Dysfunctional Electron Transport Chain Assembly in COXPD8

Abstract

Combined oxidative phosphorylation deficiency type 8 (COXPD8) is an autosomal recessive mitochondrial disorder caused by a mutation of the nuclear encoded mitochondrial alanyl-tRNA synthetase gene (AARS2). Clinical manifestations of COXPD8 include lethal infantile hypertrophic cardiomyopathy, pulmonary hypoplasia, generalized muscle weakness, and neurological involvement. We report a patient with COXPD8 caused by two mutations in the AARS2 gene. The c.1738 C>G mutation has not been previously reported, while the c.2872 C>T mutation has been associated with pulmonary hypoplasia and hypertrophic cardiomyopathy. Cardiac tissue, obtained through the LungMAP program, showed that, compared to other patients of similar ages, these two mutations affect not only the assembly of functional monomeric complexes (Cx) I and IV of the electron transport chain (ETC) but also limit the formation of respiratory supercomplexes. This patient had altered expression of some ETC proteins but normal expression of several enzymes of the tricarboxylic acid cycle. We also show that one of the control/comparison patients had an undiagnosed ETC Cx IV deficiency. In conclusion, our data demonstrate that the two mutations of the AARS2 gene are associated with failed assembly of Cx I and Cx IV and reduced formation of respiratory supercomplexes of the ETC, likely leading to acute bioenergetic stress.

Keywords: COXPD8; electron transport chain; hypertrophic cardiomyopathy; mitochondrial disease; mitochondrial supercomplexes.

Figure 1

Mitochondrial respiration in COXPD8 mitochondria. (A) Photo of a section of heart tissue of the patient with COXPD8. Red boxed areas indicate LV wall used to isolate mitochondria. RV and LV: right and left ventricle, both measured approximately 1 cm thick; IS interventricular septum, measured approximately 1.5 cm thick. (B) Original oxygraphy recordings show that isolated LV mitochondria did not respond to substrates (ADP: adenosine diphosphate; Mal/glut: malate/glutamate; Succ: succinate).

Figure 2

Clear native electrophoresis indicates the absence of Cx I and IV in the COXPD8 patient. (A) Coomassie stain of the COXPD8 and 5 unrelated comparison heart tissues and densitometric analysis in the bottom panel. (B) In-gel assays (IGA) for Cx I shows severely decreased NADH oxidase activity in the COXPD8 patient. Bottom panel: Densitometric analysis of the monomer (Mono) of Cx I and supercomplexes containing Cx I. (C) IGAs for detection of Cx III and Cx IV activity. (D) An aliquot of each sample used for native electrophoresis was analyzed by SDS electrophoresis and labeled for VDAC as housekeeping protein. 20 µg protein was loaded in each lane. COX: COXPD8 patient, CTR: control patients, Mono: monomer, SC supercomplexes.

Figure 3

Immunoblotting after clear native electrophoresis confirms the absence of Cx I and Cx IV. (A) Detection and analysis of Cx I subunit NDUFB6 expression (in green) confirms absence of monomeric Cx I and almost no supercomplexes in the COXPD8 patient, while the detection of Cx II subunit SDHA (in red) was similar in all samples. (B) The monomer of Cx III was detected in the COXPD8 patient, but supercomplexes were not when labeled for UQCRC1). (C) Faint detection of Cx IV subunit MtCO1 in monomers and supercomplexes in COXPD8 patient compared to controls. An additional signal for MtCO1 at ≈200 kD in patient D216 is indicated by *. (D) Detection of the Cx V subunit ATP5G was not affected by COXPD8. 20 µg protein was loaded in each lane of these 4–10% native gels. VDAC detection for loading control is presented in Figure 2D. COX: COXPD8 patient, CTR: control patients, mono: monomer, SC: supercomplexes.

Figure 4

SDS electrophoresis of mitochondria from COXPD8 and control hearts. (A): Immunoblotting shows the expression of a subunit of every ETC complex relative to VDAC expression. (B): Expression of the Cx IV subunits MtCO1 and MtCO2 in COXPD8 (COX) and control (CTR) patients. (C–E): Expression of Cx IV subunits MtCO1 and MtCO2 was lowest in COXPD8 patient and highest in patient D216 (●). (F–H): Expression of various TCA cycle enzymes was not affected in the COXPD8 patient. 5 µg protein were separated per lane on a 15% SDS gel. COX: COXPD8 patient, CTR: control patients, MDH2: malate dehydrogenase 2; PDH: pyruvate dehydrogenase; OSCP: oligomycin sensitivity conferring protein.

Figure 5

Enzymatic activity of ETC complexes, citrate synthase, and lactate dehydrogenase. Enzymatic activity of ETC Cx I-V (A–G), citrate synthase (H), and lactate dehydrogenase (I). Enzymatic activities of patient D216 are highlighted in red (●). All activities are presented in µM/min/mg. NADH-UQ DH: NADH-ubiquinone dehydrogenase; NADH-Cyt c OR: NADH-cytochrome c oxidoreductase; Succ-UQ DH: succinate-ubiquinone dehydrogenase; Succ-Cyt c OR: succinate-cytochrome c oxidoreductase; Cyt c Oxidase: cytochrome c oxidase; ATPase: oligomycin-sensitive ATPase activity of Cx V; CS citrate synthase; LDH lactate dehydrogenase.