Background
Cardiac hypertrophy has become an independent and predictive risk factor for adverse cardiovascular events [
1]. Despite an increasing understanding toward the pathophysiological process of cardiac hypertrophy, progress in mechanism remains sluggish in recent years. Many factors are involved in the pathophysiological process of cardiac hypertrophy, of which inflammation and oxidative stress play crucial mediating role [
2,
3]. Among cardiovascular diseases, obesity has a great impact on the cardiac remodeling and function in terms of hemodynamic load, myocardial fibrosis and impaired ventricular contractility, which leads to the progression of heart failure [
4,
5]. Obesity has also been characterized as an inflammatory state [
6,
7], and chronic low-grade inflammation and oxidative stress play important roles in the pathogenesis of obesity-related complications. It has been demonstrated both in vitro and in vivo that the increased free fatty acids in obesity can activate the nuclear factor-kappaB (NF-kB) pathway, subsequently increasing the expression of several pro-inflammatory cytokines and inducing cellular oxidative stress [
8]. However, the precise molecular mechanisms involved in obesity-induced cardiac hypertrophy process still need further elucidation.
It is well established that fibronectin type III domain containing 5 (FNDC5) and its cleaved and secreted fragment, irisin, are beneficial to improve metabolic diseases in both humans and mice [
9]. Recent studies in our lab have shown that FNDC5 overexpression ameliorates hyperlipemia and enhances lipolysis in adipose tissues of mice [
10], and prevents high fat diet (HFD)-induced hyperlipemia, hepatic lipid accumulation and impaired fatty acid oxidation in liver [
11]. Irisin improves glucose homoeostasis by reducing gluconeogenesis and increasing glycogenesis via PI3K/Akt pathway in HepG2 cells [
12]. These findings of our previous studies confirmed the beneficial effects of FNDC5/irisin in metabolic diseases. Many studies have highlighted the role of exercise in the heart, of which serves as an important organ regulating metabolism. Exercise training has various physiologic effects on the cardiovascular system, most notably multiple metabolic changes in the myocardium resulting in improved tolerance for ischemia and reperfusion injury, a reduction of resting heart rate attributed to increased parasympathetic tone and an improved vascular endothelial function with augmented flow-mediated vasodilation during exercise [
13‐
15]. Nevertheless, as an up-regulated myokine after exercise, the exact role of FNDC5 in cardiomyocytes remains unclear.
Growing evidence points to the regulatory capacity of JAK2/STAT3 pathway on inflammation-related diseases and cardiac hypertrophy [
16‐
19]. STAT3 is the most important effector of the JAK2 in the initiation and development of cardiac hypertrophy. After phosphorylation, STAT3 translocates from the cytoplasm to the nucleus and promotes the transcription of multiple hypertrophic factors including natriuretic peptide type A and natriuretic peptide type B [
20]. Therefore, it is worthy to examine whether JAK2/STAT3 pathway is involved in molecular mechanisms of obesity-induced cardiac hypertrophy. We recently reported the anti-inflammatory effects of FNDC5 in adipose tissue of mice [
21]. Here, the aim of this study is to evaluate the role and molecular mechanism of FNDC5 on cardiac function, inflammatory and oxidative stress responses in HFD-induced obese mice, which would be used for future therapeutic interventions in obesity-related complications.
Methods
Animals
Male wild-type (WT) mice and FNDC5
−/− mice on a C57BL/6 background (Nanjing BioMedical Research Institute of Nanjing University, Nanjing, China) were used in the study as previously reported [
11]. Mice were housed at 22–26 °C temperature, 40% ± 5% humidity and 12-h light/dark cycle. At the age of 6 weeks either WT or FNDC5
−/− mice were randomly divided to fed a normal chow (Ctrl, 14.7 kJ/g, 13% of energy as fat) or a HFD (21.8 kJ/g, 60% of energy as fat) for 20 weeks, respectively. The lard-based HFD were obtained from Nanjing Junke Biotechnology Corporation, Ltd (Nanjing, China).
FNDC5 overexpression in mice
As we previously described, WT and FNDC5
−/− mice at the age of 6 weeks were fed with HFD for 20 weeks to induce obesity. FNDC5 overexpression were induced by lentivirus (1 × 10
8 TU/ml, 100 μl) intravenously injection [
10]. The recombinant lentivirus expressing FNDC5 or vector were injected into the mice at the end of the 14th week after HFD. Acute experiments were carried out 6 weeks after the lentivirus injection.
Echocardiographic measurements
Cardiac function was measured by echocardiography (Vevo 2100, VisualSonic, Canada). WT and FNDC5
−/− mice were anesthetized using 2.0% isoflurane and then two-dimensional echocardiographic views of the mid-ventricular short axis were obtained at the level of the papillary muscle tips below the mitral valve. Interventricular septal thickness (IVS), left ventricular internal dimensions (LVID) and left ventricular posterior wall dimensions (LVPW) were measured at the end of diastole and systole [
22].
Western blot analysis
Tissues or cardiomyocytes were sonicated in RIPA lysis buffer containing 1% NonidetP-40, 0.1% SDS, protease inhibitor, while adding phosphatase inhibitors cocktail (Bimake, Houston, USA), and then homogenized. The nuclear and cytosolic protein were extracted by commercially available kit (Beyotime Biotechnology, Shanghai, China). Briefly, the cells expanded sufficiently under low osmotic pressure, then the cell membrane ruptured, releasing cytoplasmic proteins. Nucleus precipitation could be centrifuged to obtain after then. Finally, the nuclear protein was extracted by high salt nucleoprotein extraction reagent. Equal quantities of tissues or cardiomyocytes protein lysates were subjected to SDS-PAGE (Bio-Rad), and transferred onto PVDF membrane. Blocking was made at room temperature with 5% nonfat milk powder prepared in Tris-buffered saline containing 0.1% Tween 20. Then, membranes were incubated overnight at 4 °C with corresponding antibodies, and followed by incubation with appropriate secondary HRP-conjugated antibodies. Antibodies against p38 (#8690), P-p38 (#4511), NFκB-p65 (#8242), ERK (#4695), P-ERK (#4370), P-Stat3 (#9145), Stat3 (#4904) and GAPDH (#2118) were obtained from Cell signaling Technology (Danvers, USA). Antibodies against FNDC5 (ab174833), P-JAK2 (ab195055), JAK2 (ab108596), Nox2 (ab129068), Nox4 (ab154244) were obtained from Abcam (Cambridge, UK).
Quantitative real-time PCR analysis
Total RNA was separated using a Trizol reagent (Life Technologies, USA) according to the manufacturer’s protocols. RNA concentrations and purity were assessed by the measurement of optical density at 260 and 280 nm. The purified total-RNA was reverse transcribed with reverse transcription reagent kit (Takara, Japan) and quantitative real-time PCR was performed on Stepone Plus system using the SYBR Green Master Mix (Takara, Japan). The fold changes in the genes were analyzed based on comparative ΔΔCt method. The sequences of primers were listed in Additional file
1: Table S1.
Tissue embedding and H&E staining
The mice were anesthetized and the heart were quickly removed. Heart tissues were rinsed in ice-cold PBS, and incubated overnight in formalin. After washed and dehydrated by successive incubation in 70 to 100% ethanol solutions, heart tissues were fixed and embedded in paraffin, and slices were cut every 6 μm. The sections were stained with hematoxylin–eosin (H&E) and used for histopathological analysis. The degree of pathological changes was evaluated microscopically by measuring the cardiomyocyte area.
Cell culture and treatment
Neonatal primary cardiomyocytes (CMs) from 1 to 3-day-old WT or FNDC5
−/− mice were prepared as reported [
23]. Briefly, neonatal hearts were dissociated in cell suspension using the Neonatal Heart Dissociation Kit (MACS, Bergisch Gladbach, Germany). Dissociated cells were neutralized with DMEM and 4.5 g/l glucose supplemented with 7.5% FBS and passed through a 70-µm cell strainer. Then the dissociated cells were preplated at 37 °C for 1 h to remove fibroblasts and endothelial cells, and nonadhered cells were collected as CMs. CMs were plated in DMEM supplemented with 15% FBS, 1% penicillin, and 1% streptomycin in a humidified atmosphere of 5% CO
2 at 37 °C. Embryonic rat heart-derived cell line H9c2 were obtained from Costar Corning Inc. (Corning, CA, USA). H9c2 was cultured in DMEM/F12 medium containing 2.25 g/l glucose medium supplemented with 10% FBS, 1% of penicillin, and 1% of streptomycin. CMs and H9c2 cells were treated with palmitate (PA, obtained from Sigma Aldrich) at 400 μM (bound to BSA at a ratio of 5:1) for 24 h to mimic high lipid stimulation and to induce inflammation. PA was administrated 4 h after exogenous FNDC5 (200 nM, Sigma Aldrich) pretreatment. For WP1066 (5 μM, Med Chem Express) treatment experiments in H9c2 cells, WP1066 was added 2 h before FNDC5 pretreatment, while PA was added 4 h after FNDC5 pretreatment. The measurement for phosphorylated protein level was made 12 h after PA administration, while for mRNA levels or protein levels were made 24 h after PA administration.
Small interfering RNA transfection
H9c2 cells were pre-treated with specific small interfering RNA (siRNA) against FNDC5 (FNDC5-siRNA). The FNDC5 specific siRNA (sense sequence: 5′GAUGGCCUCUAAGAACAAA3′; antisense sequence: 3′CUACCGGAGAUUCUUGUUU5′) was purchased from RiboBio (Guangzhou, China). Negative siRNAs (NA-siRNA) with no sequence homology to any known rat genes were used as a control. H9c2 cells were transfected with 50 nM siRNA using lipofectamine 3000 reagent (Invitrogen). Cells were lysed, 48 h after transfection, to analyze FNDC5 protein expression to examine knock-down efficiencies.
Oxidative stress detection
The heart tissues or cells were washed and added to phosphate-buffered saline, ground into homogenates, and centrifuged to collect the supernatant. The activities of superoxide dismutase 1 (SOD) and the content of malondialdehyde (MDA) were detected by commercially available kits purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Griess Reagent Kit (Thermo Fisher Scientific, Waltham, USA) was used to determine the estimation level of Nitric Oxide (NO) production in cell culture medium of CMs according to the manufacturer’s protocol.
ELISA
TNF-α, IL-1β levels in heart tissues were measured with mouse TNF-α and IL-1β ELISA Kits (Excell Bio, Shanghai, China). IL-6 levels in heart tissues were measured with mouse IL-6 ELISA Kit (Beyotime Biotechnology, Shanghai, China). ELISA kit for FNDC5 was obtained from Phoenix pharmaceuticals (EK-067-19, Burlingame, USA).
Statistical analysis
All values were presented as mean ± SEM. Student’s t-test was used for 2-group comparisons. Experiments with > 2 groups were compared by 1-way or 2-way ANOVA followed by Bonferroni’s multiple comparisons test. Statistical analyses were performed with SPSS software. A value of P < 0.05 was considered statistically significant.
Discussion
Obesity has been recognized as the cause of various forms of cardiac abnormalities and development of heart failure [
31,
32]. The primary novel findings in our study are that FNDC5 deficiency deteriorates, while FNDC5 overexpression attenuates HFD-induced cardiac hypertrophy, cardiac inflammation and oxidative stress. Supporting these in vivo observations, our in vitro studies also demonstrated that FNDC5 inhibits PA-induced cardiomyocytes hypertrophy via inactivated JAK2/STAT3 signaling-related inflammation and oxidative stress. Our results identify the protective role of FNDC5 in obesity-induced cardiac hypertrophy, which shed additional light on future FNDC5-based therapeutic interventions in obesity-related cardiac abnormalities.
Low grade inflammation serves as a proximate trigger, as well as an ultimate modulator of cardiac dysfunction in obesity and cardiovascular disease [
33,
34]. Increased oxidative stress coupled with activation of down-stream pro-inflammatory cytokines have pivotal roles in the development of obesity-related complications [
35,
36]. As we known, lipid overload is often associated with increased production and release of pro-inflammatory cytokines, and these cytokines activate a series of intracellular signaling pathways including NFκB, which up-regulate the transcription of more cytokines and exaggerate the inflammatory response. These events further trigger macrophage activation and tissue filtration, leading to tissue injury [
37]. According to our data, FNDC5 gene deletion not only exacerbated HFD-induced enhancement of the pro-inflammatory cytokines levels, oxidative stress and NOX4 expression in heart, but also promoted NFκB activation and cardiac inflammatory signaling activation. The anti-inflammatory effects by FNDC5 were not only demonstrated in heart of HFD-fed mice, but also in PA-stimulated CMs and H9c2 cells. These data confirm the strong anti-inflammatory effects of FNDC5 on cardiomyocytes, which may contribute to its cardio-protective effects in the present study.
To support the direct role of fatty acid (FA) in cardiomyocyte hypertrophy, in vitro exposure of cardiomyocytes to PA was investigated in our study. Both CMs and H9c2 cells under high lipid treatment showed upregulated inflammatory responses as well as oxidative stress levels, which were also attenuated by exogenous FNDC5 pretreatment, indicating that FNDC5 is involved in the cardio-protective effects against PA-induced inflammation and oxidative stress in vitro. It has been shown that FNDC5 overexpression facilitates the process of cardiogenesis [
38]. A recently study found that irisin, the secreted protein derived from FNDC5, protects heart against ischemia–reperfusion injury through a mitochondria mechanism [
39]. Although previous studies reported some evidences of the positive role of FNDC5 in heart, the exact role in inflammation and oxidative stress on cardiomyocytes were first investigated in our present study.
According to our in vitro and in vivo data, FNDC5 absence/knockdown led to an enhanced phosphorylation level of JAK2 and STAT3 under high lipid treatment (HFD/PA) accompanied by aggravated inflammation, oxidative stress and cardiac hypertrophy. Phospho-JAK2/STAT3 level were downregulated in FNDC5 OE mice, suggesting that the protective role of FNDC5 in cardiac inflammation are associated with JAK2/STAT3 pathway inhibition. As we known, obesity results in marked alterations in cardiac energy metabolism, with a prominent effect being an increase in fatty acid uptake and oxidation by the heart [
40]. Peroxisome proliferator activator receptor (PPAR)-α is expressed in abundant levels in the heart and enhanced PPAR-α signaling is especially associated with increased fatty acid oxidation in cardiomyocytes. Interestingly, PPAR-α acts downstream of FNDC5 [
9]. We recently found that FNDC5 deficiency causes impairment in AMPK/PPAR-α-mediated fatty acid oxidation in hepatocytes [
11]. However, it is unclear whether FNDC5 is involved in cardiac fatty acid oxidation during obesity, and the crosstalk between PPAR-α and JAK2/STAT3 signaling or other signal transduction molecules needs further investigation. Elucidation of the precise molecular mechanisms underpinning FNDC5 signaling involved in the cardiac hypertrophy process may prove useful in understanding the role of FNDC5 in metabolic-related cardiovascular diseases.
Adipose tissue inflammation induce systemic inflammation and insulin resistance, resulting in subcellular low-grade inflammation, and in turn, subcellular component abnormalities such as oxidative stress, mitochondrial dysfunction in the heart. The vicious cycle of increased subcellular component abnormalities, inflammatory cell infiltration, neurohumoral activation induce the adverse cardiac remodeling in metabolic-induced cardiomyopathy. Taken together the findings of our present and previous studies, FNDC5 not only ameliorated systemic inflammation, but also had direct effects on cardiomyocytes, which may both contribute to the cardio-protective actions. Although it is still difficult to unmask the probable effectors (receptors) of endogenous FNDC5 and its cleaved fragment, irisin, we considered that FNDC5 may exert its protective function through both direct and indirect pathways. Our data show that HFD did not lead to any significant change in FNDC5 expression level in heart. Similarly, FNDC5 overexpression only increased circulating and muscle expression but not FNDC5 in heart. It is possible that, circulating but not cardiac-derived FNDC5 contributes to the beneficial and cardio-protective effects in vivo.
Skeletal muscle can act as an endocrine organ, secreting diverse myokines in a context-dependent manner [
41,
42]. Such myokines may exert specific endocrine effects on visceral fat, or work locally within the muscle and exert their effects on signaling pathways involved in fat oxidation and glucose uptake, contributing to its beneficial effects on obesity and related diseases. Sundarrajan et al. showed that exogenous irisin, the cleaved fragment of FNDC5, increased diastolic volume, heart rate and cardiac output in zebrafish by regulating cardiovascular physiology-associated modulators such as myostatin a/b, troponin C expression [
43]. Also, it has been reported that irisin plays a protective role against ischemia–reperfusion injury to heart through a SOD2-dependent mitochondria mechanism [
39]. These studies indicate significant translational value of myokines for therapeutic approaches in cardiovascular diseases. Our study highlights the important myokines as novel targets of FNDC5/irisin. Future studies will be required to elucidate the secretory and metabolic pathways for regulation of FNDC5/irisin levels in circulation, which may present significant translational value for clinical implications.
Authors’ contributions
ZG and XQX planned the experiments. ZG, WYF and YT performed the experiments. BZ and CY analyzed the data. XQX and YBZ wrote the paper. All authors read and approved the final manuscript.