{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T05:26:41Z","timestamp":1766554001344,"version":"3.48.0"},"reference-count":55,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T00:00:00Z","timestamp":1766361600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Glucagon is an endogenous peptide that is produced in the pancreas. Via glucagon receptors, glucagon increases the beating rate in cultured rat neonatal cardiomyocytes and also in isolated right atrial preparations from adult rats. Moreover, in living adult mice, injections of glucagon can elevate the heart rate. It is unknown whether these effects of glucagon in living adult mice are mediated via central glucagon receptors or via a direct effect on cardiac glucagon receptors. Thus, we tested the hypothesis that glucagon can exert a direct positive chronotropic effect in the adult mouse heart. We measured the contractile effects of cumulatively increasing concentrations of glucagon (0.1\u2013100 nM) in isolated paced (1 Hz) left atrial preparations, in isolated spontaneously beating right atrial preparations and in isolated spontaneously beating retrogradely perfused whole hearts. We detected in isolated right atrial preparations time- and concentration-dependent positive chronotropic effects of glucagon that were reversed by the glucagon receptor antagonists SC203972 and desglucagon. The positive chronotropic effects of glucagon were also attenuated by 1 \u00b5M of ivabradine, an inhibitor of the hyperpolarization-activated cation channels (HCN), but not by 100 nM rolipram, a phosphodiesterase 4 inhibitor, nor by 10 \u00b5M of propranolol, a \u03b2-adrenoceptor antagonist. Moreover, the positive chronotropic effects of glucagon were also attenuated by stimulation of the A1-adenosine receptor or muscarinic receptors. Glucagon decreased the force of contraction in right atrial preparations. In left atrial preparations, glucagon failed to alter the force of contraction. In isolated adult mouse hearts perfused in the Langendorff mode, 10 nM of glucagon increased the beating rate and reduced left ventricular force of contraction. The gene expression of the glucagon receptors was lowest in the left atrium, higher in the ventricle and highest in the right atrium of adult mice. In summary, glucagon exerted a positive chronotropic effect in the mouse heart via glucagon receptors, mediated, at least in part, via HCN channels in the sinus node.<\/jats:p>","DOI":"10.3390\/ijms27010126","type":"journal-article","created":{"date-parts":[[2025,12,22]],"date-time":"2025-12-22T16:19:26Z","timestamp":1766420366000},"page":"126","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Contractile Effects of Glucagon in Mouse Cardiac Preparations"],"prefix":"10.3390","volume":"27","author":[{"given":"Joachim","family":"Neumann","sequence":"first","affiliation":[{"name":"Institute for Pharmacology and Toxicology, Medical Faculty, Martin-Luther-University Halle-Wittenberg, Magdeburger Stra\u00dfe 4, D-06097 Halle, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Franziska","family":"Schmidt","sequence":"additional","affiliation":[{"name":"Institute for Pharmacology and Toxicology, Medical Faculty, Martin-Luther-University Halle-Wittenberg, Magdeburger Stra\u00dfe 4, D-06097 Halle, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pauline","family":"Braekow","sequence":"additional","affiliation":[{"name":"Institute for Pharmacology and Toxicology, Medical Faculty, Martin-Luther-University Halle-Wittenberg, Magdeburger Stra\u00dfe 4, D-06097 Halle, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2838-2579","authenticated-orcid":false,"given":"Uwe","family":"Kirchhefer","sequence":"additional","affiliation":[{"name":"Institute for Pharmacology and Toxicology, Medical Faculty, University M\u00fcnster, Domagkstra\u00dfe 12, D-48149 M\u00fcnster, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jan","family":"Klimas","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Odboj\u00e1rov 10, SK-832 32 Bratislava, Slovakia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7055-9561","authenticated-orcid":false,"given":"Katarina","family":"Hadova","sequence":"additional","affiliation":[{"name":"Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University in Bratislava, Odboj\u00e1rov 10, SK-832 32 Bratislava, Slovakia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6986-485X","authenticated-orcid":false,"given":"Ulrich","family":"Gergs","sequence":"additional","affiliation":[{"name":"Institute for Pharmacology and Toxicology, Medical Faculty, Martin-Luther-University Halle-Wittenberg, Magdeburger Stra\u00dfe 4, D-06097 Halle, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,12,22]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"2807","DOI":"10.1021\/ja01568a038","article-title":"The amino acid sequence of glucagon V. Location of amide groups; acid degradation studies and summary of sequential evidence","volume":"79","author":"Bromer","year":"1957","journal-title":"J. Am. Chem. Soc."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"328","DOI":"10.1006\/bbrc.1994.1046","article-title":"Cloning and expression of a human glucagon receptor","volume":"198","author":"MacNeil","year":"1994","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1016\/0378-1119(95)00472-I","article-title":"Cloning and sequence analysis of the murine glucagon receptor-encoding gene","volume":"164","author":"Burcelin","year":"1995","journal-title":"Gene"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Janah, L., Kjeldsen, S., Galsgaard, K.D., Winther-S\u00f8rensen, M., Stojanovska, E., Pedersen, J., Knop, F.K., Holst, J.J., and Wewer Albrechtsen, N.J. (2019). Glucagon receptor signaling and glucagon resistance. Int. J. Mol. Sci., 20.","DOI":"10.3390\/ijms20133314"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"203","DOI":"10.1016\/0378-1119(94)90545-2","article-title":"The human glucagon receptor encoding gene: Structure; cDNA sequence and chromosomal localization","volume":"140","author":"Lok","year":"1994","journal-title":"Gene"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Neumann, J., Hofmann, B., Dhein, S., and Gergs, U. (2023). Glucagon and its receptors in the mammalian heart. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241612829"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"1077","DOI":"10.1016\/S0021-9258(19)77423-7","article-title":"Fractionation and characterization of a cyclic adenine ribonucleotide formed by tissue particles","volume":"232","author":"Sutherland","year":"1958","journal-title":"J. Biol. Chem."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"49","DOI":"10.1016\/S0022-3565(25)25785-8","article-title":"Studies on the pharmacology of glucagon","volume":"129","author":"Farah","year":"1960","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2250","DOI":"10.1172\/JCI107411","article-title":"Maturation of responsiveness to cardioactive drugs: Differential effects of acetylcholine, norepinephrine, theophylline, tyramine, glucagon, and dibutyryl cyclic AMP on atrial rate in hearts of fetal mice","volume":"52","author":"Wildenthal","year":"1973","journal-title":"J. Clin. Investig."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"551","DOI":"10.1172\/JCI108310","article-title":"Responsiveness to glucagon in fetal hearts: Species variability and apparent disparities between changes in beating; adenylate cyclase activation; and cyclic AMP concentration","volume":"57","author":"Wildenthal","year":"1976","journal-title":"J. Clin. Investig."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1161\/01.RES.38.3.162","article-title":"Changes in cyclic nucleotide levels and contractile force in the isolated hypoxic rat heart during perfusion with glucagon","volume":"38","author":"Busuttil","year":"1976","journal-title":"Circ. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1210\/endo-99-1-23","article-title":"Evidence that cyclic AMP is not involved in the chronotropic action of glucagon in the adult mouse heart","volume":"99","author":"Clark","year":"1976","journal-title":"Endocrinology"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"e00112","DOI":"10.1002\/phy2.112","article-title":"Oxyntomodulin increases intrinsic heart rate through the glucagon receptor","volume":"1","author":"Mukharji","year":"2013","journal-title":"Physiol. Rep."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1278","DOI":"10.1038\/s42003-022-04242-7","article-title":"Evaluation of commercially available glucagon receptor antibodies and glucagon receptor expression","volume":"5","author":"Bomholt","year":"2022","journal-title":"Commun. Biol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1016\/j.molmet.2014.11.005","article-title":"Cardiomyocyte glucagon receptor signaling modulates outcomes in mice with experimental myocardial infarction","volume":"4","author":"Ali","year":"2014","journal-title":"Mol. Metab."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1438","DOI":"10.1073\/pnas.0237106100","article-title":"Lower blood glucose; hyperglucagonemia; and pancreatic alpha cell hyperplasia in glucagon receptor knockout mice","volume":"100","author":"Gelling","year":"2003","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"614","DOI":"10.1161\/CIRCRESAHA.124.324706","article-title":"Glucagon receptor antagonist for heart failure with preserved ejection fraction","volume":"135","author":"Gao","year":"2024","journal-title":"Circ. Res."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12933-019-0806-4","article-title":"Targeting the glucagon receptor improves cardiac function and enhances insulin sensitivity following a myocardial infarction","volume":"18","author":"Karwi","year":"2019","journal-title":"Cardiovasc. Diabetol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"161","DOI":"10.1016\/j.jacbts.2018.11.001","article-title":"Glucagon receptor antagonism ameliorates progression of heart failure","volume":"4","author":"Gao","year":"2019","journal-title":"JACC Basic Transl. Sci."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"100002","DOI":"10.1124\/pharmrev.123.001045","article-title":"Emerging pharmacotherapies for obesity: A systematic review","volume":"77","author":"Kokkorakis","year":"2025","journal-title":"Pharmacol. Rev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1038\/sj.bjp.0705012","article-title":"Pertussis toxin sensitive and insensitive effects of adenosine and carbachol in murine atria overexpressing A(1)-adenosine receptors","volume":"138","author":"Neumann","year":"2003","journal-title":"Br. J. Pharmacol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1016\/j.pneurobio.2013.10.001","article-title":"Neurophysiology of HCN channels: From cellular functions to multiple regulations","volume":"112","author":"He","year":"2014","journal-title":"Prog. Neurobiol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"A14259","DOI":"10.1161\/circ.148.suppl_1.14259","article-title":"Contractile effects of glucagon in atrial preparations from transgenic mice and men","volume":"148","author":"Neumann","year":"2023","journal-title":"Circulation"},{"key":"ref_24","first-page":"S34","article-title":"Contractile effects of glucagon in mouse atrial preparations","volume":"397","author":"Schmidt","year":"2024","journal-title":"Naunyn-Schmiedeberg\u2019s Arch. Pharmacol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3267","DOI":"10.2337\/diabetes.53.12.3267","article-title":"A novel glucagon receptor antagonist inhibits glucagon-mediated biological effects","volume":"53","author":"Qureshi","year":"2004","journal-title":"Diabetes"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1111\/j.1399-3011.1988.tb01374.x","article-title":"Potentiation of glucose-induced insulin release in islets by desHis1[Glu9]glucagon amide","volume":"32","author":"Kofod","year":"1988","journal-title":"Int. J. Pept. Protein Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"15496","DOI":"10.1016\/S0021-9258(19)49562-8","article-title":"Inhibition by glucagon of the cGMP-inhibited low-Km cAMP phosphodiesterase in heart is mediated by a pertussis toxin-sensitive G-protein","volume":"267","author":"Brechler","year":"1992","journal-title":"J. Biol. Chem."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"158","DOI":"10.1038\/345158a0","article-title":"Glucagon stimulates the cardiac Ca2+ current by activation of adenylyl cyclase and inhibition of phosphodiesterase","volume":"345","author":"Brechler","year":"1990","journal-title":"Nature"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"90","DOI":"10.1038\/s41569-022-00756-z","article-title":"Cyclic nucleotide phosphodiesterases as therapeutic targets in cardiac hypertrophy and heart failure","volume":"20","author":"Kamel","year":"2023","journal-title":"Nat. Rev. Cardiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"1215","DOI":"10.1007\/s00210-021-02052-y","article-title":"Phosphodiesterases 2, 3 and 4 can decrease cardiac effects of H2-histamine-receptor activation in isolated atria of transgenic mice","volume":"394","author":"Neumann","year":"2021","journal-title":"Naunyn-Schmiedeberg\u2019s Arch. Pharmacol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/j.ejphar.2005.01.056","article-title":"The phosphodiesterase 3 inhibitor cilostamide enhances inotropic responses to glucagon but not to dobutamine in rat ventricular myocardium","volume":"512","author":"Vargas","year":"2005","journal-title":"Eur. J. Pharmacol."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"6965","DOI":"10.1038\/ncomms7965","article-title":"In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease","volume":"6","author":"Sprenger","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"278","DOI":"10.1124\/pharmrev.120.000086","article-title":"Subcellular Organization of the cAMP Signaling Pathway","volume":"73","author":"Zaccolo","year":"2021","journal-title":"Pharmacol. Rev."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Merino, B., Quesada, I., and Hern\u00e1ndez-Cascales, J. (2015). Glucagon increases beating rate but not contractility in rat right atrium. Comparison with isoproterenol. PLoS ONE, 10.","DOI":"10.1371\/journal.pone.0132884"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1159\/000320454","article-title":"Inotropic effects of ivabradine in the mammalian heart","volume":"86","author":"Boldt","year":"2010","journal-title":"Pharmacology"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"247","DOI":"10.1085\/jgp.201010488","article-title":"Phosphorylation and modulation of hyperpolarization-activated HCN4 channels by protein kinase A in the mouse sinoatrial node","volume":"136","author":"Liao","year":"2010","journal-title":"J. Gen. Physiol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1124\/jpet.118.255711","article-title":"Initial characterization of transgenic mice overexpressing human histamine H2 receptors","volume":"369","author":"Gergs","year":"2019","journal-title":"J. Pharmacol. Exp. Ther."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"e220278","DOI":"10.1530\/JOE-22-0278","article-title":"Translational aspects of glucagon: Current use and future prospects","volume":"257","author":"Kaur","year":"2023","journal-title":"J. Endocrinol."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1016\/j.ejphar.2018.04.013","article-title":"Mechanism underlying the contractile activity of UTP in the mammalian heart","volume":"830","author":"Gergs","year":"2018","journal-title":"Eur. J. Pharmacol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"763","DOI":"10.1042\/bj3120763","article-title":"Cross-talk between glucagon- and adenosine-mediated signalling systems in rat hepatocytes: Effects on cyclic AMP-phosphodiesterase activity","volume":"312","author":"Allende","year":"1995","journal-title":"Biochem. J."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"199","DOI":"10.1007\/BF00499230","article-title":"Desensitization of kitten atria to chronotropic, inotropic and adenylyl cyclase stimulating effects of (-)isoprenaline","volume":"293","author":"Kaumann","year":"1976","journal-title":"Naunyn Schmiedebergs Arch. Pharmacol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1161\/01.RES.22.6.777","article-title":"Cardiac actions of glucagon","volume":"22","author":"Lucchesi","year":"1968","journal-title":"Circ. Res."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"855","DOI":"10.1016\/S0021-5198(19)32031-1","article-title":"Correlation of cyclic AMP and cyclic GMP levels with changes in contractile force of dog ventricular myocardium during cholinergic antagonism of positive inotropic actions of histamine; glucagon; theophylline and papaverine","volume":"29","author":"Endoh","year":"1979","journal-title":"Jpn. J. Pharmacol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"C155","DOI":"10.1152\/ajpcell.00315.2020","article-title":"Protective and anti-inflammatory effects of acetylcholine in the heart","volume":"320","author":"Prado","year":"2021","journal-title":"Am. J. Physiol. Cell Physiol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"488","DOI":"10.1210\/endo-117-2-488","article-title":"Effects of adenosine analogs on glucagon-stimulated adenosine 3\u2032 5\u2032-monophosphate formation in Sertoli cell cultures from immature rats","volume":"117","author":"Eikvar","year":"1985","journal-title":"Endocrinology"},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"68","DOI":"10.1038\/323068a0","article-title":"Activation of two signal-transduction systems in hepatocytes by glucagon","volume":"323","author":"Wakelam","year":"1986","journal-title":"Nature"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1042\/bj3040205","article-title":"Adenosine A1 receptors mediate chronic ethanol-induced increases in receptor-stimulated cyclic AMP in cultured hepatocytes","volume":"304","author":"Nagy","year":"1994","journal-title":"Biochem. J."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1413","DOI":"10.1016\/j.bbamem.2010.11.016","article-title":"Adenosine and its receptors in the heart: Regulation; retaliation and adaptation","volume":"1808","author":"Headrick","year":"2011","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1016\/0014-2999(82)90588-X","article-title":"Glucagon-induced densensitization: Correlation between cyclic AMP levels and contractile force","volume":"79","author":"Yao","year":"1982","journal-title":"Eur. J. Pharmacol."},{"key":"ref_50","doi-asserted-by":"crossref","unstructured":"Neumann, J., Schmidt, F., Hofmann, B., and Gergs, U. (2025). Glucagon can increase force of contraction via glucagon receptors in the isolated human atrium. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26020698"},{"key":"ref_51","doi-asserted-by":"crossref","unstructured":"Ye, J., Coulouris, G., Zaretskaya, I., Cutcutache, I., Rozen, S., and Madden, T.L. (2012). Primer-BLAST: A tool to design target-specific primers for polymerase chain reaction. BMC Bioinform., 13.","DOI":"10.1186\/1471-2105-13-134"},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1570","DOI":"10.1210\/en.2018-00004","article-title":"GLP\u22121 Receptor Expression Within the Human Heart","volume":"159","author":"Baggio","year":"2018","journal-title":"Endocrinology"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"e45","DOI":"10.1093\/nar\/29.9.e45","article-title":"A new mathematical model for relative quantification in real-time RT-PCR","volume":"29","author":"Pfaffl","year":"2001","journal-title":"Nucleic Acids Res."},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"17147","DOI":"10.1007\/s00210-025-04335-0","article-title":"Contractile effects of retatrutide in isolated mouse atrial preparations","volume":"398","author":"Neumann","year":"2025","journal-title":"Naunyn-Schmiedeberg\u2019s Arch. Pharmacol."},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Neumann, J., Ahlrep, U., Hofmann, B., and Gergs, U. (Naunyn-Schmiedeberg\u2019s Arch. Pharmacol., 2025). Inotropic effects of retatrutide in isolated human atrial preparations, Naunyn-Schmiedeberg\u2019s Arch. Pharmacol., online ahead of print.","DOI":"10.1007\/s00210-025-04421-3"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/1\/126\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,24]],"date-time":"2025-12-24T05:23:05Z","timestamp":1766553785000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/27\/1\/126"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,12,22]]},"references-count":55,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2026,1]]}},"alternative-id":["ijms27010126"],"URL":"https:\/\/doi.org\/10.3390\/ijms27010126","relation":{},"ISSN":["1422-0067"],"issn-type":[{"type":"electronic","value":"1422-0067"}],"subject":[],"published":{"date-parts":[[2025,12,22]]}}}