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(Circulation. 2005;111:1045-1053.)
© 2005 American Heart Association, Inc.

Molecular Cardiology

Targeted Proteolysis Sustains Calcineurin Activation

Natalie Burkard, MSc; Jan Becher; Cornelia Heindl, TA; Ludwig Neyses, MD; Kai Schuh, PhD; Oliver Ritter, MD

From the Department of Medicine (N.B., J.B., C.H., O.R.) and Institute of Clinical Biochemistry and Pathobiochemistry (K.S.), University of Wuerzburg, Wuerzburg, Germany, and University Department of Medicine, Manchester Royal Infirmary, Manchester, UK (L.N.).

Correspondence to Oliver Ritter, MD, Department of Medicine, University of Wuerzburg, Josef Schneider Str 2, 97080 Wuerzburg, Germany. E-mail Ritter_O{at}klinik.uni-wuerzburg.de

Received April 1, 2004; revision received October 11, 2004; accepted October 15, 2004.

Background— Calcineurin (CnA) is important in the regulation of myocardial hypertrophy. We demonstrated that targeted proteolysis of the CnA autoinhibitory domain under pathological myocardial workload leads to increased CnA activity in human myocardium. Here, we investigated the proteolytic mechanism leading to activation of CnA.

Methods and Results— In patients with diseased myocardium, we found strong nuclear translocation of CnA. In contrast, in normal human myocardium, there was a cytosolic distribution of CnA. Stimulation of rat cardiomyocytes with angiotensin (Ang) II increased calpain activity significantly (433±11%; P<0.01; n=6) and caused proteolysis of the autoinhibitory domain of CnA. Inhibition of calpain by a membrane-permeable calpain inhibitor prevented proteolysis. We identified the cleavage site of calpain in the human CnA sequence at amino acid 424. CnA activity was increased after Ang II stimulation (310±29%; P<0.01; n=6) and remained high after removal of Ang II (214±17%; P<0.01; n=6). Addition of a calpain inhibitor to the medium decreased CnA activity (110±19%; P=NS; n=6) after removal of Ang II. Ang II stimulation of cardiomyocytes also translocated CnA into the nucleus as demonstrated by immunohistochemical staining and transfection assays with GFP-tagged CnA. Calpain inhibition and therefore suppression of calpain-mediated proteolysis of CnA enabled CnA exit from the nucleus.

Conclusions— Ang II stimulation of cardiomyocytes increased calpain activity, leading to proteolysis of the autoinhibitory domain of CnA. This causes an increase in CnA activity and results in nuclear translocation of CnA. Loss of the autoinhibitory domain renders CnA constitutively nuclear and active, even after removal of the hypertrophic stimulus.

Key Words: calcium • myocardium • hypertrophy • molecular biology

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