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SUMMARYMitochondrial calcium (mtCa2+) uptake via the Mitochondrial Calcium Uniporter (MCU) couples the regulation of calcium homeostasis to energy production. mtCa2+uptake is rate-limiting for mitochondrial activation during muscle contraction, but how MCU is affected during physiopathology and whether it can be stimulated therapeutically remains largely uncharacterized. By profiling human and preclinical aging of skeletal muscle, we discovered a conserved down-regulation of MCUR1 during aging that decreases mtCa2+uptake and drives sarcopenia. Through a screen of 5000 bioactive nutrients, we identify the natural polyphenol Oleuropein as a specific MCU activator that stimulates mitochondrial respiration via binding to MICU1. Oleuropein activates mtCa2+uptake and oxidative energy metabolism to enhance endurance and limit fatigue in vivo both in young and aged. These effects of Oleuropein are mediated by an MCU-dependent mechanism in skeletal muscle as they are lost upon muscle-specific MCU KO. Our work demonstrates that impaired mtCa2+uptake causes mitochondrial dysfunction during aging and establishes Oleuropein as a novel nutrient that specifically targets MCU to stimulate mitochondrial bioenergetics and muscle performance.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are enriched at postsynaptic membrane compartments of the neuromuscular junction (NMJ), surrounding the subsynaptic nuclei and close to nicotinic acetylcholine receptors (nAChRs) of the motor endplate. At the endplate level, it has been proposed that nerve-dependent electrical activity might trigger IP3-associated, local Ca2+ signals not only involved in excitation–transcription (ET) coupling but also crucial to the development and stabilization of the NMJ itself. The present study was undertaken to examine whether denervation affects the subsynaptic IP3R distribution in skeletal muscles and which are the underlying mechanisms. Fluorescence microscopy, carried out on in vivo denervated muscles (following sciatectomy) and in vitro denervated skeletal muscle fibers from flexor digitorum brevis (FDB), indicates that denervation causes a reduction in the subsynaptic IP3R1-stained region, and such a decrease appears to be determined by the lack of muscle electrical activity, as judged by partial reversal upon field electrical stimulation of in vitro denervated skeletal muscle fibers.

Mitochondria participate in the maintenance of cellular homeostasis. Firstly, mitochondria regulate energy metabolism through oxidative phosphorylation. In addition, they are involved in cell fate decisions by activating the apoptotic intrinsic pathway. Finally, they work as intracellular signaling hubs as a result of their tight regulation of ion and metabolite concentrations and other critical signaling molecules such as ROS. Aging is a multifactorial process triggered by impairments in different cellular components. Among the various molecular pathways involved, mitochondria are key regulators of longevity. Indeed, mitochondrial deterioration is a critical signature of the aging process. In this scenario, we will focus specifically on the age-related decrease in CoQ levels, an essential component of the electron transport chain (ETC) and an antioxidant, and how CoQ supplementation could benefit the aging process. Generally, any treatment that improves and sustains mitochondrial functionality is a good candidate to counteract age-related mitochondrial dysfunctions. In recent years, heightened attention has been given to natural compounds that modulate mitochondrial function. One of the most famous is resveratrol due to its ability to increase mitochondrial biogenesis and work as an antioxidant agent. This review will discuss recent clinical trials and meta-analyses based on resveratrol and CoQ supplementation, focusing on how these compounds could improve mitochondrial functionality during aging.

Polyglutamine (polyQ)-expanded androgen receptor (AR) causes spinobulbar muscular atrophy. Skeletal muscle is a primary site of polyQ-expanded AR toxicity, however it remains to be established what the early pathological processes are and how they unfold during disease progression. Using transgenic, knock-in SBMA mice and patient-derived muscle biopsies, we show that polyQ-expanded AR alters the generation of intrinsic muscle force prior to denervation. The pattern of expression of genes encoding key components of the excitation-contraction coupling (ECC) machinery is altered in the muscles of presymptomatic mice and patients. Altered mitochondrial respiration is another early event followed by stimulus-dependent accumulation of calcium into mitochondria during disease onset and structural organization alterations of the muscle triad. Surgical castration and AR silencing prevented early pathological processes. These observations show that androgen-dependent deregulations of ECC and defective mitochondrial respiration are early but reversible events followed by altered muscle force, contraction dynamics, and calcium dyshomeostasis.

Skeletal muscle mitochondria are placed in close proximity of the sarcoplasmic reticulum (SR), the main intracellular Ca

Skeletal muscle mitochondria are placed in close proximity of the sarcoplasmic reticulum (SR), the main intracellular Ca2+ store. During muscle activity, excitation of sarcolemma and of T-tubule triggers the release of Ca2+ from the SR initiating myofiber contraction. The rise in cytosolic Ca2+ determines the opening of the mitochondrial calcium uniporter (MCU), the highly selective channel of the inner mitochondrial membrane (IMM), causing a robust increase in mitochondrial Ca2+ uptake. The Ca2+-dependent activation of TCA cycle enzymes increases the synthesis of ATP required for SERCA activity. Thus, Ca2+ is transported back into the SR and cytosolic [Ca2+] returns to resting levels eventually leading to muscle relaxation. In recent years, thanks to the molecular identification of MCU complex components, the role of mitochondrial Ca2+ uptake in the pathophysiology of skeletal muscle has been uncovered. In this chapter, we will introduce the reader to a general overview of mitochondrial Ca2+ accumulation. We will tackle the key molecular players and the cellular and pathophysiological consequences of mitochondrial Ca2+ dyshomeostasis. In the second part of the chapter, we will discuss novel findings on the physiological role of mitochondrial Ca2+ uptake in skeletal muscle. Finally, we will examine the involvement of mitochondrial Ca2+ signaling in muscle diseases.

Mitochondrial activity warrants energy supply to oxidative myofibres to sustain endurance workload. The maintenance of mitochondrial homeostasis is ensured by the control of fission and fusion processes and by the mitophagic removal of aberrant organelles. Many diseases are due to or characterized by dysfunctional mitochondria, and altered mitochondrial dynamics or turnover trigger myopathy per se. In this review, we will tackle the role of mitochondrial dynamics, turnover and metabolism in skeletal muscle, both in health and disease.

Recently, the role of mitochondrial activity in high-energy demand organs and in the orchestration of whole-body metabolism has received renewed attention. In mitochondria, pyruvate oxidation, ensured by efficient mitochondrial pyruvate entry and matrix dehydrogenases activity, generates acetyl CoA that enters the TCA cycle. TCA cycle activity, in turn, provides reducing equivalents and electrons that feed the electron transport chain eventually producing ATP. Mitochondrial Ca2+ uptake plays an essential role in the control of aerobic metabolism. Mitochondrial Ca2+ accumulation stimulates aerobic metabolism by inducing the activity of three TCA cycle dehydrogenases. In detail, matrix Ca2+ indirectly modulates pyruvate dehydrogenase via pyruvate dehydrogenase phosphatase 1, and directly activates isocitrate and α-ketoglutarate dehydrogenases. Here, we will discuss the contribution of mitochondrial Ca2+ uptake to the metabolic homeostasis of organs involved in systemic metabolism, including liver, skeletal muscle, and adipose tissue. We will also tackle the role of mitochondrial Ca2+ uptake in the heart, a high-energy consuming organ whose function strictly depends on appropriate Ca2+ signaling.

We report a method for ex vivo measurements of Ca2+ transients in skeletal muscle fibers, both in the sarcoplasma and into the mitochondria. These measurements are based on the use of genetically encoded probes. Addition of targeting DNA sequences, in frame with the probe encoding sequence, ensures protein expression in specific compartments. The use of probes with different excitation spectra allows the simultaneous determination of cytosolic and mitochondrial Ca2+ transients in the same fiber. Probe encoding plasmids are expressed in flexor digitorum brevis (FDB) muscles by means of the in vivo electroporation technique. Measurements are then performed ex vivo in isolated single myofibers.

Skeletal muscle mitochondria readily accumulate Ca2+ in response to SR store-releasing stimuli thanks to the activity of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ uptake. MCU positively regulates myofiber size in physiological conditions, and counteracts pathological loss of muscle mass. Here, we show that skeletal muscle-specific MCU deletion inhibits myofiber mitochondrial Ca2+ uptake, impairs muscle force and exercise performance, and determines a slow-to fast switch in MHCs expression. Mitochondrial Ca2+ uptake is required for effective glucose oxidation, as demonstrated by the fact that in muscle-specific MCU-/- myofibers oxidative metabolism is impaired and glycolysis rate is increased. Although defective, mitochondrial activity is partially sustained by increased fatty acid (FA) oxidation. In MCU-/- myofibers, PDP2 overexpression drastically reduces FA-dependency, demonstrating that decreased PDH activity is the main trigger of the metabolic rewiring of MCU-/- muscles. Accordingly, PDK4 overexpression in MCUfl/fl myofibers is sufficient to increase FA-dependent respiration. Finally, as a result of the muscle-specific MCU deletion, a systemic catabolic response impinging on both liver and adipose tissue metabolism occurs.

Introduction: Elucidating the mechanisms that link fiber contraction with ATP synthesis is important to understand skeletal muscle function. Mitochondria exhibit a particular architecture in skeletal muscle fibers. A large fraction resides along the I band, in close proximity to myofibrils where ATP production is essential for contraction, and closely interact with the triad structures. Sub-sarcolemmal mitochondria have a different distribution, and contain different components of the metabolic protein complexes. Objective: We studied mitochondrial Ca2 transients following depolarization by potassium (or electrical stimulation) of single skeletal muscle fibers, and their relation with metabolic output and membrane potential changes in mitochondria. Results: In isolated muscle fibers from FBD muscle, depolarization increased both cytoplasmic and mitochondria Ca2 levels. Mitochondrial Ca2 uptake required functional IP3R and RyR1 channels. Moreover, inhibition of either one decreased basal O2 consumption rate but only RyR1 inhibition decreased ATP-linked O2 consumption. Depolarization-induced Ca2 signals in sub-sarcolemmal mitochondria were accompanied by a reduction in mitochondria membrane potential; Ca2 signals propagated towards intermyofibrillar mitochondria, where mitochondrial membrane potential increased. Likewise, oligomycin induced propagation of mitochondria membrane potential from the surface towards the center of the fiber. Results are compatible with Ca2 -dependent propagation of mitochondrial membrane potential from the surface towards the center of the fiber. Conclusion: Ca2 -dependent propagation of mitochondrial membrane potential from the surface towards the center of the fiber could have a critical role in control of mitochondria metabolism both at rest and after depolarization as part of an "excitation-metabolism" coupling in skeletal muscle fibers.

Mitochondrial Ca2+ is involved in heterogeneous functions, ranging from the control of metabolism and ATP production to the regulation of cell death. In addition, mitochondrial Ca2+ uptake contributes to cytosolic [Ca2+] shaping thus impinging on specific Ca2+-dependent events. Mitochondrial Ca2+ concentration is controlled by influx and efflux pathways: the former controlled by the activity of the mitochondrial Ca2+ uniporter (MCU), the latter by the Na+/Ca2+ exchanger (NCLX) and the H+/Ca2+ (mHCX) exchanger. The molecular identities of MCU and of NCLX have been recently unraveled, thus allowing genetic studies on their physiopathological relevance. After a general framework on the significance of mitochondrial Ca2+ uptake, this review discusses the structure of the MCU complex and the regulation of its activity, the importance of mitochondrial Ca2+ signaling in different physiological settings, and the consequences of MCU modulation on organ physiology.

The mitochondrial calcium uniporter (MCU) gene codifies for the inner mitochondrial membrane (IMM) channel responsible for mitochondrial Ca(2 +) uptake. Cytosolic Ca(2 +) transients are involved in sarcomere contraction through cycles of release and storage in the sarcoplasmic reticulum. In addition cytosolic Ca(2 +) regulates various signaling cascades that eventually lead to gene expression reprogramming. Mitochondria are strategically placed in close contact with the ER/SR, thus cytosolic Ca(2 +) transients elicit large increases in the [Ca(2 +)] of the mitochondrial matrix ([Ca(2 +)]mt). Mitochondrial Ca(2 +) uptake regulates energy production and cell survival. In addition, we recently showed that MCU-dependent mitochondrial Ca(2 +) uptake controls skeletal muscle trophism. In the same report, we dissected the effects of MCU-dependent mitochondrial Ca(2 +) uptake on gene expression through microarray gene expression analysis upon modulation of MCU expression by in vivo AAV infection. Analyses were performed on single skeletal muscle fibers at two time points (7 and 14 days post-AAV injection). Raw and normalized data are available on the GEO database (http://www.ncbi.nlm.nih.gov/geo/) (GSE60931).

Age‐related sarcopenia is characterized by a progressive loss of muscle mass with decline in specific force, having dramatic consequences on mobility and quality of life in seniors. The etiology of sarcopenia is multifactorial and underlying mechanisms are currently not fully elucidated. Physical exercise is known to have beneficial effects on muscle trophism and force production. Alterations of mitochondrial Ca2+ homeostasis regulated by mitochondrial calcium uniporter (MCU) have been recently shown to affect muscle trophism in vivo in mice. To understand the relevance of MCU‐dependent mitochondrial Ca2+ uptake in aging and to investigate the effect of physical exercise on MCU expression and mitochondria dynamics, we analyzed skeletal muscle biopsies from 70‐year‐old subjects 9 weeks trained with either neuromuscular electrical stimulation (ES) or leg press. Here, we demonstrate that improved muscle function and structure induced by both trainings are linked to increased protein levels of MCU. Ultrastructural analyses by electron microscopy showed remodeling of mitochondrial apparatus in ES‐trained muscles that is consistent with an adaptation to physical exercise, a response likely mediated by an increased expression of mitochondrial fusion protein OPA1. Altogether these results indicate that the ES‐dependent physiological effects on skeletal muscle size and force are associated with changes in mitochondrial‐related proteins involved in Ca2+ homeostasis and mitochondrial shape. These original findings in aging human skeletal muscle confirm the data obtained in mice and propose MCU and mitochondria‐related proteins as potential pharmacological targets to counteract age‐related muscle loss.