Pyridostigmine alleviates cardiac dysfunction via improving mitochondrial cristae shape in a mouse model of metabolic syndrome

Free Radical Biology and Medicine 134 (2019) 119–132

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Original article

Pyridostigmine alleviates cardiac dysfunction via improving mitochondrial cristae shape in a mouse model of metabolic syndrome

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Run-Qing Xue, Xiao-Jiang Yu, Ming Zhao, Man Xu, Qing Wu, Yan-Ling Cui, Si Yang, ⁎ ⁎ Dong-Ling Li , Wei-Jin Zang Department of Pharmacology, School of Basic Medical Sciences, Xi’an Jiaotong University Health Science Center, PO Box 77#, No.76 Yanta West Road, Xi'an City, 710061, Shaanxi Province, PR China

A R T I C LE I N FO

A B S T R A C T

Keywords: Metabolic syndrome Cardiac dysfunction Mitochondrial cristae shape Pyridostigmine

Insulin resistance and autonomic imbalance are important pathological processes in metabolic syndrome–induced cardiac remodeling. Recent studies determined that disruption of mitochondrial cristae shape is associated with myocardial ischemia; however, the change in cristae shape in metabolic syndrome–induced cardiac remodeling remains unclear. This study determined the effect of pyridostigmine (PYR), which reversibly inhibits cholinesterase to improve autonomic imbalance, on high-fat diet (HFD)–induced cardiac insulin resistance and explored the potential effect on the shape of mitochondrial cristae. Feeding of a HFD for 22 weeks led to an irregular and even lysed cristae structure in cardiac mitochondria, which contributed to decreased mitochondrial content and ATP production and increased oxygen species production, ultimately impairing insulin signaling and lipid metabolism. Interestingly, PYR enhanced vagal activity by increasing acetylcholine production and exerted mito-protective effects by activating the LKB1/AMPK/ACC signal pathway. Specifically, PYR upregulated OPA1 and Mfn1/2 expression, promoted the formation of the mitofilin/CHCHD3/Sam50 complex, and decreased p-Drp1 and Fis1 expression, resulting in tight and parallel cristae and increasing cardiac mitochondrial complex subunit expression and ATP generation as well as decreasing release of cytochrome C from mitochondria and oxidative damage. Furthermore, PYR improved glucose and insulin tolerance and insulin-stimulated Akt phosphorylation, decreased lipid toxicity, and ultimately ameliorated HFD-induced cardiac remodeling and dysfunction. In conclusion, PYR prevented cardiac and insulin insensitivity and remodeling by stimulating vagal activity to regulate mitochondrial cristae shape and function in HFD-induced metabolic syndrome in mice. These results provide novel insights for the development of a therapeutic strategy for obesityinduced cardiac dysfunction that targets mitochondrial cristae.

1. Introduction The prevalence of metabolic syndrome, defined as the clustering of obesity, impaired glucose tolerance, insulin resistance, hypertension, and dyslipidemia, is increasing worldwide [1], with a corresponding increase in the population at high risk for cardiovascular disease. Although much progress has been made in characterizing and understanding the cardiovascular consequences of metabolic syndrome, the underlying mechanisms are still incompletely understood. The onset of symptoms of metabolic syndrome has been linked to high-fat diet (HFD)–induced obesity [2]. Obesity-induced abnormal lipid metabolism often leads to the accumulation of lipotoxic products in heart muscle, which inhibits the metabolic insulin/Akt signaling and further aggravates metabolic disorder and cardiomyocyte apoptosis [3].

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Therefore, novel pharmacological therapeutics that affect lipid metabolism and insulin signaling may provide more effective protection against HFD-induced cardiac remodeling. It is well established that mitochondrial dysfunction contributes to myocardial dysfunction and progression of cardiac insulin resistance [4]. Studies in recent years have illustrated how mitochondria adapt their shape to sustain necessary cellular functions, including metabolism, apoptosis, ATP production, and reactive oxygen species (ROS) detoxification, with these various functions performed in the mitochondrial cristae [5,6]. Therefore, maintenance of the proper cristae shape is crucial for mitochondrial function. A family of proteins, known as the mitochondria-shaping proteins, have been shown to orchestrate mitochondrial cristae shape. These proteins include optic atrophy-1 (OPA1), mitofusin 1/2 (Mfn1/2), dynamin-related protein 1 (Drp1),

Corresponding authors. E-mail addresses: [email protected] (D.-L. Li), [email protected] (W.-J. Zang).

https://doi.org/10.1016/j.freeradbiomed.2019.01.011 Received 19 October 2018; Received in revised form 7 January 2019; Accepted 7 January 2019 Available online 10 January 2019 0891-5849/ © 2019 Elsevier Inc. All rights reserved.

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and mitochondrial fission 1 (Fis1), which are involved in mitochondrial fusion and fission, as well as mitochondrial contact site and cristaeorganizing system (MICOS) and sorting and assembly machinery 50 (Sam50), which are involved in fixing the cristae structure [7–9]. Disturbance of mitochondria-shaping proteins destroys cristae shape and disrupts mitochondrial and cellular function [10,11]. For this reason, mitochondrial cristae shape may be a promising target for modulating metabolic and cardiac dysfunction. Nevertheless, the role of mitochondrial cristae and the associated mitochondria-shaping proteins in metabolic syndrome–induced cardiac remodeling has not been previously reported. Accumulating evidence indicates that autonomic imbalance and diminished vagus nerve activity occur frequently in humans and animal models with obesity [12,13]. Interestingly, clinical and experimental studies have demonstrated that enhancing vagal activity can ameliorate heart failure, and vagal nerve stimulation can decrease metabolic disorder and improve cardiac function by preventing mitochondrial dysfunction in obese rats [14]. However, the effect of vagal activation on mitochondrial cristae remains unknown. Acetylcholine (ACh), the principal neurotransmitter of the vagus nerve, is important to vagal activity. Previous studies have shown that serum acetylcholinesterase (AChE) levels are significantly higher in patients with advanced metabolic syndrome than in patients with mild metabolic syndrome and in controls [15,16], and thus, inhibitors of AChE represent promising new pharmacotherapy agents for metabolic syndrome-induced cardiomyopathy. In the present study, we applied a cholinesterase inhibitor, pyridostigmine (PYR), to stimulate vagal activation in HFD-fed mice and study the effects on reduced insulin resistance, with a focus on the shape of mitochondrial cristae and expression of mitochondria-shape proteins.

2.3. Insulin tolerance test and glucose tolerance test For glucose and insulin tolerance tests (GTT and ITT, respectively), all the mice were fasted overnight (12 h) at the end of 22 weeks, and the GTT and ITT were done after an intraperitoneal (i.p.) injection of either 2 g/kg glucose (Sigma-Aldrich, St. Louis, MO, USA) or 0.75 U/kg insulin (Sigma-Aldrich). Blood glucose levels were measured at 0, 30, 60, 90, 120, and 180 min with an Accucheck glucometer (Roche, Basel, Switzerland). 2.4. Blood and tissue collection and biochemical analysis After the mice were anesthetized, blood samples were obtained via the abdominal aortic method, and then liver and heart tissues were removed and washed. The serum was separated by centrifugation for 10 min at 4000 × g. Serum concentrations of free fatty acids, total cholesterol, triglycerides, high-density lipoprotein, low-density lipoprotein, cardiac AChE, and ACh were detected with a biochemical detecting system (AU2700; Olympus Melville, NY, USA). Serum cardiac ceramide and norepinephrine were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's standards and protocols. 2.5. Histological analysis of heart tissue Mice heart tissues were fixed in formalin and embedded in paraffin for sectioning into 5-μm-thick sections. Sections were stained with hematoxylin and eosin (H&E) and Masson's trichrome (Heart Biological Technology Co., Ltd., Xi’an, China) and analyzed for morphological changes.

2. Materials and methods

2.6. Immunohistochemical analyses

2.1. Animals and treatments

For immunohistochemical analysis, sections were passed through xylene and ethanol series to deparaffinize. All sections were boiled in 10 mmol/L sodium citrate antigen retrieval buffer at 95 °C for 20 min, and the slides were washed three times with PBS. Sections were exposed in 3% hydrogen peroxide for 15 min to quench endogenous peroxidase activity and then the slides were washed three times with PBS. Sections were blocked with 10% goat serum for 1 h and then incubated overnight 4 °C with anti-bax (1:200 dilution; Bioworld, Minnesota, USA), anti-bcl-2 (1:200 dilution; Bioworld), anti-cleavedcaspase 3 (1:100 dilution; Bioworld), anti-CD36 (1:200 dilution; Bioworld), anti-glut4 (1:100 dilution; Bioworld), and anti-8-OHdG (1:200 dilution; Abcam, Cambridge, UK). After three washes with PBS, then incubated in secondary antibody for 30 min at 37 °C, followed by three times washing with PBS. Diaminobenzidine was used to develop the antibody stain followed by a hematoxylin counterstain to visualize nuclei. Images were observed under a light microscope (BX53, Olympus, Japan).

Male C57BL/6J mice were purchased from the experimental animal center of Xi’an Jiao Tong University at 4 weeks old and housed in a temperature-controlled room with ad libitum access to water and food, unless otherwise indicated. All procedures involving animals were conducted in accordance with the institutional guidelines for the care and use of laboratory animals and approved by the ethics committee of Xi'an Jiao Tong University. After 2 weeks of acclimatization, mice were randomly divided into three groups: mice fed a normal diet (control group, 12% kcal content), mice fed a HFD (HFD group, 45% kcal fat content), and mice fed a HFD with a daily oral gavage of PYR (HFD +PYR group, 3 mg/kg/day). Food intake and body weight were recorded weekly. After 22 weeks of feeding, the mice were fasted overnight and sacrificed (Fig. 1A).

2.2. Echocardiographic measurement 2.7. Analysis of insulin signaling in tissues For examination of cardiac function, after 22 weeks of feeding, the mice were anesthetized with 1–2% isoflurane in O2 gas and then placed on a heated imaging platform. Echocardiographic measurements were taken using a Vevo 2100 high-resolution in vivo imaging system (Visual Sonics Inc., Toronto, Canada). The parameters of echocardiographic assessment included the left ventricular internal dimension in systole and diastole (LVIDs and LVIDd), the thickness of the interventricular septum in systole and diastole (IVSs and IVSd), the thickness of the LV posterior wall in systole and diastole (LVPWs and LVPWd), the LV ejection fraction (EF), the LV fractional shortening (FS), the end-systolic and end-diastolic LV volume (LVEDV and LVESV), the LV mass, and the LV mass corrected.

After 22 weeks of feeding, mice from each group (control, HFD, and HFD+PYR, n = 12/group) were starved overnight and then injected intraperitoneally with NaCl (0.9%) or insulin (10 mU/mice in NaCl 0.9%). Mice were euthanized 15 min later. The heart and liver were excised and frozen for western blot analysis of Akt phosphorylation as described previously [17]. 2.8. Evaluation of cardiac autonomic tone Tonic sympathetic and vagal influences on the mouse heart were evaluated according to the changes in heart rate (HR) induced by sympathetic blockade with propranolol (4 mg/kg, i.p.), and 120

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(caption on next page)

and the basal HR were calculated.

parasympathetic blockade with atropine (1 mg/kg, i.p.). Electrocardiography (ECG) and HR data for each mouse were collected continuously before, during, and after administration of each blocking agent. The differences in the HR during the drug's peak effect period

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Fig. 1. PYR improved cardiac ultrastructure and cristae morphology. (A) Experimental protocol. (B) Transmission electron microscopy (TEM) images of lipid droplets and mitochondria in heart tissue from mice in all groups. Yellow arrows indicate individual mitochondria, and red arrows indicate lipid droplets. The black frames indicate the areas magnified in panel G. Scale bar is 2 µm. (C–G) Lipid droplet and mitochondrial volume density as well as mitochondrial area, diameter and perimeter on TEM sections were determined for heart tissue samples from 6 separate mice. (H) Magnified TEM images showing mitochondrial cristae in heart tissue of mice in all groups. Scale bar is 200 nm. (I) Schematic diagram of mitochondrial cristae structure. Cj, crista conjunctions; cm, cristae membrane; om, outer membrane; imb, inner membrane boundary; ma, mitochondrial matrix. (J) Distance between cristae measured on TEM images of heart tissue samples from at least three separate mice. (K) Average cristae sinuosity quantified as the ratio of the actual cristae length to the length of a straight line linking each extremity of the cristae. (L) For each analyzed mitochondrion, the total length of cristae membrane per mitochondrion was obtained by adding the lengths of all the cristae together and multiplying by 2. The ratio of cristae membrane to outer membrane was determined by dividing the total length of the cristae membrane by the mitochondrion perimeter. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01, ###P < 0.001 vs HFD group.

1:1000 dilution; Invitrogen), anti-brain natriuretic peptide (BNP, 1:1000 dilution; AbSci, Vancouver, WA, USA), anti-β-myosin heavy chain (β-MHC, 1:1000 dilution; AbSci), OPA1 (1:1000 dilution; Abcam), anti-Mfn1 (1:1000 dilution; Millipore, Burlington, MA, USA), anti-Mfn2 (1:1000 dilution; Millipore), anti-Fis1 (1:500 dilution; Fantibody, Chongqing, China), anti-phospho-Drp1 (1:500 dilution; Cell Signaling Technology), anti-cytochrome C (1:500 dilution; Cell Signaling Technology), anti-CHCHD3 (1:1000 dilution; Invitrogen), anti-mitofilin (1:500 dilution; Invitrogen), anti-Sam50 (1:500 dilution; Proteintech), anti-complex I (1:2000 dilution; Invitrogen), anti-complex II (1:2000 dilution; Invitrogen), anti-complex III (1:2000 dilution; Invitrogen), anti-complex IV (1:2000 dilution; Invitrogen), anti-complex V (1:2000 dilution; Invitrogen), anti-liver kinase B1 (LKB1, 1:200 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), antiphospho-LKB1 (Ser426; 1:200 dilution; Santa Cruz Biotechnology), anti-AMP-activated protein kinase (AMPK, 1:500 dilution, Cell Signaling Technology), anti-phospho-AMPK (Thr172; 1:500 dilution, Cell Signaling Technology), anti-acetyl-CoA carboxylase (ACC, 1:500 dilution, Cell Signaling Technology), anti-phospho-ACC (Ser79; 1:500 dilution, Cell Signaling Technology), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH, 1:5000 dilution, Sinopept, Beijing, China). The membranes were then washed six times with Tris-buffered saline and incubated for 40 min at room temperature with the appropriate peroxidase-conjugated secondary antibody (1:5000 dilution, Sinopept, China). The bands were visualized with an enhanced chemiluminescence (ECL) reagent (Beyotime) and exposed to X-ray film. Band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD, USA).

2.9. Heart rate variability measurement ECG was performed using the Powerlab system. Frequency domain parameters were obtained using power spectrum analysis with low frequency (LF: 0.1–1.75 m s2) and high frequency (HF: 1.75–5.0 m s2) shown in normalized units, and the ratio of LF and HF was calculated. 2.10. Isolation of cytosolic and mitochondrial fractions The procedure for mitochondrial isolation was adapted from previously described procedures [18]. All steps were performed at 4 °C. Briefly, the tissue was mixed in 50 mM Tris–HCl (pH 7.4) containing 0.25 M sucrose and homogenized. The homogenate was centrifuged at 1000 × g for 10 min, and then the supernatant was centrifuged at 12,000 × g for 15 min. The resultant supernatant was designated as the cytosol fraction, and the pellet, which was considered the mitochondrial fraction, was washed and resuspended in 10 mM Tris–HCl (pH 7.8) containing 0.25 M sucrose. 2.11. Measurement of superoxide generation Fresh myocardial tissue was embedded in optimum cutting temperature (O.C.T.) compound and then cut into 10-μm sections at − 20 °C. Superoxide levels were measured by incubation of heart tissue sections in 5 mM dihydroethidium (DHE; Beyotime, Haimen, China) within a light-impermeable chamber at 37 °C for 30 min. After this incubation period, the sections were washed three times for 15 min each in darkness with PBS [19,20]. Images of stained sections were acquired using a confocal microscope (Olympus Fluoview, Hicksville, NY, USA).

2.14. Measurement of MDA-protein adducts content 2.12. Determination of ATP content The ATP content of mouse cardiac specimens was assessed using an enhanced ATP assay kit (Beyotime) according to the manufacturer's instructions.

The MDA content in heart tissue was measured using a commercial ELISA test kit (Abcam). After adding the stop solution to halt the enzymatic reaction, absorbance at 450 nm was detected using a spectrophotometer.

2.13. Immunoprecipitation and western blot analysis

2.15. Superoxide dismutase activity assay

Proteins within cardiac speciments were extracted with radioimmunoprecipitation (RIPA) buffer (Beyotime) containing 2 mmol/l phenylmethylsulfonyl fluoride, and supernatants as sample proteins were acquired by centrifugation of sample lysate at 4 °C for 15 min at 12,000 × g. For immunoprecipitation, supernatants were incubated with anti-CHCHD3 (1:100 dilution; Invitrogen, Carlsbad, CA, USA) or anti-mitofilin (1:100 dilution; Invitrogen) overnight and then with protein A beads (Beyotime) for 4 h at 4 °C. The beads were washed three times with RIPA buffer and resuspended in sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) sample loading buffer for western blotting analysis. After separation on these gels, the proteins were transferred to polyvinylidene difluoride membranes. After blocking with 5% fat-free milk in Tris-buffered saline supplemented with 0.1% Tween 20 for 1 h at room temperature, the membranes were incubated at 4 °C overnight with anti-Akt (1:500 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Akt (Ser473; 1:500 dilution, Cell Signaling Technology), anti-atrial natriuretic peptide (ANP,

Superoxide dismutase activity in heart tissue was assessed with the colorimetric Superoxide Dismutase Assay Kit with WST-8 [2-(2methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2Htetrazolium, monosodium salt] (Beyotime). Briefly, superoxide anions acted on WST-8 to produce a water-soluble formazan dye which can be detected in absorbance at 450 nm. One unit of SOD activity was defined as the amount of the enzyme causing 50% inhibition of WST-8 reduction. 2.16. Determination of H2O2 content The concentration of hydrogen peroxide (H2O2) in the heart tissue was measured using a commercially available kit (Abcam) following the colorimetric assay protocol. Individual heart tissues (40 mg) were homogenized in the assay buffer on ice. The homogenates were then centrifuged at 13,000 × g for 5 min at 4 °C and the supernatant was transferred to clear tube and deproteinized by adding ice-cold 4 M 122

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Fig. 2. PYR treatment maintained cristae shape by restoring the expression of cardiac fission and fusion proteins and promoting the formation of mitofilin/CHCHD3/ Sam50 complex. (A) Mitochondrial cristae shape is regulated by mitochondria-shaping proteins, which include dynamic proteins (division and fusion proteins: drp1, Fis1, OPA1 and Mfn1/2) and structural proteins (mitofilin/CHCHD3/Sam50 complex; OPA1 is both a dynamic and structural protein.). (B–G) Western blot analysis of expression of mitochondrial fission proteins p-Drp1 and Fis1 and fusion proteins OPA1, Mfn1 and Mfn2. (H&I) Immunoprecipitation and western blot analyses for associations among mitochondrial cristae junction-related proteins CHCHD3, mitofilin and Sam50. n = 6. *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; # P < 0.05, ##P < 0.01, ###P < 0.001 vs HFD group.

perchloric acid (PCA) to a final concentration of 1 M for 5 min on ice. The samples were centrifuged at 13,000 × g for 5 min at 4 °C. The excess PCA in the supernatant was precipitated by adding ice-cold 2 M KOH that equals 34% of the supernatant to the sample. The samples were centrifuged at 13,000 × g for 15 min at 4 °C, and the resulting supernatants were used to detect the H2O2 levels following the manufacturer's protocol. Briefly, 50 μl reaction mix, which contained 2 μl

OxiRed Pribe and 2 μl Horse Radish Peroxidase (HRP) in 46 μl assay buffer, reacted with H2O2 in 50 μl samples or standards in 96 well plates for 10 min at room temperature in dark. The products were determined in absorbance at 570 nm.

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structural protein complex mitofilin/CHCHD3/Sam50 by cross coimmunoprecipitation with anti-mitofilin and anti-CHCHD3 antibodies. HFD feeding led to a decrease in interactions among mitofilin, CHCHD3 and Sam50 compared with those observed in control mice, and PYR treatment promoted protein–protein interactions among mitofilin, CHCHD3 and Sam50 in mice of the HFD+PYR group (Fig. 2H and I). Notably, OPA1 is also involved in fixing mitochondrial cristae [10], and PYR treatment enhanced the expression of OPA1 (Fig. 2E), which benefited cristae formation in the HFD+PYR group. Overall, these results suggest that PYR regulated improved mitochondrial cristae shape in HFD-fed mice by regulating the expression of dynamic and structural mitochondria-shaping proteins.

2.17. Transmission electron microscopy Small pieces of fresh LV tissue were fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer and post-fixed in 1% OsO4. After dehydration with a gradient of increasing ethanol concentrations, the samples were exposed to propylene oxide to allow infiltration of the embedding medium, Epon 812 resin. After cutting with an ultramicrotome, thin sections were stained with acidified uranyl acetate, followed by a modification of Sato's triple lead stain, and viewed with a transmission electron microscope (TEM; H-7650; Hitachi, Tokyo, Japan). The images were analyzed using ImageJ software. 2.18. Statistical analysis

3.3. PYR treatment alleviated mitochondrial dysfunction and oxidative damage in the heart of HFD-fed mice

Data are presented as means ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism version 5.01 (GraphPad Software, La Jolla, CA, USA). Significant differences among the data were identified using one-way analysis of variance (ANOVA) followed by least significant difference post-hoc analyses. Differences were considered statistically significant if P < 0.05.

The results described above indicate that PYR regulated the shape of mitochondrial cristae. However, we still sought to determine whether mitochondrial function was improved with PYR treatment. It was found that consumption of the HFD led to reduced expression of the complex subunits I, II, III, IV and V in heart tissue of mice as well as decreased cardiac ATP content compared with that in the heart tissue of control mice (Fig. 3A–C). These abnormalities were greatly restored by PYR administration, which demonstrates that PYR improved mitochondrial function in HFD-fed mice. Accumulating evidence indicates that approximately 85% of total cytochrome C is stored in mitochondrial cristae; therefore, tight and steady mitochondrial cristae result in reduced cytochrome C release from the mitochondria to the cytosol [22]. In the current study, the ratio of mitochondrial to cytosolic cytochrome C in HFD-fed mice was markedly lower than that in control mice, and PYR treatment restored this ratio (Fig. 3D–E), indicating that PYR suppressed the release of mitochondrial cytochrome C. Mitochondria are the major source of cellular ROS, which lead to mitochondrial dysfunction and oxidative stress, ultimately causing cardiac insulin resistance [23]. In the heart tissue of HFD-fed mice, DHE staining showed increased production of superoxide anion and immunohistochemical staining revealed an increased number of 8OHdG–positive cells. However, the DHE fluorescence intensity and number of 8-OHdG–positive cells were reduced significantly in the HFD +PYR group (Fig. 3F–I). Furthermore, the HFD group resulted in elevated MDA-protein adducts and H2O2 content and reduced SOD activity in the heart, which was partially alleviated by PYR treatment (Fig. 3J–L). These data indicate that PYR reduced cardiac mitochondrial dysfunction and oxidative damage in the heart of HFD-fed mice.

3. Results 3.1. PYR treatment improved mitochondrial size and cristae structure in HFD-fed mice Mitochondrial dysfunction plays an important role in the development of insulin resistance and metabolic disorder [4]. Therefore, we first investigated whether mitochondria are destroyed in HFD-fed mice. Under electron microscopy, HFD feeding led to lipid accumulation, as evidenced by an increased lipid droplet number in Fig. 1B. In addition, the mitochondrial volume density was also increased in heart tissue, whereas the mitochondrial area, diameter and perimeter per mitochondrion were decreased compared with those in control mice. However, all of the alterations to these parameters observed in HFD-fed mice were partially relieved by PYR treatment (Fig. 1B–G). Cristae, fundamental structures for mitochondria, are the site of oxidative phosphorylation, and therefore, cristae integrity must be maintained for proper mitochondrial function [5,21]. Fig. 1H shows enlarged images of the areas framed by the black squares in Fig. 1B, for observation of the mitochondrial cristae. Quantification of mitochondrial shape parameters on electron microscopy sections revealed that the distance between cristae was longer in mitochondria from the heart tissue of HFD-fed mice than that in heart tissue mitochondria of control mice. In addition, the cristae in the HFD group were curved and nonparallel, and the HFD also induced a decrease in the length ratio of mitochondrial cristae membrane and outer membrane (a parameter of mitochondrial cristae density). However, cristae in the HFD+PYR group were quite straight, parallel, and compact, and the evaluated parameters describing mitochondrial cristae were improved by PYR treatment (Fig. 1H–L).

3.4. PYR increased cardiac vagal activity and activated AMPK in HFD-fed mice Research has confirmed that enhanced vagal activity can decrease mitochondrial dysfunction [24]. To further verify how PYR plays a protective role in mitochondria of HFD-fed mice, the alteration of vagal activity in all groups was examined. As expected, in the frequency domain, high-frequency (HF, vagal tone) power was lower, whereas low-frequency power (LF, sympathetic tone) and the LF/HF ratio were higher in the HFD group than in the control group. In addition, augmented HF and normalized LF and LF/HF ratio were observed in the HFD+PYR group (Fig. 4A–C). To assess the activity of the cardiac autonomic system, we monitored changes in HR with the inhibition of vagal and sympathetic nervous tone in all groups. The difference between the HR measured at baseline and the HR after propranolol administration was considered as the sympathetic tone, and the difference between the HR measured at baseline and the HR after methyl atropine administration was considered as the vagal tone (Fig. 4D). HFD-fed mice exhibited an autonomic imbalance characterized by increase cardiac sympathetic tone and decreased vagal tone compared with

3.2. PYR treatment regulated expression of mitochondria-shaping proteins in HFD-fed mice Mitochondrial cristae shape is controlled by the mitochondriashaping proteins, which include dynamic and structural proteins as shown in Fig. 2A. We first examined the dynamic proteins controlling mitochondrial number and size. HFD feeding significantly increased the expression of p-Drp1 (Ser616) and Fis1 and decreased the expression of OPA1 and Mfn1/2. PYR treatment inhibited the HFD-induced increase in p-Drp1 (Ser616) and Fis1 expression and also effectively increased the expression of OPA1and Mfn1/2 in mice of the HFD+PYR group (Fig. 2B–G). To further explore the mechanism by which PYR protects mitochondrial shape in HFD-fed mice, we examined the presence of the 124

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Fig. 3. PYR treatment ameliorated HFD-induced mitochondrial dysfunction and oxidative damage. (A and B) Protein expression of mitochondrial complex subunits I, II, III, IV and V. n = 6. (C) ATP content in heart tissue. n = 12. (D and E) Western blot analysis of cytochrome C expression in mitochondria and cytosol, separately. n = 6. (F and H) Superoxide levels in heart tissue based on DHE staining. Scale bar is 100 µm. n = 6. (G and I) Representative images of immunohistochemical staining of 8-OHdG and quantification of 8-OHdG–positive cells in heart tissue sections. Scale bar is 100 µm. n = 6. (J) MDA-protein adducts content, (K) Concentration of H2O2 in heart tissue and (L) SOD activity in heart tissue. n = 8. *P < 0.05, **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01, ### P < 0.001 vs HFD group.

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Fig. 4. PYR activated the vagus nerve and the AMPK-associated pathway by increasing the ACh content in HFD-fed mice. (A–C) Normalized LF and HF and absolute values of the LF/HF ratio. (D–F) Diagram and histogram analysis of the difference in HR between before and after administration of the parasympathetic blockade atropine (1 mg/kg, i.p.) or sympathetic blockade propranolol (4 mg/kg, i.p.). (G–I) Cardiac AChE activity and ACh and NE levels. (J-L) Representative western blot showing protein expression and quantitative analysis of ACC, AMPK and LKB1 phosphorylation. n = 12. **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ## P < 0.01, ###P < 0.001 vs HFD group.

Previous studies identified an important role for AMPK activation in regulating metabolism and mitochondrial form and function [25], and in our previous study, AMPK is responsible for mito-protection of vagal nerve stimulation and ACh in cardiovascular ischemia/reperfusion injury. To further confirm the mechanisms responsible for the mito-protection offered by PYR, we examined the phosphorylation of AMPK, ACC (a substrate of AMPK), and LKB1 (an upstream of AMPK) in heart tissue by western blot analysis and found that the expression of phosphorylated AMPK, ACC and LKB1 was effectively reduced in HFD-fed mice compared with control mice. However, these altered levels of phosphorylated proteins were ameliorated by PYR administration

control mice, and these changes were partially prevented in the HFD +PYR group (Fig. 4E and F). To further determine the effect of a HFD and PYR on vagal activity, cardiac AChE, ACh and NE levels were evaluated. The results revealed that AChE activity was notably augmented in the HFD group, a change that was reduced by PYR administration (Fig. 4G). Meanwhile, the concentration of cardiac ACh was lower in the HFD group than in the control group but increased in the HFD+PYR group (Fig. 4H). Additionally, the cardiac NE concentration was higher in the HFD group than in the control group, but PYR had no significant effect on the NE concentration (Fig. 4I). Together, these data suggest that PYR improved vagal tone by suppressing AChE activity. 126

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Fig. 5. PYR decreased body weight and improved insulin sensitivity in HFD-fed mice. (A) Changes in body weight over 22-week feeding period. n = 18. (B) Serum glucose concentrations. (C and D) Intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) results. n = 9. (E and F) Representative western blots of insulin-stimulated p-Akt and Akt in the heart and liver. n = 6. **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ##P < 0.01, ### P < 0.001 vs HFD group.

mitochondrial cristae and function, and mitochondria are the therapeutic target of metabolic disorder. Therefore, we further examined glucose and lipid metabolism in HFD-fed mice. Although the mice were fed the same volume of food, the HFD provided an increase in weekly energy intake compared with the control diet, and PYR treatment had no effect on food intake or energy intake of HFD-fed mice (Supplementary Table 1). As expected, HFD-fed mice were obese after 22 weeks. As shown in Fig. 5A, the body weight of HFD-fed mice began

(Fig. 4J–L), which suggests that the LKB1-AMPK-ACC pathway may participate in the process by which PYR activated vagal tone to prevent changes in the cardiac mitochondrial cristae shape in obese mice. 3.5. PYR treatment improved insulin sensitivity and thus decreased metabolic disorder in HFD-fed mice The results described above indicate that PYR regulated the shape of 127

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fragments, simultaneously accompanied by mitochondrial dysfunction and oxidative stress, which eventually led to impaired cardiac insulin sensitivity; (2) PYR enhanced vagal activity by suppressing AChE and normalized HFD-induced changes in cardiac metabolism, thereby improving glucose and insulin tolerance, increasing insulin-stimulated Akt phosphorylation in both liver and heart, and restoring cardiac hypertrophy, fibrosis, apoptosis and dysfunction; and (3) more importantly, as a bridge connecting cardiac metabolism and function, mitochondria was protected by PYR from HFD-induced both structural and functional damage. Specifically, PYR activated AMPK and enhanced mitochondrial cristae shape via regulation of mitochondria-shaping proteins, including upregulation of OPA1 and Mfn1/2, downregulation of p-Drp1 and Fis1, and enhanced formation of the mitochondrial cristae structure-associated protein complex mitofilin/CHCHD3/Sam50. These PYR-induced changes in cristae shape improved the content of mitochondria and the production of ATP, and attenuated oxidative damage, and finally reversed HFD-induced cardiac remodeling (Fig. 8). Taken together, these findings show that PYR alleviated the HFD-induced impairment of insulin sensitivity, likely through vagal activation to regulate mitochondrial cristae shape, and prevented HFD-induced cardiac remodeling. Mitochondrial dysfunction has been suggested to be an important contributor to the metabolic disorder. In obesity, increased fatty acid levels in heart may result in increased fat uptake, which may increase ROS production and further lead to activation of uncoupling proteins. The resulting increase in mitochondrial uncoupling promotes disassembly of the mitochondrial respiratory chain and finally breakdown of the mitochondrial cristae structure, leading to decreased ATP synthesis and reduced cardiac efficiency [26,27]. Therefore, in addition to reducing mitochondrial uncoupling, which has been the concern of most research, exploring the change in mitochondrial structure and its associated mechanism may help provide a new target for preventing cardiac metabolic damage in obesity. Mitochondrial cristae are the true bioenergetic structure [5], and it has been demonstrated that cristae morphology determines the assembly and stability of respiratory chain supercomplexes and respiratory efficiency [28]. Based on the available data, mitochondrial reorganize the shape of the cristae in response to a change in the nutrient status [29]. Cristae disorganization occurs in pathological syndromes, such as aging and Barth syndrome [30,31]. Jeong et al. found that a HFD induced increases in the number of cardiac mitochondria and fragments [32], which is consistent with our observations. In addition, we observed reduced, loose, unparallel, and even lysed cristae in the heart of HFD-fed mice, and this was accompanied by decreased expression of mitochondrial respiratory chain complex I–V and ATP generation in the cardiac tissue of HFD-fed mice. These phenomena are similar with findings in T cells that divided mitochondria lead to loose cristae and imbalanced redox and fused mitochondria result in tight cristae and efficient OXPHOS [33]. More importantly, mitochondria-shaping proteins were also altered in the HFD group, including enhanced expression of Drp1 and Fis1, inhibition of OPA1 and Mfn1/2, and formation of the mitofilin/CHCHD3/Sam50 complex. These novel findings suggest that mitochondrial cristae perturbations play an important role in cardiac dysfunction induced by long-term HFD feeding, and regulation of mitochondria-shaping proteins might represent a possible strategy to protect mitochondrial health in metabolic disorder. Autonomic imbalance, characterized by increased sympathetic activity and suppressed vagal activity, is correlated with cardiac damage in diabetic and obese patients [34], which suggests that activating the vagus may benefit cardiac energy metabolism. As is known, mitochondria are the center of metabolism, and a growing body of evidence suggests that elevated vagal activity and its neurotransmitter ACh exert considerable defensive effects against cardiac mitochondrial dysfunction, preventing processes such as mitochondrial proliferation, mitophagy, ROS production, and mitochondrial Ca2+ overload while also inhibiting MPTP opening and decreasing mitochondrial swelling

to show a significant increase after 10 weeks of feeding compared with that of control mice. PYR was administered intragastrically at 3 mg/kg/ d throughout the 22 weeks of the HFD feeding protocol, and in the HFDfed mice treated with PYR, a significant decrease in body weight was observed from weeks 12–22 (Fig. 5A). To evaluate glucose metabolism, fasting blood glucose concentrations were measured in mice of all groups. As expected, serum glucose levels were elevated in HFD-fed mice, and PYR treatment significantly decreased serum glucose levels in mice of the HFD+PYR group (Fig. 5B). Moreover, the IPGTT and IPITT results revealed diminished glucose and insulin tolerance under HFD treatment (Fig. 5C and D), indicating metabolic disorder and impaired insulin sensitivity in HFDfed mice. However, in HFD-fed mice treated with PYR, glucose and insulin tolerance were restored (Fig. 5C and D). In addition, while HFD feeding altered insulin-stimulated Akt phosphorylation in both liver and heart muscle, PYR improved the insulin response in both liver and heart muscle, indicating that the drug had a positive effect on insulin sensitivity in both organs (Fig. 5E and F). Together, these results demonstrate that PYR treatment reduced weight gain and enhanced insulin sensitivity in HFD-fed mice. At the end of the 22-week feeding period, the serum concentrations of free fatty acids, triglycerides, and total cholesterol were significantly higher and the ratio of high-density lipoprotein to low-density lipoprotein was lower in the HFD group compared with the control group, and these parameters of lipid metabolism were partially normalized in the HFD+PYR group (Fig. 6A–D). Furthermore, HFD feeding led to down-regulation of the glucose transporter Glut4, up-regulation of CD36 expression, and eventually accumulation of lipotoxic components (ceramide), and these effects were partially corrected by PYR treatment (Fig. 6E–H). These data indicate that PYR alleviated dyslipidemia and mitigated cardiac lipotoxicity in HFD-fed mice. 3.6. PYR improved cardiac remodeling and function in HFD-fed mice Lastly, we examined the effect of PYR on HFD-induced cardiac dysfunction. Compared with that in control mice, HFD-fed mice showed decreased cardiac Bcl-2 expression and increased Bax and cleaved caspase 3 expression, and PYR treatment ameliorated these alterations and restored mitochondria-related apoptosis (Fig. 7A–F). Meanwhile, the areas occupied by cardiomyocytes and fibrosis were greater in the HFD group than in the control group and reduced by PYR administration (Fig. 7G–K). Moreover, western blot analysis revealed greater expression of ANP, BNP and β-MHC (markers of cardiac hypertrophy) in HFD-fed mice than in control mice, but the expression of all of these proteins was restored in mice of the HFD+PYR group (Supplementary Fig. 1). ECG was performed to evaluate cardiac function after 22 weeks of feeding. As shown in Supplementary Table 2, mice in the HFD group exhibited significant increases in the LVIDd, LVIDs, LVEDV, LVESV, IVSd, IVSs, LV mass, and LV mass corrected, compared with those in the control group, which again proved that the HFD induced cardiac hypertrophy. PYR administration led to marked improvements in the LVESV, IVSd, IVSs, LV mass, and LV mass and slight improvements in the other indices. Furthermore, EF and FS were reduced in the HFD group, and these effects were reversed in the HFD+PYR group. Taken together, these data demonstrate that PYR improved cardiac remodeling in HFD-fed mice. 4. Discussion Long-term high-fat diet consumption leads to cardiac insulin resistance, which plays a vital role in the development of heart disease. Therefore, novel pharmacological agents to treat such impaired insulin signaling are urgently needed. The present study demonstrated that: (1) the structure of cardiac mitochondria in HFD-fed mice was abnormal with loose, nonparallel, and even lysed cristae and mitochondrial 128

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Fig. 6. PYR improved lipid metabolism and decreased cardiac lipotoxicity. (A–C) Serum concentrations of free fatty acids, triglycerides, and total cholesterol. (D) Ratio of serum concentration of high-density lipoprotein (HDL) to low-density lipoprotein (LDL). n = 9. (E–G) Immunohistochemical analysis of Glut4 and CD36 expression in heart tissue. Scale bar is 100 µm. n = 6. (H) Ceramide content in heart tissue. n = 9. **P < 0.01, ***P < 0.001 vs control group; #P < 0.05, ## P < 0.01, ###P < 0.001 vs HFD group.

Considering that AMPK is involved in the regulation of mitochondrial morphology and function [38], it is reasonable to suppose that activation of AMPK to regulate mitochondria-shaping proteins via vagal activation may be responsible for the protective effect of PYR on optimizing mitochondrial cristae shape and function. Further research is required to explore these issues. Reinforced mitochondrial cristae tightness not only maintained mitochondria to utilize lipids effectively, but also reduced apoptosis and insulin resistance. Interestingly, mitochondria are more than just a powerhouse; at the same time, they play an important role in cellular signal communication. A substantial amount of evidence has indicated that strengthening cristae tightness by overexpression OPA1 reduces cytochrome C release and ROS production [10,39]. It is well known that cytochrome C in the cytoplasm triggers apoptosis, and the current study showed that PYR reduced the release of cytochrome C from

[18,35]. However, the underlying mechanisms by which activating the vagal nerve can protect mitochondrial function remain unknown. AMPK activators salicylate and AICA riboside inhibit Drp1-mediated mitochondrial fission and therefore reduce intracellular and mitochondrial ROS in palmitate-treated endothelial cells [36]; similarly, Wang et al. found metformin alleviates diabetes- accelerated atherosclerosis via preventing Drp1-mediated mitochondrial fission [37]. In keeping with these results, the present study showed that PYR improved the parameters of vagal activity, as evidenced by elevated HF and ΔHR in response to atrophine and activated AMPK and decreased mitochondrial fragments by inhibiting Drp1 and Fis1 in HFD mice. However, more attractively, PYR also improved mitochondrial fusion by promoting OPA1 and Mfn1/2 and strengthened the stability of mitochondrial cristae by increasing mitofilin/CHCHD3/Sam50 complex, ultimately relieving HFD-induced mitochondrial dysfunction. 129

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Fig. 7. PYR attenuated cardiac remodeling and apoptosis in HFD-fed mice. (A–F) Immunohistochemical analysis of Bax, Bcl-2 and cleaved caspase 3 expression in heart tissue. (G-K) Representative images of H&E and Masson's trichrome staining of LV sections and analysis of cardiomyocyte area and cardiac fibrosis. Scale bar is 100 µm. n = 6. ***P < 0.001 vs control group; ##P < 0.01, ###P < 0.001 vs HFD group.

stimulation could improve cardiac function by exerting an anti-inflammatory effect in obese insulin-resistant rats, which may be mediated via the cholinergic anti-inflammatory pathway [40]. Lu et al. explored how activating the vagus by PYR enhanced white adipose tissue browning and brown adipose tissue activation via up-regulating SIRT1/AMPK/PGC1α signaling, and finally protected against HFD-induced cardiomyopathy in rats [41]. Additionally, our study showed that PYR corrected the transportation and uptake of metabolic substrates as evidenced by the up-regulation of cardiac Glut4 and down-regulation of CD36 expression in HFD-fed mice, which is irrelevant to mito-

mitochondrial cristae, which resulted in the inhibition of cardiomyocyte apoptosis and cardiac remodeling. On the other hand, it has been confirmed that ROS have a causal role in insulin resistance [23]. Our research found that PYR, probably due to reduced ROS generation, improved insulin sensitivity and glucose tolerance and enhanced Akt phosphorylation after insulin stimulation in both heart and liver of HFD-fed mice. Despite these findings, it must be admitted that mitochondrial cristae are not the only target for achieving protection by vagus activation against HFD-induced cardiac dysfunction. In fact, vagus nerve 130

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Fig. 8. PYR increased vagal activity and regulated mitochondrial cristae shape, thereby improving cardiac insulin sensitivity and cardiac remodeling in HFD-fed mice. ACh: acetylcholine; AChE: acetylcholinesterase; AMPK: AMP-activated protein kinase; ATP: adenosine triphosphate; CD36: a transporter protein involved in fatty acid uptake; CHCHD3: coiledcoil helix coiled-coil helix domain-containing protein 3; cyt C: cytochrome C; Drp1: dynamin-related peptide 1; FFA: free fatty acids; Fis1: mitochondrial fission protein 1; Glut4: glucose transporter type 4; HDL: high-density lipoprotein; HF: high-frequency; HFD: high-fat diet; IPITT: intraperitoneal insulin tolerance test; LDL: low-density lipoprotein; LF: low frequency; MDA: malonaldehyde; Mfn1/2: mitofusin 1/2; Mitofilin: inner membrane mitochondrial protein; NE: norepinephrine; OPA1: optic atrophy type 1; PYR: pyridostigmine; ROS: reactive oxygen species; Sam50: sorting and assembly machinery 50; SOD: superoxide dismutase; T-CHO: total cholesterol; TG: triglycerides; 8-OHdG: 8-hydroxyguanosine.

shape, consequent reduction of mitochondrial dysfunction and oxidative stress, and normalization of metabolic disorder and cardiac remodeling. Overall, our study provides evidence for the role of mitochondria cristae shape in metabolic syndrome-induced cardiac dysfunction and novel insights for the development of therapeutic strategies related to vagal activation.

protection. Nevertheless, this benefits muscle insulin sensitivity and lipid consumption [42,43]. However, further studies of the detailed mechanism by which PYR induces the changes in metabolic substrates and how this process correlates with vagal activity are warranted. PYR has been tested repeatedly in humans and was first used in clinical practice for treating patients with myasthenia gravis. In recent years, some evidence has reported that PYR activates the vagal nerve to elicit favorable effects in myocardial ischemia, myocardial infarction, and heart failure in both animal and clinical experiments [44,45]. Moreover, treatment with PYR is an atraumatic approach compared with direct vagal nerve stimulation. Intriguingly, we treated the mice in the present study with a PYR dose of 3 mg/kg/day to activate the vagus and explored new positive effects on an aspect of metabolic disorder. Particularly, treatment with this dosage recovered the ACh content from about 56.69% in the HFD group to nearly 95.78% in the control group, which is nearly the normal ACh content and probably could not result in accumulation of ACh in the synaptic space in the HFD+PYR group. According to the formula for dose translation based on body surface area, this dose is equivalent to about 17 mg/day for a person with 70-kg body weight [46]. This calculated dosage is greatly less than the dosage of 90 mg/day in patients with heart failure and 630 mg/day in patient with myasthenia gravis, which should correspond to fewer side effects of PYR, such as nausea and stomach cramping. Thus, our results should encourage further studies on the long-term effects of lowdose PYR on cardiac damage in metabolic syndrome patients. In conclusion, our results indicate that a HFD resulted in cardiac autonomic imbalance and metabolic disorder, which was associated with mitochondrial dysfunction. Importantly, PYR enhanced vagal activation and exerted positive effects on insulin resistance in the HFD mouse model. Moreover, the primary mechanisms responsible for these observations involved activation of AMPK signaling, regulation of mitochondria-shaping proteins, preservation of mitochondrial cristae

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