title: Phospholamban as a crucial determinant of the inotropic response of human pluripotent stem cell-derived ventricular cardiomyocytes and engineered 3-dimensional tissue constructs.
abstract: 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.
  
  
  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.
authors.affiliation: 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.).
authors.forename: Gaopeng
authors.initials: G
authors.lastname: Chen
authors.affiliation: 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.).
authors.forename: Sen
authors.initials: S
authors.lastname: Li
authors.affiliation: 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.).
authors.forename: Ioannis
authors.initials: I
authors.lastname: Karakikes
authors.affiliation: 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.).
authors.forename: Lihuan
authors.initials: L
authors.lastname: Ren
authors.affiliation: 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.).
authors.forename: Maggie Zi-Ying
authors.initials: MZ
authors.lastname: Chow
authors.affiliation: 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.).
authors.forename: Anant
authors.initials: A
authors.lastname: Chopra
authors.affiliation: 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.).
authors.forename: Wendy
authors.initials: W
authors.lastname: Keung
authors.affiliation: 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.).
authors.forename: Bin
authors.initials: B
authors.lastname: Yan
authors.affiliation: 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.).
authors.forename: Camie W Y
authors.initials: CW
authors.lastname: Chan
authors.affiliation: 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.).
authors.forename: Kevin D
authors.initials: KD
authors.lastname: Costa
authors.affiliation: 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.).
authors.forename: Chi-Wing
authors.initials: CW
authors.lastname: Kong
authors.affiliation: 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.).
authors.forename: Roger J
authors.initials: RJ
authors.lastname: Hajjar
authors.affiliation: 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.).
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Chen
authors.affiliation: 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.). ronaldli@hku.hk.
authors.forename: Ronald A
authors.initials: RA
authors.lastname: Li
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D018920:Coculture Techniques; D053595:Embryonic Stem Cells / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000187:drug effects / Q000378:metabolism*; D007545:Isoproterenol / Q000494:pharmacology; D009154:Mutation; D009200:Myocardial Contraction / Q000187:drug effects; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D015398:Signal Transduction; D023822:Tissue Engineering / Q000379:methods*; D014161:Transduction, Genetic; D014162:Transfection; D015854:Up-Regulation; D000099211:Phospholamban
chemical_list: D000318:Adrenergic beta-Agonists; D002135:Calcium-Binding Proteins; D007545:Isoproterenol; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
keywords: adrenergic effects; phospholamban; pluripotent stem cells; tissues