Mitochondria capture and subsequently release Ca(2+) ions, thereby sensing and shaping cellular Ca(2+) signals. The Ca(2+) uniporter MCU mediates Ca(2+) uptake, whereas NCLX (mitochondrial Na/Ca exchanger) and LETM1 (leucine zipper-EF-hand-containing transmembrane protein 1) were proposed to exchange Ca(2+) against Na(+) or H(+), respectively. Here we study the role of these ion exchangers in mitochondrial Ca(2+) extrusion and in Ca(2+)-metabolic coupling. Both NCLX and LETM1 proteins were expressed in HeLa cells mitochondria. The rate of mitochondrial Ca(2+) efflux, measured with a genetically encoded indicator during agonist stimulations, increased with the amplitude of mitochondrial Ca(2+) ([Ca(2+)]mt) elevations. NCLX overexpression enhanced the rates of Ca(2+) efflux, whereas increasing LETM1 levels had no impact on Ca(2+) extrusion. The fluorescence of the redox-sensitive probe roGFP increased during [Ca(2+)]mt elevations, indicating a net reduction of the matrix. This redox response was abolished by NCLX overexpression and restored by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The [Ca(2+)]mt elevations were associated with increases in the autofluorescence of NAD(P)H, whose amplitude was strongly reduced by NCLX overexpression, an effect reverted by Na(+)/Ca(2+) exchange inhibition. We conclude that NCLX, but not LETM1, mediates Ca(2+) extrusion from mitochondria. By controlling the duration of matrix Ca(2+) elevations, NCLX contributes to the regulation of NAD(P)H production and to the conversion of Ca(2+) signals into redox changes.

Mitochondrial calcium (mtCa²⁺) uptake via the mitochondrial calcium uniporter (MCU) couples calcium homeostasis and energy metabolism. mtCa²⁺ uptake via MCU is rate-limiting for mitochondrial activation during muscle contraction, but its pathophysiological role and therapeutic application remain largely uncharacterized. By profiling human muscle biopsies, patient-derived myotubes, and preclinical models, we discovered a conserved downregulation of mitochondrial calcium uniporter regulator 1 (MCUR1) during skeletal muscle aging that associates with human sarcopenia and impairs mtCa²⁺ uptake and mitochondrial respiration. Through a screen of 5,000 bioactive molecules, we identify the natural polyphenol oleuropein as a specific MCU activator that stimulates mitochondrial respiration via mitochondrial calcium uptake 1 (MICU1) binding. Oleuropein activates mtCa²⁺ uptake and energy metabolism to enhance endurance and reduce fatigue in young and aged mice but not in muscle-specific MCU knockout (KO) mice. Our work demonstrates that impaired mtCa²⁺ uptake contributes to mitochondrial dysfunction during aging and establishes oleuropein as a novel food-derived molecule that specifically targets MCU to stimulate mitochondrial bioenergetics and muscle performance.

Background and Purpose: Quinic acid (QA) is an abundant natural compound from plant sources which may improve metabolic health. However, little attention has been paid to its effects on pancreatic beta‐cell functions, which contribute to the control of metabolic health by lowering blood glucose. Strategies targeting beta‐cell signal transduction are a new approach for diabetes treatment. This study investigated the efficacy of QA to stimulate beta‐cell function by targeting the basic molecular machinery of metabolism–secretion coupling. Experimental Approach: We measured bioenergetic parameters and insulin exocytosis in a model of insulin‐secreting beta‐cells (INS‐1E), together with Ca ²⁺ homeostasis, using genetically encoded sensors, targeted to different subcellular compartments. Islets from mice chronically infused with QA were also assessed. Key Results: QA triggered transient cytosolic Ca ²⁺ increases in insulin‐secreting cells by mobilizing Ca ²⁺ from intracellular stores, such as endoplasmic reticulum. Following glucose stimulation, QA increased glucose‐induced mitochondrial Ca ²⁺ transients. We also observed a QA‐induced rise of the NAD(P)H/NAD(P) ⁺ ratio, augmented ATP synthase‐dependent respiration, and enhanced glucose‐stimulated insulin secretion. QA promoted beta‐cell function in vivo as islets from mice infused with QA displayed improved glucose‐induced insulin secretion. A diet containing QA improved glucose tolerance in mice. Conclusions and Implications: QA modulated intracellular Ca ²⁺ homeostasis, enhancing glucose‐stimulated insulin secretion in both INS‐1E cells and mouse islets. By increasing mitochondrial Ca ²⁺ , QA activated the coordinated stimulation of oxidative metabolism, mitochondrial ATP synthase‐dependent respiration, and therefore insulin secretion. Bioactive agents raising mitochondrial Ca ²⁺ in pancreatic beta‐cells could be used to treat diabetes.

Mitochondrial dysfunction is linked to numerous pathological states, in particular related to metabolism, brain health and ageing. Nuclear encoded gene polymorphisms implicated in mitochondrial functions can be analyzed in the context of classical genome wide association studies. By contrast, mitochondrial DNA (mtDNA) variants are more challenging to identify and analyze for several reasons. First, contrary to the diploid nuclear genome, each cell carries several hundred copies of the circular mitochondrial genome. Mutations can therefore be present in only a subset of the mtDNA molecules, resulting in a heterogeneous pool of mtDNA, a situation referred to as heteroplasmy. Consequently, detection and quantification of variants requires extremely accurate tools, especially when this proportion is small. Additionally, the mitochondrial genome has pseudogenized into numerous copies within the nuclear genome over the course of evolution. These nuclear pseudogenes, named NUMTs, must be distinguished from genuine mtDNA sequences and excluded from the analysis. Here we describe a novel method, named MitoRS, in which the entire mitochondrial genome is amplified in a single reaction using rolling circle amplification. This approach is easier to setup and of higher throughput when compared to classical PCR amplification. Sequencing libraries are generated at high throughput exploiting a tagmentation-based method. Fine-tuned parameters are finally applied in the analysis to allow detection of variants even of low frequency heteroplasmy. The method was thoroughly benchmarked in a set of experiments designed to demonstrate its robustness, accuracy and sensitivity. The MitoRS method requires 5 ng total DNA as starting material. More than 96 samples can be processed in less than a day of laboratory work and sequenced in a single lane of an Illumina HiSeq flow cell. The lower limit for accurate quantification of single nucleotide variants has been measured at 1% frequency. The MitoRS method enables the robust, accurate, and sensitive analysis of a large number of samples. Because it is cost effective and simple to setup, we anticipate this method will promote the analysis of mtDNA variants in large cohorts, and may help assessing the impact of mtDNA heteroplasmy on metabolic health, brain function, cancer progression, or ageing.

Sustained ryanodine receptor (RyR) Ca²⁺ leak is associated with pathological conditions such as heart failure or skeletal muscle weakness. We report that a single session of sprint interval training (SIT), but not of moderate intensity continuous training (MICT), triggers RyR1 protein oxidation and nitrosylation leading to calstabin1 dissociation in healthy human muscle and in in vitro SIT models (simulated SIT or S-SIT). This is accompanied by decreased sarcoplasmic reticulum Ca²⁺ content, increased levels of mitochondrial oxidative phosphorylation proteins, supercomplex formation and enhanced NADH-linked mitochondrial respiratory capacity. Mechanistically, (S-)SIT increases mitochondrial Ca²⁺ uptake in mouse myotubes and muscle fibres, and decreases pyruvate dehydrogenase phosphorylation in human muscle and mouse myotubes. Countering Ca²⁺ leak or preventing mitochondrial Ca²⁺ uptake blunts S-SIT-induced adaptations, a result supported by proteomic analyses. Here we show that triggering acute transient Ca²⁺ leak through RyR1 in healthy muscle may contribute to the multiple health promoting benefits of exercise.