CaMKIIδ Isoforms Differentially Affect Calcium Handling but Similarly Regulate HDAC/MEF2 Transcriptional Responses

The δB and δC splice variants of Ca2+/calmodulin-dependent protein kinase II (CaMKII), which differ by the presence of a nuclear localization sequence, are both expressed in cardiomyocytes. We used transgenic (TG) mice and CaMKII expression in cardiomyocytes to test the hypothesis that the CaMKIIδC isoform regulates cytosolic Ca2+ handling and the δB isoform, which localizes to the nucleus, regulates gene transcription. Phosphorylation of CaMKII sites on the ryanodine receptor (RyR) and on phospholamban (PLB) were increased in CaMKIIδC TG. This was associated with markedly enhanced sarcoplasmic reticulum (SR) Ca2+ spark frequency and decreased SR Ca2+ content in cardiomyocytes. None of these parameters were altered in TG mice expressing the nuclear-targeted CaMKIIδB. In contrast, cardiac expression of either CaMKIIδB or δC induced transactivation of myocyte enhancer factor 2 (MEF2) gene expression and up-regulated hypertrophic marker genes. Studies using rat ventricular cardiomyocytes confirmed that CaMKIIδB and δC both regulate MEF2-luciferase gene expression, increase histone deacetylase 4 (HDAC4) association with 14-3-3, and induce HDAC4 translocation from nucleus to cytoplasm, indicating that either isoform can stimulate HDAC4 phosphorylation. Finally, HDAC4 kinase activity was shown to be increased in cardiac homogenates from either CaMKIIδB or δC TG mice. Thus CaMKIIδ isoforms have similar effects on hypertrophic gene expression but disparate effects on Ca2+ handling, suggesting distinct roles for CaMKIIδ isoform activation in the pathogenesis of cardiac hypertrophy versus heart failure. The δB and δC splice variants of Ca2+/calmodulin-dependent protein kinase II (CaMKII), which differ by the presence of a nuclear localization sequence, are both expressed in cardiomyocytes. We used transgenic (TG) mice and CaMKII expression in cardiomyocytes to test the hypothesis that the CaMKIIδC isoform regulates cytosolic Ca2+ handling and the δB isoform, which localizes to the nucleus, regulates gene transcription. Phosphorylation of CaMKII sites on the ryanodine receptor (RyR) and on phospholamban (PLB) were increased in CaMKIIδC TG. This was associated with markedly enhanced sarcoplasmic reticulum (SR) Ca2+ spark frequency and decreased SR Ca2+ content in cardiomyocytes. None of these parameters were altered in TG mice expressing the nuclear-targeted CaMKIIδB. In contrast, cardiac expression of either CaMKIIδB or δC induced transactivation of myocyte enhancer factor 2 (MEF2) gene expression and up-regulated hypertrophic marker genes. Studies using rat ventricular cardiomyocytes confirmed that CaMKIIδB and δC both regulate MEF2-luciferase gene expression, increase histone deacetylase 4 (HDAC4) association with 14-3-3, and induce HDAC4 translocation from nucleus to cytoplasm, indicating that either isoform can stimulate HDAC4 phosphorylation. Finally, HDAC4 kinase activity was shown to be increased in cardiac homogenates from either CaMKIIδB or δC TG mice. Thus CaMKIIδ isoforms have similar effects on hypertrophic gene expression but disparate effects on Ca2+ handling, suggesting distinct roles for CaMKIIδ isoform activation in the pathogenesis of cardiac hypertrophy versus heart failure. Ca2+/calmodulin-dependent protein kinase II δ (CaMKIIδ) 4The abbreviations used are: CaMKII, Ca2+/calmodulin-dependent protein kinase II; ANF, atrial natriuretic factor; BNP, brain natriuretic peptide; dn, dominant negative; GST, glutathione S-transferase; HDAC, histone deacetylase; MEF2, myocyte enhancer factor 2;β-MHC,β-myosin heavy chain; NLS, nuclear localization sequence; NRVMs, neonatal rat ventricular myocytes; PE, phenylephrine; PLB, phospholamban; RyR2, cardiac ryanodine receptor; SK.Actin, α-skeletal actin; SR, sarcoplasmic reticulum; TG, transgenic; WT, wild type; PBS, phosphate-buffered saline; HA, hemagglutinin. is the predominant isoform of CaMKII in the heart. Splice variants differing in the presence of a nuclear localization sequence (NLS) show distinct subcellular targeting to either cytoplasmic or nuclear compartments (1Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 89-101Crossref PubMed Scopus (159) Google Scholar, 2Srinivasan M. Edman C.F. Schulman H. 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We recently demonstrated that both δB and δC CaMKII are activated in response to pressure overload induced by thoracic aortic banding but that expression of these isoforms is differentially regulated (4Zhang T. Maier L.S. Dalton N.D. Miyamoto S. Ross Jr., J. Bers D.M. Brown J.H. Circ. Res. 2003; 92: 912-919Crossref PubMed Scopus (479) Google Scholar). The possibility that there are discrete roles for these two isoforms in regulating Ca2+ homeostasis and gene transcription has not yet been explored. CaMKII has long been implicated as a regulator of Ca2+ homeostasis and excitation-contraction (E-C) coupling in ventricular myocytes. This enzyme has been shown to phosphorylate proteins involved in sarcoplasmic reticulum (SR) Ca2+ handling including the cardiac ryanodine receptors (RyR2) and phospholamban (PLB) (4Zhang T. Maier L.S. Dalton N.D. Miyamoto S. Ross Jr., J. Bers D.M. Brown J.H. Circ. Res. 2003; 92: 912-919Crossref PubMed Scopus (479) Google Scholar, 5Bassani R.A. Mattiazzi A. 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Cell Cardiol. 2002; 34: 919-939Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 11Marks A.R. J. Mol. Cell Cardiol. 2001; 33: 615-624Abstract Full Text PDF PubMed Scopus (156) Google Scholar) while phosphorylation of PLB by CaMKII can regulate SR Ca2+ uptake (10Maier L.S. Bers D.M. J. Mol. Cell Cardiol. 2002; 34: 919-939Abstract Full Text PDF PubMed Scopus (243) Google Scholar, 12Mattiazzi A. Mundina-Weilenmann C. Guoxiang C. Vittone L. Kranias E. Cardiovasc. Res. 2005; 68: 366-375Crossref PubMed Scopus (115) Google Scholar). Altered intracellular Ca2+ handling plays an important role in the pathogenesis of heart failure with changes in Ca2+ cycling preceding cardiac dysfunction. An emerging body of evidence has demonstrated that altered function of the RyR2, possibly due to increased phosphorylation by PKA, contributes to cardiac dysfunction in heart failure (13Marx S.O. Reiken S. Hisamatsu Y. Jayaraman T. Burkhoff D. Rosemblit N. Marks A.R. Cell. 2000; 101: 365-376Abstract Full Text Full Text PDF PubMed Scopus (1686) Google Scholar, 14Yano M. Ono K. Ohkusa T. Suetsugu M. Kohno M. Hisaoka T. Kobayashi S. Hisamatsu Y. Yamamoto T. Kohno M. Noguchi N. Takasawa S. Okamoto H. Matsuzaki M. Circulation. 2000; 102: 2131-2136Crossref PubMed Scopus (198) Google Scholar, 15Wehrens X.H. Lehnart S.E. Marks A.R. Annu. Rev. Physiol. 2005; 67: 69-98Crossref PubMed Scopus (298) Google Scholar, 16Yano M. Yamamoto T. Ikemoto N. Matsuzaki M. Pharmacol. Ther. 2005; 107: 377-391Crossref PubMed Scopus (61) Google Scholar). Our previous studies demonstrated that expression of CaMKIIδC in transgenic mice increased RyR2 phosphorylation and Ca2+ leak from the SR, and suggested that these were causal events in the development of heart failure and premature death (4Zhang T. Maier L.S. Dalton N.D. Miyamoto S. Ross Jr., J. Bers D.M. Brown J.H. Circ. Res. 2003; 92: 912-919Crossref PubMed Scopus (479) Google Scholar). Increased CaMKII expression and activation in the RyR2 complex also contributes to the enhanced RyR2 phosphorylation and diastolic SR Ca2+ leak observed in an arrhythmogenic rabbit model of heart failure (17Ai X. Curran J.W. Shannon T.R. Bers D.M. Pogwizd S.M. Circ. Res. 2005; 97: 1314-1322Crossref PubMed Scopus (574) Google Scholar). Whether the CaMKIIδC isoform has more privileged access to and selectively phosphorylates these cytosolic substrates has not been addressed. CaMKII has also been implicated in the transcriptional regulation associated with development of cardiac hypertrophy. We first reported that transient expression of δB CaMKII in neonatal rat ventricular myocytes induces atrial natriuretic factor (ANF) gene expression and results in enhanced transcriptional activation of an ANF-luciferase reporter gene (3Ramirez M.T. Zhao X. Schulman H. Brown J.H. J. Biol. Chem. 1997; 272: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). These data are consistent with the observation that the monomeric CaM kinases, CaMKI and CaMKIV, which like CaMKIIδB can all enter the nucleus, also induce hypertrophic responses in cardiomyocytes in vitro (18Passier R. Zeng h. Frey N. Naya F.J. Nicol R.L. McKinsey T.A. Overbeek P.A. Richardson J.A. Grant S.R. Olson E.N. J. Clin. Investig. 2000; 105: 1395-1406Crossref PubMed Scopus (422) Google Scholar). Several transgenic (TG) mouse models have now confirmed a role for CaMK in activation of the hypertrophic gene program and development of hypertrophy in vivo. Transgenic mice overexpressing calmodulin were found to develop severe cardiac hypertrophy (19Gruver C.L. DeMayo F. Goldstein M.A. Means A.R. Endocrinology. 1993; 133: 376-388Crossref PubMed Scopus (135) Google Scholar) which was subsequently shown to be associated with an increase in the activity of CaMKII in vivo (20Colomer J.M. Means A.R. Mol. Endocrinol. 2000; 14: 1125-1136Crossref PubMed Scopus (47) Google Scholar). Pronounced hypertrophy also develops in transgenic mice that overexpress CaMKIV (18Passier R. Zeng h. Frey N. Naya F.J. Nicol R.L. McKinsey T.A. Overbeek P.A. Richardson J.A. Grant S.R. Olson E.N. J. Clin. Investig. 2000; 105: 1395-1406Crossref PubMed Scopus (422) Google Scholar) although CaMKIV is not a major CaMK isoform in the heart nor is it required for pressure overload induced hypertrophy (1Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 89-101Crossref PubMed Scopus (159) Google Scholar, 21Miyano O. Kameshita I. Fujisawa H. J. Biol. Chem. 1992; 267: 1198-1203Abstract Full Text PDF PubMed Google Scholar, 22Colomer J.M. Mao L. Rockman H.A. Means A.R. Mol. Endocrinol. 2003; 17: 183-192Crossref PubMed Scopus (90) Google Scholar). Our laboratory has shown that transgenic mice that overexpress the nuclear targeted cardiac CaMKIIδB isoform develop hypertrophy, accompanied by cardiomyocyte enlargement and significant increases in hypertrophic gene expression (23Zhang T. Johnson E.N. Gu Y. Morissette M.R. Sah V.P. Gigena M.S. Belke D.D. Dillmann W.H. Rogers T.B. Schulman H. Ross Jr., J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). CaMKIIδB is present and highly concentrated in cardiomyocyte nuclei of these TG mice (23Zhang T. Johnson E.N. Gu Y. Morissette M.R. Sah V.P. Gigena M.S. Belke D.D. Dillmann W.H. Rogers T.B. Schulman H. Ross Jr., J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar), as it is when expressed in isolated cardiomyocytes (3Ramirez M.T. Zhao X. Schulman H. Brown J.H. J. Biol. Chem. 1997; 272: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The transcription factor myocyte enhancer factor 2 (MEF2) has been suggested to act as a common end point for hypertrophic signaling pathways in the myocardium (24Kolodziejczyk S.M. Wang L. Balazsi K. DeRepentigny Y. Kothary R. Megeney L.A. Curr. Biol. 1999; 9: 1203-1206Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar, 25Lu J. McKinsey T.A. Nicol R.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075Crossref PubMed Scopus (424) Google Scholar). Histone deacetylases (HDACs) have been shown to associate with and repress MEF2 activation (25Lu J. McKinsey T.A. Nicol R.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075Crossref PubMed Scopus (424) Google Scholar, 26Miska E.A. Karlsson C. Langley E. Nielsen S.J. Pines J. Kouzarides T. EMBO J. 1999; 18: 5099-5107Crossref PubMed Scopus (472) Google Scholar, 27Youn H.D. Grozinger C.M. Liu J.O. J. Biol. Chem. 2000; 275: 22563-22567Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar) and signal-dependent dissociation of HDACs from MEF2 results in its activation (25Lu J. McKinsey T.A. Nicol R.L. Olson E.N. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4070-4075Crossref PubMed Scopus (424) Google Scholar, 27Youn H.D. Grozinger C.M. Liu J.O. J. Biol. Chem. 2000; 275: 22563-22567Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar). MEF2 was demonstrated to be a downstream target for CaMK in CaMKIV TG (18Passier R. Zeng h. Frey N. Naya F.J. Nicol R.L. McKinsey T.A. Overbeek P.A. Richardson J.A. Grant S.R. Olson E.N. J. Clin. Investig. 2000; 105: 1395-1406Crossref PubMed Scopus (422) Google Scholar) where MEF2 activation was suggested to occur through phosphorylation and dissociation of class II HDACs from MEF2. Whether there is a selective ability of the nuclear-targeted CaMKIIδB isoform to control HDAC phosphorylation, MEF2 activation and hypertrophic gene expression in vivo and specifically in cardiomyocytes has not been explored. The goal of the current study was to determine whether there are specific functions for CaMKIIδB and CaMKIIδC isoforms in the heart. We hypothesized that cytoplasmic CaMKIIδ (composed of δC subunits) participates in phosphorylation and regulation of Ca2+ handling proteins, whereas nuclear CaMKIIδ (composed of NLS-containing δB subunits) is selectively involved in regulation of HDAC phosphorylation and MEF2 activation associated with cardiomyocyte hypertrophy. Surprisingly, our findings demonstrate that whereas the δB and δC CaMKII isoforms have distinct effects on phosphorylation of Ca2+-handling proteins and Ca2+ regulation, the two isoforms similarly affect HDAC-mediated MEF2 and hypertrophic gene expression. These data suggest that the pattern of CaMKII isoform activation determines the propensity for development of alterations in gene expression and Ca2+ handling that underly cardiac hypertrophy and heart failure. Transgenic Mice—Transgenic mice expressing either the cytoplasmic CaMKIIδC or the nuclear CaMKIIδB in the heart were generated as described previously (4Zhang T. Maier L.S. Dalton N.D. Miyamoto S. Ross Jr., J. Bers D.M. Brown J.H. Circ. Res. 2003; 92: 912-919Crossref PubMed Scopus (479) Google Scholar, 23Zhang T. Johnson E.N. Gu Y. Morissette M.R. Sah V.P. Gigena M.S. Belke D.D. Dillmann W.H. Rogers T.B. Schulman H. Ross Jr., J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The creation of MEF2 indicator mice harboring a lacZ transgene controlled by three tandem copies of the MEF2 binding site (from the desmin enhancer) has been described elsewhere (28Naya F.J. Wu C. Richardson J.A. Overbeek P. Olson E.N. Development. 1999; 126: 2045-2052Crossref PubMed Google Scholar). CaMKIIδB or δC TG mice were crossed with MEF2 indicator mice and offspring of WT and CaMKIIδB or δC TG mice harboring MEF2-LacZ transgene were used for experiments. All mice used in the present study were at 4-5 weeks of age, unless otherwise noted. All procedures were performed in accordance with Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care and Use Committee. Western Blotting and Immunoprecipitation—Cardiac homogenates were prepared and Western blot analysis and immunoprecipitation carried out as described previously (4Zhang T. Maier L.S. Dalton N.D. Miyamoto S. Ross Jr., J. Bers D.M. Brown J.H. Circ. Res. 2003; 92: 912-919Crossref PubMed Scopus (479) Google Scholar, 23Zhang T. Johnson E.N. Gu Y. Morissette M.R. Sah V.P. Gigena M.S. Belke D.D. Dillmann W.H. Rogers T.B. Schulman H. Ross Jr., J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). The antibodies used for immunoblotting and immunoprecipitation were as followings: mouse anti-RyR (Affinity Bioreagents), rabbit Ser2815 phospho-RyR2 antibody (a gift from Andy Marks laboratory, Columbia University, New York, NY), mouse anti-PLB (Upstate Biotechnology), rabbit anti-phospho-PLB (Thr17) (Cyclacel, Dundee, UK), rabbit anti-GFP, and mouse anti-14-3-3β (Santa Cruz Biotechnology). Isolation, Ca2+ Measurements and Immunocytochemical Staining of Adult Mouse Ventricular Myocytes—Mouse ventricular myocytes were isolated for Ca2+ spark frequency and SR Ca2+ content measurements as described previously (29Maier L.S. Zhang T. Chen L. DeSantiago J. Brown J.H. Bers D.M. Circ. Res. 2003; 92: 904-911Crossref PubMed Scopus (391) Google Scholar). For immunocytochemical staining, isolated ventricular myocytes were plated on laminin-coated chamber slides and incubated for 1 h at room temperature. The cells were fixed using 100% ethanol (20 min at -20 °C), rinsed (three times) in phosphate-buffered saline (PBS) and then blocked for 1 h with 5% bovine serum albumin in PBS. Cells were incubated with primary antibody (HA antibody, Roche, dilution 1:60) in 1% bovine serum albumin in PBS containing 0.5% Triton X-100 overnight at 4 °C, then rinsed in PBS, and incubated with secondary antibody goat anti-mouse (Texas Red, Jackson Immuno Research) in 0.5% bovine serum albumin in PBS for 2 h at room temperature (dilution 1:200). Coverslips were mounted on slides by using Vectashield (Vector). β-Galactosidase Staining and Activity Assays—Hearts from CaMKIIδB or δC TG mice harboring MEF2-LacZ transgene or from mice expressing the MEF2-LacZ transgene but not CaMKII were collected and fixed in 4% paraformaldehyde buffered with PBS. Hearts were then immersed in Bluo-gal stain (3.1 mm ferricyanide, 3.1 mm ferrocyanide, 10 mm sodium phosphate, pH 7.2, 0.15 m NaCl, 1.0 mm MgCl2, 1 mg/ml Bluo-gal in dimethylformamide) overnight at room temperature. β-Galactosidase activity assays were performed on ventricular extracts using a β-galactosidase assay kit from Stratagene under conditions of linearity with respect to time and protein concentration. Culture, Transfection, and Adenoviral Infection of Neonatal Rat Ventricular Myocytes—Neonatal rat ventricular myocytes (NRVMs) were prepared and transiently transfected or infected with adenoviruses as described previously (30Ramirez M.T. Post G.R. Sulakhe P.V. Brown J.H. J. Biol. Chem. 1995; 270: 8446-8451Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 31Adams J.W. Sakata Y. Davis M.G. Sah V.P. Wang Y. Liggett S.B. Chien K.R. Brown J.H. Dorn G.W. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 10140-10145Crossref PubMed Scopus (472) Google Scholar). Briefly, NRVMs were plated at a density of 4 × 105 cells per well of a 6-well plate, 4.5 × 106 cells per 10-cm dish or 5 × 105 cells per 3.5-cm dish and cultured overnight in serum-containing media. Cells were then washed and incubated in serum-containing medium for 2-4 h prior to transfections. Cells were cotransfected for 16-18 h with 3×MEF2-luciferase reporter gene along with vector alone or vector encoding various CaMKII isoforms using a modified calcium phosphate technique (30Ramirez M.T. Post G.R. Sulakhe P.V. Brown J.H. J. Biol. Chem. 1995; 270: 8446-8451Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Myocytes were washed, the hypertrophic agonist phenylephrine (10 μm) plus 2 μm propranolol to block β-adrenergic response was added, and cells were incubated for an additional 48 h in serum-free medium. Luciferase activity in cell lysates was measured and normalized to total protein. For adenoviral infection, cells were washed after overnight culture and the medium was replaced with serum-free medium supplemented with insulin/transferrin/selenium (ITS). Cells were infected with AdCMV, CaMKIIδB, CaMKIIδC, and/or GFP-HDAC4 adenoviruses at 200-500 viral particles/cell for 16-18 h. The GFP-HDAC4 adenovirus was a gift from Martin Schneider's laboratory, University of Maryland, Baltimore, MD. Cells were subsequently washed and maintained in serum-free medium with supplements. After an additional 24-36 h, cells were harvested for immunoprecipitation studies or fixed for immunocytochemical staining with an anti-HA antibody (Roche Applied Sciences, 1:100 dilution) or an anti-GFP antibody (Santa Cruz Biotechnology, 1:100 dilution). Localization of the expressed HDAC4 was examined by immunostaining using confocal microscopy. RNA Isolation and Real-time Reverse Transcription (RT)-PCR—Total RNA was prepared from mouse ventricular tissue using TRIzol reagent (Invitrogen). First-strand cDNA synthesis was performed using the SuperScript III First-Strand Synthesis System (Invitrogen) according to manufacturer's instructions. Real-time RT-PCR for the expression of hypertrophic marker genes was performed using TaqMan probes and primers from Applied Biosystems and the relative levels of expression were normalized to GAPDH. HDAC4 Kinase Activity Assay—HDAC4 kinase activity assays were performed in ventricular homogenates as described previously (32Backs J. Song K. Bezprozvannaya S. Chang S. Olson E.N. J. Clin. Investig. 2006; 116: 1853-1864Crossref PubMed Scopus (410) Google Scholar) with minor modification. Briefly, a glutathione S-transferase (GST)-HDAC4 fusion protein (amino acids 419-670 of HDAC4) was used as a substrate. Another GST fusion protein with a R601F mutation in HDAC4 to prevent docking to CaMKII was used as control (32Backs J. Song K. Bezprozvannaya S. Chang S. Olson E.N. J. Clin. Investig. 2006; 116: 1853-1864Crossref PubMed Scopus (410) Google Scholar). The GST-HDAC proteins (500 ng) were conjugated to glutathione-agarose beads. GST-HDAC-bound beads were incubated with ventricular lysates (100 μg potein) in lysis buffer (20 mm Tris pH 7.4, 150 mm NaCl, 0.5% Nonidet P-40, and protease inhibitors) for 4 h at 4 °C. Beads were washed once with the same buffer. Beads were then resuspended in kinase reaction buffer (30 μl; 25 mm HEPES pH 7.6, 10 mm MgCl2, and 0.1 mm CaCl2) containing 12.5 μm ATP and 5 μCi [γ-32P]ATP and reactions were allowed to proceed for 30 min at room temperature. Samples were boiled, and phosphoproteins were resolved by SDS-PAGE, visualized by autoradiography, and quantified using a phosphorimager. Statistical Analysis—All data are reported as mean ± S.E. Statistical significance of difference was determined using unpaired two-tailed Student's t test. p value <0.05 was considered statistically significant. Phosphorylation of RyR2 and PLB at the CaMKII Sites in CaMKII TG Mice—To determine whether cytoplasmic and nuclear CaMKII differentially regulate the phosphorylation of Ca2+ regulatory proteins in vivo we examined phosphorylation of RyR2 and PLB in ventricular homogenates from CaMKIIδB and δC TG mice. Our published studies used 3-4-month-old animals in which changes secondary to the development of hypertrophy or heart failure could obscure analysis of early signaling events. Accordingly in this study we examined young mice (4-5-week-old) and also directly compared two lines expressing similar amounts of either CaMKIIδB or CaMKIIδC. The phosphorylation of the RyR2, assessed using an antibody to the CaMKII phosphorylation site (Ser2815 RyR2), was increased 2.2-fold in TG mice expressing the δC isoform of CaMKII (Fig. 1A). In contrast there was no increase in RyR2 phosphorylation in TG mice expressing the nuclear-targeted CaMKIIδB (Fig. 1A). Phosphorylation of PLB at the CaMKII site (Thr17) was also significantly increased in CaMKIIδC TG mice (Fig. 1B) but not in hearts from mice expressing nuclear-targeted CaMKIIδB (Fig. 1B). This confirms our previous data suggesting that RyR2 and PLB are not in vivo targets for CaMKIIδB but are targets for cytosolic CaMKIIδC activity (4Zhang T. Maier L.S. Dalton N.D. Miyamoto S. Ross Jr., J. Bers D.M. Brown J.H. Circ. Res. 2003; 92: 912-919Crossref PubMed Scopus (479) Google Scholar, 23Zhang T. Johnson E.N. Gu Y. Morissette M.R. Sah V.P. Gigena M.S. Belke D.D. Dillmann W.H. Rogers T.B. Schulman H. Ross Jr., J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar). SR Ca2+ Sparks and SR Ca2+ Content in CaMKII TG Cardiomyocytes—To determine whether the selective effects of the CaMKII isoforms on RyR2 and PLB phosphorylation resulted in functional differences in Ca2+ handling, adult cardiomyocytes were isolated from CaMKIIδB and δC TG mice and Ca2+ spark frequency and SR Ca2+ content were assessed. The CaMKIIδB transgene product was confirmed to be localized in the nucleus using anti-HA staining; that for CaMKIIδC was shown to be concentrated in the cytoplasm of the isolated cardiomyocytes (Fig. 2A). Increased Ca2+ spark frequency (Fig. 2B) and decreased SR Ca2+ content (Fig. 2C) were observed in cardiomyocytes from the CaMKIIδC TG mice, as we demonstrated earlier (4Zhang T. Maier L.S. Dalton N.D. Miyamoto S. Ross Jr., J. Bers D.M. Brown J.H. Circ. Res. 2003; 92: 912-919Crossref PubMed Scopus (479) Google Scholar). The effects of nuclear-targeted CaMKII on these parameters were not previously reported. Here we show by direct comparison that spark frequency and SR Ca2+ changes are not observed in myocytes expressing CaMKIIδB (Fig. 2, B and C), which localizes to the nucleus and fails to increase RyR2 phosphorylation. The cytoplasmic isoform of CaMKIIδC thus appears to have a selective ability to directly alter Ca2+ handling via phosphorylation of Ca2+ regulatory proteins that are likewise confined to the cytosolic compartment. In Vivo Measurement of MEF2 Activation by CaMKII—To determine whether the transcription factor MEF2 is an in vivo target for CaMKIIδ, and explore whether its activation is specific for CaMKII signaling in the nucleus the CaMKIIδB or δC TG mice were crossed with MEF2/β-galactosidase indicator mice (28Naya F.J. Wu C. Richardson J.A. Overbeek P. Olson E.N. Development. 1999; 126: 2045-2052Crossref PubMed Google Scholar). MEF2 activation was detected by β-galactosidase staining of mouse hearts and by enzymatic assay of β-galactosidase activity in extracts from ventricles of WT, CaMKIIδB and CaMKIIδC TG mice harboring the MEF2-LacZ transgene. Young mice (4-5 weeks of age) which had not yet developed increased heart to body weight ratios or chamber enlargement were examined to ensure that observed changes in MEF2 activation were not secondary to these phenotypic changes. Hearts from WT mice expressing the MEF2/β-galactosidase gene showed background β-galactosidase staining (Fig. 3A), reflecting basal MEF2 activity. β-Galactosidase staining of mouse hearts expressing CaMKII was markedly increased compared with WT (Fig. 3A). Unexpectedly, in vivo expression of either δB or δC isoforms of CaMKII increased the activation of MEF2 (Fig. 3A). To provide more quantative assessment of β-galactosidase activity, ventricular extracts were prepared and β-galactosidase activity measured enzymatically as described under "Experimental Procedures." Equivalent increases (∼7-fold over WT) were seen in both the CaMKIIδB and CaMKIIδC expressing MEF2 reporter mice (Fig. 3B). The level of MEF2 protein expression was also assessed by immunoblotting and found not to differ in WT and TG mouse hearts (supplemental Fig. S1). Thus the observed effects of CaMKII on β-galactosidase activity indicate activation of MEF2-mediated transcription, not changes in MEF2 expression. These data demonstrate that increased expression of either δB or δC isoforms of CaMKII can drive MEF2-dependent genes in vivo. In Vitro Measurement of MEF2 Activation by CaMKII—To more directly demonstrate that MEF2 can be activated by either CaMKIIδB or CaMKIIδC, MEF2-luciferase assays were performed in neonatal rat ventricular myocytes (NRVMs). MEF2-luciferase activity, induced by the hypertrophic agonist phenylephrine (PE), was measured in the presence or absence of co-expressed WT or dominant negative (dn, K43A) forms of CaMKIIδB or δC. The activation of MEF2 was enhanced by expression of either δB or δC CaMKII and inhibited by either dn CaMKIIδB or δC (Fig. 4A). The involvement of HDAC in MEF2 activation by PE was also assessed by co-transfection of HDAC4. The ability of PE to stimulate MEF2 luciferase was inhibited by HDAC4 and the repression of MEF2 by HDAC4 was significantly attenuated by co-expression of either CaMKIIδB or δC (Fig. 4B). These data indicate that CaMKII affects MEF2 activity through derepression of HDAC. Induction of Hypertrophic Marker Genes