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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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

Results 1-7 (7)