Rob C. I. Wüst

Kinetic control of oxygen consumption during contractions in self-perfused skeletal muscle

Non‐technical summary  The ability to sustain exercise is dependent on the ability to match muscular energy supply fuelled by oxygen to the energy demands of the activity (the ‘currency’ of biological energy is termed ATP). Experiments using muscle samples in a test tube suggest that the activation of muscle oxygen consumption is caused by accumulation of ADP (a breakdown product of ATP), which signals the need to increase ATP supply. The mechanism of this signalling process in vivo, however, is not well understood. We investigated the mechanism controlling oxidative ATP supply activation in canine muscle, by simultaneous measurements of oxygen consumption and ADP at the onset of muscle contractions. At the start of contractions muscle oxygen consumption increased more rapidly than predicted from the measured accumulation of ADP. These data suggest that an additional process (or processes) occurs to activate muscle oxidative ATP provision at the onset of exercise in vivo.

FIBER CAPILLARY SUPPLY RELATED TO FIBER SIZE AND OXIDATIVE CAPACITY IN HUMAN AND RAT SKELETAL MUSCLE

The capillary supply of a muscle fiber is thought to be determined by its type, oxidative capacity, size and metabolic surrounding. Size and oxidative capacity, however, differ between fiber types. To investigate which of these factors determines the capillary supply of a myofiber most we analysed in sections from human vastus lateralis (n = 11) and rat plantaris muscle (n = 8) the type, succinate dehydrogenase activity (SDH), reflecting oxidative capacity, and capillary supply of individual fibers. Capillary fiber density differed between fiber types in rat (P < 0.03) but not in human muscle. In human muscle only, the local capillary to fiber ratio (LCFR) correlated with the integrated SDH (fiber cross-sectional area x SDH) of a fiber (R = 0.62; P < 0.001). Backward multiple regression revealed, however, that the LCFR was primarily determined by fiber size, type (R = 0.71, human) and surrounding of the fiber (R = 0.62; rat plantaris muscle), i.e. whether it came from the deep or superficial region of the muscle (all P < 0.001) and not SDH. In conclusion, size, type and metabolic surrounding rather than mitochondrial activity determine the capillary supply to a muscle fiber.

Regional skeletal muscle remodeling and mitochondrial dysfunction in right ventricular heart failure

Exercise intolerance is a cardinal symptom of right ventricular heart failure (RV HF) and skeletal muscle adaptations play a role in this limitation. We determined regional remodeling of muscle structure and mitochondrial function in a rat model of RV HF induced by monocrotaline injection (MCT; 60 mg·kg(-1); n = 11). Serial sections of the plantaris were stained for fiber type, succinate dehydrogenase (SDH) activity and capillaries. Mitochondrial function was assessed in permeabilized fibers using respirometry, and isolated complex activity by blue native gel electrophoresis (BN PAGE). All measurements were compared with saline-injected control animals (CON; n = 12). Overall fiber cross-sectional area was smaller in MCT than CON: 1,843 ± 114 vs. 2,322 ± 120 μm(2) (P = 0.009). Capillary-to-fiber ratio was lower in MCT in the oxidative plantaris region (1.65 ± 0.09 vs. 1.93 ± 0.07; P = 0.03), but not in the glycolytic region. SDH activity (P = 0.048) and maximal respiratory rate (P = 0.012) were each ∼15% lower in all fibers in MCT. ADP sensitivity was reduced in both skeletal muscle regions in MCT (P = 0.032), but normalized by rotenone. A 20% lower complex I/IV activity in MCT was confirmed by BN PAGE. MCT-treatment was associated with lower mitochondrial volume density (lower SDH activity), quality (lower complex I activity), and fewer capillaries per fiber area in oxidative skeletal muscle. These features are consistent with structural and functional remodeling of the determinants of oxygen supply potential and utilization that may contribute to exercise intolerance and reduced quality of life in patients with RV HF.

On-off asymmetries in oxygen consumption kinetics of single Xenopus laevis skeletal muscle fibres suggest higher-order control.

Skeletal muscles increase oxygen consumption to produce energy during exercise; however, the processes controlling the rate of adaptation (its kinetics) at exercise onset and offset are not well understood. Here we measure kinetics in single frog skeletal muscle fibres using a unique experimental system that allows features of intracellular control mechanisms to be elucidated. We show for the first time that at contractions onset skeletal muscle kinetics are best described by a biphasic ‘activation’ and ‘exponential’ profile, whereas at cessation recovers with a single smooth exponential. Additionally these features were dependent on oxidative capacity and the intensity of stimulated contractions. These data show that the intracellular processes that activate oxidative energy provision at the onset of contractions are far more complex than previously suggested.

Breath-by-breath changes of lung oxygen stores at rest and during exercise in humans

When measuring breath-by-breath (BbB) oxygen uptake at the mouth (V MO2 ) as the difference between the amount of inspired and expired oxygen, BbB variation in lung oxygen stores due to BbB variation in end-expiratory lung volume (VLET) introduces an error leading to a decreased signal-to-noise ratio when compared to oxygen uptake at the alveolo-capillary interface (V AO2 ). Conventional BbB measurement of oxygen uptake does not measure BbB changes in lung oxygen stores. Opto-electronic plethysmography(OEP) allows accurate monitoring of absolute lung volume changes and BbB quantification of change in pulmonary oxygen stores. To quantify BbB variation of lung oxygen stores and to assess variability in V MO2 due to BbB variation of lung oxygen stores, we measured, V MO2 and V AO2 in parallel, at rest,during transients and during steady state cycling exercise at 60, 90 and 120W in 7 healthy male subjects. Average V MO2 and V AO2 at steady state were not different (p = 0.328).Direct measurement of V AO2 reduced overall BbB variability by 24% (p < 0.0001) and variance of the difference between V MO2 and V AO2 could be explained for 55% by BbB changes in VLET and expiratory oxygen fraction. V AO2 was higher than V MO215 and 30 s after exercise onset (p < 0.01). We conclude that (1) by taking into account changes in lung oxygen stores BbB variability of oxygen uptake is reduced, (2) alveolar oxygen stores change rapidly during transients to exercise, and (3) changes in alveolar oxygen stores affect BbB oxygen uptake measured at the mouth during the cardio-dynamic phase I.

Slowed muscle oxygen uptake kinetics with raised metabolism are not dependent on blood flow or recruitment dynamics.

A slow adjustment of skeletal muscle oxygen uptake ( V̇O2 ) to produce energy during exercise predisposes to early fatigue. In human studies, V̇O2 kinetics are slow when exercise is initiated from an elevated baseline; this is proposed to reflect slow blood flow regulation and/or recruitment of muscle fibres containing few mitochondria. To investigate this, we measured V̇O2 kinetics in canine muscle, with experimental control over muscle activation and blood flow. We found that V̇O2 kinetics remained slow when contractions were initiated from an elevated baseline despite experimentally increased blood flow and uniform fibre activation. These data challenge our current understanding of the control of muscle V̇O2 and demand consideration of new alternative mediators for V̇O2 control.

Synergistic role of ADP and Ca2+ in diastolic myocardial stiffness

Diastolic dysfunction in heart failure patients is evident from stiffening of the passive properties of the ventricular wall. Increased actomyosin interactions may significantly limit diastolic capacity, however, direct evidence is absent. From experiments at the cellular and whole organ level, in humans and rats, we show that actomyosin‐related force development contributes significantly to high diastolic stiffness in environments where high ADP and increased diastolic [Ca2+] are present, such as the failing myocardium. Our basal study provides a mechanical mechanism which may partly underlie diastolic dysfunction.

Mitochondrial complex I dysfunction and altered NAD(P)H kinetics in rat myocardium in cardiac right ventricular hypertrophy and failure

AIMS In cardiac hypertrophy (CH) and heart failure (HF), alterations occur in mitochondrial enzyme content and activities but the origin and implications of these changes for mitochondrial function need to be resolved. METHODS AND RESULTS Right ventricular CH or HF was induced by monocrotaline injection, which causes pulmonary artery hypertension, in rats. Results were compared with saline injection (CON). NAD(P)H and FAD autofluorescence were recorded in thin intact cardiac trabeculae during transitions in stimulation frequency, to assess mitochondrial complex I and complex II function, respectively. Oxygen consumption, mitochondrial morphology, protein content, and enzymatic activity were assessed. NAD(P)H autofluorescence upon an increase in stimulation frequency showed a rapid decline followed by a slow recovery. FAD autofluorescence followed a similar time course, but in opposite direction. The amplitude of the early rapid change in NAD(P)H autofluorescence was severely depressed in CH and HF compared with CON. The rapid changes in FAD autofluorescence in CH and HF were reduced to a lesser extent. Complex I-coupled respiration showed an ∼3.5-fold reduction in CH and HF; complex II-coupled respiration was depressed two-fold in HF. Western blot analyses revealed modest reductions in complex I protein content in CH and HF and in complex I activity in supercomplexes in HF. Mitochondrial volume density was similar, but mitochondrial remodelling was evident from changes in ultrastructure and fusion/fission indices in CH and HF. CONCLUSION These results suggest that the alterations in mitochondrial function observed in right ventricular CH and HF can be mainly attributed to complex I dysfunction.

Decreased creatine kinase is linked to diastolic dysfunction in rats with right heart failure induced by pulmonary artery hypertension

Our objective was to investigate the role of creatine kinase in the contractile dysfunction of right ventricular failure caused by pulmonary artery hypertension. Pulmonary artery hypertension and right ventricular failure were induced in rats by monocrotaline and compared to saline-injected control animals. In vivo right ventricular diastolic pressure–volume relationships were measured in anesthetized animals; diastolic force–length relationships in single enzymatically dissociated myocytes and myocardial creatine kinase levels by Western blot. We observed diastolic dysfunction in right ventricular failure indicated by significantly steeper diastolic pressure–volume relationships in vivo and diastolic force–length relationships in single myocytes. There was a significant reduction in creatine kinase protein expression in failing right ventricle. Dysfunction also manifested as a shorter diastolic sarcomere length in failing myocytes. This was associated with a Ca2 +-independent mechanism that was sensitive to cross-bridge cycling inhibition. In saponin-skinned failing myocytes, addition of exogenous creatine kinase significantly lengthened sarcomeres, while in intact healthy myocytes, inhibition of creatine kinase significantly shortened sarcomeres. Creatine kinase inhibition also changed the relatively flat contraction amplitude–stimulation frequency relationship of healthy myocytes into a steeply negative, failing phenotype. Decreased creatine kinase expression leads to diastolic dysfunction. We propose that this is via local reduction in ATP:ADP ratio and thus to Ca2 +-independent force production and diastolic sarcomere shortening. Creatine kinase inhibition also mimics a definitive characteristic of heart failure, the inability to respond to increased demand. Novel therapies for pulmonary artery hypertension are needed. Our data suggest that cardiac energetics would be a potential ventricular therapeutic target.

Differential regulation of perineuronal nets in the brain and spinal cord with exercise training

Perineuronal nets (PNNs) are lattice like structures which encapsulate the cell body and proximal dendrites of many neurons and are thought to be involved in regulating synaptic plasticity. It is believed that exercise can enhance the plasticity of the Central Nervous System (CNS) in healthy and dysfunctional states by shifting the balance between plasticity promoting and plasticity inhibiting factors in favor of the former. Recent work has focused on exercise effects on trophic factors but its effect on other plasticity regulators is poorly understood. In the present study we investigated how exercise regulates PNN expression in the lumbar spinal cord and areas of the brain associated with motor control and learning and memory. Adult, female Sprague-Dawley rats with free access to a running wheel for 6 weeks had significantly increased PNN expression in the spinal cord compared to sedentary rats (PNN thickness around motoneurons, exercise=15.75±0.63μm, sedentary=7.98±1.29μm, p<0.01). Conversely, in areas of the brain associated with learning and memory there was a significant reduction in perineuronal net expression (number of neurons with PNN in hippocampus CA1-exercise 21±0.56 and sedentary 24±0.34, p<0.01, thickness-exercised=2.37±0.13μm, sedentary=4.27±0.21μm; p<0.01). Our results suggest that in response to exercise, PNNs are differentially regulated in select regions of the CNS, with a general decreased expression in the brain and increased expression in the lumbar spinal cord. This differential expression may indicate different regulatory mechanisms associated with plasticity in the brain compared to the spinal cord.