The force-frequency relationship is an intrinsic modulator of cardiac contractility and relaxation. Force of contraction increases with frequency, while simultaneously a frequency-dependent acceleration of relaxation occurs. While frequency dependency of calcium handling and sarcoplasmic reticulum calcium load have been well described, it remains unknown whether frequency-dependent changes in myofilament calcium sensitivity occur. We hypothesized that an increase in heart rate that results in acceleration of relaxation is accompanied by a proportional decrease in myofilament calcium sensitivity. To test our hypothesis, ultrathin right ventricular trabeculae were isolated from New Zealand White rabbit hearts and iontophorically loaded with the calcium indicator bis-fura 2. Twitch and intracellular calcium handling parameters were measured and showed a robust increase in twitch force, acceleration of relaxation, and rise in both diastolic and systolic intracellular calcium concentration with increased frequency. Steady-state force-intracellular calcium concentration relationships were measured at frequencies 1, 2, 3, and 4 Hz at 37°C using potassium-induced contractures. EC50 significantly and gradually increased with frequency, from 475 ± 64 nM at 1 Hz to 1,004 ± 142 nM at 4 Hz (P < 0.05) and correlated with the corresponding changes in half relaxation time. No significant changes in maximal active force development or in the myofilament cooperativity coefficient were found. Myofilament protein phosphorylation was assessed using Pro-Q Diamond staining on protein gels of trabeculae frozen at either 1 or 4 Hz, revealing troponin I and myosin light chain-2 phosphorylation associated with the myofilament desensitization. We conclude that myofilament calcium sensitivity is substantially and significantly decreased at higher frequencies, playing a prominent role in frequency-dependent acceleration of relaxation.
- force-frequency relationship
- calcium transient
- diastolic dysfunction
the ability of the heart to modulate its contractility ensures adequate cardiac output during shifts of physical activity. The principal modulator of cardiac contractile function acts through changes in heart rate. The Bowditch effect, a frequency-dependent gain in contractility, is an intrinsic property of cardiac muscle present in all mammals and allows for greater contractile force [positive force-frequency relation (FFR)] and faster relaxation [frequency-dependent acceleration of relaxation (FDAR)] at higher pacing frequencies (7).
It is well known that sarcoplasmic reticulum (SR) calcium handling is enhanced with increasing frequency, resulting in greater calcium transient amplitude with a faster decline (6, 13, 18, 25). While it is undisputed that SR calcium transport plays a pivotal role in the FFR and FDAR, it remains unresolved whether modulations in myofilament properties are also involved in FDAR. Myofilament calcium sensitivity has been argued to be increased (9), unchanged (15), or decreased (24) with increasing pacing frequency, but has generally been overlooked as a possible modulator of FDAR. Mainly due to technical limitations, a possible frequency dependency of myofilament calcium sensitivity remained unresolved. In addition, previous experiments performed to assess FFR and FDAR have largely been done in rats (3, 6, 13, 15) and mice (4, 23, 24), where the excitation-contraction coupling process shows major differences from larger mammals, such as the rabbit, dog, and human (19). Hence it remains unresolved whether myofilament calcium sensitivity is affected by heart rate and whether it plays a role in FDAR.
Upon β-adrenergic stimulation, it is well known that relaxation is accelerated, in part, due to phosphorylation of troponin I (TnI) (16), allowing for faster relaxation. Preliminary data in the rabbit for this study indicated that systolic calcium (like all mammals) and diastolic calcium (unlike small rodents) both rose while still maintaining FDAR. We, therefore, hypothesize that incremental changes in relaxation rates associated with stepwise increases in frequency (and diastolic calcium) must be accompanied by proportional decreases in myofilament calcium sensitivity to prevent diastolic dysfunction that would otherwise likely occur with increases in diastolic calcium. Our results indeed indicate that a substantial and significant desensitization in myofilament calcium sensitivity occurs as heart rate increases.
This protocol was approved by the institutional animal care and use committee of The Ohio State University. Male New Zealand White rabbits were anesthetized with 50 mg/kg pentobarbital and injected with 5,000 U/kg heparin. After bilateral thoracotomy, hearts were rapidly excised, and thin uniform trabeculae (average dimensions 155 ± 13 μm wide by 93 ± 8 μm thick and 1.56 ± 0.2 mm long) were carefully dissected and mounted in the setup, as previously described for rat (26). Trabeculae were iontophorically loaded (at 25°C) with the calcium indicator bis-fura 2, as described previously (2, 14, 26). This method of dye loading has previously been used for rat and mouse muscle by us (14, 26) and others (2, 17). In this study, we successfully adapted the technique for rabbit myocardium. We observed some minor technical differences between the species. Compared with rat, we have a slightly lower success rate in getting a muscle loaded. However, when loaded, the dye leakage that has been reported to be 15%/10 min in the rat at 37°C (although less fast at room temperature) was several times slower, often only 20% over the duration of the entire protocol. As a result, dye leakage was never to an extent where total fluorescence was less than two to three times the background. This expanded the experimental time to ∼30 min, thus allowing for a calibration of the signals that was precluded in rat (26) and mouse (22) muscles at 37°C. For these reasons, we used bis-fura 2. This dye has twice the fluorescence intensity compared with its calcium binding, and thus we were able to load more dye initially (preventing from getting close to background), while steering clear of significant dye buffering effects that can occur when loading too much indicator.
Because of time limitations of each experiment and the time needed for calibration, the muscle was assigned to one of two protocols: myofilament calcium sensitivity and twitch force-intracellular calcium concentration ([Ca2+]i) was assessed at 37°C, either at 1 and 3 Hz (n = 7), or at 2 and 4 Hz (n = 7). After equilibration at a given frequency, a slowly forming contracture was induced by introducing modified Krebs-Henseleit solution of 110 mM K+/40 mM Na+ for 40 s, and force and fluorescence signals were recorded, as described previously (27). Bis-fura 2 emissions were calibrated to [Ca2+]i by determining the minimum ratio (Rmin) and maximum ratio (Rmax) in each muscle (14). Force-[Ca2+]i data were plotted, and this curve was fit with the Hill equation using an iterative fitting procedure. EC50, maximal developed force (Fmax), and the Hill coefficient (nHill) were calculated for each data set. In a subset of experiments where dye leakage was extremely small, a third contracture was performed at the initial frequency. These control experiments allowed us to show that changes in the observed curves were indeed caused by frequency and not a time-dependent phenomenon; curves obtained at 1 Hz before and after assessment at 3 Hz were identical. To obtain paired data on twitch [Ca2+]i and force across the entire investigated range of frequencies (1–4 Hz), an additional series of experiments were conducted in which twitch kinetics and calcium transients were collected in the same muscle at all four frequencies (n = 4). We chose the 1- to 4-Hz range, because it encompasses most of the in vivo rabbit range, which is roughly 2–5 Hz. We did not obtain data at the very high end of the spectrum (5 Hz), because of the use of isometric contractions, which are slightly longer than ones in which shortening is allowed. This would likely cause a diastolic elevation of force at 5 Hz that would not reflect the in vivo situation. In addition, because of the high metabolic demand at 5 Hz, rundown is greater, and this may compromise the data.
To rule out, or potentially unmask, a role of pH in the modulation of FDAR, we measured intracellular pH using the ratiometric pH indicator BCECF (excitation 440 nm and 495 nm, emissions at 535 nm) at all four frequencies. The ratio 440/495 correlates with H+ concentration. The indicator was iontophorically loaded using the same protocol for loading bis-fura 2.
Because myofilament protein phosphorylations have, in the past, been shown to alter myofilament calcium sensitivity, phosphorylation analysis was carried out using Pro-Q Diamond stain technology. Trabeculae twitching at either 1 or 4 Hz was doused with liquid nitrogen until frozen and rapidly removed from the experimental setup. The tissue was then homogenized in an SDS protein lysis buffer and loaded on a 13% 8 × 10-cm SDS-PAGE gel (0.75 mm thickness, 4% stacking gel, 5 wells). The gel was then run for 45 min at 175 V. The gel was fixed overnight and stained using the ProQ Diamond phosphoprotein basic staining protocol (Invitrogen). Following staining and destaining, the gel was imaged in a Typhoon variable mode scanner (GE Healthcare) using an excitation wavelength of 532 nm and a 610-nm (BP30) emission filter at a photomultiplier tube setting of 450. After imaging, the gel was washed in water for 1 h and stained for total protein following the Sypro Ruby basic staining protocol (Invitrogen). Following staining and destaining, the gel was imaged in a Typhoon scanner using an excitation wavelength of 488 nm and a 610-nm (BP30) emission filter at a photomultiplier tube setting of 425. The gel was then stained using Coomassie Brilliant Blue, destained, and imaged with a desktop scanner. Densitometric analysis was performed on each band, and the ratio of Pro-Q stain intensity to total protein intensity was calculated.
Data obtained were analyzed with a paired or unpaired t-test (where applicable), with two-tailed P value of <0.05 being considered significant. A one-way ANOVA with repeated measures was used to analyze the FFR data [developed force, half relaxation time (RT50), diastolic and systolic calcium], where all four frequencies were measured in a single muscle. For protein data, ANOVA was used to detect differences, followed by a post hoc t-test. Data are depicted as means ± SE.
Figure 1, A and B, shows representative force and calcium tracings at 2 and 4 Hz, showing typical FFR and FDAR behavior. Notice that, although calcium decline is faster at 4 Hz, calcium is higher at all time points in the 4-Hz muscle. The difference between 2 and 4 Hz was similar to the difference between 1 and 3 Hz. Figure 1C shows the corresponding phase plane plots of the twitch force-calcium between 2 and 4 Hz. The rightward shift of the relaxation trajectory at the two frequencies (which is absent in small rodents) (14) initiated suspicions of a strong myofilament calcium sensitivity role in the FDAR.
Figure 2A shows the peak twitch force at all four frequencies measured in muscles where all frequencies were investigated. These data were taken from four muscles that did not undergo K+ contracture protocols (due to time limitations regarding assessment of fluorescence at 37°C). The respective average relaxation times from peak to 50% of peak (RT50) are shown in Fig. 2B. All trabeculae exhibited the expected normal positive FFR and normal FDAR generally found in the mammalian myocardium. When switching from one frequency to another (from 2 to 4, 4 to 2, 1 to 3, or 3 to 1), we found that it took 45 ± 4 s to reach a new steady-state twitch force. This is significantly slower than what is generally observed for smaller rodents (5–15 s typically), under otherwise near identical conditions.
Figure 3 shows the diastolic and peak systolic [Ca2+]i values for calcium transients at all four frequencies. Calcium transient amplitude increased substantially as frequency was increased. Diastolic [Ca2+]i also significantly rose with increasing stimulation frequency. With increasing frequency, the amplitude of the [Ca2+]i rose, due to a larger rise in systolic vs. diastolic calcium.
Figure 4A shows a chart of a typical experiment when the high-potassium solution was superfused onto the muscle: the twitch contractions (2 Hz in this example) began to disappear (due to loss of membrane potential), and diastolic calcium levels (measured right before each stimulation pulse, at end of diastole) and force began to rise. We tracked [Ca2+]i (bis-fura 2 ratios) and force all the way to the peak of the contracture (occurring in 20–30 s from initial rise). The downward leg of the K+ contracture was analyzed as well in most of the experiments, and generally no hysteresis was observed (superimposable on the upward leg). In the few muscles in which some hysteresis was observed, this was well past peak (at or past halfway down), resulting in the downward curve diverging from the upward curve, possibly due to fatigue (not shown). Figure 4B shows an example of how the data were analyzed. From the fitted curve through the force-calcium data combinations, Fmax, calcium sensitivity (EC50), and cooperativity (nHill) were determined.
Figure 5A shows the raw force-calcium data from a typical experiment. A clear rightward shift of sensitivity from 1 to 3 Hz was observed, followed by a shift back to the left upon a repeat at 1 Hz. A small amount of Fmax is lost due to time-dependent rundown that can never altogether be avoided. Average values for EC50 at the two sets of frequencies measured are depicted in Figure 2B. Fmax and nHill parameters did not significantly change with frequency: nHill at 1, 2, 3, and 4 Hz amounted to 5.4 ± 0.7, 4.6 ± 0.6, 6.0 ± 1.1, and 4.6 ± 0.7; Fmax amounted to 11.2 ± 2.8, 13.0 ± 2.5, 16.6 ± 2.0, and 16.3 ± 2.2 mN/mm2, respectively.
In 14 experiments, we assessed both FDAR (denoted as RT50 measurements), as well as myofilament calcium sensitivity. Figure 6 shows the relationship between the average EC50 and RT50 values obtained in the same muscles, grouped per frequency. There is a strong linear relationship between changes in myofilament calcium sensitivity and the change in relaxation rates.
As changes in frequency may modulate sodium levels and/or an altered energetic state, this could lead to significant changes in intracellular pH. To test whether pH changes could potentially explain (in part) the desensitization of the myofilaments at increasing frequency, we did additional experiments using the pH indicator BCECF. The fluorescence ratio 440/495 nm is inversely proportional to pH (ratio decreases as pH increases). Although time limitations currently do not allow a full calibration of the signals, we observed no change in the 440/495 ratio, as frequency increased (Fig. 7). Previous experience with the indicator has shown us that a change in ratio of at least 0.010 is necessary to elicit a detectable change in force (unpublished observations). Hence, we conclude pH changes are not responsible for the shift of the frequency-induced myofilament calcium sensitization.
In an attempt to further describe a possible mechanism for frequency-based myofilament desensitization, the phosphorylation status of myofilament proteins was assessed using the Pro-Q Diamond stain. Figure 8A shows a one-dimensional SDS gel stained for total protein for three muscles that had been stimulated at 1 Hz, three muscles at 4 Hz, and two muscles at 1 Hz, while under the influence of 1 μM isoproterenol. The two muscles stimulated in the presence of isoproterenol were included as a positive control for TnI phosphorylation. Figure 8B shows the same gel stained with the Pro-Q Diamond stain. The average ratios of phosphoprotein density to total protein are shown in Fig. 8, C and D, for TnI and myosin light chain-2 (MLC-2), respectively. A significantly greater level of phosphorylation of TnI (C) and MLC-2 (D) was observed in the muscles stimulated at 4 Hz and those stimulated with isoproterenol compared with those stimulated at 1 Hz.
Myofilament calcium sensitivity modulations based on frequency have been occasionally speculated by investigators attempting to describe the mechanism of the FFR and FDAR in more detail. However, there is little agreement as to the direction of a possible shift in myofilament calcium responsiveness. Previously, myofilament calcium sensitivity has been suggested to be sensitized (9), unchanged (15), or desensitized (24) at higher frequencies, but, more often in the studies addressing FDAR, a possible myofilament role in FDAR was not addressed altogether. Differences in findings are more than likely due to differences in experimental conditions, methods, and animal models chosen. Gao et al. (9) concluded that a frequency-dependent myofilament sensitization occurred and partially accounted for the increase in force. Their conclusion was based on a leftward shift in the relaxation trajectory of phase plane twitch force-[Ca2+]i relationship. They observed a disproportional increase in force for a given calcium concentration in the dynamic relaxation phase and concluded a likely sensitization of the myofilaments to be responsible. Kassiri et al. (15) found no significant difference between 0.2- and 2-Hz stimulations in the rat at room temperature. They used high-frequency stimulations in the presence of cyclopiazonic acid to induce steady-state force-calcium relationships, and it cannot be determined whether high-frequency stimulations to evoke tetani may overshadow frequency-dependent effects, or other experimental conditions may have possibly masked frequency-dependent changes. Tong et al. (24) investigated the role of frequency on myofilament sensitivity using an analytical method of analyzing dynamic force-[Ca2+]i phase-plane loops and concluded a frequency-dependent desensitization to exist, albeit at nonphysiological temperature and subphysiological frequency. Although the conclusion of the latter investigation is similar to ours, force and calcium are only in equilibrium during certain periods of diastole and not during the bulk part of the force decline. In addition, the speed of calcium handling in small rodents far exceeds that of larger mammals and could result in the emergence of different rate-limiting steps for relaxation between species. Thus whether frequency-based modulation of myofilament calcium sensitivity occurs remained incompletely understood.
Using our recently developed force-calcium assessment protocol (26), we were able to assess myofilament calcium sensitivity in larger mammals and measure this relationship in the physiological frequency range, as well as at physiological temperature. We here show that the myofilament matrix desensitizes upon increased stimulation frequency. The K+ contractures provide unique data into steady-state myofilament calcium sensitivity that is modulated during dynamic cardiac twitches, as they do not require poisoning of the SR, nor a high-frequency stimulation that could potentially mask frequency-dependent effects. To further validate the method, we ensured that, although diastolic and systolic calcium were different between the different frequencies, the calcium (amount and speed) entering the cytoplasm during the K+ contractures was not different between the frequencies. This ensured that, as the K+ contractures developed, changes in the myofilament status associated with the rising calcium per se would be similar at both frequencies. Thus the only difference between two contractures was the frequency of stimulation before and during the contracture. Second, after performing K+ contractures at both frequencies, we repeated the first frequency to observe if the second measurement was comparable to the first. This ruled out irreversible changes that would alter the response during a second contracture, as we observed full reversibility, and the results were independent of the order in which frequencies were used. With these control experiments, combined with the relatively low intermuscle variability at the various frequencies, we are confident that the changes in myofilament calcium responsiveness were caused by changes in stimulation frequency.
FDAR has been shown to occur over a wide range of frequencies, in many species, and even in the face of a negative FFR observed in failing tissue (20). Clearly, accelerated calcium handling may in part be responsible for FDAR via abbreviation of the part of the calcium transient that is above myofilament calcium binding threshold; abbreviation of a calcium transients per se will speed up relaxation. However, during frequency-dependent activation, not only is the [Ca2+]i decline faster, the [Ca2+]i amplitude is increased. As a result, throughout the bulk of the relaxation phase, despite the faster [Ca2+]i decline, the absolute [Ca2+]i is increased at all given time points throughout the larger part of the relaxation phase. Hence, with unaltered myofilament sensitivity, the muscle would simply not be able to relax as fast. In rabbits, dogs, and humans, ∼30% of the intracellular calcium decline is achieved via the Na+/Ca2+ exchanger, unlike rats and mice, where this is only ∼2–5%. Removal of Ca2+ via Na+/Ca2+ exchanger is significantly slower than reuptake of Ca2+ into the SR via the SR Ca2+-ATPase. As a result, in larger mammals, diastolic calcium rises with increased heart rate much more than in rats and mice as we here observe, which is in close agreement to what others (1) have observed in rabbit myocytes. Therefore, it is likely that the role myofilament calcium sensitivity plays in FDAR may be more critical in larger mammals than in small rodents. To have a faster force decline at increased frequency, the myofilaments of large mammals would have to be either less sensitive to [Ca2+]i, or produce significantly less force per cross bridge to explain the accelerated relaxation at higher rates. Clearly, our results indicate that, indeed, frequency-induced desensitization occurs in healthy rabbit myocardium, and thus helps to explain how myocardium of larger mammals is able to relax at high heart rates.
Compared with calcium sensitivity in skinned fiber preparations, we observed lower EC50 values for the lower heart rates used. These values, however, are consistent with previous studies in intact myocardium, confirming that calcium sensitivity is different in intact vs. skinned myocardium (8, 15, 26). A possible limitation of our study is that assessment of myofilament calcium sensitivity takes ∼20–30 s, whereas frequency-dependent changes in calcium transients and twitch force parameters take only a few seconds to begin developing. Although it is possible that changes in myofilament calcium sensitivity could occur throughout a contracture as calcium rises (skewing the absolute values), these changes would have been similar between frequencies. Similar to shifts observed in skinned fiber data, a shift in calcium sensitivity may be more revealing of a physiological process than the absolute calcium concentration at EC50 at which this occurs, as many factors, including computer calculations of the free calcium concentration, may not always be absolute. In addition, it has been speculated that intracellular pH may be involved in the frequency-dependent changes. Higher intracellular sodium levels at higher frequencies could possibly reduce the intracellular pH via reduced Na+/H+ exchange. We observed no change in intracellular pH when measured using BCECF at the range of frequencies investigated, and the observed calcium sensitivity shifts thus cannot be explained by pH effects.
Quantitatively, the observed shift in myofilament calcium sensitivity was very substantial: between 1 and 4 Hz, a 0.32 pCa shift was observed, which is at least a similar magnitude of a shift as reported for PKA-dependent desensitization via TnI phosphorylation (5, 12, 16). It is, therefore, not entirely surprising that a significant increase in TnI phosphorylation was found at 4 Hz compared with 1 Hz and that the phosphorylation at 4 Hz was not significantly different from muscles stimulated in the presence of isoproterenol. Although it is well known that TnI phosphorylation desensitizes the myofilaments (16), the current data on TnI phosphorylation are only correlative with frequency, and a causal relationship needs yet to be established. MLC-2 was also found to be phosphorylated significantly more in 4 Hz compared with 1 Hz. This is consistent with a previous finding in isolated, perfused rabbit septum, where a fourfold increase in MLC-2 phosphorylation was found between quiescent tissue and that which was stimulated at 2 Hz (21). The implications of MLC-2 phosphorylation remain poorly understood. More than one molecular signaling pathway could possibly be involved in the mediation of this effect; both PKA and PKC can desensitize the myofilament matrix via various phosphorylation actions and have been implicated in FDAR (23). Although in TnI and MLC-2 we have identified two potential molecular targets that can be responsible for the frequency-based myofilament desensitization, a complete and conclusive dissection of the underlying mechanism is deemed well beyond the current scope of this study.
Clinically, an impaired FFR is one of the major hallmarks of cardiac failure (11), and diastolic dysfunction is present, and often even dominant, in a large percentage of heart failure patients (10). If the frequency-dependent desensitization as we here describe does not occur in failing myocardium, or to a lesser degree, then it is conceivable that this may be a major underlying and/or contributing factor to diastolic dysfunction in heart failure. Even if adequate calcium handling would be present, loss of the ability to sufficiently desensitize the myofilaments at higher heart rate may cause diastolic dysfunction in the presence of normal contractile ability.
This work was supported by National Heart, Lung, and Blood Institute Grants RO1HL-73816 and KO2HL-083957 to P. M. L. Janssen, and an American Heart Association Ohio Valley Affiliate Fellowship to K. D. Varian.
We thank Dr. Mark Ziolo for helpful discussions.
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.
- Copyright © 2007 by the American Physiological Society