Particulate Matter Exposure Exacerbates High Glucose-Induced Cardiomyocyte Dysfunction through ROS Generation

Diabetes mellitus and fine particulate matter from diesel exhaust (DEP) are both important contributors to the development of cardiovascular disease (CVD). Diabetes mellitus is a progressive disease with a high mortality rate in patients suffering from CVD, resulting in diabetic cardiomyopathy. Elevated DEP levels in the air are attributed to the development of various CVDs, presumably since fine DEP (<2.5 µm in diameter) can be inhaled and gain access to the circulatory system. However, mechanisms defining how DEP affects diabetic or control cardiomyocyte function remain poorly understood. The purpose of the present study was to evaluate cardiomyocyte function and reactive oxygen species (ROS) generation in isolated rat ventricular myocytes exposed overnight to fine DEP (0.1 µg/ml), and/or high glucose (HG, 25.5 mM). Our hypothesis was that DEP exposure exacerbates contractile dysfunction via ROS generation in cardiomyocytes exposed to HG. Ventricular myocytes were isolated from male adult Sprague-Dawley rats cultured overnight and sarcomeric contractile properties were evaluated, including: peak shortening normalized to baseline (PS), time-to-90% shortening (TPS90), time-to-90% relengthening (TR90) and maximal velocities of shortening/relengthening (±dL/dt), using an IonOptix field-stimulator system. ROS generation was determined using hydroethidine/ethidium confocal microscopy. We found that DEP exposure significantly increased TR90, decreased PS and ±dL/dt, and enhanced intracellular ROS generation in myocytes exposed to HG. Further studies indicated that co-culture with antioxidants (0.25 mM Tiron and 0.5 mM N-Acetyl-L-cysteine) completely restored contractile function in DEP, HG and HG+DEP-treated myocytes. ROS generation was blocked in HG-treated cells with mitochondrial inhibition, while ROS generation was blocked in DEP-treated cells with NADPH oxidase inhibition. Our results suggest that DEP exacerbates myocardial dysfunction in isolated cardiomyocytes exposed to HG-containing media, which is potentially mediated by various ROS generation pathways

Oxygen-dependence of mitochondrial ROS production as detected by Amplex Red assay

The initial rates of superoxide plus hydrogen peroxide (ROS) generation by intact or permeabilized rat heart mitochondria and coupled inside-out bovine heart submitochondrial particles (SMP) oxidizing NAD-dependent substrates, NADH, and succinate were measured by detecting resorufin formation in the Amplex Red assay at various oxygen concentrations. Linear dependences of the initial rates on oxygen concentration within the range of ~125–750 μM were found for all significant mitochondrial generators, i.e. the respiratory complexes and ammonium-stimulated dihydrolipoamide dehydrogenase. At lower oxygen concentrations upon its decrease from air saturation level to zero, the time-course of resorufin formation by SMP catalyzing coupled oxidation of succinate (the total ROS production by respiratory complexes II and III and by the reverse electron transfer (RET)-mediated by complex I) also corresponds to the linear dependence on oxygen with the same first-order rate constant determined in the initial rate studies. Prolonged incubation of SMP generating succinate-supported complex I-mediated ROS affected neither their NADH oxidase nor ROS generating activity. In contrast to SMP significant deviation from the first-order oxygen dependence in the time-course kinetics during coupled oxidation of succinate by intact mitochondria was evident. Complex I catalyzes the NADH:resorufin oxidoreductase reaction resulting in formation of colorless reduced resorufin. Hydrogen peroxide oxidizes reduced resorufin in the presence of peroxidase, thus showing its dihydroresorufin peroxidase activity. Combined NADH:resorufin reductase and dihydroresorufin peroxidase activities result in underestimation of the amount of hydrogen peroxide generated by mitochondria. We conclude that only initial rates of the mitochondrial ROS production, not the amount of resorufin accumulated, should be taken as the reliable measure of the mitochondrial ROS-generating activity, because of the cycling of the oxidized and reduced resorufin during Amplex Red assays fed by NADH and other possible reductant(s) present in mitochondria. Keywords: Hydrogen peroxide, Amplex Red, Resorufin, Respiratory chain, Respiratory complex I, Mitochondri

The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites

The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates-nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P. denitrificans vesicles, thus suggesting that a second, NAD+-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P. denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P. denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I50 (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase

The transition between active and de-activated forms of NADH:ubiquinone oxidoreductase (Complex I) in the mitochondrial membrane of Neurospora crassa.

The mammalian mitochondrial NADH:ubiquinone oxidoreductase (Complex I) has been shown to exist in two kinetically and structurally distinct slowly interconvertible forms, active (A) and de-activated (D) [Vinogradov and Grivennikova (2001) IUBMB Life 52, 129-134]. This work was undertaken to investigate the putative Complex I A-D transition in the mitochondrial membrane of the lower eukaryote Neurospora crassa and in plasma membrane of the prokaryote Paracoccus denitrificans, organisms that are eligible for molecular genetic manipulations. The potential interconversion between A and D forms was assessed by examination of the initial and steady-state rates of NADH oxidation catalysed by inside-out submitochondrial ( N. crassa ) and sub-bacterial ( P. denitrificans ) particles and their sensitivities to N -ethylmaleimide and Mg(2+). All diagnostic tests provide evidence that slow temperature- and turnover-dependent A-D transition is an explicit feature of eukaryotic N. crassa Complex I, whereas the phenomenon is not seen in the membranes of the prokaryote P. denitrificans. Significantly lower activation energy for A-to-D transition characterizes the N. crassa enzyme compared with that determined previously for the mammalian Complex I. Either a lag or a burst in the onset of the NADH oxidase assayed in the presence of Mg(2+) is seen when the reaction is initiated by the thermally de-activated or NADH-activated particles, whereas the delayed final activities of both preparations are the same. We conclude that continuous slow cycling between A and D forms occurs during the steady-state operation of Complex I in N. crassa mitochondria