A Mouse Model of Atrial Fibrillation in Sepsis

HomeCirculationVol. 147, No. 13A Mouse Model of Atrial Fibrillation in Sepsis Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBA Mouse Model of Atrial Fibrillation in Sepsis Aneesh Bapat, Maximilian J. Schloss, Masahiro Yamazoe, Jana Grune, Maarten Hulsmans, David J. Milan, Matthias Nahrendorf and Patrick T. Ellinor Aneesh BapatAneesh Bapat https://orcid.org/0000-0002-1996-522X Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, Boston (A.B., P.T.E.). Demoulas Center for Cardiac Arrhythmias (A.B., P.T.E.), Massachusetts General Hospital, Boston. , Maximilian J. SchlossMaximilian J. Schloss Department of Radiology (M.J.S., M.Y., M.H., M.N.), Massachusetts General Hospital, Boston. Center for Systems Biology, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston (M.J.S., M.Y., J.G., M.H., M.N.). Division of Cardiac Surgery, Brigham and Women's Hospital, Harvard Medical School, Boston, MA (M.J.S.). , Masahiro YamazoeMasahiro Yamazoe Department of Radiology (M.J.S., M.Y., M.H., M.N.), Massachusetts General Hospital, Boston. Center for Systems Biology, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston (M.J.S., M.Y., J.G., M.H., M.N.). , Jana GruneJana Grune https://orcid.org/0000-0001-6209-7416 Center for Systems Biology, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston (M.J.S., M.Y., J.G., M.H., M.N.). German Centre for Cardiovascular Research, Berlin (J.G.). Institute of Physiology, Charité-Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Germany (J.G.). Department of Cardiothoracic and Vascular Surgery, German Heart Center Charité, Berlin (J.G.). , Maarten HulsmansMaarten Hulsmans https://orcid.org/0000-0003-1009-658X Department of Radiology (M.J.S., M.Y., M.H., M.N.), Massachusetts General Hospital, Boston. Center for Systems Biology, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston (M.J.S., M.Y., J.G., M.H., M.N.). , David J. MilanDavid J. Milan https://orcid.org/0000-0003-0149-3110 Leducq Foundation, Boston, MA (D.J.M.). , Matthias NahrendorfMatthias Nahrendorf https://orcid.org/0000-0002-4021-1887 Department of Radiology (M.J.S., M.Y., M.H., M.N.), Massachusetts General Hospital, Boston. Center for Systems Biology, Massachusetts General Hospital Research Institute and Harvard Medical School, Boston (M.J.S., M.Y., J.G., M.H., M.N.). Department of Internal Medicine I, University Hospital Wuerzburg, Germany (M.N.). and Patrick T. EllinorPatrick T. Ellinor Correspondence to: Patrick T. Ellinor, MD, PhD, Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114. Email E-mail Address: [email protected] https://orcid.org/0000-0002-2067-0533 Cardiovascular Research Center, Massachusetts General Hospital and Harvard Medical School, Boston (A.B., P.T.E.). Demoulas Center for Cardiac Arrhythmias (A.B., P.T.E.), Massachusetts General Hospital, Boston. Cardiovascular Disease Initiative, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge (P.T.E.). Originally published27 Mar 2023https://doi.org/10.1161/CIRCULATIONAHA.122.060317Circulation. 2023;147:1047–1049Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia, with associated morbidity and mortality including stroke, dementia, congestive heart failure, and death. Despite the burden of disease, therapeutic advances are lagging. Animal models present an opportunity to gain better insight into the mechanistic basis for AF. Mouse models are attractive because of low cost, accessibility, and ease of genetic manipulation. However, their role in the study of AF has been limited because mice typically do not develop spontaneous AF, and most require time-dependent cardiac dysfunction and remodeling.1 Here, we present a model of sepsis-related atrial fibrillation in wild-type mice with a characteristic electrophysiologic phenotype within 1 week of insult.Studies were approved by the Institutional Animal Care and Use Committee at Massachusetts General Hospital. Cecal ligation and puncture (CLP), a commonly used animal model of polymicrobial sepsis, can be easily performed in mice.2 We subjected 10- to 20-week-old male C57BL/6 mice to either mild CLP or sham operation; a separate batch of mice was used as naive controls (Figure [A]). After 1 week, ~89% of the CLP-treated mice were alive, compared with 100% of controls. There were no significant differences in tail-cuff blood pressure, or cardiac structure/function determined by transthoracic echocardiography.Download figureDownload PowerPointFigure. Cecal ligation and puncture produces a mouse model of atrial fibrillation. A, C57BL/6 mice underwent mild cecal ligation and punction (CLP) or sham operation and were studied 7 days later. Naive C57BL/6 mice were used as additional controls. B, Representative surface ECG and right atrial intracardiac electrogram acquired during an invasive EP study and showing pacing-induced AF. C, Left, Percentage of mice with an episode of atrial fibrillation (AF) longer than 5 s. Two-sided Fisher exact test. Middle, Number of AF episodes longer than 1 s during the entirety of each EP study. One-way ANOVA, post hoc Sidak test. Right, AF burden assessed per mouse during the entire EP study. One-way ANOVA, post hoc Sidak test. D, Left, Representative action potential tracings derived from optical mapping in the right (upper) and left (lower) atrium 7 days after CLP. Right, Quantification of the action potential duration (APD) at 50%, 70%, and 90% repolarization; obtained at a pacing cycle length of 100 ms. One-way ANOVA, post hoc Sidak test. E, Action potential upstroke velocity (dV/dt) in the right (upper) and left (lower) atrium; obtained at a pacing cycle length of 100 ms. One-way ANOVA, post hoc Sidak test. F, Optical mapping–derived representative activation maps from right (upper) and left (lower) atria of mice with quantification of conduction velocity to the right; obtained at a pacing cycle length of 100 ms. One-way ANOVA, post hoc Sidak test. G, Expression of atrial connexin genes measured by qPCR. One-way ANOVA, post hoc Sidak test. H, Representative blots of connexin 40 (left) and connexin 43 (right), with quantification below. One-way ANOVA, post hoc Sidak test. I, Atrial expression of ion channel genes measured by qPCR. One-way ANOVA, post hoc Sidak test. J, Representative blots of NaV1.5 sodium channel subunit with quantification shown below. One-way ANOVA, post hoc Sidak test. K, Representative blots of phosphorylated CaMKII (calcium/calmodulin-dependent protein kinase II) with quantification shown below. One-way ANOVA, post hoc Sidak test. L, C57BL/6 mice underwent CLP and then were treated with either vehicle or a CaMKII inhibitor, KN-93, every other day (total of 4 doses) until study (left upper). Left lower, Representative blot showing decreased CaMKII phosphorylation in KN-93–treated mice. Right, Percentage of mice with an episode of AF longer than 5 s in vehicle-treated or KN-93–treated mice. Two-sided Fisher exact test. M, Left, Flow cytometry plots of the left atrium. Right, Quantification of leukocytes, endothelial cells, and fibroblasts in the left atrium. Up to 2 independent experiments. Two-tailed Student t test. N, Left, Representative blots of protein expression in the atria. Right, Quantification of relative protein expression in the atria. Two-tailed Student t test. Data are mean ± SEM with individual values for data distribution, *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. EPS indicates electrophysiology study; OM, optical mapping; qPCR, quatitative polymerase chain reaction; and WB, Western blotting.One week after CLP, invasive electrophysiologic studies were performed on the 3 groups of mice to determine electrophysiologic characteristics and arrhythmia inducibility. Inducible AF was defined by rapid atrial cycle length and concomitant irregular R-R intervals for >5 s in duration (Figure [B]). There were no significant differences in baseline electrophysiologic parameters except for a higher HR in CLP-treated mice compared with controls. CLP-treated mice had an increased incidence (Figure [C], left), frequency (Figure [C], middle), and burden (Figure [C], right) of AF compared with the controls. Optical mapping revealed a CLP-related increase in action potential duration (APD) in both atrial chambers (Figure [D]) as well as a trend toward slowed action potential upstroke velocity (Figure [E]). We next investigated the effects of sepsis on impulse propagation and found a CLP-induced slowing in atrial conduction velocity (Figure [F]).We used quantitative polymerase chain reaction and Western blotting to explore possible differences in atrial gene/protein expression that may underlie the CLP-related phenotype. Because connexin expression is critical to impulse propagation, we were interested in finding CLP-related decreases in mRNA encoding Cx40 and Cx43, as well as the protein expression of connexin 40 (Cx40; Figure [G and H]). We identified a CLP-related decrease in atrial expression of several genes that encode ion channel subunits responsible for the cardiac action potential (Figure [I]). Considering the effects on conduction velocity and action potential upstroke velocity, we noted a CLP-induced decrease in the protein expression of the voltage-gated sodium channel, NaV1.5 (Figure [J]).CaMKII (calcium/calmodulin-dependent protein kinase II) is integral to calcium homeostasis and can centrally affect action potential duration through its effects on multiple ion channels. We were interested to find a CLP-induced increase in phosphorylation of CaMKII (Figure [K]). To investigate the role of CaMKII further, we treated mice with CLP followed by intraperitoneal injections of either a CaMKII inhibitor, KN-93, or vehicle. KN-93 treatment significantly reduced AF inducibility in CLP-treated mice, suggesting that CaMKII activation is necessary for sepsis-related atrial arrhythmogenesis (Figure [L]).Because systemic inflammation is evident during sepsis, we investigated local leukocyte recruitment to the left atrium. Flow cytometry of CLP- and sham-treated left atria revealed a significant increase in inflammatory leukocytes and fibroblasts (Figure [M]). Inflammasome activity has been posited as involved in AF pathogenesis, so we assayed the components of the NLRP3 inflammasome by Western blotting. CLP significantly increased atrial expression of inflammasome components procaspase 1, cleaved caspase p20, and apoptosis-associated speck-like protein containing a CARD without a change in expression of NLRP3 itself (Figure [N]). In addition, CLP resulted in increased expression of the phosphorylated form of the NF-kB (nuclear factor kB) p65 subunit, consistent with transactivation of this pathway. These data demonstrate an atrial inflammatory milieu, as would be expected in the setting of sepsis.Here, we introduce a mouse model of sepsis-related AF caused by CLP and mediated by altered atrial electrophysiology associated with dysregulated expression of conduction-related genes and atrial inflammation. AF is the most commonly encountered arrhythmia in the setting of severe sepsis, which increases the odds of new-onset AF by nearly ~7-fold.3 Sepsis is independently associated with QT prolongation,4 and our data suggest that a similar effect on repolarization reserve may underlie atrial proarrhythmia. It is interesting that CLP-treated mice demonstrate an atrial inflammatory infiltrate resulting in both "priming" and "triggering" of the NLRP3 inflammasome, which is associated with AF.5 Commonly used mouse models of AF require transgenic mice or extensive remodeling from a chronic exposure; these would increase the cost and time required for use. Thus, this clinically relevant model provides an opportunity to perform high-throughput experimentation to better understand the role of inflammation in AF pathogenesis. The data supporting this study's findings are available from the corresponding author on reasonable request, according to Transparency and Openness Promotion guidelines.Article InformationAcknowledgmentsThe authors acknowledge Servier Medical Art (https://smart.servier.com) for cartoon components. A.B. and M.J.S. conceived the study, and designed, performed, and analyzed experiments. A.B. and M.J.S. interpreted data and created the figures. M.Y., J.G., and M.H. performed and analyzed experiments. M.N. and D.J.M. discussed results and strategy. P.T.E. provided funding and discussed results and strategy. A.B. and M.J.S. wrote the article with input from all authors.Sources of FundingThis work was funded in part by US federal funds from the National Institutes of Health (T32HL076136, 1RO1HL092577, 1R01HL157635, 5R01HL139731). A.B. was supported by National Institutes of Health grant T32HL007604. M.J.S. was funded by Deutsche Forschungsgemeinschaft (SCHL 2221/1-1). J.G. was supported by funding from the German Research Foundation (GR 5261/1-1, SFB-1470-A04), German Society for Cardiology, German Center for Cardiovascular Research, and Corona-Stiftung. M.H. was supported by an American Heart Association Career Development Award (19CDA34490005) and National Institutes of Health grant HL155097. M.N. was supported by National Institutes of Health grant HL139598. P.T.E. was supported by a grant from the American Heart Association Strategically Focused Research Networks (18SFRN34110082) and by a grant from the European Union (MAESTRIA 965286).Nonstandard Abbreviations and AcronymsAFatrial fibrillationCaMKIIcalcium/calmodulin-dependent protein kinase IICLPcecal ligation and punctureDisclosures M.N. has received funds or material research support from Alnylam, Biotronik, CSL Behring, GlycoMimetics, GSK, Medtronic, Novartis and Pfizer, as well as consulting fees from Biogen, Gimv, IFM Therapeutics, Molecular Imaging, Sigilon, and Verseau Therapeutics. P.T.E has received sponsored research support from Bayer AG, IBM Research, Bristol Myers Squibb, and Pfizer; he has also served on advisory boards or consulted for Bayer AG, MyoKardia, and Novartis. The other authors report no conflicts.Footnotes*A. Bapat and M.J. Schloss contributed equally.For Sources of Funding and Disclosures, see page 1049.Circulation is available at www.ahajournals.org/journal/circCorrespondence to: Patrick T. Ellinor, MD, PhD, Cardiovascular Research Center, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114. Email ellinor@mgh.harvard.eduReferences1. Schüttler D, Bapat A, Kääb S, Lee K, Tomsits P, Clauss S, Hucker WJ. Animal models of atrial fibrillation.Circ Res. 2020; 127:91–110. doi: 10.1161/CIRCRESAHA.120.316366LinkGoogle Scholar2. Rittirsch D, Huber-Lang MS, Flierl MA, Ward PA. Immunodesign of experimental sepsis by cecal ligation and puncture.Nat Protocols. 2009; 4:31–36. doi: 10.1038/nprot.2008.214CrossrefMedlineGoogle Scholar3. Walkey AJ, Wiener RS, Ghobrial JM, Curtis LH, Benjamin EJ. Incident stroke and mortality associated with new-onset atrial fibrillation in patients hospitalized with severe sepsis.JAMA. 2011; 306:2248–2255. doi: 10.1001/jama.2011.1615CrossrefMedlineGoogle Scholar4. Tisdale JE, Jaynes HA, Kingery JR, Mourad NA, Trujillo TN, Overholser BR, Kovacs RJ. Development and validation of a risk score to predict QT interval prolongation in hospitalized patients.Circ Cardiovasc Qual Outcomes. 2013; 6:479–487. doi: 10.1161/CIRCOUTCOMES.113.000152LinkGoogle Scholar5. Yao C, Veleva T, Scott L, Cao S, Luge L, Chen G, Jeyabal P, Pan X, Alsina KM, Abu-Taha I, et al. Enhanced cardiomyocyte NLRP3 inflammasome signaling promotes atrial fibrillation.Circulation. 2018; 138:2227–2242. doi: 10.1161/CIRCULATIONAHA.118.035202LinkGoogle Scholar eLetters(0)eLetters should relate to an article recently published in the journal and are not a forum for providing unpublished data. Comments are reviewed for appropriate use of tone and language. Comments are not peer-reviewed. Acceptable comments are posted to the journal website only. Comments are not published in an issue and are not indexed in PubMed. Comments should be no longer than 500 words and will only be posted online. References are limited to 10. Authors of the article cited in the comment will be invited to reply, as appropriate.Comments and feedback on AHA/ASA Scientific Statements and Guidelines should be directed to the AHA/ASA Manuscript Oversight Committee via its Correspondence page.Sign In to Submit a Response to This Article Previous Back to top Next FiguresReferencesRelatedDetailsCited ByLin A, Bapat A, Xiao L, Niroula A, Ye J, Wong W, Agrawal M, Farady C, Boettcher A, Hergott C, McConkey M, Flores-Bringas P, Shkolnik V, Bick A, Milan D, Natarajan P, Libby P, Ellinor P and Ebert B (2024) Clonal Hematopoiesis of Indeterminate Potential With Loss of Tet2 Enhances Risk for Atrial Fibrillation Through Nlrp3 Inflammasome Activation, Circulation, 149:18, (1419-1434), Online publication date: 30-Apr-2024. Hegemann N, Barth L, Döring Y, Voigt N and Grune J (2024) Implications for neutrophils in cardiac arrhythmias, American Journal of Physiology-Heart and Circulatory Physiology, 10.1152/ajpheart.00590.2023, 326:3, (H441-H458), Online publication date: 1-Mar-2024. Zhang M, Gyberg D, Healy C, Zhang N, Liu H, Dudley S and O'Connell T (2023) Atrial Myopathy Quantified by Speckle-tracking Echocardiography in Mice, Circulation: Cardiovascular Imaging, 16:10, (e015735), Online publication date: 1-Oct-2023. March 28, 2023Vol 147, Issue 13 Advertisement Article InformationMetrics © 2023 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.122.060317PMID: 36972346 Originally publishedMarch 27, 2023 Keywordsanimal disease modelsatrial fibrillationcardiac arrhythmiassepsisPDF download Advertisement SubjectsAnimal Models of Human DiseaseArrhythmiasAtrial FibrillationElectrophysiologyInflammation