Abstract 3281: Stim1 Modulates Pacemaking and Contractility via Regulation of Calcium Stores in the Embryonic Heart
Introduction: Abnormal intracellular Ca2+ handling plays a fundamental role in cardiac disease. Store-operated Ca2+ entry (SOCE), triggered by ER/SR Ca2+ depletion, maintains intracellular Ca2+ stores. Calcium release is critical for cardiac pacemaking and contractile function, but little is known about the role of SOCE or Stim1 in the heart. We tested the hypothesis that Stim1 knockdown in embryonic zebrafish would reduce cardiac SOCE and cause altered sarcomere development, contractile function, and pacemaking.
Methods: We employed a morpholino antisense oligonucleotide designed to generate a dominant negative Stim1 (ΔERM). Morpholino effect on splicing was confirmed by RT-PCR and cloning of alternatively spliced products. In situ hybridization confirmed cardiac expression of Stim1. We assessed in vivo contractile function using m-mode analysis in 72 hour old embryos. For pharmacologic studies, 48 hour embryos were exposed to varying doses (7.7–770μM) of thapsigargin. Twelve hours later, heart rates were measured for each treatment group (n=20) in both Stim1 knockdown and control embryos. Calcium imaging was performed in isolated hearts using Fura-2 and ratiometric imaging.
Results: Stim1 knockdown caused a >4-fold reduction in SOCE measured in calcium add-back experiments (n=4). Transmission electron micrographs revealed scarce and randomly arrayed sarcomeres in Stim1 knockdown hearts compared to controls (n=3), consistent with myopathy. Stim1 knockdown reduced contractile function (fractional shortening 24%+/−2.5% vs. 35%+/−2% in control, n=5, p<0.05) and reduced heart rate at an early developmental stage (30 hours) (22% reduction, n=30, p<0.01). The effect on heart rate was synergistic with the sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitor thapsigargin.
Conclusion: SOCE exists in the developing zebrafish heart, and targeted dysregulation of Stim1 function reduces cardiac SOCE and decreases cardiac pacemaking and contractile function. We conclude SOCE is critical for normal cardiac function during embryogenesis.