Molecular Characterization and Inhibition of a Novel Stress-Induced Mitochondrial Protecting Role for Misfolded TrkAIII in Human SH-SY5Y Neuroblastoma Cells.

Pediatric neuroblastomas (NBs) are heterogeneous, aggressive, therapy-resistant embryonal tumors that originate from cells of neural crest origin committed to the sympathoadrenal progenitor cell lineage. Stress- and drug-resistance mechanisms drive post-therapeutic relapse and metastatic progression, the characterization and inhibition of which are major goals in improving therapeutic responses. Stress- and drug-resistance mechanisms in NBs include alternative TrkAIII splicing of the neurotrophin receptor tropomyosin-related kinase A (NTRK1/TrkA), which correlates with post-therapeutic relapse and advanced-stage metastatic disease. The TrkAIII receptor variant exerts oncogenic activity in NB models by mechanisms that include stress-induced mitochondrial importation and activation. In this study, we characterize novel targetable and non-targetable participants in this pro-survival mechanism in TrkAIII-expressing SH-SY5Y NB cells, using dithiothreitol (DTT) as an activator and a variety of inhibitors by regular and immunoprecipitation Western blotting of purified mitochondria and IncuCyte cytotoxicity assays. We report that stress-induced TrkAIII misfolding initiates this mechanism, resulting in Grp78, Ca2+-calmodulin, adenosine ribosylating factor (Arf) and Hsp90-regulated mitochondrial importation. TrkAIII imported into inner mitochondrial membranes is cleaved by Omi/high temperature requirement protein A2 (HtrA2) then activated by a mechanism dependent upon calmodulin kinase II (CaMKII), alpha serine/threonine kinase (Akt), mitochondrial Ca2+ uniporter and reactive oxygen species (ROS), involving inhibitory mitochondrial protein tyrosine phosphatase (PTPase) oxidation, resulting in phosphoinositide 3 kinase (PI3K) activation of mitochondrial Akt, which enhances stress resistance. This novel pro-survival function for misfolded TrkAIII mitigates the cytotoxicity of mitochondrial Ca2+ homeostasis disrupted during integrated stress responses, and is prevented by clinically approved Trk and Akt inhibitors and also by inhibitors of 78kDa glucose regulated protein (Grp78), heat shock protein 90 (Hsp90), Ca2+-calmodulin and PI3K. This identifies Grp78, Ca2+-calmodulin, Hsp90, PI3K and Akt as novel targetable participants in this mechanism, in addition to TrkAIII, the inhibition of which has the potential to enhance the stress-induced elimination of TrkAIII-expressing NB cells, with the potential to improve therapeutic outcomes in NBs that exhibit TrkAIII expression and activation.

Distinct Effects of Mitochondrial Na+/Ca2+ Exchanger Inhibition and Ca2+ Uniporter Activation on Ca2+ Sparks and Arrhythmogenesis in Diabetic Rats.

Background Mitochondrial dysfunction contributes to the cardiac remodeling triggered by type 2 diabetes (T2D). Mitochondrial Ca2+ concentration ([Ca2+]m) modulates the oxidative state and cytosolic Ca2+ regulation. Thus, we investigated how T2D affects mitochondrial Ca2+ fluxes, the downstream consequences on myocyte function, and the effects of normalizing mitochondrial Ca2+ transport. Methods and Results We compared myocytes/hearts from transgenic rats with late-onset T2D (rats that develop late-onset T2D due to heterozygous expression of human amylin in the pancreatic ß-cells [HIP] model) and their nondiabetic wild-type (WT) littermates. [Ca2+]m was significantly lower in myocytes from diabetic HIP rats compared with WT cells. Ca2+ extrusion through the mitochondrial Na+/Ca2+ exchanger (mitoNCX) was elevated in HIP versus WT myocytes, particularly at moderate and high [Ca2+]m, while mitochondrial Ca2+ uptake was diminished. Mitochondrial Na+ concentration was comparable in WT and HIP rat myocytes and remained remarkably stable while manipulating mitoNCX activity. Lower [Ca2+]m was associated with oxidative stress, increased sarcoplasmic reticulum Ca2+ leak in the form of Ca2+ sparks, and mitochondrial dysfunction in T2D hearts. MitoNCX inhibition with CGP-37157 reduced oxidative stress, Ca2+ spark frequency, and stress-induced arrhythmias in HIP rat hearts while having no significant effect in WT rats. In contrast, activation of the mitochondrial Ca2+ uniporter with SB-202190 enhanced spontaneous sarcoplasmic reticulum Ca2+ release and had no significant effect on arrhythmias in both WT and HIP rat hearts. Conclusions [Ca2+]m is reduced in myocytes from rats with T2D due to a combination of exacerbated mitochondrial Ca2+ extrusion through mitoNCX and impaired mitochondrial Ca2+ uptake. Partial mitoNCX inhibition limits sarcoplasmic reticulum Ca2+ leak and arrhythmias in T2D hearts, whereas mitochondrial Ca2+ uniporter activation does not.

Targeting the mitochondrial Ca2+ uniporter complex in cardiovascular disease.

Cardiovascular diseases (CVDs), the leading cause of death worldwide, share in common mitochondrial dysfunction, in specific a dysregulation of Ca2+ uptake dynamics through the mitochondrial Ca2+ uniporter (MCU) complex. In particular, Ca2+ uptake regulates the mitochondrial ATP production, mitochondrial dynamics, oxidative stress, and cell death. Therefore, modulating the activity of the MCU complex to regulate Ca2+ uptake, has been suggested as a potential therapeutic approach for the treatment of CVDs. Here, the role and implications of the MCU complex in CVDs are presented, followed by a review of the evidence for MCU complex modulation, genetically and pharmacologically. While most approaches have aimed within the MCU complex for the modulation of the Ca2+ pore channel, the MCU subunit, its intra- and extra- mitochondrial implications, including Ca2+ dynamics, oxidative stress, post-translational modifications, and its repercussions in the cardiac function, highlight that targeting the MCU complex has the translational potential for novel CVDs therapeutics.

Parkin Insufficiency Accentuates High-Fat Diet-Induced Cardiac Remodeling and Contractile Dysfunction Through VDAC1-Mediated Mitochondrial Ca2+ Overload.

Mitochondrial Ca2+ overload contributes to obesity cardiomyopathy, yet mechanisms that directly regulate it remain elusive. The authors investigated the role of Parkin on obesity-induced cardiac remodeling and dysfunction in human hearts and a mouse model of 24-week high-fat diet (HFD) feeding. Parkin knockout aggravated HFD-induced cardiac remodeling and dysfunction, mitochondrial Ca2+ overload, and apoptosis without affecting global metabolism, blood pressure, and aortic stiffness. Parkin deficiency unmasked HFD-induced decline in voltage-dependent anion channel (VDAC) type 1 degradation through the ubiquitin-proteasome system but not other VDAC isoforms or mitochondrial Ca2+ uniporter complex. These data suggest that Parkin-mediated proteolysis of VDAC type 1 is a promising therapeutic target for obesity cardiomyopathy.

Sex-Specific Differences in Endothelial Function Are Driven by Divergent Mitochondrial Ca2+ Handling.

Background Sex-specific differences in vasodilation are mediated in part by differences in cytosolic Ca2+ handling, but how variations in mitochondrial Ca2+ contributes to this effect remains unknown. Here, we investigated the extent to which mitochondrial Ca2+ entry via the MCU (mitochondrial Ca2+ uniporter) drives sex differences in vasoreactivity in resistance arteries. Methods and Results Enhanced vasodilation of mesenteric resistance arteries to acetylcholine (ACh) was reduced to larger extent in female compared with male mice in 2 genetic models of endothelial MCU ablation. Ex vivo Ca2+ imaging of mesenteric arteries with Fura-2AM confirmed higher cytosolic Ca2+ transients triggered by ACh in arteries from female mice versus male mice. MCU inhibition both strongly reduced cytosolic Ca2+ transients and blocked mitochondrial Ca2+ entry. In cultured human aortic endothelial cells, treatment with physiological concentrations of estradiol enhanced cytosolic Ca2+ transients, Ca2+ buffering capacity, and mitochondrial Ca2+ entry in response to ATP or repeat Ca2+ boluses. Further experiments to establish the mechanisms underlying these effects did not reveal significant differences in the expression of MCU subunits, at either the mRNA or protein level. However, estradiol treatment was associated with an increase in mitochondrial mass, mitochondrial fusion, and the mitochondrial membrane potential and reduced mitochondrial superoxide production. Conclusions Our data confirm that mitochondrial function in endothelial cells differs by sex, with female mice having enhanced Ca2+ uptake capacity, and that these differences are attributable to the presence of more mitochondria and a higher mitochondrial membrane potential in female mice rather than differences in composition of the MCU complex.

Mitochondrial calcium uniporter involved in foodborne mycotoxin-induced hepatotoxicity.

Foodborne mycotoxins are toxic metabolites that are produced by fungi. The widespread contamination of food and its by-products by mycotoxins is a global food safety problem that potentially threatens public health and other exposed animals. Most foodborne mycotoxins induce hepatotoxicity. However, only few studies have investigated the regulatory mechanisms of mitochondrial calcium transport monomers in mycotoxin-induced hepatotoxicity. Therefore, according to relevant studies and reports, this review suggests that intracellular Ca(2 +) homeostasis and mitochondrial Ca(2 +) uniporter are involved in the regulation of mycotoxin-induced hepatotoxicity. This review provides some ideas for future research involving mitochondrial Ca(2 +) uniporter in the molecular targets of mycotoxin-induced hepatotoxicity, as well as a reference for the research and development of related drugs and the treatment of related diseases.

Mitochondrial Ca2+ Homeostasis: Emerging Roles and Clinical Significance in Cardiac Remodeling.

Mitochondria are the sites of oxidative metabolism in eukaryotes where the metabolites of sugars, fats, and amino acids are oxidized to harvest energy. Notably, mitochondria store Ca2+ and work in synergy with organelles such as the endoplasmic reticulum and extracellular matrix to control the dynamic balance of Ca2+ concentration in cells. Mitochondria are the vital organelles in heart tissue. Mitochondrial Ca2+ homeostasis is particularly important for maintaining the physiological and pathological mechanisms of the heart. Mitochondrial Ca2+ homeostasis plays a key role in the regulation of cardiac energy metabolism, mechanisms of death, oxygen free radical production, and autophagy. The imbalance of mitochondrial Ca2+ balance is closely associated with cardiac remodeling. The mitochondrial Ca2+ uniporter (mtCU) protein complex is responsible for the uptake and release of mitochondrial Ca2+ and regulation of Ca2+ homeostasis in mitochondria and consequently, in cells. This review summarizes the mechanisms of mitochondrial Ca2+ homeostasis in physiological and pathological cardiac remodeling and the regulatory effects of the mitochondrial calcium regulatory complex on cardiac energy metabolism, cell death, and autophagy, and also provides the theoretical basis for mitochondrial Ca2+ as a novel target for the treatment of cardiovascular diseases.

Asymmetric Opening of Mitochondrial Permeability Transition Pore in Mouse Brain Hemispheres: A Link to the Mitochondrial Calcium Uniporter Complex

Abstract The prolonged entry of large amounts of calcium into the mitochondria through the mitochondrial calcium uniporter complex (MCUC) may cause the permeability transition pore (mPTP) to open, which contributes to the pathogenesis of several diseases. Tissue-specific differences in mPTP opening due to variable expression of MCUC components may contribute to disease outcomes. We designed this study to determine differential mPTP opening in mitochondria isolated from different regions of mouse brain and kidney and to compare it with the expression of MCUC components. mPTP opening was measured using mitochondria isolated from the left/right brain hemispheres (LH/RH, respectively) and from kidney cortex/medulla, while the expression level of MCUC components was assessed from total cellular RNA. Interestingly, LH mitochondria showed less calcium-induced mPTP opening as compared to RH mitochondria at two different calcium concentrations. Conversely, mPTP opening was similar in the renal cortex and renal medulla mitochondria. However, the kidney mitochondria demonstrated bigger and faster mPTP opening as compared to the brain mitochondria. Furthermore, asymmetric mPTP opening in the LH and RH mitochondria was not associated with the expression of MCUC components. In brief, this study demonstrates thus far unreported asymmetric mPTP opening in mouse brain hemispheres that is not associated with the mRNA levels of MCUC components.

[Mitoxantrone Inhibits FMLP-Induced Degenerative Changes in Human Neutrophils].

Neutrophils fight with invading pathogens through various mechanisms including degranulation, phagocytosis, and the release of neutrophil extracellular traps (NETs). This study aimed to determine the impact of a synthetic formyl-peptide (FMLP) on human neutrophils in vitro, and to determine the role of mitoxantrone (MTX), a pharmacological blocker of mitochondrial Ca^(2+) Uniporter (MCU), on FMLP-induced alterations. Isolated neutrophils and a whole-blood preparation of neutrophils were pre-treated with MTX and then stimulated with FMLP. Field's-stained smears and brightfield microscopy were employed for morphological characterization and quantification of neutrophils. The release of cell-free DNA (cfDNA) was also measured for determining neutrophil damage. Our data demonstrated degenerative changes in neutrophils and a greater cfDNA release upon stimulation with FMLP which was negatively associated with the presence of resting platelets in whole blood preparation. Interestingly, MTX pre-treatment significantly reduced FMLP-triggered neutrophil damage and cfDNA release. Metformin, a known inhibitor of NETs formation, also decreased the FMLP-induced changes in neutrophils. In addition to confirming the degenerative potential of FMLP, this study reveals a novel contribution of MCU in regulating FMLP-induced morphological alterations in human neutrophils.

From the Identification to the Dissection of the Physiological Role of the Mitochondrial Calcium Uniporter: An Ongoing Story.

The notion of mitochondria being involved in the decoding and shaping of intracellular Ca2+ signals has been circulating since the end of the 19th century. Despite that, the molecular identity of the channel that mediates Ca2+ ion transport into mitochondria remained elusive for several years. Only in the last decade, the genes and pathways responsible for the mitochondrial uptake of Ca2+ began to be cloned and characterized. The gene coding for the pore-forming unit of the mitochondrial channel was discovered exactly 10 years ago, and its product was called mitochondrial Ca2+ uniporter or MCU. Before that, only one of its regulators, the mitochondria Ca2+ uptake regulator 1, MICU1, has been described in 2010. However, in the following years, the scientific interest in mitochondrial Ca2+ signaling regulation and physiological role has increased. This shortly led to the identification of many of its components, to the description of their 3D structure, and the characterization of the uniporter contribution to tissue physiology and pathology. In this review, we will summarize the most relevant achievements in the history of mitochondrial Ca2+ studies, presenting a chronological overview of the most relevant and landmarking discoveries. Finally, we will explore the impact of mitochondrial Ca2+ signaling in the context of muscle physiology, highlighting the recent advances in understanding the role of the MCU complex in the control of muscle trophism and metabolism.

Structural and Functional Features of Ca2+ Transport Systems in Liver Mitochondria of Rats with Experimental Hyperthyroidism.

We analyzed structural and functional features of the main mitochondrial Ca2+-transporting systems, mitochondrial Ca2+ uniporter complex (MCUC) and Ca2+-dependent cyclosporin A-sensitive mitochondrial permeability transition pore (MPT pore), in rats with hyperthyroid state. It was found that, the rate of Ca2+ accumulation by rat liver mitochondria in this pathology increases by 1.3 times, which can be associated with higher level of the channel-forming subunit of the uniporter MCU and lower content of dominant-negative subunit of this complex MCUb. At the same time, the level of the regulatory subunit MICU1 remained unchanged. It was shown that calcium retention capacity of liver mitochondria in rats with experimental hyperthyroidism decreased by 2 times in comparison with the control, which attested to reduced resistance of liver mitochondria of hyperthyroid rats to induction of the MPT pore. The observed changes are consistent with the data on increased amount of cyclophilin D, a mitochondrial matrix peptidyl-prolyl isomerase that is known to modulate the MPT pore opening and expression of the Ppif gene that encodes mitochondrial cyclophilin D in rats with experimental hyperthyroidism.

The Function of Mitochondrial Calcium Uniporter at the Whole-Cell and Single Mitochondrion Levels in WT, MICU1 KO, and MICU2 KO Cells.

Mitochondrial Ca2+ ([Ca2+]M) uptake through its Ca2+ uniporter (MCU) is central to many cell functions such as bioenergetics, spatiotemporal organization of Ca2+ signals, and apoptosis. MCU activity is regulated by several intrinsic proteins including MICU1, MICU2, and EMRE. While significant details about the role of MICU1, MICU2, and EMRE in MCU function have emerged recently, a key challenge for the future experiments is to investigate how these regulatory proteins modulate mitochondrial Ca2+ influx through MCU in intact cells under pathophysiological conditions. This is further complicated by the fact that several variables affecting MCU function change dynamically as cell functions. To overcome this void, we develop a data-driven model that closely replicates the behavior of MCU under a wide range of cytosolic Ca2+ ([Ca2+]C), [Ca2+]M, and mitochondrial membrane potential values in WT, MICU1 knockout (KO), and MICU2 KO cells at the single mitochondrion and whole-cell levels. The model is extended to investigate how MICU1 or MICU2 KO affect mitochondrial function. Moreover, we show how Ca2+ buffering proteins, the separation between mitochondrion and Ca2+-releasing stores, and the duration of opening of Ca2+-releasing channels affect mitochondrial function under different conditions. Finally, we demonstrate an easy extension of the model to single channel function of MCU.

The pore-forming subunit MCU of the mitochondrial Ca2+ uniporter is required for normal glucose-stimulated insulin secretion in vitro and in vivo in mice.

AIMS/HYPOTHESIS: Mitochondrial oxidative metabolism is central to glucose-stimulated insulin secretion (GSIS). Whether Ca2+ uptake into pancreatic beta cell mitochondria potentiates or antagonises this process is still a matter of debate. Although the mitochondrial Ca2+ importer (MCU) complex is thought to represent the main route for Ca2+ transport across the inner mitochondrial membrane, its role in beta cells has not previously been examined in vivo. METHODS: Here, we inactivated the pore-forming subunit of the MCU, encoded by Mcu, selectively in mouse beta cells using Ins1Cre-mediated recombination. Whole or dissociated pancreatic islets were isolated and used for live beta cell fluorescence imaging of cytosolic or mitochondrial Ca2+ concentration and ATP production in response to increasing glucose concentrations. Electrophysiological recordings were also performed on whole islets. Serum and blood samples were collected to examine oral and i.p. glucose tolerance. RESULTS: Glucose-stimulated mitochondrial Ca2+ accumulation (p< 0.05), ATP production (p< 0.05) and insulin secretion (p< 0.01) were strongly inhibited in beta cell-specific Mcu-null (ßMcu-KO) animals, in vitro, as compared with wild-type (WT) mice. Interestingly, cytosolic Ca2+ concentrations increased (p< 0.001), whereas mitochondrial membrane depolarisation improved in ßMcu-KO animals. ßMcu-KO mice displayed impaired in vivo insulin secretion at 5 min (p< 0.001) but not 15 min post-i.p. injection of glucose, whilst the opposite phenomenon was observed following an oral gavage at 5 min. Unexpectedly, glucose tolerance was improved (p< 0.05) in young ßMcu-KO (<12 weeks), but not in older animals vs WT mice. CONCLUSIONS/INTERPRETATION: MCU is crucial for mitochondrial Ca2+ uptake in pancreatic beta cells and is required for normal GSIS. The apparent compensatory mechanisms that maintain glucose tolerance in ßMcu-KO mice remain to be established.

Mitochondrial Ca2+ Uptake Relieves Palmitate-Induced Cytosolic Ca2+ Overload in MIN6 Cells.

Saturated fatty acids contribute to ß-cell dysfunction in the onset of type 2 diabetes mellitus. Cellular responses to lipotoxicity include oxidative stress, endoplasmic reticulum (ER) stress, and blockage of autophagy. Palmitate induces ER Ca2+ depletion followed by notable store-operated Ca2+ entry. Subsequent elevation of cytosolic Ca2+ can activate undesirable signaling pathways culminating in cell death. Mitochondrial Ca2+ uniporter (MCU) is the major route for Ca2+ uptake into the matrix and couples metabolism with insulin secretion. However, it has been unclear whether mitochondrial Ca2+ uptake plays a protective role or contributes to lipotoxicity. Here, we observed palmitate upregulated MCU protein expression in a mouse clonal ß-cell, MIN6, under normal glucose, but not high glucose medium. Palmitate elevated baseline cytosolic Ca2+ concentration ([Ca2+]i) and reduced depolarization-triggered Ca2+ influx likely due to the inactivation of voltage-gated Ca2+ channels (VGCCs). Targeted reduction of MCU expression using RNA interference abolished mitochondrial superoxide production but exacerbated palmitate-induced [Ca2+]i overload. Consequently, MCU knockdown aggravated blockage of autophagic degradation. In contrast, co-treatment with verapamil, a VGCC inhibitor, prevented palmitate-induced basal [Ca2+]i elevation and defective [Ca2+]i transients. Extracellular Ca2+ chelation as well as VGCC inhibitors effectively rescued autophagy defects and cytotoxicity. These observations suggest enhanced mitochondrial Ca2+ uptake via MCU upregulation is a mechanism by which pancreatic ß-cells are able to alleviate cytosolic Ca2+ overload and its detrimental consequences.

Inhibition of CaMKII in mitochondria preserves endothelial barrier function after irradiation.

Damage to the microvascular endothelium is an important part of normal tissue injury after radiation exposure and driven by the production of pro-oxidants. The Ca2+/calmodulin-dependent protein kinase II is present in the mitochondrial matrix (mitoCaMKII) where it regulates Ca2+ uptake via the mitochondrial Ca2+ uniporter (MCU) and pro-oxidant production. Here, we demonstrate that radiation exposure disrupts endothelial cell barrier integrity in vitro, but can be abrogated by inhibition of mitoCaMKII, MCU, or opening of the mitochondrial transition pore. Scavenging of mitochondrial pro-oxidants with mitoTEMPO before, but not after irradiation, protected barrier function. Furthermore, markers of apoptosis and mitochondrial pro-oxidant production were elevated at 24 h following irradiation and abolished by mitoCaMKII inhibition. Endothelial barrier dysfunction was detected as early as 2 h after irradiation. Despite only mildly impaired mitochondrial respiration, the intracellular ATP levels were significantly reduced 4 h after irradiation and correlated with barrier function. MitoCaMKII inhibition improved intracellular ATP concentrations by increasing glycolysis. Finally, DNA double strand break repair and non-homologous end joining, two major drivers of ATP consumption after irradiation, were greatly increased but not significantly affected by mitoCaMKII inhibition. These findings support the hypothesis that mitoCaMKII activity is linked to mitochondrial pro-oxidant production, reduced ATP production, and loss of endothelial barrier function following irradiation. The inhibition of mitoCaMKII is a promising approach to limiting radiation-induced endothelial injury.

Blockade of MCU-Mediated Ca2+ Uptake Perturbs Lipid Metabolism via PP4-Dependent AMPK Dephosphorylation.

Mitochondrial Ca2+ uniporter (MCU)-mediated Ca2+ uptake promotes the buildup of reducing equivalents that fuel oxidative phosphorylation for cellular metabolism. Although MCU modulates mitochondrial bioenergetics, its function in energy homeostasis in vivo remains elusive. Here we demonstrate that deletion of the Mcu gene in mouse liver (MCUΔhep) and in Danio rerio by CRISPR/Cas9 inhibits mitochondrial Ca2+ (mCa2+) uptake, delays cytosolic Ca2+ (cCa2+) clearance, reduces oxidative phosphorylation, and leads to increased lipid accumulation. Elevated hepatic lipids in MCUΔhep were a direct result of extramitochondrial Ca2+-dependent protein phosphatase-4 (PP4) activity, which dephosphorylates AMPK. Loss of AMPK recapitulates hepatic lipid accumulation without changes in MCU-mediated Ca2+ uptake. Furthermore, reconstitution of active AMPK, or PP4 knockdown, enhances lipid clearance in MCUΔhep hepatocytes. Conversely, gain-of-function MCU promotes rapid mCa2+ uptake, decreases PP4 levels, and reduces hepatic lipid accumulation. Thus, our work uncovers an MCU/PP4/AMPK molecular cascade that links Ca2+ dynamics to hepatic lipid metabolism.

SR-mitochondria communication in adult cardiomyocytes: A close relationship where the Ca2+ has a lot to say.

In adult cardiomyocytes, T-tubules, junctional sarcoplasmic reticulum (jSR), and mitochondria juxtapose each other and form a unique and highly repetitive functional structure along the cell. The close apposition between jSR and mitochondria creates high Ca2+ microdomains at the contact sites, increasing the efficiency of the excitation-contraction-bioenergetics coupling, where the Ca2+ transfer from SR to mitochondria plays a critical role. The SR-mitochondria contacts are established through protein tethers, with mitofusin 2 the most studied SR-mitochondrial "bridge", albeit controversial. Mitochondrial Ca2+ uptake is further optimized with the mitochondrial Ca2+ uniporter preferentially localized in the jSR-mitochondria contact sites and the mitochondrial Na+/Ca2+ exchanger localized away from these sites. Despite all these unique features facilitating the privileged transport of Ca2+ from SR to mitochondria in adult cardiomyocytes, the question remains whether mitochondrial Ca2+ concentrations oscillate in synchronicity with cytosolic Ca2+ transients during heartbeats. Proper Ca2+ transfer controls not only the process of mitochondrial bioenergetics, but also of mitochondria-mediated cell death, autophagy/mitophagy, mitochondrial fusion/fission dynamics, reactive oxygen species generation, and redox signaling, among others. Our review focuses specifically on Ca2+ signaling between SR and mitochondria in adult cardiomyocytes. We discuss the physiological and pathological implications of this SR-mitochondrial Ca2+ signaling, research gaps, and future trends.

Dimerization of MICU Proteins Controls Ca2+ Influx through the Mitochondrial Ca2+ Uniporter.

The mitochondrial Ca2+ uniporter complex (MCUC) is responsible for Ca2+ influx into the mitochondrial matrix, playing critical roles in various mitochondrial functions. Eukaryotic MCUC consists of multiple subunits, and its Ca2+ influx activity is controlled by regulatory subunits, including mitochondrial Ca2+ uptake 1 (MICU1) and its paralogs (MICU2 and MICU3). However, the underlying mechanism remains unclear. Here, we determined multiple crystal structures of MICU2 and MICU3 from Homo sapiens. Our data demonstrate that distinct MICU protein N-domains determine the specific type of MICU dimers that perform the opposing roles in mitochondrial Ca2+ uptake at low cytosolic Ca2+ levels. In contrast, at high cytosolic Ca2+ levels, all MICU proteins undergo dimer rearrangement induced by Ca2+ binding, which releases the suppression of the MCUC pore-forming subunit and promotes the influx of large amounts of Ca2+. Altogether, our results elucidate the delicate mechanism of mitochondrial Ca2+ uptake regulation by MICU proteins.