Vol. 273, Issue 5, H2498-H2507, November 1997
Sarcoplasmic reticulum function in determining
atrioventricular contractile differences in rat heart
Ave
Minajeva1,
Allen
Kaasik1,
Kalju
Paju1,
Enn
Seppet1,
Anne-Marie
Lompré2,
Vladimir
Veksler3, and
Renée
Ventura-Clapier3
1 Department of Pathological
Physiology, Medical Faculty, University of Tartu, EE2400 Tartu,
Estonia; 2 Laboratoire de
Biochimie Moléculaire et Cellulaire, Centre National de la
Recherche Scientifique Unité de Recherche Associée 1131,
Université Paris-Sud, 91405 Orsay; and
3 Laboratoire de Cardiologie
Cellulaire et Moléculaire, Institut National de la
Santé et de la Recherche Médicale U-446, Faculté de
Pharmacie Université Paris-Sud, F-92296 Châtenay-Malabry,
Cedex, France
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ABSTRACT |
The relationships between the contractile
characteristics and the sarcoplasmic reticulum (SR) function of rat
atrial and ventricular trabeculae were compared. The isometric
developed tension (DT) and the rates of contraction
(+dT/dt) and
relaxation
(
dT/dt) normalized to cross-sectional area were 3.7, 2.2, and 1.8 times lower,
respectively, in intact atrial strips compared with ventricular strips,
whereas +dT/dt and
dT/dt
(normalized to DT) were 2.3 and 2.8 times higher, respectively, in
atria. Atria exhibited a maximal potentiation of DT after shorter rest
periods than ventricles and a lower reversal for prolonged rest
periods. Caffeine-induced tension transients in saponin-permeabilized
fibers suggested that the Ca2+
concentration released in atrial myofibrils reached a lower maximum and
decayed more slowly than in ventricular preparations. However, the
tension-time integrals indicated an equivalent capacity of sequestrable
Ca2+ in SR from both tissues. In
atrial, as in ventricular myocardium, the SR
Ca2+ uptake was more efficiently
supported by ATP produced by the SR-bound MM form of creatine kinase
(CK; MM-CK) than by externally added ATP, suggesting a tight functional
coupling between the SR Ca2+
adenosinetriphosphatase (ATPase) and MM-CK. The maximal rate of
oxalate-supported Ca2+ uptake was
two times higher in atrial than in ventricular tissue homogenates. The
SR Ca2+-ATPase 2a mRNA content
normalized to 18S RNA was 38% higher in atria than in ventricles,
whereas the amount of mRNA encoding the 
-myosin heavy chain,
calsequestrin, and the ryanodine receptor was similar in both tissues.
Thus a lower amount of readily releasable Ca2+ together with a faster uptake
rate may partly account for the shorter time course and lower tension
development in intact atrial myocardium compared with ventricular
myocardium.
sarcoplasmic reticulum calcium uptake; skinned fibers; calsequestrin; ryanodine receptor; calcium pump
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INTRODUCTION |
IT IS KNOWN THAT atria contract and relax faster than
ventricles, both isotonically and isometrically (1, 2, 16), but the
underlying mechanisms for these differences are not yet completely
understood. Besides a shorter action potential (2, 16), the faster
kinetics of atrial contraction have been attributed to a higher
proportion of the fast -myosin heavy chain (MHC) isoform compared
with ventricles. However, this seems not to be an explanation in small
mammals such as rats, in which both atria and ventricles express
-MHC in a similarly high proportion (28), but atria still contract
faster than ventricles (16). While addressing this issue, we recently
observed that skinned left atrial and ventricular preparations from
adult rat heart developed similar levels of maximal force and
stiffness, displaying also similar
Ca2+ sensitivity and tension
kinetics (31). Conversely, faster cross-bridge kinetics have been
observed in right atria of hyperthyroid rats compared with ventricles
and have been attributed to the differences in myosin light chain
content between the two tissues (11). Several observations, however,
suggest that differences in Ca2+
handling at the level of the sarcoplasmic reticulum (SR) may contribute
to the atrioventricular differences in contractile function. In mouse
heart, both volume fraction and surface area of total SR per cell
volume are higher in atria, mainly due to a higher longitudinal SR
content (10). Therefore, atrial contraction has been proposed to be
more dependent on Ca2+ release by
the SR than ventricular contraction (1, 9, 22). Atrial SR has been
shown to exhibit a 4.2-fold lower ratio of phospholamban to
Ca2+-adenosinetriphosphatase
(ATPase) than ventricular SR, and the lower
phospholamban-to-Ca2+-ATPase ratio
has been suggested to increase SR
Ca2+ uptake and, consequently,
lead to faster relaxation in rat atria (17). Unlike in ventricular
cardiomyocytes, a nonsynchronous and biphasic
Ca2+-induced
Ca2+ release has been observed in
atrial cardiomyocytes (8, 14).
Atrioventricular differences in contractile function may also reflect
the different metabolic profiles of these tissues. In comparison with
ventricles, atria are characterized by lower activities of glycolytic
and citric acid cycle enzymes (4). Lower oxidative capacities in atria are associated with decreased activities of both
total creatine kinase (CK) and mitochondrial CK (mito-CK) compared with
ventricles (31). It has also been shown in atria that
mito-CK is not coupled to the adenine nucleotide translocase (31).
These findings suggest fundamentally different mechanisms of energy
transport in atrial compared with ventricular cells. In this respect, a
question arises concerning the interactions between CK and the SR
Ca2+-ATPase in atria. The MM form
of CK (MM-CK) bound to the myofilaments exhibited the same efficacy in
controlling the ATP-to-ADP ratio in atria and ventricles (31).
Characterization of SR function in isolated membrane preparations and
in skinned fibers from ventricular myocardium has revealed a functional
coupling between SR Ca2+ uptake
and MM-CK bound to the SR membranes (18, 24). However, nothing is known
about the role of CK in SR Ca2+
uptake in atria.
This work was undertaken to study and compare the relationships between
the contractile parameters, the expression of SR proteins, the SR
function, and the functional characteristics of MM-CK bound to SR in
atria and ventricles from rats. It was found that, compared with
ventricles, atria exhibit a higher expression of SR
Ca2+-ATPase (SERCA) 2a associated
with faster SR Ca2+ uptake but
prolonged caffeine-induced Ca2+
release. These properties of SR are accompanied by a faster rate of
filling during interbeat pauses as well as a reduced loss of SR
Ca2+ during pause decay in intact
atria. At the same time, the SR calsequestrin (Cals) and ryanodine
receptor (RyR) expression as well as the SR capacity of releasable
Ca2+ appear similar in atria and
ventricles. Like in ventricles, the SR
Ca2+ uptake is highly dependent on
ATP generated locally in the CK reaction, thus indicating a coupling of
MM-CK to the SR Ca2+ pump in
atria.
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MATERIALS AND METHODS |
Muscle preparations.
Wistar rats of both sexes (average body wt 260 g) were treated
according to the recommendations of the institutional animal care
committee (INSERM, Paris, France). They were anesthetized by
intraperitoneal injection of urethan (0.2 g/100 g body wt). For
contractility measurements, papillary muscles from right ventricles or
fibers from papillary muscles of left ventricles and atria with similar
cross-sectional areas were dissected in a solution containing (in mM)
120 NaCl, 5.4 KCl, 0.6 CaCl2, 0.42 NaH2PO4, 1.05 MgCl2, 5 glucose, 30 2,3-butanedione monoxime, 0.05 Na2
EDTA, and 20 tris(hydroxymethyl)aminomethane
(Tris) · HCl, gassed with 100% oxygen, pH 7.4.
Fibers to be used for permeabilization of the sarcolemma (diameter
150-240 µm) were dissected from left ventricular papillary muscle or from left atria in ice-cold
zero-Ca2+ Krebs solution
containing (in mM) 118 NaCl, 4.7 KCl, 25 NaHCO3, 1.2 KH2PO4,
and 1.2 MgSO4 equilibrated with
95% O2-5%
CO2. Specific permeabilization of
the sarcolemma was obtained by incubating the fibers in relaxing
solution
A (see Table 1) containing 50 µg/ml
saponin in the presence of 20 µM leupeptin at +4°C for 30 min
(13).
Contractile function measurements.
Muscle preparations with silk thread tied to one end were placed
horizontally in a 0.2-ml perfusion chamber between a force transducer
(6MX1C) and a needle that was attached to a length adjustment device.
Preparations were field stimulated by a pair of platinum-plate
electrodes using rectangular current pulses (1 Hz, 5 ms, 1.5-threshold
voltage) generated by an electronic stimulator (C-50-1). During
an equilibration period of 90 min, the muscles were superfused at the
rate of 5 ml/min with a solution containing (in mM) 145 NaCl, 5.9 KCl,
2.5 CaCl2, 1.2 MgCl2, 11 glucose, 1.1 mannitol,
and 5 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.4 at 30°C, aerated with 100%
O2. The isometric contractile parameters of muscle preparation were registered at the peak of the
length-tension curve using on-line PC-AT 486 with Atrium software designed by Dr. U. Braun. The length of each fiber was measured by
means of a micrometer in a dissecting microscope. At the end of the
experiment, the fiber was cut off between the silk thread and the
needle and weighed. Mean cross-sectional area for each preparation was
calculated from the weight and length of the trabeculae, assuming a
muscle density of one. Tension values were expressed in millinewtons
per square millimeter.
The dependence of postrest potentiation on the rest interval at
extracellular Ca2+ concentrations
([Ca2+]o)
of 1 and 2.5 mM was used to compare the SR function in intact myocardium. In these experiments, the basic stimulation at
1 Hz was interrupted for 3-600 s. In each preparation, the values
of the developed tension (DT) of the first postrest twitches were normalized to the maximal value of potentiation, usually gained after a
60- to 120-s pause.
Preparation of tissue homogenates and estimation of SR
45Ca uptake.
Rats were anesthetized with thiopental sodium (50 mg/kg), and hearts
were excised and rinsed rapidly in ice-cold isotonic saline solution.
The whole atrial auricles and ventricular apex region were isolated and
weighed. The homogenization was carried out with an Ultra-Turrax
homogenizer (3 × 20 s, 24,000 revolutions/min) in 50 vol of
ice-cold homogenization buffer containing (in mM) 250 sucrose, 20 Tris
(pH 6.8), 2 MgCl2, 0.01 leupeptin,
0.01 phenylmethylsulfonyl fluoride, 1 dithiothreitol, and 2 benzamidine. The final homogenate was further treated with a
glass-glass homogenizer (10 strokes).
The Ca2+ accumulation in the
tissue homogenate was determined at 30°C in stirred medium
containing (in mM) 6 ATP, 6 MgCl2,
120 KCl, 3 sodium azide (NaN3),
60 imidazole (pH 7.0), 6 potassium oxalate, and a
45Ca-labeled
CaCl2-ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) buffer containing 0.58 mM EGTA. Appropriate concentrations of CaCl2 were added to the medium
to obtain different free Ca2+
concentrations
([Ca2+]). The reaction
was started by addition of 50 µl of homogenate per 0.5 ml of medium.
After a 2-min incubation, the samples were filtered through 0.45-µm
Schleicher & Schuell (Keene, NH) glass microfiber filters using a
vacuum pump. Radioactivity associated with the membranes was counted in
Optiphase "HiSafe" 3 (Wallac, Turku, Finland). After subtraction
of unspecific binding, the Ca2+
uptake by the homogenate was entirely blocked by 30 µM cyclopiazonic acid, a specific inhibitor of SR
Ca2+-ATPase that shows that
Ca2+ uptake was restricted to the
SR Ca2+ pump. Protein
concentration in the homogenate was determined by the biuret method and
was between 104 and 166 µg per uptake assay.
Estimation of SR
Ca2+ uptake in
saponin-permeabilized fibers.
SR Ca2+ uptake in permeabilized
fibers was estimated by analyzing the tension transients due to
caffeine-induced Ca2+ release
after various periods of SR loading (13, 30), as described recently
(24). The fibers were mounted between a length-adjustment device and a
force transducer (AE 801; Aker's Microelectronics, Horton, Norway).
Fibers were immersed in 2.5-ml chambers arranged around a disk. The
chambers were placed in a temperature-controlled bath positioned on a
magnetic stirrer. All experiments were performed at 22°C. The
immersion solutions were calculated as described previously and are
listed in Table 1. All solutions contained (in mM) 0.8 free Mg2+, 30.6 Na+, 60 N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic
acid (pH 7.1), 3.16 MgATP, 12 phosphocreatine (PCr), and 0.3 dithiothreitol. The ionic strength was adjusted to 160 mM with
potassium methanesulfonate. All solutions contained 2 mM
NaN3 to inhibit possible
Ca2+ uptake by mitochondria and 20 µM leupeptin to inhibit the proteases. To study the energy
requirement for SR Ca2+ loading,
solutions with modified adenine nucleotides and PCr content were
designed.
At the beginning of each experiment, the fiber was stretched to 120%
of the slack length in the relaxing
solution
A. The maximal Ca2+-activated tension
(Tmax) was estimated in the
activating solution B, pCa 4.5. The fiber was then relaxed
in solution
A. To empty the SR of
Ca2+, 5 mM caffeine was applied
for 2 min in the relaxing solution (A1)
followed by the washout of caffeine for 2 min in the same solution
(A). SR loading was carried out at
pCa 6.5 and 10 mM [EGTA]
(solution
C) for different time periods, so
that under control conditions the peak force never reached the upper
saturating part of the pCa-tension relationship and the
Ca2+ release was in the
quasi-linear part of the pCa-tension relationship. To wash out
Ca2+ and EGTA after loading, the
prerelease solution (D) containing 0.25 mM [EGTA] was applied for 1.5 min. The fiber was then
passed into another prerelease solution
(E) with 0.2 mM [EGTA]
for 30 s. Finally, Ca2+ was
released from the SR by applying 5 mM caffeine in a solution of the
same composition
(R1),
which resulted in a tension transient. The peak of relative tension
(T/Tmax) and the area under
isometric tension
(ST) were
measured. The pCa-tension relationship in the presence of 5 mM caffeine
in conditions identical to those of the release (except that 10 mM EGTA
was present to adequately buffer free
Ca2+) was obtained at the end of
each experiment by sequentially exposing the fibers to a set of
solutions with decreasing pCa until
Tmax was reached (at pCa 4.5). DT
at each pCa was normalized with respect to
Tmax. The data from each fiber
were fitted by the Hill equation using linear regression analysis, and
the pCa required to produce 50% of maximal activation
(pCa50) and the Hill coefficient
(nH) were
determined. The same Ca2+-release
protocol was always applied to enable comparison of SR Ca2+ uptake in different fibers or
loading conditions.
Evaluation of the role of bound CK in SR
Ca2+ uptake by
saponin-permeabilized atrial fibers.
To evaluate the role of bound CK in providing the energy for SR
Ca2+ uptake in situ, SR loading
was carried out at pCa 6.5 in the presence of 0.25 mM MgADP and 12 mM
PCr, so that ATP generated in the CK reaction was the only source of
energy for the Ca2+ pump.
Alternatively, the load in the presence of 3.16 mM MgATP alone was
carried out to compare the efficacy of externally added ATP with the
internally produced ATP. Loading in the presence of 3.16 mM MgATP and
12 mM PCr served as a control. [EGTA] in all test solutions
was 10 mM. Before loading, a 2-min preequilibration period was used at
pCa 9, with the same conditions of substrates as those of loading, to
equilibrate the concentration of compounds inside the fiber. To permit
comparison between different loading conditions,
Ca2+ release by 5 mM caffeine was
always induced at constant conditions of substrates and ions.
Evaluation of the Ca2+ uptake
under different conditions was done by analyzing tension transients due
to caffeine-induced Ca2+ release,
as described above.
Isolation of total RNA and mRNA dot-blot analysis.
Eight hearts were collected from adult Wistar rats. The atria were
isolated, and the ventricles were cut into pieces. The tissues were
blotted dry, frozen in liquid nitrogen, and kept at 80°C
until RNA preparation. Both atria from each rat and a piece of
ventricle with approximately the same weight were used for RNA
preparation. Total RNA was extracted by the guanidinium isothiocyanate
procedure using RNA quick (Bioprobe) and kept at 20°C in
70% ethanol, 0.3% sodium acetate, pH 5.2. Specific mRNA species were
quantified by slot-blot hybridization. One, two, and four micrograms of
total RNA from atria and ventricles as well as from liver and fast
skeletal muscle (extensor digitorum longus) and yeast tRNA were
denatured in 15× standard saline citrate (SSC; 1× SSC
contained 0.15 M sodium chloride and 0.015 M sodium citrate) and 3%
formaldehyde at 65°C for 15 min and rapidly cooled on ice. Liver,
skeletal muscle RNA, and tRNA were used as negative controls to check
for specificity of the various probes. The samples were directly
spotted onto the nylon membrane using a minifold apparatus (Schleicher
& Schuell). The RNA was cross-linked to the membrane by ultraviolet
irradiation, and the membranes were prehybridized at 42°C for >4
h in the presence of 50% formamide, 0.1% bovine serum albumin, 0.1%
Ficoll, 0.1% polyvinylpyrolidone, 0.05 M sodium phosphate (pH 6.5),
5× SSC, 0.1% sodium dodecyl sulfate (SDS), and 250 µg/ml
salmon sperm DNA.
The specific SERCA2 mRNA was detected using a probe [from
nucleotide (nt) 2616 to 3120] of the rat heart
Ca2+-ATPase mRNA (20). The rat RyR
probe (RyR-2) was obtained by reverse transcription of rat cardiac
total RNA and subsequent amplification of the RyR-2 mRNA using primers
derived from the sequence of the rabbit cardiac RyR-2 (25). The probe
extends from nt 8604 to 9144 of the rabbit sequence (25) with few
silent mutations. The dog Cals probe was a gift from Dr. B. Nadal-Ginard and was used as described by Lompré et al. (21). The
-MHC probe has been previously described by Schiaffino et al. (29). The cDNA probes were labeled by use of random primers, DNA polymerase I
(Klenow fragment) and
[-32P]dATP (3,000 Ci/mmol), and the specific activity was 1-3 × 109 disintegrations/min
(dpm)/µg. Hybridization was done in the same conditions as
prehybridization. Excess of probe was eliminated by washing in
0.5× SSC at 55°C for SERCA2, at 60°C for RyR-2, and at
42°C for Cals and in 1× SSC at 45°C for MHC.
After each hybridization, the blots were dehybridized by boiling in
0.1% SDS and then rehybridized as described above. To normalize to the
amount of total RNA present on the membrane, the blots were
rehybridized with a 24-mer oligonucleotide complementary to the rat 18S
ribosomal RNA. The oligonucleotide was labeled at its 5' end by
use of T4 polynucleotide kinase and
[
-32P]ATP and
diluted with cold oligonucleotide to a specific activity of
105 dpm/µg. It was hybridized in
the medium described above but in the absence of formamide. The washing
conditions were 2× SSC at room temperature. After washing, the
membranes were exposed to X-ray film for 1 day to 1 wk. Unsaturated
autoradiograms were analyzed by densitometry (Molecular Dynamics). The
specific mRNA level was corrected for the total RNA present on the
membrane by calculating the ratio of the signals obtained with the
specific probe and the 18S probe for the three dilutions of each
sample. Specific mRNA levels were expressed in arbitrary units (AU) as means ± SE. The relative proportion of SERCA2 and SERCA2b mRNA was
determined by ribonuclease (RNase) protection assay using the SERCA2
probe described above and the Ambion kit protocol (Clinisciences, Montrouge, France).
Chemicals.
Caffeine was purchased from Merck-Clevenot. PCr (Neoton, Schiapparelli
Searle, Turin, Italy) was a kind gift from Prof. E. Strumia. Other
chemicals were obtained from Sigma Chemical.
Statistical analysis.
The data are expressed as means ± SE or as representative tracings
in a single experiment. Statistical analysis is presented in
RESULTS. Differences at
P < 0.05 were considered
significant.
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RESULTS |
Contractile parameters.
Figure 1 shows that atrial contractions are
characterized by a weaker twitch DT but a faster time course than
ventricular contractions. The mean values of contractile
characteristics are presented in Table 2
and compared using unpaired Student's
t-test. DT in atrial preparations was
3.7 times less than that in ventricular preparations. The time to peak
tension (TPT) and the half-relaxation time
(RT50) were significantly less
in atria. The maximal rates of contraction
(+dT/dt) and relaxation
(dT/dt) also appeared to be
lower in atria than in ventricles. However, if normalized to DT, these
parameters [(+dT/dt)/DT and
(dT/dt)/DT] became
higher in atria, evidencing faster contractile kinetics.
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Fig. 1.
Superimposed twitch tension recordings from atrial (a)
and ventricular (v) trabeculae at a steady-state stimulation rate of 1 Hz with 2.5 mM extracellular Ca2+
concentration. Diameter of atrial trabecula, 325 µm; diameter of
ventricular trabecula, 350 µm.
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Table 2.
Mean characteristics and contractile parameters obtained at 1-Hz
stimulation, [Ca2+]o 2.5 mM,
of fibers dissected from rat atria and ventricles
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Postrest potentiation.
Postrest potentiation of twitch tension was compared in atria and
papillary muscles at two external
[Ca2+] (1 and 2.5 mM)
to assess the function of SR in providing activator Ca2+ in vivo (3, 19; for review
see Ref. 9). The absolute mean values of pretest steady-state and
maximal potentiation in both tissues are presented in Table
3. In Fig. 2, values were normalized to maximal potentiation to compare the two tissues. The values were
analyzed using analysis of variance (ANOVA) followed by Dunnett's test. Figure 2A shows that, at 1 mM
[Ca2+]o,
increasing the rest duration was accompanied by potentiation of the
first postrest twitch in both atrial and papillary muscle preparations. However, the maximal level of potentiation
was achieved after significantly shorter rest intervals (15 s) in
atrial than in papillary muscles (60 s). In addition, the magnitude of
potentiation was less in atria than in ventricles. Further increase in
rest duration led to significant reversal of potentiation in papillary but not in atrial muscles. Figure 2B
demonstrates that these differences between atrial and papillary
muscles were abolished when
[Ca2+]o
was increased from 1 to 2.5 mM.
Oxalate-supported
45Ca2+
uptake in tissue homogenates.
The oxalate-supported
45Ca2+
uptake rates were estimated in tissue homogenates in conditions (see
MATERIALS AND METHODS) under which
the Ca2+ uptake has been
previously defined to be restricted to SR vesicles (26). In our
experiments, the specific SR
Ca2+-ATPase inhibitor
cyclopiazonic acid (30 µM) inhibited ~98% of Ca2+ uptake. This also confirms
that the intrinsic SR Ca2+ uptake
was assayed.
The net SR Ca2+-uptake rates in
tissue homogenates of both atria and ventricles were assayed in the
presence of oxalate over a wide range of free
[Ca2+], corresponding
to the range of cytosolic free
[Ca2+] during the
contraction-relaxation cycle (Fig. 3). Data were fitted
using the Hill equation, and the results were compared using Student's
t-test. The net maximal
Ca2+-uptake rate was more than
twofold higher in atria than in ventricles (4.17 ± 0.72 and 2.03 ± 0.42 nmol
Ca2+ · mg tissue
protein
1 · min1,
respectively; P < 0.05). However,
the free Ca2+ concentration that produces half-maximal
activation of the SR Ca2+ pump
(0.22 ± 0.03 and 0.22 ± 0.04 µM in atria and
ventricles, respectively) as well as the
nH (1.55 ± 0.13 and 1.68 ± 0.34 in atria and ventricles, respectively) were
similar in atria and ventricles.
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Table 3.
Absolute mean values of maximally potentiated and steady-state twitch
amplitudes in atria and ventricles
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Fig. 2.
Rest potentiation in atria ( ,
n = 8) and ventricles ( ,
n = 10) at 1 (A) and 2.5 mM
(B) external
Ca2+ concentrations. Values have
been normalized to maximal potentiation.
** P < 0.01;
*** P < 0.001, ventricles vs.
atria.
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Ca2+ uptake
in skinned cardiac fibers.
To compare the SR Ca2+ handling in
atrial and ventricular myocardium in situ, saponin-permeabilized fibers
with similar mean diameters were used. Table 4 shows
that atrial and ventricular preparations exhibited similar values of
Tmax normalized per
cross-sectional area and of myofilament
Ca2+ sensitivity in the presence
of 5 mM caffeine. In addition, no effect of caffeine on
Tmax was observed (results not
shown).
The similar responsiveness of myofibrils from atrial and ventricular
preparations to Ca2+ in the
presence of 5 mM caffeine allows us to compare the time courses of
caffeine-induced tension transients in the two tissues. The SR
Ca2+ uptake was estimated by
loading tissues with Ca2+ for
different time periods and subsequent liberation by caffeine. Figure
4 shows that, after a 10-min load at pCa 6.5, a typical caffeine-induced contracture in atrial fibers was characterized by
lower peak tension and a substantial tonic component of the contracture, whereas ventricular muscle promptly returned to baseline tension. In Fig. 5, mean data
as a function of loading time are presented and compared using ANOVA
followed by Dunnett's test. Figure 5A
shows that, in response to increased periods of loading, the peak
tension of caffeine-induced contractures increased in both atrial and
ventricular fibers. However, the peak tension values normalized to
Tmax reached a lower level (50%)
in atrial than in ventricular fibers (80%). The
RT50 of caffeine-induced contracture increased progressively with the loading time in both tissues (Fig. 5B), reaching,
however, significantly higher levels in atrial than in ventricular
preparations at loading times 
10 min. To eliminate the possibility
that these differences were due to limited diffusion of caffeine at a
concentration of 5 mM, application of 25 mM caffeine was used in some
experiments. The results (not shown) indicated that the higher caffeine
did not diminish the atrioventricular differences in the time course of tension transient. In addition, when a second application of 5 mM
caffeine was made immediately after the first, no increase in tension
was observed in either tissue, suggesting that the slow phase of
tension transient in atria was due to a slow phase of
Ca2+ release. Further analysis of
tension-time integrals
(ST) calculated over the whole tension transient revealed that the area
under the caffeine-induced tension transient was the same in atrial and
ventricular fibers (Fig. 5C). Thus,
despite the different shape of the caffeine-elicited tension transients
and a faster SR Ca2+-uptake rate
in atria (Fig. 3), atria seemed to exhibit at least equal SR
Ca2+ capacity in situ as
ventricles.
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Fig. 3.
Sarcoplasmic reticulum (SR)
Ca2+ uptake rate in 4 different
preparations of atrial () and ventricular () homogenates at
different pCa (expressed in nmol
Ca2+ · min1 · mg
protein1). Each curve was
drawn using mean values of 4 preparations. Continuous curves obtained
from Hill equation were fitted by least-square fitting procedure.
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Fig. 4.
Superimposed recordings of atrial (a) and ventricular (v) tension
transients elicited by 5 mM caffeine in saponin-permeabilized fibers
after loading at pCa 6.5 for 10 min. Diameter of atrial fiber, 240 µm; diameter of ventricular fiber, 160 µm.
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Fig. 5.
Mean data of caffeine-elicited tension transients from atrial () and
ventricular () saponin-permeabilized fibers, as a function of
loading time at pCa 6.5. A: peak
tension of caffeine-induced contracture normalized to maximal
Ca2+-activated tension
(T/Tmax) at pCa 4.5 for each
fiber. B: time to half-relaxation of
tension transient (RT50).
C: tension-time integral
(ST). Each
value is mean of 3-7 determinations in different fibers.
* P < 0.05;
** P < 0.01;
*** P < 0.001; ventricles vs. atria.
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Energy dependence of SR
Ca2+ uptake in
situ in atria.
To evaluate the efficiency of the MM-CK bound to SR in providing the SR
Ca2+ pump with ATP in situ, we
compared the SR Ca2+ loading in
the presence of MgADP and PCr (when ATP generated by the bound CK was
the only source of energy for the
Ca2+ pump) with that supported by
MgATP alone. The level of SR Ca2+
loading in the presence of 3.16 mM MgATP and 12 mM PCr served as
control. Data were compared using ANOVA followed by Dunnett's test.
Figure 6A
shows that when SR was loaded either in the presence of ADP and PCr or
ATP and PCr the time dependence of relative peak tension for these two
conditions had similar shape, both reaching their equivalent maximal
values (51 and 47% of Tmax, respectively) within 1 min. Further increase in loading time did not
alter the values of T/Tmax. In
contrast, the curve obtained in the presence of ATP alone tended to
decrease if the time of loading was prolonged for >1 min. As a
result, the values of T/Tmax became significantly less than those in control conditions.
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Fig. 6.
Parameters of caffeine-elicited tension transient in
saponin-permeabilized atrial fibers as a function of loading time.
Loading was performed in presence of 12 mM phosphocreatine (PCr) and
3.16 mM MgATP (control, ), 12 mM PCr and 0.25 mM MgADP () and
3.16 mM MgATP ( ) in atrial skinned fibers.
A:
T/Tmax of each fiber.
B:
ST. Each value
is mean of 3-7 determinations in different fibers. * and **,
P < 0.05 and P < 0.01, respectively, vs. control (ATP-PCr).
|
|
Figure 6B shows the dependency of the
tension-time integral on the time of loading for different conditions
of energy supply. It can be seen that
ST
increased progressively with the time of loading, being 11.2 ± 3.2 mN · s/mm2 in
the presence of ATP and PCr and not significantly different when ATP
was replaced by ADP for a 7-min load. In contrast, when SR was loaded
in the presence of ATP alone, the value of
ST dramatically decreased (1.31 ± 0.16 mN · s/mm2,
P < 0.05) for the same loading time.
These results show that the amount of
Ca2+ released from the SR by
caffeine was much higher in conditions when SR
Ca2+ uptake was supported by ATP
produced by the SR-bound CK than when externally added ATP was used as
the only source of energy for the SR
Ca2+ pump.
Expression of mRNA.
To relate the observed differences in SR function
between the two cardiac tissues to the level of expression of the
principal SR proteins (SERCA, RyR, and Cals) and the -MHC, the
tissue amounts of corresponding mRNA relative to 18S were estimated by
slot blot (Fig. 7,
A and
B). Figure
7A shows that no signal was observed with any of our probes with liver RNA, whereas 18S RNA was present, indicating no nonspecific binding of the probes. This is also attested
by the negative signal in tRNA samples. As expected, the -MHC and
the RyR-2 probes were specific for cardiac RNA and were not detected in
skeletal muscle, whereas the SERCA2 and Cals mRNA were present in both
muscle types. Quantification of slot blot (Fig.
7B) indicates that the expression of
the -MHC relative to 18S was not significantly different between
atria and ventricles [1,000 ± 90 and 974 ± 81 AU,
respectively]. Relative to 18S RNA, the content of RyR and Cals
mRNAs in the two tissues was not different, whereas the amount of
SERCA2 mRNA was 38% higher in atria than in ventricles (220 ± 21 and 160 ± 18 AU, respectively, P < 0.05). The relative proportion of SERCA2a and SERCA2b mRNA was
determined by RNase protection analysis. As already shown in the
ventricle, the majority of the SERCA2 mRNA was of the 2a type (21). An identical pattern was observed in the atria (data not shown). Relative
to -MHC mRNA, the amount of SERCA2 mRNA was lower in ventricles than
in atria (0.165 ± 0.010 and 0.234 ± 0.049 AU, respectively),
but the difference did not reach significance. This suggests that
higher SR Ca2+-pump activity was
due to a higher expression of pump protein in atria than in ventricles.
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Fig. 7.
Quantification of 18S RNA, SR Ca2+-ATPase
2 (SERCA2) mRNA, ryanodine receptor (RyR) mRNA, calsequestrin (Cals)
mRNA, and -myosin heavy chain (-MHC) mRNA levels in atria and
ventricles by slot-blot analysis. A:
representative results of slot blot with 2 samples from atria and 2 samples from ventricles. Liver RNA, tRNA, and RNA from extensor
digitorum longus (EDL) muscle were used as negative controls.
B: level of each mRNA species relative
to 18S RNA. Specific mRNA levels were expressed in arbitrary units as
means ± SE. Differences between independent samples were tested for
significance by a nonparametric transformation of unpaired
t-test:
Wilcoxon-Mann-Whitney U-test.
* P < 0.05, atria vs. ventricles.
|
|
| |
DISCUSSION |
Contractile parameters in atria and ventricles.
In comparison with papillary muscles, atrial preparations exhibited
smaller absolute values of DT. However, the kinetic parameters (TPT and
RT50) and
+dT/dt and
dT/dt normalized to DT showed
that peak isometric tension in atria reached its maximum and relaxed faster than in ventricles, in accordance with earlier studies (1, 2,
17). A low DT in left atria is strikingly in contrast to
the observation that both maximum
Ca2+-activated tension and
Ca2+ sensitivity of myofibrils
were similar in atrial and ventricular saponin-treated preparations
(Table 3) (31). This could be the result of a smaller amount of
activator Ca2+ in intact atria
compared with ventricles. On the other hand, the process could be
limited also by a higher rate of
Ca2+ resequestration by SR.
Rest potentiation.
Rest potentiation has been suggested to result from the increase in
Ca2+ release from the SR as a
result of either the increase in SR Ca2+ content in rat cardiomyocytes
(3, 7, 10, 19) or the increase in fractional SR
Ca2+ release (6, 12). Our data
indicate that, at low
[Ca2+]o,
although relative potentiation is lower in atria, the mechanisms responsible for maximal postrest potentiation need about four times
less time to become saturated in atrial (15 s) than in ventricular (60 s) myocardium. This suggests that SR
Ca2+ filling saturates earlier and
faster in atria. These features of atrial myocardium could be
associated with a faster SR
Ca2+-uptake rate, although it is
not clear whether the amount of SR-releasable Ca2+ is indeed increased. An
increase in rest duration for 10 min led to significant reversal of
potentiation in papillary muscles but not in atria. The decline in rest
potentiation and rest decay has been attributed to SR
Ca2+ loss due to its extrusion
through the sarcolemmal Na/Ca exchanger (6, 9). This may indicate that
the SR in rat ventricular myocardium loses more
Ca2+ during pauses. As for rest
potentiation, this could be attributed to a faster SR
Ca2+ uptake rate in atria. On the
other hand, atrial myocytes are characterized by higher intracellular
sodium concentration due to a lower
Na+-K+
ATPase content (33). This may lead to less pronounced
Ca2+ extrusion via the Na/Ca
exchanger, resulting in the less pronounced rest decay. Indeed, under
high
[Ca2+]o,
which minimizes the Ca2+ extrusion
via Na/Ca exchange, there were no differences in pause-dependent potentiation between the atrial and ventricular myocardium. This may
suggest that Na/Ca exchange plays an important role in the reversal of
rest potentiation.
Differences in SR
Ca2+ handling in
atrial and ventricular myocardium.
The oxalate-supported Ca2+ uptake
rate in tissue homogenate, which reflects the intrinsic
Ca2+ transport by SR
Ca2+-ATPase (26), was taken to
estimate the SR function in vitro. Our data indicate that the
oxalate-supported Ca2+ uptake rate
(expressed per milligram of protein) is two times higher in atria than
in ventricles. This may be partly explained by the 30% higher level of
the SERCA2 mRNA relative to 18S RNA observed in atria (Fig. 7) and by
the lower expression of phospholamban (17), favoring a more efficient
Ca2+ uptake rate in atria than in
ventricles. The similar free Ca2+ concentration that
produces half-maximal activation of the SR Ca2+ pump in atria and ventricles
is in agreement with the observation of an unchanged SERCA2a-to-SERCA2b
ratio.
To evaluate the interaction of SR with the contractile apparatus, the
SR function was analyzed in situ, in saponin-permeabilized fibers. In
both atria and ventricles, caffeine-induced
Ca2+ release was observed to occur
in two phases. The fast phase of Ca2+ release was evidenced by
T/Tmax, which saturated faster and
appeared lower in atria than in ventricles. This smaller fast phase of Ca2+ release might be the basis
for the lower DT observed in atria. However, a further increase in the
loading time induced a prolongation of the tension transient as
evidenced by the progressive increase in the
ST. This
prolongation of the tension transient was more marked in atria than in
ventricles. The time course of the caffeine-induced tension transient
will depend on 1) the amount of
released Ca2+,
2) the rate of SR
Ca2+ release,
3) the diffusion of
Ca2+ away from the myofibrils,
4) the
Ca2+ buffering by EGTA and
proteins, 5) the possible reuptake
and release of Ca2+ and,
6) the
Ca2-sensitizing effect of
caffeine. Because atrial and ventricular fibers have similar diameter
and composition, it is unlikely that differences will arise from
different Ca2+ buffering.
Similarly, atrial and ventricular fibers exhibited the same sensitivity
to Ca2+ (31) and similar
sensitivity to Ca2+ in the
presence of caffeine (this study), allowing the comparison of force
transients. Moreover, increasing caffeine concentration or reapplying
caffeine quickly after the first application did not modify
atrioventricular differences, suggesting that these differences did not
arise from incomplete Ca2+ release
or differences in caffeine sensitivity. The appearance of a second slow
phase of tension could be considered as a result of prolonged
Ca2+ release, suggesting different
mechanisms of Ca2+ release in
atria and ventricles. On the other hand, the similar tension-time
integrals suggested that the amount of releasable Ca2+ appeared to be the same in
the two tissues, whatever the speed of
Ca2+ release or the duration of SR
loading. These results, together with the similar amount of mRNA for
Cals, the main Ca2+-buffering
protein in SR, suggest that the capacity of the SR Ca2+ pool is similar in atrial and
ventricular myocardium.
Slow SR Ca2+ release in atria
appeared not to be due to a lower amount of SR
Ca2+ channel, because the same
degree of expression of RyR mRNA suggested a similar amount of
Ca2+ release channels in both
tissues. An alternative explanation may be morphological differences
between atria and ventricles. In human atrial cells, it was shown that
the intracellular Ca2+ transient
triggered by membrane depolarization is not entirely controlled by the
Ca2+ current and results from the
activation of two components of Ca2+ signals (14). Berlin (8),
using confocal microscopy, showed that, in guinea pig atrial myocytes
devoid of t tubules, stimulated increases in internal
Ca2+ can be observed to arise in
focal regions of the cell before spreading to the cell interior. In
intact rabbit ventricular cardiomyocytes, Bassani et al. (7) showed
that one-half of the Ca2+ in the
SR is released during a twitch, whereas the beat-dependent depletion of
SR Ca2+ is biexponential. They
suggest that the slower phase might represent a caffeine-sensitive pool
of Ca2+ not normally released
during a twitch and speculated that the Ca2+ in the corbular SR could
represent such a pool. In comparison with ventricular
cardiomyocytes, atrial cells are characterized by the absence of a t
tubular system (23). They contain only peripheral junctional SR
connected to sarcolemma and a higher proportion of corbular SR within
the cytoplasm. Because corbular SR is not connected to sarcolemmal
membrane, Ca2+ release from these
stores must be triggered by a diffusible agent (15). The delayed phase
of Ca2+ release in atria could
thus represent Ca2+ released from
these internal cisternae by a slower
Ca2+-induced
Ca2+-release mechanism.
CK and SR
Ca2+ uptake in
atria.
The rapid work of the SR Ca2+ pump
in atria critically depends on adequate energy supply and effective
withdrawal of ATPase reaction products. The isolated SR membranes of
several muscle types have been shown to contain strongly anchored MM-CK
that is functionally coupled to the
Ca2+ pump (32). Recently, we have
confirmed the coupling between bound CK and SR
Ca2+-ATPase in situ in
saponin-skinned ventricular fibers with preserved local architecture by
showing that localized regeneration of ATP at the expense of PCr and
ADP is more efficient to meet the requirement of
Ca2+-ATPase than external ATP
(24). The role of CK in providing energy for the SR
Ca2+ pump in situ could be
demonstrated by efficient loading of the SR in the presence of PCr and
ADP. These results showed that local ATP generated by the CK reaction
was sufficient to completely meet the ATP requirements of the SR
Ca2+-ATPase in atria. In contrast,
ATP alone was not able to sustain control loading of the SR. Our study
clearly demonstrates that, in atria, the CK system bound to structures
at sites of energy utilization is at least as efficient as in
ventricles to provide energy for SR
Ca2+ uptake and to maintain a
favorable local ATP-to-ADP ratio in the vicinity of the ATPase.
In conclusion, fast and efficient CK-supported SR
Ca2+ uptake together with limited
or slow Ca2+ release and diffusion
for contraction could be proposed as the main modulator of the shorter
time course and lower tension development in rat intact atrial
myocardium compared with that in ventricular myocardium, despite a
similar total capacity of releasable
Ca2+ in both tissues.
| |
ACKNOWLEDGEMENTS |
The authors express gratitude to Dr. Urmo Braun and Patrick
Lechêne for excellent technical assistance, E. Boehm for careful reading of the manuscript, and R. Fischmeister and J.-P. Mazat for
continuous support.
| |
FOOTNOTES |
Financial support from Eesti Teadusfond is gratefully acknowledged. R. Ventura-Clapier was supported by Centre National de la Recherche
Scientifique, and A.-M. Lompré was supported by Institut National
de la Santé et de la Recherche Médicale. A. Minajeva was the recipient of a grant from "Réseau Formation Recherche" of the "Ministère de l'Enseignement
Supérieur et de la Recherche."
Address for reprint requests: V. Veksler, Laboratoire de Cardiologie
Cellulaire et Moléculaire, INSERM U-446, Faculté de
Pharmacie, Université Paris-Sud, 5 rue Jean-Baptiste
Clément, F-92296 Châtenay-Malabry, Cedex, France.
Received 16 January 1997; accepted in final form 10 July 1997.
| |
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Abstract
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