Changes In Cytosolic Ca2+ Have Greater Effects On SR Ca2+ Leak Than Changes In SR Ca2+

Abstract

Attention has recently focused on preventing arrhythmias by controlling sarcoplasmic reticulum (SR) Ca2+ “leak”. Increased leak in ventricular myocytes is associated with regenerative Ca2+ waves and delayed afterdepolarizations, leading to arrhythmias. Studies that have measured SR Ca2+ leak have not examined changes in [Ca2+]SR independent of changes in [Ca2+]i, causing a degree of uncertainty as to which factor plays a greater role. Our current work explores the possibility that changes in [Ca2+]i have a greater effect on leak than changes in [Ca2+]SR.In quiescent rat ventricular myocytes, we recorded steady-state Ca2+ levels, then blocked the ryanodine receptors (RyRs) with a saturating concentration of tetracaine. Using the calcium indicator fluo-3, we recorded changes in [Ca2+]i using a confocal microscope and analyzed the data using leak calculations that took into account underlying assumptions about cytosolic and SR buffers.When extracellular Ca2+ ([Ca2+]e) was increased from 0.5 mM to 1.0 mM at rest, leak increased 37% (9±1.019 vs. 12.3±1.108 μM/s), [Ca2+]i increased 6.6% (98.9 ±0.09 vs. 105.4±3.2 nM, and [Ca2+]SR decreased 5.2% (489±21 vs. 464±23 μM). We also compared leak in resting cells versus leak in the same cells immediately after pacing for 10 s at 1 Hz. At 1 mM [Ca2+]e, pacing increased leak by 17.9% (12.3±1.108 vs. 14.5±8.8 μM/s), increased [Ca2+]i by 9.4% (105.4±3.2 vs. 115.3±5.7 nM), but increased [Ca2+]SR by only 1.0% (463.7±22.7 vs. 468.2±26.8 μM). Qualitatively similar results were obtained after pacing in 0.5 mmol [Ca2+]e. These results suggest that [Ca]i plays a larger role in determining diastolic SR Ca2+ leak than [Ca]SR.