Do changes in mitochondrial quality contribute to increases in skeletal muscle oxidative capacity following endurance training?

Endurance training improves skeletal muscle oxidative capacity, but the molecular adaptations that drive this process are not fully understood. Specifically, whether oxidative capacity is improved solely by augmentations in mitochondrial quantity, or by enhanced mitochondrial quality as well, is unclear. For example, compared to active individuals, elite endurance athletes exhibit superior in vitro oxidative capacity even when normalised to citrate synthase (CS) activity, a common marker of mitochondrial quantity (Jacobs & Lundby, 2013). Two important questions emerge from these results: (1) what molecular or enzymatic aspects of mitochondrial composition might allow rates of in vitro oxidative capacity to increase for a given mitochondrial volume, and (2) what endurance training methods are best suited to stimulate these positive adaptations?

Adaptations to endurance training have traditionally been studied following a period of moderate intensity continuous training (MICT), which is characterised by prolonged periods of aerobic activity at submaximal workloads (i.e. high volume training). More recently, interest has shifted towards high intensity interval training (HIIT), which involves repeated bouts of vigorous‐intensity exercise interspersed with periods of recovery. Daussin et al. (2008) reported that in vitro oxidative capacity is significantly improved by HIIT, but not by MICT. Larsen et al. (2013) demonstrated that as few as six sessions of HIIT are sufficient to improve in vivo markers of skeletal muscle oxidative capacity, measured as the maximal rate of phosphocreatine resynthesis. However, these studies did not “normalise” oxidative capacity measurements to mitochondrial quantity, nor did they measure changes in mitochondrial enzymatic composition, making it difficult to infer the molecular mechanisms that control measures of oxidative capacity and how training intensity may influence these changes.

In an article in The Journal of Physiology, MacInnis et al. (2016) attempted to address this gap in the literature by comparing changes in whole muscle and mitochondria‐specific in vitro oxidative capacity after 2 weeks of MICT and HIIT. To compare the different training modalities, they used single‐leg cycle ergometry, which allowed all participants (n=10 young males) to perform both MICT and HIIT over the same training period and serve as their own controls. Peak aerobic capacity (W _peak) was measured on each leg using a ramp protocol before and after 2 weeks of endurance training. Participants completed six sessions of single‐leg MICT (30 min at 50% W _peak) and HIIT (4 bouts of 5 min at 65% W _peak and 2.5 min at 20% W _peak). Muscle biopsies were taken from the vastus lateralis of each leg pre‐ and post‐training to measure markers of mitochondrial composition and mitochondrial oxidative capacity. Markers of mitochondrial composition included CS (used as a marker for mitochondrial quantity), cytochrome c oxidase subunit 4 (COXIV), NADH:ubiquinone oxidoreductase subunit A9 (NDUFA9), and mitofusin 2 (MFN2); the protein content of the latter three was measured in myosin heavy chain (MHC) I and IIA fibres. Oxidative capacity of permeabilised muscle fibres was measured in vitro using a substrate uncoupler inhibitor titration protocol that allowed maximal O₂‐respiratory rates ($J_{{\mathrm{O}}_2}$) through complexes I and II of the electron transport chain to be determined separately. Mass‐specific $J_{{\mathrm{O}}_2}$ was calculated as $J_{{\mathrm{O}}_2}$/muscle biopsy mass, whereas mitochondria‐specific $J_{{\mathrm{O}}_2}$ was calculated as the mass‐specific $J_{{\mathrm{O}}_2}$ normalised to CS content.

The increase in whole muscle CS activity was significantly greater following HIIT compared to MICT (+39% vs. +11%, respectively). HIIT also produced significantly greater improvements in mass‐specific $J_{{\mathrm{O}}_2}$ through complex I (HIIT +22% vs. MICT –7%) and complex I+II (HIIT +22% vs. MICT –9%). In contrast, neither training method stimulated improvements in mitochondria‐specific $J_{{\mathrm{O}}_2}$. And although MICT and HIIT both stimulated increases in COXIV, NDUFA9 and MFN2 protein content, none of these increases appeared to be fibre‐type specific.

Consistent with the authors’ hypotheses, HIIT was more effective than MICT for improving skeletal muscle mitochondrial quantity (i.e. CS activity), which coincided with greater increases in mass‐specific $J_{{\mathrm{O}}_2}$. However, neither training method stimulated significant improvements in mitochondria‐specific $J_{{\mathrm{O}}_2}$ (i.e. mitochondrial quality). Based on these observations, it is possible that endurance training stimulates changes in mitochondrial abundance prior to mitochondrial quality. Alternatively stated, perhaps improvements in mitochondrial quality only occur once mitochondrial abundance has increased to the limit allowed by the spatial constraints of the muscle fibre. It would be interesting to track these changes incrementally over the course of several weeks to see if changes in mass‐ and mitochondria‐specific $J_{{\mathrm{O}}_2}$ are time‐dependent.

We were also intrigued that training method did not differentially impact changes in COXIV, NDUFA9 and MFA2 protein content within MHC I and IIA muscles fibres. Although previous studies have shown that six sessions of HIIT are sufficient to produce improvements in skeletal muscle oxidative capacity (Daussin et al. 2008; Larsen et al. 2013), the on‐interval exercise intensity in these studies has typically been much greater than the 65% of W _peak employed by MacInnis et al. (2016). Thus, the relatively modest difference in training intensity prescribed for HIIT and MICT (65 vs. 50% W _peak, respectively) may have been insufficient to elicit different energetic overloads between the two training methods. Perhaps increasing the intensity of the HIIT protocol (e.g. ∼80% W _peak) and shortening the on‐interval duration would have elicited different responses between the two training protocols.

It is also possible that enzymes other than those of the electron transport chain are limiting to $J_{{\mathrm{O}}_2}$ and oxidative capacity. For example, during in vitro respirometry, mitochondrial $J_{{\mathrm{O}}_2}$ is often stimulated with large, saturating boluses of ADP. Under these conditions, maximal $J_{{\mathrm{O}}_2}$ is essentially limited by the rate of ADP transport into the mitochondrial matrix. As such, the molecular mechanism that limits in vitro oxidative capacity, and mitochondrial quality, may be the activity or abundance of adenine nucleotide translocase (ANT). It would be interesting to see if normalising mass‐ and mitochondria‐specific $J_{{\mathrm{O}}_2}$ to ANT abundance eliminates the differences in oxidative capacity observed between recreationally active and elite trained athletes (Jacobs & Lundby, 2013), or between individuals undergoing HIIT vs. MICT (Daussin et al. 2008). On the other hand, characterising ANT's impact on in vivo oxidative capacity will be more difficult, as ANT function in vivo is closely linked to that of mitochondrial creatine kinase (i.e. the creatine kinase shuttle).

Taking into account the comments described above, we feel the study design employed by MacInnis et al. (2016) is one that had several strengths. The within‐subject design afforded by using single‐leg cycle ergometry provided adequate statistical power despite a small sample size (n=10), and reduced the number of confounding factors that are typically a concern with between‐subject interventions. Consequently, we feel there are several areas where this design would be beneficial. For example, the United States National Institutes of Health has identified sex‐based differences as an area of human research that warrants more attention. Considering that men and women exhibit different inherent preferences for MHC I and IIA phenotypes, the single‐leg cycle ergometry design employed by MacInnis et al. (2016) may be especially useful for eliminating between‐subject variability in sex‐based training studies. Tarnopolsky et al. (2007) reported that 7 weeks of MICT increased muscle CS activity in men (26%) and women (3%), although the change in CS activity did not differ between sexes. However, Talanian et al. (2007) reported a greater relative change in CS activity (20%) in women who completed a 2‐week HIIT cycling protocol (10 bouts of 4 min at ∼90% ${\dot{V}}_{{\mathrm{O}}_2\mathrm{peak}}$ with 2 min of rest between bouts). Thus, at this point, whether or not sex impacts mitochondrial adaptations to MICT and/or HIIT remains incompletely characterised and warrants further study.

In conclusion, HIIT is superior to MICT for inducing rapid improvements in skeletal muscle mitochondrial quantity and oxidative capacity. However, additional research is needed to better understand the molecular mechanisms that control oxidative capacity measurements, as well as how endurance training stimulates adaptations to these molecular mechanisms to improve mitochondrial quality. MacInnis et al. (2016) have begun to pave the way by using single‐leg cycle ergometry to demonstrate that HIIT produces greater increases in oxidative capacity than MICT, which is driven by greater augmentations in mitochondrial content (i.e. CS activity). Conversely, neither MICT nor HIIT affected mitochondrial quality (i.e. mitochondria‐specific $J_{{\mathrm{O}}_2}$), although this may be impacted by the training intensity of HIIT, or length of the training period. Moving forward, it will be important to identify the molecular mechanisms that limit oxidative capacity in vitro, and translate that knowledge into an understanding of how they control oxidative capacity in vivo. This knowledge will allow us to directly compare and contrast the effect(s) MICT and HIIT have on changes to mitochondrial quality, regardless of whether the changes are being studied between muscle fibre types, individuals with different training statuses, or across genders.

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Competing interests

None declared.

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