Impaired muscle relaxation and mitochondrial fission associated with genetic ablation of cytoplasmic actin isoforms

Abstract

While α-actin isoforms predominate in adult striated muscle, skeletal muscle-specific knockouts (KOs) of nonmuscle cytoplasmic β - or γ -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 β - and γ -actin KO mice. We found β - and γ -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 β - and/or γ -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 β - and γ -actin KO mice. Instead, we found that the maximal rate of relaxation after isometric contraction was significantly slowed in muscles of 12-month-old β - and γ -actin muscle-specific KO mice. Our data suggest that impaired Ca re-uptake may presage development of the observed SR morphological changes in aged mice while providing a potential pathological mechanism for the observed myopathy.

Essential nucleotide- and protein-dependent functions ofActb/β-actin

“…12) contrast with conventional cre-mediated knockout of a floxed Actb allele, where exons 2 and 3 were deleted, and which caused embryonic lethality in mice (8). Tissue-specific knockout of the same allele also resulted in a variety of phenotypes with muscle showing progressive myofiber degeneration/regeneration, muscle weakness, impaired mitochondrial fission (17,21), and the CNS showing tissue morphology defects that correlated with hyperactivity and cognitive behavior impairments (16). Additionally, isolated MEFs failed to divide or migrate, suggesting β-actin was required for fundamental cellular processes (6,8).…”

Induced in vivo knockdown of the Brca1 gene in skeletal muscle results in skeletal muscle weakness

“…Permeabilized muscle fibre bundles were prepared from portions of the medial gastrocnemius muscle as a result of its oxidative phenotype and ease of mechanical fibre separation, as described previously (O'Rourke et al . ). Medial gastrocnemius muscle was used because of its predominantly red phenotype.…”

“…Mitochondrial H 2 O 2 emission was measured fluorometrically in permeabilized muscle fibre bundles, as described previously (O'Rourke et al . ). Briefly, muscle fibre bundles were placed in 1 mL cuvettes containing Amplex Ultra Red (25 μ m ), horseradish peroxidase (1 U mL –1 ) and blebbistatin (25 μ m ) at 37°C, and mixed using a magnetic stirrer.…”

“…High-resolution respirometry measurements were made on permeabilized muscle fibre bundles using the OROBOROS Oxygraph-2K (Oroboros Instruments, Innsbruck, Austria) as described previously (O'Rourke et al 2018). Experiments were conducted at 37°C with a starting oxygen concentration of ß300-350 μM in buffer Z, containing 20 mM creatine monohydrate and 25 μM blebbistatin.…”

The makings of the ‘actin code': regulation of actin's biological function at the amino acid and nucleotide level

“…Mice lacking β-actin die in early embryogenesis, prior to embryonic day 8.5 (E8.5) (Bunnell et al, 2011). Conditional knockout of β-actin in the skeletal muscle leads to quadriceps myopathy (Prins et al, 2011) and impairs muscle relaxation (O'Rourke et al, 2018). Two mutations in the human β-actin gene have been characterized: E364 K mutation, which has been correlated to neutrophil dysfunction (Nunoi et al, 1999), and R183W, linked to developmental malformations, dystonia and deafness (Procaccio et al, 2006).…”