Engineering physiologically controlled pacemaker cells with lentiviral HCN4 gene transfer

Background Research on biological pacemakers for the heart has so far mainly focused on short-term gene and cell therapies. To develop a clinically relevant biological pacemaker, long-term function and incorporation of autonomic modulation are crucial. Lentiviral vectors can mediate long-term gene expression, while isoform 4 of the Hyperpolarization-activated Cyclic Nucleotide-gated channel (encoded by HCN4) contributes to pacemaker function and responds maximally to cAMP, the second messenger in autonomic modulation. Material and Methods Action potential (AP) properties and pacemaker current (If) were studied in single neonatal rat ventricular myocytes that overexpressed HCN4 after lentiviral gene transduction. Autonomic responsiveness and cycle length stability were studied using extracellular electrograms of confluent cultured monolayers. Results Perforated patch-clamp experiments demonstrated that HCN4-transduced single cardiac myocytes exhibited a 10-fold higher If than non-transduced single myocytes, along with slow diastolic depolarization, comparable to pacemaker cells of the sinoatrial node, the dominant native pacemaker. HCN4-transduced monolayers exhibited a 47% increase in beating rate, compared to controls. Upon addition of DBcAMP, HCN4-transduced monolayers had beating rates which were 54% faster than baseline and significantly more regular than controls. Conclusions Lentiviral vectors efficiently transduce cardiac myocytes and mediate functional gene expression. Because HCN4-transduced myocytes demonstrate an increase in spontaneous beating rate and responsiveness to autonomic modulation, this approach may be useful to create a biological pacemaker. Copyright (c) 2008 John Wiley & Sons, Ltd.