Temperature dependence and thermodynamic properties of Ca2+ sparks in rat cardiomyocytes

Abstract

To elucidate the temperature dependence and underlying thermodynamic determinants of the elementary Ca2+ release from the sarcoplasmic reticulum, we characterized Ca2+ sparks originating from ryanodine receptors (RyRs) in rat cardiomyocytes over a wide range of temperature. From 35 degrees C to 10 degrees C, the normalized fluo-3 fluorescence of Ca2+ sparks decreased monotonically, but the Delta[Ca2+]i were relatively unchanged due to increased resting [Ca2+]i. The time-to-peak of Ca2+ sparks, which represents the RyR Ca2+ release duration, was prolonged by 37% from 35 degrees C to 10 degrees C. An Arrhenius plot of the data identified a jump of apparent activation energy from 5.2 to 14.6 kJ/mol at 24.8 degrees C, which presumably reflects a transition of sarcoplasmic reticulum lipids. Thermodynamic analysis of the decay kinetics showed that active transport plays little role in early recovery but a significant role in late recovery of local Ca2+ concentration. These results provided a basis for quantitative interpretation of intracellular Ca2+ signaling under various thermal conditions. The relative temperature insensitivity above the transitional 25 degrees C led to the notion that Ca2+ sparks measured at a "warm room" temperature are basically acceptable in elucidating mammalian heart function.

FIGURE 1

Confocal imaging of spontaneous Ca²⁺ sparks. (A). Representative line-scan images recorded in a rat ventricular myocyte at 35°C, 25°C, and 15°C (from left to right), respectively. For each image, time runs horizontally, and space runs vertically. (B). Temperature dependence of Ca²⁺ spark frequency (n = 17 cells each; p < 0.05). The frequency was calculated by dividing the total number of Ca²⁺ sparks by the product of distance (μm) and time (s) of line scanning.

FIGURE 2

Two-dimensional and three-dimensional demonstration of the averages of 64 original Ca²⁺ sparks recorded at 35°C, 25°C, and 15°C, respectively. The original events were aligned by their takeoff times and spatial mass centers.

FIGURE 3

Temperature dependence of spark amplitude. (A). An overlay of the spatial profiles at the peak of the averaged sparks shown in Fig. 2. (B). Temperature dependence of spark amplitude (F/F₀). Each data point represents an average of more than 240 sparks from 17 cells in six animals (p < 0.05). (C). Temperature dependence of resting [Ca²⁺]_i in rat ventricular myocytes (n = nine cells from three animals; p < 0.01). (D). Ca²⁺ spark amplitude in terms of Δ[Ca²⁺]_i was calculated from the data in B and C using Eq. 1.

FIGURE 4

Temperature dependence of the temporal and spatial properties Ca²⁺ sparks. (A). An overlay of the time courses of the averaged sparks shown in Fig. 2. (B). Temperature dependence of FDHM of spark amplitude (F/F₀) (p < 0.01). (C). Temperature dependence of FWHM of Ca²⁺ sparks (p > 0.05).

FIGURE 5

Thermodynamic analysis of RyR Ca²⁺ release duration. (A). Histograms showing the distributions of time-to-peak of Ca²⁺ sparks at 35°C, 25°C, and 15°C, respectively. (B). Temperature dependence of the time-to-peak of Ca²⁺ sparks (p < 0.01). Note the break of trend at ∼25°C. (C). Arrhenius plot of the reciprocal of time-to-peak. The data were fitted by a combination of two lines with Eq. 3. Best fit was achieved by lines intersecting at 24.8°C.

FIGURE 6

Temperature dependence of the spatial dispersion of spark wavefronts at bottom 10%. (A). Spatiotemporal wave front profiles of the Ca²⁺ sparks, each from the average of 64 original events. The curves represent the fitting to the data with Eq. 4. (B). Temperature dependence of the apparent diffusion coefficient D_a determined as in A (p < 0.01). (C). Thermodynamic analysis of D_a. The solid lines represent linear fitting with Eq. 5.

FIGURE 7

Thermodynamic analysis of recovery kinetics of Ca²⁺ sparks. (A and C). Temperature dependence of half-decay time (A) and late time constant (C) of Ca²⁺ sparks (p < 0.01). (B and D). Arrhenius plot of the reciprocal of half-decay time (B) and late time constant (D). The solid lines represent linear fitting with Eq. 3.