ʻO ka ʻokoʻa o nā olonā ʻiʻo iwi kanaka ma mua o ke kaulahao kaumaha myosin

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He ʻiʻo ʻokoʻa ka ʻiʻo iwi i haku ʻia me nā myofibrils, kahi i hoʻokaʻawale pinepine ʻia i loko o ke kanaka i ʻekolu ʻano: hoʻokahi "lohi" (ʻano 1) a ʻelua "wikiwiki" (ʻano 2A a me 2X). Eia nō naʻe, ʻaʻole maopopo loa ka heterogeneity ma waena a ma loko o nā ʻano myofibril kuʻuna. Ua hoʻopili mākou i nā ʻano transcriptomic a me proteomic i 1050 a me 1038 myofibrils pākahi mai ka vastus lateralis kanaka, kēlā me kēia. Ua komo pū ka haʻawina proteomic i nā kāne, a ua komo pū ka haʻawina transcriptomic i 10 mau kāne a me 2 mau wahine. Ma waho aʻe o nā isoforms kaulahao kaumaha myosin, ua ʻike mākou i nā protein metabolic, nā protein ribosomal, a me nā protein junctional cellular ma ke ʻano he kumu o ka loli multidimensional intermyofibril. Eia kekahi, ʻoiai ka ʻike ʻana i nā hui o nā olonā lohi a wikiwiki, hōʻike kā mākou ʻikepili ʻaʻole hiki ke hoʻokaʻawale ʻia nā olonā ʻano 2X mai nā olonā wikiwiki ʻē aʻe. Eia kekahi, ʻaʻole lawa ka hoʻokaʻawale ʻana ma muli o ke kaulahao kaumaha myosin e wehewehe i ka phenotype myofiber i nā myopathies nemaline. Ma keʻano holoʻokoʻa, hōʻike kā mākou ʻikepili i ka heterogeneity myofiber multidimensional, me nā kumu o ka loli e hoʻonui ana ma mua o nā isoforms kaulahao kaumaha myosin.
ʻO ka heterogeneity cellular kahi hiʻohiʻona kūlohelohe o nā ʻōnaehana olaola āpau, e ʻae ana i nā cell e loea e hoʻokō i nā pono like ʻole o nā ʻiʻo a me nā cell.1 ʻO ka manaʻo kuʻuna o ka heterogeneity fiber muscle skeletal ʻo ia ka wehewehe ʻana o nā neurons motor i ke ʻano fiber i loko o kahi ʻāpana motor, a ʻo ke ʻano fiber (ʻo ia hoʻi, ʻano 1, ʻano 2A, a me ke ʻano 2X i loko o ke kanaka) ua hoʻoholo ʻia e nā ʻano o nā isoforms myosin heavy chain (MYH).2 Ua hoʻokumu mua ʻia kēia ma kā lākou pH ATPase instability,3,4 a ma hope ma kā lākou hōʻike molekala o MYH.5 Eia naʻe, me ka ʻike ʻana a me ka ʻae ʻana o nā fibers "hui pū ʻia" e hōʻike pū ana i nā MYH he nui i nā ʻano like ʻole, ua ʻike nui ʻia nā fibers muscle skeletal ma ke ʻano he continuum ma mua o nā ʻano fiber ʻokoʻa.6 ʻOiai kēia, hilinaʻi nui ke kahua iā MYH ma ke ʻano he mea hoʻokaʻawale mua no ka hoʻokaʻawale ʻana o myofiber, kahi manaʻo i hoʻopili ʻia e nā palena a me nā manaʻo koʻikoʻi o nā haʻawina rodent mua nona nā ʻano hōʻike MYH a me ka laulā o nā ʻano fiber ʻokoʻa mai nā kānaka.2 Ua paʻakikī hou ke kūlana ma ka ʻoiaʻiʻo e hōʻike ana nā ʻiʻo iwi kanaka like ʻole i nā ʻano fiber like ʻole.7 He ʻiʻo i hui pū ʻia ka vastus lateralis me kahi ʻano hōʻike MYH waena (a no laila he ʻelele).7 Eia kekahi, ʻo kona maʻalahi o ka laʻana e lilo ia i ʻiʻo i aʻo maikaʻi loa ʻia i loko o ke kanaka.
No laila, he mea koʻikoʻi ka noiʻi kūʻokoʻa o ka ʻokoʻa o nā fiber muscle skeletal me ka hoʻohana ʻana i nā mea hana "omics" ikaika akā he paʻakikī hoʻi, ma muli o ke ʻano multinucleated o nā fibers muscle skeletal. Eia nō naʻe, ua loli ka ʻenehana transcriptomics8,9 a me proteomics10 i ka ʻike i nā makahiki i hala iho nei ma muli o nā holomua ʻenehana like ʻole, e ʻae ana i ka nānā ʻana i nā ʻiʻo skeletal ma ka hoʻonā fiber hoʻokahi. ʻO ka hopena, ua holomua nui ka wehewehe ʻana i ka ʻokoʻa fiber hoʻokahi a me kā lākou pane ʻana i nā stimuli atrophic a me ka ʻelemakule11,12,13,14,15,16,17,18. ʻO ka mea nui, loaʻa i kēia mau holomua ʻenehana nā noi lapaʻau, e ʻae ana i ka wehewehe kikoʻī a pololei o ka dysregulation pili i ka maʻi. No ka laʻana, ʻo ka pathophysiology o ka nemaline myopathy, kekahi o nā maʻi ʻiʻo i hoʻoilina maʻamau (MIM 605355 a me MIM 161800), he paʻakikī a huikau hoʻi.19,20 No laila, ʻo ka wehewehe maikaʻi ʻana o ka dysregulation o nā fibers muscle skeletal hiki ke alakaʻi i nā holomua koʻikoʻi i ko mākou ʻike ʻana i kēia maʻi.
Ua hoʻomohala mākou i nā ʻano hana no ka loiloi transcriptomic a me ka proteomic o nā olonā ʻiʻo iwi hoʻokahi i hoʻokaʻawale lima ʻia mai nā ʻano biopsy kanaka a hoʻopili iā lākou i nā tausani o nā olonā, e ʻae ana iā mākou e noiʻi i ka heterogeneity cellular o nā olonā ʻiʻo iwi kanaka. I ka wā o kēia hana, ua hōʻike mākou i ka mana o ka phenotyping transcriptomic a me ka proteomic o nā olonā ʻiʻo a ua ʻike i nā protein metabolic, ribosomal, a me nā cellular junctional ma ke ʻano he kumu koʻikoʻi o ka loli interfiber. Eia kekahi, me ka hoʻohana ʻana i kēia kahe hana proteomic, ua wehewehe mākou i ka pilina lapaʻau o ka myopathy nematode i nā olonā ʻiʻo iwi hoʻokahi, e hōʻike ana i kahi neʻe i hoʻonohonoho ʻia i nā olonā non-oxidative kūʻokoʻa o ke ʻano fiber e pili ana iā MYH.
No ka noiʻi ʻana i ka heterogeneity o nā olonā ʻiʻo iwi kanaka, ua hoʻomohala mākou i ʻelua mau kahe hana e hiki ai i ka transcriptome a me ka proteome analysis o nā olonā ʻiʻo iwi hoʻokahi (Kiʻi 1A a me ke Kiʻi Hoʻohui 1A). Ua hoʻomohala a hoʻomaikaʻi mākou i kekahi mau ʻanuʻu methodological, mai ka mālama ʻana i nā laʻana a me ka mālama ʻana i ka RNA a me ka pono o ka protein a hiki i ka hoʻonui ʻana i ka throughput no kēlā me kēia ʻano. No ka nānā ʻana i ka transcriptome, ua hoʻokō ʻia kēia ma ka hoʻokomo ʻana i nā barcodes molekala kikoʻī ma ka ʻanuʻu mua o ka transcription reverse, e ʻae ana i 96 mau olonā e hōʻuluʻulu ʻia no ka hana ʻana ma lalo. ʻO ka hoʻonohonoho hohonu (± 1 miliona heluhelu no kēlā me kēia fiber) i hoʻohālikelike ʻia me nā ʻano kuʻuna o nā cell hoʻokahi i hoʻonui hou aku i ka ʻikepili transcriptome. 21 No nā proteomics, ua hoʻohana mākou i kahi gradient chromatographic pōkole (21 mau minuke) i hui pū ʻia me ka loaʻa ʻana o ka ʻikepili DIA-PASEF ma kahi spectrometer mass timsTOF e hoʻomaikaʻi i ka hohonu o ka proteome me ka mālama ʻana i ka throughput kiʻekiʻe. 22,23 No ka noiʻi ʻana i ka ʻokoʻa o nā olonā ʻiʻo iwi olakino, ua wehewehe mākou i nā transcriptomes o 1,050 mau olonā pākahi mai 14 mau mea hāʻawi makua olakino a me nā proteomes o 1,038 mau olonā mai 5 mau mea hāʻawi makua olakino (Papa Hoʻohui 1). Ma kēia pepa, ua kapa ʻia kēia mau ʻikepili he 1,000-fiber transcriptomes a me proteomes, kēlā me kēia. Ua ʻike kā mākou ʻano hana i ka huina o 27,237 mau transcripts a me 2,983 mau protein i loko o nā loiloi transcriptomic a me proteomic 1,000-fiber (Kiʻi 1A, Nā ʻIkepili Hoʻohui 1-2). Ma hope o ke kānana ʻana i nā ʻikepili transcriptomic a me proteomic no >1,000 mau genes i ʻike ʻia a me 50% mau waiwai kūpono no kēlā me kēia olonā, ua hana ʻia nā loiloi bioinformatics ma hope no 925 a me 974 mau olonā i loko o ka transcriptome a me ka proteome, kēlā me kēia. Ma hope o ke kānana ʻana, ua ʻike ʻia ka awelika o 4257 ± 1557 mau genes a me 2015 ± 234 mau protein (awelika ± SD) no kēlā me kēia fiber, me ka palena o ka loli ma waena o kēlā me kēia kanaka (Nā Kiʻi Hoʻohui 1B–C, Nā ʻIkepili Hoʻohui 3–4). Eia nō naʻe, ʻoi aku ka ʻike ʻia o ka loli i loko o ke kumuhana ma waena o nā mea komo, ma muli paha o nā ʻokoʻa o ka hua RNA/protein ma waena o nā fibers o nā lōʻihi like ʻole a me nā wahi ʻāpana. No ka hapa nui o nā protein (>2000), ʻo ke koina o ka loli ma lalo o 20% (Kiʻi Hoʻohui 1D). Ua ʻae nā ʻano hana ʻelua i ka hopu ʻana i kahi ākea ākea o nā transcripts a me nā protein me nā pūlima i hōʻike nui ʻia no ka ʻūlū ʻana o nā ʻiʻo (e.g., ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Nā Kiʻi Hoʻohui 1E–F). ʻO ka hapa nui o nā hiʻohiʻona i ʻike ʻia he mea maʻamau ma waena o nā ʻikepili transcriptomic a me proteomic (Kiʻi Hoʻohui 1G), a ʻo ka awelika o ka ikaika UMI/LFQ o kēia mau hiʻohiʻona ua pili pono (r = 0.52) (Kiʻi Hoʻohui 1H).
Kaʻina hana o ka transcriptomics a me ka proteomics (i hana ʻia me BioRender.com). BD Nā piʻo laulā dinamika no MYH7, MYH2, a me MYH1, a me nā paepae i helu ʻia no ka hāʻawi ʻana i ke ʻano fiber. E, F Ka hoʻolaha ʻana o ka hōʻike ʻana o MYH ma waena o nā fibers i nā ʻikepili transcriptomics a me proteomics. G, H Nā kiʻi hoʻohālikelike like ʻole a me ka Projection (UMAP) no nā transcriptomics a me proteomics i kala ʻia e ke ʻano fiber i hoʻokumu ʻia ma MYH. I, J Nā kiʻi hiʻohiʻona e hōʻike ana i ka hōʻike ʻana o MYH7, MYH2, a me MYH1 i nā ʻikepili transcriptomics a me proteomics.
Ua hoʻomaka mua mākou e hāʻawi i ke ʻano fiber MYH i kēlā me kēia fiber me ka hoʻohana ʻana i kahi ala i hoʻomaikaʻi ʻia e hoʻohana ana i ka ʻike kiʻekiʻe a me ka laulā dynamic o ka hōʻike ʻana o MYH i nā ʻikepili omics. Ua hoʻohana nā haʻawina mua i nā paepae kūpono ʻole e lepili i nā fibers ma ke ʻano maʻemaʻe 1, ʻano 2A, ʻano 2X, a i ʻole i hui pū ʻia ma muli o kahi pakeneka paʻa o ka hōʻike ʻana o nā MYHs like ʻole11,14,24. Ua hoʻohana mākou i kahi ala ʻokoʻa kahi i hoʻonohonoho ʻia ai ka hōʻike ʻana o kēlā me kēia fiber e nā MYHs a mākou i hoʻohana ai e paʻi i nā fibers: MYH7, MYH2, a me MYH1, e pili ana i ke ʻano 1, ʻano 2A, a me nā fibers ʻano 2X, kēlā me kēia. A laila ua helu mākou i ka helu makemakika i ka helu inflection haʻahaʻa o kēlā me kēia piʻo hopena a hoʻohana iā ia ma ke ʻano he paepae e hāʻawi i nā fibers ma ke ʻano he maikaʻi (ma luna o ka paepae) a i ʻole maikaʻi ʻole (ma lalo o ka paepae) no kēlā me kēia MYH (Kiʻi 1B–D). Hōʻike kēia mau ʻikepili he ʻoi aku ka ʻokoʻa o nā ʻaoʻao hōʻike ʻana o MYH7 (Kiʻi 1B) a me MYH2 (Kiʻi 1C) ma ka pae RNA i hoʻohālikelike ʻia me ka pae protein. ʻOiaʻiʻo, ma ka pae protein, he kakaikahi loa nā olonā i hōʻike ʻole i ka MYH7, a ʻaʻohe olonā i loaʻa ka hōʻike MYH2 100%. Ma hope mai, ua hoʻohana mākou i nā paepae hōʻike i hoʻoholo mua ʻia e hāʻawi i nā ʻano olonā e pili ana i ka MYH i nā olonā āpau i kēlā me kēia ʻikepili. No ka laʻana, ua hāʻawi ʻia nā olonā MYH7+/MYH2-/MYH1- i ke ʻano 1, ʻoiai ua hāʻawi ʻia nā olonā MYH7-/MYH2+/MYH1+ i ke ʻano hui ʻia 2A/2X (e ʻike i ka Papa Hoʻohui 2 no ka wehewehe piha). I ka hui pū ʻana o nā olonā āpau, ua ʻike mākou i kahi hoʻolaha like loa o nā ʻano olonā e pili ana i ka MYH ma nā pae RNA (Kiʻi 1E) a me ka protein (Kiʻi 1F), ʻoiai ʻokoʻa ka hoʻohui ʻana o nā ʻano olonā e pili ana i ka MYH ma waena o nā kānaka, e like me ka mea i manaʻo ʻia (Kiʻi Hoʻohui 2A). Ua hoʻokaʻawale ʻia ka hapa nui o nā olonā ma ke ʻano maʻemaʻe 1 (34–35%) a i ʻole ke ʻano 2A (36–38%), ʻoiai ua ʻike ʻia kekahi helu nui o nā olonā ʻano hui ʻia 2A/2X (16–19%). ʻO kahi ʻokoʻa koʻikoʻi, ʻo ia ka hiki ke ʻike ʻia nā olonā ʻano 2X maʻemaʻe ma ka pae RNA wale nō, akā ʻaʻole ma ka pae protein, e hōʻike ana ua hoʻoponopono ʻia ka hōʻike wikiwiki ʻana o MYH ma hope o ke kākau ʻana.
Ua hōʻoia mākou i kā mākou ʻano hana hoʻopili ʻana i ka fiber MYH e pili ana i ka proteomics me ka hoʻohana ʻana i ka dot blotting e pili ana i ka antibody, a ua loaʻa i nā ʻano hana ʻelua ka 100% ʻaelike i ka ʻike ʻana i nā fibers maʻemaʻe type 1 a me type 2A (e nānā i ke Kiʻi Hoʻohui 2B). Eia nō naʻe, ʻoi aku ka maʻalahi o ke ʻano e pili ana i ka proteomics, ʻoi aku ka maikaʻi i ka ʻike ʻana i nā fibers i hui pū ʻia, a me ka helu ʻana i ka hapa o kēlā me kēia gene MYH i kēlā me kēia fiber. Hōʻike kēia mau ʻikepili i ka pono o ka hoʻohana ʻana i kahi ala e pili ana i ka omics e wehewehe i nā ʻano fiber muscle skeletal.
A laila ua hoʻohana mākou i ka ʻike i hui pū ʻia i hāʻawi ʻia e ka transcriptomics a me ka proteomics e hoʻokaʻawale pono i nā myofibers ma muli o kā lākou transcriptome piha a i ʻole proteome. Ma ka hoʻohana ʻana i ke ʻano hana hoʻokokoke a me ka projection manifold (UMAP) e hōʻemi i ka dimensionality i ʻeono mau ʻāpana nui (Nā Kiʻi Hoʻohui 3A–B), ua hiki iā mākou ke ʻike i ka loli o ka myofiber i loko o ka transcriptome (Kiʻi 1G) a me ka proteome (Kiʻi 1H). ʻIke ʻia, ʻaʻole i hoʻohui ʻia nā myofibers e nā mea komo (Nā Kiʻi Hoʻohui 3C–D) a i ʻole nā ​​lā hoʻāʻo (Kiʻi Hoʻohui 3E) i loko o nā ʻikepili transcriptomics a i ʻole proteomics, e hōʻike ana he ʻoi aku ka kiʻekiʻe o ka loli o loko o nā olonā ʻiʻo skeletal ma mua o ka loli ma waena o nā kumuhana. Ma ka palapala UMAP, ua puka mai ʻelua mau hui like ʻole e hōʻike ana i nā myofibers "wikiwiki" a me "lohi" (Nā Kiʻi 1G–H). Ua hoʻākoakoa ʻia nā myofibers MYH7+ (lohi) ma ka pou maikaʻi o UMAP1, ʻoiai ua hoʻākoakoa ʻia nā myofibers MYH2+ a me MYH1+ (wikiwiki) ma ka pou maikaʻi ʻole o UMAP1 (Nā Kiʻi 1I–J). Eia naʻe, ʻaʻohe ʻokoʻa i hana ʻia ma waena o nā ʻano fiber twitch wikiwiki (ʻo ia hoʻi, ʻano 2A, ʻano 2X, a i ʻole 2A/2X i hui pū ʻia) ma muli o ka hōʻike ʻana o MYH, e hōʻike ana ʻaʻole ʻokoʻa ka hōʻike ʻana o MYH1 (Kiʻi 1I–J) a i ʻole nā ​​māka myofiber 2X maʻamau e like me ACTN3 a i ʻole MYLK2 (Nā Kiʻi Hoʻohui 4A–B) ma waena o nā ʻano myofiber like ʻole i ka wā e noʻonoʻo ai i ka transcriptome a i ʻole proteome holoʻokoʻa. Eia kekahi, i hoʻohālikelike ʻia me MYH2 a me MYH7, ua pili pono kekahi mau transcripts a i ʻole nā ​​​​proteins me MYH1 (Nā Kiʻi Hoʻohui. 4C–H), e hōʻike ana ʻaʻole hōʻike piha ka nui o MYH1 i ka transcriptome/proteome myofiber. Ua hoʻokō ʻia nā hopena like i ka loiloi ʻana i ka hōʻike hui ʻia o nā isoforms MYH ʻekolu ma ka pae UMAP (Nā Kiʻi Hoʻohui. 4I–J). No laila, ʻoiai hiki ke ʻike ʻia nā olonā 2X ma ka pae transcript ma muli o ka quantification MYH wale nō, ʻaʻole hiki ke hoʻokaʻawale ʻia nā olonā MYH1+ mai nā olonā wikiwiki ʻē aʻe i ka noʻonoʻo ʻana i ka transcriptome holoʻokoʻa a i ʻole proteome.
Ma ke ʻano he noiʻi mua ʻana i ka heterogeneity fiber lohi ma mua o MYH, ua loiloi mākou i ʻehā mau protein kikoʻī ʻano fiber lohi i hoʻokumu ʻia: TPM3, TNNT1, MYL3, a me ATP2A22. Ua hōʻike nā ʻano fiber lohi i nā pilina Pearson kiʻekiʻe, ʻoiai ʻaʻole kūpono, i nā transcriptomics (Kiʻi Hoʻohui 5A) a me proteomics (Kiʻi Hoʻohui 5B). Ma kahi o 25% a me 33% o nā fiber lohi ʻaʻole i hoʻokaʻawale ʻia he fiber lohi maʻemaʻe e nā ʻano gene/protein āpau i transcriptomics (Kiʻi Hoʻohui 5C) a me proteomics (Kiʻi Hoʻohui 5D), kēlā me kēia. No laila, ʻo ka hoʻokaʻawale ʻana o ka fiber lohi e pili ana i nā ʻano gene/protein he nui e hoʻolauna i ka paʻakikī hou aʻe, ʻoiai no nā protein i ʻike ʻia he ʻano fiber kikoʻī. Hōʻike kēia ʻaʻole hiki i ka hoʻokaʻawale ʻana o ka fiber e pili ana i nā isoforms o kahi ʻohana gene/protein hoʻokahi ke hōʻike pono i ka heterogeneity maoli o nā fibers muscle skeletal.
No ka ʻimi hou ʻana i ka loli phenotypic o nā olonā ʻiʻo iwi kanaka ma ke ana o ke kumu hoʻohālike omics holoʻokoʻa, ua hana mākou i ka hoʻemi ʻana o ka dimensionality unbiased o ka ʻikepili me ka hoʻohana ʻana i ka loiloi ʻāpana nui (PCA) (Kiʻi 2A). E like me nā kiʻi UMAP, ʻaʻole i hoʻopili ka mea komo a i ʻole ka lā hoʻāʻo i ka hui ʻana o ka fiber ma ka pae PCA (Nā Kiʻi Hoʻohui 6A-C). Ma nā ʻikepili ʻelua, ua wehewehe ʻia ke ʻano fiber MYH e PC2, kahi i hōʻike ai i kahi hui o nā olonā ʻano 1 lohi a me kahi hui ʻelua e loaʻa ana ke ʻano 2A wikiwiki, ʻano 2X, a me nā olonā 2A/2X i hui pū ʻia (Kiʻi 2A). Ma nā ʻikepili ʻelua, ua hoʻopili ʻia kēia mau hui ʻelua e kahi helu liʻiliʻi o nā olonā ʻano 1/2A i hui pū ʻia. E like me ka mea i manaʻo ʻia, ua hōʻoia ka loiloi overrepresentation o nā mea hoʻokele PC nui ua hoʻokele ʻia ʻo PC2 e nā pūlima contractile a me metabolic (Kiʻi 2B a me nā Kiʻi Hoʻohui 6D-E, Nā ʻIkepili Hoʻohui 5-6). Ma keʻano holoʻokoʻa, ua ʻike ʻia ua lawa ke ʻano fiber MYH e wehewehe i ka loli mau ma ka PC2, koe wale nō nā fibers i kapa ʻia ʻo 2X i hoʻolaha ʻia ma o ka transcriptome i loko o ka hui wikiwiki.
A. Nā kiʻi loiloi ʻāpana nui (PCA) o nā ʻikepili transcriptome a me proteome i kala ʻia e like me ke ʻano fiber e pili ana iā MYH. B. Ka nānā ʻana i ka hoʻonui ʻana o nā mea hoʻokele transcript a me ka protein ma PC2 a me PC1. Ua hana ʻia ka loiloi helu me ka hoʻohana ʻana i ka pūʻolo clusterProfiler a me nā waiwai p i hoʻoponopono ʻia e Benjamini-Hochberg. C, D. Nā kiʻi PCA i kala ʻia e like me nā huaʻōlelo ontology gene adhesion intercellular (GO) i loko o ka transcriptome a me nā huaʻōlelo costamere GO i loko o ka proteome. Hōʻike nā pua i nā mea hoʻokele transcript a me ka protein a me kā lākou mau kuhikuhi. E, F. Nā kiʻi hiʻohiʻona manifold approximation and projection (UMAP) o nā hiʻohiʻona pili i ka clinically e hōʻike ana i nā gradients expression kūʻokoʻa o ke ʻano fiber lohi/wikiwiki. G, H. Nā pilina ma waena o nā mea hoʻokele PC2 a me PC1 i nā transcriptomes a me nā proteomes.
ʻO ka mea i manaʻo ʻole ʻia, ua wehewehe wale ke ʻano myofiber i hoʻokumu ʻia ma MYH i ke kekelē lua kiʻekiʻe loa o ka loli (PC2), e hōʻike ana he kuleana koʻikoʻi nā mea olaola ʻē aʻe i pili ʻole i ke ʻano myofiber i hoʻokumu ʻia ma MYH (PC1) i ka hoʻoponopono ʻana i ka heterogeneity fiber muscle skeletal. Ua hōʻike ka loiloi overrepresentation o nā mea hoʻokele kiʻekiʻe ma PC1 ua hoʻoholo mua ʻia ka loli ma PC1 e ka hoʻopili ʻana o nā cell-cell a me ka ʻike ribosome i loko o ka transcriptome, a me nā costameres a me nā protein ribosomal i loko o ka proteome (Kiʻi 2B a me nā Kiʻi Hoʻohui 6D–E, Hoʻonohonoho ʻIkepili Hoʻohui 7). I loko o nā ʻiʻo skeletal, hoʻopili nā costameres i ka Z-disc i ka sarcolemma a pili i ka hoʻoili ikaika a me ka hōʻailona. 25 Ua hōʻike nā kiʻi PCA i hōʻike ʻia me ka hoʻohana ʻana i ka hoʻopili ʻana o nā cell-cell (transcriptome, Kiʻi 2C) a me nā hiʻohiʻona costamere (proteome, Kiʻi 2D) i kahi neʻe hema ikaika ma PC1, e hōʻike ana ua hoʻonui ʻia kēia mau hiʻohiʻona i kekahi mau fibers.
Ua hōʻike ʻia kahi nānā kikoʻī o ka hui ʻana o ka myofiber ma ka pae UMAP ua hōʻike ka hapa nui o nā hiʻohiʻona i kahi gradient hōʻike MYH-based myofiber ma mua o ka myofiber subcluster-specific. Ua ʻike ʻia kēia hoʻomau no kekahi mau genes e pili ana i nā kūlana pathological (Kiʻi 2E), e like me CHCHD10 (maʻi neuromuscular), SLIT3 (atrophy muscle), CTDNEP1 (maʻi muscle). Ua ʻike ʻia kēia hoʻomau ma waena o ka proteome, me nā protein e pili ana i nā maʻi neurological (UGDH), insulin signaling (PHIP), a me ka transcription (HIST1H2AB) (Kiʻi 2F). Ma ke ʻano hui, hōʻike kēia mau ʻikepili i ka hoʻomau ʻana i ka heterogeneity twitch lohi/wikiwiki kūʻokoʻa o ka fiber ma waena o nā myofibers like ʻole.
ʻO ka mea hoihoi, ua hōʻike nā genes hoʻokele ma PC2 i ka pilina transcriptome-proteome maikaʻi (r = 0.663) (Kiʻi 2G), e hōʻike ana he lohi a wikiwiki hoʻi nā ʻano fiber twitch, a ʻoi aku hoʻi nā waiwai contractile a me metabolic o nā olonā ʻiʻo skeletal, i hoʻoponopono ʻia e ka transcriptionally. Eia nō naʻe, ʻaʻole i hōʻike nā genes hoʻokele ma PC1 i ka pilina transcriptome-proteome (r = -0.027) (Kiʻi 2H), e hōʻike ana he nui nā ʻano like ʻole i pili ʻole i nā ʻano fiber lohi/wikiwiki i hoʻoponopono ʻia ma hope o ka transcriptionally. No ka mea, ua wehewehe nui ʻia nā ʻano like ʻole ma PC1 e nā huaʻōlelo ontology gene ribosomal, a no ka mea he kuleana koʻikoʻi a kūikawā nā ribosomes i loko o ke kelepona ma o ke komo ikaika ʻana a me ka hoʻohuli ʻana i ka unuhi protein,31 ua hoʻomaka mākou e noiʻi i kēia heterogeneity ribosomal i manaʻo ʻole ʻia.
Ua hoʻoluʻu mua mākou i ka palapala loiloi ʻāpana nui o ka proteomics e like me ka nui o nā protein i loko o ka huaʻōlelo GOCC "cytoplasmic ribosome" (Kiʻi 3A). ʻOiai ua hoʻonui ʻia kēia huaʻōlelo ma ka ʻaoʻao maikaʻi o PC1, e hopena ana i kahi gradient liʻiliʻi, hoʻokele nā ​​​​protein ribosomal i ka māhele ʻana ma nā ʻaoʻao ʻelua o PC1 (Kiʻi 3A). ʻO nā protein Ribosomal i hoʻonui ʻia ma ka ʻaoʻao maikaʻi ʻole o PC1 me RPL18, RPS18, a me RPS13 (Kiʻi 3B), ʻoiai ʻo RPL31, RPL35, a me RPL38 (Kiʻi 3C) nā mea hoʻokele nui ma ka ʻaoʻao maikaʻi o PC1. ʻO ka mea hoihoi, ua hōʻike nui ʻia ʻo RPL38 a me RPS13 i loko o nā ʻiʻo iwi i hoʻohālikelike ʻia me nā ʻiʻo ʻē aʻe (Kiʻi Hoʻohui 7A). ʻAʻole i ʻike ʻia kēia mau pūlima ribosomal ʻokoʻa ma PC1 i loko o ka transcriptome (Kiʻi Hoʻohui 7B), e hōʻike ana i ka hoʻoponopono post-transcriptional.
A. Ka hoʻolālā ʻāpana nui (PCA) i hoʻoluʻu ʻia e like me nā huaʻōlelo cytoplasmic ribosomal gene ontology (GO) ma waena o ka proteome. Hōʻike nā pua i ke kuhikuhi o ka loli i hoʻopili ʻia e ka protein i ka hoʻolālā PCA. Pili ka lōʻihi o ka laina i ka helu ʻāpana nui no kahi protein i hāʻawi ʻia. B, C. Nā hoʻolālā hiʻohiʻona PCA no RPS13 a me RPL38. D. Ka nānā ʻana i ka hierarchical clustering i mālama ʻole ʻia o nā protein ribosomal cytoplasmic. E. Ke kumu hoʻohālike o ka ribosome 80S (PDB: 4V6X) e hōʻike ana i nā protein ribosomal me nā ʻano like ʻole i loko o nā olonā ʻiʻo skeletal. F. Nā protein Ribosomal me nā stoichiometry like ʻole i kūloko kokoke i ke kahawai puka mRNA.
Ua hāpai mua ʻia nā manaʻo o ka heterogeneity ribosomal a me ka specialization, kahi e hiki ai i ke alo o nā subpopulations ribosome ʻokoʻa (ribosomal heterogeneity) ke hoʻohuli pololei i ka unuhi protein i nā ʻiʻo like ʻole32 a me nā cell33 ma o ka unuhi koho o nā waihona transcript mRNA kikoʻī34 (ribosome specialization). No ka ʻike ʻana i nā subpopulations o nā protein ribosomal i hōʻike pū ʻia i loko o nā olonā ʻiʻo skeletal, ua hana mākou i kahi loiloi clustering hierarchical unsupervised o nā protein ribosomal i loko o ka proteome (Kiʻi 3D, Hoʻonohonoho ʻIkepili Hoʻohui 8). E like me ka mea i manaʻo ʻia, ʻaʻole i hui pū nā protein ribosomal e ke ʻano fiber ma muli o MYH. Eia nō naʻe, ua ʻike mākou i ʻekolu mau hui like ʻole o nā protein ribosomal; ʻo ka hui mua (ribosomal_cluster_1) ua hui pū ʻia me RPL38 a no laila ua hoʻonui ʻia ka hōʻike ʻana i nā fibers me kahi ʻaoʻao PC1 maikaʻi. ʻO ka hui ʻelua (ribosomal_cluster_2) ua hui pū ʻia me RPS13 a ua hoʻokiʻekiʻe ʻia i loko o nā fibers me kahi ʻaoʻao PC1 maikaʻi ʻole. ʻAʻole hōʻike ka hui ʻekolu (ribosomal_cluster_3) i ka hōʻike ʻokoʻa i hoʻonohonoho ʻia i loko o nā olonā ʻiʻo skeletal a hiki ke manaʻo ʻia ʻo ia ka protein ribosomal ʻiʻo skeletal "core". Loaʻa i nā hui ribosomal 1 a me 2 nā protein ribosomal i hōʻike mua ʻia e hoʻoponopono i ka unuhi ʻē aʻe (e laʻa, RPL10A, RPL38, RPS19, a me RPS25) a hoʻohuli i ka hana i ka hoʻomohala ʻana (e laʻa, RPL10A, RPL38).34,35,36,37,38 E like me nā hopena PCA, ua hōʻike pū ka hōʻike heterogeneous i ʻike ʻia o kēia mau protein ribosomal ma waena o nā olonā i ka hoʻomau (Kiʻi Hoʻohui 7C).
No ka ʻike ʻana i kahi o nā protein ribosomal heterogeneous i loko o ka ribosome, ua hoʻohana mākou i kahi kumu hoʻohālike o ka ribosome 80S kanaka (Protein Data Bank: 4V6X) (Kiʻi 3E). Ma hope o ka hoʻokaʻawale ʻana i nā protein ribosomal no nā hui ribosomal like ʻole, ʻaʻole i kūlike loa ko lākou mau wahi, e hōʻike ana ʻaʻole i hāʻawi kā mākou ala i ka hoʻonui ʻana no kekahi mau ʻāpana/hapa o ka ribosome. Eia naʻe, he mea hoihoi, ʻo ka hapa o nā protein subunit nui i loko o ka hui 2 ua haʻahaʻa ma mua o nā hui 1 a me 3 (Kiʻi Hoʻohui 7D). Ua ʻike mākou ua hoʻokomo nui ʻia nā protein me ka stoichiometry i hoʻololi ʻia i loko o nā olonā ʻiʻo skeletal i ka ʻili ribosome (Kiʻi 3E), e kūlike me ko lākou hiki ke launa pū me nā mea komo ribosome kūloko (IRES) i loko o nā heluna mRNA like ʻole, no laila e hoʻonohonoho ana i ka unuhi koho. 40, 41 Eia kekahi, he nui nā protein me ka stoichiometry i hoʻololi ʻia i loko o nā olonā ʻiʻo skeletal i loaʻa kokoke i nā ʻāpana hana e like me ke tunnel puka mRNA (Kiʻi 3F), kahi e hoʻoponopono koho ai i ka elongation translational a me ka hopu ʻana o nā peptides kikoʻī. 42 I ka hōʻuluʻulu manaʻo, hōʻike kā mākou ʻikepili e hōʻike ana ka stoichiometry o nā protein ribosomal ʻiʻo skeletal i ka heterogeneity, e hopena ana i nā ʻokoʻa ma waena o nā olonā ʻiʻo skeletal.
A laila ua hoʻomaka mākou e ʻike i nā pūlima fiber wikiwiki a lohi a ʻimi i nā ʻano hana o kā lākou hoʻoponopono transcriptional. Ke hoʻohālikelike nei i nā hui fiber wikiwiki a lohi i wehewehe ʻia e UMAP i nā ʻikepili ʻelua (Nā Kiʻi 1G-H a me 4A-B), ua ʻike nā loiloi transcriptomic a me proteomic i nā hiʻohiʻona nui he 1366 a me 804, kēlā me kēia (Nā Kiʻi 4A-B, Nā ʻIkepili Hoʻohui 9-12). Ua nānā mākou i nā ʻokoʻa i manaʻo ʻia i nā pūlima e pili ana i nā sarcomeres (e laʻa, tropomyosin a me troponin), ka hoʻopili ʻana o ka excitation-contraction (SERCA isoforms), a me ka metabolism ikehu (e laʻa, ALDOA a me CKB). Eia kekahi, ua hōʻike ʻokoʻa ʻia nā transcripts a me nā protein e hoʻoponopono ana i ka ubiquitination protein i nā fiber wikiwiki a lohi (e laʻa, USP54, SH3RF2, USP28, a me USP48) (Nā Kiʻi 4A-B). Eia kekahi, ʻo ka gene protein microbial RP11-451G4.2 (DWORF), ka mea i hōʻike mua ʻia ua ʻokoʻa ka hōʻike ʻana ma waena o nā ʻano fiber muscle hipa43 a hoʻonui i ka hana SERCA i loko o ka ʻiʻo cardiac44, ua hoʻonui nui ʻia i loko o nā fibers muscle skeletal lohi (Kiʻi 4A). Pēlā nō, ma ka pae fiber pākahi, ua ʻike ʻia nā ʻokoʻa koʻikoʻi i nā pūlima i ʻike ʻia e like me nā isoforms lactate dehydrogenase e pili ana i ka metabolism (LDHA a me LDHB, Kiʻi 4C a me ke Kiʻi Hoʻohui 8A)45,46 a me nā pūlima kikoʻī fiber-type i ʻike ʻole ʻia ma mua (e like me IRX3, USP54, USP28, a me DPYSL3) (Kiʻi 4C). Aia kahi overlap koʻikoʻi o nā hiʻohiʻona i hōʻike ʻokoʻa ʻia ma waena o nā ʻikepili transcriptomic a me proteomic (Kiʻi Hoʻohui 8B), a me kahi pilina hoʻololi pelu i hoʻokele nui ʻia e ka hōʻike ʻokoʻa koʻikoʻi o nā hiʻohiʻona sarcomere (Kiʻi Hoʻohui 8C). ʻO ka mea nui, ua hōʻike kekahi mau pūlima (e laʻa me USP28, USP48, GOLGA4, AKAP13) i ka hoʻoponopono post-transcriptional ikaika ma ka pae proteomic wale nō a he mau ʻaoʻao hōʻike kikoʻī o ke ʻano fiber twitch lohi/wikiwiki (Kiʻi Hoʻohui 8C).
ʻO nā kiʻi pele A a me B e hoʻohālikelike ana i nā hui lohi a me ka wikiwiki i ʻike ʻia e nā kiʻi hoʻohālikelike like a me ka projection (UMAP) ma nā Kiʻi 1G-H. Hōʻike nā kiko kala i nā transcripts a i ʻole nā ​​​​protein i ʻokoʻa nui ma FDR <0.05, a hōʻike nā kiko ʻeleʻele i nā transcripts a i ʻole nā ​​​​protein i ʻokoʻa nui ma ka loli log > 1. Ua hana ʻia ka loiloi helu ʻelua ala me ka hoʻohana ʻana i ka hoʻāʻo DESeq2 Wald me nā waiwai p i hoʻoponopono ʻia e Benjamini-Hochberg (transcriptomics) a i ʻole ke ʻano hoʻohālike linear Limma me ka loiloi Bayesian empirical i ukali ʻia e ka hoʻoponopono ʻana o Benjamini-Hochberg no nā hoʻohālikelike he nui (proteomics). C Nā kiʻi pūlima o nā genes a i ʻole nā ​​​​protein i hōʻike ʻokoʻa ʻia ma waena o nā olonā lohi a me ka wikiwiki. D Ka loiloi hoʻonui ʻana o nā transcripts a me nā protein i hōʻike ʻokoʻa nui ʻia. Hoʻonui ʻia nā waiwai overlapping i nā ʻikepili ʻelua, hoʻonui ʻia nā waiwai transcriptome i loko wale nō o ka transcriptome, a hoʻonui ʻia nā waiwai proteome i loko wale nō o ka proteome. Ua hana ʻia ka loiloi helu me ka hoʻohana ʻana i ka pūʻolo clusterProfiler me nā waiwai p i hoʻoponopono ʻia e Benjamini-Hochberg. E. Nā kumu hoʻopaʻa kikoʻī o ke ʻano fiber i ʻike ʻia e SCENIC ma muli o nā helu kikoʻī o ka mea hoʻoponopono i loaʻa mai SCENIC a me ka ʻokoʻa o ka hōʻike ʻana o ka mRNA ma waena o nā ʻano fiber. F. Ke hoʻohālikelike ʻana i nā kumu hoʻopaʻa i koho ʻia i hōʻike ʻokoʻa ʻia ma waena o nā fibers lohi a me ka wikiwiki.
A laila ua hana mākou i kahi loiloi overrepresentation o nā genes a me nā protein i hōʻike ʻokoʻa ʻia (Kiʻi 4D, Hoʻonohonoho ʻIkepili Hoʻohui 13). ʻO ka hoʻonui ʻana i ke ala no nā hiʻohiʻona i ʻokoʻa ma waena o nā ʻikepili ʻelua i hōʻike i nā ʻokoʻa i manaʻo ʻia, e like me ka fatty acid β-oxidation a me nā kaʻina hana metabolism ketone (nā olonā lohi), ka myofilament/muscle contraction (nā olonā wikiwiki a lohi, kēlā me kēia), a me nā kaʻina hana catabolic carbohydrate (nā olonā wikiwiki). Ua hoʻokiʻekiʻe ʻia ka hana phosphatase protein serine/threonine i nā olonā wikiwiki, i hoʻokele ʻia e nā hiʻohiʻona e like me nā subunits phosphatase hoʻoponopono a me catalytic (PPP3CB, PPP1R3D, a me PPP1R3A), i ʻike ʻia e hoʻoponopono i ka metabolism glycogen (47) (Nā Kiʻi Hoʻohui 8D–E). ʻO nā ala ʻē aʻe i hoʻonui ʻia i loko o nā olonā wikiwiki e komo pū ana me nā kino hana (P-) (YTHDF3, TRIM21, LSM2) i loko o ka proteome (Kiʻi Hoʻohui 8F), hiki ke komo i ka hoʻoponopono post-transcriptional (48), a me ka hana transcription factor (SREBF1, RXRG, RORA) i loko o ka transcriptome (Kiʻi Hoʻohui 8G). Ua hoʻonui ʻia nā olonā lohi i ka hana oxidoreductase (BDH1, DCXR, TXN2) (Kiʻi Hoʻohui 8H), ka hoʻopaʻa ʻana o ka amide (CPTP, PFDN2, CRYAB) (Kiʻi Hoʻohui 8I), matrix extracellular (CTSD, ADAMTSL4, LAMC1) (Kiʻi Hoʻohui 8J), a me ka hana receptor-ligand (FNDC5, SPX, NENF) (Kiʻi Hoʻohui 8K).
No ka loaʻa ʻana o ka ʻike hou aku i ka hoʻoponopono transcriptional ma lalo o nā ʻano ʻano olonā ʻiʻo lohi/wikiwiki, ua hana mākou i ka loiloi hoʻonui kumu transcription me ka hoʻohana ʻana iā SCENIC49 (Supplementary Data Set 14). Ua hoʻonui nui ʻia nā kumu transcription ma waena o nā olonā ʻiʻo wikiwiki a lohi (Kiʻi 4E). Ua komo pū kēia me nā kumu transcription e like me MAFA, ka mea i hoʻopili mua ʻia i ka hoʻomohala ʻana o ka olonā ʻiʻo wikiwiki,50 a me kekahi mau kumu transcription i pili ʻole i nā papahana gene kikoʻī ʻano olonā ʻiʻo. Ma waena o kēia mau mea, ʻo PITX1, EGR1, a me MYF6 nā kumu transcription i hoʻonui nui ʻia i nā olonā ʻiʻo wikiwiki (Kiʻi 4E). I ka hoʻohālikelike ʻana, ʻo ZSCAN30 a me EPAS1 (i ʻike ʻia ʻo HIF2A) nā kumu transcription i hoʻonui nui ʻia i nā olonā ʻiʻo lohi (Kiʻi 4E). E kūlike me kēia, ua hōʻike ʻia ʻo MAFA ma nā pae kiʻekiʻe ma ka ʻāpana UMAP e pili ana i nā olonā ʻiʻo wikiwiki, ʻoiai ʻo EPAS1 ke ʻano hōʻike kū'ē (Kiʻi 4F).
Ma waho aʻe o nā genes protein-coding i ʻike ʻia, aia kekahi mau biotypes RNA non-coding e pili ana paha i ka hoʻoponopono ʻana i ka ulu ʻana o ke kanaka a me nā maʻi. 51, 52 I loko o nā ʻikepili transcriptome, hōʻike kekahi mau RNA non-coding i ke ʻano kikoʻī o ka fiber (Kiʻi 5A a me ka Supplementary Dataset 15), me LINC01405, kahi mea kikoʻī loa no nā fibers lohi a ua hōʻike ʻia ua emi iho i loko o nā ʻiʻo mai nā poʻe maʻi me ka mitochondrial myopathy. 53 I ka hoʻohālikelike ʻana, hōʻike ʻo RP11-255P5.3, e pili ana i ka gene lnc-ERCC5-5 (https://lncipedia.org/db/transcript/lnc-ERCC5-5:2) 54, i ke ʻano kikoʻī o ka fiber wikiwiki. Hōʻike ʻo LINC01405 (https://tinyurl.com/x5k9wj3h) a me RP11-255P5.3 (https://tinyurl.com/29jmzder) i ke ʻano kikoʻī o nā ʻiʻo iwi (Nā Kiʻi Hoʻohui 9A–B) a ʻaʻohe o lākou mau genes contractile i ʻike ʻia i loko o ko lākou kaiāulu genomic 1 Mb, e hōʻike ana he kuleana kūikawā ko lākou i ka hoʻoponopono ʻana i nā ʻano fiber ma mua o ka hoʻoponopono ʻana i nā genes contractile e pili ana. Ua hōʻoia ʻia nā ʻano hōʻike kikoʻī o ke ʻano fiber lohi/wikiwiki o LINC01405 a me RP11-255P5.3, me ka hoʻohana ʻana iā RNAscope (Nā Kiʻi 5B–C).
A. Hoʻoponopono nui ʻia nā palapala RNA non-coding i nā olonā ʻiʻo lohi a me ka wikiwiki. B. Nā kiʻi RNAscope hōʻike e hōʻike ana i ke ʻano fiber lohi a me ka wikiwiki o LINC01405 a me RP11-255P5.3, kēlā me kēia. Pā unahi = 50 μm. C. Ka helu ʻana o ka hōʻike RNA non-coding kikoʻī myofiber e like me ka mea i hoʻoholo ʻia e RNAscope (n = 3 biopsies mai nā poʻe kūʻokoʻa, e hoʻohālikelike ana i nā olonā ʻiʻo wikiwiki a lohi i loko o kēlā me kēia kanaka). Ua hana ʻia ka loiloi helu me ka hoʻohana ʻana i kahi t-test Student ʻelua-tailed. Hōʻike nā kiʻi pahu i ka median a me nā quartiles mua a me ke kolu, me nā whiskers e kuhikuhi ana i nā waiwai liʻiliʻi a me ka nui loa. D. Ka hana hana ʻike protein microbial de novo (i hana ʻia me BioRender.com). E. Hōʻike kikoʻī ʻia ka protein microbial LINC01405_ORF408:17441:17358 i nā olonā ʻiʻo iwi lohi (n=5 biopsies mai nā mea komo kūʻokoʻa, e hoʻohālikelike ana i nā olonā ʻiʻo wikiwiki a lohi i kēlā me kēia mea komo). Ua hana ʻia ka loiloi helu me ka hoʻohana ʻana i ke ʻano hoʻohālike linear Limm i hui pū ʻia me kahi ʻano Bayesian empirical, a ukali ʻia e ke ʻano Benjamini-Hochberg no nā hoʻohālikelike he nui me ka hoʻoponopono p-value. Hōʻike nā kiʻi pahu i ka median, nā quartiles mua a me ke kolu, me nā whiskers e kuhikuhi ana i nā waiwai kiʻekiʻe/haʻahaʻa loa.
I kēia mau lā iho nei, ua hōʻike ʻia nā haʻawina he nui nā transcripts non-coding putative e hoʻopili i nā protein microbial i kākau ʻia, ʻo kekahi o ia mau mea e hoʻoponopono i ka hana ʻiʻo. 44, 55 No ka ʻike ʻana i nā protein microbial me ka hiki ke ʻano fiber specificity, ua ʻimi mākou i kā mākou ʻikepili proteome fiber 1000 me ka hoʻohana ʻana i kahi faila FASTA maʻamau e loaʻa ana nā moʻo o nā transcripts non-coding (n = 305) i loaʻa i ka ʻikepili transcriptome fiber 1000 (Kiʻi 5D). Ua ʻike mākou i 197 mau protein microbial mai 22 mau transcripts like ʻole, 71 o ia mau mea i hoʻoponopono ʻokoʻa ʻia ma waena o nā olonā ʻiʻo skeletal lohi a me ka wikiwiki (Kiʻi Hoʻohui 9C a me ka Pūʻulu ʻIkepili Hoʻohui 16). No LINC01405, ua ʻike ʻia ʻekolu mau huahana protein microbial, ʻo kekahi o ia mau mea i hōʻike i ka fiber lohi like me kāna transcript (Kiʻi 5E a me ke Kiʻi Hoʻohui 9D). No laila, ua ʻike mākou iā LINC01405 ma ke ʻano he gene e hoʻopili ana i kahi protein microbial kūikawā no nā olonā ʻiʻo skeletal lohi.
Ua hoʻomohala mākou i kahi kaʻina hana piha no ka wehewehe ʻana i ka proteomic nui o nā olonā ʻiʻo pākahi a ua ʻike i nā mea hoʻoponopono o ka heterogeneity fiber i nā kūlana olakino. Ua hoʻopili mākou i kēia kaʻina hana e hoʻomaopopo pehea e hoʻopilikia ai nā myopathies nemaline i ka heterogeneity fiber muscle skeletal. ʻO nā myopathies Nemaline nā maʻi ʻiʻo i hoʻoilina ʻia e hoʻoulu ai i ka nāwaliwali o nā ʻiʻo a, i nā keiki i hoʻopilikia ʻia, hōʻike ʻia me nā pilikia like ʻole e pili ana i ka pilikia hanu, scoliosis, a me ka neʻe palena o nā lālā. 19,20 ʻO ka maʻamau, i nā myopathies nemaline, nā ʻano pathogenic i loko o nā genes e like me ka actin alpha 1 (ACTA1) e hopena i ka nui o ka haku mele myofiber fiber lohi-twitch, ʻoiai he heterogeneous kēia hopena. ʻO kahi ʻokoʻa koʻikoʻi ʻo troponin T1 nemaline myopathy (TNNT1), nona ka nui o nā olonā wikiwiki. No laila, ʻo ka hoʻomaopopo maikaʻi ʻana i ka heterogeneity ma lalo o ka dysregulation fiber muscle skeletal i ʻike ʻia i nā myopathies nemaline e kōkua paha i ka wehe ʻana i ka pilina paʻakikī ma waena o kēia mau maʻi a me ke ʻano myofiber.
I ka hoʻohālikelike ʻia me nā kaohi olakino (n = 3 no kēlā me kēia hui), ua hōʻike nā myofibers i hoʻokaʻawale ʻia mai nā maʻi nemaline myopathy me nā mutations i loko o nā genes ACTA1 a me TNNT1 i ka atrophy myofiber a i ʻole dystrophy (Kiʻi 6A, Papa Hoʻohui 3). Ua hōʻike kēia i nā pilikia loea koʻikoʻi no ka nānā ʻana i ka proteomic ma muli o ka palena o nā mea i loaʻa. ʻOiai kēia, ua hiki iā mākou ke ʻike i 2485 mau protein i loko o 272 mau myofibers skeletal. Ma hope o ke kānana ʻana no ka liʻiliʻi he 1000 mau protein i helu ʻia no kēlā me kēia fiber, ua hoʻokomo ʻia he 250 mau fibers i ka nānā ʻana o ka bioinformatics ma hope. Ma hope o ke kānana ʻana, ua helu ʻia ka awelika o 1573 ± 359 mau protein no kēlā me kēia fiber (Kiʻi Hoʻohui 10A, Nā Sets ʻIkepili Hoʻohui 17-18). ʻIke ʻia, ʻoiai ka emi nui ʻana o ka nui o ka fiber, ua emi iki ka hohonu o ka proteome o nā laʻana maʻi nemaline myopathy. Eia kekahi, ʻo ka hana ʻana i kēia ʻikepili me ka hoʻohana ʻana i kā mākou mau faila FASTA ponoʻī (me nā palapala hoʻohui ʻole) i hiki ai iā mākou ke ʻike i ʻelima mau protein microbial i loko o nā myofibers skeletal mai nā maʻi myopathy nemaline (Supplementary Data Set 19). ʻOi aku ka laulā o ka dynamic range o ka proteome, a ua pili pono nā protein āpau i loko o ka hui kaohi me nā hopena o kahi loiloi proteome 1000-fiber ma mua (Supplementary Fig. 10B–C).
A. Nā kiʻi microscopic e hōʻike ana i ka atrophy fiber a i ʻole ka dystrophy a me ka nui o nā ʻano fiber like ʻole e pili ana iā MYH ma ACTA1 a me TNNT1 nemaline myopathies (NM). Pā unahi = 100 μm. No ka hōʻoia ʻana i ka hana hou ʻana o ka staining i nā maʻi ACTA1 a me TNNT1, ua stained ʻia ʻekolu mau biopsies maʻi ʻelua a ʻekolu manawa (ʻehā mau ʻāpana no kēlā me kēia hihia) ma mua o ke koho ʻana i nā kiʻi ʻelele. B. Nā ʻano fiber i nā mea komo e pili ana iā MYH. C. Kiʻi loiloi ʻāpana nui (PCA) o nā fibers ʻiʻo skeletal i nā maʻi me nā myopathies nemaline a me nā kaohi. D. Nā fibers ʻiʻo skeletal mai nā maʻi me nā myopathies nemaline a me nā kaohi i hoʻolālā ʻia ma kahi kiʻi PCA i hoʻoholo ʻia mai nā fibers 1000 i kālailai ʻia ma ke Kiʻi 2. No ka laʻana, nā kiʻi lua pele e hoʻohālikelike ana i nā ʻokoʻa ma waena o nā mea komo me nā myopathies nemaline ACTA1 a me TNNT1 a me nā kaohi, a ma waena o nā mea komo me nā myopathies nemaline ACTA1 a me TNNT1. Hōʻike nā pōʻai kala i nā protein i ʻokoʻa nui ma π < 0.05, a hōʻike nā kiko ʻeleʻele i nā protein i ʻokoʻa nui ma FDR < 0.05. Ua hana ʻia ka loiloi helu me ka hoʻohana ʻana i ke ʻano hoʻohālike linear Limma a me nā ʻano Bayesian empirical, a ukali ʻia e ka hoʻoponopono p-value no nā hoʻohālikelike he nui me ka hoʻohana ʻana i ke ʻano Benjamini-Hochberg. H. Ka loiloi hoʻonui ʻana o nā protein i hōʻike ʻia me ka ʻokoʻa nui ma o ka proteome holoʻokoʻa a ma nā olonā ʻano 1 a me 2A. Ua hana ʻia ka loiloi helu me ka hoʻohana ʻana i ka pūʻolo clusterProfiler a me nā p-values ​​i hoʻoponopono ʻia e Benjamini-Hochberg. I, J. Nā kiʻi loiloi ʻāpana nui (PCA) i kala ʻia e ka matrix extracellular a me nā huaʻōlelo mitochondrial gene ontology (GO).
No ka mea hiki i nā myopathies nemaline ke hoʻololi i ka hapa o nā ʻano myofiber e hōʻike ana i ka MYH i loko o nā ʻiʻo iwi,19,20 ua nānā mua mākou i nā ʻano myofiber e hōʻike ana i ka MYH i nā maʻi me nā myopathies nemaline a me nā kaohi. Ua hoʻoholo mākou i ke ʻano myofiber me ka hoʻohana ʻana i kahi ʻano hana kūʻokoʻa i wehewehe mua ʻia no ka 1000 myofiber assay (Supplementary Figs. 10D–E) a ua hiki ʻole ke ʻike i nā myofibers 2X maʻemaʻe (Kiʻi 6B). Ua ʻike mākou i kahi hopena heterogeneous o nā myopathies nemaline ma ke ʻano myofiber, ʻoiai ʻelua mau maʻi me nā mutations ACTA1 i loaʻa kahi hapa nui o nā myofibers ʻano 1, ʻoiai ʻelua mau maʻi me ka TNNT1 nemaline myopathy i loaʻa kahi hapa emi o nā myofibers ʻano 1 (Kiʻi 6B). ʻOiaʻiʻo, ua emi ka hōʻike ʻana o MYH2 a me nā isoforms troponin wikiwiki (TNNC2, TNNI2, a me TNNT3) i nā myopathies ACTA1-nemaline, ʻoiai ua emi ka hōʻike ʻana o MYH7 i nā myopathies TNNT1-nemaline (Kiʻi Hoʻohui 11A). He kūlike kēia me nā hōʻike mua o ka hoʻololi ʻana o ke ʻano myofiber heterogeneous i nā myopathies nemaline.19,20 Ua hōʻoia mākou i kēia mau hopena ma o ka immunohistochemistry a ua ʻike ʻia he nui nā myofibers type 1 i loaʻa i nā maʻi me ACTA1-nemaline myopathy, ʻoiai ʻo nā maʻi me TNNT1-nemaline myopathy he ʻano ʻē aʻe (Kiʻi 6A).
Ma ka pae proteome fiber hoʻokahi, ua hui pū nā olonā ʻiʻo iwi mai nā maʻi myopathy nemaline ACTA1 a me TNNT1 me ka hapa nui o nā olonā kaohi, me nā olonā myopathy nemaline TNNT1 ka mea i hoʻopilikia nui ʻia (Kiʻi 6C). Ua ʻike nui ʻia kēia i ka wā e hoʻolālā ana i nā kiʻi loiloi ʻāpana nui (PCA) o nā olonā pseudo-inflated no kēlā me kēia maʻi, me nā maʻi myopathy nemaline TNNT1 2 a me 3 e ʻike ʻia ana ma kahi mamao loa mai nā laʻana kaohi (Kiʻi Hoʻohui 11B, Hoʻonohonoho ʻIkepili Hoʻohui 20). No ka hoʻomaopopo maikaʻi ʻana i ke ʻano o ka hoʻohālikelike ʻana o nā olonā mai nā maʻi myopathy me nā olonā olakino, ua hoʻohana mākou i ka ʻike kikoʻī i loaʻa mai ka loiloi proteomic o 1,000 mau olonā mai nā mea komo makua olakino. Ua hoʻolālā mākou i nā olonā mai ka ʻikepili myopathy (nā maʻi a me nā kaohi o ka ACTA1 a me TNNT1 nemaline myopathy) ma ka pakuhi PCA i loaʻa mai ka loiloi proteomic 1000-fiber (Kiʻi 6D). Ua like ka hoʻolaha ʻana o nā ʻano fiber MYH ma ka ʻaoʻao o PC2 i nā fibers kaohi me ka hoʻolaha fiber i loaʻa mai ka loiloi proteomic 1000-fiber. Eia nō naʻe, ua neʻe ka hapa nui o nā fibers i nā maʻi nemaline myopathy i lalo o PC2, e uhi ana me nā fibers wikiwiki olakino, me ka nānā ʻole i ko lākou ʻano fiber MYH maoli. No laila, ʻoiai ua hōʻike nā maʻi me ACTA1 nemaline myopathy i kahi neʻe ʻana i nā fibers ʻano 1 i ka wā i helu ʻia ai me ka hoʻohana ʻana i nā ʻano hana MYH, ua hoʻoneʻe nā ACTA1 nemaline myopathy a me TNNT1 nemaline myopathy i ka proteome fiber muscle skeletal i nā fibers wikiwiki.
A laila ua hoʻohālikelike pololei mākou i kēlā me kēia hui maʻi me nā kaohi olakino a ua ʻike i 256 a me 552 mau protein i hōʻike ʻokoʻa ʻia ma ACTA1 a me TNNT1 nemaline myopathies, kēlā me kēia (Kiʻi 6E-G a me ke Kiʻi Hoʻohui 11C, Hoʻonohonoho ʻIkepili Hoʻohui 21). Ua hōʻike ka loiloi hoʻonui gene i ka emi ʻana o nā protein mitochondrial (Kiʻi 6H-I, Hoʻonohonoho ʻIkepili Hoʻohui 22). ʻO ka mea kupanaha, ʻoiai ke ʻano nui o nā ʻano fiber ma ACTA1 a me TNNT1 nemaline myopathies, ua kūʻokoʻa loa kēia emi ʻana mai ke ʻano fiber MYH (Kiʻi 6H a me nā Kiʻi Hoʻohui 11D-I, Hoʻonohonoho ʻIkepili Hoʻohui 23). Ua hoʻoponopono pū ʻia ʻekolu mau protein microbial ma ACTA1 a i ʻole TNNT1 nemaline myopathies. ʻElua o kēia mau microproteins, ʻo ENSG00000215483_TR14_ORF67 (i ʻike ʻia hoʻi ʻo LINC00598 a i ʻole Lnc-FOXO1) a me ENSG00000229425_TR25_ORF40 (lnc-NRIP1-2), i hōʻike i ka nui o ka ʻokoʻa ma nā myofibers ʻano 1 wale nō. Ua hōʻike mua ʻia ʻo ENSG00000215483_TR14_ORF67 e pāʻani i kahi kuleana i ka hoʻoponopono ʻana i ke kaʻina hana cell. 56 Ma ka ʻaoʻao ʻē aʻe, ua hoʻonui ʻia ʻo ENSG00000232046_TR1_ORF437 (e pili ana iā LINC01798) ma nā myofibers ʻano 1 a me ke ʻano 2A ma ka myopathy ACTA1-nemaline i hoʻohālikelike ʻia me nā kaohi olakino (Kiʻi Hoʻohui 12A, Hoʻonohonoho ʻIkepili Hoʻohui 24). I ka hoʻohālikelike ʻana, ʻaʻole i hoʻopilikia nui ʻia nā protein ribosomal e ka nemaline myopathy, ʻoiai ua hoʻohaʻahaʻa ʻia ʻo RPS17 ma ka ACTA1 nemaline myopathy (Kiʻi 6E).
Ua hōʻike pū ka loiloi hoʻonui i ka hoʻonui ʻana o nā kaʻina hana o ka ʻōnaehana pale ma ACTA1 a me TNNT1 nemaline myopathies, ʻoiai ua hoʻonui ʻia ka hoʻopili ʻana o nā cell ma TNNT1 nemaline myopathy (Kiʻi 6H). Ua hōʻike ʻia ka hoʻonui ʻana o kēia mau mea extracellular e nā protein matrix extracellular e hoʻololi ana iā PCA ma PC1 a me PC2 i kahi ala maikaʻi ʻole (ʻo ia hoʻi, i nā fibers i hoʻopilikia nui ʻia) (Kiʻi 6J). Ua hōʻike nā hui maʻi ʻelua i ka hoʻonui ʻia o ka hōʻike ʻana o nā protein extracellular i komo i nā pane pale a me nā ʻano hana hoʻoponopono sarcolemmal, e like me annexins (ANXA1, ANXA2, ANXA5)57,58 a me kā lākou protein e launa pū ana S100A1159 (Nā Kiʻi Hoʻohui 12B–C). Ua hōʻike mua ʻia kēia kaʻina hana e hoʻonui ʻia i nā dystrophies muscular60 akā, i ko mākou ʻike, ʻaʻole i pili mua me nā myopathies nemaline. Pono ka hana maʻamau o kēia mīkini molekala no ka hoʻoponopono sarcolemmal ma hope o ka hōʻeha a no ka hui ʻana o nā myocytes hou i hoʻokumu ʻia me myofibers58,61. No laila, ʻo ka hoʻonui ʻia o ka hana o kēia kaʻina hana i nā hui maʻi ʻelua e hōʻike ana i kahi pane hoʻoponopono i ka hōʻeha i hoʻokumu ʻia e ka myofiber instability.
Ua pili pono nā hopena o kēlā me kēia nemaline myopathy (r = 0.736) a ua hōʻike i ka overlap kūpono (Nā Kiʻi Hoʻohui 11A–B), e hōʻike ana he like nā hopena o ACTA1 a me TNNT1 nemaline myopathy ma ka proteome. Eia nō naʻe, ua hoʻoponopono wale ʻia kekahi mau protein ma ACTA1 a i ʻole TNNT1 nemaline myopathy (Nā Kiʻi Hoʻohui 11A a me C). ʻO ka protein profibrotic MFAP4 kekahi o nā protein i hoʻonui nui ʻia ma TNNT1 nemaline myopathy akā ua noho mau ʻole ma ACTA1 nemaline myopathy. ʻO SKIC8, kahi ʻāpana o ka hui PAF1C nona ke kuleana no ka hoʻoponopono ʻana i ka hoʻololi ʻana o ka gene HOX, ua hoʻohaʻahaʻa ʻia ma TNNT1 nemaline myopathy akā ʻaʻole i hoʻopilikia ʻia ma ACTA1 nemaline myopathy (Kiʻi Hoʻohui 11A). Ua hōʻike ka hoʻohālikelike pololei ʻana o ACTA1 a me TNNT1 nemaline myopathy i ka emi nui ʻana o nā protein mitochondrial a me ka hoʻonui ʻana o nā protein ʻōnaehana pale ma TNNT1 nemaline myopathy (Kiʻi 6G–H a me nā Kiʻi Hoʻohui 11C a me 11H–I). Kūlike kēia ʻikepili me ka atrophy/dystrophy nui i ʻike ʻia ma TNNT1 nemaline myopathy i hoʻohālikelike ʻia me TNNT1 nemaline myopathy (Kiʻi 6A), e hōʻike ana he ʻano ʻoi aku ka koʻikoʻi o ka maʻi ka TNNT1 nemaline myopathy.
No ka loiloi ʻana inā mau nā hopena i ʻike ʻia o ka nemaline myopathy ma ka pae ʻiʻo holoʻokoʻa, ua hana mākou i kahi loiloi proteomic nui o nā biopsies ʻiʻo mai ka hui like o nā maʻi TNNT1 nemaline myopathy a hoʻohālikelike iā lākou me nā kaohi (n = 3 no kēlā me kēia hui) (Kiʻi Hoʻohui 13A, Hoʻonohonoho ʻIkepili Hoʻohui 25). E like me ka mea i manaʻo ʻia, ua pili loa nā kaohi i ka loiloi ʻāpana nui, ʻoiai ua hōʻike nā maʻi TNNT1 nemaline myopathy i ka ʻokoʻa intersample kiʻekiʻe e like me ka mea i ʻike ʻia ma ka loiloi fiber hoʻokahi (Kiʻi Hoʻohui 13B). Ua hana hou ka loiloi nui i nā protein i hōʻike ʻokoʻa ʻia (Kiʻi Hoʻohui 13C, Hoʻonohonoho ʻIkepili Hoʻohui 26) a me nā kaʻina hana olaola (Kiʻi Hoʻohui 13D, Hoʻonohonoho ʻIkepili Hoʻohui 27) i hōʻike ʻia ma ka hoʻohālikelike ʻana i nā fibers pākahi, akā ua nalowale ka hiki ke hoʻokaʻawale ma waena o nā ʻano fiber like ʻole a ʻaʻole i helu i nā hopena maʻi heterogeneous ma waena o nā fibers.
I ka hui pū ʻia ʻana, hōʻike kēia mau ʻikepili e hiki i nā proteomics single-myofiber ke wehewehe i nā hiʻohiʻona olaola lapaʻau i ʻike ʻole ʻia e nā ʻano i kuhikuhi ʻia e like me ka immunoblotting. Eia kekahi, hōʻike kēia mau ʻikepili i nā palena o ka hoʻohana ʻana i ka actin fiber typing (MYH) wale nō e wehewehe i ka hoʻololi ʻana o ka phenotypic. ʻOiaʻiʻo, ʻoiai ʻokoʻa ka hoʻololi ʻana o ke ʻano fiber ma waena o ka actin a me ka troponin nemaline myopathies, hoʻokaʻawale nā ​​​​myopathies nemaline ʻelua i ka MYH fiber typing mai ka metabolism fiber muscle skeletal i kahi proteome muscle wikiwiki a emi iho ka oxidative.
He mea koʻikoʻi ka heterogeneity o ke kelepona no nā ʻiʻo e hoʻokō i kā lākou mau koi like ʻole. I loko o nā ʻiʻo iwi, ua wehewehe pinepine ʻia kēia ʻano fiber i hōʻike ʻia e nā kekelē like ʻole o ka hana ikaika a me ka luhi. Eia nō naʻe, ua maopopo he wahi liʻiliʻi wale nō kēia e wehewehe ana i ka loli o ke fiber muscle iwi, ʻoi aku ka loli, ka paʻakikī a me ka multifaceted ma mua o ka mea i manaʻo ʻia ma mua. Ua hoʻomālamalama nā holomua ʻenehana i nā mea e hoʻoponopono ana i nā fibers muscle iwi. ʻOiaʻiʻo, hōʻike kā mākou ʻikepili ʻaʻole paha he ʻano fiber muscle iwi ʻokoʻa nā fibers type 2X. Eia kekahi, ua ʻike mākou i nā protein metabolic, nā protein ribosomal a me nā protein e pili ana i ke kelepona ma ke ʻano he mau mea hoʻoholo nui o ka heterogeneity fiber muscle iwi. Ma ka hoʻopili ʻana i kā mākou kahe hana proteomic i nā laʻana maʻi me ka nematode myopathy, ua hōʻike hou mākou ʻaʻole hōʻike piha ke ʻano fiber MYH i ka heterogeneity muscle iwi, ʻoiai ke hoʻopilikia ʻia ka ʻōnaehana. ʻOiaʻiʻo, me ka nānā ʻole i ke ʻano fiber MYH, hopena ka nematode myopathy i kahi neʻe ʻana i nā fibers oxidative wikiwiki a emi iho.
Ua hoʻokaʻawale ʻia nā olonā ʻiʻo iwi mai ke kenekulia 19. Ua ʻae nā loiloi omics hou iā mākou e hoʻomaka e hoʻomaopopo i nā ʻano hōʻike o nā ʻano olonā MYH like ʻole a me kā lākou pane ʻana i nā stimuli like ʻole. E like me ka mea i wehewehe ʻia ma ʻaneʻi, loaʻa i nā ʻano omics ka pono o ka ʻike nui ʻana no ka helu ʻana i nā māka ʻano olonā ma mua o nā ʻano hana antibody kuʻuna, me ka hilinaʻi ʻole i ka helu ʻana o hoʻokahi (a i ʻole kekahi mau māka) e wehewehe i kahi ʻano olonā ʻiʻo iwi. Ua hoʻohana mākou i nā kahe hana transcriptomic a me proteomic complementary a hoʻohui i nā hopena e nānā i ka hoʻoponopono transcriptional a me ka post-transcriptional o ka heterogeneity fiber i nā olonā ʻiʻo iwi kanaka. Ua hopena kēia kahe hana i ka hiki ʻole ke ʻike i nā olonā ʻano 2X maʻemaʻe ma ka pae protein i loko o ka vastus lateralis o kā mākou cohort o nā kāne ʻōpio olakino. He kūlike kēia me nā haʻawina olonā hoʻokahi ma mua i loaʻa <1% nā olonā 2X maʻemaʻe i loko o ka vastus lateralis olakino, ʻoiai pono e hōʻoia ʻia kēia i nā ʻiʻo ʻē aʻe i ka wā e hiki mai ana. He mea kupanaha ka ʻokoʻa ma waena o ka ʻike ʻana o nā olonā 2X kokoke maʻemaʻe ma ka pae mRNA a me nā olonā 2A/2X i hui pū ʻia ma ka pae protein. ʻAʻole circadian ka hōʻike ʻana o ka MYH isoform mRNA,67 e hōʻike ana ʻaʻole paha mākou i "nalowale" i ka hōʻailona hoʻomaka MYH2 i loko o nā olonā 2X maʻemaʻe ma ka pae RNA. ʻO kahi wehewehe hiki, ʻoiai he kuhiakau wale nō, hiki ke ʻokoʻa i ka protein a/a i ʻole ke kūpaʻa mRNA ma waena o nā isoform MYH. ʻOiaʻiʻo, ʻaʻohe olonā wikiwiki he 100% maʻemaʻe no kekahi isoform MYH, a ʻaʻole maopopo inā e hopena nā pae hōʻike MYH1 mRNA ma ka pae 70-90% i ka nui like o MYH1 a me MYH2 ma ka pae protein. Eia nō naʻe, i ka noʻonoʻo ʻana i ka transcriptome a i ʻole proteome holoʻokoʻa, hiki i ka loiloi cluster ke ʻike wiwo ʻole i ʻelua mau hui like ʻole e hōʻike ana i nā olonā ʻiʻo iwi lohi a wikiwiki, me ka nānā ʻole i kā lākou haku mele MYH pololei. He kūlike kēia me nā loiloi e hoʻohana ana i nā ʻano transcriptomic nucleus hoʻokahi, kahi e ʻike pinepine ai i ʻelua mau hui myonuclear like ʻole. 68, 69, 70 Eia kekahi, ʻoiai ua ʻike ʻia nā noiʻi proteomic ma mua i nā olonā ʻano 2X, ʻaʻole hui pū kēia mau olonā mai ke koena o nā olonā wikiwiki a hōʻike wale i kahi helu liʻiliʻi o nā protein nui like ʻole i hoʻohālikelike ʻia me nā ʻano olonā ʻē aʻe e pili ana iā MYH. 14 Hōʻike kēia mau hopena e pono mākou e hoʻi i ka nānā ʻana o ke kenekulia 20 o ka hoʻokaʻawale ʻana o nā olonā ʻiʻo, ka mea i hoʻokaʻawale i nā olonā ʻiʻo iwi kanaka ʻaʻole i ʻekolu mau papa like ʻole e pili ana iā MYH, akā i ʻelua mau hui e pili ana i ko lākou mau waiwai metabolic a me nā contractile. 63
ʻOi aku ka mea nui, pono e noʻonoʻo ʻia ka heterogeneity myofiber ma nā ana he nui. Ua kuhikuhi nā haʻawina "omics" ma mua i kēia ala, e hōʻike ana ʻaʻole hana nā olonā ʻiʻo iwi i nā hui discrete akā ua hoʻonohonoho ʻia ma kahi hoʻomau. 11, 13, 14, 64, 71 Maanei, ke hōʻike nei mākou, me ka ʻokoʻa o nā waiwai contractile a me metabolic o ka ʻiʻo iwi, hiki ke hoʻokaʻawale ʻia nā myofibers e nā hiʻohiʻona e pili ana i nā pilina cell-cell a me nā ʻano unuhi. ʻOiaʻiʻo, ua ʻike mākou i ka heterogeneity ribosome i loko o nā olonā ʻiʻo iwi e hāʻawi ana i ka heterogeneity kūʻokoʻa o nā ʻano fiber lohi a wikiwiki. ʻO ke kumu kumu o kēia heterogeneity myofiber nui, kūʻokoʻa o ke ʻano fiber lohi a wikiwiki, ʻaʻole maopopo, akā hiki ke kuhikuhi i ka hoʻonohonoho spatial kūikawā i loko o nā fascicles ʻiʻo e pane maikaʻi i nā ikaika a me nā ukana kikoʻī,72 ka kamaʻilio kūikawā cellular a i ʻole ke kamaʻilio kikoʻī me nā ʻano cell ʻē aʻe i loko o ka microenvironment ʻiʻo73,74,75 a i ʻole nā ​​​​ʻokoʻa i ka hana ribosome i loko o nā myofibers pākahi. ʻOiaʻiʻo, ua hōʻike ʻia ka ribosomal heteroplasmy, ma o ka hoʻololi paralogous o RPL3 a me RPL3L a i ʻole ma ka pae o 2′O-methylation o rRNA, e pili ana me ka hypertrophy ʻiʻo skeletal76,77. ʻO nā noi multi-omic a me spatial i hui pū ʻia me ke ʻano hana o nā myofibers pākahi e hoʻonui hou i ko mākou ʻike i ka biology ʻiʻo ma ka pae multi-omic78.
Ma ka nānā ʻana i nā proteomes o nā myofibers hoʻokahi mai nā maʻi me nā myopathies nemaline, ua hōʻike pū mākou i ka pono, ka pono, a me ka hoʻohana ʻana o nā proteomics myofiber hoʻokahi e wehewehe i ka pathophysiology lapaʻau o ka ʻiʻo skeletal. Eia kekahi, ma ka hoʻohālikelike ʻana i kā mākou kahe hana i ka loiloi proteomic honua, ua hiki iā mākou ke hōʻike e loaʻa i nā proteomics myofiber hoʻokahi ka hohonu like o ka ʻike e like me nā proteomics kiko honua a hoʻonui i kēia hohonu ma ka helu ʻana i ka heterogeneity interfiber a me ke ʻano myofiber. Ma waho aʻe o nā ʻokoʻa i manaʻo ʻia (ʻoiai he loli) i ka ratio ʻano fiber i ʻike ʻia ma ACTA1 a me TNNT1 nemaline myopathies i hoʻohālikelike ʻia me nā kaohi olakino,19 ua ʻike pū mākou i ka oxidative a me ka extracellular remodeling kūʻokoʻa o ka hoʻololi ʻano fiber MYH-mediated. Ua hōʻike mua ʻia ʻo Fibrosis ma nā myopathies nemaline TNNT1.19 Eia nō naʻe, kūkulu kā mākou loiloi ma luna o kēia ʻike ma ka hōʻike ʻana i nā pae i hoʻonui ʻia o nā protein pili i ke kaumaha i hoʻokuʻu ʻia ma waho o ka pūnaewele, e like me nā annexins, i komo i nā hana hoʻoponopono sarcolemmal, i loko o nā myofibers mai nā maʻi me ACTA1 a me TNNT1 nemaline myopathies.57,58,59 I ka hopena, ʻo ka piʻi ʻana o nā pae annexin i loko o nā myofibers mai nā maʻi me ka nemaline myopathy e hōʻike paha i kahi pane cellular e hoʻoponopono i nā myofibers atrophic koʻikoʻi.
ʻOiai ʻo kēia haʻawina ke hōʻike nei i ka loiloi nui loa o ka ʻiʻo holoʻokoʻa o ka fiber hoʻokahi o nā kānaka a hiki i kēia lā, ʻaʻole ia he mea ʻole. Ua hoʻokaʻawale mākou i nā olonā ʻiʻo iwi mai kahi laʻana liʻiliʻi a like o nā mea komo a me kahi ʻiʻo hoʻokahi (ka vastus lateralis). No laila, ʻaʻole hiki ke kāpae i ke ola ʻana o nā heluna olonā kikoʻī ma waena o nā ʻano ʻiʻo a ma nā ʻaoʻao kiʻekiʻe o ka physiology ʻiʻo. No ka laʻana, ʻaʻole hiki iā mākou ke kāpae i ka hiki ke puka mai kahi ʻāpana o nā olonā ultrafast (e laʻa, nā olonā 2X maʻemaʻe) i nā sprinter i hoʻomaʻamaʻa nui ʻia a/a i ʻole nā ​​​​​​mea pāʻani ikaika79 a i ʻole i nā wā o ka hana ʻole o ka ʻiʻo66,80. Eia kekahi, ʻo ka nui o ka laʻana palena o nā mea komo i pale iā mākou mai ka noiʻi ʻana i nā ʻokoʻa wahine ma ka heterogeneity fiber, ʻoiai ua ʻike ʻia nā lakio ʻano fiber e ʻokoʻa ma waena o nā kāne a me nā wahine. Eia kekahi, ʻaʻole hiki iā mākou ke hana i nā loiloi transcriptomic a me proteomic ma nā olonā ʻiʻo like a i ʻole nā ​​​​laʻana mai nā mea komo like. I ko mākou a me nā poʻe ʻē aʻe e hoʻomau nei i ka hoʻonui ʻana i nā loiloi cell-hoʻokahi a me ka myofiber hoʻokahi me ka hoʻohana ʻana i ka loiloi omics e hoʻokō ai i ka hoʻokomo laʻana haʻahaʻa loa (e like me ka mea i hōʻike ʻia ma aneʻi i ka nānā ʻana o nā fibers mai nā maʻi me ka mitochondrial myopathy), ʻike ʻia ka manawa kūpono e hoʻohui i nā ʻano multi-omics (a me ka hana) i loko o nā fibers ʻiʻo hoʻokahi.
Ma keʻano holoʻokoʻa, ʻike a wehewehe kā mākou ʻikepili i nā mea hoʻokele transcriptional a me post-transcriptional o ka heterogeneity muscle skeletal. Ma ke ʻano kikoʻī, hōʻike mākou i ka ʻikepili e hoʻokūkū nei i kahi dogma lōʻihi i ka physiology muscle skeletal e pili ana me ka wehewehe kuʻuna MYH-based o nā ʻano fiber. Manaʻolana mākou e hoʻohou i ka hoʻopaʻapaʻa a noʻonoʻo hou i ko mākou ʻike i ka hoʻokaʻawale ʻana o ka fiber muscle skeletal a me ka heterogeneity.
Ua ʻae manawaleʻa nā mea komo he ʻumikumamāhā o ke Kulanui o Caucasian (12 kāne a me 2 wahine) e komo i kēia haʻawina. Ua ʻāpono ʻia ke aʻo ʻana e ke Kōmike Ethics o ka Halemai Kulanui ʻo Ghent (BC-10237), ua hoʻokō i ka 2013 Helsinki Declaration, a ua hoʻopaʻa inoa ʻia ma ClinicalTrials.gov (NCT05131555). Hōʻike ʻia nā ʻano laulā o nā mea komo ma ka Papa Hoʻohui 1. Ma hope o ka loaʻa ʻana o ka ʻae waha a me ke kākau ʻana, ua hana ʻia kahi hoʻokolokolo lapaʻau i nā mea komo ma mua o ka hoʻokomo hope ʻana i loko o ke aʻo ʻana. He ʻōpio nā mea komo (22-42 mau makahiki), olakino (ʻaʻohe maʻi, ʻaʻohe mōʻaukala puhipaka), a ikaika kino. Ua hoʻoholo ʻia ka lawe ʻana o ka oxygen kiʻekiʻe loa me ka hoʻohana ʻana i kahi ergometer ʻanuʻu no ka loiloi ʻana i ke olakino kino e like me ka mea i wehewehe mua ʻia. 81
Ua hōʻiliʻili ʻia nā laʻana biopsy ʻiʻo i ka wā hoʻomaha a me ke kūlana hoʻokē ʻai ʻekolu manawa, he 14 mau lā ka mamao. Ma muli o ka hōʻiliʻili ʻia ʻana o kēia mau laʻana ma ke ʻano he ʻāpana o kahi noiʻi nui aʻe, ua ʻai nā mea komo i ka placebo (lactose), kahi antagonist H1-receptor (540 mg fexofenadine), a i ʻole kahi antagonist H2-receptor (40 mg famotidine) 40 mau minuke ma mua o ka biopsy. Ua hōʻike mua mākou ʻaʻole pili kēia mau antagonist receptor histamine i ke olakino ʻiʻo iwi i ka hoʻomaha81, a ʻaʻohe hui e pili ana i ke kūlana i ʻike ʻia ma kā mākou mau hoʻolālā kaohi maikaʻi (Nā Kiʻi Hoʻohui 3 a me 6). Ua mālama ʻia kahi ʻai maʻamau (41.4 kcal/kg kaumaha o ke kino, 5.1 g/kg kalapona kaumaha o ke kino, 1.4 g/kg protein kaumaha o ke kino, a me 1.6 g/kg momona kaumaha o ke kino) no 48 mau hola ma mua o kēlā me kēia lā hoʻokolohua, a ua ʻai ʻia kahi ʻaina kakahiaka maʻamau (1.5 g/kg kalapona kaumaha o ke kino) i ke kakahiaka o ka lā hoʻokolohua. Ma lalo o ka anesthesia kūloko (0.5 ml 1% lidocaine me ka ʻole o ka epinephrine), ua loaʻa nā biopsies ʻiʻo mai ka ʻiʻo vastus lateralis me ka hoʻohana ʻana i ka aspiration Bergström percutaneous.82 Ua hoʻokomo koke ʻia nā laʻana ʻiʻo i loko o RNAlater a mālama ʻia ma 4°C a hiki i ka dissection fiber manual (a hiki i 3 lā).
Ua hoʻoili ʻia nā pūʻolo myofiber i hoʻokaʻawale hou ʻia i kahi medium RNAlater hou i loko o kahi kīʻaha moʻomeheu. A laila ua ʻoki lima ʻia nā myofibers pākahi me ka hoʻohana ʻana i kahi stereomicroscope a me nā tweezers maikaʻi. Ua ʻoki ʻia he iwakāluakūmālima mau olonā mai kēlā me kēia biopsy, e uku ana i ka nānā kūikawā i ke koho ʻana i nā olonā mai nā wahi like ʻole o ka biopsy. Ma hope o ka ʻoki ʻana, ua hoʻokomo mālie ʻia kēlā me kēia olonā i loko o 3 μl o ka lysis buffer (SingleShot Cell Lysis Kit, Bio-Rad) e loaʻa ana nā enzymes proteinase K a me DNase e wehe i nā protein a me ka DNA i makemake ʻole ʻia. A laila ua hoʻomaka ʻia ka lysis cell a me ka wehe ʻana o ka protein/DNA e ka vortexing pōkole, ka milo ʻana i ka wai i loko o kahi microcentrifuge, a me ka incubation ma ka mahana o ka lumi (10 min). A laila ua hoʻomoʻa ʻia ka lysate i loko o kahi thermal cycler (T100, Bio-Rad) ma 37°C no 5 min, 75°C no 5 min, a laila mālama koke ʻia ma -80°C a hiki i ka hana hou ʻana.
Ua hoʻomākaukau ʻia nā waihona RNA polyadenylated i kūpono iā Illumina mai 2 µl o ka myofiber lysate me ka hoʻohana ʻana i ka QuantSeq-Pool 3′ mRNA-Seq Library Prep Kit (Lexogen). Hiki ke loaʻa nā ʻano kikoʻī ma ka manual a ka mea hana. Hoʻomaka ke kaʻina hana me ka synthesis cDNA kaula mua ma o ka transcription reverse, kahi e hoʻolauna ʻia ai nā mea hōʻike molekala kūikawā (UMIs) a me nā barcode i1 kikoʻī no ka laʻana e hōʻoia i ka hui ʻana o nā laʻana a hōʻemi i ka loli loea i ka wā o ka hana ʻana ma hope. A laila hui pū ʻia ka cDNA mai 96 myofibers a hoʻomaʻemaʻe ʻia me nā momi magnetic, ma hope o ka wehe ʻia ʻana o ka RNA a hana ʻia ka synthesis kaula ʻelua me ka hoʻohana ʻana i nā primers random. Hoʻomaʻemaʻe ʻia ka waihona me nā momi magnetic, hoʻohui ʻia nā lepili i5/i7 kikoʻī no ka waihona, a hoʻonui ʻia ka PCR. Hana kahi hana hoʻomaʻemaʻe hope loa i nā waihona puke kūpono iā Illumina. Ua loiloi ʻia ke ʻano o kēlā me kēia waihona waihona waihona me ka hoʻohana ʻana i ka High Sensitivity Small Fragment DNA Analysis Kit (Agilent Technologies, DNF-477-0500).
Ma muli o ka helu ʻana o Qubit, ua hoʻohui hou ʻia nā waihona ma nā ʻano equimolar (2 nM). A laila ua hoʻonohonoho ʻia ka waihona hopena ma kahi mea hana NovaSeq 6000 ma ke ʻano maʻamau me ka hoʻohana ʻana i ka NovaSeq S2 Reagent Kit (1 × 100 nucleotides) me ka hoʻouka ʻana o 2 nM (4% PhiX).
Hoʻokumu ʻia kā mākou pipeline ma ka pipeline loiloi ʻikepili QuantSeq Pool a Lexogen (https://github.com/Lexogen-Tools/quantseqpool_analysis). Ua wehe mua ʻia ka ʻikepili me bcl2fastq2 (v2.20.0) ma muli o ka helu kuhikuhi i7/i5. A laila ua wehe ʻia ka heluhelu 2 me idemux (v0.1.6) ma muli o ka barcode laʻana i1 a ua unuhi ʻia nā moʻo UMI me umi_tools (v1.0.1). A laila ua ʻoki ʻia nā heluhelu me cutadapt (v3.4) i nā pōʻai he nui e wehe i nā heluhelu pōkole (<20 ka lōʻihi) a i ʻole nā ​​heluhelu i haku wale ʻia me nā moʻo adapter. A laila ua hoʻohālikelike ʻia nā heluhelu i ka genome kanaka me ka hoʻohana ʻana iā STAR (v2.6.0c) a ua helu ʻia nā faila BAM me SAMtools (v1.11). Ua wehe ʻia nā heluhelu pālua me ka hoʻohana ʻana iā umi_tools (v1.0.1). ʻO ka hope loa, ua hana ʻia ka helu ʻana i ke ʻano me ka hoʻohana ʻana i nā featureCounts ma Subread (v2.0.3). Ua hana ʻia ka kaohi maikaʻi me ka hoʻohana ʻana iā FastQC (v0.11.9) ma kekahi mau pae waena o ka pipeline.
Ua hana ʻia nā hana hoʻoponopono a me ka nānā ʻana i nā bioinformatics hou aʻe ma R (v4.2.3), me ka hoʻohana nui ʻana i ke kaʻina hana Seurat (v4.4.0). 83 No laila, ua hoʻololi ʻia nā waiwai UMI pākahi a me nā matrices metadata i mau mea Seurat. Ua wehe ʻia nā genes i hōʻike ʻia ma lalo o 30% o nā fibers āpau. Ua wehe ʻia nā laʻana haʻahaʻa ma muli o ka paepae liʻiliʻi o 1000 mau waiwai UMI a me 1000 mau genes i ʻike ʻia. I ka hopena, ua hala nā fibers 925 i nā ʻanuʻu kānana mana maikaʻi āpau. Ua hoʻonohonoho ʻia nā waiwai UMI me ka hoʻohana ʻana i ke ʻano Seurat SCTransform v2, 84 me nā hiʻohiʻona 7418 i ʻike ʻia, a ua hoʻihoʻi ʻia nā ʻokoʻa ma waena o ka poʻe komo. Hiki ke loaʻa nā metadata pili āpau ma ka Supplementary Dataset 28.