The purpose 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 is crucial. Lentiviral vectors can mediate long-term gene expression. The purpose of the present study was to determine whether canine mesenchymal stem cells (cMSCs) provide impulse initiation over 2 weeks without the use of immunosuppression.
Methods pacemaker current (If) were studied in cMSCs that overexpressed isoform 4 of the Hyperpolarization-activated Cyclic Nucleotide-gated channel (encoded by HCN4) after lentiviral gene transduction. HCN4 protein expression was confirmed by cellular immunofluorescence staining and western blotting. 3×106 cMSCs transfected with either control plasmid (EGFP) or HCN4 gene construct were injected subepicardially in the canine right ventricular wall in situ.
Results Perforated patch-clamp experiments demonstrated that HCN4- transduced single canine mesenchymal stem cells exhibited a fivefold higher if than non-transduced single cMSCs, expressed high levels of Cs-sensitive current, confirming the expressed current as If-like. After cMSCs injected 2 weeks, during sinus arrest, all control (EGFP) hearts had spontaneous atrioventricular node rhythms; In the EGFP-mHCN4 group, 4 of 6 animals developed spontaneous ventricular rhythms; 2 of 6 animals developed spontaneous atrioventricular node rhythms. Moreover, immunostaining of the injected regions demonstrated the presence of cMSCs forming gap junctions with adjacent myocytes and manifested no cellular or humoural rejection at that time.
Conclusion These studies demonstrate that genetically modified cMSCs can express functional HCN4 channels in vitro and in vivo, mimicking overexpression of HCN4 genes in cardiac myocytes, and represent a novel delivery system for pacemaker genes into the heart.
- Biological pacemakers
- HCN4 gene
- lentiviral vectors