Journal: Redox Biology

Article Title: DKK3 as a diagnostic marker and potential therapeutic target for sarcopenia in chronic obstructive pulmonary disease

doi: 10.1016/j.redox.2024.103434

Figure Lengend Snippet: Upregulation of DKK3 in skeletal muscles due to CS-induced muscle atrophy (a) Schematic diagram illustrating the induction of emphysema and muscle atrophy in male C57BL/6J mice (aged 8–10 weeks) by chronic cigarette smoke (CS) exposure. Each exposure session involved 20 cigarettes (Nicotine 0.8 mg, Tar 10 mg, carbon monoxide 10 mg) twice daily, six days a week, over a 12-week period. (b) Representative H&E staining of lung tissue from CS-exposed and control mice, highlighting the pathological differences. Scale bar: 100 μm. (c) Quantification of mean linear intercept (MLI) and mean alveolar number (MAN) in lung tissues of CS and control mice. (d) Comparison of grip strength between CS-exposed and control mice, showing significant reduction in the former. (e) Representative dissected skeletal muscles among the CS mice and control mice, including Quad (quadriceps), Gas (gastrocnemius) and TA (tibialis anterior). (f) Proportion of Quad, Gast, TA muscle weight in CS-exposed versus control mice. (g, h) Representative H&E staining of TA muscle sections, displaying distribution of myofiber cross-section area (CSA) in CS and control mice. Scale bar: 100 μm. (i) Heatmap of significantly differentially expressed genes in TA muscles, accompanied by Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes. (j, k) Immunoblots and quantification of DKK3 levels in TA muscles of CS and control mice. (l, m) Immunoblots and quantification of whole-cell protein lysates from fully differentiated myotubes treated with CSE for 48h, showing decreased MyHC protein levels and increased expression of DKK3. (n) Elevated plasma levels of DKK3 in CS-exposed mice, as measured by ELISA. (o) Elevated plasma levels of DKK3 in healthy controls (n = 23) and patients with COPD (n = 53), as measured by ELISA. At least five visual fields were randomly selected for each of the biological replicate in pathological analysis. Data are presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Two-tailed unpaired Student's t -test.

Article Snippet: For the DKK3 treatment, C2C12 myotubes that differentiated for four days were treated with recombinant human DKK3 (rhDKK3, 150 ng/mL) (HY–P7812, MCE, China) for 48 h after transiently transfected with siRNA.

Techniques: Muscles, Staining, Control, Comparison, Western Blot, Expressing, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Redox Biology

Article Title: DKK3 as a diagnostic marker and potential therapeutic target for sarcopenia in chronic obstructive pulmonary disease

doi: 10.1016/j.redox.2024.103434

Figure Lengend Snippet: Elevated Plasma DKK3 Levels Correlate with Disease Severity and Muscle atrophy in Patients with COPD (a) Plasma DKK3 levels in patients with COPD with and without sarcopenia. (b, c) Correlation between plasma DKK3 levels and exercise tolerance in patients with COPD, assessed by 6-min walk distance (6MWD) and five-time sit-to-stand test (5STS). (d, e) Correlation between plasma DKK3 levels and muscle strength, measured by quadriceps muscular strength (QMS) and handgrip strength (HGS). (f, g) Correlation between plasma DKK3 levels and skeletal muscle mass, measured by rectus femoris diameter (RFthick) and cross-sectional area (RFcsa). (h) Receiver operating characteristic (ROC) curve analysis of plasma DKK3 levels for predicting sarcopenia in patients with COPD in the training set. (i) ROC curve analysis of plasma DKK3 levels for predicting sarcopenia in patients with COPD in the validated set. Data represent mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: For the DKK3 treatment, C2C12 myotubes that differentiated for four days were treated with recombinant human DKK3 (rhDKK3, 150 ng/mL) (HY–P7812, MCE, China) for 48 h after transiently transfected with siRNA.

Techniques: Clinical Proteomics

Journal: Redox Biology

Article Title: DKK3 as a diagnostic marker and potential therapeutic target for sarcopenia in chronic obstructive pulmonary disease

doi: 10.1016/j.redox.2024.103434

Figure Lengend Snippet: DKK3 over-expression leads to myotube atrophy and mitochondrial dysfunction in cultured myocytes (a) Representative immunofluorescence images of MyHC (green) and DAPI (blue) in C2C12 myotubes transfected with pcDNA or DKK3 plasmid. Scale bar: 50 μm. (b) Quantification of the average diameters of the control myotubes or DKK3 over-expressing myotubes. (c–g) Immunoblots and quantification of whole-cell protein lysates from fully differentiated myotubes showing decreased MyHC protein levels, increased expression of muscle atrophy markers Atrogin-1 and MuRF1, and increased DKK3 protein level in DKK3-overexpressed cells compared to controls. The data represent three independent experiments. (h) Representative immunofluorescence images of JC-1 staining in C2C12 cells transfected with control or DKK3 plasmids. Scale bar: 100 μm. (i) Quantitative results show the ratio of JC-1 aggregate (red) to monomeric (green) forms, indicating a significant decrease in mitochondrial membrane potential in DKK3-overexpressed myotubes. At least five fields were randomly selected for each of the four biological replicates in immunofluorescence analysis. (j) Cellular oxygen consumption rate (OCR) in C2C12 cells transfected with control or DKK3 plasmids. (k) Basal, ATP-linked, maximal and reserved OCR were quantified and analyzed. DKK3 over-expression significantly suppresses basal OCR and ATP-linked OCR, impaired maximum and reserved OCR. Data are presented as mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. Two-tailed unpaired Student's t -test. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the DKK3 treatment, C2C12 myotubes that differentiated for four days were treated with recombinant human DKK3 (rhDKK3, 150 ng/mL) (HY–P7812, MCE, China) for 48 h after transiently transfected with siRNA.

Techniques: Over Expression, Cell Culture, Immunofluorescence, Transfection, Plasmid Preparation, Control, Expressing, Western Blot, Staining, Membrane, Two Tailed Test

Journal: Redox Biology

Article Title: DKK3 as a diagnostic marker and potential therapeutic target for sarcopenia in chronic obstructive pulmonary disease

doi: 10.1016/j.redox.2024.103434

Figure Lengend Snippet: DKK3 knockdown rescues CSE-induced myotube atrophy and mitochondrial dysfunction (a) Representative immunofluorescence images of MyHC and DAPI in C2C12 myotubes stably transduced with shNC or shDKK3 lentivirus prior to CSE treatment. Scale bar: 50 μm. (b) Quantification of the average diameters of myotubes differentiated from shNC or shDKK3 C2C12 cells with or without CSE-treatment. At least three fields were randomly selected for each of the five biological replicates. (c–g) Immunoblots and quantification of relative MyHC protein levels, muscle atrophy markers Atrogin-1 and MuRF1 and DKK3 protein levels in whole-cell protein lysates from fully differentiated myotubes under different conditions. The data represent three independent experiments. (h) Representative immunofluorescence images of JC-1 staining in C2C12 cells transfected with shNC or shDKK3 prior to CSE treatment. DKK3 knockdown preserves mitochondrial membrane potential in CSE-treated cells. Scale bar: 100 μm. (i) Quantitative results showing the ratio of JC-1 aggregate (red) to monomeric (green) forms. At least five fields for each of the four biological replicates per group. (j) Cellular oxygen consumption rate (OCR) in C2C12 cells transfected with shNC or shDKK3 prior to CSE treatment. (k) Basal, ATP-linked, maximal and reserved OCR were quantified and analyzed. Data represent mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. One-way ANOVA with multiple comparisons. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the DKK3 treatment, C2C12 myotubes that differentiated for four days were treated with recombinant human DKK3 (rhDKK3, 150 ng/mL) (HY–P7812, MCE, China) for 48 h after transiently transfected with siRNA.

Techniques: Knockdown, Immunofluorescence, Stable Transfection, Transduction, Western Blot, Staining, Transfection, Membrane

Journal: Redox Biology

Article Title: DKK3 as a diagnostic marker and potential therapeutic target for sarcopenia in chronic obstructive pulmonary disease

doi: 10.1016/j.redox.2024.103434

Figure Lengend Snippet: Intramuscular DKK3 knockdown rescues CS-induced skeletal muscle atrophy in mice (a) Schematic representation of intramuscular injection of AAV-shDKK3 or AAV-shCtrl into TA muscles of CS and control mice. (b) Representative dissected skeletal muscles among the four groups: CS mice with AAV-shDKK3, CS mice with AAV-shCtrl, control mice with AAV-shDKK3, and control mice with AAV-shCtrl, including Quad (quadriceps), Gast (gastrocnemius), TA (tibialis anterior), EDL (extensor digitorum longus), and sol (soleus); (c) Representative immunofluorescence images of DKK3 (red) and DAPI (blue) in TA muscles, confirming efficient knockdown of DKK3 expression by AAV-shDKK3. Scale bar: 50 μm. (d) Quantification of grip strength in CS and control mice treated with AAV-shDKK3 or AAV-shCtrl (n = 5 per group, 5 measuremeants per mouse) (e) Comparison of the proportion of TA muscle weights among the four groups (n = 5 per group). (f) Representative H&E staining of TA muscle sections showing myofiber cross-sectional area (CSA) from the four groups. Scale bar: 100 μm. (g, h) The distribution and quantification of myofiber CSA in TA muscles from the different groups (n = 5 per group, at least 5 fields per mouse). (i–k) Immunoblots and quantification of relative Atrogin-1 and MuRF1 protein levels in TA muscle lysates from the four groups (n = 5 per group). Data represent mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. One-way ANOVA with multiple comparisons. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the DKK3 treatment, C2C12 myotubes that differentiated for four days were treated with recombinant human DKK3 (rhDKK3, 150 ng/mL) (HY–P7812, MCE, China) for 48 h after transiently transfected with siRNA.

Techniques: Knockdown, Injection, Muscles, Control, Immunofluorescence, Expressing, Comparison, Staining, Western Blot

Journal: Redox Biology

Article Title: DKK3 as a diagnostic marker and potential therapeutic target for sarcopenia in chronic obstructive pulmonary disease

doi: 10.1016/j.redox.2024.103434

Figure Lengend Snippet: DKK3-mediated paracrine effects contribute to myotube atrophy (a) Schematic outline of conditioned medium (CM) harvested from C2C12 myotubes transfected with pcDNA3.1 plasmids encoding DKK3 (OEDKK3) or empty pcDNA3.1 (control). The CM was used to treat myotubes derived from C2C12 cells. (b) Relative fold change of DKK3 expression level in the CM from control and OEDKK3 C2C12 myotubes. (c) Representative immunofluorescence images of MyHC (green) and DAPI (blue) in myotubes treated with CM harvested from OEDKK3 or control myotubes. Scale bar: 50 μm. (d) Quantification of the average diameters of myotubes of each group. (e–h) Immunoblots and quantification of relative MyHC and muscle atrophy markers protein levels in the total cell protein lysates from myotubes as described in b. (i) Representative immunofluorescence images of MyHC (green) and DAPI (blue) in myotubes subjected to CSE alone or in combination with monoclonal antibody against DKK3 (mAb-DKK3, 60 ng/mL), Scale bar: 50 μm. (j) Quantification of the average diameters of myotubes of each group. (k) Immunoblot analysis and quantification of MyHC in total cell protein lysates from myotubes treated with CSE alone or in combination with mAb-DKK3. Data represent mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. One-way ANOVA with multiple comparisons. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the DKK3 treatment, C2C12 myotubes that differentiated for four days were treated with recombinant human DKK3 (rhDKK3, 150 ng/mL) (HY–P7812, MCE, China) for 48 h after transiently transfected with siRNA.

Techniques: Transfection, Control, Derivative Assay, Expressing, Immunofluorescence, Western Blot

Journal: Redox Biology

Article Title: DKK3 as a diagnostic marker and potential therapeutic target for sarcopenia in chronic obstructive pulmonary disease

doi: 10.1016/j.redox.2024.103434

Figure Lengend Snippet: DKK3 physically interacts with CKAP4 to induce skeletal muscle atrophy (a) Schematic experimental procedure depicting the workflow for identifying DKK3-interacting proteins via immunoprecipitation and mass spectrometry in C2C12 cells. (b) Venn diagram analysis showing the overlap among DKK3-interacting proteins, plasma membrane proteins, and cell surface proteins. (c) Co-immunoprecipitation experiments confirmed the physical interaction between DKK3 and CKAP4. (d–h) Representative immunoblots and quantification showing MyHC protein levels, muscle atrophy markers Atrogin-1 and MuRF1 and CKAP4 protein in C2C12 myotubes treated with rhDKK3, with or without CKAP4 knockdown. (i) JC-1 staining demonstrating that CKAP4 knockdown mitigates the DKK3-induced reduction in mitochondrial membrane potential. Scale bar: 5 μm. (j) Quantitative results of JC-1 aggregate (red)/monomeric (green) forms are shown. At least five fields were randomly selected for each of the four biological replicates. Data represent mean ± SD, ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001. One-way ANOVA with multiple comparisons. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For the DKK3 treatment, C2C12 myotubes that differentiated for four days were treated with recombinant human DKK3 (rhDKK3, 150 ng/mL) (HY–P7812, MCE, China) for 48 h after transiently transfected with siRNA.

Techniques: Immunoprecipitation, Mass Spectrometry, Clinical Proteomics, Membrane, Western Blot, Knockdown, Staining