Edited by: Coen Ottenheijm, VU University Medical Center, Netherlands
Reviewed by: Thomas Charles Irving, Illinois Institute of Technology, United States; Brett Colson, The University of Arizona, United States
This article was submitted to Striated Muscle Physiology, a section of the journal Frontiers in Physiology
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Myopathies are notably associated with mutations in genes encoding proteins known to be essential for the force production of skeletal muscle fibers, such as skeletal alpha-actin. The exact molecular mechanisms by which these specific defects induce myopathic phenotypes remain unclear. Hence, in the present study, to better understand actin dysfunction, we conducted a molecular dynamic simulation together with
Congenital myopathies are genetic muscle diseases that start at a very young age. Patients experience weakness in the limb, masticatory and respiratory muscles which can result in severe debilitation, loss of independence and the need for constant care (
Over the last 20 years, a large number of reports have associated these congenital myopathies with mutations in genes encoding proteins fundamental for myofiber structure and function (
When carefully looking at the 200 different single residue substitutions in actin inducing inherited myopathies, only a small portion of these is located on amino acids directly interacting with myosin, or with tropomyosin and/or troponin molecules, which are also involved with actin binding. It is then unclear how the remaining defects interfere with cross-bridge functioning (
After sacrifice, skeletal muscles (extensor digitorum longus, EDL) were dissected from 3 to 4-month old male wild-type and age-matched transgenic mice expressing the
EDL muscles were placed in relaxing solution at 4°C. Bundles of approximately 50 myofibers were dissected free and then tied with surgical silk to glass capillary tubes at slightly stretched lengths. They were then treated with membrane-permeabilizing solution (relaxing solution containing glycerol; 50:50 v/v) for 24 h at 4°C, after which they were transferred to -20°C. In addition, the muscle bundles were treated with sucrose, a cryo-protectant, within 1–2 weeks for long-term storage (
Two to three days prior to X-ray recordings, bundles were de-sucrosed, transferred to a relaxing solution and single myofibers were dissected. Arrays of approximately 30 myofibers were set up. For each myofiber, both ends were clamped to half-split gold meshes for electron microscopy (width = 3 mm), which had been glued to precision-machined ceramic chips (width = 3 mm) designed to fit to a specimen chamber. The arrays were then transferred to the skinning solution and stored at -20°C. Approximately 80 arrays were mounted (10 arrays per mouse – four knock-in and four wild-type mice – corresponding to approximately 2,400 attached fibers) (
On the day of X-ray recordings, arrays were placed in a plastic dish containing a pre-activating solution and washed thoroughly to remove the glycerol. They were then transferred to the specimen chamber, capable of manual length adjustment and force measurement (force transducer, AE801, Memscap, Bernin, France), filled with a pre-activating solution. Mean sarcomere length was measured (laser diffraction) and set to 2.50 μm. Subsequently, X-ray diffraction patterns were recorded at 15°C, first in the pre-activating solution and then in the activating solution (pCa 4.5) when maximal steady-state isometric force was reached.
For each array, approximately 20 to 30 diffraction patterns were recorded (depending on myofiber length) for each solution at the BL45XU beamline of SPring-8. The wavelength was 0.1 nm, and the specimen-to-detector distance was 3.47 m to maximize the spatial resolution to determine filament extension. As a detector, a cooled CCD camera (C4880, Hamamatsu Photonics; 1000 × 1018 pixels) was used in combination with an image intensifier (VP5445, Hamamatsu Photonics). To minimize radiation damage, the exposure time was kept low (1–2 s) and the specimen chamber was moved by 100 μm after each exposure. Moreover, we placed an aluminum plate (thickness, 0.35–0.5 mm) upstream of the specimen chamber. Following the x-ray recordings, background scattering was subtracted, and reflection intensities were determined as described elsewhere. Note that in the literature, the determination of actin spacing (or actin extensibility) is historically performed using actin layer line (ALL) reflections, that is, the 6th ALL (ALL6,
Relaxing and activating solutions contained 4 mM Mg-ATP, 1 mM free Mg2+, 20 mM imidazole, 7 mM EGTA, 14.5 mM creatine phosphate, 324 U/mL creatine phosphokinase, 1000 U/mL catalase, and KCl to adjust the ionic strength to 180 mM and pH to 7.0. Dithiothreitol (DTT) was also added (10 mM). The pre-activating solution was identical to the relaxing solution, except that the EGTA concentration was reduced to 0.5 mM. The concentrations of free Ca2+ were 10-9.0 M (relaxing and pre-activating solutions, pCa 9.0) and 10-4.5 M (activating solution, pCa 4.5).
One actin filament containing the D286G mutants composed of subunits with bound Mg2+-ADP was constructed. This was based on vertebrate (rabbit) skeletal muscle actin Protein Data Bank entry 2ZWH, with a high-affinity Mg2+ cation placed at the nucleotide-binding site and the first solvation shell of explicit waters included. The filament contained 13-monomer subunits as described in
The MD simulations were performed using NAMD 2.10 package (
The unpaired Student’s
In the present work, we studied a missense
Even though we did not observe any change in the internal structure of individual actin monomers with D286G, interestingly, we found that intra-filament distances between monomers were affected by the residue replacement. Indeed, most of the longitudinal and lateral contact distances were lower in filaments carrying D286G than in WT filaments (Table
Comparison of longitudinal and lateral contacts between filaments containing D286G and wild-type (WT) filaments.
Equilibrated distance (A) |
|||
---|---|---|---|
Residue ID | D286G filament | WT filamenta | |
D-loop to C-term | 41 vs. 374 | 12.51 (1.40) | 13.09 (1.50) |
61 vs. 374 | 22.78 (0.82) | 23.29 (1.24) | |
45 vs. 370 | 14.68 (1.54) | 14.85 (2.09) | |
D-loop to SD1 | 45 vs. 169 | 8.36 (1.73) | 9.74 (1.74) |
61 vs. 169 | 12.85 (0.81) | 12.78 (1.40) | |
D-loop to SD3 | 62 vs. 288 | 7.52 (0.77) | 7.52 (1.13) |
SD4 to SD3 | 205 vs. 286 | 9.27 (0.73) | 9.29 (0.53) |
241 vs. 322 | 11.41 (1.74) | 11.03 (1.76) | |
H-plug vs. C | 265 vs. 374 | 19.35 (0.98) | 20.13 (1.15) |
To further investigate intra-filament changes, we assessed the subunit geometries through coarse-graining residues into relatively rigid sub-groups: residues 5–33, 80–147, and 334–349 as SG1; residues 34–39 and 52–69 as SG2; residues 148–179 and 273–333 as SG3; residues 180–219 and 252–262 as SG4. For the analysis, we referred the centres of geometry (COGs) of these residues as R1, R2, R3, and R4 (
Coarse-grained (CG) representation of subunit geometries in filaments expressing D286G and wild-type (WT) filaments.
Parameters | D286G equilibrated | WT equilibratedb | |
---|---|---|---|
R1–R2a | 21.84 (0.39) | 22.99 (0.39) | |
R1–R3 | 24.92 (0.18) | 24.85 (0.28) | |
R3–R4 | 24.87 (0.19) | 24.97 (0.28) | |
R1–R3–R4 | 74.74 (1.83) | 74.42 (1.96) | |
R2–R1–R3 | 105.95 (1.57) | 102.06 (2.06) | |
R2–R1–R3–R4 | 12.01 (3.36) | 10.27 (3.41) | |
Finally, we calculated actin filament persistence length using MD simulation (Supplementary Figure
We conclude that based on our data analysis, D286G results in a decrease in the distance between the D-loop and the C-terminal region as well as in the crossover length. It also induced an increase in the persistence length. As this persistence length spans beyond the distance between adjacent subunits, it is likely that a cooperative mechanism between subunits exists in the presence of D286G allowing a negative propagation to distant subunits.
As the above results from MD simulations imply that actin filaments may have affected stabilities we measured their maximal extensibility by recording and analyzing the actin spacing via X-ray diffraction patterns. Relaxed and activated single membrane-permeabilized myofibers from WT mice and mice expressing both D286G and WT actin molecules were tested. These experiments were possible, as muscles from transgenic mice do not have any signs of sarcomeric ultrastructure (
We determined the actin spacing by using the third order meridional reflection of troponin (TN3,
To conclude, our computational study reveal that D286G disrupts the D-loop interaction with neighboring actin monomers, affecting the flexural rigidity. Despite this, when D286G is mixed with its wild-type form, our
JF, KN, HI, and JO contributed to the conception and design of the work. JF, CC, EM, KN, HI, and JO did the acquisition, analysis, and interpretation of data, drafted the work and revised it critically, approved the final version to be published, and agreed on all aspects of the work.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at: