Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis

Biochemical and Biophysical Research Communications xxx (2018) 1e8

Contents lists available at ScienceDirect

Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc

Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis Bei Liu a, Chunxia Zhao a, Hongkun Li b, Xiaoqian Chen a, Yu Ding a, Sudan Xu a, * a b

Department of Cardiology, Shanghai General Hospital, China Department of Cardiology, Heji Hospital of Changzhi Medical College, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 February 2018 Accepted 7 February 2018 Available online xxx

Heart failure (HF) is the end stage of cardiovascular disease and is characterized by the loss of myocytes caused by cell death. Puerarin has been found to improve HF clinically, and animal study findings have confirmed its anti-cell-death properties. However, the underlying mechanisms remain unclear, especially with respect to the impact on ferroptosis, a newly defined mechanism of iron-dependent non-apoptotic cell death in HF. Here, ferroptosis-like cell death was observed in erastin- or isoprenaline (ISO)-treated H9c2 myocytes in vitro and in rats with aortic banding inducing HF, characterized by reduced cell viability with increased lipid peroxidation and labile iron pool. Interestingly, the increased iron overload and lipid peroxidation observed in either rats with HF or H9c2 cells incubated with ISO were significantly blocked by puerarin administration. These results provide compelling evidence that puerarin plays a role in inhibiting myocyte loss during HF, partly through ferroptosis mitigation, suggesting a new mechanism of puerarin as a potential therapy for HF. © 2018 Published by Elsevier Inc.

Keywords: Puerarin Ferroptosis Iron Lipid peroxidation Heart failure

1. Introduction As the final stage of cardiovascular diseases, heart failure (HF) is endangering the life and health of 22.5 million people around the world, and this number is increasing at a rate of 2 million per year. Thus, the prevention and treatment of HF is currently one of the most important topics in the medical community. Under hemodynamic stress such as high blood pressure, compensated cardiac cells lead to myocardial hypertrophy, the most important pathological characteristic of HF, causing progressive cell loss and finally advancing to HF. Several kinds of cell death have been proved to be involved in cell loss, such as apoptosis, necrosis and autophagy [1]. As a new form of regulated cell death (RCD), ferroptosis was defined by Dixon in 2012. Different from other major forms of RCD, ferroptosis, characterized by cell volume shrinkage and mitochondrial membranes thickening, is mediated by iron-dependent lipid peroxide accumulation [2]. Ferroptosis was initially observed in cancer cells expressing oncogenic Ras and then discovered in other diseases, such as Huntington's disease and tubular failure [3], but few studies on the role of ferroptosis in cardiovascular disease have

* Corresponding author. Department of Cardiology, Shanghai General Hospital, No. 100 Haining Road, Hongkou District, Shanghai, 200080, China. E-mail address: [email protected] (S. Xu).

been reported. Nevertheless, the study of iron homeostasis and myocardial injury has a long history. The term iron overload cardiomyopathy has been introduced to describe a secondary form of cardiomyopathy resulting from the accumulation of iron in the myocardium, mainly because of genetically determined disorders of iron metabolism or multiple transfusions [4]. The clinical use of doxorubicin is limited by its cardiotoxicity. The possible involvement of iron in doxorubicin-induced cardiotoxicity became evident from studies in which iron chelators were shown to be cardioprotective [5]. Nitenberg et al. demonstrated that abnormal myocardial iron status may exist in diabetic patients with HF, and chelation therapy can improve the prognosis of coronary microvascular adaptation [6]. A recent study by Lapenna et al. [7] demonstrated that the levels of low-molecular-weight iron (LMWI), a redox-active catalytic form of iron, as well as the levels of lipid and protein oxidation, were higher in the hearts of aged rabbits than in those of young adult control rabbits. The above and other research results indicate that ferroptosis, characterized by altered iron status with catalytic LMWI burden and related cardiac oxidative stress, might be underlying mechanisms for cell death during cardiac dysfunction. However, iron pools as expression of iron status and related oxidative stress have not yet been well investigated in pressure-overload-induced HF, and the further exploration of effective therapies that specifically target ferroptosis

https://doi.org/10.1016/j.bbrc.2018.02.061 0006-291X/© 2018 Published by Elsevier Inc.

Please cite this article in press as: B. Liu, et al., Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.02.061

2

B. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8

in HF may represent a new and useful management strategy. Puerarin, one of the most abundant phytoestrogens with antioxidant and other properties, has been approved by the State Food and Drug Administration in China as a therapeutic agent for clinical therapy in cardiovascular and other diseases [8]. Numerous clinical trials have established the beneficial effects of puerarin on patients with HF, but the detailed mechanisms remain unclear. Our previous study showed puerarin exerting protective effects against cardiomyocyte hypertrophy and apoptosis by restoration of autophagy in rats with myocardial hypertrophy induced by pressure overload [9], which could block the progression to HF. Based on the antioxidant properties of puerarin, the details of this agent in cell death mechanisms are worthy of further exploration, especially in relation to ferroptosis. 2. Methods 2.1. Reagents

microscope. 2.5. Lipid peroxidation assay Lipid peroxidation in cultured cell lysates was determined using the classical assay of measurement of the rate of production of TBARS, expressed as pmol/mg protein. 2.6. Immunohistochemistry for 4-HNE in vivo Formaldehyde-fixed and paraffin-embedded sections were incubated in 3% hydrogen peroxide for 30 min to block endogenous peroxidase activity and then incubated overnight at 4
C with primary rabbit antibody against 4-HNE. For antigen retrieval, the sections were immunostained using the VECTASTAIN ABC kit following the manufacturer's specifications. Diaminobenzidine was used for staining development, and the sections were counterstained with hematoxylin.

Isoprenaline(ISO), puerarin, chloroquine(CQ), propidium iodide (PI) solution and MTT assay kit were purchased from Sigma Aldrich(USA). Ferrostatin-1(Fer-1) and Z-VAD-FMK were purchased from Selleck Chemicals (USA). The hematoxylin and eosin (HE) kit was obtained from Baso (Zhuhai, China). Puerarin for injection was purchased from Zhenyuan Pharmaceutical Co., Ltd (Zhejiang, China), and the Bradford assay kit was obtained from Bio-Rad(USA). The primary antibody against Nox4 was from Sigma Aldrich(USA), while those against glutathione peroxidase 4 (GPX4), ferritin heavy chain 1(FTH1) and GAPDH were from Cell Signaling Technology(USA). The thiobarbituric acid reactive substances (TBARS) assay kit was from R&D Systems (USA), the VECTASTAIN ABC kit was from Vector Laboratories Inc. (USA), and the primary rabbit antibody against 4-hydroxy-trans-2-nonenal (4-HNE) was from Abcam Inc.(USA).

2.7. Iron assay

2.2. Animal model

2.8. Western blotting

The animal experiments conformed to the Guide for the Care and Use of Laboratory Animals (US and National Institutes of Health). Male Sprague Dawley rats weighing 80e100 g were used to make the HF model induced by descending aortic banding (AB) procedure [9]. Rats receiving a similar procedure except for the arterial ligation were defined as the sham-operated (SO) group. After the procedure, echocardiography was immediately applied to confirm the arterial banding. Rats receiving subcutaneous injections of low- or high-dose puerarin (100mg/kg/day and 200mg/ kg/day, respectively) after the AB procedure were respectively defined as the Pue1 and Pue2 groups. An equal volume of normal saline was injected into the rats of the SO and AB groups.

Western blotting was performed as previously described [9]. Briefly, after collection of the supernatants of the tissue or cell lysates, protein samples (20e25 mg) were separated by sodiumdodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were probed with primary antibodies overnight. Diluted secondary antibodies were used to detect the corresponding primary antibodies. Further analysis was carried out using Image Pro Plus v6.0 (Media Cybernetics, Carlsbad, CA, USA) to quantify the protein bands.

2.3. Cell culture and treatment

Chloral hydrate was used to anesthetize the rats. An experienced technician blinded to the study groups then used an IE33 echocardiographic system (Philips Medical Systems, Nederland BV) to perform the transthoracic two-dimensionally guided M-mode echocardiography every week after the procedure.

H9c2 cardiac myoblast cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 and 95% air at 37
C. Upon reaching 50e60% confluence, the cells were treated with ISO or erastinalone, or in combination with puerarin (10, 20 and 40 mM, respectively, dissolved in dimethyl sulfoxide [DMSO]). 2.4. Cytotoxicity assays Cell viability in vitro was determined using the in MTT kit as per the manufacturer's instructions. For the detection of cell death in vivo, cardiac sections of rats were incubated with 5mg/mL PI for 30 min and then imaged using an inverted fluorescence

A fluorescence technique with the Fe sensor calcein was applied to detect the labile iron pool (LIP). Cells were washed with PBS and then treated with Chelex-100. Subsequently, 100 mL of calcein-AM solution (final concentration of 30 mM) was incubated with the cells for 30 min at 37
C. After removing the excess calcein-AM with PBS, the fluorescence (lex ¼ 450 nm, lem ¼ 515 nm) was monitored. The results were expressed as fold change. For labile iron measurements in vivo, LMWI was determined using the sensitive iron colorimetric detector ferene S as previously described [7]. The absorbance values of the ferene Seiron complex at 594 nm were then recorded spectrophotometrically against an appropriate blank, and the results were calculated as nmol iron/mg protein using a molar extinction coefficient of 35,500 [10].

2.9. Echocardiography

2.10. Histological analysis Sections were stained with H&E and examined under a light microscope (AMG EVOS FL), and the myocyte area was measured with Image Pro Plus v6.0. 2.11. Transmission electron microscopy Harvested cardiac tissues were stained en bloc with 2% uranyl

Please cite this article in press as: B. Liu, et al., Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.02.061

B. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8

acetate (UA), dehydrated in ethanol, and embedded in eponate. The sections (70e90 nm) were then placed on copper slot grids and stained with 2% UA and lead citrate. Transmission electron microscopy (TEM) images were captured using a Hitachi 7650 TEM. 2.12. Statistic analyses Continuous data were expressed as the means ± standard error (SEM). The differences in means between the groups were evaluated using a one-way analysis of variance (ANOVA) test, followed by a post hoc Tukey test. Statistical significance was set at p < 0.05. 3. Results 3.1. Ferroptosis is induced by either erastin or ISO in myocytes in vitro To determine whether ferroptotic-like death could occur in cardiovascular cells, H9c2 cells were first exposed to three different concentrations of erastin (2, 4 or 8 mM) for 24 h; erastin is a proven specific ferroptosis inducer. In the MTT assay, even the lowest concentration of erastin(2 mM) decreased the cell viability compared with control (Fig. 1A). To further explore the involvement of lipid peroxidation and cellular iron accumulation in erastinstimulated cell death, the TBARS production rate, a common measure of lipid peroxidation products, was determined. As shown in Fig. 1B, erastin treatments for 24 h dose-dependently increased the TBARS content. Looking at iron signaling in H9c2 cells, erastin stimulation significantly increased the LIP compared to DMSO control groups and acted in a dose-dependent manner (Fig. 1C). The above results showed that erastin can induce ferroptosis in cardiomyocytes. Similar to erastin, ISO (0.01, 0.1 or 1 mM) also dose-dependently reduced the cell viability accompanied by increased intracellular TBARS and LIP, compared with the control group (Fig. 1DeF), indicating that ferroptosis might also be one of the pathways for ISO-induced cardiomyocyte death. As shown in Fig. 1G, Fer1(10 mM), a ferroptosis-specific inhibitor, co-cultured with ISO (1 mM) significantly increased cardiomyocyte viability compared with ISO alone. Although the apoptosis inhibitor Z-VAD-FMK (50 mM) inhibited the ISO-induced cardiomyocyte death as expected, importantly, Z-VAD-FMK and Fer-1 treatment together cooperatively increased the ISO-induced cell viability in a synergistic manner compared with either Z-VAD-FMK or Fer-1 alone, indicating that distinct programmed cell death processes participate in ISO-induced cardiomyocyte death. Interestingly, the coincubation of CQ (10 mM, a specific autophagy inhibitor) with Fer1 treatment revealed similar effects of improving cell viability compared with either CQ or Fer-1 alone, indicating that there might be a link between autophagic cell death and ferroptosis. 3.2. Puerarin restored cell viability in erastin- or ISO-treated myocytes through ferroptosis inhibition As shown in Fig. 2A, puerarin (10, 20 or 40 mM) treatment for 24 h dose-dependently restored the cell viability reduction induced by either erastin (8 mM) or ISO (1 mM). Compared with ISO-treated cells, TBARS and LIP were significantly decreased with puerarin treatment in a dose-dependent manner (Fig. 2B and C). Furthermore, Western blotting revealed that treatment with 1 mM ISO for 48 h significantly altered the ferroptosis-related protein expression, as indicated by an increase in Nox4 expression accompanied by decreases in Gpx4 and FTH1 expression compared with control cells. Compared with ISO-treated cells, both Fer-1 (10 mM) and puerarin (20 mM) downregulated Nox4 expression and upregulated

3

Gpx4 and FTH1 expression at the protein level (Fig. 2D). 3.3. Puerarin mitigated HF induced by pressure overload in vivo As shown in Fig. 3A, impaired left ventricular (LV) function indicated by reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) was observed in AB rats at week 12. At the same time, a significant reduction in left ventricular posterior wall end-diastolic thickness (LVPWd) accompanied by an increase in left ventricular end diastolic diameter (LVIDd) was detected compared with the SO animals. After 12 weeks of therapy with different doses of puerarin (100 mg/kg/d and 200 mg/kg/d), LVEF and LVFS were increased significantly compared with AB rats with no treatment, and puerarin treatment remarkably inhibited AB-induced ventricular wall thinning and cavity enlargement. H&E staining analysis of cardiac cross sections revealed that the increased myocyte area noted in AB rats was reduced by puerarin treatment (Fig. 3B). At week 12, TEM images revealed that the muscular stripes of the SO group were clear, the Zline and M-line were clearly observed, and the structure of the mitochondria was normal. However, in the AB rats, most of the striated muscle was ruptured, the Z-line and M-line could not be clearly distinguished, the mitochondrial structure was atrophic, and the crest was thick. Puerarin treatment led to improved striated muscle arrangement and reduced mitochondrial atrophy (Fig. 3C). 3.4. Puerarin inhibited ferroptosis in rats with HF A marked increased in cardiomyocyte death, as indicated by increased PI staining, was detected at week 12 in AB rats compared with SO rats. However, PI staining reduced significantly in AB rats treated with puerarin, indicating that puerarin effectively inhibited AB-induced cardiomyocyte death (Fig. 4A). As shown in Fig. 4B and C, in the AB hearts without puerarin treatment, the levels of LMWI and 4-HNE were significantly higher than in the SO hearts. Importantly, the increased LMWI and lipid peroxidation indicated by 4-HNE in rats with AB for 12weeks could be significantly reduced by puerarin treatment. Compared with SO rats, Nox4 protein expression increased significantly in AB rats, but was downregulated in puerarin-treated AB rats [Fig. 4D]. The ferroptotic proteins Gpx4 and FTH1 exhibited the opposite changes to Nox4 among the above groups (Fig. 4D). 4. Discussion It has been suggested by accumulated evidence that ferroptosis, characterized by increased cellular iron content and related lipid peroxidation, might represent a new regulated target as a new kind of programmed cell death in various diseases. Puerarin, a proven antioxidant, has been reported to exhibit cardioprotective functions against HF on the basis of the results of clinical trials and animal studies. However, whether puerarin can act on ferroptosis and the implicated mechanism remain unexplored. In the present study, altered iron status and oxidative stress were observed in the HF model of rats with AB. In particular, the levels of LIP in H9c2 cells incubated with ISO and the levels of LMWI in rats with AB were increased, while the levels of FTH1, a kind of ferritin that can bind iron and keep it in a safe and redox-inactive form, were decreased, which is also characterized by increased lipid peroxidation. A possible mechanism might include degradation of FTH1, which prompts an increase of LMWI resulting from the expanded nonheme iron pool in failed myocardium, conceivably supplying “free” iron to the LMWI pool. Further study demonstrated that puerarin treatment significantly reduced cardiac dysfunction in rats with HF accompanied with reduced “free” iron and lipid peroxidation.

Please cite this article in press as: B. Liu, et al., Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.02.061

4

B. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8

Fig. 1. Erastin and ISO induced ferroptosis in cardiomyocytes. (A) Erastin (2 mM, 4 mM, and 8 mM for 24 h respectively) reduced H9c2 cell viability. (B) Erastin increased(2 mM, 4 mM, and 8 mM for 24 h respectively) lipid peroxidation in H9c2 cells. (C) Erastin (2 mM, 4 mM, and 8 mM for 24 h respectively) increaded LIP in H9c2 cells. (D) ISO (0.01 mM, 0.1 mM, and 1 mM for 48 h respectively) reduced H9c2 cell viability. (E) ISO (0.01 mM, 0.1 mM, and 1 mM for 48 h respectively) increased lipid peroxidation in H9c2 cells. (F) ISO (0.01 mM, 0.1 mM, and 1 mM for 48 h respectively) increaded LIP in H9c2 cells. (G) Indicated H9c2 cells were treated with either erastin (8 mM for 24 h) or ISO (1 mM for 48 h) in the absence or presence of indicated inhibitors (Fer-1, 10 mM; Z-vad-fmk, 50 mM; CQ, 10 mM) for 24 h. Cell death was assayed using the MTT assay kit. Con, control; E, erastin; ISO, Isoprenaline; Fer-1, Ferrostatin-1; CQ, chloroquine. Each of the experiments was repeated 3 times, n ¼ 3.

The current view supports the idea that cell loss caused by myocyte death is the key pathophysiology during HF progression. Different forms of cell death, such as apoptosis, necrosis, and maladaptive autophagy (also referred to as autophagic cell death), involved in cell loss have been well established. Ferroptosis is a recently discovered, new form of cell death. Since it does not cause the chromatin condensation occurring in apoptosis, the destructed integrity of plasma membrane in necrosis, or the doublemembrane-layered vacuoles in autophagy, but uniquely causes mitochondrial shrinkage and increased mitochondrial membrane density [2], ferroptosis is therefore considered a new form of programmed cell death that is distinguished from the above forms of

cell death. Recent studies have demonstrated that ferroptosis is involved in tumors [2,11e15], neurotoxicity [3,16,17], acute renal injury [18,19], drug-induced liver injury [20], and ischemiareperfusion injury [21], suggesting that it is important to elucidate the signaling pathways and metabolic characteristics of ferroptosis and related pharmacological agents. As a tissue that consumes lots of oxygen, the heart is rich in unsaturated fatty acids, which provides the pathophysiological basis for reactive oxygen species mediated myocardial injury. In addition, abundant non-heme iron in the myocardium can cause membrane lipid peroxidation through the Fenton reaction under situations of stress, resulting in myocardial edema, mitochondrial

Please cite this article in press as: B. Liu, et al., Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.02.061

B. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8

5

Fig. 2. Puerarin restored cell viability through ferroptotic inhibiton. (A) Puerarin (10 mM, 20 mM, and 40 mM for 24 h respectively) restored cell viability in Erastin (8 mM for 24 h) or ISO (1 mM for 48 h) treated H9c2 cells. (B) Puerarin (10 mM, 20 mM, and 40 mM for 24 h respectively) reduced lipid peroxidation in Erastin (8 mM for 24 h) or ISO (1 mM for 48 h) treated H9c2 cells. (C) Puerarin (10 mM, 20 mM, and 40 mM for 24 h respectively) reduced LIP in ISO (1 mM for 48 h) treated H9c2 cells. (D) Impact of Puerarin (20 mM for 24 h) on expression of Nox4 and ferroptotsis-related proteins. Con, Control; ISO, Isoprenaline; Fer-1, ferrostatin-1(10 mM); Pue, puerarin. Each of the experiments was repeated 3 times, n ¼ 3.

damage, functional impairment, and so on. Numerous studies have demonstrated that oxidative stress plays a key role in cardiomyocyte death, and there is a regional iron deposition in some types of heart disease such as myocardial infarction, aging and so on [4e7]. Importantly, iron chelators and anti-oxidation agents can effectively reduce cardiomyocyte death [4e7], which promoted us to speculate that ferroptosis might be a possible mechanism for cell death in cardiovascular diseases. Indeed, in animal models of myocardial ischemia-reperfusion injury, targeting inhibition of glutamine decomposition, a key component in ferroptosis, was found to alleviate myocardial damage [21]. A recent study from Baba et al. [22] used ferroptosis inducers to treat adult mouse cardiomyocytes, and revealed that the mechanistic target of rapamycin (mTOR), a key effector in the insulin signaling pathway that regulates not only cell metabolism but also cell survival, plays an important role in protecting cardiomyocytes against excess iron and ferroptosis. In the current study, ISO was applied to H9c2 cells for 48 h and was shown to significantly inhibit cardiomyocyte viability in a dose-dependent manner, while increasing intracellular lipid peroxidation and LIP and downregulating expression of the proteins GPX4 and FTH1. Importantly, Fer-1, a specific ferroptosis inhibitor, effectively inhibited ISO-induced cardiomyocyte death accompanied by the reduced LIP and lipid peroxides, indicating that ISO inhibited the viability of myocardial cells partly through inducing ferroptosis. We further established a rat model of pressure-overload-mediated HF by aortic banding. Significant HF with characteristic structural changes of mitochondria under the electron microscope, such as shrinking mitochondria and increased mitochondrial membrane density, was observed in rats with AB at 12 weeks postoperatively, accompanied by left ventricular enlargement and contractility impairment. Myocyte death indicated by PI staining, with increased 4-HNE expression and LIP and

downregulated GPX4 and FTH1 expression in AB rats was much more remarkable than in SO animals, indicating that cell death mediated by ferroptosis might occur in pressure-overload-induced HF. Based on the above findings, exploring new medicines or other methods based on ferroptosis regulation might represent a new strategy for HF treatment. Based on current evidence-based medicine and clinical guidelines, there is strong evidence to support the effectiveness of several kinds of medicines, such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta blockers and aldosterone receptor antagonists, in the treatment of HF. Nevertheless, the use of these medicines is limited in certain patients owing to their side effects, especially in those with multiple co-morbidities at the end stage of HF, leading to a troublesome situation in HF treatment. Therefore, efforts should continue to explore new medicines to supplement those currently available for HF. Puerarin, a type of antioxidant, has been proven to have an underlying research and development value for HF. Ai et al. [23] reported that puerarin significantly improved impaired cardiac function and angiogenesis with great increase of VEGFA, Ang-1 and Ang-2 in rats with HF induced by left anterior descending coronary artery (LAD) ligation for four weeks. In streptozocin-induced diabetic mellitus rats with ligated LAD, puerarin treatment significantly increased survival rate and improved cardiac function compared with that in sham animals [24]. The results from our previous study and others [9,25,26] also proved that puerarin significantly retarded the progression from compensated cardiac hypertrophy to decompensated HF under pressure overload induced by aortic banding. In the current study, administration of puerarin for 12 weeks in AB rats effectively restricted cardiomyocyte death and improved cardiac function. In the in vitro experiments, similar anti-cell-death effects of puerarin on ISO-induced H9c2 cells were also observed.

Please cite this article in press as: B. Liu, et al., Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.02.061

6

B. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8

Fig. 3. Puerarin mitigated heart failure in rats with aortic banding. (A) Puerarin (100 mg/kg/d and 200 mg/kg/d for 12w respectively) improved ventricular function in rats with HF indicated by LVEF, LVFS, LVPWd and LVIDd. (B) and (C) Puerarin (100 mg/kg/d and 200 mg/kg/d for 12w respectively) improved cardiac ultrastructure in rats with HF. Each of the experiments was repeated 3 times, n ¼ 3.

Importantly, puerarin effectively improved cell viability under erastin stimulation, implying that anti-ferroptosis might be an underlying mechanism of the protective effects of puerarin on cardiomyocytes. Thus, we further tested the influence of puerarin on the two key features of ferroptosis: iron homeostasis and lipid peroxidation. In puerarin-treated AB rats with HF, the iron content significantly reduced compared with that in vehicle-treated AB rats. In addition, we assayed the expression of FTH1, an ironmetabolism-related protein that is involved in the ferroptosis process. Puerarin supplementation induced a significant increase in FTH1 expression compared with vehicle-treated animals. In vitro, puerarin also lowered the LIP level under ISO co-cultivation in a

dose-dependent manner, and similar to the ferroptosis-specific inhibitor Fer-1, puerarin significantly increased FTH1 expression in ISO-treated H9c2 cells. Of note, puerarin not only functions in the regulation of iron homeostasis during HF, but also reportedly acts as a direct free radical scavenger and an indirect antioxidant. In support of this, we found that puerarin treatment was cardioprotective in the hearts of AB rats by reducing ROS content and increasing the expression level of the antioxidant enzyme GPX4. The results from our in vitro study also supported that puerarin could lower the ISOinduced lipid peroxidation level, suggesting the protective effects of puerarin are similar to previously reported desferoxaminemediated chelation [4e7].

Please cite this article in press as: B. Liu, et al., Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.02.061

B. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8

7

Fig. 4. Puerarin inhibited ferroptosis in heats of rats with aortic banding. (A) Puerarin (100 mg/kg/d and 200 mg/kg/d for 12w respectively) inhibited myocytes death in rats with HF. (B) Puerarin (100 mg/kg/d and 200 mg/kg/d for 12w respectively) inhibited lipid peroxidation in cardiac tissue with HF. (C) puerarin (100 mg/kg/d and 200 mg/kg/d for 12w respectively) reduced Nox4 expression and influenced ferroptosis-related protein Each of the experiments was repeated 3 times, n ¼ 3.

Previous studies have identified that NOX4 levels were significantly increased by pressure overload [27]. Further studies unexpectedly proved the protective effects of NOX4 against chronicload-induced cardiac stress with cardiomyocyte-targeted NOX4 overexpression or knockout of NOX4. Increased left ventricular hypertrophy, contractile dysfunction, and dilation could be observed in NOX4-knockout animals but not in NOX4overexpressing ones [28]. Furthermore, evidence has been presented that NOX4 is involved in hydrogen peroxide promoting cell death, as the inhibition of NOX4 blocked ferroptosis in kidney tubule epithelia [29]. In epidermal growth factor receptor (EGFR)mutant human mammary epithelial cells, NOX4 expression was increased significantly, and produced hydrogen peroxide. GKT136901, a specific NOX4 inhibitor, could rescue viability, and inhibit lipid ROS generation. To explain the antioxidant role as possible contributors that attenuate NOX4 pathology, NOX4 was

examined in rats with AB for 12weeks plus puerarin supplementation. Not surprisingly, the present data showed that the protein level of NOX4 in failed hearts was remarkably inhibited by puerarin treatment. Several studies on other diseases have shown that MAPK signaling and SIRT1-mediated deacetylation of NF-kB might be involved in the mechanisms for puerarin regulating NOX4. In a renal fibrosis model induced by unilateral ureteral obstruction, puerarin treatment significantly ameliorated renal fibrosis and reduced NOX4 expression. Further study proved that MAPK signaling might be involved in the mechanism of puerarin downregulating NOX4 in renal fibrosis [30]. In STZ-induced diabetic rat aorta, puerarin demonstrated remarkable protective effects with NOX4 inhibition and NF-kB regulation followed by inhibition of cell adhesion molecule expression [31]. Therefore, it is necessary to further study whether the above mechanism can explain the results we observed that puerarin inhibited ferroptosis in HF induced

Please cite this article in press as: B. Liu, et al., Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.02.061

8

B. Liu et al. / Biochemical and Biophysical Research Communications xxx (2018) 1e8

by pressure overload. We provide here the first evidence that ferroptosis is involved in cell loss during HF with pressure overload induced by AB and puerarin is a cellular inhibitor of ferroptosis. Given that puerarin is safely used clinically, our findings offer a new insight toward understanding the mechanisms of puerarin in cytoprotection during HF.

[15]

[16]

Disclosure [17]

Bei Liu, Chunxia Zhao, and Hongkun Li are joint first authors. Conflicts of interest [18]

The authors declare that there is no conflict of interests regarding the publication of this paper. Authors' contribution

[19]

Bei Liu, Chunxia Zhao and Hongkun Li made equal contributions to this work. References [20] [1] G.W. Moe, J. Marín-García, Role of cell death in the progression of heart failure, Heart Fail. Rev. 21 (2016) 157e167. [2] S.J. Dixon, K.M. Lemberg, M.R. Lamprecht, R. Skouta, E.M. Zaitsev, C.E. Gleason, D.N. Patel, A.J. Bauer, A.M. Cantley, W.S. Yang, B. Morrison 3rd, B.R. Stockwell, Ferroptosis: an iron-dependent form of nonapoptotic cell death, Cell 149 (2012) 1060e1072. [3] R. Skouta, S.J. Dixon, J. Wang, D.E. Dunn, M. Orman, K. Shimada, P.A. Rosenberg, D.C. Lo, J.M. Weinberg, A. Linkermann, B.R. Stockwell, Ferrostatins inhibit oxidative lipid damage and cell death in diverse disease models, J. Am. Chem. Soc. 136 (2014) 4551e4556. [4] D.T. Kremastinos, D. Farmakis, Iron overload cardiomyopathy in clinical practice, Circulation 124 (2011) 2253e2263. [5] Carlos J. Miranda, Hortence Makui, Ricardo J. Soares, Marc Bilodeau, Jeannie Mui, Hajatollah Vali, Richard Bertrand, Nancy C. Andrews, Manuela M. Santos, Hfe deficiency increases susceptibility to cardiotoxicity and exacerbates changes in iron metabolism induced by doxorubicin, Blood 102 (2003) 2574e2580. [6] A. Nitenberg, S. Ledoux, P. Valensi, R. Sachs, I. Antony, Coronary microvascular adaptation to myocardial metabolic demand can be restored by inhibition of iron-catalyzed formation of oxygen free radicals in type 2 diabetic patients, Diabetes 51 (2002) 813e818. [7] D. Lapenna, G. Ciofani, S.D. Pierdomenico, M.A. Giamberardino, E. Porreca, Iron status and oxidative stress in the aged rabbit heart, J. Mol. Cell. Cardiol. 114 (2017) 328e333. [8] Y.X. Zhou, H. Zhang, C. Peng, Puerarin: a review of pharmacological effects, Phytother Res. 28 (2014) 961e975. [9] B. Liu, Z. Wu, Y. Li, C. Ou, Z. Huang, J. Zhang, P. Liu, C. Luo, M. Chen, Puerarin prevents cardiac hypertrophy induced by pressure overload through activation of autophagy, Biochem. Biophys. Res. Commun. 464 (2015) 908e915. [10] D. Lapenna, S.D. Pierdomenico, G. Ciofani, S. Ucchino, M. Neri, M.A. Giamberardino, F. Cuccurullo, Association of body iron stores with low molecular weight iron and oxidant damage ofhuman atherosclerotic plaques, Free Radic. Biol. Med. 42 (2007) 492e498. [11] W.S. Yang, B.R. Stockwell, Synthetic lethal screening identified compounds activating iron dependent. Nonapoptotic cell death in oncogenic-RASharboringcancer cells, Chem. Biol. 15 (2008) 234e245. [12] W.S. Yang, R. SriRamaratnam, M.E. Welsch, K. Shimada, R. Skouta, Cheah JH. ViswanathanVS, P.A. Clemons, A.F. Shamji, C.B. Clish, L.M. Brown, A.W. Girotti, V.W. Cornish, S.L. Schreiber, B.R. Stockwell, Regulation of ferroptotic cancer cell death by GPX4, Cell 156 (2014) 317e331. [13] T. Lorincz, K. Jemnitz, T. Kardon, J. Mandl, A. Szarka, Ferroptosis is involved in acetaminophen induced cell death, Pathol. Oncol. Res. 21 (2015) 1115e1121. [14] L. Chen, X. Li, L. Liu, B. Yu, Y. Xue, Y. Liu, Erastin sensitizes glioblastoma cells

[21] [22]

[23]

[24]

[25]

[26]

[27]

[28]

[29]

[30]

[31]

totemozolomide by restraining xCT and cystathionine-gammalyase function, Oncol. Rep. 33 (2015) 1465e1474. H. Yamaguchi, J.L. Hsu, C.T. Chen, Y.N. Wang, M.C. Hsu, S.S. Chang, Caspaseindependent cell death is involved in the negative effect of EGF receptor inhibitors on cisplatin in non-small cell lung cancer cells, Clin. Canc. Res. 19 (2013) 845e854. I. Ingold, C. Berndt, S. Schmitt, S. Doll, G. Poschmann, K. Buday, A. Roveri, X. Peng, F. Porto Freitas, T. Seibt, L. Mehr, M. Aichler, A. Walch, D. Lamp,
r, N. Fradejas-Villar, M. Jastroch, S. Miyamoto, W. Wurst, F. Ursini, E.S.J. Arne U. Schweizer, H. Zischka, J.P. FriedmannAngeli, M. Conrad, Selenium utilization by GPX4 is required to prevent hydroperoxide-induced ferroptosis, Cell 8674 (2017) 31438e31441. Q.Z. Tuo, P. Lei, K.A. Jackman, X.L. Li, H. Xiong, X.L. Li, Z.Y. Liuyang, L. Roisman, S.T. Zhang, S. Ayton, Q. Wang, P.J. Crouch, K. Ganio, X.C. Wang, L. Pei, P.A. Adlard, Y.M. Lu, R. Cappai, J.Z. Wang, R. Liu, A.I. Bush, Tau-mediated iron export prevents ferroptotic damage after ischemic stroke, MolPsychiatry 22 (2017) 1520e1530. A. Linkermann, R. Skouta, N. Himmerkus, S.R. Mulay, C. Dewitz, F. De Zen, A. Prokai, G. Zuchtriegel, F. Krombach, P.S. Welz, R. Weinlich, T. VandenBerghe, P. Vandenabeele, M. Pasparakis, M. Bleich, J.M. Weinberg, €sen, U. Kunzendorf, H.J. Anders, B.R. Stockwell, D.R. Green, C.A. Reichel, J.H. Bra S. Krautwald, Synchronized renal tubular cell death involves ferroptosis, Proc Natl AcadSci USA 111 (2014) 16836e16841. J.P. FriedmannAngeli, M. Schneider, B. Proneth, Y.Y. Tyurina, V.J. Tyurin VA,Hammond, N. Herbach, M. Aichler, A. Walch, E. Eggenhofer, O. Basavarajappa D,Rådmark, S. Kobayashi, T. Seibt, H. Beck, F. Neff, I. Esposito, € rster, O. Yefremova, M. Heinrichmeyer, G.W. Bornkamm, R. Wanke, H. Fo E.K. Geissler, S.B. Thomas, B.R. Stockwell, V.B. O'Donnell, V.E. Kagan, J.A. Schick, M. Conrad, Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice, Nat. Cell Biol. 16 (2014) 1180e1191. T. Lorincz, K. Jemnitz, T. Kardon, J. Mandl, A. Szarka, Ferroptosis is involved in Acetaminophen induced cell death, Pathol. Oncol. Res. 21 (2015) 1115e1121. M. Gao, P. Monian, N. Quadri, R. Ramasamy, X. Jiang, Glutaminolysis and transferr in regulate ferroptosis, Mol. Cell 59 (2015) 298e308. Y. Baba, J.K. Higa, B.K. Shimada, K.M. Horiuchi, T. Suhara, M. Kobayashi, J.D.1 Woo, H. Aoyagi, K.S. Marh, H. Kitaoka, T. Matsui, Protective effects of the mechanistic target of rapamycin against excess iron and ferroptosis in cardiomyocytes, Am. J. Physiol. Heart Circ. Physiol. (2017), https://doi.org/ 10.1152/ajpheart.00452.2017. F. Ai, M. Chen, B. Yu, Y. Yang, G. Xu, F. Gui, Z. Liu, X. Bai, Z. Chen, Puerarin accelerates cardiac angiogenesis and improves cardiac function of myocardial infarction by upregulating VEGFA, Ang-1 and Ang-2 in rats, Int. J. Clin. Exp. Med. 8 (2015) 20821e20828. W. Cheng, P. Wu, Y. Du, Y. Wang, N. Zhou, Y. Ge, Z. Yang, Puerarin improves cardiac function through regulation of energy metabolism in StreptozotocinNicotinamide induced diabetic mice after myocardial infarction, Biochem. Biophys. Res. Commun. 463 (2015) 1108e1114. G. Chen, S.Q. Pan, C. Shen, S.F. Pan, X.M. Zhang, Q.Y. He, Puerarin inhibits angiotensin II-induced cardiac hypertrophy via the redox-sensitive ERK1/2, p38 and NF-kB pathways, Acta Pharmacol. Sin. 35 (2014) 463e475. Y. Yuan, J. Zong, H. Zhou, Z.Y. Bian, W. Deng, J. Dai, H.W. Gan, Z. Yang, H. Li, Q.Z. Tang, Puerarin attenuates pressure overload-induced cardiac hypertrophy, J. Cardiol. 63 (2014) 73e81. M. Zhang, A. Perino, A. Ghigo, E. Hirsch, A.M. Shah, NADPH oxidases in heart failure: poachers or game keepers? Antioxidants Redox Signal. 18 (2013) 1024e1041. €der, C.X.C. Santos, D.J. Grieve, M. Wang, M. Zhang, A.C. Brewer, K. Schro N. Anilkumar, B. Yu, X. Dong, S.J. Walker, R.P. Brandes, A.M. Shah, NADPH oxidase-4 mediates protection against chronic load-induced stress in mouse hearts by enhancing angiogenesis, Proc. Natl. Acad. Sci. Unit. States Am. 107 (2010) 18121e18126. I. Poursaitidis, X. Wang, T. Crighton, C. Labuschagne, D. Mason, S.L. Cramer, K. Triplett, R. Roy, O.E. Pardo, M.J. Seckl, S.W. Rowlinson, E. Stone, R.F. Lamb, Oncogene-selective sensitivity to synchronous cell death following modulation of the AminoAcid nutrient cystine, Cell Rep. 18 (2017) 2547e2556. X. Zhou, C. Bai, X. Sun, X. Gong, Y. Yang, C. Chen, G. Shan, Q. Yao, Puerarin attenuates renal fibrosis by reducing oxidative stress induced-epithelial cell apoptosis via MAPK signal pathways in vivo and in vitro, Ren. Fail. 39 (2017) 423e431. W. Li, W. Zhao, Q. Wu, Y. Lu, J. Shi, X. Chen, Puerarin improves diabetic aorta injury by inhibiting NADPH oxidase-Derived oxidative stress in STZ-induced diabetic rats, J Diabetes Res. (2016), 8541520, https://doi.org/10.1155/2016/ 8541520.

Please cite this article in press as: B. Liu, et al., Puerarin protects against heart failure induced by pressure overload through mitigation of ferroptosis, Biochemical and Biophysical Research Communications (2018), https://doi.org/10.1016/j.bbrc.2018.02.061