Role and differential expression of calpastatin mRNA and protein in cultured cardiomyocytes exposed to hypoxic stress.
We previously proposed that the calpain-mediated proteolytic pathway is activated in cultured cardiomyocytes following exposure to hypoxia (Mol Cell Biochem 214: 47, 2000). The potential role of calpastatin, the endogenous specific inhibitor of calpain, and its expression in the hypoxic state were investigated here. Hypoxia induced the expression of two calpastatin and multiple VEGF splice variants. Although cardiomyocytes and fibroblasts responded to hypoxia differentially, both cell types exhibited hypoxia-induced calpastatin transcription. The two functional calpastatin splice variants encoding the 593- and 654-amino acid calpastatin isoforms differed only in their N-terminal leader domain sequences. In spite of the increased mRNA expression, levels of the calpastatin protein doublet were not increased, but rather slightly decreased under the hypoxic condition. Cardiac hypoxia was accompanied by preferential proteolytic cleavage of troponin I (TnI), one of the major myofibrillar proteins. Forced expression of calpastatin through an adenoviral vector effectively prevented the hypoxia- and calpain-mediated TnI proteolysis. Our results highlight the discordant expression pattern of cardiac calpastatin mRNA and protein in the hypoxic state. We suggest that although induction of calpastatin gene transcription may constitute a compensatory mechanism coping with the hypoxic stress, a sustained high calpastatin protein level appears to be essential in the intervention of the activated calpain proteolytic cascade.
Lin
Huey
H
Department of Biochemistry and Centerfor Research in Cardiovascular Medicine, State University of New York, Buffalo, NY 14214, USA.
Risbood
Milish P
MP
Jain
Atul
A
Vacanti
Victor
V
Lee
Techung
T
D000256:Adenoviridae / Q000235:genetics; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology*; D002154:Calpain / Q000378:metabolism; D015687:Cell Hypoxia; D002478:Cells, Cultured; D018076:DNA, Complementary / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000473:pathology; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D019210:Troponin I / Q000378:metabolism
D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D012333:RNA, Messenger; D019210:Troponin I; C033668:calpastatin; D002154:Calpain