Download 1 Frequency Dependent Acceleration of Relaxation Involves

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Articles
in PresS. Am J Physiol Heart Circ Physiol (January 5, 2007). doi:10.1152/ajpheart.00778.2006
Frequency Dependent Acceleration of Relaxation Involves Decreased Myofilament
Calcium Sensitivity
Kenneth D. Varian and Paul M.L. Janssen
Department of Physiology and Cell Biology, College of Medicine, The Ohio State
University, 1645 Neil Avenue, Columbus, OH 43210 USA.
Running title: Frequency Dependent Myofilament Desensitization
*Correspondence to:
Paul M.L. Janssen, Ph.D.
Department of Physiology and Cell Biology
The Ohio State University
304 Hamilton Hall
1645 Neil Avenue
Columbus, OH 43210-1218
USA
Phone: 614-247-7838
Fax: 614-292-4888
[email protected]
Subject Code: 105,136
Number of words: 4659
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Copyright © 2007 by the American Physiological Society.
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Abstract
The force-frequency relationship (FFR) is an intrinsic modulator of cardiac
contractility and relaxation. Force of contraction increases with frequency, while
simultaneously a frequency dependent acceleration of relaxation (FDAR) occurs. While
frequency-dependency of calcium handling and SR 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, ultra-thin 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 [Ca2+]I with increased frequency. Steady state force[Ca2+]i 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 1004±142 nM at 4 Hz (p<0.05) and correlated
with the corresponding changes in RT50. 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 Hz or 4 Hz revealing Troponin I and Myosin Light Chain-2
phosphorylation associated with the myofilament desensitization. We conclude that
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myofilament calcium sensitivity is substantially and significantly decreased at higher
frequencies, playing a prominent role in FDAR.
Key Words: Force Frequency Relationship, Calcium Transient, Rabbit, Diastolic
Dysfunction, Contractility
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Introduction
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, or
FFR) and faster relaxation (frequency dependent acceleration of relaxation, or FDAR) at
higher pacing frequencies(7) .
It is well known that 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 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.
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Upon -adrenergic stimulation, it is well known that relaxation is accelerated, in
part, due to phosphorylation of 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.
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Methods
Male New Zealand White (NZW) rabbits were anesthetized with 50 mg/kg
pentobarbital and injected with 5,000 units/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 set-up as previously described for rat.(27) Trabeculae were
iontophorically loaded (at 25°C) with the calcium indicator bis-fura-2 as described
previously.(2, 14, 27) 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 to 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 minutes in the rat at 37 Celsius (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-three
times background. This expanded the experimental time to about 30 minutes thus
allowing for a calibration of the signals that was precluded in rat(27) and mouse(22)
muscles at 37 ºC. For these reasons, we used bis-fura-2. This dye has twice the
fluorescence intensity compared to 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.
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Due to 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-[Ca2+]i was assessed at 37 ºC 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 seconds and force and fluorescence signals were recorded as described
previously.(27) Bis-fura-2 emissions were calibrated to [Ca2+]i by determining the Rmin
and 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 sub-set 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. In order to have a twitch force and relaxation times across the entire
investigated range of frequencies (1 to 4 Hz), an additional series of experiments 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,
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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 were doused
with liquid nitrogen till frozen and rapidly removed from the experimental setup. The
tissue was then homogenized in an SDS protein lysis buffer and loaded on a 13%
8x10cm SDS-PAGE gel (.75mm thickness, 4% stacking gel, 5 wells). The gel was then
run for 45 minutes at 175V. 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 PMT
setting of 450. After imaging, the gel was washed in water for 1 hour 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 PMT setting of 425. The
gel was then stained using coomassie Brilliant Blue, destained, and imaged with a
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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 was analyzed with a paired or un-paired 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, 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 mean ± SEM.
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Results
Figure 1a and 1b show representative force and calcium tracings at 2 Hz 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 4
muscles that did not undergo K+ contracture protocols (due to time limitations regarding
assessment of fluorescence at 37 º C). The respective average relaxation times from
peak to 50% of peak (RT50) are shown in figure 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 seconds to reach a new steady state twitch
force. This is significantly slower than what is generally observed for smaller rodents (515 seconds 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 intracellular calcium concentration also
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significantly rose with increasing stimulation frequency. With increasing frequency, the
amplitude of the intracellular calcium concentration rose, due to a larger rise in systolic
versus 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 seconds 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
where some hysteresis was observed, this was well past peak (at or past half way
down), resulting in the downward curvediverging from the upward curve, possibly due
to fatigue (not shown). Figure 4b shows an example of how the data was analyzed.
From the fitted curve through the force-calcium data combinations, maximal active
developed force (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 2 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,
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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 (Figure 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 attempt to further describe a possible mechanism for frequency based
myofilament desensitization, the phosphorylation status of myofilament proteins were
assessed using the Pro-Q Diamond stain. Figure 8a shows a 1D SDS gel stained for
total protein where 3 muscles that had been stimulated at 1 Hz, 3 muscles at 4 Hz, and
2 muscles at 1 Hz while under the influence of 1 µM Isoproterenol. The 2 muscles
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stimulated in 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 Figure 8c
and 8d for TnI and MLC-2. A significantly greater level of phosphorylation of TnI (panel
C) and MLC-2 (panel D) was observed in the muscles stimulated at 4 Hz and those
stimulated with isoproterenol compared to those stimulated at 1 Hz.
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Discussion
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 Hz and 2 Hz stimulations in the rat at room
temperature. They used high-frequency stimulations in the presence of CPA to induce
steady state force-calcium relationships, and it cannot be determined whether highfrequency 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
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concluded a frequency dependent desensitization to exist, albeit at non-physiologic
temperature and sub-physiological 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(27) we were
able to assess myofilament calcium sensitivity in larger mammals, and measure this
relationship in the physiological frequency range as well as at physiologic temperature.
We here show that the myofilament matrix desensitizes upon increase 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 insured 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 insured 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
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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 inter-muscle
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 (NCX), unlike rats and mice where this is only ~2-5%. Removal of Ca2+ via
NCX is significantly slower than re-uptake 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
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than in small rodents. In order 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 to 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 versus skinned myocardium.(8, 15, 27) A possible limitation of our study is that
assessment of myofilament calcium sensitivity takes ~20-30 seconds, 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 the 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
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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 to 1 Hz and that the
phosphorylation at 4 Hz was not significantly different from muscles stimulated in
presence of isoproterenol. Although it is well known that TnI phosphorylation
desensitizes the myofilaments(16), the current data on TnI phosphorylation is only
correlative with frequency and a causal relationship needs to yet be established. MLC-2
was also found to be phosphorylated significantly more in 4 Hz compared to 1 Hz. This
is consistent with a previous finding in isolated perfused rabbit septum where a 4 fold
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 2 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.
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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, 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 presence of normal contractile ability.
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Acknowledgements
This work was supported by National Institutes of Health Grants RO1HL73816
and KO2HL083957 to PMLJ, and an American Heart Association Ohio Valley Affiliate
Fellowship to KDV. We wish to thank Dr. Mark Ziolo for helpful discussions.
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References
1.
Baartscheer A, Schumacher CA, Belterman CN, Coronel R, and Fiolet JW.
SR calcium handling and calcium after-transients in a rabbit model of heart failure.
Cardiovasc Res 58: 99-108, 2003.
2.
Backx PH and Ter Keurs HE. Fluorescent properties of rat cardiac trabeculae
microinjected with fura-2 salt. Am J Physiol Heart Circ Physiol 264: H1098-1110, 1993.
3.
Bassani RA, Mattiazzi A, and Bers DM. CaMKII is responsible for activity-
dependent acceleration of relaxation in rat ventricular myocytes. Am J Physiol 268:
H703-712, 1995.
4.
Bluhm WF, Kranias EG, Dillmann WH, and Meyer M. Phospholamban: a major
determinant of the cardiac force-frequency relationship. Am J Physiol Heart Circ Physiol
278: H249-255, 2000.
5.
de Tombe PP and Stienen GJ. Protein kinase A does not alter economy of
force maintenance in skinned rat cardiac trabeculae. Circ Res 76: 734-741, 1995.
6.
DeSantiago J, Maier LS, and Bers DM. Frequency-dependent acceleration of
relaxation in the heart depends on CaMKII, but not phospholamban. J Mol Cell Cardiol
34: 975-984, 2002.
7.
Endoh M. Force-frequency relationship in intact mammalian ventricular
myocardium: physiological and pathophysiological relevance. Eur J Pharmacol 500: 7386, 2004.
8.
Gao WD, Backx PH, Azan-Backx M, and Marban E. Myofilament Ca2+
sensitivity in intact versus skinned rat ventricular muscle. Circ Res 74: 408-415, 1994.
21
Copyright Information
Page 22 of 34
9.
Gao WD, Perez NG, and Marban E. Calcium cycling and contractile activation in
intact mouse cardiac muscle. J Physiol (Lond) 507: 175-184, 1998.
10.
Grossman W. Diastolic dysfunction and congestive heart failure. Circulation 81:
III1-7., 1990.
11.
Hasenfuss G and Pieske B. Calcium cycling in congestive heart failure. J Mol
Cell Cardiol 34: 951-969, 2002.
12.
Hofmann PA and Lange JH, 3rd.Effects of phosphorylation of troponin I and C
protein on isometric tension and velocity of unloaded shortening in skinned single
cardiac myocytes from rats. Circ Res 74: 718-726, 1994.
13.
Hussain M, Drago GA, Colyer J, and Orchard CH. Rate-dependent
abbreviation of Ca2+ transient in rat heart is independent of phospholamban
phosphorylation. Am J Physiol 273: H695-706, 1997.
14.
Janssen PML, Stull LB, and Marban E. Myofilament properties comprise the
rate-limiting step for cardiac relaxation at body temperature in the rat. Am J Physiol
Heart Circ Physiol 282: H499-H507, 2002.
15.
Kassiri Z, Myers R, Kaprielian R, Banijamali HS, and Backx PH. Rate-
dependent changes of twitch force duration in rat cardiac trabeculae: a property of the
contractile system. J Physiol (Lond) 524: 221-231, 2000.
16.
Kranias EG and Solaro RJ. Phosphorylation of troponin I and phospholamban
during catecholamine stimulation of rabbit heart. Nature 298: 182-184, 1982.
17.
Layland J and Kentish JC. Positive force- and [Ca2+]i-frequency relationships
in rat ventricular trabeculae at physiological frequencies. Am J Physiol Heart Circ
Physiol 276: H9-H18, 1999.
22
Copyright Information
Page 23 of 34
18.
Li L, Chu G, Kranias EG, and Bers DM. Cardiac myocyte calcium transport in
phospholamban knockout mouse: relaxation and endogenous CaMKII effects. Am J
Physiol 274: H1335-1347, 1998.
19.
Maier LS, Bers DM, and Pieske B. Differences in Ca(2+)-Handling and
Sarcoplasmic Reticulum Ca(2+)-Content in Isolated Rat and Rabbit Myocardium. J Mol
Cell Cardiol 32: 2249-2258, 2000.
20.
Pieske B, Kretschmann B, Meyer M, Holubarsch C, Weirich J, Posival H,
Minami K, Just H, and Hasenfuss G. Alterations in intracellular calcium handling
associated with the inverse force-frequency relation in human dilated cardiomyopathy.
Circulation 92: 1169-1178, 1995.
21.
Silver PJ, Buja LM, and Stull JT. Frequency-dependent myosin light chain
phosphorylation in isolated myocardium. J Mol Cell Cardiol 18: 31-37, 1986.
22.
Stull LB, Leppo M, Marban E, and Janssen PML. Physiological determinants
of contractile force generation and calcium handling in mouse myocardium. J Mol Cell
Cardiol 34: 1367-1376, 2002.
23.
Takimoto E, Soergel DG, Janssen PM, Stull LB, Kass DA, and Murphy AM.
Frequency- and afterload-dependent cardiac modulation in vivo by troponin I with
constitutively active protein kinase A phosphorylation sites. Circ Res 94: 496-504. Epub
2004 Jan 2015., 2004.
24.
Tong CW, Gaffin RD, Zawieja DC, and Muthuchamy M. Roles of
phosphorylation of myosin binding protein-C and troponin I in mouse cardiac muscle
twitch dynamics. J Physiol 558: 927-941, 2004.
23
Copyright Information
Page 24 of 34
25.
Valverde CA, Mundina-Weilenmann C, Said M, Ferrero P, Vittone L, Salas M,
Palomeque J, Petroff MV, and Mattiazzi A. Frequency-dependent acceleration of
relaxation in mammalian heart: a property not relying on phospholamban and SERCA2a
phosphorylation. J Physiol 562: 801-813, 2005.
26.
Varian KD, Raman S, and Janssen PM. Measurement of myofilament calcium
sensitivity at physiological temperature in intact cardiac trabeculae. Am J Physiol Heart
Circ Physiol 290: H2092-2097, 2006.
27.
Varian KD, Raman S, and Janssen PML. Measurement of Myofilament Calcium
Sensitivity at Physiological Temperature in Intact Cardiac Trabeculae. Am J Physiol
Heart Circ Physiol 290: H2092-H2097, 2006.
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Figure Legends:
Figure 1. Example calcium transients (A), force tracings (B), and the resulting phase
plane plots (C) for a trabecula stimulated at 2 and 4 Hz. As frequency increased, force
increases and relaxation is accelerated, while systolic calcium is elevated, and a
rightward shift of the relaxation trajectory in the phase plane plots is observed.
Figure 2. Panel A: Maximum twitch developed force parameters for all four frequencies.
The rabbit trabeculae exhibited a typical positive force-frequency through the pacing
range of 1-4 Hz. In addition, relaxation accelerated throughout this range, exhibiting a
positive FDAR (panel B). These data were obtained in muscles in which all four
frequencies were measured n=7, paired data (* = p<0.05 compared to 1 Hz, ** p<0.05
compared to 1 and 2 Hz).
Figure 3. Diastolic and peak systolic [Ca2+]i values for all 4 frequencies. Both diastolic
and systolic [Ca2+]i rise with frequency, whereas systolic [Ca2+]i rises disproportionately
higher (n=7) (* = p<0.05 compared to 1 Hz, ** p<0.05 compared to 1 and 2 Hz).
Figure 4. A: Typical experiment in which a rabbit trabecula was stimulated at 2 Hz, and
subjected to the high potassium solution (K+ arrow). Developed force and [Ca2+]i are
shown on the same time scale, depicting how the steady state force-[Ca2+]i data were
obtained. B: Data obtained were plotted with a curve fit. Fmax, EC50, and the nHill were
derived from the curve fit equation.
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Figure 5. A: Using the K+ contracture protocol, a steady state force-[Ca2+] relationship
was measured at 1 Hz, 3 Hz, and then repeated at 1 Hz. This shows that the sensitivity
shifts that are based on frequency are reversible. B: Average EC50 values (n=14),
*p<0.05 paired (n=7), **p<0.01, unpaired (n=7/group).
Figure 6. Relationship between the EC50 and RT50. From 14 experiments, RT50 data
(obtained from twitch contractions), correlate well with myofilament calcium sensitivity
(EC50). Data at 1 and 3 Hz were obtained in 7 trabeculae, data obtained at 2 and 4 Hz
were obtained in a separate group of 7 trabeculae, using the identical protocol and timeline.
Figure 7. The relationship between stimulation frequency and intracellular pH. The ratio
(440/495) is inversely proportional to pH and does not change with frequency (n=4).
Figure 8. SDS PAGE of muscles frozen while contracting at either at 1 Hz, 4 Hz or 1 Hz
in the presence of 1 µM isoproterenol (Iso). A: Total protein (Sypro Ruby) B:
Phosphoprotein (Pro-Q Diamond stain). The ratio of band density of phosphoprotein
over total protein for TnI (C), and MLC-2 (D). * indicates a significant difference
(p<0.05) in this ratio was found between 1 and 4 Hz, and between 1 Hz and Iso at
bands corresponding with TnI and MLC-2. Differences were not significant between 4
Hz and Iso. The trabeculae stimulated at 1 Hz with 1 µM isoproterenol were done as a
positive control for TnI phosphorylation (n=4 per group).
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Iso
C
180
97
82
66
42
29
TnI
MLC-2
18
total protein
D
kDa
2
1.5
*
1
0.5
0
1Hz
4Hz
Iso
3
180
97
82
66
42
29
TnI
MLC-2
Ratio TnI Phosph/Total (Arb Units)
B
Ratio TnI Phosph/Total (Arb Units)
Iso
4Hz
4Hz
4Hz
1Hz
1Hz
kDa
1Hz
A
MW marker
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2.5
2
*
1.5
1
0.5
18
0
phosphoprotein
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1Hz
4Hz
Iso