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1.  Evolution of the c-kit-Positive Cell Response to Pathological Challenge in the Myocardium 
Stem cells (Dayton, Ohio)  2008;26(5):1315-1324.
Cumulative evidence indicates that myocardium responds to growth or injury by recruitment of stem and/or progenitor cells that participate in repair and regenerative processes. Unequivocal identification of this population has been hampered by lack of reagents or markers specific to the recruited population, leading to controversies regarding the nature of these cells. Use of a transgenic mouse expressing green fluorescent protein driven by the c-kit promoter allows for unambiguous identification of this cell population. Green fluorescent protein (GFP) driven by the c-kit promoter labels a fraction of the c-kit + cells recognized by antibody labeling for c-kit protein. Expression of GFP by the c-kit promoter and accumulation of GFP-positive cells in the myocardium is relatively high at birth compared with adult and declines between postnatal weeks 1 and 2, which tracks in parallel with expression of c-kit protein and c-kit-positive cells. Acute cardiomyopathic injury by infarction prompts increased expression of both GFP protein and GFP-labeled cells in the region of infarction relative to remote myocardium. Similar increases were observed for c-kit protein and cells with a slightly earlier onset and decline relative to the GFP signal. Cells coexpressing GFP, c-kit, and cardiogenic markers were apparent at 1–2 weeks postinfarction. Cardiac-resident c-kit+ cell cultures derived from the transgenic line express GFP that is diminished in parallel with c-kit by induction of differentiation. The use of genetically engineered mice validates and extends the concept of c-kit+ cells participating in the response to myocardial injury.
doi:10.1634/stemcells.2007-0751
PMCID: PMC4037162  PMID: 18308948
c-kit; Heart; Infarction; Cardiac stem cell
2.  Regulation of Cardiac Hypertrophic Signaling by Prolyl Isomerase Pin1 
Circulation research  2013;112(9):1244-1252.
Rationale
Cardiac hypertrophy results from the complex interplay of differentially regulated cascades based upon the phosphorylation status of involved signaling molecules. While numerous critical regulatory kinases and phosphatases have been identified in the myocardium, the intracellular mechanism for temporal regulation of signaling duration and intensity remains obscure. In the non-myocyte context, control of folding, activity, and stability of proteins is mediated by the prolyl isomerase Pin1, but the role of Pin1 in the heart is unknown.
Objective
To establish the role of Pin1 in the heart.
Methods and Results
Here we show that either genetic deletion or cardiac over-expression of Pin1 blunts hypertrophic responses induced by transaortic constriction and consequent cardiac failure in vivo. Mechanistically, we find that Pin1 directly binds to Akt, MEK and Raf-1 in cultured cardiomyocytes following hypertrophic stimulation. Furthermore, loss of Pin1 leads to diminished hypertrophic signaling of Akt and MEK, while over-expression of Pin1 increases Raf-1 phosphorylation on the auto-inhibitory site Ser259 leading to reduced MEK activation.
Conclusions
Collectively, these data support a role for Pin1 as a central modulator of the intensity and duration of two major hypertrophic signaling pathways, thereby providing a novel target for regulation and control of cardiac hypertrophy.
doi:10.1161/CIRCRESAHA.113.301084
PMCID: PMC3742109  PMID: 23487407
Pin1; cardiomyocyte; Akt; Raf-MEK-ERK; hypertrophy; signal transduction; molecular biology; heart failure
3.  Rejuvenation of Human Cardiac Progenitor Cells With Pim-1 Kinase 
Circulation research  2013;113(10):1169-1179.
Rationale
Myocardial function is enhanced by adoptive transfer of human cardiac progenitor cells (hCPCs) into a pathologically challenged heart. However, advanced age, comorbidities, and myocardial injury in patients with heart failure constrain the proliferation, survival, and regenerative capacity of hCPCs. Rejuvenation of senescent hCPCs will improve the outcome of regenerative therapy for a substantial patient population possessing functionally impaired stem cells.
Objective
Reverse phenotypic and functional senescence of hCPCs by ex vivo modification with Pim-1.
Methods and Results
C-kit–positive hCPCs were isolated from heart biopsy samples of patients undergoing left ventricular assist device implantation. Growth kinetics, telomere lengths, and expression of cell cycle regulators showed significant variation between hCPC isolated from multiple patients. Telomere length was significantly decreased in hCPC with slow-growth kinetics concomitant with decreased proliferation and upregulation of senescent markers compared with hCPC with fast-growth kinetics. Desirable youthful characteristics were conferred on hCPCs by genetic modification using Pim-1 kinase, including increases in proliferation, telomere length, survival, and decreased expression of senescence markers.
Conclusions
Senescence characteristics of hCPCs are ameliorated by Pim-1 kinase resulting in rejuvenation of phenotypic and functional properties. hCPCs show improved cellular properties resulting from Pim-1 modification, but benefits were more pronounced in hCPC with slow-growth kinetics relative to hCPC with fast-growth kinetics. With the majority of patients with heart failure presenting advanced age, infirmity, and impaired regenerative capacity, the use of Pim-1 modification should be incorporated into cell-based therapeutic approaches to broaden inclusion criteria and address limitations associated with the senescent phenotype of aged hCPC.
doi:10.1161/CIRCRESAHA.113.302302
PMCID: PMC3999968  PMID: 24044948
aging; cell cycle proteins; heart failure; telomere lengthening
4.  Predicting the Future With Stem Cells 
Circulation  2013;129(2):136-138.
doi:10.1161/CIRCULATIONAHA.113.007045
PMCID: PMC3919528  PMID: 24249719
Editorial; coronary bypass surgery; stem cells; ventricular remodeling
5.  Enhanced Effect of Human Cardiac Stem Cells and Bone Marrow Mesenchymal Stem Cells to Reduce Infarct Size and Restore Cardiac Function after Myocardial Infarction 
Circulation  2012;127(2):213-223.
Background
As mesenchymal stem cells (MSCs) induce proliferation and differentiation of c-kit+ cardiac stem cells (CSCs) in vivo and in vitro, we hypothesized that combining human (h)MSCs with c-kit+ hCSCs produces greater infarct size reduction compared to either cell administered alone after MI.
Methods and Results
Yorkshire swine underwent balloon occlusion of the LAD coronary artery followed by reperfusion, and were immunosuppressed after MI with cyclosporine and methylprednisolone. Intramyocardial injection of either: combination hCSCs/hMSCs (1M/200M, n=5), hCSCs alone (1M, n=5), hMSCs alone (200M, n=5), or placebo (PBS, n=5) was administered to the infarct border zones at 14 days post-MI. Phenotypic response to cell therapy was assessed by cardiac MRI and micromanometer conductance catheterization hemodynamics. While each cell therapy group had reduced MI size relative to placebo (p<0.05), the MI size reduction was 2-fold greater in combination vs. either cell therapy alone (p<0.05). Accompanying enhanced MI size reduction was substantial improvement in LV chamber compliance (end-diastolic pressure volume relationship, p<0.01) and contractility (preload recruitable stroke work and dP/dtmax, p<0.05) in combination treated swine. EF was restored to baseline in cell treated pigs, while placebo pigs had persistently depressed LV function (p<0.05). Immunohistochemistry showed 7-fold enhanced engraftment of stem cells in the combination therapy group vs. either cell type alone (P<0.001).
Conclusions
Combining hMSCs and hCSCs as a cell therapeutic enhances scar size reduction, and restores diastolic and systolic function toward normal after MI. Taken together these findings illustrate important biological interactions between c-kit+ CSCs and MSCs that enhance cell-based therapeutic responses.
doi:10.1161/CIRCULATIONAHA.112.131110
PMCID: PMC3579523  PMID: 23224061
cardiac stem cells; mesenchymal stem cells; heart failure
6.  PRAS40 prevents development of diabetic cardiomyopathy and improves hepatic insulin sensitivity in obesity 
EMBO Molecular Medicine  2013;6(1):57-65.
Diabetes is a multi-organ disease and diabetic cardiomyopathy can result in heart failure, which is a leading cause of morbidity and mortality in diabetic patients. In the liver, insulin resistance contributes to hyperglycaemia and hyperlipidaemia, which further worsens the metabolic profile. Defects in mTOR signalling are believed to contribute to metabolic dysfunctions in diabetic liver and hearts, but evidence is missing that mTOR activation is causal to the development of diabetic cardiomyopathy. This study shows that specific mTORC1 inhibition by PRAS40 prevents the development of diabetic cardiomyopathy. This phenotype was associated with improved metabolic function, blunted hypertrophic growth and preserved cardiac function. In addition PRAS40 treatment improves hepatic insulin sensitivity and reduces systemic hyperglycaemia in obese mice. Thus, unlike rapamycin, mTORC1 inhibition with PRAS40 improves metabolic profile in diabetic mice. These findings may open novel avenues for therapeutic strategies using PRAS40 directed against diabetic-related diseases.
doi:10.1002/emmm.201303183
PMCID: PMC3936489  PMID: 24408966
diabetes; PRAS40; mTOR
7.  CENP-A is essential for cardiac progenitor cell proliferation 
Cell Cycle  2013;13(5):739-748.
Centromere protein A (CENP-A) is a homolog of histone H3 that epigenetically marks the heterochromatin of chromosomes. CENP-A is a critical component of the cell cycle machinery that is necessary for proper assembly of the mitotic spindle. However, the role of CENP-A in the heart and cardiac progenitor cells (CPCs) has not been previously studied. This study shows that CENP-A is expressed in CPCs and declines with age. Silencing CENP-A results in a decreased CPC growth rate, reduced cell number in phase G2/M of the cell cycle, and increased senescence associated β-galactosidase activity. Lineage commitment is not affected by CENP-A silencing, suggesting that cell cycle arrest induced by loss of CENP-A is a consequence of senescence and not differentiation. CENP-A knockdown does not exacerbate cell death in undifferentiated CPCs, but increases apoptosis upon lineage commitment. Taken together, these results indicate that CPCs maintain relatively high levels of CENP-A early in life, which is necessary for sustaining proliferation, inhibiting senescence, and promoting survival following differentiation of CPCs.
doi:10.4161/cc.27549
PMCID: PMC3979910  PMID: 24362315
CENP-A; cardiac progenitor cell; heart; cell cycle; senescence
8.  Increased Mitotic Rate Coincident With Transient Telomere Lengthening resulting from Pim-1 Overexpression in Cardiac Progenitor Cells 
Stem cells (Dayton, Ohio)  2012;30(11):2512-2522.
Cardiac regeneration following myocardial infarction rests with the potential of c-kit+ cardiac progenitor cells (CPCs) to repopulate damaged myocardium. The ability of CPCs to reconstitute the heart is restricted by patient age and disease progression. Increasing CPC proliferation, telomere length, and survival will improve the ability of autologous CPCs to be successful in myocardial regeneration. Prior studies have demonstrated enhancement of myocardial regeneration by engineering CPCs to express Pim-1 kinase, but cellular and molecular mechanisms for Pim-1 mediated effects on CPCs remain obscure. We find CPCs rapidly expand following overexpression of cardioprotective kinase Pim-1 (CPCeP), however increases in mitotic rate are short lived as late passage CPCePs proliferate similar to control CPCs. Telomere elongation consistent with a young phenotype is observed following Pim-1 modification of CPCeP; in addition telomere elongation coincides with increased telomerase expression and activity. Interestingly, telomere length and telomerase activity normalize after several rounds of passaging, consistent with the ability of Pim-1 to transiently increase mitosis without resultant oncogenic transformation. Accelerating mitosis in CPCeP without immortalization represents a novel strategy to expand the CPC population in order to improve their therapeutic efficacy.
doi:10.1002/stem.1211
PMCID: PMC3479348  PMID: 22915504
Pim-1; Telomere; Cardiac progenitor cell; Telomerase
9.  mTOR/PRAS40 interaction 
Cell Cycle  2013;12(23):3579-3580.
doi:10.4161/cc.26822
PMCID: PMC3903704  PMID: 24131922
PRAS40; mTOR; hypertrophy; mTORC1; growth; cell cycle; cardiac
10.  HUMAN CARDIAC PROGENITOR CELLS ENGINEERED WITH PIM-1 KINASE ENHANCE MYOCARDIAL REPAIR 
Objective
Enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for treatment of myocardial infarction.
Background
Regenerative potential of stem cells to repair acute infarction is limited. Improved hCPC survival, proliferation and differentiation into functional myocardium will increase efficacy and advance translational implementation of cardiac regeneration.
Methods
hCPCs isolated from myocardium of heart failure patients undergoing left ventricular assist device (LVAD) implantation are engineered to express green fluorescent protein (GFP; hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential are performed with immunocompromised mice by intramyocardial adoptive transfer injection after infarction. Myocardial structure and function is monitored by echocardiographic and hemodynamic assessment for 20 weeks following delivery. hCPCe and hCPCeP expressing luciferase are followed by bioluminesence imaging (BLI) to non-invasively track persistence.
Results
hCPCeP exhibit augmentation of reparative potential relative to hCPCe control cells as demonstrated by significantly increased proliferation coupled with amelioration of infarction injury and increased hemodynamic performance at 20 weeks post-transplantation. Concurrent with enhanced cardiac structure and function, hCPCeP demonstrate increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe is revealed by BLI at up to 8 weeks post delivery.
Conclusion
Genetic engineering of hCPCs with Pim-1 enhances repair of damaged myocardium. Ex vivo gene delivery to modify stem cells has emerged as a viable option addressing current limitations in the field. This study demonstrates that efficacy of human CPCs from the failing myocardium can be safely and significantly enhanced through expression of Pim-1 kinase, setting the stage for use of engineered cells in preclinical settings.
doi:10.1016/j.jacc.2012.04.047
PMCID: PMC3461098  PMID: 22841153
human cardiac progenitor cells; Pim-1 kinase; heart repair
11.  Activation of Notch-Mediated Protective Signaling in the Myocardium 
Circulation research  2008;102(9):1025-1035.
The Notch network regulates multiple cellular processes, including cell fate determination, development, differentiation, proliferation, apoptosis, and regeneration. These processes are regulated via Notch-mediated activity that involves hepatocyte growth factor (HGF)/c-Met receptor and phosphatidylinositol 3-kinase/Akt signaling cascades. The impact of HGF on Notch signaling was assessed following myocardial infarction as well as in cultured cardiomyocytes. Notch1 is activated in border zone cardiomyocytes coincident with nuclear c-Met following infarction. Intramyocardial injection of HGF enhances Notch1 and Akt activation in adult mouse myocardium. Corroborating evidence in cultured cardiomyocytes shows treatment with HGF or insulin increases levels of Notch effector Hes1 in immunoblots, whereas overexpression of activated Notch intracellular domain prompts a 3-fold increase in phosphorylated Akt. Infarcted hearts injected with adenoviral vector expressing Notch intracellular domain treatment exhibit improved hemodynamic function in comparison with control mice after 4 weeks, implicating Notch signaling in a cardioprotective role following cardiac injury. These results indicate Notch activation in cardiomyocytes is mediated through c-Met and Akt survival signaling pathways, and Notch1 signaling in turn enhances Akt activity. This mutually supportive crosstalk suggests a positive survival feedback mechanism between Notch and Akt signaling in adult myocardium following injury.
doi:10.1161/CIRCRESAHA.107.164749
PMCID: PMC3760732  PMID: 18369158
Notch; Akt; cardioprotection; infarction; myocardium
12.  Sca-1 Knockout Impairs Myocardial and Cardiac Progenitor Cell Function 
Circulation research  2012;111(6):750-760.
Rationale
Cardiac progenitor cells are important for maintenance of myocardial structure and function, but molecular mechanisms governing these progenitor cells remain obscure and require elucidation to enhance regenerative therapeutic approaches.
Objective
To understand consequences of stem cell antigen-1 (Sca-1) deletion upon functional properties of c-kit+ cardiac progenitor cells and myocardial performance using a Sca-1 knockout/Green Fluorescent Protein knock-in reporter mouse (ScaKI).
Methods and Results
Genetic deletion of Sca-1 results in early-onset cardiac contractile deficiency as determined by echocardiography and hemodynamics as well as age-associated hypertrophy. Resident cardiac progenitor cells in ScaKI mice do not respond to pathological damage in vivo, consistent with observations of impaired growth and survival of ScaKI cardiac progenitor cells in vitro. The molecular basis of the defect in ScaKI cardiac progenitor cells is associated with increased canonical Wnt signaling pathway activation consistent with molecular characteristics of lineage commitment.
Conclusions
Genetic deletion of Sca-1 causes primary cardiac defects in myocardial contractility and repair consistent with impairment of resident cardiac progenitor cell proliferative capacity associated with altered canonical Wnt signaling.
doi:10.1161/CIRCRESAHA.112.274662
PMCID: PMC3463406  PMID: 22800687
Sca-1; c-kit; heart; cardiac progenitor cell; infarction; myocardium; Sca-1 knock-out; β–catenin; cardiac development
13.  Preservation of Myocardial Structure is Enhanced by Pim-1 Engineering of Bone Marrow Cells 
Circulation Research  2012;111(1):77-86.
Rationale
Bone marrow derived cells to treat myocardial injury improve cardiac function and support beneficial cardiac remodeling. However, survival of stem cells is limited due to low proliferation of transferred cells.
Objective
Demonstrate long-term potential of c-kit+ bone marrow stem cells (BMCs) enhanced with Pim-1 kinase to promote positive cardiac remodeling.
Methods and Results
Lentiviral modification of c-kit+ BMCs to express Pim-1 (BMCeP) increases proliferation and expression of pro-survival proteins relative to BMCs expressing GFP (BMCe). Intramyocardial delivery of BMCeP at time of infarction supports improvements in anterior wall dimensions and prevents left ventricle dilation compared to hearts treated with vehicle alone. Reduction of the akinetic left ventricular wall was observed in BMCeP treated hearts at 4 and 12 weeks after infarction. Early recovery of cardiac function in BMCeP-injected hearts facilitated modest improvements in hemodynamic function up to 12 weeks post infarction between cell treated groups. Persistence of BMCeP is improved relative to BMCe within the infarct together with increased recruitment of endogenous c-kit+ cells. Delivery of BMC populations promotes cellular hypertrophy in the border and infarcted regions coupled with an up regulation of hypertrophic genes. Thus, BMCeP treatment yields improved structural remodeling of infarcted myocardium compared to control BMCs.
Conclusions
Genetic modification of BMCs with Pim-1 may serve as a therapeutic approach to promote recovery of myocardial structure. Future approaches may take advantage of salutary BMC actions in conjunction with other stem cell types to increase efficacy of cellular therapy and improve myocardial performance in the injured myocardium.
doi:10.1161/CIRCRESAHA.112.265207
PMCID: PMC3398618  PMID: 22619278
Pim-1; bone marrow cells; myocardial infarction; myocardial structure; hypertrophy
14.  MYOCARDIAL AKT: THE OMNIPRESENT NEXUS 
Physiological reviews  2011;91(3):1023-1070.
One of the greatest examples of integrated signal transduction is revealed by examination of effects mediated by AKT kinase in myocardial biology. Positioned at the intersection of multiple afferent and efferent signals, AKT exemplifies a molecular sensing node that coordinates dynamic responses of the cell in literally every aspect of biological responses. The balanced and nuanced nature of homeostatic signaling is particularly essential within the myocardial context, where regulation of survival, energy production, contractility, and response to pathological stress all flow through the nexus of AKT activation or repression. Equally important, the loss of regulated AKT activity is primarily the cause or consequence of pathological conditions leading to remodeling of the heart and eventual decompensation. This review presents an overview compendium of the complex world of myocardial AKT biology gleaned from more than a decade of research. Summarization of the widespread influence that AKT exerts upon myocardial responses leaves no doubt that the participation of AKT in molecular signaling will need to be reckoned with as a seemingly omnipresent regulator of myocardial molecular biological responses.
doi:10.1152/physrev.00024.2010
PMCID: PMC3674828  PMID: 21742795
15.  Notch signaling and cardiac repair 
Notch signaling is critical for proper heart development and recently has been reported to participate in adult cardiac repair. Notch resides at the cell surface as a single pass transmembrane receptor, transits through the cytoplasm following activation, and acts as a transcription factor upon entering the nucleus. This dynamic and widespread cellular distribution allows for potential interactions with many signaling and binding partners. Notch displays temporal as well as spatial versatility, acting as a strong developmental signal, controlling cell fate determination and lineage commitment, and playing a pivotal role in embryonic and adult stem cell proliferation and differentiation. This review serves as an update of recent literature addressing Notch signaling in the heart, with attention to findings from non cardiac research that provide clues for further interpretation of how the Notch pathway influences cardiac biology. Specific areas of focus include Notch signaling in adult myocardium following pathologic injury, the role of Notch in cardiac progenitor cells with respect to differentiation and cardiac repair, crosstalk between Notch and other cardiac signaling pathways, and emerging aspects of noncanonical Notch signaling in heart.
doi:10.1016/j.yjmcc.2012.03.007
PMCID: PMC3348268  PMID: 22465038
Notch; Cardioprotection; Progenitor; Regeneration; Crosstalk; Noncanonical
16.  Myocardial Isl+-land: a place with lots of rhythm, but no beat 
Circulation Research  2012;110(10):1267-1269.
doi:10.1161/CIRCRESAHA.112.268995
PMCID: PMC3397794  PMID: 22581917
Isl-1; myocardial development
17.  Asymmetric chromatid segregation in cardiac progenitor cells is enhanced by Pim-1 kinase 
Circulation Research  2012;110(9):1169-1173.
Rationale
Cardiac progenitor cells (CPCs) in the adult heart are used for cell-based treatment of myocardial damage but factors determining stemness, self-renewal and lineage commitment are poorly understood. Immortal DNA strands inherited through asymmetric chromatid segregation correlate with self-renewal of adult stem cells, but CPCs capacity for asymmetric segregation to retain immortal strands is unknown. Cardioprotective kinase Pim-1 increases asymmetric cell division in vivo but the ability of Pim-1 to enhance asymmetric chromatid segregation is unknown.
Objective
Demonstrate immortal strand segregation in CPCs and the enhancement of asymmetric chromatid distribution by Pim-1 kinase.
Methods and Results
Asymmetric segregation is tracked by incorporation of bromodeoxyuridine (BrdU). CPC DNA was labeled for several generations and then blocked in second cytokinesis during chase to determine distribution of immortal versus newly synthesized strands. Binucleated CPCs with BrdU intensity ratio of 70:30 or more between daughter nuclei indicative of asymmetric chromatid segregation occur with a frequency of 4.57% and asymmetric chromatid segregation is demonstrated at late mitotic phases. Asymmetric chromatid segregation is significantly enhanced by Pim-1 overexpression in CPCs (9.19% vs 4.79% in eGFP expressing cells, p=0.006).
Conclusions
Asymmetric segregation of chromatids in CPCs is increased nearly twofold Pim-1 kinase overexpression, indicating Pim-1 promotes self renewal of stem cells.
doi:10.1161/CIRCRESAHA.112.267716
PMCID: PMC3357328  PMID: 22441844
immortal DNA strand hypothesis; asymmetric chromatid segregation; asymmetric cell division; BrdU; cytochalasin B
18.  β-Adrenergic Regulation of Cardiac Progenitor Cell Death Versus Survival and Proliferation 
Circulation research  2012;112(3):476-486.
Rationale
Short-term β-adrenergic stimulation promotes contractility in response to stress but is ultimately detrimental in the failing heart because of accrual of cardiomyocyte death. Endogenous cardiac progenitor cell (CPC) activation may partially offset cardiomyocyte losses, but consequences of long-term β-adrenergic drive on CPC survival and proliferation are unknown.
Objective
We sought to determine the relationship between β-adrenergic activity and regulation of CPC function.
Methods and Results
Mouse and human CPCs express only β2 adrenergic receptor (β2-AR) in conjunction with stem cell marker c-kit. Activation of β2-AR signaling promotes proliferation associated with increased AKT, extracellular signal-regulated kinase 1/2, and endothelial NO synthase phosphorylation, upregulation of cyclin D1, and decreased levels of G protein–coupled receptor kinase 2. Conversely, silencing of β2-AR expression or treatment with β2-antagonist ICI 118, 551 impairs CPC proliferation and survival. β1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by metoprolol. Efficacy of β1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed in vivo by adoptive transfer of CPC into failing mouse myocardium.
Conclusions
β-adrenergic stimulation promotes expansion and survival of CPCs through β2-AR, but acquisition of β1-AR on commitment to the myocyte lineage results in loss of CPCs and early myocyte precursors.
doi:10.1161/CIRCRESAHA.112.280735
PMCID: PMC3595054  PMID: 23243208
adrenergic receptors; adrenergic regulation; adult stem cells; cardiac progenitor cells; heart failure
19.  RAGE-Dependent Activation of the Oncoprotein Pim1 Plays a Critical Role in Systemic Vascular Remodeling Processes 
Objective
Vascular remodeling diseases (VRD) are mainly characterized by inflammation and a vascular smooth muscle cells (VSMCs) proproliferative and anti-apoptotic phenotype. Recently, the activation of the advanced glycation endproducts receptor (RAGE) has been shown to promote VSMC proliferation and resistance to apoptosis in VRD in a signal transducer and activator of transcription (STAT)3-dependant manner. Interestingly, we previously described in both cancer and VRD that the sustainability of this proproliferative and antiapoptotic phenotype requires activation of the transcription factor NFAT (nuclear factor of activated T-cells). In cancer, NFAT activation is dependent of the oncoprotein provirus integration site for Moloney murine leukemia virus (Pim1), which is regulated by STAT3 and activated in VRD. Therefore, we hypothesized that RAGE/STAT3 activation in VSMC activates Pim1, promoting NFAT and thus VSMC proliferation and resistance to apoptosis.
Methods/Results
In vitro, freshly isolated human carotid VSMCs exposed to RAGE activator Nε-(carboxymethyl)lysine (CML) for 48 hours had (1) activated STAT3 (increased P-STAT3/STAT3 ratio and P-STAT3 nuclear translocation); (2) increased STAT3-dependent Pim1 expression resulting in NFATc1 activation; and (3) increased Pim1/NFAT-dependent VSMC proliferation (PCNA, Ki67) and resistance to mitochondrial-dependent apoptosis (TMRM, Annexin V, TUNEL). Similarly to RAGE inhibition (small interfering RNA [siRNA]), Pim1, STAT3 and NFATc1 inhibition (siRNA) reversed these abnormalities in human carotid VSMC. Moreover, carotid artery VSMCs isolated from Pim1 knockout mice were resistant to CML-induced VSMC proliferation and resistance to apoptosis. In vivo, RAGE inhibition decreases STAT3/Pim1/NFAT activation, reversing vascular remodeling in the rat carotid artery-injured model.
Conclusion
RAGE activation accounts for many features of VRD including VSMC proliferation and resistance to apoptosis by the activation of STAT3/Pim1/NFAT axis. Molecules aimed to inhibit RAGE could be of a great therapeutic interest for the treatment of VRD.
doi:10.1161/ATVBAHA.111.230573
PMCID: PMC3545710  PMID: 21680901
apoptosis; oncogenes; vascular biology; vascular muscle
20.  Signal Transducers and Activators of Transcription-3/Pim1 Axis Plays a Critical Role in the Pathogenesis of Human Pulmonary Arterial Hypertension 
Circulation  2011;123(11):1205-1215.
Background
Pulmonary artery hypertension (PAH) is a proliferative disorder associated with enhanced pulmonary artery smooth muscle cell proliferation and suppressed apoptosis. The sustainability of this phenotype required the activation of a prosurvival transcription factor like signal transducers and activators of transcription-3 (STAT3) and nuclear factor of activated T cell (NFAT). Because these factors are implicated in several physiological processes, their inhibition in PAH patients could be associated with detrimental effects. Therefore, a better understanding of the mechanism accounting for their expression/activation in PAH pulmonary artery smooth muscle cells is of great therapeutic interest.
Methods and Results
Using multidisciplinary and translational approaches, we demonstrated that STAT3 activation in both human and experimental models of PAH accounts for the expression of both NFATc2 and the oncoprotein kinase Pim1, which trigger NFATc2 activation. Because Pim1 expression correlates with the severity of PAH in humans and is confined to the PAH pulmonary artery smooth muscle cell, Pim1 was identified as an attractive therapeutic target for PAH. Indeed, specific Pim1 inhibition in vitro decreases pulmonary artery smooth muscle cell proliferation and promotes apoptosis, all of which are sustained by NFATc2 inhibition. In vivo, tissue-specific inhibition of Pim1 by nebulized siRNA reverses monocrotaline-induced PAH in rats, whereas Pim1 knockout mice are resistant to PAH development.
Conclusion
We demonstrated for the first time that inhibition of the inappropriate activation of STAT3/Pim1 axis is a novel, specific, and attractive therapeutic strategy to reverse PAH.
doi:10.1161/CIRCULATIONAHA.110.963314
PMCID: PMC3545712  PMID: 21382889
apoptosis; hypertension; pulmonary; proliferation
21.  Cardiac progenitor cells engineered with Pim-1 (CPCeP) develop cardiac phenotypic electrophysiological properties as they are co-cultured with neonatal myocytes 
Stem cell transplantation has been successfully used for amelioration of cardiomyopathic injury using adult cardiac progenitor cells (CPC). Engineering of mouse CPC with the human serine/threonine kinase Pim-1 (CPCeP) enhances regeneration and cell survival in vivo, but it is unknown if such apparent lineage commitment is associated with maturation of electrophysiological properties and excitation–contraction coupling. This study aims to determine electrophysiology and Ca2+-handling properties of CPCeP using neonatal rat cardiomyocyte (NRCM) co-culture to promote cardiomyocyte lineage commitment. Measurements of membrane capacitance, dye transfer, expression of connexin 43 (Cx43), and transmission of ionic currents (ICa, INa) from one cell to the next suggest that a subset of co-cultured CPCeP and NRCM becomes connected via gap junctions. Unlike NRCM, CPCeP had no significant INa, but expressed nifedipine-sensitive ICa that could be measured more consistently with Ba2+ as permeant ion using ramp-clamp protocols than with Ca2+ and step-depolarization protocols. The magnitude of ICa in CPCeP increased during culture (4–7 days vs. 1–3 days) and was larger in co-cultures with NRCM and with NRCM-conditioned medium, than in mono-cultured CPCeP. ICa was virtually absent in CPC without engineered expression of Pim-1. Caffeine and KCl-activated Ca2+-transients were significantly present in co-cultured CPCeP, but smaller than in NRCM. Conversely, ATP-induced (IP3-mediated) Ca2+ transients were larger in CPCeP than in NRCM. INCX and IATP were expressed in equivalent densities in CPCeP and NRCM. These in vitro studies suggest that CPCeP in co-culture with NRCM: a) develop ICa current and Ca2+ signaling consistent with cardiac lineage, b) form electrical connections via Cx43 gap junctions, and c) respond to paracrine signals from NRCM. These properties may be essential for durable and functional myocardial regeneration under in vivo conditions.
doi:10.1016/j.yjmcc.2012.08.008
PMCID: PMC3528406  PMID: 23010478
Stem cell; Calcium; Ion channels; Electromechanical coupling; NCX; Pim-1
22.  EMPOWERING ADULT STEM CELLS FOR MYOCARDIAL REGENERATION 
Circulation Research  2011;109(12):1415-1428.
Treatment strategies for heart failure remain a high priority for ongoing research due to the profound unmet need in clinical disease coupled with lack of significant translational progress. The underlying issue is the same whether the cause is acute damage, chronic stress from disease, or aging: progressive loss of functional cardiomyocytes and diminished hemodynamic output. To stave off cardiomyocyte losses, a number of strategic approaches have been embraced in recent years involving both molecular and cellular approaches to augment myocardial structure and performance. Resultant excitement surrounding regenerative medicine in the heart has been tempered by realizations that reparative processes in the heart are insufficient to restore damaged myocardium to normal functional capacity and that cellular cardiomyoplasty is hampered by poor survival, proliferation, engraftment and differentiation of the donated population. To overcome these limitations, a combination of molecular and cellular approaches needs to be adopted involving use of genetic engineering to enhance resistance to cell death and increase regenerative capacity. This review will highlight biological properties of approached to potentiate stem cell-mediated regeneration to promote enhanced myocardial regeneration, persistence of donated cells, and long lasting tissue repair. Optimizing cell delivery and harnessing the power of survival signaling cascades for ex vivo genetic modification of stem cells prior to reintroduction into the patient will be critical to enhance the efficacy of cellular cardiomyoplasty. Once this goal is achieved, then cell-based therapy has great promise for treatment of heart failure to combat the loss of cardiac structure and function associated with acute damage, chronic disease or aging.
doi:10.1161/CIRCRESAHA.111.243071
PMCID: PMC3266718  PMID: 22158649
regeneration; stem cell; infarction; myocardium
23.  Pim-1 Kinase Inhibits Pathological Injury by Promoting Cardioprotective Signaling 
Stem cells mediate tissue repair throughout the lifespan of an organism. However, the ability of stem cells to mitigate catastrophic damage, such as that sustained after major myocardial infarction is inadequate to rebuild the heart and restore functional capacity. However, capitalizing on the ability of these cells to attenuate damage in the myocardium, various maneuvers that enhance repair mechanisms to improve cardiac structure and function after injury are being investigated. These studies have led to discovery of various factors that mediate cardioprotection and enhance endogenous repair by 1) salvaging surviving myocardium, 2) promoting homing of stem cells and 3) increasing survival and proliferation of stem cell populations at the site of injury. Herein we report upon a downstream target of Akt kinase, named Pim-1, which promotes cardioprotective signaling and enhances cardiac structure and function after pathological injury. The compilation of studies presented here supports use of Pim-1 to enhance long-term myocardial repair after pathological damage.
doi:10.1016/j.yjmcc.2011.01.004
PMCID: PMC3130822  PMID: 21255581
24.  Mitochondrial translocation of Nur77 mediates cardiomyocyte apoptosis 
European Heart Journal  2011;32(17):2179-2188.
Aims
The cascade of events leading to compromised mitochondrial integrity in response to stress is mediated by various combinatorial interactions of pro- and anti-apoptotic molecules. Nur77, an immediate early gene that encodes a nuclear orphan receptor, translocates from the nucleus to mitochondria to induce cytochrome c release and apoptosis in cancer cells in response to various pro-apoptotic treatments. However, the role of Nur77 in the cardiac setting is still unclear. The objective of this study is to determine the physiological relevance and pathophysiological importance of Nur77 in cardiomyocytes.
Methods and results
Myocardial Nur77 is upregulated following cardiomyopathic injury and, while expressed in the postnatal myocardium, declines in level within weeks after birth. Nur77 is localized predominantly in cardiomyocyte nuclei under normal conditions where it is not apoptotic, but translocates to mitochondria in response to oxidative stress both in vitro and in vivo. Mitochondrial localization of Nur77 induces cytochrome c release and typical morphological features of apoptosis, including chromatin condensation and DNA fragmentation. Knockdown of Nur77 rescued hydrogen peroxide-induced cardiomyocyte apoptosis.
Conclusion
Translocation of Nur77 from the nucleus to the mitochondria in cardiomyocytes results in the loss of mitochondrial integrity and subsequent apoptosis in response to ischaemia/reperfusion injury. Our findings identify Nur77 as a novel mediator of cardiomyocyte apoptosis and warrants further investigation of mitochondrial Nur77 translocation as a mechanism to control cell death in the treatment of ischaemic heart diseases.
doi:10.1093/eurheartj/ehq496
PMCID: PMC3164102  PMID: 21228009
Nur77; Mitochondria; Cardiomyocyte; Apoptosis
25.  Roles for ER-associated Degradation (ERAD) and the Novel ER Stress Response Gene, Derlin-3, in the Ischemic Heart 
Circulation Research  2009;106(2):307-316.
Rationale
Stresses, such as ischemia, impair folding of nascent proteins in the rough endoplasmic reticulum (ER), activating the unfolded protein response (UPR), which restores efficient ER protein folding, thus leading to protection from stress. In part, the UPR alleviates ER stress and cell death by increasing the degradation of terminally mis-folded ER proteins via ER-associated degradation (ERAD). ERAD is increased by the ER stress modulator, activating transcription factor 6 (ATF6), which can induce genes that encode components of the ERAD machinery.
Objective
Recently, it was shown that the mouse heart is protected from ischemic damage by ATF6; however, ERAD has not been studied in the cardiac context. A recent microarray study showed that the Derlin-3 (Derl3) gene, which encodes an important component of the ERAD machinery, is robustly induced by ATF6 in the mouse heart.
Methods and Results
In the present study, activated ATF6 induced Derl3 in cultured cardiomyocytes, and in the heart, in vivo. Simulated ischemia (sI), which activates ER stress, induced Derl3 in cultured myocytes, and in an in vivo mouse model of myocardial infarction, Derl3 was also induced. Derl3 overexpression enhanced ERAD and protected cardiomyocytes from sI-induced cell death, while dominant-negative Derl3 decreased ERAD and increased sI-induced cardiomyocyte death.
Conclusions
This study describes a potentially protective role for Derl3 in the heart, and is the first to investigate the functional consequences of enhancing ERAD in the cardiac context.
doi:10.1161/CIRCRESAHA.109.203901
PMCID: PMC3330119  PMID: 19940266
ER stress; unfolded protein response; ischemia; ER-associated degradation

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