The metabolic state of the heart regulates mitochondrial supercomplex abundance in mice

Abstract

Mitochondrial supercomplexes are observed in mammalian tissues with high energy demand and may influence metabolism and redox signaling. Nevertheless, the mechanisms that regulate supercomplex abundance remain unclear. In this study, we examined the composition of supercomplexes derived from murine cardiac mitochondria and determined how their abundance changes with substrate provision or by genetically induced changes to the cardiac glucose-fatty acid cycle. Protein complexes from digitonin-solubilized cardiac mitochondria were resolved by blue-native polyacrylamide gel electrophoresis and were identified by mass spectrometry and immunoblotting to contain constituents of Complexes I, III, IV, and V as well as accessory proteins involved in supercomplex assembly and stability, cristae architecture, carbohydrate and fat oxidation, and oxidant detoxification. Respiratory analysis of high molecular mass supercomplexes confirmed the presence of intact respirasomes, capable of transferring electrons from NADH to O₂. Provision of respiratory substrates to isolated mitochondria augmented supercomplex abundance, with fatty acyl substrate (octanoylcarnitine) promoting higher supercomplex abundance than carbohydrate-derived substrate (pyruvate). Mitochondria isolated from transgenic hearts that express kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (Glyco^Lo), which decreases glucose utilization and increases reliance on fatty acid oxidation for energy, had higher mitochondrial supercomplex abundance and activity compared with mitochondria from wild-type or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-expressing hearts (Glyco^Hi), the latter of which encourages reliance on glucose catabolism for energy. These findings indicate that high energetic reliance on fatty acid catabolism bolsters levels of mitochondrial supercomplexes, supporting the idea that the energetic state of the heart is regulatory factor in supercomplex assembly or stability.

Keywords: Glycolysis; Heart; Metabolism; Mitochondria; Respirasome; Supercomplex.

Graphical abstract

Fig. 1

Standardization of BN-PAGE for examining multimeric complexes in cardiac mitochondria. (A) Representatative Coomassie-stained 1D, non-reducing Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) gel of isolated mouse cardiac mitochondria solubilized with β-dodecylmaltoside (DDM) or digitonin (DIG); (B) Representative non-reducing, Coomassie-stained gel (top), and anti-MitoProfile immunoblot of proteins separated in the 2nd dimension (bottom).

Fig. 2

Identification of mitochondrial respiratory supercomplexes in murine heart. (A) Representative image of 1D-BN-PAGE showing bands excised for LC/MS/MS protein identification. (B) Putative respiratory supercomplexes corresponding to bands 1–6 in panel A. Identified proteins having at least 10 unique spectra were used to create the models shown here. Additional proteins that may be associated with the complexes are shown based on the abundance of unique spectra are shown; and (C) In separate experiments, bands corresponding to 1–6 in Panel A were separated in the 2nd dimension under reducing and denaturing conditions and subjected to Western blot analysis using the MitoProfile antibody.

Fig. 3

Validation of functional respirasomes. As a measure of respirasome activity, digitonin-solubilized mitochondria were subjected to BN-PAGE in the absence of Coomassie Blue-G250 dye. (A) After native separation, oxygen consumption in regions corresponding to high molecular weight regions of the unstained gel was assessed by XF analysis; (B) O₂ traces after addition of 2 mM NADH, 4 mM ADP, 10 μM cytochrome c, and 2 μM rotenone (Rot) to each well; and (C) O₂ consumption rates of gel plugs corresponding to protein complexes of different molecular masses.

Fig. 4

Respiratory substrates regulate mitochondrial supercomplex abundance. Isolated cardiac mitochondria from naïve, WT mice were incubated with no substrate (none), 100 μM octanoylcarnitine, 2.5 mM malate and 1 mM ADP (OCM), or 5 mM pyruvate, 2.5 mM malate and 1 mM ADP (PM) for 10 min at 37 °C. The mitochondria were then solubilized with digitonin, and the proteins were separated by BN-PAGE. (A) Representative Coomassie-stained BN-PAGE gel of cardiac mitochondrial proteins. Shown are three representative mice from each treatment; (B) Quantification of supercomplex band density under different substrate conditions. n = 9 mice per group, **p < 0.01, ***p < 0.001; (C) Representative Western blot of BN-PAGE gel: Antibodies against the Complex I subunit, NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9), were used to detect supercomplexes containing Complex I. The blot was reprobed with antibodies against succinate dehydrogenase A (SDHA), which was used as a loading control. Shown are three representative biological replicates; (D) Quantification of NDUFA9-containing SC bands, normalized to SDHA. n = 9 mice per group, ***p < 0.001.

Fig. 5

The glucose-fatty acid cycle influences respiratory supercomplex abundance in murine heart. BN-PAGE-mediated detection of changes in mitochondrial supercomplexes from digitonin-solubilized cardiac mitochondria from WT, Glyco^Lo and Glyco^Hi mice: (A) Representative Coomassie-stained, BN-PAGE gel of isolated cardiac mitochondria from WT, Glyco^Lo, and Glyco^Hi mice. Shown are three representative samples from each genotype; (B) Quantification of supercomplex (SC) band density: SC bands were normalized to the Complex III (C-III) band in each lane; the asterisk (*) in panel A represents the C-III band to which the SC region were normalized. n = 9–12 mice per group, *p < 0.05; (C) Representative Western blot of BN-PAGE gel: Antibodies against the Complex I subunit, NADH:Ubiquinone Oxidoreductase Subunit A9 (NDUFA9), were used to detect supercomplexes containing Complex I. The blot was reprobed with antibodies against succinate dehydrogenase A (SDHA), which was used as a loading control. Shown are three representative mice from each genotype; (D) Quantification of NDUFA9-containing SC bands, normalized to SDHA. n = 9–12 mice per group, *p < 0.05, **p < 0.01; (E) In-gel mitochondrial complex activity assays: Gels were incubated with suitable chemical substrates that change color and form a precipitate due to the enzyme activities of respiratory complexes. The activities of Complex I, Complex IV, and Complex II are shown; and (F) Quantification of respiratory complex activity in the SC region of the BN-PAGE gels. The intensity of staining was normalized to the Complex III band of the gel after Coomassie blue staining. n = 3 mice per group, *p < 0.05.