In cardiac muscle, triggered (by surface Ca2+ influx) or spontaneous opening of multiple type-2 ryanodine receptors (RyR2) at discrete sarcoplasmic reticulum (SR) generates localized elemental Ca2+ release events called Ca2+ sparks. Malfunction of spark local control is known to generate arrhythmias and is implicated heart failure making it a key point for pathological failure and possible therapeutic intervention. Yet, our understanding of spark local control is far from complete. The primary and long standing unknown in spark local control is what turns off the process of Ca2+-induced Ca2+ release (CICR). Intuitively, CICR should be self-reinforcing and operate with "explosive" positive feedback (released Ca2+ triggering further release until the SR Ca2+ store is empty). This does not happen in cells. Instead, CICR is precisely controlled. Candidate cytosolic Ca2+-dependent negative control mechanisms (inactivation &adaptation) have been tested and largely dismissed. It is now clear that the intra-SR (luminal) Ca2+ level determines when CICR initiates &terminates. It is also generally believed that luminal Ca2+ changes are "sensed" by some sensor inside the SR. A popular possibility involves calsequestrin (CSQ) but CICR termination in unstressed CSQ KO animals appears quite normal (16). Thus, the CICR termination mechanism search has generated several inconclusive dead ends. Simply put, we know luminal Ca2+ is critical but not why. We have devised a new approach to address this unknown. It stems from our earlier work (9, 11, 19, 25, 30) and allows us to manipulate single RyR2 Ca2+ flux amplitude in cells independently of resting SR Ca2+ load for the first time. Preliminary results indicate spark initiation and termination track changes in RyR2 Ca2+ flux amplitude, not simply SR Ca2+ load as previously thought. This leads us to test the following hypothesis. Single RyR2 Ca2+ flux, not local luminal Ca2+ acting on an intra-SR based regulatory mechanism, is the primary determinant of spark local control. This flux control involves a critical Ca2+ flux threshold for inter-RyR2 CICR, which determines when sparks can occur and when they terminate. This hypothesis is tested using a combination of single RyR2 channel recording, laser flash photolysis, rapid solution changing, Ca2+ spark detection, permeation/flux modeling and intra-SR Ca2+ measurements.
Our specific aims are to 1) determine how single RyR2 Ca2+ flux amplitude controls spark initiation and 2) define how single RyR2 Ca2+ flux amplitude controls spark termination. Understanding how sparks initiate and terminate is significant because local sparks evoke the global waves that are known to generate arrhythmias. Sparks also contribute to the abnormal SR Ca2+ leak that is associated with heart failure. Here, spark control is explored in innovative ways with a deliberate focus on bridging the in vitro to in situ interpretive divide which has been historically a barrier to progress in our field.
Intracellular calcium release drives a myriad of cellular phenomena. Failure or malfunction of this process has severe ramifications in nearly all types of cells. Here, we define local control mechanisms that govern intracellular calcium release as these are potential sites of pathological failure and/or therapeutic intervention.
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