Cytochrome c oxidase (COX), the rate-limiting enzyme of mitochondrial respiration, is regulated by various mechanisms. Its regulation by ATP (adenosine triphosphate) appears of particular importance, since it evolved early during evolution and is still found in cyanobacteria, but not in other bacteria. Therefore the “allosteric ATP inhibition of COX” is described here in more detail. Most regulatory properties of COX are related to “supernumerary” subunits, which are largely absent in bacterial COX. The “allosteric ATP inhibition of COX” was also recently described in intact isolated rat heart mitochondria.

This study addresses the eventual consequence of cytochrome c oxidase (CytOx) inhibition by ATP at high ATP/ADP ratio in isolated rat heart mitochondria. Earlier, it has been demonstrated that the mechanism of allosteric ATP inhibition of CytOx is one of the key regulations of mitochondrial functions. It is relevant that aiming to maintain a high ATP/ADP ratio for the measurement of CytOx activity effectuating the enzymatic inhibition as well as mitochondrial respiration, optimal concentration of mitochondria is critically important. Likewise, only at this concentration, were the differences in ΔΨ_m and ROS concentrations measured under various conditions significant. Moreover, when CytOx activity was inhibited in the presence of ATP, mitochondrial respiration and ΔΨ_m both remained static, while the ROS production was markedly decreased. Consubstantial results were found when the electron transport chain was inhibited by antimycin A, letting only CytOx remain functional to support the energy production. This seems to corroborate that the decrease in mitochondrial ROS production is solely the effect of ATP binding to CytOx which results in static respiration as well as membrane potential.

Protamine sulfate (PS) is widely used in heart surgery as an antidote for heparin albeit its pharmacological effects are not fully understood and applications are often accompanied by unwanted side effects. Here, we show the effect of PS on mitochondrial bioenergetics profile resulting in mitochondrial reactive oxygen species (ROS) production. Polarographic measurements were performed in parallel to membrane potential and ROS measurements by FACS analyzer using tetramethylrhodamine ethyl ester (TMRE) and MitoSOXTM fluorescent dyes, respectively. PS inhibited intact rat heart mitochondrial respiration (stimulated by ADP) to 76% (p < 0.001) from the baseline of 51.6 ± 6.9 to 12.4 ± 2.3 nmol O2 min-1 ml-1. Same effect was found when respiration was priorly inhibited by antimycin A (101.0 ± 8.9 vs. 38.0 ± 9.9 nmol O2 min-1 ml-1, p < 0.001) and later stimulated by substrates of cytochrome oxidase (CytOx) i.e. ascorbate and tetramethyl phenylene diamine (TMPD); suggesting that PS exerted its effect through inhibition of CytOx activity. Furthermore, the inhibition of mitochondrial respiration by PS was concentration-dependent and accompanied by hyperpolarization of the mitochondrial membrane potential (Δψm) i.e. 18 % increase at 50µg/ml and a further 3.3 % increase at 250µg/ml PS compared to control. This effect was associated with a strong consequent increase in the production of ROS i.e. 85 % and 88.6 % compared to control respectively. We propose that this excessive increase in ROS concentrations results in mitochondrial dysfunction and thus might relate to the “protamine reaction” contributing to the development of various cardiovascular adverse effects. SIGNIFICANCE STATEMENT N/A

The pathogenesis of mitral valve insufficiency is not yet fully understood. Several studies stressed the role of matrix metalloproteinases (MMPs) in the emergence of valvular pathologies. The primary objective of the present study is to analyze the role of selected MMPs and their inhibitors in mitral valve insufficiency. Eighty patients (33 female/47 male, mean age 67 years) underwent cardiopulmonary bypass surgery for mitral valve reconstruction between 2007 and 2015. All patients suffered from mitral insufficiency (MI) Stages iii and iv. When tissue resection was acquired specimens were taken immediately frozen and used for histological examination. Expression of MMP-1, MMP-9, tissue inhibitor of metalloproteinase (TIMP)-1, and TIMP-2 was examined immunohistochemically and distribution was analyzed in regard to preoperative clinical, echocardiographic, and histopathological findings. A clear correlation between the MMP expression and the MI degree of severity could be shown. The expression of MMPs proved to be high in relation to mild insufficiencies and relatively weak in the case of severe ones. Additionally, the etiology of the MI was considered in the analysis and a significant difference in the expression of MMPs between the mitral valves with endocarditis and the ones featuring a degenerative disease could be shown. Within the group of valves with degenerative diseases, no significant difference could be established between the subgroups (myxoid and sclerosed valves). The increased expression of MMPs and their inhibitors in mild insufficiencies could prove that the molecular changes in the valve precede the macroscopical and thus the echocardiographically diagnosable changes. Hence, new options for early diagnosis and therapy of MIs should be examined in further studies, respectively. Herein, the correlation of the MMP blood levels with MMP tissue expression should be addressed for surgical therapeutical decisions.