Date Published: February 3, 2015
Publisher: Public Library of Science
Author(s): Bernard Korzeniewski.
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.
State 3 in isolated mitochondria was originally defined by Chance and Williams [1,2] as a state with high external (extramitochondrial) ADP, low external ATP/ADP ratio and high (maximal in isolated mitochondria without Ca2+) oxygen consumption (V’O2) and ATP synthesis (vATPs). State 4, on the other hand, was defined as a state with a very high ATP/ADP ratio, very low ADP, no ATP synthesis and V’O2 corresponding exclusively to proton leak. Originally, state 3 was set by an addition of external ADP. After some time most ADP was transformed by oxidative phosphorylation (OXPHOS) in mitochondria into ATP and the system passed to state 4. There is essentially no real steady-state in this kind of experiments, because ATP/ADP changes continuously as ADP is continuously converted to ATP. State 4 and state 3 were set in many other studies by adding to mitochondria suspension appropriate amounts of ATP and ADP.
Throughout the article I mean by ATP, ADP and Pi extramitochondrial (cytosolic in the case of intact cells/tissues) ATP, ADP and Pi and not intramitochondrial ATP, ADP and Pi.
Computer simulations demonstrate that the rest state in intact skeletal muscle does not correspond exactly to state 4id and, first of all, the work state does not correspond to state 3id. This is demonstrated in Fig. 1, which shows the simulated phenomenological dependence of V’O2 on ADP during state 4id-state 3id transition and during rest-work transition (in both cases the phenomenological V’O2-ADP relationship involves implicitly the V’O2-Pi dependence), and in Table 1. V’O2, ADP and Pi are considerably higher at rest than in state 4id. Δp and ATP/ADP are lower at rest. During moderate and intensive work V’O2, ATP/ADP and Δp are much higher than in state 3id, while ADP and Pi are lower. During intensive work V’O2, ADP and Pi are higher, while ATP/ADP and Δp are lower than during moderate work. In intermediate state V’O2 is much lower than during work. However, the values of ADP, ATP/ADP, Pi and Δp in intermediate state are quite similar to that during work. In fact, they are located between the values for moderate work and intensive work.
In the present theoretical, research-polemic study ‘idealized’ state 4, state 3 and the intermediate (in terms of ATP synthesis, V’O2, ADP, ATP/ADP, Pi and Δp) state in isolated mitochondria are compared with resting and working states in intact skeletal muscle. Computer simulations confirm the previous experimental observation  that the resting state does not correspond exactly to state 4 and, first of all, strongly suggest, again—in agreement with experimental data—that the working state is very different from state 3. It is postulated that the differences between isolated mitochondria (at least in the absence of Ca2+) and intact muscle in the mechanisms responsible for the regulation of OXPHOS when ATP demand (rate constant of ATP usage, kUT) increases are responsible for the latter difference. Therefore, it seems that isolated mitochondria (at least in the absence of Ca2+) are not a good model of the regulation of OXPHOS during work transitions in intact tissues, in particular in intact skeletal muscle.
The present theoretical, research-polemic study demonstrates that it is possible to unify the kinetic behavior of OXPHOS (oxidative phosphorylation) in isolated mitochondria and intact skeletal muscle during varying energy (ATP) demand using a unique kinetic description of the system, under assumption that the ESA (each-step activation) mechanism is present in intact muscle at work. It confirms earlier experimental findings that resting intact skeletal muscle is not exactly in state 4 and, first of all, it strongly suggests that the working state in intact muscle is very different from state 3 in isolated mitochondria. ‘Idealized’ state 4 and state 3 (state 4id and state 3id) that are intended to serve as a reference for various ‘experimental’ states 4 and state 3 are defined. Computer simulations show that V’O2, ATP/ADP and Δp are much higher, while ADP and Pi much lower at work in skeletal muscle than in state 3id in mitochondria. The phenomenological V’O2-ADP relationship during rest-work transition is much steeper than during the state 4id-state 3id transition. It is postulated that the huge differences between intact muscle and isolated mitochondria are caused by the presence of the each-step activation (ESA) mechanism in intact skeletal muscle, which is absent in isolated mitochondria (at least in the absence of Ca2+). The metabolic control over V’O2, characterized by flux control coefficients (FCCs), is dominated by proton leak in state 4 and, to a smaller extent, in the rest state. Almost all of the control is kept by OXPHOS complexes in state 3. During moderate and intensive work in intact skeletal muscle as well as in intermediate state in isolated mitochondria (a state intermediate between state 4 and state 3) ATP usage is the main controlling process. Generally, the working state in muscle resembles much more intermediate state than state 3id in isolated mitochondria in terms of ADP, Pi, ATP/ADP, Δp and FCCs, but not in terms of V’O2. The present study suggests that isolated mitochondria (especially in the absence of Ca2+) cannot serve as a good model of OXPHOS regulation and bioenergetic system behavior in intact skeletal muscle. It also shows that the computer model used for simulations and the postulated each-step activation mechanism are able to integrate and explain the (differences in the) kinetic behavior of the energy metabolism in intact skeletal muscle and isolated muscle mitochondria in response to elevated energy demand.