Discovery of new intracellular junctions: The calcium entry units (CEUs)

In 2017, Boncompagni, Michelucci et al. demonstrated that during exercise the sarcotubular system of extensor digitorum longus (EDL) fibers undergoes a profound remodeling that leads to the assembly of new junctions between T-tubule extensions at the I band and sarcoplasmic reticulum (SR) stacks. As these junctions contain colocalized STIM1 and Orai1 and enhance store-operated Ca2+ entry (SOCE), they have been named Ca2+ entry units (CEUs). In addition, it has been more recently shown that (1) CEUs disassemble following recovery, with T-tubules retraction from the I band faster than SR stacks disassembly, and (2) lack of calsequestrin-1 (CASQ1) induces a constitutive assembly of CEUs, resulting in enhanced SOCE that counteracts the SR Ca2+ depletion. We have now analyzed (1) CEUs during postnatal maturation (at 2 wk of age) and (2) whether CEUs form in slow-twitch fibers (soleus). (a) Compared with adult (4 mo) EDL fibers of resting WT mice, at 2 wk of age we found a greater longitudinal disposition of T-tubules associated to SR membranes forming junctions virtually identical to CEUs in adult EDLs of exercised WT mice, which promote increased STIM1/Orai1-mediated SOCE. (b) We also compared structure and function of soleus (which also express the cardiac isoform CASQ2) from WT mice and from mice lacking either CASQ1 (CASQ1-null) or CASQ1/2 (dCASQ-null). In soleus from both genotypes, CEUs are constitutively assembled although they appear structurally smaller than those described previously in exercised WT or CASQ1-null EDLs. A detailed EM quantitative analysis revealed that CEUs were more abundant in dCASQ-null than CASQ1-null mice. The amount of CEUs strictly correlated with the ability of soleus fibers to recover extracellular Ca2+ via SOCE to support contractility during high-frequency stimulation. These data were supported by molecular analysis of Western blots, showing that Orai1 expression was enhanced following ablation of CASQ.

Ageing Causes Ultrastructural Modification to Calcium Release Units and Mitochondria in Cardiomyocytes

Ageing is associated with an increase in the incidence of heart failure, even if the existence of a real age-related cardiomyopathy remains controversial. Effective contraction and relaxation of cardiomyocytes depend on efficient production of ATP (handled by mitochondria) and on proper Ca2+ supply to myofibrils during excitation–contraction (EC) coupling (handled by Ca2+ release units, CRUs). Here, we analyzed mitochondria and CRUs in hearts of adult (4 months old) and aged (≥24 months old) mice. Analysis by confocal and electron microscopy (CM and EM, respectively) revealed an age-related loss of proper organization and disposition of both mitochondria and EC coupling units: (a) mitochondria are improperly disposed and often damaged (percentage of severely damaged mitochondria: adults 3.5 ± 1.1%; aged 16.5 ± 3.5%); (b) CRUs that are often misoriented (longitudinal) and/or misplaced from the correct position at the Z line. Immunolabeling with antibodies that mark either the SR or T-tubules indicates that in aged cardiomyocytes the sarcotubular system displays an extensive disarray. This disarray could be in part caused by the decreased expression of Cav-3 and JP-2 detected by western blot (WB), two proteins involved in formation of T-tubules and in docking SR to T-tubules in dyads. By WB analysis, we also detected increased levels of 3-NT in whole hearts homogenates of aged mice, a product of nitration of protein tyrosine residues, recognized as marker of oxidative stress. Finally, a detailed EM analysis of CRUs (formed by association of SR with T-tubules) points to ultrastructural modifications, i.e., a decrease in their frequency (adult: 5.1 ± 0.5; aged: 3.9 ± 0.4 n./50 μm2) and size (adult: 362 ± 40 nm; aged: 254 ± 60 nm). The changes in morphology and disposition of mitochondria and CRUs highlighted by our results may underlie an inefficient supply of Ca2+ ions and ATP to the contractile elements, and possibly contribute to cardiac dysfunction in ageing.

Morphological Alterations of the Sarcotubular System in Permanent Myopathy of Hereditary Hypokalemic Periodic Paralysis with a Mutation in the CACNA1S Gene

We investigated the immunohistochemical localization of several proteins related to excitation-contraction coupling and ultrastructural alterations of the sarcotubular system in biopsied muscles from a father and a daughter in a family with permanent myopathy with hypokalemic periodic paralysis (PMPP) due to a mutation in calcium channel CACNA1S; p. R1239H hetero. Immunostaining for L-type calcium channels (LCaC) showed linear hyper-stained regions indicating proliferation of longitudinal t-tubules. The margin of vacuoles was positive for ryanodine receptor, LCaC, calsequestrin (CASQ) 1, CASQ 2, SR/ER Ca2+-ATPase (SERCA) 1, SERCA2, dysferlin, dystrophin, α-actinin, LC3, and LAMP 1. Electron microscopy indicated that the vacuoles mainly originated from the sarcoplasmic reticulum (SR). These findings indicate impairment of the muscle contraction system related to Ca2+ dynamics, remodeling of t-tubules and muscle fiber repair. We speculate that PMPP in patients with a CACNA1S mutation might start with abnormal SR function due to impaired LCaC. Subsequent induction of muscular contractile abnormalities and the vacuoles formed by fused SR in the repair process including autophagy might result in permanent myopathy. Our findings may facilitate prediction of the pathomechanisms of PMPP seen on morphological observation.

Impact of gamma irradiation on tissues of the mud crab, Scylla serrata (Forskål, 1775) (Decapoda, Portunidae) — electron microscopic study and DNA comet assay

Radiopreservation using gamma radiation is widely in use as a safe method for extending the shelf life of shellfish. This study explored the consequences of different doses of gamma radiation (0.5 kGy, 1.0 kGy and 2.0 kGy) on various tissues of Scylla serrata at cellular and nuclear level, with the aid of electron microscopy and DNA comet assay. The highly radio exposed (2.0 kGy) pyloric muscles showed a reduction in sarcomere length, disordered organization with expanded gap between adjacent myofibrils, ruptured sarcotubular system, mitochondrial swelling with crushed cristae, significant increase in nucleus size coupled with less dense nucleoplasm, etc. Comet assay on tissues such as muscle, hepatopancreas and testis irradiated with 2.0 kGy radiation also revealed a significant degree of nuclear damage by gamma irradiation in a dose-dependent manner. The tail length of the comet showed a tissue-specific tolerance level. The present study clarified the precise dose of irradiation as 1.0 kGy and the results can be relevant for commercial purposes to qualitatively categorize the irradiated crabs.

Sarcoplasmic-reticulum biogenesis in contraction-inhibited skeletal-muscle cultures

We have previously shown that inhibition of the spontaneous contractile activity of cultured embryonic-chick skeletal-muscle fibres with tetrodotoxin (TTX) leads to decreased sarcoplasmic-reticulum Ca(2+)-transport rates and steady-state concentrations of the high-energy Ca(2+)-ATPase phosphoenzyme intermediate [Charuk & Holland (1983) Exp. Cell Res. 144, 143-157]. In the present study we used a monoclonal antibody to the Ca(2+)-ATPase to show that there is a decreased amount of enzyme accumulated by contraction-inhibited myotubes. Indirect immunofluorescence microscopy using the monoclonal antibody to the Ca(2+)-ATPase also revealed a disordered subcellular organization of the sarcotubular system in contraction-inhibited myotubes. The biogenesis of sarcoplasmic-reticulum proteins in TTX-paralysed myofibres was studied by labelling cells with [35S]methionine before isolation of the active Ca(2+)-pump membrane fraction. Protein turnover was selectively increased in that fraction from TTX-treated muscle cultures. Electrophoretic analysis and quantitative fluorography confirmed that decreased accumulation of the Ca(2+)-ATPase enzyme in contraction-inhibited myotubes was associated with increased turnover of this protein. The present results demonstrate that biogenesis of the sarcoplasmic-reticulum Ca(2+)-ATPase is regulated by the contractile activity of skeletal-muscle fibres.

Effect of anabolic steroids on mitochondria and sarcotubular system of skeletal muscle

Soleus and extensor digitorum longus (EDL) mitochondria and sarcotubular system were examined in sedentary and trained (treadmill for 12 wk) male rats that were treated with fluoxymesterone or methandrostanolone (2 mg/kg, 5 days/wk, for 8 wk). Neither physical exercise nor anabolic/androgenic steroid administration resulted in a significant change in muscle wet weight. Treatment with the anabolizing androgens increased succinate dehydrogenase activity in fast-twitch muscle mitochondria; this effect was not enhanced by training and was not observed in soleus mitochondria. On the other hand, the content of the slow-twitch muscle in sarcotubular fraction was increased in sedentary rats by fluoxymesterone or methandrostanolone treatment, whereas no significant changes were found in EDL. The training program affected adenosinetriphosphatase (ATPase) activities in the sarcotubular fraction; Mg2(+)-ATPase was increased in both soleus and EDL, but Ca2(+)-ATPase was decreased only in soleus. However, in sedentary animals only the Mg2(+)-dependent activity of EDL was increased by anabolizing androgen treatment, and this change was not potentiated by additional training. The present data indicate that anabolic/androgenic steroids can affect mitochondrial and sarcotubular enzymes in skeletal muscle. The effects are muscle-type specific.

Myotubular Myopathy: Arrest of Morphogenesis of Myofibres Associated with Persistence of Fetal Vimentin and Desmin Four cases compared with fetal and neonatal muscle

ABSTRACT:Vastus lateralis muscle biopsies of four unrelated male neonates showing myotubular (i.e. centronuclear) myopathy (MM) were compared with muscle from four human fetuses in the myotubular stage of development, a 31 week preterm infant and four term neonates. The perimysium, blood vessels, spindles, myelinated intramuscular nerves, and motor end-plates in MM are as well developed as in term neonatal muscle. The cytoarchitecture of myofibres in MM is more mature than that of fetal myotubes in the spacing of central nuclei, Z-band registry, development of the sarcotubular system, and in the condensation of nuclear chromatin and nucleoli. Triads in MM may retain an immature oblique or longitudinal orientation. Myofibrillar ATPase shows normal differentiation of fibre types, consistent with nonnal innervation. Spinal motor neurons are nonnal in number and in RNA fluorescence. Immunoreactivity for vimentin and desmin in myofibres of MM is uniformly strong, as in fetal myotubes and unlike mature neonatal muscle. Maternal muscle biopsies of two cases also showed scattered small centronuclear myofibres reactive for vimentin and desmin. The arrest in morphogenesis of fibre architecture in MM is not a general arrest in muscle development. Persistence of fetal cytoskeletal proteins that preserve the immature central positions of nuclei and mitochondria may be important in pathogenesis. Vimentin/desmin studies of the infant and maternal muscle biopsies are useful in establishing the diagnosis.