PERM1 regulates mitochondrial energetics through O-GlcNAcylation in the heart

Abstract

PERM1 was initially identified as a new downstream target of PGC-1α and ERRs that regulates mitochondrial bioenergetics in skeletal muscle. Subsequently, we and other groups demonstrated that PERM1 is also a positive regulator of mitochondrial bioenergetics in the heart. However, the exact mechanisms of regulatory functions of PERM1 remain poorly understood. O-GlcNAcylation is a post-translational modification of proteins that are regulated by two enzymes: O-GlcNAc transferase (OGT) that adds O-GlcNAc to proteins; O-GlcNAcase (OGA) that removes O-GlcNAc from proteins. O-GlcNAcylation is a powerful signaling mechanism mediating cellular responses to stressors and nutrient availability, which, among other targets, may influence cardiac metabolism. We hypothesized that PERM1 regulates mitochondrial energetics in cardiomyocytes through modulation of O-GlcNAcylation. We found that overexpression of PERM1 decreased the total levels of O-GlcNAcylated proteins, concomitant with decreased OGT and increased OGA expression levels. Luciferase gene reporter assay showed that PERM1 significantly decreases the promoter activity of Ogt without changing the promoter activity of Oga. The downregulation of OGT by PERM1 overexpression was mediated through its interaction with E2F1, a known transcription repressor of Ogt. A deliberate increase of O-GlcNAcylation through Oga silencing in cardiomyocytes decreased the basal and maximal mitochondrial respiration and ATP production rates, all of which were completely restored by PERM1 overexpression. Furthermore, excessive O-GlcNAcylation caused by the loss of PERM1 led to the increase of O-GlcNAcylated PGC-1α, a master regulator of mitochondrial bioenergetics, concurrent with the dissociation of PGC-1α from PPARα, a well-known transcription factor that regulates fatty acid β-oxidation. We conclude that PERM1 positively regulates mitochondrial energetics, in part, via suppressing O-GlcNAcylation in cardiac myocytes.

Graphical Abstract

1. Introduction

O-GlcNAcylation is a dynamic and reversible post-translational modification of proteins that occurs via the addition of O-linked β-N-acetylglucosamine (O-GlcNAc) moieties to the serine and threonine residues under tight regulation by enzymes OGT and OGA. Modification of O-GlcNAcylation is altered based on the availability of nutrients and cellular stress responses [1, 2] and is critical for physiological functions. Dysregulation of O-GlcNAcylation is involved in a wide range of pathologies, including cardiovascular diseases, cancer, metabolic disorders, and Alzheimer’s disease [3–12]. The role of O-GlcNAcylation in adaptive and maladaptive responses to pathological stress in the heart is highly complex. An acute increase in O-GlcNAcylation is known to have a cardioprotective role during oxidative stress, hypoxia, and ischemia [13–19], whereas excessive or prolonged activation of O-GlcNAcylation is associated with hypertension, diabetes, hypertrophy, and heart failure [20–30]. The intricate regulation of O-GlcNAcylation in cardiac function was further highlighted by studies showing that cardiac-specific deletion of either Ogt [18, 31, 32] or Oga genes [33] exacerbates ventricular dysfunction under pathological stress. These studies suggest that genetic ablation or pharmacological inhibition of any of these enzymes may not become a viable therapeutic strategy for managing chronic cardiac diseases involving dysregulation of O-GlcNAcylation. Despite the critical role of O-GlcNAcylation in cardiac function and its potential as a therapeutic target for chronic cardiac diseases, it remains elusive how to maintain O-GlcNAcylation homeostasis under pathological stress.

Peroxisome Proliferator-activated Receptor γ Coactivator 1 [PGC-1] - and Estrogen-related Receptor [ERR]-induced Regulator in Muscle 1 (PERM1) is a striated muscle-specific regulator of mitochondrial bioenergetics. It was first discovered in 2013 as a downstream regulator of PGC-1 and ERR target genes in C2C12 myotubes [34] and was later shown to be an important regulator of mitochondrial biogenesis and oxidative capacity in skeletal muscle [35]. We have shown that PERM1 is highly expressed in the human heart and that PERM1 is downregulated in human and mouse failing hearts [36]. We further demonstrated that loss of PERM1 in cardiomyocytes leads to reduced mitochondrial respiration capacity [36]. Since then, additional work by us and other groups revealed that PERM1 regulates energy metabolism by interacting with PGC-1α and its partner transcriptional factors PPARα and ERRα [37, 38] and that PERM1 can reduce cellular damage caused by hypoxia and reoxygenation-induced stress conditions by promoting mitochondrial biogenesis [39]. Our metabolic profiling via a multisystem approach revealed that loss of PERM1 leads to downregulation of fatty acid β-oxidation (FAO) and upregulation of glycolysis pathway in the heart, latter which was associated with upregulation of glucose transporters GLUT1 and GLUT4 [38], suggesting that loss of PERM1 function promotes glucose uptake in cardiomyocytes. Other things being equal, increased glucose uptake is expected to promote O-GlcNAcylation through increased flux through the hexosamine biosynthesis pathway (HBP), an accessary pathway of glycolysis. In addition, among multiple cellular functions of O-GlcNAcylation, O-GlcNAcylation regulates mitochondrial function [29, 40, 41]. It has been reported that increased O-GlcNAcylation in human cardiomyocytes decreases activity of Complex I, II and IV [41]. These have led us to explore the relationship between PERM1 and O-GlcNAcylation in cardiomyocytes. Here, we present evidence that PERM1 is a negative regulator of O-GlcNAcylation in the heart, which acts indirectly through suppression of gene expression of glucose transporters, and directly through suppression of expression of OGT. Furthermore, we demonstrate that PERM1 overexpression reverts reduction of mitochondrial respiration capacity and FAO caused by excessive O-GlcNAcylation. Lastly, we provide evidence that PERM1 negatively regulates O-GlcNAcylation of PGC-1α, a master regulator of mitochondrial bioenergetics, which enhances FAO. Overall, this study uncovers a new mechanism by which PERM1 maintains mitochondrial energetics in the heart through suppressing O-GlcNAcylation.

2. Experimental procedures

2.1. Animals and Tissue harvest

All animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Fralin Biomedical Research Institute (FBRI) in Virginia Tech and were conducted according to the Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize pain and distress during isolation of the heart by deep anesthesia. We also minimized the number of animals used in this study. Perm1^−/− mice were developed as described previously [38]. At the age of 8–10 weeks, hearts were harvested from these animals and were immediately frozen in liquid nitrogen and stored at −80 °C until they were used for analysis. In this study, we used both male and female mice on a C57BL/6N background.

2.2. Luciferase Gene Reporter Assay

Luciferase gene reporter assay was performed using the Dual-Luciferase Reporter Assay kit from Promega (#E1910), as we previously described [42]. H9c2 cells were grown in the Dulbecco’s Modified Eagle Medium (DMEM) (GenClone #25–501) supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/ streptomycin on white 96-well plates (Genesee Scientific #91–424TW). The cells were also cultured in clear 96-well plates (Genesee Scientific #25–109) to ensure cell confluency and viability after transfection. As cells reached 70% confluency, they were treated with either Ad-LacZ (control) or Ad-PERM1 (Ad-PM1) at a multiplicity of infection (MOI) of 50. After 24hr, the cells were transfected with 50 ng/well of human promoter Ogt (SwitchGear Genomics,https://switchdb.switchgeargenomics.com/productinfo/id_702409/), promoter Oga (SwitchGear Genomics, https://switchdb.switchgeargenomics.com/productinfo/id_704843/), promoter Glut1 (SwitchGear, https://switchdb.switchgeargenomics.com/productinfo/id_721597/), promoter Glut4 (SwitchGear, https://switchdb.switchgeargenomics.com/productinfo/id_709354/), negative control (scrambled, SwitchGear Genomics, Prod#S790001), or positive control (ACTB, SwitchGear, Prod#S717678) using 8 ng/well of a pGL4 13 [luc2 SV40] vector (Promega Corporation, Cat#E6681) and incubated for 24hr. Luciferase activity was assayed using the LightSwitch Luciferase assay reagent (SwitchGear Genomics, Cat#E1910) according to the manufacturer’s instructions and measured using the Biorad ChemiDoc ™ MP imaging system.

2.3. Cell Mito Stress Test

Cell Mito Stress Test was performed to measure the oxygen consumption rate (OCR) using Agilent Seahorse XF Cell Mito Stress Test Kit (103015–100) as we previously performed [36]. Briefly, H9c2 cells (100k cells per well) were plated in 96-well Seahorse analyzer plates (Seahorse XFe96 cell culture microplates). On reaching 70% confluency, cells were treated with Ad-LacZ (control) or Ad-PM1 at an MOI of 50 for 24hr followed by treatment with 20 μM scrambled siRNA (scr-siRNA; Qiagen#1027310) or siOGA (Qiagen #1027417) for another 24hr. Two different siOGAs that are complementary to different target genes were used. After 48hr, readings were taken after injection of 1 μM oligomycin, 4 μM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) and 1 μM rotenone + antimycin A according to the manufacturer’s instructions. The OCR values were normalized by cell counting.

2.4. XF Palmitate Oxidation Stress Test

Palmitate Oxidation Stress Test was performed using the Agilent XF Palmitate Oxidation Stress Test Kit: advanced assay (103693–100). H9c2 cells (100k cells per well) were plated in 96-well Seahorse analyzer plates (Seahorse XFe96 cell culture microplates). When cell confluency reached ~70%, H9c2 cells were treated with Ad-LacZ (control) or Ad-PM1 at an MOI of 50 for 24hr followed by the transfection with 20 μM of either scrambled siRNA (scr-siRNA; Qiagen #1027310) or siOGA (Qiagen #1027417) for another 24hr. Two siRNAs were used to increase confidence in silencing the target gene (S101915074 and S101915067). One day before the assay, cells were cultured in substrate-limited growth media containing 0.5 mM glucose, 1 mM glutamine, 1% fetal bovine serum, and 0.5 mM L-carnitine. On the day of assay, substrate-limited growth media was replaced with substrate-limited assay medium containing 2 mM glucose and 0.5 mM L-carnitine. Before assay, palmitate-Bovine serum albumin (BSA) and BSA-control were added to appropriate wells. Readings were taken after injection of 4 μM etomoxir /medium, 1.5 μM oligomycin, 4 μM FCCP, and 0.5 mM rotenone + antimycin A according to the manufacturer’s instructions. The OCR values were normalized by cell counting.

2.5. Subcellular Fractionation

Freshly harvested whole heart tissue samples from WT and Perm1^−/− mice were suspended in a homogenizing buffer containing 10 mM Tris-HCl (pH 7.4), 250 mM sucrose,1 mM ethylenediamine tetraacetic acid (EDTA), supplemented with protease inhibitors (1 mM sodium vanadate [Na3VO4], 1 mM sodium fluoride [NaF], and EDTA-free protease inhibitor cocktail tablet). Tissues were homogenized using a glass-glass Dounce tissue homogenizer and centrifuged at 1,000 × g for 10 min to generate a nuclear pellet and a supernatant containing mitochondria and cytosol. The pellet was resuspended with homogenizing buffer and overlayed on a 1M sucrose cushion followed by centrifugation at 1,600 × g for 5 min to generate a pellet (nuclear fraction). The supernatant was centrifuged at 10,000 × g for 30 min and a pellet was resuspended in the storage buffer containing 1 mM NaCl, 1 mM Tris-HCl at pH7.6, 0.5 mM EDTA, NP-40, 0.5% SDS, and protease inhibitors (Na₃VO4 and NaF) (mitochondrial fraction). The supernatant was further centrifuged at 100,000 g for 60 min and the supernatant was collected to obtain the cytosolic fraction. All procedures were conducted at 4°C.

2.6. Co-immunoprecipitation (Co-IP)

For in vivo Co-IP, freshly harvested heart tissue samples from WT and Perm1^−/− mice were lysed in lysis buffer containing 0.5% Triton, 150 mM NaCl, 50 mM Tris-HCl, 1 mM NaF, 0.1 mM Na3VO4, 1 mM dithiothreitol (DTT), 1 mM EDTA, 1 mM ethylene glycol tetraacetic acid (EGTA), and EDTA-free protease inhibitor cocktail tablet. Lysate (750 μg - 1 mg) was incubated with 2 μg of either anti-O-GlcNAc antibody (RL2, Abcam #ab2739) or anti-IgG antibody (control, R&D systems #MAB002) overnight in a rotator for immunoprecipitation. It was followed by incubation with protein-G Sepharose beads for 1 hr 30 min in a rotator and centrifugation at 10,000 rpm for 2 min. The supernatant was aspirated, and the pellet was washed 4 times with lysis buffer. Sample buffer (Bio-Rad #1610737 with 2-mercaptoethanol [Bio-Rad#1610710],10 μl) was added to the washed pellet and spun down to get the supernatant that was used for immunoblotting. Anti-PGC-1α (Abcam #ab191838) was used as a primary antibody for immunoblotting.

For in-vitro Co-IP, H9c2 cells were plated in 15 cm culture dishes. Cells at ~70% confluency were transduced with Ad-PGC-1α-tagged with FLAG or Ad-LacZ (control) at a MOI of 50 for 24hr followed by treatment with 20 μM siOGA (Qiagen #1027417) or scr-siRNA (Qiagen #1027310), for another 24hr and the cells were harvested in lysis buffer. Two siRNAs were used for silencing OGA (S101915074 and S101915067). Immunoprecipitation was performed by incubating lysate (750 μg −1 mg) with 2 μg of anti-FLAG (Sigma-Aldrich#F3165) or anti-IgG (R&D systems#MAB002). The remaining steps were same as mentioned above for in vivo Co-IP. Anti-PPARα (Cayman; product code #101710) was used as a primary antibody for immunoblotting. To assess the interaction between PERM1 and E2F1, FLAG-tagged Ad-PM1 was exogenously expressed in H9c2 cells (Ad-LacZ as control). After 24 hours, cells were transfected with either siE2F1 (Qiagen # 1027417; S100073976) or scr-siRNA for 24 hours. Co-IP was performed as described above using anti-FLAG for immunoprecipitation (IgG as control). Immunoblotting was then conducted using anti-E2F1 primary antibody (Cell Signaling #3742S).

2.7. Western Blotting Analysis

Samples for Western blotting analysis was prepared and analyzed as we previously performed [38]. Briefly, for in-vivo sample preparation, left ventricle tissue samples were lysed in lysis buffer containing 50 mM Tris (pH 7.4), 10 mM EDTA, 1% sodium dodecyl sulphate (SDS), and protease inhibitors that include sodium butyrate (NaB), phenylmethyl sulphonyl fluoride (PMSF), Na3VO4, NaF and EDTA-free protease inhibitor cocktail tablet. The lysate was centrifuged at 13,000 g for 10 min at 4°C and the supernatant was subjected to SDS electrophoresis followed by western blotting using 0.2 μm nitrocellulose membrane (Bio-Rad#1620112). For in-vitro sample preparation, the H9c2 cells were scraped in the lysis buffer (mentioned above) and were sonicated and used for Western blotting in the nitrocellulose membrane. The protein levels of total O-GlcNAcylated proteins were determined using mouse antibody RL2 (Abcam #ab2739) with 1: 1000 dilution and followed by anti-mouse secondary antibody (Abcam #ab6728) with 1: 5000 dilution. The protein levels of OGT, OGA, PERM1, H3, PGC-1α, PPARα, voltage-dependent anion channel (VDAC), E2F1, GLUT4, CPT1b, CPT2 and MCAD were detected using rabbit antibodies (Cell signaling #24083, Abcam #ab124807, Sigma #HPA031711, Abcam #ab1791, Abcam #ab191838, Cayman; product code #101710, Abcam #14734, Cell Signaling #3742S, Abcam # 33780, Cell Signaling #41803S, Cell Signaling #52552S and Abcam #92461) with 1:1000 dilution followed by goat anti-rabbit secondary antibody (Jackson ImmunoResearch Laboratories, Inc. #711– 035-152) with 1:5000 dilution. The values of band intensities were normalized using β-tubulin (Abcam, #ab6046). The protein expression levels of β-tubulin were consistent in all groups that were used in this study. For the quantification of O-GlcNAc blots from cardiac tissue samples (Figures 1A and 5A), the blots were divided into two portions: bands ≥75 kDa (referred to as “HMW”) and bands <75 kDa (referred to as “LMW”). In the HMW portion, non-specific bands around 75 kDa, which resulted from IgG band staining, were excluded by re-immunoblotting the same samples using anti-O-GlcNAc antibody pre-incubated overnight with 50 mM N-acetylglucosamine, an O-GlcNAc specific sugar (see Fig. S1 for Figure 1A; other competitive blots are shown in Supplemental Materials). Then, both HMW and LMW bands were quantified separately. The O-GlcNAc blots from H9c2 cells were quantified using the same approach, except that non-specific band subtraction was not necessary, as the competitive blots showed no detectable bands (Supplementary Materials).

Figure 1. O-GlcNAcylation is elevated in Perm1^−/− hearts.

(A-B) Western blotting analysis shows that a significant increase in the expression levels of total O-GlcNAcylated proteins at high molecular weight (HMW) and OGT in Perm1^−/− hearts compared to WT hearts, with no significant change in OGA levels (n= 6 WT and n=8 Perm1^−/− except for OGA it is n=7 Perm1^−/−, mean ± SEM). Total O-GlcNAcylated proteins were quantified separately for higher molecular weights (HMW, ≥75kDa) and lower molecular weights (LMW, <75kDa) (see Methods for details). (C) Schematic of the hexosamine biosynthesis pathway (HBP) and O-GlcNAcylation. The changes in genes and proteins in Perm1^−/− hearts relative to WT hearts are indicated by red arrows. Figure created with Biorender.com. (D) qPCR shows that the mRNA levels of glucose transporters (Glut1 and Glut4), and two key enzymes in the HBP (Gfat2 and Pgm5) were significantly increased in Perm1^−/− hearts (n=10/group; mean ± SEM). All comparisons are by unpaired two-tailed t-test. *p<0.05.

Figure 5. Loss of PERM1 increases O-GlcNAcylation of PGC-1α in association with partial dissociation from PPARα.

(A-D) Subcellular fractionation of cardiac tissue from WT and Perm1^−/− hearts. Compared with WT hearts, Perm1^−/− hearts exhibited the increased levels of total O-GlcNAcylated proteins, both at HMW and LMW, as well as OGT in the nuclear fraction. In contrast, these levels were decreased in the cytosolic fraction of Perm1^−/− hearts. No significant differences in OGA expression were observed across any subcellular fractions. Nucl: nucleus, Mito: mitochondria, Cyto: cytosol. (n=3/group; mean ± SEM). (E,F) Co-immunoprecipitation (Co-IP) using anti-O-GlcNAc followed by immunoblotting using anti-PGC-1α shows the significant increase of O-GlcNAcylated PGC-1α levels in Perm1^−/− hearts as compared with those in WT hearts (n=3/group; mean ± SEM). (G,H) Co-IP of H9c2 cells shows that the deliberate increase of O-GlcNAcylation via siOGA promoted O-GlcNAcylation of PGC-1α (n=3 in scr-siRNA, n=4 in siOGA, mean ± SEM). (I,J) H9c2 cells were treated with either adenovirus-PGC-1α-FLAG (“PGC-1α-FLAG”) or adenovirus-LacZ (“LacZ”, control) followed by transfection with either scr-siRNA (control) or siOGA. Co-IP using anti-FLAG followed by immunoblotting using anti-PPARα shows the significant reduction of PGC-1α that interacted with PPARα as compared with control cells (n=3/group; mean ± SEM). IgG was used as a non-specific antibody. *p<0.05. All comparisons are by unpaired two-tailed t-test.

2.8. Gene Expression Analysis

Real-time PCR was performed using Taqman primers as previously described [38]. Briefly, RNA was extracted using TRizol (Life Technologies), followed by ethanol extraction. Reverse transcription was performed from 1μg of RNA using QuantiTect Reverse Transcription Kit according to the manufacture’s instruction (Qiagen). Taqman primers were purchased from ThermoFisher as follows: Glut1: Mm00441480_m1 and Rn01417099_m1; Glut4: Mm0043661-m1 and Rn00562597_m1; Hk1: Mm00439344_m1; Hk2: Mm00443394_m1 and Rn00562457_m1; Gpi: Mm01962484_u1 and Rn01475756_m1: Gapdh: Mm00186822_g1 and Rn01775763_g1; Ar: Mm00442688_m1; sord: Mm00455377_g1 and Rn00820764_g1; Gfat1, Mm00600127_m1 and Rn01765495_m1; Gfat2: Mm00496565_m1 and Rn01456720_m1; Gnpnat1: Mm07297511_g1 and Rn01414655_m1; Pgm3: Mm01144498_m1 and Rn01403962_m1; Uap1: Mm01281909_m1 and Rn01490642_g1; Ogt: Mm00507317_m1 and Rn00820779_m1; Oga: Mm00452409_m1 and Rn00590870_m1. The Cq values from the genes were normalized using Rpl32 (Mm07306626_gH and Rn00820748_g1).

3. Results

3.1. Loss of PERM1 in mice leads to the increase of O-GlcNAcylation in the heart.

We first examined whether O-GlcNAcylation process was affected by PERM1 loss-of-function. We measured the total levels of O-GlcNAcylated proteins and the protein expression of OGT and OGA in the hearts of Perm1^−/− mice and their age-matched wild-type (WT) littermates. We found that the total O-GlcNAcylated protein levels at high molecular weights (≥75 kDa) were significantly upregulated in Perm1^−/− hearts as compared with WT hearts, concurrent with a significant upregulation of OGT, but there was no change in OGA expression (Fig. 1A-B).

OGT catalyzes the transfer of a GlcNAc moiety from UDP-GlcNAc, a donor substrate, to serine or threonine residues in proteins, and UDP-GlcNAc is an end-product of HBP, a glycolytic accessory pathway (Fig. 1C). To determine whether the increase of O-GlcNAcylation observed in Perm1^−/− hearts was associated with upregulation of HBP and glucose transporters, real-time quantitative PCR (RT-qPCR) was performed to measure the expression of genes involved in HBP and glucose uptake in cardiomyocytes. We found that the mRNA levels of myocardial glucose transporters Glut1 and Glut4 and two enzymes in the HBP (Gfat2 and Pgm3) were significantly increased in Perm1^−/− hearts as compared to WT littermates (Fig. 1C-D). Hence, PERM1 loss-of-function causes concerted alterations in the expression of key enzymes involved in glucose uptake and the HBP, all leading to the direction of promoting the O-GlcNAcylation process.

3.2. PERM1 suppresses O-GlcNAcylation through transcriptionally repressing Ogt and glucose transporters in H9c2 cells.

The next question was whether PERM1 gain-of-function leads to the outcomes opposite to those observed in the loss-of-function model (Fig. 1). To address this question, we performed adenovirus-mediated overexpression of PERM1 (Ad-PM1) in H9c2 cardiomyocytes. PERM1 overexpression led to a significant decrease in the mRNA levels of myocardial glucose transporters (Glut1 and Glut4) and Ogt, while Oga was upregulated by PERM1 overexpression, as compared to control cells that were treated with adenovirus LacZ (Ad-LacZ, all p<0.05, Fig. 2A-C). Of note, the expression levels of genes involved in glycolysis and HBP were not altered by PERM1 overexpression, except Gfat1, which was upregulated in Ad-PM1 treated cells (Fig. 2C). Consistent with RT-qPCR data (Fig. 2A-C), western blotting analysis showed that PERM1 overexpression resulted in downregulation of OGT and upregulation of OGA at the protein levels (both p<0.05, Fig. 2D-E). Expectedly, this in turn caused a significant decrease in total O-GlcNAcylation (p<0.05, Fig. 2D-E). These results suggest that PERM1 negatively regulates O-GlcNAcylation through suppressing the expression of OGT and glucose transporters and increasing OGA expression. Given that PERM1 is localized in the nucleus and is involved in transcriptional control of metabolic genes [37], we examined whether PERM1 transcriptionally regulates the expression of Ogt, Oga and glucose transporters. Luciferase gene promoter assay was performed to measure the promoter activities of those genes in H9c2 cells. As shown in Fig. 2F, PERM1 overexpression significantly reduced the promoter activities in Ogt as compared with the Ad-LacZ (control) group, suggesting that PERM1 acts as a gene repressor of Ogt. In contrast, there was no significant change in the promoter activity of Oga (Fig. 2F). Moreover, we found that the promoter activities of Glut1 (Slc2a1) and Glut4 (Slc2a4) were significantly reduced by PERM1 overexpression (both p<0.05, Fig. 2G). Taken together, these data suggest that PERM1 suppresses O-GlcNAcylation through transcriptionally repressing Ogt and glucose transporter genes, latter which presumably limits glucose uptake in cardiomyocytes.

Figure 2. PERM1 acts as a transcriptional repressor of Ogt and glucose transporters in H9c2 cells.

(A-C) RT-qPCR shows that adenovirus-mediated overexpression of PERM1 (Ad-PM1) in H9c2 cells resulted in downregulation of glucose transporters (Glut1 and Glut4) and Ogt. PERM1 overexpression had little effects on the expression of enzymes involved in the glycolysis and hexosamine biosynthesis pathway, except the mRNA level of Gfat1, which was increased (n=8/group; mean ± SEM). (D,E) Western blotting analysis shows PERM1 overexpression decreased the levels of total O-GlcNAcylated proteins at both HMW and LMW and OGT, while upregulating OGA expression (n=7/group; mean ± SEM). (F,G) Luciferase gene promoter assay in H9c2 cells shows that PERM1 overexpression significantly decreased the promoter activities of Ogt, Glut1 and Glut4, whereas there was no change in the promoter activity of Oga (n=6/group; mean ± SEM). *p<0.05 in control vs. Ad-PM1. (H-I) Western blot analysis shows that siRNA-mediated knockdown of E2F1 (siE2F1) in H9c2 cells significantly increased OGT expression. In contrast, the PERM1 overexpression (Ad-PM1)-induced suppression of OGT was completely abolished by siE2F1. *: p<0.05 compared with control, +: p<0.05 compared with siE2F1, #: p<0.05 compared with Ad-PM1. (J) Co-immunoprecipitation assay demonstrates that exogenously expressed PERM1-FLAG in H9c2 cells was pulled down with E2F1 using anti-FLAG, indicating an interaction between PERM1 and E2F1. Comparisons in Panels A,B,C,E,F,G by unpaired two-tailed t-test. Comparisons in Panel I are by 2-way ANOVA.

The promoter of Ogt contains multiple E2F binding site consensus sequences, and E2F1 is a known transcription factor that represses the Ogt gene [43]. Since PERM1 functions as a transcriptional cofactor [34], we hypothesized that PERM1 negatively regulates Ogt expression through E2F1. To test this, H9c2 cells were treated with either siRNA targeting E2F1 (siE2F1) alone or siE2F1 in combination with Ad-PERM1. As expected, consistent with E2F1’s role as a repressor of Ogt [43], Western blot analysis showed that E2F1 knockdown via siRNA resulted in a significant increase in OGT expression (p<0.05, light green vs. light blue in Fig. 2I). In agreement with the data shown in Fig. 2D-E, PERM1 overexpression via Ad-PERM1 significantly reduced OGT expression (p<0.05, light green vs. orange in Fig. 2I). This reduction was completely abolished when E2F1 was silenced (siE2F1 + Ad-PERM1), suggesting that PERM1 requires E2F1 to repress Ogt. Gene reporter assays further confirmed this E2F1-dependent regulation of Ogt transcription, showing that the reduced promoter activity in PERM1-overexpressing cells was fully abolished by siE2F1 (Fig. S2). Importantly, siE2F1 did not affect PERM1 expression levels (p>0.05, light green vs. light blue), and Ad-PERM1 had no effect on E2F1 expression (p>0.05, light green vs. orange in Fig. 2J). Moreover, siE2F1 had no effect on Ad-PERM1-mediated downregulation of GLUT4, suggesting that PERM1 transcriptionally regulates GLUT4 in an E2F1-independent manner. Additionally, co-immunoprecipitation (Co-IP) confirmed that PERM1 interacts with E2F1 (Fig. 2J). In this experiment, FLAG-tagged PERM1 was exogenously expressed in H9c2 cells (Ad-PERM1-FLAG) and pulled down using anti-FLAG. Together, these data suggest that PERM1 negatively regulates Ogt expression through its interaction with the transcription factor E2F1.

To determine whether glucose availability in cardiomyocytes alters O-GlcNAcylation, H9c2 cells were cultured in the media that contained 0, 5 and 25 mM of glucose. The total levels of O-GlcNAcylated proteins were progressively increased as the function of glucose concentration in the cell culture media (Fig. S3). This was consistent with progressive and reciprocal changes in the protein levels of OGT and OGA, respectively (Fig. S3). These findings confirm previously reported results that glucose availability is per se a strong driver of O-GlcNAcylation process [41, 44]. Importantly, the alterations in glucose availability did not affect the protein levels of PERM1 expression (Fig. S3). This led us to a hypothesis that PERM1 stabilizes O-GlcNAcylation during the fluctuations in glucose availability as an upstream regulator of OGT and OGA. Strikingly, PERM1 overexpression (Ad-PM1) fully abolished the effect of high glucose on O-GlcNAcylation (25 mM in media), concurrent with less prominent changes in the expression of OGT and OGA as compared with 0 mM and 5 mM glucose (Fig. 3A-B, Fig. S3). These results reveal an important role of PERM1 as a stabilizer of the overall O-GlcNAcylation process in cardiomyocytes in the face of abnormal fluctuations of glucose under pathological stress (i.e., hyperglycemia).

Figure 3. PERM1 stabilizes O-GlcNAcylation during fluctuations in glucose availability as an upstream regulator of OGT and OGA.

H9c2 cells were cultured in media that contained three different glucose concentrations: 0 mM, 5 mM, and 25 mM for 24hr. Western blotting analysis shows a progressive increase in the levels of total O-GlcNAcylated proteins and OGT as a function of glucose concentrations in media (“Ad-PM1 -”). In contrast, a reciprocal change in the protein levels of OGA was observed relative to glucose concentrations in media (also see Fig. S3). PERM1 overexpression (“Ad-PM1 +”) blunted the effect caused by the increased glucose concentrations in media, suppressing O-GlcNAcylation and OGT expression. Of note, PERM1 expression was not affected by the increase of glucose concentrations in media. The data were normalized to control cells at 25 mM of glucose (indicated as “Ad-PM1 –, 25mM” in Panel B), which was set at 100% (n=4/group; mean ± SEM). *: p<0.05 by 2-way ANOVA followed by post-hoc Sidak’s multiple comparison test.

In summary, these data suggest that PERM1 suppresses O-GlcNAcylation by simultaneously downregulating OGT and limiting the substrate availability (i.e., UDP-GlcNAc from HBP) through regulating the expression of glucose transporters. A decrease in glucose availability further suppresses O-GlcNAcylation through the reciprocal alterations in OGT and OGA expression. On the other hand, the suppression of O-GlcNAcylation by PERM1 overexpression was not associated with the changes in expression of HBP enzymes (Fig. 2C). Taken together, PERM1 suppresses O-GlcNAcylation in cardiomyocytes through regulating glucose uptake, rather than via the HBP.

3.3. PERM1 rescues reduced mitochondrial respiration caused by elevated O-GlcNAcylation in H9c2 cells.

We and other groups have shown that PERM1 regulates mitochondrial energetics in cardiac and skeletal muscles [35, 36, 38, 39]. Several studies suggest that the altered O-GlcNAcylation changes the expression of genes/proteins that are involved in oxidative phosphorylation (OXPHOS) [29, 40]. However, it remains unknown how O-GlcNAcylation regulates mitochondrial respiration capacity in cardiomyocytes. To examine how the changes in O-GlcNAcylation levels affect mitochondrial function, we first tested the effects of silencing Ogt (siOGT) and Oga (siOGA) on the total levels of O-GlcNAcylated proteins in H9c2 cells. As compared with control cells that were treated with scrambled siRNA (scr-siRNA), silencing of Oga effectively increased the total O-GlcNAcylation levels (p<0.05), while there was no significant change in the total O-GlcNAcylation levels by siOGT (p>0.05, Fig. 4A-B). Of note, neither siOGA nor siOGT affected the protein levels of PERM1, indicating that PERM1 is upstream of OGT and OGA (Fig. 4A-B). Note also that silencing Oga caused a significant reduction in OGT expression, which reflects a complex regulatory coupling between these two enzymes [11]. However, despite the similar levels of reduction in OGA and OGT expression caused by siOGA, the reduction in the levels of OGA was predominant and determined the increase in the total O-GlcNAcylation levels (Fig. 4A-B). Furthermore, we found that the effect of siOGA on the total O-GlcNAcylation was much more effective than the well-known inhibitors of OGA and OGT: Thiamet G and OSMI-1, respectively (Fig. S5). Thus, we used siOGA as a tool to increase O-GlcNAcylation in the following study.

Figure 4. PERM1 rescues the reduced cellular respiration caused by excessive O-GlcNAcylation in H9c2 cells.

(A,B) H9c2 cells were transfected with siRNA-OGA (siOGA), siRNA-OGT (siOGT) or scrambled-siRNA (scr-siRNA, control) for 48hr. Western blotting analysis shows that siOGA significantly increased the levels of total O-GlcNAcylated proteins at both HMW and LMW, whereas siOGT did not affect the O-GlcNAcylated protein levels. *p<0.05 by unpaired two-tailed t-test. (n=4/group; mean ± SEM). (C,D) Adenovirus-mediated overexpression of PERM1 (Ad-PM1) fully abolished the effect of siOGA, suppressing O-GlcNAcylation and OGT expression and increasing the expression levels of OGA. *: p<0.05 compared with control, +: p<0.05 compared with siOGA, #: p<0.05 compared with Ad-PM1. (n=4/group; control: Ad-LacZ + scr-siRNA; mean ± SEM, 2-way ANOVA followed by post-hoc Sidak’s multiple comparison test). (E,F) Cell Mito Stress Test in H9c2 cells shows that PERM1 overexpression (Ad-PM1) increased the basal and maximal respiration rates as well as ATP production, compared to the control (Ad-LacZ), when pyruvate and glucose were used as substrates. In contrast, siOGA decreased the basal and maximal respiration rates, which was fully rescued by PERM1 overexpression (Ad-PM1+siOGA) *: p<0.05 compared with control, +: p<0.05 compared with siOGA, #: p<0.05 compared with Ad-PM1 (n=6/group; control: Ad-LacZ + scr-siRNA; mean ± SEM, 2-way ANOVA followed by post-hoc Sidak’s multiple comparison test).

Next, we examined whether PERM1 overexpression can suppress O-GlcNAcylation in siOGA-treated cells. Consistent with the results shown in Fig. 2D-E and Fig. 4A-B, siRNA-mediated OGT knockdown increased total O-GlcNAcylation, while adenovirus-mediated overexpression of PERM1 (Ad-PM1) decreased total O-GlcNAcylation (Fig. 4C-D). Importantly, the combined treatment of Ad-PM1 and siOGA (Ad-PM1 + siOGA) achieved the decreased levels of total O-GlcNAcylated proteins, which was associated with a significant decrease in OGT expression and increase in OGA expression (Fig. 4C-D). It is of interest that PERM1 overexpression was able to rescue OGA expression despite its rather efficient silencing by siRNA.

To determine how PERM1 and O-GlcNAcylation regulate mitochondrial respiration capacity, the Cell Mito Stress Test was performed using a Seahorse 96XFe flux analyzer. H9c2 cells were treated with either Ad-LacZ (control) or Ad-PM1 (PERM1 overexpression), followed by transfection with either scr-siRNA (control) or siOGA. Consistent with our previous study [36], PERM1 overexpression significantly increased mitochondrial respiration capacity in the presence of glucose and pyruvate as substrates, manifested by the increase of basal, maximal, spare respiration and ATP production, compared with control cells (all p<0.05 in control vs. Ad-PM1 + scr-siRNA; light green vs. orange in Fig. 4E-F). Conversely, a deliberate increase of O-GlcNAcylation via siOGA significantly reduced the basal, maximal, spare respiration and ATP production rates (all p<0.05 in Ad-LacZ + scr-siRNA vs. Ad-LacZ + siOGA; light green vs. light blue in Fig. 4E-F). Importantly, the negative impact of the elevated O-GlcNAcylation with respect to mitochondrial respiration capacity was fully overcome by PERM1 overexpression (Ad-PM1 + si-OGA, p<0.05; light blue vs. pink in Fig. 4E-F). In the presence of Ad-PM1, siOGA essentially had little effect on mitochondrial respiration (orange vs. red in Fig. 4E-F), which, in part, could be explained by the fact that PERM1 overexpression rescues OGA expression, despite silencing by siRNA, and counteracts the effect of siOGA on OGT expression (Fig. 4C-D). Notably, PERM1 overexpression led to a significant increase in respiration capacity when pyruvate was used as the sole substrate, (light green with diagonal stripes vs. orange with diagonal stripes in Fig. S4, all p<0.05). However, PERM1 overexpression did not enhance mitochondrial respiration capacity when glucose was the substrate (Fig. S4, light green vs. orange, all p>0,05). This aligns with the suppression of myocardial glucose transporters GLUT1 and GLUT4 by PERM1 (Fig. 2B, 2G), suggesting limited glucose uptake as a potential factor.

3.4. The increase of nuclear O-GlcNAcylated proteins in Perm1^−/− hearts.

O-GlcNAcylation occurs in most subcellular fractions, including cytosol, nucleus, and mitochondria [45]. Consistently, the subcellular localization of OGT and OGA in the cytosol, nucleus, and mitochondria has been reported [46]. We have previously reported that PERM1 is present in the nucleus, cytosol and mitochondria [38]. To determine where in cells the increase of O-GlcNAcylation occurs in response to loss of PERM1, we performed subcellular fractionation of cardiac tissue from WT and Perm1^−/− mice. The specificity of subcellular fractions was confirmed using specific stains for the nucleus, mitochondria, and cytoplasm (Fig. S6). Among those subcellular fractions, we found that the total levels of O-GlcNAcylated proteins were significantly increased in the nuclear fractions of Perm1^−/− hearts compared with those in WT hearts (Fig. 5A-B). Consistently, OGT expression was significantly higher in the nucleus in Perm1^−/− hearts, while there was no significant change in OGA expression in the nuclear fraction (Fig. 5C-D).

3.5. Loss of PERM1 increases O-GlcNAcylation of PGC-1α in association with downregulation of the genes involved in FAO and the partial dissociation from PPARα.

PGC-1α is a transcriptional co-factor, which is localized in the nucleus to regulate the expression of genes involved in FAO and mitochondrial bioenergetics through interacting with various transcription factors [47]. It has been reported that PGC-1α is an O-GlcNAcylation target [48, 49]. Thus, we hypothesized that the observed increase in the total levels of O-GlcNAcylated nuclear proteins in Perm1^−/− hearts is associated with the increase of O-GlcNAcylated PGC-1α. Co-immunoprecipitation (Co-IP) using anti-O-GlcNAc showed that PGC-1α is O-GlcNAcylated, which was significantly increased in Perm1^−/− hearts as compared with that in WT hearts (Fig. 5E-F). Furthermore, the increase of O-GlcNAcylation through siOGA significantly increased O-GlcNAcylated PGC-1α (Fig. 5G-H). Our previous study showed that FAO genes were downregulated in Perm1^−/− hearts [38]. These genes are regulated by PPARα, a transcription factor that interacts with PGC-1α. Thus, we hypothesize that the increase of O-GlcNAcylated PGC-1α in Perm1^−/− hearts might lead to its dissociation from PPARα. To address this hypothesis, H9c2 cells were treated with adenovirus-PGC-1α-FLAG, and Co-IP was performed using anti-FLAG to pull down exogenously expressed PGC-1α followed by immunoblotting using anti-PPARα. The increase of O-GlcNAcylation via siOGA significantly reduced the interaction of PGC1α with PPARα as compared with scr-siRNA treated control cells (Fig. 5I-J).

To determine whether the partial dissociation of PGC-1α from PPARα via excessive O-GlcNAcylation leads to reduced FAO, we measured the mitochondrial respiration driven by palmitic acid. Similar to glucose-driven mitochondrial respiration (Fig. 4E-F), PERM1 overexpression (Ad-PM1) significantly increased the basal and maximal respiration, as well as ATP production and the spare respiration capacity in the presence of palmitate as a sole exogenous substrate, as compared with Ad-LacZ (control) (dotted green bar vs. dotted orange bar in Fig. 6A-B). Furthermore, siOGA significantly decreased the basal and maximal respiration, as well as ATP production and the spare respiration capacity (Fig. 6C-D). The addition of a carnitine palmitoyl transferase 1 (CPT1) inhibitor etomoxir significantly decreased the mitochondrial respiration and ATP production in control cells (scr-siRNA + Ad-LacZ) but did not alter the effects of siOGA (Fig. S7A-D). This suggests that the suppressive effects of elevated O-GlcNAcylation on palmitate-driven respiration are restricted to the proteins residing in mitochondria. More importantly, overexpression of PERM1 fully abolished the suppressive effects of siOGA on all measured parameters of palmitate-driven mitochondrial respiration (blue bar vs. pink bar in Fig. 6C-D). To support the notion that PERM1 positively regulates FAO, overexpression of PERM1 led to an increase in the expression of PPARα and PPARα genes (CPT1b, CPT2 and MCAD, all p<0.05, Fig. 6E-F)

Figure 6. PERM1 rescues the siOGA-mediated reduction in fatty acid oxidation (FAO) capacity.

(A-D) Palmitate Oxidation Stress Test was performed in H9c2 cells to measure oxygen consumption rates (OCR) resulting from long-chain fatty acid oxidation using palmitate (PA) as a substrate. (A-B) PERM1 overexpression (Ad-PM1) significantly increased basal, maximal, and spare respiration capacity, as well as ATP production in the presence of PA, compared to BSA-treated and control cells (Ad-LacZ), indicating that PERM1 enhances FAO. *: p<0.05 compared with Ad-LacZ+BSA, +: p<0.05 compared with Ad-LacZ+PA, #: p<0.05 compared with AdPM1+BSA. (C-D) Excessive O-GlcNAcylation via siOGA reduced both basal and maximal respiration rates, whereas PERM1 overexpression (Ad-PM1) fully reversed the effect of siOGA. *: p<0.05 compared with control, +: p<0.05 compared with siOGA, #: p<0.05 compared with Ad-PM1. (n = 6/group; mean ± SEM). (E-F) Western blot analysis shows that PERM1 overexpression (Ad-PM1) significantly increased the protein levels of PPARα and its target genes involved in FAO, compared to control cells treated with Ad-LacZ (n = 6 for Ad-LacZ, n = 7 for Ad-PM; mean ± SEM). *p<0.05. (G) Schematic illustrating PERM1’s role in regulating mitochondrial energetics via O-GlcNAcylation. PERM1 stabilizes O-GlcNAcylation levels in cardiomyocytes by transcriptionally repressing the expression of OGT and glucose transporters (GLUT1 and GLUT4). This study suggests that PERM1 limits O-GlcNAcylation of PGC-1α, thereby facilitating its interaction with PPARα, ultimately promoting mitochondrial respiration and FAO. Figure created with Biorender.com. Comparisons in Panels B and D are by 2-way ANOVA followed by post-hoc Tukey’s multiple comparison test). Comparisons in Panel F by unpaired two-tailed t-test.

Overall, these data show that loss of PERM1 leads to the increase of O-GlcNAcylation of PGC-1α, which may contribute to the downregulation of FAO (Fig. 6G). By converse logic, these data suggest that the normal role of PERM1 is to limit the level of O-GlcNAcylation of PGC-1α, which would help to maintain a higher level of FAO in the face of elevated O-GlcNAcylation resulting from stress or nutrient signals.

4. Discussion

PERM1 is a striated muscle-specific regulator of mitochondrial bioenergetics. It was first discovered by the Kralli group [34] as a protein induced by exercise. PERM1 was identified in cultured C2C12 myotubes as a downstream target of PGC-1α and ERRα involved in regulating energy metabolism and contractile function [34]. The same group showed the role of PERM1 in mitochondrial bioenergetics in vivo by overexpressing PERM1 in mice skeletal muscles [35]. The role of PERM1 in the heart was not identified until we first recognized its role as an essential regulator of mitochondrial function and an upstream regulator of ERRα in cardiomyocytes [36]. Subsequently, we and other groups demonstrated that PERM1 interacts with PGC-1α enhancing transactivation of transcriptional factors PPARα [37] and ERRα [38, 39], which leads to increase in mitochondrial biogenesis and oxidative capacity.

In addition to its involvement into regulation of energy metabolism through transcriptional control, PERM1 was shown to have other molecular roles, including activation of the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), a kinase involved in excitation-contraction coupling [50], and interaction with the intracellular adaptor protein Ankyrin B and the mitochondrial contact site and cristae organizing system - mitochondrial intermembrane space bridging complex (MICOS-MIB) [51]. Recently, Cho et al. demonstrated that PERM1 overexpression increases expression of Mic60, a protein playing a central role in MICOS formation, although the authors did not specify the molecular mechanism of this regulatory effect [52]. From all these studies, PERM1 emerges as a multifunctional protein regulating bioenergetics and contractility through a variety of mechanisms, but with a common vector of regulation directed towards enhancement of striated muscle function. This current study reveals yet another function of PERM1, that of a negative regulator of O-GlcNAcylation in the heart.

O-GlcNAcylation is a post-translational modification of proteins where N-acetylglucosamine moieties are added to the serine and threonine residues of proteins residing in the cytoplasm, the nucleus, and the mitochondria. O-GlcNAcylation is regulated by two enzymes: OGT that adds O-GlcNAc to proteins; OGA that removes O-GlcNAc from proteins. The total levels of O-GlcNAcylation are determined by nutrients and metabolic flux. In fact, hyperglycemia per se is sufficient to increase O-GlcNAcylation, due to simultaneous increase in the availability of N-acetylglucosamine through the HBP, an increase in expression of OGT, and a decrease in the expression of OGA (Fig. S3). Myocardial contractile function and cardiac energetics are regulated by O-GlcNAcylation [40, 41, 53]. Chronic cardiac diseases, including heart failure and diabetic cardiomyopathy, are associated with the elevated cardiac levels of O-GlcNAcylation [29, 54]. Although the proteins in the OXPHOS pathway are known to be O-GlcNAcylated [40], the effect of increased O-GlcNAcylation on mitochondrial function and mitochondrial energetics in the heart remains not fully settled. While a majority of reports show adverse effect of elevated O-GlcNAcylation with respect to mitochondrial function [29, 41, 44], few studies showed a protective response [40] or no significant change [55]. Several studies demonstrated that excessive O-GlcNAcylation of proteins involved in the electron transport chain (ETC) complexes leads to significant decreases of activity of ETC Complexes I, III, and IV [41, 44, 54]. The various outcomes in those studies might depend on which OXPHOS proteins are O-GlcNAcylated. With this regard, it is important in our future study to identify the mitochondrial proteins that are targeted by O-GlcNAcylation through loss of PERM1.

A recent study by Umapathi et al. has shown that cardiac specific overexpression of OGT caused increased O-GlcNAcylation, dilated cardiomyopathy, and premature death. Overexpression of OGA has led to reduced myocardial O-GlcNAcylation but did not cause cardiomyopathy and conferred protection against pressure overload-induced heart failure. The authors concluded that reducing myocardial O-GlcNAcylation could be a successful therapeutic approach to heart failure [29]. Yet, cardiac-specific OGT deletion associated with significantly decreased O-GlcNAcylation exacerbated cardiac dysfunction following myocardial infarct or transverse aortic constriction in mice [32, 56]. Overall, it seems that it is quite difficult to fine-tune O-GlcNAcylation process through directly manipulating the expression of OGT/OGA for the purpose of managing the most widespread cardiac diseases. In this study, we demonstrate that PERM1 has a remarkable property of stabilizing the levels of O-GlcNAcylation in cardiac myocytes in the context of varying glucose load. Specifically, PERM1 overexpression almost fully prevented the increase in total O-GlcNAcylation induced by high glucose in the media (Fig. 3). Hence, PERM1 gain-of-function affords prevention of O-GlcNAcylation excess while disrupting its essential “basal” O-GlcNAcylation functions. Such a “smart” way by which PERM1 regulates O-GlcNAcylation may help maintain O-GlcNAcylation within an optimal range, which is critical for managing cardiac disease.

Our current study showed that OGT expression increases, while OGA expression decreases, in a direct proportion to extracellular glucose concentrations. Which regulatory steps connect glucose flux to the O-GlcNAcylation enzymes remains largely unknown. Hyperglycemia can induce oxidative stress, and a few transcription factors associated with conditions of stress are identified as transcriptional repressors and activators of OGT. Transcriptional factor, E2F1 is known as a repressor of OGT and nuclear factor erythroid 2–related factor 2 (NrF2) and NF kappa beta (NF-κB) as transcriptional activators of OGT [57]. Our study demonstrates that PERM1 negatively regulates O-GlcNAcylation by repressing Ogt promoter activity through its interaction with E2F1 (Fig. 2H-J). Additionally, PERM1 significantly decreased the promoter activity of glucose transporters Glut1 and Glut4 (Fig. 2G). Since PERM1 functions as a transcription co-activator [34], It is likely that PERM1 requires specific transcription factor(s) to repress Ogt and Glut1/Glut4. In our study, we showed that PERM1 interacts with the transcription factor E2F1, and silencing E2F1 fully abolished PERM1’s suppression of OGT expression (Fig. 2H-J). Although E2F1 is primarily recognized as a transcriptional activator, it also represses several genes, including TERT, Mcl-1, and key regulators of energy homeostasis and mitochondrial function [58]. A previous study found that E2F1 overexpression in HEK293 cells decreased OGT protein levels [43] which is consistent with our observation following PERM1 overexpression (Fig 2H-I). Furthermore, the complete abolition of PERM1’s suppression of OGT expression upon E2F1 silencing suggests that E2F1 is essential for PERM1 to repress Ogt. In breast cancer cells, E2F’s association with the retinoblastoma protein pRB inhibits the expression of E2F-regulated genes through the recruitment of repressors, such as histone deacetylases and methyltransferase [59]. It remains to be determined whether the interaction between PERM1 and E2F1 similarly recruits epigenetic modifiers to repress Ogt. It is worth to note that we did observe significant and symmetric changes in OGA expression due to PERM1 gain-of-function, which were reciprocal to changes in OGT (Fig. 2D-E). However, we found no evidence that PERM1 regulates Oga promoter activity (Fig. 2F). The changes in OGA expression may be linked to PERM1’s effects on glucose transporters, as alterations in glucose flux are sufficient to influence OGA expression (Fig. 3).

We previously demonstrated that the mRNA levels of GLUT1 and GLUT4 were significantly upregulated in Perm1-null hearts [38]. In contrast, a study by Huang et al. reported downregulation of myocardial glucose transporters and glycolytic enzymes in Perm1-KO mice [37]. The discrepancy in the effects of PERM1 loss-of-function on glucose metabolism remains unclear. In our current study, we confirmed that PERM1 negatively regulates the expression of GLUT1 and GLUT4, as shown by qPCR and luciferase reporter assays in a PERM1 gain-of-function model. Specifically, a Cell Mito Stress Test using a Seahorse XF analyzer demonstrated that PERM1 overexpression significantly increases mitochondrial respiration capacity when pyruvate is used as a substrate, while no change was observed with glucose (Fig. S4 in Supplementary Materials). These results align with both our previous findings and the current study, indicating that PERM1 enhances pyruvate oxidation while limiting glucose uptake, potentially preventing excessive flux into glycolytic accessory pathways, such as the hexosamine biosynthesis pathway, which fuels O-GlcNAcylation. Although our data revealed that PERM1-mediated repression of Ogt occurs through the transcription factor E2F1, the mechanism by which PERM1 represses Glut1 and Glut4 remains unclear. Notably, glucose flux does not regulate PERM1, suggesting that modulating PERM1 expression and function could provide a way to control cellular glucose flux and O-GlcNAcylation without causing instabilities or unpredictable outcomes due to feedback loops in the regulatory circuit.

Because PGC-1α is a prominent nucleic protein acting as a transcription coactivator that regulates mitochondrial bioenergetics and is a target of O-GlcNAcylation signaling [48], we focused on whether PERM1 regulates O-GlcNAcylation of PGC-1α. We confirmed that PGC-1α is O-GlcNAcylated at baseline conditions, and we found that both siOGA treatment and Perm1-knockout markedly increase O-GlcNAcylation of PGC-1α (Fig. 5E-H). Finally, we demonstrated that increased O-GlcNAcylation limited the interaction of PGC-1α with PPARα (Fig. 5I-J), a major transcriptional regulator of genes involved in FAO and fatty acid transport [37]. Huang et al. have recently suggested that PERM1 may interact with both PGC-1α and PPARα, promoting PPARα-mediated transcription of genes involved in fatty acid metabolism in the mouse heart [37]. Indeed, they demonstrated that a number of fatty acid metabolism enzymes are downregulated in Perm1^−/− mice [37]. Consistent with this, our gain-of-function study in H9c2 cells demonstrated that PERM1 enhances FAO, accompanied by the upregulation of PPARα and its target genes (Fig. 6). Notably, these FAO genes are also targets of PGC-1α [61]. Our current study revealed a complementary mechanism involving PERM1 in the PGC-1α /PPARα axis, namely through suppression of O-GlcNAcylation of PGC-1α. In addition, our study demonstrated that enhanced O-GlcNAcylation due to OGA silencing significantly reduced mitochondrial respiration driven by palmitate, and this was fully abolished by PERM1 overexpression (Fig. 6C-D). The fact that PERM1 affects O-GlcNAcylation in the nucleus but not in mitochondria (Fig. 5A-B) further supports the proposed regulatory axis, whereby PERM1 positively regulates fatty acid metabolism through limiting O-GlcNAcylation of PGC-1α in the nucleus. It is tempting to speculate that O-GlcNAcylation of PGC-1α is a part of the Randall cycle [62], so that an increased glycolytic flux reciprocally suppresses fatty acid metabolism through O-GlcNAcylation of PGC-1α and disruption of PGC-1α function as an activator of PPARα. If this is the case, then PERM1 would emerge as a regulator of the balance between carbohydrate and lipid oxidation in cardiomyocyte. Whether PERM1 regulates O-GlcNAcylation of other transcription (co)factors in energy metabolism need to be examined in our future study.

In conclusion, this study revealed a multi-tiered and coordinated regulation of O-GlcNAcylation by PERM1 with a prominent effect on PGC-1α and downstream energy metabolism (Fig. 6G). The components of this regulation include control of the glucose flux through glucose transporters; transcriptional control of OGT; and control of the nuclear localization of OGT and OGA. The latter regulatory mechanism is an important example of nature’s design to narrow down the extremely wide family of O-GlcNAcylation targets. With mounting evidence that excessive O-GlcNAcylation exacerbates pathogenesis of currently prevailing types of cardiac disease, the stabilizing effect of PERM1 with respect to O-GlcNAcylation makes it an interesting therapeutic target in treatments of cardiac patients.

Highlights.

• PERM1 regulates O-GlcNAcylation in the heart

• PERM1 is a transcriptional repressor of OGT, GLUT1 and GLUT4

• Loss of PERM1 increases O-GlcNAcylation of PGC-1α

• Excessively O-GlcNAcylated PGC-1α dissociates from its transcription factor PPARα

• PERM1 regulates mitochondrial respiration through O-GlcNAcylation

Data availability

All data are included in the manuscript.