KomuroKudoh, Miho Mizukami, Masaki Shimoyama, Futoshi Shibasaki, Ryozo Nagai, Yoshio Yazaki and Issei

Yunzeng Zou, Yukio Hiroi, Hiroki Uozumi, Eiki Takimoto, Haruhiro Toko, Weidong Zhu, SumiyoHypertrophy

Induced Cardiac−Calcineurin Plays a Critical Role in the Development of Pressure Overload

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Calcineurin Plays a Critical Role in the Development ofPressure Overload–Induced Cardiac Hypertrophy

Yunzeng Zou, MD, PhD; Yukio Hiroi, MD, PhD; Hiroki Uozumi, MD; Eiki Takimoto, MD;Haruhiro Toko, MD; Weidong Zhu, MD, PhD; Sumiyo Kudoh, MD, PhD; Miho Mizukami, MD;

Masaki Shimoyama, MD, PhD; Futoshi Shibasaki, PhD; Ryozo Nagai, MD, PhD;Yoshio Yazaki, MD, PhD; Issei Komuro, MD, PhD

Background—Although activation of the Ca21-dependent phosphatase calcineurin has been reported to induce cardio-myocyte hypertrophy, whether calcineurin is involved in pressure overload–induced cardiac hypertrophy remainscontroversial.

Methods and Results—We examined in the present study the role of calcineurin in pressure overload–induced cardiachypertrophy using transgenic mice that overexpress the dominant negative mutant of calcineurin specifically in the heart.There were no significant differences in body weight, blood pressure, heart rate, heart weight, and the cardiac calcineurinactivity between the transgenic mice and their littermate wild-type mice at basal state. The activity of calcineurin wasmarkedly increased by pressure overload produced by constriction of the abdominal aorta in the heart of wild-type micebut less increased in the heart of the transgenic mice. Pressure overload induced increases in heart weight, wall thicknessof the left ventricle, and diameter of cardiomyocytes; reprogramming of expressions of immediate early response genesand fetal-type genes; activation of extracellular signal–regulated protein kinases; and fibrosis. All these hypertrophicresponses were more prominent in the wild-type mice than in the transgenic mice.

Conclusions—These results suggest that calcineurin plays a critical role in the development of pressure overload–inducedcardiac hypertrophy.(Circulation. 2001;104:97-101.)

Key Words: calcineurinn hypertrophyn genesn pressure

Cardiac hypertrophy is recognized in many cardiovasculardiseases, such as hypertension, valvular diseases, and

myocardial infarction, and is an independent risk factor forcardiac morbidity and mortality.1 Cardiac hypertrophy isinduced by a variety of factors, such as vasoactive peptides,catecholamines, cytokines, and growth factors; however,mechanical stress is most important as an initial stimulus.2,3

Hypertrophic stimuli induce an increase in protein synthesiswith reprogramming of gene expression by activation ofvarious signaling molecules, such as protein kinase C, ty-rosine kinases, Ras, the mitogen-activated protein (MAP)kinase family, and the Janus kinase (JAK)/signal transducerand activator of transcription (STAT) family.2,3

See p 9

Ca21 is an important second messenger in various cellularprocesses, including cell growth and survival.4 In response togrowth stimuli, many types of cells increase their cytosolicCa21 levels,4 and the elevated Ca21 activates many effectors,including calcineurin, a Ca21/calmodulin–dependent protein

phosphatase that is highly conserved in evolution and widelydistributed in many tissues.5 Calcineurin plays pivotal roles inneuronal functions and immune responses.5 Recently, cal-cineurin has attracted great attention as a critical mediator forcardiac hypertrophy.6 Transgenic mice that overexpressedconstitutively active forms of calcineurin and of its down-stream transcription factor nuclear factor of activated T cells(NFAT) 3 showed marked cardiac hypertrophy, whereascalcineurin inhibitors, such as cyclosporin A and FK506,significantly suppressed phenylephrine- and angiotensin II–induced cardiomyocyte hypertrophy in vitro.6 The role ofcalcineurin in the development of pressure overload–inducedcardiac hypertrophy, however, is unclear. Five researchgroups, including ours, have reported that calcineurin plays acritical role in the development of pressure overload–inducedcardiac hypertrophy,7–11 and several other groups reported anopposite conclusion.12–15 Because in those studies, manyanimals lost body weight (BW) and died, possibly of severeside effects of calcineurin inhibitors, it might be difficult toreach a conclusion. To elucidate the precise role of cal-

Received October 18, 2000; revision received February 22, 2001; accepted March 6, 2001.From the Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine, Chiba (Y.Z., H.T., M.M., I.K.); the

Department of Cardiovascular Medicine, University of Tokyo Graduate School of Medicine, Tokyo (Y.H., H.U., E.T., W.Z., S.K., M.S., R.N.); TokyoMetropolitan Institute of Medical Science, Tokyo (F.S.); and the International Medical Center of Japan, Tokyo (Y.Y.), Japan.

Correspondence to Issei Komuro, MD, PhD, Department of Cardiovascular Science and Medicine, Chiba University Graduate School of Medicine,1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan. E-mail komuro-tky@umin.ac.jp

© 2001 American Heart Association, Inc.

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cineurin in the development of pressure overload–inducedcardiac hypertrophy while avoiding drug toxicity, we createdtransgenic mice that overexpress the catalytically inactiveforms of calcineurin only in the heart. We show here thataortic constriction–induced cardiac hypertrophy was attenu-ated in the transgenic mice compared with wild-type mice.

MethodsMice and TransgeneThe dominant negative (DN) mutants of human calcineurin Asubunit (CnA)16 were constructed by deleting the autoinhibitory(carboxy terminus) and the calmodulin-binding domains throughintroducing a stop codon at the N407 amino acid and by mutating thehistidine at position 160, a calcineurin active site, to glutamine.17

Hemagglutinin (HA)-tagged DNCnA was subcloned into thea-myosin heavy chain promoter–containing expression vector6 (Fig-ure 1A). The linearized DNA was injected into pronuclei of eggsfrom BDF1 mice, which were transferred into the oviducts ofpseudopregnant ICR mice. The transgene was identified by polymer-ase chain reaction with transgene-specific primers. Two independentlines of transgenic mice were successfully created. Sixteen- to18-week-old DNCnA heterozygous transgenic mice and wild-typelittermate mice were used in the present study. All protocols wereapproved by the guidelines of the University of Tokyo.

Pressure-Overload ModelThe abdominal aorta above the left renal artery was constricted aspreviously described.9 To monitor the hemodynamic effects of aorticconstriction, the right carotid artery was cannulated with a 24-gaugepolyethylene catheter. The blood pressure (BP) was increased from45615 to 98621 mm Hg and the heart rate (HR) from 550660 to700650 bpm soon after the aortic constriction in wild-type mice.The BP was 110610 mm Hg and the HR 590660 bpm at 3 weeksafter the operation. There was no significant difference betweentransgenic mice and wild-type mice in basal BP and HR and themagnitude of the BP or HR elevation after the operation.

Echocardiographic AnalysisTransthoracic echocardiographic analysis was performed with an HPSonos 100 (Hewlett-Packard Co) with a 10-MHz imaging transduceras described previously.18

Calcineurin ActivityThe activity of calcineurin was determined by use of phosphorylatedGST-RII peptide as a substrate as previously described9 with somemodifications. We separated calmodulin-bound calcineurin (activecalcineurin,.100 kDa) from free calcineurin (inactive calcineurin,,100 kDa) by centrifugation of cell lysates at 1500gfor 10 minuteswith Ultrafree-MC Centrifugal Filter Units (Millipore). The phos-phorylated GST-RII peptide was incubated with the samples in theCa21-free condition.

Western Blot Analysis of Calcineurin ExpressionThe expressions of DNCnA and endogenous CnA were analyzed byWestern blotting using an anti-HA antibody (Mitsubishi Biochemi-cal Laboratories) or an anti-CnAb antibody (Santa Cruz Biotechnol-ogy, Inc). Immunoreactivity was detected by an enhanced chemilu-minescence reaction system (Amersham) according to themanufacturer’s directions.

Northern Blot AnalysisTotal RNA (10 mg) was size-fractionated in 1.2% formaldehydeagarose gels and transferred to nylon membranes. The blots werehybridized with the [a-32P]dCTP-labeled (Du Pont-New EnglandNuclear Co) cDNA fragment as previously described.9

Assay of Extracellular Signal–Regulated ProteinKinase ActivityExtracellular signal–regulated protein kinase (ERK) activities weremeasured with myelin basic protein (MBP)–containing gel aspreviously described.19 In brief, cell lysates from LV tissue wereelectrophoresed on SDS-polyacrylamide gel containing 0.5 mg/mLMBP. Phosphorylation of MBP was detected by incubation of the gelwith [g-32P]ATP.

Histological AnalysisThe transverse diameter of cardiomyocytes stained by hematoxylinand eosin was measured by micrometers (mm) in 20 differentrandomly chosen points from a cross section of LV free wall. Theextent of LV fibrosis was measured in 8 fields randomly selectedfrom a section by calculating the ratio of azan-stained fibrosis areadivided by total myocardium area.18 Five sections of each heart weremeasured.

StatisticsAll values are expressed as mean6SEM of 6 experiments in eachinstance. Comparisons among 3 groups were made by 1-wayANOVA followed by Dunnett’s modifiedt test. Values ofP,0.05were considered statistically significant.

ResultsExpression of DNCnA in MiceTo determine the role of calcineurin in the development ofpressure overload–induced cardiac hypertrophy, we gener-ated transgenic mice that expressed the DNCnA with HA tagspecifically in the heart under the control ofa-myosin heavychain promoter (Figure 1A).20 DNCnA can bind to thecalcineurin B subunit, but it is catalytically inactive andinterferes with NFAT4 translocation.17 Northern blot analysisusing 10mg of total RNA and cDNA probe corresponding toN1 to 407 of DNCnA revealed that although endogenousCnA gene was not detected, the DNCnA gene was abundantlyexpressed in the heart of both lines of the transgenic mice

Figure 1. Expression of DNCnA in heart. A, Structure of DNCnAtransgene. HA indicates HA tag; SA, SV40 poly A. DNCnAencodes 407 amino acids; histidine at position 160 is substi-tuted by glutamine (Q). B, mRNA expression of DNCnA. mRNAof DNCnA in heart from 2 lines of transgenic mice (TG) andwild-type littermate mice (WT) was examined by Northern blotanalysis using cDNA probe of DNCnA (N1 to 407). C, Proteinexpression of DNCnA and endogenous CnA. Proteins of DNCnAand CnA in heart were examined by Western blot analysis asdescribed in Methods. Lanes 1 to 4, DNCnA; lanes 5 to 8, en-dogenous CnA. One nonspecific band was visible in lanes 3 and4, and 2 nonspecific bands were detected in lanes 5 to 8.

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(Figure 1B). The DNCnA gene was not expressed in othertissues examined, such as brain, liver, skeletal muscle, lungs,and intestine of the transgenic mice (data not shown). We alsoexamined the expression of DNCnA and the endogenousCnA at the protein level by Western blot analysis using ananti-HA antibody and an anti-CnAb antibody that was raisedagainst a carboxy terminus peptide of the CnAb. The DNCnAprotein was abundantly expressed in the heart of bothtransgenic lines (Figure 1C, lane 1 and 2). There was nosignificant difference in the expression levels of endogenousCnA between the transgenic and wild-type mice (Figure 1C,lane 5 to 8). Expression levels of DNCnA were.10-foldhigher than those of endogenous CnA in the transgenic heart.

Inhibition of Calcineurin Activity in the Heart ofTransgenic MiceTo determine whether DNCnA effectively functions as adominant negative mutant in the transgenic heart, we exam-ined the activity of calcineurin in the heart using an enzy-matic phosphatase assay after separation of activated cal-cineurin. There was no difference in the basal activity ofcalcineurin between the wild-type heart (15 52061500 cpm/mg, n55) and the transgenic heart (14 85061050 cpm/mg,n55). After 30 minutes under pressure overload, the activityof calcineurin was significantly increased in the heart ofwild-type mice, as reported before in rats9 (34 32061750cpm/mg, n55,P,0.05 versus basal activity in wild-typemice), whereas in the heart of transgenic mice, the increase ofcalcineurin activity was attenuated (21 45061520 cpm/mg,n55, P,0.05 versus wild-type mice subjected to pressureoverload). These results suggest that the cardiac-specificoverexpression of DNCnA effectively suppresses the activa-tion of calcineurin in the heart.

Suppression of Cardiac Hypertrophy inTransgenic MiceA 2D long-axis image was used to evaluate the wall thicknessof the left ventricle (LV). M-mode images taken during 10successive beats revealed that the interventricular septum andthe posterior wall of the LV were significantly thicker inwild-type mice than the transgenic mice at 3 weeks after theaortic constriction (Figure 2).

After 3 weeks of aortic banding, the heart weight (HW) andthe ratio of HW to BW (HW/BW) were significantly in-creased in wild-type mice, whereas the increases in HW andHW/BW were significantly suppressed in the transgenic mice(Table).

Histological analysis showed that after 3 weeks, aorticconstriction induced marked cardiac hypertrophy in wild-typemice, whereas the transgenic mice developed less cardiachypertrophy in response to pressure overload (Figure 3). Thetransverse diameter of cardiomyocytes was increased signif-icantly, from 10.561.3 to 19.160.8mm (P,0.05), in wild-type mice, whereas there was no significant difference intransverse diameter of cardiomyocytes between sham-treatedand banding-treated transgenic mice (sham, 10.061.2 mmversus banding, 13.061.3mm). The extent of LV fibrosis wassignificantly reduced in the transgenic mice compared withthat in wild-type mice (transgenic mice, 1.360.2% versus

wild-type mice, 2.160.5% of a whole section, n55,P,0.05).These results clearly indicate that calcineurin plays a criticalrole in pressure overload–induced cardiac hypertrophy.

Induction of specific gene expression is one of hypertro-phic responses to hemodynamic overload.2,3 We thus exam-ined the expressions of immediate early response genes andfetal genes in the hearts at 2 hours and 3 weeks after aorticconstriction, respectively (Figure 4). Pressure overload up-regulated mRNA levels of c-fos, c-jun, and brain natriureticpeptide (BNP) genes at 2 hours and atrial natriuretic peptide(ANP) and skeletala-actin (sk.a-actin) genes at 3 weeks inwild-type mice. Expression of sarcoplasmic reticulum Ca21-ATPase (SERCA2) gene was downregulated by pressureoverload in the wild-type heart. Although the expression ofc-jun and sk. a-actin genes was upregulated by pressureoverload in the transgenic mice as well, mRNA levels ofc-fos,ANP,BNP, andSERCA2genes remained unchanged inthe hearts of transgenic mice. Thus, calcineurin might play animportant role in pressure overload–induced reprogrammingof some specific genes.

Figure 2. M-mode echocardiograms. a and b, Sham operation;c and d, 3 weeks after aortic constriction. Data are expressedas mean6SEM. *P,0.05 vs sham-operated wild-type (WT)mice. †P,0.05 vs WT mice with aortic constriction. IVS indi-cates interventicular septum; LVPW, left ventricular posteriorwall.

Pressure Overload–Induced Cardiac Hypertrophy

WT TG

Sham 3 Weeks Sham 3 Weeks

n 4 8 5 10

BW, g 29.0061.00 29.0661.67 29.1861.59 28.4261.70

HW, mg 120610 190612* 110610 140611†

HW/BW, mg/g 4.160.2 6.560.3* 3.960.3 4.960.1†

WT and TG mice were sham-operated (Sham) or subjected to abdominalaortic constriction for 3 weeks as described in Methods. BW, HW, and HW/BWratio were determined. Results are shown as mean6SEM.

*P,0.05 vs sham-operated WT mice; †P,0.05 vs WT mice subjected toaortic constriction.

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Suppression of Protein Kinases in Transgenic MiceERKs play an important role in the development of cardiachypertrophy.2,3 Calcineurin has been reported to be involvedin Ca21 mobilizing agent–induced activation of ERKs inmouse M1 myeloid leukemic cells,21 and the activity of ERKswas increased in the heart of the transgenic mice, whichexpress a constitutively active form of calcineurin.22 We also

observed that calcineurin plays a critical role in isoproterenol-induced activation of ERKs in cultured cardiomyocytes ofneonatal rats (unpublished observation). We therefore exam-ined whether calcineurin is involved in pressure overload–induced activation of ERKs in the heart in vivo. Constrictionof the abdominal aorta for 30 minutes markedly activatedERKs in the heart of wild-type mice (Figure 5). The pressureoverload–induced activation of ERKs was attenuated in theheart of transgenic mice (Figure 5), suggesting that cal-cineurin is critically involved in pressure overload–inducedactivation of ERKs in the heart.

DiscussionCa21 plays an important role in various cellular processes,such as cell growth and survival,4 including cardiac hyper-trophy.23 The mechanism of how Ca21 is involved in thedevelopment of cardiac hypertrophy, however, was unclear.Recently, calcineurin has attracted great attention as a medi-ator of Ca21-induced cardiac hypertrophy.6 Overexpression ofconstitutively active mutants of calcineurin and of its down-stream transcription factor NFAT3 induced marked cardiachypertrophy in transgenic mice.6 The calcineurin inhibitorscyclosporin A and FK506 suppressed phenylephrine- andangiotensin II–induced cardiomyocyte hypertrophy in vitro.6

Although 5 research groups, including ours, have demon-strated that calcineurin plays a critical role in pressureoverload–induced cardiac hypertrophy,7–11 several othergroups have reported that cyclosporin A and FK506 had nosuppressive effect on pressure overload–induced cardiac hy-pertrophy.12–15 The reason for the discrepancy is not clear atpresent, but there are several possibilities. First, becausecalcineurin is expressed abundantly in the heart,24 it ispossible that the dose of calcineurin inhibitors was notenough to completely inhibit the activity of calcineurin in theheart. Many studies did not examine the activity of cal-cineurin in the heart before and after administration ofcalcineurin inhibitors.7,8,11–13 Although some of them mea-sured the activity of calcineurin,10,14,15 it is difficult todetermine the precise activity of calcineurin.25 In the presentstudy, we measured the activity of calcineurin after separatingactivated calcineurin from nonactivated calcineurin, whichenabled us to accurately determine the activity of endogenouscalcineurin. Second, the role of calcineurin may differ withanimal species and the model of pressure overload–inducedcardiac hypertrophy. The third and most likely possibility isthe severe cytotoxicity of calcineurin inhibitors. Both cyclo-sporin A and FK506 induce severe adverse reactions, such as

Figure 3. Cardiac hypertrophy induced by pressure overload.Hearts were excised from transgenic (TG) and wild-type (WT)mice that were subjected to pressure overload for 3 weeks orsham operation. a to c, Hearts were sectioned longitudinally andstained with hematoxylin and eosin. Cardiac hypertrophy wasobserved in WT mice subjected to aortic banding (b) but not insham-operated WT mice (a) or in TG mice (c). d to f, High-magnification views of hematoxylin and eosin–stained LV wall.Pressure overload induced an increase in cardiomyocyte size inWT mice (e) but not in sham-operated WT mice (d) or in TGmice (f). g to i, Azan staining. g, Sham-operated WT mice. Anincrease in perivascular and interstitial fibrosis was observed inWT (h) but not in TG mice subjected to aortic banding (i).

Figure 4. Specific gene expression. Wild-type (WT) and trans-genic (TG) mice were subjected to sham operation or to con-striction of abdominal aorta. Northern blot analysis was per-formed as described in Methods. A, Induction of immediateearly response genes by acute pressure overload (2 hours). B,Expression of fetal-type and SERCA2 genes by pressure over-load for 3 weeks. Ethidium bromide staining of 18S ribosomalRNA is presented to show integrity of RNA. Representative au-toradiograms from 3 independent experiments are shown.

Figure 5. ERK activity. Wild-type (WT) and transgenic (TG) micewere subjected to either sham operation or aortic constrictionfor 30 minutes, and activity of ERKs in heart was measured by“in gel assay” as described in Methods. Representative autora-diogram showing MBP phosphorylation.

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infection, renal failure, and cachexia, which could causedeath. Indeed, most animals showed marked loss of BW, andmany animals died after the operation under treatment withcalcineurin inhibitors.12–14 It should be difficult to reach aconclusion when.50% of animals died during the experi-ments.13,14 To evaluate the role of calcineurin in pressureoverload–induced cardiac hypertrophy while avoiding thesevere side effects of calcineurin inhibition, we used trans-genic mice that overexpress DNCnA specifically in the heart.

In the basal state, there was no significant difference in theactivity of calcineurin and HW between transgenic mice andtheir littermate wild-type mice, suggesting that the basalcalcineurin activity is low in the heart. In the transgenic mice,pressure overload induced less marked hypertrophic re-sponses, including attenuated increases in HW, LV wallthickness, cardiomyocyte size, and myocardial fibrosis andreprogramming of some specific genes and activation ofERKs, compared with wild-type mice. Because there was nodifference between transgenic mice and wild-type mice inBW and hemodynamic parameters, such as cardiac function,HR, and BP, these results clearly indicate that calcineurin iscritically involved in pressure overload–induced cardiac hy-pertrophy. Although it is suggested that calcineurin inducescardiac hypertrophy by activating its downstream transcrip-tion factor NFAT3,6 there may be other pathways, includingERKs, through which calcineurin induces cardiac hypertro-phy. Further studies are necessary to elucidate the down-stream mechanism of how calcineurin induces cardiachypertrophy.

AcknowledgmentsThis work was supported by a Grant-in-Aid for Scientific Research,Developmental Scientific Research, and Scientific Research onPriority Areas from the Ministry of Education, Science, Sports, andCulture and by the Program for Promotion of Fundamental Studies inHealth Sciences of the Organization for Drug ADR Relief, R&DPromotion, and Product Review of Japan (to Dr Komuro). Dr Zou isa recipient of a postdoctoral fellowship from the Japan Society forthe Promotion of Science.

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