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Park S.,Heart Genetics | Terzic A.,Heart Genetics
Journal of Structural Biology | Year: 2010

Heterodimeric nucleotide binding domains NBD1/NBD2 distinguish the ATP-binding cassette protein SUR2A, a recognized regulatory subunit of cardiac ATP-sensitive K+ (KATP) channels. The tandem function of these core domains ensures metabolism-dependent gating of the Kir6.2 channel pore, yet their structural arrangement has not been resolved. Here, purified monodisperse and interference-free recombinant particles were subjected to synchrotron radiation small-angle X-ray scattering (SAXS) in solution. Intensity function analysis of SAXS profiles resolved NBD1 and NBD2 as octamers. Implemented by ab initio simulated annealing, shape determination prioritized an oblong envelope wrapping NBD1 and NBD2 with respective dimensions of 168 × 80 × 37 Å3 and 175 × 81 × 37 Å3 based on symmetry constraints, validated by atomic force microscopy. Docking crystal structure homology models against SAXS data reconstructed the NBD ensemble surrounding an inner cleft suitable for Kir6.2 insertion. Human heart disease-associated mutations introduced in silico verified the criticality of the mapped protein-protein interface. The resolved quaternary structure delineates thereby a macromolecular arrangement of KATP channel SUR2A regulatory domains. © 2009 Elsevier Inc. All rights reserved.


VanDusen N.J.,Heart Genetics | Firulli A.B.,Heart Genetics
Differentiation | Year: 2012

The heart is a complex organ that is composed of numerous cell types, which must integrate their programs for proper specification, differentiation and cardiac morphogenesis. During cardiogenesis members of the Twist-family of basic helix-loop-helix (bHLH) transcription factors play distinct roles within cardiac lineages such as the endocardium and extra-cardiac lineages such as the cardiac neural crest (cNCC) and epicardium. While the study of these cell populations is often eclipsed by that of cardiomyocytes, the contributions of non-cardiomyocytes to development and disease are increasingly being appreciated as both dynamic and essential. This review summarizes what is known regarding Twist-family bHLH function in extra-cardiac cell populations and the endocardium, with a focus on regulatory mechanisms, downstream targets, and expression profiles. Improving our understanding of the molecular pathways that Twist-family bHLH factors mediate in these lineages will be necessary to ascertain how their dysfunction leads to congenital disease and adult pathologies such as myocardial infarctions and cardiac fibroblast induced fibrosis. Indeed, this knowledge will prove to be critical to clinicians seeking to improve current treatments. © 2012 International Society of Differentiation.


Chung S.,Heart Genetics | Arrell D.K.,Heart Genetics | Faustino R.S.,Heart Genetics | Terzic A.,Heart Genetics | Dzeja P.P.,Heart Genetics
Journal of Molecular and Cellular Cardiology | Year: 2010

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 E1α and E1β, 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. © 2009 Elsevier Ltd. All rights reserved.


Dmitrieva R.I.,Institute of Hematology | Minullina R.,Heart Genetics | Bilibina A.A.,Heart Genetics | Tarasova O.V.,Heart Genetics | And 2 more authors.
Cell Cycle | Year: 2012

Bone marrow (BM) and subcutaneous adipose tissue (Ad) are both considered prospective sources of MSC for therapeutic applications. However, functional properties and therapeutic efficacy of MSC derived from different tissues of the same patient are still poorly investigated. In our study, BM-MSC and F-MSC cultures from 43 adult donors were evaluated in successive passages for immunophenotype; secretion of VEGF, SDF1, MCP1, IL6 and TGFβ1; frequency of colony-forming units (CFU-F); frequency of adipo- and osteoprogenitors (CFU-Ad, CFU-Ost) and for onset of in vitro replicative senescence. We have demonstrated that at early passages (P2-P3 or up to 14-15 in vitro population doublings), BM- and Ad-derived MSC cultures are comparable in such important characteristics as proliferation rate (population-doubling time: 3.4 ± 0.2% in BM-MSC, 3 ± 0.3% in F-MSC), clonogenity (CFU-F frequency: 32 ± 5% in BM-MSC, 31 ± 5% in F-MSC) and differentiation potential (CFU-Ad frequency: 10.4 ± 2% in BM-MSC, 13 ± 3% in F-MSC; CFU-Ost frequency: 18.5 ± 5.5% in BM-MSC, 18 ± 5% in F-MSC) but differ significantly in abundance of CD146 + fraction within the sample (25 ± 5% in BMMSC, 7 ± 3% in F-MSC) and in levels of VEGF, SDF-1, MCP1 and TGFβ1 secretion. We have also demonstrated that BM?MSC enter senescence after P3-4, while most F-MSC did not show senescence features up to P6-8. Together, these data demonstrate that specific properties of MSC from different sources should be always taken into account when developing and optimizing the specific protocols for MSC expansion and evaluation for each particular clinical application. © 2012 Landes Bioscience.


Arrell D.K.,Marriott Heart Disease Research Program | Arrell D.K.,Molecular Therapeutics | Arrell D.K.,Heart Genetics | Terzic A.,Marriott Heart Disease Research Program | And 2 more authors.
Clinical Pharmacology and Therapeutics | Year: 2010

Systems biology provides a platform for integrating multiple components and interactions underlying cell, organ, and organism processes in health and disease. Beyond traditional approaches focused on individual molecules or pathways, bioinformatic network analysis of high-throughput data sets offers an opportunity for integration of biological complexity and multilevel connectivity. Emerging applications in rational drug discovery range from targeting and modeling disease-corrupted networks to screening chemical or ligand libraries to identification/validation of drug-target interactions for improved efficacy and safety. © 2010 American Society for Clinical Pharmacology and Therapeutics.


Vincentz J.W.,Heart Genetics | Barnes R.M.,Heart Genetics | Firulli A.B.,Heart Genetics
Birth Defects Research Part A - Clinical and Molecular Teratology | Year: 2011

Almost 15 years of careful study have established the related basic Helix-Loop-Helix (bHLH) transcription factors Hand1 and Hand2 as critical for heart development across evolution. Hand factors make broad contributions, revealed through animal models, to the development of multiple cellular lineages that ultimately contribute to the heart. They perform critical roles in ventricular cardiomyocyte growth, differentiation, morphogenesis, and conduction. They are also important for the proper development of the cardiac outflow tract, epicardium, and endocardium. Molecularly, they function both through DNA binding and through protein-protein interactions, which are regulated transcriptionally, posttranscriptionally by microRNAs, and posttranslationally through phosphoregulation. Although direct Hand factor transcriptional targets are progressively being identified, confirmed direct targets of Hand factor transcriptional activity in the heart are limited. Identification of these targets will be critical to model the mechanisms by which Hand factor bHLH interactions affect developmental pathways. Improved understanding of Hand factor-mediated transcriptional cascades will be necessary to determine how Hand factor dysregulation translates to human disease phenotypes. This review summarizes the insight that animal models have provided into the regulation and function of these factors during heart development, in addition to the recent findings that suggest roles for HAND1 and HAND2 in human congenital heart disease. © 2011 Wiley-Liss, Inc.


Zlatkovic-Lindor J.,Heart Genetics | Arrell D.K.,Heart Genetics | Yamada S.,Heart Genetics | Nelson T.J.,Heart Genetics | Terzic A.,Heart Genetics
Stem Cells | Year: 2010

Transplantation of pluripotent stem cells has proven beneficial in heart failure, yet the proteomic landscape underlying repair remains largely uncharacterized. In a genetic model of dilated cardiomyopathy elicited by pressure overload in the KCNJ11 (potassium inwardly rectifying channel, subfamily J, member 11) null mutant, proteome-wide profiles were here resolved by means of a systems approach prior to and following disease manifestation in the absence or presence of embryonic stem cell treatment. Comparative two-dimensional gel electrophoresis revealed a unique cardiomyopathic proteome in the absence of therapy, remodeled in response to stem cell treatment. Specifically, linear ion trap quadrupole-Orbitrap mass spectrometry determined the identities of 93 and 109 differentially expressed proteins from treated and untreated cardiomyopathic hearts, respectively. Mapped protein-protein relationships and corresponding neighborhoods incorporated the stem cell-dependent subproteome into a nonstochastic network with divergent composition from the stem cell-independent counterpart. Stem cell intervention produced a distinct proteome signature across a spectrum of biological processes ranging from energetic metabolism, oxidoreductases, and stress-related chaperones to processes supporting protein synthesis/degradation, signaling, and transport regulation, cell structure and scaffolding. In the absence of treatment, bioinformatic interrogation of the disease-only proteome network prioritized adverse cardiac outcomes, ablated or ameliorated following stem cell transplantation. Functional and structural measurements validated improved myocardial contractile performance, reduced ventricular size and decreased cardiac damage in the treated cohort. Unbiased systems assessment unmasked "cardiovascular development" as a prioritized biological function in stem cell-reconstructed cardiomyopathic hearts. Thus, embryonic stem cell treatment transformed the cardiomyopathic proteome to demote disease-associated adverse effects and sustain a procardiogenic developmental response, supplying a regenerative substrate for heart failure repair. © AlphaMed Press.


Buchaklian A.H.,Medical College of Wisconsin | Helbling D.,Medical College of Wisconsin | Ware S.M.,Heart Genetics | Dimmock D.P.,Medical College of Wisconsin
Molecular Genetics and Metabolism | Year: 2012

Deoxyguanosine kinase (DGUOK) (MIM#601465) deficiency was originally described as the cause of an infantile onset hepatocerebral mitochondrial disease [1]. The classic features of this disorder include significant hepatic failure with nystagmus and hypotonia. Mitochondrial DNA studies reveal significant mitochondrial DNA depletion in the affected tissues. Subsequently it has been shown that the same mutations in this gene may present with isolated acute liver failure without cerebral involvement. In this paper we studied the mitochondrial DNA depletion in cells from a patient presenting with mitochondrial myopathy caused by a novel mutation in DGUOK. Subsequently we developed the method to diagnose this condition using MyoD induced fibroblasts to study the muscle specific phenotype. In addition, supplementation of MyoD induced fibroblasts with dAMP and dGMP resulted in a restoration of mtDNA quantity. © 2012 Elsevier Inc.


Pereira T.V.,University of Ioannina | Pereira T.V.,Heart Genetics | Ioannidis J.P.A.,University of Ioannina | Ioannidis J.P.A.,Stanford University | Ioannidis J.P.A.,Tufts University
Journal of Clinical Epidemiology | Year: 2011

Objective: To assess whether nominally statistically significant effects in meta-analyses of clinical trials are true and whether their magnitude is inflated. Study Design and Setting: Data from the Cochrane Database of Systematic Reviews 2005 (issue 4) and 2010 (issue 1) were used. We considered meta-analyses with binary outcomes and four or more trials in 2005 with P < 0.05 for the random-effects odds ratio (OR). We examined whether any of these meta-analyses had updated counterparts in 2010. We estimated the credibility (true-positive probability) under different prior assumptions and inflation in OR estimates in 2005. Results: Four hundred sixty-one meta-analyses in 2005 were eligible, and 80 had additional trials included by 2010. The effect sizes (ORs) were smaller in the updating data (2005-2010) than in the respective meta-analyses in 2005 (median 0.85-fold, interquartile range [IQR]: 0.66-1.06), even more prominently for meta-analyses with less than 300 events in 2005 (median 0.67-fold, IQR: 0.54-0.96). Mean credibility of the 461 meta-analyses in 2005 was 63-84% depending on the assumptions made. Credibility estimates changed >20% in 19-31 (24-39%) of the 80 updated meta-analyses. Conclusions: Most meta-analyses with nominally significant results pertain to truly nonnull effects, but exceptions are not uncommon. The magnitude of observed effects, especially in meta-analyses with limited evidence, is often inflated. © 2011 Elsevier Inc. All rights reserved.


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