Journal: Communications Biology

Article Title: Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation

doi: 10.1038/s42003-021-01864-1

Figure Lengend Snippet: a Knock-in of the p.Ala165Val mutation in exon 6 (blue) of the mouse Ldb3 gene. Residual loxP site post- Cre recombination is seen in intron 6 (orange triangle). b Sanger sequence shows the heterozygous C > T mutation changing the codon GCT (Ala) to GTT (Val). c Immunoblotting analysis (blot and dot plot) of the LDB3 isoforms expression relative to β−actin in the vastus muscle of 8-month-old Ldb3 Ala165Val/+ mice (blue) and Ldb3 +/+ littermates (red). n = 4 each, represents triplicate assay. d Dot plot comparing maximum all paws grip force of five pulls per mouse normalized to body weight (g/g) between Ldb3 Ala165Val/+ mice (blue) and Ldb3 +/+ littermates (red) at 3, 6, and 9 months of age. Mean ± SEM: 9.2 ± 0.3 g/g versus 10.7 ± 0.3 g/g at 3 months, 6.9 ± 0.3 g/g versus 10.4 ± 0.3 g/g at 6 months, and 6.5 ± 0.2 g/g versus 7.9 ± 0.3 g/g at 9 months; n = 10–13 Ldb3 Ala165Val/+ mice and n = 8–10 Ldb3 +/+ mice per age group. The two-way ANOVA Bonferroni’s multiple comparisons test significant p values between groups are shown. e Dot plot shows maximal isometric force (mN/mm 2 ) generated by the extensor digitorum longus muscle of 6-month-old male Ldb3 Ala165Val/+ mice (blue) versus Ldb3 +/+ littermates (red) against stimulation frequencies (Hz). Mean ± SEM: 148 ± 5 mN/mm 2 versus 187 ± 3 mN/mm 2 ; 160 Hz; n = 4 Ldb3 Ala165Val/+ mice and n = 3 Ldb3 +/+ mice. The two-way ANOVA Bonferroni’s multiple comparisons test significant p values between groups are shown. f Dot plot shows the maximum holding impulse (N*s) in the four-limb wire grid holding test in 9-month-old male Ldb3 Ala165Val/+ mice and Ldb3 +/+ littermates. Mean ± SEM: 32 ± 4 N*s versus 81 ± 8 N*s; n = 9 Ldb3 Ala165Val/+ mice and n = 7 Ldb3 +/+ mice. Male mice were used in these tests as female mice showed intra- and inter-group variability. The two-tailed unpaired t -test comparison significance between groups is shown. * p < 0.05, ** p < 0.01, **** p < 0.0001. The error bars in dot plots represent Mean (SEM).

Article Snippet: The cleared lysates were incubated with either mouse anti-LDB3 antibody (Abnova) or mouse IGg2b (Cell signaling) for overnight at 4 °C and subsequently with protein A Dynabeads for 3 h at 4 °C.

Techniques: Knock-In, Mutagenesis, Sequencing, Western Blot, Expressing, Generated, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation

doi: 10.1038/s42003-021-01864-1

Figure Lengend Snippet: a Representative Gomori trichrome (top; GT) and NADH-TR (bottom) stained adjacent frozen soleus muscle transverse sections of 4- and 8-month-old Ldb3 Ala165Val/+ mice ( n = 10 and 16, respectively), and 8-month-old Ldb3 +/+ littermates ( n = 9). b Representative images are GT – stained transverse frozen vastus and tibialis anterior muscle sections of 8-month-old Ldb3 Ala165Val/+ mice ( n = 16). Muscle staining is normal in Ldb3 +/+ mice and 4-month-old Ldb3 Ala165Val/+ mice but shows sarcoplasmic aggregates and vacuoles ( a ), muscle fiber with rounded contour, hypertrophied fiber, and internal nuclei ( b ) in 8-month-old Ldb3 Ala165Val/+ mice. c Immunofluorescence staining of consecutive soleus muscle sections to those in (a) show sarcoplasmic protein accumulations in muscle fibers of 8-month-old Ldb3 Ala165Val/+ mice but not in Ldb3 +/+ mice. d – f Electron microscopy of the soleus muscle longitudinal section of 6-month-old Ldb3 +/+ mice ( n = 3) shows normal Z-disc (black arrows) and the sarcomere architecture ( d ), whereas in the Ldb3 Ala165Val/+ littermates ( n = 3) shows myofibrillar disruption at the Z-disc as streaming (black arrow; e ) and accumulation of granular material (white arrow; e ), as well as autophagic vacuoles (white arrow; f) and dislocated enlarged mitochondria (black arrow; f ). g , h Immunoblotting analysis (blot and dot plot) of LC3A/B-II and sequestosome-1 (p62) protein levels, relative to GAPDH, in the vastus muscle of 4-month-old Ldb3 Ala165Val/+ and Ldb3 +/+ littermates after three days of colchicine (COL) or PBS treatment. n = 3 mice per treatment group and triplicate assays. i Dot plot of LC3A/B-II and sequestosome-1 flux [ΔCOL − PBS]. Mean ± SD flux: LC3A/B-II: 8.4 ± 0.8 versus 3.3 ± 0.4 and sequestosome-1: 6.6 ± 0.5 versus 2.3 ± 0.6 in Ldb3 Ala165Val/+ mice and Ldb3 +/+ littermates, respectively. The two-way ANOVA Bonferroni’s multiple comparisons test ( h ) and the two-tailed unpaired t -test comparison ( i ) significant p values are shown. ** p < 0.01; *** p < 0.001; **** p < 0.0001. The error bars in dot plots represent Mean (SD). See Supplementary Fig. for full-length blots. Scale bars = 50 μm ( a – c ), 1 μm ( d – e ), and 2 μm ( f ).

Article Snippet: The cleared lysates were incubated with either mouse anti-LDB3 antibody (Abnova) or mouse IGg2b (Cell signaling) for overnight at 4 °C and subsequently with protein A Dynabeads for 3 h at 4 °C.

Techniques: Staining, Immunofluorescence, Electron Microscopy, Disruption, Western Blot, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation

doi: 10.1038/s42003-021-01864-1

Figure Lengend Snippet: a Representative immunofluorescence on frozen vastus lateralis and soleus muscle transverse section of 6-month-old Ldb3 Ala165Val/+ mice ( n = 9 and 5, respectively) and Ldb3 +/+ littermates ( n = 5) and an MFM patient ( n = 3) with the LDB3 p.Ala165Val mutation stained with filamin C and LDB3 antibodies. White arrows indicate muscle fibers with sarcoplasmic filamin C aggregates that have normal sarcoplasmic LDB3 distribution in Ldb3 Ala165Val/+ mice and relatively less extensive LDB3 accumulations in the patient. b – d Representative immunofluorescence on frozen soleus muscle serial transverse sections of 8-month-old Ldb3 +/+ mice ( b ; n = 6) and Ldb3 Ala165Val/+ littermates ( c ; n = 8), and the vastus lateralis muscle of patient ( d ; n = 3) stained with filamin C, BAG3, HSPA8, HSPB8, and ubiquitin antibodies. White arrows indicate same muscle fibers with sarcoplasmic accumulations of filamin C and the CASA proteins in Ldb3 Ala165Val/+ mice and patient. Such protein aggregates are not seen in the sections obtained from Ldb3 +/+ mice. Scale bars = 50 μm.

Article Snippet: The cleared lysates were incubated with either mouse anti-LDB3 antibody (Abnova) or mouse IGg2b (Cell signaling) for overnight at 4 °C and subsequently with protein A Dynabeads for 3 h at 4 °C.

Techniques: Immunofluorescence, Mutagenesis, Staining, Ubiquitin Proteomics

Journal: Communications Biology

Article Title: Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation

doi: 10.1038/s42003-021-01864-1

Figure Lengend Snippet: Representative immunofluorescence staining on perfused tibialis anterior muscle consecutive longitudinal sections of 4-month-old ( a – e ) and 8-month-old ( f – j ) Ldb3 Ala165Val/+ mice and their Ldb3 +/+ littermates. a Muscle sections of 4-month-old Ldb3 +/+ mice ( n = 4) show normal Z-disc staining for LDB3, filamin C, myotilin, BAG3, and HSPA8 proteins. b , c Muscle sections of 4-month-old Ldb3 Ala165Val/+ mice ( n = 6) show same muscle fiber co-stained with LDB3 and filamin C antibodies ( b ), and LDB3 and myotilin antibodies ( c ). d , e Muscle sections of 4-month-old Ldb3 Ala165Val/+ mice ( n = 6) show same muscle fiber co-stained with filamin C and HSPA8 antibodies ( d ), and BAG3 and HSPA8 antibodies ( e ). Filamin C, HSPA8, and BAG3 aggregates are seen at the Z-disc spanning multiple sarcomeres in same fiber. In contrast, the LDB3 and myotilin antibodies show normal Z-disc staining in the fiber with filamin C aggregates. f Muscle sections of 8-month-old Ldb3 +/+ mice ( n = 5) show normal Z-disc staining for LDB3, filamin C, myotilin, BAG3, and HSPA8 proteins. Top and bottom panel each shows same muscle fiber co-stained with LDB3 and filamin C antibodies ( g ), and LDB3 and myotilin antibodies ( h ) in muscle sections of 8-month-old Ldb3 Ala165Val/+ mice ( n = 7). Muscle sections of 8-month-old Ldb3 Ala165Val/+ mice ( n = 7) show same muscle fiber co-stained with LDB3 and BAG3 antibodies ( i ), and LDB3 and HSPA8 antibodies ( j ). LDB3 aggregates colocalize with filamin C, myotilin, BAG3, and HSPA8 at the Z-discs in muscle fibers. The protein aggregates are seen in muscle fibers with normal periodicity of the Z-discs and with disorganized Z-disc periodicity indicating Z-disc myofibrillar disruption. A part of adjacent muscle fiber with normal staining pattern is shown at bottom in each image for comparison. Scale bar = 10 μm and applies to all images.

Article Snippet: The cleared lysates were incubated with either mouse anti-LDB3 antibody (Abnova) or mouse IGg2b (Cell signaling) for overnight at 4 °C and subsequently with protein A Dynabeads for 3 h at 4 °C.

Techniques: Immunofluorescence, Staining, Disruption, Comparison

Journal: Communications Biology

Article Title: Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation

doi: 10.1038/s42003-021-01864-1

Figure Lengend Snippet: a Representative immunoblots showing protein levels of filamin C, BAG3, and HSPA8 relative to β -actin in the vastus muscle of 8-month-old Ldb3 Ala165Val/+ mice and Ldb3 +/+ littermates. Data represent n = 5 mice per group and triplicate assays. b Schematics of LDB3 interaction with the mechanosensing domains Ig17–21 of filamin C and its chaperone HSPA8 as identified by yeast two-hybrid (Y2H) screen of a human skeletal muscle cDNA library. See Supplementary Table . Locations of the LDB3 bait encoded by exons 8-11Δ10 (yellow) and the p.Ala165Val mutation (white; asterisk) within actin-binding domain (ABD; blue) are shown in LDB3-LΔex10 isoform , . Domain composition of the prey clones are shown. c Pairwise Y2H assays demonstrating interaction between LDB3 peptides and HSPA8. Positive interactions show yeast growth on the media deficient in HIS3 and ADE2 . Yeast cells co-transformed with empty bait vector and the HSPA8 prey show no growth (labeled –). Transformation efficiency was uniform for all constructs. Sequential tenfold yeast dilutions are shown. d GST pulldown assay shows that GST-tagged wildtype (WT) and mutant LDB3-LΔEX10 (Ala165Val) but not GST alone pulled down filamin C and its interactor the CASA cochaperone BAG3 from the vastus muscle lysates of wildtype mice. Data represent n = 3 mice and triplicate assays. e Co-immunoprecipitation (co-IP) assays show that a FLAG antibody pulled down the FLAG-tagged WT and mutant (Ala165Val) LDB3-LΔex10 together with HA-tagged filamin C rod domain Ig17–21 and HSPA8 in Cos7 cells. Data represent triplicate assays. The proteins were detected with anti-FLAG and anti-HA antibodies. f Co-IP assays show that an LDB3 antibody pulled down LDB3 isoforms together with filamin C and HSPA8 from the tibialis anterior muscle lysates of Ldb3 Ala165Val/+ and Ldb3 +/+ mice. Data represent n = 3 mice per group and triplicate assays.

Article Snippet: The cleared lysates were incubated with either mouse anti-LDB3 antibody (Abnova) or mouse IGg2b (Cell signaling) for overnight at 4 °C and subsequently with protein A Dynabeads for 3 h at 4 °C.

Techniques: Western Blot, cDNA Library Assay, Mutagenesis, Binding Assay, Clone Assay, Transformation Assay, Plasmid Preparation, Labeling, Construct, GST Pulldown Assay, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Communications Biology

Article Title: Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation

doi: 10.1038/s42003-021-01864-1

Figure Lengend Snippet: Heatmap of protein levels in the vastus muscle lysates of 4 months ( a ) and 8-month-old ( b ) Ldb3 Ala165Val/+ mice and their gender-matched Ldb3 +/+ littermates ( n = 5 per group; total 20 mice) detected by RPPA. Differentially expressed proteins with statistically significant fold changes are shown on the vertical axis (≥1.5-fold, corrected p ≤ 0.05). The levels of each protein are presented with colors, with blue for lowest and red for highest. See Supplementary Table . Only PKCα and TSC2 are known to localize at skeletal muscle Z-disc. c , d Immunoblotting analysis (blot and dot plot) of PKCα and TSC2 protein levels relative to vinculin in the vastus muscle of 4-month-old Ldb3 Ala165Val/+ mice ( n = 5 and 4, respectively) and Ldb3 +/+ littermates ( n = 4 and 3, respectively). e , f Immunoblotting analysis (blot and dot plot) for protein levels of PKCα and TSC2 relative to vinculin in the vastus muscle of 8-month-old Ldb3 Ala165Val/+ mice ( n = 4 and 5, respectively) and Ldb3 +/+ littermates ( n = 3 and 4, respectively). The error bars in dot plots are Mean (SEM) and represent triplicate assays. The two-tailed unpaired t -test comparison significant p values are shown. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The cleared lysates were incubated with either mouse anti-LDB3 antibody (Abnova) or mouse IGg2b (Cell signaling) for overnight at 4 °C and subsequently with protein A Dynabeads for 3 h at 4 °C.

Techniques: Western Blot, Two Tailed Test, Comparison

Journal: Communications Biology

Article Title: Myopathy associated LDB3 mutation causes Z-disc disassembly and protein aggregation through PKCα and TSC2-mTOR downregulation

doi: 10.1038/s42003-021-01864-1

Figure Lengend Snippet: a , b Diagrams show LDB3 – filamin C – CASA complex interactions at skeletal muscle Z-disc relevant to MFM pathogenesis. LDB3 binds to the mechanosensor Ig 17–21 repeats (orange) in filamin C where the binding sites for the MFM-associated proteins BAG3 and myotilin, as well as a myopathy-associated integrin complex have been identified , , . LDB3 binds to myotilin and PKCα, which regulates filamin C stability through phosphorylation (P) – , . LDB3 interacts with HSPA8, a BAG3 partner that together with the MFM-associated HSPB8 chaperone degrades damaged filamin C through CASA pathway (adapted from ). BAG3 cooperates with the HSPA8-associated ubiquitin ligase STUB1 and its partner UBE2D in the ubiquitination of chaperone-bound filamin C, which is recognized by sequestosome-1 leading to lysosomal disposal. These interactions are unaffected by the MFM-associated LDB3 p.Ala165Val mutation. c Model of LDB3 p.Ala165Val mutation effects on the integrity of LDB3 – filamin C – CASA complex interactome under mechanical strain. The activity of each protein and interaction is presented with color, blue for decrease and red for increase. An arrow indicates an activation, a bar at the end of an edge indicates an inhibitory interaction. A downward arrow within a box indicates decrease in the protein levels. Under normal conditions, LDB3 enables PKCα-mediated phosphorylation of filamin C and other interacting proteins at the Z-disc and protects these proteins from proteolysis by calpains. The CASA pathway constantly operates at the Z-disc mediating degradation of large cytoskeleton components including filamin C damaged during mechanical strain . The CASA activity depends on local mTORC1 inhibition through BAG3-recruited TSC2:TSC1 signaling . PKCα is modulated by TSC2-mTORC2 signaling and the kinase may regulate mTOR assemblies including spatial localization (dashed lines) – . The LDB3 p.Ala165Val downregulates PKCα and TSC2-mTOR, two signaling proteins monitoring the integrity of the Z-disc assembly. Decreased PKCα promotes proteolysis by calpains leading to aggregation of damaged proteins. Reduced TSC2-mTOR together with increased strain on capacity of degradation pathway leads to impaired CASA function aggravating damaged protein aggregation, eventually leading to the Z-disc disassembly and myofibrillar disruption.

Article Snippet: The cleared lysates were incubated with either mouse anti-LDB3 antibody (Abnova) or mouse IGg2b (Cell signaling) for overnight at 4 °C and subsequently with protein A Dynabeads for 3 h at 4 °C.

Techniques: Binding Assay, Phospho-proteomics, Ubiquitin Proteomics, Mutagenesis, Activity Assay, Activation Assay, Inhibition, Disruption

Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: (A) Overall scheme of disease-specific phosphorylated site selection. Only 47 phosphoproteins among 134 upregulated phosphoproteins were identified as phosphoproteins related to heart and muscle disease in the human disease database. Twenty-nine phosphoproteins were mapped in the heart tissue-specific expression database and literature. The changes at each phosphorylation site were compared with its protein abundance to identify phosphorylation-specific level changes. Finally, 14 target phosphoproteins and phosphorylation competition peptides for 18 phosphorylation target sites were selected for cell-based screening. (B) Cultured neonatal rat cardiomyocytes were treated with 100 nM competition peptides to inhibit site-specific phosphorylation and exposed to 100 µM PE for 24 h. The cell surface area was then determined by immunofluorescence staining of α-actinin, and bar graphs representing surface area were drawn. Statistical significance was assessed by two-tailed Student’s t -tests for each comparison group with three biological replicates. (C) Immunofluorescence images of sarcomeric α-actinin (a, untreated; b, PE; c, TAT; d, TAT + PE; e, Ldb3-S98 + PE; f, Ldb3-S121, 123 + PE; g, Ldb3-S170, 171 + PE; h, Ldb3-S179 + PE; i, Palld-S901 + PE; j, Pallad-S1146 + PE). PE, phenylephrine; TAT, transactivator of transcription. (D) Bar graphs showing changes in cell surface area following treatment with different concentrations of competition peptides targeting Ser-98 and Ser-179 of Ldb3. *** P < 0.001 vs untreated; ## P < 0.01, ### P < 0.001 vs TAT + PE; n.s., not significant; n = 3 per group.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: Selection, Expressing, Cell Culture, Immunofluorescence, Staining, Two Tailed Test

Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: Heart lysates were immunoblotted with non-phospho-antibody, Ser-98 site-specific phospho-antibody, and Ser-179 site-specific phospho-antibody in (A) 8-week-old and (B) 12-week-old JTT-1 TG mice. Bar charts show the relative intensity of site-specific phosphorylation compared with wild-type expression. P values determined using two-tailed Student’s t -tests; * P < 0.05, ** P < 0.01; n.s., not significant; n = 3 per group. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: Expressing, Two Tailed Test

Journal: Molecules and Cells

Article Title: Integrated Quantitative Phosphoproteomics and Cell-Based Functional Screening Reveals Specific Pathological Cardiac Hypertrophy-Related Phosphorylation Sites

doi: 10.14348/molcells.2021.4002

Figure Lengend Snippet: Changes in site-specific phosphorylation of Ser-98 and Ser-179 of Ldb3 were investigated in (A) physiological cardiac hypertrophy (swimming training for 2 or 4 weeks), and (B) pathological cardiac hypertrophy (TAC and sham surgery for 2 weeks). Lysates were probed with non-phospho- and phospho-antibodies (recognizing Ser-98 or Ser-179 of Ldb3) to detect changes in endogenous site-specific phosphorylation. * P < 0.05, ** P < 0.01; n.s., not significant; n = 3 per group. con, control; sw, swimming.

Article Snippet: The antibodies and dilutions used for immunoblotting were as follows: 1:10,000 phospho-Ser-98 and phospho-Ser-179 Ldb3 polyclonal antibodies (AbFrontier, Korea); 1:5,000 Cypher (sc-136380; Santa Cruz Biotechnology, USA); and 1:2,500 GAPDH (sc-32233; Santa Cruz Biotechnology).

Techniques: