Irisin alleviates pressure overload-induced cardiac hypertrophy by inducing protective autophagy via mTOR-independent activation of the AMPK-ULK1 pathway
Ru-Li Li, Si-Si Wu, Yao Wu, Xiao-Xiao Wang, Hong-Ying Chen, Juan-juan Xin, He Li, Jie Lan, Kun-Yue Xue, Xue Li, Cai-Li Zhuo, Yu-Yan Cai, Jin-Han He, Heng-Yu Zhang, Chao-Shu Tang, Wang Wang, Wei Jiang
PII: S0022-2828(18)30696-5
DOI: doi:10.1016/j.yjmcc.2018.07.250
Reference: YJMCC 8775
To appear in: Journal of Molecular and Cellular Cardiology
Received date: 25 March 2018
Revised date: 21 July 2018
Accepted date: 23 July 2018
Please cite this article as: Ru-Li Li, Si-Si Wu, Yao Wu, Xiao-Xiao Wang, Hong-Ying Chen, Juan-juan Xin, He Li, Jie Lan, Kun-Yue Xue, Xue Li, Cai-Li Zhuo, Yu-Yan Cai, Jin-Han He, Heng-Yu Zhang, Chao-Shu Tang, Wang Wang, Wei Jiang , Irisin alleviates pressure overload-induced cardiac hypertrophy by inducing protective autophagy via mTOR-independent activation of the AMPK-ULK1 pathway. Yjmcc (2018), doi:10.1016/ j.yjmcc.2018.07.250
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Irisin alleviates pressure overload-induced cardiac hypertrophy by inducing protective autophagy via mTOR-independent activation of the AMPK-ULK1 pathway
Ru-Li Lia, Si-Si Wua, Yao Wua, Xiao-Xiao Wanga, Hong-Ying Chena, Juan-juan Xina, He Lia, Jie Lana, Kun-Yue Xuea, Xue Lia, Cai-Li Zhuoa, Yu-Yan Caib, Jin-Han Hec,
Heng-Yu Zhangb, Chao-Shu Tangd, Wang Wange, Wei Jianga,*
aMolecular Medicine Research Center, State Key Laboratory of Biotherapy , West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, PR China bDepartment of Cardiology, West China Hospital, Sichuan University, Chengdu,
Sichuan, 610041, PR China
cDepartment of Pharmacy, West China Hospital, Sichuan University, Chengdu,
Sichuan, 610041, PR China
dDepartment of Pathology and Physiology, Peking University Health Science Center,
Beijing, 10038, PR China
eDepartment of Anesthesiology and Pain Medicine, Mitochondria and Metabolism Center, University of Washington, 850 Republican Street N121, Seattle, WA 98109, USA
*Corresponding author:
Wei Jiang Ph.D.
The Laboratory of Cardiovascular Diseases
Molecular Medicine Research Center, West-China Hospital The state Key Laboratories for Biotherapy
Sichuan University
Chengdu 610041, People’s Republic of China Tel: 86-028-85164103(Office), 85164092(Lab)
E-mail: [email protected]
Abstract
In hypertrophic hearts, autophagic flux insufficiency is recognized as a key pathology leading to maladaptive cardiac remodeling and heart failure. This study aimed to illuminate the cardioprotective role and mechanisms of a new myokine and adipokine, irisin, in cardiac hypertrophy and remodeling. Adult male wild-type, mouse-FNDC5 (irisin-precursor)-knockout and FNDC5 transgenic mice received 4 weeks of transverse aortic constriction (TAC) alone or combined with intraperitoneal injection of chloroquine diphosphate (CQ). Endogenous FNDC5 ablation aggravated and exogenous FNDC5 overexpression attenuated the TAC-induced hypertrophic damage in the heart, which was comparable to the protection of irisin against cardiomyocyte hypertrophy induced by angiotensin II (Ang II) or phenylephrine (PE). Accumulated autophagosome and impaired autophagy flux occurred in the TAC-treated myocardium and Ang II- or PE- insulted cardiomyocytes. Irisin deficiency caused reduced autophagy and aggravated autophagy flux failure, whereas irisin overexpression or supplementation induced protective autophagy and improved autophagy flux, which were reversed by autophagy inhibitors Atg5 siRNA, 3-MA and CQ. Irisin boosted the activity of only AMPK but not Akt and MAPK family members in hypertrophic hearts and cultured cardiomyocytes and further activated ULK1 at Ser555 but not Ser757 and did not affect the mTOR-S6K axis. Blockage of AMPK and ULK1 with compund C and SBI-0206965, respectively, both abrogated irisin’s protection against cardiomyocyte hypertrophic injury and reversed its induction of both autophagy and autophagy flux. Our results suggest that irisin
protects against pressure overload- induced cardiac hypertrophy by inducing protective autophagy and autophagy flux via activating AMPK-ULK1 signaling.
Keywords : Irisin, Cardiac hypertrophy, Transverse aortic constriction, Autophagy flux, AMPK, ULK1
Abbreviations
3-MA 3-methyladenine
AMPK adenosine 5‘-monophosphate (AMP)-activated protein kinase Ang-II angiotensin II
ANP atrial natriuretic peptide
ATG5 autophagy related 5
BNP brain natriuretic peptide
CC compound C
CK creatine kinase
CK-MB creatine kinase-MB CO cardiac output
CQ chloroquine DMSO dimethyl sulfoxide
dPmax maximal value of the first derivative of LV pressure
dPmin minimal value of the first derivative of LV pressure EDV end-diastolic volume
EF ejection fraction
ERK extracellular regulated protein kinase ESV end-systolic volume
FNDC4 fibronectin type III domain-containing protein 4 FNDC5 fibronectin type III domain-containing protein 5
FS fractional shortening
GFP green fluorescent protein
JNK c-Jun N-terminal kinase
KO knock-out
LC3B-I nonlipidated form of LC3B
LC3B-II lipidated form of LC3B
LDH lactate dehydrogenase
LV left ventricular
LVEDD LV end diastolic diameter
LVEDV LV end-diastolic volume
LVEDP LV end-diastolic pressure
LVESP LV end-systolic pressure
LVIDs LV internal dimension in systole
LVIDd LV internal dimension in diastole
LVP LV pressure
LVPWd LV posterior wall thickness at end-diastole
LVSd left ventricle systolic diameter
LVW/TL left heart ventricle weight/tibia length
mTOR mammalian target of rapamycin
NRCMs neonatal rat cardiomyocytes
PE phenylephrine
PGC-1α peroxisome proliferator-activated receptor-γ coactivator 1α
RFP red fluorescent protein
SQSTM1 sequestosome 1
SV stroke volume
TAC transverse aortic constriction
KO knockout
Tg transgenice
ULK1 Serine/threonine-protein kinase
WT wild type
⦁ INTRODUCTION
Hypertensive heart disease is characterized by the presence of pathological cardiac hypertrophy [1]. Numerous molecular elements have been implicated, including cardiomyocyte hypertrophy and injury, extracellular matrix alterations, mesenchymal fibrotic and phlogistic processes, and fetal genetic program reactivation [2]. Accumulating evidence suggests that pressure overload triggers an autocrine/paracrine mechanism known to participate in myocardial hypertrophy development [3, 4]. Mechanical stress regulates the release of growth-promoting factors such as angiotensin II (Ang II) and endothelin-1 [5] as well as growth-suppressing factors including atrial natriuretic peptide (ANP) and adrenomedullin [6,7], which forms a complex cascade or network of interactions between different cytokines, providing a second line of growth regulation [3-6]. However, the role of many cytokines in cardiomegaly pathogenesis remains elusive [3-5].
Irisin, a novel polypeptide hormone proteolytically processed from fibronectin type III domain-containing protein 5 (FNDC5), is an exercise- induced muscle-dependent myokine [8]. Irisin is expressed abundantly in the muscle, heart and adipose tissues [8, 9], and circulating irisin mainly derives from heart and skeletal muscles [10, 11]. Irisin binds to an unidentified receptor and plays an important role in metabolism by activating AMP-activated protein kinase (AMPK) [8, 9]. Recently, irisin was confirmed to be a multifunctional peptide involved in regulating cardiovascular function [8, 9], including potently dilating systemic and mesenteric
arteries in rat [12] and increasing cardiac diastolic volume, rate and output in zebrafish [13]. There is growing interest in the role of irisin in cardiovascular diseases [11-13]. Irisin supplementation protects against atherosclerosis [14], hypertension and myocardial ischemia/reperfusion injury [11, 15], which suggests that irisin could be an endogenous counter-regulatory mediator [8, 15]. However, little is known about the role of irisin in cardiac hypertrophy and failure.
Autophagy is the major intracellular degradation system by which cytoplasmic materials are delivered to and degraded in the lysosome [16]. Much evidence has revealed a close relation between cardiomyocyte autophagy and cardiac hypertrophy [17]. In the present study, we investigated the role of irisin in cardiac hypertrophy. Circulating and cardiac irisin content was markedly decreased in hypertrophic mice. Loss- and gain-of–function genetic manipulations in mice were used to test the effects of irisin on cardiac hypertrophy, injury and fibrosis. Irisin could protect against cardiac hypertrophy by directly activating AMPK-ULK1 signaling to induce protective autophagy and autophagy flux. Thus, irisin can be a novel therapeutic target for treating hypertensive diseases.
⦁ Materials and Methods
Materials and Methods are available in the online-only Data Supplement.
⦁ Animals, Determination of Cardiac Function and Histological study
FNDC5 homozygous knockout (FNDC5-KO) mice on a C57BL/6J background were created by using CRISPR/Cas- mediated genome engineering (Biocytogen Co, Beijing), and FNDC5 transgenic (FNDC5-Tg) mice on a C57BL/6J background were created by overexpressing human FNDC5 under the control of the chicken β-actin promoter and cytomegalovirus enhancer (Biomodel organism, Shanghai). Wild-type (WT) littermates were controls. The genotype identification of FNDC5 KO or Tg mice was based on PCR assay (Supplemental Data Fig.1A and 1B). Weight- matched male mice, 8 weeks old, received sham or transverse aortic constriction (TAC) surgery by tying the transverse aorta around a 26-gauge needle as described [18]. Tg mice with or without TAC received 10 mg/kg/day chloroquine (CQ) once a day by intraperitoneal injection [19]. After 4 weeks, mice underwent transthoracic echocardiography and haemodynamic measurements, then the hearts were removed and divided into three parts for RNA, protein extraction and histology to evaluate the hypertrophy, remodeling and autophagy levels [18, 20]. All experimental procedures were approved by the Animal Care and Use Committee of Sichuan University and performed in accordance with the Guidelines for Animal Experiments from the Committee of Medical Ethics, National Health Department of China.
⦁ Histology
Four weeks after TAC treatment, mice were euthanized by intraperitoneal injection with a lethal dosage of pentobarbital (at least 200 mg/kg). Hearts were arrested in diastole by injecting 0.2 mol/L KCl intravenously, then fixed with neutralized formaldehyde and cut into 4-μm sagittal or transverse sections after embedding in paraffin [20]. Sections were stained with rabbit polyclonal anti- mouse/human irisin/FNDC5 antibody, hematoxylin and eosin (H-E), Masson trichrome and wheat germ agglutinin (WGA) according to the manufacturer’s instructions [18, 20]. A Leica system (Leica Microsystems, Wetzlar, Germany) was used to capture digital images. Irisin/FNDC5 immunoreactivity in cardiomyocytes was visualized by using diaminobenzidine substrate, and cardiomyocyte size was measured on H-E– and WGA-stained sections. About 100-150 randomly chosen cardiomyocytes from each group (n=3–4) were analyzed by using Image J (NIH), and cross-sectional cardiomyocyte area was measured and presented in milliliters squared [18]. The amount of myocardial interstitial fibrosis was measured and calculated by using Image-Pro Plus (Media Cybernetics, USA) on Masson’s trichrome-stained sections [18]. About 30 to 50 randomly chosen frames from each group (n=3–4) were assayed, and the percentage fibrosis was expressed as the ratio of fibrotic area to total left ventricular (LV) area [18, 20].
⦁ Cardiomyocyte culture and treatment
Primary cardiomyocytes were cultured from hearts of 1- to 2-day-old neonatal
Sprague–Dawley rats as described [20] and pretreated with Ang II (1 µmol/L) or phenylephrine (PE, 100 nmol/L) alone or with irisin (20 nmol/L) for 48 h [18]. For inhibitor experiments, cells were pre-incubated with the mammalian target of rapamycin (mTOR) inhibitor Torin 1 (250 nmol/L), AMPK inhibitor compo und C (10 μmol/L), ULK1 inhibitor SBI-0206965 (5 µmol/L), autophagy inhibitor 3-MA (10 mmol/L) or CQ (50 nmol/L) for 30 min, then treated with Ang II or PE pre- incubated with irisin or normal saline. In a separate experiment, cardiomyocytes were transfected with Atg-5 siRNA (Thermo, USA) or control nonspecific siRNA oligonucleotides for 48 h, starved for another 24 h, then treated with Ang II (1 µmol/L) or PE (100 nmol/L) or in combination with irisin (20 nmol/L) for 48 h [18]. Cardiomyocyte size was measured in at least 100 cardiomyocytes in each group by using Image J after staining with WGA (5.0 µg/mL) for 30 min at 37°C to visualize the cytomembrane [18]. Protein/DNA ratios were calculated on the basis of protein content measured by the Lowry method and DNA content measured by a Hoechst 33258 dye fluorescent method [21].
⦁ Detection of immunoreactive irisin and insulin in cardiac tissues, plasma and cell culture supernatant
Secreted irisin and insulin was assayed by using ELISA kits (Phoenix Pharmaceutical, CA, USA for irisin, and Millipore Corp., MA, USA for insulin) according to the manufacturer’s instructions [22]. The reactivity with mouse, rat and human irisin was 100%. The secreted irisin was calculated and is expressed as
milligrams/liter in plasma, femtomoles/105 cells in supernatant, and pictograms per milligram protein for tissues, respectively. The plasma content of insulin is expressed as nanograms per milliliter.
⦁ Measurement of glucose, lactate dehydrogenase (LDH), creatine kinase (CK) and MB isoenzyme of CK (CK-MB) activities in plasma and cell culture supernatants
Collected plasma and cell culture supernatants were measured by use of a Roche Cobas 8000 Automatic Biochemical Analyzer at the West China Hospital with clinical grade reagents, and plasma glucose is presented as millimoles per liter; LDH, CK and CK-MB activity is presented as units per liter [20].
⦁ Electron microscopy and fluoresecence microscope imaging of autophagy
Cardiomyocytes receiving different treatment were embedded in 3% glutaraldehyde and examined by transmission electron microscopy (H-600, Hitachi, Japan) [23, 24]. Cardiomyocytes were seeded on glass coverslips and transfected with an mRFP-GFP-LC3B adenovirus construct following the manufacturer’s instructions [24]. The formation of fluorescent puncta was monitored under an inverted confocal microscope (A1RMP ＋ , NiKON, Japan). GFP-LC3 (green)- and mRFP-LC3
(red)-punctated dots were measured in autophagic cardiomyocytes by counting more
than 50 cells [24].
⦁ RT-PCR and Quantitative Real-Time PCR (qPCR)
Total RNA from different mouse tissues and cardiomyocytes were extracted and reverse transcribed into cDNA. The DNA targets were amplified with the use of primers (Supplementary Table 1) by RT-PCR and qPCR [20, 22]. Mouse and human
irisin, mouse BNP, APN, collagen I and III mRNA levels were normalized to GAPDH level.
⦁ Western blot analysis
Western blot analysis followed our previous method [18, 20]. The protein expression of LC3 I and II, caspase-3, P62/SQSTM1, AMPK, mTOR, ULK1, ERK, AKT, JNK, and Vps34 as well as the phosphorylation of p-AMPK, p-mTOR, p-ULK1 at Ser555 and Ser757, p-ERK, p-AKT at Thr308 and Ser473, p-JNK and p-Vps34 were determined in cardiac tissues and cardiomyocytes. β-actin was used for normalization [20].
⦁ Statistical analysis
Data are shown as mean±SEM from at least 3 independent experiments. Statistical analyses involved using GraphPad Prism 6 (Graphpad Software). Unpaired Student’s t-test (two tailed) was used for analysis of 2 groups and one or two-way ANOVA followed by Newman-Keuls multiple comparison test for 3 or more groups. P <0.05 was considered statistically significant.
⦁ RESULTS
⦁ Characteristics of irisin knockout (KO) and transgenic (Tg) mice
To address the role of irisin in cardiac diseases, we created FNDC5-Tg mice that overexpressed human FNDC5 under control of the chicken β-actin promoter and cytomegalovirus enhancer and also FNDC5-KO mice. Both Tg and KO mice appeared normal at birth, showed no evidence of growth retardation or growth acceleration, and had no apparent signs of any disorder. Body weights and activity levels were also within the normal range, with normal plasma glucose, insulin levels (Supplementary data Table 2 and Table 3) and ratios of left heart ventricle weight to
tibia length (LVW/TL) (Fig. 1A). The relative expression of irisin in different organs, including the heart, skeletal muscle, fat, liver, spleen, lung and kidney, was further evaluated by RT-PCR and real- time PCR. In WT mice, the relative mRNA expression of FNDC5 was highest in cardiac tissues and was rich in skeletal muscles, lungs and fat (Supplemental Data Fig. 1C and 1D). In FNDC5-Tg mice, the mRNA expression of exogenous human irisin was high in all tested tissues (Supplemental Data Fig. 1E and 1F), with no FNDC5 mRNA detected in tissues from FNDC5-KO mice (Supplemental Data Fig. 1C and 1D). Furthermore, as compared with WT mice, FNDC5-Tg mice showed increased content of immunoreactive irisin (including both human and mouse) in cardiac tissues and plasma, but irisin level was below the detection limit (approximately 0.1 ng/mL) in heart and plasma of FNDC5-KO mice (Fig. 1B and 1C). FNDC5 protein was further detected by immunohistochemical staining in heart sections. WT hearts showed sporadic, positive FNDC5
immunoreactive staining in cytoplasm of cardiomyocytes, which was much stronger in cardiomyocytes of FNDC5-Tg mouse hearts but almost absent in FNDC5-KO mouse hearts (Supplemental Data Fig. 2).
⦁ Irisin is downregulated by transverse aortic constriction (TAC), angiotensin II (Ang II), or phenylephrine (PE) treatment
In WT mouse hearts, FNDC5 mRNA was highly expressed and irisin content was 3.8±0.8 ng/mg protein in cardiac tissues and 152.3±39.8 µg/L in plasma (Fig. 1B and 1C). TAC treatment significantly decreased myocardial FNDC5 mRNA expression and cardiac and plasma irisin content in WT mice and FNDC5-Tg mice (Fig. 1B-1E). In cultured neonatal rat cardiomyocytes (NRCMs), 48-h exposure to Ang II (1 μM) and PE (100 nM) significantly reduced FNDC5 mRNA expression and its secretion in supernatant (Fig. 1F and 1G). Furthermore, the cardiac mRNA expression of FNDC4 (another secreted factor sharing high homology with irisin) did not differ among FNDC5-KO, WT and FNDC5-Tg mice under normal or hypertrophic conditions (Fig. 1H).
⦁ Irisin regulates left ventricular (LV) hypertrophy, remodeling and damage induced by pressure overload in vivo
Progressive dilatation of the hypertrophic left ventricle leads to depressed LV contractility and myocardial structural changes, including cellular hypertrophy, damage and interstitial fibrosis [1, 2]. Four-week TAC treatment induced a distinct
hypertrophic phenotype in WT hearts, with a significant increase in LV weight/tibia length (LVW/TL) ratio (Fig. 1A), cross-sectional area of cardiomyocytes (Fig. 2A-2C), interstitial fibrosis (Fig. 2A, 2B and 2D), mRNA levels of hypertrophy markers including brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP) (Fig. 2E), mRNA levels of collagen I and III (Fig. 2F) and plasma activity of lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase-MB (CK-MB) (Supplementary data Fig. 3). Values for these indicators of cardiac hypertrophy, fibrosis and injury were significantly higher for TAC-treated FNDC5-KO mice and significantly lower for TAC-treated FNDC5-Tg mice than hypertrophic WT mice (Fig. 1A, 2A-2F, and Supplementary data Fig. 3).
⦁ Irisin regulates pressure overload-induced LV dysfunction
Cardiac hypertrophy and remodeling are characterized by an increase in LV mass and a decrease in cardiac function [1, 2]. At baseline, FNDC5-KO, WT and FNDC5-Tg mice did not differ in echocardiographic and hemodynamic parameters (Fig. 2G, 2H and Supplementary data Table 4). As compared with hypertrophic WT
mice, for TAC-treated FNDC5-KO mice, the echocardiographic LV mass was significantly increased and was significantly reduced in TAC-treated FNDC5-Tg mice (Fig. 2G, 2H and Supplementary data Table 4). Values for the other parameters of LV
remodeling, including LV posterior wall thickness at end-diastole (LVPWTd) and LV
end-diastolic volume (LVEDV) as well as the LV preload index LV end-diastolic pressure (LVEDP), were significantly higher in TAC-treated FNDC5-KO mice but
lower in TAC-treated FNDC5-Tg mice than in hypertrophic WT mice (Fig. 2G, 2H and Supplementary data Table 4). The pressure-volume derived cardiac contractility
indices, including LV pressure (LVP), LV end-systolic pressure (LVESP) and ±dP/dt, were significantly lower in TAC-treated FNDC5-KO mice but higher in TAC-treated FNDC5-Tg mice than in TAC-treated WT mice (Fig. 2G, 2H and Supplementary data
Table 4). Consequently, the stroke volume (SV), cardiac output (CO), ejection fraction
(EF) and fractional shortening (FS) were lower in TAC-treated FNDC5-KO mice but higher in TAC-treated FNDC5-Tg mice than in hypertrophic WT mice (Fig. 2G, 2H and Supplementary data Table 4).
⦁ Irisin attenuates hypertrophic response and damage in cultured cardiomyocytes
We further evaluated irisin’s regulation of cardiac myocyte hypertrophy in primary cultured NRCMs. As compared with control cells, in NRCMs, 48 h Ang-II or PE exposure significantly increased cellular cross-sectional area (Fig. 3A and 3B), protein/DNA ratio (Fig. 3C), BNP and ANP mRNA levels (Fig. 3D), and activity of LDH, CK and CK-MB in culture supernatants (Supplementary data Fig. 4A-4C). Furthermore, irisin supplementation markedly decreased these cellular hypertrophic and damage indicators in Ang-II– or PE-treated cells (Fig. 3A-3D, Supplementary data Fig. 4A-4C).
⦁ Irisin activated protective autophagy against pressure overload-induced cardiac hypertrophy and damage
Autophagy plays an important role in cardiac hypertrophy and the transition from hypertrophy to heart failure, and impaired autophagic flux has been linked to various cardiac pathophysiological processes [16, 17]. LC3-II protein level is a reliable marker of autophagosomes [25]. p62 is selectively degraded by autophagy and can be used to monitor autophagy flux, usually defined as a measure of autophagic degradation activity [25]. As shown in Fig. 4A, TAC treatment induced a significant accumulation of both LC3-II and p62 protein in WT mouse hearts. As compared with hypertrophic WT mice, TAC-treated FNDC5-KO hearts showed significantly lower cardiac LC3-II levels but higher p62 levels and TAC-treated FNDC5-Tg hearts showed significantly higher cardiac LC3-II levels but lower p62 levels.
TAC-treated FNDC5-Tg mice were further supplemented with chloroquine (CQ)
to investigate the role of irisin- induced autophagy in cardiac hypertrophy by inhibiting the late phase autophagy [25]. As shown in Fig. 4A, in TAC-treated FNDC5-Tg mice, CQ treatment induced a significant accumulation of both LC3-II and p62 in hearts and almost abrogated the cardioprotection of irisin by inducing a marked deterioration in pathological hypertrophy, thereby significantly increasing LVW/TL ratio (Fig. 1A), myocyte cross-sectional area (Fig. 2A-2C), fibrosis (Fig. 2D), BNP and ANP mRNA levels (Fig. 2E) and plasma myocardial enzyme activity (Supplementary data Fig. 3) as well as a decrease in cardiac function (Fig. 2G, 2H and Supplementary data Table 4).
In cultured primary NRCMs, 48-h Ang II (1 μM) or PE (100 nM) induced a significant accumulation of both LC3-II and p62 (Fig. 4B and 4C), accompanied by a significantly hypertrophic response and damage (Fig. 3A-3D, Supplementary data Fig. 4A-4C). Irisin supplementation in Ang II- or PE-treated cells increased LC3-II protein level and significantly decreased p62 accumulation (Fig. 4B and 4C), accompanied by a significant attenuation of myocyte hypertrophy and damage (Fig. 3A-3D, Supplementary data Fig. 4A-4C). Different autophagy inhibitors, including Atg5 siRNA, 3-MA and CQ, pre- and co-treated to suppress irisin-activated autophagy (Fig. 4B and 4C), significantly blunted the cardioprotection of irisin in Ang II- or PE-treated NRCMs, with a significant increase in myocyte hypertrophy and damage (Fig. 3A-3D, and Supplementary data Fig. 4A -4C).
Electron microscopy and ultrastructure analysis were used to detect intercellular autophagosomes and autolysosomes in cultured NRCMs. Autophagosomes have been confirmed to be autophagic vacuoles with a typical double- layer membrane containing organelle remnants, and autolysosomes are encircled by single line and recognized by an autophagic vacuole that contain electron-dense contents [24, 25]. Control cells showed few autophagosomes and autolysosomes (Fig. 5A). Ang II or PE exposure induced a few autophagosomes but did not significantly affect autolysosomes in NRCMs (Fig. 5A). Irisin (20 nM) pre- and co- incubation significantly increased both autophagosomes and autolysosomes number in Ang II- or PE-treated cells (Fig. 5A).
LC3 conversion and turnover assays were further performed by using 20 μM CQ
to inhibit autophagic flux [25]. Supplementary data Fig. 5C shows that CQ pre- and co-incubation induced more LC3 II and p62 accumulation in NRCMs with irisin treatment alone than in control cells, which suggests that irisin increased autophagic flux. The difference in LC3-II and p62 levels between Ang II- or PE-treated cardiomyocytes with and without CQ was further compared with and without irisin supplementation [25]. CQ pre- and co- incubation induced more LC3-II and p62 accumulation in irisin-supplemented Ang II- or PE-treated NRCMs than in cells treated with Ang II or PE alone (Fig. 5B), which suggests that irisin improved the impaired autophagic flux in hypertrophic cardiomyocytes.
Next, a tandem fluorescence RFP-GFP-LC3 reporter system introduced by adeno-associated virus infection was used as an additional monitor of autophagic flux [24, 25]. RFP fluorescence is stable in the acidic lysosomal compartment, and GFP fluorescence is quenched in an acidic environment [24, 25]. Thus, flux can be detected directly by distinguishing yellow autophagosomes from red autolysosomes [24]. Ang II or PE alone increased autophagosome number but not autolysosome number in NRCMs (Fig. 5C). Irisin supplementation significantly increased both autophagosome and autolysosome numbers in Ang II- or PE-treated cardiomyocytes, whereas CQ pre- and co- incubation significantly increased autophagosome accumulation in irisin-supplemented hypertrophic cardiomyocytes, with no detectable autolysosomes (Fig. 5C).
⦁ Irisin activates autophagic flux via AMPK-ULK1–dependent but mTOR-independent signaling
ULK1 initiates autophagy and can be regulated by AMPK and mTOR via direct phosphorylation at Ser555 and Ser757, respectively [26-28]. AMPK, PI3K-Akt and MAPK can transduce the cellular actions of irisin [8, 9]. We investigated the potential regulation of these Ser/Thr protein kinases on irisin- induced autophagy by evaluating their phosphorylation in mouse hearts. Sham FNDC5-Tg hearts showed a higher degree of cardiac baseline phosphorylation of AMPK, p38, ERK, AKT at Ser308 and ULK1 at Ser555 than sham WT hearts (Fig. 6A). In WT mice, TAC treatment induced higher cardiac protein phosphorylation of AMPK, ERK, AKT at Ser308, ULK1 at both Ser555 and Ser757, and mTOR and its downstream target S6K (Fig. 6A). However, for TAC-treated FNDC5-KO hearts, only cardiac AMPK phosphorylation was lower, and TAC-treated FNDC5-Tg hearts showed higher phosphorylation of both AMPK and ULK1 at Ser555 (Fig. 6A). In cultured NRCMs, Ang II, PE or irisin alone rapidly increased the activity of AMPK and MAPK family members (p38, JNK and ERK), which persisted for at least 10 min (Fig. 6B and 6C). Irisin supplementation augmented only AMPK phosphorylation but did not affect the activity of p38, ERK, JNK, or AKT in Ang II- or PE-treated cells (Fig. 6B and 6C).
We further investigated the role of AMPK, mTOR and ULK1 in the regulation of irisin on autophagy in Ang II- or PE- treated NRCMs. Ang II or PE alone significantly activated mTOR, p70S6K and ULK1 at both Ser555 and Ser757 and caused both LC3 II and p62 accumulation (Fig. 7A and 7B). Irisin supplementation significantly
increased the phosphorylation of AMPK and ULK1 at Ser555 but not Ser757 and had no effect on mTOR and p70S6K in Ang II- or PE-treated cells. Irisin also significantly increased LC3 II level and decreased p62 level (Fig. 7A and 7B). We further used Torin1, a potent and selective ATP-competitive inhibitor of mTOR [29], to reveal whether the mTOR pathway mediated the regulation of irisin on autophagy. Torin1 (250 nM) suppressed the phosphorylation of mTOR, p70S6K and ULK1 at Ser757 but had no effect on irisin- induced activation of both ULK1 at Ser555 and autophagy or irisin- induced reduction of p62 levels with the absence and presence of Ang II or PE (Fig. 7A and 7B).
AMPK lies upstream of ULK1, and the regulation of AMPK on ULK1 is required for proper autophagy [26-28]. A special AMPK inhibitor, compound C [28], and a special ULK1 inhibitor, SBI-0206965 [30], were used to investigate the role of AMPK-ULK1 signaling in mediating the regulation of irisin on autophagy. Compound C (10 µM) almost abrogated the phosphorylation of both AMPK and ULK1 at Ser555, and SBI-0206965 (5 µM) significantly suppressed the phosphorylation of VPS34 (an autophagy component downstream of ULK1) [30] in both irisin and Ang II- or PE-treated cells (Fig. 7C-7F). Both findings were accompanied by a significant decrease in LC3 II level and increase in p62 level (Fig. 7C-7F). Both compound C and SBI-0206965 almost abolished the protection of irisin against Ang II- or PE- induced NRCM hypertrophy (Fig. 8A-8E) and damage (Supplementary data Fig. 6A-6C). Tandem fluorescence RFP-GFP-LC3 reporter-system assay confirmed that both compound C and SBI-0206965
significantly decreased both the autophagosome and autolysosome numbers in NRCMs co-incubated with irisin and Ang II or PE (Fig. 8F).
⦁ DISCUSSION
Irisin is encoded in its precursor FNDC5 [8, 9]. Cardiac tissues of mice were the richest sources of irisin in our study, and irisin was downregulated in both cardiac tissues and plasma of mice challenged with pressure overloading and in Ang II- or PE-treated cardiomyocytes. This finding is comparable to results reported by Kuloglu et al. and Aydin et al. [31, 32], showing strong immunoreactivity of irisin in cardiac muscle tissues [31] and decreased irisin level in ischemic cardiac tissues and plasma of rats treated with isoproterenol [31] and in saliva/serum of patients with acute myocardial infarction [32]. Hence, irisin is a new autocrine/paracrine mediator in myocardial tissues [11-13]. Aydin et al. further confirmed that cardiac tissue is the main irisin source rather than skeletal muscles [33], so loss of irisin-secreting cardiomyocytes after hypertrophic or ischemic stress may contribute in part to the decrease in irisin level [31-33]. In addition, an essential role of irisin/FNDC5 in cardiac hypertrophy was further recognized by the unchanged cardiac expression of FNDC4, a highly identical ortholog of irisin [34], in response to pressure overload.
Persistent pressure overload induced by systemic hypertension or TAC triggers cardiac hypertrophy and fibrosis; increasing collagen deposition contributes to the deterioration of LV compliance and the development of diastolic dysfunction [1, 2]. The present study is the first to conclusively demonstrate that irisin is critical in the development of LV remodeling and failure. We observed that FNDC5 ablation induced a heart failure phenotype in TAC-treated mice, with significant exacerbations in cardiac eccentric hypertrophy, fibrosis, injury and dysfunction; however, FNDC5
overexpression significantly attenuated these pressure overload- induced pathological changes. These in vivo observations were further supported by studies of cultured NRVMs, in which irisin supplementation significantly inhibited Ang II- or PE- induced hypertrophy and injury. Together, these data provide conclusive evidence that like other putative protective factors such as ANP [6], adrenomedullin and adiponectin [7, 35], irisin is an essential mediator in pressure overload- induced cardiac hypertrophy.
Pressure overload induced strong myocardial autophagy in TAC-induced animal
models [16, 17]. The study conducted by Hill et al. revealed significant increases in cardiac autophagosomes in mice [36]. As well, 4-week TAC treatment induced significant LC3-II accumulation in the mouse myocardium. This finding was further verified by in vitro NRVM studies showing that Ang II or PE induced significant accumulation of autophagosomes. These results suggest that pressure overload induces autophagosome accumulation in hearts, which parallels the pathological changes and dysfunction in the LV. We also inhibited TAC-induced autophagy in WT mice by intraperitoneal injection of CQ (10 mg/kg/day) once a day for 4 weeks and observed a significant deterioration in cardiac hypertrophic pathological changes and function as compared with WT mice treated with TAC alone. The treatment significantly increased LVW/TL ratio, cross-sectional area of cardiomyocytes, interstitial fibrosis, mRNA levels of BNP and ANP, and plasma myocardial enzymatic activity (data not shown). However, CQ treatment alone in WT mice had no obvious effect on cardiac morphology and function (data not shown). These results confirm
that under pressure-overload conditions, the inhibition of autophagy further aggravated TAC-induced cardiac hypertrophy and cardiomyocyte death. Growing preclinical evidence suggests that autophagy is a double-edged sword in cardiovascular diseases, acting in beneficial or maladaptive ways depending on the context [26, 36]. Hill et al. concluded that cardiomyocyte autophagy was a maladaptive response from findings that beclin-1 knockout attenuated but beclin-1 overexpression aggravated TAC-induced cardiac hypertrophy [36]. In fact, cardiac-specific deficiency of Atg5 in mice or β-adrenergic stimulation facilitates myocardial hypertrophy, whereas rapamycin- induced autophagic activation can prevent it, which suggests that the increased autophagy plays a protective role [37, 38]. Xue et al. observed that DJ-1, a traditional anti-oxidative protein, repressed both pressure overload- and PE- induced cardiac hypertrophy by activating autophagy [39]. We revealed that irisin protected against cardiac hypertrophy by inducing protective autophagy in the hypertrophic mouse myocardium. In response to hypertrophic stimuli, irisin ablation induced lower LC3 II level but irisin-overexpression/supplementation led to higher LC3 II level than in controls. Inhibition of irisin- induced autophagy with CQ almost abolished the protection of irisin in hypertrophic FNDC5-Tg mouse hearts and hypertrophic NRVMs. Similar effects were observed with other autophagy inhibitors, including Atg5 siRNA and 3-MA. These results were comparable to those reported by Gu et al., finding that the mTOR inhibitor rapamycin attenuated TAC-induced cardiac hypertrophy by promoting autophagy [40], and by Nakai et al., finding that upregulation of autophagy
was an adaptive response in TAC-induced failing hearts [37].
Rapidly increasing evidence has shown that chronic pressure overload leads to insufficiency in myocardial autophagy flux and contributes to maladaptive cardiac remodeling and heart failure [26, 36]. In the present study, 4-week TAC treatment significantly reduced autophagy flux in hypertrophic hearts, as confirmed by a significant increase in p62 level, the selective autophagy substrate. Ang II or PE also significantly increased autophagosome number rather than autophagolysosome number in NRVMs, which was further verified by the accumulation of both LC3 II and p62. Similar results were reported by Oka et al., finding that insufficient lysosomal removal of autophagosomes was detrimental to cardiomyocytes [41]. Furthermore, Li et al. reported that blocking autophagic flux by doxorubicin induced cardiotoxicity [24]. These results indicate that chronic pressure overload leads to autophagy flux insufficiency and the accumulation of autophagic vacuoles is maladaptive and contributes to cardiac hypertrophy [16, 26, 41]. Thus, cardiac hypertrophy could be reversed by alleviating the impaired autophagy flux [16, 26].
The present study is the first to reveal that irisin improved autophagy flux in hypertrophic mouse hearts, with irisin ablation inducing the accumulation of p62 and irisin overexpression decreasing p62 level. Irisin also significantly improved autophagy flux in hypertrophic NRVMs, with a significant decrease in p62 level as well as increase in both autophagosomeand autophagolysosome numbers, which was reversed in CQ-mediated LC3 turnover assays. Our data suggest that irisin protects against cardiac hypertrophy and heart failure induced by persist pressure overload via
protective autophagy induction and autophagy flux improvement. In addition, pathological cardiac hypertrophy has complex pathophysiological changes, including suppressed angiogenesis, vascular rarefaction and endothelial dysfunction. Wu et al. observed that irisin could induce endothelial cell angiogenesis [42] and Fu et al. observed that irisin improved endothelial function [15], which suggests that irisin might antagonize cardiac hypertrophy in part by its effect on endothelium, which needs further study.
AMPK, PI3K/Akt and MAPK family members (ERK, p38 and JNK) mediate the
intracellular signaling cascades of irisin [8, 9]. The present study revealed that irisin overexpression activated AMPK, p38, ERK and AKT at Ser308 in mouse hearts, whereas exogenous irisin activated AMPK and all three MAPK family members in NRVMs. A reasonable explanation for these differences is that as compared with the simple culture environment for cells in vitro, cells in hearts live in a complex homeostasis rich in neurohumoral factors [3, 4]. Of note, irisin augmented the phosphorylation of only AMPK in TAC-, Ang II- or PE-challenged hypertrophic cardiomyocytes but had no significant effect on the other kinases. Hence, the AMPK signaling pathway mediates the protection of irisin in hypertrophic hearts.
AMPK, a ubiquitous sensor of cellular energy status, is activated in response to stresses [28]. Our findings suggest that irisin activated the AMPK pathway in hypertrophic cardiomyocytes. This observation is similar to results from Li et al. [43], finding that AMPK protected against pressure overload- induced cardiac hypertrophy by promoting autophagy via the mTOR Complex 1 (mTORC1) pathway. AMPK
activates autophagy by two different pathways: inactivating mTORC1 or directly phosphorylating a protein kinase, ULK1 [28]. ULK1 is an ortholog of yeast Atg1 [28]. AMPK phosphorylates ULK1 at Ser467 and Ser555 to initiate autophagy, whereas mTORC1 negatively regulates ULK1 via the phosphorylation of ULK1 at Ser757 to inhibit autophagy [26, 28]. The present study confirmed that irisin activated autophagy independent of Torin-1, an inhibitor of mTOR, to phosphorylate ULK1 at Ser555 but not Ser757, with an increase in LC3-II level and decrease in p62 level. However, irisin had no effect on mTOR and its downstream effector S6K in the presence and absence of hypertrophic stimuli. These results suggest that irisin activates autophagy and autophagy flux independent of mTOR. Furthermore, blocking AMPK and ULK1 with compund C and SBI-0206965, respectively, both significantly abrogated the anti- hypertrophic actions of irisin, thereby leading to autophagy dysfunction, as indicated by a decrease in LC3 II level and both autophagosome and autophagolysosome numbers and an increase in p62 level. These results suggest that irisin induces autophagy and autophagy flux via AMPK-ULK1 signaling.
Limitations
Our study focused on cardiac hypertrophy in male mice, but not females. Growing
evidence suggests that female gender confers protection from the development of
pressure overload- induced cardiac hypertrophy by inhibiting apoptosis and
hypertrophy, both of which contribute to progressive LV dilation and dysfunction [44].
In adults, as a proposed exercise hormone, irisin levels were higher in men than in
women [45]. In fact, the different physiology of women (including hormones) relative
to men may likely contribute to gender-specific autophagy regulation [46]. Thus, the
further study of irisin- induced autophagic pathways and sex differences appears
pivotal in the development of new and more appropriate anti-hypertrophic strategies
[8, 9, 46].
Conclusions
Our study reveals the unique mechanism of irisin protecting against caridac hypertrophy by inducing protective autopahgy and improving chronic pressure overload- impaired autophagy flux via activating AMPK-ULK1 signaling independent of mTOR-S6K (Fig. 8G). These findings provide definitive evidence that irisin signaling is critically important for the development of LV hypertrophy, remodeling and dysfunction during pressure overload. They also identify the heretofore unknown role of autophagy and autopahgy flux in irisin- induced AMPK activation and cardiomyocyte hypertrophy. These novel insights may be used to develop new therapeutic approaches to ameliorate hypertensive heart diseases.
Acknowledgements:
This work was supported by the National Natural Science Foundation of China (grant nos. 81670249, 31271226 and 31071001 to Dr. Wei Jiang and no. 81700229 to Dr.
Xiao-Xiao Wang).
Conflict of interest:
Potential conflicts of interest: NONE.
Disclaimers: NONE.
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Highlights
⦁ Irisin is highly expressed in mouse hearts.
⦁ Circulating and cardiac irisin content was decreased significantly in hypertrophic mice with 4-week transverse aortic constriction.
⦁ FNDC5 deletion aggravated and FNDC5 overexpression ameliorated the left ventricle hypertrophy, remodeling, injury and dysfunction induced by pressure overload.
⦁ Irisin supplementation attenuated hypertrophy in Ang II– or phenylephrine-induced cardiomyocytes.
⦁ Irisin alleviated cardiomyocyte hypertrophy in vivo and in vitro by inducing beneficial autophagy and autophagy influx via AMPK-ULK1 signaling but independent of mTOR.
Figure Legends
Figure 1. Changes in ratio of LVW/TL, FNDC5/irisin expression (mRNA and content) and FNDC4 mRNA level in cardiac tissues and plasma of WT, irisin knockout and irisin Tg mice challenged with 4-week TAC, as well as irisin mRNA level in cultured NRVMs exposed to Ang II or PE.
A. Change in LVW/TL ratio in WT, irisin-knockout and irisin-transgenic (Tg) mice challenged with 4-week TAC alone or in combination with intraperitoneal injection of CQ (10 mg/Kg/day) once a day (n=10/group).
B and C. Change in content of total irisin (including both human and mouse) in cardiac tissues and plasma of 4-week TAC-treated WT, irisin-knockout and irisin- Tg mice (n=6/group);
D and E. Change in mouse and human FNDC5/irisin mRNA expression in cardiac tissues of WT, irisin-knockout and irisin-Tg mice with 4-week TAC treatment (n=6/group);
F and G. Change in FNDC5/irisin mRNA level in cultured NRVMs and irisin content in supernatant exposed to 1 µM Ang II or 100 nM PE for 48 h (n=6/group);
H. Change in mouse FNDC4 mRNA expression in cardiac tissues of 4-week TAC-treated WT, irisin-knockout and irisin-Tg mice (n=6/group).
All data were analyzed by one-way ANOVA. *p<0.05; **P<0.01. LVW/TL, left heart ventricle weight to tibia length; WT, wild-type mice; KO, FNDC5-knockout mice; Tg, human FNDC5-overexpressing mice; TAC, transverse aortic constriction; NRVM, neonatal rat ventricular myocyte; PE, phenylephrine.
Figure 2. Irisin protected against 4-week TAC- induced cardiac hypertrophy, fibrosis and dysfunction.
⦁ Representative images of gross, sagittal (H-E) and coronal (H-E and Masson’s trichrome) sections of WT, FNDC5-KO and FNDC5-Tg mouse hearts 4 weeks after TAC treatment or combined with CQ, Scale bars=1 mm
⦁ Representative views of hematoxylin and eosin (H-E), wheat germ agglutinin (WGA) and Masson trichrome-stained cross sections (all×400, scale bars =50 μm) exhibiting cross-sectional cardiomyocyte area (top and second row panels), interstitial (third row panel) and perivascular (bottom panel) fibrosis for WT, FNDC5-KO and FNDC5-Tg mouse hearts 4 weeks after TAC treatment or combined with CQ.
C and D. Quantitative analysis of cardiomyocyte cross-sectional area with measurements of 100-150 cardiomyocytes from 6 mice per group, fibrotic area with normalizing blue Masson trichrome–stained area to total myocardial area from 30–50 randomly chosen frames from 4 mice per group (n=6/group);
⦁ Change in mRNA levels of BNP and ANP in cardiac tissues of WT, FNDC5-KO and FNDC5-Tg mice 4 weeks after TAC treatment or combined with CQ (n=6/group);
⦁ Change in mRNA levels of collagen types I and III α1 in cardiac tissues of WT, FNDC5-KO and FNDC5-Tg mice 4 weeks after TAC treatment or combined with CQ (n=6/group).
⦁ Effect of irisin ablation, overexpression and CQ (10 mg/Kg/day, ip.) on left ventricular remodeling 4 weeks after TAC treatment. Representative M-mode frames from the mid-papillary region of sham WT mice (a), irisin KO mice (c), irisin Tg
mice (e) and CQ-treated irisin Tg mice (g), as well as hypertrophic WT mice (b), irisin KO mice (d), irisin Tg mice (f) and CQ-treated irisin Tg mice (h) 4 weeks after TAC treatment. Yellow arrow indicates width of the LV chamber.
⦁ Change in LV mass, LVPWT, EF%, FS%, Max dP/dt and Min dP/dt of WT, FNDC5-KO and FNDC5-Tg mouse hearts 4 weeks after TAC treatment or combined with CQ (n=12/group).
All data were analyzed by one-way ANOVA. *p<0.05; **P<0.01. WT, wild type mice; KO, FNDC5-knockout mice; Tg, human FNDC5-overexpressing mice; TAC, transverse aortic constriction; CQ, chloroquine ; ip., intraperitoneal; LVPWd, left ventricle (LV) posterior wall dimension; EF%, ejection fraction calculated by using the Teichholz method; FS%, Left ventricular fractional shortening %; Max dP/dt, maximal value of the first derivative of LV pressure; Min dP/dt, minimal value of the first derivative of LV pressure.
Figure 3. Exogenous irisin supplementation inhibited Ang II- or PE- induced NRVM hypertrophy.
⦁ Representative WGA-Alexa 488 stained photomicrographs of NRVMs challenged with 1 μM Ang II or 100 nM PE without or with 20 nM irisin or combining irisin with autophagy inhibitors Atg 5 siRN A, 3-MA (10 mM) or CQ (50 nM) for 48 h, then cell area was assayed with Alexa Fluor 488-conjugated WGA staining. Scale bars =50 μm. Original magnifications, ×400.
⦁ Quantitative analysis of NRVM size with measurements of ≥50 NRVMs per group after Ang II- or PE treatment; data are presented as fold change relative to control
cells.
⦁ Change in protein/DNA ratio of NRVMs with Ang II or PE exposure without or with irisin, or combining irisin and the three different autopahgy inhibitors for 48 h (n=6/group).
⦁ Change in mRNA levels of BNP and ANP in NRVMs after Ang II- or PE exposure alone, or combined with irisin, or irisin with the three different autopahgy inhib itors for 48 h (n=6/group).
All data were analyzed by one-way ANOVA. *p<0.05; **P<0.01. NRVM, neonatal rat ventricular myocyte; WGA, Alexa Fluor 488-conjugated WGA stain; siCtr, control siRNA; 3-MA, 3- methyladenine; CQ, chloroquine; A, angiotensin II; I, irisin; PE, phenylephrine.
Figure 4. Irisin augmented autophagy and improved autophagy flux in TAC-treated cardiac tissues and in Ang II- or PE-insulted cultured NRVMs.
A. LC3I, LC3-II and p62 levels were detected by immunoblotting in cardiac tissues of WT, FNDC5-KO and FNDC5-Tg mice 4 weeks after TAC treatment or combined with CQ (n=5/group).
B and C. LC3I, LC3-II and p62 levels were evaluated by immunoblotting in NRVMs with Ang II (1 μM) or PE (100 nM) exposure alone, or combined with irisin, or irisin with the different autophagy inhibitors Atg 5 siRNA, 3-MA (10 mM) or CQ (50 nM) for 48 h (n=6/group).
All data were analyzed by one-way ANOVA. *P<0.05, **P<0.01. WT, wild type mice; KO, FNDC5-KO mice; Tg, human FNDC5-overexpressing mice; TAC, transverse
aortic constriction; NRVM, neonatal rat ventricular myocyte; A, angiotensin II; I, irisin; PE, phenylephrine; siCtr, control siRNA; 3-MA, 3- methyladenine; CQ, chloroquine.
Figure 5. Irisin improved autophagy flux suppressed by Ang II or PE exposure in cultured NRVMs.
⦁ Representative transmission electron microscopy images and histograms of NRVMs treated with Ang II (1 µM) or PE (100 nM) without or with irisin (20 nM) for 48 h (n=6/group). Arrows, autophagosome; Arrowhead, lysosomes/autolysosomes,
containing electron-dense contents. Scale bars, 1 µm in top panel and 0.5 µm in bottom panel images.
⦁ LC3 turnover assay, NRVMs were treated with Ang II (1 µM) or PE (100 nM) in the absence and presence of irisin (20 nM), with or without CQ (20 µM) for 48 h. At the end of treatment, cell lysates were examined by immunoblotting to detect LC3I,
LC3-II and p62 levels (n=6/group).
⦁ Representative fluorescence images of NRVM expressing RFP-GFP-LC3 and treated with Ang II (1 µM) or PE (100 nM) alone, or combined with irisin (20 nM), or irisin with the autophagy inhibitor CQ (50 nM) for 48 h. Number of autophagosomes and autolysosomes in each cell was quantified. n= 60 cells per group. Scale bar= 20
µm.
All data were analyzed by two-way ANOVA. *p<0.05; **P<0.01. NRVM, neonatal rat ventricular myocyte; A, angiotensin II; I, irisin; PE, phenylephrine; 3-MA, 3-methyladenine; CQ, chloroquine.
Figure 6. Effect of irisin deficiency, overexpression/supplementation on activity of serine/threonine protein kinases in hypertrophic myocardial tissues or NRVMs.
A. Lysates from cardiac tissues treated with 4-week TAC were analyzed by immunoblotting. Phosphorylation and expression of AMPK, p38, JNK, ERK, AKT, ULK1, mTOR, and p70/85-S6K were determined by using specific antibodies. Irisin only significantly augmented the phosphorylation of AMPK and ULK1 at Ser555 in TAC-treated FNDC5-Tg mouse hearts (n=5/group).
B and C. Effect of irisin on phosphorylation and expression of AMPK, p38, JNK, ERK and AKT induced by Ang II (1 µM) or PE (100 nM) exposure and analyzed by immunoblotting. NRVMs were treated with irisin (20 nM), Ang II (1 μM) or PE (100
nM) alone or irisin combined with Ang II or PE for 5, 10 and 15 min. Irisin induced a significant augmentation of the phosphorylation of only AMPK and ULK1 at Ser555 in Ang II or PE-treated NRVMs (n=5/group).
All data were analyzed by one-way ANOVA. *p<0.05; **P<0.01. WT, wild type mice; KO, FNDC5-KO mice; Tg, human FNDC5-overexpressing mice; TAC, transverse aortic constriction; NRVM, neonatal rat ventricular myocyte; PE, phenylephrine.
Figure 7. Irisin activated ULK1 dependent on AMPK but not mTOR.
A and B. Immunoblot analysis of phosphorylation and expression of mTOR, p70/85-S6K, ULK1, p62 and LC3II in NRVMs incubated with irisin (20 nM), Ang II (1 μM) or PE (100 nM) alone or irisin combined with Ang II or PE without or with Torin1 (250 nM) for 48 h (n=5/group).
C and D. Phosphorylation and expression of AMPK, ULK1, p62 and LC3II analyzed
by immunoblotting in NRVMs incubated with irisin (20 nM), Ang II (1 μM) or PE (100 nM) alone or irisin combined with Ang II or PE without or with the AMPK inhibitor compound C (10 μM) for 48 h (n=5/group).
E and F. Phosphorylation and expression of Vps34, p62 and LC3II analyzed by immunoblotting in NRVMs incubated with irisin (20 nM), Ang II (1μM) or PE (100 nM) alone or irisin combined with Ang II or PE without or with the ULK1 inhibitor SBI-0206965 (5 μM) for 48 h (n=5/group).
All data were analyzed by one-way ANOVA. *p<0.05; **P<0.01. NRVM, neonatal rat ventricular myocyte; PE, phenylephrine.
Figure 8. Irisin protected NRVMs against Ang II- or PE-induced hypertrophy by activating autophagy and autophagy flux through AMPK-ULK1 signaling.
⦁ Representative WGA-Alexa 488 stained photomicrographs of NRVMs challenged with 1 μM Ang II or 100 nM PE without or with 20 nM irisin or combining irisin with an AMPK inhibitor compound C (10 μM) or an ULK1 inhibitor SBI-0206965 (5 µM) for 48 h, then cell area was assayed with Alexa Fluor 488-conjugated WGA staining. Scale bars =50 μm. Original magnifications, ×400.
⦁ Quantitative analysis of NRVM size with measurements of ≥50 NRVMs per group after Ang II- or PE treatment; data are presented as fold change relative to control cells.
⦁ Change in protein/DNA ratio of NRVMs with Ang II or PE exposure without or with irisin, or combining irisin and compound C or SBI-0206965 for 48 h (n=6/group).
D and E. Change in mRNA levels of BNP and ANP in NRVMs after Ang II- or PE exposure alone, or combined with irisin, or irisin with compound C or SBI-0206965 for 48 h (n=6/group).
F. Representative fluorescence images of NRVM expressing RFP-GFP-LC3 and treated with Ang II (1 µM) or PE (100 nM) alone, or combined with irisin (20 nM), or irisin with AMPK inhibitor compound C (10 μM) or ULK1 inhibitor SBI-0206965 (5 µM) for 48 h. Number of autophagosomes and autolysosomes in each cell was quantified. n= 60 cells per group. Scale bar= 20 µm.
G. The schematic diagram summarizing the signaling pathways involved in irisin blunting pressure overload-induced cardiac hypertrophy. During hypertrophic conditions, autophagy flux is suppressed, and the accumulation of excessive autophagosomes is harmful to cardiomyocytes and contributes to the initiation and progression of cardiac hypertrophy. Irisin protects against cardiac hypertrophy by inducing protective autophagy and improving autophagy flux dependent on AMPK but not mTOR.
Data in B, C, D and E were analyzed by one-way ANOVA and in F were analyzed by two-way ANOVA. *p<0.05; **P<0.01. NRVM, neonatal rat ventricular myocyte; WGA, Alexa Fluor 488-conjugated WGA stain; CON, control; CC, compound C; SBI, SBI-0206965; A, angiotensin II; I, irisin; P, phenylephrine.
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