Does Ca2+/calmodulin-dependent protein kinase deltac activate or inhibit the cardiac ryanodine receptor ion channel?

The multifunctional Ca 2 /calmodulin-dependent protein kinase II (CaMKII ) modulates cardiac muscle function by regulating Ca 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 Ca release events (Ca sparks) and Ca 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 Ca from an intracellular Ca storing compartment, the sarcoplasmic reticulum (SR), during a cardiac muscle action potential, in a process known as excitation-contraction coupling.2 Released Ca cause cardiac muscle to contract. Sequestration of released Ca by the SR Ca -transporting ATPase and extrusion by the Na -Ca exchanger restore the myofibrillar Ca concentration from 10 10 to 10 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 Ca channels in the surface membrane and T-tubule mediate influx of Ca , which triggers massive release of Ca from SR by opening RyR2s. In addition to Ca , endogenous effectors such as Mg , 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 expressed in human embryonic kidney 293 cells. However, incorporation of more than one P 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 Ca 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 Ca channel13 and Thr17 of the SR Ca pump regulatory protein phospholamban.14 These phosphorylation events indirectly influence SR Ca release by increasing Ca entry and SR Ca 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 Ca 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 Ca 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 Mg , whereas phosphorylation of all 4 subunits by exogenous CaMKII opens the channel. Transgenic mice that overexpress CaMII C exhibit reduced contractility and altered cardiomyocyte Ca signaling. Increased phosphorylation of RyR2, coimmunoprecipitation of CaMKII and RyR2, and enhanced Ca spark activity despite reduced SR Ca 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.