In this study, we develop an innovative approach to rigorously quantify the evolving hemodynamic environment of the atrioventricular (AV) canal of avian embryos. Ultrasound generated velocity profiles were imported into Micro-Computed Tomography generated anatomically precise cardiac geometries between Hamburger-Hamilton (HH) stages 17 and 30. Computational fluid dynamic simulations were then conducted and iterated until results mimicked in vivo observations. Blood flow in tubular hearts (HH17) was laminar with parallel streamlines, but strong vortices developed simultaneous with expansion of the cushions and septal walls. For all investigated stages, highest wall shear stresses (WSS) are localized to AV canal valve forming regions. Peak WSS increased from 19.34 dynes/cm2 at HH17 to 287.18 dynes/cm2 at HH30, but spatiotemporally averaged WSS became 3.62 dynes/cm2 for HH17 to 9.11 dynes/cm2 for HH30. Hemodynamic changes often preceded and correlated with morphological changes. These results establish a quantitative baseline supporting future hemodynamic analyses and interpretations.

Key pointsr Na + current (I Na ) results from the integrated function of a molecular aggregate (the voltage-gated Na + channel complex) that includes the β subunit family. r We propose that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na + channel α subunit, can be partly consequent to disrupted intracellular Ca 2+ homeostasis.Abstract Na + current (I Na ) is determined not only by the properties of the pore-forming voltage-gated Na + channel (VGSC) α subunit, but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including cases of Brugada syndrome and sudden unexpected death in patients with epilepsy. Here, we have used Scn1b null mouse models to understand better the relation between Scn1b expression, and cardiac electrical function. Using a combination of macropatch and scanning ion conductance microscopy we show that loss of Scn1b in juvenile null animals resulted in increased tetrodotoxin-sensitive I Na but only in the cell midsection, even before full T-tubule formation; the latter occurred concurrent with increased message abundance for the neuronal Scn3a mRNA, suggesting increased abundance of tetrodotoxin-sensitive Na V 1.3 protein and yet its exclusion from the region of the intercalated disc. Ventricular myocytes from cardiac-specific adult Scn1b null animals showed increased Scn3a message, prolonged action potential repolarization, presence of delayed after-depolarizations and triggered beats, delayed Ca 2+ transients and frequent spontaneous Ca 2+ release events and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca homeostasis were prevented by 100 nM tetrodotoxin. Our results suggest that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na + channel α subunit, can be partly consequent to disrupted intracellular Ca 2+ homeostasis in ventricular myocytes.

Rapid impulse propagation in the heart is a defining property of pectinated atrial myocardium (PAM) and the ventricular conduction system (VCS) and is essential for maintaining normal cardiac rhythm and optimal cardiac output. Conduction defects in these tissues produce a disproportionate burden of arrhythmic disease and are major predictors of mortality in heart failure patients. Despite the clinical importance, little is known about the gene regulatory network that dictates the fast conduction phenotype. Here, we have used signal transduction and transcriptional profiling screens to identify a genetic pathway that converges on the NRG1-responsive transcription factor ETV1 as a critical regulator of fast conduction physiology for PAM and VCS cardiomyocytes. Etv1 was highly expressed in murine PAM and VCS cardiomyocytes, where it regulates expression of Nkx2-5, Gja5, and Scn5a, key cardiac genes required for rapid conduction. Mice deficient in Etv1 exhibited marked cardiac conduction defects coupled with developmental abnormalities of the VCS. Loss of Etv1 resulted in a complete disruption of the normal sodium current heterogeneity that exists between atrial, VCS, and ventricular myocytes. Lastly, a phenome-wide association study identified a link between ETV1 and bundle branch block and heart block in humans. Together, these results identify ETV1 as a critical factor in determining fast conduction physiology in the heart.

Understanding the relationships between genetic and microenvironmental factors that drive normal and malformed embryonic development is fundamental for discovering new therapeutic strategies. Advancements in imaging technology have enabled quantitative investigation of the organization and maturing of the body plan, but later stage embryonic morphogenesis is less clear. Chicken embryos are an attractive vertebrate animal model system for this application because of its ease of culture and surgical manipulation. Early embryos can be cultured for a short time on filter paper rings, which enables complete optical access for cell patterning and fate studies 1,2 . Studying advanced developmental processes such as cardiac morphogenesis are traditionally performed through a window of the eggshell 3-5 , but this technique limits optical access due to window size. We previously developed a simple method to culture whole embryos ex-ovo on hexagonal weigh boats for up to 10 days, which enabled high resolution imaging via ultrasonography 6,7 . These cultures were difficult to transport, limiting the types of imaging tools available for live experiments. We here present an improved shell-less culture system with a cost-effective, portable environmental chamber. Eggs were cracked onto a hammock created by a polyurethane membrane (cling wrap) affixed circumferentially to a plastic cup partially filled with sterile water. The dimensions of the circumference and depth of the hammock were both critical to maintain surface tension, while the mechanics of the hammock and water beneath helped dampen vibrations induced by transportation. A small footprint circulating water bath was also developed to enable continuous temperature control during experimentation. We demonstrate the ability to culture embryos in this way for at least 14 days without morphogenic defect or delay and employ this system in several microsurgical and imaging applications. (Figure 1a) i. Fill ¾ of 9 oz plastic cups with warm sterile water. A regular 9 oz cup has a top diameter of 8 cm. We found that this size was most suitable for long time culture application presented here. ii. Cut about 20 cm cling wrap. Affix circumferentially on top of the cup by placing a rubber band around. Plastic should be spread on top of the water. Cut the excess plastic around the band. We found cling wrap to be most appropriate for this application since it prevents adhesion of the yolk, resulting in the yolk splitting during transportation. iii. Wipe the plastic with kimwipes (Kimberly-Clark, Inc.) wetted with 70% ethanol. Prepare hammocks

Fever is a highly conserved systemic response to infection dating back over 600 million years. Although conferring a survival benefit, fever can negatively impact the function of excitable tissues, such as the heart, producing cardiac arrhythmias. Here we show that mice lacking fibroblast growth factor homologous factor 2 (FHF2) have normal cardiac rhythm at baseline, but increasing core body temperature by as little as 3 °C causes coved-type ST elevations and progressive conduction failure that is fully reversible upon return to normothermia. FHF2-deficient cardiomyocytes generate action potentials upon current injection at 25 °C but are unexcitable at 40 °C. The absence of FHF2 accelerates the rate of closed-state and open-state sodium channel inactivation, which synergizes with temperature-dependent enhancement of inactivation rate to severely suppress cardiac sodium currents at elevated temperatures. Our experimental and computational results identify an essential role for FHF2 in dictating myocardial excitability and conduction that safeguards against temperature-sensitive conduction failure.