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1Department of Physiology, Health Sciences Center, Texas Tech University, Lubbock, Texas 79430; and 2Department of Physiology, Stritch School of Medicine, Loyola University Chicago, Maywood, Illinios 60153
Submitted 3 April 2003 ; accepted in final form 7 November 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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neonate; ryanodine receptor; cardiac excitation-contraction coupling; intact heart
Although it is clear that CICR is not the predominant mechanism of E-C coupling in the neonatal myocardium, several lines of evidence suggest that RyR-mediated SR Ca2+ release might play a substantial role, even at early stages of postnatal development. For example, it has been reported that during the first day of postnatal life, ryanodine (0.1–1 µM) decreases the systolic tension up to 
50% in multicellular preparations (1, 16, 25, 28). In cultured neonate myocytes, ryanodine (0.1 µM) reduced 25% of the amplitude of intra-cellular Ca2+ transients triggered by APs (12). In the same preparation, ryanodine (10 µM) completely abolished spontaneous local Ca2+ release events as well (14). Furthermore, the robust response to caffeine reported in acutely isolated ventricular myocytes from the neonate rabbit (9) suggests a more important role of RyRs.
An additional source of uncertainty about the functional role of RyR in the neonate heart is that most of the information concerning the development of cardiac E-C coupling has been derived from studies conducted either on primary cultures or, in few cases, on acutely isolated cells. Although very valuable in nature, these experimental models pose the challenge of extrapolating the results to intact cells and to the whole organ working under physiological conditions. Factors that exist in the whole heart (e.g., cell-to-cell interactions, intact extracellular matrix, etc.) are either disturbed (mechanically or enzymatically) or simply not present in cellular models. To circumvent those obstacles, we have applied in this work the pulsed local-field fluorescence technique (5) to quantitatively define the developmental changes of the Ca2+ transients with cellular resolution but recorded in the beating whole heart (6, 15). With this approach, we have estimated how the contribution of each Ca2+ flux to the global intracellular Ca2+ transient changes during the first 3 wk of postnatal life. Specifically, we evaluated RyR-mediated SR Ca2+ release, L-type Ca2+ current, T-type Ca2+ current, and the Ca2+ current resulting from the reverse-mode operation of the Na+/Ca2+ exchanger. Furthermore, we have studied the developmental changes of the cardiac E-C coupling mechanism under experimental conditions that closely resemble those found in vivo. These conditions included the intact heart, coronary perfusion, 37°C, and spontaneous contractile activity.
Preliminary versions of this work have been previously presented at the Biophysical Society Annual Meeting (5, 6) and Iberoamerican Congress of Biophysics.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Whole heart fluorescence measurements. Fluorescence signals were recorded in perfused beating hearts by using the pulsed local field fluorescence detection technique as previously described (15). Briefly, a small (200 µm core diameter) multimode fiberoptic (3M) was used to simultaneously excite the fluorophore and collect the emitted fluorescence. A frequency-doubled, nanogreen-pulsed Nd-YAG laser (JDS Uniphase NanoLaser) was used to illuminate the tissue with short pulses (0.9 ns) of light (532 nm wavelength) at a frequency of 12 kHz. To eliminate motion artifacts associated with the contraction, the tip of the fiberoptic was inserted into a small patch-clamp glass pipette onto which gentle suction was applied. The negative pressure stabilized the tip of the fiberoptic against the moving wall of the heart. To record the fluorescence from specific regions of the heart, the tip of the fiberoptic was positioned and held in place with the aid of a micromanipulator. Emitted fluorescence was carried back through the same fiberoptic, filtered, and focused on an avalanche photodiode (EG & G) that was connected to a variable bandwidth (3.8–125 GHz) current-to-voltage converter. The voltage output of the photodiode was digitized with an analog-to-digital converter (PCI 6110, National Instruments) at a sampling frequency of 500 kHz and a bandwidth of 125 kHz. The fluorescence envelope trace was obtained offline as previously described (15) at a frequency of 12 kHz. Briefly, the peak of each fluorescence transient elicited with 0.9-ns light pulses was determined and stored in a separate file. Because only the amplitude of rhod-2 fluorescence transient depends on [Ca2+], the estimation of its maximal value allowed us to continuously monitor how the intracellular [Ca2+] changed over time. Pooled data are presented as means ± SE. Statistical significance in Figs. 3 and 5 and Table 1 was determined with two-tail P values obtained from unpaired t-tests.
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Ventricular myocytes isolation. Ventricular myocytes were enzymatically isolated from both neonate (1 day old) and juvenile (3 wk old) rat hearts by using the standard Langendorff technique. Briefly, isolated hearts were rinsed in normal Tyrode solution (composition in mM: 140 NaCl, 5 KCl, 2 CaCl2, 1.2 KH2PO4, 1 MgCl2, 20 glucose, and 10 HEPES-K; pH = 7.4). The aorta was cannulated, and the heart was mounted in a Langendorff perfusion apparatus. Flow rates varied accordingly to the rat's age: 1 ml/min for neonates and 3 ml/min for juvenile rats. Nominally Ca2+-free Tyrode solution was perfused for variable times depending on the animal's age between 3 min (neonate) and 6 min (juvenile). Enzymatic digestion of the myocardium was carried out by perfusing a mixture of collagenase (0.5 mg/ml, II, Worthington Biochem; Lakewood, NJ) and protease (0.05 mg /ml, XIV, Sigma-Aldrich; St. Louis, MO) dissolved in a low-Ca2+ (200 µM) Tyrode solution during 10 min for both neonate and juvenile hearts. Enzyme was washed out for 6 min with low-Ca2+ (200 µM) Tyrode solution, followed by a 3-min period with normal Tyrode solution. Ventricles were cut and minced. Single myocytes were obtained by gentle dispersion with a wide-bore pipette and filtering through cotton gauze. Isolated cells were placed in normal Tyrode and kept at room temperature (23°C) until used. All the solutions used for heart perfusion were at 37°C.
Single cell fluorescence measurements. Single myocytes were loaded with a fluorescent indicator by incubating the cells in 2 µM rhod-2 AM (Molecular Probes) at room temperature (23°C). Optimal loading levels were obtained when the incubation time was 30 min for adult and 45 min for neonate cells. Longer incubation times were deleterious for the cells. The incubation period was ended by rinsing the cells during 15 min with dye-free normal Tyrode solution at 23°C. Loaded myocytes were then placed on the stage of an inverted fluorescence microscope (Nikon, Diaphot) modified for flash laser imaging (4). AP-stimulated Ca2+ transients were recorded in normal Tyrode solution at 23°C. The use of higher temperatures (i.e., 37°C) normally yielded unexcitable cells. APs were triggered by field stimulation with platinum electrodes placed in the experimental chamber. Intracellular [Ca2+] was evaluated by epifluorescence detection on single ventricular myocytes from neonate and juvenile rat hearts. Fluorescence signal was digitized at a rate of 2 kHz with a 32-bit AD/DA converter and controlled by Labview-based software (National Instruments; Austin, TX) and a Pentium II-based personal computer. Results are expressed as relative fluorescence intensity.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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75% of the transient in the juvenile rat, whereas in the neonate rat the effect of large concentrations of ryanodine (100 µM) was negligible, if any. In addition, the temporal course of the Ca2+ transient in the neonate rat before and after ryanodine was considerably slower than in the juvenile rat. Time to peak, for instance, was 240 ms, whereas in the juvenile rat it was 54 ms. The possibility that nonspecific damage secondary to the isolation procedure was the cause of such a slow temporal course of the Ca2+ transient and of the absence of CICR could not be ruled out. Because we were interested in evaluating the participation of RyRs under experimental conditions where the E-C coupling machinery remains intact, we decided to conduct our studies in intact whole hearts by using the pulsed local field fluorescence detection technique.
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RyR-mediated Ca2+ release. Figure 2A illustrates the effects of ryanodine on Ca2+ transients recorded in a spontaneously beating heart from a juvenile rat. Before the addition of ryanodine, stable and regular Ca2+ transients were recorded over a period of 20 min. A few beats after the addition of ryanodine (100 µM), however, several changes in cardiac activity were observed. First, the heart rate assessed by the Ca2+ transient frequency was reduced to 50% of its original value (from 180 ± 22 to 90 ± 35 beats/min; P < 0.005; n = 3). Second, the interval between Ca2+ transients started to exhibit variable durations, indicating an alteration of the normal sinus rhythm. Finally, the most obvious effect of ryanodine was on the amplitude of the Ca2+ transient. The amplitude of the transient was rapidly reduced until it reached a stationary value, where it stayed for variable periods before dying out. This stationary state was usually reached after 1 min of the drug application. Although the transient amplitude was reduced by 88% in the presence of ryanodine, the Ca2+ transient kinetics (rising and the decaying phase) were not appreciably affected.
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Figure 2B shows a similar experiment conducted on a 5-day-old neonate rat heart. Unlike the juvenile rat heart, the effect of the same ryanodine concentration (100 µM) on the Ca2+ transient amplitude from the neonate rat was considerably smaller. Ryanodine induced only a 32% reduction of the amplitude without substantial changes of its temporal attributes. Additionally, ryanodine affected the pacemaker activity in a similar way than in the juvenile rat. This was indicated by a 20% reduction of the heart rate (from 126 ± 7 to 103 ± 7 beats/min; P < 0.05; n = 4). Interestingly, however, the extent of the heart rate reduction in neonate was 30% less than in juvenile hearts.
To define how much impact nonspecific fluorescence decay would have on the Ca2+ transient amplitude, paced activity at 0.75 Hz was recorded over prolonged periods of time. Figure 2C shows that after 40 min of continuous recording, the amplitude of Ca2+ transients was only reduced 13%. Thus loss of fluorescence had minimal impact on the ryanodine (and other agents) effects observed over considerably shorter periods of time (5–10 min).
Sarcolemmal Ca2+ entry. To define the specific contribution of the sarcolemmal Ca2+ entry to the intracellular Ca2+ transient in the neonate and juvenile stages, the effects of different pharmacological agents on the properties of the Ca2+ transient were investigated. The purpose of this strategy was to sequentially block each of the pathways through which Ca2+ ions cross the sarcolemma into the cell, namely L- and T-type Ca2+ channels and Na+/Ca2+ exchanger operating in the reverse mode. Figure 3 shows the different components of the intra-cellular Ca2+ transient in the neonate heart. The experiment shown in Fig. 3 was conducted in a heart from a 1-day-old neonate rat, and cardiac activity was externally paced at 2 Hz. Addition of ryanodine (100 µM) induced a 10.4% reduction of the Ca2+ transient amplitude without inducing substantial changes of its temporal attributes [i.e., time to peak (tpeak) and rate of decay]. This is better observed in the averaged traces shown underneath. Subsequent addition of nifedipine (10 µM) reduced 50% of the remaining Ca2+ transient amplitude (to 45% of the control value). The temporal properties of the Ca2+ transient after nifedipine are illustrated in the third trace of the bottom panel. Unlike the rising phase that remained essentially unchanged, the decay of the transient became slower, as indicated by the increase of the time constant decay (
decay). Finally, addition of 2 mM Ni2+ blocked almost the entire transient (to 5% of the control value), suggesting that both T-type Ca2+ channels and the Na+/Ca2+ exchanger contributed in an unknown extent to this last fraction (35% of the whole transient). Because of the large size of the ryanodine- and nifedipine-sensitive Ca2+ fluxes in the juvenile rat hearts, the addition of the drugs could not be conducted with a sequential protocol. Instead, the effects of each drug were evaluated separately (light bars, Fig. 3B).
In an attempt to separate the contribution of T-type Ca2+ channels from the Na+/Ca2+ exchanger to the total intracellular Ca2+ transient in the neonate, the effects of different concentrations of Ni2+ were investigated. Because Ni2+ also blocks L-type Ca2+ channels (11), Ni2+ effects on Ca2+ transients were explored in the presence of nifedipine. Saturating concentrations of nifedipine (10 µM) were used to compensate for the lack of control of the resting membrane potential in the whole heart preparation. This precaution was taken because of the well-known voltage dependence of nifedipine action (2). The results of these experiments are illustrated in Fig. 4. Addition of Ni2+ reduced the amplitude of the nifedipine-resistant component of the Ca2+ transient in a dose-dependent manner. High concentrations of Ni2+ (1 mM) induced a large reduction of the Ca2+ transient, but it was readily reversed after Ni2+ was washed out (Fig. 4A, dotted trace). The temporal attributes of the Ca2+ transient, however, were not visibly changed even when Ni2+ concentrations were in the millimolar range. The dose-response relationship shown in Fig. 4B revealed an apparent Ni2+ affinity of 220 µM and a Hill coefficient of 1. Because of the lack of specificity, this behavior could represent a combined effect of Ni2+ on the Na+/Ca2+ exchanger operating in reverse mode and on the T-type Ca2+ channels. This is graphically illustrated in Fig. 4B, where previously published data of Ni2+ effect on the Na+/Ca2+ exchanger (10; Fig. 4B, dashed line) and on T-type Ca2+ current (13; Fig. 4B, dotted line) are shown for comparison.
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To specifically determine the contribution of the T-type Ca2+ channel to the total intracellular Ca2+ transient, the selective T-type Ca2+ channel blocker mibefradil was used. Figure 5A shows a representative experiment in which the contribution of each Ca2+ flux to the intracellular Ca2+ transient was investigated by the sequential addition of different pharmacological agents. The experiment shown in Fig. 5 was conducted in a heart from a 2-day-old neonate rat, and cardiac activity was externally paced at 2 Hz. Addition of ryanodine (100 µM) induced a reduction of the Ca2+ transient amplitude of 12.5% without inducing significant changes of its temporal attributes. Subsequent addition of nifedipine (10 µM) reduced 32% of the remaining Ca2+ transient amplitude (to 60% of the control value). Finally, addition of 1.8 µM mibefradil blocked to 25% of the control value. Washout partially reversed the blockade induced by mibefradil. Ryanodine and nifedipine effects were usually irreversible. Pooled data from neonate and juvenile rats are shown in Fig. 5B. As in Fig. 3B, the effects of each drug in juvenile rats were evaluated separately.
Ca2+ transient kinetics. To correlate the developmental changes of the AP with the intracellular Ca2+ signaling, the temporal attributes of the Ca2+ transients were determined at the same ages. Figure 6 and Table 1 show how the time course of the intracellular Ca2+ transient changed during the first 3 wk of postnatal life. In Fig. 6, top, Ca2+ transients from the right atrium, right ventricle, and left ventricle recorded in 1-day-old rats are shown, whereas in Fig. 6, bottom, Ca2+ transients recorded from the same regions in a juvenile heart are illustrated. The rising phase of the Ca2+ transient accelerated with development, as indicated by the reduction of the time to peak to almost half of the value seen in the neonate. This acceleration was more evident in the ventricle. As illustrated in Fig. 6, the onset of the Ca2+ transient recorded from atrium showed modest variations with development. Interestingly, the temporal attributes of the decaying phase of the transient in both the atrium and ventricle did not show significant variations with development.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ca2+ transients in whole heart as opposed to single cells. Acutely isolated myocytes and primary cultures have been experimental models typically used for developmental studies of E-C coupling (9, 12, 14). These models, however, present a number of inconveniences that hinder a precise quantification of the Ca2+ flux that results from CICR. Examples of those problems include structural damage (particularly of the sarcolemma and/or extracellular matrix), alterations in the cell shape, and modifications of the natural differentiation pattern. The slow transient kinetics in neonate myocytes presented in Fig. 1 would be consistent with the possibility of cellular damage. This retardation was substantially larger than the one predicted from the difference in the temperature at which the experiments were conducted (23°C in single cells vs. 37°C in the whole heart). Pérez et al. (19) have recently found that the Q10 of the Ca2+ transient activation in the neonate is 2.4. Such temperature dependence would yield a time to peak of 100 ms, which is still approximately twofold slower than our measurements in the intact heart. It is possible, therefore, that enzymatically isolated neonate myocytes undergo some level of damage that could slow down the Ca2+ transient kinetics. The alternative single-cell model, primary culture, has the potential to affect CICR due to changes in relative RyR location secondary to changes of cellular shape during culture. In this regard, we have recently reported an evident exaggeration of the RyR participation in E-C coupling in primary cultures (22, 23). If the location of RyRs changes, they can become more or less responsive to activating Ca2+ because the distance to sarcolemmal Ca2+ sources (Ca2+ channels and Na+/Ca2+ exchanger) would change. To avoid all these factors, we decided to conduct our studies in intact whole hearts using the pulsed local-field fluorescence detection technique.
RyR-mediated Ca2+ release. A noticeable role of neonate RyRs has been implicated in numerous works conducted in a variety of experimental models, including multicellular preparations (1, 16, 25, 28), primary cultures (12, 14), and freshly isolated myocytes (9). Those studies have suggested based on the use of either ryanodine or caffeine, that the participation of RyRs in E-C coupling is sizeable in the neonate heart. Further support to the importance of RyRs has derived from studies of molecular biology that have demonstrated the presence of cardiac-specific RyR isoform (RyR2) mRNA in immature hearts, even as early as embryonic days 9–10 (8). Although the significance of such an early expression of RyRs is poorly understood, it is intriguing that the heart starts beating at the embryonic day 9 in the rat (gestation time of 21 days), Consequently, the possibility that RyRs could play a role, albeit minor, in E-C coupling at those embryonic stages cannot be ruled out. An additional piece of evidence on this regard stems out from a study conducted in hearts from 3-day-old neonate rabbits by Sedarat et al. (21). Those authors reported that in the neonate, 60% of the dihydropyridine receptors (DHPRs) are already colocalized with RyRs in the periphery of the cell. Because the relative position and localization of the DHPRs and RyRs are important factors to determine the extent of CICR, this observation would imply a robust RyR-mediated Ca2+ flux during E-C coupling in the neonate. This implication would be possible only if the functional properties (i.e., unitary conductance, gating kinetics, and Ca2+ dependence) of neonate RyR were similar to the adult. Taken together, all these antecedents support the notion that the functional role of RyR in the neonate heart is important. Although our results confirmed the existence of a sizeable RyR-mediated Ca2+ flux in the intact beating whole heart from a neonate rat, they also demonstrated that the contribution of this component to the global Ca2+ transient and therefore to E-C coupling (Figs. 2, 3, and 5) is considerably smaller than previously suggested.
Another support of our conclusion derives from the temporal attributes of the Ca2+ transient. In the adult, the positive feedback nature of CICR through RyRs is evident in the rapid kinetics of the Ca2+ transient rising phase. Our results clearly reveal an acceleration of the activation phase of the ventricular Ca2+ transient with development. The results shown in Fig. 6 illustrate this phenomenon as an abbreviation of the time to peak in juvenile hearts to almost half of the value seen in the neonate. This is consistent with CICR becoming progressively more important in elevating the intracellular Ca2+ concentration. Nevertheless, changes in the distribution of L-type Ca2+ channels that have been reported to occur (21) could also induce considerable changes in the kinetics of Ca2+ release. Our experimental approach did not allow us to exclude this possibility.
Our whole heart experiments showed another important role of RyR-mediated Ca2+ flux in the immature heart, pacemaker potential. The ryanodine effect on the cardiac rhythm and frequency observed in our study, particularly in juvenile rats (Fig. 2), is consistent with the functional role of the RyR in pacemaker activity. In nodal cells from the adult cat, the RyR-mediated Ca2+ release is large enough to drive the Na+/Ca2+ exchange in the forward mode (20, 30). This inward current provides a depolarizing force during the last third of the diastole to bring the membrane potential to the activation threshold of the L-type Ca2+ current responsible for the AP upstroke. Our results indicate that this mechanism could exist in rats as well. Nevertheless, the extent of this effect in the neonate was 30% smaller than in juvenile rats. Such a difference could suggest that the extent of RyR contribution to diastolic depolarization also changes during development. Whether this transformation results from developmental changes of density, location, and functional properties of RyR or from a combination of these factors was beyond the scope of this work.
Sarcolemmal Ca2+ entry. Another important conclusion from our results (Figs. 3, 4, 5) is that the relative size of each sarcolemmal Ca2+ flux changes during the first 3 wk of postnatal life. In the neonate, sarcolemmal Ca2+ flows almost equally through T- and L-type Ca2+ channels and through the Na+/Ca2+ exchanger. Our pooled data from four hearts shown in Fig. 5B indicate that the contribution to the Ca2+ transient of each of these paths is roughly 30%. In juvenile rats, however, sarcolemmal Ca2+ entry occurs predominantly through the DHP-sensitive L-type Ca2+ channel as it is indicated by the large effect of nifedipine and the rather modest reduction induced by mibefradil. Such a large effect of nifedipine should result from the combined effects on the L-type Ca2+ channel directly and from the inability to induce RyR-mediated Ca2+ release from the SR. Consequently, the amplitude difference between the experiment with ryanodine and with nifedipine is a good estimate of the component mediated by L-type Ca2+ channels. This component in the neonate accounts for 35% of the global transient, whereas in juvenile rats it is reduced to 20%. This developmental reduction indicates an important functional transition of the L-type Ca2+ channels from Ca2+ entry path to a predominantly Ca2+ trigger. Other sarcolemmal Ca2+ fluxes change with development as well. It is interesting that the mibefradil-sensitive T-type Ca2+ current practically no longer exists after 3 wk of age. This rapid disappearance of the T-type Ca2+ current coincides with the emergence of CICR mechanism of E-C coupling. Whether or not these two phenomena are related to each other we do not know, but an appealing possibility is that T-type channel could be more effective allowing Ca2+ entry to diffuse into the myoplasm than the L-type.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by Consejo Nacional de Ciencia y Tecnología Grant S1-95000493 and SEED grants (to A. L. Escobar) and by AmericanHeart Association Grant AHA-9950382N and National Heart, Lung, and Blood Institute Grant HL-62571-01 (to R. Mejía-Alvarez) R. Ribeiro-Costa was partially supported by a CAPES scholarship.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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t'ádalová I, Kolá
F, and Ot'ádal B. Inotropic effect of low extra-cellular sodium on perfused perinatal rat heart. Can J Physiol Pharmacol 73: 50–54, 1995.[ISI][Medline]
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