Impaired AMP-Activated Protein Kinase Signaling in Heart Failure With Preserved Ejection Fraction–Associated Atrial Fibrillation

HomeCirculationVol. 146, No. 1Impaired AMP-Activated Protein Kinase Signaling in Heart Failure With Preserved Ejection Fraction–Associated Atrial Fibrillation Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBImpaired AMP-Activated Protein Kinase Signaling in Heart Failure With Preserved Ejection Fraction–Associated Atrial Fibrillation Dan Tong, Gabriele G. Schiattarella, Nan Jiang, Daniel Daou, Yuxuan Luo, Mark S. Link, Sergio Lavandero, Thomas G. Gillette and Joseph A. Hill Dan TongDan Tong Correspondence to: Dan Tong, MD, PhD, Division of Cardiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, NB11.200, Dallas, TX 75390-8573. Email E-mail Address: [email protected] https://orcid.org/0000-0002-3551-2861 Department of Internal Medicine (Cardiology) (D.T., N.J., D.D., Y.L., M.L., S.L., J.A.H.), University of Texas Southwestern Medical Center, Dallas. Search for more papers by this author , Gabriele G. SchiattarellaGabriele G. Schiattarella https://orcid.org/0000-0002-7582-7171 Center for Cardiovascular Research (CCR), Department of Cardiology, Charité - Universitätsmedizin Berlin, Germany (G.G.S.). DZHK (German Center for Cardiovascular Research), Partner Site Berlin, Germany (G.G.S.). Translational Approaches in Heart Failure and Cardiometabolic Disease, Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin, Germany (G.G.S.). Division of Cardiology, Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy (G.G.S.). Search for more papers by this author , Nan JiangNan Jiang Department of Internal Medicine (Cardiology) (D.T., N.J., D.D., Y.L., M.L., S.L., J.A.H.), University of Texas Southwestern Medical Center, Dallas. Search for more papers by this author , Daniel DaouDaniel Daou https://orcid.org/0000-0002-9912-6855 Department of Internal Medicine (Cardiology) (D.T., N.J., D.D., Y.L., M.L., S.L., J.A.H.), University of Texas Southwestern Medical Center, Dallas. Search for more papers by this author , Yuxuan LuoYuxuan Luo Department of Internal Medicine (Cardiology) (D.T., N.J., D.D., Y.L., M.L., S.L., J.A.H.), University of Texas Southwestern Medical Center, Dallas. Search for more papers by this author , Mark S. LinkMark S. Link https://orcid.org/0000-0001-5277-9296 Department of Internal Medicine (Cardiology) (D.T., N.J., D.D., Y.L., M.L., S.L., J.A.H.), University of Texas Southwestern Medical Center, Dallas. Search for more papers by this author , Sergio LavanderoSergio Lavandero https://orcid.org/0000-0003-4258-1483 Advanced Center for Chronic Diseases (ACCDiS) & Center for Molecular Studies of the Cell (CEMC), Faculty of Chemical & Pharmaceutical Sciences & Faculty of Medicine, University of Chile, Santiago (S.L.). Search for more papers by this author , Thomas G. GilletteThomas G. Gillette https://orcid.org/0000-0002-7617-3544 Department of Internal Medicine (Cardiology) (D.T., N.J., D.D., Y.L., M.L., S.L., J.A.H.), University of Texas Southwestern Medical Center, Dallas. Search for more papers by this author and Joseph A. HillJoseph A. Hill Correspondence to: Joseph A. Hill, MD, PhD, Division of Cardiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, NB11.200, Dallas, TX 75390-8573. Email E-mail Address: [email protected] https://orcid.org/0000-0002-5379-1614 Department of Internal Medicine (Cardiology) (D.T., N.J., D.D., Y.L., M.L., S.L., J.A.H.), University of Texas Southwestern Medical Center, Dallas. Department of Molecular Biology (J.A.H.), University of Texas Southwestern Medical Center, Dallas. Search for more papers by this author Originally published5 Jul 2022https://doi.org/10.1161/CIRCULATIONAHA.121.058301Circulation. 2022;146:73–76In the United States, only 2 major cardiovascular disorders are increasing in prevalence: heart failure (HF) and atrial fibrillation (AF), representing 2 enormous public health challenges.1 Heart failure with preserved ejection fraction (HFpEF) accounts for ≈50% of all patients with HF and is associated with significant morbidity, mortality, and health care expenditures.1 Patients with HFpEF are uniquely predisposed to AF; approximately two-thirds of patients with HFpEF will experience AF during the course of their disease.2 Furthermore, the presence of AF is a strong and independent predictor of poor functional status, increased risk for HF hospitalization, and death.2 Thus, the convergence of these 2 disorders, each of which exacerbates the other, represents a prevalent and unique clinical challenge. To date, there are limited proven effective medical therapies for HFpEF, and little is known regarding the underlying molecular mechanisms.2We recently developed a murine model of HFpEF in which concomitant metabolic and hypertensive stress in male mice elicited by a high-fat diet coupled with inhibition of constitutive nitric oxide synthases (neuronal nitric oxide synthase, endothelial nitric oxide synthase) with Nω-nitro-l-arginine methyl ester recapitulates the numerous and myriad features of human HFpEF.3 Here, we report that these HFpEF mice are also highly susceptible to AF. After transesophageal pacing, HFpEF mice (treated with high-fat diet + Nω-nitro-l-arginine methyl ester 5–7 weeks) developed AF (Figure [A]) much more frequently (80.0%) than control mice (mice fed with chow 27.8%, high-fat diet alone 25%, or Nω-nitro-l-arginine methyl ester alone 12.5%; Figure [B]), and the AF episodes lasted significantly longer (Figure [C]).Download figureDownload PowerPointFigure. Impaired AMPK signaling in HFpEF-associated AF. All experiments involving animals were approved by the Institutional Animal Care and Use Committee of the University of Texas Southwestern Medical Center, Dallas, Texas. Statistical analysis was performed using GraphPad Prism v9. Results are presented as mean±SD. Data normality was examined using the Shapiro-Wilk test (with α=0.05). For normally distributed data, the Student t test (unpaired 2-tailed) was used for 2-group analysis, and 1-way ANOVA with Tukey multiple comparisons was used for multiple-group analysis. For nonnormally distributed data, the Mann-Whitney test was used for 2-group analysis, and the Kruskal-Wallis test with Dunn multiple comparisons test was used for multiple-group analysis. No statistical analyses were performed to predetermine sample sizes; estimates were made on the basis of our previous experience, experimental approach, and availability and feasibility required to obtain statistically significant results. When representative images are shown, the selected images are those that most accurately represent the average data obtained in all samples. A, Representative surface ECG recording showing induction of AF in HFpEF mice (bottom traces), whereas control (Chow) mice (upper traces) remain in sinus rhythm. Traces are sequentially amplified from left to right to show more detail. Clear, regular P waves (arrowhead) were persistently seen in Chow mice. Loss of P waves and irregularly irregular QRS waves were observed in HFpEF mice, suggesting AF. Scale bar, 0.2 second. B, Percentage of successful induction of sustained AF (>2 s) after each burst pacing episode. N value on bar graph indicates number of burst pacing stimuli delivered per number of mice. Each mouse received 4 stimuli (25 Hz twice, followed by 40 Hz twice; each stimulus lasts 2 seconds, with 2-minute interval in between): Chow (n=4), HFpEF (HFD+L-NAME; n=4), HFD only (n=2), and L-NAME only (n=2). C, Duration of all induced AF episodes. Nonparametric Kruskal-Wallis analysis followed by Dunn multiple comparisons. D, Representative echocardiographic images showing enlarged left atria (LA) in HFpEF mice compared with mice fed with chow. Scale bar, 2 mm. E, Quantification of LA area (LAA) in HFpEF (n=10) vs Chow mice (n=6). Mann-Whitney test. F, Quantification of LA fraction area change (FAC) in HFpEF (n=9) vs Chow mice (n=6). Unpaired t test. FAC=(LAAmax – LAAmin)/LAAmax×100%. G, Representative images of Mason trichrome staining (Top) and Picrosirius red staining (Middle) of LA tissue from Chow and HFpEF mice. Bottom, Mason trichrome staining of left ventricular tissue. Scale bar, 250 μm. H, Quantification of fibrosis signal in LA tissue of HFpEF (n=4) vs Chow mice (n=4) by Picrosirius red staining. Data were normalized to the average of Chow group. Unpaired t test. I, Quantification of mRNA expression of collagen I (ColI), transforming growth factor b (Tgfb), α-smooth muscle actin (αSMA) in LA tissue of Chow (n=4) and HFpEF mice (n=6). The delta-delta Ct (2 ΔΔCT) relative quantification method, using 18S for normalization, was used to estimate the amount of target mRNA in samples, and fold ratios were calculated relative to mRNA levels from control samples. J and K, Representative Western blot and quantification of Cx43 and Cx40 levels in LA tissue of Chow (n=5) and HFpEF mice (n=6) normalized to GAPDH. Data were normalized to the average of Chow group. Unpaired t test. L, Representative immunofluorescence staining of Cx40 (red, Top) and Cx43 (red, Bottom) in LA tissue of Chow and HFpEF mice. Slides were counterstained with α-actinin (green). Representative areas (dotted square) were amplified in the images on the right side. Scale bar, 20 μm. Arrowheads show abnormal intracellular distribution. M, Representative Western blot and quantification of phosphorylated S6 (pS6), S6, phosphorylated 4EBP1(p4EBP1), and 4EBP1 (eIF4E-binding protein) in LA tissue of Chow and HFpEF mice. N, Quantification of pS6/S6 in LA of Chow (n=4) and HFpEF mice (n=3). O, Quantification of p4EBP1/4EBP1 in LA of Chow (n=5) and HFpEF mice (n=6). Data were normalized to the average of the chow group. Unpaired t test. P, Quantification of mRNA abundance of TNFα (tumor necrosis factor α), IL1β (interleukin 1β), and IL6 (interleukin 6) in LA tissue of Chow (n=4) and HFpEF mice (n=8). Unpaired t test. Q and R, Representative Western blot and quantification of phosphorylated AMPKα (Thr172), total AMPKα, phosphorylated acetyl-CoA carboxylase (ACC), total ACC in LA of Chow (n=9) and HFpEF mice (n=9). Normalized to GAPDH. Data were normalized to the average of Chow group. Unpaired t test. S, Experimental design. T and U, Representative Western blot and quantification of phosphorylated AMPKα (Thr172) and total AMPKα in LA of Chow (n=10), HFpEF mice treated with placebo (Pbo; n=8), and metformin (Met; n=5), V, Percentage of successful induction of sustained AF (>2 s) after each burst pacing. N value on bar graph indicates number of burst pacing stimuli (4 stimuli per mouse) delivered per number of mice in Chow mice treated with metformin (Met; n=3), HFpEF mice treated with placebo (Pbo; n=2) and with metformin (Met; n=6). W, Duration of all induced AF episodes. Nonparametric Kruskal-Wallis analysis followed by Dunn multiple comparisons. The data that support the findings of this study are available from the corresponding author on reasonable request. AF indicates atrial fibrillation; AMPK, AMP-activated protein kinase; Cx40, connexin40; Cx43, connexin43; HFD, high-fat diet; HFpEF, heart failure with preserved ejection fraction; and L-NAME, Nω-nitro-l-arginine methyl ester.Echocardiographic evaluation revealed that, in addition to left ventricular hypertrophy and diastolic dysfunction as reported previously,3 significant left atrial (LA) enlargement (Figure [D and E]), another hallmark of human HFpEF, was observed in the HFpEF mice. LA contractility was impaired as demonstrated by reduced fractional LA area changes (Figure [F]). Atrial fibrosis is frequently observed in various AF models and is considered a critical contributor to arrhythmia pathogenesis. However, at this time point (5–7 weeks of treatment), despite full HFpEF presentation and increased AF inducibility, we did not observe significantly increased LA tissue fibrosis by either Masson trichrome or Picrosirius red staining (Figure [G and H, top and middle]). This was confirmed by quantitative reverse transcription polymerase chain reaction to evaluate transcript levels of major fibrosis markers (Figure [I]). In contrast, we did observe moderately increased patchy fibrosis in the left ventricle of HFpEF mice (Figure [G, bottom]) as reported previously.3 These data suggest that LA fibrosis is unlikely to be a major contributor to AF predisposition in this HFpEF model.Gap junction–mediated intercellular communication is essential for proper electrical signal conduction in the heart. We observed significant downregulation of Cx43 (connexin43) and Cx40 (connexin40) protein levels, 2 major gap junction proteins in the atria (Figure [J and K]). Immunofluorescence staining revealed heterogeneous distribution with increased intracellular signals in HFpEF LAs (Figure [L]), suggesting possible aberrant signal conduction in the atria. Consistent with LA enlargement and hypertrophy, Western blot analysis revealed activation of mTOR (mechanistic target of rapamycin) as demonstrated by increases in phosphorylated S6 ribosomal protein and 4EBP1 (eIF4E-binding protein; Figure [M–O]). Furthermore, markers of inflammation, particularly interleukin 6 (IL6; Figure [P]), were elevated in the HFpEF LA, suggesting that metabolic inflammation plays a role in the atrial remodeling observed in HFpEF hearts.Previous studies have revealed an essential role of LKB1-AMPK (liver kinase B1-AMP-activated protein kinase) signaling in maintaining normal atrial structure and function, because cardiomyocyte-specific LKB1 or AMPKβ1/β2 knockout mice develop dramatic LA remodeling and spontaneous AF.4,5 LA tissue from HFpEF mice revealed a significant decrease in AMPK activity compared with controls, as measured by phosphorylation of AMPK (Figure [Q and R]). After treating HFpEF mice with metformin, a widely used antidiabetic agent and known AMPK activator (Figure [S]), we observed significantly increased AMPK signaling (Figure [T and U]), which was associated with attenuated AF preponderance as evidenced by significantly reduced AF inducibility and duration (Figure [V and W]) in metformin-treated mice compared with the placebo group.In summary, we have demonstrated that our 2-hit HFpEF mice are highly susceptible to pacing-induced AF, providing a unique tool to unveil molecular mechanisms of AF preponderance in clinical HFpEF. Significant atrial remodeling, characterized by hypertrophy, inflammation, and aberrant intercellular communication, likely predisposes HFpEF mice to AF. Contrary to many other AF models, atrial fibrosis is not a major contributor in this model, suggesting unique pathophysiology. Our data further indicate that impaired AMPK signaling likely plays an essential role in HFpEF-associated AF, and metformin effectively attenuated AF preponderance. Further study is warranted to confirm these findings in other preclinical HFpEF models and in well-designed clinical studies, as well.Article InformationSources of FundingThis work was supported by grants from the National Institutes of Health: HL126012, HL128215, HL147933, S10RR023729 (Dr Hill), HL155765 (Drs Hill and Gillette), F32HL142244 (Dr Tong); American Heart Association: 14SFRN20510023 (Dr Hill), CDA851313 (Dr Tong); American Heart Association and the Theodore and Beulah Beasley Foundation: 18POST34060230 (Dr Schiattarella); University Federico II of Naples and Compagnia di San Paolo STAR program (Dr Schiattarella); Cancer Prevention and Research Institute of Texas: RP110486P3 (Dr Hill); and Agencia Nacional de Investigacion y Desarrollo (ANID, Chile): FONDAP 15130011 and FONDECYT 1200490 (Dr Lavandero).Nonstandard Abbreviations and AcronymsAFatrial fibrillationAMPKAMP-activated protein kinaseHFheart failureHFpEFheart failure with preserved ejection fractionLAleft atrialDisclosures Drs Schiattarella, Gillette, and Hill are coinventors on a patent application (PCT/US/2017/037019) that was filed in June 2017 (provisional application filed in June 2016). The patent relates to the diet used for modeling HFpEF. The other authors report no conflicts.FootnotesCirculation is available at www.ahajournals.org/journal/circThis manuscript was sent to Ju Chen, Guest Editor, for review by expert referees, editorial decision, and final disposition.For Sources of Funding and Disclosures, see page 76.Correspondence to: Joseph A. Hill, MD, PhD, Division of Cardiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, NB11.200, Dallas, TX 75390-8573. Email joseph.[email protected]eduCorrespondence to: Dan Tong, MD, PhD, Division of Cardiology, University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd, NB11.200, Dallas, TX 75390-8573. Email dan.[email protected]eduReferences1. Borlaug BA. The pathophysiology of heart failure with preserved ejection fraction.Nat Rev Cardiol. 2014; 11:507–515. doi: 10.1038/nrcardio.2014.83CrossrefMedlineGoogle Scholar2. Kotecha D, Lam CS, Van Veldhuisen DJ, Van Gelder IC, Voors AA, Rienstra M. Heart failure with preserved ejection fraction and atrial fibrillation: vicious twins.J Am Coll Cardiol. 2016; 68:2217–2228. doi: 10.1016/j.jacc.2016.08.048CrossrefMedlineGoogle Scholar3. 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 Scholar4. Ozcan C, Battaglia E, Young R, Suzuki G. LKB1 knockout mouse develops spontaneous atrial fibrillation and provides mechanistic insights into human disease process.J Am Heart Assoc. 2015; 4:e001733. doi: 10.1161/JAHA.114.001733LinkGoogle Scholar5. Sung MM, Zordoky BN, Bujak AL, Lally JS, Fung D, Young ME, Horman S, Miller EJ, Light PE, Kemp BE, et al. AMPK deficiency in cardiac muscle results in dilated cardiomyopathy in the absence of changes in energy metabolism.Cardiovasc Res. 2015; 107:235–245. doi: 10.1093/cvr/cvv166CrossrefMedlineGoogle 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. 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July 5, 2022Vol 146, Issue 1 Advertisement Article InformationMetrics © 2022 American Heart Association, Inc.https://doi.org/10.1161/CIRCULATIONAHA.121.058301PMID: 35858165 Originally publishedJuly 5, 2022 Keywordsheart failure, diastolicatrial fibrillationPDF download Advertisement SubjectsHeart Failure