This month’s installment of Generally Physiological focuses on mitochondria, exploring circadian regulation of mitochondrial oxidative metabolism, what happens when you eliminate the mitochondrial calcium uniporter, and Ca2+ transport by the inner mitochondrial membrane protein Letm1.
Circadian regulation of mitochondrial bioenergetics
Circadian clocks synchronize physiological processes with the day/night cycle, enabling coordination of feeding/fasting and activity/rest cycles with the appropriate time of day. The coenzyme nicotinamide adenine dinucleotide (NAD+), which plays a crucial role in metabolic redox reactions, participates in a feedback loop with the core clock machinery: NAD+ regulates the transcription of clock genes through the NAD+-dependent deacetylase SIRT1, while expression of the rate-limiting enzyme in NAD+ biosynthesis is controlled by the clock (see Rey and Reddy, 2013). Noting that NAD+ regulates the mitochondrial deacetylase SIRT3, Peek et al. (2013) explored the possibility that oscillations in NAD+ could also provide a mechanism for the circadian regulation of mitochondrial oxidative metabolism. Liver fatty acid oxidation and NAD+ abundance showed synchronized oscillations in fasted mice maintained in darkness, with peaks occurring toward the end of the rest cycle. Similarly, cultured mouse myoblasts showed rhythmic oscillations in NAD+ accumulation, fatty acid oxidation, oxygen consumption, and glucose oxidation. Livers from circadian mutant mice lacking the clock gene Bmal1 showed decreased fatty acid oxidation and total and mitochondrial NAD+ compared with that from wild-type mice, and isolated mitochondria from the Bmal1−/− mice showed decreased oxygen consumption when supplied with fatty acids or pyruvate. Moreover, Bmal1−/− fibroblasts produced less ATP than wild-type fibroblasts under conditions favoring oxidative metabolism. Acetylation of various mitochondrial oxidative enzymes was increased in Bmal1−/− mice, whereas their activity was decreased, and a mitochondrial SIRT3 target showed rhythmic oscillations in acetylation—although the abundance of SIRT3 itself remained constant. Increasing NAD+ rescued deacetylation of a SIRT3 substrate, activity of medium-chain acyl dehydrogenase (a mitochondrial enzyme that showed circadian oscillations in acetylation in wild-type mice as well as increased acetylation and decreased activity in mutants), and mitochondrial oxidative defects in circadian mutants. The authors thus conclude that circadian oscillations in NAD+ regulate mitochondrial function through the SIRT3-dependent deacetylation of oxidative enzymes, to coordinate mitochondrial bioenergetics with daily rhythms in feeding and activity.
Circadian regulation of mitochondrial oxidative metabolism, and what happens when you eliminate the mitochondrial calcium uniporter
Exploring the roles of mitochondrial calcium
Energized mitochondria can rapidly sequester cytosolic calcium, which crosses the outer and inner mitochondrial membranes to enter the mitochondrial matrix, traversing the latter by way of the mitochondrial calcium uniporter (MCU)—a process driven by the mitochondrial membrane potential generated by electron transport. Matrix calcium, in turn, is thought to regulate various mitochondrial functions and—when present in excess—to contribute to cell death through a mechanism involving opening of the permeability transition pore (PTP) and the consequent collapse of the mitochondrial membrane potential. Pan et al. (2013) used a gene trap strategy to create mice lacking the MCU as a model system with which to explore MCU function and the physiological roles of matrix calcium.
Although slightly smaller than wild-type mice, MCU−/− mice had no other gross phenotypic abnormalities. However, calcium imaging of embryonic fibroblasts, cardiac myocytes, and isolated mitochondria from wild-type or mutant mice indicated that MCU−/− mitochondria failed to rapidly accumulate calcium, and various functional tests revealed impaired skeletal muscle peak performance. Although basal oxygen consumption was comparable in wild-type and MCU−/− embryonic fibroblasts, isolated mitochondria, and mice, the robust calcium-dependent stimulation of oxygen consumption in mitochondria isolated from wild-type mice was absent in MCU−/− mitochondria. There was a marked decrease in basal matrix calcium in MCU−/− skeletal muscle mitochondria and, consistent with a role for the calcium-sensitive phosphatase in regulating pyruvate dehydrogenase (PDH) activity, PDH phosphorylation was increased in skeletal muscle of starved MCU−/− mice compared with wild-type mice, whereas PDH activity was decreased. Whereas exposure to 500 µM calcium stimulated opening of the PTP in wild-type mitochondria, it failed to do so with MCU−/− mitochondria. However, MCU−/− embryonic fibroblasts failed to show resistance to cell death in response to various stimuli (oxidative stress from hydrogen peroxide, ER stress from tunicamycin, the antineoplastic agent doxorubicin, C2-ceramide—which activates apoptotic and necrotic pathways—or thapsigargin). Similarly, MCU−/− mice showed no evidence of protection against cardiac ischemia-reperfusion injury, although, intriguingly, unlike wild-type mice, the PTP inhibitor cyclosporine A failed to protect them against cardiac damage.
Defining Letm1 function
The functions of Letm1 (leucine zipper, EF hand–containing transmembrane protein 1), another inner mitochondrial membrane protein, have been controversial. Initially implicated in mitochondrial volume regulation and proposed to play a role in K+/H+ exchange, Letm1 was later found to be involved in mitochondrial Ca2+ homeostasis and in mediating Ca2+/H+ exchange. Here, Tsai et al. (2014) reconstituted human Letm1 (purified from an Escherichia coli expression system) in liposomes to rigorously investigate its transport function. Whereas a flux assay using 86Rb+ as a K+ surrogate failed to reveal Letm1-dependent 86Rb+ uptake, assays using either 45Ca2+ or a fluorescent Ca2+ indicator indicated that Letm1 mediates Ca2+ transport. Mn2+ inhibited Ca2+ transport, with an apparent Ki of 32 µM; moreover, Letm1 could mediate Ca2+-Mn2+ exchange. The lanthanides Gd3+ and La3+ also inhibited Ca2+ transport, whereas Mg2+ did not. Letm1-mediated Ca2+ transport was independent of membrane potential, indicating that it was electroneutral. Investigation of possible transport partners eliminated K+, Na+, and Cl−: Ca2+ transport was similar in the presence of 120 mM KCl or 80 mM Na2SO4 and was unaffected by manipulation of the K+, Na2+, or Cl− gradients. In contrast, acidification of the extraliposomal medium promoted rapid Ca2+ efflux, leading the authors to conclude that Letm1functions as a Ca2+/H+ antiporter, with a 2:1 H+/Ca2+ exchange ratio.