Abstract 388: Inhibition of Nuclear Import of Calcineurin prevents Myocardial Hypertrophy
Introduction: Calcineurin (CnA) plays a major role in the development of myocardial hypertrophy. Beside a permanent increased Ca2+ level the phosphatase is actived by targeted proteolysis of the autoinhibitory domain (AID). The resulting consitiutive active calcineurin is translocated into the nucleus. In the following project a potential nuclear localization sequence (NLS) and a nuclear export sequence (NES) are identified and their mechanism described. Especially a completely new method for blocking CnA nuclear uptake has been found utilizing blocking peptide IBP as a NLS analogon (aa 172–183 of CnA).
Methods: Subcellular localization of CnA in normal and heart failure heart (human and rat) is illustrated by immunostainings. To identify potential NLS and NES EGFP tagged CnA mutants were transfected into neonatal rat cardiomyocytes. The CnA activity and the inhibitory effect of IBP in untreated and stimulated cardiomyocytes (AngII) were measured on the transcriptional (NF-ATc reporter assay) and the translational level (3H-Leucin incorporation). Using co-immunoprecipitation assays the importin of CnA could be identified.
Results: We demonstrated a nuclear localization of CnA in hypertrophied myocardium. All CnA mutants lacking the sequence between aa 420 – 445 are constitutive nuclear. The NES was defined as the motive LTLKGLTPTGML and Crm1 works as the adequate Exportin. The NLS was localized at the aa sequence 172–183. The constitutive nuclear localization by AngII stimulation increases the transcriptional activity of NF-ATc significantly, whereas IBP blocked this effect (227 % ± 11 vs. 133 % ± 8, p < 0.01). The same effect was demonstrated on the translational level (159 % ± 9 vs. 111 % ± 11, p < 0.01). On a cellular level, inhibition of Calcineurin nuclear import prevented myocardial hypertrophy. Finally Importin β1 was identified as the specific importin of CnA.
Conclusion: Proteolysis of the autoinhibitory domain of Calcineurin results in a constitutive activation and a permanent nuclear localization. The transport across the nuclear membran is mediated by a NLS and NES. Blocking the interaction of CnA and Importin β1 with a synthetic inhibitory peptide (IBP) suppresses nuclear entry of CnA. This prevents development of myocardial hypertrophy.