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In continuously beating cells like cardiac myocytes, there are rapid alterations of cytosolic Ca 2+ levels. We therefore hypothesize that decoding Ca 2+ signals for hypertrophic signaling requires intracellular Ca 2+ microdomains that are partly independent from cytosolic Ca 2+ . Furthermore, there is a need for a Ca 2+ sensor within these microdomains that translates Ca 2+ signals into hypertrophic signaling. Recent evidence suggested that the nucleus of cardiac myocytes might be a Ca 2+ microdomain and that calcineurin, once translocated into the nucleus, could act as a nuclear Ca 2+ sensor. We demonstrate that nuclear calcineurin was able to act as a nuclear Ca 2+ sensor detecting local Ca 2+ release from the nuclear envelope via IP 3 R. Nuclear calcineurin mutants defective for Ca 2+ binding failed to activate NFAT-dependent transcription. Under hypertrophic conditions Ca 2+ transients in the nuclear microdomain were significantly higher than in the cytosol providing a basis for sustained calcineurin/NFAT-mediated signaling uncoupled from cytosolic Ca 2+ . Measurements of nuclear and cytosolic Ca 2+ transients in IP 3 sponge mice showed no increase of Ca 2+ levels during diastole as we detected in wild-type mice. Nuclei, isolated from ventricular myocytes of mice after chronic Ang II treatment, showed an elevation of IP 3 R2 expression which was dependent on calcineurin/NFAT signaling and persisted for 3 weeks after removal of the Ang II stimulus. These data provide an explanation how Ca 2+ and calcineurin might regulate transcription in cardiomyocytes in response to neurohumoral signals independently from their role in cardiac contraction control. KEY MESSAGES: • Calcineurin acts as an intranuclear Ca 2+ sensor to promote NFAT activity. • Nuclear Ca 2+ in cardiac myocytes increases via IP 3 R2 upon Ang II stimulation. • IP 3 R2 expression is directly dependent on calcineurin/NFAT.

Canonical protein phosphatase 3/calcineurin signaling is central to numerous physiological processes. Here we provide evidence that calcineurin plays a pivotal role in controlling systemic energy and body weight homeostasis. Knockdown of calcineurin in Drosophila melanogaster led to a decrease in body weight and energy stores, and increased energy expenditure. In mice, global deficiency of catalytic subunit Ppp3cb, and tissue-specific ablation of regulatory subunit Ppp3r1 from skeletal muscle, but not adipose tissue or liver, led to protection from high-fat-diet-induced obesity and comorbid sequelæ. Ser637 hyperphosphorylation of dynamin-related protein 1 (Drp1) in skeletal muscle of calcineurin-deficient mice was associated with mitochondrial elongation into power-cable-shaped filaments and increased mitochondrial respiration, but also with attenuated exercise performance. Our data suggest that calcineurin acts as highly conserved pivot for the adaptive metabolic responses to environmental changes such as high-fat, high-sugar diets or exercise., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background & Aims: Radiocontrast agents are required for radiographic procedures, but these agents can injure tissues by unknown mechanisms. We investigated whether exposure of pancreatic tissues to radiocontrast agents during endoscopic retrograde cholangiopancreatography (ERCP) causes pancreatic inflammation, and studied the effects of these agents on human cell lines and in mice., Methods: We exposed mouse and human acinar cells to the radiocontrast agent iohexol (Omnipaque; GE Healthcare, Princeton, NJ) and measured intracellular release of Ca(2+), calcineurin activation (using a luciferase reporter), activation of nuclear factor-κB (NF-κB, using a luciferase reporter), and cell necrosis (via propidium iodide uptake). We infused the radiocontrast agent into the pancreatic ducts of wild-type mice (C57BL/6) to create a mouse model of post-ERCP pancreatitis; some mice were given intraperitoneal injections of the calcineurin inhibitor FK506 before and after infusion of the radiocontrast agent. CnAβ(-/-) mice also were used. This experiment also was performed in mice given infusions of adeno-associated virus 6-NF-κB-luciferase, to assess activation of this transcription factor in vivo., Results: Incubation of mouse and human acinar cells, but not HEK293 or COS7 cells, with iohexol led to a peak and then plateau in Ca(2+) signaling, along with activation of the transcription factors NF-κB and nuclear factor of activated T cells. Suppressing Ca(2+) signaling or calcineurin with BAPTA, cyclosporine A, or FK506 prevented activation of NF-κB and acinar cell injury. Calcineurin Aβ-deficient mice were protected against induction of pancreatic inflammation by iohexol. The calcineurin inhibitor FK506 prevented contrast-induced activation of NF-κB in pancreata of mice, this was observed by live imaging of mice given infusions of adeno-associated virus 6-NF-κB-luciferase., Conclusions: Radiocontrast agents cause pancreatic inflammation in mice, via activation of NF-κB, Ca(2+) signaling, and calcineurin. Calcineurin inhibitors might be developed to prevent post-ERCP pancreatitis in patients., (Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Nine years ago, we published an article that suggested a specialized role for calcineurin–nuclear factor of activated T-cells (NFAT) signaling in regulating pathological cardiac hypertrophy preferentially over physiological growth and, in fact, the later response was associated with reduced calcineurin-NFAT activity. Since this time we and others have continued to uncover how this signaling effector pathway functions in the heart in regulating specific aspects of the growth response during disease and with exercise.

Insulin resistance, hyperlipidemia, and cardiovascular complications are common dysregulations of metabolic syndrome. Transplant patients treated with immunosuppressant drugs such as cyclosporine A (CsA), an inhibitor of calcineurin phosphatase, frequently develop similar metabolic complications. Although calcineurin is known to mediate insulin sensitivity by regulating β-cell growth and adipokine gene transcription, its role in lipid homeostasis is poorly understood. Here, we examined lipid homeostasis in mice lacking calcineurin Aβ (CnAβ(-/-)). We show that mice lacking calcineurin Aβ are hyperlipidemic and develop age-dependent insulin resistance. Hyperlipidemia found in CnAβ(-/-) mice is, in part, due to increased lipolysis in adipose tissues, a process mediated by β-adrenergic G-protein-coupled receptor signaling pathways. CnAβ(-/-) mice also exhibit additional pathophysiological phenotypes caused by the potentiated GPCR signaling pathways. A cell autonomous mechanism with sustained cAMP/PKA activation is found in CnAβ(-/-) mice or upon CsA treatment to inhibit calcineurin. Increased PKA activation and cAMP accumulation in CnAβ(-/-) mice, however, are sensitive to phosphodiesterase inhibitor. Indeed, we show that calcineurin regulates degradation of phosphodiesterase 3B, in addition to phosphodiesterase 4D. These results establish a role for calcineurin in lipid homeostasis. These data also indicate that potentiated cAMP signaling pathway may provide an alternative molecular pathogenesis for the metabolic complications elicited by CsA in transplant patients.

Biliary pancreatitis is the leading cause of acute pancreatitis in both children and adults. A proposed mechanism is the reflux of bile into the pancreatic duct. Bile acid exposure causes pancreatic acinar cell injury through a sustained rise in cytosolic Ca(2+). Thus, it would be clinically relevant to know the targets of this aberrant Ca(2+) signal. We hypothesized that the Ca(2+)-activated phosphatase calcineurin is such a Ca(2+) target. To examine calcineurin activation, we infected primary acinar cells from mice with an adenovirus expressing the promoter for a downstream calcineurin effector, nuclear factor of activated T-cells (NFAT). The bile acid taurolithocholic acid-3-sulfate (TLCS) was primarily used to examine bile acid responses. TLCS caused calcineurin activation only at concentrations that cause acinar cell injury. The activation of calcineurin by TLCS was abolished by chelating intracellular Ca(2+). Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (acetoxymethyl ester) (BAPTA-AM) or the three specific calcineurin inhibitors FK506, cyclosporine A, or calcineurin inhibitory peptide prevented bile acid-induced acinar cell injury as measured by lactate dehydrogenase leakage and propidium iodide uptake. The calcineurin inhibitors reduced the intra-acinar activation of chymotrypsinogen within 30 min of TLCS administration, and they also prevented NF-κB activation. In vivo, mice that received FK506 or were deficient in the calcineurin isoform Aβ (CnAβ) subunit had reduced pancreatitis severity after infusion of TLCS or taurocholic acid into the pancreatic duct. In summary, we demonstrate that acinar cell calcineurin is activated in response to Ca(2+) generated by bile acid exposure, bile acid-induced pancreatic injury is dependent on calcineurin activation, and calcineurin inhibitors may provide an adjunctive therapy for biliary pancreatitis.

Acute pancreatitis is a major health burden for which there are currently no targeted therapies. Premature activation of digestive proenzymes, or zymogens, within the pancreatic acinar cell is an early and critical event in this disease. A high-amplitude, sustained rise in acinar cell Ca(2+) is required for zymogen activation. We previously showed in a cholecystokinin-induced pancreatitis model that a potential target of this aberrant Ca(2+) signaling is the Ca(2+)-activated phosphatase calcineurin (Cn). However, in this study, we examined the role of Cn on both zymogen activation and injury, in the clinically relevant condition of neurogenic stimulation (by giving the acetylcholine analog carbachol) using three different Cn inhibitors or Cn-deficient acinar cells. In freshly isolated mouse acinar cells, pretreatment with FK506, calcineurin inhibitory peptide (CiP), or cyclosporine (CsA) blocked intra-acinar zymogen activation (n = 3; P < 0.05). The Cn inhibitors also reduced leakage of lactate dehydrogenase (LDH) by 79%, 62%, and 63%, respectively (n = 3; P < 0.05). Of the various Cn isoforms, the β-isoform of the catalytic A subunit (CnAβ) was strongly expressed in mouse acinar cells. For this reason, we obtained acinar cells from CnAβ-deficient mice (CnAβ-/-) and observed an 84% and 50% reduction in trypsin and chymotrypsin activation, respectively, compared with wild-type controls (n = 3; P < 0.05). LDH release in the CnAβ-deficient cells was reduced by 50% (n = 2; P < 0.05). The CnAβ-deficient cells were also protected against zymogen activation and cell injury induced by the cholecystokinin analog caerulein. Importantly, amylase secretion was generally not affected by either the Cn inhibitors or Cn deficiency. These data provide both pharmacological and genetic evidence that implicates Cn in intra-acinar zymogen activation and cell injury during pancreatitis.

Mice with three amino acid mutations in the calmodulin binding domain of type-2 ryanodine receptor ion channel (Ryr2(ADA/ADA) mice) have impaired intracellular Ca(2+) handling and cardiac hypertrophy with death at an early age. In this report, the role of signaling molecules implicated in cardiac hypertrophy of Ryr2(ADA/ADA) mice was investigated. Calcineurin A-β (CNA-β) and nuclear factor of activated T cell (NFAT) signaling were monitored in mice carrying either luciferase transgene driven by NFAT-dependent promoter or knockout of CNA-β. NFAT transcriptional activity in Ryr2(ADA/ADA) hearts was not markedly upregulated at embryonic day 16.5 compared with wild-type but significantly increased at postnatal days 1 and 10. Ablation of CNA-β extended the life span of Ryr2(ADA/ADA) mice and enhanced cardiac function without improving sarcoplasmic reticulum Ca(2+) handling or suppressing the expression of genes implicated in cardiac hypertrophy. Embryonic day 16.5 Ryr2(ADA/ADA) mice had normal heart weights with no major changes in Akt1 and class II histone deacetylase phosphorylation and myocyte enhancer factor-2 activity. In contrast, phosphorylation levels of Erk1/2, p90 ribosomal S6 kinases (p90RSKs), and GSK-3β were increased in hearts of embryonic day 16.5 homozygous mutant mice. The results indicate that an impaired calmodulin regulation of RyR2 was neither associated with an altered CNA-β/NFAT, class II histone deacetylase (HDAC)/MEF2, nor Akt signaling in embryonic day 16.5 hearts; rather increased Erk1/2 and p90RSK phosphorylation levels likely leading to reduced GSK-3β activity were found to precede development of cardiac hypertrophy in mice expressing dysfunctional ryanodine receptor ion channel.

Calcineurin is an important signalling protein that regulates a number of molecular and cellular processes. Previously, we found that inhibition of calcineurin with cyclosporine reduced renal hypertrophy and blocked glomerular matrix expansion in the diabetic kidney. Isoforms of the catalytic subunit of calcineurin are reported to have tissue specific expression and functions. In particular, the β isoform has been implicated in cardiac and skeletal muscle hypertrophy. Therefore, we examined the role of calcineurin β in diabetic renal hypertrophy and glomerular matrix expansion. Type I diabetes was induced in wild-type and β(-/-) mice and then renal function, extracellular matrix expansion and hypertrophy were evaluated. The absence of β produced a significant decrease in total calcineurin activity in the inner medulla (IM) and reduced nuclear factor of activated T-cells (NFATc) activity. Loss of β did not alter diabetic renal dysfunction assessed by glomerular filtration rate, urine albumin excretion and blood urea nitrogen. Similarly, matrix expansion in the whole kidney and glomerulus was not different between diabetic wild-type and β(-/-) mice. In contrast, whole kidney and glomerular hypertrophy were significantly reduced in diabetic β(-/-) mice. Moreover, β(-/-) renal fibroblasts demonstrated impaired phosphorylation of Erk1/Erk2, c-Jun N-terminal kinases (JNK) and mammalian target of rapamycin (mTOR) following stimulation with transforming growth factor-β and did not undergo hypertrophy with 48 hrs culture in high glucose. In conclusion, loss of the β isoform of calcineurin is sufficient to reproduce beneficial aspects of cyclosporine on diabetic renal hypertrophy but not matrix expansion. Therefore, while multiple signals appear to regulate matrix, calcineurin β appears to be a central mechanism involved in organ hypertrophy., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Aims: Proteasome functional insufficiency (PFI) may play an important role in the progression of congestive heart failure but the underlying molecular mechanism is poorly understood. Calcineurin and nuclear factor of activated T-cells (NFAT) are degraded by the proteasome, and the calcineurin-NFAT pathway mediates cardiac remodelling. The present study examined the hypothesis that PFI activates the calcineurin-NFAT pathway and promotes maladaptive remodelling of the heart., Methods and Results: Using a reporter gene assay, we found that pharmacological inhibition of 20S proteasomes stimulated NFAT transactivation in both mouse hearts and cultured adult mouse cardiomyocytes. Proteasome inhibition stimulated NFAT nuclear translocation in a calcineurin-dependent manner and led to a maladaptive cell shape change in cultured neonatal rat ventricular myocytes. Proteasome inhibition facilitated left ventricular dilatation and functional decompensation and increased fatality in mice with aortic constriction while causing cardiac hypertrophy in the sham surgery group. It was further revealed that both calcineurin protein levels and NFAT transactivation were markedly increased in the mouse hearts with desmin-related cardiomyopathy and severe PFI. Expression of an aggregation-prone mutant desmin also directly increased calcineurin protein levels in cultured cardiomyocytes., Conclusions: The calcineurin-NFAT pathway in the heart can be activated by proteasome inhibition and is activated in the heart of a mouse model of desmin-related cardiomyopathy that is characterized by severe PFI. The interplay between PFI and the calcineurin-NFAT pathway may contribute to the pathological remodelling of cardiomyocytes characteristic of congestive heart failure.

Although the calcium/calmodulin-activated phosphatase calcineurin may dephosphorylate many endocytic proteins, it is not considered a key molecule in mediating the major forms of endocytosis at synapses-slow, clathrin-dependent and the rapid, clathrin-independent endocytosis. Here we studied the role of calcineurin in endocytosis by reducing calcium influx, inhibiting calmodulin with pharmacological blockers and knockdown of calmodulin, and by inhibiting calcineurin with pharmacological blockers and knock-out of calcineurin. These manipulations significantly inhibited both rapid and slow endocytosis at the large calyx-type synapse in 7- to 10-d-old rats and mice, and slow, clathrin-dependent endocytosis at the conventional cultured hippocampal synapse of rats and mice. These results suggest that calcium influx during nerve firing activates calcium/calmodulin-dependent calcineurin, which controls the speed of both rapid and slow endocytosis at synapses by dephosphorylating endocytic proteins. The calcium/calmodulin/calcineurin signaling pathway may underlie regulation of endocytosis by nerve activity and calcium as reported at many synapses over the last several decades.

Dilated cardiomyopathy (DCM) is a relatively common disease with a poor prognosis. Given that the only meaningful treatment for DCM is cardiac transplantation, investigators have explored the underlying molecular mechanisms of this disease in the hopes of identifying novel therapeutic targets. One such target is the serine-threonine phosphatase calcineurin, a Ca2+-activated signaling factor that is known to regulate the cardiac hypertrophic program, although its role in DCM is currently unknown. In order to address this issue, we crossed muscle lim protein (MLP) knock-out mice-a murine model of DCM-with calcineurin A beta ko mice, which lack the stress responsive isoform of calcineurin that critically regulates the cardiac hypertrophic response. Interestingly, the majority (73%) of the MLP/calcineurin A beta double knock-out mice died within 20 days of birth with signs of cardiomyopathy. Ultrastructural examination revealed enhanced cardiomyocyte apoptosis and necrosis in the postnatal myocardium of these mice. The MLP/calcineurin A beta double knock-out mice that survived until adulthood showed reduced left ventricular function, enhanced apoptotic and necrotic cardiomyocyte death and augmented myocardial fibrosis compared to various control groups. Antithetically, mild overexpression of activated calcineurin in the mouse heart improved function and adverse remodeling in MLP knock-out mice. Collectively, these results reveal an important and previously unrecognized protective function of endogenous myocardial calcineurin in a mouse model of dilated cardiomyopathy., ((c) 2009 Elsevier Ltd. All rights reserved.)

Calcineurin is a protein phosphatase that is uniquely regulated by sustained increases in intracellular Ca(2+) following signal transduction events. Calcineurin controls cellular proliferation, differentiation, apoptosis, and inducible gene expression following stress and neuroendocrine stimulation. In the adult heart, calcineurin regulates hypertrophic growth of cardiomyocytes in response to pathologic insults that are associated with altered Ca(2+) handling. Here we determined that calcineurin signaling is directly linked to the proper control of cardiac contractility, rhythm, and the expression of Ca(2+)-handling genes in the heart. Our approach involved a cardiomyocyte-specific deletion using a CnB1-LoxP-targeted allele in mice and three different cardiac-expressing Cre alleles/transgenes. Deletion of calcineurin with the Nkx2.5-Cre knock-in allele resulted in lethality at 1 day after birth due to altered right ventricular morphogenesis, reduced ventricular trabeculation, septal defects, and valvular overgrowth. Slightly later deletion of calcineurin with the alpha-myosin heavy chain Cre transgene resulted in lethality in early mid adulthood that was characterized by substantial reductions in cardiac contractility, severe arrhythmia, and reduced myocyte content in the heart. Young calcineurin heart-deleted mice died suddenly after pressure overload stimulation or neuroendocrine agonist infusion, and telemetric monitoring of older mice showed arrhythmia leading to sudden death. Mechanistically, loss of calcineurin reduced expression of key Ca(2+)-handling genes that likely lead to arrhythmia and reduced contractility. Loss of calcineurin also directly impacted cellular proliferation in the postnatal developing heart. These results reveal multiple mechanisms whereby calcineurin regulates cardiac development and myocyte contractility.

Rationale: Pathological cardiac myocyte hypertrophy is thought to be induced by the persistent increases in intracellular Ca(2+) needed to maintain cardiac function when systolic wall stress is increased. Hypertrophic Ca(2+) binds to calmodulin (CaM) and activates the phosphatase calcineurin (Cn) and CaM kinase (CaMK)II. Cn dephosphorylates cytoplasmic NFAT (nuclear factor of activated T cells), inducing its translocation to the nucleus where it activates antiapoptotic and hypertrophic target genes. Cytoplasmic CaMKII regulates Ca(2+) handling proteins but whether or not it is directly involved in hypertrophic and survival signaling is not known., Objective: This study explored the hypothesis that cytoplasmic CaMKII reduces NFAT nuclear translocation by inhibiting the phosphatase activity of Cn., Methods and Results: Green fluorescent protein-tagged NFATc3 was used to determine the cellular location of NFAT in cultured neonatal rat ventricular myocytes (NRVMs) and adult feline ventricular myocytes. Constitutively active (CaMKII-CA) or dominant negative (CaMKII-DN) mutants of cytoplasmic targeted CaMKII(deltac) were used to activate and inhibit cytoplasmic CaMKII activity. In NRVM CaMKII-DN (48.5+/-3%, P<0.01 versus control) increased, whereas CaMKII-CA decreased (5.9+/-1%, P<0.01 versus control) NFAT nuclear translocation (Control: 12.3+/-1%). Cn inhibitors were used to show that these effects were caused by modulation of Cn activity. Increasing Ca(2+) increased Cn-dependent NFAT translocation (to 71.7+/-7%, P<0.01) and CaMKII-CA reduced this effect (to 17.6+/-4%). CaMKII-CA increased TUNEL and caspase-3 activity (P<0.05). CaMKII directly phosphorylated Cn at Ser197 in CaMKII-CA infected NRVMs and in hypertrophied feline hearts., Conclusion: These data show that activation of cytoplasmic CaMKII inhibits NFAT nuclear translocation by phosphorylation and subsequent inhibition of Cn.

The calcium-activated protein phosphatase calcineurin is controlled by regulator of calcineurin (RCAN) in organisms ranging from yeast to mammals. Here we performed a yeast two-hybrid screen with RCAN1 as bait, identifying TAK1 binding protein 2 (TAB2) as an interacting partner. TAB2 interacted directly with RCAN1 in vitro and in vivo, recruiting TAK1, TAB1 and calcineurin, forming a macromolecular signalling complex. Overexpression of TAK1 and TAB1, or active TAK1(DeltaN), promoted direct phosphorylation of RCAN1 in vitro and in vivo. TAK1 phosphorylated RCAN1 at Ser 94 and Ser 136, converting RCAN1 from an inhibitor to a facilitator of calcineurin-NFAT signalling, and enhancing NFATc1 nuclear translocation, NFAT transcriptional activation and the hypertrophic growth of cultured cardiomyocytes. The TAK1-TAB1-TAB2 and the calcineurin-NFAT signalling modules did not interact in Rcan1/2- or Tab2-deficient mouse embryonic fibroblast (MEF) cultures. Calcineurin activation also dephosphorylated and inhibited TAK1 and TAB1, an effect that was absent in Rcan1/2 deficient MEFs. Functionally, TAK1 was indispensable for the cardiomyocyte growth response induced by pro-hypertrophic stimuli through calcineurin. These results describe a signalling relationship between two central regulatory pathways in which TAK1-TAB1-TAB2 selectively induces calcineurin-NFAT signalling through direct phosphorylation of RCAN1, while calcineurin activation diminishes TAK1 signalling by dephosphorylation of TAK1 and TAB1.

Aims: Cyclic GMP (cGMP)-specific phosphodiesterase 5 (PDE5) inhibition by sildenafil (SIL) activates myocardial cGMP-dependent protein kinase G (PKG) and blunts cardiac hypertrophy. To date, the only documented target of PKG in myocardium is the serine-threonine phosphatase calcineurin (Cn), which is central to pathological cardiac hypertrophy. We tested whether Cn suppression is necessary in order to observe anti-hypertrophic effects of SIL., Methods and Results: Mice lacking the Cn-Abeta subunit (CnAbeta(-/-)) and wild-type (WT) controls were subjected to transverse aorta constriction (TAC) with or without SIL (200 mg/kg/day, p.o.) for 3 weeks. TAC-induced elevation of Cn expression and activity in WT was absent in CnAbeta(-/-) hearts, and the latter accordingly developed less cardiac hypertrophy (50 vs. 100% increase in heart weight/tibia length, P < 0.03) and chamber dilation. SIL remained effective in CnAbeta(-/-) mice, increasing PKG activity similarly as in WT, suppressing hypertrophy and fetal gene expression, and enhancing heart function without altering afterload. TAC-stimulated calcium-calmodulin kinase II, Akt, and glycogen synthase kinase 3beta in both groups (the first rising more in CnAbeta(-/-) hearts), and SIL also suppressed these similarly. Activation of extracellular signal-regulated kinase observed in WT-TAC but not CnAbeta(-/-) hearts was also suppressed by SIL., Conclusion: PDE5A inhibition and its accompanying PKG activation blunt hypertrophy and improve heart function even without Cn activation. This occurs by its modulation of several alternative pathways which may result from concomitant distal targeting, or activity against a common proximal node.

Left ventricular (LV) hypertrophy commonly develops in response to chronic hypertension and is a significant risk factor for heart failure and death. The serine-threonine phosphatase calcineurin (Cn)A plays a critical role in the development of pathological hypertrophy. Previous experimental studies in murine models show that estrogen limits pressure overload-induced hypertrophy; our purpose was to explore further the mechanisms underlying this estrogen effect. Wild-type, ovariectomized female mice were treated with placebo or 17beta-estradiol (E2), followed by transverse aortic constriction (TAC), to induce pressure overload. At 2 weeks, mice underwent physiological evaluation, immediate tissue harvest, or dispersion of cardiomyocytes. E2 replacement limited TAC-induced LV and cardiomyocyte hypertrophy while attenuating deterioration in LV systolic function and contractility. These E2 effects were associated with reduced abundance of CnA. The primary downstream targets of CnA are the nuclear factor of activated T-cell (NFAT) family of transcription factors. In transgenic mice expressing a NFAT-activated promoter/luciferase reporter gene, E2 limited TAC-induced activation of NFAT. Moreover, the inhibitory effects of E2 on LV hypertrophy were absent in CnA knockout mice, supporting the notion that CnA is an important target of E2-mediated inhibition. In cultured rat cardiac myocytes, E2 inhibited agonist-induced hypertrophy while also decreasing CnA abundance and NFAT activation. Agonist stimulation also reduced CnA ubiquitination and degradation that was prevented by E2; all in vitro effects of estrogen were reversed by an estrogen receptor (ER) antagonist. These data support that E2 reduces pressure overload induced hypertrophy by an ER-dependent mechanism that increases CnA degradation, unveiling a novel mechanism by which E2 and ERs regulate pathological LV and cardiomyocyte growth.

In noncontractile cells, a sustained increase in total cytoplasmic Ca(2+) concentration is typically needed to activate the intracellular protein phosphatase calcineurin, leading to dephosphorylation of the transcription factor nuclear factor of activated T cells (NFAT), its nuclear translocation, and induction of gene expression. It remains a mystery exactly how Ca(2+)-dependent signaling pathways, such as that mediated by calcineurin-NFAT, are regulated in contracting cardiac myocytes given the highly specialized manner in which Ca(2+) concentration rhythmically cycles in excitation-contraction coupling. Here, we critically review evidence that supports the hypothesis that calcineurin-NFAT signaling is regulated by contractile Ca(2+) transients in cardiac myocytes.

Overexpression of calcineurin (CLN) in the mouse heart induces severe hypertrophy that progresses to heart failure, providing an opportunity to define the relationship between energetics and contractile performance in the severely failing mouse heart. Contractile performance was studied in isolated hearts at different pacing frequencies and during dobutamine challenge. Energetics were assessed by 31P-NMR spectroscopy as ATP and phosphocreatine concentrations ([ATP] and [PCr]) and free energy of ATP hydrolysis (|Delta G( approximately ATP)|). Mitochondrial and glycolytic enzyme activities, myocardial O2 consumption, and myocyte ultrastructure were determined. In transgenic (TG) hearts at all levels of work, indexes of systolic performance were reduced and [ATP] and capacity for ATP synthesis were lower than in non-TG hearts. This is the first report showing that myocardial [ATP] is lower in a TG mouse model of heart failure. [PCr] was also lower, despite an unexpected increase in the total creatine pool. Because Pi concentration remained low, despite lower [ATP] and [PCr], |Delta G( approximately ATP)| was normal; however, chemical energy did not translate to systolic performance. This was most apparent with beta-adrenergic stimulation of TG hearts, during which, for similar changes in |Delta G( approximately ATP)|, systolic pressure decreased, rather than increased. Structural abnormalities observed for sarcomeres and mitochondria likely contribute to decreased contractile performance. On the basis of the increases in enzyme activities of proteins important for ATP supply observed after treatment with the CLN inhibitor cyclosporin A, we also conclude that CLN directed inhibition of ATP-producing pathways in non-TG and TG hearts.

Elevated endogenous cholecystokinin (CCK) release induced by protease inhibitors leads to pancreatic growth. This response has been shown to be mediated by the phosphatase calcineurin, but its downstream effectors are unknown. Here we examined activation of calcineurin-regulated nuclear factor of activated T-cells (NFATs) in isolated acinar cells, as well as in an in vivo model of pancreatic growth. Western blotting of endogenous NFATs and confocal imaging of NFATc1-GFP in pancreatic acini showed that CCK dose-dependently stimulated NFAT translocation from the cytoplasm to the nucleus within 0.5-1 h. This shift in localization correlated with CCK-induced activation of NFAT-driven luciferase reporter and was similar to that induced by a calcium ionophore and constitutively active calcineurin. The effect of CCK was dependent on calcineurin, as these changes were blocked by immunosuppressants FK506 and CsA and by overexpression of the endogenous protein inhibitor CAIN. Parallel NFAT activation took place in vivo. Pancreatic growth was accompanied by an increase in nuclear NFATs and subsequent elevation in expression of NFAT-luciferase in the pancreas, but not in organs unresponsive to CCK. The changes also required calcineurin, as they were blocked by FK506. We conclude that CCK activates NFATs in a calcineurin-dependent manner, both in vitro and in vivo.

Calcineurin (Cn) is a Ca2+/calmodulin-dependent phosphatase that dephosphorylates and activates NFAT, a transcription factor essential for T cell activation. T lymphocytes predominantly express the calcineurin Abeta (CnAbeta) isoform, and the deletion of the CnAbeta gene results in defective T cell proliferation and IL-2 production in response to TCR stimulation. In this study, we show that CnAbeta enhances the spontaneous survival of naive T cells by maintaining high levels of Bcl-2, a critical homeostatic survival factor for naive T cells. T cells obtained from CnAbeta-/- mice displayed accelerated spontaneous apoptosis. The observed apoptosis of the CnAbeta-/- T cells was prevented by IL-7 and IL-15, two cytokines critical for the homeostatic survival of naive T cells. Furthermore, CD4+ or CD8+ single positive CnAbeta-/- thymocytes also underwent accelerated apoptosis. However, no obvious difference in the apoptosis of CD4+CD8+ double positive thymocytes was observed between CnAbeta-/- and wild-type mice, suggesting a specific function of CnAbeta in the survival of single positive T cells. Bcl-2 levels were found to be significantly lower in CnAbeta-/- T cells. Transgenic expression of Bcl-xL restored the survival of the CnAbeta-/- T cells. Thus, in addition to its role in mediating TCR signals essential for T cell activation, CnAbeta is also required for the homeostatic survival of naive T cells.

Calcineurin (Cn) is a Ca(2+)/calmodulin-dependent serine/threonine phosphatase that regulates differentiation-specific gene expression in diverse tissues, including the control of fiber-type switching in skeletal muscle. Recent studies have implicated Cn signaling in diminishing skeletal muscle pathogenesis associated with muscle injury or disease-related muscle degeneration. For example, use of the Cn inhibitor cyclosporine A has been shown to delay muscle regeneration following toxin-induced injury and inhibit regeneration in the dystrophin-deficient mdx mouse model of Duchenne muscular dystrophy. In contrast, transgenic expression of an activated mutant of Cn in skeletal muscle was shown to increase utrophin expression and reduce overall disease pathology in mdx mice. Here we examine the effect of altered Cn activation in the context of the delta-sarcoglycan-null (scgd(-/-)) mouse model of limb-girdle muscular dystrophy. In contrast to results discussed in mdx mice, genetic deletion of a loxP-targeted calcineurin B1 (CnB1) gene using a skeletal muscle-specific cre allele in the scgd(-/-) background substantially reduced skeletal muscle degeneration and histopathology compared with the scgd(-/-) genotype alone. A similar regression in scgd-dependent disease manifestation was also observed in calcineurin Abeta (CnAbeta) gene-targeted mice in both skeletal muscle and heart. Conversely, increased Cn expression using a muscle-specific transgene increased cardiac fibrosis, decreased cardiac ventricular shortening, and increased muscle fiber loss in the quadriceps. Our results suggest that inhibition of Cn may benefit select types of muscular dystrophy.

Fast transient outward potassium currents (I(to,f)) are critical determinants of regional heterogeneity of cardiomyocyte repolarization as well as cardiomyocyte contractility. Additionally, I(to,f) densities are markedly down-regulated in cardiac hypertrophy and heart disease, conditions associated with activation of the serine/threonine phosphatase calcineurin (Cn). In this study, we investigated the regulation of I(to,f) expression by Cn in cultured neonatal rat ventricular myocytes (NRVMs) with and without alpha(1)-adrenoreceptor stimulation with phenylephrine (PE). Overexpression of constitutively active Cn in NRVMs induced hypertrophy and caused profound increases in I(to,f) density as well as Kv4.2 mRNA and protein expression and promoter activity, without affecting Kv4.3 or KChIP2 levels. The effects of Cn on hypertrophy, I(to,f), and Kv4.2 transcription were associated with NFAT activation and were abrogated by NFAT inhibition. Despite activating Cn and inducing hypertrophy in NRVMs, PE resulted in profound down-regulation of I(to,f) densities as well as Kv4.2, Kv4.3, and KChIP2 expression. Although hypertrophy and NFAT activation were inhibited by the Cn inhibitory peptide CAIN, I(to,f) and Kv4.2 expression were further reduced by CAIN, whereas Cn overexpression eliminated PE-induced reductions in I(to,f) and Kv4.2 expression without affecting Kv4.3 or KChIP2 levels. We conclude that Cn increases cardiac I(to,f) densities by positively regulating Kv4.2 gene transcription. Consistent with this conclusion, we found that I(to,f) was increased in myocytes isolated from young mice overexpressing Cn prior to the development of heart disease. This positive regulation of Kv4.2 transcription by Cn activation is expected to minimize the reductions in I(to,f) and Kv4.2 expression observed in hypertrophic cardiomyocytes.

Objective: To determine the role of calcineurin and Src tyrosine kinase in the regulation of inducible nitric oxide synthase (iNOS) expression and protection in cardiomyocytes., Methods: iNOS expression was studied in isolated neonatal rat ventricular myocyte cultures in response to bacterial lipopolysaccharide (LPS) or following transfection with constitutively active calcineurin or Src and in hearts isolated from wild-type or calcineruin Abeta knockout mice. Cell injury in response to simulated ischemia-reperfusion was studied following overexpression of active calcineurin. Regulation of the iNOS gene promoter by calcineurin was studied using promoter-luciferase reporter and chromatin immunoprecipitation assays., Results: Overexpression of constitutively active Src co-operated with [Ca2+]c elevation to induce iNOS expression, and LPS-induced iNOS expression was abrogated by pharmacological inhibition of calcineurin or tyrosine kinase. LPS also induced tyrosine kinase-dependent but calcineurin-independent phosphorylation of Src Tyr418. LPS induced myocardial iNOS expression in wild-type but not calcineurin Abeta knockout mice. Overexpression of constitutively active calcinuerin in isolated cardiomyocytes caused dephosphorylation and nuclear accumulation of the c1 isoform of nuclear factor of activated T-cells (NFATc1), induced strong iNOS expression, and induced NOS-dependent protection against simulated ischemia-reperfusion prior to cardiomyocyte hypertrophy. Co-transfection of a mouse iNOS promoter-luciferase reporter in combination with active calcineurin and wild-type or dominant negative Src confirmed that constitutive activation of calcineurin was sufficient for transactivation. Chromatin immunoprecipitation confirmed calcineurin-dependent in vivo binding of NFATc1 to consensus sites within the iNOS promoter., Conclusions: These results support a cardioprotective role for calcineurin mediated by NFAT-dependent induction of iNOS expression and co-operativity between calcineurin and Src.

The manner in which Ca2+-sensitive signaling proteins are activated in contracting cardiomyocytes is an intriguing theoretical problem given that the cytoplasm is continually bathed with systolic Ca2+ concentrations that should maximally activate most Ca2+-sensitive signaling kinases and phosphatases. Store-operated Ca2+ entry, partially attributed to transient receptor potential (TRP) proteins, can mediate activation of the Ca2+-sensitive phosphatase calcineurin in nonexcitable cells. Here we investigated the gain-of-function phenotype associated with TRPC3 expression in the mouse heart using transgenesis to examine the potential role of store-operated Ca2+ entry in regulating cardiac calcineurin activation and ensuing hypertrophy/myopathy. Adult myocytes isolated from TRPC3 transgenic mice showed abundant store-operated Ca2+ entry that was inhibited with SKF96365 but not verapamil or KB-R7943. Associated with this induction in store-operated Ca2+ entry, TRPC3 transgenic mice showed increased calcineurin-nuclear factor of activated T cells (NFAT) activation in vivo, cardiomyopathy, and increased hypertrophy after neuroendocrine agonist or pressure overload stimulation. The cardiomyopathic phenotype and increased hypertrophy after pressure overload stimulation were blocked by targeted disruption of the calcineurin Abeta gene. Thus, enhanced store-operated Ca2+ entry in the heart can regulate calcineurin-NFAT signaling in vivo, which could secondarily impact the hypertrophic response and cardiomyopathy.

The calcium-activated phosphatase calcineurin is regulated by a binding cofactor known as modulatory calcineurin-interacting protein (MCIP) in yeast up through mammals. The physiologic function of MCIP remains an area of ongoing investigation, because both positive and negative calcineurin regulatory effects have been reported. Here we disrupted the mcip1 and mcip2 genes in the mouse and provide multiple lines of evidence that endogenous MCIP functions as a calcineurin facilitator in vivo. Mouse embryonic fibroblasts deficient in both mcip1/2 showed impaired activation of nuclear factor of activated T cells (NFAT), suggesting that MCIP is required for efficient calcineurin-NFAT coupling. Mice deficient in mcip1/2 showed a dramatic impairment in cardiac hypertrophy induced by pressure overload, neuroendocrine stimulation, or exercise, similar to mice lacking calcineurin Abeta. Moreover, simultaneous deletion of calcineurin Abeta in the mcip1/2-null background did not rescue impaired hypertrophic growth after pressure overload. Slow/oxidative fiber-type switching in skeletal muscle after exercise stimulation was also impaired in mcip1/2 mice, similar to calcineurin Abeta-null mice. Moreover, CD4(+) T cells from mcip1/2-null mice showed enhanced apoptosis that was further increased by loss of calcineurin Abeta. Finally, mcip1/2-null mice displayed a neurologic phenotype that was similar to calcineurin Abeta-null mice, such as increased locomotor activity and impaired working memory. Thus, a loss-of-function analysis suggests that MCIPs serve either a permissive or facilitative function for calcineurin-NFAT signaling in vivo.

The calcium-calmodulin-activated protein phosphatase calcineurin functions as a key mediator of diverse biologic processes, including differentiation, apoptosis, growth, and adaptive responses, in part through dephosphorylation and activation of nuclear factor of activated T-cell (NFAT) transcription factors. Apoptosis signal-regulating kinase 1 (ASK1) is an upstream component of the mitogen-activated protein kinases that serves as a pivotal regulator of cytokine-, oxidative-, and stress-induced cell death. Here, we performed a yeast two-hybrid screen with calcineurin B as bait, which identified ASK1 as a direct physical interacting partner. The C-terminal 218 amino acids of ASK1 were sufficient to mediate interaction with calcineurin B in yeast, as well as in mammalian cell lysates. Importantly, endogenous calcium binding B subunit (CnB) protein interacted with endogenous ASK1 protein in cardiomyocytes at baseline, suggesting that the interaction observed in yeast was of potential biologic relevance. Indeed, calcineurin directly dephosphorylated ASK1 at serine 967 using purified proteins or mammalian cell lysates. Dephosphorylation of ASK1 serine 967 by calcineurin promoted its disassociation from 14-3-3 proteins, resulting in ASK1 activation. Calcineurin and ASK1 cooperatively enhanced cardiomyocyte apoptosis, while expression of a dominant negative ASK1 blocked calcineurin-induced apoptosis. Mouse embryonic fibroblasts deficient in ask1 were also partially resistant to calcineurin- or ionomycin-induced apoptosis. Finally, ASK1 negatively regulated calcineurin-NFAT signaling indirectly through c-Jun NH2-terminal kinase (JNK)- and p38-mediated phosphorylation of NFAT, which blocked calcineurin- and agonist-dependent hypertrophic growth of cardiomyocytes. Thus, ASK1 and calcineurin-NFAT constitute a feedback regulatory circuit in which calcineurin positively regulates ASK1 through direct dephosphorylation, while ASK1 negatively regulates calcineurin-NFAT signaling through p38- and JNK-mediated NFAT phosphorylation.

The calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway has been shown to be of critical importance in regulating the growth response of cardiac myocytes. We have previously demonstrated that calcineurin A(beta) (CnA(beta)) mRNA and protein are increased in response to growth stimulation, although the precise regulatory mechanism underlying CnA(beta) upregulation is not clear. Here, we isolated the mouse CnA(beta) promoter and characterized its responsiveness to growth stimuli in vitro and in vivo. A 2.3-kb promoter fragment was strongly activated by phenylephrine and endothelin-1 stimulation and by cotransfection with constitutively active CnA, NFATc4, and GATA4. Using chromatin immunoprecipitation, sequence regions were identified within the 2.3-kb promoter that associated with NFAT and GATA4, as well as with acetylated histone H3, following agonist stimulation. Consistent with the chromatin immunoprecipitation experiments, deletion of the distal half of the CnA(beta) promoter severely reduced NFAT, GATA4, and hypertrophic agonist-mediated activation. To investigate in vivo activity, we generated beta-galactosidase (LacZ) containing transgenic mice under the control of the CnA(beta) 2.3-kb promoter. CnA(beta)-LacZ mice showed expression in the heart that was cyclosporine sensitive, as well as expression in the central nervous system and skeletal muscle from early embryonic stages through adulthood. CnA(beta)-LacZ mice were subjected to cardiac pressure overload stimulation and crossbreeding with mice containing cardiac-specific transgenes for activated calcineurin and NFATc4, which revealed inducible expression in the heart. These results indicate that the CnA(beta) 2.3-kb promoter is specifically activated by hypertrophic stimuli through a positive feedback mechanism involving NFAT and GATA4 transcription factors, suggesting transcriptional induction of CnA(beta) expression as an additional means of regulating calcineurin activity in the heart.

We uncovered a new pathway of interplay between calreticulin and myocyte-enhancer factor (MEF) 2C, a cardiac-specific transcription factor. We establish that calreticulin works upstream of calcineurin and MEF2C in a Ca(2+)-dependent signal transduction cascade that links the endoplasmic reticulum and the nucleus during cardiac development. In the absence of calreticulin, translocation of MEF2C to the nucleus is compromised. This defect is reversed by calreticulin itself or by a constitutively active form of calcineurin. Furthermore, we show that expression of the calreticulin gene itself is regulated by MEF2C in vitro and in vivo and that, in turn, increased expression of calreticulin affects MEF2C transcriptional activity. The present findings provide a clear molecular explanation for the embryonic lethality observed in calreticulin-deficient mice and emphasize the importance of calreticulin in the early stages of cardiac development. Our study illustrates the existence of a positive feedback mechanism that ensures an adequate supply of releasable Ca(2+) is maintained within the cell for activation of calcineurin and, subsequently, for proper functioning of MEF2C.

Adverse left ventricular (LV) remodeling after myocardial infarction (MI) is a major cause for heart failure. Molecular modifiers of the remodeling process remain poorly defined. Patients with heart failure after MI have reduced LV expression levels of muscle LIM protein (MLP), a component of the sarcomeric Z-disk that is involved in the integration of stress signals in cardiomyocytes. By using heterozygous MLP mutant (MLP+/-) mice, we explored the role of MLP in post-MI remodeling. LV dimensions and function were similar in sham-operated WT and MLP+/- mice. After MI, however, MLP+/- mice displayed more pronounced LV dilatation and systolic dysfunction and decreased survival compared with WT mice, indicating that reduced MLP levels predispose to adverse LV remodeling. LV dilatation in MLP+/- mice was associated with reduced thickening but enhanced elongation of cardiomyocytes. Activation of the stress-responsive, prohypertrophic calcineurin-nuclear factor of activated T-cells (NFAT) signaling pathway was reduced in MLP+/- mice after MI, as shown by a blunted transcriptional activation of NFAT in cardiomyocytes isolated from MLP+/-/NFAT-luciferase reporter gene transgenic mice. Calcineurin was colocalized with MLP at the Z-disk in WT mice but was displaced from the Z-disk in MLP+/- mice, indicating that MLP is essential for calcineurin anchorage to the Z-disk. In vitro assays in cardiomyocytes with down-regulated MLP confirmed that MLP is required for stress-induced calcineurin-NFAT activation. Our study reveals a link between the stress sensor MLP and the calcineurin-NFAT pathway at the sarcomeric Z-disk in cardiomyocytes and indicates that reduced MLP-calcineurin signaling predisposes to adverse remodeling after MI.

MEK1, a member of the mitogen-activated protein kinase (MAPK) cascade that directly activates extracellular signal-regulated kinase (ERK), induces cardiac hypertrophy in transgenic mice. Calcineurin is a calcium-regulated protein phosphatase that also functions as a positive regulator of cardiac hypertrophic growth through a direct mechanism involving activation of nuclear factor of activated T-cell (NFAT) transcription factors. Here we determined that calcineurin-NFAT and MEK1-ERK1/2 signaling pathways are interdependent in cardiomyocytes, where they directly coregulate the hypertrophic growth response. For example, genetic deletion of the calcineurin Abeta gene reduced the hypertrophic response elicited by an activated MEK1 transgene in the heart, while inhibition of calcineurin or NFAT in cultured neonatal cardiomyocytes also blunted the hypertrophic response driven by activated MEK1. Conversely, targeted inhibition of MEK1-ERK1/2 signaling in cultured cardiomyocytes attenuated the hypertrophic growth response directed by activated calcineurin. However, targeted inhibition of MEK1-ERK1/2 signaling did not directly affect calcineurin-NFAT activation, nor was MEK1-ERK1/2 activation altered by targeted inhibition of calcineurin-NFAT. Mechanistically, we show that MEK1-ERK1/2 signaling augments NFAT transcriptional activity independent of calcineurin, independent of changes in NFAT nuclear localization, and independent of alterations in NFAT transactivation potential. In contrast, MEK1-ERK1/2 signaling enhances NFAT-dependent gene expression through an indirect mechanism involving induction of cardiac AP-1 activity, which functions as a necessary NFAT-interacting partner. As a second mechanism, MEK1-ERK1/2 and calcineurin-NFAT proteins form a complex in cardiac myocytes, resulting in direct phosphorylation of NFATc3 within its C terminus. MEK1-ERK1/2-mediated phosphorylation of NFATc3 directly augmented its DNA binding activity, while inhibition of MEK1-ERK1/2 signaling reduced NFATc3 DNA binding activity. Collectively, these results indicate that calcineurin-NFAT and MEK1-ERK1/2 pathways constitute a codependent signaling module in cardiomyocytes that coordinately regulates the growth response through two distinct mechanisms.

Calcium and phosphate regulate PTH mRNA stability through differences in binding of parathyroid (PT) proteins to a minimal 63-nucleotide (nt) cis-acting instability element in its 3'-untranslated region. One of these proteins is adenosine-uridine-rich binding factor (AUF1), whose levels are not regulated in PT extracts from rats fed the different diets. However, two-dimensional gels showed posttranslational modification of AUF1 that included phosphorylation. There is no PT cell line, but in HEK 293 cells the 63-nt element is recognized as an instability element, and RNA interference for AUF1 decreased human PTH secretion in cotransfection experiments. Stably transfected cells with a chimeric GH gene containing the PTH 63-nt cis-acting element were used to study the signal transduction pathway that regulates AUF1 modification and chimeric gene mRNA stability. Cyclosporine A, the calcineurin inhibitor, regulated AUF1 posttranslationally, and this correlated with an increase in the stability of GH-PTH 63-nt mRNA but not of the control GH mRNA. Mice with genetic deletion of the calcineurin Abeta gene had markedly increased PTH mRNA levels that were still regulated by low calcium and phosphorus diets. Therefore, calcineurin regulates AUF1 posttranslationally in vitro and PTH gene expression in vivo but still allows its physiological regulation by calcium and phosphate.

The sensing and response to extracellular phosphate (Pi) concentration is preserved from prokaryotes to mammals and ensures an adequate supply of Pi in the face of large differences in its availability. In mammals, the kidneys are central to Pi homeostasis. Renal Pi reabsorption is mediated by a Na/Pi co-transporter that is regulated by a renal Pi sensing system and humoral factors. The signal transduction by which Pi regulates type II Na/Pi activity is largely unknown. It is shown that calcineurin inhibitors specifically and dramatically decrease type II Na/Pi gene expression in a proximal tubule cell line and in vivo. Mice with genetic deletion of the calcineurin Abeta gene had a marked decrease in type II Na/Pi mRNA levels and remarkably did not show the expected increase in type II Na/Pi mRNA levels after the challenge of a low-Pi diet. In contrast, the regulation of renal 25(OH)-vitamin D 1alpha-hydroxylase gene expression by Pi was intact. This is the first demonstration that calcineurin has a crucial role in the signal transduction pathway regulating renal Pi homeostasis both in vitro and in vivo. These results suggest that the use of calcineurin inhibitors contributes to the renal Pi wasting seen in renal transplant patients.

Cardiac hypertrophy is a leading predicator of progressive heart disease that often leads to heart failure and a loss of cardiac contractile performance associated with profound alterations in intracellular calcium handling. Recent investigation has centered on identifying the molecular signaling pathways that regulate cardiac myocyte hypertrophy, as well as the mechanisms whereby alterations in calcium handling are associated with progressive heart failure. One potential focal regulator of cardiomyocyte hypertrophy that also responds to altered calcium handling is the calmodulin-activated serine/threonine protein phosphatase calcineurin (PP2B). Once activated by increases in calcium, calcineurin mediates the hypertrophic response through its downstream transcriptional effector nuclear factor of activated T cells (NFAT), which is directly dephosphorylated by calcineurin resulting in nuclear translocation. While previous studies have convincingly demonstrated the sufficiency of calcineurin to mediate cardiac hypertrophy and progressive heart failure, its necessity remains an area of ongoing investigation. Here we weigh an increasing body of literature that suggests a causal link between calcineurin signaling and the cardiac hypertrophic response and heart failure through the use of pharmacologic inhibitors (cyclosporine A and FK506) and genetic approaches. We will also discuss the manner in which calcineurin-NFAT signaling is negatively regulated in the heart through a diverse array of kinases and inhibitory proteins. Finally, we will discuss emerging theories as to the mechanisms whereby alterations in intracellular calcium handling might stimulate calcineurin within the context of a contractile cell continually experiencing calcium flux., (Copyright 2004 Elsevier Inc.)

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., (Copryright 2004 European Society of Cardiology)

The serine/threonine phosphatase calcineurin is an important regulator of calcium-activated intracellular responses in eukaryotic cells. In higher eukaryotes, calcium/calmodulin-mediated activation of calcineurin facilitates direct dephosphorylation and nuclear translocation of the transcription factor nuclear factor of activated T-cells (NFAT). Recently, controversy has surrounded the role of calcineurin in mediating skeletal muscle cell hypertrophy. Here we examined the ability of calcineurin-deficient mice to undergo skeletal muscle hypertrophic growth following mechanical overload (MOV) stimulation or insulin-like growth factor-1 (IGF-1) stimulation. Two distinct models of calcineurin deficiency were employed: calcineurin Abeta gene-targeted mice, which show a approximately 50% reduction in total calcineurin, and calcineurin B1-LoxP-targeted mice crossed with a myosin light chain 1f cre knock-in allele, which show a greater than 80% loss of total calcineurin only in skeletal muscle. Calcineurin Abeta-/- and calcineurin B1-LoxP(fl/fl)-MLC-cre mice show essentially no defects in muscle growth in response to IGF-1 treatment or MOV stimulation, although calcineurin Abeta-/- mice show a basal defect in total fiber number in the plantaris and a mild secondary reduction in growth, consistent with a developmental defect in myogenesis. Both groups of gene-targeted mice show normal increases in Akt activation following MOV or IGF-1 stimulation. However, overload-mediated fiber-type switching was dramatically impaired in calcineurin B1-LoxP(fl/fl)-MLC-cre mice. NFAT-luciferase reporter transgenic mice failed to show a correlation between IGF-1- or MOV-induced hypertrophy and calcineurin-NFAT-dependent signaling in vivo. We conclude that calcineurin expression is important during myogenesis and fiber-type switching, but not for muscle growth in response to hypertrophic stimuli.

T cell development is dependent on the integration of multiple signaling pathways, although few links between signaling cascades and downstream nuclear factors that play a role in thymocyte differentiation have been identified. We show here that expression of the HMG box protein TOX is sufficient to induce changes in coreceptor gene expression associated with beta-selection, including CD8 gene demethylation. TOX expression is also sufficient to initiate positive selection to the CD8 lineage in the absence of MHC-TCR interactions. TOX-mediated positive selection is associated with up-regulation of Runx3, implicating CD4 silencing in the process. Interestingly, a strong T cell receptor-mediated signal can modify this cell fate. We further demonstrate that up-regulation of TOX in double positive thymocytes is calcineurin dependent, linking this critical signaling pathway to nuclear changes during positive selection.

Cardiovascular disease is the leading cause of mortality and morbidity within the industrialized nations of the world, with coronary heart disease (CHD) accounting for as much as 66% of these deaths. Acute myocardial infarction is a typical sequelae associated with long-standing coronary heart disease resulting in large scale loss of ventricular myocardium through both apoptotic and necrotic cell death. In this study, we investigated the role that the calcium calmodulin-activated protein phosphatase calcineurin (PP2B) plays in modulating cardiac apoptosis after acute ischemia-reperfusion injury to the heart. Calcineurin Abeta gene-targeted mice showed a greater loss of viable myocardium, enhanced DNA laddering and TUNEL, and a greater loss in functional performance compared with strain-matched wild-type control mice after ischemia-reperfusion injury. RNA expression profiling was performed to uncover potential mechanisms associated with this loss of cardioprotection. Interestingly, calcineurin Abeta-/- hearts were characterized by a generalized downregulation in gene expression representing approximately 6% of all genes surveyed. Consistent with this observation, nuclear factor of activated T cells (NFAT)-luciferase reporter transgenic mice showed reduced expression in calcineurin Abeta-/- hearts at baseline and after ischemia-reperfusion injury. Finally, expression of an activated NFAT mutant protected cardiac myocytes from apoptotic stimuli, whereas directed inhibition of NFAT augmented cell death. These results represent the first genetic loss-of-function data showing a prosurvival role for calcineurin-NFAT signaling in the heart.

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 Abeta 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.

The c-Jun N-terminal kinase (JNK) branch of the mitogen-activated protein kinase (MAPK) signaling pathway regulates cellular differentiation, stress responsiveness and apoptosis in multicellular eukaryotic organisms. Here we investigated the functional importance of JNK signaling in regulating differentiated cellular growth in the post-mitotic myocardium. JNK1/2 gene-targeted mice and transgenic mice expressing dominant negative JNK1/2 were determined to have enhanced myocardial growth following stress stimulation or with normal aging. A mechanism underlying this effect was suggested by the observation that JNK directly regulated nuclear factor of activated T-cell (NFAT) activation in culture and in transgenic mice containing an NFAT-dependent luciferase reporter. Moreover, calcineurin Abeta gene targeting abrogated the pro-growth effects associated with JNK inhibition in the heart, while expression of an MKK7-JNK1 fusion protein in the heart partially reduced calcineurin-mediated cardiac hypertrophy. Collectively, these results indicate that JNK signaling antagonizes the differentiated growth response of the myocardium through direct cross-talk with the calcineurin-NFAT pathway. These results also suggest that myocardial JNK activation is primarily dedicated to modulating calcineurin-NFAT signaling in the regulation of differentiated heart growth.

Calcineurin is a calcium-regulated serine-threonine protein phosphatase that controls developmental and inducible biological responses in diverse cell types, in part through activation of the transcription factor nuclear factor of activated T cells (NFAT). In skeletal muscle, calcineurin has been implicated in the regulation of myoblast differentiation, hypertrophy of mature myofibers, and fiber type switching in response to alterations in intracellular calcium concentration. However, considerable disagreement persists about the functional role of calcineurin signaling in each of these processes. Here we evaluated the molecular phenotypes of skeletal muscle from both calcineurin Aalpha and calcineurin Abeta gene-targeted mice. Calcineurin Aalpha was observed to be the predominant catalytic isoform expressed in nearly all skeletal muscles examined. Neither calcineurin Aalpha or Abeta null mice showed any gross growth-related alterations in skeletal muscle, nor was fiber size or number altered in glycolytic/fast muscle types. In contrast, both calcineurin Aalpha and Abeta gene-targeted mice demonstrated an alteration in myofiber number in the soleus, an oxidative/slow-type muscle. More significantly, calcineurin Aalpha and Abeta gene-targeted mice showed a dramatic down-regulation in the oxidative/slow fiber type program in multiple muscles (both slow and fast). Associated with this observation, NFAT-luciferase reporter transgenic mice showed significantly greater activity in slow fiber-containing muscles than in fast. However, only calcineurin Aalpha null mice showed a defect in NFAT nuclear occupancy or NFAT-luciferase transgene activity in vivo. Collectively, our results suggest that calcineurin signaling plays a critical role in regulating skeletal muscle fiber type switching but not hypertrophy. Our results also suggest that fiber type switching occurs through an NFAT-independent mechanism.

The MAPKs are important transducers of growth and stress stimuli in virtually all eukaryotic cell types. In the mammalian heart, MAPK signaling pathways have been hypothesized to regulate myocyte growth in response to developmental signals or physiologic and pathologic stimuli. Here we generated cardiac-specific transgenic mice expressing dominant-negative mutants of p38alpha, MKK3, or MKK6. Remarkably, attenuation of cardiac p38 activity produced a progressive growth response and myopathy in the heart that correlated with the degree of enzymatic inhibition. Moreover, dominant-negative p38alpha, MKK3, and MKK6 transgenic mice each showed enhanced cardiac hypertrophy following aortic banding, Ang II infusion, isoproterenol infusion, or phenylephrine infusion for 14 days. A mechanism underlying this enhanced-growth profile was suggested by the observation that dominant-negative p38alpha directly augmented nuclear factor of activated T cells (NFAT) transcriptional activity and its nuclear translocation. In vivo, NFAT-dependent luciferase reporter transgenic mice showed enhanced activation in the presence of the dominant-negative p38alpha transgene before and after the onset of cardiac hypertrophy. More significantly, genetic disruption of the calcineurin Abeta gene rescued hypertrophic cardiomyopathy and depressed functional capacity observed in p38-inhibited mice. Collectively, these observations indicate that reduced p38 signaling in the heart promotes myocyte growth through a mechanism involving enhanced calcineurin-NFAT signaling.

Calcineurin is a calcium-activated protein phosphatase involved in multiple aspects of cardiac and skeletal muscle development and disease. Genes encoding calcineurin subunit proteins are highly conserved among animal species. Toward the goal of identifying new calcineurin-interacting loci that function in myogenic processes, we expressed an activated form of mouse calcineurin A in Drosophila and screened for suppressors of the phosphatase-induced lethality. Here, we demonstrate that a mutation in the canB2 gene, which encodes a regulatory subunit of Drosophila calcineurin, can suppress a pupal developmental arrest phenotype to adult viability. As canB2 is an essential gene and rare homozygous escapers are flightless, we further analyzed canB2 expression and function in pupae and adults. The gene is expressed in the forming indirect flight muscles and central nervous system during pupal development. A canA gene is comparably expressed in these tissues. Consistent with the observed muscle expression, canB2 mutants exhibit severe defects in the organization of their indirect flight muscles, a phenotype that is likely caused by muscle hypercontractility. Together, these findings demonstrate a vital role for the phosphatase in this specific facet of Drosophila myogenesis and show conserved fly and vertebrate calcineurin genes contribute prominently to fundamental processes of muscle formation and function.

Background: Overexpression of calcineurin in transgenic (TG) mice results in cardiac hypertrophy and unexpected deaths., Methods and Results: None of the TG survived beyond 24 weeks (n=38) whereas all of the wildtype (WT, n=47) survived. Prolongation of repolarization preceded the development of sustained pleomorphic ventricular tachycardia and high degree atrioventricular block, which occurred during spontaneous sudden deaths. Since depolarization-activated K(+) channels contribute dominantly to repolarization in mice, we hypothesized that the TG would decrease these K(+) currents and that the in vivo administration of cyclosporin A (CsA), a calcineurin inhibitor, would reduce this effect. CsA reversed cardiac hypertrophy: capacitance measurements of WT left ventricular myocytes (127+/-7 pF; n=45) and CsA-treated TG (129+/-14 pF; n=17) were significantly lower than in placebo-treated TG (220+/-11 pF; n=41; P<0.001 by ANOVA). Independent of whether the data fit a bi- or a tri-exponential model, the density of I(tof) was significantly reduced in TG versus WT and CsA reversed this effect. While I(tos) and I(Kslow) were also reduced in TG, CsA does not reverse this change because long-term in vivo CsA treatment of WT also reduces I(tos) and I(Kslow.) To assess whether the decreased 'repolarization reserve' contributed to arrhythmogenesis, the residual I(Kr) was blocked by dofetilide precipitating pleomorphic ventricular tachycardias., Conclusion: Since the downregulation of I(tof) was observed with overexpression of calcineurin and was also reversed by the calcineurin inhibitor CsA, we conclude that downregulation of I(tof) is a consequence of calcineurin overexpression.

Introduction of the constitutively active calcineurin gene into neonatal rat cardiomyocytes by adenovirus resulted in decreased mitochondrial membrane potential (P < 0.05). Infection of H9c2 cells with calcineurin adenovirus resulted in increased superoxide production (P < 0.001). Transgenic mice with cardiac-specific expression of a constitutively active calcineurin cDNA (CalTG mice) exhibit a two- to threefold increase in heart size that progresses to heart failure. We prepared mitochondria enriched for the subsarcolemmal population from the hearts of CalTG mice and transgene negative littermates (control). Intact, well-coupled mitochondria prepared from one to two mouse hearts at a time yielded sufficient material for functional studies. Mitochondrial oxygen consumption was measured with a Clark-type oxygen electrode with substrates for mitochondrial complex II (succinate) and complex IV [tetramethylpentadecane (TMPD)/ascorbate]. CalTG mice exhibited a maximal rate of electron transfer in heart mitochondria that was reduced by approximately 50% (P < 0.002) without a loss of respiratory control. Mitochondrial respiration was unaffected in tropomodulin-overexpressing transgenic mice, another model of cardiomyopathy. Western blotting for mitochondrial electron transfer subunits from mitochondria of CalTG mice revealed a 20-30% reduction in subunit 3 of complex I (ND3) and subunits I and IV of cytochrome oxidase (CO-I, CO-IV) when normalized to total mitochondrial protein or to the adenine nucleotide transporter (ANT) and compared with littermate controls (P < 0.002). Impaired mitochondrial electron transport was associated with high levels of superoxide production in the CalTG mice. Taken together, these data indicate that calcineurin signaling affects mitochondrial energetics and superoxide production. The excessive production of superoxide may contribute to the development of cardiac failure.

The calcium-activated phosphatase calcineurin has been implicated as a critical intracellular signal transducer of cardiomyocyte hypertrophy. Although previous data suggested the nuclear factor of activated T-cells (NFAT) as its sole transcriptional effector, the absolute requirement of NFAT as a mediator of calcineurin signaling has not been examined in the heart. We therefore investigated the expression and activation profile of NFAT genes in the heart. Four members (NFATc1-c4) are expressed in cardiomyocytes, elicit nuclear translocation upon calcineurin activation, and are able to drive transactivation of cardiac promoter luciferase constructs. To define the necessary function of NFAT factors as hypertrophic transducers, a dominant negative NFAT construct was created, encompassing part of the N-terminal region of NFATc4 containing a conserved calcineurin-binding motif. Cotransfection of this construct dose-dependently abrogated promoter activation, irrespective of the NFAT isoform used, whereas a control construct with the calcineurin-binding motif mutated displayed no such effects. Adenoviral gene transfer of dominant negative NFAT rendered cardiomyocytes resistant toward all aspects of calcineurin or agonist-induced cardiomyocyte hypertrophy, whereas adenoviral gene transfer of the control construct had no discernable effect on these parameters. These results indicate that multiple NFAT isoforms are expressed in cardiomyocytes where they function as necessary transducers of calcineurin in facilitating cardiomyocyte hypertrophy.

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.

Nuclear factor of activated T-cells (NFAT) and calcineurin are essential regulators of immune cell and mesenchymal cell differentiation. Here we show that elevated intracellular calcium induces chondrogenesis through a calcineurin/NFAT signaling axis that activates bone morphogenetic protein (BMP) expression. The calcium ionophore, ionomycin, induced chondrogenesis through activation of calcineurin. The calcineurin substrate, NFAT4, also induced chondrogenesis and chondrocyte gene expression. Significantly, the BMP antagonist, noggin, or dominant negative BMP receptors blocked the effects of elevated intracellular calcium on chondrogenesis. This suggested that calcineurin/NFAT4 activates BMP expression. Consistent with this, BMP2 gene expression was increased by ionomycin and suppressed by the calcineurin inhibitor, cyclosporine A. Furthermore, activated NFAT4 induced BMP2 gene expression. These results have important implications for the effects of NFATs during development and adaptive responses.

Recent investigation has focused on identifying signaling pathways that inhibit cardiac hypertrophy, a major risk factor for cardiovascular morbidity and mortality. In this context, nitric oxide (NO), signaling via cGMP and cGMP-dependent protein kinase type I (PKG I), has been recognized as a negative regulator of cardiac myocyte (CM) hypertrophy. However, the underlying mechanisms are poorly understood. Here, we show that PKG I inhibits CM hypertrophy by targeting the calcineurin-NFAT signaling pathway. Calcineurin, a Ca2+-dependent phosphatase, promotes hypertrophy in part by activating NFAT transcription factors which induce expression of hypertrophic genes, including brain natriuretic peptide (BNP). Activation of PKG I by NO/cGMP in CM suppressed NFAT transcriptional activity, BNP induction, and cell enlargement in response to alpha(1)-adrenoreceptor stimulation but not in response to adenoviral expression of a Ca2+-independent, constitutively active calcineurin mutant, thus demonstrating NO-cGMP-PKG I inhibition of calcineurin-NFAT signaling upstream of calcineurin. PKG I suppressed single L-type Ca2+-channel open probability, [Ca2+]i transient amplitude, and, most importantly, L-type Ca2+-channel current-induced NFAT activation, indicating that PKG I targets Ca2+-dependent steps upstream of calcineurin. Adenoviral expression of PKG I enhanced NO/cGMP inhibitory effects upstream of calcineurin, confirming that PKG I mediates NO/cGMP inhibition of calcineurin-NFAT signaling. In CM overexpressing PKG I, NO/cGMP also suppressed BNP induction and cell enlargement but not NFAT activation elicited by constitutively active calcineurin, which is consistent with additional, NFAT-independent inhibitory effect(s) of PKG I downstream of calcineurin. Inhibition of calcineurin-NFAT signaling by PKG I provides a framework for understanding how NO inhibits cardiac myocyte hypertrophy.

The calcium-dependent phosphatase calcineurin and its downstream transcriptional effector nuclear factor of activated T cells (NFAT) are important regulators of inducible gene expression in multiple cell types. In T cells, calcineurin-NFAT signaling represents a critical event for mediating cellular activation and the immune response. The widely used immunosuppressant agents cyclosporin and FK506 are thought to antagonize the immune response by directly inhibiting calcineurin-NFAT signal transduction in lymphocytes. To unequivocally establish the importance of calcineurin signaling as a mediator of the immune response, we deleted the gene encoding the predominant calcineurin isoform expressed in lymphocytes, calcineurin A beta (CnA beta). CnA beta(-/-) mice were viable as adults, but displayed defective T cell development characterized by fewer total CD3 cells and reduced CD4 and CD8 single positive cells. Total peripheral T cell numbers were significantly reduced in CnA beta(-/-) mice and were defective in proliferative capacity and IL-2 production in response to PMA/ionomycin and T cell receptor cross-linking. CnA beta(-/-) mice also were permissive to allogeneic tumor-cell transplantation in vivo, similar to cyclosporin-treated wild-type mice. A mechanism for the compromised immune response is suggested by the observation that CnA beta(-/-) T cells are defective in stimulation-induced NFATc1, NFATc2, and NFATc3 activation. These results establish a critical role for CnA beta signaling in regulating T cell development and activation in vivo.