Gross ER Page 1

Supplemental Material:

Nociceptive-induced Myocardial Remote Conditioning Is Mediated By

Neuronal Gamma Protein Kinase C

Eric R. Gross, MD, PhD1, Anna K. Hsu, BS2, Travis J. Urban, BS1,3,Daria Mochly-Rosen, PhD3, Garrett J. Gross, PhD2

1 Department of Anesthesiology, Stanford University, School of Medicine, Stanford, CA 94305

2 Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, WI 53226

3 Department of Chemical and Systems Biology, Stanford University, School of Medicine, Stanford, CA 94305-5174

Email:

Supplemental Detailed Methods:

In vivo myocardial ischemia-reperfusion. Eight to ten week old male Sprague-Dawley rats (Harlan) were anesthetized with intra-peritoneal injection of thiobutabarbital sodium (Inactin, 100mg/kg IP, Sigma, St. Louis, MO). A tracheotomy was performed, with the lungs mechanically ventilated at 38–45 breaths per minute using an air-oxygen mixture (model 683, Harvard Instruments, Holliston, MA). The respiratory rate and tidal volume were adjusted on the basis of arterial pH, PCO2, and PO2 measurements obtained during baseline and 1 hour after myocardial reperfusion. Body temperature was measured rectally and maintained between 36.5°C and 37.5°C with a heating pad and surgical lamps. The left jugular vein was cannulated for negative staining of the area at risk (AAR). The left carotid artery was used to measure arterial blood pressure and heart rate via a PE23pressure transducer (Gould, Cleveland, OH) connected to a polygraph. Blood pressure and heart rate were monitored continuously throughout the experiment. A left-sided anterior thoracotomy was performed at the fifth intercostal space, the pericardium excised, and a silk ligature placed around the proximal left anterior descending coronary artery just distal to the left atrial appendage.

Heart rate, mean arterial blood pressure, and rate pressure product were measured at baseline, after drug administration, after 15 minutes of myocardial ischemia, and 2 hours after myocardial reperfusion. Ischemia was induced by tightening the ligature to occlude the left anterior descending coronary artery. Coronary occlusion was confirmed by the presence of dyskinesia and cyanosis distal to the occluded coronary artery. After 30 minutes, the ischemic myocardium was reperfused by loosening the ligature. After 2 hours of reperfusion, the left anterior descending coronary artery was again occluded and patent blue dye was injected into the jugular vein, staining blue the myocardial tissue outside the AAR. The heart was then excised, dissected into 4 slices and separated into the blue-stained normal zone and the AAR for injury. The myocardial pieces were then incubated in 1% 2,3,5-triphenyltetrazolium chloride for 15 min at 37°C staining viable tissue red, whereas nonviable infarct tissue failed to stain and remained white. The heart was then placed overnight in formaldehyde. The next day the infarct tissue was dissected from the AAR under a microscope. Infarct size and the AAR were assessed gravimetrically and expressed as a percent.

Supplemental Table 1.Hemodynamic results for in vivo studies. Hemodynamic results for in vivo experimental groups measured at baseline, 10 minutes after final drug administration or abdominal incision, at 15 minutes of ischemia and at 2 hours of reperfusion. No significant changes were noted between groups for each intervention phase. Abbreviations for the table are as follows: HR = heart rate (beats/min), MAP = mean arterial pressure (mmHg), RPP = rate pressure product (mmHg/1000).

Supplemental Fig.1.Area at risk per left ventricle percentage(AAR/LV%) for each experimental group. No significant differences were seen between groups. Panels a, b and c correspond to infarct size data presented in Figures 2, 3 and 4, respectively.

Supplemental Table 2.Hemodynamic results for ex vivo studies. Hemodynamic results for isolated heart experimental groups measured at baseline, 10 minutes after final drug administration, and at 15 and 90 minutes of reperfusion. Abbreviations for the table are as follows: HR = heart rate (beats/min), EDP = left ventricular end diastolic pressure (mmHg), LVDP = left ventricular developed pressure (mmHg).EDP at 15 minutes and LVDP after 90 minutes reperfusion were significantly different for the PKC activator *P<0.05.

Supplemental Table 3.Hemodynamic results for in vivo remote conditioning studies with treatments given during or after reperfusion. Hemodynamic results for experimental groups measured at baseline, at 15 minutes of ischemia and at 2 hours of reperfusion. No significant changes were noted between groups for each intervention phase. Abbreviations for the table are as follows: HR = heart rate (beats/min), MAP = mean arterial pressure (mmHg), RPP = rate pressure product (mmHg/1000).

Supplemental Fig. 2. Area at risk per left ventricle for additional experimental groups. No significant differences were seen for these groups when compared to control and TAT groups. However, the PKC or PKC activator groups did have significantly larger area at risk per left ventricle (AAR/LV%) sizes when compared to the T10 and T10 + ↑PKC groups. This larger AAR/LV% for the PKC or PKC activator groups likely caused an underestimation of the myocardial salvage effects of these agents, since a larger AAR/LV% has traditionally shown to produce a larger infarct size.