Direct measurement of SR release flux 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, Kd approximately 31 microM), 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 (ICa). Using ICa as a known source of Ca2+ influx, SR flux can be calibrated in vivo by a linear extrapolation of the ICa-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 microM 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.
Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA.
L S
LS
Song
J S
JS
Sham
M D
MD
Stern
E G
EG
Lakatta
H
H
Cheng
D000465:Algorithms; D000818:Animals; D020013:Calcium Signaling / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004558:Electric Stimulation; D004594:Electrophysiology; D005456:Fluorescent Dyes; D006321:Heart / Q000502:physiology*; D066298:In Vitro Techniques; D008564:Membrane Potentials / Q000502:physiology; D018613:Microscopy, Confocal; D009206:Myocardium / Q000166:cytology*; D018408:Patch-Clamp Techniques; D051381:Rats; D017207:Rats, Sprague-Dawley; D012519:Sarcoplasmic Reticulum / Q000502:physiology*
D002614:Chelating Agents; D005456:Fluorescent Dyes; D004533:Egtazic Acid