Journal article
Direct measurement of SR release flux by tracking ‘Ca2+ spikes’ in rat cardiac myocytes
The Journal of physiology, Vol.512(Pt 3), pp.677-691
11/01/1998
DOI: 10.1111/j.1469-7793.1998.677bd.x
PMCID: PMC2231234
PMID: 9769413
Abstract
Ca
2+
release flux across the sarcoplasmic reticulum (SR) during cardiac excitation-contraction coupling was investigated using a novel fluorescence method. Under whole-cell voltage-clamp conditions, rat ventricular myocytes were dialysed with a high concentration of EGTA (4.0 m
m
, 150 n
m
free Ca
2+
), to minimize the residence time of released Ca
2+
in the cytoplasm, and a low-affinity, fast Ca
2+
indicator, Oregon Green 488 BAPTA-5N (OG-5N; 1.0 m
m
,
K
d
≈ 31 μ
m
), to optimize the detection of localized high [Ca
2+
] in release site microdomains. Confocal microscopy was employed to resolve intracellular [Ca
2+
] at high spatial and temporal resolution.
Analytical and numerical analyses indicated that, under conditions of high EGTA concentration, the free [Ca
2+
] change is the sum of two terms: one major term proportional to the SR release flux/Ca
2+
influx, and the other reflecting the running integral of the released Ca
2+
.
Indeed, the OG-5N transients in EGTA-containing cells consisted of a prominent spike followed by a small pedestal. The OG-5N spike closely resembled the first derivative (d[Ca
2+
]/d
t
) of the conventional Ca
2+
transient (with no EGTA), and mimicked the model-derived SR Ca
2+
release function reported previously. In SR Ca
2+
-depleted cells, the OG-5N transient also closely followed the waveform of L-type Ca
2+
current (
I
Ca
). Using
I
Ca
as a known source of Ca
2+
influx, SR flux can be calibrated
in vivo
by a linear extrapolation of the
I
Ca
-elicited OG-5N signal.
The OG-5N image signal was localized to discrete release sites at the Z-line level of sarcomeres, indicating that the local OG-5N spike arises from ‘Ca
2+
spikes’ at transverse (T) tubule-SR junctions (due to the imbalance between calcium ions entering the cytosol and the buffer molecules).
Both peak SR release flux and total amount of released Ca
2+
exhibited a bell-shaped voltage dependence. The temporal pattern of SR release also varied with membrane voltage: Ca
2+
release was most synchronized and produced maximal peak release flux (4.2 m
m
s
−1
) at 0 mV; in contrast, maximal total release occurred at −20 mV (71
versus
61 μ
m
at 0 mV), but the localized release signals were partially asynchronous. Since the maximal conventional [Ca
2+
] transient and contraction were elicited at 0 mV, it appears that not only the amount of Ca
2+
released, but also the synchronization among release sites affects the whole-cell Ca
2+
transient and the Ca
2+
-myofilament interaction.
Details
- Title: Subtitle
- Direct measurement of SR release flux by tracking ‘Ca2+ spikes’ in rat cardiac myocytes
- Creators
- Long-Sheng SongJames S K ShamMichael D SternEdward G LakattaHeping Cheng
- Resource Type
- Journal article
- Publication Details
- The Journal of physiology, Vol.512(Pt 3), pp.677-691
- DOI
- 10.1111/j.1469-7793.1998.677bd.x
- PMID
- 9769413
- PMCID
- PMC2231234
- NLM abbreviation
- J Physiol
- ISSN
- 0022-3751
- eISSN
- 1469-7793
- Publisher
- Blackwell Science Inc
- Language
- English
- Date published
- 11/01/1998
- Academic Unit
- Cardiovascular Medicine; Fraternal Order of Eagles Diabetes Research Center; Biochemistry and Molecular Biology; Internal Medicine
- Record Identifier
- 9984094767002771
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