MCU genetically altered mice suggest how mitochondrial Ca²⁺ regulates metabolism

Abstract

Skeletal muscle has a major impact on total body metabolism and obesity and is characterized by dynamic regulation of substrate utilization. While it is accepted that acute increases in mitochondrial matrix Ca²⁺ increase carbohydrate usage to augment ATP production, recent studies in gene-deleted mice for components of the mitochondrial Ca²⁺ uniporter (MCU) complex have suggested a more complicated regulatory scenario. Indeed, mice deleted for the Mcu gene in muscle, which lack acute mitochondrial Ca²⁺ uptake, have greater fatty acid oxidation and less adiposity. In contrast, mice deleted for the inhibitory Mcub gene in skeletal muscle, which have greater acute mitochondrial Ca²⁺ uptake, antithetically display reduced fatty acid oxidation and progressive obesity. Here we discuss the emerging concept that dynamic fluxing of mitochondrial matrix Ca²⁺ regulates metabolism.

Skeletal muscle exhibits metabolic substrate flexibility during rest and exercise

In adult mammals, skeletal muscle requires approximately 30% of the total body energy expenditure at rest [1]. Energy consumption in skeletal muscle is derived from various substrate pathways, including carbohydrates, fatty acids, and amino acids [2]. During rest, both carbohydrates and fatty acids are important substrates contributing to caloric utilization of mammalian skeletal muscle. However, during low-intensity exercise (25% of maximal O₂ uptake, VO₂ max, see Glossary) fatty acid contributes to more than 80% of overall calories used, while during extreme high-intensity exercise (85% of VO₂ max) the oxidation of carbohydrates, and muscle glycogen becomes the primary substrate for energy production [3,4]. Thus, skeletal muscle is characterized by profound metabolic flexibility in terms of substrate use through a wide range of activity states.

In this review, we summarize the studies on mitochondrial Ca²⁺ dynamics and related metabolic changes, offering a unique perspective as to how long-term mitochondrial matrix Ca²⁺ levels controls substrate selection in response to various metabolic conditions. More specifically, we review data generated in vivo in genetically altered mice for the MCU complex, which suggests a new perspective on mitochondrial Ca²⁺-regulated energy metabolism.

Mitochondrial Ca²⁺ levels contribute to fuel selection

Mitochondria are critical organelles that underlie the majority of energy production in mammals through the formation of adenosine triphosphate (ATP). ATP generation by mitochondria results from the oxidative phosphorylation across the inner mitochondrial membrane due to the H⁺ ion gradient generated by the electron transport chain components (Figure 1). This electron transport process is fueled by the tricarboxylic acid (TCA) cycle driven by select substrate molecules from fatty acid oxidation (FAO), amino acids and glycolysis (generates acetyl coenzyme A, acetyl CoA), which pumps H⁺ ions outside the inner membrane so that the ATP synthase can flow 4 H⁺ ions inward in converting ADP to ATP [5].

Figure 1. Striated muscle Ca²⁺ cycling and excitation-contraction coupling.

In skeletal muscle the action potential from the plasma membrane propagates along the transverse tubule (T-tubule) and directly activates L-type Ca²⁺ channels by mechanical coupling with the ryanodine receptor (RyR) in the sarcoplasmic reticulum (SR) membrane, while in heart there is chemical coupling by Ca²⁺ influx through L-type Ca²⁺ channels that stimulates Ca²⁺ release form over-riding RyR channels, which dumps copious amounts of Ca²⁺ into the cytosol. Elevated cytosolic Ca²⁺ levels trigger muscle contraction by facilitating the binding of Ca²⁺ to the myofilament protein troponin C, and ATP produced by mitochondria enables the power-stroke whereby the myosin heads mechanically pull actin thin filaments. Resting cytosolic Ca²⁺ levels are restored by reuptake into the SR via sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) pump, and a minor role for export of Ca²⁺ out of the cell through the Na⁺/Ca²⁺ exchanger (NCX). Mitochondrial Ca²⁺ influx occurs through the MCU complex and efflux is thought to occur through Na⁺/Ca²⁺/Li⁺ exchanger (NCLX). Acute elevation in mitochondrial matrix Ca²⁺ levels during contraction can enhance the activity of pyruvate dehydrogenase (PDH), malate dehydrogenase (MDH), 2-oxoglutarate dehydrogenase (OGDH), and NADP⁺-dependent isocitrate dehydrogenase (ICDH) complexes, as well as augment the activity of the electron transport chain components. Mitochondria located adjacent to the SR (tethering proteins) experience a much larger effective concentration of Ca²⁺ that can effect MCU influx. Adapted from “Cardiomyocyte Energetics”, by BioRender.com (2024). Retrieved from https://app.biorender.com/biorender-templates.

The physiological role of mitochondrial matrix Ca²⁺ levels has been investigated for decades. In general, acute Ca²⁺ influx activates energy production by stimulating several dehydrogenases involved in the tricarboxylic acid cycle (TCA cycle) and by enhancing the maximal enzymatic rate of ATP synthase [6]. In addition to boosting ATP production, acute mitochondrial Ca²⁺ influx plays a vital role in fuel (substrate) selection. Mitochondrial Ca²⁺ regulates glycolysis via activation of pyruvate dehydrogenase (PDH) that converts pyruvate to acetyl CoA, one of the rate-limiting steps in glucose oxidation [7,8]. The activity of the PDH complex is regulated by pyruvate dehydrogenase kinases (PDKs) and pyruvate dehydrogenase phosphatases (PDPs) [9,10]. Mitochondrial Ca²⁺ directly activates the PDPs, particularly PDP isoform 1, which then dephosphorylates PDH, leading to increased PDH activity and greater conversion of pyruvate to acetyl CoA to increase energy production from carbohydrates. Thus, acute increases in mitochondrial Ca²⁺ influx, such as with initiation of muscle usage and Ca²⁺-dependent contraction, leads to loss of PDH phosphorylation, increases in PDH activity, and greater acetyl CoA production.

While mitochondrial matrix Ca²⁺ regulates glucose metabolism through PDH activation, the direct impact of mitochondrial Ca²⁺ on fatty acid metabolism has not been identified. One hypothesis suggests that the regulation of mitochondrial Ca²⁺ on fatty acid utilization is secondary to its direct effect on glucose metabolism, a phenomenon known as the Randle cycle. The Randle cycle, also known as the glucose-fatty acid cycle, describes the competition between glucose and fatty acids for oxidative metabolism [11,12]. FAO impairs glucose metabolism through the short-term inhibition of glycolytic pathways, including the inhibition of PDH activity as well as 6-phosphofructo-1-kinase [11]. In contrast, FAO can be inhibited via glucose-mediated malonyl CoA regulation. More specifically, malonyl CoA is a highly regulated intermediate metabolite and it is converted from citrate during glucose oxidation, and this increase in malonyl CoA directly inhibits carnitine palmitoyltransferase, which regulates mitochondrial importation of long-chain fatty acids [11,12]. Indeed, mitochondrial matrix Ca²⁺ levels can affect malonyl CoA levels and subsequent FAO [13]. However, it is unclear whether this effect is a direct result of mitochondrial Ca²⁺ binding and changing the activity of an enzyme involved in FAO, or whether its indirect effect through the Randle cycle and effects from altered glycolytic activity.

Muscle Ca²⁺ cycling and energetics during muscle contraction

Ca²⁺ plays an essential role in skeletal muscle contraction through excitation-contraction coupling (E-C coupling) [14] (Figure 1). During rest, the concentration of cytosolic Ca²⁺ is maintained within the low nanomolar (nM) range. However, with contraction intracellular Ca²⁺ increases >10-fold, with even higher levels immediately adjacent to the sarcoplasmic reticulum (SR) where many mitochondria are found in close approximation. Indeed, in skeletal muscle the SR serves as the internal storage compartment for >99% of the Ca²⁺ required for sarcomeric cross-bridge cycling of the myofilament proteins initiated by direct binding to troponin C [19], as well as metabolic coupling with mitochondria [14–16]. During relaxation, cytosolic Ca²⁺ concentration returns to its resting levels by uptake into the SR through the activity of the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) pump, which leads to the dissociation of Ca²⁺ from troponin C and myofilament contractile protein relaxation (Figure 1). Interestingly, mitochondria are thought to be largely passive to Ca²⁺ that fluxes upwards and downwards during contraction and relaxation, although some buffering activity has been reported [14,17]. The duel role of cytosolic Ca²⁺ fluctuations with both muscle contraction and mitochondrial energetics highlights the pivotal role of Ca²⁺ plays as a second messenger on many functional levels in striated muscle (Figure 1) [18].

Mitochondrial Ca²⁺ uptake has been documented to occur rapidly during E-C coupling in skeletal muscle, directly reflecting the magnitude of the Ca²⁺ transient [19], which can then regulate oxidative phosphorylation and ATP production to meet energy demand during bouts of muscle contraction [20] (Figure 2, Key Figure). This process has been termed excitation-metabolism coupling in muscle, whereby muscle oxidative metabolism is based on beat-to-beat Ca²⁺ transients that can be correlated with level of mitochondrial matrix Ca²⁺ [21]. Given these established regulatory paradigms it is predicted that inhibition of mitochondrial Ca²⁺ uniporter (MCU) activity, or deletion of the Mcu gene itself, which is thought to be the primary means of acute Ca²⁺ uptake into mitochondria, would lead to loss of metabolic activity of this organelle and possibly even lethality in null mice (Figure 2, Key Figure). However, Mcu null mice can be viable as adults on the outbred CD1 background [22], and recent genetic studies discussed below have shown no deficit in mitochondrial oxidative-phosphorylation without MCU complex activity, and even a contradictory relationship of greater FAO and loss of fat mass in mice with aging. Moreover, mice that lack a gene that normally restricts Ca²⁺ influx into mitochondria (Mcub) showed less mitochondrial FAO and increased fat mass with aging.

Figure 2. Key Figure. Acute vs long-term Ca²⁺ regulation on substrate use through MCU.

(Left panel), MCU acutely promotes increased mitochondrial matrix Ca²⁺ influx, enhancing PDH activity while decreasing PDK4 levels, and eventually leading to increased glycolysis and glucose oxidation. (Right panel), inhibiting MCU reduces acute mitochondrial Ca²⁺ influx, impairing acute PDH activity while increasing PDK4 levels, leading to impaired glycolysis. However, this reduced mitochondrial Ca²⁺ flux rewires substrate use towards greater fatty acid oxidation (FAO) by reducing malonyl CoA level and relieving inhibition of the carnitine palmitoyltransferase-I (CPT-I) enzyme to allow greater FA influx. This figure was created using BioRender (https://biorender.com/).

Genes regulating mitochondrial matrix Ca²⁺ dynamics

Ca²⁺ enters the mitochondrial matrix through the MCU complex, and in 2010 the first protein directly involved in this complex, mitochondrial Ca²⁺ uptake 1 (MICU1), was discovered [23]. Since then, many genes contributing to the MCU complex have been identified [24–30]. The MCU protein complex is composed of the Mcu Ca²⁺ pore-forming subunit gene, in competition with the inhibitory Mcub gene, which reduces Ca²⁺ influx [24,25,30]. In 2011 the Mcu gene was discovered as an essential component of the MCU complex that directly permeates Ca²⁺ in an acute manner [24,30], while the Mcub gene product, which is 50% similar in sequence to Mcu, serves to inhibit Ca²⁺ influx [25]. Several regulators of the MCU complex have also been identified, including mitochondrial calcium uptake 1/2/3 (MICU1/2/3), MCU regulator 1 (MCUR1) and the essential MCU regulator (EMRE) [23,26–28]. Together, these components regulate the opening of the MCU pore, leading to Ca²⁺ influx into the mitochondrial matrix. Na⁺/Ca²⁺/Li⁺ exchanger (NCLX) is thought to serve as a primary mediator for mitochondrial Ca²⁺ efflux in maintaining overall mitochondrial Ca²⁺ dynamics during the contractile cycle in striated muscle [29].

Acute- versus chronic mitochondrial Ca²⁺ in skeletal muscle energetics

Analysis of human subjects for mitochondrial Ca²⁺ function in skeletal muscle has suggested key relationships with energetics. In human patients, mutations in MICU1 and MICU2 have been identified as causes of muscle fatigue, myopathy, and neurodevelopmental disorders [31–33]. In mice, deleting Micu1 led to neurological and myopathic deficits with decreased muscle strength at 1-month of age [34]. Mice with skeletal muscle-specific Micu1 deletion exhibited muscle fatigue, weakness, and myofiber damage during physical activity [35] (Figure 2). Similarly, mice deleted for Micu3 showed muscle fatigue, impaired exercise capacity, and fiber type switching with more glycolytic capacity in the soleus muscle [36]. MICU3 has also been associated with aging, as the level of MICU3 in aged mice is reduced, leading to decreased ongoing myogenesis and increased apoptosis [37]. Germline Mcu null mice exhibited impaired exercise capacity, as well as isometric and concentric muscle contraction with inhibition of mitochondrial Ca²⁺ uptake [22]. Manipulating MCU expression in skeletal muscle through overexpression/silencing approaches has also suggested that MCU influences muscle size, with a likely role in regulating muscle atrophy [38].

Recent studies have suggested a prominent role for mitochondrial Ca²⁺ in preferentially affecting substrate utilization in skeletal muscle (Figure 2). Germline deleted Mcu mice showed increased PDH phosphorylation and decreased PDH activity in skeletal muscle after 16h starvation [22]. Silencing MCU expression in skeletal muscle resulted in decreased PDH activity, while overexpressing MCU showed no impact [38]. Furthermore, skeletal muscle-specific Mcu deletion rewired substrate utilization towards increased FAO, leading to a decline in whole-body fat mass with aging [39]. Skeletal muscle from Mcu deleted mice had decreased PDH activity and reduced malonyl CoA levels compared with controls following intense exercise [39] (Figure 2). Skeletal muscle from these mice also showed increased oxygen consumption rate in palmitate-supported respiration, suggesting an enhancement in FAO [39]. Elevated PDH phosphorylation was reported to decrease glucose use and increase fatty acid use in skeletal muscle-specific Mcu deleted mice [40]. These authors also observed that overexpression of PDP isoform 2, the Ca²⁺-independent PDH phosphatase, triggered PDH dephosphorylation and shifted the substrate use towards glucose oxidation.

Skeletal muscle specific Mcub deleted mice showed increased whole body fat accumulation with aging [13]. These mice showed reduced mitochondrial Ca²⁺ retention capacity but no deficit in mitochondrial function, although PDH activity was enhanced (reduced phosphorylation) with increased muscle malonyl CoA levels under baseline conditions [13] (Figure 2). Isolated flexor digitorum brevis (FDB) myofibers from these muscle-specific Mcub deleted mice showed enhanced glucose use as well as reduced fatty acid use compared to the controls. Interestingly, obese muscle-specific Mcub deleted mice also showed glucose intolerance, suggesting an onset of a type of metabolic syndrome. With respect to physiologic regulation, mitochondrial dynamic and functional changes are observed in response to nutrient-limited conditions [13,41,42]. Indeed, MCUb expression was the only component of the MCU complex that was transcriptionally regulated during fasting, where presumably the gene product is induced in skeletal muscle to reduce mitochondrial Ca²⁺ fluxing and facilitate metabolic switching towards FAO [13]. Taken together, these results suggest that the MCU complex itself is an important regulator of metabolism that responds to dynamic Ca²⁺ fluxing in skeletal muscle. Moreover, inducible MCUb overexpression in skeletal muscle had the same effect as starvation by increasing FAO, decreasing glucose use, leading to less fat accumulation with aging [13]. The fact that germline Mcu null mice are even viable in the first place, coupled with the results discussed here in genetically altered MCU component mice suggests that a primary function of this uniporter is for fine-tuning metabolism through substrate utilization regulated by the dynamic fluxing of matrix Ca²⁺ [22].

How loss of dynamic mitochondrial matrix Ca²⁺ fluxing might reprogram substrate utilization downstream of genetic modulation of MCU activity is still not clear (Figure 2). However, recent studies observed that mice with genetic alterations in MCU activity have altered PDH activity which is sufficient to trigger preferential substrate use in skeletal muscle [13,40]. PDH activity can be regulated by pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), which inhibits PDH through phosphorylation, thereby contributing to the regulation of glucose oxidation [43]. PDK4 was upregulated in skeletal muscle of both Mcu muscle-specific deleted mice and mice with skeletal muscle specific overexpression of MCUb, again resulting in greater FAO and less fat accumulation with aging. Conversely, PDK4 expression was downregulated in Mcub deleted muscle, which showed the opposite effect of impaired FAO [13]. Indeed, overexpression of PDK4 in muscle of Mcub muscle specific deleted mice showed increased fatty acid use and reversion of substrate use back towards normal [13,40]. Interestingly, PDK4 has been suggested to enhance mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) that directly link mitochondria to SR Ca²⁺ levels, potentially underlying development of select metabolic disorders [44,45]. Further studies are needed to better understand how PDK4 functions through MAMs to control fuel selection in skeletal muscle.

Studies in other tissues have also implicated regulation of mitochondrial Ca²⁺ in affecting preferential fuel selection. For example, inhibition of mitochondrial Ca²⁺ uptake by Mcu gene deletion specifically in the heart increased FAO in response to isoproterenol challenge [46]. Mcu deletion in hepatocytes showed decreased phosphorylation of 5’ AMP-activated protein kinase (AMPK) via extra-mitochondrial Ca²⁺-dependent protein phosphatase-4 (PP4), which causes increased hepatic lipid accumulation [47]. Inhibiting NCLX in astrocytes with CGP-37157, which decreased mitochondrial Ca²⁺ accumulation, showed enhanced glycolysis with increased glucose consumption as well as lactate production [48]. Thus, it appears that mitochondrial Ca²⁺ is involved in regulating substrate use across many tissues (Summarized in Table 1).

Table 1.

Summary of mouse models with altered MCU complex components or presumed Ca²⁺ efflux effectors with effects on substrate usage

Mouse model	Tissue	Phenotype	Enzymatic changes	Reference
Mcu−/−	Global	Exercise capacity ↓ Muscle contraction ↓	p-PDH ↑PDH activity ↓	[22]
AAV-MCU, shMCU	Skeletal muscle	AAV-MCU → fiber size ↑ shMCU → fiber size ↓	AAV-MCU → PDH activity = p-Akt ↑ shMCU → PDH activity ↓ p-Akt ↓	[38]
Mcufl/fl-Myod-Cre, Mcufl/fl -skaMCM	Skeletal muscle	Exercise capacity ↓ Fatigue ↑ Fat Mass ↓	p-PDH/PDH ↑ Malonyl CoA ↓	[39]
Mcufl/fl-Mlc1f-Cre	Skeletal muscle	Fiber size ↓ Type I myofiber ↓ Type IIa myofiber ↑ Glucose use ↓ FA use ↑	p-PDH ↑p-ACC ↑ p-Akt ↓p-GSK3 ↓	[40]
Mcubfl/fl-Myod-Cre, Col1a1MCUb-Myod-Cre	Skeletal muscle	Mcub KO → Fat Mass ↑ Glucose use ↑ FA use ↓ Mcub OE → Fat Mass ↓ Glucose use ↓FA use ↑	Mcub KO → p-PDH/PDH ↓ PDH activity ↑ PDK4 ↓ Malonyl CoA ↑ p-ACC ↓ post fasting Mcub OE → PDK4 ↑	[13]
Mcufl/fl-MCM	Heart	Cardiac function ↑ Glucose oxidation ↑ with insulin FA oxidation ↑ with insulin & ISO	Malonyl CoA ↓ with insulin & ISO Acetyl CoA/CoA ↑ with insulin & ISO Complex I & II ↓ NAD+ ↓ NAD+/NADH ↓ Ac-β-HAD ↑ β-HAD activity ↑ Ac-MCD ↑	[46]
McuΔhep	liver	Body Fat↑ Hepatic lipids↑ Liver TAG ↑ FAO-coupled OCR ↓	ATP ↓ AMP/ATP ↑ p-AMPKα ↓ p-ACC ↓	[47]
NCLX inhibitor CGP-37157	brain	Glycolysis ↑ ATP production ↓ Glucose use ↑	Lactate ↑	[48]

Abbreviations: Ac, acetylation; ACC, acetyl-CoA carboxylase; AMP, adenosine monophosphate; AMPK, AMP-activated protein kinase; ATP, adenosine triphosphate; β-HAD, β-hydroxyacyl CoA dehydrogenase; Col1a1^MCUb, Col1a1 gene with Mcub cDNA inserted under LoxP site control; FA, fatty acid; FAO, fatty acid oxidation; GSK3, glycogen synthase kinase 3; ISO, isoproterenol; KO, knockout; MCD, malonyl CoA decarboxylase; Mcu^Δhep, Mcu gene deleted in hepatocytes of mice; Myod-Cre, muscle-specific Cre expressing mouse line; NAD⁺, nicotinamide adenine dinucleotide; NADH, reduced nicotinamide adenine dinucleotide; OCR, oxygen consumption rate; OE, overexpression; PDH, pyruvate dehydrogenase; PDK4, pyruvate dehydrogenase lipoamide kinase isozyme 4; shMCU, shRNA against MCU; TAG, triglycerides.

MCU-dependent regulation of diabetic cardiomyopathy

Diabetic cardiomyopathy refers to alterations in the structure and function of the myocardium associated with diabetes, independent of other confounding factors such as coronary artery disease (CAD) or hypertension [49]. The heart is an interesting tissue given its ability to use multiple substrates, with a preference for fatty acids. In the diabetic heart it is thought that increased FAO substrate usage over other fuel sources contributes to pathogenesis, including a deficit in glucose utilization [49,50]. Indeed, AT-100 treatment that inhibits aldose reductase and conversion of glucose to sorbitol, also reduced myocardial FAO in vitro, and mitigated diabetic cardiomyopathy with reduced cardiac fibrosis and hypertrophy in vivo [51]. Conversely, impairment of PDH activity has been identified to initiate metabolic inflexibility and contribute to the progress of diabetic cardiomyopathy [52]. Inhibition of FAO and activation of PDH have also been shown to improve hemodynamics in Heart failure with preserved ejection fraction (HFpEF) patients [53,54].

Several MCU components are altered in the diabetic heart; indeed, reductions in MCU and EMRE levels and increases in MCUb have been observed [55]. Restoration of MCU in the diabetic heart improved mitochondrial Ca²⁺ handling with restored mitochondrial Ca²⁺ uptake and cardiac function, as well as restoration of glucose oxidation and reduction in FAO [55]. MCUb induction was observed in cardiomyocytes from type 2 diabetic (T2D) mice with substrate shifting towards FAO [56]. Overexpression of MCUb in the hearts of mice recapitulated aspects of the T2D-like phenotype, while expressing dominant-negative MCUb improved cardiac function [56]. They also showed that MCUb expression was regulated by nuclear receptor co-repressor 2 (Ncor2) through transcriptional repression. Downregulation of MICU1 was observed in the myocardial microvascular endothelial cells of diabetic mice, contributing to the progression of diabetic cardiomyopathy through mitochondrial Ca²⁺ overload-induced endothelial damage and inflammation [57]. Downregulation of MICU1 was also observed in diabetes mutant Lepr^db (db/db) mouse hearts, and overexpressing MICU1 in diabetic db/db mice preserved heart function, reduced cardiac hypertrophy, and prevented the development of diabetic cardiomyopathy [58]. Given the observation that MICU1 potentiates the activity of the MCU complex, further investigation is needed to understand if the effect of MICU1 on diabetic cardiomyopathy is related to mitochondrial Ca²⁺-regulated FAO alterations [59].

Long-term mitochondrial Ca²⁺ regulation and link to obesity

Obesity, characterized by an excess accumulation of body fat, has emerged as a global epidemic affecting individuals across diverse age groups. As per the World Health Organization, over 650 million adults were classified as obese globally in 2016, and in 2020, approximately 39 million children under the age of 5 were either overweight or obese [60]. Accumulation of fat in obesity heightens the risk of various health issues, such as type 2 diabetes and cardiovascular disease [61,62]. Lipid accumulation in skeletal muscle has been linked to insulin resistance and hindered FAO, directly contributing to the onset and progression of type 2 diabetes [63].

Although preventing insulin resistance has been a widely studied therapeutic target for addressing obesity, recent research suggests that targeting FAO could also represent a viable approach in the treatment of obesity [64–68]. Muscle-specific overexpression of carnitine palmitoyltransferase-1 (M-CPT-1), a critical enzyme in fatty acid β-oxidation, has been documented to enhance FAO and ameliorate insulin resistance induced by high-fat treatment [69]. Studies have also suggested that targeting mitochondrial Ca²⁺ dynamics in adipose tissue could be a new approach in preventing obesity. Deleting Mcu in adipose tissue ameliorated high-fat diet-induced fat deposition with increased energy expenditure and thermogenesis [70]. Indeed, MCU-EMRE has been shown to form a thermoporter with uncoupling protein 1 (UCP-1), playing an important role in thermogenesis in brown adipose tissue [71]. Mitochondrial Ca²⁺ homeostasis in brown adipose tissue has also been suggested to play a role in obesity-associated hypertension [72].

The findings on mitochondrial Ca²⁺ regulation of fuel selection in skeletal muscle indicate that targeting mitochondrial Ca²⁺ dynamics in this tissue could be a therapeutic approach to antagonize obesity and related metabolic disorders. It is possible that by driving muscle to use greater levels of fatty acids at rest or with bouts of exercise, systemic metabolism will be benefitted. The open question is if targeting mitochondrial Ca²⁺ dynamic fluxing, such as by inhibiting the MCU complex, will also be safe and effective in attacking obesity. For example, the compound Ru265, which is a cell-permeable MCU inhibitor based on Ru360, has been shown to prevent mitochondrial Ca²⁺ overload induced cell death in mice [73,74]. Drugs like Ru265 could thus represent a novel approach for future investigation in mouse models of obesity or metabolic syndrome disease.

Concluding Remarks and Future Perspectives

Here we discussed the importance of mitochondrial Ca²⁺ regulation in fuel selection in skeletal muscle and its implication in affecting total body metabolism and obesity. Given that skeletal muscle is the largest metabolic reservoir in the body, it would be interesting to investigate if manipulating mitochondrial Ca²⁺ levels independent of the MCU complex would also have an immediate impact on substrate preference and a systemic impact on total energy metabolism. For instance, recent studies showed MICU1 regulates mitochondrial Ca²⁺ dynamics mostly in a MCU-independent manner [75]. Hence, further studies will be needed to understand if MICU1 regulates fuel selection, and whether this occurs through a MCU-dependent or independent manner. Moreover, investigation into the transcriptional regulation of MCU complex genes could suggest the metabolic regulatory pathways that lie upstream of mitochondrial Ca²⁺-mediated fuel selection.

Here we discussed the concept that mitochondrial Ca²⁺-regulated metabolism is more complex than current models originally proposed whereby as acute influx of mitochondrial Ca²⁺ in striated muscle due to contraction immediately impacts ATP production. Rather, emerging data in genetically altered mice indicates a more sophisticated control mechanism wherein mitochondrial Ca²⁺ integrated over long time periods impacts differential substrate usage that then secondarily affects whole-body metabolism. Further studies are needed to investigate the underlying mechanisms whereby fuel selection is directly affected by either acute or chronic mitochondrial Ca²⁺-changes in striated muscle. It will also be important to understand how mitochondrial Ca²⁺ regulation occurs in other tissues and how that compares with substrate preference and total body metabolism. [See Outstanding Questions].

Outstanding Questions.

1. How does Ca²⁺ enter and exit mitochondria during activity periods versus the resting state? Acute increases in mitochondrial matrix Ca²⁺ levels in skeletal muscle parallel the increase in cytosolic Ca²⁺ induced by contractile cycles, but the precise mechanism remains unclear.

2. What are the MCU-independent Ca²⁺ influx pathways regulating mitochondrial Ca²⁺ dynamics? Inhibition or loss of MCU in mice, which blocks essentially all acute mitochondrial Ca²⁺ uptake, mysteriously does not impact baseline levels of mitochondrial matrix Ca²⁺ or oxidative phosphorylation.

3. How do mitochondria and the ER/SR directly communicate within MAMs to regulate substrate use associated with mitochondrial Ca²⁺ levels, especially versus mitochondria that are not associated with MAMs? How these 2 subsets of mitochondria in striated muscle function in facilitating sustained ATP production and how this impacts total body metabolic balance is unknown.

4. How does Ca²⁺ directly impact the process of FAO in skeletal muscle mitochondria? It remains unclear how Ca²⁺ directly impacts mitochondrial fatty acid import and effective oxidation of substrates and if this is direct or through the Randle cycle.

The interplay between the endoplasmic reticulum (ER) or SR and mitochondria, which largely occurs at mitochondria-associated ER membrane (MAM) domains, impacts mitochondrial Ca²⁺ homeostasis and oxidative phosphorylation, as well as regulating a variety of other cytosolic processes [76]. Studies have shown that Seipin, a protein promoting ER Ca²⁺ homeostasis, facilitates the storage of lipids in adipose tissue by augmenting Ca²⁺-dependent mitochondrial metabolism [77]. The reorganization of MAMs in hepatocytes has been recognized in obesity, causing mitochondrial Ca²⁺ overload and subsequently leading to oxidative stress [78]. Chronic disruption of MAMs has been shown to contribute to hepatic metabolic inflexibility and muscle insulin resistance [79,80]. Future studies will be critical to better understand how mitochondria and the ER/SR directly communicate to regulate substrate use associated with mitochondrial Ca²⁺ and why the dynamic fluxing of mitochondrial Ca²⁺ is so critical for proper metabolic balance.

It is also interesting that Mcu or Mcub null mice still present with normal mitochondrial matrix Ca²⁺ levels at baseline (fed state), suggesting the existence of alternative mitochondrial Ca²⁺ uptake channels or exchangers that can compensate for the loss of acute mitochondrial Ca²⁺ influx through the MCU complex [13,22]. Indeed, mitochondria Ca²⁺ overload induced necrosis of myofibers in muscular dystrophy mice was observed to be unaffected by either skeletal muscle specific deletion of Mcu or Mcub, and that another critical Ca²⁺ influx pathway exists that operates in parallel to MCU in setting baseline matrix Ca²⁺ levels in many tissues [81]. Identifying the alternative channels or exchangers that also directly control mitochondrial matrix Ca²⁺ influx or efflux will be important in more fully understanding how mitochondrial Ca²⁺ is regulated during both physiological and pathological conditions and how this impacts metabolism.

Highlights.

• Mitochondria acutely respond to elevations in cytosolic Ca²⁺ levels leading to increases in mitochondrial matrix Ca²⁺ and the increase in oxidative phosphorylation rates.

• Mitochondrial activity and substrate usage within skeletal muscle, which is the largest energy requiring tissue, can impact total body metabolism and obesity.

• Mitochondrial deletion of the MCU complex in skeletal muscle blocks acute matrix Ca²⁺ uptake in genetically altered mice, which paradoxically increases fatty acid oxidation and reduces obesity.

• Deletion of the MCU inhibitor gene Mcub in skeletal muscle mitochondria increases acute matrix Ca²⁺ uptake in genetically altered mice, which decreases fatty acid oxidation and promotes obesity.

Glossary

Acetyl CoA

acetyl coenzyme A, is a molecule derived from carbohydrates, fatty acids, and amino acids oxidation.

ATP

Adenosine triphosphate, a nucleotide that provides chemical energy.

FAO

Fatty acid oxidation, the breakdown of fatty acids to generate acetyl CoA for TCA cycle usage and ATP generation.

Glucose intolerance

a term to describe metabolic conditions that results in higher blood glucose level than normal condition, including prediabetes and diabetes.

Glycolysis

The metabolic pathway that converts glucose into pyruvate.

MAMs

mitochondrial-associated endoplasmic reticulum (ER) membranes.

MCU complex

The protein complex within the mitochondrial inner membrane that facilitates matrix Ca²⁺ uptake.

Mcu gene

Encodes pore-forming subunit of the MCU complex, responsible for acute mitochondrial matrix Ca²⁺ influx.

Mcub gene

Encodes inhibitory subunit of the MCU complex, to reduce Ca²⁺ influx into the mitochondrial matrix.

NCLX

Na⁺/Ca²⁺/Li⁺ exchanger, mediates Na⁺-dependent Ca²⁺ efflux.

PDH

Pyruvate dehydrogenase complex, an enzyme that regulates a rate-limiting step in glycolysis.

PDK4

Pyruvate dehydrogenase lipoamide kinase isozyme 4, an enzyme that phosphorylates PDH to inhibit its activity.

TCA cycle

tricarboxylic acid cycle, also known as the Kreb Cycle, is a cascade of biochemical reactions within mitochondria that ultimately generates ATP from select fuel substrates.

VO₂ max

Maximal oxygen consumption.