3.1. Mitochondria and Cellular Ca²⁺ Homeostasis

Calcium signaling plays a crucial role in many molecular processes and a range of cellular functions. Accumulating evidence indicates that Ca²⁺ strictly controls cellular senescence [6]. An elevation of intracellular Ca²⁺ levels has been observed in response to different types of stresses. Mitochondria, equipped with Ca²⁺ uniporters and a negative membrane potential (about −180 mV), are able to function as a potent Ca²⁺ buffering organelle when there is an elevation in intracellular Ca²⁺ concentration ([Ca²⁺]_i) [30]. Furthermore, mitochondrial Ca²⁺ signaling increases when an advanced age is reached ^[31] [31]. The ER and mitochondria are the two major organelles that govern the intracellular storage, sequestration, and release of [Ca²⁺]_i ^[32][33][32,33]. An elevation in [Ca²⁺]_i is usually attributable to a Ca²⁺ influx from the extracellular milieu via plasma membrane Ca²⁺ channels or from intracellular stores of Ca²⁺ present in the ER [6]. Any release of Ca²⁺ from ER elevates the cytosolic Ca²⁺ and this occurs in combination with ever larger cycles of mitochondrial Ca²⁺ uptake [34]. These increases in mitochondrial Ca²⁺ concentration leads to a drop in mitochondrial membrane potential, which in turn enhances the production of ROS. As a result, oxidative stress increases further, triggering senescence [6], as well as organ dysfunction, during the aging process [31]. Mitochondrial Ca²⁺ uniporter (MCU) and H⁺/Ca²⁺ exchanger LETM1 are the two main transporters responsible for mitochondrial Ca²⁺ uptake ^[35][36][35,36]; on the other hand, NCX (Na⁺/Ca²⁺ exchanger) and H⁺/Ca²⁺ exchanger are the two transporters that export Ca²⁺ back to cytoplasm ^[30][37][30,37].

3.2. ER-Mitochondrial Ca²⁺ Dysregulation and Cardiac Ageing

The ER, which consists of a spatially extended membranous network, is often positioned in close proximity to other cellular organelles and these areas form membrane contact sites [38]. The sites of physical interaction and communication between the ER and mitochondria, namely the mitochondria-associated ER membranes (MAMs), represents a platform that is fundamentally important to the modulation of Ca²⁺ homeostasis, autophagy, apoptosis, lipid metabolism, metabolic diseases, and tumor growth; these events are mediated via the exchange of lipids and Ca²⁺ ions ^{[5][16][39][40][41][42]}[5,16,39,40,41,42]. The conserved structures of MAMs found across eukaryotic phyla are key determinants of cell survival and their functionality, which involves the bidirectional trafficking of factors between the two organelles ^[43][44][43,44]. This means that MAMs are dynamic structures that are sensitive to the physiological conditions within cells [16].

Four major functions have been identified for MAMs. These are (a) control of Ca²⁺ signaling, (b) regulation of mitochondrial division, (c) accommodation of lipid biosynthesis, and (d) coordination of the dynamic interactions between mitochondria and the ER [44]. Importantly, MAMs provide an efficient way for Ca²⁺ traffic to take place between the ER and mitochondria and this allows the creation of a higher Ca²⁺ concentration close to the MAMs compared to the surrounding cytoplasm. The Ca²⁺ released from the ER via the MAMs to the mitochondria means that this high local concentration of Ca²⁺ is able to activate various Ca²⁺-dependent processes. These include the tricarboxylic acid (TCA) cycle, oxidative phosphorylation, the regulation of ROS signaling, cell death, and the stimulation of mitochondrial division ^{[14][44][45][46]}[14,44,45,46]. A functional protein complex is essential for the tethering of ER-mitochondria and for maintaining the MAM structure; this complex is formed via inositol 1,4,5-trisphosphate receptors (IP3Rs) at ER, voltage dependent anion-selective channel (VDAC) protein in the mitochondrion, and the chaperone 75 kDa glucose regulated protein (GRP75) [14]. In cardiac and skeletal muscle, the Ca²⁺ channel in the ER, namely the ryanodine receptor (RYR), also allows Ca²⁺ traffic from ER to mitochondria close to the MAMs [47]. Many other tether complexes of MAMs had also been reported [38]. Interestingly, when the hearts of Mfn2KO mice are examined, the contact lengths of MAMs are reduced by 30%; this is accompanied by an increased level of ROS, and reduced cardiac contractility [48]. Using the norepinephrine-induced cardiac hypertrophic model, it has been shown that the distance between the ER and mitochondria is increased and that this is accompanied by impaired Ca²⁺ trafficking between the two organelles [49]. Indeed, alterations in the contact length and gap distance of MAMs had been reported to dysregulate Ca²⁺ signaling and affect cardiac metabolism, thereby accelerating cardiac ageing and promoting the development of cardiac hypertrophy, as well as heart failure [50].

3.3. Cisd2 Maintains Ca²⁺ Homeostasis

Cisd2 is mainly located in the mitochondrial outer membrane and the ER. In particular, Cisd2 is highly enriched in the MAMs. Previous studies by our group and other researchers have revealed that the intracellular localization and binding partners of Cisd2 vary with the cell type and/or with the experimental conditions [51]. This scenario helps to explain why different proteins have been found to interact with Cisd2 under a variety of circumstances ^{[18][19][21][22][23][52][53]}[18,19,21,22,23,52,53]. For example, in the skeletal muscle, Cisd2 appears to associate with Bcl-2/Bcl-XL and IP3R when participating in the regulation of the ER Ca²⁺ store. Loss of Cisd2 in myocytes results in a dysregulation of Ca²⁺ homeostasis that is accompanied by augmented autophagy and the presence of degenerated ER ^[52][53][52,53]. On the other hand, in the liver, Cisd2 interacts directly with Serca2b, which is a Ca²⁺-pump that transports Ca²⁺ from the cytosol into the ER. Furthermore, investigations have revealed that Cisd2 directly binds to Serca2a in cardiomyocytes. In both cases, this allows there to be control of Ca²⁺ pump activity by these two proteins via modulation of their oxidative modification. This in turn regulates ER Ca²⁺ uptake by Serca2a and Serca2b, which then sustains Ca²⁺ homeostasis in the heart and liver, respectively ^[18][19][18,19]. Finally, in adipocytes, our research has revealed that Cisd2 interacts directly with Gimap5 (GTPase of immune-associated protein 5) on the mitochondrial and ER membranes in order to modulate the Ca²⁺ buffering capacity of mitochondria, thereby maintaining the intracellular Ca²⁺ homeostasis of adipocytes. Loss of Cisd2 increases the cytosolic level of Ca²⁺, and induces Ca²⁺-calcineurin-dependent signaling that inhibits adipogenesis [21].

Mechanistically, one important question is how Cisd2 modulates intracellular Ca²⁺ homeostasis via its interaction with its partner proteins. Here we use Serca2 as an example to illustrate the potential molecular mechanism behind the regulation of Ca²⁺ via Serca2a in the cardiomyocytes and Serca2b in the hepatocytes ^[18][19][18,19]. Three possibilities may explain how the redox status of Serca2a and Serca2b is affected by Cisd2. First, the only defined functional domain of the Cisd2 protein is the protein’s CDGSH domain, which binds a redox-active 2Fe-2S cluster and is oriented toward the cytosol. It seems likely that Cisd2 directly interacts with Serca2 to help maintain Serca2b in a reduced state via the redox capacity of the CDGSH domain present on Cisd2. Second, Cisd2 may cover a part of Serca2 via protein–protein interaction, thus reducing the accessibility of certain tyrosines and cysteines present on Serca2 to attack by enzymes; as a consequence, this may prevent Serca2 from undergoing irreversible oxidative modification (cysteine sulfonation and tyrosine nitration) during oxidative stress. Finally, the redox-active CDGSH domain of Cisd2 may contribute to the maintenance of the general redox status of the cell. This will act to reduce the overall oxidative stress in the cell and indirectly protect Serca2 activity from ROS-mediated and/or RNS-mediated protein oxidation that can bring about a reduction in the protein’s Ca²⁺ pumping activity. These possibilities are not exclusive, but rather all may contribute to maintaining the redox homeostasis of the Serca2b protein [18].

3.4. Cisd2 Protects the Interfibrillar and Subsarcolemmal Mitochondria from Age-Related Damage

Mitochondria can be divided into three distinct populations based on their location and proximity to Ca²⁺ release sites ^[54] [54]. These are, firstly, perinuclear mitochondria (PNMs), which are arranged in clusters and located adjacent to the nucleus; their function is presumably associated with transcription. Secondly, there are subsarcolemmal mitochondria (SSMs), which are also arranged in clusters, but are located just beneath the sarcolemma; their function is presumably associated with the functioning of ion channels and various signaling pathways. Thirdly, there are the interfibrillar mitochondria (IFMs), which are arranged in rows alongside the myofibrils and are located very close to the Ca²⁺ release sites of the SR; their function is presumably to generate ATP that then contributes to the contractile function of myofibrils, as well as to participate in the Ca²⁺ signaling within cardiomyocytes [55].

The IFMs are probably the most susceptible mitochondria to age-related damage. Previous studies have revealed that, during the ageing of cardiomyocytes, there is an increase in mitochondrial ROS and a decrease in antioxidant capacity that leads to an increased accumulation of oxidative damage with the DNA [56]; notably this is almost exclusively associated with the IFMs [57]. The abundance of IFMs and their physically close association with myofilaments suggests that IFMs provide the energy needed for cardiomyocyte contraction and thus have a higher respiratory capacity than SSMs [58]. Therefore, any loss of IFMs is likely to adversely affect muscle contractility by limiting the ATP production required by myosin ATPases. Moreover, the increased ROS level associated with aged IFMs is likely to exacerbate mitochondria-derived oxidant injury to the myofilaments; this in turn will lead to increased fibrosis and/or fiber disarrangement, thereby decreasing the contractile capacity of the heart [59]. Additionally, a previous study has shown that decreased mitophagy is accompanied by mitochondrial fragmentation in aging hearts [60]. Intriguingly, these ultrastructural alterations to the IFMs, namely the presence of fragmented mitochondria, the appearance of disarranged and degenerative myofibrils, and the presence of myofibril/organelle necrotic debris, seem to be absent in 24-mo old Cisd2TG mice, which is not the case in naturally aged 24-mo old WT mice (Figure 1C,D).

The function of SSMs is presumably related to ATP production that allows electrolyte and protein transport across the sarcolemma [58]. The exclusive localization of Connexin 43, which is the major component of gap junctions for cell–cell communication, within the SSMs further pinpoints their important role in electrical conductance within the heart [61]. In the naturally aged (24-mo old WT) and prematurely aged (3-mo old Cisd2KO) mice, the SSMs have largely disappeared with only a few degenerating SSMs remaining detectable. This seems to explain the presence of the age-related cardiac conductance abnormalities that occur during ageing (Figure 2A). Remarkably, in the long-lived mice (24-mo old Cisd2TG), a persistently higher level of Cisd2 is able to eliminate age-related damage to the SSMs in the cardiomyocytes; the amount and structural integrity of SSM in the 24-mo old Cisd2TG is similar to that of 3-mo old young WT mice (Figure 2A). This result is consistent with our previous study ^[19] [19] wherein a higher level of Cisd2 was found to preserve functional mitochondria. This will, in turn, provide a stable MAM microenvironment that ensures efficient communication between the ER and mitochondria, thereby maintaining intracellular Ca²⁺ homeostasis in the cardiomyocytes. This means that any age-related functional decline of an aging heart, namely changes to mechanical contractility and electrical conductance, are attenuated by the presence of the Cisd2 protein.

Figure 2. Cisd2 preserves the integrity of subsarcolemmal mitochondria and maintains the ultrastructure of mitochondria-associated ER membrane (MAM). (A) Cisd2 preserves the integrity of subsarcolemmal mitochondria and cardiac myofibrils of left ventricle. In 3-mo WT mice, there is an abundance of normal subsarcolemmal mitochondria and normal cardiac myofibrils (A-1 and A-1′). In 3-mo Cisd2KO mice (prematurely aged), notably, a low density of subsarcolemmal mitochondria, and degeneration of mitochondria and myofibrils are found (A-2 and A-2′). In 24-mo WT mice (naturally aged), similar ultrastructural damages are found as those observed in the 3-mo Cisd2KO mice (A-3 and A-3′). Remarkably, in 24-mo Cisd2TG mice, a high level of Cisd2 preserves the density and integrity of subsarcolemmal mitochondria as well as maintains the ultrastructure of cardiac myofibrils (A-4 and A-4′). (A-1′) to (A-4′) are schematic presentations for ultrastructure of left ventricle shown in (A-1) to (A-4). SL: sarcolemma, IS: intercellular space, M: mitochondria, Cm: cardiac myofibril, ICD: intercalated disc, MD: mitochondrial degeneration, Cmd: cardiac myofibril degeneration. (B) Cisd2 is essential to maintaining the integrity of MAM. In 3-mo WT mice, at MAM, mitochondria closely attach to ER (B-1 and B-1′). In 3-mo Cisd2KO mice, notably, a longer distance between mitochondria and ER is found; in addition, mitochondria degeneration is also found (B-2 and B-2′). In 24-mo WT mice, a longer distance between mitochondria and ER is found; this is similar to that observed in the 3-mo Cisd2KO mice (B-3 and B-3′). Intriguingly, in 24-mo Cisd2TG mice, a high level of Cisd2 maintains a relatively normal MAM, where the mitochondria closely attach to the ER (B-4 and B-4′). (B-1′) to (B-4′) are schematic presentations for the ultrastructure of left ventricle shown in (B-1) to (B-4). The inset shows a higher magnification to provide a better illustration for MAM. The inset shows a higher magnification of the selected area (yellow squares) of the middle panel to provide a better illustration for MAM.

3.5. Cisd2 Maintains the MAMs during Cardiac Ageing

Normal functioning of MAMs is associated with there being an appropriate width of the cleft between the mitochondrial outer membrane (OMM) and the ER [62]. Certain structural arrangements within the space that separates the ER and mitochondria are able to support an optimal Ca²⁺ transfer via the Ca²⁺ transport channels [46]. Thus, there is an optimal distance of ≈30 nm between ER and mitochondria that allows effective Ca²⁺ transfer from ER, through the Ca²⁺ channel IP₃R located in ER, to the mitochondria, through the Ca²⁺ channel MCU located in mitochondria. This Ca²⁺ transfer contributes to the generation of physiologically relevant cytosolic Ca²⁺ oscillations [63]. In addition, a previous study has defined the role of this microenvironment as being the key compartment driving cytosolic Ca²⁺ oscillations. The microenvironment between the ER and mitochondria plays a critical role in Ca²⁺ dynamics by modulating the activity of IP3Rs and allowing the shuttling of Ca²⁺ between the ER and mitochondria [64]. A disruption of ER-mitochondria integrity and communication and a consequential dysregulation of Ca²⁺ homeostasis have been linked with pathogenesis, aberrant metabolism ^[38][42][38,42], neurodegenerative disease [38], a decreased lifespan [65], and ageing ^[15][41][15,41].

If the cleft between OMM and ER is too narrow, this causes various abnormalities. For example, under ER stress, MAMs become closer to the mitochondria, with a 25% decrease in distance and a 60% increase in contact length between the two organelles [39]. This results in an enhanced transport of Ca²⁺ from the ER to the adjacent mitochondrial network, which stimulates oxidative metabolism and induces the apoptotic program ^[16][34][16,34]. Additionally, during long-term cultured neuron, which is a neuronal senescence model, an increased Ca²⁺ transport from the ER to mitochondria has been associated with an upregulation of the MCU [66]. Thus, it seems that increased Ca²⁺ transfer to mitochondria and an accumulation of Ca²⁺ within the mitochondria seems to serve as one of the mechanisms underlying the loss of mitochondrial membrane potential and the induction of cell senescence [67].

If the cleft between the OMM and ER is too wide, this also causes problems. A previous study has revealed that abnormally high cytosolic Ca²⁺ develops when the distance is greater than the optimum [63]. Remarkably, our studies have revealed that Cisd2 deficiency or a decreased expression of Cisd2 to less than 50%, namely haploinsufficient, disrupts Ca²⁺ homeostasis and causes elevated cytosolic Ca²⁺ levels. This Cisd2-mediated Ca²⁺ dysregulation is accompanied by abnormal dilation of the ER and a breakdown of the mitochondrial outer membrane ^[17][18][19] [17,18,19] in many different tissues and organs, including brown adipose tissue [68], skeletal muscles ^[22][53][22,53], during neurodegenerative Alzheimer’s disease [23], and during cardiac dysfunction [19].

In naturally aged WT mice at 24-mo old, in which the level of Cisd2 has decreased to less than 50% in the heart, and in prematurely aged Cisd2KO mice at 3-mo old, in which Cisd2 expression is completely eliminated in the heart, the MAM gap distance is obviously increased (Figure 2B); this seems to provide an explanation for the differences in cardiac metabolism and Ca²⁺ signaling that occur with these two mouse models [19]. Intriguingly, in the long-lived Cisd2TG mice at 24-mo old, in which Cisd2 is 2-fold higher in the heart, the compactness and proximity of the ER and mitochondria appears to be well preserved (Figure 2B). This probably ensures that there is correct contact of the mitochondria with the MAM, which then allows normal Ca²⁺ trafficking between these two organelles to be maintained. The cardiac phenotypes of Cisd2KO mice and Cisd2TG mice are summarized in Table 1.