This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 99/9/1012    most recent 01.RES.0000248753.30340.afv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Narolska, N. A. Articles by Stienen, G. J.M. Search for Related Content PubMed PubMed Citation Articles by Narolska, N. A. Articles by Stienen, G. J.M. Related Collections Contractile function Heart failure - basic studies Ischemic biology - basic studies

(Circulation Research. 2006;99:1012.)
© 2006 American Heart Association, Inc.

Integrative Physiology

Impaired Diastolic Function After Exchange of Endogenous Troponin I With C-Terminal Truncated Troponin I in Human Cardiac Muscle

Nadiya A. Narolska, Nicoletta Piroddi, Alexandra Belus, Nicky M. Boontje, Beatrice Scellini, Sascha Deppermann, Ruud Zaremba, Rene J. Musters, Cris dos Remedios, Kornelia Jaquet, D. Brian Foster, Anne M. Murphy, Jennifer E. van Eyk, Chiara Tesi, Corrado Poggesi, Jolanda van der Velden, Ger J.M. Stienen

From the Laboratory for Physiology (N.A.N., N.M.B., R.Z., R.J.M., J.v.d.V., G.J.M.S.), Institute for Cardiovascular Research, VU Medical Center, Amsterdam, the Netherlands; Dipartimento di Scienze Fisiologiche (N.P., A.B., B.S., C.T., C.P.), Università di Firenze, Italy; Research Laboratory of Molecular Cardiology (S.D., K.J.), Bergmannsheil/St. Josef-Hospital, Medical School of the Ruhr–University of Bochum, Germany; Muscle Research Unit (C.d.R.), Institute for Biomedical Research, The University of Sydney, Australia; and Department of Medicine (D.B.F., A.M.M., J.E.v.E.), Johns Hopkins University, Baltimore, Md.

Correspondence to G. J. M. Stienen, PhD, Laboratory for Physiology, VU University Medical Center, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. E-mail g.stienen{at}vumc.nl

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI_1–192) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 µm, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic ß-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI_1–192 in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca²⁺ sensitivity of the contractile apparatus was increased in preparations containing cTnI_1–192 (pCa₅₀: 5.73±0.03 versus 5.52±0.03 for cTnI_1–192 and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI_1–192-exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.

Key Words: cardiac function • contractility • cardiomyocytes • cardiomyopathy • ischemia

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Ischemic myocardial disease is manifested in a mild form of reversible contractile dysfunction and a very serious, irreversible form (necrosis). The latter may be prevented only by timely restoration of coronary perfusion, but, even then, reversible contractile dysfunction is frequently apparent. The specific and selective proteolysis of the myofilament protein cardiac troponin I (cTnI) has been proposed to play a key role in human myocardial ischemia/reperfusion injury, including stunning^1–3 and in acute pressure overload.⁴ cTnI is part of the cardiac troponin (cTn) complex that, in concert with tropomyosin (TM), regulates muscle contraction in response to a rise in intracellular Ca²⁺. The troponin complex consists of 3 subunits: troponin C (cTnC), the Ca²⁺ binding protein; cTnI, which inhibits the actin/myosin interaction; and troponin T (cTnT), which transduces the Ca²⁺ binding signal to TM. On Ca²⁺ binding to the regulatory site of cTnC, the C-terminal regulatory segment of cTnI (residues 137 to 210) moves away from the actin filament,^5,6 thereby altering the orientation and/or flexibility of cTn and TM relative to the actin filament.⁵ Hence, cTnI plays a pivotal role in activation of cardiac myofilaments, and truncation of the cTnI C terminus, as observed in human ischemic cardiac disease, might alter the Ca²⁺-induced force development as well as the inhibition of force development at low Ca²⁺ concentrations.

In rodents, McDonough et al⁷ showed that with moderate ischemia/reperfusion, cTnI is cleaved at its C terminus, resulting in a degraded subunit cTnI_1–193. Ischemia-induced cTnI degradation has been proposed to be attributable to activation of the Ca²⁺-dependent proteolytic enzyme calpain-1 during Ca²⁺ overload that occurs during reperfusion.⁸ Increased preload in the absence of ischemia has also been shown to induce cTnI degradation.⁴ In larger mammalian models, such as a postinfarct pig⁹ and dog¹⁰ model, minor degradation of cTnI (<4%) was observed in the viable remodeled left ventricle. Whereas in the dog, cTnI degradation did not correlate with the in vivo cardiac dysfunction in the whole heart,¹⁰ in the pig, the maximal force generating capacity of the individual myofilaments was reduced compared with myocardium of sham-operated animals.⁹ In advanced human heart failure, serum cTnI elevations have been reported.¹¹ In addition, cTnI degradation has been found in human cardiac tissue from coronary artery disease patients with different degrees of heart failure and a history of myocardial infarction.^1–3 The primary cTnI degradation product found in these hearts was identified as cTnI_1–192 (equivalent to cTnI_1–193 in rodent myocardium⁷). Hence, also in humans, C-terminal truncation of cTnI may underlie impaired cardiac function in ischemia-induced cardiomyopathy and heart failure. However, the direct effects of cTnI proteolysis on contractile function of human cardiac myofilaments and isolated cardiac myocytes have not been investigated.

The contractile apparatus responds quickly to increases in hemodynamic load (via the Frank–Starling mechanism) and to ß-adrenergic stimulation, resulting in an increase and decrease in myofilament Ca²⁺ sensitivity, respectively. In failing hearts, both processes are blunted. Evidence suggests^12,13 that cTnI plays an important role in these regulatory processes, by modulating length-dependent activation and by its phosphorylation through protein kinase A (PKA) of Ser23/24 in the N terminus.⁶ For instance, it has been shown¹⁴ that transduction of the PKA-induced phosphorylation signal from cTnI to the regulatory site of cTnC involves a global change in cTnI structure. Hence it is of interest to study whether or not the changes in Ca²⁺ sensitivity associated with the Frank–Starling mechanism and ß-adrenergic stimulation are perturbed by C-terminal truncation of cTnI in human isolated myocytes and myofibrils.

In this study, protein engineering was combined with functional measurements to assess the direct effects of degradation of the cTnI C terminus in human myocardium. Force measurements were performed in human cardiac myofibrils and cardiomyocytes, in which endogenous troponin complexes were partly replaced by complexes, denoted by cTn_FL and cTn_1–192, containing human full-length cTnI or truncated cTnI_1–192, respectively. The advantage of this technique is that it allows to investigate the direct effect of cTnI degradation on contractility of human cardiac preparations without degradation of other contractile proteins (as eg, follows calpain-1 treatment)^15,16 or other compensatory protein changes, which may develop in transgenic animals.

Force development at maximal and submaximal [Ca²⁺] and kinetic parameters of force activation and relaxation in preparations containing cTn_1–192 were compared with those containing cTn_FL. In the presence of cTn_1–192, maximal isometric force, length-dependent activation and the effect of PKA were preserved, whereas Ca²⁺ sensitivity was increased and the kinetics of activation and relaxation of human cardiac preparations were slowed. These results indicate that C-terminal truncation of cTnI mainly impairs diastolic properties of human myocardium, whereas the maximum force generating capacity, the Frank–Starling effect and the ß-adrenergic responsiveness are preserved.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
An expanded Materials and Methods section can be found in the online data supplement, available at http://circres.ahajournals.org.

Preparation of Recombinant Troponin Complexes
The human cardiac full-length cTnI and truncated cTnI (cTnI_1–192) were prepared as described previously.¹⁷

Isolation of Human Myofibrils and Cardiomyocytes
Left ventricular tissue was obtained from healthy donor hearts (n=4). Samples were obtained with approval of the local ethical committees and after informed written consent. Single myofibrils, bundles of a few myofibrils or single cardiac myocytes were prepared from these biopsies, as described previously.^18,19

Exchange of Human Cardiac Troponin Complex
Exchange of human cTn complex into human myofibrils and myocytes was performed according previously published methods.^18,20 Evaluation of both cTn_FL and cTn_1–192 exchange into human cardiac preparations was based on the amount of cTnI_1–192 exchange.

Force Measurements in Exchanged Human Myofibrils and Cardiomyocytes
The protocols used for rapid solution changes and to record force in single myofibrils and cardiomyocytes were as described previously.^18,19,21,22

Microscopy
To determine whether the cTn complex was homogeneously incorporated into cardiomyocytes, cTn_1–192-exchanged cardiomyocytes were studied using a Marianas digital imaging microscopy workstation.

Data Analysis
Values are given as means±SEM of n myofibrils or myocytes. Differences between preparations with cTn_1–192 and cTn_FL were tested by means of unpaired 2-tailed Student’s t tests at a level of significance of 0.05. The effects of sarcomere length and PKA treatment were tested with paired Student’s t tests.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Amount and Distribution of Troponin Complex Exchange
Figure 1A shows an example of a gel used for the calculation of the amount of exchange in cardiac myofibrils. Replacement of the endogenous troponin complex with cTn_1–192 amounted to 54±6% (n=4) in human cardiac myofibrils and to 50±2% (n=5) in the isolated human cardiomyocytes. These results confirmed by anti-cTnI Western blot analysis (Figure 1B for myofibrils and Figure 1C for cardiomyocytes).

View larger version (28K): [in this window] [in a new window]   Figure 1. A, One-dimensional SDS-PAGE of suspensions of human myofibrils after the exchange procedure with full-length cTnI (cTnIFL), degraded (cTnI1–192), or without exogenous cTn complex (control condition [CTR]). B, Subsequent western immunoblot of a SDS-PAGE gel from human myofibrils using an antibody recognizing the full-length (cTnI) as well as the truncated (cTnI1–192) form. C, Western immunoblot of cTnI in exchanged human cardiomyocytes. D, Image obtained by summation of plane intensities along the z-axis of a single cardiomyocyte exchanged with cTn1–192, incubated with an antibody directed against C terminus of cTnI. Scale bar=10 µm. E, Distribution of the fluorescence intensity within the cardiomyocyte. Planes were acquired along the z-axis (in depth) from the top of the myocyte (plane 1) to its bottom (plane 31). F, Distribution of the fluorescence intensity along the vertical (y-axis) of the cardiomyocyte in its central part (planes 11 to 21). MW indicates molecular weight marker.

To monitor the cTn distribution in cardiomyocytes following the exchange, a novel method was used in which human cardiomyocytes exchanged with cTn_1–192 were stained with a specific antibody P45-3 and studied using 3D digital imaging microscopy. P45-3 reacts only with full-length cTnI and not with truncated cTnI_1–192. Thus, staining with this antibody will reveal any inhomogeneity of cTn_1–192 exchange into the cardiomyocytes. A summation in the z-axis of the x-y images of a representative cardiomyocyte is shown in Figure 1D. Fluorescence intensity was averaged per xy plane and plotted against plane number in Figure 1E. This panel illustrates that with overnight incubation, the penetration of cTn into the cardiomyocyte is not limited by diffusion, because in that case, a parabolic intensity profile would be expected with a minimum in the core of the preparation. The fluorescence intensity distribution in the y-axis direction (Figure 1F) in the central part of the myocyte also reveals a rather homogeneous distribution of cTn complex within the exchanged cardiomyocyte. Three-dimensional image reconstruction performed in 3 typical cTn_1–192-exchanged cardiomyocytes showed similar results. This indicates that the protocol used ensures a uniform exchange of cTn_1–192 inside the cardiomyocytes (and in the much thinner myofibrils, where diffusion would be even less critical).

Force Measurements in Exchanged Human Myofibrils
Representative recordings of force production in myofibrils at saturating [Ca²⁺] are shown in Figure 2. Maximal isometric tension (ie, force at pCa 3.5 divided by cross-sectional area) was not significantly different between cTn_FL-and cTn_1–192-exchanged myofibrils (Figure 3A). However, the rate of tension rise (k_ACT) and the rate of force redevelopment (k_TR) were both significantly decreased in cTn_1–192-exchanged myofibrils compared with cTn_FL-exchanged myofibrils by 24% and 31%, respectively (P<0.05) (Figure 3B).

View larger version (22K): [in this window] [in a new window]   Figure 2. A, Image of a thin bundle of cTnFL-exchanged human cardiac myofibrils length between the attachments 40 µm. B, Representative traces of tension activation. C, Relaxation in cTnFL- and cTn1–192-exchanged human cardiac myofibrils. After the myofibril was transferred into activating solution (pCa 3.5) and active force developed, the myofibril was shortened by 20% of its length. Relaxation kinetics were obtained after return of the myofibril into relaxing solution (pCa 8).

View larger version (20K): [in this window] [in a new window]   Figure 3. A, Mean values of maximal isometric force. B, Rate of force development. C, Duration of relaxation slow phase. D, Rate of relaxation of cTnFL- or cTn1–192-exchanged myofibrils. Maximal force: kACT and kTR were obtained at maximal [Ca2+] (pCa 3.5). Duration: (tSLOW), kREL,S and kREL,F were obtained at pCa 8. Values are given as mean±SEM of 22 cTnFL- and 25 cTn1–192-exchanged myofibrils. *Significant at P<0.05, cTnFL- vs cTn1–192-exchanged myofibrils.

After a rapid switch from activating (pCa 3.5) to relaxing (pCa 9) solution, isometric force started to decline (Figure 2). Force relaxation takes place in 2 phases: a slow initial linear phase, with a rate constant k_REL,S calculated from its slope and duration t_SLOW; and a rapid exponential phase characterized by the rate constant k_REL,F (Figure 3C).²² The maximum force and kinetic parameters in cTn_FL-exchanged myofibrils did not differ significantly from the values obtained in untreated myofibrils. However, compared with cTn_FL-exchanged myofibrils, the duration of the slow phase t_SLOW was significantly increased in cTn_1–192-exchanged myofibrils (Figure 3D), whereas k_REL,S remained unchanged (Figure 3C). The k_REL,F significantly decreased in cTn_1–192-exchanged myofibrils (Figure 3C). An overview of averaged data are presented in Table 1.

View this table: [in this window] [in a new window]   Table 1. Functional Data of Human Myofibrils

Force Measurements in Exchanged Human Cardiomyocytes
The maximal isometric tension and its Ca²⁺ sensitivity could be determined more accurately in cardiomyocytes than in myofibrils, because the cross-sectional area could be determined with a higher precision in cardiomyocytes than in myofibrils (Figure 4A). Representative force recordings of cTn_FL- and cTn_1–192-exchanged myocytes are presented in Figure 4B. Maximal isometric tension and mean passive tension were not significantly different between cTn_FL- and cTn_1–192-exchanged myofibrils (Figure 5A). However, cTn_1–192 cardiomyocytes had a significantly higher Ca²⁺ sensitivity than cTn_FL-exchanged cardiomyocytes (pCa₅₀: 5.79±0.02 and 5.54±0.02 for cTn_1–192 and cTn_FL, respectively; P<0.05) (Figure 5B). Moreover, the cooperativity of force development (nH) was significantly decreased in cTn_1–192-exchanged cardiomyocytes (1.95±0.06 and 2.95±0.14 for cTn_1–192 and cTn_FL, respectively; P<0.05).

View larger version (45K): [in this window] [in a new window]   Figure 4. A, Images of single cardiomyocytes after exchange with cTnFL and cTn1–192. Scale bar=20 µm. B, Force recordings in cardiomyocytes with cTnFL and cTn1–192 at pCa 4.5. During activation, the myocyte was shortened rapidly to determine the baseline of the force transducer. After return into the relaxing solution (pCa 9), the myocyte was shortened again for 10 s to determine passive force.

View larger version (11K): [in this window] [in a new window]   Figure 5. A, Mean values of maximal isometric force (Fmax) and passive force (Fpas) with cTnFL and cTn1–192 obtained at saturating [Ca2+] (pCa 4.5) and at low [Ca2+] (pCa 9), respectively. B, The Ca2+ sensitivity of isometric force with cTnFL and cTn1–192. Isometric force at submaximal activating [Ca2+] was normalized to control force at saturating [Ca2+]. Ca2+ sensitivity with cTn1–192 was significantly higher than with cTnFL. Values are given as mean of 12 cardiomyocytes with cTnFL and 15 with cTn1–192. Error bars indicate SEM.

Sarcomere Length Dependence
To investigate the effect of cTnI proteolysis on the Frank–Starling mechanism, force development and its Ca²⁺ sensitivity was studied in cardiomyocytes after exchange. Measurements were performed near slack length, at a sarcomere length of 1.9 µm and after adjustment of sarcomere length to 2.2 µm. As expected on the basis of the sarcomere length/tension relation, maximum force increased for both cTn_FL and cTn_1–192 cardiomyocytes. Passive tension increased as well. Ca²⁺ sensitivity also increased with sarcomere length (Figure 6A), but the steepness of the force/pCa relation (nH) remained the same. The mean shifts in Ca²⁺ sensitivity (pCa₅₀) were very similar and amounted to 0.10±0.02 and 0.14±0.02 for cTn_1–192 and cTn_FL, respectively (P=0.2). An overview of averaged data are presented in Table 2.

View larger version (22K): [in this window] [in a new window]   Figure 6. A, Ca2+ sensitivity of isometric force of cardiomyocytes with cTnFL and with cTn1–192 at 1.9-µm (open symbols) and 2.2-µm (closed symbols) sarcomere length. Isometric force at submaximal activating [Ca2+] was normalized to the control force found at saturating [Ca2+] (pCa 4.5). In both cases, Ca2+ sensitivity at 2.2 µm was significantly higher than at 1.9 µm. Values are given as mean of 10 cardiomyocytes with cTnFL and 12 with cTn1–192. B, Ca2+ sensitivity of isometric force of cardiomyocytes with cTnFL and with cTn1–192 before (open symbols) and after (closed symbols) PKA treatment, determined at a sarcomere length of 2.2 µm. Isometric force at submaximal activating [Ca2+] was normalized to the control force at saturating [Ca2+] (pCa 4.5). In both cases, Ca2+ sensitivity after PKA treatment was significantly reduced. Values are given as mean of 10 cardiomyocytes with cTnFL and 13 with cTn1–192. Error bars indicate SEM.

View this table: [in this window] [in a new window]   Table 2. Functional Data of Exchanged Human Cardiomyocytes

Effects of PKA
To assess the effect of PKA phosphorylation of cTnI and its truncated fragment, cardiomyocytes after cTn exchange were incubated with the catalytic subunit of protein kinase A. Addition of PKA resulted in a decrease in Ca²⁺ sensitivity and, for cTn_1–192 cardiomyocytes, in a small but significant increase in steepness of the force/pCa relation (Figure 6B). The mean shifts in Ca²⁺ sensitivity (pCa₅₀) were very similar and amounted to 0.08±0.01 and 0.09±0.01 for cTn_1–192 and cTn_FL, respectively (P=0.5). Maximum forces were not influenced by PKA. Mean passive forces were slightly reduced after PKA treatment, but the differences were not significant. An overview of the averaged data are included in Table 2.

Comparison Between Properties of cTn_FL-Exchanged Cardiomyocytes and Untreated Controls
Measurements were also performed in untreated cardiomyocytes from the same donors as used for the exchange experiments. The maximum force in untreated cardiomyocytes 26.9±3.2 kN/m² (n=4) did not differ significantly from the value in cTn_FL-exchanged cardiomyocytes: 31.5±3.2 kN/m² (n=12; Figure 5B), whereas the pCa₅₀ values observed were consistent with the observed difference in cTnI phosphorylation (online data supplement), ie, Ca²⁺ responsiveness was significantly higher in exchanged preparations containing dephosphorylated full-length cTnI (pCa₅₀=5.54±0.02) than in untreated cardiomyocytes (pCa₅₀=5.48±0.01) in which the amount of dephosphorylated cTnI was lower.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
This study describes the first exchange experiments in human cardiac preparations, in which the direct effects of cTnI degradation are assessed. The results obtained indicate that (1) cTnI degradation at the C terminus enhances myofilament Ca²⁺ sensitivity and slows force development as well as its relaxation, whereas (2) maximal isometric force development, passive force, length-dependent activation (the Frank–Starling effect), and the responsiveness to ß-adrenergic stimulation appear to be preserved.

Comparison With Previous Studies on cTnI_1–192 Function
The decrease in maximal force generating capacity of cardiac muscle is among the main features of reversible ischemia/reperfusion injury (stunning) in rodents and has been attributed to degradation of cTnI, primarily at its C terminus.^7,8,23 In a transgenic mouse line expressing 9% to 17% of truncated human cTnI,^1,24 maximal force development of intact trabeculae was approximately 40% depressed compared with nontransgenic mice. In view of these previous findings, it is surprising that in our study, the maximum force was not affected in human preparations containing truncated cTnI. However, it should be noted that the processes involved in stunning differ in small and large animals.²⁵ The reduction in force in animal models could, at least in part, also be attributable to an additional degradation or modification of other, ultrastructural proteins¹⁵ or to secondary reactive oxygen species–related effects.²⁶ Moreover, other compensatory protein changes, which may develop in transgenic animals, and species differences may be involved as well.

An in vitro motility assay, with reconstituted actin filaments attached to anchoring -actinin, showed a 24% decrease in an index of force development of filaments consisting of 100% human cTnI_1–192, compared with filaments with full-length cTnI.¹⁷ This reduction probably would have been less with partial replacement of human cTnI_1–192, as was the case in our experiments. Exchange of the human cTn_1–192 complex into rabbit psoas myofibrils under the same experimental conditions as used in this study, revealed a 26% decrease in maximal force development in comparison with cTn_FL preparations.²⁷ A similar reduction of maximal tension was observed by Tachampa and coworkers,^28,29 where the endogenous rat cTn complexes were partly exchanged with truncated human or mice cTn. The main difference in these exchange experiments is that chimerical thin filaments were formed, whereas in the present experiments human cardiac proteins were exchanged into human cardiac tissue. It is conceivable that, despite 95% identity of the human, mice, rat, and rabbit cTn amino acid sequences, signal transduction and transmission between troponin subunits and other contractile proteins is isoform and species dependent. This might result in diverse functional changes on cTnI_1–192 exchange in matching or nonmatching species, but further experiments are required to test this hypothesis.

In viable remote remodeled tissue after infarction in pigs, minor degradation of cTnI (<4%) was observed, whereas the maximal force-generating capacity was reduced.⁹ If our findings in humans are characteristic for the situation in large animal models, this would suggest that other factors than cTnI degradation would be responsible for this loss in force.

The results on kinetics and Ca²⁺ sensitivity observed in the present study are in line with those found in exchange experiments using rabbit psoas myofibrils and rat cardiac trabeculae.^27–29 This indicates that the impact of cTnI degradation on regulatory and kinetic aspects of contractile function in rodent and human tissue are shared. However, caution should be exerted with the interpretation of these findings within the context of stunning. For instance Gao and coworkers^30,31 have observed an increase in k_TR and also provided evidence suggesting that the rate-limiting step of the transition from force-generating to non–force-generating states was accelerated in stunned myocardium. Moreover, the direction of the change in the maximum shortening velocity in stunned myocardium is unclear.³²

Structural and Biochemical Considerations on cTnI_1–192 Function
Previous studies indicate that the presence of cTn and TM enhance force development and also influence kinetic parameters of cross-bridge interaction.^33–35 This suggests that cTnI degradation may affect the maximum force-generating capacity as well as the kinetics of activation and relaxation of force. However, if steric hindrance of cross-bridge interaction by the C-terminal end of cTnI would be important, as suggested previously to explain the increase in ATPase activity,¹⁷ its truncation would tend to increase the number of interacting cross-bridges. This would result in an increase in force rather than in a decrease.

The increase in Ca²⁺ sensitivity observed can be caused by (1) an increase in Ca²⁺ affinity of cTnC, which promotes the transition from blocked to closed state; and/or (2) enhancement of strong myosin binding to actin, the transition from closed to open state.^36,37 It has been shown using various truncated forms of cTnI that progressive deletion of parts of the C terminus impairs the ability of cTnI to bind to actin/tropomyosin at low [Ca²⁺], promoting the availability of actin for binding with myosin.³⁸ This is consistent with the increase in Ca²⁺ sensitivity observed in our experiments. The nH of cardiomyocytes exchanged with cTn_1–192 was significantly lower than that of cTn_FL-exchanged cardiomyocytes. This may reflect a decrease in cooperativity of thin filament activation, in agreement with the destabilization of TM on actin, but it could also be caused by the increase in Ca²⁺ sensitivity because Ca²⁺ activation of force becomes more dependent on Ca²⁺ binding to troponin C, which is a relatively noncooperative process.

Both k_ACT and k_TR of the cTnI_1–192-exchanged cardiomyocytes were significantly decreased. Evidence suggests that k_ACT and k_TR are mainly determined by the kinetics of cross-bridge attachment.²¹ The parallel changes in k_ACT and k_TR observed is these experiments are most likely attributable to cooperative changes within the thin filament that lead to the transition to the open state. Thus, the decreased cooperativity of thin filament regulation may play a role in the slowing of the activation kinetics.

It has been shown that during the first slow linear phase of relaxation, all sarcomeres remain isometric, whereas the fast relaxation phase starts when the first mechanically weakest sarcomere "gives."^22,39 The slow k_REL,S is determined largely by the isoform of myosin heavy chain.²² This may explain the lack of a significant effect of cTnI degradation on this parameter in our experiments. The duration of the slow phase t_SLOW was, however, increased in cTn_1–192-exchanged preparations, ie, sarcomeres remain isometric for a longer time during relaxation. This might be a consequence of persistent enhanced activation caused by the increased Ca²⁺ sensitivity. Moreover, it has been shown that the fast k_REL,F depends inversely on the final steady-state force after relaxation.²¹ Because cTnI_1–192 incorporation into myofilaments increases Ca²⁺ sensitivity, the force during relaxation is larger and accordingly the fast k_REL,F will be decreased. Thus, changes in both t_SLOW and fast k_REL,F may be linked to the increased Ca²⁺ sensitivity.

In a simple 2-state model for cross-bridge interaction the maximum isometric force is proportional to the number of cross-bridges in the force generating state (N_att), ie, f/(f+g) and k_TR=f+g, where f equals the rate of cross-bridge attachment and g equals the rate of cross-bridge detachment.²¹ If we assume that at saturating Ca²⁺ concentrations g=k_REL,S, N_att amounts to 0.6, both for cTn_1–192 and cTnI_FL. Hence, the kinetic values obtained are consistent with the preservation of the maximum force-generating capacity observed.

The reduction in the kinetic parameters cannot be reconciled easily with the increase in ATPase rate observed earlier.¹⁷ However, it should be noted that the ATPase rate reported characterizes the activity during unloaded shortening, whereas our observations characterize the isometric steady state.

Modulation of Contractile Function
Evidence suggests that cTnI, the N-terminal region in particular, is involved in length-dependent activation (the Frank–Starling mechanism).⁴⁰ Our results indicate that the sarcomere length dependency of maximum force development and its Ca²⁺ sensitivity in cTnI_1–192-containing cardiomyocytes were very similar to those containing full-length cTnI. The location of the putative length sensor, the mechanism underlying the Frank–Starling mechanism as well as the involvement of other proteins such as titin, myosin-binding protein C, and myosin light chain 2 still need to be addressed in human myocardium. However, our data suggest that the C-terminal truncated part of cTnI is not essential for signal transmission of length-dependent activation.

The effects of ß-adrenergic stimulation were mimicked using PKA. In a previous study, it has been shown that the PKA sites of the endogenous cTnI in donor tissue were almost completely phosphorylated and that cTnT was partially monophosphorylated (60%).⁴¹ These endogenous complexes were partly exchanged in our experiments with dephosphorylated cTnI (and cTnT). Consistent with these earlier results, the shift in pCa₅₀ on PKA treatment was intermediate between the shift observed in donor and end-stage failing heart tissue.⁴¹ However, because treatment of both complexes was identical, the overall phosphorylation levels will be the same, as was confirmed by the Western blots shown in the online data supplement. Hence, our experiments revealed that the desensitizing effect of PKA was preserved in cTnI_1–192-containing cardiomyocytes. This indicates that the transduction of the PKA-induced phosphorylation signal from cTnI to the regulatory site of cTnC is preserved.

Recently, Metzger and colleagues⁴² provided evidence that replacement of alanine with histidine at position 163 in human cTnI may represent a therapeutic avenue to improve myocardial performance in the ischemic and failing heart. Because alanine 163 is within the region retained after cTnI proteolysis and our data show that the regulatory aspects of cTnI_1–192 are preserved, the engineering of the histidine button may be protective also in the presence of degraded cTnI.

Implications for Cardiac Function In Vivo
The amount of cTn exchange into cardiac preparations in our experiments was approximately 50%, whereas the degree of cTnI degradation observed in failing human myocardium was less (up to 26%).² Thus, we expect that the slowing of the kinetics and the increase in Ca²⁺ sensitivity in failing human myocardium will be less pronounced than observed in our study.

The slowing of relaxation after cTn_1–192 exchange observed in the present study suggests that cTnI degradation in vivo might impair cardiac relaxation and contribute to diastolic dysfunction, whereas the increased myofilament Ca²⁺ sensitivity with preserved maximal force generating capacity implies that at low Ca²⁺ concentrations present during systole, more force will be developed. However in vivo, during a twitch, this effect will be counteracted by the slowing of force development in preparations with cTn_1–192. The net effect during systole might thus be rather small. Hence, our data indicate that diastolic dysfunction in ischemic and failing human myocardium^43,44 might, at least in part, be attributable to cTnI proteolysis.

	Acknowledgments

 
Sources of Funding

This study was supported by the Netherlands Organisation for Scientific Research (VENI grant 2002) and Telethon Italy (grant GGP-02428).

Disclosures

None.

	Footnotes

 
This manuscript was sent to Hans Michael Piper, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Original received July 24, 2006; revision received September 19, 2006; accepted September 25, 2006.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 
1. Murphy AM, Kogler H, Georgakopoulos D, McDonough JL, Kass DA, Van Eyk JE, Marban E. Transgenic mouse model of stunned myocardium. Science. 2000; 287: 488–491.[Abstract/Free Full Text]

2. McDonough JL, Labugger R, Pickett W, Tse MY, MacKenzie S, Pang SC, Atar D, Ropchan G, Van Eyk JE. Cardiac troponin I is modified in the myocardium of bypass patients. Circulation. 2001; 103: 58–64.[Abstract/Free Full Text]

3. Neagoe C, Kulke M, del Monte F, Gwathmey JK, de Tombe PP, Hajjar RJ, Linke WA. Titin isoform switch in ischemic human heart disease. Circulation. 2002; 106: 1333–1341.[Abstract/Free Full Text]

4. Feng J, Schaus BJ, Fallavollita JA, Lee TC, Canty JM Jr. Preload induces troponin I degradation independently of myocardial ischemia. Circulation. 2001; 103: 2035–2037.[Abstract/Free Full Text]

5. Takeda S, Yamashita A, Maeda K, Maeda Y. Structure of the core domain of human cardiac troponin in the Ca²⁺-saturated form. Nature. 2003; 424: 35–41.[CrossRef][Medline] [Order article via Infotrieve]

6. Kobayashi T, Solaro RJ. Calcium, thin filaments, and the integrative biology of cardiac contractility. Annu Rev Physiol. 2005; 67: 39–67.[CrossRef][Medline] [Order article via Infotrieve]

7. McDonough JL, Arrell DK, Van Eyk JE. Troponin I degradation and covalent complex formation accompanies myocardial ischemia/reperfusion injury. Circ Res. 1999; 84: 9–20.[Abstract/Free Full Text]

8. Gao WD, Atar D, Liu Y, Perez NG, Murphy AM, Marban E. Role of troponin I proteolysis in the pathogenesis of stunned myocardium. Circ Res. 1997; 80: 393–399.[Medline] [Order article via Infotrieve]

9. Van der Velden J, Merkus D, Klarenbeek BR, James AT, Boontje NM, Dekkers DH, Stienen GJM, Lamers JM, Duncker DJ. Alterations in myofilament function contribute to left ventricular dysfunction in pigs early after myocardial infarction. Circ Res. 2004; 95: e85–e95.[Abstract/Free Full Text]

10. Colantonio DA, Van Eyk JE, Przyklenk K. Stunned peri-infarct canine myocardium is characterized by degradation of troponin T, not troponin I. Cardiovasc Res. 2004; 63: 217–225.[Abstract/Free Full Text]

11. Chen YN, Wei JR, Zeng LJ, Wu MY. Monitoring of cardiac troponin I in patients with acute heart failure. Ann Clin Biochem. 1999; 36: 433–437.[Medline] [Order article via Infotrieve]

12. Sakthivel S, Finley NL, Rosevear PR, Lorenz JN, Gulick J, Kim S, VanBuren P, Martin LA, Robbins J. In vivo and in vitro analysis of cardiac troponin I phosphorylation. J Biol Chem. 2005; 280: 703–714.[Abstract/Free Full Text]

13. Takimoto E, Soergel DG, Janssen PM, Stull LB, Kass DA, Murphy AM. Frequency- and afterload-dependent cardiac modulation in vivo by troponin I with constitutively active protein kinase A phosphorylation sites. Circ Res. 2004; 94: 496–504.[Abstract/Free Full Text]

14. Chandra M, Dong WJ, Pan BS, Cheung HC, Solaro RJ. Effects of protein kinase A phosphorylation on signaling between cardiac troponin I and the N-terminal domain of cardiac troponin C. Biochemistry. 1997; 36: 13305–13311.[CrossRef][Medline] [Order article via Infotrieve]

15. Papp Z, van der Velden J, Stienen GJM. Calpain-I induced alterations in the cytoskeletal structure and impaired mechanical properties of single myocytes of rat heart. Cardiovasc Res. 2000; 45: 981–993.[Abstract/Free Full Text]

16. Barta J, Toth A, Edes I, Vaszily M, Papp JG, Varro A, Papp Z. Calpain-1-sensitive myofibrillar proteins of the human myocardium. Mol Cell Biochem. 2005; 278: 1–8.[CrossRef][Medline] [Order article via Infotrieve]

17. Foster DB, Noguchi T, VanBuren P, Murphy AM, Van Eyk JE. C-terminal truncation of cardiac troponin I causes divergent effects on ATPase and force: implications for the pathophysiology of myocardial stunning. Circ Res. 2003; 93: 917–924.[Abstract/Free Full Text]

18. Piroddi N, Tesi C, Pellegrino MA, Tobacman LS, Homsher E, Poggesi C. Contractile effects of the exchange of cardiac troponin for fast skeletal troponin in rabbit psoas single myofibrils. J Physiol. 2003; 552: 917–931.[Abstract/Free Full Text]

19. Van der Velden J, Klein LJ, van der Bijl M, Huybregts MA, Stooker W, Witkop J, Eijsman L, Visser CA, Visser FC, Stienen GJM. Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue. Cardiovasc Res. 1998; 38: 414–423.[Abstract/Free Full Text]

20. Brenner B, Kraft T, Yu LC, Chalovich JM. Thin filament activation probed by fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle. Biophys J. 1999; 77: 2677–2691.[Medline] [Order article via Infotrieve]

21. Tesi C, Piroddi N, Colomo F, Poggesi C. Relaxation kinetics following sudden Ca²⁺ reduction in single myofibrils from skeletal muscle. Biophys J. 2002; 83: 2142–2151.[Medline] [Order article via Infotrieve]

22. Poggesi C, Tesi C, Stehle R. Sarcomeric determinants of striated muscle relaxation kinetics. Pflugers Arch. 2005; 449: 505–517.[CrossRef][Medline] [Order article via Infotrieve]

23. Van Eyk JE, Powers F, Law W, Larue C, Hodges RS, Solaro RJ. Breakdown and release of myofilament proteins during ischemia and ischemia/reperfusion in rat hearts: identification of degradation products and effects on the pCa-force relation. Circ Res. 1998; 82: 261–271.[Abstract/Free Full Text]

24. Kögler H, Soergel DG, Murphy AM, Marban E. Maintained contractile reserve in a transgenic mouse model of myocardial stunning. Am J Physiol. 2001; 280: H2623–H2630.

25. Kim SJ, Depre C, Vatner SF. Novel mechanisms mediating stunned myocardium. Heart Fail Rev. 2003; 8: 143–153.[CrossRef][Medline] [Order article via Infotrieve]

26. Duncan JG, Ravi R, Stull LB, Murphy AM. Chronic xanthine oxidase inhibition prevents myofibrillar protein oxidation and preserves cardiac function in a transgenic mouse model of cardiomyopathy. Am J Physiol. 2005; 289: H1512–H1518.

27. Narolska NA, Belus A, Piroddi N, Scellini B, Deppermann S, Jaquet K, Foster DB, Van Eyk JE, Tesi C, Van der Velden J, Stienen GJM, Poggesi C. C-terminal truncation of cardiac troponin I impairs force generating capacity and relaxation in rabbit psoas myofibrils. 49th Annual Meeting of the Biophysical Society, February 12–16, 2005, Long Beach, Calif (Abstract).

28. Tachampa K, Allen EJ, Rundell VL, Martin AF, de Tombe PP. Impact of C-terminal truncation of cardiac troponin I on myofilament chemo-mechanical transduction: implications for the decreased cardiac function in stunned myocardium. 78th Scientific Sessions of the American Heart Association, November 13–16, 2005, Dallas, Tex (Abstract).

29. Tachampa K, Allen EJ, Biesiadecki B, Martin AF, de Tombe PP. Similar effect of C-terminal truncation of mouse and human cardiac troponin I on myofilament chemo-mechanical transduction. 50th Annual Meeting of the Biophysical Society, February 18–22, 2006, Salt Lake City, Utah (Abstract).

30. Gao WD, Atar D, Backx PH, Marban E. Relationship between intracellular calcium and contractile force in stunned myocardium. Direct evidence for decreased myofilament Ca²⁺ responsiveness and altered diastolic function in intact ventricular muscle. Circ Res. 1995; 76: 1036–1048.[Abstract/Free Full Text]

31. Gao WD, Dai T, Nyhan D. Increased cross-bridge cycling rate in stunned myocardium. Am J Physiol. 2006; 290: H886–H893.

32. McDonald KS, Mammen PP, Strang KT, Moss RL, Miller WP. Isometric and dynamic contractile properties of porcine skinned cardiac myocytes after stunning. Circ Res. 1995; 77: 964–972.[Abstract/Free Full Text]

33. VanBuren P, Palmiter KA, Warshaw DM. Tropomyosin directly modulates actomyosin mechanical performance at the level of a single actin filament. Proc Natl Acad Sci U S A. 1999; 96: 12488–12493.[Abstract/Free Full Text]

34. Homsher E, Lee DM, Morris C, Pavlov D, Tobacman LS. Regulation of force and unloaded sliding speed in single thin filaments: effects of regulatory proteins and calcium. J Physiol. 2000; 524: 233–243.[Abstract/Free Full Text]

35. Fujita H, Lu X, Suzuki M, Ishiwata S, Kawai M. The effect of tropomyosin on force and elementary steps of the cross-bridge cycle in reconstituted bovine myocardium. J Physiol. 2004; 556: 637–649.[Abstract/Free Full Text]

36. Gordon AM, Homsher E, Regnier M. Regulation of contraction in striated muscle. Physiol Rev. 2000; 80: 853–924.[Abstract/Free Full Text]

37. Kobayashi T, Solaro RJ. Increased Ca²⁺ affinity of cardiac thin filaments reconstituted with cardiomyopathy-related mutant cardiac troponin I. J Biol Chem. 2006; 281: 13471–13477.[Abstract/Free Full Text]

38. Rarick HM, Tu XH, Solaro RJ, Martin AF. The C-terminus of cardiac troponin I is essential for full inhibitory activity and Ca²⁺ sensitivity of rat myofibrils. J Biol Chem. 1997; 272: 26887–26892.[Abstract/Free Full Text]

39. Huxley AF, Simmons RM. Rapid ‘give’ and the tension ‘shoulder’ in the relaxation of frog muscle fibres. J Physiol. 1970; 210: 32P–33P.[Medline] [Order article via Infotrieve]

40. Arteaga GM, Palmiter KA, Leiden JM, Solaro RJ. Attenuation of length dependence of calcium activation in myofilaments of transgenic mouse hearts expressing slow skeletal troponin I. J Physiol. 2000; 526: 541–549.[Abstract/Free Full Text]

41. Van der Velden J, Narolska NA, Lamberts RR, Boontje NM, Borbely A, Zaremba R, Bronzwaer JG, Papp Z, Jaquet K, Paulus WJ, Stienen GJM. Functional effects of protein kinase C-mediated myofilament phosphorylation in human myocardium. Cardiovasc Res. 2006; 69: 876–887.[Abstract/Free Full Text]

42. Day SM, Westfall MV, Fomicheva EV, Hoyer K, Yasuda S, La Cross NC, D’Alecy LG, Ingwall JS, Metzger JM. Histidine button engineered into cardiac troponin I protects the ischemic and failing heart. Nat Med. 2006; 12: 181–189.[CrossRef][Medline] [Order article via Infotrieve]

43. Wijns W, Serruys PW, Slager CJ, Grimm J, Krayenbuehl HP, Hugenholtz PG, Hess OM. Effect of coronary occlusion during percutaneous transluminal angioplasty in humans on left ventricular chamber stiffness and regional diastolic pressure-radius relations. J Am Coll Cardiol. 1986; 7: 455–463.[Abstract]

44. Williamson BD, Lim MJ, Buda AJ. Transient left ventricular filling abnormalities (diastolic stunning) after acute myocardial infarction. Am J Cardiol. 1990; 66: 897–903.[CrossRef][Medline] [Order article via Infotrieve]

This article has been cited by other articles:

				T. Kobayashi, S. E. Patrick, and M. Kobayashi Ala Scanning of the Inhibitory Region of Cardiac Troponin I J. Biol. Chem., July 24, 2009; 284(30): 20052 - 20060. [Abstract] [Full Text] [PDF]

				D. J. Duncker, N. M. Boontje, D. Merkus, A. Versteilen, J. Krysiak, G. Mearini, A. El-Armouche, V. J. de Beer, J. M.J. Lamers, L. Carrier, et al. Prevention of Myofilament Dysfunction by {beta}-Blocker Therapy in Postinfarct Remodeling Circ Heart Fail, May 1, 2009; 2(3): 233 - 242. [Abstract] [Full Text] [PDF]

				A. Molnar, A. Borbely, D. Czuriga, S. M. Ivetta, S. Szilagyi, Z. Hertelendi, E. T. Pasztor, A. Balogh, Z. Galajda, T. Szerafin, et al. Protein Kinase C Contributes to the Maintenance of Contractile Force in Human Ventricular Cardiomyocytes J. Biol. Chem., January 9, 2009; 284(2): 1031 - 1039. [Abstract] [Full Text] [PDF]

				X.-Y. Lu, L. Chen, X.-L. Cai, and H.-T. Yang Overexpression of heat shock protein 27 protects against ischaemia/reperfusion-induced cardiac dysfunction via stabilization of troponin I and T Cardiovasc Res, August 1, 2008; 79(3): 500 - 508. [Abstract] [Full Text] [PDF]

				K. Tachampa, T. Kobayashi, H. Wang, A. F. Martin, B. J. Biesiadecki, R. J. Solaro, and P. P. de Tombe Increased Cross-bridge Cycling Kinetics after Exchange of C-terminal Truncated Troponin I in Skinned Rat Cardiac Muscle J. Biol. Chem., May 30, 2008; 283(22): 15114 - 15121. [Abstract] [Full Text] [PDF]

				N. Hamdani, V. Kooij, S. van Dijk, D. Merkus, W. J. Paulus, C. d. Remedios, D. J. Duncker, G. J.M. Stienen, and J. van der Velden Sarcomeric dysfunction in heart failure Cardiovasc Res, March 1, 2008; 77(4): 649 - 658. [Abstract] [Full Text] [PDF]

				J. P. Davis and S. B. Tikunova Ca2+ exchange with troponin C and cardiac muscle dynamics Cardiovasc Res, March 1, 2008; 77(4): 619 - 626. [Abstract] [Full Text] [PDF]

				B. Iorga, N. Blaudeck, J. Solzin, A. Neulen, I. Stehle, A. J. L. Davila, G. Pfitzer, and R. Stehle Lys184 deletion in troponin I impairs relaxation kinetics and induces hypercontractility in murine cardiac myofibrils Cardiovasc Res, March 1, 2008; 77(4): 676 - 686. [Abstract] [Full Text] [PDF]

				M. C. de Waard, J. van der Velden, V. Bito, S. Ozdemir, L. Biesmans, N. M. Boontje, D. H.W. Dekkers, K. Schoonderwoerd, H. C.H. Schuurbiers, R. d. Crom, et al. Early Exercise Training Normalizes Myofilament Function and Attenuates Left Ventricular Pump Dysfunction in Mice With a Large Myocardial Infarction Circ. Res., April 13, 2007; 100(7): 1079 - 1088. [Abstract] [Full Text] [PDF]

				R. Stehle, B. Iorga, and G. Pfitzer Calcium regulation of troponin and its role in the dynamics of contraction and relaxation Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2007; 292(3): R1125 - R1128. [Full Text] [PDF]

This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 99/9/1012    most recent 01.RES.0000248753.30340.afv1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Narolska, N. A. Articles by Stienen, G. J.M. Search for Related Content PubMed PubMed Citation Articles by Narolska, N. A. Articles by Stienen, G. J.M. Related Collections Contractile function Heart failure - basic studies Ischemic biology - basic studies