Ca2+ sparks and Ca2+ waves in saponin-permeabilized rat ventricular myocytes - PubMed (original) (raw)

Ca2+ sparks and Ca2+ waves in saponin-permeabilized rat ventricular myocytes

V Lukyanenko et al. J Physiol. 1999.

Abstract

1. We carried out confocal Ca2+ imaging in myocytes permeabilized with saponin in 'internal' solutions containing: MgATP, EGTA and fluo-3 potassium salt. 2. Permeabilized myocytes exhibited spontaneous Ca2+ sparks and waves similar to those observed in intact myocytes loaded with fluo-3 AM. 3. In the presence of 'low' [EGTA] (0.05 mM), Ca2+ waves arose regularly, even at relatively low [Ca2+] (50-100 nM, free). Increasing [EGTA] resulted in decreased frequency and propagation velocity of Ca2+ waves. Propagating waves were completely abolished at [EGTA] > 0.3 mM. 4. The frequency of sparks increased as a function of [Ca2+] (50-400 nM range) with no sign of a high affinity Ca2+-dependent inactivation process. 5. The rate of occurrence of Ca2+ sparks was increased by calmodulin and cyclic adenosine diphosphate-ribose (cADPR).

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Figures

Figure 1

Figure 1. The effects of permeabilization on Ca2+ sparks and Ca2+ waves in ventricular myocytes

A, images of a cardiac myocyte obtained in transmitted light before and after permeabilization with saponin. B, line-scan images of fluorescence in a portion of the same cell pre-loaded with fluo-3 AM measured before permeabilization (a, [Ca2+]o= 5 m

m

), after permeabilization in an internal solution with no dye (b) and after addition to the internal solution 30 μ

m

fluo-3 potassium salt in the presence of 0.1 (c) or 0.5 m

m

EGTA (d) (pCa 7). Calibration bars: horizontal 10 μm, vertical 0.4 s, the colour bar represents changes in units of absolute fluorescence. C, surface plots of Ca2+ sparks measured before permeabilization (a) and after permeabilization in the presence of 0.1 or 0.5 m

m

EGTA (b and c, respectively). Each plot was obtained by averaging 10 individual events.

Figure 2

Figure 2. Effects of calcium buffering on Ca2+ waves in saponin-permeabilized myocytes

A, B, C and D, representative line-scan images of fluorescence recorded in a permeabilized myocyte in the presence of 0.05 m

m

(A), 0.1 m

m

(B), 0.2 m

m

(C) and 0.3 m

m

EGTA (D). Free [Ca2+] in all cases was adjusted to 100 n

m

. Calibration bars: horizontal 15 μm, vertical 0.35 s.

Figure 3

Figure 3. Effects of [Ca2+]i on Ca2+ sparks and Ca2+ waves in saponin-permeabilized myocytes

A, representative line-scan images of fluorescence recorded in a permeabilized myocyte at various [Ca2+]i levels (indicated at the top of the respective images) in the presence of 0.1 m

m

EGTA. B, line-scan images of Ca2+ sparks corrected for increases of background fluorescence at various [Ca2+]i levels (indicated at the top of the respective images) in the presence of 0.5 m

m

EGTA. Calibration bars: horizontal 15 μm (A) and 20 μm (B), vertical 0.5 s (A) and 0.1 s (B). C, Ca2+ spark frequency (blue) and amplitude (light grey) as a function of time before and after elevating [Ca2+]i to indicated levels for the experiment shown in B. D, Ca2+ spark frequency as a function of [Ca2+]i. The values are represented as means ±

s.e.m.

obtained in 5 experiments. The lines were obtained by fitting the data according to the equation _f_=_f_max{[Ca2+]n/([Ca2+]n+K_D_n)}, where _f_max= 10 000 events s−1 (100 μm)−1, _K_D= 9.9 μ

m

and _n_= 1.6 (blue line); _f_max= 20 000 events s−1 (100 μm)−1; _K_D= 15 μ

m

and _n_= 1.6 (red line); and _f_max= 30 000 events s−1 (100 μm)−1; _K_D= 20 μ

m

and _n_= 1.6 (green line).

Figure 4

Figure 4. The effects of elevating [Ca2+]i on sparking activity in the presence of thapsigargin

A, representative line-scan images of fluorescence acquired before (a and b) and at different times (2 and 3 min) after increasing [Ca2+]i from 80 n

m

to 250 n

m

in the presence of thapsigargin (c and d, respectively). Thapsigargin (10 μ

m

) was introduced into the bathing solution 1 min before elevating [Ca2+]i. Calibration bars: horizontal 20 μm, vertical 0.15 s. B, Ca2+ spark frequency as a function of time before and after the addition of thapsigargin into the bathing solution in the same experiment. The experimental protocol is presented schematically at the top.

Figure 5

Figure 5. Effects of calmodulin and cADPR on Ca2+ sparks

A, representative line-scan images of fluorescence changes acquired under control conditions (left-hand panel), 15 min after exposure of the cells to 5 μ

m

calmodulin (middle panel), and 10 min after changing back to the control solution (right-hand panel). Calibration bars: horizontal 10 μm, vertical 0.2 s. B, caffeine-induced Ca2+ transients measured in the same cell at the same stages of the experiment as in A. Caffeine (20 m

m

) was applied for 2 s. C, representative line-scan images of fluorescence changes measured under control conditions (left-hand panel), 2 min after exposure of the cells to 5 μ

m

cADPR (middle panel), and 5 min after reverting back to the control solution (right-hand panel). Calibration bars: horizontal 10 μm, vertical 0.2 s.

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