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Mitochondria continuously change shape, position, and matrix configuration for optimal metabolite exchange. It is well established that changes in mitochondrial metabolism influence mitochondrial shape and matrix configuration. We demonstrated previously that inhibition of mitochondrial complex I (CI or NADH:ubiquinone oxidoreductase) by rotenone accelerated matrix protein diffusion and decreased the fraction and velocity of moving mitochondria. In the present study, we investigated the relationship between inherited CI deficiency, mitochondrial shape, mobility, and matrix protein diffusion. To this end, we analyzed fibroblasts of two children that represented opposite extremes in a cohort of 16 patients, with respect to their residual CI activity and mitochondrial shape. Fluorescence correlation spectroscopy (FCS) revealed no relationship between residual CI activity, mitochondrial shape, the fraction of moving mitochondria, their velocity, and the rate of matrix-targeted enhanced yellow fluorescent protein (mitoEYFP) diffusion. However, mitochondrial velocity and matrix protein diffusion in moving mitochondria were two to three times higher in patient ceils than in control cells. Nocodazole inhibited mitochondrial movement without altering matrix EYFP diffusion, suggesting that both activities are mutually independent. Unexpectedly, electron microscopy analysis revealed no differences in mitochondrial ultrastructure between control and patient cells. It is discussed that the matrix of a moving mitochondrion in the CI-deficient state becomes less dense, allowing faster metabolite diffusion, and that fibroblasts of CI-deficient patients become more glycolytic, allowing a higher mitochondrial velocity. NADH: ubiquinone oxidoreductase deficiency; human skin fibroblasts; fluorescence correlation spectroscopy: mitochondrial motility.

Malfunction of NADH:ubiquinone oxidoreductase or complex I (CI), the first and largest complex of the mitochondrial oxidative phosphorylation system, has been implicated in a wide variety of human disorders. To demonstrate a quantitative relationship between CI amount and activity and mitochondrial shape and cellular reactive oxygen species (ROS) levels, we recently combined native electrophoresis and confocal and video microscopy of dermal fibroblasts of healthy control subjects and children with isolated CI deficiency. Individual mitochondria appeared fragmented and/or less branched in patient fibroblasts with a severely reduced CI amount and activity (class I), whereas patient cells in which these latter parameters were only moderately reduced displayed a normal mitochondrial morphology (class II). Moreover, cellular ROS levels were significantly more increased in class I compared with class II cells. We propose a mechanism in which a mutation-induced decrease in the cellular amount and activity of CI leads to enhanced ROS levels, which, in turn, induce mitochondrial fragmentation when not appropriately counterbalanced by the cell's antioxidant defense systems. complex I; reactive oxygen species; microscopy; fluorescence doi:10.1152/ajpcell.00194.2006

A study to demonstrate the mitochondrial function for Ecsit (an evolutionary conserved signaling intermediate in Toll pathways) in the assembly of mitochondrial complex I (NADH: ubiquinone oxidoreductase) was conducted. The observation supports the function for Ecsit in the assembly or stability of mitochondrial complex I which possibly links assembly of oxidative phosphorylation complexes to inflammatory response and embryonic development.

Previously, we reported that both the bradykinin (Bk)-induced increase in mitochondrial ATP concentration ([ATP]M) and the rate of cytosolic [Ca.sup.2+] removal are significantly decreased in skin fibroblasts from a patient with an isolated complex I deficiency. Here we demonstrate that the mitochondrial [Ca.sup.2+] indicator rhod-2 can be used to selectively buffer the Bk-induced increase in mitochondrial [Ca.sup.2+] concentration ([[Ca.sup.2+]]M) and, consequently, the [Ca.sup.2+]-stimulated increase in [[ATP].sub.M], thus allowing studies of how the increase in [[ATP].sub.M] and the cytosolic [Ca.sup.2+] removal rate are related. Luminometry of healthy fibroblasts expressing either aequorin or luciferase in the mitochondrial matrix showed that rhod-2 dose dependently decreased the Bk-induced increase in [[Ca.sup.2+]]M and [ATP]M by maximally 80 and 90%, respectively. Digital imaging microscopy of cells coloaded with the cytosolic [Ca.sup.2+] indicator fura-2 revealed that, in parallel, rhod-2 maximally decreased the cytosolic [Ca.sup.2+] removal rate by 20%. These findings demonstrate that increased mitochondrial ATP production is required for accelerating cytosolic [Ca.sup.2+] removal during stimulation with a [Ca.sup.2+]-mobilizing agonist. In contrast, complex I-deficient patient fibroblasts displayed a cytosolic [Ca.sup.2+] removal rate that was already decreased by 40% compared with healthy fibroblasts. Rhod-2 did not further decrease this rate, indicating the absence of mitochondrial ATP supply to the cytosolic [Ca.sup.2+] pumps. This work reveals the usefulness of rhodamine-based [Ca.sup.2+] indicators in examining the role of intramitochondrial [Ca.sup.2+] in mitochondrial (patho) physiology. human skin fibroblast; OXPHOS disease; calcium ion extrusion; rhod-2; CGP-37157 doi:10.1152/ajpcell.00561.2005

Complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation system, and its malfunction is associated with a wide variety of clinical syndromes ranging from highly progressive, often early lethal, encephalopathies to neurodegenerative disorders in adult life. The changes in mitochondrial structure and function that are at the basis of the clinical symptoms are poorly understood. Video-rate confocal microscopy of cells pulse-loaded with mitochondria-specific rhodamine 123 followed by automated analysis of form factor (combined measure of length and degree of branching), aspect ratio (measure of length), and number of revealed marked differences between primary cultures of skin fibroblasts from 13 patients with an isolated complex I deficiency. These differences were independent of the affected subunit, but plotting of the activity of complex I, normalized to that of complex IV, against the ratio of either form factor or aspect ratio to number revealed a linear relationship. Relatively small reductions in activity appeared to be associated with an increase in form factor and never with a decrease in number, whereas relatively large reductions occurred in association with a decrease in form factor and/or an increase in number. These results demonstrate that complex I activity and mitochondrial structure are tightly coupled in human isolated complex I deficiency. To further prove the relationship between aberrations in mitochondrial morphology and pathological condition, fibroblasts from two patients with a different mutation but a highly fragmented mitochondrial phenotype were fused. Full restoration of the mitochondrial network demonstrated that this change in mitochondrial morphology was indeed associated with human complex I deficiency. mitochondria; oxidative phosphorylation; rhodamine 123; fibroblast

Recent evidence indicates that oxidative stress is central to the pathogenesis of a wide variety of degenerative diseases, aging, and cancer. Oxidative stress occurs when the delicate balance between production and detoxification of reactive oxygen species is disturbed. Mammalian cells respond to this condition in several ways, among which is a change in mitochondrial morphology. In the present study, we have used rotenone, an inhibitor of complex I of the respiratory chain, which is thought to increase mitochondrial [O.sup.-.sub.2] production, and mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to investigate the relationship between mitochondrial [O.sup.-.sub.2] production and morphology in human skin fibroblasts. Video-rate confocal microscopy of cells pulse loaded with the mitochondria-specific cation rhodamine 123, followed by automated analysis of mitochondrial morphology, revealed that chronic rotenone treatment (100 nM, 72 h) significantly increased mitochondrial length and branching without changing the number of mitochondria per cell. In addition, this treatment caused a twofold increase in lipid peroxidation as determined with C11-BODIP[Y.sup.581/591]. Finally, digital imaging microscopy of cells loaded with hydroethidine, which is oxidized by [O.sup.-.sub.2]* to yield fluorescent ethidium, revealed that chronic rotenone treatment caused a two-fold increase in the rate of [O.sup.-.sub.2] production. MitoQ (10 nM, 72 h) did not interfere with rotenone-induced ethidium formation but abolished rotenone-induced outgrowth and lipid peroxidation. These findings show that increased mitochondrial [O.sup.-.sub.2]* production as a consequence of, for instance, complex I inhibition leads to mitochondrial outgrowth and that MitoQ acts downstream of this [O.sup.-.sub.2]* to prevent alterations in mitochondrial morphology. rhodamine 123; video-rate confocal microscopy: superoxide; MitoQ

Koopman, Werner J. H., Michel Renders, Arie Oosterhof, Toin H. van Kuppevelt, Baziel G. M. van Engelen, and Peter H. G. M. Willems. Upregulation of [Ca.sup.2.sup.+] removal in human skeletal muscle: a possible role for [Ca.sup.2.sup.+]-dependent priming of mitochondrial ATP synthesis. Am J Physiol Cell Physiol 285: C1263-C1269, 2003. First published July 2, 2003; 10.1152/ajpcell.00097.2003.--In muscle, ATP is required for the powerstroke of the myosin head, the detachment of actin and myosin filaments, and the reuptake of [Ca.sup.2.sup.+] into the sarcoplasmic reticulum. During contraction-relaxation, large amounts of ATP are consumed at the sites of action of the myosin-ATPase and sarcoplasmic reticulum [Ca.sup.2.sup.+]-ATPase. The present study addresses the consequences of a reduction in mitochondrial ATP production capacity on sarcoplasmic [Ca.sup.2.sup.+] handling. To this end, myotubes were cultured from patient quadriceps with a biochemically defined decrease in the maximal rate of mitechondrial ATP production and were loaded with indo 1 for imaging of sarcoplasmic [Ca.sup.2.sup.+] changes in real time by confocal microscopy. Myotubes were field-stimulated with 10-ms pulses of 16 V to evoke transient rises in sarcoplasmic [Ca.sup.2.sup.+] concentration ([Ca.sup.2.sup.+].sub.s]). Three single pulses, two pulse trains (1 Hz), and one single pulse were applied in succession to mimic changing workloads. Control myotubes displayed [Ca.sup.2.sup.+].sub.s] transients with an amplitude that was independent of the strength of the stimulus. Intriguingly, the rate of sarcoplasmic [Ca.sup.2.sup.+] removal (CRR) was significantly upregulated during the second and subsequent transients. In myotubes with a reduced mitochondrial ATP production capacity, the amplitude of the [Ca.sup.2.sup.+] is transients was markedly increased at higher stimulus intensities. Moreover, upregulation of the CRR was significantly decreased compared with control. Taken together, these results are in good agreement with a tight coupling between mitochondrial ATP production and sarcoplasmic [Ca.sup.2.sup.+] handling. Moreover, they support the existence of a relatively long-lasting mitochondrial memory for sarcoplasmic [Ca.sup.2.sup.+] rises. This memory, which manifested itself as an increase in CRR upon recurrent stimulation, was impaired in patient myotubes with a reduced mitochondrial ATP production capacity. sarcoplasmic [Ca.sup.2.sup.+] removal; video-rate imaging; indo 1; electrical stimulation; mitochondrial memory