Abstract-Calcineurin (PP2B) is a calcium/calmodulin-activated, serine-threonine phosphatase that transmits signals to the nucleus through the dephosphorylation and translocation of nuclear factor of activated T cell (NFAT) transcription factors. Whereas calcineurin-NFAT signaling has been implicated in regulating the hypertrophic growth of the myocardium, considerable controversy persists as to its role in maintaining versus initiating hypertrophy, its role in pathological versus physiological hypertrophy, and its role in heart failure. To address these issues, NFAT-luciferase reporter transgenic mice were generated and characterized. These mice showed robust and calcineurin-specific activation in the heart that was inhibited with cyclosporin A. In the adult heart, NFAT-luciferase activity was upregulated in a delayed, but sustained manner throughout eight weeks of pathological cardiac hypertrophy induced by pressure-overload, or more dramatically following myocardial infarction-induced heart failure. In contrast, physiological hypertrophy as produced in two separate models of exercise training failed to show significant calcineurin-NFAT coupling in the heart at multiple time points, despite measurable increases in heart to body weight ratios. Moreover, stimulation of hypertrophy with growth hormone-insulin-like growth factor-1 (GH-IGF-1) failed to activate calcineurin-NFAT signaling in the heart or in culture, despite hypertrophy, activation of Akt, and activation of p70 S6K. Calcineurin A␤ gene-targeted mice also showed a normal hypertrophic response after GH-IGF-1 infusion. Lastly, exercise-or GH-IGF-1-induced cardiac growth failed to show induction of hypertrophic marker gene expression compared with pressure-overloaded animals. Although a direct cause-and-effect relationship between NFAT-luciferase activity and pathological hypertrophy was not proven here, our results support the hypothesis that separable signaling pathways regulate pathological versus physiological hypertrophic growth of the myocardium, with calcineurin-NFAT potentially serving a regulatory role that is more specialized for maladaptive hypertrophy and heart failure.

Members of the mitogen-activated protein kinase (MAPK) cascade such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 are implicated as important regulators of cardiomyocyte hypertrophic growth in culture. However, the role that individual MAPK pathways play in vivo has not been extensively evaluated. Here we generated nine transgenic mouse lines with cardiac-restricted expression of an activated MEK1 cDNA in the heart. MEK1 transgenic mice demonstrated concentric hypertrophy without signs of cardiomyopathy or lethality up to 12 months of age. MEK1 transgenic mice showed a dramatic increase in cardiac function, as measured by echocardiography and isolated working heart preparation, without signs of decompensation over time. MEK1 transgenic mice and MEK1 adenovirus-infected neonatal cardiomyocytes each demonstrated ERK1/2, but not p38 or JNK, activation. MEK1 transgenic mice and MEK1 adenovirus-infected cultured cardiomyocytes were also partially resistant to apoptotic stimuli. The results of the present study indicate that the MEK1-ERK1/2 signaling pathway stimulates a physiologic hypertrophy response associated with augmented cardiac function and partial resistance to apoptotsis.

Abstract-In response to pathophysiological stress, the adult heart undergoes hypertrophic enlargement characterized by an increase in the cross-sectional area of individual myofibers. Although cardiac hypertrophy is initially a compensatory response, sustained hypertrophy is a leading predictor for the development of heart failure. At the molecular level, disease-related stimuli invoke endocrine, paracrine, and autocrine regulatory circuits, which directly influence cardiomyocyte hypertrophy, in part, through membrane bound G protein-coupled receptors and receptor tyrosine kinases. These membrane receptors activate intermediate signal transduction pathways within the cytoplasm such as mitogen-activated protein kinases (MAPKs), protein kinase C (PKC), and calcineurin, which directly modify transcriptional regulatory factors promoting alterations in cardiac gene expression. This review will weigh an increasing body of literature implicating the intermediate signaling pathway consisting of MEK1 and extracellular signal-regulated kinases (ERK1/2) as important regulators of cardiac hypertrophy and myocyte survival. The MEK1-ERK1/2 pathway likely occupies a central regulatory position in the signaling hierarchy of a cardiac myocyte given its unique ability to respond to virtually every characterized hypertrophic agonist and stress stimuli examined to date and based on its ability to promote myocyte growth in vitro and in vivo. Key Words: heart Ⅲ hypertrophy Ⅲ failure Ⅲ signaling Ⅲ mitogen-activated protein kinase M itogen-activated protein kinase (MAPK) signaling pathways consist of a sequence of successively acting kinases that ultimately result in the dual phosphorylation and activation of terminal kinases such as p38, c-Jun N-terminal kinases (JNKs), and extracellular signal-regulated kinases (ERKs) (see review 1 ) (Figure 1). The MAPK signaling cascade is initiated in cardiac myocytes by G proteincoupled receptors (angiotensin II, endothelin-1, and adrenergic receptors), receptor tyrosine kinases (insulin-like growth factor, transforming growth factor-␤, and fibroblast growth factor receptors), cardiotrophin-1 (gp130 receptor), and by stress stimuli. 2 Once activated, p38, JNKs, and ERKs each phosphorylate of a wide array of intracellular targets that includes numerous transcription factors resulting in the reprogramming of cardiac gene expression as part of the hypertrophic program.At least five different ERK proteins have been identified in mammalian cells, ERK1 to 5 (see reviews 1,3 ). ERK5 is regulated by the upstream kinase MAPK kinase 5 (MEK5), whereas ERK3 and ERK4 are related family members with unknown upstream regulators. 3 The more highly studied and abundantly expressed ERK family members, ERK1 and ERK2, are directly regulated by two MAPK kinases, MEK1 and MEK2. ERK1/2 proteins are directly phosphorylated by MEK1/2 at both a threonine and adjacent tyrosine residue within a dual specificity motif (Thr-Glu-Tyr). p38 kinases are directly activated by MKK6 and MKK3, whereas JNKs are directly activated by M...

The zinc finger-containing transcription factor GATA4 has been implicated as a critical regulator of multiple cardiac-expressed genes as well as a regulator of inducible gene expression in response to hypertrophic stimulation. Here we demonstrate that GATA4 is itself regulated by the mitogen-activated protein kinase signaling cascade through direct phosphorylation. Site-directed mutagenesis and phospho-specific GATA4 antiserum revealed serine 105 as the primary site involved in agonist-induced phosphorylation of GATA4. Infection of cultured cardiomyocytes with an activated MEK1-expressing adenovirus induced robust phosphorylation of serine 105 in GATA4, while a dominant-negative MEK1-expressing adenovirus blocked agonistinduced phosphorylation of serine 105, implicating extracellular signal-regulated kinase (ERK) as a GATA4 kinase. Indeed, bacterially purified ERK2 protein directly phosphorylated purified GATA4 at serine 105 in vitro. Phosphorylation of serine 105 enhanced the transcriptional potency of GATA4, which was sensitive to U0126 (MEK1 inhibitor) but not SB202190 (p38 inhibitor). Phosphorylation of serine 105 also modestly enhanced the DNA binding activity of bacterially purified GATA4. Finally, induction of cardiomyocyte hypertrophy with an activated MEK1-expressing adenovirus was blocked with a dominant-negative GATA4-engrailed-expressing adenovirus. These results suggest a molecular pathway whereby MEK1-ERK1/2 signaling regulates cardiomyocyte hypertrophic growth through the transcription factor GATA4 by direct phosphorylation of serine 105, which enhances DNA binding and transcriptional activation.

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues1. Here we report that genetic loss of APJ confers resistance to chronic pressure overload by dramatically reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechano-sensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of β-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and for the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.

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Calcineurin is a calcium-calmodulin-regulated, serine-threonine phosphatase that functions as a key inducer of stress responsive gene expression in multiple cell types through a direct activation of nuclear factor of activated T cells and myocyte enhancer factor 2 transcription factors. In cardiomyocytes, calcineurin signaling has been implicated in the regulation of the hypertrophic response caused by pressure overload or neuroendocrine stimulation. Three separate genes encode the catalytic subunit of calcineurin in mammalian cells, CnA␣, CnA␤, and CnA␥. To evaluate the necessary function of calcineurin as a hypertrophic regulatory factor, the CnA␤ gene was disrupted in the mouse. CnA␤-deficient mice were viable, fertile, and overtly normal well into adulthood, but displayed a 80% decrease in calcineurin enzymatic activity in the heart that was associated with a 12% reduction in basal heart size. CnA␤-deficient mice were dramatically impaired in their ability to mount a productive hypertrophic response induced by pressure overload, angiotensin II infusion, or isoproterenol infusion. Analysis of marker genes associated with the hypertrophic response revealed a partial defect in the molecular program of hypertrophy. Collectively, these data solidify the hypothesis that calcineurin functions as a central regulator of the cardiac hypertrophic growth response in vivo.

A calcineurin-nuclear factor of activated T cells (NFAT) regulatory pathway has been implicated in the control of cardiac hypertrophy, suggesting one mechanism whereby alterations in intracellular calcium handling are linked to the expression of hypertrophy-associated genes. Although recent studies have demonstrated a necessary role for calcineurin as a mediator of cardiac hypertrophy, the potential involvement of NFAT transcription factors as downstream effectors of calcineurin signaling has not been evaluated. Accordingly, mice with targeted disruptions in NFATc3 and NFATc4 genes were characterized. Whereas the loss of NFATc4 did not compromise the ability of the myocardium to undergo hypertrophic growth, NFATc3-null mice demonstrated a significant reduction in calcineurin transgene-induced cardiac hypertrophy at 19 days, 26 days, 6 weeks, 8 weeks, and 10 weeks of age. NFATc3-null mice also demonstrated attenuated pressure overload-and angiotensin II-induced cardiac hypertrophy. These results provide genetic evidence that calcineurin-regulated responses require NFAT effectors in vivo.Cardiac hypertrophy is defined by an increase in ventricular wall thickness accompanied by an increase in cardiomyocyte cell volume. Hypertrophic enlargement is precipitated by increased workload or by decreased efficiency within the heart, conditions that are associated with hypertension, ischemic heart disease, valvular insufficiency, neuroendocrine disruptions, or intrinsic defects in contractile proteins (reviewed in reference 30). Although initially compensatory, sustained cardiac hypertrophy predisposes an individual to sudden death, arrhythmias, functional decompensation, and overt heart failure (30).Numerous regulatory pathways have been implicated in the transduction of hypertrophic signaling, linking neuroendocrine and mechanical stress stimuli to altered cardiac gene expression (reviewed in reference 40). Although numerous hypertrophic regulatory pathways have been identified, the recent characterization of the calcium-regulated phosphatase calcineurin as an important signaling factor in the heart has generated considerable interest. Transgenic mice expressing an activated form of calcineurin in the heart developed robust hypertrophy that quickly transitioned to dilation and failure (41). Subsequently, the calcineurin inhibitory drugs cyclosporine (Cs) and FK506 were shown to inhibit or attenuate cardiac hypertrophy or cardiomyopathy in most, but not all, rodent models of heart disease, suggesting a necessary regulatory role for this signaling pathway in the heart (reviewed in reference 39). More recently, transgenic mice expressing either the calcineurin inhibitory domains of Cain, AKAP79, MCIP1, or dominant-negative calcineurin were shown to have attenuated cardiac hypertrophy in response to pathophysiologic stimulation (7,52,72).Perhaps the best-characterized target of calcineurin is the nuclear factor of activated T cells (NFAT) transcription factor family. Calcineurin directly dephosphorylates NFAT transcription fa...