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. 2019 Aug 23;294(34):12846-12854.
doi: 10.1074/jbc.RA119.008330. Epub 2019 Jul 12.

Inflammasome inhibition blocks cardiac glycoside cell toxicity

Doris L LaRock  1   2   3   4 , Jenna S Sands  3   4 , Ethan Ettouati  1   2 , Marine Richard  1   2   5 , Paul J Bushway  6 , Eric D Adler  6 , Victor Nizet  7   2 , Christopher N LaRock  8   2   3   4
Affiliations

Inflammasome inhibition blocks cardiac glycoside cell toxicity

Doris L LaRock et al. J Biol Chem. .

Abstract

Chronic heart failure and cardiac arrhythmias have high morbidity and mortality, and drugs for the prevention and management of these diseases are a large part of the pharmaceutical market. Among these drugs are plant-derived cardiac glycosides, which have been used by various cultures over millennia as both medicines and poisons. We report that digoxin and related compounds activate the NLRP3 inflammasome in macrophages and cardiomyocytes at concentrations achievable during clinical use. Inflammasome activation initiates the maturation and release of the inflammatory cytokine IL-1β and the programmed cell death pathway pyroptosis in a caspase-1-dependent manner. Notably, the same fluxes of potassium and calcium cations that affect heart contraction also induce inflammasome activation in human but not murine cells. Pharmaceuticals that antagonize these fluxes, including glyburide and verapamil, also inhibit inflammasome activation by cardiac glycosides. Cardiac glycoside-induced cellular cytotoxicity and IL-1β signaling are likewise antagonized by inhibitors of the NLRP3 inflammasome or the IL-1 receptor-targeting biological agent anakinra. Our results inform on the molecular mechanism by which the inflammasome integrates the diverse signals that activate it through secondary signals like cation flux. Furthermore, this mechanism suggests a contribution of the inflammasome to the toxicity and adverse events associated with cardiac glycosides use in humans and that targeted anti-inflammatories could provide an additional adjunct therapeutic countermeasure.

Keywords: IL-1; cardiac glycoside; cardiomyocyte; caspase 1 (CASP1); cell death; inflammasome; macrophage.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Figures

Figure 1.
Figure 1.
Cardiac glycosides are cytotoxic. A, death of THP-1 macrophages enumerated by LDH release after 4-h incubation with dilutions of the cardiac glycoside drugs digoxin, digitoxin, lanatoside C, or ouabain. B, microscopic examination of THP-1 macrophages treated for 4 h with 1 μm digoxin and stained with DAPI (all cells) and PI (cells with a damaged membrane). Light microscopy (DIC) shows disrupted cell architecture at high magnification, with overall morphology consistent with pyroptosis (2). Scale bar = 5 μm. C, microscopy examination of THP-1 macrophages treated for 4 h with 1 μm digoxin and stained with DAPI (all cells) and FLICAYVAD (caspase-1/11 activity probe, green) or FLICADEVD (caspase-3/7 activity probe, green). Scale bar = 50 μm. Where applicable, data are represented as mean ± S.D. n = 4, representative of at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test.
Figure 2.
Figure 2.
Cardiac glycosides activate Interleukin-1β. A, IL-1β signaling in supernatant removed from THP-1 macrophages after 4-h incubation with dilutions of the cardiac glycoside drugs digoxin, digitoxin, lanatoside C, or ouabain, assayed using IL-1R bioreporter cells. RU, relative units. B, THP-1 macrophages incubated with 1 μm digoxin or 1 mm MSU for 4 h and assayed for the proinflammatory cytokines IL-1β, IL-6, and TNFα by ELISA. C, kinetics of bioactive IL1β release by THP-1 macrophages treated with 1 μm digoxin or 1 mm MSU. D, quantitative RT-PCR of il1b transcript levels in THP-1 macrophages treated for 4 h with 1 μm digoxin or 1 mm MSU. E, bioactive IL-1β assayed from supernatants of freshly isolated human peripheral blood monocytes treated for 4 h with 1 mm MSU or 100 nm digoxin. F, immunoblot examining proteolytic maturation of pro-IL-1β (pro-) in cells and supernatent released from THP-1 macrophages (m-) with or without 2-h LPS pretreatment and an additional 4-h incubation with 1 mm MSU, 1 μm digoxin, or 20 μm nigericin. Where applicable, data are represented as mean ± S.D. n = 4, representative of at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test. *, p < 0.05; ns, not significant.
Figure 3.
Figure 3.
Cardiac glycoside toxicity is evident in human cardiomyocytes. A, cell imaging of iPS human cardiomyocytes in culture treated with digoxin, demonstrating cell death over time (see Video S1 for contraction). Scale bars = 10 μm. DIC, differential interference contrast. B, microscopy examination of iPS human cardiomyocytes treated for 2 h with 1 μm digoxin or 1 mm MSU and stained with DAPI (all cells), PI (dead cells), and FLICAYVAD (caspase-1/11 activity probe, green). Scale bars = 10 μm. C, death of THP-1 macrophages enumerated by LDH release after 2-h incubation with 1 μm digoxin or 1 mm MSU and 300 nm MCC950 (NLRP3 inhibitor) or 10 μm VX-765 (caspase-1 inhibitor). D and E, quantification of IL-1β release by ELISA (D) and expression by real-time quantitative PCR (E) from iPS human cardiomyocytes treated with 1 μm digoxin, 1 mm MSU, or 100 ng/ml LPS compared with THP-1 macrophages. Where applicable, data are represented as mean ± S.D. n = 4, representative of at least three independent experiments. ND, none detected. Statistical significance was determined by unpaired two-tailed Student's t test. *, p < 0.05.
Figure 4.
Figure 4.
Cardiac glycoside toxicity is low toward murine cells. A and B, BMMs of the C57Bl/6 murine line were incubated for 4 h with dilutions of digoxin or 1 mm MSU, and IL-1β release was quantified by ELISA (A), or death of BMMs was quantified by LDH release (B). C, macrophages derived from the bone marrow of CD1 and BALB/c mice were incubated for 4 h with dilutions of digoxin or 1 mm MSU, and IL-1β release was quantified by ELISA. D, macrophages and cardiomyocytes of the indicated murine cell lines were treated for 4 h with dilutions of digoxin or 1 mm MSU, and IL-1β release was quantified by ELISA. Data are represented as mean ± S.D. n = 4, representative of at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.
Figure 5.
Figure 5.
Pharmacologic targets to ablate cardiac glycoside toxicity. A, model of the digoxin mechanism of action (red numbers, sequential ordering of the digoxin effect on intracellular cations) and molecular targets of cation and channel-targeting drugs. B, Venn diagram of reports in the FDA Adverse Event Database for the indicated drugs and percentage of those reports resulting in the adverse event of death. C, bioactive IL-1β assayed in the supernatant from THP-1 macrophages 4 h after addition of 1 μm digoxin or 1 mm MSU and co-administration of 20 μm glyburide, 10 μm valinomycin, 100 μm verapamil, or 10 μm BAPTA-AM. RU, relative units. D, model of bifurcation of digoxin effects on the heart and on the inflammasome. E, bioactive IL-1β assayed in the supernatant from THP-1 macrophages 4 h after addition of 1 μm digoxin or 1 mm MSU and co-administration of 300 nm MCC950, 10 μm VX-765, or 20 μg/ml anakinra. Where applicable, data are represented as mean ± S.D. n = 4, representative of at least three independent experiments. Statistical significance was determined by unpaired two-tailed Student's t test. *, p < 0.05; ***, p < 0.0005.
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