USE OF MAST CELL STABILIZER FOR THE TREATMENT OF HEART FAILURE WITH PRESERVED EJECTION FRACTION

Abstract

Heart failure with preserved ejection fraction (HFpEF) which results from diastolic dysfunction is a growing epidemiologic problem. However, the pathophysiology of this disease is poorly understood. Our goal is to investigate whether microvessel disease may promote HFpEF. To do so we have used Leptin receptor deficient (Lepr.sup.db/db) female mice as a model of HFpEF and performed a transcriptomic analysis via RNA sequencing of the cardiac vascular fraction of both these mice and their control Lepr.sup.db/+littermates. In Lepr.sup.db/db female mice, end diastolic pressure (EDP) signing diastolic dysfunction is significantly increased from 3 month of age. It is correlated with a cardiac and cardiomayocyte hypertrophy, vascular leakage, endothelial cell activation and leucocyte infiltration. As expected, the RNA sequencing analysis confirmed endothelial dysfunction. Besides, it also revealed a strong increase in several mast cell markers. We confirmed, via histology, an accumulation of mast cells in the heart of Lepr.sup.db/db mice. Importantly, it was associated with increased levels of circulating IgE. Lepr.sup.db/db mice were then treated or not with Cromolyn sodium, an inhibitor of mast cell degranulation. After a month treatment, EDP was significantly reduced in Lepr.sup.db/db mice demonstrating the critical role of mast cell in the development of diastolic dysfunction in diabetic obese mice.

Claims

1. A method of treating heart failure with preserved ejection fraction (HFPEF) in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a mast cell stabilizer.

2. The method of claim 1 wherein the mast cell stabilizer is selected from the group consisting of cromolyn, a dihydropyridine lodoxamide, nedocromil, barnidipine, YC-114, elgodipine, niguldipine, ketotifen, methylxanthines, quercetin, and pharmaceutically salts thereof.

3. The method of claim 1 wherein the mast cell stabilizer is a pharmaceutically acceptable salt of cromolyn.

4. The method of claim 1 wherein the mast cell stabilizer is azatadine, cetirizine, mizolastine, desloratadine, fexofenadine, or levocetirizine.

5. The method of claim 2, wherein the dihydropyridine is nicardipine or nifedipine.

6. The method of claim 3, wherein the pharmaceutically acceptable salt of cromolyn is cromolyn sodium, cromolyn lysinate, ammonium cromonglycate, or magnesium cromoglycate.

Description

FIGURES

[0015] FIG. 1: Cromolyn Sodium therapy prevents the occurrence of diastolic dysfunction in Lepr.sup.db/db mice. 2 month old Lepr.sup.db/db female mice were either treated with 50 mg/kg/day cromolyn sodium vs control vehicle or 4 mg/kg/day cetirizine vs control vehicle for 28 days. (A-B) At the end of the treatment end diastolic pressure was measured using a pressure catheter and compared to the one of 3 month old Lepr.sup.db/+ control mice. At 3 month of age, mice were subjected to echocardiography, LV catheterization and sacrificed. End diastolic pressure was measured using a pressure catheter (n=9-15 mice per group).

[0016] FIG. 2: Cromolyn sodium decreases vascular permeability and leucocyte infiltration in Lepr.sup.db/db female mice. 2 month old Lepr.sup.db/db female mice were treated or not with 50 mg/kg/day cromolyn sodium for 28 days. Mice were sacrificed at 3 month of age. Mice were subjected to echocardiography, LV catheterization and sacrificed. (A) Capillary density was quantified as the number of CD31+ vessels/mm.sup.2 (n=8 mice/group). (B) The mean cardiac capillary diameter was measured (n=8 mice/group). (C) Albumin extravasation was measured as the albumin+ surface area (n=8 mice/group). (D) Leucocyte infiltration was measured as the number of CD45+ cells/mm.sup.2 (n=10 mice/group). (E) The mean cardiac capillary diameter was measured (n=10 and 7 mice/group respectively). (F) Albumin extravasation was measured as the albumin+surface area (n=10 and 7 mice/group respectively).*: p≤0.05; **: p≤0.01; ***: p≤0.001. NS: not significant. Mann Whitney test.

EXAMPLE

[0017] Methods

[0018] Mice

[0019] Lepr.sup.db mice (BKS.Cg-Dock7m/+ Lepr.sup.db/+J) were obtained from Charles River laboratories and bred together to obtain Lepr.sup.db/db and control Lepr.sup.db/+ mice.

[0020] Animal experiments were performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and approved by the local Animal Care and Use Committee of Bordeaux University. Only females were used. Mice were either sacrificed by cervical dislocation or exsanguination under deep anesthesia (ketamine 100 mg/kg and xylazine 20 mg/kg, IP).

[0021] Cromolyn Sodium/Cetirizine Therapy

[0022] To prevent mast cell degranulation, mice were treated with 50 mg/kg/day cromolyn sodium (Abcam) via intra-peritoneal injections for 28 days. Untreated mice received 0.9% NaCl daily intra-peritoneal injections. To investigate the role of Histamine release by mast cell, mice were treated with 4 mg/kg/day cetirizine (Arrow Generiques) orally (in the drinking water) for 28 days.

[0023] Echocardiography

[0024] Left-ventricular ejection fraction and LV dimension will be measured on a high-resolution echocardiographic system equipped with a 30-MHz mechanical transducer (VEVO 2100, VisualSonics Inc.) as previously described 13,14. Mice were anchored to a warming platform in a supine position, limbs were taped to the echocardiograph electrodes, and chests were shaved and cleaned with a chemical hair remover to minimize ultrasound attenuation. UNI′GEL ECG (Asept Inmed), from which all air bubbles had been expelled, was applied to the thorax to optimize the visibility of the cardiac chambers. Ejection fractions were evaluated by planimetry as recommended (Schiller et al. 1989). Two-dimensional, parasternal long-axis and short-axis views were acquired, and the endocardial area of each frame was calculated by tracing the endocardial limits in the long-axis view, then the minimal and maximal areas were used to determine the left-ventricular end-systolic (ESV) and end-diastolic (EDV) volumes, respectively. The system software uses a formula based on a cylindrical-hemiellipsoid model (volume=8.Math.area.sup.2/3π/length) 15. The left-ventricular ejection fraction was derived from the following formula: (EDV−ESV)/EDV*100. The cardiac wall thickness (Left ventricular posterior wall (LVPW), Inter-ventricular septum (IVS) and left ventricular internal diameter (LVID) were calculated by tracing wall limits in both the long and short axis views.

[0025] LV Pressure/Systolic Blood Pressure Measurement

[0026] LV diastolic pressure measurement was assessed using pressure—volume conductance catheter technique. Briefly, mice will be anesthetized with Isoflurane. A Scisense pressure catheter (Transonic) will be inserted into the LV through the common carotid artery. Pressure will be recorded using (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. LabChart software. End diastolic pressure, dP/dt minimum and maximum, Tau and heart rate were automatically calculated by a curve fit through end-systolic and end-diastolic points on the pressure plot.

[0027] Immuno-Histological Assessments

[0028] Prior to staining, heart were stopped in diastole using KCl, perfused and then fixed in 10% formalin for 4 hours, paraffin embedded and cut into 7 μm thick sections. Alternatively, heart were fresh frozen in OCT, then cut into 7 μm thick sections.

[0029] ECs were identified using rat anti-CD31 antibodies (Histonova, cat #DIA-310). Albumin was stained using sheep anti-albumin antibodies (Abcam, Cat #ab8940). Pan-leucocytes were identified using rat anti-mouse CD45 antibodies (BD Pharmingen Inc, Cat #550539).

[0030] For immunofluorescence analyzes, primary antibodies were resolved with Alexa Fluor®—conjugated secondary polyclonal antibodies (Invitrogen, Cat #A-21206, A-21208, A-11077, A-11057, A-31573, A-10037) and nuclei were counterstained with DAPI (1/5000). Negative controls using secondary antibodies only were done to check for antibody specificity.

[0031] Capillary density (CD31+ vessels) was quantified in 4 pictures taken under ×260 magnification in areas where cardiomyocytes were oriented transversally.

[0032] To assess the mean capillary diameter, the diameter of 10 capillary randomly chosen in each picture was measured via Image J. Inflammatory cell density (CD45+) was quantified in 8 pictures randomly taken under ×260 magnification.

[0033] All pictures and quantifications (done using ImageJ/Fiji v2.0.0-rc-59 software (National Institute of Health, USA)) were performed by a blinded investigator. More precisely, all samples were assigned a random number prior to animal sacrifice, data collection and analysis. At the end of the experiment, the genotype/treatment for each animal was unveiled to allow data comparison and experimental conclusion.

[0034] Statistics

[0035] Results are reported as mean±SEM. Comparisons between groups were analyzed for significance with the non-parametric Mann-Whitney test or a 2 way ANOVA followed by Sidak's multiple comparison test (for kinetics analyses) using GraphPad Prism v8.0.2 (GraphPad Inc, San Diego, Calif). Differences between groups were considered significant when p≤0.05 (*: p≤0.05; **: p≤0.01; ***: p≤0.001).

Example 1

[0036] Heart failure with preserved ejection fraction (HFpEF) which results from diastolic dysfunction is a growing epidemiologic problem. However, the pathophysiology of this disease is poorly understood. Our goal is to investigate whether microvessel disease may promote HFpEF. To do so we have used Leptin receptor deficient (Lepr.sup.db/db) female mice as a model of HFpEF and performed a transcriptomic analysis via RNA sequencing of the cardiac vascular fraction of both these mice and their control Lepr.sup.db/+ littermates. In Lepr.sup.db/db female mice, end diastolic pressure (EDP) signing diastolic dysfunction is significantly increased from 3 month of age. It is correlated with a cardiac and cardiomayocyte hypertrophy, vascular leakage, endothelial cell activation and leucocyte infiltration. As expected, the RNA sequencing analysis confirmed endothelial dysfunction. Besides, it also revealed a strong increase in several mast cell markers. We confirmed, via histology, an accumulation of mast cells in the heart of Lepr.sup.db/db mice. Importantly, it was associated with increased levels of circulating IgE. Lepr.sup.db/db mice were then treated or not with Cromolyn sodium, an inhibitor of mast cell degranulation. After a month treatment, EDP was significantly reduced in Lepr.sup.db/db mice demonstrating the critical role of mast cell in the development of diastolic dysfunction in diabetic obese mice (FIG. 1A).

Example 2

[0037] Activated Cardiac Mast Cells, Via Histamine Release, Induce Cardiac Small Vessel Disease in Lepr.sup.db/db mice.

[0038] To investigate the role of mast cell degranulation in the pathophysiology of cardiac microvessel disease in Lepr.sup.db/db mice, 2 month old Lepr.sup.db/db mice (i.e. before they display increased EDP) were treated with 50 mg/Kg/day cromolyn sodium versus vehicle. Mice were sacrificed 28 days later. First, we verified cromolyn sodium therapy was effective and did decrease the percentage of degranulating mast cells in the heart (Data not shown). Then, we investigated the effect of cromolyn sodium therapy on cardiac microvessel phenotype and cardiac inflammation. Cromolyn sodium therapy did not modify cardiac microvessel density (FIG. 2A), however it did reduce capillary diameter (FIG. 2B) and permeability attested by decreased albumin extravasation (FIG. 2C). Moreover, CD45+ leucocyte recruitment was significantly decreased (FIG. 2D).

[0039] To summarize, mast cells promote cardiac capillary permeability and vasodilation, which is consistent with the well-known effect of histamine contained in mast cell granules 24. To verify cardiac capillary permeability and vasodilation was indeed due to histamine, 2 month old Lepr.sup.db/db mice were treated with 4 mg/kg/days cetirizine versus vehicle. As expected, both the diameter of cardiac capillaries (FIG. 2E) and their permeability (FIG. 2F) were significantly reduced in Lepr.sup.db/db mice treated with cetirizine vs vehicle-treated Lepr.sup.db/db mice.

[0040] In conclusion, mast cells promote cardiac small vessel disease via histamine release in Lepr.sup.db/db mice.

[0041] Activated Cardiac Mast Cells Promote the Appearance of Diastolic Dysfunction in Lepr.sup.db/db Mice.

[0042] Finally, to measure to pathophysiological consequences of histamine-induced small vessel disease on cardiac function, we investigated cardiac function in both cromolyn sodium-treated and cetirizine treated-Lepr.sup.db/db mice. Ejection fraction, which is normal in Lepr.sup.db/db mice was not modulated neither by cromolyn sodium treatment (Data not shown) neither by cetirizine treatment (Data not shown). However, EDP was significantly decreased both cromolyn sodium-treated (FIG. 1A) and cetirizine-treated (FIG. 1B) Lepr.sup.db/db mice indicating improved diastolic function. Cromolyn sodium therapy did neither modify the heart weight nor the LV posterior wall thickness nor the mean cardiomyocyte size (Data not shown), indicating that cromolyn sodium therapy does not prevent cardiac hypertrophy.

[0043] Altogether these results indicated that mast cells, via secretion of their granule content, promote the development of diastolic dysfunction, however, they do not participate in the development of cardiomyocyte hypertrophy.

[0044] Discussion

[0045] The present study supports the microvascular hypothesis of HFpEF especially in the setting of obesity and type 2 diabetes. In this paper, we used Lepr.sup.db/db female mice as a model of diastolic dysfunction. Lepr.sup.db/db female mice have the advantage of recapitulating the main risk factors for HFpEF, i.e. diabetes, obesity female gender and hypertension. Lepr.sup.db/db mice were previously shown to display diastolic dysfunction and to recapitulate significant features of human HFpEF. In the present study, we thoroughly characterized the cardiac microvascular phenotype of these mice, notably, via a transcriptomic analysis. Notably we revealed that cardiac microvessel disease is characterized by a decreased capillary density, abnormal vessel permeability and vasoconstriction of arterioles but increased capillary diameter; moreover we showed that ECs display oxidative stress and have a pro-inflammatory and pro-coagulant phenotype. Strikingly, we demonstrated for the first time that, in Lepr.sup.db/db mice, cardiac microvessel disease is associated with increased mast cell activation and proved that it participates to the pathophysiology of both cardiac microvessel disease and diastolic dysfunction (Data not shown).

[0046] The current paradigm for HFpEF proposes that myocardial remodelling and dysfunction in HFpEF results from the following sequence of events: 1) comorbidities including obesity, diabetes and/or hypertension would induce a systemic low grade pro-inflammatory state; 2) this pro-inflammatory state would induce EC dysfunction characterized by an increased ROS production, a decreased NO synthesis and an increased expression of adhesion molecules such as VCAM-1 and E-selectin; 3) EC dysfunction would lead to a compromised heart perfusion secondary to impaired NO-dependent vasodilatation, oedema and pro-inflammatory/pro-thrombotic phenotype, macrophage infiltration and fibrosis. The present data show that the Lepr.sup.db/db female mice model largely recapitulates this paradigm while adding further features. Notably, we demonstrated that mast cell activation, which is either part of the low grade pro-inflammatory state or induced by the low grade inflammatory state of diabetic obese mice, promotes microvascular dysfunction especially vascular permeability and capillary dilation and participates in the development of diastolic dysfunction. However, EC activation may precede mast cell activation (Data not shown). Consistent with a central role of inflammation and microvascular disease in the pathophysiology of HFpEF, Lepr.sup.db/db mice do not display significant cardiac fibrosis or major cardiomyocyte abnormalities. Notably, we found that cardiomyocyte hypertrophy does not seem to promote the increased EDP. Indeed, although cromolyn sodium therapy prevents increased EDP, this effect is not associated to cardiomyocyte hypertrophy and dedifferentiation after the onset of diastolic dysfunction, as demonstrated by Myh7 overexpression.

[0047] Mast cells are immune cells that reside in the connective tissues including the myocardium. They are characterized by the expression of c-Kit receptors and by their granules containing active mediators including proteases, notably Cma1, Tpsab1 and histamine. Mast cells may be activated by IgEs via their receptor Fcer1a, Complement factors via Toll-like receptors, IgGs or cytokines. They have been associated with several cardiovascular diseases including atherosclerosis, myocardial infarction and aneurysms, pathologies in which mast cells are contributing to the pathogenesis essentially through the release of their granule content. Importantly, circulating Tryptase was recently suggested to be a marker for cardiovascular diseases. Moreover, mast cells have been previously involved in diastolic dysfunction induced by ovariectomy in rats and diabetic cardiomyopathy in streptozotocin-treated mice. The present study thus confirms the significant role of mast cells in cardiovascular diseases. How mast cells are activated in the setting of cardiovascular diseases remains unknown. We found that, in Lepr.sup.db/db mice, increased activation of mast cells is associated with increased circulating levels of IgEs. IgE/Fcer1a is the main route of mast cell activation in allergic diseases. Interestingly, IgEs were reported to be elevated in the serum of patients with cardiovascular diseases including coronary arterial disease and myocardial infarction. Consistently, Asthma was shown to be related to an increased incidence of coronary heart disease, particularly in women. More specifically, one study reported that coronary flow reserve, considered as an early marker of endothelial dysfunction is significantly lower in patients with high IgE levels. Altogether, these results further support that Lepr.sup.db/db mice are a relevant model of human cardiovascular diseases.

[0048] In conclusion, the present study further confirms that inflammation and cardiac microvessel disease are at the heart of HFpEF pathophysiology and identified for the first time mast cells as critical players of cardiac microvessel disease and diastolic dysfunction, making them a promising therapeutic target for HFpEF treatment.

REFERENCES

[0049] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.