T-cell infiltration and the subsequent increased intracardial chronic inflammation play crucial roles in the development of cardiac hypertrophy and heart failure (HF). A77 1726, the active metabolite of leflunomide, has been reported to have powerful anti-inflammatory and T cell-inhibiting properties. However, the effect of A77 1726 on cardiac hypertrophy remains completely unknown. Herein, we found that A77 1726 treatment attenuated pressure overload or angiotensin II (Ang II)-induced cardiac hypertrophy in vivo, as well as agonist-induced hypertrophic response of cardiomyocytes in vitro. In addition, we showed that A77 1726 administration prevented induction of cardiac fibrosis by inhibiting cardiac fibroblast (CF) transformation into myofibroblast. Surprisingly, we found that the protective effect of A77 1726 was not dependent on its T lymphocyte-inhibiting property. A77 1726 suppressed the activation of protein kinase B (AKT) signaling pathway, and overexpression of constitutively active AKT completely abolished A77 1726-mediated cardioprotective effects in vivo and in vitro. Pretreatment with siRNA targetting Fyn (si Fyn) blunted the protective effect elicited by A77 1726 in vitro. More importantly, A77 1726 was capable of blocking pre-established cardiac hypertrophy in mice. In conclusion, A77 1726 attenuated cardiac hypertrophy and cardiac fibrosis via inhibiting FYN/AKT signaling pathway.
Heart failure (HF), the common end stage of various cardiovascular diseases, remains a major clinical and public health challenge worldwide, which is associated with a high burden of hospitalization and mortality [1,2]. Most therapeutic interventions hitherto aim solely at correcting the low cardiac output and offering symptomatic relief with negligible beneficial effects on the progression and long-term outcomes of HF . Cardiac hypertrophy is now well-accepted as a key pathophysiologic process of HF, and significantly impacts the clinical course and outcome of HF . In response to chronic injury, the cardiomyocytic and non-cardiomyocytic components of the myocardium undergo a series of structural and functional alterations (i.e. cardiomyocyte growth, cardiac fibroblast (CF) proliferation and transdifferentiation, and inflammatory responses), which result in cardiomyocyte hypertrophy and interstitial fibrosis by a complex interplay of mechanisms, and ultimately elicit the initiation and progression of HF . Undoubtedly, cardiac hypertrophy plays a crucial role in contributing to the development of HF and pharmacological inhibition of cardiac hypertrophy may be of great therapeutic interest for improving the prognosis of HF.
While an exact picture of all the cell responses and pathways involved in cardiac hypertrophy is still unclear, activation of immune responses and subsequent increased inflammatory level have been recently verified to be responsible for accelerating the hypertrophic process [5–7]. Previous studies indicated that activation of the immune response by cardiac injury triggered the inflammatory cell infiltration to myocardium and enhanced the release of inflammatory cytokines [8,9]. Laroumanie et al.  reported that lack of functional CD4+ T cells prevented the transition from hypertrophy to HF in response to pressure overload. Results from our lab also demonstrated that CD4+ T cells were involved in tumor necrosis factor receptor superfamily member 4-mediated deleterious effect on pressure overload-induced cardiac hypertrophy and fibrosis . In addition, pathological stimuli resulted in the accumulation of T cells and monocytes/macrophages in the heart and promoted the release of inflammatory factors, which contributed to heart damage and dysfunction . These findings suggest that T cells contribute to hypertrophic remodeling and HF progression, and that targeting CD4+ T cells and chronic intracardial inflammation could help in developing efficacious interventions for the treatment of cardiac hypertrophy and HF.
Leflunomide is an immunosuppressive agent with antiproliferative and antiphlogistic activities that is clinically approved for the treatment of rheumatic arthritis (RA) and other autoimmune diseases . Multiple clinical and basic studies have defined leflunomide as an efficacious agent in reducing inflammatory response [14,15]. Previous studies indicated that leflunomide inhibited joint inflammation in patients with active rheumatoid arthritis via suppressing nuclear factor-κB activation [16,17]. Importantly, leflunomide blocked proliferation of T lymphocytes in a number of murine models via a reduction in dihydro-orotic acid dehydrogenase (DHODH) activity [18–20]. Leflunomide also inhibited tyrosine kinase activity [18–20]. Based on these findings, we speculated that leflunomide might be a promising candidate for suppressing intracardial inflammation and a novel therapeutic agent for protection against cardiac hypertrophy and HF.
Leflunomide is almost completely metabolized (>95%) to its active compound A77 1726 after administration, which causes the main effects of leflunomide . In the present study, we investigated for the first time the effect of A77 1726 administration on cardiac hypertrophy both in vivo and in vitro.
Materials and methods
Antibodies and reagents
Primary antibodies against the following proteins were obtained from Cell Signaling Technology (Danvers, MA, U.S.A.): p-protein kinase B (p-PKB/AKT, #4060, 1:1000), total-AKT (t-AKT, #4691, 1:1000), p-glycogen synthase kinase 3 β (p-GSK3β, #9323P, 1:1000), t-GSK3β (#9315, 1:1000), p-P70S6 kinase (p-P70, #9234P, 1:1000), t-P70 (#2708, 1:1000), p-ribosomal protein S6 (p-S6, #5364P, 1:1000), t-S6 (#2217, 1:1000), p-extracellular signal-regulated kinase (p-ERK, #4370P, 1:1000), t-ERK (#4695, 1:1000), p-P38 mitogen-activated protein kinase (MAPK) (p-P38, #4511P, 1:1000), t-P38 (#9212P, 1:1000) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 2118, 1:1000). p-FYN (sc-377555, 1:200) and t-FYN (sc-434, 1:200) were purchased from Santa Cruz Biotechnology (Dallas, TX, U.S.A.). Antibodies for α-actinin (ab68167, 1:100) and α-smooth muscle actin (α-SMA, ab5694, 1:100) were from Abcam (Cambridge, U.K.). A77 1726 (SML0936, also known as teriflunomide), transforming growth factor-β (TGF-β, T7039), angiotensin II (Ang II, A9525) and phenylephrine (PE, P6126) were purchased from Sigma–Aldrich (St. Louis, MO, U.S.A.). Adenovirus vectors carrying constitutively active AKT1 (Ad-ca. Akt) and Gfp (Ad-Gfp) were generated by Hanbio Biotechnology Co. (Shanghai, China), and the efficiency was verified by our previous study . siRNA targetting Fyn (si Fyn) and its negative control (si RNA) were obtained from RiboBio Co., Ltd (Guangzhou, China).
Animals and treatments
All animal care and experimental procedures were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University and conformed to the guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH publication, revised 2011). All experimental procedures and data analysis were performed in a blinded manner by investigators unaware of the treatment allocation.
Male C57/B6 mice (8–10 weeks old; 23.5–27.5 g) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China) and were housed and maintained in a specific pathogen-free barrier environment. After 1 week of adaptive feeding, mice were randomly assigned to undergo aortic banding (AB) operation to generate pressure overload-induced cardiac hypertrophy as previously described [23,24]. Briefly, mice were anesthetized with 3% pentobarbital sodium (50 mg/kg, Sigma) by intraperitoneal injection and then the thoracic aorta was identified and surgically dissected at the second intercostal space after left hemithoracotomy. Immediately, a 27G blunt needle was placed next to the dissected aortic segment with a 7-0 silk suture tightly tied around to produce a 70% constriction. The efficiency and adequacy of ligation were evaluated by Doppler analysis. Meanwhile, mice assigned to the sham-operated control group were subjected to a similar procedure without constricting the aorta. Temgesic (0.1 mg/kg, qd) was subcutaneously injected to relieve post-operative pain for continuous 7 days. All mice were randomly divided into four groups: sham + vehicle, sham + A77 1726, AB + vehicle and AB + A77 1726. One day after AB surgery, mice were intragastrically subjected to A77 1726 (30 mg/kg, dissolved in 1.5% CM-cellulose) or equal volume of vehicle every other day for 4 weeks according to previous articles [18,25].
Cardiac hypertrophy was also induced by constant subcutaneous infusion of Ang II at a rate of 1.4 mg/kg/day for 28 days via an osmotic minipump (Alzet model 2004, Alza Corp) as previously described ; mice in the control groups received equal volume of saline. These mice were also intragastrically given a dose of A77 1726 (30 mg/kg) or equal volume of vehicle every other day for 4 weeks.
To ascertain whether AKT inhibition was responsible for the protective effect of A77 1726, mice were given an intramyocardial injection of 1 × 109 viral genome particles (Ad-ca. Akt or Ad-Gfp, diluted in 15 μl PBS) in three separate locations in the left ventricle free wall of the heart when the AB surgery was performed according to our previous description [22,24]. Four weeks after surgery or 28 days after Ang II infusion, mice were killed by an overdose of sodium pentobarbital (200 mg/kg, i.p.) with heart and tibia collected for further evaluation.
To verify whether A77 1726 could reverse pre-established chronic hypertrophic remodeling, mice were subjected to AB surgery for 4 weeks. After that, mice orally received A77 1726 (30 mg/kg) or equal volume of vehicle every other day for 4 weeks. At 8 weeks post AB surgery, these animals were subjected to echocardiography and hemodynamic measurements, and then were killed for further molecular detection.
In vivo T-cell depletion
To determine whether the protection of A77 1726 was mediated by T cells, mice were injected intraperitoneally with a monoclonal αCD3 antibody of (300 μg/ml, BioXcell, West Lebanon, NH) or the isotype-matched control every third day for 4 weeks, which resulted in approximately 95% depletion of CD3+ T cells .
Echocardiography and hemodynamics
After anesthetization with 1.5% isoflurane, transthoracic echocardiography was performed by MyLab 30CV ultrasound (Esaote SpA, Genoa, Italy) equipped with 10-MHz phased array transducer as previously described [28–30]. 2D guided M-mode echocardiographic images at the papillary muscles level were recorded, and the corresponding parameters were calculated and averaged for five consecutive cardiac cycles.
Left ventricle invasive hemodynamic measurements were made using a PowerLab system (AD Instruments Ltd., Oxford, U.K.) equipped with a 1.4-French Millar catheter transducer (SPR-839; Millar Instruments, Houston, TX) as previously described [28–30], and the hemodynamic parameters analyzed by the PVAN data analysis software. All procedures and data analyses were performed blindly.
Cardiac tissue samples were fixed with 4% paraformaldehyde overnight, embedded in paraffin and sectioned transversely into 5-μm slices in the middle segments. Sections were prepared and stained with Hematoxylin and Eosin (HE) for the evaluation of cross-sectional areas or Picrosirius Red (PSR) for evaluating the extent of interstitial fibrosis, respectively. Data were analyzed by a digital analysis software (ImagePro Plus 6.0, Media Cybernetics, Bethesda, MD, U.S.A.) with more than 300 cells outlined per group (n=6) for detecting the cardiomyocyte area and over 60 fields per group (n=6) for the evaluation of fibrosis. All manipulations were performed blindly using standard procedures [28–30].
Liver enzyme assay
Serum concentrations of liver enzymes, alanine transaminase (ALT) and aspartate transaminase (AST), were measured using an ADVIA 2400 Chemistry System analyzer (Siemens, Tarrytown, NY, U.S.A.) at the Department of Clinical Laboratory, Renmin Hospital of Wuhan University as previously described .
Western blot and quantitative real-time PCR
Total proteins isolated from heart samples or cultured cells were electrophoresed through SDS/PAGE (10% gel) and transferred on to PVDF membranes (EMD Millipore, Billerica, MA, U.S.A.; catalog number: IPFL00010). Then the membranes were blocked with 5% non-fat milk at room temperature and incubated with indicating primary antibodies overnight at 4°C, followed with secondary antibodies at room temperature for 1 h. Images were obtained by Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, U.S.A.) and protein expression normalized to the corresponding total proteins.
Total mRNA was extracted from cardiac tissues or cells with TRIzol reagent (Invitrogen, Carlsbad, CA, U.S.A.) and reversely transcribed to cDNA with Transcriptor First Strand cDNA Synthesis Kit (#04896866001, Roche, Basel, Switzerland). The transcriptional levels of target genes were normalized to Gapdh, and the primers for quantitative real-time PCR are shown in our previous articles [22,24,28–30,32].
Cell culture and treatments
Neonatal rat cardiomyocytes (NRCMs) and adult mouse CFs were isolated and cultured as previously described [22,23]. The purities of the NRCMs and adult mouse CFs were verified by morphologic recognition and immunofluorescence staining of α-actinin and vimentin, and only cells with purity of more than 95% were used for further research. Cells were cultured with Dulbecco’s modified Eagle’s medium (DMEM)/F12 (Gibco, C11995) containing 10% (for CFs) or 15% (for NRCMs) FBS (Gibco, 10099) for 48 h and allowed for growing to 70–80% confluence. After serum derived for 16 h, NRCMs were treated with A77 1726 (100 μM, dissolved in 0.1% DMSO) or equal volume of DMSO in the presence or absence of PE (50 μM) or Ang II (1 μM) for 48 h, whereas CFs were incubated with A77 1726 (25 μM) or vehicle followed by TGF-β (10 ng/ml) for 24 h . To evaluate the role of AKT in the protective effect of A77 1726, cells were pre-infected with replication-defective adenoviral vectors (MOI = 100) carrying Ad-ca. Akt to overexpress constitutively active AKT in vitro or Ad-Gfp as a negative control . Moreover, cells were pre-incubated with si Fyn (50 nM) for 24 h to knock down the expression of FYN and siRNA was used as a negative control.
Immunofluorescence staining was performed as previously described . Briefly, coverslips were fixed with 4% formaldehyde, permeabilized in 0.2% Triton X-100, and stained with α-actinin (1:100) or α-SMA (1:100) overnight after blocking with 10% goat serum for 60 min at 37°C. Coverslips were then incubated with Alexa Fluor 488 (green) or 568 (red) goat anti-rabbit secondary antibodies (1:200) for 60 min at 37°C, with DAPI used for visualization of the nuclei. Images were obtained by a fluorescence microscope (Olympus DX51) and were analyzed via ImagePro Plus 6.0 in a blind manner.
All data were expressed as mean ± S.E.M. and analyzed by SPSS 22.0 software. Differences between two groups were compared using unpaired, two-tailed Student’s ttests, whereas one-way ANOVA followed by Tukey’s post hoc test was used for comparisons amongst three or more groups. Statistical significance was defined as P<0.05.
A weight-neutral dose of A77 1726 protects mice from developing cardiac hypertrophy and cardiac dysfunction
In view of potential side effects such as anemia, weight loss, and hepatotoxicity of leflunomide in humans, we first assessed the overall health of our animals and the liver enzyme activity in the presence of A77 1726 . Mice, treated with a weight-neutral dose of A77 1726, did not display any signs of somnolence or weakness (Supplementary Figure S1A). Moreover, serum levels of ALT and AST were not significantly different between vehicle-treated mice and A77 1726-treated mice (Supplementary Figure S1B), which were consistent with a previous study . The effects of 4-week treatment with A77 1726 on the development of cardiac hypertrophy induced by AB were shown in Figure 1A–D. AB resulted in a significant hypertrophic response in vehicle-treated mice, as estimated by heart weight (HW) to body weight (BW) ratio (HW/BW), HW to tibia length ratio (HW/TL), and cross-sectional area, which was also associated with the activation of the fetal gene program (Anp, Bnp, α-Mhc, and β-Mhc). These changes were markedly retarded in A77 1726-treated mice (Figure 1A–D). Given that fibrotic response contributes to the conversion from hypertrophy into HF by increasing myocardial stiffness , we further assessed the effect of A77 1726 on cardiac fibrosis. A77 1726 administration significantly reduced the mRNA level of fibrotic markers (Col 1 and Col 3), which paralleled the decreased collagen deposition in myocardial interstitium (Figure 1E,F). No alteration in heart rate or blood pressure was observed between vehicle-treated mice and A77 1726-treated mice (Figure 1G). Furthermore, A77 1726-treated mice exhibited a markedly increased ejection fraction (EF) and ±dP/dt, and decreased interventricular septal thickness at systole (IVSs) and left ventricle end-diastolic internal diameter (LVIDd) in response to pressure overload when compared with mice treated with vehicle (Figure 1H,I).
A77 1726 attenuated pressure overload-induced cardiac hypertrophy and fibrosis
We next established Ang II-induced hypertrophy to further confirm the protective action of A77 1726. Ang II infusion for 28 days led to a distinct cardiac hypertrophy, determined by increased cardiomyocyte hypertrophy and collagen accumulation, which were alleviated by A77 1726 supplementation (Supplementary Figure S2A–E). Consistent with the morphological improvement, the increased mRNA levels of hypertrophic and fibrotic markers were also drastically suppressed by A77 1726 (Supplementary Figure S2F,G). Correspondingly, the deteriorated cardiac function was also prevented by A77 1726 treatment (Supplementary Figure S2H,I). Taken together, these data demonstrated that A77 1726 protects against the development of cardiac hypertrophy and dysfunction in experimental murine models.
A77 1726 suppressed the activation of AKT in vivo
CD4+ T cells and inflammation have been linked to the development of cardiac hypertrophy [5,9–11]. To determine whether the beneficial effect of A77 1726 on pressure overload-induced cardiac hypertrophy was secondary to its anti-inflammatory actions, we examined the effect of the agent on intracardiac inflammation, and found no alteration in the inflammatory markers, except for a significant reduction in Il-1β in A77 1726 treated-hypertrophic hearts (Supplementary Figure S3A–D). To clarify the role of CD4+ T cells in A77 1726-mediated protection, T cells were depleted using a CD3 monoclonal antibody . In agreement with this previous study, we found that T-cells depletion alone could not affect cardiac hypertrophy and interventricular septal thickness, improving cardiac function after AB surgery (Supplementary Figure S3E–G). A77 1726 supplementation could significantly alleviate cardiac hypertrophy and cardiac dysfunction even in the absence of T cells (Supplementary Figure S3E–G), which suggested that A77 1726 exerted a protective effect on cardiac hypertrophy independent of its T cell-inhibiting capacity.
Given reports that A77 1726 inhibits the proliferation and activation of hepatic stellate cells via suppression of MAPK signaling , we explored whether MAPK was involved in the protective effect of A77 1726. Contrary to our expectations, A77 1726 did not decrease the phosphorylation of ERK and P38 in response to AB surgery (Figure 2A–G), but reduced the activation of AKT, which was corroborated by the decreased phosphorylation of GSK3β, P70, and S6 (Figure 2A–E). This finding was further confirmed by the detection of AKT and downstream targets in Ang II-induced cardiac hypertrophy (Supplementary Figure S4A,B).
Alteration of signaling pathway after A77 1726 treatment
A77 1726 blocked cardiomyocyte hypertrophy, myofibroblast transdifferentiation, and AKT activation in vitro
We further evaluated the protective effect of A77 1726 on cardiomyocyte hypertrophy, the defining characteristic of cardiac hypertrophy , in vitro. Consistent with the finding in vivo, we found that the increased cell area of NRCMs in response to PE was almost completely blocked in the presence of A77 1726 (Figure 3A,B). And the levels of hypertrophic markers (Anp and β-Mhc) were also decreased in A77 1726-treated cells (Figure 3C). Moreover, PE-induced activation of AKT signaling pathway was significantly blocked in A77 1726 treated-NRCMs, evidenced by the decreased phosphorylation of AKT, GSK3β, and P70 (Figure 3D–G). In addition, NRCMs were also stimulated with Ang II to induce cardiomyocyte hypertrophy. As shown in Supplementary Figure S5A,B, A77 1726 decreased the increased cell area and hypertrophic markers induced by Ang II stimulation, accompanied by the inactivation of AKT signaling pathway (Supplementary Figure S5C–F). These data implicated that A77 1726 could block cardiomyocyte hypertrophy in vitro.
A77 1726 attenuated cardiomyocytes hypertrophy and fibroblasts differentiation into myofibroblasts
Transdifferentiation of CFs to myofibroblasts is another hallmark of cardiac hypertrophy, which further impairs mechanical and electrical functions of the heart due to the anomalous deposition of extracellular matrix in myocardium . To evaluate the effect of A77 1726 on myofibroblast transdifferentiation in vitro, adult mouse CFs were separated and stimulated with TGF-β to induce myofibroblast transdifferentiation. And we demonstrated that stimulation of CFs with TGF-β led to a markedly increased expression of α-SMA, which was attenuated by A77 1726 (Figure 3H,I). Correspondingly, the increased Col 1 and Col 3 mRNA levels were also reduced (Figure 3J). Next we assessed whether A77 1726 could inhibit the activation of AKT in myofibroblasts. We found the activation of AKT in response to TGF-β was blunted by A77 1726 (Figure 3K–N). Based on the above findings, we concluded that A77 1726 could block the transdifferentiation of CFs to myofibroblast in vitro.
A77 1726 lost its protective effect on cardiac hypertrophy in the presence of constitutively active AKT both in vitro and in vivo
Since activation of AKT was impeded both in NRCMs and CFs after A77 1726 treatment, we speculated whether the protective effect of A77 1726 on cardiac hypertrophy was mediated by the suppression of AKT. Cultured cells and mice were infected with Ad-ca. Akt to overexpress activated AKT. As shown in Figure 4A–C, PE-induced increase in cell area and hypertrophic markers were notably decreased in A77 1726-treated NRCMs, and the protective effect was abolished in the presence of Ad-ca. Akt. Besides, A77 1726 lost its antifibrotic effect in the presence of Ad-ca. Akt in vitro, as evidenced by the non-decreased expressions of α-SMA as well as fibrotic markers after A77 1726 treatment (Figure 4D–F). Moreover, the hypertrophic responses induced by pressure overload, as evidenced by increased HW to TL ratio, cross-sectional area and collagen volume, were ameliorated by A77 1726 treatment in Ad-Gfp-infected mice but not in mice infected with Ad-ca. Akt (Figure 5A–D). In parallel with the action on morphological level, A77 1726 also lost its protective effect on cardiac dysfunction in the presence of constitutively active AKT (Figure 5E,F). We also found that chronic pressure overload increased hypertrophic and fibrotic markers, which was reversed back to control levels by A77 1726 treatment in Ad-Gfp-infected mice, but not in Ad-ca. Akt-infected mice (Figure 5G,H). Taken together, the protective effect of A77 1726 on cardiac hypertrophy was mediated by suppression of AKT signaling pathway.
A77 1726 lost its protection in cells expressing constitutively active AKT
A77 1726 lost its protection in mice with constitutively active AKT
A77 1726 inactivated AKT via suppressing the phosphorylation of FYN
We further investigated the possible mechanism involved in the inactivation of AKT by A77 1726. FYN belongs to the Src kinase family and is required for mechanical strain-induced activation of AKT, and leflunomide was supposed as an inhibitor of Src kinase family [37,38]. Therefore, we detected whether FYN was responsible for A77 1726-mediated inactivation of AKT. As shown in Figure 6A, A77 1726 treatment inhibited pressure overload-induced phosphorylation of FYN, which was coincident with inactivation of AKT. si Fyn was used to knock down FYN in vitro, and the protein expression of FYN was reduced by more than 88%, as confirmed by Western blot (Figure 6B and Supplementary Figure S6A). A77 1726 lost its inhibitory effect on AKT in FYN-deficient NRCMs (Figure 6C). Moreover, the protective effect of A77 1726 on cardiomyocyte hypertrophy was also abrogated, as evidenced by cell area and the mRNA level of Anp and β-Mhc (Figure 6D–F). Similarly, A77 1726 also lost its protective effect on myofibroblast transformation in FYN-deficient CFs (Figure 6G–I), and the inhibitory effect on AKT in CFs was also deprived (Supplementary Figure S6B). Collectively, we supposed that FYN might be responsible for A77 1726-mediated inactivation of AKT.
A77 1726 inhibited the phosphorylation of AKT via FYN
A77 1726 attenuated pre-established cardiac hypertrophy in vivo
To enhance the clinical impact of our current work, we finally investigated whether A77 1726 could reverse pre-existing cardiac hypertrophy, which is a more clinically relevant situation. Mice were subjected to AB surgery for 4 weeks to develop cardiac hypertrophy and then treated with A77 1726 for another 4 weeks. As shown in Figure 7A, A77 1726 treatment significantly reduced HW/BW and HW/TL in mice with pre-established cardiac hypertrophy. Consistent with this, A77 1726 treatment also decreased cross-sectional area and hypertrophic markers following AB insult, compared with untreated mice (Figure 7B,C). In addition, A77 1726 administration also reduced the accumulation of interstitial collagen and activation of fibrotic genes (Figure 7D,E). Correspondingly, A77 1726 supplementation restored the diminished heart function, as evidenced by improved EF, ±dp/dt, and decreased LVIDd (Figure 7F–H). Together, these data indicated that A77 1726 treatment was capable of attenuating pre-established cardiac hypertrophy in vivo.
A77 1726 reversed pre-established cardiac hypertrophy in mice
In the present study, for the first time, we defined A77 1726 as a novel therapeutic agent against cardiac hypertrophy. A77 1726 treatment attenuated pressure overload or Ang II-induced cardiac hypertrophy in vivo, as well as agonist-induced hypertrophic responses of cardiomyocytes in vitro. We also demonstrated that A77 1726 was capable of blocking pre-established cardiac hypertrophy in mice, which increases the possibility for its clinical application. In addition, we showed that A77 1726 administration prevented induction of cardiac fibrosis by inhibiting CF transformation into myofibroblast. The protective effect of A77 1726 was mediated by the inactivation of AKT, which could be abolished by overexpressing constitutively active AKT both in vivo and in vitro. And we also found that FYN suppression was essential for the inactivation of AKT. Thus, our current findings identified A77 1726 as a promising therapeutic agent against cardiac hypertrophy.
HF is still the leading contributor to human morbidity and mortality in the world, however, conventional strategies for the treatment of HF are still based on targetting its causes and subsequent neurohumoral disturbances [1,3]. Despite antineuroendocrine treatment with angiotensin-converting enzyme inhibitors, β-adrenergic blockers, and anti-aldosterone therapy which improve the outcome of HF to a certain extent, the prognosis of HF remains poor. Cardiac hypertrophy has been identified as a key process contributing to the initiation and progression of HF, and has therefore emerged as a therapeutic target in HF of all etiologies . Our present study indicated that A77 1726 treatment was capable of alleviating and even reversing the induction and progression of cardiac hypertrophy, and exerted protective effects on pressure overload or Ang II-induced cardiac dysfunction. More importantly, we observed that A77 1726 administration could attenuate pre-established cardiac hypertrophy in mice, a finding highly relevant to clinical cardiology. All the available data point toward A77 1726 as a promising and effective therapeutic agent against cardiac hypertrophy and HF.
Emerging evidence from basic and clinical trials have demonstrated that T-cells infiltration and the subsequent increased intracardial chronic inflammation played crucial roles in the development of cardiac hypertrophy and HF [12,27]. In the setting of cardiac injury, T cells are activated and recruited to cardiac interstitium, where they alter cardiomyocyte and CF function in a paracrine-dependent manner [39,40]. Leflunomide and its active metabolite A77 1726 have been reported to possess powerful anti-inflammatory properties and could also inhibit the proliferation of T cells [17–20]. Based on these findings, we speculated that A77 1726 might attenuate cardiac remodeling in mice. As expected, we found that A77 1726 exerted a protective effect against development of cardiac hypertrophy. However, in our study, A77 1726 did not have a profound effect on myocardial inflammation, which was in agreement with a previous finding that leflunomide treatment showed only a moderate or no inhibitory effect on local inflammation infiltration . Using a monoclonal CD3 antibody, we found that the protection provided by A77 1726 was independent of its T cell-inhibiting capacity. These data were supported by previous observations that the absence of CD4+ T cells only prevented cardiac dysfunction but not ventricular hypertrophy induced by pressure overload [10,27].
AKT, also known as protein kinase B, is a serine/threonine protein kinase, which has emerged as a focal point for signal transduction pathways responsible for cell survival . Short-term activation of AKT promotes growth and development of the heart, whereas constitutive AKT activation leads to the occurrence of cardiac hypertrophy, which can be ascribed to the activation of P70 and the inhibition of GSK3β . Previous studies indicated that P70 was responsible for the phosphorylation and activation of S6 protein in 40S ribosome, the one that is positively correlated to the efficiency of protein synthesis [44,45]. In addition, previous studies from our laboratory and by others have indicated that AKT-mediated phosphorylation of GSK3β was involved in the development of cardiac hypertrophy, and suppressing this phosphorylation could activate GSK3β and exert protective effects on hypertrophic remodeling [32,46,47]. Recently, we also demonstrated that inhibition of AKT/GSK3β could attenuate cardiac fibrosis in vivo and block myofibroblast transformation in vitro , which is consistent with a previous study indicating that GSK3β could interact with Smad3 and inhibit profibrotic TGF-β/Smad3 signaling in CFs . Herein, we also observed that A77 1726 exerted its cardioprotective effect mainly via suppressing the activation of AKT, whereas constitutively active AKT in vivo and in vitro practically blunted this beneficial effect. These results were consistent with a previous study revealing that A77 1726 could decrease the phosphorylation of AKT and diminish proliferation of multiple myeloma cells .
FYN kinase is a critical member of the Src kinase family of the non-receptor protein tyrosine kinases and is associated with molecular signaling in the immune system, neural system, energy metabolism, and cancer [50–52]. It has been reported that FYN can be directly activated by pressure overload, which mediates the subsequent phosphorylation of AKT, whereas genetic silencing of Fyn abrogated phosphorylation of AKT . In addition, He et al.  demonstrated that UV B-induced activation of AKT was also mediated by FYN phosphorylation, and FYN inhibition by leflunomide showed decreased phosphorylation of AKT after UV B insult. Moreover, FYN activation could phosphorylate phosphatidylinositol 3-kinase enhancer-activating AKT and promote cell survival . All these data support the hypothesis that FYN lies upstream of AKT and mediates A77 1726-induced protection. In line with these observations, our present study demonstrated that the decreased phosphorylation of FYN was responsible for A77 1726-mediated inactivation of AKT, as reflected by the data showing A77 1726 lost its protection in FYN-deficient cells.
In order to investigate the effect of A77 1726 on cardiac hypertrophy, we chose a dose of 30 mg/kg (once every 2 days) as used previously in mouse models of systemic fibrosis or endometriosis [18,25]. It has been reported that daily administration of more than 45 mg/kg of A77 1726 caused side effects such as weight loss and death . The dose of 30 mg/kg (once every 2 days) was well tolerated by mice and exerted beneficial effects on systemic sclerosis, endometriosis, and lupus nephritis [18,20,25]. At this dose, mice did not display any signs of somnolence or weakness. In addition, A77 1726 treatment did not alter the BW or the serum liver enzyme concentrations. Moreover, mice treated with A77 1726 exhibited a restricted hypertrophic response and attenuated cardiac dysfunction. The DHODH inhibitory effect occurs at a low concentration of A77 1726 (below 5 μmol/l), and inhibition of tyrosine kinase requires a comparatively high concentration of A77 1726 (above 50 μmol/l) . Clinically, the oral maintenance dose of leflunomide in RA therapy is 10–20 mg. A study reported that in the RA patients who stayed at a steady state after daily administration of leflunomide (25 mg), plasma concentration of A77 1726 was approximately 233 μmol/l . Fukushima et al.  found that even at 24 h after a single A77 1726 (30 mg/kg) administration, plasma concentration of A77 1726 was 188 μmol/l, which was higher than the dose used in our study. This suggests that protective effects of A77 1726 other than DHODH inhibition deserve more attention. In conclusion, we found that A77 1726 attenuated cardiac hypertrophy in mice via inhibiting FYN/AKT pathway. Importantly, administration with A77 1726 reversed pre-established cardiac hypertrophy, which increases its practicability for clinical application. Our findings provide evidence for the application of A77 1726 in the treatment of cardiac hypertrophy.
Inflammation was closely involved in cardiac remodeling. We investigated whether leflunomide, an immunosuppressive agent, could inhibit this hypertrophic response.
We found that A77 1726 (leflunomide) attenuated cardiac remodeling by inhibiting FYN/AKT pathway.
The data in our study provided evidence for the application of leflunomide in the treatment of cardiac hypertrophy.
The authors declare that there are no competing interests associated with the manuscript.
This work was supported by the National Natural Science Foundation of China [grant numbers 81470402, 81500184, 81700254]; the Key Project of the National Natural Science Foundation [grant number 81530012]; and the Fundamental Research Funds for the Central Universities [grant numbers 2042017kf0085, 2042015kf0073].
Z.-G.M. and Q.-Z.T. contributed to the conception of the work and designed experiments. Z.-G.M., X.Z., Y.-P.Y., and N.L. carried out experiments. P.S. and C.-Y.K. analyzed experimental results and revised the manuscript. Z.-G.M., X.Z., and Q.-Z.T. revised the manuscript. Y.-G.J. participated in the analysis and interpretation of the data.
protein kinase B
- Ang II
dihydro-orotic acid dehydrogenase
extracellular signal-regulated kinase
glyceraldehyde 3-phosphate dehydrogenase
glycogen synthase kinase 3 β
HW to BW ratio
HW to tibia length ratio
left ventricle end-diastolic internal diameter
mitogen-activated protein kinase
neonatal rat cardiomyocyte
- si Fyn
siRNA targetting Fyn
transforming growth factor-β
α-smooth muscle actin
These authors contributed equally to this work.