[Show abstract][Hide abstract] ABSTRACT: 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-alpha, enhancing the cardiogenic competence of recipient heart. The in vivo aptitude of TNF-alpha 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-alpha-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.
Journal of Experimental Medicine 03/2007; 204(2):405-20. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Sepsis, the systemic inflammatory response to infection, imposes a high demand for bodily adaptation, with the cardiovascular response a key determinant of outcome. The homeostatic elements that secure cardiac tolerance in the setting of the sepsis syndrome are poorly understood. Here, in a model of acute septic shock induced by endotoxin challenge with Escherichia coli lipopolysaccharide (LPS), knockout of the KCNJ8 gene encoding the vascular Kir6.1 K(ATP) channel pore predisposed to an early and profound survival disadvantage. The exaggerated susceptibility provoked by disruption of this stress-responsive sensor of cellular metabolism was linked to progressive deterioration in cardiac activity, ischemic myocardial damage, and contractile dysfunction. Deletion of KCNJ8 blunted the responsiveness of coronary vessels to cytokine- or metabolic-mediated vasodilation necessary to support myocardial perfusion in the wild-type (WT), creating a deficit in adaptive response in the Kir6.1 knockout. Application of a K(ATP) channel opener drug improved survival in the endotoxic WT but had no effect in the Kir6.1 knockout. Restoration of the dilatory capacity of coronary vessels was required to rescue the Kir6.1 knockout phenotype and reverse survival disadvantage in lethal endotoxemia. Thus, the Kir6.1-containing K(ATP) channel, by coupling vasoreactivity with metabolic demand, provides a vital feedback element for cardiovascular tolerance in endotoxic shock.
The FASEB Journal 12/2006; 20(13):2271-80. · 5.70 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Heart failure is a growing epidemic, with systemic hypertension a major risk factor for development of disease. However, the molecular determinants that prevent the transition from a state of hypertensive load to that of overt cardiac failure remain largely unknown. Here in experimental hypertension, knockout of the KCNJ11 gene, encoding the Kir6.2 pore-forming subunit of the sarcolemmal ATP-sensitive potassium (K(ATP)) channel, predisposed to heart failure and death. Defective decoding of hypertension-induced metabolic distress signals in the K(ATP) channel knockout set in motion pathological calcium overload and aggravated cardiac remodeling through a calcium/calcineurin-dependent cyclosporine-sensitive pathway. Rescue of the failing K(ATP) knockout phenotype was achieved by alternative control of myocardial calcium influx, bypassing uncoupled metabolic-electrical integration. The intact KCNJ11-encoded K(ATP) channel is thus a required safety element preventing hypertension-induced heart failure, with channel dysfunction a molecular substrate for stress-associated channelopathy in cardiovascular disease.
Human Molecular Genetics 09/2006; 15(15):2285-97. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac ATP-sensitive K(+) (K(ATP)) channels, gated by cellular metabolism, are formed by association of the inwardly rectifying potassium channel Kir6.2, the potassium conducting subunit, and SUR2A, the ATP-binding cassette protein that serves as the regulatory subunit. Kir6.2 is the principal site of ATP-induced channel inhibition, while SUR2A regulates K(+) flux through adenine nucleotide binding and catalysis. The ATPase-driven conformations within the regulatory SUR2A subunit of the K(ATP) channel complex have determinate linkage with the states of the channel's pore. The probability and life-time of ATPase-induced SUR2A intermediates, rather than competitive nucleotide binding alone, defines nucleotide-dependent K(ATP) channel gating. Cooperative interaction, instead of independent contribution of individual nucleotide binding domains within the SUR2A subunit, serves a decisive role in defining K(ATP) channel behavior. Integration of K(ATP) channels with the cellular energetic network renders these channel/enzyme heteromultimers high-fidelity metabolic sensors. This vital function is facilitated through phosphotransfer enzyme-mediated transmission of controllable energetic signals. By virtue of coupling with cellular energetic networks and the ability to decode metabolic signals, K(ATP) channels set membrane excitability to match demand for homeostatic maintenance. This new paradigm in the operation of an ion channel multimer is essential in providing the basis for K(ATP) channel function in the cardiac cell, and for understanding genetic defects associated with life-threatening diseases that result from the inability of the channel complex to optimally fulfill its physiological role.
Journal of Molecular and Cellular Cardiology 07/2005; 38(6):895-905. · 5.15 Impact Factor
[Show abstract][Hide abstract] 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 heart's 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.
Annals of the New York Academy of Sciences 06/2005; 1049:189-98. · 4.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metabolic-sensing ATP-sensitive K+ channels (KATP channels) adjust membrane excitability to match cellular energetic demand. In the heart, KATP channel activity has been linked to homeostatic shortening of the action potential under stress, yet the requirement of channel function in securing cardiac electrical stability is only partially understood. Here, upon catecholamine challenge, disruption of KATP channels, by genetic deletion of the pore-forming Kir6.2 subunit, produced defective cardiac action potential shortening, predisposing the myocardium to early afterdepolarizations. This deficit in repolarization reserve, demonstrated in Kir6.2-knockout hearts, translated into a high risk for induction of triggered activity and ventricular dysrhythmia. Thus, intact KATP channel function is mandatory for adequate repolarization under sympathetic stress providing electrical tolerance against triggered arrhythmia.
[Show abstract][Hide abstract] ABSTRACT: Background/Aims: Pluripotent embryonic stem cells are an emerging source for cardiotherapy, but harbor a risk for uncontrolled growth on transplantation to host tissue. This creates a need to identify a stem cell phenotype committed to the cardiac fate to achieve guided cardiogenesis for safe cell-based cardiac repair.Methods/Results: Bolus delivery of uncommitted embryonic stem cells (>1000 cells/mg myocardium) into naı¨ve hearts resulted into a teratogenic side effect. A tumor-free outcome was achieved with overexpression of the cytokine Tumor Necrosis Factor α (TNFα) which secured guided cardiogenesis of injected stem cells. Protein-based scans following TNFα stimulation demonstrated an increase in the secreted proteome, which included upregulation of downstream cardiogenic factors, such as Transforming Growth Factor β (TGFβ). With TNFα stimulation, the yield of stem cell-derived cardiomyocytes doubled, while the TGFβ antagonist, latency associated peptide, abolished the TNFα effect. The TNFα/TGFβ signaling recruited a proliferative cardiac precursor cell phenotype expressing master cardiac transcription factors, Csx/Nkx2.5 and MEF2C.Conclusions: This study identifies a novel cell phenotype devoted to the cardiac fate that is recruited and amplified by TNFα initiated TGFβ signaling. With differentiation limited to cardiopotency, such cardiopoetic stem cells loose the teratenogenic capacity thereby enhancing the therapeutic index for cell-based cardiac repair.
[Show abstract][Hide abstract] ABSTRACT: Abnormal expression of human myotonic dystrophy protein kinase (hDMPK) gene products has been implicated in myotonic dystrophy type 1 (DM1), yet the impact of distress accumulation produced by persistent overexpression of this poorly understood member of the Rho kinase-related protein kinase gene-family remains unknown. Here, in the aged transgenic murine line carrying approximately 25 extra copies of a complete hDMPK gene with all exons and an intact promoter region (Tg26-hDMPK), overexpression of mRNA and protein transgene products in cardiac, skeletal and smooth muscles resulted in deficient exercise endurance, an integrative index of muscle systems underperformance. In contrast to age-matched (11-15 months) wild-type controls, hearts from Tg26-hDMPK developed cardiomyopathic remodeling with myocardial hypertrophy, myocyte disarray and interstitial fibrosis. Hypertrophic cardiomyopathy was associated with a propensity for dysrhythmia and characterized by overt intracellular calcium overload promoting nuclear translocation of transcription factors responsible for maladaptive gene reprogramming. Skeletal muscles in distal limbs of Tg26-hDMPK showed myopathy with myotonic discharges coupled with deficit in sarcolemmal chloride channels, required regulators of hyperexcitability. Fiber degeneration in Tg26-hDMPK resulted in sarcomeric disorganization, centralization of nuclei and tubular aggregation. Moreover, the reduced blood pressure in Tg26-hDMPK indicated deficient arterial smooth muscle tone. Thus, the cumulative stress induced by permanent overexpression of hDMPK gene products translates into an increased risk for workload intolerance, hypertrophic cardiomyopathy with dysrhythmia, myotonic myopathy and hypotension, all distinctive muscle traits of DM1. Proper expression of hDMPK is, therefore, mandatory in supporting the integral balance among cytoarchitectural infrastructure, ion-homeostasis and viability control in various muscle cell types.
Human Molecular Genetics 11/2004; 13(20):2505-18. · 7.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Stress tolerance of the heart requires high-fidelity metabolic sensing by ATP-sensitive potassium (K(ATP)) 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 K(ATP) 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 K(ATP) channel phenotypes with compromised metabolic signal decoding. Defective catalysis-mediated pore regulation is thus a mechanism for channel dysfunction and susceptibility to dilated cardiomyopathy.
[Show abstract][Hide abstract] ABSTRACT: Cardiac K-ATP channels have been recently been implicated to serve a homeostatic function under catecholamine stress. Since the use of drugs that modulate K-ATP channels is common in clinical practice, the implications of channel function on cardiac electrophysiology warrant further investigation. Here, using simultaneous monophasic action potential and ECG recordings, we examined the arrhythmogenic consequences of genetic deletion of the pore-forming K-ATP channel subunit Kir6.2. Infusion of the sympathomimetic isoproterenol (1muM) readily induced early afterdepolarizations in Kir6.2 knockout (KO) but not in the wildtype (WT) hearts (incidence: 96.8% versus 1%, p<0.01). This translated into an order of 10 magnitude higher incidence of triggered activity and associated premature ventricular contraction in the KO hearts. Although there was no significant difference in perfusion flow between KO and WT hearts before or after isoproterenol infusion, the shortening of the action potential duration at 90% repolarization under sympathomimetic challenge observed in the WT (from 82 to 74 ms, p<0.01) was absent in the KO (79 to 80 ms, p>0.05). We conclude that cardiac K-ATP channels are required to secure proper repolarization reserve and prevent afterdepolarization-associated arrhythmia under sympathetic stress. Therefore, clinical conditions that are associated with reduced K-ATP channel activity, such as use of sulfonylurea drugs, may predispose to arrhythmic events.
[Show abstract][Hide abstract] ABSTRACT: Chronic hemodynamic load predisposes to cardiac dysfunction, yet the molecular components of myocardial adaptation are not completely understood. Kir6.2 is the pore-forming subunit of cardiac ATP-sensitive potassium (KATP) channels implicated in the cardiac stress response. Here, following unilateral nephrectomy, Kir6.2 knockout (KO) & C57BL/6 wild-type (WT) mice, were challenged with deoxycorticosterone acetate-salt (DOCA/salt) loading for 21 days. DOCA/salt induced similar extent of hypertension & contralateral renal hypertrophy with fibrosis in KO and WT. However, in KO, DOCA/salt challenge produced left ventricular dysfunction, pulmonary congestion, reduced exercise performance, & catecholamine-induced cardiac failure with more severe increase in left ventricular mass & more extensive interstitial fibrosis. Indeed, cyclosporine, the inhibitor of Ca2+ mediated calcineurin-dependent hypertrophic reprogramming, reversed the exaggerated cardiac restructuring in the KO. Overall, DOCA/salt-KO mice displayed significant mortality compared to DOCA/salt-WT. Treatment with the Ca2+ channel antagonist verapamil rescued the KO with prevention of cardiac pathology & associated mortality. Thus, suggesting an essential homeostatic role for cardiac KATP channels in the setting of mineralocorticoid induced hypertension. This work has implications for hypertensive diabetic patients treated with KATP channel antagonists sulfonylureas & a potential therapeutic role for KATP channel openers.
[Show abstract][Hide abstract] ABSTRACT: Adenylate kinases (AK, EC 126.96.36.199) have been considered important enzymes for energy homeostasis and metabolic signaling. To gain a better understanding of their cell-specific significance we studied the structural and functional aspects of products of one adenylate kinase gene, AK1, in mouse tissues. By combined computer database comparison and Northern analysis of mRNAs, we identified transcripts of 0.7 and 2.0 kilobases with different 5' and 3' non-coding regions which result from alternative use of promoters and polyadenylation sites. These mRNAs specify two distinct proteins, AK1 and a membrane-bound AK1 isoform (AK1beta), which differ in their N-terminal end and are co-expressed in several tissues with high-energy demand, including the brain. Immunohistochemical analysis of brain tissue and primary neurons and astrocytes in culture demonstrated that AK1 isoforms are expressed predominantly in neurons. AK1beta, when tested in transfected COS-1 and N2a neuroblastoma cells, located at the cellular membrane and was able to catalyze phosphorylation of ADP in vitro. In addition, AK1beta mediated AMP-induced activation of recombinant ATP-sensitive potassium channels in the presence of ATP. Thus, two structurally distinct AK1 isoforms co-exist in the mouse brain within distinct cellular locations. These enzymes may function in promoting energy homeostasis in the compartmentalized cytosol and in translating cellular energetic signals to membrane metabolic sensors.
Molecular and Cellular Biochemistry 01/2004; 256-257(1-2):59-72. · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
Molecular and Cellular Biochemistry 01/2004; 256-257(1-2):243-56. · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mitochondrial integrity is critical in the maintenance of bioenergetic homeostasis of the myocardium, with oxidative or metabolic challenge to mitochondria precipitating cell injury. In heart failure, where cardiac cells are exposed to elevated stress, mitochondrial vulnerability could contribute to the disease state. However, the mitochondrial response to stress is yet to be established in heart failure. Here, mitochondrial function and structure was evaluated prior and following stress using a transgenic (TG) model of heart failure, generated by cardiac overexpression of the cytokine TNFalpha. Compared to the wild type, mitochondria from TG failing hearts demonstrated impaired oxidative phosphorylation, mitochondrial DNA damage, reduced mitochondrial creatine kinase activity, abnormal calcium handling, and altered ultrastructure. Under anoxia/reoxygenation or calcium stress, mitochondria from failing hearts suffered exacerbated energetic failure with pronounced cytochrome c release. Thus, mitochondria from TNFalpha-TG failing hearts demonstrate structural and functional abnormalities, with reduced tolerance to stress manifested by impaired bioenergetics and increased susceptibility to injury. This abnormal vulnerability to stress underscores the impact of mitochondrial dysfunction in the pathobiology of heart failure.
Journal of Molecular and Cellular Cardiology 10/2003; 35(9):1161-6. · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Although ischemic preconditioning induces bioenergetic tolerance and thereby remodels energy metabolism that is crucial for postischemic recovery of the heart, the molecular components associated with preservation of cellular energy production, transfer, and utilization are not fully understood. Here myocardial bioenergetic dynamics were assessed by (18)O-assisted (31)P-NMR spectroscopy in control or preconditioned hearts from wild-type (WT) or Kir6.2-knockout (Kir6.2-KO) mice that lack metabolism-sensing sarcolemmal ATP-sensitive K(+) (K(ATP)) channels. In WT vs. Kir6.2-KO hearts, preconditioning induced a significantly higher total ATP turnover (232 +/- 20 vs. 155 +/- 15 nmol x mg protein(-1) x min(-1)), ATP synthesis rate (58 +/- 3 vs. 46 +/- 3% (18)O labeling of gamma-ATP), and ATP consumption rate (51 +/- 4 vs. 31 +/- 4% (18)O labeling of P(i)) after ischemia-reperfusion. Moreover, preconditioning preserved cardiac creatine kinase-catalyzed phosphotransfer in WT (234 +/- 26 nmol x mg protein(-1) x min(-1)) but not Kir6.2-KO (133 +/- 18 nmol x mg protein(-1) x min(-1)) hearts. In contrast with WT hearts, preconditioning failed to preserve contractile recovery in Kir6.2-KO hearts, as tight coupling between postischemic performance and high-energy phosphoryl transfer was compromised in the K(ATP)-channel-deficient myocardium. Thus intact K(ATP) channels are integral in ischemic preconditioning-induced protection of cellular energetic dynamics and associated cardiac performance.
[Show abstract][Hide abstract] ABSTRACT: ATP-sensitive potassium (K(ATP)) channels are required for maintenance of homeostasis during the metabolically demanding adaptive response to stress. However, in disease, the effect of cellular remodeling on K(ATP) channel behavior and associated tolerance to metabolic insult is unknown. Here, transgenic expression of tumor necrosis factor alpha induced heart failure with typical cardiac structural and energetic alterations. In this paradigm of disease remodeling, K(ATP) 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, K(ATP) 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 K(ATP) channel openers. Thus, disease-induced K(ATP) channel metabolic dysregulation is a contributor to the pathobiology of heart failure, illustrating a mechanism for acquired channelopathy.
The EMBO Journal 05/2003; 22(8):1732-42. · 9.82 Impact Factor