Mitochondria and calcium signaling in embryonic development

Seminars in Cell & Developmental Biology 20 (2009) 337–345

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Review

Mitochondria and calcium signaling in embryonic development Xinmin Cao ∗ , Yong Chen Institute of Molecular and Cell Biology, A*STAR (Agency for Science, Technology and Research), 61 Biopolis Drive, Singapore 138673, Singapore

a r t i c l e

i n f o

Article history: Available online 14 January 2009 Keywords: Mitochondria Calcium signaling Heart development NFAT Nkx2-5

a b s t r a c t Calcium (Ca2+ ) is a simple but critical signal for controlling various cellular processes and is especially important in fertilization and embryonic development. The dynamic change of cellular Ca2+ concentration and homeostasis are tightly regulated. Cellular Ca2+ increases by way of Ca2+ influx from extracellular medium and Ca2+ release from cellular stores of the endoplasmic reticulum (ER) and sarcoplasmic reticulum (SR). The elevated Ca2+ is subsequently sequestered by expelling it out of the cell or by pumping back to the ER/SR. Mitochondria function as a power house for energy production via oxidative phosphorylation in most eukaryotes. In addition to this well-known function, mitochondria are also recognized to regulate Ca2+ homeostasis through different mechanisms. Although critical roles of Ca2+ signaling in fertilization and embryonic development are known, the involvement of mitochondria in these processes are not fully understood. This review is focused on the role of mitochondrial respiratory chain complex I in the regulation of Ca2+ signaling pathway and gene expression in embryonic development, especially on the new findings in the cardiac development of Xenopus embryos. The data demonstrate that mitochondria modulate Ca2+ signaling and the Ca2+ -dependent NFAT pathway and its target gene which are essential for embryonic heart development. © 2009 Elsevier Ltd. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondria and cellular Ca2+ signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Cellular regulation of Ca2+ signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mitochondria and ER interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Role of mitochondrial RC in the control of Ca2+ mobilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mitochondria modulate Ca2+ signaling during embryonic development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Mitochondria-mediated Ca2+ signaling in nervous system development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Mitochondria-mediated Ca2+ signaling in heart development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Wnt/Ca2+ signaling pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Ca2+ –calcineurin–NFAT pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3. Role of mitochondria in heart development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4. Mitochondria-mediated Ca2+ –NFAT signaling pathway in heart development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5. Mitochondria-mediated Ca2+ signaling and gene expression in heart development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: OXPHOS, oxidative phosphorylation; RC, respiratory chain; ER, endoplasmic reticulum; SR, sarcoplasmic reticulum; [Ca2+ ]c , cytosolic Ca2+ concentration; [Ca2+ ]ER , Ca2+ concentration in ER; CRAC, Ca2+ release-activated Ca2+ channel; PM, plasma membrane; PLC, phospholipase C; IP3 , inositol 1,4,5-triphosphate; IP3 R, IP3 receptors; SERCA, sarco-endoplasmic reticulum ATPase; TCA, tricarboxylic acid; NFAT, nuclear factor of activated T cell; Nkx2-5, NK2 transcription factor related locus 5; GATAs, GATA binding transcription factors; MEF2, myocyte enhancer factor-2; GRIM-19, genes associated with retinoid-IFN-induced mortality-19; CsA, cyclosporine A; IL, interleukin. ∗ Corresponding author. Tel.: +65 65869657. E-mail address: [email protected] (X. Cao). 1084-9521/$ – see front matter © 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2008.12.014

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1. Introduction Calcium (Ca2+ ) is a ubiquitous intracellular signal that controls various cellular processes including proliferation, transcription, metabolism, contraction, exocytosis, and fertilization [1]. The critical role of Ca2+ signaling was first recognized during fertilization in embryonic development. Sperm entry triggers a single or repetitive Ca2+ waves which cross the egg upon fertilization. This spermtriggered Ca2+ oscillation is crucial for the initiation of embryonic developmental events such as breakdown of the nuclear membrane, mitosis, and cytokinesis [2]. As zygotes progress in division with increasing embryonic cell number, specific and diverse Ca2+ signals occurs both intra- and intercellularly. These intracellular Ca2+ pulses and intercellular Ca2+ waves are involved in axis formation in blastulae, convergent extension movement during gastrulation, and development of various tissues and organs in organogenesis [3]. However, it remains to be fully understood how the Ca2+ signaling is initiated and modulated and which pathways are involved in the Ca2+ -dependent embryonic development. Mitochondria are organelles that control “life and death” of the cell in most eukaryotes. It is well-known that mitochondrial function as a power house for energy production via oxidative phosphorylation (OXPHOS). The mitochondrial respiratory chain (RC) consists of five multi-subunit complexes (complexes I–V) localized in the inner membrane of mitochondria and two additional electron carriers, coenzyme Q10 and cytochrome c. The mitochondrial RC complexes catalyze OXPHOS by transferring two electrons from reducing substrates (NADH-FADH2 ) to molecular oxygen, and generating a proton gradient across the inner mitochondrial membrane, measured as mitochondrial membrane potential. The electrochemical energy of this gradient is then used to drive ATP synthesis by ATP synthase (complex V) [4,5]. Mitochondria are also the place for several key metabolic activities including tricarboxylic acid (TCA) cycle and ␤-oxidation, which provide substrates for ATP production in OXPHOS. In addition to the energy production that is utilized for various cellular processes, in recent years, mitochondria have been recognized to play a key role in the initiation of apoptosis that eliminates cells under both physiological and pathological conditions. Besides these crucial roles, mitochondria are also important for maintenance of cellular calcium homeostasis which is critical for cell survival and embryonic development. This review is focused on the role of mitochondrial RC complex I in the regulation of Ca2+ signaling pathway and gene expression in embryonic development, especially in cardiac development.

2. Mitochondria and cellular Ca2+ signaling Ca2+ functions as an intracellular messenger to regulate various signaling pathways and gene expression [1,6]. In general, a resting cell has Ca2+ concentration of ∼100 nM in the cytoplasm which is much lower than that in the extracellular medium (1 mM) and in the intracellular Ca2+ stores (0.1–0.8 mM) such as endoplasmic reticulum (ER), and sarcoplasmic reticulum (SR) in muscle cells. The cytoplasmic Ca2+ concentration, [Ca2+ ]c , can be raised up to 1 ␮M by Ca2+ release from the ER/SR and Ca2+ influx from the extracellular medium. The elevated [Ca2+ ]c in turn activates Ca2+ -dependent enzymes and controls gene expression. The transient elevated Ca2+ must be subsequently sequestered as prolonged high intracellular Ca2+ causes apoptosis, and the ER/SR needs to be refilled for the Ca2+ storage. This can be achieved by expelling Ca2+ out of the cell or by pumping it back to the ER/SR. The [Ca2+ ]c is therefore determined by a balance between such “on” and “off” reactions [7]. Since the spatiotemporal organization of Ca2+ signaling is crucial for diverse biological processes, it has to be precisely regulated. Mitochondria

are closely localized to the ER/SR and have crucial functions in the regulation of Ca2+ homeostasis via different mechanisms. 2.1. Cellular regulation of Ca2+ signaling In general, increase of [Ca2+ ]c is regulated by channels located at the plasma membrane (PM) which control Ca2+ entry from the extracellular space and channels on the ER and SR membranes which release pre-stored Ca2+ from the ER/SR into the cytosol. The mechanism of how the Ca2+ signaling is regulated in cells is partially illustrated in Fig. 1. The channels on the PM for Ca2+ entry include voltage-operated Ca2+ channels (VOCCs), receptor-operated Ca2+ channels (ROCCs), and Ca2+ release-activated Ca2+ channels (CRACs) [8]. VOCCs are usually activated by PM depolarization and are found mainly in excitable and contracting cells such as neurons and muscle cells [8]. The ROCCs are particularly prevalent on secretory cells and at synapses of neurons, and are activated by a wide variety of agonists such as glutamate, ATP, serotonin, and acetylcholine [8]. The CRACs, on the other hand, are activated in response to depletion of the ER’s Ca2+ store, which links intracellular Ca2+ release with Ca2+ entry from the PM [9,10]. CRACs are probably one of the most ubiquitous PM Ca2+ channels since many different cell types exhibit an enhanced Ca2+ entry following the depletion of intracellular Ca2+ store [9,11,12]. This enhanced Ca2+ entry is crucial for triggering Ca2+ -dependent gene expression. Intracellular Ca2+ release is mediated by several messengeractivated channels such as IP3 receptors (IP3 Rs) that reside primarily on the ER and ryanodine receptors (RyRs) on the SR membranes. As shown in Fig. 1, binding of stimulatory factors to the receptors on the PM leads to the activation of different isoforms of phospholipase C (PLC) that catalyze the production of inositol 1,4,5-triphosphate (IP3 ), which functions as an important second messenger in cells. IP3 subsequently diffuses from the PM to the ER/SR membrane where it binds to its receptor (IP3 R/RyR). IP3 binding causes conformational changes to the IP3 R/RyR, which triggers the opening of the Ca2+ channels to allow the release of Ca2+ from ER/SR stores to the cytoplasm. IP3 activity is also regulated by Ca2+ concentrations. Modest increase of [Ca2+ ]c (0.1–0.3 ␮M) enhances the sensitivity of IP3 R which results in a more rapid rise in the [Ca2+ ]c [13]. However, once the Ca2+ reaches a certain level (>0.3 ␮M), the IP3 R is inhibited to prevent overloading of the Ca2+ in the cytoplasm [13]. In addition to cytosolic Ca2+ , IP3 R/RyR is also sensitive to the Ca2+ level within the ER/SR [14–16]. Decrease of Ca2+ concentration in ER ([Ca2+ ]ER ) inactivates IP3 R, while elevation of [Ca2+ ]ER increases the amplitude of IP3 -triggered Ca2+ oscillations in BHK21 (hamster kidney) cells as well as Xenopus oocytes [16,17]. It has been found that ERp44, a Ca2+ binding protein localized in the ER lumen, directly interacts with and regulates the activity of IP3 R in response to [Ca2+ ]ER [15]. Another small molecule which regulates the opening of the IP3 R from cytosol is ATP. Although ATP alone is insufficient to open IP3 -gated channels, ATP or its nonhydrolyzable analogs increases the frequency of channel opening 4.8-fold in the presence of IP3, and enhances the average duration of channel opening 2.5-fold [18]. In contrast, high concentrations of ATP (>4 mM) decreases the channel activity, presumably by competing with IP3 R for receptor binding [18]. Once Ca2+ fulfills its signaling function, it has to be sequestered to a basal level. [Ca2+ ]c can be removed from the cells via Ca2+ ATPase (PMCA) pumps and Na2+ /Ca2+ exchanger on the PM, or be sequestered by the ER/SR via sarco-endoplasmic reticulum ATPase (SERCA) located on the ER/SR membranes. Since the Ca2+ level within ER modulates IP3 R sensitivity, replenishing the ER with Ca2+ is crucial for the maintenance of the potential for intracellular Ca2+ release. Thus the sequestration process not only prevents Ca2+

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Fig. 1. Model of Ca2+ signaling and mitochondrial function in heart development. Elevation of [Ca2+ ]c is elicited by either Ca2+ influx through Ca2+ channels on PM or Ca2+ release from ER/SR via IP3 receptor (IP3 R) or ryanodine receptor (RyR). The elevated [Ca2+ ]c is subsequently sequestered by expelling Ca2+ out of the cell through PMCA pumps and Na+ /Ca2+ exchangers on the PM or by refilling the ER/SR via SERCAs located on the ER/SR membranes. In heart development, non-canonical Wnt proteins may bind to their G protein-coupled receptor (GPCR), which leads to the activation of PLC and subsequent production of IP3 . IP3 binds to the receptor IP3 R, which triggers Ca2+ release from ER. Depletion of the [Ca2+ ]ER triggers the [Ca2+ ] influx via CRAC, which is crucial for the activation of the Ca2+ - and calmodulin (CaM)-dependent calcineurin. Calcineurin dephosphorylates NFATc and leads to its nuclear translocation where NFATc associates with its co-transcriptional factor, GATA, and activates the expression of downstream gene, Nkx2-5. This Ca2+ signaling is tightly controlled by mitochondria. The close proximity between mitochondria and ER facilitates mitochondrial Ca2+ uptake through uniporter, which activates enzymes of the TCA cycle and promotes ATP production by mitochondrial RC. ATP is in turn exported from mitochondria and consumed by SERCA which pumps Ca2+ back to ER and rebuilds ER Ca2+ stock. This process is crucial for establishing the potential of the IP3 -mediated Ca2+ release. Mitochondria also modulate the opening of CRAC. The mitochondial Ca2+ uptake near CRAC channels attenuates the negative feedback of Ca2+ on CRAC and prolongs the opening of CRAC. The ATP production from subplasmalemmal mitochondria also help to maintain the Ca2+ influx through CRAC and facilitates the subsequent activation of the calcineurin–NFAT signaling.

overloading but also modulates Ca2+ release. Indeed, it has been reported that overexpression of SERCA2b increases the amplitude of the IP3 R-triggered Ca2+ oscillations in Xenopus oocytes [17]. Ca2+ release and sequestration are therefore interdependent processes, both of which are regulated by mitochondrial ATP production. 2.2. Mitochondria and ER interface Mitochondria are often found to be physically in close contact with ER, which exposes mitochondria to an environment with high Ca2+ concentrations near the open mouth of the IP3 R [19]. The local Ca2+ can be taken rapidly into the mitochondrial matrix through a uniporter driven by mitochondrial inner membrane potential [20]. Once Ca2+ enters into mitochondria, it activates several key dehydrogenases in the TCA cycle, thereby increasing NADH level and ATP production [21,22]. Mitochondria in turn modulate Ca2+ release and sequestration via several ways. Firstly, active Ca2+ intake by mitochondria reduces local [Ca2+ ]c around the IP3 R opening sites. Since the local [Ca2+ ]c determines whether opening the Ca2+ channels is either stimulated or inhibited, the rate and level of Ca2+ intake by mitochondria can therefore modulate Ca2+ signaling by increasing or reducing Ca2+ release from ER [13,23]. Secondly, the ATP produced by mitochondria is in turn consumed by SERCA on the neighboring ER for sequestration of Ca2+ into the internal ER stock. Thirdly, after influx, Ca2+ exits mitochondria to the cytoplasm mainly through the Na+ /Ca2+ exchanger. This slow Ca2+ release from mitochondria increases [Ca2+ ]c , which prolongs the effect of the Ca2+ -dependent cellular processes [24,25]. Fourthly, the ATP produced from mitochondria can work as an allosteric regulator of IP3 R opening as mentioned previously. Mitochondria also help to control the Ca2+ influx via CRACs on PM. As CRACs open, the increased cytoplasmic Ca2+ around

CRACs causes inhibition of CRAC channels, a process named Ca2+ dependent slow inactivation of Ca2+ influx [26]. By buffering the flow of the incoming Ca2+ , mitochondria which are close to the CRACs on PM (subplasmalemmal mitochondria) reduce the negative feedback of Ca2+ on CRACs, which prolongs the Ca2+ influx [26]. Furthermore, the ATP production from subplasmalemmal mitochondria is recently reported to antagonize this Ca2+ -dependent inactivation of CRACs [27]. Thus, mitochondria play a crucial role in controlling Ca2+ mobilization through many ways from sequestering intracellular Ca2+ to modulating the activity of CRACs, as well as SERCAs [18,28]. 2.3. Role of mitochondrial RC in the control of Ca2+ mobilization The mitochondrial RC-mediated ATP production has been shown to be coupled with sperm-triggered Ca2+ oscillations in mouse eggs during fertilization by Dumollard et al. [29]. In their study, oscillations of NAD+ and FAD+ (the OXPHOS products from complexes I and II respectively) were observed in the eggs, and the oscillation frequency of NAD+ and FAD+ matched that of Ca2+ [29]. While inhibition of Ca2+ oscillations by a Ca2+ chelator, BAPTA, abolished the FAD+ oscillations, inhibition of OXPHOS by RC inhibitors compromised the sperm-triggered Ca2+ oscillations. These RC inhibitors also caused Ca2+ to leak from ER and impaired Ca2+ homeostasis, probably by disrupting the ATPdependent activity of SERCA [29]. These data suggest that ATP production by mitochondria is crucial for the maintenance of Ca2+ homeostasis and oscillations in mouse eggs. In agreement with these findings, glucagons-like peptide-1 has been shown to mobilize intracellular Ca2+ and stimulate mitochondrial ATP synthesis in mouse pancreatic ␤-cells [30]. Furthermore, mitochondrial depolarization results in an inhibition of IP3 -induced Ca2+ release in

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HeLa cells [31]. Thus coupling of mitochondrial RC function (ATP synthesis) and intracellular Ca2+ mobilization may be a general mechanism for the regulation of Ca2+ signaling in many organisms. Rapid depletion of ATP production in mouse eggs by a mitochondrial uncoupler p-trifluoromethoxy carbonyl cyanide phenyl hydrazone (FCCP) or RC inhibitors disrupts Ca2+ oscillation and results in a sustained elevation of [Ca2+ ]c during fertilization [29,32]. This could be due to an inhibition of Ca2+ sequestration after Ca2+ release from ER. A similar phenomenon was also observed in half of the mouse eggs incubated with medium which lacks the mitochondrial metabolic substrates, during fertilization. However, in the other half of the mouse eggs, the [Ca2+ ]c remained in the base line level with a premature arrest of Ca2+ oscillation, indicating a failure of elicitation of the Ca2+ release rather than a defect in Ca2+ sequestration [29]. In HeLa cells, disruption of the mitochondrial RC subunits by siRNA or treatment with RC inhibitor blocks histaminetriggered intracellular Ca2+ release [31,33]. A similar phenomenon was also observed in the fibroblast cells derived from patients with various mutations in the subunits of RC complex I [34]. Bradykinin stimulates intracellular Ca2+ release in the normal cells. However, eight out of fourteen fibroblast cell lines derived from patients exhibited decreased peak of [Ca2+ ]c following bradykinin treatment [34], suggesting an impaired intracellular Ca2+ release in these mutant cells. Interestingly, a decreased [Ca2+ ]ER was also observed in seven out of 12 of those RC complex I deficient cells [34]. Regression analysis showed a strong positive correlation between the complex I activity and [Ca2+ ]ER, and between [Ca2+ ]ER and peak of the Ca2+ transient [34,35]. These results suggest that a decreased [Ca2+ ]ER could result in a failure of the intracellular Ca2+ release in the RC deficient cells. In such cells, the failure of mitochondrial ATP production could compromise SERCA function and hence decrease [Ca2+ ]ER . The decreased [Ca2+ ]ER inactivates IP3 Rs and impairs the potential of intracellular Ca2+ release. Together, these data suggest that mitochondrial ATP production may play a key role in the regulation of Ca2+ dynamics by affecting both Ca2+ sequestration and Ca2+ release, although the underlying mechanisms are unclear.

3. Mitochondria modulate Ca2+ signaling during embryonic development Different Ca2+ mobilization patterns have been observed throughout embryonic development. The best studied Ca2+ event during embryonic development is the sperm-triggered Ca2+ wave in eggs, which has been studied and reviewed in detail [2,36–40] and hence would not be considered here. During development, embryonic cells are specified into different cell lineage through the activation of specific signaling pathways including the Ca2+ signaling pathway. Ca2+ signaling in dorsalventral axis specification, gastrulation, and organogenesis has been previously reviewed [3]. However, the role of mitochondria in the regulation of Ca2+ signaling in these processes is not very clear. It would be oversimplified to say that mitochondria are just passively involved in maintaining Ca2+ mobilization. Mitochondria are very dynamic organelles that constantly undergo fission and fusion, and also travel along the microtubules in the cells [41,42]. Active changes in the mitochondrial translocation and function were observed during oocyte maturation, fertilization, and embryonic development [33,36,43,44]. The spatiotemporal arrangements of mitochondria may be crucial for the regulation of the Ca2+ signaling during embryonic development. Here, we will focus on the roles of mitochondria-mediated Ca2+ signaling in the development of nerves and heart, especially pertaining to the new findings in the latter process.

3.1. Mitochondria-mediated Ca2+ signaling in nervous system development Ca2+ signaling is involved in organogenesis especially in the formation of organs rich in mitochondria, including kidney, muscle, heart, and nervous system. Development of the nervous system involves multiple steps. Firstly, the differentiation of the neural cells is triggered by induction signals in neural ectoderm. Subsequently, during neuronal differentiation, the neurites from a single neuron extend and differentiate into axon and dendrites to establish neuronal polarity. Lastly, the outgrowing axon and dendrites of individual neurons connect to other neurons through synapse and form the network of nervous system. It has been shown that Ca2+ signaling plays pivotal roles in each of these steps in the development of the nervous system. Just prior to the onset of neural induction, specific Ca2+ elevation was observed in the anterior dorsal ectoderm of Xenopus where the future neural plate forms [45]. Blockade of the Ca2+ transient reduced the expression of early neural genes, such as geminin and Zic3, and resulted in a defect in the anterior nervous system. Similarly, in zebrafish, treatment of blastomeres in 512 cell stage embryos with BAPTA, a Ca2+ chelator, decreased motoneuron formation in spinal cord [46]. Both Ca2+ spike and wave were observed in the developing neuron from spinal cord of Xenopus, and it has been shown that the Ca2+ spike promotes the expression of neurotransmitters, whereas Ca2+ wave at growth cones regulates neurite extension [47]. Furthermore, suppression of Ca2+ transient by BAPTA speeds up axon growth, while stimulation of Ca2+ transient slows axon extension [48]. Thus, a subcellular domain containing low local Ca2+ favors axon formation and helps to establish the neural polarity. It has been found that mitochondria play a role in reducing intracellular static Ca2+ and regulating axon outgrowth. In rat hippocampal neurons, mitochondria accumulate at the base of developing axons where cytoplasmic free Ca2+ is low [49]. Treatment of the hippocampal cells with ethidium bromide, an agent which disrupts mitochondrial DNA, increased intracellular static Ca2+ level and inhibited axon formation [49]. During development of the nerve network, spontaneous rhythmic Ca2+ waves are often recorded in a wide population of neurons in the embryonic brain and spinal cord [50,51]. These large-scale Ca2+ waves are associated with the electrical activity waves of neurons in the developing central nerve system (CNS). The electrical activity is characterized by membrane depolarization and is driven by synaptic transmission. Stimulation of the neuronal depolarization evokes Ca2+ waves in the embryonic CNS, whereas neurotransmitter antagonist blocks spontaneous Ca2+ waves during CNS development [51,52]. This neuronal activity-dependent Ca2+ transient is crucial for the activation of the Ca2+ -dependent enzymes and the maturation of the nerve network and synapse refinement [47,53,54]. Interestingly, this activity-dependent Ca2+ transient is found to be coupled with mitochondrial OXPHOS. In embryonic Purkinje neurons, depolarization triggers rapid intracellular Ca2+ elevation and a burst of mitochondrial oxygen consumption within 0.2 s after neuron depolarization [55]. This increased mitochondrial oxygen consumption can be completely blocked by BAPTA, which suggested that the mitochondrial OXPHOS is triggered by the activity-dependent Ca2+ transient. Although the role of this transiently activated OXPHOS is still unclear, given the crucial role of the mitochondrial ATP production in the regulation of the Ca2+ release and sequestration, mitochondria may help in shaping Ca2+ transient and subsequent Ca2+ -dependent neuronal development. In addition to this electrical activity-dependent Ca2+ signaling, an activity-independent Ca2+ oscillation was also visualized in the ventral spinal neurons of mouse embryos [56]. In contrast to the activity-dependent Ca2+ transients, such oscillatory Ca2+ transients are resistant to the blockade of membrane

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depolarization and synaptic activity. Interestingly, disruption of mitochondrial membrane potential by uncoupler carbonyl cyanide 3-chlorophenylhydrazone (CCCP) completely suppresses the Ca2+ transient. CGP-37157, a specific inhibitor of the mitochondrial Na+ /Ca2+ exchanger, also blocks this activity-independent Ca2+ oscillation [56]. Thus mitochondrial function is crucial for the maintenance of this activity-independent Ca2+ oscillation. Although the function of the activity-independent Ca2+ transient is unclear, given the synchronous activation pattern in a discrete cluster of interneurons, it has been suggested that the spontaneous activity-independent Ca2+ oscillation could function in guiding the development of synaptic connections [56]. 3.2. Mitochondria-mediated Ca2+ signaling in heart development The heart is the first definitive organ to be formed in the embryos and is essential for embryonic survival. Heart development in vertebrate embryos is initiated during gastrulation when the anterior lateral mesoderm becomes specified to a cardiac fate in response to signals from adjacent endoderm [57–59]. These signals cause the cardiac precursor cells to express a set of transcription factors that cooperate with each other to establish a positive cross-regulation network and start the cardiogenic programme by regulating their downstream cardiac gene expression [60]. The cardiac precursor cells form a linear heart tube after specification and undergo looping, segmentation, valve formation, and eventual generation of a multi-chambered heart [61,62]. 3.2.1. Wnt/Ca2+ signaling pathway Four classes of growth factors, including Wnts, activin/Nodal, bone morphogenetic proteins (BMPs), and fibroblast growth factors (FGFs), have been characterized as early signals for the cardiac specification [58,63–67]. The Wnt protein family comprises two functional groups, the canonical and non-canonical Wnts and plays a particularly complicated role in the cardiac differentiation. In general, Wnt-3a and Wnt-8, acting through a canonical ␤-catenin signaling pathway, repress cardiogenesis by inhibiting cardiac gene expression [68]. However, a recent report showed a biphasic effect of canonical Wnts on cardiac induction in mouse embryonic stem (ES) cells, whereby Wnt-3a increases the cardiac differentiation in the early stage but blocks this process at a later stage [69]. In contrast to the canonical Wnts, Wnt-11 acting via a non-canonical Wnt pathway, also called Wnt/Ca2+ pathway, stimulates cardiogenesis [63,65]. It has been shown that Ca2+ signaling plays a pivotal role in embryonic heart development. In zebrafish, high-amplitude Ca2+ spikes were observed in developing heart [70]. Injection of BAPTA buffer into embryos at one cell stage compromised heart formation, as shown by formation of non-functional hearts with small atria and ventricles [70]. Although it is unclear how the Ca2+ signaling is elicited during cardiac development, the Wnt/Ca2+ pathway is one of the possible upstream signaling pathways involved. The non-canonical Wnts bind to their receptors and activate IP3 -Ca2+ signaling which results in the release of intracellular Ca2+ and activation of Ca2+ -sensitive enzymes [71]. In agreement with this hypothesis, Wnt-11 is expressed in precardiac mesoderm in Xenopus, mouse, and avian embryos [63,64,72]. Furthermore, inhibition of Wnt-11 expression in Xenopus reduced the expression of cardiac marker Nkx2-5 and compromised heart development. In contrast, treatment of the murine embryonic carcinoma stem cell line P19 with Wnt-11 conditioned medium elicited cardiogenesis [65]. This Wnt-11-dependent heart development was found to be mediated by activation of protein kinase C (PKC) and Jun amino-terminal kinase (JNK), but not Ca2+ -CamKII [65]. Although it has not been directly demonstrated that Wnt-11 stimulates cardiogenesis via

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IP3 -Ca2+ signaling [52], another non-canonical Wnt member, Wnt5a, has been reported to stimulate the release of intracellular Ca2+ in zebrafish embryos [73]. Furthermore, in Xenopus embryos, Wnt5A also induces the activation of nuclear factor of activated T cells (NFAT), which is a Ca2+ activated-transcription factor family important for heart development [74].

3.2.2. Ca2+ –calcineurin–NFAT pathway The mouse NFAT family comprises five members including four cytosolic proteins (NFATc1–4) and a constitutive nuclear phosphoprotein (NFAT5) [75]. Increased intracellular Ca2+ regulates many Ca2+ -dependent cellular processes. The Ca2+ /calmodulindependent protein serine/threonine phosphatase, calcineurin, is one of the major targets activated by Ca2+ [76]. Activated calcineurin dephosphorylates the cytoplasmic NFATs (NFATc) and induces their nuclear translocation. In the nucleus, NFATc associates with other transcription factors and form a transcription complex to activate gene expression [77–79]. The nuclear NFATc can be rapidly phosphorylated by various kinases including GSK3, and CK1 and exported out of the nucleus [80,81]. Thus, a sustained Ca2+ signal is needed to maintain NFATc activity. It has been shown that activation of NFAT requires a high frequency of Ca2+ oscillation [82]. This high frequency of Ca2+ release depletes the intracellular Ca2+ store and triggers the opening of CRAC on the PM. This results in a prolonged Ca2+ entry that is crucial for the activation of NFATc [83]. Mutant lymphocytes with CRAC deficiency fail to promote Ca2+ through CRAC channel, resulting in a rapid transient Ca2+ release from intracellular store. NFATs are not maintained in the nucleus of the mutant cells, hence, are unable to activate the NFAT-dependent transcription of immune response genes [84]. A similar CRAC defect has also been found in patients with severe combined immunodeficiency (SCID). These patients fail to activate NFAT-regulated genes such as IL-2, IL-4 and CD40 ligand, probably due to impaired sustained Ca2+ signal [85]. Although NFAT was first identified as an important regulator in immune responses [86], it has also been reported to be involved in multiple biological processes including cardiac development in embryos and cardiac hypertrophy and heart failure in adult [79]. Deletion of NFATc1 in mice caused heart valve and septal defects which led to embryonic lethality by E13.5 due to cardiac failure [87,88]. Double knockout of NFATc3 and NAFTc4 in mice results in defects in myocardium development and vasculature patterning, and these mice die around E10.5 [89,90]. Treatment of mouse or chicken embryos with a calcineurin inhibitor, cyclosporine A (CsA), gave rise to a phenotype similar to that observed in the NFATc3/c4 null mice [90,91]. On the other hand, calcineurin/NFAT pathway may not be required for the earliest events of heart formation in these vertebrates. Mouse embryos lacking three (NFATc2–c4) of four NFATc, exhibited normal heart tube formation [92], and CsA treatment at the early stage of mouse or chicken embryos did not inhibit heart tube formation or cardiomyocyte differentiation [90,91]. The Ca2+ –calcineurin–NFAT pathway is also regulated by various intracellular Ca2+ modulators. Gene targeting of the mouse calreticulin, encoding an ER Ca2+ binding protein, inhibited the bradykinin-induced intracellular Ca2+ release and the nuclear import of NFATc [93]. The calreticulin-deficient mice exhibited a defect in the development of ventricular myocardium and embryonic lethality [93]. However, this embryonic death can be rescued by the cardiac-specific expression of a constitutively active calcineurin. This suggests that calreticulin regulates Ca2+ signaling and Ca2+ -mediated calcineurin–NFAT activities in myocardium development [94].

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Fig. 2. Mitochondrial RC regulates heart development through activation of the Ca2+ -dependent NFAT signaling pathway. (A) Knockdown (KD) of GRIM-19 results in heart defect in Xenopus embryos. (B) KD of GRIM-19 or NDUFS3 by siRNA in HeLa cells impairs histamine-triggered intracellular Ca2+ release. Arrowhead: histamine treatment; green arrow: sustained Ca2+ elevation. (C) KD of GRIM-19 decreases Nkx2-5 gene expression in the heart area of Xenopus embryos. Constitutively activated NFATc4 (CANFAT) can rescue the defect of the Nkx2-5 expression in the GRIM-19 KD embryos. The figures are adapted from [33] (Copyright ©American Society for Microbiology).

3.2.3. Role of mitochondria in heart development Mitochondrial DNA (mtDNA) encodes 13 subunits of the RC complexes. Mitochondrial transcription factor A (TFAM) regulates both mtDNA replication and transcription. Complete depletion of TFAM in mice resulted in a severe RC dysfunction and caused embryonic lethality between E8.5 and E10.5 without formation of the cardiac structure [95]. Similarly, knockout of a negative regulator of mtDNA transcription, MTERF3, in mice also compromised the RC function and caused embryonic death in midgestation (E8.5) [96]. A mouse strain with cardiac-specific knockout of either TFAM or MTERF3 has been generated by mating TfamloxP mice with myhca (␣-myosin heavy chain)-cre mice or mating Mtefr3loxP mice with Ckmm (muscle creatinine kinase)-cre mice [96,97]. These conditional knockout mice displayed compromised cardiac function and cardiomyopathy, a same phenotype seen in many human mitochondrial diseases [95,96]. These data demonstrate crucial roles of mitochondria in the mature heart function. Since myhca is expressed after E8.5 in atria of the embryonic hearts and Ckmm is expressed after E13, the role of mitochondrial RC in early heart development before E8.5 remains unknown. 3.2.4. Mitochondria-mediated Ca2+ –NFAT signaling pathway in heart development Although it is well-known that both Ca2+ signaling and mitochondria play crucial roles in embryonic development, direct evidence indicating that mitochondria control embryonic heart development via regulation of Ca2+ signaling was lacking. GRIM19 (genes associated with retinoid-IFN-induced mortality-19) was used as a tool to address the role of mitochondrial complex I in embryonic development. GRIM-19 was originally identified as a death-related gene in cancer cells [98] and subsequently found to co-purify with mitochondrial RC complex I [99]. By gene targeting in mice, we have demonstrated that GRIM-19 is essential for the assembly and electron transfer activity of RC complex I [100]. GRIM19 also plays a pivotal role in the maintenance of mitochondrial membrane potential [101]. Therefore, GRIM-19 is a newly identified functional subunit of RC complex I. Knockout of GRIM-19 in mice caused retarded embryonic development and embryonic lethality between E8.5 and E9.5 [100]. To further study the mitochondrial function in embryonic development, we knocked down GRIM-19 in Xenopus. The GRIM-19 knockdown (KD) embryos exhibited retarded growth, extensive

neuronal death, and abnormal morphology of the hindbrain, eyes, and skeletal muscles [33]. Strikingly, the most severe defect occurred in heart development. 70% of the GRIM-19 KD embryos showed no heart formation, and more than 20% developed abnormal hearts. In comparison to well-developed hearts with intact ventricles and atria in control embryos at stage 45, most GRIM19 KD embryos displayed heart tubes in the heart region (Fig. 2A). Similar phenotypes were observed in the embryos incubated with a complex I inhibitor, rotenone. Interestingly, knockdown of GRIM19 inhibited NFAT activity in both whole embryos and the cardiac region, and expression of a constitutively active form of mouse NFATc4 in these embryos rescued the heart defects. Furthermore, both the Ca2+ response to histamine (Fig. 2B), as well as Ca2+ induced NFAT activity, were impaired in the cells with knockdown of either GRIM-19 or NDUFS3, another subunit of complex I [33]. These results provide genetic evidence that mitochondrial RC function in heart development by regulating Ca2+ -NFAT signaling. 3.2.5. Mitochondria-mediated Ca2+ signaling and gene expression in heart development Heart development in vertebrates is initiated from mesoderm when it receives signals from adjacent tissues [57–59]. Upon induction, the cardiac precursor cells express a core set of evolutionarily conserved transcription factors including the homeobox protein NK2, the GATA family of zinc finger proteins, the MADS-box protein MEF2, Tbx, and Hand. These transcription factors interact with each other and with other developmentally controlled genes to establish a complex regulatory network and start the cardiogenic programme through spatiotemporal regulation of their downstream genes for muscle growth, patterning, and contractility [60,102]. Nkx2-5 (NK2transcription factor related, locus 5) is a homeoboxcontaining transcription factor and one of the earliest genes expressed in the cardiac progenitor cells upon induction [103,104]. In Drosophila, the Nkx2-5 homolog, tinman, plays an essential early role in the specification of the cardiac lineage [105]. In mice, targeted disruption of Nkx2-5 caused embryonic lethality at embryonic day 9.5–11.5 due to failure of heart tube looping and decrease in the expression of a subset of cardiac musclespecific genes [106,107]. In contrast, overexpression of Nkx2-5, initiates cardiac differentiation in P19 embryonic carcinoma stem cells [108], and results in an enlarged heart in Xenopus and zebrafish [109]. Therefore, Nkx2-5 is one of the essential genes for the car-

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diomyogenesis and heart development. The relatively late cardiac defects in the Nkx2-5 knockout mice could be due to a functional substitution by the other NK2 homeodomain proteins or transcription factors. In human, Nkx2-5 is the most commonly mutated single gene in congenital heart disease [110], and is reported to be significantly upregulated in patients with hypertrophic cardiomyopathy [111]. Studies on the Nkx2-5 transcriptional regulation identified two cardiac enhancers in a 10 kb 5 flanking sequence of Nkx2-5 gene [112–115]. Several cardiac transcription factors including GATA family and Smad proteins, which are mediators of TGF-␤ signaling, have been found to bind to the consensus site in these enhancers and regulate Nkx2-5 gene expression [116–118]. Although the NFAT functions in heart development have been known, the mechanism involved is not fully understood. NFAT was reported to regulate transcription of myocardium gene cTnl and cTnT between E14 and P0 [119]. However, knockout of NFATc3 and NFATc4 caused embryonic death at E10.5 [89]. Thus, NFATc must also regulate other early genes. Recently, we found that knockdown of GRIM-19 in Xenopus embryos inhibited Nkx2-5 expression, but this impaired expression can be rescued by a constitutively activated NFATc4 (Fig. 2C), suggesting that Nkx2-5 is a downstream gene of NFAT in the early heart development in Xenopus [33]. This has also been confirmed in mouse embryonic stem (ES) cells and embryonic carcinoma cell line P19 during cardiac differentiation [120]. Furthermore, six core NFAT binding sites were found in the 5 regulatory region of Nkx2-5 gene, and NFAT activates Nkx2-5 transcription via a specific binding site in combination with the co-factor GATA, which binds to a site adjacent to the NFAT-binding site [120]. These data suggest that Nkx2.5 is a direct target gene of NFAT. Embryos from GRIM-19 KD frogs and Nkx2-5 knockout mice displayed similar defects in heart formation, which supports the above hypothesis [33,106,107]. Together, these studies have also revealed a few additional points that are noteworthy. Firstly, most of the GRIM-19 KD embryos arrested at the heart tube stage, indicating that the defects occur from heart looping. This suggests a role of mitochondria in early heart development. Secondly, the mitochondria-mediated NFAT pathway may also play essential role in the later stage at heart valve formation. Inhibition of complex I at the stage of valve initiation led to a defect in atrioventricular valve formation, which is similar to that observed in embryos incubated with NFAT inhibitors [33,87,88]. Thirdly, inhibition of the NFATc activity by CsA at the early stage led to a heart defect at heart tube stage which is similar to that caused by knockdown of GRIM-19, suggesting that Ca2+ -NFATc is a major pathway through which mitochondrial complex I controls heart development. Fourthly, cardiac mitochondrial morphology and function are compromised in NFATc3/c4-deficient mouse embryos at E10.5, suggesting that NFAT activity is involved in mitochondrial maintenance and energy metabolism [89]. It has also been reported that NFATc4 regulates the neuronal-specific IP3 R necessary for the Ca2+ release [121]. Since Ca2+ stimulates mitochondrial energy metabolism, a positive feedback loop between mitochondria and Ca2+ –NFAT signaling may exist and play a crucial role in the maintenance of Ca2+ homeostasis necessary for the heart development. However, there are some unresolved issues. The mitochondria–Ca2+ –NFAT pathway seems to play essential roles in both early and late stages of heart development in Xenopus [33], but it is unclear whether this pathway is also required for cardiac specification before heart tube formation. In addition, in mice, the effects of NFATc seem to be at a later stage than in Xenopus. This could be due to a fundamental difference between mice and frogs for the requirement of NFAT in early cardiac development. Alternatively, it could be due to functional redundancy of the NFAT family members in mice.

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4. Concluding remarks Based on these data, a model that explains a possible mechanism for the role of mitochondrial RC complex I in heart development can be proposed (Fig. 1). Growth factors, probably non-canonical Wnts, bind to their receptors on the PM which leads to the activation of PLC and production of IP3 . IP3 binds to its receptor on the ER membrane and opens Ca2+ channels to release ER-stored Ca2+ into the cytoplasm. The [Ca2+ ]ER depletion triggers the opening of CRAC on the PM and causes Ca2+ influx. This elevated [Ca2+ ]c in turn activates Ca2+ -dependent phosphatase calcineurin that dephosphorylates and thus activates NFATc. NFATc is translocated to the nucleus, and in cooperation with GATA, stimulates the expression of target genes, such as Nkx2-5. This Ca2+ event is tightly controlled by mitochondria during cardiac development. Mitochondria provide ATP to establish [Ca2+ ]ER stock and potential for Ca2+ release. Mitochondria also directly prolong the opening of CRAC, which is important for NFAT activation. Mitochondrial RC dysfunction caused by knockdown of GRIM-19 disrupts intracellular Ca2+ mobilization, which impairs NFAT activation and causes heart defect in Xenopus embryos. These data provide the first genetic evidence for the importance of mitochondria in the modulation of Ca2+ signaling and the Ca2+ dependent NFAT pathway in heart development. However, how mitochondria regulate each stage of heart (and the other organs) development remains to be understood and requires further investigation. Acknowledgements Work in our laboratory is supported by the Agency for Science, Technology and Research of Singapore. We thank C.P. Lim for reading the manuscript. References [1] Berridge MJ, Bootman MD, Roderick HL. Calcium signalling: dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol 2003;4:517–29. [2] Ducibella T, Schultz RM, Ozil JP. Role of calcium signals in early development. Semin Cell Dev Biol 2006;17:324–32. [3] Webb SE, Miller AL. Calcium signalling during embryonic development. Nat Rev Mol Cell Biol 2003;4:539–51. [4] Hirst J, Carroll J, Fearnley IM, Shannon RJ, Walker JE. The nuclear encoded subunits of complex I from bovine heart mitochondria. Biochim Biophys Acta 2003;1604:135–50. [5] Schultz BE, Chan SI. Structures and proton-pumping strategies of mitochondrial respiratory enzymes. Annu Rev Biophys Biomol Struct 2001;30:23–65. [6] Carafoli E. Calcium signaling: a tale for all seasons. Proc Natl Acad Sci USA 2002;99:1115–22. [7] Berridge MJ, Lipp P, Bootman MD. The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 2000;1:11–21. [8] Bootman MD, Collins TJ, Peppiatt CM, Prothero LS, MacKenzie L, De Smet P, et al. Calcium signalling—an overview. Semin Cell Dev Biol 2001;12:3–10. [9] Hoth M, Penner R. Depletion of intracellular calcium stores activates a calcium current in mast cells. Nature 1992;355:353–6. [10] Putney JW, Bird GS. The signal for capacitative calcium entry. Cell 1993;75:199–201. [11] Parekh AB, Penner R. Store depletion and calcium influx. Physiol Rev 1997;77:901–30. [12] Zweifach A, Lewis RS. Mitogen-regulated Ca2+ current of T lymphocytes is activated by depletion of intracellular Ca2+ stores. Proc Natl Acad Sci USA 1993;90:6295–9. [13] Bezprozvanny I, Watras J, Ehrlich BE. Bell-shaped calcium-response curves of Ins(1,4,5)P3- and calcium-gated channels from endoplasmic reticulum of cerebellum. Nature 1991;351:751–4. [14] Gyorke I, Hester N, Jones LR, Gyorke S. The role of calsequestrin, triadin, and junctin in conferring cardiac ryanodine receptor responsiveness to luminal calcium. Biophys J 2004;86:2121–8. [15] Higo T, Hattori M, Nakamura T, Natsume T, Michikawa T, Mikoshiba K. Subtypespecific and ER lumenal environment-dependent regulation of inositol 1,4,5trisphosphate receptor type 1 by ERp44. Cell 2005;120:85–98. [16] Caroppo R, Colella M, Colasuonno A, DeLuisi A, Debellis L, Curci S, et al. A reassessment of the effects of luminal [Ca2+ ] on inositol 1,4,5-trisphosphateinduced Ca2+ release from internal stores. J Biol Chem 2003;278:39503–8. [17] Camacho P, Lechleiter JD. Increased frequency of calcium waves in Xenopus laevis oocytes that express a calcium-ATPase. Science 1993;260:226–9.

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