Naomi Anderson

Haploinsufficiency of the T-box transcription factor TBX1 is responsible for many features of 22q11.2 deletion syndrome. Tbx1 is expressed dynamically in the pharyngeal apparatus during mouse development and Tbx1 homozygous mutants display numerous severe defects including abnormal cranial ganglion formation and neural crest cell defects. These abnormalities prompted us to investigate whether parasympathetic (vagal) innervation of the heart was affected in Tbx1 mutant embryos. In this report, we used an allelic series of Tbx1 mouse mutants, embryo tissue explants and cardiac electrophysiology to characterise, in detail, the function of Tbx1 in vagal innervation of the heart. We found that total nerve branch length was significantly reduced in Tbx1^+/− and Tbx1^neo2/− mutant hearts expressing 50% and 15% levels of Tbx1. We also found that neural crest cells migrated normally to the heart of Tbx1^+/−, but not in Tbx1^neo2 mutant embryos. In addition, we showed that cranial ganglia IX^th and X^th were fused in Tbx1^neo2/− but neuronal differentiation appeared intact. Finally, we used telemetry to monitor heart response to carbachol, a cholinergic receptor agonist, and found that heart rate recovered more quickly in Tbx1^+/− animals versus controls. We speculate that this condition of decreased parasympathetic drive could result in a pro-arrhythmic substrate in some 22q11.2DS patients.

Introduction Cardiac arrhythmia is a major source of morbidity and mortality. More effective diagnosis and treatment requires a deeper understanding of the underlying pathophysiology of arrhythmia. Aberrant calcium handling is implicated in arrhythmogenesis. This study describes the development of a novel system to simultaneously measure calcium and electrophysiology at the tissue level. Existing research primarily employs single cell or whole heart models, but there is a translational gap between these levels of study which this model is intended to bridge. Calcium transients in left atrial murine tissue under electrical stimulation. Changes in fluorescence coloured green and overlaid over base grayscale image Methods A combined calcium fluorescence and solid-state electrical recording system was set up on an inverted microscope. Samples of murine tissue were loaded with a fluorescent calcium indicator dye (Fluo-4 AM). Intracellular calcium transients (elicited by electrical stimulation via external electrodes) were recorded by a CMOS digital camera, which measured emission light from samples excited with a narrow wavelength LED. The validity of this calcium imaging system was assessed by measuring the effects of decreased cycle length and pharmacological agents on calcium transients. Electrical and fluorescence data were then obtained simultaneously. Electrical data were recorded by contact electrodes in a multi-electrode array. Results Tissue was successfully loaded with fluorescent dye and calcium transients (observed as increases in green fluorescence) elicited by electrical stimulation were recorded. Calcium transient height and duration decreased by 19% (p < 0.001) and 16 ms (95% CI 13–20) respectively when coupling intervals were reduced from 400 ms to 200 ms (n = 5). Isoprenaline 100 nm reduced calcium transient length by 10 ms (95% CI 4.8–16) (n = 5). Increasing concentrations of nifedipine showed a dose-dependent decrease in calcium transient size (n = 1). Calcium fluorescence transients were successfully measured in tandem with electrical activity. Conclusion The current study describes the successful development of a calcium fluorescence imaging system and its integration into a multi-electrode array recording system. This experimental paradigm provides a novel multi-parametric tool for the study of arrhythmia in cardiac tissue.