SUMMARY The bioenergetics of somatic dedifferentiation into induced pluripotent stem cells remains largely unknown. Here, stemness factor mediated nuclear reprogramming reverted mitochondrial networks into cristae-poor structures. Metabolomic footprinting and fingerprinting distinguished derived pluripotent progeny from parental fibroblasts according to elevated glucose utilization and production of glycolytic end products. Temporal sampling demonstrated glycolytic gene potentiation prior to induction of pluripotent markers. Functional metamorphosis of somatic oxidative phosphorylation into acquired pluripotent glycolytic metabolism conformed to an embryonic-like archetype. Stimulation of glycolysis promoted, while blockade of glycolytic enzyme activity blunted, reprogramming efficiency. Metaboproteomics resolved upregulated glycolytic enzymes and downregulated electron transport chain complex I subunits underlying cell fate determination. Thus, the energetic infrastructure of somatic cells transitions into a required glycolytic metabotype to fuel induction of pluripotency.

SUMMARYCardiogenesis 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.

Reaction to stress requires feedback adaptation of cellular functions to secure a response without distress, but the molecular order of this process is only partially understood. Here, we report a previously unrecognized regulatory element in the general adaptation syndrome. Kir6.2, the ion-conducting subunit of the metabolically responsive ATP-sensitive potassium (KATP) channel, was mandatory for optimal adaptation capacity under stress. Genetic deletion of Kir6.2 disrupted KATP channel-dependent adjustment of membrane excitability and calcium handling, compromising the enhancement of cardiac performance driven by sympathetic stimulation, a key mediator of the adaptation response. In the absence of Kir6.2, vigorous sympathetic challenge caused arrhythmia and sudden death, preventable by calcium-channel blockade. Thus, this vital function identifies a physiological role for KATP channels in the heart. I on channels control the electrical potential across the cell membrane of all living organisms. The profile of channel expression within the cell is defined by evolution through natural selection (1, 2). Developed as channel͞enzyme multimers, K ATP channels combine properties of two different classes of protein to adjust rapidly and precisely membrane excitability according to the metabolic state of the cell (3-7). Identified in metabolically active tissues of a broad range of species, K ATP channels were discovered originally in heart muscle where they are expressed in high density (8, 9). Functional cardiac K ATP channels can be formed only through physical association of the pore-forming Kir6.2 subunit with the regulatory sulfonylurea receptor SUR2A (10-12). In this complex, which harbors an intrinsic ATPase activity, nucleotide interaction at SUR2A gates potassium permeation through Kir6.2, a property believed to be responsible for the fine metabolic modulation of membrane potential-dependent cellular functions (7,(13)(14)(15)(16).The physiological role of K ATP channels as metabolic sensors has been understood best in the regulation of hormone secretion in pancreatic ␤-cells and more recently in the hypothalamus (17-21). In the heart, definition of the function of this protein complex thus far has been limited to acute protection against ischemic events (22). In fact, under ischemia, the opening of as few as 1% of K ATP channels is sufficient to produce significant shortening of the cardiac action potential (23), manifested globally by ST-segment elevation on the electrocardiogram (24). Yet, beyond the impact in pathophysiology, a physiological role for the cardiac K ATP channel that supports its maintenance in hearts of many species is lacking (25).The general adaptation syndrome is a ubiquitous reaction vital for self-preservation under conditions of stress such as exertion or fear (26-28). Mediated by a catecholamine surge, this syndrome generates an alteration of physiologic and biochemical functions to sustain a superior level of bodily performance and allows confrontation or escape in response to threat...

Expression of p16(Ink4a) and p19(Arf) increases with age in both rodent and human tissues. However, whether these tumour suppressors are effectors of ageing remains unclear, mainly because knockout mice lacking p16(Ink4a) or p19(Arf) die early of tumours. Here, we show that skeletal muscle and fat, two tissues that develop early ageing-associated phenotypes in response to BubR1 insufficiency, have high levels of p16(Ink4a) and p19(Arf). Inactivation of p16(Ink4a) in BubR1-insufficient mice attenuates both cellular senescence and premature ageing in these tissues. Conversely, p19(Arf) inactivation exacerbates senescence and ageing in BubR1 mutant mice. Thus, we identify BubR1 insufficiency as a trigger for activation of the Cdkn2a locus in certain mouse tissues, and demonstrate that p16(Ink4a) is an effector and p19(Arf) an attenuator of senescence and ageing in these tissues.

Background-Nuclear reprogramming provides an emerging strategy to produce embryo-independent pluripotent stem cells from somatic tissue. Induced pluripotent stem cells (iPS) demonstrate aptitude for de novo cardiac differentiation, yet their potential for heart disease therapy has not been tested. Methods and Results-In this study, fibroblasts transduced with human stemness factors OCT3/4, SOX2, KLF4, and c-MYC converted into an embryonic stem cell-like phenotype and demonstrated the ability to spontaneously assimilate into preimplantation host morula via diploid aggregation, unique to bona fide pluripotent cells. In utero, iPS-derived chimera executed differentiation programs to construct normal heart parenchyma patterning. Within infarcted hearts in the adult, intramyocardial delivery of iPS yielded progeny that properly engrafted without disrupting cytoarchitecture in immunocompetent recipients. In contrast to parental nonreparative fibroblasts, iPS treatment restored postischemic contractile performance, ventricular wall thickness, and electric stability while achieving in situ regeneration of cardiac, smooth muscle, and endothelial tissue.

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.

Objective The goal of this study was to guide bone marrow-derived human mesenchymal stem cells (hMSC) into a cardiac progenitor phenotype, and assess therapeutic benefit in chronic myocardial infarction. Background Adult stem cells, delivered in their naïve state, demonstrate a limited benefit in patients with ischemic heart disease. Preemptive lineage pre-specification may optimize therapeutic outcome. Methods hMSC were harvested from a coronary artery disease patient cohort. A recombinant cocktail consisting of TGFβ1, BMP-4, Activin-A, retinoic acid, IGF-1, FGF-2, α-thrombin and IL-6 was formulated to engage hMSC into cardiopoiesis. Derived hMSC were injected into the myocardium of a nude infarcted murine model, and followed over 1-year for functional and structural end-points. Results While the majority of patient-derived hMSC in their native state demonstrated limited effect on ejection fraction, stem cells from rare individuals harbored a spontaneous capacity to improve contractile performance. This reparative cytotype was characterized by high expression of Nkx2.5, Tbx5, Mesp-1 and Mef2C, markers of cardiopoiesis. Recombinant cardiogenic cocktail guidance secured the cardiopoietic phenotype across the patient cohort. Compared to unguided counterparts, cardiopoietic hMSC delivered into infarcted myocardium achieved superior functional and structural benefit without adverse side effects. Engraftment into murine hearts was associated with increased human-specific nuclear, sarcomeric and gap junction content along with induction of myocardial cell cycle activity. Conclusions Guided cardiopoiesis thus enhances the therapeutic benefit of bone marrow-derived human mesenchymal stem cells in chronic ischemic cardiomyopathy.

Conventional therapies for myocardial infarction attenuate disease progression without contributing significantly to repair. Because of the capacity for de novo cardiogenesis, embryonic stem cells are considered a potential source for myocardial regeneration, yet limited information is available on their ultimate therapeutic value. We treated infarcted rat hearts with CGR8 embryonic stem cells preexamined for cardiogenicity, serially probed left ventricular function, and determined final pathological outcome. Stem cell delivery generated new cardiomyocytes of embryonic stem cell origin that integrated with host myocardium within infarct regions. This resulted in a functional benefit within 3 wk that remained sustained over 12 wk of continuous follow-up and included a vigorous inotropic response to beta-adrenergic challenge. Integration of stem cell-derived cardiomyocytes was associated with normalized ventricular architecture, little scar, and a decrease in signs of myocardial necrosis. In contrast, sham-treated infarcted hearts exhibited ventricular cavity dilation and aneurysm formation, poor ventricular function, and a lack of response to beta-adrenergic stimulation. No evidence of graft rejection, ectopy, sudden cardiac death, or tumor formation was observed after therapy. These findings indicate that embryonic stem cells, through differentiation within the host myocardium, can contribute to a stable beneficial outcome on contractile function and ventricular remodeling in the infarcted heart.