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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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Computer model geometry.(A) shows a region of a cardiac myocyte and presents the size of a typical junction relative to a confocal PSF and the computer model. A transverse tubule (purple) extends in between myofilaments (pink) that are wrapped by network of SR tubules (yellow surface, red lumen). A flattened disc of SR wraps around the T-tubule to form a Ca release junction. The size of a typical confocal PSF is shown by an ellipse in x-z orientation, at 2·FWHM. (B) shows the transverse, stylised view of (A), where the SR can be seen as an ‘X’ shape that curves around the myofibrils. The jSR is shown as a white circle and assumed to be in the centre of the PSF (opaque ellipse). The spherical mesh of the computer model is also shown (grey dashed lines), with the radius at 4 µm, which should be sufficiently large to capture a Ca spark without boundary effects. (C) shows a simplified diagram of the computer model elements (0≤i≤39) and the two compartments: the cytosol (blue) and SR (orange). The locations of mobile and immobile Ca buffers are shown. See text and Table 1 for details.
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pcbi-1002931-g001: Computer model geometry.(A) shows a region of a cardiac myocyte and presents the size of a typical junction relative to a confocal PSF and the computer model. A transverse tubule (purple) extends in between myofilaments (pink) that are wrapped by network of SR tubules (yellow surface, red lumen). A flattened disc of SR wraps around the T-tubule to form a Ca release junction. The size of a typical confocal PSF is shown by an ellipse in x-z orientation, at 2·FWHM. (B) shows the transverse, stylised view of (A), where the SR can be seen as an ‘X’ shape that curves around the myofibrils. The jSR is shown as a white circle and assumed to be in the centre of the PSF (opaque ellipse). The spherical mesh of the computer model is also shown (grey dashed lines), with the radius at 4 µm, which should be sufficiently large to capture a Ca spark without boundary effects. (C) shows a simplified diagram of the computer model elements (0≤i≤39) and the two compartments: the cytosol (blue) and SR (orange). The locations of mobile and immobile Ca buffers are shown. See text and Table 1 for details.

Mentions: As shown in Fig. 1B, the SR contained Fluo-5F, while only the jSR compartment contained calsequestrin (CSQ) and was able to release Ca (“R”), which entered the first cytosolic element. The rate of SR uptake (“U”) were set so that when [Ca]i was transiently increased to 10 µM, return to rest occurred in a half-time of ∼160 ms:(5)where Vmax was 300 µM/s and Km was 0.3 µM [17]. The leak flux (“L”) was set so that [Ca]i at rest was 100 nM [19]. Cytosolic buffers included ATP, calmodulin (CaM), Fluo-4, troponin-C (both high and low affinity sites, TnCI and TnCII, respectively) and SR membrane binding sites (SRm). The junctional space also included sarcolemmal membrane binding sites (SLm) and excluded Fluo-4 [10]. [Mg] was set to 1 mM in all compartments [20].


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)

Computer model geometry.(A) shows a region of a cardiac myocyte and presents the size of a typical junction relative to a confocal PSF and the computer model. A transverse tubule (purple) extends in between myofilaments (pink) that are wrapped by network of SR tubules (yellow surface, red lumen). A flattened disc of SR wraps around the T-tubule to form a Ca release junction. The size of a typical confocal PSF is shown by an ellipse in x-z orientation, at 2·FWHM. (B) shows the transverse, stylised view of (A), where the SR can be seen as an ‘X’ shape that curves around the myofibrils. The jSR is shown as a white circle and assumed to be in the centre of the PSF (opaque ellipse). The spherical mesh of the computer model is also shown (grey dashed lines), with the radius at 4 µm, which should be sufficiently large to capture a Ca spark without boundary effects. (C) shows a simplified diagram of the computer model elements (0≤i≤39) and the two compartments: the cytosol (blue) and SR (orange). The locations of mobile and immobile Ca buffers are shown. See text and Table 1 for details.
© Copyright Policy
Related In: Results  -  Collection

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

pcbi-1002931-g001: Computer model geometry.(A) shows a region of a cardiac myocyte and presents the size of a typical junction relative to a confocal PSF and the computer model. A transverse tubule (purple) extends in between myofilaments (pink) that are wrapped by network of SR tubules (yellow surface, red lumen). A flattened disc of SR wraps around the T-tubule to form a Ca release junction. The size of a typical confocal PSF is shown by an ellipse in x-z orientation, at 2·FWHM. (B) shows the transverse, stylised view of (A), where the SR can be seen as an ‘X’ shape that curves around the myofibrils. The jSR is shown as a white circle and assumed to be in the centre of the PSF (opaque ellipse). The spherical mesh of the computer model is also shown (grey dashed lines), with the radius at 4 µm, which should be sufficiently large to capture a Ca spark without boundary effects. (C) shows a simplified diagram of the computer model elements (0≤i≤39) and the two compartments: the cytosol (blue) and SR (orange). The locations of mobile and immobile Ca buffers are shown. See text and Table 1 for details.
Mentions: As shown in Fig. 1B, the SR contained Fluo-5F, while only the jSR compartment contained calsequestrin (CSQ) and was able to release Ca (“R”), which entered the first cytosolic element. The rate of SR uptake (“U”) were set so that when [Ca]i was transiently increased to 10 µM, return to rest occurred in a half-time of ∼160 ms:(5)where Vmax was 300 µM/s and Km was 0.3 µM [17]. The leak flux (“L”) was set so that [Ca]i at rest was 100 nM [19]. Cytosolic buffers included ATP, calmodulin (CaM), Fluo-4, troponin-C (both high and low affinity sites, TnCI and TnCII, respectively) and SR membrane binding sites (SRm). The junctional space also included sarcolemmal membrane binding sites (SLm) and excluded Fluo-4 [10]. [Mg] was set to 1 mM in all compartments [20].

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