Pim-1 kinase antagonizes aspects of myocardial hypertrophy and compensation to pathological pressure overload

Abstract

Pim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge. Similarly, Pim-1 overexpression in neonatal rat cardiomyocyte cultures inhibits hypertrophy induced by endothelin-1. On the cellular level, hearts of Pim-WT mice show enhanced incorporation of BrdU into myocytes and a hypercellular phenotype compared to wild-type controls after hypertrophic challenge. In comparison, transgenic overexpression of Pim-DN leads to dilated cardiomyopathy characterized by increased apoptosis, fibrosis, and severely depressed cardiac function. Furthermore, overexpression of Pim-DN leads to reduced contractility as evidenced by reduced Ca²⁺ transient amplitude and decreased percentage of cell shortening in isolated myocytes. These data support a pivotal role for Pim-1 in modulation of hypertrophy by impacting responses on molecular, cellular, and organ levels.

The serine/threonine kinase AKT serves as a nodal regulatory point for wide-ranging responses that mediate paracrine factor production, apoptotic stimuli, mechanical stress, hormones, energy utilization, protein synthesis, proliferation, differentiation, motility, and gene transcription (to name a select few) (1–6). Influence of AKT in the myocardial context upon hypertrophic responses is well known, either as an inducer (7, 8) or inhibitor (9, 10) depending upon experimental design. Our group previously focused upon myocardial nuclear accumulation of AKT as a mediator of cell survival (10), inhibitor of hypertrophic remodeling (9), facilitator of stem cell proliferation (11), and upstream activator of Pim-1 kinase (12). The close interrelationship between AKT and Pim-1 activities has been delineated in the context of hematopoesis leading to a greater understanding of Pim-1 function (13, 14), but activities of Pim-1 in the myocardium remain obscure.

Pim-1 is a protooncogenic serine/threonine kinase and proviral integration site for Moloney murine leukemia virus (15). Original descriptions in the hematopoetic system indicate the kinase potently increases cell survival and proliferation (reviewed in ref. 15). Recently, overexpression of Pim-1 was found to protect the myocardium following infarction injury and cardiomyocytes from apoptotic challenge by increasing cell-survival signaling (12). Genetic ablation of Pim-1 results in larger infarct size and exacerbated cardiac failure following pressure-overload aortic banding (12). These properties of Pim-1 were similar to AKT-mediated effects, but the impact of Pim-1 upon hypertrophic signaling and remodeling remained to be determined. Results show Pim-1 blunts hypertrophy, consistent with a role for Pim-1 expression downstream of nuclear AKT activity.

Results

Cardiac-Specific Pim-1 Transgenesis.

Wild-type human Pim-1 (Pim-WT) and a kinase-dead mutant that functions as a dominant-negative protein (Pim-DN) (16) were fused to GFP under control of the cardiac-specific α-myosin heavy chain promoter [supporting information (SI) Fig. S1a]. PCR constructs show incorporation of the transgenes into the genome (Fig. S1b). Whole heart lysates from transgenic samples reveal a 64-kDa GFP-Pim-1 fusion protein that is recognized by both Pim-1 and GFP antibodies (Fig. S1c). Bona fide inhibitory function of the Pim-DN construct was validated using the ability of Pim-1 to activate GATA-1 transcription (N.M., unpublished data). Pim-WT phosphorylates the transcription factor GATA-1 and induces GATA-1 luciferase reporter expression in C2C12 myoblasts, with increasing titration of Pim-DN inhibiting GATA-1 activity (Fig. S1d). On the basis of previous studies that showed Pim-1 phosphorylates p21 (17), in vitro kinase assays confirmed activity of Pim-WT construct using whole heart lysates, were prepared from GFP-Pim-1-WT, GFP-Pim-1-DN transgenic mice, and nontransgenic (NTG) mice. GFP-Pim-1 proteins [WT or kinase dead (KD)] were immunoprecipitated from whole heart lysates and incubated in the presence of [γ-³²P]ATP with GST-p21 as substrates. Pim-WT overexpression phosphorylates p21 while this activity was abolished in the Pim-DN construct (Fig. S1e).

Pim-1 Inactivation Increases Cardiomyocyte Apoptosis and Fibrosis.

Hearts created with genetic deletion of Pim-1 (Pim-1 KO) exhibit increased apoptosis in myocytes relative to NTG controls but show no evidence of overt cardiomyopathic remodeling (12). In comparison, Pim-DN overexpressing mice suffer from cardiomyopathy characterized by progressive wall thinning beginning at 3–4 months of age (Fig. 1A and B, *, P < 0.05, **, P < 0.01). Heart:body weight ratio at 10 and 22 weeks after birth is also significantly increased (Fig. 1C, **, P < 0.01). Because Pim-DN overexpression induces cardiomyocyte apoptosis in vitro (12), assessment of apoptotic myocytes in the myocardium of Pim-DN animals was performed by TUNEL staining. Pim-DN animals exhibit a twofold increase in apoptotic cardiomyocytes per mm² relative to age-matched controls (1.2/mm² and 2.4/mm², respectively, Fig. 1D, **, P < 0.01) resulting in increased fibrosis and collagen deposits in the left ventricle (Fig. 1E, blue). In addition, the amount of necrosis (Fig. S2a, **, P < 0.01) was significantly increased at basal levels.

Fig. 1.

Inactivation of Pim-1 in the myocardium increases apoptosis and fibrosis. (A and B) Echocardiographic measurement of posterior (A) and anterior (B) wall dimension (PWD and AWD, respectively) in NTG (n = 5) and Pim-DN (n = 7) animals at 2-week intervals (*, P < 0.05, **, P < 0.01). (C) Heart weight to body weight ratios in NTG and Pim-DN animals at 10 and 22 weeks of age (n = 6, **, P < 0.01). (D) Histogram representing counts of TUNEL positive myocytes per mm² in 17–22-week-old NTG and Pim-DN transgenics (n = 3, **, P < 0.01). (E) Masson trichrome staining of sections from paraffin embedded NTG and Pim-DN hearts at 17 weeks of age.

Pim-DN Hearts Exhibit Depressed Cardiac Function.

Hearts of Pim-DN mice show progressive dilation from 17 weeks of age (Fig. 2A, *, P < 0.05) with depression of fractional shortening and ejection fraction (36.6 and 74.2%, respectively) by 27 weeks of age (Fig. 2B and C, *, P < 0.05, **, P < 0.01) by echocardiographic analyses. Morphometric analysis on both NTG and Pim-DN hearts confirmed that Pim-DN hearts were significantly dilated (Table S1). In vivo hemodynamic assessments verified impaired hemodynamics with diminished ±dP/dt, increased left ventricular end diastolic pressure (LVEDP), and decreased left ventricular developed pressure (LVDP) (Fig. 2 D–F, **, P < 0.01). Mechanistically, Pim-DN myocytes displayed reduced Ca²⁺ transient amplitude with decreased percentage of cell shortening with respect to NTG myocytes (Fig. 2G). Additionally, the time constant (τ) of the Ca²⁺ transient decay was larger in Pim-DN myocytes (Fig. 2G). These results indicate that depressed contractile function of Pim-DN myocytes is mediated in part by a decline in Ca²⁺ release from the sarcoplasmic reticulum together with a slower reuptake. Thus inactivation of Pim-1 by Pim-DN in the myocardium has a negative effect on cardiac function.

Fig. 2.

Pim-DN hearts exhibit dilation and depressed function. (A–C) Echocardiographic assessment of end-diastolic diameter (EDD, A), percentage of fractional shortening (FS, B), and ejection fraction (EF, C) at 27 weeks of age in NTG (n = 5) and Pim-DN (n = 7) hearts (*, P < 0.05, **, P < 0.01). (D–F) In vivo hemodynamic measurements in 27-week-old NTG and Pim-DN animals (n = 4). Positive and negative change in pressure/change in time (±dP/dt, D), left ventricular developed pressure (LVDP, E), and left ventricular end diastolic pressure (LVEDP, F) are represented (*, P < 0.05, **, P < 0.01). (G) Calcium handling and contractile function of isolated myocytes from NTG and Pim-DN animals (*, P < 0.05).

Overexpression of Pim-1 Inhibits Hypertrophy in Vitro.

Induction of Pim-1 in the damaged myocardium is thought to be a protective survival response (12) occurring in cardiomyocytes in the border zone where Pim-1 colocalizes to cells expressing atrial natriuretic peptide (ANP, Fig. 3A). ANP is both antihypertrophic and cardioprotective (24), the coincidence of these proteins prompted assessment of the role that Pim-1 accumulation plays in mitigation of hypertrophic signaling.

Fig. 3.

Pim-1 overexpression protects cardiomyocytes from endothelin-1-induced hypertrophy. (A) Pim-1 and ANP colocalization in the border zone of pathologically challenged wild-type mice. Arrows indicate cells of interest where Pim-1, ANP, and tropomyosin are represented in green, red, and blue, respectively. (B) Individual cell surface area measurements from uninfected control, EGFP, and Pim-WT-infected neonatal rat cardiomyocyte cultures treated and untreated with endothelin-1 (ET-1) (n = 4, *, P < 0.05, **, P < 0.01). (C) qRT–PCR of molecular markers of hypertrophy in neonatal rat cardiomyocyte cultures infected with EGFP or Pim-WT. Fold change compared to uninfected control represented (n = 6, **, P < 0.01 vs. EGFP infected). (D) Levels of hypertrophic molecular marker mRNAs following infection and treatment with ET-1 (##, P < 0.01 vs. EGFP infected).

Pim-WT overexpression upon cardiomyocyte hypertrophy was examined using neonatal rat cardiomyocytes (NRCMs) infected with adenoviruses encoding EGFP-Pim-WT or EGFP protein followed by stimulation with endothelin-1 (ET-1) for 24 h. Pim-WT overexpression inhibits ET-1-induced hypertrophy (Fig. 3B, *, P < 0.05, **, P < 0.01) as assessed by cell surface area measurements relative to the increase in cell size seen in control and EGFP-infected myocytes treated with ET-1. Molecular profiling of the hypertrophic signature of untreated cultures shows that Pim-WT expression decreases mRNA levels for ANP by 60.6% and B-type natriuretic peptide (BNP) by 39.8% while increasing α-skeletal actin levels 89% compared to EGFP-infected controls (Fig. 3C, **, P < 0.01 vs. EGFP). However, upon treatment with ET-1, Pim-WT cultures exhibit a 2.5-fold increase in ANP levels and 10.2-fold decrease in β-myosin heavy chain levels vs. ET-1-treated EGFP controls (Fig. 3D, P < 0.01 vs. EGFP control). Unfortunately, cultured cardiomyocytes overexpressing Pim-DN protein show diminished viability after the necessary time course to infect and treat with ET-1 as evidenced by high levels of TUNEL positive cells. As cardiomyocytes overexpressing Pim-DN protein round up and detach, their morphology is changed, preventing an accurate assessment of cell size (12). However, the effect of Pim-DN on hypertrophy can be seen using the specific Pim-1 activity inhibitor quercetagetin. Pim-1-expressing NRCM cultures were treated for 1 h with or without 10 nM quercetagetin before a 48-h incubation with ET-1 and cell size assessment. Cells treated with quercetagetin and ET-1 had significantly larger surface compared to ET-1-stimulated cells where Pim-1 activity was not blunted by inhibitor (Fig. S3). These results support an antihypertrophic role when Pim-1 is overexpressed, albeit at levels well above normal physiological induction in this cell culture system.

Pim-1 Overexpression Inhibits Remodeling Induced by Pressure-Overload Hypertrophy.

Consequences of Pim-1 overexpression upon hypertrophy in vivo was assessed with Pim-WT mice subjected to transaortic constriction (TAC) to induce pressure overload relative to age and gender matched NTG controls. Results show that TAC of control NTG hearts prompts remodeling at 2 weeks after challenge evidenced by anterior and posterior wall thickening (Fig. 4A and B, *P < 0.05, **, P < 0.01). In comparison, Pim-WT animals do not show significant increases in wall thickness for up to 14 weeks after challenge (Fig. 4A and B). Similarly, NTG controls show left ventricular chamber enlargement measured by end diastolic diameter (EDD) within 8 weeks after banding, and end systolic diameter (ESD) increases significantly within 4 weeks. Neither EDD nor ESD parameters show significant changes in Pim-WT transgenics throughout the same time period (Fig. 4C and D, **, P < 0.01 vs. sham, *, P < 0.01 vs. Pim-WT TAC). Furthermore, NTG controls show marked decreases in both fractional shortening (FS) and ejection fraction (EF) after challenge, while myocardial function is maintained in Pim-WT hearts (Fig. 4E and F, **, P < 0.01 vs. Pim-WT TAC). Interestingly, although decreases in cardiac function are seen in NTG animals, Pim-1 protein is modestly elevated in response to pressure overload during early hypertrophy and progression to heart failure. Endogenous levels of Pim-1 expression increase early during adaptive hypertrophy and decline shortly thereafter (Fig. S3b) while during late phase hypertrophy (9 weeks post-TAC), Pim-1 appears localized to nuclei within vasculature (Fig. S3c). These observations support the notion that Pim-1 is induced in response to stress.

Fig. 4.

Pim-WT transgenic animals are resistant to pressure-overload-induced hypertrophy. (A–F) Line graphs representing weekly echocardiographic assessment of NTG and Pim-WT sham and TAC-banded hearts for anterior wall dimension (AWD, A), posterior wall dimension (PWD, B), end-diastolic dimension (EDD, C), end-systolic dimension (ESD, D), percentage of fractional shortening (FS, E), and ejection fraction (EF, F) (NTG sham n = 6, Pim-WT sham n = 6, NTG TAC n = 9, Pim-WT TAC n = 9; *, P < 0.05, **, P < 0.01).

Pim-WT Hearts Are Resistant to TAC-Induced Hypertrophy.

NTG mice exhibit significant increases in heart size and succumb faster compared to Pim-WT transgenic mice following TAC challenge (Fig. 5A and B, **, P < 0.01, **, P < 0.01). Molecular mRNA markers of hypertrophy including ANP, BNP, α-skeletal actin (α-SKA), β-myosin heavy chain (β-MHC), and c-fos are significantly increased in NTG TAC-challenged hearts compared to shams (Fig. 5C, *, P < 0.05, **, P < 0.01). In comparison, molecular hypertrophic markers are not significantly increased in hearts of Pim-WT mice subjected to TAC challenge, although Pim-WT hearts do express more c-fos mRNA under basal conditions (Fig. 5C, ψP < 0.01). Quantitation of apoptotic myocytes by TUNEL labeling reveals a 3.72-fold increase in NTG TAC-challenged hearts compared to shams (3.2/mm² and 0.86/mm², respectively), whereas Pim-WT animals exhibit no significant increase in TUNEL positive cells (1.31/mm² vs. 1.05/mm²) (Fig. 5D, **, P < 0.01). Consistent with improved myocardial viability, Pim-WT TAC-challenged hearts show decreased perivascular fibrosis (Fig. 5E) and decreased necrosis (Fig. 5F) relative to NTG TAC-challenged counterparts. Additionally, Pim-WT TAC-banded hearts have significantly increased levels of antiapoptotic proteins including Bcl-xl, Bcl-2, and increased phosphorylation of BAD relative to NTG counterparts (Fig. 5G). These data support that protection afforded by Pim-1 overexpression is due in part to increased survival signaling.

Fig. 5.

Function is maintained in Pim-WT animals after TAC. (A) Kaplan–Meier survival curve of NTG and Pim-WT animals following TAC or sham operation (NTG sham n = 6, Pim-WT sham n = 5, NTG TAC n = 8, Pim-WT TAC n = 8; **, P < 0.01 vs. Pim-WT TAC). (B) Histogram depicting heart weight/body weight ratios of NTG and Pim-WT sham and TAC hearts 4 weeks after the procedure (NTG sham n = 3, NTG TAC n = 4, Pim-WT sham n = 4, Pim-WT TAC n = 6; **, P < 0.01). (C) qRT–PCR analysis of NTG and Pim-WT hearts 4 weeks after TAC banding for molecular markers of hypertrophy (n = 4, *, P < 0.05, **, P < 0.01 vs. sham; ψ, P < 0.01 vs. NTG sham). (D) Histogram representing the number of TUNEL positive myocytes per mm² in NTG and Pim-WT hearts after banding (n = 3, **, P < 0.01). (E) Masson trichrome staining of NTG and Pim-WT paraffin sections 4 weeks after banding and quantitation of collagen. (F) Quantitation of necrotic nuclei in NTG and Pim-WT animals pre- and post-TAC banding **, P < 0.01. (G) Analysis of antiapoptotic protein expression in NTG and Pim-WT whole heart lysates post-TAC. Quantitation of results for Bcl-xl, Bcl-2, and phosphorylated Bad (pBAD) levels in NTG and Pim-WT hearts. GAPDH was used as a loading control. n = 3, *, P < 0.05, **, P < 0.01 vs. GAPDH control. (G) Quantitation of Pim-WT hearts compared to NTG hearts post-TAC banding.

Pim-WT Hearts Exhibit Increased Contractile Function in Response to TAC Banding.

Decreased fibrosis is present in Pim-WT hearts after TAC banding (Fig. 5E), suggesting Pim-1 overexpression preserves contractile function. Actions of Pim-1 overexpression upon cardiac contractility were examined using Pim-WT and NTG controls assessed by in vivo hemodynamic measurements at 4 weeks and 10 weeks after TAC challenge. While contractile function is depressed in NTG TAC-challenged hearts at both time points, Pim-WT hearts possess better function after TAC challenge with slight decreases in +dP/dt and no significant change in −dP/dt compared to sham-operated NTG controls (Fig. 6A, *, P < 0.05 and **, P < 0.01 vs. sham, *, P < 0.05 and **, P < 0.01 vs. NTG TAC). Comparison of 4-week and 10-week dP/dt assessments show significant decreases in function for both NTG and Pim-WT TAC-challenged hearts, although performance of Pim-WT TAC-challenged hearts is relatively improved (Fig. 6A **, P < 0.01 vs. 4 weeks). Measurements reveal increases in left ventricular developed pressure and end diastolic pressure in NTG hearts 4 and 10 weeks after TAC, but Pim-WT hearts show relative preservation of LVDP (Fig. 6B, Pim-WT 19.75% increase, **, P < 0.01) and no change in LVEDP (Fig. 6C **, P < 0.01, **, P < 0.01 vs. NTG TAC). Hemodynamic function reflected in ±dP/dt and LVDP is improved in Pim-WT hearts compared to NTG at 4- and 10-week time points (Fig. 6A and B, $P < 0.05, $$P < 0.01). The mechanistic basis for preservation of contractile function in Pim-WT hearts may rest with the cellular response in TAC-challenged animals. NTG and Pim-WT groups injected with BrdU for 10 days were used to assess stimulation of DNA synthesis and potential cellular proliferation after TAC challenge. Pim-WT hearts possess 67% more BrdU+ myocytes relative to NTG controls after TAC challenge (Fig. S4a, *, P < 0.05). The majority of BrdU+ cells in Pim-WT hearts post-TAC are diploid (Fig. S4b) supporting the premise that increases in BrdU+ cells stemmed from new myocyte formation and not enhanced DNA synthesis in preexisting cells. We now show that in addition to increased SERCA2a levels (12), Pim-WT hearts also show increased levels of phosphorylated phospholamban (PLB) while Pim-DN animals show significant decreases in phospho-PLB compared to NTG control animals (Fig. S5). Interestingly, the ratio of phospho-PLB over total PLB in Pim-WT animals is similar to the ratio observed in NTG animals, while in Pim-DN animals this ratio is significantly decreased. These results suggest that in the face of decreased cardiac function, overexpression of Pim-1 allows the heart to maintain function through increased contractility through elevation of SERCA2a (18) although phosphorylated PLB does not seem to play a major role in this protection.

Fig. 6.

Pim-1 enhances cardiac function. (A–C) In vivo hemodynamic assessment of NTG and Pim-WT hearts 4 and 10 weeks (black and gray bars, respectively) after sham or TAC operation (14 and 20 weeks of age, respectively). ±dP/dt measurements (A), left ventricular developed pressure (LVDP, B), left ventricular end diastolic pressure (LVEDP, C). For 4-week animals NTG sham n = 4, NTG TAC n = 3, Pim-WT sham n = 4, Pim-WT TAC n = 4. For 10-week animals NTG sham n = 5, NTG TAC n = 10, Pim-WT sham n = 14, Pim-WT TAC n = 7 (*, P < 0.05, **, P < 0.01 vs. sham, ##, P < 0.01 vs. 4-week TAC, $, P < 0.05, $$, P < 0.01 vs. NTG TAC, ψ, P < 0.05, ψψ, P < 0.01 vs. NTG sham).

Pim-1 Increases Myocardial Cellularity.

The volume and cellularity of myocytes resulting from myocardial Pim-1 overexpression was assessed by quantitation of myocyte volume distribution. Results show Pim-WT hearts possess an increased percentage of small myocytes (Fig. S6d) relative to NTG controls that is also reflected in decreased average myocyte size in these hearts (Fig. S6 a and b, **, P < 0.01), resulting in a hypercellular phenotype of ≈33% more myocytes in Pim-WT compared to NTG (Fig. S6c). Additionally, isolated Pim-DN myocytes were 11% larger than NTG myocytes (Fig. S7) indicating an inverse effect wherein impaired Pim-1 activity prompts formation of larger myocytes in the transgenic heart.

Discussion

The initial identification and characterization of myocardial Pim-1 demonstrated this kinase is regulated by nuclear AKT accumulation and is cardioprotective in response to infarction challenge (12). Because nuclear Akt also possesses antihypertrophic properties (9), Pim-1 was circumstantially associated with possible inhibition of hypertrophy. Inhibition of hypertrophy in vivo and in vitro indicates Pim-1 contributes to Akt-mediated blunting of hypertrophic remodeling. Pim-1 is upregulated in localized regions close to acute injury or damage and is not increased throughout the myocardium until initiation of transit to end-stage failure. Thus, Pim-1 likely serves as a survival and protective response to blunt maladaptive hypertrophic remodeling in early phases of reactive signaling. In comparison, Pim-1 elevation occurring in late-stage decompensation probably represents a terminal effort to preserve function, although beneficial effects are overridden by the sequelae of end-stage failure. The differential expression of endogenous Pim-1 during transition from adaptive to maladaptive hypertrophy possibly represents a mechanism by which Pim-1 is partially protective. Targets of Pim-1 interaction contributing to protection are likely to include mediators of mitochondrial protection (12) and recent published findings regarding induction and stabilization of c-myc through Pim-1 (19), with ongoing studies exploring these mechanisms to determine their participation in the Pim-1 response.

Similar to our findings with nuclear AKT (9), Pim-1 overexpression in cardiomyocytes inhibits ET-1-induced hypertrophy in vitro (Fig. 3) (Fig. S3a) and in vivo (Fig. 4). In addition, overexpression of Pim-1 increases ANP transcript levels following endothelin-1 administration while reducing β-MHC transcription and inhibiting increases in other molecular markers of hypertrophy (Fig. 3D), reminiscent of effects observed using nuclear AKT (9). However, upon further analysis, ANP protein levels are significantly decreased in both Pim-WT hearts and Pim-WT-infected NRCMs. We therefore believe that although ANP is induced in an acute area surrounding the region of insult (Fig. 3A), on a global organ level ANP is not induced and therefore not a primary downstream target that mediates the Pim-1 protective response. Additionally, we investigated potential downstream targets of Pim-1 within the MAP kinase pathway, as both JNK and ERK have been proposed to be cardioprotective. Although in vitro data do not seem to suggest specific involvement of the MAPK pathway (Fig. S8b), in vivo data indicates that both downstream targets JNK and ERK are significantly affected by Pim-1 overexpression, whereby the ratio of phosphorylated to total MAPK expression is significantly increased compared to NTG controls post-TAC banding (Fig. S8a). Collectively these results support the antihypertrophic actions of Pim-1, although overexpression models are not truly representative of physiological induction with respect to either localization or level.

Nuclear AKT delayed but did not overcome compensatory remodeling after TAC challenge (9), but Pim-WT transgenic hearts exhibit persistent blunting of myocardial hypertrophy (Fig. 4) without increases in apoptosis, changes in hypertrophic signaling markers (Fig. 5), or deterioration of function (Fig. 6). In addition, Pim-WT transgenic hearts perform significantly better functionally than NTG counterparts at 14 and 20 weeks of age (Fig. 6). A potential basis for this remarkable resiliency to pressure overload is in part related to Pim-1-mediated induction of sarco/endoplasmic reticulum Ca⁺²ATP-ase 2a (SERCA2a) (12) and PLB expression. Studies have shown that relatively minor but significant 45% increases in SERCA2a expression postaortic banding are sufficient to recover function following pressure overload (20). Furthermore, transgenic mice overexpressing constitutively activated AKT in the myocardium exhibit increased contractility because of a 6.6-fold increase in SERCA2a (21). In comparison, Pim-WT hearts exhibit a 4.89-fold increase in SERCA2a (12). Phosphorylation of phospholamban is significantly increased in hearts from Pim-WT mice relative to NTG controls, while Pim-DN and Pim-KO animals have significantly reduced levels (Fig. S5). Decreases in phospholamban accompany heart failure and overexpression of phosphorylated phospholamban improves cardiac function through activation of SERCA2a and decreases fibrosis in pathologically challenged myocardium (22). In addition, phospho-PLB:PLB ratio in Pim-DN animals is significantly decreased compared to NTG and Pim-WT animals. Thus, as Pim-DN negative animals have impaired contractile function (Fig. 2G), this decreased ratio of phospho-PLB to total PLB may ultimately negatively affect SERCA activity and represents a potential mechanism for the depressed cardiac phenotype observed in the Pim-DN animals. Furthermore, Pim-DN overexpression leads to impaired contractile function and altered calcium handling (Fig. 2G), whereas overexpression of Pim-1 leads to hypercontractile hearts with hyperdynamic calcium handling in response to pathological challenge (12). Thus, increased SERCA2a expression in Pim-WT animals provides a plausible explanation for the preservation of cardiac function observed in response to TAC challenge, although phospho-PLB does not appear to play a major role (Fig. S5).

Overexpression of Pim-DN in the myocardium increases myocyte apoptosis (Fig. 1D) with dilated cardiomyopathy, whereas genetic deletion of Pim-1 also results in increased apoptosis although dilated cardiomyopathy is not observed (12). The cardiomyopathic consequences of Pim-DN overexpression suggest this kinase is critical to cardiomyocyte viability, as compensatory upregulation of Pim-2 occurring in the Pim-1 knockout line could help account for the lack of cardiomyopathic changes (12). These findings are also consistent with our previous study that shows the dominant-negative form of Pim-1 induces PARP and caspase-3 cleavage, increasing cardiomyocyte apoptosis in vitro (12). Furthermore for the effect on hypertrophy, Pim-DN seems to be able to mount a hypertrophic response, evidenced by increased anterior wall thickness 1 week after TAC challenge (data not shown). Unfortunately, as we have previously reported (12) Pim-KO animals preclude an accurate assessment of the antihypertrophic response elicited by Pim-1 because of the potential compensatory actions of Pim-2 in this system. At present dissecting the effect of Pim-1 inhibition in the in vivo context will require an inducible inhibition system because of the potential confounding effects of Pim-DN expression.

Overexpression of Pim-1 in the pathologically challenged myocardium results in numerous salutary effects including decreased apoptosis (Fig. 5D), increased expression of antiapoptotic proteins (Fig. 5G), and decreased fibrosis (Fig. 5E) and necrosis (Fig. S2). Pim-1 also increases the percentage of small myocytes and an overall increase in the number of myocytes constituting the myocardium (Fig. S6c). Taken together, these data point to another potential mechanism to account for increased capacity to withstand TAC challenge by virtue of increased cell numbers of small cells and decreased cell death. These results were also previously postulated for the effect of nuclear AKT in the myocardium, which increases the resident cardiac progenitor pool, induces proproliferative cytokine expression, and increases the number of myocytes in the heart (11) (9). Future studies will more thoroughly elucidate the connection between Pim-1 and cardiac stem cell pools to determine whether Pim-1, like AKT, increases the resident pools of these cells.

On the basis of the collective results thus far, Pim-1 can be considered a potent cardioprotective and antihypertrophic molecule in the myocardium with effects mediated through increased cell survival, decreased myocyte size, increased myocyte number, and increased Ca⁺² reuptake. All of these properties have been previously ascribed to AKT kinase (8–11, 21, 23), so with the connection of Pim-1 as a downstream mediator of AKT activity it will be important to determine the contribution of Pim-1 to activities traditionally associated with AKT. By providing cardioprotective actions offered by AKT without undesired consequences for metabolic alterations and hypertrophic remodeling (8, 24), Pim-1 could become an important therapeutic agent in myocardial repair and prevention of cardiac failure.

Materials and Methods

Generation of Transgenic Animals and Animal Use.

Pim-WT and Pim-DN cDNA fragments (16) were subcloned into the α-MHC plasmid for transgenesis. Prior publications describe methods for TAC banding and echo (9), and HW:BW ratio determination and hemodynamics (12). Further details are provided in the online supplement. All animal studies were approved by the Institutional Animal Use and Care Committee.

Confocal Microscopy, Immunoblotting, and Assays.

GFP-Pim-1 proteins immunoprecipitated from heart lysates were used in an in vitro kinase assay with GST-p21 as substrate. For luciferase assays, C2C12 cells transfected with indicated plasmids and pGATA-Luc reporter construct were analyzed for GATA-dependent luciferase activity. Prior publications describe methods for immunofluorescence microscopy (25), immunoblotting (26), quantitative RT–PCR and TUNEL staining (9). Details are provided in SI Text.

In Vitro Cell Culture and Analyses.

Neonatal rat cardiomyocyte cultures were prepared as described previously (10). Adult myocyte isolation, volume calculations, cell shortening, and Ca²⁺ transient experiments were performed as previously described (12, 22, 27). Additional details are provided in SI Text.

Statistical Analysis.

Error bars are represented as plus and minus standard error of the mean (SEM), except where indicated. Statistical analysis was performed using Student's t test and ANOVA with Tukey's posthoc. P < 0.05 considered significant.