title: Induction of diverse cardiac cell types by reprogramming fibroblasts with cardiac transcription factors.
abstract: Various combinations of cardiogenic transcription factors, including Gata4 (G), Hand2 (H), Mef2c (M) and Tbx5 (T), can reprogram fibroblasts into induced cardiac-like myocytes (iCLMs) in vitro and in vivo. Given that optimal cardiac function relies on distinct yet functionally interconnected atrial, ventricular and pacemaker (PM) cardiomyocytes (CMs), it remains to be seen which subtypes are generated by direct reprogramming and whether this process can be harnessed to produce a specific CM of interest. Here, we employ a PM-specific Hcn4-GFP reporter mouse and a spectrum of CM subtype-specific markers to investigate the range of cellular phenotypes generated by reprogramming of primary fibroblasts. Unexpectedly, we find that a combination of four transcription factors (4F) optimized for Hcn4-GFP expression does not generate beating PM cells due to inadequate sarcomeric protein expression and organization. However, applying strict single-cell criteria to GHMT-reprogrammed cells, we observe induction of diverse cellular phenotypes, including those resembling immature forms of all three major cardiac subtypes (i.e. atrial, ventricular and pacemaker). In addition, we demonstrate that cells induced by GHMT are directly reprogrammed and do not arise from an Nxk2.5(+) progenitor cell intermediate. Taken together, our results suggest a remarkable degree of plasticity inherent to GHMT reprogramming and provide a starting point for optimization of CM subtype-specific reprogramming protocols.
authors.affiliation: Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA Department of Medicine, Division of Cardiovascular Medicine, Vanderbilt University, Nashville, TN 37232, USA.
authors.forename: Young-Jae
authors.initials: YJ
authors.lastname: Nam
authors.affiliation: Department of Internal Medicine, Division of Cardiology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Christina
authors.initials: C
authors.lastname: Lubczyk
authors.affiliation: Department of Internal Medicine, Division of Cardiology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Minoti
authors.initials: M
authors.lastname: Bhakta
authors.affiliation: Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Tong
authors.initials: T
authors.lastname: Zang
authors.affiliation: Department of Internal Medicine, Division of Cardiology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Antonio
authors.initials: A
authors.lastname: Fernandez-Perez
authors.affiliation: Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: John
authors.initials: J
authors.lastname: McAnally
authors.affiliation: Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA.
authors.forename: Rhonda
authors.initials: R
authors.lastname: Bassel-Duby
authors.affiliation: Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA Hamon Center for Regenerative Science and Medicine, UT Southwestern Medical Center, Dallas, TX 75390, USA eric.olson@utsouthwestern.edu nikhil.munshi@utsouthwestern.edu.
authors.forename: Eric N
authors.initials: EN
authors.lastname: Olson
authors.affiliation: Department of Molecular Biology, UT Southwestern Medical Center, Dallas, TX 75390, USA Department of Internal Medicine, Division of Cardiology, UT Southwestern Medical Center, Dallas, TX 75390, USA McDermott Center for Human Growth and Development, UT Southwestern Medical Center, Dallas, TX 75390, USA eric.olson@utsouthwestern.edu nikhil.munshi@utsouthwestern.edu.
authors.forename: Nikhil V
authors.initials: NV
authors.lastname: Munshi
mesh_terms: D000200:Action Potentials / Q000502:physiology; D000704:Analysis of Variance; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D017931:DNA Primers / Q000235:genetics; D004627:Embryonic Induction / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism / Q000502:physiology; D050991:GATA4 Transcription Factor / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006321:Heart / Q000196:embryology*; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels / Q000378:metabolism; D007150:Immunohistochemistry; D064326:MEF2 Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D032383:Myocytes, Cardiac / Q000166:cytology / Q000502:physiology*; D060888:Real-Time Polymerase Chain Reaction; D020825:T-Box Domain Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D050991:GATA4 Transcription Factor; D049452:Green Fluorescent Proteins; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D064326:MEF2 Transcription Factors; D020825:T-Box Domain Proteins; D014157:Transcription Factors; C484171:Gata4 protein, mouse; C498766:Hand2 protein, mouse; C578706:Hcn4 protein, mouse; C578041:Mef2c protein, mouse; C103172:T-box transcription factor 5
keywords: Cardiomyocyte; Direct reprogramming; Mouse; Pacemaker