Structural differences in the subfragment 1 and rod portions of myosin isozymes from adult and developing rat skeletal muscles

During development of fast contracting skeletal muscle in the rat hindleg, embryonic and neonatal forms of the myosin heavy chain are present prior to the accumulation of the adult fast type ( Whalen , R. G., Sell, S. M., Butler-Browne, G.S., Schwartz, K., Bouveret, P., and Pinset -H arstr ?m, I. (1981) Nature (Lond.) 292, 805-809). Polypeptide mapping of the heavy chain subunit using partial proteolysis in the presence of sodium dodecyl sulfate has shown differences in the cleavage patterns for these various heavy chains. Using this technique, we have now examined subfragments, which represent functional domains, from several different myosin isozymes. The heavy chains of the S-1 subfragments containing either light chain 1 or light chain 3 are indistinguishable for the neonatal or fast myosin isozymes. We also isolated the S-1 fragments and the alpha-helical COOH-terminal half of the molecule (rod) from rat embryonic, neonatal, and adult fast and slow myosin, as well as myosin from cardiac ventricles. All of these S-1 and rod fragments were different, indicating that the previously reported differences among these different myosin heavy chain isozymes are located in both the S-1 and rod subfragments for all myosins examined. However, the polypeptide maps of neonatal and adult fast S-1 show clear similarities, as do the maps of slow and cardiac S-1. These similarities in the two pairs of polypeptide maps were confirmed by the results of immunoblotting experiments using antibodies to adult fast and to slow myosin.     

Human desmin-coding gene: complete nucleotide sequence, characterization and regulation of expression during myogenesis and development

A recombinant clone encoding for the human desmin gene (des) has been isolated and characterized and its complete nucleotide sequence has been determined. The 8.4-kb gene has nine exons separated by introns ranging in size from 0.1-2.2 kb. Comparison of the human des gene with that of the hamster has shown that there is a full correspondence in position, size and sequence of the exons. There are eight introns in both the human and the hamster des genes. Although the nucleotide sequence of the introns reveals a large divergence, splice junction sequence signals are conserved. A particularly striking feature of the human des gene is the 1.2-kb repetitive sequence found in the introns. These sequences all belong to the human AluI family. When the 5'- and 3'-untranslated regions of the human vim and des genes were compared it was found that there was a 16-mer consensus element similar to that described by Quax et al. [Cell 43 (1985) 327-338] for the hamster and an 11-bp sequence with homology to the distal regulatory sequence of human and mouse alpha-cardiac actin-coding genes [Minty and Kedes, Mol. Cell. Biol. 6 (1986) 2125-2136] in the 5'-flanking region. The 3'-untranslated region of the human des gene was found to be conserved when compared to the hamster des gene. Only one species of desmin RNA of 2.2 kb was found in human striated and smooth muscle both in vivo and in vitro.     

Mitochondrial Dysfunction Reveals the Role of mRNA Poly(A) Tail Regulation in Oculopharyngeal Muscular Dystrophy Pathogenesis

Oculopharyngeal muscular dystrophy (OPMD), a late-onset disorder characterized by progressive degeneration of specific muscles, results from the extension of a polyalanine tract in poly(A) binding protein nuclear 1 (PABPN1). While the roles of PABPN1 in nuclear polyadenylation and regulation of alternative poly(A) site choice are established, the molecular mechanisms behind OPMD remain undetermined. Here, we show, using Drosophila and mouse models, that OPMD pathogenesis depends on affected poly(A) tail lengths of specific mRNAs. We identify a set of mRNAs encoding mitochondrial proteins that are down-regulated starting at the earliest stages of OPMD progression. The down-regulation of these mRNAs correlates with their shortened poly(A) tails and partial rescue of their levels when deadenylation is genetically reduced improves muscle function. Genetic analysis of candidate genes encoding RNA binding proteins using the Drosophila OPMD model uncovers a potential role of a number of them. We focus on the deadenylation regulator Smaug and show that it is expressed in adult muscles and specifically binds to the down-regulated mRNAs. In addition, the first step of the cleavage and polyadenylation reaction, mRNA cleavage, is affected in muscles expressing alanine-expanded PABPN1. We propose that impaired cleavage during nuclear cleavage/polyadenylation is an early defect in OPMD. This defect followed by active deadenylation of specific mRNAs, involving Smaug and the CCR4-NOT deadenylation complex, leads to their destabilization and mitochondrial dysfunction. These results broaden our understanding of the role of mRNA regulation in pathologies and might help to understand the molecular mechanisms underlying neurodegenerative disorders that involve mitochondrial dysfunction.     

Expression of slow myosin heavy chain during muscle regeneration is not always dependent on muscle innervation and calcineurin phosphatase activity

In the literature, there is an ambiguity as to the respective roles played by calcineurin phosphatase activity (CPA) and muscle innervation in the reestablishment of the slow-twitch muscle phenotype after muscle regeneration in different species. In this study, we wanted to determine the role of calcineurin and muscle innervation on the appearance and maintenance of the slow phenotype during mouse muscle regeneration. The pattern of myosin expression and CPA was analyzed in adult (n=15), regenerating (n=45) and denervated-regenerating (n=32) slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles. Moreover, in a second group of denervated-regenerating mice (n=9), the animals were treated with a calcineurin inhibitor. Regeneration was induced by injection of cardiotoxin and in the denervated-regenerating group, denervation was carried out by cutting the sciatic nerve before the administration of cardiotoxin. In innervated-regenerating soleus muscle, CPA increased continuously after 10 days postinjury and by 21 days, there was a 3.5-fold increase in CPA compared with adult basal level, whereas in innervated-regenerating EDL muscle, CPA remained unchanged. Moreover, our results show that in denervated-regenerating muscles, the MyHC profile was identical in spite of the functional differences inherent in these muscles. In long-term denervated-regenerating muscles, a slow muscle phenotype was reexpressed both in the presence or absence of calcineurin inhibitor. Our results show that although in innervated-regenerating mouse muscle, the appearance of a slow phenotype is correlated with a peak of CPA, in denervated-regenerating muscles, a slow phenotype is triggered and maintained in a calcineurin- and nerve-independent manner.     

Telomerase can extend the proliferative capacity of human myoblasts,but does not lead to their immortalization

Normal cells in culture display a limited capacity to divide and reach a non-proliferative state called cellular senescence. Spontaneous escape from senescence resulting in an indefinite life span is an exceptionally rare event for normal human cells and viral oncoproteins have been shown to extend the replicative life span but not to immortalize them. Telomere shortening has been proposed as a mitotic clock that regulates cellular senescence. Telomerase is capable of synthesizing telomere repeats onto chromosome ends to block telomere shortening and to maintain human fibroblasts in proliferation beyond their usual life span. However, the consequence of telomerase expression on the life span of human myoblasts and on their differentiation is unknown. In this study, the telomerase gene and the puromycin resistance gene were introduced into human satellite cells, which are the natural muscle precursors (myoblasts) in the adult and therefore, a target for cell-mediated gene therapy. Satellite cells expressing telomerase were selected, and the effects of the expression of the telomerase gene on proliferation, telomere length, and differentiation were investigated. Our results show that the telomerase-expressing cells are able to differentiate and to form multinucleated myotubes expressing mature muscle markers and do not form tumors in vivo. We also demonstrated that the expression of hTERT can extend the replicative life of muscle cells although these failed to undergo immortalization.     

Shorter leukocyte telomere length is associated with severity of COVID-19 infection.

The infection by COVID-19 is a serious global public health problem. An efficient way to improve this disease's clinical management would be to characterize patients at higher risk of progressing to critically severe infection using prognostic biomarkers. The telomere length could be used for this purpose. Telomeres are responsible for controlling the number of maximum cell divisions. The telomere length is a biomarker of aging and several diseases. We aimed to compare leukocyte telomere length (LTL) between patients without COVID-19 and patients with different clinical severity of the infection. Were included 53 patients who underwent SARS-CoV-2 PCR divided in four groups. The first group was composed by patients with a negative diagnosis for COVID-19 (n = 12). The other three groups consisted of patients with a confirmed diagnosis of COVID-19 divided according to the severity of the disease: mild (n = 15), moderate (n = 17) and severe (n = 9). The LTL was determined by Q-PCR. The severe group had the shortest LTL, followed by the moderate group. The negative and mild groups showed no differences. There is an increase of patients with hypertension (p = 0.0099) and diabetes (p = 0.0067) in moderate and severe groups. Severe group was composed by older patients in comparison with the other three groups (p = 0.0083). Regarding sex, there was no significant difference between groups (p = 0.6279). In an ordinal regression model, only LTL and diabetes were significantly associated with disease severity. Shorter telomere length was significantly associated with the severity of COVID-19 infection, which can be useful as a biomarker or to better understand the SARS-CoV-2 pathophysiology.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

Effects of Piper hispidinervum on spermatogenesis and histochemistry of ovarioles of Spodoptera frugiperda

The fall armyworm, Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), not only damages crops, but controlling its population also requires synthetic insecticides, which leads to selection of resistant populations and environmental contamination. Essential oils are an alternative for controlling this insect. There are few studies of the effects of these oils on the insect's reproductive system. We evaluated the effects of the long pepper, Piper hispidinervum, essential oil on the gonads of the armyworm and tested its possible influence on the fertility of this insect. Dosages of 30 and 50 mg/ml were tested in 3^rd instar caterpillars using the leaf immersion method. Testes and ovarioles were collected, fixed with 10% formalin and embedded in Historesin. The sections were stained with toluidine blue and Mallory trichrome to detect connective tissue, periodic acid-Schiff to detect neutral carbohydrates, and bromophenol blue to detect proteins. We found that the long pepper essential oil affected negatively the spermatogenesis and altered the histochemistry of the ovarioles of S. frugiperda. The effects of long pepper oil suggest that it is a promising tool for controlling the armyworm pest.