{"gene":"CKM","run_date":"2026-04-28T17:28:52","timeline":{"discoveries":[{"year":1991,"finding":"CK-M (CKM) mRNA levels in the diabetic rat heart are markedly reduced (to 46.5% of control) and are insulin-responsive: acute insulin injection produced a 1.6-fold increase in CK-M mRNA within 5 hours, with full restoration to normal within 12 hours, demonstrating direct transcriptional/post-transcriptional regulation of CKM by insulin signaling.","method":"Northern blot quantitation of CK-M mRNA in streptozotocin-diabetic rat hearts before and after insulin administration","journal":"The American journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 — clean in vivo model with defined molecular readout (mRNA quantitation), single lab, single method","pmids":["1887884"],"is_preprint":false},{"year":1987,"finding":"CK-MM accumulation is specifically and preferentially impaired in innervated, contracting cultured muscle fibers from Duchenne muscular dystrophy (DMD) patients compared to control fibers and to non-innervated DMD fibers, while other muscle-specific isozymes (glycogen phosphorylase, phosphoglycerate mutase, LDH) are unaffected, indicating a selective defect in CKM expression or stability linked to the dystrophic state and requiring innervation/contraction to manifest.","method":"Long-term innervated contracting cultured muscle fiber model with CK-MM isozyme activity assay","journal":"Life sciences","confidence":"Medium","confidence_rationale":"Tier 2 — defined cellular phenotype with isozyme specificity controls, single lab","pmids":["3613854"],"is_preprint":false},{"year":1989,"finding":"Following release from injured myocardium, the CK-MM3 tissue isoform is post-translationally converted in the circulation to MM2 and then MM1 isoforms by carboxypeptidase action, generating a time-dependent isoform profile (MM3/MM1 ratio) that peaks 2–6 hours after AMI onset, earlier than total CK or CK-MB.","method":"Anion-exchange liquid chromatography and high-voltage electrophoresis of serial blood samples from AMI patients; immunoinhibition with monoclonal antibody confirming tissue vs. serum isoforms","journal":"Clinics in laboratory medicine / Rinsho byori","confidence":"Medium","confidence_rationale":"Tier 2 — biochemical separation and immunoinhibition across multiple studies demonstrating the post-translational conversion mechanism","pmids":["2686906","1762189"],"is_preprint":false},{"year":1987,"finding":"CK-MM3 (tissue isoform) rises and peaks earlier than total CK or CK-MB after acute myocardial infarction, and during successful coronary reperfusion the MM3/MM1 ratio peaks even earlier with a more rapid rate of rise, establishing CK-MM isoform patterns as indicators of the time course of myocardial necrosis and reperfusion.","method":"Serial blood sampling with anion-exchange liquid chromatography and high-voltage electrophoresis of CK-MM sub-types in 35 AMI patients including thrombolysis-treated subgroups","journal":"Clinical chemistry","confidence":"Medium","confidence_rationale":"Tier 2 — prospective clinical study with orthogonal assay methods and controlled reperfusion subgroups","pmids":["3815799"],"is_preprint":false},{"year":1988,"finding":"Exercise-induced release of CK-MM from skeletal muscle is sex-linked in rats: male rats show a ~678% increase in plasma CK-MM after treadmill running versus ~114% in females, while CK-BB increases similarly in both sexes (~35–41%), demonstrating that sex-dependent differences in CK blood levels after exercise are attributable specifically to differential leakage of the CKM-encoded CK-MM isoform from skeletal muscle.","method":"CK isoenzyme profiling of plasma and muscle/liver homogenates before and after treadmill exercise in male and female rats","journal":"Pflugers Archiv : European journal of physiology","confidence":"Medium","confidence_rationale":"Tier 2 — isoenzyme-specific assay with tissue source identification and sex-comparison controls","pmids":["3174399"],"is_preprint":false},{"year":2006,"finding":"CK-MM autoantibodies form immune complexes with circulating CK-MM after muscle injury, and administration of CK-MM antibodies to mice reduces plasma CK activity by 11–32% following a bolus CK injection, establishing that CK-MM autoantibodies modulate the clearance rate of CKM protein from the circulation.","method":"ELISA for CK-MM autoantibodies; protein A-sepharose pulldown to quantify immune complexes; mouse in vivo antibody administration experiment","journal":"Muscle & nerve","confidence":"Medium","confidence_rationale":"Tier 2 — in vivo functional experiment with mechanistic follow-up (immune complex quantitation), single lab","pmids":["16810680"],"is_preprint":false},{"year":1995,"finding":"A point mutation at codon 54 of CKM (Exon 2, Asp→Gly) in a patient with acute myocardial infarction without CK elevation was associated with depressed CKM mRNA and absence of CK-MM protein in serum and myocardial tissue, indicating that coding-region mutations can abrogate CKM protein production.","method":"Genomic DNA sequencing and cDNA analysis of CKM from myocardial tissue; Northern blot for mRNA; protein measurement in serum and tissue","journal":"Rinsho byori. The Japanese journal of clinical pathology","confidence":"Low","confidence_rationale":"Tier 3 — single case report with sequencing and mRNA data, no functional reconstitution","pmids":["7884961"],"is_preprint":false},{"year":1991,"finding":"CK-MM isoform MM3 is completely inhibited by a monoclonal antibody against the tissue isoform, MM2 is ~57% inhibited, and MM1 (serum isoform) is not inhibited; EDTA inhibition and heat lability of MM3 indicate that CKM is a metal-dependent enzyme, and the tissue-to-serum post-translational conversion is confirmed as the basis of isoform patterns after AMI.","method":"Monoclonal antibody immunoinhibition assay; EDTA inhibition; heat lability testing of CK-MM isoforms","journal":"Rinsho byori. The Japanese journal of clinical pathology","confidence":"Medium","confidence_rationale":"Tier 2 — multiple biochemical methods (immunoinhibition, metal chelation, heat lability) characterizing enzymatic properties and isoform conversion","pmids":["1762189"],"is_preprint":false},{"year":1991,"finding":"The coding segment of the CKM gene is not the cause of myotonic dystrophy: sequencing of CKM cDNA from skeletal muscle of a DM patient revealed two novel polymorphisms but no translationally significant mutation, ruling out a CKM coding defect as the pathogenic mechanism in this family.","method":"CKM cDNA isolation and sequencing from DM patient skeletal muscle","journal":"Human genetics","confidence":"Medium","confidence_rationale":"Tier 2 — direct sequencing of disease-linked gene with clear mechanistic exclusion conclusion","pmids":["2016086"],"is_preprint":false},{"year":2023,"finding":"In chicken primary myoblasts, CKM overexpression inhibits proliferation, promotes apoptosis and differentiation, demonstrating a defined functional role for CKM in regulating myogenesis; transcriptome analysis after CKM knockdown identified differentially expressed genes linked to myogenesis pathways.","method":"Overexpression and RNA interference in chicken primary myoblasts; qPCR; RNA-seq transcriptome analysis; proliferation/apoptosis/differentiation assays","journal":"Animals : an open access journal from MDPI","confidence":"Medium","confidence_rationale":"Tier 2 — gain- and loss-of-function with multiple cellular phenotype readouts and transcriptomic follow-up, single lab","pmids":["37508090"],"is_preprint":false},{"year":2022,"finding":"The CKM gene promoter drives muscle-specific expression and can simultaneously initiate expression of CKM and an integrated exogenous gene in porcine satellite cells upon differentiation, establishing the CKM locus as a functional muscle-specific regulatory element usable as a safe harbor for gene integration.","method":"CRISPR/Cas9-mediated homologous directed repair knock-in of exogenous genes into the CKM locus in porcine satellite cells; differentiation induction; gene expression assays","journal":"Genes","confidence":"Low","confidence_rationale":"Tier 3 — demonstrates promoter activity and muscle-specificity but is primarily a gene engineering study, single lab","pmids":["35627307"],"is_preprint":false}],"current_model":"CKM encodes the muscle-specific MM isoform of creatine kinase; after release from injured muscle or myocardium, the CK-MM3 tissue isoform undergoes sequential post-translational carboxypeptidase-mediated conversion to MM2 and MM1 in the circulation, generating a diagnostic time-dependent isoform profile; CKM expression is positively regulated by insulin at the mRNA level, is selectively impaired in dystrophic muscle under innervated/contracting conditions, and functions in myoblast biology to inhibit proliferation while promoting differentiation, with circulating CK-MM levels further modulated by autoantibody-mediated immune complex formation that accelerates clearance."},"narrative":{"teleology":[{"year":1987,"claim":"Whether CKM expression was selectively affected in muscular dystrophy was unknown; innervated contracting DMD muscle fibers showed specific impairment of CK-MM accumulation while other muscle isozymes were unaffected, establishing that the dystrophic defect selectively targets CKM and requires innervation/contraction to manifest.","evidence":"Long-term innervated contracting cultured muscle fibers from DMD patients with CK-MM isozyme activity assay","pmids":["3613854"],"confidence":"Medium","gaps":["Mechanism by which dystrophin absence selectively impairs CKM expression or stability is unknown","Not tested in animal models of DMD","Whether impairment is transcriptional, translational, or due to protein instability was not resolved"]},{"year":1987,"claim":"The kinetics of CK-MM isoform conversion after myocardial infarction and reperfusion were undefined; serial sampling showed that the tissue isoform MM3 peaks earlier than total CK or CK-MB and that the MM3/MM1 ratio shifts even more rapidly during successful reperfusion, establishing isoform conversion kinetics as a mechanistic readout of myocardial necrosis timing.","evidence":"Serial blood sampling with anion-exchange chromatography and high-voltage electrophoresis in 35 AMI patients including thrombolysis subgroups","pmids":["3815799"],"confidence":"Medium","gaps":["Identity of the specific carboxypeptidase(s) responsible for in vivo conversion was not definitively established","Clearance kinetics of individual isoforms were not measured"]},{"year":1988,"claim":"Whether sex differences in post-exercise CK elevation were isoform-specific was unknown; isoenzyme profiling after treadmill exercise revealed that the sex-linked difference in plasma CK is attributable specifically to CK-MM (not CK-BB), with males showing ~6-fold greater CK-MM release than females.","evidence":"CK isoenzyme profiling of plasma and tissue homogenates before and after treadmill exercise in male and female rats","pmids":["3174399"],"confidence":"Medium","gaps":["Hormonal or membrane-integrity mechanism underlying sex-differential CK-MM leakage was not identified","Not confirmed in human subjects"]},{"year":1989,"claim":"The biochemical basis of CK-MM isoform heterogeneity was clarified: the tissue isoform MM3 is converted to MM2 and MM1 by carboxypeptidase action in the circulation, as demonstrated by differential monoclonal antibody inhibition, metal dependence (EDTA sensitivity), and heat lability of MM3.","evidence":"Monoclonal antibody immunoinhibition, EDTA chelation, and heat-lability assays of CK-MM isoforms; anion-exchange chromatography of serial AMI patient samples","pmids":["2686906","1762189"],"confidence":"Medium","gaps":["Specific carboxypeptidase isoform responsible was not identified","Structural basis of how C-terminal cleavage alters charge and activity was not resolved"]},{"year":1991,"claim":"Whether CKM expression is directly regulated by insulin was untested; insulin injection in diabetic rats restored CK-M mRNA from ~47% of control to normal levels within 12 hours, establishing CKM as an insulin-responsive gene in the heart.","evidence":"Northern blot quantitation of CK-M mRNA in streptozotocin-diabetic rat hearts after insulin administration","pmids":["1887884"],"confidence":"Medium","gaps":["Whether regulation is transcriptional or post-transcriptional was not distinguished","Signaling pathway (PI3K, MAPK, etc.) mediating insulin effect was not identified","Not confirmed in human cardiac tissue"]},{"year":1991,"claim":"CKM was investigated as a candidate gene for myotonic dystrophy due to its chromosomal proximity; sequencing of CKM cDNA from DM patient muscle excluded coding-region mutations as the cause, helping to resolve that CKM is not the DM disease gene.","evidence":"Full cDNA sequencing of CKM from DM patient skeletal muscle","pmids":["2016086"],"confidence":"Medium","gaps":["Regulatory region variants in CKM were not assessed","Whether CKM expression levels are altered in DM was not quantified"]},{"year":2006,"claim":"Whether circulating CK-MM is subject to immune regulation was unknown; CK-MM autoantibodies were shown to form immune complexes with CK-MM and to accelerate its clearance from the circulation by 11–32% in vivo, establishing an immunological mechanism that modulates serum CK-MM levels.","evidence":"ELISA for CK-MM autoantibodies; protein A-sepharose pulldown for immune complexes; in vivo mouse antibody administration experiment","pmids":["16810680"],"confidence":"Medium","gaps":["Fc receptor or reticuloendothelial mechanism of immune complex clearance was not defined","Clinical significance for diagnostic CK interpretation in autoimmune patients not established"]},{"year":2023,"claim":"Beyond its enzymatic role, CKM's function in myoblast fate decisions was uncharacterized; overexpression in primary myoblasts inhibited proliferation and promoted differentiation while knockdown altered myogenesis-associated gene networks, establishing CKM as a functional regulator of myogenesis.","evidence":"Overexpression and RNAi in chicken primary myoblasts with proliferation, apoptosis, differentiation assays and RNA-seq transcriptomics","pmids":["37508090"],"confidence":"Medium","gaps":["Mechanism by which CKM enzymatic activity (or a non-catalytic function) regulates proliferation/differentiation is unknown","Not validated in mammalian myoblasts","Downstream signaling pathways were identified transcriptomically but not functionally validated"]},{"year":null,"claim":"Key unresolved questions include the identity of the specific carboxypeptidase(s) that convert CK-MM3 to MM1 in vivo, the signaling pathway through which insulin regulates CKM transcription, and whether CKM's role in myoblast differentiation is catalytic or reflects a moonlighting function.","evidence":"","pmids":[],"confidence":"Low","gaps":["No structural model of the CK-MM isoform conversion site","Non-enzymatic functions of CKM in differentiation have not been mechanistically dissected","In vivo genetic loss-of-function models (knockout) in mammals are poorly characterized in the literature"]}],"mechanism_profile":{"molecular_activity":[{"term_id":"GO:0016740","term_label":"transferase activity","supporting_discovery_ids":[2,3,7]}],"localization":[{"term_id":"GO:0005829","term_label":"cytosol","supporting_discovery_ids":[2,4]},{"term_id":"GO:0005576","term_label":"extracellular region","supporting_discovery_ids":[2,3,5]}],"pathway":[{"term_id":"R-HSA-1430728","term_label":"Metabolism","supporting_discovery_ids":[0,2,7]},{"term_id":"R-HSA-1266738","term_label":"Developmental Biology","supporting_discovery_ids":[9]}],"complexes":[],"partners":[],"other_free_text":[]},"mechanistic_narrative":"CKM encodes the muscle-type (M) subunit of creatine kinase, a central enzyme in cellular energy homeostasis in skeletal and cardiac muscle that catalyzes the reversible transfer of phosphate between ATP and creatine. The tissue-form homodimer CK-MM3, upon release from injured myocardium or exercised skeletal muscle, undergoes sequential carboxypeptidase-mediated post-translational conversion in the circulation to MM2 and then MM1, generating a time-dependent isoform ratio that reflects the kinetics of muscle damage and reperfusion [PMID:2686906, PMID:3815799]. CKM expression is transcriptionally regulated in a muscle-specific manner, is positively controlled by insulin signaling in the heart, and is selectively impaired in dystrophic muscle under innervated/contracting conditions [PMID:1887884, PMID:3613854]. Beyond its enzymatic role, CKM functions in myoblast biology by inhibiting proliferation and promoting differentiation, and circulating CK-MM levels are further modulated by autoantibody-mediated immune complex formation that accelerates its clearance [PMID:37508090, PMID:16810680]."},"prefetch_data":{"uniprot":{"accession":"P06732","full_name":"Creatine kinase M-type","aliases":["Creatine kinase M chain","Creatine phosphokinase M-type","CPK-M","M-CK"],"length_aa":381,"mass_kda":43.1,"function":"Reversibly catalyzes the transfer of phosphate between ATP and various phosphogens (e.g. creatine phosphate). Creatine kinase isoenzymes play a central role in energy transduction in tissues with large, fluctuating energy demands, such as skeletal muscle, heart, brain and spermatozoa","subcellular_location":"Cytoplasm","url":"https://www.uniprot.org/uniprotkb/P06732/entry"},"depmap":{"release":"DepMap","has_data":true,"is_common_essential":false,"resolved_as":"","url":"https://depmap.org/portal/gene/CKM","classification":"Not Classified","n_dependent_lines":1,"n_total_lines":1208,"dependency_fraction":0.0008278145695364238},"opencell":{"profiled":false,"resolved_as":"","ensg_id":"","cell_line_id":"","localizations":[],"interactors":[{"gene":"CKB","stoichiometry":0.2}],"url":"https://opencell.sf.czbiohub.org/search/CKM","total_profiled":1310},"omim":[{"mim_id":"615671","title":"SET DOMAIN-CONTAINING PROTEIN 3; SETD3","url":"https://www.omim.org/entry/615671"},{"mim_id":"608099","title":"MUSCULAR DYSTROPHY, LIMB-GIRDLE, AUTOSOMAL RECESSIVE 3; LGMDR3","url":"https://www.omim.org/entry/608099"},{"mim_id":"606009","title":"DOUBLE HOMEOBOX PROTEIN 4; DUX4","url":"https://www.omim.org/entry/606009"},{"mim_id":"605921","title":"STROMAL INTERACTION MOLECULE 1; STIM1","url":"https://www.omim.org/entry/605921"},{"mim_id":"604559","title":"PROGRESSIVE FAMILIAL HEART BLOCK, TYPE IB; PFHB1B","url":"https://www.omim.org/entry/604559"}],"hpa":{"profiled":true,"resolved_as":"","reliability":"","locations":[],"tissue_specificity":"Group enriched","tissue_distribution":"Detected in many","driving_tissues":[{"tissue":"skeletal muscle","ntpm":68908.9},{"tissue":"tongue","ntpm":24326.4}],"url":"https://www.proteinatlas.org/search/CKM"},"hgnc":{"alias_symbol":[],"prev_symbol":["CKMM"]},"alphafold":{"accession":"P06732","domains":[{"cath_id":"1.10.135.10","chopping":"3-96","consensus_level":"high","plddt":95.9328,"start":3,"end":96},{"cath_id":"3.30.590.10","chopping":"111-368","consensus_level":"medium","plddt":94.9278,"start":111,"end":368}],"viewer_url":"https://alphafold.ebi.ac.uk/entry/P06732","model_url":"https://alphafold.ebi.ac.uk/files/AF-P06732-F1-model_v6.cif","pae_url":"https://alphafold.ebi.ac.uk/files/AF-P06732-F1-predicted_aligned_error_v6.png","plddt_mean":94.81},"mouse_models":{"mgi_url":"https://www.informatics.jax.org/marker/summary?nomen=CKM","jax_strain_url":"https://www.jax.org/strain/search?query=CKM"},"sequence":{"accession":"P06732","fasta_url":"https://rest.uniprot.org/uniprotkb/P06732.fasta","uniprot_url":"https://www.uniprot.org/uniprotkb/P06732/entry","alphafold_viewer_url":"https://alphafold.ebi.ac.uk/entry/P06732"}},"corpus_meta":[{"pmid":"17478608","id":"PMC_17478608","title":"CK-MM and ACE genotypes and physiological prediction of the creatine kinase response to exercise.","date":"2007","source":"Journal of applied physiology (Bethesda, Md. : 1985)","url":"https://pubmed.ncbi.nlm.nih.gov/17478608","citation_count":82,"is_preprint":false},{"pmid":"2309701","id":"PMC_2309701","title":"A long-range restriction map of the human chromosome 19q13 region: close physical linkage between CKMM and the ERCC1 and ERCC2 genes.","date":"1990","source":"American journal of human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/2309701","citation_count":52,"is_preprint":false},{"pmid":"3174399","id":"PMC_3174399","title":"Creatine kinase isoenzyme profiles after exercise in the rat: sex-linked differences in leakage of CK-MM.","date":"1988","source":"Pflugers Archiv : European journal of physiology","url":"https://pubmed.ncbi.nlm.nih.gov/3174399","citation_count":51,"is_preprint":false},{"pmid":"3815799","id":"PMC_3815799","title":"Early diagnosis of acute myocardial infarction by rapid analysis of creatine kinase isoenzyme-3 (CK-MM) sub-types.","date":"1987","source":"Clinical chemistry","url":"https://pubmed.ncbi.nlm.nih.gov/3815799","citation_count":51,"is_preprint":false},{"pmid":"16037885","id":"PMC_16037885","title":"Is there an association between ACE and CKMM polymorphisms and cycling performance status during 3-week races?","date":"2005","source":"International journal of sports medicine","url":"https://pubmed.ncbi.nlm.nih.gov/16037885","citation_count":50,"is_preprint":false},{"pmid":"2703233","id":"PMC_2703233","title":"Myotonic dystrophy is closely linked to the gene for muscle-type creatine kinase (CKMM).","date":"1989","source":"Human genetics","url":"https://pubmed.ncbi.nlm.nih.gov/2703233","citation_count":49,"is_preprint":false},{"pmid":"21059062","id":"PMC_21059062","title":"Association of sequence variants in CKM (creatine kinase, muscle) and COX4I2 (cytochrome c oxidase, subunit 4, isoform 2) genes with racing performance in Thoroughbred horses.","date":"2010","source":"Equine veterinary journal. Supplement","url":"https://pubmed.ncbi.nlm.nih.gov/21059062","citation_count":41,"is_preprint":false},{"pmid":"25214527","id":"PMC_25214527","title":"CKM and LILRB5 are associated with serum levels of creatine kinase.","date":"2014","source":"Circulation. 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ATP as an analytical tool for rapid detection of creatine kinase (CK-MM).","date":"2018","source":"Analytica chimica acta","url":"https://pubmed.ncbi.nlm.nih.gov/29776542","citation_count":17,"is_preprint":false},{"pmid":"22567844","id":"PMC_22567844","title":"[Association of the muscle-specific creatine kinase (CKMM) gene polymorphism with physical performance of athletes].","date":"2012","source":"Fiziologiia cheloveka","url":"https://pubmed.ncbi.nlm.nih.gov/22567844","citation_count":16,"is_preprint":false},{"pmid":"39580049","id":"PMC_39580049","title":"Bilirubin bioconversion to urobilin in the gut-liver-kidney axis: A biomarker for insulin resistance in the Cardiovascular-Kidney-Metabolic (CKM) Syndrome.","date":"2024","source":"Metabolism: clinical and experimental","url":"https://pubmed.ncbi.nlm.nih.gov/39580049","citation_count":15,"is_preprint":false},{"pmid":"2016086","id":"PMC_2016086","title":"Assessment of a creatine kinase isoform M defect as a cause of myotonic 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a Risk Factor Across Cardiovascular-Kidney-Metabolic (CKM) Syndrome: Unpacking the Burden in People with Type 2 Diabetes.","date":"2025","source":"Diabetes therapy : research, treatment and education of diabetes and related disorders","url":"https://pubmed.ncbi.nlm.nih.gov/40343683","citation_count":10,"is_preprint":false},{"pmid":"35225934","id":"PMC_35225934","title":"Evaluation of the GSP Creatine Kinase-MM Assay and Assessment of CK-MM Stability in Newborn, Patient, and Contrived Dried Blood Spots for Newborn Screening for Duchenne Muscular Dystrophy.","date":"2022","source":"International journal of neonatal screening","url":"https://pubmed.ncbi.nlm.nih.gov/35225934","citation_count":10,"is_preprint":false},{"pmid":"37415116","id":"PMC_37415116","title":"CKM and TERT dual promoters drive CRISPR-dCas9 to specifically inhibit the malignant behavior of osteosarcoma cells.","date":"2023","source":"Cellular & molecular biology 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Comparison of five fixation methods and three immunohistochemical techniques.","date":"1985","source":"The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society","url":"https://pubmed.ncbi.nlm.nih.gov/3902962","citation_count":7,"is_preprint":false},{"pmid":"20157874","id":"PMC_20157874","title":"CK-MM gene polymorphism does not influence the blood CK activity levels after exhaustive eccentric exercise.","date":"2010","source":"International journal of sports medicine","url":"https://pubmed.ncbi.nlm.nih.gov/20157874","citation_count":7,"is_preprint":false},{"pmid":"28790154","id":"PMC_28790154","title":"CKM Glu83Gly Is Associated With Blunted Creatine Kinase Variation, but Not With Myalgia.","date":"2017","source":"Circulation. 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quantitation of CK-M mRNA in streptozotocin-diabetic rat hearts before and after insulin administration\",\n      \"journal\": \"The American journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — clean in vivo model with defined molecular readout (mRNA quantitation), single lab, single method\",\n      \"pmids\": [\"1887884\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"CK-MM accumulation is specifically and preferentially impaired in innervated, contracting cultured muscle fibers from Duchenne muscular dystrophy (DMD) patients compared to control fibers and to non-innervated DMD fibers, while other muscle-specific isozymes (glycogen phosphorylase, phosphoglycerate mutase, LDH) are unaffected, indicating a selective defect in CKM expression or stability linked to the dystrophic state and requiring innervation/contraction to manifest.\",\n      \"method\": \"Long-term innervated contracting cultured muscle fiber model with CK-MM isozyme activity assay\",\n      \"journal\": \"Life sciences\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — defined cellular phenotype with isozyme specificity controls, single lab\",\n      \"pmids\": [\"3613854\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1989,\n      \"finding\": \"Following release from injured myocardium, the CK-MM3 tissue isoform is post-translationally converted in the circulation to MM2 and then MM1 isoforms by carboxypeptidase action, generating a time-dependent isoform profile (MM3/MM1 ratio) that peaks 2–6 hours after AMI onset, earlier than total CK or CK-MB.\",\n      \"method\": \"Anion-exchange liquid chromatography and high-voltage electrophoresis of serial blood samples from AMI patients; immunoinhibition with monoclonal antibody confirming tissue vs. serum isoforms\",\n      \"journal\": \"Clinics in laboratory medicine / Rinsho byori\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — biochemical separation and immunoinhibition across multiple studies demonstrating the post-translational conversion mechanism\",\n      \"pmids\": [\"2686906\", \"1762189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1987,\n      \"finding\": \"CK-MM3 (tissue isoform) rises and peaks earlier than total CK or CK-MB after acute myocardial infarction, and during successful coronary reperfusion the MM3/MM1 ratio peaks even earlier with a more rapid rate of rise, establishing CK-MM isoform patterns as indicators of the time course of myocardial necrosis and reperfusion.\",\n      \"method\": \"Serial blood sampling with anion-exchange liquid chromatography and high-voltage electrophoresis of CK-MM sub-types in 35 AMI patients including thrombolysis-treated subgroups\",\n      \"journal\": \"Clinical chemistry\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — prospective clinical study with orthogonal assay methods and controlled reperfusion subgroups\",\n      \"pmids\": [\"3815799\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1988,\n      \"finding\": \"Exercise-induced release of CK-MM from skeletal muscle is sex-linked in rats: male rats show a ~678% increase in plasma CK-MM after treadmill running versus ~114% in females, while CK-BB increases similarly in both sexes (~35–41%), demonstrating that sex-dependent differences in CK blood levels after exercise are attributable specifically to differential leakage of the CKM-encoded CK-MM isoform from skeletal muscle.\",\n      \"method\": \"CK isoenzyme profiling of plasma and muscle/liver homogenates before and after treadmill exercise in male and female rats\",\n      \"journal\": \"Pflugers Archiv : European journal of physiology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — isoenzyme-specific assay with tissue source identification and sex-comparison controls\",\n      \"pmids\": [\"3174399\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2006,\n      \"finding\": \"CK-MM autoantibodies form immune complexes with circulating CK-MM after muscle injury, and administration of CK-MM antibodies to mice reduces plasma CK activity by 11–32% following a bolus CK injection, establishing that CK-MM autoantibodies modulate the clearance rate of CKM protein from the circulation.\",\n      \"method\": \"ELISA for CK-MM autoantibodies; protein A-sepharose pulldown to quantify immune complexes; mouse in vivo antibody administration experiment\",\n      \"journal\": \"Muscle & nerve\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — in vivo functional experiment with mechanistic follow-up (immune complex quantitation), single lab\",\n      \"pmids\": [\"16810680\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1995,\n      \"finding\": \"A point mutation at codon 54 of CKM (Exon 2, Asp→Gly) in a patient with acute myocardial infarction without CK elevation was associated with depressed CKM mRNA and absence of CK-MM protein in serum and myocardial tissue, indicating that coding-region mutations can abrogate CKM protein production.\",\n      \"method\": \"Genomic DNA sequencing and cDNA analysis of CKM from myocardial tissue; Northern blot for mRNA; protein measurement in serum and tissue\",\n      \"journal\": \"Rinsho byori. The Japanese journal of clinical pathology\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — single case report with sequencing and mRNA data, no functional reconstitution\",\n      \"pmids\": [\"7884961\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"CK-MM isoform MM3 is completely inhibited by a monoclonal antibody against the tissue isoform, MM2 is ~57% inhibited, and MM1 (serum isoform) is not inhibited; EDTA inhibition and heat lability of MM3 indicate that CKM is a metal-dependent enzyme, and the tissue-to-serum post-translational conversion is confirmed as the basis of isoform patterns after AMI.\",\n      \"method\": \"Monoclonal antibody immunoinhibition assay; EDTA inhibition; heat lability testing of CK-MM isoforms\",\n      \"journal\": \"Rinsho byori. The Japanese journal of clinical pathology\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — multiple biochemical methods (immunoinhibition, metal chelation, heat lability) characterizing enzymatic properties and isoform conversion\",\n      \"pmids\": [\"1762189\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 1991,\n      \"finding\": \"The coding segment of the CKM gene is not the cause of myotonic dystrophy: sequencing of CKM cDNA from skeletal muscle of a DM patient revealed two novel polymorphisms but no translationally significant mutation, ruling out a CKM coding defect as the pathogenic mechanism in this family.\",\n      \"method\": \"CKM cDNA isolation and sequencing from DM patient skeletal muscle\",\n      \"journal\": \"Human genetics\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — direct sequencing of disease-linked gene with clear mechanistic exclusion conclusion\",\n      \"pmids\": [\"2016086\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2023,\n      \"finding\": \"In chicken primary myoblasts, CKM overexpression inhibits proliferation, promotes apoptosis and differentiation, demonstrating a defined functional role for CKM in regulating myogenesis; transcriptome analysis after CKM knockdown identified differentially expressed genes linked to myogenesis pathways.\",\n      \"method\": \"Overexpression and RNA interference in chicken primary myoblasts; qPCR; RNA-seq transcriptome analysis; proliferation/apoptosis/differentiation assays\",\n      \"journal\": \"Animals : an open access journal from MDPI\",\n      \"confidence\": \"Medium\",\n      \"confidence_rationale\": \"Tier 2 — gain- and loss-of-function with multiple cellular phenotype readouts and transcriptomic follow-up, single lab\",\n      \"pmids\": [\"37508090\"],\n      \"is_preprint\": false\n    },\n    {\n      \"year\": 2022,\n      \"finding\": \"The CKM gene promoter drives muscle-specific expression and can simultaneously initiate expression of CKM and an integrated exogenous gene in porcine satellite cells upon differentiation, establishing the CKM locus as a functional muscle-specific regulatory element usable as a safe harbor for gene integration.\",\n      \"method\": \"CRISPR/Cas9-mediated homologous directed repair knock-in of exogenous genes into the CKM locus in porcine satellite cells; differentiation induction; gene expression assays\",\n      \"journal\": \"Genes\",\n      \"confidence\": \"Low\",\n      \"confidence_rationale\": \"Tier 3 — demonstrates promoter activity and muscle-specificity but is primarily a gene engineering study, single lab\",\n      \"pmids\": [\"35627307\"],\n      \"is_preprint\": false\n    }\n  ],\n  \"current_model\": \"CKM encodes the muscle-specific MM isoform of creatine kinase; after release from injured muscle or myocardium, the CK-MM3 tissue isoform undergoes sequential post-translational carboxypeptidase-mediated conversion to MM2 and MM1 in the circulation, generating a diagnostic time-dependent isoform profile; CKM expression is positively regulated by insulin at the mRNA level, is selectively impaired in dystrophic muscle under innervated/contracting conditions, and functions in myoblast biology to inhibit proliferation while promoting differentiation, with circulating CK-MM levels further modulated by autoantibody-mediated immune complex formation that accelerates clearance.\"\n}\n```","stage2_raw":"```json\n{\n  \"mechanistic_narrative\": \"CKM encodes the muscle-type (M) subunit of creatine kinase, a central enzyme in cellular energy homeostasis in skeletal and cardiac muscle that catalyzes the reversible transfer of phosphate between ATP and creatine. The tissue-form homodimer CK-MM3, upon release from injured myocardium or exercised skeletal muscle, undergoes sequential carboxypeptidase-mediated post-translational conversion in the circulation to MM2 and then MM1, generating a time-dependent isoform ratio that reflects the kinetics of muscle damage and reperfusion [PMID:2686906, PMID:3815799]. CKM expression is transcriptionally regulated in a muscle-specific manner, is positively controlled by insulin signaling in the heart, and is selectively impaired in dystrophic muscle under innervated/contracting conditions [PMID:1887884, PMID:3613854]. Beyond its enzymatic role, CKM functions in myoblast biology by inhibiting proliferation and promoting differentiation, and circulating CK-MM levels are further modulated by autoantibody-mediated immune complex formation that accelerates its clearance [PMID:37508090, PMID:16810680].\",\n  \"teleology\": [\n    {\n      \"year\": 1987,\n      \"claim\": \"Whether CKM expression was selectively affected in muscular dystrophy was unknown; innervated contracting DMD muscle fibers showed specific impairment of CK-MM accumulation while other muscle isozymes were unaffected, establishing that the dystrophic defect selectively targets CKM and requires innervation/contraction to manifest.\",\n      \"evidence\": \"Long-term innervated contracting cultured muscle fibers from DMD patients with CK-MM isozyme activity assay\",\n      \"pmids\": [\"3613854\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which dystrophin absence selectively impairs CKM expression or stability is unknown\", \"Not tested in animal models of DMD\", \"Whether impairment is transcriptional, translational, or due to protein instability was not resolved\"]\n    },\n    {\n      \"year\": 1987,\n      \"claim\": \"The kinetics of CK-MM isoform conversion after myocardial infarction and reperfusion were undefined; serial sampling showed that the tissue isoform MM3 peaks earlier than total CK or CK-MB and that the MM3/MM1 ratio shifts even more rapidly during successful reperfusion, establishing isoform conversion kinetics as a mechanistic readout of myocardial necrosis timing.\",\n      \"evidence\": \"Serial blood sampling with anion-exchange chromatography and high-voltage electrophoresis in 35 AMI patients including thrombolysis subgroups\",\n      \"pmids\": [\"3815799\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Identity of the specific carboxypeptidase(s) responsible for in vivo conversion was not definitively established\", \"Clearance kinetics of individual isoforms were not measured\"]\n    },\n    {\n      \"year\": 1988,\n      \"claim\": \"Whether sex differences in post-exercise CK elevation were isoform-specific was unknown; isoenzyme profiling after treadmill exercise revealed that the sex-linked difference in plasma CK is attributable specifically to CK-MM (not CK-BB), with males showing ~6-fold greater CK-MM release than females.\",\n      \"evidence\": \"CK isoenzyme profiling of plasma and tissue homogenates before and after treadmill exercise in male and female rats\",\n      \"pmids\": [\"3174399\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Hormonal or membrane-integrity mechanism underlying sex-differential CK-MM leakage was not identified\", \"Not confirmed in human subjects\"]\n    },\n    {\n      \"year\": 1989,\n      \"claim\": \"The biochemical basis of CK-MM isoform heterogeneity was clarified: the tissue isoform MM3 is converted to MM2 and MM1 by carboxypeptidase action in the circulation, as demonstrated by differential monoclonal antibody inhibition, metal dependence (EDTA sensitivity), and heat lability of MM3.\",\n      \"evidence\": \"Monoclonal antibody immunoinhibition, EDTA chelation, and heat-lability assays of CK-MM isoforms; anion-exchange chromatography of serial AMI patient samples\",\n      \"pmids\": [\"2686906\", \"1762189\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Specific carboxypeptidase isoform responsible was not identified\", \"Structural basis of how C-terminal cleavage alters charge and activity was not resolved\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"Whether CKM expression is directly regulated by insulin was untested; insulin injection in diabetic rats restored CK-M mRNA from ~47% of control to normal levels within 12 hours, establishing CKM as an insulin-responsive gene in the heart.\",\n      \"evidence\": \"Northern blot quantitation of CK-M mRNA in streptozotocin-diabetic rat hearts after insulin administration\",\n      \"pmids\": [\"1887884\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Whether regulation is transcriptional or post-transcriptional was not distinguished\", \"Signaling pathway (PI3K, MAPK, etc.) mediating insulin effect was not identified\", \"Not confirmed in human cardiac tissue\"]\n    },\n    {\n      \"year\": 1991,\n      \"claim\": \"CKM was investigated as a candidate gene for myotonic dystrophy due to its chromosomal proximity; sequencing of CKM cDNA from DM patient muscle excluded coding-region mutations as the cause, helping to resolve that CKM is not the DM disease gene.\",\n      \"evidence\": \"Full cDNA sequencing of CKM from DM patient skeletal muscle\",\n      \"pmids\": [\"2016086\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Regulatory region variants in CKM were not assessed\", \"Whether CKM expression levels are altered in DM was not quantified\"]\n    },\n    {\n      \"year\": 2006,\n      \"claim\": \"Whether circulating CK-MM is subject to immune regulation was unknown; CK-MM autoantibodies were shown to form immune complexes with CK-MM and to accelerate its clearance from the circulation by 11–32% in vivo, establishing an immunological mechanism that modulates serum CK-MM levels.\",\n      \"evidence\": \"ELISA for CK-MM autoantibodies; protein A-sepharose pulldown for immune complexes; in vivo mouse antibody administration experiment\",\n      \"pmids\": [\"16810680\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Fc receptor or reticuloendothelial mechanism of immune complex clearance was not defined\", \"Clinical significance for diagnostic CK interpretation in autoimmune patients not established\"]\n    },\n    {\n      \"year\": 2023,\n      \"claim\": \"Beyond its enzymatic role, CKM's function in myoblast fate decisions was uncharacterized; overexpression in primary myoblasts inhibited proliferation and promoted differentiation while knockdown altered myogenesis-associated gene networks, establishing CKM as a functional regulator of myogenesis.\",\n      \"evidence\": \"Overexpression and RNAi in chicken primary myoblasts with proliferation, apoptosis, differentiation assays and RNA-seq transcriptomics\",\n      \"pmids\": [\"37508090\"],\n      \"confidence\": \"Medium\",\n      \"gaps\": [\"Mechanism by which CKM enzymatic activity (or a non-catalytic function) regulates proliferation/differentiation is unknown\", \"Not validated in mammalian myoblasts\", \"Downstream signaling pathways were identified transcriptomically but not functionally validated\"]\n    },\n    {\n      \"year\": null,\n      \"claim\": \"Key unresolved questions include the identity of the specific carboxypeptidase(s) that convert CK-MM3 to MM1 in vivo, the signaling pathway through which insulin regulates CKM transcription, and whether CKM's role in myoblast differentiation is catalytic or reflects a moonlighting function.\",\n      \"evidence\": \"\",\n      \"pmids\": [],\n      \"confidence\": \"Low\",\n      \"gaps\": [\"No structural model of the CK-MM isoform conversion site\", \"Non-enzymatic functions of CKM in differentiation have not been mechanistically dissected\", \"In vivo genetic loss-of-function models (knockout) in mammals are poorly characterized in the literature\"]\n    }\n  ],\n  \"mechanism_profile\": {\n    \"molecular_activity\": [\n      {\"term_id\": \"GO:0016740\", \"supporting_discovery_ids\": [2, 3, 7]}\n    ],\n    \"localization\": [\n      {\"term_id\": \"GO:0005829\", \"supporting_discovery_ids\": [2, 4]},\n      {\"term_id\": \"GO:0005576\", \"supporting_discovery_ids\": [2, 3, 5]}\n    ],\n    \"pathway\": [\n      {\"term_id\": \"R-HSA-1430728\", \"supporting_discovery_ids\": [0, 2, 7]},\n      {\"term_id\": \"R-HSA-1266738\", \"supporting_discovery_ids\": [9]}\n    ],\n    \"complexes\": [],\n    \"partners\": [],\n    \"other_free_text\": []\n  }\n}\n```"}