43 research outputs found

    A 3D diffusional-compartmental model of the calcium dynamics in cytosol, sarcoplasmic reticulum and mitochondria of murine skeletal muscle fibers

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    <div><p>Variations of free calcium concentration ([Ca<sup>2+</sup>]) are powerful intracellular signals, controlling contraction as well as metabolism in muscle cells. To fully understand the role of calcium redistribution upon excitation and contraction in skeletal muscle cells, the local [Ca<sup>2+</sup>] in different compartments needs to be taken into consideration. Fluorescent probes allow the determination of [Ca<sup>2+</sup>] in the cytosol where myofibrils are embedded, the lumen of the sarcoplasmic reticulum (SR) and the mitochondrial matrix. Previously, models have been developed describing intracellular calcium handling in skeletal and cardiac muscle cells. However, a comprehensive model describing the kinetics of the changes in free calcium concentration in these three compartments is lacking. We designed a new 3D compartmental model of the half sarcomere with radial symmetry, which accounts for diffusion of Ca<sup>2+</sup> into the three compartments and simulates its dynamics at rest and at various rates of stimulation in mice skeletal muscle fibers. This model satisfactorily reproduces both the amplitude and time course of the variations of [Ca<sup>2+</sup>] in the three compartments in mouse fast fibers. As an illustration of the applicability of the model, we investigated the effects of Calsequestrin (CSQ) ablation. CSQ is the main Ca<sup>2+</sup> buffer in the SR, localized in close proximity of its calcium release sites and near to the mitochondria. CSQ knock-out mice muscles still preserve a near-normal contractile behavior, but it is unclear whether this is caused by additional SR calcium buffering or a significant contribution of calcium entry from extracellular space, via stored-operated calcium entry (SOCE). The model enabled quantitative assessment of these two scenarios by comparison to measurements of local calcium in the cytosol, the SR and the mitochondria. In conclusion, the model represents a useful tool to investigate the impact of protein ablation and of pharmacological interventions on intracellular calcium dynamics in mice skeletal muscle.</p></div

    Simulation of the free calcium concentrations in CSQ-KO with an AB buffering capacity of 10% of that of CSQ.

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    <p>Simulation of the free calcium concentrations in the three compartments at 1 and 60 Hz as predicted by the model without CSQ (CSQ-KO) with a AB buffering capacity of 10% of that of CSQ and with doubled P<sub>max</sub> value. Red lines represent the mean value (“m” for the cytosolic space) or the steady value before the beginning of the next stimulus (“s” in SR and mitochondrion). Above each panel the values for m or s are reported.</p

    Experimental average [Ca<sup>2+</sup>] in μM for CSQ-KO mouse muscle fibers measured in the steady state attained during stimulation trains at various rates (1, 5, 20, 60 Hz).

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    <p>Experimental average [Ca<sup>2+</sup>] in μM for CSQ-KO mouse muscle fibers measured in the steady state attained during stimulation trains at various rates (1, 5, 20, 60 Hz).</p

    Schematic representation of the 3D compartmental diffusional model of murine skeletal muscle.

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    <p>The unit cell of the skeletal muscle fiber, the half sarcomere, is approximated as a cylinder, so that rotational symmetry allows for analysis in 2D space. This space is occupied by three main compartments: sarcoplasmic reticulum, cytosolic space and mitochondrion. The sarcoplasmic reticulum is divided into m longitudinal sub-compartments and the cytosolic space is divided into m longitudinal and n-1 radial sub-compartments. Diffusion of Ca<sup>2+</sup> ions is analyzed between sub-compartments. The mitochondrial compartment is close, but external, to extramyofibrillar space (EF). The mitochondrial calcium uniporter (MCU in black) and sodium calcium exchanger (NCE in red) sense the [Ca<sup>2+</sup>] in the sub-compartment 125 nm away from the RyR, toward Z-line. Several buffers are present in distinct sub-compartments: calsequestrin (CSQ) and additional buffers (AB) in sarcoplasmic reticulum, parvalbumin (PVA) in all cytosolic spaces, troponin in the myofibrillar spaces, and a mitochondrial buffer (B) in the mitochondrial matrix. Store operated Ca<sup>2+</sup> entry (SOCE) is depicted by the vertical arrows from the T-tubular network extended toward the Z-line (green).</p

    Simulation of the free calcium concentrations.

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    <p>Simulation of the free calcium concentrations in the three compartments at 1 and 60 Hz in the WT model at the beginning and at the end of the train of stimulation. Red lines represent the mean value (“m” for the cytosolic space) or the steady value before the beginning of the next stimulus (“s” in SR and mitochondrion). Above each panel the values for m or s are reported.</p

    Comparison between the simulation and experiments in CSQ-KO with an AB buffering capacity of 20% of that of CSQ.

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    <p>Comparison between the simulated steady state [Ca<sup>2+</sup>] in the three compartments and the experimental data for 1, 5, 20 and 60 Hz in CSQ-KO mouse with increased AB buffering to 20% of the capacity of CSQ. [Ca<sup>2+</sup>] is reported in μM in cytosol or mitochondrion (black and red lines, respectively) or in mM for SR (blue line). Dotted lines stand for experimental data, continuous lines for simulations.</p

    Experimental data and model simulations of calcium transients in a 2 s 60Hz stimulation train.

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    <p>Experimental data (left column, from [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201050#pone.0201050.ref012" target="_blank">12</a>] and [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201050#pone.0201050.ref013" target="_blank">13</a>] [Ca<sup>2+</sup>] calculated from Fura 2 ratio and YFP/CFP ratios according to the equations reported in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201050#pone.0201050.s008" target="_blank">S5 File</a>) and simulations (right column) of the [Ca<sup>2+</sup>] transients in the cytosol (top), SR (middle) and mitochondrion (bottom) during a 2s stimulation train at 60 Hz. It can be seen that the steady-state values reached in the experiments agree well with the model simulations. In addition the time courses agree rather well considering that the model is based solely on previously published parameters. The amplitude of the oscillations in the model simulations are larger than in the experimental recordings, but this is as expected because the experimental recordings were smoothed by an x-point running average to reduce noise. In the inset of the cytosolic calcium simulation (top-right) the local Ca<sup>2+</sup> concentration is shown (dotted line) in the element (microdomain) in front of MCU and NCE and compared to the average cytosolic [Ca<sup>2+</sup>], showing an approximately two-fold increase. The lower-right panel shows the asymmetry in the rising phase (at the beginning of the train of stimuli), and at the decay phase (at the end of the train of stimuli) in the simulated mitochondrial [Ca<sup>2+</sup>].</p

    Simulated/Experimental ratio for [Ca<sup>2+</sup>] in μM for WT and CSQ-KO fibers, assuming the amount bound by secondary buffer capacity at 10% or at 20%.

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    <p>The values are calculated at the steady state during the trains of stimulations, as in the Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201050#pone.0201050.g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201050#pone.0201050.g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201050#pone.0201050.g007" target="_blank">7</a>.</p

    Comparison between the simulation and experiments in WT muscle fibers.

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    <p>The simulated [Ca<sup>2+</sup>]reached at steady state in the three compartments and the experimental data for 1, 5, 20 and 60 Hz in WT mouse fibers are compared with experimental values. The free model parameters are adjusted to fit the data at 60 Hz. [Ca<sup>2+</sup>] is reported in μM in cytosol or mitochondrion (black and red lines, respectively) or in mM for SR (blue line). Dotted lines: experimental data; continuous lines: model predictions.</p

    Comparison between the simulation and experiments in CSQ-KO with an AB buffering capacity of 10% of that of CSQ.

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    <p>Comparison between the simulated steady state [Ca<sup>2+</sup>] in the three compartments and the experimental data for 1, 5, 20 and 60 Hz in CSQ-KO mouse with 10% of total calcium bound in the SR at rest accounted by AB and with doubled Pmax value. [Ca<sup>2+</sup>] is reported in μM in cytosol or mitochondrion (black and red lines, respectively) or in mM for SR (blue line). Dotted lines stand for experimental data, continuous lines for simulations. The model without CSQ (CSQ-KO), with 10% of AB capacity and double Pmax fails to match the observed values in all three compartments, with predicted values lower than experimental data.</p
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