Distinct Electrogram Features and Ventricular Arrhythmia Induction Modes Between Repolarization and Conduction Heterogeneities.

BACKGROUND: Recent clinical studies have indicated the presence of localized electrical abnormalities in idiopathic ventricular fibrillation and J-wave syndrome patients. OBJECTIVES: This study aims to characterize the specific electrical signatures of localized repolarization and conduction heterogeneities and their respective role in vulnerability to arrhythmias. METHODS: Optical mapping was performed in porcine right ventricles with local: 1) repolarization shortening; 2) conduction slowing; or 3) structural heterogeneity induced by locally perfusing: 1) pinacidil (20 µmol/L, n = 13); or 2) flecainide (2 µmol/L, n = 13) via an epicardial catheter; or 3) by local epicardial tissue destruction (9 radiofrequency lesions n = 12). Electrograms were recorded (n = 5 in each group) and spontaneous and induced arrhythmias were quantified and optically mapped. RESULTS: Electrograms were normal in (1) but showed local fragmentation in 40% of preparations in (2) with greater effects observed at high pacing frequencies dependent on the wavefront direction. In (3), the structural substrate alone increased the width and number of peaks in the electrograms, and addition of flecainide induced pronounced fragmentation (≥3 peaks and ≥70 ms) in all cases. Occurrence of spontaneous arrhythmias was significantly increased in (1) and (2) (P < 0.0001 and 0.05, respectively, vs baseline) and were triggered by ectopies. Vulnerability to arrhythmias at high pacing frequencies (≥2 Hz) was the lowest in (1) and greatest in (2). CONCLUSIONS: Microstructural substrates have the most pronounced impact on electrograms, especially when combined with sodium channel blockers, whereas local action potential duration shortening does not lead to electrogram fragmentation even though it is associated with the highest prevalence of spontaneous arrhythmias.

Identification of Greb1l as a genetic determinant of crisscross heart in mice showing torsion of the heart tube by shortage of progenitor cells.

Despite their burden, most congenital defects remain poorly understood, due to lack of knowledge of embryological mechanisms. Here, we identify Greb1l mutants as a mouse model of crisscross heart. Based on 3D quantifications of shape changes, we demonstrate that torsion of the atrioventricular canal occurs together with supero-inferior ventricles at E10.5, after heart looping. Mutants phenocopy partial deficiency in retinoic acid signaling, which reflect overlapping pathways in cardiac precursors. Spatiotemporal gene mapping and cross-correlated transcriptomic analyses further reveal the role of Greb1l in maintaining a pool of dorsal pericardial wall precursor cells during heart tube elongation, likely by controlling ribosome biogenesis and cell differentiation. Consequently, we observe growth arrest and malposition of the outflow tract, which are predictive of abnormal tube remodeling in mutants. Our work on a rare cardiac malformation opens novel perspectives on the origin of a broader spectrum of congenital defects associated with GREB1L in humans.

Functional Epicardial Conduction Disturbances Due to a SCN5A Variant Associated With Brugada Syndrome.

BACKGROUND: Brugada syndrome is a significant cause of sudden cardiac death (SCD), but the underlying mechanisms remain hypothetical. OBJECTIVES: This study aimed to elucidate this knowledge gap through detailed ex vivo human heart studies. METHODS: A heart was obtained from a 15-year-old adolescent boy with normal electrocardiogram who experienced SCD. Postmortem genotyping was performed, and clinical examinations were done on first-degree relatives. The right ventricle was optically mapped, followed by high-field magnetic resonance imaging and histology. Connexin-43 and NaV1.5 were localized by immunofluorescence, and RNA and protein expression levels were studied. HEK-293 cell surface biotinylation assays were performed to examine NaV1.5 trafficking. RESULTS: A Brugada-related SCD diagnosis was established for the donor because of a SCN5A Brugada-related variant (p.D356N) inherited from his mother, together with a concomitant NKX2.5 variant of unknown significance. Optical mapping demonstrated a localized epicardial region of impaired conduction near the outflow tract, in the absence of repolarization alterations and microstructural defects, leading to conduction blocks and figure-of-8 patterns. NaV1.5 and connexin-43 localizations were normal in this region, consistent with the finding that the p.D356N variant does not affect the trafficking, nor the expression of NaV1.5. Trends of decreased NaV1.5, connexin-43, and desmoglein-2 protein levels were noted; however, the RT-qPCR results suggested that the NKX2-5 variant was unlikely to be involved. CONCLUSIONS: This study demonstrates for the first time that SCD associated with a Brugada-SCN5A variant can be caused by localized functionally, not structurally, impaired conduction.

Author Correction: A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris.

A Correction to this paper has been published: https://doi.org/10.1038/s42003-020-01488-x.

A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris.

The synthesis of 3,5-dicaffeoylquinic acid (3,5-DiCQA) has attracted the interest of many researchers for more than 30 years. Recently, enzymes belonging to the BAHD acyltransferase family were shown to mediate its synthesis, albeit with notably low efficiency. In this study, a new enzyme belonging to the GDSL lipase-like family was identified and proven to be able to transform chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA, CGA) in 3,5-DiCQA with a conversion rate of more than 60%. The enzyme has been produced in different expression systems but has only been shown to be active when transiently synthesized in Nicotiana benthamiana or stably expressed in Pichia pastoris. The synthesis of the molecule could be performed in vitro but also by a bioconversion approach beginning from pure 5-CQA or from green coffee bean extract, thereby paving the road for producing it on an industrial scale.

In vitro plant regeneration and Agrobacterium-mediated genetic transformation of a carnivorous plant, Nepenthes mirabilis.

In nutrient-poor habitats, carnivorous plants have developed novel feeding strategies based on the capture and digestion of prey and the assimilation of prey-derived nutrients by specialized traps. The Nepenthes genus, comprising nearly 160 species, presents a remarkable pitcher-shaped trap, leading to great interest among biologists, but the species of this genus are listed as threatened. In this work, we developed a protocol for reproducing Nepenthes mirabilis through shoot regeneration from calli. The cultivation of stem segments of N. mirabilis on MS medium containing thidiazuron induced organogenic calli after 10 weeks. Subcultured calli exposed to 6-benzylaminopurine showed shoot regeneration in 3 weeks with considerable yields (143 shoots/g of calli). Excised shoots transferred to medium with indole-3-butyric acid allowed rooting in 4 weeks, and rooted plantlets had a 100% survival rate. Based on this method, we also developed an Agrobacterium-mediated genetic transformation protocol using calli as explants and ipt as a positive method of selection. Twelve weeks post infection, regenerated shoots were observed at the surface of calli. Their transgenic status was confirmed by PCR and RT-PCR. In conclusion, this study provides an efficient method for regenerating Nepenthes and the first protocol for its stable genetic transformation, a new tool for studying carnivory.