Denice M. Hodgson

Stem cell differentiation requires a paracrine pathway in the heart

Members of the transforming growth factor pβ (TGF‐β) superfamily‐namely, TGF‐β and BMP2—applied to undifferentiated murine embryonic stem cells up‐regulated mRNA of mesodermal (Brachyury) and cardiac specific transcription factors (Nkx2.5, MEF2C). Embryoid bodies generated from stem cells primed with these growth factors demonstrated an increased potential for cardiac differentiation with a significant increase in beating areas and enhanced myofibrillogenesis. In an environment of postmitotic cardiomyocytes, stem cells engineered to express a fluorescent protein under the control of a cardiac promoter differentiated into fluorescent ventricular myocytes beating in synchrony with host cells, a process significantly enhanced by TGF‐β or BMP2. In vitro, disruption of the TGF‐β/BMP signaling pathways by latency‐associated peptide and/or noggin prevented differentiation of stem cells. In fact, only host cells that secrete a TGF‐β family member induced a cardiac phenotype in stem cells. In vivo, transplantation of stem cells into heart also resulted in cardiac differentiation provided that TGF‐β/BMP2 signaling was intact. In infarcted myocardium, grafted stem cells differentiated into functional cardiomyocytes integrated with surrounding tissue, improving contractile performance. Thus, embryonic stem cells are directed to differentiate into cardiomyocytes by signaling mediated through TGF‐β/BMP2, a cardiac paracrine pathway required for therapeutic benefit of stem cell transplantation in diseased heart.—Behfar, A., Zingman, L. V., Hodgson, D. M., Rauzier, J.‐M., Kane, G. C., Terzic, A., Pucéat, M. Stem cell differentiation requires a paracrine pathway in the heart. FASEB J. 16, 1558–1566 (2002)

ABCC9 mutations identified in human dilated cardiomyopathy disrupt catalytic KATP channel gating

Stress tolerance of the heart requires high-fidelity metabolic sensing by ATP-sensitive potassium (KATP) channels that adjust membrane potential–dependent functions to match cellular energetic demand. Scanning of genomic DNA from individuals with heart failure and rhythm disturbances due to idiopathic dilated cardiomyopathy identified two mutations in ABCC9, which encodes the regulatory SUR2A subunit of the cardiac KATP channel. These missense and frameshift mutations mapped to evolutionarily conserved domains adjacent to the catalytic ATPase pocket within SUR2A. Mutant SUR2A proteins showed aberrant redistribution of conformations in the intrinsic ATP hydrolytic cycle, translating into abnormal KATP channel phenotypes with compromised metabolic signal decoding. Defective catalysis-mediated pore regulation is thus a mechanism for channel dysfunction and susceptibility to dilated cardiomyopathy.

Kir6.2 is required for adaptation to stress

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.

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.

Adenylate kinase phosphotransfer communicates cellular energetic signals to ATP-sensitive potassium channels

Transduction of energetic signals into membrane electrical events governs vital cellular functions, ranging from hormone secretion and cytoprotection to appetite control and hair growth. Central to the regulation of such diverse cellular processes are the metabolism sensing ATP-sensitive K+ (KATP) channels. However, the mechanism that communicates metabolic signals and integrates cellular energetics with KATP channel-dependent membrane excitability remains elusive. Here, we identify that the response of KATP channels to metabolic challenge is regulated by adenylate kinase phosphotransfer. Adenylate kinase associates with the KATP channel complex, anchoring cellular phosphotransfer networks and facilitating delivery of mitochondrial signals to the membrane environment. Deletion of the adenylate kinase gene compromised nucleotide exchange at the channel site and impeded communication between mitochondria and KATP channels, rendering cellular metabolic sensing defective. Assigning a signal processing role to adenylate kinase identifies a phosphorelay mechanism essential for efficient coupling of cellular energetics with KATP channels and associated functions.

Signaling in Channel/Enzyme Multimers: ATPase Transitions in SUR Module Gate ATP-Sensitive K+ Conductance

ATP-sensitive potassium (K(ATP)) channels are bifunctional multimers assembled by an ion conductor and a sulfonylurea receptor (SUR) ATPase. Sensitive to ATP/ADP, K(ATP) channels are vital metabolic sensors. However, channel regulation by competitive ATP/ADP binding would require oscillations in intracellular nucleotides incompatible with cell survival. We found that channel behavior is determined by the ATPase-driven engagement of SUR into discrete conformations. Capture of the SUR catalytic cycle in prehydrolytic states facilitated pore closure, while recruitment of posthydrolytic intermediates translated in pore opening. In the cell, channel openers stabilized posthydrolytic states promoting K(ATP) channel activation. Nucleotide exchange between intrinsic ATPase and ATP/ADP-scavenging systems defined the lifetimes of specific SUR conformations gating K(ATP) channels. Signal transduction through the catalytic module provides a paradigm for channel/enzyme operation and integrates membrane excitability with metabolic cascades.

Bacterial enterotoxins are associated with resistance to colon cancer

One half million patients suffer from colorectal cancer in industrialized nations, yet this disease exhibits a low incidence in under-developed countries. This geographic imbalance suggests an environmental contribution to the resistance of endemic populations to intestinal neoplasia. A common epidemiological characteristic of these colon cancer-spared regions is the prevalence of enterotoxigenic bacteria associated with diarrheal disease. Here, a bacterial heat-stable enterotoxin was demonstrated to suppress colon cancer cell proliferation by a guanylyl cyclase C-mediated signaling cascade. The heat-stable enterotoxin suppressed proliferation by increasing intracellular cGMP, an effect mimicked by the cell-permeant analog 8-br-cGMP. The antiproliferative effects of the enterotoxin and 8-br-cGMP were reversed by L-cis-diltiazem, a cyclic nucleotide-gated channel inhibitor, as well as by removal of extracellular Ca2+, or chelation of intracellular Ca2+. In fact, both the enterotoxin and 8-br-cGMP induced an L-cis-diltiazem-sensitive conductance, promoting Ca2+ influx and inhibition of DNA synthesis in colon cancer cells. Induction of this previously unrecognized antiproliferative signaling pathway by bacterial enterotoxin could contribute to the resistance of endemic populations to intestinal neoplasia, and offers a paradigm for targeted prevention and therapy of primary and metastatic colorectal cancer.

Nucleotide-gated KATP channels integrated with creatine and adenylate kinases: Amplification, tuning and sensing of energetic signals in the compartmentalized cellular environment

Transmission of energetic signals to membrane sensors, such as the ATP-sensitive K+ (KATP) channel, is vital for cellular adaptation to stress. Yet, cell compartmentation implies diffusional hindrances that hamper direct reception of cytosolic energetic signals. With high intracellular ATP levels, KATP channels may sense not bulk cytosolic, but rather local submembrane nucleotide concentrations set by membrane ATPases and phosphotransfer enzymes. Here, we analyzed the role of adenylate kinase and creatine kinase phosphotransfer reactions in energetic signal transmission over the strong diffusional barrier in the submembrane compartment, and translation of such signals into a nucleotide response detectable by KATP channels. Facilitated diffusion provided by creatine kinase and adenylate kinase phosphotransfer dissipated nucleotide gradients imposed by membrane ATPases, and shunted diffusional restrictions. Energetic signals, simulated as deviation of bulk ATP from its basal level, were amplified into an augmented nucleotide response in the submembrane space due to failure under stress of creatine kinase to facilitate nucleotide diffusion. Tuning of creatine kinase-dependent amplification of the nucleotide response was provided by adenylate kinase capable of adjusting the ATP/ADP ratio in the submembrane compartment securing adequate KATP channel response in accord with cellular metabolic demand. Thus, complementation between creatine kinase and adenylate kinase systems, here predicted by modeling and further supported experimentally, provides a mechanistic basis for metabolic sensor function governed by alterations in intracellular phosphotransfer fluxes.

Cellular remodeling in heart failure disrupts KATP channel‐dependent stress tolerance

ATP‐sensitive potassium (KATP) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on KATP channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor α induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, KATP channels responded aberrantly to metabolic signals despite intact intrinsic channel properties, implicating defects proximal to the channel. Indeed, cardiomyocytes from failing hearts exhibited mitochondrial and creatine kinase deficits, and thus a reduced potential for metabolic signal generation and transmission. Consequently, KATP channels failed to properly translate cellular distress under metabolic challenge into a protective membrane response. Failing hearts were excessively vulnerable to metabolic insult, demonstrating cardiomyocyte calcium loading and myofibrillar contraction banding, with tolerance improved by KATP channel openers. Thus, disease‐induced KATP channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.

Administration of Allogenic Stem Cells Dosed to Secure Cardiogenesis and Sustained Infarct Repair

Abstract: The mitotic capacity of heart muscle is too limited to fully substitute for cells lost following myocardial infarction. Emerging stem cell‐based strategies have been proposed to overcome the self‐renewal shortfall of native cardiomyocytes, yet there is limited evidence for their capability to achieve safe de novo cardiogenesis and repair. We present our recent experience in treating long‐term, infarcted hearts with embryonic stem cells, a prototype source for allogenic cell therapy. The cardiogenic potential of the engrafted murine embryonic stem cell colony was pre‐tested by in vitro differentiation, with derived cells positive for nuclear cardiac transcription factors, sarcomeric proteins and functional excitation‐contraction coupling. Eight weeks after infarct, rats were randomized into sham‐ or embryonic stem cell‐treated groups. Acellular sham controls or embryonic stem cells, engineered to express enhanced cyan fluorescent protein (ECFP) under control of the cardiac actin promoter, were injected through a 28‐gauge needle at three sites into the peri‐infarct zone for serial assessment of functional and structural impact. In contrast to results with sham‐treated animals, stem cell therapy yielded, over the 5‐month follow‐up period, new ECFP‐positive cardiomyocytes that integrated with the infarcted myocardium. The stem cell‐treated group showed a stable contractile performance benefit with normalization of myocardial architecture post infarction. Transition of embryonic stem cells into cardiomyocytes required host signaling to support cardiac‐specific differentiation and could result in tumorigenesis if the stem cell dose exceeded the hearts cardioinductive capacity. Supported by the host environment, proper dosing and administration of embryonic stem cells is thus here shown useful in the chronic management of cardiac injury promoting sustained repair.