Mitochondrial dysfunction in the type 2 diabetic heart is associated with alterations in spatially distinct mitochondrial proteomes

Abstract

Cardiac complications and heart failure are the leading cause of death in type 2 diabetic patients. Mitochondrial dysfunction is central in the pathogenesis of the type 2 diabetic heart. However, it is unclear whether this dysfunction is specific for a particular subcellular region. The purpose of this study was to determine whether mitochondrial dysfunction in the type 2 diabetic heart is specific to a spatially distinct subset of mitochondria. We investigated mitochondrial morphology, function, and proteomic composition of subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM) in 18-wk-old db/db mice. Oxidative damage was assessed in subpopulations through the measurement of lipid peroxidation byproducts and nitrotyrosine residues. Proteomic profiles and posttranslational modifications were assessed in mitochondrial subpopulations using iTRAQ and multi-dimensional protein identification technologies, respectively. SSM from db/db hearts had altered morphology, including a decrease in size and internal complexity, whereas db/db IFM were increased in internal complexity. Db/db SSM displayed decreased state 3 respiration rates, electron transport chain activities, ATP synthase activities, and mitochondrial membrane potential and increased oxidative damage, with no change in IFM. Proteomic assessment revealed a greater impact on db/db SSM compared with db/db IFM. Inner mitochondrial membrane proteins, including electron transport chain, ATP synthesis, and mitochondrial protein import machinery, were predominantly decreased. We provide evidence that mitochondrial dysfunction in the type 2 diabetic heart is associated with a specific subcellular locale. Furthermore, mitochondrial morphological and functional indexes are impacted differently during type 2 diabetic insult and may result from the modulation of spatially distinct mitochondrial proteomes.

Fig. 1.

Mitochondrial subpopulation size [forward scatter (FSC)] and internal complexity [side scatter (SSC)] in wild-type (WT) and db/db hearts. The relative size and internal complexity of distinct mitochondrial subpopulations were determined using flow cytometric analyses. Mitochondrial subpopulations were stained with mitotracker deep red 633, a membrane potential (ΔΨ_m)-dependent dye, and gated based on incorporation of the dye. Analysis of FSC and SSC were calculated per 20,000 gated events for all mitochondrial subpopulations. A–D: representative histograms of FSC (A and C) and SSC (B and D) in WT subsarcolemmal mitochondrial (SSM) and interfibrillar mitochondria (IFM) (green) versus db/db SSM and IFM (blue). E and F: FSC (E) and SSC (F) are expressed in arbitrary units (AU). Values are ± SE; n = 4 for each group. *P < 0.05 for WT vs. db/db.

Fig. 2.

Respiratory capacity of mitochondrial subpopulations in WT and db/db hearts. A–G: state 3 and state 4 respiration rates with glutamate + malate (A and B), palmitoylcarnitine (C and D), electron transport chain (ETC) complex activities (E and F), and ATP synthase activity (G) were assessed in isolated mitochondrial subpopulations [SSM (A, C, and E) and IFM (B, D, and F)] from hearts of 18-wk-old WT and db/db mice. State 3 and state 4 respiration rates were determined in the presence of the substrates glutamate + malate as well as palmitoylcarnitine, and state 3 respiration was examined upon the addition of ADP. ETC complex I, III, and IV activities were assessed spectrophotometrically by measuring the oxidation of NADH (complex I), reduction of cytochrome c (complex III), and oxidation of cytochrome c (complex IV). Respiration rates are expressed in nmol·min⁻¹·mg protein⁻¹, whereas enzymatic activities are expressed in activity·min⁻¹·mg protein⁻¹. Values are means ± SE; n = 6 for each group. *P < 0.05 for WT vs. db/db.

Fig. 3.

Flow cytometric analysis of mitochondrial subpopulation ΔΨ_m in WT and db/db hearts. ΔΨ_m was assessed by staining mitochondrial subpopulations with JC-1 dye and assessing the shift from green to orange fluorescence with flow cytometry. A–D: representative histograms showing green and orange fluorescence in WT SSM (A) versus db/db SSM (B) and WT IFM (C) versus db/db IFM (D). E: ΔΨ_m was calculated based on orange-to-green fluorescence ratios in WT versus db/db cardiac mitochondrial subpopulations. Orange-to-green fluorescence ratios are expressed in AU. Values are means ± SE; n = 4 for each group. *P < 0.05 for WT vs. db/db.

Fig. 4.

Adenine nucleotide translocase (ANT) protein expression in mitochondrial subpopulations from WT and db/db hearts. A and B: representative Western blots (top) and densitometric analyses (bottom) for total ANT protein in SSM (A) and IFM (B) from WT and db/db hearts. Values are means ± SE; n = 5 for each group. *P < 0.05 for WT vs. db/db.

Fig. 5.

Oxidative damage in mitochondrial subpopulations from WT and db/db hearts. Oxidative damage to lipids was assessed in mitochondrial subpopulations by measuring lipid peroxidation byproducts [malondialdehyde (MDA) and 4-hydroxyalkenal (4-HAE)] using a colorimetric assay and compared against a standard curve of known 4-HAE and MDA concentrations. Data are expressed in μmol MDA + 4-HAE/mg mitochondrial protein (A). Oxidative damage to proteins was assessed by quantifying 3-nitrotyrosine (NT) residues through a sandwich ELISA, and concentrations were determined by comparing results against a known protein NT standard curve. Results are expressed in nmol NT/mg protein (B). Values for both lipid peroxidation and protein nitrosylation are means ± SE; n = 5 for each group. *P < 0.05 for WT vs. db/db.

Fig. 6.

Representative iTRAQ spectra and Western blot analysis. Isolated mitochondrial subpopulations from WT and db/db hearts were labeled with iTRAQ reagents [114 (db/db SSM), 115 (db/db IFM), 116 (WT SSM), and 117 (WT IFM)] and combined for analysis with mass spectrometry. A: representative spectra of the simultaneous quantitation of cytochrome c oxidase subunit VIIa1 peptide in WT and db/db mitochondrial subpopulations. B: spectra for the reporter groups of the iTRAQ reagents (114, 115, 116, and 117) from cytochrome c oxidase subunit VIIa1 peptide mass spectrometry (MS)/MS spectra. These spectra were used along with other peptides to simultaneously quantify cytochrome c oxidase subunit VIIa1 in WT and db/db mitochondrial subpopulations. C and D: representative Western blots (top) and densitometric analyses (bottom) of cytochrome c oxidase subunit VIIa1 in SSM (C) and IFM (D) from WT and db/db hearts. Values are means ± SE; n = 5 for each group. *P < 0.05 for WT vs. db/db.