Development and Physiology of the Intrinsic Neurons of the Heart – HEARTBRAIN

The heart functions under dual autonomic modulation, sympathetic and parasympathetic. A classical view has been that autonomic ganglia serve as simple relay stations for two antagonistic influences on the heart: stimulating through the sympathetic outflow, inhibiting through the parasympathetic outflow. However, evidence has slowly accrued that the autonomic circuits to the heart are more complex than this description accounts for. Intrinsic ganglia of are known to contain of variety of morphological and neurochemical phenotypes and, most likely, not only post-ganglionic parasympathetic neurons (some of which also receive a sympathetic input) but also others, that don’t receive central innervation and might be interneurons or sensory neurons. Limited electrophysiological data suggest that these ganglia have a computing or integrating role or even are capable of autonomous action, a view championed by John Armour who coined the expression “the little brain in the heart”. This project, in three parts, is designed to decisively advance our knowledge of this “little brain” with the tools of developmental genetics in mouse, in a manner that has proved fruitful in other parts of the nervous system such as the spinal cord or hindbrain. To achieve this, we formed a consortium of two groups specialized in complementary aspects of the autonomic nervous system, one bringing expertise in mouse developmental genetics, the other in the field of axonal guidance and cardiovascular physiology.
In Part I, using the mouse as experimental system and recent imaging techniques on cleared thick tissue samples, we will revisit the fine structure of cardiac ganglia and, for the first time, explore their connectivity. We will produce a map of their neuronal diversity by documenting in 3D the pattern of expression of known and novel markers of autonomic neurons. We will search for additional markers, by profiling the transcriptome of dissociated single cardiac ganglionic cells. We will use a novel intersectional transgenic tool, developed by one of the partner labs to map in 3D and in developmental time the connectivity of cardiac ganglia. We will also map the projections of these ganglia to the cardiac muscle, conductive tissue, and blood vessels, a topic still debated. Collectively, we expect the results from Part I to produce an unprecedented understanding of the development and anatomical complexity of the intrinsic cardiac nervous system and of its interface with the extrinsic one, a prerequisite for the understanding of their physiology.
In Part II, We will knockout two tandem paralogous transcription factors, Hmx2 and Hmx3, which we recently identified as markers of parasympathetic differentiation. In parallel, we will create a Flpo-dependent Hmx3::Cre allele that, mated with an available Phox2b::Flpo and available Cre-dependant alleles, will enable the genetic manipulation, specifically, of parasympathetic ganglia (including cardiac ones), in particular with the Designer Receptor Exclusively Activated by Designer Drugs technology.
In Part III, we will monitor cardiovascular function of the genetic models created in Part II, where intrinsic cardiac ganglia are absent, or acutely and reversibly silenced or activated. i) On Langendorff preparations. ii) In anesthetized mice, we will record heartbeat and rate by ultrasound, the ECG, the morphology(ventricles thickness and ejection fraction). iii) by telemetry in freely moving animals, we will measure continuously heartbeat and rate, blood pressure, activity, temperature. iv) We will measure heart rate variability, a non-invasive predictor of heart failure in a variety of contexts v) The autonomic modulation of the heart being probably most important in conditions of physiological adaptation to stress, we will duplicate these analyses in graded conditions of stress. vi) Finally, we will evaluate the role of cardiac ganglia in atrial fibrillation, where the source of ectopic activities is uncertain.