Enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for treatment of myocardial infarction.
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.
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.
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.
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.
human cardiac progenitor cells; Pim-1 kinase; heart repair
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.
Notch; Akt; cardioprotection; infarction; myocardium
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.
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.
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.
Sca-1; c-kit; heart; cardiac progenitor cell; infarction; myocardium; Sca-1 knock-out; β–catenin; cardiac development
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.
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.
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.
Pim-1; bone marrow cells; myocardial infarction; myocardial structure; hypertrophy
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.
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.
Notch; Cardioprotection; Progenitor; Regeneration; Crosstalk; Noncanonical
Isl-1; myocardial development
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.
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).
Asymmetric segregation of chromatids in CPCs is increased nearly twofold Pim-1 kinase overexpression, indicating Pim-1 promotes self renewal of stem cells.
immortal DNA strand hypothesis; asymmetric chromatid segregation; asymmetric cell division; BrdU; cytochalasin B
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.
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.
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.
adrenergic receptors; adrenergic regulation; adult stem cells; cardiac progenitor cells; heart failure
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.
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.
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.
apoptosis; oncogenes; vascular biology; vascular muscle
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.
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.
apoptosis; hypertension; pulmonary; proliferation
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.
Stem cell; Calcium; Ion channels; Electromechanical coupling; NCX; Pim-1
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.
regeneration; stem cell; infarction; myocardium
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.
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.
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.
Nur77; Mitochondria; Cardiomyocyte; Apoptosis
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.
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.
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.
ER stress; unfolded protein response; ischemia; ER-associated degradation
Stem cell therapies to regenerate damaged cardiac tissue represent a novel approach to treat heart disease. However, the majority of adoptively transferred stem cells delivered to damaged myocardium do not survive long enough to impart protective benefits, resulting in modest functional improvements. Strategies to improve survival and proliferation of stem cells show promise for significantly enhancing cardiac function and regeneration.
Determine if injected cardiac progenitor cells (CPCs) genetically modified to overexpress nuclear Akt (CPCeA) increase structural and functional benefits to infarcted myocardium relative to control CPCs.
Methods and Results
CPCeA exhibit significantly increased proliferation and secretion of paracrine factors compared to CPCs. However, CPCeA exhibit impaired capacity for lineage commitment in vitro. Infarcted hearts receiving intramyocardial injection of CPCeA have increased recruitment of endogenous c-kit cells compared to CPCs, but neither population provides long-term functional and structural improvements compared to saline injected controls. Pharmacologic inhibition of Akt alleviated blockade of lineage commitment in CPCeA.
Although overexpression of nuclear Akt promotes rapid proliferation and secretion of protective paracrine factors, the inability of CPCeA to undergo lineage commitment hinders their capacity to provide functional or structural benefits to infarcted hearts. Despite enhanced recruitment of endogenous CPCs, lack of functional improvement in CPCeA treated hearts demonstrates CPC lineage commitment is essential to the regenerative response. Effective stem cell therapies must promote cellular survival and proliferation without inhibiting lineage commitment. Since CPCeA exhibit remarkable proliferative potential, an inducible system mediating nuclear Akt expression could be useful to augment cell therapy approaches.
Cardiac Progenitor Cell; Proliferation; Differentiation; Akt
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.
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.
These findings define a mechanistic basis for enhanced myocardial regeneration in transgenic mice overexpressing Pim-1 kinase.
Pim-1; Progenitor Cells; Asymmetric Division
Cardioprotective signaling mediates anti-apoptotic actions through multiple mechanisms including maintenance of mitochondrial integrity. Pim-1 kinase is an essential downstream effector of AKT-mediated cardioprotection but the mechanistic basis for maintenance of mitochondrial integrity by Pim-1 remains unexplored. This study details anti-apoptotic actions responsible for enhanced cell survival in cardiomyocytes with elevated Pim-1 activity.
The purpose of this study is to demonstrate that the cardioprotective kinase Pim-1 acts to inhibit cell death by preserving mitochondrial integrity in cardiomyocytes.
Methods and Results
A combination of biochemical, molecular, and microscopic analyses demonstrate beneficial effects of Pim-1 upon mitochondrial integrity. Pim-1 protein level increases in the mitochondrial fraction with a corresponding decrease in the cytosolic fraction of myocardial lysates from hearts subjected to 30 minutes of ischemia followed by 30 minutes of reperfusion. Cardiac-specific overexpression of Pim-1 results in higher levels of anti-apoptotic Bcl-XL and Bcl-2 compared to samples from normal hearts. In response to oxidative stress challenge Pim-1 preserves the inner mitochondrial membrane potential (ΔΨm). Ultrastructure of the mitochondria is maintained by Pim-1 activity, which prevents swelling induced by calcium overload. Finally, mitochondria isolated from hearts created with cardiac-specific overexpression of Pim-1 show inhibition of cytochrome c release triggered by a truncated form of pro-apoptotic Bid.
Cardioprotective action of Pim-1 kinase includes preservation of mitochondrial integrity during cardiomyopathic challenge conditions, thereby raising the potential for Pim-1 kinase activation as a therapeutic interventional approach to inhibit cell death by antagonizing pro-apoptotic Bcl-2 family members that regulate the intrinsic apoptotic pathway.
Pim-1; mitochondria; cardiomyocyte; apoptosis
The anthracycline doxorubicin (DOX) is an effective chemotherapeutic agent used to treat pediatric cancers, but is associated with cardiotoxicity which can manifest many years after the initial exposure. To date, very little is known about the mechanism of this late onset cardiotoxicity.
Methods and Results
To understand this problem, we developed a pediatric model of late onset DOX-induced cardiotoxicity, where juvenile mice were exposed to DOX, using a cumulative dose that did not induce acute cardiotoxicity. These mice developed normally and had no obvious cardiac abnormalities as adults. However, evaluation of the vasculature revealed that juvenile DOX exposure impaired vascular development resulting in abnormal vascular architecture in the hearts with less branching and decreased capillary density. Both physiological and pathological stress induced late onset cardiotoxicity in the adult DOX mice. Moreover, adult mice subjected to myocardial infarction (MI) developed rapid heart failure which correlated with a failure to increase capillary density in the injured area. Progenitor cells participate in regeneration and blood vessel formation after an MI, but DOX mice had fewer progenitor cells in the infarct border zone. Interestingly, DOX treatment reduced proliferation and differentiation of the progenitor cells into cells of cardiac lineages.
Our data suggest that anthracycline treatment impairs vascular development as well as progenitor cell function in the young heart, resulting in an adult heart that is more susceptible to stress.
anthracyclines; heart failure; vascularization; cardiac progenitor cells
Enterovirus infection in newborn infants is a significant cause of aseptic meningitis and encephalitis. Using a neonatal mouse model, we previously determined that coxsackievirus B3 (CVB3) preferentially targets proliferating neural stem cells located in the subventricular zone within 24 hours after infection. At later time points, immature neuroblasts, and eventually mature neurons, were infected as determined by expression of high levels of viral protein. Here, we show that blood-derived Mac3+ mononuclear cells were rapidly recruited to the CNS within 12 hours after intracranial infection with CVB3. These cells displayed a myeloid-like morphology, were of a peripheral origin based on GFP-tagged adoptive cell transplant examination, and were highly susceptible to CVB3 infection during their migration into the central nervous system (CNS). Serial immunofluorescence images suggested that the myeloid cells enter the CNS via the choroid plexus, and that they may be infected during their extravasation and passage through the choroid plexus epithelium; these infected myeloid cells ultimately penetrate into the parenchyma of the brain. Prior to their migration through the ependymal cell layer (ECL), a subset of these infected myeloid cells expressed detectable levels of nestin, a marker for neural stem and progenitor cells. As these nestin+ myeloid cells infected with CVB3 migrated through the ECL, they revealed distinct morphological characteristics typical of type B neural stem cells. The recruitment of these novel myeloid cells may be specifically set in motion by the induction of a unique chemokine profile in the CNS induced very early after CVB3 infection, which includes upregulation of CCL12. We propose that intracranial CVB3 infection may lead to the recruitment of nestin+ myeloid cells into the CNS which might represent an intrinsic host CNS repair response. In turn, the proliferative and metabolic status of recruited myeloid cells may render them attractive targets for CVB3 infection. Moreover, the migratory ability of these myeloid cells may point to a productive method of virus dissemination within the CNS.
Despite numerous studies demonstrating efficacy of cellular adoptive transfer for therapeutic myocardial regeneration, problems remain for donated cells with regard to survival, persistence, engraftment, and long-term benefits. This study redresses these concerns by enhancing the regenerative potential of adoptively transferred cardiac progenitor cells (CPCs) via genetic engineering to overexpress Pim-1, a cardioprotective kinase that enhances cell survival and proliferation.
Methods and Results
Intramyocardial injections of CPCs overexpressing Pim-1 were given to infarcted female mice. Animals were monitored over 4, 12, and 32-weeks to assess cardiac function and engraftment of Pim-1 CPCs using echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 show increased proliferation and expression of markers consistent with cardiogenic lineage commitment following dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produce greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32-weeks post-delivery. Salutary effects include reduction of infarct size, greater number of c-kit+ cells, and increased vasculature in the damaged region.
Myocardial repair is significantly enhanced by genetic engineering of CPCs using Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.
Gene therapy; infarction; echocardiography; hemodynamics
The recently discovered PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) selectively dephosphorylates Akt at Ser473 and terminates Akt signaling in cancer cells. The regulatory role of PHLPP-1 in the heart has not been considered.
To test the hypothesis that blockade/inhibition of PHLPP-1 could constitute a novel way to enhance Akt signals and provide cardioprotection.
Methods and Results
PHLPP-1 is expressed in neonatal rat ventricular myocytes (NRVMs) and in adult mouse ventricular myocytes (AMVMs). PHLPP-1 knockdown by small interfering RNA significantly enhances phosphorylation of Akt (p-Akt) at Ser473, but not at Thr308, in NRVMs stimulated with leukemia inhibitory factor (LIF). The increased phosphorylation is accompanied by greater Akt catalytic activity. PHLPP-1 knockdown enhances LIF-mediated cardioprotection against doxorubicin and also protects cardiomyocytes against H2O2. Direct Akt effects at mitochondria have been implicated in cardioprotection and mitochondria/cytosol fractionation revealed a significant enrichment of PHLPP-1 at mitochondria. The ability of PHLPP-1 knockdown to potentiate LIF-mediated increases in p-Akt at mitochondria and an accompanying increase in mitochondrial hexokinase-II was demonstrated. We generated PHLPP-1 knockout (KO) mice and demonstrate that AMVMs isolated from KO mice show potentiated p-Akt at Ser473 in response to agonists. When isolated perfused hearts are subjected to ischemia/reperfusion, p-Akt in whole-heart homogenates and in the mitochondrial fraction is significantly increased. Additionally in PHLPP-1 KO hearts, the increase in p-Akt elicited by ischemia/reperfusion is potentiated and, concomitantly, infarct size is significantly reduced.
These results implicate PHLPP-1 as an endogenous negative regulator of Akt activity and cell survival in the heart.
Akt; PHLPP; phosphatase; heart; protection
Cardiac stem cells (CSCs) show potential as a cellular therapeutic approach to blunt tissue damage and facilitate reparative and regenerative processes after myocardial infarction. Despite multiple published reports of improvement, functional benefits remain modest using normal stem cells delivered by adoptive transfer into damaged myocardium. The goal of this study is to enhance survival and proliferation of CSCs that have undergone lineage commitment in early phases as evidenced by expression of proteins driven by the α-myosin heavy chain (αMHC) promoter. The early increased expression of survival kinases augments expansion of the cardiogenic CSC pool and subsequent daughter progeny.
Materials & methods
Normal CSCs engineered with fluorescent reporter protein constructs under control of the αMHC promoter show transgene protein expression, confirming activity of the promoter in CSCs. Cultured CSCs from both nontransgenic and cardiac-specific transgenic mice expressing survival kinases driven by the αMHC promoter were analyzed to characterize transgene expression following treatments to promote differentiation in culture.
Results & conclusion
Therapeutic genes controlled by the αMHC promoter can be engineered into and expressed in CSCs and cardiomyocyte progeny with the goal of improving the efficacy of cardiac stem cell therapy.
AKT; cardiac regeneration; cardiac stem cell; heart; Pim-1; survival kinase; transgene
The phosphoinositide-3-kinase (PI3K) / phosphoinositide dependent kinase 1 (PDK1) signaling pathway exerts cardioprotective effects in the myocardium through activation of key proteins including Akt. Activated Akt accumulates in nuclei of cardiomyocytes suggesting that biologically relevant targets are located in that subcellular compartment. Nuclear Akt activity could be potentiated in both intensity and duration by the presence of a nuclear-associated PI3K / PDK1 signaling cascade as has been described in other non-myocyte cell types. PI3K / PDK1 distribution was determined in vitro and in vivo by immunostaining and nuclear extraction of cultured rat neonatal cardiomyocytes or transgenic mouse hearts. Results show that PI3K and PDK1 are present at a basal level in cardiomyocytes nuclei and that cardioprotective stimulation with atrial natriuretic peptide (ANP) increases their nuclear localization. In comparison, overexpression of nuclear-targeted Akt does not mediate increased translocation of either PI3K or PDK1 indicating that accumulation of Akt does not drive PI3K or PDK1 into the nuclear compartment. Furthermore, PI3K and phospho-Akt473 show parallel temporal accumulation in the nucleus following (MI) infarction challenge. These findings demonstrate the presence of a dynamically regulated nuclear-associated signaling cascade involving PI3K and PDK that presumably influences nuclear Akt activation.
Nucleus; PI3K; PDK1; cardiomyocyte; Akt; phosphoinositide