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1.  Circulating Cell-Free DNA Enables Noninvasive Diagnosis of Heart Transplant Rejection 
Science translational medicine  2014;6(241):241ra77.
Monitoring allograft health is an important component of posttransplant therapy. Endomyocardial biopsy is the current gold standard for cardiac allograft monitoring but is an expensive and invasive procedure. Proof of principle of a universal, noninvasive diagnostic method based on high-throughput screening of circulating cell-free donor-derived DNA (cfdDNA) was recently demonstrated in a small retrospective cohort. We present the results of a prospective cohort study (65 patients, 565 samples) that tested the utility of cfdDNA in measuring acute rejection after heart transplantation. Circulating cell-free DNA was purified from plasma and sequenced (mean depth, 1.2 giga–base pairs) to quantify the fraction of cfdDNA. Through a comparison with endomyocardial biopsy results, we demonstrate that cfdDNA enables diagnosis of acute rejection after heart transplantation, with an area under the receiver operating characteristic curve of 0.83 and sensitivity and specificity that are comparable to the intrinsic performance of the biopsy itself. This noninvasive genome transplant dynamics approach is a powerful and informative method for routine monitoring of allograft health without incurring the risk, discomfort, and expense of an invasive biopsy.
doi:10.1126/scitranslmed.3007803
PMCID: PMC4326260  PMID: 24944192
2.  Introduction to the Series: Challenges and Opportunities in Pediatric Heart Failure and Transplantation 
Circulation  2014;129(1):112-114.
doi:10.1161/CIRCULATIONAHA.113.006102
PMCID: PMC3893708  PMID: 24396014
pediatric; heart failure; heart transplantation
3.  The Effect of Bioengineered Acellular Collagen Patch on Cardiac Remodeling and Ventricular Function post Myocardial Infarction 
Biomaterials  2013;34(36):10.1016/j.biomaterials.2013.08.017.
Regeneration of the damaged myocardium is one of the most challenging fronts in the field of tissue engineering due to the limited capacity of adult heart tissue to heal and to the mechanical and structural constraints of the cardiac tissue. In this study we demonstrate that an engineered acellular scaffold comprising type I collagen, endowed with specific physiomechanical properties, improves cardiac function when used as a cardiac patch following myocardial infarction. Patches were grafted onto the infarcted myocardium in adult murine hearts immediately after ligation of left anterior descending artery and the physiological outcomes were monitored by echocardiography, and by hemodynamic and histological analyses four weeks post infarction. In comparison to infarcted hearts with no treatment, hearts bearing patches preserved contractility and significantly protected the cardiac tissue from injury at the anatomical and functional levels. This improvement was accompanied by attenuated left ventricular remodeling, diminished fibrosis, and formation of a network of interconnected blood vessels within the infarct. Histological and immunostaining confirmed integration of the patch with native cardiac cells including fibroblasts, smooth muscle cells, epicardial cells, and immature cardiomyocytes. In summary, an acellular biomaterial with specific biomechanical properties promotes the endogenous capacity of the infarcted myocardium to attenuate remodeling and improve heart function following myocardial infarction.
doi:10.1016/j.biomaterials.2013.08.017
PMCID: PMC3809823  PMID: 23992980
cardiac tissue engineering; scaffold; collagen; heart; cardiomyocyte; angiogenesis
4.  Temporal Response of the Human Virome to Immunosuppression and Antiviral Therapy 
Cell  2013;155(5):1178-1187.
Summary
There are few substantive methods to measure the health of the immune system, and the connection between immune strength and the viral component of the microbiome is poorly understood. Organ transplant recipients are treated with a post-transplant therapy that combines immunosuppressive and antiviral drugs, offering a window into the effects of immune modulation on the virome. We used sequencing of cell-free DNA in plasma to investigate drug-virome interactions in a cohort of organ transplant recipients (656 samples, 96 patients), and find that antivirals and immunosuppressants strongly affect the structure of the virome in plasma. We observe marked virome compositional dynamics at the onset of the therapy and find that the total viral load increases with immunosuppression, whereas the bacterial component of the microbiome remains largely unaffected. The data provide insight into the relationship between the human virome, the state of the immune system, and the effects of pharmacological treatment, and offer a potential application of the virome state to predict immunocompetence.
doi:10.1016/j.cell.2013.10.034
PMCID: PMC4098717  PMID: 24267896
5.  DELETION OF THE β2-ADRENERGIC RECEPTOR PREVENTS THE DEVELOPMENT OF CARDIOMYOPATHY IN MICE 
Aims
Beta adrenergic receptor (β-AR) subtypes act through diverse signaling cascades to modulate cardiac function and remodeling. Previous in vitro studies suggest that β1-AR signaling is cardiotoxic whereas β2-AR signaling is cardioprotective, and may be the case during ischemia/reperfusion in vivo. The objective of this study was to assess whether β2-ARs also played a cardioprotective role in the pathogenesis of non-ischemic forms of cardiomyopathy.
Methods and Results
To dissect the role of β1 vs β2-ARs in modulating MLP (Muscle LIM Protein) cardiomyopathy, we crossbred MLP−/− with β1−/− or β2−/− mice. Deletion of the β2-AR improved survival, cardiac function, exercise capacity and myocyte shortening; in contrast haploinsufficency of the β1-AR reduced survival. Pathologic changes in Ca2+ handling were reversed in the absence of β2-ARs: peak Ca2+ and SR Ca2+ were decreased in MLP−/− and β1+/− /MLP−/− but restored in β2−/−MLP−/−. These changes were associated with reversal of alterations in troponin I and phospholamban phosphorylation. Gi inhibition increased peak and baseline Ca2+, recapitulating changes observed in the β2−/−/MLP−/−. The L-type Ca2+ blocker verapamil significantly decreased cardiac function in β2−/−MLP−/− vs WT. We next tested if the protective effects of β2-AR ablation were unique to the MLP model using TAC-induced heart failure. Similar to MLP, β2−/− mice demonstrated delayed progression of heart failure with restoration of myocyte shortening and peak Ca2+ and Ca2+ release.
Conclusion
Deletion of β2-ARs prevents the development of MLP−/− cardiomyopathy via positive modulation of Ca2+ due to removal of inhibitory Gi signaling and increased phosphorylation of troponin I and phospholamban. Similar effects were seen after TAC. Unlike previous models where β2-ARs were found to be cardioprotective, in these two models, β2-AR signaling appears to be deleterious, potentially through negative regulation of Ca2+ dynamics.
doi:10.1016/j.yjmcc.2013.07.016
PMCID: PMC3791213  PMID: 23920331
Adrenergic receptors; cardiomyopathy; excitation-contraction coupling; signal transduction
6.  “Queasy does it”: False alcohol beliefs and memories may lead to diminished alcohol preferences 
Acta psychologica  2013;143(1):14-19.
Studies have shown that false memories can be implanted via innocuous suggestions, and that these memories can play a role in shaping people’s subsequent attitudes and preferences. The current study explored whether participants (N=147) who received a false suggestion that they had become ill drinking a particular type of alcohol would increase their confidence that the event had occurred, and whether their new-found belief would subsequently affect their alcohol preferences. Results indicated that participants who received a suggestion that they had gotten sick drinking rum or vodka before the age of 16 reported increased confidence that the suggested experience had occurred. Moreover, participants who received a false alcohol suggestion also showed a strong trend to report diminished preference for the specified type of alcohol after the false suggestion. Implantation of a false memory related to one’s past drinking experiences may influence current drink preferences and could be an important avenue for further exploration in the development of alcohol interventions.
doi:10.1016/j.actpsy.2013.01.017
PMCID: PMC3627832  PMID: 23500110
7.  People with Easier to Pronounce Names Promote Truthiness of Claims 
PLoS ONE  2014;9(2):e88671.
When people make judgments about the truth of a claim, related but nonprobative information rapidly leads them to believe the claim–an effect called “truthiness” [1]. Would the pronounceability of others’ names also influence the truthiness of claims attributed to them? We replicated previous work by asking subjects to evaluate people’s names on a positive dimension, and extended that work by asking subjects to rate those names on negative dimensions. Then we addressed a novel theoretical issue by asking subjects to read that same list of names, and judge the truth of claims attributed to them. Across all experiments, easily pronounced names trumped difficult names. Moreover, the effect of pronounceability produced truthiness for claims attributed to those names. Our findings are a new instantiation of truthiness, and extend research on the truth effect as well as persuasion by showing that subjective, tangential properties such as ease of processing can matter when people evaluate information attributed to a source.
doi:10.1371/journal.pone.0088671
PMCID: PMC3935838  PMID: 24586368
8.  Sacrificial layer technique for axial force post assay of immature cardiomyocytes 
Biomedical microdevices  2013;15(1):171-181.
Immature primary and stem cell-derived cardiomyocytes provide useful models for fundamental studies of heart development and cardiac disease, and offer potential for patient specific drug testing and differentiation protocols aimed at cardiac grafts. To assess their potential for augmenting heart function, and to gain insight into cardiac growth and disease, tissue engineers must quantify the contractile forces of these single cells. Currently, axial contractile forces of isolated adult heart cells can only be measured by two-point methods such as carbon fiber technique s, which cannot be applied to neonatal and stem cell-derived heart cells because they are more difficult to handle and lack a persistent shape. Here we present a novel axial technique for measuring the contractile forces of isolated immature cardiomyocytes. We overcome cell manipulation and patterning challenges by using a thermoresponsive sacrificial support layer in conjunction with arrays of widely separated elastomeric microposts. Our approach has the potential to be high-throughput, is functionally analogous to current gold-standard axial force assays for adult heart cells, and prescribes elongated cell shapes without protein patterning. Finally, we calibrate these force posts with piezoresistive cantilevers to dramatically reduce measurement error typical for soft polymer-based force assays. We report quantitative measurements of peak contractile forces up to 146 nN with post stiffness standard error (26 nN) far better than that based on geometry and stiffness estimates alone. The addition of sacrificial layers to future 2D and 3D cell culture platforms will enable improved cell placement and the complex suspension of cells across 3D constructs.
doi:10.1007/s10544-012-9710-3
PMCID: PMC3545035  PMID: 23007494
Force posts; thermoresponsive; sacrificial layer; cardiomyocytes; PDMS; stem cells
9.  Role of Telomere Dysfunction in Cardiac Failure in Duchenne Muscular Dystrophy 
Nature cell biology  2013;15(8):895-904.
Duchenne Muscular Dystrophy (DMD), the most common inherited muscular dystrophy of childhood, leads to death due to cardiorespiratory failure. Paradoxically, mdx mice with the same genetic deficiency of dystrophin, exhibit minimal cardiac dysfunction, impeding the development of therapies. We postulated that the difference between mdx and DMD might result from differences in telomere lengths in mice and humans. We show here that, like DMD patients, mice that lack dystrophin and have shortened telomeres (mdx/mTRKO) develop severe functional cardiac deficits including ventricular dilation, contractile and conductance dysfunction, and accelerated mortality. These cardiac defects are accompanied by telomere erosion, mitochondrial fragmentation, and increased oxidative stress. Treatment with anti-oxidants significantly retards the onset of cardiac dysfunction and death of mdx/mTRKO mice. In corroboration, of four DMD patients analyzed, all had 45% shorter telomeres in their cardiomyocytes relative to age- and sex-matched controls. We propose that the demands of contraction in the absence of dystrophin coupled with increased oxidative stress conspire to accelerate telomere erosion culminating in cardiac failure and death. These findings provide strong support for a link between telomere length and dystrophin deficiency in the etiology of dilated cardiomyopathy in DMD and suggest preventive interventions.
doi:10.1038/ncb2790
PMCID: PMC3774175  PMID: 23831727
10.  Multi-institutional Study of Outcomes After Pediatric Heart Transplantation: Candidate Gene Polymorphism Analysis of ABCC2 
OBJECTIVES
Earlier studies have indicated that the pharmacokinetics of mycophenolic acid (MPA) is influenced by polymorphisms of ABCC2, which encodes for the membrane transporter MRP2. The ABCC2 rs717620 A allele has been associated with enterohepatic recirculation of MPA, and our previous work had correlated the discontinuance of MPA with this allele in pediatric heart transplant patients. Therefore, we hypothesized that the ABCC2 rs717620 A allele would be associated with poorer outcomes including rejection with hemodynamic compromise (RHC), graft failure, and death in the pediatric heart transplant (PHTx) population receiving MPA.
METHODS
PHTx recipients from 6 institutions in the Pediatric Heart Transplantation Study (PHTS) from the period of 1993–2009, receiving MPA therapy, were genotyped for ABCC2 rs717620. Genotyping was accomplished by direct sequencing. Demographic and outcome data were limited to the data routinely collected as part of the PHTS and included RHC and mortality.
RESULTS
Two hundred ninety patients were identified who received MPA at some point post transplantation, of which 200 carried the GG genotype, 81 carried the AG genotype, and 9 carried the AA genotype. Follow-up time after transplantation was 6 years. RHC occurred in 76 patients and 18 patients died. In the 281 patients followed up more than 1 year, late RHC (>1 year post transplantation) occurred in 42 patients. While both RHC and late RHC were associated with the ABCC2 rs717620 GG genotype (hazard ratios: 1.80 and 4.57, respectively, p<0.05) in all patients, this association was not significant in PHTx patients receiving only MPA as the antiproliferative agent from the time of transplant (n=142).
CONCLUSIONS
ABCC2 rs717620 polymorphisms varied within racial groups. As a candidate gene assessment, the ABCC2 rs717620 AG and AA genotypes may be associated with improved, rather than poorer, RHC in PHTx patients receiving MPA therapy. ABCC2 rs717620 polymorphisms should be included in any expanded pharmacogenomic analysis of outcomes after pediatric heart transplantation.
doi:10.5863/1551-6776-19.1.16
PMCID: PMC3998963  PMID: 24782687
ABCC2 polymorphisms; heart transplantation; mycophenolic acid; pediatrics; pharmacogenetics; rejection
11.  Loss of Adenomatous Poliposis Coli-α3 Integrin Interaction Promotes Endothelial Apoptosis in Mice and Humans 
Circulation research  2012;111(12):10.1161/CIRCRESAHA.112.267849.
Rationale
Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins (BMPs) promote pulmonary angiogenesis by recruiting the Wnt/βcatenin pathway, we proposed that βcatenin activation could reduce loss and/or induce regeneration of small PAs and attenuate PH.
Objective
This study aims to establish the role of β–catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis following injury.
Methods and Results
To assess the impact of β-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (ApcMin/+) mouse, where reduced APC causes constitutive β–catenin elevation. Surprisingly, hypoxic ApcMin/+ mice displayed greater PH and small PA loss compared to control C57Bl6J (C57) littermates. Pulmonary artery endothelial cells (PAECs) isolated from ApcMin/+ demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase (ILK-1) and pAkt. We found that PAECs from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC.
Conclusions
We show that APC is integral to PAEC adhesion and survival and is reduced in PAECs from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.
doi:10.1161/CIRCRESAHA.112.267849
PMCID: PMC3821702  PMID: 23011394
Adenomatous poliposis coli; Wnt signaling; integrin signaling; angiogenesis; pulmonary hypertension
12.  Renal Function and Genetic Polymorphisms in Pediatric Heart Transplant Recipients 
Background
Common genetic variations influence rejection, infection, drug metabolism, and side effect profiles after pediatric heart transplantation. Reports in adults suggest that genetic background may influence post-transplant renal function. In this multicenter study we investigated the association of genetic polymorphisms (GP) in a panel of candidate genes on renal function in 453 pediatric heart transplant recipients.
Methods
We performed genotyping for functional GPs in 19 candidate genes. Renal function was determined annually after transplantation by calculation of glomerular filtration rate (eGFR). Mixed effects and Cox proportional hazard models were used to assess recipient characteristics and the effect of GPs on longitudinal eGFR and time to eGFR <60 mL/min/1.73m2.
Results
Mean age at transplantation was 6.2 ± 6.1 years and mean follow-up was 5.1 ± 2.5 years. Older age at transplant and black race were independently associated with post-transplant renal dysfunction. In univariate analyses, FASL (C-843T) T allele (p=0.014) and HO-1 (A326G) G allele (p=0.0017) were associated with decreased renal function. After adjusting for age and race, these associations were attenuated [FASL (p=0.075), HO-1 (p=0.053)]. We found no associations of other GPs, including GPs in TGFβ1, CYP3A5, ABCB1, and ACE, with post-transplant renal function.
Conclusions
In this multicenter, large sample of pediatric heart transplant recipients we found no strong associations between GPs in 19 candidate genes and post-transplant renal function. Our findings contradict reported associations of CYP3A5 and TGFβ1 with renal function and suggest that genotyping for these GPs will not facilitate individualized immunosuppression for the purpose of protecting renal function after pediatric heart transplantation.
doi:10.1016/j.healun.2012.05.010
PMCID: PMC3428223  PMID: 22789135
13.  FK506 activates BMPR2, rescues endothelial dysfunction, and reverses pulmonary hypertension 
The Journal of Clinical Investigation  2013;123(8):3600-3613.
Dysfunctional bone morphogenetic protein receptor-2 (BMPR2) signaling is implicated in the pathogenesis of pulmonary arterial hypertension (PAH). We used a transcriptional high-throughput luciferase reporter assay to screen 3,756 FDA-approved drugs and bioactive compounds for induction of BMPR2 signaling. The best response was achieved with FK506 (tacrolimus), via a dual mechanism of action as a calcineurin inhibitor that also binds FK-binding protein-12 (FKBP12), a repressor of BMP signaling. FK506 released FKBP12 from type I receptors activin receptor-like kinase 1 (ALK1), ALK2, and ALK3 and activated downstream SMAD1/5 and MAPK signaling and ID1 gene regulation in a manner superior to the calcineurin inhibitor cyclosporine and the FKBP12 ligand rapamycin. In pulmonary artery endothelial cells (ECs) from patients with idiopathic PAH, low-dose FK506 reversed dysfunctional BMPR2 signaling. In mice with conditional Bmpr2 deletion in ECs, low-dose FK506 prevented exaggerated chronic hypoxic PAH associated with induction of EC targets of BMP signaling, such as apelin. Low-dose FK506 also reversed severe PAH in rats with medial hypertrophy following monocrotaline and in rats with neointima formation following VEGF receptor blockade and chronic hypoxia. Our studies indicate that low-dose FK506 could be useful in the treatment of PAH.
doi:10.1172/JCI65592
PMCID: PMC3726153  PMID: 23867624
14.  SERPINE1 Expression Discriminates Site-Specific Metastasis in Human Melanoma 
Experimental Dermatology  2012;21(7):551-554.
Depth of invasion, a quantifier of vertical growth, is a major cutaneous melanoma staging factor. Stromal penetrance requires pericellular proteolysis regulated by the serine protease and matrix metalloproteinase cascades. The serine protease inhibitor SERPINE1, a poor prognosis biomarker in various cancers, promotes tumor progression likely by titrating the extent and local of plasmin-initiated matrix remodeling. SERPINE1 in human melanoma was assessed using tissue arrays that included primary/ metastatic tumors and normal skin. SERPINE1 was basal layer-restricted in the normal epidermis. SERPINE1 immunoreactivity was evident in 27/28 primary (96%) and 24/26 metastatic tumors (92%); cutaneous metastases (80%) had significantly elevated SERPINE1 levels compared to low signals characteristic of lymph node lesions. Moderate SERPINE1 expression was a general finding in primary melanoma whereas reduced or increased SERPINE1 immunolocalization typified metastatic deposits. The amplitude of SERPINE1 expression may impact melanoma site-specific dissemination, with cutaneous metastases representing a high-SERPINE1 tumor subtype.
doi:10.1111/j.1600-0625.2012.01523.x
PMCID: PMC3383094  PMID: 22716255
Dermatopathology; Melanoma; PAI-1; SERPINE1; Metastasis
15.  Hindsight Bias and Developing Theories of Mind 
Child development  2007;78(4):1374-1394.
Although hindsight bias (the “I knew it all along” phenomenon) has been documented in adults, its development has not been investigated. This is despite the fact that hindsight bias errors closely resemble the errors children make on theory of mind (ToM) tasks. Two main goals of the present work were to (a) create a battery of hindsight tasks for preschoolers, and (b) assess the relation between children’s performance on these and ToM tasks. In two experiments involving 144 preschoolers, 3-, 4-, and 5-year olds exhibited strong hindsight bias. Performance on hindsight and ToM tasks was significantly correlated independent of age, language ability, and inhibitory control. These findings contribute to a more comprehensive account of perspective taking across the lifespan.
doi:10.1111/j.1467-8624.2007.01071.x
PMCID: PMC3649066  PMID: 17650144
16.  In Vivo Functional and Transcriptional Profiling of Bone Marrow Stem Cells after Transplantation into Ischemic Myocardium 
Objective
Clinical trials of bone marrow-derived stem cell therapy for the heart have yielded variable results. The basic mechanism(s) that underlie their potential efficacy remains unknown. In the present study, we evaluate the survival kinetics, transcriptional response, and functional outcome of intramyocardial bone marrow mononuclear cell (BMMC) transplantation for cardiac repair in murine myocardial infarction model.
Methods and Results
We utilized molecular-genetic bioluminescence imaging and high throughput transcriptional profiling to evaluate the in vivo survival kinetics and gene expression changes of transplanted BMMCs after their engraftment into ischemic myocardium. Our results demonstrate short-lived survival of cells following transplant, with less than 1% of cells surviving by 6 weeks post-transplantation. Moreover, transcriptomic analysis of BMMCs revealed non-specific upregulation of various cell regulatory genes with a marked downregulation of cell differentiation and maturation pathways. BMMC therapy caused limited improvement of heart function as assessed by echocardiography, invasive hemodynamics, and positron emission tomography (PET). Histological evaluation of cell fate further confirmed findings of the in vivo cell tracking and transcriptomic analysis.
Conclusions
Collectively, these data suggest that BMMC therapy, in its present iteration, may be less efficacious than once thought. Additional refinement of existing cell delivery protocols should be considered to induce better therapeutic efficacy.
doi:10.1161/ATVBAHA.111.238618
PMCID: PMC3241895  PMID: 22034515
stem cells; bone marrow mononuclear cells; transcriptional profiling; molecular imaging; myocardial infarction
17.  Pharmacological inhibition of βIIPKC is cardioprotective in late-stage hypertrophy 
We previously found that in the hearts of hypertensive Dahl salt-sensitive rats, βIIPKC levels increase during the transition from compensated cardiac hypertrophy to cardiac dysfunction. Here we showed that a six-week treatment of these hypertensive rats with a βIIPKC-specific inhibitor, βIIV5-3, prolonged their survival by at least six weeks, suppressed myocardial fibrosis and inflammation, and delayed the transition from compensated hypertrophy to cardiac dysfunction. In addition, changes in the levels of the Ca2+-handling proteins, SERCA2 and the Na+/Ca2+ exchanger, as well as troponin I phosphorylation, seen in the control-treated hypertensive rats were not observed in the βIIPKC-treated rats, suggesting that βIIPKC contributes to the regulation of calcium levels in the myocardium. In contrast, treatment with the selective inhibitor of βIPKC, an alternative spliced form of βIIPKC, had no beneficial effects in these rats. We also found that βIIV5-3, but not βIV5-3, improved calcium handling in isolated rat cardiomyocytes and enhanced contractility in isolated rat hearts. In conclusion, our data using an in vivo model of cardiac dysfunction (late-phase hypertrophy), suggest that βIIPKC contributes to the pathology associated with heart failure and thus an inhibitor of βIIPKC may be a potential treatment for this disease.
doi:10.1016/j.yjmcc.2011.08.025
PMCID: PMC3418885  PMID: 21920368
18.  β2-Adrenergic Receptors Mediate Cardioprotection through Crosstalk with Mitochondrial Cell Death Pathways 
Aims
β-adrenergic receptors (β-ARs) modulate cardiotoxicity/cardioprotection through crosstalk with multiple signaling pathways. We have previously shown that β2-ARs are cardioprotective during exposure to oxidative stress induced by doxorubicin (DOX). DOX cardiotoxicity is mediated in part through a Ca2+-dependent opening of the mitochondrial permeability transition (MPT), however the signals linking a cell surface receptor like the β2-AR to regulators of mitochondrial function are not clear. The objective of this study was to assess mechanisms of crosstalk between β2-ARs and mitochondrial cell death pathways.
Methods and Results
DOX administered to WT mice resulted in no acute mortality, however 85% of β2-/- mice died within 30 min. Several pro- and anti-survival pathways were altered. The pro-survival kinase, εPKC, was decreased by 64% in β2-/- after DOX vs WT (p<0.01); the εPKC activator ψεRACK partially rescued these mice (47% reduction in mortality). Activity of the pro-survival kinase Akt decreased by 76% in β2-/- after DOX vs WT (p<0.01). The α1-antagonist prazosin restored Akt activity to normal and also partially reversed the mortality (45%). Deletion of the β2-AR increased rate of Ca2+ release by 75% and peak [Ca2+]i by 20% respectively in isolated cardiomyocytes; the Ca2+ channel blocker verapamil also partially rescued the β2-/- (26%). Mitochondrial architecture was disrupted and complex I and II activities decreased by 40.9% and 34.6% respectively after DOX only in β2-/-. The MPT blocker cyclosporine reduced DOX mortality by 41% and prazosin plus cyclosporine acted synergistically to decrease mortality by 85%.
Conclusion
β2-ARs activate pro-survival kinases and attenuate mitochondrial dysfunction during oxidative stress; absence of β2-ARs enhances cardiotoxicity via negative regulation of survival kinases and enhancement of intracellular Ca2+, thus predisposing the mitochondria to opening of the MPT.
doi:10.1016/j.yjmcc.2011.06.019
PMCID: PMC3184305  PMID: 21756913
Adrenergic receptors; cardiomyopathy; mitochondria; signal transduction; protein kinases
19.  NEW DIRECTIONS IN BASIC RESEARCH IN HYPERTROPHY AND HEART FAILURE: RELEVANCE FOR PEDIATRIC CARDIOLOGY 
doi:10.1016/j.ppedcard.2011.06.002
PMCID: PMC3171751  PMID: 21927547
Cardiac hypertrophy; heart failure; pediatric cardiomyopathy; cell signaling
20.  Hindsight Bias from 3 to 95 Years of Age 
Upon learning the outcome to a problem, people tend to believe that they knew it all along (hindsight bias). Here we report the first study to trace the development of hindsight bias across the lifespan. 194 participants aged 3 to 95 years completed three tasks designed to measure visual and verbal hindsight bias. All age groups demonstrated hindsight bias on all three tasks; however, preschoolers and the elderly exhibited more bias than older children and younger adults. Multinomial processing tree analyses of these data revealed that preschoolers’ enhanced hindsight bias resulted from them substituting the correct answer for their original answer in their recall (a qualitative error). Conversely, older adults’ enhanced hindsight bias resulted from them forgetting their original answer and recalling an answer closer to, but not equal to, the correct answer (a quantitative error). We discuss these findings in relation to mechanisms of memory, perspective-taking, theory of mind, and executive function.
doi:10.1037/a0021971
PMCID: PMC3084020  PMID: 21299327
Hindsight bias; lifespan cognitive development; mathematical models of cognition; executive function; perspective taking
21.  THE ROLE OF β-ADRENERGIC RECEPTORS IN HEART FAILURE: DIFFERENTIAL REGULATION OF CARDIOTOXICITY AND CARDIOPROTECTION 
β-adrenergic receptor blockers have demonstrated significant survival benefit and have become standard therapy for adults with dilated cardiomyopathy, although their efficacy in pediatric patients is still unproven. Recent data suggests that the two major cardiac β-adrenergic receptor subtypes (β1 and β2) couple differentially to intracellular signaling pathways regulating contractility and remodeling. This has led some to suggest that the β1 receptor is the “cardiotoxic subtype” whereas the β2 receptor is “cardioprotective.” Given this paradigm, there could be situations where subtype selective β-blockade or even subtype selective β-stimulation might be beneficial. However, since most of these studies have been performed in isolated cardiomyocytes, their application to clinical practice is unclear. To better understand the roles of β1- vs. β2-receptors in the pathogenesis of clinical cardiomyopathy, we and others have taken advantage of several well-characterized murine models of cardiovascular disease. These studies demonstrate that β-receptor regulation of the balance between cardioprotection and cardiotoxicity is even more complex than previously appreciated: the role of each β-receptor subtype may vary depending on the specific cardiac stressor involved (e.g. ischemia, pressure overload, genetic mutation, cardiotoxin). Furthermore, the remodeling effects of β-receptor signaling have a temporal component, depending on whether a cardiac stress is acute vs. chronic.
doi:10.1016/j.ppedcard.2010.11.007
PMCID: PMC3135901  PMID: 21765627
Cardiomyopathy; adrenergic receptor; cell signaling; β-blocker; heart failure
22.  Hypercholesterolemia Impairs Exercise Capacity in Mice 
Objective
We previously reported an attenuation of both exercise hyperemia and measures of aerobic capacity in hypercholesterolemic mice. In this study we expanded upon the previous findings by examining the temporal and quantitative relationship of hypercholesterolemia to aerobic and anaerobic capacity and by exploring several potential mechanisms of dysfunction.
Methods
Eight-week old wild type (n=123) and apoE knockout (n=79) C57BL/6J mice were divided into groups with distinct cholesterol levels by feeding regular or high fat diets. At various ages the mice underwent treadmill ergospirometry. To explore mechanisms, aortic ring vasodilator function and nitrate (NOx) activity, urinary excretion of NOx, running muscle microvascular density and citrate synthase activity, as well as myocardial mass and histologic evidence of ischemia were measured.
Results
At 8 weeks of age, all mice had similar measures of exercise capacity. All indices of aerobic exercise capacity progressively declined at 12 and 20 weeks of age in the hypercholesterolemic mice as cholesterol levels increased while indices of anaerobic capacity remained unaffected. Across the 4 cholesterol groups, the degree of aerobic dysfunction was related to serum cholesterol levels; a relationship that was maintained after correcting for confounding factors. Associated with the deterioration in exercise capacity was a decline in measures of nitric oxide-mediated vascular function while there was no evidence of aberrations in functional or oxidative capacities or in other components of transport capacity.
Conclusion
Aerobic exercise dysfunction is observed in murine models of genetic and diet-induced hypercholesterolemia and is associated with a reduction in vascular nitric oxide production.
doi:10.1177/1358863X08100040
PMCID: PMC3140166  PMID: 19651675
Endothelial function; nitric oxide; oxygen consumption; cholesterol
23.  Differential cardiotoxic/cardioprotective effects of β-adrenergic receptor subtypes in myocytes and fibroblasts in doxorubicin cardiomyopathy 
β-Adrenoceptor (β-AR) subtypes act through different signaling pathways to regulate cardiac function and remodeling. Previous in vivo data show a markedly enhanced cardiotoxic response to doxorubicin in β2−/− mice, which is rescued by the additional deletion of the β1-AR. We determined whether this differential response was myocyte specific by examining the effects of doxorubicin in myocytes and fibroblasts from WT and β1, β2 and β1/β2−/− mice. Cells were exposed to doxorubicin at 1–50 µM and viability and apoptosis assessed at 6, 24 and 48 h. WT myocytes showed a time and dose-dependent decrease in viability (42% decrease at 1 µM after 24 h). β2−/− Myocytes showed a greater decrease in viability vs. WT (20.8% less at 6 h; 14% less at 24 h, P < 0.05); β1−/− and β1/β2−/− myocytes showed enhanced survival (β1−/− 11%; β1/β2−/− 18% greater than WT, P < 0.05). TUNEL staining demonstrated a similar differential susceptibility (WT 26% apoptotic nuclei, β2−/− 45.9%, β1/β2−/− 16.8%, P < 0.05). β2−/− Fibroblasts also showed enhanced toxicity. Pertussis toxin pretreatment of WT cells decreased survival similar to the β2−/−, suggesting a role for Gi signaling. JNK was differentially activated in β2−/− myocytes after doxorubicin and its inhibition increased cardiotoxicity. In conclusion, the differential cardioprotective/cardiotoxic effects mediated by β1 vs. β2-AR subtypes in knockout mice are recapitulated in myocytes isolated from these mice. β2-ARs appear to play a cardioprotective role, whereas β1-ARs a cardiotoxic role.
doi:10.1016/j.yjmcc.2005.12.004
PMCID: PMC3140223  PMID: 16458323
Cardiomyopathy; Signal transduction; Apoptosis; Adrenergic receptors; Anthracyclines
24.  GENOTYPIC VARIATION AND PHENOTYPIC CHARACTERIZATION OF GRANZYME B GENE POLYMORPHISMS 
Transplantation  2009;87(12):1801-1806.
Granzyme B has been associated with allograft rejection in solid organ transplantation. Single nucleotide polymorphisms (SNPs) in the Granzyme B gene might impact its expression. The aims of this study were 1) to establish the frequency of two Granzyme B SNPs (A-295G; Q-55R) in pediatric heart transplant (PHTx) recipients and 2) to determine their phenotypic expression in healthy individuals.
Methods
396 PHTx patients (245 White non-Hispanic, 49 Black non-Hispanic, 82 Hispanics, 20 others) and 52 healthy controls were screened for Q-55R and A-295G. For the control samples, we assessed the frequency of Granzyme B positive cells by ELISPOT assay following mitogen stimulation.
Results
Among the PHTx recipients, 57% percent of the population carried the Q/Q genotype, while 6 % were R/R homozygotes. Seven of 49 (14%) Black non-Hispanics (14%) were R/R homozygotes, while 13 out of 245 (5%) of White non-Hispanics and 5 out of 82 (6%) Hispanics carried the R/R genotype (p=0.02). The A allele frequency of Granzyme B A-295G (49.6%) was similar to that of the G allele (50.4%). However, 80% of Black non-Hispanics were A allele carriers compared to 68% of White non-Hispanics (p < 0.0001).
Following mitogen stimulation, the frequency of Granzyme B positive cells was higher in the Q/Q homozygotes compared to R/R carriers (p=0.006), while a similar frequency of Granzyme B positive cells was noticed among the genotypes of A-295G SNP.
Conclusions
These data indicate that 55 Q/Q genotype is associated with increased in vitro expression of Granzyme B.
doi:10.1097/TP.0b013e3181a755a4
PMCID: PMC2739874  PMID: 19543056
Granzyme B; gene polymorphism; transplantation; pediatric; heart
25.  The endogenous peptide apelin potently improves cardiac contractility and reduces cardiac loading in vivo 
Cardiovascular research  2005;65(1):73-82.
Objective
The endogenous peptide apelin is differentially regulated in cardiovascular disease but the nature of its role in cardiac function remains unclear.
Methods
We investigated the functional relevance of this pathway using ECG and respiration gated magnetic resonance imaging, conductance catheter pressure-volume hemodynamic measurements, and echocardiography in vivo. In addition, we carried out histology and immunohistochemistry to assess cardiac hypertrophy and to localize apelin and APJ in the adult and embryonic mouse heart.
Results
Intraperitoneal injection of apelin12 (300µg/kg) resulted in a decrease in left ventricular end diastolic area (pre: 0.122±0.007; post: 0.104±0.005 cm2, p=0.006) and an increase in heart rate (pre: 537±20; post 559±19 beats per minute, p=0.03). Hemodynamic measurements revealed a marked increase in ventricular elastance (pre: 3.7±0.9; post: 6.5±1.4 mmHg/RVU, p=0.018) and preload recruitable stroke work (pre: 27.4±8.0; post: 51.8±3.1, p=0.059) with little change in diastolic parameters following acute infusion of apelin. Chronic infusion (2mg/kg/day) resulted in significant increases in the velocity of circumferential shortening (baseline: 5.36±0.401; 14 days: 6.85±0.358 circ/sec, p=0.049) and cardiac output (baseline: 0.142±0.019; 14 days: 0.25±0.019 l/min, p=0.001) as determined by 15MHz echocardiography. Post mortem corrected heart weights were not different between apelin and saline groups (p=0.5) and histology revealed no evidence of cellular hypertrophy in the apelin group (nuclei per unit area, p=0.9). Immunohistochemistry studies revealed APJ staining of myocardial cells in all regions of the adult mouse heart. Antibody staining, as well as quantitative real time polymerase chain reaction identified expression of both APJ and apelin in embryonic myocardium as early as embryonic day 13.5.
Conclusions
Apelin reduces left ventricular preload and afterload, and increases contractile reserve without evidence of hypertrophy. These results associate apelin with a positive hemodynamic profile and suggest it as an attractive target for pharmacotherapy in the setting of heart failure.
doi:10.1016/j.cardiores.2004.08.018
PMCID: PMC2517138  PMID: 15621035
Apelin; APJ; angiotensin; heart failure; pressure-volume hemodynamics

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