Sustained pressure overload leads to compensatory myocardial hypertrophy and subsequent heart failure, a leading cause of morbidity and mortality. Further unraveling of the cellular processes involved is essential for development of new treatment strategies. We have investigated the hypothesis that the transmembrane Z-disc proteoglycan syndecan-4, a co-receptor for integrins, connecting extracellular matrix proteins to the cytoskeleton, is an important signal transducer in cardiomyocytes during development of concentric myocardial hypertrophy following pressure overload. Echocardiographic, histochemical and cardiomyocyte size measurements showed that syndecan-4−/− mice did not develop concentric myocardial hypertrophy as found in wild-type mice, but rather left ventricular dilatation and dysfunction following pressure overload. Protein and gene expression analyses revealed diminished activation of the central, pro-hypertrophic calcineurin-nuclear factor of activated T-cell (NFAT) signaling pathway. Cardiomyocytes from syndecan-4−/−-NFAT-luciferase reporter mice subjected to cyclic mechanical stretch, a hypertrophic stimulus, showed minimal activation of NFAT (1.6-fold) compared to 5.8-fold increase in NFAT-luciferase control cardiomyocytes. Accordingly, overexpression of syndecan-4 or introducing a cell-permeable membrane-targeted syndecan-4 polypeptide (gain of function) activated NFATc4 in vitro. Pull-down experiments demonstrated a direct intracellular syndecan-4-calcineurin interaction. This interaction and activation of NFAT were increased by dephosphorylation of serine 179 (pS179) in syndecan-4. During pressure overload, phosphorylation of syndecan-4 was decreased, and association between syndecan-4, calcineurin and its co-activator calmodulin increased. Moreover, calcineurin dephosphorylated pS179, indicating that calcineurin regulates its own binding and activation. Finally, patients with hypertrophic myocardium due to aortic stenosis had increased syndecan-4 levels with decreased pS179 which was associated with increased NFAT activation. In conclusion, our data show that syndecan-4 is essential for compensatory hypertrophy in the pressure overloaded heart. Specifically, syndecan-4 regulates stretch-induced activation of the calcineurin-NFAT pathway in cardiomyocytes. Thus, our data suggest that manipulation of syndecan-4 may provide an option for therapeutic modulation of calcineurin-NFAT signaling.

SN provided incremental prognostic information to established risk indices in acute HF and ventricular arrhythmia-induced cardiac arrest.

Prostaglandins (PG) are released during tissue injury and inflammation, and inhibit immune responses at many points. PG may be one of several factors that protect not only against injury-induced, but also spontaneous, organ-specific autoimmune disease. Here we show that the production of PGE(2), normally produced at a very low rate in islets of Langerhans, is significantly increased in inflamed islets of non-obese diabetic (NOD) mice. We investigated a possible role of PGE(2) in controlling TCR-dependent release of IFN-gamma from islet-reactive NOD CD8(+) T cells. PGE(2) inhibited anti-TCR antibody-triggered release of IFN-gamma from CD8(+) T cell clone 8D8 and from polyclonal cytotoxic T lymphocytes (CTL). Using receptor subtype selective agonists, we present evidence that the effect of PGE(2) is mediated by EP(2) and EP(4) receptors, both of which are coupled to an increase in intracellular cAMP production. The cAMP analogs 8-Br-cAMP and Sp-cAMPS mimic the effect of EP(2)/EP(4) receptor agonists, inhibiting TCR-triggered IFN-gamma release from NOD CD8(+) T cells in a dose-dependent manner. The inhibitory effect of PGE(2) was largely reversed by IL-2 added at the time of culture initiation and decreased with increasing strength of stimulation through the TCR. Resting CTL were more sensitive to PGE(2) than recently expanded CTL and NOD CD8(+) T cells remained insensitive to PGE(2) for a longer time than BALB/c cells. Our study suggests that PGE(2) may be part of a regulatory network that controls local activation of T cells and may play a role in the balance between the development of islet autoimmunity or maintenance of tolerance.

Background: The sarcoplasmic reticulum (SR) Ca 2+ -ATPase 2 (SERCA2) mediates a 2+ -reuptake into SR and thereby promotes cardiomyocyte relaxation, whereas the ryanodine receptor (RYR) mediates a 2+ -release from SR and triggers contraction. a 2+ /calmodulin (CaM)-dependent protein kinase II (CaMKII) regulates activities of SERCA2 through phosphorylation of phospholamban (PLN) and RYR through direct phosphorylation. However, the mechanisms for CaMKIIδ anchoring to SERCA2-PLN and RYR and its regulation by local a 2+ -signals remain elusive. The objective of this study was to investigate CaMKIIδ anchoring and regulation at SERCA2-PLN and RYR. Methods: A role for A-kinase anchoring protein 18δ (AKAP18δ) in CaMKIIδ anchoring and regulation was analyzed by bioinformatics, peptide arrays, cell-permeant peptide technology, immunoprecipitations, pull-downs, transfections, immunoblotting, proximity ligation, FRET-based CaMKII activity and ELISA-based assays, whole cell and SR-vesicle fluorescence imaging, high-resolution microscopy, adenovirus transduction, adeno-associated virus injection, structural modeling, surface plasmon resonance and alpha screen technology. Results: Our results show that AKAP18δ anchors and directly regulates CaMKIIδ activity at SERCA2-PLN and RYR, via two distinct AKAP18δ regions. An N-terminal region (AKAP18δ-N) inhibited CaMKIIδ through binding of a region homologous to natural CaMKII inhibitor peptide and Thr17-PLN region. AKAP18δ-N also bound CaM, introducing a second level of control. Conversely, AKAP18δ-C, which shares homology to neuronal CaMKIIα activator peptide (N2B-s), activated CaMKIIδ by lowering the apparent a 2+ -threshold for kinase activation and inducing CaM trapping. While AKAP18δ-C facilitated faster a 2+ -reuptake by SERCA2 and a 2+ -release through RYR, AKAP18δ-N had opposite effects. We propose a model where the two unique AKAP18δ regions fine-tune a 2+ -frequency-dependent activation of CaMKIIδ at SERCA2-PLN and RYR. Conclusions: AKAP18δ anchors and functionally regulates CaMKII activity at PLN-SERCA2 and RYR, indicating a crucial role of AKAP18δ in regulation of the heartbeat. To our knowledge this is the first protein shown to enhance CaMKII activity in heart and also the first AKAP reported to anchor a CaMKII isoform, defining AKAP18δ also as a CaM-KAP.

Background— Chromogranin A (CgA) levels have previously been found to predict mortality in heart failure (HF), but currently no information is available regarding CgA processing in HF and whether the CgA fragment catestatin (CST) may directly influence cardiomyocyte function. Methods and Results— CgA processing was characterized in postinfarction HF mice and in patients with acute HF, and the functional role of CST was explored in experimental models. Myocardial biopsies from HF, but not sham-operated mice, demonstrated high molecular weight CgA bands. Deglycosylation treatment attenuated high molecular weight bands, induced a mobility shift, and increased shorter CgA fragments. Adjusting for established risk indices and biomarkers, circulating CgA levels were found to be associated with mortality in patients with acute HF, but not in patients with acute exacerbation of chronic obstructive pulmonary disease. Low CgA-to-CST conversion was also associated with increased mortality in acute HF, thus, supporting functional relevance of impaired CgA processing in cardiovascular disease. CST was identified as a direct inhibitor of CaMKIIδ (Ca 2+ /calmodulin-dependent protein kinase IIδ) activity, and CST reduced CaMKIIδ-dependent phosphorylation of phospholamban and the ryanodine receptor 2. In line with CaMKIIδ inhibition, CST reduced Ca 2+ spark and wave frequency, reduced Ca 2+ spark dimensions, increased sarcoplasmic reticulum Ca 2+ content, and augmented the magnitude and kinetics of cardiomyocyte Ca 2+ transients and contractions. Conclusions— CgA-to-CST conversion in HF is impaired because of hyperglycosylation, which is associated with clinical outcomes in acute HF. The mechanism for increased mortality may be dysregulated cardiomyocyte Ca 2+ handling because of reduced CaMKIIδ inhibition.

Background: Circulating chromogranin A (CgA) levels have been found associated with clinical outcomes in cardiovascular disease, but whether the CgA fragment catestatin (CST) may directly modulate cardiomyocyte Ca 2+ handling is not known. Methods: The prognostic utility of circulating CgA levels were compared between patients with acute HF (n=143) and chronic acute obstructive pulmonary disease (COPD, n=84). Functional effects of CST were assessed in isolated cardiomyocyte and explanted hearts. Results: CgA levels were associated with mortality in acute HF patients, but not in acute COPD. (Figure; patients stratified according to CgA quartiles on hospital admission). Admission CgA levels were also associated with mortality after adjusting for other risk factors in multivariate analysis. We found CST to interact with Ca 2+ /calmodulin (CaM)-dependent protein kinase II δ (CaMKIIδ) and to inhibit CaMKIIδ activity. CST also reduced CaMKIIδ-dependent phosphorylation of the ryanodine receptor 2 and phospholamban. In line with CaMKIIδ inhibition, CST reduced Ca 2+ spark and wave frequency, attenuated Ca 2+ sparkdimensions, increased sarcoplasmic reticulum Ca 2+ content, andaugmented magnitude and kinetics of cardiomyocyte Ca 2+ transients and contractions. Frequency dependent acceleration of relaxation was most pronounced in the Control group, an indication of CaMKIIδ activation, and this was attenuated by CST. Conclusions: CgA regulates cardiomyocyte Ca 2+ handling via CaMKIIδ inhibition, which makes CgA an interesting CV biomarker and potential compensatory mechanism in situations of enhanced CaMKIIδ activity.