doi: 10.1113/jphysiol.2008.153577. Epub 2008 Jun 12.
Restricted N-terminal truncation of cardiac troponin T: a novel mechanism for functional adaptation to energetic crisis
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- PMID:18556368
- PMCID: PMC2538805
- DOI: 10.1113/jphysiol.2008.153577
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Restricted N-terminal truncation of cardiac troponin T: a novel mechanism for functional adaptation to energetic crisis
Han-Zhong Feng et al. J Physiol..
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Abstract
The N-terminal variable region of cardiac troponin T (TnT) is a regulatory structure that can be selectively removed during myocardial ischaemia reperfusion by mu-calpain proteolysis. Here we investigated the pathophysiological significance of this post-translational modification that removes amino acids 1-71 of cardiac TnT. Working heart preparations were employed to study rat acute myocardial infarction and transgenic mouse hearts over-expressing the N-terminal truncated cardiac TnT (cTnT-ND). Ex vivo myocardial infarction by ligation of the left anterior descending coronary artery induced heart failure and produced cTnT-ND not only in the infarct but also in remote zones, including the right ventricular free wall, indicating a whole organ response in the absence of systemic neurohumoral mechanisms. Left ventricular pressure overload in mouse working hearts produced increased cTnT-ND in both ventricles, suggesting a role of haemodynamic stress in triggering an acute whole organ proteolytic regulation. Transgenic mouse hearts in which the endogenous intact cardiac TnT was partially replaced by cTnT-ND showed lowered contractile velocity. When afterload increased from 55 mmHg to 90 mmHg, stroke volume decreased in the wild type but not in the transgenic mouse hearts. Correspondingly, the left ventricular rapid-ejection time of the transgenic mouse hearts was significantly longer than that of wild type hearts, especially at high afterload. The restricted deletion of the N-terminal variable region of cardiac troponin T demonstrates a novel mechanism by which the thin filament regulation adapts to sustain cardiac function under stress conditions.
Figures
Regional ischaemia reperfusion was produced in isolated rat working hearts by reversible ligation of the left anterior descending (LAD) coronary artery.A, the SDS-PAGE and Western blots using anti-TnT mAb CT3 on total myocardial protein extracts from perfused control, ischaemia and ischaemia–reperfusion groups demonstrated that the ischaemic area had significant production of cTnT-ND. In the ischaemia-reperfused hearts, cTnT-ND was also produced in remote zones of the myocardia, indicating anex vivo whole organ response.B, quantification of the Western blots showed that the levels of cTnT-NDversus total cTnT increased in the left ventricular ischaemic area after LAD ligation and in both left and right ventricles after LAD ischaemia–reperfusion (I-R).n = 3 in each group. **P < 0.01versus perfused controls;##P < 0.01versus right ventricle after LAD ischaemia.
A, the SDS-PAGE and mAb CT3 Western blot showed that in working heart preparations from wild type C57B/L6 mice, high left ventricular pressure load (120–145 mmHg afterload at 10 mmHg preload for 40 min) induced the production of cTnT-ND in both left and right ventricles as compared with the controls at 55 mmHg afterload and 10 mmHg preload or 55 mmHg afterload and 20 mmHg preload.B, Western blots using mAb 3G7 against the N-terminal region of cTnT showed that the cTnT fragments produced in rat and mouse hearts recognized by mAb CT3 lacks the N-terminal epitope, same as the cTnT-ND protein expressed in the transgenic mouse heart.C, densitometry quantification of the Western blots showed that left ventricular (LV) pressure overload induced increased levels of cTnT-ND in both LV and right ventricle (RV) as compared with the 55 mmHg afterload controls (*P < 0.05;n = 3 in each group).
A, SDS-PAGE and Western blot using mAb CT3 demonstrated the high level expression of cTnT-ND in the transgenic mouse hearts.B, densitometry analysis showed a proportional incorporation of cTnT-ND into the myofibrils of the transgenic mouse hearts.C, H&E staining of ventricular cross sections showed no apparent hypertrophy or myocardial degeneration in the cTnT-ND transgenic mouse hearts.
A, SDS-PAGE and Western blot using mAb CT3 demonstrated the high level expression of cTnT-ND in the transgenic mouse hearts.B, densitometry analysis showed a proportional incorporation of cTnT-ND into the myofibrils of the transgenic mouse hearts.C, H&E staining of ventricular cross sections showed no apparent hypertrophy or myocardial degeneration in the cTnT-ND transgenic mouse hearts.
When the afterload increased from 55 mmHg to 90 mmHg in mouse working experiments, the stroke volume (SV) of wild type (WT) hearts decreased nearly 25% whereas cTnT-ND transgenic mouse hearts had only a minimum decrease from the baseline level, showing a significant tolerance to the pressure overload.n = 5 for WT andn = 4 for cTnT-ND. *P < 0.05versus wild type.
A, left ventricular pressure (LVP), aortic pressure (AP) and aortic dP/dt traces simultaneously collected in working heart preparations from wild type and cTnT-ND mice at 55 mmHg and 90 mmHg afterloads are shown together using the Origin software. In each cardiac cycle, the opening and closing of aorta valve were identified by the reversing points of aortic dP/dt trace (indicated by the arrowheads). The duration between the opening and closing points represents the ventricular ejection time in which the rapid ejection phase is defined as the duration from the opening of aortic valve to the peak of left ventricular pressure and the reduced ejection phase is from the peak of left ventricular pressure to the closing of aortic valve. The representative traces showed that cTnT-ND hearts had a longer ventricular rapid ejection time in comparison with that of the wild type hearts.B, the ejection time data are summarized to show that when afterload was increased from 55 mmHg to 90 mmHg, the ejection time was shortened in both wild type and cTnT-ND hearts. However, the reduction of rapid ejection phase in cTnT-ND hearts was significantly less than that in WT hearts, demonstrating a tolerance to pressure overload.n = 5 for WT andn = 4 for cTnT-ND. Statistical significance was analysed by paired two-tailed Student'st tests except for noted. *P < 0.05versus WT control; †P < 0.05versus 55 mmHg afterload; ‡P < 0.01versus 55 mmHg afterload;#P < 0.05versus WT control (by unpaired one-tailed Student'st test).
A, when the afterload was increased from 55 mmHg to 90 mmHg, stroke work significantly increased in both wild type and cTnT-ND hearts. Although statistic significance was not established (P = 0.063 in unpaired one-tailed Student'st test), cTnT-ND hearts showed a trend of greater increase in stroke work than that of wild type hearts.B, aortic TTI as an indicator of oxygen consumption was increased when the afterload was increased with no significant difference between wild type and cTnT-ND hearts.C, cardiac efficiency calculated as stroke workversus aortic TTI showed that the decrease resulted from increasing afterload from 55 to 90 mmHg was significantly less in cTnT-ND heart than that in wild type hearts.n = 5 for wild type andn = 4 for cTnT-ND. *P < 0.05versus wild type; †P < 0.05versus 55 mmHg afterload; ‡P < 0.01versus 55 mmHg afterload (by paired Student'st test).
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