Pim-1 regulates cardiomyocyte survival downstreamof AktJohn A Muraski1, Marcello Rota2, Yu Misao2, Jenna Fransioli1, Christopher Cottage1, Natalie Gude1,Grazia Esposito2, Francesca Delucchi2, Michael Arcarese2, Roberto Alvarez1, Sailay Siddiqi1,Gregory N Emmanuel1, Weitao Wu1, Kimberlee Fischer1, Joshua J Martindale1, Christopher C Glembotski1,Annarosa Leri2, Jan Kajstura2, Nancy Magnuson3, Anton Berns4, Remus M Beretta5, Steven R Houser5,Erik M Schaefer6, Piero Anversa2 & Mark A Sussman1

The serine-threonine kinases Pim-1 and Akt regulate cellular proliferation and survival. Although Akt is known to be a crucial

signaling protein in the myocardium, the role of Pim-1 has been overlooked. Pim-1 expression in the myocardium of mice

decreased during postnatal development, re-emerged after acute pathological injury in mice and was increased in failing hearts

of both mice and humans. Cardioprotective stimuli associated with Akt activation induced Pim-1 expression, but compensatory

increases in Akt abundance and phosphorylation after pathological injury by infarction or pressure overload did not protect the

myocardium in Pim-1–deficient mice. Transgenic expression of Pim-1 in the myocardium protected mice from infarction injury,

and Pim-1 expression inhibited cardiomyocyte apoptosis with concomitant increases in Bcl-2 and Bcl-XL protein levels, as well

as in Bad phosphorylation levels. Relative to nontransgenic controls, calcium dynamics were significantly enhanced in Pim-

1–overexpressing transgenic hearts, associated with increased expression of SERCA2a, and were depressed in Pim-1–deficient

hearts. Collectively, these data suggest that Pim-1 is a crucial facet of cardioprotection downstream of Akt.

Intracellular molecular signaling networks communicate throughkinases that phosphorylate target substrates to regulate crucial aspectsof growth and survival. Pim-1, a proto-oncogenic serine-threoninekinase, was originally discovered as the proviral integration site forMoloney murine leukemia virus1. Pim-1 expression can be induced bycytokines and growth factors including LIF, GM-CSF, EGF and mostinterleukins (reviewed in ref. 2), consistent with a role for Pim-1 in theproliferation and survival of hematopoietic cells. Pim-1 mediatesproliferative signaling through phosphorylation of multiple targetsubstrates, resulting in cell-cycle transition, and also mediates protec-tive effects through phosphorylation of multiple targets including Badat Ser112 (ref. 3).

Recently, induction of Pim-1 expression has been linked to Aktactivation in hematopoietic cells4. Like Pim-1, Akt is a serine-threonine kinase involved in cell proliferation and survival. Aftergrowth factor or cytokine receptor activation, Akt is phosphorylated,resulting in a conformational change that releases Akt from themembrane, allowing it to transit through the cytosol and eventuallyto the nucleus, where it affects the transcription of target genes5 andexerts cardioprotective activity6. The Akt-mediated induction ofPim-1 in nonmyocardial contexts after prolactin administration and

the direct binding of Akt to a prolactin response element on the Pim-1promoter7,8 support a role for Akt in regulation of Pim-1 activity, butwhether such a relationship also exists in the myocardium has notbeen assessed. This study was designed to investigate the potentialcardioprotective role of Pim-1.

RESULTS

Developmental regulation of Pim-1 cardiac expression

As previously described9, the 34-kDa isoform of Pim-1 is visualized astwo bands corresponding to the phosphorylated, higher moiety andunphosporylated, lower moiety forms in whole-cell lysate immuno-blots of mouse hearts (Supplementary Fig. 1a online). Pim-1 expres-sion decreased with age in myocardial lysates from mice, with neonatalheart samples showing 6.3-fold more Pim-1 than 30-week-old mice(Fig. 1a). Postnatal expression levels declined, but remained signifi-cantly elevated until 8 weeks of age, when protein levels becomecomparable to 30-week-old hearts (Fig. 1a). Similarly, a 3.5-foldincrease in Pim1 mRNA levels occurred in neonatal hearts, and theydecreased significantly by 8 weeks of age (Supplementary Fig. 1b). Asinduction of Pim-1 in the hematopoietic system is linked to Aktactivity4, we performed in vitro kinase assays on whole-heart lysates

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Received 22 August; accepted 24 September; published online 25 November 2007; doi:10.1038/nm1671

1San Diego State University Heart Institute, San Diego State University, 5500 Campanile Drive, San Diego, California 92182, USA. 2Cardiovascular Research Institute,New York Medical College, Vosburgh Pavilion, Valhalla, New York 10595, USA. 3School of Molecular Biosciences, Washington State University, Pullman, Washington99164, USA. 4Division of Molecular Genetics, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. 5Cardiovascular Research Center, TempleUniversity School of Medicine, 3420 North Broad Street, Philadelphia, Pennsylvania 19140, USA. 6BioSource Cytokines & Signaling, Invitrogen Corporation 94 SouthStreet, Hopkinton, Maryland 01748, USA. Correspondence should be addressed to M.A.S. (sussman@heart.sdsu.edu).

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(Supplementary Fig. 1i). Akt activity remained consistently low inpostnatal (o1 week), juvenile (8 weeks) and adult myocardial samples(30 weeks). Confocal microscopy and subcellular fractionation studiesshowed that Pim-1 expression is predominantly nuclear in neonates,becomes increasingly cytosolic in early adulthood, and is virtuallyabsent in 30-week-old adults (Fig. 1a). Compared to 30-week-oldmouse myocardium, nuclear Pim-1 expression was increased by 10.5-fold and 5.2-fold, and cytosolic Pim-1 expression was decreased by 80%and 78.3%, in neonatal and 8-week-old hearts, respectively (Fig. 1a).

PIM-1 is upregulated in failing human myocardium

We obtained cardiac explants from human donors (SupplementaryTable 1 online). In normal adult human myocardium, PIM-1 expres-sion was present throughout the cytoplasm (Fig. 1b). In contrast,PIM-1 adopted a predominantly nuclear localization in samplesfrom failing human hearts (Fig. 1b). PIM-1 protein expression was2.65-fold higher in failing human myocardium than in normal controlmyocardium (P o 0.05). Quantification of nuclear PIM-1 accumula-tion in failing human heart samples by immunoblot was unsuccessful,a problem we attribute to differences between the preparation ofhuman cardiac explant tissues and of murine tissue samples. PIM1mRNA transcript levels were more than 50-fold higher in the failinghuman heart than in normal controls (Supplementary Fig. 1c). Weobserved similar results in a mouse model of chronic heart failure, the

tropomodulin-overexpressing transgenic mouse (TOT)10, in whichPim-1 protein was predominantly nuclear and its level of expressionwas increased by 5.9-fold compared to nontransgenic (NTG) myo-cardial samples (Fig. 1c); Pim1 mRNA levels were increased 2.5-fold inTOT hearts (Supplementary Fig. 1d).

Pim-1 expression is reactivated after pathological injury

We examined Pim-1 localization and expression 4 d after challengeby trans-aortic constriction (TAC) pressure overload–induced hyper-trophy or myocardial infarction (MI). TAC increased Pim-1 immuno-reactivity in cardiomyocytes surrounding major vessels, showinga perinuclear distribution (Fig. 1d). Similarly, perinuclear Pim-1immunoreactivity increased in border-zone cardiomyocytes after MI(Fig. 1d) but was unchanged in remote myocardium (data notshown). Pim1 transcript levels increased twofold in mouse heart 4 dafter TAC (Supplementary Fig. 1e). However, no significant increasein Pim1 mRNA was evident 4 d after MI (Supplementary Fig. 1f).Pim-1–positive border-zone cardiomyocytes were negative for TUNELlabeling and showed increased Bcl-XL expression, consistent withcardioprotective signaling (Supplementary Fig. 1g,h).

Pim-1 is cardioprotective in vitro

We assessed the role of Pim-1 in cardioprotection using recombinantadenoviral vectors carrying GFP-tagged cDNAs encoding wild-type

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Figure 1 Pim-1 is expressed in the human and mouse myocardium. (a) Representative confocal

micrographs of paraffin-embedded sections from wild-type mouse hearts at o1 week, 8 weeks and

30 weeks stained for Pim-1 (green), tropomyosin (red) and nuclei (blue). n ¼ 4; boxed regions in

left micrographs are shown at higher magnification at right. Arrowheads in grayscale and arrows in

overlay of top right panel indicate cardiomyocytes with Pim-1 nuclear localization. Bar, 40 mm, left;

10 mm, right. Right, representative immunoblot of whole-heart lysates for Pim-1, with actin as a

loading control. Below, histogram depicting developmental time course immunoblot quantification.*P o 0.05, **P o 0.01 versus 30 weeks, n ¼ 4 for each time point. Also shown is a representative

immunoblot of nuclear and cytosolic fractions of hearts from o1-, 8- and 30-week-old mice blotted

for Pim-1, GAPDH (as cytosolic loading control) and histone 3 (as nuclear loading control). Graph

below shows quantification of these results from n ¼ 6 mice (*P o 0.05, **P o 0.001 compared

to 30 weeks). (b) Immunohistochemistry and immunoblotting of normal and failing human hearts for

Pim-1. Representative confocal scans are shown of paraffin-embedded normal and failing human

heart sections stained for Pim-1, tropomyosin and nuclei (green, red and blue, respectively, in

overlays; n ¼ 4). Arrowheads in grayscale and overlay indicate cardiomyocyte nuclei expressing Pim-1. Bar, 40 mm. Immunoblot of human heart whole-cell

lysates is shown at right. (c) Immunohistochemistry and immunoblotting of non-transgenic (NTG) and tropomodulin-overexpressing transgenic (TOT) hearts.

Representative confocal scans are shown for Pim-1, tropomyosin and nuclei (green, red and blue, respectively, in overlays). Arrowheads in grayscale and

overlay indicate cardiomyocyte nuclei expressing Pim-1. Bar, 47.62 mm. A representative (n ¼ 3) blot of fractionated (C, cytosolic; N, nuclear) NTG and TOT

myocardium is shown at right. (d) Representative confocal micrographs of 4-d sham, MI and TAC mouse hearts labeled for Pim-1 (green), tropomyosin (red)

and nuclei (blue) at 20� (top row) and 63� (bottom row) magnification of areas boxed in top panels. LV, left ventricle; I, infarct; EP, epicardium; V, vessel.

Bars, 40 mm, top; 5 mm, bottom; n ¼ 6.

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Pim-1 (Pim-WT) or a kinase-dead ATP binding site mutant (K67M)previously characterized as a functional dominant negative(Pim-DN)11. Cultures of neonatal rat cardiomyocytes (NRCMs)expressing GFP–Pim-WT or GFP–Pim-DN accumulated 64 kDaGFP–Pim-1 fusion proteins recognized by GFP– and Pim-1–specificantibodies (Fig. 2a). NRCM overexpressing GFP–Pim-WT showed astatistically significant decrease in TUNEL labeling compared tocontrol EGFP-expressing cells under basal conditions (Fig. 2b).GFP–Pim-WT overexpression protected NRCMs from apoptoticchallenge with doxorubicin or deoxyglucose (Fig. 2b). In com-parison, GFP–Pim-DN overexpression induced a 30.8% increase inapoptotic cardiomyocytes under normal culture conditions and exa-cerbated the effects of apoptotic stimuli induced by doxorubicin ordeoxyglucose. Consistent with these results, GFP–Pim-DN inducedboth caspase 3 (3.6-fold) and PARP (80%) cleavage (Fig. 2c). GFP-Pim-WT overexpression mediated cardioprotective signaling: weobserved significant increases in Bcl-XL and Bcl-2 expression (2.2-fold and 25.4-fold, respectively) compared to control EGFP over-expression (Fig. 2d). GFP–Pim-WT also increased phosphorylation ofBad(Ser112) by 16.7-fold as compared to the EGFP-overexpressingcontrol, whereas total Bad abundance remained unchanged (Fig. 2e).Levels of STAT3 expression or STAT3 activation by phosphorylation atTyr705 were unaffected by overexpression of either GFP–Pim-WT orGFP–Pim-DN (Supplementary Fig. 2 online).

Pim-1 induction by cardioprotective stimuli

Cardioprotective agents including leukemia inhibitory factor (LIF),insulin-like growth factor (IGF-1), dexamethasone (Dex) and phorbol12-myristate 13-acetate (PMA) induced Pim-1 immunoreactivity inNRCMs after 2 h of treatment (Supplementary Fig. 3 online). Quanti-fication of immunoblots showed that LIF, IGF-1, PMA and Dexinduced Pim-1 expression by 2.8-, 2.7- 2.3- and 2.0-fold, respectively(Fig. 3a,b). In contrast, Pim-1 immunoreactivity was not induced byphenylephrine, endothelin-1, forskolin or estradiol (SupplementaryFig. 3). Pim1 mRNA levels rose by 5.1- and 4.0-fold after treatment ofNRCMs with PMA or Dex, respectively (Fig. 3b). Inhibition of Akt

signaling using the K179M Akt mutant (Akt-DN) reduced Pim-1 ex-pression to basal levels after treatment with IGF-1 or Dex (Fig. 3c,d).Furthermore, the elevated Pim1 mRNA levels induced by PMA or Dexreturned to basal levels in the presence of Akt-DN (Fig. 3d).

Nuclear-targeted Akt induces Pim-1

IGF-1 stimulation promotes nuclear accumulation of Akt12, which, inturn, promotes cardioprotection6. Indeed, nuclear-targeted Akt (Akt-nuc) overexpression increased Pim-1 levels in NRCMs by 2.0-foldcompared to control overexpression of EGFP (Fig. 4a). Expression ofAkt-nuc in NRCMs induced increased nuclear localization of Pim-1(Supplementary Fig. 4a online). In comparison, wild-type Akt (Akt-WT) overexpression decreased Pim-1 level by 30% as compared touninfected control (*P o 0.05), whereas infection with Akt-DNdecreased expression 42% (Fig. 4a, **P o 0.01). Pim1 mRNA levelswere induced 3.1- and 4.7-fold by overexpression of Akt-WT or theconstitutively activated myr-Akt, respectively, whereas both Akt-nucand Akt-DN decreased Pim1 transcript levels (SupplementaryFig. 4b). NRCM apoptosis resulting from exposure to doxorubicinand deoxyglucose was exacerbated by Akt-DN expression (Fig. 4b).Pim-1 overexpression inhibited NRCM death despite blockade of Aktsignaling by concurrent Akt-DN overexpression. Akt-nuc–mediatedprotection from apoptotic challenge in NRCMs (ref. 6) was blocked byoverexpression of GFP–Pim-DN (Fig. 4b). Consistent with thesein vitro results, hearts from transgenic mice with cardiac-specificexpression of nuclear-targeted Akt showed increased Pim-1 immuno-reactivity and nuclear localization compared to nontransgenic wild-type controls (Fig. 4c) as well as increased Pim1 mRNA expression(Supplementary Fig. 4).

Pim-1 and Akt exhibit feedback relationships

Akt expression and phospho-Akt(Ser473) levels increased inresponse to GFP–Pim-DN overexpression in NRCMs (Fig. 4d),demonstrating reciprocal signaling between Akt and Pim-1. Similarly,increased levels of phospho-Akt(Ser473) were present in myocardialsections from mice with global genetic deletion of Pim1 (ref. 13;

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Figure 2 Pim-1 induces expression of antiapoptotic proteins and protects against apoptosis. (a) Representative immunoblot from adenovirus-infected NRCM

lysates (n ¼ 3) immunoblotted for the GFP–Pim1-wt (Pim-WT) and GFP–Pim1-DN (Pim-DN) fusion proteins using both Pim-1– and GFP-specific antibodies,

with a-actinin as a loading control. Antibodies used are listed at left and fusion protein and molecular weight at right. (b) Percentage of TUNEL-positive

NRCMs after adenoviral infection and exposure to doxorubicin or deoxyglucose treatment. n ¼ 3; *P o 0.05, **P o 0.01 versus EGFP control.(c) Representative immunoblot and corresponding quantification for caspase 3 or PARP cleavage after adenoviral infection. a-actinin was used as a loading

control. n ¼ 5, *P o 0.05 versus EGFP control. (d) A representative immunoblot and graph of quantification results for Bcl-2 and Bcl-XL levels in

adenovirally infected NRCM lysates. GAPDH was used as a loading control. n ¼ 5, **P o 0.01 versus EGFP control. (e) Representative immunoblot and

quantification of phospho-Bad(Ser112) levels in adenovirally infected NRCM lysates. GAPDH was used as a loading control. n ¼ 5, **P o 0.001 versus

EGFP control. rfu, relative fluorescence units.

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NATURE MEDICINE ADVANCE ONLINE PUBLICATION 3

Pim-KO; Fig. 4e); increased levels of phospho-Akt(Ser473),phospho-Akt(Thr308) and total Akt were seen in whole-heart lysatesfrom Pim-KO samples (Fig. 4f).

Genetic ablation of Pim-1 increases infarction injury

We examined the protective effects of Pim-1 after MI in Pim-KO mice.Left ventricular free wall infarct size was 22.7% greater in Pim-KOhearts than in wild-type controls (Fig. 5a). Pim-KO mice showed a

minor but significant increase in TUNEL-positive myocytes in the leftventricle relative to wild-type controls (Supplementary Table 2 online;Po 0.01), and this difference was exacerbated after MI, with a 4.0-foldincrease in TUNEL-positive myocytes relative to wild-type samples(Fig. 5b; P o 0.01). Under basal conditions, the hemodynamicperformances of Pim-KO and wild-type control mice were comparable(Supplementary Fig. 5 online). After MI, however, developedventricular pressure was depressed and end-diastolic pressure was

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Figure 3 Cardioprotective stimuli induce Pim-1 expression. (a) Representative immunoblot for

phospho-STAT3(Tyr705), total STAT3, phospho-Akt(Ser473) and phospho-Akt(Thr308), total Akt andPim-1, with GAPDH as loading control, from whole-cell lysates (n ¼ 3). Treatments are indicated

above each lane. (b) Quantification of Pim-1 protein expression from immunoblots (left) and Pim1

mRNA (right) by qRT-PCR after treatments shown in a. rfu, relative fluorescence units. n ¼ 3;

*P o 0.05, **P o 0.01 compared to control. (c) Immunoblot of cultured NRCMs preinfected with

Akt-DN, stimulated with cardioprotective agents for 2 h as indicated above each lane and probed for

phospho-Akt(Ser473), phospho-Akt(Thr308), Akt and Pim-1, with GAPDH as a loading control,

n ¼ 3. (d) Quantification of immunoblots (top) and mRNA from qRT-PCR (bottom) from experiments

as described in c. All fold changes for mRNA experiments were standardized to uninfected controls.

*P o 0.05, **P o 0.01 compared to control.

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Figure 4 Expression of Pim-1 is Akt dependent. (a) Representative immunoblot of NRCM lysates infected with EGFP, wild-type Akt (Akt-WT), myristoylated

Akt (myr-Akt), Akt-nuc and Akt-DN with quantification shown below. n ¼ 4; *P o 0.05, **P o 0.01 versus EGFP control. (b) Percentage of TUNEL-positive

cardiomyocytes in NRCM cultures infected with EGFP, Akt-DN, Akt-DN and GFP–Pim-WT, Akt-nuc or Akt-nuc and GFP–Pim-DN and then treated with

doxorubicin or deoxyglucose. n ¼ 3, *P o 0.05 and **P o 0.01 versus EGFP; #P o 0.01 versus Akt-DN; $P o 0.01 versus Akt-nuc. (c) Representative

confocal micrographs of paraffin-embedded sections from 6-month-old NTG and Akt-nuc transgenic mouse hearts stained for tropomyosin (red), Pim-1(green) and nuclei (blue). Nuclear localization of Pim-1 staining indicated (red arrowheads in grayscale and white arrows in overlay). n ¼ 3; scale bar,

40 mm. (d) Representative immunoblot of lysates from infected neonatal rat cardiomyocytes for total Akt, phospho-Akt(Ser473) and phospho-Akt(Thr308)

with GAPDH as loading control; n ¼ 3. (e) Confocal micrographs of NTG and Pim-1 KO mouse hearts stained for phospho-Akt(Ser473) (green), tropomyosin

(red) and nuclei (blue). Cardiomyocyte staining for phospho-Akt(Ser473) signal are indicated (arrows); n ¼ 4. (f) Immunoblot of whole-heart lysates from

2-month-old Pim-1 KO and NTG controls for phospho-Akt(Ser473), phospho-Akt(Thr308) and total Akt with GAPDH as loading control; n ¼ 4.

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increased in Pim-KO as compared to wild-type control mice (Fig. 5c).Further, diastolic wall stress was significantly increased in both leftventricular free wall and septum after infarction in Pim-KO hearts(Supplementary Fig. 5; P o 0.01). Notably, Pim-KO mice haddecreased lymphocyte proliferation and hematopoietic cell differentia-tion13–15 that could possibly decrease inflammatory responses afterMI, but no significant differences were found in circulating c-Kit+ cellnumber after MI or in c-Kit+Sca-1+ bone marrow cell number eitherbefore or after MI (Supplementary Fig. 5). Likewise, inflammatorycell recruitment after MI, as indicated by CD45 staining, was compar-able between Pim-KO and control hearts (Supplementary Fig. 5).

Pim-KO hearts exhibit altered protective signaling

Pim-1 may be a relatively promiscuous kinase, given its minimal targetsubstrate recognition sequence requirements16 and capacity for auto-phosphorylation17, so we examined molecular mechanisms responsiblefor Pim-1–mediated cardioprotection. Relative to wild-type samples,Pim-KO heart samples showed increases in the levels of phospho-Akt (Thr308) (90.7%), phospho-Akt(Ser473) (2.76-fold), totalAkt (2.10-fold), phospho-STAT3(Tyr705) (2.61-fold), total STAT3(68.6%) and Pim-2 (4.6-fold). However, we observed no increase inthe expression of Bcl-2, Bcl-XL, phospho-Bad(Ser112) or Pim-3 inPim-KO samples as compared to wild-type control samples (Fig. 5d).We also examined these survival-signaling molecules 7 d after MI;

relative to sham-operated controls, Pim-KO mice showed a 2.57-foldincrease in Pim-3 expression but decreases in the levels of Bcl-XL (2.1-fold), phospho-Bad(Ser112) (75.9%), phospho-Akt(Thr308) (92.6%),phospho-Akt(Ser473) (2.24-fold), total Akt (73.7%), phospho-STAT3(Tyr705) (2.72-fold) and total STAT3 (2.0-fold), with no significantchanges in Bcl-2 or Pim-2 expression (Fig. 5e). In comparison, relativeto sham-operated controls, wild-type mice after MI showed significantincreases in Bcl-XL (57.0%), phospho-Bad(Ser11) (64.6%), phospho-Akt(Thr308) (98.3%), phospho-Akt(Ser473) (2.81-fold), total Akt(2.26-fold), phospho-STAT3(Tyr705) (3.43-fold) and total STAT3(2.02-fold), with no change in Pim-2 or Pim-3 expression (Fig. 5e).Thus, as compared to wild-type hearts, Pim-KO hearts after MI showedsignificant increases in Pim-2 and Pim-3 levels, but profound decreasesin the levels of other survival signaling molecules (Fig. 5e).

Myocardial overexpression of Pim-1 blunts infarction injury

We created transgenic mice to assess the cardioprotection afforded bycardiac-specific expression18 of Pim-1 after MI. Evaluation of infarctsize 10 d after MI showed a 41.9% decrease in left ventricular free wallinfarct size in Pim-WT transgenic hearts as compared to infarctednon-transgenic hearts; there was no significant difference betweeninfarcted Pim-WT transgenic and non-transgenic hearts in the num-ber of TUNEL-labeled cells in the left ventricle (Fig. 5f,g). Althoughwe observed no difference in cardiac function in the basal state

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Figure 5 Pim-1 protects against infarction injury. (a) Infarct size 7 d after MI as a percentage of left ventricular free wall in Pim-KO hearts. n ¼ 4;

*P o 0.05 versus NTG MI. (b) Number of TUNEL-positive myocytes per mm2 7 d after MI in Pim-KO hearts. n ¼ 3; **P o 0.01 versus NTG MI. (c) In vivo

hemodynamic measurements of NTG and Pim-KO mice 5 d after MI. n ¼ 5; *P o 0.05 versus NTG. Left ventricular developed pressure (LVDP), left ventri-

cular end-diastolic pressure (LVEDP), and ± change in pressure over change in time (dP/dt). (d) Quantification of immunoblots for levels of protective

proteins in Pim-KO mice versus NTG mice. n ¼ 4; *P o 0.05, **P o 0.01. rfu, relative fluorescence units. (e) Immunoblot quantification of survivalprotein levels 7 d after MI in Pim-KO and NTG control hearts. n ¼ 4; *P o 0.05 versus sham, #P o 0.01 versus sham, $P o 0.01 versus NTG MI.

(f) Infarct size measurements 10 d after MI. n ¼ 3. **P o 0.01 versus NTG MI. (g) Number of TUNEL-labeled myocytes per square millimeter in the left

ventricle 10 d after MI. (h) In vivo hemodynamic evaluation of NTG and Pim-WT hearts after sham operation or MI. n ¼ 5, **P o 0.01 versus sham,

*P o 0.05 versus NTG sham, #P o 0.001 versus NTG sham, $P o 0.01 versus NTG MI.

ART ICL ES

NATURE MEDICINE ADVANCE ONLINE PUBLICATION 5

(Supplementary Fig. 5), wild-type mice had depressed hemodynamicperformance after MI (Fig. 5h). In contrast, contractile function wasmaintained in Pim-WT transgenic mice after infarction (Fig. 5h).

Response to TAC in Pim-KO is impaired

We challenged Pim-KO mice with TAC to assess the effects of Pim-1genetic ablation on hypertrophy. Pim-KO mice rapidly succumbed tothe challenge (Supplementary Table 2), with thinning anterior andposterior walls compared to wild-type hearts. There were moreapoptotic myocytes in the left ventricle of Pim-KO mice comparedto wild-type mice under basal conditions, with a 92% increase inTUNEL-positive nuclei (1.274 ± 0.133 versus 0.664 ± 0.131 TUNEL-positive cells per mm2, Supplementary Table 2). Four weeks afterTAC challenge, Pim-KO mice showed 54.3% more TUNEL-positivemyocytes than their wild-type TAC counterparts (1.766 ± 0.147 versus1.144 ± 0.103 TUNEL-positive cells per mm2, SupplementaryTable 2). Thus, Pim-KO mice had a higher rate of apoptotic celldeath than wild-type controls, both before and after TAC challenge.

We measured TAC-induced hypertrophy by normalizing heartweight (HW) to tibia length (TL), as Pim-KO mice showed

significantly lower body mass compared to wild-type mice (20.95 gversus 27.18 g, respectively; Supplementary Table 2; P o 0.01). TheHW/TL ratio was comparably increased in wild-type and Pim-KOhearts at 4 weeks after TAC (Supplementary Table 2). Morphometricanalyses of unoperated Pim-KO hearts showed preservation of leftventricular diameter size, similar to that of wild-type hearts undernormal conditions when corrected for wall thickness. However, echo-cardiographic assessment of Pim-KO hearts showed increases in end-diastolic and end-systolic dimensions, in marked contrast to wild-typemice (Supplementary Table 2). Furthermore, whereas hearts of wild-type TAC mice maintained contractility after 4 weeks, Pim-KO TAChearts showed significant depression of both fractional shortening andejection fraction (Supplementary Table 2).

We compared the molecular signature of TAC-induced hypert-rophy of Pim-KO and wild-type mice by quantitative RT-PCRanalyses. Pim-KO hearts lacked hypertrophic gene induction of atrialnatriuretic peptide (ANP) and b-myosin heavy chain (b-MHC),unlike wild-type controls in which these genes were significantlyupregulated after banding (Supplementary Table 2). However,levels of the mRNAs for brain natriuretic peptide (BNP) and

a-skeletal actin (aSKA) showed similarincreases after TAC in both Pim-KO andwild-type hearts. We also examined hyper-trophic markers in normal, unchallengedmyocardium and found that levels of themRNAs encoding BNP and aSKA weredecreased in Pim-KO mice compared towild-type mice (by 2.22- and 5.55-fold,respectively; Supplementary Table 2).

Pim-1 enhances calcium dynamics and

sarcomeric shortening

Pim-KO myocytes showed significant prolon-gation of both calcium decay and sarcomericrelaxation periods, accompanied by 71% and50% decreases in sarcoendoplasmic reticulumCa2+ATP-ase (SERCA) and sodium-calciumexchanger (NCX) expression relative towild-type myocytes, respectively (Fig. 6). Incontrast, Pim-1 overexpression decreasedcalcium decay and relaxation periods whileincreasing sarcomeric shortening in isolatedmyocytes; additionally, SERCA expressionwas increased 4.9-fold compared to that inwild-type hearts (Fig. 6).

DISCUSSION

The discovery that Pim-1 is involved incardiac protection elucidates a new facet ofsignaling with implications for the regulationof cell survival and proliferation downstreamof Akt. Taken together, the data presentedhere provide evidence of the protective effectsof Pim-1 expression in the myocardium,demonstrating that Pim-1 operates down-stream of Akt and that a feedback mechanismexists involving the two proteins.4 On thebasis of these results, the contribution ofPim-1 to effects previously ascribed to Aktalone in the context of the myocardium willneed to be evaluated.

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transient traces obtained from isolated NTG, Pim-KO and Pim-WT myocytes. Superimposed traces

(inset) indicate the slower transient decay in Pim-KO myocytes (blue) and faster decay in Pim-WT

myocytes (red). NTG, n ¼ 153 cells; Pim-KO, n ¼ 184 cells; Pim-WT, n ¼ 176 cells; with 5 animals

per group for all experiments. (b) Histograms showing Ca2+ transient amplitude, time to 50% and

90% decay, and t. *P o 0.05, **P o 0.01, ***P o 0.001 compared to NTG. (c) Sarcomeric

shortening traces from NTG, Pim-KO and Pim-WT isolated myocytes with histograms displayed in d.

*P o 0.05, ***P o 0.001 versus NTG. (e) Immunoblot of whole-heart lysates from NTG, Pim-KO and

Pim-WT animals for SERCA, sodium/calcium exchanger (NCX) with GAPDH as control; quantification is

shown at right. n ¼ 4, *P o 0.05, **P o 0.01 versus NTG.

ART ICL ES

6 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

In the hematopoietic system, Pim-1 and Akt work in concert tocontrol cell survival and proliferation4. To our knowledge, Pim-1 hasnot previously been studied in the myocardium. Similarly to Akt,Pim-1 defended against apoptosis induced by cardiomyopathic injury.Although Pim-1 expression was developmentally downregulated,expression reappeared in cardiomyocytes after pressure overload orinfarction challenge. Pim-1 is one of several proto-oncogenes partici-pating in the ‘immediate early response’ gene profile expressed aftercardiac injury19, which also includes c-Fos, c-Myc20, Raf and Ras.Pim-1 cooperates with c-Myc in activation of c-Myb–dependentcellular proliferation in other tissues21–23, suggesting that synergisticeffects between oncogenes may help preserve the myocardium inresponse to injury.

Nuclear localization of Pim-1 in Burkitt’s lymphoma is respon-sible for increased Mdm2 expression, phosphorylation and p53degradation23. Additionally, two nuclear targets of Pim-1, NFATc1(ref. 24) and p21Cip1/Waf1 (ref. 25), are involved in cardiac develop-ment26,27 and failure28,29, suggesting plausible roles for nuclear Pim-1in the myocardium.

Consistent with findings in noncardiac cells22,30, Pim-1 overexpres-sion protected NRCMs from apoptosis associated with induction ofBcl-2 and Bcl-XL expression, as well as phosphorylation of Bad.Notably, Pim-1 had protective effects regardless of the activationstate of Akt. Pim-1 may serve as the downstream effector of Akt-induced p53 inhibition, as Akt protects against apoptosis through ap53-dependent mechanism31. Inactivation of Pim-1 induced apopto-tic signaling in cardiomyocytes, and this effect was not reversed byexpression of nuclear-localized Akt in vitro or by compensatoryupregulation of Pim-2 protein levels and Akt and STAT3 activationin vivo. Pim-1 inactivation may increase apoptotic activity throughincreasing generation of reactive oxygen species and mitochondrialpore permeability, as found in other cellular contexts30.

Several cardioprotective factors including LIF, PMA, Dex and IGF-1significantly increased Pim-1 expression, consistent with reportsshowing induction of Pim-1 by PMA treatment of T cells32 as wellas gp130 receptor ligands including interleukin-6 and LIF (reviewed inrefs. 1,33). These ligands and their cognate receptors are increased inthe failing and developing heart34. Reports studying hematopoieticcells describe Pim-1 as both a downstream effector and a feedbackinhibitor of STAT proteins after gp130 activation33, but we wereunable to replicate these findings in the heart. In contrast to thefinding that STAT activated Pim-1 expression downstream of LIF, weobserved that LIF activation of Pim-1 expression was downregulatedin the presence of inactivated Akt, suggesting that Akt is responsiblefor Pim-1 activation under these conditions in cardiomyocytes. To ourknowledge, Pim-1 induction by IGF-1 has not been previouslypublished; Pim-1 expression induced by IGF-1 (or Dex) was Aktdependent, and nuclear-targeted Akt expression mediated a significantincrease in Pim-1 expression. Pim1 mRNA levels were increased byoverexpression of cytosolic activated Akt; however, increased Pim-1protein expression required nuclear localization of Akt. It is temptingto speculate that the basis for this discrepancy rests with nuclear Akt-regulated activation of translational machinery through inactivation of4E-BP1; this would release inhibition of eIF-4E, consequently indu-cing mRNA export from the nuclear compartment and allowing fortranslation35. In the cytosol, activated Akt induces a number of targetsubstrates that result in transcriptional activation of other genes,including NF-kB36, which induces Pim-1 transcription in B cells37,and mTOR, which activates Pim-1 translation downstream of Akt4.Conversely, inactivation or ablation of Pim-1 expression induced Aktexpression and activation, but these increases in Akt activation did not

enhance recovery or reduce apoptosis after infarction or TAC chal-lenge in Pim-deficient animals. Thus, the cardioprotective effects ofAkt depend, in part, upon Pim-1. Definitive examination of themechanism of Pim-1 induction by Akt is beyond the scope of thisinitial report, but future studies will examine connections betweenthese two molecules.

Consistent with protective effects of Pim-1 in other contexts3,38, theabsence of Pim-1 in the myocardium decreased levels of protectivemitochondrial proteins after MI. Although hearts from Pim-1–defi-cient and wild-type mice were functionally comparable under basalconditions, cellular analyses showed considerable changes in myocytecalcium handling, with impaired calcium reuptake and sarcomericrelaxation presumably caused by decreased SERCA2a and NCXexpression. Presumably as a result of these defects in calcium handling,adaptation to pressure overload in Pim-1–deficient mice wasimpaired; adaptation may also have been compromised by increasedmyocyte death and lack of a compensatory increase in the fetal fastmyosin isoform b-myosin heavy chain . Increased levels of Pim-2 andPim-3 after infarction in Pim-deficient mice were insufficient tocompensate for the decreased expression of other survival signalingmolecules observed after MI.

Myocardial overexpression of Pim-1 decreased infarct size andmaintained contractility after MI, with no significant change in thelevel of myocyte apoptosis. Cardioprotection could be a consequenceof increased surviving myocardium after infarction, enhanced cardio-myocyte progenitor proliferation, or both. Myocardial nuclear Aktoverexpression increases resident cardiac progenitor pools, inducesproproliferative cytokine expression and increases myocytenumbers39,40. Likewise, expression of Pim-1 in the myocardium mayserve as the downstream mediator of these effects ascribed tonuclear Akt.

Previous studies indicate that decreased SERCA2a expression has acrucial role in the hypertrophic response41–43. Restoration of functioncan be achieved through minimal increases in SERCA2a expressionafter pressure overload44,45 or infarction46. Myocardial overexpressionof constitutively activated Akt increases contractility, with 6.6-foldincreases in SERCA2a expression47, whereas Pim-1–deficient miceshowed decreases in SERCA2a and NCX levels. In contrast, myocardialoverexpression of Pim-1 increased SERCA2a levels without elevatingNCX expression, suggesting that hearts overexpressing Pim-1 may beresistant to TAC-induced hypertrophy through SERCA2a-enhancedcontractility. Increased SERCA2a may also help blunt the detrimentalcalcium overload of mitochondria that occurs in pathological condi-tions characterized by high diastolic calcium levels. Thus, Pim-1 hasan essential role in calcium homeostasis to maintain function aftercardiac injury.

Our results indicate that Pim-1 is a potent mediator of cardiopro-tection downstream of Akt signaling. Its cardioprotective effects,together with its heightened expression in both postnatal or juvenilemyocardium and pathologically challenged hearts, implicate Pim-1 inthe promotion of phenotypic characteristics typically associated with ayouthful myocardium. Indeed, cytokine expression in neonatal myo-cardium shares marked similarities with that in Akt-nuc transgenichearts39. Beneficial effects previously ascribed to Akt-nuc6,12,48 maydepend in part upon induction of Pim-1 because of similar substratespecificity shared by Pim-1 and Akt16. Also, as the widely used PI3Kinhibitor LY294002 binds to and inhibits Pim-1 activity49, previousstudies involving LY294002 require reinterpretation in the context ofAkt-dependent Pim-1 signaling. Future studies will elucidate the roleof Pim-1 in the Akt pathway and its effects on development, protec-tion and hypertrophy of the myocardium.

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METHODSNeonatal rat cardiomyocyte cultures infections and treatments. We prepared

NRCM cultures as described previously6. Additional details are provided in the

Supplementary Methods online.

Quantitative RT-PCR. We extracted RNA from all samples using Trizol

(Invitrogen) per the manufacturer’s protocol. We generated cDNA and carried

out real-time PCR using the cDNA preparation kit and SYBR real-time PCR

(Applied Biosystems) according to the manufacturer’s protocol. We calculated

differences using the DDC(T) method. A full list of primers is provided in the

Supplementary Methods.

Sample preparation and immunoblotting. We prepared NRCMs, whole

mouse heart and human heart lysates as described previously40. We performed

immunoblotting as described previously50. Additional details are provided in

the Supplementary Methods.

Myocardial infarction and cardiac hemodynamics. We performed myocardial

infarction and cardiac hemodynamics experiments under ketamine-aceproma-

zine anesthesia, and induced MI in male NTG, Pim-KO and Pim-WT

mice through permanent occlusion of the left anterior coronary artery. We

anesthetized mice using chloral hydrate (400 mg/kg body weight, intraperito-

neal) and cannulated the right carotid artery with a microtip pressure

transducer (SPR-671, Millar Instruments, or FT111B, Scisense) connected to

an A/D converter (iWorx 214or FV892A, Scisense) for data collection. We

advanced the catheter into the left ventricular chamber for the evaluation of

pressures and positive and negative dP/dt in closed chest48. After hemo-

dynamic measurements, we arrested hearts in diastole and perfused them with

phosphate-buffered formalin48.

Myocyte contractility and Ca2+ transients. We placed isolated myocytes

obtained from NTG, Pim-KO and Pim-WT hearts in a bath on the

stage of an inverted microscope (Axiovert, Zeiss) for contractility and

Ca2+ transient measurements. We conducted experiments at room temper-

ature (20–251C) as previously described48. Additional details are provided in

Supplementary Methods.

Doxorubicin and deoxyglucose induction of apoptosis. We treated NRCMs

with 1 mM doxorubicin or 1 mM deoxyglucose for 16 h then labeled for

TUNEL using the in situ Cell Death Detection Kit, TMR red (Roche Applied

Science), according to the manufacturer’s instructions.

Animal and human subjects. We performed all animal procedures

under protocols approved by the Institutional Animal Care and Use Commit-

tees at San Diego State University (SDSU) and New York Medical College.

Informed consent for the use of human tissue samples was obtained

for all human samples and use was approved by the SDSU Institutional

Review Board.

Statistical analysis. Statistical analysis was performed using Student’s t-test,

and ANOVA as appropriate, with Tukey or Bonferroni post hoc tests. P values

o0.05 were considered significant.

Note: Supplementary information is available on the Nature Medicine website.

ACKNOWLEDGMENTSThis work was supported by US National Institutes of Health grants(5R01HL067245, 1R01HL091102 and 1P01HL085577) to M.A.S. and a USNational Heart, Lung, and Blood Institute grant 1P01AG023071 to P.A. J.A.M.and N.G. are Fellows of the Rees-Stealy Research Foundation and the San DiegoState University Heart Institute. We appreciate the contribution of P. Bonine foroutstanding administrative assistance.

AUTHOR CONTRIBUTIONSJ.A.M. planned and performed experiments and wrote the manuscript; M.R. andY.M. planned and performed experiments; J.F., C.C., G.E., F.D., M.A., R.A. andS.S. performed experiments; G.N.E. and W.W. performed surgeries; K.F., J.J.M.,C.C.G., A.L. and J.K. performed experiments and advised on the experimentalapproach; N.M. provided Pim1 cDNAs and advised on the technical proposal;A.B. provided Pim-KO animals; R.M.B. and S.R.H. provided human samples and

advice; E.M.S. provided technical advice and advice on the experimentalapproach; P.A. provided advice on the experimental approach, rewriting andediting, and laboratory resources for experiments; M.A.S. supervised allexperimental procedures and edited and composed the manuscript.

Published online at http://www.nature.com/naturemedicine

Reprints and permissions information is available online at http://npg.nature.com/

reprintsandpermissions

1. Wang, Z. et al. Pim-1: a serine/threonine kinase with a role in cell survival, proliferation,differentiation and tumorigenesis. J. Vet. Sci. 2, 167–179 (2001).

2. Bachmann, M. & Moroy, T. The serine/threonine kinase Pim-1. Int. J. Biochem. CellBiol. 37, 726–730 (2005).

3. Aho, T.L. et al. Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein byphosphorylating it on the Ser112 gatekeeper site. FEBS Lett. 571, 43–49 (2004).

4. Hammerman, P.S., Fox, C.J., Birnbaum, M.J. & Thompson, C.B. Pim and Aktoncogenes are independent regulators of hematopoietic cell growth and survival.Blood 105, 4477–4483 (2005).

5. Pekarsky, Y. et al. Tcl1 enhances Akt kinase activity and mediates its nucleartranslocation. Proc. Natl. Acad. Sci. USA 97, 3028–3033 (2000).

6. Shiraishi, I. et al. Nuclear targeting of Akt enhances kinase activity and survival ofcardiomyocytes. Circ. Res. 94, 884–891 (2004).

7. Krumenacker, J.S., Narang, V.S., Buckley, D.J. & Buckley, A.R. Prolactin signaling topim-1 expression: a role for phosphatidylinositol 3-kinase. J. Neuroimmunol. 113,249–259 (2001).

8. Krishnan, N., Pan, H., Buckley, D.J. & Buckley, A. Prolactin-regulated pim-1 transcrip-tion: identification of critical promoter elements and Akt signaling. Endocrine 20,123–130 (2003).

9. Koike, N., Maita, H., Taira, T., Ariga, H. & Iguchi-Ariga, S.M. Identification ofheterochromatin protein 1 (HP1) as a phosphorylation target by Pim-1 kinase andthe effect of phosphorylation on the transcriptional repression function of HP1(1).FEBS Lett. 467, 17–21 (2000).

10. Sussman, M.A. et al. Myofibril degeneration caused by tropomodulin overexpressionleads to dilated cardiomyopathy in juvenile mice. J. Clin. Invest. 101, 51–61 (1998).

11. Bhattacharya, N. et al. Pim-1 associates with protein complexes necessary for mitosis.Chromosoma 111, 80–95 (2002).

12. Camper-Kirby, D. et al. Myocardial Akt activation and gender: increased nuclearactivity in females versus males. Circ. Res. 88, 1020–1027 (2001).

13. Domen, J., van der Lugt, N.M., Laird, P.W., Saris, C.J. & Berns, A. Analysis of Pim-1function in mutant mice. Leukemia 7(Suppl. 2), S108–S112 (1993).

14. Konietzko, U. et al. Pim kinase expression is induced by LTP stimulation and requiredfor the consolidation of enduring LTP. EMBO J. 18, 3359–3369 (1999).

15. Domen, J. et al. Pim-1 levels determine the size of early B lymphoid compartments inbone marrow. J. Exp. Med. 178, 1665–1673 (1993).

16. Bullock, A.N., Debreczeni, J., Amos, A., Knapp, S. & Turk, B.E. Structure andsubstrate specificity of the Pim-1 kinase. J. Biol. Chem. 280, 41675–41682 (2005).

17. Palaty, C.K. et al. Identification of the autophosphorylation sites of the Xenopus laevisPim-1 proto-oncogene-encoded protein kinase. J. Biol. Chem. 272, 10514–10521(1997).

18. Subramaniam, A. et al. Tissue-specific regulation of the alpha-myosin heavy chaingene promoter in transgenic mice. J. Biol. Chem. 266, 24613–24620 (1991).

19. Sugden, P.H. & Clerk, A. Cellular mechanisms of cardiac hypertrophy. J. Mol. Med. 76,725–746 (1998).

20. Izumo, S., Nadal-Ginard, B. & Mahdavi, V. Protooncogene induction and reprogram-ming of cardiac gene expression produced by pressure overload. Proc. Natl. Acad. Sci.USA 85, 339–343 (1988).

21. Katakami, N. et al. Role of pim-1 in smooth muscle cell proliferation. J. Biol. Chem.279, 54742–54749 (2004).

22. Hoefnagel, J.J. et al. Distinct types of primary cutaneous large B-cell lymphomaidentified by gene expression profiling. Blood 105, 3671–3678 (2004).

23. Ionov, Y. et al. Pim-1 protein kinase is nuclear in Burkitt’s lymphoma: nuclearlocalization is necessary for its biologic effects. Anticancer Res. 23, 167–178 (2003).

24. Evans, K.E. & Fox, S.W. Interleukin-10 inhibits osteoclastogenesis by reducing NFATc1expression and preventing its translocation to the nucleus. BMC Cell Biol. 8, 4 (2007).

25. Barre, B., Avril, S. & Coqueret, O. Opposite regulation of myc and p21waf1 transcrip-tion by STAT3 proteins. J. Biol. Chem. 278, 2990–2996 (2003).

26. Poolman, R.A., Gilchrist, R. & Brooks, G. Cell cycle profiles and expressions ofp21CIP1 and P27KIP1 during myocyte development. Int. J. Cardiol. 67, 133–142(1998).

27. Phoon, C.K. et al. Embryonic heart failure in NFATc1–/– mice: novel mechanisticinsights from in utero ultrasound biomicroscopy. Circ. Res. 95, 92–99 (2004).

28. Torella, D. et al. Cardiac stem cell and myocyte aging, heart failure, and insulin-likegrowth factor-1 overexpression. Circ. Res. 94, 514–524 (2004).

29. Dupays, L. et al. Dysregulation of connexins and inactivation of NFATc1 in the cardio-vascular system of Nkx2–5 null mutants. J. Mol. Cell. Cardiol. 38, 787–798 (2005).

30. Lilly, M., Sandholm, J., Cooper, J.J., Koskinen, P.J. & Kraft, A. The PIM-1 serine kinaseprolongs survival and inhibits apoptosis-related mitochondrial dysfunction in partthrough a bcl-2-dependent pathway. Oncogene 18, 4022–4031 (1999).

31. Fujiwara, Y. et al. Inhibition of the PI3 kinase/Akt pathway enhances doxorubicin-induced apoptotic cell death in tumor cells in a p53-dependent manner. Biochem.Biophys. Res. Commun. 340, 560–566 (2006).

©20

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/nat

ure

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icin

eART ICL ES

8 ADVANCE ONLINE PUBLICATION NATURE MEDICINE

32. Wingett, D., Long, A., Kelleher, D. & Magnuson, N.S. pim-1 proto-oncogene expressionin anti-CD3-mediated T cell activation is associated with protein kinase C activationand is independent of Raf-1. J. Immunol. 156, 549–557 (1996).

33. Rahman, Z., Yoshikawa, H., Nakajima, Y. & Tasaka, K. Down-regulation of Pim-1 andBcl-2 is accompanied with apoptosis of interleukin-6-depleted mouse B-cell hybri-doma 7TD1 cells. Immunol. Lett. 75, 199–208 (2001).

34. Eiken, H.G. et al. Myocardial gene expression of leukaemia inhibitory factor, inter-leukin-6 and glycoprotein 130 in end-stage human heart failure. Eur. J. Clin. Invest.31, 389–397 (2001).

35. Khaleghpour, K., Pyronnet, S., Gingras, A.C. & Sonenberg, N. Translational home-ostasis: eukaryotic translation initiation factor 4E control of 4E-binding protein 1 andp70 S6 kinase activities. Mol. Cell. Biol. 19, 4302–4310 (1999).

36. Hacker, H. & Karin, M. Regulation and function of IKK and IKK-related kinases. Sci.STKE 2006, re13 (2006).

37. Zhu, N. et al. CD40 signaling in B cells regulates the expression of the Pim-1 kinasevia the NF-kappa B pathway. J. Immunol. 168, 744–754 (2002).

38. Krumenacker, J.S. et al. Prolactin-regulated apoptosis of Nb2 lymphoma cells: pim-1,bcl-2, and bax expression. Endocrine 9, 163–170 (1998).

39. Gude, N. et al. Akt promotes increased cardiomyocyte cycling and expansion of thecardiac progenitor cell population. Circ. Res. 99, 381–388 (2006).

40. Tsujita, Y. et al. Nuclear targeting of Akt antagonizes aspects of cardiomyocytehypertrophy. Proc. Natl. Acad. Sci. USA 103, 11946–11951 (2006).

41. Takizawa, T. et al. Transcription of the SERCA2 gene is decreased in pressure-overloaded hearts: A study using in vivo direct gene transfer into living myocardium.J. Mol. Cell. Cardiol. 31, 2167–2174 (1999).

42. Prasad, A.M. et al. Phenylephrine hypertrophy, Ca2+ -ATPase (SERCA2), and Ca2+signaling in neonatal rat cardiac myocytes. Am. J. Physiol. Cell Physiol. 292,C2269–C2275 (2007).

43. Asahi, M. et al. Cardiac-specific overexpression of sarcolipin inhibits sarco(endo)plas-mic reticulum Ca2+ ATPase (SERCA2a) activity and impairs cardiac function in mice.Proc. Natl. Acad. Sci. USA 101, 9199–9204 (2004).

44. Suarez, J. et al. Doxycycline inducible expression of SERCA2a improves calciumhandling and reverts cardiac dysfunction in pressure overload-induced cardiac hyper-trophy. Am. J. Physiol. Heart Circ. Physiol. 287, H2164–H2172 (2004).

45. Sakata, S. et al. Restoration of mechanical and energetic function in failing aortic-banded rat hearts by gene transfer of calcium cycling proteins. J. Mol. Cell. Cardiol. 42,852–861 (2007).

46. del Monte, F. et al. Abrogation of ventricular arrhythmias in a model of ischemia andreperfusion by targeting myocardial calcium cycling. Proc. Natl. Acad. Sci. USA 101,5622–5627 (2004).

47. Kim, Y.K. et al. Mechanism of enhanced cardiac function in mice with hypertrophyinduced by overexpressed Akt. J. Biol. Chem. 278, 47622–47628 (2003).

48. Rota, M. et al. Nuclear targeting of Akt enhances ventricular function and myocytecontractility. Circ. Res. 97, 1332–1341 (2005).

49. Jacobs, M.D. et al. Pim-1 ligand-bound structures reveal the mechanism of serine/threonine kinase inhibition by LY294002. J. Biol. Chem. 280, 13728–13734(2005).

50. Kato, T. et al. Atrial natriuretic peptide promotes cardiomyocyte survival by cGMP-dependent nuclear accumulation of zyxin and Akt. J. Clin. Invest. 115, 2716–2730(2005).

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