011 Stabilisation of SR leak in heart failure after SERCA2A gene therapy

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) Ca 2+ levels by SERCA2a gene therapy may alter SR Ca 2+ leak in the failing heart. A critical question is whether SERCA2a predisposes to increased SR leak and cellular triggered activity by restoring SR Ca 2+ 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 Ca 2+ 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 Ca 2+ load. The ratio of sparks:SR load and Leak Index reflecting total SR Ca 2+ 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 Ca 2+ load of failing myocytes (HF+S 5.86±0.64 vs HF 4.24±0.32 p 2+ transient relaxation kinetics (R50 (ms): HF+S 174±14, HF 292±42, NF 179±23 p Conclusion SERCA2a gene therapy reduced total SR Ca 2+ leak of failing myocytes whilst concurrently increasing SR Ca 2+ 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 Ca 2+ 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.