Adiponectin up‐regulates the decrease of myocardial autophagic flux induced by β1‐adrenergic receptor autoantibody partly dependent on AMPK

Abstract Cardiomyocytes autophagy is essential for maintaining cardiac function. Our previous studies have found that β1‐adrenergic receptor autoantibody (β1‐AA) induced the decreased myocardial autophagic flux, which resulted in cardiomyocyte death and cardiac dysfunction. And other studies demonstrated that β1‐AA induced the decrease of AMPK phosphorylation, the key hub of autophagy pathway, while adiponectin up‐regulated autophagic flux mediated by AMPK. However, it is not clear whether adiponectin improves the inhibition of myocardial autophagic flux induced by β1‐AA by up‐regulating the level of AMPK phosphorylation. In this study, it has been confirmed that β1‐AA induced the decrease of AMPK phosphorylation level in both vivo and vitro. Moreover, pretreatment of cardiomyocytes with AMPK inhibitor Compound C could further reduce the autophagic flux induced by β1‐AA. Adiponectin deficiency could aggravate the decrease of myocardial AMPK phosphorylation level, autophagic flux and cardiac function induced by β1‐AA. Further, exogenous adiponectin could reverse the decline of AMPK phosphorylation level and autophagic flux induced by β1‐AA and even reduce cardiomyocyte death. While pretreated with the Compound C, the adiponectin treatment did not improve the decreased autophagosome formation, but still improved the decreased autophagosome clearance induced by β1‐AA in cardiomyocytes. This study is the first time to confirm that β1‐AA could inhibit myocardial autophagic flux by down‐regulating AMPK phosphorylation level. Adiponectin could improve the inhibition of myocardial autophagic flux induced by β1‐AA partly dependent on AMPK, so as to provide an experimental basis for the treatment of patients with β1‐AA‐positive cardiac dysfunction.


| INTRODUC TI ON
With the development of the society, the morbidity of cardiac dysfunction is rising year by year, which has become a serious public health concern. 1 Immune disorder is one of the important causes of cardiac dysfunction. 2 In 1987, the researcher first discovered β 1 -adrenergic receptor autoantibody (β 1 -AA) as a product of immune disorder in serum of patients with dilated cardiomyopathy. 3 Subsequently, other researchers found that about 40%-60% of patients with cardiac dysfunction were detected with β 1 -AA in serum. 4 It can bind with β 1 -adrenoceptor (β 1 -AR) on the surface of cardiomyocyte membrane to cause persistent injury of cardiomyocytes, induce cardiomyocyte death and eventually lead to cardiac dysfunction. 4 Our team originally found that β 1 -AA could induce the decrease of myocardial autophagic flux, which contributed to cardiac dysfunction. 5 Autophagy is a conserved intracellular degradation system, which maintains myocardial function and improves cardiac dysfunction by clearing intracellular degenerative and ageing proteins or organelles. 6,7 However, it is not clear how β 1 -AA induces the decreased myocardial autophagic flux.
AMP-dependent protein kinase (AMP)-activated protein kinase (AMPK) is an important signal molecule in the upstream of autophagy, 8 which plays a positive role in regulating autophagic flux. 9 AMPK phosphorylation activated autophagy via Unc-51 like autophagy activating kinase 1 (Ulk1)-Beclin1 signalling pathway and through inhibiting mammalian target of rapamycin (mTOR) activity. 10 In primary neonatal rat cardiomyocytes, β 1 -AA induced a decrease in AMPK phosphorylation. 11 However, whether the decrease of AMPK phosphorylation level is involved in the decline of myocardial autophagic flux induced by β 1 -AA, and whether the increase of AMPK phosphorylation level could improve the decreased myocardial autophagic flux induced by β 1 -AA remains to be explored.
As well known, as a key protein of energy metabolism, adiponectin is closely related to the energy metabolism hub AMPK. 12 It has been reported that adiponectin combined to adiponectin receptor on the surface of breast cancer cells, which led to the decrease of intracellular ATP content, activation of LKB1, then induced AMPK phosphorylation, activation of downstream Ulk1, finally initiation of autophagy. 12,13 According to the published papers, patients with cardiac dysfunction have hypoadiponectinaemia and adiponectin knockout mice (APN-KO mice) have serious cardiac dysfunction.
Thus, we speculate that adiponectin deficiency leads to cardiac dysfunction. Moreover, adiponectin is an endogenous product of human, which has a wide range of myocardial protection. 14-16 Therefore, we speculate that adiponectin may promote myocardial autophagic flux by up-regulating the decrease of AMPK phosphorylation level induced by β 1 -AA, thus improving cardiac dysfunction.
This study has confirmed for the first time that the decrease of

| Experimental animals
Six-to eight-week-old male APN-KO mice (weight 18-20 g) were obtained from Professor Bian Yunfei, Second Hospital of Shanxi Medical University. Six-to eight-week-old male C57BL/6 mice (weight 18-20 g) were obtained from Animal Center of Shanxi Medical University. The mice with free diet were placed in a suitable temperature and humidity environment throughout the experiment.
If the myocardial tissue of mice was taken, 10 % chloral hydrate was used for anaesthesia. All procedures related to animals in this study were approved by the Ethics Committee of Shanxi Medical University and followed the People's Republic of China's Guidelines for the Care and Use of Laboratory Animals.

| Agarose gel electrophoresis
The genotypes of APN-KO mice were identified by agarose gel electrophoresis. The mouse tail tissue about 5 × 10 −3 m was put into the EP tube; then, the tail lysate was added and put into the 55 ℃ constant temperature water bath for the night. The DNA of mice tail tissue was extracted and amplified, and the primer sequences were as follows: P1: GGCTCTCTGGGAGAGGCGAG, P2: CCATCACGGCCTGGTGTGCC, P3: TTCGCCATTCAGGCTGCGCA.
The samples were agarose gel electrophoresis, after electrophoresis, the glue was placed in the automatic exposure instrument. 17 dependent on AMPK, so as to provide an experimental basis for the treatment of patients with β 1 -AA-positive cardiac dysfunction.

K E Y W O R D S
Adiponectin, AMPK, Autophagic flux, Cardiac dysfunction, β 1 -AA 2.3 | Establishment of β 1 -AA actively immunized mouse model Six-to eight-week-old male APN-KO mice and WT (C57BL/6) mice were randomly divided into actively immunized group and solvent control group, with 8 mice in each group. The peptide of β 1 -AR-ECII (GLS, Shanghai, Chinese) was dissolved and diluted with Na 2 CO 3 solution (100 mM, pH 11.0) and then mixed with Freund's complete adjuvant (Sigma-Aldrich). After 1:1 emulsification and mixing, the mice were immunized with multiple injection subcutaneously into the back (0.4 μg/g) during the first immunization. Subsequently, diluted peptide of β 1 -AR-ECII emulsified with incomplete Freund's adjuvant (Sigma-Aldrich), and single subcutaneous injection was used to strengthen immunization once every 2 weeks for 12 weeks. In the solvent control group, the same amount of Na 2 CO 3 solution was used to replace the antigen solution. Compound C group is pretreated with Compound C (0.4 μg/g, IV) (s7840, Selleck, USA) for 10 min before immunization ( Figure 1A, Figure S1). 18

| SA-ELISA
SA-ELISA was used to detect the level of β 1 -AA in the serum of actively immunized mice described previously. 5 The 96-well plates were used for antigen coating, and blank control group, solvent control group, positive control and actively immunized group were set up. β 1 -AR-ECII was dissolved in Na 2 CO 3 (100 mM, PH = 11.0) to prepare a solution with the final concentration of 10 μg/mL, and 50 μL was added into each well. Except for the blank control group, the other groups were coated with antigen at 4℃ overnight. The wells were saturated with PMT (1% (w/v) bovine serum albumin, 0.1% (v/v) Tween 20 in PBS, PH 7.4). The solvent control group, positive control, blank control and serum to be tested were diluted and added to 96-well plates. Then, the 96-well plates were added with biotinylated antibody (

| Affinity chromatography
First, a β 1 -AA-positive animal model was established by actively immunized rats with β 1 -AR-ECII, as described in the previous studies. 19 Then, animal serum from actively immunized rats and the control group was collected and extracted using MAbTrap Kit (GE Healthcare, Uppsala, Sweden) for affinity and purification of IgG.

| Cell culture
H9c2 cells were purchased from Chinese Academy of Sciences Cell Bank (Shanghai, China). H9c2 cells were cultured in a complete medium containing 10 % foetal bovine serum (Sijiqing, Shanghai, China) and 100 U/mL penicillin and 100 μg/mL streptomycin (Solarbio, P1400-100, Beijing, China) and were incubated at 37 ℃ in a humidi- the blots were exposed and the grey value was analysed by ImageJ.
GAPDH was used as internal references to calculate the relative expression of different proteins. 18

| Real-time PCR
We detected the mRNA levels of autophagy-related genes Beclin1 and LC3B by real-time PCR. Firstly, total RNA was extracted by Trizol method; then, the total RNA was reverse-transcribed to cDNA. We Bonferroni post hoc test for more than two samples. P < 0.05 was considered statistically significant.

| β 1 -AA inhibited autophagic flux in myocardial tissues by reducing AMPK phosphorylation
Firstly, the effect of β 1 -AA on myocardial AMPK phosphorylation was investigated. The results showed that after active immunization with β 1 -AR-ECII for 8 weeks, the AMPK phosphorylation level (p-AMPK/AMPK) of myocardial tissues was considerably decreased ( Figure 2A). We also found that the AMPK phosphorylation level (p-AMPK/AMPK) of H9c2 cells was also significantly decreased after stimulation of β 1 -AA for 24 hours ( Figure 2B). The results showed that compared with control group, the phosphorylation level of AMPK in Compound C group was significantly decreased both in vitro and in vivo (Figure 2A,B).
In order to clarify the role of decreased AMPK phosphorylation in the decline of autophagic flux induced by β 1 -AA, H9c2 cells were pretreated with Compound C, an AMPK inhibitor, and then treated with β 1 -AA for 24 hours. The results showed that the inhibition of AMPK by Compound C further aggravated the decline of Beclin1 and LC3B mRNA levels ( Figure 2C) and LC3 II protein levels induced by β 1 -AA ( Figure 2D), and aggravated the accumulation of p62 protein ( Figure 2D). C had no effect on p62 protein expression in WT group and APN-KO group ( Figure 3B).

| Adiponectin deficiency aggravated the decline of autophagic flux induced by β 1 -AA in myocardial tissues
In order to clarify the role of adiponectin in the decrease of myocar-  Figure 5E). But there was no significant difference between WT actively immunized mice and the WT group 8 weeks ( Figure 5E). At 12 weeks, compared with WT actively immunized mice, the expression of collagen I and collagen III was further increased in APN-KO actively immunized ( Figure 5E). The Masson trichrome staining results showed that the degree of myocardial fibrosis (blue-stained area) in APN-KO actively immunized mice was more severe than that of WT actively immunized mice ( Figure 5F).

| DISCUSS ION
We have provided the first evidence that β 1 -AA induced a decrease As an important upstream signal molecule of autophagy, AMPK could directly activate autophagy initiation complex Ulk1 and phosphorylate downstream Beclin1 to start autophagy, or inhibit mTOR activity to activate autophagy. 10 AMPK was originally defined as a protein kinase derived from rat liver. It exists as a trimeric complex consisting of catalytic α-subunits and regulatory β-and γsubunits. 25 When the cellular energy status changes, the upstream kinase liver kinase B1 (LKB1) phosphorylates the threonine residues (Thr-172) in the kinase domain to activate AMPK. 26 The study revealed that LPS induced a decrease in AMPK phosphorylation in acute lung injury, which resulted in autophagy inhibition. While the phosphorylation level of AMPK was restored, the decreased autophagy was also improved. 27 Therefore, the decrease of AMPK phosphorylation level may be an important reason for autophagy inhibition. This study and other studies have confirmed that β 1 -AA could induce a decrease in the phosphorylation of myocardial AMPK in vivo and in vitro. 11 We speculated that β 1 -AA induced a decline in myocardial autophagic flux due to inhibiting the phosphorylation of AMPK. In order to verify this speculation, we used AMPK inhibitor Compound C to pretreat H9c2 cardiomyocytes before adding function. 43 The increased LVPW (d) represents cardiac hypertrophy. 44 The results showed that at 8 weeks of active immunization, AMPK is a possible regulatory mechanism still needs to be further verified.
Then, we pretreated H9c2 cells with adiponectin before adding improved the accumulation of p62 protein, which was unaffected by Compound C, suggesting that adiponectin promoted autophagosomes clearance independent on AMPK. There is evidence that in the intestinal tract of drosophila melanogaster, excessive ROS production could cause p62 protein accumulation, leading to autophagy deficiency. 46 Other studies have also shown that adiponectin receptor agonist could increase the clearance of autophagosome by reducing the production of ROS in myocardial ischaemia and reperfusion of mice. 33 Therefore, we speculate that adiponectin may increase the clearance of autophagosome by reducing the production of ROS. Taken together, adiponectin improved the decreased autophagic flux induced by β 1 -AA partly dependent on AMPK. It has been reported that adiponectin could improve myocardial ischaemia-reperfusion injury in rats through AMPK pathway. 47 Furthermore, we also found that adiponectin improved myocardial cell death induced by β 1 -AA, suggesting adiponectin played a protective role in the myocardial.

| CON CLUS ION
This study is the first to confirm that adiponectin could improve the decrease of myocardial autophagic flux induced by β 1 -AA, which is partly dependent on AMPK. Firstly, the decrease of AMPK phosphorylation is an important mechanism of the decrease of myocar-

ACK N OWLED G EM ENTS
The authors thank Dr. Jimin Cao and Dr. Jin Wang for their generous help in the detection of cardiac function in mice.

CO N FLI C T O F I NTE R E S T S
The authors declare that they have no competing interest.

E TH I C A L A PPROVA L
Not applicable.

F I G U R E 7
The proposed molecular pathway. β 1 -AA induced a decrease in myocardial autophagic flux by lowering AMPK phosphorylation. However, adiponectin improved the inhibited formation of autophagosomes induced by β 1 -AA dependent on AMPK, but adiponectin promoted autophagosomes clearance independent on AMPK