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Imisipha yamathambo iyisicubu esingafani esakhiwe kakhulu ngama-myofibril, okuthi kubantu ahlukaniswe ngezinhlobo ezintathu: eyodwa "ehamba kancane" (uhlobo 1) kanye nezimbili "ezisheshayo" (izinhlobo 2A kanye no-2X). Kodwa-ke, ukungafani phakathi nangaphakathi kwezinhlobo zendabuko ze-myofibril kusalokhu kungaqondakali kahle. Sisebenzise izindlela ze-transcriptomic kanye ne-proteomic kuma-myofibril angu-1050 kanye no-1038 avela kuma-vastus lateralis abantu, ngokulandelana. Ucwaningo lwe-proteomic lwaluhlanganisa amadoda, kanti ucwaningo lwe-transcriptomic lwaluhlanganisa amadoda ayi-10 nabesifazane ababili. Ngaphezu kwama-isoforms e-myosin heavy chain, sithole amaprotheni e-metabolic, amaprotheni e-ribosomal, kanye namaprotheni e-cellular junctional njengemithombo yokuguquguquka kwe-intermyofibril okunezilinganiso eziningi. Ngaphezu kwalokho, naphezu kokuhlonza amaqoqo e-fiber ehamba kancane nesheshayo, idatha yethu iphakamisa ukuthi imicu yohlobo lwe-2X ayihlukaniseki ngokwe-phenotypic kwezinye imicu eshintsha ngokushesha. Ngaphezu kwalokho, ukuhlukaniswa okusekelwe ku-myosin heavy chain akwanele ukuchaza uhlobo lwe-myofiber kuma-nemaline myopathies. Sekukonke, idatha yethu iphakamisa ukungafani kwe-myofiber okunezilinganiso eziningi, kanye nemithombo yokwehluka edlula ama-isoform e-myosin heavy chain.
Ukungafani kwamaseli kuyisici esingokwemvelo sazo zonke izinhlelo zezinto eziphilayo, okuvumela amaseli ukuthi akhetheke ukuze ahlangabezane nezidingo ezahlukene zezicubu namaseli.1 Umbono wendabuko wokungafani kwama-fiber emisipha yamathambo ube ukuthi ama-motor neurons achaza uhlobo lwe-fiber ngaphakathi kweyunithi ye-motor, nokuthi uhlobo lwe-fiber (okungukuthi, uhlobo 1, uhlobo 2A, kanye nohlobo 2X kubantu) lunqunywa izici ze-myosin heavy chain (MYH) isoforms.2 Lokhu ekuqaleni kwakusekelwe ekungazinzini kwe-pH ATPase yabo, 3,4 futhi kamuva ekubonakalisweni kwabo kwama-molecule kwe-MYH.5 Kodwa-ke, ngokuhlonza nokwamukelwa okulandelayo kwama-fiber "axubile" aveza ama-MYH amaningi ngokulingana okuhlukahlukene, ama-fiber emisipha yamathambo abhekwa kakhulu njenge-continuum kunokuba abe yizinhlobo ze-fiber ezihlukile.6 Naphezu kwalokhu, insimu isathembele kakhulu ku-MYH njenge-classifier eyinhloko yokuhlukaniswa kwe-myofiber, umbono okungenzeka uthonywe yimikhawulo kanye nokubandlulula okubalulekile kwezifundo zakuqala zamagundane amaphrofayili awo okubonakaliswa kwe-MYH kanye nobubanzi bezinhlobo ze-fiber ahlukile kulawo angabantu.2 Isimo siyinkimbinkimbi kakhulu ngokuthi imisipha yamathambo yabantu ehlukene ibonisa ububanzi obuhlukahlukene be-fiber izinhlobo.7 I-vastus lateralis iyimisipha exubile enephrofayili yokubonakaliswa kwe-MYH ephakathi (ngakho-ke imele).7 Ngaphezu kwalokho, ukulula kwayo kokuthatha isampula kwenza kube yimisipha efundwe kahle kakhulu kubantu.
Ngakho-ke, uphenyo olungenabandlululo ngokuhlukahlukana kwe-fiber yemisipha yamathambo kusetshenziswa amathuluzi anamandla e-"omics" lubalulekile kodwa futhi luyinselele, ngokwengxenye ngenxa yemvelo ye-multinucleated ye-fiber yemisipha yamathambo. Kodwa-ke, ubuchwepheshe be-transcriptomics8,9 kanye ne-proteomics10 bushintshe kakhulu ekuzweleni eminyakeni yamuva ngenxa yentuthuko ehlukahlukene yezobuchwepheshe, okuvumela ukuhlaziywa kwemisipha yamathambo ngesisombululo se-fiber eyodwa. Ngenxa yalokho, kwenziwe intuthuko enkulu ekuchazeni ukuhlukahluka kwe-fiber eyodwa kanye nokusabela kwabo ezicini ze-atrophic kanye nokuguga11,12,13,14,15,16,17,18. Okubalulekile, lokhu kuthuthuka kwezobuchwepheshe kunezinhlelo zokusebenza zezokwelapha, okuvumela ukuchazwa okuningiliziwe nokunembile kokungapheleli okuhlobene nesifo. Isibonelo, i-pathophysiology ye-nemaline myopathy, enye yezifo ezivame kakhulu zemisipha ezizuzwe njengefa (MIM 605355 kanye ne-MIM 161800), iyinkimbinkimbi futhi iyadida.19,20 Ngakho-ke, ukuchazwa okungcono kokungapheleli kwe-fiber yemisipha yamathambo kungaholela ekuthuthukisweni okukhulu ekuqondeni kwethu lesi sifo.
Sakha izindlela zokuhlaziya i-transcriptomic kanye ne-proteomic yezintambo zomzimba ezisodwa ezihlukaniswe ngesandla kusuka kumasampula e-biopsy yomuntu futhi sazisebenzisa ezinkulungwaneni zezintambo, okwasivumela ukuthi sihlole ukungafani kwamaseli kwezintambo zomzimba zomzimba womuntu. Phakathi nalo msebenzi, sibonise amandla e-transcriptomic kanye ne-proteomic phenotyping yezintambo zomzimba futhi sathola amaprotheni e-metabolic, ribosomal, kanye namaseli njengemithombo ebalulekile yokuguquguquka kwe-interfiber. Ngaphezu kwalokho, sisebenzisa lo msebenzi wokusebenza kwe-proteomic, sichaze ukubaluleka kwemitholampilo kwe-nematode myopathy kuma-fiber omzimba omzimba owodwa, sembula ushintsho oluhlanganisiwe oluya kuma-fiber angewona ama-oxidative azimele ngohlobo lwe-fiber olusekelwe ku-MYH.
Ukuze sihlole ukungafani kwemicu yemisipha yamathambo abantu, sakha izindlela ezimbili zokusebenza ukuze sikwazi ukuhlaziya i-transcriptome kanye ne-proteome yemicu yemisipha yamathambo eyodwa (Isithombe 1A kanye nesithombe esengeziwe 1A). Sakha futhi salungisa izinyathelo eziningana zendlela, kusukela ekugcinweni kwesampula kanye nokulondolozwa kwe-RNA kanye nobuqotho bamaprotheni kuya ekwenzeni ngcono ukuphuma kwenqubo ngayinye. Ekuhlaziyweni kwe-transcriptome, lokhu kufezwe ngokufaka amabhakhodi e-molecule athile esampula esinyathelweni sokuqala sokubhalwa kabusha, okuvumela imicu engu-96 ukuthi ihlanganiswe ukuze kucutshungulwe kahle phansi. Ukulandelana okujulile (±1 million reads per fiber) uma kuqhathaniswa nezindlela zendabuko zeseli elilodwa kwathuthukisa idatha ye-transcriptome. 21 Kuma-proteomics, sisebenzise i-gradient emfushane ye-chromatographic (imizuzu engu-21) ehlanganiswe nokutholwa kwedatha ye-DIA-PASEF ku-timsTOF mass spectrometer ukuze kuthuthukiswe ukujula kwe-proteome ngenkathi kugcinwa ukuphuma okuphezulu. 22,23 Ukuze sihlole ukungafani kwezintambo zemisipha yamathambo ezinempilo, sichaze ama-transcriptome ezintambo eziyi-1,050 ngazinye ezivela kubanikeli abadala abayi-14 abanempilo kanye nama-proteome ezintambo eziyi-1,038 ezivela kubanikeli abadala abayi-5 abanempilo (Ithebula Elingeziwe 1). Kuleli phepha, lawa masethi edatha abizwa ngokuthi ama-transcriptome nama-proteome ayi-1,000-fiber, ngokulandelana. Indlela yethu ithole ama-transcriptome angu-27,237 kanye namaprotheni angu-2,983 ekuhlaziyweni kwe-transcriptomic nama-proteomic ayi-1,000-fiber (Isithombe 1A, Amasethi Edatha Angeziwe 1–2). Ngemva kokuhlunga amasethi edatha e-transcriptomic nama-proteomic ukuze kutholakale izakhi zofuzo ezingaphezu kuka-1,000 ezitholakele kanye namanani avumelekile angu-50% ngentambo ngayinye, kwenziwa ukuhlaziywa kwe-bioinformatics okulandelayo kuma-fiber angu-925 kanye no-974 ku-transcriptome kanye ne-proteome, ngokulandelana. Ngemva kokuhlunga, kwatholakala isilinganiso sama-gene angu-4257 ± 1557 kanye namaprotheni angu-2015 ± 234 (isilinganiso ± SD) ngefayibha ngayinye, ngokuhlukahluka okulinganiselwe phakathi komuntu ngamunye (Izibalo Ezingeziwe 1B–C, Amasethi Edatha Angeziwe 3–4). Kodwa-ke, ukuhlukahluka ngaphakathi kwesihloko kwakuvelele kakhulu kubahlanganyeli, cishe ngenxa yokwehluka kokukhiqizwa kwe-RNA/protein phakathi kwamafayibha anobude obuhlukene kanye nezindawo ezinqamulayo. Kumaprotheni amaningi (>2000), i-coefficient of variation yayingaphansi kuka-20% (Isibalo Esingeziwe 1D). Zombili izindlela zavumela ukubamba uhla olubanzi oluguquguqukayo lwemibhalo kanye namaprotheni anezimpawu ezivelele kakhulu ezibalulekile ekuncipheni kwemisipha (isb., ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Izibalo Ezingeziwe 1E–F). Iningi lezici ezitholiwe zazivamile phakathi kwamasethi edatha e-transcriptomic kanye ne-proteomic (Isithombe Esingeziwe 1G), futhi amandla aphakathi e-UMI/LFQ alezi zici ayehlobene kahle (r = 0.52) (Isithombe Esingeziwe 1H).
Ukuhamba komsebenzi we-Transcriptomics kanye ne-proteomics (kudalwe nge-BioRender.com). Ama-BD Dynamic range curves e-MYH7, MYH2, kanye ne-MYH1, kanye nemingcele ebalwe yokunikezwa kohlobo lwe-fiber. E, F Ukusatshalaliswa kokubonakaliswa kwe-MYH kuwo wonke ama-fiber kuma-dataset e-transcriptomics kanye ne-proteomics. G, H Izizalo ze-Uniform Diversity Approximation and Projection (UMAP) zama-transcriptomics kanye ne-proteomics ezifakwe umbala ngohlobo lwe-fiber olusekelwe ku-MYH. I, J Izizalo zesici ezibonisa ukubonakaliswa kwe-MYH7, MYH2, kanye ne-MYH1 kuma-dataset e-transcriptomics kanye ne-proteomics.
Ekuqaleni saqala ukwabela uhlobo lwefayibha olusekelwe ku-MYH ku-fiber ngayinye sisebenzisa indlela elungiselelwe kahle esebenzisa ukuzwela okuphezulu kanye nobubanzi obuguquguqukayo bokubonakaliswa kwe-MYH kumasethi edatha e-omics. Izifundo zangaphambilini zisebenzise imingcele engahleliwe ukulebula i-fiber njengohlobo oluhlanzekile 1, uhlobo 2A, uhlobo 2X, noma ixutshwe ngokusekelwe kumaphesenti amisiwe okubonakaliswa kwe-MYHs11,14,24 ehlukene. Sisebenzise indlela ehlukile lapho ukubonakaliswa kwefayibha ngayinye kwahlelwa khona yi-MYHs esasisebenzisa ukuthayipha i-fibers: MYH7, MYH2, kanye ne-MYH1, ehambisana nohlobo 1, uhlobo 2A, kanye ne-fibers yohlobo 2X, ngokulandelana. Sabe sesibala ngokwezibalo iphuzu eliphansi lokuguquka kwejika ngalinye elivelayo futhi salisebenzisa njengomkhawulo wokwabela i-fibers njenge-positive (ngaphezu komkhawulo) noma i-negative (ngaphansi komkhawulo) we-MYH ngayinye (Isithombe 1B–D). Le datha ibonisa ukuthi i-MYH7 (Isithombe 1B) kanye ne-MYH2 (Isithombe 1C) zinamaphrofayili okubonakaliswa okuvula/okuvala ahlukile kakhulu ezingeni le-RNA uma kuqhathaniswa nezinga leprotheni. Ngempela, ezingeni leprotheni, imicu embalwa kakhulu ayizange iveze i-MYH7, futhi akukho micu eyayinokubonakaliswa okungu-100% kwe-MYH2. Sisebenzise ngokulandelayo imingcele yokubonakaliswa enqunyiwe ukuze sinikeze izinhlobo ze-fiber ezisekelwe ku-MYH kuzo zonke imicu kusethi ngayinye yedatha. Isibonelo, imicu ye-MYH7+/MYH2-/MYH1- yabelwe uhlobo 1, kuyilapho imicu ye-MYH7-/MYH2+/MYH1+ yabelwe uhlobo oluxubile lwe-2A/2X (bheka Ithebula Elingeziwe 2 ukuze uthole incazelo egcwele). Sihlanganisa yonke imicu, sibone ukusatshalaliswa okufanayo okuphawulekayo kwezinhlobo ze-fiber ezisekelwe ku-MYH kokubili emazingeni e-RNA (Isithombe 1E) kanye ne-protein (Isithombe 1F), kuyilapho ukwakheka okuhlobene kwezinhlobo ze-fiber ezisekelwe ku-MYH kwahlukahluka kubantu ngabanye, njengoba bekulindelekile (Isithombe Esingeziwe 2A). Imicu eminingi yahlukaniswa njengohlobo 1 oluhlanzekile (34-35%) noma uhlobo 2A (36-38%), yize inani elikhulu lemicu ye-2A/2X exubile nayo yatholakala (16-19%). Umehluko ophawulekayo ukuthi imicu emsulwa yohlobo lwe-2X ingatholakala kuphela ezingeni le-RNA, kodwa hhayi ezingeni leprotheni, okuphakamisa ukuthi ukubonakaliswa okusheshayo kwe-MYH okungenani kulawulwa ngokwengxenye ngemva kokubhalwa.
Siqinisekisile indlela yethu yokuthayipha i-MYH fiber esekelwe ku-proteomics sisebenzisa i-antibody-based dot blotting, futhi zombili lezi zindlela zafinyelela ukuvumelana okungu-100% ekuboneni imicu yohlobo 1 emsulwa kanye nohlobo 2A (bheka uMfanekiso Ongeziwe 2B). Kodwa-ke, indlela esekelwe ku-proteomics yayinozwelo kakhulu, isebenza kahle ekuboneni imicu exubile, kanye nokulinganisa isilinganiso sejini ngalinye le-MYH ku-fiber ngayinye. Le datha ibonisa ukusebenza kahle kokusebenzisa indlela enengqondo, ezwela kakhulu esekelwe ku-omics ukuze kuchazwe izinhlobo ze-fiber yemisipha yamathambo.
Sabe sesisebenzisa ulwazi oluhlanganisiwe olunikezwe yi-transcriptomics kanye ne-proteomics ukuze sihlukanise ama-myofibers ngokufanele ngokusekelwe ku-transcriptome noma i-proteome yawo ephelele. Sisebenzisa indlela ye-uniform manifold approximation and projection (UMAP) ukunciphisa ubukhulu kube yizingxenye eziyisithupha eziyinhloko (Izibalo Ezingeziwe 3A–B), sikwazile ukubona ngeso lengqondo ukuguquguquka kwe-myofiber ku-transcriptome (Isithombe 1G) kanye ne-proteome (Isithombe 1H). Okuphawulekayo ukuthi ama-myofibers awazange aqoqwe ngabahlanganyeli (Izibalo Ezingeziwe 3C–D) noma izinsuku zokuhlola (Isithombe Esingeziwe 3E) kumasethi edatha e-transcriptomics noma e-proteomics, okuphakamisa ukuthi ukuguquguquka okungaphakathi kwesihloko kuma-fiber emisipha yamathambo kuphakeme kunokuguquguquka okuphakathi kwesihloko. Esihlokweni se-UMAP, kwavela amaqoqo amabili ahlukene amelela ama-myofibers “asheshayo” kanye “anejubane” (Izithombe 1G–H). Ama-myofiber e-MYH7+ (ahamba kancane) ahlanganiswe endaweni eqondile ye-UMAP1, kuyilapho ama-myofiber e-MYH2+ kanye ne-MYH1+ (ashesha) ahlanganiswe endaweni engezansi ye-UMAP1 (Izithombe 1I–J). Kodwa-ke, akukho mehluko owenziwe phakathi kwezinhlobo ze-fiber ezishintsha ngokushesha (okungukuthi, uhlobo 2A, uhlobo 2X, noma i-2A/2X exubile) ngokusekelwe ekubonakalisweni kwe-MYH, okuphakamisa ukuthi i-MYH1 (Isithombe 1I–J) noma ezinye izimpawu ze-myofiber ze-2X zakudala ezifana ne-ACTN3 noma i-MYLK2 (Izithombe Ezingeziwe 4A–B) azihlukanisi phakathi kwezinhlobo ezahlukene ze-myofiber lapho kucatshangelwa i-transcriptome noma i-proteome yonke. Ngaphezu kwalokho, uma kuqhathaniswa ne-MYH2 kanye ne-MYH7, ama-transcript ambalwa noma amaprotheni ahlobene kahle ne-MYH1 (Izithombe Ezingeziwe 4C–H), okuphakamisa ukuthi ubuningi be-MYH1 abubonisi ngokugcwele i-myofiber transcriptome/proteome. Kufinyelelwe eziphethweni ezifanayo lapho kuhlolwa ukuvezwa okuxubile kwama-isoform amathathu e-MYH ezingeni le-UMAP (Izithombe Ezingeziwe. 4I–J). Ngakho-ke, yize imicu engu-2X ingabonakala ezingeni le-transcript ngokusekelwe ekulinganisweni kwe-MYH kuphela, imicu ye-MYH1+ ayihlukaniseki kwezinye imicu esheshayo uma kucatshangelwa i-transcriptome noma i-proteome yonke.
Njengokuhlola kokuqala kokungafani kwe-fiber okuhamba kancane ngale kwe-MYH, sihlole amaprotheni amane asunguliwe aqondene nohlobo lwe-fiber oluhamba kancane: i-TPM3, i-TNNT1, i-MYL3, kanye ne-ATP2A22. Izinhlobo ze-fiber ezihamba kancane zibonise ukuhlangana okuphezulu, yize kungaphelele, kwe-Pearson ne-MYH7 kuzo zombili i-transcriptomics (Isithombe Esingeziwe 5A) kanye ne-proteomics (Isithombe Esingeziwe 5B). Cishe u-25% no-33% wezintambo ezihamba kancane azizange zihlukaniswe njengezintambo ezihlanzekile ezihamba kancane yizo zonke izinhlobo zezakhi zofuzo/amaprotheni ku-transcriptomics (Isithombe Esingeziwe 5C) kanye ne-proteomics (Isithombe Esingeziwe 5D), ngokulandelana. Ngakho-ke, ukuhlukaniswa kwe-fiber okuhamba kancane okusekelwe kuzinhlobo eziningi zezakhi zofuzo/amaprotheni kuletha ubunzima obengeziwe, ngisho nakumaprotheni aziwa ngokuthi aqondene nohlobo lwefayibha. Lokhu kusikisela ukuthi ukuhlukaniswa kwefayibha okusekelwe kuma-isoform omndeni owodwa wezakhi zofuzo/amaprotheni kungase kungabonisi ngokwanele ukungafani kwangempela kwezintambo zemisipha yamathambo.
Ukuze siqhubeke sihlola ukuguquguquka kwe-phenotypic kwezintambo zemisipha yamathambo abantu esikalini semodeli yonke ye-omics, senze ukunciphisa kobukhulu obungachemile kwedatha sisebenzisa ukuhlaziywa kwengxenye eyinhloko (i-PCA) (Isithombe 2A). Ngokufana nezindawo ze-UMAP, umhlanganyeli noma usuku lokuhlola azange kuthonye ukuhlanganiswa kwezintambo ezingeni le-PCA (Izithombe Ezingeziwe 6A–C). Kuwo womabili amasethi edatha, uhlobo lwentambo olusekelwe ku-MYH luchazwe yi-PC2, olubonise iqoqo lezintambo zohlobo 1 ezihamba kancane kanye neqembu lesibili eliqukethe izintambo zohlobo 2A ezihamba ngokushesha, uhlobo 2X, kanye nezintambo ezihlanganisiwe ze-2A/2X (Isithombe 2A). Kuwo womabili amasethi edatha, la maqoqo amabili axhunywe yinani elincane lezintambo zohlobo 1/2A ezihlanganisiwe. Njengoba bekulindelekile, ukuhlaziywa kokumelwa ngokweqile kwabashayeli abayinhloko be-PC kuqinisekisile ukuthi i-PC2 iqhutshwa yizimpawu zokubopha kanye ne-metabolic (Isithombe 2B kanye nezithombe Ezingeziwe 6D–E, Amasethi Edatha Angeziwe 5–6). Sekukonke, uhlobo lwefayibha olusekelwe ku-MYH lutholakale lwanele ukuchaza ukuguquguquka okuqhubekayo ku-PC2, ngaphandle kwalokho okubizwa ngokuthi amafayibha angu-2X asakazwa kulo lonke i-transcriptome ngaphakathi kweqembu elisheshayo.
A. Iziqephu zokuhlaziywa kwezingxenye eziyinhloko (i-PCA) zamasethi edatha e-transcriptome kanye ne-proteome afakwe umbala ngokohlobo lwefayibha ngokusekelwe ku-MYH. B. Ukuhlaziywa kokunotha kwabashayeli be-transcript kanye namaprotheni ku-PC2 kanye ne-PC1. Ukuhlaziywa kwezibalo kwenziwe kusetshenziswa iphakheji ye-clusterProfiler kanye namanani e-p alungisiwe e-Benjamini-Hochberg. C, D. Iziqephu ze-PCA ezifakwe umbala ngokohlobo lwe-intercellular adhesion gene ontology (GO) ngokwemigomo ye-transcriptome kanye ne-costamere GO ku-proteome. Imicibisholo imelela abashayeli be-transcript kanye namaprotheni kanye neziqondiso zabo. E, F. Ukusondela kanye nokubikezela kwe-Uniform manifold (UMAP) kufaka iziqephu zezici ezihambisana nomtholampilo ezibonisa ama-gradients okubonakaliswa azimele ohlotsheni lwefayibha ehamba kancane/esheshayo. G, H. Ubudlelwano phakathi kwabashayeli be-PC2 kanye ne-PC1 ku-transcriptomes kanye ne-proteomes.
Ngokungalindelekile, uhlobo lwe-myofiber olusekelwe ku-MYH luchaze kuphela izinga lesibili eliphakeme kakhulu lokuguquguquka (i-PC2), okuphakamisa ukuthi ezinye izici zebhayoloji ezingahlobene nohlobo lwe-myofiber olusekelwe ku-MYH (i-PC1) zidlala indima ebalulekile ekulawuleni ukungafani kwe-fiber yemisipha yamathambo. Ukuhlaziywa kokumelwa ngokweqile kwabashayeli abaphezulu ku-PC1 kwembule ukuthi ukuguquguquka ku-PC1 kwakunqunywa kakhulu ukunamathela kweseli-seli kanye nokuqukethwe kwe-ribosome ku-transcriptome, kanye nama-costameres nama-ribosomal protein ku-proteome (Isithombe 2B kanye neZithombe Ezingeziwe 6D–E, Isethi Yedatha Engeziwe 7). Emisipheni yamathambo, ama-costameres axhuma i-Z-disc ku-sarcolemma futhi ahilelekile ekudlulisweni kwamandla kanye nokubonisana. 25 Ama-PCA plots anezincazelo asebenzisa ukunamathela kweseli-seli (i-transcriptome, Isibalo 2C) kanye nezici ze-costamere (i-proteome, Isibalo 2D) zembule ukushintsha kwesobunxele okunamandla ku-PC1, okubonisa ukuthi lezi zici zicebile kuma-fiber athile.
Ukuhlolwa okuningiliziwe kwe-myofiber clustering ezingeni le-UMAP kwembule ukuthi izici eziningi zibonise i-gradient yokubonakaliswa kwe-MYH esekwe kuhlobo lwe-myofiber esikhundleni se-myofiber subcluster-specific. Lokhu kuqhubeka kwabonwa kwezakhi zofuzo eziningana ezihlotshaniswa nezimo ze-pathological (Isithombe 2E), njenge-CHCHD10 (isifo se-neuromuscular), i-SLIT3 (i-muscle atrophy), i-CTDNEP1 (isifo se-muscle). Lokhu kuqhubeka kwabonwa nakuyo yonke i-proteome, kufaka phakathi amaprotheni ahlotshaniswa nezinkinga ze-neurological (UGDH), i-insulin signaling (PHIP), kanye ne-transcription (HIST1H2AB) (Isithombe 2F). Ngokubambisana, le datha ikhombisa ukuqhubeka kokungafani kwe-fiber okuzimele/okusheshayo kohlobo lwe-fiber kuzo zonke izinhlobo ze-myofiber.
Ngokuthakazelisayo, izakhi zofuzo zomshayeli ku-PC2 zibonise ukuhlangana okuhle kwe-transcriptome-proteome (r = 0.663) (Isithombe 2G), okuphakamisa ukuthi izinhlobo zefayibha ezihamba kancane futhi ezisheshayo, futhi ikakhulukazi izakhiwo zokubopha kanye nezokuguquguquka kwemisipha yamathambo, zilawulwa ngokwemibhalo. Kodwa-ke, izakhi zofuzo zomshayeli ku-PC1 azibonisanga ukuhlangana kwe-transcriptome-proteome (r = -0.027) (Isithombe 2H), okuphakamisa ukuthi ukuguquguquka okungahlobene nezinhlobo zefayibha ezihamba kancane/ezisheshayo kulawulwa kakhulu ngemva kokulotshwa. Ngoba ukuguquguquka ku-PC1 kwachazwa ngokuyinhloko ngamagama e-ribosomal gene ontology, futhi njengoba ama-ribosome edlala indima ebalulekile nekhethekile esitokisini ngokuhlanganyela ngenkuthalo ekuhumusheni amaprotheni nasekuthonyeni,31 ngokulandelayo saqala ukuphenya lokhu kungafani okungalindelekile kwe-ribosomal.
Siqale safaka umbala ku-proteomics principal component analysis plot ngokuya ngobuningi bamaprotheni egameni le-GOCC elithi “cytoplasmic ribosome” (Isithombe 3A). Nakuba leli gama licebile ohlangothini oluhle lwe-PC1, okuholela ekuqhekekeni okuncane, amaprotheni e-ribosomal aqhuba ukuhlukaniswa kuzo zombili izinhlangothi ze-PC1 (Isithombe 3A). Amaprotheni e-ribosomal acebile ohlangothini olubi lwe-PC1 afaka i-RPL18, i-RPS18, kanye ne-RPS13 (Isithombe 3B), kuyilapho i-RPL31, i-RPL35, kanye ne-RPL38 (Isithombe 3C) kwakuyizona ezibangela phambili ohlangothini oluhle lwe-PC1. Ngokuthakazelisayo, i-RPL38 kanye ne-RPS13 zazibonakala kakhulu emisipheni yamathambo uma kuqhathaniswa nezinye izicubu (Isithombe Esingeziwe 7A). Lezi zimpawu ezihlukile ze-ribosomal ku-PC1 azibonwanga ku-transcriptome (Isithombe Esingeziwe 7B), okubonisa umthethonqubo wangemuva kokubhalwa.
A. Isakhiwo sokuhlaziywa kwengxenye eyinhloko (i-PCA) esifakwe umbala ngokwemigomo ye-cytoplasmic ribosomal gene ontology (GO) kulo lonke i-proteome. Imicibisholo ikhombisa indlela yokwehluka okubangelwa yiprotheni kusakhiwo se-PCA. Ubude bomugqa buhambisana nesilinganiso sengxenye eyinhloko yeprotheni ethile. B, C. Isakhiwo se-PCA se-RPS13 kanye ne-RPL38. D. Ukuhlaziywa kwe-hierarchical clustering okungaqondiswanga kwamaprotheni e-cytoplasmic ribosomal. E. Imodeli yesakhiwo se-80S ribosome (PDB: 4V6X) eqokomisa amaprotheni e-ribosomal anobuningi obuhlukene kuma-fiber emisipha yamathambo. F. Amaprotheni e-ribosomal ane-stoichiometry ehlukene atholakala eduze kwesiteshi sokuphuma se-mRNA.
Imibono yokungafani kwe-ribosomal kanye nokukhethekile iye yaphakanyiswa ngaphambilini, lapho ukuba khona kwezinhlobo ezincane ze-ribosomal (ukungafani kwe-ribosomal) kungathonya ngqo ukuhumusha amaprotheni ezicutshini ezahlukene32 kanye namaseli33 ngokuhumusha okukhethiwe kwe-mRNA transcript pools34 ethile (ukukhethekile kwe-ribosome). Ukuze sithole izinhlobo ezincane zamaprotheni e-ribosomal aboniswa ndawonye kuma-fiber emisipha yamathambo, senze ukuhlaziywa kwe-hierarchical clustering okungaqondiswanga kwamaprotheni e-ribosomal ku-proteome (Isithombe 3D, Isethi Yedatha Engeziwe 8). Njengoba bekulindelekile, amaprotheni e-ribosomal awazange ahlangane ngohlobo lwe-fiber ngokusekelwe ku-MYH. Kodwa-ke, sithole amaqoqo amathathu ahlukene amaprotheni e-ribosomal; iqembu lokuqala (i-ribosomal_cluster_1) lihlanganiswe ne-RPL38 ngakho-ke linokuvezwa okwandisiwe kuma-fiber anephrofayili ye-PC1 enhle. Iqembu lesibili (i-ribosomal_cluster_2) lihlanganiswe ne-RPS13 futhi liphakanyisiwe kuma-fiber anephrofayili ye-PC1 engemihle. Iqembu lesithathu (i-ribosomal_cluster_3) alibonisi ukubonakaliswa okuhlukile okuhlanganisiwe kuma-fiber emisipha yamathambo futhi lingabhekwa njengeprotheyini ye-ribosomal yemisipha yamathambo "eyinhloko". Womabili amaqoqo e-ribosomal 1 no-2 aqukethe amaprotheni e-ribosomal aboniswe ngaphambilini ukuthi alawula ukuhumusha okunye (isb., RPL10A, RPL38, RPS19, kanye ne-RPS25) futhi athonya intuthuko ngokusebenzayo (isb., RPL10A, RPL38).34,35,36,37,38 Ngokuhambisana nemiphumela ye-PCA, ukumelwa okungafani okubonwe kwalawa maprotheni e-ribosomal kuwo wonke ama-fiber nakho kubonise ukuqhubeka (Isithombe Esingeziwe 7C).
Ukuze sibone indawo yamaprotheni e-ribosomal angafani ngaphakathi kwe-ribosome, sisebenzise imodeli yesakhiwo se-ribosome yomuntu engu-80S (i-Protein Data Bank: 4V6X) (Isithombe 3E). Ngemva kokuhlukanisa amaprotheni e-ribosomal angamaqoqo e-ribosomal ahlukene, izindawo zawo azizange zihambisane eduze, okuphakamisa ukuthi indlela yethu yehlulekile ukunikeza ukucebisa izifunda/izingxenyana ezithile ze-ribosome. Ngokuthakazelisayo, nokho, isilinganiso samaprotheni amakhulu e-subunit eqenjini lesi-2 sasiphansi kunasezingxeni 1 no-3 (Isithombe Esingeziwe 7D). Sibone ukuthi amaprotheni ane-stoichiometry eshintshiwe kuma-fiber emisipha yamathambo ayetholakala kakhulu ebusweni be-ribosome (Isithombe 3E), ehambisana nekhono lawo lokuxhumana nezakhi zangaphakathi ze-ribosome entry site (IRES) ezixukwini ezahlukene ze-mRNA, ngaleyo ndlela ehlanganisa ukuhumusha okukhethiwe. 40, 41 Ngaphezu kwalokho, amaprotheni amaningi ane-stoichiometry eshintshiwe kuma-fiber emisipha yamathambo ayetholakala eduze kwezindawo ezisebenzayo njenge-mRNA exit tunnel (Isithombe 3F), elawula ngokukhetha ukunwebeka kokuhumusha kanye nokuboshwa kwama-peptide athile. 42 Ngamafuphi, idatha yethu iphakamisa ukuthi i-stoichiometry yamaprotheni e-ribosomal muscle abonisa ukungafani, okuholela ekwehlukeni phakathi kwemicu yemisipha yamathambo.
Ngokulandelayo saqala ukuhlonza izisayini zefayibha ezisheshayo nezihamba kancane futhi sihlole izindlela zokulawulwa kwazo kokubhalwa kwemibhalo. Uma siqhathanisa amaqoqo efayibha asheshayo nezihamba kancane achazwe yi-UMAP kumasethi amabili edatha (Izithombe 1G–H kanye no-4A–B), ukuhlaziywa kwe-transcriptomic kanye ne-proteomic kuthole izici eziyi-1366 kanye no-804 ezigcwele ngokuhlukile, ngokulandelana (Izithombe 4A–B, Amasethi Edatha Angeziwe 9–12). Sibone umehluko olindelekile kumasayini ahlobene nama-sarcomere (isb., i-tropomyosin kanye ne-troponin), i-excitation-contraction coupling (i-SERCA isoforms), kanye ne-energy metabolism (isb., i-ALDOA kanye ne-CKB). Ngaphezu kwalokho, ama-transcript kanye namaprotheni alawula i-protein ubiquitination abonakaliswe ngokuhlukile kuma-fiber asheshayo nezihamba kancane (isb., i-USP54, i-SH3RF2, i-USP28, kanye ne-USP48) (Izithombe 4A–B). Ngaphezu kwalokho, i-gene ye-microbial protein RP11-451G4.2 (DWORF), eye yabonakala ngaphambilini ukuthi ivezwa ngokuhlukile kuzo zonke izinhlobo ze-fiber yemisipha yemvu43 futhi ithuthukisa umsebenzi we-SERCA emisipheni yenhliziyo44, yakhushulwa kakhulu kuma-fiber emisipha ehamba kancane (Isithombe 4A). Ngokufanayo, ezingeni le-fiber ngayinye, umehluko omkhulu wabonwa kuma-signatures aziwayo njenge-lactate dehydrogenase isoforms ehlobene ne-metabolism (i-LDHA ne-LDHB, Isithombe 4C kanye ne-Supplementary Figure 8A)45,46 kanye nama-signatures angaziwa ngaphambili e-fiber-type-specific (njenge-IRX3, i-USP54, i-USP28, kanye ne-DPYSL3) (Isithombe 4C). Kwakukhona ukunqwabelana okuphawulekayo kwezici ezivezwe ngokuhlukile phakathi kwamasethi edatha e-transcriptomic kanye ne-proteomic (Isithombe Esingeziwe 8B), kanye nokuxhumana kokushintsha kwe-fold okuqhutshwa ikakhulukazi ukubonakaliswa okuhlukile okuvelele kwezici ze-sarcomere (Isithombe Esingeziwe 8C). Okuphawulekayo ukuthi ezinye izimpawu (isb. USP28, USP48, GOLGA4, AKAP13) zibonise ukulawulwa okuqinile kwangemva kokubhalwa kuphela ezingeni le-proteomic futhi zazinamaphrofayili okubonakaliswa kohlobo lwe-fiber oluhamba kancane/olusheshayo (Isithombe Esingeziwe 8C).
Iziqephu zentaba-mlilo i-A ne-B eziqhathanisa amaqoqo ahamba kancane nasheshayo ahlonzwe yiziqephu ze-uniform manifold approximation and projection (UMAP) ku-Figures 1G–H. Amachashazi anombala amelela ama-transcript noma amaprotheni ahluke kakhulu ku-FDR < 0.05, kanti amachashazi amnyama amelela ama-transcript noma amaprotheni ahluke kakhulu ekushintsheni kwelogi > 1. Ukuhlaziywa kwezibalo okunezindlela ezimbili kwenziwe kusetshenziswa ukuhlolwa kwe-DESeq2 Wald ngamanani e-p alungisiwe e-Benjamini-Hochberg (ama-transcriptomics) noma indlela yemodeli ye-Limma linear enokuhlaziywa kwe-empirical Bayesian okulandelwa ukulungiswa kwe-Benjamini-Hochberg kokuqhathanisa okuningi (ama-proteomics). C Iziqephu zesiginesha zezakhi zofuzo noma amaprotheni akhethiwe achazwe ngokuhlukile phakathi kwemicu ehamba kancane nesheshayo. D Ukuhlaziywa kokunotha kwama-transcript nama-protein achazwe ngokuhlukile kakhulu. Amanani ahambisanayo anothekile kuzo zombili izisetho zedatha, amanani e-transcriptome anothekile kuphela ku-transcriptome, kanti amanani e-proteome anothekile kuphela ku-proteome. Ukuhlaziywa kwezibalo kwenziwa kusetshenziswa iphakheji ye-clusterProfiler enamanani e-p alungisiwe e-Benjamini-Hochberg. E. Izici zokuloba eziqondene nohlobo lwefayibha ezitholwe yi-SCENIC ngokusekelwe kumaphuzu okucaciswa komlawuli osuselwa ku-SCENIC kanye nokubonakaliswa kwe-mRNA okuhlukile phakathi kwezinhlobo zefayibha. F. Ukuhlelwa kwephrofayili yezinto zokuloba ezikhethiwe ezivezwe ngokuhlukile phakathi kwezintambo ezihamba kancane nezisheshayo.
Sabe sesenza ukuhlaziywa kokumelwa ngokweqile kwezakhi zofuzo namaprotheni amelwe ngokuhlukile (Isithombe 4D, Isethi Yedatha Engeziwe 13). Ukucebisa indlela yezici ezahluka phakathi kwamasethi amabili edatha kwembule umehluko olindelekile, njengezinqubo ze-fatty acid β-oxidation kanye ne-ketone metabolism (imicu ehamba kancane), ukufinyela kwe-myofilament/muscle (imicu ehamba ngokushesha nehamba kancane, ngokulandelana), kanye nezinqubo ze-carbohydrate catabolic (imicu ehamba ngokushesha). Umsebenzi we-serine/threonine protein phosphatase nawo waphakanyiswa kuma-fast fibers, oqhutshwa izici ezifana nama-regulatory and catalytic phosphatase subunits (PPP3CB, PPP1R3D, kanye ne-PPP1R3A), aziwa ngokulawula i-glycogen metabolism (47) (Izibalo Ezingeziwe 8D–E). Ezinye izindlela ezicebile ngemicu esheshayo zazihlanganisa imizimba yokucubungula (P-) (YTHDF3, TRIM21, LSM2) ku-proteome (Isifanekiso Esingeziwe 8F), okungenzeka ukuthi ihilelekile ekulawulweni kwe-post-transcriptional (48), kanye nomsebenzi we-transcription factor (SREBF1, RXRG, RORA) ku-transcriptome (Isifanekiso Esingeziwe 8G). Imicu ethambile yacetshiswa ngomsebenzi we-oxidoreductase (BDH1, DCXR, TXN2) (Isifanekiso Esingeziwe 8H), ukubopha i-amide (CPTP, PFDN2, CRYAB) (Isifanekiso Esingeziwe 8I), i-extracellular matrix (CTSD, ADAMTSL4, LAMC1) (Isifanekiso Esingeziwe 8J), kanye nomsebenzi we-receptor-ligand (FNDC5, SPX, NENF) (Isifanekiso Esingeziwe 8K).
Ukuze sithole ukuqonda okwengeziwe ngokulawulwa kokubhalwa phansi kwemisipha okusekelwe ezicini zohlobo lwe-fiber yemisipha ehamba kancane/esheshayo, senze ukuhlaziywa kokunongwa kwezinto zokubhalwa phansi sisebenzisa i-SCENIC49 (Isethi Yedatha Engeziwe 14). Izici eziningi zokubhalwa phansi zacetshiswa kakhulu phakathi kwezintambo zemisipha ezisheshayo nezihamba kancane (Isithombe 4E). Lokhu kwakuhlanganisa izici zokubhalwa phansi njenge-MAFA, ebilokhu ixhunyaniswe nokuthuthukiswa kwe-fiber yemisipha okusheshayo,50 kanye nezici eziningana zokubhalwa phansi ezazingahlotshaniswa ngaphambili nezinhlelo zezakhi zofuzo ezithile zohlobo lwe-fiber yemisipha. Phakathi kwalezi, i-PITX1, i-EGR1, kanye ne-MYF6 kwakuyizici zokubhalwa phansi ezicetshiswe kakhulu kwizintambo zemisipha ezisheshayo (Isithombe 4E). Ngokuphambene nalokho, i-ZSCAN30 kanye ne-EPAS1 (eyaziwa nangokuthi i-HIF2A) kwakuyizici zokubhalwa phansi ezicetshiswe kakhulu kwizintambo zemisipha ehamba kancane (Isithombe 4E). Ngokuvumelana nalokhu, i-MAFA ivezwe emazingeni aphezulu esifundeni se-UMAP esihambisana nezintambo zemisipha ezisheshayo, kuyilapho i-EPAS1 yayinephethini yokuveza ephambene (Isithombe 4F).
Ngaphezu kwezakhi zofuzo ezaziwayo zokubhala amaprotheni, kunezinhlobo eziningi ze-RNA ezingabhalisi ezingase zihileleke ekulawulweni kwentuthuko yabantu kanye nezifo. 51, 52 Kumasethi edatha e-transcriptome, ama-RNA amaningana angabhalisi abonisa ukucaciswa kohlobo lwefayibha (Isithombe 5A kanye neSethi Yedatha Engeziwe 15), kufaka phakathi i-LINC01405, ecacile kakhulu kumafayibha ahamba kancane futhi kubikwa ukuthi yehla emisipheni evela ezigulini ezine-mitochondrial myopathy. 53 Ngokuphambene nalokho, i-RP11-255P5.3, ehambisana nesakhi sofuzo se-lnc-ERCC5-5 (https://lncipedia.org/db/transcript/lnc-ERCC5-5:2) 54, ibonisa ukucaciswa kohlobo lwefayibha okusheshayo. Zombili i-LINC01405 (https://tinyurl.com/x5k9wj3h) kanye ne-RP11-255P5.3 (https://tinyurl.com/29jmzder) zibonisa ukucaciswa kwemisipha yamathambo (Izibalo Ezingeziwe 9A–B) futhi azinazo izakhi zofuzo ezikwaziyo ngaphakathi kwendawo yazo ye-genomic engu-1 Mb, okuphakamisa ukuthi zidlala indima ekhethekile ekulawuleni izinhlobo zefayibha kunokulawula izakhi zofuzo eziseduze ezikwaziyo ukuhambisana. Amaphrofayili okubonakaliswa kohlobo lwefayibha oluhamba kancane/olusheshayo lwe-LINC01405 kanye ne-RP11-255P5.3, ngokulandelana, aqinisekiswe kusetshenziswa i-RNAscope (Izibalo 5B–C).
A. Ama-transcript e-RNA angabhalisi alawulwa kakhulu kuma-fiber emisipha ahamba kancane futhi ashesha. B. Izithombe ze-RNAscope ezimele ezibonisa ukucaciswa kohlobo lwe-fiber oluhamba kancane futhi olushesha lwe-LINC01405 kanye ne-RP11-255P5.3, ngokulandelana. Ibha yesikali = 50 μm. C. Ukulinganiswa kokubonakaliswa kwe-RNA engabhalisi uhlobo oluthile lwe-myofiber njengoba kunqunywe yi-RNAscope (n = ama-biopsies angu-3 avela kubantu abazimele, kuqhathaniswa ama-fiber emisipha asheshayo nahamba kancane ngaphakathi komuntu ngamunye). Ukuhlaziywa kwezibalo kwenziwe kusetshenziswa ukuhlolwa kwe-t komfundi okunemisila emibili. Ama-box plots akhombisa ama-quartile aphakathi nendawo kanye neyokuqala neyesithathu, kanye nama-whiskers akhomba amanani amancane naphezulu. D. De novo microbial protein identification workflow (edalwe nge-BioRender.com). E. Iphrotheni yamagciwane i-LINC01405_ORF408:17441:17358 ivezwa ngqo kuma-fiber emisipha ehamba kancane (n=5 biopsies evela kubahlanganyeli abazimele, kuqhathaniswa ama-fiber emisipha esheshayo nehamba kancane kubahlanganyeli ngamunye). Ukuhlaziywa kwezibalo kwenziwe kusetshenziswa indlela yemodeli ye-Limm eqondile ehlanganiswe nendlela ye-Bayesian engokoqobo, kulandelwa indlela ye-Benjamini-Hochberg yokuqhathanisa okuningi nokulungiswa kwe-p-value. Ama-box plots abonisa ama-quartiles aphakathi nendawo, okuqala kanye nawesithathu, anezindevu ezikhomba amanani aphezulu/aphansi.
Muva nje, izifundo zibonise ukuthi imibhalo eminingi engabonakali engabhalwanga ifaka ikhodi kumaprotheni abhalwe phansi abhalwe phansi, amanye awo alawula ukusebenza kwemisipha. 44, 55 Ukuze sithole amaprotheni amagciwane anohlobo oluthile lwefayibha, siseshe isethi yethu yedatha ye-1000 fiber proteome sisebenzisa ifayela le-FASTA elenziwe ngokwezifiso eliqukethe ukulandelana kwemibhalo engelona ikhodi (n = 305) etholakala kusethi yedatha ye-1000 fiber transcriptome (Isithombe 5D). Sithole amaprotheni amagciwane angu-197 avela kumibhalo ehlukene engu-22, engu-71 yayo eyayilawulwa ngokuhlukile phakathi kwemicu yemisipha yamathambo ehamba kancane nesheshayo (Isithombe Esingeziwe 9C kanye neSethi Yedatha Engeziwe 16). Ku-LINC01405, kutholakale imikhiqizo emithathu yamaprotheni amagciwane, enye yayo ibonise ukucaciswa kwefayibha okuhamba kancane okufanayo nombhalo wayo (Isithombe 5E kanye neSethi Engeziwe 9D). Ngakho-ke, sithole i-LINC01405 njenge-gene efaka ikhodi kuphrotheni yamagciwane ethile yemicu yemisipha yamathambo ehamba kancane.
Sakha uhlelo lokusebenza oluphelele lokuchaza i-proteomic enkulu yemicu yemisipha yomuntu ngamunye kanye nabalawuli ababona ukungafani kwe-fiber ezimweni ezinempilo. Sisebenzise lolu hlelo lokusebenza ukuqonda ukuthi i-nemaline myopathies ithinta kanjani ukungafani kwe-fiber yemisipha yamathambo. I-Nemaline myopathies yizifo zemisipha ezizuzwe njengefa ezibangela ubuthakathaka bemisipha futhi, ezinganeni ezithintekile, zinezinkinga eziningi okuhlanganisa ukucindezeleka kokuphefumula, i-scoliosis, kanye nokuhamba okulinganiselwe kwezitho. 19,20 Ngokuvamile, kuma-nemaline myopathies, izinhlobo ezibangela izifo ezizakhi zofuzo ezifana ne-actin alpha 1 (ACTA1) ziholela ekubunjweni kwe-fiber myofiber ehamba kancane, yize lo mphumela ungafani. Okuhlukile okuphawulekayo yi-troponin T1 nemaline myopathy (TNNT1), enokubunjwa kwemicu esheshayo. Ngakho-ke, ukuqonda kangcono ukungafani okuyisisekelo sokungasebenzi kahle kwe-fiber yemisipha yamathambo okubonwa kuma-nemaline myopathies kungasiza ekuhlukaniseni ubudlelwano obuyinkimbinkimbi phakathi kwalezi zifo nohlobo lwe-myofiber.
Uma kuqhathaniswa nezilawuli ezinempilo (n=3 ngeqembu), ama-myofiber ahlukaniswe ezigulini ze-nemaline myopathy ezinezinguquko kuma-genes e-ACTA1 kanye ne-TNNT1 abonise i-myofiber atrophy noma i-dystrophy ephawulekayo (Isithombe 6A, Ithebula Elingeziwe 3). Lokhu kuveze izinselele ezinkulu zobuchwepheshe zokuhlaziywa kwe-proteomic ngenxa yenani elilinganiselwe lezinto ezitholakalayo. Naphezu kwalokhu, sikwazile ukuthola amaprotheni angu-2485 kuma-myofiber angu-272 amathambo. Ngemva kokuhlunga okungenani amaprotheni ayi-1000 alinganisiwe nge-fiber ngayinye, ama-fiber angu-250 ahlolwe nge-bioinformatics kamuva. Ngemva kokuhlunga, isilinganiso samaprotheni angu-1573 ± 359 nge-fiber ngayinye salinganiswa (Isithombe Esingeziwe 10A, Amasethi Edatha Engeziwe 17–18). Okuphawulekayo ukuthi, naphezu kokwehla okukhulu kosayizi we-fiber, ukujula kwe-proteome kwamasampula eziguli ze-nemaline myopathy kwancishiswa kancane. Ngaphezu kwalokho, ukucubungula le datha sisebenzisa amafayela ethu e-FASTA (kufaka phakathi imibhalo engeyona eyokubhala ikhodi) kwasivumela ukuthi sithole amaprotheni amahlanu amagciwane kuma-myofibers amathambo avela ezigulini ze-nemaline myopathy (Isethi Yedatha Engeziwe 19). Ububanzi obuguqukayo be-proteome babubanzi kakhulu, futhi amaprotheni aphelele eqenjini lokulawula ahlobene kahle nemiphumela yokuhlaziywa kwangaphambilini kwe-1000-fiber proteome (Isithombe Esingeziwe 10B–C).
A. Izithombe ezincane ezibonisa i-fiber atrophy noma i-dystrophy kanye nokugqama kwezinhlobo ezahlukene ze-fiber ngokusekelwe ku-MYH ku-ACTA1 kanye ne-TNNT1 ne-nemaline myopathies (NM). Ibha yesikali = 100 μm. Ukuqinisekisa ukuphindaphindwa kokudaya ezigulini ze-ACTA1 kanye ne-TNNT1, ama-biopsies eziguli amathathu afakwe amabala kabili kuya kathathu (izingxenye ezine ngecala ngalinye) ngaphambi kokukhetha izithombe ezimele. B. Ukulinganiswa kohlobo lwe-fibre kubahlanganyeli ngokusekelwe ku-MYH. C. Isakhiwo sokuhlaziywa kwengxenye eyinhloko (i-PCA) sezintambo zemisipha yamathambo ezigulini ezine-nemaline myopathies kanye nezilawuli. D. Izintambo zemisipha yamathambo ezivela ezigulini ezine-nemaline myopathies kanye nezilawuli eziboniswe kusakhiwo se-PCA esinqunywe kuzintambo eziyi-1000 ezihlaziywe kuMfanekiso 2. Isibonelo, izigcawu zentaba-mlilo eziqhathanisa umehluko phakathi kwabahlanganyeli abane-ACTA1 kanye ne-TNNT1 ne-nemaline myopathies kanye nezilawuli, kanye naphakathi kwabahlanganyeli abane-ACTA1 kanye ne-TNNT1 ne-nemaline myopathies. Izindilinga ezinemibala zikhomba amaprotheni ayehluke kakhulu ku-π < 0.05, kanti amachashazi amnyama akhomba amaprotheni ayehluke kakhulu ku-FDR < 0.05. Ukuhlaziywa kwezibalo kwenziwe kusetshenziswa indlela yemodeli eqondile ye-Limma kanye nezindlela ze-empirical Bayesian, kulandelwe ukulungiswa kwenani le-p ukuze kuqhathaniswe okuningi kusetshenziswa indlela ye-Benjamini-Hochberg. H. Ukuhlaziywa kokucebisa amaprotheni achazwe ngokuhlukile kakhulu kulo lonke i-proteome kanye naku-type 1 kanye ne-2A fibers. Ukuhlaziywa kwezibalo kwenziwe kusetshenziswa iphakheji ye-clusterProfiler kanye namanani e-p alungisiwe e-Benjamini-Hochberg. I, J. Iziza zokuhlaziywa kwengxenye eyinhloko (PCA) ezifakwe umbala yi-extracellular matrix kanye ne-mitochondrial gene ontology (GO) terms.
Ngenxa yokuthi ama-nemaline myopathies angathonya isilinganiso sezinhlobo ze-MYH-expressing myofiber emisipheni yamathambo, 19,20 siqale sahlola izinhlobo ze-MYH-expressing myofiber ezigulini ezine-nemaline myopathies kanye nezilawuli. Sithole uhlobo lwe-myofiber sisebenzisa indlela engachemile echazwe ngaphambilini ku-1000 myofiber assay (Izithombe Ezingeziwe. 10D–E) futhi saphinde sehluleka ukuthola ama-2X myofibers amsulwa (Isithombe 6B). Sibone umphumela ongafani we-nemaline myopathies ohlotsheni lwe-myofiber, njengoba iziguli ezimbili ezine-ACTA1 mutations zazinesilinganiso esandisiwe sama-myofibers ohlobo 1, kanti iziguli ezimbili ezine-TNNT1 nemaline myopathy zazinesilinganiso esinciphile sama-myofibers ohlobo 1 (Isithombe 6B). Ngempela, ukuvezwa kwe-MYH2 kanye ne-fast troponin isoforms (TNNC2, TNNI2, kanye ne-TNNT3) kwehle kuma-myopathies e-ACTA1-nemaline, kanti ukuvezwa kwe-MYH7 kwehle kuma-myopathies e-TNNT1-nemaline (Isithombe Esingeziwe 11A). Lokhu kuhambisana nemibiko yangaphambilini yokushintsha kohlobo lwe-myofiber olungafani kuma-myopathies e-nemaline.19,20 Siqinisekisile le miphumela nge-immunohistochemistry futhi sathola ukuthi iziguli ezine-myopathies ye-ACTA1-nemaline zazine-myofibers yohlobo 1, kanti iziguli ezine-myopathies ye-TNNT1-nemaline zazinephethini eliphambene (Isithombe 6A).
Ezingeni le-single-fiber proteome, imicu yemisipha yamathambo evela ezigulini ze-ACTA1 kanye ne-TNNT1 nemaline myopathy ihlanganiswe neningi lemicu yokulawula, kanti imicu ye-TNNT1 nemaline myopathy ngokuvamile iyona ethinteke kakhulu (Isithombe 6C). Lokhu kwabonakala kakhulu lapho kuhlelwa izigcawu zokuhlaziywa kwengxenye enkulu (i-PCA) zemicu efakwe umoya wesiguli ngasinye, kanti iziguli ze-TNNT1 nemaline myopathy 2 kanye no-3 zibonakala zikude kakhulu namasampula okulawula (Isithombe Esingeziwe 11B, Isethi Yedatha Engeziwe 20). Ukuze siqonde kangcono ukuthi imicu evela ezigulini ze-myopathy iqhathaniswa kanjani nemicu enempilo, sisebenzise ulwazi oluningiliziwe olutholwe ekuhlaziyweni kwe-proteomic kwemicu eyi-1,000 evela kubantu abadala abanempilo. Sibikezele imicu evela kusethi yedatha ye-myopathy (iziguli ze-ACTA1 kanye ne-TNNT1 nemaline myopathy kanye nezilawuli) ku-PCA plot etholwe ekuhlaziyweni kwe-proteomic ye-1000-fiber (Isithombe 6D). Ukusatshalaliswa kwezinhlobo ze-fiber ze-MYH ku-PC2 kuma-fiber okulawula kwakufana nokusatshalaliswa kwe-fiber okutholwe ekuhlaziyweni kwe-proteomic ye-fiber eyi-1000. Kodwa-ke, iningi lama-fiber ezigulini ze-nemaline myopathy lashintsha phansi i-PC2, lihambisana nama-fiber anempilo asheshayo, kungakhathaliseki ukuthi hlobo luni lwe-fiber yazo ye-MYH. Ngakho-ke, yize iziguli ezine-ACTA1 nemaline myopathy zibonise ukushintshela kuma-fiber ohlobo 1 lapho zilinganiswa kusetshenziswa izindlela ezisekelwe ku-MYH, zombili i-ACTA1 nemaline myopathy kanye ne-TNNT1 nemaline myopathy zashintsha i-skeletal muscle fiber proteome zaya kuma-fiber asheshayo.
Sabe sesiqhathanisa ngqo iqembu ngalinye leziguli nezilawuli ezinempilo futhi sathola amaprotheni angu-256 kanye no-552 achazwe ngokuhlukile ku-ACTA1 kanye ne-TNNT1 nemaline myopathies, ngokulandelana (Isithombe 6E–G kanye nesithombe esengeziwe 11C, Isethi Yedatha Engeziwe 21). Ukuhlaziywa kokucebisa izakhi zofuzo kwembule ukwehla okuhlanganisiwe kwamaprotheni e-mitochondrial (Isithombe 6H–I, Isethi Yedatha Engeziwe 22). Ngokumangazayo, naphezu kokugqama okuhlukile kwezinhlobo zefayibha ku-ACTA1 kanye ne-TNNT1 nemaline myopathies, lokhu kwehla bekuzimele ngokuphelele ohlotsheni lwefayibha olusekelwe ku-MYH (Isithombe 6H kanye nezithombe esengeziwe 11D–I, Isethi Yedatha Engeziwe 23). Amaprotheni amathathu amagciwane nawo alawulwa ku-ACTA1 noma ku-TNNT1 nemaline myopathies. Amabili alawa ma-microprotein, i-ENSG00000215483_TR14_ORF67 (eyaziwa nangokuthi i-LINC00598 noma i-Lnc-FOXO1) kanye ne-ENSG00000229425_TR25_ORF40 (lnc-NRIP1-2), abonise ubuningi obuhlukile kuphela kuma-myofiber ohlobo 1. I-ENSG00000215483_TR14_ORF67 ibikwe ngaphambilini ukuthi idlala indima ekulawulweni komjikelezo weseli. 56 Ngakolunye uhlangothi, i-ENSG00000232046_TR1_ORF437 (ehambisana ne-LINC01798) yanda kokubili kuma-myofiber ohlobo 1 kanye nohlobo 2A ku-ACTA1-nemaline myopathy uma kuqhathaniswa nezilawuli ezinempilo (Isithombe Esingeziwe 12A, Isethi Yedatha Engeziwe 24). Ngokuphambene nalokho, amaprotheni e-ribosomal awazange athintwe kakhulu yi-nemaline myopathy, yize i-RPS17 yehlisiwe ku-ACTA1 nemaline myopathy (Isithombe 6E).
Ukuhlaziywa kokucebisa kuphinde kwembule ukwanda kwezinqubo zesistimu yomzimba kuma-myopathies e-ACTA1 kanye ne-TNNT1 nemaline, kuyilapho ukunamathela kwamaseli kwanda naku-myopathies ye-nemaline ye-TNNT1 (Isithombe 6H). Ukuceba kwalezi zinto ezingaphandle kweseli kwabonakala ngamaprotheni e-matrix engaphandle kweseli ashintsha i-PCA ku-PC1 kanye ne-PC2 ngendlela engemihle (okungukuthi, aye kuma-fiber athinteke kakhulu) (Isithombe 6J). Womabili amaqembu eziguli abonise ukwanda kokubonakaliswa kwamaprotheni e-extracellular ahilelekile ekuphenduleni komzimba kanye nezindlela zokulungisa i-sarcolemmal, njenge-anxins (ANXA1, ANXA2, ANXA5)57,58 kanye neprotheni yabo esebenzisana ne-S100A1159 (Izithombe Ezingeziwe 12B–C). Le nqubo ibikwe ngaphambilini ukuthi ithuthukisiwe kuma-dystrophies emisipha60 kodwa, ngokwazi kwethu, ayizange ihlotshaniswe nama-myopathies e-nemaline. Umsebenzi ojwayelekile walo mshini wama-molecule uyadingeka ekulungisweni kwe-sarcolemmal ngemuva kokulimala kanye nokuhlanganiswa kwama-myocytes asanda kwakheka nama-myofibers58,61. Ngakho-ke, ukwanda komsebenzi wale nqubo kuwo womabili amaqembu eziguli kusikisela impendulo yokubuyisela ekulimaleni okubangelwa ukungazinzi kwe-myofiber.
Imiphumela ye-nemaline myopathy ngayinye yayihlobene kahle (r = 0.736) futhi yabonisa ukufana okunengqondo (Izibalo Ezingeziwe 11A–B), okubonisa ukuthi i-ACTA1 kanye ne-nemaline myopathy ye-TNNT1 zinemiphumela efanayo ku-proteome. Kodwa-ke, amanye amaprotheni alawulwa kuphela ku-ACTA1 noma i-TNNT1 nemaline myopathy (Izibalo Ezingeziwe 11A kanye no-C). Iphrotheni ye-profibrotic MFAP4 yayingenye yamaprotheni akhushulwe kakhulu ku-TNNT1 nemaline myopathy kodwa ayizange ishintshe ku-ACTA1 nemaline myopathy. I-SKIC8, ingxenye ye-PAF1C complex ephethe ukulawula ukuqoshwa kwezakhi zofuzo ze-HOX, yehlisiwe ku-TNNT1 nemaline myopathy kodwa ayizange ithintwe ku-ACTA1 nemaline myopathy (Isithombe Esingeziwe 11A). Ukuqhathaniswa okuqondile kwe-ACTA1 kanye ne-TNNT1 ne-nemaline myopathy kwembule ukwehla okukhulu kwamaprotheni e-mitochondrial kanye nokwanda kwamaprotheni esistimu yomzimba ku-TNNT1 nemaline myopathy (Isithombe 6G–H kanye neZithombe ezengeziwe 11C kanye no-11H–I). Le datha ihambisana ne-atrophy/dystrophy enkulu ebonwe ku-TNNT1 nemaline myopathy uma kuqhathaniswa ne-TNNT1 nemaline myopathy (Isithombe 6A), okuphakamisa ukuthi i-TNNT1 nemaline myopathy imelela uhlobo olubi kakhulu lwesifo.
Ukuze sihlole ukuthi imiphumela ebonwe ye-nemaline myopathy iyaqhubeka yini ezingeni lonke lemisipha, senze ukuhlaziywa kwe-bulk proteomic kwe-muscle biopsies evela eqenjini elifanayo leziguli ze-TNNT1 nemaline myopathy futhi saziqhathanisa nezilawuli (n=3 ngeqembu) (Isithombe Esingeziwe 13A, Isethi Yedatha Engeziwe 25). Njengoba bekulindelekile, izilawuli zazihlobene kakhulu ekuhlaziyweni kwezingxenye eziyinhloko, kanti iziguli ze-TNNT1 nemaline myopathy zibonise ukuhlukahluka okuphezulu phakathi kwesampula okufana nalokho okubonwe ekuhlaziyweni kwe-single fiber (Isithombe Esingeziwe 13B). Ukuhlaziywa kwe-bulk kuphinde kwaveza amaprotheni achazwe ngokuhlukile (Isithombe Esingeziwe 13C, Isethi Yedatha Engeziwe 26) kanye nezinqubo zebhayoloji (Isithombe Esingeziwe 13D, Isethi Yedatha Engeziwe 27) eqokonyiswe ngokuqhathanisa imicu ngayinye, kodwa yalahlekelwa ikhono lokuhlukanisa phakathi kwezinhlobo ezahlukene ze-fiber futhi yehluleka ukubheka imiphumela yesifo ehlukahlukene kuyo yonke imicu.
Uma kuhlanganiswa, le mininingwane ikhombisa ukuthi ama-proteomics e-single-myofiber angacacisa izici zebhayoloji zezokwelapha ezingabonakali ngezindlela eziqondiwe ezifana nokuvimbela i-immunoblotting. Ngaphezu kwalokho, le datha iqokomisa ukulinganiselwa kokusebenzisa i-actin fiber typing (MYH) kuphela ukuchaza ukuguquguquka kwe-phenotypic. Ngempela, yize ukushintsha kohlobo lwe-fiber kuhlukile phakathi kwe-actin kanye ne-troponin nemaline myopathies, womabili ama-myopathies e-nemaline ahlukanisa ukuthayipha kwe-MYH fiber kusuka ku-metabolism ye-skeletal muscle fiber kuya ku-proteome yemisipha esheshayo nengena-oxidative.
Ukwehlukahlukana kwamaseli kubalulekile ukuze izicubu zihlangabezane nezidingo zazo ezahlukahlukene. Emisipheni yamathambo, lokhu kuvame ukuchazwa njengezinhlobo zefayibha ezibonakala ngamazinga ahlukene okukhiqizwa kwamandla kanye nokukhathala. Kodwa-ke, kusobala ukuthi lokhu kuchaza ingxenye encane kuphela yokwehlukahlukana kwefayibha yemisipha yamathambo, okuguquguqukayo kakhulu, okuyinkimbinkimbi futhi okunezinhlangothi eziningi kunalokho obekucatshangwa ngaphambili. Intuthuko yezobuchwepheshe manje isikhanyise ngezici ezilawula ifayibha yemisipha yamathambo. Ngempela, idatha yethu isikisela ukuthi ifayibha yohlobo lwe-2X kungenzeka ingabi uhlobo oluhlukile lwefayibha yemisipha yamathambo. Ngaphezu kwalokho, sithole amaprotheni e-metabolic, amaprotheni e-ribosomal kanye namaprotheni ahlobene neseli njengezici eziyinhloko zokwehlukahlukana kwefayibha yemisipha yamathambo. Ngokusebenzisa ukuhamba kwethu kwe-proteomic kumasampula eziguli ezine-nematode myopathy, sibonise ukuthi ukuthayipha ifayibha esekelwe ku-MYH akubonisi ngokugcwele ukwehlukahlukana kwemisipha yamathambo, ikakhulukazi lapho uhlelo luphazamisekile. Ngempela, kungakhathaliseki ukuthi uhlobo lwefayibha olusekelwe ku-MYH, i-nematode myopathy iphumela ekushintsheni iye kufayibha yemisipha esheshayo nencane.
Imicu yemisipha yamathambo ibilokhu ihlukaniswa kusukela ngekhulu le-19. Ukuhlaziywa kwamuva kwe-omics kusivumele ukuthi siqale ukuqonda amaphrofayili okubonakaliswa kwezinhlobo ezahlukene ze-MYH fiber kanye nezimpendulo zazo ezinkingeni ezahlukene. Njengoba kuchaziwe lapha, izindlela ze-omics nazo zinenzuzo yokuzwela okukhulu kokulinganisa izimpawu zohlobo lwe-fiber kunezindlela zendabuko ezisekelwe kuma-antibody, ngaphandle kokuthembela ekulinganisweni kwezimpawu zodwa (noma ezimbalwa) ukuchaza uhlobo lwe-fiber yemisipha yamathambo. Sisebenzise imisebenzi yokusebenza ye-transcriptomic kanye ne-proteomic ehambisanayo futhi sahlanganisa imiphumela ukuze sihlole ukulawulwa kokubhalwa kanye nokwangemva kokubhalwa kokungafani kwe-fiber kuma-fiber emisipha yamathambo abantu. Lokhu kuhamba komsebenzi kuholele ekwehlulekeni ukuhlonza imicu ye-2X emsulwa ezingeni leprotheni ku-vastus lateralis yeqembu lethu lezinsizwa eziphilile. Lokhu kuhambisana nezifundo zangaphambilini ze-single fiber ezathola <1% imicu ye-2X emsulwa ku-vastus lateralis enempilo, yize lokhu kufanele kuqinisekiswe kweminye imisipha esikhathini esizayo. Ukungafani phakathi kokutholwa kwemicu ye-2X ecishe ibe msulwa ezingeni le-mRNA kanye nemicu ye-2A/2X exubile kuphela ezingeni leprotheni kuyadida. Ukuvezwa kwe-MYH isoform mRNA akuyona i-circadian, 67 okuphakamisa ukuthi akunakwenzeka ukuthi "siphuthelwe" isignali yokuqala ye-MYH2 kuma-fibers angu-2X abonakala emsulwa ezingeni le-RNA. Enye incazelo engaba khona, nakuba icatshangelwa nje, ingaba umehluko ekuzinzeni kweprotheni kanye/noma kwe-mRNA phakathi kwama-isoforms e-MYH. Ngempela, ayikho i-fiber esheshayo emsulwa ngo-100% kunoma iyiphi i-isoform ye-MYH, futhi akucaci ukuthi amazinga okuvezwa kwe-MYH1 mRNA ebangeni lama-70-90% angaholela enanini elilinganayo le-MYH1 kanye ne-MYH2 ezingeni leprotheni. Kodwa-ke, uma ucabangela yonke i-transcriptome noma i-proteome, ukuhlaziywa kwamaqoqo kungakhomba ngokuqiniseka amaqoqo amabili ahlukene kuphela amelela ama-fiber emisipha yamathambo ahamba kancane futhi asheshayo, kungakhathaliseki ukuthi akhiwe kanjani i-MYH enembile. Lokhu kuhambisana nokuhlaziywa okusebenzisa izindlela ze-transcriptomic ze-single-nucleus, ezivame ukukhomba amaqoqo amabili ahlukene e-myonuclear. 68, 69, 70 Ngaphezu kwalokho, yize izifundo zangaphambilini ze-proteomic zithole imicu yohlobo lwe-2X, le micu ayiqoqeki ngokwehlukana neminye imicu esheshayo futhi ikhombisa inani elincane kuphela lamaprotheni amaningi ngokuhlukile uma kuqhathaniswa nezinye izinhlobo zemicu ngokusekelwe ku-MYH. 14 Le miphumela isikisela ukuthi kufanele sibuyele embonweni wokuqala wekhulu lama-20 wokuhlukaniswa kwemicu yemisipha, okwahlukanisa imicu yemisipha yamathambo yomuntu hhayi ngezigaba ezintathu ezihlukene ngokusekelwe ku-MYH, kodwa ngamaqoqo amabili ngokusekelwe ezimpahleni zawo ze-metabolic kanye ne-contractile. 63
Okubaluleke kakhulu, ukungafani kwe-myofiber kufanele kucatshangelwe ngezindlela eziningi. Izifundo zangaphambilini ze-"omics" zikhombe kulokhu, ziphakamisa ukuthi imicu yemisipha yamathambo ayakhi amaqoqo ahlukene kodwa ihlelwe ngokuqhubekayo. 11, 13, 14, 64, 71 Lapha, sibonisa ukuthi, ngaphezu kokwehluka kwezakhiwo zokubopha kanye ne-metabolic zemisipha yamathambo, ama-myofiber angahlukaniswa ngezici ezihlobene nokusebenzisana kwamaseli namaseli kanye nezindlela zokuhumusha. Ngempela, sithole ukungafani kwe-ribosome kuma-fiber emisipha yamathambo okunikela ekungafani kuzimele kwezinhlobo ze-fiber ezihamba kancane nezisheshayo. Imbangela eyisisekelo yalokhu kungafani okukhulu kwe-myofiber, okungazimele kohlobo lwe-fiber oluhamba kancane nolusheshayo, ayikacaci, kodwa ingakhomba ekuhleleni okukhethekile kwendawo ngaphakathi kwama-fascicle emisipha aphendula kahle emandleni athile kanye nemithwalo,72 ukuxhumana okukhethekile kweseli noma kwezitho ezithile nezinye izinhlobo zamaseli endaweni encane yemisipha73,74,75 noma umehluko emsebenzini we-ribosome ngaphakathi kwama-myofiber ngamanye. Ngempela, i-ribosomal heteroplasmy, kungaba ngokushintshaniswa kwe-RPL3 ne-RPL3L noma ezingeni le-2′O-methylation ye-rRNA, kuye kwabonakala kuhlotshaniswa ne-skeletal muscle hypertrophy76,77. Ukusetshenziswa kwe-multi-omic kanye ne-spatial kuhlanganiswe nokucaciswa kokusebenza kwe-myofibers ngayinye kuzothuthukisa ukuqonda kwethu i-biology yemisipha ezingeni le-multi-omic78.
Ngokuhlaziya ama-proteome ama-single myofibers avela ezigulini ezine-nemaline myopathies, siphinde sabonisa ukusebenza, ukusebenza kahle, kanye nokusebenza kwama-single myofiber proteomics ukuze kucaciswe i-pathophysiology yemicu yamathambo. Ngaphezu kwalokho, ngokuqhathanisa ukuhamba kwethu komsebenzi nokuhlaziywa kwe-proteomic yomhlaba wonke, sikwazile ukukhombisa ukuthi ama-single myofiber proteomics akhiqiza ukujula okufanayo kolwazi njenge-global tissue proteomics futhi andisa lokhu kujula ngokubala ukungafani kwe-interfiber kanye nohlobo lwe-myofiber. Ngaphezu komehluko olindelekile (nakuba uguquguqukayo) ku-fiber type ratio obonwe ku-ACTA1 kanye ne-TNNT1 nemaline myopathies uma kuqhathaniswa nezilawuli ezinempilo,19 siphinde sabona ukuguqulwa kwe-oxidative kanye ne-extracellular ngaphandle kokushintsha kohlobo lwe-fiber oluqondiswa yi-MYH. I-Fibrosis ibike ngaphambilini ku-TNNT1 nemaline myopathies.19 Kodwa-ke, ukuhlaziywa kwethu kwakha kulokhu okutholakele ngokuveza amazinga akhuphukile amaprotheni ahlobene nokucindezeleka akhishwe ngaphandle kwe-extracellular, njenge-annexins, ehilelekile ezindleleni zokulungisa i-sarcolemmal, kuma-myofibers avela ezigulini ezine-ACTA1 kanye ne-TNNT1 nemaline myopathies.57,58,59 Ekuphetheni, amazinga akhuphukile e-annexin kuma-myofibers avela ezigulini ezine-nemaline myopathy angase amele impendulo yeselula ekulungiseni ama-myofibers atrophic kakhulu.
Nakuba lolu cwaningo lumelela ukuhlaziywa okukhulu kakhulu kwemisipha yonke ye-single-fiber kubantu kuze kube manje, alunamingcele. Sihlukanise imicu yemisipha yamathambo kusampula encane nefanayo yabahlanganyeli kanye nomsipha owodwa (i-vastus lateralis). Ngakho-ke, akunakwenzeka ukukhipha ukuba khona kwezinhlobo ezithile zemicu kuzo zonke izinhlobo zemisipha kanye nasezimweni eziphakeme kakhulu zomzimba wemisipha. Isibonelo, asikwazi ukukhipha amathuba okuba nengxenye encane yemicu esheshayo (isb. imicu emsulwa engu-2X) evela kubagijimi abaqeqeshwe kakhulu kanye/noma abanamandla79 noma ngezikhathi zokungasebenzi kwemisipha66,80. Ngaphezu kwalokho, usayizi wesampula olinganiselwe wabahlanganyeli usivimbele ekuphenyeni umehluko wobulili ekuhlukeni kwe-fiber, njengoba izilinganiso zohlobo lwe-fiber zaziwa ukuthi ziyahluka phakathi kwabesilisa nabesifazane. Ngaphezu kwalokho, asikwazanga ukwenza ukuhlaziywa kwe-transcriptomic kanye ne-proteomic kuma-fiber emisipha afanayo noma amasampula avela kubahlanganyeli abafanayo. Njengoba thina nabanye siqhubeka nokwenza ngcono ukuhlaziywa kweseli elilodwa kanye ne-single-myofiber sisebenzisa ukuhlaziywa kwe-omics ukuze kufezwe ukufakwa kwesampula okuphansi kakhulu (njengoba kuboniswe lapha ekuhlaziyweni kwezintambo ezivela ezigulini ezine-mitochondrial myopathy), ithuba lokuhlanganisa izindlela ze-multi-omics (nezisebenzayo) ngaphakathi kwezintambo zemisipha eyodwa liyabonakala.
Sekukonke, idatha yethu ihlonza futhi ichaze abashayeli bokukopisha kanye nabangemva kokubhalwa kombhalo bokungafani kwemisipha yamathambo. Ngokukhethekile, sethula idatha ephonsela inselelo imfundiso yesikhathi eside ku-physiology yemisipha yamathambo ehlotshaniswa nencazelo yakudala yezinhlobo zefayibha esekelwe ku-MYH. Sithemba ukuvuselela ingxoxo futhi ekugcineni sicabange kabusha ukuqonda kwethu ukuhlukaniswa kwefayibha yemisipha yamathambo kanye nokungafani.
Abahlanganyeli abayi-14 baseCaucasian (abesilisa abayi-12 nabesifazane ababili) bavuma ngokuzithandela ukubamba iqhaza kulolu cwaningo. Ucwaningo lwavunyelwa yiKomidi Lokuziphatha laseGhent University Hospital (BC-10237), lwahambisana neSimemezelo saseHelsinki sika-2013, futhi lwabhaliswa ku-ClinicalTrials.gov (NCT05131555). Izici ezijwayelekile zabahlanganyeli zethulwe kuThebula Elingeziwe 1. Ngemva kokuthola imvume enolwazi ngomlomo nangokubhaliwe, abahlanganyeli bahlolwe ngokwezokwelapha ngaphambi kokufakwa kokugcina ocwaningweni. Abahlanganyeli babesebancane (abaneminyaka engu-22-42), bephilile (bengenazo izimo zezokwelapha, bengenawo umlando wokubhema), futhi besebenza ngokomzimba ngokulinganisela. Ukuthathwa komoya-mpilo okuphezulu kwanqunywa kusetshenziswa i-ergometer yesinyathelo sokuhlola ukufaneleka komzimba njengoba kuchaziwe ngaphambilini. 81
Amasampula e-biopsy yemisipha aqoqwe ngesikhathi sokuphumula futhi esimweni sokuzila ukudla kathathu, izinsuku ezingu-14 zihlukene. Ngenxa yokuthi lawa masampula aqoqwe njengengxenye yocwaningo olukhulu, ababambiqhaza badle i-placebo (i-lactose), i-H1-receptor antagonist (540 mg fexofenadine), noma i-H2-receptor antagonist (40 mg famotidine) imizuzu engu-40 ngaphambi kwe-biopsy. Sibonise ngaphambilini ukuthi lawa ma-histamine receptor antagonists awathinti ukuqina kwemisipha yokuphumula81, futhi akukho ukuhlangana okuhlobene nesimo okubonwe ezigabeni zethu zokulawula ikhwalithi (Izibalo Ezingeziwe 3 no-6). Ukudla okujwayelekile (isisindo somzimba esingu-41.4 kcal/kg, ama-carbohydrate angu-5.1 g/kg, amaprotheni esisindo somzimba angu-1.4 g/kg, kanye namafutha esisindo somzimba angu-1.6 g/kg) kwagcinwa amahora angu-48 ngaphambi kosuku ngalunye lokuhlola, futhi kwadliwa isidlo sasekuseni esijwayelekile (i-1.5 g/kg yesisindo somzimba) ekuseni ngosuku lokuhlola. Ngaphansi kwe-anesthesia yendawo (i-0.5 ml ye-1% ye-lidocaine ngaphandle kwe-epinephrine), ama-biopsies emisipha atholakale emsipheni we-vastus lateralis kusetshenziswa i-percutaneous Bergström aspiration.82 Amasampula emisipha afakwa ngokushesha ku-RNAlater futhi agcinwa ku-4°C kuze kube yilapho kuhlukaniswa i-fiber ngesandla (kuze kube yizinsuku ezi-3).
Ama-myofiber bundles asanda kuhlukaniswa adluliselwa ku-RNAlater medium entsha esitsheni sokukhuliswa. Ama-myofiber ngamanye abe esehlukaniswa ngesandla kusetshenziswa i-stereomicroscope kanye nama-tweezers amahle. Ama-fiber angamashumi amabili nanhlanu ahlukaniswa kusuka ku-biopsy ngayinye, enaka kakhulu ukukhetha ama-fiber ezindaweni ezahlukene ze-biopsy. Ngemva kokuhlukaniswa, i-fiber ngayinye yacwiliswa ngobumnene ku-3 μl ye-lysis buffer (i-SingleShot Cell Lysis Kit, i-Bio-Rad) equkethe ama-enzyme e-proteinase K kanye ne-DNase ukuze kususwe amaprotheni angafuneki kanye ne-DNA. Ukususwa kwe-cell lysis kanye nokususwa kwe-protein/DNA kwabe sekuqaliswa ngokugoqa okufushane, ukujikelezisa uketshezi ku-microcentrifuge, kanye nokufakwa kwe-incubation ekushiseni kwegumbi (imizuzu eyi-10). I-lysate yabe isifakwa ku-thermal cycler (T100, Bio-Rad) ku-37°C imizuzu emi-5, 75°C imizuzu emi-5, bese igcinwa ngokushesha ku--80°C kuze kube yilapho kucutshungulwa okwengeziwe.
Amalabhulali e-RNA ahambisana ne-Illumina alungiselelwe kusuka ku-2 µl ye-myofiber lysate kusetshenziswa i-QuantSeq-Pool 3′ mRNA-Seq Library Prep Kit (Lexogen). Izindlela ezinemininingwane zingatholakala encwadini yomenzi. Inqubo iqala ngokuhlanganiswa kwe-cDNA ye-strand yokuqala ngokubhalwa kabusha, lapho kungeniswa khona ama-identifier ama-molecule ahlukile (i-UMI) kanye nama-barcode e-i1 aqondile esampula ukuqinisekisa ukuhlanganiswa kwamasampula nokunciphisa ukuguquguquka kobuchwepheshe ngesikhathi sokucubungula okungezansi. I-cDNA evela kuma-myofiber angu-96 bese ihlanganiswa futhi ihlanzwe ngama-magnetic beads, ngemva kwalokho i-RNA isusiwe bese kwenziwa ukuhlanganiswa kwe-strand yesibili kusetshenziswa ama-primer angahleliwe. Umtapo wolwazi uhlanzwa ngama-magnetic beads, kwengezwa amathegi e-i5/i7 aqondile echibini, bese kwandiswa i-PCR. Isinyathelo sokugcina sokuhlanza sikhiqiza amalabhulali ahambisana ne-Illumina. Ikhwalithi yechibi ngalinye lomtapo wolwazi ihlolwe kusetshenziswa i-High Sensitivity Small Fragment DNA Analysis Kit (Agilent Technologies, DNF-477-0500).
Ngokusekelwe ekulinganisweni kwe-Qubit, amachibi ahlanganiswa futhi ngamanani alinganayo (2 nM). Idamu elivelayo labe selihlelwa kuthuluzi le-NovaSeq 6000 kwimodi ejwayelekile kusetshenziswa i-NovaSeq S2 Reagent Kit (1 × 100 nucleotides) enomthwalo we-2 nM (4% PhiX).
Ipayipi lethu lisekelwe kupayipi lokuhlaziya idatha le-QuantSeq Pool likaLexogen (https://github.com/Lexogen-Tools/quantseqpool_analysis). Idatha yaqala yahlukaniswa nge-bcl2fastq2 (v2.20.0) ngokusekelwe kunkomba ye-i7/i5. I-Read 2 yabe isihlukaniswa nge-idemux (v0.1.6) ngokusekelwe kubhakhodi yesampula ye-i1 kanye nokulandelana kwe-UMI kwakhishwa nge-umi_tools (v1.0.1). Ukufundwa kwabe sekunqunywa nge-cutadapt (v3.4) ngezindilinga eziningi ukususa ukufundwa okufushane (<20 ubude) noma ukufundwa okuhlanganisa kuphela ukulandelelana kwe-adapter. Ukufundwa kwabe sekuqondaniswa ne-genome yomuntu kusetshenziswa i-STAR (v2.6.0c) kanye namafayela e-BAM afakwa ku-SAMtools (v1.11). Ukufundwa okuphindwe kabili kwasuswa kusetshenziswa i-umi_tools (v1.0.1). Ekugcineni, ukubalwa kokuqondanisa kwenziwa kusetshenziswa i-featureCounts ku-Subread (v2.0.3). Ukulawulwa kwekhwalithi kwenziwe kusetshenziswa i-FastQC (v0.11.9) ezigabeni eziningana eziphakathi zepayipi.
Konke ukucubungula okwengeziwe kwe-bioinformatics kanye nokuboniswa kwenziwa ku-R (v4.2.3), ikakhulukazi kusetshenziswa ukuhamba komsebenzi kwe-Seurat (v4.4.0). 83 Ngakho-ke, amanani e-UMI ngamanye kanye nama-metadata matrices aguqulwa aba yizinto ze-Seurat. Izakhi zofuzo ezivezwe ngaphansi kwama-30% azo zonke izintambo zasuswa. Amasampula ekhwalithi ephansi asusiwe ngokusekelwe emkhawulweni omncane wamanani e-UMI ayi-1000 kanye nezakhi zofuzo ezitholiwe eziyi-1000. Ekugcineni, izintambo ezingu-925 zidlule kuzo zonke izinyathelo zokuhlunga zokulawula ikhwalithi. Amanani e-UMI alungiswa kusetshenziswa indlela ye-Seurat SCTransform v2, angu-84 afaka zonke izici ezitholiwe ezingu-7418, kanye nomehluko phakathi kwabahlanganyeli wabuyiselwa emuva. Yonke i-metadata efanele ingatholakala ku-Supplementary Dataset 28.
Isikhathi sokuthunyelwe: Septhemba-10-2025