Mitochondrial function

In chapter three, it was shown that acidosis decreases the apparent Km of mitochondria for ADP, with a concomitant decrease in maximal respiration, however, the decrease in maximal respiration was challenged by the results in chapter four, where no decrease in maximal respiration was seen over a larger pH range. As identified in chapter three, a central premise of these results is that consumption of oxygen provides an index of mitochondrial function, with the assumption that accelerated oxygen consumption rates are indicative of increased function. However, consumption of oxygen occurs via the electron transport system, which serves to pump protons to support ATP synthesis. Thus if 1) ATP synthesis can be supported by an influx of protons from the cytosol to the intermembrane space; and 2) ATP synthase activity rate is not rate limiting to ATP synthesis, then the following results are explained; firstly, affinity for ADP is increased at pH ≤ 6.5 (table 3.1, figure 3.1; [517]), and leak respiration, but not maximal ADP-stimulated respiration, is depressed at pH 6.4 [517]. However, this would not explain the frequent observation that uncoupling the electron transport system from oxidative phosphorylation can result in higher rates of oxygen consumption. Measurement of ATP synthesis rates at different pH remains something worth further investigation. This would effectively test the premise that an acidotic intracellular space could contribute to a mitochondrial inner membrane potential solely through proton diffusion through the outer mitochondrial membrane.

It is also possible that intracellular acidosis may contribute indirectly to ionic membrane potential through the multitude of mitochondrial ion transport proteins (reviewed in [37]), some of which are proton symporters/antiporters. This means that accumulation of hydrogen ions may

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potentially affect other ionic gradients, either through acting as a substrate for sodium/potassium - hydrogen exchangers, or simply altering the enzyme activity of any other ion pumps. This in turn would affect the ionic component of membrane potential, independent of any effect on hydrogen ion distribution. There are numerous fluorescent dyes available that could be used to infer the effect of acidosis on membrane potential (for example JC-1/safranin to measure net membrane potential [271]). Further mechanistic information could be determined using ion-selective fluorophores to investigate changes in specific transmembrane ion gradients (for example calcium-green™). Collectively, this would provide information on whether an acidotic intracellular space may alter a) ATP synthesis, b) membrane potential, and in the event membrane potential is affected, c) which ion(s) contribute to any difference in membrane potential seen.

In chapter four, it was shown that voluntary exercise activity resulted in increased mitochondrial respiration and ROS emission, but in most cases did not affect the response of mitochondria to acute changes in pH. One notable interaction was that ROS emission from mitochondria during maximal leak respiration was maintained as pH dropped in muscle from exercised animals, but dropped in muscle from sedentary animals. This effect may be mediated by complex III, as activity assays indicate a pH-induced decline in complex III activity in both sedentary and exercised groups, but the rate of decline was half as much in the exercised cohort compared to sedentary. This suggests that the voluntary exercise protocol in some way made complex III activity less sensitive to changes in pH, and the mechanism(s) mediating this effect merit further investigation. One important thing to note is that permeabilised muscle has no intact circulation, and should have lost intracellular material – so any exercise-induced increase in carnosine or bicarbonate buffering capacity from the exercise training in chapter 4 should be washed out during the tissue preparation. This means that the decreased sensitivity to pH seen in the exercise-trained rats may be an innate property of the mitochondria themselves, rather than due to an increase in muscle buffering capacity.

One finding in chapter four which contrasts with previous findings was that mass-specific mitochondrial ROS emission was higher in exercised rats (figure 4.8), where previous studies have either shown no change, or a decrease in ROS emission [246]. It is difficult to compare results given

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the voluntary nature of the intervention, and as such an obvious further addition to this study would be the inclusion of an exercise training group, to compare the magnitude of effect between voluntary activity and a more fixed training stimulus. This could further be divided into a prescribed endurance training protocol compared to high-intensity interval type training.

Chapters three and four do not show agreement on whether acidosis does indeed depress mitochondrial respiration. In chapter three, maximal ADP-stimulated respiration was depressed at pH 6.5 compared to pH 7.1, but was not different at pH 6.9 or pH 6.7. As this suggested there may be a threshold for an effect of pH, this range was extended in chapter four. However, no depression of respiration was evident during phosphorylating states in a pH range from 7.1 to 6.2. In looking collectively at the data, the conclusion could be drawn that the difference seen in chapter 3 between pH 7.1 and pH 6.5 could just be a statistical anomaly – the lower limit of the 95% confidence interval of the difference is 0.2 pmol O2.mg-1.s-1, which indicates that though statistically significant, the

confidence limits of the difference mean it may actually not be meaningful. Coupled with no difference seen at pH 6.2 in chapter four, this suggests lower pH does not affect respiration during oxidative phosphorylation. However, one key difference between the two chapters was that in chapter three, maximal respiration was determined by curve-fitting following titration of ADP up to 2.5 mM, while in chapter four maximal respiration was determined following addition of a single 5 mM bolus of ADP. So the final concentration of ADP in each experiment is different, and there is a different time course over which ADP was provided.

Voluntary exercise also resulted in higher mean activities of all mitochondrial enzyme complex activities; however, complex II and complex IV showed greater differences than complex I or III. This suggests there is preferential increase in complex II and IV activity with exercise, and the mechanisms responsible warrant further attention. It should also be noted that no effort was made to look at supercomplex assembly, and this could be an interesting addition – particularly in comparison with a training intervention. Mitochondrial function is ultimately a synergy between different enzyme complexes, and the formation of ‘supercomplexes’, which are formed when respiratory complexes associate together [466], represents another level of control of mitochondrial function. Exercise

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increases supercomplex formation [171], however how supercomplex assembly and/or composition is affected by acidosis is unknown.