Ca²⁺ signaling and flux regulate numerous cellular physiological processes, including neuronal excitability, muscle contraction, nuclear gene expression, and mitochondrial integrity, function, and dynamics [27]. Accumulated data have shown that mitochondrial Ca²⁺ content is low under basal conditions, while increases in cytosolic free Ca²⁺ in response to various agents (nutrients, hormones, neurotransmitters, etc.) elevates mitochondrial Ca²⁺ levels [28,29]. This enhances the activity of tricarboxylic acid (TCA) cycle dehydrogenase (pyruvate-, isocitrate-, and α-ketoglutarate dehydrogenase) required for oxidative phosphorylation [30]. Thus, activation of oxidative metabolism via enhanced Ca²⁺ flux can increase the supply of redox cofactors, such as a reduced form of nicotinamide adenine dinucleotide (NAD) and flavin adenine dinucleotide (FAD) to drive ETC and adenosine triphosphate (ATP) synthesis (Fig. 2).

TFAM regulates the mitochondrial membrane potential (ΔΨm) and increases calcium/calmodulin-dependent protein kinase kinase β (CaMKKβ) [10]. In turn, ΔΨm can regulate cellular Ca²⁺ flux [31], while CaMKKβ is activated in a calcium-dependent manner [32]. Thus, TFAM-driven ΔΨm controls Ca²⁺ flux in the cell via various Ca²⁺ channels. Perturbation of TFAM expression cannot protect mtDNA [18], while damaged mtDNA has been reported to increase ER stress [33]. This suggests that TFAM may be involved in the mitochondria-ER interactions.

The lumen of the ER is the main storage site of Ca²⁺ and is constantly refilled via the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) pump [34,35]. When skeletal muscle contraction is initiated, cytosolic Ca²⁺ concentration is rapidly increased by 3 to 4-fold, with the sarcoplasmic reticulum (SR), an ER in skeletal muscle, optimizing the coupling of excitation and contraction of muscle fibers. SR has channels for efflux of Ca²⁺ into the cytosol in response to depolarization, while SERCA transports cytosolic Ca²⁺ in an ATP-dependent manner into the ER lumen, thereby terminating the contraction. The close link between the mitochondria and SR/ER allows for rapid and potent local Ca²⁺ signaling (Fig. 2) [6,36].

Mitochondrial and endoplasmic reticulum contacts (MERC) orchestrate cellular physiological functions such as mitochondrial Ca²⁺ signaling and dynamics. Under physiological stimulation requiring intercommunication between mitochondria and ER, mitofusin 2 (MFN2) tethers to stabilize both organelles [31,37,38]. Meanwhile, inositol 1,4,5-trisphosphate (IP3) binds to the IP3 receptor (IP3R), triggering Ca²⁺ release from the ER through IP3R, which can directly release Ca²⁺ into the cytosol or mitochondria [36]. Released Ca²⁺ from the ER enters the mitochondrial intermembrane space by first passing through the outer mitochondrial membrane via the voltage-dependent anion channel (VDAC) [39], then the mitochondrial calcium uniporter (MCU) transporter across the inner mitochondrial membrane [40,41]. TFAM is linked to Ca²⁺ regulation via MFN2 [42]. Ca²⁺ flux is essential for mitochondrial bioenergetic processes, which is linked to the ΔΨm (Figs. 1 and 2).

Mitochondria are key organelles in Ca²⁺ flux regulation in cells. While Ca²⁺ flux in mitochondria is one of the most significant processes in regulating energy production and communication with other organelles, it mediates various pathologies associated with metabolic disease.

Mitochondrial Ca²⁺ uptake is directly linked to mitochondrial bioenergetics, where depletion of mitochondrial ΔΨm abrogates mitochondrial Ca²⁺ uptake, and defects in the respiratory chain have been linked to the decreased ability of mitochondria to pump Ca²⁺. Energetic substrates derived by the TCA cycle are serially reduced to equivalents by ETC, and these redox reactions are coupled with protons pumping from the matrix into the intermembrane space [43]. The proton electrochemical gradient induced by ΔΨm and pH gradient is necessary to produce ATP (Fig. 1). Thus, ΔΨm can maintain mitochondrial Ca²⁺ uptake and physiological functions (Figs. 1 and 2) [44,45].

Lack of TFAM decreases Serca2a expression from the nucleus [46], indicating that TFAM in the mitochondria signals to the nucleus to transcribe Serca2a and coordinate with ER to maintain Ca²⁺ homeostasis. The ER can regulate the Ca²⁺ efflux channels [36], which could affect cytosolic Ca²⁺ and force during contraction; hence, a reduced ER Ca²⁺ level in TFAM knockout muscle results in a decrease in cytosolic Ca²⁺ [47], which leads to a lower force in single fibers during contraction [47]. In TFAM knockout mice, an increase in mitochondrial Ca²⁺ occurs during repeated contraction [47], which may induce Ca²⁺ overload in the mitochondria and oxidative stress. In contrast with TFAM knockouts, Serca2 gene can enhance contractile function and restore electrical stability in a heart failure model caused by ER(SR) Ca²⁺ leak [48]. Watanabe et al. [46] have shown that TFAM regulates Serca2 gene transcription; however, it is not clear how TFAM in mitochondria regulates nuclear Serca2a transcription (Fig. 3).

As mentioned above, mice with muscle-specific deletion of TFAM exhibit Ca²⁺ overload in the mitochondria, which might be caused by the low Ca²⁺ efflux into the cytosol by the mitochondria. MCU is the main channel transporting Ca²⁺ from the cytosol, and Na+/Ca²⁺ exchanger (NCLX) regulates efflux of Ca²⁺ from mitochondria, both of which are located at the inner mitochondrial membrane where they coordinate to regulate mitochondrial Ca²⁺ homeostasis. Mice with cardiac-specific deletion of TFAM showed how a failure in the coordination between MCU and NCLX in mitochondria can lead to cardiomyopathy [49]. TFAM deletion in cardiac muscle decrease Ca²⁺ efflux from mitochondria due to low NCLX gene and protein levels, with the reduced efflux potentially inducing mitochondrial Ca²⁺ overload [49]. Ca²⁺ flux through the MCU can be regulated by the voltage gradient across the inner mitochondrial membrane, while the opening probability of MCU is decreased based on ΔΨm depolarization [50]. Moreover, protonophore-induced depolarization of ΔΨm almost fully suppresses Ca²⁺ influx into the mitochondria [51]. These findings indicate that the large electrical driving force that arises from the negative potential across the inner ΔΨm is a major factor in regulating the influx of Ca²⁺ through MCU (Fig. 3).

Depolarization of ΔΨm or lack of mtDNA prevents mitochondrial Ca²⁺ uptake, elevating the cytoplasmic Ca²⁺ level and leading to mitochondrial retrograde signaling into the nucleus to activate Ca²⁺-mediated transcription mechanisms involved in calcineurin and Ca²⁺/calmodulin-dependent protein kinase (CaMK) [44,45]. Human TFAM (hTFAM) transgenic mice exhibit mild ΔΨm uncoupling when fatty acids are used as a substrate for skeletal muscle mitochondria, despite mtDNA in skeletal muscle being increased by hTfam [10]. These alterations by hTfam result in an increase in mild uncoupling in ΔΨm, leading to an increase in CaMKKβ expression from the nucleus [10]. Therefore, TFAM regulates ΔΨm and mediates retrograde signaling to the nucleus by Ca²⁺ transmission. Interestingly, mild uncoupling of ΔΨm by hTFAM decreased ROS [10]. Since a lack of TFAM can mediate Ca²⁺ overload in the cytoplasm, it not only induces retrograde signaling to the nucleus but also increases ROS and apoptosis [45]. ROS produced by lack of TFAM in cells may be induced by an overload of Ca²⁺ [45]; however, hTFAM overexpression can decrease ROS and oxidative stress in tissue with mild uncoupling of ΔΨm [10]. Therefore, TFAM may mediate mild uncoupling of ΔΨm that regulates Ca²⁺ retrograde signaling to tightly control cellular ROS (Fig. 3).

The nuclear genome encodes approximately 1,500 proteins that are necessary for mitochondrial function and integrity [52,53]. Intercommunication between mtDNA and the nuclear genome is necessary for mitochondrial biogenesis and normal function. To facilitate the interplay between the organelles, TFAM functions signaling transmitter to the mitochondria from the nucleus, and sends signals back to the nucleus according to the state of the mitochondria by regulating Ca²⁺. This signaling pathway is important for the regulation of normal mitochondrial biogenesis via peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which orchestrates various transcription factors including NRF-1 and peroxisome proliferator-activated receptor delta (PPARδ) (Fig. 3).

Intracellular Ca²⁺ binds CaM, thereby mediating various cellular functions [3,54]. In skeletal muscle, this Ca²⁺/CaM complex contract the muscle fiber [55]. The activation of Ca²⁺/CaMKKβ enhances AMPK activation [32], which facilitates mitochondrial biogenesis via enhanced expression of PGC-1α [56]. Muscle-specific hTFAM transgenic mice exhibit higher levels of CaMKKβ and activated AMPK [10]. To maintain and manage mtDNA as well as to regulate mitochondrial function, it is also necessary to increase the amount of TFAM as the mitochondrial number increases. It has been shown that TFAM overexpression in skeletal muscle enhances the expression of NRF-1, which is an upstream transcription factor for itself [10] and PPARδ expression. PPARδ regulates glucose transporter type 4 (GLUT4) expression and glucose uptake in muscle tissue [57]. A higher level of PGC-1α and PPARδ in tissue is beneficial in improving metabolic diseases such as insulin resistance and T2DM (Figs. 3 and 4).

Skeletal muscle, liver, and adipose are metabolic and insulin-sensitive tissues. Mitochondrial dysfunction in those tissues is associated with lower levels of TFAM in various metabolic diseases such as obesity, insulin resistance, and T2DM, which mediate abnormal Ca²⁺ flux.

Tethering between mitochondria and ER plays a key role in Ca²⁺ homeostasis, which regulates energy metabolism, transportation of lipids, and apoptosis [58]. The influx of Ca²⁺ mediates insulin-stimulated glucose uptake in skeletal muscle [4]. The authors reported that a decrease in Ca²⁺ influx by IP3R inhibition improved insulin-stimulated glucose uptake in skeletal muscle without AKT signaling [4]. In adipocytes, cytosolic Ca²⁺ levels were increased by insulin stimulation [59]. The inhibition of Ca²⁺ signaling in adipocytes [59] and lack of IP3R in primary rat cardiomyocytes [60] decreased GLUT4 translocation and glucose uptake upon insulin stimulation, respectively. Moreover, mitochondrial dysfunction [61], dysregulation of lipid and Ca²⁺ homeostasis [62], and ER stress [63] have been reported to be closely linked to insulin resistance in the liver. Palmitate is known to reduce insulin sensitivity; it has been shown that disruption of the interaction between mitochondria and ER by palmitate induces insulin resistance in human’s and mice’s hepatocytes. However, an increase in these associations between organelles can prevent palmitate-induced insulin resistance [64]. Loss of IP3R1 decreases MERC formation and induces mitochondrial dysfunction and insulin resistance; however, restoration of MERC has been shown to improve palmitate-induced insulin resistance in hepatocytes [64]. It has been reported that AKT phosphorylates the IP3R channel, resulting in decreased Ca²⁺ release capacity through IP3R [65,66]. Moreover, loss of IP3R activity has been shown to change the integrity of MERC [64]. Although defects in MERC have been suggested to play a role in insulin resistance and T2DM [37,38,42], MERC-mediated Ca²⁺ flux for glucose homeostasis is complicated and tightly regulated; hence, other specific mechanisms might be involved in this process. Thus, further studies are required to demonstrate the relationship between insulin resistance and MERC, as well as the mechanisms involved in this process. ER-associated mitochondrial division sites are spatially linked to mitochondrial nucleoids, which suggests a specific role for mitochondria-ER contacts in mtDNA maintenance [67]. ER-mitochondria contacts coordinate mtDNA synthesis with division to distribute newly replicated nucleoids into daughter mitochondria [68]. MFNs, proteins acting between mitochondria and ER [69], are also involved in metabolic diseases. Mitochondrial dysfunction is reportedly caused by depletion and point mutations of mtDNA in mice with muscle-specific deletion of MFN1 and MFN2 [69], while MFN2 expression in skeletal muscle is lower in patients with obesity or T2DM [37]. In contrast, overexpression of MFN2 has been shown to improve diet-induced insulin resistance [70], and MFN2 is necessary for normal glucose homeostasis [38]. Whether TFAM is directly involved in the process of forming or maintaining ER-mitochondria contacts remains to be determined, however, the loss of MFN1 and MFN2 decreases mtDNA that is maintained by TFAM [69], while PGC-1α, a transcription factor for Tfam, can increase the MFN2 expression levels via estrogen-related receptor-α (ERRα) [71]. Moreover, TFAM is involved in packaging mtDNA into a nucleoid [72-74]; hence, it is possible that TFAM indirectly participates in this process of MERC-mediated glucose homeostasis. hTFAM overexpression in skeletal muscle has been reported to prevent high-fat diet-induced insulin resistance along with preserving higher levels of mtDNA [10].

SERCA tightly regulates cytosolic Ca²⁺, leading to glucose oxidation [75]. An increase in SERCA has been reported to improve diabetic cardiomyopathy [7,76]. A high-fat diet decreases Serca2a expression due to a lack of TFAM; however, overexpression of TFAM inhibits hydrogen peroxide-induced decrease of Serca2a mRNA [46]. An increase in SERCA2a by TFAM can reduce levels of cytoplasmic Ca²⁺ and calpain and mitochondrial apoptosis factor, thereby improving glucose uptake in cardiac muscle [77]. Increased expression of Serca improves metabolic syndrome [78].

The MCU complex consists of a pore-forming MCU subunit, a regulatory subunit, and mitochondrial Ca²⁺ uptake proteins 1-3 (MICU1-3), which regulate the channel activity [79-82]. Indeed, a splicing variant of muscle-specific MICU1 with higher Ca²⁺ affinity facilitates mitochondrial Ca²⁺ uptake and ATP production, which is required for muscle contraction [83], whereas a deletion of MCU in skeletal muscle in mice decreases muscle force, indicating that through regulating Ca²⁺ uptake, MCU plays a vital role in energy production for contraction [84]. Deletion of the muscle-specific MCU induced lower activity of pyruvate dehydrogenase (PDH), which is sufficient to shift the preference of the substrate toward fatty acids from carbohydrates [85] since PDH converts pyruvate to acetyl-CoA. In addition, loss of MCU causes defects in Ca²⁺-sensitive TCA cycle enzymes such as isocitrate and α-ketoglutarate dehydrogenases. Therefore, MCU-deleted mitochondria in skeletal muscle cannot sustain respiration without TCA cycle support, although MCU-deleted muscle mostly relies on fatty acids as substrate for mitochondrial respiration [85]. Thus, fatty acids do not completely oxidize and accumulate in muscle, leading to insulin resistance in skeletal muscle [86]. MCU is on the mitochondrial inner membrane that is dependent on ΔΨm, and hTFAM overexpression in skeletal muscle induces mild uncoupling of ΔΨm, which may change mitochondrial Ca²⁺ flux. This alteration shifts the preferred substrate for mitochondrial respiration from glucose to fatty acids [10]. However, the hTFAM transgenic model is different from the MCU deletion since muscle-specific hTFAM overexpression increases glucose uptake and prevents high-fat diet-induced insulin resistance (Figs. 3 and 4) [10].

The NCLX plays a vital role in mitochondrial efflux into the cytosol [87]. A decrease in NCLX function leads to the accumulation of Ca²⁺ in mitochondria, while impaired NCLX function has been reported in diabetic rat hearts [87,88]. A previous study has shown that deletion of TFAM in mouse cardiomyocytes decreases NCLX transcription [49], and TFAM regulates ΔΨm in skeletal muscle; therefore, TFAM seems to clearly link to mitochondrial Ca²⁺ efflux via ΔΨm (Figs. 3 and 4).

Overloaded Ca²⁺ levels have been reported in adipocytes from patients with obesity with insulin resistance [89] and from T2DM rats [90], which may link to the abnormal functions of mitochondria and ER which are unable to maintain Ca²⁺ homeostasis in cells. ΔΨm depolarization enhances ROS production [91-93]; however, hTFAM overexpression prevents fatty acid-induced ΔΨm depolarization in muscle cells and blocks ROS production in mouse skeletal muscle [10]. Moreover, mild ΔΨm uncoupling induced by hTFAM increases glucose uptake in skeletal muscle and improves high-fat diet-induced insulin resistance [10]. Further studies are required to determine how hTFAM regulates Ca²⁺ flux in mitochondria via ΔΨm.