Specific degradation of troponin T and I by μ-calpain and its modulation by substrate phosphorylation

Abstract

The degradation of troponin (Tn) subunits by calpain was studied by incubating either isolated cardiac Tns or myocardial cryosections with two different calpain isoenzymes isolated from rat skeletal muscle. Western-blot analysis with monoclonal antibodies against TnI and TnT showed that mu-calpain was at least ten times more active than m-calpain in degrading TnI and TnT both in vitro and in situ. TnC was completely resistant to both proteinase forms. Phosphorylation by cyclic AMP-dependent protein kinase (PKA) isolated from rat skeletal muscle reduced the sensitivity of TnI to degradation. This effect in combination with an increased efficiency of the endogenous inhibitor [Salamino, De Tullio, Michetti, Mengotti, Melloni and Pontremoli (1994) Biochem. Biophys. Res. Commun. 199, 1326-1332] probably reduces the proteolytic activity of calpain in cells on PKA stimulation. Conversely, phosphorylation by protein kinase C (PKC) resulted in a twofold increase in the degradation of TnI. Degradation by m-calpain was not modified by Tn phosphorylation. The different sensitivity to mu-calpain might be related to changes in TnI oligomeric structure. Indeed, on PKC phosphorylation, the apparent molecular mass of TnI calculated from the distribution coefficient of Tn complex in Sephadex G-100 matrix was reduced from 90 to 30 kDa suggesting dissociation of the Tn complex.

Cardiac myosin binding protein c phosphorylation is cardioprotective

“…cMyBP-C AllPϩ was also highly resistant to the peptide cleavage associated with ischemia (17). Similar data have been obtained for PKA-mediated cardiac troponin I phosphorylation, which significantly reduced troponin I proteolysis (28). These data provide further support for the hypothesis that sarcomere-based gain of function provides long-term benefits in heart failure (29), although additional studies are needed to understand fully the role that cMyBP-C phosphorylation might play in cardioprotection.…”

Hydroxyl Radical-Induced Acute Diastolic Dysfunction Is Due to Calcium Overload via Reverse-Mode Na + -Ca 2+ Exchange

“…24 Previously, it has been shown that the calcium-dependent protease calpain I can cause different degradations of the troponin complex. 22,25 In the present work, degradation of TnT in the rabbit was prevented by MDL-28.170, a membrane-permeating inhibitor of calpain I. 18 Despite prevention of TnT-degradation with MDL-28.170, the acute diastolic dysfunction still developed.…”

“…Although from a biochemical point of view it is possible that calpain I proteolyses TnI as well as TnT, 25 the mechanisms of the species dependency remain open and are important to be addressed in future studies. However, this does not impact on the central conclusion of the present study, ie, that myofilament alterations are not involved in the acute diastolic dysfunction; in presence of calpain I this myofilament proteolysis can be inhibited, but the acute diastolic dysfunction is still present.…”

Myofilament proteins: From cardiac disorders to proteomic changes

“…Similar to concomitant phosphorylation, protein degradation can also occur simultaneously. Selective degradation of TnI and TnT by calpain was detected both in vitro [124] and in vivo after ischemia/ reperfusion [31,125]. Interestingly, phosphorylation of TnI by PKA and PKC changed its susceptibility to calpain-induced degradation conversely [124].…”

“…Selective degradation of TnI and TnT by calpain was detected both in vitro [124] and in vivo after ischemia/ reperfusion [31,125]. Interestingly, phosphorylation of TnI by PKA and PKC changed its susceptibility to calpain-induced degradation conversely [124]. As another example of communications between different types of modifications, degradation of MLC-2 and TnC after ischemia/reperfusion could be prevented by the addition of an oxygen-free radical scavenger [46].…”