Published online before print January 18, 2007, doi: 10.1161/01.RES.0000258172.74570.e6
© 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
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
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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
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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 (Po)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 colleagues8,9 find that PKA greatly increases RyR Po, Valdivia et al17 found reduced steady-state Po 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.
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Materials and Methods |
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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.
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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.
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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).
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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).
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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.
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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, Ki=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.
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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%.
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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 (ICa), 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 (
JSRCaRel/ICa; Figure 6C). We therefore conclude that the effects on SR Ca release (JSRCaRel) predominate over the direct effect of increased ICa to the transient. That is, both JSRCaRel and ICa 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 JSRCaRel (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 JSRCaRel 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.
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Discussion |
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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 ICa, and therefore the SL-dependent influx and efflux, the enhanced SR Ca pump function (and ICa) 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 ICa and SR Ca load may differ. Thus, enhanced [Ca]SRT (and ICa) 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.
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Acknowledgments |
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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.
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Footnotes |
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Original received August 14, 2006; revision received December 28, 2006; accepted January 5, 2007.
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References |
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1. Bers DM. Excitation-Contraction Coupling and Cardiac Contractile Force. 2nd ed. Dordrecht: Kluwer Academic Publishers; 2001.
2. Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium-calcium exchange, inward rectifier potassium current, and residual beta-adrenergic responsiveness. Circ Res. 2001; 88: 1159–1167.
3. Shannon TR, Pogwizd SM, Bers DM. Elevated sarcoplasmic reticulum Ca2+ leak in intact ventricular myocytes from rabbits in heart failure. Circ Res. 2003; 93: 592–594.
4. Nuss HB, Johns DC, Kaab S, Tomaselli GF, Kass D, Lawrence JH, Marban E. Reversal of potassium channel deficiency in cells from failing hearts by adenoviral gene transfer: a prototype for gene therapy for disorders of cardiac excitability and contractility. Gene Ther. 1996; 3: 900–912.[Medline] [Order article via Infotrieve]
5. Bers DM, Pogwizd SM, Schlotthauer K. Upregulated Na/Ca exchange is involved in both contractile dysfunction and arrhythmogenesis in heart failure. Basic Res Cardiol. 2002; 97 (suppl 1): I36–I42.[Medline] [Order article via Infotrieve]
6. Lehnart SE, Wehrens XH, Marks AR. Defective ryanodine receptor interdomain interactions may contribute to intracellular Ca2+ leak: a novel therapeutic target in heart failure. Circulation. 2005; 111: 3342–3346.
7. Wehrens XH, Lehnart SE, Huang F, Vest JA, Reiken SR, Mohler PJ, Sun J, Guatimosim S, Song LS, Rosemblit N, D’Armiento JM, Napolitano C, Memmi M, Priori SG, Lederer WJ, Marks AR. FKBP12.6 deficiency and defective calcium release channel (ryanodine receptor) function linked to exercise-induced sudden cardiac death. Cell. 2003; 113: 829–840.[CrossRef][Medline] [Order article via Infotrieve]
8. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101: 365–376.[CrossRef][Medline] [Order article via Infotrieve]
9. Wehrens XH, Lehnart SE, Reiken SR, Marks AR. Ca2+/calmodulin-dependent protein kinase II phosphorylation regulates the cardiac ryanodine receptor. Circ Res. 2004; 94: e61–e70.
10. Ai X, Curran JW, Shannon TR, Bers DM, Pogwizd SM. Ca2+/calmodulin-dependent protein kinase modulates cardiac ryanodine receptor phosphorylation and sarcoplasmic reticulum Ca2+ leak in heart failure. Circ Res. 2005; 97: 1314–1322.
11. Hain J, Onoue H, Mayrleitner M, Fleischer S, Schindler H. Phosphorylation modulates the function of the calcium release channel of sarcoplasmic reticulum from cardiac muscle. J Biol Chem. 1995; 270: 2074–2081.
12. Witcher DR, Kovacs RJ, Schulman H, Cefali DC, Jones LR. Unique phosphorylation site on the cardiac ryanodine receptor regulates calcium channel activity. J Biol Chem. 1991; 266: 11144–11152.
13. Currie S, Loughrey CM, Craig MA, Smith GL. Calcium/calmodulin-dependent protein kinase IIdelta associates with the ryanodine receptor complex and regulates channel function in rabbit heart. Biochem J. 2004; 377: 357–366.[CrossRef][Medline] [Order article via Infotrieve]
14. Guo T, Zhang T, Mestril R, Bers DM. Ca2+/calmodulin-dependent protein kinase II phosphorylation of ryanodine receptor does affect calcium sparks in mouse ventricular myocytes. Circ Res. 2006; 99: 398–406.
15. Kohlhaas M, Zhang T, Seidler T, Zibrova D, Dybkova N, Steen A, Wagner S, Chen L, Brown JH, Bers DM, Maier LS. Increased sarcoplasmic reticulum calcium leak but unaltered contractility by acute CaMKII overexpression in isolated rabbit cardiac myocytes. Circ Res. 2006; 98: 235–244.
16. Maier LS, Zhang T, Chen L, DeSantiago J, Brown JH, Bers DM. Transgenic CaMKIIdeltaC overexpression uniquely alters cardiac myocyte Ca2+ handling: reduced SR Ca2+ load and activated SR Ca2+ release. Circ Res. 2003; 92: 904–911.
17. Valdivia HH, Kaplan JH, Ellis-Davies GC, Lederer WJ. Rapid adaptation of cardiac ryanodine receptors: modulation by Mg2+ and phosphorylation. Science. 1995; 267: 1997–2000.
18. Stange M, Xu L, Balshaw D, Yamaguchi N, Meissner G. Characterization of recombinant skeletal muscle (Ser-2843) and cardiac muscle (Ser-2809) ryanodine receptor phosphorylation mutants. J Biol Chem. 2003; 278: 51693–51702.
19. Li Y, Kranias EG, Mignery GA, Bers DM. Protein kinase A phosphorylation of the ryanodine receptor does not affect calcium sparks in mouse ventricular myocytes. Circ Res. 2002; 90: 309–316.
20. Shannon TR, Ginsburg KS, Bers DM. Quantitative assessment of the SR Ca2+ leak-load relationship. Circ Res. 2002; 91: 594–600.
21. Li L, Satoh H, Ginsburg KS, Bers DM. The effect of Ca2+-calmodulin-dependent protein kinase II on cardiac excitation-contraction coupling in ferret ventricular myocytes. J Physiol. 1997; 501: 17–31.
22. Bassani JW, Bassani RA, Bers DM. Relaxation in rabbit and rat cardiac cells: species-dependent differences in cellular mechanisms. J Physiol. 1994; 476: 279–293.
23. Ginsburg KS, Bers DM. Modulation of excitation-contraction coupling by isoproterenol in cardiomyocytes with controlled SR Ca2+ load and Ca2+ current trigger. J Physiol. 2004; 556: 463–480.
24. Wang W, Zhu W, Wang S, Yang D, Crow MT, Xiao RP, Cheng H. Sustained beta1-adrenergic stimulation modulates cardiac contractility by Ca2+/calmodulin kinase signaling pathway. Circ Res. 2004; 95: 798–806.
25. Zhu WZ, Wang SQ, Chakir K, Yang D, Zhang T, Brown JH, Devic E, Kobilka BK, Cheng H, Xiao RP. Linkage of beta1-adrenergic stimulation to apoptotic heart cell death through protein kinase A-independent activation of Ca2+/calmodulin kinase II. J Clin Invest. 2003; 111: 617–625.[CrossRef][Medline] [Order article via Infotrieve]
26. Ginsburg KS, Bers DM. Isoproterenol does not enhance Ca-dependent Na/Ca exchange current in intact rabbit ventricular myocytes. J Mol Cell Cardiol. 2005; 39: 972–981.[CrossRef][Medline] [Order article via Infotrieve]
27. Shannon TR, Ginsburg KS, Bers DM. Potentiation of fractional sarcoplasmic reticulum calcium release by total and free intra-sarcoplasmic reticulum calcium concentration. Biophys J. 2000; 78: 334–343.[Medline] [Order article via Infotrieve]
28. Trafford AW, Sibbring GC, Diaz ME, Eisner DA. The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes. Cell Calcium. 2000; 28: 269–276.[CrossRef][Medline] [Order article via Infotrieve]
29. Venetucci LA, Trafford AW, Diaz ME, O’Neill SC, Eisner DA. Reducing ryanodine receptor open probability as a means to abolish spontaneous Ca2+ release and increase Ca2+ transient amplitude in adult ventricular myocytes. Circ Res. 2006; 98: 1299–1305.
30. Shannon TR, Wang F, Bers DM. Regulation of cardiac sarcoplasmic reticulum Ca release by luminal [Ca] and altered gating assessed with a mathematical model. Biophys J. 2005; 89: 4096–4110.[CrossRef][Medline] [Order article via Infotrieve]
31. Bers DM, Eisner DA, Valdivia HH. Sarcoplasmic reticulum Ca2+ and heart failure: roles of diastolic leak and Ca2+ transport. Circ Res. 2003; 93: 487–490.
32. Zhang R, Khoo MS, Wu Y, Yang Y, Grueter CE, Ni G, Price EE Jr, Thiel W, Guatimosim S, Song LS, Madu EC, Shah AN, Vishnivetskaya TA, Atkinson JB, Gurevich VV, Salama G, Lederer WJ, Colbran RJ, Anderson ME. Calmodulin kinase II inhibition protects against structural heart disease. Nat Med. 2005; 11: 409–417.[CrossRef][Medline] [Order article via Infotrieve]
33. Van Driest SL, Ommen SR, Tajik AJ, Gersh BJ, Ackerman MJ. Yield of genetic testing in hypertrophic cardiomyopathy. Mayo Clin Proc. 2005; 80: 739–744.
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