Does Ca2+/Calmodulin-Dependent Protein Kinase δc Activate or Inhibit the Cardiac Ryanodine Receptor Ion Channel?
- Ca2+/calmodulin dependent protein kinase II
- cardiac ryanodine receptor
- protein phosphorylation
- heart failure
See related article, pages 399–407
The multifunctional Ca2+/calmodulin-dependent protein kinase IIδ (CaMKIIδ) modulates cardiac muscle function by regulating Ca2+ transport proteins and nuclear signaling molecules. Aberrant activity of CaMKIIδ is implicated in heart disease. In this issue, Yang et al1 report that acute overexpression of constitutively active splice variant CaMKIIδC 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 CaMKIIδC 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), calmodulin-dependent 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 expressed 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 CaMIIδ splice molecules has been extensively studied in cardiomyocytes. The CaMIIδB variant has a nuclear localization signal and transcriptionally regulates signaling pathways that contribute to cardiac myopathies.11,12 The cytosolic variant CaMIIδC 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 WT-RyR2 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 CaMIIδC 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 CaMKIIδC 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 patch-clamped 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 CaMKIIα or CaMKIIδC are summarized in the Table. Conflicting results 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 wild-type–CaMKIIδC 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 β-adrenergic receptor agonist isoproterenol results in a CaMKII-dependent but cAMP- and PKA-independent increase in diastolic SR C2+ leak by a signaling mechanism that remains to be determined.
The functional role of CaMKIIδC in normal and diseased heart remains to be determined. Yang et al1 suggest that a CaMKIIδC-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 CaMKIIδC autophopshorylation and RyR2 phosphorylation and activity, and thereby contractile function.9 In failing heart, CaMKIIδC-dependent RyR2 phosphorylation may have no major role1 or result in a leaky SR Ca2+ channel and contractile dysfunction.22 The role of CaMKIIδ in failing hearts is likely more complex because its cytosolic variant not only modulates the activity of key Ca2+ transport proteins in excitation-contraction 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.
The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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