Abstract

   Biological roles of obscurin and its close homolog Obsl1 (obscurin-like
   1) have been enigmatic. While obscurin is highly expressed in striated
   muscles, Obsl1 is found ubiquitously. Accordingly, obscurin mutations
   have been linked to myopathies, whereas mutations in Obsl1 result in
   3M-growth syndrome. To further study unique and redundant functions of
   these closely related proteins, we generated and characterized Obsl1
   knockouts. Global Obsl1 knockouts are embryonically lethal. In
   contrast, skeletal muscle-specific Obsl1 knockouts show a benign
   phenotype similar to obscurin knockouts. Only deletion of both proteins
   and removal of their functional redundancy revealed their roles for
   sarcolemmal stability and sarcoplasmic reticulum organization. To gain
   unbiased insights into changes to the muscle proteome, we analyzed
   tibialis anterior and soleus muscles by mass spectrometry, uncovering
   additional changes to the muscle metabolism. Our analyses suggest that
   all obscurin protein family members play functions for muscle membrane
   systems.

   Subject terms: Mechanisms of disease, Membrane proteins, Molecular
   medicine, Experimental models of disease
     __________________________________________________________________

   Blondelle & Marrocco et al. show that deletion of both Obsl1 and
   obscurin in skeletal muscle leads to severe alterations in sarcolemma
   organization, protein expression profiles, and cellular calcium
   handling. These alterations affected muscle physiology and suggest
   redundant roles for Obsl1 and obscurin in murine muscle function.

Introduction

   Giant muscle proteins have been known for a long time to play important
   functions for skeletal and cardiac development and function, as well as
   for pathology of myopathies. Among the best characterized of these
   cellular giants are titin (also called connectin; approx. 3.5 MDa), and
   obscurin (approx. 800 kDa). Similar to titin, proteins of the obscurin
   protein family combine structural with signaling functions. Obscurin,
   the biggest mammalian member of this protein family, consists of
   serially arranged immunoglobulin-like and fibronectin-type III domains
   that are interspersed with signaling domains^[50]1,[51]2. Unlike titin,
   obscurin contains in addition to its protein kinase domains also a
   calcium/calmodulin-binding IQ motif, as well as a RhoGEF domain triplet
   (SH3-DH-PH domains). Extensive splicing of the obscurin gene results in
   at least three main transcripts, the giant obscurin-A and obscurin-B
   splice isoforms, and the obscurin kinase only (also known as KIAA1639
   or Obsc-kin), which originates from a separate promoter^[52]3. Recent
   reports indicate the presence of smaller obscurin splice
   variants^[53]4, their expression in non-muscle tissues, and important
   functions for tumorigenicity and metastasis^[54]5–[55]7. Other members
   of the obscurin protein family are the ubiquitously expressed
   obscurin-like 1 (Obsl1)^[56]8 as well as the striated muscle/atrial
   preferentially expressed protein kinase (Speg, also known as
   Apeg)^[57]9. Evolutionary, all members of the obscurin family are
   thought to have originated from one ancestral gene^[58]2,[59]8. This
   idea is supported by the fact that invertebrates like Caenorhabditis
   elegans have one obscurin family ortholog called unc-89^[60]10. In
   addition, Obsl1 and Speg show sequence similarity to the obscurin N and
   C terminus, respectively, and at least for Obsl1 also a certain degree
   of functional redundancy^[61]8,[62]11.

   Knockout models for obscurin and Speg have been helpful to delineate
   biological functions of these genes/proteins. While the knockout for
   obscurin resulted in a mild skeletal myopathy, changes to the
   sarcoplasmic reticulum (SR) and membrane fragility after
   exercise^[63]12–[64]14, Speg knockouts displayed a prominent dilated
   cardiomyopathy, disruption of the junctional SR membrane, and
   centronucleolar myopathy^[65]15–[66]17.

   Lately, it emerged that mutations in the human Obsl1 result in
   3M-growth syndrome in affected patients. On the molecular level, many
   of the human Obsl1 mutations are thought to result in nonsense-mediated
   decay of its messenger RNA (mRNA) and ultimately loss of the protein.
   However, owing to the extensive splicing displayed by Obsl1^[67]8
   (Supplementary Fig. [68]1a), detailed investigations into which
   isoforms are affected/unaffected and their respective expression levels
   in patient tissues remain to be done.

   The sarcomeric proteins titin and myomesin-1 have been identified as
   interaction partners for both obscurin and Obsl1. Titin offers two
   binding sites to obscurin: the titin C-terminal Ig-domain M10 interacts
   with obscurin Ig-domain 1^[69]11, while titin domains Z9–Z10 were
   identified to bind to obscurin Ig domains Ig48-Ig49 (also called
   Ig58-Ig59, depending on the obscurin splice isoform)^[70]2. Interaction
   of obscurin with the titin C terminus is the predominant binding site
   in mature myofilaments, giving rise to the prominent M-band
   colocalization of obscurin. The titin binding site in Ig-domain 1 of
   obscurin is evolutionary conserved for Obsl1 Ig-domain 1, albeit with a
   higher affinity compared to obscurin^[71]18. Differences in the side
   chains in obscurin vs. Obsl1 that generate the titin interaction
   interface and account for the differential binding affinity also
   contribute to the slightly different intracellular sorting of obscurin
   vs. Obsl1. Mutations in titin Ig-domain M10 that are known to cause
   limb-girdle muscular dystrophy 2J in affected patients were shown to
   disrupt the interaction with obscurin or Obsl1^[72]11. Indeed,
   biochemical analyses of the various titin mutations found in titin
   Ig-domain M10 indicated that the severity of the muscular dystrophy
   correlates with the degree of loss of interaction to obscurin or Obsl1.

   The functional redundancy between Obsl1 and obscurin can also be seen
   in their association with myomesin-1^[73]11. Recent advances in the
   co-crystallization of this interaction revealed that binding of
   myomesin-1 to obscurin or Obsl1 Ig3 is necessary for proper folding of
   their Ig domains in a hitherto unprecedented trans-complementation
   mechanism^[74]19.

   Another well-characterized binding site for a muscle-specific isoform
   of ankyrin-1 (sAnk1.5) is located within the obscurin-A isoform C
   terminus^[75]20,[76]21. Complex formation between obscurin, sAnk1.5,
   and tropomodulin-3 was demonstrated to be important for SR architecture
   and function^[77]12,[78]22,[79]23, and stability of sAnk1.5 itself.
   Indeed, we demonstrated that loss of obscurin leads to increased
   sAnk1.5 turnover in a cullin-3/KCTD6-dependent manner^[80]24.
   Intriguingly, the functional property that obscurins may regulate the
   stability and turnover of their interaction partners may be conserved
   within this protein family, as well as evolutionary: Obsl1 interacts
   with cullin-7^[81]25,[82]26, and dysfunction of this E3-ligase complex
   by mutations in cullin-7 or Obsl1 have been linked to the development
   of 3M-growth syndrome in patients^[83]27,[84]28. Moreover, the
   invertebrate obscurin homolog unc-89 directly interacts with cullin-1,
   and regulates myosin filament organization in a MEL-26/cullin-3- and
   MEI-1 (katanin)-dependent way^[85]24,[86]29.

   In this study, we set out to further investigate biological functions
   for obscurin proteins for skeletal muscles, with a special emphasis to
   uncover functional redundancies between obscurin and Obsl1. As Obsl1 is
   ubiquitously expressed and its mutations lead to a growth disorder in
   affected patients, we also investigated the phenotype of global
   Obsl1-knockout mice. Our studies uncovered that global Obsl1 knockouts
   are embryonically lethal, while skeletal muscle-specific Obsl1
   knockouts show a mild phenotype similar to obscurin knockouts. Only
   deletion of both proteins in skeletal muscles uncovered their role for
   SR, cellular calcium storage and handling, dystrophin–sarcoglycan (DSG)
   complex, and sarcolemmal integrity, as well as changes to the muscle
   metabolism.

Results

Global knockout mice for Obsl1 are embryonically lethal

   We generated the Obsl1 conditional knockout mouse to test the effect
   that ablation of Obsl1 would have on the whole organism, and on
   skeletal muscle organization and function. Due to its extensive
   splicing^[87]8 and the presence of alternative start codons, we decided
   to place coding exons 1 through 4 of the murine Obsl1 locus within LoxP
   sites (Supplementary Fig. [88]1a). This strategy should result in the
   total ablation of any Obsl1 isoform (Supplementary Fig. [89]1b). By
   crossing gene-targeted mice, with protamine-Cre recombinase
   mice^[90]30, we generated global Obsl1 knockouts. Surprisingly, global
   ablation of Obsl1 in mice results in embryonic lethality before
   embryonic day E8 (Table [91]1), while mice heterozygous for Obsl1
   developed normally. These results are in contrast to reports on human
   patients carrying various mutations in Obsl1, which are thought to
   result in nonsense-mediated decay of its mRNA in a majority of cases.
   Clinically, these patients present with the development of 3M-growth
   syndrome^[92]28,[93]31.

Table 1.

   Genotype analysis of global Obsl1-knockout mice
   Genotype Number and % of embryos/animals in genotyping analysis
   Expected Mendelian ratios
   E7–E8.5 E10.5–E13.5 E17–E18 P21
   Control (+/+) 7 (30%) 15 (44%) 4 (33%) 37 (36%) 25%
   Heterozygous (+/−) 16 (70%) 19 (56%) 8 (67%) 66 (64%) 50%
   Knockout (−/−) 0 (0%) 0 (0%) 0 (0%) 0 (0%) 25%
   [94]Open in a new tab

   Obsl1 obscurin-like 1

Obsl1 and/or obscurin are not required for sarcomerogenesis and skeletal
muscle development

   To overcome the lethality, we crossed conditional Obsl1 mice with mice
   expressing Cre recombinase under control of the myogenin
   promoter^[95]32. These mice do not develop gross morphological
   abnormalities or display premature death. However, it was shown that
   obscurin and Obsl1 display functional redundancy for two of their
   common interaction partners, namely myomesin-1 (Myom1) and titin’s
   C-terminal domain M10^[96]11. Loss of obscurin was shown to result in a
   mild myopathy phenotype, without adversely affecting the animals at
   baseline^[97]12. To investigate if the lack of a more pronounced
   skeletal muscle phenotype might be due to a rescue by Obsl1, we
   generated skeletal muscle-specific double knockouts (dKO; Fig. [98]1a).
   dKO mice were viable, fertile, and developed normally. Deletion of
   obscurin led to slightly increased protein levels in Obsl1 (normalized
   protein levels 2.5 ± 0.1 in obscurin vs. 1.0 ± 0.02 in CTL; p < 0.001,
   n = 4), while obscurin expression was unchanged in Obsl1-knockout TA
   muscles (Fig. [99]1a, Supplementary Fig. [100]1c). Microscopic analysis
   of myofilament structure using sarcomeric α-actinin 2 indicated normal
   sarcomerogenesis (Fig. [101]1b, c, Supplementary Fig. [102]1d, h). Loss
   of Obsl1 or obscurin (Obsc) does not alter localization of the other
   obscurin protein family member in tibialis anterior (TA) muscles
   (Fig. [103]1b, c). However, we noticed that two knockout validated
   Obsl1 antibodies recognizing different epitopes within the protein
   displayed differential localizations of Obsl1 within the sarcomere. An
   antibody that was raised against N-terminal Ig-domain 1 of the
   protein^[104]11 displayed M-band and Z-disc localization
   (Fig. [105]1c), while a commercially available antibody that recognizes
   Ig-domain 14 of Obsl1 localized almost exclusively to Z-discs
   (Supplementary Fig. [106]1d).

Fig. 1.

   Fig. 1
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   Expression and localization of obscurin and Obsl1 (obscurin-like 1) in
   tibialis anterior (TA) muscles. a Expression levels of obscurin and
   Obsl1 in TA muscles from obscurin-knockout (Obsc), Obsl1 skeletal
   muscle-knockout (Obsl1), and double-knockout (dKO) mice, compared to
   controls (CTL). Asterisk denotes non-specific cross-reactivity of the
   secondary antibody with myosin. Myosin band in Ponceau stain as a
   loading control. b, c Sarcomeric localization of obscurin (b, IQ-64
   epitope antibody) or Obsl1 (c, Ig1 epitope antibody) in Obsc, Obsl1,
   and dKO, as well as control (CTL) TA muscles. Arrows indicate Z-disc
   localizations as indicated by α-actinin 2 counterstain, while
   arrowheads signify the sarcomeric M-band. Please note that some
   background staining caused by non-specific cross-reactivity of the
   antibodies is observed in knockout tissues. Scale bars = 10 µm

   Loss of unc-89 in Caenorhabditis elegans and in Drosophila results in
   the disruption of myosin lattice organization, partially due to loss of
   the RhoGEF activity in the invertebrate obscurin
   homolog^[108]33,[109]34. While searching for novel interaction partners
   for Obsl1 by yeast two-hybrid screening, we identified the C terminus
   of myosin (MyH6), dystonin (Dst), and filamin-C (FlnC) as putative
   binding partners (Supplementary Fig. [110]1a, lower panel). Both
   dystonin and FlnC were previously identified in another high-throughput
   affinity assay as putative Obsl1 interaction partners^[111]35. Analysis
   of myosin binding to Obsl1 by co-immunoprecipitation validated their
   association (Supplementary Fig. [112]1e). However, looking at protein
   expression and localization for some of the newly and previously
   reported putative interaction partners like myomesin-1, no overt
   changes in protein levels, organization, and subcellular localization
   were observable in immunoblots or immunofluorescence images of
   longitudinal TA sections (Supplementary Fig. [113]1f, g). In addition,
   unlike in C. elegans or Drosophila, loss of obscurin and/or Obsl1 did
   not result in markedly altered Z-disc and M-band structures
   (Supplementary Fig. [114]1h).

   We next wondered what impact the loss of Obsl1 and/or obscurin would
   have on muscle mass and cross-sectional muscle area. Normalized muscle
   masses for TA, soleus (Sol) and gastrocnemius (Gas), was unchanged in
   4-month-old animals. We observed only a small increase in the mass of
   extensor digitorum longus (EDL) muscle of Obsl1-knockout animals
   (Fig. [115]2a). No significant changes were observed in the number of
   centralized nuclei in TA muscle at 4 months of age (Fig. [116]2b, c),
   confirming earlier observations for muscles from obscurin
   knockouts^[117]12. Study of cross-sectional areas in sections of TA
   muscle revealed increases in fiber sizes in Obsl1 and obscurin-knockout
   mice, probably responsible for the slight increase in muscle mass of
   the Obsl1 animals, while dKO mice displayed decreases in fiber sizes,
   specifically of fibers with high cross-sectional areas (Fig. [118]2d,
   e).

Fig. 2.

   [119]Fig. 2
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   Loss of obscurin and/or Obsl1 (obscurin-like 1) does not influence
   muscle weights, and has little impact on cross-sectional area and
   muscle regeneration. a Muscle weight to tibia-length ratios of tibialis
   anterior (TA), soleus (Sol), gastrocnemius (GC), and extensor digitorum
   longus (EDL) muscles in control (CTL), obscurin knockout (Obsc), Obsl1
   skeletal muscle-knockout (Obsl1), and double-knockout mice (dKO).
   Sample sizes (n) are indicated in the figure; ***p < 0.001 vs. CTL. b–e
   Wheat germ agglutinin (WGA)- (magenta) and
   4′,6-diamidino-2-phenylindole (DAPI)- (green) stained
   immunofluorescence of frozen TA cross-sections (b) used to determine
   fibers with centralized nuclei (c) and cross-sectional fiber areas for
   each group (d, e). Scale bar = 200 µm. Muscle from three mice per group
   were analyzed, counted fibers (n) and p values are indicated in figure
   (c–e). *p < 0.05, ***p < 0.001 vs. CTL. Box and whisker plots in c and
   d depict 5–95 percentile, averages (as cross) and outliers

Loss of obscurin/Obsl1 impacts the DSG complex and its associated proteins

   Obscurin has been reported to be important for sarcolemmal integrity,
   and loss of the protein results in changes to the subsarcolemmal
   microtubule network, increased membrane fragility, and reduced running
   performance of mice^[121]13,[122]14. We investigated if changes to
   sarcolemmal integrity and the DSG complex and its associated proteins
   are exacerbated in Obsl1 and/or dKO mice. Loss of both obscurin and
   Obsl1 reduced protein levels of some DSG components, while levels of
   tubulin increased slightly (p = 0.06). In addition, overall levels of
   dystrophin and β1D- integrin remained unchanged (Fig. [123]3a,
   Supplementary Figs. [124]2a, [125]7a). However, analysis of
   subsarcolemmal dystrophin localization in longitudinal sections of TA
   muscles revealed a patchy and abnormal distribution of the protein in
   obscurin knockouts, but more so in dKO mice, indicating enhanced
   membrane fragility even at baseline (Fig. [126]3b, Supplementary
   Fig. [127]2b). Indeed, when investigated for the presence of mouse
   immunoglobulins in immunofluorescence assays, a significant portion of
   muscle fibers in dKO mice stained positive (Fig. [128]3c, Supplementary
   Fig. [129]2c). Increased membrane fragility in dKO muscles also leads
   to significantly enhanced expression of dysferlin (Dysf) and FlnC,
   proteins involved in repair and compensatory mechanisms (Fig. [130]3d,
   Supplementary Figs. [131]2d, [132]7a).

Fig. 3.

   [133]Fig. 3
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   Loss of obscurin and Obsl1 (obscurin-like 1) impacts
   dystrophin–sarcoglycan (DSG) complex and its associated proteins,
   membrane integrity and membrane repair mechanisms. a Expression levels
   of DSG complex proteins dystrophin, utrophin, β-dystroglycan, and
   α-dystrobrevin, as well as α-tubulin and β1D-integrin in whole protein
   lysates of tibialis anterior (TA) muscles of control (CTL),
   obscurin-knockout (Obsc), Obsl1 skeletal muscle-knockout (Obsl1), and
   double-knockout (dKO) mice. Ponceau-stained actin band is shown as a
   loading control. b Subsarcolemmal dystrophin localization in
   longitudinal sections of CTL, Obsc, Obsl1, and dKO TA muscles.
   Sarcomeric α-actinin 2 was used as counterstain. Scale bar = 10 µm. c
   Mouse immunoglobulin (magenta) and 4′,6-diamidino-2-phenylindole (DAPI)
   stain (green) of frozen cross-sections of CTL, Obsc, Obsl1, and dKO TA
   muscles. Arrowheads indicate immunoglobulin G (IgG)-positive muscle
   fibers. Scale bar = 200 µm. d, e Protein expression levels of dysferlin
   and Filamin-C (d), as well as DSG-associated proteins α-syntrophin,
   Trpc1, caveolin-1, neuronal nitric oxidase (nNOS), and l-type calcium
   channel DHPR α-2 subunit (e) in whole lysates of CTL, Obsc, Obsl1, and
   dKO TA muscles. Ponceau-stained actin is shown as a loading control

   The DSG complex also regulates expression and function of
   sarcolemma-based membrane channels and signaling. Specifically,
   α-syntrophin has been demonstrated to establish a link between
   dystrophin/the DSG complex and sarcolemmal ion channels, such as Trpc1
   as well as caveolae components and neuronal nitric oxidase (nNOS)
   signaling. When investigated, we noticed reduced caveolin-1 and Trpc1
   levels in TA muscles of dKO mice, while nNOS remained unchanged
   (Fig. [135]3e, Supplementary Figs. [136]2e, 7a). Levels of L-type
   calcium channel remained also comparable with controls, indicating
   potential changes to DSG-associated, but not T-tubule-based ion
   channels.

Proteome analysis of knockout muscles

   To reveal quantitative changes to the muscle proteome between control,
   obscurin-knockout, Obsl1-knockout, or dKO mice, we isolated proteins
   from TA and Sol muscles. We specifically focused on changes between
   control and dKO muscles to eliminate compensatory mechanisms between
   Obsl1 and obscurin that could potentially obfuscate results. Of the
   1503 and 1597 detected proteins in TA and Sol of all genotypes, we
   identified 260 and 181 proteins that were significantly deregulated
   between dKO and control muscles, respectively (Fig. [137]4a, b,
   Supplementary Figs. [138]8, [139]9, Supplementary Data File [140]1).
   Gene ontology (GO) term and pathway analysis of significantly changed
   proteins from all investigated muscles, or Sol and TA alone revealed
   enrichment of oxidative phosphorylation, TCA (tricarboxylic acid)
   cycle, and electron transport chain, as well as muscle contraction
   processes (Supplementary Fig. [141]3a–c, Supplementary Data
   Files [142]2–[143]4). Among the significantly upregulated proteins in
   TA muscles of dKO mice are FlnC and Dysf, corroborating results
   obtained in our immunoblot analyses (Figs. [144]3d, [145]4c,
   Supplementary Fig. [146]2d). Comparison of significantly changed
   proteins between TA and Sol revealed 15 proteins as commonly
   upregulated and 33 proteins as commonly downregulated in both analyzed
   muscle types, including calsequestrin-2 (Casq2), Hspb1 (also known as
   Hsp27), myotilin, or calmodulin-1 (Fig. [147]4b, Supplementary Data
   File [148]1). Intriguingly, we also noticed several proteins that were
   differentially regulated between Sol and TA, including αB-crystallin
   (Fig. [149]4c, d, Supplementary Figs. [150]2f, [151]7b). Indeed, when
   studied in immunoblot assays of protein lysates from TA and Sol muscles
   from all groups, only TA muscle of dKO mice showed significantly
   increased αB-crystallin levels, while αB-crystallin levels in Sol
   muscles remained largely unchanged (Fig. [152]4e, Supplementary
   Figs. [153]2f, [154]7b). These muscle type-specific differences may be
   reflective of the differential expression levels of obscurin splice
   isoforms observed in these muscles, and/or on the muscle type (fast vs.
   slow twitch)^[155]12,[156]36.

Fig. 4.

   [157]Fig. 4
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   Proteome analysis of soleus (Sol) and tibialis anterior (TA) muscles. a
   Hierarchical clustering of significantly altered proteins identified in
   TA and Sol muscles of control (CTL), obscurin-knockout (Obsc), skeletal
   muscle-specific Obsl1 (obscurin-like 1)-knockout (Obsl1), and
   double-knockout (dKO) mice. b Grouping of significantly changed
   proteins in both muscles identified 15 proteins as common upregulated,
   while 33 proteins were found to be common downregulated between Sol and
   TA. The analysis also identified seven proteins that are differentially
   regulated between TA and Sol muscles. All commonly and differentially
   regulated proteins are identified in the boxes. c, d Volcano plot of
   significantly altered proteins between control (CTL) and dKO TA (c) and
   Sol (d) muscles. e Protein levels of αB-crystallin in whole TA and Sol
   muscle lysates from CTL, Obsc, Obsl1, and dKO mice. Ponceau-stained
   actin band is shown as a loading control

Removal of obscurin/Obsl1 alters SR protein levels

   Our enrichment analysis revealed several proteins involved in muscle
   contraction, calcium handling, and signaling as significantly changed
   in dKO muscles. Among them, the SR proteins sarcoendoplasmic reticulum
   Ca^2+ ATPase (Serca), sarcalumenin, triadin, JP-45 and junctin were
   found to be deregulated in dKO muscles (Fig. [159]5a). We corroborated
   suspected changes to the SR and its associated proteins by immunoblot
   analyses, and found a significant reduction of Serca levels, while
   ryanodine receptors (RyRs) were upregulated (Fig. [160]5b,
   Supplementary Figs. [161]2g, [162]7c). We also investigated levels of
   sAnk1.5, which was shown to be a binding partner for the obscurin-A C
   terminus. As shown before, loss of obscurin resulted in a decrease in
   sAnk1.5 levels due to increased turnover of the protein. Loss of Obsl1
   did not affect sAnk1.5 levels, while muscles from dKO mice displayed
   sAnk1.5 levels comparable with those found in obscurin knockouts
   (Fig. [163]5b, d). Our proteome data also indicated changes to
   calcium-binding proteins located within the lumen of the SR. We tested
   protein levels of Casq1, Casq2, and sarcalumenin by immunoblot analyses
   of whole muscle lysates. Sarcalumenin levels decreased significantly
   between control and dKO muscles, while Casq2 protein levels increased,
   and Casq1 remained unchanged (Fig. [164]5c, e, Supplementary
   Figs. [165]2g, [166]7c).

Fig. 5.

   [167]Fig. 5
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   Changes to sarcoplasmic reticulum (SR) and associated proteins. a
   Volcano plots of significantly altered SR and its associated proteins
   identified in our proteome analysis of double-knockout (dKO) vs.
   control tibialis anterior (TA) and soleus (Sol) muscles. b–e Immunoblot
   analysis and quantification of SR-associated proteins in TA muscles,
   including small ankyrin-1.5 (sAnk1.5; b, d), sarcoendoplasmic reticulum
   Ca^2+ ATPase (Serca1; b), and ryanodine receptor (RyR; b), as well as
   lumenal calcium-binding proteins sarcalumenin (c, e) and calsequestrin
   1 and 2 (c). Ponceau-stained actin band is shown as a loading control
   (b, c). d–g Sample sizes (n) and p values are indicated in the figure.
   f, g Analysis of extensor digitorum longus muscle “time to peak” (f)
   and “half-relaxation time” (g). The full evaluation of twitch
   parameters and their statistical analyses are shown in Table [169]2

   These data indicate changes to cellular calcium handling in dKO
   muscles, which may be reflected in defective excitation contraction
   coupling and muscle contraction. To investigate if these molecular
   changes alter muscle physiology, we studied EDL twitch parameters in
   all groups (Table [170]2). EDL muscles of obscurin-knockout and dKO
   mice displayed increased time-to-peak (TtP) values compared to muscles
   from control and Obsl1 knockouts (Fig. [171]5f). However,
   half-relaxation times remained unchanged (Fig. [172]5g).

Table 2.

   Twitch parameter analysis on 4-month-old mice
   Parameters CTL Obsc Obsl1 dKO
   Number of analyzed muscles (females/males) 10 (6/4) 7 (3/4) 9 (4/5) 10
   (4/6)
   TtP (ms) 21.45 ± 1.02 26.14 ± 0.91** 21.56 ± 0.92 25.3 ± 0.74**
   HRT (ms) 43.3 ± 2.81 42.86 ± 3.20 48.00 ± 5.68 46.5 ± 1.99
   Full-width at half-maximum (ms) 57 ± 3.4 60 ± 3.9 63 ± 6.3 64 ± 2.6
   Stress during tetanus (kPa) 71 ± 3.8 99 ± 5** 79 ± 6.2 70 ± 5.8
   PCSA (mm^2) 0.51 ± 0.027 0.61 ± 0.023 0.6 ± 0.034 0.55 ± 0.03
   EDL muscle mass (mg) 3.4 ± 0.19 4 ± 0.22 4.3 ± 0.28** 3.9 ± 0.18
   Fiber length (mm) 6.2 ± 0.11 6.1 ± 0.16 6.7 ± 0.13* 6.5 ± 0.2
   [173]Open in a new tab

   Values are presented as averages and standard error of mean. Samples
   sizes are shown in the table; p values are **p  < 0.01 or *p <   0.05
   vs. CTL

   TtP time to peak, HRT half-relaxation time, PCSA physiological
   cross-sectional area, CTL control, Obsl1 obscurin-like 1, dKO double
   knockout, EDL extensor digitorum longus

Metabolic changes in obscurin/Obsl1-knockout mice

   A surprising result of the enrichment analysis was the large number of
   significantly changed proteins belonging to metabolic pathways and the
   mitochondrial electron transport chain (Fig. [174]6a). Specifically, we
   found increased levels of Gbe1 (1,4-α-glucan branching enzyme 1) and
   Stbd1 (starch binding domain 1) in dKO Sol and TA muscles. Both
   proteins are involved in glycogen metabolism, increasing solubility of
   glycogen by branching, and its breakdown through glycophagy,
   respectively. Conversely, the rate determining enzyme responsible for
   glycogen breakdown, muscle glycogen phosphorylase, was significantly
   decreased in dKO mice. We also found significantly decreased levels of
   monoamine oxidase A and B (Maoa, Maob) in TA muscles of dKO mice, while
   Sol muscle of dKO mice had increased levels of catalase, perhaps
   indicative of increased oxidative stress. Another finding was that
   Gapdh protein levels were decreased in dKO mice, a result that we also
   consistently observed in Obsl1-knockout cells and tissues
   (Supplementary Figs. [175]1b, [176]4a, 4b), necessitating the use of
   total protein, actin or myosin, for normalization. Most intriguingly,
   however, almost all identified mitochondrial electron transport chain
   proteins were downregulated in dKO mouse Sol and TA muscles
   (Fig. [177]6b, Supplementary Fig. [178]4c). Analysis of whole TA and/or
   Sol muscle extracts using the oxphos antibody cocktail or a Uqcrb
   antibody substantiated slightly or significantly decreased levels of
   mitochondrial complex I, II, III, IV, and V proteins in dKO mice
   (Fig. [179]6c, Supplementary Fig. [180]4d–f). Analysis of Prdx3 protein
   levels, a mitochondrially targeted peroxiredoxin family member^[181]37
   that was also detected in our proteome analysis as significantly
   altered, indicating that the effect of Obsl1/obscurin loss on the
   mitochondria may go well beyond electron transport chain proteins
   (Fig. [182]6c, Supplementary Fig. [183]4e, f). Further analysis of the
   proteome data revealed that downregulation of electron transport chain
   proteins is more associated with loss of obscurin than Obsl1
   (Supplementary Fig. [184]4c). However, only the loss of both proteins,
   obscurin and Obsl1, resulted in the substantial reduction in
   mitochondrial proteins. It remains to be demonstrated if these changes
   are mirrored on the physiological level, and if dKO muscles display
   altered reactive oxygen species (ROS) levels caused by mitochondrial
   insufficiency. Our proteome data showing changes to other
   peroxiredoxins, Sod2, and other proteins involved in ROS scavenging and
   signaling (Supplementary Fig. [185]5e; Supplementary Data File [186]1)
   are highly suggestive of this possibility.

Fig. 6.

   [187]Fig. 6
   [188]Open in a new tab

   Analysis of changes to the muscle metabolism and mitochondria. a
   Volcano plot of significantly altered proteins between control (CTL)
   and double-knockout (dKO) tibialis anterior (TA; left panel) and soleus
   muscles (Sol; right panel) involved in energy metabolism (blue) and
   mitochondria (light blue). b Volcano plot of electron transport chain
   proteins identified in our proteome analysis as significantly altered
   between CTL and dKO TA and Sol muscles. The number of significantly
   changed proteins is indicated in brackets. c Analysis of mitochondrial
   electron transport chain complex protein levels as well as
   mitochondrially located peroxiredoxin-3 (Prdx3) in TA and Sol muscles
   from CTL, obscurin-knockout (Obsc), skeletal muscle-specific Obsl1
   (obscurin-like 1)-knockout (Obsl1), and dKO mice. Ponceau-stained total
   actin band is shown as a loading control

Discussion

   Our study set out to investigate biological roles for Obsl1, with a
   specific focus on redundant functions between obscurin and Obsl1 for
   the development of skeletal muscles. For this, we generated conditional
   Obsl1-knockout mice using a strategy that prevents the expression of
   all Obsl1 isoforms. Surprisingly, our global Obsl1 knockouts display an
   embryonic lethality phenotype (Table [189]1).

   Obsl1 has been shown to be responsible for ~20% of cases of 3M-growth
   syndrome, while mutations in cullin-7 and Ccdc8 account for the
   remainder of the patients^[190]27,[191]31. Most of the Obsl1 patients
   suffer from mutations that result in a truncated protein due to
   frameshift mutations^[192]31,[193]38. These findings led to the
   speculation that the Obsl1 mRNA in patients may be prematurely degraded
   by nonsense-mediated decay, resulting in complete absence of the
   protein^[194]31. However, similar to cullin-7 knockouts^[195]39, our
   global Obsl1 knockouts do not recapitulate the 3M-growth syndrome
   exhibited by patients.

   Closer analysis showed that all Obsl1 mutations are located well 3′ of
   the sequence encoding for Ig domain 1 of Obsl1^[196]31. This leaves the
   potential open for low-level expression of C terminally truncated Obsl1
   protein variants and alternative splice isoforms that circumvent some
   of the deleterious mutations in Obsl1, which is not possible in our
   mouse model. However, immunoblot analysis of Obsl1 using validated
   antibodies has not been extensively done on patient samples.

   It is difficult to compare data from the 3M-growth syndrome
   transcriptome and the Obsl1 interactome^[197]35,[198]40 with changes
   observed in our proteome analyses, as there are functional redundancies
   between Obsl1 and obscurin, which may not be present in skin
   fibroblasts and other non-muscle tissues (Supplementary Fig. [199]6a).
   Moreover, the complete dataset for the transcriptome analysis of
   fibroblasts from 3M-growth syndrome patients is not readily
   available^[200]40. However, there are several striking similarities
   between our proteome and the transcriptome and interactome datasets
   (Supplementary Fig. [201]5a, b; Supplementary Data File [202]5).
   Specifically, both studies found significant deregulation of
   genes/proteins involved in cellular transcription (e.g., Hnrnpf,
   Eef1a1, Ybx transcription factors), translation (small and large
   ribosomal subunits), and in genes involved in metabolic pathways
   downstream of IGF1 (insulin-like growth factor 1) and mTOR (mammalian
   target of rapamycin) signaling (Supplementary Fig. [203]5c). Several
   proteins that form part of membrane and vesicle trafficking complexes
   show up in both large datasets, including coatomer protein complex
   subunit alpha (Copa) or clathrin (Cltc) (Supplementary Fig. [204]5a,
   d). In addition, other proteins involved in vesicle and protein
   trafficking were found significantly altered, such as caveolin-3,
   muscle-related coiled-coil protein (MuRC/cavin-4)^[205]41, transferrin
   receptor (Tfrc)^[206]42, intersectin-1 (Itsn1)^[207]43, Rab8b^[208]44,
   non-clathrin-related endocytosis (pacsin3^[209]45, EHD3^[210]46), COP1-
   or COP2-related protein shuttling pathways (Sec31a^[211]47,[212]48,
   archain-1 (Arcn1)^[213]49), and several tubulin isoforms, which were
   also deregulated in our proteome analysis (Supplementary Data [214]1).
   Deficiencies in several of these pathways have been implicated in
   growth retardation and developmental delays, including COP-related
   protein shuttling pathways^[215]49–[216]51 or clathrin-dependent
   trafficking^[217]52. Another striking overlap between the datasets are
   proteins that are involved in metabolism, mitochondrial function (e.g.,
   electron transport chain complex proteins), and generation/scavenging
   of ROS). Specifically, we found deregulation of Gapdh and Prdx3, both
   identified as putative interaction partners for Obsl1^[218]35
   (Supplementary Figs. [219]5a, [220]6c). Maoa was also found
   downregulated in both the transcriptome and our proteome datasets.
   Patients with Maoa/Maob deficiency display loss of muscle tone and
   short statue^[221]53,[222]54, while global Maoa/Maob dKO mice show
   developmental delays, behavior, and locomotion problems^[223]55. Closer
   examination of changes to muscles and exercise capacity in monoamine
   oxidase-deficient mice remains to be done.

   It is thought that 3M-growth syndrome is caused by a deficiency in
   protein turnover due to the finding that a majority of patients with
   3M-growth disorder display mutations in cullin-7, Obsl1, and Ccdc8.
   Indeed, Obsl1 was identified as a binding partner for the E3-ubiquitin
   ligase cullin-7^[224]25,[225]26,[226]56. However, our proteome data did
   not reveal significant changes to any of the other proteins associated
   with the disease, that is, cullin-7, Ccdc8, Fbw8, Igfbp2, or Igfbp5.
   These findings raise the possibility that functional deficiencies
   associated with the disorder may be tissue specific and spare skeletal
   muscles. Closer examination of expression patterns for 3M-growth
   syndrome associated proteins could reveal tissues types where this
   complex performs its major functions. Indeed, looking at expression
   profiles in Protein Atlas ([227]https://www.proteinatlas.org/) reveals
   variable expression for Fbw8 (and many of the other F-box-containing
   substrate adaptor proteins for cullin-7), as well as Igfbp2 and Igfbp5
   across tissues. The usage of cre lines that delete Obsl1 in tissues
   other than skeletal muscles and at earlier time-points will shed
   further light into non-muscle functions for Obsl1 and the development
   of 3M-growth syndrome.

   Another surprise was the lack of baseline phenotype for skeletal
   muscle-specific Obsl1 knockouts, and the comparatively mild
   physiological phenotype observed for the dKO mice. Indeed, looking at
   the substantial molecular changes to the DSG complex, alterations to
   sarcolemmal integrity and to SR-associated proteins that the dKO mice
   display, it was unexpected to observe no changes to the number of
   centralized nuclei in muscle cross-sections as a sign for active muscle
   regeneration, and largely unchanged physiological twitch parameters.
   However, when investigating specific alterations to skeletal muscles
   upon loss of Obsl1 and obscurin redundant functions for both proteins
   start to emerge.

   Despite extensive interactions of obscurin and Obsl1 (or its
   invertebrate homolog unc-89) with sarcomeric
   proteins^[228]8,[229]11,[230]33,[231]57, loss of either or both
   obscurin family proteins had negligible effects on myofibrillogenesis
   and sarcomere structure. Indeed, apart from a slight reduction in the
   levels of myomesin-2 (Supplementary Fig. [232]1e), we were unable to
   detect any overt changes for myomesin-1, tropomyosin, α-actinin 2,
   titin-M8, and myosin localization or expression. These data indicate
   that obscurin and Obsl1 in vertebrate muscles are sarcomere-associated
   proteins that are not required for sarcomere assembly. Indeed, our
   experiments investigating muscle twitch characteristics only found
   increase in TtP values for loss of obscurin, indicative of problems
   with the SR and calcium-induced calcium release. However, no changes
   were found in other parameters measured in these experiments, with the
   exception of stress during tetanus values for obscurin knockouts. Obsl1
   has been shown to target to several subcellular localizations in heart
   and skeletal muscles, including M-band, Z-disc, and intercalated
   discs^[233]8,[234]11. While not all antibodies in these studies have
   been verified against the knockout, our experiments using two verified
   Obsl1 antibodies that target different Obsl1 epitopes corroborate
   several of these findings. Known and novel interactions may explain the
   differential Obsl1 localization to sarcomeric M-bands and Z-discs
   (Fig. [235]1c, Supplementary Figs. [236]1a, 1d, 1e, [237]6b). It
   remains to be demonstrated how splice variants of Obsl1, and binding
   affinities to interactors modifies the subcellular localization.

   Obscurin knockouts display altered ankyrin-B and dystrophin
   localization at costameres, and increased membrane fragility combined
   with reduced muscle exercise tolerance^[238]13,[239]14.
   Morpholino-mediated knockdown of obscurin-A in zebrafish also resulted
   in abnormal dystrophin and α-dystroglycan localization, suggesting
   evolutionary conserved functions of obscurin for DSG complex
   organization and sarcolemmal integrity^[240]58. Following these
   studies, we investigated expression and localization of DSG components
   and found changes at baseline predominantly in dKO mice. While dKO
   muscles did not display changes to dystrophin levels, its
   subsarcolemmal localization appeared patchy and its distribution
   disturbed. Immunofluorescence imaging of subsarcolemmal dystrophin
   indicated that obscurin and Obsl1 may partially modulate the membrane
   targeting of dystrophin, and help it to spread evenly over the
   sarcolemmal membrane (Fig. [241]3b, Supplementary Fig. [242]2b).

   Muscles of dKO mice also displayed a surprising slight decrease in
   utrophin (p = 0.06), and several other investigated DSG components,
   including α-dystrobrevin. Moreover, DSG-linked signaling via ion
   channels (Trpc1) may also be altered in dKO mice only, based on reduced
   expression levels of this channel. These data suggest that both Obsl1
   and obscurin have redundant functions for DSG complex assembly and
   stability. Loss of both proteins resulted in impaired sarcolemmal
   integrity in a substantial proportion of muscles in mice at baseline
   (not exercised), as demonstrated by positive immunoglobulin labeling of
   muscle fibers in dKO mice (Fig. [243]3c, Supplementary Fig. [244]2c).

   Muscles of dKO mice also show upregulation of proteins involved in
   muscle repair processes, including Dysf and FlnC^[245]59–[246]61.
   Future experiments that further characterize membrane damage and
   studies that investigate repair mechanisms through FlnC and Dysf should
   clarify the extent of the sarcolemmal integrity impairment in dKO mice.

   While some of the functions that obscurin exerts on the DSG complex are
   due to interactions with ankyrin isoforms, spectrin and
   FlnC^[247]13,[248]20,[249]21,[250]62,[251]63, we and others identified
   that Obsl1 may interact with other proteins linked to the DSG complex
   and its associated components^[252]35, including FlnC, tubulin,
   dystonin, plectin, ahnak, and utrophin (Supplementary Figs. [253]1a,
   [254]5a, [255]6c). Obsl1/obscurin function has also been implicated for
   microtubule integrity, by promoting microtubule assembly and/or
   stability^[256]13,[257]14,[258]64. Microtubule action is important for
   SR organization^[259]65, sarcolemmal integrity and
   repair^[260]66,[261]67, cell division, cellular shape, and the
   transport of vesicles over longer distances, in addition requiring
   actions of kinesin and dynein as molecular motors^[262]68. When looking
   at the proteome data, we noticed increases in α- and β-tubulins, which
   can be verified in immunoblot analyses of dKO TA muscles (Fig. [263]3a,
   Supplementary Fig. [264]2a). Alterations in the microtubule network
   have also been linked to the development of muscular dystrophy in mdx
   mice^[265]69. Dystrophin was shown to interact with
   microtubules^[266]70,[267]71, and its loss increased the amounts of
   both α- and β-tubulins^[268]69, a finding that is similar to what is
   seen in our dKO mice.

   One of the key findings in skeletal muscles of obscurin knockouts was
   the importance of the protein for SR architecture through its link with
   the small muscle-specific ankyrin-1 isoform sAnk1.5 that is embedded in
   the SR membrane^[269]20,[270]21. While loss of Obsl1 has little or no
   impact on protein levels of SR-associated proteins, muscles of dKO mice
   displayed profound alterations to levels of lumenal SR calcium-binding
   proteins sarcalumenin and Casq2, as well as Serca. Our proteome and
   expression data also indicated increases to junctional SR proteins like
   triadin, junctin, or RyRs in dKO muscles, something not previously
   observed for obscurin knockouts^[271]12. One might speculate based on
   the predominant subcellular localizations of obscurin at the M-band and
   Obsl1 at the M-band and Z-disc that both the longitudinal and
   junctional SR are affected in dKO muscles. Data on DSG-associated ion
   channels (Trpc1), the junctional (RyR) or longitudinal SR membranes
   (Serca), suggests that loss of obscurin/Obsl1 alters calcium handling
   in dKO muscles. These data in combination with published findings on
   Speg^[272]17 and the invertebrate homolog unc-89^[273]72 substantiates
   the finding that the regulation of SR architecture and function is a
   major biological role for all obscurin protein family members.

   Many questions remain unanswered as to the exact molecular origins for
   the observed effects in dKO muscles. Our proteome analyses may provide
   clues for some of the molecular mechanisms at play that could result in
   alterations to the cellular metabolism, changes to membrane stability
   and repair mechanisms, and the impact on calcium handling and Serca
   function/regulation. However, none of the well-characterized
   muscle-specific obscurin and Obsl1 interaction partners may account for
   all of the observed phenotypical
   changes^[274]2,[275]11,[276]20,[277]21,[278]25,[279]26,[280]57,[281]73.
   Indeed, it remains to be demonstrated for the majority of the more than
   600 identified binding partners of obscurin and Obsl1^[282]35, whether
   they play a role in muscles, and how loss of both obscurin protein
   family members affect their expression, localization, and cellular
   functions.

Methods

Gene targeting and generation of Obsl1-knockout mice, analysis of animal
physiology

   The targeting construct was generated by subcloning isogenic 129svj
   genomic DNA fragments of the murine Obsl1 gene into a targeting vector,
   and placing coding exons 1 to 4 between loxP sites (Supplementary
   Fig. [283]1a). The construct was linearized, and electroporated into R1
   embryonic stem cells. G418-resistant clones were screened for correct
   homologous recombination by Southern blot analysis described
   elsewhere^[284]12. Germline transmission was tested by appearance of
   the agouti fur color phenotype of 129-derived ES-cells, after which
   animals were backcrossed into the black swiss background. Genotying was
   done by PCR using oligonucleotides shown in Supplementary Table [285]1.
   Removal of neomycin cassette was done by crossing floxed Obsl1 mice
   with mice carrying Flpase recombinase^[286]74,[287]75. Generation of
   global Obsl1 knockouts was done by crossing floxed Obsl1 mice with mice
   carrying Cre recombinase under control of the protamine
   promoter^[288]30. Finally, generation of skeletal muscle-specific Obsl1
   knockouts was achieved by crossing floxed Obsl1 mice with mice carrying
   Cre recombinase under control of the myogenin promoter^[289]32.
   Successful recombination of Obsl1 gene and generation of skeletal
   muscle knockouts was validated by immunoblot analyses (Fig. [290]1a).
   Generation of obscurin/Obsl1 myogenin-cre dKOs was done by
   crossbreeding into the obscurin knockout background^[291]12. If not
   explicitly stated, all investigations were done with tissues from
   animals of both sexes. Experiments using the fifth toe of the EDL
   muscle from 4-month-old mice was done as described previously^[292]12.
   All procedures involving genetically modified animals have been
   approved by the UC San Diego institutional oversight committee.

Cell culture

   Culture of cos-1 cells was done as previously
   described^[293]76,[294]77. To test Obsl1 antibodies and to determine if
   effects seen in dKO and Obsl1-knockout muscles are also seen in
   non-muscle Obsl1-knockout cells, we isolated cells from lungs of
   heterozygous global Obsl1 knockouts (+/−) and from mice that have a
   knockout and a floxed allele for Obsl1 (flox/−), using a
   collagenase/dispase enzyme mix (Roche). Isolated cells were cultured
   and propagated in growth medium (10% fetal calf serum, 1%
   penicillin/streptomycin, Dulbecco’s modified Eagle’s medium [Corning]).
   To generate Obsl1 knockouts, cells were transduced with a lentivirus
   carrying a cre-blasticidin-RFP expression cassette under control of a
   CMV promoter (#LVP013; Gentarget Inc.), allowing for recombination of
   the Obsl1-floxed allele and selection of transduced cells by 5 µg/ml
   blasticidin (Thermo Fisher). After transduction, cells were kept in
   growth medium supplemented with blasticidin until harvest for analysis
   of proteins.

Protein analysis, immunofluorescence, histology, and antibodies

   For analysis of protein levels, muscles or cells were lysed directly
   into sample buffer (3.7 M urea, 134.6 mM Tris-HCl, pH 6.8, 5.4% sodium
   dodecyl sulfate (SDS), 2.3% NP-40, 4.45% β-mercaptoethanol, 4%
   glycerol, and 6 mg/100 ml bromophenol blue). Loading was normalized by
   densitometry of Coomassie-stained gels using either actin or myosin
   bands. Once normalized, muscle lysates were loaded onto
   SDS-polyacrylamide gel electrophoresis (PAGE) gels (8, 12, or 15%
   acrylamide concentration) or SDS-Agarose gels^[295]12. Proteins were
   transferred onto nitrocellulose membranes, and detection of proteins
   was done using antibodies as described elsewhere^[296]77. If not stated
   otherwise, biological replicates were used for immunoblot analyses.
   Sample sizes for quantification of protein levels are given in the
   figure or figure legend. Uncropped blot images for all western blot
   data are presented in Supplementary Data File [297]6.

   For immunofluorescence, muscles were isolated, snap frozen while
   submerged in isobutane, and embedded into cryogenic molds using optimal
   cutting temperate (OCT) medium. Longitudinal or cross-sections of
   muscles were done by cryosectioning using a cryostat (Leica). Sections
   were collected on surface-treated microscopy glass slides (Colorfrost
   Plus, Fisher), dried, and stored at −80 °C until further use.
   Immunofluorescent staining of sections was done by fixing sections in
   ice-cold acetone (5 min at −20 °C), rehydration of tissue using 1×
   phosphate-buffered saline (PBS) (5 min), permeabilization (1× PBS, 0.2%
   Triton X-100; 5 min), and blocking with 1% bovine serum albumin (BSA)
   fraction V and 5% normal donkey serum diluted in Gold Buffer (155 mM
   NaCl, 2 mM EGTA, 2 mM MgCl[2], 20 mM Tris-HCl, pH 7.5) for 30 min.
   After blocking of non-specific binding sites, tissue sections were
   incubated with primary antibody diluted in Gold Buffer overnight at
   4 °C. Following three washes with PBS (5 min each), sections were
   incubated with fluorescently labeled secondary antibodies combined with
   either 4′,6-diamidino-2-phenylindole (DAPI) and/or fluorescently
   labeled wheat germ agglutinin (WGA) (Sigma) diluted into Gold Buffer
   for 2 h at room temperature. After washing with 1× PBS (5 min each),
   sections were embedded using fluorescent mounting medium (DAKO), and
   covered with coverslips. Immunofluorescently labeled tissues were
   imaged using a Fluoview 1000 confocal microscope (Olympus), in
   sequential scanning mode using ×10 air or ×40 oil objectives and zoom
   rates between 1× and 3×. For analysis of cross-sectional areas, images
   were analyzed using the area-measuring tool in ImageJ.

   Primary antibodies used in this study are listed in Supplementary
   Table [298]2. If not noted otherwise, all secondary antibodies were
   either from DAKO or Jackson ImmunoResearch. Fluorescently labeled WGA
   was obtained from Invitrogen/Thermo Fisher. DAPI was purchased from
   Sigma-Aldrich.

Mass spectrometry and proteome analysis

   Proteins were isolated form whole muscle and lysed into ice-cold
   isolation buffer (300 mM KCl, 30 mM PIPES pH 6.6, 0.5% NP-40, 1×
   protease inhibitor (Roche), 1× Phos-stop (Roche)). Insoluble proteins
   were removed by centrifugation (14,000 rpm, 10 min at 4 °C), and the
   supernatant was diluted 1:4 with ice-cold dilution buffer (1× Phos-stop
   (Roche), 0.5% NP-40, 1 mM dithiothreitol (DTT)). Precipitation of
   acto-myosin components was done by centrifugation (14,000 rpm, 15 min
   at 4 °C), and the remaining supernatant snap frozen for further
   analysis by mass spectrometry and immunoblot analyses.

   Analysis and identification of peptides via mass spectrometry was done
   as described previously^[299]78. Briefly, immediately prior to mass
   spectrometry, protein solutions were diluted in TNE buffer (50 mM
   Tris-HCl, pH 8.0, 100 mM NaCl, 1 mM EDTA). RapiGest SF reagent (Waters
   Corp.) was added to the mix to a final concentration of 0.1% and
   samples were boiled for 5 min. TCEP (Tris (2-carboxyethyl) phosphine)
   was added to a final concentration of 1 mM, and the samples were
   incubated at 37 °C for 30 min. Subsequently, the samples were
   carboxymethylated with 0.5 mg/ml of iodoacetamide for 30 min at 37 °C,
   followed by neutralization with 2 mM TCEP (final concentration).
   Protein samples prepared as above were digested with trypsin
   (trypsin:protein ratio of 1:50) overnight at 37 °C. RapiGest was
   degraded and removed by treating the samples with 250 mM HCl at 37 °C
   for 1 h, followed by centrifugation at 14,000 rpm for 30 min at 4 °C.
   The soluble fraction was then added to a new tube and the peptides were
   extracted and desalted using C18 desalting tips (Thermo Scientific).

   The trypsinized samples (eight samples) were labeled with isobaric tags
   (iTRAQ8, ABSCIEX)^[300]79, where each sample was labeled with a
   specific tag to its peptides as described in the manufacturer’s
   instructions. Each set of experiments were then pooled and fractionated
   using high pH reverse phase chromatography (HPRP-Xterra C18 reverse
   phase, 4.6 mm × 10 mm, 5 µm particle [Waters]). The chromatography
   conditions were as follows: the column was heated to 37 °C and a linear
   gradient from 5 to 35% B (Buffer A—20 mM ammonium formate pH 10
   aqueous, Buffer B—20 mM ammonium formate pH 10 in 80% acetonitrile
   (ACN)-water), followed by 5 min at 100% B was applied for 80 min at
   0.5 ml/min flow rate. A total of 42 fractions of 0.5 ml volume were
   collected. For liquid chromatography with tandem mass spectrometry
   (LC-MS/MS) analysis, some fractions were pooled to create a final 16
   pooled samples. Each of the pooled fractions were analyzed by high
   pressure liquid chromatography coupled with LC-MS/MS using nano-spray
   ionization.

   Nano-spray ionization experiments were performed with a TripleTof 5600
   hybrid mass spectrometer (ABSCIEX) interfaced with nano-scale
   reversed-phase UPLC (Waters Corporation, nanoACQUITY) using a 20 cm ×
   75 µm ID glass capillary packed with 2.5 µm C18 (130) CSHTM beads
   (Waters Corporation). A linear gradient (5–80%) of ACN was used to
   elute the peptides from the C18 column into the mass spectrometer at a
   flow rate of 250 μl/min for 1 h. The ACN gradient was created with the
   following buffers: Buffer A (98% H[2]O, 2% ACN, 0.1% formic acid, and
   0.005% trifluoroacetic acid (TFA)) and Buffer B (100% ACN, 0.1% formic
   acid, and 0.005% TFA). MS/MS data were acquired in a data-dependent
   manner in which the MS1 data was acquired for 250 ms at m/z of 400 to
   1250 Da and the MS/MS data was acquired from m/z of 50 to 2000 Da.
   Independent data acquisition parameters were as follows: MS1-TOF (mass
   spectrometry imaging-time of flight) acquisition time of 250 ms,
   followed by 50 MS2 events of 48 ms acquisition time for each event. The
   threshold to trigger MS2 event was set to 150 counts when the ion had
   the charge state +2, +3, and +4. The ion exclusion time was set to 4 s.
   The collision energy was set to iTRAQ experiment setting. Finally, the
   collected data were analyzed using Protein Pilot 5.0 (ABSCIEX) for
   peptide identifications and Peaks^[301]80. Bioinformatic enrichment and
   pathway analysis was done using Metascape
   ([302]http://metascape.org/)^[303]81, Morpheus
   ([304]https://software.broadinstitute.org/morpheus/), the BioGRID
   ([305]https://thebiogrid.org/)^[306]82, and Venny
   ([307]http://bioinfogp.cnb.csic.es/tools/venny).

Eukaryotic expression constructs, yeast two-hybrid screening, and
co-immunoprecipitation

   Constructs for green fluorescent protein (GFP)-tagged Obsl1 fragments
   and yeast two-hybrid screening were generated as described
   elsewhere^[308]24. In short, coding sequences for human Obsl1 fragments
   (NCBI access no: [309]NM_178884) were subcloned to enhanced GFP (EGFP)
   (Clontech) or pLex vectors to produce in-frame fusion between GFP or
   the DNA-binding domain and the insert. All expression constructs were
   sequenced to determine correct integration into the vector. The yeast
   two-hybrid screening was done as described previously^[310]77. Briefly,
   L40 yeasts were transformed with bait constructs (pLex-Obsl1 domain
   truncations) and empty prey vector (pAct2) to perform auto-activation
   test. pLex-Obsl1 constructs that were negative for auto-activation were
   selected for further yeast two-hybrid screening. L40 yeasts were
   transformed with selected pLex-Obsl1 constructs, and positive
   transformants were used to perform a library transformation. Yeasts
   were plated on selective dropout plates, and colonies were allowed to
   form for 5 days. Yeast clones positive for β-galactosidase activity in
   an X-gal overlay assay were further amplified, and library plasmid DNA
   was extracted and analyzed by sequencing.

   Transfection of Cos-1 cells was done as described previously^[311]77.
   Transfected cells were lysed into ice-cold immunoprecipitation (IP)
   buffer (100 mM NaCl, 10 mM Tris-HCl, pH 8, 1× Complete Protease
   Inhibitor Cocktail [Roche], 1 mM DTT, and 0.5% NP-40) 2 days after
   transfection. Lysates were briefly sonicated, centrifugated for 10 min
   at 14,000 rpm (at 4 °C) to remove insoluble proteins, and supernatants
   were used to perform immunoprecipitation. Briefly, 5 µg GFP antibody
   (Roche) were added to lysates, and incubated overnight at 4 °C to allow
   for formation of immunocomplexes. Subsequently, magnetic
   protein-G-coated beads (Dynabeads, Life Technologies) were added and
   incubated for 3 h at 4 °C on a shaker. Bound protein immunocomplexes
   were separated from unbound proteins by magnetic separation, and the
   beads were washed three times with ice-cold wash buffer (1× PBS, 0.2%
   NP-40). Analysis of input and bound protein immunocomplexes was done by
   SDS-PAGE, followed by immunoblot analysis.

Bioinformatics and statistical analysis

   The following bioinformatics packages and programs were used to perform
   analyses in this manuscript: analysis of proteome data was done using
   Peaks Studio ([312]http://www.bioinfor.com/peaks-studio/)^[313]80;
   pathway enrichment analysis was done with the help of MetaScape
   ([314]http://metascape.org/)^[315]81; hierarchical clustering of
   proteome data was done using Morpheus
   ([316]https://software.broadinstitute.org/morpheus/); image analysis
   was done using ImageJ with the LOCI BioFormats and Image5D plugins (by
   the Open Microscopy Environment consortium and Joachim Walter,
   respectively); sequence alignments and domain structure analysis were
   done using NIH/NCBI BLAST
   ([317]https://blast.ncbi.nlm.nih.gov/Blast.cgi), SDSC Biology Workbench
   ([318]http://workbench.sdsc.edu/), and SMART
   ([319]http://smart.embl-heidelberg.de/)^[320]83. With the exception for
   built-in statistics by the Peaks Studio software, statistical analysis
   of all data was done by using analysis of variance comparison, followed
   by Dunnett’s multiple comparisons test, or unpaired t test, performed
   using Excel (Microsoft) or GraphPad Prism version 7 for Mac (GraphPad
   Software, [321]http://www.graphpad.com). Violin plots were generated
   using BoxPlotR ([322]http://shiny.chemgrid.org/boxplotr/). Results are
   presented as means ± standard error. P values of p < 0.05 were
   considered statistically significant. Sample sizes are indicated in the
   figure or figure legend. If not stated otherwise, both sexes were
   analyzed in the experimental procedures. We used biological replicates
   to showcase biological variability and reproducibility of findings.
   Whenever appropriate, different methods were used to verify a finding
   (e.g., interaction found in yeast two-hybrid screening was verified by
   co-immunoprecipitation). No data were excluded in the generation of
   figures/tables, with the exception of two lanes in Supplementary
   Fig. [323]7b (upper panel) that did not pass the quality control as
   judged by Ponceau stain.

Reporting summary

   Further information on experimental design is available in
   the [324]Nature Research Reporting Summary linked to this article.

Supplementary information

   [325]Supplementary Information^ (17MB, pdf)
   [326]42003_2019_405_MOESM2_ESM.docx^ (13.8KB, docx)

   Description of Additional Supplementary Files
   [327]Reporting Summary^ (70.1KB, pdf)
   [328]Supplementary Data 1^ (367.2KB, xlsx)
   [329]Supplementary Data 2^ (107.3KB, xlsx)
   [330]Supplementary Data 3^ (98.2KB, xlsx)
   [331]Supplementary Data 4^ (76.1KB, xlsx)
   [332]Supplementary Data 5^ (75.9KB, xlsx)
   [333]Supplementary Data 6^ (18.2MB, pdf)

Acknowledgements