Sphk1 involves in the regulation of autophagy process in cardiac myocyte cells at high glucose condition

Diabetic cardiomyopathy is the myocardium disorders caused by diabetes mellitus, which has become a key concern bringing heavy burden to the public health. Autophagy is one of activities involving in the pathogenesis of diabetic cardiomyopathy. Sphk1 gene plays a crucial role in cell survival and growth, and regulation of many diseases including diabetes and cardiovascular disease. However, the pathogenesis of diabetic cardiomyopathy remains poorly understood. To this aim, the study established adenovirus vectors expressing Sphk1 and shSphk1 to investigate the effects of Sphk1 on autophagy and cell survival in myocardial cells under high glucose conditions (25 mM). It was found that overexpression of Sphk1 promoted autophagy activity in H9c2 cells under high glucose treatment measured by various methodologies including qRT-PCR, western blot, fluorescence microscope, and so on. Inhibition of autophagy decreased cell vitality under high glucose condition. A broadly used medicine silibinin was demonstrated to induce autophagy in a dose-dependent manner. Herein, the findings in the present study may provide useful reference for unveiling the pathogenesis of diabetic cardiomyopathy and developing novel therapies treating diabetic cardiomyopathy.


Introduction
Diabetic cardiomyopathy (DCM) is defined as the myocardium disorders caused by diabetes mellitus, which was first to be reported in 1881 [1]. It is reported that diabetes become a key risk causing heat failure, and DCM is characterized by initial impairment of left ventricular (LV) relaxation followed by LV contractile dysfunction [2]. Notably, Finck and co-workers found that a transcription factor, peroxisome proliferator-activated receptor (PPAR)-α, closely regulated DCM in combination with its transcriptional targets [3]. This excellent study opened a window for studies towards the transcriptional mechanisms of DCM. This disease has been brought severe burden to the public health.
However, the exact mechanisms underlying DCM remain unclear by most physicians, even cardiologists and diabetologists.
As an important cellular process, autophagy is reported to be crucial for various diseases.
It was reported that autophagy plays an essential role in alleviating the development of insulin resistance and diabetes [4]. Autophagy also involves in muscle glucose uptake and mitigates endoplasmic reticulum (ER) stress in β cells under diabetogenic conditions [4].
As one of important components in autophagy process, lysosome is able to govern energy metabolism and its disorders disrupt autophagic activity, thus causing the accumulation of lipid within lysosomes [5]. In addition, autophagy plays an important role in maintaining intracellular homeostasis in a variety of cardiovascular derived cells including cardiomyocytes [5]. Autophagy has been confirmed to benefit for the growth of several cell types, for example, the induction of autophagy is able to promote cell survival and growth in cancer cells [6].
As a kinase producing sphingosine-1-phosphate (S1P), sphingosine kinase 1 (Sphk1), plays an essential role in cell survival and growth [7]. Sphk1 has been demonstrated to be functionally relevant to many diseases including cancer [7], Alzheimer's disease [8], inflammatory response [9], and Huntington's Disease [10]. In addition, Sphk1 is closely tied to type 2 diabetes [11]. Therefore, studies toward Sphk1 should be helpful for dissecting pathogenesis of diabetes. It is attractive to figure out the relationship between diabetes and cardiovascular disease, and ascertain the underlying mechanisms. Thus, the present study aims to explore the regulation of Sphk1 gene on autophagy in cardiac myocyte cells at high glucose circumstance, and corresponding mechanism, which should provide useful reference for developing novel therapies treating diabetic cardiomyopathy.

Cell culture
The embryonic rat heart-derived myogenic cell line H9c2 were purchased from the American Type Culture Collection (ATCC), and the cells were maintained in DMEM containing 10% FBS (V/V), 100 U/ml penicillin, and 100 μg/ml streptomycin in an atmosphere of 5% CO 2 at 37°C, as described previously [12,13]. All cells used in the present study were below the 35 th passage.
Afterwards, adenoviruses of Ad-Sphk1 and Ad-shSphk1 with multiplicity of infection (MOI) of 100 were used to transduce cells, followed by being selected in growth medium containing 1 mg/mL G418. All experiments were conducted in the absence of serum at 50% confluence. The transduction was verified by western blot and observation under fluorescence microscope.

The mRNA expression using quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from H9C2 cells using TRIzol (Invitrogen) according to the manufacturer's instructions [15,16]. The RNA reverse transcript (RT) was executed using  Table 1.

Lentiviral expression of green fluorescence protein bound to LC3
H9c2 Cells were cultured on a round cover slide on the bottom of well of a 6-well-plate with culture medium and transduced with lentiviral particles carrying a construct of TagGFP2-LC3 driven by the elongation factor-1 promoter (Millipore LentiBrite) for 48 h, followed by replacing cell culture medium containing drugs and treated for indicated time period. Green punctate representing autophagy was assessed by taking images using fluorescence microscope (Mshot, Guangzhou, China).

Monodansylcadaverine (MDC) staining
To assess autophagy, H9c2 Cells (10 5 cells/well) were plated in wells of 6-well plates and were treated with indicated treatments for indicated time. Then, cells were subjected to MDC staining buffer for 45 min at room temperature (Nanjing KeyGen Biotech co., Ltd.) according to the manufacturer's protocol. Subsequently, the fluorescence was measured using a fluorescence microscope (Mshot, Guangzhou, China). The activated autophagosome represented acidic vesicles, which was tested by measuring the level of green fluorescence.
To further measure autophagy, after that cells were stained using MDC, the cells were analyzed by a flow cytometry (Altra; Beckman), and 10000 events were recorded per sample at wave length of 355 nm, and data were analyzed by Flow Jo software (Version 7.6.1).

Statistical analysis
All data were analyzed using GraphPad software (version: GraphPad Prism5). Data are presented as mean ± SEM and the standard errors of the mean in this study were carried out in triplicates. Statistical comparisons between 2 groups were performed by t test. P < 0.05 was considered to be a statistically significant difference.

Evaluation of transduce efficiency of Ad-Sphk1 and Ad-shSphk1
Sphk1 gene plays an important role in the regulation of autophagy in several cell types [17], and this gene was also reported to be associated with coronary artery disease [18].
Therefore, it is interesting to probe the effects of Sphk1 on autophagy in H9C2 cells. To better dissect the functions of Sphk1, the overexpression and knockdown adenovirus vectors including Ad-Sphk1 and Ad-shSphk1 were generated, respectively. To confirm the success of adenovirus vectors, the virus particles of Ad-Sphk1, Ad-Sphk1-ctrl, Ad-shSphk1, and Ad-shSphk1-ctrl were used to infect H9C2 cells, which showed potent fluorescence intensity in cells infected by all four vectors (Figure 1A). Since Ad-Sphk1 vector carries chimera cDNA encoding DDDDK, thus to further verify, DDDDK protein was measured using western blot method, which indicated that Ad-Sphk1 adenovirus infected H9C2 cells expressed DDDDK protein compared with Ad-Sphk1-ctrl and negative control (NC) ( Figure   1B).  Figure 3A). The similar results were observed by using flow cytometry ( Figure 3B).

Silibinin increased autophagy in glucose supplemented H9C2 cell model
Silibinin is a key component of Silymarin that is an extract isolated from herb Silybum marianum (milk thistle), and this chemical exerts anti-oxidant and anti-inflammatory effects to protect many types of cells from injury caused by various risks [19]. To check the effects of autophagy on vitality of cardiomyocytes under high glucose condition, different concentrations of silibinin were used to treat lentivirus expressing both GPF and LC3B infected H9C2 cells, which indicated that silibinin enhanced autophagy in a dose dependent manner ( Figure 4A). To further investigate, beclin1 and LC3B protein level were measured in Ad-Sphk1, Ad-shSphk1, glucose and silibinin co-treated cells using western blot. Interestingly, it was found that higher glucose (25 mM) decreased beclin1 and LC3B level irrespective of silibinin, Ad-Sphk1 and Ad-shSphk1 treatments compared with lower glucose (5.5 mM) group ( Figure 4B). Notably, higher glucose increased cytotoxicity and decreased cell vitality, and inhibition of autophagy by autophagy inhibitors 3-MA and Z-DEVD-FMK increased cytotoxicity and decreased cell vitality ( Table   2). Above results manifested that autophagy closely regulated cell vitality of cardiomyocytes under high glucose circumstance, silibinin was able to promote autophagy in cardiomyocytes, and might be a candidate therapy for DCM disease.

Discussion
Along life quality improvement, DCM disease becomes a commonplace problem all over the world, and is bringing a severe burden for the public health. Thus the development of novel and effective medicines or therapies is warranted to defense the problematic disease. In the present study, we generated gain and loss-of functions of Sphk1 gene adenovirus vectors to transduce H9C2 cells. Sphk1 was confirmed to promote autophagy, high glucose remarkably decreased autophagy and cell vitality. Silibinin enhanced autophagy in H9c2 cells under high glucose condition, and might be a promising medicine to treat DCM.

Sphk1 gene plays an important role in a variety of diseases including diabetes mellitus
and vascular complications [20,21], although the underlying mechanism remains poorly clear. This gene has been reported to be tightly associated to autophagy process in several cell types. The previous study has been demonstrated that Sphk1 was capable of promoting autophagy in human peritoneal mesothelial cells (HPMCs), and this process aided the adhesion and invasion of HPMCs [17]. Wang and co-workers demonstrated that microRNA-506-3p was able to inhibit autophagy through targeting Sphk1 in osteosarcoma cells [22]. In another study, authors demonstrated that Sphk1/ERK/p-ERK pathway involved in the induction of autophagy in colon cancer cells [23]. Inconsistently, it was reported that the inhibition of Sphk1 expression could induce autophagy in head and neck squamous cell carcinoma cells [24]. The conflict might be due to that the physiological variation exists in different cell types. In the present study, we confirmed that activation of Sphk1 was able to induce autophagy and silence of the gene was capable of inhibiting autophagy in myocardial cells at high glucose condition (Figure 2 and Figure 3). Importantly, the present study found that autophagy had capacity to ameliorate cytotoxicity ( Table 2). Autophagy is a cellular non-specific, bulk degradation process, which is in charge of defending against nutrition environmental stress via removing damaged macromolecules and organelles [15]. Thus autophagy is closely related to the cell survival and growth. In dead, many studies have been found that autophagy supports cell proliferation and growth. For examples, a mostly recent study performed by Yang and co-authors indicated that autophagy was capable of protecting nucleus pulposus cells from the apoptosis induced cyclic mechanical tension [25]. Autophagy is also reported to potently increase human hepatocellular carcinoma HepG2 cell proliferation, by using this notion, Okubo et al screened approximately 130 kinds of crude drugs to discovery effective crude drugs supporting cell growth via regulating autophagy process [26].
Emerging evidence indicate that diabetes mellitus is linked to cardiovascular disease. To dissect the pathogenesis and develop effective therapies against diabetes induced cardiovascular disease. A glucose supplemented myocardial cell model was used in the present study. Importantly, our study confirmed high glucose inhibited autophagy and promoted cytotoxicity in myocardial cells (Figure 2 and Table 2). Sphk1 was demonstrated to induce autophagy in high glucose treated cells. These results are consistent with previous studies, in which, authors stated that Sphk1 was a modulator to closely regulate angiotensin II-induced vascular dysfunction in vascular tissues [27].
Importantly, our study confirmed that silibinin exerted induction of autophagy in myocardial cells (Figure 4). Interestingly, a previous study found the similar results, in which authors demonstrated that silibinin exerted protective effect on pancreatic β-cells from inflammation factors including TNFα-or IL-1β-induced cell death [27]. Consistently, Rezabakhsh et al reported glucose promoted the death of human endothelial cells, while silibinin was able to protect human endothelial cells from high glucose-induced cytotoxicity by inducing autophagy [28]. In another study, authors observed the similar phenomenon that silybin exerted cytoprotective effects on human, rat, chicken hepatoma cells and rat myoblasts, although the protection effect was not related to the autophagy process [29]. Similarly, silibinin was stated to reduce arsenic-induced oxidative stress

Conflicts of Interest
We have declared that no competing interests exist. The authors alone are responsible for the content and writing of the paper.

Ethical approval
The study was approved by the Ethics Committee of Shenzhen Nanshan People's Hospital and The six affiliated Hospital of Shenzhen University Health Center.