Résumé :
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SRF (Serum Response Factor) is a MADS box transcription factor that regulates the expression of numerous genes involved in contraction, signaling or energy metabolism. Cardiac-specific Cre/loxP mediated inactivation of SRF leads to reduced contractility and dilated cardiomyopathy (DCM). Affymetrix arrays analysis showed that 2 genes encoding -integrin binding proteins were regulated in an opposite way in the SRF deficient heart: Melusin (Itgb1bp2) was decreased 5 fold and MIBP (Muscle Integrin Binding Protein, Itgb1bp3), was increased 30 fold. Melusin is required for cardiac hypertrophy, but the role of MIBP in the heart is unknown. MIBP was recognized as the homolog of human Nrk2 gene encoding a nicotinamide riboside (NR) kinase. Nrk2 is involved in the biosynthetic pathway for NAD, a crucial cofactor in energy metabolism, but also a substrate of Sirtuins deacetylases and poly-ADP-ribose polymerases. We found that MIBP accumulates in the overstretched intercalated disks that are characteristic of DCM.Our aims are: (1) to analyze the regulation of the MIBP promoter, (2) to understand the role of MIBP in DCM and NAD biosynthesis. To achieve the first task, we cloned a 600 bp fragment upstream of the TATA-box, which is conserved between several species, into a Luciferase reporter plasmid. We found that MIBP600-Luc is activated during differentiation of C2C12 cells. Site-directed mutagenesis of CArG like elements present in the promoter reduced rather than increased the activity, suggesting that MIBP induction in the SRF deficient heart is not due to a direct repressive effect of SRF. Experiments are ongoing to identify inducers of MIBP promoter. To achieve the second task, we started by measuring the concentration of NAD and the activity of Sirtuins and PARP in the SRF deficient heart. All three were reduced suggesting that NAD biosynthetic pathway is defective in the DCM. We cloned a HA-tag form of MIBP into a recombinant adenovirus and infected rat neonatal cardiomyocytes. We found that MIBP colocalize with vinculin at the level of focal adhesion sites. In addition, we demonstrated that MIBP overexpression increases the cellular NAD concentration when NR is added to the culture medium. Our results suggest that NAD biosynthesis is affected during DCM and that MIBP overexpression may be an attempt of compensation. Further research will be required to determine how to sustain NAD level in failing heart and the links between integrin signaling and NAD.
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