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Selective deletion of the NH2-terminal variable region of cardiac troponin T in ischemia reperfusion by myofibril-associated mu-calpain cleavage - PubMed

  • ️Sun Jan 01 2006

Selective deletion of the NH2-terminal variable region of cardiac troponin T in ischemia reperfusion by myofibril-associated mu-calpain cleavage

Zhiling Zhang et al. Biochemistry. 2006.

Abstract

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.

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Figures

FIGURE 1
FIGURE 1

A cTnT fragment found in ischemia-reperfused cardiac muscle. A. While there was no detectable degradation of cTnT in rat heart after 105 min ex vivo perfusion, CT3 mAb Western blot showed that a specific cTnT fragment was produced in working rat heart preparations after ischemia-reperfusion treatment. B. Cardiac muscle samples from adult C57BL/6 mice were prepared at 0, 2, 4, and 8 hours postmortem and analyzed by SDS-PAGE and CT3 mAb Western blotting. The results showed that there was no detectable degradation of cardiac TnT due to the postmortem ischemia.

FIGURE 2
FIGURE 2

Isolation of the cTnT fragment from ischemia-reperfused mouse cardiomyocytes to determine the NH2-terminal sequence. A. The cTnT fragment was produced in isolated mouse cardiomyocytes that had undergone ischemia-reperfusion conditions. Samples from transgenic mouse hearts expressing embryonic cTnT and/or exon 7-deleted cTnT that differ from the endogenous adult cTnT in the NH2-terminal region (Figure 5) showed a cTnT fragment identical in size to that produced in the wild type adult mouse cardiomyocytes, indicating that a removal of the NH2-terminal variable region produces the cTnT fragment. B. The cTnT fragment band isolated from low cross-linker preparative SDS-PAGE gel (see Materials and Methods) was resolved on a high cross-linker SDS-gel and transferred to PVDF membrane for NH2-terminal sequencing. Amido Black staining revealed the yield of the cTnT fragment that is confirmed by mAb CT3 Western blot.

FIGURE 3
FIGURE 3

NH2-terminal truncations of cTnT. NH2-terminal amino acid sequencing of the cTnT fragment revealed a single truncation site between residues Arg71 and Leu72. The Met in bracket indicates the addition of an initiation codon for expressing cTnT-ND72-291 in E. coli (Figure 4). Amino acid sequence alignment demonstrated that the cTnT NH2-terminal truncation removes the entire variable region (Figure 5), similar to the NH2-terminal truncated fast TnT previously isolated from rabbit skeletal muscle (rabbit 26-kDa fsTnT, 32) and a model protein previously studied (Rat cTnT77-298, 16). In contrast, a caspase cleavage-produced cTnT fragment (Rat 25-kDa cTnT, 29) involves the deletion of a part of the conserved region. The number of amino acids (A.A.) and calculated molecular weights of these proteins are indicated after the sequences.

FIGURE 4
FIGURE 4

Bacterial expression of the reconstructed cTnT fragment. 5’-truncated mouse cTnT cDNA was constructed according to the NH2-terminal truncation site for protein expression in E. coli (see Materials and Methods). S/D and Tφ in the pAED4 expression vector indicate the Shine-Dalgarno ribosomal binding site and the transcription termination sequence, respectively. The cTnT fragment expressed from the truncated cDNA shows a size identical to that of the cTnT fragment produced in ischemia-reperfused cardiac muscle (the slightly slower gel mobility seen in the blot may be due to the addition of an NH2-terminal Met in the expression construct), indicating that the NH2-terminal truncation is the only primary structure modification.

FIGURE 5
FIGURE 5

Structural comparison of the NH2-terminal truncated cTnT with cTnT splicing variants. A cTnT mRNA map noted with exon boundaries is shown on the top. Protein primary structural maps of the cTnT variants studied are aligned with the coding region of the mRNA map. The NH2-terminal and adjacent regions of intact wild type adult mouse cTnT and two alternatively spliced variants are compared with the NH2-terminal truncated cTnT. The NH2-terminal truncation specifically removes of the entire variable region that is alternatively spliced to produce the adult mouse cardiac TnT (Adult cTnT-4, exclusion of exons 4 and 5), embryonic isoform (Emb cTnT-1, contains all the exons), and E7-deleted mouse cardiac TnT (E7-deleted TnT, exclusion of exons 4, 5 and exon 7). The central and COOH-terminal conserved regions of TnT contain the core functional structure that binds other thin filament regulatory proteins, TnI, TnC and tropomyosin (Tm). These protein binding sites and the portion of TnT with X-ray crystallography structure available are outlined. The NH2-terminal truncated cardiac (McTnT-ND72-291) retains the integrity of the conserved core structure, implying a functional role in myocardial ischemia-reperfusion.

FIGURE 6
FIGURE 6

Triton X-100 extraction promotes the production of the NH2-terminal truncated cTnT. Rat (A) and mouse (B) ventricular muscle strips before and after Triton X-100 extraction were examined by SDS-PAGE and Western blot using anti-TnT mAb CT3. The results revealed that Triton X-100 extraction induced the production of a specific cTnT fragment in both rat and mouse cardiac muscle samples. In the mouse model, adult, exon 7 (E7)-deleted and embryonic (Emb) cTnTs with different NH2-terminal structures produced a single fragment that has the same size as the reconstructed McTnT-ND72–291 protein, consistent with a selective NH2-terminal truncation. The production of NH2-terminal truncated cTnT (cTnT-ND) by Triton X-100 extraction suggests the role of activation of a myofibril-associated protease. The accompanying SDS-PAGE showed comparable amounts of protein loading normalized to that of actin and that other major myofibril proteins were not affected by the Triton treatment.

FIGURE 7
FIGURE 7

Calpain inhibitors reduce the production of NH2-terminal truncated cTnT by myofibril associated proteolytic activity. A. Western blots were used to examine Triton X-100 extracted exon 7 (E7)-deleted transgenic mouse ventricular muscle strips in the absence or presence of 100 μM hydrogen peroxide. Densitometric analysis showed that cTnT-ND production was reduced to 65% in the presence of hydrogen peroxide (*, P < 0.01), indicating an effect of inhibiting the activity of μ-calpain (

). B. Calpain-specific inhibitors, PD150606 and PD151746, also decreased the production of cTnT-ND in Triton X-100 extracted mouse cardiac muscle strips (to 63% and 50%, respectively, P < 0.01), further supporting the role of myofibril associated μ-calpain in the production of cTnT-ND. Data are shown as mean ± S.D. The results were summarized from the three individual experiments.
FIGURE 8
FIGURE 8

μ-Calpain treatment of cardiac myofibrils reproduced the NH2-terminal truncated cTnT fragment. A. Western blot using anti-cTnT mAb CT3 showed that μ-calpain treatment degraded purified bovine cTnT as that reported previously (

), but did not produce a specific fragment. B. In contrast, μ-calpain treatment of bovine cardiac myofibrils effectively reproduced the cTnT-ND fragment. The results demonstrate that the cTnT-ND modification by μ-calpain is dependent on the myofibril structure. Densitometry traces of the accompanying SDS-PAGE gel showed no apparent degradation of other major myofibrillar proteins, including myosin, actin and Tm, under the μ-calpain treatment conditions.
FIGURE 9
FIGURE 9

μ-Calpain production of NH2-terminal truncated cTnT suggests a restricted proteolytic modification. Densitometry analysis of multiple copies of mAb CT3 Western blots (Figure 8) determined the relative amounts of intact and NH2-terminal truncated cTnT in μ-calpain treated bovine cardiac myofibrils. The results show that intact cTnT decreased from 100% to 1.5% when the concentration of μ-calpain increased from 0 U/ml to 20 U/ml (A), while the amount of the NH2-terminal truncated cardiac TnT, cTnT ND, increased from 0% to 98% of the total cTnT (truncated plus intact) (B). The reverse exponential concentration responses of the cleavage of intact cTnT as shown by the semi-log plot inserts and the specific production of the NH2-terminal truncated cTnT by μ-calpain treatment suggest a selective and restricted proteolytic modification.

FIGURE 10
FIGURE 10

Isolation and μ-calpain treatment of bovine cardiac troponin complex. A. SDS-PAGE (15% high cross-linker gel) showed the effective isolation of troponin (Tn) complex from bovine ventricular muscle homogenate by immunoaffinity chromatography. The TnI-1 mAb affinity column fraction contained 1:1:1 ratio of TnT, TnI and TnC. Some tropomyosin (Tm) was co-isolated with the troponin complex, reflecting the native binding between troponin and tropomyosin in the thin filament. B. 14% Low cross-linker SDS-PAGE gel shows the isolated bovine troponin complex contains both of the two adult cTnT isoforms (

), indicating a native state. C. Western blot using anti-cTnT mAb 2C8 showed that μ-calpain treatment of isolated bovine cardiac troponin reproduced a single cTnT-ND fragment from the two NH2-terminal alternatively spliced cTnT isoforms. The insert table shows relative amounts of cTnT-ND produced. It is worth noting that while the troponin structure preserved the TnT core structure against calpain digestion in contrast to that in free cTnT, the protection was less effective than that in intact myofibrils (Figure 8B). Altogether, the results are consistent with the role of cTnT conformation in determining this selective structural modification under physiological conditions.
FIGURE 11
FIGURE 11

Similar NH2-terminal truncation of cardiac and fast skeletal muscle TnT by μ-calpain modification. A. Western blot using polyclonal antibody RATnT raised against chicken fast TnT and mAb 2C8 recognizing both cardiac and fast TnTs detected TnT-ND fragments (indicated by the arrowheads) in Triton X-100-treated transgenic mouse cardiac muscle strips containing endogenous cTnT and transgenic-expressed chicken fast skeletal muscle TnT (fsTnT) (

). While the blot using low cross-linker SDS-gel (upper panel) showed only one TnT fragment band, the blot using high cross-linker SDS-gel resolved two TnT fragments with distinct immunoreactivities to RATnT and 2C8 antibodies, indicating their cTnT and fast TnT origins. μ-Calpain-treated chicken breast muscle myofibrils, reconstructed mouse cTnT-ND and mouse fast TnT-ND were used as controls. B. Aligned amino acid sequences of the NH2-terminal regions of mouse cardiac and chicken fast skeletal muscle TnTs flanking the truncation sites are shown. The predicted molecular weights of the NH2-terminal truncated cTnT and fast TnT proteins are in agreement with the SDS-gel mobility of the protein fragments detected in A. The observation that cTnT and fast skeletal TnT were modified similarly by μ-calpain cleavage suggests a dependence on myofibril structure rather than the amino acid sequences at the cutting sites.
FIGURE 12
FIGURE 12

Removal of the NH2-terminal variable region preserves cTnT’s binding to TnI and tropomyosin (Tm) with altered affinities. ELISA protein binding curves demonstrate that the NH2-terminal truncated cTnT has an increased binding affinity for TnI as compared to that of intact cTnT (A). The concentrations of TnI required to reach 50% of maximum binding were 8.73±1.15 nM for cTnT ND and 15.33±1.36 nM for intact cTnT, P < 0.005. No significant difference was seen between the maximum bindings of cTnT-ND and intact cTnT to TnI (Panel A insert). The binding of cTnT-ND to α-tropomyosin also exhibits a higher affinity than that of intact cTnT (B). The concentrations of α-tropomyosin dimer for 50% maximum binding of cTnT ND and intact cTnT were 9.73±0.185 nM and 13.50±1.38 nM, respectively (P < 0.01) The level of maximum binding was not significantly changed (Panel B insert). The decrease of pH from 7.0 to 6.2 did not result in significant change in the binding of both intact and NH2-terminal truncated cTnT to TnI or α-tropomyosin.

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