What this is
- Gout is linked to atrial fibrillation (AF) and involves inflammation triggered by monosodium urate (MSU) crystals.
- This research investigates how the Kv1.5 potassium channel influences activation in macrophages and electrical changes in cardiomyocytes.
- The study utilizes mouse and human cell lines to explore the mechanisms of MSU-induced inflammation and its potential implications for AF.
Essence
- Kv1.5 channels mediate MSU-induced activation in macrophages, which in turn affects electrical remodeling in atrial myocytes, linking gout to atrial fibrillation.
Key takeaways
- MSU activates the in macrophages through Kv1.5-mediated potassium efflux, reducing intracellular potassium levels and enhancing caspase-1 and IL-1β production.
- Conditioned medium from MSU-stimulated macrophages increases Kv1.5 expression and currents in HL-1 cardiomyocytes, shortening action potential duration and potentially promoting AF.
- Inhibition of Kv1.5 using DPO-1 or siRNA against Kv1.5 effectively suppresses MSU-induced inflammasome activation and related electrical changes in cardiomyocytes.
Caveats
- The study primarily uses mouse cell lines, raising questions about the applicability of the findings to human primary cells and in vivo conditions.
- The specific factors in the conditioned medium responsible for NLRP3 activation in cardiomyocytes remain unidentified, necessitating further investigation.
- The role of other potassium channels in the observed effects was not explored, which could influence the interpretation of Kv1.5's specific contributions.
Definitions
- NLRP3 inflammasome: A protein complex that activates inflammatory responses, particularly in response to certain stimuli like MSU crystals.
- Kv1.5 channel: A potassium ion channel that regulates electrical activity in cells, particularly in cardiac and immune cells.
AI simplified
Introduction
Gout is the most common form of inflammatory arthritis, affecting 3–6% of men and 1–2% of women in developed countries. Gout is associated with hyperuricemia [1]. The ionized forms of uric acid (UA) form monosodium urate (MSU). MSU deposits in joints and surrounding tissues are uptaken by macrophages, causing inflammation via activation of NLRP3 (nucleotide-binding domain, leucine-rich repeats, and pyrin domain-containing protein 3) inflammasome, a cytosolic multiprotein platform [2].
The NLRP3 inflammasome is a major signaling pathway of the innate immune system, and is associated with gout, atherosclerosis, atrial fibrillation (AF), and other diseases [3, 4]. Upon activation by stimulatory particles, such as MSU or cholesterol crystals, NLRP3 interacts with the adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase-1 recruitment domain) and pro-caspase-1 to generate bioactive capsase-1(p20) [2]. Cleavage of precursor pro-IL-1β by capsase-1 yields active IL-1β (p17). Phagocytosis of particulate matters induces K+ efflux, thus reducing cytosolic K+. K+ efflux ultimately serves as an upstream requirement to activate the NLRP3 inflammasome [5]. However, the mechanism underlying K+ efflux remains unknown.
Kv1.5 channels, encoded by KCNA5, confer ultra-rapid delayed-rectifier potassium outward current (IKur). Kv1.5 is expressed in murine atrial myocytes and in macrophages [6, 7]. Uric acid enhances Kv1.5 protein expression in HL-1 mouse atrial myocytes via post-transcriptional modification by Hsp70, resulting in increases of the channel function [8]. Hyperuricemia and gout are related to the incidence of paroxysmal or persistent AF [9, 10]. Excess NLRP3 signaling in atrial myocytes augments Kv1.5 expression and IKur, which results in abbreviated atrial effective refractory period and enhanced AF susceptibility [4].
The purpose of this study was to clarify the role of Kv1.5 channel in MSU-induced activation of NLRP3 inflammasome in macrophages and electrical remodeling in atrial myocytes.
Materials and methods
Cell culture and channel treatments
Mouse macrophage cell line J774.1 and human macrophage cell line THP-1 (JCRB cell bank) were cultured in RPMI medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. HL-1 mouse cardiomyocytes were cultured in Claycomb medium (Sigma Aldrich) as previously described [6]. The macrophages were plated into 6-well tissue culture plates (1.5 X106 cells per well in 2 ml medium) overnight then primed with 1 μg/ml lipopolysaccharide (LPS) (from Sigma) in 1 ml serum-free RPMI medium for 6 h. After priming, the culture medium was replaced with 1 ml OPTI-MEM (Life Technologies) containing MSU (500 μg/ml) (Nacalai Tesque, Inc) for 6 h. K+ channel inhibitors or the inflammasome inhibitor MCC950 (50 nM) (Cayman Chemical Co.) were added to OPTI-MEM 30 min prior to the addition of MSU (Nacalai Tesque, INC). The following K+ channel inhibitors were used: non-selective voltage-gated K+ channel inhibitor, 4-aminopyridine (4-AP) (6 mM) (Tokyo Chemical Industry Co, LTD); a selective Kv1.5 channel inhibitor, diphenyl phosphine oxide-1 (DPO-1) (1 μM) (R&D systems); a selective Kv1.3 channel inhibitor, 5-(4-phenoxybutoxy) psoralen (PAP-1) (50 nM) (Chemscene LLC); a TWIK2 K+ channel blocker, quinine (1 μM) (Fuji Film, Japan); an inward-rectifier K+ channel blocker, BaCL2 (0.1 mM, 1 mM) (Fuji Film, Japan); and a ATP-sensitive K+ channel inhibitor, glibenclamide (1 μM) (Bio Vision, Milpitas, CA, USA). The conditioned medium (CM) from untreated J774.1 cells or LPS-primed and MSU-treated cells was added to 6 X 105 HL-1 cells in 6-well plates overnight.
Immunoblot analysis
Cells were harvested and lysed by sonication in a buffer (PBS supplemented with 1% polyoxyethylene octyiphenyl ether (NP-40), 0.5% sodium deoxycholate, 0.1% SDS, 1.5 mM aprotinin, 21 mM leupeptine, 15 mM pepstain, and 1 mM phenylmethylsulfonyl fluoride). After removal of insoluble materials by centrifugation, protein concentrations were determined using a bicinchoninic acid (BCA) protein assay kit (Pierce, Biotechnology, Rockford, IL, USA). Culture supernatants were collected after inflammasome stimulation by MSU. The proteins in the supernatants were concentrated using StrataClean resin (Agilent Technologies, Santa Clara, CA, Cat# 400714) according to the manufacturer’s instructions. Bound proteins were recovered by incubation at 95 °C for 5 min in a sample buffer. Proteins were separated by SDS-PAGE and electrotransferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Tween and were subsequently subjected to immunoblot (IB) analysis with a primary antibody. The antibodies used were as follows: NLRP3 (Cell Signaling, Cat# 15101), ASC (AdipoGen, Cat# AG-25B-0006) and caspase-1 (AdipoGen, Cat# AG-20B-0042, AG-20B-0048), IL-1β (R& D systems, Cat# AF-401-NA), Kv1.5 and Kv1.3 (alomone Labs, Cat# APC-004, APC-002), Na+/K+ ATPase α-1 (upstate, Cat# 05-369), Hsp70 (Stressgen, Cat# SPA-810), and β-actin (Calbiochem, Cat# CP01). The blots were developed using an ECL system (Amersham, Biosciences, Piscataway, NJ, USA) and band densities were quantified using NIH Image J software. For the chase experiment, J774.1 cells (1.5 X106 cells per well in 2 ml medium) were passaged into 6-well culture plates. cycloheximide (60 μg/ml) (Fujifilm, JP) was added to the culture medium after the LPS-primed cells were stimulated with MSU for 6 h. Cells were harvested at the indicated time points then analyzed by IB.
Subcellular fractionation
Cell fractionation was performed as described previously [11]. LPS-primed cells were treated with MSU for 6 h. Lysates from LPS-primed MSU-treated cells and control cells (without LPS and MSU treatments) were obtained and separated into cytosolic and plasma membrane fractions using the Plasma Membrane Protein Extraction Kit (BioVision, Milpitas, CA, USA, Cat# K268-50) according to the manufacturer’s instructions. Each fraction was subjected to 7.5% SDS-PAGE and analyzed by IB. Na+/K+-ATPase and β-actin were used as markers of the plasma membrane and cytosolic subcellular fraction, respectively.
RNA-mediated interference
| Sense | Antisense | |
|---|---|---|
| Kv1.5-1 | 5′ UCA UCA AGG AAG AGG AGA AUU 3′ | 5′ UUC UCC UCU UCC UUG AUG AUU 3′ |
| Kv1.5-2 | 5′ ACC UAA AGG CCA AGA GCA AUU 3′ | 5′ UUG CUC UUG GCC UUU AGG UUU 3′ |
| Kv1.3-1 | 5′ GGA CAG ACG CUG AAG GCU UUU 3′ | 5′ AAG CCU UCA GCG UCU GUC CUU 3′ |
| Kv1.3-2 | 5′ CCA GAA AUA UCA UGA ACU UUU 3 | 5′ AAG UUC AUG AUA UUU CUG GUU 3′ |
| Hsp70-1 | 5′ CUG GAG AUC GAC UCU CUG UUC 3′ | 5′ ACA GAG AGU CGA UCU CCA GGC 3′ |
| Scramble | 5′ GAA GCG AGA UAU CCC UGA CTT 3′ | 5′ GUC AGG GAU AUC UCG CUU CTT 3′ |
Immunofluorescence
Cells were seeded at 1 × 105 per slide onto gelatin-coated coverslips in a 3.5 cm dish. After LPS-priming, DPO-1 (1 μM) was applied to the cells 30 min prior to MSU stimulation for 6 h. The cells were fixed with 4% paraformaldehyde/PBS and permeabilized with 0.5% Triton X-100. The cells were then incubated for 1 h at room temperature with a primary antibody (ASC, 1:200, Enzo, Cat# ADI-905-173). After blocking in 3% albumin, bound antibodies were visualized with an Alexa Fluor 488-conjugated rabbit secondary antibody (1:1000) (ThermoFisher scientific, Cat# A32731). Images were obtained using a Bio-Rad MRC 1024 confocal microscope.
ELISA
IL-1 β concentrations in culture supernatants of J774.1 or THP-1 cells, and HL-1 cell lysates were measured by ELISA according to the manufacturer’s instructions (R& D System, cat#: MLB00C and DLB50).
Measurement of intracellular potassium concentration
The intracellular K+ concentration of J774.1 cells was measured using the FluxOR™ potassium ion channel assay (Life Technologies, Cat# F10017↗) as previously described [12]. Briefly, J774.1 cells were seeded in 6-well plates and primed with LPS (1 μg/ml) for 6 h. The LPS-primed cells were treated with MSU (500 μg/ml) for 1 h to prevent the cell death. Fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 530 nm.
Electrophysiological recordings
Kv1.5-mediated currents corresponding to Ikur in J774.1 and HL-1 cells were measured at 37 °C using whole-cell patch-clamp techniques with an Axopatch-200 amplifier (Axon Instruments, USA). The procedures for the current measurement were essentially the same as described previously [6]. The extracellular solution contained (mM): NaCl 140, KCl 4, CaCl2 1.8, MgCl2 0.53, NaH2PO4 0.33, glucose 5.5, and HEPES 5, with a pH adjusted to 7.4. The internal pipette solution contained (mM) K-aspartate 100, KCl 20, CaCl2 1, Mg-ATP 5, EGTA 5, HEPES 5, and creatine phosphate dipotassium salt 5 (pH 7.2 with KOH). The currents were elicited every 6-s by 500-ms test pulses ranging from − 60 to + 40 mV ( in 10 mV increments) with a holding potential of − 60 mV. DPO-1 (1 μM) was added to the bath solution to block Kv1.5 channel currents. The DPO-1 sensitive currents were obtained by digitally subtracting the current traces in the presence of DPO-1 from those in the absence of DPO-1. Action potentials (APs) of HL-1 cells were elicited at 0.5 Hz by 5-ms square current pulses of 1nA, and sampled at 20 kHz. AP durations (APDs) were measured at 20%, 50% and 90% repolarization (APD20, APD50 and APD90), respectively.
Statistical analysis
All data are presented as the mean ± SEM. Student’s t test and repeated measures analysis of variance (two-way ANOVA) were used for comparisons of two and multiple (more than two) groups, with p values < 0.05 considered statistically significant.
Results
Kv1.5 channel is required for MSU-induced NLRP3 inflammasome activation in J774.1 cells
Several K+ channels such as Kv1.3, Kir2.1 (inward-rectifier K+ channel), TWIK2 (two-pore domain K+ channel) and Kir6.2 (ATP-sensitive K+ channel) are expressed in J774.1 cells [12, 14, 17, 18]. To determine whether Kv1.3 regulates activation of the NLRP3 inflammasome, the selective Kv1.3 blocker PAP-1 was used [19]. PAP-1 at 2–50 nM failed to alter the levels of caspase-1 and secretion of IL-1β (Supplementary Fig. 4a–c). Two siRNAs against Kv1.3 (Kv1.3-1 and 2) both decreased the protein levels of Kv1.3 but did not reduce the expression of caspase-1 and IL-1β proteins (Supplementary Fig. 5a, b). PAP-1 effects on caspase-1 and IL-1β productions were recapitulated in human THP-1 cells (Supplementary Figs. 3b, 6). The inward-rectifier K+ channel inhibitor BaCl2 [20] was used to examine the roles of inward-rectifier K+ channels. BaCl2 at 0.1–1 mM did not change the levels of capase-1 and IL-1β (Supplementary Fig. 7a). TWIK2 and ATP-sensitive K+ channels mediate ATP- and glucose-induced NLRP3 inflammasome activation, respectively, in J774.1 cells [12, 17]. However, the TWIK2 channel inhibitor quinine and the ATP-sensitive K+ channel inhibitor glibenclamide did not alter capsase-1 and IL-1β levels in MSU-treated cells (Supplementary Fig. 7b). These results indicate that Kv1.5 regulates MSU-induced activation of the NLRP3 inflammasome in macrophages.
Effects of Kchannel blockers and a NLRP3 inflammasome inhibitor on MSU-induced activation of the NLRP3 inflammasome in J774.1 cells. The cells primed with LPS (1 μg/ml, 6 h) and subsequently stimulated with MSU (500 μg/ml, 6 h) in the presence and absence of the nonspecific Kchannel blocker 4-AP (6 mM), or the selective NLRP3 inflammasome inhibitor MCC950 (MCC) (50 nM) () or DPO-1 (1 μM) (). The inhibitors were added 30 min before the addition of MSU. The densities of caspase-1 (p20) and IL-1β (p17) were normalized to those determined for untreated cells, as indicated by the bar graph (n = 4–8).IL-1β concentrations in culture supernatants of the cells measured by ELISA (n = 5–10). *< 0.01,< 0.05 vs. LPS-primed and MSU-treated cells without administration of the inhibitors + + † a c b p p
Concentration dependent effects of DPO-1 and Kv.1.5 knockdown on MSU-induced NLRP3 inflammasome activation in J774.1 cells. DPO-1 (0–100 μM) were added 30 min before the addition of MSU (). 24 h after the introduction of one of two siRNAs against Kv1.5 (Kv1.5-1 and Kv1.5-2) or a scramble siRNA, LPS-primed cells were stimulated by MSU (). Shown are representative blots. (n = 4). *< 0.01,< 0.05. IL-1β in the culture supernatant of the LPS-primed MSU-stimulated cells treated with DPO-1 () or transfected with one of the siRNAs against Kv1.5 or the scramble siRNA () were analyzed by ELISA (n = 4–6). *< 0.01,p < 0.05 vs.DPO-1(−) a c b d p p p † †
Kv1.5 regulates MSU-induced activation of NLRP3 inflammasome via reductions in intracellular Klevels +
Whether Kv1.5 directly interacts with NLRP3 and/or ASC to affect NLRP3 inflammasome complex formation, we examined the association of Kv1.5 with NLRP3 and ASC by immunoprecipitation. The anti-Kv1.5 immunoprecipitates (IPs) in J774.1 cells contained neither ASC nor NLRP3. Kv1.5 was not present in either anti-ASC or anti-NLRP3 IPs (Supplementary Fig. 8a, b). Thus, Kv1.5 activates the NLRP3 inflammasome not by direct interactions with NLRP3 or ASC.j
Effects of Kv1.5 on intracellular Kconcentration, ASC oligomerization and speck formation in J774.1 cells. Intracellular Kconcentrations of LPS-primed cells stimulated with MSU in either the presence or absence of DPO-1 (1 μM) or the selective Kv1.3 channel blocker PAP-1 (50 nM) (), and those with the introduction of a siRNA against Kv1.5 or Kv1.3 or a scramble siRNA 24 h before the LPS and MSU treatments () (n = 5–11). *< 0.01, †< 0.05;: not statistically significant.The LPS-primed and subsequently stimulated with MSU cells were incubated with OPTI-MEM containing different Kconcentrations (0–60 mM) overnight (n = 5). Shown are the representative blot. *< 0.01 vs. LPS + MSU (+) & K0 mM.IBs of disuccinimidyl suberate (DSS)-crosslinked ASC oligomers in lysates from untreated cells and LPS-primed cells treated with DPO-1 (1 μM) or vehicle 30 min before MSU stimulation (n = 5).Representative immunofluorescence images of ASC speck formation in LPS-primed MSU-stimulated cells with and without DPO-1 (1 μM) treatment. Arrows denote ASC specks. Averaged numbers of specked cells (% total) are shown by the bar graph (n = 5–6 fields). *< 0.01 + + + + a b c d e p p NS p p
Kv1.5 promotes ASC oligomerization and speck formation in J774.1 cells
ASC oligomerization and speck formation are key events of the NLRP3 inflammasome activation [3, 21]. We examined the effects of Kv1.5 channel inhibition on ASC oligomerization and speck formation. When the cytosolic fraction from cell lysates was cross-linked using disuccinimidyl suberate (DSS), ASC dimers and oligomers were detected in LPS-primed and MSU-stimulated cells but not in that of untreated cells. DPO-1 inhibited ASC oligomerization (Fig. 3d). Immunofluorescence analyses showed that MSU stimulation of LPS-primed cells induced formation of ASC specks (Fig. 3e). DPO-1 treatment reduced the number of ASC specks.
MSU enhances Kv1.5 protein expression and channel function via Hsp70
MSU effects on protein expression of Kchannels and Hsp70, degradation of Kv1.5 proteins and Kv1.5 channel currents in J774.1 cells.IB analysis of Kv1.5 and Kv1.3 proteins and Hsp70 in the cytosolic and membrane fraction of LPS-primed and MSU-stimulated cells and untreated cells. Na/KATPase and β-actin were used as the plasma membrane and protein loading control, respectively.Degradation of Kv1.5 proteins. The LPS-primed MSU-treated cells (LPS + MSU) or untreated cells (none) were chased for the indicated times after an addition of cycloheximide. Representative blots are shown. The densities of Kv1.5 was normalized to the density at time 0 and β-actin. Bar graph shows the half-life of the Kv1.5 proteins (n = 4). *< 0.01.Effects of MSU on Kv1.5 channel currents measured as DPO-1-sensitive currents. Whole-cell membrane currents were recorded from a single J774.1 cell without LPS and MSU treatments (none) and that with LPS priming and MSU stimulation (LPS + MSU). Shown are representative DPO-1-sensitive currents that were obtained by digital subtraction. Current–voltage relationships of currents are also shown (n = 6–8). *< 0.01,< 0.05 vs. None + + + † a b c p p p
Effects of a siRNA against Hsp70 and heat shock (HS) on MSU-induced NLRP3 inflammasome activation and the expression of Kv1.5 protein. The LPS-primed J774.1 cells treated with MSU 24 h after the introduction of either a scramble siRNA or a siRNA against Hsp70 () (n = 4–5) or pre-treated at 42 °C for 1 h () (n = 5–9). Representative blots are shown. Image densities were normalized to those in the MSU-untreated cells transfected with a scramble siRNA or non-HS control cells.< 0.05, *< 0.01 a b † p p
Effects of CM from MSU-activated macrophages on NLRP3 inflammasome activity in HL-1 mouse atrial myocytes
Effects of the CM from LPS-primed and MSU-stimulated macrophages on activation of NLRP3 inflammasome and Kv1.5 in HL-1 atrial myocytes. HL-1 cells were incubated with Claycomb, the CM from J774.1 cells treated with vehicle (LPS/MSU−) or the CM from LPS-primed MSU-treated cells (LPS/MSU+) overnight. Shown are representative IBs () (n = 5–8).IL-1β concentrations in the cell lysates measured by ELISA (n = 4–11). HL-1 cells were treated with DPO-1 (1 μM) or a vehicle 30 min before the incubation with CM + LPDS + MSU overnight. Show are representative blots () (n = 9) and IL-1β concentrations in the cell lysates measured by ELISA () (n = 6). *< 0.01. HL-1 cells were transfected with a scramble siRNA or a siRNA against Kv1.5 24 h before incubation with CM + LPS + MSU. Show are representative blots () (n = 5–11) and IL-1β concentrations measured by ELISA () (n = 6).< 0.05, *< 0.01 a b c d e f p p p †
Effects of the CM + LPS + MSU on DPO-1-sensitive currents and APD in HL-1 cells.Whole-cell membrane currents recorded from a single HL-1 cell before and after application of DPO-1(1 μM). DPO-1-seseitive currents were obtained by digital subtraction. Current recordings were performed after incubation with the untreated control CM (CM) or CM + LPS + MSU overnight.Current–voltage relationships of the peak DPO-1-sensitive current (n = 9–15).< 0.05, *< 0.01 vs. the control CM.Action potentials recorded after incubation with the control CM or CM + LPS + MSU overnight.APD, APDand APDvalues summarized as a bar graph (n = 8–15). *< 0.01 a b c d † p p p 20 50 90
Discussion
MSU-induced gout flares are characterized by activation of the NLRP3 inflammasome which requires K+ efflux [5]. Mouse bone marrow-derived macrophages (BMDMs) were incubated in K+-free medium without any stimuli inducing IL-1β secretion, indicating that K+ efflux mediates activation of the NLRP3 inflammasome [5]. Kv1.5 and other K+ channels are expressed in macrophages [12–15, 17, 18] and involved in immune responses [22, 23]. K+ efflux through Kv1.5 channels forms outward IKur that regulates APD [4, 6]. In the current study, MSU-triggered decline in the intracellular K+ concentration accompanied enhanced caspase-1 formation and IL-1β release. Blocking Kv1.5 by the selective inhibitor DPO-1 decreased outward K+ currents corresponding to IKur and attenuated MSU-induced production of caspase-1 and IL-1β (Fig. 1b, c). The role of Kv1.5 in MSU-induced activation of NLRP3 inflammsome was confirmed by siRNA knockdown of Kv1.5 and was recapitulated in human macrophages. Kv1.5 protein expression is closely related to IKur and intracellular K+ concentrations in macrophages (Figs. 3b, 4a and c). DPO-1 and siRNAs against Kv1.5 similarly and effectively prevented all of them (Figs. 1c, 2c), even though DPO-1 did not significantly change Kv1.5 protein expression (Supplementary Fig. 10). Furthermore, extracellular solutions of higher K+ concentrations (30–60 mM) also clearly abolished MSU-induced NLRP3 inflammsome activation (Fig. 3c). Taken together, Kv1.5 mediated K+ efflux that decreases intracellular K+ concentrations is responsible for the MSU-induced activation of the NLRP3 inflammasome.
Molecular chaperones participate in the biogenesis of K+ channels, such as Kv1.5 and hERG. Hsp70 stabilizes these channel proteins and enhances K+ currents by post-transcriptional modifications [24, 25]. The direct interaction of Hsp70 and Kv1.5 proteins has been confirmed (Supplementary Fig. 8c). HS and cell stress induce Heat shock proteins (Hsps), including Hsp70. Hsp70 expressions are increased by soluble uric acid and IL-1β in HL-1 and Hela cells, respectively [8, 26]. MSU-treated macrophages contained more than 30 times higher concentrations of IL-1β than that in untreated macrophages (Fig. 1b). IL-1β produced by activated NLRP3 inflammasome may further enhance Hsp70 expression which exaggerates NLRP3 inflammsome activation by increasing Kv1.5 expression.
Enhanced cardiomyocyte NLPR3 inflammasome activity promotes AF [4]. MSU directly activates NLRP3 inflammasome in macrophages [2]. MSU did not directly induce capsase-1 expression and IL-1β secretion in HL-1 cells (Supplementary Fig. 9), whereas the CM from macrophages treated with LPS and MSU increased capsase-1 expression, IL-1β secretion, Kv1.5 protein expression and Kv1.5 channel currents, and shortened APD in HL-1 cells (Figs. 6a, b, 7a–d). These results suggest that activation of NLRP3 inflammasome and electrical remodeling in HL-1 cells are induced by MSU-stimulated macrophages. In vivo conditions cardiomyocytes contact with resident and infiltrated macrophages. Increased infiltration of macrophages and elevated IL-1β production have been found in the atrial tissue of AF patients [27]. Macrophages are polarized by LPS to be differentiated into pro-inflammatory type [27]. Administration of LPS for two weeks increased pro-inflammatory macrophage in the atrium and AF incidence with shortened atrial effective refractory period in both canine and mouse AF models [27]. Thus, pro-inflammatory macrophages play a key role in the occurrence of NLRP3 inflammasome-related AF. The resident macrophages in various tissues exert distinct properties from monocyte-derived macrophages. In general monocyte-derived macrophages, such as J774.1 cells, have an inflammatory phenotype by production of IL-1β, whereas the tissue resident macrophages, such as cardiac resident macrophages, possess an anti-inflammatory phenotype [28, 29]. We assume that the CM from LPS-primed and MSU-treated inflammatory phenotype macrophages mimic the effect of gout-activation of NLRP3 inflammsome in macrophages on cardiomyocytes in vitro.
The active NLRP3 promotes automaticity of atrial myocytes and shortens atrial refractory period by enhancing RYR2-mediated Ca2+ release and Kv1.5-mediated IKur [4]. In a mouse model, high fat diet induced activation of the NLRP3 inflammasome and upregulation of Kv1.5 protein expression in atrial tissue with the abbreviation of atrial-effective refractory period [30]. CM + LPS + MSU enhanced- Kv1.5 expression and channel currents, which activate NLRP3 inflammasome with shortening of APD in HL-1 cells (Figs. 6, 7). These alterations of electrical properties are involved in the development of AF and suggest that MSU-induced activation of the NLRP3 inflammasome in macrophages upregulates Kv1.5 expression in HL-1 cells, which plays a major role in APD shortening and the development of AF in atrial myocytes under hyperuricemia.
Several limitations of the present study should be addressed. First, we mainly used mouse macrophage and atrial myocyte cell lines. Whether our conclusions are also applicable to human primary macrophages and cardiomyocytes and gout in vivo models is needed to be further defined in future studies. Second, the exact factors in the CM + LPS + MSU responsible for activation of NLRP3 inflammasome in cardiomyocytes remain to be elucidated. Acute application of IL-1β causes NLRP3 inflammasome activation in HL-1 cells [31]. IL-1β probably exerts a major contribution to activation of NLRP3 inflammasome and enhancement of Kv1.5 expression in cardiomyocytes. However, the CM + LPS + MSU may contain unknown multiple biological mediators other than IL-1β. Rather than using recombinant or synthetic cytokines, the use of CM + LPS + MSU may evoke more biologically relevant responses. Third, several K+ channels are expressed on the HL-1 cell membrane. The roles of other K+ channels should be examined in more detail. Fourth, how K+ efflux leads to activation of the NLRP3 inflammasome remains unknown.
In summary, MSU activates the NLRP3 inflammasome in macrophages via enhancement of Kv1.5 mediated K+ efflux. MSU-stimulated macrophages activate the NLRP3 inflammasome and promote electrical remodeling and inflammation in atrial myocytes. Kv1.5 may be a potential therapeutic target for gout-related inflammation and AF.
Supplementary Information
Below is the link to the electronic supplementary material. Supplementary file1 (DOCX 31 kb) Supplementary file2 (PPTX 7013 kb)