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(Circulation Research. 1995;77:943.)
© 1995 American Heart Association, Inc.

Articles

Cardiac Sarcoplasmic Reticulum Phosphorylation Increases Ca²⁺ Release Induced by Flash Photolysis of Nitr-5

Jitandrakumar R. Patel, Roberto Coronado, Richard L. Moss

From the Department of Physiology, University of Wisconsin Medical School, Madison.

Correspondence to Dr Richard L. Moss, Department of Physiology, University of Wisconsin Medical School, 1300 University Ave, Madison, WI 53706.

	Abstract

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Abstract Effects on Ca²⁺-induced Ca²⁺ release due to phosphorylation of sarcoplasmic reticulum (SR) proteins were investigated in isoproterenol-treated saponin-permeabilized trabeculae from rat ventricles. In these experiments, Ca²⁺ release from the SR was induced by a rapid change in concentration of free Ca²⁺ (ie, trigger Ca²⁺) achieved by flash photolysis of nitr-5, and the amount of Ca²⁺ released was assessed by measuring isometric tension. Ca²⁺ uptake by the SR was more rapid, and the amount of Ca²⁺ released by a given concentration of trigger Ca²⁺ was greater in isoproterenol-treated trabeculae compared with control trabeculae. However, under the same conditions of Ca²⁺ loading, the amplitudes of caffeine-elicited tension transients in control trabeculae were similar to those in isoproterenol-treated trabeculae, suggesting that the Ca²⁺ available for release was similar in the two cases. Control experiments showed that there were no significant differences in Ca²⁺ sensitivity of tension between isoproterenol-treated and control trabeculae. Also, application of alkaline phosphatase to trabeculae that had previously been treated with isoproterenol returned SR Ca²⁺ release to control levels. We conclude that the greater release of Ca²⁺ in isoproterenol-treated trabeculae in response to a given concentration of trigger Ca²⁺ is due to phosphorylation of SR proteins, most likely the Ca²⁺ release channel.

Key Words: ventricular trabeculae • sarcoplasmic reticulum • Ca²⁺-induced Ca²⁺ release • flash photolysis • phosphorylation

	Introduction

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In mammalian heart, the influx of Ca²⁺ via sarcolemmal L-type Ca²⁺ channels induces release of Ca²⁺ from the SR by activating ryanodine-sensitive Ca²⁺ release channels in the junctional SR.^{1 2} Ca²⁺ released by this mechanism then diffuses to the thin filaments and activates muscle contraction. Ultimately, there is relaxation of tension developed during the cardiac twitch due to active translocation of Ca²⁺ into the SR via the ATP-dependent Ca²⁺ pump in the longitudinal SR and presumably also by inactivation of the Ca²⁺ release channels.^{3 4}

The amplitude and duration of the cardiac twitch change dramatically during ß-adrenergic stimulation of the heart, and these responses are likely to have several contributing mechanisms. ß-Adrenergic stimulation increases intracellular concentrations of cAMP, thereby activating cAMP-dependent protein kinase and causing phosphorylation of several myofibrillar proteins, including sarcolemmal L-type Ca²⁺ channels,^{5 6} troponin I,⁷ and phospholamban.⁸ Phosphorylation-induced increases in the open probability of L-type Ca²⁺ channels^{9 10} facilitate increased influx of Ca²⁺ into the cytosol during the action potential. Increased Ca²⁺ entering the cell may directly activate the myofilaments or may induce increased release of Ca²⁺ from the SR either as a consequence of the graded nature of CICR^{11 12} or of the increased Ca²⁺ content of the SR. Phosphorylation of phospholamban stimulates activity of the SR Ca²⁺ pump, resulting in rapid translocation of Ca²⁺ from the cytosol to the SR,^{8 13} and can also increase the amount of Ca²⁺ in the SR. Thus, the increase in amplitude and the more rapid decline of the Ca²⁺ transient in ß-agonist-treated myocardium might be explained on the basis of phosphorylation of L-type Ca²⁺ channels and phospholamban. However, recent evidence indicates that there is PKA-dependent phosphorylation of ryanodine-sensitive Ca²⁺ release channels,^{14 15 16} but it is not known whether phosphorylation of the release channel has a significant role in altering the Ca²⁺ transient during ß-adrenergic stimulation.

The present study was undertaken to investigate possible effects of ß-adrenergic stimulation on Ca²⁺ release from intact SR in skinned myocardium. To induce cAMP-dependent phosphorylation of intracellular proteins, trabeculae from rat ventricles were permeabilized with saponin, a cholesterol-solubilizing reagent, in the presence of a ß-adrenergic agonist. Effects on Ca²⁺ release were then assessed by measuring the tension responses that followed sudden photogeneration of trigger Ca²⁺ from a chemically caged precursor, nitr-5.¹⁷

	Materials and Methods

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Dissection and Permeabilization of Rat Ventricular Trabeculae
Wistar rats of either sex (160 to 280 g) were killed by cervical dislocation, and their hearts were rapidly excised. The hearts were placed in warm oxygenated Tyrode’s solution containing (mmol/L) NaCl 118, KCl 4.8, CaCl₂ 2, HEPES 25, KH₂PO₄ 2, MgCl₂ 1.2, pyruvate 5, insulin 0.001, and glucose 11, pH 7.4. Thin (80 to 180 µm in diameter) unbranched trabeculae, running between the right ventricular free wall and the atrial valve, were removed under a dissecting microscope and placed for 30 minutes (22°C to 24°C) in relaxing solution contain 50 µg/mL saponin, either in the presence or absence of 1 µmol/L isoproterenol. The trabeculae were subsequently bathed in relaxing solution for 10 minutes to wash out saponin and isoproterenol. The permeabilized trabeculae were then transferred to relaxing solution in an experimental trough, which in turn was placed in an experimental apparatus, as described by Moss et al.¹⁸

Experimental Protocol
Average sarcomere length was adjusted to 2.3 to 2.4 µm by adjusting the overall length of each trabecula (resting tension=0.06 to 0.10 P_o). Before the trabeculae were bathed in MA (pCa 4.5) to determine the maximum tension-generating capability, each trabecula was sequentially incubated for 2 minutes in PA containing 0.1 mmol/L EGTA (PA1), followed by 1 minute in PA containing 0.01 mmol/L EGTA (PA2). This was done to reduce the Ca²⁺-buffering capacity of relaxing solution, which sped the rate of tension development once the trabecula was placed in activating solutions.¹⁹ After tension in the MA reached a plateau, trabeculae were returned to the relaxing solution.

Trabeculae were then treated in order to load the SR with Ca²⁺ and then to subsequently release Ca²⁺ by rapid photogeneration of trigger Ca²⁺ from nitr-5. This was done by sequentially bathing the trabeculae in (1) Ca²⁺-depleting solution containing 25 mmol/L caffeine and 5 mmol/L EGTA for 1 minute, (2) PA1 for 1 minute, (3) PA2 for 1 minute, and (4) Ca²⁺-loading solution, containing 0.030 mmol/L total Ca²⁺ buffered with 0.1 mmol/L nitr-5 or 0.31 to 0.375 mmol/L total Ca²⁺ buffered with 0.7 mmol/L nitr-5, for 5 minutes. Trabeculae were then exposed to a flash of UV light (330 nm, 85 to 100 mJ) from a xenon flash lamp to generate trigger Ca²⁺, which induced transient CICR from the SR. Once the tension transient was completed, the residual Ca²⁺ content of the SR was assessed by placing the trabeculae in caffeine solution containing 10 mmol/L caffeine and 0.05 mmol/L EGTA. Finally, the trabeculae were returned to normal relaxing solution for at least 2 minutes.

This protocol was repeated several times by using different loading solutions applied in random order. Changes in maximum tension-generating capability in MA were used to assess any decline in performance of the trabeculae. In some experiments, trabeculae were also incubated for 15 minutes in relaxing solution containing ryanodine or for 30 minutes in relaxing solution containing alkaline phosphatase (type VII-NL from bovine intestinal mucosa) or both in order to assess the effects of these agents on tension transients elicited by flash photolysis of nitr-5. Experiments were done at room temperature (22°C to 24°C).

Tension-pCa relations were also obtained by measuring tension generated by both control and isoproterenol-treated trabeculae when transferred from a solution containing free Ca²⁺ of pCa 9 to a solution containing a range of free Ca²⁺ from pCa 4.5 to 5.9. Tensions at submaximal Ca²⁺ concentrations were expressed as a fraction of the tension generated by the same trabeculae at pCa 4.5. The relation between mean relative force and pCa were fitted with a Hill equation by nonlinear least-squares regression analysis: Mean Relative Force=Maximum Forcex[Ca²⁺]ⁿ/(Kⁿ+[Ca²⁺]ⁿ), where n is the Hill coefficient and K is a dissociation constant.

Experimental Solutions
All chemicals were purchased from Sigma Chemical Co except nitr-5 and ryanodine, which were purchased from Calbiochem. Relaxing, PA1, PA2, MA, calcium-depleting, and caffeine-activating solutions contained (mmol/L) BES 100, creatine phosphate 25, MgATP 5.3, free Mg²⁺ 1, and dithiothreitol 1; ionic strength was 180 mmol/L and pH was 7 at 22°C. In addition, relaxing solution contained 5 mmol/L EGTA and 2.6 mmol/L potassium propionate, PA1 contained 0.1 mmol/L EGTA and 16.8 mmol/L potassium propionate, PA2 contained 0.01 mmol/L EGTA and 17.1 mmol/L potassium propionate, and MA contained 5 mmol/L EGTA and 0.032 mmol/L free Ca²⁺ (pCa 4.5). Ca²⁺-depleting solution was relaxing solution plus 25 mmol/L caffeine, and caffeine-activating solution had 0.05 mmol/L EGTA, 16.96 mmol/L potassium propionate, and 10 mmol/L caffeine. Ca²⁺-loading solution contained 100 mmol/L BES, 14.5 mmol/L creatine phosphate, 4 mmol/L MgATP, 1 mmol/L free Mg²⁺, and 1 mmol/L dithiothreitol plus a combination of 0.1 mmol/L nitr-5, 0.02 or 0.03 mmol/L total Ca²⁺, and 58.4 mmol/L potassium propionate or a combination of 0.7 mmol/L nitr-5, 0.31, 0.325, 0.35, or 0.375 mmol/L total Ca²⁺, and 56.9 mmol/L potassium propionate. When a K_d of 145 nmol/L was assumed for nitr-5, the free [Ca²⁺] in the loading solutions buffered with 0.1 and 0.7 mmol/L nitr-5 was calculated to be 36 and 62 nmol/L and 115, 126, 145, and 167 nmol/L, respectively.

Submaximally activating solutions had pCa values ranging from pCa 4.5 to 5.9 and were prepared by mixing appropriate volumes of solutions of pCa 4.5 (MA) and pCa 9.0 (relaxing solution). The apparent stability constant for CaEGTA was corrected to 22°C and pH 7.0.²⁰ The computer program of Fabiato²⁰ was used to calculate the concentration of each metal, ligand, and metal-ligand complex.

	Results

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Oscillatory Contractions During Ca²⁺ Loading
Spontaneous oscillatory contractions were observed in almost all saponin-permeabilized trabeculae during incubation in loading solution containing 0.03 mmol/L Ca²⁺ buffered with 0.1 mmol/L nitr-5. These oscillations were presumably due to overloading of the SR with Ca²⁺,²¹ and thus, the lag time before the start of the oscillations represents the time required for Ca²⁺ loading of the SR to reach the threshold for its release. Comparison of the time course of oscillations in control and isoproterenol-treated trabeculae under these loading conditions (Fig 1) showed that the lag time was shorter and that the overall amplitude of the oscillatory contractions was greater in isoproterenol-treated (n=3) than in control (n=4) trabeculae.

View larger version (23K): [in this window] [in a new window]   Figure 1. Oscillatory contractions during Ca2+ loading of the SR in trabeculae permeabilized with saponin in the absence and presence of isoproterenol. The experimental protocols were as described in "Materials and Methods." Changes in tension were recorded while trabeculae were incubated in loading solution (L, 0.03 mmol/L Ca2+ and 0.1 mmol/L nitr-5). In each case, the lag time is the time between the start of incubation in the loading solution and the appearance of the first oscillatory contraction. Tension is expressed relative to the maximum tension developed at pCa 4.5 (Po). The slow upward drift in baseline tension is most likely due to active tension development as a result of the low Ca2+-buffering capacity of the loading solution.

To assess the Ca²⁺ content of the SR under these loading conditions, trabeculae were transferred to caffeine-containing solution after 5 minutes of incubation in Ca²⁺-loading solution. The amplitudes of the tension transient elicited by caffeine in the control and isoproterenol-treated trabeculae were 0.91±0.04 P_o (n=4) and 0.88±0.01 P_o (n=3), respectively. The similarity of responses indicates that the 5-minute loading period was adequate in both cases for the SR to accumulate nearly saturating amounts of Ca²⁺.

Tension Transient Elicited by Flash Photolysis of Nitr-5
In initial experiments, tension transients elicited by flash photolysis of nitr-5 were recorded in trabeculae preincubated in loading solution containing 0.02 or 0.03 mmol/L Ca²⁺ and 0.1 mmol/L nitr-5, but under these conditions, it was difficult to generate a tension transient without interference from oscillatory contractions. However, the oscillatory contractions during Ca²⁺ loading were prevented by increasing Ca²⁺ buffering capacity of the loading solution by increasing the concentration of nitr-5 from 0.1 to 0.7 mmol/L. Photolysis-induced tension transients were observed in both control and isoproterenol-treated trabeculae after Ca²⁺ loading in solutions of 0.31 mmol/L Ca²⁺ and 0.7 mmol/L nitr-5 (Fig 2A). Furthermore, the amplitude of caffeine-induced tension recorded after flash photolysis of nitr-5 in control trabeculae (0.75±0.06 P_o, n=10) was similar to that in isoproterenol-treated trabeculae (0.86±0.04 P_o, n=13), indicating that the SR was similarly loaded in the two preparations. Application of caffeine induced similar tensions after Ca²⁺ loading in solutions of 0.325, 0.35, and 0.375 mmol/L Ca²⁺ and 0.7 mmol/L nitr-5.

View larger version (21K): [in this window] [in a new window]   Figure 2. Tension transients elicited by flash photolysis of nitr-5 in trabeculae permeabilized with saponin in the absence and presence of isoproterenol. A, Tension transients were obtained in trabeculae permeabilized in the absence or presence of 1 µmol/L isoproterenol. To obtain the transients, trabeculae were incubated for 5 minutes in loading solution (L, 0.31 mmol/L Ca2+ and 0.7 mmol/L nitr-5) before exposure to a flash of UV light (F). B, The amplitudes of tension transients evoked by flash photolysis of nitr-5 were expressed relative to Po and were plotted against the total Ca2+ in the loading solution, which was buffered with 0.7 mmol/L nitr-5. Circles represent data from trabeculae permeabilized with saponin in the presence (, n=13) and absence (•, n=10) of isoproterenol, respectively. C, Cumulative plot of mean relative tension is shown as a function of pCa from trabeculae permeabilized with saponin in the presence (, n=6) and absence (•, n=4) of isoproterenol. Each data point is the mean, and error bars represent SEM.

Fig 2B presents records of normalized tension upon flash photolysis after periods of loading in solutions of 0.31, 0.325, 0.35, or 0.375 mmol/L Ca²⁺ and 0.7 mmol/L nitr-5. The respective amplitudes of the tension transients elicited by flash photolysis of nitr-5 were 0.22±0.07, 0.25±0.06, 0.57±0.08, and 0.79±0.06 P_o in control trabeculae (n=10) and 0.55±0.08, 0.65±0.06, 0.84±0.05, and 0.86±0.04 P_o in isoproterenol-treated trabeculae (n=13). Thus, when concentrations of Ca²⁺ in the loading solution were low or intermediate, the amount of Ca²⁺ released by CICR was greater in isoproterenol-treated than in control trabeculae.

Control experiments were performed to investigate whether the tension transients were affected by ß-agonist–induced changes in Ca²⁺ sensitivity of tension. Fig 2C shows mean tension-pCa relations from control and isoproterenol-treated trabeculae. The pCa for half-maximal tension (pCa₅₀) was 5.53±0.2 (n=4) for control trabeculae and 5.48±0.02 (n=6) for isoproterenol-treated trabeculae. However, the apparent difference in pCa₅₀ was not statistically different (P>.05, unpaired t test, after passing Kolmogorov-Smirnov normality test). This result indicates that the increase in tension after flash photolysis recorded in isoproterenol-treated trabeculae was not due to an increased Ca²⁺ sensitivity of the myofilament.

Effect of Alkaline Phosphatase on Tension Transients Elicited by Photolysis of Nitr-5 in Isoproterenol-Treated Trabeculae
One possibility to account for the results of the present study is that phosphorylation of SR proteins in isoproterenol-treated trabeculae mediates greater release of Ca²⁺ from the SR, even though the amount of photolysis-generated trigger Ca²⁺ is unchanged. This idea was investigated by applying alkaline phosphatase to isoproterenol-treated trabeculae in an attempt to dephosphorylate SR proteins. The phosphatase used in the present study was previously shown by Puceat et al²² to dephosphorylate troponin I that was previously phosphorylated by PKA. In the present study, tension transients were recorded from isoproterenol-treated trabeculae both before and after treatment with alkaline phosphatase. Fig 3 shows data from four experiments in which tension transients were recorded before phosphatase treatment, after 30 minutes of incubation in the presence of alkaline phosphatase that was previously heated to 100°C, and then after 30 minutes of incubation with normal alkaline phosphatase. Whereas the heated phosphatase caused a small but significant reduction in the amplitudes of the tension transients, treatment with unheated phosphatase largely reversed the isoproterenol-induced increase in the tension transient. From this result, we conclude that the isoproterenol-induced increase in CICR was mediated by a protein phosphorylation and presumably an SR protein.

View larger version (13K): [in this window] [in a new window]   Figure 3. Effect of alkaline phosphatase on tension transients due to flash photolysis of nitr-5 in trabeculae permeabilized with saponin in the presence of isoproterenol. The amplitudes of the tension transients after flash photolysis of nitr-5 relative to Po were plotted versus total [Ca2+] in the loading solution, which was buffered with 0.7 mmol/L nitr-5. The tension transients were recorded before treatment with phosphatase (), after 30 minutes of incubation in relaxing solution containing alkaline phosphatase (50 µg/mL) preheated to 100°C for 4 minutes (•), and after incubation with normal alkaline phosphatase (). Each data point is the mean of four experiments, and the error bars represent SEM.

Effect of Ryanodine and Alkaline Phosphatase on Tension Transients Elicited by Flash Photolysis of Nitr-5
After photolysis of nitr-5, the incomplete return of tension to the preflash baseline (Fig 2A) suggests that the trigger Ca²⁺ released by flash photolysis of nitr-5 not only induces Ca²⁺ release from the SR but may activate the myofilament directly. Thus, the total amplitude of the tension transients would be composed of Ca²⁺ from two sources, although the relative magnitudes of the tension transients and the steady postflash tension suggests that the SR is the predominant source of Ca²⁺. This idea was investigated by applying ryanodine, which eliminates Ca²⁺ release from the SR but does not affect the Ca²⁺ sensitivity of the myofibril.²³ Depending on its concentration, ryanodine works by maintaining the Ca²⁺ release channels in either a closed state or in an open subconductance state.²⁴ Fig 4 shows effects on the flash-induced tension transient that were due to incubation of isoproterenol-treated trabeculae first in ryanodine and then in alkaline phosphatase. The latter treatment was done to determine whether phosphorylation of troponin I has any effect on the steady postflash tension in ryanodine-treated trabeculae. Ryanodine treatment resulted in a tension transient that was presumably entirely due to photogeneration of trigger Ca²⁺ from nitr-5. There was a small increase in the amplitude of postflash steady tension after ryanodine treatment, probably as a result of the increased concentration of Ca²⁺–nitr-5 complexes available for photolysis. The mean amplitude of the tension recorded after loading in solutions of 0.31, 0.325, 0.35, and 0.375 mmol/L Ca²⁺ buffered with 0.7 mmol/L nitr-5 decreased from 0.49, 0.62, 0.78, and 0.84 P_o in isoproterenol-treated preparations to 0.08, 0.11, 0.25, and 0.46 P_o, respectively, after ryanodine treatment. After subsequent treatment with phosphatase, the small increase in the amplitude of the tension at each concentration of trigger Ca²⁺ was not statistically significant (P>.05, unpaired t test, after passing Kolmogorov-Smirnov normality test).

View larger version (14K): [in this window] [in a new window]   Figure 4. Effect of ryanodine on the tension transients elicited by flash photolysis of nitr-5 in trabeculae permeabilized with saponin in the presence of isoproterenol. The amplitudes of tension transients elicited by flash photolysis of nitr-5 relative to Po were plotted against total [Ca2+] in the loading solution, which was buffered with 0.7 mmol/L nitr-5. Amplitudes of transients were recorded from isoproterenol treated-trabeculae before ryanodine treatment (), after 15 minutes of incubation in relaxing solution containing 0.1 mmol/L ryanodine (•), and then after 30 minutes of incubation in 50 µg alkaline phosphatase/mL (). Each data point is the mean of four experiments, and error bars represent SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Whole cells loaded with Ca²⁺ indicators,^{11 25 26} single skinned myocytes,³ or isolated SR vesicles^{27 28} are often used to study CICR from cardiac SR. These preparations are advantageous in such studies because diffusion barriers in multicellular preparations, such as trabeculae or papillary muscle, dampen the rapid rise in trigger Ca²⁺ that is required to evoke CICR from the SR.²⁹ However, as we show in the present study, the problem of diffusion can be overcome by using the caged Ca²⁺ chelator nitr-5.^{30 31}

One difficulty of using nitr-5 in these experiments is that free nitr-5 generated by photolysis of Ca²⁺–nitr-5 can rebind Ca²⁺. Thus, nitr-5 buffers some of the Ca²⁺ released from the SR and from caged Ca²⁺ after flash photolysis. Although the extent of buffering can be reduced by using low concentrations of nitr-5, eg, 0.1 mmol/L,³⁰ in the loading solution, it was necessary in the present study to increase the concentration of nitr-5 in the loading solution from 0.1 to 0.7 mmol/L to prevent oscillatory contractions due to the spontaneous release of Ca²⁺ from the SR. Even though oscillatory contractions were inhibited by 0.7 mmol/L nitr-5, the amplitudes of caffeine-induced tension transients were similar to those recorded when using 0.1 mmol/L nitr-5, indicating that the amount of Ca²⁺ available for release from the SR was not much affected by the amount of nitr-5 in the loading solution. Still, we cannot eliminate the possibility that 0.7 mmol/L nitr-5 to some extent buffered the amount of Ca²⁺ released from the SR.

In the present study, we assessed the effect of a ß-agonist on the efficacy of a given concentration of trigger Ca²⁺ to induce Ca²⁺ release from the SR. By comparing data from isoproterenol-treated trabeculae exposed to ryanodine and alkaline phosphatase (Fig 4) with the data from control trabeculae (Fig 2), we conclude that the amount of Ca²⁺ released by a given amount of trigger Ca²⁺ was greater after treatment with isoproterenol. Our conclusion that phosphorylation was involved is strongly supported by our finding that the increased amplitudes of tension transients in isoproterenol-treated trabeculae reversed to control values after treatment with alkaline phosphatase (Fig 3).

A potential complication in these experiments is that the Ca²⁺ sensitivity of tension has been shown previously to be significantly reduced in rat skinned myocytes that were preincubated in isoproterenol before rapid skinning using Triton X-100.^{22 32} In the present study, we observed no significant change in Ca²⁺ sensitivity of tension between control and isoproterenol-treated trabeculae (Fig 2C). This discrepancy between single cells and trabeculae in their responses to isoproterenol may indicate that there is lesser responsiveness of myofilaments compared with membrane-bound proteins to ß-adrenergic stimulation. Such an effect would be exaggerated in our protocols, since in the relatively short time required for skinning, the effective concentration of isoproterenol is likely to be less in the center of our multicellular preparation than it would be in the vicinity of single cells suspended in solution. Whatever the basis for the discrepancy, the increase in amplitude of the tension transients elicited after flash photolysis in isoproterenol-treated trabeculae cannot be due to phosphorylation of myofilament proteins but instead is due to increased release of Ca²⁺ from the SR.

Isoproterenol-induced increases in Ca²⁺ release from the SR could be due to increases in the Ca²⁺ content of the SR, in the Ca²⁺-induced opening of ryanodine receptors in the SR, or both. With regard to the first possibility, we observed that the time to load the SR with Ca²⁺ was much shorter in isoproterenol-treated trabeculae (this loading time was assessed as the interval to the first tension oscillations once the trabeculae were placed in loading solution) (Fig 1). This observation is consistent with the increased rate of Ca²⁺ uptake observed in isolated SR vesicles after phosphorylation of phospholamban with cAMP-dependent protein kinase.³³ More recently, Luo et al³⁴ reported significant increases in contractile parameters of phospholamban-deficient mice compared with wild-type mice, and these increases were similar to those observed in isoproterenol-treated hearts from wild-type mice. On the basis of these observations, Luo et al concluded that phosphorylation of phospholamban is a key system in mediating the inotropic responses of the heart to ß-adrenergic stimulation. A similar conclusion was reached by Sham et al,³⁵ who assessed cellular function in intact myocytes that were infused via patch pipettes with a monoclonal antibody to phospholamban. Thus, there is clear evidence that phospholamban plays an important role in ß-adrenergic effects on contractility, presumably as a result of an increased rate of Ca²⁺ uptake into the SR due to phosphorylation of phospholamban.

Although it is likely that an increased rate of Ca²⁺ loading of the SR plays an important role in inotropic responses in vivo especially when the beat frequency increases, it is unlikely that the increased rate of Ca²⁺ loading plays an important role in the increase in photolysis-induced tension transients in the present study. Our results show that the steady state loading of the SR is maximal and similar in both control and isoproterenol-treated trabeculae, since caffeine induced similar tension transients in the two cases. Of course, we cannot eliminate the possibility that caffeine does not release SR Ca²⁺ that is loaded in response to PKA-induced phosphorylation of phospholamban, but we are unaware of any evidence indicating that this is the case.

Because the SR appears to be similarly loaded in our control and isoproterenol-treated preparations, we conclude that the isoproterenol-induced increase in the release of Ca²⁺ in response to the same concentration of trigger Ca²⁺ is due to the increased opening of ryanodine receptors. Recent evidence indicates that PKA phosphorylates the ryanodine-sensitive Ca²⁺ release channel,^{14 15 16} leading to increased opening of the channel.³⁶ Thus, phosphorylation of the ryanodine receptor could account for the increased amplitudes of the tension transients after photolysis of nitr-5. Phosphorylation-induced increases in channel openings could also account for ß-agonist–induced increases in the amplitude and frequency of oscillatory contractions during Ca²⁺ loading. Our conclusion that phosphorylation is involved in the greater response of skinned trabeculae to trigger Ca²⁺ is supported by our finding that the amplitudes of the photolysis-induced tension transients in isoproterenol-treated trabeculae reverted to control values after treatment with alkaline phosphatase (Fig 3). Since both PKA and Ca²⁺-calmodulin–dependent protein kinase are known to phosphorylate phospholamban and the ryanodine receptor,^{8 13 14 15 16 33 34 35 36 37 38} it is conceivable that phosphorylation-mediated effects induced by both kinases could increase both Ca²⁺ loading and Ca²⁺ release in vivo. However, since isoproterenol was applied in our experiments in the absence of extracellular Ca²⁺, it is unlikely that the effects we observed were due to the activation of Ca²⁺-calmodulin-dependent protein kinase.

Besides depending on the amount of Ca²⁺ available for release, the amplitude of CICR depends on the concentration of trigger Ca²⁺.²⁹ In the present study, the amount of Ca²⁺ released from the SR increased when the concentration of trigger Ca²⁺ was increased in either control or isoproterenol-treated trabeculae, supporting the conclusion that CICR in myocardium is graded.^{11 12} This phenomenon would be expected to play an important role in cardiac responses to ß-adrenergic stimulation in vivo, since increased Ca²⁺ entry via L-type Ca²⁺ channels would elicit increased release of Ca²⁺ from the SR.

In summary, our results show that in trabeculae permeabilized with saponin, ß-adrenergic stimulation increased the rate of uptake of Ca²⁺ into the SR, presumably by phosphorylation of phospholamban, but the steady state Ca²⁺ load of the SR did not appear to differ from the control value. The amount of Ca²⁺ released from the SR in response to a given amount of trigger Ca²⁺ increased when trabeculae were treated with isoproterenol; this increase was most likely due to ß-agonist–induced phosphorylation of the Ca²⁺ release channel. From the magnitude of the increase in Ca²⁺ release, we conclude that increased sensitivity of the Ca²⁺ release channel to trigger Ca²⁺ is a major mechanism by which ß-adrenergic stimulation increases the Ca²⁺ transient during the myocardial twitch.

	Selected Abbreviations and Acronyms

 

CICR = Ca2+-induced Ca2+ release MA = maximally activating solution PA = preactivating solution PKA = protein kinase A Po = maximum active tension elicited in solution of pCa 4.5 SR = sarcoplasmic reticulum

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-25861 to Dr Moss and GM-36852 to Dr Coronado.

Received August 25, 1994; accepted July 13, 1995.

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