Myocardial AKT: the omnipresent nexus.

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.

Pim-1 preserves mitochondrial morphology by inhibiting dynamin-related protein 1 translocation.

Mitochondrial morphological dynamics affect the outcome of ischemic heart damage and pathogenesis. Recently, mitochondrial fission protein dynamin-related protein 1 (Drp1) has been identified as a mediator of mitochondrial morphological changes and cell death during cardiac ischemic injury. In this study, we report a unique relationship between Pim-1 activity and Drp1 regulation of mitochondrial morphology in cardiomyocytes challenged by ischemic stress. Transgenic hearts overexpressing cardiac Pim-1 display reduction of total Drp1 protein levels, increased phosphorylation of Drp1-(S637), and inhibition of Drp1 localization to the mitochondria. Consistent with these findings, adenoviral-induced Pim-1 neonatal rat cardiomyocytes (NRCMs) retain a reticular mitochondrial phenotype after simulated ischemia (sI) and decreased Drp1 mitochondrial sequestration. Interestingly, adenovirus Pim-dominant negative NRCMs show increased expression of Bcl-2 homology 3 (BH3)-only protein p53 up-regulated modulator of apoptosis (PUMA), which has been previously shown to induce Drp1 accumulation at mitochondria and increase sensitivity to apoptotic stimuli. Overexpression of the p53 up-regulated modulator of apoptosis-dominant negative adenovirus attenuates localization of Drp1 to mitochondria in adenovirus Pim-dominant negative NRCMs promotes reticular mitochondrial morphology and inhibits cell death during sI. Therefore, Pim-1 activity prevents Drp1 compartmentalization to the mitochondria and preserves reticular mitochondrial morphology in response to sI.

Asymmetric chromatid segregation in cardiac progenitor cells is enhanced by Pim-1 kinase.

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 the capacity of CPCs 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: We aimed to 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. The CPC DNA was labeled for several generations and then blocked in second cytokinesis during chase to determine distribution of immortal versus newly synthesized strands. Intensity ratios of binucleated CPCs with bromodeoxyuridine of ≥70:30 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 versus 4.79 in eGFP-expressing cells; P=0.006). CONCLUSIONS: Asymmetric segregation of chromatids in CPCs is increased nearly two-fold with Pim-1 kinase overexpression, indicating that Pim-1 promotes self-renewal of stem cells.

Nucleolar stress is an early response to myocardial damage involving nucleolar proteins nucleostemin and nucleophosmin.

Nucleolar stress, characterized by loss of nucleolar integrity, has not been described in the cardiac context. In addition to ribosome biogenesis, nucleoli are critical for control of cell proliferation and stress responses. Our group previously demonstrated induction of the nucleolar protein nucleostemin (NS) in response to cardiac pathological insult. NS interacts with nucleophosmin (NPM), a marker of nucleolar stress with cytoprotective properties. The dynamic behavior of NS and NPM reveal that nucleolar disruption is an early event associated with stress response in cardiac cells. Rapid translocation of NS and NPM to the nucleoplasm and suppression of new preribosomal RNA synthesis occurs in both neonatal rat cardiomyocytes (NRCM) and cardiac progenitor cells (CPC) upon exposure to doxorubicin or actinomycin D. Silencing of NS significantly increases cell death resulting from doxorubicin treatment in CPC, whereas NPM knockdown alone induces cell death. Overexpression of either NS or NPM significantly decreases caspase 8 activity in cultured cardiomyocytes challenged with doxorubicin. The presence of altered nucleolar structures resulting from myocardial infarction in mice supports the model of nucleolar stress as a general response to pathological injury. Collectively, these findings serve as the initial description of myocardial nucleolar stress and establish the postulate that nucleoli acts as sensors of stress, regulating the cellular response to pathological insults.

Increased mitotic rate coincident with transient telomere lengthening resulting from pim-1 overexpression in cardiac progenitor cells.

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.

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 alpha-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 alpha-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.

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. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure."

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

Pim-1 kinase exerts potent cardioprotective effects in the myocardium downstream of AKT, but the participation of Pim-1 in cardiac hypertrophy requires investigation. Cardiac-specific expression of Pim-1 (Pim-WT) or the dominant-negative mutant of Pim-1 (Pim-DN) in transgenic mice together with adenoviral-mediated overexpression of these Pim-1 constructs was used to delineate the role of Pim-1 in hypertrophy. Transgenic overexpression of Pim-1 protects mice from pressure-overload-induced hypertrophy relative to wild-type controls as evidenced by improved hemodynamic function, decreased apoptosis, increases in antihypertrophic proteins, smaller myocyte size, and inhibition of hypertrophic signaling after challenge. Similarly, Pim-1 overexpression in neonatal rat cardiomyocyte cultures inhibits hypertrophy induced by endothelin-1. On the cellular level, hearts of Pim-WT mice show enhanced incorporation of BrdU into myocytes and a hypercellular phenotype compared to wild-type controls after hypertrophic challenge. In comparison, transgenic overexpression of Pim-DN leads to dilated cardiomyopathy characterized by increased apoptosis, fibrosis, and severely depressed cardiac function. Furthermore, overexpression of Pim-DN leads to reduced contractility as evidenced by reduced Ca(2+) transient amplitude and decreased percentage of cell shortening in isolated myocytes. These data support a pivotal role for Pim-1 in modulation of hypertrophy by impacting responses on molecular, cellular, and organ levels.

Pim1 maintains telomere length in mouse cardiomyocytes by inhibiting TGFß signalling.

AIMS: Telomere attrition in cardiomyocytes is associated with decreased contractility, cellular senescence, and up-regulation of proapoptotic transcription factors. Pim1 is a cardioprotective kinase that antagonizes the aging phenotype of cardiomyocytes and delays cellular senescence by maintaining telomere length, but the mechanism remains unknown. Another pathway responsible for regulating telomere length is the transforming growth factor beta (TGFß) signalling pathway where inhibiting TGFß signalling maintains telomere length. The relationship between Pim1 and TGFß has not been explored. This study delineates the mechanism of telomere length regulation by the interplay between Pim1 and components of TGFß signalling pathways in proliferating A549 cells and post-mitotic cardiomyocytes. METHODS AND RESULTS: Telomere length was maintained by lentiviral-mediated overexpression of PIM1 and inhibition of TGFß signalling in A549 cells. Telomere length maintenance was further demonstrated in isolated cardiomyocytes from mice with cardiac-specific overexpression of PIM1 and by pharmacological inhibition of TGFß signalling. Mechanistically, Pim1 inhibited phosphorylation of Smad2, preventing its translocation into the nucleus and repressing expression of TGFß pathway genes. CONCLUSION: Pim1 maintains telomere lengths in cardiomyocytes by inhibiting phosphorylation of the TGFß pathway downstream effectors Smad2 and Smad3, which prevents repression of telomerase reverse transcriptase. Findings from this study demonstrate a novel mechanism of telomere length maintenance and provide a potential target for preserving cardiac function.

Enhancement of myocardial regeneration through genetic engineering of cardiac progenitor cells expressing Pim-1 kinase.

BACKGROUND: Despite numerous studies demonstrating the 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 with echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 showed increased proliferation and expression of markers consistent with cardiogenic lineage commitment after dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produced greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32 weeks after delivery. Salutary effects include reduction of infarct size, greater number of c-kit(+) cells, and increased vasculature in the damaged region. CONCLUSIONS: Myocardial repair is significantly enhanced by genetic engineering of CPCs with Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.

Activation of Notch-mediated protective signaling in the myocardium.

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.

Evolution of the c-kit-positive cell response to pathological challenge in the myocardium.

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.

Targeting p16-induced senescence prevents cigarette smoke-induced emphysema by promoting IGF1/Akt1 signaling in mice.

Senescence is a mechanism associated with aging that alters tissue regeneration by depleting the stem cell pool. Chronic obstructive pulmonary disease (COPD) displays hallmarks of senescence, including a diminished stem cell population. DNA damage from cigarette smoke (CS) induces senescence via the p16 pathway. This study evaluated the contribution of p16 to CS-associated lung pathologies. p16 expression was prominent in human COPD lungs compared with normal subjects. CS induces impaired pulmonary function, emphysema, and increased alveolar epithelial cell (AECII) senescence in wild-type mice, whereas CS-exposed p16-/- mice exhibit normal pulmonary function, reduced emphysema, diminished AECII senescence, and increased pro-growth IGF1 signaling, suggesting that improved lung function in p16-/- mice was due to increased alveolar progenitor cell proliferation. In conclusion, our study suggests that targeting senescence may facilitate alveolar regeneration in COPD emphysema by promoting IGF1 proliferative signaling.

Pharmacodynamics of telomerase inhibition and telomere shortening by noncytotoxic suramin.

We reported that suramin is an effective chemosensitizer at noncytotoxic concentrations (<50 µM); this effect was observed in multiple types of human xenograft tumors in vitro and in vivo. Clinical evaluation of noncytotoxic suramin is ongoing. Because (a) suramin inhibits reverse transcriptase, (b) telomerase is a reverse transcriptase, and (c) inhibition of telomerase enhances tumor chemosensitivity, we studied the pharmacodynamics of noncytotoxic suramin on telomerase activity and telomere length in cultured cells and tumors grown in animals. In three human cancer cells that depend on telomerase for telomere maintenance (pharynx FaDu, prostate PC3, breast MCF7), suramin inhibited telomerase activity in cell extracts and intact cells at concentrations that exhibited no cytotoxicity (IC50 of telomerase was between 1 and 3 µM vs. >60 µM for cytotoxicity), and continuous treatment at 10-25 µM for 6 weeks resulted in gradual telomere shortening (maximum of 30%) and cell senescence (measured by ß-galactosidase activity and elevation of mRNA levels of two senescence markers p16 and p21). In contrast, noncytotoxic suramin did not shorten the telomere in telomerase-independent human osteosarcoma Saos-2 cells. In mice bearing FaDu tumors, treatment with noncytotoxic suramin for 6 weeks resulted in telomere erosion in >95% of the tumor cells with an average telomere shortening of >40%. These results indicate noncytotoxic suramin inhibits telomerase, shortens telomere and induces cell senescence, and suggest telomerase inhibition as a potential mechanism of its chemosensitization.

Human cardiac progenitor cells engineered with Pim-I kinase enhance myocardial repair.

OBJECTIVES: The goal of this study was to demonstrate the enhancement of human cardiac progenitor cell (hCPC) reparative and regenerative potential by genetic modification for the 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 the myocardium of heart failure patients undergoing left ventricular assist device implantation were engineered to express green fluorescent protein (hCPCe) or Pim-1-GFP (hCPCeP). Functional tests of hCPC regenerative potential were performed with immunocompromised mice by using intramyocardial adoptive transfer injection after infarction. Myocardial structure and function were monitored by echocardiographic and hemodynamic assessment for 20 weeks after delivery. hCPCe and hCPCeP expressing luciferase were observed by using bioluminescence imaging to noninvasively track persistence. RESULTS: hCPCeP exhibited augmentation of reparative potential relative to hCPCe control cells, as shown 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 demonstrated increased cellular engraftment and differentiation with improved vasculature and reduced infarct size. Enhanced persistence of hCPCeP versus hCPCe was revealed by bioluminescence imaging at up to 8 weeks post-delivery. CONCLUSIONS: Genetic engineering of hCPCs with Pim-1 enhanced 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 hCPCs 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 pre-clinical settings.

Cardiac stem cell genetic engineering using the alphaMHC promoter.

AIMS: 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 alpha-myosin heavy chain (alphaMHC) 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 alphaMHC 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 alphaMHC promoter were analyzed to characterize transgene expression following treatments to promote differentiation in culture. RESULTS & CONCLUSION: Therapeutic genes controlled by the alphaMHC promoter can be engineered into and expressed in CSCs and cardiomyocyte progeny with the goal of improving the efficacy of cardiac stem cell therapy.