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.
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.
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.
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.
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.
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.