Optogenetic Modulation Of Cardiac Sympathetic Nerve Activity To Prevent Ventricular Arrhythmia

Background:Malignant ventricular arrhythmias(VAs)remain a major contributor to sudden cardiac death in patients with cardiac disease.It is well established that the cardiac sympathetic nervous system,particularly the left stellate ganglion(LSG),plays a prominent role in modulating ventricular electrophysiology and arrhythmias.Left cardiac sympathetic denervation(LCSD)has been applied to treat animals or patients with life-threatening ventricular arrhythmias such as post-myocardial infarction(MI)refractory VAs or electrical storm.However,there are some side effects associated with this traumatizing and irreversible operation such as unilateral hand dryness,abnormal sweating,chest pain,etc.Optogenetics is a novel powerful neuromodulatory technology that combines optics and genetics to achieve spatiotemporal precise activation or inhibition of targeted neurons.When Arch T,an inhibitory light-sensitive opsin,is genetically expressed in targeted cells and activated by illumination with the appropriate wavelength,it induces hyperpolarizing currents,thus silencing the cells.Aims:1.To investigate whether optogenetics could precisely modulate cardiac sympathetic nerve activity;2.To investigate whether optogenetic modulation of cardiac sympathetic nerve activity could stablize ventricular electrophysiological properties and protect against acute MI-induced VAs;3.To develop a fully implantable,battery-free wireless LED device to achieve long-term safe and effective wireless optogenetic cardiac sympathetic modulation in awake,freely moving canines,and investigate whether chronic wireless optogenetic cardiac sympathetic modulation could improve post-MI cardiac remodeling and VAs.Methods:All canines were anesthetized with Na-pentobarbital(30 mg/kg)and ventilated with room air by a positive pressure respirator.Additional maintenance doses of 60 mg/h Na-pentobarbital were administered during the procedure.Normal saline at 100 m L/h was infused to replace spontaneous fluid loss.Left femoral arterial catheterization was performed to monitor systemic arterial pressure.Body surface electrocardiograph(ECG)was recorded with a computer-based Laboratory System.A heating pad was used to maintain a core body temperature of 36.5 ± 0.5 °C.1.Twenty male beagles were randomly divided into the optogenetics group(n=10)and control group(n=10).The virus AAV2/9-CAG-Arch T-GFP was chosen for transfecting the LSG.A similar construct(AAV2/9-CAG-GFP)without Arch T was used as a control.Four weeks after AAV injection to transfect LSG neurons,the LSG function and neural activity and heart rate variability(HRV)were measured in the absence or presence of LED illumination(565nm).At the end of the experiment,LSG tissue was collected to analyze the expression of GFP/Arch T-GFP expression.2.Twenty male beagles were randomized into the optogenetics group(n = 10,AAV2/9-CAG-Arch T-GFP microinjected into LSG)and control group(n = 10,AAV2/9-CAG-GFP microinjected into LSG).Four weeks after virus injection,the LSG neural activity,HRV,ventricular action potential duration(APD)and effective refractory period(ERP)were measured in the absence or presence of LED illumination(565nm).Acute MI was induced by left anterior descending coronary artery ligation and 1 h of ECG was recorded for VAs analysis.LSG tissue was collected to analyze the expression of c-fos and nerve growth factor(NGF)in sympathetic neurons.3.Eighteen beagle canines were randomly divided into a normal control group(n=6,AAV2/9-hsyn-GFP-WPRE-p A injection with sham MI),a MI group(n=6,AAV2/9-hsyn-Arch T-GFP-WPRE-p A injection with MI),and an optogenetics group(n=6,AAV2/9-hsyn-Arch T-GFP-WPRE-p A injection with LED illumination and MI).Four weeks after AAV microinjection to transfect LSG neurons,the wireless LED device was implanted on top of the LSG to perform illumination(565nm)for optogenetic modulation.Left anterior descending coronary artery was ligated to induce MI and then light illumination was performed for 1h immediately after MI and daily for four weeks.Cardiac function,LSG function and neural activity,HRV,plasma norepinephrine,ventricular ERP and APD,VA inducibility and ventricular fibrillation threshold were measured.At the end of the experiment,LSG and cardiac tissue were collected to measure infarction size,cardiac fibrosis and sympathetic remodeling.Results:1.Arch T was successfully and extensively expressed in all canines.Transient LED illumination significantly suppressed the LSG function,LSG neural activity and sympathetic nerve indices of HRV only in the optogenetics group.Thirty-minute illumination further enhanced these changes in the optogenetics group.Importantly,all these changes returned to baseline within two hours after illumination was turned off.GFP/Arch T-GFP expression or LED illumination alone did not induce obvious changes in LSG function and neural activity.2.Transient LED illumination significantly prolonged left ventricular ERP and APD90 in the optogenetics group.Thirty-minute illumination further enhanced these changes in the optogenetics group.Importantly,all these changes returned to baseline within two hours after illumination was turned off.Optogenetic modulation significantly suppressed the acute MI-induced LSG hyperactivity and improve HRV.Moreover,the acute MI-induced VAs were significantly suppressed by illumination only in the optogenetics group.3.In freely-moving post-MI canines,chronic wireless optogenetic modulation using the implantable LED device significantly inhibited MI-induced LSG hyperactivity and neural remodeling,improved HRV and decreased plasma norepinephrine concentration.Chronic wireless optogenetic cardiac sympathetic inhibition improved ventricular electrophysiological stability as reflected by prolonged ERP and APD with reduced dispersion,suppressed VA inducibility and increased ventricular fibrillation threshold.In addition,chronic optogenetic modulation significantly improved LV dysfunction and suppressed MI-induced cardiac remodeling with reduced infarct size and fibrosis.Conclusions:1.The present study firstly proved that optogenetics could precisely and reversibly inhibit LSG function and neural activity.2.Optogenetic modulation of cardiac sympathetic nerve activity could significantly increase cardiac electrophysiological stability and prevent acute MI-induced VAs.3.This fully implantable,battery-free wireless optogenetics system enables chronic wireless optogenetic cardiac neuromodulation in awake freely-moving canines.Chronic wireless optogenetic inhibition of cardiac sympathetic hyperactivity significantly improved MI-induced cardiac autonomic,electrical,functional and structural remodeling and thus reduced VA susceptibility.