Direct measurement of SR release nux by tracking 'Ca²⁺ spikes' in rat cardiac myocytes

1. Ca²⁺ release flux across the sarcoplasmic reticulum (SR) during cardiac excitation-contraction coupling was investigated using a novel fluorescence method. Under whole-cell voltage-clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 mM, 150 nM free Ca²⁺), to minimize the residence time of released Ca²⁺ in the cytoplasm, and a low-affinity, fast Ca²⁺ indicator, Oregon Green 488 BAPTA-5N (OG-5N; 1.0 mM, K(d) ≃31 μM), to optimize the detection of localized high [Ca²⁺] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca²⁺] at high spatial and temporal resolution. 2. Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca²⁺] change is the sum of two terms: one major term proportional to the SR release flux/Ca²⁺ influx, and the other reflecting the running integral of the released Ca²⁺ 3. Indeed, the OG-5N transients in EGTA-containing cells consisted of a prominent spike followed by a small pedestal. The OG-5N spike closely resembled the first derivative (d[Ca²⁺]/dt) of the conventional Ca²⁺ transient (with no EGTA), and mimicked the model derived SR Ca²⁺ release function reported previously. In SR Ca²⁺-depleted cells, the OG-5N transient also closely followed the waveform of L-type Ca²⁺ current (I(Ca)). Using I(Ca) as a known source of Ca²⁺ influx, SR flux can be calibrated in vivo by a linear extrapolation of the I(Ca)-elicited OG-5N signal. 4. The OG-5N image signal was localized to discrete release sites at the Z-line level of sarcomeres, indicating that the local OG-5N spike arises from 'Ca²⁺ spikes' at transverse (T) tubule-SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules). 5. Both peak SR release flux and total amount of released Ca²⁺ exhibited a bell-shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca²⁺ release was most synchronized and produced maximal peak release flux (4.2 mM s^-1) at 0 mV; in contrast, maximal total release occurred at -20 mV (71 versus 61 μM at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca²⁺] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca²⁺ released, but also the synchronization among release sites affects the whole-cell Ca²⁺ transient and the Ca²⁺-myofilament interaction.