Calcineurin and hypertrophic heart disease: novel insights and remaining questions

Abstract

In the past 2 years, an emerging body of research has focused on a novel transcriptional pathway involved in the cardiac hypertrophic response. Ever since its introduction, the significance of the calcineurin–NFAT module has been subject of controversy. The aim of this review is to provide both an update on the current status of knowledge and discuss the remaining issues regarding the involvement of calcineurin in hypertrophic heart disease. To this end, the molecular biology of calcineurin and its direct downstream transcriptional effector NFAT are discussed in the context of the genetic studies that established the existence of this signaling paradigm in the heart. The pharmacological mode-of-action and specificity of the calcineurin inhibitors cyclosporine A (CsA) and FK506 is dicussed, as well as their inherent limitations to study the biology of calcineurin. A critical interpretation is given on studies aimed at analyzing the role of calcineurin in cardiac hypertrophy using systemic immunosuppression. To eliminate the controversy surrounding CsA/FK506 usage, recent studies employed genetic inhibitory strategies for calcineurin, which confirm the pivotal role for this signal transduction pathway in the ventricular hypertrophy response. Finally, unresolved issues concerning the role of calcineurin in cardiac pathobiology are discussed based upon the information available, including its controversial role in cardiomyocyte viability, the reciprocal relationship between myocyte Ca²⁺ homeostasis and calcineurin activity and the relative importance of calcineurin in relation to other hypertrophic signaling cascades.

Apoptosis, Calcium (cellular), Cardiomyopathy, Gene expression, Heart failure, Hypertrophy, Signal transduction

Time for primary review 27 days.

1. Molecular biology of calcineurin

The calcineurin–NFAT pathway was one of the first signaling paradigms that provided molecular insight how extracellular signals travel from the cell membrane into the nucleus. The precise components of the pathway were defined by working backwards from the T-lymphocyte nucleus to the cell membrane. The regulatory region in the interleukin-2 (IL-2) gene was found to be under control of a transcriptional factor designated nuclear factor of activated T-cells (NFAT), which shuttled between cytoplasmic and nuclear components under influence of a Ca²⁺ signal. Subsequently, nuclear NFAT import was defined to be regulated by dephosphorylation steps catalyzed by the phosphatase calcineurin, which in its turn was subject to regulation by Ca²⁺-calmodulin binding [1–3]. Finally, calcineurin was identified as the cellular target of the immunosuppressive agents CsA and FK506 [1,2].

Calcineurin itself is a heterodimer composed of two distinct subunits, designated calcineurin A, a 58–59 kDa subunit which contains the catalytic site of the enzyme, and a small ∼19 kDa calcineurin B subunit which contains the Ca²⁺-binding regulatory domain of the holoenzyme. Three different mammalian genes, designated α, β and γ, encode calcineurin A, and share substantial homology in the domain encoding the catalytic site. Of these isoforms, calcineurin Aγ displays a testis-restricted expression pattern, while calcineurin Aα and Aβ exist in an overlapping fashion in multiple tissues, including the heart. Multiple alternative splice isoforms of calcineurin Aα and Aβ have been reported [4]. The smaller calcineurin B subunit is encoded by two genes, each having alternative splice isoforms [5]. Only mutant mice lacking calcineurin Aα have been created and these animals display a subtle defect in the antigen-specific T-cell response [6], accumulation of a hyperphosphorylated form of tau in the mossy fibers of the hippocampus, accompanied by cytoskeletal changes and altered synaptic plasticity in the central nervous system [7,8]. The phenotype of the calcineurin Aα knockout is surprising in that it does not reveal the full scale of phenotypic characteristics associated with CsA or FK506 administration, suggesting functional redundancy between the calcineurin Aα and Aβ isoforms. The creation and phenotypic characterization of genetic mouse models mutant for either calcineurin Aβ or calcineurin B will be instrumental for resolving these issues and efforts along this line are in progress (Bueno and Molkentin, Unpublished observations).

To date, five genes encoding NFAT complexes have been identified and designated NFATc1 (NFATc or NFAT2), NFATc2 (NFATp or NFAT1), NFATc4 (NFAT3), NFATc3 (NFAT4 or NFATx) and NFAT5, of which only the latter appears to be constitutively nuclear and not subject to regulation by calcineurin [9,10]. The NFATc members share high homology within their DNA binding region, which is related to the Rel domain present in the transcription factor NF-κB [9]. NFATc members transactivate target genes by interacting with other transcription factors like c-Maf, activator protein-1 (AP-1), and GATA-4 [11–15].

Calcineurin physically interacts with NFATc members and dephosphorylates serine residues within SP repeats and the serine-rich region. Dephosphorylation of these residues results in the unmasking of two nuclear localization sequences required for its nuclear import [16]. Nuclear export, on the other hand, is dependent upon rephosphorylation of these same residues by glycogen synthase kinase-3β (GSK3β) [17] and the p38 and c-Jun NH₂-terminal protein kinase (JNK) members of the mitogen-activated protein kinase (MAPK) superfamily [18,19]. The biological significance of the individual NFATc members is rapidly being elucidated due to the availability of murine models deficient for the single NFATc isoforms [9]. Of interest to the cardiovascular field, mice lacking NFATc1 die in utero from failure to develop semi lunar cardiac valves [20,21], while the combined disruption of NFATc3 and NFATc4 results in embryonic lethality due to abberant vascular patterning [22].

2. First evidence for a role of calcineurin in cardiac hypertrophy

Molkentin and colleagues first established the existence of a myocardial calcineurin-NFAT module [11]. Using a yeast-two-hybrid screening of interacting factors for the cardiac embryonic transcription factor GATA-4, a specific interaction was observed between zinc finger domains of GATA-4 with the DNA binding domain of NFATc4 (NFAT3). Since interaction between NFATc4 and GATA-4 drove synergistic activation of a brain natriuretic peptide (BNP) promoter-reporter construct, a specific role for NFATc4 activation and hypertrophic gene induction was suggestive. (Fig. 1) [11]

In line with this hypothesis, CsA (calcineurin inhibition) prevented the hypertrophic growth response of cultured cardiomyocytes in response to humoral factors as angiotensin II (AngII) and phenylephrine (PE) [11]. To elucidate the functional significance of the calcineurin-NFATc4 pathway in vivo, several lines of transgenic mice were generated containing either truncated activated mutants of NFATc4 or the calcineurin Aα subunit specifically in the heart. Persistent calcineurin activation was sufficient to promote a hypertrophic response in 11 separate TG founder lines, varying from relatively benign forms of concentric hypertrophy to severe forms of dilated cardiomyopathy and early lethality in low and high copy number TG mice, respectively. Pharmacological inhibition of calcineurin through CsA injections (25 mg/kg per day) resulted in complete prevention of the pathology [11]. NFATc4 TG mice also demonstrated a hypertrophic myopathy associated with re-expression of fetal genes. As a testimony to the specificity of the proposed pathway, CsA was unable to prevent the morphologic pathology of the NFATc4 TG animals, which express a calcineurin-independent form of the transcription factor [23].

This initial study was soon followed by one which analyzed whether calcineurin may also be involved in the ventricular growth response in other animal models of cardiomyopathy and LV hypertrophy. Accordingly, CsA administration prevented hypertrophy in tropomodulin overexpressing mice, a model of dilated cardiomyopathy due to sarcomeric disruption [24–27]. CsA also proved to be effective in TG mice expressing a non-phosphorylatable form of myosin light chain 2v (MLC-2v), which display hypertrophic cardiomyopathy (HCM) as a result of inefficient cross-bridge cycling [24,28,29], and in mice overexpressing β-tropomyosin, a model of hypertrophic cardiomyopathy and defective calcium handling [24,30,31]. CsA was ineffective, however, in a retinoic acid receptor (RAR) TG driven pathology [24,32], indicating the possibility that calcineurin activation might act as a molecular driving force behind some, but not all forms of ventricular myopathies. The authors were further able to demonstrate activation of calcineurin enzymatic activity concurrent with a 27% increase in indexed heart weight in a rat model of pressure overload created by surgical constriction of the abdominal aorta. CsA administration resulted in complete prevention of calcineurin activation and LV hypertrophy 6 days following coarctation [24].

Fig. 1

Model for calcineurin function in cardiac hypertrophy as postulated by Molkentin and colleagues in 1998 [12]. In this scheme, Ca²⁺/calmodulin-dependent phosphatase calcineurin is activated by hypertrophic agonists such as Angiotensin II (AngII) or phenylephrine (PE) or by intrinsic defects, which trigger an increase in intracellular Ca²⁺. Upon activation, calcineurin dephosphorylates one single nuclear factor of activated T-cell (NFATc) target, which translocates to the nucleus upon activation. NFATc4 (NFAT3) physically associates with the transcription factor GATA-4 to synergistically activate gene transcription of hypertrophic genes such as the brain natriuretic peptide gene (BNP).

These two initial studies suggested several novel implications for cardiac pathobiology. (1) The existence of a conservational preservation of a calcineurin–NFATc pathway in the myocardium, such as defined earlier in T lymphocytes; (2) the potential of this transcriptional pathway to activate a fetal gene expression profile consistent with LV hypertrophy (Fig. 1); and (3) pharmacological inhibition of (transgenically driven levels) of activated calcineurin prevents the histopathology resulting from its activation. These initial studies immediately evoked questions related to the significance of this novel pathway in view of the complexity and hierarchy of the multitude of hypertrophic signaling pathways known to govern cardiac hypertrophy. To address these issues the vast majority of these studies, unfortunately, made use of the pharmacological inhibitors of calcineurin. The inconsistent outcome of these studies ignited controversy regarding the requirement of this pathway in the LV hypertrophic response.

3. Pharmacological inhibitors of calcineurin

Csa (Sandimmune) and FK506 or tacrolimus (Prograf) are the most important immunosuppressive agents used in organ transplantation and in the treatment of diverse immune disorders. These agents produce similar effects on signal transduction pathways in T lymphocytes, however, they do not share similar chemical structure nor do they react with the same target. CsA is a neutral, lipophilic and very hydrophobic, cyclic polypeptide of 11 amino acids extracted from Tolypocladium inflatum Gams. Following oral or intravenous administration, CsA distributes rapidly between blood cells and plasma with an apparent large volume of distribution of 4 to 8 l/kg [33]. CsA accumulates in erythrocytes (50–60%) and in leukocytes (10–20%), the remainder of the drug is bound to plasma lipoproteins. Tissue drug concentrations depend on actual cyclophilin and lipid content. CsA is metabolized predominantly in the liver by cytochrome P-450IIIA enzymes to more than 30 metabolites. These metabolites do not contribute significantly to the immunosuppressive effects of CsA since the most active ones only retain 10–20% of the activity of the parent compound. CsA does not cross the blood–brain barrier, but it crosses the placenta and it can be detected in amniotic fluid and fetal blood. The elimination half-life of CsA has been estimated of approximately 6 h [33].

FK506 is a macrolide antibiotic extracted from Streptomyces tsukubaensis. FK506 can be administered orally or intravenously and its elimination half-life is approximately 20 h. It is extensively metabolized in the liver since less than 1% of the drug is excreted unchanged. FK506 is approximately 100 times more potent than CsA in its calcineurin inhibitory characteristics.

The mechanism of action of these two immunosuppressive agents has been investigated extensively in immune cells. CsA and FK506 bind with high affinity to the ubiquitous cytosolic peptidyl-propyl isomerases cyclophilin and FK506-binding protein-12 (FKBP12), respectively. The complex of CsA-cyclophilin or FK506-FKBP12 associates with calcineurin and inhibits its phosphatase activity as well as its interaction with a variety of substrates. CsA and FK506 also inhibit the peptidyl-propyl isomerase activity of cyclophilin and FKBP12, but this effect is not involved in the immunosuppressive mechanism of these drugs since CsA analogues with no effect on T-cell activation are still able to block the peptidyl-propyl isomerase activity.

It is generally accepted that calcineurin inhibition by CsA and FK506 blocks the dephosphorylation and subsequent nuclear translocation of the NFATc transcription factors [34,35]. However, novel, calcineurin-independent mechanisms of action for CsA and FK506 have recently been proposed. It has been hypothesized that part of the immunosuppressive effects of CsA are mediated through TGFβ1, a cytokine with immunosuppressive effects in diverse cells and tissues [36,37]. However, this is still a matter of controversy since other studies have failed to show an induction of TGF-β1 production during CsA treatment [38]. This issue could potentially be of interest to the current topic in view of the recent demonstration that selective TGFβ-activated kinase (TAK1) activation can result in a cardiomyopathic phenotype in mice [39]. CsA was also found to inhibit the activation of some family members of the mitogen-activated protein kinases (MAPK) in different cell types, although this may still be an indirect response to calcineurin inhibition [40,41]. Chronic CsA administration produces changes in the properties of the sarcoplasmatic reticulum (SR) Ca²⁺-release channel [42] and in isolated guinea-pig cardiomyocytes alterations in the kinetics of L-type Ca²⁺ channels [43].

The ryanodine receptor (RyR2) is a multiprotein complex including several phosphatases, kinases, achoring proteins and FKBP12.6. Altered RyR2 channel function has been postulated to play a role in cardiomyopathy, since hyperphosphorylation of the complex was observed in human heart failure biopsies, which results in dissociation of FKBP12.6 from its cognate receptor. These events result in increased Ca²⁺ sensitivity for activation, elevated channel open probability, and impaired myocyte Ca²⁺ homeostasis [44]. Genetic ablation of FKBP12 resulted in severe septum defects and dilated cardiomyopathy in mice [45]. Much less is known about the cardiac biology of the target of CsA, the cyclophilin A-D family.

Accordingly, it is becoming increasingly clear that CsA and FK506 have calcineurin-independent effects in multiple organs, and do not constitute the optimal tool to test for a potential role of calcineurin in the setting of cardiac hypertrophy. If these agents are to be used in a systemic fashion, precaution should be taken for dosage, mode of delivery, and severe extra-cardiac toxicity on major target organs besides the heart muscle (e.g. neural tissue, smooth muscle, kidney), which may influence the outcome of hypertrophy studies.

4. CsA and FK506: evidence in support of a role for calcineurin in hypertrophy

Endothelin-1 (ET-1) is an established humoral factor that induces cardiac hypertrophy. ET-1 was reported to increase the activity of CaMKII and calcineurin in cultured cardiac myocytes and pretreatment with KN62 (CaMK inhibitor) or CsA strongly suppressed ET-1-induced increases in phenylalanine uptake and in cell size. Pharmacologic inhibition of calmodulin, CaMK or calcineurin or overexpression of dominant-negative mutants of CaMKII and calcineurin strongly suppressed hypertrophic gene activation [46]. Similarly, leukemia inhibitory factor (LIF), a hypertrophic cytokine employing gp130 receptors for transmembrane signaling, enhanced both calcineurin and CaMK activities. KN62 and CsA administration significantly reduced LIF-induced hypertrophy and fetal gene expression, suggesting a crucial role for these Ca²⁺-activated pathways in LIF-mediated cardiomyocyte growth [47] (Table 1).

CsA demonstrated to be highly effective in tropomodulin overexpressing mice, in mice expressing a non-phosphorylatable form of myosin light chain 2v (MLC-2v) and in mice overexpressing β-tropomyosin [24]. An additional genetic model of hypertrophy, the Gqα overexpressing mouse, was found to be partially sensitive to comparable doses of CsA [48].

Shimoyama and colleagues analyzed the effectiveness of FK506 in a rat model of pressure overload hypertrophy. A drug dosage-effect preceded this study to minimize toxic side-effects on parameters as blood pressure, cardiac hemodynamics, and operative mortality. Accordingly, a dosage of 1 mg/kg per day FK506 injected intramuscularly was found to have negligible side-effects, yet still inhibited LV calcineurin activation. A 3-week treatment regimen on abdominal aortic constricted animals was associated with a near complete prevention of the increase in (indexed) heart size, fibrosis formation, and hypertrophic marker gene expression [49].

Using the same rat model, Lim and colleagues demonstrated that a 14-day CsA regimen resulted in a dose-dependent prevention of the morphologic, histological and molecular aspects of LV hypertrophy. Although this study suffered from a moderate post-operative mortality (∼25%), mortality was unaffected by the drug. One striking and clinically relevant finding demonstrated that treatment of CsA (20 mg/kg per day) was associated with a regression of established myocardial hypertrophy resulting from a 14-day pressure overload [50].

Hill and colleagues were able to demonstrate a progressive and reproducible hypertrophic response following a 5-week period thoracic aortic banding protocol in mice (45% increase in HW/BW). A relatively high dose of CsA resulted in a near complete blockade of this response (Table 1). The authors found no evidence of deleterious effects of CsA on myocardial hemodynamics, the transstenotic pressure gradient, weight gain, physical activity or mortality [51]. Mice with renovascular hypertension, created by a two kidney one clip method, also demonstrated substantial LV calcineurin activation and CsA prevented hypertrophy in this model (Table 1) [52].

Table 1

Studies using CsA or FK506 to analyze the role of calcineurin on the development of cardiac hypertrophy

Model	Stimulus	Calcineurin inhibition			Hypertrophy		Genetic
		Mode	Dosage (per day)	Duration	Prevention	Side effects	strain
Cultured cardiomyocytes (Ref.)
Kato et al. [47]	LIF	CsA	50 ng ml−1	1 day	Yes	None	Wistar
Molkentin et al. [11]	PE/AngII	CsA/FK506	500/150 ng ml−1	3 days	Yes	None	NR
Zhu et al. [46]	ET-1	CsA/dn-CnA	500 nM/NA	2 days	Yes	None	Wistar
Boluyt et al. [57]	PE	FK506	250 ng ml−1	1 day	No	HDa	Wistar
Mouse
Hill et al. [51]	Transverse AC	CsA	50 mg kg−1	5 weeks	Yes	None	C57BL/6
Molkentin et al. [11]	ca-CnA TG	CsA	50 mg kg−1	14 days	Yes	None	FVB/ICR
Murat et al. [52]	Two-kidney-one-Clip	CsA	50 mg kg−1	4 weeks	Yes	None	C57BL/6
Sussman et al. [25]	β-tropomyosin	CsA	30	8 days	Yes	None	FVB
Sussman et al. [25]	MLC-2v* TG	CsA	30	6 weeks	Yes	None	FVB
Sussman et al. [25]	Tropomodulin TG	CsA/FK506	30/6 mg kg−1	2 weeks	Yes	None	FVB
Ding et al. [59]	Ascending AC	CsA	50 mg kg−1	4 weeks	No	HF	C57BL/6
Fatkin et al. [66]	αMHC403−/−	CsA/FK506	30/6 mg kg−1	5 weeks	No	HD, OCM, AM	C57BL6/SV129
Lim et al. [23]	ca-NFATc4	CsA	30 mg kg−1	4 weeks	No	WL	FVB
Meguro et al. [60]	Transverse AC	CsA	25 mg kg−1	3 weeks	No	HF	CD-1
Sussman et al. [25]	RAR TG	CsA/FK506	30	6 weeks	No	None	FVB
Muller et al. [58]	Transverse AC	CsA	50 mg kg−1	3 weeks	No	NR	C57BL/6
Rat
Eto et al. [55]	Exercise	CsA	20 mg kg−1	10 weeks	Yes	None	Wistar
Lim et al. [50]	Abdominal AC	CsA	20 mg kg−1	2 weeks	Yes	WL	Sprague–Dawley
Mervaala et al. [54]	Angiotensin/Renin TG	CsA	5 mg kg−1	3 weeks	Yes	None	NR
Oie et al. [56]	Myocardial infarction	CsA	50 mg kg−1	2 weeks	Yes	HF	Wistar
Sakata et al. [53]	DS hypertension	FK506	1 mg kg−1	12 weeks	Yes	DPS	Dahl–Issei
Shimoyama et al. [67]	DS hypertension	FK506	0.1/0.01 mg kg−1	6 weeks	Yes	None	Dahl–Issei
Sussman et al. [25]	Abdominal AC	CsA	20 mg kg−1	6 days	Yes	M	Sprague–Dawley
Shimoyama et al. [49]	Abdominal AC	FK506	1 mg kg−1	3 weeks	Yes	None	Wistar
Eto et al. [55]	Ascending AC	CsA	20 mg kg−1	4 weeks	Yes	None	Wistar
Hayashida et al. [63]	DS hypertension	FK506	1 mg kg−1	6 weeks	No	PI, WL	Dahl–Issei
Zhang et al. [62]	Abdominal AC	CsA	10 mg kg−1	4 weeks	No	HF, WL	Sprague–Dawley
Luo et al. [61]	Abdominal AC	CsA/FK506	40/4 mg kg−1	2 weeks	No	M	Sprague–Dawley
Zhang et al. [62]	SHR hypertension	CsA	5 mg kg−1	6 weeks	No	None	SHR

LIF, Leukemia Inhibitory Factor; PE, phenylephrine; AngII, angiotensin II; ET-1, endothelin-1; HD, hypertrophy development; AC, aortic constriction; ca, constitutively active; RAR, retinoic acid receptor; TG, transgenic; CnA, calcineurin; DS, Dahl-salt sensitive; SHR, spontaneously hypertensive rat; CsA, cyclosporine A; NA, not applicable; NR, not reported; HF, heart failure; OCM, obstructive cardiomyopathy; AM, accelerated mortality; WL, weight loss; DPS, decreased blood pressure; PI, pulmonary infection; FHC, familial hypertrophic cardiomyopathy; MLC-2v*, non-phosphorylatable myosin light chain-2v.

a

FK506 induces hypertrophy when used in combination with rapamyicin.

Using the Dahl salt-sensitive rat model, which develops a rapid onset hypertension and both pressure and volume overload hypertrophy, Shimoyama and colleagues demonstrated a substantial reduction in indexed left ventricular weight using only very low doses of FK506 (0.1 and 0.01 mg/kg per day) over a 6-week period of severe hypertension. In fact, cardiac enzymatic calcineurin activity was elevated at any time point examined in this hypertensive model. FK506 also prevented fibrosis deposition and expression of certain fetal-type cardiac genes. Sakata et al. demonstrated LV blockade and prevention to heart failure development in the same model when FK506 (1 mg/kg per day) was administered early (from 8 weeks), but no attenuation of heart failure remodeling when administered late in life (from 17 weeks) [53].

Using TG rats harboring both the human renin and angiotensin genes, Mervaala et al. investigated CsA effectiveness to protect against Ang II-induced myocardial and renal damage. In their model, CsA completely prevented cardiovascular death, decreased 24-h albuminuria by 90%, lowered systolic blood pressure by 35 mmHg, and protected against the development of cardiac hypertrophy [54].

Calcineurin activation may also play a role in distinct hypertrophic stimuli [55]. Following 10 weeks of voluntary exercise training to evoke a physiological adaptive hypertrophy response in rats, indexed LV weight and LV calcineurin activity were increased by 20% and 2.5 fold, respectively, and CsA was effective in preventing both parameters [55]. Post-infarction failure is usually associated with a strong volume overload stimulus. Rats subjected to chronic myocardial infarction (MI) and administered CsA over a 14-day post-MI recovery period demonstrated a significant attenuation of cardiac hypertrophy and α-skeletal actin gene induction. However, the inhibition of hypertrophy led to an increased incidence of LV dilation and reduced hemodynamic performance, suggesting acceleration of the heart failure development in this model [56].

5. CsA and FK506: no correlation between calcineurin and cardiac hypertrophy

Boluyt and colleagues demonstrated significant activation of 70-kDa S6 kinase (P70^S6K) following phenylephrine-stimulation, and rapamycin pretreatment prevented this effect [57]. Rapamycin mediates its effects through specific binding to the intracellular immunophilin FKBP. Since FK506 and rapamycin both bind FKBP and act mutually antagonistic, the authors used FK506 at a 10-fold molar excess to competitively reverse the anti-hypertrophic, rapamycin-mediated effects. This study implicates that FK506 may have both anti-hypertrophic (calcineurin inhibition) as well as pro-hypertrophic properties, albeit that latter effects only play a role at excess concentrations, and emphasizes the need for correct CsA or FK506 dosage use to study calcineurin biology.

In the initial report of Sussman and coworkers, the RAR overexpressing TG model failed to morphologically respond to CsA [24]. This model was created to study the function of excessive retinoid signaling during cardiac morphogenesis, and resulted in severe heart failure in neonates and juvenile mice when a consititutively activated RAR was driven to the embryonic ventricle by the β-MyHC promoter. Interestingly, postnatal ventricular expression of the same receptor (driven by the α-MyHC promoter) did not result in cardiac pathology, suggesting a critical role for retinoid signaling during developmental stages of ventriculogenesis. Nevertheless, the failure of the RAR model to respond to systemic immunosuppressive therapy suggests that retinoid signaling acts independently from calcineurin during cardiomorphogenesis.

Muller and coworkers found that CsA administration of 25 mg/kg twice daily did not attenuate cardiac hypertrophy in mice with transverse aortic constriction, but, unfortunately, calcineurin activity assays were not provided [58]. Ding and colleagues investigated CsA effectiveness in a murine model of ascending aortic constriction [59]. Morphometric analyses of hearts subjected to this very severe form of pressure overload revealed no statistical differences between non-treated and CsA-treated, banded animals. In fact, calcineurin activity assays revealed a lower enzymatic activity following pressure overload compared to sham operated animals. Given this, one would not expect a significant impact from further CsA administration (calcineurin inhibition), yet the authors reported development of heart failure by drug treatment [59].

Meguro et al. found a substantial prevention of LV hypertrophy by CsA treatment in transverse aortic constricted mice over a period of 3 weeks. However, a disproportionate number of premature deaths in the CsA treated group was observed, all within the first 7 days of the study and accompanied by pleural efflusion. Invasive LV hemodynamic analysis revealed a significant lower ascending as well as abdominal systolic aortic pressure in the presence of comparable trans stenotic pressure gradient, suggesting both an intrinsic myocardial depressive and a distal blood pressure lowering effect by CsA. The authors concluded that inhibition of LV hypertrophy might be of detriment to the heart, and could accelerate decompensation and heart failure [60].

Luo and colleagues investigated a similar pressure overload model in the rat by constriction of the abdominal aorta as initially employed by multiple groups [24,49,50] and randomized their groups to receive different doses of CsA or FK506 [61]. No effect of CsA or FK506 on the development of LV hypertrophy was observed, but a significant increase in mortality was evident with increasing dosages of CsA or FK506. In fact, the highest FK506 dose (4 mg/kg per day) was associated with 90% mortality [61].

Zhang and colleagues demonstrated that 6 week CsA treatment (5 mg/kg per day) was associated with a significant elevation of blood pressure in the spontaneously hypertensive rat (SHR) model, but this additive blood pressure increase was not associated with increased heart weight [62]. To avoid interpretative complications due to the genetic component of the SHR model, the authors next turned to normotensive rats to address the effect of CsA on pressure overload induced hypertrophy. Two weeks of CsA treatment resulted in a 28% increase in indexed LV weights in abdominal aorta constricted rats compared to a 38% increase in vehicle-treated, banded animals, a difference that was not found to be statistical significant. Four weeks of aortic banding resulted in an increase of 46% in vehicle treated animals, while CsA treated banded animals demonstrated an increase of 27 and 22% at CsA dosages of 10 and 20 mg/kg per day, respectively. Again, the difference was not indicated as statistically significant, even though CsA appeared to have dose-dependent effects on the development of LV hypertrophy. This particular study was also associated with a substantial post-operative mortality, ranging from 33 to 67%, and a reduction in pressure gradients at 4 weeks, most notably in CsA treated groups [62].

Hayashida and coworkers observed increased LV calcineurin activity in hypertensive hypertrophied Dahl-Issei rat strain, but not during the later congestive heart failure phase. However, a CsA regimen started at 11 weeks of age did not prevent LV hypertrophy, nor heart failure development [63].

Finally, in a murine model of familial hypertrophic cardiomyopathy (FHC) bearing a knock-in missense mutation in the cardiac myosin heavy chain (αMyHC^403/+) [64], both CsA and FK506 resulted in accentuated LV hypertrophy and worsening of pathology [65]. Pre-treatment with diltiazem (I_Ca antagonist) prevented the exaggerated pathology in αMyHC^403/+ mice. Long term treatment with a K⁺-channel agonist, minoxidil, mimicked the morphologic pattern associated with CsA and FK506 in this model. These changes were attributed to drug-induced elevation of diastolic Ca²⁺ concentration [65].

6. Interpreting the CsA and FK506 studies

How can we reconcile these differing accounts on CsA/FK506 effectiveness? By far the easiest explanation would be to assume some level of model-dependency for calcineurin involvement, implicating that other signaling modules may take over in relative importance to the expense of calcineurin. Indeed, it has been postulated that calcineurin inhibition effectively blocks Ang II-mediated cardiac hypertrophy, such as that developing in response to abdominal aortic banding or following reduced renal perfusion in renovascular hypertension. In support of this view, all reports involving models with AngII activation (abdominal aortic constriction [49,50]; renin-angiotensin TG rat [54]; two-kidney-one-clip [52]) reported a higher degree of CsA/FK506 effectiveness (Table 1). This view must be an oversimplification, since constriction of the aortic arch (transverse aortic constriction) does not activate the renin-angiotensin system [66], yet one report still demonstrated near complete prevention of hypertrophy by CsA administration [51] (Table 1).

Genetic variability between the animals and their sensitivity to CsA/FK506 may play an underestimated role and coincides with substantial variability in drug dosage. To date, Sprague–Dawley rats, Wistar rats and FVB/N mice exhibited a relatively higher degree of drug tolerance than Dahl–Iwai rats and C57BL/6 mice (Table 1). The most striking example of drug tolerance and strain dependency is provided by the studies of Shimoyama and colleagues [49,67], where Wistar rats tolerated 1 mg/kg per day FK506 [49], while the same group found a 10-fold lower whole body upper limit tolerance in rats with the Dahl–Iwai background [67]. This contrasts with dosages of up to 4 mg/kg per day FK506 used in Sprague–Dawley rats (40- to 400-fold excess vs. Shimoyama et al. [67]) which, not surprisingly, resulted in up to 90% mortality in pressure-overloaded animals. Clearly, the vast variations in genetic background and drug dosage must have influenced the interpretation of calcineurin involvement in cardiac hypertrophy.

Careful inspection further suggests some degree of variance associated with surgical procedure, peri-operative milieu and route of drug administration. A number of studies have reported a high degree of drug-associated premature mortality, up to 90% in drug administered animals, an effect which might have preselected interindividual variations. In fact, careful inspection suggests an inverse relation between high mortality and drug-effectiveness (Table 1).

Differences in timing, route and duration of drug administration could be another factor relating to some of the differences between the studies. Ding et al. reported no drug effectiveness following 4 weeks of administration, while Eto et al., employing a similar model of ascending aortic constriction and comparable CsA dosage, demonstrated a clear effect on the early phase (1 week post-surgery), but not on later stages (4 weeks post-surgery) of hypertrophy development. Two groups reported efficient hypertrophy prevention in the Dahl-Salt sensitive model using low doses of FK506 and starting drug administration early in life (6 or 8 weeks) [53,67], while another reported no effectiveness when they started CsA injections at 11 weeks of age [63]. Luo and colleagues reported high mortality and no CsA effectiveness in rats administered the drug in the drinking water [61]. The majority of studies, however, have injected CsA or FK506 subcutaneously (often twice daily) due to its low solubility in aqueous solutions and limited half-life.

A final issue concerns the phenotypic interpretation following drug administration. Meguro et al. reported high mortality in their CsA banded animal groups, interpreted as development of heart failure since a disproportionate number of animals displayed pleural effusion upon autopsy [60]. It is unlikely, however, that heart failure was the true cause of death. Only a 28 and 16% decrease in LV dP/dt_max and LV ejection fraction in CsA-treated, banded vs. vehicle-treated, banded animals was observed, respectively [60]. A more plausible explanation for their observations would be enhanced susceptibility to pleural infection in treated animals [67] due to the systemic immunosuppressive therapy [67].

Conclusively, the vast majority of studies, regardless of their qualitative outcome, were hindered to some degree by intra-and extracardiac side effects of the drugs. Latter notion underscores the problematic situation of using CsA or FK506 as experimental devices, virtually excludes their potential as future treatment options for patients with hypertrophic heart disease, and motivated some research groups to find alternative experimental strategies to elucidate the myocardial (patho)physiological role of calcineurin.

7. Genetic inhibition of calcineurin

In recent years, different classes of proteins with calcineurin inhibitory properties have been described. AKAP79 (for -inase nchoring rotein) was the first protein discovered to have calcineurin inhibitory characterstics [68]. AKAP79 acts as a scaffold protein, which binds protein kinase A and protein kinase C besides calcineurin and is thought to anchor multiple classes of signaling modules in the vicinity of substrates to facilitate their proper and timed activation [68]. One interesting class of calcineurin inhibitors are the products of the (Down's syndrome critical region) DSCR1 gene and its relatives, DSCR2 or ZAK14[69]. DSRC1 is located on human chromosome 21 in the so-called Down's syndrome critical region. The small gene products of DSCR1 and DSCR2, designated MCIP1 (for yocyte-enriched alcineurin nteracting rotein-1) and MCIP2, respectively, are remarkably potent inhibitors of calcineurin activity in striated muscle [70]. Interestingly, it was demonstrated that calcineurin activity upregulates the MCIP1 gene product in cardiomyocytes through an intragenic segment in the MCIP1 gene, which includes a dense cluster of consensus NFAT binding sites [71]. MCIP1 may therefore participate in a negative feedback circuit to diminish potentially deleterious effects of unrestrained calcineurin activity in striated muscle cell lineages. Two additional classes of cellular calcineurin inhibitors are Cain (for lcineurin hibitory protein) and the alcineurin B omologous rotein (CHP). Cain (alos known as Cabin) is a large 240-kDa protein with multiple binding domains that functions as a scaffolding protein attaching numerous proteins besides calcineurin [72]. Cain is a noncompetetive inhibitor of calcineurin phosphatase activity with a K_i of 440 nM and antagonizes NFATc translocation [73,74]. CHP has a high similarity to the calcineurin B subunit and is able to compete with the calcineurin B subunit for binding the calcineurin A subunit [75,76].

The identification of specific cellular antagonists of calcineurin have provided an excellent platform to design experimentation that circumvents the issue of drug specificity and whole body toxicity. This strategy was first reported by Taigen and co-workers, who created adenoviral vectors expressing the specific calcineurin binding domains of the Cain and AKAP79 proteins (designated ΔCain and ΔAKAP, respectively) [77]. AdΔCain and AdΔAKAP infection resulted in attenuation of AngII, PE and growth factor-induced calcineurin activity, cardiomyocyte hypertrophy, and atrial natriuretic factor (ANF) expression (Fig. 2). The same non-competitive calcineurin inhibitors were recently overexpressed in a cardiac-restricted manner in mice. ΔCain and ΔAKAP79 TG animals demonstrated stable transgene expression in the cardiac compartment, reduced cardiac calcineurin activity and a significant attenuation of hypertrophy in response to catecholamine infusion or pressure overload stimuli [78](Fig. 2). In the same study, the ΔCain adenovirus was used for viral-mediated gene transfer of the ΔCain peptide into the adult rat myocardium. Overexpression of the ΔCain protein resulted in inhibition of pressure-induced calcineurin activation and cardiac hypertrophy [78] and supports the feasibility to acutely intervene in reactive hypertrophic signaling using gene therapy approaches [79].

As an alternative approach to inhibit calcineurin, transgenic mice expressing a truncated form of human MCIP1 in a cardiac-selective manner were created. Remarkably, unstressed MCIP1 TG animals revealed a 5–10% smaller heart size, establishing a role for calcineurin in normal, developmental myocardial growth. MCIP1 overexpression prevented the massive hypertrophic response, fetal gene induction and progression to dilated cardiomyopathy in the calcineurin TG mouse [11]. MCIP1 TG mice demonstrated resistance towards cardiac hypertrophy as a result of long-term β-adrenergic stimulation and exercise training. Finally, myocardial overexpression of a dominant negative (dn) form of calcineurin provided protection against pressure overload hypertrophy following abdominal aortic banding in mice [80]. The results from these three reports utilizing four distinct genetic strategies to inhibit calcineurin activity (ΔCain, ΔAKAP79, MCIP1 and dn-calcineurin) make it hard to dispute that calcineurin is a required component of hypertrophic signaling following diverse stimuli [78,80,81].

The existence of viable calcineurin Aα and Aβ somatic knockout mice may provide a means of obtaining further genetic evidence for the involvement of calcineurin in the hypertrophic response. Because the viability of double-null calcineurin Aα and Aβ mice is still uncertain, the establishment of transgenic animals with a dominant-negative calcineurin inhibition based approach [78,80,81] may still prove to be of significant value. A preferential approach would be to identify the critical calcineurin A isoform and ablate it in an inducible, ventricular myocyte cell lineage-dependent fashion, which would also permit definitive insight into the temporal aspects of calcineurin involvement in the distinct stages of heart failure development.

Still many other questions remain and concern calcineurin's precise pathobiological role in terms of biochemical properties, subcellular localization, crosstalk with other notorious hypertrophic signaling modules, its suggested role in myocyte viability and its precise contribution in the progression of human heart failure. The current status of knowledge of these issues is discussed below.

Fig. 2

Genetic inhibition of calcineurin with calcineurin inhibitory protein (Cain) abrogates the LV hypertrophy response [77,78]. The left panel summarizes the in vitro results obtained with adenoviral ΔCain gene transfer (AdΔCain) in cardiomyocytes. Phenylephrine (PE) induces a massive increase in cardiomyocyte size in cultures infected with a control adenovirus (Adβgal) compared to uninfected, non-stimulated cells (top left and middle left panels). In contrast, blockade of calcineurin in the presence of ΔCain prevents PE-induced hypertrophy (lower left panel). Cardiomyocytes were visualized by immunocytochemical α-actinin detection (red), nuclei were visualized with bisbenzamide (white). Hematoxylin-Eosin stained cross-sections demonstrate that similar effects were observed following ΔCain overexpression in vivo. A 14-day pressure overload stimulus resulted in a visible increase in heart weight in wildtype animals, as compared to sham-operated, wildtype mice (top right and middle right panels). Mice overexpressing the non-competetive ΔCain protein in the cardiomyocyte component are protected against this pressure-overload stimulus (lower right panel).

8. Reciprocal relationship between Ca²⁺ homeostasis and calcineurin activity

One fundamental question relates to the processes leading to myocardial calcineurin activation. How can the cardiac myocyte distinguish between changes in Ca²⁺ that result in calmodulin activation versus the vast fluctuations in Ca²⁺ that occur upon each cycle of contraction and relaxation? Studies in other cell types have demonstrated that NFATc remains nuclear only in response to prolonged, low-amplitude Ca²⁺ signals and is insensitive to transient, high-amplitude Ca²⁺ alterations [82]. Interestingly, the activity of CaMK, another important Ca²⁺/calmodulin regulated hypertrophic signal transducer [83], was reported to be uniquely sensitive to transient, high amplitude Ca²⁺ fluctuations [82]. The differential response of two major Ca²⁺/calmodulin-regulated hypertrophic signal transducers to fundamentally distinct Ca²⁺ fluctuations may implicate that they fulfill specialized pathophysiological functions within the cardiomyocyte. Whether calcineurin and CaMK also differentially respond to distinct Ca²⁺ alterations in striated muscle cell types and what these functions might be remains to be explored.

Furthermore, the actual source of Ca²⁺ that activates calcineurin (or other Ca²⁺/calmodulin signaling modules for that matter) is incompletely understood. Studies using nicardipine and verapamil suggest a critical involvement of the L-type Ca²⁺ channel, which may respond downstream of G-protein coupled receptor (GPCR) agonists such as ET-1, AngII or PE [11,46] and gp130 receptor agonists like LIF [47]. LIF enhances intracellular Ca²⁺ transients through an increase in L-type Ca²⁺ current (I_{Ca, L}) in adult cardiomyocytes [84]. LIF-mediated activation of I_{Ca, L} resulted in increased calcineurin and CamK activities and nicardipine and verapamil pretreatment fully prevented their activation, suggesting at least one plausible source of Ca²⁺ that activates calcineurin and CaMK.

Findings in other cell types suggest a reciprocal relation between calcineurin on the one hand, and the RyR, IP3 receptor and L-type Ca²⁺ channel, on the other [85,86]. In line with this notion, we have previously reported that adenoviral expression of calcineurin in neonatal cardiomyocytes resulted in positive inotropy and increased Ca²⁺ transients [87]. In addition, adult myocytes derived from the calcineurin TG mouse exhibited increased I_Ca,L amplitude and density and shortened time to half decay compared to wildtype myocytes [88]. Since the total number of L-type Ca²⁺ channel α1 and β2 subunits was unaltered, the increased density of I_Ca,L is due to an increased fraction of channels that open during the repolarization phase. Interestingly, therapeutic doses of CsA had no effects on I_Ca,L in wildtype mice, suggesting that calcineurin indirectly affects L-type Ca²⁺ channel properties. This contrasts observations in the mammalian brain, where calcineurin is the major phosphatase responsible for L-type Ca²⁺ channel inactivation [89]. One plausible explanation for the effects of myocardial calcineurin on the profile of myocyte Ca²⁺ handling may relate to SR Ca²⁺ handling. Indeed, Janssen and coworkers demonstrated that CsA induces sustained SR Ca²⁺ leakage from adult rabbit and human ventricular myocytes at therapeutic doses [90]. Collectively, calcineurin inhibition may have the potential to alter myocardial intracellular Ca²⁺ homeostasis and influence the susceptibility to the occurrence of lethal ventricular arrhythmias.

The finding that CsA exacerbated the hypertrophic response in a murine FHC model [65] has further complicated our understanding of cardiomyocyte Ca²⁺ homeostasis and Ca²⁺/calmodulin-regulated signaling in the LV hypertrophy response [91]. Fatkin and coworkers observecd an increase in Ca²⁺ transient in wildtype myocytes, and, to a lesser extent, in myocytes from mice carrying the FHC mutation. Pretreatment with diltiazem, a L-type Ca²⁺ channel blocker, prevented the wosening of the phenotype in mutant mice, suggesting that CsA infavourably modulated I_Ca,L properties [65]. Latter interpretation appears to contrast their finding that CsA subtly increased the Ca²⁺ transient of αMyHC^403/+ myocytes. Moreover, Yatani and coworkers [88] failed to observe a response of CsA on L-type Ca²⁺ channel kinetics in wildtype myocytes, and Janssen and coworkers observed a reduction of the Ca²⁺ transient in human failing trabeculae following CsA administration [90].

Nevertheless, latter study poses the interesting question whether blockade of calcineurin is of detriment to FHC-associated LV hypertrophy yet effective to reduce acquired forms of LV hypertrophy and dilated cardiomyopathies. The finding that CsA and FK506 proved to be highly effective in rescuing the cardiac phenotype of tropomodulin or MLC-2v transgenic mice [24], while exacerbating the phenotype of αMyHC^403/+ animals suggests several possibilities. Alterations at the sarcomere may either induce cardiomyopathy through fundamentally distinct alterations in intracellular Ca²⁺ homeostasis, and/or these studies might have suffered from the short-comings of the particular animal model investigated, and/or the findings are related to calcineurin-unrelated effects of the drugs investigated. Genetic approaches to inhibit calcineurin in these and other models of FHC [92–96] may become helpful in answering these questions.

As a result of these uncertainties regarding momentous calcineurin activation, a major experimental problem relates to the current assessment of cardiac calcineurin activity. The calcineurin activity assay depends upon inclusion of several phosphatase inhibitors to reduce background phosphatase activity and enhance specificity, since different phosphatases besides calcineurin exist in cardiac tissue. One required inhibitor (okadaic acid) also partially inhibits calcineurin activity, leading to an inherent reduction of output. Furthermore, calcineurin is subject to rapid oxidation in vitro, which underscores the notion that calcineurin may be several folds more active in situ compared with purified protein extracts. Thus, regardless whether the complicated technical aspects of the assay are performed correctly, the relevance of the information it provides is still questionable. Other assays are required to objectively monitor instantaneous calcineurin activation in situ.

The identification of MCIP1, as part of a self-promoting negative feedback loop of calcineurin biology, may provide an indirect readout of calcineurin activation status. Before such an assay could be implemented, additional investigation is required to assess whether MCIP1 is specifically induced upon calcineurin (NFATc translocation) activation or whether it is a more general marker of hypertrophy status. Another approach could be to use the intragenic MCIP1 region, which contains an unusual dense cluster of NFAT consensus binding sites [71], and link this to reporter genes to create transgenic models that provide a constant readout of (myocardial) calcineurin activity. Alternatively, transgenic models with multimerized NFATc consensus binding sites linked to a reporter output [97] may provide an alternative to monitor the temporal dynamics of calcineurin activation during the progression of LV hypertrophy.

Studies on calcineurin involvement in failing human hearts further underscored the complex pathobiology of calcineurin. Human hypertrophic biopsies revealed that calcineurin activity (as measured by the enzymatic assay) correlated well with absolute calcineurin Aβ protein levels (as measured by immunoblot analyses) [98]. It appears that in addition to an acute increase in enzymatic activity of pre-existing protein (Ca²⁺ activation), activity may also be subject to positive feedback mechanisms at the transcriptional and/or translational level. Therefore, future calcineurin assays may simply become based upon assessment of absolute myocardial calcineurin Aβ protein levels.

9. Calcineurin and cardiomyocyte viability

Mitochondria occupy an unusual large fraction of intracellular volume within adult cardiomyocytes. Multiple death signals impinge upon the mitochondrial membrane potential, resulting in hallmark apoptotic events such as loss of mitochondrial matrix components and activation of caspases. Since cardiomyocytes are limited in their ability to enter the cell cylce reentry, cummulative loss of single cardiomyocytes is regarded as a contributing factor in the genesis of human heart failure [99]. Given this, remarkably few studies have assessed whether calcineurin activity may influence muscle cell viability.

To test whether programmed cell death may contribute to the rapid transition from hypertrophy to overt heart failure in the calcineurin TG mice, calcineurin adenoviral infected cardiomyocytes were tested for their viability. Morphological analyses and TUNEL assays provided evidence that calcineurin activtion afforded protection against 2-deoxyglucose and staurosporine-mediated apoptosis. Moreover, endogenous calcineurin activation through PE stimulation [77] resulted in substantial protected cardiomyocytes from apoptosis, and this effect could be antagonized by targeted inhibition of calcineurin by adenoviral ΔCain gene transfer [77]. The mode of protection was found to be partially associated with NFATc translocation and protein kinase B (Akt) activation. In support of these findings in vitro, it was demonstrated that massively hypertrophic hearts from adult calcineurin TG animals displayed a remarkable level of resistance against ischemia/reperfusion-induced TUNEL laddering. Conversely, CsA treatment did not render cardiomyocytes more vulnerable towards programmed cell death, not even in the apoptosis prone Gqα-overexpressing, cardiomyopathic mouse model, suggesting that endogenous calcineurin activation promotes cardiomyocyte viability [100]. These findings were largely supported by a recent report from Kakita and colleagues, who were able to demonstrate that ET-1-mediated protection of cardiomyocyte apoptosis requires calcineurin activation, since CsA and FK506 negated ET-1-induced protection against apoptotic cell death and expression of the anti-apoptotic factor Bcl-2 [101].

In contrast, Saito and co-workers demonstrated a 3-fold increase in calcineurin enzymatic activity following isoproteronol exposure concomitant with DNA fragmentation, while CsA and FK506 reversed this effect. TG animals expressing a dominant-negative mutant of calcineurin in the heart were resistant towards isoproteronol-induced cardiomyocyte apoptosis, suggesting that calcineurin may act downstream of catecholamine-induced apoptosis [102].

Although these findings appear to be in contradiction with one another, it is known that calcineurin can activate opposing pathways that either suppress or induce apoptosis in the same cell type [103]. Indeed, Saito et al. reported that myocardial ischemia/reperfusion (oxidative stress) resulted in significantly more cardiomyocyte apoptosis in their dominant negative-calcineurin TG model than in wildtype littermates [102], supporting the findings of the initial two reports [100]. Conclusively, it seems reasonable to hypothesize that calcineurin-induced hypertrophy protects the heart from apoptotic death, depending upon the actual death signal and (sub)cellular context. The precise molecular mechanisms behind the anti-apoptotic properties of calcineurin are most complex and could well be associated with the the initiation of the hypertrophic response itself [104].

10. Calcineurin and (human) heart failure

The vast majority of reports to date have remained focussed upon assessing the involvement of calcineurin on the initiation and early progression phase of LV hypertrophy. Although justified in view of the intimate relationship between LV hypertrophy and progression of congestive heart failure, remarkably few studies have directly addressed the more clinically relevant question whether altering the balance of calcineurin activity directly alters the pathogenesis of (experimentally induced) cardiac failure.

Current experimental efforts have been limited to the demonstration that CsA prevents morphological remodeling in the tropomodulin or MLC-2v overexpressing mice with dilated cardiomyopathy [26,27], and hemodynamic recordings of the rescued phenotypes are still lacking. In light of recent evidence that CsA has direct cardio-depressive actions on rabbit and human cardiomyocytes [90], an assessment whether genetic calcineurin inhibition per se has negative inotropic/lusitropic effects in these and other valuable murine models of severe heart failure [105–109].

A recent study established the activation of calcineurin in biopsies of patients with hypertrophy secondary to hypertension (‘compensatory hypertrophy’) and patients with heart failure secondary to coronary artery disease or idiopathic DCM. One interesting feature of the study is that both increased specific calcineurin activation and calcineurin Aβ protein was evident in patients with ‘compensatory hypertrophy’, while calcineurin activation in the failing hearts seemed to be due to only a relative increase in protein content. These findings were supported by Lim and Molkentin, who reported increased can isoform expression in the failing human heart [110]. In contrast, Tsao and colleagues reported lower CnAβ mRNA levels in human failing hearts, but the probe used was directed against a minor splice isoform of gene of interest [111].

Due to their substantial extra-cardiac side-effects in humans, the use of CsA or FK506 as treatment options for hypertrophic heart disease in patients is doubtful. In fact, long term CsA treatment is associated with renal toxicity and hypertension, which lead to cardiac hypertrophy in certain subjects. The concept that CsA or FK506 may have direct pro-cardiohypertrophic properties in humans [112] (and calcineurin inhibition associated with myocardial growth) is incorrect. More likely, systemic immunosuppression may induce hypertension in select subjects, which influences myocardial growth secondarily. In addition, the dose of CsA and FK506 required to prevent cardiac hypertrophy in experimental animals studies is approximately 10-fold higher than used to achieve immunosupression in humans, a phenomenon related to a higher myocardial calcineurin content, differential tissue accessibility or the higher metabolic rate of small rodents.

11. Integrated signal transduction: calcineurin, crosstalk and transcriptional events

Various reports suggest that multiple signaling networks play a role in hypertrophic remodeling [113]. For example, altering the balance of myocardial RSG4 expression, or cardiac introduction of a dominant inhibitory G_qα peptide or disruption of the JNK MAPK pathway have been demonstrated to impact on hypertrophic remodeling. How is it possible that inhibition of separate signaling networks can have such dramatic whole organ effects? Clearly, myocardial growth is not regulated by the additive activation of signaling modules, but by a more complex mechanism, involving the activation of multiple (intergrated) transduction pathways (Fig. 3).

Fig. 3

Contemporary model for (patho)physiological calcineurin signaling in the cardiac myocyte. Several stimuli can trigger activation of calcineurin, among which intrinsic defects of sarcomere function, multiple humoral factors (e.g. AngII, ET-1, LIF, PE), which directly or indirectly (by transient Ca²⁺ release from SR stores through ryanodine sensitive receptors (RyR) or through L-type Ca²⁺ channels (VLDC) in T-tubuli) elevate free cytosolic Ca²⁺. The Ca²⁺/calmodulin complex initiates parallel activation of intracellular signaling targets such as Calmodulin activated protein kinase (CaMK) isoforms, calcineurin, myosin light chain kinase (MLCK) or conventional protein kinase C (PKC) isoforms, each of which have been implicated in cardiomyocyte hypertrophy. Calcineurin differentially alters the activation status of certain members of the mitogen-activated protein kinase (MAPK) or PKC superfamilies, most probably as a reinforcement of cellular signaling. Activated calcineurin may recognize up to four nuclear factor of activated T-cell (NFAT) isoforms in the heart and, in addition, increases myocyte-enhancing factor (MEF2) activation status to direct hypertrophic gene expression remodeling. One particularly interesting genetic target of calcineurin is myocyte enriched calcineurin interacting protein-1 (MCIP1), which acts as a myocyte specific calcineurin inhibitor. Similarly, activition of glycogen synthase kinase 3β (GSK3β) counteracts the activation status of NFAT isoforms induced by calcineurin. Calcineurin also positively influences the viability of cardiac muscle cells by effects on the mitochondrial permeability transition (MPT) and anti-apoptotic processes, probably depending upon the actual death signal. Finally, an underrepresented feature of calcineurin function concerns its localization. It has been reported to localize either in the cytosol or nucleus, associated with specialized anchoring proteins [68], in the vicinity of the sarcoplasmatic reticulum (SR) or close to sarcomeres by its specialized docking protein calsarcin [124].

One aspect that undoubtedly contributes to the integration of cardiac signaling is the interdependence or crosstalk between parallel signaling pathways. We have previously demonstrated that hearts from calcineurin TG mice demonstrated robust JNK MAPK activation, and activation of several PKC isoforms. Adenoviral expression of calcineurin resulted in marked hypertrophy in cultured myocytes [87,100], and was prevented by JNK or PKC inhibition [87]. Also, CsA treatment of pressure overloaded rat hearts prevented PKCα, PKCθ and JNK MAPK activation [87]. Later studies supported this concept of interconnectiveness between calcineurin, PKC and MAPK factors [52,114], which coincide with the finding that PKCθ and calcineurin synergize to activate the JNK MAPK pathway in control of the interleukin-2 promoter in T-cells [115]. Collectively, these studies suggest the existence of a conserved, interconnected, regulatory circuit between calcineurin, PKC and JNK MAPK that controls myocardial growth, and it suggests that inhibition of either component may abrogate the entire integrated cascade (Fig. 3) [87].

Some level of functional diversity and hierarchy among the multitude of hypertrophic pathways may still be anticipated. Transgenic lines expressing a constitutively activated mutant of the ERK1/2 MAPK-selective activator MEK1 display a physiological form of hypertrophy with increased contractility parameters throughout their complete life span [116], while activation of another MAPK member, ERK5, results in pure eccentric hypertrophy and heart failure [117]. Transgenic overexpression of protein kinase CβII (PKCβII) sufficed to result in juvenile lethality and cardiac hypertrophy/failure in mice [118], but its targeted disruption failed to significantly abrogate pressure overload-induced myocardial growth [119], suggesting that certain networks are not a required component of the hypertrophic response.

Given the above notions and data from contemporary literature, how should we define the role of calcineurin in cardiac hypertrophy? Given the massive hypertrophic response in calcineurin (and CaMK) TG animals, the overall success of CsA/FK506 studies to inhibit myocardial growth, and the recent demonstration that genetic calcineurin inhibition strategies successfully abrogate hypertrophic growth, it is difficult to dispute that calcineurin plays both a sufficient as well as an indispensable role in transducing hypertrophic signals following most pathophysiological stimuli. In addition, it seems reasonable to assume that calcineurin may be a crucial factor especially during the early phases of hypertrophic heart disease based upon the following notions. (1) The rise in intracellular Ca²⁺ in cardiomyocytes most probably constitutes an early phenomenon during the LV hypertrophy response [120,121]; and (2) calcineurin activation was observed predominantly in hearts from patients in a compensatory hypertrophic phase and to a lesser extent in overtly failing patients [98].

A more fundamental aspect of calcineurin signaling involves the contribution of downstream transcriptional effectors. NFATc4 (NFAT3) is still viewed upon as the critically effector downstream of myocardial calcineurin activation, based upon its ability to interact with GATA-4 and since NFATc4 TG animals display a massive hypertrophic phenotype [11]. However, given the overlapping expression patterns and redundant functions of single NFATc isoforms in other cell types, it is conceivable that some level of NFATc isoform redundancy may also exist in the heart. In that case, a simple genetic loss-of-function approach for each NFATc isoforms may prove to be insufficient to assess whether NFATc activity is the sole downstream transcriptional effector of calcineurin, due to the co-existence of up to four cardiac NFATc family members with largely overlapping functions. Recent evidence indeed suggests that at least NFATc1 (NFATc) is also present in the ventricular myocyte, can associate with GATA-4 and responds to calcineurin activation, in a manner analogous to NFATc4. Dominant negative NFATc approaches that simultaneously abrogate NFATc function may become useful to circumvent such complications as recently demonstrated for T lymphocytes [122]. Alternative downstream transcriptional effectors may also be responsible for the nuclear events downstream of calcineurin. Recent evidence implicates myocyte enhancing factor-2 (MEF-2), nuclear factor-κB and Elk-1 [83,123].

Collectively, the present review provided an update on the role of calcineurin in the pathogenesis of cardiac hypertrophy. An emerging concept indicates that interference with distinct hypertrophic signaling pathways may significantly abrogate the LV hypertrophy response [113]. Therefore, future efforts in this field requires a further analyses to identify the key integrated cascades through which maladaptive hypertrophic signals are channeled to achieve the ultimate goal of a better treatment of heart failure. It appears that calcineurin will continue to be the focus of ongoing research, providing us with a platform to understand the complexity of Ca²⁺/calmodulin signaling on cardiac morphology and function.

Acknowledgements

O.B., E.v.R. and P.A.D. are supported by NIH grant F32-HL-10336, the Netherlands Heart Foundation (NHS 99-114 to E.v.R. and NHS 2000-160 to P.A.D.) and the Interuniversitary Cardiology Institute Netherlands (ICIN), respectively. J.D.M. is supported in part by National Institutes of Health (NIH) grants HL60562 and HL07382 and a Scholar Award from the Pew Foundation. L.J.D.W. is supported by a Bekales Foundation Award in Cardiology and the Netherlands Foundation for Scientific Research (NWO 902-16-275).