda bwino poyerekeza ndi Ctrl, koma zidalephera kukhalabe zotheka, kukhulupirika kwadongosolo, komanso kuwonetsa mtima kwamasiku 12 pachikhalidwe. Poyerekeza ndi Ctrl, kuwonjezera kwa T3 ndi Dex ku CTCM (MT) zikhalidwe zinasintha bwino ndikusunga mbiri yofananira, kukhulupirika kwadongosolo, ndi zochitika za metabolic ndi minofu yatsopano yamtima kwa masiku 12. Kuonjezera apo, poyang'anira kuchuluka kwa minofu yotambasula, chitsanzo cha hyperextension-choyambitsa mtima hypertrophy chinapangidwa pogwiritsa ntchito STCM, kusonyeza kusinthasintha kwa dongosolo la STCM. Tiyenera kuzindikira kuti ngakhale kuti kukonzanso mtima ndi fibrosis nthawi zambiri kumaphatikizapo ziwalo zowonongeka zomwe maselo ozungulira amatha kupereka ma cytokines oyenerera komanso phagocytosis ndi zinthu zina zokonzanso, zigawo za mtima zimathabe kutsanzira ndondomeko ya fibrotic poyankha kupsinjika maganizo ndi kupwetekedwa mtima. mu myofibroblasts. Izi zawunikidwa kale mu chitsanzo ichi cha gawo la mtima. Tiyenera kuzindikira kuti magawo a CTCM amatha kusinthidwa mwa kusintha kupanikizika / matalikidwe amagetsi ndi mafupipafupi kuti ayese zinthu zambiri monga tachycardia, bradycardia, ndi mechanical circulatory support (mechanical unloaded heart). Izi zimapangitsa kuti dongosololi likhale lapakati poyesera mankhwala. Kuthekera kwa CTCM kuwonetsa hypertrophy yamtima yopitilira muyeso-induced cardiac hypertrophy imatsegula njira yoyesera dongosololi la chithandizo chamunthu payekha. Pomaliza, kafukufuku wapano akuwonetsa kuti kutambasula kwamakina ndi kukondoweza kwamasewera ndikofunikira kwambiri pakusunga chikhalidwe cha zigawo zamtima.
Ngakhale zomwe zafotokozedwa pano zikusonyeza kuti CTCM ndi nsanja yodalirika kwambiri yowonetsera bwino myocardium, njira ya chikhalidweyi ili ndi malire. Cholepheretsa chachikulu cha chikhalidwe cha CTCM ndikuti chimapangitsa kuti pakhale kupanikizika kosalekeza kwamakina pamagawo, zomwe zimalepheretsa kuwunika kwapang'onopang'ono kwa magawo amtima panthawi iliyonse. Kuonjezera apo, chifukwa chazing'ono za zigawo za mtima (7 mm), kukwanitsa kuyesa ntchito ya systolic kunja kwa machitidwe a chikhalidwe pogwiritsa ntchito mphamvu zamagetsi ndizochepa. M'mipukutu yamakono, timagonjetsa pang'ono izi poyesa magetsi owoneka ngati chizindikiro cha ntchito ya contractile. Komabe, kuchepetsa kumeneku kudzafuna ntchito yowonjezereka ndipo kungayankhidwe m'tsogolomu poyambitsa njira zowunikira ntchito ya magawo a mtima mu chikhalidwe, monga mapu a kuwala pogwiritsa ntchito calcium ndi utoto wosamva magetsi. Cholepheretsa china cha CTCM ndikuti mawonekedwe ogwirira ntchito sagwiritsa ntchito kupsinjika kwa thupi (kutsitsa ndikutsitsa). Mu CTCM, kupanikizika kunapangitsidwa motsatira njira zosiyana kuti zibereke 25% kutambasula kwa thupi mu diastole (kutambasula kwathunthu) ndi systole (kutalika kwapakati panthawi ya kukondoweza kwa magetsi) m'magulu akuluakulu kwambiri. Kuchepetsa kumeneku kuyenera kuchotsedwa m'mapangidwe amtsogolo a CTCM ndi kukakamiza kokwanira kwa minofu yamtima kuchokera kumbali zonse ziwiri ndikugwiritsa ntchito maubwenzi enieni a kupanikizika-kuchuluka komwe kumachitika m'zipinda za mtima.
Kukonzanso kopitilira muyeso komwe kumanenedwa mumpukutuwu kumangokhala kutsanzira ma hypertrophic hyperstretch sign. Choncho, chitsanzochi chingathandize pofufuza zizindikiro za hypertrophic zotambasula popanda kufunikira kwa zinthu zamatsenga kapena zamatsenga (zomwe sizilipo m'dongosolo lino). Maphunziro owonjezera akufunika kuti awonjezere kuchuluka kwa CTCM, mwachitsanzo, kugwirizanitsa ndi maselo a chitetezo cha mthupi, kuyendayenda kwa plasma humoral factor, komanso kukhala ndi moyo wokhazikika pamene kugwirizana ndi maselo a neuronal kudzasintha mwayi wowonetsera matenda ndi CTCM.
Nkhumba khumi ndi zitatu zinagwiritsidwa ntchito pa kafukufukuyu. Njira zonse za nyama zidachitidwa motsatira malangizo a mabungwe ndipo zidavomerezedwa ndi University of Louisville Institutional Animal Care and Use Committee. Mng'oma wa aortic unatsekedwa ndipo mtima udaphatikizidwa ndi 1 L ya sterile cardioplegia (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U / mL heparin, pH mpaka 7.4); mitima inasungidwa mu ayezi-ozizira cardioplegic solution mpaka kutumizidwa ku labu pa ayezi omwe nthawi zambiri amakhala <10 min. mitima inasungidwa mu ayezi-ozizira cardioplegic solution mpaka kutumizidwa ku labu pa ayezi omwe nthawi zambiri amakhala <10 min. сердца хранили в ледяном кардиоплегическом растворе до транспортировки pa лабораторию на льду, что обычно занимает <10 мин. mitima inasungidwa mu ayezi-ozizira cardioplegic yankho mpaka zoyendera kwa labotale pa ayezi, amene nthawi zambiri amatenga <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. Держите сердца pa ледяной кардиоплегии до транспортировки pa лабораторию pa льду, обычно <10 мин. Sungani mitima pa ice cardioplegia mpaka mayendedwe opita ku labotale pa ayezi, nthawi zambiri <10 min.
Chipangizo cha CTCM chinapangidwa mu pulogalamu ya SolidWorks computer-aid design (CAD). Zipinda zachikhalidwe, zogawa ndi zipinda zam'mlengalenga zimapangidwa ndi pulasitiki ya CNC clear acrylic. Mphete yobwerera m'mimba ya 7mm imapangidwa ndi polyethylene yapamwamba kwambiri (HDPE) pakatikati ndipo ili ndi poyambira o-ring kuti agwirizane ndi silicone o-ring yomwe imagwiritsidwa ntchito kusindikiza media pansi. Kakhungu kakang'ono ka silika kamalekanitsa chipinda cha chikhalidwe ndi mbale yolekanitsa. Nembanemba ya silikoni ndi laser yodulidwa kuchokera ku 0.02 ″ wandiweyani pepala la silikoni ndipo ili ndi kuuma kwa 35A. Pansi ndi pamwamba pa silicone gaskets ndi laser odulidwa kuchokera 1/16 ″ wandiweyani pepala silikoni ndipo ali ndi kuuma kwa 50A. 316L zomangira zitsulo zosapanga dzimbiri ndi mtedza wa mapiko zimagwiritsidwa ntchito kumangirira chipika ndikupanga chisindikizo chopanda mpweya.
Gulu lodzipatulira losindikizidwa (PCB) lapangidwa kuti liphatikizidwe ndi dongosolo la C-PACE-EM. Zolumikizira zolumikizira makina a swiss pa PCB zimalumikizidwa ndi maelekitirodi a graphite ndi mawaya amkuwa okhala ndi siliva ndi zomangira zamkuwa 0-60 zomangika mu maelekitirodi. Bolodi yosindikizidwa imayikidwa pachikuto cha chosindikizira cha 3D.
Chipangizo cha CTCM chimayang'aniridwa ndi pulogalamu ya pneumatic actuator (PPD) yomwe imapanga kuthamanga kwa kayendedwe ka kayendedwe kake kofanana ndi kayendedwe ka mtima. Pamene kupanikizika mkati mwa chipinda cha mpweya kumawonjezeka, nembanemba ya silicone yosinthika imakula mmwamba, kukakamiza sing'anga pansi pa malo a minofu. Dera la minofu lidzatambasulidwa ndi kutulutsa madzimadzi, kutsanzira kukula kwamtima kwa mtima pa diastole. Pachimake cha kumasuka, kukondoweza kwa magetsi kunagwiritsidwa ntchito kudzera mu ma electrode a graphite, omwe amachepetsa kupanikizika mu chipinda cha mpweya ndikupangitsa kuti zigawo za minofu. Mkati mwa chitolirocho muli valavu ya hemostatic yokhala ndi sensor yokakamiza kuti izindikire kupanikizika kwa mpweya. Kupanikizika komwe kumamveka ndi sensa yothamanga kumagwiritsidwa ntchito kwa wosonkhanitsa deta wolumikizidwa ndi laputopu. Izi zimathandiza kuwunika mosalekeza kupanikizika mkati mwa chipinda cha gasi. Pamene kuthamanga kwakukulu kwa chipinda kunafikiridwa (muyezo 80 mmHg, 140 mmHg OS), chipangizo chotengera deta chinalamulidwa kutumiza chizindikiro ku C-PACE-EM system kuti apange chizindikiro cha biphasic voltage kwa 2 ms, chokhazikitsidwa ku 4 V.
Zigawo za mtima zinapezedwa ndipo zikhalidwe za chikhalidwe m'zitsime za 6 zinachitidwa motere: Tumizani mitima yokolola kuchokera ku chotengera chotengera kupita ku tray yomwe ili ndi kuzizira (4 ° C.) cardioplegia. Kumanzere ventricle analekanitsidwa ndi tsamba wosabala ndi kudula mu zidutswa 1-2 cm3. Minofu imeneyi imamangiriridwa ku zomangira za minofu ndi zomatira za minofu ndikuyikidwa mu bafa yogwedeza ya microtome yomwe ili ndi njira ya Tyrode ndi mpweya wosalekeza (3 g/L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g) . ), 6 mM KCl (0.610) glukosi (0.101M) D-Glucose mM HEPES (2.38 g), 1 mM MgCl2 (1 ml 1 M yankho), 1.8 mM CaCl2 (1.8 ml 1 M yankho), mpaka 1 L ddH2O). Microtome yogwedezeka idakhazikitsidwa kuti idulire magawo 300 µm wandiweyani pafupipafupi 80 Hz, matalikidwe opingasa a 2 mm, ndi kuchuluka kwa 0.03 mm/s. Malo osambiramo a minofu adazunguliridwa ndi ayezi kuti madziwo azikhala ozizira ndipo kutentha kumasungidwa pa 4 ° C. Chotsani zigawo za minofu kuchokera ku bafa la microtome kupita ku bafa yoyatsira yomwe imakhala ndi madzi oundana a Tyrode pa ayezi mpaka magawo okwanira apezeka mbale imodzi yachikhalidwe. Pazikhalidwe za transwell, magawo a minofu adalumikizidwa ku 6 mm wide polyurethane supports ndikuyikidwa mu 6 ml ya optimized medium (199 medium, 1x ITS supplement, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-alkaline ndi 2X antibiotic-antifungal). Kukondoweza kwamagetsi (10 V, pafupipafupi 1.2 Hz) kunagwiritsidwa ntchito ku zigawo za minofu kupyolera mu C-Pace. Pazinthu za TD, T3 yatsopano ndi Dex zidawonjezedwa pa 100 nM ndi 1 μM pakusintha kulikonse. Sing'anga imadzaza ndi okosijeni isanalowe m'malo katatu patsiku. Magawo a minofu adakulitsidwa mu chofungatira pa 37 ° C ndi 5% CO2.
Kwa zikhalidwe za CTCM, magawo a minofu adayikidwa pa chosindikizira chopangidwa ndi 3D mu mbale ya Petri yomwe ili ndi yankho la Tyrode losinthidwa. Chipangizocho chimapangidwa kuti chiwonjezere kukula kwa gawo la mtima ndi 25% ya dera la mphete yothandizira. Izi zimachitidwa kotero kuti zigawo za mtima zisatambasulidwe pambuyo posamutsidwa kuchokera ku njira ya Tyrode kupita ku sing'anga ndi pa diastole. Pogwiritsa ntchito guluu wa histoacrylic, zigawo 300 µm zokhuthala zidakhazikika pa mphete yothandizira 7 mm m'mimba mwake. Mukalumikiza zigawo za minofu ku mphete yothandizira, dulani minyewa yowonjezereka ndikuyikanso zigawo za minyewa mumadzi osambira a Tyrode pa ayezi (4 ° C) mpaka magawo okwanira atakonzedwa pa chipangizo chimodzi. Nthawi yonse yopangira zida zonse sayenera kupitilira maola awiri. Pambuyo pa magawo a minofu ya 6 ataphatikizidwa ku mphete zawo zothandizira, chipangizo cha CTCM chinasonkhanitsidwa. Chipinda cha chikhalidwe cha CTCM chimadzazidwa ndi 21 ml pre-oxygenated medium. Tumizani zigawo za minofu ku chipinda cha chikhalidwe ndikuchotsa mosamala mavuvu aliwonse a mpweya ndi pipette. Gawo la minofu limalowetsedwa mu dzenje ndikukanikizidwa pang'onopang'ono m'malo mwake. Pomaliza, ikani kapu ya elekitirodi pa chipangizo ndi kusamutsa chipangizo chofungatira. Kenako gwirizanitsani CTCM ku chubu cha mpweya ndi dongosolo la C-PACE-EM. The actuator pneumatic imatsegula ndipo valavu ya mpweya imatsegula CTCM. Dongosolo la C-PACE-EM linakonzedwa kuti lipereke 4 V pa 1.2 Hz panthawi ya biphasic pacing kwa 2 ms. Sing'anga idasinthidwa kawiri pa tsiku ndipo ma elekitirodi amasinthidwa kamodzi patsiku kuti apewe kudzikundikira kwa graphite pamagetsi. Ngati kuli kofunikira, zigawo za minofu zimatha kuchotsedwa ku zitsime za chikhalidwe chawo kuti zitulutse thovu lililonse la mpweya lomwe lingakhale litagwera pansi pawo. Pazikhalidwe za chithandizo cha MT, T3/Dex idawonjezedwa mwatsopano ndikusintha kulikonse kwapakati ndi 100 nM T3 ndi 1 μM Dex. Zida za CTCM zidakulitsidwa mu chofungatira pa 37°C ndi 5% CO2.
Kuti apeze njira zotambasula za magawo a mtima, makina apadera a kamera adapangidwa. Kamera ya SLR (Canon Rebel T7i, Canon, Tokyo, Japan) idagwiritsidwa ntchito ndi Navitar Zoom 7000 18-108mm macro lens (Navitar, San Francisco, CA). Kuwona kunkachitika kutentha kwa chipinda pambuyo posintha sing'anga ndi sing'anga yatsopano. Kamera imayikidwa pamtunda wa 51 ° ndipo kanema amajambulidwa pazithunzi za 30 pa sekondi iliyonse. Choyamba, mapulogalamu otsegula (MUSCLEMOTION43) adagwiritsidwa ntchito ndi Image-J kuti ayese kusuntha kwa magawo a mtima. Chigobacho chinapangidwa pogwiritsa ntchito MATLAB (MathWorks, Natick, MA, USA) kuti afotokoze madera okonda kumenya magawo a mtima kuti apewe phokoso. Masks omwe amagawika pamanja amayikidwa pazithunzi zonse motsatizana ndikudutsa ku pulagi ya MUSCLEMOTION. Muscle Motion imagwiritsa ntchito kuchuluka kwa ma pixel mu chimango chilichonse kuti iwonetse kusuntha kwake molingana ndi chimango. Deta inalembedwa, kusefedwa ndi kugwiritsidwa ntchito kuwerengera nthawi yozungulira ndikuyesa kutambasula kwa minofu panthawi ya mtima wamtima. Kanema wojambulidwayo adakonzedwanso pogwiritsa ntchito fyuluta ya digito ya gawo loyamba la zero. Kuti muyese kutambasula kwa minofu (pamwamba-pa-nsonga), kusanthula kwapamwamba kunachitidwa kuti kusiyanitsa pakati pa nsonga ndi nsonga mu chizindikiro chojambulidwa. Kuphatikiza apo, detrending imachitika pogwiritsa ntchito 6th order polynomial kuti athetse kusuntha kwa ma sign. Khodi ya pulogalamu idapangidwa mu MATLAB kuti idziwe kayendedwe ka minofu yapadziko lonse, nthawi yozungulira, nthawi yopumula, ndi nthawi yopumira (Supplementary Program Code 44).
Pofufuza zovuta, pogwiritsa ntchito mavidiyo omwewo omwe adapangidwa kuti ayese kuwongolera makina, tidayang'ana koyamba zithunzi ziwiri zoyimira nsonga zoyenda (zokwera kwambiri (zapamwamba) ndi zotsika (zotsika) molingana ndi pulogalamu ya MUSCLEMOTION. Kenaka tinagawaniza zigawo za minofu ndikugwiritsira ntchito mawonekedwe a shading algorithm ku minofu yogawidwa (Supplementary Fig. 2a). Minofu yogawidwayo inagawidwa m'magulu khumi, ndipo kupanikizika pamtunda uliwonse kunawerengedwa pogwiritsa ntchito equation yotsatirayi: Kupsyinjika = (Sup-Sdown) / Sdown, kumene Sup ndi Sdown ndi mtunda wa mawonekedwe kuchokera pamwamba ndi pansi pamithunzi ya nsalu, motero (Supplementary Fig. .2b).
Magawo a mtima adakhazikitsidwa mu 4% paraformaldehyde kwa maola 48. Minofu yokhazikika idachotsedwa madzi mu 10% ndi 20% sucrose kwa ola limodzi, kenako 30% sucrose usiku wonse. Zigawozo zidaphatikizidwa muzabwino kwambiri zodulira kutentha (OCT compound) ndipo pang'onopang'ono amaundana mumadzi osambira a isopentane / owuma. Sungani midadada yoyika ya OCT pa -80 °C mpaka mutapatukana. Masilayidi adakonzedwa ngati magawo okhala ndi makulidwe a 8 μm.
Kuti muchotse OCT m'magawo amtima, tenthetsani ma slide pa chotenthetsera pa 95 ° C kwa mphindi 5. Onjezani 1 ml PBS pa slide iliyonse ndikuumirira kwa mphindi 30 pa kutentha kwapakati, kenako lowetsani zigawozo poyika 0.1% Triton-X mu PBS kwa mphindi 15 kutentha kwa chipinda. Kuti muteteze ma antibodies osakhala enieni kuti asamangidwe ku chitsanzo, onjezerani 1 ml ya 3% BSA yankho ku slide ndikuumirira kwa ola limodzi pa kutentha kwapakati. BSA idachotsedwa ndipo zithunzizo zidatsukidwa ndi PBS. Chongani chitsanzo chilichonse ndi pensulo. Ma antibodies oyambira (ochepetsedwa 1:200 mu 1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) ndi troponin-T (Thermo Scientific; #MA5-12960) adawonjezedwa kupitilira mphindi 90, kenako ma anti-1% mbewa (Alexaluted 1%) Fluor 488 (Thermo Scientific; #A16079), motsutsana ndi kalulu Alexa Fluor 594 (Thermo Scientific; #T6391) kwa mphindi zina 90 Kutsukidwa katatu ndi PBS Kuti tisiyanitse madontho a chandamale kuchokera kumbuyo, tidagwiritsa ntchito antibody yachiwiri monga chowongolera Pomaliza, DAPIld adayikidwa mu nyukiliya Laboratories) ndikusindikizidwa ndi polishi ya misomali -x magnification) ndi maikulosikopu a Keyence okhala ndi 40x magnification.
WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) pa 5 μg/ml mu PBS idagwiritsidwa ntchito pakudetsa kwa WGA ndikuyika magawo okhazikika kwa mphindi 30 kutentha kwachipinda. Ma slidewo adatsukidwa ndi PBS ndipo Sudan wakuda adawonjezedwa pa slide iliyonse ndikuumirira kwa mphindi 30. Ma slidewo adatsukidwa ndi PBS ndipo sing'anga yoyika vectashield idawonjezedwa. Ma Slide adawonetsedwa pa microscope ya Keyence pakukula kwa 40x.
OCT inachotsedwa ku zitsanzo monga tafotokozera pamwambapa. Mukachotsa OCT, mizani ma slide mu yankho la Bouin usiku wonse. Kenako zithunzizo zimachapidwa ndi madzi osungunuka kwa ola limodzi ndikuyika mu Bibrich aloe acid fuchsin solution kwa mphindi 10. Ndiye zithunzi anali osambitsidwa ndi madzi osungunuka ndi kuikidwa mu njira ya 5% phosphomolybdenum/5% phosphotungstic asidi kwa mphindi 10. Popanda kutsuka, sinthani zithunzizo molunjika mu njira yabuluu ya aniline kwa mphindi 15. Kenako zithunzizo zimatsukidwa ndi madzi osungunuka ndikuyika mu 1% acetic acid solution kwa mphindi ziwiri. Ma slide adawumitsidwa mu 200 N Mowa ndikusamutsidwa ku xylene. Makanema opaka utoto adawonetsedwa pogwiritsa ntchito maikulosikopu a Keyence okhala ndi cholinga cha 10x. Chigawo cha Fibrosis chinayesedwa pogwiritsa ntchito pulogalamu ya Keyence Analyzer.
CyQUANT™ MTT Cell Viability Assay (Invitrogen, Carlsbad, CA), nambala ya catalog V13154, malinga ndi ndondomeko ya opanga ndi zosintha zina. Makamaka, nkhonya ya opaleshoni yokhala ndi mainchesi a 6 mm idagwiritsidwa ntchito kutsimikizira kukula kwa minofu yofananira pakuwunika kwa MTT. Minofu inayikidwa payekhapayekha m'zitsime za mbale ya 12 yomwe ili ndi gawo lapansi la MTT malinga ndi ndondomeko ya wopanga. Zigawozo zimayikidwa pa 37 ° C. kwa maola a 3 ndipo minofu yamoyo imatulutsa gawo la MTT kuti likhale lofiira la formazan. Bwezerani njira ya MTT ndi 1 ml DMSO ndi kuumitsa pa 37 °C kwa mphindi 15 kuti mutenge formazan yofiirira kuchokera kumadera amtima. Zitsanzo zidasungunuka 1:10 mu DMSO mu mbale 96 zomveka bwino zapansi ndi utoto wofiirira woyezedwa pa 570 nm pogwiritsa ntchito Cytation plate reader (BioTek). Kuwerenga kunali kofanana ndi kulemera kwa gawo lililonse la mtima.
Makanema amtundu wamtima adasinthidwa ndi media okhala ndi 1 μCi/ml [5-3H] -glucose (Moravek Biochemicals, Brea, CA, USA) pakuyesa kugwiritsa ntchito shuga monga tafotokozera kale. Pambuyo pa mawola anayi a incubation, onjezerani 100 µl wapakati ku chubu lotseguka la microcentrifuge lomwe lili ndi 100 µl ya 0.2 N HCl. Kenaka chubucho chinayikidwa mu chubu chotsitsimula chomwe chili ndi 500 μl ya dH2O kuti isungunuke [3H]2O kwa maola 72 pa 37°C. Kenaka chotsani chubu cha microcentrifuge ku chubu cha scintillation ndikuwonjezera 10 ml ya scintillation fluid. Kuwerengera kwa scintillation kunachitika pogwiritsa ntchito Tri-Carb 2900TR liquid scintillation analyzer (Packard Bioscience Company, Meriden, CT, USA). Kugwiritsiridwa ntchito kwa shuga ndiyeno kuwerengedwa poganizira [5-3H] -shuga yomwe ili yeniyeni, kusalingana kosakwanira ndi maziko ake, kuchepetsedwa kwa [5-3H] -kuchokera ku glucose wosalembedwa, ndi scintillation counter performance. Zomwe zimasinthidwa zimasinthidwa kukhala magawo ambiri a mtima.
Pambuyo pa homogenization ya minofu ku Trizol, RNA idasiyanitsidwa ndi magawo amtima pogwiritsa ntchito Qiagen miRNeasy Micro Kit #210874 molingana ndi protocol ya wopanga. Kukonzekera kwa library ya RNAsec, kutsata ndi kusanthula deta kunachitika motere:
1 μg ya RNA pachitsanzo chilichonse idagwiritsidwa ntchito ngati zoyambira pokonzekera laibulale ya RNA. Ma library otsatizana adapangidwa pogwiritsa ntchito NEBNext UltraTM RNA Library Preparation Kit for Illumina (NEB, USA) kutsatira malingaliro a wopanga, ndipo ma index a index adawonjezedwa pazotsatira zachitsanzo chilichonse. Mwachidule, mRNA idayeretsedwa ku RNA yonse pogwiritsa ntchito mikanda yamaginito yolumikizidwa ndi oligonucleotides ya poly-T. Kugawikana kumachitika pogwiritsa ntchito ma divalent cations pa kutentha kwakukulu mu NEBNext First Strand Synthesis Reaction Buffer (5X). CDNA yoyamba ya chingwe idapangidwa pogwiritsa ntchito zida zoyambira za hexamer ndi M-MuLV reverse transcriptase (RNase H-). CDNA yachiwiri ya chingwe imapangidwa pogwiritsa ntchito DNA polymerase I ndi RNase H. Zowonjezera zotsalira zimasinthidwa kukhala malekezero osamveka ndi ntchito ya exonuclease/polymerase. Pambuyo pa kuchotsedwa kwa 3 ′ mapeto a DNA fragment, NEBNext Adapter yokhala ndi hairpin loop structure imamangiriridwapo kuti ikonzekere kusakanikirana. Posankha zidutswa za cDNA zautali wokonda 150-200 bp. zidutswa za laibulale zidayeretsedwa pogwiritsa ntchito dongosolo la AMPure XP (Beckman Coulter, Beverly, USA). Kenako, 3 μl USER Enzyme (NEB, USA) yokhala ndi cDNA yosankhidwa kukula yolumikizidwa ndi adaputala idagwiritsidwa ntchito kwa mphindi 15 pa 37 ° C kenako kwa mphindi 5 pa 95 ° C pamaso pa PCR. PCR idachitidwa pogwiritsa ntchito Phusion High-Fidelity DNA polymerase, ma PCR primers, ndi zoyambira za Index (X). Pomaliza, zinthu za PCR zidatsukidwa (AMPure XP system) ndipo mtundu wa library unayesedwa pa Agilent Bioanalyzer 2100 system. Laibulale ya cDNA idasinthidwa pogwiritsa ntchito sequencer ya Novaseq. Mafayilo azithunzi aiwisi ochokera ku Illumina adasinthidwa kukhala zowerengeka zosaphika pogwiritsa ntchito CASAVA Base Calling. Raw Data imasungidwa mumtundu wa FASTQ(fq) womwe uli ndi mawerengedwe owerengera komanso zoyambira zofananira. Sankhani HISAT2 kuti mufanane ndi masanjidwe osankhidwa ndi Sscrofa11.1 genome. Nthawi zambiri, HISAT2 imathandizira ma genomes amtundu uliwonse, kuphatikiza ma genomes akulu kuposa maziko mabiliyoni 4, ndipo zosintha zosasinthika zimayikidwa pamagawo ambiri. Splicing imawerengedwa kuchokera ku data ya RNA Seq imatha kulumikizidwa bwino pogwiritsa ntchito HISAT2, njira yofulumira kwambiri yomwe ilipo, yolondola kapena yolondola kuposa njira ina iliyonse.
Kuchuluka kwa zolembedwa kumawonetsa mwachindunji kuchuluka kwa mawu amtundu. Miyezo ya mawu a jini imawunikidwa ndi kuchuluka kwa zolembedwa (zowerengera zotsatizana) zomwe zimalumikizidwa ndi ma genome kapena ma exons. Kuchuluka kwa zowerengedwa kumayenderana ndi milingo ya jini, kutalika kwa jini, ndi kuya kwa kutsatizana. FPKM (zidutswa pa chikwi chimodzi cha zolembedwa zotsatiridwa pa miliyoni miliyoni) zidawerengedwa ndipo ma P-mawonekedwe osiyanitsa adatsimikiziridwa pogwiritsa ntchito phukusi la DESeq2. Kenako tinawerengera kuchuluka kwa zopezeka zabodza (FDR) pamtengo uliwonse wa P pogwiritsa ntchito njira ya Benjamini-Hochberg9 potengera R-function "p.adjust".
RNA yolekanitsidwa ndi magawo a mtima idasinthidwa kukhala cDNA pamlingo wa 200 ng/μl pogwiritsa ntchito SuperScript IV Vilo Master mix kuchokera ku Thermo (Thermo, cat. no. 11756050). Quantitative RT-PCR inkachitidwa pogwiritsa ntchito Applied Biosystems Endura Plate Microamp 384-well transparent reaction plate (Thermo, cat. no. 4483319) ndi microamp optical adhesive (Thermo, cat. no. 4311971). Kusakaniza kwake kunali 5 µl Taqman Fast Advanced Master mix (Thermo, mphaka # 4444557), 0.5 µl Taqman Primer ndi 3.5 µl H2O wosakaniza pachitsime. Zozungulira zokhazikika za qPCR zidayendetsedwa ndipo mayendedwe a CT adayezedwa pogwiritsa ntchito chida cha Applied Biosystems Quantstudio 5 nthawi yeniyeni ya PCR (384-well module; product # A28135). Zoyambira za Taqman zidagulidwa ku Thermo (GAPDH (Ss03375629_u1), PARP12 (Ss06908795_m1), PKDCC (Ss06903874_m1), CYGB (Ss06900188_m1), RGL1 (Ss06908795_m1) (Ss01009508_mH), GATA4 (Ss03383805_u1), GJA1 (Ss03374839_u1), COL1A2 (Ss03375009_u1 ), COL3A1 (Ss04323794_m1), 084A2mS 5 ACT2m (5 ACT2m) zitsanzo zonse zidasinthidwa kukhala jini yosunga nyumba GAPDH.
Kutulutsidwa kwa media kwa NT-ProBNP kunayesedwa pogwiritsa ntchito zida za NT-ProBNP (nkhumba) (Cat. No. MBS2086979, MyBioSource) molingana ndi protocol ya wopanga. Mwachidule, 250 µl yachitsanzo chilichonse ndi muyezo zidawonjezedwa mowirikiza pachitsime chilichonse. Mukangowonjezera chitsanzo, onjezani 50 µl wa Assay Reagent A pachitsime chilichonse. Pang'onopang'ono gwedezani mbale ndikusindikiza ndi sealant. Kenako mapiritsiwo adakulungidwa pa 37 ° C kwa ola limodzi. Kenaka tenthetsani madziwo ndikutsuka zitsime kanayi ndi 350 µl wa 1X wash solution, ndikumangirira njira yochapira kwa mphindi 1-2 nthawi iliyonse. Kenako onjezani 100 µl wa Assay Reagent B pachitsime chilichonse ndikusindikiza ndi chosindikizira mbale. Piritsi idagwedezeka pang'onopang'ono ndikuyika pa 37 ° C kwa mphindi 30. Thirani madziwo ndikutsuka zitsime kasanu ndi 350 µl wa 1X wash solution. Onjezani 90 µl wa njira yapansi panthaka pa chitsime chilichonse ndikusindikiza mbaleyo. Ikani mbale pa 37 ° C kwa mphindi 10-20. Onjezani 50 µl Stop Solution pachitsime chilichonse. Mbaleyo idayezedwa nthawi yomweyo pogwiritsa ntchito chowerengera cha Cytation (BioTek) chomwe chili pa 450 nm.
Kufufuza kwamphamvu kunachitika kuti asankhe magulu akuluakulu omwe angapereke> 80% mphamvu kuti azindikire 10% kusintha kotheratu kwa parameter ndi 5% Type I error rate. Kufufuza kwamphamvu kunachitika kuti asankhe magulu akuluakulu omwe angapereke> 80% mphamvu kuti azindikire 10% kusintha kotheratu kwa parameter ndi 5% Type I error rate. Zomwe zalembedwa pansipa > 80% zapadziko lonse lapansi 10% zapadziko lonse lapansi 10% частотой ошибок типа I. Kusanthula mphamvu kunachitidwa kuti asankhe magulu akuluakulu omwe angapereke> 80% mphamvu kuti azindikire 10% mtheradi kusintha kwa parameter ndi 5% mtundu wolakwika wa mtundu wa I.进行功效分析以选择将提供> 80％功效以检测参数中10％绝对变化和5％I型错误率的进行功效分析以选择将提供> 80％功效以检测参数中10％绝对变化和5％I型错误率的 Был проведен анализ мощности для выбора размера группы, который обеспечил бы > 80% kuthandizira kuthandizira 10% yapadziko lonse lapansi 5%. Kusanthula mphamvu kunachitika kuti asankhe kukula kwa gulu komwe kungapereke> 80% mphamvu kuti azindikire 10% kusintha kokwanira kwa parameter ndi 5% mtundu wolakwika wa I.Magawo a minofu adasankhidwa mwachisawawa asanayesedwe. Zowunikira zonse zinali zakhungu ndipo zitsanzo zidasinthidwa pokhapokha deta yonse itawunikidwa. Pulogalamu ya GraphPad Prism (San Diego, CA) idagwiritsidwa ntchito kusanthula zonse zowerengera. Paziwerengero zonse, ma p-values amawonedwa ngati ofunika pamtengo <0.05. Paziwerengero zonse, p-values idawonedwa ngati yofunika pamtengo <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Paziwerengero zonse, p-values idawonedwa ngati yofunika pamtengo <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. Paziwerengero zonse, p-values idawonedwa ngati yofunika pamtengo <0.05.Mayeso a t a Wophunzira a michira iwiri adachitidwa pa data ndikuyerekeza 2 kokha. ANOVA yanjira imodzi kapena iwiri idagwiritsidwa ntchito kudziwa kufunikira pakati pamagulu angapo. Pochita mayeso a post hoc, kuwongolera kwa Tukey kudagwiritsidwa ntchito poyerekezera kangapo. Deta ya RNAsec ili ndi malingaliro apadera owerengera powerengera FDR ndi p.adjust monga momwe tafotokozera mu gawo la Njira.
Kuti mumve zambiri pamapangidwe amaphunziro, onani Nature Research Report abstract yolumikizidwa ndi nkhaniyi.
Nthawi yotumiza: Sep-28-2022
