Stx4 is required to regulate cardiomyocyte Ca2+ handling during vertebrate cardiac development

April 27, 2022

Eliyahu Perl 1 2 3, Padmapriyadarshini Ravisankar 3, Manu E Beerens 4, Lejla Mulahasanovic 5 6, Kelly Smallwood 7, Marion Bermúdez Sasso 8, Carina Wenzel 9, Thomas D Ryan 10 11, Matej Komár 12, Kevin E Bove 10 13 14, Calum A MacRae 4 15 16, K Nicole Weaver 7 10, Carlos E Prada 7 10, Joshua S Waxman 3 10 17

Abstract

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.

Keywords: SNARE; Syntaxin 4; calcium handling; conduction defects; congenital heart disease; dilated cardiomyopathy; vesicular transport; zebrafish.

  1. Medical Scientist Training Program, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
  2. Molecular and Developmental Biology Graduate Program, University of Cincinnati College of Medicine, Cincinnati, OH, USA.
  3. Division of Molecular Cardiovascular Biology, The Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA.
  4. Cardiovascular Medicine Division, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.
  5. Praxis für Humangenetik, Tübingen, Baden-Württemberg, Germany.
  6. CeGaT GmbH, Tübingen, Baden-Württemberg, Germany.
  7. Division of Human Genetics, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA.
  8. Institute for Clinical Genetics, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Saxony, Germany.
  9. Institute of Pathology, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Germany.
  10. Department of Pediatrics, University of Cincinnati, College of Medicine, Cincinnati, OH, USA.
  11. Heart Institute, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA.
  12. Department of Gynecology and Obstetrics, University Hospital Carl Gustav Carus at the Technische Universität Dresden, Dresden, Saxony, Germany.
  13. Division of Pathology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA.
  14. Department of Pathology and Laboratory Medicine, University of Cincinnati, College of Medicine, Cincinnati, OH, USA.
  15. Genetics and Network Medicine Divisions, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA.
  16. Harvard Stem Cell Institute, Boston, MA, USA.
  17. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA.