In Vivo Electrophysiologic Studies in Endothelial Nitric Oxide Synthase (eNOS)-Decient Mice AMIT RAKHIT, M.D., COLIN T. MAGUIRE, B.S., HIROKO WAKIMOTO, M.D., PH.D., JOSEF GEHRMANN, M.D., GORDON K. LI, M.D.,* RALPH A. KELLY, M.D.,* THOMAS MICHEL, M.D., PH.D.,* and CHARLES I. BERUL, M.D. From the Department of Cardiology, Children’s Hospital, Department of Pediatrics, Harvard Medical School, and the *Department of Cardiology, Brigham & Women’s Hospital, Department of Medicine, Harvard Medical School, Boston, Massachusetts Electrophysiologic Studies in eNOS-Decient Mice. Introduction: Endothelial nitric oxide synthase (eNOS) mediates attenuation of the L-type calcium channel and modulates myocyte contractility. Arrhythmogenic afterdepolarizations are seen in vitro in ouabain-treated isolated myocytes from eNOS- decient mice. The aim of these studies was to characterize the baseline electrophysiologic (EP) phenotype of eNOS-decient mice and their potential susceptibility to cardiac conduction abnormalities and induc- ible arrhythmias. Methods and Results: Surface ECG and in vivo intracardiac EP studies were performed in 27 mice lacking the eNOS gene and 21 wild-type littermate control mice. Baseline studies were performed in 10 eNOS-decient mice and 10 wild-type controls. Subsequently, 17 eNOS-decient mice and 11 wild-type controls were pretreated with digoxin, and ECG and EP testing were repeated. Data analysis revealed no signicant differences in ECG intervals or cardiac conduction parameters, except sinus cycle length was higher in eNOS-decient mice than wild-type mice (P < 0.01). After digoxin pretreatment, 7 of 17 eNOS-decient mice had inducible ventricular tachycardia and 2 others had frequent ventricular pre- mature beats, compared with only 3 of 11 wild-type mice with inducible ventricular tachycardia. In addition, 2 digoxin-treated eNOS-decient mice and 1 wild-type mouse had inducible nonsustained atrial brillation. Conclusion: Mice with a homozygous targeted disruption of the eNOS gene have slower heart rates but no other distinguishable EP characteristics under basal sedated conditions. Partial inhibition of the Na 1 /K 1 ATPase pump with digoxin administration increases ventricular ectopic activity in eNOS 2/ 2 mice, a phenotype analogous to afterdepolarizations seen in vitro in this eNOS-decient mouse model. (J Cardiovasc Electrophysiol, Vol. 12, pp. 1295-1301, November 2001) nitric oxide synthase, arrhythmia, digoxin, animal model Introduction Nitric oxide (NO) has been implicated in affecting a wide range of hemodynamic functions in the mammalian heart, including ischemia-reperfusion injury, 1-5 cardiac mor- phogenesis, 6,7 and heart rate control through autonomic signaling. 8-11 NO is produced in the body by NO synthase, of which there are three major isoforms: neuronal NO synthase (nNOS) and its subtype neuronal NO synthase m (nNOSm); inducible NO synthase (iNOS); and endothelial NO synthase (eNOS). NO modulates the cardiac L-type calcium channel and thus inuences the transsarcoplasmic reticular calcium cur- rent. 12 Utilizing a cyclic adenosine monophosphate (cAMP)-dependent phosphorylation pathway, the calcium current plays a major role in cardiac contractility and is affected by various autocrine controls. Control of these pathways is due in part to the ability of NO to induce soluble guanylyl cyclase to produce cyclic guanosine mono- phosphate (cGMP). 13 In experimental in vitro studies, NO has been shown to modulate cholinergic effects on automa- ticity in isolated myocytes. 14 In vitro studies performed in ouabain-treated isolated myocytes of eNOS-decient mice have shown an increased incidence of arrhythmogenic af- terdepolarizations as compared to wild-type counterparts. No studies describing the electrophysiologic (EP) pheno- type and the susceptibility to inducible arrhythmias have been described in an in vivo model of eNOS-decient mouse. The aim of the present study was to assess the effect of eNOS deciency on in vivo cardiac electrophysiology and the potential vulnerability to inducible or spontaneous arrhythmias. Methods Complete ECG and in vivo EP studies (EPS) were per- formed in 48 mice inbred in the 129/BS background: 27 homozygous eNOS-decient mice (eNOS 2/2 ) and 21 wild- type controls (eNOS 1/1 ). The studies were performed in a prospective, blinded fashion following an in vivo EPS pro- cedure described previously. 15 The animals were obtained from a colony established at the Cardiovascular Division Research Laboratories at Brigham & Women’s Hospital, Boston, MA. All animals were maintained in accordance with the guidelines of the Institutional Animal Care and Use Dr. Berul is supported in part by National Institutes of Health Grants K08-HL03607 and P50-HL61036. Address for correspondence: Charles I. Berul, M.D., Department of Car- diology, Children’s Hospital, Boston, 300 Longwood Avenue, Boston, MA 02115. Fax: 617-739-9058; E-mail: berul@cardio.tch.harvard.edu Manuscript received 28 June 2001; Accepted for publication 25 September 2001. 1295 Reprinted with permission from JOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Volume 12, No. 11, November 2001 Copyright ©2001 by Futura Publishing Company, Inc., Armonk, NY 10504-0418