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Comparative Study
. 2018 Feb;285(3):481-500.
doi: 10.1111/febs.14367. Epub 2018 Jan 8.

Impaired Muscle Relaxation and Mitochondrial Fission Associated With Genetic Ablation of Cytoplasmic Actin Isoforms

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Free PMC article
Comparative Study

Impaired Muscle Relaxation and Mitochondrial Fission Associated With Genetic Ablation of Cytoplasmic Actin Isoforms

Allison R O'Rourke et al. FEBS J. .
Free PMC article

Abstract

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic βcyto - or γcyto -actin each cause a mild, but progressive myopathy effected by an unknown mechanism. Using transmission electron microscopy, we identified morphological abnormalities in both the mitochondria and the sarcoplasmic reticulum (SR) in aged muscle-specific βcyto - and γcyto -actin KO mice. We found βcyto - and γcyto -actin proteins to be enriched in isolated mitochondrial-associated membrane preparations, which represent the interface between mitochondria and sarco-endoplasmic reticulum important in signaling and mitochondrial dynamics. We also measured significantly elongated and interconnected mitochondrial morphologies associated with a significant decrease in mitochondrial fission events in primary mouse embryonic fibroblasts lacking βcyto - and/or γcyto -actin. Interestingly, mitochondrial respiration in muscle was not measurably affected as oxygen consumption was similar in skeletal muscle fibers from 12 month-old muscle-specific βcyto - and γcyto -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old βcyto - and γcyto -actin muscle-specific KO mice. Our data suggest that impaired Ca2+ re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy.

Keywords: isoforms; mitochondrial dynamics; β-actin; γ-actin.

Figures

Figure 1
Figure 1. Altered sarcoplasmic reticulum and mitochondrial morphology in aged Actg1 and Actb KO skeletal muscle
(A–I) Longitudinal sections of TA from control, Actg1-msKO, and Actb-msKO mice at 6 (A–C), 12 (D–F), and 22–25 (G–I) months of age. White arrowheads denote normal SR, white arrows denote dilated SR. Scale bars = 1µm. (P) Quantification of the percentage of images examined that displayed dilated SR. (J–L) Transverse sections of TA from control, Actg1-msKO, and Actb-msKO mice at 22–25 months of age. Scale bars = 1µm. (M–O) High magnification image of mitochondria internal structure, white arrow heads denote cristae. Scale bar = 250nm. (Q) Quantification of mitochondrial length:width ratio. 1-way ANOVA with Tukey’s post hoc test were performed to determine significance. ** is 0.01
Figure 2
Figure 2. No shift in Z-disk Alignment in Actg1 and Actb KO skeletal muscle
Z-disk alignment of adjacent sarcomere pairs at 6 months (A) and 22–25 months (B). 2-way ANOVA determined no significant difference across genotypes.
Figure 3
Figure 3. Unaltered desmin abundance and localization in Actg1 and Actb KO skeletal muscle
(A–B) Western blot against desmin in quadriceps muscle from control and Actg1-msKO and Actb-msKO animals and quantification of western blot, normalized to GAPDH and relative to the average of the control samples. 1-way ANOVA with Tukey’s post hoc test determined no significant difference. (C–E) Representative isolated EDL muscle fibers stained for desmin localization in green and DAPI in blue. Scale bars = 20µm.
Figure 4
Figure 4. Enrichment of γcyto- and βcyto-actin in isolated mitochondrial associated membrane
Shown are identical western blots loaded with equal amounts of protein from the fractions obtained during the preparation of isolated mitochondrial associated membranes from mouse liver and stained with antibodies to the indicated protein.
Figure 5
Figure 5. Ablation of γcyto- or βcyto-actin results in increased mitochondrial area and decreased fission
(A–F) Mitochondria appear elongated in Actg1 KO Actb KO, and Actb/Actg1 dKO MEFs compared to control (CT). Scale bar = 10µm. (G) Surface area of each individual mitochondrion per genotype. (H) The number of individual (non-branched) mitochondria per genotype. (I) The number of network (branched) mitochondria per genotype. (J) The mean length of each branch off of a network mitochondria. (K) The mean number of branches on a network mitochondria. (L–M). Quantitation of fission and fusion frequencies in CT, Actg1 KO, Actb KO, and Actb/Actg1 dKO MEFs. 1-way ANOVA with Tukey’s post hoc test determined significance * is 0.01
Figure 6
Figure 6. Unaltered abundance of mitochondrial dynamics proteins in isolated mitochondrial from Actg1 and Actb KO skeletal muscles
Western blots and quantification of abundance relative to control average for Fis1 (A–B), MFN2 (C–D), and OPA1 (E–F). 1-way ANOVA with Tukey’s post hoc test determined no significant difference.
Figure 7
Figure 7. Unaltered abundance of electron transport chain complex proteins in Actg1 and Actb KO skeletal muscle
(A) Representative western blot showing immunoreactivity to proteins from complex II, III, and V. (B) Quantification of SDHA, a complex II protein, immunoreactivity normalized to GAPDH and relative to Actg1-msCT or Actb-msCT mean. (C) Quantification of UQCR, a complex III protein, immunoreactivity normalized to GAPDH and relative to Actg1-msCT or Actb-msCT mean. (D) Quantification of ATP5A, a complex V protein, immunoreactivity normalized to GAPDH and relative to Actg1-msCT or Actb-msCT mean. 1-way ANOVA with Tukey’s post hoc test determined no significant difference.
Figure 8
Figure 8. Mitochondrial function is unaffected in Actg1 and Actb KO skeletal muscle
(A–J) Oxygen consumption parameters measured in isolated gastrocnemius fibers. (A) Complex I leak (CI leak), JO2 was measured in the presence of pyruvate, malate, and glutamate (state 2). (B) Complex I respiration rate (CI), JO2 was measured in the presence of ADP (state 3). (C) Complex I+II respiration rate (CI+II), JO2 was measured in the presence of succinate. (D) Complex II respiration rate (CII), JO2 was measured in the presence of rotenone. (E) Complex IV respiration rate, JO2 was measured in the presence of antimycin A, ascorbic acid, and TMPD. (F) Complex I control is the ratio of (JO2 Complex I+II - JO2 Complex II)/JO2 Complex I+II. (G) Succinate Control (JO2 CI+II-JO2 CI)/JO2 CI+II. (H) RCR is the respiratory control ratio it is the ratio of JO2 CI/JO2 CI leak. (I) Biochemical coupling efficiency is the ratio of (JO2 CI - JO2 CI leak)/JO2 CI. (J) E- supercomplex is the ratio of JO2 CI+II/ (JO2 CI + JO2 CII). 1-way ANOVA with Tukey’s post hoc test determined no significant difference.
Figure 9
Figure 9. Unaffected whole body respiration in 22–25 month old muscle-specific Actb KO mice
(A–E) Whole body mitochondrial respirometry of 22–25 month old mice. (A) O2 consumption. (B) CO2 consumption. (C) Heat released. (D) Respiratory exchange ratio of O2 to CO2. (E) Ambulation. (F) Body composition of mice used in study.
Figure 10
Figure 10. Unaltered H2O2 Production in Actg1 and Actb KO skeletal muscle
(A) Succinate stimulated H2O2 production in 12 month old animals in the absence or presence (AF/BCNU) of thioredoxin reductase inhibitor AF (auranofin) and glutathione reductase inhibitor BCNU (Carmustine). (B) The scavenger index is the percent increase in succinate induced H2O2 production relative to H2O2 production when inhibitors are present.
Figure 11
Figure 11. Impaired relaxation rates in Actg1 and Actb KO skeletal muscle
(A–B) Representative isometric contraction force tracings of tetanically-stimulated 12 month old control, Actg1-msKO (A), and Actb-msKO (B) EDL muscles. (C) Peak specific forces generated during maximal isometric tetanic contractions. (D) Maximal rates of relaxation. 1-way ANOVA with Tukey’s post hoc test were performed to determine significance. # or * is 0.01<p<0.05, ** is 0.01<p<0.001, and *** is p<0.001. Error bars are S.E.M.
Figure 12
Figure 12. Unaltered SERCA 1 and SR protein abundance in Actg1 and Actb KO skeletal muscle
(A) Quantification of SERCA 1 immunoreactivity normalized to GAPDH relative to control average. (B) Small Ankyrin 1 representative western blot on SR fractions isolated from skeletal muscle. (C) Quantification of small ankryin 1 protein by western blot, normalized to protein load, relative to control average. (D) Calsequestrin representative western blot on SR fractions isolated from skeletal muscle. (E) Quantification of calsequestrin protein by western blot, normalized to protein load, relative to control average. 1-way ANOVA with Tukey’s post hoc test determined no significant difference.
Figure 13
Figure 13. Unaffected SERCA ATPase Activity in SR from Actg1 and Actb KO skeletal muscle
Activity of SERCA (A) Actb-msKO in high (pCa5) calcium, low (pCa8) calcium and Vmax (pCa5-pCa8). SERCA activity relative to SERCA abundance as measured by western blot for (B) Actb-msKO. 1-way ANOVA with Tukey’s post hoc test determined no significant difference.
Figure 14
Figure 14. ER Stress Response Pathway Proteins are not upregulated in muscle-specific Actb KO mice at 12 months old
Western blot showing immunoreactivity to ER stress pathway proteins. Quantification of immunoreactivity to PDI (A), BiP (B), PERK (C), and IRE1α (D). All samples were normalized to GAPDH and relative to Actg1-msCT or Actb-msCT mean. 1-way ANOVA with Tukey’s post hoc test determined no significant difference.

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