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. 2017 May 11;12(5):e0176676.
doi: 10.1371/journal.pone.0176676. eCollection 2017.

The cardiac glycoside ouabain activates NLRP3 inflammasomes and promotes cardiac inflammation and dysfunction

Motoi Kobayashi  1 , Fumitake Usui-Kawanishi  1 , Tadayoshi Karasawa  1 , Hiroaki Kimura  1 , Sachiko Watanabe  1 , Nathan Mise  2 , Fujio Kayama  2 , Tadashi Kasahara  1 , Naoyuki Hasebe  3 , Masafumi Takahashi  1
Affiliations

The cardiac glycoside ouabain activates NLRP3 inflammasomes and promotes cardiac inflammation and dysfunction

Motoi Kobayashi et al. PLoS One. .

Abstract

Cardiac glycosides such as digoxin are Na+/K+-ATPase inhibitors that are widely used for the treatment of chronic heart failure and cardiac arrhythmias; however, recent epidemiological studies have suggested a relationship between digoxin treatment and increased mortality. We previously showed that nucleotide-binding oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasomes, which regulate caspase-1-dependent interleukin (IL)-1β release, mediate the sterile cardiovascular inflammation. Because the Na+/K+-ATPase is involved in inflammatory responses, we investigated the role of NLRP3 inflammasomes in the pathophysiology of cardiac glycoside-induced cardiac inflammation and dysfunction. The cardiac glycoside ouabain induced cardiac dysfunction and injury in wild-type mice primed with a low dose of lipopolysaccharide (LPS), although no cardiac dysfunction was observed in mice treated with either ouabain or LPS alone. Ouabain also induced cardiac inflammatory responses, such as macrophage infiltration and IL-1β release, when mice were primed with LPS. These cardiac manifestations were all significantly attenuated in mice deficient in IL-1β. Furthermore, deficiency of NLRP3 inflammasome components, NLRP3 and caspase-1, also attenuated ouabain-induced cardiac dysfunction and inflammation. In vitro experiments revealed that ouabain induced NLRP3 inflammasome activation as well as subsequent IL-1β release from macrophages, and this activation was mediated by K+ efflux. Our findings demonstrate that cardiac glycosides promote cardiac inflammation and dysfunction through NLRP3 inflammasomes and provide new insights into the mechanisms underlying the adverse effects of cardiac glycosides.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. IL-1β deficiency attenuates ouabain-induced cardiac dysfunction and injury.
WT and IL-1β−/− mice were treated with ouabain (2 mg/kg) 12 h after LPS (3 mg/kg) administration. Echocardiography was performed, and mice were sacrificed 12 h after ouabain treatment. (A) Two-dimensional M-mode echocardiograms are shown. (B) Cardiac function [%FS and EF (%)] and dimension [LVDs (mm) and LVEDs (mm)] were assessed (n = 8–13 for WT mice, n = 4–7 for NLRP3−/− mice). (C and D) Plasma CPK and cardiac IL-1β protein levels were assessed (n = 4–8 for each). (E) The heart sections were stained with hematoxylin and eosin. Arrowheads indicate infiltrated inflammatory cells. Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01. #P < 0.05 vs. vehicle (WT).
Fig 2
Fig 2. IL-1β deficiency decreases ouabain-induced macrophage infiltration.
WT and IL-1β−/− mice were treated with ouabain (2 mg/kg) 12 h after LPS (3 mg/kg) administration and then sacrificed 12 h after ouabain treatment. (A and C) Heart sections were immunohistochemically stained for CD45 and CD68. (B and D) The number of CD45+ (leukocytes) and CD68+ (macrophages) cells was quantified (n = 4 for each). Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01. #p < 0.05 vs. vehicle (WT).
Fig 3
Fig 3. Deficiency in NLRP3 and Casp1 attenuates ouabain-induced cardiac dysfunction.
WT, NLRP3−/−, and Casp1−/− mice were treated with ouabain (2 mg/kg) 12 h after LPS (3 mg/kg) administration. Echocardiography was performed, and mice were sacrificed 12 h after ouabain treatment. (A) Two-dimensional M-mode echocardiograms are shown. (B) Cardiac function [%FS and EF (%)] and dimension [LVDs (mm) and LVEDs (mm)] were assessed [n = 13 (WT), 8 (NLRP3−/−), 6 (Casp1−/−)]. (C and D) Plasma CPK and cardiac IL-1β protein levels were assessed [n = 8 (WT), 6 (NLRP3−/−), and 5 (Casp1−/−)]. (E) Heart sections were stained with hematoxylin and eosin. Arrowheads indicate infiltrated inflammatory cells. Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01.
Fig 4
Fig 4. Deficiency in NLRP3 and Casp1 decreases ouabain-induced macrophage infiltration.
WT, NLRP3−/−, and Casp1−/− mice were treated with ouabain (2 mg/kg) 12 h after LPS (3 mg/kg) administration, and then sacrificed 12 h after ouabain treatment. (A and C) Heart sections were immunohistochemically stained for CD45 and CD68. (B and D) The number of CD45+ (leukocytes) and CD68+ (macrophages) cells was quantified (n = 4 for each). Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01.
Fig 5
Fig 5. Ouabain induces NLRP3 inflammasome activation and IL-1β release in macrophages.
(A–C) After priming with or without LPS (100 ng/mL) for 6 h, J774 macrophages were treated with ouabain (A, 50–100 μM; B and C, 100 μM) for 3 h. (A) IL-1β levels in the supernatants were assessed (n = 6 for each). (B) Heart Il1b and Nlrp3 mRNA levels were assessed by real-time RT-PCR analysis (n = 4 for each). (C) The processing of pro-IL-1β in the lysates and supernatants was assessed by western blot analysis. ATP was used as a positive control. (D) LPS-primed J774 macrophages were treated with ouabain (100 μM) for 3 h in the presence or absence of YVAD-FMK (20 μM). IL-1β levels in the supernatants were assessed (n = 4 for each). (E and F) After priming with or without LPS (100 ng/mL) for 6 h, primary murine macrophages from WT and NLRP3−/− mice were treated with ouabain (E, 100 μM; F, 50–100 μM) for 3 h. (E) IL-1β levels in the supernatants were assessed (n = 4 for each). (F) Cell death was assessed by LDH activity in the supernatants (n = 4 for each). Data are expressed as the mean ± SEM. *P < 0.05 and **p < 0.01. #P < 0.05 vs. vehicle. $P < 0.05 vs. LPS alone and §P < 0.05 vs. Ouabain with LPS.
Fig 6
Fig 6. Ouabain-induced NLRP3 inflammasome activation is mediated through K+ efflux.
(A) After priming with or without LPS (100 ng/mL) for 6 h, J774 macrophages were treated with ouabain (100 μM) for 3 h or nigericin (3.4 μM) for 2 h. The cells were lysed and intracellular K+ concentrations were assessed by using an atomic absorption spectrometry (n = 4 for each). (B) LPS-primed J774 macrophages were treated with ouabain (100 μM) for 3 h in the presence or absence of KCl (130 mM), NaCl (130 mM), or glibenclamide (10 and 50 μM). IL-1β levels in the supernatants were assessed (n = 4 for each). Data are expressed as the mean ± SEM. *P < 0.05 and **P < 0.01. #p < 0.05 vs. vehicle and §P < 0.05 vs. Ouabain with LPS.
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References

    1. Ziff OJ, Kotecha D. Digoxin: The good and the bad. Trends Cardiovasc Med. 2016;26(7):585–95. 10.1016/j.tcm.2016.03.011 - DOI - PubMed
    1. Ahmed A, Rich MW, Fleg JL, Zile MR, Young JB, Kitzman DW, et al. Effects of digoxin on morbidity and mortality in diastolic heart failure: the ancillary digitalis investigation group trial. Circulation. 2006;114(5):397–403. 10.1161/CIRCULATIONAHA.106.628347 - DOI - PMC - PubMed
    1. Whitbeck MG, Charnigo RJ, Khairy P, Ziada K, Bailey AL, Zegarra MM, et al. Increased mortality among patients taking digoxin—analysis from the AFFIRM study. Eur Heart J. 2013;34(20):1481–8. 10.1093/eurheartj/ehs348 - DOI - PubMed
    1. Washam JB, Stevens SR, Lokhnygina Y, Halperin JL, Breithardt G, Singer DE, et al. Digoxin use in patients with atrial fibrillation and adverse cardiovascular outcomes: a retrospective analysis of the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF). Lancet. 2015;385(9985):2363–70. 10.1016/S0140-6736(14)61836-5 - DOI - PubMed
    1. Xie Z, Askari A. Na(+)/K(+)-ATPase as a signal transducer. Eur J Biochem. 2002;269(10):2434–9. - PubMed

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Grants and funding

This study was supported by grants from the Japan Society for the Promotion of Science (JSPS) through the Grant-in-Aid for Scientific Research (MT:15K09146), MEXT-supported program for the Strategic Foundation at Private Universities (MT:S1311029), Agency for Medical Research and Development-Core Research for Evolutional Science and Technology (AMED-CREST) (MT:16gm0610012h0103), and the Salt Science Research Foundation (MT:No.1541).

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