PGAM5 expression levels in heart failure and protection ROS-induced oxidative stress and ferroptosis by Keap1/Nrf2

ABSTRACT

Objectives

As a common and frequently occurring disease, heart failure has been paid more and more attention, but the mechanism of its occurrence and development is still unclear. This study investigated that PGAM5 expression levels in heart failure and its underlying mechanisms in vivo and in vitro.

Methods

The inhibition of PGAM5 mRNA expression levels in patients with heart failure was compared with the normal group.

Results

The serum of PGAM5 mRNA expression was negative correlation with collagen I and collagen III in patients with heart failure. PGAM5 mRNA and protein expression in the heart tissue of mice with heart failure were down-regulated at a time-dependent rate. The inhibition of PGAM5 presented heart failure in the model. PGAM5 reduced inflammation and inhibited ROS-induced oxidative stress in models of heart failure. PGAM5 reduced Ferroptosis in models of heart failure. PGAM5 regulated Keap1/Nrf2 signaling pathway. IP also showed that PGAM5 protein combined with the Keap1 protein. PGAM5 could increase Keap1 protein ubiquitination. Keap1 inhibition affected the effects of PGAM5 in model of heart failure.

Conclusions

We conclude that the protection of PGAM5 reduced ROS-induced oxidative stress and ferroptosis by the Keap1/Nrf2 signaling pathway in heart failure, suggesting that targeting this mechanism of PGAM5 may be a feasible strategy to treat heart failure.

KEYWORDS:

• PGAM5
• Keap1
• Nrf2
• ferroptosis
• heart failure

Introduction

Heart failure is a complex clinical syndrome that leads to ventricular filling or impaired ejection capacity due to changes in cardiac structure and function (1). At present, the number of patients with heart failure in the world has reached 22.5 million, increasing at the rate of 2 million cases per year, and the 5-year survival rate is equivalent to that of malignant tumors (2). Heart failure has become a major public health problem in many countries (3). It not only has a high disability rate and mortality rate, which affects the quality of life of patients, but also aggravates the national medical burden year by year (3).

Ferroptosis is different from apoptosis, necrosis, and autophagy in morphology, biochemistry, and genetics (4). The main feature is a large amount of iron-dependent lipid peroxide accumulation. Its morphological characteristics are mainly manifested in mitochondria, such as the inhibition of mitochondrial volume, the increase in membrane density, and the loss of cristae (5,6). In terms of mechanism, the disorder of redox balance mediated by glutathione (GSH) and glutathione peroxidase 4 (GPx4) is a unique feature of Ferroptosis (4). Studies have shown that ferroptosis plays an important role in tumors, neurodegenerative diseases, renal failure, and cardiovascular diseases (5,6).

PGAM5 is an atypical mitochondrial serine/threonine phosphatase homologous to phosphoglycerate mutase (7). However, it lacks the corresponding enzyme catalytic function and was initially found to interact with BCL-XL. Recent studies have found that PGAM5 regulates multiple cell death pathways, including apoptosis and necrosis (8). For example, PGAM5/Bcl-XL regulated mitochondrial autophagy induced by hypoxia, so as to control apoptosis (9). This study investigated that PGAM5 expression levels in heart failure and its underlying mechanisms in vivo and in vitro.

Materials and methods

Clinical data analysis

Patients with atrial fibrillation were checked using electrocardiograms and were selected from our hospital. The protocol was approved by the Ethics Committee of our hospital. Informed written consent was obtained from all participants. The general clinical data of the patients in the two groups are given in Table 1.

Table 1. Feeding operation of rats.

Group	Feed	Drinking water	Gavage
Control	Normal	Normal	Water
Model	High-fat	Normal	Water
High-Liguid	High-fat	Normal	221 mg/kg/d supernatant lyophilized powder
Med-Liguid	High-fat	Normal	442 mg/kg/d supernatant lyophilized powder
Low-Liguid	High-fat	Normal	884 mg/kg/d supernatant lyophilized powder
High-Drug residue	High-fat	Normal	221 mg/kg/d precipitate lyophilized powder
Med-Drug residue	High-fat	Normal	442 mg/kg/d precipitate lyophilized powder
Low-Drug residue	High-fat	Normal	884 mg/kg/d precipitate lyophilized powder

RT-PCR assay and microarray analysis

Briefly, total RNA was extracted from human serum samples or mice heart tissue samples or cell samples using RIzol reagent (Invitrogen, Thermo Fisher Scientific, Inc., Waltham, MA, USA). Total RNA was then reverse transcribed into cDNAs using the Reverse Transcription System Kit (Takara, Dalian, China). The cDNAs were amplified by qRT-PCR using TB Green™ Premix Ex Taq™ II (Takara, Dalian, China) on a StepO-nePlus Real-Time PCR System. Fold changes in mRNA levels were calculated using 2 -ΔΔCt methods.

Total RNA was extracted from serum samples, and the amount of RNA was quantified by use of NanoDrop 1000. Total RNA of each sample was used for reverse transcription using an Invitrogen SuperScript double stranded cDNA synthesis kit. Double-stranded cDNA was executed with a NimbleGen one-color DNA labeling kit and then executed for array hybridization using a NimbleGen hybridization system and washed with the NimbleGen wash buffer kit. Axon GenePix 4000B microarray scanner (Molecular Devices) was used for scanning.

Histological examination and enzyme-linked immunosorbent assay (ELISA)

The tissue was fixed in 4% paraformaldehyde for 24 h and cut into about 5 μM thick transverse slices. The paraffin-embedded tissues were then sectioned at 4-μm and subjected to H&E staining or Masson staining. The sections were analyzed under an Olympus BH-2 light microscope (Olympus Corporation). MDA, ROS production, GSH, GSH-px, and SOD levels were measured using ELISA kit (Beyotime) according to the manufacturer’s protocol.

Cell transfection and culture of adult mouse atrial fibroblasts

To down-regulation PGAM5, sh-PGAM5 virus (sc-41670, Santa Cruz Biotechnology) was used in mice. Adult mouse atrial fibroblasts were isolated as we previously described (10). Hearts from C57BL/6 J mice (age of 5–6 weeks, weighing 18–20 g) were rapidly excised and submerged in cold PBS. The atria were cut into pieces using a tissue chopper and washed with cold PBS. The cell was then dissociated using collagenase II (1 mg/mL, Worthington Biochemical, USA) for 30 min at 37°C. Isolated cells were collected and centrifuged at 1500 rpm for 5 min and were resuspended in DMEM containing 10% FBS for 2 h in a 5% CO2 incubator at 37°C. After removing the myocyte-enriched medium, the cell was cultured for 3–4 days before being passaged. Cells were transfected with their respective constructs using Lipofectamine™ 2000 (Invitrogen, USA), following the manufacturer’s instructions. After 4 h of transfection, the cell was treated with 1 μM Ang II (Sigma-Aldrich, St. Louis, MO, USA) and used to other experiment at 48 h of incubation.

Animal experiment

C57BL/6 J mice (age of 5–6 weeks, weighing 18–20 g) were randomly divided into two groups (n = 8/group): sham and model or model and model + UBQLN1. Mice from the sham group received intraperitoneal injections of normal saline. Mice from the model group received an intraperitoneal injection of Ang II (1.5 μg/g/day; Sigma-Aldrich). After 4 weeks, mice were sacrificed under anesthesia. Next, Mice of model + UBQLN1 group received an intraperitoneal injection of Ang II (1.5 μg/g/day; Sigma-Aldrich) and UBQLN1 RNA vectors (4 × 10⁷ TU/mice/week). After 4 weeks, mice were sacrificed under anesthesia. The lentiviral vectors carrying UBQLN1 RNA and a negative control were designed and chemically synthesized by Hanyin Biotechnology Limited Company (Shanghai, China). The animal procedures were approved by the Ethics Committee of the Renmin Hospital of Wuhan University. Left ventricular internal diameter, left ventricular ejection fraction, left ventricular fractional shortening, and left ventricular stroke volume were obtained from Millar pressure-volume system (MPVS-400).

Western blot assay

Proteins were extracted from heart tissue or cell samples in a protein lysis buffer (Keygen Biotech, Nanjing, China). Protein content was quantified with the BCA reagent kit. Protein lysates were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore Corp. Billerica, MA, USA). The PVDF was incubated by UBQLN1, PGC1β, Nlrp3, and β-actin at 4°C over-night. PVDF was incubated with a horseradish peroxidase-conjugated secondary antibody (Beyotime, 1:5000) for 2 h. The signal was tested with a chemiluminescence system (Amersham Pharmacia).

Immunofluorescence

Cells were fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.1% Triton X-100 in PBS for 15 min at room temperature. Cells were blocked with 5% BSA for 30 min at 37°C and were treated with primary antibodies anti-PGAM5 (1:100) and anti-Keap1 (1:100) at 4°C overnight. Cells were then incubated with Cy3-conjugated goat anti-rabbit or goat anti-mouse IgG DyLight 488-conjugated secondary antibodies (1:500) for 1 h at 37°C. Nuclei were stained with DAPI, and cells were viewed by fluorescent confocal microscopy (Nikon, Tokyo, Japan).

Data and statistical analysis

Statistical analyses were performed using the SPSS 16.0 statistical software (SPSS, Inc., Chicago, IL, USA) and reported as the mean ± SD. The experiments have been repeated at least three times. The unpaired Student’s t-test was used to analyze differences between the two groups. A one-way ANOVA was employed for the comparison of data between groups. p < .05 was considered to indicate a statistically significant difference.

Results

PGAM5 expression levels in heart failure

The experiment examined the changes of PGAM5 levels in patients with heart failure. As shown in Figure 1A, there was inhibition of PGAM5 mRNA expression levels in patients with heart failure, compared with the normal group. The serum of PGAM5 mRNA expression was negative correlation with collagen I and collagen III in patients with heart failure (Figure 1B-1C). The receiver operating characteristic (ROC) curve was constructed to assess the diagnostic value of PGAM5 in patients with heart failure (Figure 1D). PGAM5 mRNA expression in heart tissue of mice with heart failure was down-regulated at a time-dependent rate (Figure 1E). Importantly, the protein expression of PGAM5 in heart tissue of mice with heart failure was down-regulated (Figure 1F).

Figure 1. PGAM5 expression levels in Heart failure.

PGAM5 mRNA expression levels (a), the serum of PGAM5 mRNA expression was negative correlation with collagen I and collagen III (b and c), ROC curve (d) in patients with Heart failure; PGAM5 mRNA expression (e) and PGAM5 protein expression (f) in heart tissue of mice with heart failure. Normal, normal volunteer group; Patients, patients with Heart failure; Sham, sham control group; model, mice with Heart failure. The number of patients = 24; the number of mice model = 6;^##p < .01 compared with normal volunteer group or sham control group.

The inhibition of PGAM5 presented heart failure in model

To further examine the function of PGAM5 in model of heart failure, sh-PGAM5 virus induced into mice with heart failure. Sh-PGAM5 virus enhanced left ventricular internal diameter (LVID), reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS), and inhibited left ventricular stroke volume (LVSV) and myocardial fibrosis (HE staining or Masson staining) in mice with heart failure (Figure 2A-2E). Meanwhile, sh-PGAM5 virus increased CK and LDH activity levels, and promoted the mRNA expression of collagen I, collagen III, and TGF-β1 in heart tissue of mice with heart failure (Figure 2F-2J).

Figure 2. The inhibition of PGAM5 presented heart failure in model.

LVID, LVEF, LVFS, and LVSV (a, b, c, and d), myocardial fibrosis (HE staining or Masson staining, e), CK and LDH activity levels (f and g), the mRNA expression of collagen I, collagen III, and TGF-β1 (h, i, and j) in mice with heart failure. Model, mice with Heart failure; sh-PGAM5, sh-PGAM5 virus; the number of mice model = 6;^##p < .01 compared with Model group.

PGAM5 reduced inflammation in model of heart failure

The experiment evaluated the effects of PGAM5 on inflammation in a model of heart failure using in vitro models. PGAM5 plasmid increased the mRNA expression of PGAM5, and si-PGAM5 mimics reduced PGAM5 mRNA expression in vitro model of heart failure (Figure 3A). PGAM5 up-regulation reduced collagen I and collagen III mRNA expressions in in vitro models (Figure 3B-3C). PGAM5 down-regulation increased collagen I and collagen III mRNA expression in in vitro models (Figure 3D-3E). Then, sh-PGAM5 virus increased the serum and heart tissue of IL-1β levels in mice with heart failure (Figure 3F). PGAM5 up-regulation reduced IL-1β levels in vitro model by Ang II, and PGAM5 down-regulation increased IL-1β levels in vitro model by Ang II (Figure 3G-3H).

Figure 3. PGAM5 reduced inflammation in model of heart failure.

PGAM5 mRNA expression (a), collagen I and collagen III (b and c) in vitro model by over-expression of PGAM5, collagen I and collagen III (d and e) in vitro model by down-regulation of PGAM5, IL-1β level in mice with heart failure (f), IL-1β level (g) in vitro model by over-expression of PGAM5, IL-1β level (h) in vitro model by down-regulation of PGAM5. Control, control group; PGAM5, PGAM5 up-regulation; si-PGAM5, PGAM5 down-regulation group; the number of vitro model = 3; ^##p < .01 compared with control group.

PGAM5 reduced ROS-induced oxidative stress in model of heart failure

The experiment validated the effects of PGAM5 on ROS-induced oxidative stress in models of heart failure. In mice with heart failure, sh-PGAM5 virus reduced SOD, GSH, and GSH-px levels, and increased MDA level (Figure 4A-4D). PGAM5 up-regulation reduced ROS production level and MDA level and increased SOD, GSH, and GSH-px levels in in vitro models (Figure 4E-4I). PGAM5 down-regulation increased ROS production level and MDA level and inhibited SOD, GSH, and GSH-px levels in in vitro models (Figure 4J-4N).

Figure 4. PGAM5 reduced ROS-induced oxidative stress in model of heart failure.

SOD, GSH, GSH-PX, and MDA (a, b, c, and d) in mice with heart failure; ROS, MDA, SOD, GSH, and GSH-PX levels (e, f, g, h, and i) in vitro model by over-expression of PGAM5; ROS, MDA, SOD, GSH, and GSH-PX levels (j, k, l, m, and n) in vitro model by down-regulation of PGAM5. Control, control group; PGAM5, PGAM5 up-regulation; si-PGAM5, PGAM5 down-regulation group; the number of mice model = 6; the number of vitro model = 3; ^##p < .01 compared with control group.

PGAM5 reduced ferroptosis in model of heart failure

To further investigate the mechanism of PGAM5 on heart failure, we analyzed the effects of PGAM5 regulated cardiomyocyte death in a model of heart failure. PGAM5 up-regulation promoted cell viability and reduced LDH activity levels in in vitro models of heart failure (Figure 5A, 5C). PGAM5 down-regulation reduced cell viability and promoted LDH activity levels in in vitro models of heart failure (Figure 5B-5C). PGAM5 up-regulation increased JC-1 disaggregation and calcein levels and reduced PI positive cells and iron concentration levels in in vitro models of heart failure (Figure 5D-5H). PGAM5 down-regulation reduced JC-1 disaggregation and calcein levels and promoted PI positive cells and iron concentration levels in in vitro models of heart failure (Figure 5D-5H).

Figure 5. PGAM5 reduced Ferroptosis in model of heart failure.

Cell viability (a and b), LDH activity level (c), JC-1 disaggregation (d), calcein-AM/CoCl2 levels (e), calcein/PI (f), PI positive cells (g), and iron concentration levels (h). Control, control group; PGAM5, PGAM5 up-regulation; si-PGAM5, PGAM5 down-regulation group; the number of vitro model = 3; ^##p < .01 compared with the control group.

PGAM5 regulated Keap1/Nrf2 signaling pathway

The experiment determined the mechanism of PGAM5 on ROS-induced oxidative stress and ferroptosis of heart failure using microarray analysis. We found that PGAM5 up-regulated Nrf2 expression and suppressed Keap1 expression in the model (Figure 6A-6B). Then, sh-PGAM5 virus suppressed Nrf2 mRNA expression, and induced Keap1 mRNA expression in mice with heart failure (Figure 6C-6D). Certainly, PGAM5 up-regulation suppressed Keap1 mRNA expression, and induced Nrf2 mRNA expression in vitro model of heart failure (Figure 6E-6F). PGAM5 down-regulation also induced Keap1 mRNA expression and suppressed Nrf2 mRNA expression in in vitro model of heart failure (Figure 6G-6H).

Figure 6. PGAM5 regulated Keap1/Nrf2 signaling pathway.

Results of microarray analysis (a), KEGG terms (b), Keap1/Nrf2 mRNA expression in mice with heart failure (c and d), Keap1/Nrf2 mRNA expression in vitro model by over-expression of PGAM5 (e and f); Keap1/Nrf2 mRNA expression in vitro model by down-regulation of PGAM5 (g and h). Model, mice with Heart failure; sh-PGAM5, sh-PGAM5 virus; Control, control group; PGAM5, PGAM5 up-regulation; si-PGAM5, PGAM5 down-regulation group; the number of vitro model = 3; ^##p < .01 compared with Model or control group.

Next, sh-PGAM5 virus-induced Keap1 protein expression, and suppressed Nrf2 protein expression in mice with heart failure (Figure 7A-7B). PGAM5 up-regulation induced PGAM5 and Nrf2 protein expressions and suppressed Keap1 protein expression in in vitro model of heart failure (Figure 7C-7E). PGAM5 down-regulation suppressed PGAM5 and Nrf2 protein expressions and induced the Keap1 protein expression in in vitro model of heart failure (Figure 7F-7H). Confocal results showed that PGAM5 up-regulation reduced Keap1 expression in in vitro model of heart failure (Figure 7I). IP also showed that PGAM5 protein combined with Keap1 protein (Figure 7J). PGAM5 could increase Keap1 protein Ubiquitination (Figure 7K).

Figure 7. PGAM5 protein interlinking Keap1 protein.

Keap1/Nrf2 protein expression in mice with heart failure (a and b), PGAM5/Keap1/Nrf2 protein expression in vitro model by over-expression of PGAM5 (c, d, and e), PGAM5/Keap1/Nrf2 protein expression in in vitro model by down-regulation of PGAM5 (f, g, and h), PGAM5/Keap1 expression in in vitro model by over-expression of PGAM5 (Confocal, i), PGAM5 protein interlinking Keap1 protein (IP, j), Keap1 protein Ubiquitination (k). Model, mice with Heart failure; sh-PGAM5, sh-PGAM5 virus; Control, control group; PGAM5, PGAM5 up-regulation; si-PGAM5, PGAM5 down-regulation group; the number of vitro model = 3; ^##p < .01 compared with Model or control group.

The inhibition of Keap1 affected the effects of PGAM5 in model of heart failure

Furthermore, we analyzed the role of Keap1 in the effects of PGAM5 in model of heart failure. Keap1 plasmid induced Keap1 protein expression and suppressed Nrf2 protein expression in in vitro model by over-expression of PGAM5 (Figure 8A-8B). Keap1 up-regulation increased MDA and ROS production levels and reduced SOD, GSH, and GSH-px levels in in vitro model by over-expression of PGAM5 (Figure 8C-8G). Keap1 up-regulation induced collagen I, collagen III, and TGF-β1 mRNA expressions in in vitro model by over-expression of PGAM5 (Figure 8H-8J).

Figure 8. The regulation of Keap1 affected the effects of PGAM5 on ROS-induced oxidative stress in model of heart failure.

Keap1/Nrf2 protein expression (a and b); MDA, ROS, SOD, GSH, and GSH-PX levels (c, d, e, f, and g); mRNA expression of collagen I, collagen III, and TGF-β1 (h, i, and j) in vitro model by over-expression of PGAM5; Keap1/Nrf2 protein expression (k and l); MDA, ROS, SOD, GSH, and GSH-PX levels (m, n, o, p, and q); mRNA expression of collagen I, collagen III, and TGF-β1 (r, s, and t) in vitro model by down-regulation of PGAM5. Control, control group; PGAM5, PGAM5 up-regulation; Keap1, over-expression of Keap1; si-PGAM5, PGAM5 down-regulation group; Keap1 i, Keap1 inhibitor. The number of mice model = 6; the number of vitro model = 3; ^##p < .01 compared with the model or control group.

The Keap1 inhibitor (5 μM of KI696) reduced the effects of si-PGAM5 on Keap1 and Nrf2 protein expression, MDA and ROS production levels, the SOD/GSH/GSH-px levels, and collagen I/collagen III/TGF-β1 mRNA expressions in vitro model (Figure 8K-8T).

Then, Keap1 up-regulation reduced cell viability, increased LDH activity level, promoted PI positive cells and iron concentration levels, and inhibited JC-1 disaggregation and calcein levels in in vitro models by over-expression of PGAM5 (Figure 9A-9F). However, the Keap1 inhibitor (5 μM of KI696) increased cell viability, reduced LDH activity levels, inhibited PI positive cells and iron concentration levels, and promoted JC-1 disaggregation and calcein levels in in vitro models by down-regulation of PGAM5 (Figure 9G-9L).

Figure 9. The regulation of Keap1 affected the effects of PGAM5 on ferroptosis in model of heart failure.

Cell viability (a), LDH activity level (b), PI positive cells (c), JC-1 disaggregation (d), calcein-AM/CoCl2 levels (e), and iron concentration levels (f) in vitro model by over-expression of PGAM5; Cell viability (g), LDH activity level (h), PI positive cells (i), JC-1 disaggregation (j), calcein-AM/CoCl2 levels (k), and iron concentration levels (l) in vitro model by down-regulation of PGAM5. Control, control group; PGAM5, PGAM5 up-regulation; Keap1, over-expression of Keap1; si-PGAM5, PGAM5 down-regulation group; Keap1 i, Keap1 inhibitor. The number of mice model = 6; the number of vitro model = 3; ^##p < .01 compared with the model or control group.

Discussion

According to epidemiological statistics, there are 330 million patients with cardiovascular diseases in China, and the data show that the mortality rate of cardiovascular diseases in China remained the first in 2017 (11). The mortality rate of cardiovascular diseases still ranks first, higher than that of high-risk diseases such as tumors (12). The global epidemiological survey report in 2020 shows that nearly 30 million patients worldwide are troubled by heart failure, and there are about 4.5 million patients with heart failure in China (13). We first found that PGAM5 expression levels in the model of heart failure were down-regulated. Yang et al. suggested that PGAM5 expression decreased in cardiomyocytes after hypoxia/reoxygenation (14). Taken together, PGAM5 may be a novel regulator of mitochondrial dynamics for heart failure, a possibility that is currently under active investigation.

Ferroptosis is a new type of iron dependent programmed cell death (15). It is caused by the increase in fatty acid lipid peroxidation induced by cell iron content overload and the accumulation of reactive oxygen species (16). Ferroptosis is closely related to the occurrence and development of tumors, neurodegenerative diseases, metabolic diseases, aging diseases, and other major diseases (16). Ferroptosis is involved in many diseases, such as cardiomyopathy, myocardial infarction, heart failure, coronary atherosclerosis, myocardial ischemia reperfusion injury, and diabetic cardiomyopathy (16). Our study indicated that PGAM5 reduced Ferroptosis in models of heart failure. Ma et al. indicated that PGAM5 regulate mitochondrial cell death (17). Taken together, these results indicate that PGAM5 function in heart failure is necessary for heart cell-mediated ferroptosis.

Oxidative stress refers to the excessive production of ROS, which weakens the endogenous antioxidant defense function and leads to cell damage Iron usually exists in the form of trivalent iron (Fe3 +) in human body (18). Under the action of some enzymes and transporters, it enters the cytoplasm and mitochondria from the circulatory system and becomes divalent iron (Fe2 +) with redox activity (19). These active irons will catalyze the production of ROS through Fenton reaction, and the iron-dependent ROS will react with lipids to induce Ferroptosis (20,21). This experiment demonstrated that PGAM5 reduced ROS-induced oxidative stress in a model of heart failure. Lo et al. showed that PGAM5 reduced oxidative stress by Keap1 (17). Thus, PGAM5 is also critical for ROS-induced oxidative stress in models of heart failure.

The expression of GPx4 is regulated by Nrf2. When stimulated by external oxidative stress factors, Nrf2 and its inhibitory protein Keap1 dissociate and activate, enter the nucleus, start the expression of its downstream target genes such as SOD, GPx4, XCT, and HO-1, and play an antioxidant role in inactivation (22). The inhibition and knockdown of Nrf2 gene will enhance Ferroptosis; the activating Nrf2 signaling pathway will reduce ferroptosis and can improve heart failure (23,24). Importantly, we found that PGAM5 protein combined with Keap1 protein, and PGAM5 could increase Keap1 protein ubiquitination. Lo et al. suggest that PGAM5 tethers a ternary complex containing Keap1 and Nrf2 to changes in mitochondrial function (25). These data together demonstrated that PGAM5 is a substrate for Keap1/Nrf2 signaling pathways and is efficiently targeted for ferroptosis and oxidative stress in models of heart failure.

In conclusion, our study showed that the PGAM5 was down-regulated in heart failure. The protection of PGAM5 reduced ROS-induced oxidative stress and ferroptosis by the Keap1/Nrf2 signaling pathway in heart failure. The PGAM5 resulted in alterations and stabilization of important factors for ferroptosis and oxidative stress in models of heart failure. Since PGAM5 might be a clinical factor for the treatment of heart failure.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This study was funded by Health Commission of Sichuan Province (19PJ105).