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...

Members of the transforming growth factor beta1 (TGF-beta) superfamily--namely, TGF-beta 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-beta or BMP2. In vitro, disruption of the TGF-beta/BMP signaling pathways by latency-associated peptide and/or noggin prevented differentiation of stem cells. In fact, only host cells that secrete a TGF-beta 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-beta/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-beta/BMP2, a cardiac paracrine pathway required for therapeutic benefit of stem cell transplantation in diseased heart.

Atrial fibrillation is a rhythm disorder characterized by chaotic electrical activity of cardiac atria. Predisposing to stroke and heart failure, this common condition is increasingly recognized as a heritable disorder. To identify genetic defects conferring disease susceptibility, patients with idiopathic atrial fibrillation, lacking traditional risk factors, were evaluated. Genomic DNA scanning revealed a nonsense mutation in KCNA5 that encodes Kv1.5, a voltage-gated potassium channel expressed in human atria. The heterozygous E375X mutation, present in a familial case of atrial fibrillation and absent in 540 unrelated control individuals, introduced a premature stop codon disrupting the Kv1.5 channel protein. The truncation eliminated the S4-S6 voltage sensor, pore region and C-terminus, preserving the N-terminus and S1-S3 transmembrane domains that secure tetrameric subunit assembly. Heterologously expressed recombinant E375X mutant failed to generate the ultrarapid delayed rectifier current I(Kur) vital for atrial repolarization and exerted a dominant-negative effect on wild-type current. Loss of channel function translated into action potential prolongation and early after-depolarization in human atrial myocytes, increasing vulnerability to stress-provoked triggered activity. The pathogenic link between compromised Kv1.5 function and susceptibility to atrial fibrillation was verified, at the organism level, in a murine model. Rescue of the genetic defect was achieved by aminoglycoside-induced translational read-through of the E375X premature stop codon, restoring channel function. This first report of Kv1.5 loss-of-function channelopathy establishes KCNA5 mutation as a novel risk factor for repolarization deficiency and atrial fibrillation.

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.

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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 K ATP 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 K ATP 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 K ATP channels and associated functions. D elivery of metabolic signals to intracellular compartments is a critical determinant of cellular homeostasis. In particular, efficient communication between cellular energetics and membrane metabolic sensors is required for regulation of cell excitability and associated functions (1, 2). Plasmalemmal ATPsensitive K ϩ (K ATP ) channels, formed by the Kir6.2 potassium channel and the sulfonylurea receptor (SUR), are unique nucleotide sensors that adjust membrane potential in response to intracellular metabolic oscillations (2-5). Transition of the SUR subunit from the ATP to the ADP-liganded state promotes K ϩ permeation through Kir6.2 and defines K ATP channel activity (5-7). However, the mechanism that facilitates nucleotide exchange in the K ATP channel environment and promotes coupling of membrane electrical events with cellular metabolic pathways remains unknown.Cellular phosphotransfer reactions catalyze nucleotide exchange facilitating communication between sites of ATP generation and ATP utilization (8-11). In this way, the phosphotransfer enzyme adenylate kinase (AK) amplifies metabolic signals and promotes intracellular phosphoryl transfer by catalyzing the reaction ATP ϩ AMP 7 2ADP (12, 13). Adenylate kinase has a distinct signaling role in setting the cellular response to stress through activation of AMP-dependent processes (12-15). Deletion of the major AK isoform (AK1) results in disturbed muscle energetic economy and decreased tolerance to metabolic stress (14, 15). Mutations in AK compromise nucleotide export from mitochondria (16), as well as the function of ATP-binding cassette transporters (17). Conversely, stimulation of AK phosphotransfer promotes nucleotide-dependent membrane functions (18,19). However, the actual significance of AK phosphotransfer in communicating energetic signals to membrane metabolic sensors, such as the K ATP cha...

One half million patients suffer from colorectal cancer in industrialized nations, yet this disease exhibits a low incidence in underdeveloped 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 cellpermeant 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 Ca 2؉ , or chelation of intracellular Ca 2؉ . In fact, both the enterotoxin and 8-br-cGMP induced an L-cis-diltiazem-sensitive conductance, promoting Ca 2؉ 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.

Summary Atrial fibrillation is a common arrhythmia that is hereditary in a small subgroup of patients. In a family with 11 clinically affected members, we mapped an atrial fibrillation locus to chromosome 1p36-p35 and identified a heterozygous frameshift mutation in the gene encoding atrial natriuretic peptide. Circulating chimeric atrial natriuretic peptide (ANP) was detected in high concentration in subjects with the mutation, and shortened atrial action potentials were seen in an isolated heart model, creating a possible substrate for atrial fibrillation. This report implicates perturbation of the atrial natriuretic peptide–cyclic guanosine monophosphate (cGMP) pathway in cardiac electrical instability.