Zaniar Ghazizadeh1, Tuomas Kiviniemi1,2,3, Sigurast Olafsson1, David Plotnick1, Manu E Beerens1, Kun Zhang1, Leah Gillon1, Michael J Steinbaugh4, Victor Barrera4, Shannan Ho Sui4, Andreas A Werdich1, Sunil Kapur1, Antti Eranti2,3, Jarmo Gunn2,3, Juho Jalkanen5, Juhani Airaksinen2,3, Andre G Kleber6, Maija Hollmén5, Calum A MacRae1,7,8. 1. Cardiovascular Medicine Division (Z.G., T.K., S.O., D.P., M.E.B., K.Z., L.G., A.A.W., S.K., C.A.M.), Brigham and Women's Hospital, Harvard Medical School, Boston, MA. 2. Heart Center, Turku University Hospital (T.K., A.E., J.G., J.A.), Harvard T.H. 3. University of Turku, Finland (T.K., A.E., J.G., J.A.). Harvard T.H. 4. Chan School of Public Health, Boston, MA (M.J.S., V.B., S.H.S.). 5. Medicity Research Laboratories (J.J., M.H.), Harvard T.H. 6. Department of Pathology, Beth Israel Deaconess Medical Center Harvard Medical School, Boston, MA (A.G.K.). 7. Genetics and Network Medicine Divisions (C.A.M.), Brigham and Women's Hospital, Harvard Medical School, Boston, MA. 8. Harvard Stem Cell Institute, Boston, MA (C.A.M.).
Abstract
BACKGROUND: Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. METHODS: Using an MYH6:mCherry knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell-derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled human MYL4 mutants, one of the few definitive genetic causes of AF. To explore non-cell-autonomous components of AF substrate, we also created a zebrafish Myl4 knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. RESULTS: There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)-related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in MYL4 mutants, as well as in atrial biopsies from unselected forms of human AF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. CONCLUSIONS: These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.
BACKGROUND:Atrial fibrillation (AF) is the most common clinical arrhythmia and is associated with heart failure, stroke, and increased mortality. The myocardial substrate for AF is poorly understood because of limited access to primary human tissue and mechanistic questions around existing in vitro or in vivo models. METHODS: Using an MYH6:mCherry knock-in reporter line, we developed a protocol to generate and highly purify human pluripotent stem cell-derived cardiomyocytes displaying physiological and molecular characteristics of atrial cells. We modeled humanMYL4 mutants, one of the few definitive genetic causes of AF. To explore non-cell-autonomous components of AF substrate, we also created a zebrafishMyl4 knockout model, which exhibited molecular, cellular, and physiologic abnormalities that parallel those in humans bearing the cognate mutations. RESULTS: There was evidence of increased retinoic acid signaling in both human embryonic stem cells and zebrafish mutant models, as well as abnormal expression and localization of cytoskeletal proteins, and loss of intracellular nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide + hydrogen. To identify potentially druggable proximate mechanisms, we performed a chemical suppressor screen integrating multiple human cellular and zebrafish in vivo endpoints. This screen identified Cx43 (connexin 43) hemichannel blockade as a robust suppressor of the abnormal phenotypes in both models of MYL4 (myosin light chain 4)-related atrial cardiomyopathy. Immunofluorescence and coimmunoprecipitation studies revealed an interaction between MYL4 and Cx43 with altered localization of Cx43 hemichannels to the lateral membrane in MYL4 mutants, as well as in atrial biopsies from unselected forms of humanAF. The membrane fraction from MYL4-/- human embryonic stem cell derived atrial cells demonstrated increased phospho-Cx43, which was further accentuated by retinoic acid treatment and by the presence of risk alleles at the Pitx2 locus. PKC (protein kinase C) was induced by retinoic acid, and PKC inhibition also rescued the abnormal phenotypes in the atrial cardiomyopathy models. CONCLUSIONS: These data establish a mechanistic link between the transcriptional, metabolic and electrical pathways previously implicated in AF substrate and suggest novel avenues for the prevention or therapy of this common arrhythmia.
Authors: Maarten Aj De Smet; Alessio Lissoni; Timur Nezlobinsky; Nan Wang; Eef Dries; Marta Pérez-Hernández; Xianming Lin; Matthew Amoni; Tim Vervliet; Katja Witschas; Eli Rothenberg; Geert Bultynck; Rainer Schulz; Alexander V Panfilov; Mario Delmar; Karin R Sipido; Luc Leybaert Journal: J Clin Invest Date: 2021-04-01 Impact factor: 14.808
Authors: Shinwan Kany; Bruno Reissmann; Andreas Metzner; Paulus Kirchhof; Dawood Darbar; Renate B Schnabel Journal: Cardiovasc Res Date: 2021-06-16 Impact factor: 10.787
Authors: Ryan M Samuel; Homa Majd; Mikayla N Richter; Zaniar Ghazizadeh; Seyedeh Maryam Zekavat; Albertas Navickas; Jonathan T Ramirez; Hosseinali Asgharian; Camille R Simoneau; Luke R Bonser; Kyung Duk Koh; Miguel Garcia-Knight; Michel Tassetto; Sara Sunshine; Sina Farahvashi; Ali Kalantari; Wei Liu; Raul Andino; Hongyu Zhao; Pradeep Natarajan; David J Erle; Melanie Ott; Hani Goodarzi; Faranak Fattahi Journal: Cell Stem Cell Date: 2020-11-17 Impact factor: 24.633
Authors: Alessio Lissoni; Nan Wang; Timur Nezlobinskii; Maarten De Smet; Alexander V Panfilov; Nele Vandersickel; Luc Leybaert; Katja Witschas Journal: Int J Mol Sci Date: 2020-10-05 Impact factor: 5.923