Nuclear Calcium in Cardiac Myocytes

Abstract

Calcium (Ca²⁺) is a universal second messenger involved in the regulation of various cellular processes including electrical signaling, contraction, secretion, memory, gene transcription and cell death. In heart, Ca²⁺ governs cardiomyocytes contraction, is central in electrophysiological properties and controls major signaling pathway implicated in gene transcription. How cardiomyocytes decode Ca²⁺ signal to regulate gene expression without interfering with, or being controlled by “contractile” Ca²⁺ that floods the entire cytosol during each heart beat is still elusive. In this review we summarize recent findings on nuclear Ca²⁺ regulation and its downstream signaling in cardiomyocytes. We will address difficulties in reliable quantification of nuclear Ca²⁺ fluxes and discuss its role in the development and progression of cardiac hypertrophy and heart failure. We also point out key open questions to stimulate future work.

1. Introduction

Calcium (Ca²⁺) is a universal second messenger underlying key cellular processes varying from gene transcription to cell death.¹ In cardiac muscle, Ca²⁺ is best known for its role in beat-to-beat contractile activation. During each heartbeat, a transient rise in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_cyto) is followed by Ca²⁺ removal, completing the Ca²⁺ cycle that governs contraction and relaxation. In addition to this fundamental role in mediating cardiac myocyte contraction, in recent years a broader role for Ca²⁺ in cellular signaling has emerged.² It is intriguing how a cardiomyocyte decodes Ca²⁺ signal to regulate gene expression without interfering with, or being controlled by “contractile” Ca²⁺, given the prevailing conditions in which [Ca²⁺]_cyto increases up to 20-fold (100 nM in diastole to 1–2 μM in systole) and Ca²⁺ floods the entire cytosol during each contractile cycle. It became evident that Ca²⁺-dependent signaling regulation works via specific Ca²⁺-binding proteins, but how molecular components involved in these processes may distinguish contractile vs. signaling Ca²⁺ still remains unknown and even controversial. On one hand, Ca²⁺ oscillations can vary in frequency, baseline, amplitude and duration, providing a biological signal with unlimited combinations for encoding information. On the other hand, a growing body of evidence suggests that such discrimination is attained by triggering spatially segregated Ca²⁺ release, generating subcellular microdomains for minutely regulated local Ca²⁺-signaling events.³

In this review we will discuss nuclear Ca²⁺ signaling in cardiomyocytes, with focus on current understanding of its regulation and role in gene transcription. We will also address difficulties in reliably measuring nuclear Ca²⁺ concentration, [Ca²⁺]_nuc, and tackle its involvement in development and progression of cardiac diseases. In addition, we will identify some unanswered questions to encourage further work.

2. Role of Nuclear Calcium in Cardiac Myocytes

Maintaining cardiac output, which is generally proportional to the tissues' need for oxygen, is one of the most intricate functions of circulatory system. Acute cardiac adaptation to increased oxygen demand is ensured by specific neurohormonal mediators (such as endothelin-1, angiotensin II, epinephrine and norepinephrine) which can very rapidly increase myocardial contractility and heart rate.^4–6 On the other hand, long-term responses require complex cellular mechanisms allowing cardiomyocytes to reprogram their gene expression profile to meet changing cardiac demand. The mechanism of long-term cardiac reprograming initiation and maintenance, as well as how it turns into maladaptive remodeling, is still not fully understood. However, over the past two decades, activation of Ca²⁺-dependent transcription factors in a process termed excitation-transcription coupling (ETC), has emerged as a connecting link integrating extracellular signaling information and subsequent cardiomyocyte reprograming. Pioneering work identified activation of key transcription factors via Ca²⁺-dependent signaling pathways in adult cardiomyocytes, including nuclear factor of activated T cells (NFAT) responding to calcineurin (CaN) activation and myocyte enhancer factor 2 (MEF2) responding to CaMKII activation and histone deacetylase (HDAC) phosphorylation.^{7, 8} Since the known Ca²⁺-dependent targets for these transcription factors (CaN-NFAT and CaMKII-HDAC) exist both in the cytosol and nucleus and can translocate, their transcriptional activation effects may be influenced by both [Ca²⁺]_cyto and [Ca²⁺]_nuc. There is also evidence of locally regulated (peri)nuclear Ca²⁺-signaling events and strategic organization of molecular components involved in ETC on the nuclear envelope and in the perinuclear regions.^9–11 Local increase in [Ca²⁺]_nuc, derived from Ca²⁺ released inside, or in the close proximity to the nucleus, in contrast to global increase in [Ca²⁺]_cyto, is currently believed to have a central role in the regulation of gene expression in cardiomyocytes.¹² Another transcription factor, cyclic-AMP-response element-binding protein (CREB), and its co-activator CREB-binding protein (CBP) are known to decode Ca²⁺ signals across the nucleus. Their activation requires high-amplitude changes in [Ca²⁺]_nuc ¹³ and they enhance expression of genes important in anti-oxidative and anti-apoptotic processes.¹⁴ However, we are not aware of direct evidence for [Ca²⁺]_nuc activation of CREB in adult cardiomyocytes. Therefore, better understanding of the regulation as well as reliable quantification of [Ca²⁺]_nuc in single cardiomyocytes is essential for understanding ongoing physiological and pathophysiological processes in the heart.

3. Nuclear Calcium Regulation

Although the nucleus is an autonomous subcellular compartment, well-defined by the nuclear envelope (NE), numerous nuclear pores complexes (NPCs) penetrate the NE and allow bidirectional passive diffusion of ions (including Ca²⁺) making the nucleoplasm only partly insulated from the surrounding cytoplasm. Thus, each cytoplasmic [Ca²⁺] transient (CaT) also elicits a nucleoplasmic CaT.¹⁵ Similarly, any stimuli that cause an elevation of [Ca²⁺]_cyto (i.e. mechanical stretch, increased heart rate or non-excitatory stimulation) will also lead to an increase in [Ca²⁺]_nuc.

On the other hand, accumulating evidence suggests that nucleoplasmic CaTs follow distinct kinetics and may – in part – be regulated independently from cytoplasmic CaTs.^{15, 16} A central role in the independent regulation of [Ca²⁺]_nuc, is attributed to the NE which, beside its contribution to nuclear structure, also acts as a functional Ca²⁺ store to regulate [Ca²⁺]_nuc.¹⁷ Even though recent studies provided compelling evidence that both mechanisms of [Ca²⁺]_nuc regulation co-exist in cardiomyocytes, their relative contribution under different conditions, as well as functional consequences of the specific message they transmit, remain elusive.

3.1 Passive diffusion

It was initially showed that Ca²⁺ ions can enter the nucleus via passive diffusion of cytoplasmic Ca²⁺ through NPCs transversing the NE. The NE consists of the inner and outer nuclear membrane with the space between them acting as a functional Ca²⁺ store, akin to, and with its lumen connected to, the sarcoplasmic reticulum (SR).¹⁸ The nuclear membranes fuse at many sites to form pores that harbor the NPCs. These multiprotein complexes are the major gateway for ions (including Ca²⁺) and small molecules to diffuse along their concentration gradients between the cytosol and nucleoplasm, given the approximate pore diameter of 8 nm.¹⁹ Although the conductance of NPCs may change in response to factors such as Ca²⁺ and ATP, therefore influencing the kinetics of Ca²⁺ traffic,²⁰ the pore itself does not entirely close, allowing punctate passages for Ca²⁺ between the cytoplasm and the nucleus.

In addition, NE of numerous cell types (including cardiomyocytes) is interrupted by infoldings that reach deep into the nucleoplasm and may even traverse the nucleus completely (for review see ²¹). The NE invaginations are lined by both, the inner and the outer nuclear membranes and filled with cytosol, SR and even mitochondria.²² Recently demonstrated presence of NPCs on the NE invaginations ensures effective nucleo-cytoplasmic ion diffusion and cargo transport in regions that would otherwise be remote from the nuclear periphery, by decreasing the diffusion delay and by increasing membrane surface area.²³

Whole cell Ca²⁺ oscillations

The Ca²⁺ cycle in cardiomyocytes that governs contraction and relaxation on a beat-to-beat basis consists of a transient rise and decay of [Ca²⁺]_cyto. During the cardiac action potential, myocyte membrane depolarization leads to opening of the voltage-dependent L-type Ca²⁺ channels, inducing an inward Ca²⁺ current. The increase in local intracellular [Ca²⁺] triggers the release of Ca²⁺ stored in the SR through Ca²⁺ release channels – ryanodine receptors (RyR) – in a positive feedback fashion. The transient increase in cytoplasmic free [Ca²⁺] allows Ca²⁺ binding to the myofilament protein troponin C. The conformational change of troponin regulatory complex leads to initiation of cross-bridge formation between actin and myosin, causing myocyte contraction. For relaxation to occur, free cytoplasmic [Ca²⁺] has to decline and allow Ca²⁺ to dissociate from troponin. Intracellular Ca²⁺ is mostly taken up by an ATP-dependent Ca²⁺ pump, the sarco-endoplasmic reticulum calcium-ATPase (SERCA), into the SR. To a quantitatively smaller extent, cytoplasmic Ca²⁺ is removed from the cell by the electrogenic sodium-calcium exchanger. About 1% of the activating Ca²⁺ is taken up by mitochondria or extruded via the sarcolemmal Ca²⁺-ATPase.²⁴ The dynamic regulation of Ca²⁺ transport mechanisms is essential at varying heart rates.²⁵ To achieve this, several cellular kinases and phosphatases regulate proteins involved in excitation-contraction coupling, providing their suitable activation and inhibition under different physiological conditions. In healthy hearts, Ca²⁺-handling proteins are regulated by α- and β-adrenergic receptor (α-AR and β-AR) stimulation upon the activation of sympathetic nerves, coupled to G_q and G_s protein-coupled signaling cascades, respectively. β-AR stimulation activates PKA which causes increased Ca²⁺ entry via L-type Ca²⁺ channels and enhances SR Ca²⁺ uptake (by phospholamban phosphorylation and dis-inhibition of SERCA). α-AR stimulation can also promote Ca²⁺ release from the SR and NE via inositol-1,4,5-trisphosphate receptors (IP₃R) located on the SR and NE. However, in ventricular myocytes the IP₃R-mediated Ca²⁺ release has greater impact on [Ca²⁺]_nuc vs. the rise and fall of [Ca²⁺]_cyto that drives contraction and relaxation. On the other hand, local IP₃R-mediated Ca²⁺ release can influence signaling (see below).

It is well established that due to the passive diffusion of Ca²⁺ ions through NPCs, each cytoplasmic CaT also elicits a nucleoplasmic CaT.¹⁵ It has been proposed that NE can function as ‘diffusion filter’ allowing only a limited fraction of cytoplasmic Ca²⁺ to enter the nucleoplasm, as well as introducing a kinetic delay in the equilibration of [Ca²⁺]_nuc and [Ca²⁺]_cyto.¹²

Based on NE invaginations and NPC, SERCA and RyR distribution around nucleus,²³ the rise in [Ca²⁺]_nuc is driven mainly by the RyR-dependent rise in [Ca²⁺]_cyto and Ca²⁺ diffusion via NPCs (including invaginations) which slows rise times and peak [Ca²⁺]_nuc (Figure 1). As SERCA is mainly expressed on the outer NE facing the cytoplasm (where there is also much more SERCA on SR), most Ca²⁺ probably has to diffuse out of the nucleus to be pumped back into the NE and SR, accounting for the much slower [Ca²⁺]_nuc decline. In line with this hypothesis, quantitative analysis of nucleoplasmic vs. cytoplasmic CaTs revealed significant nucleoplasmic-to-cytoplasmic [Ca²⁺] gradients during the cardiac cycle. Higher diastolic [Ca²⁺] and lower systolic [Ca²⁺] in the nucleus was explained by significantly slower CaTs kinetics in the nucleoplasm than in the cytoplasm.¹⁶ Still, the amplitude of the nuclear CaT was found to be proportional to the amplitude of the cytoplasmic CaT, providing evidence for the passive nature of nuclear CaT. One of the most important consequences of markedly slower kinetics of nucleoplasmic CaTs is the non-proportional increase in diastolic [Ca²⁺]_nuc vs. [Ca²⁺]_cyto when diastole is shortened at higher heart rates. An increase in pacing frequency is, therefore, enough to cause profound differences in the level of [Ca²⁺]_nuc vs. [Ca²⁺]_cyto.¹⁶

Figure 1. Nucleoplasmic [Ca²⁺] regulation.

[Ca²⁺]_nuc is the sum of Ca²⁺ that enters the nucleoplasm by diffusion from the cytoplasm through nuclear pores (red arrows) and Ca²⁺ that is released from the NE in a specific and regulated manner upon GPCRs stimulation (blue arrows). ECM, extracellular matrix; ET-1, endothelin-1; ATII, angiotensin II; NE, norepinephrine; GPCR, G protein-coupled receptor; PLC, phospholipase C; IP₃, inositol-1,4,5-trisphosphate; IP₃R, IP₃ receptors; RyR, ryanodine receptor; SERCA, SR-Ca²⁺-ATPase; DAG, diacylglycerol; TRPC, transient receptor potential cation channel; LCC, L-type calcium channel.

In this model, the degree of the kinetic delay might be subject to modulation, therefore regulating the portion of cytoplasmic Ca²⁺ diffusing to the nucleus upon specific stimuli. First of all, a greater expression of NPCs would allow a more rapid equilibration of [Ca²⁺]_i and [Ca²⁺]_nuc with a tendency to minimize nucleoplasmic-to-cytoplasmic [Ca²⁺] gradients. Increased permeability of NPCs would have similar effect on Ca²⁺ traffic between the nucleus and the cytoplasm. Additionally, changes in Ca²⁺ buffering capacity of the nucleus could favor or diminish nucleoplasmic CaTs propagation. It is also tempting to speculate that – similar to T-tubular sarcolemmal invaginations that are critical for coordinated Ca²⁺ cycling throughout the myocyte – NE invaginations may be critical for fine control of nucleoplasmic Ca²⁺ regulation. Fewer nuclear invaginations, as we see in heart failure, may also magnify such cytosol-nuclear [Ca²⁺] gradients.²³ However, further experimental work is necessary to confirm or dismiss these possibilities in cardiomyocytes.

Local Ca²⁺ oscillations - SR Ca²⁺ leak

In addition to changes of [Ca²⁺]_nuc as a result of whole cell CaTs, brief, spontaneous, local Ca²⁺ release events from the SR – so-called Ca²⁺ sparks (RyR release) and Ca²⁺ puffs (IP₃R release) – may also give rise to nuclear Ca²⁺ signal. Bootman et al. showed that if the spontaneous Ca²⁺ release occurs within the 5 µM distance from the nucleus, it may briefly increase [Ca²⁺]_nuc.²⁶ The advantage of controlling specific cellular processes by elementary Ca²⁺ signals is their rapid local action at relatively low energy cost to the cells, in contrast to global Ca²⁺ changes. Since the elementary Ca²⁺ signals have only a limited spatial spread (usually 2–4 µm), and the [Ca²⁺] declines sharply with distance from the site of origin, regulation of cellular activities relies on close localization of the Ca²⁺ channels and their targets. Existence of structural elements that correspond to these requirements were recently described in cardiomyocytes.²⁷ Subcellular organizational units formed by an association between the SR and the T-tubule segments of plasma membrane (dyads) were found to be frequently located in the space between the nearest myofibril and the NE. The linear distance between RyRs from the nearest dyads and the NE is as low as ≈200 nm or occasionally less. Ca²⁺ released from these dyads would reach the NE in less than 1 ms, in that way creating an effective perinuclear microdomain that can influence [Ca²⁺]_nuc.

Although we have convincing structural and functional evidence showing that [Ca²⁺]_nuc may be regulated by local cytoplasmic Ca²⁺ release events in the close proximity to the nucleus, how this affects downstream signaling and cellular processes under different physiological and pathophysiological conditions, requires future studies.

3.2 Receptor Stimulated regulation of Nuclear Calcium

In addition to [Ca²⁺]_nuc regulation via passive diffusion of Ca²⁺ ions from the cytoplasm, several lines of evidence suggest that there is an additional, entirely independent source of Ca²⁺ in the nucleus. First, the nucleus is a cellular compartment with its own perinuclear Ca²⁺ store (NE) that can actively store and release Ca²⁺.²³ Second, the NE expresses Ca²⁺-regulating proteins, including IP₃R Ca²⁺ release channels (facing the nucleoplasm and the cytoplasm) and Ca²⁺-buffering proteins.^{23, 28, 29} Third, specific stimuli may – via IP₃R-mediated Ca²⁺ release from the NE – increase [Ca²⁺]_nuc independently from [Ca²⁺]_cyto.^{10, 15}

Key component of the nuclear Ca²⁺-regulatory machinery independent of [Ca²⁺]_cyto is the G-protein coupled receptor (GPCR)-dependent activation of phospholipase C, followed by the generation of IP₃ and IP₃R stimulation (Figure 1). The high affinity type 2 IP₃R (IP₃R2) is the predominant subtype in cardiac myocytes, which in ventricular myocytes is concentrated in the NE.³⁰ One of the first experimental evidence of this concept in cardiomyocytes came from the work of Zima et al., showing that treatment of isolated nuclei with IP₃ leads to NE Ca²⁺ depletion via IP₃Rs, that is paralleled by an increase of [Ca²⁺]_nuc.³¹ Further studies revealed that a number of agonists such as ET-1,¹⁰ angiotensin II (ATII) ³² or insulin-like growth factor 1 (IGF-1) ³³ may use various signaling mechanisms to stimulate specific GPCRs upstream of IP₃ generation. The structural organization of this signaling may also involve either receptors and IP₃ production at T-tubule membranes that come very close to the nucleus,³³ or even GPCR-complexes right on the NE (Figure 2).³² Clearly, local IP₃R-mediated Ca²⁺ release can modify [Ca²⁺]_nuc that is driven by global CaT. Mechanisms of receptor-specific ligand action will be discussed next.

Figure 2. Three models of membrane receptor signaling to IP₃-dependent Ca²⁺ signals.

(A) In the traditional model, receptors (as for ET-1 in this example) activate IP₃ production at the cell periphery. The IP₃ has to diffuse a long way to the nuclear IP₃R to cause local [Ca²⁺]_nuc elevation by release from the Ca²⁺ store in the nuclear envelope. (B) The model indicating the insulin-like growth factor 1 receptor (IGF-1R) complex in plasma membrane invaginations, reducing the diffusional distance for IP₃ to reach the nucleus. (C) A third model where functional G-protein coupled receptors can exist near or on the nuclear envelope.

ET-1 receptors

Several years ago, Wu and colleagues were the first to show that ET-1, which activates GPCRs to produce IP₃, elicits local nuclear envelope Ca²⁺ release via IP₃R.¹⁰ They proposed the mechanism in which receptor signaling complex including the GPCR, phospholipase C and IP₃ precursor phosphatidylinositol 4,5-bisphosphate is located on the plasma membrane and IP₃R is located on the NE accessing local nuclear Ca²⁺ store. The communication between these two signaling hubs is achieved via IP₃ diffusion. Fluorescence resonance energy transfer-based IP₃ biosensors confirmed that ET-1 triggers IP₃ production at the plasma membrane that precedes the rise of IP₃ level in the nucleus and that most of the IP₃R in adult ventricular myocytes is largely localized to the NE.³⁴ These findings confirm the ability of cytoplasmic IP₃ to diffuse to the nucleus and this sequence of events correspond well to the kinetics of ET-1-induced rise in [Ca²⁺]_nuc. That is consistent with the longer range of IP₃ signaling (≈50 µm; greater than the radius of a myocyte),³⁵ although IP₃ is also hydrolyzed to its inactive forms such as IP₂.³⁶ An obvious question is why does cell produce excess of IP₃ far from its target? Whether it is to fine tune the nuclear Ca²⁺ response in terms of its kinetics or amplitude, or for the interplay with cytoplasmic IP₃R, complete understanding of regulatory mechanism behind this seemingly inefficient cellular strategy needs further clarification.

At the same time, a fraction of GPCRs stimulated by ET-1 appear to be localized at the nuclear membrane, where intracrine ET-1 signaling evokes IP₃-dependent increases in [Ca²⁺]_nuc.³⁷ In this regulatory model, ET-1 receptors are targeted directly to the NE following biosynthesis ³⁸ and activated selectively by endogenous ET-1, which is produced, stored and secreted by cardiomyocytes.³⁹ Similar to the previous mechanism, ET-1 stimulation resulted in an IP₃R-mediated increase in [Ca²⁺]_nuc.

Although we know that two different ET-1 receptor subtypes are responsible for these two different regulatory mechanisms, with type A ET-1 receptor being located at sarcolemma and responding to exogenous ET-1 ⁴⁰ and type B ET-1 receptor found at the NE and being activated by intracellular ET-1,³⁷ functional importance and activation pattern for each mechanism is still not fully understood.

Angioensin II receptors

Two different subtypes of functional ATII receptors, ATII receptor 1 and ATII receptor 2, have been detected in cardiomyocytes.⁴¹ Similarly to ET-1 receptors, ATII receptors can be located at the T-tubular network of the plasma membrane and on the NE. Again, the fraction of the receptor found on the NE is a result of intracellular synthesis and trafficking, rather than endocytosis. Exposure of isolated nuclei to ATII transiently raised [Ca²⁺]_nuc, whereas the use of subtype specific agonists revealed that ATII receptor 1 is specifically responsible for nuclear [Ca²⁺] response.³² When a specific inhibitor of IP₃R was applied, ATII-mediated increase in [Ca²⁺]_nuc was completely blocked, consistent with the ATII effects being IP₃-dependent. In isolated cardiomyocytes, application of ATII increased diastolic [Ca²⁺] in both the cytoplasm and the nucleus. The increase in [Ca²⁺]_nuc was much larger than the increase in diastolic [Ca²⁺]_cyto and it was prevented by cardiomyocytes pretreatment with IP₃R blocker.²³

Although blockers of the renin-angiotensin system are widely used in the treatment of hypertension and heart failure, the exact molecular mechanism of how activation of different ATII receptor subtypes at different subcellular localization is orchestrated still remains elusive.

α1-Adrenergic Receptors

Presence and signaling function of another GPCR, α-AR, on the NE has been demonstrated in cardiomyocytes.^{42, 43} When two different subtypes of functional α-ARs (α1A and α1B) were expressed as GFP fusion proteins in α1AB-KO cardiomyocytes, both could be observed at nuclear and perinuclear locations.⁴⁴ Although evidence for colocalization of both α1A and α1B subtypes with G-protein and PLC in the nuclear membrane is available, the involvement of α1-AR in the regulation of [Ca²⁺]_nuc remains unknown.

β-Adrenergic Receptors

In a series of experiments, Boivin et al. presented strong evidence for the presence and functional role of β-ARs on the NE.⁴⁵ The best investigated downstream effector of β-ARs is the enzyme adenylyl cyclase, which catalyzes the conversion of ATP to cAMP, and is also identified in the NE.⁴⁶ Rise in cAMP levels activates cAMP-dependent protein kinases (PKA). In turn, PKA increases the phosphorylation state of target proteins involved in the regulation of Ca²⁺ cycling (L-type Ca²⁺ channels, phospholamban, RyR). Some of these phosphorylation events increase the Ca²⁺ content of the cardiomyocytes, in this way not only elevating force but also hastening relaxation.⁴⁷ As NE contains components for regulation of the Ca²⁺ equilibrium, we cannot exclude partial role of nuclear β-ARs in the regulation of [Ca²⁺]_nuc.

IGF-1 receptors

In a recent study, Ibarra et al. proposed an exciting extension to the previously described models of receptor-mediated [Ca²⁺]_nuc regulation.³³ They provided evidence that an entire signaling complex, in this case IGF-1 receptor, G-protein, phospholipase C, and IP₃ production, is situated in the close proximity to the nucleus due to deep plasma membrane infoldings towards the nuclear envelope. IP₃ production close to the IP₃R at the NE greatly reduce the amount of IP₃ required for increase in [Ca²⁺]_nuc and provides spatial insulation of nuclear Ca²⁺ signals from large cytoplasmic Ca²⁺ oscillations.

There is now sufficient evidence for the existence of NE-proximal pathways (Figure 2). However, it remains to be clarified what the relative importance of different structural pathways is vs. the traditional surface membrane IP₃ production and if these pathways have functionally discrete physiological functions.

4. Nuclear Ca²⁺ in hypertrophy and heart failure

Heart failure (HF) is characterized by impaired systolic and diastolic function with abnormalities of Ca²⁺ handling in cardiomyocytes underlying contractile dysfunction in failing hearts.^{48, 49} Current research is focused on better understanding the early triggers for alterations of intracellular Ca²⁺ homeostasis, as well as whether Ca²⁺-dependent signaling pathways (e.g. via CaMKII and calcineurin) may initiate and/or facilitate the progression of cardiac remodeling (such as hypertrophy) to severe HF. Although substantial progress has been made towards understanding the role of altered cytoplasmic Ca²⁺ homeostasis in hypertrophy and HF,⁵⁰ not as much data are available on nucleoplasmic Ca²⁺ homeostasis. It may, however, be a critical component to cardiac remodeling by influencing protein expression via nuclear Ca²⁺-dependent gene transcription.¹⁰

As previously mentioned, NE contains invaginations that reach deep into the nucleoplasm and may facilitate intranuclear regulation of ions in regions that would otherwise be remote from the nuclear periphery.²¹ Our most recent work showed that there is a progressive decrease in NE invagination density in hypertrophied and failing hearts, associated with alterations of [Ca²⁺]_nuc in electrically stimulated cardiomyocytes. Changes in nuclear CaTs occurred at an earlier disease stage than did cytoplasmic CaT changes, with an onset so early that they may well be involved in the development and progression of hypertrophy and HF.²³ As expected, due to the slower kinetics of nucleoplasmic CaTs, the frequency-dependent higher increase in diastolic [Ca²⁺]_nuc vs. diastolic [Ca²⁺]_cyto was even more pronounced in cardiomyocytes from hypertrophied and failing hearts. Higher pacing frequencies may, therefore, be causally involved in the remodeling processes leading to HF, in particular when considering the often elevated heart rates of patients with HF.

Significant changes in specific proteins of the NPC in cardiomyocytes from failing human hearts were recently reported.⁵¹ These changes, related to ventricular function, could be accompanied by alterations in the nucleo-cytoplasmic transport and regulation of gene expression. In addition, mutations in genes encoding proteins of the inner and outer nuclear membrane as well as nucleoskeleton cause early onset of cardiomyopathy with altered nuclear positioning, shape, and chromatin organization.^{52, 53} Indeed, such changes could also interfere with the nucleoplasmic Ca²⁺ handling and nuclear Ca²⁺-mediated gene expression.

Increased Ca²⁺ leak via RyRs is another well-established feature of HF.⁵⁴ Even more, RyR-mediated SR Ca²⁺ leak itself promotes myocardial remodeling, including eccentric hypertrophy, dilatation and contractile dysfunction in RyR2^R4496C+/− mice under pressure overload.⁵⁵ It is tempting to speculate that RyR leak in perinuclear dyads may have a critical role in promoting hypertrophy as it may directly increase [Ca²⁺]_nuc.

GPCRs (α-adrenergic, ET-1 and ATII receptors) that induce IP₃ production are also more activated during the development and progression of HF.^{56, 57} There are also higher levels of IP₃R2 expression in HF, as well as downstream activation of nuclear CaMKII-dependent nuclear signaling.^{58, 59} Elevated [Ca²⁺]_nuc may enhance the IP₃ sensitivity of IP₃ receptors ⁶⁰ and could well synergize with other factors which elevate [Ca²⁺]_nuc in HF, as we have shown for AII and high pacing frequency.²³ Indeed, increased IP₃R2 expression was proposed to be essential during hypertrophy and HF.⁶¹ In line with this notion, we recently observed increased perinuclear IP₃R2 expression, and higher diastolic [Ca²⁺]_nuc vs. [Ca²⁺]_cyto in HF cardiomyocytes treated with ATII.²³

Complex regulation of [Ca²⁺]_nuc in cardiomyocytes seems to be an important determinant of cardiac remodeling and may contribute to the development and progression of hypertrophy and HF. Normalization of nucleoplasmic Ca²⁺ levels and its regulation may, therefore, be a novel therapeutic approach for preventing adverse cardiac remodeling.

5. Measuring nuclear [Ca²⁺]

As [Ca²⁺]_nuc in cardiomyocytes regulates transcription and its alterations are implicated in remodelling processes leading to HF, quantification of subcellularly resolved Ca²⁺ signals is essential for understanding physiological and pathological processes in the heart.

Advancement of confocal laser microscopy together with the development of chemical fluorescent Ca²⁺ indicators provides the basis for visualization of whole cell and subcellular [Ca²⁺] fluxes. In principle, upon excitation, these indicators emit light at particular wavelengths and the emitted fluorescence intensity or emission spectrum is changed in a Ca²⁺ bound state.⁶²

The dissociation constant (K_d) as a measure of Ca²⁺ binding affinity is crucial for the selection of the appropriate Ca²⁺ indicator for the cellular compartment of interest. Low affinity, high K_d dyes (e.g. Fluo-5N and Mag-fluo-4) are used for the visualization of [Ca²⁺] changes in the SR or NE, whereas, high affinity, low K_d indicators (e.g. Fluo-3 and Fluo-4) are preferred for measuring changes in cytosolic and nucleoplasmic free [Ca²⁺]. The use of fluorescent Ca²⁺ indicators for simultaneous quantification of cytoplasmic and nucleoplasmic Ca²⁺ fluctuations gets complicated somewhat as properties of fluorescent Ca²⁺-indicators in intracellular compartments may differ, due to the specific physicochemical properties of the environment. In situ calibration of the most frequently used Ca²⁺ indicator, Fluo-4, revealed significant differences in the apparent K_d between cytoplasm and nucleoplasm of adult cardiomyocytes,¹⁶ confirming that without proper independent determination of the indicator properties in the nucleoplasm vs. the cytoplasm, any quantitative analysis of [Ca²⁺]_nuc versus [Ca²⁺]_cyto is not reliable.

Targeted molecular tools, engineered in such way that their mode of action is restricted to a specific subcellular compartment is a promising new strategy to avoid artefacts of commonly used chemical Ca²⁺ indicators.⁶³ Protein-based Ca²⁺ probes such as cameleons,⁶⁴ pericams⁶⁵ or aequorins⁶⁶ have been successfully targeted to the nucleus. Differences in behaviour between [Ca²⁺]_nuc and [Ca²⁺]_cyto have been reported in neurons using cameleon,⁶⁷ and ratiometric pericam has been used to monitor [Ca²⁺]_nuc in beating neonatal cardiomyocytes.⁶⁸ However, improvement in dynamic range, in combination with ultra-fast kinetics needed to reliably monitor Ca²⁺ changes in cardiomyocytes is a continuing challenge.

6. Conclusion

At present, due to limited tools for measuring and selectively blocking/buffering nuclear Ca²⁺, we have only begun to understand how a cardiomyocyte decodes a complex array of nuclear Ca²⁺ signals to reprogram gene expression profile and meet constantly varying cardiac demand. It is striking that a simple ion such as Ca²⁺ can regulate so many different cellular processes and it seems that current research has opened more questions than the answers it has provided. Thus, understanding the regulation of nuclear Ca²⁺ in cardiomyocytes will undoubtedly remain an exciting research focus and a topic of debate for some time.