title: Peptide-enhanced mRNA transfection in cultured mouse cardiac fibroblasts and direct reprogramming towards cardiomyocyte-like cells.
abstract: The treatment of myocardial infarction is a major challenge in medicine due to the inability of heart tissue to regenerate. Direct reprogramming of endogenous cardiac fibroblasts into functional cardiomyocytes via the delivery of transcription factor mRNAs has the potential to regenerate cardiac tissue and to treat heart failure. Even though mRNA delivery to cardiac fibroblasts has the therapeutic potential, mRNA transfection in cardiac fibroblasts has been challenging. Herein, we develop an efficient mRNA transfection in cultured mouse cardiac fibroblasts via a polyarginine-fused heart-targeting peptide and lipofectamine complex, termed C-Lipo and demonstrate the partial direct reprogramming of cardiac fibroblasts towards cardiomyocyte cells. C-Lipo enabled the mRNA-induced direct cardiac reprogramming due to its efficient transfection with low toxicity, which allowed for multiple transfections of Gata4, Mef2c, and Tbx5 (GMT) mRNAs for a period of 2 weeks. The induced cardiomyocyte-like cells had α-MHC promoter-driven GFP expression and striated cardiac muscle structure from α-actinin immunohistochemistry. GMT mRNA transfection of cultured mouse cardiac fibroblasts via C-Lipo significantly increased expression of the cardiomyocyte marker genes, Actc1, Actn2, Gja1, Hand2, and Tnnt2, after 2 weeks of transfection. Moreover, this study provides the first direct evidence that the stoichiometry of the GMT reprogramming factors influence the expression of cardiomyocyte marker genes. Our results demonstrate that mRNA delivery is a potential approach for cardiomyocyte generation.
authors.affiliation: Department of Bioengineering, University of California, Berkeley, CA, USA ; UC Berkeley and UCSF Joint Graduate Program in Bioengineering, Berkeley/San Francisco, CA, USA.
authors.forename: Kunwoo
authors.initials: K
authors.lastname: Lee
authors.affiliation: Gladstone Institute of Cardiovascular Disease, San Francisco, CA, USA.
authors.forename: Pengzhi
authors.initials: P
authors.lastname: Yu
authors.affiliation: Department of Bioengineering, University of California, Berkeley, CA, USA.
authors.forename: Nithya
authors.initials: N
authors.lastname: Lingampalli
authors.affiliation: Department of Bioengineering, University of California, Berkeley, CA, USA.
authors.forename: Hyun Jin
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Bioengineering, University of California, Berkeley, CA, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Tang
authors.affiliation: Department of Bioengineering, University of California, Berkeley, CA, USA ; UC Berkeley and UCSF Joint Graduate Program in Bioengineering, Berkeley/San Francisco, CA, USA.
authors.forename: Niren
authors.initials: N
authors.lastname: Murthy
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D065150:Cellular Reprogramming / Q000502:physiology*; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology*; D005786:Gene Expression Regulation; D005819:Genetic Markers; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008055:Lipids / Q000008:administration & dosage / Q000737:chemistry / Q000633:toxicity; D008822:Mice, Transgenic; D009206:Myocardium / Q000166:cytology / Q000648:ultrastructure; D032383:Myocytes, Cardiac / Q000166:cytology* / Q000502:physiology; D009842:Oligopeptides / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D020825:T-Box Domain Proteins / Q000235:genetics; D014162:Transfection / Q000379:methods*
chemical_list: D005819:Genetic Markers; D008055:Lipids; C086724:Lipofectamine; D009842:Oligopeptides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D020825:T-Box Domain Proteins; C103172:T-box transcription factor 5; C507807:cysteinyl-arginyl-prolyl-prolyl-arginine; D049452:Green Fluorescent Proteins
keywords: cardiac fibroblast transfection; cardiac regeneration; direct cardiac reprogramming; mRNA transfection
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