Ca2+/Calmodulin Kinase II-Dependent Phosphorylation of Ryanodine Receptors Suppresses Ca2+ Sparks and Ca2+ Waves in Cardiac Myocytes
The multifunctional Ca2+/calmodulin-dependent protein kinase II δC (CaMKIIδC) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca2+ release channels in the heart. However, the functional role of CaMKII-dependent phosphorylation of RyR2 is highly controversial. To address this issue, we expressed wild-type, constitutively active, or dominant-negative CaMKIIδC via adenoviral gene transfer in cultured adult rat ventricular myocytes. CaMKII-mediated phosphorylation of RyR2 was reduced, enhanced, or unaltered by dominant-negative, constitutively active, or wild-type CaMKIIδC expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing β-galactosidase (β-gal), respectively. In parallel with the phospholamban phosphorylation, the decay kinetics of global Ca2+ transients was slowed, accelerated, or unchanged, whereas spontaneous Ca2+ spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKIIδC groups, respectively. When challenged by high extracellular Ca2+, both wild-type and constitutively active CaMKIIδC protected the cells from store overload-induced Ca2+ release, manifested by a ≈60% suppression of Ca2+ waves (at 2 to 20 mmol/L extracellular Ca2+) in spite of an elevated sarcoplasmic reticulum Ca2+ content, whereas dominant-negative CaMKIIδC promoted Ca2+ wave production (at 20 mmol/L Ca2+) with significantly depleted sarcoplasmic reticulum Ca2+. Taken together, our data support the notion that CaMKIIδC negatively regulates RyR2 activity and spontaneous sarcoplasmic reticulum Ca2+ release, thereby affording a negative feedback that stabilizes local and global Ca2+-induced Ca2+ release in the heart.
- Ca2+/calmodulin-dependent protein kinase II
- Ryanodine receptor
- Ca2+ sparks
- Ca2+ waves
- Ca2+-induced Ca2+ release
The type 2 ryanodine receptor (RyR2) Ca2+ release channels in the sarcoplasmic reticulum (SR) constitute a key component of excitation-contraction (EC) coupling in cardiac myocytes. Activated by the Ca2+-induced Ca2+ release (CICR) mechanism, they are responsible for generating 70% to 90% of the transient increase of intracellular Ca2+ that drives the heartbeat.1 Under Ca2+ overload conditions, RyRs mediate spontaneous Ca2+ waves2,3 or store overload-induced Ca2+ release (SOICR),4 which is thought to be arrhythmogenic for disturbance of cardiac electrical activity. Given its central role in cardiac EC coupling and Ca2+ signaling, RyR2 activity is under the exquisite control of an array of molecular partners found in the RyR2 macromolecular signaling complex, including protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), protein kinase C, and protein phosphatases 1, 2A, and 2B (calcineurin).5,6 Among these, CaMKII-dependent phosphorylation of RyR2 is of particular interest, because the waxing and waning of high microdomain Ca2+ accompanying the channel gating might activate and deactivate this kinase, affording a local feedback regulation to SR Ca2+ release.7
Early studies have suggested that RyR2 possesses a unique CaMKII phosphorylation site at Ser2809, which is also phosphorylated by PKA to a lesser extent.8 In contrast, more recent studies proposed that Ser2809 is a PKA-specific phosphorylation site in RyR2,6,9 whereas our own data support that Ser2809 is not an exclusive PKA-dependent phosphorylation site, but is also phosphorylated by CaMKII and other kinases.10 Furthermore, a 4:1 stoichiometry for CaMKII:PKA phosphorylation of RyR2 has been inferred from metabolic labeling.11 With the recent identification of Ser 2030 as a novel PKA-specific site,10 ≈8 CaMKII sites per RyR monomer are anticipated. To date, both Ser2809 (nonexclusive site) and Ser28159 have been shown to be phosphorylated by CaMKII.
The functional consequences of RyR2 phosphorylation in the healthy and diseased heart are highly controversial, and the conflicting literature falls into 4 categories: enhancement, inhibition, no function, or complicated regulation, with the enhancement view being prevalent.12 Using canine cardiac junctional SR vesicles or partially purified RyR2 fused into planar bilayers, it has been shown that CaMKII phosphorylation activates the channel by increasing open channel probability (Po).8,13 However, Hain et al14 found that activation of endogenous CaMKII led to RyR2 channel closure, whereas exogenous CaMKII activated the channel. Using photolytic Ca2+ steps, Valdivia et al15 showed that RyR2 response to CaMKII- or PKA-dependent phosphorylation is rather complex, characterized by a higher peak Po, a markedly accelerated “adaptation,” and a reduced steady-state Po. The results from studies using swine and rabbit SR vesicles suggested that the intracellular signaling pathway leading to CaMKII activation reduced Ca2+ efflux from the SR by inhibiting RyR2 channel activity.16 By inclusion of constitutive active CaMKII in the patch pipette under whole-cell patch-clamp conditions, Anderson and colleagues demonstrated that CaMKII reduced depolarization-triggered Ca2+ release from the SR and elevated SR Ca2+ content.17 In a series of studies, Marks and colleagues advocated the idea that PKA phosphorylation at Ser2809 displaces calstabin (FKBP 12/12.6) from the channel complex, that CaMKII phosphorylation at Ser2815 does not, and that these 2 distinct mechanisms both result in an increase in Ca2+ sensitivity, causing Ca2+ instability that might contribute to arrhythmias and contractile dysfunction in heart failure.6,9,18 In contrast, Valdivia and colleagues demonstrated that, in the canine heart failure model and human failing hearts, there were no detectable changes in RyR phosphorylation, calstabin dissociation, or single-channel properties.19 Measurements of single-channel activity of mutant RyR2 have also challenged the view that phosphorylation of Ser2809 alters the RyR2 channel function.20,21 On the other hand, overexpression of CaMKIIδC in transgenic mice, the predominant cytosolic isoform in the heart,22 results in dilated cardiomyopathy and heart failure,23 whereas myocytes isolated from these hearts exhibit hyperactive Ca2+ sparks and a “leaky” SR.24 CaMKII activation Ca2+ sparks was further demonstrated in permeabilized cardiac myocytes isolated from phospholamban (PLB) knockout mice using pharmacological approaches.25 In the present study, we used acute adenoviral gene transfer in intact cultured adult cardiac myocytes from rat, which complements and extends previous studies. Specifically, we implemented bidirectional manipulation of CaMKII activity and exploited Ca2+ sparks, Ca2+ transients, and Ca2+ waves as the physiological readouts in an attempt to further delineate the possible role of CaMKIIδC phosphorylation in regulating RyR function in cardiac myocytes.
Materials and Methods
Construction of Viral Vectors
Hemagglutinin (HA)-tagged constitutively active CaMKIIδC (CA CaMKII) was generated by replacing the residue Thr287 with aspartic acid (T287D) using the transformer site directed mutagenesis kit (Clontech). Dominant-negative CaMKIIδC (DN CaMKII) was generated by replacing the residue Lys43 with alanine (K43A).26 The generation and amplification of adenoviruses harboring the target gene were performed in HEK293 cells.
Cell Culture and Adenoviral Infection
Single cardiac myocytes were isolated from the hearts of 2- to 3-month-old Sprague-Dawley rats using a standard enzymatic technique, then cultured and infected with adenoviral vectors at a multiplicity of infection (moi) of 100.27 Myocytes were plated at a density of 0.5 to 1′104/cm2 on coverslips or in dishes precoated with 10 μg/mL laminin. The culture medium was medium 199 (Sigma) in the presence of (in mmol/L) creatine 5, l-carnitine 2, taurine 5, insulin/transferrin/selenium-X 0.1%, penicillin and streptomycin 1%, and HEPES 25 (pH=7.4 at 37°C). Experiments were performed in cells cultured for 24 hours following infection. Procedures were performed according to Guiding Principles in the Care and Use of the Animals approved by the Council of the American Physiological Society.
Western Blotting and Immunostaining
To quantify the expression of wild-type (WT) and mutant CaMKIIδC, cell lysates (30≈50 μg protein) were loaded in a Ca2+-free loading buffer containing 20 mmol/L EDTA and immunoblotted using anti-HA antibody (1:5000, Berkeley Antibody Co) and horseradish peroxidase-conjugated secondary antibody (Bio-Rad). Other antibodies include the site-specific antibody reacting with PLB phosphorylated at Thr17 (1:10 000, Badrilla, Leeds, UK), anti-total PLB antibody (1:5000, Badrilla), anti-RyR2 antibody (1:5000, ABR), and site-specific antibodies recognizing the phosphorylated RyR2 at Ser2809 (1:5000, Badrilla, UK) and at Ser2030.10
For immunostaining, cells were incubated with anti-HA antibody (1:1000) at 4°C overnight after fixation and blocking and then stained with Cy5-conjugated secondary antibody (1:500, Jackson ImmunoResearch). Confocal images were obtained using a Zeiss LSM 510 confocal microscope (Carl Zeiss Inc, Thornewood, NY) with 633-laser line to excite the Cy5 fluorophore.
CaMKII Activity Assay
To measure CaMKII activity, cell lysate (200 μg of protein) was first immunoprecipitated with anti-CaMKII antibody (1:100) in a medium containing (in mmol/L) Tris-HCl 20, NaCl 150, Na2-EDTA 1, EGTA 1, Triton 1%, sodium pyrophosphate 2.5, β-glycerophosphate 1, Na3VO4 1, leupeptin 1 μg/mL (pH=7.4). The precipitated proteins were then incubated with a specific peptide substrate (KKALRRQETVDAL) to evaluate the kinase activity according to the recommendations of the manufacturer (Upstate Biotechnology Inc), as previously described.27
Myocytes were loaded with the Ca2+ indicator fluo-4 acetoxymethyl ester (fluo-4 AM) (20 μmol/L, 30 minutes) (Invitrogen) and Ca2+ sparks, Ca2+ transients, and Ca2+ waves were observed with Zeiss confocal microscope.3 To measure Ca2+ transients, myocytes were paced at 1.0 Hz with perfusion solution containing (in mmol/L) NaCl 137, KCl 4.9, CaCl2 1, MgSO4 1.2, NaH2PO4 1.2, glucose 15, and HEPES 20 (pH=7.4). For ratiometric measurement of cytosolic free Ca2+ concentration, cells were loaded with 25 μmol/L indo-1/AM for 30 minutes (Invitrogen). To measure the SR Ca2+ content with either indo-1 or fluo-4 AM loading, 20 mmol/L caffeine was rapidly applied to the cell locally. For spontaneous contractile wave measurement, the perfusion solution contained (in mmol/L) NaCl 138.2, KCl 4.6, MgCl2 1.2, glucose 15, and HEPES 20 (pH=7.4) at various Ca2+ concentrations (1, 2, 5, 10, or 20), without readjusting the osmolarity and ionic strength. All measurements were performed at room temperature (22°C to 23°C). Image processing, data analysis, and presentation were performed using IDL software (version 6.1, Research Systems Inc Co).
Back-Phosphorylation of RyR2
Homogenates (500 μg) were suspended in 0.5 mL of buffer (50 mmol/L Tris-HCl, pH 7.4), 0.9% NaCl, 0.5 mmol/L NaF, 0.5 mmol/L Na3VO4, 0.25% Triton X-100, and protease inhibitors and immunoprecipitated with anti-RyR2 antibody (1:300). The immunoprecipitated RyR2 was then incubated for 45 minutes at room temperature in 10 μL of preactivated 1× CaMKII reaction buffer (50 U; BioLabs), MgATP (100 μmol/L), and 10% [32P]ATP for back-phosphorylation assay.9 The CaMKII inhibitor KN-93 (10 μmol/L) was added to the reaction solution as negative control. After SDS-PAGE, the level of RyR2 protein and the associated radioactivity were determined using an anti-RyR2 antibody (1:5000) and autoradiography, respectively. The densities of total RyR2 and autoradioactivity were quantified using Multi-Analyst (Bio-Rad).
Data were reported as mean±SEM. Student’s t test, paired t test, or Kruskal-Wallis test was applied, when appropriate, to determine statistical significance of the differences. A probability value of less than 0.05 was considered statistically significant.
Manipulation of CaMKIIδC Activity in Cardiac Myocytes
To up- and downregulate CaMKIIδC activity, ventricular myocytes were infected with adenoviral vectors carrying the target gene for WT and mutant CaMKIIδC at moi of 100 and cultured for 24 hours. Confocal immunocytochemical imaging (Figure 1A) visualized that the WT and mutant CaMKIIδC were similarly localized to the cytoplasm and the surface membrane including the transverse tubules, consistent with previous reports.28,29 Using Western blotting with an antibody recognizing the HA epitope, we found comparable expression for the mutants (CA and DN CaMKIIδC) and higher expression for CaMKIIδC (Figure 1B). Direct measurement of the Ca2+-independent kinase activity was assessed in preactivated form in resting cells at 1 mmol/L extracellular Ca2+. In the DN, WT, and CA CaMKIIδC groups, the Ca2+-dependent kinase activity relative to the β-gal group was 0.19±0.08 (n=3, P<0.01), 1.8±0.3 (n=3, P<0.05), and 4.0±0.4 (n=4, P<0.01), respectively. PLB, a key physiological regulator of SR Ca2+-ATPase (SERCA), bears a PKA-specific phosphorylation site Ser16 and a CaMKII-specific phosphorylation site Thr17. Phosphorylation of either is sufficient to remove PLB inhibition of SERCA, leading to accelerated SR Ca2+ recycling and hastened cardiac relaxation.30 Compared with cells infected with adeno-β-gal at the same moi, the expression of CA and DN CaMKIIδC resulted in a 0.6-fold increase and 0.3-fold decrease, respectively, of PLB Thr17 phosphorylation, whereas WT CaMKIIδC expression did not affect the PLB phosphorylation (Figure 1C and 1D). Notably, total PLB level was unchanged among the groups, suggesting no compensatory change in PLB expression.
Effect of CaMKIIδC on the Decay of Ca2+ Transients
Using the aforementioned cell models, we examined the overall effects of CaMKIIδC activation on intracellular Ca2+ transients and contraction. Figure 2A shows typical examples of fluo-4 line-scan images and their corresponding time courses from cells expressing WT, CA, and DN CaMKIIδC. CA CaMKIIδC expression induced a profound positive relaxant effect at the single-cell level (Figure 2A and 2D). The T50 was reduced by ≈15% compared with WT CaMKIIδC or β-gal. Conversely, DN CaMKIIδC expression prolonged the T50 by ≈15% (Figure 2A and 2D). Enhanced CaMKII activity accelerates cardiac relaxation and reduced CaMKII activity slows the decay of Ca2+ transients. These changes occurred in parallel with the changes in the status of PLB Thr17 phosphorylation (Figure 1C and 1D), in agreement with previous reports.31,32
Interestingly, we found no significant effects of CaMKIIδC (WT, CA, or DN) on the amplitudes of the Ca2+ transients (Figure 2B) or contraction (Figure 2C). Because the CaMKII-mediated enhancement of L-type Ca2+ currents (Figure I in the online data supplement, available at http://circres.ahajournals.org)28,32 and SR Ca2+ recycling30,31,33 (Figure 2D) would predict a positive (or negative) inotropic effect in response to enhanced (or reduced) CaMKII activity, this result provided the first clue that CaMKII may also negatively regulate some key component(s) of EC coupling, giving rise to a net null effect on cardiac inotropy.
Phosphorylation of RyR2 by CaMKIIδC
We measured the status of RyR2 phosphorylation in response to the expression of WT and mutant CaMKIIδC by using the back-phosphorylation assay. Figure 3A shows a representative autoradiograph of [γ-32P]ATP incorporation into RyR2 after CaMKII phosphorylation in vitro and Western blotting analysis of the corresponding RyR2 protein. As a negative control, we showed that inhibition of CaMKII by KN-93 completely blocked back-phosphorylation of RyR2 (Figure 3A and 3B). We found no difference in the 32P-RyR/total RyR densitometric ratio between β-gal control (0.653±0.046, n=6) and WT CaMKIIδC (0.630±0.078, n=6) but a significant decrease in the CA CaMKIIδC group (0.159±0.017, n=6, P<0.001) and an increase in the DN CaMKIIδC group (0.857±0.054, n=6, P<0.05), respectively. Hence, RyR2 was hyperphosphorylated in the CA CaMKIIδC group and hypophosphorylated in the DN CaMKIIδC group compared with WT CaMKIIδC and β-gal.
To test the possibility that RyR2 phosphorylation by PKA might also be altered via crosstalk between CaMKII and PKA pathways, we used a site-specific antibody reacting with phospho-Ser2030, a PKA-specific site in cardiac myocytes.10 Western blotting clearly showed that there is no detectable signal in all 4 groups (Figure 3B, bottom). In the positive control group, the same antibody detected PKA phosphorylation of purified RyR2 expressed in HEK 293 cells.
Using a phospho-Ser2809–specific antibody, we found significant basal phosphorylation of RyR2 in the β-gal group, whereas no significant changes were found among the WT and DN CaMKIIδC and β-gal groups (Figure 3B), except for a slight hyperphosphorylation in the CA CaMKIIδC group (2.18±0.16 versus 1.55±0.09 of β-gal control; n=5 for both groups, P<0.05). This observation supports the notion that Ser2809 retains some sensitivity to CaMKII.21 Unlike the CaMKIIδC transgenic animal model,24 acute CaMKIIδC manipulation did not alter the RyR protein level (Figure 3A and 3B).
CaMKIIδC Modulation of Ca2+ Sparks
Characteristics of Ca2+ sparks34 are informative in characterizing CICR sensitivity, RyR gating, and SR Ca2+ leakage in intact cells. The frequency of spontaneous Ca2+ sparks was diminished from 2.75±0.30 (n=35 cells from 6 hearts) in the β-gal group to 1.64±0.17 Hz/100 μm (n=50, P<0.05) in CA CaMKIIδC group (Figure 4B). In contrast, DN CaMKIIδC induced hyperactive Ca2+ sparks, increasing the spark frequency to 4.21±0.45 Hz/100 μm (n=56, P<0.01 versus β-gal) (Figure 4B). In WT CaMKIIδC, there was a trend of decreasing spark frequency (2.33±0.28, n=60), which did not reach statistical significance. Properties of individual Ca2+ sparks were largely unchanged among groups, with 2 exceptions (Figure 4C and supplemental Figures II and III). The spark rising time, which reflects the termination kinetics of the release channel opening, was significantly prolonged in the DN CaMKIIδC compared with the WT group (21.12±0.44 ms, n=611, versus 19.44±0.60 ms, n=274, P<0.01) (supplemental Figure II). DN CaMKIIδC also slightly reduced spark amplitude (Figure 4C) despite the longer rising time and unchanged SR Ca2+ content (Figure 5C), suggesting smaller Ca2+ release fluxes in Ca2+ sparks on CaMKII inhibition. Taken together, CaMKII inhibition and RyR hypophosphorylation cause a “leaky” SR, whereas CaMKII activation and RyR hyperphosphorylation suppress spontaneous SR Ca2+ release.
Acute manipulation of CaMKII activity in cultured cells did not affect the overall Ca2+ homeostasis in quiescent cells at 1 mmol/L extracellular Ca2+. Ratiometric Ca2+ measurements with indo-1 as the Ca2+ indicator revealed no significant change in either the resting Ca2+ level (r410/490) (Figure 5B) or the caffeine-liable SR Ca2+ content (Figure 5C) among the groups (see below for results at 20 mmol/L). Moreover, the half-decay time of caffeine-induced Ca2+ transients, an index of Ca2+ clearance by Na+/Ca2+ exchange, showed no detectable difference either (Figure 5D).
CaMKIIδC Suppresses Ca2+ Waves
The above results suggest that CaMKII stabilizes, rather than destabilizes, Ca2+ signaling. If such stabilization were the case, it might be expected that CaMKII activation would reduce the occurrence of spontaneous Ca2+ waves or SOICR.4,35,36 To test this possibility, we challenged the cells with increasing concentrations of extracellular Ca2+ to create Ca2+-overload conditions. Resting cells expressing β-gal exhibited a low frequency of Ca2+ waves at 1 mmol/L Ca2+ (0.19±0.07 min−1, n=42 cells from 3 hearts). By raising extracellular Ca2+ concentrations gradually to 2, 5, 10, and 20 mmol/L, the wave frequency was increased progressively and reversibly, up to 11.8±1.1 min−1 (Figure 6A and 6B). CA CaMKIIδC markedly reduced the genesis of Ca2+ waves at all Ca2+ concentrations higher than 2 mmol/L (Figure 6B). Interestingly, in the WT CaMKIIδC group, the Ca2+ wave frequency was suppressed to ≈30% of that in β-gal, as if WT CaMKIIδC became activated at high extracellular Ca2+. DN CaMKIIδC exerted an opposite effect on Ca2+ stability, evidenced by a 37% increase in Ca2+ wave frequency at 20 mmol/L Ca2+ (Figure 6A and 6B). Nevertheless, the propagation velocity and amplitude of Ca2+ waves were almost identical in all groups (supplemental Figure IV).
Although no significant changes in the SR Ca2+ content were observed at 1 mmol/L Ca2+ (Figure 5C), high Ca2+ challenge did bring out the differences: increasing the SR content by WT and CA CaMKIIδC and depleting it by DN CaMKIIδC, as measured at 20 mmol/L Ca2+ (Figure 6C). The reciprocal regulation of Ca2+ wave frequency and SR Ca2+ content suggests that CaMKII phosphorylation of RyR2 raises the threshold of SR luminal Ca2+ concentration at which SOICR occurs.
In the present study, we have provided experimental evidence pointing to the unexpected finding that phosphorylation of RyR2 by CaMKII negatively regulates Ca2+ spark and Ca2+ wave activities, thus serving as a stabilizing factor for SR Ca2+ release in intact cardiac myocytes. This result is in sharp contrast to the prevalent view that RyR2 phosphorylation by CaMKII enhances RyR2 Ca2+ sensitivity and causes leaky SR and Ca2+ instability.24,33
Models of Altered CaMKII Activity
The controversy of CaMKII modulation on RyR function may arise in part from methodological limitations. Results from studies using cell-free system may not be directly relevant to intact cells, and experiments using intracellular CaMKII dialysis may not be readily reproducible because of technical difficulties. The transgenic animal approach also has its own limitations as a result of plasticity, adaptation, and maladaptation typical of any biological systems. Indeed, transgenic CaMKIIδC overexpression is accompanied by a substantial remodeling of the Ca2+ signaling system, characterized by remarkable downregulation of RyR2, PLB, and SERCA and substantial upregulation of Na+/Ca2+ exchange.24 In this study, we have established 3 cell models expressing WT, CA, and DN CaMKIIδC, respectively. These ex vivo cellular models allow for bidirectional genetic manipulation of CaMKII activity, with minimal compensatory changes in the other components of the Ca2+ signaling system. Similar efforts have also been made previously to express WT CaMKIIδC in cultured rabbit ventricular myocytes,33 although an opposite conclusion on CaMKII modulation of RyR function has been drawn (see below).
Validating criteria of the current models include CaMKII expression and localization, direct measurement of the kinase activity, and CaMKII phosphorylation of PLB and RyR2. Physiological readouts, including the decay time of action potential-elicited Ca2+ transients (Figure 2A and 2D) demonstrate either an enhanced or depressed SR Ca2+ recycling on CaMKII activation or inhibition, as expected. As an advantage of acute genetic manipulation, we detected little unwanted alterations in the Ca2+ signaling system: the expression levels of PLB and RyR2 remain unchanged and the Na+/Ca2+ exchange activity indexed by the decay kinetics of caffeine-induced Ca2+ transients remains intact in all groups. A combination of multiple models further permitted us to detect reciprocal changes in response to up- or down-manipulation of CaMKII activity. Hence, the present cell models should be useful for investigation of direct CaMKII actions with minimal secondary long-term effects caused by remodeling of the Ca2+ signaling system.
Negative Regulation of RyR2 Activity by CaMKIIδC
One of the main findings of the present study is that enhanced CaMKII activation reduces the rate of occurrence of spontaneous Ca2+ sparks and Ca2+ waves, whereas inhibition of CaMKII activity exerts an opposite effect. Examination of spark properties has led to the insight that CaMKII modulation of sparks is predominantly via the frequency-dependent modulation mechanism. At the cellular level, the CaMKII-mediated inhibitory effect on SR Ca2+ release should negate the CaMKII effects on enhancing L-type Ca2+ channel current28,32 and SR Ca2+ recycling,30,31,33 explaining the lack of change in the peak of Ca2+ transients elicited by action potentials.
In cultured rabbit ventricular myocytes expressing WT CaMKIIδC, however, Kohlhaas et al33 detected a large reduction of the SR Ca2+ content and, after “normalizing” the spark frequency by the corresponding depleted SR Ca2+ content, an 88% increase in spark production, reaching a conclusion that is opposite from ours. The discrepancies between the rat and rabbit results may reflect genuine species-dependent differences. To this end, it is well known that cardiac Ca2+ transients and contraction display postrest potentiation in the rat and postrest depression in the rabbit. Nevertheless, the normalization procedure used needs to be justified because our previous studies in the rat have shown that unloading the SR Ca2+ below its physiological level does not affect Ca2+ spark production.37
The most compelling evidence for CaMKII-mediated negative regulation of spontaneous SR Ca2+ release came from investigating global Ca2+ release. We found that CaMKII activation protects myocytes from arrhythmogenic Ca2+ waves under Ca2+-overload conditions, and the opposite is true for CaMKII inhibition. The protective effect is so powerful that the Ca2+ wave frequency is reduced by more than 50% over a wide range of extracellular Ca2+ (2 to 20 mmol/L) in cells expressing CA or WT CaMKII. Because the wave frequency reduction occurs in spite of an elevated SR Ca2+ load, CaMKII activation apparently increases the threshold of SOICR. Thus, based on both Ca2+ spark and Ca2+ wave measurements, we conclude that CaMKII negatively regulates the SR Ca2+ release. This conclusion is in general agreement with the observation that intracellular dialysis of constitutively active CaMKII suppresses SR Ca2+ release17 and that protein phosphatases PP1 and PP2A enhance spontaneous Ca2+ sparks in chemically permeabilized myocytes.38
How can the vast discrepancies across different studies (see the introduction) reconciled? One possibility is that CaMKII phosphorylation and functional modulation of RyR2 might be more complex than had been thought previously. If there were 8 CaMKII phosphorylation sites per monomer as suggested by metabolic labeling,11 there would be 9 (0 to 8) phosphorylation levels and 256 combinations of phosphorylation patterns. The possibilities are expanded to 33 (0 to 32) phosphorylation levels and ≈1010 patterns of phosphorylation for a tetrameric RyR and are increased astronomically for some 50 to 200 RyRs assembled in a Ca2+ release unit.39 Added to the complexity, the functionality of CaMKII phosphorylation might be context sensitive, depending on PKA phosphorylation of the channel, binding of calstabin and other regulatory proteins, and the species of animals. The distinct phenotypes in transgenic animal models and ex vivo cell models reported here testify that it is also necessary to isolate direct CaMKII effects from those that are convoluted with changes of the Ca2+ signaling system. In perspective, there is still much to be learned about the intricacy and exquisiteness of CaMKII regulation of cardiac RyR2 function.
Possible Physiological Significance
The CICR, with its inherent positive-feedback nature, provides the speed, the sensitivity, and the high-gain amplification that are much needed for cardiac Ca2+ signaling. Equally important, it is necessary to enforce signaling stability for local Ca2+ release events (spark termination, refractory to reactivation) and global Ca2+ transients (suppression of unwanted Ca2+ waves). In this scenario, our data indicate that CaMKII affords a molecular mechanism that negates the positive feedback of the CICR. Activated by high local Ca2+, CaMKII phosphorylates RyR2 and consequentially reduces the Ca2+ sensitivity of the channel and increases the threshold for SOICR, imposing a frequency-dependent modulation of local and global spontaneous SR Ca2+ release.
The above finding shows that CaMKII-dependent RyR2 phosphorylation might not be the underlying cause for the dysregulation and instability of Ca2+ signaling common to many types of heart failure. Nevertheless, caution should be excised when extrapolating the cell results to situations in failing hearts, where extensive functional and structural remodeling may undermine Ca2+ stability by mechanisms other than RyR2 phosphorylation.40,41 It is also noteworthy that CaMKII is multifunctional, and its cellular phenotypes would be context dependent. A comprehensive understanding of CaMKII in cardiac Ca2+ physiology and pathophysiology awaits future investigation using diverse cellular and animal models.
We thank Bruce Ziman and Dr Harold A. Spurgeon for technical support.
Sources of Funding
This work was supported by the Intramural Research Programs of the NIH, National Institute on Aging (to R.-P.X., H.C., and E.G.L.); Major State Basic Research Development Program and Natural Science Foundation of China (to R.-P.X. and H.C.); and research grants from the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Alberta, Northwest Territories and Nunavut (to S.R.W.C.).
Original received June 29, 2006; resubmission received November 21, 2006; revised resubmission received December 22, 2006; accepted January 4, 2007.
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