Stx4 is required to regulate cardiomyocyte Ca2+ handling during vertebrate cardiac development
Eliyahu Perl,
Padmapriyadarshini Ravisankar,
Manu E. Beerens,
Lejla Mulahasanovic,
Kelly Smallwood,
Marion Bermúdez Sasso,
Carina Wenzel,
Thomas D. Ryan,
Matej Komár,
Kevin E. Bove,
Calum A. MacRae,
K. Nicole Weaver,
Carlos E. Prada,
Joshua S. Waxman
Affiliations
Eliyahu Perl
Medical Scientist Training Program, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Molecular and Developmental Biology Graduate Program, University of Cincinnati College of Medicine, Cincinnati, OH, USA; Division of Molecular Cardiovascular Biology, The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Padmapriyadarshini Ravisankar
Division of Molecular Cardiovascular Biology, The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Manu E. Beerens
Cardiovascular Medicine Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA
Lejla Mulahasanovic
Praxis für Humangenetik, Tübingen, Baden-Württemberg, Germany; CeGaT GmbH, Tübingen, Baden-Württemberg, Germany
Kelly Smallwood
Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Marion Bermúdez Sasso
Institute for Clinical Genetics, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Saxony, Germany
Carina Wenzel
Institute of Pathology, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Germany
Thomas D. Ryan
Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH, USA; Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA
Matej Komár
Department of Gynecology and Obstetrics, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Saxony, Germany
Kevin E. Bove
Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH, USA; Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Department of Pathology and Laboratory Medicine, University of Cincinnati, College of Medicine, Cincinnati, OH, USA
Calum A. MacRae
Cardiovascular Medicine Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; Genetics and Network Medicine Divisions, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA; Harvard Stem Cell Institute, Boston, MA, USA
K. Nicole Weaver
Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH, USA
Carlos E. Prada
Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH, USA
Joshua S. Waxman
Division of Molecular Cardiovascular Biology, The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH, USA; Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; Corresponding author
Summary: Requirements for vesicle fusion within the heart remain poorly understood, despite the multitude of processes that necessitate proper intracellular trafficking within cardiomyocytes. Here, we show that Syntaxin 4 (STX4), a target-Soluble N-ethylmaleimide sensitive factor attachment receptor (t-SNARE) protein, is required for normal vertebrate cardiac conduction and vesicular transport. Two patients were identified with damaging variants in STX4. A patient with a homozygous R240W missense variant displayed biventricular dilated cardiomyopathy, ectopy, and runs of non-sustained ventricular tachycardia, sensorineural hearing loss, global developmental delay, and hypotonia, while a second patient displayed severe pleiotropic abnormalities and perinatal lethality. CRISPR/Cas9-generated stx4 mutant zebrafish exhibited defects reminiscent of these patients’ clinical presentations, including linearized hearts, bradycardia, otic vesicle dysgenesis, neuronal atrophy, and touch insensitivity by 3 days post fertilization. Imaging of Vamp2+ vesicles within stx4 mutant zebrafish hearts showed reduced docking to the cardiomyocyte sarcolemma. Optical mapping of the embryonic hearts coupled with pharmacological modulation of Ca2+ handling together support that zebrafish stx4 mutants have a reduction in L-type Ca2+ channel modulation. Transgenic overexpression of zebrafish Stx4R241W, analogous to the first patient’s STX4R240W variant, indicated that the variant is hypomorphic. Thus, these data show an in vivo requirement for SNAREs in regulating normal embryonic cardiac function and that variants in STX4 are associated with pleiotropic human disease, including cardiomyopathy.
