Oral Presentation ASSCR, AGCTS, ISCT ANZ and Friends Joint Scientific Conference 2019

Reprogramming Cardiomyocytes into Pacemaker Cells Using Gene Therapy (#8)

Melad Farraha 1 2 , Renuka Rao 2 , Sindhu Igoor 2 , Loan Le 2 , Michael Barry 3 , Christopher Davey 2 4 , Cindy Kok 2 , James Chong 1 2 3 , Eddy Kizana 1 2 3
  1. Sydney Medical School, The University of Sydney, Sydney, NSW, Australia
  2. Centre For Heart Research, The Westmead Institute for Medical Research, Westmead, NSW, Australia
  3. Department of Cardiology, Westmead Hospital, Westmead, NSW, Australia
  4. Physics, The University of Sydney, Sydney, NSW, Australia

Introduction

Electronic pacemakers are currently the only viable option for the treatment of pacemaker conduction issues. They are however limited due to lack of biological control, high cost of revision surgeries and risk of lead and device related complications. We therefore aimed to develop a biological alternative to electronic devices using gene therapy to demonstrate reprogramming of cardiomyocytes into pacemaker cells. This was achieved by overexpression of the transcription factor hTBX18 using recombinant adeno-associated viral vectors.

Methods

Neonatal rat ventricular cardiomyocytes were isolated and transduced with recombinant adeno-associated virus vector 6 encoding either hTBX18 or GFP and maintained for 3 weeks. At the endpoint, mRNA and protein were collected for qPCR and western blot analysis. Immunocytochemistry was used to visualize changes in markers of pacemaker cells. Changes in cell morphology were imaged via microscopy. Voltage sensing vectors (Arclight) were used to characterise functional changes of the cells.

Results

Analysis of cardiomyocytes transduced with hTBX18 showed faithful recapitulation of the distinctive characteristics of pacemaker cells.

Comparison of hTBX18 treatment and GFP control using qPCR, western blot and immunocytochemistry showed changes in markers specific to pacemaker cells: hTBX18 and HCN4 were significantly upregulated. Cx45 was upregulated, though not statistically significant. Cx43 was significantly down regulated and Sarcomeric α-actinin remained unchanged.

Cardiomyocytes transduced with hTBX18 acquired the distinctive tapered morphology of pacemaker cells, as compared to the block like, striated appearance of cardiomyocytes.

Analysis of the action potentials generated via Arclight showed phase 4 depolarisation and decrease in the APD50 of the hTBX18 transduced cells, highlighting a functional change.

Discussion

We have successfully demonstrated that hTBX18 gene transfer to ventricular cardiomyocytes results in morphological, molecular, physiological and functional changes, recapitulating the native pacemaker phenotype. The generation of these reprogrammed pacemaker cells opens up new prospects for biological pacemakers in a clinically translatable setting.

  • Have you presented your abstract at another international meeting?: No