Abstract 18006: Nuclear Calcineurin is a Sensor for Detecting Ca2+ Release From the Nuclear Envelope via InsP3R

Abstract

Background: Recent evidence demonstrates that not only NFAT, but also calcineurin (CN) is translocated to the nucleus upon hypertrophic stimulation. It was also shown that calpain-mediated degradation caused a constitutive active CN. We hypothesised 1) that nuclear CN is an intranuclear Ca2+ sensor, 2) that the nucleus in cardiac myocytes is a Ca2+ microdomain largely independent of cytosolic Ca2+ alterations and that 3) inhibition of nuclear translocation of CN is a therapeutic strategy to prevent hypertrophy. Methods: Employing a transgene mouse model with conditional calpastatin overexpression (resulting in calpain inhibition), different adenoviral CN mutants and confocal microscopy in isolated adult cardiac myocytes we investigated CN translocation and nuclear Ca2+ transients. Assessment of cardiac function in transgenic animals was performed by MRI. Results: We could demonstrate that chronic Ang II stimulation of mice caused calpain-mediated degradation of CN resulting in a constitutive active CN with nuclear translocation. The constitutive active CN in the nucleus escaped degradation by the UPS and sustained an ongoing hypertrophic response, even after removal of Ang II. Transgenic inhibition of calpain by calpastatin overexpression prevented proteolysis of CN and allowed for relocation of CN from the nucleus back to the cytosol and regression of hypertrophy after removal of Ang II. Inhibition of the interaction between CN and the transport protein importin ß1 by an inhibitory peptide prevented nuclear translocation of activated CN. The synthetic peptide prevented myocardial hypertrophy in vivo. We were able to demonstrate that Ang II increases nuclear Ca2+ transients via nuclear InsP3 receptors. We also showed that Ca2+ levels are constantly elevated during diastole (up to 0.2 μM) in Ang II stimulated cardiac myocytes and that CN is able to act as nuclear Ca2+ sensor detecting this local Ca2+ release from the nuclear envelope via InsP3R. Nuclear CN mutants that are defective for Ca2+ activation failed to activate NFAT dependent transcription. Conclusion: These results provide an explanation how Ca2+ and CN can regulate transcription in cardiac myocytes in response to neurohumoral signals apart from Ca2+ changes in contraction regulation.