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Cardiovascular Research 1998 40(3):580-590; doi:10.1016/S0008-6363(98)00164-3
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Copyright © 1998, European Society of Cardiology

Alterations of cross-bridge kinetics in human atrial and ventricular myocardium

Thorsten Rufa, Heiner Schulte-Baukloha, Jens Lüdemanna, Herbert Posivalc, Friedhelm Beyersdorfb, Hanjörg Justa and Christian Holubarscha,*

aDepartment of Cardiology and Angiology, Internal Medicine, University of Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany
bDepartment of Cardiovascular Surgery, University of Freiburg, Hugstetter Strasse 55, 79106 Freiburg, Germany
cDepartment of Cardiovascular Surgery and Heart Transplantation, 32545 Bad Oeynhausen, Germany

* Corresponding author. Tel.: +761-270-3283; fax: +761-270-3611.

Received 13 February 1998; accepted 6 May 1998


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Condensed abstract: We analyzed actomyosin cross-bridge kinetics in human atrial and ventricular muscle strip preparations by using sinusoidal length changes from 0.1 to 60 Hz. The minimum stiffness frequency was higher in atrial than in ventricular human myocardium and lower in failing than in non-failing left ventricular human myocardium. β-Adrenergic stimulation increased the minimum stiffness frequency by 18±3% (p<0.05). Cross-bridge kinetics are temperature-dependent, with a Q10 of at least 2.7. Background: Dynamic stiffness measurements have revealed acute and chronic alterations of actomyosin cross-bridge kinetics in cardiac muscles of a variety of different animal species. We studied dynamic stiffness in right atrial and left ventricular preparations of non-failing and failing human hearts and tested the influence of the temperature and β-adrenergic stimulation on cross-bridge kinetics. Methods and Results: Muscle strips were prepared from right atria and left ventricles from human non-failing and failing hearts. After withdrawal of calcium, steady contracture tension was induced by the addition of 1.5 mM barium chloride. Sinusoidal length oscillations of 1% muscle length were applied, with a frequency spectrum of between 0.1 and 60 Hz. Dynamic stiffness was calculated from the length change and the corresponding force response amplitude. The specific minimum stiffness frequency, which indicates the interaction between cross-bridge recruitment and cross-bridge cycling dynamics, was analyzed for each condition: (1) The minimum stiffness frequency was 0.78±0.04 Hz in left ventricular myocardium and 2.80±0.31 Hz in right atrial myocardium (p<0.01) at 27°C. (2) The minimum stiffness frequency was 41% higher in non-failing compared to failing left ventricular human myocardium. (3) Over a wide range of experimental temperatures, the minimum stiffness frequency changed, with a Q10 of at least 2.7. (4) β-Adrenergic stimulation significantly (p<0.05) increased the minimum stiffness to 18±3% higher frequencies and significantly (p<0.05) lowered contracture tension by 7±1%. Conclusions: The contractility of human heart muscle is not only regulated by excitation–contraction coupling but also by modulation of intrinsic properties of the actomyosin system. Acute and chronic alterations of cross-bridge kinetics have been demonstrated, which play a significant role in the physiology and pathophysiology of the human heart.

KEYWORDS Cross-bridge kinetics; Actomyosin; Barium contracture; Cardiomyopathy; Experimental temperature


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Changes in myocardial force generation may occur acutely by mechanisms such as (1) adrenergic stimulation [1, 2], (2) sarcomere length [3, 4], (3) heart rate alterations [5, 6], (4) vasoactive peptides [7, 8]and (5) some pharmacological interventions [9, 10]. Chronically, a number of genes have been shown to be differentially expressed during cardiac hypertrophy and failure in a quantitative and qualitative manner, thereby influencing the contractility of the heart [11–16].

Independent of whether acute or chronic alterations of contractility are involved, four basic mechanisms may contribute to alterations in the strength of the heart:

1. Due to an enhanced intracellular amount of calcium, the number of activated actomyosin cross-bridges may be increased [17].
2. Physiological [18]and pharmacological [19]interventions may sensitize the regulatory properties of the contractile proteins towards calcium, thereby increasing the number of activated cross-bridges, without causing any changes in calcium transients.
3. Acute and chronic stimulating factors may basically alter the interaction between myosin and actin, i.e., kinetics of the actomyosin cross-bridge cycling [20–28].
4. As a result of impaired ATP generation in failing myocardium, a decreased energy reserve may limit the ability of the myocardium to perform work [29, 30].
An insufficient energy supply in the failing myocardium may also alter the kinetics of cross-bridge cycling.

In order to study the first two of the four mechanisms, one would prefer measurements of the intracellular calcium transients using calcium indicators or skinned fiber preparations. To investigate the latter two mechanisms, i.e., alterations of cross-bridge cycling mechanics, sophisticated mechanical measurements of either shortening velocity or of dynamic stiffness due to rapid length changes are necessary [31–33].

Because those studies are limited to the myocardium of animal species, with the exception of one study using human myocardium [34], we performed dynamic stiffness measurements by applying sinusoidal oscillating length changes to human muscle preparations subjected to barium contracture. This approach allowed us to investigate the impact of the following variables on myocardial cross-bridge mechanics:

(1) Experimental temperature (37, 27 and 17°C), (2) tissue specificity (atrial versus ventricular myocardium), (3) failing versus non-failing myocardium and (4) the influence of isoproterenol.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Source of tissues
Left ventricular myocardium was obtained from patients suffering from idiopathic dilative cardiomyopathy and undergoing orthotopic heart transplantation (Bad Oeynhausen, Cardiac Transplantation Center, Nordrhein-Westfalen, Germany). These patients were in end-stage heart failure (New York Heart Association classes III and IV). Preoperative medical treatment consisted of digitalis (digoxin or digitoxin), angiotensin converting enzyme inhibitors (captopril, enalapril, lisinopril) and diuretics (furosemide, piretanide). One third of the patients received intravenous heparin and/or a low dose of dopamine. The latter was applied at low concentrations (4 µg/min/kg body weight). In addition, three donor hearts were obtained that could not be used for heart transplantation for technical reasons. Pre-explantation echocardiography revealed a normal ejection fraction of the left ventricle in all three patients. Atrial appendices were excised during the cannulation procedure for extracorporeal circulation from patients undergoing routine aortocoronary bypass grafting (Department of Cardiovascular Surgery, University of Freiburg, Germany). The left ventricular ejection fraction was normal in all of these patients, and no clinical signs of congestive heart failure were present. All patients had given written informed consent for using explanted myocardial tissues scientifically. The study protocol had been approved by the local Ethics Committee of Freiburg University.

2.2 Transportation and storage of tissues
The tissues were stored in Krebs-Ringer solution containing 30 mM 2,3-butanedione monoxime (BDM) for cardioplegia [35]and were bubbled constantly with a 95% O2–5% CO2 gas mixture. The transportation time for left ventricular tissues was about 8 h and for normal atrial myocardial tissue was about 15 min.

2.3 Preparation procedure and experimental protocol
Long and thin preparations were cut along the fiber directions using a biocular microscope (Olympus), microscissors and forceps [36].

The isolated preparations (length, 4.2±0.4 mm; diameter, 0.6±0.2 mm) were fixed, using a steel tweezers, to a force transducer and a linear motor (Muscle Research System, Scientific Instruments, Heidelberg, Germany) driven by a sinus function generator (HAMEG HM 8130).

The preparations were stimulated electrically at a rate of 60/min (end-to-end square wave pulse, 6 V; 5 ms) and carefully stretched to the optimal fiber length, lmax, in steps of 0.05 mm in an isometric contraction mode. The muscles were allowed to stabilize for 15 min before calcium was washed out with Ca2+-free Krebs-Ringer solution at 27°C. During Ca2+-wash-out, the force of the contraction decreased continuously (Fig. 1). As the mechanical activity was completely abolished, contracture was induced by superfusion of a calcium-free Krebs-Ringer solution containing 1.5 mM barium chloride.


Figure 1
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  		Fig. 1 Typical force record of a representative experiment. Reduction of the calcium concentration from 2.5 to 0 mM in the bathing solution abolishes the force of twitch contraction (left part of the original record), and the addition of 1.5 mM Ba2+ leads to a tonic and stable force development (right part of the original record). Muscle length at 95% lmax was 4.8 mm and the cross-sectional area was 0.40 mm2; experimental temperature, 27°C.

 
After reducing the muscle length to 95% lmax, sinusoidal length perturbations were performed with frequencies from 0.1 to 60 Hz and an amplitude of 1% muscle length. The length oscillations and the resulting force signal were simultaneously registered on a strip chart recorder (Graphtec linearcorder WR3310, resonance frequency higher than 60 Hz).

The oscillation protocol was repeated at 17 and 37°C or, alternatively, with solutions containing 10–5 M isoproterenol or 30 mM BDM.

2.4 Rigor experiments
Additional experiments (n=5) were performed in which cross-bridge rigor bonds were induced by poisoning the muscle preparations with iodoacetate (1.25 mM) and atractyloside (5 mM), thereby blocking anaerobic and aerobic glycolysis. In contrast to the BaCl2-contracture, three different phenomena were observed:

1. Rigor tension was 28% higher than BaCl2-contracture tension, on average (Table 1).
2. Dynamic stiffness (0.3 Hz) was 113% higher in the rigor state compared to the BaCl2-contracture state (Table 1).
3. In these rigor states, no dip frequency could be found at all (as shown in Fig. 4).
From these experiments, it can be concluded that the contracture tension is generated mainly by actively cycling cross-bridges.


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  		Table 1 Rigor experiments

 

Figure 4
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  		Fig. 4 Typical example of experiments in which the developed force was abolished by 30 mM BDM. No dip frequency was detectable during increasing oscillating frequencies. In the same way, no dip frequencies were detectable in passive muscles (unstimulated, no-contracture) and in rigor-state muscles.

 
2.5 Calculation of dynamic stiffness and cross-sectional area
Dynamic stiffness is calculated at each oscillating frequency as the measured change in force divided by cross-sectional area of the preparation and divided by the measured change in length. In this procedure, the resting tension of the muscle preparation was not taken into consideration, because (1) the resting tension was small at 95% of optimum length compared to contracture tension and (2) it did not interfere with the minimum stiffness frequency. Nevertheless, increased passive stiffness of failing myocardium [7]may explain the higher dynamic stiffness values in those preparations.

Cross-sectional area was determined as follows: At optimum length, lmax, the muscle length was measured microscopically. At the end of each experiment, the muscle mass of the preparation was measured by weighing the muscle after blotting it three times. Using the following two equations, the cross-sectional area was calculated:


Formula 1

(1)


Formula 2

(2)
When A=area, V=volume, l=length, M=mass, {delta}=specific gravity, and assuming the specific gravity to be 1 g/cm3.

2.6 Statistics
Data are given as mean values±SEM. The paired and unpaired Student's t-test was used when applicable. When repeated measurements within groups were performed, the paired t-test and the Bonferroni–Holm procedure was used [37].


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Tension development of the isometric twitch and the Ba2+-induced contracture
At lmax, the muscle preparations were allowed to stabilize at 37°C and 1 Hz for 30 min in the isometric contraction mode. Exposure of the muscle strips to 1.5 mM BaCl2 in Krebs-Ringer solution after calcium wash-out lead to a continuous and stable force development (Fig. 1). Steady-state levels were obtained 6–10 min after the superfusion with BaCl2 was started. The tension of contracture reached 118% of the peak twitch tension for preparations from failing left ventricles, 121% for non-failing left ventricles, and 114% for atrial myocardium (Table 2).


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  		Table 2 Developed force of muscle preparations

 
3.2 Dynamic stiffness spectra
With a dynamic stiffness spectrum recorded for sinusoidal length perturbations throughout the frequency range from 0.1 to 60 Hz, we observed a characteristic minimum stiffness (Fig. 2) accompanied by a sudden phase shift (Fig. 3) in each individual experiment.


Figure 2
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  		Fig. 2 Constancy of length change at different oscillation frequencies (0.1–60 Hz) is shown in the lower panel and the corresponding force change is shown in the upper panel. The minimum of stiffness is observed at a frequency of 0.8 Hz (dip frequency). The experimental temperature was 27°C; muscle dimensions: 95% lmax was 4.8 mm and the cross-sectional area was 0.40 mm2.

 

Figure 3
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  		Fig. 3 Sudden phase shift at 0.8 Hz, using the same preparation as in Fig. 2.

 
In addition, superfusion of the cardioplegic agent BDM (30 mM) abolished the dip shape occurring at the frequency of minimum stiffness (Fig. 4). At all frequencies, the stiffness was drastically reduced with BDM; both phase shift and stiffness increases at high frequencies were absent in the presence of BDM.

3.3 Temperature dependence and tissue specificity
The dip frequencies were 0.18±0.03 Hz (17°C), 0.78±0.04 Hz (27°C) and 2.69±0.15 Hz (37°C) for ventricular myocardium (Fig. 5a).


Figure 5
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  		Fig. 5 Typical experiment using a ventricular muscle strip preparation. With increasing temperatures, the minimum dynamic stiffness was shifted to higher frequencies. (b) Typical experiment using an atrial muscle strip preparation. With increasing temperatures, the minimum dynamic stiffness was shifted to higher frequencies. Note that the bandwidth of dynamic stiffness was less pronounced.

 
In normal left ventricular human myocardium (three donor hearts), the dip frequencies at 27°C were 1.0, 1.1 and 1.2 Hz, indicating an average dip frequency that was 41% higher compared to that found in failing left ventricular myocardium (Table 3).


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  		Table 3 Left ventricular human myocardium: Frequency of minimum stiffness, fmin, and stiffness spectra at 0.1 Hz (S0.1), fmin (Smin), and 60 Hz (S60) at experimental temperature of 27°C.

 
In atrial human myocardium (Fig. 5bTable 4), the dip frequencies were significantly (p<0.01) higher (17°C, 1.05±0.0.17; 27°C, 2.80±0.31) than those obtained in ventricular myocardium. At 37°C, reliable stiffness minima were detectable only in half of the atrial preparations investigated (Fig. 5b).


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  		Table 4 Right atrial and left ventricular myocardium: Frequency of minimum stiffness, fmin, and stiffness spectra at 0.1 Hz (S0.1), fmin (Smin), and 60 Hz (S60) at a temperature of 27°C

 
3.4 Influence of β-adrenergic stimulation
In the presence of 10–5 M isoproterenol, the minimum stiffness and the phase shift occurred at higher frequencies (+18±3%, p<0.05). Fig. 6 demonstrates the frequency shift in each single experiment performed in left ventricular human muscle strips. The contracture force, however, declined by 7±1% after the application of isoproterenol (p<0.05).


Figure 6
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  		Fig. 6 Shift of dip frequency due to isoproterenol in all of the preparations investigated.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Methods to study cross-bridge kinetics
In general, in order to study cross-bridge kinetics, four different methods may be used: (1) The myothermal method allows one to determine the relationship between the heat released from and the force developed by a muscle preparation [16, 22, 23, 38, 39]. A prerequisite for these kinds of experiments is an experimental separation between the resting heat rate, tension-independent heat, tension-dependent heat and recovery heat. Making certain assumptions, the single cross-bridge force–time integral can then be calculated. (2) The slack length test, which was originally developed by Edman [40], measures the maximum unloaded shortening velocity during contraction. This parameter indicates the speed of cross-bridges going through several cycles, thereby producing shortening under unloaded conditions. (3) In skinned fiber preparations, the relationship between isometric ATPase activity and isometric force may be used to analyse cross-bridge kinetics [41]. (4) Because we were especially interested in the influence of isoproterenol on cross-bridge kinetics, an intact fiber preparation was needed. Since the length perturbation method or dynamic stiffness analysis can be used in activated skinned fiber preparations as well as in intact myocardial preparations exposed to barium chloride, we have chosen the latter method in intact human muscle preparations, as has been described for animal model experiments [20, 21, 24, 31–33, 42, 43].

In contrast to previous reports [43, 44], in which the minimum stiffness frequency was thought to be determined solely by the cross-bridge cycling rate, Berman et al. [20]discussed and Campbell et al. [45]mathematically analyzed the phenomenon of the minimum stiffness frequency as the interaction between cross-bridge recruitment dynamics and cross-bridge cycling dynamics: At low oscillating frequencies, the ratio between force generating and non-force-generating cross-bridges is stabile. Therefore, the changes in force are proportional to the changes in length due to recruitment of a greater number of cross-bridges, according to the Frank–Starling mechanism. When the oscillating frequency increases, there is a critical frequency at which the time for complete recruitment will be too short within a single oscillation. As a consequence, stiffness must decrease because of the reduced ratio between force-generating and non-force-generating cross-bridges. At higher oscillating frequencies, beyond the dip frequency, cycling cross-bridges are significantly distorted by rapid length changes before cross-bridge detachment can occur. In this context, it is noteworthy to mention that the phenomenon of the minimum stiffness frequency was found to be similar both in papillary muscles and in the left ventricular chambers of rabbit hearts [45]. Additionally, the minimum stiffness frequency was shown to be independent of the degree of calcium activation [25, 44], on the one hand, but greatly dependent on temperature and myosin enzyme distribution [21, 24, 25], on the other. Furthermore, one may argue that crush trauma at the muscular attachments may have unexpected effects on stiffness measurements. However, as has been shown previously, crush trauma influences the magnitude of dynamic stiffness, but does not invalidate the minimum stiffness frequency as an indicator of the dynamic elastance [34, 46].

4.2 Influence of temperature
Because biochemically driven processes, like myosin ATPase or cross-bridge activity, must be temperature-dependent, we first studied the influence of temperature on the minimum stiffness frequency in left ventricular and right atrial human cardiac muscle preparations. Covering a wide range of temperatures, Q10-values of at least 2.7 or higher can be calculated (Fig. 5a–b). This finding in human atrial and ventricular preparations is in good agreement with our previous studies in rat myocardium using the myothermal method [47, 48], as well as with those reported by Shibata et al. [33]. These authors, who also used the length perturbation method, preferred 28°C as the highest temperature, being afraid of the induction of rigor and thereby loosing the dip of dynamic stiffness, as observed earlier [32]. In contrast, in our study, this problem occurred only in some human atrial preparations but not in human left ventricular failing preparations at 37°C. Nevertheless, one could argue that contracture tension, as induced by the application of barium chloride, is mainly generated by rigor bonds. We, therefore, performed experiments in which the preparations were poisoned, to block glycolysis completely. These results (Table 1) clearly demonstrate that dynamic stiffness in the rigor state is very different from that found in contracture tension, and dip frequency is only observed in muscles with actively cycling cross-bridges. These data show that we worked with qualitatively good and metabolically well-supported muscle preparations. The difference between right atrial and left ventricular muscle preparations is explained by a greater energy demand of atrial muscle preparations (see below).

4.3 Tissue specificity
With respect to the absolute minimum of the stiffness spectrum, substantial differences between atrial and ventricular myocardium were found: The oscillation frequency at which the minimum stiffness occurred was 2.5 to 3.0-fold higher in atrial than in ventricular myocardium.

These findings can be interpreted as a consequence of different patterns of myosin isoenzymes. The regulation of cross-bridge cycling is influenced by the distribution of these myosin isoenzymes, which depends on the species, the developmental and the hormonal state. In ventricular human myocardium, nearly all of the myosin has been thought to be of the V3-type, whereas in atrial tissue, V1-myosin is also expressed to a considerable extent (30–40%) [49–51]. The functional consequences of the different isoenzyme pattern are reflected by different stiffness spectra in our experiments: The cross-bridge cycling rate is significantly higher in atrial than in ventricular myocardium. These data correspond to those reported by Morano et al. [41], who demonstrated a more than two-fold higher ATPase–force relation of atrial fibers compared to ventricular fibers of human hearts.

4.4 Failing versus non-failing left ventricular human myocardium
Because of the general difficulty in obtaining human cardiac tissues from normal donor hearts for scientific purposes, we could investigate only three preparations obtained from three non-failing ventricles. The minimum stiffness frequencies were 54, 41 and 28% higher than the average value of the failing myocardium. Although our observations are restricted to three human donor hearts, and the difference in the minimum frequencies between non-failing and failing myocardium is low compared to the difference between atrial and ventricular myocardium (see Table 4), these data provide evidence for altered dynamic elastance in the failing human myocardium. This decrease in the minimum stiffness frequency may be interpreted in two different ways: Either recruitment dynamics of cross-bridges during length oscillations are reduced or cross-bridge cycling dynamics are slowed down. We favour the second interpretation for the following reasons: (1) Under the specific experimental conditions of barium contracture, activation of cross-bridges is maximum, guaranteeing a constant number of cross-bridges being activated. (2) A length-dependent shift of the force–pCa-activation curve, i.e. the physiological basis of the Frank–Starling mechanism, has been postulated to be attenuated or even abolished in the failing human myocardium [52], which might be a cause for reduced recruitment of cross-bridges with increasing oscillating frequencies. Recently, however, we have shown that the Frank–Starling mechanism is fully maintained on the sarcomere, tissue and organ level [4].

Hajjar and Gwathmey [34]reported an average minimum frequency of 0.78 Hz for normal non-failing myocardium and of 0.42 Hz for human failing myocardium. This quantitative difference between their and our data may be explained by the different experimental conditions used: an experimental temperature of 27°C in our study and of 22°C in their study; unskinned preparations in barium contracture versus skinned fibers with maximum calcium activation [34].

If it is assumed that such a decrease in the minimum stiffness frequency of the failing myocardium is due to a reduced off-rate of the cross-bridges from a force-generating into a non-force-generating state, then such a change in cross-bridge kinetics is expected to be associated with an increased economy of isometric force generation. Indeed, using myothermal methods, Hasenfuss et al. showed an increased cross-bridge force-time integral for failing [39]and volume-overload hypertrophied myocardium [38].

However, the presented data do not seem to be consistent with other contributions in the literature: Ca2+–myosin ATPase was not found to differ between normal and hypertrophied or failing human myocardium [53–55]. In addition, in vitro myosin motility was found to be unaltered in human mitral regurgitation heart failure compared to normal [55]. Despite these findings, the presented data and those published by Hajjar and Gwathmey [34]have to be interpreted in the light of two very recent contributions by Lowes et al. [56]and Nakao et al. [57]. These authors showed, either in specimens obtained from explanted hearts [57]or in biopsies taken from patients in vivo [56], a profound gene expression of {alpha}-myosin heavy chain mRNA, which was found to be almost completely downregulated in hypertrophied failing human myocardium. This alteration in gene expression of myosin heavy chain isoforms would explain the observed decrease in the minimum stiffness frequency very well, provided that this gene expression of {alpha}-myosin heavy chain is translated into protein expression, and conventional analysis of myosin isoforms, e.g. pyrophosphate gel electrophoresis, is not sensitive enough to separate {alpha}- and β-myosin isoforms properly.

In addition to altered gene expression of {alpha}-myosin heavy chain, other molecular changes in the contractile proteins may contribute to altered cross-bridge dynamics in the failing human myocardium: Anderson et al. [58]have found a change in the troponin T (TnT) isoform composition in failing human ventricles that was associated with a significant reduction in the myofibrillar ATPase activity. In addition to different TnT isoforms, the level of light chain 2 phosphorylation may alter the mechanics of cross-bridge cycling and be responsible for the differences observed between failing and non-failing human myocardium. Morano [59]has described the complete dephosphorylation of light chain 2 in myocardium from dilated hearts. However, because we have used BDM (30 mM/l) for transportation and preparation, light chain 2 is likely to be completely dephosphorylated in our experiments.

4.5 β-Adrenergic effect on the minimum stiffness frequency
Isoproterenol shifted the minimum stiffness of both failing and non-failing human myocardium to higher oscillation frequencies, accompanied by a concordant slight decrease in contracture tension and high frequency stiffness. Whereas the rightward shift of the minimum stiffness frequency is in good agreement with previous studies from mammalian myocardium [20, 21], there are two possible explanations for the latter findings: (1) Isoproterenol leads to a phosphorylation of TnI [60, 61]and thereby a desensitization of contractile proteins towards calcium or barium ions, respectively [62]. With a lower amount of Ba2+ bound to TnC, the activation of the actomyosin would fall to submaximal levels. (2) The observed increase in the overall cycling rate under conditions of catecholamines is a consequence of an increased off-rate of the cross-bridges from the force-generating state. With an increased off-rate, the steady-state between the force-generating and non-force-generating state of cross-bridge would be shifted in favour of the non-force-generating state and thereby lessen the total number of force-generating cross-bridges per unit of time.

We favour the second explanation because this view is consistent with our data from previous studies using the myothermal method: In these studies [22, 23, 63], the force–time integral of a single cross-bridge was calculated from the tension-dependent heat per half sarcomere, the enthalpy of ATP hydrolysis and the force–time integral of a twitch contraction [23], assuming that one molecule of ATP is hydrolysed during one cross-bridge cycle and that the force–time integral of the twitch is the sum of the force–time integrals of all cross-bridges. Using this approach, we found an approximately 40% reduction in the force–time integral of a single cross-bridge under β1-adrenoceptor-stimulating conditions. This finding is best explained by a reduction of the on-time of the force-generating state, i.e., an increase in the off-rate, gapp. An increase in the off-rate, gapp, however, would not only explain the reduced isometric economy of contraction seen with β1-adrenoceptor-stimulators, but also the overall increase in cross-bridge cycling rates observed with the dynamic stiffness measurement in failing and non-failing left ventricular myocardium.

4.6 Physiological and pathophysiological implications
The magnitude of the cycling rate has physiological and pathophysiological implications for both systole and diastole. At first glance, in failing human myocardium, slowed cross-bridge dynamics associated with an increased on-time of force-generating cross-bridges may be thought to be advantageous, by increasing the force–time integral and thereby improving economy. This may help to compensate for the decreased number of activated cross-bridges due to disturbances in calcium turnover in the failing heart. However, this compensation may well be associated with reduced power output. Regarding diastole, a reduced cross-bridge cycling frequency may contribute to impaired relaxation and increased diastolic stiffness. This diastolic failure may be aggravated further when either the β-adrenoceptor system is blunted or a further reduction in the cross-bridge cycling frequency comes into play, as with ischaemia or hypothermia. Therefore, therapeutics that aim to normalize cross-bridge kinetics by (1) increasing {alpha}-myosin heavy chain gene expression on the one hand and (2) by upregulating the β-adrenoceptor/adenylate-cyclase system, on the other, may become feasible.

Time for primary review 31 days.


   Acknowledgements
 
Supported by the Deutsche Forschungsgemeinschaft (Ho 915/4–3).


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

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