Qhov sib txawv ntawm cov leeg nqaij pob txha tib neeg dhau ntawm cov saw hlau myosin hnyav

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Cov leeg nqaij pob txha yog cov nqaij sib txawv uas muaj cov myofibrils feem ntau, uas hauv tib neeg feem ntau raug faib ua peb hom: ib qho "qeeb" (hom 1) thiab ob qho "ceev" (hom 2A thiab 2X). Txawm li cas los xij, qhov sib txawv ntawm thiab hauv cov hom myofibril ib txwm tseem tsis tau nkag siab zoo. Peb tau siv cov txheej txheem transcriptomic thiab proteomic rau 1050 thiab 1038 tus kheej myofibrils los ntawm tib neeg vastus lateralis, raws li. Kev tshawb fawb proteomic suav nrog cov txiv neej, thiab kev tshawb fawb transcriptomic suav nrog 10 tus txiv neej thiab 2 tus poj niam. Ntxiv rau myosin hnyav saw isoforms, peb tau txheeb xyuas cov protein metabolic, ribosomal proteins, thiab cellular junctional proteins ua qhov chaw ntawm ntau qhov sib txawv intermyofibril. Ntxiv mus, txawm hais tias muaj kev txheeb xyuas cov pawg ntawm cov fibers qeeb thiab ceev, peb cov ntaub ntawv qhia tias hom 2X fibers yog phenotypically indistinguishable los ntawm lwm cov fibers ceev-twitch. Ntxiv mus, myosin hnyav saw-raws li kev faib tawm tsis txaus los piav qhia txog myofiber phenotype hauv nemaline myopathies. Zuag qhia tag nrho, peb cov ntaub ntawv qhia txog ntau yam myofiber heterogeneity, nrog rau qhov chaw ntawm kev hloov pauv txuas ntxiv mus dhau myosin heavy chain isoforms.
Qhov sib txawv ntawm cov hlwb yog ib qho tseem ceeb ntawm txhua lub cev muaj sia, uas tso cai rau cov hlwb tshwj xeeb los ua kom tau raws li qhov xav tau sib txawv ntawm cov nqaij thiab cov hlwb.1 Lub tswv yim ib txwm muaj ntawm cov leeg nqaij pob txha sib txawv yog tias cov hlwb ntawm lub cev txhais cov hom fiber hauv ib lub cev muaj zog, thiab tias hom fiber (piv txwv li, hom 1, hom 2A, thiab hom 2X hauv tib neeg) yog txiav txim siab los ntawm cov yam ntxwv ntawm myosin heavy chain (MYH) isoforms.2 Qhov no tau pib raws li lawv cov pH ATPase tsis ruaj khov,3,4 thiab tom qab ntawd ntawm lawv cov kev qhia molecular ntawm MYH.5 Txawm li cas los xij, nrog rau kev txheeb xyuas thiab kev lees txais tom qab ntawm cov fibers "sib xyaw" uas sib koom ua ke ntau MYHs hauv ntau qhov sib txawv, cov leeg nqaij pob txha tau pom ntau dua li cov fiber sib txawv.6 Txawm li cas los xij, thaj chaw tseem vam khom MYH ua tus thawj coj rau kev faib tawm myofiber, ib qho kev xav yuav cuam tshuam los ntawm cov kev txwv thiab kev ntxub ntxaug tseem ceeb ntawm kev tshawb fawb thaum ntxov ntawm cov nas uas cov qauv qhia MYH thiab ntau yam ntawm cov hom fiber sib txawv ntawm cov neeg hauv tib neeg.2 Qhov xwm txheej nyuaj dua los ntawm qhov tseeb tias cov leeg nqaij pob txha tib neeg sib txawv qhia ntau yam fiber. hom.7 Lub vastus lateralis yog cov leeg nqaij sib xyaw nrog qhov nruab nrab (thiab yog li ntawd sawv cev) MYH qhia tawm.7 Ntxiv mus, nws qhov yooj yim ntawm kev kuaj ua rau nws yog cov leeg nqaij zoo tshaj plaws uas tau kawm hauv tib neeg.
Yog li, kev tshawb nrhiav tsis muaj kev ntxub ntxaug ntawm cov leeg nqaij fiber ntau ntau yam siv cov cuab yeej "omics" muaj zog yog qhov tseem ceeb tab sis kuj nyuaj, ib feem vim yog qhov xwm txheej ntawm cov leeg nqaij fiber ntau. Txawm li cas los xij, transcriptomics8,9 thiab proteomics10 thev naus laus zis tau dhau los ua kev hloov pauv hauv kev nkag siab hauv xyoo tas los no vim muaj ntau yam kev nce qib hauv kev siv tshuab, tso cai rau kev tshuaj xyuas cov leeg nqaij ntawm ib qho kev daws teeb meem. Yog li ntawd, kev nce qib tseem ceeb tau ua tiav hauv kev piav qhia txog kev sib txawv ntawm ib qho fiber thiab lawv cov lus teb rau atrophic stimuli thiab kev laus11,12,13,14,15,16,17,18. Qhov tseem ceeb, cov kev nce qib hauv kev siv tshuab no muaj cov ntawv thov hauv kev kho mob, tso cai rau kev piav qhia ntxaws ntxiv thiab meej txog kev cuam tshuam ntawm tus kab mob. Piv txwv li, pathophysiology ntawm nemaline myopathy, ib qho ntawm cov kab mob leeg uas tau txais los ntawm cov noob caj noob ces (MIM 605355 thiab MIM 161800), yog qhov nyuaj thiab tsis meej pem.19,20 Yog li ntawd, kev piav qhia zoo dua ntawm kev cuam tshuam ntawm cov leeg nqaij fiber ntau tuaj yeem ua rau muaj kev nce qib tseem ceeb hauv peb txoj kev nkag siab txog tus kab mob no.
Peb tau tsim cov txheej txheem rau kev tshuaj xyuas transcriptomic thiab proteomic ntawm cov leeg nqaij ib leeg uas tau rho tawm los ntawm cov qauv biopsy tib neeg thiab siv rau ntau txhiab tus fibers, uas tso cai rau peb tshawb xyuas qhov sib txawv ntawm cov leeg nqaij tib neeg. Hauv txoj haujlwm no, peb tau ua pov thawj lub zog ntawm transcriptomic thiab proteomic phenotyping ntawm cov leeg nqaij thiab txheeb xyuas cov metabolic, ribosomal, thiab cellular junctional proteins ua qhov tseem ceeb ntawm kev hloov pauv interfiber. Ntxiv mus, siv cov txheej txheem proteomic no, peb tau piav qhia txog qhov tseem ceeb ntawm kev kho mob ntawm nematode myopathy hauv cov leeg nqaij ib leeg, qhia txog kev hloov pauv mus rau cov fibers tsis-oxidative ywj pheej ntawm hom fiber raws li MYH.
Txhawm rau tshawb nrhiav qhov sib txawv ntawm cov leeg nqaij pob txha tib neeg, peb tau tsim ob txoj haujlwm ua haujlwm los pab kom muaj kev tshuaj xyuas transcriptome thiab proteome ntawm cov leeg nqaij pob txha ib leeg (Daim Duab 1A thiab Daim Duab Ntxiv 1A). Peb tau tsim thiab ua kom zoo dua ntau kauj ruam kev ua haujlwm, los ntawm kev khaws cov qauv thiab kev khaws cia ntawm RNA thiab cov protein kom zoo dua rau kev ua kom zoo dua rau txhua txoj hauv kev. Rau kev tshuaj xyuas transcriptome, qhov no tau ua tiav los ntawm kev ntxig cov qauv tshwj xeeb molecular barcodes ntawm kauj ruam pib ntawm kev sau rov qab, tso cai rau 96 fibers kom sib sau ua ke rau kev ua tiav downstream. Kev sib sau ua ke tob dua (± 1 lab nyeem ib fiber) piv rau cov txheej txheem ib leeg-cell ib txwm muaj ntxiv cov ntaub ntawv transcriptome. 21 Rau proteomics, peb siv cov chromatographic gradient luv luv (21 feeb) ua ke nrog DIA-PASEF cov ntaub ntawv tau txais ntawm timsTOF pawg spectrometer los ua kom zoo dua qhov tob ntawm proteome thaum tswj hwm qhov siab throughput. 22,23 Txhawm rau tshawb nrhiav qhov sib txawv ntawm cov leeg nqaij pob txha noj qab haus huv, peb tau piav qhia txog cov ntawv sau ntawm 1,050 tus kheej cov fibers los ntawm 14 tus neeg pub dawb noj qab haus huv thiab cov proteomes ntawm 1,038 cov fibers los ntawm 5 tus neeg pub dawb noj qab haus huv (Cov Lus Qhia Ntxiv 1). Hauv daim ntawv no, cov ntaub ntawv no raug xa mus rau 1,000-fiber transcriptomes thiab proteomes, feem. Peb txoj hauv kev tau pom tag nrho 27,237 cov ntawv sau thiab 2,983 cov protein hauv 1,000-fiber transcriptomic thiab proteomic kev tshuaj xyuas (Daim Duab 1A, Cov Ntaub Ntawv Ntxiv 1–2). Tom qab lim cov ntaub ntawv transcriptomic thiab proteomic rau> 1,000 cov noob pom thiab 50% tus nqi siv tau ib lub fiber, kev tshuaj xyuas bioinformatics tom qab ntawd tau ua rau 925 thiab 974 cov fibers hauv transcriptome thiab proteome, feem. Tom qab lim dej, qhov nruab nrab ntawm 4257 ± 1557 noob thiab 2015 ± 234 cov protein (nruab nrab ± SD) tau pom ib lub fiber, nrog rau kev hloov pauv ntawm tus kheej tsawg (Cov Duab Ntxiv 1B-C, Cov Ntaub Ntawv Ntxiv 3-4). Txawm li cas los xij, kev hloov pauv hauv cov ntsiab lus tau pom tseeb dua thoob plaws cov neeg koom nrog, tej zaum vim muaj qhov sib txawv ntawm RNA / protein tawm ntawm cov fibers ntawm qhov ntev sib txawv thiab thaj chaw hla. Rau feem ntau cov protein (> 2000), tus lej ntawm kev hloov pauv qis dua 20% (Daim Duab Ntxiv 1D). Ob txoj kev tso cai rau kev ntes ntau yam kev hloov pauv ntawm cov ntawv sau thiab cov protein nrog cov cim qhia siab heev tseem ceeb rau kev cog lus ntawm cov leeg (piv txwv li, ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Cov Duab Ntxiv 1E-F). Feem ntau ntawm cov yam ntxwv uas tau txheeb xyuas tau yog sib xws ntawm cov ntaub ntawv transcriptomic thiab proteomic (Daim Duab Ntxiv 1G), thiab qhov nruab nrab UMI/LFQ qhov sib zog ntawm cov yam ntxwv no tau sib raug zoo (r = 0.52) (Daim Duab Ntxiv 1H).
Cov txheej txheem ua haujlwm ntawm Transcriptomics thiab proteomics (tsim nrog BioRender.com). BD Dynamic range curves rau MYH7, MYH2, thiab MYH1, thiab cov thresholds xam rau kev faib hom fiber. E, F Kev faib tawm ntawm MYH kev qhia thoob plaws cov fibers hauv cov ntaub ntawv transcriptomics thiab proteomics. G, H Uniform Diversity Approximation thiab Projection (UMAP) plots rau transcriptomics thiab proteomics xim los ntawm MYH-raws li hom fiber. I, J Feature plots qhia MYH7, MYH2, thiab MYH1 kev qhia hauv cov ntaub ntawv transcriptomics thiab proteomics.
Thaum xub thawj peb tau teeb tsa los muab hom fiber raws li MYH rau txhua lub fiber siv txoj hauv kev zoo tshaj plaws uas siv qhov muaj zog siab thiab ntau yam ntawm MYH kev qhia tawm hauv cov ntaub ntawv omics. Cov kev tshawb fawb yav dhau los tau siv cov kev txwv tsis pub dhau los sau cov fibers ua hom 1 ntshiab, hom 2A, hom 2X, lossis sib xyaw raws li feem pua ​​​​​​ntawm kev qhia tawm ntawm ntau yam MYHs11,14,24. Peb tau siv txoj hauv kev sib txawv uas kev qhia tawm ntawm txhua lub fiber tau raug xaiv los ntawm MYHs peb siv los ntaus cov fibers: MYH7, MYH2, thiab MYH1, sib raug rau hom 1, hom 2A, thiab hom 2X fibers, raws li. Tom qab ntawd peb tau suav lej qhov qis dua ntawm txhua qhov nkhaus thiab siv nws ua qhov txwv los muab cov fibers ua qhov zoo (saum toj ntawm qhov txwv) lossis tsis zoo (hauv qab qhov txwv) rau txhua MYH (Daim Duab 1B-D). Cov ntaub ntawv no qhia tau tias MYH7 (Daim Duab 1B) thiab MYH2 (Daim Duab 1C) muaj cov qauv qhia tawm ntawm / tawm sib txawv ntawm qib RNA piv rau qib protein. Tseeb tiag, ntawm theem protein, ob peb lub fibers tsis qhia MYH7, thiab tsis muaj fiber muaj 100% MYH2 kev qhia. Tom ntej no peb siv cov kev txwv kev qhia ua ntej los muab MYH-raws li hom fiber rau txhua lub fibers hauv txhua cov ntaub ntawv. Piv txwv li, MYH7+/MYH2-/MYH1- fibers tau muab rau hom 1, thaum MYH7-/MYH2+/MYH1+ fibers tau muab rau hom sib xyaw 2A/2X (saib Cov Lus Qhia Ntxiv 2 rau cov lus piav qhia tag nrho). Los ntawm kev muab tag nrho cov fibers, peb pom muaj kev faib tawm zoo sib xws ntawm MYH-raws li hom fiber ntawm ob qho tib si RNA (Daim Duab 1E) thiab protein (Daim Duab 1F), thaum cov khoom sib piv ntawm MYH-raws li hom fiber sib txawv ntawm cov tib neeg, raws li xav tau (Daim Duab Ntxiv 2A). Feem ntau cov fibers tau muab faib ua hom 1 ntshiab (34–35%) lossis hom 2A (36–38%), txawm hais tias muaj ntau hom sib xyaw 2A/2X fibers kuj tau pom (16–19%). Qhov txawv txav yog tias cov fibers ntshiab hom 2X tsuas yog pom tau ntawm RNA xwb, tab sis tsis pom ntawm cov protein, qhia tias kev qhia tawm MYH sai sai tsawg kawg yog ib feem tswj hwm tom qab kev sau ntawv.
Peb tau lees paub peb txoj kev siv proteomics-based MYH fiber typing siv cov tshuaj antibody-based dot blotting, thiab ob txoj kev tau ua tiav 100% kev pom zoo hauv kev txheeb xyuas cov fibers ntshiab hom 1 thiab hom 2A (saib Daim Duab Ntxiv 2B). Txawm li cas los xij, txoj hauv kev raws li proteomics muaj kev nkag siab ntau dua, ua haujlwm tau zoo dua hauv kev txheeb xyuas cov fibers sib xyaw, thiab ntsuas qhov sib piv ntawm txhua tus gene MYH hauv txhua tus fiber. Cov ntaub ntawv no qhia txog kev ua haujlwm zoo ntawm kev siv txoj hauv kev omics-based uas muaj lub hom phiaj, muaj kev nkag siab zoo los piav qhia txog cov hom fibers ntawm cov leeg nqaij.
Tom qab ntawd peb siv cov ntaub ntawv sib xyaw ua ke los ntawm transcriptomics thiab proteomics los faib cov myofibers raws li lawv cov transcriptome lossis proteome tiav. Siv txoj kev sib npaug ntawm cov manifold approximation thiab projection (UMAP) los txo qhov dimensionality rau rau lub ntsiab lus tseem ceeb (Cov Duab Ntxiv 3A-B), peb tau pom qhov sib txawv ntawm myofiber hauv transcriptome (Daim Duab 1G) thiab proteome (Daim Duab 1H). Qhov tseem ceeb, myofibers tsis tau muab faib ua pawg los ntawm cov neeg koom nrog (Cov Duab Ntxiv 3C-D) lossis hnub xeem (Daim Duab Ntxiv 3E) hauv cov ntaub ntawv transcriptomics lossis proteomics, qhia tias qhov sib txawv ntawm cov leeg nqaij pob txha siab dua qhov sib txawv ntawm cov neeg koom nrog. Hauv daim duab UMAP, ob pawg sib txawv uas sawv cev rau "ceev" thiab "qeeb" myofibers tau tshwm sim (Cov Duab 1G-H). MYH7+ (qeeb) myofibers tau sib sau ua ke ntawm qhov zoo ntawm UMAP1, thaum MYH2+ thiab MYH1+ (ceev) myofibers tau sib sau ua ke ntawm qhov tsis zoo ntawm UMAP1 (Cov Duab 1I–J). Txawm li cas los xij, tsis muaj qhov sib txawv ntawm cov hom fiber ceev ceev (piv txwv li, hom 2A, hom 2X, lossis sib xyaw 2A/2X) raws li MYH kev qhia tawm, qhia tias MYH1 (Daim Duab 1I–J) lossis lwm cov cim myofiber 2X xws li ACTN3 lossis MYLK2 (Cov Duab Ntxiv 4A–B) kev qhia tawm tsis sib txawv ntawm cov hom myofiber sib txawv thaum xav txog tag nrho cov transcriptome lossis proteome. Ntxiv mus, piv nrog MYH2 thiab MYH7, ob peb cov transcripts lossis cov protein tau sib raug zoo nrog MYH1 (Cov Duab Ntxiv 4C–H), qhia tias MYH1 qhov ntau tsis qhia tag nrho cov myofiber transcriptome/proteome. Cov lus xaus zoo sib xws tau raug ncav cuag thaum soj ntsuam qhov kev sib xyaw ua ke ntawm peb lub MYH isoforms ntawm qib UMAP (Cov Duab Ntxiv 4I-J). Yog li, thaum 2X fibers tuaj yeem txheeb xyuas tau ntawm qib transcript raws li MYH quantification ib leeg, MYH1 + fibers tsis sib txawv ntawm lwm cov fibers ceev thaum xav txog tag nrho cov transcriptome lossis proteome.
Ua ib qho kev tshawb nrhiav thawj zaug ntawm qhov sib txawv ntawm cov fiber qeeb dhau ntawm MYH, peb tau soj ntsuam plaub hom protein tshwj xeeb uas tau tsim los ntawm cov fiber qeeb: TPM3, TNNT1, MYL3, thiab ATP2A22. Cov subtypes fiber qeeb tau qhia tias muaj kev sib raug zoo siab, txawm tias tsis zoo tag nrho, Pearson correlations nrog MYH7 hauv ob qho tib si transcriptomics (Daim Duab Ntxiv 5A) thiab proteomics (Daim Duab Ntxiv 5B). Kwv yees li 25% thiab 33% ntawm cov fiber qeeb tsis tau muab faib ua cov fiber qeeb ntshiab los ntawm txhua hom gene/protein hauv transcriptomics (Daim Duab Ntxiv 5C) thiab proteomics (Daim Duab Ntxiv 5D), raws li. Yog li ntawd, kev faib cov fiber qeeb raws li ntau hom gene/protein qhia txog qhov nyuaj ntxiv, txawm tias rau cov protein uas paub tias yog hom fiber tshwj xeeb. Qhov no qhia tau hais tias kev faib cov fiber raws li isoforms ntawm ib tsev neeg gene/protein yuav tsis qhia txog qhov sib txawv ntawm cov leeg nqaij pob txha.
Txhawm rau tshawb nrhiav ntxiv txog qhov sib txawv ntawm cov leeg nqaij ntawm tib neeg ntawm qhov ntsuas ntawm tag nrho cov qauv omics, peb tau ua qhov kev txo qis ntawm cov ntaub ntawv siv kev tshuaj xyuas cov khoom tseem ceeb (PCA) (Daim Duab 2A). Zoo ib yam li UMAP plots, tsis yog tus neeg koom nrog lossis hnub sim cuam tshuam rau kev sib sau ua ke ntawm cov fiber ntawm PCA theem (Cov Duab Ntxiv 6A-C). Hauv ob qho tib si datasets, MYH-based fiber hom tau piav qhia los ntawm PC2, uas qhia txog ib pawg ntawm cov fibers qeeb-twitch hom 1 thiab pawg thib ob uas muaj cov fibers ceev-twitch hom 2A, hom 2X, thiab sib xyaw 2A/2X (Daim Duab 2A). Hauv ob qho tib si datasets, ob pawg no tau txuas nrog los ntawm ib tug me me ntawm cov fibers sib xyaw hom 1/2A. Raws li xav tau, kev tshuaj xyuas overrepresentation ntawm cov tsav tsheb PC tseem ceeb tau lees paub tias PC2 tau tsav los ntawm cov cim contractile thiab metabolic (Daim Duab 2B thiab Cov Duab Ntxiv 6D-E, Cov Ntaub Ntawv Ntxiv 5-6). Zuag qhia tag nrho, hom fiber MYH-raws li pom tias txaus los piav qhia txog kev hloov pauv tas mus li raws PC2, tshwj tsis yog cov fibers hu ua 2X uas tau faib thoob plaws hauv cov ntawv sau hauv pawg ceev ceev.
A. Cov phiaj xwm tseem ceeb ntawm kev tshuaj xyuas cov khoom tseem ceeb (PCA) ntawm cov ntaub ntawv sau ua ke thiab cov ntaub ntawv proteome uas muaj xim raws li hom fiber raws li MYH. B. Kev tshuaj xyuas kev nplua nuj ntawm cov tsav tsheb sau ua ke thiab cov protein hauv PC2 thiab PC1. Kev tshuaj xyuas cov ntaub ntawv tau ua tiav siv pob clusterProfiler thiab Benjamini-Hochberg kho p-values. C, D. Cov phiaj xwm PCA muaj xim raws li cov lus sib txuas ntawm cov noob caj noob ces (GO) hauv cov lus sau ua ke thiab cov lus sau ua ke GO hauv cov proteome. Cov xub sawv cev rau cov tsav tsheb sau ua ke thiab cov protein thiab lawv cov lus qhia. E, F. Kev kwv yees sib xws thiab kev kwv yees (UMAP) muaj cov phiaj xwm ntawm cov yam ntxwv tseem ceeb hauv kev kho mob uas qhia txog kev hloov pauv ntawm cov qauv fiber qeeb / ceev. G, H. Kev sib raug zoo ntawm PC2 thiab PC1 tsav tsheb hauv cov ntawv sau ua ke thiab cov proteomes.
Tsis tau xav txog, MYH-raws li myofiber hom piav qhia tsuas yog qib thib ob siab tshaj plaws ntawm kev hloov pauv (PC2), qhia tias lwm yam kab mob tsis muaj feem cuam tshuam rau MYH-raws li myofiber hom (PC1) ua lub luag haujlwm tseem ceeb hauv kev tswj hwm cov leeg nqaij fiber heterogeneity. Kev tshuaj xyuas overrepresentation ntawm cov tsav tsheb saum toj kawg nkaus hauv PC1 tau qhia tias kev hloov pauv hauv PC1 feem ntau yog txiav txim siab los ntawm cell-cell adhesion thiab ribosome cov ntsiab lus hauv transcriptome, thiab costameres thiab ribosomal proteins hauv proteome (Daim Duab 2B thiab Cov Duab Ntxiv 6D-E, Cov Ntaub Ntawv Ntxiv 7). Hauv cov leeg nqaij pob txha, costameres txuas Z-disc rau sarcolemma thiab koom nrog kev xa mus rau lub zog thiab kev taw qhia. 25 Cov phiaj xwm PCA sau tseg siv cell-cell adhesion (transcriptome, Daim Duab 2C) thiab costamere (proteome, Daim Duab 2D) nta tau qhia txog kev hloov pauv sab laug muaj zog hauv PC1, qhia tias cov yam ntxwv no tau nplua nuj hauv qee cov fibers.
Kev tshuaj xyuas kom ntxaws ntxiv ntawm myofiber clustering ntawm UMAP theem tau qhia tias feem ntau cov yam ntxwv tau qhia txog myofiber hom-ywj pheej MYH-raws li kev qhia tawm gradient es tsis yog myofiber subcluster-specific. Qhov kev sib txuas no tau pom rau ntau lub noob caj noob ces cuam tshuam nrog cov mob pathological (Daim Duab 2E), xws li CHCHD10 (mob neuromuscular), SLIT3 (mob leeg atrophy), CTDNEP1 (mob leeg). Qhov kev sib txuas no kuj tau pom thoob plaws hauv proteome, suav nrog cov protein cuam tshuam nrog cov kab mob neurological (UGDH), insulin signaling (PHIP), thiab transcription (HIST1H2AB) (Daim Duab 2F). Ua ke, cov ntaub ntawv no qhia txog kev sib txuas hauv cov fiber hom-ywj pheej qeeb/ceev twitch heterogeneity thoob plaws ntau yam myofibers.
Qhov nthuav yog, cov noob tsav tsheb hauv PC2 tau qhia txog kev sib raug zoo ntawm transcriptome-proteome (r = 0.663) (Daim Duab 2G), qhia tias cov hom fiber qeeb thiab ceev ceev, thiab tshwj xeeb tshaj yog cov khoom contractile thiab metabolic ntawm cov leeg nqaij pob txha, yog tswj hwm transcriptional. Txawm li cas los xij, cov noob tsav tsheb hauv PC1 tsis qhia txog kev sib raug zoo ntawm transcriptome-proteome (r = -0.027) (Daim Duab 2H), qhia tias kev hloov pauv tsis cuam tshuam rau cov hom fiber qeeb / ceev ceev feem ntau yog tswj hwm tom qab transcriptional. Vim tias kev hloov pauv hauv PC1 tau piav qhia feem ntau los ntawm cov lus hais txog ribosomal gene ontology, thiab muab tias ribosomes ua lub luag haujlwm tseem ceeb thiab tshwj xeeb hauv lub cell los ntawm kev koom tes thiab cuam tshuam rau kev txhais lus protein,31 tom ntej no peb tau teeb tsa los tshawb xyuas qhov kev hloov pauv ribosomal tsis tau xav txog no.
Peb xub pleev xim rau daim phiaj tshuaj xyuas cov khoom tseem ceeb ntawm proteomics raws li qhov muaj ntau ntawm cov protein hauv GOCC lo lus "cytoplasmic ribosome" (Daim Duab 3A). Txawm hais tias lo lus no tau nplua nuj rau sab zoo ntawm PC1, ua rau muaj qhov sib txawv me me, cov protein ribosomal tsav kev faib ua ob qho kev taw qhia ntawm PC1 (Daim Duab 3A). Cov protein ribosomal nplua nuj rau sab tsis zoo ntawm PC1 suav nrog RPL18, RPS18, thiab RPS13 (Daim Duab 3B), thaum RPL31, RPL35, thiab RPL38 (Daim Duab 3C) yog cov tsav tsheb tseem ceeb rau sab zoo ntawm PC1. Qhov nthuav yog, RPL38 thiab RPS13 tau qhia ntau heev hauv cov leeg nqaij pob txha piv rau lwm cov nqaij (Daim Duab Ntxiv 7A). Cov cim tshwj xeeb ribosomal no hauv PC1 tsis tau pom hauv cov ntawv sau (Daim Duab Ntxiv 7B), qhia txog kev tswj hwm tom qab sau ntawv.
A. Daim phiaj xwm tsom xam cov khoom tseem ceeb (PCA) uas muaj xim raws li cov lus qhia ntawm cytoplasmic ribosomal gene ontology (GO) thoob plaws hauv proteome. Cov xub qhia txog kev coj ua ntawm kev hloov pauv ntawm cov protein hauv daim phiaj xwm PCA. Qhov ntev ntawm kab sib raug rau qhov qhab nia tseem ceeb rau ib qho protein uas tau muab. B, C. Cov phiaj xwm PCA rau RPS13 thiab RPL38. D. Kev tsom xam cov pawg neeg tsis muaj kev saib xyuas ntawm cov protein cytoplasmic ribosomal. E. Tus qauv ntawm 80S ribosome (PDB: 4V6X) qhia txog cov protein ribosomal nrog ntau yam sib txawv hauv cov leeg nqaij pob txha. F. Cov protein ribosomal nrog cov stoichiometry sib txawv nyob ze ntawm mRNA tawm channel.
Cov tswv yim ntawm ribosomal heterogeneity thiab kev tshwj xeeb tau raug thov ua ntej lawm, qhov twg qhov muaj cov ribosome subpopulations sib txawv (ribosomal heterogeneity) tuaj yeem cuam tshuam ncaj qha rau kev txhais lus protein hauv cov ntaub so ntswg sib txawv 32 thiab cell 33 los ntawm kev txhais lus xaiv ntawm cov pas dej mRNA transcript tshwj xeeb 34 (ribosome specialization). Txhawm rau txheeb xyuas cov subpopulations ntawm ribosomal proteins co-expressed hauv cov leeg nqaij pob txha, peb tau ua qhov kev tshuaj xyuas hierarchical clustering tsis muaj kev saib xyuas ntawm ribosomal proteins hauv proteome (Daim Duab 3D, Supplementary Data Set 8). Raws li xav tau, ribosomal proteins tsis tau cluster los ntawm hom fiber raws li MYH. Txawm li cas los xij, peb tau txheeb xyuas peb pawg sib txawv ntawm ribosomal proteins; thawj pawg (ribosomal_cluster_1) yog coregulated nrog RPL38 thiab yog li ntawd muaj kev nce qib hauv cov fibers nrog qhov zoo PC1 profile. Pawg thib ob (ribosomal_cluster_2) yog coregulated nrog RPS13 thiab yog siab hauv cov fibers nrog qhov tsis zoo PC1 profile. Pawg thib peb (ribosomal_cluster_3) tsis qhia txog kev sib koom ua ke ntawm cov leeg nqaij pob txha thiab tuaj yeem suav hais tias yog "core" skeletal muscle ribosomal protein. Ob pawg ribosomal 1 thiab 2 muaj cov protein ribosomal uas tau pom tias tswj hwm lwm txoj kev txhais lus (piv txwv li, RPL10A, RPL38, RPS19, thiab RPS25) thiab cuam tshuam rau kev loj hlob (piv txwv li, RPL10A, RPL38).34,35,36,37,38 Sib xws nrog cov txiaj ntsig PCA, qhov kev sawv cev heterogeneous pom ntawm cov protein ribosomal no thoob plaws cov fibers kuj qhia txog kev sib txuas (Daim Duab Ntxiv 7C).
Txhawm rau pom qhov chaw ntawm cov protein heterogeneous ribosomal hauv ribosome, peb tau siv cov qauv qauv ntawm tib neeg 80S ribosome (Protein Data Bank: 4V6X) (Daim Duab 3E). Tom qab cais cov protein ribosomal uas koom nrog cov pawg ribosomal sib txawv, lawv qhov chaw tsis sib haum xeeb, qhia tias peb txoj hauv kev ua tsis tau muab kev nplua nuj rau qee thaj chaw / feem ntawm ribosome. Txawm li cas los xij, qhov nthuav yog, qhov feem pua ​​​​​​ntawm cov protein subunit loj hauv pawg 2 qis dua li hauv pawg 1 thiab 3 (Supplementary Daim Duab 7D). Peb tau pom tias cov protein nrog kev hloov pauv stoichiometry hauv cov leeg nqaij pob txha tau feem ntau nyob rau ntawm qhov chaw ribosome (Daim Duab 3E), sib xws nrog lawv lub peev xwm los cuam tshuam nrog cov ntsiab lus nkag mus rau sab hauv ribosome (IRES) hauv cov pej xeem mRNA sib txawv, yog li sib koom ua ke kev txhais lus xaiv. 40, 41 Ntxiv mus, ntau cov protein uas muaj kev hloov pauv stoichiometry hauv cov leeg nqaij pob txha tau nyob ze rau thaj chaw ua haujlwm xws li mRNA tawm qhov av (Daim Duab 3F), uas xaiv tswj kev txhais lus elongation thiab ntes cov peptides tshwj xeeb. 42 Hauv kev xaus, peb cov ntaub ntawv qhia tias stoichiometry ntawm cov leeg nqaij pob txha ribosomal proteins qhia txog heterogeneity, ua rau muaj qhov sib txawv ntawm cov leeg nqaij pob txha.
Tom ntej no peb tau teeb tsa los txheeb xyuas cov cim qhia fiber ceev thiab qeeb thiab tshawb nrhiav cov txheej txheem ntawm lawv txoj kev tswj hwm transcriptional. Piv rau cov pawg fiber ceev thiab qeeb uas tau txhais los ntawm UMAP hauv ob daim ntawv teev cov ntaub ntawv (Cov Duab 1G-H thiab 4A-B), kev tshuaj xyuas transcriptomic thiab proteomic tau txheeb xyuas 1366 thiab 804 cov yam ntxwv sib txawv, raws li (Cov Duab 4A-B, Cov Ntaub Ntawv Ntxiv 9-12). Peb tau pom qhov sib txawv ntawm cov cim qhia cuam tshuam nrog sarcomeres (piv txwv li, tropomyosin thiab troponin), kev sib txuas lus excitation-contraction (SERCA isoforms), thiab kev ua haujlwm ntawm lub zog (piv txwv li, ALDOA thiab CKB). Ntxiv mus, cov ntawv sau thiab cov protein tswj hwm protein ubiquitination tau qhia sib txawv hauv cov fiber ceev thiab qeeb (piv txwv li, USP54, SH3RF2, USP28, thiab USP48) (Cov Duab 4A-B). Ntxiv mus, cov noob caj noob ces protein microbial RP11-451G4.2 (DWORF), uas tau pom tias tau qhia sib txawv thoob plaws cov leeg nqaij yaj hom43 thiab txhim kho SERCA kev ua haujlwm hauv cov leeg nqaij plawv44, tau nce siab heev hauv cov leeg nqaij pob txha qeeb (Daim Duab 4A). Ib yam li ntawd, ntawm qib fiber ib leeg, qhov sib txawv tseem ceeb tau pom hauv cov cim paub xws li cov metabolism-related lactate dehydrogenase isoforms (LDHA thiab LDHB, Daim Duab 4C thiab Daim Duab Ntxiv 8A)45,46 nrog rau cov cim tshwj xeeb fiber-hom tsis paub yav dhau los (xws li IRX3, USP54, USP28, thiab DPYSL3) (Daim Duab 4C). Muaj qhov sib tshooj tseem ceeb ntawm cov yam ntxwv sib txawv ntawm cov ntaub ntawv transcriptomic thiab proteomic (Daim Duab Ntxiv 8B), nrog rau kev hloov pauv ntawm qhov sib piv uas tsav los ntawm qhov sib txawv ntawm cov yam ntxwv sarcomere (Daim Duab Ntxiv 8C). Qhov tseem ceeb, qee cov cim kos npe (piv txwv li USP28, USP48, GOLGA4, AKAP13) tau qhia txog kev tswj hwm tom qab kev sau ntawv muaj zog tsuas yog ntawm qib proteomic thiab muaj cov qauv qhia txog cov fiber qeeb / ceev ceev (Daim Duab Ntxiv 8C).
Cov phiaj xwm roob hluav taws A thiab B piv rau cov pawg qeeb thiab ceev uas tau txheeb xyuas los ntawm cov phiaj xwm sib xws manifold approximation thiab projection (UMAP) hauv Cov Duab 1G-H. Cov xim sawv cev rau cov ntawv sau lossis cov protein uas sib txawv ntawm FDR <0.05, thiab cov xim tsaus dua sawv cev rau cov ntawv sau lossis cov protein uas sib txawv ntawm kev hloov cav> 1. Kev tshuaj xyuas ob txoj kev suav lej tau ua tiav siv DESeq2 Wald xeem nrog Benjamini-Hochberg kho p tus nqi (transcriptomics) lossis Limma linear qauv txoj kev nrog empirical Bayesian kev tshuaj xyuas ua raws li Benjamini-Hochberg kev kho rau ntau qhov kev sib piv (proteomics). C Cov phiaj xwm kos npe ntawm cov noob caj noob ces lossis cov protein xaiv sib txawv ntawm cov fibers qeeb thiab ceev. D Kev tshuaj xyuas kev txhim kho ntawm cov ntawv sau thiab cov protein uas sib txawv. Cov nqi sib tshooj tau nplua nuj hauv ob qho tib si datasets, cov nqi transcriptome tsuas yog nplua nuj hauv transcriptome, thiab cov nqi proteome tsuas yog nplua nuj hauv proteome. Kev tshuaj xyuas kev suav lej tau ua tiav siv pob clusterProfiler nrog Benjamini-Hochberg kho p-tus nqi. E. Cov yam ntxwv sau ntawv tshwj xeeb ntawm hom fiber uas SCENIC tau txheeb xyuas raws li cov qhab nia tshwj xeeb ntawm SCENIC thiab kev qhia tawm mRNA sib txawv ntawm cov hom fiber. F. Kev txheeb xyuas cov yam ntxwv sau ntawv xaiv uas tau qhia sib txawv ntawm cov fiber qeeb thiab cov fiber ceev.
Tom qab ntawd peb tau ua qhov kev tshuaj xyuas ntau dhau ntawm cov noob caj noob ces thiab cov protein uas sawv cev sib txawv (Daim Duab 4D, Cov Ntaub Ntawv Ntxiv 13). Txoj kev txhim kho rau cov yam ntxwv uas sib txawv ntawm ob daim ntawv teev lus qhia tau qhia txog qhov sib txawv uas xav tau, xws li cov txheej txheem fatty acid β-oxidation thiab ketone metabolism (cov fibers qeeb), myofilament / cov leeg nqaij contraction (cov fibers ceev thiab qeeb, feem), thiab cov txheej txheem carbohydrate catabolic (cov fibers ceev). Serine / threonine protein phosphatase kev ua ub no kuj tau nce siab hauv cov fibers ceev, tsav los ntawm cov yam ntxwv xws li cov kev tswj hwm thiab catalytic phosphatase subunits (PPP3CB, PPP1R3D, thiab PPP1R3A), uas paub tias tswj hwm glycogen metabolism (47) (Cov Duab Ntxiv 8D-E). Lwm txoj kev uas muaj cov fibers ceev ceev suav nrog kev ua cov protein (P-) lub cev (YTHDF3, TRIM21, LSM2) hauv cov proteome (Daim Duab Ntxiv 8F), tej zaum yuav koom nrog kev tswj hwm tom qab kev sau ntawv (48), thiab kev ua haujlwm ntawm cov ntawv sau (SREBF1, RXRG, RORA) hauv cov ntawv sau (Daim Duab Ntxiv 8G). Cov fibers qeeb qeeb tau nplua nuj hauv kev ua haujlwm oxidoreductase (BDH1, DCXR, TXN2) (Daim Duab Ntxiv 8H), amide binding (CPTP, PFDN2, CRYAB) (Daim Duab Ntxiv 8I), extracellular matrix (CTSD, ADAMTSL4, LAMC1) (Daim Duab Ntxiv 8J), thiab kev ua haujlwm ntawm receptor-ligand (FNDC5, SPX, NENF) (Daim Duab Ntxiv 8K).
Yuav kom nkag siab ntxiv txog kev tswj hwm kev sau ntawv uas yog lub hauv paus ntawm cov yam ntxwv ntawm cov leeg nqaij qeeb/ceev, peb tau ua qhov kev tshuaj xyuas cov yam ntxwv ntawm kev sau ntawv siv SCENIC49 (Supplementary Data Set 14). Muaj ntau yam kev sau ntawv tau nplua nuj ntawm cov leeg nqaij ceev thiab qeeb (Daim Duab 4E). Qhov no suav nrog cov yam ntxwv ntawm kev sau ntawv xws li MAFA, uas yav dhau los tau txuas nrog kev txhim kho cov leeg nqaij ceev,50 nrog rau ntau yam kev sau ntawv uas tsis tau cuam tshuam nrog cov kev pab cuam gene tshwj xeeb ntawm cov leeg nqaij. Ntawm cov no, PITX1, EGR1, thiab MYF6 yog cov yam ntxwv ntawm kev sau ntawv uas nplua nuj tshaj plaws hauv cov leeg nqaij ceev (Daim Duab 4E). Qhov sib txawv, ZSCAN30 thiab EPAS1 (tseem hu ua HIF2A) yog cov yam ntxwv ntawm kev sau ntawv uas nplua nuj tshaj plaws hauv cov leeg nqaij qeeb (Daim Duab 4E). Sib xws nrog qhov no, MAFA tau qhia tawm ntawm cov qib siab dua hauv thaj tsam UMAP uas sib raug rau cov leeg nqaij ceev, thaum EPAS1 muaj tus qauv qhia tawm sib txawv (Daim Duab 4F).
Ntxiv rau cov noob caj noob ces uas paub tias yog protein-coding, muaj ntau cov RNA biotypes uas tsis yog coding uas yuav koom nrog kev tswj hwm tib neeg txoj kev loj hlob thiab kab mob. 51, 52 Hauv cov ntaub ntawv transcriptome, ntau cov RNAs uas tsis yog coding qhia txog hom fiber tshwj xeeb (Daim Duab 5A thiab Cov Ntaub Ntawv Ntxiv 15), suav nrog LINC01405, uas yog qhov tshwj xeeb heev rau cov fibers qeeb thiab tau tshaj tawm tias tau txo qis hauv cov leeg nqaij los ntawm cov neeg mob uas muaj mitochondrial myopathy. 53 Hauv kev sib piv, RP11-255P5.3, sib raug rau lnc-ERCC5-5 gene (https://lncipedia.org/db/transcript/lnc-ERCC5-5:2) 54, qhia txog hom fiber ceev ceev. Ob qho tib si LINC01405 (https://tinyurl.com/x5k9wj3h) thiab RP11-255P5.3 (https://tinyurl.com/29jmzder) qhia txog cov leeg nqaij tshwj xeeb (Cov Duab Ntxiv 9A-B) thiab tsis muaj cov noob caj noob ces contractile hauv lawv thaj tsam 1 Mb genomic, qhia tias lawv ua lub luag haujlwm tshwj xeeb hauv kev tswj cov hom fiber ntau dua li kev tswj cov noob caj noob ces contractile ze. Cov qauv qhia txog hom fiber qeeb/ceev ntawm LINC01405 thiab RP11-255P5.3, raws li, tau lees paub siv RNAscope (Cov Duab 5B-C).
A. Cov ntawv sau RNA uas tsis yog coding raug tswj hwm zoo hauv cov leeg nqaij qeeb thiab ceev ceev. B. Cov duab sawv cev RNAscope qhia txog qhov tshwj xeeb ntawm hom fiber qeeb thiab ceev ceev ntawm LINC01405 thiab RP11-255P5.3, raws li. Scale bar = 50 μm. C. Kev ntsuas ntawm cov myofiber hom tshwj xeeb RNA uas tsis yog coding raws li txiav txim siab los ntawm RNAscope (n = 3 biopsies los ntawm cov tib neeg ywj pheej, piv cov leeg nqaij ceev thiab qeeb hauv txhua tus neeg). Kev tshuaj xyuas cov ntaub ntawv tau ua tiav siv ob-tailed Student's t-test. Cov phiaj xwm thawv qhia qhov nruab nrab thiab thawj thiab thib peb quartiles, nrog whiskers taw qhia rau qhov tsawg kawg nkaus thiab siab tshaj plaws. D. De novo microbial protein identification workflow (tsim nrog BioRender.com). E. Cov protein microbial LINC01405_ORF408:17441:17358 yog qhia tshwj xeeb hauv cov leeg nqaij pob txha qeeb (n = 5 biopsies los ntawm cov neeg koom nrog ywj pheej, piv cov leeg nqaij ceev thiab qeeb hauv txhua tus neeg koom nrog). Kev tshuaj xyuas lej tau ua tiav siv txoj kev qauv Limm linear ua ke nrog txoj hauv kev Bayesian empirical, ua raws li txoj kev Benjamini-Hochberg rau ntau qhov kev sib piv nrog kev hloov kho p-value. Cov phiaj xwm thawv qhia cov median, thawj thiab thib peb quartiles, nrog cov whiskers taw rau qhov siab tshaj plaws / tsawg kawg nkaus tus nqi.
Tsis ntev los no, cov kev tshawb fawb tau qhia tias ntau cov ntawv sau tsis-coding encodes cov protein microbial transcribed, qee qhov uas tswj cov leeg nqaij ua haujlwm. 44, 55 Txhawm rau txheeb xyuas cov protein microbial nrog cov fiber hom tshwj xeeb, peb tau tshawb nrhiav peb cov ntaub ntawv 1000 fiber proteome siv cov ntaub ntawv FASTA kev cai uas muaj cov kab ke ntawm cov ntawv sau tsis-coding (n = 305) pom nyob rau hauv 1000 fiber transcriptome dataset (Daim Duab 5D). Peb tau txheeb xyuas 197 microbial proteins los ntawm 22 cov ntawv sau sib txawv, 71 ntawm cov uas tau tswj hwm sib txawv ntawm cov leeg nqaij qeeb thiab ceev (Daim Duab Ntxiv 9C thiab Cov Ntaub Ntawv Ntxiv 16). Rau LINC01405, peb cov khoom protein microbial tau txheeb xyuas, ib qho uas qhia txog kev tshwj xeeb qeeb qeeb rau nws cov ntawv sau (Daim Duab 5E thiab Daim Duab Ntxiv 9D). Yog li, peb tau txheeb xyuas LINC01405 ua ib lub noob encoding ib qho protein microbial tshwj xeeb rau cov leeg nqaij qeeb qeeb.
Peb tau tsim ib txoj haujlwm ua tiav rau kev piav qhia txog cov protein loj ntawm cov leeg nqaij thiab txheeb xyuas cov neeg tswj hwm ntawm cov fiber heterogeneity hauv cov xeev noj qab haus huv. Peb tau siv txoj haujlwm no los nkag siab tias nemaline myopathies cuam tshuam li cas rau cov leeg nqaij pob txha heterogeneity. Nemaline myopathies yog cov kab mob leeg uas tau txais los ntawm leej twg uas ua rau cov leeg tsis muaj zog thiab, hauv cov menyuam yaus uas raug kev txom nyem, muaj ntau yam teeb meem suav nrog kev ua pa nyuaj, scoliosis, thiab kev txav mus los ntawm ceg tsawg. 19,20 Feem ntau, hauv nemaline myopathies, cov kab mob sib txawv hauv cov noob xws li actin alpha 1 (ACTA1) ua rau muaj kev sib xyaw ua ke ntawm cov leeg nqaij qeeb qeeb, txawm hais tias qhov cuam tshuam no yog heterogeneous. Ib qho kev zam tshwj xeeb yog troponin T1 nemaline myopathy (TNNT1), uas muaj kev sib xyaw ua ke ntawm cov fibers ceev ceev. Yog li, kev nkag siab zoo dua ntawm qhov sib txawv hauv qab cov leeg nqaij pob txha dysregulation pom hauv nemaline myopathies yuav pab daws qhov kev sib raug zoo ntawm cov kab mob no thiab hom myofiber.
Piv nrog cov neeg tswj kev noj qab haus huv (n = 3 ib pawg), myofibers cais los ntawm cov neeg mob nemaline myopathy uas muaj kev hloov pauv hauv ACTA1 thiab TNNT1 noob qhia tau tias muaj myofiber atrophy lossis dystrophy (Daim Duab 6A, Cov Lus Qhia Ntxiv 3). Qhov no tau nthuav tawm cov teeb meem kev siv tshuab tseem ceeb rau kev tshuaj xyuas proteomic vim muaj cov khoom siv tsawg. Txawm li cas los xij, peb tau pom 2485 cov protein hauv 272 myofibers pob txha. Tom qab lim rau tsawg kawg 1000 cov protein uas tau ntsuas ib lub fiber, 250 cov fiber tau raug rau kev tshuaj xyuas bioinformatics tom qab. Tom qab lim, qhov nruab nrab ntawm 1573 ± 359 cov protein ib lub fiber tau ntsuas (Daim Duab Ntxiv 10A, Cov Ntaub Ntawv Ntxiv 17–18). Qhov tseem ceeb, txawm tias muaj kev txo qis ntawm qhov loj me ntawm cov fiber, qhov tob ntawm cov proteome ntawm cov qauv neeg mob nemaline myopathy tsuas yog txo qis me ntsis. Ntxiv mus, kev ua cov ntaub ntawv no siv peb cov ntaub ntawv FASTA (suav nrog cov ntawv sau tsis yog coding) tau tso cai rau peb txheeb xyuas tsib cov protein microbial hauv cov myofibers pob txha los ntawm cov neeg mob nemaline myopathy (Cov Ntaub Ntawv Ntxiv 19). Qhov ntau ntawm cov proteome tau dav dua, thiab tag nrho cov protein hauv pawg tswj hwm tau sib raug zoo nrog cov txiaj ntsig ntawm kev tshuaj xyuas proteome 1000-fiber yav dhau los (Daim Duab Ntxiv 10B-C).
A. Cov duab me me uas qhia txog cov fiber atrophy lossis dystrophy thiab qhov muaj ntau hom fiber sib txawv raws li MYH hauv ACTA1 thiab TNNT1 nemaline myopathies (NM). Scale bar = 100 μm. Txhawm rau kom ntseeg tau tias muaj kev rov ua dua ntawm kev pleev xim rau cov neeg mob ACTA1 thiab TNNT1, peb daim biopsies ntawm tus neeg mob tau pleev xim ob mus rau peb zaug (plaub ntu ib rooj plaub) ua ntej xaiv cov duab sawv cev. B. Cov feem pua ​​ntawm cov fiber hauv cov neeg koom nrog raws li MYH. C. Kev tshuaj xyuas cov khoom tseem ceeb (PCA) daim phiaj ntawm cov leeg nqaij pob txha hauv cov neeg mob uas muaj nemaline myopathies thiab cov neeg tswj. D. Cov leeg nqaij pob txha los ntawm cov neeg mob uas muaj nemaline myopathies thiab cov neeg tswj tau tso rau ntawm daim phiaj PCA txiav txim siab los ntawm 1000 fibers uas tau tshuaj xyuas hauv Daim Duab 2. Piv txwv li, cov phiaj xwm volcano piv qhov sib txawv ntawm cov neeg koom nrog ACTA1 thiab TNNT1 nemaline myopathies thiab cov neeg tswj, thiab ntawm cov neeg koom nrog ACTA1 thiab TNNT1 nemaline myopathies. Cov voj voog xim qhia txog cov protein uas sib txawv ntawm π <0.05, thiab cov dots tsaus qhia txog cov protein uas sib txawv ntawm FDR <0.05. Kev tshuaj xyuas cov ntaub ntawv tau ua tiav siv txoj kev Limma linear model thiab cov txheej txheem empirical Bayesian, ua raws li kev hloov kho p-value rau ntau qhov kev sib piv siv txoj kev Benjamini-Hochberg. H. Kev tshuaj xyuas kev nplua nuj ntawm cov protein uas qhia tawm sib txawv thoob plaws tag nrho cov proteome thiab hauv hom 1 thiab 2A fibers. Kev tshuaj xyuas cov ntaub ntawv tau ua tiav siv pob clusterProfiler thiab Benjamini-Hochberg kho p-values. I, J. Kev tshuaj xyuas cov khoom tseem ceeb (PCA) plots xim los ntawm extracellular matrix thiab mitochondrial gene ontology (GO) cov lus.
Vim tias nemaline myopathies tuaj yeem cuam tshuam rau qhov sib piv ntawm MYH-expressing myofiber hom hauv cov leeg pob txha,19,20 peb thawj zaug tshuaj xyuas MYH-expressing myofiber hom hauv cov neeg mob uas muaj nemaline myopathies thiab cov neeg tswj. Peb tau txiav txim siab hom myofiber siv txoj kev tsis ncaj ncees uas tau piav qhia ua ntej rau 1000 myofiber assay (Supplementary Figs. 10D-E) thiab dua tsis tau txheeb xyuas 2X myofibers ntshiab (Daim Duab 6B). Peb tau pom qhov cuam tshuam heterogeneous ntawm nemaline myopathies ntawm hom myofiber, vim tias ob tus neeg mob uas muaj ACTA1 mutations muaj qhov sib piv ntawm hom 1 myofibers ntau dua, thaum ob tus neeg mob uas muaj TNNT1 nemaline myopathy muaj qhov sib piv ntawm hom 1 myofibers tsawg dua (Daim Duab 6B). Tseeb tiag, qhov kev qhia tawm ntawm MYH2 thiab cov kab mob troponin ceev ceev (TNNC2, TNNI2, thiab TNNT3) tau txo qis hauv ACTA1-nemaline myopathies, thaum MYH7 kev qhia tawm tau txo qis hauv TNNT1-nemaline myopathies (Daim Duab Ntxiv 11A). Qhov no yog sib xws nrog cov ntawv ceeb toom yav dhau los ntawm kev hloov pauv hom myofiber hauv nemaline myopathies.19,20 Peb tau lees paub cov txiaj ntsig no los ntawm immunohistochemistry thiab pom tias cov neeg mob uas muaj ACTA1-nemaline myopathy muaj feem ntau ntawm hom 1 myofibers, thaum cov neeg mob uas muaj TNNT1-nemaline myopathy muaj tus qauv sib txawv (Daim Duab 6A).
Ntawm theem proteome ib leeg-fiber, cov leeg nqaij pob txha los ntawm ACTA1 thiab TNNT1 nemaline myopathy cov neeg mob sib sau ua ke nrog feem ntau ntawm cov fibers tswj, nrog rau TNNT1 nemaline myopathy fibers feem ntau yog qhov cuam tshuam loj tshaj plaws (Daim Duab 6C). Qhov no tau pom tseeb tshwj xeeb thaum kos duab cov ntsiab lus tseem ceeb (PCA) cov phiaj xwm ntawm pseudo-inflated fibers rau txhua tus neeg mob, nrog rau TNNT1 nemaline myopathy cov neeg mob 2 thiab 3 tshwm sim nyob deb tshaj plaws ntawm cov qauv tswj (Daim Duab Ntxiv 11B, Cov Ntaub Ntawv Ntxiv 20). Txhawm rau kom nkag siab zoo dua li cas cov fibers los ntawm cov neeg mob myopathy piv rau cov fibers noj qab haus huv, peb siv cov ntaub ntawv ntxaws ntxaws tau los ntawm kev tshuaj xyuas proteomic ntawm 1,000 fibers los ntawm cov neeg koom nrog laus noj qab haus huv. Peb tau kwv yees cov fibers los ntawm cov ntaub ntawv myopathy (ACTA1 thiab TNNT1 nemaline myopathy cov neeg mob thiab cov neeg tswj) rau ntawm PCA phiaj xwm tau los ntawm kev tshuaj xyuas proteomic 1000-fiber (Daim Duab 6D). Qhov kev faib tawm ntawm MYH fiber hom raws PC2 hauv cov fiber tswj tau zoo ib yam li qhov kev faib tawm fiber tau los ntawm 1000-fiber proteomic tsom xam. Txawm li cas los xij, feem ntau cov fibers hauv cov neeg mob nemaline myopathy tau hloov mus rau PC2, sib tshooj nrog cov fibers noj qab haus huv ceev-twitch, tsis hais lawv cov hom MYH fiber ib txwm muaj. Yog li, txawm hais tias cov neeg mob uas muaj ACTA1 nemaline myopathy tau qhia txog kev hloov pauv mus rau hom 1 fibers thaum ntsuas siv cov txheej txheem MYH, ob qho tib si ACTA1 nemaline myopathy thiab TNNT1 nemaline myopathy tau hloov cov leeg nqaij fiber proteome mus rau cov fibers ceev-twitch.
Tom qab ntawd peb tau piv ncaj qha rau txhua pawg neeg mob nrog cov neeg tswj kev noj qab haus huv thiab tau txheeb xyuas 256 thiab 552 cov protein uas qhia sib txawv hauv ACTA1 thiab TNNT1 nemaline myopathies, raws li (Daim Duab 6E-G thiab Daim Duab Ntxiv 11C, Cov Ntaub Ntawv Ntxiv 21). Kev tshuaj xyuas cov noob caj noob ces tau qhia txog kev txo qis hauv cov protein mitochondrial (Daim Duab 6H-I, Cov Ntaub Ntawv Ntxiv 22). Qhov xav tsis thoob, txawm hais tias muaj ntau hom fiber hauv ACTA1 thiab TNNT1 nemaline myopathies, qhov kev txo qis no yog ywj pheej kiag li ntawm MYH-raws li hom fiber (Daim Duab 6H thiab Cov Duab Ntxiv 11D-I, Cov Ntaub Ntawv Ntxiv 23). Peb cov protein microbial kuj tau tswj hwm hauv ACTA1 lossis TNNT1 nemaline myopathies. Ob ntawm cov microproteins no, ENSG00000215483_TR14_ORF67 (tseem hu ua LINC00598 lossis Lnc-FOXO1) thiab ENSG00000229425_TR25_ORF40 (lnc-NRIP1-2), tau qhia txog qhov sib txawv ntawm qhov muaj ntau hauv hom 1 myofibers. ENSG00000215483_TR14_ORF67 tau tshaj tawm yav dhau los tias ua lub luag haujlwm hauv kev tswj hwm lub voj voog ntawm tes. 56 Ntawm qhov tod tes, ENSG00000232046_TR1_ORF437 (sib raug rau LINC01798) tau nce ntxiv hauv ob qho tib si hom 1 thiab hom 2A myofibers hauv ACTA1-nemaline myopathy piv nrog cov neeg tswj hwm noj qab haus huv (Daim Duab Ntxiv 12A, Cov Ntaub Ntawv Ntxiv 24). Qhov sib txawv, cov protein ribosomal feem ntau tsis raug cuam tshuam los ntawm nemaline myopathy, txawm hais tias RPS17 tau downregulated hauv ACTA1 nemaline myopathy (Daim duab 6E).
Kev tshuaj xyuas kev txhim kho kuj tau qhia txog kev nce ntxiv ntawm cov txheej txheem tiv thaiv kab mob hauv ACTA1 thiab TNNT1 nemaline myopathies, thaum lub cell adhesion kuj tau nce ntxiv hauv TNNT1 nemaline myopathy (Daim Duab 6H). Kev nplua nuj ntawm cov yam ntxwv extracellular no tau pom los ntawm cov protein extracellular matrix hloov PCA hauv PC1 thiab PC2 hauv qhov tsis zoo (piv txwv li, mus rau cov fibers cuam tshuam tshaj plaws) (Daim Duab 6J). Ob pawg neeg mob tau qhia txog kev nce ntxiv ntawm cov protein extracellular uas koom nrog kev teb tiv thaiv kab mob thiab cov txheej txheem kho sarcolemmal, xws li annexins (ANXA1, ANXA2, ANXA5)57,58 thiab lawv cov protein sib cuam tshuam S100A1159 (Cov Duab Ntxiv 12B-C). Cov txheej txheem no tau tshaj tawm yav dhau los tias tau txhim kho hauv cov leeg nqaij dystrophies60 tab sis, rau peb txoj kev paub, tsis tau cuam tshuam nrog nemaline myopathies yav dhau los. Kev ua haujlwm ib txwm ntawm cov tshuab molecular no yog qhov yuav tsum tau ua rau kev kho sarcolemmal tom qab raug mob thiab rau kev sib xyaw ntawm cov myocytes tshiab tsim nrog myofibers58,61. Yog li, qhov kev ua ub no ntxiv ntawm cov txheej txheem no hauv ob pawg neeg mob qhia txog kev kho kom zoo rau kev raug mob los ntawm myofiber instability.
Cov teebmeem ntawm txhua tus nemaline myopathy tau sib raug zoo (r = 0.736) thiab qhia tau tias muaj kev sib tshooj tsim nyog (Cov Duab Ntxiv 11A-B), qhia tias ACTA1 thiab TNNT1 nemaline myopathy muaj cov teebmeem zoo sib xws rau cov proteome. Txawm li cas los xij, qee cov protein tau tswj hwm tsuas yog hauv ACTA1 lossis TNNT1 nemaline myopathy (Cov Duab Ntxiv 11A thiab C). Cov protein profibrotic MFAP4 yog ib qho ntawm cov protein uas tau nce siab tshaj plaws hauv TNNT1 nemaline myopathy tab sis tseem tsis tau hloov pauv hauv ACTA1 nemaline myopathy. SKIC8, ib feem ntawm PAF1C complex uas yog lub luag haujlwm rau kev tswj hwm HOX gene transcription, tau downregulated hauv TNNT1 nemaline myopathy tab sis tsis cuam tshuam hauv ACTA1 nemaline myopathy (Daim Duab Ntxiv 11A). Kev sib piv ncaj qha ntawm ACTA1 thiab TNNT1 nemaline myopathy qhia tau tias muaj kev txo qis ntau dua hauv cov protein mitochondrial thiab kev nce ntxiv ntawm cov protein ntawm lub cev tiv thaiv kab mob hauv TNNT1 nemaline myopathy (Daim Duab 6G-H thiab Cov Duab Ntxiv 11C thiab 11H-I). Cov ntaub ntawv no sib xws nrog qhov atrophy/dystrophy ntau dua uas pom hauv TNNT1 nemaline myopathy piv rau TNNT1 nemaline myopathy (Daim Duab 6A), qhia tias TNNT1 nemaline myopathy sawv cev rau hom kab mob hnyav dua.
Yuav kom ntsuam xyuas seb cov teebmeem pom ntawm nemaline myopathy puas tseem nyob ntawm tag nrho cov leeg nqaij, peb tau ua qhov kev tshuaj xyuas proteomic ntau ntawm cov leeg nqaij biopsies los ntawm tib pawg neeg ntawm TNNT1 nemaline myopathy cov neeg mob thiab piv rau lawv nrog cov neeg tswj (n = 3 ib pawg) (Daim Duab Ntxiv 13A, Cov Ntaub Ntawv Ntxiv 25). Raws li xav tau, cov neeg tswj tau muaj feem cuam tshuam ze rau hauv kev tshuaj xyuas cov khoom tseem ceeb, thaum cov neeg mob TNNT1 nemaline myopathy tau pom qhov sib txawv ntawm cov qauv zoo ib yam li qhov pom hauv kev tshuaj xyuas ib leeg (Daim Duab Ntxiv 13B). Kev tshuaj xyuas ntau dua tau rov ua dua cov protein sib txawv (Daim Duab Ntxiv 13C, Cov Ntaub Ntawv Ntxiv 26) thiab cov txheej txheem biological (Daim Duab Ntxiv 13D, Cov Ntaub Ntawv Ntxiv 27) tau hais txog los ntawm kev sib piv cov fibers ib leeg, tab sis poob lub peev xwm los sib txawv ntawm cov fiber sib txawv thiab tsis ua tiav los suav nrog cov teebmeem kab mob heterogeneous thoob plaws cov fibers.
Thaum muab cov ntaub ntawv no ua ke, qhia tau tias cov proteomics ib leeg-myofiber tuaj yeem piav qhia txog cov yam ntxwv ntawm kev kho mob uas tsis tuaj yeem pom los ntawm cov txheej txheem tsom xws li immunoblotting. Ntxiv mus, cov ntaub ntawv no qhia txog cov kev txwv ntawm kev siv actin fiber typing (MYH) ib leeg los piav qhia txog kev hloov pauv phenotypic. Tseeb tiag, txawm hais tias kev hloov pauv hom fiber sib txawv ntawm actin thiab troponin nemaline myopathies, ob qho tib si nemaline myopathies decouple MYH fiber typing los ntawm cov leeg nqaij pob txha metabolism mus rau qhov ceev dua thiab tsawg dua oxidative cov leeg nqaij proteome.
Qhov sib txawv ntawm cov hlwb yog qhov tseem ceeb rau cov ntaub so ntswg kom tau raws li lawv cov kev xav tau sib txawv. Hauv cov leeg nqaij pob txha, qhov no feem ntau piav qhia tias yog cov hom fiber uas muaj ntau qib ntawm kev tsim lub zog thiab kev qaug zog. Txawm li cas los xij, nws yog qhov tseeb tias qhov no piav qhia tsuas yog ib feem me me ntawm cov leeg nqaij pob txha sib txawv, uas yog ntau yam sib txawv, nyuaj thiab ntau yam dua li yav dhau los xav. Kev nce qib thev naus laus zis tam sim no tau ua rau pom tseeb rau cov yam ntxwv tswj cov leeg nqaij pob txha. Tseeb tiag, peb cov ntaub ntawv qhia tias hom 2X fibers yuav tsis yog cov leeg nqaij pob txha sib txawv. Ntxiv mus, peb tau txheeb xyuas cov protein metabolic, ribosomal proteins thiab cov protein cuam tshuam nrog cell ua cov ntsiab lus tseem ceeb ntawm cov leeg nqaij pob txha sib txawv. Los ntawm kev siv peb cov txheej txheem proteomic rau cov neeg mob kuaj nrog nematode myopathy, peb tau qhia ntxiv tias MYH-raws li fiber typing tsis qhia tag nrho cov leeg nqaij pob txha sib txawv, tshwj xeeb tshaj yog thaum lub cev raug cuam tshuam. Tseeb tiag, tsis hais txog MYH-raws li hom fiber, nematode myopathy ua rau hloov mus rau cov fibers sai dua thiab tsawg dua oxidative.
Txij li thaum xyoo pua 19th, cov leeg nqaij tau raug cais tawm. Cov kev tshuaj xyuas omics tsis ntev los no tau tso cai rau peb pib nkag siab txog cov qauv qhia tawm ntawm ntau hom MYH fiber thiab lawv cov lus teb rau ntau yam kev txhawb nqa. Raws li tau piav qhia ntawm no, cov txheej txheem omics kuj muaj qhov zoo ntawm kev nkag siab ntau dua rau kev ntsuas cov cim hom fiber ntau dua li cov txheej txheem ib txwm muaj antibody, yam tsis tas yuav cia siab rau kev ntsuas ntawm ib qho (lossis ob peb) cim los txhais cov hom leeg nqaij. Peb tau siv cov txheej txheem transcriptomic thiab proteomic sib xyaw ua ke thiab koom ua ke cov txiaj ntsig los tshuaj xyuas kev sau ntawv thiab kev tswj hwm tom qab kev hloov pauv ntawm cov fiber heterogeneity hauv cov leeg nqaij tib neeg. Cov txheej txheem no ua rau tsis tuaj yeem txheeb xyuas cov fibers 2X-hom ntshiab ntawm qib protein hauv vastus lateralis ntawm peb pawg neeg ntawm cov txiv neej hluas noj qab haus huv. Qhov no yog sib xws nrog cov kev tshawb fawb fiber ib leeg yav dhau los uas pom <1% cov fibers 2X ntshiab hauv vastus lateralis noj qab haus huv, txawm hais tias qhov no yuav tsum tau lees paub hauv lwm cov leeg yav tom ntej. Qhov sib txawv ntawm kev kuaj pom cov fibers 2X yuav luag ntshiab ntawm qib mRNA thiab tsuas yog sib xyaw 2A / 2X fibers ntawm qib protein yog qhov xav tsis thoob. MYH isoform mRNA kev qhia tawm tsis yog circadian,67 qhia tias peb tsis zoo li yuav "tsis nco qab" lub cim pib MYH2 hauv cov fibers 2X uas zoo li ntshiab ntawm RNA theem. Ib qho kev piav qhia tau, txawm hais tias tsuas yog hypothetical xwb, tej zaum yuav yog qhov sib txawv ntawm cov protein thiab/lossis mRNA ruaj khov ntawm MYH isoforms. Tseeb tiag, tsis muaj fiber ceev ceev yog 100% ntshiab rau txhua MYH isoform, thiab nws tsis meej tias MYH1 mRNA kev qhia tawm theem hauv 70-90% ntau npaum li cas yuav ua rau muaj MYH1 thiab MYH2 ntau sib npaug ntawm cov protein theem. Txawm li cas los xij, thaum xav txog tag nrho cov transcriptome lossis proteome, kev tshuaj xyuas pawg tuaj yeem txheeb xyuas tau tsuas yog ob pawg sib txawv uas sawv cev rau cov leeg nqaij qeeb thiab ceev, tsis hais lawv cov khoom MYH meej. Qhov no yog sib xws nrog kev tshuaj xyuas siv cov txheej txheem transcriptomic ib leeg, uas feem ntau txheeb xyuas tsuas yog ob pawg myonuclear sib txawv. 68, 69, 70 Ntxiv mus, txawm hais tias cov kev tshawb fawb proteomic yav dhau los tau txheeb xyuas hom 2X fibers, cov fibers no tsis sib sau ua ke sib cais ntawm cov fibers ceev thiab tsuas yog qhia me me ntawm cov protein ntau sib txawv piv rau lwm hom fiber raws li MYH. 14 Cov txiaj ntsig no qhia tias peb yuav tsum rov qab mus rau qhov kev pom thaum ntxov xyoo pua 20th ntawm kev faib cov leeg nqaij fiber, uas faib cov leeg nqaij tib neeg tsis yog peb pawg sib txawv raws li MYH, tab sis ua ob pawg raws li lawv cov khoom metabolic thiab contractile. 63
Qhov tseem ceeb tshaj, myofiber heterogeneity yuav tsum tau txiav txim siab raws ntau qhov ntau thiab tsawg. Cov kev tshawb fawb "omics" yav dhau los tau taw qhia rau hauv qhov kev taw qhia no, qhia tias cov leeg nqaij pob txha tsis tsim cov pawg sib cais tab sis tau teeb tsa raws li kev sib txuas ntxiv. 11, 13, 14, 64, 71 Ntawm no, peb qhia tias, ntxiv rau qhov sib txawv ntawm cov khoom cog lus thiab cov khoom siv metabolic ntawm cov leeg nqaij pob txha, myofibers tuaj yeem sib txawv los ntawm cov yam ntxwv cuam tshuam nrog kev sib cuam tshuam ntawm tes-cell thiab cov txheej txheem txhais lus. Tseeb tiag, peb pom ribosome heterogeneity hauv cov leeg nqaij pob txha uas pab txhawb rau heterogeneity ywj pheej ntawm cov hom fiber qeeb thiab ceev. Qhov ua rau muaj qhov sib txawv ntawm myofiber heterogeneity no, ywj pheej ntawm cov hom fiber qeeb thiab ceev, tseem tsis meej, tab sis nws yuav taw qhia rau lub koom haum tshwj xeeb hauv cov leeg nqaij uas teb zoo rau cov zog tshwj xeeb thiab cov khoom thauj, 72 kev sib txuas lus tshwj xeeb ntawm tes lossis lub cev nrog lwm hom cell hauv cov leeg nqaij microenvironment 73, 74, 75 lossis qhov sib txawv ntawm ribosome kev ua ub no hauv tus kheej myofibers. Tseeb tiag, ribosomal heteroplasmy, los ntawm kev hloov pauv paralogous ntawm RPL3 thiab RPL3L lossis ntawm qib 2′O-methylation ntawm rRNA, tau pom tias cuam tshuam nrog cov leeg nqaij pob txha hypertrophy76,77. Kev siv ntau yam thiab qhov chaw ua ke nrog kev ua haujlwm ntawm cov myofibers ib tus zuj zus yuav ua rau peb nkag siab ntxiv txog cov leeg nqaij biology ntawm qib ntau yam78.
Los ntawm kev tshuaj xyuas cov proteomes ntawm cov myofibers ib leeg los ntawm cov neeg mob uas muaj nemaline myopathies, peb kuj tau ua pov thawj txog kev siv tau zoo, kev ua haujlwm zoo, thiab kev siv tau ntawm cov myofiber proteomics ib leeg los piav qhia txog kev kho mob ntawm cov leeg pob txha. Ntxiv mus, los ntawm kev sib piv peb cov txheej txheem ua haujlwm rau kev tshuaj xyuas proteomic thoob ntiaj teb, peb tau ua pov thawj tias cov myofiber proteomics ib leeg muab cov ntaub ntawv tob ib yam li cov ntaub so ntswg thoob ntiaj teb thiab nthuav qhov tob no los ntawm kev suav txog interfiber heterogeneity thiab myofiber hom. Ntxiv rau qhov kev xav tau (txawm tias hloov pauv) qhov sib txawv ntawm cov fiber hom piv pom hauv ACTA1 thiab TNNT1 nemaline myopathies piv rau cov neeg tswj kev noj qab haus huv,19 peb kuj tau pom oxidative thiab extracellular remodeling ywj pheej ntawm MYH-mediated fiber hom switching. Fibrosis tau raug tshaj tawm ua ntej hauv TNNT1 nemaline myopathies.19 Txawm li cas los xij, peb qhov kev tshuaj xyuas ua raws li qhov kev tshawb pom no los ntawm kev qhia txog cov protein uas muaj kev ntxhov siab ntau ntxiv, xws li annexins, uas koom nrog cov txheej txheem kho sarcolemmal, hauv myofibers los ntawm cov neeg mob uas muaj ACTA1 thiab TNNT1 nemaline myopathies.57,58,59 Hauv kev xaus, cov theem annexin ntau ntxiv hauv myofibers los ntawm cov neeg mob uas muaj nemaline myopathy yuav sawv cev rau kev teb ntawm tes los kho cov myofibers uas puas tsuaj loj heev.
Txawm hais tias txoj kev tshawb fawb no sawv cev rau qhov kev tshuaj xyuas cov leeg nqaij loj tshaj plaws ntawm tib neeg txog niaj hnub no, nws tsis yog tsis muaj kev txwv. Peb tau rho tawm cov leeg nqaij pob txha los ntawm cov qauv me me thiab sib xws ntawm cov neeg koom nrog thiab ib leeg (vastus lateralis). Yog li ntawd, nws tsis yooj yim sua kom tsis txhob muaj cov pej xeem fiber tshwj xeeb thoob plaws cov leeg nqaij thiab ntawm qhov kawg ntawm cov leeg nqaij physiology. Piv txwv li, peb tsis tuaj yeem tsis txhob muaj qhov ua tau ntawm ib pawg ntawm cov fibers ultrafast (piv txwv li, cov fibers ntshiab 2X) tshwm sim hauv cov neeg khiav ceev thiab / lossis cov neeg ncaws pob muaj zog 79 lossis thaum lub sijhawm tsis muaj zog 66,80. Ntxiv mus, qhov loj me ntawm cov neeg koom nrog tau tiv thaiv peb los ntawm kev tshawb nrhiav qhov sib txawv ntawm poj niam txiv neej hauv kev sib txawv ntawm cov fiber, vim tias cov qauv fiber paub tias txawv ntawm txiv neej thiab poj niam. Ntxiv mus, peb tsis tuaj yeem ua qhov kev tshuaj xyuas transcriptomic thiab proteomic ntawm cov leeg nqaij lossis cov qauv los ntawm cov neeg koom nrog tib yam. Thaum peb thiab lwm tus txuas ntxiv mus rau qhov zoo tshaj plaws ntawm ib lub cell thiab ib leeg-myofiber kev tshuaj xyuas siv omics kev tshuaj xyuas kom ua tiav cov qauv qis heev (raws li tau pom ntawm no hauv kev tshuaj xyuas cov fibers los ntawm cov neeg mob uas muaj mitochondrial myopathy), lub sijhawm los sib xyaw ua ke ntau-omics (thiab kev ua haujlwm) hauv cov leeg nqaij ib leeg yuav pom tseeb.
Zuag qhia tag nrho, peb cov ntaub ntawv txheeb xyuas thiab piav qhia txog cov tsav tsheb sau ntawv thiab tom qab sau ntawv ntawm cov leeg nqaij pob txha sib txawv. Tshwj xeeb, peb nthuav qhia cov ntaub ntawv uas tawm tsam lub tswv yim ntev hauv kev ua haujlwm ntawm cov leeg nqaij pob txha uas cuam tshuam nrog cov lus txhais ntawm cov hom fiber raws li MYH. Peb vam tias yuav rov ua dua qhov kev sib cav thiab thaum kawg rov xav txog peb txoj kev nkag siab txog kev faib tawm ntawm cov leeg nqaij pob txha thiab kev sib txawv.
Kaum plaub tus neeg koom nrog Caucasian (12 tus txiv neej thiab 2 tus poj niam) tau txaus siab koom nrog txoj kev tshawb fawb no. Txoj kev tshawb fawb no tau txais kev pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm Ghent University Hospital (BC-10237), ua raws li 2013 Helsinki Declaration, thiab tau sau npe rau ntawm ClinicalTrials.gov (NCT05131555). Cov yam ntxwv dav dav ntawm cov neeg koom nrog tau nthuav tawm hauv Daim Ntawv Ntxiv 1. Tom qab tau txais kev pom zoo hais lus thiab sau ntawv, cov neeg koom nrog tau kuaj mob ua ntej koom nrog txoj kev tshawb fawb zaum kawg. Cov neeg koom nrog yog cov hluas (22-42 xyoo), noj qab nyob zoo (tsis muaj mob, tsis muaj keeb kwm haus luam yeeb), thiab muaj zog nruab nrab. Kev nqus pa oxygen siab tshaj plaws tau txiav txim siab siv lub tshuab ergometer rau kev ntsuas kev noj qab haus huv ntawm lub cev raws li tau piav qhia ua ntej. 81
Cov qauv kuaj nqaij tau sau thaum so thiab thaum yoo mov peb zaug, sib nrug 14 hnub. Vim tias cov qauv no tau sau ua ib feem ntawm kev tshawb fawb loj dua, cov neeg koom nrog tau noj placebo (lactose), H1-receptor antagonist (540 mg fexofenadine), lossis H2-receptor antagonist (40 mg famotidine) 40 feeb ua ntej kev kuaj nqaij. Peb tau ua pov thawj ua ntej tias cov histamine receptor antagonists no tsis cuam tshuam rau kev noj qab haus huv ntawm cov leeg nqaij thaum so81, thiab tsis muaj kev sib sau ua ke hauv peb cov phiaj xwm tswj xyuas zoo (Cov Duab Ntxiv 3 thiab 6). Kev noj zaub mov zoo (41.4 kcal/kg qhov hnyav, 5.1 g/kg qhov hnyav carbohydrate, 1.4 g/kg qhov hnyav protein, thiab 1.6 g/kg qhov hnyav rog) tau khaws cia rau 48 teev ua ntej txhua hnub sim, thiab noj tshais zoo (1.5 g/kg qhov hnyav carbohydrate) tau noj thaum sawv ntxov ntawm hnub sim. Nyob rau hauv qab tshuaj loog hauv zos (0.5 ml 1% lidocaine tsis muaj epinephrine), cov leeg nqaij biopsies tau txais los ntawm cov leeg vastus lateralis siv percutaneous Bergström aspiration.82 Cov qauv leeg tau tam sim ntawd embedded rau hauv RNAlater thiab khaws cia ntawm 4 ° C kom txog thaum manual fiber dissection (txog li 3 hnub).
Cov myofiber bundles tshiab raug rho tawm tau raug xa mus rau RNAlater medium tshiab hauv lub tais kab lis kev cai. Cov myofibers ib leeg tau raug txiav tes siv lub stereomicroscope thiab cov tweezers zoo. Nees nkaum tsib lub fibers tau raug txiav los ntawm txhua qhov biopsy, them sai sai rau kev xaiv cov fibers los ntawm ntau qhov chaw ntawm biopsy. Tom qab kev txiav, txhua lub fiber tau maj mam muab tso rau hauv 3 μl ntawm lysis buffer (SingleShot Cell Lysis Kit, Bio-Rad) uas muaj cov proteinase K thiab DNase enzymes kom tshem tawm cov protein thiab DNA tsis xav tau. Cell lysis thiab protein/DNA tshem tawm tau pib los ntawm kev vortexing luv luv, tig cov kua hauv microcentrifuge, thiab incubation ntawm chav tsev kub (10 feeb). Lub lysate tau raug incubated hauv lub thermal cycler (T100, Bio-Rad) ntawm 37 ° C rau 5 feeb, 75 ° C rau 5 feeb, thiab tom qab ntawd khaws cia tam sim ntawd ntawm -80 ° C kom txog thaum ua tiav ntxiv.
Cov tsev qiv ntawv RNA polyadenylated uas sib xws nrog Illumina tau npaj los ntawm 2 µl ntawm myofiber lysate siv QuantSeq-Pool 3′ mRNA-Seq Library Prep Kit (Lexogen). Cov txheej txheem ntxaws ntxaws tuaj yeem pom hauv phau ntawv qhia ntawm cov chaw tsim khoom. Cov txheej txheem pib nrog thawj-strand cDNA synthesis los ntawm kev sau rov qab, thaum lub sijhawm uas cov cim tshwj xeeb molecular (UMIs) thiab cov qauv tshwj xeeb i1 barcodes tau qhia kom ntseeg tau tias muaj kev sib sau ua ke ntawm cov qauv thiab txo qhov sib txawv ntawm kev ua haujlwm thaum lub sijhawm ua tiav. cDNA los ntawm 96 myofibers tom qab ntawd tau sib sau ua ke thiab ntxuav nrog cov hlaws dai sib nqus, tom qab ntawd RNA raug tshem tawm thiab kev sib sau ua ke thib ob tau ua tiav siv cov primers random. Lub tsev qiv ntawv tau ntxuav nrog cov hlaws dai sib nqus, cov cim npe i5 / i7 tshwj xeeb rau pas dej tau ntxiv, thiab PCR amplified. Kauj ruam kawg ntawm kev ntxuav tsim cov tsev qiv ntawv Illumina-compatible. Qhov zoo ntawm txhua lub pas dej hauv tsev qiv ntawv tau soj ntsuam siv High Sensitivity Small Fragment DNA Analysis Kit (Agilent Technologies, DNF-477-0500).
Raws li Qubit quantification, cov pas dej tau ntxiv rau ntawm equimolar concentrations (2 nM). Cov pas dej tau raug sequenced ntawm NovaSeq 6000 ntsuas hauv hom txheem siv NovaSeq S2 Reagent Kit (1 × 100 nucleotides) nrog 2 nM loading (4% PhiX).
Peb cov kav dej yog raws li Lexogen's QuantSeq Pool cov ntaub ntawv tshuaj xyuas kav dej (https://github.com/Lexogen-Tools/quantseqpool_analysis). Cov ntaub ntawv tau demultiplexed ua ntej nrog bcl2fastq2 (v2.20.0) raws li i7/i5 index. Nyeem 2 ces tau demultiplexed nrog idemux (v0.1.6) raws li i1 qauv barcode thiab UMI sequences tau rho tawm nrog umi_tools (v1.0.1). Nyeem ces tau trimmed nrog cutadapt (v3.4) hauv ntau qhov puag ncig kom tshem tawm cov nyeem luv luv (<20 ntev) lossis nyeem tsuas yog muaj cov adapter sequences. Nyeem ces tau sib phim rau tib neeg genome siv STAR (v2.6.0c) thiab BAM cov ntaub ntawv tau indexed nrog SAMtools (v1.11). Nyeem ob zaug tau tshem tawm siv umi_tools (v1.0.1). Thaum kawg, kev suav sib phim tau ua tiav siv featureCounts hauv Subread (v2.0.3). Kev tswj xyuas zoo tau ua tiav los ntawm kev siv FastQC (v0.11.9) ntawm ntau theem nruab nrab ntawm cov kav dej.
Txhua qhov kev ua tiav bioinformatics thiab kev pom ntxiv tau ua tiav hauv R (v4.2.3), feem ntau siv Seurat (v4.4.0) workflow. 83 Yog li ntawd, tus kheej UMI tus nqi thiab metadata matrices tau hloov mus rau hauv Seurat cov khoom. Cov noob caj noob ces qhia hauv tsawg dua 30% ntawm tag nrho cov fibers tau raug tshem tawm. Cov qauv qis zoo tau raug tshem tawm raws li qhov tsawg kawg nkaus ntawm 1000 UMI tus nqi thiab 1000 pom cov noob caj noob ces. Thaum kawg, 925 fibers dhau tag nrho cov kauj ruam tswj kev lim dej zoo. UMI tus nqi tau normalized siv Seurat SCTransform v2 txoj kev, 84 suav nrog tag nrho 7418 cov yam ntxwv pom, thiab qhov sib txawv ntawm cov neeg koom nrog tau rov qab los. Txhua metadata cuam tshuam tuaj yeem pom hauv Supplementary Dataset 28.