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Abstract
011 Stabilisation of SR leak in heart failure after SERCA2A gene therapy
  1. A R Lyon1,
  2. M L Bannister1,
  3. E Coward1,
  4. S S Dubb1,
  5. E Garcia1,
  6. E Kohlbrenner2,
  7. R J Hajjar2,
  8. S E Harding1,
  9. K M Macleod1
  1. 1Imperial College, London, UK
  2. 2Mount Sinai School of Medicine, New York, US

Abstract

Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure, and is under evaluation in a clinical trial. Restoration of sarcoplasmic reticulum (SR) Ca2+ levels by SERCA2a gene therapy may alter SR Ca2+ leak in the failing heart. A critical question is whether SERCA2a predisposes to increased SR leak and cellular triggered activity by restoring SR Ca2+ levels?

Methods Cardiomyocytes were studied isolated from rats with heart failure 4–6 weeks post in vivo AAV9SERCA (2×1011 drp) gene transfer (HF+S). Cells from untreated failing (HF) and non failing (NF) hearts served as controls. Spontaneous Ca2+ spark generation was measured in fluo4-loaded cells by scanning confocal microscopy during a quiescent 30 s period after baseline stimulation. Field stimulated calcium transients were measured at 0.5 Hz, followed by caffeine application to measure SR Ca2+ load. The ratio of sparks:SR load and Leak Index reflecting total SR Ca2+ leak (spark frequency × amplitude × width × duration) were calculated. Baseline and isoproterenol (ISO)-induced triggered activity were measured in parallel cell studies, and ryanodine receptor (RyR) phosphorylation was measured.

Results SERCA2a gene transfer normalised SR Ca2+ load of failing myocytes (HF+S 5.86±0.64 vs HF 4.24±0.32 p<0.05 (NF 5.34±0.38)) with recovery of Ca2+ transient relaxation kinetics (R50 (ms): HF+S 174±14, HF 292±42, NF 179±23 p<0.01). Spark frequency was unchanged between HF+S (2.9±0.4) and HF (2.5±0.4), with both significantly higher than NF (1.2±0.2 p<0.01). Interestingly spark amplitude was significantly reduced in HF+S compared with HF, with no change in spark decay kinetics, leading to a significant reduction in total SR leak. Leak index: NF234±76, HF 1630±358, HF+S 683±192 (p<0.05). Total RyR was reduced by 20% in failing hearts, with a significant increase in the proportion of RyR-P2809 and RyR-P2815 compared to non-failing controls (p<0.05). These RyR-P and total RyR changes were reversed to normal after SERCA2a gene transfer to failing hearts. Despite increased SR content and unaltered spark frequency, myocytes from HF+S hearts were more stable in vitro, with significantly fewer basal and ISO-induced aftercontractions compared to HF (p<0.05).

Conclusion SERCA2a gene therapy reduced total SR Ca2+ leak of failing myocytes whilst concurrently increasing SR Ca2+ load to normal levels. Leak reduction was dependent upon altered spark characteristics as spontaneous spark frequency was unchanged, and reduced RyR phosphorylation supports resetting of SR Ca2+ leak threshold after SERCA2a gene transfer. SERCA2a gene therapy reduced ISO-induced triggered activity in vitro and ISO-induced arrhythmias in vivo, and may represent a novel antiarrhythmic strategy in heart failure.

  • SERCA2a gene therapy
  • SR calcium leak
  • arrhythmias

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