Proarrhythmia

QT interval

Type 2 diabetes

Endocrinology

Cardiology

Empagliflozin

Heart failure

Internal medicine

Medicine

Ejection fraction

Diabetes mellitus

Circulation

HomeCirculationVol. 145, No. 13Empagliflozin Reverses Late Na+ Current Enhancement and Cardiomyocyte Proarrhythmia in a Translational Murine Model of Heart Failure With Preserved Ejection Fraction Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBEmpagliflozin Reverses Late Na+ Current Enhancement and Cardiomyocyte Proarrhythmia in a Translational Murine Model of Heart Failure With Preserved Ejection Fraction Bence Hegyi, MD, PhD, Juliana Mira Hernandez, DVM, MSc, Erin Y. Shen, BSc, Nima R. Habibi, BSc, Julie Bossuyt, DVM, PhD and Donald M. Bers, PhD Bence HegyiBence Hegyi https://orcid.org/0000-0003-3113-221X Department of Pharmacology, University of California, Davis (B.H., J.M.H., E.Y.S., N.R.H., J.B., D.M.B.). Search for more papers by this author , Juliana Mira HernandezJuliana Mira Hernandez Department of Pharmacology, University of California, Davis (B.H., J.M.H., E.Y.S., N.R.H., J.B., D.M.B.). Research Group in Veterinary Medicine (GIVET), School of Veterinary Medicine, University Corporation Lasallista (Unilasallista), Caldas, Antioquia, Colombia (J.M.H.). Search for more papers by this author , Erin Y. ShenErin Y. Shen Department of Pharmacology, University of California, Davis (B.H., J.M.H., E.Y.S., N.R.H., J.B., D.M.B.). Search for more papers by this author , Nima R. HabibiNima R. Habibi Department of Pharmacology, University of California, Davis (B.H., J.M.H., E.Y.S., N.R.H., J.B., D.M.B.). Search for more papers by this author , Julie BossuytJulie Bossuyt Department of Pharmacology, University of California, Davis (B.H., J.M.H., E.Y.S., N.R.H., J.B., D.M.B.). Search for more papers by this author and Donald M. BersDonald M. Bers Correspondence to: Donald M. Bers, PhD, Department of Pharmacology, University of California, Davis, 451 Health Sciences Dr, Davis, CA 95616. Email E-mail Address: [email protected] https://orcid.org/0000-0002-2237-9483 Department of Pharmacology, University of California, Davis (B.H., J.M.H., E.Y.S., N.R.H., J.B., D.M.B.). Search for more papers by this author Originally published28 Mar 2022https://doi.org/10.1161/CIRCULATIONAHA.121.057237Circulation. 2022;145:1029–1031Empagliflozin, a sodium-glucose cotransporter-2 inhibitor, has been recently shown to improve cardiovascular outcomes in patients who have heart failure with preserved ejection fraction (HFpEF), independent of diabetes status.1 In addition to its advantageous renal and hemodynamic effects, empagliflozin can directly affect heart cells, and preclinical studies have demonstrated empagliflozin effects in ex vivo perfused hearts and isolated cardiomyocytes.2 However, the exact mechanism by which empagliflozin affects the heart remains elusive. In cardiomyocytes, sodium-glucose cotransporter-2 expression is virtually absent, suggesting off-target beneficial effects. Although controversial, the sodium-hydrogen exchanger-1 has been suggested as a potential empagliflozin target in cardiomyocytes, especially during ischemia. Philippaert et al3 recently showed that empagliflozin directly inhibited late Na+ current (INaL) in a transverse aortic constriction–induced heart failure murine model and in recombinant human NaV1.5 channels in HEK cells carrying long-QT3 mutations or treated with H2O2. Moreover, empagliflozin reduces reactive oxygen species production2 and Ca2+/calmodulin-dependent kinase II (CaMKII) activity,4 important known regulators of INaL in heart diseases. Here, we tested for empagliflozin effects on INaL and consequent action potential (AP) changes in a translational 2-hit murine model of HFpEF.5HFpEF was induced in C57BL/6J wild-type mice (male, 10-week-old, Jackson Laboratory) by combining high-fat diet (D12492, Research Diets) and inhibition of nitric oxide synthases with L-NG-nitroarginine methyl ester (L-NAME, 0.5 g/L, Sigma-Aldrich) in drinking water, whereas control mice were fed normal chow (5k52, LabDiet) and water. After 15 weeks of HFpEF treatment, consistent with the previous report,5 morphometric and echocardiographic evaluations (Figure [A]) revealed significant obesity, concentric cardiac hypertrophy, preserved ejection fraction, and significant diastolic dysfunction, as demonstrated by increased heart mass, left ventricular remodeling index, and elevated E/e′. Empagliflozin effects were studied in freshly isolated left ventricular myocytes using patch-clamp at 37 °C. In voltage-clamp INaL measurements, pipette solution contained (mmol/L): 110 CsCl, 20 tetraethylammonium chloride, 5 MgATP, 10 HEPES, 5 phosphocreatine-Na2, 0.0001 calmodulin, 10 EGTA, 4.1 CaCl2 (free [Ca2+]=100 nmol/L), pH=7.20. Bath solution contained (mmol/L): 140 NaCl, 4 CsCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, 5 Na-HEPES, 5.5 glucose, 5 4-aminopyridine, 0.01 nifedipine, pH=7.40.Download figureDownload PowerPointFigure. Empagliflozin reduces late Na+ current and action potential prolongation in a preclinical model of heart failure with preserved ejection fraction (HFpEF). Wild-type mice (10-week-old) on high-fat diet+L-NG-nitroarginine methyl ester for 15 weeks were compared with those fed normal chow (10 animals per group). A, Body weight, heart weight to tibial length ratio (HW/TL), and membrane capacitance (Cm) of single ventricular cardiomyocytes. Left ventricular (LV) ejection fraction (EF), ratio between mitral E wave and e′ wave (E/e′), and LV remodeling index LVRI (LV mass/LV end-diastolic internal diameter, LVM/LVIDd). B, Representative late Na+ current (INaL) traces in control and HFpEF myocytes after acute empagliflozin (EMPA) and subsequent tetrodotoxin (TTX) applications. Insets shows voltage protocol, comparison of peak vs late INa, and the time course of EMPA and TTX effects on INaL. Peak INa was off-scale as shown. C, Four-hour preincubation with empagliflozin reversed INaL upregulation in HFpEF. D, Action potential (AP) measurements in HFpEF myocytes paced at 1 Hz. Top left, Representative AP traces in control and HFpEF myocytes without or with preincubation with empagliflozin and autocamtide-2–related inhibitory peptide (AIP). Bottom left, Fifty consecutive AP durations at 90% repolarization (APD90) for control and HFpEF as above. Right, Average data on APD90 and short-term variability of APD90 (STV). E, Concentration-dependent acute effect of empagliflozin on H2O2 (100 μmol/L, 5 minutes)-induced INaL in wild-type and CaMKII MM281/282VV (4 animals per group). Data are presented as mean±SEM. Two-tailed Welch t test was used for A. ANOVA followed by the Tukey multiple-comparisons test was used for B, C, and D. All data, P values, and n numbers are shown. A/F indicates Ampere per Farad; CaMKII, Ca2+/calmodulin-dependent kinase II; IC50, half-maximal inhibitory concentration; and INaT, transient Na+ current.Acute empagliflozin treatment (1 μmol/L, 3 minutes), in contrast to a previous report,3 did not change INaL in control and HFpEF, whereas tetrodotoxin (10 μmol/L) readily inhibited INaL in both cases (Figure [B]). INaL density (current amplitude normalized to cell capacitance) was significantly increased in HFpEF. However, empagliflozin preincubation (4 hours) versus vehicle (0.01% dimethyl sulfoxide) reversed the tetrodotoxin-sensitive INaL upregulation in HFpEF to the control level but had no effects in control myocytes (Figure [C]).We also measured APs to assess antiarrhythmic properties of empagliflozin. For physiological AP measurements, Cs+ was replaced with K+ in pipette and bath solutions without K+ and Ca2+ current inhibitors. Pipette EGTA was reduced to 10 μmol/L and CaCl2 was omitted. In line with upregulated INaL, myocyte AP duration was significantly prolonged, and the short-term AP duration variability was markedly increased in HFpEF. These proarrhythmic AP duration changes were reversed in cells preincubated with empagliflozin (Figure [D]). Because CaMKII is an important regulator of INaL in heart diseases, we also tested the effect of cell pretreatment with the selective CaMKII inhibitor autocamtide-2–related inhibitory peptide (myristoylated, 1 μmol/L). CaMKII inhibition also reversed AP duration prolongation and temporal variability in HFpEF (Figure [D]).Last, we measured concentration-response effects of empagliflozin on reactive oxygen species–promoted INaL. Empagliflozin acutely inhibited the H2O2-induced INaL, but significantly only >3 µmol/L with a half-maximal inhibition at 5.68 μmol/L (Figure [E]). Critically, myocytes with oxidation-resistant mutations in CaMKII (MM281/282VV) prevented both the H2O2-induced increase in INaL and the empagliflozin effect (Figure [E]).Our data show that INaL is elevated in a preclinical murine model of HFpEF and that empagliflozin at a clinically relevant 1 μmol/L concentration reverts INaL upregulation and arrhythmogenic AP changes. However, this empagliflozin effect on the Na+ channel in HFpEF is not acute inhibition (unlike the direct acute selective INaL blocker tetrodotoxin) but requires drug preincubation, and this effect is mimicked by CaMKII inhibition and absent when CaMKII lacks 2 critical oxidation sites. Moreover, empagliflozin acutely inhibits H2O2-induced INaL with a 7-times higher half-maximal inhibition in cardiomyocytes than in HEK cells3 (5.68 μmol/L versus 0.79 μmol/L). Thus, we suggest that empagliflozin likely regulates INaL in HFpEF by an indirect mechanism that may involve CaMKII. That agrees with results that empagliflozin treatment for 24 hours (but not 30 minutes) suppresses CaMKII activity and sarcoplasmic reticulum Ca2+ leak, and enhanced Ca2+ transients.4 Mechanisms could include reduced reactive oxygen species and its ability to activate CaMKII. Although the key molecular targets of empagliflozin in cardiomyocytes remain unclear, INaL and CaMKII suppression may be important mediators of its beneficial effects that merit further investigation. Our study is limited in that we studied empagliflozin effects in isolated murine myocytes and focused on cell electrophysiology. Further studies are required to confirm these findings in human cardiomyocytes and on other phenotypic aspects of HFpEF.All procedures involving animals were approved by institutional and national authorities. All supporting data are available within the article.Article InformationAcknowledgmentsThe authors thank M. E. Anderson (Johns Hopkins University) for providing CaMKIIδ-MM281/282VV knock-in mice, and D. Smoliarchuk, A. Mandel, E. Spencer, and A. Wilder for their help in animal care, cell isolation, and laboratory tasks.Sources of FundingThis work was supported by grants from the National Institutes of Health: P01-HL141084 (Dr Bers) and R01-HL142282 (Drs Bers and Bossuyt), and Minciencias–Fulbright Colombia Scholarship (Dr Mira Hernandez).Nonstandard Abbreviations and AcronymsAPaction potentialCaMKIICa2+/calmodulin-dependent kinase IIHFpEFheart failure with preserved ejection fractionDisclosures None.FootnotesFor Sources of Funding and Disclosures, see page 1031.https://www.ahajournals.org/journal/circCorrespondence to: Donald M. Bers, PhD, Department of Pharmacology, University of California, Davis, 451 Health Sciences Dr, Davis, CA 95616. Email [email protected]eduReferences1. Anker SD, Butler J, Filippatos G, Ferreira JP, Bocchi E, Böhm M, Brunner-La Rocca HP, Choi DJ, Chopra V, et al.; EMPEROR-Preserved Trial Investigators. Empagliflozin in heart failure with a preserved ejection fraction.N Engl J Med. 2021; 385:1451–1461. doi: 10.1056/NEJMoa2107038CrossrefMedlineGoogle Scholar2. Cowie MR, Fisher M. SGLT2 inhibitors: mechanisms of cardiovascular benefit beyond glycaemic control.Nat Rev Cardiol. 2020; 17:761–772. doi: 10.1038/s41569-020-0406-8CrossrefMedlineGoogle Scholar3. Philippaert K, Kalyaanamoorthy S, Fatehi M, Long W, Soni S, Byrne NJ, Barr A, Singh J, Wong J, Palechuk T, et al.. Cardiac late sodium channel current is a molecular target for the sodium/glucose cotransporter 2 inhibitor empagliflozin.Circulation. 2021; 143:2188–2204. doi: 10.1161/CIRCULATIONAHA.121.053350LinkGoogle Scholar4. Mustroph J, Wagemann O, Lücht CM, Trum M, Hammer KP, Sag CM, Lebek S, Tarnowski D, Reinders J, Perbellini F, et al.. Empagliflozin reduces Ca/calmodulin-dependent kinase II activity in isolated ventricular cardiomyocytes.ESC Heart Fail. 2018; 5:642–648. doi: 10.1002/ehf2.12336CrossrefMedlineGoogle Scholar5. Schiattarella GG, Altamirano F, Tong D, French KM, Villalobos E, Kim SY, Luo X, Jiang N, May HI, Wang ZV, et al.. Nitrosative stress drives heart failure with preserved ejection fraction.Nature. 2019; 568:351–356. doi: 10.1038/s41586-019-1100-zCrossrefMedlineGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetails March 29, 2022Vol 145, Issue 13 Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.121.057237PMID: 35344407 Originally publishedMarch 28, 2022 Keywordsarrhythmia, cardiacheart failure, diastolicempagliflozinsodium-glucose transporter 2 inhibitorssodium channelsPDF download Advertisement SubjectsAnimal Models of Human DiseaseBasic Science ResearchElectrophysiologyHeart FailureIon Channels/Membrane Transport

Empagliflozin Reverses Late Na <sup>+</sup> Current Enhancement and Cardiomyocyte Proarrhythmia in a Translational Murine Model of Heart Failure With Preserved Ejection Fraction

Empagliflozin Reverses Late Na + Current Enhancement and Cardiomyocyte Proarrhythmia in a Translational Murine Model of Heart Failure With Preserved Ejection Fraction

Empagliflozin Reverses Late Na ⁺ Current Enhancement and Cardiomyocyte Proarrhythmia in a Translational Murine Model of Heart Failure With Preserved Ejection Fraction