Reply to Rutter et al.: The roles of cytosolic and intramitochondrial Ca²⁺ and the mitochondrial Ca²⁺-uniporter (MCU) in the stimulation of mammalian oxidative phosphorylation

Each model used in the work referred to by Rutter et al. (1) addressed certain aspects of mitochondrial biology, and together, they fully support the conclusions made. Please note that we describe Ca²⁺-mediated regulation of oxidative phosphorylation (OXPHOS) fluxes (2, 3) and do not question Ca²⁺-responsiveness of pyruvate dehydrogenase en-zyme activity (4). To address concerns such as those raised by Rutter et al. (1), we studied glutamate/malate-dependent OXPHOS in the absence of exogenous pyruvate in mitochondria, omitted pyruvate from cell experiments, and implemented the working rat heart model perfused by Krebs–Henseleit (glucose) buffer. This unequivocally demonstrates in a broad range of models that MAS (malate-aspartate shuttle) inhibition induces a state of mitochondrial pyruvate starvation (3).

An unresolved observation is that mitochondria of MCU knockout mice show negligible activity of Ca²⁺-uptake (5), which we confirm (3). We attributed this activity to residual expression of wild-type Mcu transcripts (3) as the result of a rare event of gene-trap excision during mRNA splicing, since this activity was sensitive to ruthenium red, an inhibitor of the MCU. Besides, please also note the low MCU Ca²⁺ affinity (6). In vivo, the endoplasmic reticulum is thought to facilitate the generation of microcompartments of high Ca²⁺ concentration to allow Ca²⁺ uptake via MCU (6). This mechanism is compromised in MCU knockout mice and can be ruled out in isolated mitochondria. Thus, our data support the notion that, depending on tissue, model system and pathophysiological status, a combination of mechanisms (e.g., mitochondrial gas pedal and MCU) control OXPHOS substrate supply.

Notably, the viability of MCU knockout mice (3, 5), albeit living in a laboratory cage, indicates that MCU-dependent pathways are dispensable for a sedentary life. It remains interestingto elucidate, however, why MCU-dependent activation of matrix dehydrogenases is indispensable for high activity states (7) and how this may allow stressful life in the wild.