Randolph S. Faustino

Mitochondrial oxidative metabolism is required for the cardiac differentiation of stem cells

Cardiogenesis within embryos or associated with heart repair requires stem cell differentiation into energetically competent, contracting cardiomyocytes. While it is widely accepted that the coordination of genetic circuits with developmental bioenergetics is critical to phenotype specification, the metabolic mechanisms that drive cardiac transformation are largely unknown. Here, we aim to define the energetic requirements for and the metabolic microenvironment needed to support the cardiac differentiation of embryonic stem cells. We demonstrate that anaerobic glycolytic metabolism, while sufficient for embryonic stem cell homeostasis, must be transformed into the more efficient mitochondrial oxidative metabolism to secure cardiac specification and excitation–contraction coupling. This energetic switch was programmed by rearrangement of the metabolic transcriptome that encodes components of glycolysis, fatty acid oxidation, the Krebs cycle, and the electron transport chain. Modifying the copy number of regulators of mitochondrial fusion and fission resulted in mitochondrial maturation and network expansion, which in turn provided an energetic continuum to supply nascent sarcomeres. Disrupting respiratory chain function prevented mitochondrial organization and compromised the energetic infrastructure, causing deficient sarcomerogenesis and contractile malfunction. Thus, establishment of the mitochondrial system and engagement of oxidative metabolism are prerequisites for the differentiation of stem cells into a functional cardiac phenotype. Mitochondria-dependent energetic circuits are thus critical regulators of de novo cardiogenesis and targets for heart regeneration.

Cardiopoietic programming of embryonic stem cells for tumor-free heart repair

Embryonic stem cells have the distinct potential for tissue regeneration, including cardiac repair. Their propensity for multilineage differentiation carries, however, the liability of neoplastic growth, impeding therapeutic application. Here, the tumorigenic threat associated with embryonic stem cell transplantation was suppressed by cardiac-restricted transgenic expression of the reprogramming cytokine TNF-α, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-α to promote cardiac differentiation was recapitulated in embryoid bodies in vitro. The procardiogenic action required an intact endoderm and was mediated by secreted cardio-inductive signals. Resolved TNF-α–induced endoderm-derived factors, combined in a cocktail, secured guided differentiation of embryonic stem cells in monolayers produce cardiac progenitors termed cardiopoietic cells. Characterized by a down-regulation of oncogenic markers, up-regulation, and nuclear translocation of cardiac transcription factors, this predetermined population yielded functional cardiomyocyte progeny. Recruited cardiopoietic cells delivered in infarcted hearts generated cardiomyocytes that proliferated into scar tissue, integrating with host myocardium for tumor-free repair. Thus, cardiopoietic programming establishes a strategy to hone stem cell pluripotency, offering a tumor-resistant approach for regeneration.

CXCR4+/FLK-1+ Biomarkers Select a Cardiopoietic Lineage from Embryonic Stem Cells

Pluripotent stem cells demonstrate an inherent propensity for unrestricted multi‐lineage differentiation. Translation into regenerative applications requires identification and isolation of tissue‐specified progenitor cells. From a comprehensive pool of 11,272 quality‐filtered genes, profiling embryonic stem cells at discrete stages of cardiopoiesis revealed 736 transcripts encoding membrane‐associated proteins, where 306 were specifically upregulated with cardiogenic differentiation. Bioinformatic dissection of exposed surface biomarkers prioritized the chemokine receptor cluster as the most significantly over‐represented gene receptor family during pre cardiac induction, with CXCR4 uniquely associated with mesendoderm formation. CXCR4+ progenitors were sorted from the embryonic stem cell pool into mesoderm‐restricted progeny according to co‐expression with the early mesoderm marker Flk‐1. In contrast to CXCR4−/Flk‐1− cells, the CXCR4+/Flk‐1+ subpopulation demonstrated overexpressed cardiac lineage transcription factors (Mef2C, Myocardin, Nkx2.5), whereas pluripotent genes (Oct4, Fgf4, Sox2) as well as neuroectoderm (Sox1) and endoderm alpha‐fetoprotein markers were all depleted. In fact, the CXCR4+/Flk‐1+ biomarker combination identified embryonic stem cell progeny significantly enriched with Mesp‐1, GATA‐4, and Tbx5, indicative of pre cardiac mesoderm and the primary heart field. Although the CXCR4+/Flk‐1+ transcriptome shared 97% identity with the CXCR4−/Flk‐1− counterpart, the 818 divergent gene set represented predominantly cardiovascular developmental functions and formed a primitive cardiac network. Differentiation of CXCR4+/Flk‐1+ progenitors yielded nuclear translocation of myocardial transcription factors and robust sarcomerogenesis with nascent cardiac tissue demonstrating beating activity and calcium transients. Thus, the CXCR4/Flk‐1 biomarker pair predicts the emergence of cardiogenic specification within a pluripotent stem cell pool, enabling targeted selection of cardiopoietic lineage.

Protection conferred by myocardial ATP-sensitive K+ channels in pressure overload-induced congestive heart failure revealed in KCNJ11 Kir6.2-null mutant

Ventricular load can precipitate development of the heart failure syndrome, yet the molecular components that control the cardiac adaptive response to imposed demand remain partly understood. Compromised ATP‐sensitive K+ (KATP) channel function renders the heart vulnerable to stress, implicating this metabolic sensor in the homeostatic response that would normally prevent progression of cardiac disease. Here, pressure overload was imposed on the left ventricle by transverse aortic constriction in the wild‐type and in mice lacking sarcolemmal KATP channels through Kir6.2 pore knockout (Kir6.2‐KO). Despite equivalent haemodynamic loads, within 30 min of aortic constriction, Kir6.2‐KO showed an aberrant prolongation of action potentials with intracellular calcium overload and ATP depletion, whereas wild‐type maintained ionic and energetic handling. On catheterization, constricted Kir6.2‐KO displayed compromised myocardial performance with elevated left ventricular end‐diastolic pressure, not seen in the wild‐type. Glyburide, a KATP channel inhibitor, reproduced the knockout phenotype in the wild‐type, whereas the calcium channel antagonist, verapamil, prevented abnormal outcome in Kir6.2‐KO. Within 48 h following aortic constriction, fulminant biventricular congestive heart failure, characterized by exercise intolerance, cardiac contractile dysfunction, hepatopulmonary congestion and ascites, halved the Kir6.2‐KO cohort, while no signs of organ failure or mortality were seen in wild‐type. Surviving Kir6.2‐KO developed premature and exaggerated fibrotic myocardial hypertrophy associated with nuclear up‐regulation of calcium‐dependent pro‐remodelling MEF2 and NF‐AT pathways, precipitating chamber dilatation within 3 weeks. Thus, KATP channels appear mandatory in acute and chronic cardiac adaptation to imposed haemodynamic load, protecting against congestive heart failure and death.

Glycolytic network restructuring integral to the energetics of embryonic stem cell cardiac differentiation

Decoding of the bioenergetic signature underlying embryonic stem cell cardiac differentiation has revealed a mandatory transformation of the metabolic infrastructure with prominent mitochondrial network expansion and a distinctive switch from glycolysis to oxidative phosphorylation. Here, we demonstrate that despite reduction in total glycolytic capacity, stem cell cardiogenesis engages a significant transcriptome, proteome, as well as enzymatic and topological rearrangement in the proximal, medial, and distal modules of the glycolytic pathway. Glycolytic restructuring was manifested by a shift in hexokinase (Hk) isoforms from Hk-2 to cardiac Hk-1, with intracellular and intermyofibrillar localization mapping mitochondrial network arrangement. Moreover, upregulation of cardiac-specific enolase 3, phosphofructokinase, and phosphoglucomutase and a marked increase in glyceraldehyde 3-phosphate dehydrogenase (GAPDH) phosphotransfer activity, along with apparent post-translational modifications of GAPDH and phosphoglycerate kinase, were all distinctive for derived cardiomyocytes compared to the embryonic stem cell source. Lactate dehydrogenase (LDH) isoforms evolved towards LDH-2 and LDH-3, containing higher proportions of heart-specific subunits, and pyruvate dehydrogenase isoforms rearranged between E1alpha and E1beta, transitions favorable for substrate oxidation in mitochondria. Concomitantly, transcript levels of fetal pyruvate kinase isoform M2, aldolase 3, and transketolase, which shunt the glycolytic with pentose phosphate pathways, were reduced. Collectively, changes in glycolytic pathway modules indicate active redeployment, which would facilitate connectivity of the expanding mitochondrial network with ATP utilization sites. Thus, the delineated developmental dynamics of the glycolytic phosphotransfer network is integral to the remodeling of cellular energetic infrastructure underlying stem cell cardiogenesis.

Guided stem cell cardiopoiesis: Discovery and translation

Over 1000 patients have participated worldwide in clinical trials exploring the therapeutic value of bone marrow-derived cells in ischemic heart disease. Meta-analysis evaluation of this global effort indicates that adult stem cell therapy is in general safe, but yields a rather modest level of improvement in cardiac function and structural remodeling in the setting of acute myocardial infarction or chronic heart failure. Although promising, the potential of translating adult stem cell-based therapy from bench to bedside has yet to be fully realized. Inter-trial and inter-patient variability contribute to disparity in the regenerative potential of transplanted stem cells with unpredictable efficacy on follow-up. Strategies that mimic the natural embryonic program for uniform recruitment of cardiogenic progenitors from adult sources are currently tested to secure consistent outcome. Guided cardiopoiesis has been implemented with mesenchymal stem cells obtained from bone marrow of healthy volunteers, using a cocktail of secreted proteins that recapitulate components of the endodermal secretome critical for cardiogenic induction of embryonic mesoderm. With appropriate validation of this newly derived cardiopoietic phenotype, the next generation of trials should achieve demonstrable benefit across patient populations.

Cardioinductive Network Guiding Stem Cell Differentiation Revealed by Proteomic Cartography of Tumor Necrosis Factor α‐Primed Endodermal Secretome

In the developing embryo, instructive guidance from the ventral endoderm secures cardiac program induction within the anterolateral mesoderm. Endoderm‐guided cardiogenesis, however, has yet to be resolved at the proteome level. Here, through cardiopoietic priming of the endoderm with the reprogramming cytokine tumor necrosis factor α (TNFα), candidate effectors of embryonic stem cell cardiac differentiation were delineated by comparative proteomics. Differential two‐dimensional gel electrophoretic mapping revealed that more than 75% of protein species increased >1.5‐fold in the TNFα‐primed versus unprimed endodermal secretome. Protein spot identification by linear ion trap quadrupole (LTQ) tandem mass spectrometry (MS/MS) and validation by shotgun LTQ‐Fourier transform MS/MS following multidimensional chromatography mapped 99 unique proteins from 153 spot assignments. A definitive set of 48 secretome proteins was deduced by iterative bioinformatic screening using algorithms for detection of canonical and noncanonical indices of secretion. Protein‐protein interaction analysis, in conjunction with respective expression level changes, revealed a nonstochastic TNFα‐centric secretome network with a scale‐free hierarchical architecture. Cardiovascular development was the primary developmental function of the resolved TNFα‐anchored network. Functional cooperativity of the derived cardioinductive network was validated through direct application of the TNFα‐primed secretome on embryonic stem cells, potentiating cardiac commitment and sarcomerogenesis. Conversely, inhibition of primary network hubs negated the procardiogenic effects of TNFα priming. Thus, proteomic cartography establishes a systems biology framework for the endodermal secretome network guiding stem cell cardiopoiesis.

Genomic chart guiding embryonic stem cell cardiopoiesis

BackgroundEmbryonic stem cells possess a pluripotent transcriptional background with the developmental capacity for distinct cell fates. Simultaneous expression of genetic elements for multiple outcomes obscures cascades relevant to specific cell phenotypes. To map molecular patterns critical to cardiogenesis, we interrogated gene expression in stem cells undergoing guided differentiation, and defined a genomic paradigm responsible for confinement of pluripotency.ResultsFunctional annotation analysis of the transcriptome of differentiating embryonic stem cells exposed downregulated components of DNA replication, recombination and repair machinery, cell cycling, cancer mechanisms, and RNA post-translational modifications. Concomitantly, cardiovascular development, cell-to-cell signaling, cell development and cell movement were upregulated. These simultaneous gene ontology rearrangements engaged a repertoire switch that specified lineage development. Bioinformatic integration of genomic and gene ontology data further unmasked canonical signaling cascades prioritized within discrete phases of cardiopoiesis. Examination of gene relationships revealed a non-stochastic network anchored by integrin, WNT/β-catenin, transforming growth factor β and vascular endothelial growth factor pathways, validated by manipulation of selected cascades that promoted or restrained cardiogenic yield. Moreover, candidate genes within anchor pathways acted as nodes that organized correlated expression profiles into functional clusters, which collectively orchestrated and secured an overall cardiogenic theme.ConclusionThe present systems biology approach reveals a dynamically integrated and tractable gene network fundamental to embryonic stem cell specification, and represents an initial step towards resolution of a genomic cardiopoietic atlas.

Stem cells transform into a cardiac phenotype with remodeling of the nuclear transport machinery

Nuclear transport of transcription factors is a critical step in stem cell commitment to a tissue-specific lineage. While it is recognized that nuclear pores are gatekeepers of nucleocytoplasmic exchange, it is unknown how the nuclear transport machinery becomes competent to support genetic reprogramming and cell differentiation. Here, we report the dynamics of nuclear transport factor expression and nuclear pore microanatomy during cardiac differentiation of embryonic stem cells. Cardiac progeny derived from pluripotent stem cells displayed a distinct proteomic profile characterized by the emergence of cardiac-specific proteins. This profile correlated with the nuclear translocation of cardiac transcription factors. The nuclear transport genes, including nucleoporins, importins, exportins, transportins, and Ran-related factors, were globally downregulated at the genomic level, streamlining the differentiation program underlying stem cell-derived cardiogenesis. Establishment of the cardiac molecular phenotype was associated with an increased density of nuclear pores spanning the nuclear envelope. At nanoscale resolution, individual nuclear pores exhibited conformational changes resulting in the expansion of the pore diameter and an augmented probability of conduit occupancy. Thus, embryonic stem cells undergo adaptive remodeling of the nuclear transport infrastructure associated with nuclear translocation of cardiac transcription factors and execution of the cardiogenic program, underscoring the plasticity of the nucleocytoplasmic trafficking machinery in accommodating differentiation requirements.

Nuclear Transport: Target for Therapy

Drugs directed at plasma membrane receptors target environment–cell interactions and are the mainstay of clinical pharmacology. Decoding mechanisms that govern intracellular signaling has recently opened new therapeutic avenues for interventions at cytosol–organellar interfaces. The nuclear envelope and nuclear transport machinery have emerged central in the discovery and development of experimental therapeutics capable of modulating cellular genetic programs. Insight into nucleocytoplasmic exchange has unmasked promising anticancer, antiviral, and anti‐inflammatory strategies.