Minding the store of Ca2+ during ischaemia/reperfusion.

Cardiac ischaemia puts a tremendous stress on cardiac myocytes, and abrupt reperfusion causes dramatic changes during the first seconds to minutes that profoundly affect cell survival vs. cell death. – 3 Myocyte ionic changes can contribute to these critical effects. During ischaemia, the concentration of intracellular Naþ ([Na]i) rises because of a combination of increased Naþ entry via Naþ/Hþ exchange and tetrodotoxin-sensitive Naþ channels and reduced extrusion via Naþ/Kþ-ATPase (as ATP and phosphocreatine are gradually depleted and DGATP declines). The rise in [Na ]i contributes to cellular Ca2þ gain via shifts in Naþ/Ca2þ exchange, and thus elevation of diastolic [Ca]i. Contractile force and pressure development are abolished in the first minute or so of ischaemia because of rapidly developing acidosis. More gradually, phosphate rises and eventually rigor crossbridges form as [ATP] is dissipated. Reperfusion causes an abrupt normalization of extracellular pH, which drives a rapid rise in [Na]i via Na þ/Hþ exchange. This is thought to drive a rapid further rise in [Ca]i (via Na þ/Ca2þ exchange), which along with pH normalization causes a rise in diastolic force. Spontaneous Ca2þ transients, oscillations, and arrhythmias are also observed upon reperfusion, and agents that inhibit Naþ and Ca2þ loading can improve the long-term recovery after reperfusion. Despite intensive study of ischaemia/reperfusion, remarkably little is known about the dynamic changes in sarcoplasmic reticulum (SR) Ca2þ handling at the critical moment of reperfusion. In this issue, Valverde et al. address this problem with innovative direct measurement of intra-SR free [Ca2þ] ([Ca]SR) and [Ca ]i in intact mouse hearts subjected to brief periods (12 min) of ischaemia and subsequent reperfusion. SR and cytosolic Ca2þ were studied in these intact hearts with the novel and robust pulsed local field fluorescence (PLFF) technique, with SR-selective loading of the low-affinity fluorophore MagFura2 and cytosolic Rhod2, respectively. PLFF resolves a single cell’s fluorescence via photometry through a flexible light guide held in place with a suction pipette. This eliminates motion artifacts (verified with blebbistatin controls) without need for immobilization, which could alter electrophysiology and Ca2þ handling. Pulsed laser excitation (nsec duration) also limits photobleaching, and fast excitationsynchronized photodetection enhances the signal-to-noise ratio. One limitation, as the authors mention, is that PLFF detects fluorescence from only the epicardial surface, whereas reperfusion-related arrhythmias may have complex origins related to transmural heterogeneity. Cytosolic [Ca]i levels during ischaemia and reperfusion changed as expected. During ischaemia, diastolic [Ca]i increased more than systolic, within 30 s, and pacing-induced Ca2þ transients diminished to ,20% of preischemic size within 4 min. Consistent with acidotic inhibition of SR Ca2þ-ATPase and inhibition of SR Ca2þ release, Ca2þ transient rise, and decay rates slowed dramatically. Upon reperfusion, a distinct ‘bump’ in the already-high diastolic [Ca2þ] signal occurred reliably in the first 30 s. Diastolic Ca2þ recovered gradually (4–5 min) whereas Ca2þ transient amplitude and decay rate recovered by 15 min. However, release rise rate never recovered, implying SR uptake function and loading were restored but release was not. SR function was first inferred via cytosolic Ca2þ responses to caffeine. Although this approach is indirect and especially difficult to interpret quantitatively in intact hearts, the data suggested that SR Ca2þ content were reduced at 6 min of reperfusion vs. the initial preischemic level. In isolated cardiomyocytes, SR Ca2þ has been quantified directly with low-affinity Ca2þ indicators, dynamically during pacing-induced twitches and SR Ca2þ depletion induced by caffeine. SR Ca2þ buffering has also been measured. Valverde et al. compared cytosolic and SR Ca2þ time courses in parallel experiments in intact hearts. Before ischaemia, pacing caused SR Ca2þ depletions of 25% of the caffeine-releasable amount. Within 2 min of the start of ischaemia, SR content rose, although release (depletion transients) dwindled to essentially nothing, paralleling cytosolic [Ca2þ] increase, and loss of transiency. The dramatic [Ca]i increase and hypercontracture upon reperfusion has historically been attributed to influx. However, in the present study it was shown that within 30 s of the start of reperfusion, diastolic SR [Ca2þ] decreased suddenly and severely. Twitch SR depletions disappeared along with loss of SR Ca2þ (and/or electrical