Roles of calcineurin and calcium/calmodulin-dependent protein kinase IIin pressure overload-induced cardiac hypertrophy
Tetsuya Saito a, Jun Fukuzawa a,*, Junzo Osaki a, Hitoshi Sakuragi a, Naoyuki Yao a,Takashi Haneda b, Takayuki Fujino c, Nobutaka Wakamiya d, Kenjiro Kikuchi a,
Naoyuki Hasebe a
a First Department of Medicine, Asahikawa Medical College, 2-1-1-1 Midorigaoka-Higashi, Asahikawa 078 8510, Japanb Nemuro Municipal Hospital, 1-2 Ari-iso, Nemuro 078-8686, Japanc Department of Pharmacology, Asahikawa Medical College, Japan
d Department of Microbiology and Immunochemistry, Asahikawa Medical College, Japan
Received 8 January 2003; received in revised form 27 June 2003; accepted 2 July 2003
Abstract
Calcineurin and calcium/calmodulin-dependent protein kinase (CaMK) II have been suggested to be the signaling molecules in cardiachypertrophy. It was not known, however, whether these mechanisms are involved in cardiac hypertrophy induced by pressure overload withoutthe influences of blood-derived humoral factors, such as angiotensin II. To elucidate the roles of calcineurin and CaMK II in this situation, weexamined the effects of calcineurin and CaMK II inhibitors on pressure overload-induced expression of c-fos, an immediate-early gene, andprotein synthesis using heart perfusion model. The hearts isolated from Sprague–Dawley rats were perfused according to the Langendorfftechnique, and then subjected to the acute pressure overload by raising the perfusion pressure. The activation of calcineurin was evaluated byits complex formation with calmodulin and by its R-II phosphopeptide dephosphorylation. CaMK II activation was evaluated by itsautophosphorylation. Expression of c-fos mRNA and rates of protein synthesis were measured by northern blot analysis and by 14C-phenylalanine incorporation, respectively. Acute pressure overload significantly increased calcineurin activity, CaMK II activity, c-fosexpression and protein synthesis. Cyclosporin A and FK506, the calcineurin inhibitors, significantly inhibited the increases in both c-fosexpression and protein synthesis. KN62, a CaMK II inhibitor, also significantly prevented the increase in protein synthesis, whereas it failedto affect the expression of c-fos. These results suggest that both calcineurin and CaMK II pathways are critical in the pressure overload-inducedacceleration of protein synthesis, and that transcription of c-fos gene is regulated by calcineurin pathway but not by CaMK II pathway.
Keywords: Calcium/calmodulin-dependent protein kinase; Ca2+-dependent protein phosphatase; Cardiac hypertrophy; Gene expression; Isolated perfused ratheart; Pressure overload; Protein synthesis
1. Introduction
The hypertrophic response of the cardiomyocyte is char-acterized by cellular events, such as gene expression andincrease in protein synthesis [1]. Intracellular Ca2+ plays arole in these events via the activation of Ca2+-dependentprotein kinases and phosphatases in a variety of cell typesincluding the cardiomyocyte [2]. In cardiomyocytes, how-ever, it was not clear how Ca2+ regulates these events. Re-cently, calcineurin, (a Ca2+-dependent protein phosphatase),
and nuclear factor of activated T cells (NFAT)-c4, (a down-stream transcription factor of calcineurin), have been re-ported to participate in the development of cardiac hypertro-phy. Transgenic expression of constitutive active form ofcalcineurin or NFAT-c4 in mice caused marked cardiac hy-pertrophy and eventually congestive heart failure [3], whichwere prevented by cyclosporin A (CsA) and FK506, cal-cineurin inhibitors, suggesting that the activation of cal-cineurin leads to cardiac hypertrophy. Moreover, calcineurininhibitors prevented pressure overload-induced cardiac hy-pertrophy in vivo [4–9], indicating that calcineurin plays acrucial role in the development of cardiac hypertrophy in-duced by pressure overload. On the other hand, KN62, a
calcium/calmodulin-dependent protein kinase (CaMK) II in-hibitor, prevented the hypertrophy induced by humoral fac-tors, such as endothelin-1 and leukemia inhibitory factor(LIF), in rat cardiomyocytes [10,11], suggesting that theCaMK II pathway plays some roles in the development ofcardiac hypertrophy, in which humoral factors play the majorrole. However, it has not been fully elucidated whether cal-cineurin and CaMK II pathways would be activated by pres-sure overload in the heart, and if so, how these pathways areinvolved in the expression of c-fos and acceleration of proteinsynthesis.
In perfused heart model, acute pressure overload to theventricular wall induces expression of c-fos mRNA and ac-celerates protein synthesis [12]. Furthermore, this model isuseful to evaluate the mechanism of the pressure overload-induced response in the heart without the influence of circu-lating humoral factors, which occur in vivo and would beinduced by hemodynamic changes and could modulate theresponse. In the model, however, the roles of calcineurin andCaMK II in pressure overload-induced responses are largelyunknown, although the participation of protein kinase (PK) A(PKA) and C (PKC) pathways have been reported [12–16].In the present study, using the isolated perfused rat heart, weexamined:
• whether acute pressure overload could activate the cal-cineurin and CaMK II pathways;
• whether the calcineurin and CaMK II pathways areinvolved in the pressure overload-induced expression ofc-fos mRNA and acceleration of protein synthesis.
2. Materials and methods
2.1. Animals and heart perfusion
Male Sprague–Dawley rats of 10–12 weeks old wereused. The isolated heart was perfused according to the Lan-gendorff method as described [14]. During the first 10 min ofperfusion, remaining blood in the heart was washed out withKrebs–Henseleit buffer containing the amino acids described[17] and 15 mM glucose at a constant pressure of 50 mmHg.Then, the heart was perfused in a recirculation manner withthe buffer containing 0.1% bovine serum albumin (FractionV, Sigma Chemical, USA) at a perfusion pressure of60 mmHg. After the stabilization period of 5 min, 9 µg/ml oftetrodotoxin (Seikagaku Co., Japan) was added to the perfu-sate to arrest the heart, because this procedure eliminates theeffect of cardiac contractility itself on the cardiomyocytes[18]. CsA (0.1–100 µmol/l), FK506 (10 µmol/l), and KN62(1 µmol/l) were added to the perfusate 5 min after the appli-cation of tetrodotoxin. Five minutes after the addition of theinhibitor, the perfusion pressure was elevated from 60 to120 mmHg to reproduce an acute pressure overload. At thetime indicated, the heart was frozen immediately as de-scribed [19], and stored at –80 °C until use. During theexperiment, the buffer was equilibrated with gas (95% O2,5% CO2) at 37°C.
2.2. Sample preparation for western blot analysis
The sample for western blot analysis was prepared fromthe frozen heart which was pulverized in a mortar, lysed witha lysis buffer (50 mM Tris–HCl, 0.1 mM EDTA, 1 mMEGTA, 1 mM DTT, 0.2% NP 40, pH 7.5) and then 300 mg ofsample was homogenized with a polytron for 20 s and soni-cated for 3 s at 4 °C. The homogenates were centrifuged at100,000 g for 30 min at 4 °C. After centrifugation, the upperphase was used as a sample.
2.3. Assays for activated calcineurin
The amount of activated calcineurin was determined ac-cording to the reported method [9,20]. The calmodulin-associated calcineurin was measured as an active form, be-cause calcineurin could be activated through the associationwith calmodulin. The complex was immunoprecipitated withan antibody against calmodulin, and then calcineurin wasdetected by immunoblot with an antibody against cal-cineurin. For immunoprecipitation, the samples were incu-bated with a calmodulin rabbit polyclonal antibody (ZymedLaboratory Inc., USA) followed by the addition of proteinA/G agarose (Santa Cruz Biotechnology, Inc., USA). Then,the immunoprecipitates were harvested by centrifugation,washed three times with a buffer, and subjected to sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferredto nitrocellulose membrane (Amersham Life Science, USA).The membrane was then incubated with calcineurin antibody(Transduction Laboratory, USA) or calmodulin antibody fol-lowed by the incubation with horseradish peroxidase-conjugated secondary antibody (Cell Signaling Technology,Inc., USA). To detect proteins, the membrane was incubatedin LumiGLO chemiluminescent reagent (Cell SignalingTechnology, Inc., USA). For quantification, the intensity ofbands on the blots was scanned densitometrically.
2.4. Determination of R-II phosphopeptidedephosphorylation rate
R-II phosphopeptide dephosphorylation rate was deter-mined using a kit (BIOMOL Research Laboratory, Inc.,USA), in which the detection of the released free phosphatewas based on the classic malachite green assay [21,22]. Afterthe samples prepared as described above were incubated withR-II phosphopeptide [23,24], a substrate of calcineurin, for30 min at 37 °C, the released phosphates were measuredphotometrically with the absorption at 620 nm.
2.5. Assays for CaMK II autophosphorylation
After the sample was prepared, SDS-PAGE was per-formed. Electrophoresed proteins were transferred to thenitrocellulose membrane. The western blot analysis was per-formed to detect the autophosphorylation of CaMK II withusing antibodies against CaMK II or phospho-specific
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CaMK II, respectively (Santa Cruz). To detect the proteins,the membrane was incubated with using LumiGLO chemilu-minescent reagent.
2.6. Determination of c-fos expression by northern blotanalysis
At 30 and 60 min of perfusion, the expression of c-fosmRNA was determined by northern blot analysis as de-scribed [12,16,25]. For standardization, the relative intensityof c-fos mRNA expression was compared to that of b-actinmRNA expression, which was determined using a probemade from Hin f I/Hin f I fragment of human b-actin (WakoSeiyaku Co., Japan).
2.7. Estimation of rates of protein synthesis
Rates of protein synthesis were estimated by incorpora-tion of L-[U-14C] phenylalanine (Amersham Japan Co., Ja-pan) into the heart protein according to the reported method[14]. L-[U-14C] phenylalanine, 0.1 µCi/ml, was added to theperfusate 1 h after the perfusion pressure was raised to120 mmHg, and the sample was prepared at 1 h after theaddition of the isotope.
2.8. Statistical analysis
All values are expressed as mean ± SEM. Statistical analy-sis was performed using one-way analysis of variance(ANOVA) followed by a post hoc analysis (Scheffe). Theresults were considered to be significant when P < 0.05.
3. Results
3.1. Calcineurin is activated by pressure overload inperfused heart
We first examined whether acute pressure overload couldactivate calcineurin in perfused heart. Elevation of perfusionpressure from 60 to 120 mmHg significantly increased thecontent of activated calcineurin, the calcineurin–calmodulincomplex, at 30 min of perfusion. It reached a peak at 60 minand was twice that in the control heart. At 120 min ofperfusion it decreased to a level higher than basal level(Fig. 1A,B). In the control heart, the levels of activatedcalcineurin and calmodulin were unchanged during the per-fusion (Fig. 1A,B). We also examined the effect of the pres-sure overload on the dephosphorylation rate of the R-IIphosphopeptide, a synthetic substrate of calcineurin. Thepressure overload significantly increased the dephosphoryla-tion rate at 30 min of perfusion, and at 60 min it reached apeak of 2.2 times that in the control heart. It then declined toa level higher than basal level at 120 min of perfusion. Basallevel of dephosphorylation rate was unchanged during theperfusion (Fig. 2).
3.2. CsA and FK506 inhibit the pressure overload-inducedactivation of calcineurin
We next examined the effects of CsA and FK506, cal-cineurin inhibitors, on the pressure overload-induced activa-tion of calcineurin. CsA (0.1–100 µmol/l) inhibited the pres-sure overload-induced increases in the amount of activatedcalcineurin and in the dephosphorylation of R-II phospho-peptide at 60 min of perfusion in a dose-dependent manner(Fig. 3); this inhibitory effect was almost complete above theconcentration of 10 µmol/l. FK506 also inhibited the pres-sure overload-induced activation of calcineurin in a similarmanner with CsA (Fig. 4). The level of calmodulin did notchange under any conditions.
3.3. CaMK II is autophosphorylated by pressure overloadin perfused rat heart
CaMK II autophosphorylation as its activity were exam-ined in pressure-overloaded perfused rat heart. Elevation of
Fig. 1. Calcineurin was activated by pressure overload in perfused hearts.Time course of the activation of calcineurin is presented. (A) A representa-tive data of western blot analysis of the samples immunoprecipitated with ananti-calmodulin antibody. (B) The summary data of the time course of theactivation of calcineurin. The values are expressed as fold increase of thecontent of calcineurin in the control at the start of the perfusion.* P < 0.05 vs. control, n = 5.
Fig. 2. R-II phosphopeptide dephosphorylation was increased by pressureoverload in perfused hearts. R-II phosphopeptide dephophorylation wasevaluated at each time of perfusion. The value of 60 mmHg at 0 min ofperfusion was designated as 1.0. * P < 0.05 vs. control, n = 5.
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perfused pressure from 60 to 120 mmHg significantly acti-vated autophosphorylation of CaMK II at 5 min of perfusion.It reached a peak at 15 min and was a 6.5-fold increase as thatin the control heart. At 120 min of perfusion it decreased to alevel higher than basal level (Fig. 5A,B). KN62, a specificCaMK II inhibitor, suppressed the pressure overload-inducedautophosphorylation of CaMK II in a dose-dependent man-ner (0.1 nmol/l–1 µmol/l) at 15 min (Fig. 5C,D).
3.4. Pressure overload-induced increases in the expressionof c-fos mRNA are inhibited by CsA and FK506 but not byKN62
We examined whether the pressure overload-induced ex-pression of c-fos mRNA is affected by the calcineurin inhibi-tors and the CaMK II inhibitor in perfused heart. The pres-sure overload significantly increased the expression of c-fosmRNA at 30 and 60 min of perfusion to a level of almostthree times that of the control (Fig. 6A). CsA and FK506inhibited the increases almost completely, suggesting a piv-otal role of calcineurin in this event. KN62, however, failed to
affect the increases, suggesting a minor role if any in CaMKII in the heart (Fig. 6B,C).
3.5. CsA, FK506 and KN62 prevent the pressureoverload-induced acceleration of protein synthesis
Finally, to determine the roles of calcineurin and CaMK IIpathway in the protein synthesis, we examined the effects ofthe calcineurin inhibitors and the CaMK II inhibitor on pres-sure overload-induced acceleration of protein synthesis inperfused heart. Pressure overload significantly acceleratedthe rates of protein synthesis during second hour of perfusionby 78%; this increase was reduced markedly by CsA, FK506and KN62 by 74%, 73% and 69%, respectively (Fig. 7),suggesting roles for both calcineurin and CaMK II in theacceleration of protein synthesis. CsA, FK506 and KN62 didnot affect the rates of protein synthesis under control condi-tions (Fig. 7).
Fig. 3. Effects of CsA on the pressure overload-induced activation of calci-neurin in perfused hearts. (A,B) Concentration-dependent effect of CsA onthe contents of activated calcineurin. (C) Concentration-dependent effect ofCsA on the R-II phosphopeptide dephosphorylation. The samples wereprepared at 1 h of acute pressure overload. * P < 0.05 vs. control,# P < 0.05 vs. the heart in the absence of CsA, n = 5 for each group.
Fig. 4. Effect of FK506 on the pressure overload-induced activation ofcalcineurin in perfused hearts. (A,B) Inhibitory effect of CsA and FK506 onthe contents of activated calcineurin. (C) Effect of CsA and FK506 on theR-II phophopeptide dephosphorylation. * P < 0.05 vs. control, # P < 0.05 vs.the heart in the absence of CsA, n = 5 for each group.
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4. Discussion
Cardiac hypertrophy has been regarded as an end result ofadaptation of the heart to the stress. It can be induced by avariety of factors, such as hemodynamic overload, humoralfactors and genetic defects [26]. Among these factors, hemo-dynamic overload is the most frequent cause of cardiachypertrophy in clinical medicine [26] because of the highincidence of hypertension. After sustained pressure overload,the hypertrophied heart often develops dilatation and eventu-ally falls into congestive failure [26]. Moreover, recent epi-demiological studies showed that cardiac hypertrophy is oneof the independent risk factors for arrhythmia, ischemic heartdisease and sudden death [27]. Therefore, to understand themechanism of the development of cardiac hypertrophy espe-cially that induced by pressure overload, it is essential for
effective prevention and management of these pathophysi-ological conditions.
Mechanical stress to the heart induces a series of hyper-trophic responses, of which the first is the reprogramming ofgene expressions; these are represented by the expression ofc-fos, an immediate-early gene, and those of atrial natriuretic
Fig. 5. CaMK II autophosphorylation was stimulated by pressure overloadand KN62 inhibited its stimulation in perfused heart. Time course of theautophosphorylation of CaMK II is presented. (A) A representative data ofwestern blot analysis of the samples with an anti-phospho (upper) and total(lower) CaMK II antibodies. (B) The summary data of the time course of theautophosphorylaton of CaMK II. The values are expressed as fold increaseof the content of CaMK II-corrected phospho to CaMK II in the control atthe start of the perfusion. * P < 0.05 vs. control, n = 5. (C) Concentration-dependent effect of KN62 on the autophosophorylation of CaMK II. Thesamples were prepared at 15 min of acute pressure overload. (D) Thesummary data of the concentration dependency of the CaMK II autophos-phorylation by pressure overload. * P < 0.05 vs. control, # P < 0.05 vs. theheart in the absence of KN62, n = 5 for each group.
Fig. 6. Effects of CsA, FK506 and KN62 on the expression of c-fos mRNAin perfused hearts. (A) Pressure overload-induced increase in the expressionof c-fos mRNA. (B,C) Effects of CsA, FK506 and KN62 on the expressionof c-fos mRNA. * P < 0.05 vs. an aortic pressure of 60 mmHg, # P < 0.05 vs.the heart in the absence of inhibitors, n = 5 for each group.
Fig. 7. Inhibitory effects of CsA, FK506 and KN62 on protein synthesisrates in perfused hearts. The protein synthesis rates were measured fromtime 60 to 120 min (during second hour of perfusion). * P < 0.05 vs. an aorticpressure of 60 mmHg, # P < 0.05 vs. the heart in the absence of the inhibitors,n = 5 hearts for each group.
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peptide, skeletal muscle-type a-actin and b-myosin heavychain, the fetal-type genes [28]. This reprogramming of thegenes is brought about through the activation of severalintracellular signaling molecules, which reportedly includeseveral key enzymes such as PKC [29,30], PKA [12,14],mitogen-activated protein kinase (MAPK) [29,31] and ty-rosine kinase(s) [30]. Another signaling molecule, cytosolicfree Ca2+, has also been proposed as a contributor to cardiachypertrophy [32], although there is little evidence that Ca2+
plays a major role in the pressure overload-induced hypertro-phy [33]. A number of recent reports have suggested a linkbetween the Ca2+ signaling and the hypertrophic responseand have suggested that calcineurin and CaMK II, the Ca2+-dependent enzymes, are crucial molecules in the pressureoverload [3–11,34–36]. The precise mechanisms leading tothe activation of calcineurin and CaMK II in the pressureoverload-induced cardiac hypertrophy, however, are largelyunknown, because pressure overload itself elicits severalkinds of stresses to the heart. Among these stresses, mechani-cal stress to the myocardium and humoral factors derivedfrom hemodyanamic change, such as angiotensin-II andendothelin-1, seem to be the two most important.
To clarify whether pressure overload without the influenceof blood-derived humoral factors could induce the activationof calcineurin and CaMK II, and whether the activation ofthese enzymes would contribute to the reprogramming of thegenes and succeeding acceleration of the protein synthesis,we performed the present study using the perfusion model ofisolated heart. The model in which a drain was not insertedinto the left ventricle was reported to have the same intraven-tricular pressure as aortic pressure (peak intraventricularpressures of 67 ± 3 and 121 ± 7 mmHg) at 60 and 120 mmHg,respectively. A coronary flow changed from 78.8 ± 5.1 to131 ± 11 ml/min/g by raising aortic pressure from 60 to120 mmHg. Neither peak intraventricular pressure nor coro-nary flow changed in the heart arrested by tetrodotoxin in thesame perfusion pressure. Deceased intraventricular volumeby insertion of drain caused no changes in protein synthesisrate compared with non-drained control heart [18]. Tetrodot-oxin was applied to inhibit all mechanical activity of the heartin the present models. The arrest of all mechanical activitywith tetrodotoxin in aerobic condition did not reduce the rateof protein synthesis. This leads to the conclusion that cardiacwork could be excluded as the parameters accelerating rate ofprotein synthesis in Langendorff preparation. However, toeliminate the possibilities that calcineurin and CaMK II path-way could be regulated by the cardiac work changed byelevated aortic pressure, we used tetrodotoxin to arrest theheart in the present study [18]. Under the experimental con-dition, we clearly showed that pressure overload induced thecardiac hypertrophic responses in the absence of blood-bornhumoral factors, which result, however, do not exclude thepossible contribution of humoral factors produced locally inthe heart upon the pressure overload. In fact, there are severalreports showing mechanical stretch-induced local produc-
tion of humoral factors such as angiotensin-II andendothelin-1 [37,38].
We found that pressure overload alone indeed activatedcalcineurin, induced the expression of c-fos, and acceleratedthe rate of protein synthesis, all of which were preventedsignificantly by the calcineurin inhibitors, indicating that thecalcineurin pathway plays a critical role in these eventsinduced by pressure overload. We also demonstrated thepressure overload-induced CaMK II activation and furtherexamined the effects of the CaMK II inhibitor on the expres-sion of c-fos and acceleration of protein synthesis, as the roleof the CaMK II pathway in the development of cardiachypertrophy has not been clear. We found that pressureoverload-induced CaMK II activation, and KN62 preventedthe pressure overload-induced acceleration of protein syn-thesis, although it could not inhibit the expression of c-fos.These results indicate that CaMK II also plays an importantrole in the acceleration of protein synthesis, and that CaMKII signaling is not indispensable for c-fos gene transcriptionin pressure overload-induced cardiac hypertrophy. Althoughthe reason of the differential effects of inhibitors of cal-cineurin and CaMK II on c-fos mRNA expression in spite oftheir similar inhibitory effect on protein synthesis rate is notclear, the present results suggest that these two pathwaysactivate different sets of transcription factors leading to up-regulation of protein synthesis. The c-fos promoter containsseveral cis-elements, such as the SRE, CRE and SIE [39].Some transcription factors and their cofactors includingNFAT-c4, a target molecule of calcineurin, can bind to andstimulate these cis-elements, and iduce c-fos expression. Ifthe c-fos promoter contains a cis-element responsible to atranscription factor activated by both calcineurin and CaMKII pathways along with the one that couples NFAT-c4, aCaMK II inhibitor would not prevent c-fos gene transcrip-tion, because it could not inhibit the NFAT-c4-mediated acti-vation pathway. In contrast, calcineurin inhibitor would in-hibit both pathways mediated by a common transcriptionfactor and NFAT-c4, leading to an inhibition of c-fos expres-sion. The precise mechanism for differential effects of inhibi-tors of calcineurin and CaMK II on c-fos expression, how-ever, remains to be clarified. Interestingly, a potent inhibitoryeffect of KN62 and no effect of CsA and FK506 on LIF-induced expressions of the marker genes for cardiac hyper-trophy were reported [11]; these are not consistent with ourresults, suggesting that the roles of calcineurin and CaMK IIin cardiac hypertrophy might differ according to the kinds ofstress factors conferred on the heart. Therefore, therapeutictargets would be different according to the cause inducingcardiac hypertrophy. These results will lead the future clini-cal management for cardiac hypertrophy to more specificmechanism-based treatment.
In summary, we have demonstrated that calcineurin andCaMK II are activated by pressure overload in isolated per-fused heart, and that both calcineurin and CaMK II pathwaysare critical in the acceleration of protein synthesis induced by
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pressure overload. Moreover, whereas c-fos gene expressionis regulated by calcineurin pathway, the CaMK II pathwaydoes not regulate it.
Acknowledgements
This work was supported by a Grant-in-Aid for ScientificResearch from Ministry of Education, Science, Sports andCulture of Japan. This work was also supported by a grant forresearch on cardiovascular disease from the Japan HeartFoundation/Pfizer Pharmaceuticals Inc. and a grant fromMitsui Life Social Welfare Foundation. The authors alsowish to acknowledge to Ms. Mika Yashima for her excellenttechnical assistance.
References
[1] Aoki H, Izumo S, Sadoshima J. Angiotensin II activates RhoA incardiac myocytes, a critical role of RhoA in angiotensin II-inducedpremyofibril formation. Circ Res 1998;82:666–76.
[2] Berridge MJ. Calcium signaling and cell proliferation. Bioessays1995;17:491–500.
[3] Molkentin JD, Lu JR, Antos CL, Markham B, Richardson J, Rob-bins J, et al. A calcineurin-dependent transcriptional pathway forcardiac hypertrophy. Cell 1998;93:215–28.
[4] Sussman MA, Lim HW, Gude N, Taigen T, Olson EN, Robbins J, et al.Prevention of cardiac hypertrophy in mice by calcineurin inhibition.Science 1998;281:1690–3.
[5] Meguro T, Hong C, Asai K, Takagi G, McKinsey TA, Olson EN, et al.Cyclosporin attenuates pressure-overload hypertrophy in mice whileenhancing susceptibility to decompensation and heart failure. CircRes 1999;84:735–40.
[6] Shimoyama M, Hayashi D, Takimoto E, Zou Y, Oka T,Uozumi H, et al. Calcineurin plays a critical role in pressure overload-induced cardiac hypertrophy. Circulation 1999;100:2449–54.
[7] Shimoyama M, Hayashi D, Zou Y, Takimoto E, Mizukami M,Monzen K, et al. Calcineurin inhibitor attenuates the development andinduces the regression of cardiac hypertrophy in rats with salt-sensitive hypertension. Circulation 2000;102:1996–2004.
[8] Passier R, Zeng H, Frey N, Naya FJ, Nicol RL, McKinsey TA, et al.CaM kinase signaling induces cardiac hypertrophy and activates theMEF2 transcription factor in vivo. J Clin Invest 2000;105:1395–406.
[9] Lim HW, Windt LJ, Steinberg L, Taigen T, Witt SA, Kimball TR, et al.Calcineurin expression, activation, and function in cardiac pressure-overload hypertrophy. Circulation 2000;101:2431–7.
[10] Zhu W, Zou Y, Shiojima I, Kudoh S, Aikawa R, Hayashi D, et al.Ca2+/calmodulin dependent kinase II and calcineurin play criticalroles in endothelin-1 induced cardiomyocyte hypertrophy. J BiolChem 2000;275:15239–45.
[11] Kato T, Sano M, Miyoshi S, Sato T, Hakuno D, Ishida H, et al.Calmodulin kinases II and IV and calcineurin are involved in leuke-mia inhibitory factor-induced cardiac hypertrophy in rats. Circ Res2000;87:937–45.
[12] Osaki J, Haneda T, Sakai H, Kikuchi K. cAMP-mediated c-fos expres-sion in pressure-overloaded acceleration of protein synthesis in adultrat heart. Cardiovasc Res 1997;33:631–40.
[13] Xenophontos XP, Watson PA, Chua BHL, Haneda T, Morgan HE.Increased cyclic AMP content accelerates protein synthesis in ratheart. Circ Res 1989;65:647–56.
[14] Fukuzawa J, Osaki J, Haneda T. Differential effects of amiloride onthe basal rate and the pressure overload-induced increase in proteinsynthesis in perfused rat heart. Clin Exp Hypertens 1994;16:835–52.
[15] Watson PA, Haneda T, Morgan HE. Effect of higher aortic pressure onribosome formation and cAMP content in rat heart. Am J Physiol1989;256:C1257–61.
[16] Osaki J, Haneda T, Kashiwagi Y, Oi S, Fukuzawa J, Sakai H, et al.Pressure-induced expression of heat shock protein 70 mRNA in adultrat heart is coupled both to protein kinase A-dependent and proteinkinase C-dependent systems. J Hypertens 1998;16:1193–200.
[17] Williams LH, Chua BHL, Sahms RH, Siehl D, Morgan HE. Effects ofdiabetes on protein turnover in cardiac muscle. Am J Physiol 1980;239:E178–85.
[18] KiraY, Kochel PJ, Gordon EE, Morgan HE. Aortic perfusion pressureas a determinant of cardiac protein synthesis. Am J Physiol 1984;246:C247–58.
[19] Fukuzawa J, Haneda T, Kikuchi K. Arginine vasopressin increases therate of protein synthesis in isolated perfused adult rat heart via the V1receptor. Mol Cell Biochem 1999;195:93–8.
[20] Lim HW, Windt LJ, Mante J, Kimball TR, Witt SA, Suss-man MA, et al. Reversal of cardiac hypertrophy in transgenic diseasemodels by calcineurin inhibition. J Mol Cell Cardiol 2000;32:697–709.
[21] Martin B, Pallen CJ, Wang JH, Graves DJ. Use of fluorinated tyrosinephosphates to probe the substrate specificity of the low molecularweight phosphatase activity of calcineurin. J Biol Chem 1985;260:14932–7.
[22] Harder KW, Owen P, Wong LK, Aebersold R, Clark-Lewis I, Jirik FR.Characterization and kinetic analysis of the intracellular domain ofhuman protein tyrosine phosphatase beta (HPTP beta) using syntheticphosphopeptides. Biochem J 1994;298:395–401.
[23] Enz A, Shapiro G, Chappuis A, Dattler A. Nonradioactive assay forprotein phosphatase 2B (calcineurin) activity using a partial sequenceof the subunit of cAMP-dependent protein kinase as substrate. AnalBiochem 1994;216:147–53.
[24] Donella-Deana A, Krinks MH, Ruzzene M, Klee C, Pinna LA.Dephosphorylation of phosphopeptide by calcineurin (protein phos-phatase 2B). Eur J Biochem 1994;219:109–17.
[25] Chomczynski P, Sacchi N. Single-step method of RNA isolation byacid guanidinium thiocynate–phenol–chloroform extraction. AnalBiochem 1987;162:156–9.
[26] Katz AM. Cardiomyopathy of overload. A major determinant ofprognosis in congestive heart failure. New Engl J Med 1990;322:100–10.
[27] Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognos-tic implications of echocardiographically determined left ventricularmass in the Framingham heart study. New Engl J Med 1990;322:1561–6.
[28] Komuro I,YazakiY. Control of cardiac gene expression by mechanicalstress. Annu Rev Physiol 1993;55:55–75.
[29] Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Mizuno T, et al.Mechanical stress activates protein kinase cascade of phosphorylationin neonatal rat cardiac myocytes. J Clin Invest 1995;96:438–46.
[30] Sadoshima J, Izumo S. Mechanical stretch rapidly activates multiplesignal transduction pathways in cardiac myocytes: potential involve-ment of an autocrine/paracrine mechanism. EMBO J 1993;12:1681–92.
[31] Davis RJ. The mitogen-activated protein kinase signal transductionpathway. J Biol Chem 1993;268:14553–6.
[32] Morgan HE, Baker KM. Cardiac hypertrophy. Mechanical, neural,and endocrine dependence. Circulation 1991;83:13–25.
[33] Sadoshima J, Takahashi T, Jahn L, Izumo S. Roles of mechanosensitive ion channels, cytoskeleton, and contractile activity instretch-induced immediate-early gene expression and hypertrophy ofcardiac myocytes. Proc Natl Acad Sci USA 1992;89:9905–9.
[34] Zou Y, Hiroi Y, Uozumi H, Takimoto E, Toko H, Zhu W, et al.Calcineurin plays a critical role in the development of pressureoverload-induced cardiac hypertrophy. Circulation 2001;104:97–101.
1159T. Saito et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 1153–1160
[35] De Windt LJ, Lim HW, Bueno OF, Liang Q, Delling U, Braz JC, et al.Targeted inhibition of calcineurin attenuates cardiac hypertrophy invivo. Proc Natl Acad Sci USA 2001;98:3322–7.
[36] Rothermel BA, McKinsey TA, Vega RB, Nicol RL, Mammen P,Yang J, et al. Myocyte-enriched calcineurin-interacting protein,MCIP1, inhibits cardiac hypertrophy in vivo. Proc Natl Acad Sci USA2001;98:3328–33.
[37] Sadoshima J, Izumo S. The cellular and molecular response of cardiacmyocytes to mechanical stress. Annu Rev Physiol 1997;59:551–71.
[38] Yamazaki T, Komuro I, Kudoh S, Zou Y, Shiojima I, Hiroi Y, et al.Endothelin-1 is involved in mechanical stress-induced cardiomyocytehypertrophy. J Biol Chem 1996;271:3221–8.
[39] Janknecht R, Cahill MA, Nordheim A. Signal integration at the c-fospromoter. Carcinogenesis 1995;16:443–50.
1160 T. Saito et al. / Journal of Molecular and Cellular Cardiology 35 (2003) 1153–1160
