Abstract
Using a combination of experimental and numerical approaches, we have
tested two different approaches to calculating the sarcoplasmic reticulum
(SR) Ca$^2+$ release flux, which gives rise to cardiac muscle
Ca$^2+$ sparks. By using two-photon excited spot photolysis of
DM-Nitrophen, known Ca$^2+$ release flux time courses were generated
to provide the first experimental validation of spark flux reconstruction
algorithms. These artificial Ca$^2+$ sparks show that it is possible
to calculate the SR Ca$^2+$ release waveform with reasonable
accuracy, provided the flux equations reasonably reflect the properties
of the experimental system. Within cardiac muscle cells, we show
that Ca$^2+$ flux reconstruction is complicated by the substantial
dye binding to proteins, a factor that has not been adequately addressed
in previous flux reconstruction algorithms. Furthermore, our numerical
experiments suggest that the calculated time course of release flux
inactivation based on conventional flux reconstruction algorithms
is likely to be in error. We therefore developed novel algorithms
based on an explicit dye binding scheme. When these algorithm were
applied to evoked Ca$^2+$ sparks in rat cardiac ventricular myocytes,
the reconstructed Ca$^2+$ release waveform peaked in ~5 ms and
decayed with a halftime of approximately 5 ms. The peak flux magnitude
was 7-12 pA, suggesting that sparks must arise from clusters of >15
ryanodine receptors.
- acid,
- action
- adaptation,
- algorithms,
- analysis,
- animals,
- antibodies,
- array
- bilayers,
- biological,
- biosensing
- butyric
- c57bl,
- calcium
- calcium,
- cardiac,
- cardiovascular,
- carrier
- cell
- cells,
- channel
- channels,
- communication,
- comparative
- computer
- computer-assisted,
- conductivity,
- confocal,
- connexins,
- contraction,
- crystalline,
- crystallins,
- culture
- cultured,
- diagnostic
- differentiation,
- diffusion,
- dyes,
- electric
- electrochemistry,
- electrophysiology,
- enhancement,
- epithelial
- epitopes,
- feasibility
- feedback,
- fluorescein,
- fluorescein-5-isothiocyanate,
- fluorescence,
- fluorescent
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