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Am J Physiol Heart Circ Physiol 273: H2498-H2507, 1997;
0363-6135/97 $5.00
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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

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -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.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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(beta -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.

                              
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  		Table 1.   Composition of solutions for estimating Ca2+ uptake in skinned cardiac fibers

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 alpha -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 [alpha -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 [gamma -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.

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

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 · min-1, 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 (black-square, n = 8) and ventricles (bullet , 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.

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 (black-square) and ventricular (bullet ) homogenates at different pCa (expressed in nmol Ca2+ · min-1 · mg protein-1). 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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  		Table 4.   Characteristics of saponin-permeabilized atrial and ventricular fibers in presence of 5 mM caffeine


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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 (black-square) and ventricular (bullet ) 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.

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, black-square), 12 mM PCr and 0.25 mM MgADP (bullet ) and 3.16 mM MgATP (black-triangle) 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 alpha -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 alpha -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 alpha -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 alpha -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 alpha -myosin heavy chain (alpha -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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AJP Heart Circ Physiol 273(5):H2498-H2507
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