Martin Hey-Mogensen

Biomarkers of mitochondrial content in skeletal muscle of healthy young human subjects

•  Several biochemical measures of mitochondrial components are used as biomarkers of mitochondrial content and muscle oxidative capacity. However, no studies have validated these surrogates against a morphological measure of mitochondrial content in human subjects. •  The most commonly used markers (citrate synthase activity, cardiolipin content, mitochondrial DNA content (mtDNA), complex I–V protein, and complex I–IV activity) were correlated with a measure of mitochondrial content (transmission electron microscopy) and muscle oxidative capacity (respiration in permeabilized fibres). •  Cardiolipin content followed by citrate synthase activity and complex I activity were the biomarkers showing the strongest association with mitochondrial content. •  mtDNA was found to be a poor biomarker of mitochondrial content. •  Complex IV activity was closely associated with mitochondrial oxidative phosphorylation capacity.

The 2-Oxoacid Dehydrogenase Complexes in Mitochondria Can Produce Superoxide/Hydrogen Peroxide at Much Higher Rates Than Complex I

Background: At the redox potential of NADH/NAD+, at least four mitochondrial sites produce superoxide/H2O2. Results: We compare their capacities in situ in isolated mitochondria. Conclusion: Maximum capacities of complexes were 2-oxoglutarate dehydrogenase > pyruvate dehydrogenase > branched-chain 2-oxoacid dehydrogenase > complex I. Significance: H2O2 production from 2-oxoacid dehydrogenases can be considerable but may previously have been misattributed to complex I. Several flavin-dependent enzymes of the mitochondrial matrix utilize NAD+ or NADH at about the same operating redox potential as the NADH/NAD+ pool and comprise the NADH/NAD+ isopotential enzyme group. Complex I (specifically the flavin, site IF) is often regarded as the major source of matrix superoxide/H2O2 production at this redox potential. However, the 2-oxoglutarate dehydrogenase (OGDH), branched-chain 2-oxoacid dehydrogenase (BCKDH), and pyruvate dehydrogenase (PDH) complexes are also capable of considerable superoxide/H2O2 production. To differentiate the superoxide/H2O2-producing capacities of these different mitochondrial sites in situ, we compared the observed rates of H2O2 production over a range of different NAD(P)H reduction levels in isolated skeletal muscle mitochondria under conditions that favored superoxide/H2O2 production from complex I, the OGDH complex, the BCKDH complex, or the PDH complex. The rates from all four complexes increased at higher NAD(P)H/NAD(P)+ ratios, although the 2-oxoacid dehydrogenase complexes produced superoxide/H2O2 at high rates only when oxidizing their specific 2-oxoacid substrates and not in the reverse reaction from NADH. At optimal conditions for each system, superoxide/H2O2 was produced by the OGDH complex at about twice the rate from the PDH complex, four times the rate from the BCKDH complex, and eight times the rate from site IF of complex I. Depending on the substrates present, the dominant sites of superoxide/H2O2 production at the level of NADH may be the OGDH and PDH complexes, but these activities may often be misattributed to complex I.

Simvastatin Effects on Skeletal Muscle: Relation to Decreased Mitochondrial Function and Glucose Intolerance

OBJECTIVES Glucose tolerance and skeletal muscle coenzyme Q(10) (Q(10)) content, mitochondrial density, and mitochondrial oxidative phosphorylation (OXPHOS) capacity were measured in simvastatin-treated patients (n = 10) and in well-matched control subjects (n = 9). BACKGROUND A prevalent side effect of statin therapy is muscle pain, and yet the basic mechanism behind it remains unknown. We hypothesize that a statin-induced reduction in muscle Q(10) may attenuate mitochondrial OXPHOS capacity, which may be an underlying mechanism. METHODS Plasma glucose and insulin concentrations were measured during an oral glucose tolerance test. Mitochondrial OXPHOS capacity was measured in permeabilized muscle fibers by high-resolution respirometry in a cross-sectional design. Mitochondrial content (estimated by citrate synthase [CS] activity, cardiolipin content, and voltage-dependent anion channel [VDAC] content) as well as Q(10) content was determined. RESULTS Simvastatin-treated patients had an impaired glucose tolerance and displayed a decreased insulin sensitivity index. Regarding mitochondrial studies, Q(10) content was reduced (p = 0.05), whereas mitochondrial content was similar between the groups. OXPHOS capacity was comparable between groups when complex I- and complex II-linked substrates were used alone, but when complex I + II-linked substrates were used (eliciting convergent electron input into the Q intersection [maximal ex vivo OXPHOS capacity]), a decreased (p < 0.01) capacity was observed in the patients compared with the control subjects. CONCLUSIONS These simvastatin-treated patients were glucose intolerant. A decreased Q(10) content was accompanied by a decreased maximal OXPHOS capacity in the simvastatin-treated patients. It is plausible that this finding partly explains the muscle pain and exercise intolerance that many patients experience with their statin treatment.

Skeletal muscle mitochondrial H2O2 emission increases with immobilization and decreases after aerobic training in young and older men

Currently, it is not known whether impaired mitochondrial function contributes to human ageing or whether potential impairments in mitochondrial function with age are secondary to physical inactivity. The present study investigated mitochondrial respiratory function and reactive oxygen species emission at a predefined membrane potential in young and older men subjected to 2 weeks of one‐leg immobilization followed by 6 weeks of aerobic cycle training. Immobilization increased reactive oxygen species emission and decreased ATP generating respiration. Subsequent aerobic training reversed these effects. By contrast, age had no effect on the measured variables. The results of the present study support the notion that increased mitochondrial reactive oxygen species production mediates the detrimental effects seen after physical inactivity and that ageing per se does not cause mitochondrial dysfunction.

The influence of age and aerobic fitness: effects on mitochondrial respiration in skeletal muscle

Mitochondrial function has previously been studied in ageing, but never in humans matched for maximal oxygen uptake ( V·O2max ). Furthermore, the influence of ageing on mitochondrial substrate sensitivity is not known.

Decreased mitochondrial oxidative phosphorylation capacity in the human heart with left ventricular systolic dysfunction

Heart failure (HF) with left ventricular systolic dysfunction (LVSD) is associated with a shift in substrate utilization and a compromised energetic state. Whether these changes are connected with mitochondrial dysfunction is not known. We hypothesized that the cardiac phenotype in LVSD could be caused by reduced mitochondrial oxidative phosphorylation (OXPHOS) capacity and reduced mitochondrial creatine kinase (miCK) capacity. The study aim was to test mitochondrial OXPHOS capacity in LVSD myocardium compared with OXPHOS capacity in a comparable patient group without LVSD.

Two weeks of one-leg immobilization decreases skeletal muscle respiratory capacity equally in young and elderly men.

Physical inactivity affects human skeletal muscle mitochondrial oxidative capacity but the influence of aging combined with physical inactivity is not known. This study investigates the effect of two weeks of immobilization followed by six weeks of supervised cycle training on muscle oxidative capacity in 17 young (23±1years) and 15 elderly (68±1years) healthy men. We applied high-resolution respirometry in permeabilized fibers from muscle biopsies at inclusion after immobilization and training. Furthermore, protein content of mitochondrial complexes I-V, mitochondrial heat shock protein 70 (mtHSP70) and voltage dependent anion channel (VDAC) were measured in skeletal muscle by Western blotting. The elderly men had lower content of complexes I-V and mtHSP70 but similar respiratory capacity and content of VDAC compared to the young. In both groups the respiratory capacity and protein content of VDAC, mtHSP70 and complexes I, II, IV and V decreased with immobilization and increased with retraining. Moreover, there was no overall difference in the response between the groups. When the intrinsic mitochondrial capacity was evaluated by normalizing respiration to citrate synthase activity, the respiratory differences with immobilization and training disappeared. In conclusion, aging is not associated with a decrease in muscle respiratory capacity in spite of lower complexes I-V and mtHSP70 protein content. Furthermore, immobilization decreased and aerobic training increased the respiratory capacity and protein contents of complexes I-V, mtHSP70 and VDAC similarly in the two groups. This suggests that inactivity and training alter mitochondrial biogenesis equally in young and elderly men.

Obesity augments the age-induced increase in mitochondrial capacity for H2O2 release in Zucker fatty rats

Aim:  Mitochondrial dysfunction has been suggested to play a significant role in obesity and insulin resistance. The aim of the present study was to investigate if changes in obesity and insulin resistance were related to similar changes in mitochondrial capacity for hydrogenperoxide release in Zucker diabetic fatty rats and their lean littermates.

Plasticity in mitochondrial cristae density allows metabolic capacity modulation in human skeletal muscle

In human skeletal muscles, the current view is that the capacity for mitochondrial energy production, and thus endurance capacity, is set by the mitochondria volume. However, increasing the mitochondrial inner membrane surface comprises an alternative mechanism for increasing the energy production capacity. In the present study, we show that mitochondrial inner membranes in leg muscles of endurance‐trained athletes have an increased ratio of surface per mitochondrial volume. We show a positive correlation between this ratio and whole body oxygen uptake and muscle fibre mitochondrial content. The results obtained in the present study help us to understand modulation of mitochondrial function, as well as how mitochondria can increase their oxidative capacity with increased demand.

Impaired mitochondrial function in chronically ischemic human heart.

Chronic ischemic heart disease is associated with myocardial hypoperfusion. The resulting hypoxia potentially inflicts damage upon the mitochondria, leading to a compromised energetic state. Furthermore, ischemic damage may cause excessive production of reactive oxygen species (ROS), producing mitochondrial damage, hereby reinforcing a vicious circle. Ischemic preconditioning has been proven protective in acute ischemia, but the subject of chronic ischemic preconditioning has not been explored in humans. We hypothesized that mitochondrial respiratory capacity would be diminished in chronic ischemic regions of human myocardium but that these mitochondria would be more resistant to ex vivo ischemia and, second, that ROS generation would be higher in ischemic myocardium. The aim of this study was to test mitochondrial respiratory capacity during hyperoxia and hypoxia, to investigate ROS production, and finally to assess myocardial antioxidant levels. Mitochondrial respiration in biopsies from ischemic and nonischemic regions from the left ventricle of the same heart was compared in nine human subjects. Maximal oxidative phosphorylation capacity in fresh muscle fibers was lower in ischemic compared with nonischemic myocardium (P < 0.05), but the degree of coupling (respiratory control ratio) did not differ (P > 0.05). The presence of ex vivo hypoxia did not reveal any chronic ischemic preconditioning of the ischemic myocardial regions (P > 0.05). ROS production was higher in ischemic myocardium (P < 0.05), and the levels of antioxidant protein expression was lower. Diminished mitochondrial respiration capacity and excessive ROS production demonstrate an impaired mitochondrial function in ischemic human heart muscle. No chronic ischemic preconditioning effect was found.