Reduced Respiratory Control with ADP and Changed Pattern of Respiratory Chain Enzymes as a Result of Selective Deficiency of the Mitochondrial ATP Synthase

Abstract

The F o F 1 -ATPase, a multisubunit protein complex of the inner mitochondrial membrane, produces most of the ATP in mammalian cells. Mitochondrial diseases as a result of a dysfunction of ATPase can be caused by mutations in mitochondrial DNA-encoded ATPase subunit a or rarely by an ATPase defect of nuclear origin. Here we present a detailed functional and immunochemical analysis of a new case of selective and generalized ATPase deficiency found in an Austrian patient. The defect manifested with developmental delay, muscle hypotonia, failure to thrive, ptosis, and varying lactic acidemia (up to 12 mmol/L) beginning from the neonatal period. A low-degree dilated cardiomyopathy of the left ventricle developed between the age of 1 and 2 y. A Ͼ90% decrease in oligomycin-sensitive ATPase activity and an 86% decrease in the content of the ATPase complex was found in muscle mitochondria. It was associated with a significant decrease of ADP-stimulated respiration of succinate (1.5-fold) and respiratory control with ADP (1.7-fold) in permeabilized muscle fibers, and with a slight decrease of the respiratory chain complex I and compensatory increase in the content of complexes III and IV. The same ATPase deficiency without an increase in respiratory chain complexes was found in fibroblasts, suggesting a generalized defect with tissue-specific manifestation. Absence of any mutations in mitochondrial ATP6 and ATP8 genes indicates a nuclear origin of the defect. Mitochondrial disorders caused by impairment of mitochondrial oxidative phosphorylation (OXPHOS) affect predominantly tissues with high-energy demands: muscle, brain, and heart. Subunits of OXPHOS complexes are encoded in two separate genomes: nuclear and mitochondrial. A pathogenic mutation in both genomes can cause an OX-PHOS defect. Defects in complex V-mitochondrial F o F 1 -ATPase are less frequent than the defects of the respiratory chain complexes, but they are mostly very severe and can be caused by mitochondrial DNA (mtDNA) mutations or by mutations in nuclear genes.Mitochondrial ATPase is a multisubunit complex composed of 16 different subunits (1). Six of these compose the globular F 1 part, which is responsible for enzymatic catalysis of ATP synthesis or hydrolysis. Ten remaining subunits form the membrane-spanning F o part, which performs H ϩ translocation across the inner mitochondrial membrane. Four subunits of F o form stalks that connect F 1 and F o parts. Only two subunits from F o part are encoded by mitochondrial genome: subunits a and A6L (subunits 6 and 8) (2). All of the other 14 subunits of the ATPase are encoded by the nuclear genes.Specific defects in mitochondrial ATPase are caused mainly by mtDNA mutations that affect subunit a; no mutations in subunit A6L have been described so far. The most frequent mutation in subunit a is T8993G mutation (3-7) or T8993C mutation (8)

Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

“…Oligomycin sensitive ATPase activity of complex V was determined using buffer conditions described by Rustin et al (26), but by applying sonification of the whole reaction mixture for 10 s with an ultra-sonifier (Bio cell disruptor 250, Branson, Vienna, Austria) at the lowest energy output (27)…”

Mitochondrial diseases and ATPase defects of nuclear origin

“…Transmitochondrial cybrid cells made of patient's fibroblasts fully complemented the ATPase defect and confirmed the nuclear origin of impaired biogenesis of the enzyme complex [27]. Later on, two other similar cases of selective ATPase deficiency were found in the Czech Republic [22] and one in Austria [28]. Most recently, two patients with selective ATPase deficiency were described in Belgium [29].…”

“…Pronounced reduction of ATPase content in patient's fibroblasts strongly decreased the synthesis of ATP, ADPstimulated respiration, and the discharge of mitochondrial membrane potential which showed even higher steady state values than the control cells [22,27,28]. However, even in cells with 90% decrease of ATPase content, a significant ATP production could be seen [28].…”

“…However, even in cells with 90% decrease of ATPase content, a significant ATP production could be seen [28]. As apparent from inhibitor titration studies [45,46], individual OXPHOS enzymes of mitochondrial energy metabolism can be inhibited to a certain extent without noticeable reduction of the mitochondrial coupled respiration rate.…”

93 Loss of complex I due to mitochondrial DNA mutations in renal oncocytoma

“…Solubilized mitochondrial membranes were prepared from post nuclear supernatant (30 Ag protein) of kidney and oncocytoma tissues, as described previously (21). Briefly, post nuclear supernatants were sedimented by centrifugation at 13,000 Â g for 15 min.…”