Publications by authors named "Anant Chopra"

19 Publications

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Probing the subcellular nanostructure of engineered human cardiomyocytes in 3D tissue.

Microsyst Nanoeng 2021 27;7:10. Epub 2021 Jan 27.

Department of Mechanical Engineering, Boston University, Boston, MA 02215 USA.

The structural and functional maturation of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is essential for pharmaceutical testing, disease modeling, and ultimately therapeutic use. Multicellular 3D-tissue platforms have improved the functional maturation of hiPSC-CMs, but probing cardiac contractile properties in a 3D environment remains challenging, especially at depth and in live tissues. Using small-angle X-ray scattering (SAXS) imaging, we show that hiPSC-CMs matured and examined in a 3D environment exhibit a periodic spatial arrangement of the myofilament lattice, which has not been previously detected in hiPSC-CMs. The contractile force is found to correlate with both the scattering intensity (  = 0.44) and lattice spacing (  = 0.46). The scattering intensity also correlates with lattice spacing (  = 0.81), suggestive of lower noise in our structural measurement than in the functional measurement. Notably, we observed decreased myofilament ordering in tissues with a myofilament mutation known to lead to hypertrophic cardiomyopathy (HCM). Our results highlight the progress of human cardiac tissue engineering and enable unprecedented study of structural maturation in hiPSC-CMs.
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http://dx.doi.org/10.1038/s41378-020-00234-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8433147PMC
January 2021

Filamin C Cardiomyopathy Variants Cause Protein and Lysosome Accumulation.

Circ Res 2021 Sep 18;129(7):751-766. Epub 2021 Aug 18.

Department of Genetics (R.A., C.N.T., Q.Z., J.G., S.R.D., C.E.S., J.G.S.), Harvard Medical School, Boston, MA.

[Figure: see text].
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http://dx.doi.org/10.1161/CIRCRESAHA.120.317076DOI Listing
September 2021

Switching a HO···π Interaction to a Nonconventional OH···π Hydrogen Bond: A Completed Crystallographic Puzzle.

J Org Chem 2020 08 17;85(15):9801-9807. Epub 2020 Jul 17.

Department of Chemistry, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, United States.

In this article, we present crystallographic and spectroscopic evidence of a tunable system wherein a HO···π interaction switches incrementally to a nonconventional OH···π hydrogen bonding (HB) interaction. More specifically, we report the synthesis of substituted forms of model system to study the effects of aryl ring electronic density on the qualitative characteristics of OH···π hydrogen bonds therein. The OH stretch in experimental infrared data, in agreement with density-functional theory (DFT) calculations, shows continuous red-shifts as the adjacent ring becomes more electron rich. For example, the OH stretch of an amino-substituted analogue is red-shifted by roughly 50 cm compared to the same stretch in the CF analogue, indicating a significantly stronger HB interaction in the former. Moreover, DFT calculations (ωB97XD/6-311+G**) predict that increasing electronic density on the adjacent ring reduces the aryl π-OH σ* energy gap with a concomitant enhancement of the OH n-π* energy gap. Consequently, a dominant π-σ* interaction in the amino substituted analogue locks the system in the -form while a favorable n-π* interaction reverses the orientation of the oxygen-bound hydrogen in its protonated form. Additionally, the H NMR data of various analogues reveal that stronger OH···π interactions in systems with electron-rich aromatic rings slow exchange of the alcoholic proton, thereby revealing coupling with the geminal proton. Finally, X-ray crystallographic analyses of a spectrum of analogues clearly visualize the three distinct stages of "switch"-starting with exclusive HO···π, to partitioned HO···π/OH···π, and finally to achieving exclusive OH···π forms. Furthermore, the crystal structure of the amino analogue reveals an interesting feature in which an extended HB network, involving two conventional (NH···O) and two nonconventional (OH···π) HBs, dimerizes and anchors the molecule in the unit cell.
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http://dx.doi.org/10.1021/acs.joc.0c01121DOI Listing
August 2020

Myosin Sequestration Regulates Sarcomere Function, Cardiomyocyte Energetics, and Metabolism, Informing the Pathogenesis of Hypertrophic Cardiomyopathy.

Circulation 2020 03 27;141(10):828-842. Epub 2020 Jan 27.

Department of Genetics, Harvard Medical School, Boston, MA (C.N.T., A.C.G., G.V., H.W., G.R., A.S., R.A., A.C.P., J.G.S., C.E.S.).

Background: Hypertrophic cardiomyopathy (HCM) is caused by pathogenic variants in sarcomere protein genes that evoke hypercontractility, poor relaxation, and increased energy consumption by the heart and increased patient risks for arrhythmias and heart failure. Recent studies show that pathogenic missense variants in myosin, the molecular motor of the sarcomere, are clustered in residues that participate in dynamic conformational states of sarcomere proteins. We hypothesized that these conformations are essential to adapt contractile output for energy conservation and that pathophysiology of HCM results from destabilization of these conformations.

Methods: We assayed myosin ATP binding to define the proportion of myosins in the super relaxed state (SRX) conformation or the disordered relaxed state (DRX) conformation in healthy rodent and human hearts, at baseline and in response to reduced hemodynamic demands of hibernation or pathogenic HCM variants. To determine the relationships between myosin conformations, sarcomere function, and cell biology, we assessed contractility, relaxation, and cardiomyocyte morphology and metabolism, with and without an allosteric modulator of myosin ATPase activity. We then tested whether the positions of myosin variants of unknown clinical significance that were identified in patients with HCM, predicted functional consequences and associations with heart failure and arrhythmias.

Results: Myosins undergo physiological shifts between the SRX conformation that maximizes energy conservation and the DRX conformation that enables cross-bridge formation with greater ATP consumption. Systemic hemodynamic requirements, pharmacological modulators of myosin, and pathogenic myosin missense mutations influenced the proportions of these conformations. Hibernation increased the proportion of myosins in the SRX conformation, whereas pathogenic variants destabilized these and increased the proportion of myosins in the DRX conformation, which enhanced cardiomyocyte contractility, but impaired relaxation and evoked hypertrophic remodeling with increased energetic stress. Using structural locations to stratify variants of unknown clinical significance, we showed that the variants that destabilized myosin conformations were associated with higher rates of heart failure and arrhythmias in patients with HCM.

Conclusions: Myosin conformations establish work-energy equipoise that is essential for life-long cellular homeostasis and heart function. Destabilization of myosin energy-conserving states promotes contractile abnormalities, morphological and metabolic remodeling, and adverse clinical outcomes in patients with HCM. Therapeutic restabilization corrects cellular contractile and metabolic phenotypes and may limit these adverse clinical outcomes in patients with HCM.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.042339DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077965PMC
March 2020

Sarco/endoplasmic reticulum Ca-ATPase is a more effective calcium remover than sodium-calcium exchanger in human embryonic stem cell-derived cardiomyocytes.

Am J Physiol Heart Circ Physiol 2019 11 26;317(5):H1105-H1115. Epub 2019 Jul 26.

Dr. Li Dak-Sum Research Centre, The University of Hong Kong, Pokfulam, Hong Kong.

Human pluripotent stem cell (hPSCs)-derived ventricular (V) cardiomyocytes (CMs) display immature Ca-handing properties with smaller transient amplitudes and slower kinetics due to such differences in crucial Ca-handling proteins as the poor sarco/endoplasmic reticulum Ca-ATPase (SERCA) pump but robust Na-Ca exchanger (NCX) activities in human embryonic stem cell (ESC)-derived VCMs compared with adult. Despite their fundamental importance in excitation-contraction coupling, the relative contribution of SERCA and NCX to Ca-handling of hPSC-VCMs remains unexplored. We systematically altered the activities of SERCA and NCX in human embryonic stem cell-derived ventricular cardiomyocytes (hESC-VCMs) and their engineered microtissues, followed by examining the resultant phenotypic consequences. SERCA overexpression in hESC-VCMs shortened the decay of Ca transient at low frequencies (0.5 Hz) without affecting the amplitude, SR Ca content and Ca baseline. Interestingly, short hairpin RNA-based NCX suppression did not prolong the transient decay, indicating a compensatory response for Ca removal. Although hESC-VCMs and their derived microtissues exhibited negative frequency-transient/force responses, SERCA overexpression rendered them less negative at high frequencies (>2 Hz) by accelerating Ca sequestration. We conclude that for hESC-VCMs and their microtissues, SERCA, rather than NCX, is the main Ca remover during diastole; poor SERCA expression is the leading cause for immature negative-frequency/force responses, which can be partially reverted by forced expression. Combinatorial approach to mature calcium handling in hESC-VCMs may help shed further mechanistic insights. In this study of human pluripotent stem cell-derived cardiomyocytes, we studied the role of sarco/endoplasmic reticulum Ca-ATPase (SERCA) and Na-Ca exchanger (NCX) in Ca handling. Our data support the notion that SERCA is more effective in cytosolic calcium removal than the NCX.
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http://dx.doi.org/10.1152/ajpheart.00540.2018DOI Listing
November 2019

SarcTrack.

Circ Res 2019 04;124(8):1172-1183

From the Department of Genetics (C.N.T., A.S., A.C.G., M.N., J.A.L.W., R.A., M.S., J.R., O.P., J.G.S., C.E.S.), Harvard Medical School, Boston, MA.

Rationale: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in combination with CRISPR/Cas9 genome editing provide unparalleled opportunities to study cardiac biology and disease. However, sarcomeres, the fundamental units of myocyte contraction, are immature and nonlinear in hiPSC-CMs, which technically challenge accurate functional interrogation of contractile parameters in beating cells. Furthermore, existing analysis methods are relatively low-throughput, indirectly assess contractility, or only assess well-aligned sarcomeres found in mature cardiac tissues.

Objective: We aimed to develop an analysis platform that directly, rapidly, and automatically tracks sarcomeres in beating cardiomyocytes. The platform should assess sarcomere content, contraction and relaxation parameters, and beat rate.

Methods And Results: We developed SarcTrack, a MatLab software that monitors fluorescently tagged sarcomeres in hiPSC-CMs. The algorithm determines sarcomere content, sarcomere length, and returns rates of sarcomere contraction and relaxation. By rapid measurement of hundreds of sarcomeres in each hiPSC-CM, SarcTrack provides large data sets for robust statistical analyses of multiple contractile parameters. We validated SarcTrack by analyzing drug-treated hiPSC-CMs, confirming the contractility effects of compounds that directly activate (CK-1827452) or inhibit (MYK-461) myosin molecules or indirectly alter contractility (verapamil and propranolol). SarcTrack analysis of hiPSC-CMs carrying a heterozygous truncation variant in the myosin-binding protein C ( MYBPC3) gene, which causes hypertrophic cardiomyopathy, recapitulated seminal disease phenotypes including cardiac hypercontractility and diminished relaxation, abnormalities that normalized with MYK-461 treatment.

Conclusions: SarcTrack provides a direct and efficient method to quantitatively assess sarcomere function. By improving existing contractility analysis methods and overcoming technical challenges associated with functional evaluation of hiPSC-CMs, SarcTrack enhances translational prospects for sarcomere-regulating therapeutics and accelerates interrogation of human cardiac genetic variants.
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http://dx.doi.org/10.1161/CIRCRESAHA.118.314505DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6485312PMC
April 2019

Force Generation via β-Cardiac Myosin, Titin, and α-Actinin Drives Cardiac Sarcomere Assembly from Cell-Matrix Adhesions.

Dev Cell 2018 01 8;44(1):87-96.e5. Epub 2018 Jan 8.

Department of Biomedical Engineering, Boston University, Boston, MA 02215, USA; The Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, MA 02115, USA. Electronic address:

Truncating mutations in the sarcomere protein titin cause dilated cardiomyopathy due to sarcomere insufficiency. However, it remains mechanistically unclear how these mutations decrease sarcomere content in cardiomyocytes. Utilizing human induced pluripotent stem cell-derived cardiomyocytes, CRISPR/Cas9, and live microscopy, we characterize the fundamental mechanisms of human cardiac sarcomere formation. We observe that sarcomerogenesis initiates at protocostameres, sites of cell-extracellular matrix adhesion, where nucleation and centripetal assembly of α-actinin-2-containing fibers provide a template for the fusion of Z-disk precursors, Z bodies, and subsequent striation. We identify that β-cardiac myosin-titin-protocostamere form an essential mechanical connection that transmits forces required to direct α-actinin-2 centripetal fiber assembly and sarcomere formation. Titin propagates diastolic traction stresses from β-cardiac myosin, but not α-cardiac myosin or non-muscle myosin II, to protocostameres during sarcomerogenesis. Ablating protocostameres or decoupling titin from protocostameres abolishes sarcomere assembly. Together these results identify the mechanical and molecular components critical for human cardiac sarcomerogenesis.
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http://dx.doi.org/10.1016/j.devcel.2017.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6421364PMC
January 2018

Integrative Analysis of PRKAG2 Cardiomyopathy iPS and Microtissue Models Identifies AMPK as a Regulator of Metabolism, Survival, and Fibrosis.

Cell Rep 2016 12;17(12):3292-3304

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA; Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA; Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA. Electronic address:

AMP-activated protein kinase (AMPK) is a metabolic enzyme that can be activated by nutrient stress or genetic mutations. Missense mutations in the regulatory subunit, PRKAG2, activate AMPK and cause left ventricular hypertrophy, glycogen accumulation, and ventricular pre-excitation. Using human iPS cell models combined with three-dimensional cardiac microtissues, we show that activating PRKAG2 mutations increase microtissue twitch force by enhancing myocyte survival. Integrating RNA sequencing with metabolomics, PRKAG2 mutations that activate AMPK remodeled global metabolism by regulating RNA transcripts to favor glycogen storage and oxidative metabolism instead of glycolysis. As in patients with PRKAG2 cardiomyopathy, iPS cell and mouse models are protected from cardiac fibrosis, and we define a crosstalk between AMPK and post-transcriptional regulation of TGFβ isoform signaling that has implications in fibrotic forms of cardiomyopathy. Our results establish critical connections among metabolic sensing, myocyte survival, and TGFβ signaling.
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http://dx.doi.org/10.1016/j.celrep.2016.11.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5193246PMC
December 2016

Non-cell autonomous cues for enhanced functionality of human embryonic stem cell-derived cardiomyocytes via maturation of sarcolemmal and mitochondrial K channels.

Sci Rep 2016 Sep 28;6:34154. Epub 2016 Sep 28.

Stem Cell &Regenerative Medicine Consortium, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong.

Human embryonic stem cells (hESCs) is a potential unlimited ex vivo source of ventricular (V) cardiomyocytes (CMs), but hESC-VCMs and their engineered tissues display immature traits. In adult VCMs, sarcolemmal (sarc) and mitochondrial (mito) ATP-sensitive potassium (K) channels play crucial roles in excitability and cardioprotection. In this study, we aim to investigate the biological roles and use of sarcK and mitoK in hESC-VCM. We showed that SarcI in single hESC-VCMs was dormant under baseline conditions, but became markedly activated by cyanide (CN) or the known opener P1075 with a current density that was ~8-fold smaller than adult; These effects were reversible upon washout or the addition of GLI or HMR1098. Interestingly, sarcI displayed a ~3-fold increase after treatment with hypoxia (5% O). MitoI was absent in hESC-VCMs. However, the thyroid hormone T3 up-regulated mitoI conferring diazoxide protective effect on T3-treated hESC-VCMs. When assessed using a multi-cellular engineered 3D ventricular cardiac micro-tissue (hvCMT) system, T3 substantially enhanced the developed tension by 3-folds. Diazoxide also attenuated the decrease in contractility induced by simulated ischemia (1% O). We conclude that hypoxia and T3 enhance the functionality of hESC-VCMs and their engineered tissues by selectively acting on sarc and mitoI.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5039730PMC
http://dx.doi.org/10.1038/srep34154DOI Listing
September 2016

HEART DISEASE. Titin mutations in iPS cells define sarcomere insufficiency as a cause of dilated cardiomyopathy.

Science 2015 Aug;349(6251):982-6

Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA. Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.

Human mutations that truncate the massive sarcomere protein titin [TTN-truncating variants (TTNtvs)] are the most common genetic cause for dilated cardiomyopathy (DCM), a major cause of heart failure and premature death. Here we show that cardiac microtissues engineered from human induced pluripotent stem (iPS) cells are a powerful system for evaluating the pathogenicity of titin gene variants. We found that certain missense mutations, like TTNtvs, diminish contractile performance and are pathogenic. By combining functional analyses with RNA sequencing, we explain why truncations in the A-band domain of TTN cause DCM, whereas truncations in the I band are better tolerated. Finally, we demonstrate that mutant titin protein in iPS cell-derived cardiomyocytes results in sarcomere insufficiency, impaired responses to mechanical and β-adrenergic stress, and attenuated growth factor and cell signaling activation. Our findings indicate that titin mutations cause DCM by disrupting critical linkages between sarcomerogenesis and adaptive remodeling.
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http://dx.doi.org/10.1126/science.aaa5458DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4618316PMC
August 2015

Proteomic Analysis of Human Pluripotent Stem Cell-Derived, Fetal, and Adult Ventricular Cardiomyocytes Reveals Pathways Crucial for Cardiac Metabolism and Maturation.

Circ Cardiovasc Genet 2015 Jun 10;8(3):427-36. Epub 2015 Mar 10.

From the Stem Cell and Regenerative Medicine Consortium (E.P., W.K., B.Y., Z.W., O.T. W., K.R.B., R.A.L.) and Department of Physiology, LKS Faculty of Medicine (E.P., W.K., B.Y., Z.W., O.T.W., K.R.B., R.A.L.), University of Hong Kong, Hong Kong, P.R. China; Departments of Biology and Chemistry (Y.M.L., R.R., Y.W.L.) and Computer Science (H.S.W.), City University of Hong Kong, Hong Kong, P.R. China; Department of Computer Science, Guangzhou University, Guangzhou, P.R. China (S.Z.); Department of Bioengineering, Boston University, MA (A.C., C.C.); Harvard Wyss Institute for Biologically Inspired Engineering, Boston, MA (A.C., C.C.); Department of Cell Biology and Human Anatomy, University of California, Davis (J.M., A.H., D.K.L.); Cardiovascular Research Center, Mount Sinai School of Medicine, New York (D.K.L., R.A.L.); and Division of Cardiology, Johns Hopkins University, Baltimore, MD (G.F.T., K.R.B.).

Background: Differentiation of pluripotent human embryonic stem cells (hESCs) to the cardiac lineage represents a potentially unlimited source of ventricular cardiomyocytes (VCMs), but hESC-VCMs are developmentally immature. Previous attempts to profile hESC-VCMs primarily relied on transcriptomic approaches, but the global proteome has not been examined. Furthermore, most hESC-CM studies focus on pathways important for cardiac differentiation, rather than regulatory mechanisms for CM maturation. We hypothesized that gene products and pathways crucial for maturation can be identified by comparing the proteomes of hESCs, hESC-derived VCMs, human fetal and human adult ventricular and atrial CMs.

Methods And Results: Using two-dimensional-differential-in-gel electrophoresis, 121 differentially expressed (>1.5-fold; P<0.05) proteins were detected. The data set implicated a role of the peroxisome proliferator-activated receptor α signaling in cardiac maturation. Consistently, WY-14643, a peroxisome proliferator-activated receptor α agonist, increased fatty oxidative enzyme level, hyperpolarized mitochondrial membrane potential and induced a more organized morphology. Along this line, treatment with the thyroid hormone triiodothyronine increased the dynamic tension developed in engineered human ventricular cardiac microtissue by 3-fold, signifying their maturation.

Conclusions: We conclude that the peroxisome proliferator-activated receptor α and thyroid hormone pathways modulate the metabolism and maturation of hESC-VCMs and their engineered tissue constructs. These results may lead to mechanism-based methods for deriving mature chamber-specific CMs.
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http://dx.doi.org/10.1161/CIRCGENETICS.114.000918DOI Listing
June 2015

Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.

Circ Arrhythm Electrophysiol 2015 Feb 10;8(1):193-202. Epub 2014 Dec 10.

From the Cardiovascular Research Center, Icahn School of Medicine at Mount Sinai, Manhattan, NY (G.C., I.K., K.D.C., R.J.H., R.A.L.); Department of Physiology (G.C., S.L., L.R., M.Z.-Y.C., W.K., C.-W.K., R.A.L.), Stem Cell and Regenerative Medicine Consortium (G.C., S.L., L.R., M.Z.-Y.C., W.K., B.Y., C.W.Y.C., C.-W.K., R.A.L.), Department of Anatomy (C.W.Y.C.), LKS Faculty of Medicine, University of Hong Kong, Pokfulam, Hong Kong; Department of Bioengineering, Boston University, MA (A.C., C.S.C.); Harvard Wyss Institute for Biologically Inspired Engineering, Boston, MA (A.C., C.S.C.); and Department of Biology, Hong Kong Baptist University, Hong Kong (B.Y.).

Background: Human (h) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) serve as a potential unlimited ex vivo source of cardiomyocytes (CMs). However, a well-accepted roadblock has been their immature phenotype. hESC/iPSC-derived ventricular (v) CMs and their engineered cardiac microtissues (hvCMTs) similarly displayed positive chronotropic but null inotropic responses to β-adrenergic stimulation. Given that phospholamban (PLB) is robustly present in adult but poorly expressed in hESC/iPSC-vCMs and its defined biological role in β-adrenergic signaling, we investigated the functional consequences of PLB expression in hESC/iPSC-vCMs and hvCMTs.

Methods And Results: First, we confirmed that PLB protein was differentially expressed in hESC (HES2, H9)- and iPSC-derived and adult vCMs. We then transduced hES2-vCMs with the recombinant adenoviruses (Ad) Ad-PLB or Ad-S16E-PLB to overexpress wild-type PLB or the pseudophosphorylated point-mutated variant, respectively. As anticipated from the inhibitory effect of unphosphorylated PLB on sarco/endoplasmic reticulum Ca2+-ATPase, Ad-PLB transduction significantly attenuated electrically evoked Ca2+ transient amplitude and prolonged the 50% decay time. Importantly, Ad-PLB-transduced hES2-vCMs uniquely responded to isoproterenol. Ad-S16E-PLB-transduced hES2-vCMs displayed an intermediate phenotype. The same trends were observed with H9- and iPSC-vCMs. Directionally, similar results were also seen with Ad-PLB-transduced and Ad-S16E-transduced hvCMTs. However, Ad-PLB altered neither the global transcriptome nor ICa,L, implicating a PLB-specific effect.

Conclusions: Engineered upregulation of PLB expression in hESC/iPSC-vCMs restores a positive inotropic response to β-adrenergic stimulation. These results not only provide a better mechanistic understanding of the immaturity of hESC/iPSC-vCMs but will also lead to improved disease models and transplantable prototypes with adult-like physiological responses.
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http://dx.doi.org/10.1161/CIRCEP.114.002049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5884688PMC
February 2015

Force measurement tools to explore cadherin mechanotransduction.

Cell Commun Adhes 2014 Jun 23;21(3):193-205. Epub 2014 Apr 23.

Department of Chemical and Biomolecular Engineering, University of Pennsylvania , Philadelphia, PA , USA.

Cell-cell adhesions serve to mechanically couple cells, allowing for long-range transmission of forces across cells in development, disease, and homeostasis. Recent work has shown that such contacts also play a role in transducing mechanical cues into a wide variety of cellular behaviors important to tissue function. As such, understanding the mechanical regulation of cells through their adhesion molecules has become a point of intense focus. This review will highlight the existing and emerging technologies and models that allow for exploration of cadherin-based adhesions as sites of mechanotransduction.
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http://dx.doi.org/10.3109/15419061.2014.905929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7079283PMC
June 2014

Augmentation of integrin-mediated mechanotransduction by hyaluronic acid.

Biomaterials 2014 Jan 10;35(1):71-82. Epub 2013 Oct 10.

Dept. of Cardiothoracic Surgery, Drexel Univ. College of Med, Philadelphia, PA, USA; Dept. of Bioengineering, Univ. of Pennsylvania, Philadelphia, PA, USA.

Changes in tissue and organ stiffness occur during development and are frequently symptoms of disease. Many cell types respond to the stiffness of substrates and neighboring cells in vitro and most cell types increase adherent area on stiffer substrates that are coated with ligands for integrins or cadherins. In vivo cells engage their extracellular matrix (ECM) by multiple mechanosensitive adhesion complexes and other surface receptors that potentially modify the mechanical signals transduced at the cell/ECM interface. Here we show that hyaluronic acid (also called hyaluronan or HA), a soft polymeric glycosaminoglycan matrix component prominent in embryonic tissue and upregulated during multiple pathologic states, augments or overrides mechanical signaling by some classes of integrins to produce a cellular phenotype otherwise observed only on very rigid substrates. The spread morphology of cells on soft HA-fibronectin coated substrates, characterized by formation of large actin bundles resembling stress fibers and large focal adhesions resembles that of cells on rigid substrates, but is activated by different signals and does not require or cause activation of the transcriptional regulator YAP. The fact that HA production is tightly regulated during development and injury and frequently upregulated in cancers characterized by uncontrolled growth and cell movement suggests that the interaction of signaling between HA receptors and specific integrins might be an important element in mechanical control of development and homeostasis.
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http://dx.doi.org/10.1016/j.biomaterials.2013.09.066DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3930571PMC
January 2014

α-Catenin localization and sarcomere self-organization on N-cadherin adhesive patterns are myocyte contractility driven.

PLoS One 2012 15;7(10):e47592. Epub 2012 Oct 15.

Department of Cardiothoracic Surgery, Drexel University College of Medicine, Philadelphia, Pennsylvania, United States of America.

The N-cadherin (N-cad) complex plays a crucial role in cardiac cell structure and function. Cadherins are adhesion proteins linking adjacent cardiac cells and, like integrin adhesions, are sensitive to force transmission. Forces through these adhesions are capable of eliciting structural and functional changes in myocytes. Compared to integrins, the mechanisms of force transduction through cadherins are less explored. α-catenin is a major component of the cadherin-catenin complex, thought to provide a link to the cell actin cytoskeleton. Using N-cad micropatterned substrates in an adhesion constrainment model, the results from this study show that α-catenin localizes to regions of highest internal stress in myocytes. This localization suggests that α-catenin acts as an adaptor protein associated with the cadherin mechanosensory apparatus, which is distinct from mechanosensing through integrins. Myosin inhibition in cells bound by integrins to fibronectin-coated patterns disrupts myofibiril organization, whereas on N-cad coated patterns, myosin inhibition leads to better organized myofibrils. This result indicates that the two adhesion systems provide independent mechanisms for regulating myocyte structural organization.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0047592PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3471892PMC
April 2013

Reprogramming cardiomyocyte mechanosensing by crosstalk between integrins and hyaluronic acid receptors.

J Biomech 2012 Mar 24;45(5):824-31. Epub 2011 Dec 24.

Department of Biomedical Engineering, Drexel University, Philadelphia, PA 19102, USA.

The elastic modulus of bioengineered materials has a strong influence on the phenotype of many cells including cardiomyocytes. On polyacrylamide (PAA) gels that are laminated with ligands for integrins, cardiac myocytes develop well organized sarcomeres only when cultured on substrates with elastic moduli in the range 10 kPa-30 kPa, near those of the healthy tissue. On stiffer substrates (>60 kPa) approximating the damaged heart, myocytes form stress fiber-like filament bundles but lack organized sarcomeres or an elongated shape. On soft (<1 kPa) PAA gels myocytes exhibit disorganized actin networks and sarcomeres. However, when the polyacrylamide matrix is replaced by hyaluronic acid (HA) as the gel network to which integrin ligands are attached, robust development of functional neonatal rat ventricular myocytes occurs on gels with elastic moduli of 200 Pa, a stiffness far below that of the neonatal heart and on which myocytes would be amorphous and dysfunctional when cultured on polyacrylamide-based gels. The HA matrix by itself is not adhesive for myocytes, and the myocyte phenotype depends on the type of integrin ligand that is incorporated within the HA gel, with fibronectin, gelatin, or fibrinogen being more effective than collagen I. These results show that HA alters the integrin-dependent stiffness response of cells in vitro and suggests that expression of HA within the extracellular matrix (ECM) in vivo might similarly alter the response of cells that bind the ECM through integrins. The integration of HA with integrin-specific ECM signaling proteins provides a rationale for engineering a new class of soft hybrid hydrogels that can be used in therapeutic strategies to reverse the remodeling of the injured myocardium.
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http://dx.doi.org/10.1016/j.jbiomech.2011.11.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3386849PMC
March 2012

Intercellular and extracellular mechanotransduction in cardiac myocytes.

Pflugers Arch 2011 Jul 25;462(1):75-87. Epub 2011 Mar 25.

Department of Cardiothoracic Surgery, Drexel University College of Medicine, 245 North 15th Street, MS 111, Philadelphia, PA 19102, USA.

Adult cardiomyocytes are terminally differentiated with minimal replicative capacity. Therefore, long-term preservation or enhancement of cardiac function depends on structural adaptation. Myocytes interact with the extracellular matrix, fibroblasts, and vascular cells and with each other (end to end; side to side). We review the current understanding of the mechanical determinants and environmental sensing systems that modulate and regulate myocyte molecular machinery and its structural organization. We feature the design and application of engineered cellular microenvironments to demonstrate the ability of cardiac cells to remodel their cytoskeletal organization and shape, including sarcomere/myofibrillar architectural topography. Cell shape-dependent functions result from complex mechanical interactions between the cytoskeleton architecture and external conditions, be they cell-cell or cell-extracellular matrix (ECM) adhesion contact-mediated. This mechanobiological perspective forms the basis for viewing the cardiomyocyte as a mechanostructural anisotropic continuum, exhibiting constant mechanosensory-driven self-regulated adjustment of the cytoskeleton through tight interplay between its force generation activity and concurrent cytoarchitectural remodeling. The unifying framework guiding this perspective is the observation that these emerging events and properties are initiated by and respond to cytoskeletal reorganization, regulated by cell-cell and cell-ECM adhesion and its corresponding (mutually interactive) signaling machinery. It is important for future studies to elucidate how cross talk between these mechanical signals is coordinated to control myocyte structure and function. Ultimately, understanding how the highly interactive mechanical signaling can give rise to phenotypic changes is critical for targeting the underlying pathways that contribute to cardiac remodeling associated with various forms of dilated and hypertrophic myopathies, myocardial infarction, heart failure, and reverse remodeling.
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http://dx.doi.org/10.1007/s00424-011-0954-1DOI Listing
July 2011

Cardiac myocyte remodeling mediated by N-cadherin-dependent mechanosensing.

Am J Physiol Heart Circ Physiol 2011 Apr 21;300(4):H1252-66. Epub 2011 Jan 21.

Department of Biomedical Engineering, Drexel University, Philadelphia, PA 19102, USA.

Cell-to-cell adhesions are crucial in maintaining the structural and functional integrity of cardiac cells. Little is known about the mechanosensitivity and mechanotransduction of cell-to-cell interactions. Most studies of cardiac mechanotransduction and myofibrillogenesis have focused on cell-extracellular matrix (ECM)-specific interactions. This study assesses the direct role of intercellular adhesion, specifically that of N-cadherin-mediated mechanotransduction, on the morphology and internal organization of neonatal ventricular cardiac myocytes. The results show that cadherin-mediated cell attachments are capable of eliciting a cytoskeletal network response similar to that of integrin-mediated force response and transmission, affecting myofibrillar organization, myocyte shape, and cortical stiffness. Traction forces mediated by N-cadherin were shown to be comparable to those sustained by ECM. The directional changes in predicted traction forces as a function of imposed loads (gel stiffness) provide the added evidence that N-cadherin is a mechanoresponsive adhesion receptor. Strikingly, the mechanical sensitivity response (gain) in terms of the measured cell-spread area as a function of imposed load (adhesive substrate rigidity) was consistently higher for N-cadherin-coated surfaces compared with ECM protein-coated surfaces. In addition, the cytoskeletal architecture of myocytes on an N-cadherin adhesive microenvironment was characteristically different from that on an ECM environment, suggesting that the two mechanotransductive cell adhesion systems may play both independent and complementary roles in myocyte cytoskeletal spatial organization. These results indicate that cell-to-cell-mediated force perception and transmission are involved in the organization and development of cardiac structure and function.
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http://dx.doi.org/10.1152/ajpheart.00515.2010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3075038PMC
April 2011
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