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Nr2f2 Overexpression Aggravates Ferroptosis and Mitochondrial Dysfunction by Regulating the PGC-1α Signaling in Diabetes-Induced Heart Failure Mice

Diabetes is well recognized to increase the risk of heart failure, which is associated with higher mortality and morbidity. It is important for the development of novel therapeutic methods targeting heart failure in diabetic patients. Ferroptosis, an iron-dependent regulated cell death, has been implicated in the progression of diabetes-induced heart failure (DIHF). This study was designed to investigate the contribution of Nr2f2 to the activation of ferroptosis and mitochondrial dysfunction in DIHF. We established a diabetic model by a high-fat feeding diet combined with an intraperitoneal injection of streptozotocin. After 16 weeks, Nr2f2 expression was increased in heart tissue of DIHF mice. In vivo, DIHF mice overexpressing Nr2f2 (AAV9-cTNT-Nr2f2) exhibited severe heart failure and enhanced cardiac ferroptosis compared with DIHF control mice (AAV9-cTNT-ctrl), accompanied by mitochondrial dysfunction and aggravated oxidative stress reaction. In vitro, Nr2f2 knockdown ameliorated ferroptosis and mitochondrial dysfunction by negatively regulating PGC-1α, a crucial metabolic regulator. PGC-1α knockdown counteracted the protective effect of Nr2f2 knockdown. These data suggest that Nr2f2 promotes heart failure and ferroptosis in DIHF by modulating the PGC-1α signaling. Our study provides a new idea for the treatment of diabetes-induced heart failure.


Introduction
Heart failure (HF) is a major cause of cardiovascular morbidity and mortality in individuals with diabetes, including type 1 diabetes (T1DM) and type 2 diabetes (T2DM), which is the main clinical manifestation of diabetic cardiomyopathy (DCM) in end-stage describing myocardial dysfunction in the absence of hypertension, atherosclerotic heart disease, and valvular heart disease in patients with diabetes. Globally, an estimated 700 million people are expected to be living with diabetes by 2045. The understanding of pathophysiology and underlying mechanism of diabetes-induced heart failure (DIHF) is still limited. Due to the rapidly increasing prevalence of diabetes worldwide, there is a need to better understand the pathogenesis of DIHF and develop novel therapies [1].
Nuclear receptor subfamily 2, group F, member 2 (Nr2f2) (also known as COUP-TFII), belongs to a family of nuclear orphan receptors. Nr2f2 plays a significant role in energy metabolism, stem cell differentiation, tumor progression, and the development of the cardiovascular system [13]. Nr2f2 is expressed at low levels in the adult heart [13], but expression increased under certain pathological conditions, such as heart failure [14]. In diabetes, Nr2f2 may be involved in the regulation of glycometabolism, oxidative stress, and inflammation in the pancreas [15]. Previous studies have demonstrated that elevated Nr2f2 expression is generally accompanied by mitochondrial dysfunction, increased reactive oxygen species (ROS) production, and alterations in energy metabolism [13,16,17]. Although the role of mitochondria in ferroptosis has been controversial, the occurrence of ferroptosis is always accompanied by abnormal mitochondrial dysfunction. Evidence suggested that inhibition of mitochondrial TCA cycle or electron transfer chain mitigated mitochondrial membrane potential hyperpolarization, lipid-ROS accumulation, and ferroptosis [18]. Hence, we hypothesized that Nr2f2 might participate in the regulation of ferroptosis in DIHF.
In this study, we investigated the contribution of Nr2f2 to the cardiac function of diabetic mice and the activation of ferroptosis in vivo and in vitro. Findings showed that Nr2f2 expression was increased in heart tissue of diabetic mice by feeding a high-fat diet and streptozotocin intraperitoneal injection. In vivo, overexpressing Nr2f2 exhibited symptoms of severe heart failure, ferroptosis, mitochondrial dysfunction, and oxidative stress in diabetes mice. In vitro, Nr2f2 knockdown ameliorated ferroptosis and mitochondrial dysfunction in palmitic acid-(PA-) treated neonatal rat cardiomyocytes (NRCMs). Mechanically, Nr2f2 promoted ferroptosis and mitochondrial dysfunction by regulating PGC-1α negatively. These data suggest that Nr2f2 is associated with diabetes-induced heart failure and ferroptosis by modulating the PGC-1α signaling pathway, providing a novel therapeutic target.

Materials and Methods
2.1. Animal Model. All animal procedures were approved by the animal care and use committee of Huashan Hospital of Fudan University (no. 201802049S). Male C57BL/6 mice (weight approx. 20 g, age 4-5 weeks) were obtained from the animal experimental center of Fudan University. Mice were housed in a specific pathogen-free animal facility with a standard temperature range and feeding regimen. Prior to feeding a high-fat (D12492, 60% kcal fat, Research Diets Company, NJ, USA) or normal diet, fasting blood glucose levels (Roche, Mannheim, Germany) and body weight were measured. Con (control) mice and DIHF mice were fed a normal or high-fat diet for one month; subsequently, Con and DIHF mice received an intraperitoneal injection of dilu-ent or 100 mg/kg streptozotocin (STZ) (Sigma-Aldrich, San Luis, MO, USA). Fasting blood-glucose levels were measured to confirm diabetes (fasting blood glucose > 11 nmol/L). DIHF mice were fed a high-fat diet for a total of 4 months, and the fasting blood-glucose level and body weight were measured once a month.

Histology.
Heart tissue was fixed with 4% PFA, embedded in paraffin, and cut into 5 mm sections for hematoxylin & eosin (H&E) staining. H&E staining was performed according to a protocol of the H&E staining kit (Solarbio, G1120, China). The heart sections were also subjected to Masson staining using a Masson Trichrome staining kit (Solarbio, G1346, China) according to the manufacturers' instructions. 4-4-HNE immunohistochemistry staining was conducted to measure the lipid peroxidation product 4hydroxynonenal (1 : 200, ab46545, Abcam, Hong Kong, China). Images were captured using a Nikon Eclipse microscope (Nikon, Tokyo, Japan) and NIS Elements software.

Transmission Electron
Microscope. The morphology of mitochondria was observed using a transmission electron microscope (TEM). Heart tissue was fixed with 2.5% glutaraldehyde for 1 hour at room temperature and then overnight at 4°C. Fixed tissues were embedded in Embed-812 resin and sectioned into 1 mm 3 blocks. Sections were stained with uranyl acetate and lead citrate for 30 and 15 min, respectively. Ultrathin sections were observed using a JEOL JEM-2100 transmission electron microscope (Tokyo, Japan).

PGC-1α Acts Downstream of Nr2f2 in Regulating
Ferroptosis and Mitochondrial Dysfunction. PGC-1α is an important coregulator of mitochondrial energy metabolism and oxidative stress. In order to investigate the regulatory relationship between Nr2f2 and PGC-1α, we constructed the Nr2f2 overexpression plasmid and found that Nr2f2 overexpression ( Figure S2) negatively regulated PGC-1α in NRCMs (Figure 4(a)). Further, PGC-1α knockdown ( Figure S3) exacerbated the ferroptosis and mitochondrial dysfunction induced by PA treatment and partially diminished the protective effect of Nr2f2 downregulation in PA-treated NRCMs (Figures 4(b)-4(g)). Overall, Nr2f2, acting as an upstream factor, negatively regulates PGC-1α and promotes ferroptosis and mitochondrial dysfunction induced by PA treatment in NRCMs.

Discussion
Diabetes is associated with a high incidence of heart failure and cardiac death, resulting in substantial morbidity and mortality in individuals [20]. Currently, there are no effective treatments for diabetes-induced heart failure (DIHF). Except for antidiabetes, the management of heart failure is   (n = 4, means ± SDs, * p < 0:05 and * * p < 0:01, two-way ANOVA). (d) Lipid ROS was assessed by C11 BODIPY 581/591 staining coupled with flow cytometry analysis (n = 3, means ± SDs, * * * p < 0:005, two-way ANOVA). (e) Cell viability was measured by CCK8 (n = 6, means ± SDs, * * * p < 0:005, two-way ANOVA). (f) Representative JC-1 staining (scale bar = 50 μm, n = 6, means ± SDs; ns: not significant, * * * p < 0:005, two-way ANOVA). (g) Intracellular ATP level in each group (n = 6, means ± SDs, * * * p < 0:005, two-way ANOVA). 10 Mediators of Inflammation also important. In the present study, we investigated the role of Nr2f2 in regulating ferroptosis in diabetes-induced heart failure mice by feeding a high-fat diet and streptozotocin intraperitoneal injection and the underlying mechanism. Findings revealed that Nr2f2 is increased in DIHF mice and Nr2f2 overexpression aggravated heart failure and promoted ferroptosis and mitochondrial dysfunction. Nr2f2 knockdown attenuated the ferroptosis and mitochondrial dysfunction induced by PA in NRCMs. As a regulator of transcription, Nr2f2 plays an important role in many pathological and physiological processes, including organ development, metabolism, and cancer. Previous studies demonstrated that Nr2f2 is necessary for the development and functioning of the cardiovascular system. Altered Nr2f2 expression may lead to congenital heart defects (CHD) and susceptibility to atherosclerosis due to the impact of Nr2f2 on lipid metabolism and inflammation [16,21,22]. Specifically, Nr2f2 overexpression appears detrimental to the cardiovascular system by a mechanism linked to oxidative stress and mitochondrial dysfunction [14,17]. Mice overexpressing Nr2f2 eventually develop heart failure due to impaired glycolipid metabolism and mitochondrial dysfunction [14]. In this study, we revealed that Nr2f2 expression was increased in heart tissue of DIHF mice and mice overexpressing Nr2f2 exhibited symptoms of severe heart failure. Recent evidence suggests that inhibition of Nr2f2 with a small molecular compound could suppress the progression and metastasis of prostate cancer [23], which reflects the application prospect of targeting Nr2f2.
Suppression of programmed cell death may be an effective strategy in the treatment of cardiovascular disease [24]. Distinct types of cell death that lead to cardiac contractile dysfunction and remodeling have been implicated in diabetes-induced heart failure. Long-term hyperglycemia can lead to apoptosis of renal cells and cardiomyocytes in patients with diabetes [25]. Ferroptosis, an iron-dependent, nonapoptotic kind of cell death, is characterized by lipid ROS accumulation and mitochondrial iron overload [26]. Accumulating evidence shows the contribution of ferroptosis to the development of cardiovascular diseases, and inhibition of ferroptosis may relieve diabetic myocardial ischemic reperfusion injury [7,27,28] and cardiomyopathy [12]. NRF2 (nuclear factor-erythroid 2 p45-related factor 2), another crucial transcription factor, is essential for preventing oxidative damage in diabetic cardiomyopathy by modulating several genes involved in ferroptosis, including those required for iron metabolism and lipid peroxidation [29]. Although, it is very close in terms of nomenclature, no direct evidence is available to indicate that the elevated Nr2f2 and cardiac ferroptosis are related. In our present study, diabetic mice overexpressing Nr2f2 showed obvious ferroptosis and an enhanced oxidative stress response in heart tissue, including upregulated NRF2/HO-1 gene expression, which is involved in the intracellular iron metabolism and ferroptosis in doxorubicin-induced cardiomyopathy [8,30,31].
Mitochondria play a critical role in metabolism and energy regulation. As a major site of iron metabolism and ROS production, mitochondria likely contribute to ferroptosis [18,32]. Ferroptosis is characterized by changes in mitochondrial morphology, with smaller than normal mitochondria and decreased mitochondria cristae. Diabetes-induced heart failure is associated with altered fatty acid metabolism and mitochondrial oxidative stress, such that targeting mitochondria has become a popular therapeutic strategy [33,34]. Evidence suggests that Nr2f2 overexpression disrupts mitochondrial pathways [14]. In the present study, we verified that Nr2f2 knockdown could relieve mitochondrial damage and ferroptosis in PA-treated NRCMs. This study concludes that Nr2f2 may promote ferroptosis by affecting intracellular mitochondrial function, although the role of mitochondria in ferroptosis has been a matter of considerable debate. However, further research is required to confirm the association between PA-induced ferroptosis and mitochondrial dysfunction in NRCMs.
Previous reports showed that Nr2f2 directly modulates genes in the mitochondrial metabolic regulators, including ERRα, ERRγ, PPARα, and PGC-1α [14]. In this study, we found that Nr2f2 knockdown mitigated PA-induced mitochondrial dysfunction by activating PGC-1α and PGC-1β. PGC-1α is a coregulator of mitochondrial metabolism, specifically involved in lipid and fatty acid metabolism. In the cardiovascular system, PGC-1α may regulate cardiomyocyte metabolism by coactivating PPARs, ERRs, and NRFs [35]. In diabetes, PGC-1α expression is downregulated resulting in cardiac complications [36]. PGC-1α knockout in mouse heart may damage the mitochondrial ETC and decrease ATP synthesis [37]. In this single study, Nr2f2 overexpression was associated with reduced PGC-1α expression, while suppressing Nr2f2 was associated with PGC-1α upregulation, suggesting that PGC-1α may serve as a crucial downstream target of Nr2f2. One of this study's limitations is that we did not investigate the function of PGC-1β in regulating ferroptosis which needed to further study.

Conclusions
In summary, our data indicate that expression of Nr2f2 was elevated in diabetic failing heart and Nr2f2 overexpression accentuated symptoms of heart failure and ferroptosis in diabetic mice. Nr2f2 knockdown mitigated the ferroptosis and mitochondrial dysfunction induced by PA treatment of NRCMs by downstream regulatory actions on PGC-1α.