{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,1]],"date-time":"2026-05-01T08:19:45Z","timestamp":1777623585538,"version":"3.51.4"},"reference-count":58,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2022,2,16]],"date-time":"2022-02-16T00:00:00Z","timestamp":1644969600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["422681845"],"award-info":[{"award-number":["422681845"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Connexins (Cx) are a large family of membrane proteins that can form intercellular connections, so-called gap junctions between adjacent cells. Cx43 is widely expressed in mammals and has a variety of different functions, such as the propagation of electrical conduction in the cardiac ventricle. Despite Cx43 knockout models, many questions regarding the biology of Cx43 in health and disease remain unanswered. Herein we report the establishment of a Cre-inducible Cx43 overexpression system in murine embryonic stem (ES) cells. This enables the investigation of the impact of Cx43 overexpression in somatic cells. We utilized a double reporter system to label Cx43-overexpressing cells via mCherry fluorescence and exogenous Cx43 via fusion with P2A peptide to visualize its distribution pattern. We proved the functionality of our systems in ES cells, HeLa cells, and 3T3-fibroblasts and demonstrated the formation of functional gap junctions based on dye diffusion and FRAP experiments. In addition, Cx43-overexpressing ES cells could be differentiated into viable cardiomyocytes, as shown by the formation of cross striation and spontaneous beating. Analysis revealed faster and more rhythmic beating of Cx43-overexpressing cell clusters. Thus, our Cx43 overexpression systems enable the investigation of Cx43 biology and function in cardiomyocytes and other somatic cells.<\/jats:p>","DOI":"10.3390\/cells11040694","type":"journal-article","created":{"date-parts":[[2022,2,16]],"date-time":"2022-02-16T20:26:41Z","timestamp":1645043201000},"page":"694","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Generation and Characterization of an Inducible Cx43 Overexpression System in Mouse Embryonic Stem Cells"],"prefix":"10.3390","volume":"11","author":[{"given":"Pia","family":"Niemann","sequence":"first","affiliation":[{"name":"Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, 53127 Bonn, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miriam","family":"Schiffer","sequence":"additional","affiliation":[{"name":"Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, 53127 Bonn, Germany"},{"name":"Department of Cardiac Surgery, University Hospital Bonn, 53127 Bonn, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9034-6555","authenticated-orcid":false,"given":"Daniela","family":"Malan","sequence":"additional","affiliation":[{"name":"Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, 53127 Bonn, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Sabine","family":"Gr\u00fcnberg","sequence":"additional","affiliation":[{"name":"Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, 53127 Bonn, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wilhelm","family":"Roell","sequence":"additional","affiliation":[{"name":"Department of Cardiac Surgery, University Hospital Bonn, 53127 Bonn, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Caroline","family":"Geisen","sequence":"additional","affiliation":[{"name":"Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, 53127 Bonn, Germany"},{"name":"Department of Cardiac Surgery, University Hospital Bonn, 53127 Bonn, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9202-8363","authenticated-orcid":false,"given":"Bernd K.","family":"Fleischmann","sequence":"additional","affiliation":[{"name":"Institute of Physiology I, Life and Brain Center, Medical Faculty, University of Bonn, 53127 Bonn, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2022,2,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1007\/s00395-009-0007-5","article-title":"Presence of connexin 43 in subsarcolemmal, but not in interfibrillar cardiomyocyte mitochondria","volume":"104","author":"Boengler","year":"2009","journal-title":"Basic Res. Cardiol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"H2124","DOI":"10.1152\/ajpheart.01028.2001","article-title":"Paradoxical overexpression and translocation of connexin43 in homocysteine-treated endothelial cells","volume":"282","author":"Li","year":"2002","journal-title":"Am. J. Physiol. Heart Circ. Physiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1016\/j.cardiores.2003.11.013","article-title":"Gap junctions and the connexin protein family","volume":"62","author":"Willecke","year":"2004","journal-title":"Cardiovasc. Res."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Boengler, K., Rohrbach, S., Weissmann, N., and Schulz, R. (2021). Importance of Cx43 for Right Ventricular Function. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22030987"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"527","DOI":"10.1042\/BJ20051922","article-title":"Life cycle of connexins in health and disease","volume":"394","author":"Laird","year":"2006","journal-title":"Biochem. J."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"48","DOI":"10.1016\/j.bbamem.2017.05.008","article-title":"The connexin 43 C-terminus: A tail of many tales","volume":"1860","author":"Leithe","year":"2018","journal-title":"Biochim. Biophys. Acta Biomembr."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1193","DOI":"10.1016\/j.febslet.2014.02.025","article-title":"Mix and match: Investigating heteromeric and heterotypic gap junction channels in model systems and native tissues","volume":"588","author":"Koval","year":"2014","journal-title":"FEBS Lett."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2377","DOI":"10.1016\/j.yexcr.2010.05.026","article-title":"Cell membrane permeabilization via connexin hemichannels in living and dying cells","volume":"316","author":"Schalper","year":"2010","journal-title":"Exp. Cell Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"3501","DOI":"10.1242\/dev.127.16.3501","article-title":"Loss of connexin45 causes a cushion defect in early cardiogenesis","volume":"127","author":"Kumai","year":"2000","journal-title":"Development"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"319","DOI":"10.1038\/30639","article-title":"Connexin 26 gene linked to a dominant deafness","volume":"393","author":"Denoyelle","year":"1998","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"192","DOI":"10.1016\/j.bbamem.2017.04.024","article-title":"Human diseases associated with connexin mutations","volume":"1860","author":"Srinivas","year":"2018","journal-title":"Biochim. Biophys. Acta Biomembr."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1638","DOI":"10.1161\/01.RES.68.6.1638","article-title":"Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart\/\/Spatial distribution of connexin43, the major cardiac gap junction protein, in the developing and adult rat heart","volume":"68","author":"Fromaget","year":"1991","journal-title":"Circ. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1528","DOI":"10.1161\/CIRCRESAHA.112.270561","article-title":"Connexin45 provides optimal atrioventricular nodal conduction in the adult mouse heart","volume":"111","author":"Frank","year":"2012","journal-title":"Circ. Res."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"2677","DOI":"10.1056\/NEJMoa052800","article-title":"Somatic mutations in the connexin 40 gene (GJA5) in atrial fibrillation","volume":"354","author":"Gollob","year":"2006","journal-title":"N. Engl. J. Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"539","DOI":"10.1093\/hmg\/ddm329","article-title":"The conditional connexin43G138R mouse mutant represents a new model of hereditary oculodentodigital dysplasia in humans","volume":"17","author":"Dobrowolski","year":"2008","journal-title":"Hum. Mol. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1093\/cvr\/cvn133","article-title":"Remodelling of gap junctions and connexin expression in diseased myocardium","volume":"80","author":"Severs","year":"2008","journal-title":"Cardiovasc. Res."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Dhein, S., and Salameh, A. (2021). Remodeling of Cardiac Gap Junctional Cell-Cell Coupling. Cells, 10.","DOI":"10.3390\/cells10092422"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"489","DOI":"10.4161\/chan.5.6.18523","article-title":"Residual Cx45 and its relationship to Cx43 in murine ventricular myocardium","volume":"5","author":"Bao","year":"2011","journal-title":"Channels"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1035","DOI":"10.1161\/01.RES.0000148664.33695.2a","article-title":"Modulation of cardiac gap junction expression and arrhythmic susceptibility","volume":"95","author":"Danik","year":"2004","journal-title":"Circ. Res."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1557","DOI":"10.1093\/cvr\/cvaa082","article-title":"Heritable arrhythmia syndromes associated with abnormal cardiac sodium channel function: Ionic and non-ionic mechanisms","volume":"116","author":"Rivaud","year":"2020","journal-title":"Cardiovasc. Res."},{"key":"ref_21","doi-asserted-by":"crossref","unstructured":"Rodr\u00edguez-Sinovas, A., S\u00e1nchez, J.A., Valls-Lacalle, L., Consegal, M., and Ferreira-Gonz\u00e1lez, I. (2021). Connexins in the Heart: Regulation, Function and Involvement in Cardiac Disease. Int. J. Mol. Sci., 22.","DOI":"10.3390\/ijms22094413"},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Marsh, S.R., Williams, Z.J., Pridham, K.J., and Gourdie, R.G. (2021). Peptidic Connexin43 Therapeutics in Cardiac Reparative Medicine. J. Cardiovasc. Dev. Dis., 8.","DOI":"10.3390\/jcdd8050052"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"704","DOI":"10.1161\/CIRCRESAHA.110.235747","article-title":"A peptide mimetic of the connexin43 carboxyl terminus reduces gap junction remodeling and induced arrhythmia following ventricular injury","volume":"108","author":"Palatinus","year":"2011","journal-title":"Circ. Res."},{"key":"ref_24","unstructured":"Reynolds, J. (2021, December 20). Zealand Announces Results of a Phase II Proof-of-Concept Trial with Danegaptide for Cardiac Reperfusion Injuries. FierceBiotech, Available online: https:\/\/www.fiercebiotech.com\/biotech\/zealand-announces-results-of-a-phase-ii-proof-of-concept-trial-danegaptide-for-cardiac."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"C1112","DOI":"10.1152\/ajpcell.00097.2007","article-title":"Modulation of membrane channel currents by gap junction protein mimetic peptides: Size matters","volume":"293","author":"Wang","year":"2007","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1831","DOI":"10.1126\/science.7892609","article-title":"Cardiac malformation in neonatal mice lacking connexin43","volume":"267","author":"Reaume","year":"1995","journal-title":"Science"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1016\/S1050-1738(99)00015-8","article-title":"Gap Junction Communication and the Modulation of Cardiac Neural Crest Cells","volume":"9","author":"Lo","year":"1999","journal-title":"Trends Cardiovasc. Med."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1048","DOI":"10.1161\/01.CIR.0000117402.70689.75","article-title":"Slow conduction and enhanced anisotropy increase the propensity for ventricular tachyarrhythmias in adult mice with induced deletion of connexin43","volume":"109","author":"Eckardt","year":"2004","journal-title":"Circulation"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1242\/dev.124.7.1281","article-title":"Heart and neural tube defects in transgenic mice overexpressing the Cx43 gap junction gene","volume":"124","author":"Ewart","year":"1997","journal-title":"Development"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"133","DOI":"10.1038\/nn.2467","article-title":"A robust and high-throughput Cre reporting and characterization system for the whole mouse brain","volume":"13","author":"Madisen","year":"2010","journal-title":"Nat. Neurosci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2886","DOI":"10.1128\/JVI.73.4.2886-2892.1999","article-title":"Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors","volume":"73","author":"Zufferey","year":"1999","journal-title":"J. Virol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4455","DOI":"10.1073\/pnas.0609277104","article-title":"Developmental and adult phenotyping directly from mutant embryonic stem cells","volume":"104","author":"George","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1161\/01.RES.0000027865.61704.32","article-title":"Differentiation of pluripotent embryonic stem cells into cardiomyocytes","volume":"91","author":"Boheler","year":"2002","journal-title":"Circ. Res."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-021-23858-7","article-title":"Overexpression of human BAG3P209L in mice causes restrictive cardiomyopathy","volume":"12","author":"Kimura","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","unstructured":"Raulf, A., Voeltz, N., Korzus, D., Fleischmann, B.K., and Hesse, M. (2017). Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes. J. Vis. Exp., 120.","DOI":"10.3791\/55204-v"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1038\/5007","article-title":"Generalized lacZ expression with the ROSA26 Cre reporter strain","volume":"21","author":"Soriano","year":"1999","journal-title":"Nat. Genet."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-018-25147-8","article-title":"Overexpression of Cx43 in cells of the myocardial scar: Correction of post-infarct arrhythmias through heterotypic cell-cell coupling","volume":"8","author":"Roell","year":"2018","journal-title":"Sci. Rep."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"522","DOI":"10.1002\/ijc.2910510404","article-title":"Expression of Cx26, Cx32 and Cx43 gap junction proteins in normal and neoplastic human tissues","volume":"51","author":"Wilgenbus","year":"1992","journal-title":"Int. J. Cancer"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-019-09322-7","article-title":"Optogenetic stimulation of Gs-signaling in the heart with high spatio-temporal precision","volume":"10","author":"Makowka","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/ncomms2089","article-title":"Direct visualization of cell division using high-resolution imaging of M-phase of the cell cycle","volume":"3","author":"Hesse","year":"2012","journal-title":"Nat. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1080\/00313020701230807","article-title":"A histological survey of green fluorescent protein expression in \u2018green\u2019 mice: Implications for stem cell research","volume":"39","author":"Biankin","year":"2007","journal-title":"Pathology"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"1513","DOI":"10.1101\/gad.5.9.1513","article-title":"Promoter traps in embryonic stem cells: A genetic screen to identify and mutate developmental genes in mice","volume":"5","author":"Friedrich","year":"1991","journal-title":"Genes Dev."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"11388","DOI":"10.1073\/pnas.1434298100","article-title":"Gating and regulation of connexin 43 (Cx43) hemichannels","volume":"100","author":"Contreras","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Brokamp, C., Todd, J., Montemagno, C., and Wendell, D. (2012). Electrophysiology of single and aggregate Cx43 hemichannels. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0047775"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"255","DOI":"10.1038\/s41576-019-0205-4","article-title":"Engineering adeno-associated virus vectors for gene therapy","volume":"21","author":"Li","year":"2020","journal-title":"Nat. Rev. Genet."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"80","DOI":"10.1016\/j.yjmcc.2020.11.016","article-title":"What we know about cardiomyocyte dedifferentiation","volume":"152","author":"Zhu","year":"2021","journal-title":"J. Mol. Cell. Cardiol."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"360","DOI":"10.1161\/01.RES.82.3.360","article-title":"Heart Defects in Connexin43-Deficient Mice\/\/Heart defects in connexin43-deficient mice","volume":"82","author":"Ya","year":"1998","journal-title":"Circ. Res."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Kim, J.H., Lee, S.-R., Li, L.-H., Park, H.-J., Park, J.-H., Lee, K.Y., Kim, M.-K., Shin, B.A., and Choi, S.-Y. (2011). High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. PLoS ONE, 6.","DOI":"10.1371\/journal.pone.0018556"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1006\/mthe.2000.0050","article-title":"IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector","volume":"1","author":"Mizuguchi","year":"2000","journal-title":"Mol. Ther."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"2556","DOI":"10.1073\/pnas.050588497","article-title":"Clustering of connexin 43-enhanced green fluorescent protein gap junction channels and functional coupling in living cells","volume":"97","author":"Bukauskas","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"211","DOI":"10.1080\/cac.10.4-6.211.214","article-title":"Fusion of GFP to the carboxyl terminus of connexin43 increases gap junction size in HeLa cells","volume":"10","author":"Hunter","year":"2003","journal-title":"Cell Commun. Adhes."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"5686","DOI":"10.1091\/mbc.e05-08-0737","article-title":"Zonula occludens-1 alters connexin43 gap junction size and organization by influencing channel accretion","volume":"16","author":"Hunter","year":"2005","journal-title":"Mol. Biol. Cell"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"1516","DOI":"10.1091\/mbc.e10-06-0548","article-title":"Connexin 43 connexon to gap junction transition is regulated by zonula occludens-1","volume":"22","author":"Rhett","year":"2011","journal-title":"Mol. Biol. Cell"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1083\/jcb.115.5.1357","article-title":"Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques","volume":"115","author":"Musil","year":"1991","journal-title":"J. Cell Biol."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Solan, J.L., and Lampe, P.D. (2020). Src Regulation of Cx43 Phosphorylation and Gap Junction Turnover. Biomolecules, 10.","DOI":"10.3390\/biom10121596"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"862","DOI":"10.1016\/j.stem.2020.05.004","article-title":"Human-iPSC-Derived Cardiac Stromal Cells Enhance Maturation in 3D Cardiac Microtissues and Reveal Non-cardiomyocyte Contributions to Heart Disease","volume":"26","author":"Giacomelli","year":"2020","journal-title":"Cell Stem Cell"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"819","DOI":"10.1038\/nature06321","article-title":"Engraftment of connexin 43-expressing cells prevents post-infarct arrhythmia","volume":"450","author":"Roell","year":"2007","journal-title":"Nature"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"2687","DOI":"10.1007\/s11064-021-03236-x","article-title":"Astrocytes and Epilepsy","volume":"46","author":"Binder","year":"2021","journal-title":"Neurochem. Res."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/11\/4\/694\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T22:20:52Z","timestamp":1760134852000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/11\/4\/694"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,2,16]]},"references-count":58,"journal-issue":{"issue":"4","published-online":{"date-parts":[[2022,2]]}},"alternative-id":["cells11040694"],"URL":"https:\/\/doi.org\/10.3390\/cells11040694","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,2,16]]}}}