Unravelling the complexity of the mitochondrial Ca2+ uniporter: regulation, tissue specificity, and physiological implications.

Calcium (Ca2+) signalling acts a pleiotropic message within the cell that is decoded by the mitochondria through a sophisticated ion channel known as the Mitochondrial Ca2+ Uniporter (MCU) complex. Under physiological conditions, mitochondrial Ca2+ signalling is crucial for coordinating cell activation with energy production. Conversely, in pathological scenarios, it can determine the fine balance between cell survival and death. Over the last decade, significant progress has been made in understanding the molecular bases of mitochondrial Ca2+ signalling. This began with the elucidation of the MCU channel components and extended to the elucidation of the mechanisms that regulate its activity. Additionally, increasing evidence suggests molecular mechanisms allowing tissue-specific modulation of the MCU complex, tailoring channel activity to the specific needs of different tissues or cell types. This review aims to explore the latest evidence elucidating the regulation of the MCU complex, the molecular factors controlling the tissue-specific properties of the channel, and the physiological and pathological implications of mitochondrial Ca2+ signalling in different tissues.

Neither too much nor too little: mitochondrial calcium concentration as a balance between physiological and pathological conditions.

Ca2+ ions serve as pleiotropic second messengers in the cell, regulating several cellular processes. Mitochondria play a fundamental role in Ca2+ homeostasis since mitochondrial Ca2+ (mitCa2+) is a key regulator of oxidative metabolism and cell death. MitCa2+ uptake is mediated by the mitochondrial Ca2+ uniporter complex (MCUc) localized in the inner mitochondrial membrane (IMM). MitCa2+ uptake stimulates the activity of three key enzymes of the Krebs cycle, thereby modulating ATP production and promoting oxidative metabolism. As Paracelsus stated, "Dosis sola facit venenum,"in pathological conditions, mitCa2+ overload triggers the opening of the mitochondrial permeability transition pore (mPTP), enabling the release of apoptotic factors and ultimately leading to cell death. Excessive mitCa2+ accumulation is also associated with a pathological increase of reactive oxygen species (ROS). In this article, we review the precise regulation and the effectors of mitCa2+ in physiopathological processes.

The Splicing of the Mitochondrial Calcium Uniporter Genuine Activator MICU1 Is Driven by RBFOX2 Splicing Factor during Myogenic Differentiation.

Alternative splicing, the process by which exons within a pre-mRNA transcript are differentially joined or skipped, is crucial in skeletal muscle since it is required both during myogenesis and in post-natal life to reprogram the transcripts of contractile proteins, metabolic enzymes, and transcription factors in functionally distinct muscle fiber types. The importance of such events is underlined by the numerosity of pathological conditions caused by alternative splicing aberrations. Importantly, many skeletal muscle Ca2+ homeostasis genes are also regulated by alternative splicing mechanisms, among which is the Mitochondrial Ca2+ Uniporter (MCU) genuine activator MICU1 which regulates MCU opening upon cell stimulation. We have previously shown that murine skeletal muscle MICU1 is subjected to alternative splicing, thereby generating a splice variant-which was named MICU1.1-that confers unique properties to the mitochondrial Ca2+ uptake and ensuring sufficient ATP production for muscle contraction. Here we extended the analysis of MICU1 alternative splicing to human tissues, finding two additional splicing variants that were characterized by their ability to regulate mitochondrial Ca2+ uptake. Furthermore, we found that MICU1 alternative splicing is induced during myogenesis by the splicing factor RBFOX2. These results highlight the complexity of the alternative splicing mechanisms in skeletal muscle and the regulation of mitochondrial Ca2+ among tissues.

Identification and functional validation of FDA-approved positive and negative modulators of the mitochondrial calcium uniporter.

The mitochondrial calcium uniporter (MCU), the highly selective channel responsible for mitochondrial Ca2+ entry, plays important roles in physiology and pathology. However, only few pharmacological compounds directly and selectively modulate its activity. Here, we perform high-throughput screening on a US Food and Drug Administration (FDA)-approved drug library comprising 1,600 compounds to identify molecules modulating mitochondrial Ca2+ uptake. We find amorolfine and benzethonium to be positive and negative MCU modulators, respectively. In agreement with the positive effect of MCU in muscle trophism, amorolfine increases muscle size, and MCU silencing is sufficient to blunt amorolfine-induced hypertrophy. Conversely, in the triple-negative breast cancer cell line MDA-MB-231, benzethonium delays cell growth and migration in an MCU-dependent manner and protects from ceramide-induced apoptosis, in line with the role of mitochondrial Ca2+ uptake in cancer progression. Overall, we identify amorolfine and benzethonium as effective MCU-targeting drugs applicable to a wide array of experimental and disease conditions.

Parvalbumin affects skeletal muscle trophism through modulation of mitochondrial calcium uptake.

Parvalbumin (PV) is a cytosolic Ca2+-binding protein highly expressed in fast skeletal muscle, contributing to an increased relaxation rate. Moreover, PV is an "atrogene" downregulated in most muscle atrophy conditions. Here, we exploit mice lacking PV to explore the link between the two PV functions. Surprisingly, PV ablation partially counteracts muscle loss after denervation. Furthermore, acute PV downregulation is accompanied by hypertrophy and upregulation by atrophy. PV ablation has a minor impact on sarcoplasmic reticulum but is associated with increased mitochondrial Ca2+ uptake, mitochondrial size and number, and contacts with Ca2+ release sites. Mitochondrial calcium uniporter (MCU) silencing abolishes the hypertrophic effect of PV ablation, suggesting that mitochondrial Ca2+ uptake is required for hypertrophy. In turn, an increase of mitochondrial Ca2+ is required to enhance expression of the pro-hypertrophy gene PGC-1α4, whose silencing blocks hypertrophy due to PV ablation. These results reveal how PV links cytosolic Ca2+ control to mitochondrial adaptations, leading to muscle mass regulation.

The molecular complexity of the Mitochondrial Calcium Uniporter.

The role of mitochondria in regulating cellular Ca2+ homeostasis is crucial for the understanding of different cellular functions in physiological and pathological conditions. Nevertheless, the study of this aspect was severely limited by the lack of the molecular identity of the proteins responsible for mitochondrial Ca2+ uptake. In 2011, the discovery of the gene encoding for the Mitochondrial Calcium Uniporter (MCU), the selective channel responsible for mitochondrial Ca2+ uptake, gave rise to an explosion of studies aimed to characterize the composition, the regulation of the channel and its pathophysiological roles. Here, we summarize the recent discoveries on the molecular structure and composition of the MCU complex by providing new insights into the mechanisms that regulate MCU channel activity.

Crosstalk between Calcium and ROS in Pathophysiological Conditions.

Calcium ions are highly versatile intracellular signals that regulate many cellular processes. The key to achieving this pleiotropic role is the spatiotemporal control of calcium concentration evoked by an extensive molecular repertoire of signalling components. Among these, reactive oxygen species (ROS) signalling, together with calcium signalling, plays a crucial role in controlling several physiopathological events. Although initially considered detrimental by-products of aerobic metabolism, it is now widely accepted that ROS, in subtoxic levels, act as signalling molecules. However, dysfunctions in the mechanisms controlling the physiological ROS concentration affect cellular homeostasis, leading to the pathogenesis of various disorders.

Publisher Correction: Parkin-dependent regulation of the MCU complex component MICU1.

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Parkin-dependent regulation of the MCU complex component MICU1.

The mitochondrial Ca2+ uniporter machinery is a multiprotein complex composed by the Ca2+ selective pore-forming subunit, the mitochondrial uniporter (MCU), and accessory proteins, including MICU1, MICU2 and EMRE. Their concerted action is required to fine-tune the uptake of Ca2+ into the mitochondrial matrix which both sustains cell bioenergetics and regulates the apoptotic response. To adequately fulfil such requirements and avoid impairment in mitochondrial Ca2+ handling, the intracellular turnover of all the MCU components must be tightly regulated. Here we show that the MCU complex regulator MICU1, but not MCU and MICU2, is rapidly and selectively degraded by the Ubiquitin Proteasome System (UPS). Moreover, we show that the multifunctional E3 ubiquitin ligase Parkin (PARK2), whose mutations cause autosomal recessive early-onset Parkinson's disease (PD), is a potential candidate involved in this process since its upregulation strongly decreases the basal level of MICU1. Parkin was found to interact with MICU1 and, interestingly, Parkin Ubl-domain, but not its E3-ubquitin ligase activity, is required for the degradation of MICU1, suggesting that in addition to the well documented role in the control of Parkin basal auto-inhibition, the Ubl-domain might exert important regulatory functions by acting as scaffold for the proteasome-mediated degradation of selected substrates under basal conditions, i.e. to guarantee their turnover. We have found that also MICU2 stability was affected upon Parkin overexpression, probably as a consequence of increased MICU1 degradation. Our findings support a model in which the PD-related E3 ubiquitin ligase Parkin directly participates in the selective regulation of the MCU complex regulator MICU1 and, indirectly, also of the MICU2 gatekeeper, thus indicating that Parkin loss of function could contribute to the impairment of the ability of mitochondria to handle Ca2+ and consequently to the pathogenesis of PD.

Mitochondrial calcium uptake in organ physiology: from molecular mechanism to animal models.

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.

Increased mitochondrial calcium uniporter in adipocytes underlies mitochondrial alterations associated with insulin resistance.

Intracellular calcium influences an array of pathways and affects cellular processes. With the rapidly progressing research investigating the molecular identity and the physiological roles of the mitochondrial calcium uniporter (MCU) complex, we now have the tools to understand the functions of mitochondrial Ca2+ in the regulation of pathophysiological processes. Herein, we describe the role of key MCU complex components in insulin resistance in mouse and human adipose tissue. Adipose tissue gene expression was analyzed from several models of obese and diabetic rodents and in 72 patients with obesity as well as in vitro insulin-resistant adipocytes. Genetic manipulation of MCU activity in 3T3-L1 adipocytes allowed the investigation of the role of mitochondrial calcium uptake. In insulin-resistant adipocytes, mitochondrial calcium uptake increased and several MCU components were upregulated. Similar results were observed in mouse and human visceral adipose tissue (VAT) during the progression of obesity and diabetes. Intriguingly, subcutaneous adipose tissue (SAT) was spared from overt MCU fluctuations. Furthermore, MCU expression returned to physiological levels in VAT of patients after weight loss by bariatric surgery. Genetic manipulation of mitochondrial calcium uptake in 3T3-L1 adipocytes demonstrated that changes in mitochondrial calcium concentration ([Ca2+]mt) can affect mitochondrial metabolism, including oxidative enzyme activity, mitochondrial respiration, membrane potential, and reactive oxygen species formation. Finally, our data suggest a strong relationship between [Ca2+]mt and the release of IL-6 and TNFα in adipocytes. Altered mitochondrial calcium flux in fat cells may play a role in obesity and diabetes and may be associated with the differential metabolic profiles of VAT and SAT.

A MICU1 Splice Variant Confers High Sensitivity to the Mitochondrial Ca2+ Uptake Machinery of Skeletal Muscle.

Skeletal muscle is a dynamic organ, characterized by an incredible ability to rapidly increase its rate of energy consumption to sustain activity. Muscle mitochondria provide most of the ATP required for contraction via oxidative phosphorylation. Here we found that skeletal muscle mitochondria express a unique MCU complex containing an alternative splice isoform of MICU1, MICU1.1, characterized by the addition of a micro-exon that is sufficient to greatly modify the properties of the MCU. Indeed, MICU1.1 binds Ca2+ one order of magnitude more efficiently than MICU1 and, when heterodimerized with MICU2, activates MCU current at lower Ca2+ concentrations than MICU1-MICU2 heterodimers. In skeletal muscle in vivo, MICU1.1 is required for sustained mitochondrial Ca2+ uptake and ATP production. These results highlight a novel mechanism of the molecular plasticity of the MCU Ca2+ uptake machinery that allows skeletal muscle mitochondria to be highly responsive to sarcoplasmic [Ca2+] responses.

Molecular structure and pathophysiological roles of the Mitochondrial Calcium Uniporter.

Mitochondrial Ca(2+) uptake regulates a wide array of cell functions, from stimulation of aerobic metabolism and ATP production in physiological settings, to induction of cell death in pathological conditions. The molecular identity of the Mitochondrial Calcium Uniporter (MCU), the highly selective channel responsible for Ca(2+) entry through the IMM, has been described less than five years ago. Since then, research has been conducted to clarify the modulation of its activity, which relies on the dynamic interaction with regulatory proteins, and its contribution to the pathophysiology of organs and tissues. Particular attention has been placed on characterizing the role of MCU in cardiac and skeletal muscles. In this review we summarize the molecular structure and regulation of the MCU complex in addition to its pathophysiological role, with particular attention to striated muscle tissues. This article is part of a Special Issue entitled: Mitochondrial Channels edited by Pierre Sonveaux, Pierre Maechler and Jean-Claude Martinou.

MICU1 and MICU2 finely tune the mitochondrial Ca2+ uniporter by exerting opposite effects on MCU activity.

Mitochondrial calcium accumulation was recently shown to depend on a complex composed of an inner-membrane channel (MCU and MCUb) and regulatory subunits (MICU1, MCUR1, and EMRE). A fundamental property of MCU is low activity at resting cytosolic Ca(2+) concentrations, preventing deleterious Ca(2+) cycling and organelle overload. Here we demonstrate that these properties are ensured by a regulatory heterodimer composed of two proteins with opposite effects, MICU1 and MICU2, which, both in purified lipid bilayers and in intact cells, stimulate and inhibit MCU activity, respectively. Both MICU1 and MICU2 are regulated by calcium through their EF-hand domains, thus accounting for the sigmoidal response of MCU to [Ca(2+)] in situ and allowing tight physiological control. At low [Ca(2+)], the dominant effect of MICU2 largely shuts down MCU activity; at higher [Ca(2+)], the stimulatory effect of MICU1 allows the prompt response of mitochondria to Ca(2+) signals generated in the cytoplasm.