Editorials
See related article, pages 399 – 407
Does Ca2ⴙ/Calmodulin-Dependent Protein Kinase ␦c Activate
or Inhibit the Cardiac Ryanodine Receptor Ion Channel?
Naohiro Yamaguchi, Gerhard Meissner
T
Downloaded from http://circres.ahajournals.org/ by guest on July 12, 2017
he multifunctional Ca2⫹/calmodulin-dependent protein kinase II␦ (CaMKII␦) modulates cardiac muscle
function by regulating Ca2⫹ transport proteins and
nuclear signaling molecules. Aberrant activity of CaMKII␦ is
implicated in heart disease. In this issue, Yang et al1 report
that acute overexpression of constitutively active splice variant CaMKII␦C phosphorylates the cardiac ryanodine receptor
ion channel (RyR2) to decrease the rate of occurrence of local
Ca2⫹ release events (Ca2⫹ sparks) and Ca2⫹ waves in cultured
rat cardiomyocytes. A dominant negative form of CaMKII␦C
was shown to have opposite effects.
The cardiac ryanodine receptors are cation selective channels that release Ca2⫹ from an intracellular Ca2⫹ storing
compartment, the sarcoplasmic reticulum (SR), during a
cardiac muscle action potential, in a process known as
excitation-contraction coupling.2 Released Ca2⫹ cause cardiac
muscle to contract. Sequestration of released Ca2⫹ by the SR
Ca2⫹-transporting ATPase and extrusion by the Na⫹-Ca2⫹
exchanger restore the myofibrillar Ca2⫹ concentration from
10-6 - 10-5 to ⬇10-7 M, causing muscle to relax. The RyR2s are
regulated by a variety of effectors.3 During a cardiac action
potential, closely apposed dihydropyridine-sensitive L-type
Ca2⫹ channels in the surface membrane and T-tubule mediate
influx of Ca2⫹, which triggers massive release of Ca2⫹ from
SR by opening RyR2s. In addition to Ca2⫹, endogenous
effectors such as Mg2⫹, ATP, reactive oxygen and nitrogen
molecules regulate RyR2. RyR2 is also regulated by calmodulin, cAMP-dependent protein kinase A (PKA), calmodulindependent kinase II (CaMKII), protein kinase C, and protein
phosphatases 1 and 2A. Phosphorylation of RyR2-Ser2030 by
PKA4 and Ser2809 by PKA5,6 and CaMKII5 has been
described. Marks and colleagues6 report that PKA-mediated
phosphorylation of RyR2-Ser2809 causes a small subunit,
FKBP12.6 or calstabin 2, to dissociate from RyR2, which
results in a “leaky” SR channel, aberrant contractile function,
and heart failure. But other laboratories fail to support
this.4,7,8 Wehrens et al9 identified a third RyR2 phosphorylation site. Mutagenesis suggests that CaMKII uniquely phosphorylates Ser2815 near S2809 on recombinant RyR2 ex-
pressed in human embryonic kidney 293 cells. However,
incorporation of more than one 32P per monomer into the
native, immunoprecipitated receptor indicates the presence of
another CaMKII site in RyR2, in partial agreement with
Rodriguez et al10 that there are 4 CaMKII phosphorylation
sites per PKA site or 8 sites based on 2 PKA sites per RyR2
monomer.4
In the presence of CaM and elevated local Ca2⫹ concentrations, the multimeric CaMKIIs are autophosphorylated to
become constitutively active. The function of 2 CaMII␦
splice molecules has been extensively studied in cardiomyocytes. The CaMII␦B variant has a nuclear localization signal
and transcriptionally regulates signaling pathways that contribute to cardiac myopathies.11,12 The cytosolic variant
CaMII␦C phosphorylates, not only RyR2, but also the
voltage-dependent L-type Ca2⫹ channel13 and Thr17 of the
SR Ca2⫹ pump regulatory protein phospholamban.14 These
phosphorylation events indirectly influence SR Ca2⫹ release
by increasing Ca2⫹ entry and SR Ca2⫹ content, and thereby
RyR2 activity.
The functional consequences of CaMKII-mediated RyR2
phosphorylation are less clear. Single channel experiments
indicate that phosphorylation by CaMKII increases WTRyR2 activity5,9 and Ca2⫹ sensitivity but not of the mutant
RyR2-S2815A that lacks the RyR2 CaMKII phosphorylation
site.9 Other groups report more complex regulation by protein
kinases. Valdivia et al15 suggest PKA regulates RyR2 by
increasing its responsiveness to photo-released Ca2⫹ that
results in reduced levels of the steady state open channel.
Hain et al16 speculate that phosphorylation of one subunit of
the tetrameric RyR2 by endogenous CaMKII results in
channel blockade by Mg2⫹, whereas phosphorylation of all 4
subunits by exogenous CaMKII opens the channel. Transgenic mice that overexpress CaMII␦C exhibit reduced contractility and altered cardiomyocyte Ca2⫹ signaling. Increased
phosphorylation of RyR2, coimmunoprecipitation of CaMKII
and RyR2, and enhanced Ca2⫹ spark activity despite reduced
SR Ca2⫹ content taken together imply that CaMKII␦C RyR2
phosphorylation results in the formation of a leaky SR
channel.17,18
It is perplexing that some laboratories report that CaMKII
RyR2 phosphorylation inhibits the RyR2 ion channel. The
Table compares the results by Kohlhaas,19 Guo,20 Wu21 and
Yang1 and colleagues, using intact, permeabilized or patchclamped adult rabbit, mouse or rat cardiomyocytes. Isolated
cardiomyocytes were used to minimize the effects of overexpressing CaMKII for prolonged times in an animal model.
The effects of acute overexpression or perfusion of wild-type,
constitutively active or dominant negative CaMKII␣ or
CaMKII␦C are summarized in the Table. Conflicting results
The opinions expressed in this editorial are not necessarily those of the
editors or of the American Heart Association.
From the Departments of Biochemistry and Biophysics (N.Y., G.M.),
and Cell and Molecular Physiology (G.M.), University of North Carolina,
Chapel Hill, NC.
Correspondence to Gerhard Meissner, Department of Biochemistry
and Biophysics, University of North Carolina, Chapel Hill, NC 275997260. E-mail [email protected]
(Circ Res. 2007;100:293-295.)
© 2007 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/01.RES.0000259327.56377.55
293
294
Circulation Research
February 16, 2007
Effects of CaMKII
Kohlhaas et al19
Cardiomyocytes
CaMKII
SR Ca2⫹ content
Ca2⫹ transient amplitude
Ca2⫹ sparks
Resting Ca2⫹
Rabbit
Guo et al20
⫺/⫺
Mouse WT and PLB
permeabilized
Wu et al21
Yang et al1
Rabbit patch-clamped
Rat
acute overexpression of WT
CaMKII␦C
end. CaMKII & exo. preactivated
CaMII␣
CA CaMKII␣
acute overexpression of WT, CA
and DN CaMKII␦C
decreased
WT increased
PLB⫺/⫺ unchanged
increased
unchanged
decreased (not significant)
N.D.
decreased
unchanged
increased frequency
increased duration and frequency
N.D.
WT unchanged, CA decreased, DN
increased frequency
unchanged
variable
N.D.
unchanged
S2815 increased
S2809 increased
increased
N.D.
WT unchanged, CA increased, DN
decreased
PLB Thr17⬃P
increased
WT increased
N.D.
WT unchanged, CA increased, DN
deceased
ICa current
increased
N.D.
increased
CA increased
RyR2⬃P
Downloaded from http://circres.ahajournals.org/ by guest on July 12, 2017
N.D., not determined; PLB, phospholamban; CA, constitutively active; DN, dominant negative
were obtained with regard to SR Ca2⫹ content, SR Ca2⫹
release and RyR2 phosphorylation. How can then these
differences be explained? Yang et al1 suggest species dependent differences between rat and rabbit or use of intact versus
perfused myocardiocytes. Indeed, overexpression of wildtype–CaMKII␦C increased RyR2 phosphorylation and activity (measured as Ca2⫹ sparks) in rabbit19 but not rat cardiomyocytes.1 The constitutively activated CaM kinase was
required for increased RyR2 phosphorylation; however, this
correlated with a decrease in Ca2⫹ spark frequency, a result
opposite to that obtained with rabbit cardiomyocytes. A
second plausible explanation is that phospholamban Thr17
phosphorylation is responsible for the differences by causing
de-inhibition of the SR Ca2⫹ transport ATPase and increased
SR Ca2⫹ content. However against this possibility argues that
phospholamban KO cardiomyocytes exhibit increased Ca2⫹
spark frequency and duration despite unchanged SR Ca2⫹
content.20 Moreover, intact cardiomyocytes display increased
Ca2⫹ spark frequency despite a decreased SR Ca2⫹ content.19
A third explanation we favor is that RyR2 phosphorylation
(as a measurement of CaMKII activity) does not correlate
with RyR2 activity. Most studies report relative RyR phosphorylation changes that depending on the control RyR2
phosphorylation level can represent a small or large increase
in RyR2 phosphorylation status. As noted above, the extent of
RyR2 phosphorylation may affect its activity.16
In this issue in a related study, Curran et al16a use a
pharmacological approach to show in accordance with their
previous work that CaMKII increases RyR2 activity. A new
finding is that the ␤-adrenergic receptor agonist isoproterenol
results in a CaMKII-dependent but cAMP- and PKAindependent increase in diastolic SR C2⫹ leak by a signaling
mechanism that remains to be determined.
The functional role of CaMKII␦C in normal and diseased
heart remains to be determined. Yang et al1 suggest that a
CaMKII␦C-dependent decrease in RyR2 Ca2⫹ sensitivity in
the normal heart provides a mechanism that compensates the
effects of increased Ca2⫹ influx (ICa, Table). An opposing
view is that an increased heart rate enhances CaMKII␦C
autophopshorylation and RyR2 phosphorylation and activity,
and thereby contractile function.9 In failing heart, CaMKII␦Cdependent RyR2 phosphorylation may have no major role1 or
result in a leaky SR Ca2⫹ channel and contractile dysfunction.22 The role of CaMKII␦ in failing hearts is likely more
complex because its cytosolic variant not only modulates the
activity of key Ca2⫹ transport proteins in excitationcontraction but also has a role in gene regulation.23
Sources of Funding
Support by National Institutes of Health Grants HL073051 and
AR018687 is gratefully acknowledged.
Disclosures
None.
References
1. Yang D, Zhu WZ, Xiao B, Brochet DXP, Chen SRW, Lakatta EG, Xiao
RP, Chang H. Ca2⫹/calmodulin kinase II-dependent phosphorylation of
ryanodine receptors suppresses Ca2⫹ sparks and Ca2⫹ waves in cardiac
myocytes. Circ Res. 2007;100:399 – 407.
2. Bers DM. Cardiac excitation-contraction coupling. Nature. 2002;415:
198 –205.
3. Meissner G. Molecular regulation of cardiac ryanodine receptor ion
channel. Cell Calcium. 2004;35:621– 628.
4. Xiao B, Jiang MT, Zhao M, Yang D, Sutherland C, Lai FA, Walsh MP,
Warltier DC, Cheng H, Chen SRW. 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.
5. 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.
6. Wehrens XH, Lehnart SE, Marks AR. Intracellular calcium release and
cardiac disease. Annu Rev Physiol. 2005;67:69 –98.
7. 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.
Yamaguchi and Meissner
Downloaded from http://circres.ahajournals.org/ by guest on July 12, 2017
8. 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.
9. Wehrens XH, Lehnart SE, Reiken SR, Marks AR. Ca2⫹/calmodulindependent protein kinase II phosphorylation regulates the cardiac
ryanodine receptor. Circ Res. 2004;94:e61– e70.
10. 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.
11. Zhang T, Johnson EN, Gu Y, Morissette MR, Sah VP, Gigena MS,
Belke DD, Dillmann WH, Rogers TB, Schulman H, Ross J, Brown JH.
The cardiac-specific nuclear delta(B) isoform of Ca2⫹/calmodulindependent protein kinase II induces hypertrophy and dilated cardiomyopathy associated with increased protein phosphatase 2A activity. J Biol
Chem. 2002;277:1261–1267.
12. Li B, Dedman JR, Kaetzel MA. Nuclear Ca2⫹/calmodulin-dependent
protein kinase II in the murine heart. Biochim Biophys Acta. 2006;1763:
1275–1281.
13. Grueter CE, Abiria SA, Dzhura I, Wu Y, Ham AJ, Mohler PJ, Anderson
ME, Colbran RJ. L-type Ca2⫹ channel facilitation mediated by phosphorylation of the beta subunit by CaMKII. Mol Cell. 2006;23:641– 650.
14. 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.
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.
16. 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.
16a. Curran J, Hinton MJ, Ríos Eduardo, Bers DM, Shannon TR.
␤-adrenergic enhancement of sarcoplasmic reticulum Ca2⫹ leak in
17.
18.
19.
20.
21.
22.
23.
CaMKII Regulation of RyR2
295
cardiac myocytes is mediated by Ca2⫹/calmodulin dependent protein
kinase. Circ Res. 2007;100:391–398.
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.
Zhang T, Maier LS, Dalton ND, Miyamoto S, Ross J, 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.
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.
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.
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.
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.
Backs J, Song K, Bezprozvannaya S, Chang S, Olson EN. CaM kinase
II selectively signals to histone deacetylase 4 during cardiomyocyte
hypertrophy. J Clin Invest. 2006;116:1853–1864.
KEY WORDS: Ca2⫹/calmodulin dependent protein kinase II
ryanodine receptor 䡲 protein phosphorylation 䡲 heart failure
䡲
cardiac
Does Ca2+/Calmodulin-Dependent Protein Kinase δc Activate or Inhibit the Cardiac
Ryanodine Receptor Ion Channel?
Naohiro Yamaguchi and Gerhard Meissner
Downloaded from http://circres.ahajournals.org/ by guest on July 12, 2017
Circ Res. 2007;100:293-295
doi: 10.1161/01.RES.0000259327.56377.55
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2007 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/100/3/293
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.
Reprints: Information about reprints can be found online at:
http://www.lww.com/reprints
Subscriptions: Information about subscribing to Circulation Research is online at:
http://circres.ahajournals.org//subscriptions/