Anthony Yiu‐Ho Woo

Ca2+-calmodulin-dependent protein kinase II (CaMKII) is expressed in many mammalian cells, with the δ isoform predominantly expressed in cardiomyocytes. Previous studies have shown that inhibition of CaMKII protects cardiomyocytes against β1-adrenergic receptor-mediated apoptosis. However, it is unclear whether activation of CaMKII is sufficient to cause cardiomyocyte apoptosis and whether CaMKII signaling is important in heart muscle cell apoptosis mediated by other stimuli. Here, we specifically enhanced or suppressed CaMKII activity using adenoviral gene transfer of constitutively active (CA-CaMKIIδC) or dominant negative (DN-CaMKIIδC) mutants of CaMKIIδC in cultured adult rat cardiomyocytes. Expression of CA-CaMKIIδC promoted cardiomyocyte apoptosis that was associated with increased mitochondrial cytochrome c release and attenuated by co-expression of Bcl-XL. Importantly, isoform-specific suppression of CaMKIIδC with the DN-CaMKIIδC mutant similar to nonselective CaMKII inhibition by the pharmacological inhibitors (KN-93 or AIP) not only prevented CA-CaMKIIδC-mediated apoptosis but also protected cells from multiple death-inducing stimuli. Thus, activation of CaMKIIδC constitutes a common intermediate by which various death-inducing stimuli trigger cardiomyocyte apoptosis via the primary mitochondrial death pathway. Ca2+-calmodulin-dependent protein kinase II (CaMKII) is expressed in many mammalian cells, with the δ isoform predominantly expressed in cardiomyocytes. Previous studies have shown that inhibition of CaMKII protects cardiomyocytes against β1-adrenergic receptor-mediated apoptosis. However, it is unclear whether activation of CaMKII is sufficient to cause cardiomyocyte apoptosis and whether CaMKII signaling is important in heart muscle cell apoptosis mediated by other stimuli. Here, we specifically enhanced or suppressed CaMKII activity using adenoviral gene transfer of constitutively active (CA-CaMKIIδC) or dominant negative (DN-CaMKIIδC) mutants of CaMKIIδC in cultured adult rat cardiomyocytes. Expression of CA-CaMKIIδC promoted cardiomyocyte apoptosis that was associated with increased mitochondrial cytochrome c release and attenuated by co-expression of Bcl-XL. Importantly, isoform-specific suppression of CaMKIIδC with the DN-CaMKIIδC mutant similar to nonselective CaMKII inhibition by the pharmacological inhibitors (KN-93 or AIP) not only prevented CA-CaMKIIδC-mediated apoptosis but also protected cells from multiple death-inducing stimuli. Thus, activation of CaMKIIδC constitutes a common intermediate by which various death-inducing stimuli trigger cardiomyocyte apoptosis via the primary mitochondrial death pathway. Ca2+-calmodulin-dependent protein kinase II (CaMKII) 2The abbreviations used are: CaMKII, Ca2+-calmodulin-dependent protein kinase II; AR, adrenergic receptor; CA, constitutively active; DN, dominant negative; SR, sarcoplasmic reticulum; PLB, phospholamban; HA, hemagglutinin; WT, wild type; m.o.i., multiplicity of infection; β-gal, β-galactosidase; TUNEL, deoxynucleotidyltransferase-mediated dUTP nick end labeling. is a ubiquitous and multifunctional serine/threonine protein kinase family that is activated upon binding of Ca2+ and calmodulin (1Braun A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Crossref PubMed Scopus (741) Google Scholar). CaMKII mediates phosphorylation of a wide range of target proteins involved in vital cellular processes such as Ca2+ handling, cell growth, and cell death, particularly in the heart and the brain. There are about 30 distinct members of CaMKII family encoded by four different genes (α, β, δ, and γ). The δ isoform of CaMKII family is predominantly expressed in the heart of many mammalian species, including humans (2Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 89-101Crossref PubMed Scopus (161) Google Scholar, 3Baltas L.G. Karczewski P. Krause E.G. FEBS Lett. 1995; 373: 71-75Crossref PubMed Scopus (55) Google Scholar, 4Hoch B. Wobus A.M. Krause E.G. Karczewski P. J. Cell. Biochem. 2000; 79: 293-300Crossref PubMed Scopus (21) Google Scholar, 5Hoch B. Meyer R. Hetzer R. Krause E.G. Karczewski P. Circ. Res. 1999; 84: 713-721Crossref PubMed Scopus (279) Google Scholar). Two primary splicing variants of the δ isoform, CaMKIIδB and CaMKIIδC, have been cloned from rat heart (2Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 89-101Crossref PubMed Scopus (161) Google Scholar). Compared with CaMKIIδC, CaMKIIδB has an additional 11-amino acid sequence that contains a nuclear targeting signal (6Srinivasan M. Edman C.F. Schulman H. J. Cell Biol. 1994; 26: 839-852Crossref Scopus (241) Google Scholar). Thus, CaMKIIδB and CaMKIIδC localize to the nuclear and the cytosolic compartments, respectively, in cardiac myocytes (6Srinivasan M. Edman C.F. Schulman H. J. Cell Biol. 1994; 26: 839-852Crossref Scopus (241) Google Scholar). Over the past decade, a number of studies have been focused on the role of CaMKII (without reference to specific isoforms) in regulating cardiac excitation-contraction coupling. Most if not all of the characterized CaMKII target proteins in the heart are involved in the regulation of intracellular Ca2+ and excitation-contraction coupling. These include sarcoplasmic reticulum (SR) Ca2+ release channels known as the ryanodine receptor (RyR2) (7Witcher D.R. Kovacs R.J. Schulmans H. Cefali D.C. Jones L.R. J. Biol. Chem. 1991; 266: 11144-11152Abstract Full Text PDF PubMed Google Scholar), SR Ca2+ pump and its regulator phospholamban (PLB) (8Bruce A. Davis S.Q. Schwartz A. Samaha R.J. Kranias E.G. J. Biol. Chem. 1983; 258: 13587-13591Abstract Full Text PDF PubMed Google Scholar, 9Wegener A.D. Simmererman H.K.B. Lindemann J.P. Jones L.R. J. Biol. Chem. 1989; 264: 11468-11474Abstract Full Text PDF PubMed Google Scholar), and sarcolemmal L-type Ca2+ channels (10Xiao R.P. Cheng P. Lederer W.J. Suzuki T. Lakatta E.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9659-9663Crossref PubMed Scopus (180) Google Scholar, 11Yuan W Bers D.M. Am. J. Physiol. 1994; 267: H982-H993PubMed Google Scholar, 12Dzhura I. Wu Y. Colbran R.J. Balser J.R. Anderson M.E. Nat. Cell Biol. 2000; 2: 173-177Crossref PubMed Scopus (302) Google Scholar, 13Grueter C.E. Abiria S.A. Dzhura I. Wu Y. Ham A.J. Mohler P.J. Anderson M.E. Colbran R.J. Mol. Cell. 2006; 23: 641-650Abstract Full Text PDF PubMed Scopus (170) Google Scholar). Phosphorylation of these substrates plays an essential role in regulating diverse and important cardiac functions such as the well established frequency-dependent accelerations in contractile relaxation and Ca2+ transient decay (14Bassani R.A. Mattiazzi A. Bers D.M. Am. J. Physiol. 1995; 268: H703-H712Crossref PubMed Google Scholar, 15Hagemann D. Kuschel M. Kuramochi T. Zhu W. Cheng H. Xiao R.P. J. Biol. Chem. 2000; 275: 22532-22536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) and cardiac pacemaker activity (16Vinogradova T.M. Zhou Y.Y. Bogdanov K.Y. Yang D. Kuschel M. Cheng H. Xiao R.P. Circ. Res. 2000; 87: 760-767Crossref PubMed Scopus (153) Google Scholar). However, CaMKII activation might be abnormally enhanced under certain pathological conditions that disrupt intracellular Ca2+ mobilization, such as cardiac ischemia, acidosis, myocardial infarction, and excessive β-adrenergic receptor (βAR) stimulation. Because of its positive feedback biochemical nature (i.e. autophosphorylation) (1Braun A.P. Schulman H. Annu. Rev. Physiol. 1995; 57: 417-445Crossref PubMed Scopus (741) Google Scholar), the exaggerated CaMKII activation or expression has been implicated in the pathogenesis of arrhythmia (17Anderson M.E. Braun A.P. Wu Y. Lu T. Wu Y. Schulman H. Sung R.J. J. Pharmacol. Exp. Therap. 1998; 287: 996-1006PubMed Google Scholar, 18Anderson M.E. Trends Cardiovasc. Med. 2004; 14: 152-161Crossref PubMed Scopus (68) Google Scholar), cardiac hypertrophy (19Ramirez M.T. Zhao X. Schulman H. Brown J.H. J. Biol. Chem. 1997; 272: 31203-31208Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 20Zhu W. Zou Y. Shiojima I. Kudoh S. Aikawa R. Hayashi D. Mizukami M. Toko H. Shibasaki F. Yazaki Y. Nagai R. Komuro I. J. Biol. Chem. 2000; 275: 15239-15245Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 21Zhang 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 J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 22Zhang T. Brown J.H. Cardiovasc. Res. 2004; 63: 476-486Crossref PubMed Scopus (252) Google Scholar), and cardiomyopathy (5Hoch B. Meyer R. Hetzer R. Krause E.G. Karczewski P. Circ. Res. 1999; 84: 713-721Crossref PubMed Scopus (279) Google Scholar, 22Zhang T. Brown J.H. Cardiovasc. Res. 2004; 63: 476-486Crossref PubMed Scopus (252) Google Scholar, 23Xu X. Yang D. Ding J.H. Wang W. Chu P.H. Dalton N.D. Wang H.Y. Bermingham Jr., J.R. Ye Z. Liu F. Rosenfeld M.G. Manley J.L. Ross Jr., J. Chen J. Xiao R.P. Cheng H. Fu X.D. Cell. 2005; 120: 59-72Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 24Ai X. Curran J.W. Shannon T.R. Bers D.M. Pogwizd S.M. Circ. Res. 2005; 97: 1314-1322Crossref PubMed Scopus (578) Google Scholar). It has been demonstrated that CaMKII is persistently activated during sustained βAR stimulation in cardiac myocytes both in cell culture and in vivo (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar, 26Zhang R. Khoo M.S.C. Wu Y. Yang Y. Grueter C.E. Ni G. Price Jr., E.E. Thiel W. Guatimosim S. Song L.S. Madu E.C. Shah A.N. Vishnivetskaya T.A. Atkinson J.B. Gurevich V.V. Salama G. Lederer W.J. Colbran R.J. Anderson M.E. Nat. Med. 2005; 11: 409-417Crossref PubMed Scopus (488) Google Scholar, 27Wang W. Zhu W.Z. Wang S.Q. Yang D. Crow M.T. Xiao R.P. Cheng H. Circ. Res. 2004; 95: 798-806Crossref PubMed Scopus (165) Google Scholar) and that this activation is part of the signaling pathway relaying β1AR-evoked apoptotic signals in the heart (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar). In this model, inhibition of CaMKII prevents β1AR-induced cardiac myocyte apoptosis, whereas overexpression of CaMKIIδC enhances the β1AR pro-apoptotic effect (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar). It is also noteworthy that the cytosolic isoform, CaMKIIδC, is selectively up-regulated in a rabbit heart failure model (24Ai X. Curran J.W. Shannon T.R. Bers D.M. Pogwizd S.M. Circ. Res. 2005; 97: 1314-1322Crossref PubMed Scopus (578) Google Scholar), whereas there is little or no change in the expression or activity of the nuclear isoform (CaMKIIδB). Despite these studies implicating CaMKIIδC in the β1AR-induced cell death, it is currently unclear whether activation of the cytosolic isoform CaMKIIδC is sufficient to cause heart muscle cell apoptosis and whether other death-inducing stimuli require CaMKII activity to cause cell Because apoptosis is a cause of heart the of involved in heart muscle cell has in the past decade, an important in the of and The of the to whether activation of the cardiac cytosolic isoform, CaMKIIδC, is sufficient to trigger cardiac myocyte apoptosis if to whether CaMKIIδC is a intermediate in the death signaling by stimuli other stimulation of these we used cultured adult rat cardiac myocytes in with adenoviral gene transfer of constitutively active (CA-CaMKIIδC) or dominant negative (DN-CaMKIIδC) mutants of to or CaMKIIδC that enhanced activation of CaMKIIδC is sufficient to trigger cardiac myocyte apoptosis via the mitochondrial apoptotic pathway and that inhibition of CaMKII protects cardiomyocytes not only from β1AR-induced apoptosis, as (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar), but from a number of other death-inducing stimuli as These that activation of CaMKIIδC is an important common intermediate in the death signaling pathway of many different stimuli that apoptosis in heart muscle of constitutively active CaMKIIδC was by the with using the whereas dominant negative CaMKIIδC was by the with as W. Zhu W.Z. Wang S.Q. Yang D. Crow M.T. Xiao R.P. Cheng H. Circ. Res. 2004; 95: 798-806Crossref PubMed Scopus (165) Google Scholar). (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar) was by Brown the of of The and of the target gene in cells W. Zhu W.Z. Wang S.Q. Yang D. Crow M.T. Xiao R.P. Cheng H. Circ. Res. 2004; 95: 798-806Crossref PubMed Scopus (165) Google Scholar). was from the of of Cell and cardiac myocytes from the of using a and with adenoviral a multiplicity of as W. Zhu W.Z. Wang S.Q. Yang D. Crow M.T. Xiao R.P. Cheng H. Circ. Res. 2004; 95: 798-806Crossref PubMed Scopus (165) Google Scholar, Y.Y. Wang S.Q. Zhu W.Z. A. Kobilka B.K. B. Wang S. Lakatta E.G. Cheng H. Xiao R.P. Am. J. Physiol. 2000; PubMed Google Scholar). myocytes a of to on or in with The culture was and and gene transfer was by adenoviral rat or the culture The with cells cultured and was by cardiomyocytes with an with in of M. R. Xiao R.P. J. 2005; PubMed Scopus Google Scholar). cultured adult rat cardiomyocytes with the in the or of of cells with a CaMKII or or expression of In of cultured cardiac myocytes with or as in the or of with or expression of Cell apoptosis was by deoxynucleotidyltransferase-mediated dUTP nick end as (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar). The cells was by cardiac myocytes in culture was also by as (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar). phosphorylation of was by using a D. Kuschel M. Kuramochi T. Zhu W. Cheng H. Xiao R.P. J. Biol. Chem. 2000; 275: 22532-22536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 27Wang W. Zhu W.Z. Wang S.Q. Yang D. Crow M.T. Xiao R.P. Cheng H. Circ. Res. 2004; 95: 798-806Crossref PubMed Scopus (165) Google Scholar). the expression of and mutant CaMKIIδC, cell of in a and using or and with a the to the and with a The used as protein was with an of CaMKII activity was as W. Zhu W.Z. Wang S.Q. Yang D. Crow M.T. Xiao R.P. Cheng H. Circ. Res. 2004; 95: 798-806Crossref PubMed Scopus (165) Google Scholar). cell of was with in a β-gal, and The proteins with a specific to the kinase activity by the to the as W. Zhu W.Z. Wang S.Q. Yang D. Crow M.T. Xiao R.P. Cheng H. Circ. Res. 2004; 95: 798-806Crossref PubMed Scopus (165) Google Scholar). and of was using a A. H. A. S. Circ. Res. 2000; 87: PubMed Scopus Google Scholar). the and mitochondrial used c or a cytosolic a mitochondrial and from whereas and was from are as the was by of or of was to be of CaMKIIδC in studies have shown that CaMKII activation has been implicated in β1AR-induced apoptosis of adult cardiomyocytes (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar) and Ca2+ apoptosis of cardiomyocytes X. Zhang X. H. D.M. J. R. Circ. Res. 2005; 97: PubMed Scopus Google Scholar). Here, we to whether there is a the activation of CaMKIIδC and heart muscle cell apoptosis. specifically enhanced or CaMKIIδC activity by adenoviral gene transfer of a constitutively active (CA-CaMKIIδC) or a dominant negative CaMKIIδC mutant The expression of and DN-CaMKIIδC was by with an or an in cultured adult rat cardiomyocytes The activity of CaMKII, kinase by a was by in cardiomyocytes with whereas it was suppressed by in cardiomyocytes with the of CaMKII we with the kinase activity the expression of CA-CaMKIIδC and DN-CaMKIIδC a and respectively, in the phosphorylation of D. Zhu W. Xiao B. Chen Lakatta E.G. Xiao R.P. Cheng H. Circ. Res. PubMed Scopus Google Scholar). These that we specifically or CaMKIIδC activity using adenoviral gene transfer in cultured adult rat cardiac CaMKIIδC to Cell the effect of CaMKIIδC signaling on myocyte expression of CA-CaMKIIδC increased cardiomyocyte apoptosis, by increased cells and and by whereas overexpression of not apoptosis in these cells and with the kinase activity under the conditions CA-CaMKIIδC-mediated myocyte apoptosis adenoviral gene transfer and increased in a of CaMKII activity with the or co-expression of the DN-CaMKIIδC mutant suppressed apoptosis CA-CaMKIIδC protein and the kinase activity with the of to the cultured cells and with the of myocyte apoptosis with the of CaMKII activity and the activity of expressed CA-CaMKIIδC and its apoptosis in cultured cardiac of of with in of the of cells, as in CaMKII activity by of cells with and of mediated myocyte apoptosis and the in the activity of CaMKII was with the in the of apoptotic cells with of in by CaMKIIδC whether the mitochondrial death pathway is involved in heart muscle cell apoptosis, cytochrome c release the was in by that cytochrome c was increased in the cytosolic but in mitochondrial in cardiomyocytes but not in DN-CaMKIIδC or and that enhanced CaMKIIδC activity to cytochrome c release from the The of mitochondrial death signaling was by the that overexpression of the family suppressed CA-CaMKIIδC-mediated cardiac myocyte apoptosis and of CaMKII by the that other cell death-inducing such as increased intracellular Ca2+ acidosis, and CaMKII to myocyte cell phosphorylation of PLB, the regulator of SR phosphorylation as an intracellular of increased CaMKII was by in myocytes with or intracellular from cells with an with a of the M. R. Xiao R.P. J. 2005; PubMed Scopus Google Scholar), and by the of with cells that CaMKII activity was in to The promoted myocyte apoptosis as by increased the of apoptotic cells was increased intracellular or the of cells and of CaMKII with or protected cells not only from the but also against and myocyte apoptosis and In an no effect the of CaMKIIδC that of CaMKIIδB to the cell death, we suppressed CaMKIIδC activity using adenoviral gene transfer of inhibition of CaMKIIδC with the DN-CaMKIIδC similar to inhibition of CaMKII with myocyte apoptosis Thus, increased CaMKII activity is associated with the of a number of different cell death-inducing stimuli. Expression of DN-CaMKIIδC is to the kinase activity and the associated cell death as that activation of CaMKIIδC is a common intermediate in the signaling by multiple stimuli that myocyte apoptotic inhibition of CaMKIIδC by adenoviral gene transfer of DN-CaMKIIδC and cell death in cultured adult rat cardiomyocytes. culture and adenoviral gene transfer of DN-CaMKIIδC or a the cells with or an and was Expression of DN-CaMKIIδC both and intracellular cell as in cells from to In the there are isoform-specific activation of CaMKIIδC by expression of a constitutively active mutant is sufficient to trigger myocyte apoptosis. inhibition of CaMKII protects myocytes not only from adenoviral gene transfer of but also from that by increased intracellular intracellular acidosis, and These the that enhanced activation of CaMKIIδC in cardiac myocytes is a common intermediate in the death signaling by diverse cell death-inducing stimuli. of CaMKIIδC as a a of is the cells, apoptotic signaling of cardiac myocytes be the receptor-mediated pathway activation of and and the mitochondrial or the of which the release of cytochrome of apoptotic and the activation of and M.T. K. Circ. Res. 2004; 95: PubMed Scopus Google Scholar, M. C.E. Ding T. X. G. Cell. Full Text PDF PubMed Scopus Google Scholar). In to the pathway that death signaling from a of death the pathway a of and intracellular that signals on the to the release of a number of proteins that or The has shown that activation of the cardiac cytosolic CaMKII isoform, CaMKIIδC, is a common intermediate that various apoptotic stimuli and the apoptotic signals to the mitochondrial death as by the in mitochondrial cytochrome c release and the effect of an important of family Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). the have a intermediate involved in heart muscle cell and on of the pathogenesis of heart failure and to a important myocyte or the of heart failure by various CaMKII has demonstrated that stimulation of such as to cardiac myocyte hypertrophy via a signaling pathway nuclear Ca2+ activation of nuclear CaMKII and activation of Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). this nuclear hypertrophy signaling pathway be activated by the Ca2+ that cause myocyte X. Zhang T. J. X. T.A. J.R. E.N. Chen J. Brown J.H. Bers D.M. J. Clin. Investig. 2006; PubMed Scopus Google Scholar). Thus, it is to that the distinct intracellular of cardiac CaMKIIδB and CaMKIIδC myocytes to and Ca2+ signals and different is by studies in CaMKII overexpression of the CaMKIIδC isoform to of substrates involved in cardiac excitation-contraction in SR Ca2+ and cardiomyopathy T. L.S. Dalton N.D. S. Ross J. Bers D.M. Brown J.H. Circ. Res. 2003; PubMed Scopus Google Scholar, L.S. Zhang T. Chen J. Brown J.H. Bers D.M. Circ. Res. 2003; PubMed Scopus Google Scholar), whereas overexpression of the nuclear CaMKIIδB isoform to cardiac gene expression 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 J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). These that the intracellular of the kinase its and to and pathological stimuli. studies have shown that overexpression of CaMKIIδC but not CaMKIIδB enhances cardiac myocyte apoptosis (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar). The has demonstrated that increased activation of CaMKIIδC is sufficient to trigger cardiac myocyte apoptosis, whereas isoform-specific inhibition of CaMKIIδC is to cardiomyocytes against multiple death-inducing These an the of heart failure and death in cardiac CaMKIIδC T. L.S. Dalton N.D. S. Ross J. Bers D.M. Brown J.H. Circ. Res. 2003; PubMed Scopus Google Scholar, L.S. Zhang T. Chen J. Brown J.H. Bers D.M. Circ. Res. 2003; PubMed Scopus Google Scholar) with cardiac CaMKIIδB 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 J. Brown J.H. J. Biol. Chem. 2002; 277: 1261-1267Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). is to the distinct of these cardiac CaMKII and regulation under a of or pathological of CaMKII in the of of that of CaMKII as an important heart this kinase plays a role in cardiac excitation-contraction and pacemaker activity (10Xiao R.P. Cheng P. Lederer W.J. Suzuki T. Lakatta E.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9659-9663Crossref PubMed Scopus (180) Google Scholar, 11Yuan W Bers D.M. Am. J. Physiol. 1994; 267: H982-H993PubMed Google Scholar, 12Dzhura I. Wu Y. Colbran R.J. Balser J.R. Anderson M.E. Nat. Cell Biol. 2000; 2: 173-177Crossref PubMed Scopus (302) Google Scholar, 13Grueter C.E. Abiria S.A. Dzhura I. Wu Y. Ham A.J. Mohler P.J. Anderson M.E. Colbran R.J. Mol. Cell. 2006; 23: 641-650Abstract Full Text PDF PubMed Scopus (170) Google Scholar, R.A. Mattiazzi A. Bers D.M. Am. J. Physiol. 1995; 268: H703-H712Crossref PubMed Google Scholar, 15Hagemann D. Kuschel M. Kuramochi T. Zhu W. Cheng H. Xiao R.P. J. Biol. Chem. 2000; 275: 22532-22536Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, T.M. Zhou Y.Y. Bogdanov K.Y. Yang D. Kuschel M. Cheng H. Xiao R.P. Circ. Res. 2000; 87: 760-767Crossref PubMed Scopus (153) Google Scholar). studies have demonstrated that sustained β1AR a of the cardiomyocyte apoptosis via activation of CaMKII of the protein kinase pathway (25Zhu W.Z. Wang S.Q. Chakir K. Yang D. Zhang T. Brown J.H. Devic E. Kobilka B.K. Cheng H. Xiao R.P. J. Clin. Investig. 2003; 111: 617-625Crossref PubMed Scopus (378) Google Scholar). CaMKII expression is increased in the of humans and (5Hoch B. Meyer R. Hetzer R. Krause E.G. Karczewski P. Circ. Res. 1999; 84: 713-721Crossref PubMed Scopus (279) Google Scholar, 22Zhang T. Brown J.H. Cardiovasc. Res. 2004; 63: 476-486Crossref PubMed Scopus (252) Google Scholar, 23Xu X. Yang D. Ding J.H. Wang W. Chu P.H. Dalton N.D. Wang H.Y. Bermingham Jr., J.R. Ye Z. Liu F. Rosenfeld M.G. Manley J.L. Ross Jr., J. Chen J. Xiao R.P. Cheng H. Fu X.D. Cell. 2005; 120: 59-72Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 24Ai X. Curran J.W. Shannon T.R. Bers D.M. Pogwizd S.M. Circ. Res. 2005; 97: 1314-1322Crossref PubMed Scopus (578) Google Scholar). overexpression of the cytosolic isoform CaMKIIδC heart failure T. L.S. Dalton N.D. S. Ross J. Bers D.M. Brown J.H. Circ. Res. 2003; PubMed Scopus Google Scholar), which is associated with enhanced SR Ca2+ SR Ca2+ and enhanced SR Ca2+ release T. L.S. Dalton N.D. S. Ross J. Bers D.M. Brown J.H. Circ. Res. 2003; PubMed Scopus Google Scholar). β1AR-induced gene a of cardiac is mediated by a in cultured rat cardiac myocytes M.R. Am. J. Physiol. 2006; PubMed Scopus Google Scholar). In inhibition of CaMKII activity prevents cardiac and a heart (17Anderson M.E. Braun A.P. Wu Y. Lu T. Wu Y. Schulman H. Sung R.J. J. Pharmacol. Exp. Therap. 1998; 287: 996-1006PubMed Google Scholar, 18Anderson M.E. Trends Cardiovasc. Med. 2004; 14: 152-161Crossref PubMed Scopus (68) Google Scholar). CaMKII inhibition in vivo cardiac and and myocardial cardiac R. Khoo M.S.C. Wu Y. Yang Y. Grueter C.E. Ni G. Price Jr., E.E. Thiel W. Guatimosim S. Song L.S. Madu E.C. Shah A.N. Vishnivetskaya T.A. Atkinson J.B. Gurevich V.V. Salama G. Lederer W.J. Colbran R.J. Anderson M.E. Nat. Med. 2005; 11: 409-417Crossref PubMed Scopus (488) Google Scholar). These studies have demonstrated that of CaMKII signaling is involved in many of cardiac hypertrophy and heart CaMKII as a target the of heart

OBJECTIVES: Baicalin and its aglycone baicalein are the major flavonoid components of the root of Scutellaria baicalensis. Recent studies have shown that they can attenuate oxidative stress in various in vitro models as they possess potent antioxidant activities. This study investigated alternative protective mechanisms of baicalein in a cardiomyocyte model. METHODS: Neonatal rat cardiomyocytes pretreated with the test compound were subjected to hypoxia/reoxygenation. The extent of cellular damage was accessed by the amount of released lactate dehydrogenase RESULTS: Pretreatment with baicalein up to 10 microM reduced lactate dehydrogenase release significantly (P<0.001), while pretreatment with baicalin up to 100 microM was ineffective. The cardioprotective effect of baicalein is not due to its antioxidant effect, because an adverse effect rather than a protective effect was observed when baicalein was present during hypoxia. Cotreatment with N-acetylcysteine attenuated the protective effect of baicalein and concomitantly increased intracellular reactive oxygen species level and the cytotoxic effect of baicalein, but N-acetylcysteine alone did not have such effects. In addition, cotreatment with catalase, but not superoxide dismutase or mannitol, reversed the cardioprotective effect of baicalein, suggesting the involvement of hydrogen peroxide in the cardioprotective mechanism. The NAD(P)H:quinone oxidoreductase inhibitors dicoumarol and chrysin also abolished the cardioprotective effect of baicalein. While pretreatment with baicalein did not increase antioxidant enzyme activities, it alleviated calcium accumulation in cardiomyocytes undergoing simulated ischemia. CONCLUSION: These results highlight the important role of hydrogen peroxide produced during the auto-oxidation of baicalein in the cardioprotective effect of baicalein.