Modeling the dependence of the period of intracellular Ca2+ waves on SERCA expression

Abstract

Contrary to intuitive expectations, overexpression of sarco-endoplasmic reticulum (ER) Ca(2+) ATPases (SERCAs) in Xenopus oocytes leads to a decrease in the period and an increase in the amplitude of intracellular Ca(2+) waves. Here we examine these experimental findings by modeling Ca(2+) release using a modified Othmer-Tang-model. An increase in the period and a reduction in the amplitude of Ca(2+) wave activity are obtained when increases in SERCA density are simulated while keeping all other parameters of the model constant. However, Ca(2+) wave period can be reduced and the wave amplitude and velocity can be significantly increased when an increase in the luminal ER Ca(2+) concentration due to SERCA overexpression is incorporated into the model. Increased luminal Ca(2+) occurs because increased SERCA activity lowers cytosolic Ca(2+), which is partially replenished by Ca(2+) influx across the plasma membrane. These simulations are supported by experimental data demonstrating higher luminal Ca(2+) levels, decreased periods, increased amplitude, and increased velocity of Ca(2+) waves in response to increased SERCA density.

FIGURE 1

Simulated spiral waves at different degrees of SERCA expression (top left: P^max = 0.594 μMs⁻¹; top right: P^max = 1.194 μMs⁻¹; bottom left: P^max = 3.550 μMs⁻¹; bottom right: P^max = 5.394 μMs⁻¹). The resting level of Ca²⁺ is 69.0 nM. The examples are on the solid lines in Fig. 2. The size of the area shown is 620 × 620 μm².

FIGURE 2

Characteristics of simulated spiral waves in dependence on pump strength P^max. Left panel: [IP₃] = 0.3 μM, resting level of free Ca²⁺ in the cytosol is 69.0 nM (solid line) and 40.2 nM (dashed line). Right panel: [IP₃] = 0.08 μM, resting level of free Ca²⁺ in the cytosol is 32.4 nM (solid line) and 21.8 nM (dashed line). C_T was calculated according to Eq.4. The curves in Figs. 2 and 6 are not smooth everywhere. The reason for the fluctuations is meandering of the spiral tip. Meandering is a motion of the spiral tip on a trajectory outlining a petal-like pattern and causes modulation of amplitude and period (Falcke et al., 1999b). The time T_1/2 is the time for the Ca²⁺ concentration to decrease from the maximum to 50% of the maximum in the back of the pulse.

FIGURE 3

Western blot of SERCA2b overexpressing oocytes. Oocyte lysates were prepared and run on 12% SDS PAGE. Following transfer, the membranes were probed with an anti-SERCA2 antibody (gift of J. Lytton). Lanes were loaded as follows: (a) endogenous SERCA2b (no mRNA control); (b) 3.25 ng; (c) 6.5 ng; (d) 13 ng; and (e) 26 ng. Normalizing the intensity relative to the endogenous control (0 ng mRNA) gives increases in relative protein levels of 1 (0 ng), 1.5 (3.2 ng), 6.3-fold (6.5 ng), 7.5-fold (13 ng), and 11.1-fold (26 ng).

FIGURE 4

Dependence of Ca²⁺ wave period, T_1/2, amplitude and velocity on SERCA2b density. Ca²⁺ wave parameters are plotted as mean ± SE.

FIGURE 5

Spatio-temporal plots of Ca²⁺ wave activity in zero extracellular Ca²⁺. Panels A and B show experimental results; panels C and D show simulations. Oocytes were injected with low (3.2 ng) (panel A) and high (26 ng) (panel B) concentrations of mRNA encoding SERCA. Representative wave patterns are shown next to the time series. The ΔF/F₀ values were recorded at the spot marked in the wave pattern. Panels C and D show simulated spiral wave amplitudes recorded approximately one spiral revolution off the spiral core. The loss of Ca²⁺ through the plasma membrane was modeled by an exponential decay of C_T. Panel C shows the low SERCA density case C_T = 11.565 μM e^−t/1000s, P^max = 4.97 μMs⁻¹, panel D shows results with high SERCA density C_T = 141.35 μM e^−t/500s, P^max = 54.6 μMs⁻¹. The base line of the simulated concentrations decreases from 69 nM to 27 nM in panel C and from 48 nM to 30 nM in panel D in the course of the simulation.

FIGURE 6

Characteristics of simulated spiral waves in dependence on pump strength of additionally expressed SERCAs with a dissociation constant K₁ different from that one of the endogenous pumps. The parameter P₁^max indicates the degree of expression. Left panel: [IP₃] = 0.3 μM and different dissociation constants K₁: 0.112 μM (thick solid line); 0.136 μM (thick dashed line); 0.164 μM (thin solid line); 0.188 μM (thin dashed line). The resting level of free Ca²⁺ in the cytosol is 69.0 nM. Right panel: [IP₃] = 0.08 μM and different dissociation constants K₁: 0.112 μM (thick solid line); 0.124 μM (thick dashed line); 0.136 μM (thin solid line), 0.188 μM (thin dashed line). The resting level of free Ca²⁺ in the cytosol is 28.1 nM.