Fret imaging and optogenetics shed light on neurocardiac regulation in vitro and in vivo

The heart is densely innervated by sympathetic neurons (SN) that regulate cardiac function both through chronotropic and inotropic effects. During exercise and stress, SN-released norepinephrine activates cardiac beta adrenergic receptors (beta-ARs) on both the conduction and contractile systems. Increased cardiac sympathetic activity leads to arrhythmias in acquired (e.g. myocardial ischemia) or inherited conditions, including Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT), possibly via development of Ca2+ overload-dependent early- or delayed-afterdepolarizations (EAD, DAD, respectively). The DAD would serve as arrhythmogenic focus, leading to the onset of triggered activity in discrete groups of cardiac cells. Unbalanced sympathetic discharge to different regions of the heart has been identified as a potent arrhythmogenic condition 1. In addition to the direct cardiomyocyte damage, alteration in presynaptic NE reuptake from the autonomic neuron endings, leading to catecholamine spillover in the failing myocardium 2, inducing is an arrhythmic event. These data support a model in which autonomic control of cardiac function relies on specialized sites of direct interaction between the neurons and their target cardiomyocytes (CM). The aims of the project are: 1. To investigate whether specific cell-cell interactions have a role in the dynamics of intercellular signaling between SN and CM, aims to understand how unbalanced SN activity leads to arrhythmic condition. 2. To understand whether the unbalanced SN modulation of a limited group of cardiac cells could be involved in generating arrhythmias in vivo, based on an optogenetic approach 3. To study in vivo, non-invasively, the critical mass of myocardium necessary to generate an arrhythmogenic focus, using optogenetics. In the first part of the project, we used an in vitro model of sympathetic neurons/cardiomyocytes (SN-CM) co-cultures to analyze the dynamics of intercellular signaling. Upon NGF treatment, SNs extend their axons and establish direct contact with CMs. NE-synthesizing terminals developed on SN at the contact site, and beta1-ARs were enriched on the CM membrane in correspondence of the active release areas 3. We performed real-time imaging using the FRET-based biosensors EPAC1-camps and AKAR3 to assess intracellular cAMP and PKA activity, respectively. Stimulation of SN was achieved using KCl or bradykinin. We observed that activation of a specific SN lead to cAMP increase in the interacting CM (ΔR/R0 = 5.6% ± 1% mean ± SEM, n = 8, AKAR3 ΔR/R0= 5.3% ± 1.5%, mean ± SEM, n=6). The cAMP response in cardiomyocytes was not due to NE released in the medium, and was absent in cells not in direct contact with the activated neuron. We showed that in cells without SN coupled the intracellular cAMP and PKA activity were not affected. To estimate the [NE] acting on the CM beta-AR at the contact site, we compared the amplitude of the FRET signal evoked by SN activation (ΔR/R0= 2.6 % ± 0.6%, mean ± SEM, n=13 ) to that elicited by different [NE] administered to the cell bathing solution, and we observed that the increase in the CFP/YFP ratio achieved by SN-released NE is comparable to that obtained with 3.5e-10 M NE to whole cell. Using the competitive beta-antagonist propranolol we determined the effective [NE] in the ‘synaptic’ cleft. Competition antagonism of neuronal stimulation to CM was obtained with [Propranolol] equal to that antagonizing 100 nM of NE, indicating that such concentration is achieved in the ‘synaptic cleft’. Moreover, by calculating the fraction of occupancy of the receptor at different concentration of NE we calculated that the fraction of beta-ARs activated by the SN-released NE is < 1%. 2. In the second part of the project we used an optogenetic-based strategy to modulate cardiac sympathetic neurons activity non invasively in vivo. ChR2 is a light-gated cation channel that becomes permeable mainly to Na+ upon light-stimulation, shown to enable control of neuronal activity both in vitro and in the intact brain. We generated a mouse model expressing ChR2 in SN under the tyrosine hydroxilase (TOH) promoter. Photostimulation of the stellate ganglia neurons (SGN) was obtained in an anesthetized, open-chest model using a fiber optic to locally (1mm) deliver light (470nm) generated from a LED. ECG recording demonstrates a rapid (100-150 ms) increase (40%±6%) in heart rate (HR) upon SGN stimulation. The extremely short activation time of the cardiac response upon ChR2 depolarization of the neurons support a model in which NE acts in a short range, consistent with direct interaction between SN and CM. 3. We used ChR2 to modulate cardiac electrophysiology. We determined in cultured neonatal cardiomyocytes that photostimulation allows triggering action potential (AP). Moreover depending on when the light pulses were given we generated normal AP, early- or delayed-aferdepolarizations (EAD or DAD). We generated a mouse model with cardiac expression of ChR2, driven by the α-MHC promoter. Optical control of cardiomyocyte membrane potential was obtained with a fiber optic, while recording the ECG in the anesthesized mouse. Stimulation was applied to different regions of the heart. Atrial illumination was used to obtain non-invasive atrial pacing resulting in tachycardia with unchanged QRS, indicating as expected that the cardiac activation wave followed the natural conduction system. Ventricular photoactivation, on the contrary, bypassing the natural conduction system gave rise to premature ventricular beats. We provide evidence of the existence of a ‘synaptic’ contact between SN and CM that forms a high agonist concentration, diffusion-restricted space allowing potent activation of a small fraction of beta-ARs on the CM membrane upon neuronal stimulation. SN stimulation leads to a rapid increase of the HR supporting the idea of the existence of the synaptic contact between SN and CM. This close interaction has the potential of fast control of local CM signalling, suggesting that SNs control locally discrete groups of myocardial cells. Stimulation of a small fraction of the cardiac cells (< 200 microm-wide area) induced ectopic beats conducted to the whole heart