Biochemical changes in rat muscle tissue with prolonged use of simvastatin

Aim. Analysis of biochemical changes in rat muscle tissue after prolonged use of simvastatin. Methods. The study was conducted on mongrel male rats. Three groups were identified: control group (intact animals), comparison group (animals with induced hypercholesterolemia not reeciving the drugs), and experimental group (animals with induced hypercholesterolemia receiving simvastatin 0.0012 g/100 g of weight once a day for 2 months as an aqueous suspension through the esophageal probe). Metabolite concentration of glycolysis (pyruvic acid and lactate), activity of antioxidant protection enzymes (reduced glutathione, superoxide dismutase, catalase, glutathione reductase, glutathione peroxidase), titin isoforms and proteolytic fragments of titin and nebulin concentration were determined in the muscles of animals. Results. After administration of simvastatin to animals with induced hypercholesterolemia, a decrease in the concentration of glycolysis metabolites (pyruvic acid and lactate) compared to comparison group was revealed, as well as multidirectional changes in the activity of antioxidant protection enzymes (decrease in activity of superoxide dismutase, glutathione peroxidase, and glutathione reductase, decreased concentration of reduced glutathione, but catalase activity remained unchanged). The analysis of structural changes in animal muscle tissue after administration of simvastatin revealed a decrease in the concentration of NT- and N2A-titin isoforms and practically complete absence of nebulin compared to the animals from the comparison group. At the same time an increase in the concentration of proteolytic fragments of titin (T2) by 1.3 times was recorded. Conclusion. The study showed that the basis of myotoxicity of statins in their long-term use is disintegration of enzyme antioxidant processes, as well as tissue hypoxia, leading to destruction of muscle fibers and prevalence of proteolytic processes.

The increase in non-cross-bridge forces after stretch of activated striated muscle is related to titin isoforms

Skeletal muscles present a non-cross-bridge increase in sarcomere stiffness and tension on Ca2+ activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca2+-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus, and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments, and treatment with the actomyosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths varying between 1.93 and 3.37 μm for the psoas, 2.68 and 4.21 μm for the soleus, and 1.51 and 2.86 μm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca2+-dependent change in titin properties and not associated with changes in titin-actin interactions.

Removal of immunoglobulin-like domains from titin’s spring segment alters titin splicing in mouse skeletal muscle and causes myopathy

Titin is a molecular spring that determines the passive stiffness of muscle cells. Changes in titin’s stiffness occur in various myopathies, but whether these are a cause or an effect of the disease is unknown. We studied a novel mouse model in which titin’s stiffness was slightly increased by deleting nine immunoglobulin (Ig)-like domains from titin’s constitutively expressed proximal tandem Ig segment (IG KO). KO mice displayed mild kyphosis, a phenotype commonly associated with skeletal muscle myopathy. Slow muscles were atrophic with alterations in myosin isoform expression; functional studies in soleus muscle revealed a reduced specific twitch force. Exon expression analysis showed that KO mice underwent additional changes in titin splicing to yield smaller than expected titin isoforms that were much stiffer than expected. Additionally, splicing occurred in the PEVK region of titin, a finding confirmed at the protein level. The titin-binding protein Ankrd1 was highly increased in the IG KO, but this did not play a role in generating small titin isoforms because titin expression was unaltered in IG KO mice crossed with Ankrd1-deficient mice. In contrast, the splicing factor RBM20 (RNA-binding motif 20) was also significantly increased in IG KO mice, and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Thus, increasing titin’s stiffness triggers pathological changes in skeletal muscle, with an important role played by RBM20.