Letter to the editor: “Does a reduction in ADP-limited respiration indicate impaired mitochondrial function?”

to the editor: In a recent study, Dabkowski et al. (7) investigated the differential influence of streptozotocin-induced diabetes mellitus on interfibrillar and subsarcolemmal mitochondria in mice. They found decreased mitochondrial size, increased superoxide production, increased oxidative damage, and reduced complex activities. This was associated with impaired respiration rates (state 4) and impaired contractile function in diabetic mouse hearts. While the results appear consistent and the conclusion creditable, some technical modalities may affect the interpretation of the results, perspectives not considered in the article.

First, the authors measured mitochondrial respiratory activity in isolated mitochondria as basal, non-ADP-stimulated respiration, i.e., state 4 according to Chance and Williams (5). This respiration rate is not the maximal respiratory capacity, which is usually measured under ADP stimulation and termed “state 3 respiration” (4). It is the common notion that state 3 respiration reflects the maximal activity of the respiratory chain in isolated conditions when not limited by substrate, oxygen, or ADP (5). In contrast, state 4 respiration, as measured by Dabkowski et al. (7), is regarded as ADP-limited respiration (5) and is influenced by several factors, including the integrity of the inner mitochondrial membrane (8), the permeability of the outer membrane for protons (9), and the dissipation of the proton gradient through uncoupling proteins (2). The interpretation of isolated changes in state 4 respiration is still a matter of dispute. Increased state 4 respiration may indicate loose coupling (11) and may be regarded as beneficial (1, 6, 10) or detrimental (3). Irrespective of the interpretation, the nature of the changes in state 4 cannot really be assessed without knowing state 3 respiration. Since Dabkowski et al. (7) did not present the state 3 respiration rate, the true value of their finding of a decreased state 4 respiration cannot be determined. In theory, if a decrease in state 4 respiration, as described by the investigators, were associated with a normal state 3 respiration, the results would indicate a greater degree of coupling of the mitochondria, which would not be considered detrimental. This would significantly affect the interpretation of all the results.

Second, the authors measured mitochondrial function on complex I, II, and IV by assessing respiratory rates with complex-specific substrates and determined complex III activity with a biochemical spectroscopic assay. As respiratory rates test the function of the complete oxidative chain beyond the entrance of the electrons, they do not measure single complex activities. It was shown by Fannin et al. (8) that there may be significant differences between biochemically determined complex activities and mitochondrial respiration rates on one complex. These differences may be due to an altered inner membrane environment (8) or a different assembly of complexes into supercomplexes (12). Thus the supplementation of complex activity assessment by mixing the results of the two methods may lead to an erroneous interpretation.