Abstract

Calcium ions are the most ubiquitous and versatile signaling molecules in eukaryotic cells. Calcium homeostasis and signaling systems are crucial for both the normal growth of the budding yeast Saccharomyces cerevisiae and the intricate working of the mammalian heart. In this paper, we make a detailed comparison between the calcium homeostasis/signaling networks in yeast cells and those in mammalian cardiac myocytes. This comparison covers not only the components, structure and function of the networks but also includes existing knowledge on the measured and simulated network dynamics using mathematical models. Surprisingly, most of the factors known in the yeast calcium homeostasis/signaling network are conserved and operate similarly in mammalian cells, including cardiac myocytes. Moreover, the budding yeast S. cerevisiae is a simple organism that affords powerful genetic and genomic tools. Thus, exploring and understanding the calcium homeostasis/signaling system in yeast can provide a shortcut to help understand calcium homeostasis/signaling systems in mammalian cardiac myocytes. In turn, this knowledge can be used to help treat relevant human diseases such as pathological cardiac hypertrophy and heart failure.

Introduction

In eukaryotic cells, the calcium ion (Ca2+) functions as a ubiquitous intracellular messengers regulating a myriad of biological processes such as cell proliferation, muscle contraction, programmed cell death, etc. (Berridge, 1998, 2000, 2003; Putney, 2005). Calcium homeostasis systems are highly regulated metabolic pathways used by cells to maintain Ca2+ within optimal concentration ranges in the cytosol and other organelles. For example, in normal-growing yeast (Saccharomyces cerevisiae), cytosolic Ca2+ is maintained in the range of 50–200 nM, in the presence of environmental Ca2+ concentrations ranging from <1 μM to >100 mM, through the functioning of an elaborate calcium homeostasis system as shown in Fig. 1a (Bonilla & Cunningham, 2002; Cui & Kaandorp, 2006; Cui, 2009). Extracellular stimuli cause a sudden increase in the cytosolic Ca2+ level, activating the effector proteins to exert a cellular response.

1

(a) Calcium homeostasis system in Saccharomyces cerevisiae. CaM, calmodulin; CaN,calcineurin; ER, endoplasmic reticulum. (b) Calcium homeostasis system in mammalian cardiac myocytes (this graph is modified after Fig. 1 in Bers, 2002). The schematic inset at the down-right corner shows the action potential (solid line) and the bulk cytosolic Ca2+ transient (dashed line) (this inset is modified after fig. 1c in Shannon, 2004). LTCC, L-Type Ca2+ channel; ATPase (ATP); NCX, Na+/Ca2+ exchanger; SR, sarcoplasmic reticulum; SERCA, SR Ca2+-ATPase; PLB, phospholamban; RyR, ryanodine receptor; mRyR, mitochondrial ryanodine receptor; UP, mitochondrial uniporter; RaM, rapid-mode uptake pathway.

1

(a) Calcium homeostasis system in Saccharomyces cerevisiae. CaM, calmodulin; CaN,calcineurin; ER, endoplasmic reticulum. (b) Calcium homeostasis system in mammalian cardiac myocytes (this graph is modified after Fig. 1 in Bers, 2002). The schematic inset at the down-right corner shows the action potential (solid line) and the bulk cytosolic Ca2+ transient (dashed line) (this inset is modified after fig. 1c in Shannon, 2004). LTCC, L-Type Ca2+ channel; ATPase (ATP); NCX, Na+/Ca2+ exchanger; SR, sarcoplasmic reticulum; SERCA, SR Ca2+-ATPase; PLB, phospholamban; RyR, ryanodine receptor; mRyR, mitochondrial ryanodine receptor; UP, mitochondrial uniporter; RaM, rapid-mode uptake pathway.

Extracellular Ca2+ normally enters the cytosol of yeast cells through an unknown transporter X (Locke, 2000). Cytosolic Ca2+ is then transported into large vacuoles through a vacuolar calcium ATPase Pmc1 and a vacuolar Ca2+/H+ exchanger Vcx1. A calcium ATPase termed Pmr1 is responsible for pumping cytosolic Ca2+ into the endoplasmic reticulum and Golgi, and excessive Ca2+ in the secretory pathway is then extruded from the cell via exocytosis. Another transporter on the plasma membrane, transporter M, has been detected recently. This transporter transports Ca2+ into the yeast cell in response to very high extracellular Ca2+ (Cui, 2009). A plasma membrane voltage-gated Ca2+ channel, composed of Cch1 and Mid1, becomes activated only under abnormal conditions such as membrane depolarization, depletion of secretory Ca2+ (Bonilla & Cunningham, 2003), pheromone stimulation (Muller, 2001; Zhang, 2006) and hypotonic shock (Batiza, 1996). In response to extracellular hypertonic shock, vacuolar Ca2+ is released into the cytosol through Yvc1, a distant homolog of mammalian TRPC-type Ca2+ channels (Denis & Cyert, 2002). In response to sudden increases in extracellular Ca2+, or the sudden removal of extracellular Mg2+ (Wiesenberger, 2007), the universal Ca2+ sensor protein calmodulin can bind and activate the protein phosphatase calcineurin. In turn, this inhibits the function of Vcx1 and induces the expression of Pmc1 and Pmr1 via activation of the Crz1 transcription factor and its subsequent translocation into the nucleus (Stathopoulos-Gerontides, 1999). In addition to these slow calcineurin-dependent feedback networks, rapid cytosolic Ca2+-dependent feedback also inhibits the activities of transporters M and X. However, the details of the constituents and the mechanisms of these rapid feedback inhibition pathways remain to be identified (Cui, 2009). The activity of calcineurin is regulated by Rcn1, whose function is regulated by Mck1, a member of the GSK-3 family of protein kinases (Hilioti, 2004).

In contrast to the calcium homeostasis process in yeast, calcium homeostasis in cardiac myocytes is inseparable from the calcium-signaling process involved in excitation–contraction coupling, the process that links electrical excitation of the myocytes to contraction of the whole heart (Barry & Bridge, 1993; Bers, 2002). Cardiac myocytes must achieve a resting cytosolic Ca2+ concentration of <200 nM if the contractile elements in the cells are to relax. As shown in Fig. 1b, when the cardiac myocyte is in a resting relaxed state, extracellular calcium enters the cytosol through a Ca2+ leak channel, while the sarcolemmal Ca2+-ATPase (PMCA) and a sarcolemmal Na+–Ca2+ exchanger (NCX) are responsible for cellular Ca2+ efflux. During the cardiac action potential, Ca2+ enters the cell through L-type Ca2+ channels (LTCC), which are located primarily at sarcolemmal–sarcoplasmic reticulum (SR) junctions, where the SR Ca2+ release channels [i.e. ryanodine receptors (RyRs)] exist. In addition, a small amount of extracellular Ca2+ will enter the cytosol through the NCX (normally, this exchanger functions to pump calcium out of the cell, but it can also function in the reverse direction). These Ca2+ entries trigger Ca2+ release from SR through RyRs. As a result, the cytosolic free Ca2+ concentration is increased, which facilitates the binding of cytosolic Ca2+ with the myofilament protein troponin C to switch on the cell's contractile machinery. Later, cytosolic Ca2+ is removed by the SR Ca2+-ATPase (SERCA; its function is regulated by phospholamban), sarcolemmal NCX, sarcolemmal Ca2+-ATPase and the mitochondrial Ca2+ uniporter, or other mitochondrial transport pathways [rapid-mode uptake pathway (RaM) and mitochondrial ryanodine receptor (mRyR)], such that its concentration is reduced. In turn, this decreased cytosolic Ca2+ concentration leads to the dissociation of Ca2+ from troponin C and subsequent cardiac relaxation (Bers, 2002). Mitochondrial RaM and mRyR are thought to underlie this phenomenon of excitation–metabolism coupling (Brookes, 2004).

Comparison of relevant calcium networks

Regulated membrane ion transport constitutes the main scheme used by cells to maintain optimal concentrations of metal ions in their cytosol. The names, types and regulations of the various calcium transport proteins involved in calcium homeostasis/signaling systems in yeast cells and mammalian cardiac myocytes are listed in Table 1. A concise summary of the functional protein counterparts in these two systems is shown in Table 2. As we can see from Tables 1 and 2, most proteins involved in calcium homeostasis and signaling in yeast have functional counterparts in the corresponding system in mammalian cardiac myocytes. In fact, the functional expression of heterologous proteins in yeast calcium homeostasis machinery has been recently shown to be a feasible, and elegant way, to investigate the function of mammalian Ca2+-transport proteins. For example, it has been demonstrated that heterologous expression of mammalian PMCA, SERCA and hSPCA1 pumps all fully complemented the BAPTA hypersensitivity of K616 (Δpmr1Δpmc1Δcnb1), a yeast mutant-lacking endogenous Ca2+ pumps (Ton & Rao, 2004; Ton, 2002).

1

Calcium transport proteins that affect calcium homeostasis in yeast cells and cardiac myocytes

 Protein name Type Regulation Other notes 
Budding yeast Transporter X Transporter Regulated by the extracellular Mg2+ level and by the rapid cytosolic Ca2+-dependent feedback inhibition pathway independent of calcineurin (Cui, 2009 
Transporter M Transporter Regulated by the extracellular Mg2+ level and by the rapid cytosolic Ca2+-dependent feedback inhibition pathway independent of calcineurin (Cui, 2009Opens in case of high extracellular Ca2+ 
Cch1-Mid1 VGCC-type channel Regulated by plasma membrane potential and some other unidentified factors (Locke, 2000; Muller, 2001). Its activity is partially inhibited by L-type VGCC blockers nifedipine and verapamil and increased by diltiazem (Teng, 2008Activated only under some abnormal conditions 
Pmc1 ATPase Regulated by a slow calcineurin-dependent gene expression feedback control pathway through Crz1 (Kingsbury & Cunningham, 2000; Stathopoulos-Gerontides, 1999Critical for long-term calcium homeostasis 
Vcx1 Ca2+/H+ exchanger Regulated by vacuolar pH and by calcineurin through a presumably post-translational mechanism (Bonilla & Cunningham, 2002; Kingsbury & Cunningham, 2000Critical for short-term calcium homeostasis 
Pmr1 P-type ATPase Regulated by a slow calcineurin-dependent gene expression feedback control pathway through Crz1 (Kingsbury & Cunningham, 2000Mediating the high-affinity transport of Ca2+ and Mn2+ 
Yvc1 Channel Regulated by cytosolic calcium level, calmodulin and cytosolic pH (Bertl & Slayman 1992; Su, 2009 
Mammalian Cardiac Myocyte Sarcolemmal Leak Channel Regulated by sarcolemmal membrane potential (Shannon, 2004 
LTCC Channel Regulated by sarcolemmal membrane potential and by CaMKII and PKA through phosphorylation (Bers, 2002; Sipido & Eisner, 2005 
Sarcolemmal NCX 3Na+/Ca2+ exchanger Regulated by sarcolemmal membrane potential and by both extracellular and the cytosolic Na+ and Ca2+ concentrations and by cytosolic Ca2+-dependent gene expression feedback control pathway (Bers, 2002, 2008; Shannon, 2004; Prasad & Inesi, 2009Rapid removal of cytoplasmic Ca2+(contribution: 28%); reversible 
Sarcolemmal Ca2+-ATPase ATPase Regulated trivially by cytosolic Ca2+ (Shannon, 2004Slow removal of cytoplasmic Ca2+ (1%) 
RyR Channel Regulated by cytosolic calcium through a CICR mechanism and by numerous proteins (e.g. calmodulin, CaMKII, FK-506 binding protein, PKA, phosphatases 1 and 2A and sorcin) and by a cytosolic Ca2+-dependent gene expression feedback control pathway (Bers, 2002, 2008A total of more than 100 RyRs are arranged in large organized arrays 
SERCA ATPase Regulated by PLB, whose phosphorylation state is regulated by PKA and CaMKII. The expressions of both SERCA and PLB may be regulated by cytosolic Ca2+-dependent gene expression feedback control pathways (Bers, 2002; Sipido & Eisner, 2005; Prasad & Inesi, 2009Rapid removal of cytoplasmic Ca2+ (70%) 
Mitochondrial Uniporter Transporter Regulated by mitochondrial membrane potential and by a p38 MAP kinase-dependent pathway (Brookes, 2004Slow removal of cytoplasmic Ca2+ (<1%) 
Mitochondrial NCX 2Na+/Ca2+ exchanger Regulated by mitochondrial membrane potential and by both mitochondrial and cytosolic Na+ and Ca2+ concentrations (Brookes, 2004 
mRyR Channel Regulated by cytosolic calcium through a calcium-induced Ca2+-uptake mechanism (Brookes, 2004Rapid removal of cytoplasmic Ca2+ (<1%) 
RaM Possibly channel Regulated by cytosolic Ca2+ concentration, strongly inhibited by AMP and affected by the cytosolic ADP level in a biphasic way (Buntinas, 2001Quick removal of cytoplasmic Ca2+ (<1%) 
 Protein name Type Regulation Other notes 
Budding yeast Transporter X Transporter Regulated by the extracellular Mg2+ level and by the rapid cytosolic Ca2+-dependent feedback inhibition pathway independent of calcineurin (Cui, 2009 
Transporter M Transporter Regulated by the extracellular Mg2+ level and by the rapid cytosolic Ca2+-dependent feedback inhibition pathway independent of calcineurin (Cui, 2009Opens in case of high extracellular Ca2+ 
Cch1-Mid1 VGCC-type channel Regulated by plasma membrane potential and some other unidentified factors (Locke, 2000; Muller, 2001). Its activity is partially inhibited by L-type VGCC blockers nifedipine and verapamil and increased by diltiazem (Teng, 2008Activated only under some abnormal conditions 
Pmc1 ATPase Regulated by a slow calcineurin-dependent gene expression feedback control pathway through Crz1 (Kingsbury & Cunningham, 2000; Stathopoulos-Gerontides, 1999Critical for long-term calcium homeostasis 
Vcx1 Ca2+/H+ exchanger Regulated by vacuolar pH and by calcineurin through a presumably post-translational mechanism (Bonilla & Cunningham, 2002; Kingsbury & Cunningham, 2000Critical for short-term calcium homeostasis 
Pmr1 P-type ATPase Regulated by a slow calcineurin-dependent gene expression feedback control pathway through Crz1 (Kingsbury & Cunningham, 2000Mediating the high-affinity transport of Ca2+ and Mn2+ 
Yvc1 Channel Regulated by cytosolic calcium level, calmodulin and cytosolic pH (Bertl & Slayman 1992; Su, 2009 
Mammalian Cardiac Myocyte Sarcolemmal Leak Channel Regulated by sarcolemmal membrane potential (Shannon, 2004 
LTCC Channel Regulated by sarcolemmal membrane potential and by CaMKII and PKA through phosphorylation (Bers, 2002; Sipido & Eisner, 2005 
Sarcolemmal NCX 3Na+/Ca2+ exchanger Regulated by sarcolemmal membrane potential and by both extracellular and the cytosolic Na+ and Ca2+ concentrations and by cytosolic Ca2+-dependent gene expression feedback control pathway (Bers, 2002, 2008; Shannon, 2004; Prasad & Inesi, 2009Rapid removal of cytoplasmic Ca2+(contribution: 28%); reversible 
Sarcolemmal Ca2+-ATPase ATPase Regulated trivially by cytosolic Ca2+ (Shannon, 2004Slow removal of cytoplasmic Ca2+ (1%) 
RyR Channel Regulated by cytosolic calcium through a CICR mechanism and by numerous proteins (e.g. calmodulin, CaMKII, FK-506 binding protein, PKA, phosphatases 1 and 2A and sorcin) and by a cytosolic Ca2+-dependent gene expression feedback control pathway (Bers, 2002, 2008A total of more than 100 RyRs are arranged in large organized arrays 
SERCA ATPase Regulated by PLB, whose phosphorylation state is regulated by PKA and CaMKII. The expressions of both SERCA and PLB may be regulated by cytosolic Ca2+-dependent gene expression feedback control pathways (Bers, 2002; Sipido & Eisner, 2005; Prasad & Inesi, 2009Rapid removal of cytoplasmic Ca2+ (70%) 
Mitochondrial Uniporter Transporter Regulated by mitochondrial membrane potential and by a p38 MAP kinase-dependent pathway (Brookes, 2004Slow removal of cytoplasmic Ca2+ (<1%) 
Mitochondrial NCX 2Na+/Ca2+ exchanger Regulated by mitochondrial membrane potential and by both mitochondrial and cytosolic Na+ and Ca2+ concentrations (Brookes, 2004 
mRyR Channel Regulated by cytosolic calcium through a calcium-induced Ca2+-uptake mechanism (Brookes, 2004Rapid removal of cytoplasmic Ca2+ (<1%) 
RaM Possibly channel Regulated by cytosolic Ca2+ concentration, strongly inhibited by AMP and affected by the cytosolic ADP level in a biphasic way (Buntinas, 2001Quick removal of cytoplasmic Ca2+ (<1%) 

The quantitative importance of various routes (SERCA, sarcolemmal NCX, sarcolemmal Ca2+-ATPase, mitochondrial Ca2+-sequestering pathways) for the removal of cytoplasmic Ca2+ varies between species. The proportions (70%, 28%, 1%, 1%) shown in Table 1 are for rabbit ventricular myocytes (Bers, 2002).

VGCC, voltage-gated Ca2+ channel.

2

Functional counterparts of proteins in calcium homeostasis and signaling systems in yeast and cardiac myocytes

Yeast protein Cardiac protein in human Similarity Equivalence and complementation 
Functional similarity Gene sequence similarity (%) 
Transporter X Sarcolemmal leak Both mediate constitutive Ca2+ influx Not known Not known 
Cch1 Ca(v)1.2 (α 1C subunit of LTCC) Both mediate additional Ca2+ influx in most cases of calcium signaling and are inhibited by L-type VGCC blockers nifedipine and verapamil (Teng, 200845 Equivalent 
Vcx1 NCX1 Exchangers mediating the removal of cytoplasmic Ca2+ 31 No 
Pmc1 PMCA1 ATPases mediating the removal of cytoplasmic Ca2+ 50 Equivalent, complementation carried out (Ton & Rao, 2004
Pmr1 SERCA2 Both mediate the sequestration of cytoplasmic Ca2+ into ER/SR and are regulated by cytosolic Ca2+-dependent gene expression feedback control pathways (Locke, 2000; Cui & Kaandorp, 2006; Prasad & Inesi, 200948 Partially equivalent, complementation carried out. Mammalian SERCA complements Pmr1 on its Ca2+ transport activity, but fails to complement its Mn2+ transport activity (Ton & Rao, 2004; Ton et al., 2002) 
Cmd1 CAMI, CAMII Calmodulin (Ca2+ sensor proteins) 66 for Cmd1 with CAMI; 63 for Cmd1 with CAMII Equivalent, complementation carried out (Cyert, 2001
Cna1 CNA1 Catalytic subunit of calcineurin 54 (Cyert, 1991Equivalent 
Cnb1 CNBII Regulatory subunit of calcineurin 56 (Cyert & Thorner, 1992Equivalent 
Rcn1 RCAN1 Regulator of calcineurin 43 Equivalent, complementation carried out (Kingsbury & Cunningham, 2000
Mck1 Gsk3β Kinases mediating the phosphorylation of Rcn1 and RCAN1 proteins 49 Equivalent 
Crz1 NFATc4 Calcineurin-dependent transcription factor 40 No 
Yvc1 TRPC1 Transient receptor potential (TRP) Ca2+ channels 50 Equivalent 
Yeast protein Cardiac protein in human Similarity Equivalence and complementation 
Functional similarity Gene sequence similarity (%) 
Transporter X Sarcolemmal leak Both mediate constitutive Ca2+ influx Not known Not known 
Cch1 Ca(v)1.2 (α 1C subunit of LTCC) Both mediate additional Ca2+ influx in most cases of calcium signaling and are inhibited by L-type VGCC blockers nifedipine and verapamil (Teng, 200845 Equivalent 
Vcx1 NCX1 Exchangers mediating the removal of cytoplasmic Ca2+ 31 No 
Pmc1 PMCA1 ATPases mediating the removal of cytoplasmic Ca2+ 50 Equivalent, complementation carried out (Ton & Rao, 2004
Pmr1 SERCA2 Both mediate the sequestration of cytoplasmic Ca2+ into ER/SR and are regulated by cytosolic Ca2+-dependent gene expression feedback control pathways (Locke, 2000; Cui & Kaandorp, 2006; Prasad & Inesi, 200948 Partially equivalent, complementation carried out. Mammalian SERCA complements Pmr1 on its Ca2+ transport activity, but fails to complement its Mn2+ transport activity (Ton & Rao, 2004; Ton et al., 2002) 
Cmd1 CAMI, CAMII Calmodulin (Ca2+ sensor proteins) 66 for Cmd1 with CAMI; 63 for Cmd1 with CAMII Equivalent, complementation carried out (Cyert, 2001
Cna1 CNA1 Catalytic subunit of calcineurin 54 (Cyert, 1991Equivalent 
Cnb1 CNBII Regulatory subunit of calcineurin 56 (Cyert & Thorner, 1992Equivalent 
Rcn1 RCAN1 Regulator of calcineurin 43 Equivalent, complementation carried out (Kingsbury & Cunningham, 2000
Mck1 Gsk3β Kinases mediating the phosphorylation of Rcn1 and RCAN1 proteins 49 Equivalent 
Crz1 NFATc4 Calcineurin-dependent transcription factor 40 No 
Yvc1 TRPC1 Transient receptor potential (TRP) Ca2+ channels 50 Equivalent 

The gene sequence similarity is analyzed using the global pairwise alignment tool named as stretcher available from the website http://mobyle.pasteur.fr/cgi-bin/portal.py. A detailed introduction of this popular tool can be found at http://bioweb2.pasteur.fr/docs/EMBOSS/stretcher.html. The sequence similarity score calculated using stretcher are in accordance with the reported values in the literature (for example, the similarity score for Cna1 and CNA1 is 54%; the similarity score for Cnb1 and CNBII is 56%).

Another important observation from Table 1 is that the phosphorylation of several key proteins (phospholamban, LTCC and RyR), by kinases such as protein kinase A (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII), plays a critical role in the calcium homeostasis process in cardiac myocytes (Bers, 2002; Sipido & Eisner, 2005). This is because the short duration of a heart beat (typically <1 s) requires very rapid regulation processes such as protein phosphorylation, whose speed can never be achieved by an expression control process, which usually requires >3 min (Bers, 2002; Cui, 2009). This also explains why yeast cells use slow calcineurin-dependent expression control pathways (see Fig. 1a) to regulate only the long-term rather than the short-term calcium homeostasis process (Cui & Kaandorp, 2006; Cui, 2009). From this comparison, we can speculate that, besides the possibility of involvement of direct inhibition by calmodulin, the rapid Ca2+-dependent feedback inhibition of transporters M and X may also involve kinase-dependent phosphorylation (Cui, 2009).

Measured dynamics and mathematical modeling

Protein-based indicators such as aequorin have been widely used to monitor the cytosolic-free calcium concentration in populations of yeast cells due to the ease of recombinant expression in these unicellular organisms (Miseta, 1999; Denis & Cyert, 2002; Cui, 2009). A simple algebraic relationship has been experimentally established between the light emission of aequorin and the calcium concentration (Allen, 1977). For example, Fig. 2a describes cytosolic calcium transients that were measured using aequorin for the yvc1 cch1 mutant under conditions of a very strong extracellular calcium shock (800 mM), together with an extracellular Mg2+ challenge (Cui, 2009). In cardiac myocytes, nonratiometric dyes, such as fluo and rhod dyes, are used widely to determine the calcium concentration (Cheng, 1996; Weber, 2002; Rudolf, 2003). Compared with the rapid change in the cardiac calcium concentration shown in the inset of Fig. 1b (the dashed line), the change of yeast cytosolic calcium concentration (see Fig. 2a) seems to be much slower.

2

(a) Measured aequorin luminescence curves for yeast yvc1 cch1 mutant cells in response to the sudden step-like increase of the extracellular calcium concentration (800 mM), together with the step-like increase of extracellular Mg2+ to various concentrations (0, 0.1, 0.3, 1 and 3 mM). (b) The corresponding simulated response curves.

2

(a) Measured aequorin luminescence curves for yeast yvc1 cch1 mutant cells in response to the sudden step-like increase of the extracellular calcium concentration (800 mM), together with the step-like increase of extracellular Mg2+ to various concentrations (0, 0.1, 0.3, 1 and 3 mM). (b) The corresponding simulated response curves.

A mathematical model has been constructed to reproduce these measured calcium transients in yeast cells, and the corresponding simulated response curves are shown in Fig. 2b. In the model (Cui, 2009), standard Michaelis–Menten kinetics were used to describe the uptake behavior of cytosolic Ca2+-sequestering proteins (Pmc1, Pmr1 and Vcx1), while the experimentally noted Mg2+-sensitive Ca2+ influx was modeled using Michaelis–Menten kinetics with Mg2+ competitive inhibition. The necessity of having two Mg2+-sensitive influx pathways (transporters M and X), rather than having only one Mg2+-sensitive influx pathway (i.e. transporter X) was demonstrated by both theoretical analysis and optimal fitting to the experimental data using a hybrid optimization algorithm. The validity of the model was further confirmed by its ability to reproduce the experimentally determined effects of Vcx1 on cytosolic-free Ca2+ dynamics and to simulate the effects of extracellular Mg2+ removal (Wiesenberger, 2007; Cui, 2009).

In yeast cells, calcium signals are usually in the form of transients (see Fig. 2a) whereas in cardiac myocytes various forms exist, including calcium oscillations, sparks and transients (Cheng, 1996). In part, this difference can be explained by the relatively large size of cardiac myocytes relative to yeast cells. In turn, spatial effects such as diffusion play an important role in cardiac myocytes. For example, it has been experimentally noted that the calcium concentration in sarcolemmal–SR junctions controlling RyR and LTCC differs considerably from the local submembrane calcium concentration that controls sarcolemmal NCX (Bers, 2002; Weber, 2002). In addition, the spatial organization of RyRs in cardiac myocytes and their regulatory mechanisms involving a combination of positive [i.e. calcium-induced Ca2+ release (CICR)] and negative feedback controls by cytosolic Ca2+ (see Fig. 1b and Table 1) further account for the difference between the cells (Bers, 2002; Kim, 2008). These structural and functional differences between the cells also translate to the mathematical models of calcium dynamics in yeast and cardiac myocytes. Because of the biological complexity of cardiac myocytes, the mathematical models describing calcium homeostasis/signaling in these cells (Sobie, 2002; Shannon, 2004; Niederer, 2006; Cui & Kaandorp, 2008; Groff & Smith, 2008) are usually much more complicated than those for yeast cells (Cui & Kaandorp, 2006; Cui, 2009). Besides ordinary differential equations (ODEs), partial differential equations are frequently used to model the calcium homeostasis and signaling processes in cardiac myocytes. Moreover, in order to approximate microdomain and stochastic calcium signaling events in cardiac myocytes, more sophisticated and novel methods such as molecular dynamics simulations and stochastic simulation methods are required (Bers, 2002; Beckstein, 2003; Groff & Smith, 2008).

As an example of the complexity of calcium systems in cardiac myocytes, the calcium–calcineurin–regulator of calcineurin (RCAN)–nuclear factor of activated T cells (NFAT) signaling network is shown in Fig. 3. This describes the experimentally found calcineurin-dependent signaling pathways controlling heart growth under conditions of hypertrophic stress, such as pressure overload (PO) and active calcineurin (CaN*) overexpression (Cui & Kaandorp, 2008). This network is a part of the recently characterized group of signal-transduction pathways implicated in the regulation of cardiac hypertrophy (Heineke & Molkentin, 2006). The activation of different pathways seems to be stimulus specific (Sipido & Eisner, 2005).

3

A schematic graph depicting the calcium–calcineurin–RCAN–NFAT signaling network in cardiac myocytes (this figure is modified after the graph in the cellml version of the model developed by Cui and Kaandorp, see http://models.cellml.org/exposure/8e4d38d104176cb9a0d2e126ea23941c/cui_kaandorp_2008_c.cellml.index.html (Lloyd, 2008). As shown in the top left corner, the increased level of cytosolic calcium incurred by stress is sensed by calmodulin. Ca2+-bound calmodulin binds to CaN to activate it. CaN* can bind to RCAN to form Complex1 (Vega, 2003a, b) and can also dephosphorylate NFATP to produce NFAT. GSK3β catalyzes the phosphorylation of NFAT to produce NFATP, which can bind to protein 14-3-3 to form Complex3 (Liao, 2005). Such phosphorylation of NFAT and dephosphorylation of NFATP also occur in the nucleus (Hallhuber, 2006). Cytosolic NFAT is imported into the nucleus and nuclear NFATP is exported into the cytosol. Nuclear NFAT is responsible for initiating the transcription of hypertrophic genes and also the gene encoding RCAN (more precisely, RCAN1, a form of RCAN). Both GSK3β and CaN* are shuttled between the nucleus and the cytosol. As shown in the top right corner, certain stresses, such as PO, can activate BMK1 (Takeishi, 2001), which phosphorylates RCAN on serine 112 to produce RCANP. RCANP can be further phosphorylated by GSK3β to produce RCANPP. The dephosphorylation of RCANPP into RCANP is again mediated by CaN*. Protein 14-3-3 can bind RCANPP to form Complex2 (Abbasi, 2006). CaN, calcineurin; CaN*, active calcineurin; NFAT, nuclear factor of activated T-cells; NFATP, phosphorylated NFAT (please note that NFAT has multiple phosphorylation sites. Here, for simplicity, we denote phosphorylated NFAT as NFATP. This is similar in the case of Crz1); RCAN, regulator of calcineurin (also named as calcipressin, down syndrome critical region or modulatory calcineurin-interacting protein) (Davies, 2007); RCANP, phosphorylated RCAN on serine 112; RCANPP, phosphorylated RCAN on both serine 112 and serine 108; BMK1 (also called ERK5), big mitogen-activated protein kinase 1; GSK3β, glycogen synthase 3β; Complex1, the complex formed by RCAN and calcineurin; Complex2, the complex formed by RCANPP and 14-3-3; Complex3, the complex formed by NFATP and 14-3-3; PO, pressure overload; stress, hypertrophic stimuli.

3

A schematic graph depicting the calcium–calcineurin–RCAN–NFAT signaling network in cardiac myocytes (this figure is modified after the graph in the cellml version of the model developed by Cui and Kaandorp, see http://models.cellml.org/exposure/8e4d38d104176cb9a0d2e126ea23941c/cui_kaandorp_2008_c.cellml.index.html (Lloyd, 2008). As shown in the top left corner, the increased level of cytosolic calcium incurred by stress is sensed by calmodulin. Ca2+-bound calmodulin binds to CaN to activate it. CaN* can bind to RCAN to form Complex1 (Vega, 2003a, b) and can also dephosphorylate NFATP to produce NFAT. GSK3β catalyzes the phosphorylation of NFAT to produce NFATP, which can bind to protein 14-3-3 to form Complex3 (Liao, 2005). Such phosphorylation of NFAT and dephosphorylation of NFATP also occur in the nucleus (Hallhuber, 2006). Cytosolic NFAT is imported into the nucleus and nuclear NFATP is exported into the cytosol. Nuclear NFAT is responsible for initiating the transcription of hypertrophic genes and also the gene encoding RCAN (more precisely, RCAN1, a form of RCAN). Both GSK3β and CaN* are shuttled between the nucleus and the cytosol. As shown in the top right corner, certain stresses, such as PO, can activate BMK1 (Takeishi, 2001), which phosphorylates RCAN on serine 112 to produce RCANP. RCANP can be further phosphorylated by GSK3β to produce RCANPP. The dephosphorylation of RCANPP into RCANP is again mediated by CaN*. Protein 14-3-3 can bind RCANPP to form Complex2 (Abbasi, 2006). CaN, calcineurin; CaN*, active calcineurin; NFAT, nuclear factor of activated T-cells; NFATP, phosphorylated NFAT (please note that NFAT has multiple phosphorylation sites. Here, for simplicity, we denote phosphorylated NFAT as NFATP. This is similar in the case of Crz1); RCAN, regulator of calcineurin (also named as calcipressin, down syndrome critical region or modulatory calcineurin-interacting protein) (Davies, 2007); RCANP, phosphorylated RCAN on serine 112; RCANPP, phosphorylated RCAN on both serine 112 and serine 108; BMK1 (also called ERK5), big mitogen-activated protein kinase 1; GSK3β, glycogen synthase 3β; Complex1, the complex formed by RCAN and calcineurin; Complex2, the complex formed by RCANPP and 14-3-3; Complex3, the complex formed by NFATP and 14-3-3; PO, pressure overload; stress, hypertrophic stimuli.

In order to accurately simulate the calcium dynamics using a mathematical model, the whole network was broken down into its composite reactions, which were automatically transformed into 28 equations using cellerator software (Cui & Kaandorp, 2008). A similar procedure can be generally applied to the ODE modeling of various signal transduction networks (Shapiro, 2003; Hilioti, 2004; Cui, 2008).

The model simulation results demonstrate that the inhibiting effects of RCAN, under conditions of CaN* overexpression, are due to the buffering effects of RCAN on calcineurin. Meanwhile, the seemingly positive influence of RCAN on calcineurin signaling, in the presence of PO, can be explained by the second compensatory buffering effect of phosphorylated RCAN (i.e. RCANPP) on protein 14-3-3, which otherwise would buffer NFATP and prevent recognition by calcineurin (Vega, 2003b; Cui & Kaandorp, 2008).

Besides this mathematical model, there exist several other published computational studies, that focus on the paradoxically dual role of RCAN. For example, the model by Shin (2006) assumes the existence of a threshold concentration of nuclear NFAT, above which RCAN is switched on, and the apparent facilitating role of RCAN under PO is explained as the effect of the release of CaN from the CaN/RCAN complex (i.e. Complex1) by some protein kinase. Compared with the work by Shin (2006), the current model integrates more recent and extensive experimental findings such as the function of active calcineurin in the nucleus and the buffering effect of 14-3-3 on NFATP (Liao, 2005; Hallhuber, 2006).

Similarly, in yeast, the paradoxical role of Rcn1 in calcineurin activity has been both reported experimentally and modeled computationally. Using this combination of techniques, Hilioti (2004) demonstrated that the facilitating role of phosphorylated Rcn1 in calcineurin signaling involves the phosphorylation of a conserved serine residue of Rcn1 by Mck1. Later, the experimental work by Kishi (2007) suggested that this Mck1-dependent phosphorylation targets Rcn1 for degradation through the SCFCdc4 complex, which relieves the inhibition of calcineurin, thereby stimulating calcineurin. Lastly, Mehta (2009) showed that a low expression of Rcn1 stimulates calcineurin signaling in a docking-dependent and proteolysis-dependent fashion, which strongly suggests that calcineurin signaling benefits from a transient interaction with Rcn1. Based on this observation, Mehta (2009) argued that Rcn1 may function as a suicidal molecular chaperone of calcineurin and could regulate its activity in a manner similar to the regulation of type-1 protein phosphatase (PP1) by inhibitor-2 (I-2).

Specificity encoding in calcium signaling

The central question in the field of calcium signaling is related to its specificity. How can a single intracellular messenger (Ca2+) link different extracellular signals with strict specificity to the corresponding cellular responses? The calcium signature hypothesis attempts to explain this phenomenon by assuming that signals are encoded in the temporal and spatial nature, and the amplitude of the cytosolic calcium concentration changes (the calcium signature), which are later decoded by the effectors (Berridge, 1998; Thomine, 2001; Petersen, 2005). Experimental evidence to support this hypothesis has been found in many different types of mammalian cells as well as in Arabidopsis guard cells (Dolmetsch, 1997; Li, 1998; Allen, 2000; Mackenzie, 2004). For example, it has been demonstrated experimentally that Ca2+/calmodulin-dependent protein kinase II (CaMKII) and PKC are able to decode frequency-modulated Ca2+ signals (Koninck & Schulman, 1998; Oancea & Meyer, 1998; Allen, 2000). In yeast cells, although the cytosolic calcium concentration hardly oscillates, it has been reported recently that the nuclear localization of the downstream transcription factor Crz1 (see Fig. 1a) exhibits a frequency-modulated burst behavior to coordinate gene regulation under conditions of calcium stress (Cai, 2008).

In contrast, Scrase-Field & Knight (2003) argued that calcium may merely be an essential chemical switch and that the calcium signature hypothesis describes the exception rather than the rule. Their arguments for the calcium switch hypothesis include experimentally documented phenomena in plant calcium signaling, such as similar calcium signatures with different end responses, as well as similar end responses with different calcium signatures. Similar phenomena have also been found experimentally in yeast cells. For example, Wiesenberger (2007) reported that Mg2+ starvation and Ca2+ stress can induce very similar cytosolic calcium transients in yeast. In addition, this experimental observation has been supported by the simulation results of a mathematical model (Cui, 2009). Further, a striking portion of the genes that are upregulated under conditions of Mg2+ depletion, including ENA1 (encoding a P-type ATPase sodium pump) and PHO89 (encoding a sodium/phosphate cotransporter), are also induced by Ca2+ stress. However, there are many other genes (such as SUL1, ARA1, MDH2 and STF2) that are upregulated under conditions of Mg2+ depletion, but are not induced by Ca2+ stress (Wiesenberger, 2007).

Because both the calcium signature hypothesis and the calcium switch hypothesis are supported by experimental evidence, it will be important to discriminate between the cases in which calcium is responsible for encoding specificity, from those in which it functions only as a chemical switch. If we consider this viewpoint: the end response is usually the result of an interaction between multiple signaling pathways, we can more easily understand why the signal specificity can be encoded by signaling components other than calcium. Indeed, the cardiac calcium signaling network presented in the previous section illustrates how different stimuli can trigger their respective end response through the complex cross-interaction of different signaling pathways. This is because PO induces cardiac hypertrophy by activating both the calcium–calcineurin–NFAT pathway and the BMK1/ERK5 signaling pathway whereas the BMK1/ERK5 signaling pathway, is not activated in the case of CaN* overexpression (Takeishi, 2001; Hayashi & Lee, 2004). We predict that accurate mathematical model descriptions of the calcium-signaling systems will help us to uncover the mystery of specificity encoding in calcium signaling networks.

Conclusions

Both yeast cells and mammalian cardiac myocytes are equipped with calcium-signaling tool kits composed of channels, pumps, exchangers and other relevant components (such as sensors like calmodulin, and effectors like calcineurin). These function to maintain calcium homeostasis and also perform the task of calcium signaling. Compared with the same systems in mammalian cardiac myocytes, calcium homeostasis and signaling systems in yeast cells are relatively simple and easy to approximate. The extreme complexity of calcium homeostasis/signaling processes in cardiac myocytes arises from their coupling with other ion homeostasis processes, important spatial and stochastic effects (e.g. the local and microdomain calcium signals), the huge number of factors involved in the systems and their sophisticated regulatory mechanisms (Bers, 2002, 2008; Heineke & Molkentin, 2006).

Surprisingly, most of the factors known in the yeast calcium homeostasis/signaling network are conserved and operate in a similar manner in mammalian cells, including cardiac myocytes (see Table 2 and Fig. 4; Ton & Rao, 2004; Dolinski & Botstein, 2007). For example, as shown in Fig. 4, NFAT translocation in cardiac myocytes is strikingly similar to the process of Crz1 translocation in yeast (Stathopoulos-Gerontides, 1999). Further, RCAN regulation on calcineurin signaling in cardiac myocytes is similar to Rcn1 regulation on calcineurin signaling in yeast (Hilioti, 2004). Thus, our knowledge of the calcium homeostasis/signaling system in a simple organism, such as the budding yeast (S. cerevisiae) can help us to better understand the calcium homeostasis/signaling systems in mammalian cardiac myocytes, and treat relevant human diseases such as pathological cardiac hypertrophy and heart failure.

4

Highlighting the striking similarity between the Ca2+ homeostasis/signaling systems of yeast cells and cardiac myocytes. In yeast, Ca2+ induces the expression of RCN1 (the gene encoding Rcn1) through the calcineurin-dependent activation of Crz1. After the phosphorylation of serine 117 by an unknown priming kinase, Rcn1P can be further phosphorylated by MCK1 on serine 113. The dephosphorylation of Rcn1PP into Rcn1P is mediated by calcineurin (Kishi, 2007). All these components and mechanisms are conserved in the corresponding system in mammalian cardiac myocytes (Cui & Kaandorp, 2008). Crz1P, phosphorylated Crz1; Rcn1P, phosphorylated Rcn1 on serine 117; Rcn1PP, phosphorylated Rcn1 on both serine 117 and serine 113.

4

Highlighting the striking similarity between the Ca2+ homeostasis/signaling systems of yeast cells and cardiac myocytes. In yeast, Ca2+ induces the expression of RCN1 (the gene encoding Rcn1) through the calcineurin-dependent activation of Crz1. After the phosphorylation of serine 117 by an unknown priming kinase, Rcn1P can be further phosphorylated by MCK1 on serine 113. The dephosphorylation of Rcn1PP into Rcn1P is mediated by calcineurin (Kishi, 2007). All these components and mechanisms are conserved in the corresponding system in mammalian cardiac myocytes (Cui & Kaandorp, 2008). Crz1P, phosphorylated Crz1; Rcn1P, phosphorylated Rcn1 on serine 117; Rcn1PP, phosphorylated Rcn1 on both serine 117 and serine 113.

In order to accelerate our progress in the field of yeast calcium homeostasis/signaling systems, MS-based proteomics, complemented with yeast two-hybrid assays, can provide a very powerful technique for searching for the missing components in the networks, detecting and determining the protein interactions and quantifying the concentrations of proteins (Causier, 2004; Olsen, 2006; Cox & Mann, 2007). Functional assays based on gene-knockout techniques can provide a useful method for checking the validity of mathematical models. In order to determine the rate constants for such models, various methods exist, such as surface plasmon resonance analysis (Slepak, 2000; Portmann, 2004). Electrophysiological recordings of ion channel activity in the plasma membrane of live yeast cells have been proven to be difficult to obtain (Putney, 2005). An alternative method to achieve such measurements is to express the corresponding genes in mammalian cells, where electrophysiological recordings are much easier to obtain. Effective collaborations among scientists who are proficient in genetics, proteomics and computational science will result in the eventual completion of the whole-yeast calcium homeostasis/signaling system and an improved understanding of its dynamics. This will be achieved via high-throughput experimental and computational methods, and then back through the iterative systems biology procedure (i.e. experiment→model→experiment) (Finkelstein, 2004).

Acknowledgements

We would like to thank Prof. Kyle W. Cunningham (Johns Hopkins University) for his valuable data and stimulating discussions. We sincerely acknowledge all the other colleagues and collaborators who were involved in the work presented in this paper, including Y. Fomekong Nanfack, Olufisayo O. Ositelu, Veronica Beaudry and Alicia Knight. We thank two anonymous referees for their valuable comments and suggestions. J.C. was firstly funded by the Dutch Science Foundation on his PhD research, later supported by two grants from the EC on MORPHEX (NEST contract no. 043322) and QosCos projects and lastly by an NUS grant (R-252-000-350-112) for his postdoctoral project ‘Decomposition and composition of large signaling pathway models with emphasis on parameter estimation.’

References

Abbasi
S
Lee
JD
Su
B
Chen
X
Alcon
JL
Yang
J
Kellems
RE
Xia
Y
(
2006
)
Protein kinase-mediated regulation of calcineurin through the phosphorylation of modulatory calcineurin-interacting protein 1
.
J Biol Chem
 
281
:
7717
7726
.
Allen
DG
Blinks
JR
Prendergast
FG
(
1977
)
Aequorin luminescence: relation of light emission to calcium concentration – a calcium-independent component
.
Science
 
195
:
996
998
.
Allen
GJ
Chu
SP
Schumacher
K
et al
(
2000
)
Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutant
.
Science
 
289
:
2338
2342
.
Barry
WH
Bridge
JH
(
1993
)
Intracellular calcium homeostasis in cardiac myocytes
.
Circulation
 
87
:
1806
1815
.
Batiza
AF
Schulz
T
Masson
PH
(
1996
)
Yeast respond to hypotonic shock with a calcium pulse
.
J Biol Chem
 
271
:
23357
23362
.
Beckstein
O
Biggin
PC
Bond
P
Bright
JN
Domene
C
Grottesi
A
Holyoake
J
Sansom
MSP
(
2003
)
Ion channel gating: insights via molecular simulations
.
FEBS Lett
 
555
:
85
90
.
Berridge
MJ
Bootman
MD
Lipp
P
(
1998
)
Calcium – a life and death signal
.
Nature
 
395
:
645
648
.
Berridge
MJ
Lipp
P
Bootman
MD
(
2000
)
The versatility and universality of calcium signaling
.
Nat Rev Mol Cell Bio
 
1
:
11
21
.
Berridge
MJ
Bootman
MD
Roderick
HL
(
2003
)
Calcium signaling: dynamics, homeostasis and remodeling
.
Nat Rev Mol Cell Bio
 
4
:
517
529
.
Bers
DM
(
2002
)
Cardiac excitation–contraction coupling
.
Nature
 
415
:
198
205
.
Bers
DM
(
2008
)
Calcium cycling and signaling in cardiac myocytes
.
Annu Rev Physiol
 
70
:
23
49
.
Bertl
A
Slayman
CL
(
1992
)
Complex modulation of cation channels in the tonoplast and plasma membrane of Saccharomyces cerevisiae: single-channel studies
.
J Exp Biol
 
172
:
271
287
.
Bonilla
M
Cunningham
KW
(
2002
)
Calcium release and influx in yeast: TRPC and VGCC rule another kingdom
.
Science's STKE
 
127
:
pe17
.
Bonilla
M
Cunningham
KW
(
2003
)
MAP kinase stimulation of Ca2+ signaling is required for survival of endoplasmic reticulum stress in yeast
.
Mol Biol Cell
 
14
:
4296
4305
.
Brookes
PS
Yoon
Y
Robotham
JL
Anders
MW
Sheu
S-S
(
2004
)
Calcium, ATP, and ROS: a mitochondrial love–hate triangle
.
Am J Physiol-Cell Ph
 
287
:
C817
C833
.
Buntinas
L
Gunter
KK
Sparagna
GC
Gunter
TE
(
2001
)
The rapid mode of calcium uptake into heart mitochondria (RaM): comparison to RaM in liver mitochondria
.
Biochim Biophys Acta
 
1504
:
248
261
.
Cai
L
Dalal
KJ
Elowitz
MB
(
2008
)
Frequency-modulated nuclear localization bursts coordinate gene regulation
.
Nature
 
455
:
485
490
.
Causier
B
(
2004
)
Studying the interactome with the yeast two-hybrid system and mass spectrometry
.
Mass Spectrom Rev
 
23
:
350
367
.
Cheng
H
Lederer
MR
Lederer
WJ
Cannell
MB
(
1996
)
Calcium sparks and [Ca2+]i waves in cardiac myocytes
.
Am J Physiol
 
270
:
C148
C159
.
Cox
J
Mann
M
(
2007
)
Is proteomics the new genomics?
Cell
 
130
:
395
398
.
Cui
J
Kaandorp
JA
(
2006
)
Mathematical modeling of calcium homeostasis in yeast cells
.
Cell Calcium
 
39
:
337
348
.
Cui
J
Kaandorp
JA
(
2008
)
Simulating complex calcium–calcineurin signaling network
.
Lecture Notes in Computer Science
 , Vol.
5103
(
Bubak
M
Van Albada
GD
Dongarra
J
Sloot
PMA
, eds), pp.
110
119
.
Springer-Verlag
,
Berlin
.
Cui
J
Kaandorp
JA
Lloyd
CM
(
2008
)
Simulating in vitro transcriptional response of zinc homeostasis system in Escherichia coli
.
BMC Syst Biol
 
2
:
89
.
Cui
J
Kaandoorp
J
Ositelu
OO
Beaudry
V
Knight
A
Nanfack
YF
Cunningham
KW
(
2009
)
Simulating calcium influx and free calcium concentrations in yeast
.
Cell Calcium
 
45
:
123
132
.
Cyert
MS
(
2001
)
Genetic analysis of calmodulin and its targets in Saccharomyces cerevisiae
.
Annu Rev Genet
 
35
:
647
72
.
Cyert
MS
Thorner
J
(
1992
)
Regulatory subunit (CNB1 gene product) of yeast Ca2+/calmodulin-dependent phosphoprotein phosphatases is required for adaptation to pheromone
.
Mol Cell Biol
 
12
:
3460
3469
.
Cyert
MS
Kunisawa
R
Kaim
D
Thorner
J
(
1991
)
Yeast has homologs (CNA1 and CNA2 geneproducts) of mammalian calcineurin, a calmodulin-regulated phosphoprotein phosphatase
.
P Natl Acad Sci USA
 
88
:
7376
7380
.
Davies
KJ
Ermak
G
Rothermel
BA
et al
(
2007
)
Renaming the DSCR1/Adapt78 gene family as RCAN: regulators of calcineurin
.
FASEB J
 
21
:
3023
3028
.
Denis
V
Cyert
MS
(
2002
)
Internal Ca2+ release in yeast is triggered by hypertonic shock and mediated by a TRP channel homologue
.
J Cell Biol
 
156
:
29
34
.
Dolinski
K
Botstein
D
(
2007
)
Orthology and functional conservation in eukaryotes
.
Annu Rev Genet
 
41
:
465
507
.
Dolmetsch
RE
Lewis
RS
Goodnow
CC
Healy
JI
(
1997
)
Differential activation of transcription factors induced by Ca2+ response amplitude and duration
.
Nature
 
386
:
855
858
.
Finkelstein
A
Hetherington
J
Li
L
Margoninski
O
Saffrey
P
Seymour
R
Warner
A
(
2004
)
Computational challenges of systems biology
.
Computer
 
37
:
26
33
.
Groff
JR
Smith
GD
(
2008
)
Ryanodine receptor allosteric coupling and the dynamics of calcium sparks
.
Biophys J
 
95
:
135
154
.
Hallhuber
M
Burkard
N
Wu
R
Buch
MH
Engelhardt
S
Hein
L
Neyses
L
Schuh
K
Ritter
O
(
2006
)
Inhibition of nuclear import of calcineurin prevents myocardial hypertrophy
.
Circ Res
 
99
:
626
635
.
Hayashi
M
Lee
JD
(
2004
)
Role of the BMK1/ERK5 signaling pathway: lessons from knockout mice
.
J Mol Med
 
82
:
800
808
.
Heineke
J
Molkentin
JD
(
2006
)
Regulation of cardiac hypertrophy by intracellular signaling pathways
.
Nat Rev Mol Cell Bio
 
7
:
589
600
.
Hilioti
Z
Gallagher
DA
Low-Nam
ST
Ramaswamy
P
Gajer
P
Kingsbury
TJ
Birchwood
CJ
Levchenko
A
Cunningham
KW
(
2004
)
GSK-3 kinases enhance calcineurin signaling by phosphorylation of RCNs
.
Gene Dev
 
18
:
35
47
.
Kim
J-R
Yoon
Y
Cho
K-H
(
2008
)
Coupled feedback loops form dynamic motifs of cellular networks
.
Biophys J
 
94
:
359
365
.
Kingsbury
TK
Cunningham
KW
(
2000
)
A conserved family of calcineurin regulators
.
Gene Dev
 
14
:
1595
1604
.
Kishi
T
Ikeda
A
Nagao
R
Koyama
N
(
2007
)
The SCFCdc4 ubiquitin ligase regulates calcineurin signaling through degradation of phosphorylated Rcn1, an inhibitor of calcineurin
.
P Natl Acad Sci USA
 
104
:
17418
17423
.
Koninck
PD
Schulman
H
(
1998
)
Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations
.
Science
 
279
:
227
230
.
Li
WH
Llopis
J
Whitney
M
Zlokarnik
G
Tsien
RY
(
1998
)
Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression
.
Nature
 
392
:
936
941
.
Liao
W
Wang
S
Han
C
Zhang
Y
(
2005
)
14-3-3 Proteins regulate glycogen synthase3β phosphorylation and inhibit cardiomyocyte hypertrophy
.
FEBS J
 
272
:
1845
1854
.
Lloyd
CM
Lawson
JR
Hunter
PJ
Nielsen
PF
(
2008
)
The CellML model repository
.
Bioinformatics
 
24
:
2122
2123
.
Locke
EG
Liang
L
Bonilla
M
Takita
Y
Cunningham
KW
(
2000
)
A homolog of voltage-gated channels stimulated by depletion of Ca2+ secretory pools in yeast
.
Mol Cell Biol
 
20
:
6686
6694
.
Mackenzie
L
Roderick
HL
Berridge
MJ
Conway
SJ
Bootman
MD
(
2004
)
The spatial pattern of atrial cardiomyocyte calcium signalling modulates contraction
.
J Cell Sci
 
117
:
6327
6337
.
Mehta
S
Li
H
Hogan
PG
Cunningham
KW
(
2009
)
Domain architecture of the regulators of calcineurin (RCANs) and identification of a divergent RCAN in yeast
.
Mol Cell Biol
 
29
:
2777
2793
.
Miseta
A
Kellermayer
R
Aiello
DP
Fu
L
Bedwell
DM
(
1999
)
The vacuolar Ca2+/H+ exchanger Vcx1p/Hum1p tightly controls cytosolic Ca2+ levels in S. cerevisiae
.
FEBS Lett
 
451
:
132
136
.
Muller
E
Locke
EG
Cunningham
KW
(
2001
)
Differential regulation of two Ca2+ influx systems by pheromone signaling in Saccharomyces cerevisiae
.
Genetics
 
159
:
1527
1538
.
Niederer
SA
Hunter
PJ
Smith
NP
(
2006
)
A quantitative analysis of cardiac myocyte relaxation: a simulation study
.
Biophys J
 
90
:
1697
1722
.
Oancea
E
Meyer
T
(
1998
)
Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals
.
Cell
 
95
:
307
318
.
Olsen
JV
Blagoev
B
Gnad
F
Macek
B
Kumar
C
Mortensen
P
Mann
M
(
2006
)
Global, in vivo, and site-specific phosphorylation dynamics in signaling networks
.
Cell
 
127
:
635
648
.
Petersen
OH
Michalak
M
Verkhratsky
A
(
2005
)
Calcium signaling: past, present and future
.
Cell Calcium
 
38
:
161
169
.
Portmann
R
Magnani
D
Stoyanov
JV
Schmechel
A
Multhaup
G
Solioz
M
(
2004
)
Interaction kinetics of the copper-responsive CopY repressor with the cop promoter of Enterococcus hirae
.
J Biol Inorg Chem
 
9
:
396
402
.
Prasad
AM
Inesi
G
(
2009
)
Effects of thapsigargin and phenylephrine on calcineurin and protein kinase C signaling functions in cardiac myocytes
.
Am J Physiol-Cell Ph
 
296
:
C992
C1002
.
Putney
JW
Jr
(ed) (
2005
)
Calcium Signaling
 .
CRC Press
,
Boca Raton
.
Rudolf
R
Mongillo
M
Rizzuto
R
Pozzan
T
(
2003
)
Looking forward to seeing calcium
.
Nat Rev Mol Cell Bio
 
4
:
579
586
.
Scrase-Field
SAMG
Knight
MR
(
2003
)
Calcium: just a chemical switch?
Curr Opin Plant Biol
 
6
:
500
506
.
Shannon
TR
Wang
F
Puglisi
J
Webber
C
Bers
DM
(
2004
)
A mathematical treatment of integrated Ca dynamics within the ventricular myocyte
.
Biophys J
 
87
:
3351
3371
.
Shapiro
BE
Levchenko
A
Meyerowitz
EM
Wold
BJ
Mjolsness
ED
(
2003
)
Cellerator: extending a computer algebra system to include biochemical arrows for signal transduction simulations
.
Bioinformatics
 
19
:
677
678
.
Shin
S-Y
Choo
S-M
Kim
D
Baek
SJ
Wolkenhauer
O
Cho
K-H
(
2006
)
Switching feedback mechanisms realize the dual role of RCAN in the regulation of calcineurin activity
.
FEBS Lett
 
580
:
5965
5973
.
Sipido
KR
Eisner
D
(
2005
)
Something old, something new: changing views on the cellular mechanisms of heart failure
.
Cardio Res
 
68
:
167
174
.
Slepak
VZ
(
2000
)
Application of surface plasmon resonance for analysis of protein–protein interactions in the G protein-mediated signal transduction pathway
.
J Mol Recognit
 
13
:
20
26
.
Sobie
EA
Dilly
KW
Dos Santos Cruz
J
Lederer
WJ
Jafri
MS
(
2002
)
Termination of cardiac Ca2+ sparks: an investigative mathematical model of calcium-induced calcium release
.
Biophys J
 
83
:
59
78
.
Stathopoulos-Gerontides
A
Guo
JJ
Cyert
MS
(
1999
)
Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation
.
Gene Dev
 
13
:
798
803
.
Su
Z
Zhou
X
Loukin
SH
Saimi
Y
Kung
C
(
2009
)
Mechanical force and cytoplasmic Ca2+ activate yeast TRPY1 in parallel
.
J Membrane Biol
 
227
:
141
150
.
Takeishi
Y
Huang
Q
Abe
J-i
et al
(
2001
)
Src and multiple MAP kinase activation in cardiac hypertrophy and congestive heart failure under chronic pressure-overload: comparison with acute mechanical stretch
.
J Mol Cell Cardio
 
33
:
1637
1648
.
Teng
J
Goto
R
Iida
K
Kojima
I
Iida
H
(
2008
)
Ion-channel blocker sensitivity of voltage-gated calcium-channel homologue Cch1 in Saccharomyces cerevisiae
.
Microbiology
 
154
:
3775
3781
.
Thomine
S
(
2001
)
Cracking the calcium code
.
Trends Plant Sci
 
6
:
501
.
Ton
V-K
Mandal
D
Vahadji
C
Rao
R
(
2002
)
Functional expression in yeast of the human secretory pathway Ca2+, Mn2+-ATPase defective in Hailey-Hailey disease
.
J Biol Chem
 
277
:
6422
6427
.
Ton
V-K
Rao
R
(
2004
)
Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases
.
Am J Physiol-Cell Ph
 
287
:
C580
C589
.
Vega
RB
Bassel-Duby
R
Olson
EN
(
2003a
)
Control of Cardiac growth and function by calcineurin signaling
.
J Biol Chem
 
278
:
36981
36984
.
Vega
RB
Rothermel
BA
Weinheimer
CJ
Kovacs
A
Naseem
RH
Bassel-Duby
R
Williams
RS
Olson
EN
(
2003b
)
Dual roles of modulatory calcineurin-interacting protein 1 in cardiac hypertrophy
.
P Natl Acad Sci USA
 
100
:
669
674
.
Weber
CR
Piacentino
V
Ginsburg
KS
Houser
SR
Bers
DM
(
2002
)
Na+–Ca2+ exchange current and submembrane [Ca2+] during the cardiac action potential
.
Circ Res
 
90
:
182
189
.
Wiesenberger
G
Steinleitner
K
Malli
R
Graier
WF
Vormann
J
Schweyen
RJ
Stadler
JA
(
2007
)
Mg2+ deprivation elicits rapid Ca2+ uptake and activates Ca2+/calcineurin signaling in Saccharomyces cerevisiae
.
Euk Cell
 
6
:
592
599
.
Zhang
NN
Dudgeon
DD
Paliwal
S
Levchenko
A
Grote
E
Cunningham
KW
(
2006
)
Multiple signaling pathways regulate yeast cell death during the response to mating pheromones
.
Mol Biol Cell
 
17
:
3409
3422
.

Author notes

Editor: Teun Boekhout