Comparative Proteomics Reveals Dysregulated Mitochondrial O-GlcNAcylation in Diabetic Hearts

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification on serine and threonine residues of many proteins, plays crucial regulatory roles in diverse biological events. As a nutrient sensor, O-GlcNAc modification (O-GlcNAcylation) on nuclear and cytoplasmic proteins underlies the pathology of diabetic complications including cardiomyopathy. However, mitochondrial O-GlcNAcylation, especially in response to chronic hyperglycemia in diabetes, has been poorly explored. We performed a comparative O-GlcNAc profiling of mitochondria from control and streptozotocin (STZ)-induced diabetic rat hearts by using an improved β-elimination/Michael addition with isotopic DTT reagents (BEMAD) followed by tandem mass spectrometric analysis. In total, 86 mitochondrial proteins, involved in diverse pathways, were O-GlcNAcylated. Among them, many proteins have site-specific alterations in O-GlcNAcylation in response to diabetes, which suggests that protein O-GlcNAcylation is a novel layer of regulation mediating adaptive changes in mitochondrial metabolism during the progression of diabetic cardiomyopathy.

Keywords: O-GlcNAcome; O-GlcNAcylation; diabetic cardiomyopathy; mass spectrometry; mitochondria; proteomics; pyruvate dehydrogenase.

Figure 1.

Impaired cardiac mitochondrial function in diabetic hearts. Mitochondrial oxygen consumption rates were determined with the presence of glutamate/malate (G/M; A) and succinate (Succ.; B). State 4, without the addition of ADP; State 3, with the addition of ADP. (C, D) Mitochondrial membrane potential was determined with either (C) G/M or (D) Succ. (E) Respiratory control ratios (RCRs) of isolated mitochondria were determined from the rates of State 3 and State 4 oxygen consumption in the presence of G/M. (F) Calculated Ca²⁺ uptake capacity prior to permeability transition pore opening in mitochondria isolated from control and diabetic rats (N ≥ 4; the 2-tailed unpaired Student t test was used to compare different groups, with p < 0.05 being considered significant).

Figure 2.

Altered abundance of proteins in several major metabolic pathways in mitochondria between control and diabetic hearts. Proteins with decreased abundance are shown in green, proteins with increased abundance are shown in red, while proteins without apparent changes are shown in blue. HK1, hexokinase-1; MPC1, mitochondrial pyruvate carrier 1; PDH, pyruvate dehydrogenase; PDC, pyruvate dehydrogenase complex; PDK4, pyruvate dehydrogenase kinase 4; CPT1, carnitine O-palmitoyltransferase 1; ACADL, very long-chain specific acyl-CoA dehydrogenase; ECHS, enoyl-CoA hydratase, mitochondrial precursor; HADHA, trifunctional enzyme subunit alpha; HADHB, trifunctional enzyme subunit beta; CS, citrate synthase; ACO2, aconitate hydratase; IDH, isocitrate dehydrogenase; OGDH, 2-oxoglutarate dehydrogenase; SUCLA2, succinyl-CoA ligase; SDHA; succinate dehydrogenase A; SDHB, succinate dehydrogenase B; MDH, malate dehydrogenase; FUM, fumarate hydratase.

Figure 3.

O-GlcNAcylation occurs on many mitochondria proteins. Highly purified cardiac mitochondrial proteins from (A) control rats and (B) diabetic rats were separated with two-dimensional gel (IEF/SDS-PAGE), transferred to PVDF membranes, and immunoblotted with CTD 110.6.

Figure 4.

O-GlcNAcylated proteins are involved in multiple mitochondrial pathways. O-GlcNAcylated proteins are shown in red, with number of sites shown in parentheses. PDH, pyruvate dehydrogenase; CPT1, carnitine O-palmitoyltransferase 1; CPT2, carnitine O-palmitoyltransferase 2; ACADL, very long-chain specific acyl-CoA dehydrogenase; ECHS, enoyl-CoA hydratase, mitochondrial precursor; HADHA, trifunctional enzyme subunit alpha; HADHB, trifunctional enzyme subunit beta; CS, citrate synthase; ACO2, aconitate hydratase; IDH, isocitrate dehydrogenase; OGDH, 2-oxoglutarate dehydrogenase; SUCLA2, succinyl-CoA ligase; SDHA; succinate dehydrogenase A; SDHB, succinate dehydrogenase B; MDH, malate dehydrogenase; FUM, fumarate hydratase; CI, complex I; CII, complex II; CIII, complex III; CIV, complex IV; CV, complex V; SLC24A4, ADP/ATP translocase 1; SLC25A3, phosphate carrier protein; MCU, mitochondrial Ca²⁺ uniporter; VDAC1/2, voltage-dependent anion-selective channel protein 1/2; SOD2, superoxide dismutase [Mn]; PRDX3, thioredoxin-dependent peroxide reductase; HSP60, 60 kDa heat shock protein; SAM50, sorting and assembly machinery component 50 homologue; CKMT2, creatine kinase S-type, mitochondrial precursor. Please refer to Table S2 for detailed description of the corresponding O-GlcNAc peptides.

Figure 5.

Site-specific O-GlcNAc dynamics on enzymes in (A) pyruvate decarboxylation and TCA cycle and (B) fatty acid utilization in control and diabetic hearts. Only proteins with ≥ 2 O-GlcNAc sites were shown. The dashed line denotes an ROR = 1. PDHA1, pyruvate dehydrogenase subunit alpha; DLAT, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex; CPT1B, carnitine O-palmitoyltransferase 1, muscle isoform; HADHA, trifunctional enzyme subunit alpha; HADHB, trifunctional enzyme subunit beta; ACO2, aconitate hydratase; SDHA; succinate dehydrogenase A.

Figure 6.

Validation of O-GlcNAcylation of PDHA1 in cardiac mitochondria from control and diabetic rats. Pyruvate dehydrogenase complexes were immunopurified from lysates of cardiac mitochondria from control and diabetic rats. After extensive washing, the immunoprecipitates were treated with or without CpNag J as indicated, then separated with SDS-PAGE, and transferred to PVDF membranes for blotting with PDHA1 and RL2. Blots in the presence of 1 M GlcNAc were used as a competitive assay.