Dissecting the Calcium Transient Refractoriness in Mouse Ventricular Myocytes

The activation cascade of Ca2+ induced Ca2+ release (CICR) to produce a [Ca2+]i transient has been well described; on the other hand, mechanisms responsible for its termination and refractoriness are still a matter of debate. It is known that the ryanodine receptor (RyR) state and sarcoplasmic reticulum Ca2+ content ([Ca2+]SR) are major determinates in the termination and refractoriness of Ca2+ sparks at room temperature. The goal of this work was to evaluate [Ca2+]i transient refractoriness (CaTR) when RyR state and/or [Ca2+]SR was altered by pharmacological means at physiological temperature (36±1oC). CaTR was measured in mouse ventricular myocytes loaded with Fluo-5F and paced at 2Hz (field stimulation or depolarization through patch pipette) followed by an extra-stimuli with a decreased interval (ESI). This enabled us to measure a [Ca2+]i transient restitution curves. The [Ca2+]i transient peak recovered exponentially as ESI increased (τ ∼ 137ms). L-type Ca2+ channel current (ICa) recovery from inactivation was measured (τ ∼ 53ms, Vhold = −80mV) and found to be much faster than CaTR; suggesting a minimal role. Interestingly, decreasing RyR cytosolic Ca2+ sensitivity with tetracaine strongly shifted CaTR curve to the right. While increasing RyR Ca2+ sensitivity with caffeine only moderately shifted the CaTR curve to the left. These results were then compared to [Ca2+]SR measurements under similar conditions and times. The roles of RyR state and SR Ca2+ load in determining CaTR will be discussed. These findings are relevant to an investigation of the mechanisms of Ca2+ signaling instability and SR Ca2+ leak in arrhythmic conditions including those attributable to myocardial infarction, heart failure and inheritable diseases including catecholaminergic polymorphic ventricular tachycardia (CPVT).