Ua tsaug rau koj tuaj xyuas Nature.com.Qhov browser version koj siv tau txwv CSS kev txhawb nqa.Rau qhov kev paub zoo tshaj plaws, peb xav kom koj siv qhov browser tshiab (lossis lov tes taw Compatibility Hom hauv Internet Explorer).Nyob rau lub sijhawm no, txhawm rau ua kom muaj kev txhawb nqa txuas ntxiv, peb yuav ua rau lub xaib tsis muaj qauv thiab JavaScript.
Yuav tsum muaj kev txhim khu kev qha hauv vitro uas tuaj yeem tsim kho lub cev ib puag ncig ntawm lub plawv rau kev kuaj tshuaj.Kev txwv tsis pub muaj ntawm tib neeg lub plawv kab lis kev cai kab lis kev cai tau ua rau kev txhais lus tsis raug ntawm cov tshuaj mob plawv.Ntawm no, peb tau tsim cov qauv kab lis kev cai plawv (CTCM) uas electromechanically nkoos lub plawv hlais thiab ua rau lub cev ncab thaum lub sij hawm systolic thiab diastolic theem ntawm lub plawv voj voog.Tom qab 12 hnub ntawm kab lis kev cai, txoj hauv kev no tau txhim kho ib feem ntawm kev ua haujlwm ntawm lub plawv, tab sis tsis tau khaws tag nrho lawv cov qauv kev ncaj ncees.Yog li ntawd, tom qab kev tshuaj ntsuam xyuas me me, peb pom tias qhov sib ntxiv ntawm 100 nM triiodothyronine (T3) thiab 1 μM dexamethasone (Dex) rau peb qhov nruab nrab tswj cov microstructure ntawm cov seem rau 12 hnub.Ua ke nrog T3 / Dex kev kho mob, CTCM system tswj hwm cov ntaub ntawv hloov pauv, muaj peev xwm ua tau, metabolic kev ua haujlwm, thiab kev ncaj ncees ntawm tib theem li cov ntaub so ntswg tshiab rau 12 hnub.Tsis tas li ntawd, kev ncab ntau dhau ntawm cov ntaub so ntswg hauv kab lis kev cai induces hypertrophic cardiac signaling, muab pov thawj rau lub peev xwm ntawm CTCM rau mimic hypertrophic mob tshwm sim los ntawm lub plawv stretch.Hauv kev xaus, CTCM tuaj yeem ua qauv rau lub cev thiab pathophysiology ntawm lub plawv hauv kab lis kev cai nyob rau lub sijhawm ntev, ua rau muaj kev tshuaj xyuas tshuaj txhim khu kev qha.
Ua ntej kev tshawb fawb soj ntsuam, txhim khu kev qha nyob rau hauv vitro systems yog xav tau uas yuav tsum tau muaj tseeb reproduce lub physiological ib puag ncig ntawm tib neeg lub siab.Cov tshuab no yuav tsum ua raws li kev hloov kho txhua yam stretch, lub plawv dhia, thiab electrophysiological zog.Cov qauv tsiaj feem ntau yog siv los ua qhov kev tshuaj ntsuam xyuas rau lub plawv physiology uas muaj kev ntseeg siab tsawg hauv kev xav txog cov teebmeem ntawm cov tshuaj hauv tib neeg lub siab 1,2.Thaum kawg, Lub Tswv Yim Zoo Tshaj Plaws Kev Cuam Tshuam Kab Mob Kab Mob Kab Mob (CTCM) yog ib qho qauv uas muaj kev nkag siab zoo thiab tshwj xeeb rau ntau yam kev kho mob thiab tshuaj kho mob, ua kom rov tsim kho lub cev thiab kab mob ntawm tib neeg lub siab 3.Qhov tsis muaj xws li cov txheej txheem txwv qhov kev tshawb pom ntawm cov kev kho mob tshiab rau lub plawv tsis ua hauj lwm 4,5 thiab tau ua rau cov tshuaj cardiotoxicity yog ib qho laj thawj tseem ceeb rau kev tawm hauv kev lag luam 6.
Xyoo kaum xyoo dhau los, yim cov tshuaj uas tsis yog mob plawv tau raug tshem tawm los ntawm kev siv tshuaj kho mob vim tias lawv ua rau QT lub sijhawm ncua ntev ua rau lub plawv tsis ua haujlwm thiab tuag sai sai7.Yog li, muaj kev xav tau ntau ntxiv rau cov tswv yim kev tshuaj ntsuam xyuas preclinical txhim khu kev qha los ntsuas kev ua haujlwm ntawm lub plawv thiab toxicity.Kev siv tsis ntev los no ntawm tib neeg-induced pluripotent qia cell-derived cardiomyocytes (hiPS-CM) hauv kev tshuaj xyuas tshuaj thiab tshuaj toxicity muab ib feem kev daws teeb meem rau qhov teeb meem no.Txawm li cas los xij, qhov tsis paub qab hau ntawm hiPS-CMs thiab tsis muaj ntau lub cellular complexity ntawm cov ntaub so ntswg plawv yog cov kev txwv loj ntawm txoj kev no.Cov kev tshawb fawb tsis ntev los no tau pom tias qhov kev txwv no tuaj yeem dhau los ua ib feem los ntawm kev siv hiPS-CM thaum ntxov los tsim cov ntaub so ntswg hydrogels sai tom qab qhov pib ntawm kev sib cog lus thiab maj mam nce hluav taws xob stimulation dhau sij hawm.Txawm li cas los xij, cov kab mob hiPS-CM no tsis muaj qhov paub tab electrophysiological thiab contractile zog ntawm cov neeg laus myocardium.Tsis tas li ntawd, tib neeg lub plawv cov ntaub so ntswg muaj ib tug complex qauv, muaj ib tug heterogeneous sib tov ntawm ntau hom cell, nrog rau cov endothelial hlwb, neurons, thiab stromal fibroblasts, interconnected los ntawm tej pawg ntawm extracellular matrix proteins.Qhov no heterogeneity ntawm non-cardiomyocyte pejxeem11,12,13 nyob rau hauv cov neeg laus mammalian lub plawv yog ib tug loj teeb meem rau modeling cardiac cov ntaub so ntswg siv ib tug neeg hom cell.Cov kev txwv loj no hais txog qhov tseem ceeb ntawm kev tsim cov txheej txheem rau kev loj hlob ntawm cov ntaub so ntswg myocardial nyob rau hauv physiological thiab pathological mob.
Kab lis kev cai nyias (300 µm) ntu ​​ntawm tib neeg lub siab tau ua pov thawj tias yog tus qauv pheej hmoo ntawm tib neeg myocardium.Txoj kev no muab kev nkag mus rau qhov ua tiav 3D multicellular system zoo ib yam li tib neeg lub plawv cov ntaub so ntswg.Txawm li cas los xij, kom txog rau thaum 2019, kev siv lub plawv kab lis kev cai raug txwv los ntawm luv luv (24 teev) kab lis kev cai ciaj sia.Qhov no yog vim muaj ntau yam xws li qhov tsis muaj lub cev-txheej txheem ncab, huab cua-kua interface, thiab siv cov xov xwm yooj yim uas tsis txhawb cov kev xav tau ntawm cov ntaub so ntswg.Hauv xyoo 2019, ntau pab pawg tshawb fawb pom tau hais tias kev sib koom ua ke ntawm cov khoom siv kho tshuab rau hauv cov kab mob hauv lub plawv tuaj yeem ua rau lub neej kev coj noj coj ua, txhim kho kev mob plawv, thiab ua raws li cov kab mob plawv.Ob txoj kev tshawb fawb zoo nkauj 17 thiab 18 qhia tias uniaxial mechanical loading muaj txiaj ntsig zoo rau lub plawv phenotype thaum kab lis kev cai.Txawm li cas los xij, cov kev tshawb fawb no tsis tau siv lub zog peb-dimensional physico-mechanical loading ntawm lub voj voog ntawm lub plawv, txij li cov seem ntawm lub plawv tau ntim nrog cov isometric tensile rog 17 lossis linear auxotonic loading 18.Cov txheej txheem ntawm cov ntaub so ntswg stretching ua rau muaj kev cuam tshuam ntawm ntau lub plawv cov noob caj noob ces los yog overexpression ntawm cov noob cuam tshuam nrog cov lus teb txawv txav.Piv txwv li, Pitoulis et al.19 tau tsim lub plawv dhia kab lis kev cai da dej rau lub plawv voj voog rov tsim kho siv lub zog transducer tawm tswv yim thiab nro drives.Txawm hais tias qhov system no tso cai rau kom muaj tseeb ntau dua hauv vitro cardiac cycle modeling, qhov nyuaj thiab qis dhau ntawm txoj kev txwv tsis pub siv cov kab ke no.Peb lub chaw kuaj mob tsis ntev los no tau tsim cov kab lis kev cai yooj yim uas siv hluav taws xob stimulation thiab qhov nruab nrab zoo kom tswj tau qhov muaj feem ntawm cov porcine thiab tib neeg lub plawv cov ntaub so ntswg ntev txog 6 hnub 20,21.
Hauv cov ntawv sau tam sim no, peb piav qhia txog tus qauv kab lis kev cai plawv (CTCM) siv cov ntu ntawm lub plawv porcine uas suav nrog kev humoral cues kom rov hais dua peb-dimensional cardiac physiology thiab pathophysiological mob thaum lub sij hawm lub plawv dhia.Qhov CTCM no tuaj yeem ua kom qhov tseeb ntawm kev tshuaj xyuas ua ntej kev tshuaj ntsuam xyuas mus rau ib qib uas tsis tau ua tiav los ntawm kev muab cov txiaj ntsig zoo, nruab nrab ntawm lub plawv uas ua rau lub cev muaj zog / pathophysiology ntawm lub plawv mammalian rau kev kuaj tshuaj ua ntej.
Hemodynamic mechanical signals ua lub luag haujlwm tseem ceeb hauv kev tswj hwm cardiomyocyte kev ua haujlwm hauv vitro 22,23,24.Hauv cov ntawv sau tam sim no, peb tau tsim CTCM (Daim duab 1a) uas tuaj yeem ua raws li cov neeg laus lub plawv ib puag ncig los ntawm kev txhawb nqa hluav taws xob thiab tshuab hluav taws xob ntawm lub cev muaj zog (1.2 Hz, 72 neeg ntaus ib feeb).Txhawm rau kom tsis txhob muaj cov ntaub so ntswg ntau dhau thaum lub sijhawm diastole, 3D tshuab luam ntawv tau siv los ua kom cov ntaub so ntswg loj dua 25% (Fig. 1b).Hluav taws xob pacing induced los ntawm C-PACE system tau teem sij hawm pib 100 ms ua ntej systole siv cov ntaub ntawv nrhiav tau los ua kom tag nrho cov kab mob plawv.Cov kab lis kev cai ntawm cov ntaub so ntswg siv lub programmable pneumatic actuator (LB Engineering, Lub teb chaws Yelemees) txhawm rau txhawm rau txhawm rau nthuav dav cov ntaub nplaum silicone ua kom nthuav dav ntawm lub plawv hauv lub tsev sab saud.Lub kaw lus tau txuas nrog cov kab hluav taws xob sab nraud los ntawm lub tshuab hluav taws xob siab, uas ua rau nws tuaj yeem kho qhov siab (± 1 mmHg) thiab lub sijhawm (± 1 ms) (Daim duab 1c).
a Txuas cov ntaub so ntswg mus rau lub nplhaib txhawb nqa 7 hli, qhia hauv xiav, hauv kab lis kev cai ntawm lub cuab yeej.Cov kab lis kev cai chamber yog sib cais los ntawm huab cua chamber los ntawm nyias nyias nyias silicone membrane.Muab lub gasket nruab nrab ntawm txhua lub chamber kom tiv thaiv kom tsis txhob yaig.Lub hau ntawm lub cuab yeej muaj graphite electrodes uas muab hluav taws xob stimulation.b Schematic sawv cev ntawm cov khoom siv ntaub so ntswg loj, coj lub nplhaib thiab txhawb nqa lub nplhaib.Cov ntaub so ntswg (xim av) tau muab tso rau ntawm cov khoom siv loj nrog lub nplhaib qhia tau muab tso rau hauv qhov zawj ntawm sab ntug ntawm lub cuab yeej.Siv cov lus qhia, ua tib zoo tso lub nplhaib txhawb nqa nrog cov ntaub so ntswg acrylic nplaum hla ntu ntawm cov ntaub so ntswg.c Daim duab qhia lub sij hawm ntawm hluav taws xob stimulation raws li kev ua haujlwm ntawm huab cua chamber siab tswj los ntawm programmable pneumatic actuator (PPD).Cov cuab yeej nrhiav tau cov ntaub ntawv tau siv los synchronize hluav taws xob stimulation siv lub siab sensors.Thaum lub siab nyob rau hauv cov kab lis kev cai chamber ncav cuag qhov chaw pib, lub mem tes teeb liab raug xa mus rau C-PACE-EM kom ua rau hluav taws xob stimulation.d Daim duab ntawm plaub CTCMs muab tso rau ntawm lub txee incubator.Plaub lub cuab yeej txuas nrog rau ib qho PPD ntawm lub pneumatic Circuit Court, thiab lub siab sensors tau muab tso rau hauv hemostatic valve los saib xyuas lub siab hauv pneumatic circuit.Txhua lub cuab yeej muaj rau cov ntaub so ntswg.
Siv ib lub pneumatic actuator, peb muaj peev xwm tswj tau 4 CTCM li, txhua tus muaj peev xwm tuav 6 cov ntaub so ntswg (Fig. 1d).Nyob rau hauv CTCM, cov huab cua siab nyob rau hauv cov huab cua chamber hloov mus rau synchronous siab nyob rau hauv cov kua chamber thiab induces physiological expansion ntawm lub plawv daim (Daim duab 2a thiab Supplementary Movie 1).Kev ntsuam xyuas ntawm cov ntaub so ntswg stretch ntawm 80 mm Hg.Art.pom stretching ntawm cov ntaub so ntswg los ntawm 25% (Fig. 2b).Qhov feem pua ​​​​ntawm qhov stretch tau pom tias sib haum rau lub cev sarcomere ntev ntawm 2.2-2.3 µm rau ib txwm mob plawv contractility17,19,25.Cov ntaub so ntswg txav tau raug ntsuas los ntawm kev siv lub koob yees duab kev cai (Cov duab ntxiv 1).Lub amplitude thiab ceev ntawm cov ntaub so ntswg txav (Fig. 2c, d) sib raug mus rau stretch thaum lub sij hawm lub plawv thiab lub sij hawm thaum lub sij hawm systole thiab diastole (Fig. 2b).Kev ncab thiab nrawm ntawm cov ntaub so ntswg ntawm lub plawv thaum lub sij hawm contraction thiab so nyob twj ywm rau 12 hnub hauv kab lis kev cai (Fig. 2f).Txhawm rau ntsuas qhov cuam tshuam ntawm hluav taws xob stimulation ntawm contractility thaum lub sij hawm kab lis kev cai, peb tau tsim ib txoj kev rau kev txiav txim siab active deformity siv ib tug shading algorithm (Supplementary Fig. 2a, b) thiab muaj peev xwm paub qhov txawv ntawm deformities nrog thiab tsis muaj hluav taws xob stimulation.Tib seem ntawm lub siab (Fig. 2f).Hauv cheeb tsam txav tau ntawm qhov txiav (R6-9), qhov hluav taws xob thaum lub sij hawm hluav taws xob stimulation yog 20% ​​siab dua thaum tsis muaj hluav taws xob stimulation, uas qhia tau hais tias qhov kev pab cuam ntawm hluav taws xob stimulation rau contractile muaj nuj nqi.
Cov neeg sawv cev ntawm cov huab cua chamber siab, kua chamber siab, thiab cov ntaub so ntswg txav kev ntsuas pom tau hais tias chamber siab hloov cov kua chamber siab, ua rau ib tug sib thooj txav ntawm cov ntaub so ntswg daim ntaub.b Cov neeg sawv cev ntawm qhov feem pua ​​stretch (xiav) ntawm cov ntaub so ntswg sib txuas mus rau feem pua ​​stretch (txiv kab ntxwv).c Qhov ntsuas ntsuas ntawm lub plawv hlais yog ua raws li qhov ntsuas ceev ntawm kev txav.(d) Cov neeg sawv cev ntawm kev sib tw ntawm kev txav mus los (xiav kab) thiab kev tshaj tawm (txiv kab ntxwv dotted kab) nyob rau hauv ib daim ntawm lub plawv.e Quantification of cycle time (n = 19 slices per group, from different pigs), contraction time (n = 19 slices per group), so lub sij hawm (n = 19 slices per group, from different pigs), tissue move (n = 25).slices) / pab pawg los ntawm cov npua sib txawv), lub siab tshaj plaws systolic tshaj tawm (n = 24(D0), 25(D12) daim / pab pawg los ntawm cov npua sib txawv) thiab qhov siab so siab siab (n = 24(D0), 25(D12) daim / pab pawg los ntawm cov npua sib txawv).Ob tug tub-ntxhais kawm ntawv qhov t-test tsis muaj qhov sib txawv tseem ceeb hauv ib qho kev ntsuas.f Cov neeg sawv cev kev soj ntsuam cov kab mob ntawm cov ntaub so ntswg nrog (liab) thiab tsis muaj (xiav) hluav taws xob stimulation, kaum cheeb tsam ntawm cov ntaub so ntswg los ntawm tib seem.Cov vaj huam sib luag hauv qab qhia txog qhov ntau ntawm qhov feem pua ​​​​ntawm qhov sib txawv ntawm cov kab mob hauv cov ntaub so ntswg nrog thiab tsis muaj hluav taws xob stimulation nyob rau hauv kaum cheeb tsam los ntawm ntau seem. (n = 8 daim / pab pawg los ntawm cov npua sib txawv, Ob-tailed Student t-test yog ua; ****p <0.0001, **p <0.01, *p <0.05). (n = 8 daim / pab pawg los ntawm cov npua sib txawv, Ob-tailed Student t-test yog ua; ****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<0,01, (n = 8 ntu/pab pawg los ntawm cov npua sib txawv, ob-tailed Student's t-test; ****p<0.0001, **p<0.01, *p<0.05). （n = 8 片/组，来自不同的猪，进行双尾学生t 检验； ****p <0.0001，**p <0.01，*p <0.05). （n = 8 片/组，来自不同的猪，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 ntu/pab pawg, los ntawm cov npua sib txawv, ob-tailed Tub Ntxhais Kawm T-test; ****p<0.0001, **p<0.01, *p<0.05).Cov kab yuam kev sawv cev qhov txhais tau tias ± tus qauv sib txawv.
Nyob rau hauv peb yav dhau los zoo li qub biomimetic lub plawv daim kab lis kev cai kab lis kev cai [20, 21], peb khaws cia qhov kev muaj peev xwm, muaj nuj nqi, thiab kev ncaj ncees ntawm lub plawv rau 6 hnub los ntawm kev siv hluav taws xob stimulation thiab optimizing nruab nrab muaj pes tsawg leeg.Txawm li cas los xij, tom qab 10 hnub, cov nuj nqis no poob qis heev.Peb yuav xa mus rau ntu kab lis kev cai hauv peb cov kab lis kev cai qub qub biomimetic kab lis kev cai 20, 21 tswj cov xwm txheej (Ctrl) thiab peb yuav siv peb qhov kev ua tau zoo yav dhau los raws li MC cov xwm txheej thiab kab lis kev cai nyob rau hauv ib txhij txhua yam thiab hluav taws xob stimulation (CTCM).hu ua.Ua ntej, peb tau txiav txim siab tias qhov kev txhawb zog tsis muaj hluav taws xob tsis txaus los tswj cov ntaub so ntswg rau 6 hnub (Cov duab ntxiv 3a, b).Interestingly, nrog rau kev taw qhia ntawm lub cev-mechanical thiab hluav taws xob stimulation siv STCM, kev ua tau zoo ntawm 12-hnub lub plawv seem tseem zoo ib yam li hauv lub plawv tshiab nyob rau hauv MS tej yam kev mob, tab sis tsis nyob rau hauv Ctrl tej yam kev mob, raws li qhia los ntawm MTT tsom xam (Fig. 1).3 a).Qhov no qhia tau hais tias txhua yam stimulation thiab simulation ntawm lub voj voog ntawm lub plawv tuaj yeem ua kom cov ntaub so ntswg siv tau ob zaug ntev npaum li tau tshaj tawm hauv peb cov kab lis kev cai qub qub.Txawm li cas los xij, kev ntsuam xyuas ntawm cov qauv kev ncaj ncees ntawm cov ntaub so ntswg los ntawm immunolabeling ntawm cardiac troponin T thiab connexin 43 qhia tau hais tias connexin 43 qhia tau ntau dua hauv MC cov ntaub so ntswg hnub 12 dua li kev tswj hwm tib hnub.Txawm li cas los xij, kev sib xyaw connexin 43 qhia thiab Z-disc tsim tsis tau khaws cia tag nrho (Daim duab 3b).Peb siv lub tswv yim kev txawj ntse (AI) kom muaj nuj nqis ntawm cov ntaub so ntswg kev ncaj ncees26, ib qho duab-raws li kev kawm sib sib zog nqus raj raws troponin-T thiab connexin staining43 kom tau txais qhov ntsuas qhov kev ncaj ncees thiab fluorescence ntawm lub plawv hlais raws li lub zog ntawm thaj chaw.Txoj kev no siv Convolutional Neural Network (CNN) thiab lub hauv paus kev kawm sib sib zog nqus kom ntseeg tau tias muaj kev ntseeg siab ntawm cov ntaub so ntswg hauv kev siv tshuab thiab tsis muaj kev ywj pheej, raws li tau piav qhia hauv kev siv.26. MC cov ntaub so ntswg tau pom zoo cov qauv zoo sib xws rau hnub 0 piv rau cov seem tswj zoo li qub.Tsis tas li ntawd, Masson's trichrome staining qhia qhov feem pua ​​​​ntawm cov fibrosis qis dua hauv MS cov xwm txheej piv rau kev tswj hwm hnub 12 ntawm kab lis kev cai (Fig. 3c).Thaum CTCM tau nce qhov kev muaj peev xwm ntawm cov ntaub so ntswg hauv plawv hnub 12 mus rau qib zoo ib yam li cov ntaub so ntswg tshiab, nws tsis tau txhim kho cov qauv kev ncaj ncees ntawm lub plawv.
ib daim duab qhia tau hais tias muaj nuj nqis ntawm MTT kev muaj peev xwm ntawm lub plawv tshiab (D0) lossis lub plawv hlais kab lis kev cai rau 12 hnub nyob rau hauv kab lis kev cai zoo li qub (D12 Ctrl) lossis hauv CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) slices / pawg # NO #0 # pVA test. rau D0 thiab **p <0.01 piv rau D12 Ctrl). ib daim duab qhia tau hais tias muaj nuj nqis ntawm MTT kev muaj peev xwm ntawm lub plawv tshiab (D0) lossis lub plawv hlais kab lis kev cai rau 12 hnub nyob rau hauv kab lis kev cai zoo li qub (D12 Ctrl) lossis hauv CTCM (D12 MC) (n = 18 (D0), 15 (D12 Ctrl), 12 (D12 MC) slices / ib pawg #0 # NO. piv rau D0 thiab **p <0.01 piv rau D12 Ctrl).lub histogram qhia txog qhov muaj peev xwm ntawm MTT tshiab lub plawv ntu (D0) lossis kab lis kev cai ntawm lub plawv ntu rau 12 hnub hauv kab lis kev cai zoo li qub (D12 tswj) lossis CTCM (D12 MC) (n = 18 (D0), 15 (D12 tswj). ) ), 12 (D12 MC) ntu ​​npuag / ib pawg ua haujlwm sib txawv.####p <0,0001 по сравнению с D0 и **p < 0,01 по сравнению с D12 Ctrl). ####p <0.0001 piv rau D0 thiab **p <0.01 piv rau 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) 中新鲜心脏切片(1幇)天的MTT 活力的量化), 来自不同猪的12 (D12 MC) 切片/组，进行单向 ANOVA 测试；与D0 縛#0.#0比,**p <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) 中新鲜心脏切牸(D0) ，D0 ) 切片/组，进行单向 ANOVA 测试；与D0 相比，####p < 0.0001，与D12 Ctrl 相比，**p.)histogram qhia qhov muaj nuj nqis ntawm MTT kev muaj peev xwm nyob rau hauv lub plawv tshiab (D0) los yog lub plawv kab lis kev cai rau 12 hnub hauv kab lis kev cai zoo li qub (D12 tswj) lossis CTCM (D12 MC) (n = 18 (D0), 15 (D12 tswj)) , 12 (D12-MC) ntu ​​/ pawg los ntawm kev sib txawv ntawm npua NOVA,####p <0,0001 по сравнению с D0, **p < 0,01 по сравнению с D12 Ctrl). ####p <0.0001 piv rau D0, **p <0.01 piv rau D12 Ctrl).b Troponin-T (ntsuab), connexin 43 (liab) thiab DAPI (xiav) nyob rau hauv freshly cais lub plawv seem (D0) los yog lub plawv seem kab lis kev cai nyob rau hauv zoo li qub (Ctrl) los yog CTCM tej yam kev mob (MC) rau 12 hnub) ntawm tus neeg sawv cev immunofluorescence dluab (dawb teev = 100 µm). Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) slices/group each from different pig, one-way ANOVA test is performed; ####p < 0.0001 piv rau D0 thiab ****p < 0.01201 Ctrl). Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) slices/paub txhua tus los ntawm txawv npua, ib txoj kev ANOVA xeem yog ua; ####p < 0.0001 piv rau D0 thiab ****p < Ctrl ) . Количественная оценка структурной целостности сердечной ткани искуственным интеллектом (n = 7 (D52) , 7 (D0), 7 пп от разных свиней, проводится однофакторный тест ANOVA; ####p < 0,0001 по сравнению с D0 и ****p < 0,0001). Kev suav ntawm cov qauv kev ncaj ncees ntawm cov ntaub so ntswg los ntawm kev txawj ntse (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) ntu ​​/ pawg los ntawm cov npua sib txawv, ib txoj kev ANOVA xeem tau ua; ####p <0.0001 vs. nrog D0 thiab ****p < Ctrl 1.00).人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) slices/pawg txhua tus npua sib txawv, ib-txoj kev ANOVA xeem; ####0D 曯 0 . .0001 与D12 Ctrl 相比).人工智能量化心脏组织结构完整性（n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) slices/paub txhua tus npua sib txawv, ib-txoj kev ANOVA xeem;###. .0001 与D12 Ctrl 相比). Искусственный интеллект для количественной оценки структурной целостности сердечной ткани (n = 7р 2 MC 1 / 7), группу каждой из разных свиней, односторонний тест ANOVA; ####p <0,0001 vs. D0 Для сравнения ****p < 0,0001 всрни 2 нерния . Artificial txawj ntse kom quantify lub structural integrity ntawm cardiac cov ntaub so ntswg (n = 7 (D0), 7 (D12 Ctrl), 5 (D12 MC) seem/pab pawg ntawm cov sib txawv npua, ib-txoj kev ANOVA xeem; ####p<0.0001 vs .D0 Rau kev sib piv ****p <10. c Cov duab sawv cev (sab laug) thiab quantification (txoj cai) rau lub plawv slices stained nrog Masson's trichrome stain (Scale liab qab = 500 µm) (n = 10 daim / pab pawg los ntawm txawv npua, ib-txoj kev ANOVA xeem yog ua; ####p <0.0001 piv rau D0). c Cov duab sawv cev (sab laug) thiab quantification (txoj cai) rau lub plawv slices stained nrog Masson's trichrome stain (Scale liab qab = 500 µm) (n = 10 slices/paub txhua tus los ntawm txawv npua, ib-txoj kev ANOVA xeem yog ua; #### p < 0.0001 piv rau D0). c Репрезентативные изображения (слева) и количественная оценка (справа) срезов сердца, окрашенныр тронам а (масштаб без покрытия = 500 мкм) (n = 10 срезов/группу от разных свиней, выполняется односторонтай, VA #0пронний ; нению с D0 и ***p < 0,001 по сравнению с D12 Ctrl). c Cov duab sawv cev (sab laug) thiab quantification (txoj cai) ntawm lub plawv seem stained nrog Masson's trichrome stain (uncoated scale = 500 µm) (n = 10 seem / pawg los ntawm cov npua sib txawv, ib-txoj kev ANOVA xeem ua; #### p < 0 .0001 piv rau D0. 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 µ切片片片。来自不同的猪，进行单向ANOVA 测试；#### p < 0.0001 与D0 相比，***p < 0.001 与D12 Ctrl 相比). C 用 masson 三色染料的心脏 切片的代表性（左左）量化（右）裸尺度 裸尺度尺尸 0 m²） （n = 10 个切片组每组来自 不同猪，进行单向 单向 Anova 测试； ，#### p < 0.0001 *** 与 0.0001 D12 Ctrl 相比). C резентаиаые the изобения (слеваیй и споличественный) (срашеныхров сона (. ООT А а;;;;;;;;;;;;;;;;;;;;;;;;;;;;;; Ог; Ог #p <0,0001 пО сО сО сО сО сО,,, D0, *** P <0,001 c Cov duab sawv cev (sab laug) thiab quantitation (sab xis) ntawm lub plawv seem stained nrog Masson's trichrome stain (blank = 500 µm) (n = 10 ntu / pawg, txhua tus los ntawm ib qho txawv npua, sim los ntawm ib txoj kev tsom xam ntawm qhov sib txawv ;### # p < 0.0001 piv rau D0.Cov kab yuam kev sawv cev qhov txhais tau tias ± tus qauv sib txawv.
Peb xav tias los ntawm kev ntxiv cov me me rau hauv kab lis kev cai nruab nrab, cardiomyocyte kev ncaj ncees tuaj yeem txhim kho thiab kev txhim kho fibrosis txo qis thaum CTCM kab lis kev cai.Yog li ntawd, peb tau soj ntsuam rau cov me me molecules siv peb cov kab lis kev cai zoo li qub 20,21 vim yog qhov me me ntawm qhov tsis sib haum xeeb.Dexamethasone (Dex), triiodothyronine (T3), thiab SB431542 (SB) raug xaiv rau lub vijtsam no.Cov me me molecules yav dhau los tau siv nyob rau hauv hiPSC-CM kab lis kev cai los txhawb kev loj hlob ntawm cardiomyocytes los ntawm kev nce sarcomere ntev, T-tubules, thiab conduction tshaj tawm.Tsis tas li ntawd, ob qho tib si Dex (ib qho glucocorticoid) thiab SB paub tias yuav txo qis kev mob 29,30.Yog li ntawd, peb tau sim seb qhov kev koom ua ke ntawm ib lossis ib qho ntawm cov me me molecules yuav txhim kho cov qauv kev ncaj ncees ntawm lub plawv.Rau kev tshuaj ntsuam thawj zaug, cov koob tshuaj ntawm txhua qhov sib xyaw tau raug xaiv raws li qhov muaj feem ntau siv hauv cov qauv kab lis kev cai ntawm tes (1 μM Dex27, 100 nM T327, thiab 2.5 μM SB31).Tom qab 12 hnub ntawm kab lis kev cai, kev sib xyaw ua ke ntawm T3 thiab Dex ua rau pom kev zoo ntawm cardiomyocyte structural kev ncaj ncees thiab kev hloov kho fiber ntau tsawg (Cov duab ntxiv 4 thiab 5).Tsis tas li ntawd, kev siv ob npaug los yog ob npaug ntawm cov concentrations ntawm T3 thiab Dex ua rau muaj kev cuam tshuam zoo dua piv rau cov concentrations ib txwm muaj (Cov Lus Qhia Ntxiv 6a,b).
Tom qab kev tshuaj ntsuam thawj zaug, peb tau ua qhov sib piv ntawm lub taub hau ntawm 4 kab lis kev cai (Daim duab 4a): Ctrl: lub plawv kab lis kev cai hauv peb cov kab lis kev cai yav dhau los tau piav qhia zoo li qub siv peb qhov nruab nrab optimized;20.21 TD: T3 thiab Ctrl s Ntxiv Dex rau hnub Wednesday;MC: lub plawv kab lis kev cai hauv CTCM siv peb qhov nruab nrab optimized yav dhau los;thiab MT: CTCM nrog T3 thiab Dex ntxiv rau qhov nruab nrab.Tom qab 12 hnub ntawm kev cog qoob loo, qhov ua tau zoo ntawm MS thiab MT cov ntaub so ntswg tseem zoo ib yam li hauv cov ntaub so ntswg tshiab tau soj ntsuam los ntawm MTT kev soj ntsuam (Fig. 4b).Interestingly, qhov sib ntxiv ntawm T3 thiab Dex rau kev hloov pauv kab lis kev cai (TD) tsis ua rau muaj kev txhim kho tseem ceeb hauv kev muaj txiaj ntsig zoo piv rau Ctrl tej yam kev mob, qhia txog lub luag haujlwm tseem ceeb ntawm kev ua kom lub cev muaj zog hauv kev tswj hwm kev muaj peev xwm ntawm lub plawv.
Ib daim duab tsim kev sim piav qhia txog plaub kab lis kev cai siv los ntsuas qhov cuam tshuam ntawm kev siv tshuab thiab T3 / Dex supplementation ntawm nruab nrab rau 12 hnub. b Bar graph qhia qhov muaj nuj nqis ntawm kev muaj peev xwm 12 hnub tom qab kab lis kev cai hauv tag nrho 4 kab lis kev cai (Ctrl, TD, MC, thiab MT) piv rau lub plawv tshiab (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD thiab D12 MT), 12 (D12 MC) cov hlais / ib pawg #0. 01, ###p <0.001 piv rau D0 thiab **p <0.01 piv rau D12 Ctrl). b Bar graph qhia qhov muaj nuj nqis ntawm kev muaj peev xwm 12 hnub tom qab kab lis kev cai hauv tag nrho 4 kab lis kev cai (Ctrl, TD, MC, thiab MT) piv rau lub plawv tshiab (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD thiab D12 MT), 12 (D12 MC) cov hlais / ib pawg #0. 01, ###p <0.001 piv rau D0 thiab **p <0.01 piv rau D12 ctrl). b пстогра Оказывает кизественнкуеннкуенности культи во во воех 4 улильвияхияхияхияхияхияхияхияхияхияхияхиях Eования (Td, Mc thiab MT) разных свинеи тровортсий тест тренний тавнению и и D0 с ** ** P <0,01 Ya D12 Ctrl). b Daim duab qhia txog qhov muaj peev xwm ntawm 12 hnub tom qab kab lis kev cai nyob rau hauv tag nrho 4 kab lis kev cai (tswj, TD, MC, thiab MT) piv rau lub plawv tshiab (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD, thiab D12 MT), 12 (D12 MT), 12 (D12 MC nce los ntawm qhov sib txawv . #pig # , # # 0 # / # / # / # / # / pig # 0001, ###p <0.001 vs. D0 thiab **p <0.01 los ntawm piv rau 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, D1 MT) Ctrl 1, D同猪的12 (D12 MC) 切片/组，进行单向ANOVA 测试；####p < 0.0001，###p < 0.001 与D0 相毸，0.D 1*2.)b 4 12 (D12 MC) b Гистограмма, показывающая все 4 условия культивирования (контроль, TD, MC и MT) по сравнению со сравнению (1 зравнению со свижижием) 8 (D0), 15 (D12 Ctrl, D12 TD thiab D12 MT), от разных свиней 12 (D12 MC) срезы/группа, односторонний тест ANOVA; ##0#0#p <нересторонний тест ANOVA; ##0#0#0 нию с D0, **p <0,01 по сравнению с контролем D12). b Histogram qhia tag nrho 4 kab lis kev cai (tswj, TD, MC thiab MT) piv rau cov ntu tshiab lub plawv (D0) (n = 18 (D0), 15 (D12 Ctrl, D12 TD thiab D12 MT), los ntawm cov npua sib txawv 12 (D12 MC) ntu ​​/ pab pawg, ib-txoj kev ANOVA xeem <0.####p0. ** p <0.01 vs. tswj D12). c Bar graph qhia qhov muaj nuj nqis ntawm qabzib flux 12 hnub tom qab kab lis kev cai hauv tag nrho 4 kab lis kev cai (Ctrl, TD, MC, thiab MT) piv rau lub plawv tshiab (D0) (n = 6 daim / pab pawg los ntawm cov npua sib txawv, ib txoj kev ANOVA xeem yog ua; ###p <0.001, piv rau D0 thiab *** Ctrl 1. c Bar graph qhia qhov muaj nuj nqis ntawm qabzib flux 12 hnub tom qab kab lis kev cai hauv tag nrho 4 kab lis kev cai (Ctrl, TD, MC, thiab MT) piv rau lub plawv tshiab (D0) (n = 6 daim / pab pawg los ntawm cov npua sib txawv, ib txoj kev ANOVA xeem yog ua; ###p <0.001, piv rau D0 thiab *** Ctrl 1. c Гистограмма показывает количественную оценку потока глюкозы через 12 дней после культивховавсья во 4 ания (контроль, TD, MC и MT) по сравнению со свежими срезами сердца (D0) (n = 6 срезов/группоу от разнный с винтрей , ся тест ANOVA; ###p < 0,001 по сравнению с D0 и ***p < 0,001 по сравнению с D12 Ctrl). c Histogram qhia tias muaj pes tsawg leeg ntawm qabzib flux 12 hnub tom qab kab lis kev cai nyob rau hauv tag nrho 4 kab lis kev cai tej yam kev mob (tswj, TD, MC thiab MT) piv rau lub plawv tshiab (D0) (n = 6 seem / pawg los ntawm txawv npua, ib-txoj kev ANOVA xeem ua ; ###p <0.001 piv rau D0. Ctrl ***p 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，培养倎的鏑n = 6 片/组，来自不同猪，单向执行ANOVA 测试；###p < 0.001，与D0 相比，***p < 0.001 与D12 Ctrl ） C 条形图 显示所有 4 种 条件 ((ctrl 、 td 、 mc 和 mt ) 新鲜心脏 切片 切片 切片 君 君天的通量 定量（n = 6 片/组，来自猪，，，，，，，，，，，，，猪猪单向执行ANOVA <片与D0 ， #0***与 #0 ， #0*** .001 与D12 Ctrl 相比). c Гистограмма, показывающая количественную оценку потока глюкозы через 12 дней после култ культивирляния ирования (контроль, TD, MC и MT) по сравнению со свежими срезами сердца (D0) (n = 6 срезов/группа, от равсниныхнона, от равсиныхный, проведены тесты ANOVA; ###p < 0,001 по сравнению с D0, ***p < 0,001 по сравнению с D12 (контроль). c Histogram uas qhia qhov muaj nuj nqis ntawm cov piam thaj hauv 12 hnub tom qab kab lis kev cai rau tag nrho 4 kab lis kev cai (tswj, TD, MC, thiab MT) piv rau cov hlab plawv tshiab (D0) (n = 6 ntu / pawg, los ntawm cov npua sib txawv, unilateral Were ANOVA kuaj tau ua, ###p < 0.0012c piv rau D.d Strain tsom xam cov phiaj xwm tshiab (xiav), hnub 12 MC (ntsuab), thiab hnub 12 MT (liab) cov ntaub so ntswg ntawm kaum cov ntaub so ntswg hauv cheeb tsam cov ntsiab lus (n = 4 daim / pab pawg, ib-txoj kev ANOVA xeem; tsis muaj qhov sib txawv ntawm pawg).e Volcano zaj duab xis uas qhia qhov sib txawv ntawm cov noob nyob rau hauv lub plawv tshiab (D0) piv rau cov kab mob plawv nyob rau hauv cov xwm txheej zoo li qub (Ctrl) lossis hauv qab MT tej yam kev mob (MT) rau 10-12 hnub.f Heatmap ntawm sarcomere noob rau lub plawv kab lis kev cai raws li txhua yam kab lis kev cai.Cov kab yuam kev sawv cev qhov txhais tau tias ± tus qauv sib txawv.
Metabolic dependence ntawm kev hloov ntawm fatty acid oxidation mus rau glycolysis yog ib qho cim ntawm cardiomyocyte dedifferentiation.Tsis paub qab hau cardiomyocytes feem ntau siv cov piam thaj rau ATP ntau lawm thiab muaj hypoplastic mitochondria nrog ob peb cristae5,32.Kev txheeb xyuas kev siv cov piam thaj pom tau tias nyob rau hauv MC thiab MT tej yam kev mob, kev siv cov piam thaj zoo ib yam li hnub 0 cov ntaub so ntswg (Daim duab 4c).Txawm li cas los xij, Ctrl cov qauv qhia pom qhov nce ntxiv hauv kev siv cov piam thaj piv rau cov ntaub so ntswg tshiab.Qhov no qhia tau hais tias kev sib xyaw ntawm CTCM thiab T3 / Dex txhim kho cov ntaub so ntswg muaj peev xwm thiab khaws cov metabolic phenotype ntawm 12-hnub kab lis kev cai hauv plawv.Tsis tas li ntawd, kev soj ntsuam kab mob pom tau tias cov kab mob tseem nyob zoo ib yam li hauv cov ntaub so ntswg tshiab rau 12 hnub nyob rau hauv MT thiab MS tej yam kev mob (Fig. 4d).
Txhawm rau txheeb xyuas qhov cuam tshuam tag nrho ntawm CTCM thiab T3 / Dex ntawm lub ntiaj teb transcriptional toj roob hauv pes ntawm daim tawv nqaij daim tawv nqaij, peb tau ua RNAseq ntawm cov hlab plawv los ntawm tag nrho plaub qhov kev coj noj coj ua (Cov Ntaub Ntawv Ntxiv 1).Interestingly, MT seem tau pom qhov zoo sib xws ntawm cov ntaub so ntswg plawv tshiab, tsuas yog 16 qhov sib txawv ntawm 13,642 cov noob.Txawm li cas los xij, raws li peb tau pom ua ntej, Ctrl daim duab qhia 1229 qhov sib txawv ntawm cov noob tom qab 10-12 hnub hauv kab lis kev cai (Fig. 4e).Cov ntaub ntawv no tau lees paub los ntawm qRT-PCR ntawm lub plawv thiab fibroblast genes (Ntxiv daim duab 7a-c).Interestingly, Ctrl seem qhia downregulation ntawm lub plawv thiab cell voj voog noob thiab ua kom cov inflammatory gene program.Cov ntaub ntawv no qhia tias dedifferentiation, uas ib txwm tshwm sim tom qab kev coj noj coj ua mus sij hawm ntev, tau ua tiav tag nrho raws li MT tej yam kev mob (Ntxiv Fig. 8a,b).Ua tib zoo kawm txog sarcomere noob tau pom tias tsuas yog nyob rau hauv MT tej yam kev mob yog cov noob encoding lub sarcomere (Fig. 4f) thiab ion channel (Supplementary Fig. 9) khaws cia, tiv thaiv lawv los ntawm kev quab yuam nyob rau hauv Ctrl, TD, thiab MC tej yam kev mob.Cov ntaub ntawv no qhia tau hais tias nrog kev sib xyaw ua ke ntawm kev siv tshuab thiab kev lom zem (T3 / Dex), lub plawv hlais cov ntaub ntawv tuaj yeem nyob zoo ib yam li lub plawv tshiab tom qab 12 hnub hauv kab lis kev cai.
Cov kev tshawb pom no tau txais kev txhawb nqa los ntawm qhov tseeb tias cov qauv kev ncaj ncees ntawm cardiomyocytes hauv cov hlab plawv yog qhov zoo tshaj plaws khaws cia nyob rau hauv MT tej yam kev mob rau 12 hnub, raws li qhia los ntawm qhov tsis zoo thiab hauv zos connexin 43 (Fig. 5a).Tsis tas li ntawd, fibrosis nyob rau hauv lub plawv seem nyob rau hauv MT tej yam kev mob tau txo qis piv rau Ctrl thiab zoo ib yam li tshiab lub plawv seem (Fig. 5b).Cov ntaub ntawv no qhia tau hais tias kev sib xyaw ua ke ntawm kev siv tshuab thiab T3 / Dex kho tau zoo khaws cov qauv plawv hauv plawv hauv kab lis kev cai.
Ib tus neeg sawv cev immunofluorescence dluab ntawm troponin-T (ntsuab), connexin 43 (liab), thiab DAPI (xiav) nyob rau hauv freshly cais lub plawv seem (D0) los yog kab lis kev cai rau 12 hnub nyob rau hauv tag nrho plaub lub plawv kab lis kev cai tej yam kev mob (scale bar = 100 µm).). Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0 and D12 Ctrl), 5 (D12 TD, D12 MC thiab D12 MT) slices/pawg los ntawm txawv npua, ib-txoj kev ANOVA xeem yog ua; ####p < 0.0001 < piv rau D0. * 0. Ctrl). Artificial intelligence quantification of the heart tissue structural integrity (n = 7 (D0 and D12 Ctrl), 5 (D12 TD, D12 MC thiab D12 MT) slices/pawg los ntawm txawv npua, ib-txoj kev ANOVA xeem yog ua; #### p < 0.0001 < piv rau D0. 0 * 0. Ctrl). Количественная оценка структурной целостности ткани сердца с помощью искусственного интеллекта D5D 1D 1 MC (n = 7) thiab D12 MT) срезов/группу от разных свиней, проведен однофакторный тест ANOVA; #### p < 0,0001 по сравнинию 0,0*0 * 0 * 0 * 0 * 0 * 0 Ua raws li D12 Ctrl). Quantification ntawm cov qauv kev ncaj ncees ntawm lub plawv cov ntaub so ntswg siv dag zog txawj ntse (n = 7 (D0 thiab D12 Ctrl), 5 (D12 TD, D12 MC thiab D12 MT) seem / pab pawg los ntawm txawv npua, ib-txoj kev ANOVA xeem ua; #### p < 0.0001 piv rau D0 thiab *p 0. * 0. * 0.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/焇鿷进）行单向ANOVA 测试；#### p < 0.0001 与D0 和*p < 0.05 相比，或****p < 0.0001 与D12 Ctrl 相比).对不同猪的心脏结构 完整性（n = 7 (d0 和 d12 ctrl）（5）（d12 td、d12 mc 和 d12 mt）巺茄）行 单向 单向 测试 ； ########### p < 0.0001 与D0 和*p < 0.05 相比，或 ****p < 0.0001 .)Quantification ntawm cov qauv kev ncaj ncees ntawm cov ntaub so ntswg uas siv cov kev txawj ntse hauv cov npua sib txawv (n = 7 (D0 thiab D12 Ctrl), 5 (D12 TD, D12 MC thiab D12 MT) ntu ​​/ pawg) nrog rau ib txoj kev ANOVA xeem;#### p < 0,0001 по сравнению с D0 и *p < 0,05 или ****p < 0,0001 по сравнению с D12 Ctrl). #### p <0.0001 piv rau D0 thiab *p <0.05 los yog ****p <0.0001 piv rau D12 Ctrl). b Cov duab sawv cev thiab kom muaj nuj nqis rau lub plawv slices stained nrog Masson's trichrome stain (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, thiab D12 MC), 9 (D12 MT) slices / pawg los ntawm cov npua sib txawv, ib-txoj kev yog 0 #0 NO. p <0.001, los yog ****p <0.0001 piv rau D12 Ctrl). b Cov duab sawv cev thiab kom muaj nuj nqis rau lub plawv slices stained nrog Masson's trichrome stain (Scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD, thiab D12 MC), 9 (D12 MT) slices / pawg los ntawm cov npua sib txawv, ib-txoj kev yog 0 #0 NO. p <0.001, los yog ****p <0.0001 piv rau D12 Ctrl). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных тримромным блсаси инейка = 500 мкм) (n = 10 (D0, D12 Ctrl, D12 TD и D12 MC), 9 (D12 MT) срезов/группу от разных свиней, выполнятся A ##нолнятся A 0,0001 по сравнению с D0 и ***p < 0,001 или ****p < 0,0001 по сравнению с D12 Ctrl). b Cov duab sawv cev thiab kom muaj nuj nqis ntawm lub plawv seem stained nrog Masson's trichrome stain (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD thiab D12 MC), 9 (D12 MT) ntu ​​/ pab pawg los ntawm cov npua sib txawv, ua ib qho ###00# ANOp; .001 los yog ****p < 0.0001 vs. D12 Ctrl). b 用Masson 三色染料染色的心脏切片的代表性图像和量化（比例尺 = 500 µm) (n = 10 (D Ctrl 1, MC 2) 1)自不同猪的9个（D12 MT）切片/组，进行单因素方差分析；####p < 0.0001 与D0 与 D0 相戈 0.0 ***p <1.0*0. Ctrl 相比). b 用 masson 三色染料的心脏 切片的代表性和 量化 (比例尺尺 尺 = 500 µm) (d 1 0 µm)和 d12 mc) 来自 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 切片 切片 切片/组，进行单因素方差分析；####p < 0.0001 爇片 / 组，进行单因素方差分析；####p < 0.0001 与 * 0 * 0 . 001 与D12 Ctrl 相比). b Репрезентативные изображения и количественная оценка срезов сердца, окрашенных трихромом мнм 5лисяташ км) (n = 10 (D0, D12 Ctrl, D12 TD и D12 MC), 9 (D12 MT) срезов от разных свиней / группы, один- способ ANOVA; ####0#p <0 0,001 или ****p < 0,0001 по сравнению с D12 Ctrl). b Cov duab sawv cev thiab kom muaj nuj nqis ntawm lub plawv seem stained nrog Masson's trichrome (scale bar = 500 µm) (n = 10 (D0, D12 Ctrl, D12 TD thiab D12 MC), 9 (D12 MT) seem los ntawm cov npua sib txawv / pab pawg, ib qho ANOVA * 0, piv rau 1 los yog #0. ***p <0.0001 piv rau D12 Ctrl).Cov kab yuam kev sawv cev qhov txhais tau tias ± tus qauv sib txawv.
Thaum kawg, lub peev xwm ntawm CTCM rau kev ua raws li kev mob plawv hypertrophy tau soj ntsuam los ntawm kev ua kom cov nqaij mos stretch.Hauv CTCM, qhov siab tshaj plaws cua chamber siab tau nce los ntawm 80 mmHg txog 80 mmHg.Art.(ib txwm stretch) mus txog 140 mmHg Art.(Fig. 6a).Qhov no sib haum mus rau 32% nce nyob rau hauv stretch (Daim duab. 6b), uas yav tas los qhia raws li cov feem pua ​​ntawm stretch yuav tsum tau rau lub plawv seem kom ua tau ib tug ntev sarcomere zoo ib yam li pom nyob rau hauv hypertrophy.Kev ncab thiab nrawm ntawm cov ntaub so ntswg ntawm lub plawv thaum lub sij hawm contraction thiab so kom txaus nyob twj ywm nyob rau hauv 6 hnub ntawm kab lis kev cai (Fig. 6c).Lub plawv cov ntaub so ntswg los ntawm MT tej yam kev mob tau raug mus rau ib txwm stretch (MT (Nco)) los yog overstretch mob (MT (OS)) rau rau hnub.Twb tau tom qab plaub hnub hauv kab lis kev cai, hypertrophic biomarker NT-ProBNP tau nce siab nyob rau hauv nruab nrab raws li MT (OS) cov xwm txheej piv rau MT (ib txwm) cov xwm txheej (Fig. 7a).Tsis tas li ntawd, tom qab 6 hnub ntawm kev coj noj coj ua, lub xov tooj ntawm tes hauv MT (OS) (Daim duab 7b) tau nce ntau dua piv rau ntu ntawm MT lub plawv (ib txwm).Tsis tas li ntawd, NFATC4 nuclear translocation tau nce ntau hauv cov ntaub so ntswg overstretched (Fig. 7c).Cov txiaj ntsig no qhia txog kev txhim kho ntawm kev txhim kho pathological tom qab hyperdistension thiab txhawb lub tswv yim tias CTCM cov cuab yeej siv tau los ua lub platform los kawm txog kev ncab ntawm lub plawv hypertrophy signaling.
Cov neeg sawv cev ntawm cov huab cua chamber siab, kua chamber siab, thiab cov ntaub so ntswg txav kev ntsuas pom tau hais tias chamber siab hloov cov kua chamber siab, ua rau ib tug sib thooj txav ntawm cov ntaub so ntswg daim ntaub.b Cov neeg sawv cev ncab feem pua ​​​​thiab stretch tus nqi nkhaus rau ib txwm ncab (txiv kab ntxwv) thiab overstretched (xiav) cov ntaub so ntswg.c Bar graph qhia lub sij hawm voj voog (n = 19 daim rau ib pab pawg, los ntawm cov npua sib txawv), lub sij hawm cog lus (n = 18-19 daim rau ib pab pawg, los ntawm cov npua sib txawv), lub sij hawm so (n = 19 daim ib pab pawg, los ntawm cov npua sib txawv)), amplitude ntawm cov nqaij mos txav (n = 14 daim / pab pawg, los ntawm cov npua sib txawv), peak systolic peak 1 (peaks / peaks) thiab peaksystolic peak so npaum li cas (n = 14 (D0), 15 (D6) ) ntu ​​/ pawg) los ntawm cov npua sib txawv), ob-tailed Tub Ntxhais Kawm T-test tsis muaj qhov sib txawv tseem ceeb hauv ib qho kev ntsuas, qhia tias cov kev txwv no tseem nyob tas li thaum 6 hnub ntawm kab lis kev cai nrog overvoltage.Cov kab yuam kev sawv cev qhov txhais tau tias ± tus qauv sib txawv.
Bar graph quantification ntawm NT-ProBNP concentration nyob rau hauv kab lis kev cai xov xwm los ntawm lub plawv slices cultured nyob rau hauv MT ib txwm stretch (Norm) los yog overstretching (OS) tej yam kev mob (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, thiab D4 MTOS) slices / pawg los ntawm txawv npua, Ob-txoj kev ncab ANOVA. a Bar graph quantification ntawm NT-ProBNP concentration nyob rau hauv kab lis kev cai xov xwm los ntawm lub plawv slices cultured nyob rau hauv MT ib txwm stretch (Norm) los yog overstretching (OS) tej yam kev mob (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, thiab D4 MTOS) slices / pawg los ntawm txawv npua, ob-txoj kev yog 1.** NO.Quantitative histogram ntawm NT-ProBNP concentration nyob rau hauv kab lis kev cai nruab nrab ntawm lub plawv slices cultured nyob rau hauv tej yam kev mob ntawm ib txwm MT stretch (ib txwm) los yog overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm, thiab D4).MTOS) slices / pab pawg los ntawm txawv npua, ob-factor tsom xam ntawm variance;**p <0,01 по сравнению с нормальным растяжением). ** p <0.01 piv rau ib txwm ncab). 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 浓度的匢(2nm) 、 3 (D2 MTOS, D4 MTNorm 和D4 MTOS) 来自不同猪的切片/组，进行双向方差分析；。 Qhov Kev Ntsuas ntawm NT-ProBNP concentration hauv lub plawv hlais kab lis kev cai nyob rau hauv MT ib txwm stretch (Norm) lossis overstretch (OS) tej yam kev mob (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm和D4 MTOS) los ntawm txawv 猪的切片/疄幑可piv nrog ib txwm ncab, p <0.01).histogram Quantification of NT-ProBNP concentrations in heart slices cultured under conditions of normal MT stretch (norm) or overstretch (OS) (n = 4 (D2 MTNorm), 3 (D2 MTOS, D4 MTNorm) and D4 MTOS) slices/group from different pigs, two-way analysis of variance;**p <0,01 по сравнению с нормальным растяжением). ** p <0.01 piv rau ib txwm ncab). b Cov duab sawv cev rau lub plawv daim kab xev stained nrog troponin-T thiab WGA (sab laug) thiab cell size quantification (txoj cai) (n = 330 (D6 MTOS), 369 (D6 MTNorm) hlwb / pab pawg los ntawm 10 qhov sib txawv ntawm cov npua sib txawv, Ob-tailed Student t-test yog ua piv txwv li 0. * 0. b Cov duab sawv cev rau lub plawv daim kab xev stained nrog troponin-T thiab WGA (sab laug) thiab cell size quantification (txoj cai) (n = 330 (D6 MTOS), 369 (D6 MTNorm) hlwb / pab pawg los ntawm 10 qhov sib txawv ntawm cov npua sib txawv, Ob-tailed Student t-test yog piv rau 0. b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗ П (слева) и количесятрвено клеток (справа) (n = 330 (D6 MTOS), 369 (D6 MTNorm) клеток/группу из 10 разных срезов от разных свиовоней, дртре-t ий Стьюдента; ****p < 0,0001 по сравнению с нормальным растяжением). b Cov duab sawv cev ntawm cov seem hauv plawv stained nrog troponin-T thiab AZP (sab laug) thiab cell size quantification (right) (n = 330 (D6 MTOS), 369 (D6 MTNorm) cells/pab pawg los ntawm 10 qhov sib txawv ntawm cov npua sib txawv, ob-tailed Student's piv rau t-0 *** stra. b 用肌钙蛋白-T 和WGA（左）和细胞大小量化（右）染色的心脏切片的代表性图像（n = 6 OS 130） 0 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，0.*0.*0. b Cov duab sawv cev ntawm lub plawv hlais stained nrog calcarein-T thiab WGA (sab laug) thiab cell loj (txoj cai) (n = 330 (D6 MTOS), 369 los ntawm 10 qhov sib txawv (D6 MTNorm)) Cells/组，两方法有尾学0p 生t test. b Репрезентативные изображения срезов сердца, окрашенных тропонином-Т и АЗ П (слева) и количлест ванкест венкест венкест (справа) (n = 330 (D6 MTOS), 369 (D6 MTNorm) из 10 различных срезов от разных свиней Клетки/групки, двуськини ***двуськини ***двуськини 0,0001 по сравнению с нормальным растяжением). b Cov duab sawv cev ntawm lub plawv cov seem uas muaj troponin-T thiab AZP (sab laug) thiab qhov ntau ntawm cov xov tooj ntawm tes (txoj cai) (n = 330 (D6 MTOS), 369 (D6 MTNorm) los ntawm 10 qhov sib txawv ntawm cov npua sib txawv) Cells / pawg, ob-tailed criterion Tub kawm ntawv****p <0.0001 piv rau ib txwm lim). c Cov duab sawv cev rau hnub 0 thiab hnub 6 MTOS lub plawv slices immunolabeled rau troponin-T thiab NFATC4 thiab qhov kom muaj nuj nqis ntawm kev hloov ntawm NFATC4 mus rau lub nuclei ntawm CMs (n = 4 (D0), 3 (D6 MTOS) slices / pawg los ntawm txawv npua, Ob-tailed tub ntxhais kawm ua . * 0. c Cov duab sawv cev rau hnub 0 thiab hnub 6 MTOS lub plawv slices immunolabeled rau troponin-T thiab NFATC4 thiab kom muaj nuj nqis ntawm translocation ntawm NFATC4 mus rau lub nuclei ntawm CMs (n = 4 (D0), 3 (D6 MTOS) slices / pab pawg los ntawm txawv npua, Ob-tailed yog 0). c Репрезентативные изображения для срезов сердца 0 и 6 дней MTOS, иммуномеченых для тропонинена-Тл и NFATC транслокации NFATC4 в ядра кавернозных клеток (n = 4 (D0), 3 (D6 MTOS) срезов/группу от разных свиней , выртсятня ий Стьюдента; *p <0,05). c Cov duab sawv cev rau lub plawv ntu ntawm 0 thiab 6 hnub MTOS, immunolabeled rau troponin-T thiab NFATC4, thiab kom muaj nuj nqis ntawm NFATC4 translocation nyob rau hauv lub nucleus ntawm cavernous hlwb (n = 4 (D0), 3 (D6 MTOS) slices / pab pawg neeg ntawm txawv npua) ua ob-tailed*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 心脏切的片的代表怪的第6 天MTOS 心脏切的片的代表怪囸像，易位至CM 细胞核的量化(n = 4 (D0), 3 (D6 MTOS) 切片/组, 进行双尾学生t 检验；*p <0.05). c Cov duab sawv cev ntawm calcanin-T thiab NFATC4 immunolabeling 第0天和第6天MTOS lub plawv hlais, thiab NFATC4 los ntawm txawv NFATC4 易位至CM cell nucleus 的quantity 化 (n = 4 (D0), 現閄 珶 列 珶 珶 匄 MT尾学生et 电影；*p <0.05). c Репрезентативные изображения срезов сердца MTOS ntawm 0 thiab 6 день для иммуномаркировки тропонином-Т и NFATC4 ранслокации NFATC4 в ядра CM от разных свиней (n = 4 (D0), 3 (D6 MTOS) срез/группа, два- хвостатый t-критеритерий; 5. c Cov duab sawv cev ntawm MTOS lub plawv slices ntawm hnub 0 thiab 6 rau troponin-T thiab NFATC4 immunolabeling thiab kom muaj nuj nqis ntawm NFATC4 translocation nyob rau hauv lub nucleus ntawm CM los ntawm txawv npua (n = 4 (D0), 3 (D6 MTOS) slices/pab pawg, ob-tailed t's. 5).Cov kab yuam kev sawv cev txhais tau tias ± tus qauv sib txawv.
Kev txhais cov hlab plawv kev tshawb fawb xav tau cov qauv ntawm tes uas raug tsim kho lub plawv ib puag ncig.Nyob rau hauv txoj kev tshawb no, ib tug CTCM ntaus ntawv tau tsim thiab tus yam ntxwv uas yuav txhawb nqa ultrathin seem ntawm lub plawv.CTCM system muaj xws li physiologically synchronized electromechanical stimulation thiab T3 thiab Dex kua enrichment.Thaum lub plawv porcine tau nthuav tawm rau cov xwm txheej no, lawv txoj kev muaj peev xwm, kev ncaj ncees ntawm cov txheej txheem, kev ua haujlwm hauv metabolic, thiab kev hloov pauv cov lus qhia tseem zoo ib yam li hauv cov ntaub so ntswg tshiab tom qab 12 hnub ntawm kab lis kev cai.Tsis tas li ntawd, kev ncab ntau dhau ntawm cov ntaub so ntswg tuaj yeem ua rau lub siab hypertrophy los ntawm hyperextension.Zuag qhia tag nrho, cov txiaj ntsig no txhawb nqa lub luag haujlwm tseem ceeb ntawm lub cev kab lis kev cai hauv kev tswj hwm lub plawv ib txwm muaj phenotype thiab muab lub platform rau kev tshuaj xyuas tshuaj.
Muaj ntau yam ua rau tsim kom muaj ib puag ncig zoo rau kev ua haujlwm thiab muaj sia nyob ntawm cardiomyocytes.Qhov pom tseeb tshaj plaws ntawm cov xwm txheej no muaj feem xyuam nrog (1) kev sib cuam tshuam ntawm cov cellular, (2) electromechanical stimulation, (3) humoral yam, thiab (4) metabolic substrates.Physiological cell-rau-cell kev sib cuam tshuam yuav tsum tau complex peb-dimensional tes hauj lwm ntawm ntau hom cell txhawb los ntawm ib tug extracellular matrix.Xws li kev sib cuam tshuam ntawm tes tsis yooj yim rau kev tsim kho hauv vitro los ntawm kev sib koom ua ke ntawm ib tus neeg ntawm tes, tab sis tuaj yeem ua tiav tau yooj yim siv qhov xwm txheej ntawm lub plawv.
Txhua yam stretch thiab hluav taws xob stimulation ntawm cardiomyocytes yog ib qho tseem ceeb rau kev tswj lub plawv phenotype33,34,35.Thaum lub tshuab stimulation tau dav siv rau hiPSC-CM txias thiab maturation, ob peb elegant kev tshawb fawb tsis ntev los no tau sim mechanical stimulation ntawm lub plawv slices nyob rau hauv kab lis kev cai siv uniaxial loading.Cov kev tshawb fawb no qhia tau hais tias 2D uniaxial mechanical loading muaj txiaj ntsig zoo rau phenotype ntawm lub plawv thaum kab lis kev cai.Nyob rau hauv cov kev tshawb fawb no, ntu ntawm lub plawv yog loaded nrog isometric tensile force17, linear auxotonic loading18, los yog lub voj voog ntawm lub plawv tau rov tsim dua siv lub zog transducer tawm tswv yim thiab nro drives.Txawm li cas los xij, cov txheej txheem no siv cov ntaub so ntswg uniaxial stretch yam tsis muaj kev ua kom zoo ib puag ncig, ua rau muaj kev cuam tshuam ntawm ntau lub plawv cov noob los yog overexpression ntawm cov noob cuam tshuam nrog cov lus teb txawv txav.CTCM tau piav qhia ntawm no muab 3D electromechanical stimulus uas ua raws li lub voj voog ntawm lub plawv raws li lub sij hawm voj voog thiab lub cev nyhav (25% stretch, 40% systole, 60% diastole, thiab 72 neeg ntaus ib feeb).Txawm hais tias qhov kev txhawb nqa peb-dimensional mechanical stimulation ib leeg tsis txaus los tswj cov ntaub so ntswg kev ncaj ncees, kev sib xyaw ua ke ntawm kev lom zem thiab kev siv tshuab siv T3 / Dex yuav tsum tau ua kom cov ntaub so ntswg muaj peev xwm, muaj nuj nqi, thiab kev ncaj ncees.
Humoral yam tseem ceeb ua lub luag haujlwm tseem ceeb hauv kev hloov kho tus neeg laus lub plawv phenotype.Qhov no tau qhia meej hauv HiPS-CM cov kev tshawb fawb uas T3 thiab Dex tau ntxiv rau kab lis kev cai xov xwm kom ceev cov cell loj hlob.T3 tuaj yeem cuam tshuam rau kev thauj mus los ntawm cov amino acids, suab thaj thiab calcium hla cov cell membranes36.Tsis tas li ntawd, T3 txhawb nqa MHC-α qhia thiab MHC-β downregulation, txhawb kev tsim ntawm ceev nrooj myofibrils hauv cov laus cardiomyocytes piv rau qeeb twitch myofibrils hauv fetal CM.T3 deficiency nyob rau hauv cov neeg mob hypothyroid ua rau poob ntawm myofibrillar bands thiab txo tus nqi ntawm lub suab loj hlob37.Dex ua rau glucocorticoid receptors thiab tau pom tias yuav ua rau kom myocardial contractility nyob rau hauv ib leeg perfused lub siab;38 qhov kev txhim kho no yog xav tias muaj feem xyuam rau cov txiaj ntsig ntawm calcium deposit-driven entry (SOCE) 39,40.Tsis tas li ntawd, Dex khi rau nws cov receptors, ua rau cov lus teb dav hauv lub cev uas ua rau lub cev tsis muaj zog thiab mob 30.
Peb cov txiaj ntsig tau qhia tias kev txhawb zog ntawm lub cev (MS) txhim kho tag nrho cov kab lis kev cai kev ua tau zoo piv rau Ctrl, tab sis ua tsis tau raws li kev muaj peev xwm, kev ncaj ncees ntawm cov qauv, thiab kev mob plawv tshaj 12 hnub hauv kab lis kev cai.Piv nrog rau Ctrl, qhov sib ntxiv ntawm T3 thiab Dex rau CTCM (MT) kab lis kev cai txhim kho kev muaj peev xwm thiab tswj tau cov ntaub ntawv zoo sib xws, kev ncaj ncees, thiab kev ua haujlwm metabolic nrog cov ntaub so ntswg tshiab rau 12 hnub.Tsis tas li ntawd, los ntawm kev tswj cov qib ntawm cov ntaub so ntswg stretch, ib tug hyperextension-induced cardiac hypertrophy qauv tau tsim siv STCM, piv txwv li versatility ntawm STCM system.Nws yuav tsum raug sau tseg tias txawm hais tias kev kho mob plawv thiab fibrosis feem ntau cuam tshuam nrog cov kab mob hauv lub cev uas nws cov hlwb tuaj yeem muab cov cytokines tsim nyog nrog rau phagocytosis thiab lwm yam kev hloov kho, cov ntu ntawm lub plawv tseem tuaj yeem ua raws li cov txheej txheem fibrotic hauv kev teb rau kev ntxhov siab thiab kev raug mob.nyob rau hauv myofibroblasts.Qhov no tau raug soj ntsuam yav dhau los hauv daim qauv ntawm lub plawv.Nws yuav tsum raug sau tseg tias CTCM tsis tuaj yeem hloov kho los ntawm kev hloov pauv siab / hluav taws xob amplitude thiab zaus los simulate ntau yam mob xws li tachycardia, bradycardia, thiab kev txhawb nqa txhua yam (lub plawv tsis muaj zog).Qhov no ua rau lub kaw lus nruab nrab ntawm cov khoom siv rau kev sim tshuaj.Lub peev xwm ntawm CTCM los ua qauv overexertion-induced cardiac hypertrophy paves txoj kev rau kev sim no system rau tus kheej txoj kev kho.Hauv kev xaus, txoj kev tshawb fawb tam sim no qhia tau hais tias kev siv tshuab ncab thiab kev lom zem yog qhov tseem ceeb rau kev tswj hwm kab lis kev cai ntawm cov kab mob plawv.
Txawm hais tias cov ntaub ntawv nthuav tawm ntawm no qhia tias CTCM yog qhov muaj txiaj ntsig zoo rau kev ua qauv zoo li myocardium, qhov kab lis kev cai no muaj qee qhov kev txwv.Lub ntsiab kev txwv ntawm CTCM kab lis kev cai yog tias nws imposes dynamic mechanical stresses ntawm lub slices, uas txwv tsis pub muaj peev xwm mus saib xyuas lub plawv daim hlab contractions nyob rau hauv txhua lub voj voog.Tsis tas li ntawd, vim qhov me me ntawm cov hlab plawv (7 hli), lub peev xwm los soj ntsuam systolic muaj nuj nqi sab nraud ntawm kab lis kev cai siv cov tsoos quab yuam sensors raug txwv.Nyob rau hauv daim ntawv tam sim no, peb ib nrab kov yeej qhov kev txwv no los ntawm kev ntsuas qhov muag qhov hluav taws xob raws li qhov taw qhia ntawm kev ua haujlwm contractile.Txawm li cas los xij, qhov kev txwv no yuav xav tau kev ua haujlwm ntxiv thiab yuav raug hais nyob rau yav tom ntej los ntawm kev qhia txog kev kho qhov muag ntawm kev ua haujlwm ntawm lub plawv hlais hauv kab lis kev cai, xws li optical mapping siv calcium thiab voltage-sensitive dyes.Lwm qhov kev txwv ntawm CTCM yog tias tus qauv ua haujlwm tsis tswj kev ntxhov siab ntawm lub cev (preload thiab afterload).Nyob rau hauv CTCM, lub siab raug ntxias nyob rau hauv cov lus qhia rov qab los tsim 25% physiological stretch nyob rau hauv diastole (tag nrho stretch) thiab systole (ntev ntawm contraction thaum lub sij hawm hluav taws xob stimulation) nyob rau hauv cov ntaub so ntswg loj heev.Qhov kev txwv no yuav tsum raug tshem tawm yav tom ntej CTCM cov qauv tsim los ntawm kev txaus siab ntawm cov ntaub so ntswg ntawm ob sab thiab los ntawm kev siv cov kev sib raug zoo ntawm lub siab-ntim uas tshwm sim hauv cov chav hauv plawv.
Qhov kev hloov kho overstretch-induced tau tshaj tawm hauv phau ntawv no tsuas yog ua raws li cov cim hypertrophic hyperstretch.Yog li, tus qauv no tuaj yeem pab hauv kev kawm txog kev ncab-txhim kho hypertrophic signaling yam tsis tas yuav tsum muaj kev lom zem lossis neural yam (uas tsis muaj nyob hauv qhov system no).Cov kev tshawb fawb ntxiv yog xav tau los ua kom muaj ntau yam ntawm CTCM, piv txwv li, kev sib koom ua ke nrog lub cev tiv thaiv kab mob, ua rau cov ntshav plasma humoral yam, thiab innervation thaum co-culturing nrog cov hlwb neuronal yuav txhim kho qhov muaj peev xwm ntawm cov kab mob ua qauv nrog CTCM.
Kaum peb npua tau siv rau hauv txoj kev tshawb no.Tag nrho cov txheej txheem tsiaj tau ua raws li cov txheej txheem hauv tsev thiab tau pom zoo los ntawm University of Louisville Institutional Animal Care and Use Committee.Lub aortic arch tau clamped thiab lub plawv tau perfused nrog 1 L ntawm sterile cardioplegia (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 U / mL heparin, pH txog 7.4); lub siab tau khaws cia rau hauv cov dej khov-txias cardioplegic tov kom txog thaum thauj mus rau qhov chaw kuaj dej khov uas feem ntau yog <10 min. lub siab tau khaws cia rau hauv cov dej khov-txias cardioplegic tov kom txog thaum thauj mus rau qhov chaw kuaj dej khov uas feem ntau yog <10 min. сердца хранили в ледяном кардиоплегическом растворе до транспортировки в лабораторию на льду, что обаторовки в лабораторию на льду, что обаты lub siab tau muab khaws cia rau hauv cov dej khov-txias cardioplegic tov kom txog thaum thauj mus rau qhov chaw kuaj dej khov, uas feem ntau siv sijhawm <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. Khaws lub siab ntawm cov dej khov cardioplegia kom txog thaum thauj mus rau qhov chaw kuaj dej khov, feem ntau <10 min.
CTCM ntaus ntawv tau tsim nyob rau hauv SolidWorks computer-aided design (CAD) software.Kab lis kev cai chamber, dividers thiab cua chambers yog ua los ntawm CNC ntshiab acrylic yas.Lub 7 hli txoj kab uas hla rov qab lub nplhaib yog ua los ntawm high density polyethylene (HDPE) nyob rau hauv nruab nrab thiab muaj ib tug o-ring zawj kom haum rau silicone o-ring siv los kaw cov xov xwm hauv qab.Ib lub silica nyias nyias cais cov kab lis kev cai chamber los ntawm cov phaj sib cais.Silicone daim nyias nyias yog laser txiav los ntawm 0.02 ″ tuab silicone daim ntawv thiab muaj ib tug hardness ntawm 35A.Hauv qab thiab sab saum toj silicone gaskets yog laser txiav los ntawm 1/16 ″ tuab silicone ntawv thiab muaj ib tug hardness ntawm 50A.316L stainless hlau screws thiab tis txiv ntseej yog siv rau fastening lub thaiv thiab tsim ib tug airtight foob.
Lub rooj tsav xwm tshwj xeeb luam tawm Circuit Board (PCB) yog tsim los ua ke nrog C-PACE-EM system.Lub tshuab swiss connector sockets ntawm PCB txuas nrog graphite electrodes los ntawm cov nyiaj-plated tooj liab xov hlau thiab tooj liab 0-60 screws screwed rau hauv cov electrodes.Lub rooj tsavxwm luam tawm tau muab tso rau hauv lub hau ntawm lub tshuab luam ntawv 3D.
Cov cuab yeej CTCM yog tswj los ntawm lub programmable pneumatic actuator (PPD) uas tsim kom muaj kev tswj cov hlab ntshav zoo ib yam li lub voj voog plawv.Raws li lub siab nyob rau hauv cov huab cua chamber nce, lub saj zawg zog silicone membrane expands upward, yuam cov nruab nrab nyob rau hauv cov ntaub so ntswg qhov chaw.Lub cheeb tsam ntawm cov ntaub so ntswg yuav raug stretched los ntawm no expulsion ntawm kua, ua raws li lub physiological expansion ntawm lub plawv thaum lub sij hawm diastole.Thaum lub ncov ntawm so, hluav taws xob stimulation tau siv los ntawm graphite electrodes, uas txo cov siab nyob rau hauv cov huab cua chamber thiab ua rau contraction ntawm cov ntaub so ntswg.Nyob rau hauv cov yeeb nkab yog lub hemostatic valve nrog lub siab sensor los xyuas lub siab nyob rau hauv cov huab cua system.Lub siab xav tau los ntawm lub siab sensor yog siv rau cov ntaub ntawv khaws cia txuas nrog lub laptop.Qhov no tso cai rau kev soj ntsuam tas li ntawm lub siab hauv cov pa roj chamber.Thaum lub siab tshaj plaws chamber siab tau mus txog (tus qauv 80 mmHg, 140 mmHg OS), cov ntaub ntawv tau txais cov cuab yeej raug yuam kom xa cov teeb liab mus rau C-PACE-EM system los tsim ib lub teeb liab biphasic voltage rau 2 ms, teem rau 4 V.
Lub plawv ntu tau txais thiab kab lis kev cai hauv 6 lub qhov dej tau ua raws li hauv qab no: Hloov lub siab lub siab los ntawm lub nkoj hloov mus rau lub tais uas muaj qhov txias (4 ° C.) cardioplegia.Sab laug ventricle raug rho tawm nrog cov hniav tsis muaj menyuam thiab txiav rau hauv daim ntawm 1-2 cm3.Cov ntaub so ntswg tau txuas nrog cov ntaub so ntswg txhawb nqa nrog cov ntaub nplaum thiab muab tso rau hauv lub vibrating microtome cov ntaub so ntswg da dej uas muaj Tyrode cov tshuaj thiab tsis tu ncua oxygenated (3 g / L 2,3-butanedione monooxime (BDM), 140 mM NaCl (8.18 g) . ), 6 mM KCl (0.447 g.-18 mEP), 10 mM KCl (0.447 g.-18 mEP), 38 g), 1 mM MgCl2 (1 ml 1 M tov), ​​1.8 mM CaCl2 (1.8 ml 1 M tov), ​​mus txog 1 L ddH2O).Lub vibrating microtome tau teem caij txiav 300 µm tuab tuab ntawm qhov zaus ntawm 80 Hz, kab rov tav vibration amplitude ntawm 2 hli, thiab tus nqi ua ntej ntawm 0.03 mm / s.Cov ntaub so ntswg da dej nyob ib puag ncig los ntawm cov dej khov ua kom cov kua dej txias thiab khaws cia ntawm 4 ° C.Hloov cov ntaub so ntswg los ntawm microtome da dej mus rau ib qho chaw incubation da dej uas tsis tu ncua oxygenated Tyrode tov rau dej khov kom txog thaum txaus seem tau txais rau ib kab lis kev cai phaj.Rau transwell kab lis kev cai, cov ntaub so ntswg tau txuas rau sterile 6 mm wide polyurethane kev txhawb nqa thiab muab tso rau hauv 6 ml ntawm optimized nruab nrab (199 nruab nrab, 1x ITS ntxiv, 10% FBS, 5 ng / ml VEGF, 10 ng / ml FGF-alkaline thiab 2X tshuaj tua kab mob-antifungal).Hluav taws xob stimulation (10 V, zaus 1.2 Hz) tau siv rau cov ntaub so ntswg los ntawm C-Pace.Rau TD cov xwm txheej, T3 tshiab thiab Dex tau ntxiv ntawm 100 nM thiab 1 μM ntawm txhua qhov kev hloov pauv nruab nrab.Qhov nruab nrab yog saturated nrog oxygen ua ntej hloov 3 zaug ib hnub twg.Cov ntaub so ntswg tau coj mus rau hauv qhov chaw tsim khoom ntawm 37 ° C thiab 5% CO2.
Rau CTCM kab lis kev cai, cov ntaub so ntswg tau muab tso rau ntawm lub tshuab luam ntawv 3D kev cai hauv Petri tais uas hloov kho Tyrode cov tshuaj.Cov cuab yeej tsim los ua kom qhov loj ntawm daim hlais ntawm lub plawv los ntawm 25% ntawm thaj tsam ntawm lub nplhaib txhawb nqa.Qhov no yog ua kom cov ntu ntawm lub plawv tsis ncab tom qab tau pauv los ntawm Tyrode cov tshuaj mus rau qhov nruab nrab thiab thaum lub sij hawm diastole.Siv cov kua nplaum histoacrylic, ntu 300 µm tuab tau tsau rau ntawm lub nplhaib txhawb nqa 7 hli inch.Tom qab txuas cov ntaub so ntswg mus rau lub nplhaib txhawb nqa, txiav tawm cov ntaub so ntswg ntau dhau thiab tso cov ntaub so ntswg txuas rov qab rau hauv da dej ntawm Tyrode tov ntawm dej khov (4 ° C) kom txog thaum cov seem txaus tau npaj rau ib lub cuab yeej.Lub sijhawm ua haujlwm tag nrho rau txhua yam khoom siv yuav tsum tsis pub tshaj 2 teev.Tom qab 6 cov ntaub so ntswg tau txuas nrog lawv lub nplhaib txhawb nqa, cov khoom siv CTCM tau sib sau ua ke.CTCM kab lis kev cai chamber yog pre-filled nrog 21 ml pre-oxygenated nruab nrab.Hloov cov ntaub so ntswg mus rau hauv kab lis kev cai chamber thiab ua tib zoo tshem tawm cov cua npuas nrog lub pipette.Cov ntaub so ntswg yog ces coj mus rau hauv lub qhov thiab maj mam nias rau hauv qhov chaw.Thaum kawg, tso lub hau electrode rau ntawm lub cuab yeej thiab hloov lub cuab yeej mus rau qhov chaw tsim khoom.Tom qab ntawd txuas CTCM rau lub raj cua thiab C-PACE-EM system.Lub pneumatic actuator qhib thiab cua valve qhib lub CTCM.C-PACE-EM system tau teeb tsa kom xa 4 V ntawm 1.2 Hz thaum lub sijhawm biphasic pacing rau 2 ms.Qhov nruab nrab tau hloov ob zaug ib hnub twg thiab cov electrodes tau hloov ib zaug ib hnub kom tsis txhob tsub zuj zuj ntawm graphite ntawm cov electrodes.Yog tias tsim nyog, cov ntaub so ntswg tuaj yeem raug tshem tawm ntawm lawv cov kab lis kev cai kom tshem tawm cov npuas huab cua uas yuav poob rau hauv lawv.Rau MT kev kho mob, T3 / Dex tau ntxiv tshiab nrog txhua qhov kev hloov pauv nruab nrab nrog 100 nM T3 thiab 1 μM Dex.Cov khoom siv CTCM tau coj mus rau hauv qhov chaw tsim khoom ntawm 37 ° C thiab 5% CO2.
Txhawm rau kom tau txais kev nthuav dav ntawm lub plawv hlais, lub koob yees duab tshwj xeeb tau tsim.Lub koob yees duab SLR (Canon Rebel T7i, Canon, Tokyo, Nyiv) tau siv nrog Navitar Zoom 7000 18-108mm macro lens (Navitar, San Francisco, CA).Kev pom pom tau ua nyob rau hauv chav tsev kub tom qab hloov qhov nruab nrab nrog qhov nruab nrab tshiab.Lub koob yees duab yog positioned ntawm 51 ° lub kaum sab xis thiab yees duab kaw ntawm 30 ntas ib ob.Ua ntej, qhib qhov software (MUSCLEMOTION43) tau siv nrog Image-J los ntsuas qhov kev txav ntawm lub plawv.Lub npog ntsej muag tau tsim los siv MATLAB (MathWorks, Natick, MA, USA) los txhais cov cheeb tsam txaus siab rau kev ntaus lub plawv kom tsis txhob muaj suab nrov.Manually segmented qhov ncauj qhov ntswg yog siv rau tag nrho cov duab nyob rau hauv ib tug thav duab ib theem zuj zus thiab ces dhau mus rau MUSCLEMOTION plug-in.Muscle Motion siv qhov nruab nrab qhov siv ntawm cov pixels nyob rau hauv txhua lub thav duab los ntsuas nws qhov kev txav mus los ntawm tus qauv siv.Cov ntaub ntawv raug kaw, lim thiab siv los ntsuas lub sijhawm voj voog thiab ntsuas cov ntaub so ntswg stretch thaum lub voj voog plawv.Cov yees duab kaw tau tom qab ua tiav siv thawj zaug xaj xoom-theem digital lim.Txhawm rau kom muaj nuj nqis ntawm cov ntaub so ntswg stretch (peak-to-peak), ncov-rau-peak tsom xam tau ua kom paub qhov txawv nruab nrab ntawm peaks thiab troughs hauv cov teeb liab kaw.Tsis tas li ntawd, detrending yog ua los ntawm 6th kev txiav txim polynomial kom tshem tawm cov teeb liab drift.Txoj cai lij choj tau tsim nyob rau hauv MATLAB los txiav txim siab thoob ntiaj teb cov ntaub so ntswg txav, lub sij hawm voj voog, lub sij hawm so, thiab lub sij hawm sib cog lus (Cov Txheej Txheem Ntxiv 44).
Rau kev tsom xam, siv tib cov vis dis aus tsim rau kev ntsuas kev sib tw, peb thawj zaug taug qab ob daim duab uas sawv cev rau qhov siab tshaj plaws (sab sauv) thiab qis tshaj (qis) cov ntsiab lus ntawm cov lus tsa suab) raws li MUSCLEMOTION software.Peb mam li segmented cov ntaub so ntswg cheeb tsam thiab siv ib daim ntawv ntawm shading algorithm rau cov segmented cov ntaub so ntswg (Ntxiv daim duab 2a).Cov ntaub so ntswg segmented tau muab faib ua kaum subsurfaces, thiab kev ntxhov siab ntawm txhua qhov saum npoo tau suav nrog kev sib npaug hauv qab no: Strain = (Sup-Sdown) / Sdown, qhov twg Sup thiab Sdown yog qhov kev ncua deb ntawm cov duab los ntawm sab saum toj thiab hauv qab ntxoov ntxoo ntawm cov ntaub, raws li (Supplementary Fig. .2b).
Lub plawv ntu tau kho hauv 4% paraformaldehyde rau 48 teev.Cov ntaub so ntswg qhuav tau qhuav hauv 10% thiab 20% sucrose rau 1 h, tom qab ntawd hauv 30% sucrose thaum hmo ntuj.Cov seem tau muab tso rau hauv qhov zoo tshaj plaws txiav qhov kub thiab txias (OCT compound) thiab maj mam khov rau hauv ib qho isopentane / qhuav dej khov da dej.Khaws OCT embedding blocks ntawm -80 ° C kom txog thaum sib cais.Slides tau npaj ua ntu nrog lub thickness ntawm 8 μm.
Txhawm rau tshem tawm OCT ntawm lub plawv, ua kom sov cov slides rau ntawm qhov ntsuas kub ntawm 95 ° C rau 5 min.Ntxiv 1 ml PBS rau txhua qhov swb thiab ua rau 30 feeb ntawm chav sov, tom qab ntawd permeate cov ntu los ntawm kev teeb tsa 0.1% Triton-X hauv PBS rau 15 feeb ntawm chav tsev kub.Txhawm rau tiv thaiv tsis muaj cov tshuaj tiv thaiv tshwj xeeb los ntawm kev khi rau cov qauv, ntxiv 1 ml ntawm 3% BSA tov rau swb thiab incubate rau 1 teev ntawm chav tsev kub.Tom qab ntawd BSA raug tshem tawm thiab cov swb raug ntxuav nrog PBS.Kos txhua tus qauv nrog ib tug cwj mem.Thawj cov tshuaj tiv thaiv (diluted 1: 200 hauv 1% BSA) (connexin 43 (Abcam; # AB11370), NFATC4 (Abcam; # AB99431) thiab troponin-T (Thermo Scientific; # MA5-12960) tau ntxiv ntau dua 901 nas this (tom qab ntawd cov tshuaj tiv thaiv BSA thib ob) Fluor 488 (Thermo Scientific; #A16079), tawm tsam luav Alexa Fluor 594 (Thermo Scientific; #T6391) ntxiv rau 90 feeb Ntxuav 3 zaug nrog PBS Kom paub qhov txawv ntawm lub hom phiaj staining los ntawm keeb kwm yav dhau, peb tsuas yog siv cov tshuaj tiv thaiv kab mob thib ob ua tus tswj hwm. Thaum kawg, cov khoom siv hluav taws xob tau muab tso rau hauv qhov chaw ua haujlwm PI. thiab kaw nrog ntsia thawv polish. -x magnification) thiab Keyence microscope nrog 40x magnification.
WGA-Alexa Fluor 555 (Thermo Scientific; #W32464) ntawm 5 μg / ml hauv PBS tau siv rau WGA staining thiab siv rau qhov chaw ruaj khov rau 30 feeb ntawm chav tsev kub.Tom qab ntawd cov slides raug ntxuav nrog PBS thiab Sudan dub tau ntxiv rau txhua qhov swb thiab incubated rau 30 feeb.Cov slides raug ntxuav nrog PBS thiab vectashield embedding nruab nrab tau ntxiv.Slides tau pom ntawm Keyence microscope ntawm 40x magnification.
OCT raug tshem tawm ntawm cov qauv raws li tau piav qhia saum toj no.Tom qab tshem tawm OCT, raus cov slides hauv Bouin cov tshuaj thaum hmo ntuj.Tom qab ntawd cov slides tau muab yaug nrog dej distilled rau 1 teev thiab muab tso rau hauv Bibrich aloe acid fuchsin tov rau 10 feeb.Tom qab ntawd cov slides tau ntxuav nrog dej distilled thiab muab tso rau hauv ib qho kev daws ntawm 5% phosphomolybdenum / 5% phosphotungstic acid rau 10 feeb.Tsis tas yuav yaug, hloov cov slides ncaj qha mus rau hauv cov tshuaj aniline xiav rau 15 feeb.Tom qab ntawd cov swb raug ntxuav nrog dej distilled thiab muab tso rau hauv 1% acetic acid tov rau 2 feeb.Cov swb tau qhuav hauv 200 N ethanol thiab xa mus rau xylene.Stained slides tau pom pom siv Keyence microscope nrog lub hom phiaj 10x.Fibrosis cheeb tsam feem pua ​​​​tau suav nrog Keyence Analyzer software.
CyQUANT™ MTT Cell Viability Assay (Invitrogen, Carlsbad, CA), tus lej xov tooj V13154, raws li cov chaw tsim khoom raws tu qauv nrog qee qhov kev hloov kho.Tshwj xeeb, ib qho kev phais phais nrog txoj kab uas hla ntawm 6 hli tau siv los xyuas kom meej cov ntaub so ntswg loj thaum MTT tsom xam.Cov ntaub so ntswg tau muab tso rau hauv ib lub qhov dej ntawm 12-zoo phaj uas muaj MTT substrate raws li cov chaw tsim khoom raws tu qauv.Cov seem yog incubated ntawm 37 ° C. rau 3 teev thiab cov ntaub so ntswg nyob metabolizes MTT substrate los ua ib tug ntshav formazan compound.Hloov cov tshuaj MTT nrog 1 ml DMSO thiab incubate ntawm 37 ° C rau 15 feeb kom rho tawm cov ntshav formazan ntawm lub plawv.Cov qauv tau diluted 1:10 hauv DMSO hauv 96-zoo meej hauv qab daim hlau thiab cov xim liab liab ntsuas ntawm 570 nm siv Cytation phaj nyeem ntawv (BioTek).Kev nyeem ntawv tau normalized rau qhov hnyav ntawm txhua daim ntawm lub plawv.
Lub plawv hlais tawm tau hloov nrog cov xov xwm uas muaj 1 μCi / ml [5-3H]-glucose (Moravek Biochemicals, Brea, CA, USA) rau kev siv cov piam thaj raws li tau piav qhia dhau los.Tom qab 4 teev ntawm incubation, ntxiv 100 µl ntawm nruab nrab mus rau ib tug qhib microcentrifuge raj uas muaj 100 µl ntawm 0.2 N HCl.Tom qab ntawd cov raj tau muab tso rau hauv ib lub raj xaim hluav taws xob uas muaj 500 μl ntawm dH2O kom evaporate [3H] 2O rau 72 teev ntawm 37 ° C.Tom qab ntawd tshem tawm cov raj microcentrifuge los ntawm lub raj xa dej thiab ntxiv 10 ml ntawm cov kua dej tawm.Scintillation suav tau siv lub Tri-Carb 2900TR kua scintillation analyzer (Packard Bioscience Company, Meriden, CT, USA).Kev siv cov piam thaj hauv qab no suav nrog suav nrog [5-3H] - qabzib cov haujlwm tshwj xeeb, ua tsis tiav qhov sib npaug thiab keeb kwm yav dhau, dilution ntawm [5-3H] - rau cov piam thaj uas tsis muaj npe, thiab cov tshuaj tiv thaiv kev ua haujlwm.Cov ntaub ntawv yog normalized rau qhov loj ntawm seem ntawm lub plawv.
Tom qab cov ntaub so ntswg homogenization hauv Trizol, RNA raug cais tawm ntawm lub plawv siv Qiagen miRNeasy Micro Kit #210874 raws li cov chaw tsim khoom raws tu qauv.RNAsec lub tsev qiv ntawv npaj, ua ntu zus thiab txheeb xyuas cov ntaub ntawv tau ua raws li hauv qab no:
1 μg ntawm RNA ib qho piv txwv tau siv los ua cov khoom pib rau kev npaj ntawm lub tsev qiv ntawv RNA.Cov tsev qiv ntawv Sequencing tau tsim los siv NEBNext UltraTM RNA Library Preparation Kit rau Illumina (NEB, USA) ua raws li cov chaw tsim khoom pom zoo, thiab cov lej ntsuas tau ntxiv rau cov khoom sib txuas rau txhua tus qauv.Luv luv, mRNA raug ntxuav los ntawm tag nrho RNA siv cov hlaws sib nqus txuas nrog poly-T oligonucleotides.Fragmentation yog nqa tawm siv divalent cations ntawm qhov kub thiab txias hauv NEBNext First Strand Synthesis Reaction Buffer (5X).Thawj strand cDNA tau tsim los siv random hexamer primers thiab M-MuLV thim rov qab transcriptase (RNase H-).Qhov thib ob strand cDNA yog ces synthesized siv DNA polymerase I thiab RNase H. Cov seem overhangs yog hloov dua siab tshiab rau blunt kawg los ntawm exonuclease/polymerase kev ua.Tom qab adenylation ntawm 3' kawg ntawm DNA tawg, NEBNext Adapter nrog lub voj voos plaub hau txuas rau nws los npaj nws rau hybridization.Rau kev xaiv ntawm cDNA fragments ntawm nyiam ntev 150-200 bp.cov khoom siv hauv tsev qiv ntawv tau huv si siv AMPure XP system (Beckman Coulter, Beverly, USA).Tom qab ntawd, 3 μl USER Enzyme (NEB, USA) nrog qhov loj-xaiv cDNA ligated nrog lub adapter tau siv rau 15 feeb ntawm 37 ° C thiab tom qab ntawd rau 5 feeb ntawm 95 ° C ua ntej PCR.PCR tau ua tiav siv Phusion High-Fidelity DNA polymerase, universal PCR primers, thiab Index (X) primers.Thaum kawg, PCR cov khoom raug purified (AMPure XP system) thiab tsev qiv ntawv zoo soj ntsuam ntawm Agilent Bioanalyzer 2100 system.Lub tsev qiv ntawv cDNA tau ua raws li kev siv Novaseq sequencer.Raw duab cov ntaub ntawv los ntawm Illumina tau hloov dua siab tshiab rau cov ntawv nyeem siv CASAVA Base Hu.Cov ntaub ntawv nyoos yog khaws cia rau hauv FASTQ(fq) cov ntaub ntawv uas muaj cov ntawv nyeem thiab cov hauv paus zoo.Xaiv HISAT2 kom phim cov lim dej sib txuas nyeem rau Sscrofa11.1 siv genome.Feem ntau, HISAT2 txhawb nqa genomes ntawm txhua qhov loj me, suav nrog cov genomes loj dua 4 billion bases, thiab cov nqi qub tau teeb tsa rau feem ntau qhov tsis muaj.Splicing nyeem los ntawm RNA Seq cov ntaub ntawv tuaj yeem ua tiav zoo siv HISAT2, qhov ceev tshaj plaws uas tam sim no muaj, nrog tib yam lossis qhov tseeb dua li lwm txoj hauv kev.
Kev nplua nuj ntawm cov ntawv sau ncaj qha qhia txog qib ntawm cov noob qhia.Gene qhia qib raug soj ntsuam los ntawm ntau cov ntaub ntawv sau tseg (ntau zaus suav) cuam tshuam nrog cov genome lossis exons.Tus naj npawb ntawm nyeem yog proportional rau gene qhia theem, gene ntev, thiab sequencing qhov tob.FPKM (fragments ib txhiab lub hauv paus khub ntawm cov ntawv sau ua ke ntawm ib lab lub hauv paus khub) raug xam thiab P-tus nqi ntawm kev qhia sib txawv tau txiav txim siab siv pob DESeq2.Peb mam li xam qhov kev tshawb pom tsis tseeb (FDR) rau txhua tus nqi P siv Benjamini-Hochberg txoj kev 9 raws li kev ua haujlwm R-function "p.adjust".
RNA cais tawm ntawm lub plawv tau hloov mus rau cDNA ntawm qhov siab ntawm 200 ng / μl siv SuperScript IV Vilo Master mix los ntawm Thermo (Thermo, cat. no. 11756050).Quantitative RT-PCR tau ua los ntawm Applied Biosystems Endura Plate Microamp 384-zoo pob tshab cov tshuaj tiv thaiv phaj (Thermo, cat. no. 4483319) thiab microamp optical nplaum (Thermo, cat. no. 4311971).Cov tshuaj tiv thaiv sib tov muaj 5 µl Taqman Fast Advanced Master mix (Thermo, miv # 4444557), 0.5 µl Taqman Primer thiab 3.5 µl H2O tov rau ib qhov dej.Standard qPCR cycles tau khiav thiab CT qhov tseem ceeb tau ntsuas siv qhov Applied Biosystems Quantstudio 5 real-time PCR ntsuas (384-zoo module; khoom # A28135).Taqman primers tau muas los ntawm Thermo (GAPDH (Ss03375629_u1), PARP12 (Ss06908795_m1), PKDCC (Ss06903874_m1), CYGB (Ss06900188_m1), RGL1 (Ss06900188_m1), RGL1 (Ss0809068) mH), GATA4 (Ss03383805_u1), GJA1 (Ss03374839_u1), COL1A2 (Ss03375009_u1 ), COL3A1 (Ss04323794_m1), ACTA2 (Ss0424551) GATT cov nqi hauv tsev PDH.
Kev tshaj tawm xov xwm ntawm NT-ProBNP tau soj ntsuam siv cov khoom siv NT-ProBNP (pig) (Cat. No. MBS2086979, MyBioSource) raws li cov chaw tsim khoom raws tu qauv.Luv luv, 250 µl ntawm txhua tus qauv thiab tus qauv tau ntxiv rau hauv qhov sib npaug rau txhua qhov dej.Tam sim ntawd tom qab ntxiv cov qauv, ntxiv 50 µl ntawm Assay Reagent A rau txhua qhov dej.Maj mam co lub phaj thiab kaw nrog sealant.Tom qab ntawd cov ntsiav tshuaj tau incubated ntawm 37 ° C rau 1 teev.Tom qab ntawd yaug cov tshuaj thiab ntxuav lub qhov dej 4 zaug nrog 350 µl ntawm 1X tshuaj ntxuav, incubating cov tshuaj ntxuav rau 1-2 feeb txhua zaus.Tom qab ntawd ntxiv 100 µl ntawm Assay Reagent B rau ib qhov dej thiab muab khi nrog phaj sealant.Cov ntsiav tshuaj tau maj mam shaken thiab incubated ntawm 37 ° C rau 30 feeb.Aspirate cov tshuaj thiab ntxuav lub qhov dej 5 zaug nrog 350 µl ntawm 1X ntxuav kua.Ntxiv 90 µl ntawm substrate tov rau txhua qhov dej thiab kaw lub phaj.Ci lub phaj ntawm 37 ° C rau 10-20 feeb.Ntxiv 50 µl Stop Solution rau txhua qhov dej.Lub phaj tau ntsuas tam sim ntawd siv Cytation (BioTek) phaj nyeem ntawv teeb tsa ntawm 450 nm.
Kev txheeb xyuas lub zog tau ua los xaiv cov pab pawg ntau thiab tsawg uas yuav muab> 80% lub zog txhawm rau txheeb xyuas 10% kev hloov pauv hauv qhov ntsuas nrog 5% Hom I yuam kev. Kev txheeb xyuas lub zog tau ua los xaiv cov pab pawg ntau thiab tsawg uas yuav muab> 80% lub zog txhawm rau txheeb xyuas 10% kev hloov pauv hauv qhov ntsuas nrog 5% Hom I yuam kev. Анализ мощности был выполнен для выбора размеров групп, которые обеспечат >80% мощности для обята для обята нения параметра с 5% частотой ошибок типа I. Kev tshuaj xyuas lub zog tau ua los xaiv cov pab pawg ntau thiab tsawg uas yuav muab> 80% lub zog txhawm rau txheeb xyuas 10% kev hloov pauv tsis meej nrog 5% Hom I yuam kev.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尯野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尯野 Был проведен анализ мощности для выбора размера группы, который обеспечил бы > 80% мощности для обнона для изменения параметров и 5% частоты ошибок типа I. Kev tshuaj xyuas lub zog tau ua los xaiv ib pawg loj uas yuav muab> 80% lub zog txhawm rau txheeb xyuas 10% kev hloov pauv tsis muaj tseeb thiab 5% hom I yuam kev.Cov ntaub so ntswg raug xaiv xaiv ua ntej qhov kev sim.Tag nrho cov kev ntsuam xyuas yog qhov muag tsis pom thiab cov qauv raug txiav txim tsuas yog tom qab tag nrho cov ntaub ntawv tau txheeb xyuas.GraphPad Prism software (San Diego, CA) tau siv los ua txhua yam kev ntsuas ntsuas. Rau txhua qhov kev txheeb cais, p-tus nqi raug suav tias yog qhov tseem ceeb ntawm qhov tseem ceeb <0.05. Rau txhua qhov kev txheeb cais, p-tus nqi tau suav tias yog qhov tseem ceeb ntawm tus nqi <0.05. Для всей статистики p-значения считались значимыми при значениях <0,05. Rau txhua qhov kev txheeb cais, p-tus nqi tau suav tias yog qhov tseem ceeb ntawm tus nqi <0.05.对于所有统计数据，p 值在值 <0.05 时被认为是显着的。对于所有统计数据，p 值在值 <0.05 时被认为是显着的。 Для всей статистики p-значения считались значимыми при значениях <0,05. Rau txhua qhov kev txheeb cais, p-tus nqi tau suav tias yog qhov tseem ceeb ntawm tus nqi <0.05.Ob tug tub-ntxhais kawm ntawv qhov t-test tau ua ntawm cov ntaub ntawv nrog tsuas yog 2 kev sib piv.Ib txoj kev lossis ob txoj kev ANOVA tau siv los txiav txim qhov tseem ceeb ntawm ntau pawg.Thaum ua qhov kev xeem tom qab hoc, Tukey qhov kev kho tau raug siv los suav rau ntau qhov kev sib piv.RNAsec cov ntaub ntawv muaj kev txiav txim siab tshwj xeeb thaum xam FDR thiab p.hloov raws li tau piav qhia hauv Tshooj Lus.
Yog xav paub ntxiv txog kev tsim qauv, saib Nature Research Report abstract linked to this article.