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NLRP3 Inflammasome Activation Through Heart-Brain Interaction Initiates Cardiac Inflammation and Hypertrophy During Pressure Overload

Originally publishedhttps://doi.org/10.1161/CIRCULATIONAHA.122.060860Circulation. 2023;147:338–355

Abstract

Background:

Mechanical stress on the heart, such as high blood pressure, initiates inflammation and causes hypertrophic heart disease. However, the regulatory mechanism of inflammation and its role in the stressed heart remain unclear. IL-1β (interleukin-1β) is a proinflammatory cytokine that causes cardiac hypertrophy and heart failure. Here, we show that neural signals activate the NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3) inflammasome for IL-1β production to induce adaptive hypertrophy in the stressed heart.

Methods:

C57BL/6 mice, knockout mouse strains for NLRP3 and P2RX7 (P2X purinoceptor 7), and adrenergic neuron-specific knockout mice for SLC17A9, a secretory vesicle protein responsible for the storage and release of ATP, were used for analysis. Pressure overload was induced by transverse aortic constriction. Various animal models were used, including pharmacological treatment with apyrase, lipopolysaccharide, 2′(3′)-O-(4-benzoylbenzoyl)-ATP, MCC950, anti–IL-1β antibodies, clonidine, pseudoephedrine, isoproterenol, and bisoprolol, left stellate ganglionectomy, and ablation of cardiac afferent nerves with capsaicin. Cardiac function and morphology, gene expression, myocardial IL-1β and caspase-1 activity, and extracellular ATP level were assessed. In vitro experiments were performed using primary cardiomyocytes and fibroblasts from rat neonates and human microvascular endothelial cell line. Cell surface area and proliferation were assessed.

Results:

Genetic disruption of NLRP3 resulted in significant loss of IL-1β production, cardiac hypertrophy, and contractile function during pressure overload. A bone marrow transplantation experiment revealed an essential role of NLRP3 in cardiac nonimmune cells in myocardial IL-1β production and cardiac phenotype. Pharmacological depletion of extracellular ATP or genetic disruption of the P2X7 receptor suppressed myocardial NLRP3 inflammasome activity during pressure overload, indicating an important role of ATP/P2X7 axis in cardiac inflammation and hypertrophy. Extracellular ATP induced hypertrophic changes of cardiac cells in an NLRP3- and IL-1β–dependent manner in vitro. Manipulation of the sympathetic nervous system suggested sympathetic efferent nerves as the main source of extracellular ATP. Depletion of ATP release from sympathetic efferent nerves, ablation of cardiac afferent nerves, or a lipophilic β-blocker reduced cardiac extracellular ATP level, and inhibited NLRP3 inflammasome activation, IL-1β production, and adaptive cardiac hypertrophy during pressure overload.

Conclusions:

Cardiac inflammation and hypertrophy are regulated by heart-brain interaction. Controlling neural signals might be important for the treatment of hypertensive heart disease.

Clinical Perspective

What Is New?

  • The nervous system controls cardiac inflammation and hypertrophy during pressure overload through NOD-like receptor pyrin domain-containing protein 3 inflammasome activation.

  • Extracellular ATP released from sympathetic efferent nerve terminals activates the NOD-like receptor pyrin domain-containing protein 3 inflammasome in cardiac nonimmune cells through stimulation of the P2X7 purinergic receptor.

  • Pressure overload is sensed by cardiac afferent nerves to activate sympathetic efferent nerves for ATP release.

What Are the Clinical Implications?

  • Controlling neural signals might have therapeutic potential for the treatment of hypertensive heart disease.

  • Lipophilic β-adrenergic receptor blockers might act on the brain as well as on the heart to inhibit signals of the sympathetic nervous system and cardiac inflammation with its ability to cross the blood-brain barrier.

  • Our mechanism might link psychological distress to heart disease.

Cardiac hypertrophy occurs as an adaptive response to pathological stimuli to maintain cardiac function through reduction in wall stress and energy expenditure.1 However, persistent stress responses lead to contractile dysfunction and heart failure.2 Although inflammation is involved in these processes,3 very little is known about the mechanism that controls cardiac inflammation and hypertrophy.

Inflammation is a complex process in which both immune cells and nonimmune cells are involved. Proinflammatory cytokines, including IL-1β (interleukin-1β), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α), induce cellular responses, such as cardiomyocyte hypertrophy and fibroblast and immune cell activation‚ that lead to cardiac hypertrophy and heart failure.3 It has been reported that nonimmune cells, including cardiomyocytes, initiate inflammation by recognizing the endogenous molecules termed danger-associated molecular patterns in the stressed heart, such as heat shock proteins and mitochondrial DNA (mtDNA), through innate immune receptors such as Toll-like receptors (TLRs).4,5 Innate immune receptor signaling activates the transcription factor NF-κB (nuclear factor-κB) for the expression of proinflammatory cytokines, which activate NF-κB again through their receptors in an autocrine and paracrine manner for further inflammatory responses.6 Immune cells such as macrophages and lymphocytes interact with proinflammatory cytokines to modulate their expression pattern.7–9

The NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3) inflammasome is a cytosolic multiprotein complex that mediates active IL-1β production.10 IL-1β is a proinflammatory cytokine that is critically involved in the pathophysiology of hypertrophic heart disease.4,11 This complex consists of the Nod-like receptor family protein NLRP3, the ASC (apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain), and procaspase-1. These components are upregulated through the activation of NF-κB, which is called NLRP3 inflammasome priming. On activation of NLRP3 by exogenous pathogens or endogenous danger signals, procaspase-1 is cleaved into 10-kDa and 20-kDa subunits to form the active enzyme, caspase-1. Subsequently, caspase-1 cleaves inactive pro–IL-1β into active IL-1β. Although the NLRP3 inflammasome in immune cells and nonimmune cells has been implicated in cardiovascular stress and disease,9,12–17 how it is activated and contributes to cardiac hypertrophy remain poorly understood.

Here we show that neural signals contribute to NLRP3 inflammasome activation in cardiac nonimmune cells that initiates inflammation and the adaptive programs in the stressed heart through IL-1β production. We find that pressure overload to the left ventricle activates sympathetic efferent nerves (SENs) to secrete extracellular ATP. Extracellular ATP stimulates the purinergic receptor P2X7 (P2X purinoceptor 7) for NLRP3 inflammasome activation in cardiomyocytes, fibroblasts, and vascular endothelial cells. This mechanism is essential for IL-1β production that causes cardiac adaptive hypertrophy in response to mechanical stress. We also demonstrate that cardiac afferent nerve signals contribute to ATP secretion from SEN terminals. These data collectively reveal that cardiac inflammation and hypertrophy are controlled through heart-brain interaction.

Methods

For detailed description of methods, please see the Supplemental Material. The data that support the findings of this study are available from the corresponding author on reasonable request.

Animal Study

All experiments were approved by the University of Tokyo Ethics Committee for Animal Experiments and strictly adhered to the guidelines for animal experiments of the University of Tokyo. Eight- to 12-week-old male mice were used. Wild-type C57BL/6 mice were purchased from Takasugi Experimental Animal Supply. Nlrp3–/– mice were provided by the laboratory of Dr Tschopp (The University of Lausanne, Switzerland).18P2rx7–/– and Slc17a9flox/flox mice were purchased from the Jackson Laboratory. DBH-Cre mice were provided by RIKEN BRC through the National Bio-Resource Project of MEXT, Japan.19

Pressure overload was induced by transverse aortic constriction (TAC) as described previously.4 Apyrase (4 units, Sigma), lipopolysaccharide (2 mg/kg, InvivoGen), 2′(3′)-O-(4-benzoylbenzoyl)-ATP (BzATP; 5 mg/kg, Sigma), MCC950 (10 mg/kg, Selleck Chemicals), and clonidine (10 μg/kg, Sigma) were injected intraperitoneally daily. Anti–IL-1β antibody or control antibody (100 μg per mouse, R&D Systems) was injected intravenously once. Pseudoephedrine (20 mg·kg–1·d–1, Ieda Chemicals) and bisoprolol hemifumarate (5 mg·kg–1·d–1, Tokyo Chemical Industry) were orally administered to mice by gavage daily. Isoproterenol (Sigma-Aldrich) was administered to mice through an osmotic minipump (30 mg·kg–1·d–1, ALZET mini-osmotic pump, DURECT Corporation). Left stellate ganglionectomy was performed just before TAC or sham operation.20 To ablate primary afferent neurons, subepicardial injection of capsaicin (50 mg/mL, Sigma) dissolved in olive oil (Wako) was performed at 2 weeks before induction of TAC.21

Human Samples

The use of previously obtained human heart biopsy samples from patients with heart failure for daily practice was approved by the Institutional Review Board of the University of Tokyo Hospital, and consent was obtained from all subjects. These samples were fixed in 10% formalin and embedded in paraffin.

Statistical Analyses

Statistical analyses were performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), which is a graphical user interface for R (The R Foundation for Statistical Computing).22 Differences in means between 2 groups were analyzed by unpaired 2-tailed t test. More than 2 groups were compared using parametric or nonparametric 1-way ANOVA followed by Holm test or Dunnett test for multiple comparisons. The Kaplan-Meier method with log-rank test was used for survival analysis. Values of P<0.05 were considered statistically significant.

Results

NLRP3 Inflammasome Is Activated in the Stressed Heart

To investigate the involvement of the NLRP3 inflammasome in hypertrophic heart disease, we first examined gene and protein expression of NLRP3 inflammasome components and IL-1β, and caspase-1 activity and cleavage in myocardial tissue. In wild-type mice, NLRP3 mRNA and protein were upregulated during pressure overload caused by TAC (Figure 1A and Figure S1A and S1B). Immunohistochemical staining demonstrated that NLRP3 is heterogeneously expressed in both cardiomyocytes and noncardiomyocytes in the murine pressure-overloaded heart and the human failing heart (Figure 1B). Gene and protein expression levels of other inflammasome components, such as ASC and procaspase-1, and pro–IL-1β were also upregulated in the pressure-overloaded heart, indicating activation of priming signals (Figure S1). Caspase-1 activity and cleaved caspase-1 level were increased during pressure overload and peaked at day 14 after TAC (Figure 1C and Figure S1B). Protein expression of IL-1β was significantly increased during the adaptive hypertrophic phase until day 14 after TAC (Figure 1D). These results indicate that the NLRP3 inflammasome is activated in the stressed heart, especially during the adaptive hypertrophic phase.

Figure 1.

Figure 1. Genetic disruption of NLRP3 inhibits adaptive cardiac hypertrophy. A, Time course of Nlrp3 gene expression during pressure overload in wild-type (WT) mice (n=5 per group). Quantitative reverse transcription polymerase chain reaction was performed. Expression level of Nlrp3 was normalized to that of Gapdh. B, Immunohistochemical staining for NLRP3 in WT murine heart subjected to 14 days of TAC and in human endomyocardial biopsy sample from patient with heart failure. C and D, Caspase-1 activity (C, n=4 per group) and IL-1β protein level (D, n=5 per group) in pressure-overloaded WT hearts. IL-1β protein was detected by ELISA. Values were normalized to total protein level. E, Time course of echocardiographic parameters in WT and Nlrp3 knockout (KO) mice during pressure overload (n=5 per group). F, Histological analysis of cardiomyocyte cross-sectional area (CSA), collagen volume fraction, macrophage density, and capillary density in pressure-overloaded WT and KO hearts (n=5 per group). G, Survival curve after TAC operation in WT and KO mice (n=20 for WT mice; n=16 for KO mice). H, Hemodynamic parameters in WT and KO hearts subjected to sham or 14 days of TAC (n=5 per group). P values were calculated by 1-way ANOVA with the Dunnett test (A, C, and D) or Holm test (E, F, and H) and Kaplan-Meier method with log-rank test (G). For A, C, and D, *P<0.05 versus day 0. For E and F, *P<0.05 versus day 0; †P<0.05 versus WT mice at the same time point; ‡P<0.05 versus day 4; §P<0.05 versus day 7; ∥P<0.05 versus day 14. For H, *P<0.05 versus sham. †P<0.05 versus WT mice. All error bars represent SEM. BP indicates blood pressure; IL-1β, interleukin-1β; IVSth, interventricular septum thickness; LVEDd, left ventricle end-diastolic diameter; LVEDP, left ventricle end-diastolic pressure; LVPWth, left ventricular posterior wall thickness; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3; and TAC, transverse aortic constriction.

Genetic Disruption of NLRP3 Inhibits IL-1β Production and Adaptive Cardiac Hypertrophy

To examine the role of the NLRP3 inflammasome in hypertrophic heart disease, we induced pressure overload by TAC in Nlrp3+/+ and Nlrp3–/– mice.18 We confirmed deletion of NLRP3 protein in hearts from Nlrp3–/– mice by Western blot analysis (Figure S2A). In the absence of pressure overload, cardiac function and morphology did not differ between Nlrp3+/+ and Nlrp3–/– mice (Figure 1E and 1F and Figure S2B). In Nlrp3+/+ mice, pressure overload induced cardiac hypertrophy with preserved contractile function until day 14 (adaptive phase) and heart failure at day 28 after TAC (Figure 1E and Figure S2B). On the other hand, Nlrp3–/– hearts showed attenuated cardiac hypertrophy, but greater left ventricular dilation with impaired contractile function compared with Nlrp3+/+ hearts in the adaptive phase after TAC (Figure 1E and Figure S2B). In the heart-failure phase, cardiac function and chamber size were similar between Nlrp3+/+ and Nlrp3–/– hearts, whereas cardiac hypertrophy remained attenuated in Nlrp3–/– hearts (Figure 1E). Histological assessment demonstrated significantly smaller cardiomyocytes and reduced cardiac fibrosis and macrophage infiltration in Nlrp3–/– hearts compared with Nlrp3+/+ hearts during pressure overload (Figure 1F and Figure S2C–S2E). In the adaptive phase, angiogenesis was suppressed in Nlrp3–/– hearts (Figure 1F and Figure S2F). Mortality after TAC was higher in Nlrp3–/– mice than in Nlrp3+/+ mice (Figure 1G), whereas all sham-operated Nlrp3+/+ and Nlrp3–/– mice survived (n=8 for each). All deaths of Nlrp3–/– mice were observed in the adaptive phase. Hemodynamic measurement revealed higher left ventricle end-diastolic pressure and smaller absolute values of maximum and minimum dp/dt in Nlrp3–/– mice than in Nlrp3+/+ mice after 14 days of pressure overload, whereas blood pressure in the ascending aorta was similar between these mice (Figure 1H). There were no significant differences in hemodynamic parameters between Nlrp3+/+ and Nlrp3–/– mice at 28 days after TAC or sham operation (Figure S2G). These results collectively indicate that genetic disruption of NLRP3 prevented pathological cardiac remodeling but might have impaired cardiac adaptative response to pressure overload.

At 14 days after TAC, the mRNA levels of hypertrophic marker genes (Nppa and Myh7), a fibrosis-related gene (Col1a1), an angiogenesis-related gene (Vegfa), and inflammation-related genes (Il6, Mcp1, and Tnfa) were consistently upregulated in wild-type hearts, whereas they were suppressed in Nlrp3–/– hearts (Figure 2A). Caspase-1 activity and cleaved caspase-1 and IL-1β levels were lower in Nlrp3–/– hearts than in wild-type hearts (Figure 2B–2D). Mitochondrial dysfunction is one of the characteristics of failing cardiomyocytes. To examine mitochondrial function, we assessed mtDNA content and oxidative damage and expression levels of genes associated with mitochondrial oxidative phosphorylation in the heart (Figure 2E and Figure S3).23 Although Nlrp3+/+ failing hearts showed the decrease in mtDNA content and expression levels of some of the oxidative phosphorylation genes and the increase in mtDNA oxidative damage, no significant changes in these indicators of mitochondrial function were observed in Nlrp3–/– hearts during pressure overload. These results indicate that mitochondrial function during pressure overload was not impaired in Nlrp3–/– hearts. Thus, contractile dysfunction in Nlrp3–/– mice during pressure overload might not be attributable to pathological changes of cardiomyocytes, but to insufficient hemodynamic adaptation to pressure overload.

Figure 2.

Figure 2. NLRP3 inflammasome activation contributes to pathological cardiac remodeling during pressure overload. A, Expression levels of hypertrophic marker genes such as Nppa and Myh7, a fibrosis-related gene such as Col1a1, an angiogenesis-related gene such as Vegfa, and inflammation-related genes such as Il6, Mcp1, and Tnfa in wild-type (WT) and Nlrp3 knockout (KO) hearts. Mice were subjected to sham or 14 days of TAC (n=5 per group). Expression level of each gene was determined by quantitative reverse transcription polymerase chain reaction, and normalized to that of Gapdh. B, Caspase-1 activity with and without a specific caspase-1 inhibitor in pressure-overloaded hearts (n=4 per group). Values were normalized to total protein level. C, Western blot for procapase-1, cleaved caspase-1 (p20), and β-actin in pressure-overloaded hearts. D, IL-1β protein level in WT and KO hearts subjected to sham or 14 days of TAC (n=5 per group). IL-1β protein was detected by ELISA. Values were normalized to total protein level. E, Mitochondrial DNA (mtDNA) content in pressure-overloaded hearts (n=3 per group). DNA extracted from myocardial tissues was subjected to quantitative real-time polymerase chain reaction with specific primer sets for cytochrome b (Cytb; mtDNA) and β-actin (Actb; nuclear DNA). mtDNA to nuclear DNA ratios were measured. F and G, Myocardial caspase-1 activity (F) and IL-1β protein level (G) in WT mice with WT bone marrow (W→W) or KO bone marrow (K→W) subjected to sham or 14 days of TAC. IL-1β protein was detected by ELISA. Values were normalized to total protein level. For F, n=4 per group. For G, n=3 for sham-operated W→W mice; n=4 for sham-operated K→W mice; n=5 for TAC-operated W→W and K→W mice. P values were calculated by parametric or nonparametric 1-way ANOVA with Holm test. For A, B, and D, *P<0.05 versus sham. †P<0.05 versus WT mice. For E, *P<0.05 versus day 14; †P<0.05 versus WT mice. For F and G, *P<0.05 versus sham. All error bars represent SEM. IL-1β indicates interleukin-1β; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3; and TAC, transverse aortic constriction.

To dissect the role of NLRP3 in immune cells from that in nonimmune cells, we performed a bone marrow transplantation experiment (Figures 2F and 2G and Figure S4). Caspase-1 activation and IL-1β production in the heart did not differ between wild-type mice transplanted with Nlrp3+/+ and Nlrp3–/– bone marrow during pressure overload (Figure 2F and 2G and Figure S4A). Both mice showed cardiac hypertrophy with preserved contractile function after 14 days of TAC to a similar extent (Figure S4B and S4C). Cardiomyocyte hypertrophy, fibrosis, macrophage infiltration, and angiogenesis in pressure-overloaded hearts were consistently similar between wild-type mice with Nlrp3+/+ and Nlrp3–/– bone marrow (Figure S4D–S4G). No significant differences in hemodynamic parameters were observed between these mice (Figure S4H). These data collectively indicate that NLRP3 inflammasome activation in cardiac nonimmune cells initiate inflammation and adaptive cardiac hypertrophy in response to pressure overload.

ATP/P2X7 Axis Contributes to NLRP3 Inflammasome Activation and Cardiac Hypertrophy During Pressure Overload

Three models of NLRP3 inflammasome activation have been widely suggested: potassium efflux through the P2X7 purinergic receptor stimulated by extracellular ATP, reactive oxygen species–dependent dissociation of TXNIP (thioredoxin-interacting protein) from thioredoxin, and cathepsin B release through lysosomal rupture attributable to mechanical insult by crystalline ligands.24 We focused on the first mechanism because, during pressure overload, TXNIP level was not upregulated in the heart (Figure S5A) and crystalline ligands are considered not to be produced.

To investigate the involvement of extracellular ATP and the P2X7 receptor in NLRP3 inflammasome activation and cardiac hypertrophy, we induced pressure overload in wild-type mice treated with an ATP diphosphohydrolase, apyrase,25 and P2rx7–/– mice. In these mice, caspase-1 activation and IL-1β production were suppressed during pressure overload compared with wild-type mice treated with vehicle or P2rx7+/+ mice (Figure 3A–3D and Figure S5B and S5C). We also found attenuated cardiac hypertrophy, greater ventricular dilation and contractile dysfunction with smaller cardiomyocytes, reduced cardiac fibrosis and macrophage infiltration, and lower capillary density in these mice compared with control mice on day 14 after TAC (Figure 3E and 3F and Figures S5D, S5E, and S6A–S6H). Hemodynamic measurement showed that apyrase treatment or genetic disruption of the P2X7 receptor led to higher left ventricle end-diastolic pressure and smaller maximum dp/dt in pressure-overloaded hearts without affecting blood pressure in the ascending aorta (Figure S6I and S6J). These data indicate that extracellular ATP and the P2X7 receptor are required for NLRP3 inflammasome activation and adaptive cardiac hypertrophy in response to pressure overload.

Figure 3.

Figure 3. ATP/P2X7 axis is involved in NLRP3 inflammasome activation and cardiac hypertrophy during pressure overload. A and B, Caspase-1 activity (A) and IL-1β protein level (B) in vehicle-treated (V-treated) or apyrase-treated (APR-treated) wild-type (WT) hearts subjected to sham or 14 days of TAC. IL-1β protein was detected by ELISA. Values were normalized to total protein level. For A, n=4 per group. For B, n=3 for sham; n=5 for TAC. C and D, Caspase-1 activity (C) and IL-1β protein level (D) in WT and P2rx7 knockout (KO) hearts subjected to sham or 14 days of TAC. For C, n=4 per group. For D, n=5 per group. E and F, Echocardiographic parameters in V- or APR-treated WT mice (n=6 for sham; n=7 for TAC; E), and WT and KO mice (n=5 for sham; n=7 for TAC; F). G through I, Cell surface area of cardiomyocytes after 48-hour stimulation (G) and proliferation of cardiac fibroblasts (H) and human microvascular endothelial cells from the heart (HMVEC-C; I) after 24-hour stimulation with Pam3CSK4, ATP, or both under treatment with NLRP3 siRNA or scrambled siRNA. G, Cell surface area was measured in specimens by anti–sarcomeric α-actinin staining (n=40 per group). Scale bars, 20 μm. H and I, Cell proliferation was assessed by MTS (dimethylthiazol-carboxymethoxyphenyl-sulfophenyl-tetrazolium) assay. The percentage of the absorbance of wells with cells treated with scrambled siRNA and vehicle was calculated (n=7 per group for fibroblasts; n=5 per group for HMVEC-C). Data are expressed as a fold-change relative to the control group. P values were calculated by parametric or nonparametric 1-way ANOVA with Holm test. For A through F, *P<0.05 versus sham. †P<0.05 versus vehicle-treated (V-treated; A, B, and E) or WT (C, D, and F) mice. For G through I, *P<0.05 versus vehicle; †P<0.05 versus Pam3CSK4; ‡P<0.05 versus ATP; §P<0.05 versus scrambled siRNA. All error bars represent SEM. IL-1β indicates interleukin-1β; IVSth, interventricular septum thickness; LVEDd, left ventricle end-diastolic diameter; LVPWth, left ventricular posterior wall thickness; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3; siRNA, small interfering RNA; and TAC, transverse aortic constriction.

Role of ATP/P2X7 Axis in the Heart

To clarify the role of the ATP/P2X7 axis and the NLRP3 inflammasome in cardiac cells, we assessed their effect on cardiomyocyte hypertrophy and proliferation of fibroblasts and vascular endothelial cells, which are important processes in cardiac hypertrophy.2,4 We previously showed that TLR2 signaling is essential for IL-1β mRNA production in the stressed heart.4 A specific ligand for TLR2, Pam3CSK4,4 induced cardiomyocyte hypertrophy and proliferation of fibroblasts and vascular endothelial cells (Figure 3G–3I). Treatment with ATP in combination with Pam3CSK4 resulted in further hypertrophy and proliferation. Pharmacological inhibition of the P2X7 receptor, Nlrp3 knockdown, or anti–IL-1β neutralization antibody treatment inhibited the synergistic effects of ATP and Pam3CSK4 on cardiomyocytes, fibroblasts, and vascular endothelial cells (Figure 3G–3I and Figure S7). These data indicate that the ATP/P2X7 axis contributes to hypertrophic responses of cardiac cells through the NLRP3 inflammasome activation and IL-1β production.

We next examined whether activation of the P2X7 receptor, with or without an inflammasome priming signal, induces cardiac hypertrophy in vivo. Wild-type mice treated with a combination of BzATP, a P2X7 receptor agonist, and lipopolysaccharide, a ligand of TLR4, for 14 days showed cardiac hypertrophy and increased myocardial caspase-1 activity and IL-1β level with preserved systolic function and chamber size, compared with those treated with vehicle (Figure S8A–S8C). Histological assessment demonstrated cardiomyocyte hypertrophy and augmented interstitial fibrosis in wild-type mice treated with the combination (Figure S8D–S8F), which was confirmed by the increase in expression levels of Myh7 and Col1a1(Figure S8G–S8I). At our dose, BzATP or lipopolysaccharide alone did not induce cardiac hypertrophy (Figure S8A). Treatment with BzATP alone increased myocardial caspase-1 activity, but to a lesser extent than the combination treatment, which did not lead to IL-1β production (Figure S8B and S8C). Treatment with MCC950, a potent and specific NLRP3 inhibitor, or anti-IL-1β antibodies suppressed caspase-1 activity, IL-1β level, and cardiac hypertrophy without affecting systolic function or chamber size in wild-type mice treated with the combination of BzATP and lipopolysaccharide, compared with control treatment (Figure S8A–S8I). Blood pressure was not affected by these treatments (Figure S8J). The P2X7 receptor has been implicated in cardiomyocyte hypertrophy and cellular survival through NLRP3 inflammasome-independent mechanisms.26 Our data suggest that P2X7 receptor signaling might regulate cardiac hypertrophic changes in vivo mainly through the NLRP3 inflammasome activation and IL-1β production, rather than through NLRP3 inflammasome-independent mechanisms. In addition, IL-1β might affect myocardial caspase-1 activity in a positive-feedback manner through NF-κB activation.

SENs Release ATP for NLRP3 Inflammasome Activation

To examine the dynamics of extracellular ATP during pressure overload, we measured its concentration in the heart with an ATP-sensing electrode (Figure 4A and Figure S9A).27 We found that extracellular ATP was increased in wild-type hearts on day 14 after TAC compared with sham-operated mice, whereas apyrase treatment reduced its concentration (Figure 4B and Figure S9B–S9D). We also visualized extracellular ATP in vivo by expressing the engineered firefly luciferase, called pmeLUC, which localizes to the outer aspect of the plasma membrane with the catalytic site facing the extracellular environment,28 in cardiomyocytes through the use of adeno-associated virus, and confirmed the increase of extracellular ATP by pressure overload (Figures 4C–4F). As a danger signal, ATP is released by necrotic or apoptotic cells in damaged organs. In the pressure-overloaded heart, however, massive cell death does not occur; thus, extracellular ATP may be released by active transport from living cells. It is reported that cardiac cells can release ATP only below a physiologically active concentration.29 Because ATP is a neurotransmitter and is secreted from nerve terminals,30 we hypothesized that the autonomic nervous system may be the main source of extracellular ATP for NLRP3 inflammasome activation in the heart during pressure overload.

Figure 4.

Figure 4. SENs Release ATP for NLRP3 inflammasome activation. A, Schematic of extracellular ATP measurement using enzyme-based biosensors. Electric current between the reference electrode and the ATP biosensor in the left ventricle (LV) was measured through a potentiostat and transformed into ATP level on the basis of the calibration curve. B, Extracellular ATP level in the heart in sham-operated wild-type mice (WT sham), TAC-operated WT mice (WT TAC), TAC-operated mice with left stellate ganglionectomy (SGN TAC), pseudoephedrine-treated mice (PE), isoproterenol-treated mice (ISO), TAC-operated mice treated with apyrase (APR TAC), clonidine (CLN TAC), or capsaicin (CAP TAC), and TAC-operated Slc17a9flox/flox;DBH-Cre mice (CWT TAC) and Slc17a9flox/flox;DBH-Cre+ mice (CKO TAC; n=3 mice per group). Mice were subjected to 14 days of treatment. C, Schematic of adeno-associated virus vector harboring an engineered firefly luciferase, called pmeLUC, that localizes to the outer aspect of the plasma membrane with the catalytic site facing the extracellular environment, in the downstream of the chicken cardiac troponin T promoter. This protein contains the Myc epitope tag. D, Western blot for pmeLUC and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in murine hearts with and without the transduction of AAV9 vector harboring pmeLUC. The pmeLUC protein was detected by anti–c-Myc antibody. E and F, Representative image (E) and data analysis (F) of extracellular ATP detection by the IVIS luminometer in WT hearts subjected to sham or 14 days of TAC. The average of luminescent signals within region of interest (ie, the heart) was calculated for comparison (n=3 per group). G, Immunohistochemical staining for neurofilament (NF) and tyrosine hydroxylase (TH) in WT murine heart subjected to sham or 14 days of TAC with consecutive slices. Nerve fibers are stained by anti-NF antibody. Catecholaminergic nerve fibers are stained by anti-TH antibody. Total areas of epicardial TH-positive nerve fibers were measured for comparison (n=3 per group). H through K, Myocardial caspase-1 activity (H) and IL-1β protein level (I), ejection fraction (J), and cardiomyocyte cross-sectional area (CSA; K) in sham or TAC-operated mice with sham (S) or SGN and with vehicle (V) or CLN. H, n=4 per group. I, For SGN experiment, n=3 per group. For CLN experiment, n=3 for sham; n=5 for V TAC; n=6 for CLN TAC. J, For SGN experiment, n=7 per group. For CLN experiment, n=6 for sham; n=7 for TAC. K, n=5 per group. L, Caspase-1 activity and IL-1β protein level in WT and Nlrp3 knockout (KO) hearts treated with vehicle or PE (n=4 per group). IL-1β protein was detected by ELISA. Values were normalized to total protein level. M, Cardiomyocyte CSA (n=6 per group) and myocardial caspase-1 activity (n=4 per group) and IL-1β protein level (n=4 per group) in WT and KO mice treated with vehicle or ISO. IL-1β protein was detected by ELISA. Values were normalized to total protein level. P values were calculated by parametric or nonparametric 1-way ANOVA with Holm test or unpaired 2-tailed t test. For B, *P<0.05 versus WT sham. For F, *P<0.05 versus sham. For H through K, *P<0.05 versus sham. †P<0.05 versus mice treated with S or V. For L and M, *P<0.05 versus vehicle. †P<0.05 versus WT mice. All error bars represent S.E.M. DBH indicates dopamine β-hydroxylase; GPI, glycosylphosphatidylinositol; IL-1β, interleukin-1β; ITR, inverted terminal repeat; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3; SEN, sympathetic efferent nerve; and TAC, transverse aortic constriction.

We first examined the role of the sympathetic nervous system (SNS), which innervates the left ventricle more abundantly than does the parasympathetic nervous system, in ATP release and NLRP3 inflammasome activation in the pressure-overloaded heart.31 Immunohistochemistry confirmed the presence of catecholaminergic nerve fibers in the epicardial nerve bundles around the left ventricle (Figure 4G). No significant difference in total areas of epicardial catecholaminergic nerve fibers was observed between sham-operated and TAC-operated hearts at day 14 after the operations. We measured extracellular ATP level in the left ventricle of wild-type mice treated with ablation of the left stellate ganglion,20 the main source of the SEN terminals in the left ventricle, and clonidine, an SNS suppressant through stimulation of the α2-adrenergic receptor in the central nervous system. In these mice, extracellular ATP level during pressure overload was suppressed compared with wild-type mice without any treatment (Figure 4B and Figure S9E and S9F). In addition, these mice showed impairment of the cardiac adaptive response to pressure overload, with suppressed caspase-1 activation and IL-1β production, whereas systolic blood pressure was not affected by ablation of the left stellate ganglionectomy or clonidine treatment (Figure 4H–4K and Figure S10 and S11). These data indicate that SEN signals to the heart are required for the increase of extracellular ATP level and NLRP3 inflammasome activation to induce cardiac adaptive hypertrophy during pressure overload. In addition, it is suggested that the central nervous system is involved in this mechanism.

Next, we investigated whether SNS activation is sufficient for the increase of extracellular ATP level and NLRP3 inflammasome activation in the heart. Sympathetic activation by pseudoephedrine increased extracellular ATP level and caspase-1 activity in wild-type hearts (Figure 4B and 4L and Figure S9G and S12A). In pseudoephedrine-treated Nlrp3–/– hearts, caspase-1 activation was suppressed compared with that in pseudoephedrine-treated wild-type hearts (Figure 4L and Figure S12A). We did not find significant differences in systolic blood pressure and heart rate between pseudoephedrine-treated Nlrp3+/+ and Nlrp3–/– mice, although pseudoephedrine slightly increased systolic blood pressure in these mice compared with vehicle (Figure S12B and S12C). Thus, sympathetic activation induces extracellular ATP release and NLRP3 inflammasome-dependent caspase-1 activation in the heart.

At our dose, pseudoephedrine did not induce IL-1β production and cardiac hypertrophy in both wild-type and Nlrp3–/– mice, although pseudoephedrine slightly increased macrophage infiltration (Figure 4L and Figure S12D–S12I). These findings suggest that NLRP3 inflammasome activation by the SNS alone might not be sufficient for IL-1β production. Other signaling pathways such as TLR signaling, which upregulates IL-1β mRNA, might be necessary for IL-1β production and cardiac hypertrophy.

We next investigated the effect of adrenergic signals on extracellular ATP level and NLRP3 inflammasome activation in the heart. Infusion of isoproterenol, a β-adrenergic receptor agonist, induced cardiac hypertrophy with fibrosis, macrophage infiltration, and angiogenesis in both wild-type and Nlrp3–/– mice (Figure 4M and Figure S13A–S13F). No significant differences in systolic blood pressure and heart rate were observed between isoproterenol-treated Nlrp3+/+ and Nlrp3–/– mice, although isoproterenol slightly increased systolic blood pressure and heart rate compared with vehicle (Figure S13G and S13H). Norepinephrine, a main neurotransmitter of the SENs, induced cardiomyocyte hypertrophy and proliferation of cardiac fibroblasts and vascular endothelial cells in an NLRP3 inflammasome-independent manner in vitro (Figure S14). Isoproterenol did not significantly increase extracellular ATP level in the heart (Figure 4B and Figure S9H). Caspase-1 activity and cleaved caspase-1 level were increased in both isoproterenol-treated wild-type and Nlrp3–/– mice (Figure 4M and Figure S13I). IL-1β level was not increased in these mice (Figure 4M).These data collectively suggest that, in the pressure-overloaded heart, ATP might be released mainly from SEN terminals rather than from cardiac cells, stimulated by adrenergic signals. In addition, adrenergic signals might be able to induce caspase-1 activation and cardiac hypertrophy in an NLRP3 inflammasome-independent manner.

ATP released from the sympathetic nerve terminals has been reported to modulate presynaptic norepinephrine release through activation of purinergic receptors.32,33 To examine the effect of extracellular ATP on presynaptic norepinephrine release and the role of norepinephrine on NLRP3 inflammasome activation and cardiac phenotype in the pressure-overloaded heart, we next measured myocardial and plasma norepinephrine levels. We found that pressure overload by TAC increases myocardial and plasma norepinephrine levels in wild-type mice (Figure S15). No significant differences in myocardial and plasma norepinephrine levels were detected between TAC-operated Nlrp3+/+ and Nlrp3–/– mice. Apyrase treatment or genetic disruption of the P2X7 receptor did not affect norepinephrine level in the heart or plasma on day 14 after TAC or sham operation. We did not observe the increase of myocardial norepinephrine level after TAC in wild-type mice treated with ablation of the left stellate ganglion, whereas this treatment did not have an effect on plasma norepinephrine level, which might reflect the contribution of the left stellate ganglion to sympathetic innervation in the left ventricle. These data suggest that ATP might regulate presynaptic norepinephrine release with various positive and negative feedbacks through purinergic receptors, including P2X and P2Y receptors,32 which might explain why myocardial norepinephrine level was not altered by ATP depletion or genetic disruption of the P2X7 receptor. In addition, our data indicate that norepinephrine might not contribute to NLRP3 inflammasome activation and cardiac phenotype in our TAC model. Furthermore, pressure overload might increase systemic sympathetic neural activity and norepinephrine in the blood, whereas the increase of norepinephrine in the pressure-overloaded heart might be attributable mainly to its release from the SEN terminals in the heart.

ATP From SENs Is Essential for Cardiac Hypertrophy

Vesicular nucleotide transporter (also known as Slc17a9) is a secretory vesicle protein that is responsible for the storage and release of ATP in neurons.34 To clearly dissect the role of ATP release from that of norepinephrine release by the SNS in the pressure-overloaded heart, we crossed mice bearing an Slc17a9flox allele with transgenic mice expressing Cre recombinase under the control of the dopamine β-hydroxylase (DBH) promoter to generate Slc17a9flox/flox;DBH-Cre+ (Slc17a9–/–) mice.19 The DBH promoter drives gene expression in noradrenergic and adrenergic cell groups, including postganglionic neurons in the SNS (Figure 5A). In Slc17a9–/– mice, ATP release from SEN terminals is inhibited, whereas norepinephrine release remains intact. We used Slc17a9flox/flox;DBH-Cre- (Slc17a9+/+) littermates as controls. Slc17a9–/– mice did not display a cardiac structural or functional deficit at baseline. In Slc17a9–/– mice, extracellular ATP release, caspase-1 activation, and IL-1β production in the heart were suppressed compared with those in Slc17a9+/+ mice on day 14 after TAC (Figures 4B and 5B–5D and Figure S9I and S9J). Myocardial and plasma norepinephrine levels were comparable between Slc17a9+/+ and Slc17a9–/– mice (Figure 5E and 5F). Slc17a9–/– mice showed attenuated cardiac hypertrophy and contractile dysfunction, with reduced cardiomyocyte hypertrophy, fibrosis, capillary density, and macrophage infiltration compared with Slc17a9+/+ mice, indicating impairment of the adaptive mechanisms in response to pressure overload (Figure 5G–5L). Higher left ventricle end-diastolic pressure and smaller maximum dp/dt in pressure-overloaded hearts were consistently observed in Slc17a9–/– mice compared with Slc17a9+/+ mice, although blood pressure in the ascending aorta was comparable between these mice (Figure 5M). Collectively, SEN signals directly regulate NLRP3 inflammasome activation and cardiac adaptive hypertrophy through ATP release.

Figure 5.

Figure 5. ATP from sympathetic efferent nerves is essential for cardiac hypertrophy. A, Gene expression of Slc17a9 in left stellate ganglion of Slc17a9flox/flox;DBH-Cre- (WT) and Slc17a9flox/flox;DBH-Cre+ (CKO) mice determined by quantitative reverse transcription polymerase chain reaction (n=5 per group). Expression level of Slc17a9 was normalized to that of Gapdh. *P<0.05 versus WT mice. B through M, WT and CKO mice were subjected to sham or 14 days of TAC. B, Myocardial caspase-1 activity (n=4 per group). Values were normalized to total protein level. C, Western blot for procapase-1, cleaved caspase-1 (p20), and β-actin in the heart. D, Myocardial IL-1β protein level (n=5 per group). IL-1β protein was detected by ELISA. Values were normalized to total protein level. E and F, Myocardial and plasma norepinephrine (NE) concentration (n=4 per group). NE was detected by ELISA. Myocardial NE levels were normalized to tissue weight. G, Heart weight (HW) to body weight (BW) ratio (n=5 for sham; n=7 for TAC). H, Echocardiographic analysis of ejection fraction, left ventricle end-diastolic diameter (LVEDd), interventricular septum thickness (IVSth), and left ventricular posterior wall thickness (LVPWth; n=5 for sham; n=7 for TAC). I through L, Histological analysis of cardiomyocyte cross-sectional area (CSA; I), collagen volume fraction (CVF; J), macrophage density (K), and capillary density (L; n=5 for each group). Quantification of cardiomyocyte CSA and CVF was performed in specimens stained with hematoxylin and eosin or Sirius Red dye, respectively. Quantification of macrophage and capillary density was performed by immunohistochemical staining for Mac3 and CD31, respectively. Representative images are shown for each analysis. Scale bars, 20 μm for I, K, and L. Scale bars, 50 μm for J. M, Hemodynamic parameters (n=5 per group). P values were calculated by parametric or nonparametric 1-way ANOVA with Holm test. *P<0.05 versus sham. †P<0.05 versus WT mice. All error bars represent SEM. BP indicates blood pressure; CKO, conditional knockout; DBH, dopamine β-hydroxylase; IL-1β, interleukin-1β; LVEDP, left ventricle end-diastolic pressure; TAC, transverse aortic constriction; and WT, wild type.

Role of Cardiac Afferent Nerves in ATP Release From SENs and NLRP3 Inflammasome Activation

We next assessed whether afferent input signals from the heart contribute to ATP release from SENs and NLRP3 inflammasome activation. No significant difference in total areas of epicardial primary afferent nerve fibers was observed between sham-operated and TAC-operated hearts at day 14 after the operations (Figure 6A). We treated the heart with capsaicin, an agonist for the transient receptor potential vanilloid type 1 channel that is predominantly expressed on the terminals of primary sensory neurons, to ablate afferent nerves from the heart.8,21 Immunohistochemical staining confirmed ablation of primary sensory nerve fibers without depletion of catecholaminergic nerve fibers consistently with the previous reports (Figure 6B). Cardiac function and morphology did not differ between sham-operated wild-type mice treated with capsaicin or vehicle (Figures 6C–6H). Capsaicin-treated hearts showed reduced extracellular ATP level and suppressed caspase-1 activation and IL-1β production during pressure overload compared with vehicle-treated mice (Figures 4B and 6I–6K and Figure S9K). Ablation of cardiac afferent nerves attenuated cardiac hypertrophy but resulted in greater left ventricular dilation and impaired systolic function with higher left ventricle end-diastolic pressure and smaller absolute values of maximum and minimum dp/dt on day 14 after TAC, but this treatment did not affect systolic blood pressure in the ascending aorta (Figure 6C, 6D, and 6L). Histological assessment revealed reduced cardiomyocyte hypertrophy, interstitial fibrosis, capillary density, and macrophage infiltration by capsaicin treatment during pressure overload (Figure 6E–6H). Thus, ablation of cardiac afferent nerves impaired adaptive cardiac hypertrophy. These data indicate that afferent nerve signals are required for ATP release from SEN terminals and NLRP3 inflammasome activation for cardiac adaptive hypertrophy during pressure overload.

Figure 6.

Figure 6. Role of cardiac afferent nerves in ATP release from sympathetic efferent nerves and NLRP3 inflammasome activation. A, Immunohistochemical staining for neurofilament (NF) and calcitonin gene–related peptide (CGRP) in wild-type murine heart subjected to sham or 14 days of TAC with consecutive slices. Nerve fibers are stained by anti-NF antibody. Primary afferent nerve fibers are stained by anti-CGRP antibody. Total areas of epicardial CGRP-positive nerve fibers were measured for comparison (n=3 per group). B, Immunohistochemical staining for NF, tyrosine hydroxylase (TH), and CGRP in murine hearts treated with olive oil (O) or capsaicin (CAP) for ablation of the cardiac afferent nerves with consecutive slices. Catecholaminergic nerve fibers are stained by anti-TH antibody. C through L, Wild-type mice treated with O or CAP were subjected to sham or 14 days of TAC. C, Heart weight (HW) to body weight (BW) ratio (n=6 for sham; n=8 for TAC). D, Echocardiographic analysis of ejection fraction, left ventricle end-diastolic diameter (LVEDd), interventricular septum thickness (IVSth), and left ventricular posterior wall thickness (LVPWth; n=6 for sham; n=8 for TAC). E through H, Histological analysis of cardiomyocyte cross-sectional area (CSA; E), collagen volume fraction (CVF; F), macrophage density (G). and capillary density (H) in the heart (n=5 for each group). Quantification of cardiomyocyte CSA and CVF was performed in specimens stained with hematoxylin and eosin or Sirius Red dye, respectively. Quantification of macrophage and capillary density was performed by immunohistochemical staining for Mac3 and CD31, respectively. Representative images are shown for each analysis. Scale bars, 20 μm for E, G, and H. Scale bars, 50 μm for F. I, Myocardial caspase-1 activity (n=4 per group). Values were normalized to total protein level. J, Western blot for procapase-1, cleaved caspase-1 (p20), and β-actin in the heart. K, Myocardial IL-1β protein level (n=5 per group). IL-1β protein was detected by ELISA. Values were normalized to total protein level. L, Hemodynamic parameters (n=5 per group). Values were normalized to total protein level. P values were calculated by parametric or nonparametric 1-way ANOVA with Holm test or unpaired 2-tailed t-test. *P<0.05 versus sham. †P<0.05 versus wild-type mice treated with O. All error bars represent SEM. BP indicates blood pressure; IL-1β, interleukin-1β; LVEDP, left ventricle end-diastolic pressure; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3; and TAC, transverse aortic constriction.

NLRP3 Inflammasome Activity in Isolated Perfused Hearts Ex Vivo

To further examine the significance of neural signals in NLRP3 inflammasome activation in the pressure-overloaded heart, we performed an ex vivo experiment using the Langendorff perfused heart model.35 In this experimental model, input and output neural signals in the heart are completely ablated. We found no significant differences in caspase-1 activity and IL-1β production between hearts with and without a pressure overload of ≈40 mm Hg for 60 minutes (Figure S16). Our data suggest that pressure overload itself might not be sufficient for NLRP3 inflammasome activation and IL-1β production, although our experiment could assess only short-term responses.

Bisoprolol Inhibits Extracellular ATP Release and NLRP3 Inflammasome Activation

Lipophilic β-adrenergic receptor blockers are used as standard therapy for cardiac remodeling and heart failure.36 These drugs can cross the blood-brain barrier. To investigate the effect of lipophilic β-adrenergic receptor blockers on extracellular ATP release and NLRP3 inflammasome activation in the heart, we induced pressure overload in wild-type mice treated with a lipophilic β-adrenergic receptor blocker, bisoprolol, or vehicle. Our tested dose of bisoprolol lowered heart rate, but it did not change systolic blood pressure in wild-type mice (Figure 7A). We assessed extracellular ATP levels by using the pmeLUC system and found that bisoprolol reduced extracellular ATP in TAC-operated hearts (Figure 7B and 7C). In bisoprolol-treated mice, caspase-1 activation and IL-1β production were suppressed during pressure overload compared with vehicle-treated mice (Figure 7D and 7E). We observed attenuated cardiac hypertrophy, greater ventricular dilation, and reduced ejection fraction with smaller cardiomyocytes, reduced cardiac fibrosis and macrophage infiltration, and lower capillary density in bisoprolol-treated mice compared with control mice at day 14 after TAC (Figure 7F–7K). Ejection fraction remained >50% in bisoprolol-treated mice. These results indicate that bisoprolol ameliorated cardiac inflammation and pathological cardiac remodeling, possibly in part, by inhibiting extracellular ATP release from the sympathetic nerve terminals, although our tested dose of bisoprolol suppressed systolic function under persistent pressure overload.

Figure 7.

Figure 7. Bisoprolol inhibits extracellular ATP release, NLRP3 inflammasome activation, and cardiac hypertrophy. A, Systolic blood pressure (BP) and heart rate in wild-type mice treated with vehicle (V) or bisoprolol (BIS) for 14 days (n=4 per group). B through K, Wild-type mice treated with V or BIS were subjected to sham or 14 days of TAC. B and C, Representative image (B) and data analysis (C) of extracellular ATP detection by the IVIS luminometer. The average of luminescent signals within region of interest (ie, the heart) was calculated for comparison (n=4 for sham; n=5 for TAC). D and E, Myocardial caspase-1 activity (D, n=4 per group) and IL-1β protein level (E, n=4 for sham; n=6 for TAC). IL-1β protein was detected by ELISA. Values were normalized to total protein level. F, Heart weight (HW) to body weight (BW) ratio (n=4 for sham; n=6 for TAC). G, Echocardiographic analysis of ejection fraction, left ventricle end-diastolic diameter (LVEDd), interventricular septum thickness (IVSth), and left ventricular posterior wall thickness (LVPWth; n=4 for sham; n=6 for TAC). H through K, Histological analysis of cardiomyocyte cross-sectional area (CSA; H), collagen volume fraction (CVF; I), macrophage density (J), and capillary density (K; n=4 for sham; n=6 for TAC). Quantification of cardiomyocyte CSA and CVF was performed in specimens stained with hematoxylin and eosin or Sirius Red dye, respectively. Quantification of macrophage and capillary density was performed by immunohistochemical staining for Mac3 and CD31, respectively. Representative images are shown for each analysis. Scale bars, 20 μm for H, J, and K. Scale bars, 50 μm for I. P values were calculated by parametric or nonparametric 1-way ANOVA with Holm test or unpaired 2-tailed t test. For A, *P<0.05 versus V. For C through K, *P<0.05 versus sham. †P<0.05 versus wild-type mice treated with V. All error bars represent SEM. IL-1β indicates interleukin-1β; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3; and TAC, transverse aortic constriction.

Discussion

In this study, we demonstrated that neural signals control cardiac inflammation and hypertrophy through NLRP3 inflammasome activation and IL-1β production during pressure overload (Figure 8). IL-1β is a key proinflammatory cytokine that contributes to the pathophysiology of hypertrophic heart disease.4,11 Active IL-1β production is tightly controlled in a 2-step process.10 The first step upregulates the inactive precursor, pro–IL-1β, by promoting the transcription of IL-1β gene. The second step processes pro–IL-1β into active IL-1β by activating caspase-1. In the heart, the first step is activated by local proinflammatory mechanisms, in which danger-associated molecular patterns from damaged cardiac cells stimulate innate immune receptors for NF-κB activation in an autocrine and paracrine manner.4,5 Our data show that the second step is regulated by the nervous system, including the central nervous system, through NLRP3 inflammasome activation. Thus, both local mechanisms and organ communications are necessary for proinflammatory process in the heart. Together with the previous reports that showed the involvement of the central nervous system in the pathophysiology of heart disease,8,21,37 our mechanism might link psychological distress to heart disease.38

Figure 8.

Figure 8. Proposed regulatory mechanism of cardiac inflammation and homeostasis during pressure overload. Pressure overload is sensed by cardiac afferent nerves to activate sympathetic efferent nerves for ATP release, possibly through the central nervous system. ATP released from sympathetic efferent nerve terminals activates the NLRP3 inflammasome in cardiac nonimmune cells through stimulation of the P2X7 receptor, which, together with Toll-like receptor signaling, leads to IL-1β production to induce cardiac adaptive hypertrophy. Cardiac inflammation and homeostasis are controlled through heart-brain interaction. IL-1β indicates interleukin-1β; NLRP3, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3; P2X7R, P2X purinoceptor 7; and SNS, sympathetic nervous system.

Inflammation is a double-edged sword that has both protective and harmful effects under various pathological conditions.39,40 In our study, genetic disruption of NLRP3 resulted in contractile dysfunction and hemodynamic maladaptation with high mortality after TAC, whereas it inhibited cardiac hypertrophy and progression of pathological cardiac remodeling during pressure overload. NLRP3 inflammasome inhibitors have recently been suggested to be effective for the treatment of cardiovascular disease.12,15,41,42 Our findings suggest that, similarly to β-adrenergic receptor blockers, careful titration of NLRP3 inflammasome inhibitors might be necessary for inhibition of pathological cardiac remodeling without hemodynamic maladaptation.

Our results suggest that ATP, rather than norepinephrine, might be the main neurotransmitter that initiates cardiac inflammation and hypertrophy, at least in the adaptive phase of pressure overload, although β-adrenergic signals in the heart might have an interaction with the NLRP3 inflammasome and contribute to pathological cardiac remodeling in both NLRP3 inflammasome-dependent and -independent manners. Taken together with the data on bisoprolol-treated mice, lipophilic β-adrenergic receptor blockers, which are used as standard therapy for cardiac remodeling and heart failure,36 might act on the brain as well as on the heart to inhibit SNS signals and cardiac inflammation with its ability to cross the blood-brain barrier. Further studies are needed to clarify the crosstalk between NLRP3 inflammasome activation and β-adrenergic signals in hypertensive heart disease.

In conclusion, NLRP3 inflammasome activation through heart-brain interaction initiates cardiac inflammation and hypertrophy during pressure overload. The nervous system could be a therapeutic target for the treatment of hypertensive heart disease, although fine-tuning might be necessary.

For a comprehensive discussion of the study, see the Expanded Discussion in the Supplemental Material.

Article Information

Acknowledgments

The authors are grateful to Dr Tschopp (The University of Lausanne, Switzerland) and Dr Di Virgilio (The University of Ferrara, Italy) for providing Nlrp3–/– mice and the plasmid harboring pmeLUC, respectively. Drs Higashikuni and Sata conceived the study. Dr Higashikuni designed and performed experiments and analyzed data. Drs Liu and Y. Tanaka assisted with animal experiments and data analysis. Drs Numata and Takimoto established and performed the Langendorff heart experiment. Drs K. Tanaka, Fukuda, and Hirata assisted with histological analysis and discussed analyses and results of all experiments. Dr Imamura assisted with the analysis of human samples. Drs Komuro and Sata supervised the research and provided scientific guidance and analysis. Drs Higashikuni and Sata wrote the manuscript with input from all authors. All authors approved the article.

Supplemental Material

Expanded Methods

Expanded Discussion

Figures S1–S16

Table S1

References 43–50

Nonstandard Abbreviations and Acronyms

ASC

apoptosis-associated speck-like protein containing a C-terminal caspase recruitment domain

IL

interleukin

mtDNA

mitochondrial DNA

NF-κB

nuclear factor-κB

NLRP3

nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing 3

SEN

sympathetic efferent nerve

TAC

transverse aortic constriction

TLR

Toll-like receptor

TNF-α

tumor necrosis factor-α

TXNIP

thioredoxin-interacting protein

Disclosures None.

Footnotes

Supplemental Material is available at https://www.ahajournals.org/doi/suppl/10.1161/CIRCULATIONAHA.122.060860.

For Sources of Funding and Disclosures, see page 354.

Circulation is available at www.ahajournals.org/journal/circ

Correspondence to: Yasutomi Higashikuni, MD, PhD, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8655, Japan, Email
Masataka Sata, MD, PhD, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan, Email

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