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Basic science: Cardiovascular disease basic research
e0030 Mesenchymal stem cells transfected with HCN4 genes by LentiV to create cardiac pacemakers
  1. Zhou Yafeng1,
  2. Yang Xiangjun2
  1. 1Department of Cardiology, The First Affiliated Hospital of Soochow University
  2. 2Department of Cardiology

Abstract

Objective The study was to test proof-of-principle if genetically engineered mesenchymal stem cells (MSCs) transfected with HCN4 genes can be modified to be cardiac pacemaker cells.

Methods 1. MSCs of rabbit were isolated from the posterior iliac crest of rabbit and were used from passages 2 to 4. 2. The self-inactivating HIV1-based lentiviral vector (LentiV) was used as transgene delivery, which was constructed with plasmid hHCN4/pcDNA3. 3. Total RNA was extracted from control MSCs and those transfected with hHCN4, and RT-PCR was performed. 4. Membrane proteins were extracted from control MSCs and those transfected with hHCN4. Western blot analysis was performed. 5. Whole-cell patch clamp was used to study membrane currents. After the If was recorded, cells were superfused with extracellular solution containing 4 mM caesium chloride and the currents were measured accordingly. 6. MSCs transfected with either GFP alone or GFP-hHCN4 were cocultured with neonatal rabbit ventricular myocytes. The coculture beating rate of cardiac myocytes was measured after 3 days of coculture.

Results 1. In addition to expressing characteristic hHCN4 protein, mHCN4-transfected hMSCs also express an anticipated high level of hHCN4 gene by RT-PCR and Western blot analysis. 2. If was elicited using hyperpolarizing steps in 10-mV increments from −40 mV to −140 mV, and it was significantly inhibited by 4 mM caesium chloride. 3. The coculture beating rate of cardiac myocytes was 87±11 bpm when MSCs were transfected with control plasmid (expressing only GFP) and 149±14 bpm when MSCs were expressing both GFP+hHCN4 (p<0.05).

Conclusion The MSC expressing hHCN4 is a demonstration of feasibility of preparing MSC-based biological pacemaker cells. The study provides a platform for further investigation of the possibility of modifying cardiac excitability with genetically engineered MSCs transfected with HCN4 gene by LentiV. MSCs transfected with hHCN4 genes by LentiV are potentially capable of actively pacing ventricular cardiac myocytes and can potentially be modified to be cardiac pacemaker cells.

  • Mesenchymal stem cells
  • HCN4 genes
  • cardiac pacemakers

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