MICU1’s calcium sensing beyond mitochondrial calcium uptake

Abstract

The discovery of MICU1 as gatekeeper of mitochondrial calcium (_mCa²⁺) entry has transformed our understanding of _mCa²⁺ flux. Recent studies revealed an additional role of MICU1 as a Ca²⁺ sensor at MICOS (mitochondrial contact site and cristae organizing system). MICU1’s presence at MICOS suggests its involvement in coordinating Ca²⁺ signaling and mitochondrial ultrastructure. Besides its role in Ca²⁺ regulation, MICU1 influences cellular signaling pathways including transcription, epigenetic regulation, metabolism, and cell death signaling pathways, thereby affecting human health. Here, we summarize recent findings on MICU1’s canonical and noncanonical functions, and its relevance to human health and diseases.

1. Introduction

Calcium (Ca²⁺) signaling plays a fundamental role in many physiological cellular processes, including signal transduction, cell metabolism and proliferation, synaptic plasticity, muscle contraction, and cell death pathways. Mitochondria are key components in maintaining Ca²⁺ homeostasis and modulating cytoplasmic Ca²⁺ (_cCa²⁺) signaling. Ca²⁺ flux across the inner mitochondrial membrane (IMM) is critical for decoding _cCa²⁺ signals and increasing ATP production to match the energy demand of various cellular processes. While the influx of Ca²⁺ into the mitochondrial matrix (MM) is necessary to sustain cellular bioenergetics, excessive mitochondrial calcium (_mCa²⁺) uptake has detrimental effects including oxidative stress and activation of cell death signaling pathways. Hence, precise regulation of _mCa²⁺flux across the IMM is imperative for cellular physiology and survival.

Ca²⁺ enters the MM via the mitochondrial Ca²⁺ uniporter complex (mtCU). Beyond its role as a conduit for Ca²⁺ entry, the mtCU decodes dynamic _cCa²⁺ signals by reducing _mCa²⁺ uptake under resting conditions and increasing uptake in response to physiological _cCa²⁺ elevation. This dynamic response prevents futile energy usage and _mCa²⁺ overload, while promoting ATP production and signal transmission. Due to the large electrochemical gradient (the driving force for _mCa²⁺ uptake) generated by the electron transport chain (ETC) at the IMM, the mtCU requires a regulatory mechanism to prevent continuous Ca²⁺ uptake and _mCa²⁺ overload. The mtCU complex is composed of several distinct subunits, including a pore-forming subunit called the Mitochondrial Calcium Uniporter (MCU), and several associated subunits which are thought to play various regulatory roles [1, 2]. Of these subunits, Mitochondrial Calcium Uptake 1 (MICU1) acts as a Ca²⁺ sensor for the MCU [3, 4], by setting the threshold for _mCa²⁺ uptake, while concurrently supporting cooperative activation of the MCU as _cCa²⁺ concentrations rise [5]. Thus, MICU1-mediated regulation of _mCa²⁺ uptake is critical for effective _cCa²⁺ signaling and preventing _mCa²⁺ overload.

Recent studies revealed MICU1’s role as a Ca²⁺ sensor at the mitochondrial contact site and cristae organizing system (MICOS) [6], demonstrating its multifaceted impact on mitochondrial function and homeostasis (Figure 1). Beyond participating in mitochondrial calcium dynamics, MICU1 influences diverse cell signaling pathways, including transcription, epigenetic regulation, metabolism, and cell death signaling pathways. Notably, several human diseases, including neurological, muscular, and metabolic disorders are linked to MICU1 mutations (Table 1). Aberrant MICU1 expression is also implicated in tumor progression [7–9], cardiac dysfunction and disease [10, 11], as well as impaired cell differentiation [12, 13] and tissue regeneration [12, 14, 15] (Table 2). Consequently, MICU1 emerges as a key player in the orchestration of _mCa²⁺ dynamics, with critical roles in diverse cellular processes and human disease pathogenesis.

Figure 1. MICU1 regulates mitochondrial ultrastructure and function by interacting with MICOS.

MICU1 is a component of mtCU complex which plays a critical role in regulating _mCa²⁺ homeostasis. At low _cCa²⁺ levels, MICU1 acts as a gatekeeper, preventing MCU channel opening. As _cCa²⁺ levels rise, MICU1 promotes channel opening, allowing influx of Ca²⁺ into the mitochondrial matrix. _mCa²⁺ controls bioenergetics by regulating the production TCA cycle metabolites. The reducing equivalents generated from TCA cycle are delivered to ETC components and energy is produced in the form of ATP. However, excess _mCa²⁺ can lead to MPTP opening, followed by collapse of mitochondrial membrane potential and activation of cell death signaling pathways. Hence, MICU1 plays a crucial role in regulating mitochondrial bioenergetics and cell survival. Beyond its canonical role in maintaining _mCa²⁺ homeostasis, the non-canonical role of MICU1 is to maintain structure and function of cristae junctions (CJ) by associating with components of the mitochondrial contact site and cristae organizing system (MICOS). MICU1 mediated Ca²⁺ sensing at MICOS plays an important role in maintaining cristae structure and function, ultimately regulating mitochondrial ultra-structure and apoptotic cell death signaling. Electron transfer within the ETC can also generate ROS which mediate apoptotic and ferroptotic cell death.

Table 1.

Genetic abnormalities in MICU1 and associated diseases/disorders

S. No	Disease/Disorder/Symptom	Abnormality type	Exon affected/deleted	References
1	Congenital muscular dystrophy	c.386G>C; p.(Arg129Pro)	Exon 3	[35]
2.	Learning disability and delayed motor skills	c.553C>T (p.Q185*)	Exon 4	[39]
3.	Fatigue and lethargy in childhood	Deletion (within a 2,755-base pair deletion)	Exon 1	[34]
4.	Myopathy with extrapyramidal signs (MPXPS)	C.386G > C (R129P) c.161+1G > A	Exon 2	[40]
5.	Myopathy with extrapyramidal signs (MPXPS)	c.1295delA	Exon 13	[42]
6.	Neuromuscular disorder	c.1078-1G>C c.741+1G>A	Exon 10 Exon 8	[33]
7.	Myopathy with extrapyramidal signs (MPXPS)	c.1078-1G>C	Exon 10	[37]

Table 2.

Role of MICU1 in cellular processes in different in vitro and in vivo disease model

S. No	Disease model	Phenotype	MICU1 expression	Reference
1.	Diabetic cardiomyopathy (DCM)	Exacerbated the levels of cardiac hypertrophy and myocardial fibrosis in myocardial microvascular endothelial cells (CMECs) of diabetic mice Loss of MICU1 in db/db mouse hearts contributes to myocardial apoptosis in diabetes, which is restored by MICU1 expression	Reduced expression MICU1 deficiency	[10] [11]
2.	Ovarian cancer	Silencing MICU1 in vitro increases oxygen consumption, decreases lactate production, inhibits clonal growth, migration and invasion of ovarian cancer cells, whereas silencing in vivo inhibits tumor growth, increases cisplatin efficacy and OS miR-195 is under-expressed in ovarian cancer which leads to aberrant MICU1 levels in ovarian cancer	Elevated expression Elevated expression	[7] [9]
3.	Lung regeneration	Reduced AT2-to-AT1 cell differentiation, which is important for lung tissue maintenance and alveolar epithelial regeneration after bacterial pneumonia.	MICU1 deficiency	[14]
4.	Neurodegeneration	Degeneration of motor neurons in the spinal cord and the cortex of KO mouse. Increased cell death in KO neurons and MICU1-deficient patient-derived cells.	MICU1 deficiency	[43]
5.	Bacterial infections	Mice exhibited lower bacterial burdens in the heart with increased survival during systemic S. aureus infection.	MICU1 deficiency	[33]
6.	Muscle Weakness and Wasting	Impaired mCa2+ signaling, energy metabolism, and membrane repair, leading to muscle weakness, fatigue, myofiber damage, and high CK levels in KO mice	MICU1 deficiency	[47]
7.	Myopathy with extrapyramidal signs (MPXPS)	Human fibroblasts lacking MICU1 increases resting mCa2+ uptake and induces mitochondrial fragmentation	MICU1 deficiency	[49]

2. Discovery of MICU1 as the guardian of mitochondrial calcium entry

Perocchi et al. initially identified MICU1 as a crucial regulator of the mtCU [3]. Using HeLa cells, they found that loss of MICU1 impairs _mCa²⁺ uptake in response to elevated _cCa²⁺ concentration. Thus, they proposed that MICU1 plays a significant role in cooperative activation of the mtCU in response to rising _cCa²⁺ levels. MICU1, recognized as a Ca²⁺-binding protein, contains two canonical EF-hand Ca²⁺ binding domains which are crucial for _mCa²⁺ uptake regulation. In a landmark study, Mallilankaraman et al. established MICU1 as the gatekeeper of the mtCU [4]. Loss of MICU1 resulted in constitutive _mCa²⁺ uptake through the MCU, leading to excessive reactive oxygen species (ROS) generation and susceptibility to cell death. The authors concluded that MICU1 sets a threshold (~3μM in most cell types) that inhibits excessive _mCa²⁺ uptake under resting conditions, thereby preventing detrimental effects associated with _mCa²⁺ overload (Figure 2). Subsequently, Csordás et al. confirmed that MICU1 plays a key role in both setting the threshold for _mCa²⁺ uptake and supporting cooperative activation of the MCU [5]. This dual role in regulating _mCa²⁺ uptake is necessary to prevent _mCa²⁺ overload and superfluous energy expenditure, while also ensuring that mitochondria respond effectively to physiological _cCa²⁺ fluctuations.

Figure 2. MICU1 deficiency affects _mCa²⁺ uptake and cristae structure.

(A) Loss of MICU1 causes unregulated MCU channel opening, leading impaired _mCa²⁺ dynamics. This representation of a calcium uptake assay in permeabilized WT and MICU1^−/− cells demonstrates increased Ca²⁺ clearance (bath calcium: outside the mitochondria) after inhibition of SERCA with Thapsigargin (Tg) (1), reduced Ca²⁺ threshold for mtCU channel opening (2), enhanced _cCa²⁺ clearance (3), and increased matrix free Ca²⁺ content (4) compared to WT. (B) MICU1^−/− cells show altered mitochondrial ultra-structure, including increased cristae junction width (1), cristae junction distance (2), and decreased mitochondrial membrane potential (3) compared to WT.

The mechanisms through which MICU1 regulates _mCa²⁺ uptake remain a matter of debate. While several studies demonstrate that MICU1 binds to the core of the mtCU complex, including the MCU [16, 17] and Essential MCU Regulator (EMRE) [17–20], the functional implications of these interactions remain unclear. MICU1 also interacts with its two paralogs, MICU2 (in most cell types) and MICU3 (in neurons) [16], but the role of these proteins has yet to be fully elucidated. Structural and biochemical studies provide evidence for an “occlusion” mechanism, where MICU1 seals off the MCU pore when _cCa²⁺ levels are low, preventing Ca²⁺ from entering the matrix [19–23]. This model posits that as _cCa²⁺ levels rise, Ca²⁺ binds to the EF-hands of MICU1 and MICU2, leading to conformational rearrangement of the MICU proteins which unblocks the pore. Conversely, Garg et al. propose a “potentiation” model where MICU1 enhances the activity of MCU as _cCa²⁺ levels rise [24]. They demonstrate reduced Ca²⁺ currents in MICU1-KO mitochondria under conditions of high Ca²⁺ and suggest that MICU1 increases the open state probability of MCU, thereby potentiating its activity when _cCa²⁺ levels increase. Additional evidence contradicting the “occlusion” model is provided by the observation that MICU1 and MCU have different spatial distributions within mitochondria under resting conditions [25], making it unlikely that MICU1 physically blocks the MCU pore. These discrepancies may stem from variations in mitochondrial preparations, protein constructs, and other experimental conditions used across studies. In addition, the limitations of various techniques employed underscore the need for cautious consideration of alternative interpretations of experimental results. Therefore, further research is warranted to reconcile these conflicting findings and determine whether the “occlusion” model, “potentiation” model, or a modification of these models accurately reflects the regulatory mechanisms of the mtCU.

3. Noncanonical role of MICU1 as calcium sensor at MICOS

Recently, studies have begun to unravel other vital functions of MICU1, which are independent from its role in regulating mtCU-mediated Ca²⁺ uptake. Tufi et al. suggested an mtCU-independent role for MICU1 in Drosophila, where loss of MICU1 is lethal [26]. They postulated that if the lethal phenotype resulted from loss of MCU gating by MICU1 and subsequent _mCa²⁺ overload, abolishing _mCa²⁺ uptake would rescue this phenotype. Deletion of MCU or EMRE successfully abolished _mCa²⁺ uptake and did not impact viability. However, loss of MICU1 in combination with loss of MCU or EMRE did not rescue the lethality of MICU1 deletion. This led to the conclusion that the lethal phenotype in Drosophila with a MICU1 loss-of-function mutation was not due to _mCa²⁺ overload and suggested that MICU1 plays a role in mitochondrial function and survival that is mtCU-independent.

MICU1 was later shown to play a pivotal role in determining mitochondrial ultrastructure, particularly at the cristae junctions (CJ) [25]. This structural aspect is crucial as mitochondrial morphology is intricately linked to its function. The IMM is made up of 2 domains that are physically and functionally distinct: the cristae membrane (CM) and the inner boundary membrane (IBM). The CM includes the IMM invaginations into the matrix, called cristae, which increases the surface area available for the ETC and oxidative phosphorylation. Cristae junctions are bottleneck membrane structures that separate individual cristae from the IBM, restricting ion/metabolites flow and membrane potential generated at the CM. The CJ consist of the MICOS, which is essential for cristae structure and integrity, and consequently, mitochondrial function. MICU1 localizes to the IBM, where it was shown to interact with the MICOS complex and stabilize the CJ [6, 25, 27] (Figure 1). Loss of MICU1 affected CJ, cristae organization [6, 25], and mitochondrial membrane potential dynamics [6, 27] (Figure 2). Moreover, these interactions are independent of the mtCU but require the Ca²⁺-sensing EF-hand motifs of MICU1. This led to the hypothesis that MICU1 acts as a Ca²⁺ sensor for the CJ and MICOS complex. Through interactions with the mtCU and MICOS, MICU1 maintains a balance of bioenergetic processes and cell death signaling. MICU1-regulated _mCa²⁺ flux through the mtCU primarily influences cell death by regulating the mitochondrial permeability transition pore (MPTP) opening [15]. On the other hand, through MICOS, MICU1 controls the release of apoptogens from cristae bottlenecks [6] (Figure 1). These findings provide insight into the lethal phenotype observed in MICU1 knockout mice [15, 28] and fly models [26] The absence of MICU1 can trigger cell death through both necrotic and apoptotic pathways, which may underlie the severe phenotypes linked to MICU1 mutations or genetic loss. This revelation marks a paradigm shift, providing key insights into how Ca²⁺ regulates mitochondrial structure and function, and elucidating cellular signaling mechanisms involved in mitochondrial remodeling across various disease states.

4. MICU1 in cellular signaling

MICU1 has emerged as a key player in diverse cellular processes, providing further evidence for its multifaceted role in cell function and survival. Proper cristae organization and morphology, mediated in part by MICOS, are essential for preventing the release of apoptotic factors. In fact, MICU1 was found to play a crucial role in initiating cell death pathways [6, 15, 25]. Loss of MICU1 in mouse hepatocytes resulted in _mCa²⁺ overload induced MPTP opening, leading to necrosis and impaired hepatocyte regeneration [15]. Furthermore, Tomar et al. demonstrated that loss of MICU1 in HEK293T cells results in an MPTP-independent increase in cytochrome c release, thus priming cells for initiation of apoptosis [6]. MICU1 has also been shown to contribute to lipid peroxidation and ferroptosis in response to cold stress [29]. Similarly, Arvanil, a synthetic capsaicin analog, was found to disrupt MICU1 gating of _mCa²⁺ uptake, leading to excessive _mCa²⁺ accumulation and ferroptosis in hepatocellular carcinoma [30]. Marmolejo-Garza et al. used MICU1-binding compounds MCU-i4 and MCU-i11 to disrupt MICU1 function in human dopaminergic neurons, immortalized mouse hippocampal neurons (HT22 cells), and mouse primary cortical neurons [31]. These manipulations resulted in increased ferroptosis, providing further evidence that MICU1 protects against initiation of various cell death signaling mechanisms. Nemani et al. demonstrated that nutrient stress leads to increased expression of MICU1, reducing _mCa²⁺ uptake in various cell types including HeLa, HEK293T, HepG2 (human liver cancer), primary mouse hepatocytes, and mouse embryonic fibroblasts [32]. Increased MICU1 impairs mitochondrial bioenergetics, prompting autophagy. Therefore, this could be a survival mechanism, limiting bioenergetic crisis during nutrient stress and preventing cell death.

In addition, MICU1 was found to play a key role in cell differentiation through MCU-dependent mechanisms. Lombardi et al. demonstrated that MICU1 expression rapidly increases in response to fibrotic agonists, driving fibroblast to myofibroblast differentiation [12], which is vital for the cellular response to injury and sustaining wound healing. MICU1 overexpression triggers a metabolic shift in fibroblasts, boosting glycolysis and glutaminolysis, thereby increasing α-ketoglutarate (αKG) levels. This rise in αKG induces essential epigenetic modifications and transcriptional switches, facilitating the fibroblast to myofibroblast transition. Similarly, differential regulation of MICU1 was found in tissues from multiple organs of developing mouse embryos [13]. During embryonic development, MICU1 expression was suppressed compared to neonatal developmental stages. Using human induced pluripotent stem cells (iPSCs) and primary neonatal myocytes, they demonstrated that this repression of MICU1 was under the control of transcription factor FOXD1. Moreover, restoration of MICU1 levels promoted cellular differentiation and maturation. These studies establish that MICU1 can be regulated by different signaling cascades, and moreover, plays a key role in critical cellular processes.

5. MICU1 in human health and diseases

The discovery of MICU1 mutations in various human disease conditions provided further evidence of the critical role MICU1 plays in regulating _mCa²⁺ homeostasis (Table 1). Logan et al. described a cohort of children who presented with proximal skeletal muscle weakness, chorea, tremors, ataxia, and learning disabilities [33]. Genetic analysis revealed an autosomal recessive loss-of-function mutation in MICU1. Fibroblasts from these patients demonstrated mitochondrial dysfunction, including an increase in _mCa²⁺ uptake at low _cCa²⁺ concentrations and reduced _cCa²⁺ signaling. Another study described two cousins with homozygous MICU1 loss-of-function mutations who presented with lethargy, fatigue, and muscle weakness [34]. Fibroblasts from these patients also demonstrated impaired _mCa²⁺ handling, which was rescued by overexpression of MICU1. Subsequently, MICU1 mutations were identified in other disorders characterized by developmental delays and neuromuscular impairments. Many of these patients presented with congenital muscular dystrophy [35], myopathy with extrapyramidal signs, learning disabilities and fatigue [36-42]. Notably, these symptoms are consistent with those of other conditions associated with mitochondrial dysfunction and damage.

Furthermore, these symptoms were accompanied by severe encephalopathy and other developmental brain abnormalities in a patient harboring two distinct heterozygous MICU1 mutations [40]. To further investigate the effects of MICU1 deletion on neural and cognitive function, Singh et al. recently created a neuron-specific MICU1 knock-out (KO) mouse [43]. MICU1-KO mice demonstrated abnormal motor and cognitive phenotypes. Neurons from MICU1-KO mice and MICU1-deficient patient-derived cells exhibited susceptibility to _mCa²⁺ overload, excitotoxicity, and cell death. Moreover, loss of MICU1 expression has been reported in the frontal cortex of Alzheimer’s disease patients and mouse models [44]. Taken together, these studies suggest that the loss of MICU1 and subsequent impairments in _mCa²⁺ homeostasis are causally linked to both neuromuscular and cognitive impairments in several human disease conditions.

Beyond its role in neuromuscular and cognitive health, MICU1 has been found to play a role in cardiac physiology and disease. Heart tissue from patients with ischemic heart failure demonstrated elevated mRNA and protein levels for both MICU1 and MICU2 [45]. Paillard et al. found that increased MICU1 expression correlated with decreased cardiac contractile function in these patients. On the other hand, decreased MICU1 expression has been implicated in diabetic cardiomyopathy and myocardial ischemia-reperfusion injury. MICU1 was found to be downregulated in a mouse model of diabetes, contributing to cardiac hypertrophy, fibrosis, and reduced ventricular function [10, 11]. Overexpression of MICU1 in these mice preserved cardiac function and inhibited the development of diabetic cardiomyopathy. MICU1 was also found to be reduced in mitochondria following myocardial ischemia-reperfusion injury in mice [46]. In this context, MICU1 KO exacerbated _mCa²⁺ overload, mitochondrial dysfunction, and myocardial injury.

Further evidence for MICU1’s role in tissue homeostasis and regeneration has been demonstrated in lung, liver, and skeletal muscle. Recently, Ali et al. explored the role MICU1 plays in cell differentiation and regeneration in the lung [14]. They inhibited or deleted MICU1 in mouse alveolar type 2 cells, which play a critical role in lung cell differentiation, epithelial repair, and regeneration. Loss of MICU1 led to decreased cell differentiation, and impaired alveolar epithelial repair and regeneration after lung injury (Table 2). Similarly, mice with a liver specific MICU1 KO demonstrated increased susceptibility to liver injury and impaired liver regeneration [15]. A skeletal muscle specific MICU1 KO mouse resulted in impaired skeletal muscle repair [47]. Notably, these mice exhibited proximal muscle weakness and atrophy, recapitulating the observations in human patients with MICU1 mutations. On the other hand, MICU1 overexpression has been implicated in the development of cancer. MICU1 overexpression has been observed in ovarian cancer cell lines and correlated with poor overall survival [7, 9]. Cancer cells demonstrated chemoresistance and metabolic abnormalities such as increased glycolysis, decreased oxygen consumption, and decreased lactate production. Importantly, these changes were reversed by decreasing MICU1 expression. Mechanistically, Marchi et al. found that phosphorylation of the N-terminal region of MICU1 leads to an increase in basal _mCa²⁺ levels, ROS production, and tumor progression in several different human cancer cell lines [8]. Collectively, these studies provide evidence that altered Ca²⁺ sensing by MICU1 plays a key role in the initiation and/or progression of several human diseases, underscoring the crucial role MICU1 plays in cell physiology and homeostasis.

6. Future perspective and open questions

The regulation of _mCa²⁺ uptake by MICU1 represents a robust and intricate process crucial for fine-tuning _mCa²⁺ homeostasis. Disruptions in this process results in _mCa²⁺ accumulation within the matrix, leading to MPTP opening, subsequent mitochondrial depolarization, and ultimately cell death. Given the detrimental consequences of MICU1 mutations and aberrant expression in various human diseases, investigating MICU1’s role in regulating Ca²⁺-linked mitochondrial processes becomes imperative. In our previous study, we found mtCU independent MICU1 interaction with MICOS components and distinct alterations in mitochondrial morphology and cristae structure in MICU1-deficient cells [6]. However, the precise pathological significance of these alterations requires further characterization. As an essential component of MICOS, the impact of MICU1 deficiency on MICOS assembly remains an open question. Further clarification of MICU1’s role in stabilizing MICOS will provide a deeper understanding of mitochondrial ultrastructure and function. Additionally, more research is needed to resolve the discrepancies in proposed models (occlusion and potentiation) for MICU1’s action on MCU.

Given that MICU1 serves as the gatekeeper for _mCa²⁺ uptake, pharmacological targeting of MICU1 emerges as a possible strategy for situations where fine tuning of _mCa²⁺ uptake threshold is desired. While various compounds targeting different MCU components have been screened to block _mCa²⁺ uptake, only a few compounds specifically targeting MICU1 have been identified (such as MCU-i4 and MCU-i11) [48]. Unfortunately, these compounds prove ineffective at blocking _mCa²⁺ uptake when MICU1 harbors mutations in critical amino acids of the binding cleft. Consequently, there is a pressing need to identify and screen novel compounds capable of achieving fine tuning of _mCa²⁺ uptake, thus preventing pathological _mCa²⁺ overload in disease conditions while maintaining the basal bioenergetic functions.

Highlights.

1. MICU1, a mitochondrial Ca²⁺ sensor, plays a key role in decoding cytoplasmic Ca²⁺ signals.

2. Through the mtCU, MICU1 modulates mitochondrial Ca²⁺ influx.

3. MICU1 functions extend beyond mtCU, impacting mitochondrial ultrastructure via MICOS.

4. Loss of MICU1 leads to bioenergetics failure and increased susceptibility to various cell death pathways.

5. MICU1 mutations are linked to neuromuscular, cognitive, and cardiac impairments.