This Article Abstract Full Text (PDF) Data Supplement All Versions of this Article: 100/3/399    most recent 01.RES.0000258022.13090.55v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yang, D. Articles by Cheng, H. Search for Related Content PubMed PubMed Citation Articles by Yang, D. Articles by Cheng, H. Related Collections Calcium cycling/excitation-contraction coupling Cell signalling/signal transduction Imaging Related Article

(Circulation Research. 2007;100:399.)
© 2007 American Heart Association, Inc.

Cellular Biology

Ca²⁺/Calmodulin Kinase II-Dependent Phosphorylation of Ryanodine Receptors Suppresses Ca²⁺ Sparks and Ca²⁺ Waves in Cardiac Myocytes

Dongmei Yang, Wei-Zhong Zhu, Bailong Xiao, Didier X.P. Brochet, S.R. Wayne Chen, Edward G. Lakatta, Rui-Ping Xiao, Heping Cheng

From the Laboratory of Cardiovascular Science (D.Y., W.-Z.Z., D.X.P.B., E.G.L., R.-P.X., H.C.), National Institute on Aging, NIH, Baltimore, Md; and Cardiovascular Research Group (B.X., S.R.W.C.), Department of Physiology and Biophysics, University of Calgary, Canada; and Institute of Molecular Medicine (R.-P.X. and H.C.), Peking University, Beijing, China. Present address for D.X.P.B.: Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, Md. Present address for R.-P.X. and H.C.: Institute of Molecular Medicine, Peking University, Beijing, China.

Correspondence to Heping Cheng, PhD, Institute of Molecular Medicine, Peking University, Beijing 100871, China. E-mail chengp{at}pku.edu.cn; and Wei-Zhong Zhu, MD, PhD, Laboratory of Cardiovascular Science, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224. E-mail zhuw@grc.nia.nih.gov

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The multifunctional Ca²⁺/calmodulin-dependent protein kinase II _C (CaMKII_C) 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 CaMKII_C 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 CaMKII_C expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing ß-galactosidase (ß-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 CaMKII_C groups, respectively. When challenged by high extracellular Ca²⁺, both wild-type and constitutively active CaMKII_C 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 CaMKII_C promoted Ca²⁺ wave production (at 20 mmol/L Ca²⁺) with significantly depleted sarcoplasmic reticulum Ca²⁺. Taken together, our data support the notion that CaMKII_C 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.

Key Words: Ca²⁺/calmodulin-dependent protein kinase II • Ryanodine receptor • Ca²⁺ sparks • Ca²⁺ waves • Ca²⁺-induced Ca²⁺ release

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
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⁺ waves^2,3 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 (P_o).^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 P_o, a markedly accelerated "adaptation," and a reduced steady-state P_o. 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 CaMKII_C 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 CaMKII_C phosphorylation in regulating RyR function in cardiac myocytes.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Construction of Viral Vectors
Hemagglutinin (HA)-tagged constitutively active CaMKII_C (CA CaMKII) was generated by replacing the residue Thr287 with aspartic acid (T287D) using the transformer site directed mutagenesis kit (Clontech). Dominant-negative CaMKII_C (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 CaMKII_C, cell lysates (3050 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, ß-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

Ca²⁺ Measurements
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 1x 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).

Statistics
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.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Manipulation of CaMKII_C Activity in Cardiac Myocytes
To up- and downregulate CaMKII_C activity, ventricular myocytes were infected with adenoviral vectors carrying the target gene for WT and mutant CaMKII_C at moi of 100 and cultured for 24 hours. Confocal immunocytochemical imaging (Figure 1A) visualized that the WT and mutant CaMKII_C 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 CaMKII_C) and higher expression for CaMKII_C (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 CaMKII_C groups, the Ca²⁺-dependent kinase activity relative to the ß-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-ß-gal at the same moi, the expression of CA and DN CaMKII_C resulted in a 0.6-fold increase and 0.3-fold decrease, respectively, of PLB Thr17 phosphorylation, whereas WT CaMKII_C 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.

View larger version (30K): [in this window] [in a new window]   Figure 1. Adenovirus-mediated expression of WT and mutant CaMKIIC in rat ventricular myocytes. A, Confocal micrographs of immunofluorescence staining showing the intracellular distribution of HA-tagged WT and mutant CaMKIIC 24 hours after adv-WT, CA, and DN CaMKIIC infection with adv-ß-gal as control. B, Western blot analysis of WT, CA, and DN CaMKIIC expression using anti-HA antibodies, with ß-actin as protein loading control. C and D, Western blotting of total and phospho-Thr17 (P-Thr17) and average data expressed as the fold of that in ß-gal (n=3). *P<0.05 vs ß-gal.

Effect of CaMKII_C on the Decay of Ca²⁺ Transients
Using the aforementioned cell models, we examined the overall effects of CaMKII_C 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 CaMKII_C. CA CaMKII_C 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 CaMKII_C or ß-gal. Conversely, DN CaMKII_C 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

View larger version (30K): [in this window] [in a new window]   Figure 2. CaMKII modulation of Ca2+ transients elicited by action potentials. A, Confocal fluo-4 images of Ca2+ transients at 1 mmol/L Ca2+ and 1.0-Hz field stimulation. The corresponding time courses of Ca2+ transients are shown beneath the images. B through D, Statistics of peak Ca2+ transient (F/F0) (B), contraction amplitude (C), and decay time (T50) (D): time from peak to 50% decrease of Ca2+ transient (n=29 to 32 cells from 8 to 9 hearts for each group; *P<0.05 vs ß-gal or WT CaMKIIC).

Interestingly, we found no significant effects of CaMKII_C (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⁺ recycling^30,31,33 (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 CaMKII_C
We measured the status of RyR2 phosphorylation in response to the expression of WT and mutant CaMKII_C by using the back-phosphorylation assay. Figure 3A shows a representative autoradiograph of [-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 ß-gal control (0.653±0.046, n=6) and WT CaMKII_C (0.630±0.078, n=6) but a significant decrease in the CA CaMKII_C group (0.159±0.017, n=6, P<0.001) and an increase in the DN CaMKII_C group (0.857±0.054, n=6, P<0.05), respectively. Hence, RyR2 was hyperphosphorylated in the CA CaMKII_C group and hypophosphorylated in the DN CaMKII_C group compared with WT CaMKII_C and ß-gal.

View larger version (30K): [in this window] [in a new window]   Figure 3. Phosphorylation of RyR2 by CaMKII or PKA on expression of ß-gal, WT, CA, or DN CaMKIIC. A, Back-phosphorylation assay. In the negative control (NC) group, CaMKII in the reaction solution was inhibited by 10 µmol/L KN-93. a, Representative autoradiograph of [-32P]ATP incorporation into RyR2 by CaMKII in vitro (top) and Western blotting of the total amount of RyR protein (bottom). b, Densitometric ratio of the autoradiograph and total RyR protein in Western blot data (*P<0.05 and ***P<0.001 vs ß-gal or WT CaMKIIC; n=6 for each group). B, Representative Western blotting gels of total RyR2 (top), phospho-Ser2809 (P-Ser2809) RyR2 (middle), and phospho-Ser2030 (P-Ser2030) RyR2 (bottom). Positive control used PKA-phosphorylated RyR2 purified from HEK 293 cells.

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 ß-gal group, whereas no significant changes were found among the WT and DN CaMKII_C and ß-gal groups (Figure 3B), except for a slight hyperphosphorylation in the CA CaMKII_C group (2.18±0.16 versus 1.55±0.09 of ß-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 CaMKII_C transgenic animal model,²⁴ acute CaMKII_C manipulation did not alter the RyR protein level (Figure 3A and 3B).

CaMKII_C 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 ß-gal group to 1.64±0.17 Hz/100 µm (n=50, P<0.05) in CA CaMKII_C group (Figure 4B). In contrast, DN CaMKII_C induced hyperactive Ca²⁺ sparks, increasing the spark frequency to 4.21±0.45 Hz/100 µm (n=56, P<0.01 versus ß-gal) (Figure 4B). In WT CaMKII_C, 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 CaMKII_C 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 CaMKII_C 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.

View larger version (36K): [in this window] [in a new window]   Figure 4. CaMKII modulation of Ca2+ sparks. A, Confocal line-scan images showing spontaneous Ca2+ sparks in cells expressing WT, CA, or DN CaMKIIC. B and C, Statistics of spark frequency (n=35 to 60 cells from 6 hearts for each group; *P<0.05 and **P<0.01 vs ß-gal or WT CaMKIIC) (B) and spark amplitude (F/F0) (n=250 to 800 sparks for each group; ***P<0.001 vs ß-gal, WT, and CA CaMKIIC) (C).

View larger version (31K): [in this window] [in a new window]   Figure 5. CaMKII modulation of resting Ca2+ homeostasis. A, Typical fluo-4 confocal image and its time course of caffeine-elicited Ca2+ transient in a cell expressing DN CaMKIIC. B, Resting cytosolic Ca2+ level, indexed by indo-1 fluorescent ratio r410/490 (n=20 to 37 cells from 2 hearts). C, The amplitude of caffeine-elicited Ca2+ transients, indexed by the difference between peak and basal indo-1 fluorescent ratio (r410/490). D, The 50% decay time (T50) of caffeine-elicited Ca2+ transients in fluo-4–loaded cells (n=34 to 40 cells from 5 to 6 hearts).

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 (r_410/490) (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).

CaMKII_C 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 ß-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 CaMKII_C markedly reduced the genesis of Ca²⁺ waves at all Ca²⁺ concentrations higher than 2 mmol/L (Figure 6B). Interestingly, in the WT CaMKII_C group, the Ca²⁺ wave frequency was suppressed to 30% of that in ß-gal, as if WT CaMKII_C became activated at high extracellular Ca²⁺. DN CaMKII_C 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).

View larger version (32K): [in this window] [in a new window]   Figure 6. CaMKII modulation of Ca2+ waves. A, Typical confocal line-scan images of Ca2+ waves at 20 mmol/L extracellular Ca2+. B, Ca2+ wave frequency in response to elevated extracellular Ca2+ in dye-free cells. (#P<0.01 vs ß-gal control; &P<0.001 of both WT and CA CaMKIIC vs ß-gal and DN CaMKIIC; n=42 to 47 from 3 to 4 hearts for each data point.) C, SR Ca2+ content (at 20 mmol/L extracellular Ca2+) measured by caffeine-induced (20 mmol/L) Ca2+ transient (n=30 cells from 3 hearts for each group; *P<0.05, **P<0.01, ***P<0.001 vs ß-gal control).

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 CaMKII_C and depleting it by DN CaMKII_C, 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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
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 CaMKII_C 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 CaMKII_C, 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 CaMKII_C 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 CaMKII_C
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 current^28,32 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 CaMKII_C, 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.

	Acknowledgments

 
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.).

Disclosures

None.

	Footnotes

 
Original received June 29, 2006; resubmission received November 21, 2006; revised resubmission received December 22, 2006; accepted January 4, 2007.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002; 415: 198–205.[CrossRef][Medline] [Order article via Infotrieve]
2. Kort AA, Capogrossi MC, Lakatta EG. Frequency, amplitude, and propagation velocity of spontaneous Ca²⁺-dependent contractile waves in intact adult rat cardiac muscle and isolated myocytes. Circ Res. 1985; 57: 844–855.[Abstract/Free Full Text]
3. Cheng H, Lederer MR, Lederer WJ, Channell MB. Calcium sparks and [Ca²⁺]_i waves in cardiac myocytes. Am J Physiol. 1996; 270: C148–C159.[Medline] [Order article via Infotrieve]
4. Jiang D, Xiao B, Yang D, Wang R, Choi P, Zhang L, Cheng H, Chen SRW. RyR2 mutations linked to ventricular tachycardia and sudden death reduce the threshold for store overload induced Ca²⁺ release (SOICR). Proc Natl Acad Sci U S A. 2004; 101: 13062–13067.[Abstract/Free Full Text]
5. Meissner G. Regulation of mammalian ryanodine receptors. Front Biosci. 2001; 7: d2071–d2080.
6. 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]
7. Hook SS, Means AR. Ca²⁺/CaM-dependent kinases: from activation to function. Annu Rev Pharmaco Toxicol. 2001; 41: 471–505.
8. 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.[Abstract/Free Full Text]
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.[Abstract/Free Full Text]
10. Xiao B, Jiang MT, Zhao M, Yang D, Sutherland C, Lai FA, Walsh MP, Warltier DC, Cheng H, Chen SR. Characterization of a novel PKA phosphorylation site, serine-2030, reveals no PKA hyperphosphorylation of the cardiac ryanodine receptor in canine heart failure. Circ Res. 2005; 96: 847–855.[Abstract/Free Full Text]
11. Rodriguez P, Bhogal MS, Colyer J. Stoichiometric phosphorylation of cardiac ryanodine receptor on serine 2809 by calmodulin-dependent kinase II and protein kinase A. J Biol Chem. 2003; 278: 38593–38600.[Abstract/Free Full Text]
12. Danila CI, Hamilton SL. Phosphorylation of ryanodine receptors. Biol Res. 2004; 37: 521–525.[Medline] [Order article via Infotrieve]
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. 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.[Abstract/Free Full Text]
15. 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.[Abstract/Free Full Text]
16. Lokuta AJ, Rogers TB, Lederer WJ, Valdivia HH. Modulation of cardiac ryanodine receptors of swine and rabbit by a phosphorylation-dephosphorylation mechanism. J Physiol. 1995; 487: 609–622.[Medline] [Order article via Infotrieve]
17. Wu Y, Colbran RJ, Anderson ME. Calmodulin kinase is a molecular switch for cardiac excitation-contraction coupling. Proc Natl Acad Sci U S A. 2001; 98: 2877–2881.[Abstract/Free Full Text]
18. Reiken S, Gaburjakova M, Guatimosim S, Gomez AM, D’Armiento J, Burkhoff D, Wang J, Vassort G, Lederer WJ, Marks AR. Protein kinase A phosphorylation of the cardiac calcium release channel (ryanodine receptor) in normal and failing hearts. Role of phosphatases and response to isoproterenol. J Biol Chem. 2003; 278: 444–453.[Abstract/Free Full Text]
19. Jiang MT, Lokuta AJ, Farrell EF, Wolff MR, Haworth RA, Valdivia HH. Abnormal Ca²⁺ release, but normal ryanodine receptors, in canine and human heart failure. Circ Res. 2002; 91: 1015–1022.[Abstract/Free Full Text]
20. 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.[Abstract/Free Full Text]
21. Xiao B, Sutherland C, Walsh MP, Chen SRW. Protein kinase A phosphorylation at serine-2808 of the cardiac Ca²⁺-release channel (ryanodine receptor) does not dissociate 12.6-kDa FK506-binding protein (FKBP12.6). Circ Res. 2004; 94: 487–495.[Abstract/Free Full Text]
22. Hudmon A, Schulman H. Structure/function of the multifunctional Ca²⁺/calmodulin-dependent protein kinase II. Biochem J. 2002; 364: 593–611.[CrossRef][Medline] [Order article via Infotrieve]
23. Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J Jr, Bers DM, Brown JH. The _C isoform of CaMKII is activated in cardiac hypertrophy and induces dilated cardiomyopathy and heart failure. Circ Res. 2003; 92: 912–919.[Abstract/Free Full Text]
24. Maier LS, Zhang T, Chen L, DeSantiago J, Brown JH, Bers DM. Transgenic CaMKII_C overexpression uniquely alters cardiac myocyte Ca²⁺ handling: reduced SR Ca²⁺ load and activated SR Ca²⁺ release. Circ Res. 2003; 92: 904–911.[Abstract/Free Full Text]
25. 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.[Abstract/Free Full Text]
26. Wang W, Zhu W, Wang S, Yang D, Crow MT, Xiao RP, Cheng H. Sustained ß₁-adrenergic stimulation modulates cardiac contractility by Ca²⁺/calmodulin kinase signaling pathway. Circ Res. 2004; 95: 798–806.[Abstract/Free Full Text]
27. Yang D, Song LS, Zhu WZ, Chakir K, Wang W, Wu C, Wang Y, Xiao RP, Chen SR, Cheng H. Calmodulin regulation of excitation-contraction coupling in cardiac myocytes. Circ Res. 2003; 92: 659–667.[Abstract/Free Full Text]
28. Xiao RP, Cheng H, Lederer WJ, Suzuki T, Lakatta EG. Dual regulation of Ca²⁺/calmodulin-dependent kinase II activity by membrane voltage and by calcium influx. Proc Natl Acad Sci U S A. 1994; 91: 9659–9663.[Abstract/Free Full Text]
29. 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]
30. Hagemann D, Kuschel M, Kuramochi T, Zhu W, Cheng H, Xiao RP. Frequency-encoding Thr¹⁷ phospholamban phosphorylation is independent of Ser¹⁶ phosphorylation in cardiac myocytes. J Biol Chem. 2000; 275: 22532–22536.[Abstract/Free Full Text]
31. Mattiazzi A, Mundina-Weilenmann C, Guoxiang C, Vittone L, Kranias E. Role of phospholamban phosphorylation on Thr¹⁷ in cardiac physiological and pathological conditions. Cardiovasc Res. 2005; 68: 366–375.[Abstract/Free Full Text]
32. Dzhura I, Wu Y, Colbran RJ, Balser JR, Anderson ME. Calmodulin kinase determines calcium-dependent facilitation of L-type calcium channels. Nat Cell Biol. 2000; 2: 173–177.[CrossRef][Medline] [Order article via Infotrieve]
33. Kohlhaas M, Zhang T, Seidler T, Zibrova D, Dybkova N, Steen A, Wagner S, Chen L, Broen 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.[Abstract/Free Full Text]
34. Cheng H, Lederer WJ, Cannell MB. Calcium sparks: Elementary events underlying excitation-contraction coupling in heart muscle. Science. 1993; 262: 740–744.[Abstract/Free Full Text]
35. Diaz ME, Trafford AW, O’Neill SC, Eisner DA. Measurement of sarcoplasmic reticulum Ca²⁺ content and sarcolemmal Ca²⁺ fluxes in isolated rat ventricular myocytes during spontaneous Ca²⁺ release. J Physiol. 1997; 501: 3–16.[CrossRef][Medline] [Order article via Infotrieve]
36. Orchard CH, Lakatta EG. Intracellular calcium transients and developed tension in rat heart muscle. A mechanism for the negative interval-strength relationship. J Gen Physiol. 1985; 86: 637–651.[Abstract/Free Full Text]
37. Song LS, Sham JS, Stern MD, Lakatta EG, Cheng H. Direct measurement of SR release flux by tracking ‘Ca2+ spikes’ in rat cardiac myocytes. J Physiol. 1998; 512: 677–691.[Abstract/Free Full Text]
38. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Terentyeva R, Gyorke S. Protein phosphatases decrease sarcoplasmic reticulum calcium content by stimulating calcium release in cardiac myocytes. J Physiol. 2003; 552: 109–118.[Abstract/Free Full Text]
39. Franzini-Armstrong C, Protasi F, and Ramesh V. Shape, size, and distribution of Ca²⁺ release units and couplons in skeletal and cardiac muscles. Biophys J. 1999; 77: 1528–1539.[Abstract/Free Full Text]
40. Song LS, Sobie EA, McCulle S, Lederer WJ, Balke CW, Cheng H. Orphaned ryanodine receptors in the failing heart. Proc Natl Acad Sci U S A. 2006; 103: 4305–4310.[Abstract/Free Full Text]
41. Terentyev D, Viatchenko-Karpinski S, Gyorke I, Volpe P, Williams SC, Gyorke S. Calsequestrin determines the functional size and stability of cardiac intracellular calcium stores: Mechanism for hereditary arrhythmia. Proc Natl Acad Sci U S A. 2003; 100: 11759–11764.[Abstract/Free Full Text]

Related Article:

Does Ca²⁺/Calmodulin-Dependent Protein Kinase _c Activate or Inhibit the Cardiac Ryanodine Receptor Ion Channel?

Naohiro Yamaguchi and Gerhard Meissner
Circ. Res. 2007 100: 293-295. [Full Text] [PDF]

This article has been cited by other articles:

				L. F. Couchonnal and M. E. Anderson The Role of Calmodulin Kinase II in Myocardial Physiology and Disease Physiology, June 1, 2008; 23(3): 151 - 159. [Abstract] [Full Text] [PDF]

				X. Chen, X. Zhang, D. M. Harris, V. Piacentino III, R. M. Berretta, K. B. Margulies, and S. R. Houser Reduced effects of BAY K 8644 on L-type Ca2+ current in failing human cardiac myocytes are related to abnormal adrenergic regulation Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H2257 - H2267. [Abstract] [Full Text] [PDF]

				B. Gellen, M. Fernandez-Velasco, F. Briec, L. Vinet, K. LeQuang, P. Rouet-Benzineb, J.-P. Benitah, M. Pezet, G. Palais, N. Pellegrin, et al. Conditional FKBP12.6 Overexpression in Mouse Cardiac Myocytes Prevents Triggered Ventricular Tachycardia Through Specific Alterations in Excitation- Contraction Coupling Circulation, April 8, 2008; 117(14): 1778 - 1786. [Abstract] [Full Text] [PDF]

				T. Banyasz, I. Lozinskiy, C. E. Payne, S. Edelmann, B. Norton, B. Chen, Y. Chen-Izu, L. T. Izu, and C. W. Balke Transformation of adult rat cardiac myocytes in primary culture Exp Physiol, March 1, 2008; 93(3): 370 - 382. [Abstract] [Full Text] [PDF]

				Y. Ikeda, M. Hoshijima, and K. R. Chien Toward Biologically Targeted Therapy of Calcium Cycling Defects in Heart Failure Physiology, February 1, 2008; 23(1): 6 - 16. [Abstract] [Full Text] [PDF]

				N. Liu and S. G. Priori Disruption of calcium homeostasis and arrhythmogenesis induced by mutations in the cardiac ryanodine receptor and calsequestrin Cardiovasc Res, January 15, 2008; 77(2): 293 - 301. [Abstract] [Full Text] [PDF]

				C. H. George Sarcoplasmic reticulum Ca2+ leak in heart failure: mere observation or functional relevance? Cardiovasc Res, January 15, 2008; 77(2): 302 - 314. [Abstract] [Full Text] [PDF]

				S. Gyorke and D. Terentyev Modulation of ryanodine receptor by luminal calcium and accessory proteins in health and cardiac disease Cardiovasc Res, January 15, 2008; 77(2): 245 - 255. [Abstract] [Full Text] [PDF]

W. Zhu, A. Y.-H. Woo, D. Yang, H. Cheng