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Cardiovascular Research 2003 57(2):505-514; doi:10.1016/S0008-6363(02)00662-4
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Copyright © 2003, European Society of Cardiology

The effect of myosin light chain 2 dephosphorylation on Ca2+-sensitivity of force is enhanced in failing human hearts

J van der Veldena,*, Z Pappb, N.M Boontjea, R Zarembaa, J.W de Jongc, P.M.L Janssend, G Hasenfussd and G.J.M Stienena

aLaboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
bUDMHSC Department of Cardiology, Debrecen, Hungary
cThorax Center, Erasmus University, Rotterdam, The Netherlands
dDepartment of Cardiology and Pneumology, Georg-August-University Goettingen, Goettingen, Germany

* Corresponding author. Tel.: +31-20-444-8121; fax: +31-20-444-8255 j.van_der_velden.physiol{at}med.vu.nl

Received 11 March 2002; accepted 10 September 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Phosphorylation of the myosin light chain 2 (MLC-2) isoform expressed as a percentage of total MLC-2 was decreased in failing (21.1±2.0%) compared to donor (31.9±4.8%) hearts. To assess the functional implications of this change, we compared the effects of MLC-2 dephosphorylation on force development in failing and non-failing (donor) human hearts. Methods: Cooperative effects in isometric force and rate of force redevelopment (Ktr) were studied in single Triton-skinned human cardiomyocytes at various [Ca2+] before and after protein phosphatase-1 (PP-1) incubation. Results: Maximum force and Ktr values did not differ between failing and donor hearts, but Ca2+-sensitivity of force (pCa50) was significantly higher in failing myocardium ({Delta}pCa50=0.17). Ktr decreased with decreasing [Ca2+], although this decrease was less in failing than in donor hearts. Incubation of the myocytes with PP-1 (0.5 U/ml; 60 min) decreased pCa50 to a larger extent in failing (0.20 pCa units) than in donor cardiomyocytes (0.10 pCa units). A decrease in absolute Ktr values was found after PP-1 in failing and donor myocytes, while the shape of the Ktr–Ca2+ relationships remained unaltered. Conclusions: Surprisingly, the contractile response to MLC-2 dephosphorylation is enhanced in failing hearts, despite the reduced level of basal MLC-2 phosphorylation. The enhanced response to MLC-2 dephosphorylation in failing myocytes might result from differences in basal phosphorylation of other thin and thick filament proteins between donor and failing hearts. Regulation of Ca2+-sensitivity via MLC-2 phosphorylation may be a potential compensatory mechanism to reverse the detrimental effects of increased Ca2+-sensitivity and impaired Ca2+-handling on diastolic function in human heart failure.

KEYWORDS Cardiomyopathy; Contractile apparatus; Contractile function; Myocytes; Signal transduction


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Ca2+ binding to the thin filament initiates the actin–myosin interaction, the cross-bridge cycle. Phosphorylation of thin filament proteins (e.g., troponin) plays a key role in regulation of Ca2+-sensitivity of this interaction [1–3]. However, the effect of myosin light chain 2 (MLC-2) phosphorylation on Ca2+-responsiveness is also of interest, as it relates to a thick filament-based regulatory process, which is complementary to and possibly interfering with that of the thin filament.

Myosin is composed of two heavy chains and two pairs of light chains. Each pair of light chains consists of an essential light chain (myosin light chain 1) and a regulatory light chain (myosin light chain 2) [4]. Two isoforms of the ventricular MLC-2 are known (MLC-2 and MLC-2*) [4], which may both be phosphorylated by Ca2+/calmodulin-dependent myosin light chain kinase (MLCK) [5] and protein kinase C (PKC) [6] and dephosphorylated by light chain phosphatase [7]. In human heart failure increased activity has been reported for PKC [8] and for Ca2+/calmodulin-dependent protein kinase (CaM-kinase), which may phosphorylate and activate MLCK [9]. In addition, type 1 phosphatase activity was increased in failing human myocardium [10]. Recently a decreased MLC-2 phosphorylation was observed in end-stage failing human hearts [11,12].

During the past years several animal studies have been performed concerning the effect of MLC-2 phosphorylation on myocardial contraction [13–16]. Phosphorylation of MLC-2 has been found to increase Ca2+-responsiveness of force [13–16] and the rate of force redevelopment (Ktr) [7]. Based on this latter finding it was proposed that the increase in Ca2+-responsiveness after MLC-2 phosphorylation resulted from a change in cross-bridge cycling kinetics [7]. However, data in human myocardium concerning the effect of MLC-2 phosphorylation on Ca2+-responsiveness of force are limited to the atrium [7]. Therefore, in the present study the effects of altered MLC-2 phosphorylation on Ca2+-sensitivity of force and Ktr were determined in human ventricular myocardium.

In failing human myocardium changes have been found in both thin and thick filament proteins [11,12,17–19], which might influence the effect of MLC-2 (de)phosphorylation on cross-bridge cycling kinetics in heart failure. Since basal MLC-2 phosphorylation is decreased in end-stage human heart failure [11,12], we hypothesized that the response to MLC-2 dephosphorylation is decreased in failing compared to non-failing donor myocardium. To test this hypothesis, the effect of MLC-2 dephosphorylation on isometric force development and rate of force redevelopment were studied at various [Ca2+] in single Triton-skinned myocytes from donor and end-stage failing human hearts.

Surprisingly, our results indicate that the response to MLC-2 dephosphorylation is enhanced in human heart failure, despite the decreased basal level of MLC-2 phosphorylation. Changes in the endogenous phosphorylation status of other thin and thick filament proteins in human heart failure may have altered the response to protein phosphatase-1 (PP-1).


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Biopsies
Left ventricular biopsies were obtained during heart transplantation surgery from six explanted end-stage failing (New York Heart Association class IV) hearts (five males, one female; age range 41–61 years) and from three non-failing donor hearts (one male, two females; age range 26–46 years). Heart failure resulted from ischemic (n=3) or dilated (n=3) cardiomyopathy. All patients received angiotensin converting enzyme inhibitors and diuretics. Some patients also received anti-arrhythmic agents, anticoagulants, digitoxin and/or nitrates. The tissue was transported in cardioplegic solution (ranging from half an hour to 14 h) and upon arrival in the laboratory, stored in liquid nitrogen. Samples were obtained after informed consent and with approval of the local Ethical Committees. The investigation conforms with the principles outlined in the Declaration of Helsinki (Cardiovascular Research 1997;35:2–3).

2.2 Two-dimensional gel electrophoresis
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) [7,13] was performed on donor and failing heart samples to determine the phosphorylation status of MLC-2 and troponin T (TnT). Tissue samples were treated with trichloroacetic acid (TCA) to fixate the phosphorylation status of contractile proteins [7,13]. Samples (600 µg dry weight) were loaded on immobiline strips with a pH gradient of 4.5 to 5.5 (Amersham Pharmacia Biotech, Uppsala, Sweden). In the second dimension proteins were separated by sodium dodecyl sulfate (SDS)–PAGE [19]. Gels were stained with Coomassie Blue, scanned and analyzed using Image Quant (Molecular Dynamics) [11]. We have checked the linearity of the Coomassie-staining of the protein spots to be analyzed. When 300 and 600 µg of tissue were loaded on the gels the density of all protein spots of interest were in the linear range.

To investigate PP-1 specificity a suspension of Triton-skinned donor cardiomyocytes was incubated in 1 ml of relaxing solution with and without PP-1 (0.5 U/ml, lot no. 16757; Upstate Biotechnology) for 60 min at room temperature. Subsequently, TCA-treated cells (75 µg dry weight) were analyzed by 2D-PAGE. These gels were silver-stained to enhance resolution.

2.3 Myocyte isolation
Cardiomyocytes were mechanically isolated and mounted in the experimental set-up as described previously [20]. Before mechanical isolation, tissue was defrosted in relaxing solution (pH 7.0; in mmol/l: free Mg2+ 1, KCl 145, EGTA 2, ATP 4, imidazole 10). During the isolation the tissue was kept on ice. Isolated myocytes were immersed for 5 min in relaxing solution containing 1% Triton X-100. Triton removes soluble and membrane-bound kinases and phosphatases and thereby arrests the phosphorylation status of myofibrillar proteins. To exclude an effect of kinases/phosphatases during the isolation, we have analyzed the protein composition in a human ventricular tissue sample before and after the complete isolating procedure (mechanical isolation and skinning in Triton). The 2D-gels did not reveal differences in the phosphorylation status of contractile proteins between the samples taken before and after myocyte isolation (not shown).

Triton-skinned myocytes enable the study of myofibrillar contractile properties under standardized conditions (i.e. composition of the intracellular medium, sarcomere length) without disturbing factors present in the intact heart (i.e. hormonal factors, variable [Ca2+]). To remove Triton, cells were washed twice in relaxing solution. Thereafter, a single myocyte was attached between a force transducer and a piezoelectric motor.

2.4 Experimental protocol
Isometric force measurements were performed at 15 °C and a sarcomere length, measured in relaxing solution, of 2.2 µm. The composition of relaxing and activating solutions used during force measurements was calculated as described by Fabiato [21]. The pCa, i.e. –log10[Ca2+], of the relaxing and activating solution (pH 7.1) were, respectively, 9 and 4.5. Solutions with intermediate free [Ca2+] were obtained by mixing of the activating and relaxing solutions. After the first control activation at saturating (maximal) [Ca2+] (pCa=4.5), resting sarcomere length was readjusted to 2.2 µm, if necessary. The second control measurement was used to calculate maximal isometric tension (i.e. force divided by cross-sectional area). The next four to five measurements were carried out at submaximal [Ca2+] followed by a control measurement. Force values obtained in solutions with submaximal [Ca2+] were normalized to the interpolated control values. After the initial force–pCa series, the myocyte was incubated in relaxing solution containing 0.5 U/ml PP-1 and 6 mM dithiothreitol (DTT) for 60 min at 20 °C. Thereafter, the force–pCa series was repeated.

In addition to MLC-2, troponin I (TnI) may be dephosphorylated by PP-1. To investigate if dephosphorylation of TnI had occurred after PP-1, a number of myocytes were treated with protein kinase A (PKA), which is known to phosphorylate TnI and to decrease Ca2+-sensitivity of force [3]. After the second force–pCa series, the myocytes were incubated in relaxing solution containing the catalytic subunit of PKA (3 µg/ml, batch no. 35H9522; Sigma) and 6 mM DTT for 40 min at 20 °C. This treatment was followed by a third series of force–pCa measurements.

Rate of force redevelopment (Ktr) was determined at pCa values ranging from 4.5 to 5.4 using the slack-test as described previously [22]. Briefly, when a steady level of force was developed in activating solution the myocyte was rapidly slackened and re-stretched by 20% of its length. Upon slackening force drops to zero and upon re-stretch force redevelopment occurs to the initial steady level. Force redevelopment was fitted to a single exponential to estimate Ktr. At low [Ca2+] (pCa>5.4) force redevelopment could not be fitted accurately due to the low signal-to-noise ratio.

2.5 Data analysis
Force–pCa relations were fit to a modified Hill equation:

Formula
F is steady-state force, F0 denotes the steady force at saturating [Ca2+], nH reflects the steepness of the relationship, and Ca50 (or pCa50) represents the midpoint of the relation. Force–pCa relations obtained after PP-1 contained a small component approximated by an offset, k. To determine nH at high (pCa<pCa50; n1) and low (pCa>pCa50; n2) [Ca2+], force–pCa data were analyzed using a Hill plot transformation [3]:

Formula
Frel is the relative force, F(Ca2+)/F0.

Values are given as means±S.E.M. of n experiments. Mean values for donor and failing samples were compared using an unpaired Student t-tests. Paired Student t-tests were used when comparing maximal force, Ca2+-sensitivity and Ktr of single cardiomyocytes before and after PP-1 treatment. A two-tailed P-value of less than 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Myosin light chain 2 phosphorylation
Fig. 1 shows Coomassie-stained 2D-gels from a donor and a failing heart illustrating the difference observed in MLC-2 phosphorylation. 2D-PAGE analysis revealed decreased phosphorylation of the MLC-2 isoform (MLC-2P) expressed as a percentage of total MLC-2 in failing compared to non-failing ventricular tissue (P<0.05; Table 1) from 31.9 to 21.1%. The percentage of phosphorylated and unphosphorylated MLC-2* isoform did not differ between failing and donor myocardium. Troponin T (TnT) phosphorylation was also visible on these gels. The amount of monophosphorylated TnT, in percentage of total TnT, did not differ significantly between donor (69.7±3.1%) and failing (72.3±3.0%) hearts. To investigate PP-1 specificity donor myocytes were incubated with and without PP-1 and analyzed on silver-stained 2D-gels. The densitometric scans shown in Fig. 1C indicate that MLC-2 was completely dephosphorylated by incubation of myocytes in PP-1 containing (0.5 U/ml; 60 min) relaxing solution (dashed line), while incubation in relaxing solution without PP-1 did not alter MLC-2 phosphorylation (continuous line). Since silver-staining is only linear in a narrow concentration range, these gels are not optimal for quantitative analysis. Therefore, the densitometric scans shown in panel (C) cannot be used quantitatively. Phosphorylation of TnT and MLC-1 was preserved after PP-1 treatment (not shown).


Figure 1
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  		Fig. 1 2-D gels to illustrate MLC-2 composition in a donor (A) and a failing (B) heart. MLC-2 phosphorylation was decreased in end-stage failing myocardium. (C) shows densitometric scans of MLC-2 composition in donor cardiomyocytes treated without (continuous line) and with (dashed line) PP-1. PP-1 specifically dephosphorylated MLC-2, while TnT and MLC-1 phosphorylation were preserved (not shown). IEF, isoelectric focussing; TnT and TnTP, unphosphorylated and monophosphorylated troponin T; MLC-1, myosin light chain 1; MLC-2, myosin light chain 2 composed of two isoforms (2 and 2*), which are both partly phosphorylated (respectively, 2P and 2*P). The proteins have been identified using commercially available monoclonal antibodies in Western immunoblotting.

 

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  		Table 1 MLC-2 phosphorylation

 
3.2 Force measurements
Recordings of force development during maximal (pCa 4.5) and submaximal activation (pCa 5.6) before and after incubation in PP-1 are shown in Fig. 2. Maximal isometric tension amounted to 34.8±5.8 and 34.7±6.7 kN/m2 in donor (17 myocytes) and failing (19 myocytes) hearts, respectively. Passive tension did not differ between donor (1.8±0.2 kN/m2) and failing (2.4±0.5 kN/m2) myocardium.


Figure 2
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  		Fig. 2 Recordings of isometric force development during maximal (A) and submaximal (B) activation before (continuous recording) and after (dashed recording) PP-1 incubation in a failing myocyte. The abrupt changes in force mark the transitions of the preparation through the interface between solution and air. The dashed horizontal lines indicate the passive force level. To determine Ktr the myocyte was slackened when force development reached a steady level and restretched (slack-test). Registrations of force redevelopment at maximal [Ca2+] before and after incubation with PP-1 are shown in (C).

 
The average force–pCa relationships obtained in donor and failing hearts before PP-1 treatment are shown in Fig. 3A. It can be noted that Ca2+-responsiveness of force was significantly higher in end-stage failing hearts (pCa50=5.65±0.03) than in donor myocardium (pCa50=5.48±0.01) (P<0.05). The force–pCa relation tended to be less steep in failing (nH=2.34±0.22) than in donor (nH=3.02±0.16) hearts, although the difference did not reach statistical significance. To determine steepness at high [Ca2+] (n1) and at low [Ca2+] (n2) the force–pCa data were analyzed using a Hill plot transformation (inset Fig. 3A). Statistical analysis revealed that the steepness of the force–pCa relation at high [Ca2+] (n1) was significantly lower in failing than in donor hearts. Moreover, in donor hearts n1 was significantly higher than n2.


Figure 3
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  		Fig. 3 Force (A) and Ktr (B) as a function of pCa in donor (n=3) and failing (n=6) hearts before PP-1 treatment. The myocyte values per donor/patient were averaged to obtain individual values for donor and patient hearts. Force at submaximal [Ca2+] was normalized to the control force at saturating [Ca2+]. Ca2+-sensitivity of force was significantly increased in failing myocardium compared to donor hearts ({Delta}pCa50=0.17). Ktr decreased with decreasing [Ca2+], although this decrease was less steep in failing than in donor hearts. A, inset: Hill plot transformations revealed that steepness was significantly different at high [Ca2+] in donor (n1=3.26±0.39) and in failing (n1=1.72±0.15) myocardium (P<0.05), while at low [Ca2+] values were similar (n2=respectively, 1.46±0.24 and 1.65±0.10 in donor and failing hearts). (C) The relation between Ktr and relative force was approximately linear in both donor and failing hearts. *P<0.05, donor versus failing.

 
The rate of force redevelopment was determined at pCa values ranging from 4.5 to 5.4. Maximal Ktr did not differ significantly between donor (0.77±0.02 1/s) and failing (0.86±0.05 1/s) hearts. The dependency of Ktr on [Ca2+] in donor and failing hearts is presented in Fig. 3B. Ktr decreased with increasing pCa, although this decrease was less pronounced in failing myocardium. At pCa 5.2 and 5.4 a significant difference in Ktr was found between donor and failing hearts. In both donor and failing hearts approximately linear relations were found between Ktr and relative force (Fig. 3C).

3.3 Effect of PP-1 on Ca2+-sensitivity of force
Fig. 2 illustrates that maximal force (pCa 4.5) decreased by approximately 10% after PP-1. This decline is attributable to the duration of the incubation period as it was also found in control experiments described below. However, PP-1 significantly decreased Ca2+-sensitivity of force in donor (n=13) and failing (n=15) cardiomyocytes (Fig. 4). The shift in pCa50 after PP-1 treatment was significantly larger in failing (0.20 pCa units) than in donor (0.10 pCa units) cardiomyocytes. Hence PP-1 reduced the difference existing in Ca2+-responsiveness between donor and failing hearts from 0.17 to 0.08 pCa units (P<0.05). It should be noted that at low [Ca2+] (pCa>6) relative force values were somewhat elevated after PP-1 in both donor and failing myocytes. PP-1 did not alter the steepness of the force–pCa relationships. In Table 2 pCa50 and nH values before and after PP-1 are summarized.


Figure 4
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  		Fig. 4 Ca2+-sensitivity of force before and after PP-1 in donor (A; n=13) and failing (B; n=15) cardiomyocytes. Force was measured at different [Ca2+] before and after PP-1 in the same myocyte. Force at submaximal [Ca2+] was normalized to the control force at saturating [Ca2+]. PP-1 decreased pCa50 by 0.10 and 0.20 units in donor and failing cardiomyocytes, respectively. *P<0.05, before versus after PP-1.

 

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  		Table 2 pCa50 and nH values before and after PP-1

 
To investigate if the decrease in Ca2+-responsiveness could be attributed to PP-1 or was due to time-dependent alterations during the incubation period, two donor cardiomyocytes were incubated in relaxing solution for 60 min at 20 °C without addition of PP-1 (time-control). No alterations were observed in the midpoint (5.44±0.02 and 5.45±0.01) and steepness (2.71±0.29 and 2.76±0.13) of the force–pCa relationship before and after incubation in relaxing solution, respectively.

To assess whether the PP-1-induced shift in Ca2+-sensitivity was maximal, two failing cardiomyocytes were incubated for 60 min with a 10-fold higher dose of PP-1 (5 U/ml). The decrease in pCa50 (0.21 pCa units) was similar to the decrease after incubation with 0.5 U/ml PP-1.

In addition to MLC-2, troponin I may be dephosphorylated by PP-1. TnI dephosphorylation would increase Ca2+-sensitivity of force and would interfere with the effect of MLC-2 dephosphorylation on Ca2+-responsiveness. To investigate if TnI dephosphorylation occurred, myocytes were incubated with PKA after PP-1 treatment. In a previous study [23], we observed a small non-significant decrease in Ca2+-responsiveness of force after PKA in donor cardiomyocytes ({Delta}pCa50=0.02), but a significant decrease in failing ({Delta}pCa50=0.24) cells. Hence, if dephosphorylation of TnI would occur by PP-1 the effect of PKA on Ca2+-sensitivity of force would be enlarged. However, after PP-1 treatment, PKA did not significantly alter Ca2+-responsiveness of force in donor cardiomyocytes (n=4), while the decrease in Ca2+-responsiveness after PKA in failing cardiomyocytes (n=4) was smaller ({Delta}pCa50=0.10) than in the absence of PP-1 [23]. From these data we consider it likely that PP-1 did not alter the phosphorylation status of TnI. Hence, the changes in myofilament contractility after PP-1 may be fully attributed to MLC-2 dephosphorylation. These experiments also showed that the difference in Ca2+-responsiveness between donor and failing myocytes was completely abolished by the combined action of PP-1 and PKA.

3.4 Effect of PP-1 on rate of force redevelopment
Fig. 2C illustrates force redevelopment after a slack-test, before and after incubation with PP-1 at maximal [Ca2+]. The mean Ktr values as a function of pCa are summarized in Fig. 5. Ktr values were not significantly altered in the time-control. PP-1 treatment decreased Ktr values at maximal and submaximal [Ca2+] in both donor and failing cardiomyocytes (Fig. 5A and B). However, relative Ktr values (normalized to Ktr at pCa 4.5) remained unaltered after PP-1 indicating that MLC-2 dephosphorylation did not alter Ca2+-responsiveness of Ktr. The linear relationships between absolute Ktr and relative force shown in Fig. 6 reveal that PP-1 decreased the force-dependency of Ktr in both donor and failing myocytes.


Figure 5
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  		Fig. 5 Absolute Ktr values as a function of pCa before and after PP-1 in donor (A; n=13) and failing (B; n=15) cardiomyocytes. *P<0.05, before versus after incubation.

 

Figure 6
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  		Fig. 6 Ktr as a function of relative force before and after PP-1 in donor (A, n=13) and failing (B, n=15) cardiomyocytes. Force at submaximal [Ca2+] was normalized to values at saturating [Ca2+]. Force-dependence of Ktr decreased after PP-1 in both donor and failing myocytes.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Although the basal level of MLC-2 phosphorylation was decreased in failing myocardium, the response to MLC-2 dephosphorylation was enhanced in human heart failure. Therefore our hypothesis ‘the response to MLC-2 dephosphorylation is decreased in failing compared to non-failing hearts’ has to be discarded.

4.1 Differences between donor and failing myocardium
The present study confirms and extends our previous observation [11,12] that the phosphorylation level of the MLC-2 isoform is decreased in end-stage human heart failure. However, no significant difference was found in phosphorylation of the less abundant MLC-2* isoform between donor and failing hearts. It was mentioned previously by Morano et al. [17] that incubation in cardioplegic solution would dephosphorylate MLC-2. However, although our biopsies were transported in cardioplegic solution, we did not observe complete dephosphorylation of MLC-2. In fact, in this study and in a previous study [12] we observed that the level of TnI, TnT and MLC-2 phosphorylation was quite homogeneous in the donor and failing group.

In accordance with previous studies on human hearts, no difference was observed in maximal isometric tension, while Ca2+-responsiveness of force was significantly increased in end-stage failing hearts [17,18]. In a recent study [12], we have shown that the increased Ca2+-responsiveness in failing human hearts most likely results from altered phosphorylation of myosin light chain 2 (MLC-2) and troponin I (TnI).

In previous studies on rabbit skeletal and rat cardiac muscle [24–26], the force–pCa relationship was found to be biphasic, with the strongest cooperativity present at low [Ca2+] (n1<n2), i.e. the force–pCa relation is steeper at low [Ca2+] than at high [Ca2+]. It should be noted that albeit our data at low Ca2+ were quite noisy, the opposite (n1n2) was found in donor hearts (inset Fig. 3A). In failing human hearts no difference in n1 and n2 was found, but n1 was significantly less than in donor hearts, which indicates that the cooperativity at high [Ca2+] is somewhat depressed in human heart failure.

In addition to increased force levels at submaximal [Ca2+], Ktr tended to be higher in failing than in donor hearts, with a significant difference at pCa≥5.2 (Fig. 3B). These findings indicate that, like an enhanced Ca2+-sensitivity of force, the responsiveness of Ktr to Ca2+ is also increased in human heart failure.

Ktr values in human ventricular myocytes were 10- to 20-fold lower than Ktr values found previously in rat ventricular myocardium [22,27,28], but similar to those found in pig ventricular tissue [29]. The differences in Ktr may be attributed to differences in myosin heavy chain (MHC) composition which is a determinant of the rate of tension recovery [30]. In adult rats almost exclusively the fast {alpha}-MHC is present, while the slow β-MHC isoform predominates in human [19,31] and pig [31] ventricular myocardium.

In agreement with previous studies on myocardial tissue [25,27,28], Ktr decreased with increasing pCa in both donor and failing hearts, indicating that redevelopment of force gets slower with decreasing [Ca2+]. When Ktr values were plotted against relative force at different [Ca2+] (Fig. 3C), an approximately linear relationship was obtained in donor and failing hearts. In skeletal muscle a curvilinear relationship between Ktr and force has been observed [26,32]. Data concerning the Ktr–force relationship in cardiac tissue have been conflicting. Wolff et al. [27] observed a linear relationship between Ktr and force in rat trabeculae, while others [25,28] found a curvilinear relationship in rat and guinea-pig myocardium. Since we could not accurately determine Ktr at low [Ca2+], it remains to be established if the relation between Ktr and force in human myocardium is linear or becomes curvilinear at high pCa values.

4.2 Effect of PP-1
Surprisingly, PP-1 decreased Ca2+-responsiveness of force to a larger extent in failing than in donor cardiomyocytes, although the endogenous level of MLC-2 phosphorylation was significantly lower in failing than in donor hearts. After PP-1 the difference in Ca2+-responsiveness between failing and donor myocardium was diminished, while it was completely abolished after subsequent treatment with PKA. These results suggest that the difference in Ca2+-responsiveness does not reflect intrinsic differences in contractile protein isoform composition, but rather differences in endogenous phosphorylation status of thin and thick filament proteins. The enhanced response to MLC-2 dephosphorylation in failing myocytes may result from differences in TnI phosphorylation levels between donor and failing hearts [12]. Since in non-failing donor hearts Ca2+-responsiveness is lower compared to failing hearts, the desensitizing effect of PP-1 may already be saturated, while failing hearts may be more susceptible to MLC-2 dephosphorylation due to the enhanced Ca2+-sensitivity of force.

An alternative explanation for the difference in responsiveness to MLC-2 dephosphorylation between donor and failing hearts could be differences in the phosphorylation level of myosin binding protein-C (MyBP-C). It has been suggested that phosphorylation of MyBP-C performs a permissive role in contraction of the heart muscle [33,34]. Phosphorylation of MyBP-C would decrease the restriction of myosin and would thereby facilitate changes in the actin–myosin interaction upon (de)phosphorylation of MLC-2 [33]. Thus, a difference in MyBP-C phosphorylation between donor and failing hearts could alter the response to MLC-2 (de)phosphorylation. An increased activity of Ca2+/calmodulin-dependent protein kinase, which may phosphorylate MyBP-C [35], has been found in idiopathic dilated cardiomyopathy [9]. Knowledge on the phosphorylation status of MyBP-C in healthy and failing myocardium is of great interest and warrants further study, particular in human tissue.

The decrease in Ca2+-sensitivity of force and in absolute Ktr values after dephosphorylation of MLC-2 are in agreement with the results obtained after phosphorylation of MLC-2 in cardiac and skeletal muscle [7,13]. Huiting-Hollander et al. [16] reported no difference in Ca2+-responsiveness of Ktr between control mice expressing endogenous MLC-2, in which 39% of the total MLC-2 was phosphorylated, and knock-in mice expressing a non-phosphorylable form of MLC-2. In accordance, we did not observe an effect of PP-1 on Ca2+-dependency of relative Ktr values. Since Ca2+-sensitivity of force development was decreased, while Ca2+-dependency of Ktr remained unaffected, the force-dependency of Ktr in both donor and failing myocytes was decreased after PP-1. The increase in Ktr upon MLC-2 phosphorylation in rabbit psoas fibers might be due to an increase in fapp [7]. The effect of MLC-2 dephosphorylation on force development and Ktr in human cardiomyocytes observed in the present study both may be explained by a decrease in fapp. It has been proposed that upon MLC-2 dephosphorylation myosin heads move toward the backbone of the thick filament away from the thin filament, diminishing the probability of actin to myosin binding [36]. Hence, the decrease in cross-bridge attachment may originate from a structural change in the thick filament.

4.3 Clinical relevance
Our results indicate that the enhanced response to MLC-2 dephosphorylation in failing myocytes, despite the decreased basal level of MLC-2 phosphorylation, might result from differences in endogenous phosphorylation of thin and thick filament proteins between donor and failing hearts. By and large, decreased MLC-2 phosphorylation in end-stage human heart failure may be a compensatory mechanism in order to improve myocardial contractility by opposing the detrimental effects of increased Ca2+-responsiveness of force and impaired Ca2+-handling on diastolic function.

Time for primary review 28 days.


   Acknowledgements
 
Supported by the Netherlands Heart Foundation (grant 99.155).


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

  1. Garvey J.L, Kranias E.G, Solaro R.J. Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts. Biochem J (1988) 249:709–714.[ISI][Medline]
  2. Noland T.A, Raynor R.L, Kuo J.F. Identification of sites phosphorylated in bovine cardiac troponin I and troponin T by protein kinase C and comparative substrate activity of synthetic peptides containing the phosphorylation sites. J Biol Chem (1989) 264:20778–20785.[Abstract/Free Full Text]
  3. Strang K.T, Sweitzer N.K, Greaser M.L, Moss R.L. β-Adrenergic receptor stimulation increases unloaded shortening velocity of skinned single ventricular myocytes from rats. Circ Res (1994) 74:542–549.[Abstract/Free Full Text]
  4. Morano I, Wankerl M, Böhm M, Erdman E, Rüegg J.C. Myosin P-light chain isoenzymes in the human heart: evidence for diphosphorylation of the atrial P-light chain isoform. Basic Res Cardiol (1989) 84:298–305.[CrossRef][ISI][Medline]
  5. Frearson N, Perry S.V. Phosphorylation of the light-chain components of myosin from cardiac and red skeletal muscles. Biochem J (1975) 151:99–107.[ISI][Medline]
  6. Venema R.C, Raynor R.L, Noland T.A, Kuo J.F. Role of protein kinase C in the phosphorylation of cardiac myosin light chain 2. Biochem J (1993) 294:401–406.[ISI][Medline]
  7. Morano I. Effects of different expression and posttranslational modifications of myosin light chains on contractility of skinned human cardiac fibers. Basic Res Cardiol (1992) 87:129–141.[ISI][Medline]
  8. Bowling N, Walsh R.A, Song G, et al. Increased protein kinase C activity and expression of Ca2+-sensitive isoforms in the failing human heart. Circulation (1999) 99:384–391.[Abstract/Free Full Text]
  9. Kirchhefer U, Schmitz W, Scholz H, Neumann J. Activity of cAMP-dependent protein kinase and Ca2+/calmodulin-dependent protein kinase in failing and nonfailing human hearts. Cardiovasc Res (1999) 42:254–261.[Abstract/Free Full Text]
  10. Neumann J, Eschenhagen T, Jones L.R, et al. Increased expression of cardiac phosphatases in patients with end-stage heart failure. J Mol Cell Cardiol (1997) 29:265–272.[CrossRef][ISI][Medline]
  11. Van der Velden J, Klein L.J, Zaremba R, et al. Effects of calcium, inorganic phosphate and pH on isometric force in single skinned cardiomyocytes from donor and failing human hearts. Circulation (2001) 104:1140–1146.[Abstract/Free Full Text]
  12. Van der Velden J, Papp Z, Zaremba R, et al. Increased Ca2+-sensitivity of the contractile apparatus in end-stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res (in press).
  13. Morano I, Arndt H, Bächle-Stolz C, Rüegg J.C. Further studies on the effects of myosin P-light chain phosphorylation on contractile properties of skinned cardiac fibers. Basic Res Cardiol (1986) 81:611–619.[CrossRef][ISI][Medline]
  14. Noland T.A, Kuo J.F. Phosphorylation of cardiac myosin light chain 2 by protein kinase C and myosin light chain kinase increases Ca2+-stimulated actomyosin MgATPase activity. Biochem Biophys Res Commun (1993) 193:254–260.[CrossRef][ISI][Medline]
  15. Hollander M.S, Moss R.L. Dephosphorylation of regulatory light chain by protein phosphatase-1 decreases the Ca2+-sensitivity of tension in rat myocardium. Biophys J (1999) 76:A311.
  16. Huiting-Hollander M.S, Chen J, Chu P, Moss R.L. Modulation of mechanical properties due to regulatory light chain phosphorylation in myocardium. Biophys J (2001) 80:91a.
  17. Morano I, Hädicke K, Haase H, Böhm M, Erdmann E, Schaub M.C. Changes in essential myosin light chain isoform expression provide a molecular basis for isometric tension regulation in the failing human heart. J Mol Cell Cardiol (1997) 29:1177–1187.[CrossRef][ISI][Medline]
  18. Wolff M.R, Buck S.H, Stoker S.W, Greaser M.L, Mentzer R.M. Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies. J Clin Invest (1996) 98:167–176.[ISI][Medline]
  19. Van der Velden J, Klein L.J, van der Bijl M, et al. Isometric tension development and its calcium sensitivity in skinned myocyte-sized preparations from different regions of the human heart. Cardiovasc Res (1999) 42:706–719.[Abstract/Free Full Text]
  20. Van der Velden J, Klein L.J, van der Bijl M, et al. Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue. Cardiovasc Res (1998) 38:414–423.[Abstract/Free Full Text]
  21. Fabiato A. Myoplasmic free calcium concentration reached during the twitch of an intact isolated cardiac cell and during calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned cardiac cell from the adult rat or rabbit ventricle. J Gen Physiol (1981) 78:457–497.[Abstract/Free Full Text]
  22. Papp Z, van der Velden J, Stienen G.J.M. Calpain-1 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]
  23. Van der Velden J, de Jong J.W, Owen V.J, Burton P.B.J, Stienen G.J.M. Effect of protein kinase A on calcium sensitivity of force and its sarcomere length dependence in human cardiomyocytes. Cardiovasc Res (2000) 46:487–495.[Abstract/Free Full Text]
  24. Sweitzer N.K, Moss R.L. The effect of altered temperature on Ca2+-sensitive force in permeabilized myocardium and skeletal muscle. J Gen Physiol (1990) 96:1221–1245.[Abstract/Free Full Text]
  25. Vannier C, Chevassus H, Vassort G. Ca2+-dependence of isometric force kinetics in single skinned ventricular cardiomyocytes from rats. Cardiovasc Res (1996) 32:580–586.[Abstract/Free Full Text]
  26. Fitzsimons D.P, Patel J.R, Campbell K.S, Moss R.L. Cooperative mechanisms in the activation dependence of the rate of force redevelopment in rabbit skinned skeletal muscle fibers. J Gen Physiol (2001) 117:133–148.[Abstract/Free Full Text]
  27. Wolff M.R, McDonald K.S, Moss R.L. Rate of tension development in cardiac muscle varies with level of activator calcium. Circ Res (1995) 76:154–160.[Abstract/Free Full Text]
  28. Palmer S, Kentish J.C. Roles of Ca2+ and crossbridge kinetics in determining the maximum rates of Ca2+ activation and relaxation in rat and guinea pig skinned trabeculae. Circ Res (1998) 83:179–186.[Abstract/Free Full Text]
  29. Morano I, Österman A, Arner A. Rate of active tension development from rigor in skinned atrial and ventricular cardiac fibers from swine following photolytic release of ATP from caged ATP. Acta Physiol Scand (1995) 154:343–353.[ISI][Medline]
  30. Ventura-Clapier R, Mekhfi H, Oliviero P, Swynghedauw B. Pressure overload changes cardiac skinned-fiber mechanics in rats, not in guinea-pigs. Am J Physiol (1988) 254:H517–H524.[ISI][Medline]
  31. Morano I, Arndt H, Gärtner C, Rüegg J.C. Skinned fibers of human atrium and ventricle: myosin isoenzymes and contractility. Circ Res (1998) 62:632–639.
  32. Brenner B. Effect of Ca2+ on cross-bridge turnover kinetics in skinned single rabbit psoas fibers: Implications for regulation of muscle contraction. Proc Natl Acad Sci (1988) 85:3265–3269.[Abstract/Free Full Text]
  33. Winegrad S. Cardiac myosin binding protein C. Circ Res (1999) 84:1117–1126.[Abstract/Free Full Text]
  34. McClellan G, Kulikovskaya I, Winegrad S. Changes in cardiac contractility related to calcium-mediated changes in phosphorylation of myosin-binding protein C. Biophys J (2001) 81:1083–1092.[Abstract/Free Full Text]
  35. Schlender K.K, Bean L.J. Phosphorylation of chicken cardiac C-protein by calcium/calmodulin-dependent protein kinase II. J Biol Chem (1991) 266:2811–2817.[Abstract/Free Full Text]
  36. Levine R.J, Kensler R.W, Yang Z, Stull J.T, Sweeney H.L. Myosin light chain phosphorylation affects the structure of rabbit skeletal muscle thick filaments. Biophys J (1996) 71:898–907.[Abstract/Free Full Text]

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