Selective deletion of the NH2-terminal variable region of cardiac troponin T in ischemia reperfusion by myofibril-associated mu-calpain cleavage.
The structure of the NH2-terminal region of troponin T (TnT) is hypervariable among the muscle type-specific isoforms and is also regulated by alternative RNA splicing. This region does not contain binding sites for other thin filament proteins, but alteration of its structure affects the Ca2+ regulation of muscle contraction. Here we report a truncated cardiac TnT produced during myocardial ischemia reperfusion. Amino acid sequencing and protein fragment reconstruction determined that it is generated by a posttranslational modification selectively removing the NH2-terminal variable region and preserving the conserved core structure of TnT. Triton X-100 extraction of cardiac muscle fibers promoted production of the NH2-terminal truncated cardiac TnT (cTnT-ND), indicating a myofibril-associated proteolytic activity. Mu-calpain is a myofibril-associated protease and is known to degrade TnT. Supporting a role of mu-calpain in producing cTnT-ND in myocardial ischemia reperfusion, calpain inhibitors decreased the level of cTnT-ND in Triton-extracted myofibrils. Mu-calpain treatment of the cardiac myofibril and troponin complex specifically reproduced cTnT-ND. In contrast, mu-calpain treatment of isolated cardiac TnT resulted in nonspecific degradation, suggesting that this structural modification is relevant to physiological structures of the myofilament. Triton X-100 treatment of transgenic mouse cardiac myofibrils overexpressing fast skeletal muscle TnT produced similar NH2-terminal truncations of the endogenous and exogenous TnT, despite different amino acid sequences at the cleavage site. With the functional consequences of removing the NH2-terminal variable region of TnT, the mu-calpain-mediated proteolytic modification of TnT may act as an acute mechanism to adjust muscle contractility under stress conditions.
Zhang
Zhiling
Z
Section of Molecular Cardiology, Evanston Northwestern Healthcare and Northwestern University Feinberg School of Medicine, Evanston, Illinois 60201, USA.
Biesiadecki
Brandon J
BJ
Jin
Jian-Ping
JP
D000595:Amino Acid Sequence; D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D066298:In Vitro Techniques; D051379:Mice; D008969:Molecular Sequence Data; D018656:Muscle Fibers, Fast-Twitch / Q000187:drug effects; D009206:Myocardium / Q000378:metabolism; D009210:Myofibrils / Q000201:enzymology*; D017830:Octoxynol / Q000494:pharmacology; D010447:Peptide Hydrolases / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D015427:Reperfusion Injury / Q000139:chemically induced / Q000378:metabolism*; D017384:Sequence Deletion; D014335:Tropomyosin / Q000378:metabolism; D019210:Troponin I / Q000378:metabolism; D020107:Troponin T / Q000737:chemistry* / Q000235:genetics
D014335:Tropomyosin; D019210:Troponin I; D020107:Troponin T; D017830:Octoxynol; D010447:Peptide Hydrolases; D002154:Calpain; C449002:mu-calpain