Differential effects of and mechanisms underlying the protection of cardiomyocytes by liver-X-receptor subtypes against high glucose stress-induced injury

Qing He 1, Fengdan Wang 1, Yuqi Fan, Changqian Wang, Junfeng Zhang

Abstract

Liver-X-receptors (LXRs) are ligand-activated transcription factors belonging to the nuclear receptor superfamily. The two popular homologous receptor subtypes, LXRa and LXRb, exhibit differential expression patterns, thereby probably playing different roles in different contexts. This study aimed to evaluate the different roles of the two LXR subtypes and the mechanisms underlying their protection of cardiomyocytes against high-glucose stress. Silencing of LXRa, but not LXRb impaired normal LXRmediated cardioprotective effects against high glucose-induced oxidative stress, apoptosis, and inflammation. Mechanistically, silencing of small ubiquitin-like modifier (SUMO)1 or SUMO2/3 did not affect LXRmediated cardioprotective effects; however, these were impaired in response to nuclear receptor corepressor (NCoR) silencing. Together, these findings indicate that LXRa, but not LXRb, protects against high glucose-induced cardiomyocyte injury, probably via the NCoR-dependent transrepression of downstream target genes.

Keywords:
Liver-X-receptors
Glucose
Expression
Cardiomyocytes Oxidative stress

1. Introduction

According to the World Health Organization, the global prevalence of diabetes mellitus (DM) among adults has increased considerably in recent decades and is estimated to cause approximately 1.5 million deaths annually, largely via DM-induced cardiovascular disease, particularly diabetic cardiomyopathy (DCM). In this condition, DM-induced hyperglycemia disrupts normal metabolism and directly affects cardiomyocytes, endothelial cells, and microcirculation, thereby inducing structural and functional changes eventually resulting in cardiac failure and mortality [1]. In particular, the induction of cardiomyocyte apoptosis in response to high-glucose stress is a key feature of DCM pathogenesis and is the primary cause of diabetic heart failure and mortality [2].
Hyperglycemia also induces the accumulation of reactive oxygen species (ROS), which result in oxidative protein modifications leading to further tissue injury and dysfunction. ROS also react with nitric oxide (overproduced by activated inducible nitric oxide synthase [iNOS]) to generate significant amounts of the reactive nitrogen species (RNS), especially the peroxynitrite anion (ONOOˉ). Moreover, increasing evidence from experimental and clinical studies suggest that ROS/RNS critically mediate the pathogenesis of diabetic cardiovascular diseases. Furthermore, they also trigger cardiomyocytic apoptosis, and damage mitochondrial membranes via endogenous or exogenous signaling pathways, thereby initiating the harmful “ROS-induced ROS release” positive-feedback loop [3,4]. Thus, studies on novel DCM therapies have long focused on the identification of therapeutic targets and development of new methods to attenuate high glucose stressinduced injury. Figs. 1e4.
Liver-X-receptors (LXRs), comprising two different but highly homologous LXR isoforms (LXRa and LXRb), are ligand-activated transcriptional factors belonging to the nuclear receptor superfamily. LXRa (NR1H3) is highly expressed in metabolically active tissue and cell types, including those of the liver, intestine, adipose tissue, and macrophages, whereas LXRb (NR1H2) is ubiquitously expressed throughout the body [5]. Endogenous LXRs are also expressed in the cardiovascular system and play important roles in several cardiac diseases [6e8]. Recent evidence suggests that LXRs serve as functional nuclear receptors during cellular responses to high glucose stress-induced injury. For example, LXR activation protects against high glucose-induced apoptosis in H9C2 cardiac muscle cells in vitro, via inhibition of ROS production, mitochondrial death, and nuclear factor (NF)-kB activation [9]. Similarly, our previous in vivo study reported that LXR activation protects against DCM by attenuating insulin resistance and reducing both the induced oxidative/nitrative stress and the inflammatory response [10]. However, the specific roles of the two LXR subtypes in the regulation of cardiomyocyte function under high-glucose stress and the mechanisms underlying LXR-mediated protection of cardiomyocytes against DCM are presently unknown. Interestingly, different types of SUMOylation reportedly affect the two LXR subtypes and downregulate downstream inflammatory genes in macrophages and neuroglial cells [11,12]. Furthermore, LXRs direct the ligand-dependent transrepression of numerous endogenous inflammatory-response genes in primary macrophages in a SUMOylation- and/or NCoR-dependent manner [13]. Together, these findings led us to hypothesize that the SUMOylation and NCoR-dependent pathways may underlie the LXR-induced inhibition of target gene expression in cardiomyocytes. Thus, the present study aimed to investigate the specific role and the mechanisms underlying the protection of cardiomyocytes by the two LXR subtypes in DCM.

2. Materials and methods

2.1. Materials

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin (pen/strep, 10,000 U/ml each) were purchased from Gibco (Carlsbad, CA, USA). Synthetic LXRa/b dual agonist 3-[3-[N-(2-Chloro-3-trifluoromethylbenzyl)(2,2-diphenylethyl) amino] propyloxy] phenylacetic acid hydrochloride (GW3965) was kindly provided by Jon Collins (GlaxoSmithKline, Research Triangle Park, NC, USA). TRIzol Reagent was purchased from Life Technologies (Carlsbad, CA, USA). A mouse monoclonal anti-LXRa antibody and rabbit polyclonal anti-LXRb and anti-iNOS antibodies were obtained from Abcam (Cambridge, UK). Rabbit anti-nuclear factor kappa-light-chain-enhancer of activated B cell p65 (NF-kB p65), rabbit anti-caspase-3, rabbit anticytochrome c (Cyt-c), and rabbit anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology (Beverly, MA, USA). IRDye 800CW goat anti-mouse and anti-rabbit IgG secondary antibodies were obtained from LI-COR Biosciences (Lincoln, NE, USA). All other chemicals were sourced commercially.

2.2. Cell culture and lentiviral shRNA transfection

H9C2 rat ventricular myocardial cells (American Type Culture Collection [ATCC]; Rockville, MD, USA) were maintained (37 C, 5% CO2 in air) in DMEM supplemented with 10% FBS, and 1% penicillin/ streptomycin. High glucose (HG) stress was induced by replacing the culture medium with 33 mM D-glucose, as described previously [14]. Control cells were treated with 33 mM D-mannitol (M) to exclude the potential effects of osmolarity. The shRNAs targeting LXRa (50GGATAGGGTTGGAGTCATC-30), LXRb (50eCCTGCCAGATGGATGCCTT30), NCoR (50-GCCTCGGACAAGGATGCAA-30), SUMO1 (50-GGAAGAAGACGTGATTGAA-30), SUMO2/3 (50-CGACGAGAAACCCAAGGAA-30), and a scramble shRNA (50-TTCTCCGAACGTGTCACGT-30) were designed, synthesized, and cloned into the pGCSIL-GFP vector at GeneChem Corporation (Shanghai, China). The pGCSIL-shRNA-GFP, pHelper1.0, and pHelper2.0 were mixed and transfected into 293 T cells, using Lipofectamine 2000 (Invitrogen, Camarillo, CA, USA) in accordance with the manufacturer’s instructions. Culture supernatants were harvested 48 h after transfection, concentrated, and used as virus stocks. For stable infection, H9C2 cell lines were cultured in 6-well plates, and infected with either a shRNA-expressing, or a nonsilencing shRNA-expressing (control) lentivirus, with a multiplicity of infection (MOI) of 10. The achieved shRNA knockdown efficiency was analyzed via real-time quantitative polymerase chain reaction (RT-qPCR) and western blot analyses.

2.3. Western blot analysis

In brief, proteins were extracted using a standard protocol, and protein lysate concentrations were determined using the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). To prepare mitochondrial or cytosolic fractions, protein lysates were harvested using a Mitochondria Isolation Kit (Thermo Scientific). Protein samples from cell lysates were separated via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), electrotransferred onto nitrocellulose membranes, and incubated with primary antibodies against LXRa, LXRb, iNOS, caspase-3, NFkB p65, and Cyt-c. After incubation with the corresponding secondary antibodies, protein bands were detected using an Odyssey® IR scanner (LI-COR Biosciences, Lincoln, NE, USA).

2.4. RT-qPCR

The expression levels of LXRa, LXRb, SUMO1, SUMO2/3, and NCoR were determined via RT-qPCR. Total RNA was isolated from cells, using the TRIzol Reagent and reverse-transcribed using the Omniscript RT Kit (Qiagen, Hilden, Germany). The resultant cDNA was amplified using the SYBR® Premix Ex Taq™ Perfect Real Time Kit (Takara BIO, Otsu, Japan), and the LightCycler® 480 Real-Time PCR System (Roche Applied Science, Indianapolis, IN, USA). LightCycler 480 Data Analysis software was used to analyze and represent the generated data as Ct values. Expression fold changes of target genes were calculated and normalized to that of GAPDH, using the 2 eDDCt method [10].

2.5. Flow cytometry analysis

Apoptotic cells were detected via flow cytometric analysis performed using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (KeyGen Biotech, Nanjing, China). Following treatments, cells were harvested via trypsinization, washed with cold phosphate-buffered saline (PBS), and resuspended in Annexin V binding buffer. They were then double-stained with Annexin V FITC and PI in accordance with the manufacturer’s instructions. Apoptotic cells were finally visualized using a FACSAria flow cytometer (BD Biosciences, San Jose, CA, USA).

2.6. Statistical analysis

All experiments were performed in triplicate and quantitative data are expressed as the mean ± standard error of the mean (SEM) values. Statistically significant differences among multiple treatments were ascertained using either a one- or two-way analysis of variance (ANOVA), using GraphPad Prism 7 software (San Diego, CA, USA). Probability values of 0.05 were considered to indicate statistical significance.

3. Results

3.1. Changes in LXR subtype expression under high glucose conditions

Both LXRa and LXRb were downregulated under high glucose, but not under high osmotic conditions. Conversely, LXRa, but not LXRb, was upregulated in a dose-dependent manner when cardiomyocytes were treated with the LXRa/b dual agonist GW3965 under high glucose conditions. Since GW3965 doses greater than 2 mM caused cell death (data not shown), 2 mM was selected as the highest dose level for all further GW3965 treatments.

3.2. Effect of LXRa/LXRb knockdown on cellular oxidative stress, inflammation, and apoptosis under high glucose conditions

H9C2 cells were exposed to high glucose conditions and transfected with shLXRa and shLXRb to silence both LXR subtypes. Control cells were transfected with the scramble shRNA. Different cell groups (i.e., the LXRa knockdown, LXRb knockdown, and control groups), were then further stimulated with GW3965. The LXRa knockdown cells exhibited increased production of iNOS (a regulatory enzyme in oxidative stress), NF-kB (a transcriptional molecular in inflammation), Caspase-3 (an apoptosis marker), and Cytochrome C (Cyt-c; a mitochondrial injury marker) compared to control cells. Thus, LXRa silencing repressed LXR-mediated protective responses to oxidative stress, inflammation, and apoptosis in cardiomyocytes under high glucose conditions. In contrast, the LXRb knockdown cells expressed iNOS, NF-kB, Caspase-3, and Cyt-c at the same levels as those in the control cells. These results indicate that the cardioprotective effects of LXR activation are probably mediated primarily by the LXRa subtype.

3.3. Effect of SUMO1 or SUMO2/3 silencing on cellular oxidative stress, inflammation, and apoptosis under high glucose conditions

SUMO1 and SUMO2/3 were silenced, and the H9C2 cells were treated with GW9365 under high glucose conditions. Western blot analyses revealed no significant differences in the production of iNOS, NF-kB, Caspase-3, nor cytoplasmic Cyt-c in the either the SUMO1- or SUMO2/3-silenced cells, compared to the control cells. Thus SUMO1 or SUMO2/3 silencing did not affect LXR-mediated cardiomyocyte protection.

3.4. Effect of NCoR knockdown on cellular oxidative stress, inflammation, and apoptosis under high glucose conditions

NCoR was silenced via transfection with shNCoR before the H9C2 cells were treated with GW9365 under high glucose conditions. Western blot analysis revealed that this treatment significantly enhanced the production of iNOS, NF-kB, Caspase-3, and cytoplasmic Cyt-c, as compared to the control group, implicating the role of NCoR in normal LXR-mediated cellular protection.

4. Discussion

In the present study, we first reported that both LXRa and LXRb were downregulated under high glucose conditions and conversely, stimulation with the LXRa/b dual agonist GW3965 selectively upregulated LXRa, but not LXRb. Second, the cardioprotective effects of LXR activation were weakened upon siRNA-mediated silencing of LXRa, but not LXRb. Finally, NCoR knockdown also impaired LXR-mediated protective effects, whereas silencing of SUMOylation-related genes did not. Taken together, these results suggest that activation of LXRa, but not LXRb, protects cardiomyocytes under high glucose conditions via the NCoRdependent transrepression of downstream target genes.
We previously reported that the LXR agonist GW3965 can ameliorate hyperglycemia-induced oxidative stress and apoptosis in vivo [10]; however, the specific roles of the two LXR subtypes in cardiomyocyte protection under high glucose conditions were not elucidated. In the present study, we demonstrated that LXRa (but not LXRb) protects cardiomyocytes against high glucose stressinduced injury. This finding is concurrent with that of a previous study investigating the role of LXRs in ischemia/reperfusion injury [15]. Furthermore, LXRa is a direct target of miR-1 and is involved in high glucose-induced apoptosis in cardiomyocytes; miR-1 overexpression abrogated the inhibitory effect of GW3965 on glucoseinduced apoptosis in H9C2 cells [16]. However, in contrast, other previous studies have reported that LXRb, but not LXRa, mediates pro-proliferative and anti-ischemic effects in neural progenitor cells [17,18] and that LXRb activation decreases platelet aggregation [19]. Together, these data indicate that the LXR subtypes exert tissue-specific effects.
The ubiquitin family member SUMO plays an important role in numerous cellular processes including DNA repair, transcription, and cell division [20]. SUMO1 and SUMO2/3 share overlapping targets and have distinct substrates, suggesting that they play different roles in cellular processes [21]. For example, both LXRa and LXRb are modified by SUMO2/3 in macrophages, while in brain astrocytes, LXRa is modified by SUMO2/3, and LXRb is targeted by SUMO-1 [12,13]. These tissue-specific effects are probably associated with SUMOylation activity levels, since the two LXR subtypes undergo different patterns of SUMOylation, and consequent suppression, in different tissues. Hence, in the present study, we evaluated whether SUMOylation also mediates the protective role of LXRs in cardiomyocytes under high glucose conditions. However, SUMO1 or SUMO2/3 silencing did not affect LXR-mediated cardiomyocyte protection. Since desumoylation of SUMO1 and SUMO2/3 in cardiomyocytes is mediated by a class of enzymes called sentrin-specific proteases (SENPs), this results in congenital heart malformations and cardiac dysfunction [22]. Simultaneous silencing of SUMO1 and SUMO2/3 sould have completely eliminated cardiomyocytes. Therefore, we inferred that the cardioprotective effect of LXRs depends on both subtypes of SUMO isoforms.
NCoR is reportedly associated with LXRs to regulate inflammation by transrepressing the expression of LXR downstream target genes [23]. In the present study, NcoR silencing suppressed the LXR protective effect in cardiomyocytes, suggesting that NcoR and LXRa may function together to protect cardiomyocytes against high glucose-induced stress. However, the deeper molecular mechanisms responsible for the regulation of high glucose-induced oxidative stress, apoptosis, and inflammation by LXRa are likely complex and multifactorial, and remain unidentified. Future studies are required to determine whether LXRa directly regulates iNOS and Caspase-3 and to identify additional downstream LXR target genes.
In summary, the present study confirmed that LXRa (previously considered to primarily be a metabolic regulatory factor) has a markedly stronger cardiac phenotype than the ubiquitously expressed LXRb subtype. However, LXRa, but not LXRb protects cardiomyocytes against high-glucose-induced injury via NCoRdependent transrepression of downstream target genes.

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