Te rerekētanga o ngā muka uaua anga tangata i tua atu i te mekameka taumaha myosin

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He kiko rerekē te uaua angaanga, he mea hanga te nuinga ki ngā myofibril, ā, i roto i te tangata ka whakarōpūtia kia toru ngā momo: tētahi "pūhoi" (momo 1) me ētahi atu "tere" (momo 2A ​​me 2X). Heoi, kāore anō kia tino mārama te rerekētanga i waenga i ngā momo myofibril tuku iho me roto hoki. I whakamahia e mātou ngā huarahi transcriptomic me te proteomic ki ngā myofibril takitahi 1050 me 1038 mai i te vastus lateralis tangata. I whakaurua ki te rangahau proteomic ngā tāne, ā, i whakaurua ki te rangahau transcriptomic ngā tāne 10 me ngā wāhine 2. Haunga ngā momo mekameka taumaha myosin, i tautuhia e mātou ngā pūmua pākia, ngā pūmua ribosomal, me ngā pūmua hononga pūtau hei pūtake o te rerekētanga intermyofibril maha-ahu. I tua atu, ahakoa te tautuhi i ngā tautau o ngā muka puhoi me te tere, e kī ana ā mātou raraunga kāore e taea te wehewehe i ngā muka momo 2X mai i ētahi atu muka tere-twitch. I tua atu, kāore i te rawaka te whakarōpūtanga e hangai ana ki te mekameka taumaha myosin hei whakaahua i te momo myofiber i roto i ngā myopathies nemaline. I te nuinga, e kī ana ā mātou raraunga he rerekētanga maha-ahu o ngā muka myosin, me ngā pūtake rerekētanga e toro atu ana ki tua atu i ngā momo mekameka taumaha myosin.
Ko te rerekētanga pūtau he āhuatanga tūturu o ngā pūnaha koiora katoa, e āhei ai ngā pūtau ki te motuhake hei whakatutuki i ngā hiahia rerekē o ngā kiko me ngā pūtau.1 Ko te tirohanga tuku iho mō te rerekētanga o te muka uaua angaanga ko ngā neuron motuka e tautuhi ana i te momo muka i roto i te waeine motuka, ā, ko te momo muka (arā, te momo 1, te momo 2A, me te momo 2X i roto i te tangata) ka whakatauhia e ngā āhuatanga o ngā isoforms myosin heavy chain (MYH).2 I te tuatahi i ahu mai tēnei i tō rātou pH ATPase kore pumau,3,4 ā, i muri mai i tō rātou whakaaturanga ngota o MYH.5 Heoi, me te tautuhi me te whakaae i muri mai o ngā muka "whakaranu" e whakaatu tahi ana i ngā MYH maha i roto i ngā ōwehenga rerekē, ka nui haere te tirohanga o ngā muka uaua angaanga hei kaupapa tonu kaua hei momo muka motuhake.6 Ahakoa tēnei, kei te whakawhirinaki tonu te mara ki te MYH hei whakarōpū matua mō te whakarōpūtanga myofiber, he tirohanga pea i awehia e ngā herenga me ngā whakaaro nui o ngā rangahau kiore o mua e rerekē ana ngā kōtaha whakaaturanga MYH me te whānuitanga o ngā momo muka i ērā i roto i te tangata.2 Ka uaua ake te āhuatanga nā te mea he rerekē ngā momo muka e whakaatuhia ana e ngā uaua angaanga tangata rerekē.7 He uaua whakauru te vastus lateralis me te āhua whakapuakanga MYH waenga (nō reira he tohu).7 Hei tāpiri, nā te ngāwari o te tauira, koinei te uaua tino rangahauhia i roto i te tangata.
Nō reira, he mea nui te rangahau kore whakaaro mō te kanorau o ngā muka uaua angaanga mā te whakamahi i ngā taputapu "omics" kaha, engari he wero anō hoki, nā te āhua maha-kaupapa o ngā muka uaua angaanga. Heoi, kua huri te aro o ngā hangarau transcriptomics8,9 me te proteomics10 i ngā tau tata nei nā ngā whanaketanga hangarau maha, e āhei ai te tātari i te uaua angaanga i te whakataunga muka kotahi. Nō reira, kua nui te ahunga whakamua ki te whakaahua i te kanorau muka kotahi me tā rātou urupare ki ngā whakaohooho ngoikore me te koroheketanga11,12,13,14,15,16,17,18. He mea nui, he tono haumanu ēnei whanaketanga hangarau, e āhei ai te whakaahua taipitopito me te tika o te korenga o te whakahaere e pā ana ki te mate. Hei tauira, he uaua, he whakapoauau hoki te pathophysiology o te nemaline myopathy, tētahi o ngā mate uaua tuku iho tino noa (MIM 605355 me te MIM 161800).19,20 Nō reira, ko te whakaahua pai ake i te korenga o te whakahaere o ngā muka uaua angaanga ka arahi pea ki ngā whanaketanga nui i roto i tō tātou māramatanga ki tēnei mate.
I whakawhanakehia e mātou ngā tikanga mō te tātaritanga transcriptomic me te proteomic o ngā muka uaua angaanga kotahi i wehea ā-ringa mai i ngā tauira koiora tangata, ā, i whakamahia ki ngā mano tini muka, kia taea ai e mātou te rangahau i te rerekētanga pūtau o ngā muka uaua angaanga tangata. I roto i tēnei mahi, i whakaaturia e mātou te kaha o te phenotyping transcriptomic me te proteomic o ngā muka uaua, ā, i tautuhia ngā pūmua metabolic, ribosomal, me te cellular junctional hei pūtake nui o te rerekētanga interfiber. I tua atu, mā te whakamahi i tēnei rerenga mahi proteomic, i whakaahuahia e mātou te hiranga haumanu o te myopathy nematode i roto i ngā muka uaua angaanga kotahi, e whakaatu ana i te nekehanga taurite ki ngā muka kore-waikura motuhake i runga i te momo muka i runga i te MYH.
Hei tirotiro i te rerekētanga o ngā muka uaua angaanga tangata, i whakawhanakehia e mātou ngā rerenga mahi e rua hei whakahohe i te tātaritanga transcriptome me te proteome o ngā muka uaua angaanga kotahi (Pikitia 1A me te Pikitia Tāpiri 1A). I ​​whakawhanakehia e mātou, ā, i arotauhia hoki ētahi mahi tikanga, mai i te rokiroki tauira me te tiaki i te RNA me te pono o te pūmua ki te arotau i te putanga mō ia huarahi. Mō te tātaritanga transcriptome, i tutuki tēnei mā te whakauru i ngā paewaehere ngota ngota motuhake ki te tauira i te taahiraa tuatahi o te tāruatanga whakamuri, e taea ai te whakakotahi i ngā muka 96 mō te tukatuka whai hua i raro iho. Ko te raupapatanga hohonu ake (±1 miriona pānui mō ia muka) ki te whakataurite ki ngā huarahi pūtau-kotahi tuku iho i whakarei ake i ngā raraunga transcriptome. 21 Mō ngā proteomics, i whakamahia e mātou he gradient chromatographic poto (21 meneti) i honoa ki te kohikohinga raraunga DIA-PASEF i runga i te spectrometer papatipu timsTOF hei arotau i te hohonutanga o te proteome me te pupuri tonu i te putanga teitei. 22,23 Hei tirotiro i te rerekētanga o ngā muka uaua angaanga hauora, i whakaahuahia e mātou ngā tuhinga o ngā muka takitahi 1,050 mai i ngā kaituku pakeke hauora 14 me ngā pūmua o ngā muka 1,038 mai i ngā kaituku pakeke hauora 5 (Ripanga Tāpiri 1). I roto i tēnei pepa, ka kiia ēnei huinga raraunga ko ngā tuhinga me ngā pūmua muka 1,000. I kitea e tā mātou huarahi he 27,237 ngā tuhinga katoa me te 2,983 ngā pūmua i roto i ngā tātaritanga tuhinga me te pūmua muka 1,000 (Pikitia 1A, Huinga Raraunga Tāpiri 1–2). I muri i te tātari i ngā huinga raraunga tuhinga me te pūmua mō ngā ira >1,000 kua kitea me te 50% o ngā uara tika mō ia muka, i mahia ngā tātaritanga koiora mō ngā muka 925 me te 974 i roto i te tuhinga me te pūmua. I muri i te tātari, i kitea he toharite o te 4257 ± 1557 ngā ira me te 2015 ± 234 ngā pūmua (toharite ± SD) mō ia muka, me te iti o te rerekētanga i waenga i ngā tāngata takitahi (Ngā Pikitia Tāpiri 1B–C, Ngā Huinga Raraunga Tāpiri 3–4). Heoi, i kitea ake te rerekētanga i roto i ngā kaupapa puta noa i ngā kaiuru, nā te rerekētanga pea o te hua RNA/pūmua i waenga i ngā muka o ngā roa rerekē me ngā horahanga whakawhiti-wāhanga. Mō te nuinga o ngā pūmua (>2000), i raro iho i te 20% te tauwehenga rerekētanga (Pikitia Tāpiri 1D). Nā ngā tikanga e rua i taea ai te hopu i te whānuitanga hihiri o ngā tuhinga me ngā pūmua me ngā waitohu tino kitea he mea nui mō te whakaheke uaua (hei tauira, ACTA1, MYH2, MYH7, TNNT1, TNNT3) (Ngā Pikitia Tāpiri 1E–F). Ko te nuinga o ngā āhuatanga i tautuhia he mea noa i waenga i ngā huinga raraunga transcriptomic me te proteomic (Pikitia Tāpiri 1G), ā, ko te kaha toharite o te UMI/LFQ o ēnei āhuatanga he pai te hononga (r = 0.52) (Pikitia Tāpiri 1H).
Te reremahi o te transcriptomics me te proteomics (i hangaia ki BioRender.com). BD Ngā kōpiko whānuitanga hihiri mō MYH7, MYH2, me MYH1, me ngā paepae kua tatauhia mō te tohatoha momo muka. E, F Te tohatoha o te whakaaturanga MYH puta noa i ngā muka i roto i ngā huinga raraunga transcriptomics me te proteomics. G, H Ngā kauwhata Whakatata me te Matapae Kanorau Kotahi (UMAP) mō ngā transcriptomics me te proteomics kua taehia e te momo muka e hangai ana ki te MYH. I, J Ngā kauwhata āhuatanga e whakaatu ana i te whakaaturanga MYH7, MYH2, me MYH1 i roto i ngā huinga raraunga transcriptomics me te proteomics.
I te tuatahi i whakatakoto mātou ki te tuku momo muka MYH ki ia muka mā te whakamahi i tētahi huarahi kua arotauhia e whakamahi ana i te tairongo teitei me te whānuitanga hihiri o te whakaaturanga MYH i roto i ngā huinga raraunga omics. Kua whakamahia e ngā rangahau o mua ngā paepae matapōkere hei tapanga i ngā muka hei momo parakore 1, momo 2A, momo 2X, he ranunga rānei i runga i te ōrau pumau o te whakaaturanga o ngā MYH rerekē11,14,24. I whakamahia e mātou tētahi huarahi rerekē i whakarōpūhia ai te whakaaturanga o ia muka e ngā MYH i whakamahia e mātou hei pato i ngā muka: MYH7, MYH2, me MYH1, e rite ana ki ngā muka momo 1, momo 2A, me te momo 2X. Kātahi ka tatauhia e mātou te pūwāhi piko o raro o ia pihi hua, ā, i whakamahia hei paepae hei tuku muka hei pai (kei runga ake i te paepae) hei kino rānei (kei raro iho i te paepae) mō ia MYH (Pikitia 1B–D). E whakaatu ana ēnei raraunga he rerekē ake ngā kōtaha whakaaturanga whakakā/whakaweto a MYH7 (Pikitia 1B) me MYH2 (Pikitia 1C) i te taumata RNA ki te taumata pūmua. Ina hoki, i te taumata pūmua, he iti noa ngā muka kāore i whakaatu i te MYH7, ā, kāore he muka i 100% te whakaaturanga MYH2. I muri mai ka whakamahia e mātou ngā paepae whakaaturanga kua whakaritea hei tohu i ngā momo muka e hangai ana ki te MYH ki ngā muka katoa i roto i ia huinga raraunga. Hei tauira, i tohua ngā muka MYH7+/MYH2-/MYH1- ki te momo 1, ko ngā muka MYH7-/MYH2+/MYH1+ i tohua ki te momo whakauru 2A/2X (tirohia te Ripanga Tāpiri 2 mō te whakaahuatanga katoa). I te whakakotahi i ngā muka katoa, i kite mātou i te tohatoha tino ōrite o ngā momo muka e hangai ana ki te MYH i ngā taumata RNA (Pikitia 1E) me te pūmua (Pikitia 1F), i te mea he rerekē te hanganga whanaunga o ngā momo muka e hangai ana ki te MYH puta noa i ngā tāngata takitahi, e ai ki te tumanako (Pikitia Tāpiri 2A). Ko te nuinga o ngā muka i whakarōpūtia hei momo 1 parakore (34–35%), hei momo 2A ​​rānei (36–38%), ahakoa he maha ngā muka momo whakauru 2A/2X i kitea hoki (16–19%). He rerekētanga whakamiharo ko ngā muka momo 2X parakore ka kitea anake i te taumata RNA, engari kāore i te taumata pūmua, e kī ana ko te tere o te whakaputanga MYH ka whakahaeretia i muri i te tuhi.
I whakamanahia e mātou tā mātou tikanga momo muka MYH e ahu mai ana i te proteomics mā te whakamahi i te ira-ira e ahu mai ana i te antibody, ā, i tutuki i ngā tikanga e rua te 100% o te whakaae ki te tautuhi i ngā muka momo 1 me te momo 2A ​​parakore (tirohia te Pikitia Tāpiri 2B). Heoi, he tairongo ake te huarahi e ahu mai ana i te proteomics, he pai ake te tautuhi i ngā muka whakauru, me te ine i te ōwehenga o ia ira MYH i roto i ia muka. E whakaatu ana ēnei raraunga i te whai huatanga o te whakamahi i tētahi huarahi omics whai kiko, tino tairongo ki te tautuhi i ngā momo muka uaua anga.
I muri mai ka whakamahia e mātou ngā mōhiohio kua whakakotahitia e te transcriptomics me te proteomics hei whakarōpū i ngā myofibers i runga i tō rātou transcriptome, proteome rānei katoa. Mā te whakamahi i te tikanga uniform manifold approximation and projection (UMAP) hei whakaiti i te whānuitanga ki ngā wāhanga matua e ono (Pikitia Tāpiri 3A–B), i taea e mātou te whakaatu i te rerekētanga o ngā myofibers i roto i te transcriptome (Pikitia 1G) me te proteome (Pikitia 1H). He mea nui, kāore i whakarōpūhia ngā myofibers e ngā kaiuru (Pikitia Tāpiri 3C–D) me ngā rā whakamātautau (Pikitia Tāpiri 3E) i roto i ngā huinga raraunga transcriptomics, proteomics rānei, e kī ana he teitei ake te rerekētanga o ngā muka uaua angaanga i roto i te kaupapa i te rerekētanga i waenga i te kaupapa. I roto i te kauwhata UMAP, e rua ngā rōpū motuhake e tohu ana i ngā myofibers "tere" me te "pōturi" i puta mai (Pikitia 1G–H). I whakarōpūhia ngā muka mio MYH7+ (pōturi) ki te pou pai o UMAP1, ko ngā muka mio MYH2+ me MYH1+ (tere) i whakarōpūhia ki te pou kino o UMAP1 (Pikitia 1I–J). Heoi, kāore i wehewehea i waenga i ngā momo muka tere-twitch (arā, momo 2A, momo 2X, 2A/2X whakauru rānei) i runga i te whakaaturanga MYH, e kī ana ko te whakaaturanga MYH1 (Pikitia 1I–J) me ētahi atu tohu muka mio 2X tawhito pērā i te ACTN3, MYLK2 rānei (Pikitia Tāpiri 4A–B) kāore e wehewehe i waenga i ngā momo muka mio rerekē ina whakaarohia te transcriptome katoa, te proteome rānei. Heoi anō, ki te whakatauritea ki te MYH2 me te MYH7, he iti noa ngā tuhinga, ngā pūmua rānei i hono pai ki te MYH1 (Pikitia Tāpiri 4C–H), e kī ana ko te nui o te MYH1 kāore e whakaata katoa i te transcriptome/proteome o te muka mio. I tae atu ngā whakatau ōrite i te aromatawai i te whakaaturanga whakauru o ngā momo MYH e toru i te taumata UMAP (Ngā Pikitia Tāpiri 4I–J). Nō reira, ahakoa ka taea te tautuhi i ngā muka 2X i te taumata tuhinga i runga i te ine MYH anake, kāore e taea te wehewehe i ngā muka MYH1+ mai i ētahi atu muka tere ina whakaarohia te transcriptome katoa, te proteome rānei.
Hei tūhuratanga tuatahi mō te rerekētanga o te muka puhoi i tua atu i te MYH, i arotakehia e mātou ētahi pūmua motuhake e whā o te muka puhoi: TPM3, TNNT1, MYL3, me ATP2A22. I whakaatuhia e ngā momo muka puhoi he hononga Pearson teitei, ahakoa kāore i tino tika, ki te MYH7 i roto i ngā mahi transcriptomics (Pikitia Tāpiri 5A) me ngā mahi proteomics (Pikitia Tāpiri 5B). Tata ki te 25% me te 33% o ngā muka puhoi kāore i whakarōpūtia hei muka puhoi parakore e ngā momo ira/pūmua katoa i roto i ngā mahi transcriptomics (Pikitia Tāpiri 5C) me ngā mahi proteomics (Pikitia Tāpiri 5D). Nō reira, ko te whakarōpūtanga muka puhoi i runga i ngā momo ira/pūmua maha ka whakauru i te uauatanga tāpiri, ahakoa mō ngā pūmua e mōhiotia ana he momo muka motuhake. E kī ana tēnei ko te whakarōpūtanga muka i runga i ngā momo isoforms o tētahi whānau ira/pūmua kotahi, kāore pea e whakaata tika i te rerekētanga tūturu o ngā muka uaua angaanga.
Hei tūhura atu i te rerekētanga āhuahanga o ngā muka uaua anga tangata i te tauine o te tauira omics katoa, i whakahaerehia e mātou he whakaiti i te whānuitanga kore whakaaro o ngā raraunga mā te whakamahi i te tātaritanga wāhanga matua (PCA) (Pikitia 2A). Pērā i ngā kauwhata UMAP, kāore te kaiuru me te rā whakamātautau i awe i te whakarōpū muka i te taumata PCA (Ngā Pikitia Tāpiri 6A–C). I roto i ngā huinga raraunga e rua, i whakamāramahia te momo muka e hangai ana ki te MYH e te PC2, i whakaatu i te rōpū muka momo 1 puhoi-ngoikore me te rōpū tuarua kei roto ko te momo 2A ​​tere-ngoikore, momo 2X, me ngā muka 2A/2X whakauru (Pikitia 2A). I ​​roto i ngā huinga raraunga e rua, i honoa ēnei rōpū e rua e te iti o ngā muka momo 1/2A whakauru. E ai ki te tumanako, i whakaū te tātaritanga nui rawa o ngā taraiwa PC matua i peia a PC2 e ngā waitohu kirimana me te pākia (Pikitia 2B me ngā Pikitia Tāpiri 6D–E, Huinga Raraunga Tāpiri 5–6). I te nuinga, i kitea he rawaka te momo muka MYH hei whakamārama i te rerekētanga tonu i te taha o te PC2, haunga ngā muka e kiia nei ko te 2X i horahia puta noa i te transcriptome i roto i te tautau tere.
A. Ngā kauwhata tātaritanga wāhanga matua (PCA) o ngā huinga raraunga transcriptome me te proteome i taehia kia rite ki te momo muka i runga i te MYH. B. Te tātaritanga whakarei ake i ngā taraiwa transcriptome me te pūmua i roto i te PC2 me te PC1. I whakahaerehia te tātaritanga tatauranga mā te whakamahi i te kete clusterProfiler me ngā uara-p kua whakatikatikaina e Benjamini-Hochberg. C, D. Ngā kauwhata PCA i taehia kia rite ki ngā kupu ontology ira pirihono (GO) i roto i ngā kupu transcriptome me te costamere GO i roto i te proteome. Ko ngā pere e tohu ana i ngā taraiwa transcriptome me te pūmua me ō rātou ahunga. E, F. Ngā kauwhata āhuatanga whakatata me te whakaaturanga manifold ōrite (UMAP) o ngā āhuatanga e pā ana ki te haumanu e whakaatu ana i ngā rōnaki whakapuakanga motuhake mai i te momo muka puhoi/tere. G, H. Ngā hononga i waenga i ngā taraiwa PC2 me te PC1 i roto i ngā transcriptome me ngā proteome.
I te mea ohorere, ko te momo myofiber e hangai ana ki te MYH te mea i whakamārama noa i te tuarua o ngā taumata rerekētanga teitei (PC2), e kī ana ko ētahi atu āhuatanga koiora kāore e pā ana ki te momo myofiber e hangai ana ki te MYH (PC1) he mea nui ki te whakahaere i te rerekētanga o ngā muka uaua angaanga. Nā te tātaritanga nui rawa atu o ngā taraiwa matua i roto i te PC1 i whakaatu ko te rerekētanga i roto i te PC1 i whakatauhia tuatahitia e te piri o te pūtau-pūtau me te ihirangi ribosome i roto i te transcriptome, me ngā costamere me ngā pūmua ribosomal i roto i te proteome (Pikitia 2B me ngā Pikitia Tāpiri 6D–E, Huinga Raraunga Tāpiri 7). I roto i te uaua angaanga, ka hono ngā costamere i te kōpae-Z ki te sarcolemma, ā, ka uru ki te tuku kaha me te tohu. 25 Ngā kauwhata PCA kua whakamāramahia mā te whakamahi i te piri o te pūtau-pūtau (transcriptome, Pikitia 2C) me ngā āhuatanga costamere (proteome, Pikitia 2D) i whakaatu i te nekehanga maui kaha i roto i te PC1, e tohu ana kei roto i ētahi muka ēnei āhuatanga.
I roto i tētahi tirotiro taipitopito ake i te whakarōpūtanga o ngā muka myofiber i te taumata UMAP, i kitea ko te nuinga o ngā āhuatanga he rerekētanga whakapuakanga e ahu mai ana i te momo muka myofiber, kaua i te whakarōpūtanga iti o ngā muka myofiber. I kitea tēnei haere tonu mō ētahi ira e pā ana ki ngā āhuatanga mate (Pikitia 2E), pērā i te CHCHD10 (mate neuromuscular), te SLIT3 (te ngoikore o te uaua), te CTDNEP1 (mate uaua). I kitea anō hoki tēnei haere tonu puta noa i te proteome, tae atu ki ngā pūmua e pā ana ki ngā mate neurological (UGDH), te tohu insulin (PHIP), me te tuhi (HIST1H2AB) (Pikitia 2F). I te katoa, e tohu ana ēnei raraunga i te haere tonu o te rerekētanga o te tere/puku puhoi e kore e uru ki te momo muka puta noa i ngā muka myofiber rerekē.
He mea whakamīharo, i whakaatuhia e ngā ira taraiwa i roto i te PC2 he hononga pai o te transcriptome-proteome (r = 0.663) (Pikitia 2G), e whakaatu ana ko ngā momo muka puhoi me te tere-tītaha, me ngā āhuatanga whakapūmau me te pākia o ngā muka uaua angaanga, ka whakahaeretia e te whakawhitiwhitinga. Heoi, kāore i whakaatuhia e ngā ira taraiwa i roto i te PC1 he hononga transcriptome-proteome (r = -0.027) (Pikitia 2H), e whakaatu ana ko ngā rerekētanga kāore e pā ana ki ngā momo muka puhoi/tere-tītaha ka whakahaeretia i muri i te whakawhitiwhitinga. Nā te mea ko ngā rerekētanga i roto i te PC1 i whakamāramahia e ngā kupu ontology ira ribosomal, ā, i te mea he mahi nui, he mahi motuhake hoki tā ngā ribosome i roto i te pūtau mā te whai wāhi kaha ki roto me te awe i te whakamaoritanga pūmua,31 ka tīmata mātou i muri mai ki te tirotiro i tēnei rerekētanga ribosomal ohorere.
I te tuatahi i taehia e mātou te kauwhata tātaritanga wāhanga matua o te proteomics i runga i te nui o ngā pūmua i roto i te kupu GOCC "cytoplasmic ribosome" (Pikitia 3A). Ahakoa he nui ake te whakanikoniko o tēnei kupu i te taha pai o PC1, ka hua ake he iti te pikinga, ko ngā pūmua ribosomal te mea e akiaki ana i te wehewehenga i ngā taha e rua o PC1 (Pikitia 3A). Ko ngā pūmua ribosomal i nui ake i te taha kino o PC1 ko RPL18, RPS18, me RPS13 (Pikitia 3B), ko RPL31, RPL35, me RPL38 (Pikitia 3C) ngā mea matua e akiaki ana i te taha pai o PC1. He mea whakamīharo, he nui te whakaaturanga o RPL38 me RPS13 i roto i te uaua angaanga ki te whakataurite ki ētahi atu kiko (Pikitia Tāpiri 7A). Kāore i kitea ēnei waitohu ribosomal motuhake i roto i te PC1 i roto i te transcriptome (Pikitia Tāpiri 7B), e tohu ana i te whakahaere i muri i te whakawhiti.
A. Kauwhata tātaritanga wāhanga matua (PCA) kua taehia kia rite ki ngā kupu ontology ira ribosomal cytoplasmic (GO) puta noa i te proteome. E tohu ana ngā pere i te ahunga o te rerekētanga e whakahaerehia ana e te pūmua i roto i te kauwhata PCA. E rite ana te roa o te rārangi ki te kaute wāhanga matua mō tētahi pūmua kua hoatu. B, C. Kauwhata āhuatanga PCA mō te RPS13 me te RPL38. D. Tātaritanga rōpū hierarchical kāore i tirotirohia o ngā pūmua ribosomal cytoplasmic. E. Tauira hanganga o te ribosome 80S (PDB: 4V6X) e whakaatu ana i ngā pūmua ribosomal me ngā momo nui rerekē i roto i ngā muka uaua angaiwi. F. Ngā pūmua ribosomal me ngā stoichiometry rerekē e tata ana ki te hongere putanga mRNA.
Kua whakaarohia kētia ngā ariā o te rerekētanga me te motuhaketanga o te ribosomal, e taea ai e te noho o ngā taupori ribosome motuhake (te rerekētanga o te ribosomal) te awe tika i te whakamaoritanga o te pūmua i roto i ngā kiko rerekē32 me ngā pūtau33 mā te whakamaoritanga whiriwhiri o ngā puna tuhinga mRNA motuhake34 (te motuhaketanga o te ribosome). Hei tautuhi i ngā taupori iti o ngā pūmua ribosomal e whakaatuhia ana i roto i ngā muka uaua angaiwi, i whakahaerehia e mātou he tātaritanga whakarōpū hierarchical kore tirotiro o ngā pūmua ribosomal i roto i te proteome (Pikitia 3D, Huinga Raraunga Tāpiri 8). E ai ki te tumanako, kāore ngā pūmua ribosomal i whakarōpūhia e te momo muka i runga i te MYH. Heoi, i tautuhia e mātou e toru ngā rōpū motuhake o ngā pūmua ribosomal; ko te rōpū tuatahi (rōpū_ribosomal_1) e whakahaerehia tahi ana me te RPL38, nō reira kua piki ake te whakaaturanga i roto i ngā muka me te kōtaha PC1 pai. Ko te rōpū tuarua (rōpū_ribosomal_2) e whakahaerehia tahi ana me te RPS13, ā, kua teitei ake i roto i ngā muka me te kōtaha PC1 kino. Kāore te rōpū tuatoru (rōpū_ribosomal_3) e whakaatu i te whakaaturanga rerekētanga taurite i roto i ngā muka uaua angaanga, ā, ka taea te kī ko te pūmua ribosomal uaua angaanga "matua". Kei roto i ngā rōpū ribosomal 1 me te 2 ngā pūmua ribosomal kua whakaaturia i mua hei whakahaere i te whakamaoritanga rerekē (hei tauira, RPL10A, RPL38, RPS19, me te RPS25) ā, he awe mahi ki te whanaketanga (hei tauira, RPL10A, RPL38).34,35,36,37,38 E rite ana ki ngā hua PCA, ko te whakaaturanga kanorau o ēnei pūmua ribosomal puta noa i ngā muka i whakaatu hoki i te haere tonu (Pikitia Tāpiri 7C).
Hei tiro i te taunga o ngā pūmua ribosomal rerekē i roto i te ribosome, i whakamahia e mātou he tauira hanganga o te ribosome 80S tangata (Pūtake Raraunga Pūmua: 4V6X) (Pikitia 3E). Whai muri i te wehewehe i ngā pūmua ribosomal nō ngā tautau ribosomal rerekē, kāore i tino ōrite ō rātou taunga, e kī ana kāore i taea e tā mātou huarahi te whakarato i te whakarei ake mō ētahi rohe/wāhanga o te ribosome. Heoi, he mea whakamīharo, ko te ōwehenga o ngā pūmua wāhanga nui i roto i te tautau 2 he iti iho i ngā tautau 1 me te 3 (Pikitia Tāpiri 7D). I kitea e mātou ko ngā pūmua me te stoichiometry kua whakarerekētia i roto i ngā muka uaua angaiwi i te nuinga o te wā kei te mata o te ribosome (Pikitia 3E), e rite ana ki tō rātou kaha ki te taunekeneke me ngā huānga wāhi whakauru ribosome ā-roto (IRES) i roto i ngā taupori mRNA rerekē, nā reira ka whakarite i te whakamaoritanga whiriwhiri. 40, 41 I tua atu, he maha ngā pūmua me te stoichiometry kua whakarerekētia i roto i ngā muka uaua angaiwi i tata ki ngā rohe mahi pēnei i te kauhanga putanga mRNA (Pikitia 3F), e whakahaere whiriwhiri ana i te roa o te whakamaoritanga me te aukati i ngā peptides motuhake. 42 Hei whakarāpopototanga, e kī ana ā mātou raraunga he rerekē te stoichiometry o ngā pūmua ribosomal uaua angaiwi, ā, ka hua ake he rerekētanga i waenga i ngā muka uaua angaiwi.
I muri mai ka tīmata mātou ki te tautuhi i ngā waitohu muka tere-me-te-piko-ā-roto, me te tūhura i ngā tikanga o tā rātou whakahaere i te whakawhitiwhiti kōrero. I te whakataurite i ngā tautau muka tere-me-te-piko-ā-roto i tautuhia e UMAP i roto i ngā huinga raraunga e rua (Pikitia 1G–H me 4A–B), i kitea e ngā tātaritanga whakawhitiwhiti kōrero me te proteomic he 1366 me te 804 ngā āhuatanga rerekē te nui, ia (Pikitia 4A–B, Huinga Raraunga Tāpiri 9–12). I kitea e mātou ngā rerekētanga e tumanakohia ana i roto i ngā waitohu e pā ana ki ngā sarcomeres (hei tauira, te tropomyosin me te troponin), te hononga whakaoho-whakaiti (ngā momo SERCA), me te pākia o te pūngao (hei tauira, te ALDOA me te CKB). I tua atu, ko ngā whakawhitiwhiti kōrero me ngā pūmua e whakahaere ana i te ubiquitination pūmua i whakaatuhia i roto i ngā muka tere-me-te-piko-ā-roto (hei tauira, te USP54, SH3RF2, USP28, me te USP48) (Pikitia 4A–B). I tua atu, ko te ira pūmua moroiti RP11-451G4.2 (DWORF), kua whakaaturia i mua he rerekē te whakaaturanga puta noa i ngā momo muka uaua reme43 me te whakarei ake i te mahi SERCA i roto i te uaua ngākau44, i tino piki ake i roto i ngā muka uaua angaanga puhoi (Pikitia 4A). Waihoki, i te taumata muka takitahi, i kitea ngā rerekētanga nui i roto i ngā waitohu e mōhiotia ana pērā i ngā isoforms lactate dehydrogenase e pā ana ki te metabolism (LDHA me te LDHB, Pikitia 4C me te Pikitia Tāpiri 8A)45,46 me ngā waitohu motuhake momo muka kāore i mōhiotia i mua (pērā i te IRX3, USP54, USP28, me te DPYSL3) (Pikitia 4C). He nui te taupatupatu o ngā āhuatanga rerekē te whakaaturanga i waenga i ngā huinga raraunga transcriptomic me te proteomic (Pikitia Tāpiri 8B), me te hononga huringa paparanga i akiakihia e te whakaaturanga rerekētanga nui ake o ngā āhuatanga sarcomere (Pikitia Tāpiri 8C). He mea nui, ko ētahi waitohu (hei tauira, USP28, USP48, GOLGA4, AKAP13) i whakaatu i te kaha o te whakahaere i muri i te whakawhiti i te taumata proteomic anake, ā, he puhoi/tere hoki ngā kōtaha whakapuakanga motuhake mō te momo muka tītaha (Pikitia Tāpiri 8C).
Ngā kauwhata puia A me B e whakataurite ana i ngā tautau puhoi me ngā tautau tere i tautuhia e ngā kauwhata whakatata me te whakaaturanga manifold ōrite (UMAP) i roto i ngā Pikitia 1G–H. Ko ngā ira tae e tohu ana i ngā tuhinga, i ngā pūmua rānei he tino rerekē i te FDR < 0.05, ā, ko ngā ira pōuri ake e tohu ana i ngā tuhinga, i ngā pūmua rānei he tino rerekē i te huringa takiuru > 1. I whakahaerehia te tātari tatauranga rua-ara mā te whakamahi i te whakamātautau DESeq2 Wald me ngā uara p kua whakatikatikaina e Benjamini–Hochberg (transcriptomics) me te tikanga tauira raina Limma rānei me te tātari Bayesian empirical i whai ake ko te whakatikatika Benjamini–Hochberg mō ngā whakataurite maha (proteomics). C Ngā kauwhata waitohu o ngā ira, ngā pūmua rānei kua rerekē te whakaaturanga i waenga i ngā muka puhoi me te tere. D Te tātari whakarei ake i ngā tuhinga me ngā pūmua kua tino rerekē te whakaaturanga. Ka whakareihia ngā uara taupatupatu i roto i ngā huinga raraunga e rua, ka whakareihia ngā uara transcriptome i roto i te transcriptome anake, ā, ka whakareihia ngā uara proteome i roto i te proteome anake. I whakahaerehia te tātari tatauranga mā te whakamahi i te mōkihi clusterProfiler me ngā uara-p kua whakatikatikaina e Benjamini-Hochberg. E. Ngā āhuatanga whakawhiti motuhake ki te momo muka i tautuhia e SCENIC i runga i ngā kaute motuhake o te kaiwhakahaere i ahu mai i te SCENIC me te rerekētanga o te whakaaturanga mRNA i waenga i ngā momo muka. F. Te whakaahua i ngā āhuatanga whakawhiti kua tīpakohia kua rerekē te whakaaturanga i waenga i ngā muka puhoi me ngā muka tere.
I muri mai ka whakahaerehia e mātou he tātaritanga nui rawa atu o ngā ira me ngā pūmua kua rerekē te whakaaturanga (Pikitia 4D, Huinga Raraunga Tāpiri 13). Ko te whakarei ake i te ara mō ngā āhuatanga i rerekē i waenga i ngā huinga raraunga e rua i whakaatu i ngā rerekētanga e tumanakohia ana, pērā i te β-oxidation waikawa ngako me ngā tukanga pākia ketone (ngā muka puhoi), te myofilament/te whakahekenga uaua (ngā muka tere me te puhoi), me ngā tukanga catabolic warowaihā (ngā muka tere). I piki ake hoki te mahi phosphatase pūmua serine/threonine i roto i ngā muka tere, i akiakihia e ngā āhuatanga pērā i ngā waeine phosphatase whakahaere me te catalytic (PPP3CB, PPP1R3D, me PPP1R3A), e mōhiotia ana hei whakahaere i te pākia glycogen (47) (Ngā Pikitia Tāpiri 8D–E). Ko ētahi atu ara i whakarei ake i ngā muka tere ko ngā tinana tukatuka (P-) (YTHDF3, TRIM21, LSM2) i roto i te proteome (Pikitia Tāpiri 8F), tera pea ka uru ki te whakahaere i muri i te whakawhiti (48), me te mahi tauwehenga whakawhiti (SREBF1, RXRG, RORA) i roto i te transcriptome (Pikitia Tāpiri 8G). I whakarei ake ngā muka puhoi i te mahi oxidoreductase (BDH1, DCXR, TXN2) (Pikitia Tāpiri 8H), te here amide (CPTP, PFDN2, CRYAB) (Pikitia Tāpiri 8I), te matrix extracellular (CTSD, ADAMTSL4, LAMC1) (Pikitia Tāpiri 8J), me te mahi receptor-ligand (FNDC5, SPX, NENF) (Pikitia Tāpiri 8K).
Hei whiwhi atu i te māramatanga ki te whakahaere i te whakawhitiwhiti kōrero e pā ana ki ngā āhuatanga momo muka uaua puhoi/tere, i whakahaerehia e mātou he tātaritanga whakarei ake i ngā āhuatanga whakawhitiwhiti kōrero mā te whakamahi i te SCENIC49 (Huinga Raraunga Tāpiri 14). He maha ngā āhuatanga whakawhitiwhiti kōrero i tino whakarei ake i waenga i ngā muka uaua tere me ngā muka uaua puhoi (Pikitia 4E). I roto i tēnei ko ngā āhuatanga whakawhitiwhiti kōrero pēnei i te MAFA, kua honoa i mua ki te whanaketanga muka uaua tere,50 me ētahi āhuatanga whakawhitiwhiti kōrero kāore i honoa i mua ki ngā kaupapa ira motuhake mō te momo muka uaua. I roto i ēnei, ko te PITX1, EGR1, me MYF6 ngā āhuatanga whakawhitiwhiti kōrero tino whakarei ake i roto i ngā muka uaua tere (Pikitia 4E). He rerekē, ko ZSCAN30 me EPAS1 (e mōhiotia ana ko HIF2A) ngā āhuatanga whakawhitiwhiti kōrero tino whakarei ake i roto i ngā muka uaua puhoi (Pikitia 4E). E rite ana ki tēnei, i whakaatuhia te MAFA i ngā taumata teitei ake i te rohe UMAP e rite ana ki ngā muka uaua tere, ko te EPAS1 he tauira whakaaturanga rerekē (Pikitia 4F).
Haunga ngā ira e mōhiotia ana e whakakōrero ana i te pūmua, he maha ngā momo RNA kore-kōrero e whai wāhi ana ki te whakahaere i te whanaketanga me te mate o te tangata. 51, 52 I roto i ngā huinga raraunga transcriptome, he maha ngā RNA kore-kōrero e whakaatu ana i te motuhaketanga momo muka (Pikitia 5A me te Huinga Raraunga Tāpiri 15), tae atu ki a LINC01405, he tino motuhake mō ngā muka puhoi, ā, e kīia ana kua heke iho i roto i te uaua mai i ngā tūroro e pāngia ana e te myopathy mitochondrial. 53 He rerekē, ko te RP11-255P5.3, e rite ana ki te ira lnc-ERCC5-5 (https://lncipedia.org/db/transcript/lnc-ERCC5-5:2) 54, e whakaatu ana i te motuhaketanga momo muka tere. E whakaatu ana a LINC01405 (https://tinyurl.com/x5k9wj3h) me RP11-255P5.3 (https://tinyurl.com/29jmzder) i te motuhaketanga o te uaua angaanga (Ngā Pikitia Tāpiri 9A–B) ā, kāore he ira whakapūmau e mōhiotia ana i roto i tō rātou rohe ira 1 Mb, e kī ana he mahi motuhake tā rātou ki te whakahaere i ngā momo muka kaua ki te whakahaere i ngā ira whakapūmau tata. I whakaūhia ngā kōtaha whakapuakanga momo muka puhoi/tere o LINC01405 me RP11-255P5.3, ia, mā te whakamahi i te RNAscope (Ngā Pikitia 5B–C).
A. He nui te whakahaere i ngā tuhinga RNA kore-waehere i roto i ngā muka uaua puhoi me te tere-tītaha. B. Ngā whakaahua RNAscope e whakaatu ana i te motuhaketanga momo muka puhoi me te tere-tītaha o LINC01405 me RP11-255P5.3, ia. Pae tauine = 50 μm. C. Te ine i te whakaaturanga RNA kore-waehere motuhake momo myofiber e ai ki te RNAscope (n = 3 ngā koiora mai i ngā tāngata takitahi, e whakataurite ana i ngā muka uaua tere me te puhoi i roto i ia tāngata takitahi). I whakahaerehia te tātari tatauranga mā te whakamahi i te whakamātautau-t a te Tauira e rua-waewae. E whakaatu ana ngā kauwhata pouaka i te tau waenga me ngā hauwhā tuatahi me te tuatoru, me ngā ūpoko e tohu ana ki ngā uara iti rawa me te mōrahi. D. Te rere mahi tautuhi pūmua moroiti de novo (i hangaia ki BioRender.com). E. Ko te pūmua moroiti LINC01405_ORF408:17441:17358 e whakaatuhia ana i roto i ngā muka uaua angaanga puhoi (n=5 ngā koiora mai i ngā kaiuru motuhake, e whakataurite ana i ngā muka uaua tere me te puhoi i roto i ia kaiuru). I whakahaerehia te tātari tatauranga mā te whakamahi i te tikanga tauira raina Limm me te huarahi Bayesian empirical, ā, i whai ake ko te tikanga Benjamini-Hochberg mō ngā whakatairite maha me te whakatikatika uara-p. E whakaatu ana ngā kauwhata pouaka i te waenga, te hauwhā tuatahi me te tuatoru, me ngā ūkui e tohu ana ki ngā uara mōrahi/iti rawa.
Nō nā tata nei, kua whakaatuhia e ngā rangahau he maha ngā tuhinga kore-waehere e whakapaetia ana e whakamārama ana i ngā pūmua moroiti kua tāruatia, ko ētahi o ēnei e whakahaere ana i te mahi uaua. 44, 55 Hei tautuhi i ngā pūmua moroiti me te momo muka motuhake pea, i rapu mātou i tā mātou huinga raraunga pūmua muka 1000 mā te whakamahi i tētahi kōnae FASTA ritenga kei roto ngā raupapatanga o ngā tuhinga kore-waehere (n = 305) i kitea i roto i te huinga raraunga tuhinga muka 1000 (Pikitia 5D). I tautuhia e mātou he 197 ngā pūmua moroiti mai i te 22 ngā tuhinga rerekē, 71 o ēnei i rerekē te whakahaere i waenga i ngā muka uaua angaanga puhoi me te tere (Pikitia Tāpiri 9C me te Huinga Raraunga Tāpiri 16). Mō LINC01405, e toru ngā hua pūmua moroiti i tautuhia, ko tētahi o ēnei i whakaatu i te motuhaketanga muka puhoi rite ki tana tuhinga (Pikitia 5E me te Pikitia Tāpiri 9D). Nō reira, i tautuhia e mātou a LINC01405 hei ira e whakamārama ana i tētahi pūmua moroiti motuhake mō ngā muka uaua angaanga puhoi.
I whakawhanakehia e mātou he rerenga mahi whānui mō te whakaahuatanga proteomic whānui o ngā muka uaua takitahi, ā, i tautuhia ngā kaiwhakahaere o te rerekētanga o te muka i roto i ngā āhua hauora. I whakamahia e mātou tēnei rerenga mahi hei mārama ki te pānga o ngā myopathies nemaline ki te rerekētanga o te muka uaua angaiwi. Ko ngā myopathies nemaline he mate uaua tuku iho e puta ai te ngoikore o te uaua, ā, i roto i ngā tamariki e pāngia ana, ka puta mai he maha ngā raruraru tae atu ki te uaua manawa, te scoliosis, me te nekehanga iti o ngā peka. 19,20 I te nuinga o te wā, i roto i ngā myopathies nemaline, ko ngā momo mate i roto i ngā ira pēnei i te actin alpha 1 (ACTA1) ka hua ake he nui ake te hanganga myofiber muka puhoi-tītaha, ahakoa he rerekē tēnei pānga. Ko tētahi mea motuhake ko te troponin T1 nemaline myopathy (TNNT1), he nui ake ngā muka tere. Nō reira, ko te mārama ake ki te rerekētanga kei raro i te koretake o te whakahaere muka uaua angaiwi i kitea i roto i ngā myopathies nemaline ka āwhina pea ki te wetewete i te whanaungatanga uaua i waenga i ēnei mate me te momo myofiber.
Ki te whakatauritea ki ngā mana hauora (n=3 mō ia rōpū), ko ngā muka mio i wehea mai i ngā tūroro myopathy nemaline me ngā whakarerekētanga ira i roto i ngā ira ACTA1 me TNNT1 i whakaatu i te ngoikore, te ngoikore rānei o ngā muka mio (Pikitia 6A, Ripanga Tāpiri 3). He wero hangarau nui tēnei mō te tātari proteomic nā te iti o ngā rauemi e wātea ana. Ahakoa tēnei, i taea e mātou te kite i te 2485 pūmua i roto i te 272 muka mio angaanga. I muri i te tātari mō te iti rawa 1000 pūmua kua inehia mō ia muka, 250 muka i tukuna ki te tātari bioinformatics i muri mai. I muri i te tātari, he toharite o te 1573 ± 359 pūmua mō ia muka i inehia (Pikitia Tāpiri 10A, Huinga Raraunga Tāpiri 17–18). He mea nui, ahakoa te whakahekenga nui o te rahi o te muka, he iti noa te hekenga o te hohonutanga o te proteome o ngā tauira tūroro myopathy nemaline. Heoi anō, mā te tukatuka i ēnei raraunga mā te whakamahi i ā mātou ake kōnae FASTA (tae atu ki ngā tuhinga kore-waehere) i taea ai e mātou te tautuhi i ētahi pūmua moroiti e rima i roto i ngā muka angaanga mai i ngā tūroro myopathy nemaline (Huinga Raraunga Tāpiri 19). He whānui ake te whānuitanga hihiri o te proteome, ā, he pai te hononga o ngā pūmua katoa i roto i te rōpū whakahaere ki ngā hua o te tātaritanga proteome muka-1000 o mua (Pikitia Tāpiri 10B–C).
A. Ngā whakaahua mōkitokito e whakaatu ana i te ngoikore o te muka, te ngoikore rānei, me te kaha o ngā momo muka rerekē i runga i te MYH i roto i ngā mate pukupuku nemaline ACTA1 me te TNNT1 (NM). Pae tauine = 100 μm. Hei whakarite i te taea o te whakahou i te tae i roto i ngā tūroro ACTA1 me te TNNT1, e toru ngā koiora tūroro i peitahia kia rua, kia toru rānei ngā wā (e whā ngā wāhanga mō ia take) i mua i te whiriwhiri i ngā whakaahua māngai. B. Ngā ōwehenga momo muka i roto i ngā kaiuru i runga i te MYH. C. Kauwhata tātari wāhanga matua (PCA) o ngā muka uaua angaanga i roto i ngā tūroro me ngā mate pukupuku nemaline me ngā mana whakahaere. D. Ngā muka uaua angaanga mai i ngā tūroro me ngā mate pukupuku nemaline me ngā mana whakahaere i whakaaturia ki runga i tētahi kauwhata PCA i whakatauhia mai i ngā muka 1000 i tātarihia i te Pikitia 2. Hei tauira, ngā kauwhata puia e whakataurite ana i ngā rerekētanga i waenga i ngā kaiuru me ngā mate pukupuku nemaline ACTA1 me te TNNT1 me ngā mana whakahaere, me waenga i ngā kaiuru me ngā mate pukupuku nemaline ACTA1 me te TNNT1. Ko ngā porowhita tae e tohu ana i ngā pūmua i tino rerekē i te π < 0.05, ā, ko ngā ira pouri e tohu ana i ngā pūmua i tino rerekē i te FDR < 0.05. I whakahaerehia te tātaritanga tatauranga mā te whakamahi i te tikanga tauira raina Limma me ngā tikanga Bayesian empirical, ā, i whai ake ko te whakatikatika uara-p mō ngā whakataurite maha mā te whakamahi i te tikanga Benjamini-Hochberg. H. Tātaritanga whakarei ake i ngā pūmua rerekē te whakaaturanga puta noa i te proteome katoa me ngā muka momo 1 me te 2A. I whakahaerehia te tātaritanga tatauranga mā te whakamahi i te kete clusterProfiler me ngā uara-p kua whakatikatikaina e Benjamini-Hochberg. I, J. Ngā kauwhata tātaritanga wāhanga matua (PCA) i taehia e te matrix extracellular me ngā kupu ontology ira mitochondrial (GO).
Nā te mea ka taea e ngā myopathies nemaline te awe i te ōwehenga o ngā momo myofiber e whakaatu ana i te MYH i roto i te uaua angaiwi,19,20 i tirotirohia tuatahitia e mātou ngā momo myofiber e whakaatu ana i te MYH i roto i ngā tūroro he myopathies nemaline me ngā mana whakahaere. I whakatauhia e mātou te momo myofiber mā te whakamahi i tētahi tikanga kore whakaaro i whakaahuatia i mua mō te whakamātautau myofiber 1000 (Ngā Pikitia Tāpiri 10D–E) ā, i rahua anō ki te tautuhi i ngā myofibers 2X parakore (Pikitia 6B). I kitea e mātou he pānga rerekē o ngā myopathies nemaline ki te momo myofiber, i te mea e rua ngā tūroro he whakarerekētanga ACTA1 he nui ake te ōwehenga o ngā myofibers momo 1, engari e rua ngā tūroro he myopathy nemaline TNNT1 he iti iho te ōwehenga o ngā myofibers momo 1 (Pikitia 6B). Ina hoki, i heke te whakaaturanga o MYH2 me ngā momo troponin tere (TNNC2, TNNI2, me te TNNT3) i roto i ngā myopathies ACTA1-nemaline, engari i heke te whakaaturanga MYH7 i roto i ngā myopathies TNNT1-nemaline (Pikitia Tāpiri 11A). He rite tēnei ki ngā pūrongo o mua mō te whakawhiti momo myofiber heterogeneous i roto i ngā myopathies nemaline.19,20 I whakaūhia e mātou ēnei hua mā te immunohistochemistry, ā, i kitea ko ngā tūroro me te myopathy ACTA1-nemaline he nui ake te myofibers momo 1, engari ko ngā tūroro me te myopathy TNNT1-nemaline he tauira rerekē (Pikitia 6A).
I te taumata proteome muka-kotahi, i huihui ngā muka uaua angaanga mai i ngā tūroro myopathy nemaline ACTA1 me TNNT1 ki te nuinga o ngā muka whakahaere, ko ngā muka myopathy nemaline TNNT1 te nuinga o te wā i tino pāngia (Pikitia 6C). I tino kitea tēnei i te wā i tuhia ai ngā kauwhata tātaritanga wāhanga matua (PCA) o ngā muka kua pupuhihia mō ia tūroro, me ngā tūroro myopathy nemaline TNNT1 2 me 3 i puta mai i tawhiti rawa atu i ngā tauira whakahaere (Pikitia Tāpiri 11B, Huinga Raraunga Tāpiri 20). Hei mārama ake ki te whakataurite o ngā muka mai i ngā tūroro myopathy ki ngā muka hauora, i whakamahia e mātou ngā mōhiohio taipitopito i whiwhi mai i te tātaritanga proteomic o ngā muka 1,000 mai i ngā kaiuru pakeke hauora. I matapaetia e mātou ngā muka mai i te huinga raraunga myopathy (ngā tūroro myopathy nemaline ACTA1 me TNNT1 me ngā mana whakahaere) ki runga i te kauwhata PCA i whiwhi mai i te tātaritanga proteomic muka 1000 (Pikitia 6D). He rite te tohatoha o ngā momo muka MYH i te taha o te PC2 i roto i ngā muka whakahaere ki te tohatoha muka i whiwhi mai i te tātaritanga proteomic muka-1000. Heoi, ko te nuinga o ngā muka i roto i ngā tūroro myopathy nemaline i neke ki raro i te PC2, e taupatupatu ana ki ngā muka tere-twitch hauora, ahakoa te momo muka MYH taketake. Nō reira, ahakoa i whakaatuhia e ngā tūroro me te myopathy nemaline ACTA1 he nekehanga ki ngā muka momo 1 i te wā i inehia ai mā te whakamahi i ngā tikanga MYH, i nekehia e te myopathy nemaline ACTA1 me te myopathy nemaline TNNT1 te proteome muka uaua angaiwi ki ngā muka tere-twitch.
Kātahi ka whakatauritea tika e mātou ia rōpū tūroro ki ngā mana hauora, ā, ka tautuhia e mātou he 256 me te 552 ngā pūmua rerekē te whakaputanga i roto i ngā mate pukupuku nemaline ACTA1 me te TNNT1, ia (Pikitia 6E–G me te Pikitia Tāpiri 11C, Huinga Raraunga Tāpiri 21). I whakaatuhia e te tātari whakarei ira he hekenga taurite o ngā pūmua mitochondrial (Pikitia 6H–I, Huinga Raraunga Tāpiri 22). He mea whakamiharo, ahakoa te rerekētanga o te mana o ngā momo muka i roto i ngā mate pukupuku nemaline ACTA1 me te TNNT1, he tino motuhake tēnei hekenga i te momo muka e hangai ana ki te MYH (Pikitia 6H me ngā Pikitia Tāpiri 11D–I, Huinga Raraunga Tāpiri 23). E toru ngā pūmua moroiti i whakahaeretia hoki i roto i ngā mate pukupuku nemaline ACTA1, TNNT1 rānei. E rua o ēnei pūmua iti, ko ENSG00000215483_TR14_ORF67 (e mōhiotia ana ko LINC00598, ko Lnc-FOXO1 rānei) me ENSG00000229425_TR25_ORF40 (lnc-NRIP1-2), i whakaatu i te nui rerekētanga i roto i ngā muka pūtau momo 1 anake. Kua pūrongohia i mua ko ENSG00000215483_TR14_ORF67 he mahi tāna ki te whakahaere i te huringa pūtau. 56 I tētahi atu taha, i piki ake te ENSG00000232046_TR1_ORF437 (e rite ana ki a LINC01798) i roto i ngā muka pūtau momo 1 me te momo 2A ​​i roto i te myopathy ACTA1-nemaline ki te whakataurite ki ngā mana hauora (Pikitia Tāpiri 12A, Huinga Raraunga Tāpiri 24). He rerekē, kāore i tino pāngia ngā pūmua ribosomal e te nemaline myopathy, ahakoa i heke te RPS17 i ​​roto i te ACTA1 nemaline myopathy (Pikitia 6E).
I whakaatuhia hoki e te tātari whakarei ake te pikinga ake o ngā tukanga pūnaha ārai mate i roto i ngā mate pukupuku nemaline ACTA1 me TNNT1, i te mea i piki ake te piri o ngā pūtau i roto i te mate pukupuku nemaline TNNT1 (Pikitia 6H). Ko te whakarei ake o ēnei āhuatanga o waho i whakaatahia e ngā pūmua matrix o waho e neke ana i te PCA i roto i te PC1 me te PC2 i te ahunga kino (arā, ki ngā muka tino pāngia) (Pikitia 6J). I whakaatuhia e ngā rōpū tūroro e rua te pikinga ake o te whakaaturanga o ngā pūmua o waho e uru ana ki ngā urupare ārai mate me ngā tikanga whakatika sarcolemmal, pērā i ngā annexins (ANXA1, ANXA2, ANXA5)57,58 me tā rātou pūmua taunekeneke S100A1159 (Ngā Pikitia Tāpiri 12B–C). Kua pūrongohia i mua ko tēnei tukanga ka whakarei ake i roto i ngā mate pukupuku uaua60 engari, ki tā mātou mōhio, kāore i mua i honoa ki ngā mate pukupuku nemaline. Ko te mahi noa o tēnei mīhini ngota e hiahiatia ana mō te whakatika sarcolemmal i muri i te whara me te whakakotahitanga o ngā myocytes hou kua hangaia me ngā myofibers58,61. Nō reira, ko te pikinga ake o te ngohe o tēnei tukanga i roto i ngā rōpū tūroro e rua e tohu ana i tētahi urupare whakatikatika ki te whara i puta mai i te kore pumau o te myofiber.
He pai te hononga o ngā pānga o ia nemaline myopathy (r = 0.736) ā, i whakaatuhia he taupatupatu whaitake (Ngā Pikitia Tāpiri 11A–B), e tohu ana he rite tonu ngā pānga o te ACTA1 me te TNNT1 nemaline myopathy ki te proteome. Heoi, ko ētahi pūmua i whakahaeretia i roto i te ACTA1, TNNT1 rānei nemaline myopathy (Ngā Pikitia Tāpiri 11A me C). Ko te pūmua profibrotic MFAP4 tētahi o ngā pūmua tino piki ake i roto i te TNNT1 nemaline myopathy engari i noho tonu te rerekētanga i roto i te ACTA1 nemaline myopathy. Ko SKIC8, he wāhanga o te matatini PAF1C e kawenga ana mō te whakahaere i te tuhi ira HOX, i heke iho i roto i te TNNT1 nemaline myopathy engari kāore i pāngia i roto i te ACTA1 nemaline myopathy (Pikitia Tāpiri 11A). I te whakataurite tika i waenga i te ACTA1 me te TNNT1 nemaline myopathy, i kitea he nui ake te whakahekenga o ngā pūmua mitochondrial me te pikinga o ngā pūmua pūnaha ārai mate i roto i te TNNT1 nemaline myopathy (Pikitia 6G–H me ngā Pikitia Tāpiri 11C me 11H–I). He rite ēnei raraunga ki te nui ake o te atrophy/dystrophy i kitea i roto i te TNNT1 nemaline myopathy ki te whakataurite ki te TNNT1 nemaline myopathy (Pikitia 6A), e kī ana ko te TNNT1 nemaline myopathy te momo kino rawa atu o te mate.
Hei aromatawai mēnā kei te mau tonu ngā pānga o te nemaline myopathy i kitea i te taumata uaua katoa, i whakahaerehia e mātou he tātaritanga pūmua nui o ngā koiora uaua mai i te rōpū kotahi o ngā tūroro TNNT1 nemaline myopathy, ā, i whakaritea ki ngā mana whakahaere (n=3 mō ia rōpū) (Pikitia Tāpiri 13A, Huinga Raraunga Tāpiri 25). E ai ki te whakaaro, he tata te whanaungatanga o ngā mana whakahaere i roto i te tātaritanga wāhanga matua, engari ko ngā tūroro TNNT1 nemaline myopathy i whakaatu i te rerekētanga i waenga i ngā tauira teitei ake e rite ana ki te mea i kitea i roto i te tātaritanga muka kotahi (Pikitia Tāpiri 13B). I tāruatia e te tātaritanga nui ngā pūmua rerekē te whakaaturanga (Pikitia Tāpiri 13C, Huinga Raraunga Tāpiri 26) me ngā tukanga koiora (Pikitia Tāpiri 13D, Huinga Raraunga Tāpiri 27) i whakaatuhia mā te whakataurite i ngā muka takitahi, engari i ngaro te kaha ki te wehewehe i waenga i ngā momo muka rerekē, ā, i rahua ki te whakaaro mō ngā pānga mate rerekē puta noa i ngā muka.
Ina huihuia, e whakaatu ana ēnei raraunga ka taea e ngā proteomics myofiber kotahi te whakamārama i ngā āhuatanga koiora haumanu kāore e kitea e ngā tikanga kua tohua pēnei i te immunoblotting. Heoi anō, e whakaatu ana ēnei raraunga i ngā herenga o te whakamahi i te momo muka actin (MYH) anake hei whakaahua i te urutau phenotypic. Ina hoki, ahakoa he rerekē te whakawhiti momo muka i waenga i te actin me te troponin nemaline myopathies, ka wehea e ngā nemaline myopathies e rua te momo muka MYH mai i te pākia o te muka uaua angaiwi ki tētahi proteome uaua tere ake, iti iho hoki te oxidative.
He mea nui te rerekētanga o ngā pūtau kia tutuki ai ngā hiahia kanorau o ngā kiko. I roto i te uaua angaanga, ka whakaahuahia tēnei hei momo muka e tohuhia ana e ngā taumata rerekē o te whakaputa kaha me te ngenge. Heoi, e mārama ana he iti noa te wāhanga o te rerekētanga o ngā muka uaua angaanga e whakamāramahia ana e tēnei, he mea tino rerekē, he uaua, he maha ngā taha i te whakaaro o mua. Kua whakamāramahia e ngā whanaketanga hangarau ngā āhuatanga e whakahaere ana i ngā muka uaua angaanga. Inaa, e kī ana ā mātou raraunga kāore pea ngā muka momo 2X i te momo muka uaua angaanga motuhake. I tua atu, i tautuhia e mātou ngā pūmua pākia, ngā pūmua ribosomal me ngā pūmua e hono ana ki te pūtau hei whakatau matua mō te rerekētanga o ngā muka uaua angaanga. Mā te whakamahi i tā mātou rerenga mahi proteomic ki ngā tauira tūroro me te myopathy nematode, i whakaaturia anō e mātou kāore te momo muka e hangai ana ki te MYH e whakaata katoa ana i te rerekētanga o ngā uaua angaanga, inā koa ka raruraru te pūnaha. Inaa, ahakoa te momo muka e hangai ana ki te MYH, ka puta te myopathy nematode ki te neke ki ngā muka tere me te iti ake o te waikura.
Kua whakarōpūtia ngā muka uaua angaanga mai i te rautau 19. Nā ngā tātaritanga omics hou nei i taea ai e tātou te tīmata ki te mārama ki ngā kōtaha whakaaturanga o ngā momo muka MYH rerekē me ā rātou urupare ki ngā whakaohooho rerekē. E ai ki te whakaahuatanga i konei, he painga anō hoki ngā huarahi omics ko te aro nui ake mō te ine i ngā tohu momo muka i ngā tikanga tuku iho e hangai ana ki te paturopi, me te kore e whakawhirinaki ki te ine i tētahi tohu kotahi (e torutoru rānei) hei tautuhi i tētahi momo muka uaua angaanga. I whakamahia e mātou ngā rerenga mahi transcriptomic me te proteomic tautoko, ā, i whakauruhia ngā hua ki te tirotiro i te whakahaeretanga whakawhiti me te whai-whakawhiti o te rerekētanga muka i roto i ngā muka uaua angaanga tangata. Nā tēnei rerenga mahi i kore ai e taea te tautuhi i ngā muka momo-2X parakore i te taumata pūmua i roto i te vastus lateralis o tā mātou rōpū o ngā tāne taitamariki hauora. He rite tēnei ki ngā rangahau muka kotahi o mua i kitea he <1% ngā muka 2X parakore i roto i te vastus lateralis hauora, ahakoa me whakaū tēnei i roto i ētahi atu uaua ā muri ake nei. He mea whakamīharo te rerekētanga i waenga i te kitenga o ngā muka 2X tata parakore i te taumata mRNA me ngā muka 2A/2X whakauru anake i te taumata pūmua. Ehara te whakaaturanga mRNA isoform MYH i te circadian,67 e kī ana kāore pea tātou i "ngaro" i te tohu tīmatanga MYH2 i roto i ngā muka 2X āhua parakore i te taumata RNA. Ko tētahi whakamārama pea, ahakoa he whakapae noa iho, ko ngā rerekētanga o te pūmua me/ranei te pumau mRNA i waenga i ngā isoform MYH. Ina hoki, kāore he muka tere e 100% parakore mō tētahi isoform MYH, ā, kāore i te mārama mēnā ka hua ake he nui ōrite o MYH1 me MYH2 i te taumata pūmua i ngā taumata whakaaturanga mRNA MYH1 i roto i te awhe 70–90%. Heoi, ina whakaarohia te transcriptome katoa, te proteome rānei, ka taea e te tātaritanga rōpū te tautuhi māia i ngā rōpū motuhake e rua e tohu ana i ngā muka uaua angaanga puhoi me te tere, ahakoa te hanganga MYH tika. He rite tēnei ki ngā tātaritanga e whakamahi ana i ngā huarahi transcriptomic kotahi-karu, e tautuhi ana i ngā rōpū myonuclear motuhake e rua anake. 68, 69, 70 Haunga tēnā, ahakoa kua tautuhia e ngā rangahau proteomic o mua ngā muka momo 2X, kāore ēnei muka e kohikohi motuhake mai i ngā muka tere katoa, ā, he iti noa iho ngā pūmua rerekē te nui e whakaatuhia ana ki ērā atu momo muka i runga i te MYH. 14 E kī ana ēnei hua me hoki tātou ki te tirohanga o te tīmatanga o te rautau 20 mō te whakarōpūtanga o ngā muka uaua, i wehea ai ngā muka uaua angiangi tangata kia kaua e toru ngā akomanga motuhake i runga i te MYH, engari kia rua ngā rōpū i runga i ō rātou āhuatanga pākia me te whakawhāiti. 63
He mea nui ake, me whai whakaaro ki te rerekētanga o ngā muka uaua i runga i ngā taha maha. Kua tohu ngā rangahau "omics" o mua ki tēnei huarahi, e kī ana kāore ngā muka uaua angaanga e hanga i ngā tautau motuhake engari e whakaritea ana i runga i te haere tonu. 11, 13, 14, 64, 71 I konei, e whakaatu ana mātou, i tua atu i ngā rerekētanga o ngā āhuatanga whakawhāiti me te pākia o te uaua angaanga, ka taea te wehewehe i ngā muka uaua mā ngā āhuatanga e pā ana ki ngā taunekeneke pūtau-pūtau me ngā tikanga whakamāori. Ina hoki, i kitea e mātou te rerekētanga o ngā ribosome i roto i ngā muka uaua angaanga e whai wāhi ana ki te rerekētanga motuhake i ngā momo muka puhoi me te tere. Ko te take matua o tēnei rerekētanga nui o ngā muka uaua, motuhake i te momo muka puhoi me te tere, kāore anō kia mārama, engari tera pea e tohu ki te whakaritenga ā-wāhi motuhake i roto i ngā uaua uaua e aro pai ana ki ngā kaha me ngā kawenga motuhake,72 te whakawhitiwhiti kōrero motuhake ki te pūtau, ki te okana rānei me ētahi atu momo pūtau i roto i te taiao moroiti uaua73,74,75 ngā rerekētanga rānei o te mahi ribosome i roto i ngā muka uaua takitahi. Ina hoki, kua whakaatuhia te hononga o te heteroplasmy ribosomal, mā te whakakapinga paralogous o RPL3 me RPL3L, i te taumata rānei o te 2′O-methylation o te rRNA, ki te hypertrophy uaua angaanga76,77. Mā ngā tono maha-omic me te ā-wāhi me te whakaahuatanga mahi o ngā myofibers takitahi ka whakatairanga ake i tō tātou māramatanga ki te koiora uaua i te taumata maha-omic78.
Mā te tātari i ngā proteom o ngā myofibers takitahi mai i ngā tūroro he nemaline myopathies, i whakaaturia hoki e mātou te whai huatanga, te whai huatanga, me te whaihua o ngā proteomics myofiber takitahi hei whakamārama i te pathophysiology haumanu o te uaua angaanga. I tua atu, mā te whakataurite i tā mātou rerenga mahi ki te tātari proteomic ā-ao, i taea e mātou te whakaatu he rite te hohonutanga o ngā mōhiohio e puta mai ana i ngā proteomics myofiber takitahi ki ngā proteomics kiko ā-ao, ā, ka whakawhānui ake i tēnei hohonutanga mā te whakaaro ki te rerekētanga o ngā muka me te momo myofiber. Haunga ngā rerekētanga e tumanakohia ana (ahakoa he rerekē) i roto i te ōwehenga momo muka i kitea i roto i ngā myopathies nemaline ACTA1 me TNNT1 ki te whakataurite ki ngā mana hauora,19 i kitea anō hoki e mātou te hangarua oxidative me te extracellular motuhake mai i te whakawhiti momo muka MYH-mediated. Kua pūrongohia i mua te mate pukupuku o te uaua (Fibrosis) i roto i ngā mate pukupuku o te uaua TNNT1 nemaline.19 Heoi, ka hangahia e tā mātou tātaritanga tēnei kitenga mā te whakaatu anō i ngā taumata kua piki ake o ngā pūmua e pā ana ki te ahotea i tukuna mai i waho o te pūtau, pērā i ngā annexins, e whai wāhi ana ki ngā tikanga whakatika sarcolemmal, i roto i ngā muka uaua mai i ngā tūroro e pāngia ana e ngā mate pukupuku o te uaua ACTA1 me te TNNT1 nemaline.57,58,59 Hei whakamutunga, ko te pikinga o ngā taumata annexin i roto i ngā muka uaua mai i ngā tūroro e pāngia ana e te mate pukupuku o te uaua he urupare pūtau ki te whakatika i ngā muka uaua tino ngoikore.
Ahakoa ko tēnei rangahau te tātaritanga uaua-katoa muka kotahi nui rawa atu o te tangata tae noa ki tēnei wā, kāore i te kore he herenga. I wehea e mātou ngā muka uaua angaanga mai i tētahi tauira iti, ōrite hoki o ngā kaiuru me tētahi uaua kotahi (te vastus lateralis). Nō reira, kāore e taea te whakakore i te noho o ngā taupori muka motuhake puta noa i ngā momo uaua me ngā pito rawa o te mahi uaua. Hei tauira, kāore e taea e mātou te whakakore i te tūponotanga o tētahi huinga o ngā muka tino tere (hei tauira, ngā muka 2X parakore) e puta ake ana i roto i ngā kaitākaro tere tino whakangungua me/ranei ngā kaitākaro kaha79, i ngā wā kore mahi uaua rānei66,80. I tua atu, ko te iti o te tauira o ngā kaiuru i aukati i a mātou ki te rangahau i ngā rerekētanga ira tangata i roto i te rerekētanga o te muka, i te mea e mōhiotia ana he rerekē ngā ōwehenga momo muka i waenga i ngā tāne me ngā wāhine. I tua atu, kāore i taea e mātou te whakahaere i ngā tātaritanga transcriptomic me te proteomic i runga i ngā muka uaua kotahi, i ngā tauira rānei mai i ngā kaiuru kotahi. I a mātou me ētahi atu e haere tonu ana ki te arotau i ngā tātaritanga pūtau-kotahi me te myofiber-kotahi mā te whakamahi i te tātaritanga omics hei whakatutuki i te whakaurunga tauira tino-iti (e whakaaturia ana i konei i roto i te tātaritanga o ngā muka mai i ngā tūroro e pāngia ana e te mitochondrial myopathy), ka kitea te whai wāhitanga ki te whakakotahi i ngā huarahi maha-omics (me te mahi) i roto i ngā muka uaua takitahi.
I te nuinga o te wā, ka tautuhia, ka whakamāramahia hoki e ā mātou raraunga ngā take whakawhiti me ngā take muri-whakawhiti o te rerekētanga o te uaua angaanga. Ina koa, ka whakaatuhia e mātou ngā raraunga e wero ana i tētahi whakapono tawhito i roto i te mahi uaua angaanga e pā ana ki te whakamāramatanga matarohia o ngā momo muka e ahu mai ana i te MYH. Ko tā mātou tumanako ka whakahoutia te tautohetohe, ā, ka whakaarohia anō tō mātou māramatanga ki te whakarōpūtanga me te rerekētanga o ngā muka uaua angaanga.
Tekau mā whā ngā tāngata Pākehā (12 ngā tāne me 2 ngā wāhine) i whakaae noa ki te whai wāhi atu ki tēnei rangahau. I whakaaetia te rangahau e te Komiti Matatika o te Hōhipera o te Whare Wānanga o Ghent (BC-10237), i tutuki i te Whakapuakitanga o Helsinki 2013, ā, i rēhitatia ki ClinicalTrials.gov (NCT05131555). Kei te Ripanga Tāpiri 1 ngā āhuatanga whānui o ngā tāngata whai wāhi. I muri i te whiwhinga whakaaetanga ā-waha me te ā-tuhi, i tirotirohia ngā tāngata whai wāhi i mua i te whakaurunga whakamutunga ki te rangahau. He taitamariki ngā tāngata whai wāhi (22–42 tau), he hauora (kāore he mate hauora, kāore he hītori momi hikareti), ā, he āhua kaha te tinana. I whakatauhia te nui o te hāora e tangohia ana mā te whakamahi i te ergometer hikoi hei aromatawai i te oranga tinana e ai ki te whakaahuatanga o mua. 81
E toru ngā wā i kohia ai ngā tauira koiora uaua i te wā okioki me te wā nohopuku, 14 ngā rā te wehe. Nā te mea i kohia ēnei tauira hei wāhanga o tētahi rangahau nui ake, i kai ngā kaiuru i te placebo (lactose), he kaitukino H1-receptor (540 mg fexofenadine), he kaitukino H2-receptor rānei (40 mg famotidine) 40 meneti i mua i te koiora. Kua whakaaturia e mātou i mua kāore ēnei kaitukino histamine receptor e pā ki te hauora o te uaua angaanga i te wā okioki81, ā, kāore i kitea he rōpū e pā ana ki te āhua i roto i ā mātou kauwhata whakahaere kounga (Ngā Pikitia Tāpiri 3 me te 6). I puritia he kai paerewa (41.4 kcal/kg taumaha tinana, 5.1 karamu/kg warowaihā taumaha tinana, 1.4 karamu/kg pūmua taumaha tinana, me te 1.6 karamu/kg ngako taumaha tinana) mō ngā hāora 48 i mua i ia rā whakamātautau, ā, i kainga he parakuihi paerewa (1.5 karamu/kg warowaihā taumaha tinana) i te ata o te rā whakamātautau. I raro i te rongoā whakamoe ā-rohe (0.5 ml 1% lidocaine kāore he epinephrine), i tangohia ngā koiora uaua mai i te uaua vastus lateralis mā te whakamahi i te werohanga Bergström percutaneous.82 I whakauruhia tonutia ngā tauira uaua ki roto i te RNA i muri mai, ā, i rongoatia ki te 4°C tae noa ki te wetewete ā-ringa i ngā muka (tae atu ki te 3 rā).
I whakawhitia ngā paihere miofiber hou kua wehea ki te reo RNA hou i roto i tētahi rihi ahurea. Kātahi ka wetewetehia ā-ringa ngā miofiber takitahi mā te whakamahi i tētahi stereomicroscope me ngā tweezers angiangi. E rua tekau mā rima ngā muka i wetewetehia mai i ia koiora, me te aro nui ki te whiriwhiri i ngā muka mai i ngā wāhi rerekē o te koiora. I muri i te wetewete, ka rumakina mārire ia muka ki roto i te 3 μl o te lysis buffer (SingleShot Cell Lysis Kit, Bio-Rad) kei roto ko ngā whākōkī proteinase K me te DNase hei tango i ngā pūmua me te DNA kāore e hiahiatia. Kātahi ka tīmata te lysis pūtau me te tango pūmua/DNA mā te vortexing poto, te hurihuri i te wai i roto i tētahi microcentrifuge, me te whaowhia i te pāmahana rūma (10 meneti). Kātahi ka whaowhia te lysate ki roto i tētahi thermal cycler (T100, Bio-Rad) i te 37°C mō te 5 meneti, 75°C mō te 5 meneti, kātahi ka rongoa tonu ki te -80°C tae noa ki te tukatuka anō.
I whakaritea ngā whare pukapuka RNA polyadenylated hototahi-Illumina mai i te 2 µl o te myofiber lysate mā te whakamahi i te QuantSeq-Pool 3′ mRNA-Seq Library Prep Kit (Lexogen). Kei roto i te pukapuka a te kaihanga ngā tikanga taipitopito. Ka tīmata te tukanga me te hanga cDNA aho-tuatahi mā te whakawhiti whakamuri, i reira ka whakauruhia ngā tohu tautuhi ngota ahurei (UMI) me ngā waehere pae i1 motuhake-tauira hei whakarite i te kohikohinga o ngā tauira me te whakaiti i te rerekētanga hangarau i te wā tukatuka iho. Kātahi ka kohikohia te cDNA mai i te 96 myofibers ka purea ki ngā piripiri aukume, ā, muri iho ka tangohia te RNA ka mahia te hanga aho-tuarua mā te whakamahi i ngā paninga matapōkere. Ka purea te whare pukapuka ki ngā piripiri aukume, ka tāpirihia ngā tohu i5/i7 motuhake-pūnaha, ā, ka whakanuia te PCR. Ka puta he whare pukapuka hototahi-Illumina i te taahiraa purenga whakamutunga. I aromatawaihia te kounga o ia pūnaha whare pukapuka mā te whakamahi i te High Sensitivity Small Fragment DNA Analysis Kit (Agilent Technologies, DNF-477-0500).
I runga i te inenga Qubit, i whakaemihia anō ngā puna i ngā kukū equimolar (2 nM). Kātahi ka raupapahia te puna hua i runga i te taputapu NovaSeq 6000 i te aratau paerewa mā te whakamahi i te Kete Tauhohenga NovaSeq S2 (1 × 100 nucleotides) me te uta 2 nM (4% PhiX).
Ko tā mātou pūnaha tātari raraunga QuantSeq Pool a Lexogen e hangai ana ki te pūnaha tātari raraunga QuantSeq Pool (https://github.com/Lexogen-Tools/quantseqpool_analysis). I tuatahitia te wetewete i ngā raraunga ki te bcl2fastq2 (v2.20.0) i runga i te taupū i7/i5. Kātahi ka wetewetehia te Pānui 2 ki te idemux (v0.1.6) i runga i te tauira waehere i1, ā, i tangohia ngā raupapa UMI ki te umi_tools (v1.0.1). Kātahi ka tapahia ngā pānui ki te cutadapt (v3.4) i roto i ngā rauna maha hei tango i ngā pānui poto (<20 te roa) ngā pānui rānei he raupapa urutau anake. Kātahi ka whakaritea ngā pānui ki te ira tangata mā te whakamahi i te STAR (v2.6.0c) ā, i tohua ngā kōnae BAM ki te SAMtools (v1.11). I tangohia ngā pānui tāruarua mā te whakamahi i te umi_tools (v1.0.1). Hei whakamutunga, i whakahaerehia te tatau whakaritenga mā te whakamahi i ngā featureCounts i roto i te Subread (v2.0.3). I whakahaerea te whakahaere kounga mā te whakamahi i te FastQC (v0.11.9) i ētahi wāhanga waenga o te paipa.
I whakahaerehia ngā tukatuka me ngā whakaaturanga koiora katoa i roto i te R (v4.2.3), mā te whakamahi i te tukanga mahi Seurat (v4.4.0). 83 Nō reira, i hurihia ngā uara UMI takitahi me ngā matihiko raraunga hei mea Seurat. I tangohia ngā ira i whakaatuhia i roto i te iti iho i te 30% o ngā muka katoa. I tangohia ngā tauira kounga iti i runga i te paepae iti rawa o te 1000 uara UMI me te 1000 ira i kitea. I te mutunga, 925 ngā muka i eke ki ngā mahi tātari mana kounga katoa. I whakatauritea ngā uara UMI mā te whakamahi i te tikanga Seurat SCTransform v2, 84 tae atu ki ngā āhuatanga katoa e 7418 i kitea, ā, i tangohia ngā rerekētanga i waenga i ngā kaiuru. Ka kitea ngā raraunga katoa e tika ana i roto i te Huinga Raraunga Tāpiri 28.