Dataset Information

Ca2+ sparks and embers of mammalian muscle. Properties of the sources.

ABSTRACT: Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were "lone embers." Average spatial width was approximately 2 microm in the rat and 1.5 microm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differences were highly significant. The greater spatial width was not a consequence of greater open time of the rat source, and was greatest at the shortest rise times, suggesting a wider Ca2+ source. In the rat, but not the frog, spark width was greater in scans transversal to the fiber axis. These features suggested that rat spark sources were elongated transversally. Ca2+ release was calculated in averages of sparks with long embers. Release current during the averaged ember started at 3 or 7 pA (depending on assumptions), whereas in lone embers it was 0.7 or 1.3 pA, which suggests that embers that trail sparks start with five open channels. Analysis of a spark with leading ember yielded a current ratio ranging from 37 to 160 in spark and ember, as if 37-160 channels opened in the spark. In simulations, 25-60 pA of Ca2+ current exiting a point source was required to reproduce frog sparks. 130 pA, exiting a cylindric source of 3 microm, qualitatively reproduced rat sparks. In conclusion, sparks of rat muscle require a greater current than frog sparks, exiting a source elongated transversally to the fiber axis, constituted by 35-260 channels. Not infrequently, a few of those remain open and produce the trailing ember.

SUBMITTER: Zhou J

PROVIDER: S-EPMC2234470 | biostudies-literature | 2003 Jul

REPOSITORIES: biostudies-literature

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Ca2+ sparks and embers of mammalian muscle. Properties of the sources.

Zhou J J Brum G G Gonzalez A A Launikonis B S BS Stern M D MD Rios E E

The Journal of general physiology 20030701 1

Ca2+ sparks of membrane-permeabilized rat muscle cells were analyzed to derive properties of their sources. Most events identified in longitudinal confocal line scans looked like sparks, but 23% (1,000 out of 4,300) were followed by long-lasting embers. Some were preceded by embers, and 48 were "lone embers." Average spatial width was approximately 2 microm in the rat and 1.5 microm in frog events in analogous solutions. Amplitudes were 33% smaller and rise times 50% greater in the rat. Differen ...[more]

PMID: 12835473

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Project description:RATIONALE:The development of a refractory period for Ca2+ spark initiation after Ca2+ release in cardiac myocytes should inhibit further Ca2+ release during the action potential plateau. However, Ca2+ release sites that did not initially activate or which have prematurely recovered from refractoriness might release Ca2+ later during the action potential and alter the cell-wide Ca2+ transient. OBJECTIVE:To investigate the possibility of late Ca2+ spark (LCS) activity in intact isolated cardiac myocytes using fast confocal line scanning with improved confocality and signal to noise. METHODS AND RESULTS:We recorded Ca2+ transients from cardiac ventricular myocytes isolated from rabbit hearts. Action potentials were produced by electric stimulation, and rapid solution changes were used to modify the L-type Ca2+ current. After the upstroke of the Ca2+ transient, LCSs were detected which had increased amplitude compared with diastolic Ca2+ sparks. LCS are triggered by both L-type Ca2+ channel activity during the action potential plateau, as well as by the increase of cytosolic Ca2+ associated with the Ca2+ transient itself. Importantly, a mismatch between sarcoplasmic reticulum load and L-type Ca2+ trigger can increase the number of LCS. The likelihood of triggering an LCS also depends on recovery from refractoriness that appears after prior activation. Consequences of LCS include a reduced rate of decline of the Ca2+ transient and, if frequent, formation of microscopic propagating Ca2+ release events (Ca2+ ripples). Ca2+ ripples resemble Ca2+ waves in terms of local propagation velocity but spread for only a short distance because of limited regeneration. CONCLUSIONS:These new types of Ca2+ signaling behavior extend our understanding of Ca2+-mediated signaling. LCS may provide an arrhythmogenic substrate by slowing the Ca2+ transient decline, as well as by amplifying maintained Ca2+ current effects on intracellular Ca2+ and consequently Na+/Ca2+ exchange current.

Project description:Patch-clamp electrophysiology is widely used to characterize neuronal electrical phenotypes. However, there are no standard experimental conditions for in vitro whole cell patch-clamp electrophysiology, complicating direct comparisons between data sets. In this study, we sought to understand how basic experimental conditions differ among laboratories and how these differences might impact measurements of electrophysiological parameters. We curated the compositions of external bath solutions (artificial cerebrospinal fluid), internal pipette solutions, and other methodological details such as animal strain and age from 509 published neurophysiology articles studying rodent neurons. We found that very few articles used the exact same experimental solutions as any other, and some solution differences stem from recipe inheritance from advisor to advisee as well as changing trends over the years. Next, we used statistical models to understand how the use of different experimental conditions impacts downstream electrophysiological measurements such as resting potential and action potential width. Although these experimental condition features could explain up to 43% of the study-to-study variance in electrophysiological parameters, the majority of the variability was left unexplained. Our results suggest that there are likely additional experimental factors that contribute to cross-laboratory electrophysiological variability, and identifying and addressing these will be important to future efforts to assemble consensus descriptions of neurophysiological phenotypes for mammalian cell types. NEW & NOTEWORTHY This article describes how using different experimental methods during patch-clamp electrophysiology impacts downstream physiological measurements. We characterized how methodologies and experimental solutions differ across articles. We found that differences in methods can explain some, but not all, of the study-to-study variance in electrophysiological measurements. Explicitly accounting for methodological differences using statistical models can help correct downstream electrophysiological measurements for cross-laboratory methodology differences.

Dataset Information

Ca2+ sparks and embers of mammalian muscle. Properties of the sources.

Publications

Ca2+ sparks and embers of mammalian muscle. Properties of the sources.

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