Organ explant culture of neonatal rat ventricles: a new model to study gene and cell therapy.

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

Organ explant cultures can be used to study the effects of gene and cell therapy.

<p><b>A,</b> EGFP expression in organ explant cultures transduced with LV-<i>Egfp</i>. <b>B,</b> Percentage of preparations showing spontaneous beating, comparing organ explant cultures alone with organ explants co-cultured with spontaneously beating neonatal rat ventricular cardiomyocytes. <b>C,</b> Isochronal activation map of organ explant culture with spontaneously beating neonatal rat ventricular cardiomyocytes, constructed from the moment of maximal action potential upstroke velocity. Spontaneous beating originates from the monolayer, with capture of organ explant culture. Numbers are activation times in ms.*−* statistically different from each other, p<0.05.</p

Summary data from intracellular recordings.

<p>Values are n (%) or mean ± standard error of the mean. RMP, resting membrane potential; MDP, maximal diastolic potential. *−* or †−† statistically different from each other, p<0.05.</p

Summary data from optical mapping.

<p>Values are n (%) or mean ± standard error of the mean. CL, cycle length; CV, conduction velocity; d(%APA)/dt<i>_max</i>_,, maximal upstroke velocity. *−* or †−† statistically different from each other, p<0.05.</p

Organ explant cultures are (immuno) histologically similar to freshly isolated tissue.

<p>From left to right: organ explant cultures, freshly isolated tissue, and monolayers. <b>A,</b> Hematoxylin-eosin staining. <b>B,</b> Immunohistochemistry for α-actinin (green). <b>C,</b> Immunohistochemistry for connexin-43 (green). <b>D,</b> Immunohistochemistry for α-smooth muscle actin (green). Nuclei are stained with sytox orange (red).</p

Isochronal activation maps.

<p>Isochronal activation maps constructed from the moment of maximal action potential upstroke velocity. Numbers are activation times in ms. <b>A,</b> Organ explant culture. <b>B,</b> Freshly isolated tissue. <b>C,</b> Monolayer.</p

Optical mapping results comparing monolayers cultured in monolayer medium or organ explant medium.

<p><b>A,</b> Percentage of monolayers showing spontaneous beating activity. <b>B,</b> Cycle length of spontaneously beating monolayers (ms; mean ± SEM). <b>C,</b> Conduction velocity (cm/s; mean ± SEM). <b>D,</b> Action potential duration at 20%, 50%, 70% and 90% repolarization (ms; mean ± SEM). <b>E,</b> Percentage of monolayers showing propagated action potentials when stimulated at 500, 400, 300, 200, 150, and 100 ms cycle length.</p

Typical examples from intracellular recordings.

<p>Typical examples of action potentials as measured with intracellular recordings when stimulated at 500 ms cycle length.</p

Cardiomyocyte progenitor cells as a functional gene delivery vehicle for long-term biological pacing

Sustained pacemaker function is a challenge in biological pacemaker engineering. Human cardiomyocyte progenitor cells (CMPCs) have exhibited extended survival in the heart after transplantation. We studied whether lentivirally transduced CMPCs that express the pacemaker current If (encoded by HCN4) can be used as functional gene delivery vehicle in biological pacing. Human CMPCs were isolated from fetal hearts using magnetic beads coated with Sca-1 antibody, cultured in nondifferentiating conditions, and transduced with a green fluorescent protein (GFP)- or HCN4-GFP-expressing lentivirus. A patch-clamp analysis showed a large hyperpolarization-activated, time-dependent inward current (−20 pA/pF at −140 mV, n = 14) with properties typical of If in HCN4-GFP-expressing CMPCs. Gap-junctional coupling between CMPCs and neonatal rat ventricular myocytes (NRVMs) was demonstrated by efficient dye transfer and changes in spontaneous beating activity. In organ explant cultures, the number of preparations showing spontaneous beating activity increased from 6.3% in CMPC/GFP-injected preparations to 68.2% in CMPC/HCN4-GFP-injected preparations (P < 0.05). Furthermore, in CMPC/HCN4-GFP-injected preparations, isoproterenol induced a significant reduction in cycle lengths from 648 ± 169 to 392 ± 71 ms (P < 0.05). In sum, CMPCs expressing HCN4-GFP functionally couple to NRVMs and induce physiologically controlled pacemaker activity and may therefore provide an attractive delivery platform for sustained pacemaker function

Cardiomyocyte progenitor cells as a functional gene delivery vehicle for long-term biological pacing

Sustained pacemaker function is a challenge in biological pacemaker engineering. Human cardiomyocyte progenitor cells (CMPCs) have exhibited extended survival in the heart after transplantation. We studied whether lentivirally transduced CMPCs that express the pacemaker current If (encoded by HCN4) can be used as functional gene delivery vehicle in biological pacing. Human CMPCs were isolated from fetal hearts using magnetic beads coated with Sca-1 antibody, cultured in nondifferentiating conditions, and transduced with a green fluorescent protein (GFP)- or HCN4-GFP-expressing lentivirus. A patch-clamp analysis showed a large hyperpolarization-activated, time-dependent inward current (−20 pA/pF at −140 mV, n = 14) with properties typical of If in HCN4-GFP-expressing CMPCs. Gap-junctional coupling between CMPCs and neonatal rat ventricular myocytes (NRVMs) was demonstrated by efficient dye transfer and changes in spontaneous beating activity. In organ explant cultures, the number of preparations showing spontaneous beating activity increased from 6.3% in CMPC/GFP-injected preparations to 68.2% in CMPC/HCN4-GFP-injected preparations (P < 0.05). Furthermore, in CMPC/HCN4-GFP-injected preparations, isoproterenol induced a significant reduction in cycle lengths from 648 ± 169 to 392 ± 71 ms (P < 0.05). In sum, CMPCs expressing HCN4-GFP functionally couple to NRVMs and induce physiologically controlled pacemaker activity and may therefore provide an attractive delivery platform for sustained pacemaker function