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(Circulation Research. 1997;80:345-353.)
© 1997 American Heart Association, Inc.

Articles

Different Compartments of Sarcoplasmic Reticulum Participate in the Excitation-Contraction Coupling Process in Human Atrial Myocytes

Stephane N. Hatem, Agnes Benardeau, Catherine Rucker-Martin, Isabelle Marty, Patricia de Chamisso, Michel Villaz, Jean-Jacques Mercadier

the Laboratoire de Cardiologie Moleculaire et Cellulaire (S.N.H., A.B., C.R.-M., J.-J.M.), Universite de Paris XI-CNRS URA 1159, Hopital Marie Lannelongue, Le Plessis Robinson, France; the Laboratoire de Biophysique Moleculaire et Cellulaire (I.M., M.V.), Grenoble, France; and the Laboratoire de Cancerologie Experimentale (P. de C.), CEA, Fontenay-aux-Roses, France.

Correspondence to Dr J.-J. Mercadier, CNRS-URA 1159, Hopital Marie Lannelongue, 133 Avenue de la Resistance, 92350 Le Plessis Robinson, France. E-mail jjmercad@pratique.fr

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The excitation-contraction coupling process of human atrial myocytes was studied in voltage-clamped myocytes isolated from right atrial appendages obtained during cardiac surgery. Intracellular Ca²⁺ transients (Ca_i transients) were monitored with 0.1 mmol/L indo 1 added to the internal dialyzing solution. Ryanodine receptors (RyRs) and sarcomeric -actinin were stained with specific antibodies and visualized using plane and confocal microscopy. L-Type Ca²⁺ current (I_Ca) elicited a prolonged Ca_i transient, with an initial rapidly activating phase (slope 1, 23.6±1.2 s^-1) followed by a slowly activating phase (slope 2, 5.8±0.4 s^-1; P<.001 versus slope 1), resulting in a dome-shaped Ca_i transient. Ryanodine (100 µmol/L) inhibited 79±6% of the Ca_i transient, indicating that it was due essentially to sarcoplasmic reticulum Ca²⁺ release. During step depolarizations, maximal activation of the Ca_i transient or tail current (I_tail) (in cells dialyzed with Ca²⁺ buffer–free internal solution) preceded that of I_Ca and did not follow its voltage dependence (n=12). Test pulses lasting from 5 to 150 milliseconds elicited a similar time course of both Ca_i transient and I_tail (n=5). In a given cell, the two components of the Ca_i transient could be dissociated by altering the intracellular Ca²⁺ load, by increasing the stimulation rate from 0.1 to 1 Hz, or by varying the amplitude of I_Ca. Immunostaining of atrial sections and isolated myocytes showed that a large number of RyRs were located not only in a subsarcolemmal position but also deeper inside the cell, in a regularly spaced transverse band pattern at the level of Z lines. Together, our results indicate that, in human atrial myocytes, I_Ca only partially controls the activation of RyRs, with the prolonged and dome-shaped Ca_i transient of these cells probably reflecting the activation of RyRs not coupled to L-type Ca²⁺ channels.

Key Words: intracellular Ca²⁺ transient • sarcoplasmic reticulum • human cardiac myocyte

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In mammalian cardiac myocytes, activation of contraction is caused by the release of Ca²⁺ from the SR, which occurs during membrane depolarization. There is abundant evidence that this EC coupling process is mainly governed by the entry of Ca²⁺ through sarcolemmal L-type Ca²⁺ channels (DHPRs), which control the activation of the SR Ca²⁺-release channels (RyRs).^{1 2} This process, known as "Ca²⁺-induced Ca²⁺ release," where Ca²⁺ triggers its own SR release through an amplification mechanism,³ should provide a highly regenerative system but in fact produces a graded response.⁴ This paradox has been explained by "local control" models in which dynamic changes of Ca²⁺ occur in a well-defined "domain," such as the Ca²⁺ release from SR is controlled by a few DHPRs rather than the macroscopic I_Ca.⁵ Recently, elementary SR Ca²⁺-release events called "Ca²⁺ sparks" have been reported in cardiac myocytes.^{6 7} These Ca²⁺ sparks reflect the activation of a small number of RyRs that can be evoked by the opening of a few or even a single Ca²⁺ channel.^{8 9 10} These results are in keeping with the close spatial coupling between DHPRs and RyRs, which are concentrated in the transverse tubules (T tubules) and terminal cisternae of the SR, respectively.^{11 12 13 14} These channels constitute "release units" not connected to the neighboring release units, thus preventing propagated Ca²⁺-induced Ca²⁺ release. These characteristics of EC coupling emphasize the role of the intracellular microarchitecture and the spatial relationships between DHPRs and RyRs in the nature of the EC coupling in cardiac myocytes.

Interestingly, immunolocation of DHPRs and RyRs has revealed marked differences between atrial and ventricular myocytes.¹⁵ In ventricular myocytes, these proteins are in close association, whereas in atrial myocytes, many RyRs are not associated with the DHPRs. In human right atrial myocytes, it was also reported that an extensive T-tubule system is lacking and that a number of terminal cisternae of the SR not directly associated with the sarcolemma are present.¹⁶ Together, these studies raise questions as to the mechanism of activation of these "uncoupled" RyRs and their role in the EC coupling of atrial myocytes. In the present study, this question was examined in isolated human right atrial myocytes by means of physiological and immunocytochemical techniques. We provide strong evidence for the presence of distinct compartments of SR, which can explain the specificities of EC coupling of human atrial myocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Myocyte Isolation
With approval from the ethics committee of our institution (GREBB, Hopital de Bicetre, Universite de Paris XI), specimens of human right atrial appendages were obtained from 22 patients (4.5 to 77 years; mean, 43±4 years) undergoing heart surgery for coronary artery disease (n=13), valve disease (n=4), or congenital heart defects (n=5). Myocytes were isolated as follows: small pieces of atrial appendages were cut up and washed in Ca²⁺-free Krebs-Ringer solution containing (mmol/L) NaCl 35, KCl 4.75, KH₂PO₄ 1.19, Na₂HPO₄ 16, HEPES 10, glucose 10, NaHCO₃ 25, saccharose 134, and BDM 30 (pH was adjusted to 7.4 with NaOH), gassed with 95% O₂/5% CO₂, and maintained at 37°C. Pieces were reincubated in this solution without BDM and containing BSA (5 mg/mL, Hoescht-Behring), 200 IU/mL collagenase (type IV, Sigma Chemical Co), and 6 IU/mL protease (type XXIV, Sigma). After 30 minutes of digestion, the enzyme solution was replaced by the same solution containing only collagenase (400 IU/mL). Isolated myocytes were suspended in a bicarbonate-buffered Tyrode's solution containing 2 mmol/L Ca²⁺ and incubated at 37°C with continuous gassing with 21% O₂/5% CO₂ for at least 1 hour before use. An average of 550 000±88 600 (n=19) myocytes per gram of tissue was obtained, and 34.6±3.6% of the isolated myocytes were Ca²⁺ tolerant, ie, rod-shaped, well striated, and devoid of spontaneous beating.

Current Measurements
Currents were recorded with the patch-clamp technique in the whole-cell configuration, using borosilicate glass pipettes with a tip resistance of 1 to 2 M, connected to the input stage of a patch-clamp amplifier (Axoclamp 200A, Axon Instruments). Currents filtered at 5 kHz were digitized by a Labmaster (Lab Rac, Scientific Solution) and stored on the hard disk of a personal computer. Data were acquired and analyzed using a program written for our laboratory (Acquis, G. Sadock). Resistance in series, but not the capacitive or leakage current, was compensated to obtain the fastest capacity transient current. Membrane capacitance was calculated using the fit of the capacity transient decay. Pipettes were filled with an internal solution containing (mmol/L) potassium aspartate 115, KCl 10, MgATP 3, glucose 10, NaCl 5, and HEPES 10; pH was adjusted to 7.2 with KOH. In some experiments potassium was replaced by cesium (CsCl, 135 mmol/L) (referred to as Cs⁺ internal solution in the figure legends). The external solution contained (mmol/L) NaCl 136, KCl 5.4, CaCl₂ 2, glucose 10, MgCl₂ 1.06, NaH₂PO₄ 0.33, and HEPES 10; pH was adjusted to 7.4 with NaOH. Experiments were carried out at room temperature (22°C to 24°C). Data are expressed as mean±SEM.

Ca_i Transient Measurements
Ca_i transients were monitored using 0.1 mmol/L indo 1 pentapotassium salt (Molecular Probes) added to the intrapipette medium. Myocytes were excited at 340 nm with a xenon UV lamp; emission was measured at 405 and 480 nm using two photomultipliers attached to an inverted microscope (Nikon TDM, Diaphot). The [Ca²⁺]_i was calculated using the following equation: [Ca²⁺]_i=K_d·ß(R-R_min)/(R_max-R), where R represents the ratio of fluorescence emitted at 405 and 480 nm after subtraction of background fluorescence, R_min and R_max are the fluorescence ratios obtained in the absence of Ca²⁺ and at saturating [Ca²⁺], respectively, and ß is the ratio of the 480-nm signal in the absence of Ca²⁺ and at saturating [Ca²⁺]; the dissociation constant of indo 1 (K_d) is assumed to be 213 nmol/L.^{17 18}

The following protocol was used to fill SR with Ca²⁺ and maintain a stable SR Ca²⁺ load: after establishing the patch, myocytes were depolarized by a train of 150-millisecond test pulses from -60 to 0 mV at 0.1 Hz to elicit a maximal I_Ca. Full stable SR Ca²⁺ loading and steady state intracellular dialysis of the Ca²⁺ dye were considered to be achieved when the time course and amplitude of the two fluorescence signals reached a plateau and remained constant. Between 5 and 10 minutes was required to reach this equilibrium, and the experiments lasted from 15 to 30 minutes, a shorter time than that required for I_Ca rundown.

Immunostaining
RyRs were labeled with polyclonal antibodies raised in rabbits against RyRs purified from pig skeletal muscle.¹⁹ Their specificity against human cardiac RyRs has been shown by Western blot analysis of homogenates of human ventricles.²⁰ Indirect immunofluorescence was performed on frozen sections (5 µm) of atrial appendages and on freshly isolated myocytes fixed in acetone at -20°C for 10 minutes. Briefly, after they were washed in PBS at pH 7.4 (four changes, 5 minutes each), sections or myocytes were incubated in PBS containing 5% BSA for 60 minutes to block nonspecific binding sites, followed by overnight incubation with anti-RyR antibodies (1/200) at 4°C. After two washes of 10 minutes each in PBS, sections and myocytes were incubated in goat biotinylated anti-rabbit IgG secondary antibodies (1/30, Vector Laboratories) in a humid chamber for 1 hour at room temperature. After they were washed in PBS (two changes of 5 minutes each), sections and isolated myocytes were incubated in the same conditions with streptavidin-FITC (1/30, Amersham). For double immunolabeling, after they were washed in PBS (two changes of 5 minutes each), longitudinal sections were incubated with mouse anti–-actinin antibodies (1/200, Sigma) for 1 hour at room temperature, washed in PBS, and incubated with sheep Texas red anti-mouse IgG secondary antibodies (1/30, Amersham) as described above. After a final wash in PBS, coverslips were mounted in mounting medium (Fluoprep, bioMerieux). Slides were examined with a Leica DMLB fluorescence microscope set for fluorescein and Texas red fluorescence and connected to a Sony CCD DXC 930P color camera. The resulting images were printed on a Sony UP5600 video color printer. In control experiments, the incubation steps with anti-RyR and anti–-actinin antibodies were omitted.

Confocal Microscopy
Observations were carried out with an MRC-1024 (Bio-Rad) confocal scanning laser with a microscope (Nikon Optiphot Fluorescence) using Lasersharp version 2.0 software (Bio-Rad). Discrete photon counting allowed a sharp visualization of weak label, even with the highest (x,y) calibration (6.2 pixels·µm^-1). A multiple-line krypton-argon-ion laser beam (Bio-Rad) was operated at full power (15 mW) and attenuated with a neutral density filter to obtain 30% of maximal laser intensity. The microscope was operated in the fluorescent mode. The detection pinhole was set to minimum to give the thinnest possible optical section. An excitation filter at 568 nm and an emission filter at 605 nm were used for Texas red fluorescence with a gain of 1200 V and an iris of 2.1 mm. For FITC fluorescence, the settings were as follows: excitation, 488 nm; emission, 522 nm; gain, 1173 V; and iris, 2.1 mm. For the comparison of staining patterns, digitized images from the FITC and Texas red channels were acquired from the same area of the section and merged to determine overlap in the staining patterns arising from the two antibodies.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characteristics of Ca_i Transient in Human Atrial Myocytes
In the vast majority of myocytes studied, independent of the donor's age or the cardiac disease,^{21 22} activation of I_Ca by a 150-millisecond depolarizing test pulse from -60 to 0 mV elicited a Ca_i transient characterized by an initial rapidly activating phase (slope 1, 23.6±1.2 s^-1; n=36) followed by a slowly activating phase (slope 2, 5.8±0.4 s^-1; n=36, P<.001 versus slope 1) resulting in a prolonged dome-shaped Ca_i transient (Fig 1A and 1B). The contribution of the Ca²⁺ released by the SR in the activation of the Ca_i transient was examined by testing the effects of ryanodine. As shown in Fig 1C, activation of I_Ca by step depolarizations from -60 mV upon external application of ryanodine (100 µmol/L, a concentration required to obtain a rapid and almost complete suppression of the Ca_i transient) caused a gradual rise in [Ca²⁺]_i of much smaller magnitude (79±6% of inhibition, n=5) than that observed in control conditions (Fig 1D), and the remaining Ca_i transient was characterized by a marked inhibition of both the rapidly and slowly activating phases (Fig 1D). Inhibition of the Ca_i transient by ryanodine was associated with a decrease in the rate of inactivation of I_Ca, indicating a marked contribution of Ca²⁺ released by the SR in the Ca²⁺-dependent inactivation of I_Ca (Fig 1D). These results indicated that (1) the Ca_i transient recorded during the activation of I_Ca activated as two rapid and slow phases, (2) most of the Ca_i transient was caused by the release of Ca²⁺ by the SR, and (3) there is a functional coupling between RyRs and DHPRs in human atrial myocytes.

View larger version (28K): [in this window] [in a new window]   Figure 1. Characteristics of the Cai transient in human atrial myocytes. A, The Cai transient elicited by ICa (test pulse [TP], from a holding potential [HP] of -60 to 0 mV) was characterized by an initial rapidly activating phase (arrow 1) followed by a slower activating phase (arrow 2) (membrane capacitance, 59 pF; K+ internal solution). B, Rate of release of the rapidly and slowly activating phases of Cai transient were obtained by linear regression analysis of the two phases, followed by normalization of the resulting slopes to the maximal amplitude of Cai transients. Slopes were compared using the t test adapted for slope comparison. *P<.001 (n=36). C, Traces of ICa and Cai transient were elicited by 10-mV incremental test pulses upon ryanodine (100 µmol/L) exposure. D, Superimposition of ICa and Cai transients is shown in control () and ryanodine () conditions (for panels C and D: membrane capacitance, 118 pF; Cs+ internal solution).

Partial Control of SR Ca²⁺ Release by I_Ca
The voltage relationship of the Ca_i transient and I_Ca is illustrated in Fig 2A. Myocytes were depolarized by 10-mV incremental depolarizing test pulses from a holding potential of -60 mV. Both the original traces (Fig 2A) and the average values (Fig 2B) showed that an almost complete activation of the Ca_i transient occurred before that of I_Ca (at -30 mV, 74±4% of the Ca_i transients were activated versus 16±3% for I_Ca [n=12]). For potentials above -10 mV, the Ca_i transient reached a plateau distinct from the bell-shaped voltage dependence of I_Ca. Note that both the rapidly and slowly activating phases of the Ca_i transient were observed independently from the amplitude of I_Ca (see traces 6 and 9 of Fig 2A). Moreover, depolarizing test pulses positive to the reversal potential for Ca²⁺ (+60 mV in our experimental conditions) failed to elicit the Ca_i transient, which nevertheless activated with the rapid I_tail at the termination of the test pulse (trace 10 of Fig 2A), indicating that it was Ca²⁺ entering through the Ca²⁺ channels that gated SR Ca²⁺ release. A similar voltage relationship of the Ca_i transient and I_Ca was obtained when cells were dialyzed with Na⁺-free internal solution (n=4), eliminating a significant contribution of the reverse Na⁺-Ca²⁺ exchange in the voltage dependence of Ca_i transients (not shown). Moreover, not only the amplitude but also the duration of I_Ca had little effect on the time course of the Ca_i transient, since interruption of the test pulse at 5, 20, 50, 100, and 150 milliseconds did not significantly modify the time course or amplitude of the Ca_i transient (Fig 3). Together, these results indicated that although I_Ca triggered Ca_i transients in human atrial myocytes, it had little influence on the on-going Ca²⁺ release process, which developed in an all-or-none manner. To examine if this feature of EC coupling could be caused by the Ca²⁺ buffering properties of indo 1, in some experiments we used an internal solution without indo 1, and changes in [Ca²⁺]_i were estimated by the amplitude of the slow I_tail recorded at membrane repolarization, the activation of which depends on [Ca²⁺]_i.²³ As in the voltage relationship of the Ca_i transient, the activation of I_tail preceded that of I_Ca (elicited by a 20-millisecond test pulse) and then rapidly reached a plateau that did not follow the voltage dependence of I_Ca (Fig 4A and 4B). In addition, in myocytes dialyzed with a Ca²⁺ buffer–free internal solution, both the amplitude and the time course of I_tail were not significantly modified by the duration of the test pulse (Fig 4C). Together, these results indicated that the activation of RyRs was not entirely controlled by I_Ca.⁴

View larger version (32K): [in this window] [in a new window]   Figure 2. Voltage relationship of ICa and Cai transient. A, Traces of ICa and Cai transients were recorded during 10-mV incremental step depolarizations (membrane capacitance, 120 pF; Cs+ internal solution). HP indicates holding potential. B, Normalized ICa and Cai transients are plotted against membrane potential; each point is the average value of 12 experiments.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effects of interruption of the voltage-clamp depolarization on ICa and Cai transient (membrane capacitance, 110 pF; Cs+ internal solution).

View larger version (23K): [in this window] [in a new window]   Figure 4. Relationship between the activation of ICa and Itail in myocytes dialyzed with Ca2+ buffer–free internal solution. A, ICa and Itail elicited by 10-mV step depolarizations of 20 milliseconds from a holding potential (HP) of -60 mV. TP indicates test pulse. B, Same traces of currents as in panel A displayed on an expanded scale of current amplitude. C, Effects of interruption of the voltage-clamp depolarization on ICa and Itail (for panels A and B: membrane capacitance, 150 pF; Cs+ internal solution).

Two Ca_i Transient Components Activate in Human Atrial Myocytes
The observation of rapidly and slowly activating phases of the Ca_i transient suggested that it resulted from the fusion of two Ca²⁺ components, which we attempted to separate by using various experimental procedures. Fig 5 illustrates the effects on the Ca_i transient of changes in SR Ca²⁺ load induced by the rapid (<30 seconds) external application of 5 mmol/L caffeine during a train of test pulses to 0 mV (n=4). Caffeine markedly reduced the amplitude of the Ca_i transient, thus confirming that most of the Ca_i transient that occurred during a depolarizing test pulse was caused by the release of Ca²⁺ from the SR. Refilling of the SR during caffeine washout led to a gradual increase in the Ca_i transient, characterized first by a rapidly activating phase (Fig 5, trace 3), followed by the emergence of (trace 4) and gradual increase in (traces 5 and 6) a slowly activating phase. Additional evidence of the relationship between SR Ca²⁺ load and activation of the slowly activating component of the Ca_i transient was provided by another set of experiments in which isoproterenol (1 µmol/L; apparent IC₅₀, 10^-7 mol/L) was used to increase the intracellular Ca²⁺ load. In these experiments the isoproterenol-induced increase in SR Ca²⁺ load resulted in a progressive increase in the amplitude of the slowly activating component (n=5, data not shown). Conversely, increasing cell stimulation from 0.1 to 1 Hz induced a marked decrease in the slope and amplitude of the slowly activating component of the Ca_i transient, resulting in a fall in the overall Ca_i transient, which still exhibited a rapidly activating phase with a slope similar to that observed at 0.1 Hz (Fig 6A and 6B). In these experiments (n=5), a high intracellular Ca²⁺ load was maintained by using an external solution containing 5 mmol/L Ca²⁺ and isoproterenol. In addition, when the membrane of a given cell was depolarized at the subthreshold potential for I_Ca (-30 mV, Fig 6C), short rapidly activating or prolonged slowly activating Ca_i transients could be observed (n=4). Together, these data suggested that the dome-shaped Ca_i transient of human atrial myocytes resulted from the fusion of a rapidly and a slowly activating component, the activation threshold of the latter being higher than that of the former and possibly depending on the amplitude and kinetics of the rapidly activating component and/or the SR Ca²⁺ load.

View larger version (10K): [in this window] [in a new window]   Figure 5. Traces of ICa and Cai transient elicited by a train of depolarizing test pulses in control conditions, upon application of caffeine, and during its washout. Arrow indicates the appearance and gradual increase in the slow component (membrane capacitance, 78 pF; Cs+ internal solution).

View larger version (29K): [in this window] [in a new window]   Figure 6. A, Effects on Cai transient of increasing stimulation from 0.1 to 1 Hz (membrane capacitance, 120 pF; K+ internal solution). B, Rate of release of the rapidly and slowly activating phases of Cai transient obtained by linear regression analysis measured at 0.1 and 1 Hz. Slopes were compared using the t test adapted for slope comparison. *P<.05 (n=5). C, Cai transients activated by small ICa elicited by subthreshold depolarization at -30 mV (membrane capacitance, 78 pF; Cs+ internal solution).

RyRs Are Located Both at the Periphery and Inside the Cell
Fig 7 shows the labeling pattern (by anti-RyR antibodies) of frozen-longitudinal (Fig 7A) and transverse (Fig 7B) sections of a right atrial appendage and freshly isolated myocytes (Fig 7C). A very similar staining pattern characterized by a regular striation was observed both in longitudinal sections (Fig 7A, arrowhead) and in isolated myocytes (Fig 7C), suggesting that RyRs were located not solely at the cell border in a subsarcolemmal position but also deeper inside the cells, organized as regularly spaced transverse bands. In addition, examination of isolated myocytes allowed us to visualize the expected RyR labeling in its subsarcolemmal location (Fig 7C, arrowheads). This dual location was confirmed on atrial cross sections (Fig 7B), in which RyR labeling appeared both at the periphery (arrowhead) and in the cell interior. Double immunostaining of longitudinal sections with antibodies against RyRs and -actinin, observed by means of confocal microscopy, confirmed that a large number of RyRs were located deep inside the cell in a regularly spaced transverse band pattern at the level of the Z lines and resulting in yellow regularly spaced transverse bands (Fig 8C). There were also a few discrete longitudinal green lines at the cell periphery (Fig 8A, arrowhead), not visible in the -actinin staining (Fig 8B) and barely visible in the merged image (Fig 8C) because of the preponderance of the resulting yellow color over the specific green staining. This suggested the presence of subsarcolemmal RyRs. Control experiments omitting the anti–-actinin and/or anti-RyR antibodies did not show any detectable staining.

View larger version (106K): [in this window] [in a new window]   Figure 7. Immunolocation of RyRs in human atrial myocardium. A and B, Frozen longitudinal (A) and transverse (B) section of atrial appendage showing both a striated staining pattern inside the cell (arrow, A) and a staining at the periphery of the cell (arrow, B). C, Immunostaining of myocytes with anti-RyR antibodies. The predominant staining pattern appears organized as regularly spaced transverse bands in addition with a staining at the periphery of the cell (arrow, C). Patient ages were 48 and 68 years for the atrial section and isolated myocyte studies, respectively. Bar=20 µm.

View larger version (67K): [in this window] [in a new window]   Figure 8. A and B, Double immunostaining observed by means of confocal microscopy with antibodies against RyRs (FITC) with a discrete staining at the periphery of the cell (arrows) (A) and sarcomeric -actinin (Texas red) (B). C, Merged images of the same area of the section from FITC and Texas red channels to determine the overlap (yellow) in the staining patterns yielded by the two antibodies. Patient age was 48 years. Bar=20 µm.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we show that in human atrial myocytes the Ca_i transient that activated during membrane depolarization is not entirely controlled by I_Ca and results from the activation of two components of Ca²⁺ signals. These characteristics of EC coupling differ from those of ventricular myocytes and could reflect specificities in the intracellular microarchitecture of atrial myocytes, such as a poorly developed T-tubular system associated with an abundant corbular SR.^{15 16}

In human atrial myocytes, most of the Ca_i transient observed after the activation of I_Ca is caused by the release of Ca²⁺ from the SR, as indicated by its inhibition by ryanodine and caffeine. Thus, the prolonged Ca_i transients elicited by I_Ca in these cells suggest either a slow SR Ca²⁺-release process or a reduced capacity of Ca²⁺ uptake by the SR. It is unlikely that the temperature used in our experiments (22°C to 24°C) is solely responsible for the prolonged Ca_i transients, which are markedly different from those observed in ventricular myocytes of various species also studied at room temperature.^{1 4} Furthermore, in human atrial myocytes, the I_Ca-gated Ca_i transient was characterized by both rapidly and slowly activating phases despite the low temperature. An effect of excessive Ca²⁺ buffering by the fluorescent dye has been ruled out by experiments performed in the absence of intracellular Ca²⁺ buffer, which yielded similar data (Fig 4), and experiments carried out with Cs⁺- or K⁺-containing internal solution.²⁴ A reduced capacity of Ca²⁺ uptake by the SR is inconsistent with the activation in the same cells of both short and prolonged Ca_i transients (Fig 5) and with the rapidly decreasing phase of the Ca_i transient. Of note, it has been reported recently that the lower expression of phospholamban in mice atrial myocardium (and thus the lower negative regulation of the SR Ca²⁺-ATPase) compared with ventricles is responsible for a short twitch contraction, suggesting a very rapid SR Ca²⁺ uptake in atrial myocytes.²⁵ Therefore, the most likely explanation for the prolonged Ca_i transient in human atrial myocytes is a slow SR Ca²⁺ release process, which probably reflects specificities of the EC coupling mechanism in these cells, resulting in a partial control of the SR Ca²⁺ release by I_Ca.

Several pieces of evidence indicate that DHPRs are functionally coupled to RyRs in human atrial myocytes. These include (1) the high efficiency of I_Ca in triggering Ca²⁺ release and (2) the significant contribution of Ca²⁺ released by the SR in the inactivation of I_Ca.¹⁸ This is also in keeping with the immunolocation of DHPRs and RyRs in rabbit right atrial myocytes, which shows that these two channel types are closely associated.¹⁵ In contrast, the same authors observed that a substantial proportion of RyRs formed regularly spaced transverse bands that were at or near the Z lines and not associated with DHPRs, indicating that a large amount of SR (corbular SR) is not coupled to the sarcolemma.²⁶ In human atrial myocytes, we also found that, in addition to a subsarcolemmal location, a large number of RyRs were located in the cell interior, organized as a regularly spaced transverse bands at the level of the Z lines, in keeping with SR terminal cisternae overlying sarcomeres also at the level of the Z lines in electronic microscopy studies.¹⁶ These observations, together with the poorly developed T-tubular system in human atrial myocytes,¹⁶ strongly suggest that many RyRs are not associated with DHPRs, which could result, as in rabbit atrial myocytes, in an abundant central or corbular SR. This SR compartment, which is not closely connected to DHPRs, may provide a structural explanation for the original characteristics of EC coupling of human atrial myocytes reported in the present study. Indeed, the two components of the Ca_i transient could reflect the fact that I_Ca-gated Ca²⁺ release from peripheral (junctional) SR compartments, in turn, activates, in cascade, a secondary Ca²⁺ release from central (corbular) SR, although low temperature may have led to exaggerated resolution of the two components.²⁷ Moreover, other experimental conditions, such as dialyzing the cell with the low-affinity Ca²⁺ citrate buffer, induced functional uncoupling between peripheral and central release SR compartments, resulting in "regenerative Ca²⁺ release" in cultured guinea pig atrial myocytes.^{28 29}

In keeping with the idea of an abundant corbular SR in human atrial myocytes, a number of RyRs should not be localized in subsarcolemmal microdomains where Ca²⁺ accumulates in a restricted space. This may lead to an apparent high threshold and low gain of the Ca²⁺ release system⁵ and explain why, when the magnitude of I_Ca or SR Ca²⁺ load is insufficient, the first component of the Ca_i transient fails to trigger total SR Ca²⁺ release. In these circumstances, Ca²⁺ released by the peripheral (junctional) compartments might not reach sufficiently high concentrations and/or occur sufficiently rapidly to activate the RyRs of central (corbular) compartments of the SR. This is in full agreement with the recent observation, in cat atrial myocytes, of the presence of peripheral coupling SR, which induces Ca²⁺ release from central SR stores.³⁰ Conversely, when the threshold of activation of the central SR is reached, the Ca²⁺ release process tends to develop in an all-or-none manner that is poorly controlled by membrane electrical activity (Fig 3). The involvement of other mechanisms, such as the Na⁺-Ca²⁺ exchange, in modulating the Ca_i transient at longer membrane depolarization times than that used in the present study (150 milliseconds) cannot be ruled out, as reported in rat ventricular myocytes.³¹ Interestingly, in a previous study of immature human atrial myocytes, we found that the voltage dependence of Ca_i transients followed that of I_Ca.³² This discrepancy with the results of the present study (Fig 2) may be explained by low SR Ca²⁺ loading due to a reduced capacity of the immature myocyte SR to store Ca²⁺, resulting in the activation of essentially superficial stores.

Our observation of distinct Ca²⁺ compartments of SR in human atrial myocytes raises questions as to their physiological role. In a recent study, we found that the Na⁺-Ca²⁺ exchange current, the activation of which depends on [Ca²⁺]_i, contributes markedly to the shape and duration of action potentials in these cells.²³ Consequently, depending on the intracellular Ca²⁺ load or heart rate, the time course of the Ca_i transient may have different effects on action potential duration, resulting, for instance, in alternating electrical phenomenon or contributing to the high incidence of delayed afterdepolarizations observed in these cells. Additional studies of the role of these distinct SR compartments in other aspects of atrial myocyte physiology and pathophysiology are now required.

	Selected Abbreviations and Acronyms

 

BDM = 2,3-butanedione monoxime Cai transient = intracellular Ca2+ transient DHPR = L-type Ca2+ channel EC coupling = excitation-contraction coupling ICa = L-type Ca2+ current Itail = tail current RyR = ryanodine receptor SR = sarcoplasmic reticulum

	Acknowledgments

 
This study was supported by grants from the Institut National de la Sante et de la Recherche Medicale (INSERM, CRE 930410), Ministere de la Recherche et de l'Enseignement Superieur (ACC-SV9), Glaxo-Wellcome Laboratories (France), and Fondation de France. Dr Benardeau was supported by a grant from Association Francaise contre les Myopathies; Dr Hatem, by a grant from Institut National de la Sante et de la Recherche Medicale (poste d'accueil). We thank the surgical teams headed by Profs Jean-Yves Neveux and Claude Planche for providing atrial samples. We are also indebted to David Young for his help in restyling this manuscript.

Received June 13, 1996; accepted December 9, 1996.

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