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Articles in PresS. Am J Physiol Heart Circ Physiol (December 23, 2016). doi:10.1152/ajpheart.00644.2016 1 2 3 4 5 6 CARDIAC SYMPATHETIC INNERVATION VIA THE MIDDLE CERVICAL AND STELLATE GANGLIA AND ANTI-ARRHYTHMIC MECHANISM OF BILATERAL STELLECTOMY 7 8 9 10 Running Title: Anti-arrhythmic Mechanism of Bilateral Stellectomy 11 12 13 Tadanobu Irie,1,2* Kentaro Yamakawa,2* David Hamon,1,2 Keijiro Nakamura,1,2 Kalyanam 14 Shivkumar,1,2 Marmar Vaseghi1,2 15 16 17 1 UCLA Cardiac Arrhythmia Center & 2 Neurocardiology Research Center of Excellence, 18 Los Angeles, CA 19 *These authors contributed equally 20 21 22 Drs. Irie created the infarct model, helped perform the terminal experiments, assisted in data analysis 23 and interpretation, and drafted the manuscript. Dr. Yamakawa performed the terminal experiments, 24 data analysis, and interpretation. Drs. Hamon and Nakamura participated in creating the infarct model 25 and performing the terminal experiments. Dr. Shivkumar participated in the design of the study, helped 26 with funding of the study, and edited and revised the manuscript. Dr. Vaseghi designed and funded the 27 study, assisted in performing the experiments, data analysis and interpretation, and drafted and 28 revised the manuscript. 29 30 31 Address for Correspondence 32 Marmar Vaseghi, MD, PhD 33 UCLA Cardiac Arrhythmia Center, 34 100 Medical Plaza, Suite 660, 35 Los Angeles, CA 90095 36 Tel:+1-310-206-2235 37 Fax:+1-310-825-2092 38 Email: [email protected] 39 Copyright © 2016 by the American Physiological Society. 40 ABSTRACT 41 Cardiac sympathetic denervation, CSD, is reported to reduce burden of ventricular 42 tachy-arrhythmias (VT/VF) in cardiomyopathy patients, but the mechanisms behind this 43 benefit are unknown. In addition, the relative contribution to cardiac innervation of the middle 44 cervical ganglia (MCG), which may contain cardiac neurons, and is not removed during this 45 procedure, is unclear. 46 the heart via the MCG vs. stellate ganglia, assess effects of bilateral CSD on cardiac function 47 and VT/VF, and determine changes in cardiac sympathetic innervation after CSD to elucidate 48 mechanisms of benefit in 6 normal and 18 infarcted pigs. 49 hemodynamic parameters were evaluated at baseline, during bilateral stellate and during 50 bilateral MCG stimulation in 6 normal and 12 infarcted animals. Bilateral CSD (removal of 51 bilateral stellates and T2 ganglia) was then performed, and MCG stimulation repeated. In 52 addition, in 18 infarcted animals VT/VF inducibility was assessed pre- and post-CSD. 53 infarcted hearts, MCG stimulation resulted in greater chronotropic and inotropic response 54 than stellate ganglia stimulation. Bilateral CSD acutely reduced VT/VF inducibility by 50% in 55 infarcted hearts and prolonged global activation recovery interval. CSD mitigated effects of 56 MCG stimulation on dispersion of repolarization and T-peak to T-end interval in infarcted 57 hearts, without causing hemodynamic compromise. This data demonstrate that the MCG 58 provides significant cardiac sympathetic innervation before CSD and adequate sympathetic 59 normal after CSD, maintaining hemodynamic stability. 60 inducibility by improving electrical stability in infarcted hearts in the setting of sympathetic 61 activation. The purpose of this study was to compare sympathetic innervation of Electrophysiological and In Bilateral CSD reduces VT/VF 62 63 1 64 65 66 67 68 69 NEW AND NOTEWORTHY Sympathetic activation in myocardial infarction leads arrhythmias and worsens heart failure. Bilateral cardiac sympathetic denervation reduces VT/VF inducibility and mitigates effects of sympathetic activation on dispersion of repolarization and T-peak to T-end interval in infarcted hearts. Hemodynamic stability is maintained, as innervation via the middle cervical ganglia is not interrupted. 70 2 71 KEYWORDS 72 Autonomic; 73 sympathetic nervous system cardiac sympathetic denervation; stellectomy, ventricular arrhythmias, 74 75 3 76 INTRODUCTION 77 Myocardial infarction (MI) increases risk of sudden cardiac death due to ventricular 78 tachy-arrhythmias (VT/VF),(13) by causing pathological cardiac (16, 34, 41) and neural 79 remodeling.(15, 44, 53) 80 increases T-peak to T-end (Tp-Te) interval, a marker of sudden cardiac death (8, 33) and 81 increases dispersion of repolarization, predisposing to VT/VF.(27, 44, 45, 50) Left cardiac 82 sympathetic denervation (CSD) has been reported to decrease burden of VT/VF in patients 83 with hereditary channelopathies, including long QT syndrome and catecholaminergic 84 polymorphic ventricular tachycardia, who have structurally normal hearts.(4, 17, 38) In a 85 small series of patients with structural heart disease, bilateral CSD has shown promise in 86 reducing burden of internal cardioverter defibrillator (ICD) shocks. This study also suggested 87 that bilateral CSD may lead to a greater ICD shock free survival compared to left CSD in 88 patients with cardiomyopathy.(43) However, electrophysiological mechanisms behind the 89 reduction in life-threatening arrhythmias with CSD are not clear. In addition, it is unknown how 90 much of cardiac sympathetic innervation, that may be required for beat-to-beat function, is 91 preserved after bilateral CSD. The middle cervical ganglia (MCG), located anatomically 92 superior to the stellate ganglia, have been reported to contain cardiac sympathetic neurons,(7, 93 21) 94 electrophysiological effects have not been characterized. The purpose of this study was to 95 delineate (1) sympathetic innervation of the heart via the MCG compared to the stellate 96 ganglia (2) electrophysiological and anti-arrhythmic effects of CSD, including VT inducibility, 97 and (3) alterations in functional cardiac sympathetic innervation after CSD and its 98 anti-arrhythmic mechanisms in a porcine model. and could provide Sympathetic activation via the right or left stellate ganglion sympathetic innervation after CSD. However, their 99 100 101 METHODS Study procedures were approved by UCLA Institutional Animal Research Committee 4 102 and performed in compliance with the National Institutes of Health Guide for the Care and 103 Use of Laboratory Animals. 104 105 Creation of Myocardial Infarcts 106 Eighteen Yorkshire pigs were sedated (telazol 6-10 mg/kg and fentanyl 2-5 μg/kg), 107 intubated, and placed under general anesthesia with inhaled isoflurane (0.5-1.5%). After 108 femoral arterial access was obtained using an 8F sheath, an Amplatz guide catheter (Boston 109 Scientific, Marlborough, MA) was advanced over a J tipped guide wire under fluoroscopic 110 guidance and used to cannulate the left main coronary artery from the left femoral artery. A 111 coronary angiogram was performed to delineate the branches of the left anterior descending 112 coronary artery. A 3 mm luminal angioplasty balloon (Armada 35 PTA, Abbott Vascular, 113 Temecula, CA) was advanced past the first diagonal branch of the left anterior descending 114 artery over a 0.014 inch coronary guide wire (Balance Middle Weight wire, Abbott Vascular, 115 Temecula, CA) and inflated. Polystyrene microspheres (5.0-7.5 ml, Polysciences, Inc. 116 Warrington, PA) were injected through the lumen of the percutaneous balloon catheter.(26) 117 Repeat coronary angiography after microsphere injection showed confirmed poor blood flow 118 in the LAD after the first diagonal branch. Continuous electrocardiogram (ECG) monitoring 119 was performed. ECG changes including ST segment elevation and or T wave inversions were 120 also used to confirm myocardial infarction acutely. If sustained VT/VF was observed, 121 resuscitation with chest compressions and external direct current cardioversion was 122 performed. Animals were then extubated and monitored until they could ambulate without 123 assistance. 124 Surgical Preparation 125 Four to six weeks after MI, 18 infarcted (48 ± 2 kg) and six normal animals (43 ± 1 126 kg) were sedated (telazol 6-10 mg/kg and fentanyl 2-5 μg/kg) and placed under general 127 anesthesia with isoflurane (0.8-1.5%). Normal animals had not undergone a percutaneous 5 128 interventional procedure prior to terminal experiments. Median sternotomy was performed 129 and isolation of bilateral stellate ganglia and T2 thoracic ganglia (all animals) and bilateral 130 MCG (12 of 18 animals) were performed. Presence and extent of scar on the epicardium was 131 assessed in infarcted animals grossly, figure 1, as well as by using bipolar voltage 132 measurements using a 2-2-2 duodecapolar catheter (2-2-2 mm spacing, St. Jude Medical, St. 133 Paul, MN). Regions with a voltage less than 0.5 mV were defined as scar, those with voltage 134 of 0.5 to 1.5 mV were defined as border zone (BZ), and electrodes overlying regions with 135 voltage greater than 1.5mV were defined as viable/normal.(14, 23) The location and extent of 136 infarct was marked on the heart. 137 subclavian artery, at the inlet of the thorax, figure 1. After completion of the surgical portion of 138 the procedure, isoflurane was discontinued, and an intravenous infusion of α-chloralose 139 (10-30 mg/kg/h) was begun.(5, 10, 11) During anesthesia with both α-chloralose and 140 isoflurane, heart rate as well as jaw tone and eye blink reflexes were monitored every 10-15 141 minutes to ensure adequate sedation. 142 Stellate and Middle Cervical Ganglia Stimulation The MCG were identified often above and behind the 143 Bipolar needle electrodes (figure 1) were used for bilateral stellate ganglia 144 stimulation and bilateral middle cervical ganglia stimulation using a Grass Stimulator (Model 145 S88, Grass Technologies, Warwick, RI) in both infarcted (n =12) and normal (n = 6) animals. 146 Threshold was defined as current needed to increase heart rate (HR) or systolic blood 147 pressure by 10% (at 4Hz, 4 ms). 148 Stimulation was performed at 1.5 times threshold for 30 seconds. In normal animals, 149 the stimulation current used for right stellate ganglion was 2.4±0.5 mA and for the right MCG 150 was 2.3±0.4 mA. The stimulation current used for the left stellate ganglion was 5.6±0.8 mA 151 and for the left MCG was 6.0±0.7 mA in normal animals. For infarcted animals, the stimulation 152 current used for the right stellate ganglion was 6.7±1.0 mA and for the right MCG was 5.9±1.2 153 mA; the current for left stellate ganglion stimulation was 9.8±1.0 mA and for left MCG was 6 154 10.0±1.0 mA. In normal animals, a second stellate and MCG stimulation was performed prior 155 to CSD for 60 seconds and electrophysiological effects were compared at 5, 10, 15, 20, 30, 156 45, and 60 seconds. A 30-minute period was allowed for return of hemodynamic parameters 157 to baseline in between stimulations. The timeline for the experimental protocol in normal 158 animals is shown in figure 2. 159 Hemodynamic and Electrocardiogram Recordings 160 Hemodynamic parameters were continuously recorded with a 12-pole conductance 161 pressure catheter in the left ventricle (LV), connected to a MPVS Ultra® Pressure Volume 162 Loop System (Millar Instruments, Houston, TX). ECG was continuously obtained using the 163 GE Cardiolab System (GE Healtcare). Tp-Te interval was assessed in the inferior leads with 164 the clearest T wave. Tp-Te interval was assessed in the inferior leads with the clearest T wave 165 off-line manually from the electrocardiograms obtained from the Cardiolab System at 400 mm/sec 166 paper speed. 167 peak of the T wave was visually determined, and the end of the T wave was defined as the 168 intersection of the tangent to the slope of the T wave and the isoelectric line, when not followed by 169 a U wave, figure 1. If a U wave followed the T wave, the off-set of the T-wave was measured as 170 the nadir between the T and U waves. This is a similar to what has been described in the literature. 171 (29, 50) 172 Bilateral Cardiac Sympathetic Denervation Tp-Te interval was measured from the peak of T wave to the end of T wave. The 173 Bilateral CSD was performed in 18 infarcted and 6 normal animals after isolation of 174 bilateral stellate and T2 thoracic ganglia. T3 and T4 thoracic ganglia were not removed as it 175 has been previously shown that cardiac fibers from these ganglia traverse through the 176 stellate and T2 ganglia before reaching the heart.(12) The lungs on each side were retracted, 177 the sympathetic chain again identified, and its connections to the spinal cord and 178 cardiopulmonary nerves were transected. Both stellate ganglia and T2 thoracic ganglia were 179 removed. A 30-minute stabilization period after CSD was allowed prior to repeat stimulation 7 180 or inducibility testing. VT/VF inducibility was tested pre- and post-CSD. 181 Inducibility Testing for Ventricular Tachy-arrhythmias 182 In 18 infarcted hearts, programmed ventricular stimulation up to triple extra-stimuli to 183 a minimum coupling interval of 200 ms or effective refractory period was performed before 184 and after CSD first from the right ventricular endocardium at the mid to apical septum using 185 an endocardial quadripolar electrophysiology catheter connected to a MicroPace stimulator 186 (MicroPace EP Inc. Santa Ana, CA). Current was set at two times the ventricular capture 187 threshold in each animal and pulse duration of 1 ms. If VT/VF was not induced from the RV 188 endocardium at baseline pre-CSD, a second site on the LV epicardium close to the scar was 189 used pre-CSD. The same or a very similar site was used for induction of VT/VF post-CSD 190 that had been used to induce VT/VF pre-CSD. The induction protocol was as follows: an 191 extra-stimulus was placed after 8 beats of drive cycle length (at 500 ms) at an interval of 400 192 ms (S2). The premature extra-stimulus interval (S2) was reduced by 10 ms until either an 193 interval of 200 ms or effective refractory period (ERP) was reached or VT/VF was induced. 194 If VT/VF was not induced, then, S2 was fixed at 20 ms above the effective refractory period or 195 at 220 ms (if ERP was < 200 ms), and a second premature extra-stimulus (S3) was added at 196 an interval of 400 ms. The S3 interval was then reduced by 10 ms until an S2-S3 interval of 197 200 ms, or effective refractory period was reached, or VT/VF was induced. Finally, if no 198 VT/VF was induced, then S3 interval was set at 220 ms (if ERP was < 200 ms) or 20 ms 199 above effective refractory period, and an S4 interval (triple extra-stimulus) added at an S3-S4 200 interval of 400 ms. This S4 interval was then reduced by 10 ms until a coupling interval of 200 201 ms or effective period was reached, or VT/VF was induced. Inducibility was defined as 202 hemodynamically tolerated VT that lasted greater than 30 seconds or that degenerated into 203 VF requiring direct cardioversion. 204 Measurement of Norepinephrine Levels 205 Norepinephrine (NE) levels in the inferior vena cava and coronary sinus (CS) were 8 206 measured to confirm adequate ganglia stimulation. Venous blood was obtained from a 207 luminal catheter inserted in the IVC, superior to the adrenal veins and close to the right atrium. 208 CS blood was obtained by cannulation of the CS with a luminal catheter (St. Jude Medical, St. 209 Paul, MN) from the right external jugular vein. Blood samples at baseline and in the last 5 210 seconds of stellate ganglia and MCG stimulation pre-CSD were immediately centrifuged 211 (3000 rpm, 15 minutes) to separate the plasma portion. Quantification of NE was performed 212 using an ultrasensitive enzyme linked immunoassay (ELISA, BA E-5200, sensitivity 1.3 pg/ml, 213 Labor Diagnostika Nord GmbH & Co.KG, Nordhorn, Germany) and an ELISA microplate 214 reader (Fisher Scientific, Waltham, MA). 215 ARI Recordings 216 In 12 infarcted and 6 normal animals, detailed electrophysiological mapping was 217 performed. A 56-electrode sock (figure 1) was placed over the ventricles to assess activation 218 recovery interval (ARI), a surrogate of local action potential duration. Unipolar electrograms 219 were recorded (0.05-500 Hz) using the GE Cardiolab System. ARIs were calculated with 220 customized software (Scaldyn, University of Utah, Salt Lake City, UT) as previously 221 described.(44, 46) Briefly, activation time (AT) was defined as the interval from electrogram 222 onset to most negative dV/dt of the activation wave, and repolarization time (RT) was defined 223 as the interval from electrogram onset to the most positive dV/dt of the repolarization wave. 224 ARI was defined as the difference between RT and AT. Map3D software (University of Utah) 225 was used to map epicardial pattern of activation and ARI using a sock-electrode polar map 226 template, figure 1F. Global dispersion of repolarization (DOR) was calculated as the variance 227 of ARIs recorded across all epicardial electrodes. 228 In this manuscript, anterior refers to ventral and posterior refers to dorsal aspect of 229 the heart. In normal animals, mean ARIs in the following regions were analyzed based on 230 electrode location: LV apex, LV anterior, LV lateral, LV posterior wall and right ventricular (RV) 231 anterior, lateral, posterior wall, and RV outflow tract. A median of 3 electrodes (range 2-4) per 9 232 region was used for regional ARI and AT analysis. 233 Atrial and Ventricular Pacing 234 To evaluate effect of CSD on ARI during ventricular pacing compared to atrial pacing, 235 ventricular pacing was performed at baseline and during MCG stimulation before and after 236 CSD (pacing cycle length = 400 ms or 500 ms, depending on HR during stimulation) in 6 237 normal and 5 infarcted animals. To compare differences to ventricular pacing, right atrial 238 pacing at the same cycle length was performed in normal animals at baseline and during 239 BMCGS, pre- and post-CSD. In addition, Map3D software (University of Utah, Salt Lake City, 240 UT, http://www.sci.utah.edu/cibc-software/map3d.html) was used to map epicardial pattern of 241 activation and ARI during scar pacing in 5 infarcted hearts. The template, with sock electrode 242 locations, used for creation of these polar maps is shown in figure 1. 243 Pre-ganglionic vs. Post-ganglionic Neural Fibers Within the MCG 244 To evaluate presence of pre- vs. post-ganglionic fibers within the MCG in normal 245 animals after CSD, bilateral MCG stimulation was repeated after infusion of hexamethonium, 246 a nicotinic receptor blocker. Right vagal nerve stimulation (VNS) was also performed pre and 247 post hexamethonium infusion to ensure nicotinic receptor blockade, as vagal efferent 248 parasympathetic fibers are known to be preganglionic. The right cervical vagal trunk was 249 isolated via a lateral neck cut-down and bipolar needle electrodes (Cyberonics, Houston, TX) 250 attached to a Grass Stimulator used for right VNS (10 Hz, 1 ms). 251 the current at which HR decreased by 10%, and stimulation performed at 1.2 times threshold 252 for 20 seconds, before and after hexamethonium. Hexamethonium was infused for 30 253 minutes (0.025-0.2 mg/kg/min, dose titrated by response to right VNS in each animal). 254 Statistical Analysis Threshold was defined as 255 Unless specified otherwise, data are presented as mean±standard error (SE). Global 256 ARI was calculated as the mean ARI across all 56 electrodes. Global dispersion of 257 repolarization (DOR) was calculated as the variance in ARIs across all 56 electrodes. For 10 258 comparison of paired variables, Wilcoxon signed-rank or paired t-test was used was used. 259 Sample size of infarcted animals was driven by the assumption of a 40% reduction in VT/VF 260 inducibility after CSD with 80% power to detect this reduction at alpha of 0.05. McNemar test 261 was used to compare VT inducibility before and after CSD. Sample size of normal animals 262 was driven by 80% power to detect a 10% difference in the effects of MCG vs. stellate ganglia 263 stimulation on global ARI in the same animal at an alpha of 0.05. The Mann-Whitney test was 264 used for comparison of continuous variables in different groups. Percentage change in 265 regional ARIs for stellate ganglia vs. MCG stimulation were analyzed using repeated 266 measure ANOVA after controlling for a false discovery rate at 5%. SPSS (version 22, IBM, 267 Armonk, NY) was used for statistical analysis. P<0.05 was considered statistically significant. 268 269 RESULTS 270 Thoracic Sympathetic Innervation in Normal Hearts 271 Timeline for the experimental protocol in normal animals is shown in figure 2. In 272 normal hearts bilateral stellate ganglia and bilateral MCG stimulation significantly increased 273 HR, maximum dP/dt, and left ventricular end-systolic pressure Table 1. Comparison of 274 bilateral stellate ganglia to bilateral MCG stimulation demonstrated no differences in the HR 275 or maximum dP/dt increase. However, bilateral stellate ganglia stimulation had a greater 276 effect on left ventricular end-systolic pressures (LVESP) than bilateral MCG stimulation. 277 Coronary sinus NE levels increased in a similar fashion during bilateral stellate and bilateral 278 MCG stimulation compared to pre-stimulation values, Table 2. 279 Both bilateral stellate and MCG stimulation decreased epicardial ARI, figure 2. There 280 was no significant difference in the time course of ARI shortening during the 60 seconds of 281 stimulation between the stellate ganglia and MCG, figure 3, and no differences in regional 282 ARIs were detected between bilateral stellate and bilateral MCG stimulation figure 3. 283 Compared to right atrial pacing, LV apical pacing at baseline and during MCG 11 284 stimulation at the same cycle length led to significantly shorter epicardial ARIs, P<0.05, 285 suggesting that ventricular pacing alone led to cardiac sympathetic activation, figure 3. 286 287 CSD and Residual Sympathetic Innervation in Normal Hearts 288 CSD did not cause hemodynamic deterioration in normal hearts, table 1. 289 global ARI was observed before compared to after CSD, figure 4. 290 No difference in MCG continued to exert significant sympathetic effects after CSD. Global ARI 291 continued to decrease during MCG stimulation after CSD, figure 4. 292 stimulation after CSD on HR and dP/dt max were unchanged compared to before CSD, table 293 1. Of note, comparison of atrial to ventricular pacing at the same cycle length showed that the 294 differences in ARI shortening between RA pacing and LV pacing were no longer significant 295 after CSD, with or without MCG stimulation, figure 3. DOR and Tp-Te interval, which 296 increased during MCG stimulation, were unaffected by CSD in normal hearts, figure 4. 297 Furthermore, CSD had no effect on activation time (pre-CSD: baseline 26.1±1.5 ms vs. MCG 298 stimulation 30.4±4.1 ms, P=0.3, post-CSD: baseline 26.1±1.1 ms vs. MCG stimulation 299 28.2±1.5 ms, P=0.2). Effect of bilateral MCG 300 To evaluate presence of preganglionic fibers within the MCG, response to 301 hexamethonium was evaluated. Right VNS was also performed to assure adequate nicotinic 302 receptor blockade. 303 hexamethonium, figure 5. After hexamethnoium infusion, there was no change in global ARI 304 during right VNS. However, effects of MCG stimulation after hexamethonium were 305 unchanged, suggesting presence of predominantly post-ganglionic sympathetic fibers in the 306 MCG, figure 5. 307 Thoracic Sympathetic Innervation in Infarcted Hearts Global ARI significantly increased during right VNS prior to 308 Timeline for the experimental protocol in infarcted animals is shown in figure 6. In 309 infarcted animals, changes in ARI were greater during MCG stimulation compared to stellate 12 310 ganglia stimulation (MCG stimulation: 24.7±3.5% vs. stellate stimulation: 12.0±2.8%), P=0.02, 311 figure 6. The increase in HR was also greater during bilateral MCG stimulation compared to 312 bilateral stellate gangila stimulation, table 3, while the increase in LVESP was greater with 313 stellate ganglia stimulation. Finally, the rise in coronary sinus NE levels was similar during 314 bilateral MCG compared to bilateral stellate ganglia stimulation, table 2. 315 CSD and Residual Sympathetic Innervation in Infarcted Hearts 316 In infarcted hearts, CSD led to a prolongation of global ARI (pre-CSD: 385±17 ms vs. 317 post-CSD: 392±17 ms, P<0.05), figure 7. CSD had no effect on HR or dP/dt max. However, a 318 decrease in LVESP was observed after CSD in infarcted hearts, P=0.02, table 3. 319 In 66.7% of MI animals (12/18), VT/VF was inducible prior to CSD. After CSD, VT/VF 320 was inducible in 6 of these 12 animals, reducing VT inducibility by 50% (P<0.05), figure 7. Of 321 note, the 6 animals, which were not inducible for VT/VF before CSD, remained non-inducible 322 after CSD. 323 Residual Cardiac Sympathetic Pathways After CSD 324 In infarcted animals, global ARI continued to decrease during MCG stimulation, figure 8. No 325 significant differences in the effects of MCG stimulation on hemodynamic parameters were 326 observed pre vs. post-CSD, table 3. 327 Of note, CSD mitigated the increase in DOR during bilateral MCG stimulation in 328 infarcted animals. Before CSD, bilateral MCG stimulation increased DOR by 356±222 ms2, 329 P<0.05, figure 8. 330 longer significant, figure 8. Before CSD, MCG stimulation increased Tp-Te interval from 44±4 331 ms to 68±7 ms (mean±SE) in infarcted hearts. After CSD, MCG stimulation increased Tp-Te 332 interval from 44±5 ms to 58±6 ms. Therefore, the percentage increase in this interval after 333 CSD during MCG stimulation was significantly reduced, P<0.05, figure 8. Given that 334 premature ventricular contractions that cause ventricular arrhythmias in infarcted hearts often 335 originate from the myocardium, we qualitatively assessed activation patterns during apical However, after CSD the increase in DOR with MCG stimulation was no 13 336 pacing in 5 infarcted hearts, figure 9, which suggested improvement in epicardial 337 conduction/activation and decrease in functional block after CSD, or during MCG stimulation 338 after CSD, in two of the five infarcted hearts. 339 340 DISCUSSION 341 Major Findings 342 In this study the MCG provided significant innervation to the ventricles, and these 343 sympathetic pathways remained intact in normal and infarcted animals after CSD. 344 Furthermore, bilateral CSD reduced VT inducibility acutely in the setting of chronic MI, 345 without compromising hemodynamic stability, a finding that can have implications for 346 cardiomyopathy patients with recurrent ICD shocks. An important mechanism behind the 347 anti-arrhythmic benefit of CSD in infarcted hearts was the mitigation of the effects of 348 sympathetic activation on DOR and on prolongation of Tp-Te interval, a marker of sudden 349 cardiac death. 350 Cardiac Sympathetic Innervation 351 Contribution of the MCG to cardiac sympathetic innervation has been previously 352 overlooked. Cardiac neurons that fire in specific phases of the cardiac cycle had been 353 previously described in the MCG.(7) But the degree of functional sympathetic innervation to 354 the heart from the MCG was unknown. In this study, effects of bilateral MCG stimulation in 355 normal hearts were similar to bilateral stellate ganglia stimulation. However, in infarcted 356 animals, MCG stimulation led to significantly greater effects on ARI and HR, while stellate 357 ganglia stimulation had greater effects on LVESP. Given multiple other fibers to the head and 358 neck that traverse through the MCG, removal of the MCG, unlike the lower half of stellate 359 ganglia, is not feasible clinically. MCG stimulation continued to show significant cardiac 360 effects after hexamethonium infusion, suggesting that the cardiac neural fibers from the MCG 361 are post-ganglionic, similar to the stellate ganglia. 14 362 Anti-Arrhythmic Effects of CSD 363 Previous reports had shown that left CSD can reduce ischemia driven ventricular 364 arrhythmias.(30, 39) In this study, the rationale for performing bilateral rather just left CSD 365 was driven by emerging evidence in patients with structural disease and refractory VT/VF. It has 366 been noted that unlike patients with channelopathies, case series of patients who undergo 367 bilateral CSD for refractory ventricular arrhythmias in the setting of structural heart disease (i.e. 368 myocardial infarction) have greater ventricular tachycardia free survival compared to those 369 patients who undergo left sided only procedures.(43) Furthermore, right stellate ganglion 370 stimulation is also pro-arrhythmic, increases dispersion of repolarization(50) and T-peak to T-end 371 interval, an independent marker of sudden cardiac death in patients.(29, 33), Therefore, in this 372 model, bilateral CSD was performed both to assess hemodynamic stability after bilateral (rather 373 just left sided CSD) and to assess VT inducibility acutely after bilateral CSD. However, acute 374 effects of CSD on ventricular arrhythmias in the setting of a chronic remodeled infarct were 375 unknown. Furthermore, hemodynamic compromise with bilateral CSD remained a 376 concern.(43) In this study, hemodynamic parameters were not compromised while arrhythmia 377 inducibility decreased after CSD. 378 Although this study was performed in the swine model, significant similarities 379 between canine and human autonomic nervous system exist. As in canines and in humans, 380 the sympathetic chain on each side consists of a superior cervical, a middle cervical, and a 381 stellate ganglion, followed by a ganglion located at the level of each rib.(20) Like the canine model 382 sympathetic stimulation via the stellate ganglion increase dispersion of repolarization.(45, 50) 383 Direct and indirect sympathetic activation in humans increases dispersion of repolarization, 384 similar to pigs and dogs.(44) Vagal nerve stimulation and decentralization in pigs has similar 385 effects to canines.(6, 51) Furthermore, we have shown that in the pig, similar to the dog, intrinsic 386 cardiac ganglia remodels in the setting of myocardial infarction. In addition, pig stellate ganglia 387 show evidence of pathological neural remodeling similar to humans in the setting of myocardial 15 388 infarction.(1, 2) Of note, the cardiac neurons in the MCG were first detected in the canine 389 model.(7) The fact that coronary circulation in the pig is more similar to humans (than canines to 390 humans) is important in that the scars that are generated in the porcine myocardial infarct model 391 are very similar to scars observed in humans with ischemic cardiomyopathy, carrying the same 392 electrical signature.(25) In humans, as in pigs, the right and left stellate ganglia provide significant 393 innervation to the heart, which is also true of humans.(20, 45) Like canines, the right stellate 394 ganglion provides predominant innervation to the anterior aspect of the heart and left stellate 395 ganglion provides innervation to the posterior/dorsal aspect of the heart.(45, 52) Like canines, 396 sympathetic stimulation via the stellate ganglia in pigs increases dispersion of repolarization and 397 predisposes to arrhythmias.(28, 50) 398 In addition to interrupting efferent fibers, some of the beneficial effects of CSD are 399 likely due to interruption of afferent neurotransmission. Cardiac sympathetic afferent fibers 400 exist throughout the cardiac chambers, and sense mechano-chemical stimuli.(22) Disruption 401 of these afferent fibers reduces sympathetic outflow and has been shown to decrease the 402 number of ectopic beats that occur with brief episodes of ischemia.(36) Ventricular pacing 403 leads to activation of cardiac mechano-receptors, increased sympathetic tone, and alteration 404 processing through the intrinsic cardiac ganglia can be observed.(18, 31) Afferent fiber 405 activation subsequently increases sympathetic efferent outflow,(37) and can explain the 406 shorter ARIs that were observed with apical ventricular pacing compared to right atrial pacing 407 before CSD, despite a fixed pacing cycle length. This effect was eliminated after CSD. 408 Therefore, a portion of the anti-arrhythmic benefit of CSD is likely due to interruption of 409 afferent neurotransmission, which then alters subsequent efferent neurotransmission via the 410 stellate and middle cervical ganglia. Sympathetic activation during VT inducibility testing, (18, 411 35, 42) which by its nature involves ventricular pacing, is reduced by CSD in infarcted hearts, 412 leading to less observed arrhythmias. In fact, it has been shown that sympathetic afferent 413 responses to noxious stimuli are enhanced in heart failure dogs and increase renal 16 414 sympathetic outflow more than normal animals.(48) Furthermore, chemical cardiac 415 sympathetic de-afferentation using epicardial application of resiniferatoxin decreases cardiac 416 sympathetic nerve activity and attenuates cardiac remodeling in rats with heart failure.(47) 417 Therefore, reduction in afferent transduction leading to decrease efferent outflow could play 418 an important role in the anti-arrhythmic benefit of CSD. A framework for the current 419 understanding of the effect of CSD on cardiac neurotransmission via afferent and efferent 420 fibers is shown in figure 10. 421 Although CSD prolonged global ARI in infarcted animals, this effect was not 422 observed in normal animals. This may be due to the heightened sympathetic tone and neural 423 remodeling that occurs in infarcted hearts,(2) which can be improved by interruption of 424 efferent and afferent fibers through the stellate ganglia and removal of these cardiac neurons. 425 It has been demonstrated both in this infarct model(26) and in patients with ischemic 426 and non-ischemic cardiomyopathy, that scar regions are rarely composed of homogeneous 427 and dense fibrotic tissue (which would not give rise to any electrical signal), and contain many 428 islands of live myocardium.(24, 26, 32, 49) These regions of live myocardium or channels 429 within “scar” give rise to fractionated bipolar electrograms, which have been shown to be 430 good targets of catheter ablation in patients with ventricular tachycardia.(19, 32, 40) However, 431 these same scar regions with intermingling myocytes have heterogenous innervation(9, 44) 432 that can lead to regions of delayed activation and conduction block, allowing for reentry and 433 ventricular arrhythmias to occur.(3, 34) In this study, we noted that in two animals, regions of 434 slow activation along the septal border zones of infarcts were improved after CSD, either at 435 baseline or during MCG stimulation, suggesting that CSD allowed for more uniform cardiac 436 activation, an anti-arrhythmic effect. More homogenous activation could be either due to 437 earlier activation of “late” areas, or more likely, due to slower activation of the surrounding 438 regions as a result of disruption of sympathetic efferent fibers. 439 MCG Stimulation After CSD 17 440 A theoretical concern with performing bilateral CSD in patients with cardiomyopathy 441 had been the possibility of eliminating sympathetic pathways that may be necessary for 442 day-to-day activities and exercise. In this study, efferent innervation from the MCG remained 443 intact after CSD, and CSD did not lead to any hemodynamic compromise in the porcine 444 model. 445 Stellate ganglia stimulation has been reported to increase DOR and Tp-Te 446 interval.(45, 50) Tp-Te interval represents both transmural and whole heart DOR and 447 increases risk of sudden cardiac death.(8, 27, 33) In this study, MCG stimulation also 448 increased DOR and Tp-Te interval in infarcted hearts. Importantly, CSD mitigated the effects 449 of MCG stimulation on DOR and Tp-Te interval in the setting of chronic myocardial infarction, 450 a novel finding of this study. The electrical stability that results from CSD in infarcted hearts 451 during sympathetic activation, and the lack of hemodynamic compromise is reassuring. 452 Limitations 453 Electrograms were recorded only from ventricular epicardium. Therefore, intramural 454 and endocardial effects were not assessed. This study was performed in a porcine model, 455 and given interspecies differences, direct interpolation to humans requires additional studies. 456 However, Isoflurane, which can suppress autonomic activity and was used for general 457 anesthesia during the surgical portion of the protocol. However, anesthesia was switched to 458 α-chloralose during the stimulation and CSD portions of the protocol. In addition, ARI and 459 Tp-Te interval were not corrected for increases in HR. Therefore, increases in Tp-Te interval 460 observed with stimulation are likely a conservative estimate of the actual effects. Given 461 different stimulation currents to achieve the same hemodynamic threshold, direct comparison 462 of parameters in MI vs. normal hearts was not performed. However, stimulation currents for 463 right and left stellate ganglia vs. MCG were similar. Finally, direct comparison of ARI values at 464 baseline or after CSD in infarcted compared to normal hearts cannot be made as normal 465 animals did not undergo a sham myocardial infarction procedure, did not have VT inducibility 18 466 testing, and had an additional BSG and MCG stimulation where the time course of effects of 467 BSG stimulation to MCG stimulation was compared. Finally, VT inducibility was not tested 468 during MCG stimulation. However, VT inducibility, by the nature of its ventricular pacing, does 469 cause sympathetic activation,(18, 35, 42) suggesting that the beneficial effects of CSD are 470 most influential during a state of elevated sympathetic tone. 471 472 Conclusions 473 Both MCG and stellate ganglia contribute significantly to cardiac sympathetic 474 innervation. In addition, VT inducibility in animals with chronic MI is significantly reduced by 475 bilateral CSD, even acutely. A mechanism for this benefit is the improvement in the increase 476 in DOR and Tp-Te interval prolongation observed with sympathetic activation. Furthermore, 477 efferent innervation via the MCG remains intact after bilateral CSD and hemodynamic 478 parameters remain stable. 479 480 481 482 19 483 FUNDING/GRANTS 484 This study was supported by NIH1DP2HL132356 and AHA 11FTF755004 to MV and NHLBI 485 R01HL084261 to KS. 486 20 487 DISCLOSURES 488 None for all authors. 489 490 21 491 REFERENCES 492 493 1. 494 Shivkumar K. 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Functional distribution of right and left Zhou S, Jung BC, Tan AY, Trang VQ, Gholmieh G, Han SW, Lin SF, Fishbein MC, 667 668 26 669 670 FIGURE LEGENDS 671 672 Figure 1. Isolation of the middle cervical ganglia and Tp-Te interval and ARI recordings. 673 (A) Anatomy of MCG and surrounding tissue is shown. The MCG sits just at the thoracic inlet 674 and provides cardiopulmonary nerves that innervate the heart. (B) Customized bipolar needle 675 electrodes were placed in the isolated MCG. (C) The method for the measurement of Tp-Te 676 interval from surface ECG is shown. The peak of the T wave determined as the highest 677 voltage of the T wave, T-p. T-end (T-e) was determined by the tangent of the T wave. (D) The 678 region of scar in infarcted hearts predominantly involved the LVA apex, anterior, and 679 anterolateral walls. (E) 56-electrode sock is placed over the ventricles to obtain unipolar 680 electrograms that are used for ARI analysis. (F) The template for the polar maps used to 681 display regional ARIs from the sock electrodes is shown. MCG = middle cervical ganglion, 682 Ant = anterior, Lat = lateral, Post = posterior, RVOT = right ventricular outflow tract. 683 684 Figure 2. Experimental protocol in normal hearts and effect of bilateral stellate ganglia 685 compared to bilateral MCG stimulation (A) Timeline of the experimental protocol in 6 686 normal animals. (B) In all normal animals, bilateral MCG stimulation had a similar effect on 687 ARI compared to bilateral stellate ganglia stimulation (C) Polar maps from a normal animal 688 show the effects of MCG and stellate ganglia stimulation on ARI. Blue dashed lines indicate 689 course of left anterior descending coronary artery. Dashed circle indicates regions of scar on 690 polar maps. BL = baseline, BSGS = bilateral stellate ganglia stimulation, BMCGS = bilateral 691 middle cervical ganglia stimulation. *BMCGS or BSGS was performed in a random order, 692 LAD = left anterior descending coronary artery. 693 694 Figure 3. (A) The time course of ARI shortening with bilateral stellate ganglia stimulation was 695 similar to bilateral MCG stimulation (n = 6). (B) There were no significant regional differences 27 696 between bilateral stellate and bilateral MCG stimulation in normal hearts (P values > 0.05 for 697 all regions, n = 6). (c) Apical ventricular pacing pre-CSD demonstrated shorter global ARIs 698 than right atrial (RA) pacing in normal animals (n = 6) at the same cycle lengths. However, 699 after CSD, there was no significant difference in ARI between ventricular and atrial pacing, 700 suggesting that apical pacing may cause reflex sympathetic activation that is prevented by 701 CSD. Ant = anterior, Lat = lateral, Post = posterior wall, RV = right ventricle, LV =left ventricle, 702 RA = right atrium. BSGS = bilateral stellate ganglia stimulation. BMCGS = bilateral middle 703 cervical ganglia stimulation. 704 705 Figure 4. Effects of CSD in Normal Animals. (A) There was no difference in global ARI in 706 normal heart before compared to after CSD. (B) Bilateral MCG stimulation after CSD had 707 similar effects on global ARI compared to before CSD, with significant ARI shortening 708 observed. (C) There were no differences in the regional effects of MCG stimulation after CSD 709 compared to before CSD. (D) Before CSD, bilateral MCG stimulation increased dispersion of 710 repolarization in normal hearts, and this effect was not modified by CSD. (E) Bilateral MCG 711 stimulation in normal hearts also increased Tp-Te interval, with no differences observed in 712 this parameter before compared to after CSD in normal hearts. 713 714 Figure 5. Effects of Hexamethonium on MCG stimulation. (A) The increase in global ARI 715 during right vagal nerve stimulation was no longer observed after the infusion of 716 hexamethonium in normal animals (n = 6). (B) Global ARI during bilateral MCG stimulation 717 significantly decreased even after administration of hexamethonium in all normal hearts (n = 718 6). (C) Percent change in global ARI during right vagal nerve stimulation and bilateral MCGS 719 stimulation showed that hexamethonium significantly blocked nicotinic receptors, but did not 720 affect the response to bilateral MCG stimulation. Hex = hexamethonium, VNS = vagal nerve 721 stimulation. 28 722 723 Figure 6. Experimental protocol in infarcted hearts and effect of bilateral stellate 724 ganglia compared to bilateral MCG stimulation (A) Timeline the experimental protocol in 725 12 infarcted animals. (B) In all infarcted animals, bilateral stellate ganglia stimulation 726 significantly decreased ARI as did bilateral MCG stimulation. Bilateral MCG stimulation, 727 however, caused greater ARI shortening than bilateral stellate stimulation (C) Polar maps 728 from an infarcted animal show the effects of MCG and stellate ganglia stimulation on ARI. 729 Black dashed lines indicate region of the infarct on the polar maps. Blue dashed lines indicate 730 course of left anterior descending coronary artery. Dashed circle indicates regions of scar on 731 polar maps. BL = baseline, BSGS = bilateral stellate ganglia stimulation, BMCGS = bilateral 732 middle cervical ganglia stimulation. *BMCGS or BSGS was performed in a random order, 733 LAD = left anterior descending coronary artery. 734 735 Figure 7. Effects of CSD and VT inducibility pre and post CSD (A) Epicardial global ARI 736 from infarcted animals significantly increased with CSD (n = 12). (B) Inducibility of VT/VF was 737 reduced after CSD in infarcted animals (n = 18, 12 were inducible at baseline. Of this 12, only 738 6 were inducible after CSD). (C) An example of VT induction with ventricular extra-stimulus 739 pacing is shown. VT/VF was induced before CSD with double extra-stimuli at cycle lengths of 740 600/320/200 ms. After CSD, ERP was reached at an interval of 600/340 ms with double 741 extra-stimuli. Therefore, the S2 stimulus was increased by 20 ms to 600/360 ms (to allow for 742 consistent capture) and S3 was added and the interval reduced by 10 ms. The animal 743 reached ERP at 270 ms with triple extra-stimuli (S3). Therefore, the S3 coupling interval was 744 increased to 290 ms (to allow for consistent capture), and S4 was reduced by 10 ms, starting 745 at 600/360/290/400 ms. Despite reducing S4 extra-stimulus (triple extra-stimulus testing) to a 746 coupling interval of 200 ms, no ventricular arrhythmias could be induced. Black dashed lines 747 indicate region of the infarct on the polar maps. 29 748 749 Figure 8. MCG stimulation and its effects before and after CSD on infarcted hearts. (A) 750 Global ARI at baseline and during bilateral MCG Stimulation after CSD and percentage 751 change in ARI pre- vs. post-CSD during BMCGS in infarcted hearts (n = 12). MCG stimulation 752 decreased ARI despite CSD. (B) Examples of polar maps at baseline and during MCG 753 stimulation after CSD in infarcted hearts. Dashed-black lines indicates course of LAD. 754 Dashed blue circles indicates region of scar. (C) Although MCG stimulation significantly 755 increased DOR before CSD, this effect was mitigated after CSD in infarcted hearts (n = 12). 756 (D) The prolongation in Tp-Te interval during MCG stimulation was reduced by CSD in 757 infarcted hearts. BL = baseline, BMCGS = bilateral MCG stimulation, LAD = left anterior 758 descending coronary artery. 759 760 Figure 9. Modulation of propagation and activation time by CSD. (A) Polar maps of 761 activation time/sequence obtained during scar/apical pacing in this infarct animal 762 demonstrated a localized region at the septal border zone of the infarct that was activated 763 late as compared to it surrounding area. This area of functional block (localized regions of 764 late activation, delineated as “II”) pre-CSD was no longer observed after CSD, and the entire 765 area was activated more homogenously. (B) Polar maps of activation time/sequence during 766 scar pacing at baseline and during bilateral MCG stimulation before and after CSD in a 767 different infarcted animal are shown. 768 activation are seen at the border zone of the infarct, creating a potential isthmus or area of 769 slow conduction that could serve as the substrate for a reentrant circuits. Both of these 770 regions of late myocardial activation are no longer observed during MCG stimulation after 771 CSD. After CSD, the entire region is more uniformly activated. 772 bilateral middle cervical ganglia stimulation. Black dashed lines indicate region of the infarct 773 on the polar maps. During MCG stimulation, two localized regions of late BL = baseline, BMCGS = 30 774 775 Figure 10. Efferent and afferent cardiac sympathetic pathways. Afferent fibers from the 776 myocardium that traverse through the MCG pass through the stellate ganglia before reaching 777 the spinal cord, and some of these pathway are interrupted by CSD. In addition, 778 pre-ganglionic efferent fibers that pass from the spinal cord through the stellate ganglia and to 779 the MCG and any post-ganglionic fibers that arise from the stellate ganglia and innervate the 780 myocardium are also interrupted. However, efferent post-ganglionic fibers from MCG neurons 781 to the myocardium remain intact despite CSD. Aff = afferent neurons, Eff = efferent neurons, 782 DRG = dorsal root ganglion, IML = intermediolateral nucleus, DH = dorsal horn of the spinal 783 cord. 784 31 Figure Figure 1. 1 B A B Subclavian Artery MCG Sympathetic Chain Ansa Subclavia C Needle Electrode Cardiopulmonary Nerves MCG F E D RV Ant RV Lat RVOT Infarcted Areas LAD RV LV Ant LAD LV Infarcted Area Infarcted Area LV Lat RV Post Apex LV Post Figure 1. Isolation of the middle cervical ganglia and Tp-Te interval and ARI recordings. (A) Anatomy of MCG and surrounding tissue is shown. The MCG sits just at the thoracic inlet and provides cardiopulmonary nerves that innervate the heart. (B) Customized bipolar needle electrodes were placed in the isolated MCG. (C) The method for the measurement of Tp-Te interval from surface ECG is shown. The peak of the T wave determined as the highest voltage of the T wave, T-p. T-end (T-e) was determined by the tangent of the T wave. (D) The region of scar in infarcted hearts predominantly involved the LVA apex, anterior, and anterolateral walls. (E) 56-electrode sock is placed over the ventricles to obtain unipolar electrograms that are used for ARI analysis. (F) The template for the polar maps used to display regional ARIs from the sock electrodes is shown. MCG = middle cervical ganglion, Ant = anterior, Lat = lateral, Post = posterior, RVOT = right ventricular outflow tract. Figure 2. A. Timeline of Experimental Protocol in Normal Animals HEX CSD 30min 30min Isola7onof BMCGS MCGand orBSGS* Stellate (30sec) Ganglia 30min 30min BSGS BMCGS orBSGS* orBMCGS* (60sec) (30sec) BMCGS orBSGS* (60sec) B 400 350 300 350 300 P < 0.05 14 Change in ARI (%) P < 0.05 BL 1 BSGS 2 200 BL 1 BMCGS 2 VNS (20sec) RV LV 10 Apex BL 8 6 LAD RV 2 200 10min LAD 12 4 250 250 P = 0.4 30min BMCGS (30sec) C Global ARI (ms) Global ARI (ms) 400 30min 0 LV BSGS 1 BMCGS 2 Apex ARI (ms) 376 364 352 340 327 315 303 291 279 266 254 242 BSGS VNS BMCGS (20sec) (30sec) LAD RV LV Apex BL LAD RV LV Apex BMCGS Figure 2. Experimental protocol in normal hearts and effect of bilateral stellate ganglia compared to bilateral MCG stimulation (A) Timeline of the experimental protocol in 6 normal animals. (B) In all normal animals, bilateral MCG stimulation had a similar effect on ARI compared to bilateral stellate ganglia stimulation (C) Polar maps from a normal animal show the effects of MCG and stellate ganglia stimulation on ARI. Blue dashed lines indicate course of left anterior descending coronary artery. Dashed circle indicates regions of scar on polar maps. BL = baseline, BSGS = bilateral stellate ganglia stimulation, BMCGS = bilateral middle cervical ganglia stimulation. *BMCGS or BSGS was performed in a random order, LAD = left anterior descending coronary artery. Figure 3. Percent Change in ARI (%) BSGS A BMCGS Pre-CSD 0 -5 -10 -15 -20 -25 5sec 10sec 15sec 20sec 30sec 45sec 60sec B Change in ARI (%) 30 BSGS 25 BMCGS Pre-CSD 20 15 10 5 0 Apex Ant Lat Post Post Lat LV Ant RVOT RV RA Pacing C Global ARI (ms) 260 P < 0.05 Apical Pacing P = 0.6 250 240 P = 0.5 P < 0.05 230 220 210 200 BL Pre-CSD BMCGS Pre-CSD Pre-CSD BL Post-CSD BMCGS Post-CSD Post-CSD Figure 3. (A) The time course of ARI shortening with bilateral stellate ganglia stimulation was similar to bilateral MCG stimulation (n = 6). (B) There were no significant regional differences between bilateral stellate and bilateral MCG stimulation in normal hearts (P values > 0.05 for all regions, n = 6). (c) Apical ventricular pacing pre-CSD demonstrated shorter global ARIs than right atrial (RA) pacing in normal animals (n = 6) at the same cycle lengths. However, after CSD, there was no significant difference in ARI between ventricular and atrial pacing, suggesting that apical pacing may cause reflex sympathetic activation that is prevented by CSD. Ant = anterior, Lat = lateral, Post = posterior wall, RV = right ventricle, LV =left ventricle, RA = right atrium. BSGS = bilateral stellate ganglia stimulation. BMCGS = bilateral middle cervical ganglia stimulation. Figure 4. A B 300 250 200 Pre1 CSD Post2 CSD ARI (ms) Pre-CSD BMCGS Percent Change in ARI (%) Global ARI (ms) 350 400 Global ARI (ms) P = 0.2 400 Post-CSD 355 349 342 336 329 323 316 310 303 297 291 284 P < 0.03 350 300 250 200 BL 1 25 20 P = 0.6 15 10 5 0 1 PreCSD BMCGS 2 Post-CSD 2 PostCSD BL BMCGS Post-CSD 350 * † * 300 * * * * * 250 200 Apex Ant Lat Post Post Lat LV Ant RVOT 0 Apex Ant Lat Post Post Lat Ant RVOT RV LV 700 700 600 600 400 300 80 P<0.05 Tp-Te Interval (ms) 800 P<0.05 500 Normal Hearts Post-CSD - Normal Hearts DOR (ms2) DOR (ms2) 10 E Pre-CSD - Normal Hearts 200 BMCGS Post-CSD 20 RV D 800 BMCGS Pre-CSD 30 500 400 P=0.1 P=0.1 60 40 20 300 1 BL 2 BMCGS 200 BL 1 BMCGS 2 Percent Change in Tp-Te (%) ARI (ms) 400 Percent change in ARI (%) C P=0.9 80 60 40 20 0 0 BL BMCGS Pre-CSD BL BMCGS Post-CSD PreCSD PostCSD Figure 4. Effects of CSD in normal animals. (A) There was no difference in global ARI in normal heart before compared to after CSD. (B) Bilateral MCG stimulation after CSD had similar effects on global ARI compared to before CSD, with significant ARI shortening observed. (C) There were no differences in the regional effects of MCG stimulation after CSD compared to before CSD. (D) Before CSD, bilateral MCG stimulation increased dispersion of repolarization in normal hearts, and this effect was not modified by CSD. (E) Bilateral MCG stimulation in normal hearts also increased TpTe interval, with no differences observed in this parameter before compared to after CSD in normal hearts. Figure 5. A B Right VNS MCG Stimulation P < 0.05 350 300 250 400 P = 0.6 P < 0.05 Global ARI (ms) Global ARI (ms) 400 350 300 250 BLS%m Pre-Hex BLS%m Post-Hex Pre-HexPostHex C P < 0.05 BLS%m Pre-Hex BLS%m Post-Hex Pre-HexPostHex Percent Change in ARI (%) P = 0.3 12 10 8 P < 0.05 6 4 2 0 BLS%m RVNS RightVNS BLS%m BMCGS MCGS%mula%on Figure 5. Effects of hexamethonium on MCG stimulation. (A) The increase in global ARI during right vagal nerve stimulation was no longer observed after the infusion of hexamethonium in normal animals (n = 6). (B) Global ARI during bilateral MCG stimulation significantly decreased even after administration of hexamethonium in all normal hearts (n = 6). (C) Percent change in global ARI during right vagal nerve stimulation and bilateral MCGS stimulation showed that hexamethonium significantly blocked nicotinic receptors, but did not affect the response to bilateral MCG stimulation. Hex = hexamethonium, VNS = vagal nerve stimulation. Figure 6. A Timeline of Creation of Myocardial Infarct and Mapping Experiments in Infarcted Animals CSD 4-6weeks 30min 30min 30min Voltage VT/VF BMCGS BMCGS Mapping Induc+on orBSGS orBSGS* Myocardial Infarc+on C B 350 300 400 424 402 379 357 334 312 289 267 244 222 200 177 BL 15 300 LAD 10 250 5 200 200 0 2 BSGS LAD Apex 20 250 1 BL VT/VF BMCGS Induc+on RV 25 350 30min LV Change in ARI (%) Global ARI (ms) 400 P = 0.02 P < 0.01 P < 0.01 Global ARI (ms) 30min BL 1 BMCGS 2 RV LV BSGS 1 BMCGS 2 Apex LAD RV LV Apex BL LAD RV LV ARI (ms) BSGS Apex BMCGS Figure 6. Experimental protocol in infarcted hearts and effect of bilateral stellate ganglia compared to bilateral MCG stimulation (A) Timeline the experimental protocol in 12 infarcted animals. (B) In all infarcted animals, bilateral stellate ganglia stimulation significantly decreased ARI as did bilateral MCG stimulation. Bilateral MCG stimulation, however, caused greater ARI shortening than bilateral stellate stimulation (C) Polar maps from an infarcted animal show the effects of MCG and stellate ganglia stimulation on ARI. Black dashed lines indicate region of the infarct on the polar maps. Blue dashed lines indicate course of left anterior descending coronary artery. Dashed circle indicates regions of scar on polar maps. BL = baseline, BSGS = bilateral stellate ganglia stimulation, BMCGS = bilateral middle cervical ganglia stimulation. *BMCGS or BSGS was performed in a random order, LAD = left anterior descending coronary artery. Figure 7. A LAD Global ARI (ms) 400 350 300 250 Pre1 CSD Post2 CSD 444 432 421 409 397 385 374 362 350 339 327 315 ARI (ms) P < 0.05 RV LV Pre-CSD LAD RV VT/VF Inducibility (%) P < 0.05 0.05 B 60 40 20 0 LV pre-CSD post-CSD Pre-CSD Post-CSD Post-CSD C Post-CSD Pre-CSD 500ms 500ms III II V1 V1 V3 V3 RVD RVD RVP RVP Figure 7. Effects of CSD and VT Inducibility pre and post CSD (A) Epicardial global ARI from infarcted animals significantly increased with CSD (n = 12). (B) Inducibility of VT/VF was reduced after CSD in infarcted animals (n = 18, 12 were inducible at baseline. Of this 12, only 6 were inducible after CSD). (C) An example of VT induction with ventricular extra-stimulus pacing is shown. VT/VF was induced before CSD with double extra-stimuli at cycle lengths of 600/320/200 ms. After CSD, ERP was reached at an interval of 600/340 ms with double extra-stimuli. Therefore, the S2 stimulus was increased by 20 ms to 600/360 ms (to allow for consistent capture) and S3 was added and the interval reduced by 10 ms. The animal reached ERP at 270 ms with triple extrastimuli (S3). Therefore, the S3 coupling interval was increased to 290 ms (to allow for consistent capture), and S4 was reduced by 10 ms starting at 600/360/290/400 ms. Despite reducing S4 extra-stimulus (triple extra-stimulus testing) to a coupling interval of 200 ms, no ventricular arrhythmias could be induced. Black dashed lines indicate region of the infarct on the polar maps. Figure8. A B INFARCTED HEARTS Post-CSD BMCGS P < 0.01 Percent Change in ARI (%) P = 0.4 Global ARI (ms) 400 350 300 250 200 BL 1 BMCGS 2 25 20 15 10 5 0 1 PreCSD 2 PostCSD ARI (ms) C LAD LAD RV RV LV LV Apex Apex Post-CSD BMCGS Post-CSD D 1200 1000 1000 DOR (ms2) 1200 800 600 400 200 80 1400 P=0.03 2 BMCGS 800 600 200 P=0.02 P=0.4 400 1 BL P=0.01 BL 1 BMCGS 2 60 40 20 Percent Change in Tp-Te (%) 1400 Infarcted Hearts Post-CSD – Infarcted Hearts Tp-Te Interval (ms) Pre-CSD- Infarcted Hearts DOR (ms2) 371 353 334 316 298 279 261 243 224 206 187 169 P<0.05 80 60 40 20 0 0 BL BMCGS Pre-CSD BL BMCGS Post-CSD PreCSD PostCSD Figure 8. MCG stimulation and its effects before and after CSD on infarcted hearts. (A) Global ARI at baseline and during bilateral MCG Stimulation after CSD and percentage change in ARI pre- vs. post-CSD during BMCGS in infarcted hearts (n = 12). MCG stimulation decreased ARI despite CSD. (B) Examples of polar maps at baseline and during MCG stimulation after CSD in infarcted hearts. Dashed-black lines indicates course of LAD. Dashed blue circles indicates region of scar. (C) Although MCG stimulation significantly increased DOR before CSD, this effect was mitigated after CSD in infarcted hearts (n = 12). (D) The prolongation in Tp-Te interval during MCG stimulation was reduced by CSD in infarcted hearts. BL = baseline, BMCGS = bilateral MCG stimulation, LAD = left anterior descending coronary artery. Figure 9. A AT (ms) 78 72 66 61 55 49 43 37 32 26 20 14 Pre-CSD Post-CSD B 73 67 61 55 48 42 36 30 24 17 11 5 AT (ms) BL Pre-CSD BMCGS Pre-CSD BL Post-CSD BMCGS Post-CSD Figure 9. Modulation of propagation and activation time by CSD. (A) Polar maps of activation time/sequence obtained during scar/apical pacing in this infarct animal demonstrated a localized region at the septal border zone of the infarct that was activated late as compared to it surrounding area. This area of functional block (localized regions of late activation, delineated as “II”) pre-CSD was no longer observed after CSD, and the entire area was activated more homogenously. (B) Polar maps of activation time/sequence during scar pacing at baseline and during bilateral MCG stimulation before and after CSD in a different infarcted animal are shown. During MCG stimulation, two localized regions of late activation are seen at the border zone of the infarct, creating a potential isthmus or area of slow conduction that could serve as the substrate for a reentrant circuits. Both of these regions of late myocardial activation are no longer observed during MCG stimulation after CSD. After CSD, the entire region is more uniformly activated. BL = baseline, BMCGS = bilateral middle cervical ganglia stimulation. Blackdashedlinesindicateregionoftheinfarctonthepolarmaps. Figure 9 Figure 10. Brainstem MCG Stellate DRG Eff DH Eff Aff IML Aff DH IML HEART DH IML CSD Pre-ganglionic efferent sympathetic fibers Post-ganglionic efferent sympathetic fibers Afferent sympathetic fibers Intraganglionic Information Processing Sympathetic Chain Spinal Cord Figure 10. Efferent and afferent cardiac sympathetic pathways. Afferent fibers from the myocardium that traverse through the MCG pass through the stellate ganglia before reaching the spinal cord, and some of these pathway are interrupted by CSD. In addition, pre-ganglionic efferent fibers that pass from the spinal cord through the stellate ganglia and to the MCG and any post-ganglionic fibers that arise from the stellate ganglia and innervate the myocardium are also interrupted. However, efferent postganglionic fibers from MCG neurons to the myocardium remain intact despite CSD. Aff = afferent neurons, Eff = efferent neurons, DRG = dorsal root ganglion, IML = intermediolateral nucleus, DH = dorsal horn of the spinal cord. Table 1 Normal Animals (N = 6) BL (Pre-BSGS) BL BMCGS BSGS (PreMCGS) (Pre-CSD) BL BMCGS Pre-CSD Post-CSD (Pre-MCGS Post-CSD) (Post-CSD) HR (bpm) 84±17 98±13* 89±14 103±10* 86±12 89±8 90±12 104±10* Max dP/dt 1178±166 2391±724* 1086±116 2364±531* 1032±157 1087±296 1007±228 2232±467* Min dP/dt -1126±317 -1149±462 -1172±337 -1174±302 -1121±353 -1094±286 -984±266 -1149±382 LVESP 103±20 147±23* 99.5±21 134±24*§ 97.5±22 94±16 87±13 125±20*¥ Mean±SD reported. HR = heart rate, LVESP = left ventricular end-systolic pressure, BSGS = bilateral stellate ganglia stimulation, BMCGS = bilateral middle cervical ganglia stimulation. Values represent percent change in parameters from pre-stimulation value. *P value < 0.05 for percentage change from baseline (pre-stimulation).¥ P= 0.08 for percentage increase in LVESP with BMCGS pre vs. post CSD. § P = 0.02 for comparison of % change in LVESP for BSGS vs. BMCGS. Pre-CSD represents hemodynamic values immediately before removal of the stellate ganglia. Post-CSD values represent hemodynamic parameters after a 30 minute period of stabilization after removal of stellate ganglia Table 2 Baseline (ng/ml) BSGS (ng/ml) Normal pigs (CS) 0.38±0.09 8.70±2.28 Normal pigs (IVC) 0.58±0.22 Infarct pigs (CS) 1.35±0.41 * 1.52±0.29 * 7.47±1.48 * Infarct pigs (IVC) 1.97±0.54 2.53±0.28 Baseline (ng/ml) BMCGS (ng/ml) 0.54±0.16 13.73±4.47 1.79±0.51 * 1.61±0.29 * 7.83±2.16 * 1.89±0.46 3.78±0.95 0.55±0.07 CS = coronary sinus, IVC = inferior vena cava, BSGS = bilateral stellate ganglia stimulation, BMCGS = bilateral middle cervical ganglia stimulation, *P<0.05 compared to baseline. * Table 3 Infarcted Animals (N = 12) BL (Pre-BSGS) BL BMCGS BSGS (PreBMCGS) (Pre-CSD) BL BMCGS Pre-CSD Post-CSD (Pre-MCGS Post CSD) (Post-CSD) HR 85±10.7 94±21* 84±11.8 112±22*§ 83±13 84±13 84±13 114±24* Max dP/dt 1246±390 2127±315* 1123±332 2271±9678*¥ 984±349 880±337 878±338 1797±937* Min dP/dt -1280±582 -1157±542 -1179±537 -1093±635 -1090±571 -1025±634 -1027±630 -958±574 LVESP 90±20 123±28 86±21 99±21§ 78±25 69±22¶ 69±22 86±26 Mean±SD reported. HR = heart rate, LVESP = left ventricular end-systolic pressure, BSGS = bilateral stellate ganglia stimulation, BMCGS = bilateral middle cervical ganglia stimulation. Values represent percent change in parameters from pre-stimulation value. *P value < 0.05 for percentage change from baseline (pre-stimulation).¥ P= 0.06 for percentage increase in dP/dt max with BMCGS compared to BSGS. §P < 0.01 for comparison of BMCGS vs. BSGS. ¶ P=0.02 for comparison of pre-CSD vs. post CSD. Pre-CSD represents hemodynamic values immediately before removal of the stellate ganglia. Post-CSD values represent hemodynamic parameters after a 30 minute period of stabilization after removal of stellate ganglia