Loss of MCL-1 leads to impaired autophagy and rapid development of heart failure.

Myeloid cell leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 protein that is up-regulated in several human cancers. MCL-1 is also highly expressed in myocardium, but its function in myocytes has not been investigated. We generated inducible, cardiomyocyte-specific Mcl-1 knockout mice and found that ablation of Mcl-1 in the adult heart led to rapid cardiomyopathy and death. Although MCL-1 is known to inhibit apoptosis, this process was not activated in MCL-1-deficient hearts. Ultrastructural analysis revealed disorganized sarcomeres and swollen mitochondria in myocytes. Mitochondria isolated from MCL-1-deficient hearts exhibited reduced respiration and limited Ca(2+)-mediated swelling, consistent with opening of the mitochondrial permeability transition pore (mPTP). Double-knockout mice lacking MCL-1 and cyclophilin D, an essential regulator of the mPTP, exhibited delayed progression to heart failure and extended survival. Autophagy is normally induced by myocardial stress, but induction of autophagy was impaired in MCL-1-deficient hearts. These data demonstrate that MCL-1 is essential for mitochondrial homeostasis and induction of autophagy in the heart. This study also raises concerns about potential cardiotoxicity for chemotherapeutics that target MCL-1.

Functional Effect of Pim1 Depends upon Intracellular Localization in Human Cardiac Progenitor Cells.

Human cardiac progenitor cells (hCPC) improve heart function after autologous transfer in heart failure patients. Regenerative potential of hCPCs is severely limited with age, requiring genetic modification to enhance therapeutic potential. A legacy of work from our laboratory with Pim1 kinase reveals effects on proliferation, survival, metabolism, and rejuvenation of hCPCs in vitro and in vivo. We demonstrate that subcellular targeting of Pim1 bolsters the distinct cardioprotective effects of this kinase in hCPCs to increase proliferation and survival, and antagonize cellular senescence. Adult hCPCs isolated from patients undergoing left ventricular assist device implantation were engineered to overexpress Pim1 throughout the cell (PimWT) or targeted to either mitochondrial (Mito-Pim1) or nuclear (Nuc-Pim1) compartments. Nuc-Pim1 enhances stem cell youthfulness associated with decreased senescence-associated ß-galactosidase activity, preserved telomere length, reduced expression of p16 and p53, and up-regulation of nucleostemin relative to PimWT hCPCs. Alternately, Mito-Pim1 enhances survival by increasing expression of Bcl-2 and Bcl-XL and decreasing cell death after H2O2 treatment, thereby preserving mitochondrial integrity superior to PimWT. Mito-Pim1 increases the proliferation rate by up-regulation of cell cycle modulators Cyclin D, CDK4, and phospho-Rb. Optimal stem cell traits such as proliferation, survival, and increased youthful properties of aged hCPCs are enhanced after targeted Pim1 localization to mitochondrial or nuclear compartments. Targeted Pim1 overexpression in hCPCs allows for selection of the desired phenotypic properties to overcome patient variability and improve specific stem cell characteristics.

Human DMTF1ß antagonizes DMTF1α regulation of the p14(ARF) tumor suppressor and promotes cellular proliferation.

The human DMTF1 (DMP1) transcription factor, a DNA binding protein that interacts with cyclin D, is a positive regulator of the p14ARF (ARF) tumor suppressor. Our earlier studies have shown that three differentially spliced human DMP1 mRNAs, α, ß and γ, arise from the human gene. We now show that DMP1α, ß and γ isoforms differentially regulate ARF expression and promote distinct cellular functions. In contrast to DMP1α, DMP1ß and γ did not activate the ARF promoter, whereas only ß resulted in a dose-dependent inhibition of DMP1α-induced transactivation of the ARF promoter. Ectopic expression of DMP1ß reduced endogenous ARF mRNA levels in human fibroblasts. The DMP1ß- and γ-isoforms share domains necessary for the inhibitory function of the ß-isoform. That DMP1ß may interact with DMP1α to antagonize its function was shown in DNA binding assays and in cells by the close proximity of DMP1α/ß in the nucleus. Cells stably expressing DMP1ß, as well as shRNA targeting all DMP1 isoforms, disrupted cellular growth arrest induced by serum deprivation or in PMA-derived macrophages in the presence or absence of cellular p53. DMP1 mRNA levels in acute myeloid leukemia samples, as compared to granulocytes, were reduced. Treatment of acute promyelocytic leukemia patient samples with all-trans retinoic acid promoted differentiation to granulocytes and restored DMP1 transcripts to normal granulocyte levels. Our findings imply that DMP1α- and ß-ratios are tightly regulated in hematopoietic cells and DMP1ß antagonizes DMP1α transcriptional regulation of ARF resulting in the alteration of cellular control with a gain in proliferation.

Preservation of myocardial structure is enhanced by pim-1 engineering of bone marrow cells.

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: To 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 prosurvival proteins relative to BMCs expressing green fluorescent protein (BMCe). Intramyocardial delivery of BMCeP at time of infarction supports improvements in anterior wall dimensions and prevents left ventricle dilation compared with 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 after 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 upregulation of hypertrophic genes. Thus, BMCeP treatment yields improved structural remodeling of infarcted myocardium compared with 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.

Cardiac progenitor cell commitment is inhibited by nuclear Akt expression.

RATIONALE: 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. OBJECTIVE: To determine whether 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 with 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 with CPCs, but neither population provides long-term functional and structural improvements compared with saline-injected controls. Pharmacological inhibition of Akt alleviated blockade of lineage commitment in CPCeA. CONCLUSIONS: 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. Because CPCeA exhibit remarkable proliferative potential, an inducible system mediating nuclear Akt expression could be useful to augment cell therapy approaches.

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.

Mitochondrial translocation of Nur77 mediates cardiomyocyte apoptosis.

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.

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.

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.

Let-7 coordinately suppresses components of the amino acid sensing pathway to repress mTORC1 and induce autophagy.

Macroautophagy (hereafter autophagy) is the major pathway by which macromolecules and organelles are degraded. Autophagy is regulated by the mTOR signaling pathway-the focal point for integration of metabolic information, with mTORC1 playing a central role in balancing biosynthesis and catabolism. Of the various inputs to mTORC1, the amino acid sensing pathway is among the most potent. Based upon transcriptome analysis of neurons subjected to nutrient deprivation, we identified let-7 microRNA as capable of promoting neuronal autophagy. We found that let-7 activates autophagy by coordinately downregulating the amino acid sensing pathway to prevent mTORC1 activation. Let-7 induced autophagy in the brain to eliminate protein aggregates, establishing its physiological relevance for in vivo autophagy modulation. Moreover, peripheral delivery of let-7 anti-miR repressed autophagy in muscle and white fat, suggesting that let-7 autophagy regulation extends beyond CNS. Hence, let-7 plays a central role in nutrient homeostasis and proteostasis regulation in higher organisms.

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.

Alternative splicing of the human cyclin D-binding Myb-like protein (hDMP1) yields a truncated protein isoform that alters macrophage differentiation patterns.

We have cloned two novel, alternatively spliced messages of human cyclin D-binding Myb-like protein (hDMP1). The known, full-length protein has been named hDMP1alpha and the new isoforms, hDMP1beta and hDMP1gamma. The hDMP1alpha, -beta, and -gamma splice variants have unique expression patterns in normal hematopoietic cells; hDMP1beta mRNA transcripts are strongly expressed in quiescent CD34+ cells and freshly isolated peripheral blood leukocytes, as compared with hDMP1alpha. In contrast, activated T-cells and developing myeloid cells, macrophages, and granulocytes express low levels of hDMP1beta transcripts, and hDMP1gamma is ubiquitously and weakly expressed. Mouse Dmp1 has been shown to activate CD13/aminopeptidase N (APN) and p19ARF gene expression via binding to canonical DNA recognition sites in the respective promoters. Assessment of CD13/APN promoter responsiveness demonstrated that hDMP1alpha but not hDMP1beta and -gamma, is a transcriptional activator. Furthermore, hDMP1beta was found to inhibit the CD13/APN promoter transactivation ability of hDMP1alpha. Stable, ectopic expression of hDMP1beta and, to a lesser extent hDMP1gamma, reduced endogenous cell surface levels of CD13/APN in U937 cells. Moreover, stable, ectopic expression of hDMP1beta altered phorbol 12-myristate 13-acetate-induced terminal differentiation of U937 cells to macrophages and resulted in maintenance of proliferation. These results demonstrate that hDMP1beta antagonizes hDMP1alpha activity and suggest that cellular functions of hDMP1 may be regulated by cellular hDMP1 isoform levels.