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Pane kudiwa kweyakavimbika in vitro system inokwanisa kunyatso kuburitsa physiological nharaunda yemoyo yekuongorora zvinodhaka.Kuwanikwa kushoma kwemaitiro emoyo wematishu emagariro evanhu kwakakonzera kududzirwa kusina kururama kwemigumisiro yemishonga yemwoyo.Pano, takagadzira cardiac tissue culture model (CTCM) iyo electromechanically inomutsa zvimedu zvemwoyo uye inopinda mumuviri wakatambanudzwa panguva yesystolic uye diastolic zvikamu zvemwoyo.Mushure memazuva gumi nemaviri etsika, nzira iyi yakavandudza zvishoma kushanda kwezvikamu zvemoyo, asi haina kuchengetedza zvizere kutendeseka kwavo kwechimiro.Naizvozvo, mushure mekuongorora mamorekuru madiki, takaona kuti kuwedzera kwe100 nM triiodothyronine (T3) uye 1 μM dexamethasone (Dex) kune yedu yepakati kwakachengetedza microstructure yezvikamu kwemazuva gumi nemaviri.Mukubatana neT3/Dex kurapwa, iyo CTCM system yakachengetedza transcriptal profiles, kugona, metabolic chiitiko, uye kutendeseka kwechimiro padanho rakafanana nemoyo mutsva wemazuva gumi nemaviri.Mukuwedzera, kutambanudza zvakanyanya kwehutano hwemwoyo mutsika kunokonzera hypertrophic cardiac signing, kupa uchapupu hwekukwanisa kweCTCM kutevedzera hypertrophic mamiriro anokonzerwa nemoyo wekutambanudza.Mukupedzisa, CTCM inogona kutevedzera iyo physiology uye pathophysiology yemoyo mutsika nekufamba kwenguva yakareba, ichigonesa yakavimbika kuongororwa kwezvinodhaka.
Pamberi pekutsvagisa kwekiriniki, inovimbika in vitro masisitimu anodiwa anogona kunyatso kubereka nharaunda yehupenyu hwemoyo wemunhu.Masisitimu akadaro anofanirwa kutevedzera akachinjika mekiniki kutambanudza, kurova kwemoyo, uye electrophysiological properties.Mienzaniso yemhuka inowanzoshandiswa senzvimbo yekuongorora yehutano hwemwoyo ine kuvimbika kushoma mukuratidza mhedzisiro yezvinodhaka mumoyo wemunhu1,2.Pakupedzisira, iyo Ideal Cardiac Tissue Culture Experimental Model (CTCM) muenzaniso une hanya zvakanyanya uye wakananga kune akasiyana siyana ekurapa uye emishonga kupindira, kuburitsa nenzira kwayo iyo physiology uye pathophysiology yemoyo wemunhu3.Kusavapo kwehurongwa hwakadai kunoganhurira kuwanikwa kwemishonga mitsva yekutadza kwemoyo4,5 uye zvakonzera kudhaka cardiotoxicity sechikonzero chikuru chekubuda mumusika6.
Mumakore gumi apfuura, zvinodhaka zvisere zvisiri zvemwoyo zvakabviswa kubva mukushandiswa kwekiriniki nekuti zvinokonzeresa nguva yeQT kureba zvichitungamira kune ventricular arrhythmias uye kufa kamwe kamwe7.Nekudaro, pane kuwedzera kuri kuda kweakavimbika preclinical yekuongorora nzira dzekuongorora kushanda kwemoyo uye chepfu.Kushandiswa kwemazuva ano kwevanhu-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CM) mukuongorora zvinodhaka uye kuongororwa kwehuputi kunopa chikamu chekugadzirisa dambudziko iri.Zvisinei, hunhu husina kukura hwehiPS-CMs uye kushayikwa kwehuwandu hwemagetsi ehutano hwemwoyo ndiyo miitiro mikuru yeiyi nzira.Zvidzidzo zvenguva pfupi yapfuura zvakaratidza kuti kudzikisira uku kunogona kukundwa nekushandisa kwekutanga hiPS-CM kuumba moyo wetishu hydrogels nguva pfupi mushure mekutanga kwekuzvibata kwega uye zvishoma nezvishoma kuwedzera kukurudzira kwemagetsi nekufamba kwenguva.Nekudaro, aya hiPS-CM microtissues anoshaya yakakura electrophysiological uye contractile zvimiro zveyakura myocardium.Pamusoro pezvo, matishu emwoyo wemunhu ane chimiro chakaomesesa, chinosanganisira musanganiswa wakasiyana-siyana wemhando dzakasiyana dzemasero, anosanganisira endothelial masero, neurons, uye stromal fibroblasts, yakabatana nemaseti chaiwo eextracellular matrix proteins.Iyi heterogeneity yevasiri-cardiomyocyte populations11,12,13 mumoyo mukuru wemamalia ndicho chipingamupinyi chikuru chekuenzanisa mutsara wemwoyo uchishandisa marudzi emasero ega.Izvi zvipingamupinyi zvakakura zvinosimbisa kukosha kwekugadzira nzira dzekuvandudza myocardial tishu pasi pemamiriro ehupenyu uye pathological.
Zvikamu zvakaonda (300 µm) zvemoyo wemunhu zvakaratidza kuva muenzaniso unovimbisa wemyocardium yemunhu.Iyi nzira inopa mukana kune yakakwana 3D multicellular system yakafanana neyemoyo wenyama tishu.Nekudaro, kusvika muna 2019, kushandiswa kwezvikamu zvemoyo zvine tsika kwakaganhurirwa nehupfupi (24 h) kupona kwetsika.Izvi zvinokonzerwa nehuwandu hwezvinhu zvinosanganisira kushaikwa kwemuviri-mechanicha yakatambanudzwa, iyo air-liquid interface, uye kushandiswa kweiyo midhiya yakapfava isingatsigire zvinodiwa zvenyama yemoyo.Muna 2019, mapoka akati wandei ekutsvagisa airatidza kuti kubatanidza zvinhu zvemagetsi mukati memoyo matishu tsika masisitimu zvinogona kuwedzera hupenyu hwetsika, kunatsiridza kutaura kwemoyo, uye kutevedzera cardiac pathology.Zvidzidzo zviviri zvinoyevedza 17 uye 18 zvinoratidza kuti uniaxial mechanical loading ine mhedzisiro yakanaka pamwoyo phenotype panguva yetsika.Zvisinei, zvidzidzo izvi hazvina kushandisa simba rematatu-dimensional physico-mechanical loading yemwoyo, sezvo zvikamu zvemwoyo zvakatakurwa pamwe chete neyometric tensile forces 17 kana linear auxotonic loading 18.Idzi nzira dzekutambanudza kwenyama dzakakonzera kudzvinyirirwa kwemajini akawanda emwoyo kana kuwandisa kwemajini ane chokuita nemhinduro dzisina kujairika dzakatambanudzwa.Zvikurukuru, Pitoulis et al.19 yakagadzira inochinja yemoyo siketi tsika yekugezera yekuvakazve yemoyo kutenderera uchishandisa simba transducer mhinduro uye tension drive.Kunyangwe iyi sisitimu inobvumira kuti iwedzere kurongeka mu vitro cardiac cycle modeling, kuomarara uye kuderera kweiyo nzira inodzikamisa kushandiswa kweiyo system.Rabhoritari yedu ichangobva kugadzira yakareruka tsika sisitimu ichishandisa magetsi ekusimudzira uye yakagadziridzwa svikiro kuchengetedza kushanda kwezvikamu zveporcine uye moyo wevanhu nyama kusvika kumazuva matanhatu20,21.
Muchinyorwa chazvino, tinotsanangura cardiac tissue culture model (CTCM) tichishandisa zvikamu zveporcine heart iyo inosanganisira humoral cues to recapitulate three-dimensional cardiac physiology uye pathophysiological distension panguva ye cardiac cycle.Iyi CTCM inogona kuwedzera kurongeka kwepre-kiriniki yekufanotaura kwezvinodhaka kusvika padanho risati ramboitwa kuburikidza nekupa inodhura-inoshanda, yepakati-yepakati-kuburikidza cardiac system inotevedzera iyo physiology / pathophysiology yemoyo yemammalian yepre-kiriniki yekuongorora zvinodhaka.
Hemodynamic mechanical signals inoita basa rinokosha mukuchengetedza cardiomyocyte basa mu vitro 22,23,24.Muchinyorwa chemazuva ano, takagadzira CTCM (Mufananidzo 1a) inogona kutevedzera nharaunda yemwoyo yevanhu vakuru kuburikidza nekukurudzira zvose zvemagetsi uye zvemagetsi pamagetsi ehupenyu (1.2 Hz, 72 beats paminiti).Kuti udzivise kuwandisa kwenyama kutambanudza panguva ye diastole, chigadziro chekudhinda che 3D chakashandiswa kuwedzera kukura kwenyama ne25% (Fig. 1b).Kufamba kwemagetsi kwakakonzerwa neC-PACE system yakarongwa kuti itange 100 ms isati yasvika systole ichishandisa data acquisition system kubereka zvizere kutenderera kwemoyo.Iyo tishu tsika sisitimu inoshandisa programmable pneumatic actuator (LB Injiniya, Germany) kutenderera kuwedzera inochinjika silicone membrane kukonzera kuwedzera kwemoyo zvimedu mukamuri yepamusoro.Iyo sisitimu yakabatanidzwa kune yekunze mutsara wemhepo kuburikidza nekumanikidza transducer, izvo zvakaita kuti zvikwanise kunyatsogadzirisa kudzvinyirira (± 1 mmHg) uye nguva (± 1 ms) (Fig. 1c).
a Batanidza chikamu chetishu kune 7 mm yekutsigira mhete, inoratidzwa nebhuruu, mukati metsika yekamuri yechishandiso.Imba yetsika inoparadzaniswa kubva mukamuri yemhepo nekatete flexible silicone membrane.Isa gasket pakati pekamuri yega yega kudzivirira kubuda.Chivharo chechigadzirwa chine graphite electrodes inopa kurudziro yemagetsi.b Schematic inomiririra yemudziyo mukuru wetishu, mhete yekutungamira uye mhete yekutsigira.Zvikamu zvetishu (brown) zvinoiswa pachigadzirwa chakakura kwazvo chine mhete yekutungamira yakaiswa mugero kumucheto kwekunze kwemudziyo.Uchishandisa gwara, nyatsoisa mhete yekutsigira yakavharwa nematishu acrylic adhesive pamusoro pechikamu chetishu yemwoyo.c Girafu rinoratidza nguva yekukurudzira magetsi sekushanda kwemhepo yekamuri yekumanikidza inodzorwa neprogrammable pneumatic actuator (PPD).Chishandiso chekutora data chakashandiswa kuwiriranisa kurudziro yemagetsi uchishandisa ma sensors ekumanikidza.Kana kudzvinyirirwa mukamuri yetsika kunosvika pachikumbaridzo chakaiswa, chiratidzo chepulse chinotumirwa kuC-PACE-EM kukonzeresa kurudziro yemagetsi.d Mufananidzo wemaCTCM mana akaiswa pasherufu yeincubator.Zvishandiso zvina zvakabatana kune imwe PPD kuburikidza nepneumatic circuit, uye ma sensors ekumanikidza anopinzwa muvharuvhu yehemostatic kuti atarise kudzvanywa mumhepo yemhepo.Mudziyo wega wega une zvikamu zvitanhatu zvematishu.
Tichishandisa imwe pneumatic actuator, takakwanisa kudzora 4 CTCM michina, imwe neimwe inogona kubata 6 tishu zvikamu (Fig. 1d).MuCTCM, mhepo yemhepo muimba yemhepo inoshandurwa kuita synchronous pressure muimba yemvura uye inokonzera kuwedzera kwehutano hwemoyo wechidimbu (Mufananidzo 2a uye Supplementary Movie 1).Kuongororwa kwetishu kutambanudza pa80 mm Hg.Art.yakaratidza kutambanudza kwezvikamu zvetishu ne25% (Fig. 2b).Ichi chikamu chakatambanudzwa chakaratidzwa kuti chinoenderana nehupenyu hwesarcomere hurefu hwe2.2-2.3 µm yezvakajairwa chikamu chemoyo contractility17,19,25.Kufamba kwematishu kwakaongororwa uchishandisa tsika kamera zvigadziriso (Supplementary Figure 1).Iyo amplitude uye velocity yekufamba kwenyama (Fig. 2c, d) inopindirana nekutambanudza panguva yemwoyo uye nguva panguva ye systole uye diastole (Fig. 2b).Kutambanudza uye kukurumidza kwenyama yemwoyo panguva yekuputika uye kusununguka kwakaramba kuripo kwemazuva gumi nemaviri mutsika (Fig. 2f).Kuti tiongorore mhedzisiro yemagetsi ekusimudzira pachibvumirano panguva yetsika, takagadzira nzira yekuona kushanduka kwakashanda tichishandisa shading algorithm (Supplementary Fig. 2a, b) uye takakwanisa kusiyanisa pakati pekuremara uye pasina magetsi ekusimudzira.Chikamu chimwechete chemoyo (Fig. 2f).Munharaunda inotakurika yekucheka (R6-9), iyo voltage panguva yekusimudzira magetsi yaive 20% yakakwirira pane kusavapo kwemagetsi ekusimudzira, izvo zvinoratidza mupiro wekukurudzira kwemagetsi kune contractile basa.
Inomiririra inoteedzera yemhepo kamuri yekumanikidza, yemvura yekamuri yekumanikidza, uye zviyero zvekufamba kwetishu zvinosimbisa kuti kudzvanywa kwekamuri kunoshandura kudzvanywa kwekamuri yemvura, zvichikonzera kufamba kunoenderana kwechidimbu chetishu.b Mumiriri anoteedzera maperesenti akatambanudzwa (bhuruu) ematishu zvikamu zvinoenderana nezana kutambanudza (orenji).c Chiyereso chekufamba kwechenga chemoyo chinoenderana nekupimwa kwekukurumidza kwekufamba.(d) Inomiririra trajectories ye cyclic motion (blue line) uye velocity (orenji ine dot line) muchidimbu chemoyo.e Quantification yenguva yekutenderera (n = 19 zvimedu neboka, kubva kune nguruve dzakasiyana), nguva yekuputika (n = 19 zvimedu neboka), nguva yekuzorora (n = 19 zvimedu neboka, kubva kune nguruve dzakasiyana), kufamba kwenyama (n = 25).zvimedu)/boka kubva kunguruve dzakasiyana), peak systolic velocity (n = 24(D0), 25(D12) zvimedu/boka kubva kunguruve dzakasiyana) uye peak relaxation rate (n=24(D0), 25(D12) zvimedu/boka kubva kunguruve dzakasiyana).T-test yeMudzidzi ine miswe miviri haina kuratidza musiyano wakakosha mune chero parameter.f Representative strain analysis traces of tissue sections ne (dzvuku) uye pasina (blue) magetsi ekusimudzira, gumi ematunhu nzvimbo dzematishu zvikamu kubva muchikamu chimwe chete.Iwo epasi mapaneru anoratidza quantification yemusiyano weperesenti mukunetsa muzvikamu zvetishu zvine uye pasina kukurudzirwa kwemagetsi munzvimbo gumi kubva muzvikamu zvakasiyana. (n = 8 zvimedu/boka kubva kunguruve dzakasiyana, Two-tailed Student t-test inoitwa; ****p <0.0001, **p <0.01, *p <0.05). (n = 8 zvimedu/boka kubva kunguruve dzakasiyana, Two-tailed Student t-test inoitwa; ****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 zvikamu/boka kubva kunguruve dzakasiyana, t-test yeMudzidzi ine miswe miviri; ****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 zvikamu/boka, kubva kunguruve dzakasiyana, t-test yeMudzidzi ine miswe miviri; ****p<0.0001, **p<0.01, *p<0.05).Mabhawa ekukanganisa anomiririra zvinoreva ± kutsauka kwakajairwa.
Mune yedu yapfuura static biomimetic heart slice tsika system [20, 21], isu takachengeta kugona, kushanda, uye kurongeka kwemaitiro ezvimedu zvemoyo kwemazuva matanhatu nekushandisa kurudziro yemagetsi uye nekugonesa kuumbwa kwepakati.Zvisinei, mushure memazuva gumi, nhamba idzi dzakadzikira zvakanyanya.Tichataura nezve zvikamu zvakasimudzwa mune yedu yapfuura static biomimetic tsika system 20, 21 control mamiriro (Ctrl) uye isu tichashandisa yedu yaimbove yakagadziridzwa svikiro seMC mamiriro uye tsika pasi penguva imwe chete yemagetsi uye magetsi kukurudzira (CTCM).akadaidzwa .Kutanga, takasarudza kuti kushandiswa kwemagetsi pasina kukurudzirwa kwemagetsi kwakanga kusina kukwana kuchengetedza hutano hwemazuva 6 (Supplementary Fig. 3a, b).Zvinofadza, nekuiswa kwe physio-mechanical uye magetsi ekusimudzira uchishandisa STCM, kubudirira kwezvikamu zvemoyo zvemazuva gumi nemaviri zvakaramba zvakangofanana muzvikamu zvemwoyo mutsva pasi pemamiriro ezvinhu eMS, asi kwete pasi peCtrl mamiriro, sezvinoratidzwa neMTT kuongorora (Fig. 1).3a).Izvi zvinoratidza kuti kukurudzirwa kwemagetsi uye kuenzanisa kwekutenderera kwemwoyo kunogona kuchengetedza zvikamu zvetishu zvichishanda kwekaviri kwenguva yakareba sezvakashumwa mune yedu yapfuura static tsika system.Zvisinei, kuongororwa kwehutano hwehutano hwezvikamu zvetishu ne immunolabeling ye cardiac troponin T uye connexin 43 yakaratidza kuti connexin 43 kutaura kwaive kwakakwirira zvikuru muMC tissues pazuva 12 pane kutonga pazuva rimwe chete.Zvisinei, yunifomu connexin 43 kutaura uye kuumbwa kweZ-disc kwakanga kusina kuchengetedzwa zvakakwana (Fig. 3b).Isu tinoshandisa artificial intelligence (AI) framework kuyera kutendeseka kwetishu26, mufananidzo-wakavakirwa pakadzika wepombi yekudzidza yakavakirwa patroponin-T uye connexin staining43 kuverengera otomatiki kuvimbika kwechimiro uye fluorescence yezvimedu zvemoyo maererano nekusimba kwenzvimbo.Iyi nzira inoshandisa Convolutional Neural Network (CNN) uye yakadzama yekudzidza dhizaini kuverengera nekuvimbika kurongeka kwechimiro chetishu yemoyo mune otomatiki uye isina rusaruro, sezvakatsanangurwa mureferensi.26. MC tissue yakaratidza kuvandudzwa kwemaitiro akafanana nezuva 0 kana ichienzaniswa nezvikamu zvekugadzirisa static.Mukuwedzera, Masson's trichrome staining yakaratidza yakaderera zvakanyanya muzana yefibrosis pasi peMS mamiriro kana ichienzaniswa nekudzora mamiriro pazuva 12 yetsika (Fig. 3c).Kunyange zvazvo CTCM yakawedzera kushanda kwezvikamu zvezvinyama zvemwoyo pazuva 12 kusvika kune imwe nhanho yakafanana neyeyo yemwoyo mutsva, haina kunyatsovandudza kutendeseka kwemaitiro ezvikamu zvemwoyo.
Bhara girafu rinoratidza huwandu hweMTT inoshanda yezvimedu zvemoyo zvitsva (D0) kana tsika yemoyo zvidimbu kwemazuva gumi nemaviri ingave mune static tsika (D12 Ctrl) kana muCTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) zvimedu / boka rinoitwa kubva ku #0 # bvunzo yeD#0 inofananidzwa 0 uye ** p <0.01 zvichienzaniswa neD12 Ctrl). Bhara girafu rinoratidza huwandu hweMTT inoshanda yezvimedu zvemoyo mitsva (D0) kana tsika yemoyo zvidimbu kwemazuva gumi nemaviri ingave mune static tsika (D12 Ctrl) kana muCTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) zvimedu / boka rimwe nzira 0 # # 0 bvunzo inoitwa # # 0 bvunzo kubva kune akasiyana nguruve ##0 D0 uye ** p <0.01 zvichienzaniswa neD12 Ctrl).iyo histogram inoratidza chiyero chekushanda kweMTT mitsva yemoyo zvikamu (D0) kana tsika yezvikamu zvemoyo kwemazuva gumi nemaviri mune chero static tsika (D12 control) kana CTCM (D12 MC) (n = 18 (D0), 15 (D12 control) )), 12 (D12 MC) zvikamu / boka rinoitwa zvakasiyana-siyana kubva kunguruve;####p < 0,0001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). ####p <0.0001 zvichienzaniswa neD0 uye **p <0.01 zvichienzaniswa neD12 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) 18 戇片(D0) 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 18 戇片(D0) 18活力的量化)，來自不同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；与D0 相比，####p <0.0001, Ctrl 0.0001 . a 条形图显示在静态培养(D12 Ctrl) 或CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl) 新鲜心脏切片(D0) 中 中公片(D0) ， D12 MC组，进行单向ANOVA 测试；与D0 相比，####p <0.0001，与D12 Ctrl 相比，**p.)histogram inoratidza quantification yeMTT inobudirira muzvikamu zvemwoyo mutsva (D0) kana zvikamu zvemwoyo zvakagadzirirwa kwemazuva 12 mu static tsika (D12 control) kana CTCM (D12 MC) (n = 18 (D0), 15 (D12 control)), 12 (D12 MC) zvikamu / boka kubva kune zvakasiyana-siyana nguruve bvunzo, imwe-nzira yenguruve;####p < 0,0001 по сравнению с D0, **p < 0,01 по сравнению с D12 Ctrl). ####p <0.0001 zvichienzaniswa neD0, **p <0.01 zvichienzaniswa neD12 Ctrl).b Troponin-T (yakasvibirira), connexin 43 (tsvuku) uye DAPI (bhuruu) muzvikamu zvemwoyo zvitsva (D0) kana zvikamu zvemwoyo zvakasimudzwa pasi pemamiriro ekugadzirisa (Ctrl) kana CTCM mamiriro (MC) kwemazuva gumi nemaviri) yemifananidzo inomiririra immunofluorescence (isina chinhu chikero = 100 µm). Artificial intelligence quantification yemoyo tissue structural integrity (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) zvimedu / boka rimwe nerimwe kubva kune imwe nguruve, imwe-nzira imwe ANOVA bvunzo inoitwa; ####p <0.0001 ichienzaniswa neD0 uye ****p <ichienzaniswa neD010). Artificial intelligence quantification yemoyo tissue structural integrity (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) zvimedu / boka rimwe nerimwe kubva kune nguruve dzakasiyana, nzira imwe chete yeANOVA bvunzo inoitwa; ####p <0.0001 ichienzaniswa neD0 uye ****p <pr <0.000 ichienzaniswa neD010). Количественная оценка структурной целостности сердечной ткани искусственным интеллектом (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) срезов/ ся однофакторный тест ANOVA; ####p <0,0001 по сравнению с D0 и ****p <0,0001 по сравнению с D12 Ctrl). Quantification yehutano hwemagariro ehutano hwemwoyo nehungwaru hwekugadzira (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) zvikamu / mapoka kubva kunguruve dzakasiyana, nzira imwe chete yeANOVA bvunzo yakaitwa; ####p <0.0001 vs. neD0 uye ****00 <10 ichienzaniswa neD0 uye ****02010.人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) zvimedu/boka rimwe nerimwe renguruve, nzira imwe ANOVA bvunzo;##0#0D0D0 **** 0001 dhizaini 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) zvimedu/boka rimwe nerimwe renguruve, nzira imwe ANOVA bvunzo;##0#0D0D010 **** . 001 dhizaini D12 Ctrl 相比）. Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC/зрый) сзрка ней, односторонний тест ANOVA ####p <0,0001 vs. D0 Для сравнения ****p < 0,0001 по сравнению с D12 Ctrl). Artificial intelligence to quant structural integrity of cardiac tissue (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) zvikamu/boka rimwe nerimwe renguruve dzakasiyana, nzira imwe chete ANOVA bvunzo; ####p<0.0001 vs .D0 Kana tichienzanisa 0.0001 . c Inomiririra mifananidzo (kuruboshwe) uye quantification (kurudyi) yezvimedu zvemoyo zvakasvibiswa neMasson's trichrome stain (Scale bare = 500 µm) (n = 10 zvimedu/boka rimwe nerimwe kubva kune imwe nguruve, nzira imwe ANOVA bvunzo inoitwa; ####p <0.0001 kana ichienzaniswa neD00 uye Ctrl 20 ***p). c Inomiririra mifananidzo (kuruboshwe) uye quantification (kurudyi) yezvimedu zvemoyo zvakasvibiswa ne Masson's trichrome stain (Scale bare = 500 µm) (n = 10 zvimedu/boka rimwe nerimwe kubva kunguruve dzakasiyana, nzira imwe chete ANOVA bvunzo inoitwa; <### p <0.0001 zvichienzaniswa neD00 uye D*20). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенных трихромным красителем Масязкба = (n = 10 p. c Inomiririra mifananidzo (kuruboshwe) uye quantification (kurudyi) yezvikamu zvemoyo zvakasvibiswa ne Masson's trichrome stain (isina kuvharwa chikero = 500 µm) (n = 10 zvikamu/boka kubva kunguruve dzakasiyana, nzira imwe chete ANOVA bvunzo yakaitwa; #### p <0 .0001 zvichienzaniswa neD0 uye D00 ***p <D0 uye D0.1 ***p 1). c 用Masson 三色染料染色的心脏切片的代表性图像（左）和量化（右）（裸尺度= 500 1m）尺度= 500 µ ¥ 500组，每组來自不同的猪，进行单向ANOVA 测试；#### p <0.0001 与D0 相比，***p <0.001 与D12 Ctrl 相比。 C 用 mason 三 色 染料 心脏 切片 的 代表性 （左 左） 量化 （右） 裸尺度 裸尺度 裸尺度 裸尺度 裸尺度 裸尺度 裸尺度 裸尺度 裸尺度 左 左） 量化µm） （n = 10 个 切片 组 每 來自 不同 猪 , 进行 单向 Anova 测试；#### p <0.00001 D12 Ctrl dhizaini. c Репрезентативные изображения (слева) и количественный анализ (справа) срезов сердца, окрашенных трихромным красителем Массом =00 = 10 = срезов/группа, каждый от другой свиньи, протестировано с помощью однофакторного дисперсионного анализа ;##0 #p <0,0001ю помощью однофакторного дисперсионного анализа ; Ctrl). c Inomiririra mifananidzo (kuruboshwe) uye kuwanda (kurudyi) kwezvikamu zvemoyo zvakasvibiswa neMasson's trichrome stain (isina chinhu = 500 µm) (n = 10 zvikamu/boka, imwe neimwe kubva kune imwe nguruve, yakaedzwa nekuongororwa kweimwe nzira yekusiyana ;### # p <0.0001 ichienzaniswa neD01,0001 Ctrl ichienzaniswa neD01,*20).Mabhawa ekukanganisa anomiririra zvinoreva ± kutsauka kwakajairwa.
Isu takafungidzira kuti nekuwedzera mamorekuru kune tsika yepakati, cardiomyocyte kutendeseka kunogona kuvandudzwa uye fibrosis kukura kuderedzwa panguva yeCTCM tsika.Isu saka takaongorora mamorekuru madiki tichishandisa static control tsika20,21 nekuda kwehuwandu hudiki hwezvinhu zvinovhiringidza.Dexamethasone (Dex), triiodothyronine (T3), uye SB431542 (SB) dzakasarudzirwa skrini iyi.Aya mamorekuru madiki akamboshandiswa mune hiPSC-CM tsika kukonzeresa maturation ecardiomyocytes nekuwedzera sarcomere kureba, T-tubules, uye conduction velocity.Mukuwedzera, zvose Dex (a glucocorticoid) uye SB zvinozivikanwa kudzvinyirira kuzvimba29,30.Nokudaro, takaedza kana kuiswa kweimwe kana kusanganiswa kwemamorekuru aya maduku kwaizovandudza kutendeseka kwemaitiro ezvikamu zvemwoyo.Pakuongororwa kwekutanga, muyero wekomponi yega yega yakasarudzwa zvichienderana nehuwandu hunowanzo shandiswa mumasero tsika modhi (1 μM Dex27, 100 nM T327, uye 2.5 μM SB31).Mushure memazuva gumi nemaviri etsika, kusanganiswa kweT3 neDex kwakaguma nepamusoro pe cardiomyocyte structural kutendeseka uye zvishoma zvishoma fibrous remodeling (Supplementary Figures 4 uye 5).Pamusoro pezvo, kushandiswa kwekaviri kana kaviri izvi zvakadzika zveT3 neDex zvakaburitsa zvinodzima zvichienzaniswa neyakajairwa kuisa (Supplementary Fig. 6a,b).
Mushure mekuongorora kwekutanga, takaita musoro-ku-musoro kuenzanisa kwe4 tsika mamiriro (Mufananidzo 4a): Ctrl: zvikamu zvemwoyo zvakasimudzwa mumutauro wedu wakambotsanangurwa static uchishandisa nzira yedu yakagadziridzwa;20.21 TD: T3 uye Ctrl s Yakawedzerwa Dex neChitatu;MC: zvikamu zvemoyo zvakasimudzwa muCTCM uchishandisa yedu yaimbove yakagadziridzwa svikiro;uye MT: CTCM ine T3 uye Dex yakawedzerwa kune yepakati.Mushure memazuva e12 ekurima, kushanda kweMS uye MT tissues kwakaramba kwakafanana mumasumbu matsva akaongororwa neMTT assay (Fig. 4b).Sezvineiwo, kuwedzerwa kweT3 neDex kune transwell tsika (TD) hakuna kuita kuti kuve nekunatsiridza kukuru kwekubudirira kuchienzaniswa neCtrl mamiriro, zvichiratidza basa rakakosha rekukurudzira kwemagetsi mukuchengetedza kugona kwezvikamu zvemoyo.
dhizaini rekuyedza dhizaini rinoratidza mana etsika mamiriro anoshandiswa kuongorora mhedzisiro yekukurudzira kwemakanika uye T3 / Dex yekuwedzera pane yepakati kwemazuva gumi nemaviri. b Bhara girafu rinoratidza huwandu hwekuita 12 mazuva ekutumira tsika mune ese 4 tsika mamiriro (Ctrl, TD, MC, uye MT) zvichienzaniswa nemoyo mitsva zvimedu (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD uye D12 MT), 12 (D12 MC) zvimedu / boka rimwechete-00 nguruve. ###p <0.001 zvichienzaniswa neD0 uye **p <0.01 zvichienzaniswa neD12 Ctrl). b Bhara girafu rinoratidza huwandu hwekuita 12 mazuva ekutumira tsika mune ese 4 tsika mamiriro (Ctrl, TD, MC, uye MT) zvichienzaniswa nemoyo mitsva zvimedu (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD uye D12 MT), 12 (D12 MC) zvimedu / boka rimwechete-00 nguruve. ###p <0.001 zvichienzaniswa neD0 uye **p <0.01 zvichienzaniswa neD12 ctrl). b Гистограмма показывает количественную оценку жизнеспособности через 12 дней после культивирования во всех 4 условиях культивирования во всех 4 ю со свежими срезами сердца (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD uye D12 MT), 12 (D12 MC) срезов/группу от разных свиней #0сто#дисто#дисто#дисто#дисто#дисто#дисто#дисто#дисто#дисто#дисто#дисто#дис. ,0001, ###p < 0,001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). b The bar graph inoratidza quantification yekushanda pamazuva e12 post tsika mune zvose 4 tsika mamiriro (kutonga, TD, MC, uye MT) zvichienzaniswa nezvikamu zvemoyo zvitsva (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, uye D12 MT), 12 (D12 MC yakasiyana) # 00 ; 1, ###p <0.001 vs. D0 uye **p <0.01 nekuenzanisa neD12 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 MT MT1,D12 自和2 MC) 切片/组，进行单向ANOVA 测试；####p <0.0001,###p < 0.001 与D0 相比，**p < 0.01 与D12控制。b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC ne MT) по сравнению свежими срезами сердца (D0) (n = 18, D12), TD0 (n = 18, D1) разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA; b Histogram inoratidza zvose 4 tsika mamiriro (kutonga, TD, MC uye MT) zvichienzaniswa nemwoyo mutsva zvikamu (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD uye D12 MT), kubva kune dzakasiyana nguruve 12 (D12 MC) zvikamu / boka, imwe-nzira ANOVA bvunzo; ##0#0 vs 0. 0.01 vs. kutonga D12). c Bhara girafu rinoratidza huwandu hweglucose flux 12 mazuva post tsika mune ese 4 tsika mamiriro (Ctrl, TD, MC, uye MT) zvichienzaniswa nemoyo mutsva zvimedu (D0) (n = 6 zvimedu / boka kubva kune dzakasiyana nguruve, imwe-nzira ANOVA bvunzo inoitwa; ###p <0.001, zvichienzaniswa neD0 uye ***p <0.0 Ctrl 0 ichienzaniswa neD1). c Bhara girafu rinoratidza huwandu hweglucose flux 12 mazuva post tsika mune ese 4 tsika mamiriro (Ctrl, TD, MC, uye MT) zvichienzaniswa nemoyo mutsva zvimedu (D0) (n = 6 zvimedu / boka kubva kune dzakasiyana nguruve, imwe-nzira ANOVA bvunzo inoitwa; ###p <0.001, zvichienzaniswa neD0 uye ***p <0.0 Ctrl 0 ichienzaniswa neD1). Г Г Г Г Г Г Г Г Г Г Г Г Г Г Г Г Г Г Г к Г Г Mt) О О О О О О <0,001 по савнененененению D12 CTRL). c Histogram inoratidza quantification yeglucose flux 12 mazuva mushure-tsika pasi pese 4 tsika mamiriro (kutonga, TD, MC uye MT) zvichienzaniswa nezvikamu zvemoyo zvitsva (D0) (n = 6 zvikamu / boka kubva kune nguruve dzakasiyana, imwe-nzira ANOVA bvunzo yakaitwa; ###p <0.001 ichienzaniswa neD0 uye ***p <0.00 Ctrl <0.00). 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，培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) 相比，培养条件（Ctrl、TD、MC 和MT）与新鲜心脏切片(D0) 6 片/组，來自不同猪，单向执行ANOVA 测试；###p <0.001，与D0 相比，***p <0.001 与D12 Ctrl 相比）. C 条形图 显示 所有 4 种 条件 （ctrl 、 td 、 mc 、 mc 和 mt） 新鲜 心脏 切片 切片 切特城 1的 通量 定量 （n = 6 片/组 ， 來自 猪 , , , , , , , 猪 猪单行向执ANOVA 测试；###p <0.0001 . D12 Ctrl dhizaini. c Гистограмма, показывающая количественную оценку тока глюкозы через 12 дней после культивирования для всех 4 ю со свежими срезами сердца (D0) (n = 6 срезов/группа, от разных свиней, односторонний Были проведены тесты ANOVA; ###p < 0,000 12 (контроль). c Histogram inoratidza quantification yeglucose flux pamazuva e12 post-culture kune zvose 4 tsika mamiriro (kutonga, TD, MC, uye MT) zvichienzaniswa nezvikamu zvemoyo zvitsva (D0) (n = 6 zvikamu / boka, kubva kune nguruve dzakasiyana, unilateral Were ANOVA miedzo yakaitwa, ###p <0.001 ichienzaniswa neD01.d Strain yekuongorora zvirongwa zvezvitsva (bhuruu), zuva 12 MC (girini), uye zuva 12 MT (tsvuku) matishu panzvimbo gumi yedunhu matishu echikamu nzvimbo (n = 4 zvimedu / boka, imwe-nzira ANOVA bvunzo; pakanga pasina musiyano wakakosha pakati pemapoka).e Volcano chirongwa chinoratidza zvakasiyana-siyana majini muzvikamu zvemoyo zvitsva (D0) zvichienzaniswa nezvikamu zvemoyo zvakasimudzwa pasi pemamiriro ezvinhu akasimba (Ctrl) kana pasi peMT mamiriro (MT) kwemazuva 10-12.f Heatmap ye sarcomere genes yezvikamu zvemoyo zvakasimudzwa pasi peimwe neimwe yetsika mamiriro.Mabhawa ekukanganisa anomiririra zvinoreva ± kutsauka kwakajairwa.
Kutsamira kwemetabolic pakuchinja kubva kumafuta acid oxidation kuenda ku glycolysis chiratidzo che cardiomyocyte dedifferentiation.Immature cardiomyocytes inonyanya kushandisa glucose yeATP kugadzira uye ine hypoplastic mitochondria ine mashoma cristae5,32.Glucose kushandiswa kwekuongorora kwakaratidza kuti pasi peMC uye MT mamiriro, kushandiswa kweglucose kwakafanana neiyo muzuva 0 tishu (Mufananidzo 4c).Nekudaro, Ctrl samples yakaratidza kuwedzera kwakanyanya mukushandiswa kweglucose kana ichienzaniswa nematishu matsva.Izvi zvinoratidza kuti kusanganiswa kweCTCM uye T3 / Dex kunowedzera kushanda kwetishu uye kuchengetedza metabolic phenotype ye12-day cultured heart sections.Mukuwedzera, kuongororwa kwekutambudzika kwakaratidza kuti mazinga ehutano anoramba akafanana nemukati mutsva wemoyo kwemazuva 12 pasi peMT uye MS mamiriro (Fig. 4d).
Kuti tiongorore kukanganisa kwese kweCTCM uye T3 / Dex pane yepasi rose transcriptional landscape ye cardiac slice tissue, takaita RNAseq pazvimedu zvemwoyo kubva kune ese mana akasiyana tsika mamiriro (Supplementary Data 1).Sezvineiwo, zvikamu zveMT zvakaratidza kufanana kwepamusoro kunyorwa kune nyowani moyo tishu, ine 16 chete yakaratidzwa zvakasiyana kubva ku13,642 majini.Zvisinei, sezvatakaratidza kare, Ctrl zvimedu zvakaratidzwa 1229 zvakasiyana-siyana zvakaratidzwa majini mushure memazuva 10-12 mutsika (Fig. 4e).Iyi data yakasimbiswa neqRT-PCR yemwoyo uye fibroblast majini (Supplementary Fig. 7a-c).Sezvineiwo, zvikamu zveCtrl zvakaratidza kuderedzwa kwemoyo uye sero cycle gene uye activation yeanopisa gene zvirongwa.Iyi data inoratidza kuti kusiyanisa, iyo inowanzoitika mushure mekugara kwenguva refu, inoderedzwa zvachose pasi pemamiriro eMT (Supplementary Fig. 8a, b).Kunyatsoongorora kwemajini e sarcomere kwakaratidza kuti pasi peMT mamiriro chete ndiwo magene encoding sarcomere (Fig. 4f) uye ion channel (Supplementary Fig. 9) yakachengetedzwa, ichivadzivirira kubva pakudzvinyirira pasi peCtrl, TD, uye MC mamiriro.Idzi data dzinoratidza kuti nekubatanidzwa kwemakanika uye kusekesa kukurudzira (T3 / Dex), iyo yemoyo slice transcriptome inogona kuramba yakafanana neyemoyo mitsva zvidimbu mushure memazuva gumi nemaviri mutsika.
Izvi zvinyorwa zvinyorwa zvinotsigirwa nechokwadi chokuti chimiro chechimiro che cardiomyocytes muzvikamu zvemwoyo zvinonyatsochengetedzwa pasi pemamiriro eMT kwemazuva 12, sezvakaratidzwa neintact uye localized connexin 43 (Fig. 5a).Mukuwedzera, fibrosis muzvikamu zvemwoyo pasi pemamiriro eMT yakaderedzwa zvakanyanya kana ichienzaniswa neCtrl uye yakafanana nezvikamu zvemwoyo mutsva (Fig. 5b).Aya data anoratidza kuti kusanganiswa kwemakanika stimulation uye T3 / Dex kurapwa zvinobudirira kuchengetedza chimiro chemoyo muzvikamu zvemoyo mutsika.
a Representative immunofluorescence mifananidzo ye troponin-T (girinhi), connexin 43 (tsvuku), uye DAPI (bhuruu) muzvikamu zvemoyo zvitsva (D0) kana zvakagadzirirwa kwemazuva gumi nemaviri muzvikamu zvina zvemoyo chikamu chetsika (scale bar = 100 µm).) Artificial intelligence quantification ye heart tissue structural integrity (n = 7 (D0 uye D12 Ctrl), 5 (D12 TD, D12 MC uye D12 MT) zvimedu/boka kubva kunguruve dzakasiyana, nzira imwe chete ANOVA bvunzo inoitwa; <###p <0.0001 kana ichienzaniswa neD000 uye D002. Ctrl). Artificial intelligence quantification ye heart tissue structural integrity (n = 7 (D0 uye D12 Ctrl), 5 (D12 TD, D12 MC uye D12 MT) zvimedu/boka kubva kunguruve dzakasiyana, nzira imwe chete ANOVA bvunzo inoitwa; <### p <0.0001 kana ichienzaniswa neD000 uye D002. Ctrl). Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта (n = 7 (D0 uye D12 Ctrl), 5 (D12 TD, D12 MC/ D12 MT/ гур) ых свиней, проведен однофакторный тест ANOVA; Kuverengera kwechimiro chechimiro chetishu yemoyo uchishandisa hungwaru hwekugadzira (n = 7 (D0 uye D12 Ctrl), 5 (D12 TD, D12 MC uye D12 MT) zvikamu/boka kubva kunguruve dzakasiyana, nzira imwe ANOVA bvunzo yakaitwa; #### p <0.0001 <0.0001 ichienzaniswa neD0 p0 uye 1 **** Ctrl kana ichienzaniswa neD0 p0 uye 1202 D0 **** 5. )对不同猪的心脏组织结构完整性（n = 7（D0 和D12 Ctrl）、5（D12 TD、D12 MC 和D12 MT）切片/滄人）切片/组行单向ANOVA 测试；#### p <0.0001 与D0 和*p <0.05 相比，或****p < 0.0001 与D12 Ctrl 相比）.对 不同 猪 的 心脏 结构 完整性 （n = 7 （d0 和 d12 ctrl)化 进行 单向 单向 单向 测试 ; ########### p <0.0001 与D0 和*p <0.05 相比，或****p <0.0001 与D0 .Quantification yehutano hwemaitiro ehutano hwemwoyo uchishandisa hungwaru hwekugadzira munguruve dzakasiyana (n = 7 (D0 uye D12 Ctrl), 5 (D12 TD, D12 MC uye D12 MT) zvikamu / boka) neine nzira imwe chete yeANOVA bvunzo;#### p < 0,0001 по сравнению с D0 и *p < 0,05 или ****p < 0,0001 по сравнению с D12 Ctrl). #### p <0.0001 zvichienzaniswa neD0 uye *p <0.05 kana ****p <0.0001 zvichienzaniswa neD12 Ctrl). b mifananidzo inomiririra uye huwandu hwezvimedu zvemoyo zvakasvibiswa neMasson's trichrome stain (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, uye D12 MC), 9 (D12 MT) zvimedu/boka kubva kunguruve dzakasiyana, nzira imwe chete 0 # D0p uye 0 # D0p inofananidzwa 0.001, kana ****p <0.0001 zvichienzaniswa neD12 Ctrl). b mifananidzo inomiririra uye huwandu hwezvimedu zvemoyo zvakasvibiswa neMasson's trichrome stain (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, uye D12 MC), 9 (D12 MT) zvimedu/boka kubva kunguruve dzakasiyana, nzira imwe chete 0 # D0p uye 0 # D0p inofananidzwa 0.001, kana ****p <0.0001 zvichienzaniswa neD12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромным красителем Массона (масштабная 10D) = 10D = D10 Ctrl, D12 TD uye D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполняется односторонний тест ANOVA; ####p <0,0001 по D0, *** 1 по сравнению с D12 Ctrl). b mifananidzo inomiririra uye uwandu hwezvikamu zvemoyo zvakasvibiswa neMasson's trichrome stain (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD uye D12 MC), 9 (D12 MT) zvikamu/boka kubva kunguruve dzakasiyana, yakaitwa nenzira imwe chete.#000 uye #DANOVA #001. 1 kana ****p <0.0001 vs. D12 Ctrl). b 用 Masson 三 染料 染色 染色 的 切片 的 的 的 (n = 10 (d0, D12 TD 和 D12 MC) 1, 或 **** p <0.0001 与 D12 CTRL 相比). b 用 Masson 三 染料 的 心脏 心脏 和 (n = 10 (d0 (d0, D12 TD 和 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, 进行组 进行因素 方 差 分析; # # # # p <0.0001 与 d0 相比, *** P <0.001, 或 **** p <0.0001 与 d12 CTRL 相比). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом Массона (масштабная линейка линейка = 500 , D1 = 200, D1 = 1 м. 12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA; b mifananidzo inomiririra uye uwandu hwezvikamu zvemoyo zvakasvibiswa neMasson's trichrome (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD uye D12 MC), 9 (D12 MT) zvikamu kubva kunguruve/boka rakasiyana, imwe nzira yeANOVA; #0.#0 kana D****0, #0. p <0.0001 zvichienzaniswa neD12 Ctrl).Mabhawa ekukanganisa anomiririra zvinoreva ± kutsauka kwakajairwa.
Pakupedzisira, kugona kweCTCM kutevedzera cardiac hypertrophy yakaongororwa nekuwedzera cardiac tissue stretch.MuCTCM, peak air chamber pressure yakawedzera kubva pa80 mmHg kusvika 80 mmHg.Art.(yakajairika kutambanudza) kusvika ku140 mmHg Art.(Mufananidzo 6a).Izvi zvinopindirana ne32% kuwedzera kwekutambanudza (Fig. 6b), iyo yakamboratidzwa seyakaenzana yepesenteji yakatambanudzwa inodiwa kuti zvikamu zvemwoyo zviwane sarcomere urefu hwakafanana nehwo hunoonekwa mu hypertrophy.Kutambanudza uye kukurumidza kwenyama yemwoyo panguva yekuputika uye kusununguka kwakaramba kuripo mumazuva matanhatu etsika (Fig. 6c).Mwoyo wemoyo kubva kuMT mamiriro akaiswa pasi pemaitiro akareruka (MT (Normal)) kana overstretch mamiriro (MT (OS)) kwemazuva matanhatu.Kare mushure memazuva mana mutsika, hypertrophic biomarker NT-ProBNP yakasimudzwa zvakanyanya mukati mepakati pasi peMT (OS) mamiriro ekuenzaniswa neMT (yakajairika) mamiriro (Fig. 7a).Mukuwedzera, mushure memazuva matanhatu ekurima, ukuru hwesero muMT (OS) (Fig. 7b) yakanyanya kuwedzera kana ichienzaniswa nezvikamu zveMT mwoyo (yakajairika).Mukuwedzera, NFATC4 nyukireya translocation yakawedzera zvakanyanya mumasumbu akatambanudzwa (Fig. 7c).Iyi mhedzisiro inoratidza kufambira mberi kwekuvandudzwa kwepathological remodeling mushure me hyperdistension uye inotsigira pfungwa yekuti CTCM mudziyo unogona kushandiswa sepuratifomu yekudzidza kutambanudza-induced cardiac hypertrophy signing.
Inomiririra inoteedzera yemhepo kamuri yekumanikidza, yemvura yekamuri yekumanikidza, uye zviyero zvekufamba kwetishu zvinosimbisa kuti kudzvanywa kwekamuri kunoshandura kudzvanywa kwekamuri yemvura, zvichikonzera kufamba kunoenderana kwechidimbu chetishu.b Representative kutambanudza muzana uye kutambanudza mwero macurves kune anowanzo kutambanudzwa (orenji) uye yakatambanudzwa (yebhuruu) matissue zvikamu.c Bhara girafu rinoratidza nguva yekutenderera (n = 19 zvimedu neboka, kubva kune nguruve dzakasiyana), nguva yekuputika (n = 18-19 zvimedu neboka, kubva kune nguruve dzakasiyana), nguva yekuzorora (n = 19 zvimedu neboka, kubva kune nguruve dzakasiyana)), amplitude yekufamba kwenyama (n = 14 zvimedu / boka, kubva kune nguruve dzakasiyana), peak syrice yakasiyana-siyana / 1 peak syrice = 1 peak syrice = 1, peak syrica = 1, peak syrige (peak syrige) n = 14 (D0), 15 (D6) ) zvikamu / mapoka) kubva kune nguruve dzakasiyana-siyana), t-test yeMudzidzi miviri-tailed yakaratidza kusakosha kwakasiyana mune chero parameter, zvichiratidza kuti maparameter aya anoramba aripo panguva ye6 mazuva etsika ne overvoltage.Mabhawa ekukanganisa anomiririra zvinoreva ± kutsauka kwakajairwa.
a Bar graph quantification yeNT-ProBNP concentration mutsika midhiya kubva kumoyo zvidimbu zvakasimudzwa pasi peMT zvakajairwa kutambanudza (Norm) kana overstretching (OS) mamiriro (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, uye D4 MTOS) zvimedu / boka kubva kune nguruve dzakasiyana, Nzira mbiri ANOVA inofananidzwa neyakajairwa 0 inoitwa. **00. a Bar graph quantification ye NT-ProBNP kutariswa mutsika midhiya kubva kumoyo zvimedu zvakasimudzwa pasi peMT zvakajairwa kutambanudza (Norm) kana overstretching (OS) mamiriro (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, uye D4 MTOS) zvimedu / boka kubva kunguruve dzakasiyana, mbiri-nzira ANOVA inofananidzwa inoitwa.Quantitative histogram yeNT-ProBNP concentration mutsika yepakati kubva pamwoyo zvimedu zvakasimudzwa pasi pemamiriro ezvakajairwa MT kutambanudza (norm) kana overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, uye D4)) MTOS) zvimedu / boka kubva kune nguruve dzakasiyana, maviri-maviri kuongororwa kwekusiyana kunoitwa;**p < 0,01 по сравнению с нормальным растяжением). ** p <0.01 zvichienzaniswa neyakajairika kutambanudza). a 在MT 正常拉伸(Norm) 或过度拉伸(OS) 条件下培养的心脏切片培养基中NT-ProBNP 浓麦序的条彈归 =Nom3 （D2 MTOS、D4 MTNorm 和D4 MTOS）來自不同猪的切片/组，进行双向方差分析；**与正常拉伸相比，**与正常拉伸相比，p . a Quantification ye NT-ProBNP kusungirirwa muzvidimbu zvemwoyo zvakasimudzwa pasi peMT yakajairwa kutambanudza (Norm) kana overstretch (OS) mamiriro (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm neD4 MTOS) kubva kwakasiyana 猪的切片/组，可以双向 yakajairwa p <0.01).histogram Quantification ye NT-ProBNP yakanyanyisa muzvidimbu zvemwoyo zvakasimudzwa pasi pemamiriro ehutano hweMT kutambanudza (norm) kana kudarika (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) uye D4 MTOS) zvimedu / boka kubva kune nguruve dzakasiyana, nzira mbiri dzekuongorora kusiyana;**p < 0,01 по сравнению с нормальным растяжением). ** p <0.01 zvichienzaniswa neyakajairika kutambanudza). b Mumiririri mifananidzo yezvimedu zvemoyo zvakasvibiswa ne troponin-T uye WGA (kuruboshwe) uye sero size quantification (kurudyi) (n = 330 (D6 MTOS), 369 (D6 MTNorm) masero / boka kubva ku10 zvidimbu zvakasiyana kubva kunguruve dzakasiyana, Two-tailed Student t-test inoitwa; 0****001 inofananidzwa; b Mifananidzo inomiririra yezvimedu zvemoyo zvakasvibiswa ne troponin-T uye WGA (kuruboshwe) uye sero size quantification (kurudyi) (n = 330 (D6 MTOS), 369 (D6 MTNorm) masero / boka kubva ku10 zvidimbu zvakasiyana kubva kunguruve dzakasiyana, Two-tailed Student t-test inoitwa; 0****00 t-test inoitwa. b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественного определения размера клеток (6), 6D размера клеток (6) MTNorm) клеток/группу из 10 разных срезов от разных свиней, два- проводится хвостовой t-критерий Стьюдента; ****p <0,00001 проводится хвостовой t-критерий Стьюдента; b Mifananidzo inomiririra yezvikamu zvemoyo zvakasvibiswa ne troponin-T uye AZP (kuruboshwe) uye sero size quantification (kurudyi) (n = 330 (D6 MTOS), 369 (D6 MTNorm) masero / boka kubva kune zvikamu zve 10 zvakasiyana kubva kune nguruve dzakasiyana, maviri-tailed Mudzidzi t-test 0 yakaenzaniswa ne0. b 用肌钙蛋白-T 和WGA（左）和细胞大小量化（右)个不同切片的369（D6 MTNorm）细胞/组，两进行有尾学生t 检验；与正常拉伸相比，****p <0.00. b Inomiririra mifananidzo yezvimedu zvemoyo zvakasvibiswa ne calcarein-T uye WGA (kuruboshwe) uye sero saizi (kurudyi) (n = 330 (D6 MTOS), 369 kubva ku10 zvimedu zvakasiyana (D6 MTNorm)) Masero/组，两方法有尾学生00 akatambanudzwa. b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗП (слева) и количественная оценка размера клеток (0D69 = MT69) rm) из 10 различных срезов от разных свиней Клетки/группа, двусторонние критерий Стьюдента; ****p < 0,0001 по сравнению с номнимальные). b Mumiririri mifananidzo yezvikamu zvemoyo zvakasvibiswa ne troponin-T uye AZP (kuruboshwe) uye quantification yehukuru hwesero (kurudyi) (n = 330 (D6 MTOS), 369 (D6 MTNorm) kubva ku10 zvikamu zvakasiyana kubva kune nguruve dzakasiyana) Masero / boka, maviri-tailed criterion Student's t;****p <0.0001 zvichienzaniswa neyakajairwa kuomarara). c Mumiririri mifananidzo yezuva 0 uye zuva 6 MTOS mwoyo zvimedu immunolabeled nokuda troponin-T uye NFATC4 uye quantification of translocation yeNFATC4 kune nuclei yeCMs (n = 4 (D0), 3 (D6 MTOS) zvimedu / boka kubva kune nguruve dzakasiyana-siyana, Two-tailed 0 Mudzidzi . *p <p. c Mumiririri mifananidzo yezuva 0 uye zuva 6 MTOS mwoyo zvimedu immunolabeled nokuda troponin-T uye NFATC4 uye quantification of translocation yeNFATC4 kune nuclei yeCMs (n = 4 (D0), 3 (D6 MTOS) zvimedu / boka kubva kune nguruve dzakasiyana-siyana , Two-tasted 0 Mudzidzi. c Репрезентативные изображения для срезов сердца 0 и 6 дней MTOS, иммуномеченых для тропонина-Т и NFATC4, и количественная оценская зных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней , выполняется двусторонний t-критерий Стьюдента, *p. c Mumiririri mifananidzo yezvikamu zvemoyo pa 0 uye 6 mazuva MTOS, immunolabeled ye troponin-T uye NFATC4, uye quantification yeNFATC4 translocation mu nucleus ye cavernous cells (n = 4 (D0), 3 (D6 MTOS) zvimedu / boka kubva kune nguruve dzakasiyana) dzakaitwa-t-tailed Students';*p <0.05). c 用于肌钙蛋白-T 和NFATC4 免疫标记的第0 天和第6 天MTOS 心脏切片的代表性图像，以及來自第TC-CM4的量化（n = 4 (D0)、3 (D6 MTOS) 切片/组, 进行双尾学生t 检验；*p <0.05). c Inomiririra mifananidzo ye calcanin-T uye NFATC4 immunolabeling 第0天和第6天MTOS moyo zvimedu, uye NFATC4 kubva kwakasiyana NFATC4 易位至CM cell nucleus的quantity化 (n = 4 (D0), 3 (D6 MTOS) 其兄生et 电影；*p <0.05). c Репрезентативные изображения срезов сердца MTOS на 0 и 6 день для иммуномаркировки тропонином-Т и NFATC4 и количественная оценской TCFAC зных свиней (n = 4 (D0), 3 (D6 MTOS) срез/группа, два- хвостатый t-критерий Стьюдента; *p < 0,05). c Inomiririra mifananidzo yeMTOS heart slices pazuva 0 uye 6 ye troponin-T uye NFATC4 immunolabeling uye quantification yeNFATC4 translocation munyucleus yeCM kubva kune nguruve dzakasiyana (n = 4 (D0), 3 (D6 MTOS) zvimedu / boka, maviri-tailed t -0 chirevo < Mudzidzi <0 .Mabhawa ekukanganisa anomiririra kureva ± kutsauka kwakajairwa.
Tsvagiridzo yemoyo yemoyo inoda mamodhiyamu anoburitsa nenzira kwayo nharaunda yemoyo.Muchidzidzo ichi, chigadzirwa cheCTCM chakagadzirwa uye chinoratidzwa chinogona kukurudzira zvikamu zvemoyo zvakanyanya.Iyo CTCM system inosanganisira physiologically synchronized electromechanical stimulation uye T3 uye Dex fluid hupfumi.Apo zvikamu zvemwoyo yenguruve zvakaratidzwa kune izvi zvinhu, kushanda kwavo, kutendeseka kwechimiro, kushandiswa kwemagetsi, uye kutaura kwekunyora kwakaramba kwakafanana nemukati mutsva wemwoyo mushure memazuva gumi nemaviri etsika.Mukuwedzera, kunyanya kutambanudza kwenyama yemwoyo kunogona kukonzera hypertrophy yemoyo inokonzerwa ne hyperextension.Pakazere, mhedzisiro iyi inotsigira basa rakakosha remamiriro ehunhu hwetsika mukuchengetedza yakajairika moyo phenotype uye nekupa chikuva chekuongorora zvinodhaka.
Zvinhu zvakawanda zvinobatsira pakugadzira nharaunda yakakwana yekushanda uye kupona kwe cardiomyocytes.Zvinonyanya kuoneka zvezvinhu izvi zvine chekuita ne (1) intercellular interactions, (2) electromechanical stimulation, (3) humoral factors, uye (4) metabolic substrates.Physiological cell-to-cell kudyidzana kunoda akaomesesa matatu-dimensional network emhando dzemasero akawanda anotsigirwa neextracellular matrix.Kudyidzana kwemaserura kwakaoma kudaro kwakaoma kuvaka patsva mu vitro ne-co-tsika yemhando yega yega maseru, asi inogona kuwanikwa nyore nyore uchishandisa organotypic chimiro chemoyo zvikamu.
Mechanical stretch uye magetsi ekusimudzira cardiomyocytes zvakakosha pakuchengetedza cardiac phenotype33,34,35.Nepo kusimudzirwa kwemichina kwave kushandiswa zvakanyanya kune hiPSC-CM mamiriro uye maturation, akati wandei akanakisa zvidzidzo achangobva kuyedza kusimudzira emoyo zvimedu mutsika vachishandisa uniaxial kurodha.Izvi zvidzidzo zvinoratidza kuti 2D uniaxial mechanical loading ine mhedzisiro yakanaka pane phenotype yemoyo panguva yetsika.Muzvidzidzo izvi, zvikamu zvemoyo zvingave zvakatakurwa neisometric tensile forces17, linear auxotonic loading18, kana kutenderera kwemwoyo kwakagadzirwazve uchishandisa simba transducer mhinduro uye tension drives.Zvisinei, nzira idzi dzinoshandisa uniaxial tissue kutambanudza pasina kugadzirisa kwezvakatipoteredza, zvichiita kuti kudzvinyirirwa kwemajini akawanda emwoyo kana kuwandisa kwemajini ane chokuita nemhinduro dzisina kujairika dzakatambanudzwa.CTCM inotsanangurwa pano inopa 3D electromechanical stimulus iyo inotevedzera yakasikwa cardiac cycle maererano nenguva yekutenderera uye physiological stretch (25% kutambanudza, 40% systole, 60% diastole, uye 72 beats paminiti).Kunyange zvazvo iyi mitatu-dimensional mechanical stimulation chete haina kukwana kuchengetedza kuvimbika kwenyama, musanganiswa wekuseka uye mechanical stimulation uchishandisa T3 / Dex inodiwa kuchengetedza zvakakwana kushanda kwenyama, kushanda, uye kuvimbika.
Humoral zvinhu zvinoita basa rakakosha mukugadzirisa moyo wevakuru phenotype.Izvi zvakasimbiswa muzvidzidzo zveHiPS-CM umo T3 neDex zvakawedzerwa kune tsika midhiya kuti ikurumidze kukura kwesero.T3 inogona kukanganisa kutakurwa kweamino acids, shuga uye calcium musero membranes36.Mukuwedzera, T3 inokurudzira MHC-α kutaura uye MHC-β kuderera, kukurudzira kuumbwa kwekukurumidza twitch myofibrils mune yakakura cardiomyocytes kana ichienzaniswa neanononoka twitch myofibrils mu fetal CM.T3 inoshaya mu hypothyroid varwere inoguma nekurasikirwa kwemyofibrillar bands uye kuderedzwa kwehuwandu hwekukura kwenzwi37.Dex inobata pane glucocorticoid receptors uye yakaratidzwa kuwedzera myocardial contractility mumoyo yakasarudzika yakasarudzika;38 kuvandudzwa uku kunofungidzirwa kuti kune chekuita nemhedzisiro ye calcium deposit-driven entry (SOCE) 39,40.Uye zvakare, Dex inosunga kune yayo mareceptor, ichikonzera yakakura intracellular mhinduro inodzvanya immune function uye kuzvimba30.
Mhedzisiro yedu inoratidza kuti kurudziro yemuviri (MS) yakavandudza hunhu hwese hwekuita kana ichienzaniswa neCtrl, asi yakatadza kuchengetedza kugona, kuvimbika kwechimiro, uye kutaura kwemoyo kwemazuva gumi nemaviri mutsika.Kuenzaniswa neCtrl, kuwedzera kweT3 uye Dex kune CTCM (MT) tsika dzakavandudza kushanda uye dzakachengetedza zvakafanana zvinyorwa zvinyorwa, kutendeseka kwechimiro, uye chiitiko chemetabolism nemoyo mutsva kwemazuva gumi nemaviri.Mukuwedzera, nekudzora dhigirii yetishu yakatambanudzwa, muenzaniso we hyperextension-induced cardiac hypertrophy yakagadzirwa uchishandisa STCM, ichiratidza kushandiswa kwemaitiro e STCM.Zvinofanira kuonekwa kuti kunyange zvazvo kugadziriswa kwemwoyo uye fibrosis kunowanzobatanidza nhengo dzakasimba dzine masero anotenderera anogona kupa ma cytokines akakodzera pamwe ne phagocytosis nezvimwe zvinhu zvinogadzirisa, zvikamu zvemoyo zvinogona kuramba zvichitevedzera fibrotic process mukupindura kushushikana uye kushungurudzika.mune myofibroblasts.Izvi zvakamboongororwa mune iyi cardiac slice modhi.Zvinofanira kucherechedzwa kuti CTCM parameters inogona kugadziridzwa nekuchinja kumanikidza / magetsi amplitude uye frequency kutevedzera mamiriro mazhinji akadai se tachycardia, bradycardia, uye mechanical circulatory support (mechanical unloaded heart).Izvi zvinoita kuti sisitimu ive yepakati nepakati yekuongorora zvinodhaka.Kugona kweCTCM kuenzanisira overexertion-induced cardiac hypertrophy inovhura nzira yekuyedza iyi sisitimu yekurapa kwemunhu.Mukupedzisa, chidzidzo chemazuva ano chinoratidza kuti mechanical stretch and humoral stimulation yakakosha pakuchengetedza tsika dzezvikamu zvemwoyo.
Kunyangwe iyo data yakapihwa pano ichiratidza kuti CTCM ipuratifomu inovimbisa kwazvo yekuenzanisira intact myocardium, iyi tsika yetsika ine zvimwe zvinogumira.Mhedzisiro huru yeCTCM tsika ndeyekuti inoisa inoenderera ine simba mechani kushushikana pane zvimedu, izvo zvinodzivirira kugona kunyatso tarisa cardiac slice contractions panguva yega yega kutenderera.Pamusoro pezvo, nekuda kwehukuru hudiki hwezvikamu zvemoyo (7 mm), kugona kuongorora systolic basa kunze kwemaitiro etsika uchishandisa echinyakare simba sensors inogumira.Muchinyorwa chazvino, isu tinokunda chikamu ichi chinogumira nekuongorora optical voltage sechiratidzo chekushanda kwechibvumirano.Zvakadaro, kudzikisira uku kunoda rimwe basa uye kunogona kugadziriswa mune ramangwana nekuunza nzira dzekutarisa kwemaziso kwekushanda kwezvimedu zvemoyo mutsika, senge optical mepu uchishandisa calcium uye voltage-sensitive dhayi.Chimwe chinogumira cheCTCM ndechekuti modhi yekushanda haishandisi kushushikana kwemuviri (preload uye afterload).MuCTCM, kudzvinyirirwa kwakakonzerwa nenzira dzakasiyana kuti ibereke 25% physiological stretch in diastole (yakazara yakatambanudzwa) uye systole (kureba kwechibvumirano panguva yekusimudzira magetsi) mumasumbu makuru kwazvo.Ichi chinogumira chinofanira kubviswa mune ramangwana CTCM magadzirirwo nekumanikidza kwakakwana pamatishu emwoyo kubva kumativi ese uye nekushandisa iyo chaiyo yekumanikidza-huwandu hukama hunoitika mumakamuri emoyo.
Iko kuwedzeredza-kukonzeresa kugadziridza kwakataurwa mugwaro iri kunogumira pakutevedzera masaini ehypertrophic hyperstretch.Nekudaro, iyi modhi inogona kubatsira mukudzidza kwekutambanudza-induced hypertrophic kusaina pasina kudikanwa kwehumoral kana neural factor (iyo isipo mune ino system).Zvimwe zvidzidzo zvinodiwa kuti kuwedzere kuwanda kweCTCM, semuenzaniso, kushamwaridzana nemasero ekudzivirira muviri, kutenderera plasma humoral factor, uye innervation apo kushamwaridzana nemasero neuronal kuchavandudza mikana yechirwere chekufananidzira neCTCM.
Nguruve gumi nenhatu dzakashandiswa muchidzidzo ichi.Nzira dzose dzemhuka dzakaitwa maererano nemitemo yemasangano uye dzakabvumirwa neYunivhesiti yeLouisville Institutional Animal Care uye Komiti Yekushandisa.Iyo aortic arch yakasungirirwa uye moyo waive wakasanganiswa ne1 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 kusvika 7.4); mwoyo yakachengetedzwa muchando-inotonhora cardioplegic solution kusvika yatakurwa kuenda ku lab paaizi iyo inowanzo <10 min. mwoyo yakachengetedzwa muchando-inotonhora cardioplegic solution kusvika yatakurwa kuenda ku lab paaizi iyo inowanzo <10 min. Сердца хранили в ледяном кардиоплегическом растворе до транспортировки в лабораторию на льду, что обычно занимает <10 мин. mwoyo yakachengetwa muchando-inotonhora cardioplegic solution kusvikira kutakura kuenda ku laboratori pachando, iyo inowanzotora <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 в ледяной кардиоплегии до транспортировки в лабораторию на льду, обычно <10 мин. Chengetedza mwoyo pa ice cardioplegia kusvikira kutakura kuenda ku laboratori pachando, kazhinji <10 min.
Iyo CTCM mudziyo wakagadzirwa muSolidWorks komputa-inobatsira dhizaini (CAD) software.Dzimba dzetsika, kupatsanura uye makamuri emhepo akagadzirwa neCNC yakajeka acrylic plastiki.Iyo 7mm dhayamita yekudzosera mhete yakagadzirwa nepamusoro density polyethylene (HDPE) pakati uye ine o-ring groove yekugadzika silicone o-ring inoshandiswa kuvharira midhiya pasi.Sirica membrane yakaonda inoparadzanisa kamuri yetsika kubva mundiro yekuparadzanisa.Iyo silicone membrane ndeye laser yakachekwa kubva 0.02 ″ gobvu silicone sheet uye ine kuoma kwe35A.Pazasi nepamusoro silicone gaskets laser akachekwa kubva 1/16 ″ gobvu silicone sheet uye ane kuoma kwe50A.316L Stainless simbi screws uye mapapiro nzungu anoshandiswa kusungira bhuroko uye kugadzira chisimbiso chisina mhepo.
Yakazvipira yakadhindwa yedunhu bhodhi (PCB) yakagadzirirwa kubatanidzwa neiyo C-PACE-EM system.Iyo swiss machine connector sockets paPCB yakabatana negraphite electrode nesirivha-plated copper waya uye bhuronzi 0-60 screws akaiswa muma electrode.Bhodhi redunhu rakadhindwa rinoiswa mubutiro reprinta ye3D.
Iyo CTCM mudziyo inodzorwa neprogrammable pneumatic actuator (PPD) inogadzira inodzorwa kutenderera kwemhepo yakafanana neyemoyo kutenderera.Sezvo kudzvanywa mukati mekamuri yemhepo kunowedzera, iyo inochinjika silicone membrane inowedzera kumusoro, ichimanikidza yepakati pasi petishu saiti.Iyo nharaunda yetishu inozotambanudzwa nekudzingwa kwemvura, kutevedzera kuwedzera kwehupenyu hwemoyo panguva ye diastole.Pamusoro pekuzorora, kukurudzirwa kwemagetsi kwakashandiswa kuburikidza ne graphite electrodes, iyo yakaderedza kudzvinyirira mukamuri yemhepo uye yakakonzera kuputika kwezvikamu zvetishu.Mukati mepombi mune hemostatic valve ine pressure sensor kuti ione kumanikidzwa kwemhepo.Kudzvinyirirwa kunonzwiwa nepressure sensor kunoiswa kune anounganidza data akabatana nelaptop.Izvi zvinobvumira kuenderera mberi kwekutarisa kwekumanikidzwa mukati mekamuri yegesi.Pakasvikwa iyo yakanyanya kudzvanywa yekamuri (yakajairwa 80 mmHg, 140 mmHg OS), mudziyo wekutora data wakarairwa kutumira chiratidzo kuC-PACE-EM system kugadzira biphasic voltage siginecha ye2 ms, yakaiswa ku4 V.
Zvikamu zvemwoyo zvakawanikwa uye mamiriro etsika mumatsime e6 akaitwa sezvinotevera: Tumira mwoyo yakakohwa kubva mumudziyo wekuendesa kune tireyi ine kutonhora (4 ° C.) cardioplegia.Iyo yeruboshwe ventricle yakaparadzaniswa nebeche isina mbereko uye yakachekwa kuita zvidimbu zve 1-2 cm3.Aya matishu akasungirirwa pamatishu anotsigira ane adhesive yetishu uye akaiswa mu vibrating microtome tissue bath ine Tyrode's solution uye inoramba iine oxygenated (3 g/L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g) . ), 6 mM KCl (0.1 mPES g D) 0.401M, GM 6 DM DM DM DM 6. (2.38 g), 1 mM MgCl2 (1 ml 1 M mhinduro), 1.8 mM CaCl2 ( 1.8 ml 1 M mhinduro), kusvika ku1 L ddH2O).Iyo microtome inozunguza yakaiswa kuti icheke 300 µm gobvu zvimedu pane frequency ye80 Hz, yakachinjika vibration amplitude ye2 mm, uye chiyero chepamberi che0.03 mm/s.Iyo yekugezera yematishu yakakomberedzwa nechando kuitira kuti mushonga wacho utonhore uye tembiricha yaichengetwa iri pa4°C.Shandura zvikamu zvematishu kubva mubhavhu rekugezera remicrotome kuenda kubhavhu rekunyungudutsira rinenge richigara rakaiswa okisijeni Tyrode mhinduro paaizi kudzamara zvikamu zvakakwana zvawanikwa kune imwe tsika ndiro.Kune transwell tsika, matishu zvikamu zvakanamirwa kune isingabereki 6 mm yakafara polyurethane inotsigira uye yakaiswa mu6 ml ye optimized medium (199 yepakati, 1x ITS supplement, 10% FBS, 5 ng/ml VEGF, 10 ng/ml FGF-alkaline uye 2X antibiotic-antifungal).Kukurudzira kwemagetsi (10 V, frequency 1.2 Hz) yakashandiswa kune zvikamu zvetishu kuburikidza neC-Pace.Kune mamiriro eTD, nyowani T3 neDex yakawedzerwa pa100 nM uye 1 μM pane yega yega shanduko.Iyo yepakati inozadzwa neokisijeni isati yatsiviwa katatu pazuva.Zvikamu zvenyama zvakagadzirirwa mu incubator pa 37 ° C uye 5% CO2.
Kune tsika dzeCTCM, zvikamu zvematishu zvakaiswa pane yakasarudzika-yakagadzirwa 3D printer muPetri dish ine yakagadziridzwa mhinduro yeTyrode.Chishandiso chacho chakagadzirirwa kuwedzera saizi yechidimbu chemoyo ne25% yenzvimbo yemhete yekutsigira.Izvi zvinoitirwa kuti zvikamu zvemoyo zvisatambanuke mushure mekutamiswa kubva mugadziriso weTyrode kuenda kupakati uye panguva yediastole.Kushandisa histoacrylic glue, zvikamu 300 µm gobvu zvakaiswa paringi yekutsigira 7 mm muhupamhi.Mushure mekubatanidza zvikamu zvetishu kune mhete yekutsigira, cheka zvikamu zvenyama zvakawandisa uye woisa zvikamu zvakabatanidzwa zvakare mubhavhu reTyrode solution pachando (4 ° C) kusvikira zvikamu zvakakwana zvagadzirirwa mudziyo mumwe.Iyo yakazara yekugadzirisa nguva yemidziyo yese haifanirwe kudarika maawa maviri.Mushure mokunge 6 matishu zvikamu zvakabatanidzwa kune zvindori zvavo zvekutsigira, chigadzirwa cheCTCM chakaunganidzwa.Kamuri yetsika yeCTCM isati yazadzwa ne21 ml pre-oxygenated medium.Tumira zvikamu zvenyama kukamuri yetsika uye nyatsobvisa chero mabhubhu emhepo ane pipette.Chikamu chetishu chinobva chatungamirwa kupinda mugomba uye nekudzvanywa zvinyoro nyoro munzvimbo.Pakupedzisira, isa chivharo che electrode pachigadziro uye woendesa chigadziro kune incubator.Wobva wabatanidza iyo CTCM kune yemhepo chubhu uye iyo C-PACE-EM system.Iyo pneumatic actuator inovhura uye vheji yemhepo inovhura iyo CTCM.Iyo C-PACE-EM sisitimu yakagadziridzwa kuburitsa 4 V pa1.2 Hz panguva yebiphasic pacing ye2 ms.Iyo svikiro yakachinjwa kaviri pazuva uye ma electrode aichinjwa kamwe chete pazuva kudzivirira kuunganidzwa kwegraphite pamagetsi.Kana zvichidikanwa, zvikamu zvenyama zvinogona kubviswa kubva mumatsime emagariro avo kuti adzinge chero mabhuru emhepo angave akawira pasi pawo.Pamamiriro ekurapa eMT, T3/Dex yakawedzerwa nyowani neshanduko yega yega yepakati ne100 nM T3 uye 1 μM Dex.Midziyo yeCTCM yakagadzirwa muincubator pa37°C uye 5% CO2.
Kuti vawane nzira dzakatambanudzwa dzezvidimbu zvemwoyo, imwe yakakosha kamera system yakagadzirwa.Kamera yeSLR (Canon Rebel T7i, Canon, Tokyo, Japan) yakashandiswa neNavitar Zoom 7000 18-108mm macro lens (Navitar, San Francisco, CA).Visualization yakaitwa pakamuri tembiricha mushure mekutsiva yepakati nepakati nyowani.Iyo kamera inomira pa51 ° angle uye vhidhiyo inorekodhwa pa30 mafuremu pasekondi.Chekutanga, yakavhurika sosi software (MUSCLEMOTION43) yakashandiswa neImage-J kuyera kufamba kwezvimedu zvemoyo.Iyo mask yakagadzirwa uchishandisa MATLAB (MathWorks, Natick, MA, USA) kutsanangura matunhu anofarira kurova zvidimbu zvemoyo kudzivirira ruzha.Masiki akakamurwa nemaoko anoiswa kumifananidzo yese mukutevedzana kwefuremu yobva yapfuudzwa kuMUSCLEMOTION plug-in.Muscle Motion inoshandisa avhareji kusimba kwemapikseli mune yega furemu kuyera mafambiro ayo zvichienderana nereferensi furemu.Dhata dzakarekodhwa, dzakasefa uye dzakashandiswa kuyera nguva yekutenderera uye kuongorora tishu dzakatambanudzwa panguva yekutenderera kwemoyo.Vhidhiyo yakarekodhwa yakagadziridzwa mushure mekushandisa yekutanga-odha zero-chikamu chedhijitari sefa.Kuyera matishu akatambanudzwa (peak-to-peak), peak-to-peak ongororo yakaitwa kuti isiyanise pakati penhongonya uye zvimwiro muchiratidzo chakanyorwa.Uye zvakare, detrending inoitwa uchishandisa 6th odha polynomial kubvisa chiratidzo chekudonha.Chirongwa chekodhi chakagadzirwa muMATLAB kuti itarise mafambiro epasi rose, nguva yekutenderera, nguva yekuzorora, uye nguva yekumisikidza (Supplementary Program Code 44).
Kuongorora kuomarara, tichishandisa iwo mavhidhiyo akafanana akagadzirirwa mechanical stretch assessment, takatanga tatsvaga mifananidzo miviri inomiririra mafambiro epamusoro (yepamusoro-soro (yepamusoro) uye yakaderera (yakaderera) mapoinzi ekufamba) zvinoenderana nesoftware yeMUSCLEMOTION.Isu takazopatsanura matunhu ematishu uye takaisa fomu ye shading algorithm kune yakakamurwa tishu (Supplementary Fig. 2a).Mishonga yakakamurwa yakabva yakamurwa kuva gumi pasi, uye kushushikana pamusoro penzvimbo imwe neimwe kwakaverengwa uchishandisa equation inotevera: Strain = (Sup-Sdown) / Sdown, apo Sup uye Sdown ndiwo marefu echimiro kubva kumusoro uye pasi mimvuri yemucheka, maererano (Supplementary Fig. .2b).
Zvikamu zvemoyo zvakagadziriswa mu4% paraformaldehyde kwemaawa makumi mana nemasere.Fixed tissues akapera mvura mu10% uye 20% sucrose kwe1 h, ipapo mu30% sucrose husiku.Zvikamu zvacho zvakabva zvaiswa mune yakanakisa yekucheka tembiricha yekamu (OCT compound) uye zvishoma nezvishoma yakaomeswa mune isopentane / yakaoma ice yekugezera.Chengetedza OCT embedding block pa -80 °C kusvika paparadzana.Masiraidhi akagadzirwa sezvikamu zvine ukobvu hwe8 μm.
Kuti ubvise OCT kubva muzvikamu zvemoyo, kupisa masiraidhi pamudziyo wekudziya pa95 ° C kwemaminetsi mashanu.Wedzera 1 ml PBS kune imwe neimwe slide uye incubate kwemaminitsi makumi matatu pane imwe tembiricha, wopinda muzvikamu nekuisa 0.1% Triton-X muPBS kwemaminetsi gumi nemashanu patembiricha yekamuri.Kuti udzivise masoja ekudzivirira chirwere kubva pakusunga sampuli, wedzera 1 ml ye3% BSA mhinduro kumasiraidhi uye incuite kweawa imwe patembiricha.BSA yakabva yabviswa uye masiraidhi akagezwa nePBS.Maka muenzaniso wega wega nepenzura.Masoja ekudzivirira chirwere ekutanga (akadirwa 1:200 mu1% BSA) (connexin 43 (Abcam; #AB11370), NFATC4 (Abcam; #AB99431) uye troponin-T (Thermo F Scientific; #MA5-12960) akawedzerwa pamusoro pemaminetsi makumi mapfumbamwe, kana mamwe masoja ekudzivirira chirwere anonzi 12% ( Alexadi) 8 (Thermo Scientific; #A16079), inopesana netsuro Alexa Fluor 594 (Thermo Scientific; #T6391) kwemamwe maminetsi makumi mapfumbamwe Yakashambidzwa katatu nePBS Kusiyanisa tsvina yechinangwa kubva kumashure, takashandisa chete antibody yechipiri sekudzora. -x magnification) uye Keyence microscope ine 40x magnification.
WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) pa5 μg/ml muPBS yakashandiswa kutsvina yeWGA uye yakashandiswa kune zvikamu zvakagadziriswa kwemaminetsi makumi matatu patembiricha yekamuri.Masiraidhi acho akazogezwa nePBS uye Sudan dema yaiwedzerwa kune yega yega siraidhi uye incubated kwemaminetsi makumi matatu.Iwo masiraidhi akazogezwa nePBS uye vectashield embedding medium yakawedzerwa.Masiraidhi akaonekwa paKeyence microscope pa40x magnification.
OCT yakabviswa kubva kumasampuli sezvakatsanangurwa pamusoro apa.Mushure mekubvisa OCT, nyudza masiraidhi mumhinduro yeBouin husiku hwose.Masiraidhi acho akazosukwa nemvura yakadirwa kweawa imwe ndokuzoiswa muBibrich aloe acid fuchsin solution kwemaminitsi gumi.Zvadaro masiraidhi akashambidzwa nemvura yakasvibiswa uye akaiswa mumhinduro ye5% phosphomolybdenum/5% phosphotungstic acid kwemaminitsi gumi.Pasina kugeza, fambisa masiraidhi zvakananga muaniline yebhuruu mhinduro kwemaminitsi gumi nemashanu.Zvadaro masiraidhi akashambidzwa nemvura yakasvibiswa uye akaiswa mu 1% acetic acid solution kwemaminitsi maviri.Masiraidhi akaomeswa mu200 N ethanol uye akaendeswa kune xylene.Masiraidhi akasvibiswa akaonekwa pachishandiswa Keyence microscope ine chinangwa che10x.Fibrosis nharaunda muzana yakaverengerwa uchishandisa Keyence Analyzer software.
CyQUANT ™ MTT Cell Viability Assay (Invitrogen, Carlsbad, CA), katalogi nhamba V13154, maererano neprotocol yemugadziri ine zvimwe zvinogadziridzwa.Kunyanya, chibhakera chekuvhiya chine dhayamita ye6 mm chakashandiswa kuve nechokwadi chekukura kwemaviri mayunifomu panguva yekuongorora kweMTT.Matishu akaputirwa ega mumatsime e12-tsime ndiro ine MTT substrate zvinoenderana nemugadziri weprotocol.Zvikamu zvinoputirwa pa 37 ° C. kwemaawa matatu uye mhenyu inorarama inogadzirisa iyo MTT substrate kuti iite yepepuru formazan compound.Tsiva mhinduro yeMTT ne 1 ml DMSO woisa pa37 °C kwemaminetsi gumi nemashanu kuti ubvise formazan yepepuru kubva muzvikamu zvemoyo.Samples dzakadzikiswa 1:10 muDMSO mu96-yakanyatsojeka pasi mahwendefa uye yepepuru color intensity yakayerwa pa570 nm uchishandisa Cytation plate reader (BioTek).Kuverenga kwaiitwa kurema kwechidimbu chimwe nechimwe chemoyo.
Heart slice midhiya yakatsiviwa nenhau ine 1 μCi/ml [5-3H] -glucose (Moravek Biochemicals, Brea, CA, USA) yekuongorora kushandiswa kweglucose sezvakambotsanangurwa.Mushure memaawa mana ekukuchidzira, wedzera 100 µl yepakati kune yakavhurika microcentrifuge chubhu ine 100 µl ye0.2 N HCl.Ipapo chubhu yakaiswa muchubhu ye scintillation ine 500 μl ye dH2O kuti ibude [3H] 2O kwemaawa 72 pa37°C.Zvadaro bvisa microcentrifuge chubhu kubva kune scintillation tube uye uwedzere 10 ml ye scintillation fluid.Scintillation kuverenga kwakaitwa pachishandiswa Tri-Carb 2900TR liquid scintillation analyzer (Packard Bioscience Company, Meriden, CT, USA).Glucose kushandiswa kwakabva kwaverengerwa tichifunga nezve [5-3H] -glucose chaiyo chiitiko, kusakwana kuenzana uye kumashure, dilution ye [5-3H] -kune glucose isina kunyorwa, uye scintillation counter kunyatsoshanda.Iyo data yakajairwa kune kuwanda kwezvikamu zvemoyo.
Mushure mematishu homogenization muTrizol, RNA yakaparadzaniswa nezvikamu zvemoyo uchishandisa Qiagen miRNeasy Micro Kit #210874 zvinoenderana neprotocol yemugadziri.RNAsec kugadzirira raibhurari, kutevedzana uye kuongororwa kwedata kwakaitwa sezvinotevera:
1 μg yeRNA pamuenzaniso yakashandiswa sekutanga zvinhu kugadzirira raibhurari yeRNA.Kutevedza maraibhurari akagadzirwa uchishandisa iyo NEBNext UltraTM RNA Raibhurari Yekugadzirira Kit yeIllumina (NEB, USA) ichitevera kurudziro yemugadziri, uye macode eindex akawedzerwa kune hunhu kutevedzana kwemuenzaniso wega wega.Muchidimbu, mRNA yakacheneswa kubva kuRNA yakazara ichishandisa magineti mabedhi akabatanidzwa nepoly-T oligonucleotides.Kupatsanurwa kunoitwa uchishandisa divalent cations pakupisa kwakanyanya muNEBNext Yekutanga Strand Synthesis Reaction Buffer (5X).Chekutanga tambo cDNA yakagadziridzwa ichishandisa random hexamer primers uye M-MuLV reverse transcriptase (RNase H-).Yechipiri tambo cDNA inobva yagadzirwa uchishandisa DNA polymerase I uye RNase H. Iyo yakasara overhangs inoshandurwa kuva migumo isina kujeka ne exonuclease/polymerase chiitiko.Mushure mekuramba kweiyo 3 ′ kupera kwechimedu cheDNA, NEBNext Adapter ine hairpin loop chimiro inonamirwa pairi kuti igadzirire iyo hybridization.Kusarudzwa kwezvimedu zvecDNA zvehurefu hunodiwa 150-200 bp.zvidimbu zveraibhurari zvakacheneswa pachishandiswa AMPUre XP system (Beckman Coulter, Beverly, USA).Zvadaro, 3 μl USER Enzyme (NEB, USA) ine saizi-yakasarudzwa cDNA yakasungirirwa neadapter yakashandiswa kwemaminetsi gumi nemashanu pa37 ° C uyezve kwemaminitsi mashanu pa95 ° C pamberi pePCR.PCR yakazoitwa pachishandiswa Phusion High-Fidelity DNA polymerase, universal PCR primers, uye Index (X) primers.Pakupedzisira, zvigadzirwa zvePCR zvakacheneswa (AMPure XP system) uye mhando yeraibhurari yakaongororwa pane Agilent Bioanalyzer 2100 system.Iyo cDNA raibhurari yakazoteedzerwa ichishandisa Novaseq sequencer.Mafaera emifananidzo akaomeswa kubva kuIllumina akashandurwa kuita kuverenga kwakabikwa uchishandisa CASAVA Base Calling.Raw data inochengetwa muFASTQ(fq) fomati mafaera ane kuverenga kunotevedzana uye zvinoenderana nehunhu.Sarudza HISAT2 kuti ienderane nesefa yakateedzana kuverenga kune iyo Sscrofa11.1 referensi genome.Kazhinji, HISAT2 inotsigira genomes chero saizi, kusanganisira genomes akakura kupfuura 4 bhiriyoni mabhesi, uye default values ​​akagadzirirwa akawanda paramita.Splicing inoverenga kubva kuRNA Seq data inogona kunyatso kurongeka uchishandisa HISAT2, iyo inokurumidza sisitimu iripo parizvino, iine zvakafanana kana zvirinani kurongeka kupfuura chero imwe nzira.
Kuwanda kwezvinyorwa kunoratidza zvakananga nhanho yekutaura kwemajini.Gene kutaura mazinga anoongororwa nekuwanda kwezvinyorwa (sequencing count) yakabatana neiyo genome kana exons.Huwandu hwekuverenga hunoenderana nematanho ekutaura kwemajini, kureba kwemajini, uye kudzika kwekutevedzana.FPKM (zvimedu pazviuru zvepasi peya yezvinyorwa zvakateedzana pamiriyoni base pairi) zvakaverengerwa uye P-tsika dzekusiyanisa kutaura dzakatemerwa uchishandisa DESeq2 package.Takazoverenga chiyero chekuwanikwa kwenhema (FDR) kune imwe neimwe P kukosha tichishandisa nzira yeBenjamini-Hochberg9 yakavakirwa paR-function "p.adjust".
RNA yakaparadzaniswa nezvikamu zvemoyo yakashandurwa kuita cDNA pakusangana kwe200 ng/μl uchishandisa SuperScript IV Vilo Master mix kubva kuThermo (Thermo, katsi no. 11756050).Quantitative RT-PCR yakaitwa uchishandisa Applied Biosystems Endura Plate Microamp 384-well transparent reaction plate (Thermo, cat. no. 4483319) uye microamp optical adhesive (Thermo, cat. no. 4311971).Musanganiswa wekuita waisanganisira 5 µl Taqman Fast Advanced Master musanganiswa (Thermo, katsi # 4444557), 0.5 µl Taqman Primer uye 3.5 µl H2O yakasanganiswa patsime.Yakajairwa qPCR mitsetse yaiitwa uye CT kukosha kwakayerwa pachishandiswa Applied Biosystems Quantstudio 5 chaiyo-nguva PCR chiridzwa (384-tsime module; chigadzirwa # A28135).Taqman pranters vakatengwa kubva kuTrmo (Gapdh (ss03375629_u1), Parcc (ss0690387_m1), Rgl1 (ss06868890_m1 (SS01009508_M1), Gata4 (SS03383805_u1), Gja1 (SS03374839_u1), Col1a2 (ss043232323232332)
Kuburitswa kweMedia kweNT-ProBNP kwakaongororwa uchishandisa NT-ProBNP kit (nguruve) (Cat. No. MBS2086979, MyBioSource) maererano neprotocol yemugadziri.Muchidimbu, 250 µl yemuenzaniso wega wega uye chiyero chakawedzerwa mukudzokorora kutsime rega rega.Pakarepo mushure mekuwedzera sampuli, wedzera makumi mashanu µl yeAssay Reagent A patsime rega rega.Zvinyoro zunza ndiro uye uvhare ne sealant.Zvadaro mapiritsi acho akaiswa pa 37 ° C kweawa imwe.Wobva washuvira mushonga wogeza matsime ka4 ne350 µl ye1X wash solution, uchiisa mushonga wewash kwemaminetsi 1-2 nguva yega yega.Wobva wawedzera 100 µl yeAssay Reagent B patsime uye simbisa neplate sealant.The tablet yakazununguswa zvinyoro nyoro uye incubated pa 37°C kwemaminetsi makumi matatu.Shingairira mushonga uye geza matsime kashanu ne350 µl ye1X wash solution.Wedzera 90 µl ye substrate solution kune imwe neimwe tsime uye simbisa ndiro.Isai ndiro pa 37 ° C kwemaminitsi 10-20.Wedzera 50 µl Stop Solution kune imwe neimwe tsime.Iyo ndiro yakabva yapimwa pachishandiswa Cytation (BioTek) plate reader yakaiswa pa450 nm.
Ongororo yesimba yakaitwa kuti isarudze masaizi eboka ayo achapa> 80% simba rekuona 10% shanduko yakakwana muparameter ine 5% Type I kukanganisa mwero. Ongororo yesimba yakaitwa kuti isarudze masaizi eboka ayo achapa> 80% simba rekuona 10% shanduko yakakwana muparameter ine 5% Type I kukanganisa mwero. Анализ мощности был выполнен для выбора размеров групп, которые обеспечат >80% выполнен для выбора размеров групп, которые обеспечат >80% выполнен для выбора размеров групп, которые обеспечат >80% выполнен для выбора размеров групп, которые обеспечат >80% мощности для обнаружения 10% той ошибок типа I. Kuongorora kwesimba kwakaitwa kusarudza masaizi eboka aizopa > 80% simba rekuona 10% absolute parameter shanduko ne5% Type I kukanganisa mwero.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 обнаружности для выбора размера группы, который обеспечил бы > 80% мощности для обнаружности для выбора размера группы, который обеспечил бы > 80% мощности для обнаружения 10% астоты ошибок типа I. Ongororo yesimba yakaitwa yekusarudza saizi yeboka yaizopa > 80% simba rekuona 10% absolute parameter shanduko uye 5% mhando I kukanganisa mwero.Zvikamu zvenyama zvakasarudzwa zvisina kurongwa pamberi pekuedza.Ongororo dzese dzaive mapofu uye samples dzakatemwa chete mushure mekunge data rese raongororwa.GraphPad Prism software (San Diego, CA) yakashandiswa kuita ongororo dzese dzenhamba. Kune ese manhamba, p-values ​​aionekwa seakakosha pamitengo <0.05. Kune ese manhamba, p-maitiro aionekwa akakosha pamitengo <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Kune ese manhamba, p-maitiro aionekwa akakosha pamitengo <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. Kune ese manhamba, p-maitiro aionekwa akakosha pamitengo <0.05.T-test yeMudzidzi ine miswe miviri yakaitwa padata nekuenzanisa kaviri chete.Nzira imwe kana mbiri-nzira ANOVA yakashandiswa kuona kukosha pakati pemapoka akawanda.Paunenge uchiita bvunzo dzepost hoc, kururamisa kwaTukey kwakaiswa kuaccount yekuenzanisa kwakawanda.RNAsec data ine zviverengero zvakakosha pakuverenga FDR uye p.adjust sezvinotsanangurwa muchikamu cheNzira.
Kuti uwane rumwe ruzivo nezve dhizaini yekudzidza, ona Nature Research Report abstract yakabatana nechinyorwa ichi.