An integrated mechanism of cardiomyocyte nuclear Ca²⁺ signaling

Abstract

In cardiomyocytes, Ca²⁺ plays a central role in governing both contraction and signaling events that regulate gene expression. Current evidence indicates that discrimination between these two critical functions is achieved by segregating Ca²⁺ within subcellular microdomains: transcription is regulated by Ca²⁺ release within nuclear microdomains, and excitation–contraction coupling is regulated by cytosolic Ca²⁺. Accordingly, a variety of agonists that control cardiomyocyte gene expression, such as endothelin-1, angiotensin-II or insulin-like growth factor-1, share the feature of triggering nuclear Ca²⁺ signals. However, signaling pathways coupling surface receptor activation to nuclear Ca²⁺ release, and the phenotypic responses to such signals, differ between agonists. According to earlier hypotheses, the selective control of nuclear Ca²⁺ signals by activation of plasma membrane receptors relies on the strategic localization of inositol trisphosphate receptors at the nuclear envelope. There, they mediate Ca²⁺ release from perinuclear Ca²⁺ stores upon binding of inositol trisphosphate generated in the cytosol, which diffuses into the nucleus. More recently, identification of such receptors at nuclear membranes or perinuclear sarcolemmal invaginations has uncovered novel mechanisms whereby agonists control nuclear Ca²⁺ release. In this review, we discuss mechanisms for the selective control of nuclear Ca²⁺ signals with special focus on emerging models of agonist receptor activation.

1. Introduction

Calcium homeostasis is regulated by the combined action of a variety of channels, transporters, and binding proteins which allow cells to increase or decrease intracellular Ca²⁺ concentration on demand [1]. Ca²⁺-releasing events, or Ca²⁺ transients, occur when Ca²⁺ channels embedded within either the plasma membrane or in select internal membranes open, allowing Ca²⁺ to move down its electrochemical gradient from either external sources or intracellular Ca²⁺ stores, flooding the cytosolic compartment. Cytosolic Ca²⁺ increases a remarkable 50-fold this way with each heart beat (0.1μM in diastole to ≈5μM in systole). Then, Ca²⁺ is rapidly removed from the cytosol by Na⁺–Ca²⁺ exchangers and ATP-dependent transporters that pump Ca²⁺ out of the cell or back into intracellular stores [2]. This Ca²⁺ cycle defines the Ca²⁺ transient, whereas repeated Ca²⁺ cycles comprise a Ca²⁺ oscillation [3,4].

Ca²⁺ oscillations can be tuned in frequency, amplitude, and duration, providing a biological signal with limitless possible combinations for encoding information [5]. Cardiac contraction provides an excellent example of the importance of Ca²⁺ oscillations, and the need to maintain them under fine control [6]. Under normal conditions, the human heart beats once every second, therefore, each cardiomyocyte undergoes a full, coordinated Ca²⁺ cycle nearly 60 times per minute [2]. Many biological inputs ultimately exert control over heart rate by impacting various components governing Ca²⁺ oscillation.

Although Ca²⁺ oscillations are central to driving cardiomyocyte contraction, non-contractile Ca²⁺-dependent signaling has emerged as an important regulatory mechanism of both transcriptional control and structural remodeling in the heart. In a wonderfully intricate manner, Ca²⁺ manages to regulate these processes independent of the whole-cell Ca²⁺ oscillations that drive contraction. Ca²⁺-mediated changes in gene expression often occur in response to agonist binding to receptors at the plasma membrane, or sarcolemma [7,8]. This mechanism allows cells to reprogram their gene expression profiles to meet ever-changing cardiac demand. Ca²⁺-mediated signaling can also influence transcriptional control of cardiomyocyte development [9], differentiation [10], survival [11], hypertrophic growth [12,13], metabolism [14] and cell death [11]. At present, we are only beginning to understand how a cardiomyocyte decodes a Ca²⁺ signal to alter gene expression without interfering with, or being controlled by, the essential and ongoing process of contraction [15]. A growing body of evidence indicates that such discrimination is accomplished by triggering local Ca²⁺ release in segregated subcellular compartments (cytosol versus nucleus) or specific sub-regions of these compartments, generating microdomains of localized Ca²⁺-signaling events. In this review, we focus on mechanisms currently proposed to explain such selective control of nuclear Ca²⁺ signals.

2. Cytosolic source of nuclear Ca²⁺ signals

Although it is currently controversial whether the initiation of nuclear Ca²⁺ signals derives from cytosolic Ca²⁺ entry into the nucleus, or generated by the nuclear release of Ca²⁺, there is evidence that both mechanisms exist in cardiomyocytes (as summarized in Fig. 1). Indeed, several studies in cardiomyocytes and other cell types suggest that elevations nuclear Ca²⁺ are the direct consequence of changes in cytosolic Ca²⁺ [16–18]. On the other hand, it has also been shown in different cardiac muscle cells that changes in nuclear Ca²⁺ can be regulated independent of cytosolic Ca²⁺ and derive from Ca²⁺ released inside, or in close proximity to, the nucleus [18–20].

Fig. 1.

Nuclear and cytosolic pathways controlling nuclear Ca²⁺ homeostasis in cardiomyocytes. Different pathways can lead to nuclear Ca²⁺ signals. Pathway 1: InsP₃ is generated by PLC either in the plasma membrane or in peri/intra-nuclear compartments. Pathway 2: InsP₃ can diffuse in and out of the nucleus. Pathway 3: InsP₃ from cytosolic or nuclear origin triggers Ca²⁺ release inside and/or outside the nucleus. Pathway 4: Ca²⁺ triggers Ca²⁺ release from RyRs in the cytoplasm or in the nucleus of cardiomyocytes (nuclear RyR is present only in neonatal cardiac myocytes). Pathway 5: Ca²⁺ diffusion from the cytosol can also generate nuclear Ca²⁺ transients and vice versa.

2.1. Ca²⁺ diffusion through the nuclear pore complex

It was initially held that primary access for Ca²⁺ to the nuclear compartment occurred through passive diffusion of cytosolic Ca²⁺ through nuclear pores connecting the nucleus and the cytoplasm. This is a reasonable hypothesis given that the nuclear pore complex, a multiprotein structure integral to the nuclear envelope (NE), has an approximate diameter of 8nm although this was estimated in isolated Xenopus oocyte nuclei [21]. Although Ca²⁺ has an ionic radius of only 0.99°Å, its hydrophobicity in solution gives it an effective diameter of 12Å per hydrated ion (or 1.2nm). This would allow unlimited traffic of Ca²⁺ ions between the cytoplasm and nucleus as postulated by pioneer studies in amphibian and insect cells [22,23].

The concept of passive diffusion of Ca²⁺ into the nucleus from a cytoplasmic source is supported in cardiomyocytes by several lines of evidence, such as the observations of synchronous elevations in nuclear and cytoplasmic Ca²⁺ [16]. Indeed, the cytosolic Ca²⁺ wave propagated during cardiomyocyte contraction can invade the nucleoplasm via diffusion [16], favored by the lower Ca²⁺ buffer capacity of the nucleus [24]. In this model, the NE functions as a barrier and the nuclear pores provide the entryway for regulated diffusion of high cytosolic Ca²⁺, and this has also been observed in mouse neuroblastoma cells [25].

Like any gate, the nuclear pore is subject to regulation. Diffusion through nuclear pores is complex and regulated by several mechanisms, including passive diffusion of small molecules (up to 10kDa), Ca²⁺-regulated transport of intermediate molecules (10–70kDa) and also involves active transport for larger molecules [26]. Ca²⁺ itself can influence diffusion through nuclear pores, and Ca²⁺ store depletion decreases diffusion of intermediate but not small size molecules or ions [26–28]. Consistent with this, atomic force microscopy demonstrates that the nuclear pore complex is a dynamic structure capable of responding to changes in intracellular Ca²⁺ [29]. Indeed, some hormones that increase cytosolic Ca²⁺ levels also increase permeability of the nuclear pore complex [30]. Thus, regulation of nuclear pore function is complex, rendering it capable of either increasing or decreasing the traffic of Ca²⁺ and other middle size molecules into and out of the nucleus. The origin of a Ca²⁺ signal and its proximity to the nucleus can also be important factors influencing regulated diffusion of cytosolic Ca²⁺ into the nucleus [16,31,32].

2.2. Whole-cell Ca²⁺ oscillations

Propagation of cytosolic Ca²⁺ oscillations into the nucleus has also been reported in different cell types. In adrenal chromaffin and pituitary cells, both high and low frequency Ca²⁺ oscillations invade the nucleus, and interestingly high frequency oscillations are more able to do so than those of low frequency [33]. It is not clear whether in cardiomyocytes cytosolic Ca²⁺ diffusion into the nucleus during cardiac contraction is a function of the frequency and amplitude of cytosolic Ca²⁺ fluctuations, however, it has been proposed that during cardiomyocyte hypertrophy the nuclear machinery can decode distinct Ca²⁺ oscillatory patterns and respond specifically [34]. Distinct patterns of Ca²⁺ oscillations can alter the threshold of activation for Ca²⁺ as a second messenger by preferentially activating or inactivating distinct Ca²⁺-dependent compartments, resulting in directed, downstream changes in gene expression or cell function [35–38]. This may be of particular importance during inotropic and chronotropic responses where oscillatory patterns of Ca²⁺ handling are quickly shaped in response to hormonal stimulation.

3. Perinuclear sources of nuclear Ca²⁺ signals

A second hypothesis for the generation of nuclear Ca²⁺ signals has been proposed based on the ability of the nuclear envelope itself to function as a perinuclear Ca²⁺ store, capable of releasing Ca²⁺ into the nuclear matrix in response to stimulation [39]. This concept is supported by the identification of specific perinuclear structures that generate, modulate, and shape nuclear Ca²⁺ signals.

3.1. Perinuclear endoplasmic reticulum

Rough ER is distinguished by the abundance of attached protein-synthesizing ribosomes and is the type of ER most closely associated with the NE. Recent studies have explored the importance of a perinuclear subset of rough ER in the regulation of nucleoplasmic Ca²⁺. Over-expression of calsequestrin-2 targeted to the perinuclear rough ER promotes amplified RyR2-mediated nuclear Ca²⁺ transients in both adult and neonatal cardiomyocytes [40], suggesting that perinuclear rough ER acts as a source, in addition to the NE, for nucleoplasmic Ca²⁺ transients. For instance, in atrial cardiomyocytes, cytosolic Ca²⁺ sparks must be close to the nucleus (within 5μm) to contribute to a nuclear Ca²⁺ signal [41], demonstrating the importance of localized events in the perinuclear endoplasmic reticulum for nuclear Ca²⁺ signaling.

3.2. The nuclear envelope

Two membranes comprise the NE: the outer nuclear membrane (ONM), which is a structure continuous with the endoplasmic reticulum (ER), and the inner nuclear membrane (INM), which is in direct contact with the nuclear matrix. The perinuclear space (PS) is the space between the ONM and INM [42]. The NE of cardiomyocytes harbors Ca²⁺, which can generate Ca²⁺ signals independent of cytosolic Ca²⁺ [26,39,43]. Fluorescence recovery after photobleaching of Fluo-5N in intact adult cardiomyocytes demonstrates that the lumen of the sarcoplasmic reticulum (SR) and the PS are interconnected, enabling rapid diffusion of intraluminal Ca²⁺ ions and limiting the generation of spatial gradients during Ca²⁺ release events [20]. In other words, Ca²⁺ inside the NE is connected to cytoplasmic Ca²⁺ stores and fully available for rapid signaling, only requiring nuclear Ca²⁺ channels to trigger release.

The ONM of embryonic chicken cardiomyocytes harbors the sarco/endoplasmic reticulum Ca²⁺-ATPase 2 (SERCA2), which actively transports Ca²⁺ from the cytosol surrounding the nucleus into the PS [44]. Although Ca²⁺-ATPases have not been identified in the INM, there is a Na⁺/Ca²⁺ exchanger complex in the IMM, suggesting a mechanism for maintaining Ca²⁺ homeostasis in the nucleoplasm through Na⁺/Ca²⁺ exchange and allowing restoration of resting Ca²⁺ levels by transfer of Ca²⁺ to the NE or PS [45,46]. Thus, in addition to the intraluminal equilibrium of Ca²⁺ stores between the PS and the SR, the NE participates in active uptake of Ca²⁺ from both inside and outside the nucleus, thereby exhibiting Ca²⁺ storage capabilities similar to the ER. Buffering of cytoplasmic Ca²⁺ by binding proteins and organelles in the cytoplasm can also directly and indirectly influence nuclear Ca²⁺ levels.

3.3. The nucleoplasmic reticulum

In 2003, a model, derived from SKHep1 epithelial cells, of the dynamic interconnections between ER and NE membranes was extended to structures inside the nuclear matrix, comprising a nucleoplasmic reticulum (NR). The NR is a continuous reticular network that invaginates the INM of the NE toward the nuclear matrix [47]. Subsequently, these nuclear structures were reported in neonatal and adult cardiomyocytes [48], as well as in many other mammalian and plant cells [49]. Interestingly, this discrete Ca²⁺ rich structure can regulate Ca²⁺ signals in specific subregions of the nucleus, as Ca²⁺ releasing channels are localized to specific regions of the NR, forming nucleoplasmic Ca²⁺-release hotspots.

Invaginations of both the ONM and INM into the nuclear matrix have been described in many cell types [49]. The lumen of these structures, or “NE tunnels” as they have been called, contains both ER and mitochondrial markers [50,51], indicating that they bring cytoplasmic elements and organelles in close physical proximity to the nuclear matrix. As a result, they comprise intranuclear signaling microdomains that can segregate, generate, or amplify Ca²⁺ release from the NE, possibly in response to T-tubule invaginations.

3.4. Perinuclear mitochondria

Mitochondria can also shape the dynamics of nuclear Ca²⁺ [50,52] by acting as a perinuclear buffer, thereby influencing the magnitude and duration of nuclear Ca²⁺ signals. Mitochondria are often found in close proximity to the ER, particularly in the perinuclear region, and can rapidly uptake Ca²⁺ released from InsP₃-receptors, buffering the impact of InsP₃-mediated cytoplasmic Ca²⁺ release [53]. During E–C coupling this same process of mitochondrial Ca²⁺ uptake through the VDAC/mitochondrial Ca²⁺ uniporter (MCU) complex can buffer the amplitude of cytosolic Ca²⁺ transients, theoretically limiting the passive diffusion of cytosolic Ca²⁺ into the nucleus [54].

All the above-mentioned sources of Ca²⁺ can contribute or shape nuclear Ca²⁺, but despite the multiple sources of nuclear Ca²⁺ signals, whether the origin is nuclear or perinuclear, remains in dispute. A recent report may help reconcile these two models. In situ calibration of Ca²⁺ concentrations in the nucleus and cytosol of murine adult cardiomyocytes reveals that nuclear Ca²⁺ transients evoked by electrical stimulation consist of at least two separate components, a passive component of Ca²⁺ diffusion from the cytosol to the nucleus through nuclear pores and an active component of Ca²⁺ release via InsP₃ receptors [18]. Thus, nuclear Ca²⁺ transients in adult cardiomyocytes arising from electrical stimulation are both dependent and independent of cytosolic Ca²⁺. Together, these studies unveil the complex regulation of cardiomyocyte nuclear Ca²⁺ signals through combined actions of highly structured nuclear and perinuclear sub-compartments.

4. Nucleus-initiated nuclear Ca²⁺ release

Despite advances in our understanding of the nuclear Ca²⁺ store, the question remains whether these specialized structures can be activated independent of cytosolic Ca²⁺ release. For instance, does targeted stimulation by a hormone act directly on the nuclear Ca²⁺ release machinery to trigger Ca²⁺ release?

4.1. Nucleus-restricted molecular tools

To address this question, targeted molecular tools were engineered such that their mode of action is restricted to a specific subcellular compartment. The high-capacity, low-affinity Ca²⁺ buffer protein, parvalbumin (PV) was targeted to either the nucleus or the cytosol by addition of a peptide signal that directs the protein to a specific subcellular location [55]. These fusion proteins were used in HepG2 cells to reveal that nuclear Ca²⁺ buffering suppresses ATP induction of nuclear Ca²⁺ signals without affecting the rise in cytosolic Ca²⁺. By contrast, cytosol-targeted PV accomplished the opposite, blocking cytosolic Ca²⁺ transients but sparing nuclear transients. In the same study, EGF-mediated activation of the transcription factor Elk-1 was abolished specifically by buffering nuclear Ca²⁺, indicating that, in this context, nucleus-restricted Ca²⁺ increases drive the regulation of gene expression [56].

When this technology was applied to cardiomyocytes, global E–C coupling-related Ca²⁺ transients were largely decreased by buffering cytosolic Ca²⁺, as expected. However, buffering nuclear Ca²⁺ had the surprising effect of decreasing the amplitude of Ca²⁺ oscillations while increasing both their frequency and action potential duration [48]. Together, these findings suggest that a componcient of nuclear Ca²⁺ release helps shape global Ca²⁺ transients during the cardiac contraction cycle, likely via a nucleus-delimited E–C coupling process.

4.2. Nuclear Ca²⁺ contribution to E–C coupling

The concept that nuclear Ca²⁺ is an important component of E–C coupling was first suggested by structural studies demonstrating the existence of perinuclear T-tubules and nuclear Ca²⁺ microdomains along with RyR2 [48] and L-type Ca²⁺ release channels [57,58]. Amplification of cytosolic Ca²⁺ signals by this nuclear E–C coupling mechanism may occur through passive diffusion of Ca²⁺ from the nucleus to the cytosol through nuclear pores, and is favored by the nuclear-to-cytosolic Ca²⁺ gradient and relatively lower Ca²⁺ buffering capacity of the nucleus [18].

4.3. Nuclear Ca²⁺ signals independent of E–C coupling

Although nuclear Ca²⁺ signals coordinated with cardiac E–C coupling exhibit features both dependent and independent of changes in cytosolic Ca²⁺, activation of nuclear Ca²⁺ in response to certain stimuli can occur entirely independent of cytosolic Ca²⁺. For example, this has been demonstrated for insulin-like growth factor-1 (IGF-1) receptor signaling [59]. Treating neonatal cardiomyocytes with IGF-1 initiates an InsP₃-dependent nuclear Ca²⁺ transient that precedes the cytosolic transient [59]. Blocking the nuclear Ca²⁺ transient with targeted PV abrogated both the nuclear and cytosolic responses, whereas cytosol-localized PV blocked only the cytosolic response, indicating that the signal originates in the nucleus [58]. This cytosol-independent nuclear Ca²⁺ response is required for activation of the transcription factor MEF-2C, a mediator of IGF-1-induced cardiomyocyte hypertrophy [58]. Using a similar approach, a nucleus-restricted InsP₃ buffer inhibited the hypertrophic response of neonatal cardiomyocytes to either IGF-1 or ET-1 [60]. A different study showed that phenylephrine-stimulated cardiomyocytes showed an increased frequency of nuclear Ca²⁺ sparks leading to nuclear Ca²⁺ transients [61], which is also in favor of the notion that both InsP₃- and Ca²⁺-dependent signaling occur within the nucleus in response to hypertrophic agonists to regulate cardiac transcription, and this signaling is not dependent on cytosolic Ca²⁺ release.

Thus, current evidence suggests that, depending on whether a nuclear Ca²⁺ transient arises during an E–C coupling cycle or in response to activation of a receptor on the plasma membrane, the elevation in nuclear Ca²⁺ can be either partially dependent or fully independent of cytosolic Ca²⁺. Considering that cardiac contraction is a continuous, episodic process that itself can be shaped by extracellular stimuli, the details of the interplay between nuclear and cytoplasmic Ca²⁺ signaling are likely to be complex. With the exception of extreme situations, such as cardioplegic arrest during surgery, receptor activation in cardiomyocytes must always occur simultaneously with E–C coupling. Therefore, Ca²⁺ signals arising from nuclear stores are constantly influenced by regulatory signals that diffuse from the cytosol. How nuclear Ca²⁺ stores detect, decode, and respond appropriately to all the possible inputs delivered at a given time remains an important and unanswered question.

5. Ca²⁺ channels mediating nuclear Ca²⁺ transients

Cardiomyocytes harbor three classes of ion channels that mediate the rapid mobilization of Ca²⁺ ions from intracellular stores: inositol 1,4,5-trisphosphate receptors (InsP₃Rs), ryanodine receptors (RyRs) [10] and NAADP-sensitive endolysosomal Ca²⁺ channels [62]. While two pore channels (TPCs) have been proposed as the NAADP-sensitive Ca²⁺ channels at endolysosomal stores [63], the best-characterized channels that release Ca²⁺ from intracellular stores are InsP₃Rs and RyRs. Both of the latter families form high-conductance, low-selectivity cation channels by association of four high-molecular mass protein monomers in combination with a large number of accessory proteins [64–68]. The InsP₃R family consists of at least three distinct gene products, encoding monomers of molecular mass around 300°kDa, with three conserved and characterized domains (i.e. binding, regulatory/coupling, and channel pore). All three of these proteins bind their endogenous agonist InsP₃ and share approximately 70% amino acid identity with one another [64,65]. There is general agreement that the type-2 InsP₃R is the predominant InsP₃R isoform in cardiomyocytes [69,70], although its expression levels are nearly 100-fold lower than that of RyRs, the primary release channels during E–C coupling [71]. Thus, InsP₃Rs are not the primary source of Ca²⁺ release. That said, InsP₃R localization in cardiomyocytes is concentrated in the NE where it is found at the ONM, INM, and at the nucleoplasmic reticulum [48,72–74]. Accordingly, the introduction of InsP₃ into the nucleus has been shown to evoke Ca²⁺ release from the NE [75–77]. Release of Ca²⁺ from the NE can be inhibited by microinjection of heparin (an InsP₃R antagonist) into the nucleus, [76] or by an antibody against the InsP₃R [77,78]. InsP₃ triggers rapid nuclear Ca²⁺ signals in permeabilized atrial cardiomyocytes that can be blocked by InsP₃Rinhibition, but not by inhibition of RyR [74]. In isolated cardiomyocyte nuclei, InsP₃ triggers increases in nucleoplasmic Ca²⁺ and decreases in the PS [74]. InsP₃ increases the frequency of perinuclear Ca²⁺ sparks in permeabilized neonatal cardiomyocytes [61]. InsP₃ also induces prominent monophasic nuclear Ca²⁺ transients in immortalized HL-1 cells, where InsP₃R type-1 is localized to the perinuclear region [79]. Thus, it is not the relative abundance of InsP₃Rs, but rather their strategic localization to nuclear membranes, that renders these Ca²⁺ channels relevant for gating nuclear Ca²⁺ signals in cardiomyocytes.

Release of Ca²⁺ via the RyR has been implicated in the regulation of nuclear Ca²⁺ signals, as suggested in the proposed mechanism 4 from Fig. 1, but this has been restricted to neonatal cardiomyocytes, whereas evidence for nuclear localization of RyRs in adult cardiomyocytes has not been clearly established. Hypothetically, this could be a difference lost with differentiation, due to the importance of E-C coupling in promoting an adult cardiomyocyte phenotype. Similarly in a different myocyte cell, photo-release of caged Ca²⁺ directly within the nucleus of C2C12 myoblasts triggers localized Ca²⁺-induced Ca²⁺ release (CICR) in the nucleus which could be suppressed by the RyR inhibitor dantrolene [80]. RyR1 was found to localize to intranuclear extensions of the NE in C2C12 myoblasts. RyR2 was also found to localize to the perinuclear region of embryonic chick cardiomyocytes [44], and the nucleoplasmic reticulum of neonatal rat cardiomyocytes [48], providing structural evidence that nuclear RyRs control locally-restricted nuclear CICR in neonatal cells. Together, these studies unveil selective regulation of nuclear Ca²⁺ signals by specific InsP₃R and RyR isoforms localized to nuclear membranes, where they mediate Ca²⁺ release from perinuclear Ca²⁺ stores by the combined action of the second messengers InsP₃ and Ca²⁺. These mechanisms could play important roles in the establishment of the adult cardiac phenotype, and also may be involved in pathological conditions where neonatal gene programs are activated.

6. Diffusion of cytosolic second messengers into the nucleus

Activation of nuclear Ca²⁺ channels can occur via diffusion into the nucleus of second messengers initially generated in the cytoplasm or via second messengers generated on-site. Several second messengers traverse the nuclear pore to promote nuclear Ca²⁺ release [76,81].

6.1. Diffusion of Ca²⁺

Ca²⁺ itself is an excellent candidate for regulating the initiation and maintenance of nuclear Ca²⁺ signals. Analogous to signaling events in the cytosol, an increase in nucleoplasmic Ca²⁺ facilitates Ca²⁺ release from InsP₃Rs [82,83]. Indeed, various isoforms of InsP₃R are either activated or inhibited by high Ca²⁺ concentrations [64,65,68,84,85]. It is possible to envision a mechanism of nuclear Ca²⁺ regulation of CICR similar to that of cytoplasmic CICR, where an increase in nuclear Ca²⁺ serves as a positive modulator, initiating, amplifying and ultimately terminating the signal at NE Ca²⁺-release hotspots [18,24,86]. This positive feedback between nuclear Ca²⁺ and nuclear Ca²⁺ release channels would then operate as an amplifying mechanism to direct nucleoplasmic Ca²⁺-mediated signaling.

6.2. Diffusion of InsP₃

Evidence exists for diffusion of InsP₃ from the cytosol to the nucleus, as illustrated in Fig. 1. FRET-based InsP₃ biosensors have revealed that either stimulation with ET-1 or delivery of InsP₃ directly into the cytosol induces diffusion of InsP₃ into the nucleus, with delayed kinetics relative to cytosolic concentration [87]. Diffusion of InsP3 into the nucleus initiates a CaM/CaMK II signaling pathway ultimately controlling HDAC5 phosphorylation and nuclear export [73]. These findings confirm the ability of cytosolic InsP₃ to diffuse into the nucleus and are consistent with the documented timing of nuclear Ca²⁺ transients in response to extracellular ET-1. This is illustrated in mechanism 2 from Fig. 1. However, diffusion of InsP₃ from the cytosol is not sufficient to explain the rate of nuclear Ca²⁺ responses which exceed that of InsP₃ diffusion. InsP₃ generated at the plasma membrane has a limited capacity for traversing large distances in the cell (20–50μm) [88,89]. The cytosolic diffusion rate of InsP₃ is 15μm/s [89] and is compatible with the kinetics of the nuclear Ca²⁺ signal in response to ET-1. As with Ca²⁺, it is unclear how nuclear InsP₃Rs discriminate between InsP₃ generated in the cytoplasm versus InsP₃ generated in the nucleus. Rates of InsP₃ degradation are likely an important factor. InsP₃ is rapidly hydrolyzed to inactive forms, such as InsP₂ [90]. Therefore, cytoplasm-localized InsP₃Rs encounter the bulk of InsP₃ generated in the cytoplasm, whereas, the concentration of cytoplasmic InsP₃ at the nucleus is relatively small. This argument hints at the ability to generate InsP₃ inside the nucleus, near its site of action, as will be discussed below.

7. Generation of second messenger inside the nucleus

Increasing evidence indicates that nuclear Ca²⁺ signals can be initiated by second messengers generated in and/or around the nucleus. The nucleus of many cell types is equipped with a toolkit for inositol lipid metabolism [91], as nuclear membranes harbor enzymatic systems for the production and degradation of InsP₃: phospholipases [92,93], PIP₂ [94] and kinases [91,95]. However, and importantly, these have mainly been identified in cell types different than cardiomyocytes. Evidence for a nuclear phosphoinositide cycle in cardiomyocytes has indeed been more elusive. In particular, nuclear PLCε has been detected in neonatal cardiomyocytes, and its knock down in the nucleus inhibited the hypertrophy of neonatal cardiomyocytes induced by ET-1, isoproterenol or norepinephrine [96]. PLCβ3 and PLCγ have been observed in the nucleus in confocal studies in cardiomyocytes leading to nuclear Ca²⁺ signals, but further subcellular fractionation experiments are necessary to confirm such observations, specifically as the localization of these enzymes in perinuclear microdomains could account for the effects on nuclear Ca²⁺ [58], as proposed in mechanism 1 from Fig. 2. One study did suggest that PLCβ1 was localized to the nucleus in adult cardiomyocytes [97], although others have questioned the antibody used in this study (Santa Cruz, G12 PLCβ antibody). Conversely, Zhang et al. [98] suggested that the substrate PIP₂ is not at the nuclear membrane in neonatal cardiomyocytes, rather that PLCε is at the nucleus. However, this localization was not related to nuclear InsP₃ generation, but instead to the production of DAG from PI4P for controlling PKD phosphorylation and nuclear import [96]. Disruption of nuclear localization of PLCε did not affect nuclear Ca²⁺ transients induced by ET-1, while PLCβ knock down did affect it [98], in agreement with the notion that perinuclear PLCβ is necessary for nuclear Ca²⁺ regulation [58]. Other studies, however, have shown that PLCβ isoforms also adopt nuclear localization. Nuclear PLCβ1 have been reported in adult cardiomyocytes [97] whereas nuclear PLCβ3 and PLCγ have been shown in neonatal cardiomyocytes [58]. Moreover, stimulation of neonatal cardiomyocytes with IGF-1 induces a fast and transient increase in intracellular InsP₃ levels [59]. This effect is reversed by preincubating cells with the Gαi blocker pertussis toxin [59] or by over-expressing the Gβγ scavenger peptide βARKct [58], indicating that a G protein-sensitive PLC isoform, likely PLCβ, is involved in this response. Subcellular analysis of PLC activation by means of a fluorescent reporter demonstrates that PIP₂ hydrolysis occurs exclusively in the perinuclear region upon IGF-1 stimulation, without affecting the peripheral PIP₂ pool [58], although the dynamic range for the fluorescent PIP2 reporter in the nuclear region seems to be lower than the one observed in the plasma membrane [98], which may reflect local differences in phosphoinositide composition and availability. Thus, different nuclear PLC isoforms may contribute differently on nuclear Ca²⁺ regulation or nuclear lipid signaling in cardiomyocytes.

Fig. 2.

An integrated view of receptor-initiated nuclear Ca²⁺ signals in cardiomyocyte. Different agonists can activate receptors located in the sarcolemma (1 and 3) or intracellular receptors (2). Pathway 1: Perinuclear receptors located in deep T-tubules can lead to PLC-dependent InsP₃ generation in peri/nuclear compartments. Pathway 2: Intracellular receptors can be activated by lipophilic ligands (steroid hormones) or endocytosed ligands, leading to PLC-dependent InsP₃ generation in peri/nuclear compartments. Pathway 3: Receptors located in peripheral sarcolemma lead to generation of InsP₃ and its diffusion into the nucleus triggers nuclear Ca²⁺ signals.

A central unanswered question is how nuclear PLC isoforms are activated by an extracellular ligand. In cancer cells, ERK-mediated kinases activate nuclear PLCβ [99]. However, the kinetics of this MAPK cascade are slower (15–30min) than the fast nuclear response to IGF-1 of cardiomyocytes (<15s). This suggests that IGF-1 increases nuclear PLC activity via a different mechanism. Moreover, evidence indicates that not all agonists use the same signaling mechanisms to provoke a nuclear Ca²⁺ transient [100].

8. Working models of receptor-initiated nuclear Ca²⁺ release

As mentioned above, different extracellular ligands trigger a range of signaling mechanisms to achieve specific regulation of nuclear Ca²⁺ release p[100], as shown in Fig. 2. For example, a number of agonists induce nuclear Ca²⁺ release in cardiomyocytes. Endothelin-1 (ET-1) is perhaps the best studied and therefore much of our understanding of nuclear Ca²⁺ signaling has been built upon mechanisms triggered by ET-1 [19,73,101]. However, other agonists, such as IGF-1 [59], insulin [102], angiotensin II (Ang II) [103], ATP [53], extracellular Ca²⁺ [104], and testosterone [105], also mobilize cardiomyocyte nuclear Ca²⁺, although a full understanding of underlying mechanisms remains elusive.

8.1. Activation of sarcolemmal receptors

ET-1 receptor A (ETA) signals via the classic model for ligand activation of cell membrane receptors [100]. Binding of ET-1 to its cognate receptor in the cardiomyocyte plasma membrane triggers a dose-dependent increase in nuclear Ca²⁺ via Gαq-mediated activation of PLC, which triggers hydrolysis of sarcolemmal PIP₂ to generate InsP₃ [19,73]. InsP₃ generated in the cytosol diffuses into the nucleus and activates perinuclear Ca²⁺ release through type-2 InsP₃ receptors, as shown in pathway 3 from Fig. 2. In this model, the selective regulation of nuclear Ca²⁺ is achieved by the strategic localization of InsP₃Rs in nuclear membranes. Two physically segregated signaling hubs can be identified: one at the plasma membrane, formed by the ET-1/ET-1 receptor complex–Gq–PLCβ–PIP₂ generating InsP₃, which then diffuses through nuclear pores to the second hub, comprising the nuclear InsP₃R and the perinuclear Ca²⁺ store. This chain of events is consistent with the kinetics of ET-1-elicited nuclear Ca²⁺ transients, i.e. ranging between 30 and 60 s. It remains an intriguing question why the second messenger, InsP₃, is produced in excess far from the target site for its effect. One possibility is that by filtering the InsP₃ signal through cytosolic elements that facilitate or limit InsP₃ diffusion into the nucleus, the signal can be tuned to facilitate an appropriate response. Importantly, ET-1 receptor localization is not restricted to the sarcolemma but can also be detected in intracellular membranes, especially in the vicinity of the NE.

8.2. Perinuclear receptors

This working model is designed to explain the rapid effects of IGF-1 on nuclear Ca²⁺ release. A key component of this model is that a significant fraction of plasma membrane IGF-1R in cardiomyocytes is localized to perinuclear plasma membrane invaginations, or perinuclear T-tubules. This brings the surface receptor complex into close proximity of NE Ca²⁺ stores and provides direct access for extracellular ligands to the juxtanuclear receptor, thereby facilitating a localized nuclear response [58]. In this near-nuclear receptor model [100], plasma membrane IGF-1 receptors are directly apposed to the NE where they stimulate local production of InsP₃ mediated by Gαi-dependent activation of perinuclear PLC. InsP₃ then enters the nucleus to activate InsP₃Rs and release Ca²⁺ into the nucleoplasm [59]. This perinuclear Ca²⁺ signaling microdomain provides spatial insulation of nuclear Ca²⁺ signals from large cytosolic Ca²⁺ fluctuations [57].

This model is characterized by a single signaling hub, as all components share close proximity to the nucleus, thus avoiding the rate-limiting process of long-distance InsP₃ diffusion and degradation. This likely contributes to the fact that cardiomyocytes display the fastest nuclear Ca²⁺ response to IGF-1 of any cell type described to date, with a nuclear peak of only 2–6 s. This model is also potentially more efficient, because the total amount of InsP₃ necessary for triggering a nuclear Ca²⁺ response is significantly smaller than that required in the classic model. In summary, this model connects surface receptor activation directly with the nuclear Ca²⁺ store through a specialized and highly structured perinuclear Ca²⁺ microdomain, which is independent and physically separated from the cytosolic Ca²⁺ machinery.

8.3. Nuclear membrane receptors

A third working model situates a fraction of classical plasma membrane receptors (Ang II, ET-1 receptors or α1-adrenergic receptors) and their downstream signaling elements intracellularly, at nuclear membranes [106]. Activation of GPCRs located in nuclear membranes sets in motion a signaling cascade involving G protein–PLC–PIP₂ hydrolysis to produce InsP₃ directly in the nucleoplasm promoting a very rapid InsP₃ receptor release of nuclear Ca²⁺ from NE stores [107]. Similar to the near-nucleus receptor model, a single signaling hub can be identified in this model, as all the signaling elements are recruited to act together in close proximity in the nucleus. However, an important difference between the two models is the way in which ligands gain access to their cognate receptors. Indeed, it is a matter of debate whether the nuclear-delimited receptors are activated by the same ligands as their plasma membrane counterparts or by alternative ligands made available through intracrine mechanisms [106]. Mechanisms of ligand action may be receptor-specific, as will be discussed next.

8.3.1. Angiotensin II receptors

Functional Ang II receptors (AT1 and AT2) have been detected in the cardiomyocyte nuclear membrane [106]. Other components of the renin–angiotensin system, such as angiotensinogen, angiotensin converting enzyme (ACE) and renin are also expressed in adult cardiomyocytes and found to act intracellularly [108]. Thus, cardiomyocytes possess the potential for an entirely cell-autonomous renin–angiotensin axis. In addition, cardiomyocytes can take up and activate circulating prorenin [109]. Functional ACE has been detected in the nuclei of neonatal cardiomyocytes, suggesting that this can be a site of Ang II production [110]. Activation of AT1 receptors in nuclei isolated from cardiomyocytes triggers increases in nucleoplasmic Ca²⁺ via release from InsP₃ receptors [107]. This intracellular Ang II-mediated Ca²⁺ transient is required for downstream activation of transcription. Together, these facts provide evidence for an intracrine pathway of intracellular Ang II production which can activate Ca²⁺-dependent transcriptional events involved in the expression of specific cardiac genes through the modulation of InsP₃ levels and nuclear Ca²⁺ release.

8.3.2. ET-1 receptors

In contrast to type A ET receptors (ETA) that localize to the sarcolemma (see Section 8.1), ETB receptors localize to nuclear membranes of adult cardiomyocytes [111]. This localization is not the consequence of surface receptor internalization, as demonstrated by the absence of N-glycosylated ETB receptor in nuclear protein fractions [112]. Moreover, endocytosed ETB receptor is destined for lysosomal degradation and does not traffic to the NE, suggesting that a significant pool of ETB receptor is targeted directly to nuclear membranes following biosynthesis [112]. Photo-release of caged ET-1 induces InsP₃-dependent nucleoplasmic Ca²⁺ increases that can be selectively blocked by intracellular delivery of an ETB antagonist, but not by an extracellular exposure to the same drug [112]. It is well established that ET-1 can be produced, stored, and secreted by cardiomyocytes under basal or stimulated conditions [113], suggesting that nuclear ETB can be activated by intracellular ET-1, comprising an intracrine system of nuclear Ca²⁺ release control.

8.3.3. α1-Adrenergic receptors

Evidence is also available for the nuclear localization and signaling of α1-adrenergic receptors in cardiomyocytes. α1-Adrenergic receptors localize to the nucleus of adult mouse cardiomyocytes and colocalize with nuclear Gαq and PLCβ1 [97]. Endogenous nuclear α1-adrenergic receptor signaling complex can be activated locally by extracellular cathecolamines since cardiomyocytes uptake catecholamines through a norephinephrine-uptake mechanism. Activation of nuclear-delimited α1-adrenergic receptors leads to receptor oligomerization, ERK phosphorylation [114], nuclear PKCδ activation and phosphorylation of cardiac TnI, which in turn increase myocyte contractility [115]. Although nuclear α1-adrenergic receptor signaling has been extensively demonstrated in cardiomyocytes, the exact involvement of nuclear receptor signaling on nuclear Ca²⁺ homeostasis remains unknown and future studies are needed to clarify the link between these 2 pathways.

9. Conclusion

Nuclear Ca²⁺ signals provide a strategic mechanism which allows cardiomyocytes to segregate the control of Ca²⁺-dependent transcription from cytosolic Ca²⁺ transients mediating E–C coupling [15]. Although different models have been proposed to explain the selective control of nuclear Ca²⁺ release in response to hormone stimulation [100], current evidence suggests that cardiomyocytes harbor a variety of mechanisms to activate and regulate nuclear Ca²⁺ release (Fig. 1). Furthermore, organization and signaling through the same receptor type may be different when localized to the sarcolemma versus the nuclear environment (Fig. 2).

There are many fascinating and unresolved questions. First, it is intriguing that cardiomyocytes are equipped with multiple mechanisms for controlling a common Ca²⁺-dependent transcriptional response. As discussed earlier, it may be that the diversity of mechanisms, all culminating in nuclear Ca²⁺ release, provides a physiological filter that encodes information regarding both the cellular environment and the nature of the specific agonist. Shaping the magnitude and temporal dynamics of nuclear Ca²⁺ release could impact the constellation of genes affected and the duration of the transcriptional or translational response. This is certainly plausible considering that agonists that use different mechanisms to control nuclear Ca²⁺ release also differ in their phenotypic effects. For example, Ang II and ET-1 generally mediate the expression of maladaptive genes and pathological cardiac hypertrophy, whereas IGF-1 mediates adaptive physiological cardiac hypertrophy [116]. In addition, many of these extracellular ligands activate other pathways different than Ca²⁺ signals, and their compartmentation in different subcellular locations may be a key for achieving selective responses.

Second, when a single agonist promotes nuclear Ca²⁺ release via multiple pathways, how is this decoded by the nucleus? As an example, ET-1 can initiate a signal at a sarcolemmal receptor and/or at a nuclear-membrane receptor. Decoding these signals by the nuclear machinery may lead to divergent phenotypic responses, or be integrated to give rise to a specified phenotype. Such a response could be further tuned to the initiating signal depending upon the balance of flux between the mechanisms. Third, it is not known whether there is cross-talk among the various mechanisms controlling nuclear Ca²⁺ or how the resulting nuclear Ca²⁺ signals are coordinated with other important pathways activated by the same ligand or receptor.

Finally, current knowledge regarding mechanisms activating nuclear Ca²⁺ signals and their downstream consequences has been limited to experimental models, primarily in vitro. Applying this knowledge in the context of human heart disease may provide new therapeutic tools that harness mechanisms through which cardiomyocytes decode and respond to agonist-activated nuclear Ca²⁺ signals.

Thus, the conceptual framework of nuclear Ca²⁺ signals is in rapid flux, with exciting new insights emerging. Further elucidation of underlying mechanisms is necessary to fully define the complex roles of Ca²⁺ in the governance of contractile function and intracellular signaling in the heart.

Abbreviations

Ang II

angiotensin II

E–C

excitation–contraction

ER

endoplasmic reticulum

ET-1

endothelin-1

IGF-1

insulin-like growth factor-1

INM

inner nuclear membrane

InsP₃

inositol 1,4,5-trisphosphate

InsP₃R

InsP₃ receptor

NE

nuclear envelope

ONM

outer nuclear membrane

PI4P

phosphatidylinositol-4-phosphate

PIP₂

phosphatidylinositol bisphosphate

PLC

phospholipase C

PS

perinuclear space

RyR

ryanodine receptor

SR

sarcoplasmic reticulum