Role of oxidation of excitation-contraction coupling machinery in age-dependent loss of muscle function in Caenorhabditis elegans
- Department of Physiology and Cellular Biophysics, The Clyde and Helen Wu Center for Molecular Cardiology, United States
- PhyMedExp, Montpellier University, INSERM, CNRS, CHRU Montpellier, France
- Medical Intensive Care Unit, Montpellier University and Montpellier University Health Care Center, France
Figures
Figure 1
UNC-68 comprises a macromolecular complex comparable to its mammalian homolog ryanodine receptor (RyR); RyR (A) and FKBP (B) evolution among species was inferred by the maximum likelihood method based on the JTT matrix-based model.
(C) Homology comparison between UNC-68 and the two human RyR isoforms (RyR1 and RyR2). (D) Homology comparison between the different FKB isoforms (1–8) and the human FKBP isoforms (FKBP12 and …
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Figure 1—source data 1
Full incut gels of Figure 1.
- https://cdn.elifesciences.org/articles/75529/elife-75529-fig1-data1-v2.pdf
Figure 2
Remodeling of UNC-68 and age-dependent reduction in intracellular calcium (Ca2+) transients is accelerated in FKB-2 (ok3007) (A) Representative trace of Ca2+ transients from GCaMP2 wild type (WT) and FKB-2 KO (at day 7).
Green box denotes peak fluorescence from worm’s muscle during contraction. (B) Ca2+ transients in age-synchronized populations of WT and FKB-2 (ok3007) nematodes (at day 3 and 7); (C) UNC-68 was …
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Figure 2—source data 1
Full incut gels of Figure 2.
- https://cdn.elifesciences.org/articles/75529/elife-75529-fig2-data1-v2.pdf
Figure 3
Depleting FKB-2 from UNC-68 causes UNC-68 oxidation (A) Representative image of caffeine activated calcium transient in GCaMP2 wild type (WT) at day 5; arrow denotes peak fluorescence in body wall muscle.
(B) Intracellular calcium (Ca2+) transients in day 5 age-synchronized populations of WT and FKB-2 (ok3007) nematodes treated with 15 μM and 50 μM rapamycin and FK506, respectively (treatment was …
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Figure 3—source data 1
Full incut gels of Figure 3.
- https://cdn.elifesciences.org/articles/75529/elife-75529-fig3-data1-v2.pdf
Figure 4
UNC-68 oxidation causes defective intracellular calcium (Ca2+) handling; (A) UNC-68 was immunoprecipitated and immunoblotted using anti-ryanodine receptor (RyR), anti-calstabin, and dinitrophenyl (DNP; marker of oxidation) antibodies in nematodes acutely treated for 0, 15 min, 2 hr, or 4 hr with FK506 or paraquat (treatment was applied for 20 min) at increasing concentration (day 5).
(B–C) Quantification of the band intensity shown in (A): band intensity was defined as the ratio of either DNP (marker of UNC-68 oxidation) or FKB-2 binding over its corresponding /UNC-68’s …
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Figure 4—source data 1
Full incut gels of Figure 4.
- https://cdn.elifesciences.org/articles/75529/elife-75529-fig4-data1-v2.pdf
Figure 5
The ryanodine receptor (RyR)-stabilizing drug S107 increases body wall muscle calcium (Ca2+) transients in aged Caenorhabditis elegans; (A) UNC-68 was immunoprecipitated and immunoblotted with anti-RyR, anti-calstabin, and dinitrophenyl (DNP; marker of oxidation) in aged nematodes (Day L4, 5, and 15) with 10 µM of S107 (5 hr).
(B–C) Quantification of the band intensity shown in (A): band intensity was defined as the ratio of either DNP (marker of UNC-68 oxidation) or FKB-2 binding over its corresponding /UNC-68 …
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Figure 5—source data 1
Full incut gels of Figure 5.
- https://cdn.elifesciences.org/articles/75529/elife-75529-fig5-data1-v2.pdf
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (worms) | ok3007 | Caenorhabditis Genetics Center (University of Minnesota) | WormBase ID: WBVar00094093 | Genomic position: I: 2918075.12918967 |
| Strain, strain background (worms) | Pmyo-3:GCaMP2 worms | Kindly provided by Zhao-Wen Wang, University of Connecticut Health Center | ||
| Strain, strain background (worms) | mev-1 | Caenorhabditis Genetics Center (University of Minnesota) | WormBase ID: WBGene00003225 | Genomic position III: 10334277.10335168 |
| Strain, strain background (worms) | clk-1 | Caenorhabditis Genetics Center (University of Minnesota) | WormBase ID: WBGene00000536 | Genomic position III: 5277894.5279344 |
| Antibody | anti-RyR1 (Rabbit polyclonal) | Marks’ lab, Columbia University, NY, USA | Cat. #: 5,029 Aa 1327–1339 | WB (1:1000), (10 μl) |
| Antibody | anti-PDE4 (Rabbit monoclonal) | Kindly provided by Kenneth Miller, Oklahoma Medical Research Foundation, Oklahoma City, Oklahoma | WB (1:1000), (10 μl) | |
| Antibody | anti-PP1 (Rabbit polyclonal) | Santa Cruz | Cat. #: sc6104 | WB (1:1000), (10 μl) |
| Antibody | anti-FKBP12 (Mouse monoclonal) | Santa Cruz | Cat. #: sc6104 | WB (1:2500), (10 μl) |
| Antibody | anti-FKBP12 (Rabbit polyclonal) | Abcam | Cat. #: ab2918 | WB (1:2000), (10 μl) |
| Commercial assay or kit | Oxyblot protein oxydation detection kit | Millipore | Cat. #: S7150 | WB (1:1000), (10 μl) |
| Chemical compound, drug | Rapamacin | Sigma Aldrich | Cat. #: 37,094 | |
| Chemical compound, drug | FK506 | Sigma Aldrich | Cat. #: Y0001926 | |
| Chemical compound, drug | Paraquat | Sigma Aldrich | Cat. #: 36,541 | |
| Chemical compound, drug | S107rycal drug | Marks’ lab, Columbia University, NY, USA | ||
| Software, algorithm | GraphPad | GraphPad | V8.0 |
Additional files
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Supplementary file 1
Oxidative regulators in C. elegans vs mice vs humans.
Comparison of homologous oxidative and antioxidative genes between C. elegans, mouse and human. Criteria of comparison includes the function, the subcellular location, the enzymatic activity, mutation induces disrupted phenotype and percentage of homology.
- https://cdn.elifesciences.org/articles/75529/elife-75529-supp1-v2.docx
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Supplementary file 2
Amino acid composition of UNC-68 and Human RyR1.
Comparison of amino acid abundance in the C. elegans UNC-68 and the human RyR1 calcium channels. Number and percentage of Serines and methionines for each species are shown in red.
- https://cdn.elifesciences.org/articles/75529/elife-75529-supp2-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/75529/elife-75529-transrepform1-v2.pdf
