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

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).¹⁸ P2rx7^–/– and Slc17a9^flox/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

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 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.

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.

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.

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.

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.

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.

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.

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.

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 Slc17a9^flox allele with transgenic mice expressing Cre recombinase under the control of the dopamine β-hydroxylase (DBH) promoter to generate Slc17a9^flox/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 Slc17a9^flox/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.

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.

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.

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.

Expanded Methods

Expanded Discussion

Figures S1–S16

Table S1

References 43–50