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Minofu ya chigoba ndi minofu yosiyana yomwe imapangidwa makamaka ndi myofibrils, zomwe mwa anthu nthawi zambiri zimagawidwa m'magulu atatu: imodzi "yochedwa" (mtundu 1) ndi iwiri "yofulumira" (mitundu 2A ndi 2X). Komabe, kusiyana pakati pa mitundu ya myofibril yachikhalidwe ndi mkati mwake sikukumveka bwino. Tinagwiritsa ntchito njira za transcriptomic ndi proteomic pa 1050 ndi 1038 myofibrils payekha kuchokera ku vastus lateralis ya anthu, motsatana. Kafukufuku wa proteomic adaphatikizapo amuna, ndipo kafukufuku wa transcriptomic adaphatikizapo amuna 10 ndi akazi awiri. Kuphatikiza pa isoforms ya myosin heavy chain, tidazindikira mapuloteni a metabolic, mapuloteni a ribosomal, ndi mapuloteni olumikizana ndi maselo ngati magwero a kusiyanasiyana kwa multidimensional intermyofibril. Kuphatikiza apo, ngakhale kuti tazindikira magulu a ulusi wochedwa komanso wofulumira, deta yathu ikuwonetsa kuti ulusi wa mtundu wa 2X sungasiyanitsidwe ndi ulusi wina wofulumira. Kuphatikiza apo, kugawa kwa myosin heavy chain-based sikukwanira kufotokoza mtundu wa myofiber mu nemaline myopathies. Ponseponse, deta yathu ikuwonetsa kusiyana kwa myofiber m'magawo ambiri, pomwe magwero amitundu yosiyanasiyana amapitilira kupitirira myosin heavy chain isoforms.
Kusiyana kwa maselo ndi chinthu chodziwika bwino m'machitidwe onse a zamoyo, zomwe zimathandiza maselo kuti azidziwa bwino zosowa za minofu ndi maselo.1 Lingaliro lachikhalidwe la kusiyana kwa ulusi wa minofu ya mafupa lakhala kuti ma neuron amagetsi amatanthauzira mtundu wa ulusi mkati mwa unit yamagetsi, ndipo mtundu wa ulusi (monga, mtundu 1, mtundu 2A, ndi mtundu 2X mwa anthu) umatsimikiziridwa ndi makhalidwe a isoforms a myosin heavy chain (MYH).2 Poyamba izi zidakhazikitsidwa pa pH ATPase kusakhazikika kwawo, 3,4 kenako pa ma molecular expression awo a MYH.5 Komabe, ndi kuzindikira ndi kuvomereza pambuyo pake ulusi "wosakanikirana" womwe umatulutsa ma MYH angapo mosiyanasiyana, ulusi wa minofu ya mafupa umawonedwa ngati wopitilira m'malo mwa mitundu yosiyana ya ulusi.6 Ngakhale zili choncho, mundawu umadalirabe kwambiri MYH ngati gulu lalikulu la magulu a myofiber, lingaliro lomwe mwina limakhudzidwa ndi zofooka ndi tsankho lalikulu la maphunziro oyambirira a makoswe omwe mawonekedwe awo a MYH ndi mitundu yosiyanasiyana ya ulusi zimasiyana ndi zomwe zili mwa anthu.2 Mkhalidwewu umakhala wovuta kwambiri chifukwa chakuti minofu yosiyanasiyana ya mafupa ya anthu imawonetsa ulusi wosiyanasiyana. mitundu.7 Vastus lateralis ndi minofu yosakanikirana yokhala ndi mawonekedwe apakati (ndipo motero imayimira) a MYH.7 Kuphatikiza apo, kusavuta kwake kutengera zitsanzo kumapangitsa kuti ikhale minofu yophunziridwa bwino kwambiri mwa anthu.
Motero, kufufuza kosakondera kwa mitundu yosiyanasiyana ya minofu ya mafupa pogwiritsa ntchito zida zamphamvu za "omics" ndikofunikira komanso kovuta, chifukwa cha mtundu wa ulusi wa minofu ya mafupa womwe uli ndi nucleotide yambiri. Komabe, ukadaulo wa transcriptomics8,9 ndi proteomics10 wasintha kwambiri m'zaka zaposachedwa chifukwa cha kupita patsogolo kwaukadaulo, zomwe zimalola kusanthula kwa minofu ya mafupa pamlingo umodzi. Zotsatira zake, kupita patsogolo kwakukulu kwachitika pakulongosola mitundu yosiyanasiyana ya ulusi umodzi ndi momwe imayankhira ku zinthu zoyambitsa atrophic ndi ukalamba11,12,13,14,15,16,17,18. Chofunika kwambiri, kupita patsogolo kumeneku kwaukadaulo kuli ndi ntchito zachipatala, zomwe zimathandiza kufotokoza mwatsatanetsatane komanso molondola za kusokonekera kwa minofu komwe kumakhudzana ndi matenda. Mwachitsanzo, matenda a nemaline myopathy, omwe ndi amodzi mwa matenda ofala kwambiri a minofu (MIM 605355 ndi MIM 161800), ndi ovuta komanso osokoneza.19,20 Chifukwa chake, kufotokoza bwino kusokonekera kwa ulusi wa minofu ya mafupa kungapangitse kuti tipite patsogolo kwambiri pakumvetsetsa kwathu matendawa.
Tinapanga njira zowunikira transcriptomic ndi proteomic za ulusi umodzi wa minofu ya mafupa wochotsedwa pamanja kuchokera ku zitsanzo za biopsy ya anthu ndikuziyika pa ulusi zikwizikwi, zomwe zimatithandiza kufufuza kusiyana kwa maselo a ulusi wa minofu ya mafupa ya anthu. Mu ntchito iyi, tinawonetsa mphamvu ya transcriptomic ndi proteomic phenotyping ya ulusi wa minofu ndipo tinazindikira mapuloteni a metabolic, ribosomal, ndi ma cell junctional ngati magwero ofunikira a kusiyanasiyana kwa interfiber. Kuphatikiza apo, pogwiritsa ntchito njira iyi ya proteomic, tinazindikira kufunika kwa nematode myopathy mu ulusi umodzi wa minofu ya mafupa, ndikuwulula kusintha kogwirizana kupita ku ulusi wopanda okosijeni wodziyimira pawokha pamtundu wa ulusi kutengera MYH.
Kuti tifufuze kusiyana kwa ulusi wa minofu ya mafupa a anthu, tinapanga njira ziwiri zogwirira ntchito kuti tithe kusanthula transcriptome ndi proteome za ulusi umodzi wa minofu ya mafupa (Chithunzi 1A ndi Chithunzi Chowonjezera 1A). Tinapanga ndikuwongolera njira zingapo zoyendetsera, kuyambira kusungira zitsanzo ndi kusunga umphumphu wa RNA ndi mapuloteni mpaka kukonza bwino njira iliyonse. Pa kusanthula transcriptome, izi zidakwaniritsidwa poika ma barcode a molekyulu apadera pa sitepe yoyamba yolembera mobwerezabwereza, zomwe zidalola ulusi 96 kuti uphatikizidwe kuti ugwiritsidwe ntchito bwino pansi. Kutsata mozama (± 1 miliyoni kuwerenga pa ulusi uliwonse) poyerekeza ndi njira zachikhalidwe za selo limodzi kunawonjezeranso deta ya transcriptome. 21 Pa proteomics, tinagwiritsa ntchito gradient yaifupi ya chromatographic (mphindi 21) yophatikizidwa ndi kupeza deta ya DIA-PASEF pa timsTOF mass spectrometer kuti tiwongolere kuzama kwa proteome pamene tikusunga kupitirira kwakukulu. 22,23 Kuti tifufuze kusiyana kwa ulusi wa minofu ya mafupa yathanzi, tinafotokoza ma transcriptome a ulusi 1,050 wochokera kwa opereka 14 athanzi achikulire ndi ma proteome a ulusi 1,038 ochokera kwa opereka 5 athanzi achikulire (Table Yowonjezera 1). Mu pepalali, ma datasets awa amatchedwa ma transcriptome ndi ma proteome a ulusi 1,000, motsatana. Njira yathu idapeza ma transcriptome 27,237 ndi mapuloteni 2,983 mu ma analysis a transcriptome ndi proteome a ulusi 1,000 (Chithunzi 1A, Ma Datasets Owonjezera 1-2). Pambuyo posefa ma datasets a transcriptomic ndi proteomic kuti mupeze majini opitilira 1,000 omwe apezeka ndi ma valid valid 50% pa ulusi uliwonse, kusanthula kwa bioinformatics komwe kunachitika kunachitika pa ulusi 925 ndi 974 mu transcriptome ndi proteome, motsatana. Pambuyo posefa, pafupifupi majini 4257 ± 1557 ndi mapuloteni a 2015 ± 234 (avereji ± SD) adapezeka pa ulusi uliwonse, ndi kusiyana kochepa pakati pa munthu ndi munthu (Zithunzi Zowonjezera 1B–C, Ma Datasets Owonjezera 3–4). Komabe, kusiyana kwa mkati mwa munthu kunali koonekera kwambiri kwa ophunzira, mwina chifukwa cha kusiyana kwa RNA/protein pakati pa ulusi wa kutalika kosiyana ndi madera opingasa. Kwa mapuloteni ambiri (>2000), coefficient of variation inali pansi pa 20% (Zithunzi Zowonjezera 1D). Njira zonsezi zidalola kujambula mitundu yosiyanasiyana ya zolembedwa ndi mapuloteni okhala ndi zizindikiro zomveka bwino zofunika kwambiri pakukoka kwa minofu (monga, ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Zithunzi Zowonjezera 1E–F). Zambiri mwa zinthu zomwe zinadziwika zinali zofala pakati pa ma datasets a transcriptomic ndi proteomic (Chithunzi Chowonjezera 1G), ndipo mphamvu yapakati ya UMI/LFQ ya zinthuzi inali yogwirizana bwino (r = 0.52) (Chithunzi Chowonjezera 1H).
Mayendedwe a Transcriptomics ndi proteomics (opangidwa ndi BioRender.com). Ma curve a BD Dynamic range a MYH7, MYH2, ndi MYH1, ndi kuwerengera malire a mtundu wa fiber. E, F Kugawa kwa MYH expression kudutsa ulusi mu transcriptomics ndi proteomics datasets. G, H Uniform Diversity Approximation and Projection (UMAP) plots za transcriptomics ndi proteomics zokongoletsedwa ndi mtundu wa fiber wochokera ku MYH. I, J plots zomwe zikuwonetsa MYH7, MYH2, ndi MYH1 expression mu transcriptomics ndi proteomics datasets.
Poyamba tinayamba kugawa mtundu wa ulusi wochokera ku MYH ku ulusi uliwonse pogwiritsa ntchito njira yabwino kwambiri yomwe imagwiritsa ntchito kukhudzidwa kwakukulu ndi kuchuluka kwa mphamvu kwa MYH mu datasets a omics. Kafukufuku wakale adagwiritsa ntchito malire okhazikika kuti alembe ulusi ngati mtundu woyera 1, mtundu 2A, mtundu 2X, kapena kusakanikirana kutengera kuchuluka kokhazikika kwa MYHs11,14,24 yosiyana. Tinagwiritsa ntchito njira yosiyana momwe kufotokozera kwa ulusi uliwonse kunayikidwa m'magulu ndi MYHs zomwe tidagwiritsa ntchito kulemba ulusi: MYH7, MYH2, ndi MYH1, zofanana ndi ulusi wa mtundu 1, mtundu 2A, ndi mtundu 2X, motsatana. Kenako tinawerengera mwa masamu mfundo yotsika ya curve iliyonse yomwe idatuluka ndikuigwiritsa ntchito ngati malire kuti tigawire ulusi ngati wabwino (pamwamba pa malire) kapena woipa (pansi pa malire) pa MYH iliyonse (Chithunzi 1B–D). Deta iyi ikuwonetsa kuti MYH7 (Chithunzi 1B) ndi MYH2 (Chithunzi 1C) ali ndi ma profiles odziwika bwino a on/off expression pamlingo wa RNA poyerekeza ndi mulingo wa mapuloteni. Zoonadi, pamlingo wa mapuloteni, ulusi wochepa kwambiri sunatulutse MYH7, ndipo palibe ulusi womwe unali ndi 100% MYH2 expression. Kenako tinagwiritsa ntchito malire okonzedweratu a expression kuti tigawire mitundu ya ulusi wochokera ku MYH ku ulusi wonse mu deta iliyonse. Mwachitsanzo, ulusi wa MYH7+/MYH2-/MYH1- unaperekedwa ku mtundu 1, pomwe ulusi wa MYH7-/MYH2+/MYH1+ unaperekedwa ku mtundu wosakanikirana wa 2A/2X (onani Gome Lowonjezera 2 kuti mufotokoze bwino). Pogwirizanitsa ulusi wonse, tinawona kufalikira kofanana kwambiri kwa mitundu ya ulusi wochokera ku MYH pamlingo wa RNA (Chithunzi 1E) ndi mapuloteni (Chithunzi 1F), pomwe kapangidwe ka ulusi wochokera ku MYH kanasiyana pa anthu, monga momwe timayembekezera (Chithunzi Chowonjezera 2A). Ulusi wambiri unagawidwa ngati mtundu woyera 1 (34-35%) kapena mtundu 2A (36-38%), ngakhale kuti ulusi wambiri wosakanikirana wa 2A/2X unapezekanso (16-19%). Kusiyana kwakukulu ndi kwakuti ulusi weniweni wa 2X unkapezeka kokha pamlingo wa RNA, koma osati pamlingo wa mapuloteni, zomwe zikusonyeza kuti kufalikira kwa MYH mwachangu kumayendetsedwa pang'ono pambuyo polemba.
Tinatsimikizira njira yathu yolembera ulusi wa MYH pogwiritsa ntchito ma antibodies-based dot blotting, ndipo njira zonse ziwirizi zinagwirizana 100% pozindikira ulusi wa mtundu 1 ndi mtundu 2A (onani Chithunzi Chowonjezera 2B). Komabe, njira yolembera ulusi wa proteomics inali yothandiza kwambiri, yothandiza kwambiri pozindikira ulusi wosakanikirana, komanso yowerengera kuchuluka kwa jini iliyonse ya MYH mu ulusi uliwonse. Deta iyi ikuwonetsa kuti kugwiritsa ntchito njira yolunjika, yomvera kwambiri ya omics pofotokoza mitundu ya ulusi wa minofu ya mafupa kumagwira ntchito bwino.
Kenako tinagwiritsa ntchito mfundo zophatikizidwa zomwe zinaperekedwa ndi transcriptomics ndi proteomics kuti tigawire myofibers m'magulu molingana ndi transcriptome yawo yonse kapena proteome. Pogwiritsa ntchito njira ya uniform manifold approximation and projection (UMAP) kuti tichepetse kukula kwa myofiber kukhala zigawo zisanu ndi chimodzi zazikulu (Zithunzi Zowonjezera 3A–B), tinatha kuwona kusiyana kwa myofiber mu transcriptome (Chithunzi 1G) ndi proteome (Chithunzi 1H). Chodziwika bwino n'chakuti, myofibers sizinagawidwe m'magulu ndi ophunzira (Zithunzi Zowonjezera 3C–D) kapena masiku oyesera (Chithunzi Zowonjezera 3E) m'ma data a transcriptomics kapena proteomics, zomwe zikusonyeza kuti kusiyana kwa mkati mwa mutu mu ulusi wa minofu ya mafupa ndikokwera kuposa kusiyana kwa pakati pa mutu. Mu UMAP plot, magulu awiri osiyana omwe akuyimira myofibers "yofulumira" ndi "yochedwa" adawonekera (Zithunzi 1G–H). Ma myofiber a MYH7+ (ochedwa) adayikidwa pamodzi pamtengo wabwino wa UMAP1, pomwe ma myofiber a MYH2+ ndi MYH1+ (ofulumira) adayikidwa pamodzi pamtengo woipa wa UMAP1 (Zithunzi 1I–J). Komabe, palibe kusiyana komwe kudapangidwa pakati pa mitundu ya ulusi wothamanga mwachangu (monga, mtundu wa 2A, mtundu wa 2X, kapena 2A/2X wosakanikirana) kutengera mawonekedwe a MYH, zomwe zikusonyeza kuti mawonekedwe a MYH1 (Chithunzi 1I–J) kapena zizindikiro zina zakale za 2X myofiber monga ACTN3 kapena MYLK2 (Zithunzi Zowonjezera 4A–B) sakusiyanitsa mitundu yosiyanasiyana ya myofiber poganizira za transcriptome yonse kapena proteome. Komanso, poyerekeza ndi MYH2 ndi MYH7, zolemba zochepa kapena mapuloteni adagwirizana bwino ndi MYH1 (Zithunzi Zowonjezera 4C–H), zomwe zikusonyeza kuti kuchuluka kwa MYH1 sikukuwonetsa kwathunthu transcriptome/proteome ya myofiber. Mapeto ofanana adapezeka poyesa kusakanikirana kwa ma isoform atatu a MYH pamlingo wa UMAP (Zithunzi Zowonjezera 4I–J). Chifukwa chake, ngakhale kuti ulusi wa 2X ukhoza kuzindikirika pamlingo wa transcript kutengera kuchuluka kwa MYH kokha, ulusi wa MYH1+ sungasiyanitsidwe ndi ulusi wina wofulumira poganizira transcriptome yonse kapena proteome.
Monga kafukufuku woyamba wa kusiyana kwa ulusi wocheperako kupitirira MYH, tinayesa mapuloteni anayi omwe adakhazikitsidwa a mtundu wa ulusi wocheperako: TPM3, TNNT1, MYL3, ndi ATP2A22. Mitundu ya ulusi wocheperako inawonetsa kulumikizana kwakukulu, ngakhale kuti sikunali kwangwiro, kwa Pearson ndi MYH7 mu transcriptomics (Chithunzi Chowonjezera 5A) ndi proteomics (Chithunzi Chowonjezera 5B). Pafupifupi 25% ndi 33% ya ulusi wocheperako sunasankhidwe ngati ulusi wocheperako weniweni ndi mitundu yonse ya majini/mapuloteni mu transcriptomics (Chithunzi Chowonjezera 5C) ndi proteomics (Chithunzi Chowonjezera 5D), motsatana. Chifukwa chake, kugawa ulusi wocheperako kutengera mitundu yambiri ya majini/mapuloteni kumabweretsa zovuta zina, ngakhale kwa mapuloteni omwe amadziwika kuti ndi amtundu wa ulusi. Izi zikusonyeza kuti kugawa ulusi kutengera isoforms ya banja limodzi la majini/mapuloteni sikungawonetse mokwanira kusiyana kwenikweni kwa ulusi wa minofu ya mafupa.
Kuti tipitirize kufufuza kusiyana kwa phenotypic kwa ulusi wa minofu ya mafupa a anthu pamlingo wa chitsanzo chonse cha omics, tinachita kuchepetsa kopanda tsankho kwa deta pogwiritsa ntchito principal component analysis (PCA) (Chithunzi 2A). Mofanana ndi UMAP plots, palibe wophunzira kapena tsiku loyesera lomwe linakhudza kusonkhana kwa ulusi pamlingo wa PCA (Zithunzi Zowonjezera 6A–C). M'ma data onse awiri, mtundu wa ulusi wochokera ku MYH unafotokozedwa ndi PC2, yomwe inawonetsa gulu la ulusi wa mtundu wa 1 wopindika pang'onopang'ono ndi gulu lachiwiri lomwe lili ndi ulusi wa mtundu wa 2A wopindika mwachangu, mtundu wa 2X, ndi ulusi wosakanikirana wa 2A/2X (Chithunzi 2A). M'ma data onse awiri, magulu awiriwa adalumikizidwa ndi ulusi wosakanikirana wa mtundu wa 1/2A. Monga momwe tinkayembekezera, kusanthula kwakukulu kwa madalaivala akuluakulu a PC kunatsimikizira kuti PC2 inkayendetsedwa ndi zizindikiro za contractile ndi metabolic (Chithunzi 2B ndi Zithunzi Zowonjezera 6D–E, Ma Datasets Owonjezera 5–6). Ponseponse, mtundu wa ulusi wochokera ku MYH unapezeka kuti ndi wokwanira kufotokoza kusiyana kosalekeza pa PC2, kupatulapo ulusi wotchedwa 2X womwe unagawidwa mu transcriptome yonse mkati mwa gulu lofulumira.
A. Ma ploti a kusanthula kwa zigawo zazikulu (PCA) a ma data a transcriptome ndi proteome omwe adapakidwa utoto malinga ndi mtundu wa ulusi kutengera MYH. B. Kusanthula kwa Enrichment kwa ma transcript ndi ma protein drivers mu PC2 ndi PC1. Kusanthula kwa ziwerengero kunachitika pogwiritsa ntchito clusterProfiler package ndi Benjamini-Hochberg adjusted p-values. C, D. Ma ploti a PCA omwe adapakidwa utoto malinga ndi intercellular adhesion gene ontology (GO) terms mu transcriptome ndi costamere GO terms mu proteome. Mivi ikuyimira ma transcript ndi ma protein drivers ndi malangizo awo. E, F. Uniform manifold approximation and projection (UMAP) ili ndi ma ploti a zizindikiro zofunikira kuchipatala zomwe zikuwonetsa ma gradients osadalira mtundu wa ulusi wofulumira/wosachedwa. G, H. Kugwirizana pakati pa ma PC2 ndi ma PC1 drivers mu transcriptomes ndi proteomes.
Mosayembekezereka, mtundu wa myofiber wochokera ku MYH unafotokoza mlingo wachiwiri wapamwamba kwambiri wa kusiyana (PC2), zomwe zikusonyeza kuti zinthu zina zamoyo zomwe sizigwirizana ndi mtundu wa myofiber wochokera ku MYH (PC1) zimagwira ntchito yofunika kwambiri pakulamulira kusiyana kwa ulusi wa minofu ya mafupa. Kusanthula kochulukira kwa oyendetsa apamwamba mu PC1 kunawonetsa kuti kusiyana kwa PC1 kunkadziwika makamaka ndi kumamatirana kwa maselo ndi kuchuluka kwa ribosome mu transcriptome, ndi mapuloteni a costameres ndi ribosomal mu proteome (Chithunzi 2B ndi Zithunzi Zowonjezera 6D–E, Seti Yowonjezera ya Deta 7). Mu minofu ya mafupa, costameres amalumikiza Z-disc ku sarcolemma ndipo amagwira ntchito yotumiza mphamvu ndi ma signaling. 25 Zizindikiro za PCA zolembedwa pogwiritsa ntchito kumamatirana kwa maselo (transcriptome, Chithunzi 2C) ndi zizindikiro za costamere (proteome, Chithunzi 2D) zinawonetsa kusintha kwakukulu kumanzere mu PC1, kusonyeza kuti zinthuzi zili ndi ulusi wina.
Kufufuza mwatsatanetsatane kwa myofiber clustering pamlingo wa UMAP kwawonetsa kuti zinthu zambiri zikuwonetsa gradient ya MYH yochokera ku mtundu wa myofiber m'malo mwa myofiber subcluster-specific. Kupitiriza uku kunawonedwa m'majini angapo okhudzana ndi matenda (Chithunzi 2E), monga CCHHD10 (matenda a neuromuscular), SLIT3 (kufooka kwa minofu), CTDNEP1 (matenda a minofu). Kupitirira uku kunawonedwanso kudutsa proteome, kuphatikizapo mapuloteni okhudzana ndi matenda amitsempha (UGDH), insulin signaling (PHIP), ndi transcription (HIST1H2AB) (Chithunzi 2F). Pamodzi, deta iyi ikuwonetsa kupitirira mu kusiyana kwa kugwedezeka pang'onopang'ono/mwachangu kwa ulusi pa myofibers zosiyanasiyana.
Chochititsa chidwi n'chakuti, majini oyendetsa mu PC2 adawonetsa mgwirizano wabwino wa transcriptome-proteome (r = 0.663) (Chithunzi 2G), zomwe zikusonyeza kuti mitundu ya ulusi wopindika pang'onopang'ono komanso mwachangu, makamaka mphamvu zogwira ntchito komanso kagayidwe kachakudya ka ulusi wa minofu ya mafupa, zimayendetsedwa motsatira malamulo. Komabe, majini oyendetsa mu PC1 sanawonetse mgwirizano wa transcriptome-proteome (r = -0.027) (Chithunzi 2H), zomwe zikusonyeza kuti kusiyana kosagwirizana ndi mitundu ya ulusi wopindika pang'onopang'ono/mwachangu kumayendetsedwa kwambiri pambuyo polemba. Chifukwa kusiyana kwa PC1 kunafotokozedwa makamaka ndi mawu a ribosomal gene ontology, ndipo popeza ma ribosome amachita gawo lofunikira komanso lapadera mu selo mwa kutenga nawo mbali mwachangu mu kumasulira kwa mapuloteni,31 kenako tinayamba kufufuza kusiyana kosayembekezereka kwa ribosomal.
Choyamba tinapaka utoto wa proteomics principal component analysis plot malinga ndi kuchuluka kwa mapuloteni mu GOCC term "cytoplasmic ribosome" (Chithunzi 3A). Ngakhale kuti mawuwa ali ndi ubwino wa PC1, zomwe zimapangitsa kuti pakhale gradient yaying'ono, mapuloteni a ribosomal amachititsa kuti PC1 igawanike mbali zonse ziwiri (Chithunzi 3A). Mapuloteni a ribosomal omwe ali ndi ubwino wa PC1 anali ndi RPL18, RPS18, ndi RPS13 (Chithunzi 3B), pomwe RPL31, RPL35, ndi RPL38 (Chithunzi 3C) anali ma driver akuluakulu omwe ali ndi ubwino wa PC1. Chochititsa chidwi n'chakuti, RPL38 ndi RPS13 zinali zodziwika bwino mu minofu ya mafupa poyerekeza ndi minofu ina (Chithunzi Chowonjezera 7A). Zizindikiro zapadera za ribosomal izi mu PC1 sizinawonekere mu transcriptome (Chithunzi Chowonjezera 7B), zomwe zikusonyeza kuti pambuyo pa transcriptional regulation.
A. Chiwembu cha kusanthula kwa zigawo zazikulu (PCA) chojambulidwa molingana ndi mawu a cytoplasmic ribosomal gene ontology (GO) kudutsa proteome. Mivi imasonyeza njira ya kusintha kwa mapuloteni mu chiwembu cha PCA. Kutalika kwa mzere kumafanana ndi chiwembu cha zigawo zazikulu za puloteni yoperekedwa. B, C. Ziwembu za PCA zimasonyeza RPS13 ndi RPL38. D. Kusanthula kosayang'aniridwa kwa magulu a mapuloteni a cytoplasmic ribosomal. E. Chitsanzo cha kapangidwe ka 80S ribosome (PDB: 4V6X) chowonetsa mapuloteni a ribosomal okhala ndi kuchuluka kosiyanasiyana mu ulusi wa minofu ya mafupa. F. Mapuloteni a ribosomal okhala ndi stoichiometry yosiyana omwe ali pafupi ndi njira yotulukira ya mRNA.
Malingaliro a kusiyana kwa ribosomal ndi specialization adanenedwa kale, pomwe kukhalapo kwa magulu osiyana a ribosomal (kusiyana kwa ribosomal) kumatha kukhudza mwachindunji kumasulira kwa mapuloteni m'maselo osiyanasiyana32 ndi maselo33 kudzera mu kumasulira kosankhidwa kwa ma shelufu enaake a mRNA transcript34 (kudziwika kwa ribosome). Kuti tizindikire magulu ochepa a mapuloteni a ribosomal omwe amafotokozedwa mu ulusi wa minofu ya mafupa, tinachita kusanthula kosayang'aniridwa kwa magulu a ribosomal mu proteome (Chithunzi 3D, Seti Yowonjezera 8). Monga momwe tinkayembekezera, mapuloteni a ribosomal sanagwirizane ndi mtundu wa ulusi kutengera MYH. Komabe, tinapeza magulu atatu osiyana a mapuloteni a ribosomal; gulu loyamba (ribosomal_cluster_1) limagwirizanitsidwa ndi RPL38 ndipo motero limawonjezera mawonekedwe mu ulusi wokhala ndi mbiri yabwino ya PC1. Gulu lachiwiri (ribosomal_cluster_2) limagwirizanitsidwa ndi RPS13 ndipo limakwezedwa mu ulusi wokhala ndi mbiri yoyipa ya PC1. Gulu lachitatu (ribosomal_cluster_3) silikuwonetsa kuyanjana kosiyana mu ulusi wa minofu ya mafupa ndipo likhoza kuonedwa ngati puloteni ya "core" ya minofu ya mafupa. Magulu onse a ribosomal 1 ndi 2 ali ndi mapuloteni a ribosomal omwe adawonetsedwa kale kuti amasintha njira zina (monga, RPL10A, RPL38, RPS19, ndi RPS25) ndipo amakhudza chitukuko (monga, RPL10A, RPL38).34,35,36,37,38 Mogwirizana ndi zotsatira za PCA, mawonekedwe osiyanasiyana a mapuloteni a ribosomal awa pa ulusi adawonetsanso kupitilira (Chithunzi Chowonjezera 7C).
Kuti tiwone komwe kuli mapuloteni a ribosomal osiyanasiyana mkati mwa ribosome, tinagwiritsa ntchito chitsanzo cha kapangidwe ka ribosome ya munthu (Protein Data Bank: 4V6X) (Chithunzi 3E). Titapatula mapuloteni a ribosomal omwe ali m'magulu osiyanasiyana a ribosomal, malo awo sanagwirizane bwino, zomwe zikusonyeza kuti njira yathu sinathe kupereka zowonjezera m'madera/zigawo zina za ribosome. Komabe, chochititsa chidwi n'chakuti, chiwerengero cha mapuloteni akuluakulu a subunit mu gulu lachiwiri chinali chotsika kuposa m'magulu 1 ndi 3 (Chithunzi Chowonjezera 7D). Tinaona kuti mapuloteni okhala ndi stoichiometry yosinthika mu ulusi wa minofu ya mafupa anali opezeka makamaka pamwamba pa ribosome (Chithunzi 3E), mogwirizana ndi kuthekera kwawo kuyanjana ndi zinthu zamkati za ribosome entry site (IRES) m'magulu osiyanasiyana a mRNA, motero kulumikiza kumasulira kosankha. 40, 41 Kuphatikiza apo, mapuloteni ambiri okhala ndi stoichiometry yosinthika mu ulusi wa minofu ya mafupa anali pafupi ndi madera ogwira ntchito monga njira yotulukira ya mRNA (Chithunzi 3F), yomwe imawongolera mosankha kutalika kwa kumasulira ndi kumangidwa kwa ma peptide enaake. 42 Mwachidule, deta yathu ikusonyeza kuti stoichiometry ya mapuloteni a ribosomal minofu ya mafupa imawonetsa kusiyana kwa maselo, zomwe zimapangitsa kuti pakhale kusiyana pakati pa ulusi wa minofu ya mafupa.
Kenako tinayamba kuzindikira zizindikiro za ulusi zomwe zimasinthasintha mofulumira komanso pang'onopang'ono ndikufufuza njira zowongolera ma transcriptional. Poyerekeza magulu a ulusi omwe amasinthasintha mofulumira komanso pang'onopang'ono omwe amafotokozedwa ndi UMAP m'ma datasets awiri (Zithunzi 1G–H ndi 4A–B), kusanthula kwa transcriptomic ndi proteomic kunapeza zizindikiro 1366 ndi 804 zomwe zimakhala ndi mitundu yosiyanasiyana, motsatana (Zithunzi 4A–B, Zowonjezera za Datasets 9–12). Tinaona kusiyana komwe kumayembekezeredwa mu zizindikiro zokhudzana ndi sarcomeres (monga, tropomyosin ndi troponin), excitation-contraction coupling (SERCA isoforms), ndi energy metabolism (monga, ALDOA ndi CKB). Kuphatikiza apo, zolemba ndi mapuloteni omwe amalamulira protein ubiquitination adawonetsedwa mosiyana mu ulusi womwe umasinthasintha mwachangu komanso pang'onopang'ono (monga, USP54, SH3RF2, USP28, ndi USP48) (Zithunzi 4A–B). Komanso, jini ya puloteni ya tizilombo toyambitsa matenda ya RP11-451G4.2 (DWORF), yomwe yawonetsedwa kale kuti imawonetsedwa mosiyana pamitundu yonse ya minofu ya mwanawankhosa43 ndipo imawonjezera ntchito ya SERCA mu minofu ya mtima44, idakwezedwa kwambiri mu ulusi wa minofu yocheperako (Chithunzi 4A). Mofananamo, pamlingo wa ulusi uliwonse, kusiyana kwakukulu kunawonedwa mu zizindikiro zodziwika bwino monga lactate dehydrogenase isoforms yokhudzana ndi kagayidwe kachakudya (LDHA ndi LDHB, Chithunzi 4C ndi Chithunzi Chowonjezera 8A)45,46 komanso zizindikiro zodziwika kale za mtundu wa ulusi (monga IRX3, USP54, USP28, ndi DPYSL3) (Chithunzi 4C). Panali kuphatikizana kwakukulu kwa zizindikiro zodziwika bwino pakati pa ma datasets a transcriptomic ndi proteomic (Chithunzi Chowonjezera 8B), komanso kulumikizana kwa kusintha kwa fold komwe kumachitika makamaka ndi mawonekedwe osiyana kwambiri a sarcomere (Chithunzi Chowonjezera 8C). Chochititsa chidwi n'chakuti, zizindikiro zina (monga USP28, USP48, GOLGA4, AKAP13) zinasonyeza mphamvu yolamulira pambuyo pa kulembedwa kokha pamlingo wa proteomic ndipo zinali ndi mawonekedwe ofotokozera a ulusi pang'onopang'ono/mwachangu (Chithunzi Chowonjezera 8C).
Ma plots a A ndi B oyerekeza magulu ofulumira komanso othamanga omwe amadziwika ndi ma plots a uniform manifold approximation and projection (UMAP) mu Zithunzi 1G–H. Madontho amitundu akuyimira ma transcripts kapena mapuloteni omwe ali osiyana kwambiri pa FDR < 0.05, ndipo madontho akuda akuyimira ma transcripts kapena mapuloteni omwe ali osiyana kwambiri pakusintha kwa log > 1. Kusanthula kwa ziwerengero kwa njira ziwiri kunachitika pogwiritsa ntchito mayeso a DESeq2 Wald ndi Benjamini-Hochberg adjusted p values (transcriptomics) kapena njira ya Limma linear model yokhala ndi kusanthula kwa empirical Bayesian kutsatiridwa ndi kusintha kwa Benjamini-Hochberg kwa kufananiza kosiyanasiyana (proteomics). C Ma plots osayina a majini kapena mapuloteni osankhidwa mosiyana pakati pa ulusi wofulumira komanso wofulumira. D Kusanthula kwa zowonjezera za ma transcripts ndi mapuloteni omwe ali osiyana kwambiri. Mitengo yolumikizana imachulukitsidwa m'ma datasets onse awiri, mitengo ya transcriptome imachulukitsidwa mu transcriptome yokha, ndipo mitengo ya proteome imachulukitsidwa mu proteome yokha. Kusanthula kwa ziwerengero kunachitika pogwiritsa ntchito phukusi la clusterProfiler ndi mitengo ya p yosinthidwa ya Benjamini-Hochberg. E. Zinthu zolembera zamtundu wa fiber zomwe zimazindikiritsidwa ndi SCENIC kutengera ziwerengero za SCENIC-derived regulator specificity scores ndi kusiyana kwa mRNA pakati pa mitundu ya fiber. F. Kufotokozera zinthu zolembera zomwe zasankhidwa zomwe zimafotokozedwa mosiyana pakati pa ulusi wochedwa komanso wothamanga.
Kenako tinachita kusanthula kochulukirapo kwa majini ndi mapuloteni omwe amaimiridwa mosiyana (Chithunzi 4D, Seti Yowonjezera ya Deta 13). Kukulitsa njira ya zinthu zomwe zimasiyana pakati pa ma dataseti awiriwa kunawonetsa kusiyana komwe kumayembekezeredwa, monga mafuta acid β-oxidation ndi njira za ketone metabolism (ulusi wocheperako), myofilament/minofu contraction (ulusi wofulumira ndi wocheperako, motsatana), ndi njira za carbohydrate catabolic (ulusi wofulumira). Ntchito ya serine/threonine protein phosphatase inakwezedwanso mu ulusi wofulumira, chifukwa cha zinthu monga regulatory and catalytic phosphatase subunits (PPP3CB, PPP1R3D, ndi PPP1R3A), zomwe zimadziwika kuti zimayendetsa kagayidwe ka glycogen (47) (Zithunzi Zowonjezera 8D–E). Njira zina zomwe zinapangidwa ndi ulusi wofulumira zinali kuphatikizapo matupi opangira (P-) (YTHDF3, TRIM21, LSM2) mu proteome (Chithunzi Chowonjezera 8F), zomwe mwina zimagwira ntchito mu malamulo otsatirawa (48), ndi ntchito ya transcription factor (SREBF1, RXRG, RORA) mu transcriptome (Chithunzi Chowonjezera 8G). Ulusi wocheperako unapangidwanso mu ntchito ya oxidoreductase (BDH1, DCXR, TXN2) (Chithunzi Chowonjezera 8H), amide binding (CPTP, PFDN2, CRYAB) (Chithunzi Chowonjezera 8I), extracellular matrix (CTSD, ADAMTSL4, LAMC1) (Chithunzi Chowonjezera 8J), ndi ntchito ya receptor-ligand (FNDC5, SPX, NENF) (Chithunzi Chowonjezera 8K).
Kuti tidziwe zambiri za malamulo okhudza kulembedwa kwa minofu omwe ali ndi makhalidwe a minofu yofulumira/yofulumira, tinachita kusanthula kwa kukulitsa kwa zinthu pogwiritsa ntchito SCENIC49 (Supplementary Data Set 14). Zinthu zambiri zolembera zinawonjezeka kwambiri pakati pa ulusi wa minofu yofulumira komanso yofulumira (Chithunzi 4E). Izi zinaphatikizapo zinthu zolembera monga MAFA, zomwe kale zinkalumikizidwa ndi kukula kwa ulusi wa minofu yofulumira,50 komanso zinthu zingapo zolembera zomwe sizinagwirizane ndi mapulogalamu amtundu wa ulusi wa minofu. Pakati pa izi, PITX1, EGR1, ndi MYF6 zinali zinthu zolembera zomwe zinali zolemera kwambiri mu ulusi wa minofu yofulumira (Chithunzi 4E). Mosiyana ndi zimenezi, ZSCAN30 ndi EPAS1 (zomwe zimadziwikanso kuti HIF2A) zinali zinthu zolembera zomwe zinali zolemera kwambiri mu ulusi wa minofu yofulumira (Chithunzi 4E). Mogwirizana ndi izi, MAFA inafotokozedwa pamlingo wapamwamba m'chigawo cha UMAP chofanana ndi ulusi wa minofu yofulumira, pomwe EPAS1 inali ndi mawonekedwe osiyana (Chithunzi 4F).
Kuwonjezera pa majini odziwika bwino olembera mapuloteni, pali mitundu yambiri ya RNA yosalembera yomwe ingakhale yogwirizana ndi kayendetsedwe ka chitukuko cha anthu ndi matenda. 51, 52 Mu ma data a transcriptome, ma RNA angapo osalembera amasonyeza mtundu wa ulusi (Chithunzi 5A ndi Supplementary Dataset 15), kuphatikizapo LINC01405, yomwe ndi yeniyeni kwambiri pa ulusi wochedwa ndipo imanenedwa kuti imachepa minofu kuchokera kwa odwala omwe ali ndi mitochondrial myopathy. 53 Mosiyana ndi zimenezi, RP11-255P5.3, yofanana ndi jini ya lnc-ERCC5-5 (https://lncipedia.org/db/transcript/lnc-ERCC5-5:2) 54, imasonyeza mtundu wa ulusi wofulumira. LINC01405 (https://tinyurl.com/x5k9wj3h) ndi RP11-255P5.3 (https://tinyurl.com/29jmzder) zonse zimasonyeza kudziwika kwa minofu ya mafupa (Zithunzi Zowonjezera 9A–B) ndipo zilibe majini odziwika bwino ogwirizana mkati mwa 1 Mb genomic residency yawo, zomwe zikusonyeza kuti zimagwira ntchito yapadera polamulira mitundu ya ulusi m'malo molamulira majini ogwirizana oyandikana nawo. Ma profiles a LINC01405 ndi RP11-255P5.3 omwe ali ndi ulusi wofulumira/wofulumira, motsatana, adatsimikiziridwa pogwiritsa ntchito RNAscope (Zithunzi 5B–C).
A. Zolemba za RNA zosalemba ma code zimayendetsedwa kwambiri mu ulusi wa minofu wopindika pang'onopang'ono komanso mwachangu. B. Zithunzi zoyimira RNAscope zomwe zikuwonetsa mtundu wa ulusi wopindika pang'onopang'ono komanso mwachangu wa LINC01405 ndi RP11-255P5.3, motsatana. Scale bar = 50 μm. C. Kuyeza kwa mafotokozedwe a RNA osalemba ma code amtundu wa myofiber monga momwe RNAscope imadziwira (n = 3 biopsies kuchokera kwa anthu odziyimira pawokha, kuyerekeza ulusi wa minofu wofulumira komanso wopindika mkati mwa munthu aliyense). Kusanthula kwa ziwerengero kunachitika pogwiritsa ntchito mayeso a t-test a Student okhala ndi michira iwiri. Ma graph a mabokosi akuwonetsa ma quartiles apakati ndi oyamba ndi achitatu, ndi ndevu zomwe zikuwonetsa ku mtengo wotsika komanso wapamwamba. D. De novo microbial protein identification functionality (yopangidwa ndi BioRender.com). E. Puloteni ya tizilombo toyambitsa matenda LINC01405_ORF408:17441:17358 imafotokozedwa makamaka mu ulusi wa minofu ya mafupa yochedwa (n=5 biopsies kuchokera kwa ophunzira odziyimira pawokha, kuyerekeza ulusi wa minofu yofulumira komanso yochedwa mwa wophunzira aliyense). Kusanthula kwa ziwerengero kunachitika pogwiritsa ntchito njira ya chitsanzo cha Limm yolumikizidwa ndi njira ya Bayesian, kutsatiridwa ndi njira ya Benjamini-Hochberg yoyerekeza kangapo ndi kusintha kwa p-value. Ma box plots amasonyeza ma quartiles apakati, oyamba ndi achitatu, ndi ndevu zomwe zikuwonetsa ku ma maximum/osachepera.
Posachedwapa, kafukufuku wasonyeza kuti zolemba zambiri zosalemba ma code zimalemba mapuloteni a tizilombo toyambitsa matenda, ena mwa iwo amayang'anira ntchito ya minofu. 44, 55 Kuti tizindikire mapuloteni a tizilombo toyambitsa matenda omwe angakhale ndi mtundu wa ulusi, tinafufuza deta yathu ya 1000 fiber proteome pogwiritsa ntchito fayilo ya FASTA yokhala ndi mndandanda wa zolemba zosalemba ma code (n = 305) zomwe zapezeka mu dataset ya 1000 fiber transcriptome (Chithunzi 5D). Tinapeza mapuloteni 197 a tizilombo toyambitsa matenda kuchokera ku zolemba 22 zosiyanasiyana, 71 mwa izo zinali zolamulidwa mosiyana pakati pa ulusi wa minofu ya mafupa yofulumira komanso yofulumira (Chithunzi Chowonjezera 9C ndi Seti Yowonjezera ya Deta 16). Pa LINC01405, zinthu zitatu za mapuloteni a tizilombo toyambitsa matenda zinapezeka, chimodzi mwa izo chinawonetsa kufanana kwa ulusi wocheperako ndi zolemba zake (Chithunzi 5E ndi Chithunzi Chowonjezera 9D). Chifukwa chake, tinapeza LINC01405 ngati jini yomwe imalemba mapuloteni a tizilombo toyambitsa matenda omwe ali ndi ulusi wa minofu ya mafupa yocheperako.
Tinapanga njira yolumikizirana yonse yofotokozera ma proteomic a minofu yayikulu komanso kuzindikira owongolera kusiyana kwa ulusi m'thupi la munthu wathanzi. Tinagwiritsa ntchito njira yolumikiziranayi kuti timvetse momwe nemaline myopathies imakhudzira kusiyana kwa ulusi wa minofu ya mafupa. Nemaline myopathies ndi matenda obadwa nawo a minofu omwe amayambitsa kufooka kwa minofu ndipo, mwa ana omwe akhudzidwa, amakhala ndi zovuta zosiyanasiyana kuphatikizapo kupuma movutikira, scoliosis, komanso kuyenda pang'ono kwa miyendo. 19,20 Kawirikawiri, mu nemaline myopathies, mitundu yosiyanasiyana ya matenda m'majini monga actin alpha 1 (ACTA1) imabweretsa kuchuluka kwa kapangidwe ka ulusi wa myofiber pang'onopang'ono, ngakhale kuti izi ndizosiyana. Chosiyana chimodzi chodziwika bwino ndi troponin T1 nemaline myopathy (TNNT1), yomwe imakhala ndi ulusi wofulumira kwambiri. Chifukwa chake, kumvetsetsa bwino kusiyana komwe kumayambitsa kusokonekera kwa ulusi wa minofu ya mafupa komwe kumawoneka mu nemaline myopathies kungathandize kuthetsa ubale wovuta pakati pa matendawa ndi mtundu wa myofiber.
Poyerekeza ndi owongolera athanzi (n=3 pa gulu), ma myofibers olekanitsidwa ndi odwala omwe ali ndi matenda a nemaline myopathy omwe ali ndi kusintha kwa majini a ACTA1 ndi TNNT1 adawonetsa kuchepa kwa myofiber kapena dystrophy (Chithunzi 6A, Gome Lowonjezera 3). Izi zidabweretsa zovuta zazikulu zaukadaulo pakusanthula kwa proteomic chifukwa cha kuchuluka kochepa kwa zinthu zomwe zilipo. Ngakhale izi, tidatha kupeza mapuloteni 2485 mu ma myofibers 272 a mafupa. Titasefa mapuloteni osachepera 1000 oyezedwa pa ulusi uliwonse, ulusi 250 udayesedwa pambuyo pake. Pambuyo posefa, avareji ya mapuloteni 1573 ± 359 pa ulusi uliwonse adayesedwa (Chithunzi Chowonjezera 10A, Ma Deta Owonjezera 17-18). Chodziwika bwino, ngakhale kuchepa kwakukulu kwa kukula kwa ulusi, kuzama kwa proteome kwa zitsanzo za odwala omwe ali ndi matenda a nemaline myopathy kunachepetsedwa pang'ono. Kuphatikiza apo, kukonza deta iyi pogwiritsa ntchito mafayilo athu a FASTA (kuphatikiza zolemba zosalemba) kunatilola kuzindikira mapuloteni asanu a tizilombo toyambitsa matenda m'mafupa a myofibers ochokera kwa odwala a nemaline myopathy (Supplementary Data Set 19). Mphamvu ya proteome inali yayikulu kwambiri, ndipo mapuloteni onse omwe anali mu gulu lolamulira adagwirizana bwino ndi zotsatira za kusanthula kwakale kwa proteome ya 1000-fiber (Chithunzi Chowonjezera 10B–C).
A. Zithunzi za microscopic zomwe zikuwonetsa kufooka kwa ulusi kapena dystrophy komanso kuchuluka kwa mitundu yosiyanasiyana ya ulusi kutengera MYH mu ACTA1 ndi TNNT1 nemaline myopathies (NM). Scale bar = 100 μm. Kuti zitsimikizire kuti utoto ubwereranso mwa odwala a ACTA1 ndi TNNT1, ma biopsies atatu a odwala adapakidwa utoto kawiri kapena katatu (magawo anayi pa mlandu uliwonse) asanasankhe zithunzi zoyimira. B. Kuchuluka kwa ulusi mwa ophunzira kutengera MYH. C. Kusanthula kwa gawo lalikulu (PCA) kufotokozera ulusi wa minofu ya mafupa mwa odwala omwe ali ndi nemaline myopathies ndi zowongolera. D. Ulusi wa minofu ya mafupa kuchokera kwa odwala omwe ali ndi nemaline myopathies ndi zowongolera zomwe zawonetsedwa pa PCA kufotokozera kuchokera ku ulusi 1000 womwe wafufuzidwa mu Chithunzi 2. Mwachitsanzo, zithunzi za volcano zikuyerekeza kusiyana pakati pa ophunzira omwe ali ndi nemaline myopathies ndi zowongolera za ACTA1 ndi TNNT1, komanso pakati pa ophunzira omwe ali ndi nemaline myopathies ndi zowongolera za ACTA1 ndi TNNT1. Mabwalo okhala ndi mitundu yosiyanasiyana amaimira mapuloteni omwe anali osiyana kwambiri pa π < 0.05, ndipo madontho akuda amaimira mapuloteni omwe anali osiyana kwambiri pa FDR < 0.05. Kusanthula kwa ziwerengero kunachitika pogwiritsa ntchito njira ya Limma linear model ndi njira za Bayesian, kutsatiridwa ndi kusintha kwa p-value kwa kufananiza kosiyanasiyana pogwiritsa ntchito njira ya Benjamini-Hochberg. H. Kusanthula kwa kuchuluka kwa mapuloteni omwe amawonetsedwa mosiyana kwambiri pa proteome yonse komanso mu ulusi wa mtundu 1 ndi 2A. Kusanthula kwa ziwerengero kunachitika pogwiritsa ntchito phukusi la clusterProfiler ndi Benjamini-Hochberg adjusted p-values. I, J. Principal component analysis (PCA) plots zojambulidwa ndi extracellular matrix ndi mitochondrial gene ontology (GO) terms.
Popeza kuti ma nemaline myopathies amatha kukhudza kuchuluka kwa mitundu ya ma nemaline myofiber yomwe imatulutsa MYH mu minofu ya mafupa, 19,20 tinayamba tafufuza mitundu ya ma nemaline myofiber yomwe imatulutsa MYH mwa odwala omwe ali ndi ma nemaline myopathies ndi ma controls. Tinapeza mtundu wa ma nemaline pogwiritsa ntchito njira yopanda tsankho yomwe idafotokozedwa kale pa mayeso a 1000 myofiber (Zithunzi Zowonjezera 10D–E) ndipo tinalepheranso kuzindikira ma nemaline myofibers oyera a 2X (Chithunzi 6B). Tinaona zotsatira zosiyana za ma nemaline myopathies pa mtundu wa ma nemaline, popeza odwala awiri omwe ali ndi kusintha kwa ACTA1 anali ndi kuchuluka kwa ma nemaline myofibers amtundu woyamba, pomwe odwala awiri omwe ali ndi TNNT1 nemaline myopathies anali ndi kuchepa kwa ma nemaline myofibers amtundu woyamba (Chithunzi 6B). Zoonadi, kufotokozera kwa MYH2 ndi ma isoforms a troponin ofulumira (TNNC2, TNNI2, ndi TNNT3) kunachepa mu ACTA1-nemaline myopathies, pomwe MYH7 inachepa mu TNNT1-nemaline myopathies (Chithunzi Chowonjezera 11A). Izi zikugwirizana ndi malipoti am'mbuyomu a kusintha kwa mitundu yosiyanasiyana ya myofiber mu maline myopathies.19,20 Tinatsimikizira zotsatirazi ndi immunohistochemistry ndipo tinapeza kuti odwala omwe ali ndi ACTA1-nemaline myopathy anali ndi ma myofibers amtundu wa 1 ambiri, pomwe odwala omwe ali ndi TNNT1-nemaline myopathy anali ndi njira yosiyana (Chithunzi 6A).
Pa mlingo wa single-fiber proteome, ulusi wa minofu ya mafupa ochokera kwa odwala a ACTA1 ndi TNNT1 nemaline myopathy adasonkhana pamodzi ndi ulusi wambiri wowongolera, ndipo ulusi wa TNNT1 nemaline myopathy nthawi zambiri ndi womwe umakhudzidwa kwambiri (Chithunzi 6C). Izi zidawonekera bwino kwambiri pokonza mapulani a principal component analysis (PCA) a ulusi wopangidwa ndi pseudo-infused kwa wodwala aliyense, ndipo odwala a TNNT1 nemaline myopathy 2 ndi 3 akuwoneka kutali kwambiri ndi zitsanzo zowongolera (Chithunzi Chowonjezera 11B, Supplementary Data Set 20). Kuti timvetse bwino momwe ulusi wochokera kwa odwala a myopathy umafananira ndi ulusi wathanzi, tidagwiritsa ntchito zambiri zomwe tidapeza kuchokera ku kusanthula kwa proteomic kwa ulusi 1,000 kuchokera kwa akuluakulu athanzi omwe adachita nawo. Tidawonetsa ulusi kuchokera ku dataset ya myopathy (adwala a ACTA1 ndi TNNT1 nemaline myopathy ndi owongolera) pa PCA plot yomwe tidapeza kuchokera ku kusanthula kwa 1000-fiber proteomic (Chithunzi 6D). Kufalikira kwa mitundu ya ulusi wa MYH pamodzi ndi PC2 mu ulusi wolamulira kunali kofanana ndi kufalikira kwa ulusi komwe kunapezeka kuchokera ku kusanthula kwa proteomic ya 1000-fiber. Komabe, ulusi wambiri mwa odwala omwe ali ndi nemaline myopathy unasintha kukhala PC2, ndipo unalumikizana ndi ulusi wathanzi wothamanga mofulumira, mosasamala kanthu za mtundu wawo wa ulusi wa MYH. Chifukwa chake, ngakhale odwala omwe ali ndi ACTA1 nemaline myopathy adasintha kupita ku ulusi wa mtundu 1 akayesedwa pogwiritsa ntchito njira zochokera ku MYH, onse a ACTA1 nemaline myopathy ndi TNNT1 nemaline myopathy adasintha proteome ya minofu ya mafupa kupita ku ulusi wothamanga mofulumira.
Kenako tinayerekeza mwachindunji gulu lililonse la odwala ndi owongolera athanzi ndipo tinapeza mapuloteni 256 ndi 552 omwe amawonetsedwa mosiyana mu ACTA1 ndi TNNT1 nemaline myopathies, motsatana (Chithunzi 6E–G ndi Chithunzi Chowonjezera 11C, Seti Yowonjezera 21). Kusanthula kwa majini kunawonetsa kuchepa kogwirizana kwa mapuloteni a mitochondrial (Chithunzi 6H–I, Seti Yowonjezera 22). Chodabwitsa n'chakuti, ngakhale kuti mitundu yosiyanasiyana ya ulusi mu ACTA1 ndi TNNT1 nemaline myopathies inali yayikulu, kuchepa kumeneku kunali kosiyana kwambiri ndi mtundu wa ulusi wochokera ku MYH (Chithunzi 6H ndi Zithunzi Zowonjezera 11D–I, Seti Yowonjezera 23). Mapuloteni atatu a tizilombo toyambitsa matenda adayang'aniridwanso mu ACTA1 kapena TNNT1 nemaline myopathies. Ma microprotein awiriwa, ENSG00000215483_TR14_ORF67 (omwe amadziwikanso kuti LINC00598 kapena Lnc-FOXO1) ndi ENSG00000229425_TR25_ORF40 (lnc-NRIP1-2), adawonetsa kuchuluka kosiyana kokha mu myofibers ya mtundu 1. ENSG00000215483_TR14_ORF67 idanenedwa kale kuti imagwira ntchito pakulamulira kayendedwe ka maselo. 56 Kumbali inayi, ENSG00000232046_TR1_ORF437 (yofanana ndi LINC01798) idawonjezeka mu myofibers ya mtundu 1 ndi mtundu 2A mu ACTA1-nemaline myopathy poyerekeza ndi zowongolera zathanzi (Chithunzi Chowonjezera 12A, Seti Yowonjezera 24). Mosiyana ndi zimenezi, mapuloteni a ribosomal sanakhudzidwe kwambiri ndi nemaline myopathy, ngakhale kuti RPS17 inachepa mu ACTA1 nemaline myopathy (Chithunzi 6E).
Kusanthula kwa kukulitsa kunawonetsanso kukwera kwa njira zodzitetezera ku matenda a ACTA1 ndi TNNT1 nemaline myopathies, pomwe kumamatira kwa maselo kunawonjezekanso mu TNNT1 nemaline myopathy (Chithunzi 6H). Kuwonjezeka kwa zinthu izi zakunja kunawonetsedwa ndi mapuloteni a extracellular matrix omwe amasuntha PCA mu PC1 ndi PC2 mbali yoyipa (monga, kupita ku ulusi womwe wakhudzidwa kwambiri) (Chithunzi 6J). Magulu onse awiri odwala adawonetsa kuwonjezeka kwa kuwonetsa kwa mapuloteni akunja omwe amakhudzidwa ndi mayankho a chitetezo chamthupi ndi njira zokonzanso sarcolemmal, monga annexins (ANXA1, ANXA2, ANXA5)57,58 ndi mapuloteni awo ogwirizana S100A1159 (Zithunzi Zowonjezera 12B–C). Njirayi idanenedwa kale kuti ikuwonjezeka mu muscular dystrophies60 koma, monga momwe tikudziwira, sinagwirizanepo kale ndi nemaline myopathies. Ntchito yanthawi zonse ya makina a molekyulu iyi imafunika pakukonzanso sarcolemmal pambuyo povulala komanso kuphatikiza ma myocytes atsopano ndi myofibers58,61. Motero, kuchuluka kwa ntchito ya njirayi m'magulu onse awiri a odwala kukuwonetsa kuti pali yankho lobwezeretsa kuvulala komwe kumachitika chifukwa cha kusakhazikika kwa myofiber.
Zotsatira za nemaline myopathy iliyonse zinali zogwirizana bwino (r = 0.736) ndipo zinawonetsa kuyanjana koyenera (Zithunzi Zowonjezera 11A–B), zomwe zikusonyeza kuti nemaline myopathy ya ACTA1 ndi TNNT1 zili ndi zotsatira zofanana pa proteome. Komabe, mapuloteni ena adalamulidwa kokha mu nemaline myopathy ya ACTA1 kapena TNNT1 (Zithunzi Zowonjezera 11A ndi C). Puloteni ya profibrotic MFAP4 inali imodzi mwa mapuloteni omwe adakwera kwambiri mu nemaline myopathy ya TNNT1 koma sinasinthe mu nemaline myopathy ya ACTA1. SKIC8, gawo la PAF1C complex lomwe limayang'anira kulembedwa kwa majini a HOX, linachepetsedwa mu nemaline myopathy ya TNNT1 koma silinakhudzidwe mu nemaline myopathy ya ACTA1 (Zithunzi Zowonjezera 11A). Kuyerekeza mwachindunji kwa ACTA1 ndi TNNT1 nemaline myopathy kunawonetsa kuchepa kwakukulu kwa mapuloteni a mitochondrial ndi kuwonjezeka kwa mapuloteni a chitetezo chamthupi mu TNNT1 nemaline myopathy (Chithunzi 6G–H ndi Zithunzi Zowonjezera 11C ndi 11H–I). Deta iyi ikugwirizana ndi atrophy/dystrophy yayikulu yomwe yawonedwa mu TNNT1 nemaline myopathy poyerekeza ndi TNNT1 nemaline myopathy (Chithunzi 6A), zomwe zikusonyeza kuti TNNT1 nemaline myopathy ikuyimira mtundu woopsa wa matendawa.
Kuti tiwone ngati zotsatira za nemaline myopathy zomwe zawonedwa zikupitirirabe pamlingo wonse wa minofu, tinachita kusanthula kwa bulk proteomic kwa minofu kuchokera ku gulu lomwelo la odwala TNNT1 nemaline myopathy ndipo tinawayerekeza ndi owongolera (n=3 pa gulu lililonse) (Chithunzi Chowonjezera 13A, Seti Yowonjezera ya Deta 25). Monga momwe tinkayembekezera, owongolera anali ofanana kwambiri mu kusanthula kwa zigawo zazikulu, pomwe odwala TNNT1 nemaline myopathy adawonetsa kusiyana kwakukulu pakati pa zitsanzo zofanana ndi zomwe zimawonedwa mu kusanthula kwa single fiber (Chithunzi Chowonjezera 13B). Kusanthula kwakukulu kunabwereza mapuloteni osiyanasiyana (Chithunzi Chowonjezera 13C, Seti Yowonjezera ya Deta 26) ndi njira zamoyo (Chithunzi Chowonjezera 13D, Seti Yowonjezera ya Deta 27) yomwe yawonetsedwa poyerekeza ulusi uliwonse, koma tinataya kuthekera kosiyanitsa mitundu yosiyanasiyana ya ulusi ndipo sitinathe kufotokozera zotsatira za matenda osiyanasiyana pa ulusi uliwonse.
Ponseponse, deta iyi ikuwonetsa kuti ma proteomics a single-myofiber amatha kufotokoza bwino zamoyo zomwe sizingadziwike pogwiritsa ntchito njira zowunikira monga immunoblotting. Kuphatikiza apo, deta iyi ikuwonetsa zoletsa zogwiritsa ntchito actin fiber typing (MYH) yokha pofotokoza kusintha kwa phenotypic. Inde, ngakhale kusintha kwa mtundu wa fiber kumasiyana pakati pa actin ndi troponin nemaline myopathies, ma nemaline myopathies onsewa amachotsa MYH fiber typing kuchokera ku kagayidwe ka minofu ya mafupa kupita ku proteome ya minofu yachangu komanso yochepa.
Kusiyana kwa maselo ndikofunikira kwambiri kuti minofu ikwaniritse zosowa zawo zosiyanasiyana. Mu minofu ya mafupa, izi nthawi zambiri zimatchedwa mitundu ya ulusi yomwe imadziwika ndi kuchuluka kosiyanasiyana kwa kupanga mphamvu ndi kutopa. Komabe, n'zoonekeratu kuti izi zikufotokoza gawo laling'ono chabe la kusiyana kwa ulusi wa minofu ya mafupa, komwe kumakhala kosinthasintha kwambiri, kovuta komanso kokhala ndi mbali zambiri kuposa momwe timaganizira kale. Kupita patsogolo kwaukadaulo tsopano kwawunikira zinthu zomwe zimalamulira ulusi wa minofu ya mafupa. Zoonadi, deta yathu ikusonyeza kuti ulusi wa mtundu wa 2X sungakhale mtundu wosiyana wa ulusi wa minofu ya mafupa. Kuphatikiza apo, tazindikira mapuloteni a kagayidwe kachakudya, mapuloteni a ribosomal ndi mapuloteni okhudzana ndi maselo ngati zinthu zazikulu zomwe zimayambitsa kusiyana kwa ulusi wa minofu ya mafupa. Pogwiritsa ntchito njira yathu yogwirira ntchito ya proteomic ku zitsanzo za odwala omwe ali ndi nematode myopathy, tawonetsanso kuti kuyika ulusi wochokera ku MYH sikuwonetsa kwathunthu kusiyana kwa minofu ya mafupa, makamaka pamene dongosolo likusokonezeka. Inde, mosasamala kanthu za mtundu wa ulusi wochokera ku MYH, nematode myopathy imabweretsa kusintha kupita ku ulusi wofulumira komanso wochepa wa okosijeni.
Ulusi wa minofu ya mafupa wakhala m'magulu kuyambira m'zaka za m'ma 1800. Kusanthula kwaposachedwa kwa omics kwatithandiza kuyamba kumvetsetsa mawonekedwe a mitundu yosiyanasiyana ya ulusi wa MYH ndi mayankho awo ku zinthu zosiyanasiyana. Monga tafotokozera pano, njira za omics zilinso ndi ubwino wodziwa bwino kuchuluka kwa zizindikiro za mtundu wa ulusi kuposa njira zachikhalidwe zochokera ku ma antibodies, popanda kudalira kuchuluka kwa chizindikiro chimodzi (kapena zingapo) kuti tidziwe mtundu wa ulusi wa minofu ya mafupa. Tinagwiritsa ntchito njira zotsatizana za transcriptomic ndi proteomic ndipo tinaphatikiza zotsatira zake kuti tiwone momwe kusinthasintha kwa ulusi m'mafupa a anthu kunakhalira. Kuyenda kumeneku kunapangitsa kuti kulephere kuzindikira ulusi wamtundu wa 2X wokha pamlingo wa mapuloteni mu vastus lateralis ya gulu lathu la anyamata athanzi. Izi zikugwirizana ndi kafukufuku wakale wa ulusi umodzi womwe unapeza <1% ulusi wa 2X wokha mu vastus lateralis wathanzi, ngakhale izi ziyenera kutsimikiziridwa m'minofu ina mtsogolo. Kusiyana pakati pa kuzindikira ulusi wa 2X wokha pamlingo wa mRNA ndi ulusi wa 2A/2X wosakanikirana pamlingo wa mapuloteni ndizovuta. Kufotokozera kwa MYH isoform mRNA si kwa circadian, 67 zomwe zikusonyeza kuti sitingathe "kuphonya" chizindikiro choyamba cha MYH2 mu ulusi wa 2X wooneka ngati woyera pamlingo wa RNA. Kufotokozera kwina komwe kungatheke, ngakhale kuti ndi kongoganizira chabe, kungakhale kusiyana kwa kukhazikika kwa mapuloteni ndi/kapena mRNA pakati pa ma isoform a MYH. Zoonadi, palibe ulusi wofulumira womwe uli 100% woyera pa isoform iliyonse ya MYH, ndipo sizikudziwika ngati kuchuluka kwa MYH1 mRNA mu 70-90% kungabweretse kuchuluka kofanana kwa MYH1 ndi MYH2 pamlingo wa mapuloteni. Komabe, poganizira transcriptome yonse kapena proteome, kusanthula kwa cluster kumatha kuzindikira motsimikiza magulu awiri osiyana okha omwe akuyimira ulusi wa minofu ya mafupa oyenda pang'onopang'ono komanso othamanga, mosasamala kanthu za kapangidwe kake kolondola ka MYH. Izi zikugwirizana ndi kusanthula pogwiritsa ntchito njira za single-nucleus transcriptomic, zomwe nthawi zambiri zimazindikira magulu awiri osiyana a myonuclear. 68, 69, 70 Komanso, ngakhale kuti kafukufuku wakale wa proteomic adapeza ulusi wa mtundu wa 2X, ulusi uwu sumasonkhana padera ndi ulusi wotsalawo ndipo umangowonetsa mapuloteni ochepa okha omwe ali ndi mitundu yosiyanasiyana ya ulusi kutengera MYH. 14 Zotsatirazi zikusonyeza kuti tiyenera kubwerera kumayambiriro kwa zaka za m'ma 1900 pakuwona magulu a ulusi wa minofu, omwe adagawa ulusi wa minofu ya mafupa a anthu osati m'magulu atatu osiyana kutengera MYH, koma m'magulu awiri kutengera mphamvu zawo zamagetsi ndi zogwirira ntchito. 63
Chofunika kwambiri, kusiyana kwa myofiber kuyenera kuganiziridwa m'njira zosiyanasiyana. Kafukufuku wakale wa "omics" wasonyeza mbali iyi, zomwe zikusonyeza kuti ulusi wa minofu ya mafupa supanga magulu osiyana koma umakonzedwa motsatira continuum. 11, 13, 14, 64, 71 Pano, tikuwonetsa kuti, kuwonjezera pa kusiyana kwa mphamvu zogwira ntchito ndi kagayidwe kachakudya ka minofu ya mafupa, myofibers imatha kusiyanitsidwa ndi zinthu zokhudzana ndi kuyanjana kwa maselo ndi maselo ndi njira zosinthira. Inde, tapeza kusiyana kwa ribosome mu ulusi wa minofu ya mafupa zomwe zimapangitsa kusiyana kosiyana kosagwirizana ndi mitundu ya ulusi wofulumira komanso wofulumira. Chifukwa chachikulu cha kusiyana kwakukulu kwa myofiber kumeneku, kosagwirizana ndi mtundu wa ulusi wofulumira komanso wofulumira, sichikudziwika bwino, koma chikhoza kuloza ku dongosolo lapadera la malo mkati mwa minofu ya minofu yomwe imayankha bwino mphamvu ndi katundu winawake,72 kulumikizana kwapadera kwa maselo kapena ziwalo ndi mitundu ina ya maselo mu minofu ya microenvironment73,74,75 kapena kusiyana kwa ntchito ya ribosome mkati mwa myofibers payekhapayekha. Zoonadi, ribosomal heteroplasmy, kaya kudzera m'malo mwa RPL3 ndi RPL3L kapena pamlingo wa 2′O-methylation wa rRNA, yawonetsedwa kuti ikugwirizana ndi hypertrophy ya minofu ya mafupa76,77. Kugwiritsa ntchito ma multi-omic ndi malo pamodzi ndi mawonekedwe a ntchito ya myofibers payekha kudzapititsa patsogolo kumvetsetsa kwathu kwa biology ya minofu pamlingo wa multi-omic78.
Mwa kusanthula ma proteome a single myofibers ochokera kwa odwala omwe ali ndi nemaline myopathies, tinawonetsanso kufunika, kugwira ntchito, komanso kugwiritsidwa ntchito kwa single myofiber proteomics kuti timvetse bwino matenda a minofu ya mafupa. Kuphatikiza apo, poyerekeza njira yathu yogwirira ntchito ndi kusanthula kwa proteomic padziko lonse lapansi, tinatha kuwonetsa kuti single myofiber proteomics imapereka chidziwitso chofanana ndi global tissue proteomics ndipo imakulitsa kuzama kumeneku powerengera kusiyana kwa interfiber ndi mtundu wa myofiber. Kuphatikiza pa kusiyana komwe kumayembekezeredwa (ngakhale kosinthika) mu chiŵerengero cha mtundu wa fiber chomwe chimapezeka mu ACTA1 ndi TNNT1 nemaline myopathies poyerekeza ndi zowongolera zathanzi,19 tinawonanso kusintha kwa oxidative ndi extracellular popanda kusintha kwa mtundu wa fiber komwe kumayendetsedwa ndi MYH. Fibrosis yanenedwapo kale mu TNNT1 nemaline myopathies.19 Komabe, kusanthula kwathu kumawonjezera pa zomwe tapezazi powululanso kuchuluka kwa mapuloteni okhudzana ndi kupsinjika omwe amachokera kunja kwa thupi, monga ma annexins, omwe amagwira ntchito mu njira zokonzanso sarcolemmal, mu myofibers kuchokera kwa odwala omwe ali ndi ACTA1 ndi TNNT1 nemaline myopathies.57,58,59 Pomaliza, kuchuluka kwa annexin mu myofibers kuchokera kwa odwala omwe ali ndi nemaline myopathy kungayimire yankho la maselo pokonza ma myofibers atrophic kwambiri.
Ngakhale kuti kafukufukuyu akuyimira kusanthula kwakukulu kwa minofu yonse ya ulusi umodzi kwa anthu mpaka pano, sikuti alibe malire. Tinasiyanitsa ulusi wa minofu ya mafupa kuchokera ku chitsanzo chochepa komanso chofanana cha ophunzira ndi minofu imodzi (vastus lateralis). Chifukwa chake, n'zosatheka kuchotsa kupezeka kwa ulusi winawake m'mitundu yonse ya minofu komanso pamlingo wapamwamba kwambiri wa minofu. Mwachitsanzo, sitingathe kuchotsa kuthekera kwa ulusi wothamanga kwambiri (monga ulusi wa 2X) womwe umapezeka mwa othamanga ophunzitsidwa bwino komanso/kapena othamanga amphamvu79 kapena nthawi ya kusachita masewera olimbitsa thupi66,80. Kuphatikiza apo, kuchuluka kochepa kwa zitsanzo za ophunzira kunatilepheretsa kufufuza kusiyana kwa amuna ndi akazi pa kusiyana kwa ulusi, chifukwa ma fiber type ratio amadziwika kuti amasiyana pakati pa amuna ndi akazi. Kuphatikiza apo, sitinathe kuchita kusanthula kwa transcriptomic ndi proteomic pa ulusi wa minofu womwewo kapena zitsanzo kuchokera kwa ophunzira omwewo. Pamene ife ndi ena tikupitiriza kukonza kusanthula kwa selo limodzi ndi myofiber imodzi pogwiritsa ntchito kusanthula kwa omics kuti tipeze kulowetsedwa kochepa kwambiri kwa zitsanzo (monga momwe zasonyezedwera pano mu kusanthula kwa ulusi kuchokera kwa odwala omwe ali ndi mitochondrial myopathy), mwayi wophatikiza njira zambiri (ndi zogwira ntchito) mkati mwa ulusi umodzi wa minofu umawonekera.
Ponseponse, deta yathu imazindikira ndikufotokozera zomwe zimayambitsa kusiyana kwa minofu ya mafupa ndi yolembedwa pambuyo pa kulemba. Makamaka, tikupereka deta yomwe imatsutsa chiphunzitso cha nthawi yayitali cha physiology ya minofu ya mafupa chogwirizana ndi tanthauzo lakale la mitundu ya ulusi lochokera ku MYH. Tikukhulupirira kukonzanso mkanganowu ndikuganiziranso kumvetsetsa kwathu kwa magulu a ulusi wa minofu ya mafupa ndi kusiyana kwa ulusi.
Anthu khumi ndi anayi ochokera ku Caucasian (amuna 12 ndi akazi awiri) adavomera kutenga nawo mbali mu kafukufukuyu. Kafukufukuyu adavomerezedwa ndi Komiti ya Makhalidwe Abwino ya Chipatala cha Ghent University (BC-10237), adatsatira Chilengezo cha Helsinki cha 2013, ndipo adalembetsedwa ku ClinicalTrials.gov (NCT05131555). Makhalidwe onse a ophunzirawo aperekedwa mu Supplementary Table 1. Atalandira chilolezo cholembedwa ndi mawu, ophunzirawo adayesedwa kuchipatala asanalowe mu kafukufukuyu. Ophunzirawo anali achichepere (zaka 22-42), athanzi (osadwala, osasuta fodya), komanso ochita masewera olimbitsa thupi pang'ono. Kuchuluka kwa mpweya womwe umabwera chifukwa cha mpweya kunadziwika pogwiritsa ntchito step ergometer poyesa thanzi la thupi monga tafotokozera kale. 81
Zitsanzo za biopsy ya minofu zinasonkhanitsidwa panthawi yopuma komanso mu nthawi yosala kudya katatu, masiku 14 osiyana. Popeza zitsanzozi zinasonkhanitsidwa ngati gawo la kafukufuku wamkulu, ophunzirawo adadya placebo (lactose), H1-receptor antagonist (540 mg fexofenadine), kapena H2-receptor antagonist (40 mg famotidine) mphindi 40 biopsy isanachitike. Tawonetsa kale kuti ma histamine receptor antagonists awa samakhudza thanzi la minofu yopumula81, ndipo palibe kusonkhana kokhudzana ndi boma komwe kunawonedwa muzithunzi zathu zowongolera khalidwe (Zithunzi Zowonjezera 3 ndi 6). Zakudya zokhazikika (41.4 kcal/kg kulemera kwa thupi, 5.1 g/kg chakudya cholemera thupi, 1.4 g/kg mapuloteni olemera thupi, ndi 1.6 g/kg mafuta olemera thupi) zidasungidwa kwa maola 48 tsiku lililonse loyesera lisanafike, ndipo chakudya cham'mawa chokhazikika (1.5 g/kg chakudya cholemera thupi) chinadyedwa m'mawa wa tsiku loyesera. Pogwiritsa ntchito mankhwala oletsa ululu am'deralo (0.5 ml 1% lidocaine wopanda epinephrine), zizindikiro za minofu zinapezeka kuchokera ku minofu ya vastus lateralis pogwiritsa ntchito percutaneous Bergström aspiration.82 Zitsanzo za minofu zinayikidwa nthawi yomweyo mu RNAlater ndikusungidwa pa 4°C mpaka kudulidwa kwa ulusi wamanja (mpaka masiku atatu).
Ma myofiber bundles omwe angopatulidwa kumene adasamutsidwira ku RNAlater medium yatsopano mu mbale yopangira zokolola. Kenako ma myofibers amodzi ndi amodzi adadulidwa pamanja pogwiritsa ntchito stereomicroscope ndi ma tweezers abwino. Ulusi 25 unadulidwa kuchokera ku biopsy iliyonse, poganizira kwambiri kusankha ulusi kuchokera m'malo osiyanasiyana a biopsy. Pambuyo poduladula, ulusi uliwonse unamizidwa mofatsa mu 3 μl ya lysis buffer (SingleShot Cell Lysis Kit, Bio-Rad) yokhala ndi proteinase K ndi ma enzymes a DNase kuti achotse mapuloteni osafunikira ndi DNA. Kuduladula kwa maselo ndi kuchotsa mapuloteni/DNA kunayambitsidwa ndi vortexing yochepa, kuzunguliza madziwo mu microcentrifuge, ndikuyikamo kutentha kwa chipinda (mphindi 10). Lysate kenako idayikidwa mu thermal cycler (T100, Bio-Rad) pa 37°C kwa mphindi 5, 75°C kwa mphindi 5, kenako nkusungidwa nthawi yomweyo pa -80°C mpaka kukonzanso kwina.
Malaibulale a RNA ogwirizana ndi Illumina adakonzedwa kuchokera ku 2 µl ya myofiber lysate pogwiritsa ntchito QuantSeq-Pool 3′ mRNA-Seq Library Prep Kit (Lexogen). Njira zatsatanetsatane zitha kupezeka m'buku la wopanga. Njirayi imayamba ndi kupanga cDNA ya chingwe choyamba pogwiritsa ntchito reverse transcription, pomwe ma identifier apadera a molecular (UMIs) ndi ma barcode a i1 enieni amayambitsidwa kuti atsimikizire kuphatikiza zitsanzo ndikuchepetsa kusinthasintha kwaukadaulo panthawi yokonza. cDNA kuchokera ku 96 myofibers imaphatikizidwa ndikuyeretsedwa ndi mikanda yamaginito, pambuyo pake RNA imachotsedwa ndipo kupanga kwa chingwe chachiwiri kumachitika pogwiritsa ntchito ma primer osasinthika. Laibulale imayeretsedwa ndi mikanda yamaginito, ma tag a i5/i7 enieni amawonjezedwa, ndipo PCR imakulitsidwa. Gawo lomaliza loyeretsa limapanga malaibulale ogwirizana ndi Illumina. Ubwino wa dziwe lililonse la laibulale unayesedwa pogwiritsa ntchito High Sensitivity Small Fragment DNA Analysis Kit (Agilent Technologies, DNF-477-0500).
Kutengera kuchuluka kwa Qubit, maiwe osambira adaphatikizidwanso pamlingo wa equimolar (2 nM). Kenako dziwe lomwe linatuluka linayikidwa pa chida cha NovaSeq 6000 mu mode yokhazikika pogwiritsa ntchito NovaSeq S2 Reagent Kit (1 × 100 nucleotides) yokhala ndi 2 nM loading (4% PhiX).
Chitoliro chathu chimachokera pa njira ya Lexogen yowunikira deta ya QuantSeq Pool (https://github.com/Lexogen-Tools/quantseqpool_analysis). Deta idachotsedwa koyamba ndi bcl2fastq2 (v2.20.0) kutengera index ya i7/i5. Kenako Read 2 idachotsedwa ndi idemux (v0.1.6) kutengera i1 sample barcode ndipo UMI sequences idachotsedwa ndi umi_tools (v1.0.1). Kenako Reads idakonzedwa ndi cutadapt (v3.4) m'magawo angapo kuti ichotse ma reads afupi (<20 kutalika) kapena ma reads okhala ndi ma adapter sequences okha. Ma reads adalumikizidwa ku genome ya munthu pogwiritsa ntchito STAR (v2.6.0c) ndipo mafayilo a BAM adayikidwa ndi SAMtools (v1.11). Ma reads obwerezabwereza adachotsedwa pogwiritsa ntchito umi_tools (v1.0.1). Pomaliza, kuwerengera kwa alignment kunachitika pogwiritsa ntchito featureCounts mu Subread (v2.0.3). Kuwongolera khalidwe kunachitika pogwiritsa ntchito FastQC (v0.11.9) pa magawo angapo apakati a payipi.
Kukonza ndi kuwonetsa zinthu zina zonse za bioinformatics kunachitika mu R (v4.2.3), makamaka pogwiritsa ntchito njira ya Seurat (v4.4.0). 83 Chifukwa chake, ma UMI ndi ma metadata matrices osiyanasiyana adasinthidwa kukhala zinthu za Seurat. Majini omwe adawonetsedwa mu ulusi wosakwana 30% adachotsedwa. Zitsanzo zosalimba zidachotsedwa kutengera malire ochepera a ma UMI 1000 ndi majini 1000 omwe adapezeka. Pamapeto pake, ulusi 925 udadutsa njira zonse zosefera zaubwino. Ma UMI adasinthidwa pogwiritsa ntchito njira ya Seurat SCTransform v2, 84 kuphatikiza mawonekedwe onse 7418 omwe adapezeka, ndipo kusiyana kwa omwe ali pakati pa omwe adatenga nawo mbali kudachotsedwa. Ma metadata onse oyenera angapezeke mu Supplementary Dataset 28.
Nthawi yotumizira: Sep-10-2025