Cardiac Progenitor Cell Cycling Stimulated by Pim-1 Kinase
Rationale: Cardioprotective effects of Pim-1 kinase have been previously reported but the underlying mechanistic basis may involve a combination of cellular and molecular mechanisms that remain unresolved. The elucidation of the mechanistic basis for Pim-1 mediated cardioprotection provides important insights for designing therapeutic interventional strategies to treat heart disease.
Objective: Effects of cardiac-specific Pim-1 kinase expression on the cardiac progenitor cell (CPC) population were examined to determine whether Pim-1 mediates beneficial effects through augmenting CPC activity.
Methods and Results: Transgenic mice created with cardiac-specific Pim-1 overexpression (Pim-wt) exhibit enhanced Pim-1 expression in both cardiomyocytes and CPCs, both of which show increased proliferative activity assessed using 5-bromodeoxyuridine (BrdU), Ki-67, and c-Myc relative to nontransgenic controls. However, the total number of CPCs was not increased in the Pim-wt hearts during normal postnatal growth or after infarction challenge. These results suggest that Pim-1 overexpression leads to asymmetric division resulting in maintenance of the CPC population. Localization and quantitation of cell fate determinants Numb and α-adaptin by confocal microscopy were used to assess frequency of asymmetric division in the CPC population. Polarization of Numb in mitotic phospho-histone positive cells demonstrates asymmetric division in 65% of the CPC population in hearts of Pim-wt mice versus 26% in nontransgenic hearts after infarction challenge. Similarly, Pim-wt hearts had fewer cells with uniform α-adaptin staining indicative of symmetrically dividing CPCs, with 36% of the CPCs versus 73% in nontransgenic sections.
Conclusions: These findings define a mechanistic basis for enhanced myocardial regeneration in transgenic mice overexpressing Pim-1 kinase.
Discovery of cycling progenitor cells residing in the myocardium has challenged the paradigm that the heart is a postmitotic organ. Instead, present research indicates that the heart is a self-renewing organ comprised primarily of terminally differentiated myocytes, vascular smooth muscle cells, endothelial cells together with cardiac progenitor cells (CPCs).1 These CPCs are c-kit+, have the ability to self renew, and can differentiate into all 3 cardiac cell lineage.2–4⇓⇓ The stem cell antigen c-kit has been used to identify several types of adult stem cells including those residing in cardiac, hematopoietic, liver, brain, and pancreatic tissues.5,6⇓ The primary characteristic of commitment to the cardiogenic lineage distinguishes c-kit+ CPCs from other stem cell types.1,4⇓ CPCs reside within the myocardium in specialized niche structures where they self renew and produce daughter progeny that supply the heart with new myocytes and vessels,7 allowing for myocardial regeneration.8
Increased generation of new cardiomyocytes in postnatal development can be stimulated by cardiac-specific expression of proliferative factors or signaling proteins leading to myocardial hyperplasia.9–11⇓⇓ Specifically, cardiac specific nuclear-targeted Akt expression leads to increased cycling and ultimately an increase in the CPC population,11 which may contribute to the cardioprotective effects seen when Akt is overexpressed.12–15⇓⇓⇓ Akt is a nodal signaling kinase that influences multiple cellular processes including metabolism, cycling, cell growth and apoptosis.16–20⇓⇓⇓⇓ Akt exerts cardioprotective effects in concert with another serine/threonine kinase called Pim-1 that lies downstream of nuclear Akt accumulation. Pim-1 expression inhibits pathological damage and remodeling resulting from myocardial infarction (MI)21 and pressure overload–induced hypertrophy.9 Antiapoptotic effects of Pim-1 activity in the myocardium are linked to phosphorylation of Bad and inhibition of caspase cleavage.21 Transgenic cardiac-specific overexpression of Pim-1, like nuclear-targeted Akt, produces postnatal myocardial hyperplasia consistent with increased cardiomyocyte or CPC cycling. In neoplastic cell types, Pim-1 activity is associated with enhanced cellular proliferation. Similarly, Pim-1 induction leads to enhanced proliferation of hematopoietic stem/ progenitor cells downstream of STAT5 activation.22 Pim-1 exerts proproliferative effects through phosphorylation of p21 on Thr145,23 stabilizing c-Myc24 and increasing MDM2-mediated degradation of p53 via the proteasome.25 Subsequent loss of p53 leads to hyperproliferative phenotypes in several cancer cell lines.26–28⇓⇓ Collectively these studies point toward an important influence of Pim-1 expression to increase cell cycling, but the role of Pim-1 on CPC proliferation has yet to be elucidated. To determine whether Pim-1 enhances CPC cycling, control nontransgenic mice (NTG) were compared to 3 genetically engineered mouse lines with altered Pim-1 activity: cardiac-specific overexpression of Pim-1 (Pim-wt), a kinase dead form of Pim-1 (Pim-DN),9 and Pim-1–null mice (Pim-KO). Results indicate that Pim-1 overexpression leads to substantial increases in CPC cycling during development and after MI without an increase in overall myocardial CPC population number. Reconciliation of this apparent paradox rests in the observation of increased asymmetric division in cycling CPCs found in hearts overexpressing Pim-1. Thus we find that Pim-1 overexpression leads to increased CPC cycling, which ultimately leads to a preservation of the stem cell pool.
Construction of Pim-KO and Pim Transgenic Animals and Animal Use
Creation and characterization of Pim-1 transgenic lines has been described previously9,21⇓ with further details in the Online Data Supplement (available at http://circres.ahajournals.org). Murine surgical procedures were performed as previously described.29 All animal studies were approved by the Institutional Animal Use and Care Committee.
Immunohistochemistry, Confocal Microscopy, and Immunoblot Analysis
Formalin fixed, paraffin embedded hearts were used for immunohistochemistry as previously described11,30⇓ with details provided in the Online Data Supplement. Immunoblot methods with antibody information are detailed in the Online Data Supplement.
Adult Cardiac Progenitor Cell Isolation, Trypan Blue Exclusion Assay, CyQuant Proliferation Assay, and MTT Assay
Adult cardiac progenitor cells were isolated from nontransgenic hearts between the ages of 8 and 12 weeks as described previously.2 Trypan blue exclusion assay used a 50% trypan blue solution with hemocytometer determination. CyQuant proliferation assay (Invitrogen no. C35007) was performed as per manufacturer instructions. MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay was performed as previously described,31 with additional details in the Online Data Supplement.
All data are expressed as means±SEM. Statistical analysis was performed using Student’s t test and ANOVA with Tukey’s post hoc as appropriate. Probability values of <0.05 were considered significant.
Pim-1 Is Expressed in CPCs
Consistent with reports of Pim-1 activity in stem cells,32–34⇓⇓ confocal microscopy performed on myocardial sections of neonatal (2 day) nontransgenic (NTG) hearts shows Pim-1 colocalization with the progenitor cell marker c-kit (Figure 1A). Consequences of increased myocardial Pim-1 expression on the CPC pool was assessed by confocal microscopy of tissue sections from transgenic mice overexpressing Pim-1 and green fluorescent protein (GFP) downstream of the α-myosin heavy chain promoter (Pim-wt).21 Immunolabeling for c-kit and GFP in myocardial sections from Pim-wt hearts (Figure 1B) show colocalization of c-kit and GFP in both 2-day-old (61% colocalization) and 2-week-old (51% colocalization) hearts consistent with α-myosin heavy chain promoter activity in CPCs.35 Pim-1 expression in CPCs is corroborated by immunoblots of CPCs isolated from NTG and Pim-wt hearts, which are immunoreactive for the transgenically encoded 34 kDa Pim-1 protein (Figure 1C). CPC lysates are negative for sarcomeric desmin, indicating no cardiomyocyte contamination. Collectively, these results indicate that c-kit+ cells of cardiac origin express Pim-1 and that expression of Pim-1 as a transgenic protein can be detected in CPCs isolated from murine lines engineered with cardiac-specific overexpression of Pim-1 protein.
Pim-1 Stimulates Cardiac Progenitor Cell Cycling In Vitro and In Vivo
Consequences of Pim-1 overexpression on DNA content, CPC viability, and metabolic activity were measured in vitro using CyQuant DNA content assay, trypan blue exclusion assay, and tetrazolium salt (MTT) colorimetric reduction assay with cultures derived from NTG or Pim-wt hearts. Pim-wt CPCs show significantly increased DNA content following culture for 1 or 5 days by CyQuant assay (4.3- and 2-fold, respectively; Figure 2A). CPC viability was assessed in cultures seeded with 20,000 cells from NTG or Pim-wt hearts followed by trypan blue exclusion assay, which reveals that the number of Pim-wt CPCs increase at a significantly greater rate than NTG (3.8-fold at day 2 and 4.4-fold at day 4; Figure 2B). Involvement of Pim-1 activity in cell division and metabolic activity of CPC cultures was confirmed by subsequent treatment with the Pim-specific inhibitor quercetagetin (10 μmol/L) leading to significantly less DNA incorporation (Figure 2C). Pim-wt CPCs also show significantly higher metabolic activity compared to NTG CPCs at 3 days (1.7-fold) after plating, and lower MTT conversion following Quercetagetin exposure (Figure 2D) indicative of enhanced proliferation. Levels of c-Myc were increased 3-fold in Pim-wt CPCs and reduced by 63% with treatment of Quercetagetin (Figure 2E), consistent with previous studies linking c-Myc stabilization and increased cycling of neoplastic cells with overexpression of Pim-1.24 Increases in phosphorylation of p21 at Thr145, a direct target of Pim-1, and increased levels of Cyclin E relative to NTG CPCs are findings consistent with enhanced proliferative signaling in Pim-wt CPCs (Online Figure I). During mitosis, nuclear mitotic apparatus protein (NuMA) organizes and tethers microtubules to the spindle poles and has been shown to interact with Pim-1.36 Therefore, the localization of NuMA was investigated; enhanced proliferative signaling in Pim-wt CPCs does not correlate to changes in NuMA localization (Online Figure II).
Cell cycling in vivo was examined in NTG and Pim-wt hearts with Ki-67, PCNA, and c-kit immunolabeling of myocardial sections. Coincident Ki67+/c-kit+ labeling is significantly increased in postnatal Pim-wt hearts at 2 days (58.1% versus 41.5%) and 2 weeks (52.4% versus 26.8%) after birth compared to NTG. (Figure 3A; *P<0.05). In comparison, cycling CPC numbers were significantly decreased in myocardial sections from Pim-DN hearts at both 2 days (64.7%) and 2 weeks (55.3%) relative to NTG samples. Significant increases in PCNA+/c-kit+ cells at 2 days and 2 weeks of age (1.7- and 1.8-fold, respectively) confirms enhanced cell cycling in Pim-wt CPCs (Figure 3B). These effects on the proportion of cycling CPCs are consistent with proproliferative effects of Pim-1 in the postnatal myocardium.
To further assess physiological CPC dynamics within the developing myocardium the number of apoptotic CPCs was determined. Figure 3C demonstrates that NTG hearts have significantly more apoptotic CPCs than Pim-wt at 2 days and 2 weeks (9.75- and 5-fold respectively). Expansion of the CPC population resulting from altered Pim-1 expression was assessed by quantitation of c-kit+ cells in the left ventricles at 2 days, 2 weeks, and 3 months of age. Indeed, the number of CPCs was significantly decreased in the Pim-KO (2.1-fold less at 2 days and 3-fold less at 2 weeks), indicating that loss of Pim-1 by genetic deletion impairs CPC production. Of note however is a significant increase in PCNA+/GATA4+ cells at 2 days in the Pim-KO hearts (2.6-fold, Online Figure III) possibly in an attempt to retain homeostasis caused by the lack of CPC cycling. Curiously, an increase in total number of c-kit+ cells versus the NTG controls was not observed in Pim-wt hearts despite evidence of increased CPC cycling (Figure 3). Thus, Pim-1 expression increases the frequency of cycling CPCs (Ki67+/c-kit+) without increasing the population of CPCs (c-kit+). The increased number of CPCs observed in the Pim-DN heart relative to NTG controls (1.7-fold; Figure 3D) may reflect increased recruitment of the CPC pool in response to cardiomyopathic effects of the Pim-DN construct.9 Clearly, these results imply Pim-1-mediated effects on the CPC pool, but straightforward interpretation of these findings is challenging because of the multifaceted nature of Pim-1 mediated effects that necessitated further experiments to unravel.
Pim-1 Colocalizes With but Does Not Increase the Number of CPCs Following MI
Previous studies showed elevated levels of Pim-1 in cardiomyocytes following pathological challenge.9,21⇓ Consistent with the accumulation of c-kit+ CPCs following infarction2 Pim-1 colocalizes with c-kit+ cells in myocardial sections of NTG as well as Pim-wt mice at 7, 10, and 21 days postinfarction (Figure 4A through 4C). The impact of Pim-1 expression on accumulation of CPCs in the border zone surrounding MI was assessed at 7, 10 and 21 days following challenge. Despite severe damage from coronary ligation assessed by infarct size (Online Figure IV, A) the number of accumulated CPCs in the border zone region is comparable between heart samples of Pim-wt versus NTG at all 3 time points examined (Figure 4D). In comparison, myocardial sections from Pim-KO mice subjected to infarction show significantly fewer accumulated CPCs at the one week time point (2-fold); samples at later stages were unavailable because Pim-KO mice were unable to survive for more than one week postinfarction. Analysis of apoptotic CPCs by TUNEL staining revealed fewer TUNEL+ CPCs in Pim-wt hearts 10 days and 3 weeks after infarction (Online Figure IV, B). Thus, although loss of Pim-1 may impair the CPC response to infarction, the cardiac-specific overexpression of Pim-1 does not provide an increased benefit through increasing total CPCs generated in response to infarction.
Pim-1 Stimulates CPC Cycling After MI
Because the primary effect of cardiac-specific Pim-1 overexpression is to expand the number of cycling CPCs in the transgenic heart (Figure 3), myocardial sections from Pim-wt hearts were immunolabeled to assess the quantity of cycling CPCs following infarction challenge. CPCs with coincident labeling for Ki-67+/c-kit+ (Online Figure IV, yellow arrows) and PCNA+/c-kit+ cells in the border zone surrounding the infarct region were quantitated at 7, 10, and 21 days after infarction. Indeed, Pim-wt hearts had a significant increase in the percent of Ki-67+ (2.1-, 2.24-, and 1.80-fold, respectively) and PCNA+ (1.98-, 2.05-, and 1.59-fold, respectively) CPCs versus NTG controls at all 3 time points (Figure 5A through 5F). In contrast, Pim-KO mice show a drastic decrease in cycling c-kit+ cells seven days after infarction versus NTG controls (1.7-fold). In addition to increased CPC cycling Pim-wt hearts exhibit increased myocyte cycling measured by Ki-67+ and PCNA+ myocytes (Online Figure VI, A [yellow arrow]; Online Figure VI, B through E). In addition, Pim-wt hearts contain small myocytes with longer telomeres (2.3-fold longer), indicative of new myocytes of CPC origin (Online Figure VI, F). These findings are consistent with earlier results (Figures 2 and 3⇑) that point toward a role for Pim-1 in expansion of the cycling CPC population.
Pim-1 Stimulates 5-Bromodeoxyuridine Incorporation in CPCs After MI
CPCs undergoing DNA synthesis as defined by coincidence of 5-bromodeoxyuridine (BrdU)+/c-kit+ immunolabeling (Figure 5G) were quantitated in myocardial sections from sham operated versus infarcted hearts. Consistent with findings of increased Ki-67+ and PCNA+ CPCs in Pim-wt hearts following infarction challenge Pim-wt hearts possess significantly more BrdU+/c-kit+ CPCs (1.7-fold; Figure 5H). Pim-wt hearts also show a significant increase of BrdU+ nuclei (8.3-fold increase overall versus NTG) including BrdU+/tropomyosin+ labeled cardiomyocytes (4.4-fold increase; Online Figure VII).
Pim-1 Expression Promotes Asymmetric Division of CPCs Following Infarction
The conundrum of increased cycling CPCs without expansion of the CPC population could be reconciled by increased frequency of asymmetric division in the presence of Pim-1 overexpression. Frequency of asymmetric cell division in CPCs was assessed using cell determinant markers α-adaptin and Numb (Figure 6A and 6B). Asymmetric division is characterized by sequestration of Numb or α-adaptin immunolabeling lateralized to one side of the mitotic cell, whereas symmetric division of CPCs is characterized by uniform α-adaptin and Numb distribution.7 Cycling CPCs are identified by coincident phospho-histone+/c-kit+ immunolabeling and colocalized with the cell determinant markers. With respect to asymmetric division, Numb sequestration is significantly greater in cycling CPCs from Pim-wt mice relative to NTG samples (65% versus 26%, respectively; Figure 6C). Alternatively, symmetric division evidenced by uniform α-adaptin labeling is higher in NTG CPC relative to Pim-wt (73% versus 36%, respectively; Figure 6D). Collectively, these results indicate that cardiac-specific Pim-1 overexpression leads to increased asymmetric division of the CPC population in response to infarction challenge.
Collectively, this report delineates a previously unrecognized mechanistic cellular basis for the enhanced resistance to cardiomyopathic injury observed in mice created with cardiac-specific overexpression of Pim-1 kinase.21 In combination with the previously detailed prosurvival properties of Pim-1,9,21,33⇓⇓ the multifaceted consequences of Pim-1 actions seem well suited to the task of augmenting CPC regenerative potential. The capacity of Pim-1 to influence the CPC population through increased cycling and asymmetric division would be a valuable molecular interventional approach to potentiate CPC-based regeneration following myocardial injury by preserving the CPC pool as well as providing cardiogenic daughter cells. With the advent of regenerative medicine new possibilities are being explored to mediate myocardial repair and cellular replacement, but our understanding of CPC biology lags far behind the comparatively rapid implementation of adoptive transfer studies in experimental animal models and the clinical setting.37,38⇓ Although not surprising that such studies are being pursued, it is similarly predictable that the underlying mechanistic basis for salutary effects observed remains in debate.1,39–41⇓⇓⇓ Virtually all studies show modest engraftment, persistence, proliferation, and survival of CPCs on adoptive transfer into infarcted myocardium. Manipulation of cellular signaling to expand the CPC pool, enhance survival, and promote repopulation of damaged tissue is an attractive approach to augment the limited regenerative potential that currently hampers cellular-based intervention strategies for myocardial repair.
Pim-1 influences cell proliferation in cancer and hematopoietic stem cells. Pim-1 phosphorylates heterochromatin protein-1 and NuMA, which are crucial in spindle fiber assembly and subsequent cell division during HeLa cell mitosis.36 Phosphorylation of NuMA leads to its translocation to the spindle pole where it anchors microtubule (−) ends as a critical part of the chromosomal segregation process.42 Pim-1 cooperates with other cell cycle proteins such as c-Myc, which is stabilized by Pim-1 activity thereby promoting enhanced cell cycling.24 In stem cells, Pim-1 is implicated in the antiapoptotic and hyperproliferative phenotypes of hematopoietic progenitor cells.22 Hematopoietic progenitor cells overexpressing STAT5 induce Pim-1 protein expression, resulting in significantly enhanced proliferation.22 Proliferation is also enhanced by Pim-1 mediated phosphorylation of the cell cycle inhibitor p21,43 leading to cytoplasmic sequestration of p21 and inability to interact with cyclinE/cdk2 in the nucleus.44 The proliferative phenotype of transformed cell lines such chronic myelogenous leukemia cells K562 and BV173 is associated with increased Pim-1 expression in G1/S phase that is maintained at high levels through S phase until the end of M phase.45 Collectively, these observations indicate that Pim-1 activity enhances proliferative activity when present in cycling cells, as appears to be the case in this study where Pim-1 is regulated by the α-myosin heavy chain promoter in CPCs.
The α-myosin heavy chain promoter has been exploited to overexpress several signaling molecules in the heart of transgenic mice.46–51⇓⇓⇓⇓⇓ Cardiac overexpression of proliferative and antiapoptotic molecules such as IGF-1, cyclin D2, Bcl-2, and nuclear Akt lead to a hyperplastic phenotype and are cardioprotective.10,11,46,51⇓⇓⇓ Cardiac-specific transgenic overexpression of nuclear Akt was found to increase cell cycling, thereby expanding CPCs, indicating this population is influenced by activation of the α-myosin heavy chain promoter (demonstrated elsewhere11,12,35⇓⇓). However, because Akt is a nodal kinase with several biological functions including survival, proliferation, gene transcription, protein translation, metabolism, and differentiation,16 beneficial effects of Akt overexpression can be accompanied by deleterious consequences.52–54⇓⇓ Thus, Pim-1 with a relatively narrow spectrum of biological effects limited to cell proliferation and survival would seem a much more suitable cardioprotective molecular target for therapeutic interventional strategies, including expansion of the CPC population to promote repair and regeneration in the wake of pathological injury.
CPCs in the adult mouse heart favor asymmetric division in an attempt to maintain cardiac homeostasis and replenish myocardial cells rather than constant self-renewal.7 Similarly, asymmetric cell division creates cell type diversity during early mammalian development as observed in neuroblasts and embryonic stem cells.55–57⇓⇓ Cancer stem cells use normal stem cell characteristics, including asymmetric cell division to evade chemotherapy and promote growth.58,59⇓ However, in the context of the myocardium that is notoriously resistant to oncogenic transformation, amplifying the inherent ability of CPCs to divide asymmetrically may prove a useful tool in regenerative medicine.
Taking our findings with myocardial Pim-1 expression together in the context of the literature, a hypothetical model is proposed wherein overexpression of Pim-1 leads to increased CPC cycling during hyperplastic growth occurring during pre/postnatal development and after infarction induced stress (Figure 7). Increased cycling in Pim-wt CPCs correlates with elevated levels of c-Myc in the CPCs (Figure 2E) and previous reports of cardiac c-Myc during periods of hyperplastic growth.60–62⇓⇓ Elevated levels of c-Myc are stabilized by interaction with the overexpressed Pim-1, thereby working synergistically to promote CPC cycling. Once the myocardium matures and transitions from hyperplastic growth to hypertrophic growth, c-Myc levels decline,60 which decreases CPC cycling in the adult myocardium. During times of stress such as infarction induction of c-Myc occurs,62 which is stabilized by Pim-1, resulting in enhanced CPC cycling and subsequent protection of the myocardium. The ability of Pim-1 to increase cycling in the CPC population also prompts interesting questions regarding cellular senescence and chromosome integrity. Specifically, to sustain increased levels of mitosis the overexpression of Pim-1 may contribute to maintenance of DNA integrity and telomeric length, thereby antagonizing apoptotic cell death. In addition to such studies expanding our knowledge of the mechanistic basis for enhanced cellular proliferation, studies are already underway that demonstrate preservation of mitochondrial integrity by Pim-1 (unpublished results). The ability of Pim-1 to increase cycling, enhance survival, and promote production of differentiated progeny through asymmetric division makes Pim-1 an attractive candidate to genetically engineer CPCs with enhanced capacity to mitigate damage following MI.
We thank all members of the Sussman laboratory for helpful discussions and technical support.
Sources of Funding
K.M.F. and N.A.G. are supported by the Rees-Stealy Foundation. N.A.G. is also supported by an American Heart Association Predoctoral Training Grant. M.A.S. is supported by NIH grants R01HL067245, P01 HL085577, 1R37 HL091102, and 7P01 AG023071.
- ↵Tallini YN, Greene KS, Craven M, Spealman A, Breitbach M, Smith J, Fisher PJ, Steffey M, Hesse M, Doran RM, Woods A, Singh B, Yen A, Fleischmann BK, Kotlikoff MI. c-kit expression identifies cardiovascular precursors in the neonatal heart. Proc Natl Acad Sci U S A. 2009; 106: 1808–1813.
- ↵Ikuta K, Weissman IL. Evidence that hematopoietic stem cells express mouse c-kit but do not depend on steel factor for their generation. Proc Natl Acad Sci U S A. 1992; 89: 1502–1506.
- ↵Leri A, Kajstura J, Anversa P. Cardiac stem cells and mechanisms of myocardial regeneration. Physiol Rev. 2005; 85: 1373–1416.
- ↵Urbanek K, Cesselli D, Rota M, Nascimbene A, De Angelis A, Hosoda T, Bearzi C, Boni A, Bolli R, Kajstura J, Anversa P, Leri A. Stem cell niches in the adult mouse heart. Proc Natl Acad Sci U S A. 2006; 103: 9226–9231.
- ↵Linke A, Muller P, Nurzynska D, Casarsa C, Torella D, Nascimbene A, Castaldo C, Cascapera S, Bohm M, Quaini F, Urbanek K, Leri A, Hintze TH, Kajstura J, Anversa P. Stem cells in the dog heart are self-renewing, clonogenic, and multipotent and regenerate infarcted myocardium, improving cardiac function. Proc Natl Acad Sci U S A. 2005; 102: 8966–8971.
- ↵Muraski JA, Fischer KM, Wu W, Cottage CT, Quijada P, Mason M, Din S, Gude N, Alvarez R Jr, Rota M, Kajstura J, Wang Z, Schaefer E, Chen X, MacDonnel S, Magnuson N, Houser SR, Anversa P, Sussman MA. Pim-1 kinase antagonizes aspects of myocardial hypertrophy and compensation to pathological pressure overload. Proc Natl Acad Sci U S A. 2008; 105: 13889–13894.
- ↵Reiss K, Cheng W, Ferber A, Kajstura J, Li P, Li B, Olivetti G, Homcy CJ, Baserga R, Anversa P. Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice. Proc Natl Acad Sci U S A. 1996; 93: 8630–8635.
- ↵Gude N, Muraski J, Rubio M, Kajstura J, Schaefer E, Anversa P, Sussman MA. Akt promotes increased cardiomyocyte cycling and expansion of the cardiac progenitor cell population. Circ Res. 2006; 99: 381–388.
- ↵Shiraishi I, Melendez J, Ahn Y, Skavdahl M, Murphy E, Welch S, Schaefer E, Walsh K, Rosenzweig A, Torella D, Nurzynska D, Kajstura J, Leri A, Anversa P, Sussman MA. Nuclear targeting of Akt enhances kinase activity and survival of cardiomyocytes. Circ Res. 2004; 94: 884–891.
- ↵Tsujita Y, Muraski J, Shiraishi I, Kato T, Kajstura J, Anversa P, Sussman MA. Nuclear targeting of Akt antagonizes aspects of cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A. 2006; 103: 11946–11951.
- ↵Fujio Y, Nguyen T, Wencker D, Kitsis RN, Walsh K. Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation. 2000; 101: 660–667.
- ↵Matsui T, Tao J, del Monte F, Lee KH, Li L, Picard M, Force TL, Franke TF, Hajjar RJ, Rosenzweig A. Akt activation preserves cardiac function and prevents injury after transient cardiac ischemia in vivo. Circulation. 2001; 104: 330–335.
- ↵Condorelli G, Drusco A, Stassi G, Bellacosa A, Roncarati R, Iaccarino G, Russo MA, Gu Y, Dalton N, Chung C, Latronico MV, Napoli C, Sadoshima J, Croce CM, Ross J Jr. Akt induces enhanced myocardial contractility and cell size in vivo in transgenic mice. Proc Natl Acad Sci U S A. 2002; 99: 12333–12338.
- ↵DeBosch B, Sambandam N, Weinheimer C, Courtois M, Muslin AJ. Akt2 regulates cardiac metabolism and cardiomyocyte survival. J Biol Chem. 2006; 281: 32841–32851.
- ↵DeBosch B, Treskov I, Lupu TS, Weinheimer C, Kovacs A, Courtois M, Muslin AJ. Akt1 is required for physiological cardiac growth. Circulation. 2006; 113: 2097–2104.
- ↵Matsui T, Nagoshi T, Hong EG, Luptak I, Hartil K, Li L, Gorovits N, Charron MJ, Kim JK, Tian R, Rosenzweig A. Effects of chronic Akt activation on glucose uptake in the heart. Am J Physiol Endocrinol Metab. 2006; 290: E789–E797.
- ↵Muraski JA, Rota M, Misao Y, Fransioli J, Cottage C, Gude N, Esposito G, Delucchi F, Arcarese M, Alvarez R, Siddiqi S, Emmanuel GN, Wu W, Fischer K, Martindale JJ, Glembotski CC, Leri A, Kajstura J, Magnuson N, Berns A, Beretta RM, Houser SR, Schaefer EM, Anversa P, Sussman MA. Pim-1 regulates cardiomyocyte survival downstream of Akt. Nat Med. 2007; 13: 1467–1475.
- ↵Nosaka T, Kawashima T, Misawa K, Ikuta K, Mui AL, Kitamura T. STAT5 as a molecular regulator of proliferation, differentiation and apoptosis in hematopoietic cells. EMBO J. 1999; 18: 4754–4765.
- ↵Rodriguez-Lopez AM, Xenaki D, Eden TO, Hickman JA, Chresta CM. MDM2 mediated nuclear exclusion of p53 attenuates etoposide-induced apoptosis in neuroblastoma cells. Mol Pharmacol. 2001; 59: 135–143.
- ↵McDonnell TJ, Chari NS, Cho-Vega JH, Troncoso P, Wang X, Bueso-Ramos CE, Coombes K, Brisbay S, Lopez R, Prendergast G, Logothetis C, Do KA. Biomarker expression patterns that correlate with high grade features in treatment naive, organ-confined prostate cancer. BMC Med Genomics. 2008; 1: 1.
- ↵Gude NA, Emmanuel G, Wu W, Cottage CT, Fischer K, Quijada P, Muraski JA, Alvarez R, Rubio M, Schaefer E, Sussman MA. Activation of Notch-mediated protective signaling in the myocardium. Circ Res. 2008; 102: 1025–1035.
- ↵Leoni LM, Bailey B, Reifert J, Bendall HH, Zeller RW, Corbeil J, Elliott G, Niemeyer CC. Bendamustine (Treanda) displays a distinct pattern of cytotoxicity and unique mechanistic features compared with other alkylating agents. Clin Cancer Res. 2008; 14: 309–317.
- ↵Kim KT, Baird K, Ahn JY, Meltzer P, Lilly M, Levis M, Small D. Pim-1 is up-regulated by constitutively activated FLT3 and plays a role in FLT3-mediated cell survival. Blood. 2005; 105: 1759–1767.
- ↵Hammerman PS, Fox CJ, Birnbaum MJ, Thompson CB. Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival. Blood. 2005; 105: 4477–4483.
- ↵Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res. 2008; 103: 1204–1219.
- ↵Gnecchi M, He H, Noiseux N, Liang OD, Zhang L, Morello F, Mu H, Melo LG, Pratt RE, Ingwall JS, Dzau VJ. Evidence supporting paracrine hypothesis for Akt-modified mesenchymal stem cell-mediated cardiac protection and functional improvement. FASEB J. 2006; 20: 661–669.
- ↵Zhang Y, Wang Z, Magnuson NS. Pim-1 kinase-dependent phosphorylation of p21Cip1/WAF1 regulates its stability and cellular localization in H1299 cells. Mol Cancer Res. 2007; 5: 909–922.
- ↵Stewart ZA, Leach SD, Pietenpol JA. p21(Waf1/Cip1) inhibition of cyclin E/Cdk2 activity prevents endoreduplication after mitotic spindle disruption. Mol Cell Biol. 1999; 19: 205–215.
- ↵Limana F, Urbanek K, Chimenti S, Quaini F, Leri A, Kajstura J, Nadal-Ginard B, Izumo S, Anversa P. bcl-2 overexpression promotes myocyte proliferation. Proc Natl Acad Sci U S A. 2002; 99: 6257–6262.
- ↵Nebigil CG, Jaffre F, Messaddeq N, Hickel P, Monassier L, Launay JM, Maroteaux L. Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mitochondrial function and cardiac hypertrophy. Circulation. 2003; 107: 3223–3229.
- ↵Karpanen T, Bry M, Ollila HM, Seppanen-Laakso T, Liimatta E, Leskinen H, Kivela R, Helkamaa T, Merentie M, Jeltsch M, Paavonen K, Andersson LC, Mervaala E, Hassinen IE, Yla-Herttuala S, Oresic M, Alitalo K. Overexpression of vascular endothelial growth factor-B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy. Circ Res. 2008; 103: 1018–1026.
- ↵Urayama K, Guilini C, Messaddeq N, Hu K, Steenman M, Kurose H, Ert G, Nebigil CG. The prokineticin receptor-1 (GPR73) promotes cardiomyocyte survival and angiogenesis. FASEB J. 2007; 21: 2980–2993.
- ↵Pasumarthi KB, Nakajima H, Nakajima HO, Soonpaa MH, Field LJ. Targeted expression of cyclin D2 results in cardiomyocyte DNA synthesis and infarct regression in transgenic mice. Circ Res. 2005; 96: 110–118.
- ↵Shioi T, McMullen JR, Kang PM, Douglas PS, Obata T, Franke TF, Cantley LC, Izumo S. Akt/protein kinase B promotes organ growth in transgenic mice. Mol Cell Biol. 2002; 22: 2799–2809.
- ↵Schiekofer S, Shiojima I, Sato K, Galasso G, Oshima Y, Walsh K. Microarray analysis of Akt1 activation in transgenic mouse hearts reveals transcript expression profiles associated with compensatory hypertrophy and failure. Physiol Genomics. 2006; 27: 156–170.
- ↵Matsui T, Li L, Wu JC, Cook SA, Nagoshi T, Picard MH, Liao R, Rosenzweig A. Phenotypic spectrum caused by transgenic overexpression of activated Akt in the heart. J Biol Chem. 2002; 277: 22896–22901.
- ↵Jackson T, Allard MF, Sreenan CM, Doss LK, Bishop SP, Swain JL. The c-myc proto-oncogene regulates cardiac development in transgenic mice. Mol Cell Biol. 1990; 10: 3709–3716.
Novelty and Significance
What Is Known?
Pim-1 is a cardioprotective kinase that inhibits cell death and cardiomyocyte hypertrophy induced by pathological injury.
Myocardial regeneration is enhanced using cardiac stem cells genetically engineered to overexpress Pim-1.
What New Information Does This Article Contribute?
Pim-1 overexpression in the heart increases proliferation of cardiac progenitor cells during postnatal growth or after myocardial infarction.
Pim-1–mediated increases in cardiac progenitor cell proliferation are mirrored by an elevated rate of asymmetric cell division that produces more cardiogenic cells to populate the myocardium.
The regulatory mechanisms governing cardiac progenitor cell growth and lineage commitment are poorly understood but are critically important issues in developing therapeutic strategies for enhancing regenerative and reparative processes in the damaged heart. The cardioprotective properties of Pim-1 kinase activity render the heart resistant to injury, but the involvement of cardiac progenitor cells remains undetermined. In this study, we demonstrate that Pim-1 enhances cardiac progenitor cell cycling without increasing the progenitor cell population during physiological growth and after myocardial infarction. This apparently paradoxical outcome is reconciled by concomitant increases in progenitor cell asymmetric division in hearts overexpressing Pim-1. Higher rates of asymmetric division coupled with proliferation maintain the progenitor cell population while generating de novo cardiogenic differentiated daughter cells, which likely accounts in part for enhanced resistance to pathological injury in Pim-1 overexpressing transgenic mice. These findings, in conjunction with the ability of Pim-1–engineered cardiac progenitor cells to mediate enhanced regeneration in the damaged heart, suggest that Pim-1 kinase is an important target for therapeutic strategies aimed at augmenting the limited reparative potential of cell-based approaches in damaged myocardium.
Original received September 4, 2009; revision received December 29, 2009; accepted December 31, 2009.