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Imisipha yamathambo sisicwili esingafaniyo esakhiwe ikakhulu zii-myofibrils, ezithi ebantwini zahlulwe zibe ziintlobo ezintathu: enye "ecothayo" (uhlobo 1) kunye nezimbini "ezikhawulezayo" (iintlobo 2A kunye ne-2X). Nangona kunjalo, ukungafani phakathi nangaphakathi kweentlobo ze-myofibril zemveli akukaqondwa kakuhle. Sisebenzise iindlela ze-transcriptomic kunye ne-proteomic kwi-1050 kunye ne-1038 myofibrils nganye evela kwi-vastus lateralis yomntu, ngokulandelelana. Uphononongo lwe-proteomic luquke amadoda, kwaye uphando lwe-transcriptomic luquke amadoda ali-10 kunye nabafazi ababini. Ukongeza kwi-isoforms ze-myosin heavy chain, sichonge iiproteni ze-metabolic, iiproteni ze-ribosomal, kunye neeproteni ze-cellular junctional njengemithombo yokuguquguquka kwe-intermyofibril enemilinganiselo emininzi. Ngaphezu koko, nangona kuchongiwe amaqela ee-fibers ezicothayo nezikhawulezayo, idatha yethu icebisa ukuba ii-fibers zohlobo lwe-2X azinakwahlulwa kwezinye ii-fibers ezikhawulezayo. Ngaphezu koko, ulwahlulo olusekelwe kwi-myosin heavy chain alwanelanga ukuchaza i-myofiber phenotype kwi-nemaline myopathies. Ngokubanzi, idatha yethu ibonisa ukungafani kwe-myofiber enemilinganiselo emininzi, kunye nemithombo yokwahluka edlula i-myosin heavy chain isoforms.
Ukungafani kweeseli luphawu oluqhelekileyo kuzo zonke iinkqubo zebhayoloji, oluvumela iiseli ukuba zikhetheke ukuhlangabezana neemfuno ezahlukeneyo zezicubu kunye neeseli.1 Imbono yendabuko yokungafani kweefayibha zemisipha yamathambo ibikukuba ii-motor neurons zichaza uhlobo lwefayibha ngaphakathi kweyunithi yemoto, kwaye uhlobo lwefayibha (oko kukuthi, uhlobo 1, uhlobo 2A, kunye nohlobo 2X ebantwini) lumiselwa ziimpawu ze-myosin heavy chain (MYH) isoforms.2 Oku ekuqaleni kwakusekelwe kwi-pH ATPase instability yazo, 3,4 kwaye kamva kwi-molecular expression yazo ye-MYH.5 Nangona kunjalo, ngokuchongwa kunye nokwamkelwa okulandelayo kweefayibha "ezixutyiweyo" ezibonisa ii-MYH ezininzi ngokulinganayo, iifayibha zemisipha yamathambo zijongwa ngakumbi njenge-continuum endaweni yokuba ziintlobo zefayibha ezahlukeneyo.6 Nangona kunjalo, intsimi isaxhomekeke kakhulu kwi-MYH njenge-classifier ephambili yokuhlelwa kwe-myofiber, umbono onokuthi uphenjelelwe yimida kunye nocalucalulo olubalulekileyo lwezifundo zokuqala zeempuku ezineprofayili yokubonakaliswa kwe-MYH kunye noluhlu lweentlobo zefayibha ezahlukileyo kwezo zabantu.2 Imeko iyinkimbinkimbi ngakumbi kukuba izihlunu zamathambo zabantu ezahlukeneyo zibonisa uluhlu olwahlukeneyo lwefayibha. iintlobo.7 I-vastus lateralis sisihlunu esixutyiweyo esineprofayili yokubonakaliswa kwe-MYH ephakathi (kwaye ke ngoko imele).7 Ngaphezu koko, ukulula kwayo kokuthatha iisampulu kuyenza ibe sisihlunu esifundwe kakuhle ebantwini.
Ngoko ke, uphando olungenamkhethe malunga nokwahluka kwefayibha yemisipha yamathambo kusetyenziswa izixhobo ezinamandla ze-"omics" lubalulekile kodwa lukwanzima, ngokuyinxenye ngenxa yendalo yefayibha yemisipha yamathambo ene-nucleic eninzi. Nangona kunjalo, ubuchwepheshe be-transcriptomics8,9 kunye ne-proteomics10 buye batshintsha indlela abavakalelwa ngayo kwiminyaka yakutshanje ngenxa yophuhliso lwetekhnoloji eyahlukeneyo, okuvumela uhlalutyo lwemisipha yamathambo kwisisombululo sefayibha enye. Ngenxa yoko, kwenziwe inkqubela phambili enkulu ekuchazeni ukwahluka kwefayibha enye kunye nempendulo yazo kwi-atrophic stimuli kunye nokuguga11,12,13,14,15,16,17,18. Okubalulekileyo kukuba, olu phuhliso lwetekhnoloji lunezicelo zeklinikhi, oluvumela ukuchazwa okuneenkcukacha ngakumbi nokuchanekileyo kokwahluka kwesifo okunxulumene nesifo. Umzekelo, i-pathophysiology ye-nemaline myopathy, enye yezifo zemisipha ezizuzwe njengelifa (MIM 605355 kunye ne-MIM 161800), iyinkimbinkimbi kwaye iyadida.19,20 Ke ngoko, ukuchazwa ngcono kokwahluka kwefayibha yemisipha yamathambo kunokukhokelela ekukhuleni okukhulu ekuqondeni kwethu esi sifo.
Siphuhlise iindlela zohlalutyo lwe-transcriptomic kunye ne-proteomic lwee-single skeletal muscle fibers ezithathwe ngesandla kwiisampuli ze-biopsy yomntu saza sazisebenzisa kumawaka ee-fibers, nto leyo esivumela ukuba siphande ukungafani kwee-cellular kwee-skeletal muscle fibers. Kulo msebenzi, sibonise amandla e-transcriptomic kunye ne-proteomic phenotyping yee-muscle fibers saza sachonga ii-metabolic, ribosomal, kunye nee-cellular junctional proteins njengemithombo ebalulekileyo yokwahluka kwe-interfiber. Ngaphezu koko, sisebenzisa olu hambo lwe-proteomic, sichaze ukubaluleka kwe-nematode myopathy kwii-single skeletal muscle fibers, sityhila utshintsho oludibeneyo oluya kwii-non-oxidative fibers ezizimeleyo kuhlobo lwe-fiber olusekelwe kwi-MYH.
Ukuze sihlolisise ukungafani kweefayibha zemisipha yomntu, siphuhlise iindlela ezimbini zokusebenza ukuze sikwazi uhlalutyo lwe-transcriptome kunye ne-proteome lweefayibha zemisipha enye (Umfanekiso 1A kunye noMfanekiso oNcedisayo 1A). Siphuhlise kwaye saphucula amanyathelo aliqela eendlela, ukusuka ekugcinweni kwesampulu kunye nokugcinwa kwe-RNA kunye nokuthembeka kweproteni ukuya ekuphuculeni i-throughput yendlela nganye. Kuhlalutyo lwe-transcriptome, oku kufezekisiwe ngokufaka ii-barcode ze-molecular ezithile zesampulu kwinqanaba lokuqala lokubhalwa kwe-reverse, okuvumela iifayibha ezingama-96 ukuba zihlanganiswe ukuze kusetyenzwe kakuhle ngezantsi. Ukulandelelana okunzulu (±1 yezigidi zokufunda ngefayibha nganye) xa kuthelekiswa neendlela zendabuko zeseli enye kuphucule ngakumbi idatha ye-transcriptome. 21 Kwi-proteomics, sisebenzise i-gradient emfutshane ye-chromatographic (imizuzu engama-21) edityaniswe nokufunyanwa kwedatha ye-DIA-PASEF kwi-timsTOF mass spectrometer ukuze kuphuculwe ubunzulu be-proteome ngelixa kugcinwa i-throughput ephezulu. 22,23 Ukuze sihlolisise ukungafani kweefayibha zemisipha esempilweni, sichaze ii-transcriptomes zeefayibha eziyi-1,050 ezivela kubanikeli abadala abali-14 abasempilweni kunye neeproteomes zeefayibha eziyi-1,038 ezivela kubanikeli abadala aba-5 abanempilo (Itheyibhile eyoNgezelelweyo 1). Kweli phepha, ezi seti zedatha zibizwa ngokuba zii-transcriptomes ze-fiber eziyi-1,000 kunye neeproteomes, ngokwahlukeneyo. Indlela yethu ifumene ii-transcripts ezingama-27,237 kunye neeproteni eziyi-2,983 kuhlalutyo lwe-transcriptomic kunye ne-proteomic lwe-fiber eziyi-1,000 (Umfanekiso 1A, Iiseti zeDatha eziNgezelelweyo 1–2). Emva kokucoca iiseti zedatha ze-transcriptomic kunye ne-proteomic ze-genes ezifunyenweyo ezingaphezulu kwe-1,000 kunye namaxabiso asebenzayo angama-50% kwifayibha nganye, uhlalutyo olulandelayo lwe-bioinformatics lwenziwe kwiifayibha eziyi-925 kunye ne-974 kwi-transcriptome kunye ne-proteome, ngokwahlukeneyo. Emva kokucoca, umyinge we-4257 ± 1557 genes kunye neeproteni zika-2015 ± 234 (i-mean ± SD) zifunyenwe ngefayibha nganye, kunye nokuguquguquka okulinganiselweyo phakathi komntu ngamnye (Imifanekiso eyongezelelweyo 1B–C, Iiseti zeDatha ezongezelelweyo 3–4). Nangona kunjalo, ukutshintsha ngaphakathi kwesihloko kwabonakala ngakumbi kubo bonke abathathi-nxaxheba, mhlawumbi ngenxa yomahluko kwi-RNA/protein vield phakathi kweefayibha zobude obahlukeneyo kunye neendawo ezinqamlezileyo. Kwiiproteni ezininzi (>2000), i-coefficient of variation yayingaphantsi kwe-20% (Imifanekiso eyongezelelweyo 1D). Zombini iindlela zivumele ukubamba uluhlu olubanzi lwe-transcripts kunye neeproteni ezineempawu ezicacileyo ezibalulekileyo ekucutheni kwemisipha (umz., ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Imifanekiso eyongezelelweyo 1E–F). Uninzi lweempawu ezichongiweyo beziqhelekile phakathi kweedatha ze-transcriptomic kunye ne-proteomic (Umfanekiso oNcedisayo 1G), kwaye i-UMI/LFQ intensities ephakathi kwezi mpawu yayinxulumene kakuhle (r = 0.52) (Umfanekiso oNcedisayo 1H).
Umsebenzi we-Transcriptomics kunye ne-proteomics (wenziwe nge-BioRender.com). Ii-BD Dynamic range curves ze-MYH7, MYH2, kunye ne-MYH1, kunye nemida ebaliweyo yokunikezelwa kohlobo lwefayibha. E, F Ukusasazwa kokubonakaliswa kwe-MYH kuzo zonke iifayibha kwiiseti zedatha ze-transcriptomics kunye ne-proteomics. G, H Iiploti ze-Uniform Diversity Approximation and Projection (UMAP) ze-transcriptomics kunye ne-proteomics ezifakwe umbala ngohlobo lwefayibha olusekelwe kwi-MYH. I, J Iiploti zeempawu ezibonisa ukubonakaliswa kwe-MYH7, MYH2, kunye ne-MYH1 kwiiseti zedatha ze-transcriptomics kunye ne-proteomics.
Ekuqaleni sazimisela ukwabela uhlobo lwefayibha olusekelwe kwi-MYH kwifayibha nganye sisebenzisa indlela ephuculiweyo esebenzisa uvakalelo oluphezulu kunye noluhlu oluguquguqukayo lwe-MYH expression kwiiseti zedatha ze-omics. Izifundo zangaphambili zisebenzise imida engaqhelekanga ukulebhelisha iifayibha njengohlobo olucocekileyo 1, uhlobo 2A, uhlobo 2X, okanye ezixutyiweyo ngokusekelwe kwipesenti emiselweyo yokubonakaliswa kwee-MYHs ezahlukeneyo11,14,24. Sisebenzise indlela eyahlukileyo apho ukubonakaliswa kwefayibha nganye kwabekwa ngokwee-MYHs esizisebenzisileyo ukuchwetheza iifayibha: MYH7, MYH2, kunye ne-MYH1, ezihambelana nohlobo 1, uhlobo 2A, kunye neefayibha zohlobo 2X, ngokwahlukeneyo. Emva koko sibale ngokwezibalo inqaku eliphantsi le-inflection ye-curve nganye ephumayo kwaye sayisebenzisa njengomlinganiselo wokwabela iifayibha njengezakhayo (ngaphezulu komda) okanye ezingezizo (ngaphantsi komda) kwi-MYH nganye (Umfanekiso 1B–D). Ezi datha zibonisa ukuba i-MYH7 (Umfanekiso 1B) kunye ne-MYH2 (Umfanekiso 1C) zineprofayili zokubonakaliswa ezicacileyo ngakumbi kwinqanaba le-RNA xa kuthelekiswa nenqanaba leprotheni. Enyanisweni, kwinqanaba leproteni, zimbalwa kakhulu iifayibha ezingaziveziyo i-MYH7, kwaye akukho fayibha yayine-100% ye-MYH2 expression. Sisebenzise imida yokubonakalisa emiselweyo ukuze sinike iintlobo zefayibha ezisekelwe kwi-MYH kuzo zonke iifayibha kwiseti nganye yedatha. Umzekelo, iifayibha ze-MYH7+/MYH2-/MYH1- zabelwe uhlobo loku-1, ngelixa iifayibha ze-MYH7-/MYH2+/MYH1+ zabelwe uhlobo oluxutyiweyo lwe-2A/2X (jonga iTheyibhile eyoNgezelelweyo yesi-2 ukuze ufumane inkcazo epheleleyo). Sidibanisa zonke iifayibha, sibone ukusasazwa okufanayo okuphawulekayo kweentlobo zefayibha ezisekelwe kwi-MYH kuzo zombini i-RNA (Umfanekiso 1E) kunye neproteni (Umfanekiso 1F), ngelixa ukwakheka okunxulumeneyo kweentlobo zefayibha ezisekelwe kwi-MYH kwahluka kubantu ngabanye, njengoko bekulindelwe (Umfanekiso oNgezelelweyo wesi-2A). Uninzi lweefayibha lwahlulwa njengohlobo olucocekileyo lwe-1 (34–35%) okanye uhlobo lwe-2A (36–38%), nangona inani elikhulu leefayibha ezixutyiweyo ze-2A/2X nazo zafunyanwa (16–19%). Umahluko omkhulu kukuba iifayibha zohlobo lwe-2X ezimsulwa zinokufunyanwa kuphela kwinqanaba le-RNA, kodwa kungekhona kwinqanaba leproteni, nto leyo ebonisa ukuba ukubonakaliswa kwe-MYH okukhawulezayo ubuncinane kulawulwa ngokuyinxenye emva kokubhalwa.
Siqinisekisile indlela yethu yokubhala i-MYH fiber based proteomics sisebenzisa i-antibody-based dot blotting, kwaye zombini ezi ndlela zifikelele kwi-100% ukuvumelana ekuchongeni i-pure type 1 kunye ne-type 2A fibers (jonga uMfanekiso oNgezelelweyo 2B). Nangona kunjalo, indlela esekwe kwi-proteomics yayinobuntununtunu ngakumbi, isebenza ngakumbi ekuchongeni i-mixed fibers, kwaye ilinganisa umlinganiselo we-MYH gene nganye kwi-fiber nganye. Ezi datha zibonisa ukusebenza kakuhle kokusebenzisa indlela ejolise kakhulu, enobuntununtunu kakhulu esekelwe kwi-omics ukuchaza iintlobo ze-skeletal muscle fiber.
Emva koko sisebenzise ulwazi oludibeneyo olubonelelwe yi-transcriptomics kunye ne-proteomics ukwahlula ngokweengqiqo i-myofibers ngokusekelwe kwi-transcriptome okanye i-proteome yazo epheleleyo. Sisebenzisa indlela ye-uniform manifold approximation and projection (UMAP) ukunciphisa ubukhulu ukuya kwiinxalenye ezintandathu eziphambili (Imifanekiso eyongezelelweyo 3A–B), sikwazile ukubona umahluko we-myofiber kwi-transcriptome (Umfanekiso 1G) kunye ne-proteome (Umfanekiso 1H). Okuphawulekayo kukuba, i-myofibers ayizange ibekwe ngamaqela ngabathathi-nxaxheba (Imifanekiso eyongezelelweyo 3C–D) okanye iintsuku zovavanyo (Umfanekiso eyongezelelweyo 3E) nokuba kwiiseti zedatha ze-transcriptomics okanye ze-proteomics, nto leyo ebonisa ukuba umahluko ongaphakathi kwi-subject kwi-skeletal muscle fibers uphezulu kunomahluko ophakathi kwe-subject. Kwi-UMAP plot, kwavela amaqela amabini ahlukeneyo amele i-myofibers "ekhawulezayo" kunye "necothayo" (Imifanekiso 1G–H). Ii-myofibers ze-MYH7+ (ezicothayo) ziqokelelwe kwi-positive pole ye-UMAP1, ngelixa ii-myofibers ze-MYH2+ kunye ne-MYH1+ (ezikhawulezayo) ziqokelelwe kwi-negative pole ye-UMAP1 (Imifanekiso 1I–J). Nangona kunjalo, akukho mahluko wenziweyo phakathi kweentlobo zefayibha ezikhawulezayo (oko kukuthi, uhlobo lwe-2A, uhlobo lwe-2X, okanye i-2A/2X exutyiweyo) ngokusekelwe kwi-MYH expression, nto leyo ebonisa ukuba i-MYH1 (Umfanekiso 1I–J) okanye ezinye iimpawu ze-2X ze-myofiber zakudala ezifana ne-ACTN3 okanye i-MYLK2 (Imifanekiso eyongezelelweyo 4A–B) ayihlukanisi phakathi kweentlobo ezahlukeneyo ze-myofiber xa kujongwa i-transcriptome okanye i-proteome iyonke. Ngaphezu koko, xa kuthelekiswa ne-MYH2 kunye ne-MYH7, ii-transcripts okanye iiproteni ezimbalwa zidibene kakuhle ne-MYH1 (Imifanekiso eyongezelelweyo 4C–H), nto leyo ebonisa ukuba ubuninzi be-MYH1 abubonakalisi ngokupheleleyo i-myofiber transcriptome/proteome. Izigqibo ezifanayo zifikelelwe xa kuvavanywa ukubonakaliswa okuxutyiweyo kwee-isoforms ezintathu ze-MYH kwinqanaba le-UMAP (Iimifanekiso ezongezelelweyo. 4I–J). Ngoko ke, nangona ii-fibers ze-2X zinokuchongwa kwinqanaba le-transcript ngokusekelwe kumlinganiselo we-MYH kuphela, ii-fibers ze-MYH1+ azinakwahlulwa kwezinye ii-fibers ezikhawulezayo xa kujongwa i-transcriptome okanye i-proteome iyonke.
Njengophando lokuqala lokungafani kweefayibha ezicothayo ngaphaya kwe-MYH, sivavanye iiproteni ezine ezisekelwe kwi-slow fiber type-specific: TPM3, TNNT1, MYL3, kunye ne-ATP2A22. Ii-slow fiber subtypes zibonise ulwalamano oluphezulu, nangona lungagqibelelanga, lwePearson kunye ne-MYH7 kuzo zombini ii-transcriptomics (Umfanekiso oNcedisayo 5A) kunye neeproteomics (Umfanekiso oNcedisayo 5B). Malunga ne-25% kunye ne-33% yeefayibha ezicothayo azizange zihlelwe njengeefayibha ezicothayo ezicocekileyo kuzo zonke iifayibha/iiproteni subtypes kwi-transcriptomics (Umfanekiso oNcedisayo 5C) kunye neeproteomics (Umfanekiso oNcedisayo 5D), ngokwahlukeneyo. Ke ngoko, ukwahlulahlula iifayibha ezicothayo ngokusekelwe kwiifayibha ezininzi/iiproteni subtypes kwazisa ubunzima obongezelelweyo, nokuba kwiiproteni ezaziwa ngokuba zezohlobo oluthile lwefayibha. Oku kuthetha ukuba ukwahlulahlula iifayibha ngokusekelwe kwii-isoforms zosapho olunye lwefayibha/iiproteni kusenokungabonisi ngokwaneleyo ukungafani kokwenyani kweefayibha zemisipha yamathambo.
Ukuze sihlolisise ngakumbi ukuguquguquka kwe-phenotypic kwe-skeletal muscle fibers yomntu kwisikali semodeli ye-omics iyonke, senze ukunciphisa ubungakanani obungenamkhethe kwedatha sisebenzisa uhlalutyo lwe-principal component (PCA) (Umfanekiso 2A). Ngokufana ne-UMAP plots, akukho mntu uthatha inxaxheba okanye usuku lovavanyo oluchaphazele ukuhlanganiswa kwe-fiber kwinqanaba le-PCA (Imifanekiso eyongezelelweyo 6A–C). Kuzo zombini iiseti zedatha, uhlobo lwe-fiber olusekelwe kwi-MYH luchazwe yi-PC2, eyabonisa iqela le-fibers yohlobo lwe-1 oluhamba kancinci kunye neqela lesibini eliqulethe i-fast-twitch type 2A, uhlobo lwe-2X, kunye ne-mixed 2A/2X fibers (Umfanekiso 2A). Kuzo zombini iiseti zedatha, la maqela mabini aqhagamshelwe linani elincinci le-mixed type 1/2A fibers. Njengoko bekulindelekile, uhlalutyo olugqithisileyo lwabaqhubi be-PC abaphambili luqinisekisile ukuba i-PC2 iqhutywa ziisignatures ze-contractile kunye ne-metabolic (Umfanekiso 2B kunye ne-Supplementary Figures 6D–E, ii-Supplementary Datasets 5–6). Ngokubanzi, uhlobo lwefayibha olusekelwe kwi-MYH lufunyenwe lwanele ukuchaza umahluko oqhubekayo kwi-PC2, ngaphandle kweefayibha ezibizwa ngokuba yi-2X ezazisasazwa kuyo yonke i-transcriptome ngaphakathi kweqela elikhawulezayo.
A. Iiploti zohlalutyo lwecandelo eliphambili (PCA) zedatha ye-transcriptome kunye ne-proteome ezifakwe umbala ngokohlobo lwefayibha ngokusekelwe kwi-MYH. B. Uhlalutyo lokuphucula ii-transcript kunye nee-protein drivers kwi-PC2 kunye ne-PC1. Uhlalutyo lwezibalo lwenziwe kusetyenziswa iphakheji ye-clusterProfiler kunye ne-Benjamini-Hochberg adjusted p-values. C, D. Iiploti ze-PCA ezifakwe umbala ngokwe-intercellular adhesion gene ontology (GO) ngokwe-transcriptome kunye ne-costamere GO ngokwe-proteome. Iintolo zimele ii-transcript kunye nee-protein drivers kunye nemiyalelo yazo. E, F. Ukuhambelana kunye nokuqikelelwa kwe-Uniform manifold (UMAP) kubonisa iiploti zeempawu ezifanelekileyo zonyango ezibonisa ii-gradients zokubonakaliswa ezizimeleyo kuhlobo lwefayibha olucothayo/olukhawulezayo. G, H. Ulwalamano phakathi kwee-PC2 kunye nee-PC1 drivers kwi-transcriptomes kunye nee-proteomes.
Ngokungalindelekanga, uhlobo lwe-myofiber olusekelwe kwi-MYH luchaze kuphela inqanaba lesibini eliphezulu lokuguquguquka (i-PC2), nto leyo ebonisa ukuba ezinye izinto zebhayoloji ezingadibaniyo nohlobo lwe-myofiber olusekelwe kwi-MYH (i-PC1) zidlala indima ebalulekileyo ekulawuleni ukungafani kwefayibha yemisipha yamathambo. Uhlalutyo olugqithisileyo lwabaqhubi abaphambili kwi-PC1 lutyhile ukuba ukuguquguquka kwi-PC1 kumiselwe kakhulu kukunamathela kweseli kunye nomxholo we-ribosome kwi-transcriptome, kunye neeproteni ze-costameres kunye ne-ribosomal kwi-proteome (Umfanekiso 2B kunye neMifanekiso eyongezelelweyo 6D–E, Iseti yeDatha eyongezelelweyo 7). Kwimisipha yamathambo, ii-costameres zidibanisa i-Z-disc kwi-sarcolemma kwaye zibandakanyeka ekudlulisweni kwamandla kunye nokubonisa. 25 Iimpawu ze-PCA ezichaziweyo zisebenzisa ukunamathela kweseli (i-transcriptome, Umfanekiso 2C) kunye neempawu ze-costamere (i-proteome, Umfanekiso 2D) zityhile utshintsho olunamandla lwasekhohlo kwi-PC1, olubonisa ukuba ezi mpawu zityebile kwiifayibha ezithile.
Uvavanyo oluneenkcukacha ngakumbi lwe-myofiber clustering kwinqanaba le-UMAP lubonise ukuba uninzi lweempawu lubonise i-MYH-based expression gradient exhomekeke kuhlobo lwe-myofiber kunokuba ibe yi-myofiber subcluster-specific. Olu qhubekeko lubonwe kwiijini ezininzi ezinxulumene neemeko ze-pathological (Umfanekiso 2E), ezifana ne-CHCHD10 (isifo se-neuromuscular), i-SLIT3 (i-muscle atrophy), i-CTDNEP1 (isifo se-muscle). Olu qhubekeko lubonwe nakwi-proteome, kubandakanya iiproteni ezinxulumene neengxaki ze-neurological (UGDH), i-insulin signaling (PHIP), kunye ne-transcription (HIST1H2AB) (Umfanekiso 2F). Ngokudibeneyo, ezi datha zibonisa ukuqhubeka kwi-fiber-dependent slow/fast twitch heterogeneity kwii-myofibers ezahlukeneyo.
Okunomdla kukuba, ii-driver genes kwi-PC2 zibonise ulungelelwaniso oluhle lwe-transcriptome-proteome (r = 0.663) (Umfanekiso 2G), nto leyo ebonisa ukuba iintlobo zefayibha ezicothayo nezikhawulezayo, ngakumbi iipropati zokuqunjelwa kunye ne-metabolic ze-skeletal muscle fibers, zilawulwa ngokwe-transcription. Nangona kunjalo, ii-driver genes kwi-PC1 azibonisanga ulungelelwaniso lwe-transcriptome-proteome (r = -0.027) (Umfanekiso 2H), nto leyo ebonisa ukuba utshintsho olungahambelaniyo neentlobo zefayibha ezicothayo/ezikhawulezayo lulawulwa kakhulu emva kokubhalwa. Ngenxa yokuba utshintsho kwi-PC1 luchazwe ngokuyintloko ngamagama e-ribosomal gene ontology, kwaye ngenxa yokuba ii-ribosomes zidlala indima ebalulekileyo nekhethekileyo kwiseli ngokuthatha inxaxheba ngokukhutheleyo kunye nefuthe ekuguqulelweni kweproteni,31 ngokulandelayo siqalise ukuphanda olu hlobo lungaqhelekanga lwe-ribosomal heterogeneity.
Siqale ngombala iploti yohlalutyo lwe-proteomics principal component ngokwenani elikhulu leeproteni kwigama le-GOCC elithi “cytoplasmic ribosome” (Umfanekiso 3A). Nangona eli gama lityebile kwicala elihle le-PC1, nto leyo ebangela i-gradient encinci, iiproteni ze-ribosomal ziqhuba ukwahlulwahlulwa kuzo zombini iindlela ze-PC1 (Umfanekiso 3A). Iiproteni ze-ribosomal ezityebile kwicala elibi le-PC1 ziquka i-RPL18, i-RPS18, kunye ne-RPS13 (Umfanekiso 3B), ngelixa i-RPL31, i-RPL35, kunye ne-RPL38 (Umfanekiso 3C) yayizezona ziphambili eziqhuba icala elihle le-PC1. Okubangela umdla kukuba, i-RPL38 kunye ne-RPS13 zazibonakala kakhulu kwimisipha yamathambo xa kuthelekiswa nezinye izicubu (Umfanekiso ongezelelweyo 7A). Ezi signatures ze-ribosomal ezahlukileyo kwi-PC1 azibonwanga kwi-transcriptome (Umfanekiso ongezelelweyo 7B), nto leyo ebonisa ummiselo wokubhalwa emva kokubhalwa.
A. Iploti yohlalutyo lwecandelo eliphambili (i-PCA) enemibala ngokwemigaqo ye-cytoplasmic ribosomal gene ontology (GO) kuyo yonke iproteome. Iintolo zibonisa indlela yokwahluka okubangelwa yiproteni kwiploti ye-PCA. Ubude bomgca buhambelana nenqaku lecandelo eliphambili kwiproteni enikiweyo. B, C. Iploti zePCA zibonisa i-RPS13 kunye ne-RPL38. D. Uhlalutyo olungenalo ulawulo lwe-hierarchical clustering lweeproteni ze-cytoplasmic ribosomal. E. Imodeli yolwakhiwo lwe-80S ribosome (PDB: 4V6X) igxininisa iiproteni ze-ribosomal ezinobuninzi obahlukeneyo kwiifayibha zemisipha yamathambo. F. Iiproteni ze-ribosomal ezine-stoichiometry ezahlukeneyo ezikufutshane nomjelo wokuphuma kwe-mRNA.
Iingcamango zokungafani kwe-ribosomal kunye nokuzikhethela ziye zacetyiswa ngaphambili, apho ubukho be-subpopulations ezahlukeneyo ze-ribosomal (i-ribosomal heterogeneity) bunokuchaphazela ngokuthe ngqo ukuguqulwa kweproteni kwizicubu ezahlukeneyo32 kunye neeseli33 ngoguqulelo olukhethiweyo lwe-mRNA transcript pools34 ethile (i-ribosome specialization). Ukuchonga ii-subpopulations zeeproteni ze-ribosomal ezibonakaliswa kwi-fibers yemisipha yamathambo, senze uhlalutyo olungenalo ulawulo lwe-hierarchical clustering lweeproteni ze-ribosomal kwi-proteome (Umfanekiso 3D, Iseti yeDatha eyoNgezelelweyo 8). Njengoko bekulindelekile, iiproteni ze-ribosomal azizange ziqokelele ngohlobo lwefayibha ngokusekelwe kwi-MYH. Nangona kunjalo, sichonge amaqela amathathu ahlukeneyo eeproteni ze-ribosomal; iqela lokuqala (ribosomal_cluster_1) liqokelelene ne-RPL38 kwaye ngenxa yoko linokwanda kokubonakaliswa kwi-fibers ngeprofayili ye-PC1 elungileyo. Iqela lesibini (ribosomal_cluster_2) liqokelelene ne-RPS13 kwaye liphakanyisiwe kwi-fibers ngeprofayili ye-PC1 engalunganga. Iqela lesithathu (i-ribosomal_cluster_3) alibonisi ukubonakaliswa kokwahluka okudibeneyo kwimicu yemisipha yamathambo kwaye linokuthathwa njengeproteni ye-ribosomal yemisipha "engundoqo". Zombini ii-ribosomal clusters 1 kunye ne-2 ziqulathe iiproteni ze-ribosomal eziye zaboniswa ngaphambili ukuba zilawula uguqulelo olungelulo olunye (umz., RPL10A, RPL38, RPS19, kunye ne-RPS25) kwaye zichaphazela uphuhliso (umz., RPL10A, RPL38).34,35,36,37,38 Ngokuhambelana neziphumo ze-PCA, ukumelwa okungafaniyo kwezi proteni ze-ribosomal kuzo zonke ii-fibers kubonise ukuqhubeka (Umfanekiso ongezelelweyo 7C).
Ukuze sibone indawo yeeproteni ze-ribosomal ezahlukeneyo ngaphakathi kwi-ribosome, sisebenzise imodeli yolwakhiwo lwe-ribosome yomntu i-80S (iProtein Data Bank: 4V6X) (Umfanekiso 3E). Emva kokwahlula iiproteni ze-ribosomal ezikwiiqela ezahlukeneyo ze-ribosomal, iindawo zazo azizange zihambelane ngokusondeleyo, nto leyo ebonisa ukuba indlela yethu ayiphumelelanga ukubonelela ngokutyebisa iindawo/iinxalenye ezithile ze-ribosome. Okunomdla kukuba, nangona kunjalo, umlinganiselo weeproteni ezinkulu ze-subunit kwiqela lesi-2 wawuphantsi kunakwiqela lesi-1 nelesi-3 (Umfanekiso oNcedisayo 7D). Siqaphele ukuba iiproteni ezine-stoichiometry eguquliweyo kwiifayibha zemisipha yamathambo zazifumaneka kakhulu kumphezulu we-ribosome (Umfanekiso 3E), ngokuhambelana nokukwazi kwazo ukusebenzisana nezinto zangaphakathi ze-ribosome entry site (IRES) kwiindawo ezahlukeneyo ze-mRNA, ngaloo ndlela zilungelelanisa uguqulelo olukhethiweyo. 40, 41 Ngaphezu koko, iiproteni ezininzi ezine-stoichiometry eguquliweyo kwiifayibha zemisipha yamathambo zazifumaneka kufutshane neendawo ezisebenzayo ezifana nomjelo wokuphuma we-mRNA (Umfanekiso 3F), olawula ngokukhethayo ukunwebeka koguqulelo kunye nokubanjwa kweepeptide ezithile. 42 Ngamafutshane, idatha yethu ibonisa ukuba i-stoichiometry yeeproteni ze-ribosomal muscle skeletal muscle ibonisa ukungafani, nto leyo ebangela umahluko phakathi kweefayibha ze-muscle muscle.
Emva koko sizimisele ukuchonga iziginitsha zefayibha ezikhawulezayo nezicothayo size sihlole iindlela zokulawula kwazo ukuguqulelwa kwe-transcriptional. Ukuthelekisa amaqela efayibha akhawulezayo nezicothayo achazwe yi-UMAP kwiiseti zedatha ezimbini (Imifanekiso 1G–H kunye ne-4A–B), uhlalutyo lwe-transcriptomic kunye ne-proteomic luchonge iimpawu ezili-1366 kunye ne-804 ezifumaneka ngokwahlukileyo, ngokulandelelana (Imifanekiso 4A–B, Iiseti zeDatha ezongezelelweyo 9–12). Sibone umahluko olindelekileyo kwiziginitsha ezinxulumene nee-sarcomeres (umz., i-tropomyosin kunye ne-troponin), i-excitation-contraction coupling (i-SERCA isoforms), kunye ne-energy metabolism (umz., i-ALDOA kunye ne-CKB). Ngaphezu koko, ii-transcript kunye neeproteni ezilawula i-protein ubiquitination zibonakaliswe ngokwahlukileyo kwiifayibha ezikhawulezayo nezicothayo (umz., i-USP54, i-SH3RF2, i-USP28, kunye ne-USP48) (Imifanekiso 4A–B). Ngaphezu koko, i-microbial protein gene RP11-451G4.2 (DWORF), ebikade iboniswa ngokwahlukileyo kwiintlobo zefayibha yemisipha yemvu43 kwaye iphucula umsebenzi we-SERCA kwimisipha yentliziyo44, yandiswa kakhulu kwiifayibha zemisipha ezicothayo (Umfanekiso 4A). Ngokufanayo, kwinqanaba lefayibha nganye, umahluko omkhulu wabonwa kwiimpawu ezaziwayo ezifana ne-metabolism-related lactate dehydrogenase isoforms (LDHA kunye ne-LDHB, Umfanekiso 4C kunye noMfanekiso oNcedisayo 8A)45,46 kunye neempawu ezithile zefayibha ezazingaziwa ngaphambili (ezifana ne-IRX3, USP54, USP28, kunye ne-DPYSL3) (Umfanekiso 4C). Kwakukho ukudibana okubalulekileyo kweempawu ezichazwe ngokwahlukileyo phakathi kweeseti zedatha ze-transcriptomic kunye ne-proteomic (Umfanekiso oNcedisayo 8B), kunye nolwalamano lotshintsho lwe-fold oluqhutywa ikakhulu kukubonakaliswa okucacileyo kokwahluka kweempawu ze-sarcomere (Umfanekiso oNcedisayo 8C). Okuphawulekayo kukuba, ezinye iimpawu (umz. USP28, USP48, GOLGA4, AKAP13) zibonise ummiselo onamandla emva kokubhalwa kuphela kwinqanaba leproteomic kwaye zineprofayili zokubonakaliswa kwe-fiber type twitch ecothayo/ekhawulezayo (Umfanekiso oNcedisayo 8C).
Iiploti ze-volcano ze-A kunye ne-B ezithelekisa amaqela acothayo nakhawulezayo achongiweyo ziiploti ze-uniform manifold approximation and projection (UMAP) kwiMifanekiso 1G–H. Amachaphaza anemibala amele ii-transcripts okanye iiproteni ezahluke kakhulu kwi-FDR < 0.05, kwaye amachaphaza amnyama amele ii-transcripts okanye iiproteni ezahluke kakhulu xa kutshintshwa ilog > 1. Uhlalutyo lwezibalo oluneendlela ezimbini lwenziwe kusetyenziswa uvavanyo lwe-DESeq2 Wald kunye namaxabiso e-p alungisiweyo e-Benjamini-Hochberg (i-transcriptomics) okanye indlela yemodeli ye-Limma linear ene-empirical Bayesian analysis elandelwa luhlengahlengiso lwe-Benjamini-Hochberg lokuthelekisa okuninzi (ii-proteomics). C Iiploti zesignitsha ze-genes okanye iiproteni ezikhethiweyo ezichazwe ngokwahlukileyo phakathi kwee-fibers ezicothayo nezikhawulezayo. D Uhlalutyo lokutyebisa ii-transcripts kunye neeproteni ezichazwe ngokwahlukileyo kakhulu. Amaxabiso adibeneyo atyebiswe kuzo zombini iiseti zedatha, amaxabiso e-transcriptome atyebiswe kuphela kwi-transcriptome, kwaye amaxabiso e-proteome atyebiswe kuphela kwi-proteome. Uhlalutyo lwezibalo lwenziwe kusetyenziswa iphakheji ye-clusterProfiler ene-p-values elungisiweyo ye-Benjamini-Hochberg. E. Izinto ezichaza uhlobo oluthile lwefayibha ezichongiwe yi-SCENIC ngokusekelwe kumanqaku okuchaneka komlawuli ofunyenwe yi-SCENIC kunye nokubonakaliswa kwe-mRNA eyahlukileyo phakathi kweentlobo zefayibha. F. Ukubhala iinkcukacha zezinto ezichazayo ezichazwe ngokwahlukileyo phakathi kweefayibha ezicothayo nezikhawulezayo.
Emva koko senze uhlalutyo lokumelwa ngokugqithisileyo kweejini kunye neeproteni ezimele ngokwahlukileyo (Umfanekiso 4D, Iseti yeDatha eyoNgezelelweyo 13). Ukuphucuka kwendlela yeempawu ezahluke phakathi kwezi seti zimbini zedatha kubonise umahluko olindelekileyo, njengeenkqubo ze-fatty acid β-oxidation kunye ne-ketone metabolism (ii-fibers ezicothayo), ukucotha kwe-myofilament/muscle (ii-fibers ezikhawulezayo nezicothayo, ngokulandelanayo), kunye neenkqubo ze-carbohydrate catabolic (ii-fibers ezikhawulezayo). Umsebenzi we-serine/threonine protein phosphatase nawo uphakanyisiwe kwii-fibers ezikhawulezayo, eziqhutywa ziimpawu ezifana nee-subunits ze-regulatory kunye ne-catalytic phosphatase (PPP3CB, PPP1R3D, kunye ne-PPP1R3A), ezaziwa ngokulawula i-glycogen metabolism (47) (Iimpawu eziNcedisayo 8D–E). Ezinye iindlela ezityebiswe kwiifayibha ezikhawulezayo ziquka imizimba yokucubungula (P-) (YTHDF3, TRIM21, LSM2) kwiproteome (Umzobo oNcedisayo 8F), enokuthi ibandakanyeke kummiselo we-post-transcriptional (48), kunye nomsebenzi we-transcription factor (SREBF1, RXRG, RORA) kwi-transcriptome (Umzobo oNcedisayo 8G). Iifayibha ezicothayo zatyebiswe kumsebenzi we-oxidoreductase (BDH1, DCXR, TXN2) (Umzobo oNcedisayo 8H), i-amide binding (CPTP, PFDN2, CRYAB) (Umzobo oNcedisayo 8I), i-extracellular matrix (CTSD, ADAMTSL4, LAMC1) (Umzobo oNcedisayo 8J), kunye nomsebenzi we-receptor-ligand (FNDC5, SPX, NENF) (Umzobo oNcedisayo 8K).
Ukuze sifumane ulwazi oluthe kratya malunga nolawulo lwe-transcriptional olusekelwe kwiimpawu zohlobo lwe-muscle fiber ecothayo/ekhawulezayo, senze uhlalutyo lokuphucula i-transcription factor sisebenzisa i-SCENIC49 (Iseti yeDatha eyongezelelweyo 14). Izinto ezininzi ze-transcription zatyetyiswa kakhulu phakathi kwe-muscle fibers ekhawulezayo necothayo (Umfanekiso 4E). Oku kuquka izinto ze-transcription ezifana ne-MAFA, ebikade inxulunyaniswa nophuhliso lwe-muscle fiber olukhawulezayo,50 kunye nezinye izinto ze-transcription ezazingadibaniswanga ngaphambili neenkqubo ze-muscle fiber-specific gene. Phakathi kwezi, i-PITX1, i-EGR1, kunye ne-MYF6 yayizezona zinto ze-transcription zityetyiswe kakhulu kwi-muscle fibers ecothayo (Umfanekiso 4E). Ngokwahlukileyo koko, i-ZSCAN30 kunye ne-EPAS1 (ekwaziwa ngokuba yi-HIF2A) yayizezona zinto ze-transcription zityetyiswe kakhulu kwi-muscle fibers ecothayo (Umfanekiso 4E). Ngokuhambelana noku, i-MAFA yabonakaliswa kumanqanaba aphezulu kummandla we-UMAP ohambelana ne-muscle fibers ecothayo, ngelixa i-EPAS1 yayinepatheni yokubonakalisa eyahlukileyo (Umfanekiso 4F).
Ukongeza kwiijini ezaziwayo zokubhala iiproteni, kukho iintlobo ezininzi ze-RNA ezingabhalwanga ezinokuba negalelo ekulawuleni uphuhliso lomntu kunye nesifo. 51, 52 Kwiiseti zedatha ze-transcriptome, ii-RNA ezininzi ezingabhalwanga zibonisa uhlobo oluthile lwefayibha (Umfanekiso 5A kunye neSeti yeDatha eyongezelelweyo 15), kubandakanya i-LINC01405, echanekileyo kakhulu kwiifayibha ezicothayo kwaye kubikwa ukuba iyancipha kwizihlunu ezivela kwizigulana ezine-mitochondrial myopathy. 53 Ngokwahlukileyo koko, i-RP11-255P5.3, ehambelana ne-lnc-ERCC5-5 gene (https://lncipedia.org/db/transcript/lnc-ERCC5-5:2) 54, ibonisa uhlobo oluthile lwefayibha olukhawulezayo. Zombini i-LINC01405 (https://tinyurl.com/x5k9wj3h) kunye ne-RP11-255P5.3 (https://tinyurl.com/29jmzder) zibonisa ukucaciswa kwemisipha yamathambo (Imifanekiso eyongezelelweyo 9A–B) kwaye azinazo ii-genes ezikwaziyo zokuqunjelwa ngaphakathi kommandla wazo we-genomic we-1 Mb, nto leyo ebonisa ukuba zidlala indima ekhethekileyo ekulawuleni iintlobo zefayibha kunokulawula ii-genes ezikufutshane zokuqunjelwa. Iiprofayili zokubonakaliswa kwe-fiber ethile ecothayo/ekhawulezayo ye-LINC01405 kunye ne-RP11-255P5.3, ngokwahlukeneyo, ziqinisekisiwe kusetyenziswa i-RNAscope (Imifanekiso 5B–C).
A. Ii-transcript ze-RNA ezingabhalwanga zilawulwa kakhulu kwiifayibha zemisipha ezicothayo nezikhawulezayo. B. Imifanekiso ye-RNAscope emeleyo ebonisa uhlobo lwefayibha olucothayo nolukhawulezayo lwe-LINC01405 kunye ne-RP11-255P5.3, ngokulandelelana. Ibha yesikali = 50 μm. C. Ukulinganiswa kokubonakaliswa kwe-RNA engabhalwanga ngohlobo oluthile lwe-myofiber njengoko kumiselwe yi-RNAscope (n = 3 biopsies ezivela kubantu abazimeleyo, kuthelekiswa iifayibha zemisipha ezikhawulezayo nezicothayo ngaphakathi komntu ngamnye). Uhlalutyo lwezibalo lwenziwe kusetyenziswa uvavanyo lwe-t-lwabafundi olunemisila emibini. Iiploti zebhokisi zibonisa i-median kunye ne-quartiles yokuqala neyesithathu, kunye neentshebe ezikhomba kumaxabiso aphantsi kunye naphezulu. D. De novo microbial protein identification workflow (eyenziwe nge-BioRender.com). E. Iproteni ye-microbial LINC01405_ORF408:17441:17358 ibonakaliswa ngokukodwa kwiifayibha zemisipha ezicothayo (n=5 biopsies ezivela kubathathi-nxaxheba abazimeleyo, kuthelekiswa iifayibha zemisipha ezikhawulezayo nezicothayo kumthathi-nxaxheba ngamnye). Uhlalutyo lwezibalo lwenziwe kusetyenziswa indlela yemodeli yeLimm edityaniswe nendlela yeBayesian eqinisekisiweyo, ilandelwa yindlela yeBenjamini-Hochberg yokuthelekisa okuninzi kunye nohlengahlengiso lwexabiso le-p. Iiploti zebhokisi zibonisa i-median, i-first and third quartiles, kunye neentshebe ezikhomba kumaxabiso aphezulu/aphantsi.
Kutshanje, uphando lubonise ukuba ii-transcript ezininzi ezingasebenzisi ikhowudi zibhala iiproteni ze-microbial ezibhaliweyo, ezinye zazo ezilawula ukusebenza kwemisipha. 44, 55 Ukuze sichonge iiproteni ze-microbial ezinohlobo oluthile lwefayibha, sikhangele iseti yethu yedatha ye-1000 fiber proteome sisebenzisa ifayile ye-FASTA eyenzelwe wena equlethe ulandelelwano lwee-transcripts ezingasebenzisi ikhowudi (n = 305) ezifunyenwe kwiseti yedatha ye-1000 fiber transcriptome (Umfanekiso 5D). Sichonge iiproteni ze-microbial ezili-197 ezivela kwii-transcripts ezingama-22 ezahlukeneyo, ezingama-71 kuzo ezazilawulwa ngokwahlukileyo phakathi kwee-fibers muscle ezicothayo nezikhawulezayo (Umfanekiso ongezelelweyo 9C kunye neSeti yeDatha eyongeziweyo 16). Kwi-LINC01405, kuchongwe iimveliso ezintathu zeproteni ye-microbial, enye yazo ibonise ukucaciswa kwe-fiber ecothayo kwi-transcript yayo (Umfanekiso 5E kunye nomfanekiso ongezelelweyo 9D). Ke ngoko, sichonge i-LINC01405 njenge-gene ebhala iproteni ye-microbial ethile kwii-fibers muscle ezicothayo.
Siphuhlise umsebenzi opheleleyo wokuchonga i-proteomic enkulu yeefayibha zemisipha nganye kunye nabalawuli abachongiweyo bokungafani kwefayibha kwiimeko eziphilileyo. Sisebenzise lo msebenzi ukuze siqonde indlela i-nemaline myopathies echaphazela ngayo ukungafani kwefayibha yemisipha yamathambo. I-Nemaline myopathies zizifo zemisipha ezizuzwe njengelifa ezibangela ubuthathaka bemisipha kwaye, kubantwana abachaphazelekayo, ziba neengxaki ezahlukeneyo kubandakanya ukuphefumla, i-scoliosis, kunye nokuhamba okulinganiselweyo kwamalungu. 19,20 Ngokwesiqhelo, kwi-nemaline myopathies, iintlobo ezahlukeneyo ze-pathogenic kwiijini ezifana ne-actin alpha 1 (ACTA1) zibangela ukuba kubekho ubuninzi be-fiber myofiber ecothayo, nangona esi siphumo singafani. Omnye umahluko ophawulekayo yi-troponin T1 nemaline myopathy (TNNT1), enokubakho kakhulu kwiifayibha ezikhawulezayo. Ke ngoko, ukuqonda okungcono ukungafani okuphantsi kokungasebenzi kakuhle kwefayibha yemisipha yamathambo okubonwa kwi-nemaline myopathies kunokunceda ukusombulula ubudlelwane obunzima phakathi kwezi zifo kunye nohlobo lwe-myofiber.
Xa kuthelekiswa nolawulo olusempilweni (n=3 kwiqela ngalinye), ii-myofibers ezahlulwe kwizigulane ze-nemaline myopathy ezineenguqu kwi-ACTA1 kunye ne-TNNT1 genes zibonise i-myofiber atrophy okanye i-dystrophy ephawulekayo (Umfanekiso 6A, Itheyibhile eyoNgezelelweyo 3). Oku kuveze imingeni ebalulekileyo yobugcisa kuhlalutyo lwe-proteomic ngenxa yobuninzi bezinto ezikhoyo. Nangona kunjalo, sikwazile ukufumanisa iiproteni ezingama-2485 kwi-skeletal myofibers ezingama-272. Emva kokucoca ubuncinane iiproteni ezili-1000 ezilinganisiweyo kwi-fiber nganye, ii-fibers ezingama-250 zafakwa kuhlalutyo lwe-bioinformatics olulandelayo. Emva kokucoca, umyinge weeproteni ezili-1573 ± 359 kwi-fiber nganye zalinganiswa (Umfanekiso oNgezelelweyo 10A, Iiseti zeDatha eNgezelelweyo 17–18). Okuphawulekayo kukuba, nangona kuncitshiswe kakhulu ubungakanani be-fiber, ubunzulu be-proteome beesampulu zezigulane ze-nemaline myopathy buncitshiswe kancinci. Ngaphezu koko, ukucubungula le datha sisebenzisa iifayile zethu ze-FASTA (kuquka ii-transcripts ezingabhalwanga ngekhowudi) kusivumele ukuba sichonge iiproteni ezintlanu ze-microbial kwi-skeletal myofibers ezivela kwizigulane ze-nemaline myopathy (Iseti yeDatha eyongeziweyo 19). Uluhlu oluguquguqukayo lweproteome lwalubanzi kakhulu, kwaye iiproteni ezipheleleyo kwiqela lolawulo zidibene kakuhle neziphumo zohlalutyo lwangaphambili lwe-1000-fiber proteome (Umzobo oyongeziweyo 10B–C).
A. Imifanekiso yeMicroscopic ebonisa i-fiber atrophy okanye i-dystrophy kunye nokuxhaphaka kweentlobo ezahlukeneyo zefayibha ngokusekelwe kwi-MYH kwi-ACTA1 kunye ne-TNNT1 ne-nemaline myopathies (NM). I-Scale bar = 100 μm. Ukuqinisekisa ukuphinda kuvele i-stain kwizigulane ze-ACTA1 kunye ne-TNNT1, ii-biopsies ezintathu zezigulane zafakwa i-stain izihlandlo ezibini ukuya kwezintathu (amacandelo amane kwimeko nganye) ngaphambi kokukhetha imifanekiso emeleyo. B. Ubungakanani bohlobo lwefayibha kubathathi-nxaxheba ngokusekelwe kwi-MYH. C. Uhlalutyo lwecandelo eliphambili (PCA) lwe-skeletal muscle fibers kwizigulane ezine-nemaline myopathies kunye nolawulo. D. I-skeletal muscle fibers ezivela kwizigulane ezine-nemaline myopathies kunye nolawulo oluqikelelwe kwi-PCA plot echazwe kwii-fibers ezili-1000 ezihlalutyiweyo kuMfanekiso 2. Umzekelo, ii-volcano plot zithelekisa umahluko phakathi kwabathathi-nxaxheba abane-ACTA1 kunye ne-TNNT1 ne-nemaline myopathies kunye nolawulo, kunye naphakathi kwabathathi-nxaxheba abane-ACTA1 kunye ne-TNNT1 ne-nemaline myopathies. Izangqa ezinemibala zibonisa iiproteni ezahluke kakhulu kwi-π < 0.05, kwaye amachaphaza amnyama abonisa iiproteni ezahluke kakhulu kwi-FDR < 0.05. Uhlalutyo lwezibalo lwenziwe kusetyenziswa indlela yemodeli ethe ngqo yeLimma kunye neendlela zeBayesian ezisebenza nge-empirical, kulandele ukulungiswa kwexabiso le-p ukuze kuthelekiswe izinto ezininzi kusetyenziswa indlela yeBenjamini-Hochberg. H. Uhlalutyo lokutyebisa iiproteni ezichazwe ngokwahlukileyo kakhulu kulo lonke iproteome nakwimicu yohlobo 1 kunye ne-2A. Uhlalutyo lwezibalo lwenziwe kusetyenziswa iphakheji ye-clusterProfiler kunye ne-Benjamini-Hochberg adjusted p-values. I, J. I-Principal component analysis (PCA) plots ezifakwe umbala yi-extracellular matrix kunye ne-mitochondrial gene ontology (GO) terms.
Ngenxa yokuba i-nemaline myopathies inokuchaphazela umlinganiselo weentlobo ze-MYH-expressing myofiber kwimisipha yamathambo, 19,20 siqale sahlola iintlobo ze-MYH-expressing myofiber kwizigulane ezine-nemaline myopathies kunye nolawulo. Sichonge uhlobo lwe-myofiber sisebenzisa indlela engakhethi cala echazwe ngaphambili kwi-1000 myofiber assay (Iimifanekiso ezongezelelweyo. 10D–E) saza saphinda sahluleka ukuchonga i-2X pure myofibers (Umfanekiso 6B). Sibone isiphumo esahlukileyo se-nemaline myopathies kuhlobo lwe-myofiber, njengoko izigulane ezibini ezine-ACTA1 mutations zazinomlinganiselo owandisiweyo we-type 1 myofibers, ngelixa izigulane ezibini ezine-TNNT1 nemaline myopathy zazinomlinganiselo ophantsi we-type 1 myofibers (Umfanekiso 6B). Enyanisweni, ukubonakaliswa kwe-MYH2 kunye ne-fast troponin isoforms (TNNC2, TNNI2, kunye ne-TNNT3) kwehle kwi-ACTA1-nemaline myopathies, ngelixa ukubonakaliswa kwe-MYH7 kwehle kwi-TNNT1-nemaline myopathies (Umfanekiso ongezelelweyo 11A). Oku kuhambelana neengxelo zangaphambili zokutshintsha kohlobo lwe-myofiber kwii-nemaline myopathies.19,20 Siqinisekisile ezi ziphumo nge-immunohistochemistry kwaye safumanisa ukuba izigulane ezine-ACTA1-nemaline myopathy zazine-type 1 myofibers ezininzi, ngelixa izigulane ezine-TNNT1-nemaline myopathy zazine-pattern eyahlukileyo (Umfanekiso 6A).
Kwinqanaba le-single-fiber proteome, ii-skeletal muscle fibers ezivela kwi-ACTA1 kunye ne-TNNT1 nemaline myopathy ziqokelelene kunye ne-control fibers ezininzi, apho ii-TNNT1 nemaline myopathy fibers zihlala zichaphazeleka kakhulu (Umfanekiso 6C). Oku kwabonakala ngakumbi xa kudweliswa ii-principal component analysis (PCA) plots zee-pseudo-inflated fibers kwisigulane ngasinye, apho i-TNNT1 nemaline myopathy izigulane 2 kunye no-3 zibonakala zikude kakhulu kwiisampulu zolawulo (Umfanekiso ongezelelweyo 11B, Iseti yeDatha eyongeziweyo 20). Ukuze siqonde ngcono indlela ii-fibers ezivela kwizigulane ze-myopathy ezithelekiswa ngayo nee-fibers eziphilileyo, sisebenzise ulwazi oluneenkcukacha olufunyenwe kuhlalutyo lwe-proteomic lwee-fibers ezili-1,000 ezivela kubantu abadala abasempilweni. Siqikelele ii-fibers ezivela kwi-dataset ye-myopathy (izigulane ze-ACTA1 kunye ne-TNNT1 nemaline myopathy kunye nolawulo) kwi-PCA plot efunyenwe kuhlalutyo lwe-1000-fiber proteomic (Umfanekiso 6D). Ukusasazwa kweentlobo zefayibha ze-MYH kwi-PC2 kwiifayibha zolawulo kwakufana nokusasazwa kwefayibha okufunyenwe kuhlalutyo lwe-1000-fiber proteomic. Nangona kunjalo, uninzi lweefayibha kwizigulane ze-nemaline myopathy ziye zatshintsha i-PC2, zidibene neefayibha eziphilileyo ezikhawulezayo, kungakhathaliseki ukuba zeziphi iintlobo zefayibha ze-MYH. Ngoko ke, nangona izigulane ezine-ACTA1 nemaline myopathy zibonise utshintsho oluya kwiifayibha zohlobo 1 xa zilinganiswa kusetyenziswa iindlela ezisekelwe kwi-MYH, zombini i-ACTA1 nemaline myopathy kunye ne-TNNT1 nemaline myopathy zitshintshe i-skeletal muscle fiber proteome ukuya kwiifayibha ezikhawulezayo.
Emva koko sithelekise ngokuthe ngqo iqela ngalinye lesigulana kunye nabalawuli abasempilweni saza sachonga iiproteni ezingama-256 kunye nama-552 ezivezwe ngokwahlukileyo kwi-ACTA1 kunye ne-TNNT1 nemaline myopathies, ngokulandelelana (Umfanekiso 6E–G kunye noMfanekiso oNcedisayo 11C, Iseti yeDatha eNcedisayo 21). Uhlalutyo lokuphucula i-gene lubonise ukwehla okudibeneyo kwiproteni ze-mitochondrial (Umfanekiso 6H–I, Iseti yeDatha eNcedisayo 22). Okumangalisayo kukuba, nangona kukho umahluko phakathi kweentlobo zefayibha kwi-ACTA1 kunye ne-TNNT1 nemaline myopathies, olu nciphiso lwaluzimele ngokupheleleyo kuhlobo lwefayibha olusekelwe kwi-MYH (Umfanekiso 6H kunye neMifanekiso eNcedisayo 11D–I, Iseti yeDatha eNcedisayo 23). Iiproteni ezintathu ze-microbial nazo zilawulwa kwi-ACTA1 okanye kwi-TNNT1 nemaline myopathies. Ezimbini kwezi microproteins, i-ENSG00000215483_TR14_ORF67 (ekwaziwa ngokuba yi-LINC00598 okanye i-Lnc-FOXO1) kunye ne-ENSG00000229425_TR25_ORF40 (lnc-NRIP1-2), zibonise ubuninzi obahlukeneyo kuphela kwi-type 1 myofibers. I-ENSG00000215483_TR14_ORF67 ibike ngaphambili ukuba idlala indima ekulawuleni umjikelo weseli. 56 Kwelinye icala, i-ENSG00000232046_TR1_ORF437 (ehambelana ne-LINC01798) yonyuswe kuzo zombini iintlobo ze-1 kunye ne-type 2A myofibers kwi-ACTA1-nemaline myopathy xa kuthelekiswa nolawulo olusempilweni (Umfanekiso ongezelelweyo 12A, iSeti yeDatha eyongeziweyo 24). Ngokwahlukileyo koko, iiproteni ze-ribosomal azizange zichaphazeleke kakhulu yi-nemaline myopathy, nangona i-RPS17 yayinciphile kwi-ACTA1 nemaline myopathy (Umzobo 6E).
Uhlalutyo lokutyebisa lukwabonakalise ukunyuka kweenkqubo zenkqubo yomzimba yokuzikhusela kwi-ACTA1 kunye ne-TNNT1 nemaline myopathies, ngelixa ukunamathela kweseli kwanda nakwi-TNNT1 nemaline myopathy (Umfanekiso 6H). Ukutyebisa kwezi zinto zingaphandle kweseli kwabonakaliswa ziiproteni ze-extracellular matrix ezitshintsha i-PCA kwi-PC1 kunye ne-PC2 kwicala elingalunganga (oko kukuthi, ukuya kwiifayibha ezichaphazeleke kakhulu) (Umfanekiso 6J). Omabini amaqela ezigulana abonise ukwanda kokubonakaliswa kweeproteni ezingaphandle kweseli ezibandakanyekayo kwiimpendulo zomzimba kunye neendlela zokulungisa i-sarcolemmal, ezifana nee-anxins (ANXA1, ANXA2, ANXA5)57,58 kunye neproteni yazo esebenzisana nayo i-S100A1159 (Imifanekiso eyongezelelweyo 12B–C). Le nkqubo ibike ngaphambili ukuba iphuculwe kwi-muscular dystrophies60 kodwa, ngokolwazi lwethu, ayizange idibaniswe ngaphambili ne-nemaline myopathies. Umsebenzi oqhelekileyo wale mishini ye-molecular uyafuneka ukulungiswa kwe-sarcolemmal emva kokwenzakala kunye nokuhlanganiswa kwee-myocytes ezisandula ukwenziwa kunye ne-myofibers58,61. Ngoko ke, ukwanda komsebenzi wale nkqubo kumaqela omabini ezigulana kubonisa impendulo yokubuyisela ukwenzakala okubangelwa kukungahlaliseki kwe-myofiber.
Iziphumo ze-nemaline myopathy nganye zazihambelana kakuhle (r = 0.736) kwaye zabonisa ukudibana okufanelekileyo (Imifanekiso eyongezelelweyo 11A–B), nto leyo ebonisa ukuba i-ACTA1 kunye ne-TNNT1 nemaline myopathy zineziphumo ezifanayo kwi-proteome. Nangona kunjalo, ezinye iiproteni zazilawulwa kuphela kwi-ACTA1 okanye kwi-TNNT1 nemaline myopathy (Imifanekiso eyongezelelweyo 11A kunye no-C). Iproteni ye-profibrotic MFAP4 yayiyenye yeeproteni eziphakanyisiweyo kakhulu kwi-TNNT1 nemaline myopathy kodwa ayizange itshintshe kwi-ACTA1 nemaline myopathy. I-SKIC8, icandelo le-PAF1C complex enoxanduva lokulawula i-HOX gene transcription, yancitshiswa kwi-TNNT1 nemaline myopathy kodwa ayizange ichaphazeleke kwi-ACTA1 nemaline myopathy (Imifanekiso eyongezelelweyo 11A). Ukuthelekiswa ngokuthe ngqo kwe-ACTA1 kunye ne-TNNT1 ne-nemaline myopathy kubonise ukwehla okukhulu kwiiproteni ze-mitochondrial kunye nokwanda kweeproteni zenkqubo yomzimba kwi-TNNT1 nemaline myopathy (Umfanekiso 6G–H kunye neMifanekiso eyongezelelweyo 11C kunye ne-11H–I). Le datha iyahambelana ne-atrophy/dystrophy enkulu ebonwe kwi-TNNT1 nemaline myopathy xa ithelekiswa ne-TNNT1 nemaline myopathy (Umfanekiso 6A), nto leyo ebonisa ukuba i-TNNT1 nemaline myopathy imele uhlobo olubi kakhulu lwesi sifo.
Ukuvavanya ukuba iziphumo ezibonweyo ze-nemaline myopathy ziyaqhubeka na kwinqanaba lemisipha yonke, senze uhlalutyo lwe-bulk proteomic lwee-muscle biopsies ezivela kwi-cohort efanayo yezigulane ze-TNNT1 nemaline myopathy saza sazithelekisa nezilawuli (n=3 ngeqela ngalinye) (Umzobo oNcedisayo 13A, Iseti yeDatha eNcedisayo 25). Njengoko bekulindelekile, izilawuli zazinxulumene kakhulu kuhlalutyo lwecandelo eliphambili, ngelixa izigulana ze-TNNT1 nemaline myopathy zibonise umahluko ophezulu phakathi kwesampulu efana naleyo ibonwa kuhlalutyo lwefayibha enye (Umzobo oNcedisayo 13B). Uhlalutyo lwe-bulk luvelise iiproteni ezivezwe ngokwahlukileyo (Umzobo oNcedisayo 13C, Iseti yeDatha eNcedisayo 26) kunye neenkqubo zebhayoloji (Umzobo oNcedisayo 13D, Iseti yeDatha eNcedisayo 27) ezigqanyisiweyo ngokuthelekisa iifayibha nganye, kodwa salahlekelwa kukwazi ukwahlula phakathi kweentlobo ezahlukeneyo zefayibha kwaye sahluleka ukuqikelela iziphumo zesifo ezahlukeneyo kwiifayibha.
Xa zizonke, ezi datha zibonisa ukuba i-single-myofiber proteomics inokucacisa iimpawu zebhayoloji zeklinikhi ezingafumanekiyo ngeendlela ezijoliswe kuzo ezifana ne-immunoblotting. Ngaphezu koko, ezi datha zibonisa imida yokusebenzisa i-actin fiber typing (MYH) yodwa ukuchaza i-phenotypic adaptation. Enyanisweni, nangona utshintsho lwe-fiber type lwahlukile phakathi kwe-actin kunye ne-troponin nemaline myopathies, zombini i-nemaline myopathies zisusa i-MYH fiber typing kwi-skeletal muscle fiber metabolism ukuya kwi-proteome ye-muscle ekhawulezayo nengaxinaniswanga kakhulu.
Ukwahluka kweeseli kubalulekile ukuze izicubu zihlangabezane neemfuno zazo ezahlukeneyo. Kwimisipha yamathambo, oku kudla ngokuchazwa njengeentlobo zefayibha ezibonakaliswa ngamanqanaba ahlukeneyo okuveliswa kwamandla kunye nokudinwa. Nangona kunjalo, kuyacaca ukuba oku kuchaza inxalenye encinci yokwahluka kwefayibha yemisipha yamathambo, eguquguqukayo kakhulu, iyinkimbinkimbi kwaye ineenkalo ezininzi kunokuba bekucingelwa ngaphambili. Ukuqhubela phambili kwezobuchwepheshe ngoku kukhanyise izinto ezilawula iifayibha zemisipha yamathambo. Enyanisweni, idatha yethu ibonisa ukuba iifayibha zohlobo lwe-2X zisenokungabi yintlobo eyahlukileyo yefayibha yemisipha yamathambo. Ngaphezu koko, sichonge iiproteni ze-metabolic, iiproteni ze-ribosomal kunye neeproteni ezinxulumene neseli njengezona zinto ziphambili ezibangela ukungafani kwefayibha yemisipha yamathambo. Ngokusebenzisa umsebenzi wethu weproteomic kwiisampuli zezigulane ezine-nematode myopathy, sibonise ngakumbi ukuba uhlobo lwefayibha olusekelwe kwi-MYH alubonisi ngokupheleleyo ukungafani kwemisipha yamathambo, ngakumbi xa inkqubo iphazamisekile. Enyanisweni, nokuba loluphi uhlobo lwefayibha olusekelwe kwi-MYH, i-nematode myopathy ibangela ukutshintshela kwiifayibha ze-oxidative ezikhawulezayo nezincinci.
Iifayibha zemisipha yamathambo ziye zahlulwahlulwa ukususela kwinkulungwane ye-19. Uhlalutyo lwakutshanje lwe-omics luye lwasivumela ukuba siqale ukuqonda iiprofayili zokubonakaliswa kweentlobo ezahlukeneyo zefayibha ze-MYH kunye neempendulo zazo kwiintshukumo ezahlukeneyo. Njengoko kuchaziwe apha, iindlela ze-omics zikwanenzuzo yokuba novakalelo olukhulu lokulinganisa iimpawu zohlobo lwefayibha kuneendlela zemveli ezisekwe kwi-antibody, ngaphandle kokuxhomekeka ekulinganisweni kweempawu enye (okanye ezimbalwa) ukuchaza uhlobo lwefayibha yemisipha yamathambo. Sisebenzise imisebenzi yokusebenza ye-transcriptomic kunye ne-proteomic eyongezelelweyo kwaye sadibanisa iziphumo ukuze sihlolisise ukulawulwa kwe-transcriptional kunye ne-post-transcriptional ye-fiber heterogeneity kwiifayibha zemisipha yamathambo omntu. Olu hambo lomsebenzi lubangele ukungaphumeleli ukuchonga iifayibha ze-2X ezicocekileyo kwinqanaba leproteni kwi-vastus lateralis yeqela lethu lamadoda aselula asempilweni. Oku kuhambelana nezifundo zangaphambili zefayibha enye ezifumene <1% iifayibha ze-2X ezicocekileyo kwi-vastus lateralis enempilo, nangona oku kufanele kuqinisekiswe kwezinye izihlunu kwixesha elizayo. Umahluko phakathi kokufunyanwa kweefayibha ze-2X ezicocekileyo kwinqanaba le-mRNA kunye neefayibha ze-2A/2X ezixutyiweyo kuphela kwinqanaba leproteni kuyadida. Ukubonakaliswa kwe-MYH isoform mRNA akusiyo i-circadian, 67 oko kuthetha ukuba akunakwenzeka ukuba "siyiphosile" isignali yokuqala ye-MYH2 kwiifayibha ze-2X ezibonakala zimsulwa kwinqanaba le-RNA. Enye inkcazo enokwenzeka, nangona icingelwa nje, inokuba ngumahluko kukuzinza kweproteni kunye/okanye i-mRNA phakathi kwee-isoform ze-MYH. Enyanisweni, akukho fast fiber ecocekileyo nge-100% kuyo nayiphi na i-MYH isoform, kwaye akucaci ukuba amanqanaba okubonakaliswa kwe-MYH1 mRNA kuluhlu lwe-70-90% aya kubangela ubuninzi obulinganayo be-MYH1 kunye ne-MYH2 kwinqanaba leproteni. Nangona kunjalo, xa kujongwa yonke i-transcriptome okanye i-proteome, uhlalutyo lwe-cluster lunokuchonga ngokuzithemba kuphela amaqela amabini ahlukeneyo amele iifayibha zemisipha ezicothayo nezikhawulezayo, nokuba zinjani na izinto ezichanekileyo ze-MYH. Oku kuhambelana nohlalutyo olusebenzisa iindlela ze-single-nucleus transcriptomic, ezihlala zichonga amaqela amabini ahlukeneyo e-myonuclear. 68, 69, 70 Ngaphezu koko, nangona izifundo zangaphambili zeproteomic zichonge iifayibha zohlobo lwe-2X, ezi fayibha aziqokeleli ngokwahlukeneyo kwezinye iifayibha ezikhawulezayo kwaye zibonisa inani elincinci kuphela leeproteni ezininzi ngokwahlukileyo xa kuthelekiswa nezinye iintlobo zefayibha ezisekelwe kwi-MYH. 14 Ezi ziphumo zibonisa ukuba kufuneka sibuyele kwimbono yokuqala kwenkulungwane yama-20 yokwahlulwahlulwa kweefayibha zemisipha, eyahlula iifayibha zemisipha yamathambo yomntu kungekhona kwiiklasi ezintathu ezahlukeneyo ezisekelwe kwi-MYH, kodwa kwiiqela ezimbini ezisekelwe kwiipropati zazo ze-metabolic kunye ne-contractile. 63
Okubaluleke ngakumbi, ukungafani kwe-myofiber kufuneka kuqwalaselwe ngokwemilinganiselo emininzi. Izifundo zangaphambili ze-"omics" zikhombe kweli cala, zibonisa ukuba iifayibha zemisipha yamathambo azenzi amaqela ahlukeneyo kodwa zicwangciswe ngokuqhubekekayo. 11, 13, 14, 64, 71 Apha, sibonisa ukuba, ukongeza kumahluko kwiipropati zokuqunjelwa kunye ne-metabolic zemisipha yamathambo, ii-myofibers zinokwahlulwa ngeempawu ezinxulumene nokusebenzisana kweeseli kunye neendlela zokuguqula. Enyanisweni, sifumene ukungafani kwe-ribosome kwiifayibha zemisipha yamathambo ezinegalelo kukungafani ngaphandle kweentlobo zefayibha ezicothayo nezikhawulezayo. Isizathu esisisiseko sale heterogeneity ibalulekileyo ye-myofiber, engaxhomekekanga kuhlobo lwefayibha olucothayo nolukhawulezayo, asikacaci, kodwa sinokukhomba kulungiselelo olukhethekileyo lwendawo ngaphakathi kwee-fascicles zemisipha eziphendula ngokufanelekileyo kumandla athile kunye nemithwalo,72 unxibelelwano olukhethekileyo lweseli okanye lwelungu elithile kunye nezinye iintlobo zeseli kwindawo encinci yemisipha73,74,75 okanye umahluko kumsebenzi we-ribosome ngaphakathi kwee-myofibers zomntu ngamnye. Enyanisweni, i-ribosomal heteroplasmy, nokuba kukutshintshwa kwe-RPL3 kunye ne-RPL3L okanye kwinqanaba le-2′O-methylation ye-rRNA, kuboniswe ukuba inxulumene ne-skeletal muscle hypertrophy76,77. Ukusetyenziswa kwe-multi-omic kunye ne-spatial kunye nokuchazwa komsebenzi wee-myofibers zomntu ngamnye kuya kuphucula ngakumbi ukuqonda kwethu i-biology yemisipha kwinqanaba le-multi-omic78.
Ngokuhlalutya iiproteome ze-single myofibers ezivela kwizigulana ezine-nemaline myopathies, sikwabonise ukusetyenziswa, ukusebenza kakuhle, kunye nokusebenza kwe-single myofiber proteomics ukucacisa i-pathophysiology yeklinikhi yemisipha yamathambo. Ngaphezu koko, ngokuthelekisa indlela esisebenza ngayo nohlalutyo lwe-proteomic lwehlabathi, sikwazile ukubonisa ukuba i-single myofiber proteomics ivelisa ubunzulu bolwazi obufanayo ne-global tissue proteomics kwaye yandisa obu bunzulu ngokubala ukungafani kwe-interfiber kunye nohlobo lwe-myofiber. Ukongeza kumahluko olindelekileyo (nangona uguquguqukayo) kumlinganiselo wohlobo lwefayibha obonwe kwi-ACTA1 kunye ne-TNNT1 nemaline myopathies xa kuthelekiswa nolawulo olusempilweni,19 sikwabone ukuhlaziywa kwe-oxidative kunye ne-extracellular ngaphandle kokutshintsha kwe-MYH-mediated fiber type. I-Fibrosis ibike ngaphambili kwi-TNNT1 nemaline myopathies.19 Nangona kunjalo, uhlalutyo lwethu lwakha phezu kolu phando ngokutyhila amanqanaba aphezulu eeproteni ezinxulumene noxinzelelo oluphuma ngaphandle kweseli, ezifana nee-annexins, ezibandakanyeka kwiinkqubo zokulungisa i-sarcolemmal, kwi-myofibers ezivela kwizigulane ezine-ACTA1 kunye ne-TNNT1 nemaline myopathies.57,58,59 Ukuqukumbela, amanqanaba akhulayo e-annexin kwi-myofibers ezivela kwizigulane ezine-nemaline myopathy anokubonisa impendulo yeseli ekulungiseni i-myofibers e-atrophic kakhulu.
Nangona olu phononongo lumele uhlalutyo olukhulu lwe-single-fiber-whole-muscle-omics lwabantu ukuza kuthi ga ngoku, alunazo izithintelo. Sizahlule ii-skeletal muscle fibers kwisampulu encinci nefanayo yabathathi-nxaxheba kunye ne-muscle enye (i-vastus lateralis). Ke ngoko, akunakwenzeka ukukhupha ubukho be-fiber populations ezithile kwiintlobo zemisipha nakwi-physiology yemisipha egqithisileyo. Umzekelo, asinakukhupha amathuba okuba kubekho i-subset yee-fibers ezikhawulezayo (umz., ii-fibers ezimsulwa ze-2X) ezivela kwi-sprinters eziqeqeshwe kakhulu kunye/okanye abadlali bamandla79 okanye ngexesha lokungasebenzi kwemisipha66,80. Ngaphezu koko, ubungakanani besampulu obuncinci babathathi-nxaxheba busithintele ekuphandeni umahluko wesini kwi-fiber heterogeneity, njengoko umlinganiselo we-fiber type waziwa ngokwahluka phakathi kwamadoda nabafazi. Ngaphezu koko, asikwazanga ukwenza uhlalutyo lwe-transcriptomic kunye ne-proteomic kwi-muscle fibers efanayo okanye iisampulu ezivela kubathathi-nxaxheba abafanayo. Njengoko thina nabanye siqhubeka nokuphucula uhlalutyo lwe-single-cell kunye ne-single-myofiber sisebenzisa uhlalutyo lwe-omics ukuze kufezekiswe igalelo lesampulu eliphantsi kakhulu (njengoko kubonisiwe apha kuhlalutyo lweefayibha ezivela kwizigulana ezine-mitochondrial myopathy), ithuba lokudibanisa iindlela ze-multi-omics (kunye nezisebenzayo) ngaphakathi kweefayibha zemisipha enye liyacaca.
Ngokubanzi, idatha yethu ichonga kwaye ichaza izinto ezibangela ukungafani kwemisipha yamathambo kunye nokwasemva kokuyibhala. Ngokukodwa, sibonisa idatha echasayo imfundiso ende kwi-physiology yemisipha yamathambo enxulumene nenkcazo yakudala yeentlobo zefayibha esekelwe kwi-MYH. Sinethemba lokuhlaziya ingxoxo kwaye ekugqibeleni sicinge ngokutsha ngokuqonda kwethu ukuhlelwa kwefayibha yemisipha yamathambo kunye nokungafani kwayo.
Abathathi-nxaxheba abalishumi elinesine baseCaucasian (amadoda ali-12 kunye nabafazi ababini) bavuma ngokuzithandela ukuthatha inxaxheba kolu phononongo. Olu phononongo lwavunywa yiKomiti yoBulungisa yeSibhedlele saseGhent University (BC-10237), lwathobela iSibhengezo saseHelsinki sika-2013, kwaye lwabhaliswa kwiClinicalTrials.gov (NCT05131555). Iimpawu eziqhelekileyo zabathathi-nxaxheba ziboniswe kwiTheyibhile eyoNgezelelweyo 1. Emva kokufumana imvume yomlomo nebhaliweyo, abathathi-nxaxheba bavavanywa ngaphambi kokuba bafakwe kokugqibela kolu phononongo. Abathathi-nxaxheba babesebancinci (abaneminyaka engama-22-42 ubudala), besempilweni (abazange babe nazimeko zempilo, abazange batshaye), kwaye besebenza kakuhle emzimbeni. Ukuthathwa kweoksijini ephezulu kwamiselwa kusetyenziswa i-ergometer yokulinganisa impilo yomzimba njengoko kuchaziwe ngaphambili. 81
Iisampulu ze-biopsy yemisipha zaqokelelwa ngexesha lokuphumla nakwimeko yokuzila ukutya kathathu, iintsuku ezili-14 zihlukene. Ngenxa yokuba ezi sampuli zaqokelelwa njengenxalenye yophando olukhulu, abathathi-nxaxheba batya i-placebo (lactose), i-H1-receptor antagonist (540 mg fexofenadine), okanye i-H2-receptor antagonist (40 mg famotidine) imizuzu engama-40 ngaphambi kwe-biopsy. Ngaphambili sibonise ukuba ezi arhente ze-histamine receptor antagonists azichaphazeli ukuqina kwemisipha yokuphumla81, kwaye akukho kuhlangana okunxulumene nemeko okubonwe kwizicwangciso zethu zolawulo lomgangatho (Imifanekiso eyongezelelweyo 3 kunye no-6). Ukutya okumiselweyo (41.4 kcal/kg ubunzima bomzimba, 5.1 g/kg ubunzima bomzimba i-carbohydrate, 1.4 g/kg ubunzima bomzimba iproteni, kunye namafutha omzimba ayi-1.6 g/kg) kwagcinwa iiyure ezingama-48 ngaphambi kosuku ngalunye lovavanyo, kwaye isidlo sakusasa esimiselweyo (1.5 g/kg ubunzima bomzimba i-carbohydrate) satyiwa kusasa losuku lovavanyo. Phantsi kwe-anesthesia yendawo (i-0.5 ml ye-1% ye-lidocaine ngaphandle kwe-epinephrine), ii-biopsies zemisipha zifunyenwe kwi-vastus lateralis muscle kusetyenziswa i-percutaneous Bergström aspiration.82 Iisampuli zemisipha zafakwa kwangoko kwi-RNAlater zaza zagcinwa kwi-4°C de kube kukhutshwe i-fiber ngesandla (ukuya kuthi ga kwiintsuku ezi-3).
Iibhanti ze-myofiber ezisandula ukususwa zadluliselwa kwi-RNAlater medium entsha kwisitya sokukhuliswa. Ii-myofibers nganye zasuswa ngesandla kusetyenziswa i-stereomicroscope kunye nee-tweezers ezincinci. Iifayibha ezingamashumi amabini anesihlanu zasuswa kwi-biopsy nganye, zinika ingqalelo ekhethekileyo ekukhetheni iifayibha kwiindawo ezahlukeneyo ze-biopsy. Emva kokusikwa, ifayibha nganye yafakwa ngobunono kwi-3 μl ye-lysis buffer (i-SingleShot Cell Lysis Kit, i-Bio-Rad) equlethe i-proteinase K kunye nee-enzymes ze-DNase ukususa iiproteni ezingafunekiyo kunye ne-DNA. Ukususwa kweseli kunye nokususwa kweproteni/i-DNA kwaqaliswa ngokujikelezisa okufutshane, ukujikelezisa ulwelo kwi-microcentrifuge, kunye nokufunxwa kubushushu begumbi (imizuzu eli-10). I-lysate yafakwa kwi-thermal cycler (T100, i-Bio-Rad) kwi-37°C imizuzu emi-5, i-75°C imizuzu emi-5, emva koko yagcinwa ngoko nangoko kwi--80°C de kube kusetyenzwa ngakumbi.
Iilayibrari zeRNA ezihambelana ne-Illumina ezilungiselelwe nge-2 µl ye-myofiber lysate kusetyenziswa i-QuantSeq-Pool 3′ mRNA-Seq Library Prep Kit (Lexogen). Iindlela ezineenkcukacha zingafumaneka kwincwadi yemiyalelo yomenzi. Le nkqubo iqala ngokwenziwa kwe-cDNA ye-strand yokuqala ngokubhalwa okungasemva, apho i-unique molecular identifiers (UMIs) kunye nee-sample-specific i1 barcodes ziziswa ukuqinisekisa ukuhlanganiswa kweesampuli kunye nokunciphisa ukuguquguquka kobugcisa ngexesha lokucubungula okusezantsi. I-cDNA evela kwi-96 myofibers emva koko ihlanganiswa kwaye icocwe ngee-magnetic beads, emva koko i-RNA isusiwe kwaye ukwenziwa kwe-second-strand kwenziwa kusetyenziswa ii-random primers. Ilayibrari icocwa ngee-magnetic beads, kongezwa iithegi ze-i5/i7 ezithile kwi-pool, kwaye i-PCR iyandiswa. Inyathelo lokugqibela lokucoca livelisa iilayibrari ezihambelana ne-Illumina. Umgangatho we-library pool nganye uhlolwe kusetyenziswa i-High Sensitivity Small Fragment DNA Analysis Kit (Agilent Technologies, DNF-477-0500).
Ngokusekelwe kumlinganiselo weQubit, amachibi ahlanganiswe ngakumbi kumanqanaba e-equimolar (2 nM). Iqula eliphumayo lalandelelaniswa kwisixhobo seNovaSeq 6000 kwimo eqhelekileyo kusetyenziswa iNovaSeq S2 Reagent Kit (1 × 100 nucleotides) kunye nomthwalo we-2 nM (4% PhiX).
Umbhobho wethu usekelwe kumbhobho wohlalutyo lwedatha yeQuantSeq Pool kaLexogen (https://github.com/Lexogen-Tools/quantseqpool_analysis). Idatha yaqala yahlulwahlulwa nge-bcl2fastq2 (v2.20.0) ngokusekelwe kwisalathisi se-i7/i5. I-Read 2 yahlulwahlulwa nge-idemux (v0.1.6) ngokusekelwe kwibhakhowudi yesampulu ye-i1 kwaye ulandelelwano lwe-UMI lwakhutshwa nge-umi_tools (v1.0.1). Emva koko iiReads zahluzwa nge-cutadapt (v3.4) kwiiraundi ezininzi ukususa ii-reads ezimfutshane (<20 ubude) okanye ii-reads eziquka kuphela ii-adapter sequences. IiReads zalungelelaniswa ne-genome yomntu kusetyenziswa i-STAR (v2.6.0c) kwaye iifayile ze-BAM zafakwa kwi-SAMtools (v1.11). Ii-reads eziphindwe kabini zasuswa kusetyenziswa i-umi_tools (v1.0.1). Ekugqibeleni, ukubalwa kokulungelelaniswa kwenziwa kusetyenziswa ii-featureCounts kwi-Subread (v2.0.3). Ulawulo lomgangatho lwenziwe kusetyenziswa iFastQC (v0.11.9) kumanqanaba aliqela aphakathi ombhobho.
Zonke ezinye iindlela zokucubungula kunye nokubonisa izinto nge-bioinformatics zenziwe kwi-R (v4.2.3), kusetyenziswa kakhulu indlela yokusebenza ye-Seurat (v4.4.0). 83 Ke ngoko, amaxabiso e-UMI kunye neematrices ze-metadata nganye zaguqulwa zaba zizinto ze-Seurat. Iijini ezivezwe ngaphantsi kwe-30% yazo zonke iifayibha zasuswa. Iisampuli ezikumgangatho ophantsi zasuswa ngokusekelwe kumda omncinci wamaxabiso e-1000 e-UMI kunye neejini ezifunyenweyo ezili-1000. Ekugqibeleni, iifayibha ezingama-925 zadlula kuzo zonke iindlela zokucoca umgangatho. Amaxabiso e-UMI ahlengahlengiswa kusetyenziswa indlela ye-Seurat SCTransform v2, ezingama-84 kuquka zonke iimpawu ezifunyenweyo ezingama-7418, kwaye umahluko phakathi kwababandakanyekayo wabuyiselwa. Zonke iimetadata ezifanelekileyo zingafumaneka kwiSeti yeDatha eyongeziweyo 28.
Ixesha leposi: Septemba-10-2025