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Am J Physiol Regul Integr Comp Physiol 293: R20-R29, 2007. First published November 16, 2006; doi:10.1152/ajpregu.00537.2006
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Regulation of Cardiac Muscle Contraction

Rate of tension redevelopment is not modulated by sarcomere length in permeabilized human, murine, and porcine cardiomyocytes

István Ferenc Édes,1 Dániel Czuriga,1 Gábor Csányi,2 Stefan Chlopicki,2 Fabio A. Recchia,3,4 Attila Borbély,1 Zoltán Galajda,1 István Édes,1 Jolanda van der Velden,5 Ger J. M. Stienen,5 and Zoltán Papp1

1Division of Clinical Physiology, Institute of Cardiology, University of Debrecen, Medical and Health Science Center, Faculty of Medicine, Debrecen, Hungary; 2Department of Experimental Pharmacology, Chair of Pharmacology, Jagiellonian University Medical College, Krakow, Poland; 3Sector of Medicine, Scuola Superiore Sant’ Anna, Pisa, Italy; 4New York Medical College, Valhalla, New York; and 5Laboratory for Physiology, Institute for Cardiovascular Research, VU University Medical Center, Amsterdam, The Netherlands

Submitted 27 July 2006 ; accepted in final form 13 November 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The increase in Ca2+ sensitivity of isometric force development along with sarcomere length (SL) is considered as the basis of the Frank-Starling law of the heart, possibly involving the regulation of cross-bridge turnover kinetics. Therefore, the Ca2+ dependencies of isometric force production and of the cross-bridge-sensitive rate constant of force redevelopment (ktr) were determined at different SLs (1.9 and 2.3 µm) in isolated human, murine, and porcine permeabilized cardiomyocytes. ktr was also determined in the presence of 10 mM inorganic phosphate (Pi), which interfered with the force-generating cross-bridge transitions. The increases in Ca2+ sensitivities of force with SL were very similar in human, murine, and porcine cardiomyocytes ({Delta}pCa50: ~0.11). ktr was higher (P < 0.05) in mice than in humans or pigs at all Ca2+ concentrations ([Ca2+]) [maximum ktr (ktr,max) at a SL of 1.9 µm and pCa 4.75: 1.33 ± 0.11, 7.44 ± 0.15, and 1.02 ± 0.05 s–1, in humans, mice, and pigs, respectively] but ktr did not depend on SL in any species. Moreover, when the ktr values for each species were expressed relative to their respective maxima, similar Ca2+ dependencies were obtained. Ten millimolar Pi decreased force to ~60–65% and left {Delta}pCa50 unaltered in all three species. Pi increased ktr,max by a factor of ~1.6 in humans and pigs and by a factor of ~3 in mice, independent of SL. In conclusion, species differences exert a major influence on ktr, but SL does not appear to modulate the cross-bridge turnover rates in human, murine, and porcine hearts.

mouse; pig; heart; skinned muscle; myofilament length-dependent activation; rate of tension redevelopment; calcium

THE LENGTH-DEPENDENT INCREASE in myofilament Ca2+ sensitivity is considered to be the explanation for the improved cardiac systolic performance following an increase in end-diastolic ventricular volume, i.e., for the Frank-Starling mechanism. Despite recent advances in the understanding of various aspects of the Frank-Starling mechanism (for a recent review, see Ref. 12), the molecular mechanism of the length-dependent Ca2+ sensitization remains obscure. Ca2+ regulates force production through changes in cross-bridge turnover kinetics in both the skeletal (4) and the cardiac (2, 44) muscle; accordingly, length-dependent alterations of cross-bridge transitions may well underlie the Frank-Starling law of the heart.

Using rabbit skeletal muscle fibers, Zhao and Kawai (45) observed alterations in the rates of cross-bridge transitions during osmotic compression. These results were consistent with the length-dependent decrease in cross-bridge turnover and cross-bridge detachment at higher sarcomere length (SL), where lattice spacing was also reduced (45). However, in rat slow-twitch and rabbit fast-twitch skeletal muscle fibers the rate of tension redevelopment (ktr), a measure of the rate of cross-bridge cycling, was reduced at short SL compared with a longer SL (24). In skinned rat cardiac trabeculae, simultaneous measurements of isometric force and rates of ATP consumption at various levels of Ca2+ activation did not reveal alterations in the kinetics of the apparent detachment step in the cross-bridge cycle at various SLs (43). Furthermore, an analysis of the transfer functions of stiffness during sinusoidal length perturbations in the same type of preparations suggested that the processes that underlie the Frank-Starling mechanism are mediated mainly by the recruitment of cross bridges, rather than by alterations in cross-bridge transition kinetics (42). The absence of changes in cross-bridge kinetics with variation of SL was also indicated by measurements of ktr in permeabilized rat cardiac trabeculae (1).

The myosin heavy chain (MHC) composition of rodent ventricles and their myofibrillar activation kinetics differ from those of other mammals (29, 35); it is currently not known whether the controversy may in part relate to the length-dependent regulation in different muscle types with different myofibrillar protein compositions (19, 20, 23). In addition, little is known about factors modulating the length dependence of Ca2+ sensitivity in the human myocardium.

We therefore set out to perform a comparative investigation of the SL dependencies of force production and actin-myosin turnover rate in three different mammalian species. Isolated Triton-permeabilized human, murine, and porcine ventricular cardiomyocytes were exposed to a range of activating Ca2+ concentrations ([Ca2+]) under isometric conditions at two different SLs (1.9 or 2.3 µm) to assess Ca2+ sensitivities of isometric force production and ktr following unloaded shortening and rapid restretch. ktr reflects the isometric cross-bridge turnover kinetics: fapp + gapp, where fapp and gapp are the apparent rate constants characterizing the transitions from the non-force-generating states to the force-generating states and back to the non-force-generating states, respectively, in a simple two-state model of the actin-myosin cycle (Fig. 1; Ref. 4). This parameter has been reported to be sensitive to the level of activating Ca2+ and also to the myofibrillar protein composition (4, 29, 44), but to date no comparative studies have addressed its species-dependent characteristics during changes in SL. In one set of experiments, 10 mM inorganic phosphate (Pi) was included to probe the length dependence of force and ktr, since Pi has been suggested to decrease the number of strongly binding cross bridges and to enhance ktr and the length dependence of Ca2+-sensitive force production (14).


Figure 1
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  		Fig. 1. A scheme for the 2-state cross bridge model. The transition from detached non-force-generating cross bridges to attached force-generating cross bridges is governed by a rate constant fapp, whereas cross-bridge detachment is governed by a rate constant gapp. Since the reverse rate constants (f– and g–) are considered to be very small, the overall cycle can be approximated by first-order kinetics, and hence the rate of isometric force development is the sum of fapp and gapp.

 
Our data indicate that SL had no effect on ktr in the three different species under any of the experimental conditions. Thus we suggest that the Ca2+-dependent regulatory processes of force production link fixed cross-bridge cycling rates to various levels of Ca2+ activation, irrespective of muscle length, via the same fundamental myofibrillar mechanisms in the mammalian myocardium.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental tissue material. Adult human, murine, and porcine left ventricular cardiac tissues stored at –80°C were used in this study.

Healthy human hearts, obtained from three general organ donor patients (a 41-year-old man, a 46-year-old woman, and a 53-year-old woman) were explanted to obtain pulmonary and aortic valves as homografts for cardiac surgery. The donors did not show any sign of cardiac abnormalities and did not receive any medication except short-term dobutamine and furosemide. The cause of death was cerebral contusion and cerebral hemorrhage due to accident or subarachnoid hemorrhage due to stroke. The experiments on human tissues complied with the Helsinki Declaration of the World Medical Association and were approved by the Hungarian Ministry of Health (no. 323-8/2005-1018EKU). All biopsies were transported in cardioplegic solution (pH 7.4; in mM: 110 NaCl, 16 KCl, 1.6 MgCl2, 1.2 CaCl2, and 5 NaHCO3) and kept at 4°C for ~6–8 h before being frozen in liquid nitrogen.

FVB mice were bred in the Animal House of Jagiellonian University. Mice were maintained on 12:12-h light-dark cycles in air-conditioned rooms (22.5 ± 0.5°C, 50 ± 5% humidity) and had access to chow and water ad libitum. Female FVB mice (age 12–14 mo) were injected with fraxiparine (1,000 IU) and killed under thiopental anesthesia (100 mg/kg ip) by cervical dislocation. After opening of the thorax the whole heart was rapidly excised and quickly washed in cold (4°C) physiological saline solution, and after removal of the atria and the right ventricle the left ventricle was frozen in liquid nitrogen. The experimental procedures used in the present study were approved by the Jagiellonian University Ethical Committee on Animal Experiments (no. 1/OP/2003).

Juvenile farm pigs (weight 35–45 kg) were anesthetized with isoflurane and then euthanized with an intravenous injection of saturated KCl solution. The chest was rapidly opened, and transmural tissue samples were harvested from the left ventricular free wall and immediately frozen in liquid nitrogen. Pigs received humane care in accordance with the Italian law (DL-116, Jan. 27, 1992), which is in compliance with the National Institutes of Health publication Guide for the Care and Use of Laboratory Animals, and the scientific project, including animal care, was supervised and approved by the Italian Ministry of Health.

Force measurements in permeabilized cardiomyocyte preparations. All chemicals used for the experimental solutions were obtained from Merck (Darmstadt, Germany) unless otherwise indicated. The technique of isolation of cardiomyocyte preparations has been described elsewhere (30).

The compositions of the relaxing and activating solutions used during the force measurements were calculated as described previously (30). The pCa, i.e., –log[Ca2+], of the relaxing solution and the activating solution (pH 7.2) were 9 and 4.75, respectively. Force measurements were carried out by using two pairs of relaxing and activating solutions, one pair without added Pi and the other pair with added Pi. The compositions of the solutions were as follows: the activating solution without Pi contained (mM) 1 free Mg2+, 5 MgATP, 15 phosphocreatine, 7 calcium ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (Ca-EGTA), and 100 N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid. The ionic equivalent was adjusted to 150 mM with KCl at an ionic strength of 186 mM. The pH was adjusted to 7.2 with KOH. The composition of the Pi-free relaxing solution differed from that of the activating solution only in the use of 7 mM EGTA instead of Ca-EGTA. The second pair of solutions differed from the first pair of solutions in that 10 mM KH2PO4 was also added to both the activating and relaxing solutions. The ionic equivalent was identically adjusted to 150 mM with KCl at an ionic strength of 191 mM. All solutions were supplemented with the protease inhibitors leupeptin (40 µM; batch no. L-2303, Sigma, St. Louis, MO) and E-64 (10 µM; batch no. E-3132, Sigma).

The technique used for force measurements in single myocyte-sized preparations was described previously (3). The average SL of the preparation was determined in the relaxing solution by means of a spatial Fourier transformation, as described previously (8). The diameters of the preparations were measured microscopically in two perpendicular directions. The thickness of the preparation was determined by focusing on the lower and upper surfaces of the cardiomyocytes and measuring the displacement of the objective. The cross-sectional area was calculated by assuming an elliptical cross section.

Isometric force measurements were performed during repeated activation-relaxation cycles at 15°C (and in some human cardiomyocytes also at 25°C), first at a SL of 1.9 µm and then at a SL of 2.3 µm, in every cardiomyocyte. Force development was followed after the myocardial cell had been transferred from a droplet of relaxing solution to a droplet of activating solution (both having volumes of 50 µl) by moving the stage of the microscope laterally. When the peak force was reached, the length of the cardiomyocyte was reduced by 20% within 1 ms, and 20 ms later the original length was restored. As a result of this intervention, the force dropped from the peak level to zero, thus allowing the determination of the total force level, and then started to redevelop. About 6 s after the onset of force redevelopment, the cardiomyocyte was returned to the relaxing solution, where a shortening to 80% of original preparation length with a long slack duration (10 s) was performed to assess the passive force level. The active isometric force was calculated by subtracting the passive force from the total isometric force. The force redevelopment after the restretch was fitted to a single exponential function in order to estimate the rate constant of force redevelopment (ktr) at various [Ca2+] levels: F(t) = Fi + Fa(1 – eFormula), where F(t) is the force at any time t after the restretch at a given [Ca2+], and Fi and Fa denote the initial force value after the restretch and the amplitude of Ca2+-activated force redevelopment, respectively. Force records between Fi and complete force recoveries were included for ktr determinations for all [Ca2+] and species. At low [Ca2+] (i.e., at less than ~20% of the control maximal Ca2+-activated force), the low signal-to-noise ratio prevented the determination of ktr.

Force and length signals were monitored with an analog pen recorder and stored in a personal computer. The sampling rate was 20 Hz during the overall experiment, and during the ktr determination it was 1 kHz.

Each experimental protocol began and ended with a control activation at saturating [Ca2+] (pCa 4.75). This allowed force normalization and assessment of the rundown of the preparations through the comparison of changes in maximal Ca2+-activated force (Fo). The Fo value used to normalize submaximal force levels was obtained by linear interpolation between successive maximal activations. If at the end of the experimental session the cardiomyocyte produced less than at least 80% of the maximum force of the first contraction, that preparation was excluded from the analysis. Because of rundown, force reduced, on average, at a SL of 1.9 µm to 94.6 ± 0.1% in human cardiomyocytes (n = 23), 97.6 ± 0.7% in murine cardiomyocytes (n = 19), and 98.1 ± 0.2% in porcine cardiomyocytes (n = 16) (P > 0.05). The rundown at 2.3 µm did not differ significantly from that at 1.9 µm. Furthermore, we tested whether ktr differed because of the rundown during the first and the last control activations and also excluded experiments where the ktr for the last contractions was <80% of that of the first. The final values of ktr amounted to 92.7 ± 3.5% in human cardiomyocytes (n = 23), 99.2 ± 10.6% in murine cardiomyocytes (n = 12), and 97.8 ± 2.4% in porcine cardiomyocytes (n = 15) of the first values at a SL of 1.9 (P > 0.05). The change in SL had no significant effect on the rundown of ktr.

Data analysis. Ca2+-force relations were fitted to a modified Hill equation: F = Fo[Ca2+]Formula/(CaFormula+ [Ca2+]Formula), where F is the steady-state force at a given [Ca2+] and Fo, nH, and Ca50 (or pCa50) denote the maximal Ca2+-activated force at saturating [Ca2+] and the slope and the midpoint of the sigmoidal relationship, respectively. To check whether the Ca2+-force relations were symmetrical with respect to pCa50 and hence a single nH correctly approximated our data, a linearization procedure using the following equation was also employed: log[FFormula/(1 – FFormula)] = nH(log[Ca2+] + k), allowing for different values of nH for pCa values below and above pCa50, where FFormula is the steady-state force as a fraction of Fo, nH is the Hill coefficient, and k is the pCa50.

Each experimental preparation was fitted individually, the fitted parameters were pooled, and the mean values are reported. Statistical significance was calculated by analysis of variance (ANOVA, repeated measures) and, where applicable, by Student's t-test. Values are given as means ± SE. The number of experiments in each group was six or more from at least three different hearts. Statistical significance was accepted at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Figure 2 illustrates representative contraction/relaxation cycles in permeabilized human, murine, and porcine cardiomyocytes at [Ca2+] (pCa 6.0 for the human and porcine and pCa 6.2 for the murine preparations) resulting in approximately half-maximal force at the SL of 1.9 µm. Figure 2, A–C, shows that the force increased by 57% in human, by 59% in murine, and by 48% in porcine cardiomyocytes after the readjustment of SL from 1.9 µm to the final 2.3 µm. Figure 2, D–F, presents the corresponding traces in response to these maneuvers after force normalization for the same durations to reveal differences in force redevelopment kinetics, if present. Although the kinetics of force redevelopments in the murine cardiomyocyte were markedly higher than those in the human and porcine cardiomyocytes, the force redevelopment rates appeared to be very similar at short and long SLs within the same species. Table 1 lists the mean values of the Ca2+-activated force and Ca2+-independent passive force in the three different species at short and long SLs. At the 2.3-µm SL the passive force of murine myocytes was significantly higher than that in porcine or human cardiomyocytes, in accordance with the previously suggested high resting stiffness in murine cardiomyocytes (6).


Figure 2
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  		Fig. 2. Force responses during isometric contractions in single permeabilized cardiomyocytes of human (A and D), mouse (B and E), and pig (C and F) hearts at a short sarcomere length (SL) (1.9 µm) and subsequently at a long SL (2.3 µm). The Ca2+ concentration ([Ca2+]) for activation was selected to produce about half-maximal force at 1.9-µm SL (pCa 6.0 for A and C, pCa 6.2 for B). A–C illustrate the length-dependent enhancement in peak force levels at a slow time base, while D–F demonstrate preserved force redevelopment kinetics following brief shortening to 80% of the initial length, followed by a restretch on a fast time base. Horizontal dashes denote zero force levels before activations in A–C. To facilitate comparison of the force redevelopment kinetics at SLs of 1.9 and 2.3 µm in the 3 different species in D–F, force traces were normalized to maxima attained and illustrated for the same durations. Curve fittings to determine the rate of force redevelopment (ktr) always included complete force recoveries until reaching steady state.

 

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  		Table 1. Parameters of force-pCa and ktr-pCa relationships of isolated cardiomyocytes in human, mouse, and pig hearts in the absence or presence of Pi at 15°C

 
To compare the SL-dependent enhancement of isometric force production in the three investigated species, normalized force-pCa relations were determined under identical experimental conditions (Fig. 3A). Figure 3 shows that the change in Ca2+ sensitivity of force production (i.e., the difference in pCa50 resulting in half the maximal force for an increase in SL from 1.9 µm to 2.3 µm) was very similar in human, murine, and porcine cardiomyocytes and corresponded to ~0.11 pCa unit. Moreover, there were no statistically significant differences in nH between the three species (Table 1). When nH were derived from the force values after a linearization procedure (Fig. 3A, insets) there were also no statistically significant differences in nH between the three species. However, as suggested by the results in Fig. 2, there were major differences when the ktr values for the murine preparations were compared with those for the human or porcine cardiac preparations (Fig. 3B) at all investigated [Ca2+]. While the human and porcine cardiomyocytes exhibited comparable absolute ktr,max values (measured at pCa 4.75) at both SLs, the murine preparations displayed significantly higher ktr values at both SLs (i.e., ktr was ~6–7 times higher in mice than in humans or pigs). ktr decreased with [Ca2+] at both SLs in all preparations. However, SL adjustment did not have a statistically significant effect on ktr in any preparation at any [Ca2+]. To compare the extent of the Ca2+-dependent changes in ktr, the ktr values were normalized to their respective maxima during each experiment (Fig. 3C). This type of data representation suggests that the Ca2+ dependencies of ktr are very similar in the three investigated species.


Figure 3
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  		Fig. 3. Force-pCa (A) and ktr-pCa (B and C) relations in human, mouse, and pig cardiomyocytes at SLs of 1.9 and 2.3 µm. Force values of submaximal activations were normalized to maximal forces at both SLs in A to stress the similarities in length-dependent Ca2+ sensitization in the 3 species. Insets in A show the same data after force linearization. The Hill coefficients derived from these latter analyses were 2.09 ± 0.12 and 2.1 ± 0.11, 1.98 ± 0.10 and 2.00 ± 0.19, and 1.65 ± 0.11 and 1.67 ± 0.08 at SLs of 1.9 and 2.3 µm from human, mouse, and pig cardiomyocytes, respectively (P > 0.05).ktr is expressed in absolute values in B to illustrate distinctions in the underlying cross-bridge turnover rates and in relative terms in C to show that the extents of Ca2+-dependent changes in ktr were comparable in cardiomyocytes of human, mouse, and pig hearts at both SLs. The means of the ktr values at a SL of 1.9 µm did not differ significantly from those measured at 2.3 µm in any species or at any [Ca2+]; n = 16 cardiomyocytes for human, n = 8 cardiomyocytes for murine, and n = 6 cardiomyocytes for porcine samples.

 
To test whether the freeze-thaw cycle modified the mechanical properties of our preparations, we compared the results of measurements in eight freshly isolated cardiomyocytes (from 3 different mice) with those of frozen tissues. Active and passive forces in these additional experiments were 28.5 ± 2.3 and 3.0 ± 0.3 kN/m2 at the 1.9-µm SL and 59.6 ± 5.9 and 8.9 ± 0.4 kN/m2 at the 2.3-µm SL, respectively. ktr,max in these myocytes were 8.50 ± 0.45 s–1 at the 1.9-µm SL and 7.83 ± 0.34 s–1 at the 2.3-µm SL. None of these values was significantly different from that obtained from frozen tissue. Moreover, neither the results of the Hill plots nor the ktr values at submaximal [Ca2+] differed between freshly isolated and thawed murine preparations.

Pi is known to blunt the maximal Ca2+-activated force production through direct interference with force-generating cross-bridge transitions (17, 31, 34, 38). Accordingly, we studied the effects of 10 mM Pi on force production and ktr. Figure 4A depicts the reductions in Ca2+-activated force in the presence of 10 mM Pi. The maximal Ca2+-activated force (Fo) was decreased by 10 mM Pi to ~60–65% of the Pi-free values in all three species (i.e., to 65 ± 3%, 60 ± 1%, and 61 ± 1% in humans, mice, and pigs, respectively; P > 0.05) at a SL of 1.9 µm. Moreover, the effect of 10 mM Pi on Fo did not depend on the SL. To address the SL dependencies of Ca2+ sensitivity in the presence of Pi, the force values were also normalized to their respective maxima and the data were fitted to the Hill equation (Fig. 4B). This analysis revealed that, for all of the species, the change in pCa50 in the presence of Pi did not differ from that in the absence of Pi (Table 1), i.e., it was ~0.1 pCa unit. However, 10 mM Pi induced a dramatic acceleration in the cross-bridge turnover kinetics in all preparations. The ratio of the ktr,max values in the presence and absence of Pi was ~1.6 in both humans and pigs, whereas in mice it was nearly 3 (see Table 1 for details) at both SLs. The SL had no significant effect on ktr in the presence of Pi at any investigated [Ca2+].


Figure 4
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  		Fig. 4. Relative force-pCa (A), normalized force-pCa (B), and ktr-pCa (C) relations in human, mouse and pig cardiomyocytes at SLs of 1.9 and 2.3 µm in the presence of 10 mM inorganic phosphate (Pi). Force values of submaximal activations were normalized to maximal forces at both SLs in A to illustrate the similarities in the Pi-dependent depression in maximal Ca2+-activated force (Fo) in the 3 species. Force values of submaximal activations were normalized to maximal forces at both SLs in B to show the preserved length-dependent Ca2+ sensitization in the presence of Pi. ktr is expressed in absolute values in C to illustrate the Pi-induced parallel enhancement in cross-bridge turnover rates. Dotted and continuous lines without symbols in A and C are the corresponding sets of data in the absence of Pi. The means of the ktr values at a SL of 1.9 µm did not differ significantly from those measured at 2.3 µm in any species or at any [Ca2+] in the presence of Pi. n = 8 cardiomyocytes for human, n = 12 cardiomyocytes for murine, and n = 12 cardiomyocytes for porcine samples.

 
With increasing temperatures the Pi-induced acceleration in cross-bridge kinetics is augmented in skeletal myofibrils of the rabbit (38) and cardiac myofibrils of the mouse (33). Hence, we set out to verify whether this effect is also present in human cardiomyocytes, using a more physiological experimental temperature. Moreover, we tested whether a change in temperature affected the Pi dependence of force production and the apparent independence of ktr from SL. To these ends, Ca2+ contractures were also evoked in the presence of Pi at 25°C (Fig. 5). At a SL of 1.9 µm, maximal Ca2+-activated force at 25°C in the absence of Pi was 35.2 ± 4.2 kN/m2 (n = 10 human cardiomyocytes), while in the presence of 10 mM Pi it was 17.4 ± 2.4 kN/m2. Hence, these experiments showed that at a higher temperature the decrease in the maximal Ca2+-activated force by Pi was somewhat larger in the human myocardium. When the data were expressed in relative terms at a SL of 1.9 µm, the reduction in Fo by 10 mM Pi at 25°C was to 52.1 ± 0.9%, whereas at 15°C it was to 60.0 ± 1.9% (P < 0.05; Fig. 5A). The SL had no influence on the Pi-evoked reduction in force. The amplitudes of the Ca2+ contractures at 25°C were also normalized, and the Ca2+ sensitivities of force production were determined at both SLs (Fig. 5B). Similar to the previous results, the shift in pCa50 did not change ({Delta}pCa50 0.10 ± 0.02; P < 0.05), despite the fact that ktr was further increased with temperature (to ~5.2-fold when expressed as a function of ktr,max in the absence of Pi at 15°C). ktr,max at 25°C for human cardiomyocytes was 7.03 ± 0.32 s–1. However, no significant differences could be observed between the ktr values of the two SLs at the same activating [Ca2+] at 25°C in human cardiomyocytes (Fig. 5C).


Figure 5
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  		Fig. 5. Relative force-pCa (A), normalized force-pCa (B), and ktr-pCa (C) relations in human cardiomyocytes at SLs of 1.9 and 2.3 µm in the presence of 10 mM Pi at 25°C. Force values of submaximal activations were normalized to maximal forces at both SLs in A to illustrate the temperature (T)-dependent enhancement of Pi-induced depression on Fo. Force values of submaximal activations were normalized to maximum forces at both SLs in B to show the preserved length-dependent Ca2+ sensitization at the higher temperature. ktr is expressed in absolute values in C to illustrate the combined effects of temperature and Pi on cross-bridge turnover rates. Dotted and continuous lines without symbols in A and C are the corresponding sets of data in the absence of Pi, as indicated in A. The means of ktr values at 1.9-µm SL did not differ significantly from those measured at a SL of 2.3 µm at any [Ca2+] in the presence of Pi at 25°C; n = 12 human cardiomyocytes.

 
To compare the activation dependencies of ktr on species and Pi, ktr was replotted against the force normalized to Fo under all experimental conditions at 15°C (Fig. 6). ktr increased nonlinearly with force at each SL and in all species to an apparent maximum at Fo. In general, at the shorter SL, ktr tended to be slightly larger at equivalent submaximal force levels in all preparations and under all experimental conditions, similar to that reported previously in rat cardiac trabeculae (1). The increase in ktr at submaximal levels of force, when force was matched at both SLs, might be due to the higher [Ca2+] needed to reach the same level of force at the shorter SL. In the absence of Pi and at a SL of 2.3 µm, the range of ktr change for doubling of the force from half-maximal force to Fo was ~1.7-fold in humans, 2.2-fold in mice, and 2.4-fold in pigs. For the same increase in force, these changes in ktr were somewhat less at a SL of 1.9 µm (P > 0.05). Ten millimolar Pi shifted the ktr dependencies on force toward higher levels, but it did not change the characteristic slopes of these relationships at constant SL within the same species.


Figure 6
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  		Fig. 6. SL dependence of ktr-force relations for the data obtained from human (A), mouse (B), and pig (C) cardiomyocytes shown in Figs. 3 and 4. Data are shown with the force normalized to the Fo of each condition and SL. Open symbols denote values obtained at a SL of 1.9 µm, and closed symbols denote values at a SL of 2.3 µm. Circles denote data in the absence of Pi, and squares denote data in the presence of 10 mM Pi.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
These results indicate that ktr varies with species, [Ca2+], [Pi], and (in human cardiomyocytes) temperature, but not with SL in permeabilized cardiomyocytes. Furthermore, our results illustrate identical SL-dependent changes in the Ca2+-activated force production for human, murine, and porcine cardiomyocytes, despite marked differences in the underlying cross-bridge turnover rates. On the other hand, the Ca2+ dependencies of relative ktr values appeared to be similar in these three species. Together, our data suggest similar characteristics for the Ca2+ regulation of force and ktr, whereas changes in SL are not accompanied by alterations in ktr in the mammalian heart.

It is generally accepted that the species specificity of MHC composition and the high ratio of expressed fast {alpha}-MHC isoform versus slow beta-MHC isoform are responsible for the high cross-bridge turnover rates of rodent hearts (35) compared with large mammals. Our data suggest that the fast {alpha}-MHC does not characteristically alter the SL-dependent Ca2+ sensitization. Hence, the myosin isoenzyme switch during the development of chronic heart failure, which involves a relative increase in beta-MHC isoform expression at the expense of {alpha}-MHC expression (26), is expected not to interfere with this process. Moreover, our results suggest that the species differences in the expression of the contractile proteins do not give rise to changes in the extent of SL-dependent Ca2+ sensitization in the mammalian heart.

Pi reduces the overall free energy of MgATP hydrolysis; it reverses the Pi release step of the cross-bridge cycle through mass action and thereby decreases the proportion of cross bridges in the high-force conformation (17, 34, 38). Furthermore, the Pi-induced changes in pCa50 are considered to be a consequence of the altered distribution of actin-myosin states within the cross-bridge cycle (28, 31). Our data indicate that Pi reduced the force independently of the species and of the SL. Furthermore, Pi induced a small rightward shift in the Ca2+-force relations and did not change nH under any of the investigated conditions. Overall, these data suggested that Pi release in the cross-bridge cycle for the different species studied is highly conserved. The Pi-induced accelerations in cross-bridge turnover are in accord with previous experimental observations (33, 38). However, ktr was increased to a greater extent by Pi in murine than in human or porcine cardiomyocytes. ktr increased further when the temperature was raised to 25°C in the presence of Pi in human cardiomyocytes. Nevertheless, at a given [Ca2+] and Pi ktr did not change with SL in any of the three species. At high temperatures the reduction in Fo by Pi was smaller than at low temperatures (38) in rabbit soleus and psoas myofibrils. In contrast to this finding, our data in human cardiomyocytes and of others in mouse myofibrils (33) suggest that the Pi-dependent reduction of Fo is not attenuated at higher temperatures in the myocardium.

The molecular mechanism by which an increase in SL promotes Ca2+-activated myofibrillar force production is not fully understood. It appears reasonable to suppose, however, that at a given submaximal free [Ca2+], an increase in SL results in a cooperative amplification of the number of force-generating cross bridges (1, 10, 11, 13) and increased binding of Ca2+ to the thin filament protein troponin C (15, 16). The extent to which these two mechanisms are coordinated and how they affect the kinetics of the actin-myosin cycle is largely unknown. Theoretical considerations imply that the cooperative amplification of the number of force-generating cross bridges would decrease the turnover rate of the cross-bridge cycle (5), whereas an increased binding of Ca2+ to troponin C would increase this turnover rate (4, 29, 44). The present study revealed that at a given [Ca2+] ktr was constant despite SL-dependent alterations in force. In other words, in all three investigated species, variations in [Ca2+] and Pi (and in human hearts also in temperature) did not differentiate between these two scenarios. This might imply that the two processes are in exact opposition, but given the large variation in experimental conditions tested, this would seem highly fortuitous. Hence it appears that [Ca2+], and not SL, is the major determinant of ktr in the mammalian heart. Moreover, our findings support the suggestion that the Ca2+ binding to cardiac troponin C does not directly modulate cross-bridge steps that are rate limiting to ktr (1). This idea is consistent with the observation that the ratio of ATPase activity to force during isometric contraction at maximal Ca2+ activation does not change with decreasing SL in permeabilized cardiac trabeculae of the rat (18) until 1.95 µm and below, where restoring forces may oppose contraction (18, 43). Accordingly, it was suggested that the kinetics of the apparent detachment step (i.e., gapp) in the cross-bridge cycle do not change with SL between 2.0 and 2.2 µm (43). An unchanged gapp together with a constant ktr would then suggest that fapp does not change with SL either. Hence our results are in agreement with the proposals based on cross-bridge recruitment (42), a "switchlike" activation mechanism (9), and/or a three-state model of thin filament activation (25).

This latter model postulates that two Ca2+-controlled transitions of the thin filament would permit the separate regulation of a weak-binding cross-bridge attachment (a closed state) and force generation (an open state). To explain length-dependent activation, it was proposed that the size of the thin filament regulatory units in the closed state is length dependent (22, 36). Our results lend support to this latter hypothesis because they allow the development of a SL-dependent increase in force without apparent changes in cross-bridge kinetics.

Limitations of study. Our estimates on force per cross-sectional area are complicated by the limitations inherent to the determination of the thickness of the preparations. In addition, regional and cell-to-cell variation could also modulate the results of these force values (6, 7). Our ktr determinations in human and pig hearts gave results very similar to those of several previous studies (3, 27, 40), and our ktr values in mouse hearts are very similar to those of others using isolated cardiomyocytes (21), isolated myofibrils in mice (33), or cardiomyocytes in rats (41). However, ktr,max values of multicellular cardiac preparations were about two times higher both in murine (32, 37) and in rat (2, 29, 35) hearts than those in cardiomyocytes or myofibrils (21, 33, 41). We would like to point out, however, that a large part of this discrepancy can be ascribed to differences in experimental temperature, because ktr,max in multicellular preparations of rats ranged between 9 s–1 (11, 44) and 12 s–1 (1) at 15°C. SL was not controlled during the course of the ktr, and hence this may explain why our ktr estimates fell below this range (39, 44). Deviations from the theoretical values of force/cross-sectional area or ktr would apply to all of the preparations in all of the groups, and in our study, therefore, the above factors would not alter or mask our main finding of the independence of ktr from SL.

Our observation that ktr is unaffected by changes in SL is in accord with earlier observations in rat hearts (1) and extends them to the human, murine, and porcine myocardium. The findings that [Pi], [Ca2+], temperature, and species differences were all involved in the modulation of ktr, while SL was not, strongly suggest that kinetic alterations within the actin-myosin cycle are not prerequisites for the length-dependent Ca2+ /øsensitization in the heart.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
The work was supported by grants from the Hungarian Ministry of Health (ETT 449/2006) and the Hungarian-Polish Intergovernmental Cooperation Programme (PL-12/2005) through the National Office for Research and Technology and the Agency for Research Fund Management and Research Exploitation from the Research and Technology Innovation Fund and in part by the Compagnia di San Paolo, Torino, Italy (F. A. Recchia). Z. Papp holds a Bolyai Fellowship. G. Csányi (Department of Pathophysiology, Albert Szent-Györgyi Medical and Pharmaceutical Center, University of Szeged, Hungary) was a recipient of Jagiellonian University Fellowships, J. Dietl's fund.


   ACKNOWLEDGMENTS
 
Present address of Gabor Csanyi: Department of Pathophysiology, Albert Szent-Gyorgyi Medical and Pharmaceutical Center, University of Szeged, Semmelweis u. 1., H-6701 Szeged, Hungary.


   FOOTNOTES
 

Address for reprint requests and other correspondence: Z. Papp, Div. of Clinical Physiology, Inst. of Cardiology, Univ. of Debrecen, Medical and Health Science Center, Faculty of Medicine, PO Box 1, H-4004 Debrecen, Hungary (e-mail: pappz{at}dote.hu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R961 - R962.
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