{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,14]],"date-time":"2026-10-14T20:01:24Z","timestamp":1792008084444,"version":"4.4.0"},"reference-count":44,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2022,6,16]],"date-time":"2022-06-16T00:00:00Z","timestamp":1655337600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Deutsche Forschungsgemeinschaft(DFG)","award":["JU2859\/2-1"],"award-info":[{"award-number":["JU2859\/2-1"]}]},{"name":"Deutsche Forschungsgemeinschaft(DFG)","award":["JU2859\/7-1 (SJ)"],"award-info":[{"award-number":["JU2859\/7-1 (SJ)"]}]},{"name":"Deutsche Forschungsgemeinschaft(DFG)","award":["#01ZX1407A"],"award-info":[{"award-number":["#01ZX1407A"]}]},{"name":"Deutsche Forschungsgemeinschaft(DFG)","award":["#01ZX1708C"],"award-info":[{"award-number":["#01ZX1708C"]}]},{"name":"German Federal Ministry of Education and Research (BMBF)","award":["JU2859\/2-1"],"award-info":[{"award-number":["JU2859\/2-1"]}]},{"name":"German Federal Ministry of Education and Research (BMBF)","award":["JU2859\/7-1 (SJ)"],"award-info":[{"award-number":["JU2859\/7-1 (SJ)"]}]},{"name":"German Federal Ministry of Education and Research (BMBF)","award":["#01ZX1407A"],"award-info":[{"award-number":["#01ZX1407A"]}]},{"name":"German Federal Ministry of Education and Research (BMBF)","award":["#01ZX1708C"],"award-info":[{"award-number":["#01ZX1708C"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Valosin-containing protein (VCP) acts as a key regulator of cellular protein homeostasis by coordinating protein turnover and quality control. Mutations in VCP lead to (cardio-)myopathy and neurodegenerative diseases such as inclusion body myopathy with Paget\u2019s disease of the bone and frontotemporal dementia (IBMPFD) or amyotrophic lateral sclerosis (ALS). To date, due to embryonic lethality, no constitutive VCP knockout animal model exists. Here, we generated a constitutive CRISPR\/Cas9-induced vcp knockout zebrafish model. Similar to the phenotype of vcp morphant knockdown zebrafish embryos, we found that vcp-null embryos displayed significantly impaired cardiac and skeletal muscle function. By ultrastructural analysis of skeletal muscle cells and cardiomyocytes, we observed severely disrupted myofibrillar organization and accumulation of inclusion bodies as well as mitochondrial degeneration. vcp knockout was associated with a significant accumulation of ubiquitinated proteins, suggesting impaired proteasomal function. Additionally, markers of unfolded protein response (UPR)\/ER-stress and autophagy-related mTOR signaling were elevated in vcp-deficient embryos, demonstrating impaired proteostasis in VCP-null zebrafish. In conclusion, our findings demonstrate the successful generation of a stable constitutive vcp knockout zebrafish line that will enable characterization of the detailed mechanistic underpinnings of vcp loss, particularly the impact of disturbed protein homeostasis on organ development and function in vivo.<\/jats:p>","DOI":"10.3390\/ijms23126722","type":"journal-article","created":{"date-parts":[[2022,6,17]],"date-time":"2022-06-17T01:48:12Z","timestamp":1655430492000},"page":"6722","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["CRISPR\/Cas9-Mediated Constitutive Loss of VCP (Valosin-Containing Protein) Impairs Proteostasis and Leads to Defective Striated Muscle Structure and Function In Vivo"],"prefix":"10.3390","volume":"23","author":[{"given":"Philipp","family":"Voisard","sequence":"first","affiliation":[{"name":"Molecular Cardiology, Department of Internal Medicine II, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Federica","family":"Diofano","sequence":"additional","affiliation":[{"name":"Molecular Cardiology, Department of Internal Medicine II, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amelia A.","family":"Glazier","sequence":"additional","affiliation":[{"name":"Molecular Cardiology, Department of Internal Medicine II, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wolfgang","family":"Rottbauer","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine II, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7396-1281","authenticated-orcid":false,"given":"Steffen","family":"Just","sequence":"additional","affiliation":[{"name":"Molecular Cardiology, Department of Internal Medicine II, University of Ulm, Albert-Einstein-Allee 23, 89081 Ulm, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,6,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"182","DOI":"10.1016\/j.molcel.2017.10.028","article-title":"VCP\/p97-Mediated Unfolding as a Principle in Protein Homeostasis and Signaling","volume":"69","author":"Meyer","year":"2018","journal-title":"Mol. Cell"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"17806","DOI":"10.1074\/jbc.274.25.17806","article-title":"Identification of the cell cycle regulator VCP (p97\/CDC48) as a substrate of the band 4.1-related protein-tyrosine phosphatase PTPH1","volume":"274","author":"Zhang","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"5","DOI":"10.3724\/SP.J.1005.2012.00005","article-title":"Molecular mechanisms and functions of autophagy and the ubiquitin-proteasome pathway","volume":"34","author":"Chen","year":"2012","journal-title":"Yi Chuan"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"377","DOI":"10.1038\/ng1332","article-title":"Inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia is caused by mutant valosin-containing protein","volume":"36","author":"Watts","year":"2004","journal-title":"Nat. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"914","DOI":"10.5692\/clinicalneurol.cn-000765","article-title":"A patient with familial amyotrophic lateral sclerosis associated with a new valosin-containing protein (VCP) gene mutation","volume":"55","author":"Segawa","year":"2015","journal-title":"Rinsho Shinkeigaku"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"e25","DOI":"10.1017\/S1462399408000793","article-title":"Molecular pathology of myofibrillar myopathies","volume":"10","author":"Ferrer","year":"2008","journal-title":"Expert Rev. Mol. Med."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"381","DOI":"10.1093\/brain\/awl238","article-title":"Pathological consequences of VCP mutations on human striated muscle","volume":"130","author":"Clemen","year":"2007","journal-title":"Brain"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"109399","DOI":"10.1016\/j.celrep.2021.109399","article-title":"Neuronal VCP loss of function recapitulates FTLD-TDP pathology","volume":"36","author":"Wani","year":"2021","journal-title":"Cell Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"1082","DOI":"10.1080\/15548627.2019.1569933","article-title":"VCP maintains lysosomal homeostasis and TFEB activity in differentiated skeletal muscle","volume":"15","author":"Arhzaouy","year":"2019","journal-title":"Autophagy"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"4502","DOI":"10.1080\/15548627.2021.1985880","article-title":"Loss-of-function mutation in VCP mimics the characteristic pathology as in FTLD-TARDBP","volume":"17","author":"Wani","year":"2021","journal-title":"Autophagy"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"8918","DOI":"10.1074\/jbc.RA119.007585","article-title":"Disruption of valosin-containing protein activity causes cardiomyopathy and reveals pleiotropic functions in cardiac homeostasis","volume":"294","author":"Brody","year":"2019","journal-title":"J. Biol. Chem."},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Sun, X., Zhou, N., Ma, B., Wu, W., Stoll, S., Lai, L., Qin, G., and Qiu, H. (2021). Functional Inhibition of Valosin-Containing Protein Induces Cardiac Dilation and Dysfunction in a New Dominant-Negative Transgenic Mouse Model. Cells, 10.","DOI":"10.3390\/cells10112891"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Viswanathan, M.C., Blice-Baum, A.C., Sang, T.K., and Cammarato, A. (2016). Cardiac-Restricted Expression of VCP\/TER94 RNAi or Disease Alleles Perturbs Drosophila Heart Structure and Impairs Function. J. Cardiovasc. Dev. Dis., 3.","DOI":"10.3390\/jcdd3020019"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"119","DOI":"10.1111\/cge.13095","article-title":"Genotype-phenotype study in patients with valosin-containing protein mutations associated with multisystem proteinopathy","volume":"93","author":"Surampalli","year":"2018","journal-title":"Clin. Genet."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"459","DOI":"10.1016\/j.bbrc.2006.12.206","article-title":"Targeted deletion of p97 (VCP\/CDC48) in mouse results in early embryonic lethality","volume":"354","author":"Deinhardt","year":"2007","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1038\/sj.cdd.4400955","article-title":"Identification of ter94, Drosophila VCP, as a modulator of polyglutamine-induced neurodegeneration","volume":"9","author":"Higashiyama","year":"2002","journal-title":"Cell Death Differ."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1911","DOI":"10.1080\/15548627.2018.1491491","article-title":"Loss of the novel Vcp (valosin containing protein) interactor Washc4 interferes with autophagy-mediated proteostasis in striated muscle and leads to myopathy in vivo","volume":"14","author":"Kustermann","year":"2018","journal-title":"Autophagy"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"dev186452","DOI":"10.1242\/dev.186452","article-title":"Transcriptional adaptation: A mechanism underlying genetic robustness","volume":"147","author":"Sztal","year":"2020","journal-title":"Development"},{"key":"ref_19","doi-asserted-by":"crossref","unstructured":"Diofano, F., Weinmann, K., Schneider, I., Thiessen, K.D., Rottbauer, W., and Just, S. (2020). Genetic compensation prevents myopathy and heart failure in an in vivo model of Bag3 deficiency. PLoS Genet., 16.","DOI":"10.1371\/journal.pgen.1009088"},{"key":"ref_20","first-page":"e50925","article-title":"Analysis of skeletal muscle defects in larval zebrafish by birefringence and touch-evoke escape response assays","volume":"82","author":"Smith","year":"2013","journal-title":"J. Vis. Exp."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"507","DOI":"10.1016\/S1084952102001040","article-title":"Cardiac development in zebrafish: Coordination of form and function","volume":"13","author":"Glickman","year":"2002","journal-title":"Semin Cell Dev. Biol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"23047","DOI":"10.1074\/jbc.M111.332882","article-title":"Protective role of cell division cycle 48 (CDC48) protein against neurodegeneration via ubiquitin-proteasome system dysfunction during zebrafish development","volume":"287","author":"Imamura","year":"2012","journal-title":"J. Biol. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1038\/nrm3270","article-title":"The unfolded protein response: Controlling cell fate decisions under ER stress and beyond","volume":"13","author":"Hetz","year":"2012","journal-title":"Nat. Rev. Mol. Cell Biol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1926","DOI":"10.1101\/gad.1212704","article-title":"Upstream and downstream of mTOR","volume":"18","author":"Hay","year":"2004","journal-title":"Genes Dev."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"875","DOI":"10.1083\/jcb.200908115","article-title":"Valosin-containing protein (VCP) is required for autophagy and is disrupted in VCP disease","volume":"187","author":"Ju","year":"2009","journal-title":"J. Cell Biol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"807","DOI":"10.1101\/gad.887201","article-title":"Regulation of translation initiation by FRAP\/mTOR","volume":"15","author":"Gingras","year":"2001","journal-title":"Genes Dev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1766","DOI":"10.1038\/s41418-018-0076-9","article-title":"The mTOR-S6 kinase pathway promotes stress granule assembly","volume":"25","author":"Sfakianos","year":"2018","journal-title":"Cell Death Differ."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1168","DOI":"10.1111\/acel.12653","article-title":"The valosin-containing protein is a novel repressor of cardiomyocyte hypertrophy induced by pressure overload","volume":"16","author":"Zhou","year":"2017","journal-title":"Aging Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2469","DOI":"10.18632\/aging.101357","article-title":"VCP represses pathological cardiac hypertrophy","volume":"9","author":"Zhou","year":"2017","journal-title":"Aging (Albany NY)"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"485","DOI":"10.1042\/BST20200981","article-title":"Emerging role of VCP\/p97 in cardiovascular diseases: Novel insights and therapeutic opportunities","volume":"49","author":"Shu","year":"2021","journal-title":"Biochem. Soc. Trans."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"565","DOI":"10.1042\/EBC20170027","article-title":"mTORC1 as the main gateway to autophagy","volume":"61","author":"Otten","year":"2017","journal-title":"Essays Biochem."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s13023-022-02172-5","article-title":"Development of a standard of care for patients with valosin-containing protein associated multisystem proteinopathy","volume":"17","author":"Korb","year":"2022","journal-title":"Orphanet J. Rare Dis."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"744","DOI":"10.1016\/j.bbadis.2008.09.003","article-title":"VCP disease associated with myopathy, Paget disease of bone and frontotemporal dementia: Review of a unique disorder","volume":"1782","author":"Kimonis","year":"2008","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"217","DOI":"10.4161\/auto.6.2.11014","article-title":"VCP\/p97 is essential for maturation of ubiquitin-containing autophagosomes and this function is impaired by mutations that cause IBMPFD","volume":"6","author":"Tresse","year":"2010","journal-title":"Autophagy"},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Nalbandian, A., Llewellyn, K.J., Kitazawa, M., Yin, H.Z., Badadani, M., Khanlou, N., Edwards, R., Nguyen, C., Mukherjee, J., and Mozaffar, T. (2012). The homozygote VCP(R\u00b9\u2075\u2075H\/R\u00b9\u2075\u2075H) mouse model exhibits accelerated human VCP-associated disease pathology. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0046308"},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"B\u00fchler, A., Kustermann, M., Bummer, T., Rottbauer, W., Sandri, M., and Just, S. (2016). Atrogin-1 Deficiency Leads to Myopathy and Heart Failure in Zebrafish. Int. J. Mol. Sci., 17.","DOI":"10.3390\/ijms17020187"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"253","DOI":"10.1002\/aja.1002030302","article-title":"Stages of embryonic development of the zebrafish","volume":"203","author":"Kimmel","year":"1995","journal-title":"Dev. Dyn."},{"key":"ref_38","doi-asserted-by":"crossref","unstructured":"Alexandre-Moreno, S., Bonet-Fern\u00e1ndez, J.-M., Atienzar-Aroca, R., Aroca-Aguilar, J.-D., and Escribano, J. (2021). Null cyp1b1 Activity in Zebrafish Leads to Variable Craniofacial Defects Associated with Altered Expression of Extracellular Matrix and Lipid Metabolism Genes. Int. J. Mol. Sci., 22.","DOI":"10.20944\/preprints202106.0357.v1"},{"key":"ref_39","doi-asserted-by":"crossref","unstructured":"Hirth, S., B\u00fchler, A., B\u00fchrdel, J.B., Rudeck, S., Dahme, T., Rottbauer, W., and Just, S. (2016). Paxillin and Focal Adhesion Kinase (FAK) Regulate Cardiac Contractility in the Zebrafish Heart. PLoS ONE, 11.","DOI":"10.1371\/journal.pone.0150323"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"505","DOI":"10.1089\/zeb.2019.1761","article-title":"Protein Purification and Western Blot Detection from Single Zebrafish Embryo","volume":"16","author":"Schnabel","year":"2019","journal-title":"Zebrafish"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1007\/s00395-015-0468-7","article-title":"Loss of dihydrolipoyl succinyltransferase (DLST) leads to reduced resting heart rate in the zebrafish","volume":"110","author":"Kessler","year":"2015","journal-title":"Basic Res. Cardiol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"217","DOI":"10.1016\/j.bbrc.2015.03.149","article-title":"In vivo characterization of human myofibrillar myopathy genes in zebrafish","volume":"461","author":"Buhrdel","year":"2015","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1152\/jn.00265.2018","article-title":"Neuromuscular junction abnormalities in a zebrafish loss-of-function model of TDP-43","volume":"121","author":"Bose","year":"2019","journal-title":"J. Neurophysiol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"102876","DOI":"10.1016\/j.micron.2020.102876","article-title":"Cardiac function and blood flow hemodynamics assessment of zebrafish (Danio rerio) using high-speed video microscopy","volume":"136","author":"Benslimane","year":"2020","journal-title":"Micron"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/12\/6722\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T23:33:24Z","timestamp":1760139204000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/23\/12\/6722"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,6,16]]},"references-count":44,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2022,6]]}},"alternative-id":["ijms23126722"],"URL":"https:\/\/doi.org\/10.3390\/ijms23126722","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,6,16]]}}}