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Introduction: Transgenic Scn5a-1798insD+/- mice display atrio-ventricular conduction disturbances and develop susceptibility to arrhythmias with age. Isolated cardiomyocytes from Scn5a-1798insD+/- mouse hearts exhibit increased sustained inward sodium current which is associated with elevated intracellular sodium (Na+), intracellular diastolic calcium ([Ca2+]i) levels and Ca2+ transient amplitudes. We investigated whether the Scn5a-1798insD+/- mutation leads to activation of Ca2+-dependent calcineurin/Nfat signaling pathway, and assessed its impact on atrio-ventricular conduction. Results: Despite increased [Ca2+]i, activation of the calcineurin/Nfat pathway was not observed in Scn5a-1798insD+/- hearts at baseline (as evidenced by unaltered nuclear levels of Nfat protein as compared to wild type hearts). Wild-type and Scn5a-1798insD+/- mice displayed a similar hypertrophic response to 2 weeks of TAC (as evidenced by similar increases in heart weight and Anf mRNA levels), indicating equal activation of the calcineurin/Nfat pathway. However, Scn5a-1798insD+/–TAC mice displayed increased incidence of sudden death preceded by progressive bradycardia and AV-block. Furthermore, isolated Scn5a-1798insD+/–TAC hearts showed AV-block and significantly prolonged AV-delay as compared to wild-type-TAC hearts. Since Nfatc2 is a well-described downstream effector of the calcineurin pathway, we next investigated whether the observed electrical abnormalities after TAC could be rescued by crossing Scn5a-1798insD+/- with Nfatc2-KO mice. Scn5a-1798insD+/-/Nfatc2-KO mice showed a lower response to TAC as Scn5a-1798insD+/–TAC mice (as evidenced by lower increase in heart weight and Anf mRNA levels). The ex vivo AV-delay after TAC was attenuated in Scn5a-1798insD+/-/Nfatc2-KO TAC hearts, indicating rescue of the AV-conduction disturbances in Scn5a-1798insD+/–TAC mice by Nfatc2 deficiency. Moreover, His-ventricular conduction was unaffected by TAC, indicating that the increase in AV-delay in Scn5a-1798insD+/–TAC mice occurs proximal to the His bundle. Conclusions: Scn5a-1798insD+/- mice show intracellular Na+/Ca2+-dysregulation at baseline and severe AV-conduction abnormalities and sudden death upon increased activation of the calcineurin/Nfat-pathway by TAC challenge. Nfatc2 deficiency prevents these electrical disturbances secondary to TAC in Scn5a-1798insD+/-/Nfatc2-KO mice, indicating a role of the calcineurin/Nfat pathway on atrio-ventricular conduction.

Objectives To investigate if calcineurin pathway is involved in cardiac fibroblasts proliferation, transdifferentiation and apoptosis induced by mechanical stretch. Methods Primary cardiac fibroblasts from 1- to 2-day-old Sprague-Dawley rats were isolated by trypsinization and purified by differential anchoring velocity technology. The cultured cells of passage 2-3 were divided into: control group, mechanical stretch group (MS group), calcineurin siRNA + mechanical stretch group (CaN-siRNA + MS group), FK506 + mechanical stretch group (FK506 + MS group), calcineurin overexpression + mechanical stretch group (CaN-overex + MS group). Calcineurin and downstream NFAT3 expressions were determined by western blot. Cardiac fibroblasts proliferation was determined by cell counting kit-8 and detection of proliferating cell nuclear antigen (PCNA), cell transdifferentiation was measured by alpha-smooth muscle actin (alpha-SMA) through western blot and apoptosis by Bax/Bcl-2. Results Mechanical stretch promoted cardiac fibroblasts proliferation, as well as upregulation of alpha-SMA and increased Bax/Bcl-2 ratio, overexpression of calcineurin could promote more cardiac fibroblasts adverse remodelling similar to those induced by mechanical stretch. Pretreatment with calcineurin-siRNA lentivirus significantly attenuated cardiac fibroblasts proliferation, transdifferentiation and apoptosis when later stimulated by mechanical stretch, whereas pretreatment with FK506, a calcineurin inhibitor, only moderately inhibited cardiac fibroblasts proliferation and apoptosis with no obvious effect on cell transdifferentiation. Conclusions This study demonstrates that mechanical stretch promoted cardiac fibroblasts proliferation, transdifferentiation and apoptosis via activating calcineurin/NFAT3, thus abolishing the calcineurin pathway might be beneficial for reversing myocardial fibrosis.

Objective We aimed to study the mechanism and pathway of the adenosine A1 receptor agonist 2-chloro-N6 cyclopentyladenosine (CCPA) in the inhibition of the cardiomyocyte hypertrophy induced by angiotensin II (AngII) in vitro. Methods Cardiomyocytes from neonatal SD rats were treated with AngII to generate a cardiomyocyte hypertrophy model. The cell cultures were randomised into four groups: (1) control; (2) AngII; (3) AngII + cyclosporin A (CsA); and (4) AngII + CCPA. The MTT method was used to measure cardiomyocyte viability. Protein synthesis was monitored by measuring the incorporation of tritiated leucine (3H-Leu), and the expression of β-myosin heavy chain (β-MHC) and calcineurin Aβ (CnAβ) mRNA was measured by semi-quantitative RT-PCR. Results Forty-eight hours after AngII treatment, surface area, viability and 3H-Leu incorporation of the cardiomyocytes were significantly increased in a dose-dependent manner. Both CCPA and the calcineurin (CaN)-specific inhibitor CsA significantly inhibited AngII-induced cardiomyocyte 3H-Leu incorporation. These compounds also inhibited the mRNA expression of CnAβ and β-MHC in AngII-induced cardiomyocytes but had no significant effect on 3H-Leu incorporation in the control group. The inhibition by CCPA was dose-dependent within the range of 1 × 10–8 mol/L to 1 × 10–6 mol/L. Conclusions The adenosine A1 receptor agonist CCPA can significantly inhibit AngII-induced cardiomyocyte hypertrophy in a manner related to the CaN signalling pathway.

Yeast cells are able to tolerate and adapt to a variety of environmental stresses. An essential aspect of stress adaptation is the regulation of monovalent ion concentrations. Ion regulation determines many fundamental physiological parameters, such as cell volume, membrane potential, and intracellular pH. It is achieved through the concerted activities of multiple cellular components, including ion transporters and signaling molecules, on both short and long time scales. Although each component has been studied in detail previously, it remains unclear how the physiological parameters are maintained and regulated by the concerted action of all components under a diverse range of stress conditions. In this study, we have constructed an integrated mathematical model of ion regulation in Saccharomyces cerevisiae to understand this coordinated adaptation process. Using this model, we first predict that the interaction between phosphorylated Hog1p and Tok1p at the plasma membrane inhibits Tok1p activity and consequently reduces Na+ influx under NaCl stress. We further characterize the impacts of NaCl, sorbitol, KCl and alkaline pH stresses on the cellular physiology and the differences between the cellular responses to these stresses. We predict that the calcineurin pathway is essential for maintaining a non-toxic level of intracellular Na+ in the long-term adaptation to NaCl stress, but that its activation is not required for maintaining a low level of Na+ under other stresses investigated. We provide evidence that, in addition to extrusion of toxic ions, Ena1p plays an important role, in some cases alongside Nha1p, in re-establishing membrane potential after stress perturbation. To conclude, this model serves as a powerful tool for both understanding the complex system-level properties of the highly coordinated adaptation process and generating further hypotheses for experimental investigation., Author Summary Ion regulation is fundamental to cell physiology. The concentrations of monovalent ions, such as H+, K+ and Na+, determine many physiological parameters such as cell volume, plasma membrane potential and intracellular pH. In yeast cells, these parameters are maintained within a narrow range during the adaptation to external perturbations, including ionic, osmotic and alkaline pH stress. This is achieved by the remarkably coordinated activities of ion transporters, regulatory molecules and signaling pathways. The response characteristics of individual components in adaptation have been studied extensively. However, a coherent understanding of the coordinated adaptation process is lacking. In this study, we address this gap by constructing a mathematical model that integrates the characteristics of the ion transporters, regulatory molecules, signaling pathways and changes in cell volume. Using this model, we characterize the impact of ionic, osmotic and alkaline pH stresses on cellular physiology and analyze the role that individual components play in the cellular adaptation processes. Our results also reveal system level properties achieved by the concerted regulatory responses. Therefore, this integrated model serves as a suitable tool to understand the coordinated processes of ion regulation in response to environmental stresses, and to make predictions that are experimentally testable.

Activation through the Ca2+/calcineurin pathway is essential to the transcription of many cytokine genes. The conserved cis-acting sequence, GGAAAA, and transcription factors binding to this sequence are involved in the response to increased intracellular Ca2+ concentrations. Here we report the identification and importance of the same sequence in a non-cytokine gene, the human T cell receptor gamma (TCRG) enhancer. Results from site-directed mutations and electrophoretic mobility shift assays strongly suggest that this sequence mediates the ionomycin-induced activation of the TCRG enhancer. Our studies provide an explanation for a previous observation that TCRG mRNA levels, but not mRNA levels for T cell receptor alpha and -beta, are increased by ionomycin treatment.

Although the immediate receptors (immunophilins) of the immunosuppressants cyclosporin A (CsA) and FK506 are distinct, their similar mechanisms of inhibition of cell signaling suggest that their associated immunophilin complexes interact with a common target. We report here that the complexes cyclophilin-CsA and FKBP-FK506 (but not cyclophilin, FKBP, FKBP-rapamycin, or FKBP-506BD) competitively bind to and inhibit the Ca(2+)- and calmodulin-dependent phosphatase calcineurin, although the binding and inhibition of calcineurin do not require calmodulin. These results suggest that calcineurin is involved in a common step associated with T cell receptor and IgE receptor signaling pathways and that cyclophilin and FKBP mediate the actions of CsA and FK506, respectively, by forming drug-dependent complexes with and altering the activity of calcineurin-calmodulin.

Megakaryocytes, as precursor cells of platelets, comprise a cell population crucial for the maintenance of adult hematopoiesis. Since platelets are anuclear cellular fragments, the platelet transcriptome as well as the proteome are largely determined by the megakaryocyte. Calcium signaling in response to agonists such as collagen or thrombin has a central function in platelet activation and therefore likely represents an important signaling pathway in megakaryocytes as well. Indeed, megakaryocytes show considerable elevation of intracellular calcium levels in response to selected platelet agonists, but the downstream effector molecules of calcium signaling in these cells or the transcriptional responses induced are unknown. We here establish calcineurin and the NFAT (Nuclear Factor of Activated T cells) family of transcription factors as components of a calcium-induced signaling cascade in megakaryocytes. In resting megakaryocytes, NFAT is cytoplasmic and inactive, but can be activated by fibrillar collagen type 1, a physiological agonist of platelets and megakaryocytes known to induce a sustained increase in intracellular calcium levels in these cells. In contrast, treatment with SDF-1a, thrombopoietin, or VEGF remained without noticeable effect, presumably because of the short duration of the calcium transients induced by these agents. Collagen-induced NFAT activation in megakaryocytes requires dephosphorylation by calcineurin and is completely sensitive to the calcineurin inhibitor cyclosporin A. Activation of NFAT by collagen was paralleled by the induction of the expression of Down Syndrome Critical Region I (DSCR1) and Fas Ligand (FasL), two genes recently identified as NFAT targets in megakaryocytes. Collagen-induced expression of DSCR1 and FasL occurred in a calcineurin- and NFAT-dependent manner, as it was blocked by both cyclosporin A as well as the specific peptide inhibitor of NFAT, VIVIT. These experiments show that the calcineurin pathway is a target for selected physiological ligands capable of inducing sustained calcium mobilization in megakaryocytes and regulates megakaryocyte gene expression in a cyclosporin A- and NFAT-dependent manner.

Calcineurin signaling has been implicated in a broad spectrum of developmental processes in a variety of organ systems. Calcineurin is a calmodulin-dependent, calcium-activated protein phosphatase composed of catalytic and regulatory subunits. The serine/threonine-specific phosphatase functions within a signal transduction pathway that regulates gene expression and biological responses in many developmentally important cell types. Calcineurin signaling was first defined in T lymphocytes as a regulator of nuclear factor of activated T cells (NFAT) transcription factor nuclear translocation and activation. Recent studies have demonstrated the vital nature of calcium/calcineurin/NFAT signaling in cardiovascular and skeletal muscle development in vertebrates. Inhibition, mutation, or forced expression of calcineurin pathway genes result in defects or alterations in cardiomyocyte maturation, heart valve formation, vascular development, skeletal muscle differentiation and fiber-type switching, and cardiac and skeletal muscle hypertrophy. Conserved calcineurin genes are found in invertebrates such as Drosophila and Caenorhabditis elegans, and genetic studies have demonstrated specific myogenic functions for the phosphatase in their development. The ability to investigate calcineurin signaling pathways in vertebrates and model genetic organisms provides a great potential to more fully comprehend the functions of calcineurin and its interacting genes in heart, blood vessel, and muscle development.

SummaryThe premetastatic niche is a predetermined site of metastases, awaiting the influx of tumor cells. However, the regulation of the angiogenic switch at these sites has not been examined. Here, we demonstrate that the calcineurin and nuclear factor of activated T cells (NFAT) pathway is activated specifically in lung endothelium prior to the detection of tumor cells that preferentially metastasize to the lung. Upregulation of the calcineurin pathway via deletion of its endogenous inhibitor Dscr1 leads to a significant increase in lung metastases due to increased expression of a newly identified NFAT target, Angiopoietin-2 (ANG2). Increased VEGF levels specifically in the lung, and not other organ microenvironments, trigger a threshold of calcineurin-NFAT signaling that transactivates Ang2 in lung endothelium. Further, we demonstrate that overexpression of DSCR1 or the ANG2 receptor, soluble TIE2, prevents the activation of lung endothelium, inhibiting lung metastases in our mouse models. Our studies provide insights into mechanisms underlying angiogenesis in the premetastatic niche and offer targets for lung metastases.

We have previously reported that transcriptional induction of cyclooxygenase-2 (COX-2) isoenzyme occurs early after T cell receptor triggering, suggesting functional implications of cyclooxygenase activity in this process. Here, we identify the cis-acting elements responsible for the transcriptional activation of this gene in human T lymphocytes. COX-2 promoter activity was induced upon T cell activation both in primary resting T lymphocytes and in Jurkat cells. This induction was abrogated by inhibition of calcineurin phosphatase with the immunosuppressive drug cyclosporin A, whereas expression of an active calcineurin catalytic subunit enhanced COX-2 transcriptional activation. Moreover, cotransfection of nuclear factor of activated T cells (NFAT) wild type protein transactivated COX-2 promoter activity. Conversely, dominant negative mutants of NFATc or c-Jun proteins inhibited COX-2 induction. Electrophoretic mobility shift assays and site-directed mutagenesis allowed the identification of two regions of DNA located in the positions -117 and -58 relative to the transcriptional start site that serves as NFAT recognition sequences. These results emphasize the central role that the Ca(2+)/calcineurin pathway plays in COX-2 transcriptional regulation in T lymphocytes pointing to NFAT/activator protein-1 transcription factors as essential for COX-2 promoter regulation in these cells.

Background: NFATc1 is a transcription factor activated by the calcium/calcineurin pathway, which regulates several physiological processes. Evidence has also shown a role for NFAT transcriptions factors in oncogenesis. BRG1 and BRM are chromatinremodelling genes that are also regulated by calcium homoeostasis. Diminished expression of BRG1 is associated with poor prognosis in breast cancer, non-small-cell lung cancer, colorectal cancer, and prostate cancer. To understand the biological relationship between these two pathways, we examined the expression pattern of NFATc1, BRG1, and BRM in invasive breast cancer. Methods: Paraffin blocks of 150 cases of invasive breast cancer were retrieved from Kaiser Permanente Medical Centre database. Monoclonal antibodies against NFATc1, BRG1, and BRM were used, and expression pattern was determined as no expression, weak (1+ staining intensity), moderate (2+), and high expression (3+). Pearson Chi-Squared test was used for statistical analysis. Findings: NFATc1 was expressed in approximately 22% of the 150 cases of invasive breast cancer, whereas BRG1 was expressed in 56.7% and BRM in 52%. Both nuclear and cytoplasmic expression of NFATc1 was detected, with nuclear expression as the predominant feature. BRM1/BRM are predominantly expressed in the nucleus. Most cases expressing NFATc1 showed 2+ moderate intensity expression patterns. Statistical analysis showed that expression of NFATc1 was highly correlated with the expression of both BRG1 and BRM. Interpretation: Our preliminary data shows that NFATc1 is expressed in a subset of BRG1/BRM-positive invasive breast cancers. This correlation of two calcium homoeostasis-regulated pathways provides insight into the oncogenesis of breast cancer. Funding: Kaiser Hospital Foundation Community Benefit Grant. The author declared no conflicts of interest.