Variations in local calcium signaling in adjacent cardiac myocytes of the intact mouse heart detected with two-dimensional confocal microscopy (original) (raw)
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Computer-Aided Measurement of Cell Shortening and Calcium Transients in Adult Cardiac Myocytes
Biotechnology Progress, 2001
The contractile cycle of the cardiac myocyte is essentially controlled by the concentration of intracellular calcium ([Ca2+]i). Measurement of [Ca2+]i using Ca2+-dependent fluorescence and simultaneous monitoring of cell dynamics enable characterization of a variety of substances interacting with ion channels and contractile proteins. In this report we describe a novel method featuring up to 480 frames/s for monitoring rapid changes in cellular calcium and cell length, in which every individual cycle allows effective evaluation of major cell parameters. Computers aid in determination of time to peak (in ms), time to 50% decrease (ms), diastolic Ca2+ (relative fluorescence units, rfu), systolic Ca2+ (rfu), Ca2+ transients (rfu), ΔCa2+/Δt rise (rfu/s), and ΔCa2+/Δt fall (rfu/s). Contractile parameters are as follows: maximum cell length (μm), minimum cell length (μm), absolute cell shortening (μm), peak ΔL/Δt shortening (μm/s), and peak ΔL/Δt relaxation (μm/s). In summary, we succeeded in demonstrating that this system is a unique and valuable tool that allows simultaneous and accurate assessment of contractile parameters and of calcium movements of isolated adult cardiac myocytes.
Biophysical Journal, 2001
We have developed a distance modulated protocol for scanning ion conductance microscopy to provide a robust and reliable distance control mechanism for imaging contracting cells. The technique can measure rapid changes in cell height from 10 nm to several micrometers, with millisecond time resolution. This has been demonstrated on the extreme case of a contracting cardiac myocyte. By combining this method with laser confocal microscopy, it was possible to simultaneously measure the nanometric motion of the cardiac myocyte, and the local calcium concentration just under the cell membrane. Despite large cellular movement, simultaneous tracking of the changes in cell height and measurement of the intracellular Ca 2ϩ near the cell surface is possible while retaining the cell functionality.
Multimodal SHG-2PF Imaging of Microdomain Ca 2+ -Contraction Coupling in Live Cardiac Myocytes
Circulation Research, 2015
E xcitation-contraction coupling in cardiac myocytes is mediated by Ca 2+. During systole, an action potential opens voltage-gated Ca 2+ channels in the sarcolemma to allow Ca 2+ entry into the cell, which triggers a much larger release of Ca 2+ from the sarcoplasmic reticulum through the ryanodine receptor (RyR); this process is termed Ca 2+-induced Ca 2+ release. Synchronous Ca 2+-induced Ca 2+ release throughout the cell increases the cytosolic Ca 2+ concentration, and subsequent Ca 2+ binding to troponin C causes conformational changes in contractile machinery that results in the cross-bridge power stroke and sarcomere contraction. During diastole, cytosolic Ca 2+ is lowered to basal level by sequestration into the sarcoplasmic reticulum Ca 2+ store and extrusion from the cell, resulting in sarcomere relaxation. 1 The RyR cluster is the basic Ca 2+ release unit in cardiac myocytes. RyR responds to local rises in Ca 2+ and opens in a stochastic manner. It opens and closes in an all-or-none fashion to release a quantum amount of Ca 2+ , giving rise to New Methods in Cardiovascular Biology
Automated image analysis of cardiac myocyte Ca 2+ dynamics
Proceedings of the Annual International Conference of the IEEE Engineering in Medicine and Biology Society, EMBS, 2011
Intracellular Ca 2+ dynamics act as a key link between the electrical and mechanical activity of the heart. Here we present a method for high-throughput measurement, automated cell segmentation and signal analysis of Ca 2+ transients in isolated adult ventricular myocytes. In addition to increasing experimental throughput ~10-fold compared to conventional approaches, this approach allows the study of individual cell-cell variability and relationships between Ca 2+ signaling and cell morphology.
Confocal imaging of calcium release events in single smooth muscle cells
Acta Physiologica Scandinavica, 1998
Localized [Ca 2+ ] i transients (`sparks') ®rst directly detected in cardiac myocytes were considered to represent`elementary' Ca 2+-release events playing a key role during excitation±contraction coupling (Cheng et al. 1993). In this study we employed confocal [Ca 2+ ] i imaging to characterize subcellular calcium signalling in¯uo-3 loaded visceral and vascular smooth muscle cells. In some experiments membrane potential of the myocyte was controlled using whole-cell patch clamp technique and changes in membrane current were recorded simultaneously with [Ca 2+ ] i imaging. Some local [Ca 2+ ] i transients were very similar to`Ca 2+ sparks' observed in heart, i.e. lasting 200mswithapeakuorescenceratioof1.750.23(meanSD,n33).Ca2+sparkswerefoundtooccurincertainpreferredlocationsinthecell,termedfrequentdischargesites.Othereventswerefasterandsmaller,lastingonly200 ms with a peak uorescence ratio of 1.75 0.23 (mean SD, n 33). Ca 2+ sparks were found to occur in certain preferred locations in the cell, termed frequent discharge sites. Other events were faster and smaller, lasting only 200mswithapeakuorescenceratioof1.750.23(meanSD,n33).Ca2+sparkswerefoundtooccurincertainpreferredlocationsinthecell,termedfrequentdischargesites.Othereventswerefasterandsmaller,lastingonly40 ms with a peak normalized¯uorescence of 1.36 0.09 (mean SD, n 28). A high correlation between spontaneous transient outward currents and spark occurrence was observed. Proliferating waves of elevated [Ca 2+ ] i initiated during membrane depolarization seem to arise from spatio-temporal recruitment of local Ca 2+-release events. The spatial non-uniformity of sarcoplasmic reticulum and ryanodine receptor distribution within the cell may account for the existence of`frequent discharge sites' and the wide variation in the Ca 2+ wave propagation velocities observed.
Identification of intracellular calcium dynamics in stimulated cardiomyocytes
2010
We have developed an automatic method for the analysis and identification of dynamical regimes in intracellular calcium patterns from confocal calcium images. The method allows the identification of different dynamical patterns such as spatially concordant and discordant alternans, irregular behavior or phase-locking regimes such as period doubling or halving. The method can be applied to the analysis of different cardiac pathologies related to anomalies at the cellular level such as ventricular reentrant arrhythmias.
Structural variability of dyads relates to calcium release in rat ventricular myocytes
Scientific Reports, 2020
Cardiac excitation-contraction coupling relies on dyads, the intracellular calcium synapses of cardiac myocytes, where the plasma membrane contacts sarcoplasmic reticulum and where electrical excitation triggers calcium release. The morphology of dyads and dynamics of local calcium release vary substantially. To better understand the correspondence between the structure and the functionality of dyads, we estimated incidences of structurally different dyads and of kinetically different calcium release sites and tested their responsiveness to experimental myocardial injury in left ventricular myocytes of rats. According to the structure of dyads estimated in random electron microscopic images of myocardial tissue, the dyads were sorted into ‘compact’ or ‘loose’ types. The calcium release fluxes, triggered at local calcium release sites in patch-clamped ventricular myocytes and recorded by laser scanning confocal fluorescence microscopy, were decomposed into ‘early’ and ‘late’ componen...
Acta histochemica et cytochemica, 2014
Intracellular Ca(2+) ([Ca(2+)]i) dynamics in isolated myocytes differ between the atria and ventricles due to the distinct t-tubular distributions. Although cellular aspects of ventricular [Ca(2+)]i dynamics in the heart have been extensively studied, little is known about those of atrial myocytes in situ. Here we visualized precise [Ca(2+)]i dynamics of atrial myocytes in Langendorff-perfused rat hearts by rapid-scanning confocal microscopy. Of 16 fluo-4-loaded hearts imaged during pacing up to 4-Hz, five hearts showed spatially uniform Ca(2+) transients on systole among individual cells, whereas no discernible [Ca(2+)]i elevation developed during diastole. In contrast, the remaining hearts showed non-uniform [Ca(2+)]i dynamics within and among the cells especially under high-frequency (4 Hz) excitation, where subcellular cluster-like [Ca(2+)]i rises or wave-like [Ca(2+)]i propagation occurred on excitation. Such [Ca(2+)]i inhomogeneity was more pronounced at high-frequency pacing,...
The Keio journal of medicine, 1990
To examine the origin and spreading of the Ca2+ transient following electrical stimulation of isolated myocyte, a system capable of recording intracellular Ca2+ distribution with sufficient temporal and spatial resolution was constructed. The system consists of a fluorescence microscope with computer-controlled pulse illumination and a digital image analyzer. The results with this new equipment show that the Ca2+ transient originates from one or a few points within a myocyte, and spreads throughout the cell. During the initial 60-msec period, the distribution of Ca2+ within a myocyte was not uniform. The system may be used for better understanding of the excitation-contraction coupling mechanism occurring within a cardiac myocyte or of changes in intracellular Ca2+ concentrations in other cells in which Ca2+ plays a crucial role in signal transduction.