Critical Role of Nuclear Calcium/Calmodulin-dependent Protein Kinase IIδB in Cardiomyocyte Survival in Cardiomyopathy
2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês
10.1074/jbc.m109.003186
ISSN1083-351X
AutoresGillian H. Little, Aman Saw, Yan Bai, Joan Dow, Paul Marjoram, Boris Z. Simkhovich, Justin Leeka, Larry Kedes, Robert A. Kloner, Coralie Poizat,
Tópico(s)Cardiac electrophysiology and arrhythmias
ResumoCalcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in cardiac contractility and heart disease. However, the specific role of alternatively spliced variants of CaMKII in cardiac disease and apoptosis remains poorly explored. Here we report that the δB subunit of CaMKII (CaMKIIδB), which is the predominant nuclear isoform of calcium/calmodulin-dependent protein kinases in heart muscle, acts as an anti-apoptotic factor and is a novel target of the antineoplastic and cardiomyopathic drug doxorubicin (Dox (adriamycin)). Hearts of rats that develop cardiomyopathy following chronic treatment with Dox also show down-regulation of CaMKIIδB mRNA, which correlates with decreased cardiac function in vivo, reduced expression of sarcomeric proteins, and increased tissue damage associated with Dox cardiotoxicity. Overexpression of CaMKIIδB in primary cardiac cells inhibits Dox-mediated apoptosis and prevents the loss of the anti-apoptotic protein Bcl-2. Specific silencing of CaMKIIδB by small interfering RNA prevents the formation of organized sarcomeres and decreases the expression of Bcl-2, which all mimic the effect of Dox. CaMKIIδB is required for GATA-4-mediated co-activation and binding to the Bcl-2 promoter. These results reveal that CaMKIIδB plays an essential role in cardiomyocyte survival and provide a mechanism for the protective role of CaMKIIδB. These results suggest that selective targeting of CaMKII in the nuclear compartment might represent a strategy to regulate cardiac apoptosis and to reduce Dox-mediated cardiotoxicity. Calcium/calmodulin-dependent protein kinase II (CaMKII) plays a central role in cardiac contractility and heart disease. However, the specific role of alternatively spliced variants of CaMKII in cardiac disease and apoptosis remains poorly explored. Here we report that the δB subunit of CaMKII (CaMKIIδB), which is the predominant nuclear isoform of calcium/calmodulin-dependent protein kinases in heart muscle, acts as an anti-apoptotic factor and is a novel target of the antineoplastic and cardiomyopathic drug doxorubicin (Dox (adriamycin)). Hearts of rats that develop cardiomyopathy following chronic treatment with Dox also show down-regulation of CaMKIIδB mRNA, which correlates with decreased cardiac function in vivo, reduced expression of sarcomeric proteins, and increased tissue damage associated with Dox cardiotoxicity. Overexpression of CaMKIIδB in primary cardiac cells inhibits Dox-mediated apoptosis and prevents the loss of the anti-apoptotic protein Bcl-2. Specific silencing of CaMKIIδB by small interfering RNA prevents the formation of organized sarcomeres and decreases the expression of Bcl-2, which all mimic the effect of Dox. CaMKIIδB is required for GATA-4-mediated co-activation and binding to the Bcl-2 promoter. These results reveal that CaMKIIδB plays an essential role in cardiomyocyte survival and provide a mechanism for the protective role of CaMKIIδB. These results suggest that selective targeting of CaMKII in the nuclear compartment might represent a strategy to regulate cardiac apoptosis and to reduce Dox-mediated cardiotoxicity. Calcium/calmodulin-dependent protein kinase II (CaMKII) 2The abbreviations used are:CaMKIIcalcium/calmodulin-dependent protein kinase IIDoxdoxorubicinsiRNAsmall interfering RNANLSnuclear localization signalHAhemagglutininAd-GFPadenovirus-green fluorescent proteinFSfractional shorteningRTreverse transcriptionEMSAelectrophoretic mobility shift assayGAPDHglyceraldehyde-3-phosphate dehydrogenaseCAAcardiac α-actinHDAChistone deacetylase. is a serine/threonine kinase regulated by calcium that is implicated in numerous cellular functions. The δ subunit of CaMKII predominates in the adult heart, and two isoforms generated by alternative splicing, δB and δC, are detected at the protein level in this organ (1Edman C.F. Schulman H. Biochim. Biophys. Acta. 1994; 1221: 89-101Crossref PubMed Scopus (155) Google Scholar, 2Baltas L.G. Karczewski P. Krause E.G. FEBS Lett. 1995; 373: 71-75Crossref PubMed Scopus (55) Google Scholar, 3Hagemann D. Hoch B. Krause E.G. Karczewski P. J. Cell. Biochem. 1999; 74: 202-210Crossref PubMed Scopus (27) Google Scholar, 4Zhang T. Brown J.H. Cardiovasc. Res. 2004; 63: 476-486Crossref PubMed Scopus (240) Google Scholar, 5Zhang T. Miyamoto S. Brown J.H. Recent. Prog. Horm. Res. 2004; 59: 141-168Crossref PubMed Scopus (57) Google Scholar). In contrast, the γ isoform is expressed at very low levels in heart muscle, whereas the α and β subunits are not detected at all (6Tobimatsu T. Fujisawa H. J. Biol. Chem. 1989; 264: 17907-17912Abstract Full Text PDF PubMed Google Scholar, 7Singer H.A. Benscoter H.A. Schworer C.M. J. Biol. Chem. 1997; 272: 9393-9400Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). CaMKIIδ isoforms are highly homologous with the exception of a variable domain generated by alternative splicing (4Zhang T. Brown J.H. Cardiovasc. Res. 2004; 63: 476-486Crossref PubMed Scopus (240) Google Scholar, 5Zhang T. Miyamoto S. Brown J.H. Recent. Prog. Horm. Res. 2004; 59: 141-168Crossref PubMed Scopus (57) Google Scholar). CaMKIIδB contains an 11-amino acid nuclear localization signal (NLS) not present in the δC, which directs the enzyme to the cell nucleus (8Brocke L. Srinivasan M. Schulman H. J. Neurosci. 1995; 15: 6797-6808Crossref PubMed Google Scholar, 9Srinivasan M. Edman C.F. Schulman H. J. Cell Biol. 1994; 126: 839-852Crossref PubMed Scopus (232) Google Scholar). The relative abundance of particular subunits dictates the subcellular localization of the enzyme (9Srinivasan M. Edman C.F. Schulman H. J. 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U.S.A. 2000; 97: 14400-14405Crossref PubMed Scopus (419) Google Scholar). Recently, we and others have shown that the cardiac enzyme CaMKIIδB has characteristics distinct from CaMKI/IV. CaMKIIδB selectively transmits signals to HDAC4 and not to other class II HDACs, through phosphorylation of Ser-210, Ser-467, and Ser-632 (15Backs J. Song K. Bezprozvannaya S. Chang S. Olson E.N. J. Clin. Invest. 2006; 116: 1853-1864Crossref PubMed Scopus (390) Google Scholar, 16Little G.H. Bai Y. Williams T. Poizat C. J. Biol. Chem. 2007; 282: 7219-7231Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). CaMK signaling plays a significant role in cardiac disease (for review see Ref. 4Zhang T. Brown J.H. Cardiovasc. Res. 2004; 63: 476-486Crossref PubMed Scopus (240) Google Scholar). α-Adrenergic stimulation, endothelin-1, or leukemia inhibitory factor promote hypertrophic growth through activation of CaMK signaling in isolated cells. CaMKII inhibition in mice markedly inhibits cardiac hypertrophy and dysfunction after β-adrenergic stimulation or myocardial infarction (17Zhang R. Khoo M.S. 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 (468) Google Scholar). Increased CaMKII activity has been reported in several animal models of cardiac hypertrophy and heart failure. Decreased CaMKII activity and expression were observed in a number of animal models of myocardial infarction (18Netticadan T. Temsah R.M. Kawabata K. Dhalla N.S. Circ. Res. 2000; 86: 596-605Crossref PubMed Scopus (97) Google Scholar, 19Mishra S. Sabbah H.N. Jain J.C. Gupta R.C. Am. J. Physiol. Heart Circ. Physiol. 2003; 284: H876-H883Crossref PubMed Scopus (42) Google Scholar). Transgenic mice with high cardiac levels of CaMKIIδB or -δC develop dilated cardiomyopathy (20Zhang 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 (208) Google Scholar, 21Maier L.S. Zhang T. Chen L. DeSantiago J. Brown J.H. Bers D.M. Circ. Res. 2003; 92: 904-911Crossref PubMed Scopus (382) Google Scholar). Recently, increased activity of both the δB and δC splice variants of CaMKII were reported in patients with end-stage idiopathic dilated cardiomyopathy and ischemic cardiomyopathy (22Bossuyt J. Helmstadter K. Wu X. Clements-Jewery H. Haworth R.S. Avkiran M. Martin J.L. Pogwizd S.M. Bers D.M. Circ. Res. 2008; 102: 695-702Crossref PubMed Scopus (112) Google Scholar). Deletion of all CaMKIIδ isoforms in mouse heart decreases cardiac hypertrophy and remodeling induced by pressure overload (23Backs J. Backs T. Neef S. Kreusser M.M. Lehmann L.H. Patrick D.M. Grueter C.E. Qi X. Richardson J.A. Hill J.A. Katus H.A. Bassel-Duby R. Maier L.S. Olson E.N. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 2342-2347Crossref PubMed Scopus (316) Google Scholar). Despite clear evidence for a role of CaMKII signaling in cardiac diseases, the specific role and contribution of CaMKIIδ isoforms generated after alternative splicing still remain unclear. Doxorubicin (Dox) (adriamycin)) is one of the most effective anti-cancer agents discovered so far. Despite its high efficacy in the treatment of many neoplastic diseases, chronic administration is limited because of severe side effects that lead to cardiomyopathy and congestive heart failure (for reviews see Refs. 24Singal P.K. Iliskovic N. N. Engl. J. Med. 1998; 339: 900-905Crossref PubMed Scopus (1528) Google Scholar, 25Wallace K.B. Pharmacol. Toxicol. 2003; 93: 105-115Crossref PubMed Scopus (312) Google Scholar, 26Takemura G. Fujiwara H. Prog. Cardiovasc. Dis. 2007; 49: 330-352Crossref PubMed Scopus (626) Google Scholar). Dox cardiotoxicity is due in part to the down-regulation of contractile protein mRNAs in vivo and in primary cardiac cells (27Ito H. Miller S.C. Billingham M.E. Akimoto H. Torti S.V. Wade R. Gahlmann R. Lyons G. Kedes L. Torti F.M. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 4275-4279Crossref PubMed Scopus (231) Google Scholar). This effect is mediated by a loss of cardiac transcription factors such as Mef2C, NKX2.5 (28Poizat C. Sartorelli V. Chung G. Kloner R.A. Kedes L. Mol. Cell. Biol. 2000; 20: 8643-8654Crossref PubMed Scopus (85) Google Scholar), and GATA-4 (29Aries A. Paradis P. Lefebvre C. Schwartz R.J. Nemer M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6975-6980Crossref PubMed Scopus (222) Google Scholar). Dox side effects are also due to the proteasome-mediated degradation of the co-activator p300 in primary cardiomyocytes (28Poizat C. Sartorelli V. Chung G. Kloner R.A. Kedes L. Mol. Cell. Biol. 2000; 20: 8643-8654Crossref PubMed Scopus (85) Google Scholar), following activation of p38 mitogen-activated protein kinase (30Poizat C. Puri P.L. Bai Y. Kedes L. Mol. Cell. Biol. 2005; 25: 2673-2687Crossref PubMed Scopus (98) Google Scholar). Cardiac apoptosis is a major factor in the development of the cardiomyopathy and heart failure induced by Dox (30Poizat C. Puri P.L. Bai Y. Kedes L. Mol. Cell. Biol. 2005; 25: 2673-2687Crossref PubMed Scopus (98) Google Scholar, 31Arola O.J. Saraste A. Pulkki K. Kallajoki M. Parvinen M. Voipio-Pulkki L.M. Cancer Res. 2000; 60: 1789-1792PubMed Google Scholar, 32Ueno M. Kakinuma Y. Yuhki K. Murakoshi N. Iemitsu M. Miyauchi T. Yamaguchi I. J. Pharmacol. Sci. 2006; 101: 151-158Crossref PubMed Scopus (134) Google Scholar). There is some evidence that CaMK signaling plays a role in programmed cell death in the heart. Several studies have documented a pro-apoptotic role of CaMKII in cardiomyocyte apoptosis following β1-adrenergic stimulation (33Yang Y. Zhu W.Z. Joiner M.L. Zhang R. Oddis C.V. Hou Y. Yang J. Price E.E. Gleaves L. Eren M. Ni G. Vaughan D.E. Xiao R.P. Anderson M.E. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H3065-H3075Crossref PubMed Scopus (117) Google Scholar), ischemia-reperfusion injury (34Vila-Petroff M. Salas M.A. Said M. Valverde C.A. Sapia L. Portiansky E. Hajjar R.J. Kranias E.G. Mundiña-Weilenmann C. Mattiazzi A. Cardiovasc. Res. 2007; 73: 689-698Crossref PubMed Scopus (187) Google Scholar), and UV light-induced DNA damage (35Wright S.C. Schellenberger U. Ji L. Wang H. Larrick J.W. FASEB J. 1997; 11: 843-849Crossref PubMed Scopus (81) Google Scholar). Although two reports have demonstrated a pro-apoptotic role of the cytoplasmic δC isoform of CaMKII (36Zhu 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. Invest. 2003; 111: 617-625Crossref PubMed Scopus (365) Google Scholar, 37Zhu W. Woo A.Y. Yang D. Cheng H. Crow M.T. Xiao R.P. J. Biol. Chem. 2007; 282: 10833-10839Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), the function of the major nuclear isoform, CaMKIIδB, in cardiac apoptosis remains unknown. To further understand the molecular genetic mechanisms of Dox-mediated cardiotoxicity and apoptosis, we profiled gene expression in the heart of animals with degenerative cardiomyopathy after treatment with Dox. Here we report that chronic administration of Dox leads to the selective decrease of CaMKIIδB mRNA in vivo, which parallels the known side effects of Dox. In primary neonatal rat cardiomyocytes, siRNA-mediated depletion of CaMKIIδB results in abnormal sarcomere organization and leads to a severe loss of the anti-apoptotic protein Bcl-2, suggesting a protective role of the kinase. CaMKIIδB exerts its protective effect by regulating GATA-4 binding to the Bcl-2 promoter. Importantly, forced expression of CaMKIIδB inhibits GATA-4 and Bcl-2 down-regulation by Dox. Collectively, our results demonstrate that nuclear CaMKIIδB is a novel major target of the cardiotonic agent Dox and that a persistent level of CaMKIIδB is required for cardiomyocyte integrity and survival. The chronic rat model of Dox-induced cardiomyopathy was generated as described before (38Schwarz E.R. Pollick C. Dow J. Patterson M. Birnbaum Y. Kloner R.A. Cardiovasc. Res. 1998; 39: 216-223Crossref PubMed Scopus (68) Google Scholar). For the microarray analysis and echocardiography, a total of five female Sprague-Dawley rats were used. Three rats received weekly intravenous injections of Dox in equally divided doses for a total cumulative dose of 17–20 mg/kg over 7–10 weeks. Two rats served as controls and received weekly saline injections. Transthoracic echocardiography was performed in all animals at the beginning of the treatment (base line) at 5 weeks and 2 weeks after discontinuation of Dox administration. After the animals were anesthetized with a mixture of ketamine and xylazine, a 7.5-MHz standard pediatric transducer was connected to an echocardiographic computer console (Hewlett Packard 1500, Andover, MA). Left ventricular end-diastolic and end-systolic diameters were measured using two-dimensional guided M-mode imaging. Fractional shortening was calculated from the mean value of three measurements and was used to evaluate cardiac function of the animals during the treatment. To validate the microarray and echocardiography, we added more animals to our study and performed transthoracic echocardiography in rats injected with saline (n = 7) or Dox (n = 8) as described before. Experiments were conducted in accordance with the institutional guidelines for the use and care of laboratory animals, which conforms to the Guide for Care and Use of Laboratory Animals (National Institutes of Health Publication 85-23). Electron microscopy of the left ventricle was performed in all five rats to confirm the degree of cardiac damage induced by Dox. Rat hearts were collected at the end of Dox treatment and were fixed in 0.1 m sodium phosphate buffer containing 2.5% glutaraldehyde in 0.1 m sodium cacodylate. After washing, the samples were post-fixed in 1% osmium tetroxide, 0.1 m cacodylate, prestained in 1% uranyl acetate, dehydrated in a graded ethanol series, and then embedded in 100% epoxy resin. After sectioning, the samples were mounted on parlodian-coated grids, stained with lead citrate, and examined with a Zeiss TEM electron microscope (Microscopy Core Facility of the Doheny Eye Institute, University of Southern California). The cardiomyopathy was scored on a 1–5 scale. Gene expression profiling was performed as described previously (39Simkhovich B.Z. Marjoram P. Poizat C. Kedes L. Kloner R.A. Arch. Biochem. Biophys. 2003; 420: 268-278Crossref PubMed Scopus (37) Google Scholar). Briefly, total RNA was extracted from each heart tissue using TRIzol reagent (Invitrogen). Biotin-labeled cRNA was prepared and used to hybridize GeneChip Rat U34 array set (Affymetrix). cRNA from each heart was hybridized to one array. Analysis of gene expression was done as described previously using Dchip software (39Simkhovich B.Z. Marjoram P. Poizat C. Kedes L. Kloner R.A. Arch. Biochem. Biophys. 2003; 420: 268-278Crossref PubMed Scopus (37) Google Scholar). Replicates for each condition (duplicates for controls and triplicates for Dox-treated rats) were used to estimate mean expression levels along with associated standard errors. Genes with a fold ratio above +2 for up-regulated genes or below 2 for down-regulated genes were selected. The p value resulting from a t test for a change in the gene expression level was less than 0.05. Total RNA was extracted from heart tissue or primary cardiomyocytes in culture as above and cleaned using an RNeasy kit (Qiagen). Quantitative RT-PCR was carried out as described in Ref. 30Poizat C. Puri P.L. Bai Y. Kedes L. Mol. Cell. Biol. 2005; 25: 2673-2687Crossref PubMed Scopus (98) Google Scholar. Briefly, 2 μg of total RNA was used for first strand cDNA synthesis. Random primers were used in a 20-μl reaction volume in the presence of 10 μm dNTPs and [32P]dCTP. 10 ng of cRNA was used for PCR amplification with gene-specific primers used under condition of linear range. Primer sequences are as follows: cardiac α-actin, sense 5′-TGTGACGACGAGGAGACCACAGCT-3′ and antisense 5′-CTGAGCCTCGTCACCTACATAG-3′; cardiac troponin I, sense 5′-GATGAGAGCAGCGATGCGGCTG-3′ and antisense 5′-GCATAGGTCCTGAAGCTCTTC-3′; myosin light chain 2-a, sense 5′-GCAAGCTGCAGCCACCAA-3′ and antisense 5′-AAGGCACTCAGGATGGCT-3′; Mef2C, sense 5′-TTGGGAACTGAGCTGTGC-3′ and antisense 5′-CTGGAACAGCTTGTTGGTG-3′; NKX2.5, sense 5′-CGACGGAAGCCACGCGTGC-3′ and antisense 5′-GCTCCAGAGTCTGGTCCTG-3′; CaMKIIδB sense 5′-AGGAAGTCCAGTTCGAGTGTTC-3′ and antisense 5′-CAGGATGATAGTGTGGATTG-3′; and p300 sense 5′-GCATGCTGGTGGAGCTGC-3′ and antisense 5′-GATGGCAGTGAGCAATTGGC-3′. Ribosomal RNA primers were used as internal control and were mixed with CompetimersTM to amplify the genes of interest in the same linear range. PCR products were separated on a 5% acrylamide gel and quantitated with a STORM scanner (GE Healthcare). Results were normalized relative to 18 S RNA expression. Real time PCRs were performed after RT-PCR (RETROscriptTM, Ambion) with various dilutions of cDNA template using IQ SYBR Green and an Opticon 2 reader (Bio-Rad). Primer sequences for both quantitative RT-PCR and real time PCR are as follows: α-actin sense 5′-TCTCTTCCAGCCCTCTTTCA-3′ and antisense 5′-CCCCCAATCCAGACAGAGTA-3′; MLC2-a sense 5′-GCTGCATTGACCAGAACAGA-3′ and antisense 5′-GCTGCTTGAACTCCTCCTTG-3′; MLC2-v sense 5′-AAAGAGGCTCCAGGTCCAAT-3′ and antisense 5′-AAAAGCTGCGAACATCTGGT-3′; CaMKIIδB sense 5′-AGGAAGTCCAGTTCGAGTGTTC-3′ and antisense 5′- CAGGATGATAGTGTGGATTG-3′; CaMKIIδC sense 5′-CCGGATGGGGTAAAGGAG and antisense 5′-CAGGATGATAGTGTGGATTG-3′. Each primer pair produced a single band after amplification, and melting curves were clean. Standard curves of reverse-transcribed RNA from heart tissue were generated with 18 S primer pairs and analyzed in triplicate by serial dilutions. Calibration curves were linear over the complete range for all primers (R2 > 0.989). Concentrations were obtained by comparing Ct values of the samples to the standard curves using Opticon Monitor 2 software. Results are expressed as relative expression compared with our standard and are corrected for 18 S or GAPDH. The 1281-bp fragment of the Bcl-2 promoter (Addgene plasmid 15382) (40Heckman C.A. Mehew J.W. Ying G.G. Introna M. Golay J. Boxer L.M. J. Biol. Chem. 2000; 275: 6499-6508Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) was a gift from Dr. Linda Boxer (Stanford University). Doxorubicin was purchased from Calbiochem, and all cell treatments were at 1 μm. Ad-GFP, Ad-CaMKIIδB, siCtrl, and siCaMKIIδB were described previously (16Little G.H. Bai Y. Williams T. Poizat C. J. Biol. Chem. 2007; 282: 7219-7231Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Ad-CaMKIIδC was generously provided by Dr. Joan Heller-Brown (University of California, San Diego). Anti-HA (F-7), anti-Bcl-2 (C-2), anti-Bax (N-20), anti-P53 (FL-393), anti-GATA-4 (H-112), anti-CaMKII (H-300), and anti-GAPDH (V-18) antibodies were from Santa Cruz Biotechnology. Anti-α-actinin was from Sigma. Anti-phospho-Akt was from Cell Signaling. The anti-CaMKIIδ-specific antibody was a gift from Dr. Donald Bers (University of California, Davis). Neonatal rat cardiomyocytes were prepared as described previously (30Poizat C. Puri P.L. Bai Y. Kedes L. Mol. Cell. Biol. 2005; 25: 2673-2687Crossref PubMed Scopus (98) Google Scholar). Transfections were carried out by calcium phosphate precipitation or using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations. siRNA luciferase experiments were as described (16Little G.H. Bai Y. Williams T. Poizat C. J. Biol. Chem. 2007; 282: 7219-7231Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Nuclear and whole cell extracts were prepared from primary neonatal rat cardiomyocytes as described previously (30Poizat C. Puri P.L. Bai Y. Kedes L. Mol. Cell. Biol. 2005; 25: 2673-2687Crossref PubMed Scopus (98) Google Scholar). Briefly, extracts were resuspended in a buffer containing 50 mm Tris, pH 7.6, 150 or 500 mm NaCl, 1 mm EDTA, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, a phosphatase and a protease inhibitor mixture (Sigma). After centrifugation 30 μg of the supernatant was separated by SDS-PAGE on 4–12% Tris-glycine gradient gels. Western blotting was described previously (16Little G.H. Bai Y. Williams T. Poizat C. J. Biol. Chem. 2007; 282: 7219-7231Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar), Detection was performed with chemifluorescence (ECF reagent, Amersham Biosciences) and a Storm scanner. To establish the specificity of siCaMKIIδB, recombinant adenoviruses expressing CaMKIIδB (Ad-CaMKIIδB) and CaMKIIδC (Ad-CaMKIIδC) carrying HA tags were produced and amplified as described previously (16Little G.H. Bai Y. Williams T. Poizat C. J. Biol. Chem. 2007; 282: 7219-7231Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Primary cardiomyocytes were transfected with increasing concentrations of siCaMKIIδB or siControl using Lipofectamine. 24 h later, the cells were infected with Ad-CaMKIIδB or Ad-CaMKIIδC, and exogenous CaMKIIδB and CaMKIIδC was measured by Western blot using an HA antibody. For all other experiments, siCaMKIIδB or siControl was transfected in primary cardiomyocytes, and endogenous proteins were measured by Western blot 72 h later using the indicated specific primary antibodies. Apoptosis was measured in cardiomyocytes transfected with siCaMKIIδB or siControl and treated with Dox for 6, 12, and 24 h using the Cell Death Detection ELISAPLUS (Roche Applied Science). Immunofluorescence was performed as described previously (16Little G.H. Bai Y. Williams T. Poizat C. J. Biol. Chem. 2007; 282: 7219-7231Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 30Poizat C. Puri P.L. Bai Y. Kedes L. Mol. Cell. Biol. 2005; 25: 2673-2687Crossref PubMed Scopus (98) Google Scholar). For siRNA experiments, primary cardiomyocytes were transfected with 300 pmol of either siCtrl or siCaMKIIδB oligonucleotides 72 h before fixing or were maintained in media supplemented with Dox for the indicated times. For overexpression of CaMKIIδB, primary cardiomyocytes were infected with a control Ad-GFP or Ad-CaMKIIδB. 12 h later, half of the cells were maintained in normal media or in media supplemented with Dox for the indicated times. Sarcomeric structures were analyzed by indirect immunofluorescence using an antibody specific for α-actinin. Images were visualized by confocal microscopy. Apoptosis assays were carried out using the Cell Death ELISAPLUS kit from Roche Applied Science according to the manufacturer's protocol. Photometric analysis was carried out using Victor 3 (GE Healthcare). Electrophoretic mobility shift assays (EMSAs) and probes were as described (41Kobayashi S. Lackey T. Huang Y. Bisping E. Pu W.T. Boxer L.M. Liang Q. FASEB J. 2006; 20: 800-802Crossref PubMed Scopus (91) Google Scholar), with the exception that 10 μg of nuclear extract prepared from primary cardiomyocytes was used in each reaction (30Poizat C. Puri P.L. Bai Y. Kedes L. Mol. Cell. Biol. 2005; 25: 2673-2687Crossref PubMed Scopus (98) Google Scholar). To identify Dox-sensitive genes in heart muscle in vivo, we analyzed gene expression profiling in a chronic rat model of Dox-induced cardiomyopathy (38Schwarz E.R. Pollick C. Dow J. Patterson M. Birnbaum Y. Kloner R.A. Cardiovasc. Res. 1998; 39: 216-223Crossref PubMed Scopus (68) Google Scholar). Rats treated with Dox (animals 3–5) received weekly intravenous injections over 7–10 weeks, and control rats (animals 1 and 2) received weekly saline injections. To monitor the effect of Dox on cardiac contractility, transthoracic echocardiography was performed in all animals at the beginning of the treatment (base line) and at 5 and 2 weeks after the last dose (Fig. 1A). Fractional shortening (FS) was calculated as a reliable indicator of cardiac function. At 5 weeks, FS was similar to base line in control animals (supplemental Table S1). However, in the three animals that received Dox, FS decreased incrementally with time to 54, 75, and 71% of base line in animals 3–5, respectively. At the end of the treatment, FS decreased to 48 and 33% of base line for animals 3 and 4. Final functional parameters could not be measured in animal 5 which died of heart failure at week 7. Heart tissue from this animal was recovered immediately after death and was kept frozen until further analysis. Together, our results indicate a significant deterioration of cardiac function in animals 3–5. To evaluate the degree of cardiac damage exerted by Dox, heart sections from each animal were examined by electron microscopy (Fig. 1B and supplemental Table S2). The analysis revealed a normal ultrastructure of the two control animals. Animal 3 displayed a mostly intact myocardium with only slight sarcoplasmic edema, whereas animal 4 revealed sarcoplasmic and interstitial swelling characteristic of Dox cardiotoxicity. Animal 5 showed, in addition to intense sarcoplasmic and interstitial swelling, severe mitochondrial damage suggesting a pronounced cardiomyopathy (Fig. 1B and supplemental Table S2). Together, these data indicate different degrees of cardiac damage in the three animals chronically treated with Dox, animal 3 displaying almost no signs of cardiac damage, and animals 4 and 5 revealing a more profound cardiomyopathy. We next compared changes in gene expression between the hearts of control animals and the hearts of Dox-treated animals using Affymetrix Gene Chips (rat gene array U34A). cRNA from each heart was hybridized to two arrays twice, so that a total of four chips were used per animal. As expected, chronic exposure to Dox caused a decreased expression of structural proteins (27Ito H. Miller S.C. Billingham M.E. Akimoto H. Torti S.V. Wade R. Gahlmann R. Lyons G. Kedes L. Torti F.M. Proc. Natl. Acad. Sci. U.S.A. 1990; 87: 4275-4279Crossref PubMed Scopus (231) Google Scholar) such as myosin light chain-2a (MLC2-a), atrial myosin light chain 1, SM22, and α-actin. We also observed a significant decrease of mitochondrial genes such as cytochrome c and acetyl-CoA acetyltransferase (Table 1). The results of the gene chip analysis were then validated by quantitative RT-PCR analysis performed from the same RNA samples. Dox-sensitive mRNAs such as cardiac α-actin (CAA), cardiac troponin I, MLC2-a, and Mef2C were slightly decr
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