24-h Langendorff-perfused neonatal rat heart used to study the impact of adenoviral gene transfer

Abstract

The human genome project has increased the demand for simple experimental systems that allow the impact of gene manipulations to be studied under controlled ex vivo conditions. We hypothesized that, in contrast to adult hearts, neonatal hearts allow long-term perfusion and efficient gene transfer ex vivo. A Langendorff perfusion system was modified to allow perfusion for >24 h with particular emphasis on uncompromised contractile activity, sterility, online measurement of force of contraction, inotropic response to beta-adrenergic stimulation, and efficient gene transfer. The hearts were perfused with serum-free medium (DMEM + medium 199, 4 + 1) supplemented with hydrocortisone, triiodothyronine, ascorbic acid, insulin, pyruvate, L-carnitine, creatine, taurine, L-glutamine, mannitol, and antibiotics recirculating (500 ml/2 hearts) at 1 ml/min. Hearts from 2 day-old rats beat constantly at 135-155 beats/min and developed active force of 1-2 mN. During 24 h of perfusion, twitch tension increased to similar to165% of initial values (P < 0.05), whereas the inotropic response to isoprenaline remained constant. A decrease in total protein content of 10% and histological examination indicated moderate edema, but actin and calsequestrin concentration remained unchanged and perfusion pressure remained constant at 7-11 mmHg. Perfusion with a LacZ-encoding adenovirus at 3 x 10(8) active virus particles yielded homogeneous transfection of &SIM;80% throughout the heart and did not affect heart rate, force of contraction, or response to isoprenaline compared with uninfected controls (n = 7 each). Taken together, the 24-h Langendorff-perfused neonatal rat heart is a relatively simple, inexpensive, and robust new heart model that appears feasible as a test bed for functional genomics.