Each-step Activation Research Articles

Mechanisms of slowed V̇O2 on-kinetics in second step of two-step-incremental exercise in skeletal muscle

Simulations using a computer model of the skeletal muscle bioenergetic system demonstrate that the slowed V̇O2 on-kinetics of the second step in two-step incremental exercise (exercise initiated from elevated baseline metabolic rate) can be accounted for by a decrease in the stimulation of oxidative phosphorylation (OXPHOS) and/or increase in the stimulation of glycolysis through each-step activation (ESA) in working skeletal muscle. This effect can be caused by either a recruitment of more glycolytic type IIa, IIx and IIb fibers or metabolic regulation in already recruited fibers, or both. The elevated-glycolysis-stimulation mechanism predicts that the end-second-step pH in two-step-incremental exercise is lower than the end-exercise pH in constant-power exercise with the same work intensity (power output). The lowered-OXPHOS-stimulation mechanism predicts higher end-exercise ADP and Pi, and lower PCr in the second step of two-step-incremental than in constant-power exercise. These predictions/mechanisms can be verified or falsified in the experimental way. Data Availability StatementThere are no additional data available.

Sensitivity of V̇O2max, critical power and V̇O2 on-kinetics to O2 concentration/delivery and other factors in skeletal muscle

Relative sensitivities of the maximal oxygen uptake (V̇O2max), critical power (CP) and transition time of the primary phase II of the V̇O2 on-kinetics (t0.63) to selected factors in skeletal muscle are simulated using a computer model of the skeletal muscle bioenergetic system. In normoxia, V̇O2max is significantly positively sensitive to peak Pi (Pipeak, Pi at which exercise is terminated because of fatigue), OXPHOS (oxidative phosphorylation) activity (kOX) and ESA (each-step-activation) intensity (AOXmax), and negatively sensitive to the accessible phosphate (and total creatine) pool (Pacc). CP is additionally moderately positively sensitive to critical Pi (Picrit, Pi at which the additional ATP usage appears) and negatively sensitive to the additional ATP usage activity (kadd). t0.63 is significantly positively sensitive to Pacc and negatively to kOX and AOXmax. The positive sensitivity of V̇O2max and CP, and negative sensitivity of t0.63 to O2 concentration/delivery is low at normoxic and hyperoxic O2 levels, but increases significantly at severely hypoxic O2 levels.

Integration of mitochondrial energetics in heart with mathematical modelling.

It has been an unsolved question how cardiac mitochondrial energetics is regulated during working transition. Mathematical modelling is a powerful tool for exploring the complicated networks of mitochondrial metabolism. We summarize the recent progress and remaining questions about mitochondrial energetics in heart, especially focusing on approaches utilizing mathematical modelling. Feedback activation by ADP and/or inorganic phosphate is an old but still attractive hypothesis for explaining the regulation mechanisms of cardiac mitochondrial energetics. However, this hypothesis has not been fully validated by experiments because rises of ADP and/or inorganic phosphate concentrations during cardiac workload increase have not been detected in many experiments. The hypothesis of intracellular energetic units is an extended version of feedback activation, which has a similar problem. The each-step activation hypothesis beautifully reproduces metabolite constancy, although such master regulators have not been identified yet. Ca2+ has been the most plausible candidate because some of the mitochondrial dehydrogenases are activated by it. Recent experimental and simulation studies, however, throw doubt on its physiological relevance. Finally, we discuss issues to be solved to obtain a better view of cardiac mitochondrial energetics.

Muscle V˙O2-power output nonlinearity in constant-power, step-incremental, and ramp-incremental exercise: magnitude and underlying mechanisms

A computer model of the skeletal muscle bioenergetic system was used to simulate time courses of muscle oxygen consumption (V˙O2), cytosolic metabolite (ADP, PCr, Pi, and ATP) concentrations, and pH during whole‐body constant‐power exercise (CPE) (6 min), step‐incremental exercise (SIE) (30 W/3 min), and slow (10 W/min), medium (30 W/min), and fast (50 W/min) ramp‐incremental exercise (RIE). Different ESA (each‐step activation) of oxidative phosphorylation (OXPHOS) intensity‐ATP usage activity relationships, representing different muscle fibers recruitment patterns, gave best agreement with experimental data for CPE, and for SIE and RIE. It was assumed that the muscle V˙O2‐power output (PO) nonlinearity is related to a time‐ and PO‐dependent increase in the additional ATP usage underlying the slow component of the V˙O2 on‐kinetics minus the increase in ATP supply by anaerobic glycolysis leading to a decrease in V˙O2. The muscle V˙O2‐PO relationship deviated upward (+) or downward (−) from linearity above critical power (CP), and the nonlinearity equaled +16% (CPE),+12% (SIE), +8% (slow RIE), +1% (moderate RIE), and −2% (fast RIE) at the end of exercise, in agreement with experimental data. During SIE and RIE, changes in PCr and Pi accelerated moderately above CP, while changes in ADP and pH accelerated significantly with time and PO. It is postulated that the intensity of the additional ATP usage minus ATP supply by anaerobic glycolysis determines the size of the muscle V˙O2‐PO nonlinearity. It is proposed that the extent of the additional ATP usage is proportional to the time integral of PO ‐ CP above CP.

Mechanisms underlying extremely fast muscle V˙O2 on-kinetics in humans.

The time constant of the primary phase of pulmonary V˙O2 on‐kinetics (τ p), which reflects muscle V˙O2 kinetics during moderate‐intensity exercise, is about 30 s in young healthy untrained individuals, while it can be as low as 8 s in endurance‐trained athletes. We aimed to determine the intramuscular factors that enable very low values of t 0.63 to be achieved (analogous to τ p, t 0.63 is the time to reach 63% of the V˙O2 amplitude). A computer model of oxidative phosphorylation (OXPHOS) in skeletal muscle was used. Muscle t0.63 was near‐linearly proportional to the difference in phosphocreatine (PCr) concentration between rest and work (ΔPCr). Of the two main factors that determine t0.63, a huge increase in either OXPHOS activity (six‐ to eightfold) or each‐step activation (ESA) of OXPHOS intensity (>3‐fold) was needed to reduce muscle t 0.63 from the reference value of 29 s (selected to represent young untrained subjects) to below 10 s (observed in athletes) when altered separately. On the other hand, the effect of a simultaneous increase of both OXPHOS activity and ESA intensity required only a twofold elevation of each to decrease t 0.63 below 10 s. Of note, the dependence of t 0.63 on OXPHOS activity and ESA intensity is hyperbolic, meaning that in trained individuals a large increase in OXPHOS activity and ESA intensity are required to elicit a small reduction in τ p. In summary, we postulate that the synergistic action of elevated OXPHOS activity and ESA intensity is responsible for extremely low τ p (t 0.63) observed in highly endurance‐trained athletes.

Contribution of proton leak to oxygen consumption in skeletal muscle during intense exercise is very low despite large contribution at rest.

A computer model was used to simulate the dependence of protonmotive force (Δp), proton leak and phenomenological (involving proton leak) ATP/O2 ratio on work intensity in skeletal muscle. Δp, NADH and proton leak decreased with work intensity. The contribution of proton leak to oxygen consumption () decreased from about 60% at rest to about 3 and 1% at moderate and heavy/severe exercise, respectively, while the ATP/O2 ratio increased from 2.1 to 5.5 and 5.7. A two-fold increase in proton leak activity or its decrease to zero decreased/increased the ATP/O2 ratio by only about 3 and 1% during moderate and heavy/severe exercise, respectively. The low contribution of proton leak to in intensively working skeletal muscle was mostly caused by a huge increase in ATP usage intensity during rest-to-work transition, while OXPHOS, and thus oxidative ATP supply and related to it, was mostly stimulated by high each-step activation (ESA) of OXPHOS complexes. The contribution of proton leak to and ATP/O2 ratio in isolated mitochondria should not be directly extrapolated to working muscle, as mitochondria lack ESA, at least in the absence of Ca2+, and therefore cannot be elevated as much as in intact muscle.

Principles, insights, and potential pitfalls of the noninvasive determination of muscle oxidative capacity by near-infrared spectroscopy.

Skeletal muscle oxidative capacity is highly plastic, strongly associated with whole-body aerobic capacity (16, 18) and state of health. Loss of muscle oxidative capacity is associated with physical inactivity, aging and chronic disease (17), and has been implicated in the pathophysiology of obesity and diabetes (21). Evaluating these changes has traditionally been limited to invasive or costly assessments (biopsy or ³¹P MRS). To address this, Hamaoka and colleagues developed an innovative, non-invasive approach using near-infrared spectroscopy (NIRS) to quantitatively measure muscle oxygen consumption (mVO₂; 12) and use this to infer muscle oxidative capacity based on the mVO₂ recovery rate constant (k) (23; later modified 26). This technique has been subsequently used to interpret relative differences in oxidative capacity across a wide range of muscles, ages and disease states (Figure 1C). The purpose of this Viewpoint is to open a discussion on the principles, insights and potential pitfalls of using NIRS to measure k and infer muscle oxidative capacity.

Each-step activation of oxidative phosphorylation is necessary to explain muscle metabolic kinetic responses to exercise and recovery in humans.

The basic control mechanisms of oxidative phosphorylation (OXPHOS) and glycolysis during work transitions in human skeletal muscle are still a matter of debate. We used simulations of skeletal muscle bioenergetics to identify key system features that contribute to this debate, by comparing kinetic model outputs with experimental human data, including phosphocreatine, pH, pulmonary oxygen uptake and fluxes of ATP production by OXPHOS (vOX), anaerobic glycolysis and creatine kinase in moderate and severe intensity exercise transitions. We found that each-step activation of particular OXPHOS complexes, NADH supply and glycolysis, and strong (third-order) glycolytic inhibition by protons was required to reproduce observed phosphocreatine, pH and vOX kinetics during exercise. A slow decay of each-step activation during recovery, which was slowed further following severe exercise, was necessary to reproduce the experimental findings. Well-tested computer models offer new insight in the control of the human skeletal muscle bioenergetic system during physical exercise. To better understand muscle bioenergetic regulation, a previously-developed model of the skeletal muscle cell bioenergetic system was used to simulate the influence of: (1) each-step activation (ESA) of NADH supply (including glycolysis) and oxidative phosphorylation (OXPHOS) complexes and (2) glycolytic inhibition by protons on the kinetics of ATP synthesis from OXPHOS, anaerobic glycolysis and creatine kinase. Simulations were fitted to previously published experimental data of ATP production fluxes and metabolite concentrations during moderate and severe intensity exercise transitions in bilateral knee extension in humans. Overall, the computer simulations agreed well with experimental results. Specifically, a large (>5-fold) direct activation of all OXPHOS complexes was required to simulate measured phosphocreatine and OXPHOS responses to both moderate and severe intensity exercise. In addition, slow decay of ESA was required to fit phosphocreatine recovery kinetics, and the time constant of ESA decay was slower following severe (180s) than moderate (90s) exercise. Additionally, a strong inhibition of (anaerobic) glycolysis by protons (glycolytic rate inversely proportional to the cube of proton concentration) provided the best fit to the experimental pH kinetics, and may contribute to the progressive increase in oxidative ATP supply during acidifying contractions. During severe-intensity exercise, an 'additional' ATP usage (a 27% increase at 8min, above the initial ATP supply) was necessary to explain the observed V̇O2 slow component. Thus, parallel activation of ATP usage and ATP supply (ESA), and a strong inhibition of ATP supply by anaerobic glycolysis, were necessary to simulate the kinetics of muscle bioenergetics observed in humans.

Effects of OXPHOS complex deficiencies and ESA dysfunction in working intact skeletal muscle: implications for mitochondrial myopathies

The effects of inborn oxidative phosphorylation (OXPHOS) complex deficiencies or possible each-step activation (ESA) dysfunction on the bioenergetic system in working intact skeletal muscle are studied using a computer model of OXPHOS published previously. The curves representing the dependencies of V˙O2 and metabolite concentrations on single complex activity, entire OXPHOS activity or ESA intensity exhibit a characteristic threshold at some OXPHOS complex activity/ESA intensity. This threshold for V˙O2 of single complex activities is significantly lower in intact muscle during moderate and heavy work, than in isolated mitochondria in state 3. Metabolite concentrations and pH in working muscle start to change significantly at much higher OXPHOS complex activities/ESA intensities than V˙O2. The effect of entire OXPHOS deficiency or ESA dysfunction is potentially much stronger than the effect of a single complex deficiency. Implications of these findings for the genesis of mitochondrial myopathies are discussed. It is concluded that V˙O2 in state 3 and its dependence on complex activity in isolated mitochondria is not a universal quantitative determinant of the effect of mitochondrial dysfunctions in vivo. Moderate and severe mitochondria dysfunctions are defined: the former affect significantly only metabolite concentrations and pH, while the latter also decrease significantly V˙O2 in intact skeletal muscle during work. The dysfunction-caused decrease in V˙O2/oxidative ATP synthesis flux, disturbance of metabolite homeostasis, elevated ROS production and anaerobic glycolysis recruitment can account for such mitochondrial myopathy symptoms as muscle weakness, exercise intolerance (exertional fatigue) and lactic acidosis.

'Idealized' state 4 and state 3 in mitochondria vs. rest and work in skeletal muscle.

A computer model of oxidative phosphorylation (OXPHOS) in skeletal muscle is used to compare state 3, intermediate state and state 4 in mitochondria with rest and work in skeletal muscle. ‘Idealized’ state 4 and 3 in relation to various ‘experimental’ states 4 and 3 are defined. Theoretical simulations show, in accordance with experimental data, that oxygen consumption (V’O2), ADP and Pi are higher, while ATP/ADP and Δp are lower in rest than in state 4, because of the presence of basal ATP consuming reactions in the former. It is postulated that moderate and intensive work in skeletal muscle is very different from state 3 in isolated mitochondria. V’O2, ATP/ADP, Δp and the control of ATP usage over V’O2 are much higher, while ADP and Pi are much lower in the former. The slope of the phenomenological V’O2-ADP relationship is much steeper during the rest-work transition than during the state 4-state 3 transition. The work state in intact muscle is much more similar to intermediate state than to state 3 in isolated mitochondria in terms of ADP, ATP/ADP, Δp and metabolic control pattern, but not in terms of V’O2. The huge differences between intact muscle and isolated mitochondria are proposed to be caused by the presence of the each-step activation (ESA) mechanism of the regulation of OXPHOS in intact skeletal muscle. Generally, the present study suggests that isolated mitochondria (at least in the absence of Ca2+) cannot serve as a good model of OXPHOS regulation in intact skeletal muscle.

Regulation of oxidative phosphorylation during work transitions results from its kinetic properties

The regulation of oxidative phosphorylation (OXPHOS) during work transitions in skeletal muscle and heart is still not well understood. Different computer models of this process have been developed that are characterized by various kinetic properties. In the present research-polemic theoretical study it is argued that models belonging to one group (Model A), which predict that among OXPHOS complexes complex III keeps almost all of the metabolic control over oxygen consumption (Vo2) and involve a strong complex III activation by inorganic phosphate (Pi), lead to the conclusion that an increase in Pi is the main mechanism responsible for OXPHOS activation (feedback-activation mechanism). Models belonging to another group (Model B), which were developed to take into account an approximately uniform distribution of metabolic control over Vo2 among particular OXPHOS complexes (complex I, complex III, complex IV, ATP synthase, ATP/ADP carrier, phosphate carrier) encountered in experimental studies in isolated mitochondria, predict that all OXPHOS complexes are directly activated in parallel with ATP usage and NADH supply by some external cytosolic factor/mechanism during rest-to-work or low-to-high work transitions in skeletal muscle and heart ("each-step-activation" mechanism). Model B demonstrates that different intensities of each-step activation can account for the very different (slopes of) phenomenological Vo2-ADP relationships observed in various skeletal muscles and heart. Thus they are able to explain the differences in the regulation of OXPHOS during work transitions between skeletal muscle (where moderate changes in ADP take place) and intact heart in vivo (where ADP is essentially constant).

Metabolic control over the oxygen consumption flux in intact skeletal muscle: in silico studies

It has been postulated previously that a direct activation of all oxidative phosphorylation complexes in parallel with the activation of ATP usage and substrate dehydrogenation (the so-called each-step activation) is the main mechanism responsible for adjusting the rate of ATP production by mitochondria to the current energy demand during rest-to-work transition in intact skeletal muscle in vivo. The present in silico study, using a computer model of oxidative phosphorylation developed previously, analyzes the impact of the each-step-activation mechanism on the distribution of control (defined within Metabolic Control Analysis) over the oxygen consumption flux among the components of the bioenergetic system in intact oxidative skeletal muscle at different energy demands. It is demonstrated that in the absence of each-step activation, the oxidative phosphorylation complexes take over from ATP usage most of the control over the respiration rate and oxidative ATP production at higher (but still physiological) energy demands. This leads to a saturation of oxidative phosphorylation, impossibility of a further acceleration of oxidative ATP synthesis, and dramatic drop in the phosphorylation potential. On the other hand, the each-step-activation mechanism allows maintenance of a high degree of the control exerted by ATP usage over the ATP turnover and oxygen consumption flux even at high energy demands and thus enables a potentially very large increase in ATP turnover. It is also shown that low oxygen concentration shifts the metabolic control from ATP usage to cytochrome oxidase and thus limits the oxidative ATP production.