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Calcineurin–NFAT signaling regulates the cardiac hypertrophic response in coordination with the MAPKs

Jeffery D Molkentin
DOI: http://dx.doi.org/10.1016/j.cardiores.2004.01.021 467-475 First published online: 15 August 2004

Abstract

Prolonged cardiac hypertrophy of pathologic etiology is associated with arrhythmia, sudden death, decompensation, and dilated cardiomyopathy. In an attempt to understand the mechanisms that underlie the hypertrophic response, extensive investigation has centered on a characterization of the molecular pathways that initiate or maintain the pathologic growth of individual cardiac myocytes. While a large number of signal transduction cascades have been identified as critical regulators of cardiac hypertrophy, here the scientific evidence implicating the protein phosphatase calcineurin (PP2B) and the mitogen-activated protein kinases (MAPK) as co-regulators of reactive hypertrophy will be discussed. Gain- and loss-of-function studies in genetically altered mice and in cultured cardiomyocytes have demonstrated the necessity and sufficiency of calcineurin to regulate pathologic cardiac hypertrophy. However, using similar approaches, the hypertrophic regulatory role attributed to various branches of the MAPK signaling pathway has been less conclusive, although a loose consensus suggests that the c-Jun N-terminal kinases (JNK) and p38 kinases function as mediators of dilated cardiomyopathy, while extracellular signal-regulated kinases (ERKs) function as regulators of hypertrophy. More recently, the actions of calcineurin and MAPK signaling pathways have been shown to be co-dependent such that unitary activation of calcineurin in myocytes leads to up-regulation in ERK and JNK signaling, but down-regulation in p38 signaling. Conversely, unitary activation of JNK or p38 in cardiac myocytes leads to down-regulation of calcineurin effectiveness by directly antagonizing nuclear factor of activated T cells (NFAT) nuclear occupancy. Thus, an emerging paradigm suggests that calcineurin–NFAT and MAPK signaling pathways are inter-dependent and together orchestrate the cardiac hypertrophic response.

Keywords
  • Calcineurin
  • Cardiac hypertrophy
  • MAPK
  • Heart failure
  • Signaling

1. Calcineurin as a signaling factor in the heart

The hypertrophic growth of the myocardium is initiated and maintained by a wide array of endocrine, paracrine, and autocrine growth factors in response to increased work load, injury, or intrinsic defects in contractile performance. While initially an adaptive response that attempts to maintain cardiac output, sustained cardiac hypertrophy induced by pathological stimuli is a leading predictor for the development of heart failure and sudden death [1,2]. To begin to understand the molecular determinants of the hypertrophic response, recent investigation has focused on characterizing intracellular signal transduction pathways in the heart.

Signal transduction in cardiomyocytes is typically initiated by membrane bound receptors that respond to various neural–humoral agonists/effectors. Once activated, these membrane bound receptors promote intracellular signaling through multiple GTPase proteins, kinases, and phosphatases. One such regulator is the calcium–calmodulin-activated protein phosphatase calcineurin (PP2B). Calcineurin is a serine/threonine-specific phosphatase that is uniquely activated by sustained elevations in intracellular calcium [3–5]. Calcineurin is comprised of a 59–63-kDa catalytic subunit referred to as calcineurin A, a 19-kDa calcium binding protein referred to as calcineurin B, and the calcium binding protein calmodulin [3,4] (Fig. 1). Three mammalian calcineurin A catalytic genes (α, β, γ) and two B subunit regulatory genes (B1, B2) have been identified in vertebrates. The calcineurin Aα, and B1 gene products are each expressed in a ubiquitous pattern throughout the body, while calcineurin Aγ and B2 expression are more restricted to a smaller subset of tissues such as brain and testis [6–9].

Fig. 1

Diagrammatic representation of calcineurin–NFAT signaling. Stimuli that increase intracellular calcium concentration result in calmodulin saturation, which is turn promotes calcineurin activation and the subsequent dephosphorylation of NFAT transcription factors within the cytoplasm. Dephosphorylated NFAT translocates to the nucleus where it participates in mediating calcium-inducible gene expression in coordination with other transcription factors. Kinases such as JNK, p38, GSK3β, casein kinase I and II (CKI and CKII), protein kinase A (PKA), and possibly even ERK phosphorylate specific NFAT family members antagonizing nuclear translocation (although ERK activation can also stimulate calcineurin–NFAT signaling). Many of these same kinases likely also participate in re-phosphorylating NFAT within the nucleus aiding in extrusion. Other abbreviations: CnA, calcineurin A; CnB, calcineurin B, Cam, calmodulin; CsA, cyclosporine; CyA, cyclophilin A; FKBP12, FK506 binding protein 12.

Calcineurin catalytic activity is inhibited by the immunosuppressive drugs cyclosporine and FK506 through complexes with immunophilin proteins [3,4] (Fig. 1). The identification of calcineurin as a target for cyclosporine and FK506 suggested a critical role for this phosphatase in the regulation of T-cell reactivity and cytokine gene expression. Once activated, calcineurin directly dephosphorylates members of the nuclear factor of activated T-cells (NFAT) transcription factor family in the cytoplasm, promoting their translocation into the nucleus (Fig. 1). Once in the nucleus, NFAT family members participate in the transcriptional induction of various immune response genes in T cells, as well as genes with diverse functions in other cell types [10]. There are four calcineurin-regulated NFAT transcription factors, NFATc1–c4, each of which is expressed in the myocardium [11].

2. Calcineurin and the initiation of cardiac hypertrophy

Calcineurin was originally implicated as a hypertrophic signaling factor based on its overexpression in the hearts of transgenic mice [12]. Mice expressing an activated mutant of calcineurin demonstrated a profound hypertrophic response (2–3-fold increase in heart size) that rapidly progressed to dilated heart failure within 2–3 months. Such data implicated calcineurin as a sufficient inducer of the hypertrophic response and as a potential causative factor associated with the transition to decompensation and heart failure [12]. In vitro, infection of cultured cardiomyocytes with an activated calcineurin expressing adenovirus similarly induced a hypertrophic response, supporting the sufficiency of calcineurin as a hypertrophic mediator in two different model systems [13].

The properties underlying activation of calcineurin in response to pathophysiologic stress in vivo remains an area of on-going investigation. Calcineurin enzymatic activity and protein levels were each significantly up-regulated in hearts from juvenile tropomodulin transgenic mice, a model of dilated heart failure [14,15]. Similarly, a number of groups have reported increased cardiac calcineurin activity in hypertrophic hearts from aortic-banded rats or mice [16–21]. Exercise-induced cardiac hypertrophy, salt-sensitive hypertension-induced hypertrophy, mineralocorticoid-induced cardiac hypertrophy, and infrarenal aortic constriction-induced neuroendocrine-mediated hypertrophy were each associated with an increase in cardiac calcineurin activity [18,22–25]. A mouse model of cardiac hypertrophy secondary to systemic carnitine deficiency (JVS mouse) was also shown to have increased calcineurin activity in the heart [26]. Finally, the failing or hypertrophied human heart was associated with increased calcineurin activity or protein levels [27–29]. In contrast, one group reported no change in cardiac calcineurin activity in response to pressure overload stimulation or in human heart failure [30,31], while another group reported decreased calcineurin activity following pressure overload stimulation [32].

While most studies support the assertion that calcineurin is activated in the hypertrophic or diseased myocardium, a handful do not. It is likely that technical details related to the calcineurin enzymatic assay itself or perhaps even significant differences between animal models underlies these disparate observations. For example, the traditional biochemical calcineurin activity assay is fraught with both technical and theoretical difficulties. The assay relies on homogenized extracts in the presence of exogenously supplied calmodulin, a design that more closely approximates the Vmax of calcineurin rather than specific activity. An alternative method for measuring calcineurin activity consists of calmodulin immunoprecipitation followed by western blotting for calcineurin protein to infer activation [16]. However, this method is influenced by calcium levels in the cellular lysates, which probably does not recapitulate endogenous levels, thus altering the interaction between calmodulin and calcineurin within the in vitro assay. A potentially more relevant and meaningful measure of calcineurin activity that has proven highly reliable is the analysis of NFAT transcriptional responsiveness. Indeed, NFATc1–c4 are only activated by calcineurin, and the amount and timing of nuclear translocation are directly proportional to the degree of calcineurin activation in vivo [10]. However, it is often not possible to directly examine NFAT nuclear translocation from intact tissues or from fractionated protein extracts due to low expression levels and the relatively poor quality of commercially available antibodies [11]. For this reason, we generated and characterized transgenic mice containing an NFAT-dependent luciferase reporter, which shows specific induction in the heart by activated calcineurin, and repression by cyclosporine [33]. Using these reporter mice, we recently demonstrated that calcineurin–NFAT signaling is constitutively upregulated throughout a time course of pressure overload hypertrophy, as well as in the failing mouse heart following myocardial infarction. Collectively, the data discussed in this section suggest that calcineurin–NFAT signaling is activated in pathological cardiac hypertrophy and heart failure, consistent with the proposed functional role for this pathway in mediating these processes.

3. Use of cyclosporine and FK506 in animal models of cardiac hypertrophy

To more directly assess the cause-and-effect relationship between calcineurin activation and the initiation and propagation of cardiac hypertrophy, investigators first employed the widely used calcineurin inhibitory agents cyclosporine and FK506. These agents each prevented the phenotypic manifestations of hypertrophic and dilated cardiomyopathy in three separate transgenic mouse models of desease [14]. In the same report, cyclosporine administration to aortic-banded rats over 6 days prevented the induction of pressure overload-induced cardiac hypertrophy [14]. However, this initial positive report was closely followed by four negative reports employing cyclosporine or FK506, which concluded no regulatory role for calcineurin in aortic-banded mice or rats [30,32,34,35]. Also consistent with these negative data, a subsequent study concluded that cyclosporine was actually detrimental to disease progression in MyHC 403 mutant mice; where it increased left ventricular free wall thickness, [36]. These seemingly contradictory results spurred a great deal of additional investigation into the causal linkage between calcineurin and cardiac hypertrophy. Nearly all of the subsequent pharmacologic studies, which now number greater than 17, have supported the initial hypothesis that calcineurin–NFAT are requisite mediators of the hypertrophic response in pleiotropic rodent models [15–18,22,37–48].

However, it is worth dissecting the potential variables that might underlie at least one of the negative reports discussed above, as described by Fatkin et al. [36]. Interestingly, both cyclosporine and FK506 were reported to enhance left ventricular wall thicknesses in MyHC 403 mutant mice, implicating alterations in calcineurin signaling in the exaggerated disease phenotype. However, Fatkin et al. did not evaluate calcineurin activity at base line or after pharmacologic intervention, making it difficult to exclude nonspecific effects associated with either agent. Also to be considered, Fatkin et al. only reported hypertrophy as measured by echocardiography of the left ventricular free wall. MyHC 403 mutant mice model hypertrophic cardiomyopathy, a disease in humans that typically involves a dramatic thickening of the interventricular septum, yet this parameter was not reported at baseline or after cyclosporine treatment. Moreover, other more straightforward measures of cardiac hypertrophy were not reported, such as weighing the heart at necropsy or quantifying myocyte cellular dimensions. Considering these issues, it is difficult to interpret the extent of the “hypertrophic” phenotype associated with cyclosporine treatment in MyHC 403 mutant mice. Despite this concern, clearly MyHC 403 mutant mice have altered sensitivity to calcium, which could predispose them to pharmacologic agents that secondarily exacerbate calcium handling; as previously reported for both cyclosporine and FK506. Indeed, minoxidil, a K+ channel agonist that also alters calcium handling, similarly exaggerated thickening of the left ventricular free wall in these mice [36]. Given the potential for nonspecific effects associated with calcineurin inhibitory agents, we and other investigators have sought potentially more definitive genetic-based methods for altering calcineurin–NFAT signaling in the heart as a means of further evaluating causation (see below).

4. Targeted inhibition of calcineurin attenuates hypertrophy

Another aspect of the controversy surrounding cyclosporine and FK506 studies in animal models of hypertrophy pertains to drug pharmacodynamics, such that neither agent completely blocks calcineurin activity for all its downstream substrates, and that these agents might also function independent of calcineurin. To address these issues, the non-competitive calcineurin inhibitory domains from the calcineurin interacting proteins Cain/Cabin-1 and AKAP79 were employed [49–51]. Adenovirus expressing the inhibitory domains of Cain or AKAP blocked calcineurin activity and attenuated phenylephrine- and angiotensin II-induced hypertrophy in cultured cardiomyocytes [52]. The inhibition of hypertrophy by Cain and AKAP adenoviral infection was similar to the inhibition observed with cyclosporine and FK506, suggesting calcineurin as the determinative factor [52]. More recently, transgenic mice were generated that express the calcineurin inhibitory domains of Cain or AKAP [19]. These transgenic mice demonstrated a significant reduction in pressure overload (aortic banding) and agonist-induced (isoproterenol infusion) cardiac hypertrophy [19]. Calcineurin activity is also negatively regulated by the modulatory-calcineurin interacting proteins (MCIP/calcipressin/DSCR1/ZAKI-4), which are each highly expressed in the heart and skeletal muscle [53,54]. Transgenic mice expressing the calcineurin inhibitory domain from MCIP1 have also been recently characterized and shown to have reduced cardiac hypertrophy in response to stress stimulation [55]. Lastly, transgenic mice expressing a dominant negative mutant of calcineurin within the heart also demonstrated reduced cardiac hypertrophy to stress stimuli (aortic banding) [20]. Collectively, these four separate transgenic models from three independent laboratories quenches doubt that calcineurin is an integral regulator of the cardiac hypertrophic response. More importantly, the data obtained with these transgenic models also suggest that cyclosporine and FK506 attenuate cardiac hypertrophy through a calcineurin-dependent mechanism. However, any enthusiasm for calcineurin inhibitory agents as therapeutics in treating human heart disease should be considered within the framework of drug toxicity and lack of correlative clinical data pertaining to the effects of these agents on the myocardium. Calcineurin signaling may also provide a pro-survival signal to cardiac myocytes, suggesting that long-term inhibition of this phosphatase might have other consequences to the heart (see below).

More recently, calcineurin Aβ null mice were generated and characterized by our group. These gene-targeted mice were overtly normal but displayed a significant reduction in heart weight in the unstimulated state, suggesting that calcineurin might also regulate homeostatic heart size at baseline [56]. More importantly, calcineurin Aβ null mice failed to undergo cardiac hypertrophy in response to pressure overload, isoproterenol infusion, or angiotensin II infusion [56]. An analysis of NFAT gene-targeted mice has also been employed for causal assessment of cardiac hypertrophy. While targeted disruption of NFATc4 did not diminish the magnitude of calcineurin transgene-dependent hypertrophy or pressure overload hypertrophy, NFATc3 null mice showed a significant and long-standing reduction in calcineurin-induced hypertrophy at multiple time points up to 10 weeks of age. NFATc3 gene-targeted mice were also compromised in their ability to mount an efficient hypertrophic response following aortic banding or angiotensin II infusion [11]. These results establish NFATc3 as a critical downstream mediator of calcineurin-regulated hypertrophy in the heart and further validate the original hypothesis that calcineurin mediates myocyte hypertrophy, in part, through NFAT transcription factors. Likewise, overexpression of a dominant negative NFAT mutant in cultured cardiomyocytes also antagonized agonist- or activated calcineurin-induced hypertrophy [57]. These overall results are also supported by three studies in which cardiac myocyte hypertrophic growth was reduced by overexpression of glycogen synthase kinase 3β (GSK3β). GSK3β was previously shown to directly phosphorylate the N-terminal regulatory domain of NFATc1, thus antagonizing the action of calcineurin and inhibiting nuclear shuttling of NFAT [58]. Overexpression of GSK3β in cultured cardiomyocytes attenuated agonist-induced hypertrophy, in part, by blocking NFAT nuclear translocation [59]. More recently, transgenic mice with constitutive or inducible GSK3 expression in the heart were generated, which showed reduced cardiac hypertrophy in response to the activated calcineurin transgene, isoproterenol infusion, or pressure overload [60,61].

Taken together, these various reports discussed above indicate that calcineurin–NFAT serves within a fundamental regulatory circuit in the myocardium that programs the hypertrophic growth response. However, this assertion may not extend to all types of stimulation that induce cardiac hypertrophy. For example, we have proposed that calcineurin–NFAT signaling has little causal role in regulating physiologic cardiac growth following exercise stimulation or insulin-like growth factor stimulation in vivo [33]. In the future, it will be important to more clearly define the molecular signaling circuitry that underlies various pathological or physiological growth states of the myocardium.

A final issue that should be discussed relates to emerging data indicating a pro-survival or potentially beneficial regulatory role for calcineurin signaling in the heart. Indeed, we originally observed that calcineurin transgenic mice, despite manifesting profound cardiac hypertrophy and heart failure, were partially protected from ischemia–reperfusion injury and cellular apoptosis [13]. More recently, calcineurin Aβ gene-targeted mice showed greater susceptible to ischemic injury and subsequent apoptosis, collectively suggesting a protective role for calcineurin signaling in the heart [62]. Consistent with this report, NFAT activation protected myocytes from phenylephrine-induced myocytes apoptosis [63], and endothelin-1-mediated protection of cardiac myocytes from oxidative stress-induced apoptosis required calcineurin [64]. However, isoproterenol-induced apoptosis of cardiac myocytes was reduced with cyclosporine, suggesting a pro-apoptotic role for calcineurin in response to β-adrenergic signaling [65]. Despite the potential inconsistency surrounding β-adrenergic signaling, most studies support the notion that calcineurin signaling imparts a necessary pro-survival effect to cardiac myocytes. This assertion suggests that calcineurin serves dichotomous regulatory roles in the heart, such that it mediates pathologic cardiac hypertrophy and failure when activated long-term, despite providing an anti-apoptotic signal.

5. Role of the MAPKs in cardiac signaling

Even though calcineurin–NFAT signaling is hypothesized to serve as a pivotal or nodal control point for mediating many forms of pathologic cardiac hypertrophy, this pathway obviously functions in concert with other signaling effectors. For example, the hypertrophic response is not only characterized by alterations in cardiac gene expression, but it also requires coordinated control of protein synthesis, protein degradation, and rRNA production. It is not readily obvious how calcineurin or NFAT would directly regulate these later aspects of the cardiac hypertrophic response given the direct transcriptional regulatory function of this pathway. However, a more reasonable hypothesis is that calcineurin–NFAT signaling initiates the hypertrophic response through a mechanism involving only a handful of direct effectors, transcriptional or otherwise. These immediate downstream targets of calcineurin–NFAT then function as secondary mediators to indirectly coordinate the other necessary aspects of the hypertrophic response. For example, the mitogen-activated protein kinases (MAPKs) have been shown to both affect calcineurin–NFAT signaling as well as being affected by it.

The MAPK signaling pathway is generally sub classified into three main branches consisting of p38 kinases, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs) [66] (Fig. 2). These three branches are each comprised of a series of successively acting kinases that serve an amplification function, thereby regulating diverse biologic functions including cell growth, differentiation, proliferation, and apoptosis. The JNKs and p38 kinases generally serve as more specialized transducers of stress or injury responses, hence their sub-classification as stress-activated protein kinases (SAPKs), while the ERKs are somewhat more specialized for mitogenic and growth factor stimulation [66]. Signaling through the ERK branch is typically initiated at the cell membrane in coordination with Ras activation. In turn, Ras directly couples to the MAPK kinase kinase Raf, which then couples with the MAPK kinases MEK1 and MEK2. MEK1/2 function as dual specificity kinases that directly phosphorylate the TEY motif in ERK1 and ERK2 kinases [66] (Fig. 2). With respect to the JNK branch, activation of MAPK kinase kinases, such as MEKK1, promotes activation of the dual specificity kinases MKK4 and MKK7, which in turn directly phosphorylates the TPY motif in JNK proteins, of which there are multiple spliced isoforms produced from three separate genes, Jnk1, Jnk2, and Jnk3 [66] (Fig. 2). Finally, the p38 branch is also initiated by MAPK kinase kinases at the level of the cell membrane, which couple to the dual specificity kinases MKK3 and MKK6 that in turn phosphorylate the TGY motif in p38 kinases (Fig. 2). Four separate genes encoding p38 kinases have been identified, p38α, p38β, p38δ, and p38γ, although p38α is the major protein isoform expressed in the adult heart [67].

Fig. 2

Highly simplified diagrammatic representation of MAPK signaling pathways. Mitogenic or stress stimuli elicit activation of MAPK kinase kinases (MEKKs and Raf) which in turn activate MAPK kinases (MEK1/2, MKK3/6, and MKK4/7). Activation of the MAPK kinases facilitates binding and direct phosphorylation of ERK1/2, JNK1/2/3, and p38α/β/γ/δ. Once activated, the terminal MAPKs directly phosphorylate diverse effector proteins.

In cardiac myocytes, each of the three MAPK branches discussed above is regulated by G-protein coupled receptors (GPCRs) through neuroendocrine factors such as angiotensin II, endothelin-1, and catecholamines, although other membrane receptors and generalized stress stimuli can also elicit signaling [68,69]. Such activation characteristics suggested a causal role for MAPK signaling in mediating or otherwise modulating the cardiac hypertrophic response. While studies conducted in cultured neonatal cardiac myocytes have indeed demonstrated a prohypertrophic regulatory role for each of the three MAPK branches, this paradigm did not extend to the adult heart as investigated in various transgenic and gene-targeted mouse models [67]. For example, overexpression of activated MKK3 or activated MKK6 in the heart by transgenesis, which produced a singular and uniform activation of p38, did not induce the hypertrophic growth response of individual myocytes [70]. In fact, activated MKK3 and MKK6 transgenic mice rapidly developed heart failure as juveniles characterized by reduced functional performance, interstitial fibrosis, and thinned ventricular walls, suggesting a potential phenotype of compromised cardiac growth or overt dilation [70]. Similarly, transgenic mice expressing activated MKK7 in the heart showed specific JNK activation but not cardiac hypertrophy, which was associated with lethal cardiomyopathy in juveniles [71,72]. In contrast, transgenic mice overexpressing an activated MEK1 cDNA, which showed specific activation of only ERK1/2, were characterized by a prominent hypertrophy response [73]. Collectively, these studies suggested a revised hypothesis whereby the SAPKs do not serve as forward regulators of the cardiac hypertrophic response, in dichotomy to the prohypertrophic regulatory role proposed for the MEK1–ERK1/2 pathway. However, that p38 and JNK do not positively regulate the cardiac growth response does not exclude their potential importance in modulating the hypertrophic response or the transition towards heart failure in concert with other signaling pathways (see below).

6. Calcineurin–NFAT modulates MAPK signaling pathways

Previous investigation into the mechanisms controlling T lymphocyte reactivity has suggested a model whereby calcineurin functions in concert with MAPK and protein kinase C signaling pathways to regulate cytokine gene expression and the immune response. For example, NFAT-dependent cytokine gene expression also depends on direct interaction with the inducible transcriptional regulator AP-1, which is directly regulated by JNK phosphorylation [10]. Indeed, calcineurin A gene-targeted mice, as well as PKCθ and JNK each show a defect in aspects of T-cell reactivity or differentiation in vivo, suggesting that multiple intracellular signaling pathways are necessary for orchestrating the immune response, or that these pathways are interdependent [74–77]. A similar scenario likely applies to myocardial cell signaling, such that calcineurin–NFAT signaling is coupled with other pathways such as MAPK and/or PKC. For example, pharmacologic inhibition of calcineurin is associated with inhibition of PKCα, θ, and JNK p54 in the heart [78]. Mice expressing the activated calcineurin transgene, which are characterized by a robust hypertrophic response, showed constitutive JNK and ERK activation in the heart [78] (Fig. 3). Isoproterenol stimulation of cardiac myocytes was also shown to activate ERK signaling through a mechanism involving calcineurin [79]. Antithetically, Ras activation was shown to promote NFATc4 activity in cardiac myocytes through a MEK1–ERK-dependent pathway [80] (Fig. 3). These later two studies suggest a reciprocal, yet reinforcing signaling relationship between calcineurin and MEK1–ERK1/2, such that calcineurin activation promotes ERK activation and Ras–MEK1–ERK activation enhances NFAT activation through an unknown mechanism. In contrast, calcineurin activation was associated with down-regulation of p38 MAPK signaling through direct up-regulation of the dual-specificity phosphatase MKP-1 in cardiac myocytes [81] (Fig. 3). This result suggests that calcineurin has distinct effects on members of the MAPK signaling pathway, such that some are activated and some are inhibited. Finally, calcineurin–NFAT signaling has also been directly linked to nitric oxide and the cGMP-dependent protein kinase type I (PKG I). More specifically, inhibition of cardiac hypertrophy through nitric oxide and PKG I depended on inhibition of calcineurin–NFAT signaling [82]. Taken together, these various reports provide a consistent data set suggesting coordination of the cardiac growth response through interconnected regulatory pathways. In other words, even though calcineurin–NFAT signaling is hypothesized to serve as a nodal control point, it must still mobilize a large array of seemingly parallel signaling effector pathways to propagate such a sophisticated biologic response.

Fig. 3

Diagram of interconnectivity between calcineurin–NFAT and MAPK signaling pathways in the myocardium. Increased calcineurin activity is associated with augmented JNK and ERK phosphorylation, but reduced p38 activation. Antithetically, ERK activation is associated with increased calcineurin–NFAT signaling, while JNK and p38 activation result in direct phosphorylation of specific NFAT family members, thus antagonizing their nuclear occupancy.

7. All three branches of the MAPK cascade regulate calcineurin–NFAT signaling

While calcineurin–NFAT signaling directly or indirectly modulates the activity of numerous parallel regulatory pathways in the heart, less is understood as to how it might be co-modulated by other signaling effectors. Here, evidence will be discussed suggesting that each of the MAPK signaling branches, when singularly activated, is capable of directly modulating calcineurin–NFAT signaling. As discussed above, Ras activation through MEK1–ERK was associated with increased NFATc4 nuclear translocation and transcriptional activity in cardiac myocytes [80]. This result is also consistent with an observation made in T lymphocytes whereby Vav signaling was coupled to Ras–MEK–ERK activation, which in turn promoted NFAT activation [83]. Likewise, dominant negative and constitutively active MEK1 mutants blocked and induced NFAT activation in T lymphocytes, respectively [84]. These results suggest that MEK1–ERK signaling is capable of enhancing calcineurin–NFAT signals. However, ERK was shown to directly phosphorylate NFATc1 in vitro resulting in inhibition of nuclear translocation and transcriptional activity in COS cells [85]. Taken together, these various studies indicate that MEK1–ERK signaling can either stimulate or inhibit calcineurin–NFAT activity depending on the cell type and the specific NFAT factors that are present, or the time course that was employed. Clearly, this issue requires additional investigation to more definitively determine the potential interconnections between calcineurin–NFAT and MEK1–ERK signaling in the heart, especially since each pathway can initiate and propagate the hypertrophic growth response when unitarily activated. It remains uncertain if these two pathways function as independent and parallel regulatory pathways, or if they are somehow more coordinated and interconnected representing a single signaling unit.

JNK and p38 are capable of directly phosphorylating specific NFAT transcription factors in their N-terminal regulatory domains, resulting in net inhibition of nuclear occupancy. For example, JNK factors can directly phosphorylate NFATc1, NFATc2, and NFATc3, but not NFATc4, thus antagonizing the effects of calcium-regulated signaling mediated through calcineurin dephosphorylation [85–87]. By comparison, p38 was shown to directly phosphorylate NFATc1, NFATc2, and NFATc4, but not NFATc3, also functioning to antagonize calcineurin-mediated dephosphorylation and nuclear translocation of NFAT [85,87,88]. We have recently extended these initial observations to the context of the cardiac myocyte, which suggests that a primary function of p38 and JNK signaling appears to be dedicated to the negative regulation of NFAT transcriptional responsiveness, hence antagonizing hypertrophy (Fig. 3). For example, we recently analyzed the phenotype of Jnk1 and Jnk2 gene-targeted mice, as well as transgenic mice expressing dominant negative mutants of JNK1/2 in the heart [89]. These dominant negative JNK1/2 transgenic mice and three allele Jnk1/2 gene-targeted mice each demonstrated enhanced cardiac hypertrophic growth following pressure overload induced by aortic banding [89]. Dominant negative JNK1/2 transgenic mice and three allele JNK1/2 gene-targeted mice also showed spontaneous cardiac hypertrophy by 7 months of age, suggesting that JNK signaling normally serves to antagonize or buffer cardiac growth to both acute and chronic stimulation (if aging can be thought of as a stimulus) [89]. These results are also consistent with the previous observation showing that JNK activation in culture repressed atrial natriuretic factor promoter activity, while JNK inhibition lead to activation of this hypertrophy-sensitive reporter [90]. The mechanism underlying the enhanced hypertrophic response associated with JNK inhibition was shown to be dependent on calcineurin–NFAT signaling in the heart. Specifically, JNK-inhibited transgenic mice showed significant activation of an NFAT-dependent luciferase reporter transgene in the heart, which was synergistically activated by simultaneous pressure overload stimulation [89]. Consistent with this proposed interconnection between NFAT and JNK, calcineurin Aβ gene targeting reduced the augmented hypertrophic growth response observed in JNK-inhibited mice [89]. More significantly, overexpression of an MKK7-JNK1 fusion protein in the heart, which specifically enhanced JNK activity, reduced cardiac hypertrophy induced by the activated calcineurin transgene [89]. Lastly, JNK activation or inhibition in cultured cardiac myocytes potently inhibited or potentiated NFAT nuclear occupancy and transcriptional activity, respectively [89].

The results discussed above suggest that JNKs normally function to antagonize the cardiac hypertrophic response through inhibition of calcineurin–NFAT signaling. A nearly identical NFAT-inhibitory paradigm was also observed with p38 signaling in the myocardium. Three different molecular strategies were employed to inhibit p38 signaling in cardiac-specific transgenic mice (dominant negative mutants of p38α, MKK3, and MKK6), each of which showed specific inhibition of p38 signaling that was associated with spontaneous cardiac hypertrophy in 2–4-month-old mice [91]. As with JNK-inhibited mice, blockade of p38 signaling robustly stimulated activity of the NFAT-dependent luciferase reporter transgene in vivo [91]. Calcineurin Aβ gene targeting also reversed the pro-hypertrophic effects associated with p38 inhibition in the heart, further demonstrating that p38 signaling likewise serves an anti-hypertrophic role through antagonizing calcineurin–NFAT signaling [91]. Taken together, it is likely that both stress-activated protein kinase-signaling branches have somewhat overlapping functions in the heart aimed at counter-regulating NFAT activity, as well as other potential effectors. Once again, these results also highlight the notion that NFAT factors are central mediators of the cardiac hypertrophic response and that alterations in the activity of NFAT effector kinases can dramatically influence hypertrophic growth. As discussed earlier, transgene-mediated overexpression of another NFAT kinase in the mouse heart, GSK-3β, similarly antagonized the cardiac hypertrophic response [60,61]. However, one minor consideration is that the genetic models discussed in this section may not be optimal for discriminating effects associated with more subtle levels of signaling, such as basal levels of MAPK or calcineurin activation, or transient activation following acute stresses.

In summary, while the MAPK and calcineurin–NFAT pathways undoubtedly serve as pivotal regulators in the heart, they likely function as only a small component within a more comprehensive and integrated signaling network that ultimately directs the entire myocyte growth response. Here, a working model is proposed whereby the SAPKs (JNK/p38) are partially antagonistic towards the hypertrophic program through direct inhibition of NFAT transcription factors, while ERK MAPK signaling potentiates calcineurin–NFAT activation through an unknown mechanism to coordinately enhance myocardial growth. However, such a model does not exclude parallel regulatory roles for JNK/p38/ERK that are independent of calcineurin–NFAT signaling, but yet might still otherwise modulate the hypertrophic response. Future analysis of MAPK gene-targeted mice will permit further refinement to this model and will ultimately suggest the overall predominance of the calcineurin–NFAT–MAPK cross-talk mechanism.

Acknowledgements

J.D.M. was supported by the National Institutes of Health and by the American Heart Association through an Established Investigator Grant.

Footnotes

  • Time for primary review 28 days

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View Abstract