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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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Related in: MedlinePlus

Ca blinks and [Ca]SR signals.(A) shows the simulated line-scan images of [Ca]SR (purple), Ca-Fluo-5N (green) and Ca blink (blurred Ca-Fluo-5N, red) signals when the Ca sparks were simulated with an cell-top PSF. Left panels in (B) and (C) show the time and spatial profiles through the minimum Ca blink intensity, respectively. The right panels show scaled versions of the profiles.
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pcbi-1002931-g005: Ca blinks and [Ca]SR signals.(A) shows the simulated line-scan images of [Ca]SR (purple), Ca-Fluo-5N (green) and Ca blink (blurred Ca-Fluo-5N, red) signals when the Ca sparks were simulated with an cell-top PSF. Left panels in (B) and (C) show the time and spatial profiles through the minimum Ca blink intensity, respectively. The right panels show scaled versions of the profiles.

Mentions: Fig. 5A shows the simulated line-scan images of [Ca]SR, Ca-Fluo-5N and the Ca blink (optically blurred Ca-Fluo-5N) signals generated from using the cell-top PSF marked by purple, green and red bars, respectively. Depending on the degree of optical blurring determined by the orientation of the network SR relative to the optical axis (see Fig. 1B), Ca blink depletions ranged between 25–35%, which are within the range of values reported in other studies [3], [5], [39]. Importantly, the underlying non-blurred Ca-Fluo-5N signal showed much more extensive depletion compared to the corresponding Ca blink. For example, the Ca-Fluo-5N signal had decreased by ∼70% at its minimum, more than double that suggested by the blurred signal. Additionally, Ca-Fluo-5N also under-reported the underlying depletion of [Ca]SR, which was actually ∼90%. Overall, dye kinetics and optical blurring caused a near 3-fold under-estimation of the true extent of [Ca]SR depletion. Dye kinetics also had an effect on the ability of Ca blink signals to correctly report the time-course of [Ca]SR changes (right panel, Fig. 5B). Though there were only small distortions to the time to minimum, the time taken for [Ca]SR recovery was under-estimated by Ca-Fluo-5N by ∼40 ms and which was slightly lengthened by optical blurring. Overall, this led to a ∼35% under-estimation of [Ca]SR recovery time.


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 blinks and [Ca]SR signals.(A) shows the simulated line-scan images of [Ca]SR (purple), Ca-Fluo-5N (green) and Ca blink (blurred Ca-Fluo-5N, red) signals when the Ca sparks were simulated with an cell-top PSF. Left panels in (B) and (C) show the time and spatial profiles through the minimum Ca blink intensity, respectively. The right panels show scaled versions of the profiles.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002931-g005: Ca blinks and [Ca]SR signals.(A) shows the simulated line-scan images of [Ca]SR (purple), Ca-Fluo-5N (green) and Ca blink (blurred Ca-Fluo-5N, red) signals when the Ca sparks were simulated with an cell-top PSF. Left panels in (B) and (C) show the time and spatial profiles through the minimum Ca blink intensity, respectively. The right panels show scaled versions of the profiles.
Mentions: Fig. 5A shows the simulated line-scan images of [Ca]SR, Ca-Fluo-5N and the Ca blink (optically blurred Ca-Fluo-5N) signals generated from using the cell-top PSF marked by purple, green and red bars, respectively. Depending on the degree of optical blurring determined by the orientation of the network SR relative to the optical axis (see Fig. 1B), Ca blink depletions ranged between 25–35%, which are within the range of values reported in other studies [3], [5], [39]. Importantly, the underlying non-blurred Ca-Fluo-5N signal showed much more extensive depletion compared to the corresponding Ca blink. For example, the Ca-Fluo-5N signal had decreased by ∼70% at its minimum, more than double that suggested by the blurred signal. Additionally, Ca-Fluo-5N also under-reported the underlying depletion of [Ca]SR, which was actually ∼90%. Overall, dye kinetics and optical blurring caused a near 3-fold under-estimation of the true extent of [Ca]SR depletion. Dye kinetics also had an effect on the ability of Ca blink signals to correctly report the time-course of [Ca]SR changes (right panel, Fig. 5B). Though there were only small distortions to the time to minimum, the time taken for [Ca]SR recovery was under-estimated by Ca-Fluo-5N by ∼40 ms and which was slightly lengthened by optical blurring. Overall, this led to a ∼35% under-estimation of [Ca]SR recovery time.

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