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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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Simulation and fit of a Ca spark with on-coverslip and on-cell PSFs.(A) shows a line-scan image of a measured Ca spark. From left to right, (B) shows the on-coverslip PSF in x-y (top) and x-z (bottom) view (scale bars show 0.5 µm), the simulated Ca spark and absolute difference between the simulated and recorded events. The mean of the difference image was −0.06. (C) shows a similar dataset, but for an on-cell PSF, where the mean of the difference image was −0.07. The goodness of fit can be appreciated in the time and distance profiles are shown in (D) and (E), respectively, colored by the marks beside the Ca spark images.
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pcbi-1002931-g003: Simulation and fit of a Ca spark with on-coverslip and on-cell PSFs.(A) shows a line-scan image of a measured Ca spark. From left to right, (B) shows the on-coverslip PSF in x-y (top) and x-z (bottom) view (scale bars show 0.5 µm), the simulated Ca spark and absolute difference between the simulated and recorded events. The mean of the difference image was −0.06. (C) shows a similar dataset, but for an on-cell PSF, where the mean of the difference image was −0.07. The goodness of fit can be appreciated in the time and distance profiles are shown in (D) and (E), respectively, colored by the marks beside the Ca spark images.

Mentions: Two 3D Gaussian profiles based on our PSF measurements were used to simulate the effect of PSFs that are on the coverslip or on top of cells. A high signal-to-noise spontaneous Ca spark is shown in Fig. 3A and was used as the data set for parameter-fitting. Fig. 3B shows the coverslip PSF (shown in in x-y and x-z views) and the Ca spark generated by the computer model. The image on the right shows the absolute difference between the simulated and recorded events, showing a good fit with no systematic residuals. Fig. 3C shows the results of using the cell-top PSF, where the model could also fit the recorded event with a larger release flux (see below). The measured (black lines) and fitted time (Fig. 3D) and spatial (Fig. 3E) profiles of the Ca sparks are also shown, colored according to the markers shown in Fig. 3A–C. These profiles also show that the fitted and measured events are in reasonable agreement.


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)

Simulation and fit of a Ca spark with on-coverslip and on-cell PSFs.(A) shows a line-scan image of a measured Ca spark. From left to right, (B) shows the on-coverslip PSF in x-y (top) and x-z (bottom) view (scale bars show 0.5 µm), the simulated Ca spark and absolute difference between the simulated and recorded events. The mean of the difference image was −0.06. (C) shows a similar dataset, but for an on-cell PSF, where the mean of the difference image was −0.07. The goodness of fit can be appreciated in the time and distance profiles are shown in (D) and (E), respectively, colored by the marks beside the Ca spark images.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002931-g003: Simulation and fit of a Ca spark with on-coverslip and on-cell PSFs.(A) shows a line-scan image of a measured Ca spark. From left to right, (B) shows the on-coverslip PSF in x-y (top) and x-z (bottom) view (scale bars show 0.5 µm), the simulated Ca spark and absolute difference between the simulated and recorded events. The mean of the difference image was −0.06. (C) shows a similar dataset, but for an on-cell PSF, where the mean of the difference image was −0.07. The goodness of fit can be appreciated in the time and distance profiles are shown in (D) and (E), respectively, colored by the marks beside the Ca spark images.
Mentions: Two 3D Gaussian profiles based on our PSF measurements were used to simulate the effect of PSFs that are on the coverslip or on top of cells. A high signal-to-noise spontaneous Ca spark is shown in Fig. 3A and was used as the data set for parameter-fitting. Fig. 3B shows the coverslip PSF (shown in in x-y and x-z views) and the Ca spark generated by the computer model. The image on the right shows the absolute difference between the simulated and recorded events, showing a good fit with no systematic residuals. Fig. 3C shows the results of using the cell-top PSF, where the model could also fit the recorded event with a larger release flux (see below). The measured (black lines) and fitted time (Fig. 3D) and spatial (Fig. 3E) profiles of the Ca sparks are also shown, colored according to the markers shown in Fig. 3A–C. These profiles also show that the fitted and measured events are in reasonable agreement.

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