Cristina Bianchi

The Mitochondrial Production of Reactive Oxygen Species in Relation to Aging and Pathology

Abstract: Mitochondria are known to be strong producers of reactive oxygen species (ROS) and, at the same time, particularly susceptible to the oxidative damage produced by their action on lipids, proteins, and DNA. In particular, damage to mtDNA induces alterations to the polypeptides encoded by mtDNA in the respiratory complexes, with consequent decrease of electron transfer, leading to further production of ROS and thus establishing a vicious circle of oxidative stress and energetic decline. This deficiency in mitochondrial energetic capacity is considered the cause of aging and age‐related degenerative diseases. Complex I would be the enzyme most affected by ROS, since it contains seven of the 13 subunits encoded by mtDNA. Accordingly, we found that complex I activity is significantly affected by aging in rat brain and liver mitochondria as well as in human platelets. Moreover, due to its rate control over aerobic respiration, such alterations are reflected on the entire oxidative phosphorylation system. We also investigated the role of mitochondrial complex I in superoxide production and found that the one‐electron donor to oxygen is most probably the Fe‐S cluster N2. Short chain coenzyme Q (CoQ) analogues enhance ROS formation, presumably by mediating electron transfer from N2 to oxygen, both in bovine heart SMP and in cultured HL60 cells. Nevertheless, we have accumulated much evidence of the antioxidant role of reduced CoQ10 in several cellular systems and demonstrated the importance of DT‐diaphorase and other internal cellular reductases to reduce exogenous CoQ10 after incorporation.

Evaluation of cellular energetics by the Pasteur effect in intact cardiomyoblasts and isolated perfused hearts

This work aims at exploring changes in cellular energetics by exploiting the Pasteur effect. We assumed that lactate overproduction arising from antimycin A-induced inhibition of mitochondrial respiration (Δ-lactate = stimulated [lactate] – basal [lactate]) is indicative of the energy provided aerobically by the cell. Rat embryonal cardiomyocytes (H9c2), incubated with 2 μmol/L antimycin A, increased about 6 fold their lactate production in a manner linear with time and cell number. Antimycin A was also delivered to Langendorff-perfused rat hearts under control aerobic conditions or after 20 min-ischemia and 30 min-reperfusion. The test started at the end of each perfusion and lactate was measured into perfusate collected for further 25 min. A cardioplegic solution was also delivered during the test to exclude that lactate production was influenced by cardiac contraction. Control Δ-lactate was 20.9 ± 2.31 (S.E.M.) μg/mL and markedly decreased after reperfusion (7.66 ± 0.51, p < 0.001), showing that energy production was impaired of about 70%. The determination of oxygen consumption by mitochondria isolated from reperfused hearts also suggested that the damage to the respiratory chain was similar to that evaluated by lactate overproduction (Respiratory Control Index: 75% lower than control, p < 0.001). Moreover, when Δ-lactate was referred to the estimated cells which remained viable at the end of reperfusion (49.9%), it was 25% lower than control (p < 0.05). Therefore, we proposed this test as a tool for quantifying both physiological and pathological energetic modifications in living intact cardiomyocytes and in isolated and perfused hearts.