Myeloid differentiation protein 1 protected myocardial function against high‐fat stimulation induced pathological remodelling

Abstract Myeloid differentiation 1 (MD‐1) is a secreted protein that regulates the immune response of B cell through interacting with radioprotective 105 (RP105). Disrupted immune response may contribute to the development of cardiac diseases, while the roles of MD‐1 remain elusive. Our studies aimed to explore the functions and molecular mechanisms of MD‐1 in obesity‐induced cardiomyopathy. H9C2 myocardial cells were treated with free fatty acid (FFA) containing palmitic acid and oleic acid to challenge high‐fat stimulation and adenoviruses harbouring human MD‐1 coding sequences or shRNA for MD‐1 overexpression or knockdown in vitro. MD‐1 overexpression or knockdown transgenic mice were generated to assess the effects of MD‐1 on high‐fat diet (HD) induced cardiomyopathy in vivo. Our results showed that MD‐1 was down‐regulated in H9C2 cells exposed to FFA stimulation for 48 hours and in obesity mice induced by HD for 20 weeks. Both in vivo and in vitro, silencing of MD‐1 accelerated myocardial function injury induced by HD stimulation through increased cardiac hypertrophy and fibrosis, while overexpression of MD‐1 alleviated the effects of HD by inhibiting the process of cardiac remodelling. Moreover, the MAPK and NF‐κB pathways were overactivated in MD‐1 deficient mice and H9C2 cells after high‐fat treatment. Inhibition of MAPK and NF‐κB pathways played a cardioprotective role against the adverse effects of MD‐1 silencing on high‐fat stimulation induced pathological remodelling. In conclusion, MD‐1 protected myocardial function against high‐fat stimulation induced cardiac pathological remodelling through negative regulation for MAPK/NF‐κB signalling pathways, providing feasible strategies for obesity cardiomyopathy.


| INTRODUC TI ON
Obesity is a metabolic disorder affecting millions of people worldwide. Patients with obesity are highly vulnerable to cardiovascular dysfunctions, including hypertrophic cardiomyopathy. 1,2 Featured with impaired diastolic function and left ventricular hypertrophy, obesity cardiomyopathy (OCM) patients exhibited disrupted substrate metabolism, oxidative stress, endoplasmic reticulum stress in the early stage. [2][3][4][5][6][7] These events cause the accumulation of lipid droplets in cardiomyocytes, changing the immune response and resulting in cardiac fibrosis, remodelling and compromised cardiac functions. [8][9][10] In the process, immune system is vital in modulating the development of diabetic cardiomyopathy. 9 However, the underlying mechanism is not fully understood.
Myeloid differentiation 1 (MD-1) is a secreted glycoprotein produced by macrophages. 11 It forms complex with radioprotective 105 (RP105), a Toll-like receptor protein, to play critical roles in lipopolysaccharide (LPS) recognition by B cells. [11][12][13] Recent studies have revealed that MD-1/RP105 complex as a key regulator of high-fat diet induced chronic inflammation in obesity. 14,15 Moreover, RP105 was proved to play cardioprotective function in against myocardial ischaemia-reperfusion injury by suppressing TLP4 signalling pathways. 16,17 As an indispensable accessory molecule required for the cell surface expression of RP105, 18 whether MD-1 may also play essential roles in cardiac diseases needs to be further explored. Recently, Jiang et al reported that loss of MD1 exacerbated myocardial I/R injury and increased the susceptibility to ventricular arrhythmia, both of which are possibly related to the up-regulation of TLR4/NF-κB signalling pathway. 19 Another study found that MD1 deficiency played an important role in accelerating the development of inflammatory atrial fibrosis and increasing vulnerability to AF in mice with HFD-fed induced obesity. 20 In our previous research, we have verified MD-1 is a vital modulator of cardiac hypertrophy and fibrosis. 21 Thus, we hypothesized that MD-1 may also play vital effects in obesity-induced cardiomyopathy.
In this study, utilizing free fatty acid (FFA) containing palmitic acid and oleic acid to mimic high-fat environment in H9C2 cells, or high-fat diet (HD) administration in vivo to induce hyperlipaemia cardiomyopathy mice and cell models. Results demonstrated that MD-1 protected myocardial function against high-fat stimulation induced cardiac pathological remodelling through negative regulation for MAPK/NF-κB signalling pathways. Our studies are beneficial to better understand the mechanisms of obesity cardiomyopathy and provided a new strategy to treat obesity cardiomyopathy through targeting MD-1.

| Reagents
Palmitic acid and oleic acid were purchased from Sigma Aldrich (USA). Inhibitors U0126 and Bay 11-7082 were purchased from Selleck Chemicals (USA).
Cells were maintained in a humidified incubator with 5% CO 2 at 37°C.
Free fatty acid (FFA), containing oleic acid and palmitic acid at 2:1 molar ratio, was prepared with fat-free bovine serum albumin (BSA) as previously described 22,23 and then added into the complete medium with final concentration of 0.25, 0.5 and 1 mmol/L. The control group was treated with 1% BSA. After treated with FFA for 16, 24 or 48 hours, cells were collected for analysis.

| Oil red staining
The lipid deposition within the cells was assessed by oil red O staining. H9C2 cells were fixed with pre-cooled 4% formaldehyde. Then cells were stained with oil red O (Sigma-Aldrich) and counterstained with hematoxylin to visualize lipid vacuoles. Images were photographed with microscope (Olympus, Tokyo, Japan).

| Construction and infection of the recombinant adenovirus
The full coding region of the human MD-1 gene controlled by the cytomegalovirus (CMV) promoter was cloned into a replication-defective adenoviral vector (Ad-MD-1). Parallelly, adenoviral vector containing GFP gene (Ad-GFP) was used as control. Short hairpin RNAs (shRNAs) for MD-1 knockdown and the negative control shRNA (shRNA-NC) constructs purchased from GenePharma (Shanghai, China) were used to generate MD-1 knockdown adenovirus (Ad-shMD-1) and control (Ad-shNC) respectively. After packaging, amplification and purification, the viral titres were measured by a plaque assay with fluorescence counting in HEK-293 cells.
Cardiomyocyte H9C2 cells were infected with these adenoviruses at a multiplicity of infection (MOI) of 100.

| RNA extraction and quantification polymerase chain reaction
Total RNA was extracted from H9C2 and mouse tissues using Trizol Reagent (Invitrogen, Paisley, UK) and cDNA was synthesized by reverse transcription using PrimeScript 1st Strand cDNA Synthesis Kit (Takara). The gene expression was measured by quantification polymerase chain reaction (qPCR) with Applied Biosystems 7500 Fast Real Time qPCR machine. The relative expression levels were calculated using 2 −∆∆Ct method, and GAPDH was used as an internal reference. The sequences of primers used for qPCR were listed in Table 1.

| Western blot
Total protein was extracted and separated on 8%-10% SDS-PAGE.

| Glucose tolerance testing
Two days before end of the 20th week of HD feed, a glucose tolerance test (GTT) was performed on all these mice. Briefly, all mice were subjected to an overnight fast (≈16 hours) before test. Glucose at dose of 2 g/kg·body-weight was intraperitoneally injected into the mice. At 0 minute before glucose injection and at 15, 30, 60, 90 and 120 minutes after glucose injection, the blood glucose was measured, respectively, using a glucometer (OneTouch Ultra, Johnson & Johnson, USA) through collecting blood from tail vein.

| Echocardiography and serum lipid index analysis
At the end of treatment, mice were anesthetized by intraperitoneally injecting with 3% pentobarbital sodium at a dose of 30 mg/ kg. Half hour later, the mice were fixed on the test plate, removed the hairs and coated with conductive fluid on the heart, then echocardiography analysis was performed using an ultrasonic apparatus Mylab30CV (Esaote SpA) instrument with a 10 MHz linear array ultrasound transducer according to the manufacturer's introduction and as previously described. 21  (IVSd), systolic interventricular septum (IVSs) and fractional shortening (FS) were analysed by matched software.
After echocardiography experiments, mice were sacrificed.
Serum was collected for biochemical determination including total cholesterol (TC, mmol/L), triglyceride (mmol/L), low density lipoprotein (LDL, mmol/L) and creatine kinase-MB (CK-MB, U/L). The hearts were dissected and weighed to compare heart weight (HW, g), body weight (BW, g), the ratios of HW/BW (%) and heart weight to tibia length (HW/TL, mg/mm).

| Masson's trichrome staining and immunohistochemistry
Paraffin-embedded sections from heart tissues were stained with Masson's trichrome to determine the degree of cardiac fibrosis. Masson's trichrome staining kit was purchased from Sigma Aldrich (MO, USA) and sections were stained according to the manufacturer's instruction.
For immunohistochemistry, the slices were deparaffinized, hydrated and treated for antigen retrieval, and then incubated with NF-κB p65 primary antibody (1:500 dilution, Abcam, USA) at 4°C for overnight. The positive reactions were visualized as brown using a DAB (3, 3-diamino-benzidine) kit (Sigma, USA) and haematoxylin for nuclear counterstaining.

| Statistics analysis
Data were presented as mean ± Standard deviation (SD) based on at least three different determinations. Student's t test was employed to compare the difference between two groups. The statistical analysis between multi-groups was carried out using one-way analysis of variance (ANOVA) by Tukey's post hoc test. A two-side value of P < 0.05 was considered statistically significant. All statistical analyses were performed by GraphPad Prism 5 (GraphPad Software, La Jolla, CA).

| FFA induces lipid accumulation and MD-1 differential expression in cardiomyocytes
To  Figure 1D). Therefore, FFA as an inducer of cardiomyocytes injury is involved in promoting lipid accumulation, cell proliferation and MD-1 may play roles in these processes.

| MD-1 protected cardiomyocytes to resist pathological remodelling induced by FFA stimulation in vitro
To

| Silencing of MD-1 accelerated high-fat diet induced myocardial function injury through increased cardiac fibrosis
To  Table 2). The content of CK-MB, a common serum marker for cardiac damage, was observably elevated after HD induction, especially in MD-1 −/− mice ( Figure 3B), indicating that HD-induced damage in heart tissue, which was much more serious in MD-1 −/− mice. The BW, HW and ratio of HW/TL were raised markedly after HD induction, especially in MD-1 −/− mice ( Figure 3C,D,F). However, the HW/BW ratio was lower dramatically in MD-1 −/− mice with HD than WT mice with HD ( Figure 3E). GTT also revealed reduced glucose tolerance in MD-1 −/− mice with HD than WT mice with HD ( Figure 3G). The mRNA of hypertrophy markers ANP, BNP and β-MHC were up-regulated in heart tissues of mice for HD feed, however, there is surprisingly just slight increases in MD-1 −/− mice compared to WT mice after HD feed ( Figure 3H).

| Overexpression of MD-1 alleviated high-fat diet induced cardiac dysfunction in vivo
To further confirm the effects of MD-1 on heart function in vivo by high-fat diet feed, transgenic mice overexpressing specially in myocardial cells by the α-myosin heavy chain (α-MHC) promoter were generated. Western blot validated that the overexpression efficiency in transgenic mice (TG) compared to wild-type mice (WT), and the MD-1 protein level was almost tripled ( Figure 5A). The enhanced activity of serum CK-MB by HD feed was observably suppressed in TG mice than WT mice ( Figure 5B), indicating that cardiac damage induced by HD were alleviated by overexpression of MD-1. The serum lipid indexes for TC, triglyceride and LDL and body weight were elevated remarkedly in mice by HD feed (Table 3 and Figure 5C); however, there was no significant difference between the HD-treated TG mice and WT mice on these indexed except TC (Table 3 and Figure 5C). The HW, ratios of HW/BW and HW/TL of TG hearts were lower markedly than WT hearts after HD feed ( Figure 5D-F).
Slight reduced glucose tolerance was observed in mice with HD treatment, however, there was no difference between TG mice and   For cardiac function analysis, echocardiographic measurements were also performed in TG and WT mice. The heart rate was faster in TG mice after HD feed than ND feed; however, the increase in heart rate in TG mice compared to WT was no significant ( Figure 6A). The IVSd and IVSs were decreased in TG hearts than WT hearts after HD feed ( Figure 6B,C); however there was no significant difference in LVDd, LVDs or FS in TG hearts ( Figure 6D). The increased perivascular and interstitial fibrosis induced by HD feed was reduced remarkedly in TG hearts compared to WT hearts ( Figure 6E). Consistently, the mRNA levels of COL1A1, COL3A1 and CTGF were down-regulated significantly in TG hearts than WT hearts with HD feed ( Figure 6F). All these results indicated that overexpression of MD-1 alleviated the myocardial dysfunction induced by HD feed through inhibiting cardiac fibrosis.

| MD-1 mediated HD-induced cardiac pathological remodelling via negatively regulating MAPK and NF-κB signalling
Compared with normal diet, high-fat diet administration markedly decreased the expression of MD-1 in WT mice hearts and transgenic mice hearts; however, the effect of HD was less in TG group than in WT group ( Figure 7A,B). Since MD-1 is reported to deliver cardiac protection against cardiac hypertrophy and suppresses cardiac dysfunction during the remodelling process through modulating MEK-ERK 1/2 and NF-κB signalling pathways. 19 Thus, we investigated the expression of MAPK and NF-κB pathways in the hearts of mice

| D ISCUSS I ON
Obesity is known to contribute to the development of cardiomyopathy. 3,[24][25][26][27] Although the underlying mechanism has been studies recently, the exact reason of obesity-induced cardiac hypertrophy and fibrosis is not fully understood. Wang

ACK N OWLED G EM ENT
This work was supported by the National Natural Science Foundation of China (grant no. 81570306)

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