Repurposing Nintedanib for Pathological Cardiac Remodeling and Dysfunction

Abstract

Heart Failure (HF) is the leading cause of death worldwide. Myocardial fibrosis, one of the clinical manifestations implicated in almost every form of heart disease, contributes significantly to HF development. However, there is no approved drug specifically designed to target cardiac fibrosis. Nintedanib (NTB) is an FDA approved tyrosine kinase inhibitor for idiopathic pulmonary fibrosis (IPF) and chronic fibrosing interstitial lung diseases (ILD). The favorable clinical outcome of NTB in IPF patients is well established. Furthermore, NTB is well tolerated in IPF patients irrespective of cardiovascular comorbidities. However, there is a lack of direct evidence to support the therapeutic efficacy and safety of NTB in cardiac diseases. In this study we examined the effects of NTB treatment on cardiac fibrosis and dysfunction using a murine model of HF. Specifically, 10 weeks old C57BL/6J male mice were subjected to Transverse Aortic Constriction (TAC) surgery. NTB was administered once daily by oral gavage (50mg/kg) till 16 weeks post-TAC. Cardiac function was monitored by serial echocardiography. Histological analysis and morphometric studies were performed at 16 weeks post-TAC. In the control group, systolic dysfunction started developing from 4 weeks post-surgery and progressed till 16 weeks. However, NTB treatment prevented TAC-induced cardiac functional decline. In another experiment, NTB treatment was stopped at 8 weeks, and animals were followed till 16 weeks post-TAC. Surprisingly, NTB’s beneficial effect on cardiac function was maintained even after treatment interruption. NTB treatment remarkably reduced cardiac fibrosis as confirmed by Masson’s trichrome staining and decreased expression of collagen genes (COL1A1, COL3A1). Compared to the TAC group, NTB treated mice showed a lower HW/TL ratio and cardiomyocyte cross-sectional area. NTB treatment reduced myocardial and systemic inflammation by inhibiting pro-inflammatory subsets and promoting regulatory T cells (Tregs). Our in vitro studies demonstrated that NTB prevents myofibroblast transformation, TGFβ1-induced SMAD3 phosphorylation, and the production of fibrogenic proteins (Fibronectin-1, α-SMA). However, NTB promoted immunosuppressive phenotype in Tregs, and altered vital signaling pathways in isolated cardiac fibroblast and cardiomyocytes, suggesting that its biological effect and underlying cardiac protection mechanisms are not limited to fibroblast and fibrosis alone.

Our findings provide a proof of concept for repurposing NTB to combat adverse myocardial fibrosis and encourage the need for further validation in large animal models and subsequent clinical development for HF patients.

Graphical Abstract

1. Introduction

Nintedanib (BIBF 1120) was developed by Boehringer Ingelheim (BI) Pharmaceuticals, Inc. In 2014, the FDA approved its use for the treatment of Idiopathic Pulmonary Fibrosis (IPF). BI is marketing NTB under the brand name Ofev. Recently (October 2019) it obtained FDA’s “breakthrough therapy” designation and approval for treatment of chronic fibrosing interstitial lung diseases (ILD). Moreover, NTB is used as a second-line treatment along with other medications to treat non-small-cell lung cancer ¹, and in several phases I to III clinical trials for different types of cancer (ClinicalTrials.gov Identifier: NCT02149108, NCT02902484, NCT03377023, NCT02399215). Currently, BI is recruiting patients in a phase 3 clinical trial named NINTECOR (ClinicalTrials.gov Identifier: NCT04541680) to assess whether NTB slows the progression of lung fibrosis in COVID-19 survivors.

NTB is a potent tyrosine kinase inhibitor (TKI) that primarily targets Receptor Tyrosine Kinases (RTKs) such as vascular endothelial growth factor receptor (VEGFR) 1, 2, and 3, platelet-derived growth factor receptor (PDGFR) α and β, and fibroblast growth factor receptor (FGFR) 1, 2, and 3. It also inhibits some non-RTKs including Lck, Lyn, and Src.² Clinical trials revealed that NTB prevented the progression of fibrosis and improved lung function, leading to a better prognosis in IPF patients.³ Many pre-clinical studies have demonstrated that NTB has antifibrotic effects in disease models of different organs, including lung, liver, skin, and kidney. ^4–7 Since the fundamental mechanisms of fibrosis remain common irrespective of the etiology of the disease, it is conceivable that NTB may also prevent adverse fibrotic remodeling implicated in cardiac diseases.

Fibrosis, characterized by excessive accumulation of ECM, is associated with multiple cardiac disorders. ECM deposition causes stiffening of the cardiac muscles, increases the risk of arrhythmia, disrupts electrical and mechanical coupling of cardiac cells, and subsequently leading to functional decline and HF. Fibroblasts are the key players in ECM production and fibrosis progression.⁸ Several recent studies with FB-specific mouse models have demonstrated that activated fibroblasts and myocardial fibrosis contribute significantly to HF. ^9–14 However, at present, there is no therapy specifically designed to target myocardial fibrosis. In the current study, we determined the possibility of repurposing NTB to treat myocardial fibrosis and cardiac dysfunction. We employed a mouse model of pressure overload and examined the effect of NTB intervention on pressure overload-induced cardiac pathophysiology. We found that NTB, when administered as a preventive regimen, remarkably reduces pressure overload-induced cardiac fibrosis, hypertrophy, and dysfunction. Surprisingly these cardioprotective effects were observed despite interruption of the NTB treatment. Furthermore, we demonstrated that NTB alters fundamental signaling pathways in cardiac fibroblasts and cardiomyocytes, and modulates the phenotype of immune cells indicating multi-target mechanisms of action in the heart.

2. Materials and Methods

2.2. Mice and Nintedanib treatment

C57BL/6J mice were obtained from The Jackson Laboratories (Stock No: 000664), and the colony was maintained at the University of Alabama at Birmingham (UAB) animal care facility. All animal studies were approved by the Institutional Animal Care and Use Committee at UAB. At 10 weeks of age, mice were subjected to TAC surgery. NTB (Cat. No. N9055, LC Laboratories) was dissolved in water by heating to 50°C while intermittent stirring. NTB was administered daily by oral gavage (50mg/kg). NTB administration was started 1 day before surgery and continued as per experimental design.

2.3. Transverse Aortic Constriction (TAC) surgery in mice

TAC was performed as previously described.¹⁵ Briefly, mice were sedated with isoflurane (induction, 3%; maintenance, 1.5%) and anesthetized to a surgical plane with intraperitoneal ketamine (50 mg/kg) and xylazine (2.5 mg/kg). Anesthetized mice were intubated, and a midline cervical incision was made to expose the trachea and carotid arteries. A blunt 20-gauge needle was inserted into the trachea and connected to a volume-cycled rodent ventilator on supplemental oxygen at a rate of 1 l/min, with a respiratory rate of 140 breaths/min. Aortic constriction was performed by tying a 7–0 nylon suture ligature against a 27-gauge needle. The needle was then promptly removed to yield a constriction of approximately 0.4 mm in diameter. To confirm the efficiency of consistent TAC surgery, the pressure gradient across the aortic constriction was measured by Doppler echocardiography.

2.4. Echocardiography

Echocardiography was performed as described previously.¹⁶ In brief, transthoracic M-mode echocardiography was performed with a 12-MHz probe (VisualSonics) on mice anesthetized by inhalation of isoflurane (1–1.5%). LV end-systolic interior dimension (LVID; s), LV end-diastolic interior dimension (LVID;d), LV end-diastolic posterior wall thickness (LVPW, d), LV end-systolic posterior wall thickness (LVPW, s), ejection fraction (EF), and fractional shortening (FS) values were obtained by analyzing data using the Vevo 3100 program.

2.5. Histology

Heart tissues were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm thickness. Sections were stained with Masson Trichrome (HT15, Sigma-Aldrich) as per the manufacturer’s instruction. The images of the LV region were captured using a Nikon Eclipse E200 microscope with NIS element software version 5.20.02. The quantification of LV fibrosis and cross-sectional area of cardiomyocytes (CSA) were determined with ImageJ version 1.52a software (NIH). For fibrosis measurement, 8–10 images of the LV region were taken, and LV fibrosis was quantified as a percentage of the total LV area scanned.

2.6. Immunofluorescence studies

Cells were plated on a coverslip and treated as mentioned. Cells were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 in PBS for 10 min, blocked with 3% BSA in PBS for 1h. Cells were incubated with primary antibodies overnight at 4°C. After washing with PBS, cells were stained with secondary antibodies for 1h at RT, mounted in ProLong Gold antifade reagent with DAPI (Invitrogen, P36941).

In the case of paraffin-embedded tissue sections, deparaffinization and rehydration were carried out followed by antigen retrieval using Antigen Unmasking Solution (Vector Lab Inc., H-3300). Tissue sections were permeabilized with 0.4% Triton X-100 in PBS (PBST) for 20 min. To block non-specific antibody binding, sections were incubated with a blocking solution containing 5% goat serum in PBST for 30 min at RT. Sections were incubated with primary antibodies overnight at 4°C. After washing with PBS, sections were stained with secondary antibodies for 1h at RT, mounted in ProLong Gold antifade reagent with DAPI (Invitrogen, P36941). A list of antibodies is provided in Supplemental Table 1. Fluorescent images were taken on a KEYENCE BZ-X800 fluorescence microscope.

2.7. Western blot

LV tissues were dissected from mouse heart and homogenized with cell lysis buffer (Cell signaling Technology) having 50 mM Tris-HCl (pH7.4), 150 mM NaCl, 1mM EDTA, 0.25% sodium deoxycholate, 1% NP-40, and freshly supplemented Protease Inhibitor Cocktail (Sigma-Aldrich) and Phosphatase Inhibitor Cocktail (Sigma-Aldrich). Protein concentration was determined with the Bradford assay (Bio-Rad protein assay kit). An equal amount of proteins was denatured in SDS–PAGE sample buffer, resolved by SDS–PAGE, and transferred to Immobilon-P PVDF membrane (EMD Millipore). The membranes were blocked in Odyssey blocking buffer (LI-COR Biosciences) for 1h at RT. Primary antibody incubations were performed at different dilutions as described in Supplemental Table 2. All incubations for primary antibodies were done overnight at 4°C and followed by secondary antibody (IRDye 680RD or IRDye 800CW from LI-COR Biosciences) incubation at 1:3000 dilutions for 1h at RT. Proteins were visualized with the Odyssey Infrared Imaging System (LI-COR Biosciences). Band intensity was quantified by Image Studio version 5.2 software.

2.8. RNA extraction and quantitative PCR analysis

Total RNA was extracted from heart tissue using the RNeasy Mini Kit (74104, Qiagen) according to the manufacturer’s protocol. cDNA was synthesized using the iScript cDNA synthesis kit (170–8891, Bio-Rad) following the manufacturer’s instructions. Gene expression was analyzed by quantitative PCR (qPCR) using the TaqMan Gene Expression Master Mix (4369016, Applied Biosystems) and TaqMan gene expression assays (Applied Biosystems) on a Quant studio 3 (Applied Biosystems) Real-Time PCR Detection machine. Details of TaqMan gene expression assays used in this study are provided as Supplemental Table 3. Relative gene expression was determined by using the comparative CT method (2^−ΔΔCT) and was represented as fold change. Briefly, the first ΔCT is the difference in threshold cycle between the target and reference genes: ΔCT= CT (a target gene X) − CT (18S rRNA) while ΔΔCT is the difference in ΔCT as described in the above formula between the CTRL and NTB group, which is = ΔCT (NTB target gene X) − ΔCT (CTRL target gene X). Fold change is calculated using the 2^{− ΔΔCT} equation.¹⁷

2.9. Isolation and culture of neonatal rat cardiomyocytes and cardiac fibroblasts

Primary cultures of neonatal rat ventricular cardiomyocytes (CM) and fibroblasts (CF) were prepared from 1–2-day-old pups of Sprague-Dawley rats as described previously.¹⁸

2.10. Isolation of cardiac leukocytes and splenocytes from mouse

To isolate cardiac leukocytes, mice hearts were perfused with 1X PBS, harvested, and minced. Minced hearts were suspended in digestion media - RPMI 1640 containing collagenase I (C0130, 1mg/ml), collagenase XI (C7657, 0.1mg/ml), hyaluronidase (H3506, 0.1mg/ml) and DNase I (D4527, 1ul/ml). The tissue was digested at 37°C for 1 hour with gentle shaking. After 1 hour of digestion, the tissue digest was filtered through 70μm strainers. To stop enzyme reaction, RPMI 1640 media containing 5% FBS was added to the digest. The tissue digest was then centrifuged at 1300 rpm for 10 minutes at 4°C to pellet down the leukocytes. Cells were suspended in complete media (10% FBS containing RPMI 1640).

For adult mouse splenocytes isolation, spleens were excised from mice and macerated in RPMI 1640 (Gibco, Invitrogen, UK) containing 10% FBS to prepare a single-cell suspension. Mice splenocytes were cultured in RPMI 1640 media containing 10% FBS and used for the in vitro experiments.

2.11. Flow cytometry analyses

Mice cardiac leukocytes and splenocytes were isolated as described in previous sections. Red blood cells (RBCs) were lysed with RBC lysis buffer (Quality Biological, Inc.). For surface staining, 0.5–1×10⁶ cells were used post - Fc blocking (1μg/ml) in 3% FBS for 30 minutes over ice. For intracellular staining, 0.5–1×10⁶ cells were seeded per well in 96-well plates (Nunc, USA) then incubated for 5 hrs in stimulating RPMI media which contained Phorbol 12-myristate 13-acetate, or PMA, (0.1 mg/ml, Sigma), ionomycin (1 mg/ml, Sigma), and Golgistop/Golgiplug (1:1000, BD Biosciences). Following stimulation, cells were first surface stained for 30 minutes on ice. After washing with PBS cells were fixed and permeabilized using the BD cytofix/cytoperm kit (BD Biosciences) for 30 minutes at 4°C then washed again with BD perm wash kit (BD Biosciences) and stained with fluorescently labeled antibodies. A list of antibodies is provided in Supplemental Table 4. Fluorescence intensity of fluorochrome-labeled cells was analyzed by flow cytometry (BD LSR-II) and FACS Diva software and final data analysis was performed by Flow Jo (Tree Star, USA).

2.12. Cell migration assay

CFs were seeded in culture dishes and allowed to grow till confluency. The wound was created by scratching the monolayer of the cells. Cells were pre-treated with NTB (0.5μM) for 1h followed by agonists treatments for a total of 24h. The percentage of wound closure was monitored and quantified over the indicated time points.

2.13. Statistical analysis

Analyses were performed using GraphPad Prism (version 9.0.0). Differences between more than 2 data groups were evaluated for significance by ANOVA followed by Tukey’s multiple comparison test. All data are expressed as mean ± SEM. For all tests, a p-value of < 0.05 was considered statistically significant.

3. Results:

3.1. NTB administration prevented pressure overload-induced cardiac dysfunction and adverse cardiac remodeling

We evaluated the therapeutic efficacy of NTB in a pre-clinical mouse model of heart failure. We subjected 10 weeks old C57BL/6J mice to TAC surgery. To study the effect of NTB on pressure overload-induced cardiac pathology, the drug was administered throughout the experimental period (preventive regimen) (Figure 1A). We examined cardiac function by serial M-mode echocardiography. In the TAC group, LVEF and LVFS started to decline from 4 weeks post-surgery, indicating systolic dysfunction (Figure 1B–1C). These changes were associated with the development of structural remodeling as reflected by a significant increase in LVIDs, LVPWDs, and LV Mass in the TAC group. All these parameters were remarkably normalized due to NTB administration, indicating NTB treatment improved cardiac function and prevented adverse cardiac remodeling in TAC mice (Figure 1D–1H).

Figure 1: NTB treatment improves cardiac function in TAC mice.

(A) At 10 weeks of age, C57BL/6J mice were subjected to TAC surgery and treated with NTB (50mg/kg/day) for the indicated time. Cardiac function was assessed by serial echocardiography (B) EF: Ejection fraction, (C) FS: Fractional shortening, (D) LVID, d: LV end-diastolic dimension (E) LVID, s: LV end-systolic dimension, (F) LV Mass corrected (G) LVPW, d: LV end-diastolic posterior wall thickness (H) LVPW, s: LV end-systolic posterior wall thickness. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. n>5 per group. ^#P<0.05 for SHAM vs TAC, *P<0.05 for TAC vs.TAC + NTB.

To verify the effects of NTB treatment on adverse cardiac remodeling, we examined cardiac hypertrophy and fibrosis at 16 weeks post-TAC. We measured HW/TL ratio and cardiomyocyte cross-sectional area (CSA). Both these parameters were elevated in the TAC group revealing hypertrophic remodeling (Figure 2A & 2B). For assessment of cardiac fibrosis, we did Masson’s Trichrome staining and found excessive collagen deposition in TAC hearts (Figure 2C & 2D). Moreover, TAC hearts displayed a significant increase in α-SMA expression (Figure 2E & 2F). All these characteristics of pathological cardiac remodeling were alleviated in NTB treated TAC group. Additionally, we compared expression levels of key genes related to cardiac fibrosis (COL1A1 and COL3A1). These molecular markers were significantly augmented in the TAC group and were normalized in NTB treated TAC mice (Figure 2G & 2H).

Figure 2: NTB treatment prevents pressure overload-induced cardiac fibrosis and hypertrophy.

Morphometric studies were performed at 16 weeks after TAC surgery; (A) Heart weight (HW) to tibia length (TL) ratio, (B) Quantification of cardiomyocyte cross-sectional area (CSA), (C) Masson’s trichrome staining. Representative trichrome-stained LV regions and (D) Quantification of LV fibrosis. (E) Representative heart sections showing α-SMA +ve cells (Red) and (F) Quantification. Scale bar = 30 μm. RNA was extracted from the left ventricle of experimental animals and gene expression analysis was carried out by qPCR (G) COL1A1 (H) COL3A1. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. n=3–5 per group. ^#P<0.05 for SHAM vs. TAC, *P<0.05 for TAC vs. TAC + NTB.

3.2. Cardioprotective effects of NTB persisted despite interruption of the treatment

To test whether the efficacy of NTB is maintained even after cessation of the treatment, we treated mice with NTB for 8 weeks post-TAC, thereafter drug administration was discontinued, and animals were followed till 16 weeks post-TAC (Figure 3A). We assessed cardiac function by serial echocardiography. We found that cardiac function started to deteriorate from 4 weeks post-surgery in TAC groups. When compared to SHAM, LVIDs, LVPWDs, and LV mass were significantly increased in TAC groups indicating the development of adverse cardiac remodeling. As observed in the preventive treatment regimen, continuous NTB treatment till 8 weeks prevented TAC-induced cardiac dysfunction and remodeling. Surprisingly, these cardioprotective effects of NTB administration were sustained up even after 8 weeks of washout period (Figure 3B–3H).

Figure 3: Interruption of the treatment does not affect cardiac function improvement in NTB treated TAC mice.

(A) At 10 weeks of age, C57BL/6J mice were subjected to TAC surgery and treated with NTB (50mg/kg/day) for 8 weeks. After 8 weeks, NTB treatment was discontinued, and animals were followed for additional 8 weeks. Cardiac function was assessed by serial echocardiography (B) EF: Ejection fraction, (C) FS: Fractional shortening, (D) LVID, d: LV end-diastolic dimension (E) LVID, s: LV end-systolic dimension, (F) LV Mass corrected, (G) LVPW, d: LV end-diastolic posterior wall thickness, and (H) LVPW, s: LV end-systolic posterior wall thickness. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. n>5 per group. ^#P<0.05 for SHAM vs. TAC, *P<0.05 for TAC vs. TAC + NTB.

To further verify the cardioprotective effects of NTB treatment on cardiac remodeling, we harvested the heart of experimental animals at 8 weeks washout period for morphometrics and histological studies. TAC group showed cardiac hypertrophy as reflected by a significant increase in HW/TL and CSA compared with the SHAM group (Figure 4A & 4B). Masson’s Trichrome staining revealed excessive collagen deposition in TAC groups (Figure 4C & 4D). Importantly, cardiac hypertrophy and fibrosis were reduced in mice treated with NTB. Next, we compared the expression of genes related to cardiac fibrosis (COL1A1, COL3A1) using the qPCR method. In the TAC group, there was a significant increase in levels of these molecular markers and NTB treatment could normalize their levels (Figure 4E & 4F). All these results indicate the maintained efficacy of NTB despite the interruption of the treatment.

Figure 4: Effect of NTB treatment interruption on pressure overload induced adverse cardiac remodeling.

At 10 weeks of age, C57BL/6J mice were subject to TAC surgery and treated with NTB (50mg/kg/day) for 8 weeks. After 8 weeks, NTB treatment was discontinued for further 8 weeks. Morphometric studies were performed at the end of the washout period; (A) Heart weight (HW) to tibia length (TL) ratio, (B) Quantification of cardiomyocyte cross-sectional area (CSA), (C) Masson’s trichrome staining. Representative trichrome-stained LV regions and (D) Quantification of LV fibrosis. Scale bar = 30 μm. RNA was extracted from the left ventricle of experimental animals and gene expression analysis was carried out by qPCR (E) COL1A1 (F) COL3A1. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. n>5 per group. ^#P<0.05 for SHAM vs. TAC, *P<0.05 for TAC vs. TAC + NTB.

3.3. NTB treatment prevented profibrotic responses in cardiac fibroblasts

NTB has proven anti-fibrotic potential in disorders such as IPF. Since we observed a reduction in cardiac fibrosis in NTB treated TAC mice, we examined cardiac fibroblasts specific effects of NTB. Cardiac fibroblasts were pre-treated with NTB (0.5μM) for 1h followed by TGF-β1 treatment (10ng/ml for 1h). Our western blot results showed that NTB treatment prevented TGF-β1 mediated SMAD3 phosphorylation. However, NTB treatment had no effect on TGF-β1 induced p38 activation. These results indicate that NTB exerts its anti-fibrotic effect via canonical TGF- β1-SMAD3 pathway in a MAPK p38 independent manner (Figure 5A & 5B). NTB targets FGFR and inhibits RTKs associated with it. Since FGF/FGFR signaling is fundamental in fibroblast growth and survival, we studied the effects of NTB on basic fibroblast growth factor (bFGF) induced MAPK and AKT activation in cardiac fibroblasts. Cardiac fibroblasts were pre-treated with NTB (0.5μM) for 1h followed by bFGF treatment (25ng/ml bFGF for 10 min). As anticipated, NTB treatment prevented bFGF induced ERK and AKT activation in cardiac fibroblasts (Figure 5C & 5D). Additionally, NTB treatment inhibited TGF-β1-induced production of fibrogenic protein (α-SMA and fibronectin) (Figure 5E & 5F) and prevented agonists-induced cell migration (Figure 5G). These results indicate that NTB prevents myofibroblast transformation and fibrotic responses in cardiac fibroblasts.

Figure 5: NTB treatment prevents profibrotic responses in cardiac fibroblasts.

Neonatal rat cardiac fibroblasts (CFs) were pretreated with NTB (0.5μM) for 1h followed by agonists treatment (10ng/ml TGFβ1 for 1h or 25ng/ml bFGF for 10 min). Proteins were extracted, and Western blot analysis was carried out. Representative blots and Quantification - (A) SMAD3 (B) P38 (C) ERK (D) AKT, and (E) Fibronectin-1. (F) Representative immunofluorescence image showing α-SMA +ve cells and quantification. For cell migration assay, CFs were treated with agonists (10ng/ml TGFβ1, 25ng/ml bFGF, or 10μM AngII) in the presence or absence of NTB (0.5μM) for 24h. Wound closure was monitored from 0h-24h and % wound closure was calculated at 24h using ImageJ software (G). Data were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. n=3–4. ^#P<0.05 vs. Control, *P<0.05 vs. Agonist.

3.4. Effect of NTB treatment on cardiomyocytes

Our in vivo studies showed that NTB treatment prevented pressure overload-induced cardiac hypertrophy. To evaluate whether the reduction in cardiac hypertrophy is due to the direct effect of NTB on cardiomyocytes (CM), we treated CMs with hypertrophy inducing agents [100μM phenylephrine (PE), 25ng/ml bFGF, or 10μM Angiotensin II (AngII) ] in presence or absence of NTB (1μM) for 48h. Induction of hypertrophic response was confirmed by analyzing the expression of genes (ANP, BNP) and measuring cell surface area. We observed that NTB treatment had no significant effect on agonists -induced hypertrophic response (Figure 6A–6C). Next, we verified the effect of NTB treatment on key signaling pathways in CMs. CMs were pre-treated with NTB (1μM) for 1h followed by PE (100μM) treatment for 15min. Western blot analysis showed that PE-induced MAPK and AKT phosphorylation was inhibited by NTB (Figure 6D–6G). Taken together, these results indicate that NTB can directly modulate CM signaling underlying hypertrophic stimuli. However, this much interference is not sufficient to prevent CM hypertrophic growth.

Figure 6: NTB treatment inhibits signaling pathways underlying hypertrophic stimuli in cardiomyocytes.

To study whether NTB could prevent cardiomyocyte hypertrophy, neonatal rat cardiomyocytes (CMs) were treated with agonists (100μM PE, 25ng/ml bFGF, or 10μM AngII) in the presence or absence of NTB (1μM) for 48h, RNA was extracted and gene expression levels were compared using qPCR method (A) ANP and (B) BNP. Immunofluorescence staining of α-Actinin was carried out at 48hrs after treatment. (C) Representative immunofluorescence image and quantification of cell surface area. Scale bar = 50μm. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. n=3. ^#P<0.05 vs. Control, *P<0.05 vs. Agonist. To examine the effect of NTB on CM’s signaling, CMs were pretreated with NTB (1μM) for 1h followed by PE (100μM) treatment for 15min. Proteins were extracted, and Western blot analysis was carried out. Representative blots and Quantification - (D) ERK (E) P38 (F) AKT, and (G) GSK3α/β. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. n=3. ^#P<0.05 vs. Control, *P<0.05 vs. PE.

3.5. NTB reduces myocardial and systemic inflammation by inhibiting pro-inflammatory subsets and promoting regulatory T cells (Tregs)

Previous pre-clinical studies have demonstrated the anti-inflammatory properties of NTB.⁴ Since inflammation is one of the contributing factors in the pathogenesis of cardiac diseases, we examined whether NTB had any effect on myocardial and systemic inflammation. We assessed the subsets of infiltrating leukocytes in the mouse heart at 3 weeks post-TAC. We observed a significant reduction in CD45⁺ leukocytes and TNF-α producing CD45⁺ cells in NTB treated TAC group. However, Tregs were elevated in NTB treated TAC mice (Figure 7A–7D). Additionally, NTB administration reduced systemic inflammation as evidenced by the diminished frequency of pro-inflammatory TNF-α, IL-1β, IL-6 producing splenocytes (Figure 7E–7G).

Figure 7: NTB promotes Tregs and inhibits pro-inflammatory subsets.

At 10 weeks of age, C57BL/6J mice were subject to TAC surgery and treated with NTB (50mg/kg/day) for 3 weeks. After 3 weeks of NTB treatments flow cytometric analysis of immune cells was carried out. (A) Total percentages of CD45+ cells in the heart (B) Total calculated percentages of CD3+CD4+CD25+FOXP3+ identified as Treg in the heart (C) Representative flow cytometry plot showing CD3+CD4+ and CD25+Foxp3+ cells in the heart-based CD45+ gated population (D) Total percentages of TNF-α producing leukocytes in the heart and Representative flow cytometry plot (E) Total percentages of TNF-α producing splenocytes and Representative flow cytometry plot (F) Total percentages of IL1β producing splenocytes and Representative flow cytometry plot (G) Total percentages of IL-6 producing splenocytes and Representative flow cytometry plot. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc analysis and represented as mean ± SEM. n=4–7. To examine the direct effect of NTB on immune cells in vitro, splenocytes were isolated from C57BL/6J mice, cultured, and treated with NTB (100 nM, 500 nM) for 72 hours. Flow cytometric analysis was performed (H) Total calculated percentage of Tregs identified as CD3+CD4+CD25+Foxp3+ by flow cytometry (I) Total percentages of PD-1 expressing CD4+ T cells (J) Total percentages of CTLA-4 expressing CD4+ T cells (K) Representative flow cytometry plot confirming enhanced expression of CTLA-4 and PD-1 over CD4+ T cells. Data were analyzed using one-way ANOVA followed by Tukey’s post hoc 1 analysis and represented as mean ± SEM. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To further confirm the immunomodulatory role of NTB, we evaluated the direct effect of NTB on immune cells in in vitro setting. We treated mouse splenocytes with NTB (100 nM and 500 nM for 72h) and examined immune cell dynamics using flow cytometry. Interestingly, we observed induction of Treg frequency in response to NTB treatment (Figure 7H). One of the critical mechanism by which Treg develop immunosuppressive phenotype is the through expression of CTLA-4 and PD-1.¹⁹ Thus to ascertain whether Treg cells have acquired immunosuppressive phenotype after NTB treatment, we examined CTLA-4 and PD-1 expression in immune cells. We found that NTB treatment significantly increased CTLA-4 and PD-1 expressing CD4⁺ T cells (Figure 7I–7K). Thus, confirming the immunosuppressive phenotype of these cells.

4. Discussion

In the present study, we examined the effects of NTB treatment on pathological remodeling and cardiac dysfunction in a murine model of pressure overload. We found that when administered as a preventive regimen, NTB prevents pressure- overload induced myocardial dysfunction, adverse fibrotic remodeling, and pathological hypertrophy in mice. Importantly, the cardioprotective effects of NTB were sustained despite the cessation of the treatment.

NTB is approved for treatment in IPF patients, where its beneficial effects are attributed to its anti-fibrotic action and improvement of lung function.³ In the mdx mouse model of Duchenne Muscle Dystrophy (DMD), NTB administration reduced fibrosis and improved muscle performance.²⁰ The efficacy of NTB has been tested in the pre-clinical model of Pulmonary Arterial Hypertension (PAH) in which authors demonstrated that NTB treatment reduced fibrosis and prevented RV hypertrophy.²¹ All these studies revealed that halting the progression of adverse fibrotic remodeling facilitates the functional improvement of the organ. In line with these reports, we observed that after NTB intervention, cardiac function is improved in the murine model of heart failure. This functional gain was associated with a significant decrease in interstitial cardiac fibrosis. Nintedanib is a TKI that primarily targets PDGFR, VEGFR, and FGFR. There are multiple FDA approved TKIs with similar inhibition profiles. Most of these targets are associated with adverse cardiovascular events such as arrhythmia, PAH, and atherosclerosis.^{22, 23} Of note, we have reported that a similar kinase inhibitor, “sorafenib,” is cardiotoxic, despite an anti-fibrotic effect.²³ Paradoxically, our study demonstrated the cardioprotective effects of NTB in a pre-clinical model of HF. In fact, with our multiple years of experience researching the cardiac effects of various kinase inhibitors, particularly cancer therapeutic KIs, NTB is the first with favorable outcomes at both fronts, cancer, and heart.

Since we observed a reduction in cardiac hypertrophy in NTB treated animals, we were interested in determining if NTB directly affects cardiomyocytes apart from its well-studied fibroblast-specific anti-fibrotic mechanisms. Although NTB could not prevent agonist-induced CMs hypertrophic response, significant alteration in key signaling pathways underlying such stimulus was observed. Of note, the expression and biological function of NTB targeted receptors in CMs are well-established. Therefore, it was not surprising to observe a significant effect of NTB on major cardiomyocyte signaling pathways. Moreover, our in vitro experiments demonstrated that NTB treatment leads to induction of immunosuppressive phenotype in Treg cells. Since Treg cells are shown to effectively mitigate the progression of many cardiovascular diseases,²⁴ we believe that observed expansion of Treg population in NTB treated TAC group could be one of the mechanisms of cardio-protection. Taken together, it is conceivable that NTB’s cardioprotective effects cannot be simply attributed to its direct effect on fibroblast and fibrosis alone. Further studies are warranted to delineate detailed mechanisms of NTB’s therapeutic efficacy and define the contribution of various cells, including immune cells, fibroblast, and cardiomyocytes.

Noth et al. investigated the cardiovascular safety of NTB in IPF patients.²⁵ They performed an analysis using pooled data from the TOMORROW and INPULSIS trials and concluded that irrespective of cardiovascular risk at baseline, the incidence of major adverse cardiac events was similar between patients treated with NTB vs placebo. However, the study had limitations such as the absence of randomization at baseline and unavailability of data on cardiovascular medication. Thus, there is a need to evaluate the efficacy of NTB specifically in CVD patients. Importantly, the current standard-of-care therapeutic agents, such as angiotensin II receptor blockers (ARBs), beta-blockers, angiotensin-converting enzyme (ACE) inhibitors, statins, etc. operate through pleiotropic mechanisms and none of them are explicitly designed to target cardiac fibrosis. Hence, one can test the hypothesis that there might be synergistic benefits of NTB when given as combinatorial regimens with standard cardiac therapeutic agents. Also, future studies are needed to evaluate whether NTB is equally efficacious when given as a therapeutic regimen post-injury.

5. Conclusion

In conclusion, our study showed beneficial effects of NTB on pressure overload-induced cardiac remodeling and dysfunction in mice. We further demonstrated that the cardioprotective effects of NTB are mediated via multiple coordinated mechanisms directly affecting immune cells, cardiac fibroblasts, and cardiomyocytes. Our findings provide a proof of concept for the repurposing of NTB in cardiac diseases and suggest the need for randomized, placebo-controlled clinical studies to evaluate the safety and efficacy of NTB in HF patients.

Non-standard abbreviations and acronyms:

NTB

Nintedanib

CSA

Cross-Sectional Area

HW

Heart weight

LV

Left Ventricle

LW

Lung Weight

TL

Tibia Length

PE

Phenylephrine

bFGF

Basic Fibroblast Growth Factor