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 hear

Optogenetic determination of the myocardial requirements for extrasystoles by cell type-specific targeting of ChannelRhodopsin-2

Extrasystoles lead to several consequences, ranging from uneventful palpitations to lethal ventricular arrhythmias, in the presence of pathologies, such as myocardial ischemia. The role of working versus conducting cardiomyocytes, as well as the tissue requirements (minimal cell number) for the generation of extrasystoles, and the properties leading ectopies to become arrhythmia triggers (topology), in the normal and diseased heart, have not been determined directly in vivo. Here, we used optogenetics in transgenic mice expressing ChannelRhodopsin-2 selectively in either cardiomyocytes or the conduction system to achieve cell type-specific, noninvasive control of heart activity with high spatial and temporal resolution. By combining measurement of optogenetic tissue activation in vivo and epicardial voltage mapping in Langendorff-perfused hearts, we demonstrated that focal ectopies require, in the normal mouse heart, the simultaneous depolarization of at least 1,300–1,800 working cardiomyocytes or 90–160 Purkinje fibers. The optogenetic assay identified specific areas in the heart that were highly susceptible to forming extrasystolic foci, and such properties were correlated to the local organization of the Purkinje fiber network, which was imaged in three dimensions using optical projection tomography. Interestingly, during the acute phase of myocardial ischemia, focal ectopies arising from this location, and including both Purkinje fibers and the surrounding working cardiomyocytes, have the highest propensity to trigger sustained arrhythmias. In conclusion, we used cell-specific optogenetics to determine with high spatial resolution and cell type specificity the requirements for the generation of extrasystoles and the factors causing ectopies to be arrhythmia triggers during myocardial ischemia

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 heartIl cuore è densamente innervato dai neuroni del sistema nervoso simpatico che regolano la funzionalità cardiaca attraverso un effetto cronotropo o inotropo positivi. Durante lo stress o l’esercizio, la noradrenalina rilasciata dai neuroni attiva i β recettori cardiaci sia sul sistema di conduzione che sul muscolo contrattile. L’aumento dell’attività del sistema nervoso simpatico cardiaco sia in condizioni normali o in presenza di patologie genetiche, come per esempio la Tachicardia Catecolaminergica Polimorfica Ventricolare, porta ad aritmie presumibilmente attraverso l’insorgere di ‘DADs’. Le ‘DADs’ sono un focus di aritmia che porta a una serie di depolarizzazioni che interessano un piccolo gruppo di cellule cardiache. E’ stato identificato un rilascio di catecolamine non bilanciato in diverese regioni del cuore da parte del sistema nervoso simpatico come possibile causa di aritmia. Inoltre alterazioni del ‘reuptake’ di noradrenalina porta a una concentrazione anomala di NE nello scompenso cardiaco che può essere coinvolto in un evento aritmico. Questi dati supportano un modello in cui il controllo della funzionalità cardiaca da parte del sistema nervoso simpatico avviene attraverso un sito d’interazione diretta e specializzata fra neurone e cardiomiocita accoppiato. Gli scopi del progetto sono quindi: 1. Studiare se l’interazione fra neurone e cardiomiocita ha un ruolo nella trasmissione cardiaca del segnale, per capire come un’attività non bilanciata del sistema nervoso simpatico porta a un evento aritmico. 2. Capire se l’attività non bilanciata del sistema nervoso simpatico modulando l’attività di un piccolo gruppo di cellule cardiache, possa essere coinvolto nella generazione di un’aritmia in vivo. Per verificare quest’ipotesi ci serviremo di un approccio innovativo basato su proteine foto attivabili 3. Studiare in vivo e in maniera non invasiva la massa critica di cellule cardiache necessaria per scatenare un evento aritmico. Anche per questo tipo di studio abbiamo utilizzato una metodologia basata sull’optogenetica. Nella prima parte del progetto, abbiamo creato un modello in vitro costituito da cardiomiociti neonatali e neuroni isolati dal ganglio cervicale superiore. I neuroni in seguito a trattamento con NGF sviluppano assoni che stabiliscono contatti con i cardiomiociti. Sotto terminali che sono in contatto con le cellule cardiache si osserva un maggiore accumulo di β1 recettori [3]. Abbiamo misurato l’attivazione dei β recettori monitorando in tempo reale le variazioni di AMP ciclico e attività di PKA, attraverso l’uso di sensori geneticamente codificati e che si basano sul FRET (EPAC1-camps, che ci permette di monitorare cAMP e AKAR3 che ci permette di monitorare l’attività di PKA). I neuroni del SNS sono stati stimolati con KCl o bradichinina. Abbiamo osservato che stimolando il rilascio di noradrenalina da un neurone, l’AMP ciclico e l’attività di PKA aumentano solo nei cardiomiociti accoppiati a neurone e non nei cardiomiociti senza un contatto (ΔR/R0 = 0.056 ± 0.01 mean ± SEM, n = 8, AKAR3 ΔR/R0=5.3% ± 1.5%, mean ± SEM, n=6). Per stimare la [NE] che agisce sui β recettori nel sito di contatto abbiamo paragonato l’ampiezza del segnale FRET generato dall’attivazione neuronale (ΔR/R0= 0.026 ± SEM) con quello generato da diverse [NE] note aggiunte alla soluzione in cui si trovano le cellule. Abbiamo osservato che l’aumento del rapporto CFP/YFP ottenuto dalla noradrenalina rilasciata dai neuroni e paragonabile a quello ottenuto con 3.5e-10 M di noradrenalina che attiva tutti i recettori. Usando un antagonista competitivo dei β recettori (propranololo) abbiamo determinato la concentrazione di noradrenalina nel cleft sinaptico. La concentrazione di propranolol necessaria per abolire totalmente la risposta indotta dalla noradrenalina rilasciata dai neuroni, e pari a quella necessaria per bloccare la risposta indotta da 100 nM di noradrenalina, suggerendo che la concentrazione nel cleft sinaptico è dell’ordine di 100 nM. Sulla base di questi dati abbiamo quindi calcolato che la frazione recettoriale con cui interagisce la noradrenalina rilasciata dai neuroni che è inferiore all’1% del totale. 1.Nella seconda parte del progetto abbiamo usato una strategia che si basa sull’‘optogenetica’ per modulare l’attività del sistema nervoso simpatico in vivo e in maniera non invasiva. ChR2 è un canale la cui permeabilità è regolata dalla luce. Infatti questo canale diventa permeabile soprattutto al Na+ in seguito a stimolazione con luce blu. Negli ultimi anni è stato largamente utilizzato per il controllo dell’attività neuronale sia in vitro che in vivo [4, 5]. Abbiamo generato un modello murino che esprime ChR2 nei neuroni del sistema nervoso simpatico sotto il promotore tirosina idrossilasi. La foto stimolazione del ganglio stellato è stata ottenuta in un modello a ‘torace aperto’ di topo anestetizzato, usando una fibra ottica per indirizzare in uno specifico punto la luce generata da un LED. L’analisi dell’ECG del topo mostra un rapido (100-150 ms) aumento (40%±6%) nella frequenza di contrazione cardiaca in seguito a ‘fotostimolazione’ del ganglio stellato. Questo rapido aumento nella frequenza cardiaca supporta il modello in cui la noradrenalina agisce in uno spazio piccolo e confinato in cui neurone e cardiomiocita interagisccono direttamente. 3. Abbiamo usato ChR2 anche per modulare l’elettrofisiologia cardiaca. Abbiamo determinato che la fotostimolazione di ChR2 è sufficiente a modulare il potenziale d’azione in cardiomiociti neonatali in cultura. Inoltre a seconda di quando viene dato il pulso di luce siamo in grado di generare un battito normale, una DAD o una EAD. Abbiamo quindi generato un modello di topo che esprima ChR2 nel cuore sotto il promotore α-MHC. Abbiamo controllato tramite stimolazione luminosa il potenziale d’azione di cellule cardiache utilizzando fibre ottiche alimentate da LED, durante l’acquisizione dell’ECG del topo. La stimolazione è stata eseguita in diverse regioni del cuore. La stimolazione atriale ci ha permesso di mimare un pacing atriale sfociato poi una tachicardia. Abbiamo osservato che il QRS non ha variazioni rispetto al normale, indicando che l’onda di depolarizzazione segue il sistema di conduzione cardiaco. La foto attivazione ventricolare invece genera un battito prematuro dato che non segue il sistema di conduzione. Abbiamo qui dimostrato l’esistenza di un contatto sinaptico fra i neuroni e i cardiomiociti che forma un sito a elevata concentrazione di neurotrasmettitore, uno spazio a diffusione limitata permettendo quindi l’attivazione di un ristretto gruppo di recettori β localizzati nella membrana della cellula cardiaca. La stimolazione neuronale genera un rapido aumento nella frequenza cardiaca avvalorando l’ipotesi dell’esistenza di un contatto sinaptico fra neuroni e cardiomiociti. Questa interazione è importante per un controllo rapido del segnale locale dei cardiomiociti, suggerendo che i neuroni controllino un gruppo ristretto di cellule cardiache. La stimolazione di una frazione di cardiomiociti è sufficiente a indurre un battito condotto in tutto il cuor

Dynamics of neuroeffector coupling at cardiac sympathetic synapses

ABSTRACT: Cardiac sympathetic neurons (SNs) finely tune the rate and strength of heart contractions to match blood demand, both at rest and during acute stress, through the release of noradrenaline (NE). Junctional sites at the interface between the two cell types have been observed, although whether direct neurocardiac coupling has a role in heart physiology has not been clearly demonstrated to date. We investigated the dynamics of SN/cardiomyocyte intercellular signalling, both by fluorescence resonance energy transfer-based imaging of cAMP in co-cultures, as a readout of cardiac \u3b2-adrenergic receptor activation, and in vivo, using optogenetics in transgenic mice with SN-specific expression of Channelrhodopsin-2. We demonstrate that SNs and cardiomyocytes interact at specific sites in the human and rodent heart, as well as in co-cultures. Accordingly, neuronal activation elicited intracellular cAMP increases only in directly contacted myocytes and cell-cell coupling utilized a junctional extracellular signalling domain with an elevated NE concentration. In the living mouse, optogenetic activation of cardiac SNs innervating the sino-atrial node resulted in an instantaneous chronotropic effect, which shortened the heartbeat interval with single beat precision. Remarkably, inhibition of the optogenetically elicited chronotropic responses required a high dose of propranolol (20-50 mg kg-1 ), suggesting that sympathetic neurotransmission in the heart occurs at a locally elevated NE concentration. Our in vitro and in vivo data suggest that the control of cardiac function by SNs occurs via direct intercellular coupling as a result of the establishment of a specific junctional site