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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.

The objective of this study was to identify unknown modulators of Calcineurin (Cn)-NFAT signaling. Measurement of NFAT reporter driven luciferase activity was therefore utilized to screen a human cardiac cDNA-library (~10 7 primary clones) in C2C12 cells through serial dilutions until single clones could be identified. This extensive screening strategy culminated in the identification of SUMO2 as a most efficient Cn-NFAT activator. SUMO2-mediated activation of Cn-NFAT signaling in cardiomyocytes translated into a hypertrophic phenotype. Prohypertrophic effects were also observed in mice expressing SUMO2 in the heart using AAV9 (Adeno-associated virus), complementing the in vitro findings. In addition, increased SUMO2-mediated sumoylation in human cardiomyopathy patients and in mouse models of cardiomyopathy were observed. To decipher the underlying mechanism, we generated a sumoylation-deficient SUMO2 mutant (ΔGG). Surprisingly, ΔGG replicated Cn-NFAT-activation and the prohypertrophic effects of native SUMO2, both in vitro and in vivo, suggesting a sumoylation-independent mechanism. Finally, we discerned a direct interaction between SUMO2 and CnA, which promotes CnA nuclear localization. In conclusion, we identified SUMO2 as a novel activator of Cn-NFAT signaling in cardiomyocytes. In broader terms, these findings reveal an unexpected role for SUMO2 in cardiac hypertrophy and cardiomyopathy, which may open the possibility for therapeutic manipulation of this pathway.

The serine-threonine phosphatase calcineurin is activated in cardiac myocytes in the diseased heart and induces pathological hypertrophy. Calcineurin activity is mainly triggered by calcium/calmodulin binding but also through calpain mediated cleavage. How controlled calcineurin activation is possible in cardiac myocytes, which typically show a 10-fold difference in cytosolic calcium concentration with every heartbeat, has remained enigmatic. It is now emerging that calcineurin activation and signaling occur in subcellular microdomains, in which it is brought together with target proteins and exceedingly high concentrations of calcium in order to induce downstream signaling. We review current evidence of subcellular calcineurin mainly at the sarcolemma and the nucleus, but also in association with the sarcoplasmic reticulum and mitochondria. We also suggest that knowledge about subcellular signaling could help to develop inhibitors of calcineurin in specific microdomains to avoid side-effects that may arise from complete calcineurin inhibition., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Calcineurin (Cn), a serine/threonine phosphatase, plays a crucial role in the development of myocardial hypertrophy. Cn is a cytosolic phosphatase which dephosphorylates various target molecules, e.g. the transcriptional factor nuclear factor of activated T cells (NFAT), thereby enabling its nuclear translocation. Recently, it was demonstrated that not only NFAT, but also Cn is translocated into the nucleus. The nuclear coexistence of Cn and NFAT is important for the full transcriptional activity of the Cn-NFAT signalling cascade. Once Cn and NFAT have entered the nucleus of cardiomyocytes, the transcription of genes characteristic for myocardial hypertrophy (e.g. BNP, ANP) is initiated. The nuclear localization sequence (NLS), a region spanning amino acids 172-183 of calcineurin Abeta (CnAbeta) is essential for recognition and shuttling of Cn into the nucleus by importinbeta (1). A synthetic import blocking peptide (IBP) that mimics the NLS of Cn was tested recently. The NLS analogon IBP saturates the Cn binding site of importinbeta(1) thereby preventing binding of Cn and importin. This inhibits the translocation of Cn into the nucleus. Inhibiting the Cn/importin interaction with competing synthetic peptides is one of several new approaches to prevent the development of myocardial hypertrophy. Several patents have also been filed on molecules related to inhibition of Cn-NFAT signalling.

The time that transcription factors remain nuclear is a major determinant for transcriptional activity. It has recently been demonstrated that the phosphatase calcineurin is translocated to the nucleus with the transcription factor nuclear factor of activated T cells (NF-AT). This study identifies a nuclear localization sequence (NLS) and a nuclear export signal (NES) in the sequence of calcineurin. Furthermore we identified the nuclear cargo protein importinbeta(1) to be responsible for nuclear translocation of calcineurin. Inhibition of the calcineurin/importin interaction by a competitive peptide (KQECKIKYSERV), which mimicked the calcineurin NLS, prevented nuclear entry of calcineurin. A noninhibitory control peptide did not interfere with the calcineurin/importin binding. Using this approach, we were able to prevent the development of myocardial hypertrophy. In angiotensin II-stimulated cardiomyocytes, [(3)H]-leucine incorporation (159%+/-9 versus 111%+/-11; P<0.01) and cell size were suppressed significantly by the NLS peptide compared with a control peptide. The NLS peptide inhibited calcineurin/NF-AT transcriptional activity (227%+/-11 versus 133%+/-8; P<0.01), whereas calcineurin phosphatase activity was unaffected (298%+/-9 versus 270%+/-11; P=NS). We conclude that calcineurin is not only capable of dephosphorylating NF-AT, thus enabling its nuclear import, but the presence of calcineurin in the nucleus is also important for full NF-AT transcriptional activity.

Background: Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload leads to increased CnA activity in human myocardium. Here, we investigated the proteolytic mechanism leading to activation of CnA., Methods and Results: In patients with diseased myocardium, we found strong nuclear translocation of CnA. In contrast, in normal human myocardium, there was a cytosolic distribution of CnA. Stimulation of rat cardiomyocytes with angiotensin (Ang) II increased calpain activity significantly (433+/-11%; P<0.01; n=6) and caused proteolysis of the autoinhibitory domain of CnA. Inhibition of calpain by a membrane-permeable calpain inhibitor prevented proteolysis. We identified the cleavage site of calpain in the human CnA sequence at amino acid 424. CnA activity was increased after Ang II stimulation (310+/-29%; P<0.01; n=6) and remained high after removal of Ang II (214+/-17%; P<0.01; n=6). Addition of a calpain inhibitor to the medium decreased CnA activity (110+/-19%; P=NS; n=6) after removal of Ang II. Ang II stimulation of cardiomyocytes also translocated CnA into the nucleus as demonstrated by immunohistochemical staining and transfection assays with GFP-tagged CnA. Calpain inhibition and therefore suppression of calpain-mediated proteolysis of CnA enabled CnA exit from the nucleus., Conclusions: Ang II stimulation of cardiomyocytes increased calpain activity, leading to proteolysis of the autoinhibitory domain of CnA. This causes an increase in CnA activity and results in nuclear translocation of CnA. Loss of the autoinhibitory domain renders CnA constitutively nuclear and active, even after removal of the hypertrophic stimulus.

Unlabelled: The role of AT2-receptors has recently been subject of considerable debate. We investigated the influence of AT2-stimulation/inhibition on myocardial endothelial NO-synthase (eNOS, NOS-III) promoter activity and eNOS protein expression. Stimulation of rat cardiomyocytes with angiotensin II (AngII) increased eNOS protein expression 3.3-fold. This was blocked by Cyclosporin A (CsA). Inhibition of the AT1-receptor did not reduce AngII-mediated eNOS protein expression, whereas AT2 stimulation increased it 2.4-fold and AT2 inhibition suppressed it. The modulatory effects of the AT2-receptor on eNOS expression was confirmed in mice with a genetic deletion of the AT2-receptor (AT2-KO). In gel shift assays two putative NF-AT sites in a 1.6 kb eNOS promoter fragment showed NF-AT binding and a supershift by NF-AT2(-c1)-specific antibodies. Stimulation of transfected cells with AngII or specific AT2-receptor agonists resulted in a significant increase in eNOS promoter activity, which was blocked by CsA, MCIP1, and mutation of an upstream NF-AT site., Conclusion: 1) AngII-stimulation of the myocardium, both in vivo and in vitro, is accompanied by increased expression of eNOS. 2) This effect is mediated by the calcineurin pathway and is induced by the AT2-receptor. 3) These results define a calcineurin/NF-AT/eNOS pathway as downstream effector of AT2-receptor activation in the myocardium.

Background: In animal models, increased signaling through the calcineurin pathway has been shown to be sufficient for the development of cardiac hypertrophy. Calcineurin activity has been reported to be elevated in the myocardium of patients with congestive heart failure. In contrast, few data are available about calcineurin activity in patients with pressure overload or cardiomyopathic hypertrophy who are not in cardiac failure., Methods and Results: We investigated calcineurin activity and protein expression in 2 different forms of cardiac hypertrophy: hypertrophic obstructive cardiomyopathy (HOCM) and aortic stenosis (AS). We found that the C-terminus of calcineurin A protein containing the autoinhibitory domain was less abundant in myocardial hypertrophy than in normal heart, which suggests the possibility of proteolysis. No new splice variants could be detected by reverse-transcription polymerase chain reaction. This resulted in a significant elevation of calcineurin enzymatic activity in HOCM and AS compared with 6 normal hearts. Increased calcineurin phosphatase activity caused increased migration of NF-AT2 (nuclear factor of activated T cells 2) in SDS-PAGE compatible with pronounced NF-AT dephosphorylation in hypertrophied myocardial tissue., Conclusions: Hypertrophy in HOCM and AS without heart failure is characterized by a significant increase in calcineurin activity. This might occur by (partial) proteolysis of the calcineurin A C-terminus containing the autoinhibitory domain. Increased calcineurin activity has functional relevance, as shown by altered NF-AT phosphorylation state. Although hypertrophy in AS and HOCM may be initiated by different upstream triggers (internal versus external fiber overload), in both cases, there is activation of calcineurin, which suggests an involvement of this pathway in the pathogenesis of human cardiac hypertrophy.

An abstract of the article "Stimulation of Myocardial AT2-Rezeptors Increases Calcineurin Activity Via Calpain," by O. Ritter and colleagues is presented.

An abstract of the study "Prohibition of Calcineurin/Importin-Interaction Disrupts Calcineurin/NFAT Cascade," by O. Ritter and colleagues is presented.

An abstract of the study "Inhibition of nuclear import of calcineurin prevents myocardial hypertrophy," by O. Ritter is presented