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(Circulation Research. 2007;100:391.)
© 2007 American Heart Association, Inc.

Cellular Biology

ß-Adrenergic Enhancement of Sarcoplasmic Reticulum Calcium Leak in Cardiac Myocytes Is Mediated by Calcium/Calmodulin-Dependent Protein Kinase

Jerald Curran, Mark J. Hinton, Eduardo Ríos, Donald M. Bers, Thomas R. Shannon

From the Rush University Medical Center (J.C., M.J.H., E.R., T.R.S.), Chicago; and Loyola University Medical Center (D.M.B.), Maywood, Ill.

Correspondence to Thomas Shannon, Department of Physiology and Molecular Biophysics, Rush University Medical Center, 1750 W Harrison St, Chicago, IL 60611. E-mail tshannon{at}rush.edu

	Abstract

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Enhanced cardiac diastolic Ca leak from the sarcoplasmic reticulum (SR) ryanodine receptor may reduce SR Ca content and contribute to arrhythmogenesis. We tested whether ß-adrenergic receptor (ß-AR) agonists increased SR Ca leak in intact rabbit ventricular myocytes and whether this depends on protein kinase A or Ca/calmodulin-dependent protein kinase II (CaMKII) activity. SR Ca leak was assessed by acute block of the ryanodine receptor by tetracaine and assessment of the consequent shift of Ca from cytosol to SR (measured at various SR Ca loads induced by varying frequency). Cytosolic [Ca] ([Ca]_i) and SR Ca load ([Ca]_SRT) were assessed using fluo-4. ß-AR activation by isoproterenol dramatically increased SR Ca leak. However, this effect was not inhibited by blocking protein kinase A by H-89, despite the expected reversal of the isoproterenol-induced enhancement of Ca transient amplitude and [Ca]_i decline rate. In contrast, inhibitors of CaMKII, KN-93, or autocamtide-2–related inhibitory peptide II or ß-AR blockade reversed the isoproterenol-induced enhancement of SR Ca leak, and CaMKII inhibition could even reduce leak below control levels. Forskolin, which bypasses the ß-AR in activating adenylate cyclase and protein kinase A, did not increase SR Ca leak, despite robust enhancement of Ca transient amplitude and [Ca]_i decline rate. The results suggest that ß-AR stimulation enhances diastolic SR Ca leak in a manner that is (1) CaMKII dependent, (2) not protein kinase A dependent, and 3) not dependent on bulk [Ca]_i.

Key Words: sarcoplasmic reticulum • excitation-contraction coupling • ryanodine receptor

	Introduction

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A great deal of effort has been focused on the causes of death from heart failure (HF). Patients with chronic HF have a contractile deficit that is attributable in part to decreased [Ca]_i transient magnitude and exhibit electrical arrhythmias, which can result in sudden death. Abnormal cellular ionic handling may play a role in causing these problems.¹ Possible contributors include increased diastolic Ca leak from the sarcoplasmic reticulum (SR).

SR Ca leak is increased in isolated cardiac myocytes from rabbit in HF versus control and may lead to an exacerbation of both the decreased systolic Ca transient and the generation of arrhythmias.^2,3 The directed leak of SR Ca toward the Na/Ca exchanger (NCX) may also lead to depolarization of the sarcolemma (SL).^2,4 This may contribute to electrical instability, early or delayed afterdepolarizations, triggered arrhythmias, and sudden cardiac death.^5–7

Marks and colleagues have been the primary proponents of the idea that SR Ca leak may be increased by ryanodine receptor (RyR) phosphorylation. They showed that phosphorylation by either protein kinase A (PKA) or Ca/Calmodulin-dependent protein kinase II (CaMKII) may increase RyR activity.^8,9 In HF, there is more activated CaMKII and less phosphatase in the RyR enzyme complex, and we have shown that the enhanced diastolic SR Ca leak in HF can be prevented by CaMKII inhibition.¹⁰ CaMKII has been shown to increase both the RyR open probability (P_o)^9,11,12 and Ca spark frequency.^13–16

The details of this mechanism have been challenged, and the mechanism may be complex. For instance, whereas Marks and colleagues^8,9 find that PKA greatly increases RyR P_o, Valdivia et al¹⁷ found reduced steady-state P_o and Stange et al¹⁸ found no effect of PKA on RyR gating. Li et al¹⁹ found the alteration in Ca sparks on PKA phosphorylation in wild-type myocytes to be entirely dependent on phospholamban phosphorylation and the subsequent rise in [Ca]_SRT.

The current report focuses on the regulation of SR Ca leak. We test the hypothesis that ß-adrenergic receptor (ß-AR) activation enhances diastolic SR Ca leak via both PKA and CaMKII. SR Ca leak is measured in intact myocytes with isoproterenol (ISO). Surprisingly, the ß-AR-induced SR Ca leak was blocked by inhibition of CaMKII, but inhibition of PKA had no effect. Furthermore, direct activation of adenylate cyclase and PKA by forskolin did not increase SR Ca leak, despite the expected strong inotropic and lusitropic effects concomitant with PKA activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Additional details can be found in the online data supplement, available at http://circres.ahajournals.org. All chemicals were from Sigma Chemical Co, except as indicated. 0 Na⁺/0 Ca²⁺ normal tyrode (NT) had Li⁺ substituted for Na⁺ and 10 mmol/L EGTA with no added Ca²⁺. All experiments were conducted at 23°C on myocytes isolated from rabbit hearts as previously described.²⁰

Experimental Protocol
The protocol (Figure 1) was largely as previously described,^3,10,21 where [Ca]_i was measured using fluo-4 or fura-2 in isolated myocytes in the presence and absence of diastolic SR Ca leak. Tetracaine was used to rapidly and reversibly block the RyR, thus disrupting the pump-leak balance. The tetracaine-dependent shift of Ca from the cytosol to the SR (decrease in [Ca]_i and increase in SR Ca content) is proportional to SR Ca leak.

View larger version (23K): [in this window] [in a new window]   Figure 1. A, Illustration of protocol used to measure SR Ca leak. The SR Ca leak is proportional to the fall in [Ca]i and the resultant rise in [Ca]SRT in the presence of the RyR blocker tetracaine. The steady-state shift of Ca2+ from the cytosol to the SR in tetracaine is proportional to the SR Ca leak. The SR [Ca] is graded through the use of loading protocols in 2 mmol/L Ca2+ NT with or without the test substances. This is followed by a rapid switch to 0 Na+, 0 Ca2+ NT+tetracaine (1 mmol/L). Tetracaine is washed out 30 seconds later. SR [Ca] is measured with 10 mmol/L caffeine in 0 Na+/0 Ca2+ NT. B, Typical raw fluorescence trace of typical protocol illustrated in Figure 1A. C, Typical raw fluorescence trace of typical protocol in presence of ISO.

To activate ß-AR or adenylate cyclase, 250 nmol/L ISO or 1 µmol/L forskolin (respectively) was added to field-stimulated myocytes for 5 to 7 minutes before leak assessment. PKA inhibitor (H-89) or CaMKII inhibitor KN-93 (and its inactive analog KN-92, Calbiochem), when used, were applied (at 1 µmol/L) together with ISO. Cell permeant CaMKII inhibitor autocamtide-2–related inhibitory peptide II (AIP) (Calbiochem, San Diego, Calif) was loaded into myocytes in a 0 Ca²⁺ NT solution+250 nmol/L ISO+1 µmol/L AIP for 1 to 1.5 hours. Inhibition of ß-AR subtypes 1 and 2 in the presence of ISO was accomplished by the addition of subtype specific inhibitors CGP-20712A (500 nmol/L) and ICI 118,551 (300 nmol/L), respectively.

Balance of Ca Fluxes Analysis
Ca²⁺ fluxes during typical [Ca]_i transients were calculated as described in the online data supplement. Briefly, the decline of the total Ca transient on caffeine application was attributed to NCX transport. The decline of the steady-state total Ca transient was representative of sum of the NCX plus SR Ca-ATPase transport (so the difference indicates SR Ca-ATPase transport).

Statistical Analysis
Data were reported as mean±SEM. Student’s t test was applied when appropriate, with P<0.05 considered significant.

	Results

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ISO and Diastolic SR Ca Release
SR Ca leak was determined at different SR Ca loads attained by 5 different loading protocols (see the expanded Materials and Methods section in the online data supplement).²¹ Blocking the leak shifts Ca from the cytosol ([Ca]_i) into the SR ([Ca]_SRT) in proportion to the leak. Figure 2A shows how SR Ca leak ([Ca]_SRT, top; [Ca]_i, bottom) depends on diastolic SR [Ca]±ISO exposure. Each point on the graph represents the average [Ca]_SRT for the indicated pacing protocol. The leak/[Ca]_SRT relationship (for both [Ca]_SRT and [Ca]_i) is left-shifted by ISO, so at a given [Ca]_SRT (without tetracaine), SR Ca leak is higher with ISO than control (and reversed by ß-AR blockade).

View larger version (18K): [in this window] [in a new window]   Figure 2. A, Leak-dependent changes in SR [Ca] (top) and [Ca]i (bottom) in cells grouped together progressively by loading protocol; control cells marked for reference. At a given SR [Ca], the leak (ie, the tetracaine-dependent shift of Ca from the cytosol to the SR) is higher with ISO than control (n=7 to 14 for control, n=6 to 9 for ISO). This effect is reversed in the presence of ß blockers (n=6 to 7). B, Statistical analysis of the data in A. In data grouped such that the same SR [Ca] load is examined (right), the leak is higher in ISO (left, n=9 to 13). C, In data grouped by the same average leak (left), the SR Ca load needed to induce that leak is higher in control than ISO (right, n=8 to 12). *Significantly different from control.

Figure 2B and 2C shows data grouped (from any pacing rate) to assess leak at a comparable SR [Ca] content (Figure 2B) or to assess SR Ca content at a comparable leak (Figure 2C). SR Ca leak is significantly higher with ISO treatment (Figure 2B, right; control=6.64±0.88; ISO=12.99±1.75; ISO+ß blockade=6.50±0.99; ISO+ß₁ blockade=6.77±1.08; ISO+ß₂ blockade=10.45±1.27 µmol/L) at the same average [Ca]_SRT (124.7±3.6, 128.9±1.8, 124.2±4.2, 123.9±4.02, and 123.7±4.45 µmol/L). Figure 2C shows the converse analysis. Data are selected to average the same SR Ca leak (4.9±0.9, 4.8±0.4, 4.9±0.3, 4.9±0.2, 4.9±0.5 µmol/L [Ca]_SRT). The [Ca]_SRT at which this leak is achieved is much higher in control than in ISO-treated cells (114.8±3.0, 89.8±6.8, 112.4±18.4, 112.2±8.7, and 81.1±12.0 µmol/L). The data indicate that ß-AR stimulation increases RyR activity and diastolic SR Ca release.

The data in Figure 2B and 2C also show that ß₁-AR are sufficient to explain the ß-AR-induced increase in SR Ca leak (without requiring ß₂-AR). Thus ß₁-AR is surely involved. ß₂-AR blockade alone could not reverse the ISO-induced SR Ca leak. However, given the predominance of ß₁- versus ß₂-AR in rabbit heart, we cannot completely rule out ß₂-AR as also playing a minor role.

SR Ca Leak and CaMKII Inhibition
The CaMKII inhibitor KN-93 (1 µmol/L) prevented the effect of ISO on SR Ca leak (Figure 3A). The inactive analog KN-92 did not alter the ISO-induced shift. In Figure 3B, data were grouped to match SR Ca load (124.7±3.6, 124.8±3.6, 125.8±1.5 µmol/L for control, ISO+KN-93, and ISO+KN-92, respectively). The leak ([Ca]_SRT) at these loads was reduced by CaMKII inhibition (ISO+KN-93 5.1±1.0 µmol/L) versus ISO (ISO+KN-92, 14.5±3.0 µmol/L) and was comparable to control (6.6±0.9 µmol/L; Figure 3B). Figure 3C shows that at matched [Ca]_SRT (5.9±1.2, 6.0±0.9, 5.8±0.9 µmol/L for control, ISO+KN-93, and ISO+KN-92, respectively), the [Ca]_SRT was higher with CaMKII inhibition (ISO+KN-93, 192.4±20.0 µmol/L) versus ISO (ISO+KN-92, 97.5±8.2 µmol/L) and was comparable to control (190.2±3.4 µmol/L).

View larger version (27K): [in this window] [in a new window]   Figure 3. A, Leak-dependent changes in SR [Ca] (top) and [Ca]i (bottom) in cells grouped together by loading protocol. The left shift of the curve with ISO (Figure 2) is reversed by KN-93 but not by H-89 or KN-92 (KN-93 n=4 to 11; H-89 n=5 to 9; KN-92 n=5 to 11). B, Statistical analysis of the data in A. In data grouped such that the same SR [Ca] load is examined (right), the increase in leak with ISO is not reversed by the addition of H-89 or KN-92; only the addition of KN-93 (n=8 to 12) decreases the leak to below the control (left, analysis of data for matched control and ISO loads from Figure 2 shown for comparison). C, In data grouped to obtain the same average leak (left), the SR Ca load is lower with the addition of H-89 and KN-92, but the SR Ca load is high, as in control, with the addition of KN-93 (right, n=6 to 9). Addition of forskolin did not vary from control (n=9 to 12). *Significantly different from control.

These results suggest that CaMKII mediates the increased SR diastolic leak on ß-AR stimulation. Our working hypothesis was that CaMKII activation was secondary to PKA-dependent [Ca]_i transient enhancement (eg, increasing time averaged [Ca]_i).

To further test that the reduced SR Ca leak with KN-93 was specific to CaMKII inhibition, experiments were performed with a highly selective peptide CaMKII inhibitor, AIP (IC₅₀=4.1 nmol/L). As with KN-93, when SR Ca loads were matched (94.0±1.9 versus 91.6±10.1 µmol/L for ISO and ISO+AIP; Figure 4), the leak was lower in the presence of CaMKII inhibition by AIP (13.9±3.4 versus 6.7±2.0 µmol/L for ISO versus ISO+AIP). Similarly, when the [Ca]_SRT was matched (11.5±0.6 versus 11.5±0.4 µmol/L for ISO and ISO+AIP, respectively) the SR Ca load was increased in the presence of AIP (108.3±11.5 versus 169.0±19.2 µmol/L for ISO and ISO+AIP, respectively; Figure 4B). Each of these results indicates that when CaMKII is inhibited the ß-AR-induced SR Ca leak declines in isolated myocytes. They therefore support the KN-93 and KN-92 results (Figure 3), indicating that the KN-93 effects on leak are likely to be CaMKII specific. Moreover, the ß-AR-induced enhancement of SR Ca leak at a given [Ca]_SRT appears to be mediated by CaMKII activity.

View larger version (21K): [in this window] [in a new window]   Figure 4. A, SR Ca leak measurement using the CaMKII inhibitor AIP. When data are grouped such that the same SR [Ca] load is examined (left), the leak in ISO is higher (left, n=7 to 8). B, In data grouped to obtain the same average leak (right), the [Ca]SRT needed to induce the leak was higher in cells treated with AIP compared with ISO alone (right, n=8 to 10). The data are comparable to the effects in KN-93 (Figure 3). *Significantly different from control.

SR Ca Leak and PKA Inhibition and Stimulation
To assess whether PKA mediated the ISO-induced increase of SR Ca leak, we applied the PKA inhibitor H-89 (1 µmol/L, together with ISO, K_i=48 nmol/L) to the isolated cardiac myocytes and measured leak. The hypotheses were 2-fold: (1) that ISO, by increasing PKA activity would phosphorylate RyR and increase SR Ca leak; and (2) that stimulation of PKA would increase Ca transient magnitude, which would indirectly increase CaMKII activity and increase SR Ca leak via CaMKII activity. Our expectation was then that PKA inhibition by H-89 would inhibit the ISO-dependent leak (by either mechanism).

Surprisingly, this was not the case. H-89 had no effect on the ISO-dependent increase in SR Ca leak, yielding virtually the same relationship versus [Ca]_SRT as with ISO and ISO+KN-92 (Figure 3A). Selected data with the same average [Ca]_SRT (124.1±4.4 µmol/L) shows a leak comparable to ISO alone or ISO+KN-92 (14.6±2.0 µmol/L; Figure 3B), and data selected for match [Ca]_SRT (6.0±0.6 µmol/L) had a limited SR Ca load (67.8±6.0 µmol/L; Figure 3C). This indicates that both of our above hypotheses are incorrect.

We further tested whether the ISO-induced increase in the leak could be mimicked by activating PKA during application of forskolin (1 µmol/L) rather than ß-AR activation by ISO. Forskolin potently stimulates adenylate cyclase, elevating cAMP production and activating PKA. Importantly, forskolin bypasses the ß-AR; thereby, any potential effect on SR Ca leak could be solely attributed to the production of cAMP and PKA activation. Indeed, the addition of forskolin produced the expected increase in the peak of the [Ca]_i transient (to 314±78% of control) and a simultaneous decrease in the of the [Ca]_i transient decline versus control (to 28.9±5.0% of control). Remarkably, PKA activation by forskolin had no effect on leak. Indeed, forskolin treatment failed to shift the leak versus load relationship (Figure 3A) away from control.

When we selected data with the same average [Ca]_SRT (120.8±3.0 µmol/L) the [Ca]_SRT was no different when compared with control (6.2±0.8 µmol/L; Figure 3B). Likewise, data selected to yield the same [Ca]_SRT (5.8±1.1 µmol/L) showed that the [Ca]_SRT at which that leak occurred was no different from that in control (188.7±7.0 µmol/L; Figure 3C). Taken together with the H-89 results, these data strongly indicate that PKA does not mediate the ß-AR-induced enhancement of diastolic SR Ca leak in intact myocytes.

Mechanism of ISO-Dependent Ca/Calmodulin Protein Kinase Activity
The lack of effect of H-89 on the ISO-induced SR Ca leak led us to examine how H-89 and KN-93 influence [Ca]_i transients. ISO was clearly effective in dramatically enhancing Ca transient amplitude and the rate of Ca transient decline (Figure 5A, 5B, and 5D). It is also clear that H-89 was effective in blocking these classic PKA effects. That is, the acceleration of [Ca] decline induced by ISO was completely prevented by H-89 (Figure 5A and 5B), and the increased amplitude was almost completely prevented (Figure 5D). Thus, H-89 was effective in blocking the PKA-dependent effects of ISO. In addition, the forskolin-induced enhancement of Ca transient amplitude and decline rate (see above) were at least as potent as those induced by ISO, indicating strong PKA-dependent inotropy in both cases, but with dramatically different effects on SR Ca leak.

View larger version (29K): [in this window] [in a new window]   Figure 5. A, Typical [Ca]i transients as measured using fura-2 to ensure a calibrated fluorescent signal. Data are normalized to the peak [Ca]i. B, Statistical analysis of data in A. Average of [Ca]i transient decline at 0.5Hz. H-89 completely reversed the acceleration of the decline exhibited by ISO, whereas KN-93 did not. C, Time-averaged [Ca]i of transients at 0.5 Hz. Both H-89 and KN-93 decreased the time-averaged [Ca]i toward control levels. Although time-averaged [Ca]i was decreased in the presence of H-89, the leak remained high (Figure 3), indicating that ISO stimulation of CaMKII is at least partially [Ca]i independent. D, Averaged peak [Ca]i values in cells stimulated at 0.5 Hz. ISO nearly triples the peak value of control; both KN-93 and H-89 return the peak toward control level. E, Fractional release (100xpeak[Ca]i/[Ca]SRT) at 0.5 Hz. [Ca]SRT: 130.8±15.4 [n=7], 123.4±13.7 [n=7], 134.1±18.5 [n=7], 188.8±40.4 [n=8] for control, ISO, ISO+H-89, and ISO+KN-93, respectively. *Significantly different from control, #significantly different from ISO.

KN-93 substantially inhibited the ISO-induced enhancement of Ca transient amplitude (Figure 5D) and fractional SR Ca release (Figure 5E). This may reflect the previously described ability of endogenous CaMKII to enhance fractional SR Ca release in ventricular myocytes.²⁰ Thus, CaMKII may activate the RyR both at rest (Figures 3 and 4) and during excitation-contraction (E-C) coupling (Figure 5). Surprisingly, the higher CaMKII-dependent SR Ca leak was maintained in the presence of H-89. We assumed that any increase in CaMKII activity would occur via increased PKA activity and the resulting increased [Ca]_i. Such was apparently not the case here, because H-89 virtually abolished the ISO effects on [Ca]_i transient amplitude (Figure 5D), [Ca]_i decline (Figure 5A and 5B), and the time averaged [Ca]_i (Figure 5C) at 0.5 Hz, but did not reverse the ISO-dependent effect on SR Ca leak. We can only conclude that the increased CaMKII activity at the RyR is not dependent on bulk [Ca]_i.

Ca Flux Analysis During E-C coupling
Figure 6 analyzes the effects of ISO and kinase inhibitors using balance of Ca flux analysis by the method of Bassani et al.²² Caffeine-induced [Ca]_i transients, which were used to empty the SR Ca (for the low [Ca]_SRT protocols; see the supplemental Materials and Methods section), were examined to determine the Ca transport rates via NCX (as a function of [Ca]_i). This relationship was used to determine the Ca efflux during Ca transients at 0.5 Hz, with the remainder being SR Ca pump-mediated transport. In control myocytes (Figure 6A, top left), the SR Ca pump accounted for 59±2% of the transported Ca with the transport through the NCX, accounting for the other 41±2% of the cytosolic Ca efflux. ISO (Figure 6A, top right) significantly increased the percentage of Ca transport from the cytosol through the SR Ca pump to 85±2%, with the NCX carrying only 15±2%.

View larger version (28K): [in this window] [in a new window]   Figure 6. A, Analysis of the balance of Ca fluxes. Control cells showing characteristic integrated fluxes (top, left) where SR Ca-ATPase (SERCA) accounted for 59±2% (11.5 µmol/L, n=12) of the total cytosolic Ca efflux, with the other 41±2% (8.1 µmol/L) accounted for by NCX and other SL "slow" mechanisms. In cells treated with 250 nmol/L ISO (top, right), the SERCA pump dominates, contributing 85±2% (226.5 µmol/L, n=8) of all total Ca flux, with the NCX/slow mechanisms accounting for the remaining 15±2% (40.2 µmol/L). H-89 (bottom, left) only partially reversed the effects of ISO (SERCA=64±1%, 32.4 µmol/L; NCX/slow=36±1%, 18.4 µmol/L, n=7). KN-93 (bottom, right) also partially reversed the ISO effects, possibly because of its effect on the SR Ca pump (SERCA=77±4%, 72.3 µmol/L; NCX/slow=23±4%, 21.5 µmol/L, n=3). B, Effects of ISO on the SR Ca pump. ISO dramatically increased the SR Ca uptake (Vmax=282±17.3 µmol/L per second, Km=274.2±4.0 nmol/L, H=2.85±0.01) over control (Vmax=91.4±11.2 µmol/L per second, Km=271.5±1.2 nmol/L, H=2.89±0.02). KN-93 only slightly reduced the pump activity (Vmax=237.0±59.0 µmol/L per second, Km=274.9±0.5 nmol/L, H=2.83±0.81) in contrast to H-89, which completely reversed the ISO pump effects (Vmax=77.6±2.5 µmol/L per second, Km=268.0±0.2 nmol/L, H=3.03±0.01). NCX parameters were similar for all conditions (Vmax=60.6±5.9 µmol/L per second, 51.4±5.5, 41.1±1.5, 65.7±2.9; Km=275.1±1.8, 276.7±2.1, 282.4±0.4, 271.5±0.1; H=2.78±0.04, 2.71±0.06, 2.52±0.01, 2.86±0.00 for control, ISO, ISO+H-89, and ISO+KN-93, respectively). C, Gain of Ca-induced Ca release from the parameters in A (SR Ca flux)/(SL Ca flux). The parameter increased dramatically with ISO treatment and remained high despite the decreased leak in KN-93–treated cells. In contrast, H-89 completely reversed the effects of H-89 on the gain of E-C coupling. *Significantly different from control, #significantly different from ISO.

The data are consistent with an increased phospholamban phosphorylation and SR Ca pumping activity (Figure 6B). Ultimately, however, this flux balance reflects not only competition of Ca removal pathways during [Ca]_i decline, but also E-C coupling gain. This is because at steady state, the amount of Ca entering the cell, primarily through the L-type Ca current (I_Ca), must equal the amount of Ca that leaves the cell, primarily through NCX. Thus, the ratio of integrated Ca removal via SR versus SL is indicative of E-C coupling gain (J_SRCaRel/I_Ca; Figure 6C). We therefore conclude that the effects on SR Ca release (J_SRCaRel) predominate over the direct effect of increased I_Ca to the transient. That is, both J_SRCaRel and I_Ca are increased, but the ratio is increased.

Both H-89 and KN-93 decreased ISO-stimulated peak [Ca]_i and, therefore, total integrated Ca flux during the decline (Figure 6). H-89 completely reversed the effects of ISO on the [Ca]_i-dependent SR Ca-ATPase activity (Figure 6B) and returned E-C coupling gain to control levels (1.8±0.1; Figure 6C). KN-93+ISO caused a small decline in J_SRCaRel (77±4%) and in the gain (3.4±0.9), back toward control, which was likely related to its relatively small effects on the SR Ca pump (Figures 5A, 5B, and 6B). Note, however, that this relatively minor effect on J_SRCaRel compared with ISO takes place despite a large increase in [Ca]_SRT (123.4±13.7 and 188.8±40.4 for ISO and ISO+KN-93, respectively; Figure 3A), resulting in a larger effect on fractional release (Figure 5E) than on SR Ca Pump function (see Discussion below).

Of note in Figure 6 is that the effects of ISO, H-89, and KN-93 on the balance of fluxes (Figure 6A) and the gain of E-C coupling (Figure 6C) generally parallel the effects of these agents on the SR Ca uptake (Figure 6B). Furthermore, these seem to be relatively unaffected by the SR Ca leak. For instance, H-89 completely inhibits the effect of ISO on uptake, despite having no effect on the ISO-induced leak. The data suggest that the SR Ca leak does not greatly affect the gain of E-C coupling and that the relevance of this process may be found in other areas, eg, increased rate of release,²³ increased speed of physiological response to perturbation, or regulation of [Ca]_SR, which may be dominated by the RyR effects on larger Ca fluxes during systole⁸ or diastolic SR Ca leak, which can have arrhythmogenic consequences.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The present study was undertaken to clarify the roles of PKA and CaMKII in causing SR Ca leak. Specifically, it addresses the relationship between the relevant kinases and the RyR activity in the intact ventricular myocytes in response to ß-AR activation by ISO. ISO can activate PKA and CaMKII, both of which have been implicated in causing increased RyR activity in HF, and either of which may be selectively inhibited. Therefore, the study is significant for both pathophysiological and physiological ß-AR activation.

PKA and Diastolic SR Ca Release
ß-AR stimulation enhances several Ca handling mechanisms (ie, via L-type Ca current and phospholamban phosphorylation). Our results here are consistent with this, as shown in Figure 5. H-89 reversed these ISO-dependent changes, indicating that PKA was responsible for them.

We found that ISO dramatically increased the diastolic SR Ca leak (Figures 2 and 3) but that it was not PKA dependent. These data are consistent with that of Li et al,¹⁹ who found no PKA-dependent increase in Ca spark frequency in phospholamban knockout mice (at constant SR Ca content). It is also compatible with data indicating that H-89 could not decrease SR Ca leak in HF.¹⁰ The fact that H-89 and forskolin had their expected effects on other PKA-dependent changes leads us to conclude that PKA does not mediate the ß-AR-induced enhancement of SR Ca leak in our system.

CaMKII and Diastolic SR Ca Release
When CaMKII was inhibited in the presence of ISO, leak returned to normal levels (Figure 3). Again, this is consistent with data in HF animals, in which higher SR Ca leak associated with higher RyR phosphorylation levels was also CaMKII-dependent,¹⁰ and with data from CaMKII-overexpressing myocytes, in which the higher leak was also KN-93 sensitive.^15,16 RyR regulation by CaMKII has been shown, and phosphorylation regulates RyR by increasing its open probability.^11,12

A puzzling aspect, however, involves how the CaMKII activity is increased. We anticipated that the PKA-dependent increase in [Ca]_i (Figure 5C and 5D) might activate CaMKII effects on RyR. However, H-89 prevented the ISO-induced enhancement of Ca transients, but the increased SR Ca leak remained. Similarly, forskolin increased Ca transient amplitude and time-averaged [Ca]_i by direct stimulation of PKA, and yet the SR Ca leak was no different than that found in control. This leads us to the conclusion that ISO-dependent CaMKII activation is not dependent on increased PKA or [Ca²⁺]_i and an alternative pathway must exist. By bypassing the ß-AR pathway with forskolin we show here that direct stimulation of adenylate cyclase (and any subsequent consequences of that stimulation) is not linked to CaMKII activation. Indeed, Wang et al²⁴ and Zhu et al²⁵ have implicated the ß₁ receptor in a PKA-independent activation of CaMKII in apoptotic heart cell death. The data are consistent with the idea that ß-AR activation can lead to CaMKII activation, in a manner independent of cAMP, PKA, or bulk [Ca²⁺]_i.

Diastolic SR Ca Release and E-C Coupling
Rabbit myocytes (in which Ca flux balance is similar to human) shifted to become more SR dominated in response to ISO treatment.²⁶ Although ß-AR stimulation increases peak I_Ca, and therefore the SL-dependent influx and efflux, the enhanced SR Ca pump function (and I_Ca) increases [Ca]_SRT; and both these largely PKA-dependent effects synergize to increase SR Ca release and the size of the SR Ca transient.^23,27 The data in Figure 6 indicate that both H-89 and KN-93 shift the ISO-dependent balance of fluxes back toward control, where it was less SR dominated (Figure 6A). In each case, the degree of shift is associated with the degree of change in the SR Ca uptake parameters (Figure 6). That is, CaMKII inhibition only partially reverses the ISO-induced SR Ca-ATPase stimulation (Figure 6B) and E-C coupling gain (Figure 6C), whereas H-89 reverses both. Note that "gain" as used here is a broad definition, because both I_Ca and SR Ca load may differ. Thus, enhanced [Ca]_SRT (and I_Ca) may largely explain the ISO-dependent enhancement of E-C coupling gain (see also Ginsburg and Bers²⁶).

Conversely, the reversal of ISO-induced SR Ca leak by CaMKII inhibition was complete, whereas PKA inhibition had no effect on leak. This dichotomy, with respect to PKA inhibition, indicates that these alterations in diastolic SR Ca leak with no alteration in the largely PKA-dependent effects above have minimal effects on systolic Ca transients. This can be understood by considering the large amplitude of SR Ca fluxes during E-C coupling compared with those during diastole. Moreover, CaMKII-dependent enhancement of SR Ca leak may resemble low caffeine concentrations (which sensitize RyR to Ca and increase diastolic SR Ca leak, but do not empty the SR).²⁸ Strikingly, this caffeine-induced leak only transiently alters Ca transient amplitude, but not in the steady state.²⁸ Rather, RyR sensitization only decreases the [Ca]_SRT at which the transient is generated (but see Venetucci et al²⁹). It has also been suggested that such enhanced RyR Ca sensitivity may function to limit SR Ca overload during higher inotropic states.³⁰

Implications
Increased SR Ca leak will tend to decrease [Ca]_SRT,³⁰ and it follows that reversal of such an increase would raise SR Ca content. Indeed, a massive increase in SR Ca content was observed on blockage of CaMKII activity in our system (Figures 3 and 4). As the CaMKII-dependent effects on the RyR have no effect on the size of the Ca transient, per se, it may be that these changes are meant to regulate the SR Ca load physiologically.³⁰

Reduction of the SR Ca load ultimately causes decreased SR Ca release and contractile dysfunction in myocardial cells in HF.² SR Ca leak is higher in intact HF myocytes,³ but the mechanism by which this occurs remains controversial.³¹ Our data suggest that RyR phosphorylation by CaMKII may cause increased diastolic Ca release in HF,¹⁰ although PKA-dependent RyR phosphorylation has also been implicated.⁸ In HF, CaMKII amount and activity is upregulated in the RyR complex (where associated phosphatases are also reduced), resulting in enhanced RyR phosphorylation.¹⁰ Furthermore, ß-AR-induced HF has been shown to be prevented by CaMKII inhibition.³² Enhanced diastolic SR Ca leak may contribute to arrhythmogenesis, especially when coupled with increased NCX and decreased inward rectifier current in HF, both of which increase the ease with which depolarization from rest may be induced.² Indeed, mutations that lead to increases in SR Ca leak have been correlated with the incidence of hypertrophic cardiomyopathy³³ and cardiac sudden death.^5–7 ß-AR tone associated with stress or strenuous exercise, when added to an already higher than normal level, may exacerbate arrhythmogenesis, attributable at least in part to effects on the RyR. The increased SR Ca leak may, therefore, have implications both in terms of the pathology of HF as well as physiological regulation of the Ca content of the SR.

	Acknowledgments

 
Sources of Funding

Supported by NIH grants T32 HL 07692 (to J.C.), HL71893 (to T.R.S.), and HL 64724 and HL 30077 (to D.M.B.).

Disclosures

None.

	Footnotes

 
Original received August 14, 2006; revision received December 28, 2006; accepted January 5, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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