Reply from Joachim Nielsen, Kasper D. Gejl and Niels Ørtenblad

In our recent article (Nielsen et al. 2017) published in The Journal of Physiology, we establish that an elevated mitochondrial cristae density can be a regulatory mechanism for increasing metabolic power in human skeletal muscle. The comments offered in a Letter to the Editor by Larsen et al. (2017) relate to (1) the conclusion not being substantiated by the results and (2) the hypothesis introduced in our article that experimental studies have shown that respiration per mitochondria varies being based on false premises. We realise that Larsen et al. have made this statement based on a misreading of our text and also by neglecting two-thirds of our lines of argument that were presented in the introduction. Larsen and colleagues misread our text by stating that line 5 in the abstract, “In the present study, we confirm this hypothesis by showing that, in human skeletal muscle, and in contrast to the current view, the mitochondrial cristae density is not constant but, instead exhibits plasticity with long-term endurance training”, refers to our hypothesis on page 2. This is not the case and the sentence is taken out of the context. This line in the abstract refers to the preceding sentence in the abstract: “Modelling studies have hypothesized that this variation in respiration per mitochondria depends on plasticity in cristae density, although current evidence for such a mechanism is lacking”. Therefore, our data showing plasticity in cristae density actually confirm this hypothesis. We also state that the plasticity in cristae density is seen with long-term endurance training. This is based on the full data set, where the cross-sectional findings are combined with detailed analysis at the fibre level. Here, we observed that the frequently recruited fibres (evaluated by glycogen utilisation during 1 h exercise; see Nielsen et al. 2011) have higher cristae density than the fibres that were not recruited. This is a clear indication of a training effect and we discuss this association in the paper. However, despite this interpretation, one cannot indisputably conclude from our study that mitochondrial cristae density is trainable, which is very clearly stated throughout the article, i.e. in our conclusion on page 8 about the cross-sectional design: “Our data unequivocally demonstrate that human skeletal muscle mitochondria cristae density varies between populations with different physical activity levels”. This conclusion is unquestionably substantiated by the results. Larsen et al. (2017) stated that the hypothesis for the study is based on false premises. Our rationale for conducting the study is centred on three lines of argument. Firstly, extensive research is going on within the field of mitochondrial morphology and plasticity across different biological and medical disciplines. This has revealed several regulatory factors of the inner membrane morphology, clearly suggesting that the cristae density could be important for regulation of mitochondrial function in skeletal muscle. Secondly, we have cited three papers, which all conclude that the respiration rate of mitochondria ex vivo may not exclusively depend on the quantity of mitochondria investigated, clearly indicating qualitative differences between mitochondria from different populations or after endurance training. Thirdly, we have built the hypothesis on the ‘trade-off’ concept of the composition of the muscle fibres, whereby a high quest for both mechanical and metabolic power output could lead to volumetric constraints, and, in turn, advantage qualitative over quantitative adaptations. Larsen et al. (2017) questioned the validity of the second argument (while neglecting the two others), which we based on three experimental studies, conducted by independent research groups. Larsen et al. (2017) claim that their simple calculation of data from one of the studies (Jacobs & Lundby, 2013) shows no difference in mitochondrial respiration per mitochondria, without any specifics of the claimed miscalculation of Jacobs & Lundy. We can only comprehend this critique as referring to a calculation based on individual data on citrate synthase, but using mean values of respiration. The distribution of the individual data of respiration can influence the outcome, so that a simple calculation based on the population mean is by no means valid and therefore not sufficient to state that Jacob & Lundy’s calculation is wrong. We agree that use of mtDNA as a marker of mitochondrial content in the work by Pesta et al. (2011) was later shown not to be a good choice by some of us in collaboration with Larsen et al. (2012a). However, a fair and unbiased interpretation of the data in Larsen et al. 2012a is that all of the analysed biomarkers of mitochondrial content are indeed poor when the spectrum of mitochondrial content is narrow, as in Pesta et al. (2011) and many other publications on mitochondrial respiration (e.g. Larsen et al. 2012b; Guadalupe-Grau et al. 2015), and this indicates a high risk of getting false negative or false positive results. Thus, the results from Pesta et al. (2011) cannot stand alone, but should be placed in a context with other observations as well. In this context, the respiration rate of fatty acids has been reported to be elevated following low-intensity training, without changes in maximal respiration, indicating no change in mitochondrial content but improved ability to oxidise fatty acids (Boushel et al. 2014). This has recently been confirmed (Boushel et al. 2015). Overall, the three studies suggest that the mitochondrial respiration rate is not determined only by the mitochondrial content in skeletal muscle. Thus, there are clear indications for diverse functional changes in mitochondria relative to indices of mitochondrial volume, which together with the two other lines of argument (plasticity in mitochondrial morphology and cellular volumetric constraints), form strong premises for the hypothesis introduced in our article.