Role of CEFIP, a Novel Z-disc Protein, in Cardiac Hypertrophy and Cardiomyopathy
Final Report Abstract
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. By performing an „in silico“screen for novel cardiac specific cDNAs we discovered several previously unknown heart and skeletal muscle specific proteins, including a 230 kDa protein which we named CEFIP Cardiac Enriched FHL2 Interacting protein. Via quantitative real-time PCR (163-fold enrichment in heart, 384-fold in skeletal muscle) and Northern blot we could confirm the tissue-specific expression pattern. First we examined the differential expression of CEFIP in models of heart disease. CEFIP mRNA was upregulated in cardiomyopathy, as observed in muscle-LIM-protein-knockout mice (MLP-KO, 2.94 ± 0.5-fold, p<0.05) and calsarcin1-KO animals (CS1-KO, 1.93 ± 0.4-fold, p<0.001). Consistently, the expression level of CEFIP mRNA was significantly higher in samples of myocardial tissue from human patients suffering from ischemic (ICM) or dilated (DCM) cardiomyopathy compared with non-failing controls. To further characterize the role of CEFIP in cardiomyocytes we generated adenoviral constructs encoding for CEFIP and a synthetic miRNA targeting CEFIP to allow for gain and loss of functions experiments. Overexpression of CEFIP in cultivated neonatal rat cardiomyocytes led to induction of the hypertrophic gene markers such as ANF (Nppa, 2.36-fold) and BNP (Nppb, 1.63-fold). Conversely, miRNA-mediated 80 % downregulation of CEFIP did not modulate the expression pattern of these markers. In order to identify potential binding and interacting partners of CEFIP we performed a yeast two-hybrid screen. Several clones encoding FHL2 (Four and a Half Lim domains protein 2), a well-known titin and filamin binding protein, were identified. Coimmunoprecipitation and co-immunostaining confirmed the interaction between FHL2 and CEFIP. Of note, FHL2 has been found to repress pathological cardiac growth via calcineurin, a key phosphatase controlling cardiac hypertrophy. Therefore we further examined the effect by CEFIP in calcineurin signaling. Overexpression of CEFIP increased NFAT-luciferase-reporter activity with further signal enhancement in the presence of constitutively active calcineurin. Conversely, downregulation of CEFIP repressed NFAT-luciferase-reporter activity. Interestingly, simultaneous downregulation of the interacting partner FHL2 and overexpression of CEFIP at the same time did not show an additive increase of NFAT-luciferase-reporter activity. These in vitro data were published by us in 2017. Moreover, we could identify two serine phosphorylation sites of CEFIP, which are predicted targets for β-adrenoceptor kinase (βARK) and casein kinase II. Luciferase assays showed that activated phosphosite (gain-of-function) constructs led to an increase of luciferase activity in contrast to the inactivated (loss-of-function) mutants which repressed the activity to nearly basal level. These results were identical regardless, which phosphosites were modified. Given these in vitro data, a constitutive CEFIP-knockout mouse model was generated. The knockout mice developed normally until the age of 20 months and did not show cardiac dysfunction as assessed by echocardiography. Inducing hypertrophic stimuli in CEFIP deficient mice revealed that the absence of CEFIP can blunt the calcineurin-induced hypertrophic response in vivo. Surprisingly, the absence of CEFIP in mice undergoing TAC or isoproterenol stimulation was not beneficial as hypothesized. This shows that CEFIP is part of a more specific signaling cascade, which we currently still investigate. Investigating additional interacting partners will also help to understand the in vivo regulation of CEFIP. Once these data are established we plan another publication of the CEFIP knockout mouse. To summarize, the previously uncharacterized protein CEFIP is introduced as a new modulator of the NFAT-calcineurin signaling and hypothesized to play a critical role in calcineurin-dependent hypertrophic signal transduction. These findings provide insight into the pathogenesis of cardiac hypertrophy and could lead to new therapeutic approaches.
Publications
- CEFIP, a novel sarcomeric Z-disc protein, is modulated in cardiac hypertrophy and directly interacts with FHL2. Clin. Res. Cardiol.102:Suppl. 1:P1378
F. Dierck, C. Kuhn, C. Rohr, N. Frey
- (2016). The novel sarcomeric Z-disc protein CEFIP is a negative regulator of hypertrophic signaling in the heart. Clin. Res. Cardiol. 105:Suppl 1:P380
F. Dierck, C. Kuhn, C. Rohr, B. Warscheid, D. O. Fürst, N. Frey
- (2017). "The novel cardiac z-disc protein CEFIP regulates cardiomyocyte hypertrophy by modulating calcineurin signaling." J Biol Chem 292(37): 15180-15191
F. Dierck, C. Kuhn, C. Rohr, S. Hille, J. Braune, S. Sossalla, S. Molt, P. F. M. van der Ven, D. O. Fürst & N. Frey
(See online at https://doi.org/10.1074/jbc.M117.786764)
