Proteomic Analysis of Myocardia Containing the Obscurin R4344Q Mutation Linked to Hypertrophic Cardiomyopathy.

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Recently, mutations in OBSCN were associated with the development of different forms of cardiomyopathies, including hypertrophic cardiomyopathy (HCM). We previously reported that homozygous mice carrying the HCM-linked R4344Q obscurin mutation develop arrhythmia by 1-year of age under sedentary conditions characterized by increased heart rate, frequent incidents of premature ventricular contractions, and episodes of spontaneous ventricular tachycardia. In an effort to delineate the molecular mechanisms that contribute to the observed arrhythmic phenotype, we subjected protein lysates prepared from left ventricles of 1-year old R4344Q and wild-type mice to comparative proteomics analysis using tandem mass spectrometry; raw data are available via ProteomeXchange with identifier PXD017314. We found that the expression levels of proteins involved in cardiac function and disease, cytoskeletal organization, electropotential regulation, molecular transport and metabolism were significantly altered. Moreover, phospho-proteomic evaluation revealed changes in the phosphorylation profile of Ca2+ cycling proteins, including sAnk1.5, a major binding partner of obscurin localized in the sarcoplasmic reticulum; notably, this is the first report indicating that sAnk1 undergoes phosphorylation. Taken together, our findings implicate obscurin in diverse cellular processes within the myocardium, which is consistent with its multiple binding partners, localization in different subcellular compartments, and disease association.", "claims": [{"id": "claim-method", "statement": "A two-tailed t-test using a one-way ANOVA model was applied to evaluate the adjusted modified peptides between wild-type and R4344Q LV samples using the Partek Genomics Suite 6.6 analytic platform (Partek Inc. St. Louis, MO, United States).", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:32528308", "locator": "sections[6] \"Data Analysis\" paragraph 2", "quote": "A two-tailed t-test using a one-way ANOVA model was applied to evaluate the adjusted modified peptides between wild-type and R4344Q LV samples using the Partek Genomics Suite 6.6 analytic platform (Partek Inc. St. Louis, MO, United States)."}], "state": "complete"}, {"id": "claim-data-access", "statement": "The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.", "kind": "methodological", "target": null, "evidence": [{"asset": "source:pmid:32528308", "locator": "sections[16] \"Data Availability Statement\" paragraph 2", "quote": "The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher."}], "state": "complete"}], "gaps": []}, "experiment": {"state": "complete", "objective": null, "steps": [{"id": "read_published_estimate", "order": 1, "name": "Read the published estimate from the source", "kind": "other", "state": "complete", "blocked_on": null, "description": "The claim, method and evidence span published with the source article.", "duration_hours": null, "parameters": {}, "inputs": ["source:pmid:32528308"], "outputs": ["the published estimate and its evidence"], "instruments": [], "reagents": []}, {"id": "obtain_source_data", "order": 2, "name": "Obtain the source data from the named repository", "kind": "data-preparation", "state": "complete", "blocked_on": null, "description": "The GEO series accession resolves and its sample accessions are listed. This lane did not download the dataset or read its licence.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:PXD017314"], "outputs": ["the source data with its repository provenance"], "instruments": [], "reagents": []}, {"id": "run_analysis", "order": 3, "name": "Run the published analysis", "kind": "analysis", "state": "complete", "blocked_on": null, "description": "Apply the paper's statistical method to the resolved series and compare the result with the published target.", "duration_hours": null, "parameters": {}, "inputs": ["data:geo:PXD017314"], "outputs": ["the replicated result and its comparison"], "instruments": [], "reagents": []}], "materials": [], "instruments": [], "reagents": [], "conditions": {}, "gaps": []}, "data": {"state": "partial", "assets": [{"id": "source:pmid:32528308", "role": "manuscript", "format": "xml", "media_type": "application/xml", "uri": "https://doi.org/10.3389/fphys.2020.00478", "sha256": 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"1", ""], ["ANK1", "Small Ankyrin 1.5", "44.0(2.0,103.3)", "0.0426", "1", "3", "Cardiac output"], ["DDAH1", "Dimethylarginine dimethylaminohydrolase 1", "45.5(24.1,70.6)", "0.0028", "2", "3", ""], ["NPPA", "Natriuretic polypeptide type A", "−37.4(−60.1,−1.8)", "0.0447", "3", "3", ""], ["PKG1", "Protein kinase, cGMP-dependent, type 1 (PKG1)", "43.6(1.5,103.1)", "0.0444", "1", "5", ""], ["KCNQ1", "Potassium channel, voltage-gated, subfamily Q, member 1", "−19.0(−31.6,−4.2)", "0.0253", "1", "1", "Familial atrial fibrillation"], ["NPPA", "Natriuretic polypeptide type A", "−37.4(−60.1,−1.8)", "0.0447", "3", "3", ""], ["FHL2", "Four and a half LIM domains 2", "−56.0(−79.7,−4.6)", "0.0422", "5", "5", "Relaxation of aortic ring tissue"], ["NPPA", "Natriuretic polypeptide type A", "−37.4(−60.1,−1.8)", "0.0447", "3", "3", ""], ["CACNA1C", "Calcium channel, voltage-dependent, L-type, alpha 1C subunit", "−16.4(−26.1,−5.6)", "0.0151", "9", "9", "Supraventricular tachycardia"], ["NPPA", "Natriuretic polypeptide type A", "−37.4(−60.1,−1.8)", "0.0447", "3", "3", ""], ["GUCY1A2", "Guanylate cyclase 1, alpha 2", "−16.2(−28.1,−2.5)", "0.0317", "1", "1", "Acute heart failure"], ["MME", "Membrane metallo endopeptidase", "17.3(1.1,36.0)", "0.0407", "2", "2", ""], ["DYSF", "Dysferlin", "−34.2(−49.5,−14.2)", "0.0118", "40", "42", "Early onset cardiomyopathy"], ["NPPA", "Natriuretic polypeptide type A", "−37.4(−60.1,−1.8)", "0.0447", "3", "3", "Mild congestive heart failure"]], "row_count": 17, "column_count": 7, "numeric_cells": 74, "header_rows": 1, "locator": "section[8]/table-wrap[1]#T1"}}, {"id": "asset:table:pmid:32528308:2", "role": "table", "format": "jats-table", "media_type": "application/vnd.jats.table+json", "uri": null, "sha256": "sha256:2e2ca0488c0a60c23c25055da2fa6b60886e5c1a4491b77bbc294756b3a7a34f", "bytes": 1083, "access": "open", "state": "verified", "reader": "read_table", "reader_status": "available", "identifier": "section[11]/table-wrap[1]#T2", "version": null, "note": 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O-fucosyltransferase 2", "21.9 (1.3, 46.7)", "0.0411", "3", "3", ""], ["PKG1", "Protein kinase, cGMP-dependent, type 1 (PKG1)", "43.6 (1.5, 103.1)", "0.0444", "1", "5", ""], ["PTPN11", "Protein tyrosine phosphatase, non-receptor type 11", "20.8 (9.1, 33.8)", "0.0067", "21", "21", ""], ["RAB3B", "Rab3b", "−30.3 (−46.0, −10.0)", "0.0172", "1", "2", ""], ["SIRPA", "Signal-regulatory protein alpha", "17.8 (5.7, 31.3)", "0.0138", "2", "2", ""], ["TRAF3", "TNF receptor-associated factor 3", "9.1 (0.3, 18.7)", "0.0451", "1", "1", ""], ["GOT2", "Glutamatic-oxaloacetic transaminase 2, mitochondrial", "−16.8 (−30.0, −1.2)", "0.0408", "29", "29", "Transport of long chain fatty acids"], ["PLIN2", "Perillipin2", "16.5 (2.7, 32.2)", "0.0280", "5", "5", ""], ["SLC27A4", "Solute carrier family 27", "19.4 (0.9, 41.2)", "0.0428", "3", "4", ""], ["NPPA", "Natriuretic peptide type A", "−37.4 (−60.1, −1.8)", "0.0447", "3", "3", "Flux of water"]], "row_count": 19, "column_count": 7, "numeric_cells": 85, 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"Catabolism of lipids"], ["ACOT7", "Acyl-CoA thioesterase 7", "22.0(1.2,47.2)", "0.0418", "5", "5", ""], ["DDHD2", "DDHD domain containing 2", "30.4(0.3,69.5)", "0.0486", "1", "1", ""], ["HEXB", "Hexosaminidase B", "11.5(3.9,19.7)", "0.0129", "3", "3", ""], ["MGLL", "Monoglyceride lipase", "−14.6(−27.0,−0.1)", "0.0488", "3", "3", ""], ["SLC27A4", "Solute carrier family 27", "19.4(0.9,41.2)", "0.0428", "3", "4", ""], ["VLDLR", "Very low density lipoprotein receptor", "7.6(1.2,14.3)", "0.0291", "8", "8", ""], ["ABAT", "4-aminobutyrate aminotransferase", "−29.9(−38.6,−19.9)", "0.0018", "8", "8", "Metabolism of amino acids"], ["BCKDHB", "Branched chain ketoacid dehydrogenase E1, beta polypeptide", "−18.6(−31.3,−3.5)", "0.0284", "5", "5", ""], ["DDAH1", "Dimethylarginine dimethylaminohydrolase 1", "45.5(24.1,70.6)", "0.0028", "2", "3", ""], ["DDO", "D-aspartate oxidase", "−55.4(−74.7,−21.4)", "0.0167", "2", "2", ""], ["GOT2", "Glutamatic-oxaloacetic transaminase 2, mitochondrial", "−16.8(−30.0,−1.2)", "0.0408", "29", "29", ""], ["IVD", "Isovaleryl coenzyme A dehydrogenase", "−12.5(−20.2,−4.1)", "0.0156", "19", "19", ""], ["MCCC2", "Methylcrotonoyl-coenzyme A carboxylase 2", "−12.2(−20.3,−3.4)", "0.0198", "22", "22", ""], ["NPPA", "Natriuretic polypeptide type A", "−37.4(−60.1,−1.8)", "0.0447", "3", "3", ""], ["PTS", "6-pyruvoyl-tetrahydropterin synthase", "−13.6(−24.3,−1.4)", "0.0375", "2", "2", ""], ["ACOT7", "Acyl-CoA thioesterase 7", "22.0(1.2,47.2)", "0.0418", "5", "5", "Metabolism of coenzyme A"], ["MCCC2", "Methylcrotonoyl-coenzyme A carboxylase 2", "−12.2(−20.3,−3.4)", "0.0198", "22", "22", ""], ["SLC27A4", "Solute carrier family 27", "19.4(0.9,41.2)", "0.0428", "3", "4", ""], ["HEXB", "Hexosaminidase B", "11.6(3.9,19.7)", "0.0129", "3", "3", "Metabolism of N-acetylglucosamine"], ["NAGK", "N-acetylglucosamine kinase", "−14.7(−21.0,−7.8)", "0.0047", "5", "5", ""], ["CST3", "Cystatin C", "−32.6(−50.2,−8.7)", "0.0225", "3", "3", "Hydrolysis of protein fragments"], ["CTSS", "Cathepsin S", "−16.6(−30.2,−0.4)", "0.0470", "1", "1", ""], ["CTSZ", "Cathepsin Z", "50.0(15.8,94.1)", "0.0121", "2", "2", ""], ["FBXO6", "F-box protein 6", "−18.8(−27.6,−8.8)", "0.0075", "2", "2", ""], ["HDAC6", "Histone deacetylase 6", "−14.0(−24.5,−2.0)", "0.0324", "5", "5", ""], ["IDE", "Insulin degrading enzyme", "6.4(1.7,11.4)", "0.0188", "8", "8", ""], ["MME", "Membrane metallo endopeptidase", "17.3(1.1.36.0)", "0.0407", "2", "2", ""], ["TRAF3", "TNF receptor-associated factor 3", "9.1(0.3,18.7)", "0.0451", "1", "1", ""], ["UBE2N", "Ubiquitin-conjugating enzyme E2N", "−11.8(−20.0,−2.7)", "0.0236", "7", "7", ""], ["SLC25A20", "Solute carrier family 25 (mitochondrial carnitine/acylcarnitine translocase), member 20", "16.7(9.2,24.6)", "0.0029", "14", "14", "Canitine acyl-carnitine translocase deficiency"], ["MGLL", "Monoglyceride lipase", "−14.6(−27.0,−0.1)", "0.0488", "3", "3", "Hydrolysis of glycerol"], ["OXCT1", "3-oxoacid CoA transferase 1", 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"row_count": 38, "column_count": 7}, "table": {"label": "TABLE 3", "caption": "Altered expression levels of proteins involved in cytoskeleton organization, cellular assembly and maintenance.", "rows": [["Gene", "Protein", "% Change (95% Confidence interval)", "P-value", "Unique peptides", "Total peptides", "Function"], ["CLIP1", "CAP-GLY domain containing linker protein 1", "−16.3 (−29.3, −0.8)", "0.0435", "1", "46", "Stabilization of filaments"], ["EML2", "Echinoderm microtubule associated protein like 2", "−28.9 (−40.7, −14.7)", "0.0065", "9", "9", ""], ["GALK2", "Galactokinase 2", "−6.8 (−11.1, −2.4)", "0.0138", "2", "2", ""], ["GAP43", "Growth associated protein 43", "−32.0 (−44.5, −16.6)", "0.0063", "4", "4", ""], ["KIFC3", "Kinesin family member C3", "15.8 (1.4, 32.2)", "0.0373", "1", "1", ""], ["MAP1B", "Microtubule-associated protein 1B", "−16.9 (−28.1, −3.9)", "0.0242", "9", "9", ""], ["ACTB", "Actin, beta", "−11.7 (−22.0, −0.1)", "0.0485", "8", "28", "Microtubule dynamics"], ["CCL21A", "Chemokine (C-C motif) ligand 21A", "−21.9 (−35.9, −4.7)", "0.0260", "1", "1", ""], ["CLIP1", "CAP-GLY domain containing linker protein 1", "−16.3 (−29.3, −0.8)", "0.0435", "1", "46", ""], ["CRK", "v-crk avian sarcoma CT10 oncogene homolog", "22.2 (4.4, 43.0)", "0.0242", "10", "10", ""], ["CTNND1", "Catenin, delta 1", "−10.0 (−18.4, −0.8)", "0.0397", "1", "13", ""], ["CTSS", "Cathepsin S", "−16.6 (−30.2, −0.4)", "0.0470", "1", "1", ""], ["DDAH1", "Dimethylarginine dimethylaminohydrolase 1", "45.5 (24.1, 70.6)", "0.0028", "2", "3", ""], ["EIF4G2", "Eukaryotic translation initiation factor 4, gamma 2", "24.2 (7.9, 43.1)", "0.0130", "14", "14", ""], ["EML2", "Echinoderm microtubule associated protein like 2", "−28.9 (−40.7, −14.7)", "0.0065", "9", "9", ""], ["EPHA4", "Eph receptor A4", "14.9 (2.1, 29.3)", "0.0312", "3", "3", ""], ["GALK2", "Galactokinase 2", "−6.8 (−11.1, −2.4)", "0.0138", "2", "2", ""], ["GAP43", "Growth associated protein 43", "−32.0 (−44.5, −16.6)", 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(−20.3, −3.4)", "0.0198", "22", "22", ""], ["HSD17B10", "Hydroxysteroid (17-beta) dehydrogenase 10", "−10.2 (−15.3, −4.8)", "0.0070", "14", "14", "Fatty Acid β-oxidation"], ["IVD", "Isovaleryl coenzyme A dehydrogenase", "−12.5 (−20.2, −4.1)", "0.0156", "19", "19", ""], ["SLC27A4", "Solute carrier family 27 (fatty acid transporter), member 4", "19.4 (0.9, 41.2)", "0.0428", "3", "4", ""], ["PTPN11", "Protein tyrosine phosphatase, non-receptor type 11", "20.8 (9.1, 33.8)", "0.0067", "21", "21", "Role of JAK2 in hormone-like cytokine signaling"], ["SIRPA", "Signal-regulatory protein alpha", "17.8 (5.7, 31.3)", "0.0138", "2", "2", ""], ["NR1D1", "Nuclear receptor subfamily 1, group D, member 1", "42.0 (2.2, 97.3)", "0.0414", "2", "2", "Circadian rhythm signaling"]], "row_count": 18, "column_count": 7, "numeric_cells": 88, "header_rows": 1, "locator": "section[14]/table-wrap[1]#T6"}}, {"id": "asset:table:pmid:32528308:7", "role": "table", "format": "jats-table", "media_type": 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