Publications by authors named "Chee Chew Lim"

23 Publications

  • Page 1 of 1

Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Circ Res 2015 Dec 1;117(12):995-1000. Epub 2015 Oct 1.

From the Departments of Pharmacology (T.K.F.), Cardiovascular Medicine (Y.W.C., J.E.H., C.C.L., C.C.H.), and Department of Medicine, Divisions of Cardiovascular Medicine and Clinical Pharmacology, Oates Institute for Experimental Therapeutics (L.W., B.C.K.), Department of Molecular Physiology and Biophysics (A.G.C.), Vanderbilt University School of Medicine, Nashville, TN; Departments of Cell and Developmental Biology (C.H.W.) and Biomedical Engineering (N.B., W.D.M.), Vanderbilt University, Nashville, TN; Research Medicine, Veterans Affairs TVHS, Nashville, TN (C.C.H.); and Stanford Cardiovascular Institute, Department of Medicine, Division of Cardiology and Department of Radiology, Stanford University School of Medicine, CA (J.C.W.).

Rationale: The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery.

Objective: To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs.

Methods And Results: Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa).

Conclusions: The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.
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http://dx.doi.org/10.1161/CIRCRESAHA.115.307580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4670592PMC
December 2015

Right ventricular protein expression profile in end-stage heart failure.

Pulm Circ 2015 Sep;5(3):481-97

Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA.

Little is known about the right ventricular (RV) proteome in human heart failure (HF), including possible differences compared to the left ventricular (LV) proteome. We used 2-dimensional differential in-gel electrophoresis (pH: 4-7, 10-150 kDa), followed by liquid chromatography tandem mass spectrometry, to compare the RV and LV proteomes in 12 explanted human hearts. We used Western blotting and multiple-reaction monitoring for protein verification and RNA sequencing for messenger RNA and protein expression correlation. In all 12 hearts, the right ventricles (RVs) demonstrated differential expression of 11 proteins relative to the left ventricles (LVs), including lesser expression of CRYM, TPM1, CLU, TXNL1, and COQ9 and greater expression of TNNI3, SAAI, ERP29, ACTN2, HSPB2, and NDUFS3. Principal-components analysis did not suggest RV-versus-LV proteome partitioning. In the nonischemic RVs (n = 6), 7 proteins were differentially expressed relative to the ischemic RVs (n = 6), including increased expression of CRYM, B7Z964, desmin, ANXA5, and MIME and decreased expression of SERPINA1 and ANT3. Principal-components analysis demonstrated partitioning of the nonischemic and ischemic RV proteomes, and gene ontology analysis identified differences in hemostasis and atherosclerosis-associated networks. There were no proteomic differences between RVs with echocardiographic dysfunction (n = 8) and those with normal function (n = 4). Messenger RNA and protein expression did not correlate consistently, suggesting a major role for RV posttranscriptional protein expression regulation. Differences in contractile, cytoskeletal, metabolic, signaling, and survival pathways exist between the RV and the LV in HF and may be related to the underlying HF etiology and differential posttranscriptional regulation.
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http://dx.doi.org/10.1086/682219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4556499PMC
September 2015

Differential activation of natriuretic peptide receptors modulates cardiomyocyte proliferation during development.

Development 2014 Jan 18;141(2):335-45. Epub 2013 Dec 18.

Division of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, TN 37235, USA.

Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways.
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http://dx.doi.org/10.1242/dev.100370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3879815PMC
January 2014

26S proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells.

Biochem Biophys Res Commun 2012 Sep 7;425(4):830-5. Epub 2012 Aug 7.

Department of Pathology, Microbiology and Immunology, Vanderbilt University School of Medicine, Nashville, TN 37232-2561, USA.

Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.
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http://dx.doi.org/10.1016/j.bbrc.2012.07.162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3460693PMC
September 2012

The continuing evolution of the Langendorff and ejecting murine heart: new advances in cardiac phenotyping.

Am J Physiol Heart Circ Physiol 2012 Jul 25;303(2):H156-67. Epub 2012 May 25.

Cardiac Muscle Research Laboratory, Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA, USA.

The isolated retrograde-perfused Langendorff heart and the isolated ejecting heart have, over many decades, resulted in fundamental discoveries that form the underpinnings of our current understanding of the biology and physiology of the heart. These two experimental methodologies have proven invaluable in studying pharmacological effects on myocardial function, metabolism, and vascular reactivity and in the investigation of clinically relevant disease states such as ischemia-reperfusion injury, diabetes, obesity, and heart failure. With the advent of the genomics era, the isolated mouse heart preparation has gained prominence as an ex vivo research tool for investigators studying the impact of gene modification in the intact heart. This review summarizes the historical development of the isolated heart and provides a practical guide for the establishment of the Langendorff and ejecting heart preparations with a particular emphasis on the murine heart. In addition, current applications and novel methods of recording cardiovascular parameters in the isolated heart preparation will be discussed. With continued advances in methodological recordings, the isolated mouse heart preparation will remain physiologically relevant for the foreseeable future, serving as an integral bridge between in vitro assays and in vivo approaches.
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http://dx.doi.org/10.1152/ajpheart.00333.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3404701PMC
July 2012

Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy.

PLoS One 2012 20;7(4):e35743. Epub 2012 Apr 20.

Molecular Medicine Program, Department of Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America.

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0035743PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3332030PMC
August 2012

Neuregulin-1β regulation of embryonic endothelial progenitor cell survival.

Am J Physiol Heart Circ Physiol 2011 Apr 14;300(4):H1311-9. Epub 2011 Jan 14.

Department of Molecular Medicine, Boston University, Massachusetts, USA.

Endothelial progenitor cells (EPCs) are mobilized into the vascular space and home to damaged tissues, where they promote repair in part through a process of angiogenesis. Neuregulins (NRGs) are ligands in the epidermal growth factor family that signal through type I receptor tyrosine kinases in the erbB family (erbB2, erbB3, and erbB4) and regulate endothelial cell biology, promoting angiogenesis. Stimuli such as ischemia and exercise that promote EPC mobilization also induce cleavage and release of transmembrane NRG from cardiac microvascular endothelial cells (CMECs). We hypothesized that NRG/erbB signaling may regulate EPC biology. Using an embryonic (e)EPC cell line that homes to and repairs injured myocardium, we were able to detect erbB2 and erbB3 transcripts. Identical receptor expression was found in EPCs isolated from rat bone marrow and human whole blood. NRG treatment of eEPCs induces phosphorylation of kinases including Akt, GSK-3β, and Erk1/2 and the nuclear accumulation and transcriptional activation of β-catenin. NRG does not induce eEPC proliferation or migration but does protect eEPCs against serum deprivation-induced apoptosis. These results suggest a role for tissue-derived NRG in the regulation of EPC survival.
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http://dx.doi.org/10.1152/ajpheart.01104.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075022PMC
April 2011

Mechanisms of anthracycline cardiac injury: can we identify strategies for cardioprotection?

Prog Cardiovasc Dis 2010 Sep-Oct;53(2):105-13

Department of Medicine, Vanderbilt University, Nashville, TN, USA.

Anthracycline antibiotics have saved the lives of many cancer victims in the 50 plus years since their discovery. A major limitation of their use is the dose-limiting cardiotoxicity. Efforts focusing on understanding the biochemical basis for anthracycline cardiac effects have provided several strategies currently in clinical use: limit dose exposure, encapsulate anthracyclines in liposomes to reduce myocardial uptake, administer concurrently with the iron chelator dexrazoxane to reduce free iron-catalyzed reactive oxygen species formation; and modify anthracycline structure in an effort to reduce myocardial toxicity. Despite these efforts, anthracycline-induced heart failure continues to occur with consequences for both morbidity and mortality. Our inability to predict and prevent anthracycline cardiotoxicity is, in part, due to the fact that the molecular and cellular mechanisms remain controversial and incompletely understood. Studies examining the effects of anthracyclines in cardiac myocytes in vitro and small animals in vivo have demonstrated several forms of cardiac injury, and it remains unclear how these translate to the clinical setting. Given the clinical evidence that myocyte death occurs after anthracycline exposure in the form of elevations in serum troponin, myocyte cell death seems to be a probable mechanism for anthracycline-induced cardiac injury. Other mechanisms of myocyte injury include the development of cellular "sarcopenia" characterized by disruption of normal sarcomere structure. Anthracyclines suppress expression of several cardiac transcription factors, and this may play a role in the development of myocyte death as well as sarcopenia. Degradation of the giant myofilament protein titin may represent an important proximal step that leads to accelerated myofilament degradation. An interesting interaction has been noted clinically between anthracyclines and newer cancer therapies that target the erbB2 receptor tyrosine kinase. There is now evidence that erbB2 signaling in response to the ligand neuregulin regulates anthracycline uptake into cells via the multidrug-resistance protein. Therefore, up-regulation of cardiac neuregulin signaling may be one strategy to limit myocardial anthracycline injury. Moreover, assessing an individual's risk for anthracycline injury may be improved by having some measure of endogenous activity of this and other myocardial protective signals.
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http://dx.doi.org/10.1016/j.pcad.2010.06.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2933091PMC
September 2010

Glutathione peroxidase deficiency exacerbates ischemia-reperfusion injury in male but not female myocardium: insights into antioxidant compensatory mechanisms.

Am J Physiol Heart Circ Physiol 2009 Dec 2;297(6):H2144-53. Epub 2009 Oct 2.

Department of Cardiovascular Medicine, Vanderbilt University School of Medicine, 2220 Pierce Ave., PRB 340, Nashville, TN 37232, USA.

The female sex has been associated with increased resistance to acute myocardial ischemia-reperfusion (I/R) injury. While enhanced antioxidant capacity has been implicated in female cardioprotection, there is little evidence to support this assumption. Previous studies have shown an important role of cellular glutathione peroxidase (GPx1) in protection of the myocardium from I/R injury. Whether GPx1 is mechanistic in the protection of female myocardium, post-I/R, has not been examined. We utilized a murine model with homozygous deletion of GPx1 and examined its impact on postischemic myocardial recovery in male and female mice. Following I/R, male GPx1(-/-) hearts were more susceptible to contractile and diastolic dysfunction, and this was associated with increased protein carbonyls, a marker of oxidative stress. In contrast, GPx1 deficiency in female hearts did not exacerbate dysfunction or oxidative stress post-I/R. Both wild-type and GPx1(-/-) female hearts exhibited reduced creatine kinase leakage and a more favorable ascorbate redox status compared with males. Following I/R, female GPx1(-/-) hearts showed a comparable decrease in glutathione redox status as their male counterparts; however, they exhibited a greater decrease in nitrate-to-nitrite ratio, suggesting a higher consumption of nitrate in female GPx1(-/-) hearts. Our findings demonstrate that GPx1 is critical for cardioprotection during I/R in male, but not female, mice. The maintenance of cardioprotection in female mice lacking GPx1 post-I/R may be due to an improved ascorbate redox homeostasis and enhanced nitrate-to-nitrite conversion, which would predictably be accompanied by enhanced production of cardioprotective nitric oxide.
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http://dx.doi.org/10.1152/ajpheart.00673.2009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2793140PMC
December 2009

A novel role for tumor necrosis factor-like weak inducer of apoptosis (TWEAK) in the development of cardiac dysfunction and failure.

Circulation 2009 Apr 6;119(15):2058-68. Epub 2009 Apr 6.

Division of Cardiology, Brigham and Women's Hospital, Harvard Medical School, Boston, MA 02115, USA.

Background: Tumor necrosis factor-like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis factor superfamily, is a multifunctional cytokine known to regulate cellular functions in contexts of injury and disease through its receptor, fibroblast growth factor-inducible molecule 14 (Fn14). Although many of the processes and downstream signals regulated by the TWEAK/Fn14 pathway have been implicated in the development of cardiac dysfunction, the role of TWEAK in the cardiovascular system is completely unknown.

Methods And Results: Herein, we demonstrate that mouse and human cardiomyocytes express the TWEAK receptor Fn14. Furthermore, we determine that elevated circulating levels of TWEAK, induced via transgenic or adenoviral-mediated gene expression in mice, result in dilated cardiomyopathy with subsequent severe cardiac dysfunction. This phenotype was mediated exclusively by the Fn14 receptor, independent of tumor necrosis factor-alpha, and was associated with cardiomyocyte elongation and cardiac fibrosis but not cardiomyocyte apoptosis. Moreover, we find that circulating TWEAK levels were differentially upregulated in patients with idiopathic dilated cardiomyopathy compared with other forms of heart disease and normal control subjects.

Conclusions: Our data suggest that TWEAK/Fn14 may be important in regulating myocardial structural remodeling and function and may play a role in the pathogenesis of dilated cardiomyopathy.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.108.837286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2924152PMC
April 2009

A novel mutant cardiac troponin C disrupts molecular motions critical for calcium binding affinity and cardiomyocyte contractility.

Biophys J 2008 May 22;94(9):3577-89. Epub 2008 Jan 22.

Department of Cardiovascular Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee 37232, USA.

Troponin C (TnC) belongs to the superfamily of EF-hand (helix-loop-helix) Ca(2+)-binding proteins and is an essential component of the regulatory thin filament complex. In a patient diagnosed with idiopathic dilated cardiomyopathy, we identified two novel missense mutations localized in the regulatory Ca(2+)-binding Site II of TnC, TnC((E59D,D75Y)). Expression of recombinant TnC((E59D,D75Y)) in isolated rat cardiomyocytes induced a marked decrease in contractility despite normal intracellular calcium homeostasis in intact cardiomyocytes and resulted in impaired myofilament calcium responsiveness in Triton-permeabilized cardiomyocytes. Expression of the individual mutants in cardiomyocytes showed that TnC(D75Y) was able to recapitulate the TnC((E59D,D75Y)) phenotype, whereas TnC(E59D) was functionally benign. Force-pCa relationships in TnC((E59D,D75Y)) reconstituted rabbit psoas fibers and fluorescence spectroscopy of TnC((E59D,D75Y)) labeled with 2-[(4'-iodoacetamide)-aniline]naphthalene-6-sulfonic acid showed a decrease in myofilament Ca(2+) sensitivity and Ca(2+) binding affinity, respectively. Furthermore, computational analysis of TnC showed the Ca(2+)-binding pocket as an active region of concerted motions, which are decreased markedly by mutation D75Y. We conclude that D75Y interferes with proper concerted motions within the regulatory Ca(2+)-binding pocket of TnC that hinders the relay of the thin filament calcium signal, thereby providing a primary stimulus for impaired cardiomyocyte contractility. This in turn may trigger pathways leading to aberrant ventricular remodeling and ultimately a dilated cardiomyopathy phenotype.
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http://dx.doi.org/10.1529/biophysj.107.112896DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2292379PMC
May 2008

Enhanced calcium cycling and contractile function in transgenic hearts expressing constitutively active G alpha o* protein.

Am J Physiol Heart Circ Physiol 2008 Mar 11;294(3):H1335-47. Epub 2008 Jan 11.

Division of Cardiology, Rhode Island Hospital and The Warren Alpert Medical School of Brown University, Providence, RI 02903, USA.

In contrast to the other heterotrimeric GTP-binding proteins (G proteins) Gs and Gi, the functional role of G o is still poorly defined. To investigate the role of G alpha o in the heart, we generated transgenic mice with cardiac-specific expression of a constitutively active form of G alpha o1* (G alpha o*), the predominant G alpha o isoform in the heart. G alpha o expression was increased 3- to 15-fold in mice from 5 independent lines, all of which had a normal life span and no gross cardiac morphological abnormalities. We demonstrate enhanced contractile function in G alpha o* transgenic mice in vivo, along with increased L-type Ca2+ channel current density, calcium transients, and cell shortening in ventricular G alpha o*-expressing myocytes compared with wild-type controls. These changes were evident at baseline and maintained after isoproterenol stimulation. Expression levels of all major Ca2+ handling proteins were largely unchanged, except for a modest reduction in Na+/Ca2+ exchanger in transgenic ventricles. In contrast, phosphorylation of the ryanodine receptor and phospholamban at known PKA sites was increased 1.6- and 1.9-fold, respectively, in G alpha o* ventricles. Density and affinity of beta-adrenoceptors, cAMP levels, and PKA activity were comparable in G alpha o* and wild-type myocytes, but protein phosphatase 1 activity was reduced upon G alpha o* expression, particularly in the vicinity of the ryanodine receptor. We conclude that G alpha o* exerts a positive effect on Ca2+ cycling and contractile function. Alterations in protein phosphatase 1 activity rather than PKA-mediated phosphorylation might be involved in hyperphosphorylation of key Ca2+ handling proteins in hearts with constitutive G alpha o activation.
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http://dx.doi.org/10.1152/ajpheart.00584.2007DOI Listing
March 2008

Molecular and cellular mechanisms of anthracycline cardiotoxicity.

Cardiovasc Toxicol 2007 ;7(2):114-21

Department of Molecular Medicine, Boston University, Boston, TN, USA.

The molecular and cellular mechanisms that cause cumulative dose-dependent anthracycline-cardiotoxicity remain controversial and incompletely understood. Studies examining the effects of anthracyclines in cardiac myocytes inA vitro have demonstrated several forms of cellular injury. Cell death in response to anthracyclines can be observed by one of several mechanisms including apoptosis and necrosis. Cell death by apoptosis can be inhibited by dexrazoxane, the iron chelator that is known to prevent clinical development of heart failure at high cumulative anthracycline exposure. Together with clinical evidence for myocyte death after anthracycline exposure, in the form of elevations in serum troponin, make myocyte cell death a probable mechanism for anthracycline-induced cardiac injury. Other mechanisms of myocyte injury include the development of cellular \'sarcopenia\' characterized by disruption of normal sarcomere structure. Anthracyclines suppress expression of several cardiac transcription factors, and this may play a role in the development of myocyte death as well as sarcopenia. Degradation of the giant myofilament protein titin may represent an important proximal step that leads to accelerated myofilament degradation. Titin is an entropic spring element in the sarcomere that regulates length-dependent calcium sensitivity. Thus titin degradation may lead to impaired diastolic as well as systolic dysfunction, as well as potentiate the effect of suppression of transcription of sarcomere proteins. An interesting interaction has been noted clinically between anthracyclines and newer cancer therapies that target the erbB2 receptor tyrosine kinase. Studies of erbB2 function in viro suggest that signaling through erbB2 by the growth factor neuregulin may regulate cardiac myocyte sarcomere turnover, as well as myocyte-myocyte/myocyte-matrix force coupling. A combination of further in vitro studies, with more careful monitoring of cardiac function after exposure to these cancer therapies, may help to understand to what extent these mechanisms are at work during clinical exposure of the heart to these important pharmaceuticals.
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http://dx.doi.org/10.1007/s12012-007-0005-5DOI Listing
October 2007

Simultaneous orientation and cellular force measurements in adult cardiac myocytes using three-dimensional polymeric microstructures.

Cell Motil Cytoskeleton 2007 Sep;64(9):718-25

Laboratory for Biomedical Microsystems, Department of Biomedical Engineering, The Ohio State University, Columbus, Ohio 43210, USA.

A number of techniques have been developed to monitor contractile function in isolated cardiac myocytes. While invaluable observations have been gained from these methodologies in understanding the contractile processes of the heart, they are invariably limited by their in vitro conditions. The present challenge is to develop innovative assays to mimic the in vivo milieu so as to allow a more physiological assessment of cardiac myocyte contractile forces. Here we demonstrate the use of a silicone elastomer, poly(dimethylsiloxane) (PDMS), to simultaneously orient adult cardiac myocytes in primary culture and measure the cellular forces in a three-dimensional substrate. The realignment of adult cardiac myocytes in long-term culture (7 days) was achieved due to directional reassembly of the myofibrils along the parallel polymeric sidewalls. The cellular mechanical forces were recorded in situ by observing the deformation of the micropillars embedded in the substrate. By coupling the cellular mechanical force measurements with on-chip cell orientation, this novel assay is expected to provide a means of a more physiological assessment of single cardiac myocyte contractile function and may facilitate the future development of in vitro assembled functional cardiac tissue.
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http://dx.doi.org/10.1002/cm.20218DOI Listing
September 2007

A novel microfluidic impedance assay for monitoring endothelin-induced cardiomyocyte hypertrophy.

Biosens Bioelectron 2007 Mar 7;22(8):1688-93. Epub 2006 Sep 7.

Laboratory for Microsystems Technology, Department of Manufacturing Engineering, 15 Saint Mary's Street, Boston University, Boston, MA 02215, USA.

Cardiac hypertrophy is an established and independent risk factor for the development of heart failure and sudden cardiac death. At the level of individual cardiac myocytes (heart muscle cells), the cell morphology alters (increase in cell size and myofibrillar re-organization) and protein synthesis is activated. In this paper, a novel cardiomyocyte-based impedance sensing system with the assistance of dielectrophoresis cell concentration is reported to monitor the dynamic process of endothelin-1-induced cardiomyocyte hypertrophy. A dielectrophoresis (DEP) microfluidic device is fabricated capable of concentrating cells from a dilute sample to form a confluent cell monolayer on the surface of microelectrodes. This device can increase the sensitivity of the impedance system and also has the potential to reduce the time for detection by a significant factor. To examine the feasibility of this impedance sensing system, cardiomyocytes are treated with endothelin-1 (ET-1), a known hypertrophic agent. ET-1 induces a continuous rise in cardiomyocyte impedance, which we interpret as strengthening of cellular attachments to the surface substrate. An equivalent circuit model is introduced to fit the impedance spectrum to fully understand the impedance sensing system.
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http://dx.doi.org/10.1016/j.bios.2006.07.032DOI Listing
March 2007

Rapid electrical stimulation induces early activation of kinase signal transduction pathways and apoptosis in adult rat ventricular myocytes.

Exp Physiol 2006 Jul 4;91(4):773-80. Epub 2006 May 4.

Molecular Stress Response Unit, Whitaker Cardiovascular Institute, Department of Medicine, Boston University Medical Center, Boston, MA 02118, USA.

Chronic tachycardia in patients and rapid pacing in animal models induce myocardial dysfunction and initiate a cascade of compensatory adaptations that are ultimately unsustainable, leading to ventricular enlargement and failure. The molecular pathogenesis during the early stages of tachycardia-induced cardiomyopathy, however, remains unclear. We utilized our previously reported cell culture pacing system to directly assess phosphatidylinositol-3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signalling of adult rat ventricular myocytes (ARVM) in response to rapid electrical stimulation. Freshly isolated ARVMs were maintained quiescent (0 Hz), or continuously stimulated at 5 (normofrequency) and 8 Hz (rapid frequency). Pacing resulted in an increase in mitochondrial respiration, assessed by mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) at 48 h. Rapid pacing at 8 Hz significantly increased cell injury and death as assessed by Trypan Blue uptake, creatine phosphokinase release, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay. Pacing at 5 Hz induced early, but weak, activation of Akt and protein kinase 38 (p38). Rapid pacing further augmented the early activation of Akt and p38, and induced extracellular signal-related kinase (Erk) and c-jun amino terminal kinase (JNK) activation. Incubation of ARVM with PI3K inhibitor LY294002 resulted in a twofold increase of TUNEL-positive cells under all pacing conditions examined. In conclusion, rapid pacing has immediate and detrimental consequences for cardiomyocyte survival, with pro-apoptotic pathways (e.g. JNK, p38) able to overwhelm antiapoptotic signalling (PI3K/Akt, Erk). The rapid pacing methodology described in this report will be particularly useful in determination of cell signalling pathways associated with tachycardia-induced cardiomyopathy.
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http://dx.doi.org/10.1113/expphysiol.2006.033894DOI Listing
July 2006

The cardiotoxicology of anthracycline chemotherapeutics: translating molecular mechanism into preventative medicine.

Mol Interv 2005 Jun;5(3):163-71

Whitaker Cardiovascular Institute and Center for Molecular Stress Response, Boston University Medical Center, 650 Albany Street, Boston, MA 02118, USA.

Anthracyclines remain a mainstay of chemotherapy in spite of their well-recognized cardiotoxicity. Recent experience with trastuzumab (Herceptin) and anthracycline therapy has prompted a detailed analysis of the function of erbB2 in the heart. These studies demonstrate a cardioprotective effect of neuregulin, the endogenous ligand for the erbB4/erbB2 heterodimeric receptor complex. Although the mechanisms of cytoprotection remain incompletely understood, these studies have triggered the question of whether physiological manipulation of cardioprotective pathways that involve erbB can be used to improve outcome in patients treated with anthracyclines. The local activation of cardioprotection by cardiovascular exercise may be such a manipulation and warrants further investigation.
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http://dx.doi.org/10.1124/mi.5.3.6DOI Listing
June 2005

Modulation of cardiac function: titin springs into action.

J Gen Physiol 2005 Mar;125(3):249-52

Whitaker Cardiovascular Institute and Center for the Molecular Stress Response, Boston University School of Medicine, Boston, MA 02118, USA.

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http://dx.doi.org/10.1085/jgp.200509268DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2234017PMC
March 2005

Anthracyclines induce calpain-dependent titin proteolysis and necrosis in cardiomyocytes.

J Biol Chem 2004 Feb 14;279(9):8290-9. Epub 2003 Dec 14.

Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118, USA.

Titin, the largest myofilament protein, serves as a template for sarcomere assembly and acts as a molecular spring to contribute to diastolic function. Titin is known to be extremely susceptible to calcium-dependent protease degradation in vitro. We hypothesized that titin degradation is an early event in doxorubicin-induced cardiac injury and that titin degradation occurs by activation of the calcium-dependent proteases, the calpains. Treatment of cultured adult rat cardiomyocytes with 1 or 3 micromol/liter doxorubicin for 24 h resulted in degradation of titin in myocyte lysates, which was confirmed by a reduction in immunostaining of an antibody to the spring-like (PEVK) domain of titin at the I-band of the sarcomere. The elastic domain of titin appears to be most susceptible to proteolysis because co-immunostaining with an antibody to titin at the M-line was preserved, suggesting targeted proteolysis of the spring-like domain of titin. Doxorubicin treatment for 1 h resulted in approximately 3-fold increase in calpain activity, which remained elevated at 48 h. Co-treatment with calpain inhibitors resulted in preservation of titin, reduction in myofibrillar disarray, and attenuation of cardiomyocyte necrosis but not apoptosis. Co-treatment with a caspase inhibitor did not prevent the degradation of titin, which precludes caspase-3 as an early mechanism of titin proteolysis. We conclude that calpain activation is an early event after doxorubicin treatment in cardiomyocytes and appears to target the degradation of titin. Proteolysis of the spring-like domain of titin may predispose cardiomyocytes to diastolic dysfunction, myofilament instability, and cell death by necrosis.
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http://dx.doi.org/10.1074/jbc.M308033200DOI Listing
February 2004

Myocyte contractile activity modulates norepinephrine cytotoxicity and survival effects of neuregulin-1beta.

Am J Physiol Cell Physiol 2004 Feb 1;286(2):C222-9. Epub 2003 Oct 1.

Whitaker Cardiovascular Institute and Cardiovascular Divisions, Department of Medicine, Boston University Medical Center, Boston, MA 02118, USA.

The purpose of this study is to test the hypothesis that mechanical and electrical activity in adult rat ventricular myocytes (ARVM) alters responses to proapoptotic and prosurvival ligands. The effects of electrical stimulation on myocyte survival, stress signaling, response to beta-adrenergic receptor (beta-AR)-stimulated apoptosis, and neuregulin-1beta (NRG) were examined. Electrical stimulation (6.6 V/cm; 0, 2, and 5 Hz; 2-ms duration; alternating polarity) of ARVM resulted in more than 70% capture. Although ARVM paced for 48 h showed higher mitochondrial uptake of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (P < 0.05, 0 vs. 2 and 5 Hz), electrical stimulation had little effect on cell survival assessed by trypan blue uptake, CPK release, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining. Electrical stimulation for 24 h did not induce stress response (heat shock protein 70, 90) nor stress kinase (Erk, JNK, p38) activation. NRG stimulation of Erk and Akt was similar between paced and quiescent cells. Pacing sensitized myocytes to beta-AR-stimulated JNK phosphorylation and cell death with 0.1 microM norepinephrine (NE) in paced myocytes causing equivalent cytotoxicity to 10 microM NE in quiescent cells. NRG suppressed beta-AR-induced apoptosis through a phosphatidylinositol-3-kinase-dependent pathway in both paced and quiescent cells, although it is overwhelmed by high-NE concentration in paced cells. Thus myocyte contractility modulates both NE cytotoxicity as well as the cytoprotective effect of NRG. These results demonstrate the feasibility and importance of using electrically paced cardiomyocytes in primary culture when examining the signaling pathways of cell survival.
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http://dx.doi.org/10.1152/ajpcell.00312.2003DOI Listing
February 2004

Glucose-6-phosphate dehydrogenase modulates cytosolic redox status and contractile phenotype in adult cardiomyocytes.

Circ Res 2003 Jul 26;93(2):e9-16. Epub 2003 Jun 26.

Cardiac Muscle Research Laboratory, Whitaker Cardiovascular Institute and Evans Department of Medicine, Boston University School of Medicine, Boston, Mass, USA.

Reactive oxygen species (ROS)-mediated cell injury contributes to the pathophysiology of cardiovascular disease and myocardial dysfunction. Protection against ROS requires maintenance of endogenous thiol pools, most importantly, reduced glutathione (GSH), by NADPH. In cardiomyocytes, GSH resides in two separate cellular compartments: the mitochondria and cytosol. Although mitochondrial GSH is maintained largely by transhydrogenase and isocitrate dehydrogenase, the mechanisms responsible for sustaining cytosolic GSH remain unclear. Glucose-6-phosphate dehydrogenase (G6PD) functions as the first and rate-limiting enzyme in the pentose phosphate pathway, responsible for the generation of NADPH in a reaction coupled to the de novo production of cellular ribose. We hypothesized that G6PD is required to maintain cytosolic GSH levels and protect against ROS injury in cardiomyocytes. We found that in adult cardiomyocytes, G6PD activity is rapidly increased in response to cellular oxidative stress, with translocation of G6PD to the cell membrane. Furthermore, inhibition of G6PD depletes cytosolic GSH levels and subsequently results in cardiomyocyte contractile dysfunction through dysregulation of calcium homeostasis. Cardiomyocyte dysfunction was reversed through treatment with either a thiol-repleting agent (L-2-oxothiazolidine-4-carboxylic acid) or antioxidant treatment (Eukarion-134), but not with exogenous ribose. Finally, in a murine model of G6PD deficiency, we demonstrate the development of in vivo adverse structural remodeling and impaired contractile function over time. We, therefore, conclude that G6PD is a critical cytosolic antioxidant enzyme, essential for maintenance of cytosolic redox status in adult cardiomyocytes. Deficiency of G6PD may contribute to cardiac dysfunction through increased susceptibility to free radical injury and impairment of intracellular calcium transport. The full text of this article is available online at http://www.circresaha.org.
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http://dx.doi.org/10.1161/01.RES.0000083489.83704.76DOI Listing
July 2003

Titin determines the Frank-Starling relation in early diastole.

J Gen Physiol 2003 Feb;121(2):97-110

Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA 02118, USA.

Titin, a giant protein spanning half the sarcomere, is responsible for passive and restoring forces in cardiac myofilaments during sarcomere elongation and compression, respectively. In addition, titin has been implicated in the length-dependent activation that occurs in the stretched sarcomere, during the transition from diastole to systole. The purpose of this study was to investigate the role of titin in the length-dependent deactivation that occurs during early diastole, when the myocyte is shortened below slack length. We developed a novel in vitro assay to assess myocyte restoring force (RF) by measuring the velocity of recoil in Triton-permeabilized, unloaded rat cardiomyocytes after rigor-induced sarcomere length (SL) contractions. We compared rigor-induced SL shortening to that following calcium-induced (pCa) contractions. The RF-SL relationship was linearly correlated, and the SL-pCa curve displayed a characteristic sigmoidal curve. The role of titin was defined by treating myocytes with a low concentration of trypsin, which we show selectively degrades titin using mass spectroscopic analysis. Trypsin treatment reduced myocyte RF as shown by a decrease in the slope of the RF-SL relationship, and this was accompanied by a downward and leftward shift of the SL-pCa curve, indicative of sensitization of the myofilaments to calcium. In addition, trypsin digestion did not alter the relationship between SL and interfilament spacing (assessed by cell width) after calcium activation. These data suggest that as the sarcomere shortens below slack length, titin-based restoring forces act to desensitize the myofilaments. Furthermore, in contrast to length-dependent activation at long SLs, length-dependent deactivation does not depend on interfilament spacing. This study demonstrates for the first time the importance of titin-based restoring force in length-dependent deactivation during the early phase of diastole.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2217323PMC
http://dx.doi.org/10.1085/jgp.20028652DOI Listing
February 2003

Heterozygous cellular glutathione peroxidase deficiency in the mouse: abnormalities in vascular and cardiac function and structure.

Circulation 2002 Aug;106(9):1154-8

Evans Department of Medicine, Boston University School of Medicine, Boston, Mass, USA.

Background: Oxidant stress has been implicated in the pathogenesis of atherothrombosis and other vascular disorders accompanied by endothelial dysfunction. Glutathione peroxidases (GPx) play an important role in the cellular defense against oxidant stress by utilizing glutathione (GSH) to reduce lipid hydroperoxides and hydrogen peroxide to their corresponding alcohols. Cellular GPx (GPx-1) is the principal intracellular isoform of GPx. We hypothesized that GPx-1 deficiency per se induces endothelial dysfunction and structural vascular abnormalities through increased oxidant stress.

Methods And Results: A murine model of heterozygous deficiency of GPx-1 (GPx(+/-)) was investigated to examine this hypothesis. Mesenteric arterioles in GPx-1(+/-) mice demonstrated vasoconstriction to acetylcholine compared with vasodilation in wild-type mice (maximal change in vessel diameter, -13.0+/-2.8% versus 13.2+/-2.8%, P<0.0001). We also noted an increase in the plasma and aortic levels of the isoprostane iPF(2alpha)-III, a marker of oxidant stress, in GPx-1(+/-) mice compared with wild-type mice (170.4+/-23 pg/mL plasma versus 98.7+/-7.1 pg/mL plasma, P<0.03; 11.7+/-0.87 pg/mg aortic tissue versus 8.2+/-0.55 pg/mg aortic tissue, P<0.01). Histological sections from the coronary vasculature of GPx-1(+/-) mice show increased perivascular matrix deposition, an increase in the number of adventitial fibroblasts, and intimal thickening. These structural abnormalities in the myocardial vasculature were accompanied by diastolic dysfunction after ischemia-reperfusion.

Conclusions: These findings demonstrate that heterozygous deficiency of GPx-1 leads to endothelial dysfunction, possibly associated with increased oxidant stress, and to significant structural vascular and cardiac abnormalities. These data illustrate the importance of this key antioxidant enzyme in functional and structural responses of the mammalian cardiovascular system.
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http://dx.doi.org/10.1161/01.cir.0000026820.87824.6aDOI Listing
August 2002