Nobiletin, a Polymethoxy Flavonoid, Protects Against Cardiac Hypertrophy Induced by Pressure-Overload via Inhibition of NAPDH Oxidases and Endoplasmic Reticulum Stress

Pathological cardiac hypertrophy is associated with a complicated spectrum of pathophysiological changes, including oxidative stress, metabolic dysfunction, sarcomere disorganization, endoplasmic reticulum (ER) stress, cell apoptosis and fibrosis [1]. Extensive study has suggested that oxidative stress plays a predominant role in the progression of cardiac hypertrophy [2]. Increased reactive oxygen species (ROS) results in cellular damage, energetic deficit and cardiac dysfunction in heart failure. A large body of evidence demonstrates that ROS generated by NADPH oxidases (NOX) are a major source in cardiac hypertrophy [3, 4]. In addition, endoplasmic reticulum (ER) stress has been shown to increase in cardiac hypertrophy in a murine pressure-overload model [5]. ER and oxidative stress have close associations in ROS generation [6], and increased ER stress can initiate myocyte apoptosis [7]. NADPH oxidase activation contributes to ER stress and myocardial dysfunction in diabetic mice [8]. Thus, inhibition of oxidative stress is considered a potential therapy for cardiac hypertrophy [9].

There is some evidence, which indicates that nobiletin (NOB) is a flavonoid with anti-oxidative and anti-diabetic properties [10]. Several studies have demonstrated that NOB relieves age-related cognitive impairment, accompanied by alleviation of oxidative stress in SAMP8 mice [11]. Our previous study indicated that NOB inhibits NADPH oxidases in streptozotocin-induced diabetic cardiomyopathy [12]. Previous studies have shown that NOB inhibits LPS-induced ROS production in RAW 264.7 cells [13]. Additional studies have found that NOB can ameliorate apoptosis in SK-N-SH human neuroblastoma cells and repress TXNIP expression, which plays a crucial role in irremediable ER stress. However, there is still a paucity of data regarding the beneficial role of NOB in cardiac hypertrophy induced by aortic banding (AB). Moreover, it remains unclear whether NOB treatment mitigates ER stress via a reduction of oxidative stress in a murine cardiac pressure-overload model. In the present study, we aimed to clarify the protective role of NOB in cardiac hypertrophy.

All animal care and experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication, revised 2011) and the institutional guidelines of the Animal Care and Use Committee of Renmin Hospital of Wuhan University (Wuhan, China). Animals were housed under specific-pathogen-free (SPF) conditions in a 12-h light and 12-h dark cycle with food and water provided ad libitum. The AB operation and subsequent analyses were performed in a blind manner for all groups. Male C57BL/6 mice (8 to 10 weeks old; body weight: 25.5 ± 2 g) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences (Beijing, China). NOB was purchased from Shanghai Winherb Medical Science Co. Ltd. (Shanghai, China), and the purity of NOB was >98 % as determined by high-performance liquid chromatography (HPLC) analysis. The animals were randomly divided into four groups: sham+vehicle (VEH/SH) (n = 15), sham+NOB (NOB/SH) (n = 15), AB+vehicle (VEH/AB) (n = 15), and AB+NOB (NOB/AB) (n = 15). The mice were anesthetized with 3% sodium pentobarbital by intraperitoneal injection, and cardiac hypertrophy model was established via AB surgery as previously described [14, 15]. Three days after AB or sham procedure, mice were treated with NOB or vehicle for 4 weeks by gavage, and the dose of NOB (dissolved in normal saline including 0.05% Tween-80) was 50 mg/kg as previously described [12]. After administration of NOB or vehicle for four weeks, all experimental mice were anesthetized with 1.5% isoflurane, euthanized by cervical dislocation, and the heart was then collected and the tibia measured for length.

Cardiac function was detected using an echocardiographic system equipped with a 10-MHz transducer (Esaote SpA, Genoa, Italy). Briefly, lightly anesthetized mice (1.5% isoflurane and 98.5% O₂) were placed on a temperature-controlled warming pad (37°C). Parasternal long-axis M-mode tracings of the left ventricle (LV) were recorded at the level immediately above the papillary muscles, LV end-diastolic diameter (LVEDd), LV end-systolic diameter (LVESd) and end-diastolic interventricular septal thickness (IVSd) were detected. LV fractional shortening (LVFS) was calculated using the following formula: LVFS(%) = (LVEDd – LVESd )/LVEDd×100.

In the process of pressure-volume measurements, mice were first anesthetized with 1.5% isoflurane and maintained their core temperature by a temperature-controlled heating platform, and then a catheter transducer (Millar, Houston, USA) was inserted into the right carotid artery and advanced gradually into the left ventricle. The data was continuously recorded using a Millar Pressure-Volume system (Millar, Houston, USA), hemodynamic parameters including end-systolic pressure (ESP), end-diastolic pressure (EDP), the maximal rate of pressure development (dp/dt max) and minimal rate of pressure decay (dp/dt min) were measured by PVAN data analysis software (Millar, Houston, USA).

Cardiac tissues were fixed in 4% paraformaldehyde and paraffin-embedded, then cut into 5 µm sections. To evaluate the myocyte size, the slides were stained with hematoxylin and eosin (HE) and fluorescein isothiocyanate-labeled wheat germ agglutinin (WGA) (Invitrogen, Carlsbad, USA). WGA staining was performed to measure the accurate myocyte cross-sectional area and 4,6-diamidino-2-phenyl-indole (DAPI) (Invitrogen, Carlsbad, USA) to detect nuclei. Picrosirius red staining was performed to assess the extent of heart fibrosis, and the slides were then visualized using light microscopy and a Nikon Photo-Imaging System (H550L, Tokyo, Japan). The cross-sectional areas of the myocardium and cardiac fibrosis were measured using Image-pro Plus 6.0 (Maryland, USA).

Immunohistochemistry for myocardial 4-hydroxynonenal (4-HNE) was conducted, and the sections were deparaffinized and blocked with 8% goat serum in PBS, followed by incubation with an anti-4-HNE antibody (Abcam, Cambridge, MA) at 1:100 dilution for 12 h at 4°C. Thereafter, the sections were incubated with anti-rabbit HRP reagent (Gene Tech, Shanghai, China) for 1 h at room temperature and developed using a peroxide-based substrate DAB kit (Gene Tech, Shanghai, China). Finally, the sections were dehydrated in ethanol and cleared in xylene. Slides were examined under light microscopy and a Nikon Photo-Imaging System (H550L, Tokyo, Japan).

NRCMs were isolated from 1- to 2-day-old Sprague-Dawley rats as previously described [16]. NRCMs were plated onto gelatin-coated culture dishes in DMEM/F12 medium containing 15% fetal calf serum. After 48 hours, the medium was exchanged for serum-free DMEM/F12, and NRCMs were subsequently treated with 100 µmol/L phenylephrine (PE) (Sigma, Milwaukee, USA) or phosphate buffered saline (PBS) containing 0.1% bovine serum albumin and/or NOB (100 µmol/L) for 48 hours. NRCMs were transfected with NOX2 siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) or scrambled siRNA (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s protocol and then incubated with PE (100 µmol/L) or PBS for 24 hours.

For analysis of the, NRCMs were cultured on coverslips and fixed with 4% formaldehyde, permeabilized with 0.3% Triton X-100 in PBS and blocked with 8% goat serum solution at room temperature. The cells were subsequently stained with a primary antibody against α-actinin (Abcam, Cambridge, MA) overnight at 4°C and then incubated with a fluorescent secondary antibody. The cell surface area of NRCMs was analyzed using Image-Pro Plus 6.0 software (Maryland, USA).

Cardiac tissue slides were stained with an Apoptosis Detection Kit (Millipore, Temecula, CA) according to the manufacturer’s instructions. Briefly, the sections were initially deparaffinized and pretreated with proteinase K (20 µg/ml) and then incubated with fluorescein-labeled dUTP. Finally, DAPI (Invitrogen, Carlsbad, CA, USA) was applied to identify all nuclei on the section. The slides were viewed using fluorescence microscopy (BX51, Olympus, Japan) and measured using Image-Pro Plus 6.0 software (Maryland, USA).

To determine the superoxide dismutase 1 (SOD1) concentration of heart tissues, the mice were sacrificed, and their hearts were washed with ice-cold PBS. Cardiac tissues were ground into homogenates and centrifuged at 3,000 g for 15 min at 4°C to collect the supernatant. The concentration of SOD1 was determined using commercially available kits purchased from Beyotime Institute of Biotechnology (Haimen, China).

Intracellular ROS were detected using the reactive oxygen species assay kit (Beyotime, Haimen, China) according to the manufacturer's protocols. Briefly, NRCMs were seeded onto 96-well plates and incubated with 10 µmol/L DCFH-DA probe (100 µL/well) for 30 min at 37°C, and the plate was then washed three times with PBS to eliminate residual probe. The fluorescence intensity at 488 nm excitation wavelength and 525 nm emission wavelength was determined using a luminometer (Synergy HT, BioTek, USA).

Cardiac tissues were dissected, immediately frozen and stored in liquid nitrogen. For total RNA extraction from the left ventricles and cultured NRCMs, hearts and NRCMs were thawed in TRIzol reagent (Invitrogen, Carlsbad, CA, USA), and first strand cDNA was synthesized by reverse-transcription of 2 µg of total RNA using oligo (dT) primers and a cDNA synthesis kit (Roche, Mannheim, Germany). The mRNA expression level was quantified using a LightCycler 480 SYBR Green Master Mix (Roche, Mannheim, Germany) and normalized against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA levels. All primers utilized for amplification are shown in Table 1.

For western blotting analysis, left ventricular tissues and cultured NRCMs were homogenized in RIPA lysis buffer (containing 50 mmol/L Tris-HCl, 0.1 % sodium deoxycholate, 1 % Triton X-100, 5 mmol/L EDTA, 5 mmol/L EGTA, 150 mmol/L NaCl, 40 mmol/L NaF, 2.175 mmol/L sodium orthovanadate, 0.1 % SDS, 0.1 % aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride, pH=7.2). Subsequently, the homogenates was centrifuged at 12,000 g for 30 min at 4°C and the supernatant was collected as protein extracts. The protein concentration was examined using a BCA protein assay kit (Thermo, Waltham, MA, USA). Fifty micrograms of protein extract was separated by 10% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked in Tris-buffered saline containing 5% skim milk powder for 60 min at room temperature. The blots were then incubated with the following primary antibodies: rabbit polyclonal anti-caspase-12 and rabbit monoclonal ani-NOX2 and anti-NOX4 (Abcam, Cambrige, MA), rabbit polyclonal anti-GRP78 and mouse monoclonal anti-CHOP (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-GAPDH monoclonal antibody (Abcam, Cambridge, MA). The membranes were scanned using a two-color infrared imaging system (Odyssey; LI-COR Biosciences, Lincoln, NE, USA). Each sample was normalized against GAPDH protein levels.

All data are presented as the mean±SD. Statistical comparisons among multiple groups were examined using one-way ANOVA followed by Tukey’s post hoc test, and comparisons between two groups were determined using Student’s t-test. Statistical analysis was performed using SPSS 17.0 software (SPSS Inc., Chicago, USA). A difference of P<0.05 was considered significant.

The chemical structure of NOB is shown in Fig. 1A. To confirm the protective role of NOB in cardiac hypertrophy, we evaluated the morphological change in hearts and mRNA expression of hypertrophic biomarkers. After 4 weeks of AB surgery, the ameliorated hypertrophic response in NOB/AB mice was further confirmed by morphological examination (Fig. 1B). NOB treatment greatly alleviated the cardiomyocyte hypertrophy and extracellular matrix fibrosis in response to pressure overload, as evidenced by a smaller myocyte cross-sectional area and reduced collagen ratio (Fig. 1C, D). In addition, heart weight/body weight (HW/BW) and HW/tibia length (HW/TL) ratios were dramatically lower in NOB/AB mice compared to VEH/AB mice (Fig. 1E). Consistent with these findings, the mRNA expression of the fetal genes ANP and BNP induced by AB was markedly attenuated following administration of NOB for 4 weeks (Fig. 1F). However, there were no significant differences between the VEH/SH and NOB/SH group.

To examine whether NOB supplementation alleviated pressure-overload-induced cardiac dysfunction, we assessed cardiac dimension and function by echocardiography (Fig. 2A). NOB treatment resulted in an alleviation of cardiac dysfunction, as reflected by a reduction of LVEDd, LVESd and IVSd (Fig. 2B). Indeed, NOB treatment enhanced systolic function after AB operation, as indicated by an increase in LV fractional shorting (FS) (Fig. 2B). Invasive hemodynamic results confirmed preserved systolic (dp/dt max) and diastolic function (dp/dt min) in the NOB/AB mice following 4 weeks pressure-overload (Fig. 2C). Meanwhile, heart rate (HR), end-systolic pressure (ESP) and end-diastolic pressure (EDP) were similar in Sham and AB groups, excluding the possibility that NOB administration influences the pathogenesis of cardiac hypertrophy by affecting these parameters (Fig. 2C). However, we did not observe the statistical differences in cardiac function between the VEH/ SH and NOB/SH mice.

Increased oxidative stress aggravated cardiac hypertrophy, there was decreased SOD1concentration in the heart tissues following AB for 4 weeks. The activity of SOD1 in cardiac tissues was promoted by NOB supplementation following long-term pressure overload induced by AB (Fig. 3A). To detect the expression of NOX induced by AB surgery, we found increased mRNA and protein expression of NOX2 and NOX4. Moreover, NOB supplementation suppressed the increases in NOX2 and NOX4 expression in NOB/AB mice (Fig. 3B, C, D). Furthermore, Fig. 3E shows that myocardial levels of 4-HNE, another marker of oxidative stress, was mainly in the cytosol of the cardiomyocytes in cardiac tissues after AB operation, and the staining intensity of 4-HNE was lower in the myocardium of NOB/AB compared with VEH/AB mice (Fig. 3F). The cardioprotective effect exerted by NOB might be associated with inhibition of NOX2 and NOX4 activities. However, there were no significant differences between the VEH/SH and NOB/SH mice.

Several studies have shown that ER stress plays a crucial role in the development of pathological cardiac hypertrophy [17] and that NOB is able to mediate the process of ER stress [18]. ER chaperones GRP78 is crucial for cell survival/apoptosis under stressful conditions, and ER stress induces apoptosis through CHOP- and caspase-12 dependent signaling pathways. Thus, we investigated whether ER stress was alleviated in response to NOB supplementation after AB. GRP78, CHOP and cleaved-caspase 12/full length caspase 12 ratio were increased in the tissues of cardiac hypertrophy induced by AB, and NOB treatment led to mitigation of ER stress, as reflected by a reduction of GRP78, CHOP and cleaved-caspase 12 protein/full length caspase 12 ratio (Fig. 4A, B). Moreover, Fig. 4C and 4D revealed that the number of TUNEL-positive nuclei was dramatically increased in the cardiac sections of AB mice compared with sham mice. NOB/AB mice showed a decrease in cardiomyocyte apoptosis, as indicated by a reduced number of TUNEL-positive nuclei (Fig. 4C, D). However, we did not detect any differences between the VEH/SH and NOB/SH mice.

The hypertrophic response in NRCMs was measured by the mRNA expression of ANP. As shown in Fig. 5A, NOB could suppress PE-induced cardiomyocyte hypertrophy in a dose-dependent manner, and it was confirmed that 100 µmol/L NOB markedly inhibited the hypertrophic response in NRCMs. To determine the anti-oxidative capacity of NOB in NRCMs, the reduction in reactive oxygen species was observed in NOB-treated cells but not in saline-treated cells after stimulation with PE (Fig. 5B). Furthermore, NOB treatment inhibited PE-induced cardiomyocyte hypertrophy, as reflected by a smaller cell surface area (Fig. 5C, D).

We subsequently analyzed whether PE-induced NOX2 and NOX4 protein expression coincided with cardiac hypertrophy in vivo. PE increased NOX2 and NOX4 protein expression (Fig. 5E), however, NOB supplementary markedly decreased NOX2 protein expression and no significant difference in NOX4 expression between NOB-treated cells and saline-treated cells following stimulation with PE (Fig. 5F). RT-PCR analysis was further performed to evaluate the effect of NOB on the mRNA levels of NOX2, NOX4, GRP78 and CHOP in NRCMs stimulated by PE. Consistent with the inhibition of NOX2 expression in cardiac hypertrophy in vivo, NOB could also reduce NOX2 mRNA expression induced by PE in NRCMs. Meanwhile, there was no significant difference in NOX4 mRNA expression between NOB-treated cells and saline-treated cells following stimulation with PE (Fig. 5G). Importantly, significant decreases in ER stress, as indicated by a lower mRNA expression of GRP78 and CHOP, were detected in NOB-treated cells but not in saline-treated cells following stimulation with PE (Fig. 5H). Taken together, these results supported the role of NOB as a cardioprotective agent, which could suppress cardiomyocyte hypertrophy and ER stress induced by PE. However, no significance was observed in the PBS-stimulated group.

To further examine the effect of NOX2 on ROS generation, we measured ROS stimulated by PE following transfection with scrambled siRNA or NOX2 siRNA. A reduction in reactive oxygen species was detected in NOX2 siRNA cells but not in scrambled siRNA cells (Fig. 6A). As indicated by the data described above, NOB significantly inhibited the expression of NOX2 in NRCMs stimulated by PE and alleviated ER stress. We hypothesized a direct role of NOX2 on the ER stress. Consistent with this idea, we transfected NOX2 siRNA in NRCMs and then exposed the cells to PE. We found that knockdown of NOX2 dramatically attenuated the increased protein expression of GRP78 and CHOP. (Fig. 6B-D)

The pathophysiology of cardiac hypertrophy is clearly associated with increased oxidative stress. A large body of evidence has demonstrated that ROS generated by NOX is a major source during the progression of cardiac remodeling [3, 4]. Moreover, a flavonoid, NOB, possesses anti-oxidative properties. However, knowledge of the protective effect of NOB on cardiac hypertrophy is still lacking. Here, we found that (1) NOB treatment attenuated cardiac hypertrophy, myocardial fibrosis and LV dysfunction; (2) NOB alleviated increased ER stress and myocyte apoptosis; and (3) NOX2 knockdown in NRCMs mitigated the ER stress induced by PE. Taken together, these findings suggested that NOB may potentially be an ideal therapeutic agent for the treatment of heart failure (Fig. 7).

Under pathological conditions of pressure and/or volume overload, NADPH oxidase becomes overly stimulated, and results in the production of excess oxidants and aggravated oxidative stress [19]. NOX consists of membrane [NOX2 and p22phox] and cytosolic [p47phox, p40 phox, p67 phox] isoforms [20]. NOX2 utilizes NADPH to reduce molecular oxygen to O₂^- and is expressed in endothelial cells, cardiomyocytes, fibroblasts and vascular smooth muscle cells. Several studies have indicated that NOX2 expression is higher in the infarcted area of human cardiac tissues, and NOX2 knockout mice alleviated post-MI fibrosis and left ventricular dysfunction [21, 22]. Additional research studies have revealed that NOX2-deficient mice was less vulnerable to cardiac hypertrophy and fibrosis induced by Ang II infusion. Here, we found that NOB can alleviate cardiac dysfunction and fibrosis induced by pressure overload via inhibition of oxidative stress, a protective mechanism might be partly attributed to a reduced expression of NOX2. The capacity of NOB to alleviate cardiac hypertrophy in vitro was corroborated using NRCMs stimulated by PE, and NOB treatment could inhibit ROS generation and NOX2 expression. Previous data indicated that ROS production mediated by NOX2 was major source in phenylephrine-induced hypertrophy of H9C2 cells [23]. Furthermore, NOB attenuated the PE-induced ROS production in NRCMs, whereas the increased NOX4 mRNA and protein expression observed in NOB-treated NRCMs was not dramatically inhibited.

Another NOX isoform, NOX4, existed in the heart. Furthermore, NOX4 differs from NOX2 such that it is regulated mainly by its expression level and produces H₂O₂ via a dismutation of O₂^- [24]. Global loss of NOX4 contributed to increased susceptibility to pressure-overload-induced heart failure, which has been linked to defective angiogenesis [25]. However, cardiac-specific deficiency of NOX4 was less vulnerable to mitochondrial and cardiac dysfunction in response to pressure overload [26], and cardiomyocyte-specific loss of NOX4 resulted in increased susceptibility to myocardial ischemic injury in response to energy stress [27]. Taken together, these divergent results may indicate that NOX4 deficiency in cardiomyocytes was beneficial, whereas loss of NOX4 in the vasculature was detrimental and contributed to adaptive angiogenesis. In contrast, NOX2 knockout mice were protected against post-MI ventricular dysfunction [22] and showed alleviated cardiac hypertrophy. Collectively, these findings revealed that inhibition of NOX might be an ideal therapeutic target for heart failure. Here, we found that there was a markedly increased mRNA and protein expression of NOX4 induced by AB and that NOB treatment dramatically reduced NOX4 expression after pressure-overload stimulation for 4 weeks.

Various stimuli including ischemia, hypoxia, free-radical exposure could disturb ER homeostasis and cause ER stress. Prolonged ER stress induced by pressure overload was accompanied by the increased expression of GRP78 and CHOP [26, 27]. Moreover, GRP78 was up-regulated under stressful conditions of low oxygen and low calcium. Upon excessive ER stress, GRP78 redistributed to the ER lumen, and cleaved caspase-12 was released to initiate its activation, which could cause cell death [28]. Previous study indicated that NOB was implicated in ER stress by mediating the expression of GRP78 [18]. In the present study, we found that NOB treatment mitigated ER stress in vivo and in vitro, as indicated by decreased GRP78 expression. The alleviation of ER stress by NOB may result from the inhibition of GRP78. Moreover, ER stress participated in cardiomyocyte apoptosis, and CHOP and caspase-12 were two major signaling pathways involved in ER stress-induced apoptosis. NOX activation aggravated ER stress and involved in pro-apoptotic signaling in the progression of myocardial remodeling [5, 21, 29]. Our results showed NOB supplementation reduced CHOP expression, the ratio of cleaved-caspase 12/full length caspase12 and the number of TUNEL-positive nuclei in NOB/AB mice. The protective effect of NOB may result from the prevention of cardiomyocyte apoptosis via inhibition of NOX, GRP78 and CHOP expression. The capacity of NOB to alleviate oxidative stress in vitro was corroborated using NRCMs stimulated by PE, and NOB treatment could inhibit NOX2 expression but not dramatically blunt NOX4 expression. Inhibition of NOX2 attenuated increased ER stress in the remote non-infarcted tissues after MI [30]. Moreover, NOX2 deficiency protected ER-stressed mice from renal dysfunction [31]. In the present study, we also found that knockdown of NOX2 retarded ER stress in cultured NRCMs after exposure to PE, as indicated by the decreased expression of GRP78 and CHOP, suggesting a direct role of NOX2 in mediating ER stress in cardiomyocytes.

To the best of our knowledge, this study evaluated the cardioprotective effect of NOB on cardiac hypertrophy induced by AB. We found that NOB displayed significant inhibition of NOX2 and NOX4 expression in vivo but did not markedly reduce NOX4 expression in vitro. The decrease in oxidative stress caused by inhibition of NOX appeared in parallel with a decrease in ER stress, as well as alleviation in the number of apoptotic cardiomyocytes. Moreover, we proposed a direct role of NOX2 in regulating the ER stress-associated protein GRP78 and CHOP. Thus, inhibition of NOX by NOB in the myocardium might be considered a potential therapy for cardiac hypertrophy. However, further investigation in different animal models is under way.

This study was supported by grants obtained from the Key Project of the National Natural Science Foundation (No. 81530012), the National Natural Science Foundation of China (No. 81470516, No.81270303).

The authors declare that they have no conflicts of interest.

N. Zhang and W.-Y. Wei are joint first authors, they contributed equally to this manuscript.