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Cardiovascular Research Advance Access originally published online on December 20, 2007
Cardiovascular Research 2008 77(4):676-686; doi:10.1093/cvr/cvm113
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2007. For permissions please email: journals.permissions@oxfordjournals.org

Lys184 deletion in troponin I impairs relaxation kinetics and induces hypercontractility in murine cardiac myofibrils

Bogdan Iorga1,2,*, Natascha Blaudeck1, Johannes Solzin1,3, Axel Neulen1, Ina Stehle1, Alfredo J. Lopez Davila1, Gabriele Pfitzer1,3 and Robert Stehle1,3

1 Institute of Vegetative Physiology, University of Cologne, Robert-Koch-Strasse 39, Cologne 50931, Germany
2 Department of Physics and Applied Mathematics, Faculty of Chemistry, University of Bucharest, Bucharest 030018, Romania
3 Center for Molecular Medicine of Cologne, Cologne 50931, Germany

* Corresponding author. Tel: +49 221 478 6948; fax: +49 221 478 3538. E-mail address: bogdan.iorga{at}uni-koeln.de; robert.stehle{at}uni-koeln.de

Received 31 July 2007; revised 14 December 2007; accepted 18 December 2007

Time for primary review: 22 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Aims: To understand the functional consequences of the Lys184 deletion in murine cardiac troponin I (mcTnI{Delta}K184), we have studied the primary effects of this mutation linked to familial hypertrophic cardiomyopathy (FHC) at the sarcomeric level.

Methods and results: Ca2+ sensitivity and kinetics of force development and relaxation were investigated in cardiac myofibrils from transgenic mice expressing mcTnI{Delta}K184, as a model which co-segregates with FHC. Ca2+-dependent conformational changes (switch-on/off) of the fluorescence-labelled human troponin complex, containing either wild-type hcTnI or mutant hcTnI{Delta}K183, were investigated in myofibrils prepared from the guinea pig left ventricle. Ca2+ sensitivity and maximum Ca2+-activated and passive forces were significantly enhanced and cooperativity was reduced in mutant myofibrils. At partial Ca2+ activation, mutant but not wild-type myofibrils displayed spontaneous oscillatory contraction of sarcomeres. Both conformational switch-off rates of the incorporated troponin complex and the myofibrillar relaxation kinetics were slowed down by the mutation. Impaired relaxation kinetics and increased force at low [Ca2+] were reversed by 2,3-butanedione monoxime (BDM), which traps cross-bridges in non-force-generating states.

Conclusion: We conclude that these changes are not due to alterations of the intrinsic cross-bridge kinetics. The molecular mechanism of sarcomeric diastolic dysfunction in this FHC model is based on the impaired regulatory switch-off kinetics of cTnI, which induces incomplete inhibition of force-generating cross-bridges at low [Ca2+] and thereby slows down relaxation of sarcomeres. Ca2+ sensitization and impairment of the relaxation of sarcomeres induced by this mutation may underlie the enhanced systolic function and diastolic dysfunction at the sarcomeric level.

KEYWORDS Ca2+ sensitization; Cross-bridge kinetics; Diastolic dysfunction; Hypercontractility; Sarcomere dynamics


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
Familial hypertrophic cardiomyopathy (FHC), a frequent hereditary disease of the myocardium, is associated with mutations in genes encoding sarcomeric proteins. The clinical course of the disease is extremely variable and may range from an asymptomatic to a malignant phenotype with sudden cardiac death.1 One of the most eminent functional disorders in affected individuals is diastolic dysfunction, which may have many underlying causes such as impaired inactivation of sarcomeres, altered Ca2+ sequestration at the myocyte level, and hypertrophy and fibrosis at the organ level.1,2 Thus, in order to understand the mechanistic interpretation of the altered physiological read-outs of the organ function, it is essential to characterize first the primary genotype–phenotype relation at the sarcomeric level, which is the smallest functional contractile unit of the cardiomyocyte.

About 5% of FHC patients have mutations in the TNNI3 gene,3 which codes for the inhibitory subunit (cTnI) of the regulatory troponin complex (cTn). cTnI is essential for inactivation of cardiac contraction and hence for diastolic function. The functional consequences of cTnI mutations at the level of the contractile machinery have been investigated by several in vitro protocols such as determination of acto-myosin ATPase activity in reconstituted systems or exchange of the mutant protein against the endogenous one in skinned fibres or myofibrils.4–7 In skinned fibres, most cTnI mutations increase Ca2+ sensitivity of steady-state contraction,4 which, however, does not allow to draw conclusions regarding dynamic heart function. Especially, sarcomeric diastolic dysfunction, i.e. impairment of the relaxation kinetics, is difficult to be investigated in skinned fibres due to the limited Ca2+-buffering capacity of available caged Ca2+ chelators. Therefore, we used myofibrils, which allows visual observation of each individual sarcomere behaviour, as a favourable model to study the kinetics of the Ca2+-regulated contraction and relaxation induced by rapid, defined changes in [Ca2+].8–11 This approach can enhance the mechanistic understanding of systolic-diastolic dynamics, because the rate-limiting sarcomeric processes significantly sustain myocardial stiffness during ejection and isovolumetric relaxation.8,12

We have previously shown that several kinetic results, obtained with cardiac myofibrils isolated from transgenic mice carrying the mutation R146G in the inhibitory region of cTnI (mcTnIR146G), differed from those obtained by exchange of the endogenous wild-type troponin in myofibrils for the mutant protein.6 Based on this study, we believe that cardiac myofibrils isolated from transgenic mice are superior to exchanged myofibrils. Therefore, we generated a transgenic mouse model for one of the clinically best characterized mutations3,13 in the C-terminal mobile domain (Md) of cTnI, a Lys184 deletion (murine sequence), and analysed its functional consequences on contractile properties in myofibrils (TG myofibrils) isolated from the hearts of these transgenic mice (TG-{Delta}K184 mice).

Using this model, we confirmed the mutation-induced Ca2+ sensitization of contraction. Kinetic analysis revealed that the mutation has no effect on the maximum rate of force development, but impairs relaxation kinetics and increases passive force. The Lys184 deletion did not alter switch-off kinetics of the isolated cTn, but it slows down the switch-off when cTn was incorporated into myofibrils. These results are interpreted according to structural models, which suggest that the Md is essential for returning to a relaxed state.14 We show for the first time that the Lys184 deletion affects the inter-sarcomeric dynamics, because TG myofibrils, but not wild-type myofibrils (nTG myofibrils), exhibit spontaneous oscillatory contractions (SPOCs) of sarcomeres. In conclusion, we report here that the Lys184 deletion leads to sarcomeric diastolic dysfunction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 
2.1 Transgenic mice
We generated two transgenic mouse (C57BL/6) models (see Supplementary material online): TG-{Delta}K184 mice expressing mcTnI{Delta}K184 and TG-flag mice expressing wild-type mcTnI with an N-terminal Flag-tag (mcTnIWT-flag). We compared TG-{Delta}K184 mice with their nTG littermates to reduce transgene-independent inter-individual variability.

All animal investigations were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the US NIH (Publication No.85-23, revised 1996).

2.2 Troponin exchange in skinned fibres
Murine cTn (mcTn) containing either mcTnI{Delta}K184 or mcTnIWT was reconstituted from recombinant mcTn-subunits (see Supplementary material online) and used to replace the endogenous mcTn in skinned fibres isolated from TG-{Delta}K184-, nTG- or TG-flag mice following an exchange protocol previously described.15

2.3 Mechanical experiments with skinned fibres and myofibrils
Mice were sacrificed by cervical dislocation and the beating hearts quickly excised. Skinned fibres were dissected from left-ventricular papillary muscles, mounted and subjected to a successive Ca2+ activation/relaxation protocol as described in Refs.15,16

Myofibrils (sarcomere length, SL = 1.8–2.0 µm; diameter = 1.2–4.7 µm) for micro-mechanical experiments were prepared from papillary muscles as described in Ref.10 The experimental setup was previously described.9,10 The protocol for myofibrillar isometric force measurement and the assignation of steady-state and kinetic activation/relaxation parameters are shown in Figures 1B and C. Slack sarcomere lengths (SL0) of isometrically mounted myofibrils were measured in relaxing buffer (pCa 7.50). Experiments with fibres and myofibrils were performed at 10°C.


Figure 1
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  		Figure 1 (A) Transgenic mcTnI expression levels (% of total mcTnI protein) in myofibrils prepared from ventricles of TG-{Delta}K184-, nTG- and TG-flag mice (see Supplementary material online). (B) Representative full force transients of isolated nTG myofibrils. [Ca2+] is rapidly (10–20 ms) changed from pCa 7.50 to 4.53 and back to 7.50 to induce contraction and relaxation, respectively. Ca2+-induced force (FACT) develops mono-exponentially (kACT). Force redevelopment (kTR) is induced mechanically by slackening–restretching the myofibril during Ca2+ activation. Passive force (Fpass) is determined prior to activation by transiently slackening the myofibril from the sarcomere length of 15% above SL0. (C) The best fit (black) superimposed on the force transient (grey trace) during relaxation is a biphasic, linear-exponential function10 yielding kLIN, tLIN and kREL. (D) nTG myofibrils mounted isometrically.

 
2.4 Troponin switch kinetics
Prior to reconstitution of the human cTn (hcTn) from recombinant hcTn-subunits, hcTnC was fluorescence-labelled at Cys84 with N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD) as indicated in Ref.17 The IANBD-labelled hcTn was incorporated into left ventricular myofibrils.17

The Ca2+-dependent fluorescence change of myofibril-incorporated or isolated IANBD-labelled hcTn, containing either hcTnIWT or hcTnI{Delta}K183, was measured using a Stopped-Flow apparatus (SFM400/S-BioLogic, Claix, France) and the troponin switch-on/off kinetics were determined as previously published.11,17 Experiments were performed at 10°C.

Because the Stopped-Flow experiments require large quantities of myofibrils, they were prepared from guinea pig instead of murine ventricles.

2.5 Data analysis and modelling
The equations used for data analysis and modelling are explained in Supplementary material online. Analysis of myofibrils force transients was explained previously.8–10 We used 1 and 4 pairs of nTG- and TG-{Delta}K184 mice aged 3 and 6–7 months, respectively, and 1 pair of 3 months aged nTG- and TG-flag mice. All data are presented as mean ± SEM; n corresponds to the number of myofibrils/fibres analysed. Statistical significant differences were determined by two-tailed unpaired Student’s t-tests: *(P < 0.05), **(P < 0.01), ***(P < 0.001).


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Supplementary material
 Funding
 References
 
3.1 Transgenic protein expression levels, viability and heart weights
TG-{Delta}K184 mice were viable with normal life spans and no significant differences in body weights. Surprisingly, the mutant hearts did not exhibit hypertrophy. Rather the heart-to-body weight ratio was decreased: 3.55 ± 0.09, n = 11 for TG-{Delta}K184 mice; 4.28 ± 0.09, n = 15 for nTG mice; P < 0.001; 6 months old.

In the cardiac myofibrils isolated from TG-{Delta}K184- and TG-flag mice, ≥90% of the mcTnIWT protein was replaced by the transgenic mcTnI{Delta}K184 and mcTnIWT-flag proteins, respectively (Figure 1A), but the total mcTnI protein content was preserved (for the estimation of heart weights and protein expression levels, see Supplementary material online).

3.2 Ca2+-activated myofibrillar force
TG myofibrils developed significantly higher maximum force (Fmax) per cross-sectional area (CSA) compared with nTG myofibrils or TG-flag myofibrils, irrespective of the age of the mice (Table 1). This is due to enhancements of both passive force (Fpass) and Ca2+-induced force amplitude (FACT) (Figure 2A).


Figure 2
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  		Figure 2 (A) Passive force (left), Ca2+-induced force amplitude (middle), and total active force (right), normalized to cross-sectional area (CSA), developed by myofibrils of 6–7 months old TG-{Delta}K184 mice (grey-filled bars) and nTG mice (open bars); Fmax = FACT + Fpass. (B) Force–pCa relations for TG myofibrils (closed circles) and nTG myofibrils (open circles). The curves represent Hill-functions (equation (1), see Supplementary material online) fitted to force data normalized to Fmax at pCa 4.53. (C) Data of B are linearized (see Supplementary material online) and plotted as function of pCa. Zero-crossing values correspond to pCa50 at half-maximum force. The biphasic relation of data from nTG myofibrils indicates that the degree of cooperativity above pCa50 (nH1 = 3.77 ± 0.28) is significantly higher than below pCa50 (nH2 = 1.50 ± 0.35). Encircled points indicate data at which Ca2+-induced spontaneous oscillatory contractions (Ca-SPOCs) were detected by video-microscopy (see Supplementary material, movies). Numbers next to each data point give Ca-SPOC events detected per number of videos investigated.

 


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  		Table 1 Steady-state and kinetic parameters of cardiac myofibrils isolated from TG-{Delta}K184-, TG-flag- and nTG mice (passive force was measured at SL = 1.15 x SL0)

 
The force-pCa relation of TG myofibrils was shifted to the left (Figure 2B), indicating an increase in Ca2+ sensitivity of force development relative to nTG myofibrils (Table 1). The Hill-coefficient, reflecting the overall cooperativity of the force-pCa relation, was significantly reduced (Table 1). This loss in cooperativity occurs specifically at lower Ca2+-activated force levels (Figure 2C). Interestingly, at the same sub-half-maximal force levels SPOC of sarcomeres was observed (see Supplementary material online, movies). The occurrence of Ca2+-induced SPOC (Ca-SPOC) is indicated in Figure 2C by the encircled data points. Ca-SPOC was never observed in nTG myofibrils at any partial [Ca2+].

3.3 Force-pCa relations in skinned papillary muscle fibres
Skinned fibres isolated from TG-{Delta}K184 mice exhibited similar features in the force-pCa relation like myofibrils when compared with either nTG- or TG-flag fibres (Figure 3A and B; Table 2). There were no significant differences between force-pCa relations of nTG- and TG-flag fibres (Figure 3A).


Figure 3
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  		Figure 3 Force–pCa relations of skinned fibres from papillary muscles of 6–7 months old TG-{Delta}K184- and nTG- or TG-flag mice. (A) Force–pCa relations from TG fibres (mcTnI{Delta}K184) (closed circles), nTG fibres (mcTnIWT) (open circles) and TG-flag fibres (mcTnIWT-flag) (open squares). (B) Force data of A from TG fibres (closed circles) and nTG fibres (open circles) are linearized as in Figure 2C. The linearized relation for nTG fibres is biphasic: above pCa50, nH1 = 5.51 ± 0.45; below pCa50, nH2 = 3.02 ± 0.47. (C) Endogenous troponin-complex of either nTG fibres (open triangles) or TG fibres (closed triangles) was exchanged for recombinant mcTnWT. (D) Endogenous troponin of nTG fibres was exchanged for recombinant mcTn containing either mcTnIWT (open nablas) or mcTnI{Delta}K184 (closed nablas). (E) Western blot showing the replacement of endogenous mcTnIWT-flag by the recombinant mcTn containing either mcTnI{Delta}K184 or mcTnIWT.

 


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  		Table 2 Functional parameters of skinned fibres from the papillary muscles of 6–7 months old TG-{Delta}K184- and nTG mice before and after exchange with mcTn containing either mcTnI{Delta}K184 or mcTnIWT

 
To exclude that the mutation causes adaptive effects in the myofibrils which may affect the force-pCa relation, the endogenous mcTn in the skinned fibres of TG-{Delta}K184- and nTG mice was replaced by recombinantly expressed murine wild-type troponin complex (mcTnWT). The incorporation of mcTnWT into TG- and nTG fibres completely abolished the differences in force-pCa relations (Figure 3C; Table 2) suggesting that Ca2+ sensitization is solely meditated by the mutation. This is supported by the finding that replacement of the endogenous mcTn from skinned nTG fibres by mutant mcTn caused Ca2+ sensitization and loss in cooperativity, while replacement by mcTnWT had no significant effect (Figure 3D; Table 2).

Since endogenous mcTnI and exogeneous mcTnI{Delta}K184 or mcTnIWT cannot be separated by SDS-PAGE, we used skinned fibres isolated from TG-flag mice to estimate the efficiency of exchange: ~80% of endogenous mcTnIWT-flag protein was replaced by the recombinant mcTnI{Delta}K184 or mcTnIWT protein (Figure 3E).

3.4 Myofibrillar Ca2+-induced kinetics of force development
Force transients induced by a sudden increase in [Ca2+] from pCa 7.50 to pCa 4.53 (saturating [Ca2+]) (Figure 4A) were fitted by a mono-exponential function yielding the kinetic rate constant kACT, which was similar for TG- and nTG- or TG-flag myofibrils (Table 1). All these myofibrils displayed similar rate constants of mechanically induced force redevelopment, kTR (Table 1).


Figure 4
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  		Figure 4 Effect of mcTnI{Delta}K184 on kinetics of Ca2+-induced force development in TG myofibrils of 6–7 months old mice. (A) Following a rapid increase in [Ca2+], force transients of TG myofibrils (dark-grey trace) and nTG myofibrils (light-grey trace) are normalized to same amplitudes to illustrate their similar kinetics. Both traces are fitted to mono-exponential functions (black) yielding the same kACT. (B) Values of kACT for TG myofibrils (closed circles) and nTG myofibrils (open circles) plotted vs. pCa. Significant differences are indicated. (C) Relation of kACT vs. force normalized to its maximum value at pCa 4.53. Data of TG myofibrils (closed circles) and nTG myofibrils (open circles) are fitted by equation (2) (see Supplementary material online) yielding gapp and fappmax. Dashed curves indicate the 95%-confidence intervals of the fitting curves. (D) Electrophoretic separation of {alpha}-MHC and β-MHC isoforms. Myocardium from mice at 1 day postnatal (p.n.) containing both isoforms served as control. β-MHC is absent in both, adult nTG- and TG-{Delta}K184 mice. (E) two-states model of the cross-bridge cycle.20

 
At partial Ca2+ activation, in TG myofibrils, kACT is higher than in nTG myofibrils (Figure 4B). This effect relates to the increased Ca2+ sensitivity of contraction in TG myofibrils as shown by plotting kACT against the relative force, Fn (Figure 4C). Fitting the kACT–force data to equation (2) (see Supplementary material online) yielded similar curves for both TG myofibrils and nTG myofibrils (Figure 4C) and the extrapolation of the curves to Fn->0 gives an estimation of gapp.8

Figure 4D indicates that in adult TG-{Delta}K184 mice there is no shift of the {alpha}-MHC to the β-MHC isoform, which would result in a slower cross-bridge cycling.

3.5 Myofibrillar relaxation kinetics
Both, TG- and nTG myofibrils exhibit a biphasic force decay consisting of an initial, slow phase with the rate constant kLIN and a duration tLIN, followed by a rapid, exponential phase with the rate constant kREL (Figure 5A). We have shown previously8,9 that the sarcomeres remain isometric during the slow phase, whereas the second phase initiates from sequential sarcomere dynamics which enable the myofibril to relax faster than it contracts (kREL > kACT; Table 1).


Figure 5
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  		Figure 5 Effect of mcTnI{Delta}K184 on the kinetics of myofibrillar normalized force relaxation following rapid Ca2+-removal from pCa 4.53 to pCa 7.50 and on the passive force at pCa 7.50 (myofibrils of 6–7 (A, B) and 3 (C, D) months old mice). (A) Relaxation kinetic parameters obtained by the best fits (black) of the force decays for nTG myofibrils (light-grey trace) and for TG myofibrils (dark-grey trace). (B) Summary of the relaxation kinetic parameters for TG myofibrils (grey-filled bars) and nTG myofibrils (open bars). (C) Effect of 2,3-butanedione-monoxime (BDM) on the relaxation time constant, {tau}REL (equation (3), see Supplementary material online), of TG myofibrils (closed circles) and nTG myofibrils (open circles). (D) Passive force normalized to cross-sectional area (CSA) at SL = 2.30 ± 0.06 µm (SL = 1.15 x SL0) for TG myofibrils (closed circles) and nTG myofibrils (open circles) at different [BDM]. Significant differences at the respective [BDM] are indicated (C, D).

 
Due to a significant increase in the duration tLIN and a significant decrease in the rate constant kREL, the overall relaxation process is delayed and slower in TG myofibrils than in nTG myofibrils (Figures 5A and B; Table 1). In contrast, there was no significant difference in the rate constant kLIN (Figure 5B-left; Table 1). The overall relaxation kinetics can be expressed by the relaxation time constant, {tau}REL (equation (3), see Supplementary material online). For 3 and 6–7 months old mice, the mutation significantly increased {tau}REL by 46% and 30%, respectively (P < 0.01; Table 1). There were no significant differences between the relaxation parameters of nTG- and TG-flag myofibrils (Table 1).

To study the contribution of cross-bridges to the deceleration of the relaxation in TG myofibrils, the effect of 2,3-butanedione-monoxime (BDM) on the relaxation kinetics was investigated. BDM, an uncompetitive inhibitor of the myosin ATPase activity,18 traps cross-bridges in pre-powerstroke non-force generating states.19 BDM accelerated the relaxation kinetics of both TG- and nTG myofibrils. Increasing [BDM] in the relaxing buffer diminishes the difference of the relaxation time between TG- and nTG myofibrils (Figure 5C) and at 10 and 15 mM BDM no significant differences were observed (P > 0.05, n = 7).

In the absence of Ca2+, myofibrils were stretched by 15% of their slack sarcomere length. This imposed mechanical perturbation induced a larger increment in passive force in TG myofibrils compared with nTG- or TG-flag myofibrils (Figures 2A-left, 5D; Table 1). The enhancement was diminished by BDM in TG myofibrils but not in nTG myofibrils (Figure 5D). Hence, in the presence of 15 mM BDM, there was no significant difference in the passive force (P > 0.05, n = 7).

3.6 Ca2+-controlled switch-kinetics of isolated and myofibril-incorporated troponin
To investigate whether altered force kinetics of TG myofibrils could be explained by the effects of the mutation on the switch-on/off kinetics of troponin, recombinantly expressed hcTn, containing either hcTnIWT or hcTnI{Delta}K183, was incorporated into myofibrils. Conformational changes of the incorporated hcTn, sensed by IANBD-labelled hcTnC, were measured by mixing the myofibrils with Ca2+ (switch-on kinetics) (Figure 6A) or the Ca2+-chelator BAPTA (switch-off kinetics) (Figure 6B) as previously described.17 While the mutation did not significantly alter the observed rate constant of Ca2+-induced switch-on kinetics (kobs,+Ca) (Figure 6C), it significantly decreased the observed rate constant of switch-off kinetics (kobs,–Ca) induced by Ca2+-removal (Figure 6D). Thus, in line with the kinetics of force decay during relaxation (Table 1), the mutation only affected the inactivation (switch-off) kinetics. Both kobs,+Ca and kobs,–Ca are too fast to directly rate-limit the intrinsic cross-bridges cycling as explained previously.17 Interestingly, neither switch-on kinetics (Figure 6E) nor switch-off kinetics (Figure 6F) of isolated hcTn was significantly affected by the mutation. Thus, hcTnI{Delta}K183 impairs the switch-off kinetics only when incorporated into the sarcomeric environment.


Figure 6
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  		Figure 6 Conformational changes of the reconstituted fluorescence-labelled hcTn-complex recorded by Stopped-Flow contained hcTnT, N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (IANBD)-labelled hcTnC and either hcTnI{Delta}K183 or hcTnIWT. The hcTn-complex fluorescence transients were recorded either upon Ca2+ activation (switch-on kinetics, A) or Ca2+-removal (switch-off kinetics, B). The dark-grey and light-grey transients in A and B refer to hcTn-complex, containing hcTnI{Delta}K183 and hcTnIWT, respectively, incorporated into myofibrils. The first 2.2 ms of the traces is lost due to the dead-time of the Stopped-Flow apparatus. Prior to Ca2+-removal (B), myofibrils had been Ca2+-activated at pCa 5.0 for 91 ms as explained in Ref.17 The black curves superimposed on each grey fluorescence transient represent the best fit to mono-exponential rise (A) or decay (B). (C–F) Observed rate constants for switch-kinetics of incorporated (C, D) or isolated (E, F) hcTn-complex containing either hcTnI{Delta}K184 (grey-filled bars) or hcTnIWT (open bars). N = number of experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Supplementary material
 Funding
 References
 
One of the prominent clinical characteristics of FHC-patients carrying mutations in hcTnI is diastolic dysfunction. However, the underlying cause is not fully understood. The major finding of our study using isolated myofibrils from transgenic murine hearts carrying a Lys184 deletion in the C-terminal mobile domain of cTnI is that this mutation leads to a decrease in the switch-off kinetics of troponin, a deceleration of myofibrillar force relaxation kinetics and an increase in their passive force due to residual activated cross-bridges in the absence of Ca2+. The mutation further enhances the Ca2+ sensitivity of both myofibrils and multi-cellular skinned fibres. Troponin exchange studies showed that this is due to the mutation rather than to secondary mutation-induced effects. Interestingly, we found that the Lys184 deletion also induces SPOCs at partial Ca2+ activation, suggesting that the Ca2+-regulated actin filament accessibility for steady cross-bridge cycling is altered due to loss of cooperative inhibition.

4.1 Ca2+ sensitization is not due to the alteration in intrinsic turnover kinetics of cross-bridges
In agreement with previous studies,4,5 we found an increase in Ca2+ sensitivity of both TG myofibrils and skinned TG fibres. The effect was also mimicked in skinned fibres from nTG mice with incorporated mcTnI{Delta}K184 and was lost when the endogenous mutant cTn from skinned TG fibres was exchanged for wild-type troponin. The troponin exchange studies suggest that the Ca2+ sensitization and loss in cooperativity observed in myofibrils and skinned fibres isolated from transgenic hearts result mainly from the primary effects of the mutation on troponin function.

kACT reflects the sum of the apparent rate constants which rate-limit the turnover of cross-bridges (kACT = fapp+ gapp), whereby fapp represents the Ca2+-modulated transition from non-force-generating to force-generating states and gapp is the Ca2+-independent transition to non-force-generating states (Figure 4E).8,20,21 The similarity of kACT–force relations of TG myofibrils and nTG myofibrils implies that the intrinsic cross-bridge kinetics, which depend on the MHC isoform, were not affected by the mutation. Consistently, there was no shift in the MHC isoform. Therefore, intrinsic kinetic properties of cross-bridges are not responsible for the observed Ca2+ sensitization and reduced cooperativity in TG myofibrils.

Similar values of maximum kACT (=fmaxapp + gapp) for both isometric TG- and nTG myofibrils are consistent with the observation that maximum Ca2+-activated ATPase activity of myofibrils with incorporated hcTnI{Delta}K183 was not enhanced,4 but it appears to be at variance with the observation that the speed of regulated actin filaments sliding is enhanced by the hcTnI{Delta}K183 mutation.5 Nevertheless, the latter finding5 is not contradictory, because the rate-limiting cross-bridge cycle rates are not the same under isometric (our study) and unloaded sliding conditions (in vitro motility assay study5). This indicates that indeed both extreme isometric and unloaded kinetic parameters have to be investigated for a better understanding of the effects of the mutation.

As explained in Ref.,8 gapp becomes predominant (kACT{approx}gapp) at very low [Ca2+], i.e. when myofibrils relax. Both, gapp and kLIN (kLIN giving an independent estimation for gapp) were similar for TG- and nTG myofibrils, suggesting that the intrinsic rates of cross-bridge detachment (gapp) are unaffected by the mutation. Thus, the impairment of myofibrillar relaxation is not due to the alteration of the intrinsic cross-bridge kinetic properties.

We note that, despite of unaltered maximum kACT, maximal force is increased in TG myofibrils, but the molecular mechanism remains to be solved. However, a significant enhanced maximum force has not been previously detected in skinned fibres containing the Lys183 deletion4 or other C-terminal cTnI mutations.5 This may be due to non-homogeneity of myofibrils in the analyzed skinned fibres.

4.2 mcTnI{Delta}K184 mutation reduces cooperativity and induces spontaneous oscillatory contractions
A high slope of the force–pCa relation is an indicator for a strong cooperative behaviour. Basically, two sources of cooperativity have been proposed: (i) attachment of strongly bound cross-bridges within a regulatory troponin-tropomyosin unit turns-on the unit favouring further cross-bridge attachment and (ii) already turned-on units facilitate further turning-on of neighbouring units.

Other studies indicated that mutations which alter charges in cTnI could cause a loss of the cooperative behaviour6,7,22 as we observed in TG myofibrils, which specifically occurs in the lower half of the force–pCa relation.

Simulation of force–pCa relations, using sophisticated models which involve several types of cooperativity,23 showed that strong neighbour interactions of troponin-tropomyosin produce asymmetric relations. These were clearly observed for nTG myofibrils, presenting a higher slope at low Ca2+ activation levels, but not for TG myofibrils. Specific abolishment of the higher cooperativity at lower [Ca2+] in TG myofibrils therefore most likely indicates that the Lys184 deletion impairs the communication between neighbouring units along the thin filament.

In the same range of Ca2+ activation where cooperativity was lost, TG myofibrils exhibited the striking feature of Ca2+-induced SPOC, which did not occur in nTG myofibrils. Ca-SPOC had been first described for cardiac tissues24 and afterwards for myofibrils25 containing predominantly the slow β-MHC isoform. Recently, SPOC had been investigated in myocardium of various species.26 SPOC was found to be less pronounced in rat myocardium containing a mixture of {alpha}-MHC and β-MHC. To our knowledge there is no study describing Ca-SPOC in the murine myocardium, which might be due to the fact that it contains only the fast {alpha}-MHC isoform. Thus, it may be plausible that we do not find Ca-SPOC in nTG myofibrils.

Since MHC isoform composition was not changed in TG myofibrils, we propose that Ca-SPOC might arise from attenuated cooperative responses within the thin filaments. This is consistent with the interpretation described in Ref.27 that a diminished cooperative behaviour of partial Ca2+-activated cross-bridges along the thin filament could favour the occurrence of Ca-SPOC.

4.3 mcTnI{Delta}K184 mutation impairs relaxation kinetics of sarcomeres
It is known from studies on reconstituted thin filaments that several cTnI mutations weaken the ability of cTn to fully inhibit actin–myosin ATPase and that this incomplete inhibition is associated with weakening of cTnI–actin interactions.7,22 Analogously, we showed here that the Lys183 deletion selectively impairs the switch-off kinetics in the sarcomere without affecting the switch-on kinetics and, thus, destabilizing the off-state of the Ca2+-regulated actin filament. This effect does not occur in the isolated cTn-complex, i.e. without actin. Therefore, the presence of actin filaments, i.e. an organized sarcomeric environment, is a prerequisite for the effect of the hcTnI{Delta}K183 mutation on the troponin switch-off kinetics, suggesting that the C-terminus of cTnI has a functional importance in the rate modulating process of the actin–myosin interaction.

This interpretation is supported by previous structural studies: within the highly flexible C-terminal mobile domain (Md) of cTnI, Lys184 is located in a fragile β-bulge motif of an antiparallel β-sheet.14 During diastole, when [Ca2+] is very low, the Md and the inhibitory region (Ir) of cTnI interact strongly with actin and shield the ‘secondary’ and ‘primary’ myosin-binding sites, respectively, on the actin filaments preventing actin–myosin interaction.14 Deletion of the positively charged Lys184 breaks one of the salt-bridges involved in Md–actin interaction and destabilizes some hydrogen bonds,14 probably shifting the β-bulge motif into a more rigid β-turn (Wakabayashi, personal communications). On the other hand, the β-sheet of the Md, containing Lys184, does not interact with cTnC14 as, for example, Arg146 in the Ir does. Therefore, the Ca2+-induced conformational change (switch-on) of cTnI during systole is not affected by the Lys184 deletion, which is in agreement with our and previous interpretations from functional measurements.4

Considering the proposed mechanism in Ref.28 by which the initially intrinsically unfolded Md at high [Ca2+] nucleates by binding to actin (flycasting hypothesis) upon Ca2+-removal, Lys184 deletion could interfere with the efficient flycasting activity. As a consequence, a possible scenario is that the kinetics of the complete transfer of Ir to actin (switch-off) can be indirectly disturbed by the mutation in Md, but not the reverse transition towards cTnC (switch-on) upon Ca2+-binding to cTnC.

The hypothesis is supported by considering both, our present and previous6 functional results. In Ref.6 the mutation R146G, expressed in TG mice, slowed down both relaxation kinetics and kACT at high [Ca2+] implying that R146G in the Ir impaired both full inhibition of the force-generating actin–myosin interactions in the absence of Ca2+ (as {Delta}K184 does) and the release of this inhibition at high [Ca2+] (as {Delta}K184 does not). This is because Ir competes with myosin for the ‘primary’ myosin-binding site on actin (at low [Ca2+]) and binds to cTnC (at high [Ca2+]), which is not the case for the β-sheet, containing Lys184, and the downstream located {alpha}-helices of the Md.14

Based on this, we propose that the Lys184 deletion impairs the rapidly tumbling Md upon Ca2+ removal and, thus, restricts tropomyosin to adopt an optimal configuration for an efficient inhibition of the actin–myosin interaction, but it could also indirectly affect the inhibitory role of the Ir.

Thus, our findings are in line with the hypothesis28 that perturbations of folding of the Md primarily affect relaxation kinetics. Following a similar way of thinking and considering previous results in Ref.6 we assume that a perturbation of the folding-unfolding kinetics of the Ir (e.g. by the mutation R146G) would impair both relaxation and Ca2+ activation kinetics.

Following this mechanism, destabilization of the switched-off state leads to an impaired inhibition of force-generating actin–myosin interactions as indicated by the increased passive force in TG myofibrils and its reduction by BDM close to the passive force level recorded for nTG myofibrils.

The Lys184 deletion decreased the switch-off rate, preserved the switch-on rate and consequently increased the switch on–off equilibrium constant of troponin. This enables cross-bridges to reattach to the actin filament even at very low [Ca2+] during relaxation. The slow-down of relaxation is comparable with that caused by an incomplete Ca2+-removal from cTnC, i.e. incomplete turning-off of the thin filament: it has been shown that myofibrillar relaxation kinetics are slowed down very sensitively by turning to a lower, residual Ca2+ activation instead of a complete relaxation.8,29

When added during the relaxation process, BDM prevents the reattachment of cross-bridges by trapping them in non-force-generating states19 such that the effect of the mcTnI{Delta}K184 mutation to incompletely inhibit force-generating actin–myosin interactions becomes diminished. As a consequence, the relaxation time constant, {tau}REL, of TG myofibrils approaches that of nTG myofibrils. It had also been shown that BDM fastens the rate of cross-bridge detachment, presumably by increasing the affinity of nucleotide binding to myosin.30 Thus, targeting myosin motors with uncompetitive inhibitors, such as BDM, could accelerate the relaxation kinetics and therefore ameliorate the consequences of the incomplete inhibition of actin–myosin interaction, i.e. the slow-down in relaxation kinetics. This finding could suggest new hints concerning a potential therapeutic approach in the treatment of sarcomeric diastolic dysfunction induced by a less-functional inhibition of actin–myosin interaction in the absence of Ca2+.

A proteolytic cTnI truncation (hcTnI1–192),31 not related to FHC, exhibited features resembling those induced by mcTnI{Delta}K184: impairment of the relaxation kinetics, Ca2+ sensitization and loss in cooperativity, which probably occurred because Ca2+ activation of force becomes more dependent, via the Ir, on Ca2+-binding to cTnC, which is a relatively low cooperative process.

Similar features were reported in a recent study using cardiomyocytes expressing the mutation hcTnIR193H linked to restrictive cardiomyopathy (RCM).22 The authors concluded that the mutation R193H weakens the inhibitory function of hcTnI resulting in an elevated Ca2+-independent basal mechanical tone. Thus, an insufficient inhibition of actin–myosin interaction could be a common dysfunction caused by both RCM (e.g. hcTnIR193H) and FHC (e.g. hcTnI{Delta}K183) mutations and would accommodate the proposed mechanism.

In conclusion, TG myofibrils generate higher forces at low and high [Ca2+] and exhibit an increased Ca2+ sensitivity of contraction, which is based on the loss of cooperative inhibition rather than on facilitated activation and accompanied by Ca-SPOC. The incomplete inhibition of actin–myosin interactions in the absence of Ca2+ in TG myofibrils and not an alteration of the intrinsic cross-bridge kinetic properties, or a shift of {alpha}-MHC to the slow β-MHC, can explain the slow-down of the relaxation kinetics. Therefore, the impaired relaxation and elevated Ca2+-independent residual force of the sarcomeres carrying mcTnI{Delta}K184 could involve disturbances in ventricular relaxation, distensibility or filling of the non-hypertrophied murine transgenic heart with probable unfavourable consequences on the diastolic function.


   Supplementary material
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
Funding
 References
 
Supplementary material is available at Cardiovascular Research online.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
References
 
This work was supported by the Deutsche Forschungsgemeinschaft (SFB12-A2/070021); Center for Molecular Medicine Cologne (TV91); and Koeln-Fortune Cologne (109/2003).


   Acknowledgements
 
Stefan Zittrich and Christina Schroth are acknowledged for their technical assistance.

Conflict of interest: none declared.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Supplementary material
 Funding
 References
 

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R. J. Solaro and P. P. de Tombe
Review focus series: sarcomeric proteins as key elements in integrated control of cardiac function
Cardiovasc Res, March 1, 2008; 77(4): 616 - 618.
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