Supplement Material. chain (MLC2v) promoter to generate cardiac-specific MKK4 knockout mice (referred to

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1 Supplement Material Materials and Methods Generation of MKK Mutant Mice The mkk-flox mice (referred to as ) were previously generated and characterised. mice were mated with mice expressing Cre under a myosin light chain (MLCv) promoter to generate cardiac-specific MKK knockout mice (referred to as MKK cko ). The MLCv-Cre line (kindly provided by Dr. KR Chien, Massachusetts General Hospital, USA) is a well established model that provides ventricular specific and efficient Cre recombinase activity and is known not to cause any abnormality in cardiac morphology and function following cardiac stress. All mice used in this study were maintained in a pathogen-free facility at the University of Manchester. The animal studies were performed in accordance with the Home Office and institutional guidelines. Osmotic Minipump Infusion of Isoproterenol To generate β-adrenergic-induced cardiac hypertrophy, isoproterenol (Iso, Sigma- Aldrich) (mg/kg/day) or vehicle (ddh O) was administrated to week old male and MKK cko mice via osmotic mini-pumps (Alzet) implanted subcutaneously. Hearts were isolated and analysed for cardiac hypertrophy following 7 days of Iso infusion. In vivo Physiology Analysis In vivo hemodynamic analysis was performed using a pressure volume system (Millar Instruments). We used a.f pressure-volume catheter (SPR-9) following a protocol described previously. Maximal derived pressures were obtained during systole (dp/dt max )

2 and diastole (dp/dt min ) as indices representing cardiac contractile functions. For cardiac morphological analysis, transthoracic two dimensional and M-mode echocardiography were performed using an Acuson Sequoia C5 system (Siemens) following a protocol described previously. Parameters of intraventricular septal thickness (IVS), left ventricular posterior wall thickness (LVPW), left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD) and fractional shortening (FS) were obtained. Immunostaining of Single Cardiomyocytes Hearts from -week old mice were perfused via the aorta using a modified collagenase and protease digestion method. Dissociated cardiomyocytes were plated and fixed in % paraformaldehyde prior to being incubated with antibodies specific to MKK (:, BD Pharmingen) or to α-actinin (:5, Sigma). The secondary anti-mouse antibody conjugated to Alexa Fluoro 5 (:5, Invitrogen), or conjugated to Alexa Fluoro (:5, Invitrogen) was applied respectively to detect the immune signals, respectively. Fluorescence images were viewed with a Nikon upright confocal microscope. Histology and TUNEL Assay Freshly dissected heart tissue was fixed in % paraformaldehyde, dehydrated and embedded in paraffin. 5-7µm thick sections were cut and stained with hematoxylin & eosin or Masson s trichrome method as described 5. To calculate the mean cross-sectional area approximately 5 randomly selected cardiomyocytes were measured. 5 randomly chosen frames from Masson s trichrome stained sections were quantified to assess the degree of myocardial fibrosis using Image J software. TUNEL assay to detect apoptosis was performed on paraffin-embedded heart sections using the in situ Cell Death

3 Detection kit (Roche). Triple staining with DAPI, anti-α-actinin antibody (Sigma), and TUNEL was performed to confirm apoptotic morphology in cardiac nuclei. Preparation of Lysates and Immunoblot Analysis Proteins were homogenised and extracted from tissues in Triton lysis buffer ( mm Tris- HCl, ph 7., 7 mm NaCl, mm EDTA, % Triton X-, 5 mm ß- glycerophosphate, % glycerol, mm orthovanadate, mm phenylsulphfonyl fluoride, µg/ml leupeptin, µg/ml aprotinin). Protein extracts ( µg) were subjected to Western blot analysis with antibodies against MKK, MKK7 (BD Pharmingen); JNK, p MAPK, JunD, Bax (Santa Cruz); PKB, active caspase-, Bcl-, phospho-pkb (Ser 7), phospho-mkk, phospho-mkk7, phospho-jnk, phospho-p MAPK, GSKβ, phospho-gskβ, phosphor-cjun/jund (Ser 7) (Cell Signalling); ERK5 (Upstate); phospho-erk5 (Invitrogen); and tubulin (Sigma). Immunecomplexes were detected by enhanced chemiluminescence with anti-mouse, anti-rabbit, or anti-goat immunoglobulin G coupled to horseradish peroxidise as the secondary antibody (Amersham-Pharmacia). Protein Kinase Assay JNK and p MAPK activities were measured in ventricular tissue lysates following precipitation with glutathione S-transferase (GST)-c-Jun (the substrate for JNK) and glutathione-sepharose beads (Amersham), or with a polyclonal antibody to p MAPK (Santa Cruz) and protein A agarose beads (Sigma), respectively, for hours at C. The kinase reactions were performed at C for minutes in kinase buffer (5 mm HEPES, ph 7., 5 mm ß-glycerophosphate, 5 mm MgCl, mm dithiothreitol,.% orthovanadate) containing 5 µm [γ-p] ATP ( Ci/mmol) and µg of GSTtranscription factor (ATF) for p MAPK assays. The reactions were terminated by

4 the addition of Laemmli sample buffer. Phosphorylated substrates were examined and quantified after SDS-PAGE by phosphorimager analysis (Fuji FLA ). Quantitative Real-time PCR Total RNA was prepared from ventricular tissue using Trizol reagent, followed by the synthesis of cdna. Real-time quantitative PCRs were performed using the SYBR-green I Core Kit (Eurogentec). The primers used for ANP, BNP, Myh7, Colα and Colα were obtained from Qiagen. The primers used for RCAN. were as follows: forward primer, 5'-AGCTCCCTGATTGCCTGTGT-', reverse primer, 5'- TTTGGCCCTGGTCTCACTTT-'. PCR products were detected in the ABI-PRISM 77 sequence detection system (Applied Biosystems), and the results were analyzed using the - CT method. The level of expression of mrna was normalized to glyceraldehyde--phosphate dehydrogenase (GAPDH) mrna. sirna Transfection Primary cultures of neonatal rat cardiomyocytes (NRCMs) were prepared as previously described 7. NRCMs were transfected with sirna (nm) using Lipofectamine Plus reagent according to the manufacturer s instructions (Invitrogen). Rat MKK sirna (gene ID # 79; si genome SMART pool) was purchased from Dharmacon, sirna negative control (Si Neg) was obtained from Eurogenetec. To assess the specific effect of MKK sirna on silencing MKK expression, the protein levels of MKK and MKK7 were detected by immunoblot analysis 7h post-transfection. In addition, JNK activation upon isoproterenol stimulation (µm) was determined in sirna-treated NRCMs by immunoblot analysis.

5 Luciferase Reporter Assay h post-transfection of sirna, NRCMs were infected with recombinant adenovirus encoding NFAT-luciferase reporter gene (AdNFAT-luc) at 5 MOI in serum-free medium. At h after infection, the virus was removed. Following isoproterenol treatment (µm), aliquots of NRCM lysates were assayed for the NFAT luciferase activity using a luciferase assay kit (Promega). NFAT-Luc reporter cdna was obtained from Clontech, AdNFAT-luc was generated using the AdEasy TM adenoviral vector system (Stratagene). Figure legends Online Table I. Echocardiographic assessment of -week old and MKK cko mice at baseline. All measurements are mean ± SEM. N indicates the number of mice analysed in each group. HW/TL, the ratio of heart weight and tibia length; divs, enddiastolic interventricular septal wall thickness; dpw, end-diastolic left ventricular posterior wall thickness; LVEDD, diastolic left ventricular internal dimension; LVESD, systolic left ventricular internal dimension; FS, fraction shortening. Online Figure I. Isoproterenol (A) or swimming exercise (B) induced activation of MKK in control hearts was examined by immunoblot analysis at various time points. The ratios of phosphorylation to expression of MKK are shown in the bar graphs. Data correspond to the mean ± SEM (n= per group). * no difference found between two groups. 5

6 Online Figure II. The deletion of MKK in myocardium severely blunts JNK activity. In vitro kinase assay was performed to measure JNK and p activities after minutes of isoproterenol stimulation. Phosphorylated GST-cJun or GST-ATF, used for measuring JNK or p activity respectively were examined and quantified after SDS-PAGE by phosphorimager analysis. Black bar: control, grey bar: mins after isoproterenol treatment. n=, * no difference between two groups. Data are presented as mean ± SEM. Online Figure III. Echocardiography assessment (EF%, FS%) demonstrate moderately decreased contractility in MKK cko mice (n=). Data are presented as mean ± SEM. Online Figure IV. Quantitative real-time PCR analyses of gene markers associated with hypertrophy and fibrosis following 5 weeks TAC. The data are derived from three independent experiments performed in triplicate and are normalized to the GAPDH content and expressed relative to the mrna extracted from sham treated- mice. Data are the mean + SEM, n=. ANP, atrial natriuretic peptide; BNP, brain natriuretic peptide; Myh7, β-myosin heavy chain; Colα, procollagen type alpha ; Colα, procollagen type III alpha, RCAN., regulator of calcineurin. isoform. Online Figure V. Analysis of hypertrophic regulators in MKK cko hearts in response to TAC for week. Protein extracts from and MKK cko hearts subjected to TAC or sham operation were examined by immunoblotting for total ERK/, ERK5, PKB, GSKβ and JunD expression, as well as their phosphorylation levels using specific

7 antibodies. The ratios of phosphorylation to total expression of each protein are shown (right panel). Data are mean ± SEM, n=, * no differences between two groups. Online Figure VI. Analysis of hypertrophic regulators in MKK cko hearts in response to TAC for 5 weeks. Protein extracts from and MKK cko hearts subjected to TAC or sham operation were examined by immunoblotting for total ERK/, ERK5, PKB, GSKβ and JunD expression as well as their phosphorylation levels using specific antibodies. Bar graphs summarize the ratios of phosphorylation to total expression of each protein (right panel). Data are the mean ± SEM, n=, * no differences found between two groups. Online Figure VII. Immunoblot analyses of the phosphorylation levels of JNK, p and MKK7 together with their expression levels in and MKK cko hearts in response to week TAC (A), or 5 weeks TAC (B). MKK cko mice showed a substantial reduction in p-jnk compared to controls in both week and 5 week TAC groups. Both p and MKK7 were activated by TAC treatment, and levels of p-p and p-mkk7 were equivalent in these two groups. Bar graphs summarize the ratios of phosphorylation to total expression of each protein (right panel). Data are the mean ± SEM, n=, * no differences are found between two groups. Online Figure VIII. The loss of MKK promotes isoproterenol induced cardiac hypertrophy. (A) MKK cko mice display an enhanced cardiac hypertrophy (upper panel, scale bar: mm) following isoproterenol treatment compared to control mice. 7

8 Hematoxylin & eosin staining of heart cross-sections indicates enlarged cardiomyocytes in MKK cko mice (bottom panel, scale bar: µm). (B) HW/TL ratios of and MKK cko mice were evaluated after 7-day isoproterenol treatment. Both and MKK cko mice develop increased HW/TL ratio following treatment with isoproterenol compared the vehicle treated mice. MKK cko mice display enhanced HW/TL indicative of cardiac hypertrophy compared to controls. Data are presented as mean ± SEM, (n=). (C) Mean cross-sectional areas of cardiomyocytes in and MKK cko mice are calculated. Data correspond to the mean ± SEM (n=). Online Figure IX. Characterization of hypertrophic remodeling in MKK cko mice treated with isoproterenol. (A) Ventricular interstitial fibrosis is apparent in MKK cko hearts shown by Masson s trichrome-staining of cross-sections (left panel) (arrows are pointed to fibrosis areas, scale bar: 5µm). Quantification of the relative fibrosis area is expressed as percentage of the fibrosis area in the microscope views (right panel, data are the mean + SEM, n=). (B) In vivo hemodynamic analysis of dp/dt max (contractile response) and dp/dt min (lusitropic response) and (C) echocardiographic assessment of fractional shortening (FS) are indices of cardiac function. Data are mean ± SEM, n= for in vivo hemodynamic analysis, n= for echocardiography. * no difference between the two groups. Online Figure X. Quantitative RT-PCR analyses of marker genes for isoproterenol induced cardiac hypertrophy and ventricular remodeling. The data are derived from three independent experiments performed in triplicate and are normalized to the GAPDH content and expressed relative to the mrna extracted from mice treated with

9 vehicle. Hypertrophic marker genes: ANP, BNP, Myh7; fibrosis marker genes: Colα, Colα and RCAN.. Data are the mean + SEM, n=. Online Figure XI. JNK is not activated by swimming. Ventricular tissues prepared from and MKK cko hearts after swimming were analyzed for expression and phosphorylation levels of JNK, p and PKB. Ratios of phosphorylation to expression levels are shown in graphs (n=). Data are presented as mean ± SEM. References. Wang X, Nadarajah B, Robinson AC, McColl BW, Jin JW, Dajas-Bailador F, Boot-Handford RP, Tournier C. Targeted deletion of the mitogen-activated protein kinase kinase gene in the nervous system causes severe brain developmental defects and premature death. Mol Cell Biol. 7;7: Minamisawa S, Gu Y, Ross Jr J, Chien KR, Chen J. A post-transcriptional compensatory pathway in heterozygous ventricular myosin light chain -deficient mice results in lack of gene dosage effect during normal cardiac growth or hypertrophy. J Biol Chem. 999;7:-7.. Oceandy D, Cartwright EJ, Emerson M, Prehar S, Baudoin FM, Zi M, Alatwi N, Schuh K, Williams JC, Armesilla AL, Neyses L. Neuronal nitric oxide synthase signaling in the heart is regulated by the sarcolemmal calcium pump b. Circulation. 7;5:-9. 9

10 . Kirchhefer U, Neumann J, Baba HA, Begrow F, Kobayashi YM, Reinke U, Schmitz W, Jones lr. Cardiac hypertrophy and impaired relaxation in transgenic mice overexpressing Triadin. J Biol Chem. ;7: Wang J, Xu N, Feng X, Hou N, Zhang J, Cheng X, Chen Y, Zhang Y, Yang X. Targeted disruption of smad in cardiomyocytes results in cardiac hypertrophy and heart failure. Circ Res. 5;97:-.. Livak KL, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the - CT method. Methods. ;5:-. 7. Armesilla AL, Williams JC, Buch MH, Pickard A, Emerson M, Cartwright EJ, Oceandy D, Vos MD, Gillies S, Clark GJ, Neyses L. Novel functional interaction between the plasma membrane Ca + pump b and proapoptotic tumor suppressor Ras-associated factor (RASSF). J Biol Chem. ;79:-.

11 Online Table I Echocardiographic assessment of adult and MKK cko mice at baseline Genotype CTL (n=) KO (n=) HW/TL (mg/mm) Heart Rate (bpm) divs (mm) dpw (mm) LVEDD (mm) LVESD (mm) FS (%)

12 Online Figure I A p-mkk MKK iso 9 (min) P/T MKK ratio (arbitrary units) P< iso 9 (min) B swim p-mkk MKK rest swim ( min) P/T MKK ratio (arbitrary units).5 * *.5 swim rest swim ( min)

13 Online Figure II (min) (min) c-jun ATF P<. * * JNK activity (% of maximum) p activity (% of maximum)

14 Online Figure III P<. 5 P<. Ejection fraction (%) P<. Fractional shortening (%) P<.

15 Online Figure IV ANP P<. P<. BNP P<. P<. Myh7 P<. P<. Colα P<. P<. Colα P<. P<. RCAN. P<. P<. 5

16 P/T ERK/ ratio (arbitrary units) * * Online Figure V sham w-tac sham w-tac p-erk/ ERK/ P/T ERK5 ratio (arbitrary units) p-erk5 ERK5 p-pkb PKB P-GSKβ GSKβ p-jund JunD P/T GSKβ ratio (arbitrary units) P/T PKB ratio (arbitrary units) P/T JunD ratio (arbitrary units).5.5.5

17 P/T ERK/ ratio (arbitrary units) Online Figure VI sham 5w-TAC sham 5w-TAC p-erk/ ERK/ P/T ERK5 ratio (arbitrary units) p-erk5 ERK5 p-pkb PKB p-gskβ GSKβ p-jund JunD P/T GSKβ ratio (arbitrary units) P/T PKB ratio (arbitrary units) P/T JunD ratio (arbitrary units).5.5 7

18 A p-jnk sham w-tac sham w-tac P/T JNK ratio (arbitrary units) 5 P<. sham w-tac sham w-tac Online Figure VII JNK p-p P/T p ratio (arbitrary units) p sham w-tac sham w-tac p-mkk7 MKK7 P/T MKK7 ratio (arbitrary units) sham w-tac sham w-tac.5 P<. B sham 5w-TAC sham 5w-TAC p-jnk P/T JNK ratio (arbitrary units).5.5 sham 5w-TAC sham 5wTAC JNK p-p p P/T p ratio (arbitrary units) 5 sham 5w-TAC sham 5w-TAC p-mkk7 MKK7 P/T MKK7 ratio (arbitrary units) sham 5w-TAC sham 5w-TAC

19 Online Figure VIII A vehicle iso vehicle iso B HW/TL (mg/mm) P<. P<. P<. vehicle iso vehicle iso C Myocyte cross-sectional area (µm ) 5 P<. P<. P<. vehicle iso vehicle iso 9

20 Online Figure IX A iso Interstitial myocardial fibrosis (percentage %) P<.5 B P<. P<. dp/dt max dp/dt min C mmhg/s Fractional shortening (%) P< P<. P<. vehicle iso vehicle iso vehicle iso vehicle iso

21 Online Figure X ANP BNP Myh7 P<. P<. P<. P<. P<. P<. vehicle iso vehicle iso vehicle iso vehicle iso vehicle iso vehicle iso Colα Colα RCAN. P<. P<. P<. P<. P<. P<. vehicle iso vehicle iso vehicle iso vehicle iso vehicle iso vehicle iso

22 Online Figure XI p-jnk rest 9 rest 9 (min) P/T JNK ratio (arbitrary units) * 9 (min) JNK p-p p p-pkb P/T p ratio (arbitrary units) # 9 (min) PKB P/T PKB ratio (arbitrary units) # 9 (min)