Targeted proteolysis sustains calcineurin activation.
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.


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.
Burkard
Natalie
N
Department of Medicine, University of Wuerzburg, Wuerzburg, Germany.
Becher
Jan
J
Heindl
Cornelia
C
Neyses
Ludwig
L
Schuh
Kai
K
Ritter
Oliver
O
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D019703:Calcineurin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D006332:Cardiomegaly / Q000473:pathology; D002457:Cell Extracts / Q000737:chemistry; D002467:Cell Nucleus / Q000737:chemistry; D002478:Cells, Cultured; D004789:Enzyme Activation / Q000502:physiology; D005629:Frozen Sections; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009206:Myocardium / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism / Q000473:pathology; D032383:Myocytes, Cardiac / Q000737:chemistry / Q000166:cytology / Q000201:enzymology / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D014158:Transcription, Genetic / Q000235:genetics
D002457:Cell Extracts; D011506:Proteins; D019703:Calcineurin; D010744:Phosphoric Monoester Hydrolases; D002154:Calpain