Preserving transverse tubule function in adult cardiac myocytes in short-term culture.

Student thesis: Master's ThesisMaster of Science by Research (MScR)

Abstract

T-tubules (TTs) are invaginations of sarcolemma in cardiomyocytes important in excitation contraction coupling. Loss of TTs in short-term culture is a poorly understood phenomenon, insight into which may be valuable for development of future clinical interventions. The hypothesis that junctophilin-2 (JPH2) overexpression in adult rabbit ventricular myocytes (ARVMs) preserves TTs and calcium current (ICa) density in short-term culture was tested.

ARVMs were cultured for four days, and TT loss quantified by fast-Fourier transform analysis (‘TT-power’) of wheatgerm agglutinin (WGA) staining, which showed a threefold-reduction from day 0 at day 4. ICa density was obtained from whole-cell patch clamp recordings. Transduction with Ad-h-JPH2-GFP and Ad-GFP constructs was carried out at day 0 and culture media changed at day 2 of culture.

Over four days in culture ARVMs became more rounded, capacitance decreased
exponentially with a time constant of 1.68 days and, by day 4, internal WGA staining was absent -indicating complete loss of TTs. At culture day 1, ICa density was half that of day 0, yet by day 4 had recovered to ~85 % of day 0. The response to 100 nM isoprenaline was preserved across days in culture. Neither TT-power nor ICa density were significantly different between day 4 ARVMs transduced with either JPH2 or GFP, and the reduction in TT-power between day 0 and day 4 was similar in JPH2-transduced (JPH2+) and non-transduced ARVMs.

Despite GFP-expression indicating successful viral transduction, JPH2-staining intensity was surprisingly low in JPH2+ ARVMs and did not correlate with GFP-intensity. Similar results were obtained from preliminary data from adult rat ventricular myocytes. In conclusion, JPH2-overexpression via Ad-h-JPH2-GFP did not lead to preservation of TTs and ICa density in ARVMs in short-term culture. However, further work is needed to quantify whether a sufficient level of JPH2-overexpression had been achieved.
Date of Award19 Mar 2024
Original languageEnglish
Awarding Institution
  • The University of Bristol
SupervisorAndrew F James (Supervisor) & Stephen C Harmer (Supervisor)

Keywords

  • cell culture
  • cardiac myocytes
  • rabbit
  • rat
  • junctophillin
  • JPH2
  • junctophillin-2
  • t-tubules
  • TT

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