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Abstract
We carried out a series of experiment demonstrating the role of mitochondria in the cytosolic and mitochondrial Ca2+ transients and compared the results with those from computer simulation. In rat ventricular myocytes, increasing the rate of stimulation (1∼3 Hz) made both the diastolic and systolic [Ca2+] bigger in mitochondria as well as in cytosol. As L-type Ca2+ channel has key influence on the amplitude of Ca2+-induced Ca2+ release, the relation between stimulus frequency and the amplitude of Ca2+ transients was examined under the low density (1/10 of control) of L-type Ca2+ channel in model simulation, where the relation was reversed. In experiment, block of Ca2+ uniporter on mitochondrial inner membrane significantly reduced the amplitude of mitochondrial Ca2+ transients, while it failed to affect the cytosolic Ca2+ transients. In computer simulation, the amplitude of cytosolic Ca2+ transients was not affected by removal of Ca2+ uniporter. The application of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) known as a protonophore on mitochondrial membrane to rat ventricular myocytes gradually increased the diastolic [Ca2+] in cytosol and eventually abolished the Ca2+ transients, which was similarly reproduced in computer simulation. The model study suggests that the relative contribution of L-type Ca2+ channel to total transsarcolemmal Ca2+ flux could determine whether the cytosolic Ca2+ transients become bigger or smaller with higher stimulus frequency. The present study also suggests that cytosolic Ca2+ affects mitochondrial Ca2+ in a beat-to-beat manner, however, removal of Ca2+ influx mechanism into mitochondria does not affect the amplitude of cytosolic Ca2+ transients.
References
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Fig. 1.
Comparison of APs generated by experiment (A) and model simulation (B). (A) Myocytes were stimulated at a rate of 1 Hz and a representative trace of AP was obtained after 200 sec of stabilization period. Resting membrane potential was at –76.3±3.7 mV (n=21). APD50 and APD90 of the representative recording were 21.1 ms and 42.9 ms, respectively. Recordings were obtained at room temperature (22∼25°C). (B) Model-generated AP. Resting membrane potential was at –80.2 mV. APD50 and APD90 were 22.2 ms and 43.4 ms, respectively. Temperature was set at 24°C (Table 1).
Fig. 2.
Stimulus frequency-dependent change in cytosolic Ca2+ transients recorded by experiment (A) and model (B). (A) Myocytes were field stimulated at a rate of 0.2 Hz for 200 sec and the rate was sequentially changed from 0.2 to 1 Hz, 2 Hz, and 3 Hz. Fura-2 fluorescence ratio (F340/F380) was recorded to see change in cytosolic Ca2+ transients. Expanded trace of fluorescence ratio during 2 Hz stimulation was demonstrated on the right panel. The expanded trace was chosen from our other experimental data. (B) Model-generated cytosolic Ca2+ transients. Expanded trace during 2 Hz stimulation was also displayed on the right panel. The downward arrow indicates the point where the expanded trace was extracted.
Fig. 3.
Negative staircase in the model with reduced ICaL. (A) Model simulation with control. The rate of stimulation was switched between 0.2 and 0.8 Hz. (B) Model simulation with reduced ICaL. Channel density of L-type Ca2+ channel was reduced to 1/10 of control. The stimulation protocol is the same as that one used in A. (C) Model simulation with increased Ito. Channel density of Ca2+-independent transient outward K+ current (Ito) was increased to 10-times of control.
Fig. 4.
Stimulus frequency-dependent change in mitochondrial Ca2+ transients by experiment (A) and model simulation (B). (A) Myocytes were field stimulated and the rate was switched between 0.2 Hz and test frequency (1 Hz, 2 Hz, and 3 Hz). Rhod-2 intensity was recorded to monitor change in mitochondrial Ca2+ transients. Output intensity was normalized to diastolic level at control in order to get a pseudo ratio unit (arbitrary unit). Expanded trace of normalized Rhod-2 intensity during 2 Hz stimulation was presented on the right panel. The downward arrow indicates the point where the expanded trace was extracted. (B) Model-generated mitochondrial Ca2+ transients. Expanded trace during 2 Hz stimulation was also shown on the right panel.
Fig. 5.
The effect of ruthenium red (10μM) on mitochondrial and cytosolic Ca2+ transients. Stimulation rate is 0.2 Hz. (A) Mitochondrial Ca2+ transients during perfusion of ruthenium red. Cells were preincubated with ruthenium red for 5 min before recording. (B) Cytosolic Ca2+ transients during perfusion of ruthenium red.
Fig. 6.
The effect of Ca2+ uniporter removal on mitochondrial and cytosolic Ca2+ transients in model simulation. Stimulation rate is 0.2 Hz. Mitochondrial Ca2+ transients with (A) or without (C) Ca2+ uniporter activity. Cytosolic Ca2+ transients with (B) or without (D) Ca2+ uniporter activity.
Fig. 7.
The effect of mitochondrial protonophore on cytosolic Ca2+ transients. Stimulation rate is 0.2 Hz. 1μM FCCP eventually abolished Ca2+ transients. Cells became round-up after 5 min perfusion of FCCP. Arrow (A) indicates the point where the expanded trace before FCCP was extracted while arrow (B) indicates the point where expanded trace after FCCP was extracted.
Fig. 8.
Simulation of the effects of mitochondrial protonophore on cytosolic Ca2+ transients and ATP content. Stimulation rate is 0.2 Hz. 106-fold increase in H+ permeability through inner mitochondrial membrane was tested on the model simulation. (A) Change in cytosolic Ca2+ transients during stimulation. (B) Change in cytosolic ATP content during stimulation.
Table 1.
General parameters
Table 2.
Ca2+-binding proteins
Table 3.
Initial values of parameters related with state variables
Table 4.
Initial concentrations of ions and energy metabolites in extramitochondrial space
Table 5.
Definition and initial values of parameters in mitochondrial space
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