%0 Journal Article %1 Song_1998_677 %A Song, L. S. %A Sham, J. S. %A Stern, M. D. %A Lakatta, E. G. %A Cheng, H. %D 1998 %J J. Physiol. %K 9769413 Acid, Agents, Agonists, Algorithms, Analysis, Animal Animals, Anoxia, Biological, Blockers, Bone Bones, Calcium Calcium, Cattle, Cell Cells, Channel Channel, Channels, Chelating Clonazepam, Confocal, Cultured, Cytosol, Dietary Dyes, Dynamics, Egtazic Electric Electrophysiology, Exchanger, Feed, Female, Fluorescence, Fluorescent Gov't, Heart, In Indoles, Kinetics, Linear Male, Meat, Membrane Microscopy, Minerals, Mitochondria, Models, Myocardium, Non-U.S. Nonlinear Oxygen, P.H.S., Patch-Clamp Potentials, Proteins, Pyrroles, Rats, Receptor Red, Regression Release Research Reticulum, Rumen, Ruthenium Ryanodine Sarcoplasmic Signaling, Size, Sodium-Calcium Soybeans, Sprague-Dawley, Stimulation, Support, Techniques, U.S. Vitro, Wistar, and %P 677--691 %T Direct measurement of SR release flux by tracking 'Ca$^2+$ spikes' in rat cardiac myocytes. %U http://jp.physoc.org/cgi/content/full/512/3/677 %V 512 ( Pt 3) %X 1. Ca$^2+$ 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 Ca$^2+$), to minimize the residence time of released Ca$^2+$ in the cytoplasm, and a low-affinity, fast Ca$^2+$ indicator, Oregon Green 488 BAPTA-5N (OG-5N; 1.0 mM, Kd approximately 31 microM), to optimize the detection of localized high Ca$^2+$ in release site microdomains. Confocal microscopy was employed to resolve intracellular Ca$^2+$ at high spatial and temporal resolution. 2. Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free Ca$^2+$ change is the sum of two terms: one major term proportional to the SR release flux/Ca$^2+$ influx, and the other reflecting the running integral of the released Ca$^2+$. 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 (dCa$^2+$/dt) of the conventional Ca$^2+$ transient (with no EGTA), and mimicked the model-derived SR Ca$^2+$ release function reported previously. In SR Ca$^2+$-depleted cells, the OG-5N transient also closely followed the waveform of L-type Ca$^2+$ current (ICa). Using ICa as a known source of Ca$^2+$ 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 'Ca$^2+$ 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 Ca$^2+$ exhibited a bell-shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca$^2+$ 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 Ca$^2+$ transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca$^2+$ released, but also the synchronization among release sites affects the whole-cell Ca$^2+$ transient and the Ca$^2+$-myofilament interaction.

@article{Song_1998_677, abstract = {1. {C}a$^{2+}$ 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 {C}a$^{2+}$), to minimize the residence time of released {C}a$^{2+}$ in the cytoplasm, and a low-affinity, fast {C}a$^{2+}$ indicator, Oregon Green 488 BAPTA-5N ({OG}-5N; 1.0 mM, Kd approximately 31 microM), to optimize the detection of localized high [{C}a$^{2+}$] in release site microdomains. Confocal microscopy was employed to resolve intracellular [{C}a$^{2+}$] at high spatial and temporal resolution. 2. Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [{C}a$^{2+}$] change is the sum of two terms: one major term proportional to the SR release flux/{C}a$^{2+}$ influx, and the other reflecting the running integral of the released {C}a$^{2+}$. 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[{C}a$^{2+}$]/dt) of the conventional {C}a$^{2+}$ transient (with no EGTA), and mimicked the model-derived SR {C}a$^{2+}$ release function reported previously. In SR {C}a$^{2+}$-depleted cells, the {OG}-5N transient also closely followed the waveform of L-type {C}a$^{2+}$ current (ICa). Using ICa as a known source of {C}a$^{2+}$ 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 '{C}a$^{2+}$ 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 {C}a$^{2+}$ exhibited a bell-shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: {C}a$^{2+}$ 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 [{C}a$^{2+}$] transient and contraction were elicited at 0 mV, it appears that not only the amount of {C}a$^{2+}$ released, but also the synchronization among release sites affects the whole-cell {C}a$^{2+}$ transient and the {C}a$^{2+}$-myofilament interaction.}, added-at = {2009-06-03T11:20:58.000+0200}, author = {Song, L. S. and Sham, J. S. and Stern, M. D. and Lakatta, E. G. and Cheng, H.}, biburl = {https://www.bibsonomy.org/bibtex/23af212a9abf42557760c3836f603437d/hake}, description = {The whole bibliography file I use.}, file = {Song_1998_677.pdf:Song_1998_677.pdf:PDF}, interhash = {d3e0a9b364b6026bb1c43a60fb9077e5}, intrahash = {3af212a9abf42557760c3836f603437d}, journal = {J. Physiol.}, keywords = {9769413 Acid, Agents, Agonists, Algorithms, Analysis, Animal Animals, Anoxia, Biological, Blockers, Bone Bones, Calcium Calcium, Cattle, Cell Cells, Channel Channel, Channels, Chelating Clonazepam, Confocal, Cultured, Cytosol, Dietary Dyes, Dynamics, Egtazic Electric Electrophysiology, Exchanger, Feed, Female, Fluorescence, Fluorescent Gov't, Heart, In Indoles, Kinetics, Linear Male, Meat, Membrane Microscopy, Minerals, Mitochondria, Models, Myocardium, Non-U.S. Nonlinear Oxygen, P.H.S., Patch-Clamp Potentials, Proteins, Pyrroles, Rats, Receptor Red, Regression Release Research Reticulum, Rumen, Ruthenium Ryanodine Sarcoplasmic Signaling, Size, Sodium-Calcium Soybeans, Sprague-Dawley, Stimulation, Support, Techniques, U.S. Vitro, Wistar, and}, month = Nov, pages = {677--691}, pmid = {9769413}, timestamp = {2009-06-03T11:21:32.000+0200}, title = {Direct measurement of SR release flux by tracking '{C}a$^{2+}$ spikes' in rat cardiac myocytes.}, url = {http://jp.physoc.org/cgi/content/full/512/3/677}, volume = {512 ( Pt 3)}, year = 1998 }