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.


METHODS
Action potential (AP) properties and pacemaker current (I(f)) 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 I(f) 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.
Boink
Gerard J J
GJ
Heart Failure Research Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Verkerk
Arie O
AO
van Amersfoorth
Shirley C M
SC
Tasseron
Sara J
SJ
van der Rijt
Roos
R
Bakker
Diane
D
Linnenbank
André C
AC
van der Meulen
Jan
J
de Bakker
Jacques M T
JM
Seppen
Jurgen
J
Tan
Hanno L
HL
D000200:Action Potentials; D000818:Animals; D001683:Biological Clocks; D002460:Cell Line; D054815:Cyclic Nucleotide-Gated Cation Channels / Q000235:genetics / Q000502:physiology*; D004594:Electrophysiology; D005822:Genetic Vectors; D006801:Humans; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D016086:Lentivirus / Q000235:genetics; D009124:Muscle Proteins / Q000235:genetics / Q000502:physiology*; D009200:Myocardial Contraction; D032383:Myocytes, Cardiac / Q000378:metabolism* / Q000502:physiology; D015221:Potassium Channels; D051381:Rats; D023822:Tissue Engineering / Q000379:methods*; D014161:Transduction, Genetic / Q000379:methods*
D054815:Cyclic Nucleotide-Gated Cation Channels; C119587:HCN4 protein, human; D064428:Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels; D009124:Muscle Proteins; D015221:Potassium Channels