MD1 deletion exaggerates cardiomyocyte autophagy induced by heart failure with preserved ejection fraction through ROS/MAPK signalling pathway

Abstract In our previous studies, we reported that myeloid differentiation protein 1 (MD1) serves as a negative regulator in several cardiovascular diseases. However, the role of MD1 in heart failure with preserved ejection fraction (HFpEF) and the underlying mechanisms of its action remain unclear. Eight‐week‐old MD1‐knockout (MD1‐KO) and wild‐type (WT) mice served as models of HFpEF induced by uninephrectomy, continuous saline or d‐aldosterone infusion and a 1.0% sodium chloride treatment in drinking water for 4 weeks to investigate the effect of MD1 on HFpEF in vivo. H9C2 cells were treated with aldosterone to evaluate the role of MD1 KO in vitro. MD1 expression was down‐regulated in the HFpEF mice; HFpEF significantly increased the levels of intracellular reactive oxygen species (ROS) and promoted autophagy; and in the MD1‐KO mice, the HFpEF‐induced intracellular ROS and autophagy effects were significantly exacerbated. Moreover, MD1 loss activated the p38‐MAPK pathway both in vivo and in vitro. Aldosterone‐mediated cardiomyocyte autophagy was significantly inhibited in cells pre‐treated with the ROS scavenger N‐acetylcysteine (NAC) or p38 inhibitor SB203580. Furthermore, inhibition with the autophagy inhibitor 3‐methyladenine (3‐MA) offset the aggravating effect of aldosterone‐induced autophagy in the MD1‐KO mice and cells both in vivo and in vitro. Our results validate a critical role of MD1 in the pathogenesis of HFpEF. MD1 deletion exaggerates cardiomyocyte autophagy in HFpEF via the activation of the ROS‐mediated MAPK signalling pathway.

patients and the pathophysiology of HFpEF are poorly understood.
Moreover, there are no evidence-based therapies for HFpEF. It is important to explore the pathophysiology of HFpEF to develop novel targets for use in HFpEF therapy.
Autophagy was identified as a cytoplasmic component degradation process paramount to cellular homeostasis and survival. Microtubuleassociated protein light chain-3 (LC3) is a major component of autophagosomes. The cytoplasmic form LC3I is converted to LC3II in autophagosomes. Therefore, LC3 was identified as the main specific autophagy indicator. 6,7 Increasing evidence indicates that autophagy plays an important role in HFpEF. Essick et al reported that adiponectin suppresses HFpEF-induced autophagy by reducing the LC3II/LC3I protein expression ratio. 8 Moreover, impaired autophagy was found in mice with STZ-induced diastolic dysfunction, as indicated by increased LC3 protein expression and decreased P62 protein expression. 9 Increasing evidence suggests that overproduced reactive oxygen species (ROS) regulate autophagy. 10,11 A recent study demonstrated that ROS can induce autophagy via the MAPK signalling pathway. 12 Another study indicated that paroxetine hydrochloride blocked the autophagic flux through the ROS/MAPK signaling pathway. 13 Therefore, it is believed that the ROS/MAPK signalling pathway may contribute to HFpEF-induced autophagy. Theoretically, finding the molecules suppressing ROS/MAPK signalling pathway in HFpEF-induced autophagy would be of great benefit for developing therapies for HFpEF-induced autophagy.
Myeloid differentiation protein 1 (MD1) is a secreted glycoprotein that forms a complex with radioprotective 105 (RP105). 14 MD1-RP105 has been demonstrated to be an important TLR4 negative regulator. 15 MD1 is expressed predominantly in B cells, macrophages and other immune cells. 16 Recently, MD1 was reported to play a negative role in several cardiac pathological processes, including pressure overload-induced cardiac and electrical remodelling 17 and high-fat diet-induced atrial remodelling. 18 Moreover, MD1 also plays an important role in aortic banding-induced cardiac remodelling via the MAPK signalling pathway. 19 However, the role of MD1 in HFpEF-induced autophagy remains largely unknown. Therefore, we explored the role of MD1 in aldosterone-induced HFpEF both in vivo and in vitro.

| Reagents
Aldosterone was purchased from Sigma-Aldrich (St. Louis, MO, USA).   21 At the endpoint of treatment, all the mice were anaesthetized with 1.5% isoflurane, and echocardiographic measurements and haemodynamic analysis were performed. After that, hearts were weighed and snap-frozen in liquid nitrogen for the further detection.
M-mode images of the left ventricle at the papillary muscle level were recorded and then left ventricular ejection fraction (EF) and the fractional shortening (FS) were measured. 22 Haemodynamic variables were analysed using a Millar catheter transducer (SPR-839; Millar Instruments, Houston, TX). After stabilization for 15 min, the LV diastolic function indicators left ventricular minimum rates of pressure rise (dp/dtmin), left ventricular relaxation time constant (Tau) and end-diastolic pressure-volume relationship (EDPVR) were recorded continuously with an ARIA pressure-volume conductance system coupled with a PowerLab/4SP A/D converter. The data were analysed using LabChart 7 software.

| Histological analysis and DHE staining
The mice were anaesthetized with pentobarbital sodium (50 mg/ kg, i.p.) at 4 weeks after HFpEF and then hearts were harvested.
The tissues were fixed in 10% formalin, embedded in paraffin and sectioned at 5 μm. Left ventricular sections were stained with haematoxylin and eosin (H&E) for histopathology. Cryosections of fresh heart samples were stained with DHE (10 μM) for 30 min at 37°C to detect ROS production. Pictures were taken with an OLYMPUS DX51 fluorescence microscope (Tokyo, Japan).

| Immunofluorescence staining
Paraffin sections were also used for immunofluorescence, and slides were incubated with primary antibodies against LC3 (CST, MA, USA). Slides were washed and incubated with fluorescencelabelled secondary antibodies. The results were blindly calculated/ section. After being washed 3 times with PBS, the H9C2 cells were visualized in a blinded manner under an Olympus IX53 fluorescence microscope.

| Western blot and quantitative real-time PCR
The total proteins were extracted from the frozen heart tissues or iced cell lysates by RIPA agent (Invitrogen, Carlsbad, CA, USA).  Table 1. The mRNA data were normalized to GAPDH.

| Transmission electron microscope (TEM)
TEM for autophagy analysis was performed according to standard operating procedures. For morphological TEM, hearts from saline or d-aldosterone treated mice were fixed in 2.5% glutaraldehyde (Sigma) in phosphate buffer overnight at 4°C. After sample preparation, 90-100 nm thick sections were mounted onto a 200 mesh copper grid (Electron Microscopy Sciences, Hatfield, PA, USA) and imaged with an FEI Tecnai 12 120 kV transmission electron microscope equipped with an AMT XR80C 8 megapixel CCD camera.

| Statistical analysis
All data in the tables and figures are expressed as mean ± SEM and were analysed using GraphPad Prism 8.0 software. The differences between two groups were performed with unpaired Student's t test.
The differences were analysed with one-way analysis of variance (ANOVA) with Tukey post hoc analysis. P < .05 was considered as statistically significant.

| MD1 expression was decreased when aldosterone was administered
To investigate whether MD1 is involved in aldosterone-induced HFpEF, we first examined MD1 expression in an aldosterone-induced HFpEF model. As shown in Figure 1A-C, MD1 protein levels were significantly decreased in the HFpEF mice compared with those of the sham controls. In vitro studies showed that MD1 expression was significantly down-regulated in H9C2 cells treated with aldosterone (Aldo, 1 μM) for 12, 24 and 48 hour in a time-dependent manner ( Figure 1B-D). These results suggest that MD1 down-regulation is associated with aldosterone-induced HFpEF and that MD1 may be implicated in the development of HFpEF.

| MD1 deficiency aggravated the HFpEF phenotype acquired after aldosterone was administered to the mice
Next, we were interested in determining whether MD1 deficiency results in an altered cardiac phenotype. Left ventricular hypertrophy (LVH) plays a major pathophysiological role in HFpEF. 24 We initially investigated LVH in a HFpEF mouse model, measuring it by cardiac myocyte size. H&E staining showed that the cross-sectional area of the HFpEF-WT sample was significantly greater than that of the sham-WT and that HFpEF-MD1-KO mice had HFpEF-induced LVH (Figure 2A

| MD1 deletion accelerated the rate of HFpEFinduced autophagy
Overactivated autophagy was reported to play an important role in HFpEF. 8,9 Therefore, we focused on autophagy and tested whether MD1 deletion accelerated HFpEF-induced autophagy.
As depicted in Figure 3A, we detected the formation of autolysosomes in the HFpEF and HFpEF-MD1-KO mice by TEM analysis, indicating the occurrence of autophagy, which was confirmed by LC3 immunofluorescence staining. As shown in Figure 3B-C, the LC3 intensity was significantly increased in the HFpEF mice compared to that in the WT mice, and the MD1-KO mice exhibited enhanced HFpEF-induced autophagy. Moreover, we found that the indicators of autophagy, the LC3II/LC3I ratio and P62 level, were significantly increased and significantly decreased, respectively, in the HFpEF mice, and MD1 deletion increased the LC3II/LC3I ratio

| Elimination of MD1 caused deleterious cardiac oxidative injury in the HFpEF mice
Reactive oxygen species (ROS) have been identified as signalling molecules in various pathways that regulate cell autophagy through several mechanisms. 26,27 To investigate whether ROS plays a role in the induction of the autophagy induced by HFpEF, DHE staining was used to measure the ROS levels. As expected, the HFpEF mice exhibited excessive ROS production, and MD1 elimination increased the ROS production induced by HFpEF ( Figure 4B)

| Effect of the ROS-mediated P38/MAPK signalling pathway on HFpEF-induced autophagy
In our previous study, we demonstrated that MD1 deficiency exacerbates cardiac remodelling by activating the MAPK pathway 19 and that the ROS/MAPK signalling pathway, reported to be associated with cell autophagy, is a major pathway in this deleterious process. 28 We then sought to evaluate whether the ROS/MAPK signalling pathway contributes to the role of MD1 KO in enhancing HFpEFinduced autophagy. We first analysed the expression of the MAPK component P38 both in vivo and in vitro. As shown in Figure 5A

| Inhibition of autophagy offsets the exaggerated effects induced by knocking down MD1 in the H9C2 cells
The above results showed that knocking out MD1 may suppress HFpEF-induced autophagy. We speculated that the inhibition of  Figure 6C-E). These results were confirmed by LC3 immunofluorescence staining ( Figure 6A-B).

| Inhibition of autophagy abolished the exacerbated effects in the MD1-KO HFpEF mice
To confirm the role of autophagy in HFpEF, we treated mice with

| D ISCUSS I ON
In this study, we uncovered evidence that MD1 acts as a novel negative regulator of HFpEF-induced autophagy. The expression of MD1 HFpEF is a disease characterized by the signs and symptoms of HF with preserved LVEF, accompanied by diastolic function, and is an increasingly serious health problem, with morbidity and mortality rates similar to those of HFrEF. 29,30 In addition, the prevalence of HFpEF continues to increase. No specific therapies have been proven beneficial in terms of reducing morbidity or mortality, despite its increasing prevalence, due to our poor understanding of the pathophysiological process of HFpEF. 31 To explore potential novel therapeutic targets for HFpEF, the current study aimed to examine the role of MD1 in HFpEF. Initial experiments showed that MD1 was predominantly expressed in the innate immune system. 32,33 MD1 was found to be expressed in dendritic cells, and the loss of MD1 could weaken dextran sodium sulphate (DSS)-induced colitis by modulating the function of colonic lamina propria dendritic cells. 34 Furthermore, MD1 is also expressed in macrophages and exerts a regulatory role in lupus-prone MRL mice. 35 In our previous research, we found that MD1 is expressed in cardiomyocytes. MD1 serves as a negative regulator in cardiac remodelling. [17][18][19] Consistent with our previous report, the current study demonstrated that MD1 expression was decreased in both HFpEF mice and Aldo-treated H9C2 cells (Figure 1). We next checked whether MD1 deficiency resulted in an altered cardiac phenotype. Pathologic LVH with diastolic dysfunction is the basic feature of HFpEF, in which the fundamental problem is the inability of the ventricle to relax. In this study, we verified that the aldosterone-induced HFpEF models exhibited LVH and diastolic dysfunction ( Figure 2). MD1 deficiency significantly aggravated LVH.
Moreover, MD1 ablation enhanced LV and passive cardiac stiffness and lung congestion without affecting the LVEF (Figure 2). Our findings strongly support the supposition that MD1 serves as a negative regulator in HFpEF.
Autophagy is important in regulating cell survival, including in the healthy heart. However, autophagy can be activated under pathological conditions, including HF and LVH. 36,37 Autophagy was reported to be involved in the pathophysiological process of HFpEF. 8 In a STZ-induced HFpEF model, autophagy was enhanced, LC3 expression was increased and the P62 level was decreased. 9 The LC3 and P62 expression levels are strongly related to the extent of autophagosome formation. However, to date, no relevant study has reported that MD1 has an effect on autophagy. We sought to determine whether the deleterious effect of autophagy is attributable to the deletion of MD1. In line with previous studies, 8,9 we found that autophagy was enhanced in the  (Figures 6 and 7). However, the exact mechanism by which MD1 deficiency aggravates HFpEF-induced autophagy remains unclear.   19 On the other hand, increasing evidence has implicated the ROS-mediated MAPK signalling pathway in the autophagy induced by multiple stressors. 13,42 The

| CON CLUS IONS
In conclusion, the present study indicates that MD1 may serve as a negative regulator in the pathogenesis of HFpEF. MD1 deficiency exacerbates autophagy induced by HFpEF via activation of ROS/ MAPK signalling pathway. Thus, modulating MD1-ROS/MAPK axis may provide a promising therapeutic approach for HFpEF.

ACK N OWLED G EM ENTS
This work was supported by grants from the key R&D Program (No. 2017YFC1700504) and the National Natural Science Foundation of China (No. 81570306).

CO N FLI C T O F I NTE R E S T
The authors disclose no conflict of interest. All data generated during the current study are available from the corresponding author on reasonable request.