Abstract
Testing cardiac gene and cell therapies in vitro requires a tissue substrate that survives for several days in culture while maintaining its physiological properties. The purpose of this study was to test whether culture of intact cardiac tissue of neonatal rat ventricles (organ explant culture) may be used as a model to study gene and cell therapy. We compared (immuno) histology and electrophysiology of organ explant cultures to both freshly isolated neonatal rat ventricular tissue and monolayers. (Immuno) histologic studies showed that organ explant cultures retained their fiber orientation, and that expression patterns of α-actinin, connexin-43, and α-smooth muscle actin did not change during culture. Intracellular voltage recordings showed that spontaneous beating was rare in organ explant cultures (20%) and freshly isolated tissue (17%), but common (82%) in monolayers. Accordingly, resting membrane potential was -83.9±4.4 mV in organ explant cultures, −80.5±3.5 mV in freshly isolated tissue, and −60.9±4.3 mV in monolayers. Conduction velocity, measured by optical mapping, was 18.2±1.0 cm/s in organ explant cultures, 18.0±1.2 cm/s in freshly isolated tissue, and 24.3±0.7 cm/s in monolayers. We found no differences in action potential duration (APD) between organ explant cultures and freshly isolated tissue, while APD of monolayers was prolonged (APD at 70% repolarization 88.8±7.8, 79.1±2.9, and 134.0±4.5 ms, respectively). Organ explant cultures and freshly isolated tissue could be paced up to frequencies within the normal range for neonatal rat (CL 150 ms), while monolayers could not. Successful lentiviral (LV) transduction was shown via Egfp gene transfer. Co-culture of organ explant cultures with spontaneously beating cardiomyocytes increased the occurrence of spontaneous beating activity of organ explant cultures to 86%. We conclude that organ explant cultures of neonatal rat ventricle are structurally and electrophysiologically similar to freshly isolated tissue and a suitable new model to study the effects of gene and cell therapy.
Highlights
New therapies for various cardiovascular diseases are currently being developed with a focus on gene and cell therapy
We investigated whether organ explant cultures may be useful in evaluating the effects of cell therapy, by testing their capability to couple to other cardiomyocytes
We found the addition of 0.36106 spontaneously active neonatal rat ventricular cardiomyocytes to increase the percentage of spontaneously beating organ explant cultures to 86% (CL 605.8670.8 ms; Figure 6B)
Summary
New therapies for various cardiovascular diseases are currently being developed with a focus on gene and cell therapy. Experimental models to test gene and cell therapy for cardiovascular diseases should replicate the in vivo situation over a period of at least days. This may be achieved via animal studies, these studies have important drawbacks, including their high cost and labor intensity. Neonatal rat ventricular cardiomyocytes cultured in a syncytium (monolayer) show a flat, star-shaped morphology instead of the normal rodlike shape, along with a reduction in peak transient outward current, sodium current, and calcium current [5] These cells display a depolarized resting membrane potential (RMP), a reduced action potential upstroke velocity, and spontaneous beating [6,7]. Since myofibroblast have a more depolarized membrane potential, coupling between myocyte and myofibroblast is believed to induce depolarization in the cardiomyocyte, thereby causing depolarization-induced automaticity [11]
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