Kv1.5 channel mediates monosodium urate-induced activation of NLRP3 inflammasome in macrophages and arrhythmogenic effects of urate on cardiomyocytes

Background Gout is usually found in patients with atrial fibrillation (AF). K+ efflux is a common trigger of NLRP3 inflammasome activation which is involved in the pathogenesis of AF. We investigated the role of the K+ channel Kv1.5 in monosodium urate crystal (MSU)-induced activation of the NLRP3 inflammasome and electrical remodeling in mouse and human macrophages J774.1 and THP-1, and mouse atrial myocytes HL-1. Methods and Results Macrophages, primed with lipopolysaccharide (LPS), were stimulated by MSU. HL-1 cells were incubated with the conditioned medium (CM) from MSU-stimulated macrophages. Western blot, ELISA and patch clamp were used. MSU induced caspase-1 expression in LPS-primed J774.1 cells and IL-1β secretion, suggesting NLRP3 inflammasome activation. A selective Kv1.5 inhibitor, diphenyl phosphine oxide-1 (DPO-1), and siRNAs against Kv1.5 suppressed the levels of caspase-1 and IL-1β. MSU reduced intracellular K+ concentration which was prevented by DPO-1 and siRNAs against Kv1.5. MSU increased expression of Hsp70, and Kv1.5 on the plasma membrane. siRNAs against Hsp70 were suppressed but heat shock increased the expression of Hsp70, caspase-1, IL-1β, and Kv1.5 in MSU-stimulated J774.1 cells. The CM from MSU-stimulated macrophages enhanced the expression of caspase-1, IL-1β and Kv1.5 with increased Kv1.5-mediated currents that shortened action potential duration in HL-1 cells. These responses were abolished by DPO-1 and a siRNA against Kv1.5. Conclusions Kv1.5 regulates MSU-induced activation of NLRP3 inflammasome in macrophages. MSUrelated activation of NLRP3 inflammasome and electrical remodeling in HL-1 cells are via macrophages. Kv1.5 may have therapeutic value for diseases related to gout-induced activation of the NLRP3 inflammsome, including AF. Supplementary Information The online version contains supplementary material available at 10.1007/s11033-022-07378-1.


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 (I Kur ). 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 I Kur , 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.

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
siRNA transfection into J774.1 and HL-1 cells was performed using Lipofectamine RNAiMAX (Invitrogen, Cat# 13778150) following the manufacturer's protocols. Two pairs of siRNAs that target Kv1.5 and Kv1.3 were used, along with an siRNA against Hsp70 and a scrambled control siRNA. Table 1 shows sequences of siRNAs.

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 LPSprimed 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.

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.
These results indicate that Kv1.5 regulates MSU-induced activation of the NLRP3 inflammasome in macrophages.

Kv1.5 regulates MSU-induced activation of NLRP3 inflammasome via reductions in intracellular K + levels
Whether the K + efflux induced by Kv1.5 channels could trigger activation of the NLRP3 inflammasome in MSUtreated J774.1 cells, we evaluated the effects of MSU on the intracellular K + concentration in the presence or absence of DPO-1. MSU caused a marked decrease in intracellular K + levels (Fig. 3a). DPO-1 prevented the MSU-induced decrease in intracellular K + levels. The siRNAs against Kv1.5 introduced to LPS-primed cells mimicked the effects of DPO-1 on intracellular K + levels (Fig. 3b). To evaluate the role of K + efflux in MSU-induced NLRP3 inflammasome activation, LPS-primed cells were incubated in OPTI-MEM with various K + concentrations. MSU-induced enhancement of caspase-1 expression and IL-1β secretion was suppressed with increasing extracellular K + concentrations (Fig. 3c), demonstrating that MSU-induced activation of NLRP3 inflammasome is related to K + efflux and a decrease in intracellular K + concentrations. 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 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
To investigate how MSU enhanced Kv1.5 expression, we examined the effects of MSU on Kv1.5 protein localization and levels in LPS-primed J774.1 cells using fractionation experiment. The Kv1.5 levels on the plasma membrane, not in the cytosol, and Hsp70 expression were increased by MSU (Fig. 4a). The half-life of Kv1.5 proteins was 3.7 ± 0.1 h in untreated cells and was prolonged to 5.1 ± 0.3 h in MSU-treated cells (Fig. 4b). Kv1.5 channel currents in J774.1 cells were recorded as DPO-1-sensitive currents using the patch clamp technique. In cells without LPS and MSU treatment (none), depolarization pulses activated time-dependent outward currents, which were completely abolished by DPO-1. LPS priming followed by MSU treatment augmented DPO-1 sensitive currents (Fig. 4c).

Effects of CM from MSU-activated macrophages on NLRP3 inflammasome activity in HL-1 mouse atrial myocytes
Enhanced cardiomyocyte NLRP3 inflammasome signaling promotes AF with increased I Kur [4]. To assess the direct effects of MSU on NLPR3 inflammasome in atrial myocytes, we stimulated LPS-treated HL-1 cells with MSU for 6 h. Caspase-1 or IL-1β bands could not be detected in supernatants or cell lysates (Supplementary Fig. 9). Therefore, we hypothesized that MSU activates NLRP3 inflammasome in HL-1 cells via stimulating macrophages. HL-1 cells were incubated with Claycomb medium, the CM from untreated J774.1 cells (control CM) or the CM obtained from LPS-primed and MSU-treated J774.1 cells (CM + LPS + MSU) overnight. The bands of caspase-1 in cell lysates were undetectable in the HL-1 cells cultured with either Claycomb medium or the control CM. Exposure to CM + LPS + MSU induced caspase-1 bands and increased IL-1β expression measured by ELISA as well as enhanced Kv1.5 and Hsp70 expression (Fig. 6a,  b). DPO-1 (1 μM) was added to the medium 30 min prior to exposure to CM + LPS + MSU or an siRNA against Kv1.5 was introduced into HL-1 cells 24 h before exposure to CM + LPS + MSU. Both DPO-1 and knockdown of Kv1.5 decreased the levels of caspase-1 and IL-1β in the HL-1 cells exposed to CM + LPS + MSU without alteration of Hsp70 expression (Fig. 6c-f). Depolarizing test  pulses activated time-dependent outward currents in HL-1 cells exposed to the control CM, which were completely abolished by DPO-1 (Fig. 7a, b). HL-1 cells exposed to CM + LPS + MSU showed larger DPO-1 sensitive currents and shorter APDs than those exposed to the control CM without differences in resting membrane potentials (Fig. 7b-d). These findings suggest that as a paracrine action the CM from MSU-activated macrophages could induce activation of NLRP3 inflammasome and enhancement of Kv1.5 channel currents to shorten APD in HL-1 cells.
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 LPSprimed 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 Ca 2+ release and Kv1.5-mediated I Kur [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.