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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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Analysis of recorded Ca sparks.(A) shows 3 examples of (i) recorded and (ii) simulated Ca sparks analyzed with a cell-top PSF. These events were chosen for their high signal-to-noise ratio and high amplitude, which suggests they are in-focus. (B) shows the (i) time and (ii) spatial profiles of the recorded and fitted (smooth lines) datasets, color-coded by the bars shown in (A). The events have been offset for clarity. (C) shows the flux responsible for each event, which was used to calculate the time-course of n·PO. (D) shows the time-course of [Ca]SR in the junction (solid lines), overlaid with the corresponding Ca blink signals (dashed lines). (E) shows the time-courses of release flux and n·PO averaged from fitting a population of Ca sparks (n = 14), where the shaded regions show one S.E.M. For all events, RyRs were open longer than release flux duration, which were 24.0±13 vs. 11.2±2.2 ms, respectively, when measured from the start to the time at half maximal decay. (F) shows the corresponding average [Ca]jSR and Ca blink time-courses with the shaded areas showing one S.E.M.
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pcbi-1002931-g006: Analysis of recorded Ca sparks.(A) shows 3 examples of (i) recorded and (ii) simulated Ca sparks analyzed with a cell-top PSF. These events were chosen for their high signal-to-noise ratio and high amplitude, which suggests they are in-focus. (B) shows the (i) time and (ii) spatial profiles of the recorded and fitted (smooth lines) datasets, color-coded by the bars shown in (A). The events have been offset for clarity. (C) shows the flux responsible for each event, which was used to calculate the time-course of n·PO. (D) shows the time-course of [Ca]SR in the junction (solid lines), overlaid with the corresponding Ca blink signals (dashed lines). (E) shows the time-courses of release flux and n·PO averaged from fitting a population of Ca sparks (n = 14), where the shaded regions show one S.E.M. For all events, RyRs were open longer than release flux duration, which were 24.0±13 vs. 11.2±2.2 ms, respectively, when measured from the start to the time at half maximal decay. (F) shows the corresponding average [Ca]jSR and Ca blink time-courses with the shaded areas showing one S.E.M.

Mentions: Fig. 6A and B show further examples of model fits to a number of experimentally recorded Ca sparks with different amplitudes and time-courses. The ability of the model to fit this range of Ca sparks is notable. The computed Ca release functions are shown in Fig. 6C, where peak flux occurred before the peak of the Ca spark. The calculated SR Ca signals in Fig. 6D show that [Ca]SR depletion was large compared to their associated Ca blink signals (dashed lines). In addition, the duration of the computed release flux was always shorter than the duration of the fitted permeability function, where peak flux always occurred before RyR open probability had even begun to decline (Fig. 6E), showing jSR depletion plays an important role in reducing release flux prior to closure of the RyR channels. The mean peak flux was 7.9±2.9 pA (s.d.) for Ca sparks with maximum F/F0 of 3.0±0.6. Panel F shows average Ca blinks compared to average Ca-Fluo-5N and [Ca]SR signals, consistent with the trends described in Fig. 5. The average (blurred) Ca blink signal was ΔF/F0 = 0.24±0.098 (s.d.), compared to the corresponding SR depletion to an average minimum of 210±130 µM.


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)

Analysis of recorded Ca sparks.(A) shows 3 examples of (i) recorded and (ii) simulated Ca sparks analyzed with a cell-top PSF. These events were chosen for their high signal-to-noise ratio and high amplitude, which suggests they are in-focus. (B) shows the (i) time and (ii) spatial profiles of the recorded and fitted (smooth lines) datasets, color-coded by the bars shown in (A). The events have been offset for clarity. (C) shows the flux responsible for each event, which was used to calculate the time-course of n·PO. (D) shows the time-course of [Ca]SR in the junction (solid lines), overlaid with the corresponding Ca blink signals (dashed lines). (E) shows the time-courses of release flux and n·PO averaged from fitting a population of Ca sparks (n = 14), where the shaded regions show one S.E.M. For all events, RyRs were open longer than release flux duration, which were 24.0±13 vs. 11.2±2.2 ms, respectively, when measured from the start to the time at half maximal decay. (F) shows the corresponding average [Ca]jSR and Ca blink time-courses with the shaded areas showing one S.E.M.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002931-g006: Analysis of recorded Ca sparks.(A) shows 3 examples of (i) recorded and (ii) simulated Ca sparks analyzed with a cell-top PSF. These events were chosen for their high signal-to-noise ratio and high amplitude, which suggests they are in-focus. (B) shows the (i) time and (ii) spatial profiles of the recorded and fitted (smooth lines) datasets, color-coded by the bars shown in (A). The events have been offset for clarity. (C) shows the flux responsible for each event, which was used to calculate the time-course of n·PO. (D) shows the time-course of [Ca]SR in the junction (solid lines), overlaid with the corresponding Ca blink signals (dashed lines). (E) shows the time-courses of release flux and n·PO averaged from fitting a population of Ca sparks (n = 14), where the shaded regions show one S.E.M. For all events, RyRs were open longer than release flux duration, which were 24.0±13 vs. 11.2±2.2 ms, respectively, when measured from the start to the time at half maximal decay. (F) shows the corresponding average [Ca]jSR and Ca blink time-courses with the shaded areas showing one S.E.M.
Mentions: Fig. 6A and B show further examples of model fits to a number of experimentally recorded Ca sparks with different amplitudes and time-courses. The ability of the model to fit this range of Ca sparks is notable. The computed Ca release functions are shown in Fig. 6C, where peak flux occurred before the peak of the Ca spark. The calculated SR Ca signals in Fig. 6D show that [Ca]SR depletion was large compared to their associated Ca blink signals (dashed lines). In addition, the duration of the computed release flux was always shorter than the duration of the fitted permeability function, where peak flux always occurred before RyR open probability had even begun to decline (Fig. 6E), showing jSR depletion plays an important role in reducing release flux prior to closure of the RyR channels. The mean peak flux was 7.9±2.9 pA (s.d.) for Ca sparks with maximum F/F0 of 3.0±0.6. Panel F shows average Ca blinks compared to average Ca-Fluo-5N and [Ca]SR signals, consistent with the trends described in Fig. 5. The average (blurred) Ca blink signal was ΔF/F0 = 0.24±0.098 (s.d.), compared to the corresponding SR depletion to an average minimum of 210±130 µM.

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