Die Rolle des herzspezifischen Proteins CEFIP in der kardialen Hypertrophie und Kardiomyopathie

Die Z-Scheibe des Sarkomers ist ein subzellulärer Knotenpunkt in Kardiomyozyten, der sowohl für die Strukturerhaltung als auch für die Signaltransduktion eine zentrale Rolle spielt. Viele Z-Scheibenproteine konnten bereits im Zusammenhang mit genetischen Kardiomyopathien und Muskelerkrankungen beschrieben werden. Ziel dieser Arbeit war es das noch unbekannte herz- und skelettmuskelspezifische Protein CEFIP in der Entstehung von Hypertrophie zu charakterisieren. CEFIP konnte als neuer Modulator im Calcineurin-NFAT Signalweg identifiziert werden, was ein neuer Beitrag zum Verständnis der Myokardhypertrophie ist und einen möglichen neuen Ansatzpunkt für Therapien von Herzerkrankungen darstellt.The sarcomeric z-disc has been recognized as a nodal point in cardiomyocyte function and signaling. Numerous z-disc proteins have been associated with cardiomyopathies and muscle diseases. The main goal of this work was to characterize the novel heart- and skeletal muscle protein CEFIP and its role in hypertrophic signaling. CEFIP is introduced as a new modulator of the calcineurin-NFAT signaling and hypothesized to play a critical role in calcineurin-dependent hypertrophic signal transduction. These findings provide a novel insight into the pathogenesis of cardiac hypertrophy and could lead to new therapeutic approaches

Fibin regulates cardiomyocyte hypertrophy and causes protein-aggregate-associated cardiomyopathy in vivo

Despite the identification of numerous molecular pathways modulating cardiac hypertrophy its pathogenesis is not completely understood. In this study we define an unexpected role for Fibin (“fin bud initiation factor homolog”) in cardiomyocyte hypertrophy. Via gene expression profiling in hypertrophic murine hearts after transverse aortic constriction we found a significant induction of Fibin. Moreover, Fibin was upregulated in another mouse model of cardiac hypertrophy (calcineurin-transgenics) as well as in patients with dilated cardiomyopathy. Immunoflourescence microscopy revealed subcellular localization of Fibin at the sarcomeric z-disc. Overexpression of Fibin in neonatal rat ventricular cardiomyocytes revealed a strong anti-hypertrophic effect through inhibiting both, NFAT- and SRF-dependent signalling. In contrast, transgenic mice with cardiac-restricted overexpression of Fibin developed dilated cardiomyopathy, accompanied by induction of hypertrophy-associated genes. Moreover, Fibin overexpression accelerated the progression to heart failure in the presence of prohypertrophic stimuli such as pressure overload and calcineurin overexpression. Histological and ultrastructural analyses surprisingly showed large protein aggregates containing Fibin. On the molecular level, aggregate formation was accompanied by an induction of the unfolded protein response subsequent UPR-mediated apoptosis and autophagy. Taken together, we identified Fibin as a novel potent negative regulator of cardiomyocyte hypertrophy in vitro. Yet, heart-specific Fibin overexpression in vivo causes development of a protein-aggregate-associated cardiomyopathy. Because of close similarities to myofibrillar myopathies, Fibin represents a candidate gene for cardiomyopathy and Fibin transgenic mice may provide additional mechanistic insight into aggregate formation in these diseases

The JNK Inhibitor XG-102 Protects against TNBS-Induced Colitis

The c-Jun N-terminal kinase (JNK)-inhibiting peptide D-JNKI-1, syn. XG-102 was tested for its therapeutic potential in acute inflammatory bowel disease (IBD) in mice. Rectal instillation of the chemical irritant trinitrobenzene sulfonic acid (TNBS) provoked a dramatic acute inflammation in the colon of 7–9 weeks old mice. Coincident subcutaneous application of 100 µg/kg XG-102 significantly reduced the loss of body weight, rectal bleeding and diarrhoea. After 72 h, the end of the study, the colon was removed and immuno-histochemically analysed. XG-102 significantly reduced (i) pathological changes such as ulceration or crypt deformation, (ii) immune cell pathology such as infiltration and presence of CD3- and CD68-positive cells, (iii) the production of tumor necrosis factor (TNF)-α in colon tissue cultures from TNBS-treated mice, (iv) expression of Bim, Bax, FasL, p53, and activation of caspase 3, (v) complexation of JNK2 and Bim, and (vi) expression and activation of the JNK substrate and transcription factor c-Jun. A single application of subcutaneous XG-102 was at least as effective or even better depending on the outcome parameter as the daily oral application of sulfasalazine used for treatment of IBD

JNK2-Bim co-precipitation.

<p>JNK1 and JNK2 immunoprecipitates (IP) from colon tissue homogenates were analyzed by Western blotting with an anti-Bim antibody. Pounceau staining demonstrated equal loading (data not shown).</p

Representative CD3 and CD68 immunofluorescence.

<p>Representative CD3 (left) and CD68 (right) immunofluorescence of the distal colon from normal mice, trinitrobenzene sulfonic acid (TNBS) administration and treatment with sc. 100 µg/kg XG-102.</p

c-Jun and phospho-c-Jun.

<p>Western blot analysis of c-Jun and phospho-c-Jun in colon homogenates from untreated controls (co) and 12 h or 24 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Hematoxylin and eosin (H&E) scores.

<p>Hematoxylin and eosin (H&E) scores from distal (A) and medial (B) colon. For the tissue damage score (hatched bars), the scores of ulcer, crypts and submucosa were summed-up for each individual animal, and the mean±SEM was calculated for each group. The mean±SEM of the infiltration score (grey bar) is separately shown. ***, ** = p<0.001 and p<0.01 for all groups compared with TNBS group.</p

Tissue damage scores.

<p>Tissue damage scores.</p

Apoptosis.

<p>Western blot analysis of (A) caspase-3 and cleaved caspase-3, (B) Bax and Bim, and (C) FasL and p53 from colon extracts of untreated controls (co) 12 h, 24 h and 72 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Production of TNF-α.

<p>TNF-α release (pg/ml) into the supernatant of organic colon culture from normal mice, following trinitrobenzene sulfonic acid (TNBS) only, and treatment with sc. 100 µg/kg XG-102. ** = p<0.01 for all groups compared with TNBS group.</p