title: AAV6-βARKct gene delivery mediated by molecular cardiac surgery with recirculating delivery (MCARD) in sheep results in robust gene expression and increased adrenergic reserve.
abstract: OBJECTIVE
  Genetic modulation of heart function is a novel therapeutic strategy. We investigated the effect of molecular cardiac surgery with recirculating delivery (MCARD)-mediated carboxyl-terminus of the β-adrenergic receptor kinase (βARKct) gene transfer on cardiac mechanoenergetics and β-adrenoreceptor (βAR) signaling.
  
  
  METHODS
  After baseline measurements, sheep underwent MCARD-mediated delivery of 10(14) genome copies of self-complimentary adeno-associated virus (scAAV6)-βARKct. Four and 8 weeks after MCARD, mechanoenergetic studies using magnetic resonance imaging were performed. Tissues were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) and Western blotting. βAR density, cyclic adenosine monophosphate levels, and physiologic parameters were evaluated.
  
  
  RESULTS
  There was a significant increase in dP/dt(max) at 4 weeks: 1384 ± 76 versus 1772 ± 182 mm Hg/s; and the increase persisted at 8 weeks in response to isoproterenol (P < .05). Similarly, the magnitude of dP/dt(min) increased at both 4 weeks and 8 weeks with isoproterenol stimulation (P < .05). At 8 weeks, potential energy was conserved, whereas in controls there was a decrease in potential energy (P < .05) in response to isoproterenol. RT-qPCR confirmed robustness of βARKct expression throughout the left ventricle and undetectable expression in extracardiac tissues. Quantitative Western blot data confirmed higher expression of βARKct in the left ventricle: 0.46 ± 0.05 versus 0.00 in lung and liver (P < .05). Survival was 100% and laboratory parameters of major organ function were within normal limits.
  
  
  CONCLUSIONS
  MCARD-mediated βARKct delivery is safe, results in robust cardiac-specific gene expression, enhances cardiac contractility and lusitropy, increases adrenergic reserve, and improves energy utilization efficiency in a preclinical large animal model.
authors.affiliation: Division of Cardiovascular Surgery, Department of Surgery, the University of Pennsylvania School of Medicine, Philadelphia, PA, USA.
authors.forename: Michael G
authors.initials: MG
authors.lastname: Katz
authors.forename: Anthony S
authors.initials: AS
authors.lastname: Fargnoli
authors.forename: JaBaris D
authors.initials: JD
authors.lastname: Swain
authors.forename: Catherine E
authors.initials: CE
authors.lastname: Tomasulo
authors.forename: Michele
authors.initials: M
authors.lastname: Ciccarelli
authors.forename: Z Maggie
authors.initials: ZM
authors.lastname: Huang
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Rabinowitz
authors.forename: Charles R
authors.initials: CR
authors.lastname: Bridges
mesh_terms: D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western; D006348:Cardiac Surgical Procedures; D000242:Cyclic AMP / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D015150:Echocardiography, Doppler; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D006352:Heart Ventricles / Q000000981:diagnostic imaging / Q000187:drug effects / Q000201:enzymology*; D006439:Hemodynamics; D007545:Isoproterenol / Q000494:pharmacology; D008279:Magnetic Resonance Imaging; D008297:Male; D009200:Myocardial Contraction; D060888:Real-Time Polymerase Chain Reaction; D011943:Receptors, Adrenergic, beta / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D012756:Sheep; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D013997:Time Factors; D017725:Ventricular Pressure; D051552:beta-Adrenergic Receptor Kinases / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D000318:Adrenergic beta-Agonists; D011943:Receptors, Adrenergic, beta; D000242:Cyclic AMP; D051552:beta-Adrenergic Receptor Kinases; D007545:Isoproterenol