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Cardiovascular Research 2005 65(1):221-229; doi:10.1016/j.cardiores.2004.09.029
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Copyright © 2004, European Society of Cardiology

Myocardial contraction is 5-fold more economical in ventricular than in atrial human tissue

N.A. Narolskaa,*, R.B. van Loonb, N.M. Boontjea, R. Zarembaa, S. Eiras Penasa, J. Russella, S.R. Spiegelenbergc, M.A.J.M. Huybregtsc, F.C. Visserb, J.W. de Jongd, J. van der Veldena and G.J.M. Stienena

aLaboratory for Physiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, van der Boechorststraat 7, 1081BT Amsterdam, The Netherlands
bDepartment of Cardiology, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, The Netherlands
cDepartment of Cardiac Surgery, Institute for Cardiovascular Research (ICaR-VU), VU University Medical Center, Amsterdam, The Netherlands
dThorax Center, Erasmus MC, Rotterdam, The Netherlands

* Corresponding author. Tel.: +31 20 444 8121; fax: +31 20 444 8255. Email address: na.narolska{at}vumc.nl

Received 20 March 2004; revised 7 September 2004; accepted 28 September 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
Objective: Cardiac energetics and performance depend on the expression level of the fast ({alpha}-) and slow (β-) myosin heavy chain (MHC) isoform. In ventricular tissue, the β-MHC isoform predominates, whereas in atrial tissue a variable mixture of {alpha}- and β-MHC is found. In several cardiac diseases, the slow isoform is upregulated; however, the functional implications of this transition in human myocardium are largely unknown. The aim of this study was to determine the relation between contractile properties and MHC isoform composition in healthy human myocardium using the diversity in atrial tissue.

Methods: Isometric force production and ATP consumption were measured in chemically skinned atrial trabeculae and ventricular muscle strips, and rate of force redevelopment was studied using single cardiomyocytes. MHC isoform composition was determined by one-dimensional SDS-gel electrophoresis.

Results: Force development in ventricular tissue was about 5-fold more economical, but nine times slower, than in atrial tissue. Significant linear correlations were found between MHC isoform composition, ATP consumption and rate of force redevelopment.

Conclusion: These results clearly indicate that even a minor shift in MHC isoform expression has considerable impact on cardiac performance in human tissue.

KEYWORDS Atrial function; Ventricular function; Contractile apparatus; Myocytes; Heart failure


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Acknowledgments
 References
 
The contractile protein myosin consists of two heavy chains (MHC), which contain the actin- and ATP-binding sites, and two pairs of light chains (MLC). The motor function of myosin is performed by MHC, while the MLCs exert a regulatory role [1–3]. Two different isoforms of MHC ({alpha} and β) are expressed in mammalian cardiac muscle. Although they have 93% sequence homology [4], animal studies indicated that these isoforms possess distinct functional properties [5,6]. Studies on rat and rabbit tissue indicated that the fast {alpha}-MHC isoform exhibits a two to three times higher actin-activated ATPase activity [5] and actin filament sliding velocity [6] than the slow β-MHC isoform.

In human myocardium, the β-MHC predominates in ventricles, whereas a variable mixture of {alpha}- and β-MHC is found in atria. During human heart failure, the {alpha}-MHC fraction declines in both atria and ventricles [7,8]. However, the functional implications of this MHC switch in human hearts are still unclear. Moreover, little is known about differences in contractile features and energy consumption between atrial and ventricular tissues.

Thus, the first aim of the present study was to determine functional differences between human atrial and ventricular tissue. The second aim was to determine to what extent MHC protein composition determines human myocardial performance using the variability found in MHC composition in atrial tissue. Isometric force production, ATP utilization and their Ca2+-sensitivities were measured in chemically skinned atrial trabeculae and ventricular muscle strips from human hearts. The main advantage of skinned preparations is that they allow standardisation of the conditions (e.g. composition of intracellular milieu and sarcomere length) under which functional properties are studied. Moreover, simultaneous measurement of force and ATP consumption in skinned myocardial tissue allows unambiguous determination of the relation between contractile and energetic properties. The rate of force redevelopment was recorded in single Triton-skinned cardiomyocytes in order to obtain information on the kinetic properties of the actomyosin interaction. The MHC composition of the human atrial and ventricular biopsies was determined by one-dimensional gel electrophoresis and correlated with the functional properties measured in tissue from the same hearts.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Acknowledgments
 References
 
2.1. Preparations
Trabeculae were isolated from right atrial appendages obtained during coronary bypass surgery on patients (n=14; age 41–80 years) with normal left ventricular function. Ventricular muscle strips were isolated from left ventricular biopsies, which were obtained from healthy donor hearts (n=6; age 23–54 years) that could not be transplanted due to technical reasons, and were stored in liquid nitrogen. Samples were obtained after informed consent and with approval of the local ethics committees. The investigation conforms with the principles outlined in the Declaration of Helsinki; 20 atrial trabeculae and 13 ventricular strips were isolated in cold relaxing solution (pH 7.0; in mmol l–1: free Mg2+ 1, KCl 145, EGTA 2, ATP 4, imidazole 10) and then chemically skinned in relaxing solution with 1% Triton X-100 overnight (4 °C). Ventricular muscle strips were cut longitudinally, i.e. in parallel to the long axis of the cardiomyocytes in order to minimize damage. Mean dimensions (± S.E.M.) of the atrial and ventricular preparations amounted to 1.61 ± 0.12 and 2.36 ± 0.17 mm in length, 435 ± 25 and 472 ± 41 µm in width, and 384 ± 22 and 495 ± 34 µm in depth, respectively. Part of the biopsy was used for mechanical isolation of single cardiomyocytes as described previously [9,10]. Before mechanical isolation, tissue was defrosted in relaxing solution. During the isolation, the tissue was kept on ice. Tissue samples were mechanically disrupted within a few seconds in relaxing solution using a small glass tissue homogenizer. Isolated myocytes were immersed for 5 min in relaxing solution containing 0.5% Triton X-100. To remove the Triton, cells were washed twice in relaxing solution. Thereafter, a single myocyte was attached between a force transducer and a piezoelectronic motor. The remainder of the biopsy was used for determination of MHC composition.

2.2. MHC composition
MHC composition was analysed by one-dimensional SDS-gel electrophoresis (1D SDS-PAGE), silver staining and laser scanning densitometry as described by van der Velden et al. [10,11]. 1D SDS-PAGE was performed using an acrylamide to bis-acrylamide ratio of 200:1 in the separating gel (12% acrylamide; pH 9.3) and of 20:1 in the stacking gel (3.5% acrylamide; pH 6.8). Densitometric analysis was performed on an LKB UltroScan XL Enhanced Laser Densitometer (LKB Produkter, Bromma, Sweden) using the GelScan XL software package (Pharmacia, Uppsala, Sweden). To check for linearity, different amounts of atrial and ventricular tissue (0.2–1.0 µg) were loaded and the density of the MHC bands was analysed. Based on these determinations, 0.5 µg was chosen for sample application as it was found to be within the linear range. In one ventricular sample, containing a relatively small amount of the {alpha}-MHC isoform (Fig. 1C), the intensity profile was fit to the sum of two Gaussian curves representing the {alpha}- and β-isoforms and the ratio was calculated from the area underneath the {alpha}-peak divided by the total MHC area. For validation of MHC isoform analysis, various mixtures (3:1, 1:1, 1:3) of an atrial (84.7% {alpha}-MHC) and a ventricular (99.3% β-MHC) tissue sample were applied. The percentage of β-MHC obtained from the densitometric analysis correlated well with the amount of ventricular sample present in the mixture (r2=0.97, Fig. 1A). Since the distance on gel between {alpha}- and β-MHC bands is increased by using less total acrylamide, a comparison was made between 8% SDS-PAGE [7,8] and 12% SDS-PAGE [10,11]. No significant differences were found between the data obtained with the two gel protocols.


Figure 1
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  		Fig. 1 MHC composition in atrial and ventricular tissue. (A) Validation of MHC analysis by SDS-PAGE using various mixtures (3:1, 1:1 and 1:3) of an atrial (84.7% {alpha}-MHC) and a ventricular (99.3% β-MHC) tissue sample. The percentage of β-MHC obtained from the densitometric analysis correlated well with the amount of ventricular sample present in the mixture. Regression line: β-MHC (in %)=(21.3 ± 4.9)+(0.78 ± 0.08) x ventricular tissue sample portion (in %) (r2=0.97, P<0.05). (B) Silver stained SDS-polyacrylamide gel of MHC proteins in an atrial (A) and a ventricular (V) tissue sample. (C) Laser densitometric scan corresponding to the atrial (A) and ventricular (V) samples shown in (B). AU, arbitrary units.

 
2.3. Measurements of force, ATP consumption and rate of force redevelopment
The experimental procedures and equipment used were as described previously [12,13]. ATPase activity was measured by enzymatic coupling of ATP resynthesis to the oxidation of NADH, which could be quantified photometrically from the absorbance of near-UV light. The composition of relaxing, preactivating and activating solutions was calculated as described recently [14]. Isometric force and ATPase activity were measured at saturating and subsaturating Ca2+ concentrations at 20 ± 1 °C. Maximum force was determined when a steady-state force was reached. In a few ventricular preparations, the force values reached after approximately 3 min of activation were used as maximal values. On average force decline between the first and the last maximal control activation amounted to 18 ± 1% and 10 ± 2% for atrial and ventricular preparations, respectively. The Ca2+-activated ATPase activity was determined by subtraction of basal ATPase activity (measured in relaxing solution) from total ATPase activity measured in activating solution with various Ca2+ concentrations. The length of the preparations was adjusted on the basis of passive tension by stretching them to 1–2 kN m–2. In two trabeculae, it was possible to examine sarcomere length by means of laser diffraction. The sarcomere lengths found after adjustment of the length of the preparations amounted to 2.20 and 2.15 µm. This corresponds well with the values found by Kentish et al. [15] in rat trabeculae. The cross-sectional area of the preparations was estimated assuming an elliptical shape, i.e. cross-sectional area=(width x depth x {pi})/4.

The rate of force redevelopment was determined at saturating Ca2+ concentration in Triton-skinned single cardiomyocytes at 15 ± 1 °C [13]. In brief, when reaching a steady level of force the myocyte was rapidly shortened and after a delay of 30 ms, restretched by 20% of its length. Upon shortening force dropped to zero and upon restretch force redevelopment occurred to the initial steady level. Force redevelopment was fitted to a single exponential to estimate the rate of force redevelopment (Ktr).

2.4. Histochemical analysis
Histochemical analysis was performed on six atrial trabeculae and five ventricular preparations. At the end of the experiment, preparations were embedded in relaxing solution containing 15% (w/v) gelatin and frozen in liquid nitrogen. Tissue sections (5 µm) were cut with a cryostat (Leica CM 1850, Rijswijk ZH, Netherlands) at –22 °C. Sections were collected on slides treated with Vectabond (Vector Laboratories, Birlingame, CA), air dried for 20 min and slides were stored at –80 °C. The sections were fixed at room temperature for 5 min in a 4% formalin in 0.1 M phosphate buffer, pH 7.4. After hematoxylin and eosin staining, the sections were dehydrated and mounted in Entallan (Merck, Darmstadt, Germany). Sections were studied as described previously [16] using a Leica DMRB microscope (Wetzlar, Germany). Images were collected with a Sony XC-77CE camera (Towada, Japan) connected to a LG-3 frame grabber (Scion, Frederick, MD, USA) on personal computer, and analysed using public domain software (NIH Image 1.61). The myofibrillar area and interstitial space were determined relative to the cross-sectional area and expressed as percentages.

2.5. Data analysis
Force–pCa and ATPase–pCa relations were fit by a nonlinear fit procedure to a Hill equation [17]: P(Ca2+)/P0=[Ca2+]nHill/(Ca50nHill+[Ca2+]nHill), where P is steady state force (or ATPase activity). P0 denotes the steady force (or ATPase activity) at saturating Ca2+ concentration, nHill reflects the steepness of the relation, and Ca50 (or pCa50) represents the midpoint of the relation.

All data were averaged per patient. Values are given as means ± S.E.M. of n patients. All statistical statements are based on two-tailed Student's t-tests with a 0.05 level of significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Acknowledgments
 References
 
3.1. MHC composition
MHC isoforms were separated using 1D SDS-PAGE (Fig. 1). Densitometric analysis of the SDS-gels (Fig. 1C) revealed that atrial tissue consisted of a variable mixture of {alpha}- and β-MHC (β-MHC ranged from 6.9% to 51.3% of total MHC content), while β-MHC was the predominant isoform in ventricular tissue (Table 1). In two of the ventricular samples, low expression levels of {alpha}-MHC were detected. Low abundance in these two samples was confirmed by Western immunoblotting. Western immunoblotting, performed on all ventricular samples using a specific monoclonal antibody against {alpha}-MHC [10], yielded negative results except in these two samples.


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  		Table 1 Mean parameters in atrial and ventricular tissue

 
3.2. Functional properties
Recordings of isometric force and ATP consumption in an atrial trabecula and a ventricular muscle strip during a contraction–relaxation cycle at saturating Ca2+ concentration are shown in Fig. 2A and B. The slope of the regression line fitted to the NADH absorbance signal (Fig. 2B) was considerably steeper in atrial than in ventricular preparations. Indeed, the maximal rate of Ca2+ activated ATP consumption (i.e. ATPase activity per preparation volume) was on average 3.3 times larger in atrial tissue than in ventricular tissue (0.147 ± 0.015 and 0.045 ± 0.007 mmol l–1 s–1, respectively). Maximal isometric tension (i.e. force divided by cross-sectional area) was lower in atrial trabeculae than in ventricular muscle strips. Table 1 provides mean values of Ca2+-sensitivity (pCa50) and steepness of the relationships (nHill) of both force and ATP consumption. No significant difference was found in these parameters between atrial and ventricular preparations. Representative examples of force–pCa and ATPase activity–pCa curves obtained in one atrial trabecula are shown in Fig. 3.


Figure 2
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  		Fig. 2 Recordings of force and NADH absorbance in an atrial trabecula and a ventricular strip during a contraction–relaxation cycle. (A) Force production and (B) NADH absorbance recorded at saturating Ca2+ concentration. When the preparation was transferred to the activating solution, isometric force developed and the NADH absorbance signal started to decline. ATP consumption was determined by calculation of the slope of the dotted regression line fitted to the absorbance signal during the last 20 s of activation. After returning trabeculae into the relaxing solution, 0.5 nmol ADP was injected into the measuring bath to calibrate the absorbance signal. The zero level of the absorbance signal was arbitrarily chosen. Dimensions of the preparations were 2.5 mm in length, 420 µm in width and 380 µm in depth for atrial and 2.7 mm in length, 480 µm in width and 507 µm in depth for ventricular.

 

Figure 3
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  		Fig. 3 Ca2+-sensitivity of isometric force and ATPase activity. The force–pCa and ATPase activity–pCa relations of an atrial trabecula from one atrial biopsy are shown as representative examples. Isometric force and ATP utilization rate at submaximally activating Ca2+ concentrations are normalised to the control force/ATP utilization rate found at saturating Ca2+ concentration (pCa 4.5). Both the force–pCa and ATPase activity–pCa relations were fitted to a Hill equation.

 
Tension cost was calculated for each preparation by dividing ATP consumption per preparation volume by force per cross-sectional area. The preparation volume equals cross-sectional area multiplied by preparation length. Therefore, this measure has the advantage that it is independent of possible inaccuracy in the determination of cross-sectional area of the preparation. Tension cost is a measure of muscle economy, i.e. the rate of ATP splitting necessary for maintenance of isometric force. Tension cost (in mmol kN–1 m–1 s–1) amounted to 11.4 ± 1.4 in atrial tissue and to 2.4 ± 0.3 in ventricular tissue. This implies that the economy of force maintenance in ventricular tissue is about five times larger than in atrial tissue.

The rate of force redevelopment (Ktr) measured in single isolated cardiomyocytes was on average nine times higher in atrial than in ventricular tissue. These values are also summarized in Table 1. When ATPase activity was plotted as a function of Ktr (Fig. 4), a weak but significant linear correlation was found between these two parameters (r2=0.35; P<0.05).


Figure 4
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  		Fig. 4 Correlation between Ktr and ATPase activity. A linear relationship exists between the rate of force redevelopment and mean ATPase activity (n=12 for atria and n=6 for ventricles). Regression line: ATPase activity/volume (in mmol l–1 s–1)=(0.07 ± 0.02)+(0.008 ± 0.003) x Ktr (in s–1) (r2=0.35, P<0.05).

 
3.3. Correlation between MHC content and functional properties
Significant linear correlations were found between maximal isometric force, rate of ATP consumption, tension cost and MHC composition (Fig. 5). The rate of ATP consumption of preparations with pure (100%) {alpha}-MHC (estimated by extrapolation of the regression line) was approximately five times higher than the rate of preparations with pure β-MHC (0.181 ± 0.018 and 0.039 ± 0.014 mmol l–1 s–1, respectively, for extrapolated values ± their standard errors). In addition, a significant negative correlation was found between mean Ktr and β-MHC content (r2=0.55) (Fig. 6). The Ktr associated with pure {alpha}-MHC expression calculated from the regression line (10.76 s–1) was considerably higher than the values obtained in ventricular tissue (0.87 s–1).


Figure 5
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  		Fig. 5 Correlations between β-MHC content and isometric force, ATP consumption and tension cost. (A) Isometric force per cross-sectional area averaged per every patient (n=14 for atria and n=6 for ventricles) depended significantly on β-MHC composition. Regression line: isometric force/cross-sectional area (in kN m–2)=(12.2 ± 1.7)+(0.07 ± 0.03) x β-MHC (in %) (r2=0.22, P<0.05). (B) A significant negative correlation exists between β-MHC fraction (% of total MHC) and mean ATPase activity for every patient (n=14 for atria and n=6 for ventricles). Regression line: ATPase activity/volume (in mmol l–1 s–1)=(0.18 ± 0.02)–(0.0014 ± 0.0003) x β-MHC (in %) (r2=0.54, P<0.05). (C) A significant correlation was also found between β-MHC content and mean tension cost for every patient (n=14 for atria and n=6 for ventricles). Regression line: tension cost (in mmol kN–1 m–1 s–1)=(14.32 ± 1.62)–(0.124 ± 0.028) x β-MHC (in %) (r2=0.51, P<0.05).

 

Figure 6
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  		Fig. 6 Correlations between β-MHC content and rate of force redevelopment (Ktr). A significant correlation exists between β-MHC content and mean Ktr for every patient (n=12 for atria and n=6 for ventricles). Regression line: Ktr (in s–1)=(10.76 ± 1.49)–(0.111 ± 0.025) x β-MHC (in %) (r2=0.55, P<0.05).

 
3.4. Myofibrillar density
Histochemical analysis revealed that the average density of myofibrillar tissue was significantly lower in atrial (Fig. 7A, C) than in ventricular (Fig. 7B, D) preparations (50 ± 9% and 89 ± 2%, respectively, Table 1), mainly because in atrial tissue an outer layer of connective tissue was found and occasionally blood vessels were present (Fig. 7A). The difference in the percentage of interstitial space in atrial (20 ± 4%) and ventricular (11 ± 2%) tissue did not reach statistical significance (P=0.09; Table 1).


Figure 7
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  		Fig. 7 Cross-sectional images of preparations. Cross-sectional images of an atrial trabecula (A, C) and ventricular muscle strip (B, D) were made at 10 x (A, B) and 40 x (C, D) magnification. Atrial trabeculae have a surrounding layer of connective tissue and occasionally contain blood vessel(s). The atrial trabecula shown in panel A contains one blood vessel. Dark red structures correspond to myofibrillar tissue, while lighter parts correspond to the non-myofibrillar tissue.

 
The overall difference in amount of myofibrillar tissue between atrial and ventricular preparations may explain part of the differences in isometric force and ATP consumption between atrial and ventricular tissue. To investigate this point, data were corrected for the proportion of myofibrillar tissue. It abolished the difference in maximal tension, increased the difference in ATPase activity and slightly enhanced the relative difference in the extrapolated ATPase-values of the "pure" isoforms (Table 1). These results suggest that the difference in maximal isometric force found between atrial and ventricular preparations was due to the difference in the amount of myofibrillar tissue. Within the preparations used for histochemical analysis (n=11), no correlation was present between maximal force and MHC composition both before and after correction of the data for the proportion of myofibrillar tissue (Fig. 8A). However, the correction slightly improved the correlation between ATPase activity and β-MHC composition as reflected by an increase in the r2 value from 0.72 for the uncorrected data to 0.79 for the corrected data (Fig. 8B) but, as expected, it did not influence the values obtained for tension cost.


Figure 8
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  		Fig. 8 Correction of data for the amount of myofibrillar tissue. The correction of isometric force per cross-sectional area (A) and ATPase activity per volume (B) was performed in six atrial (open symbols) and five ventricular (closed symbols) preparations and the regression lines to the uncorrected (continuous line, circles) and corrected data (dotted line, squares) are shown. Isometric force did not depend on β-MHC composition for both uncorrected and corrected data. Regression line of uncorrected values: isometric force/cross-sectional area (in kN m–2)=(16.8 ± 3.5)+(0.03 ± 0.05) x β-MHC (in %) (r2=0.04, P=0.57). Regression line of corrected values: isometric force/cross-sectional area (in kN m–2)=(45.1 ± 10.3)–(0.2 ± 0.1) x β-MHC (in %) (r2=0.18, P=0.20). Correction enhanced the correlation between β-MHC composition and ATPase activity. Regression line of uncorrected values: ATPase activity/volume (in mmol l–1 s–1)=(0.19 ± 0.02)–(0.0015 ± 0.0003) x β-MHC (in %) (r2=0.72, P<0.05). Regression line of corrected values: ATPase activity/volume (in mmol l–1 s–1)=(0.38 ± 0.04)–(0.0032 ± 0.0006) x β-MHC (in %) (r2=0.79, P<0.05).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Acknowledgments
 References
 
This study shows that force development in human ventricular tissue is about five times more economical, though nine times slower than in atrial myocardium and provides evidence that these differences are largely attributable to the difference in myosin heavy chain isoform composition. Significant linear relations were found between the MHC composition and ATP consumption and rate of force redevelopment. Extrapolation of these relations indicated that ATP consumption of the pure {alpha}-MHC was five times higher, whereas force redevelopment was considerably faster than observed for the β-MHC. It should be noted, however, that the extrapolated values have to be considered with caution because the scatter in the data is rather high. In addition, the linear relations used for the data analysis implicitly imply that myosin molecules act as independent force generators. Evidence suggests that this might not necessarily be the case [6,18]. However, even if interaction between myosin heads does occur the functional differences between atrial and ventricular tissue would remain. In fact, the concave relationships found [6,18] would imply that the difference between the properties of the pure isoforms would become even larger. Within atrial tissue, the correlation between MHC composition and rate of force redevelopment was significant (r2=54, P<0.05), and although the correlation between MHC composition and ATP consumption for atrial samples did not reach statistical significance (P=0.18), its functional relation was rather similar to the relationship found for the combined set of atrial and ventricular data.

4.1. Methodological considerations
It should be noted that isometric force and ATP consumption were determined at 20 °C, whereas Ktr values were determined in cardiac myocytes at 15 °C. These experimental temperatures were chosen to facilitate comparison with previous studies in rat and human, from which it also became apparent that at higher temperatures sarcomere uniformity and stability of the cardiomyocytes during the measurements were not well preserved. We do not expect a difference in temperature dependency of functional properties between atrial and ventricular tissue, but caution should be exerted in extrapolation of the values found in this study to body temperature.

During activation, inorganic phosphate (Pi) accumulates in the preparations due to the ATP hydrolysis. Since Pi accumulation is proportional to the ATPase activity and the square of muscle diameter, this accumulation will be approximately 0.17 and 0.70 mM, respectively in the ventricular and atrial preparations used in this study. From the previous studies in rat [19] and human cardiac myocytes [17,20], it followed that such low amounts of Pi depress force by less than 5%, while the effect on ATPase activity will be even smaller. Since, Pi accumulation is somewhat larger in atrial than in ventricular tissue, it slightly reduces the difference in force and consequently, in tension cost between atrial and ventricular tissue. However, overall the impact of Pi accumulation on functional properties is very minor and therefore does not affect the conclusions of this study.

4.2. Ca2+-sensitivity
The Ca2+-sensitivities of force and ATPase activity observed both in atrial and in ventricular tissue were very similar (Table 1). This indicates that the difference in tension cost between atrial and ventricular tissue is independent of the free Ca2+ concentration and implies that the energetic differences observed also hold at submaximal Ca2+ concentrations found in vivo.

4.3. Effect of MHC composition
During human heart failure, a shift has been observed from the fast {alpha}-MHC isoform to the slow β-MHC isoform both in atria and ventricles [7,8]. The marked difference in economy observed in this study clearly indicates that even a minor shift is associated with an improvement of the economy of contraction, albeit at the expense of speed of force development. It can be calculated from our experiments that a 7% decline in {alpha}-MHC composition, as was found in failing human ventricles by Miyata et al. [7], will cause a 20% reduction in maximal ATPase activity. Recently, it has been shown that expression of a small amount of {alpha}-MHC (~12%) in rat cardiomyocytes significantly increases power output [21], whereas no significant differences in isometric force or actin sliding velocity were observed between nonfailing and failing human myosin with a minor (~3%) difference in the expression of fast myosin (V1) [22]. Our results in human tissue are in line with the observations in rat cardiomyocytes [21] and suggest that enhanced economy is paralleled by a reduction in maximum power output.

4.4. Effect of regulatory proteins and their phosphorylation status
Although the catalytic subdomain of ATPase activity and the actin binding region are located at the head portion of myosin, other contractile proteins, such as the myosin light chains and the troponin complex may exert a modulatory role in contraction [2,3,10,23–26]. Part of the variability in contractile properties within and between atrial and ventricular tissue may be explained by the expression of tissue-specific isoforms and/or by differences in phosphorylation status of contractile proteins. For instance, protein kinase C mediated phosphorylation of troponin has been shown to reduce both maximum tension and actomyosin Mg-ATPase activity by ~30% in mouse myocardium [24–26]. It should be noted that these effects are relatively small compared to the 3.3-fold difference in ATPase activity between atrial and ventricular tissue. Moreover, if troponin phosphorylation induces a parallel change in force and ATPase activity, it would not affect the differences in economy observed in the present study.

4.5. Speed versus economy
The correlation between ATPase activity and Ktr (Fig. 4) resembles previous observations on stable maintenance heat rate and rate of force redevelopment in skeletal muscle fibers [27]. In a simple two-state model for crossbridge interaction [28], ATPase activity is governed by the rate limiting step in the crossbridge cycle: the rate of crossbridge detachment (g) and Ktr=f+g, where f equals the rate of crossbridge attachment. The proportionality observed between ATPase activity and Ktr thus suggests that either Ktr is dominated by g (f>>g) or that f and g covary in proportion in different mixtures of fast and slow cardiac MHC isoforms (i.e. f=cg; where c is constant). In either case maximal tension, which is proportional to f/(f+g), would be expected to be similar in atrial and ventricular preparations, as was found experimentally after correction for myofibrillar tissue density (Table 1).

Cardiac adaptation to mechanical overload is likely governed by the trade-off between speed and economy of contraction [29,30]. This principle might also explain why the relatively "expensive" fast MHC-isoform predominates in the atria: the contribution of atrial contraction to ventricular filling is rather small (as are the associated energy costs) but it might gain importance, and promote cardiac output, at high heart rate. Moreover, it is noteworthy that in states of reduced ventricular compliance such as ventricular hypertrophy and heart failure the atrial contribution to ventricular filling increases by approximately 20% [31].

4.6. Conclusion
Since our data show that the slow isoform is five times more economical than the fast isoform, the switch towards the slow β isoform clearly has considerable impact on performance in human atrial and ventricular myocardium.


   Acknowledgments
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Acknowledgments
References
 
This work was supported by the Netherlands Heart Foundation (Grant 99.155), the European Union (Contract Number HPRN-CT-2000-00091) and Netherlands Organisation for Scientific Research (VENI grant).


   Notes
 
Time for primary review 24 days


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

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