A requirement for Syntaxin 4 during vertebrate development and cardiomyocyte conduction

Soluble N-ethylmaleimide-sensitive factor protein receptors (SNAREs) comprise a universally conserved complex of proteins that are key components of the cellular machinery required for intracellular membrane trafficking. Although the mechanism by which SNAREs mediate neurotransmitter release at the synaptic membrane is well-studied, their requirements during normal vertebrate development have only recently become appreciated. Indeed, mutations in SNAREs are now known to result in developmental syndromes, primarily synaptopathies—these disorders have been collectively termed “SNAREopathies”. However, SNAREs ubiquitously regulate vesicle fusion in virtually all cell lineages; therefore, extra-neuronal disease pathologies may also plausibly manifest as “SNAREopathies”.

Cardiomyocytes are among the most specialized cell types, owing to their substantial organization and the dynamic requirements for their diverse function. While it is known that cardiomyocytes heavily rely on intracellular membrane trafficking, to date remarkably few bona fide trafficking proteins have been identified as having a specific function in cardiac tissues. SNAREs are a promising candidate for elucidating how cardiac intracellular trafficking is regulated. Consequently, herein, we endeavored to understand the in vivo requirement for Syntaxin 4 (STX4), a target-SNARE, during normal vertebrate development, cardiac conduction, and cardiomyocyte vesicular transport.

This work was initiated upon the identification of two patients with damaging variants in the STX4 locus: One patient, homozygous for a R240W missense variant, presented with sensorineural hearing loss, global developmental delay, hypotonia, and biventricular dilated cardiomyopathy; ectopy; and runs of non-sustained ventricular tachycardia, requiring an orthotopic heart transplant, while a second patient with compound heterozygous truncating alleles presented with severe pleiotropic abnormalities that resulted in perinatal lethality. To understand the requirement for Stx4 in vertebrate development, we utilized CRISPR/Cas9 to generate stx4 mutant zebrafish, which exhibit defects reminiscent of these patients’ clinical presentations, including linearized hearts, bradycardia, otic vesicle dysgenesis, neuronal atrophy, and touch insensitivity.

Imaging of vesicles within stx4 mutant zebrafish hearts showed reduced docking at the cardiomyocyte sarcolemma, while optical mapping of explanted hearts coupled with pharmacological modulation of Ca²⁺ handling support that stx4 mutants have an in vivo reduction in L-type Ca²⁺ channel modulation. Additionally, ubiquitous transgenic overexpression of zebrafish Stx4^R241W alone was insufficient to rescue the cardiac dysfunction of stx4 mutants, suggesting that the R240W variant functions as a hypomorph. Altogether these data suggest that loss of STX4 function might account for the arrhythmogenic disorder observed in humans. This is notable as congenital heart diseases (CHDs), which are the most common birth defects and account for nearly one-third of all major congenital anomalies, often progress to arrhythmias by adulthood. As such disorders often present as sudden cardiac arrest and are typically lethal, the impact of these collective congenital arrhythmogenic disorders (CADs) is significant. In summary, the data presented within this dissertation demonstrate a conserved requirement for SNARE proteins in regulating normal vertebrate heart development in vivo and that previously unreported variants in STX4 are associated with pleiotropic human disease; thus, we anticipate that further study of these requirements for STX4 may potentiate novel avenues for the treatment of CHDs, CADs, or arrythmias.