Interplay Between Intracellular Ca(2+) Oscillations and Ca(2+)-stimulated Mitochondrial Metabolism

Abstract

Oscillations of cytosolic Ca(2+) concentration are a widespread mode of signalling. Oscillatory spikes rely on repetitive exchanges of Ca(2+) between the endoplasmic reticulum (ER) and the cytosol, due to the regulation of inositol 1,4,5-trisphosphate receptors. Mitochondria also sequester and release Ca(2+), thus affecting Ca(2+) signalling. Mitochondrial Ca(2+) activates key enzymes involved in ATP synthesis. We propose a new integrative model for Ca(2+) signalling and mitochondrial metabolism in electrically non-excitable cells. The model accounts for (1) the phase relationship of the Ca(2+) changes in the cytosol, the ER and mitochondria, (2) the dynamics of mitochondrial metabolites in response to cytosolic Ca(2+) changes, and (3) the impacts of cytosol/mitochondria Ca(2+) exchanges and of mitochondrial metabolism on Ca(2+) oscillations. Simulations predict that as expected, oscillations are slowed down by decreasing the rate of Ca(2+) efflux from mitochondria, but also by decreasing the rate of Ca(2+) influx through the mitochondrial Ca(2+) uniporter (MCU). These predictions were experimentally validated by inhibiting MCU expression. Despite the highly non-linear character of Ca(2+) dynamics and mitochondrial metabolism, bioenergetics were found to be robust with respect to changes in frequency and amplitude of Ca(2+) oscillations.

Figure 1. Schematic representation of the model for Ca²⁺ dynamics and mitochondrial metabolism.

Non standard abbreviations: J_o: rate of NADH oxidation; J_PDH: rate of NADH production by the pyruvate dehydrogenase, followed by the Krebs cycle; J_ANT: rate of the ATP/ADP translocator; J_x: bidirectional Ca²⁺ leak between the cytosol and mitochondria (model hypothesis); J_IPR: Ca²⁺ flux through the IP₃ receptor. J_AGC: rate of NADH production induced by the MAS NADH shuttle. See text.

Figure 2. Dynamics of Ca²⁺ exchanges between cytosol, ER and mitochondria.

Curves show the simulated changes in Ca²⁺ concentrations in the cytosol (black), the mitochondria (blue) and the ER (red). (A) Sustained oscillations triggered by 1 μM IP₃, followed by the return to a non-stimulated situation (IP₃ = 0.1 μM). (B) Detail of one Ca²⁺ peak occurring when IP₃ = 1 μM allowing a detailed comparison of the phase relationships in the model and in the experiments of Ishii et al. (2006). Parameter values are listed in Table 1.

Figure 3. The Ca²⁺ buffering capacity of mitochondria modifies the amplitude of Ca²⁺ oscillations.

(A) Effect on mitochondrial Ca²⁺. (B) Effect on cytosolic Ca²⁺. Curves indicate the minima and maxima reached during oscillations. Parameter values are listed in Table 1. IP₃ = 1 μM.

Figure 4. The rate of Ca²⁺ entry into mitochondria alters cytosolic Ca²⁺ oscillations.

(A) Relationship between the frequency of oscillations and the rate constant of the MCU. The rate constant of the NCX is the default value (black curve, V_NCX = 0.35 μM.s⁻¹) or is increased (red curve, V_NCX = 1 μM.s⁻¹). (B) Effect of the rate constant of the MCU on cytosolic Ca²⁺ oscillations, as predicted by the model. The black curve shows oscillations for the default value (V_MCU = 0.0006 μM.s⁻¹) given in Table 1, while the red curve shows oscillations obtained when V_MCU = 0. (C,D) Measurement of Ca²⁺ variations in control (C) or MCU-silenced HeLa cells (D). Cells loaded with Fluo4 were perfused with 3 μM histamine for the time shown by the black line. Experiments are representative of more than five trials. See also Supplementary Fig. S4.

Figure 5. Analysis of the bidirectional Ca²⁺ flux between the cytosol and the mitochondria, J_x.

(A) Effect of altering J_x on the simulated Ca²⁺ oscillations. The black curve shows oscillations for the default value (k_x = 0.008 s⁻¹) given in Table 1, while the red curve shows oscillations obtained when k_x = 0. In the latter case, the period of oscillations is slightly increased. IP₃ = 1 μM. (B) Experimental investigation of the effect of inhibiting the mPTP with CSA (1 μM) on the period of Ca²⁺ oscillations in Hela cells. n = 64, 18, 16, 14 for control cells (0.3 and 10 μM histamine) and CSA-treated cells (0.3 and 10 μM histamine), respectively. Two groups were compared with an unpaired student’s t-test and two-tail p-value. Results were considered statistically significant when p < 0.05 (*p < 0.05 and **p < 0.01).

Figure 6. Dynamics of mitochondrial variables.

Curves show the simulated changes in the concentrations of the variables related to mitochondrial metabolism in response to a square-wave Ca²⁺ pulse in the cytosol (1.5 μM for 10 s). In all panels, the horizontal line indicates the time of the Ca²⁺ pulse. Mitochondrial Ca²⁺ is shown in blue and the variable indicated on the vertical left axis in red. Parameter values are listed in Table 1.

Figure 7. Decreasing the glycolytic input slows down the cytosolic Ca²⁺ oscillations.

The curve in black shows cytosolic Ca²⁺ oscillations for the default values of the parameters given in Table 1, while the red curve shows the effect of decreasing the rate of the glycolytic pathway (k_GLY) to 250 μM.s⁻¹. IP₃ = 0.7 μM.

Figure 8. Effect of changing the characteristics of Ca²⁺ spikes on mitochondrial metabolism.

The color code indicates the values of [NADH]_m (A) and [ATP]_m (B) averaged over one period of the Ca²⁺ repetitive spikes. Baseline Ca²⁺ is set to 100 nM and the duration of the spikes always equals 10 s.