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 approximately 165% 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 108 active virus particles yielded homogeneous transfection of approximately 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.

Heart on a Plate: Histological and Functional Assessment of Isolated Adult Zebrafish Hearts Maintained in Culture

“…While, such preparations allow for a broad spectrum of key biochemical, morphological and pharmacological studies to be carried out on the intact heart, they have the disadvantage of requiring relatively complex perfusion techniques. Furthermore, mammalian hearts usually cannot survive beyond 24 h ex-vivo [16] although interest has re-emerged in maintaining isolated rat hearts for a number of days as a method of reseeding stem-cell derived cardiomyocytes [17].…”

Engineered Cardiac Organoid Chambers: Toward a Functional Biological Model Ventricle

“…7,8 On the other hand, twodimensional (2D) cell cultures on rigid substrates are much simpler to control and study but are intrinsically nonphysiologic; indeed, three-dimensional (3D) culture systems are providing new biological insights that were not possible using traditional Petri dishes. 9 Isolated organ and tissue preparations have also played an important role in cardiac research; however, viability of 24 h or less for an isolated working heart, 10 or even up to 72 h for isolated cardiac trabeculae in a sterile muscle culture system, 11 may not suffice for the study of growth, remodeling, and repair processes lasting weeks or more.…”

Effects of Nutrient and Hemoglobin Enriched Cell Free Perfusates Upon Ex Vivo Isolated Rat Heart Preparation

“…Our results are analogous to those of other investigators who have shown that substrate enhanced, cell free perfusion is advantageous in preventing injury during long-term ex vivo perfusion. 13,14 Use of HBOC did not improve postischemic cardiac function. In fact, the addition of HBOC to M199 resulted in inferior functional outcome compared with that seen with M199 alone.…”

“…2,[5][6][7][8][9][10][11][12] The use of such additives in ex vivo perfusates has been associated with more physiologically stable preparations and has made possible the perfusion of isolated hearts for prolonged periods. 13,14 However, such perfusates have the disadvantage of generally requiring fresh preparation at the time of each experiment and, in the case of added erythrocytes, requiring a second donor animal as well.…”