Murine obscurin and Obsl1 have functionally redundant roles in sarcolemmal integrity, sarcoplasmic reticulum organization, and muscle metabolism

Fig. 1. 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

Fig. 2. 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

Fig. 3. 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,…

Fig. 4. 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

Fig. 5. 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 Ca2+ 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 2

Fig. 6. 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