Cardiac CaM Kinase II genes δ and γ contribute to adverse remodeling but redundantly inhibit...

DOI: 10.1161/CIRCULATIONAHA.114.006185

1

The Cardiac CaMKII Genes and Contribute Redundantly to Adverse

Remodeling but Inhibit Calcineurin-Induced Myocardial Hypertrophy

Running title: Kreusser et al.; CaMKII isoforms and calcineurin

Michael M. Kreusser, MD1; Lorenz H. Lehmann, MD1; Stanislav Keranov, MD1; Marc-Oscar Hoting1; Michael Kohlhaas, PhD2; Jan-Christian Reil, MD2; Kay Neumann, MD3;

Michael D. Schneider, MD4; Joseph A. Hill, MD, PhD5; Dobromir Dobrev, MD6; Christoph Maack, MD2; Lars S. Maier, MD3; Hermann-Josef Gröne, MD7; Hugo A. Katus, MD8;

Eric N. Olson, PhD9; Johannes Backs, MD1

1Research Unit Cardiac Epigenetics, Dept of Cardiology, University of Heidelberg, Heidelberg, Germany; DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/

Mannheim, Germany; 2Dept of Cardiology, Saarland University, Homburg, Germany; 3Dept of Internal Medicine II, University of Regensburg, Regensburg, Germany; 4British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Faculty of Medicine, Imperial College London, United Kingdom; 5Dept of Internal Medicine, University of Southwestern Texas

Medical Center, Dallas, TX; 6Institute of Pharmacology, University of Duisburg-Essen, Essen, Germany; 7Dept of Molecular Pathology, German Cancer Research Center, Heidelberg, Germany; 8Dept of Cardiology, University of Heidelberg, Heidelberg, Germany; DZHK (German Centre for

Cardiovascular Research), partner site Heidelberg, Germany; 9Dept of Molecular Biology, University of Southwestern Texas Medical Center, Dallas, TX

Address for Correspondence:

Johannes Backs, MD

Research Unit Cardiac Epigenetics, Department of Cardiology

Heidelberg University

Im Neuenheimer Feld 410

69120 Heidelberg, Germany

Tel: +49-6221-56-37714

Fax: +49-6221-56-5573

E-mail: [email protected]

Journal Subject Codes: Heart failure:[110] Congestive, Myocardial biology:[155] Physiological and pathological control of gene expression, Basic science research:[130] Animal models of human disease

p g , p gy, y gg, gg,Germany; DZHK (German Centre for Cardiovascular Research), partner sitee HHHeeieiddedelblblberererg/g/g/

Mannheim, Germany; 2Dept of Cardiology, Saarland University, Homburg, Geermrmmananyy;y; 33DeDeDeptptpt oooff Internal Medicine II, University of Regensburg, Regensburg, Germany; 4British Heart FoundationCentre of Research Excellence, National Heart and Lung Institute, Faculty of Medicine, ImperialCoollllegegegeee LoLoLondndndonnn, UnUnU ited Kingdom; 5Dept of Inteernrnrnalaa Medicine, Univerersisisitytyty of Southwestern Texas

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Abstract

Background—Ca2+-dependent signaling through CaMKII and calcineurin was suggested to

contribute to adverse cardiac remodeling. However, the relative importance of CaMKII versus

calcineurin for adverse cardiac remodeling remained unclear.

Methods and Results—We generated double-knockout mice (DKO) lacking the two cardiac

CaMKII genes and specifically in cardiomyocytes. We show that both CaMKII isoforms

contribute redundantly to phosphorylation not only of phospholamban, ryanodine receptor 2,

histone deacetylase 4 but also calcineurin. Under baseline conditions, DKO mice are viable and

display neither abnormal Ca2+ handling nor functional and structural changes. Upon pathological

pressure overload and beta-adrenergic stimulation, DKO mice are protected against cardiac

dysfunction and interstitial fibrosis. But surprisingly and paradoxically, DKO mice develop

cardiac hypertrophy driven by excessive activation of endogenous calcineurin, which is

associated with a lack of phosphorylation at the auto-inhibitory calcineurin A site Ser411.

Likewise, calcineurin inhibition prevents cardiac hypertrophy in DKO. Upon exercise

performance, DKO mice show an exaggeration of cardiac hypertrophy with increased expression

of the calcineurin target gene RCAN1-4 but no signs of adverse cardiac remodeling.

Conclusions—We established a mouse model in which CaMKII’s activity is specifically and

completely abolished. By the use of this model we show that CaMKII induces maladaptive

cardiac remodeling while it inhibits calcineurin-dependent hypertrophy. These data suggest

inhibition of CaMKII but not calcineurin as a promising approach to attenuate the progression of

heart failure.

Key words: heart failure, signal transduction, calcium/calmodulin-dependent protein kinase II, calcineurin, cardiac hypertrophy

display neither abnormal Ca2+ handling nor functional and structural changes. UpUppononon pppatatathohohololologigigicac lt

pressure overload and beta-adrenergic stimulation, DKO mice are protected against cardiac

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Introduction

Heart failure is the leading cause of death in developed countries.1 It results from adverse cardiac

remodeling upon pathological stress situations such as arterial hypertension, ischemic injuries or

genetic causes. Adverse cardiac remodeling is usually described by a combined appearance of

myocardial hypertrophy, activation of a fetal gene program, cell death and interstitial fibrosis.2

Ca2+-dependent signaling pathways including CaMKII and calcineurin were both proposed to

play pivotal roles in adverse cardiac remodeling.3

The protein kinase CaMKII consists of four different isoforms with distinct expression

patterns. CaMKII and CaMKII are enriched in the brain, but CaMKII and CaMKII are

expressed ubiquitously. CaMKII is the predominant cardiac CaMKII isoform but CaMKII is

also expressed in the heart.4 In human and experimental heart failure, CaMKII expression and

activity is enhanced.4, 5 Phosphorylation of the ryanodine receptor RyR2 by CaMKII has been

reported to cause sarcoplasmic reticulum (SR) Ca2+ leak, which in turn seems to drive heart

failure development.6, 7 At the epigenetic level, CaMKII inactivates the negative regulator of

adverse cardiac remodeling histone deacetylase 4 (HDAC4), leading to transcriptional activation

of the myocyte enhancer factor 2 (MEF2), and phosphorylates histone 3.8-14 Transgenic

overexpression of the splice variants CaMKII B (localizes to the nucleus) and CaMKII C

(localizes to the cytosol) promote cardiac hypertrophy and dilated cardiomyopathy,

respectively.15, 16 CaMKII inhibitory peptides prevent structural heart disease.17, 18 Mice with a

global deletion of CaMKII were protected against adverse cardiac remodeling.7, 8 However in

all of these models, substantial phosphorylation of the target phospholamban (PLB) was still

detectable, indicating an incomplete loss-of-function.

The protein phosphatase calcineurin consists of two subunits: calcineurin A (CnA), which

expressed ubiquitously. CaMKII is the predominant cardiac CaMKII isoform bubuut CaCaCaMKMKMKIIIIII iis s

also expressed in the heart.4 In human and experimental heart failure, CaMKII expression and

acctitiivivivitytyty iiiss enene hhhancncceeded.4, 5 Phosphorylation of the ryryyanannodine receptororr RyRyRR22 2 byb CaMKII has been

eepooortr ed to caaususeee sssarcrcopopplalalasmsmsmicicic rretetticicicululumumum (SSRR))) Caaa2+++ leaaakk,k, wwwhihiichch iinn tuturnrnn sseeeemsmsms tto o drdrd ivivive e heeearart tt

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4

contains the catalytic site and calcineurin B (CnB), the small regulatory Ca2+-binding subunit.19

Transgenic CnA overexpression in mice induces massive cardiac hypertrophy by

dephosphorylation-dependent translocation of the transcription factor NFAT from the cytosol to

the nucleus.20, 21 The CnA- and CnA- isoforms are expressed in the myocardium but only

CnA was demonstrated to be stress responsive.22 Moreover, mice lacking CnA were protected

against cardiac hypertrophy.23 Inhibition of CnA has been suggested as a treatment option for

cardiac hypertrophy because studies with the calcineurin inhibitory agent cyclosporine A (CyA)

have been successful in rodents.24 On the other hand, the splicing variant CnA- 1 seems to

activate Akt-dependent cardioprotective pathways without inducing NFAT-dependent

maladaptive hypertrophy.25

We developed a cardiomyocyte-specific double knockout model, in which the two

cardiac isoforms, CaMKII and CaMKII , were both deleted. This model identifies CaMKII as

the critical regulator of maladaptive cardiac remodeling and reveals that CaMKII serves as a

negative regulator of calcineurin activity in vivo. Unexpectedly, we provide evidence that cardiac

hypertrophy due to over-activation of endogenous calcineurin does not cause systolic cardiac

dysfunction.

Methods

Mouse experiments

We generated conditional knockout mice containing cardiomyocyte-specific deletions of

CaMKII ( -CKO), CaMKII ( -CKO) or both isoforms (DKO) by crossing previously

described conditional knockout mice8, 26 to transgenic mice expressing Cre recombinase under

the control of the -MHC promoter ( MHC-Cre) or by using the mER-Cre-mER fusion protein

maladaptive hypertrophy.25

We developed a cardiomyocyte-specific double knockout model, in which the two

caardrddiaiaiaccc iisisofofofororrmss,, CCaCaMKII and CaMKII , wereee bbbootth deleted. Thihiis s momodededel l identifies CaMKII as

hhhe crc itical reggulullatatoror ooof mamamalalaladadadaptptivivvee e cacaardddiac reeemoodedeelingngng aandndd rrreveveeaealsls tthahahat CaCaaMKMKM IIIII sererervevees aasas aaa

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5

under the control of a cardiomyocyte-specific -MHC-promoter.27, 28 As controls, littermates

homozygous for the conditional CaMKII and CaMKII alleles without MHC-Cre transgene

( -FF, -FF and FFFF) were used. For some experiments, C57BL/6 mice were obtained from

Charles River (wild type mice). A detailed description of the surgical methods, the exercise

protocol and in vivo imaging as well as hemodynamic measurement techniques can be found in

Supplemental Methods. All experimental procedures were reviewed and approved by the

Institutional Animal Care and Use Committee at the Regierungspräsidium Karlsruhe, Germany.

Histology

The protocols for hematoxylin and eosin (H&E), Masson´s trichrome staining and CD31

immunostaining can be found in the Supplemental Methods.

Western Blotting, GST-pulldown and immunocytochemistry

The immunoblotting, GST-pulldown and immunocytochemistry protocols as well as the

antibodies used in this study can be found in the Supplemental Methods.

CaMKII kinase activity

CaMKII kinase activity was measured by detecting the amount of endogenous CaMKII that

associates to GST-HDAC4 419-670 or by radioactive kinase assay using 32P-ATP.10 A detailed

description can be found in the Supplemental Methods.

Gene expression analysis

The RNA isolation and quantitative RT-PCR as well as the primer sequences used in this study

can be found in the Supplemental Methods.

Culture of primary cardiomyocytes

Adenoviruses for gene transfer into cardiomyocytes were produced as described in the

Supplemental Methods. Neonatal mouse ventricular myocytes (NMVMs) and adult mouse

mmunostaining can be found in the Supplemental Methods.

Western Blotting, GST-pulldown and immunocytochemistry

ThThee e imimimmumunonon bbblototttititinngng, GST-pulldown and immunnococo yytochemistry pppror toocococolls as well as the

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6

ventricular myocytes (AMVMs) were isolated from wild type and mutant mice, neonatal rat

ventricular myocytes (NRVMs) were isolated from Wistar rats (Charles River). A detailed

description is included in the Supplemental Methods.

Epifluorescence microscopy

Freshly isolated AMVMs were incubated with Fura-2 AM or Indo-1 AM and Ca2+ transients as

well as SR Ca2+ load were measured. AMVMs were transilluminated by red light (>650 nm) to

visualize sarcomeres, and fractional shortening was measured. A detailed description is included

in the Supplemental Methods.

Statistical analysis

Data are summarized as mean±SEM. Statistical analysis was performed with the Graph-Pad

Prism Software Package Version 5.0 (GraphPad, Inc.). When two groups were compared, we

used Wilcoxon-Mann-Whitney U tests. When more than two groups were compared, a Kruskal-

Wallis test was applied. When we obtained a significant p-value we continued with pair-wise

comparisons using Wilcoxon-Mann-Whitney U tests according to the closed testing principle. A

value of P<0.05 was considered statistically significant.

Results

CaMKII and CaMKII contribute redundantly to cardiac CaMKII activity

Gene-expression analysis of the four CaMKII genes (CaMKII , CaMKII , CaMKII and

CaMKII ) in RNA samples from healthy and diseased wild type mouse hearts was performed in

relation to the expression levels in the brain. In accordance with others,4 we revealed that besides

CaMKII , CaMKII is also expressed in the heart, whereas CaMKII and CaMKII are not (Fig.

1A). Moreover, both genes were up-regulated in diseased hypertrophic hearts after transverse

Data are summarized as mean±SEM. Statistical analysis was performed with the e GrGrrapapph-hh-PaPaPad d d

Prism Software Package Version 5.0 (GraphPad, Inc.). When two groups were compared, we

ussededd WWWilililcococoxoxoxon-MaMaMannn-Whitney U tests. When mmmoroo eee than two grououupsp wwwererere e compared, a Kruskal-

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7

aortic constriction (TAC)-induced pressure overload of the left ventricle. Thus, we took

advantage of two conditional CaMKII knockout models that we developed previously,8, 26 and

generated cardiomyocyte-specific CaMKII ( -CKO), CaMKII ( -CKO) and

CaMKII /CaMKII double knockout mice (DKO) (Fig. 1B). As controls, MHC-Cre negative

littermates ( -FF, -FF and FFFF) were used. Western blot analysis using an antibody, that was

shown to recognize at least CaMKII and CaMKII ,8, 26 confirmed a marked loss of CaMKII in

cardiac extracts of -CKO and DKO (Fig. 1C). With this antibody, CaMKII was almost not

detectable in -CKO, suggesting (under the assumption that the antibody has the same affinity to

CaMKII as to CaMKII ) that CaMKII is largely more abundant than CaMKII in the heart

(Fig. 1C, see also Suppl. Fig. 1A). A CaMKII -specific antibody could detect CaMKII in -

CKO but not in -CKO and DKO mice, suggesting that CaMKII could compensate for the loss

of CaMKII in -CKO mice. Notably, phosphorylation of the CaMKII target site of PLB Thr17

was only slightly reduced in -CKO mice, whereas it was completely abolished in DKO mice,

confirming that CaMKII and CaMKII exert redundant roles and largely compensate for each

other. As a control, phosphorylation at the protein kinase A (PKA) site PLB-Ser16 was not

reduced (Fig. 1C). Thus, DKO represents a so far unique model for a complete loss of CaMKII

activity in cardiomyocytes in vivo. We also analyzed the phosphorylation level of targets of

interest. Consistent with PLB, phosphorylation of RyR2-Ser2814 and HDAC4-Ser632 was

decreased moderately in -CKO but clearly in DKO animals. Again, phosphorylation at the PKA

site RyR2-Ser2808 was not affected by CaMKII deletion (Fig. 1C).

CaMKII DKO mice are viable and do not display cardiac abnormalities

DKO mice developed normally and showed no increase in mortality or apparent morphological

abnormalities. Cardiac size as indicated by heart weight/body weight ratios, ventricular chamber

Fig. 1C, see also Suppl. Fig. 1A). A CaMKII -specific antibody could detect CaCaaMKMKMKIIIII iiin n --

CKO but not in -CKO and DKO mice, suggesting that CaMKII could compensate for the loss

off CCCaMaMaMKIKIIIII iiin C-C-CKKO mice. Notably, phosphorrylyly aattion of the CaaMKMKM IIIII tttaararget site of PLB Thr17

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8

dimensions or fractional shortening, assessed by echocardiography were unchanged in mice at

age of 12 weeks (Suppl. Fig. 1B). To study functional parameters in more detail we used a

working heart preparation to standardize pre- and afterload (Suppl. Fig. 1C). We detected slight

improvements in ESPVR (which was not significant in all experimental groups used in this

study, see also Fig. 3A) as a measure for maximal pressure that can be developed by the ventricle

at any given LV volume. Based on the observation that the CaMKII sites on PLB and RyR2 were

hypo-phosphorylated, we measured global parameters of Ca2+ handling (Suppl. Fig. 1D-E).

However, we did not detect any change in Ca2+ transients, SR Ca2+ content, diastolic Ca2+

concentration and single-cell fractional shortening, surprisingly indicating that – at least under

basal conditions – CaMKII activity is dispensable for intracellular Ca2+ handling and

cardiomyocyte contractility.

Calcineurin precedes CaMKII activation upon pathological pressure overload

We analyzed the time course of CaMKII and calcineurin activation after TAC (Fig. 2A-B).

Starting at 24 hours after TAC, CaMKII expression (CaMKII C > CaMKII B) and CaMKII

activity, as judged by its activity-dependent binding affinity to HDAC4,10 increased (~2-fold)

(Fig. 2A). We did not use the usual antibodies recognizing CaMKII autophosphorylation to

determine CaMKII activity because in DKO one of these antibodies still showed a signal

although CaMKII protein was gone (Suppl. Fig. 2A and Suppl. Fig. 1A). As another indication

for CaMKII activation, phosphorylation of HDAC4 at its CaMKII phosphorylation site Ser632

also started to increase three days after TAC. In contrast, expression of RCAN1-4, a specific

target gene of the calcineurin-NFAT pathway, which is considered as an endogenous calcineurin

reporter,29 was dramatically overexpressed (~17-fold) one day after TAC (Fig. 2B). These data

indicate that CaMKII activation and HDAC4 phosphorylation depend on upstream signals

basal conditions – CaMKII activity is dispensable for intracellular Ca2+ handlingg aandndnd

cardiomyocyte contractility.

CaCalclclcininineeueuriririn n pprecececeededes CaMKII activation upononon pppathological prprpressusuurerere overload

WeWe aanalyzed ththhee tiiimeme cououoursrsrsee e ofoof CCCaMaMaMKKKIIII anndd ccalccinnneurrrininn aaccctiivvatatioionn afafftteterr TATAACCC ((FiFiig.g. 222A-A-A BBB).).

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9

distinct from the upstream signals of calcineurin activation.

Dissociation of cardiac dysfunction and interstitial fibrosis from myocardial hypertrophy

Next, we performed TAC surgeries in DKO and FFFF mice. Strikingly, DKO were protected

against cardiac fibrosis, increased caspase 3/7 activity as marker for apoptosis and systolic

dysfunction and display an attenuated rarefication of capillarization (Fig. 3A, see also Suppl.

Fig. 3A). This protective effect was not associated with changes in intracellular Ca2+ handling or

cellular contractility (Suppl. Fig. 2C+D). Surprisingly, TAC still induced cardiac and

cardiomyocyte hypertrophy in DKO. Moreover, the gene expression changes of typical fetal

genes (ANP, BNP, MHC) and remodeling marker ( MHC) were similar in DKO and FFFF

(Fig. 3B). These data indicate a dissociation of cardiac hypertrophy and re-activation of the fetal

gene program on one hand from maladaptive cardiac remodeling including interstitial fibrosis,

apoptosis, reduced capillarization and cardiac dysfunction on the other hand. To confirm these

data in an independent model, we applied neurohormonal stress to DKO. We injected

isoproterenol (Iso) to DKO and FFFF mice. Iso led to cardiac hypertrophy (Suppl. Fig. 4A) and

fetal gene activation in DKO as observed in FFFF mice (Suppl. Fig. 4B). But again and

consistent with the TAC data, we observed less cardiac fibrosis, apoptotic markers and collagen

expression in DKO (Suppl. Fig. 4A+B, see also Suppl. Fig. 3A). These observations were

especially surprising in view of our previous finding that global deletion of one isoform,

CaMKII , attenuated cardiac hypertrophy and fetal gene activation.8 However, in our previous

model we could still detect substantial phosphorylation of the typical CaMKII target PLB-Thr17

in the myocardium because CaMKII was not deleted (see also Fig. 1C). Thus, we hypothesized

that complete CaMKII inhibition might exert dual effects on anti- and pro-hypertrophic

pathways. To substantiate this hypothesis we performed a gene dosage experiment (Fig. 3C).

Fig. 3B). These data indicate a dissociation of cardiac hypertrophy and re-activavaatitionono oof f f thththe e e feffettal

gene program on one hand from maladaptive cardiac remodeling including interstitial fibrosis,

appopopoptototosisisis,s, rrredededuccededed ccapillarization and cardiac dyyyssfs uuunction on thee ooothererr hhhaand. To confirm these

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10

Consistent with our previous findings, the homozygous deletion of either CaMKII or CaMKII

resulted in a reduction of cardiac hypertrophy upon TAC. Paradoxically, the additional

heterozygous deletion of the other CaMKII isoform resulted in a less pronounced reduction of

cardiac hypertrophy upon TAC. Furthermore, the complete cardiac-specific deletion of all four

CaMKII and CaMKII gene copies (DKO) did not prevent cardiac hypertrophy. We also

analyzed the effects of the single deletion of CaMKII and CaMKII in isolated adult

cardiomyocytes and could confirm the anti-hypertrophic effects of CaMKII and CaMKII

(Suppl. Fig. 3C). We were also interested whether CaMKII or CaMKII play a specific role for

apoptosis because our recent data suggested a specific role of CaMKII for apoptosis at least in

macrophages.30 However, TUNEL assays indicated that CaMKII deletion more than CaMKII

deletion attenuates cardiomyocyte apoptosis and that the combined deletion is even more

effective (Suppl. Fig. 4B).

CaMKII controls the calcineurin-NFAT pathway

Thus, we hypothesized that complete CaMKII deletion may result in an activation of a pro-

hypertrophic pathway. Therefore, we searched for candidate signaling molecules that are

activated by TAC or Iso. Whereas, protein kinase D (PKD) was not activated in DKO after TAC

(Suppl. Fig. 2A), we found the calcineurin reporter gene RCAN1-4 to be strongly up-regulated

in DKO mice after TAC and Iso (Fig. 4A, Suppl. Fig. 2E). This could not be observed after

deletion of only one CaMKII gene, in particular CaMKII (Suppl. Fig. 2E), indicating that

complete CaMKII deletion in the heart results in calcineurin activation. Consistently, Iso induced

a dramatic increase in nuclear NFAT translocation and NFAT activity in NMVMs from DKO

when compared to NMVMs from FFFF (Fig. 4B+C). In this regard, it was reported that CaMKII

directly phosphorylates CnA-Ser411 in vitro but it remained unclear whether this mechanism

macrophages.30 However, TUNEL assays indicated that CaMKII deletion moree thahaan CaCaCaMKMKMKIIII

deletion attenuates cardiomyocyte apoptosis and that the combined deletion is even more

efffefeectctctivivivee (((SuSuSuppppl.l. FFFigi . 4B).

CCCaMMMKII contrtroolo ss thheee ccacalclclcininneeueuririin-n-n-NFNFFAAAT pppaaathwwwaayy

ThThhususu , wewew hhhypypypotothhehessisizezedd tththata cccomommplplpletetetee CaCaCaMKMKMKIIII dddeelletettioionn n mamamayyy rereresusuultt iiin n n aanan aaactctivivivatatatioionnn ooof aaa pproro--

hypertrophicc pppatatathwhwhwayayay.. ThTT erererefefeforrre,e,e, wwweee seseseararrchchchededed ffororo ccanana didididadadatetete sisisigngngnalllinining g g momomolelelecucuuleleles ss ththhatatat are




11

was of biological significance in vivo.30, 31 Thus, using a newly synthesized specific antibody

against CnA-Ser411 (Fig. 4D) we aimed to detect endogenous CnA phosphorylation. Indeed,

overexpression of active CaMKII (T287D) in NRVMs resulted in hyper-phosphorylation of

CnA-Ser411 (Fig. 4E). In accordance, we found that CnA was strongly hypo-phosphorylated in

cardiac lysates from sham- and TAC-operated DKO hearts, which was associated with RCAN1-

4 upregulation (Fig. 4F). Moreover, as compared to DKO CnA-Ser411 hypo-phosphorylation

was less pronounced in cardiac extracts of -CKO, and not found in -CKO, explaining why

calcineurin activity was only strongly increased in DKO but not in the single knockouts (Fig.

4G).

Cardiac hypertrophy, but not dysfunction is controlled by calcineurin during pathological

and physiological hypertrophy in DKO

To test whether cardiac hypertrophy after TAC in DKO depended on calcineurin activation, we

performed CnA knockdown experiments in isolated NMVMs of mutant mice. CnA knockdown,

which resulted in approximately 50% decrease of CnA protein (Suppl. Fig. 5A), prevented Iso-

induced cellular hypertrophy and activation of fetal genes in myocytes derived from DKO but

not FFFF (Fig. 5A-B). We then repeated the TAC experiment in DKO with the calcineurin

inhibitor cyclosporine A (CyA). CyA was shown to inhibit cardiac hypertrophy after TAC in

dosages from 12.5 - 50 mg/kg body weight per day.31 Using a dosage of 4 mg/kg body weight

per day, which did not affect TAC-induced cardiac hypertrophy in FFFF, we were able to inhibit

RCAN1-4 expression as well as cardiac and cardiomyocyte hypertrophy in DKO but not FFFF

(Fig. 5C and Suppl. Fig. 5B), indicating that TAC-dependent hypertrophy in DKO depends on

calcineurin. As CnA knockdown, CyA attenuated also the expression of fetal genes in DKO

(Fig. 5B and Suppl. Fig. 5B). In contrast to the anti-hypertrophic effects of CyA in DKO, CyA

Cardiac hypertrophy, but not dysfunction is controlled by calcineurin duriinngng pppattthohohololologiggicacal

and physiological hypertrophy in DKO

Too tttesesesttt whwhwhetetethehh r cacacardrdr iac hypertrophy after TAC ininin DDDKO depended d d on cccalalalccineurin activation, we

perffforo med CnnA AA knknknocckdkdk owowownn n exexxpeperirir mmementntss in issoolatteddd NNMVMVM MsMsMs ooff f mmumutatannnt mmiciccee.e. CCnAnAnA kkknonockckckdododowwwn,

whwhhicici hhh rerer susuultltltedeed iinnn apapapprprproxxximimatatatelellyyy 505050%%% dededecrcrcreaeaeaseee ooff f CCnCnAAA prprprotototeiiin n n ((SuSuSuppppp ll.. FFFigig. . 5A5A5A))),,, prprpreveveenentteted d IIsoo-o-

nduced cellulullararar hhhypypypererrtrtrropophyhyhy aandndnd acacctitit vavaatitiononon ooof f f fefefetatatal l gegeg nenenes s ininin mymymyocoo ytytyteseses ddderererivivivededd fffrororom m m DKD O but




12

did not affect the protective effects of DKO on cardiac fibrosis or systolic function, providing

evidence that - at least in this model - calcineurin-dependent cardiac hypertrophy is not

maladaptive on the one hand but also not cardioprotective on the other hand. In line with the

latter, we could not accumulate evidence that the potential cardioprotective calcineurin-

dependent Akt pathway was activated (Suppl. Fig. 5C).

To clarify the role of the CaMKII-calcineurin pathway in physiological hypertrophy, we

performed endurance exercise training by a swimming protocol in DKO and FFFF mice (Fig.

5D-F). Interestingly, whereas CaMKII protein levels were not altered after swimming in control

mice (FFFF), CaMKII activity decreased about 30% (Fig. 5D). Remarkably, we found

exaggerated heart weight/body weight (HW/BW) ratios and increased myocyte size in DKO

mice after swimming, which was associated with increased expression of the calcineurin target

gene RCAN1-4 (Fig. 5E+F). Again, despite calcineurin activation and increased cardiac

hypertrophy no signs for systolic cardiac dysfunction were detected, indicating that activation of

endogenous calcineurin is not maladaptive.

Discussion

By generating mice lacking the two cardiac CaMKII isoforms and we introduce a new loss-

of-function model with the deletion of both cardiac CaMKII isoforms in cardiomyocytes, and

demonstrate that CaMKII - under unstressed conditions - is dispensable in cardiomyocytes with

regard to integrity, growth, cardiomyocyte contractility and Ca2+-handling. This is somewhat

surprising in light of innumerous reports about CaMKII functions. But from the drug developer’s

point of view, this indicates that CaMKII inhibition does not exert major unwanted side effects

on basal cardiac function. Furthermore, we show that - upon pathological stress conditions -

exaggerated heart weight/body weight (HW/BW) ratios and increased myocyte sisiizeee iinn n DKDKDKO OO

mice after swimming, which was associated with increased expression of the calcineurin target

gegenenene RRRCACACAN1N1N1-4 (((FFiFigg. 5E+F). Again, despite calclccinini eeeurin activationonn andndd iiinnncreased cardiac

hhyhyppepertrophy nono sssigggnsns ffororr sssysysystototolilic c cacacardrdiaiaccc dyysfffuuncttiooon wwweerere e dedeetetecccteeded, ininindiiicacatititinngng tthahahat t acactitt vavavatitiononon of

enndodod gegegenonoususus cccalalcciinneneururinin iis s nooot t mamamalalaladdadapptptivivve.ee.




13

CaMKII plays a dual role in the regulation of adverse cardiac remodeling (Fig. 6). On the one

hand, CaMKII transduces maladaptive signaling. On the other hand, CaMKII negatively

regulates calcineurin-dependent cardiac hypertrophy in vivo.

Redundancy of CaMKII and CaMKII

Because of a lack of loss-of-function models with the deletion of both cardiac CaMKII isoforms,

it was so far not possible to study all essential roles of CaMKII in myocardial disease. Moreover,

most of the reported CaMKII studies used non-genetic approaches such as overexpression of

peptides or pharmacologic compounds such as KN-93. However, off-target effects, as e.g.

described on protein kinase D or ion channels (e.g. L-type Ca2+ channels),8, 32, 33 could have

complicated the conclusions. Therefore, we focused first on the development of a specific and

complete genetic mouse model, in which the two CaMKII genes that are expressed in

cardiomyocytes are deleted. We found that CaMKII and CaMKII compensate for each other

and act redundantly on the phosphorylation of PLB-Thr17, RyR2-Ser2814, CnA-Ser411 and

HDAC4-Ser632. In contrast to previously described approaches aiming at inhibiting CaMKII,7, 8,

17, 18, 34, 35 the new DKO model results in profound hypo-phosphorylation of these typical

CaMKII phosphorylation targets.

CaMKII controls calcineurin signaling

Previous data from our lab showed that homozygous CaMKII knockout mice are protected

against cardiac hypertrophy and interstitial fibrosis.8 Here we demonstrate that the loss of more

than two cardiac CaMKII gene copies unmasks a dual role of CaMKII in regulating cardiac

hypertrophy. DKO mice appear to develop a similar hypertrophic response as FFFF littermates

after TAC and Iso, but histological, gene expression and biochemical analyses revealed that

cardiac hypertrophy in DKO depends on activation of endogenous calcineurin. CaMKII

complicated the conclusions. Therefore, we focused first on the development of f aaa spppecccififificicic aaandnnd

complete genetic mouse model, in which the two CaMKII genes that are expressed int

caardrddioioiommymyocococytytytes aaarrere deleted. We found that CaMKMKMKIIII and CaMKIKIIIII ccomommppensate for each other

anndd d aca t redunddananntllyy ononn ttthehehe pphohohospsphohohorryryllattiion offf PLLBLB--Thhrhr1717,, RRyRyRR22--SSerer2822 1414,, CnCnCnA-A-SeSeSer4r4r411111 aandndnd

HDHDDACACAC4-4-SeSeer6r6r63232.. InInn cconoontrtrt aast t totot ppprerereviviv ououuslllyy y ddedescsccririr bbbeddd apapapprprproaoaoachhhesese aimimimiinngng aaat t ininnhihihibibib titiingngng CCCaMMMKKIII,,7,7 8

777, 181818 33, 3444, 353535 thhe e nenenew w w DKDKDKO OO momomodeded l rereresusuultltl s ininin ppprororofffououundndnd hhhypypy o-o-o phphphosososphphphororylylylatatatioioion n n ofofo tthehehesesese tttypypypical ffffff




14

phosphorylates CnA-Ser411, which was shown to lead to a decrease in its phosphatase

activity.36, 37 Here, we show that this phosphorylation event occurs in vivo and in a CaMKII-

dependent manner. Moreover, we found CnA-Ser411 hypo-phosphorylation to be associated

with cardiac hypertrophy after pathological and physiological stress. Importantly and in contrast

to current paradigms, the data of this study suggest that anti-hypertrophic effects are not required

for an improved cardiac function. Moreover, a vast amount of literature suggested that

calcineurin induces pathological cardiac hypertrophy, ultimately leading to heart failure.20, 21, 38,

39 However, the latter studies used forced overexpression of a truncated CnA construct lacking

the regulatory domain that is phosphorylated by CaMKII, complicating the interpretation of

these findings. A recent CnA loss-of-function study seemed to prove the idea that calcineurin

mediates pathological cardiac hypertrophy but this study focused mainly on cardiac hypertrophy

but not on other aspects of pathological remodeling such as cardiac dysfunction.23 In fact,

histological analyses did not show a protection against cardiac fibrosis in CnA null mice.23 In

the present study we identify DKO mice as a model with activation of endogenous calcineurin.

We challenge current paradigms and conclude that - at least in the absence of CaMKII signals -

activation of endogenous calcineurin is not maladaptive. Support comes from a recent study,

demonstrating that calcineurin induces physiological hypertrophy in pregnant mice.40 Moreover,

Heineke et al. suggested that calcineurin exerts cardioprotective functions under certain

conditions: hearts of transgenic mice with mild overexpression of CnA were protected in a

murine model of dilated cardiomyopathy.41 Thus it is tempting to speculate that calcineurin

activation in DKO contributes to the observed cardioprotective effects upon TAC. Because it

was suggested that the splice variant CnA 1 exerts cardioprotection via Akt,25 we measured Akt

phosphorylation. But we found no changes. We cannot completely rule out that other

hese findings. A recent CnA loss-of-function study seemed to prove the idea thahat cccalclclcininineueueuririr n n

mediates pathological cardiac hypertrophy but this study focused mainly on cardiac hypertrophy

buutt t nononottt onn ooothththerr aaasspspects of pathological remodelililingngng such as cardiiacaca ddysyssfufufunction.23 In fact,

hhistttolo ogical ananallyysssess dddiddd nnotott ssshhohow w w aaa prproottectiiionnn aggagaiinst t t ccacardrddiaaacc fifiibrrosisis innn CnCnCnAAA nnululu l l l mmmiccece.23323 Innn

hhe e prprpresesesenenent t t stsstududdy y y wewewe iiidedeentntn ifi y y DKDKDKO O O mimicecec aaasss a a momomoddedel ll wiwiwiththh aaacctc ivivvatatioioi n n ofofof enenndodod gegegenononoususus ccalalalcicic neneneurururininn..

We challengegege cccururu rererentntn ppparara adaddigigigmsmss aaandndd cconononclclcludududeee thththatatat --- aaat leleleasasast t t ininin ttthehh aaabsbsbsenenencecece ooof f CaCaCaMKMKMKIIII signals -




15

calcineurin-dependent pathways contribute to the cardioprotective effects but our data show that

CyA-treatment after TAC in DKO did not lead to cardiac dysfunction. Thus, we conclude that -

in the setting of the current study - CaMKII-dependent but calcineurin-independent processes

mediate pathological remodeling.

CaMKII is required for pathological remodeling but not cardiac hypertrophy and fetal

gene expression

The question arises of how CaMKII regulates pathological remodeling, especially cardiac

fibrosis. We have shown that HDAC4 transmits CaMKII signals to the cardiac genome via

MEF2.8, 10, 13 Because gene deletion and overexpression studies revealed that MEF2D primarily

drives cardiac fibrosis and dysfunction rather than cardiac hypertrophy,42 the CaMKII-HDAC4-

MEF2 pathway is a likely pathway leading to adverse cardiac remodeling (see model in Fig. 6).

Likewise, HDAC4 hypo-phosphorylation was associated to the protective situation in DKO

mice. But it was remarkable that DKO mice were not protected against re-activation of certain

fetal genes including ANP, BNP and MHC. It is important to note, that fetal gene activation is

suggested to be driven not only by MEF2 but also NFAT.43 Thus, we speculate that over-

activation of the calcineurin-NFAT pathway in DKO compensates for the attenuation of the

CaMKII-HDAC4-MEF2 pathway with regard to ANP, BNP and MHC but not for attenuation

of yet unidentified genes that cause cardiac fibrosis. Natriuretic peptides are actually known not

to be causative but rather to be adaptive to combat the hemodynamic load and oxidative capacity

of muscle cells.44, 45 On the other hand, MEF2-dependent genes that cause interstitial fibrosis are

not clearly described yet. Taken together, our data indicate that fetal genes associated with

calcineurin-dependent hypertrophy do not cause cardiac dysfunction. Likewise, in a model of

physiological hypertrophy we found no activation of CaMKII but activation of calcineurin-

drives cardiac fibrosis and dysfunction rather than cardiac hypertrophy,42 the CaMaMMKIKIK II-I-HDHDHDACACAC44-

MEF2 pathway is a likely pathway leading to adverse cardiac remodeling (see model in Fig. 6).

Liikekekewiwiwisese, HDHDHDACCC444 hyh po-phosphorylation was aassssssocciated to the prprroto ecctititivveve situation in DKO

mmiccece. But it wwasas reememaarrkakaablblble e thththatat DDDKOKOKO mmmicee wwweree nnot ppprroroteteectteded aaggaininstst rre-e-t acacactititivavatititiononn oof ff cececertrtaiaa nnn

feetatatal l l gegegenenen ss s inininclcluududininng g g ANANANP,P BBBNPNPNP aaandndn MHMHMHCCC. IIIt t t isiss impmpmpororortatatanntnt ttto o o nnootetete,,, ththhatt ffetettalala gggenenne e e aca ttitivvaatitioonon iis s

uggested too bbbe e e drdrdrivivivenenen nnnotot ooonlnln y y bybyby MMMEFEFEF2 2 2 bububut t t alalalsososo NNNFAFAFAT.T.T 43 TTThuhuhus,ss wwwe e e spspspecececululu atatte e e thththatatat ooovev r-




16

NFAT signaling without signs of pathological remodeling.

CaMKII is dispensable for intracellular Ca2+ handling under unstressed conditions

Another puzzling observation of this study was the lack of effect on Ca2+ handling although

profound changes on phosphorylation on RyR2 and PLB were detected. We speculate that the

lack of CaMKII phosphorylation on Ca2+ handling proteins might be compensated by other

mechanisms such as PKA-mediated phosphorylation, although we could not detect significant

increases in phosphorylation at the PKA sites PLB-Ser16 or RyR2-Ser2808. Knockin mouse

models with a specific mutation of these sites would clarify the specific role of CaMKII

phosphorylation at PLB-Thr17 and RyR2-Ser2814. Indeed, Respress et al. described RyR2-

2814-S/A knockin mice.6 Consistent with our data, they also observed an unaltered SR Ca2+

content, Ca2+ transient decay and Ca2+ sparks at baseline, but they discovered that in the situation

of heart failure, mutant mice were protected. Thus, further experiments are warranted to

investigate DKO mice and Ca2+ handling in models of severe heart failure. However, it appears

unlikely that an attenuation of disturbed Ca2+ handling contributes to the improved cardiac

function as observed in DKO after TAC in the present study.

Summary

Taken together, we show that CaMKII is a key driver of maladaptive cardiac remodeling. This

study describes maladaptive cardiac remodeling as a process of interstitial fibrosis and cardiac

dysfunction but not cardiac hypertrophy and activation of commonly measured fetal genes such

as ANP and BNP. Under certain conditions, CaMKII inhibits cardiac hypertrophy by a

previously underestimated cross talk mechanism to calcineurin. In the absence of CaMKII

signals, calcineurin does not seem to contribute to maladaptive cardiac remodeling, highlighting

CaMKII and not calcineurin as a promising drug target to combat heart failure.

2814-S/A knockin mice.6 Consistent with our data, they also observed an unalteerereed d SRSRR CCCaaa2+ 2+2+

content, Ca2+ transient decay and Ca2+ sparks at baseline, but they discovered that in the situation

off hhheaeaeartrtrt fffaiaiailululurrre, mumumutat nt mice were protected. Thuhuhus,s further experimimmenntstss aaarer warranted to

nnveeests igate DKKOO O mmimicecee aaandndnd CCCaaa2+ 2+2+ hahaanndndliingng in mmmodeeelss of f seseevevereree hheeaarrtt ffaiaiilull rrere. HoHoHowewevevev r,r,r iiitt apapappepeearrrs

ununlililikekekelylyly tthahahat t t anan aaattttenenuuuattitionon ooof f dididistststuruurbebeed d CaCaCa222+ hhhananandldliingg g cococontntntriibububuttees s tototo tthehehe iimpmpmprorooveveedd d ccarrdrdiiaac c

function as obobbseseservrvvededed iiin n n DKDKKO O O afffteteterr TATAT C CC ininin ttthehehe ppprerer sesesentnn ssstututudydydy..




17

Acknowledgments: The authors thank Ulrike Oehl, Lonny Jürgensen, Jutta Krebs, Sylvia Katz,

Claudia Heft, Michaela Oestringer and Claudia Liebetrau for their excellent technical assistance.

We thank David Stanmore for editing the manuscript. We thank Lorenz Uhlmann for statistical

advice.

Funding Sources: J.B., H.A.K., D.D. and L.S.M. were supported by the DZHK (Deutsches

Zentrum für Herz-Kreislauf-Forschung - German Centre for Cardiovascular Research) and by

the BMBF (German Ministry of Education and Research). J.B. was supported by the Deutsche

Forschungsgemeinschaft (BA 2258/2-1, SFB 1118) and by the European Commission (FP7-

Health-2010; MEDIA-261409). L.S.M. was supported by the Deutsche Forschungsgemeinschaft

(SFB 1002, IRTG 1816) and the Fondation Leducq. L.H.L. is recipient of a HRCMM

(Heidelberg Research Center for Molecular Medicine) career development fellowship. M.M.K.

was supported by a research grant from the Ernst-und-Berta-Grimmke foundation and by a

Young Investigator grant of the University of Heidelberg. D.D. was supported by a grant from

Foundation Leducq (07CVD03).

Conflict of Interest Disclosures: None.

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g ) p pp

was supported by a research grant from the Ernst-und-Berta-Grimmke foundationonn aaandndnd bbby y y a aa

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CCConnfnflict of Innteterererestt DDDisssclclcloososururureses:: NNoNonnene..

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111. . AwAwAwadadad SSS,, KKuKunhnhnhii M, Little GH, Bai Y, An W,, BeBeBers D, Kedes L L ana d d PoPoPoizi at C. Nuclear CaMKIIenennhahaances hhisstttonene HHH33 phphphosossphphp ororylylylatatatioionn anand d reremmododeeelsss chroroomamamatit n dududurriringng ccarara diacac hhhypypyperertrtropopophyhyhy.. NNucclcleic Acids s ReReRes.. 2201010 3;3;3;414141:7:77656656-6--76767672722.

1222. LiLiLittttttlele GGGH,H,H BBaai YY, , WiWWilllliaiammsms TTT aaandndn PPPoioioizazazattt C.C.C NNNucucclelearar cccalalalciciumumum/c/calalalmomomoduduulilinn-n-dededepepependndndennnt pprprototteieinnn kinanasese IIIdIdelelta pprer fefererentn ialllly y trtranansmsmits siigngnalals s toto hhistoonene ddeeacacetetylasasee 44 ini cacarddiaiacc cellls.s. J BiBiolol Chem. 2007;2;2;2828282:7:772121219-9-9 7277 313131.




19

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2233. ZZhou XF, MMMararleley y PDPDD aaandndnd LLLivivetete tt t BBGG.. Suubsbsstanccee P momom duduulalaatetesss thhhe e titiimememe ccououoursrsee ofofof nnnicicicotttinininiccic bbbut nonoot mmum scarinnici ccatateechhoolaamiminnene seccreetitit oonn ffrommm pperffufusesed d adadadrererenaaal ggllaannds ooof f rarattt. BBrBr J PPPhhaharrmrmaacooll.199991919 ;1;1;10404:1:11595959-1166565..

24. Sussman n MAMAMA, , LiLiL m m m HWHWHW, , , GuGuGudedede NNN,, TaTaTaigigigenenen TTT,,, OlOlOlsosoon n ENENEN,, RoRoRobbbbbbininss s J,J,J, CCCololo bebeb rtrtt MMMC,C,C, GGGualberto AA,WiWiececzozorerekk DFDF aandnd MMololkekentntinin JJDD PPrereveventntioionn ofof ccarardidiacac hhypyperertrtropophyhy iinn mimicece bbyy cacalclcinineueuririnn




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28. Agah R, Frenkel PA, French BA, Michael LH, Overbeek PA and Schneider MD. Gene recombination in postmitotic cells. Targeted expression of Cre recombinase provokes cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J Clin Invest. 1997;100:169-179. 29. Frey N, Frank D, Lippl S, Kuhn C, Kogler H, Barrientos T, Rohr C, Will R, Muller OJ, Weiler H, Bassel-Duby R, Katus HA and Olson EN. Calsarcin-2 deficiency increases exercise capacity in mice through calcineurin/NFAT activation. J Clin Invest. 2008;118:3598-3608. 30. Timmins JM, Ozcan L, Seimon TA, Li G, Malagelada C, Backs J, Backs T, Bassel-Duby R, Olson EN, Anderson ME and Tabas I. Calcium/calmodulin-dependent protein kinase II links ER stress with Fas and mitochondrial apoptosis pathways. J Clin Invest. 2009;119:2925-2941.

31. Hill JA, Karimi M, Kutschke W, Davisson RL, Zimmerman K, Wang Z, Kerber RE and Weiss RM. Cardiac hypertrophy is not a required compensatory response to short-term pressure overload. Circulation. 2000;101:2863-2869. 32. Rezazadeh S, Claydon TW and Fedida D. KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N -methylbenzylamine), a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellular blocker of voltage-gated potassium channels. J Pharmacol Exp Ther. 2006;317:292-299. 33. Gao L, Blair LA and Marshall J. CaMKII-independent effects of KN93 and its inactive analog KN92: reversible inhibition of L-type calcium channels. Biochem Biophys Res Commun. 2006;345:1606-1610.

34. Ling H, Zhang T, Pereira L, Means CK, Cheng H, Gu Y, Dalton ND, Peterson KL, Chen J, Bers D and Heller Brown J. Requirement for Ca2+/calmodulin-dependent kinase II in the transition from pressure overload-induced cardiac hypertrophy to heart failure in mice. J Clin Invest. 2009;119:1230-1240. 35. Yang Y, Zhu WZ, Joiner ML, Zhang R, Oddis CV, Hou Y, Yang J, Price EE, Gleaves L, Eren M, Ni G, Vaughan DE, Xiao RP and Anderson ME. Calmodulin kinase II inhibition protects against myocardial cell apoptosis in vivo. Am J Physiol Heart Circ Physiol. 2006;291:H3065-3075. 36. Hashimoto Y, King MM and Soderling TR. Regulatory interactions of calmodulin-binding proteins: phosphorylation of calcineurin by autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Proc Natl Acad Sci U S A. 1988;85:7001-7005. 37. MacDonnell SM, Weisser-Thomas J, Kubo H, Hanscome M, Liu Q, Jaleel N, Berretta R, Chen X, Brown JH, Sabri AK, Molkentin JD and Houser SR. CaMKII negatively regulates calcineurin-NFAT signaling in cardiac myocytes. Circ Res. 2009;105:316-325. 38. Wilkins BJ and Molkentin JD. Calcium-calcineurin signaling in the regulation of cardiac hypertrophy. Biochem Biophys Res Commun. 2004;322:1178-1191.

32. Rezazadeh S, Claydon TW and Fedida D. KN-93 (2-[N-(2-hydroxyethyl)]-N-((4-4-4-methoxybenzenesulfonyl)]amino-N-(4-chlorocinnamyl)-N -methylbenzylamine)e),, , a calcium/calmodulin-dependent protein kinase II inhibitor, is a direct extracellularar bbblololockckkererer ooofff voltage-gated potassium channels. J Pharmacol Exp Ther. 2006;317:292-299.

333. . GaGaGaooo LL,L, BBBlllairr LLLAA and Marshall J. CaMKII-indndndepeppendent effectstss of KNKNKN939 and its inactiveanannalalloog KN9N9222: rrevevverere sisiblblle e inininhihibibitititiononon ooff L-L-tytyypepep ccalalcciuuum cchahahannnnnnele s.s. BiBiBiocochehem m m Biiopopphyhyhysss ReRes s CoCoC mmmmmmunu . 220000606;345:16006-6-1616161000..

3444. LiLiLingngng HHH,,, ZhZZhananng g g T,T, PPeerereie raaa LLL, MeMeMeananss s CKCKCK,,, ChChChenenng g H,H,H, GGGuuu YY,Y, DDDaalaltooon n n NDNDND, , , PePeetetetersrsr ononn KKKLL,L, CCChehennn JJ,J, Bersrs DD andnd Helellel r BrBroown n JJ. RReqequiuirementnt fforor CaCa2+2 /ccalalmomodudulilin-n dedepependndennt t kikinan sese II inin thehe ransition froom m m prprp esesessususurerere oveveverlrlrloaoaad-d-d ininndududucecec dd d cacacarrrdididiacacac hhhypypy ererrtrtrtropopophyhyhy tttooo heheheararart t t fafafailillururu e e e ininin mmmicicce.e J Clin

InInvevestst 2200009;9;11119:9:12123030 1-1242400




21

39. Wilkins BJ, Dai YS, Bueno OF, Parsons SA, Xu J, Plank DM, Jones F, Kimball TR and Molkentin JD. Calcineurin/NFAT coupling participates in pathological, but not physiological, cardiac hypertrophy. Circ Res. 2004;94:110-118.

40. Marley PD and Thomson KA. Inhibition of nicotinic responses of bovine adrenal chromaffin cells by the protein kinase C inhibitor, Ro 31-8220. Br J Pharmacol. 1996;119:416-422. 41. Heineke J, Wollert KC, Osinska H, Sargent MA, York AJ, Robbins J and Molkentin JD. Calcineurin protects the heart in a murine model of dilated cardiomyopathy. J Mol Cell Cardiol. 2010;48:1080-1087. 42. Kim Y, Phan D, van Rooij E, Wang DZ, McAnally J, Qi X, Richardson JA, Hill JA, Bassel-Duby R and Olson EN. The MEF2D transcription factor mediates stress-dependent cardiac remodeling in mice. J Clin Invest. 2008;118:124-132. 43. Kuwahara K and Nakao K. New molecular mechanisms for cardiovascular disease:transcriptional pathways and novel therapeutic targets in heart failure. J Pharmacol Sci. 2011;116:337-342. 44. Nishikimi T, Kuwahara K and Nakao K. Current biochemistry, molecular biology, and clinical relevance of natriuretic peptides. J Cardiol. 2011;57:131-140. 45. Engeli S, Birkenfeld AL, Badin PM, Bourlier V, Louche K, Viguerie N, Thalamas C, Montastier E, Larrouy D, Harant I, de Glisezinski I, Lieske S, Reinke J, Beckmann B, Langin D, Jordan J and Moro C. Natriuretic peptides enhance the oxidative capacity of human skeletal muscle. J Clin Invest. 2012;122:4675-4679. Figure Legends

Figure 1. CaMKII and CaMKII contribute redundantly to cardiac CaMKII activity. (A) Real-

time-PCR analysis of cardiac CaMKII isoforms three weeks after sham (SH) or transverse aortic

constriction (TAC) surgery in wild type mice. The relative expression levels were normalized to

CaMKII in brain tissue. All values are reported as mean±SEM (n 3, *p<0.05). (B) To generate

cardiomyocyte-specific conditional CaMKII and single knockout ( -CKO and -CKO) and

CaMKII /CaMKII double knockout mice (DKO), CaMKII loxP-exon1/2-loxP ( -FF), CaMKII loxP-

exon1/2-loxP ( -FF) and CaMKII loxP-exon1/2-loxP, CaMKII loxP-exon1/2-loxP (FFFF) mice were crossed to

2011;116:337-342.

44. Nishikimi T, Kuwahara K and Nakao K. Current biochemistry, molecular biiolollogogogyy, aaandndnd clinical relevance of natriuretic peptides. J Cardiol. 2011;57:131-140.

455. . EnEnEnggegelilili SSS, Biirkrkrkeenenfeld AL, Badin PM, Bourlieeerr r VVV, Louche K, VVVigi ueeriririee e N, Thalamas C, MMoMonntntastier EEE,,, LaLarrrrrrououy y y D,D,D, HHHararanannttt I,I,I, ddee GlGlisisezezininskskiii II,, Lieeskskskeee S,S ReReReiinnkeke JJ, , BeB ckckmamamannnnn BB,,, LaLaL ngngngininin D,Joordddana J and MMoorrooo C.C.C NNatatatriririuruureeteticic pppeepepttiiddees enhnhhanccce theee oooxixidadad titivevee ccappacaccittyy ofoff hhhumummanana sskekek leleetatal llmmmusscscle. J ClClinin Innvnveest. 200012;2;121222:464 75757 --4466679.

Figure Leggenenndsdsds




22

transgenic mice harboring Cre-recombinase under the control of the MHC promoter. (C)

Western blot analysis using antibodies directed against CaMKII, CaMKII , total PLB, phospho-

PLB-Thr17, phospho-PLB-Ser16, total RyR2, phospho-RyR2-Ser2814, phospho-RyR2-Ser2808,

total HDAC4, phospho-HDAC4-Ser632 and GAPDH (as loading control). Left ventricular

extracts from -CKO, -CKO and DKO mice and their Cre-negative littermates ( -FF, -FF and

FFFF) were analyzed (n=4 per group). Quantitative analysis of the expression and

phosphorylation of the proteins is shown at right. All values are presented as mean±SEM.

*p<0.05. n.s., not significant.

Figure 2. Calcineurin precedes CaMKII activation upon pathological pressure overload. Western

blot analysis were performed in cardiac extracts from wild type mice. Organs were harvested at

different time points after TAC surgery as indicated. (A) Western blot analysis after GST-

HDAC4 pulldown using GST-HDAC4 419–670, which contains a CaMKII-activity dependent

binding site. GST-HDAC4 2-250, which lacks a CaMKII binding domain, served as a negative

control. As an additional negative control, protein lysates from global CaMKII mice versus wild

type mice were used. GST-HDAC4 input was visualized by GST-immunoblotting, associated

endogenous CaMKII is visualized by CaMKII-immunoblotting. The degree of CaMKII binding

to GST-HDAC4 419-670 is a specific measure for CaMKII activity.10 A quantification of

CaMKII binding normalized to the input of GST-HDAC4 is shown. (B) Western blot analysis

using antibodies directed against CaMKII, HDAC4, phospho-HDAC4-Ser632, RCAN1-4 and

GAPDH. Quantitative analysis of the expression and phosphorylation of the proteins is shown.

For all experiments n 3 per group were used. All values are presented as mean±SEM. *p<0.05.

n.s., not significant.

Figure 2. Calcineurin precedes CaMKII activation upon pathological pressure ovvoverrrlloadadad.. WeWeWeststerern

blot analysis were performed in cardiac extracts from wild type mice. Organs were harvested at

didifffffererereenentt tititimememe poioioinntnts s after TAC surgery as indicatatateddd. (A) Westernnn bbloot t anananala ysis after GST-

HDHDAAC4 pulldodownwnwn uusisingngg GGGSTSTST-H-HDADADAC4C44 44419––6–670, wwhhiccch hh coconntntaiainsnss aaa CCaMaMMKKIKII-I-I-aacactitivivivitytyy ddeepepeenendedeenntnt

bibindndndininnggg sisisitetete.. GSGSST-T-T-HDHDHDACACC4 4 2-2-2-2525250,0,0, wwwhihichchch lllacacacksss aa CCCaaaMKMKMKIIIIII bbbinindidid ngngg dddomomomaiin,n,n sserere veveveddd aasas aa negegegatattivive ee

control. As ananan aaadddddditititioioionananal l nenenegagagatiiveveve ccconono tttroorol,l,l, ppprorooteteeininin lllysysysatateseses fffrororom m m glglg obobbalalal CCCaMaMaMKIKIKIIII mmmicicice e e versus willd




23

Figure 3. Dissociation of cardiac dysfunction and interstitial fibrosis from myocardial

hypertrophy. (A) DKO and FFFF mice were randomized to either TAC or SH surgery and

sacrificed after 3 weeks. Representative images of the total hearts, H&E, Masson´s trichrome and

CD31 staining, echocardiographic M-modes and quantification of heart weight/body weight

ratios, myocyte size, fibrosis area, capillary density and fractional shortening are shown (n 8 per

group). Values of left ventricular ejection fraction, isovolumetric relaxation time constant (Tau),

end systolic pressure volume relation (ESPVR) and stroke volume measured in a working heart

preparation are shown (n 7 per group). (B) Fold-changes in mRNA levels of the hypertrophic

markers ANP, BNP, MHC and MHC and fold-changes in collagen expression (n 5 per

group). (C) Heart weight/body weight ratios of SH- and TAC-operated mice with the indicated

genotypes (n 4 per group). All values are presented as mean±SEM. *p<0.05. n.s., not

significant.

Figure 4. CaMKII controls CnA activity. (A) Real-time-PCR analysis of RCAN1-4 in hearts

from animals with genotypes and treatments as indicated ( 5 per group). (B+C) NMVMs from

FFFF and DKO were used for cell-based experiments. (B) NFAT-GFP was adenovirally

expressed in NMVMs. Cells were stained for -actinin (shown in red) and with DAPI nuclear

stain (shown in blue). The percentage of cells in which NFAT-GFP was localized to the nucleus

is indicated (>100 cardiomyocytes per well; n 3 per condition). (C) NFAT-luciferase reporter

assay in NMVMs (n 3 per condition). (D) Western blot analysis was performed using lysates

from COS-cells that were transfected with CnA in the absence and presence of constitutive

active CaMKII (CaMKII-T287D). A schematic diagram of CnA indicates the position of the

CaMKII phosphorylation site at Ser411, which is located within the calmodulin-binding domain

group). (C) Heart weight/body weight ratios of SH- and TAC-operated mice withthh ttheheh iindndndicicicatatateded

genotypes (n 4 per group). All values are presented as mean±SEM. *p<0.05. n.s., not

iigngnnififificicicananntt.t.

FiFigugugurerere 44.. CaCaCaMKMKMKIIII cconontrtrrolols CnCnCnAAA acacacttitiviviitytyy.. (((AAA) RRReaeaal---tit mememe-P-P-PCRCRCR aannanalylylysisis ss offf RRRCACACAN1N11-4-44 inn n hheheararttts

from animalss wwwititi h h h gegegenononotytypepepes s s annnd d d trtrreaeae tmtmmenenentststs aaas s s ininndidid cacac teeed d d ((( 555 pepeper r r grgrrouououp)p)p). . (B(B(B+C+CC))) NNNMVMVMVMs from




24

of CnA (CnB, calcineurin B-binding domain; CaM, calmodulin-binding domain; AID,

autoinhibitory domain). (E) Adenoviral overexpression of CaMKII-T287D (adCaMKII-T287D)

in NRVMs results in hyper-phosphorylation of CnA-Ser411 compared to non infected (control)

NRVMs and NRVMs infected with GFP. (F) Western blot analysis of total CaMKII, RCAN1-4,

total CnA and, CnA-Ser411 in cardiac extracts from FFFF and DKO mice 3 weeks after SH or

TAC surgery (n 3 per group). (G) Western blot analysis of total CnA and CnA-Ser411 in -

CKO, -CKO or DKO versus their Cre-negative littermates (n=4 per group). For all Western blot

experiments, GAPDH was used as loading control. Quantitative analysis of the expression and

phosphorylation of the proteins is shown at right (F and G). All values are presented as

mean±SEM. *p<0.05. n.s., not significant.

Figure 5. Cardiac hypertrophy, but not dysfunction is controlled by calcineurin during

pathological and physiological hypertrophy in DKO. (A-B) NMVMs from FFFF and DKO mice

were transfected with two siRNAs directed against CnA or with scrambled siRNA (knockdown

efficiency is shown in Suppl. Fig. 5A). NMVMs were then treated with Iso or vehicle. (A) ANP

and BNP gene expression in NMVMs after Iso-stimulation (n 3 per condition). (B) Cells were

stained for -actinin (shown in red) and cardiomyocyte size was determined after Iso-stimulation

(>100 cardiomyocytes per well; n 3 per condition). (C) FFFF and DKO mice underwent SH or

TAC surgery and were treated with cyclosporine A (CyA; 4 mg/Kg/d, 3 weeks i.p.; n 7) or

vehicle (n 3) as indicated. CyA treatment was started at the day of TAC or SH surgery. Three

weeks later the mice were sacrificed. HW/BW ratios, myocyte cross sectional areas,

quantification of cardiac fibrosis from Masson´s trichrome-stained myocardial sections and

fractional shortening assessed by echocardiography are shown. (D-F) FFFF and DKO mice were


Fiigugugurerere 555. CaCaCarrrdiaaaccc hhypertrophy, but not dysfunctititiononon is controlled bybb ccalalalccicinen urin during

pathhholo ogical aandndnd pphyhysisis olollogoggicicicalalal hhypypypeerertrrropphy innn DKKKOOO. (A(AA-B-B))) NNNMMVVMsMs fffrorom m FFFFFFFFFF aaandndnd DDDKOKOKO mmmiici e

weweererer tttrarar nsnsfefefectccteded wwwitith h twtwwoo siiRRRNANANAsss diddirereectcttededed aagagagaininstst CCnAnAnA ooorr r wiwiwiththt scrcrcramamamblblleded sssiRiRRNANAA (((knknnoccckdkdowowown nRR

efficiency is shshshowowown n n ininin SSSupupplplpl... FiFiig.g.g. 55A)AA .. NMNMNMVMVMVMs s wewewererer tthehehen n n trtrtreaeaeateteted wiwiwiththth IIIsososo oor r r vevevehihihiclclcle.e (A) ANP




25

randomly assigned to a 14-day swimming or resting group, respectively. (D) Cardiac CaMKII

activity was determined in heart lysates by GST-HDAC4 pulldown using GST-HDAC4 419–670

(GST-HDAC4 2-250 served as specificity control). A quantification of CaMKII-activity

dependent binding as measure for CaMKII activity is given (n 11 per group). (E) Representative

images of whole hearts as well as H&E and Masson´s trichrome staining of left ventricular wall

sections. Quantification of HW/BW ratios, myocyte size, fibrosis area and fractional shortening

(n 3 per group). (F) Western blot analysis using antibodies directed against CaMKII, total CnA,

phospho-CnA-Ser411, RCAN1-4 and GAPDH. Quantitative analysis of the expression and

phosphorylation of the proteins is shown (n 7 per group). All values are presented as


Figure 6. Schematic model. Stimulation of -adrenergic receptors by norepinephrine (NE) leads

to an activation of the two Ca2+-dependent pathways calcineurin and CaMKII (mediated by the

isoforms CaMKII and CaMKII ). Activated CaMKII phosphorylates HDAC4, which leads to a

translocation of HDAC4 from the nucleus to the cytosol. Thereby, the repressive effect of

HDAC4 on transcription factors as MEF2 is interrupted and a maladaptive remodeling gene

program is initiated. As a second mechanism, CaMKII phosphorylates CnA, which leads to

repression of the calcineurin-NFAT pathway and thereby acts anti-hypertrophic. Unexpectedly,

the endogenous calcineurin-NFAT driven pro-hypertrophic pathway is not maladaptive.


Fiigugugurerere 666. ScScSchhhemamamattiticc model. Stimulation of -adrdrdrenenenergic receptorrss s byy nnnoororepinephrine (NE) leads

oo aaan n activationon ooof f thhhe e twwwo oo CaCaCa2+2+2+-d-depepepenendedeent pppattthwayayays ccaallclcininneuuuririnnn aannd d CaCC MKMKMKIIIIII ((memeedididiatatedede bbby y ththhe

ssofofofororormsmsm CCCaMaMaMKIKIII aanndnd CCCaMaMMKIKIKIIII ).).). AAccttivvvatatateeed CCCaMaMMKKKII I I phphphosososphphhororo ylyly atatateeses HHDADAAC4C4C4,,, whwhwhiichh h leeeadadsss tooo a

ranslocatioonnn ofofof HHHDADADAC4C4C4 ffrororom mm ththhe e e nununuclleuueuss s tototo ttthhhe e e cycycytototososol.l.l. TTTheheherererebybyby, thththee e rerereprprpresese sisiiveveve eeffffffecece t of



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Johannes BacksDobrev, Christoph Maack, Lars S. Maier, Hermann-Josef Gröne, Hugo A. Katus, Eric N. Olson and

Kohlhaas, Jan-Christian Reil, Kay Neumann, Michael D. Schneider, Joseph A. Hill, Dobromir Michael M. Kreusser, Lorenz H. Lehmann, Stanislav Keranov, Marc-Oskar Hoting, Michael

Inhibit Calcineurin-Induced Myocardial Hypertrophy Contribute Redundantly to Adverse Remodeling butγ and δThe Cardiac CaMKII Genes

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SUPPLEMENTAL MATERIAL

The cardiac CaMKII genes δ and γ contribute redundantly to adverse

remodeling but inhibit calcineurin-induced myocardial hypertrophy

Michael M. Kreusser, MD1; Lorenz H. Lehmann, MD1; Stanislav Keranov, MD1; Marc-Oscar Hoting1;

Michael Kohlhaas, PhD2; Jan-Christian Reil, MD2; Kay Neumann, MD3; Michael D. Schneider, MD4;

Joseph A. Hill, MD, PhD5; Dobromir Dobrev, MD6; Christoph Maack, MD2; Lars S. Maier, MD3;

Hermann-Josef Gröne, MD7; Hugo A. Katus, MD8; Eric N. Olson, PhD9; Johannes Backs, MD1, 10

1 Research Unit Cardiac Epigenetics, Department of Cardiology, University of Heidelberg, Heidelberg,

Germany; DZHK (German Centre for Cardiovascular Research), partner site Heidelberg/Mannheim,

Germany.

2 Department of Cardiology, Saarland University, Homburg, Germany

3 Department of Internal Medicine II, University of Regensburg, Regensburg, Germany

4 British Heart Foundation Centre of Research Excellence, National Heart and Lung Institute, Faculty

of Medicine, Imperial College London, United Kingdom

5 Department of Internal Medicine, University of Southwestern Texas Medical Center, Dallas, TX, USA

6 Institute of Pharmacology, University of Duisburg-Essen, Essen, Germany

7 Department of Molecular Pathology, German Cancer Research Center, Heidelberg, Germany

8 Department of Cardiology, University of Heidelberg, Heidelberg, Germany; DZHK (German Centre

for Cardiovascular Research), partner site Heidelberg, Germany

9 Department of Molecular Biology, University of Southwestern Texas Medical Center, Dallas, TX, USA

10 Address for correspondence:

Johannes Backs, MD, Research Unit Cardiac Epigenetics / Department of Cardiology, Heidelberg

University, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany. Phone: +49-6221-56-37714. Fax:

+49-6221-56-5573. e-mail: [email protected]

Supplemental Methods

Generation of DKO mice. All DKO animals (except the animals that were used for the

endurance exercise experiment) were generated by crossing FFFF mice with transgenic

mice expressing Cre-recombinase under the control of the a-MHC promoter (aMHC-Cre).1

DKO mice that were used for the endurance exercise experiment were generated by

crossing FFFF mice with transgenic mice expressing the mER-Cre-mER fusion protein under

control of a cardiomyocyte-specific αMHC-promoter.2 The latter animals were treated with

tamoxifen to achieve CaMKII deletion. Therefore, 10 mg/ml tamoxifen (Sigma) was dissolved

in a regular sunflower oil solution containing 6% ethanol. Tamoxifen was administered via

gavage for 10 consecutive days once per day in a dose of 80 mg/Kg body weight per day.

Efficient take-out of CaMKII was verified by Western blot. To avoid interferences with

transient Cre expression, experiments were conducted at least 3 weeks after the last

tamoxifen application.

Transverse aortic constriction. TAC to a 27 gauge stenosis was performed in 9-10 week-

old male DKO, δ-KO, γ-KO, CaMKII δ-/-/γ+/-, CaMKIIγ-/-/δ+/-, FFFF and wild type mice as

described previously.3, 4 A subgroup of the animals was treated twice daily by i.p. injection

with cyclosporin A (CyA; Sandimmun, Novartis) with 24 mg, 12 mg or 4 mg/kg body weight

per day for 7 days or 3 weeks, beginning at the day of surgery. CyA was diluted in 0.9%

saline solution with 12% ethanol, and the vehicle alone served as control.

Isoproterenol administration. Isoproterenol (Iso; Sigma) diluted in 0.1% ascorbic acid was

injected intraperitoneally (i.p.) once daily in 8-week-old male DKO mice and FFFF mice in a

given dose of 10 mg/kg body weight per day over 14 days. 0.1% ascorbic acid alone

(vehicle) served as control.

Swimming exercise protocol. Endurance exercise was carried out as described by others.5,

6 In brief, a ramp protocol was used starting at 10 minutes two times daily, with 10 minutes

increase each day until 90 minutes, two times per day was reached. The protocol ended after

14 days. The animals were closely observed at all times to avoid relative hypoxia. With this

protocol, there were no events of mice submerging under the water surface.

Transthoracic echocardiography and working heart preparation. Echocardiography and

studies on isolated hearts were carried out as previously described. 7, 8

Histology. Hematoxylin and eosin (H&E), Masson´s trichrome and CD31 stainings were

performed as previously described.9, 10 Cardiomyocyte size was assessed on H&E-stained

sections by using Image J software (http://rsb.info.nih.gov/ij/). More than 200 randomly

chosen cardiomyocytes from each group were analyzed to measure cross-sectional

cardiomyocyte area. To quantify cardiac fibrosis, 20 trichrome-stained sections

(magnification 20x) from the left ventricle were randomly selected, and morphometric

analysis by using Image J was performed. Capillary density was determined as the ratio of

capillaries to 100 cardiomyocytes in transversely sectioned left ventricular tissue

immunostained with CD31 and counterstained with DAPI. Photographs were acquired with

an Olympus SZH zoom stereo dissection scope with an Optronics DEI-750 CCD digital

camera. All data were analyzed by a single observer blinded to the mouse genotypes.

Western Blotting and GST-pulldown. Proteins from heart tissue and cultured

cardiomyocytes were isolated, and Western blot analysis was performed according to

protocols described previously.11 GST-pulldown experiments were performed as described.11

Besides GST-HDAC4 419-670 (which contains a CaMKII activity-dependent binding domain)

we used GST-HDAC4 2-250 (which does not contain a CaMKII binding domain) as a

negative control. Primary antibodies used were anti-CaMKII total (BD Bioscience), anti-

CaMKIIγ (Santa Cruz), anti-p-CaMKII (Ser287) (Affinity Bioreagents), anti-PKD (Cell

signaling), anti-p-PKD (Ser744) (Cell signaling), anti-CnA (Abcam), anti-p-CnA (Ser411)

(generated by Pineda antibodies), anti-GAPDH (Chemicon), anti-HDAC4 (Santa Cruz), anti-

p-HDAC4 (Ser632) (Abcam), anti-phospholamban (PLB) (Upstate), anti-p-PLB (Thr17)

(Santa Cruz), anti-p-PLB (Ser16) (Upstate), anti-RyR2 (Affinity BioReagents), anti-p-RyR2

(Ser2808) and anti-RyR2 (Ser2814) (kind gifts from Dr. Xander Wehrens, Houston, USA),

anti-Akt (Cell signaling), anti-p-Akt (Ser473) (Cell signaling), anti-RCAN1-4 (kind gift from Dr.

Timothy McKinsey, Denver, USA), and anti-α-actin (Sigma-Aldrich). Primary antibody

incubation was followed by corresponding horseradish peroxidase (HRP)-conjugated

secondary anti-mouse and anti-rabbit antibodies and ECL detection. Relative protein levels

were detected by densitometry using the Image J program.

CaMKII kinase activity. CaMKII kinase activity was measured by radioactive kinase assays

using GST-HDAC4 419-670 as a substrate and indirectly by detecting the amount of

endogenous CaMKII that associates to GST-HDAC4 419-670 (see also above under GST

pulldown). A detailed protocol has been described previously.3, 11

Caspase 3/7 activity measurements. The Caspase-Glo 3/7 Assay (Promega Corporation)

was used to measure caspase-3 and -7 activities according to the manufacturer´s

instructions. Heart lysates were mixed with reaction solution at equal volumes in a 96-well

plate. Luminescence was detected after 60 minutes of incubation at room temperature.

RNA analysis. Total RNA was isolated from ventricular tissue or from cultured

cardiomyocytes using TRIzol (Invitrogen). Total RNA was digested with DNase, and cDNA

synthesis from 500 ng of RNA was carried out using a SuperScript first-strand synthesis

system for RT-PCR (Invitrogen). Quantitative real-time PCR (qPCR) was performed with

Universal ProbeLibrary (Roche) by using TaqMan Universal PCR Mastermix (Applied

Biosystems) and detection on a 7500 Fast Cycler (Applied Biosystems). Primers and probes

were: For rat: ANP sense 5´-cccgacccagcatgg-3´ and antisense 5´-caactgctttctgaaaggggtg-

3´; For mouse: CaMKIIα sense 5´-gctgccaagattatcaacacc-3´ and antisense 5´-

cacgctccagcttctggt-3´; CaMKIIβ sense 5´-gccatcctcaccactatgct-3´ and antisense 5´-

ctccatctgctttcttgttgagt-3´; CaMKIIγ sense 5´-agttcacagggacctgaagc-3´ and antisense 5´-

cgccttgaacttctatggcta-3´; CaMKIIδ sense 5´-gtgccatcctcacaaccat-3´ and antisense 5´-

catctgacttcttgttcaataggc-3´; GAPDH sense 5´-gggttcctataaatacggactgc-3` and antisense 5´-

ccattttgtctacgggacga-3`; Col5a1 sense 5`-ctacatccgtgccctggt-3` and antisense 5`-

ccagcaccgtcttctggtag-3`; Col16a1 sense 5`-gcattgcaggagaaaatggt-3` and antisense 5`-

ccatcttgccataacctgga-3`; ANP sense 5`-cacagatctgatggatttcaaga-3` and antisense 5`-

cctcatcttctaccggcatc-3`; BNP sense 5´-gtctggccggacactcag-3` and antisense 5`-

tgcactggtgtcttcaacaac-3`; bMHC sense 5`-cgcatcaaggagctcacc-3` and antisense 5`-

ctgcagccgcagtaggtt; aMHC sense 5`-cgcatcaaggagctcacc-3` and antisense 5`-

cctgcagccgcattaagt-3`; Rcan1-4 sense 5`-actggaaggtggtgtccttgt-3` and antisense 5´-

tccagcttgggcttgactgag-3` .

Adenovirus production. Adenoviruses harboring CaMKII-T287D and GFP were generated

according to the manufacturer’s instructions (ViraPower Adenoviral Expression System;

Invitrogen). Nuclear factor of activated T-cells (NFAT) luciferase, and NFAT-GFP

adenoviruses were obtained from Seven Hills Bioreagents. After generation, the

adenoviruses were amplified, purified with the Adeno-X Purification Kit (BD Biosciences) and

its infectious units per µl were determined with the Adeno-X Rapid Titer Kit (BD Biosciences).

Cell culture of COS cells and transfection assays. COS cells were maintained in DMEM

with 10% FBS, 2 mM l-glutamine, and penicillin-streptomycin. Transfection was performed

with GeneJammer (Agilent Technologies) according to manufacturer’s instructions. The

expression constructs for CaMKII-T287D and CnA were described before.11, 12

Culture of neonatal rat ventricular cardiomyocytes (NRVMs). NRVMs were isolated from

1 to 2-day old Wistar rats as previously described.11 After isolation, NRVMs were maintained

in DMEM/199 medium (4:1) with 10% FBS, 2 mM l-glutamine, and penicillinstreptomycin.

NRVMs were infected 24 h after plating, grown 12 h later in serum-free media for another 4

h.

Culture of neonatal mouse ventricular cardiomyocytes (NMVMs). Neonatal

cardiomyocytes were isolated from 1- to 2-day-old DKO or FFFF mice according to

previously published methods.13 Cells grew for 24 h in the fresh complete medium. Some of

the cells were treated with CnA-siRNA or scrambled siRNA according to the manufacturer’s

instructions (Sigma-Aldrich). Medium was then replaced with serum-free medium 24 h prior

to adding isoproterenol (Sigma-Aldrich, 0.1 μM) for another 24 h. Cells were then harvested

for immunocytochemical staining or luciferase assay. For some of the immunostainings and

for luciferase assay, adenovirus harboring NFAT-GFP or NFAT luciferase reporter were

added, respectively.

Culture of adult mouse ventricular myocytes (AMVMs). For AMVM isolation, δ-CKO, γ-

CKO and DKO mice and their Cre-negative littermates (control), respectively, were

anesthetized with isoflurane and hearts were excised. Explanted hearts were retrogradely

perfused and digested as described before.14-16 Cells were plated onto regular cell culture

plates or superfusion chambers, with the glass bottoms treated with laminin. Cells were

immediately used for epifluorescence microscopy experiments or grew for 24 h in the fresh

complete medium. Medium was then replaced with serum-free medium 24 h prior to adding

isoproterenol (Sigma-Aldrich, 0.1 μM) for another 24 h.

Immunocytochemistry. NMVMs or AMVMs were fixed in 4 % paraformaldehyde (Sigma-

Aldrich) in PBS, permeabilized with 0.3 % Triton X-100 (Sigma-Aldrich) for 10 min at room

temperature and blocked for 30-60 min with 5 % goat serum (PAA) or BSA in PBS. Primary

antibody for α-actinin (Sigma) was applied in PBS containing 5 % goat serum or BSA and

0.05 % Triton X-100 for 1 h. Cells were treated with secondary antibody (Texas Red-coupled

anti-mouse-antibody) in PBS containing 5 % goat serum and 0.1 % Triton X-100 for 1 h.

Nuclei were labeled with DAPI (Invitrogen).

Apoptosis assay and cell size in AMVMs. TUNEL assays were performed using the in situ

cell death detection kit (Roche) according to the manufacturer’s protocol. To quantify the

number of apoptotic AMVMs, AMVMs were counterstained with sarcomeric α-actinin, and the

total numbers of AMVMs and TUNEL-positive nuclei were counted in 10 low power fields.

For each experimental condition, contiguous visual fields were counted to accumulate data

on 200 AMVMs per condition. Stained cells were normalized to total cell count as judged by

DAPI staining. More than 80% of cells were sarcomeric α-actinin positive. Cardiomyocyte

size was assessed by using Image J software (http://rsb.info.nih.gov/ij/). More than 100

randomly chosen cardiomyocytes from each group were analyzed to measure cross-

sectional cardiomyocyte area.

Epifluorescence microscopy. A Nikon Eclipse TE2000-U inverted microscope which was

provided with an IonOptix fluorescence detection system for assessment of Ca2+ handling

properties was used (IonOptix). For the studies on unstressed mice (was performed in the

Maier lab), AMVMs were loaded with Fura-2AM and analysed as described previously 14 For

the studies on sham and TAC-operated mice (was performed in the Maack lab), AMVMs ere

loaded with Indo-1AM and analysed as described previously. 15, 16

Supplemental References:

1. Agah R, Frenkel PA, French BA, Michael LH, Overbeek PA and Schneider MD. Gene

recombination in postmitotic cells. Targeted expression of Cre recombinase provokes

cardiac-restricted, site-specific rearrangement in adult ventricular muscle in vivo. J

Clin Invest. 1997;100:169-79.

2. Marley PD and Thomson KA. The Ca++/calmodulin-dependent protein kinase II

inhibitors KN62 and KN93, and their inactive analogues KN04 and KN92, inhibit

nicotinic activation of tyrosine hydroxylase in bovine chromaffin cells. Biochem

Biophys Res Commun. 1996;221:15-8.

3. Backs J, Backs T, Neef S, Kreusser MM, Lehmann LH, Patrick DM, Grueter CE, Qi

X, Richardson JA, Hill JA, Katus HA, Bassel-Duby R, Maier LS and Olson EN. The

delta isoform of CaM kinase II is required for pathological cardiac hypertrophy and

remodeling after pressure overload. Proc Natl Acad Sci U S A. 2009;106:2342-7.

4. Hill JA, Karimi M, Kutschke W, Davisson RL, Zimmerman K, Wang Z, Kerber RE

and Weiss RM. Cardiac hypertrophy is not a required compensatory response to short-

term pressure overload. Circulation. 2000;101:2863-9.

5. Kirchhefer U, Schmitz W, Scholz H and Neumann J. Activity of cAMP-dependent

protein kinase and Ca2+/calmodulin-dependent protein kinase in failing and nonfailing

human hearts. Cardiovasc Res. 1999;42:254-61.

6. Toischer K, Rokita AG, Unsold B, Zhu W, Kararigas G, Sossalla S, Reuter SP, Becker

A, Teucher N, Seidler T, Grebe C, Preuss L, Gupta SN, Schmidt K, Lehnart SE,

Kruger M, Linke WA, Backs J, Regitz-Zagrosek V, Schafer K, Field LJ, Maier LS and

Hasenfuss G. Differential cardiac remodeling in preload versus afterload. Circulation.

2010;122:993-1003.

7. Malekar P, Hagenmueller M, Anyanwu A, Buss S, Streit MR, Weiss CS, Wolf D,

Riffel J, Bauer A, Katus HA and Hardt SE. Wnt signaling is critical for maladaptive

cardiac hypertrophy and accelerates myocardial remodeling. Hypertension.

2010;55:939-45.

8. Reil JC, Hohl M, Oberhofer M, Kazakov A, Kaestner L, Mueller P, Adam O, Maack

C, Lipp P, Mewis C, Allessie M, Laufs U, Bohm M and Neuberger HR. Cardiac Rac1

overexpression in mice creates a substrate for atrial arrhythmias characterized by

structural remodelling. Cardiovasc Res. 2010;87:485-93.

9. Song K, Backs J, McAnally J, Qi X, Gerard RD, Richardson JA, Hill JA, Bassel-Duby

R and Olson EN. The transcriptional coactivator CAMTA2 stimulates cardiac growth

by opposing class II histone deacetylases. Cell. 2006;125:453-66.

10. Hamzah J, Jugold M, Kiessling F, Rigby P, Manzur M, Marti HH, Rabie T, Kaden S,

Grone HJ, Hammerling GJ, Arnold B and Ganss R. Vascular normalization in Rgs5-

deficient tumours promotes immune destruction. Nature. 2008;453:410-4.

11. Backs J, Song K, Bezprozvannaya S, Chang S and Olson EN. CaM kinase II

selectively signals to histone deacetylase 4 during cardiomyocyte hypertrophy. J Clin

Invest. 2006;116:1853-64.

12. Frey N, Richardson JA and Olson EN. Calsarcins, a novel family of sarcomeric

calcineurin-binding proteins. Proc Natl Acad Sci U S A. 2000;97:14632-7.

13. Montgomery RL, Davis CA, Potthoff MJ, Haberland M, Fielitz J, Qi X, Hill JA,

Richardson JA and Olson EN. Histone deacetylases 1 and 2 redundantly regulate

cardiac morphogenesis, growth, and contractility. Genes Dev. 2007;21:1790-802.

14. Sag CM, Kohler AC, Anderson ME, Backs J and Maier LS. CaMKII-dependent SR Ca

leak contributes to doxorubicin-induced impaired Ca handling in isolated cardiac

myocytes. J Mol Cell Cardiol. 2011;51:749-59.

15. Kohlhaas M and Maack C. Adverse bioenergetic consequences of Na+-Ca2+

exchanger-mediated Ca2+ influx in cardiac myocytes. Circulation. 2010;122:2273-80.

16. Kohlhaas M, Liu T, Knopp A, Zeller T, Ong MF, Bohm M, O'Rourke B and Maack C.

Elevated cytosolic Na+ increases mitochondrial formation of reactive oxygen species

in failing cardiac myocytes. Circulation. 2010;121:1606-13.

FFFF DKO

5 mm

5 mm

5 mm

5 mm

Fractional shortening

80

20

0

40

SH TAC SH

Fra

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on

al sh

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ing

(%

)

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60

HW/BW ratio

6

2

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4

SH TAC SH

Heart

weig

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mg

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Ca2+ transient amplitude

1.02

0.96

0.94

1.00

F

(340 n

m /

380 n

m)

0.98

FFFF

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0 200

time (ms)

SR Ca2+ content

1.40

1.55

1.45

F

(340 n

m / 3

80 n

m)

1.50

0 200

time (ms)

400

FFFF

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D

Fractional Ca2+ release

80

20

0

40

SH TAC SH

F

racti

on

al re

lease

Ca

2+ tr

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sie

nts

/ C

a2+ c

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SR Ca2+ content

1.5

0.5

0FFFF DKO

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R C

a2+

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340 n

m / 3

80 n

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0.8

0.2

0

0.6

Ca

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40 n

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380 n

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0.4

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Ca2+ transient decay 80%

0.4

0.1

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0.3

Ca

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40 n

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380 n

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0.2

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n.s. n.s.

n.s.

1 Hz 2 Hz 3 Hz

Diastolic Ca2+

1.6

KO NT

n.s.

n.s.

n.s.

1 Hz 2 Hz 3 Hz

1.2

1.0

0.8

1.4

D

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F (

340 n

m / 3

80 n

m)

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4

3

KO NT

FFFF

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1 Hz 2 Hz 3 Hz

2

1

0

F

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Fig. S1

Ejection fraction

80

20

0

40

SH TAC SH

Eje

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on

fra

cti

on

(%

)

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n.s.

60

ESPVR

8

2

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4

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Stroke volume

50

10

0

20

SH TAC SH

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15

5

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)

10

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6

40

IB: CaMKII total

IB: CaMKII

IB: GAPDH

Co

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DK

O

~35 kDa

~55 kDa

~55 kDa

~55 kDa

A

Fig. S1. Baseline characterization of DKO mice. (A) Western blot analysis of total CaMKII,

CaMKII�, phospho-CaMKII-Thr287, and GAPDH as loading control in adult ventricular

myocytes (AMVMs) from !-CKO, �-CKO and DKO mice and Cre-negative littermates (Control).

(B) Representative images of whole hearts, sections and M-mode echocardiograms from DKO

mice and floxed Cre-negative littermates (FFFF). Heart weight/body weight (HW/BW) ratios

and fractional shortening for both groups are also indicated. Values are presented as

mean±SEM (n"18). (C) Hemodynamic measurements in isolated Langendorff-perfused hearts

(n"10). Ejection fraction, end systolic pressure volume relations (ESPVR), stroke volume and

isovolumetric relaxation time constant (tau) are shown. (D) Representative single cell

measurements for Ca2+ transient amplitude, fractional Ca2+ release and sarcoplasmatic

reticulum (SR) Ca2+ content in isolated ventricular cardiomyocytes from DKO and FFFF mice,

loaded with fluo-4. (E) Ca2+ transient decay after field stimulation with 1, 2 and 3 Hz.

Measurements for intracellular diastolic Ca2+ and fractional shortening (FS) given as % of

resting cell length after stimulation with 1, 2 and 3 Hz. All measurements were conducted in

n"30 single cardiomyocytes from n"3 mice. Values are reported as mean±SEM. *p<0.05. n.s.,

not significant.

IB: P-Thr-287 CaMKII

IB: CaMKII total

IB: P-Thr-17 PLN

FFFF DKO

SH TAC SH TAC

~55 kDa

~55 kDa

~10 kDa

IB: P-Ser-744 PKD ~110 kDa

CaMKII activity

³²P-autoradiogram

Coomassie

B

A

Fractional

Ca2+ release

150

0

50

SH TAC SH TAC

Fra

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on

al

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as

e

Ca

2+

tra

ns

ien

ts /

Ca

2+

co

nte

nt

(%)

FFFF DKO

100

n.s. n.s.

SR Ca2+ content

0.8

0.2

0

0.6

SH TAC SH TAC

SR

Ca2+

c

on

ten

t

F (

40

5 n

m /

48

5 n

m)

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0.4

n.s. n.s.

Tau (s)

4

2

0SH TAC SH TAC

Ta

u (

s)

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3

n.s. n.s.

1 Hz

SH TAC SH TAC

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n.s. n.s.

Caffeine

D

Fig. S2

CaMKII activity

0

2

4

6

³²P

acti

vit

y /

to

tal

pro

tein

*

SH TAC SHSH TAC

~110 kDa

IB: GAPDH ~35 kDa

IB: PKD total

SH TAC

C

E

CaMKII

0

0.5

1.0

1.5

CaM

KII

/ G

AP

DH

pro

tein

level

SH TAC SH TAC

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n.s.

*

*

P-Thr17-PLN

0

0.5

1.0

1.5

P-T

hr-

17-P

LN

/

GA

PD

H p

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in l

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SH TAC SH TAC

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n.s.

n.s.

*

P-Thr282-CaMKII

0

0.5

1.0

1.5

2.0

2.5

P-T

hr-

282-C

aM

KII

/

GA

PD

H p

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SH TAC SH TAC

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**

n.s.

P-Ser-744-PKD

0

1

2

3

5

P-S

er-

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DK

/

PK

D p

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n.s.

*n.s.

SH TAC SH TAC

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0

2

4

6

8

MHC

Fo

ldch

an

ge T

AC

vs.

SH

n.s.

*

WT FFFF DKO

SH2

4

6

8

10

ANP

Fo

ldch

an

ge T

AC

vs.

SH

n.s.

*

0WT FFFF DKO

SH0

10

20

30

Rcan1-4

Fo

ldch

an

ge T

AC

vs.

SH

*

n.s.

SHWT FFFF DKO

SH

4

1


0.3

0.1

02 Hz

Ca

2+

tr

an

sie

nt

am

pli

tud

e

F

(40

5 n

m /

48

5 n

m)

0.2

FFFF SH

FFFF TAC

n.s.

n.s.n.s.

0.5 1 3 2 4 5 Hz

n.s. n.s.

DKO SH

DKO TAC


8

2

0

6

F

rac

tio

na

l s

ho

rte

nin

g

(%

of

res

tin

g c

ell

le

ng

ht)

4

FFFF SH

FFFF TAC

n.s.

n.s.

n.s.n.s.

n.s.

DKO SH

DKO TAC

2 Hz 0.5 1 3 2 4 5 Hz

n.s.

n.s.

Fig. S2. Ca2+-handling after TAC in DKO. (A) Western blot analysis of total CaMKII, phospho-

CaMKII-Thr287, phospho-PLB-Thr17, phospho-PKD-Ser744, total PKD and GAPDH as

loading control in cardiac extracts from FFFF and DKO mice three weeks after SH or TAC

surgery. Quantitative analysis is shown (n�3 per group). (B) Radioactive CaMKII kinase activity

measured by in vitro kinase assay using GST-HDAC4 419-670 as a substrate. Coomassie

staining was used to demonstrate equivalent GST-HDAC4 input. Representative blots and

quantification (n=4) are shown. (C+D) Intracellular Ca2+ handling and cell shortening after TAC.

Measurements were conducted in adult mouse ventricular myocytes (AMVMs) from FFFF and

DKO mice three weeks after SH or TAC surgery. AMVMs were loaded with indo-1. Averaged

data for intracellular Ca2+ transient amplitudes and fractional shortening given as % of resting

cell length after field stimulation with 0.5, 1, 2, 3, 4 and 5 Hz. Fractional Ca2+ release and

averaged sarcoplasmatic (SR) Ca2+ content were estimated from the caffeine-induced peak in

Ca2+ transients. Time constant (tau) of intracellular Ca2+ decline after field stimulation and

under caffeine. All measurements were conducted in n�25 single AMVMs from n�3 mice. (E)

Gene expression in wild type and !-KO as well as DKO and FFFF mice after TAC or SH

surgery. Animals were sacrificed after 3 weeks. Fold-changes in mRNA levels of ANP, "MHC

and Rcan1-4 (n�3 per group). All values are reported as mean±SEM. *p<0.05. n.s., not

significant.

KO KO

Iso

DKO

+ + + +

a diomyocy e apop osis

0

2

4

6

8

Tu

ep

osi

ive c

ad

iom

yo

cy

es (

%)

KO

KO

DK

O

Iso � + + + +

B

DA

PI

KO DKOKO

Ve

hIs

o

Myocy e size

0

0.5

1.0

1.5

2.0

Re

aiv

e m

yo

cy

e s

ize

KO

KO

KO

DK

O

FF

FF

Iso � + + ++

DK

O

DK

OKO

� � �

*

*

*

**

.s.2.0

0.5

0

1.0

1.5

Veh Iso Veh Iso

*

FFFF DKO

.s.

2.5

0.5

0

1.0

1.5

*

FFFF DKO

A

2.0

Fig. S3. Redundant roles for CaMKII� and � for cardiomyocyte apoptosis and

hypertrophy. (A) Quantification of cardiac caspase 3/7 activity in DKO and FFFF mice as

measure for apoptosis induced by 3 weeks after TAC or 14 days of isoproterenol (Iso)

treatment (n�3 per group). (B-C) Experiments were conducted in adult ventricular myocytes

(AMVMs) from !-CKO, "-CKO and DKO mice and their Cre-negative littermates (Control).

Cells were stimulated with Iso or treated with vehicle. (B) Cells were stained for #-actinin

(shown in red) and with DAPI nuclear stain (shown in blue). TUNEL-positive cells display green

nuclei (more than 200 myocytes were analyzed per well). Shown are selected myocytes with

TUNEL-positive and negative staining. The percentage TUNEL-positive cells is indicated (n=4

per condition). (C) Myocyte size was determined by measuring >100 cardiomyocytes per well

(n=4 per condition). All values are presented as mean±SEM. *p<0.05. n.s., not significant.

Veh

FFFF DKO

Iso Veh Iso

3 mm 3 mm 3 mm 3 mm

BA

3 mm 3 mm 3 mm 3 mm

H&

ETri

ch

rom

e

50 µm 50 µm 50 µm 50 µm

100 µm 100 µm 100 µm 100 µm

ANP

4

1

0

3

Veh Iso Veh IsoRe

lati

ve

ge

ne

ex

pre

ss

ion *

FFFF DKO

*

n.s.

MHC

1.5

0.5

0

1.0

Veh Iso Veh Iso

*

FFFF DKO

*

n.s.

Re

lati

ve

ge

ne

ex

pre

ss

ion

BNP

6

2

0Veh Iso Veh Iso

*

FFFF DKO

*

n.s.

Re

lati

ve

ge

ne

ex

pre

ss

ion

MHC

4

2

0

3

Veh Iso Veh Iso

*

FFFF DKO

*

n.s.

Re

lati

ve

ge

ne

ex

pre

ss

ion

Col5a1

6

0

4

Veh Iso Veh Iso

FFFF DKO

2

**

Re

lati

ve

ge

ne

ex

pre

ss

ion

Col16a1

Veh Iso Veh Iso

FFFF DKO

* 10

2

0

8

6

4

*

n.s.

Re

lati

ve

ge

ne

ex

pre

ss

ion

Cardiac fibrosis

Veh Iso Veh Iso

Ca

rdia

c f

ibro

tic

are

a (

%)

*

FFFF DKO

15

10

0

5

*

My

oc

yte

cro

ss

se

cti

on

al

are

a (

µm

²)

Myocyte size

500

200

0

400

Veh Iso Veh Iso

*

FFFF DKO

*

100

300

n.s.

HW/BW ratio

10

2

0Veh Iso Veh Iso

He

art

we

igh

t /

bo

dy

we

igh

t (m

g/g

)

*

FFFF DKO

*

6

4

n.s.

Fig. S4

n.s.

n.s.

2

4

1

8

Fig. S4. CaMKII-dependent dissociation of cardiac fibrosis and dysfunction from cardiac

hypertrophy and fetal gene activation after isoproterenol. (A) Representative images of

whole hearts as well as H&E and Masson´s trichrome staining of left ventricular wall sections.

Quantification of HW/BW ratios, myocyte size and fibrosis area (n>6 per group). FFFF and

DKO mice were treated i.p. with isoproterenol (Iso) 10 mg/Kg/d or vehicle for 14 days,

respectively (B) Fold-changes in mRNA levels of the hypertrophic markers ANP, BNP, �-MHC

and !-MHC and fold-changes in collagen expression (n"4 per group). All values are presented

as mean±SEM. *p<0.05. n.s., not significant.

IB: CaMKII

IB: P-Ser-473-Akt

FFFF DKO

SH TAC SH TAC

IB: Akt total

Veh

FFFF DKO

SH TAC SH TAC

~55 kDa

~60 kDa

~60 kDa

CyA

IB: GAPDH ~35 kDa

C

Ve

h

Cy

A

Figure S5

CaMKII

0

0.5

1.0

1.5

2.0

2.5

CaM

KII

/ G

AP

DH

pro

tein

level

SH TAC SH TAC

FFFF DKO

n.s.

**

CaMKII

0

1

2

CaM

KII

/G

AP

DH

pro

tein

level

SH TAC SH TAC

FFFF DKO

n.s.

**

P-Ser-473-Akt

0

1

2

P-S

er-

473-A

kt

/

Akt

pro

tein

level

SH TAC SH TAC

FFFF DKO

n.s.

n.s.

P-Ser-473-Akt

0

1

2

P-S

er-

473-A

kt

/

Akt

pro

tein

level

SH TAC SH TAC

FFFF DKO

n.s.n.s.

SH TACTACFFFF DKO

ANP

0

5

10

15

Fo

ldch

an

ge T

AC

vs.

SH

BNP

0

1

2

3

5

Fo

ldch

an

ge T

AC

vs.

SH

Rcan1-4

0

5

10

15

Fo

ldch

an

ge T

AC

vs.

SH

MHC

0

1

2

Fo

ldch

an

ge T

AC

vs.

SH

MHC

0

2

4

6

8

Fo

ldch

an

ge T

AC

vs.

SH

B

FFFF DKO

Veh CyA

SH TACTACFFFF DKO FFFF DKO

Veh CyA


Veh CyA


Veh CyA


Veh CyA

SH

SH

SH

SH

SH*

*

*

n.s.

n.s.

n.s.

n.s.

*

n.s.

*

IB: GAPDH ~35 kDa

IB: CnA ~60 kDa

non trea

ted

scra

mble

dC

nA s

iRN

A

A

*

4

Fig. S5. Normalization of fetal gene program and unaltered Akt phosphorylation after

CnA inhibition in DKO mice. (A) For data presented in Fig. 5A+B, neonatal mouse ventricular

myocytes (NMVMs) were transfected with CnA-siRNA or scrambled siRNAs, or they were not

transfected (non treated). Western blot analysis demonstrates efficient CnA knockdown.

GAPDH shows equal loading. (B+C) Analog to the experiment shown in Fig. 5C, DKO and

FFFF mice were randomized to either TAC or SH surgery and were treated with cyclosporine

A (CyA; 4 mg/Kg/d i.p.) or vehicle (n�3). Mice were sacrificed 7 days after TAC surgery. CyA-

treated DKO mice were protected from TAC-induced hypertrophy (data not shown) as

described in Fig. 5C (3 weeks after TAC). (B) Fold-changes in cardiac mRNA levels of the

hypertrophic markers ANP, BNP, !MHC and "MHC and fold-changes of RCAN 1-4 expression

(n�3 per group). (C) Western blots for CaMKII, Akt total, phospho-Akt-Ser473, and GAPDH as

loading control were performed in lysates from left ventricle. Quantitative analysis of the

expression and phosphorylation of the proteins is shown (n=3). All values are presented as


Cardiac CaM Kinase II genes δ and γ contribute to adverse remodeling but redundantly inhibit...

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