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Extraction of sub-microscopic Ca fluxes from blurred and noisy fluorescent indicator images with a detailed model fitting approach.

Kong CH, Laver DR, Cannell MB - PLoS Comput. Biol. (2013)

Bottom Line: While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level.This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms.By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.

ABSTRACT
The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This 'forward method' allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

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Ca-Fluo-4 and release flux associated with Ca spark.The (A) time profiles of the unblurred calculated Ca-Fluo-4 signals for Ca sparks at the top of the cell (black) and near the coverslip (red). The dashed lines show the corresponding simulated Ca sparks (blurred Ca-Fluo-4 signals) scaled to the amplitudes of the un-blurred signals to allow comparison of the time-courses. (B) shows the release flux required to produce the fitted simulated Ca sparks shown in Fig. 3 and 4A, where a larger current was required when optical blurring was more severe (i.e. if the Ca spark had originated from near the cell top).
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pcbi-1002931-g004: Ca-Fluo-4 and release flux associated with Ca spark.The (A) time profiles of the unblurred calculated Ca-Fluo-4 signals for Ca sparks at the top of the cell (black) and near the coverslip (red). The dashed lines show the corresponding simulated Ca sparks (blurred Ca-Fluo-4 signals) scaled to the amplitudes of the un-blurred signals to allow comparison of the time-courses. (B) shows the release flux required to produce the fitted simulated Ca sparks shown in Fig. 3 and 4A, where a larger current was required when optical blurring was more severe (i.e. if the Ca spark had originated from near the cell top).

Mentions: The un-blurred Fluo-4 dye signal at the center of the Ca spark is shown in Fig. 4A, as calculated if the recorded Ca spark occurred at the cell-top (black lines) or near the coverslip (red lines), respectively. The dashed lines correspond to the blurred dye signals, where they been scaled to the amplitude of the un-blurred signals to allow comparison of the effect of blurring on time-course. Despite being in-focus, optical blurring in both cases caused the recorded Ca spark to be slower than that of the underlying signal. For a Ca spark that originated at the cell-top, the time of peak fluorescence was increased by ∼1 ms. This effect was smaller when the Ca spark was at the coverslip. The FWHM of the Ca spark was doubled due to blurring (not shown). The fluxes that were required to produce the same recorded Ca spark (Fig. 3) were different due from using the two different PSFs (Fig. 4B). When the cell-top PSF was used, the calculated peak release flux was ∼11.8 pA, while when the smaller, coverslip PSF was used, only ∼8.7 pA was required to produce the blurred Ca spark. Since these values represent two extreme cases for Ca sparks recorded in-focus, but from different locations within a cell, it is likely that an average Ca spark would be associated with an intermediate peak current (e.g. ∼10 pA). Note that the different PSFs did not markedly change the time-course of the calculated release flux.


Extraction of sub-microscopic Ca fluxes from blurred and noisy fluorescent indicator images with a detailed model fitting approach.

Kong CH, Laver DR, Cannell MB - PLoS Comput. Biol. (2013)

Ca-Fluo-4 and release flux associated with Ca spark.The (A) time profiles of the unblurred calculated Ca-Fluo-4 signals for Ca sparks at the top of the cell (black) and near the coverslip (red). The dashed lines show the corresponding simulated Ca sparks (blurred Ca-Fluo-4 signals) scaled to the amplitudes of the un-blurred signals to allow comparison of the time-courses. (B) shows the release flux required to produce the fitted simulated Ca sparks shown in Fig. 3 and 4A, where a larger current was required when optical blurring was more severe (i.e. if the Ca spark had originated from near the cell top).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3585382&req=5

pcbi-1002931-g004: Ca-Fluo-4 and release flux associated with Ca spark.The (A) time profiles of the unblurred calculated Ca-Fluo-4 signals for Ca sparks at the top of the cell (black) and near the coverslip (red). The dashed lines show the corresponding simulated Ca sparks (blurred Ca-Fluo-4 signals) scaled to the amplitudes of the un-blurred signals to allow comparison of the time-courses. (B) shows the release flux required to produce the fitted simulated Ca sparks shown in Fig. 3 and 4A, where a larger current was required when optical blurring was more severe (i.e. if the Ca spark had originated from near the cell top).
Mentions: The un-blurred Fluo-4 dye signal at the center of the Ca spark is shown in Fig. 4A, as calculated if the recorded Ca spark occurred at the cell-top (black lines) or near the coverslip (red lines), respectively. The dashed lines correspond to the blurred dye signals, where they been scaled to the amplitude of the un-blurred signals to allow comparison of the effect of blurring on time-course. Despite being in-focus, optical blurring in both cases caused the recorded Ca spark to be slower than that of the underlying signal. For a Ca spark that originated at the cell-top, the time of peak fluorescence was increased by ∼1 ms. This effect was smaller when the Ca spark was at the coverslip. The FWHM of the Ca spark was doubled due to blurring (not shown). The fluxes that were required to produce the same recorded Ca spark (Fig. 3) were different due from using the two different PSFs (Fig. 4B). When the cell-top PSF was used, the calculated peak release flux was ∼11.8 pA, while when the smaller, coverslip PSF was used, only ∼8.7 pA was required to produce the blurred Ca spark. Since these values represent two extreme cases for Ca sparks recorded in-focus, but from different locations within a cell, it is likely that an average Ca spark would be associated with an intermediate peak current (e.g. ∼10 pA). Note that the different PSFs did not markedly change the time-course of the calculated release flux.

Bottom Line: While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level.This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms.By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology and Pharmacology, University of Bristol, Bristol, United Kingdom.

ABSTRACT
The release of Ca from intracellular stores is key to cardiac muscle function; however, the molecular control of intracellular Ca release remains unclear. Depletion of the intracellular Ca store (sarcoplasmic reticulum, SR) may play an important role, but the ability to measure local SR Ca with fluorescent Ca indicators is limited by the microscope optical resolution and properties of the indicator. This leads to an uncertain degree of spatio-temporal blurring, which is not easily corrected (by deconvolution methods) due to the low signal-to-noise ratio of the recorded signals. In this study, a 3D computer model was constructed to calculate local Ca fluxes and consequent dye signals, which were then blurred by a measured microscope point spread function. Parameter fitting was employed to adjust a release basis function until the model output fitted recorded (2D) Ca spark data. This 'forward method' allowed us to obtain estimates of the time-course of Ca release flux and depletion within the sub-microscopic local SR associated with a number of Ca sparks. While variability in focal position relative to Ca spark sites causes more out-of-focus events to have smaller calculated fluxes (and less SR depletion), the average SR depletion was to 20±10% (s.d.) of the resting level. This focus problem implies that the actual SR depletion is likely to be larger and the five largest depletions analyzed were to 8±6% of the resting level. This profound depletion limits SR release flux during a Ca spark, which peaked at 8±3 pA and declined with a half time of 7±2 ms. By comparison, RyR open probability declined more slowly, suggesting release termination is dominated by neither SR Ca depletion nor intrinsic RyR gating, but results from an interaction of these processes.

Show MeSH
Related in: MedlinePlus