Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: (A) WT and A165V mutant Ldb3 mRNA sequences are shown, highlighting the mutation site (red). Nineteen candidate siRNAs were designed to target this region; the five selected sequences (sense strand), chosen based on computational predictions and prior evidence, are indicated by arrows. siRNAs were numbered according to their position relative to the mismatch with the WT sequence and evaluated in HEK-293 cells expressing either WT or mutant LDB3. (B and C) Immunoblot analysis performed 48 h post-transfection showing effect of the siRNAs on total LDB3 protein levels in mutant- and WT-LDB3 expressing cells (n = 3 per group), including si-SC and NT controls. (D and E) AS-qPCR quantification of mutant and WT Ldb3 transcripts (n = 3 per group). Transcript levels were normalized to Gapdh using the ΔΔCt method and immunoblots were normalized to GAPDH. (F) Schematic of the miR30-based shRNA construct encoding either si10-Ldb3-A165V or si16-Ldb3-A165V (top). The construct was packaged into AAV9 for intramuscular delivery. Following cellular entry, shRNAmiR transcripts are processed through the endogenous microRNA pathway to generate mature shRNAs targeting mutant Ldb3 mRNA (bottom). (G) Schematic of the dose-optimization strategy for allele-specific Ldb3 targeting. Three-month-old Ldb3 Ala165Val/+ mice received a single intramuscular injection of AAV-shRNAmiR (sh10 or sh16), and TA muscles were analyzed 1 month later. (H) Schematic of the therapeutic disease-reversal paradigm. Ldb3 Ala165Val/+ mice were injected intramuscularly with AAV-shRNAmiR (sh10 or sh16) at 5 months of age, and outcomes were evaluated at 1 and 3 months post-injection. All data are presented as mean ± SD of three biological replicates (n = 3) with technical triplicates. Only statistically significant comparisons are shown. *P < 0.05, **P < 0.01 and ***P < 0.001, as determined by Welch’s two-sample t test. si-SC, scrambled siRNA control; NT, no-treatment control. F, G and H created from Biorender.com.

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Mutagenesis, Sequencing, Expressing, Western Blot, Transfection, shRNA, Construct, Injection, Muscles, Control

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: 5-month-old Ldb3 Ala165Val/+ mice received a single intramuscular injection of sh10 or sh16 into the TA muscle; contralateral TA muscles received scramble control (SC). (A and B) AS-qPCR analysis showing relative expression of WT (A165A) and mutant Ldb3 -A165V transcripts at 1 and 3 months post-injection compared to SC-treated controls in sh16 and sh10 treatment groups. (C and D) Representative immunoblots and densitometric quantification showing total LDB3 protein levels in the sh16 and sh10 treated muscles at 1 and 3 months post-injection compared to SC-treated controls. AS-qPCR and immunoblot data were obtained from 3-7 animals per group and triplicate assays. AS-qPCR data were normalized to Myom1 using the ΔΔCt method, and immunoblots were normalized to endogenous vinculin protein. (E and F) Adjacent transverse cryosections of frozen TA muscles harvested 3 months posttreatment was stained with modified Gomori trichrome (MGT), NADH-TR, and filamin C. Representative images show sarcoplasmic aggregates and abnormal oxidative enzyme activity (black arrowheads) in SC-treated controls (top panels) compared with sh16- and sh10-treated muscles (bottom panels). Filamin C-positive aggregates are indicated by white arrowheads in SC-treated and absence in sh16 and sh10 treated muscle. (G and H) Representative images of frozen transverse TA muscle cryosections stained with wheat germ agglutinin (WGA; cyan) showing internal nuclei (red dots in magnified insets), together with scatter bar plots quantifying the percentage of fibers containing internal nuclei in sh16- and sh10-treated muscles compared with SC-treated controls. Scale bars: MGT, NADH-TR, and filamin C: 50 µm; WGA: 250 µm. (N = 3-6 TA muscles per group). (I and J) In-vivo assessment of muscle contractility in 5-month-old Ldb3 Ala165Val/+ mice treated with sh10 for 1 month. (I) Force-frequency relationship curves showing specific isometric torque generated across increasing stimulation frequencies in WT mice, sh10-treated TA muscles, and contralateral SC-treated TA muscles. (J) Quantification of peak specific isometric torque in WT, sh10-treated TA muscles, and contralateral SC-treated TA muscles. In the force-frequency curves, matching colored rectangles and circles denote shRNA-treated TA muscles and the corresponding contralateral SC-treated TA muscles from the same mice, respectively, while purple triangles denote PBS-treated WT TA muscles used as reference controls (n = 3-4 mice per group). Data are presented as mean ± SD (A-F) and mean ± SEM (J). Statistical significance: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant, as determined by an unpaired, two-tailed Student’s t-test (A, B, I and J) and Welch’s t-test (C and D).

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Injection, Muscles, Control, Expressing, Mutagenesis, Western Blot, Staining, Modification, Activity Assay, In Vivo, Generated, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: Representative IF images of LDB3 and filamin C in PFA perfused longitudinal TA muscle sections from advanced-stage, (5-month-old) Ldb3 Ala165Val/+ mice treated with sh-SC, sh16, or sh10 ( A and B) for one month (upper panel) or three months (bottom panel) (n = 4-6 per group). (C and D) Quantification of muscle fibers containing sarcoplasmic filamin C aggregates in sh-SC control and shRNA-treated groups. Bar scatter plots represent the percentage of fibers exhibiting filamin C aggregates, quantified from at least five randomly selected areas (> 50 fibers per muscle) from immunostained TA sections (n = 3 per group). (E and F) Representative IF images of BAG3 and HSPA8 in longitudinal TA cryosections from advanced-stage, (5-month-old) Ldb3 Ala165Val/+ mice treated with sh-SC, sh16, or sh10 for 1 month (upper panels) or 3 months (lower panels) (n = 4-6 per group). Data are expressed as mean ± SEM, with statistical significance denoted by ****P < 0.0001, determined using an unpaired, two-tailed Student’s t-test. Scale bars represent 50 µm (A and B) and 20 µm (E and F).

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Control, shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: Representative immunoblots showing PKCα protein levels in frozen TA muscle from 5-monthold Ldb3 Ala165Val/+ mice treated with AAV9-shRNAmiR constructs and age-matched PBS treated WT controls. (A and B) PKCα protein levels following sh16 (A) or sh10 (B) treatment at 1 and 3 months post-injection. Bar-scatter plots below the blots show densitometric quantification of PKCα normalized to endogenous vinculin (n = 3 per group and triplicate assays). Ldb3 Ala165Val/+ mice were administered either scramble control (SC) or shRNAmiR targeting sh16 or sh10. Data are presented as mean ± SD, with statistical significance levels of **P < 0.01, ***P < 0.001, ****P < 0.0001, calculated using a two-tailed unpaired Student’s t-test.

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Western Blot, Construct, Injection, Control, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: Three-month-old Ldb3 Ala165Val/+ mice received a single intramuscular injection of sh10 or sh16 into the TA muscle; contralateral TA muscles received scramble control (SC). (A) Schematic of the early-intervention (disease-prevention) paradigm. Ldb3 Ala165Val/+ mice were injected intramuscularly with AAV-shRNAmiR (sh10 or sh16) at 3 months of age, and outcomes were evaluated at 1 and 3 months post-injection (created from Biorender.com ). (B and C) AS-qPCR analysis showing relative expression of WT (A165A) and mutant Ldb3 -A165V transcripts at 1 and 3 months post-injection compared to scramble-treated controls (SC) in sh16 and sh10 treatment groups. (D and E) Representative immunoblots and densitometric quantification showing total LDB3 protein levels in the sh16 and sh10 treated muscles at 1 and 3 months post-injection compared to SC-treated controls. AS-qPCR and immunoblot data were obtained from 3-6 animals per group and triplicate assays. AS-qPCR data were normalized to endogenous Myom1 using the ΔΔCt method, and immunoblots were normalized to endogenous vinculin protein. Data are presented as mean ± SD, with statistical significance defined as **P < 0.01, ***P < 0.001, and ****P < 0.0001; ns, not significant, as determined by unpaired, two-tailed Student’s t-test (B and C) and Welch’s t test (D and E).

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Injection, Muscles, Control, Expressing, Mutagenesis, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: (A and C) Representative IF images of LDB3 and filamin C in PFA perfused longitudinal TA muscle sections from early-stage (3-month-old) Ldb3 Ala165Val/+ mice treated with sh-SC, sh16 (A), or sh10 (C) for one month (upper panel) or three months (lower panel) (n = 4-6 per group). (B and D) Quantification of muscle fibers containing sarcoplasmic filamin C aggregates in sh-SC and shRNA-treated groups corresponding to panels (A) and (C). Bar-scatter plots represent the percentage of fibers exhibiting filamin C aggregates, quantified from at least five randomly selected fields (>50 fibers per muscle) per section (n = 3 per group). (E and F) Representative IF images of BAG3 and HSPA8 in longitudinal TA cryosections from early-stage, (3-month-old) Ldb3 Ala165Val/+ mice treated with sh-SC, sh16, or sh10 for 1 month (upper panels) or 3 months (lower panels) (n = 4-6 per group). Data are expressed as mean ± SEM, with statistical significance denoted by **P < 0.01, determined using an unpaired, two-tailed Student’s t-test. Scale bars represent 50 µm (A and C) and 20 µm (E and F).

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: shRNA, Two Tailed Test

Journal: bioRxiv

Article Title: AAV-Delivered RNAi Targeting Mutant LDB3 Prevents and Reverses Myofibrillar Myopathy through Mechanosignaling Restoration

doi: 10.64898/2026.03.28.715031

Figure Lengend Snippet: Representative immunoblots showing PKCα protein levels in frozen TA muscle from 3-monthold Ldb3 Ala165Val/+ mice treated with AAV9-shRNAmiR constructs and age-matched PBS treated WT controls. (A and B) PKCα expression following sh16 (A) or sh10 (B) treatment at 1 and 3 months post-injection. Bar-scatter plots below the blots show densitometric quantification of PKCα normalized to endogenous vinculin (n = 3 per group and triplicate assays). Ldb3 Ala165Val/+ mice were administered either scramble control (SC) or shRNAmiR targeting sh16 or sh10. Data are presented as mean ± SD, with statistical significance levels of *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, calculated using a two-tailed unpaired Student’s t-test. Phosphoproteomic profiling was performed to compare shRNA-treated and SC-treated TA muscles (n = 3 per group). (C) Volcano plot of differentially phosphorylated proteins in sh16-treated versus SC-treated TA muscle. Each point represents a phosphoprotein; red points indicate significantly upregulated phosphosites and green points indicate significantly downregulated phosphosites (adjusted P < 0.05), whereas gray points denote non-significant changes. The x axis represents log□ fold change (treatment/control), and the y axis represents −log□□ adjusted P values. A total of 35 phosphoproteins met significance criteria (adjusted P < 0.05). (D) Pathway and process enrichment analysis of differentially phosphorylated proteins. Significantly enriched MGI Phenotype and Gene Ontology (GO) Biological Process terms are shown. Bars represent−log□□ (P value); unadjusted P values are displayed. (E) Protein-protein interaction network constructed from significantly altered phosphoproteins. Nodes represent phosphoproteins; edges indicate curated interactions. PKCα is positioned within the network to visualize connectivity among structural and signaling modules. (F) KEA3 kinase enrichment analysis. Mean rank heat bar showing top-ranked upstream kinases inferred from the phosphoproteomic dataset across multiple kinase-substrate libraries (PhosD, STRING, and Kinase Library). Color scale reflects relative ranking within each database. (G) Cheng-KSIN kinase enrichment analysis. Bar plot and corresponding network visualization of kinases enriched based on motif-derived kinase–substrate interactions. Enrichment is represented as −log(P value). (H) PhosD all-kinase ranking. Top kinases ranked using kinase-substrate co-phosphorylation associations derived from the PhosD library. (I) PhosD kinase interaction mini-network illustrating inferred relationships among PRKCA (PKCα) and associated signaling nodes.

Article Snippet: For initial assessment of total Ldb3 expression, a TaqMan assay targeting constitutively spliced exons 2 and 3 of Ldb3 (NM_001039071.2; Mm01208763_m1) was used, with Gapdh (Mm99999915_g1) serving as the reference gene (Life Technologies).

Techniques: Western Blot, Construct, Expressing, Injection, Control, Two Tailed Test, shRNA, Muscles, Derivative Assay, Phospho-proteomics

Journal: Frontiers in Genetics

Article Title: Time- and Ventricular-Specific Expression Profiles of Genes Encoding Z-Disk Proteins in Pressure Overload Model of Left Ventricular Hypertrophy

doi: 10.3389/fgene.2018.00684

Figure Lengend Snippet: Oligonucleotide primers and Assay ID.

Article Snippet: Ldb3 , Rn01453313_m1.

Techniques:

Journal: Frontiers in Genetics

Article Title: Time- and Ventricular-Specific Expression Profiles of Genes Encoding Z-Disk Proteins in Pressure Overload Model of Left Ventricular Hypertrophy

doi: 10.3389/fgene.2018.00684

Figure Lengend Snippet: Altered mRNA expression of genes, encoding Z-disk proteins, in different parts of myocardium. (A) Actn2 , Cmya5 , and Nebl expression patterns after different period of aortic banding measured by qRT-PCR – downregulation after 1 week with subsequent return to normal level and further decrease after 10 weeks. Expression Ldb3 declined after 1, 2, and 10 weeks of aortic banding. (B) Altered expression of genes, encoding Z-disk proteins, in right ventricle (RV). Actn2 , Cmya5 , and Ldb3 expression was gradually increasing after 2 weeks up to 10 weeks of aortic constriction. Despite the gradual decrease of Nebl expression in RV, we showed no further Nebl reduction to the 10 week’s time point. Notably, expression pattern in RV is clearly distinct from pattern in LV. (C) Expression pattern of genes, encoding Z-disk proteins, in IVS are distinctly more close to RV than to LV profiles. Cmya and Nebl mRNA levels were elevated on later phases of hypertrophy progression (after 10 weeks). Opposite, Actn2 is upregulated only on the early phase (after 1 week). Ldb3 mRNA level was significantly increased after 1 week as well as after 10 weeks of aortic constriction. For all ∗ P < 0.05.

Article Snippet: Ldb3 , Rn01453313_m1.

Techniques: Expressing, Quantitative RT-PCR

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: STAR Protocols

Article Title: A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

doi: 10.1016/j.xpro.2021.101117

Figure Lengend Snippet: Blast of ldb3 primers for rat against the human LDB3 locus (A) The ldb3 forward primer for rat ( Guo et al., 2012 ) was blasted against the human LDB3 locus. The primer shows 3 mismatches compared to the sequence of the human exon 4. (B) The ldb3 reverse primer for rat (from Guo et al., 2012 ) was blasted against the human LDB3 locus. The primer shows 2 mismatches compared to the sequence of the human exon 7. The color code in A and B is related to the human LDB3 locus. (C) Schematic summary of localization of primers for ldb3 in rat (from Guo et al., 2012 ) in ENSRNOT0000083623.1 and the localization on the human locus in LDB3 in ENST00000623056.4.

Article Snippet: LDB3 expression exon3_4 (for normalization) qPCR Fwd: ACCTCGTGGTGGCCATTG , Microsynth , N/A.

Techniques: Sequencing

Journal: STAR Protocols

Article Title: A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

doi: 10.1016/j.xpro.2021.101117

Figure Lengend Snippet: DNA bands after PCR amplification of LDB3 using primer “LDB3 exon 4–7” for semi-quantitative RT-PCR (see ) on an agarose gel The two bands were excised from the gel, and the DNA bands were purified for Sanger sequencing. Sequencing results in combination with Clustalomega-based blasting demonstrated exons 4, 5, 6, 7 in the upper band and exons 4, 6, 7 in the lower band.

Article Snippet: LDB3 expression exon3_4 (for normalization) qPCR Fwd: ACCTCGTGGTGGCCATTG , Microsynth , N/A.

Techniques: Amplification, Quantitative RT-PCR, Agarose Gel Electrophoresis, Purification, Sequencing

Journal: STAR Protocols

Article Title: A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

doi: 10.1016/j.xpro.2021.101117

Figure Lengend Snippet: Cq-values for the quantification of LDB3 -exon 5 inclusion

Article Snippet: LDB3 expression exon3_4 (for normalization) qPCR Fwd: ACCTCGTGGTGGCCATTG , Microsynth , N/A.

Techniques: Expressing

Journal: STAR Protocols

Article Title: A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

doi: 10.1016/j.xpro.2021.101117

Figure Lengend Snippet: Relative Expression of LDB3 -exon 5 Differential mis-splicing for exon 5 in LDB3 in RBM20_S635AiPSC-CM. Every dot represents one differentiation experiment. Data is shown as bar graph with standard error (control).

Article Snippet: LDB3 expression exon3_4 (for normalization) qPCR Fwd: ACCTCGTGGTGGCCATTG , Microsynth , N/A.

Techniques: Expressing, Control

Journal: STAR Protocols

Article Title: A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

doi: 10.1016/j.xpro.2021.101117

Figure Lengend Snippet:

Article Snippet: LDB3 expression exon3_4 (for normalization) qPCR Fwd: ACCTCGTGGTGGCCATTG , Microsynth , N/A.

Techniques: Gel Extraction, Reverse Transcription, Control, Mutagenesis, Expressing, Software, Recombinant, SYBR Green Assay, Saline, Electrophoresis

Journal: STAR Protocols

Article Title: A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

doi: 10.1016/j.xpro.2021.101117

Figure Lengend Snippet:

Article Snippet: LDB3 expression exon3_4 (for normalization) qPCR Fwd: ACCTCGTGGTGGCCATTG , Microsynth , N/A.

Techniques:

Journal: STAR Protocols

Article Title: A quantitative RT-PCR protocol to adapt and quantify RBM20-dependent exon splicing of targets at the human locus

doi: 10.1016/j.xpro.2021.101117

Figure Lengend Snippet:

Article Snippet: LDB3 expression exon3_4 (for normalization) qPCR Fwd: ACCTCGTGGTGGCCATTG , Microsynth , N/A.

Techniques:

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: