An mTORC1‐Dependent Mouse Model for Cardiac Sarcoidosis
Journal of the American Heart Association
Abstract
Background
Sarcoidosis is an inflammatory, granulomatous disease of unknown cause affecting multiple organs, including the heart. Untreated, unresolved granulomatous inflammation can lead to cardiac fibrosis, arrhythmias, and eventually heart failure. Here we characterize the cardiac phenotype of mice with chronic activation of mammalian target of rapamycin (mTOR) complex 1 signaling in myeloid cells known to cause spontaneous pulmonary sarcoid‐like granulomas.
Methods and Results
The cardiac phenotype of mice with conditional deletion of the tuberous sclerosis 2 (TSC2) gene in CD11c+ cells (TSC2fl/flCD11c‐Cre; termed TSC2KO) and controls (TSC2fl/fl) was determined by histological and immunological stains. Transthoracic echocardiography and invasive hemodynamic measurements were performed to assess myocardial function. TSC2KO animals were treated with either everolimus, an mTOR inhibitor, or Bay11‐7082, a nuclear factor‐kB inhibitor. Activation of mTOR signaling was evaluated on myocardial samples from sudden cardiac death victims with a postmortem diagnosis of cardiac sarcoidosis. Chronic activation of mTORC1 signaling in CD11c+ cells was sufficient to initiate progressive accumulation of granulomatous infiltrates in the heart, which was associated with increased fibrosis, impaired cardiac function, decreased plakoglobin expression, and abnormal connexin 43 distribution, a substrate for life‐threatening arrhythmias. Mice treated with the mTOR inhibitor everolimus resolved granulomatous infiltrates, prevented fibrosis, and improved cardiac dysfunction. In line, activation of mTOR signaling in CD68+ macrophages was detected in the hearts of sudden cardiac death victims who suffered from cardiac sarcoidosis.
Conclusions
To our best knowledge this is the first animal model of cardiac sarcoidosis that recapitulates major pathological hallmarks of human disease. mTOR inhibition may be a therapeutic option for patients with cardiac sarcoidosis.
Nonstandard Abbreviations and Acronyms
- ACM
- arrhythmogenic cardiomyopathy
- CS
- cardiac sarcoidosis
- mTORC1
- mTOR complex 1
- mTOR
- mammalian target of rapamycin
- MHC
- major histocompatibility complex
- NF‐kB
- nuclear factor k‐light‐chain‐enhancer of activated B cells
- TSC2
- tuberous sclerosis complex 2
Research Perspective
What Is New?
•
Constitutive activation of mammalian target of rapamycin complex 1 signaling in CD11c‐expressing cells in mice promotes cardiac granuloma formation, recapitulating several features of human cardiac sarcoidosis including inflammatory granulomatous infiltration, gap junction remodeling, progressive interstitial, perivascular fibrosis, and diastolic dysfunction with preserved ejection fraction.
•
Mammalian target of rapamycin inhibition with everolimus reduces inflammatory infiltration and cardiac fibrosis, reverses gap junction remodeling, and improves cardiac function in this sarcoidosis model.
What Question Should Be Addressed Next?
•
Because 78% of sudden cardiac death victims with a postmortem diagnosis of cardiac sarcoidosis stain positive for mammalian target of rapamycin complex 1 activation, our findings indicate the use of mammalian target of rapamycin inhibitors as potential therapeutic option for patients with cardiac sarcoidosis and suggest the use of p‐S6 as marker for the diagnosis of high‐risk (poor prognosis) patients.
Sarcoidosis is a systemic inflammatory disease that is histologically characterized by the formation and accumulation of non‐necrotizing granulomas, primarily in the lungs, but can also manifest independently in other organs such as the heart.1, 2 Particularly during cardiac sarcoidosis (CS), unresolved granulomatous inflammation is life‐threatening when diagnosed late and untreated and can eventually lead to cardiac fibrosis, arrhythmias, and sudden cardiac death.3 Clinically apparent cardiac sarcoidosis has been reported in 5% to 7% of the patients with systemic sarcoidosis and is associated with a poor outcome.4 However, based on postmortem evaluation, cardiac involvement of sarcoidosis has been reported in 25% to 58% of the patients.5 In the Japanese population, cardiac involvement is the leading cause of death related to sarcoidosis, accounting for up to 85% of deaths.6
Often referred to as the great mimicker, sarcoidosis is a diagnosis of exclusion7 and shares histopathological and clinical phenotype with arrhythmogenic cardiomyopathy (ACM).8 Abnormal distribution of plakoglobin, a cytoplasmatic structural component of the desmosome, and connexin 43, the main ventricular gap junction protein, at the intercalated disks is a key feature of ACM and sarcoidosis and an indicator of early disease progression.9, 10 Recently, nuclear factor k‐light‐chain‐enhancer of activated B cells (NF‐kB) induced inflammation has been proposed as a driver of the key pathological features of ACM, and its inhibition with BAY11‐7082 prevents the development of the disease features in a range of in vitro, in vivo, and ex vivo models of ACM.11
Although environmental, genetic, and microbial factors have been postulated to underlie sarcoidosis, its pathogenesis remains unknown. Consequently, glucocorticoids continue to be the first‐line management option.12, 13 For patients with refractory CS, a stepwise approach, including steroid‐sparing alternatives and monoclonal antibodies, has been proposed, but randomized controlled studies to systematically evaluate the efficacy of these drugs are lacking.7 There is a pressing need to develop mechanism‐based therapies to treat CS. The development of experimental models that recapitulate the process of granuloma formation in the heart would allow for mechanistic insights into the molecular events underlying the disease and suggest novel therapeutic targets. To date, no experimental models of sarcoidosis with cardiac involvement have been reported.14
We previously showed that genetic deletion of tuberous sclerosis complex 2 (TSC2) in myeloid lineage Lyz2 expressing cells (targeting monocytes, macrophages, and granulocytes) led to spontaneous development of pulmonary sarcoid‐like granulomas in mice.15 Exome sequencing studies have also implicated genes coding for regulators of mammalian target of rapamycin (mTOR) and autophagy in familial forms of sarcoidosis.16 Here we phenotyped the heart of mice with conditional genetic deletion of TSC2 (an upstream inhibitor of mTOR complex 1 [mTORC1]) in CD11c‐expressing cells, where we also found spontaneous development of sarcoid‐like granulomas in the lungs. The aims of this study were (1) to characterize cardiac involvement in the TSC2fl/flCD11c‐Cre model of sarcoidosis, and (2) to evaluate the efficacy of 2 mechanism‐based therapies: everolimus (targeting the mTOR signaling pathway) and BAY11‐7082 (inhibiting the NF‐kB pathway).
METHODS
All data and supporting materials have been provided within the published article.
Animal Model
In this study we used a mouse model of sarcoidosis with mTORC1 constitutive expression in CD11c‐expressing cells generated by deleting its upstream inhibitor TSC2. In brief, TSC2fl/fl mice17 were crossed with transgenic mice expressing Cre recombinase under the control of the CD11c promoter, CD11c‐Cre mice.18 Both male and female mice between 13 and 54 weeks old were used. All mouse studies were approved by the official Austrian ethics committee for animal experiments (GZ.BMWF‐66.009/0163‐WF/V/3b/2016 and BMBWF GZ‐2021‐0.611.635).
In Vivo Treatments
Male and female TSC2fl/fl CD11c‐Cre (TSC2KO) mice, already displaying symptoms at 26 weeks of age were treated by oral gavage with 5 mg/kg body weight everolimus (RAD001, Selleckchem) or diluent (PBS with 0.05% DMSO, 30% PEG [Sigma 202 371], 5% Tween‐80 [Sigma P1754‐25 mL]) daily for 3 and 21 days. To study the role of NF‐κB signaling in the disease progression, another group of TSC2KO mice were injected intraperitoneally with 5 mg/kg Bay11‐7082 (Sigma 196 870) or vehicle (5% DMSO/saline) every day for 21 days. The drug and the vehicle were administered to both males and females between 24 to 26 weeks of age. At this dose, Bay11‐7082 has been shown to block NF‐κB signaling in several in vivo studies. Age‐matched TSC2fl/fl littermates were used as controls.
Histology and Immunohistochemistry of Heart Samples
Mouse hearts were fixed in ROTI Histofix 4% formaldehyde (Carl Roth), processed, and embedded in paraffin. Tissue morphology and the presence of inflammatory cells were evaluated by conventional hematoxylin and eosin staining and tissue fibrosis by picrosirius red staining. Whole heart sections from mice were stained and bright field images taken with a Nikon eclipse 80i microscope and recorded with a Nikon DS‐Fi1 camera. Protein expression and localization on mouse myocardial tissue samples were evaluated by confocal immunofluorescence. In brief, 5 μm heart sections were deparaffinized and rehydrated. Following citrate antigen retrieval, slides were incubated overnight at 4 °C with primary antibodies: anti‐Galectin 3 (Mac‐2) (Abcam, ab76245 or Cedarlane CL8942AP), rabbit monoclonal anti‐Phospho‐S6 Ribosomal Protein (p‐S6) (Ser240/244) (Cell Signaling Technology, 5364S), rabbit polyclonal anti‐N‐Cadherin (Merck, C3678), rabbit monoclonal anti‐gamma Catenin (plakoglobin) (Abcam, ab184919), rabbit polyclonal anti‐Connexin 43 (Merck, C6219), rabbit monoclonal anti‐CD3 (Abcam, ab5690), CD206 polyclonal antibody (Thermo Fisher PA5‐46994), or rat anti‐CD68 (Thermo Fisher, 14–0681‐82). The next day, heart sections were incubated with host‐specific Cy3‐conjugated secondary antibodies and mounted with ProLong Gold (Invitrogen, P36935). Immunoreactive signal was recorded by either Nikon A1R confocal microscope or Zeiss Axioscan 7. Collagen content (fibrosis) in mouse heart sections was quantified as described by Vogel et al19 and expressed as % of total tissue area. For image quantification, 5 microscopy fields were analyzed per preparation. Fluorescence image analysis was performed with Zen Blue 3.4 software (Zeiss).
Human Cardiac Sarcoidosis Samples
Human heart samples from 9 sudden cardiac death victims with a definite postmortem diagnosis of CS by an expert cardiac pathologist were obtained from the Cardiac Risk in the Young Centre for Cardiac Pathology at St. George's, University of London. Ethical approval for this study was granted by the London Stanmore National Health Service Research Ethics Committee (reference: 17/LO/0747). Informed consent was provided by next‐of‐kin at the time of autopsy. Heart tissue sections from the right ventricle (RV), left ventricle (LV), and interventricular septum from paraformaldehyde‐fixed paraffin‐embedded heart samples were immunostained with rabbit monoclonal anti‐CD68 (Abcam, ab213363), rat monoclonal anti‐mouse/human Mac‐2 (Cedarlane CL8942AP), or rabbit monoclonal anti‐Phospho‐S6 Ribosomal Protein (p‐S6) (Ser240/244) antibodies. Immunoreactive signal was recorded with Nikon A1R confocal microscope.
Echocardiography
Transthoracic echocardiography was performed using a Vevo 3100 Imaging system (Visualsonics) with a 55‐MHz transducer as described previously.20 The mice (aged 26–29 weeks of age) were anesthetized with 1% to 1.5% isoflurane. Body temperature and ECG were continuously monitored throughout the measurement via limb electrodes and rectal probe, respectively. Parasternal long‐axis view and short axis view were obtained, which were analyzed to assess the LV dimension and function. The obtained ultrasound images and videos were analyzed by Vevo LAB software where a mean of 3 cardiac cycles in each view was used for each parameter.
Hemodynamic Measurements
Mice at 26 to 29 weeks of age were anesthetized with a mixture of ketamine and xylazine, trachea cannulated, and the mice were mechanically ventilated. When the ECG was stable, the thorax was opened and a microtip catheter (Millar Instruments) was inserted first in the LV then into the RV to monitor the hemodynamic parameters at least for 10 minutes with Powerlab system with LabChart (v7.3.2) software (ADInstruments) as described previously.20
Statistical Analysis
Statistical analysis was performed using Prism software (Version 9.2.0 [283]; GraphPad Software Inc., San Diego, CA). Normality distribution of the data was assessed by Kolmogorov–Smirnov test. Data were analyzed for differences by using a Mann–Whitney test for single comparison, 1‐way analysis of variance (ANOVA) and the Newman–Keuls post‐test for multiple comparisons, Kruskal–Wallis test with Dunn post hoc (Bonferroni) multiple comparison test, and 2‐way ANOVA and the Tukey post‐test for multiple comparison. Data in the text and figures are presented as mean ±SEM. P values <0.05 were considered significant.
RESULTS
Granulomatous Disease in the Hearts of Mice With Constitutive Activation of mTORC1 in CD11c‐Expressing Myeloid Cells
Similar to our observations in the TSC2fl/fl Lyz2‐Cre mouse model15 where we found spontaneous development of pulmonary sarcoid‐like granulomas after genetic ablation of TSC2 in Lyz2 expressing myeloid cells, constitutive activation of mTORC1 by deletion of TSC2 in CD11c‐expressing cells termed TSC2KO (affecting tissue resident macrophages and dendritic cells) was also sufficient to initiate and maintain granulomas in the lung (Figure S1). In addition, TSC2KO mice showed slightly elevated tumor necrosis factor‐α levels in the serum compared with their littermate controls (Figure S1D).
Histological investigation of the hearts of adult TSC2KO animals revealed the presence of a significantly higher number of inflammatory cells in the base and midventricular areas of the left ventricular free wall, interventricular septum, RV, LV, papillary muscles, and to a lesser extent in the atria and visceral pericardium (Figure 1A; Figures S2 and S3). Increased inflammatory infiltration of the mitral and tricuspid valves was also observed in the TSC2KO sarcoidosis mice (Figure S3). Inflammatory cells were mainly distributed between cardiomyocytes (interstitial) and around the blood vessels (perivascular) (Figure 1A) and consisted mostly of activated Mac‐2+ macrophages expressing CD68 and CD206 (Figure 1B; Figures S2 and S3). Some animals showed structures reminiscent of giant cells and non‐necrotizing granulomas (Figure 1C) as observed in human patient hearts.21 Few T lymphocytes (CD3+ cells) were observed in the hearts of the TSC2KO animals (Figure 1D). Here, the presence of inflammatory infiltrates was associated with myocardial damage and increased interstitial and perivascular fibrosis (Figure 1E).
Figure 1. Histopathological characterization of TSC2KO mouse hearts at 26 weeks of age.
A, Hematoxylin and eosin staining (200× magnification). Heart sections of TSC2KO mice showed increased numbers of inflammatory cells between cardiomyocytes (interstitial) and around the blood vessels (perivascular). B, Immunoperoxidase staining with Mac‐2 (200× magnification) of control and TSC2KO heart section. 1 and 2 are close‐up images (600× magnification) showing the presence of interstitial and perivascular Mac‐2+ cells in the hearts of TSC2KO respectively. C, Structures reminiscent of human giant cells and non‐necrotizing granulomas. D, Immunoperoxidase staining CD3 (200× magnification). Inflammatory cells in the hearts of TSC2KO mice were mainly Mac‐2+ cells. E, Picrosirius red staining (400× magnification). The presence of inflammatory cells in the hearts of TSC2KO mice correlated with increased deposition of interstitial and perivascular collagen fibers (n=6 mice/group). H&E indicates hematoxylin and eosin; PSR, picrosirius red; and TSC2KO, tuberous sclerosis complex 2 knockout.
Progressive Granulomatous Disease and Fibrosis Over Time
Progressive and chronic sarcoidosis, unlike Löfgren syndrome sarcoidosis, is unlikely to resolve spontaneously with time, and such patients are also more likely to be refractory to corticosteroid treatment.22 To investigate if cardiac disease seen in these mice is chronic and progresses over time, we measured the number of Mac‐2+ macrophages, the activation of mTORC1 signaling pathway, and the amount of fibrosis in the hearts of control and TSC2KO mice at 13 and 54 weeks of age (Figure S2D).
At 13 weeks of age, we observed the presence of few Mac‐2+ cells in the pericardium, the perivascular, and interstitial areas of the myocardium of the TSC2KO animals. At 54 weeks of age, the area occupied by Mac‐2+ cells in the hearts of TSC2KO animals increased from 0.43%±0.12% to 5.58%±0.75% (P<0.0001). Mac‐2+ cells were absent in the hearts of control animals at 13 and 54 weeks of age (Figure 2B). To evaluate mTORC1 signaling pathway involvement in the progression of the granulomatous infiltration in the hearts of the TSC2KO animals, we measured the phosphorylation of the downstream effector ribosomal protein S6 (p‐S6), a hallmark of mTORC1 activation (Figure 2C). Phosphorylation levels of S6, as determined by the percentage of area occupied by cells expressing p‐S6, increased from 0.446%±0.067% in the TSC2KO animals at 13 weeks of age to 4.54%±0.39% in the TSC2KO animals at 54 weeks of age (P<0.0001; Figure 2D). The expression of p‐S6 was virtually absent in the hearts of control mice at 13 and 54 weeks of age.
Figure 2. Cardiac involvement is progressive in the TSC2KO animal model of sarcoidosis.
A, Confocal images are representative of control and TSC2KO mice hearts at 13 and 54 woa immunostained with Mac‐2 (red); autofluorescence (green) was used to calculate the area of live cells (total area) in the tissue; nuclei were counterstained with DAPI (blue). B, Mac‐2 percentage was determined with Zen Blue 3.4 software and was calculated by dividing the area occupied with Mac‐2+ cells (red) by total tissue area (green). C, Representative confocal images of control and TSC2KO mice hearts at 13 woa and 54 woa immunostained with p‐S6 (red); live cells in the tissue (green); nuclei (blue). D, p‐S6 percentage was calculated as in (B). E, Confocal images representative of control and TSC2KO mice hearts at 13 woa and 54 woa stained with picrosirius red. Collagen fibers (red) and area of live cells in the tissue (green). Images were acquired and analyzed as in Vogel et al.19 F, Percentage of fibrosis in the hearts of control and TSC2KO animals at 13 woa and 54 woa. Data are expressed as mean±SEM. (n=5/group for 13 woa animals and n=3/group for 54 woa animals). **P<0.01 and ****P<0.0001 by 1‐way ANOVA with Tukey multiple comparison test. ns indicates not significant; PSR, picrosirius red; TSC2KO, tuberous sclerosis complex 2 knockout; and woa, weeks of age.
Increasing accumulation of macrophages with activated mTOR in the hearts of TSC2KO animals over time was associated with progressive myocardial destruction and fibrotic replacement (Figure 2E). At 13 weeks of age, we observed a trend toward increased fibrosis in the hearts of TSC2KO animals when compared with 13‐week‐old control mice (Figure 2E and 2F). The percentage of fibrosis in the hearts (RV, LV, and interventricular septum) of TSC2KO mice at 54 weeks old was significantly higher than in the control mice at 54 weeks old and 13 weeks old (3.45%±0.026% versus 1.80%±0.15% and 1.92%±0.313%, respectively; P<0.01, Figure 2F).
Gap Junction Remodeling in Hearts of Sarcoidosis Mice
A high proportion of sarcoidosis‐related hospitalizations—one‐fifth reported in 1 population study23—involve patients who suffer from arrhythmias, and there are many overlaps in clinical presentation between cardiac sarcoidosis patients and patients with ACM, including a higher risk for sudden cardiac death.24, 25 Therefore, we analyzed the expression and distribution of the desmosomal protein plakoglobin and connexin 43, the main protein in cardiac ventricular gap junctions. The housekeeping protein N‐Cadherin was used as a tissue quality control, and its expression levels and distribution pattern were similar between controls and TSC2KO animals. Similar to patients with cardiac sarcoidosis and patients with ACM,9, 26 adult TSC2KO mice presented with reduced immunoreactive signal for plakoglobin at the myocardial intercalated disks (Figure 3A). Additionally, heterogeneous connexin 43 remodeling, known to constitute a threatening substrate for fatal arrhythmias,27 was observed at intercalated disks of TSC2KO mice hearts.
Figure 3. Cardiac phenotype of TSC2KO animal model of sarcoidosis.
A, Representative confocal images of control and TSC2KO mice hearts immunostained with plakoglobin, connexin 43, and N‐Cadherin (red); nuclei were counterstained with DAPI (blue). Arrows show the localization of immunoreactive signal at the intercalated disks. N‐cadherin (used as a tissue quality control) shows comparable distribution in both groups. TSC2KO animals at 34 weeks of age showed reduced immunoreactive signal for plakoglobin and connexin 43. Scale bar, 50 μm, (n=6/group). B, Heart weight, tibia length, body weight, and heart weight to tibia length ratio of control vs TSC2KO mice (26–29 weeks of age). C through D, Quantitative echocardiography and hemodynamic measurements of control and TSC2KO mice between 26 to 29 weeks of‐age. Data are expressed as mean±SEM. *P<0.05 for controls vs TSC2KO by using a Mann–Whitney test. LV indicates left ventricular; LVEDP, left ventricular end diastolic pressure; LVESP; ventricular end systolic pressure; LVIDd, left ventricle internal diameter in diastole; RVESP, right ventricular end systolic pressure; and TSC2KO, tuberous sclerosis complex 2 knockout.
TSC2KO Sarcoidosis Mice Have Diastolic Dysfunction With Preserved Ejection Fraction
Despite the presence of inflammatory infiltrates in the hearts of the TSC2KO mice, they presented with lighter hearts and decreased heart weight to tibia length ratio (Figure 3B) compared with control TSC2fl/fl mice. To understand the functional consequences that constitutive activation of mTORC1 signaling in CD11c+ cells and Mac‐2+ macrophage infiltration has on the heart of these mice, we performed echocardiography and hemodynamic pressure measurements on middle‐aged mice displaying symptoms between 26 and 29 weeks of age. At this age, the mice already exhibited significant infiltration of Mac‐2 inflammatory infiltrates and fibrosis (Figure S4). Although we did not find significant changes in left ventricle ejection fraction or fractional shortening, we saw a decrease in the left ventricular internal diameter in diastole, left ventricular end diastolic volume, left ventricle end systolic pressure, and dP/dt values in the TSC2KO mice (Figure 3C). In addition, elevated left ventricle end diastolic pressure and right ventricular end systolic pressure was observed in TSC2KO mice in comparison with their controls (Figure 3D).
Bay11‐7082 Treatment Ineffective in Reducing Granulomatous Inflammation or Gap Junction Remodeling in Sarcoidosis Mice
Having characterized the sarcoid development in the heart of TSC2KO mice, we wanted to assess whether we can therapeutically interfere with the disease. Given the clinical and molecular overlap between sarcoidosis and ACM,8 and the recent implication of NF‐kB in the pathogenesis of ACM,11 we tested the efficacy of Bay11‐7082 in reducing disease‐associated features in our model. TSC2KO animals between 24 and 26 weeks of age were treated intraperitoneally with 5 mg/kg Bay11‐7082 or vehicle daily for 21 days. Administration of Bay11‐7082 for 21 days was neither able to reduce cardiac (Figure S5A) and pulmonary granulomatous infiltration (not shown) nor inhibit the mTOR signaling pathway (Figure S5B) in the hearts of TSC2KO animals. In line with these results, expression levels of plakoglobin and connexin 43 were similar between vehicle‐treated TSC2KO animals and those receiving Bay11‐7082 for 21 days (Figure S5C).
mTOR Inhibition Reduces Cardiac Granulomatous Infiltration and Cardiac Fibrosis in Sarcoidosis Model Mice
To test whether mTOR inhibition would improve disease severity, we treated TSC2KO animals at 26 weeks of age (already displaying cardiac granulomatous infiltration and fibrosis) by oral gavage with 5 mg/kg body weight15 with everolimus, an inhibitor of mTOR, or vehicle daily for 3 and 21 days. The 3‐day treatment was chosen to evaluate the ability of everolimus to inhibit the phosphorylation of S6,28 whereas the 21‐day‐treatment regimen was selected to clarify whether administration of the mTOR inhibitor everolimus has therapeutic potential.
Vehicle‐treated TSC2KO sarcoidosis model mice exhibited 3.14%±0.38% of the heart with granulomatous Mac‐2+ cell infiltrates. This percentage was reduced to 1.9%±0.22% (relative 39% reduction) in TSC2KO animals receiving everolimus for 3 days (P <0.01) and to 0.52%±0.09% (relative 83% reduction) in TSC2KO animals receiving everolimus for 21 days (P<0.0001) (Figure 4A and 4B). Body weight, heart weight, and tibia length, heart weight to tibia length ratio, and heart rate of the mice were not affected by everolimus treatment (Figure S6A). As expected, everolimus treatment reduced the phosphorylation of the mTOR downstream effector S6 ribosomal protein. The percentage of myocardial tissue occupied by cells expressing p‐S6 was reduced by 75% and 95% in TSC2KO animals receiving everolimus for 3 and 21 days, respectively (Figure 4C and 4D). In TSC2KO animals treated with everolimus for 21 days, the granulomatous infiltrates completely resolved, while they were perfectly recognizable in the hearts of TSC2KO animals receiving vehicle. Moreover, everolimus treatment for 21 days was able to halt the progression of fibrotic lesions in the hearts TSC2KO animals (Figure 4E and 4F).
Figure 4. Mammalian target of rapamycin inhibition prevents cardiac sarcoidosis progression in the hearts of TSC2KO mice.
A, Representative confocal images of control and TSC2KO mice hearts at 29 weeks of age, treated with everolimus for 3 and 21 days, immunostained with Mac‐2 (red); autofluorescence (green) was used to calculate the area of live cells (total area) in the tissue; nuclei were counterstained with DAPI (blue). B, Mac‐2 percentage was determined with Zen Blue 3.4 software and was calculated by dividing the area occupied with Mac‐2+ cells (red) by total tissue area (green). C, Representative confocal images of control and TSC2KO mice hearts treated as previously described immunostained with p‐S6 (red); live cells in the tissue (green); nuclei (blue). D, p‐S6 percentage was calculated as in (B). E, Confocal images are representative of control and TSC2KO mice hearts treated with everolimus for 3 and 21 days stained with picrosirius red. Collagen fibers (red); area of live cells in the tissue (green). Images were acquired and analyzed as in Vogel et al.19 F, Percentage of fibrosis in the hearts of control and TSC2KO treated as previously described. Data are expressed as mean±SEM. (n=6 for control, TSC2KO+vehicle, TSC2KO+everolimus 3 days; n=4 for TSC2KO+everolimus 21 days). *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001, by 1‐way ANOVA with Tukey multiple comparison test. PSR indicates picrosirius red; and TSC2KO, tuberous sclerosis complex 2 knockout.
mTOR Inhibition Reverses Gap Junction Remodeling and Improves Cardiac Function in Sarcoidosis Model Mice
Heterogeneous plakoglobin and electrical gap junction remodeling, a substrate for fatal arrhythmias, observed in the vehicle‐treated TSC2KO animals, was resolved in the TSC2KO animals receiving everolimus for 21 days (Figure 5A). Here, we saw that plakoglobin and connexin 43 (Cx43) protein expression levels and their localization at the intercalated disk were reverted to those observed in the control group. Importantly, the echocardiographic and cardiac pressure abnormalities detected in vehicle‐treated TSC2KO animals, such as reduced left ventricular internal diameter in diastole and left ventricular end diastolic volume and increased LVESP and right ventricular end systolic pressure (Figure 5B and 5C), were normalized to control values in the TSC2KO group receiving everolimus for 21 days. No significant change in left ventricle end diastolic pressure, cardiac dP/dT (max) values, left ventricle internal diameter in systole, left ventricle end systolic volume, left ventricle posterior wall thickness in systole and diastole, or interventricular septum diameter in diastole was observed in the TSC2KO mice treated with everolimus (Figure S6B and S6C). Altogether, these results suggest that chronic activation of mTORC1 in CD11c‐expressing myeloid cells plays a role in the development of echocardiographic and cardiac pressure abnormalities. Conversely, mTOR inhibition improves cardiac function in this experimental model of sarcoidosis.
Figure 5. Mammalian target of rapamycin inhibition restores cardiac phenotype and function of the hearts of TSC2KO mice.
A, Representative confocal images of control and TSC2KO mice hearts immunostained with N‐Cad, plakoglobin, and Cx43 (red); nuclei were counterstained with DAPI (blue). Arrows show the localization of immunoreactive signal at the intercalated disks of the myocardium. N‐Cad normal distribution in all experimental groups is shown as a positive control. TSC2KO animals showed reduced immunoreactive signal for plakoglobin and Cx43 when compared with the control group. In TSC2KO animals receiving everolimus treatment for 21 days, immunoreactive signal for plakoglobin and Cx43 was restored to control levels. Scale bar, 50 μm, (n=4 to 6/group). B, Quantitative echocardiography analysis in vehicle‐ and everolimus‐treated TSC2KO mice (29 weeks of age) treated for 21 days. Group data for left ventricle internal diameter in diastole, left ventricle end diastolic volume, left ventricular ejection fraction, and fractional shortening. C, Hemodynamic pressure measurements of vehicle‐ and everolimus‐treated TSC2KO mice treated for 21 days and control mice at 29 weeks of age. Group data showing left ventricular end systolic pressure and right ventricular end systolic pressure. Data are expressed as mean±SEM (n=6 for control group, n=11 for TSC2KO+vehicle and n=9 for TSC2‐knockout+everolimus 21 days). *P<0.05 and **P<0.01, by 1‐way ANOVA with Tukey multiple comparison test except for the analysis of left ventricular end diastolic volume where Kruskal–Wallis test with Dunn post hoc (Bonferroni) multiple comparison test was performed. Cx43 indicates connexin 43; LV, left ventricle; LVEDP, left ventricular end diastolic pressure; LVESP, ventricular end systolic pressure; LVIDd, left ventricle internal diameter in diastole; N‐Cad, N‐Cadherin; RVESP, right ventricular end systolic pressure; and TSC2KO, tuberous sclerosis complex 2 knockout.
mTOR Signaling Pathway Is Active in Human Cardiac Sarcoidosis
Considering that constitutive activation of mTOR signaling in myeloid cells is a sufficient condition for granulomatous infiltration in the hearts of the TSC2KO animals generating a histopathological phenotype highly reminiscent of human CS, we decided to investigate mTOR activation in heart samples from sudden cardiac death victims with a postmortem diagnosis of CS (Figure 6A; Table). We found that non‐necrotizing granulomas in the myocardium of patients with CS consisted of CD68+ macrophages (Figure 6B). We also found Mac‐2 expressing cells among the granulomatous inflammatory infiltrates in the myocardium of the patients (Figure 6C). Additionally, we observed activation of the mTORC1 signaling pathway in 7 out of 9 hearts (78%) of decedents because of CS (Figure 6D; Figure S7). Collectively, these results indicate that mTOR activation may be a relevant mechanism underlying the pathophysiology of human CS.
Figure 6. Mammalian target of rapamycin signaling in human cardiac sarcoidosis.
A, Hematoxylin and eosin staining of human heart samples from sudden cardiac death victims with a postmortem diagnosis of cardiac sarcoidosis (200× magnification). B, Representative images of hearts immunostained with CD68 and (C) Mac‐2 and (D) p‐S6 (red); autofluorescence (green) was used to image healthy myocardial tissue; nuclei were counterstained with DAPI (blue). Images are representative of control subjects (n=6) and patients with cardiac sarcoidosis (n=9). mTOR indicates mammalian target of rapamycin.
| Patient ID | Age at death, y | Sex | Coexisting conditions | Pathological cause of death |
|---|---|---|---|---|
| 1 | 33 | Male | None | Sarcoidosis |
| 2 | 33 | Male | Tuberculosis | Sarcoidosis |
| 3 | 54 | Male | Obesity | Sarcoidosis |
| 4 | 36 | Male | None | Sarcoidosis, SUDEP |
| 5 | 53 | Male | None | Sarcoidosis |
| 6 | 44 | Male | Obesity | Sarcoidosis |
| 7 | 47 | Female | None | Sarcoidosis |
| 8 | 41 | Male | Fibromyalgia | Sarcoidosis |
| 9 | 48 | Female | Sarcoidosis | Sarcoidosis |
| Mean age, y | Male % | ||
|---|---|---|---|
| 43.2 | 77.78 |
Age, sex, coexisting diseases, and pathological cause of death are listed. SUDEP indicates sudden unexplained death in epilepsy.
DISCUSSION
In this study we provide a histopathological and functional description of the first experimental mouse model of progressive systemic sarcoidosis with cardiac involvement. Here we show that constitutive activation of mTORC1 by genetic deletion of TSC2 in CD11c‐expressing myeloid cells led to the presence of inflammatory infiltrates in the hearts of these mice, mainly composed by Mac‐2+ macrophages, which is associated with increased interstitial and perivascular fibrosis. This is accompanied by gap junction remodeling and diastolic dysfunction with preserved ejection fraction. Therapeutic administration of everolimus, an mTORC1 inhibitor, but not Bay11‐7082 was able to rescue the pathological features of CS in this model. Corroborating our mouse model observations, we also show aberrant mTOR activation in myocardial samples of 78% of sudden cardiac death victims with a postmortem diagnosis of CS.
In this mouse model, histological evaluation of the heart revealed the presence of progressive granulomatous infiltrates throughout the myocardium, mostly in the base and midventricular areas of the left ventricle free wall, interventricular septum, and LV and RV. These findings are in line with those reported by Tavora et al in a cohort of patients who died suddenly from CS where the hearts of such patients were characterized by extensive active granulomas, especially in the subepicardium and ventricular septum.29 Indeed, old animals (54 weeks of age) with a more severe phenotype also showed structures reminiscent of giant cells and non‐necrotizing granulomas. Additionally, we describe a model with cardiac fibrosis that worsens with age. This phenocopies disease progression in CS, where advanced cardiac sarcoidosis correlates with more fibrosis and worse outcomes,30 and the presence of fibrosis is associated with increased disease severity such as congestive heart failure,31, 32 arrhythmias, and sudden cardiac death.33
Patients with CS often present with echocardiographic abnormalities, which include dilated cardiomyopathy, diastolic dysfunction, pericardial effusions, and ventricular aneurysms.34, 35 While the sarcoidosis mice show diastolic dysfunction, reduced left ventricular ejection fraction, a characteristic criterion used in the diagnosis of CS, was not observed in TSC2KO mice that were 26 to 29 weeks of age. Nevertheless, TSC2KO animals of this age showed elevation of left ventricle end diastolic pressure and right ventricular systolic pressure. The precise physiological significance of the echocardiographic alterations in the hearts of our sarcoidosis model mice, and its correlation with the echocardiographic abnormalities observed in patients with CS, remain to be determined and will require further investigations. Additionally, the functional significance of mitral and tricuspid valve involvement in these mice needs to be clarified in future studies. Interestingly, CS patients with valve involvement have been reported and deserve further attention in future studies.36, 37
Although the cause underlying sarcoidosis still remains elusive, the evidence for a role of aberrant mTOR activation in sarcoidosis is mounting. Previously, we identified rare gene variants encoding for mTOR regulators and autophagy‐related proteins in patients with familial sarcoidosis.13, 16 We also described mTORC1 activation in lung biopsies of patients with progressive sarcoidosis.15 RNA sequencing of skin lesions from patients with cutaneous sarcoidosis also revealed an enrichment of genes involved in the mTORC1 signaling pathway.38, 39 Of note, spatial transcriptomics and single nucleus sequencing of frozen cardiac tissues from patients diagnosed with CS revealed the presence of 3 inflammatory macrophage populations, of which 2 populations, the human leukocyte antigen DR isotype (HLA‐DR)+ and synaptotagmin‐like 3 (SYTL3)+ macrophage population, show high mTORC1 activation.40 Additionally, differentially expressed genes that were upregulated in inflammatory myeloid cells in CS versus resident cardiac macrophages were enriched for the mTOR signaling pathway.40 This is in accordance with the observations in our mouse model, where the hearts of control TSCfl/fl mice with resident cells and little/no inflammatory infiltrates do not stain positive for p‐S6 (mTORC1 activation), while the sarcoidosis model mice show increased activation of mTORC1 and inflammatory infiltration over time, corresponding to disease severity. Moreover, we show that the mTOR signaling pathway was active in the hearts of 78% of sudden cardiac death victims attributable to CS. The specificity of the mTOR findings needs to be examined in the hearts of sudden cardiac death attributable to other pathologies and will be the topic of future investigations. Nevertheless, our results suggest the use of p‐S6 or other molecules indicating activated mTORC1 signaling as a target for diagnosis of high‐risk CS by routine immunohistochemical staining and for development of tracers for noninvasive molecular imaging as an alternative to current use of echocardiographic measurements, where echocardiographic abnormalities are often nonspecific and hinder an accurate diagnosis of the disease.41
Although CD11c is a common marker for dendritic cells, it is also expressed by tissue macrophage populations. In the myocardium, CD11c is highly expressed on mouse CCR2+ MHCII high macrophages and lowly expressed on CCR2‐MHCII high and low macrophages and Ly6C+ macrophages.42 In humans, the abundance of CCR2+ macrophage population is associated with persistent left ventricular systolic dysfunction after mechanical unloading in patients with heart failure.43 It remains to be seen what role the various macrophages play in CS pathophysiology and if a particular dendritic cell or macrophage population is responsible for the associated phenotypes such as cardiac fibrosis and diastolic dysfunction. However, it is clear that constitutive activation of mTOR signaling in CD11c‐expressing cells is able to initiate a CS‐like phenotype. Future work should characterize the mTOR high Mac‐2+ CD11c‐expressing cells in this mouse model and clarify their role in disease progression (whether they play a direct or indirect role). Additionally, it would be interesting to compare the macrophages in this model to the 3 inflammatory macrophage populations identified in human CS.40
Abnormal plakoglobin and connexin 43 distribution in the myocardium is a shared feature of sarcoidosis and ACM.9, 44 The latter abnormality, also known as gap junction remodeling, is present in many other cardiomyopathies and is recognized to be a substrate for fatal arrhythmias.9 While we show reduced plakoglobin expression and abnormal distribution of connexin 43 in our mouse model as in myocardial samples of patients with CS,9 the arrhythmogenic phenotype in this model remains to be determined. ECG monitoring of the mice should be done in future studies to characterize the role of gap junction remodeling on the cardiac conduction system in this mouse model. Previously, a mouse model of ACM showed aberrant activation of NF‐kB, and the treatment with Bay11‐7082, an inhibitor or NF‐kB, abolished all disease‐associated features.11 The clinical and molecular overlap between ACM and sarcoidosis prompted us to test the efficacy of Bay11‐7082 in preventing disease‐associated abnormalities in TSC2KO animals. Interestingly, treatment with Bay11‐7082 neither prevented granulomatous infiltration nor inhibited the mTOR signaling pathway in the hearts of TSC2KO animals. However, Bay11‐7082 reverses localization of plakoglobin and connexin 43 in an animal model of ACM.11 The fact that Bay11‐7082 does not restore physiological distribution of the plakoglobin and connexin 43 proteins in the TSC2KO animal model of sarcoidosis implicates disparate pathogenetic mechanisms underlying protein remodeling in these diseases. In prostate cancer, Dan et al indicated that mTOR can control NF‐kB activity via interaction with and stimulation of IKK.45 In fact, more recently, Dai et al showed that in macrophages mTOR deficiency suppresses NF‐κB activation induced by high glucose.46 Altogether, this evidence suggests that mTOR is upstream of NF‐κB. Consequently, targeting NF‐kB alone may not be sufficient for prevention of the CS phenotype in our animal model.
In this study we show that therapeutic administration of the mTOR inhibitor everolimus is sufficient to resolve granulomatous infiltrates and prevent the progression of fibrotic lesions in the hearts of TSC2KO animals. These changes were associated with the improvement of cardiac dysfunction in TSC2KO animals. In agreement with our findings, the administration of sirolimus to a patient with de novo systemic sarcoidosis following a liver transplant resulted in disease resolution.47 Similarly, sirolimus was recently used to successfully treat a patient with pulmonary sarcoidosis.48 mTOR inhibition was also shown to inhibit pulmonary fibrosis by regulating epithelial‐mesenchymal transition49 and that targeted inhibition of PI3K/mTOR in lung fibroblasts suppresses pulmonary fibrosis.50 Future work should clarify the role played by CD11c‐expressing macrophages or cells in the development of fibrosis in CS and their interactions with cardiac fibroblasts.
Altogether, we show that macrophages showing high mTORC1 signaling play an important role in the development and progression of CS. We also present the first animal model of sarcoidosis with cardiac involvement, recapitulating several histopathological features of human CS. This model of CS, though systemic (also affecting the lungs and skin), can be used as a preclinical model to test the efficacy of corticosteroid‐sparing alternative drugs and gain insight into the molecular mechanism behind their action. As everolimus treatment clears inflammatory infiltrates and improves cardiac function and fibrosis in this model of sarcoidosis, we propose the use of mTOR inhibitors in future clinical trials to test for efficacy in CS.
Sources of Funding
The authors thank the British Heart Foundation project grant (PG/18/27/33616) for funding all studies related to this project. A. Asimaki is also funded by the Wellcome Trust project grant (208 460/Z/17/Z) and Rosetrees Foundation Trust corn seed fund (M689). C. Bueno‐Beti is supported by the British Heart Foundation project grant (PG/18/27/33616). Research in the Weichhart laboratory is supported by funding from the Austrian Science Fund (FWF) grants P34023‐B, P34266‐B, FWF Sonderforschungsbereich F83, Vienna Science and Technology Fund (WWTF) grant LS18‐058, and the Ann Theodore Foundation Breakthrough Sarcoidosis Initiative. Dr Behr is funded by St George's Hospital Charity, RES 19‐20 002 ‘Genomics in Sudden Cardiac Death and Inherited Cardiac Conditions,’ and supported by the Robert Lancaster Memorial Fund. Drs Sheppard and Westaby are supported by the charity, Cardiac Risk in the Young (CRY).
Disclosures
None.
Footnotes
Supplemental Material is available at Supplemental Material
This manuscript was sent to Rebecca D. Levit, MD, Associate Editor, for review by expert referees, editorial decision, and final disposition.
For Sources of Funding and Disclosures, see page 12.
Supplemental Material
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© 2023 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.
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Received: 22 June 2023
Accepted: 15 August 2023
Published online: 26 September 2023
Published in print: 3 October 2023
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Funding Information
British Heart Foundation: PG/18/27/33616
Wellcome Trust: 208 460/Z/17/Z
Rosetrees Foundation Trust
Austrian Science Fund: P34266‐B, P34023‐B
Austrian Science Fund: FWFSonderforschungsbereichF83
Vienna Science and Technology Fund: LS18‐058
Ann Theodore Foundation
St George’s Hospital Charity: RES 19‐20 002
Robert Lancaster Memorial Fund
Cardiac Risk in the Young
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