FAT10 Combined with Miltefosine Inhibits Mitochondrial Apoptosis and Energy Metabolism in Hypoxia-Induced H9C2 Cells by Regulating the PI3K/AKT Signaling Pathway

Hypoxia-induced cardiomyocyte apoptosis is the main contributor to heart diseases. Human leukocyte antigen F-associated transcript 10 (FAT10), the small ubiquitin-like protein family subtype involved in apoptosis, is expressed in the heart and exhibits cardioprotective functions. This study explored the impact of FAT10 on hypoxia-induced cardiomyocyte apoptosis and the involved mechanisms. The cardiomyocyte cell line H9C2 was cultivated in hypoxia-inducing conditions (94% N2, 5% CO2, and 1% O2) and the expression of FAT10 in hypoxia-stimulated H9C2 cells was identified. For this, FAT10 overexpression/interference vectors were exposed to transfection into H9C2 cells with/without the PI3K/AKT inhibitor, miltefosine. The results indicated that hypoxia exposure decreased the FAT10 expression, suppressed H9C2 cell growth, disrupted mitochondrial metabolism, and promoted H9C2 cell apoptosis and oxidative stress. However, these impacts were reversed by the FAT10 overexpression. In addition, the inhibition of PI3K/AKT in FAT10-overexpressing cells suppressed cell proliferation, impaired mitochondrial metabolism, and promoted apoptosis and oxidative stress response. The findings demonstrated that FAT10 inhibited mitochondrial apoptosis and energy metabolism in hypoxia-stimulated H9C2 cells through the PI3K/AKT pathway. This finding can be utilized for developing therapeutic targets for treating heart disorders associated with hypoxia-induced apoptosis.


Introduction
Hypoxia is a prevalent pathophysiological event in cardiovascular diseases. Inadequate oxygen supply hinders energy metabolism in cardiomyocytes, leading to cell injury and reduced cardiac functional ability [1]. Mitochondrial damage occurring in cardiomyocytes upon hypoxic exposure induces apoptosis together with oxidative stress (OS), References [2,3]. Hypoxia-induced cardiomyocyte apoptosis is the major contributor to heart disorders, including myocardial infarction [4,5] and congenital heart disease, Reference [6]. Because cardiomyocytes are terminally differentiated, preventing their loss is the major challenge in treating heart diseases [7]. Another challenge is the complicated mechanism of hypoxia-induced cardiomyocyte apoptosis references [2]. Consequently, investigating molecular mechanisms underlying hypoxia-induced cardiomyocyte apoptosis can contribute to developing potential therapeutic strategies.
Human leukocyte antigen F-associated transcript 10 (FAT10), the small ubiquitin-like protein family subtype, is upregulated in several tumor types, including hepatocellular carcinoma (HCC), gastric carcinoma, and gynecological malignancies [8]. Additionally, FAT10 is expressed in the heart and exerts a cardioprotective effect. Reference [9] reported an enhancement in FAT10 levels in cultivated neonatal rat cardiomyocytes that experienced a hypoxia or reoxygenation condition. e elevated FAT10 level caused an enhancement in the level of B cell leukemia or lymphoma 2, also known as apoptosis regulator Bcl-2 (Bcl-2), and a decrease in the level of BCL2 associated X protein (Bax), thereby inhibiting hypoxia-or reoxygenation-induced cardiomyocyte apoptosis [10]. Furthermore, previous research studies have suggested that FAT10 regulates several cellular processes through protein kinase B (AKT) and AKT-related signaling pathways. Zou demonstrated that FAT10 contributed to bladder cancer development by promoting EGFR/AKT-mediated activation of HK2 [11], which suggested that FAT10 induced epithelial-mesenchymal transition and accelerated HCC cell metastasis by modulating the AKT/GSK3β pathway, thereby affecting the HCC prognosis [12]. Moreover, AKT refers to a downstream effector of phosphoinositide 3-kinase (PI3K); therefore, PI3K/AKT represents a critical pathway of cellular processes that are responsible for cell survival [13,14]. A study indicated that the activation of PI3K/AKT signaling inhibited hypoxiainduced apoptosis in vascular endothelial cells [15]. Apart from that, the PI3K/AKT pathway was related to the apoptosis of cardiomyocytes subjected to ischemic hypoxia [4]. Nevertheless, the impact of FAT10 on hypoxia-induced cardiomyocyte apoptosis and whether its potential action mechanism is related to PI3K/AKT signaling remain unexplored.
In this study, the cardiomyocyte cell line H9C2 was cultured in hypoxic conditions to evaluate the expression of FAT10. e FAT10 overexpression or interference vectors were co-transfected into H9C2 cells with or without the PI3K/AKT inhibitor, miltefosine. Cells were exposed to treatment with 1 μM miltefosine for 48 h [16], followed by culturing in hypoxic conditions to study the impact of FAT10 on hypoxia-induced cardiomyocyte apoptosis.

Cell Culture Procedures.
e cardiomyocyte H9C2 cells (Shanghai Institutes for Biological Sciences, Chinese Academy of Science) were cultivated in Dulbecco's Modified Eagle Medium (DMEM)-high glucose medium ( # SH30022.01, Hyclone, USA) containing 10% fetal bovine serum (FBS, # 10270-106, Gibco, USA) with 5% CO 2 as well as 95% O 2 at 37°C. In terms of hypoxia induction, H9C2 cells at the logarithmic growth phase were cultured at 37°C with 5% CO 2, 1% O 2 , and 94% N 2 for 6, 12, and 24 h. Cell viability was detected, and the FAT10 expression was determined with the application of the Cell Counting kit-8 (CCK-8) assay using quantitative RT-PCR (qRT-PCR). e H9C2 cell suspension was seeded into a 12-well plate at 1 × 10 5 cells per well, and cells were transfected once the cell density reached 70%. Subsequently, 1.6 μg of the FAT10 overexpression or interference plasmids were diluted with 100 μL opti-MEM ( # 31985070, Gibco, USA). Besides, the mixture was incubated for 5 min and mixed with the transfection reagent containing 2 μL lipofectamine 2000 reagent ( # 11668027, Invitrogen, USA) and 100 μL opti-MEM; the mixture was incubated for 20 min. Next, 800 μL of the basic culture medium and 200 μL plasmid mixtures were supplemented to each well of the 12-well plate and incubated at 37°C with 5% CO 2 for 48 h.

Cell Activity.
e CCK-8 assay was carried out with the purpose of examining H9C2 cell viability. For this, H9C2 cells (3 × 10 3 /well) were seeded in a 96-well plate, followed by overnight incubation with 5% CO 2 and 95% O 2 at 37°C. Cells were exposed to transfection with the FAT10 overexpression or interference plasmids and with or without 1 μM miltefosine, followed by culturing at hypoxic conditions for 24 h (5% CO 2, 1% O 2 , and 94% N 2 ). ereafter, CCK-8 (10 μL, # CA1210, Solarbio, China) was supplemented to each well and further cultured for 4 h. Besides, the plate was placed on a microplate reader (Allsheng, China), and the optical density was read at 450 nm.
en, cells were determined using flow cytometry (ACEA Biosciences, USA).

Observation of the Mitochondrial Ultrastructure.
e mitochondrial ultrastructure of H9C2 cells was observed with the transmission electron microscope. For this, 1 × 10 7 H9C2 cells were prefixed for 20 min with 2 mL of 2.5% glutaraldehyde, followed by 1 h post-fixation by adding 1% osmic acid.
rough dehydration, permeation, and embedding, the samples were sliced into 60 nm-thick samples, followed by 20 min staining with uranyl acetate and another 15 min staining with lead citrate in the dark. e mitochondrial ultrastructure was observed with the use of a transmission electron microscope (Hitachi).

Statistical Analysis.
Data are denoted to be mean ± SD. One-way analysis of variance (ANOVA) in combination with the Tukey test was employed with the aim of analyzing differences across diverse groups; p < 0.05 was regarded to show statistical significance. GraphPad Prism 8 software was employed for graph construction. Evidence-Based Complementary and Alternative Medicine 3

Hypoxia Downregulates the FAT10 Expression in H9C2
Cells. According to Figures 1(a) and 1(b), hypoxia suppressed H9C2 cell viability and downregulated FAT10 mRNA levels through the time-dependent manner. In comparison with control cells, a statistical difference in FAT10 mRNA expression was observed after 24 h of hypoxic culturing; therefore, we considered 24 h as the culture time for further experiments.

Effect of the FAT10 Overexpression on Proliferation of
Hypoxia-Stimulated H9C2 Cells and Apoptosis Reversion through PI3K/AKT Inhibition. As depicted in Figures 2(a) and 2(b), the FAT10 expression in H9C2 cells was silenced or overexpressed by interference and overexpression vectors, respectively, and shRNA1 was randomly chosen for the subsequent experiments. According to the CCK-8 assay, the FAT10 overexpression weakened the inhibitory effect of hypoxia in H9C2 cells, whereas this effect was strengthened by FAT10 interference or PI3K/AKT inhibition prior to the hypoxia treatment (Figure 2(c)). In addition, hypoxia-induced H9C2 cell apoptosis was accentuated by FAT10 interference or PI3K/AKT inhibition but weakened by FAT10 overexpression prior to the hypoxia treatment. Apoptosis in FAT10-overexpressing hypoxia-stimulated cells was further promoted by miltefosine treatment (Figure 2(d)). e apoptotic protein levels, analyzed through Western blot, revealed that hypoxia treatment upregulated Bax, and this effect was accentuated by FAT10 interference or PI3K/AKT inhibition but weakened by FAT10 overexpression prior to the hypoxia treatment. Bax protein levels in FAT10-overexpressing hypoxia-stimulated cells further increased after miltefosine exposure. e Bcl-2 expression exhibited the opposite trend as that of Bax (Figure 3). Figure 4, hypoxia decreased SOD, GSH, and GSH-PX levels, and this effect was strengthened by FAT10 interference or PI3K/AKT inhibition but weakened by the FAT10 overexpression prior to the hypoxia treatment. e levels of SOD, GSH, and GSH-PX in FAT10-overexpressing hypoxia-stimulated cells decreased after miltefosine treatment. e levels of MDA and LDH and cell proportion with increased ROS production demonstrated an opposite trend as that of SOD, GSH, and GSH-PX levels. (Figure 5(a)) and the expressions of DRP1, MTFP1, p-DRP1 (S616), glucose ( Figure 5(e)), and lactic acid ( Figure 5(f )); decreased MFN2, p-DRP1 (S637) (Figure 5(c)), and ATP ( Figure 5(d)) levels and disrupted the mitochondrial ultrastructure ( Figure 5(b)). e effects of hypoxia were aggravated by FAT10 interference or PI3K/AKT inhibition but mitigated by FAT10 overexpression prior to the hypoxia treatment. In addition, these effects in FAT10-overexpressing hypoxiastimulated cells were reversed by the addition of miltefosine.

FAT10 Overexpression Enhances PI3K/AKT Signal
Activation. According to the results of Western blot, hypoxia treatment reduced FAT10, p-AKT, and p-PI3K activities, and this effect was strengthened by FAT10 interference or PI3K/ AKT inhibition but weakened by FAT10 overexpression prior to the hypoxia treatment. e activities of FAT10, p-AKT, and p-PI3K in FAT10-overexpressing hypoxia-stimulated cells were suppressed by the addition of miltefosine ( Figure 6).

Discussion
Apoptosis involves highly complicated mechanisms and a series of energy-dependent molecular events [17]. In general, the mechanism of apoptosis is classified into two primary pathways: the intrinsic pathway and the extrinsic pathway. e intrinsic pathway or mitochondrial apoptosis is a mitochondria-dependent process that induces apoptosis through intracellular signals that directly target cells; these signals are produced by various nonreceptor-mediated stimuli [17]. Mitochondria are an energy-producing organelle and a dominating ROS cell source. Mitochondria manage cell survival and apoptosis principally through   Evidence-Based Complementary and Alternative Medicine fission and fusion, processes involved in homeostatic balance maintenance [18][19][20]. Several proteins that control mitochondrial fusion and fission, including optic atrophy 1, DRP1, MTFP1, and MFN1/2, are also implicated in apoptosis and are indispensable for maintaining mitochondrial morphology [21][22][23][24]. Mitochondrial ultrastructure and homeostasis are intimately associated with the crosslink between cell survival/death and bioenergetic processes [25]. Moreover, the mitochondrial ultrastructure directly influences the bioenergetic function of mitochondria [26].  Mitochondrial fragmentation is often associated with mitochondrial dysfunction and ATP depletion [26,27]. Accumulating evidence indicates that inhibiting mitochondrial fusion enhances apoptosis. Proteins associated with mitochondrial fusion, namely, MFN1 and MFN2, not only increase mitochondrial connectivity but also leads to Bax activation and Cyt-c release delay, thereby inhibiting mitochondrial apoptosis [28]. A study reported that the inhibition of MFN1 or MFN2 increased the sensitivity of apoptotic stimuli, thereby leading to mitochondrial fragmentation [28]. Mitochondria is a primary source of ROS, whose accumulation contributes to OS [29,30]. Under OS conditions, MFN2 expression declines, whereas the protein related to mitochondrial fission, DRP1, elevates, leading to mitochondrial fragmentation, decreased energy metabolism, and ultimately mitochondrial apoptosis [31][32][33]. Our results indicated that hypoxia enhanced OS response, promoted mitochondrial apoptosis, and inhibited the metabolism of mitochondrial energy in H9C2 cells; these findings conform to those of previous studies [34][35][36][37]. However, the effects of hypoxia were weakened by the FAT10 overexpression.
Pretreatment with miltefosine to inhibit PI3K/AKT aggravated hypoxia-induced damage in H9C2 cells and negated the attenuating impact of the FAT10 overexpression on hypoxia-induced H9C2 cell damage. Several studies have suggested that FAT10 participates in several cellular events through the regulation of AKT-related signaling pathways [11,12]. AKT refers to a downstream effector for PI3K, and the PI3K/AKT signaling pathway modulates cellular events, including cell growth, metabolism, and apoptosis [38][39][40] demonstrated that PI3K/ AKT activation attenuated OS-induced mitochondria-dependent apoptosis by upregulating Bcl-2 and suppressing Bax, Cyt-c, and cleaved caspase 9/3 expression [41]. PI3K/ AKT pathway activation also inhibited OS and apoptosis in streptozotocin-mediated diabetic cardiomyopathy [42]. Apart from that, PI3K/AKT activation suppressed doxorubicin-induced mitochondrial OS by reducing ROS production in H9C2 cells and prevented doxorubicin-induced insufficient mitochondrial fusion and excessive mitochondrial fission by promoting DRP1 phosphorylation (S637) [43]. Miltefosine is the most commonly used PI3K/ AKT pathway inhibitor [44].

Conclusion
In conclusion, our work revealed that FAT10 inhibited intrinsic apoptosis induced by OS and mitochondrial dysfunction in hypoxia-stimulated H9C2 cells. We suggest that the underlying mechanism is mediated by PI3K/AKT signaling activation, which was inhibited by miltefosine. ese findings define FAT10 as the therapeutic candidate to treat  Figure 6: FAT10 overexpression promotes PI3K/AKT pathway activation. Evaluation of FAT10, AKT, p-AKT, PI3K, and p-PI3K protein levels in H9C2 cells by adopting Western blot. In addition, data are denoted to be mean ± SD, n � 3, * p < 0.05.