Yvonne Mounier

Time-dependent changes in myosin heavy chain mRNA and protein isoforms in unloaded soleus muscle of rat.

Time-dependent changes in myosin heavy chain (MHC) isoform expression were investigated in rat soleus muscle unloaded by hindlimb suspension. Changes at the mRNA level were measured by RT-PCR and correlated with changes in the pattern of MHC protein isoforms. Protein analyses of whole muscle revealed that MHCI decreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showed that MHCIId(x) displayed the steepest increase after 4 days and continued to rise until 28 days, the longest time period investigated. MHCIIb mRNA followed a similar time course, although at lower levels. MHCIalpha mRNA, present at extremely low levels in control soleus, peaked after 4 days, stayed elevated until 15 days, and then decayed. Immunohistochemistry of 15-day unloaded muscles revealed that MHCIalpha was present in muscle spindles but at low amounts also in extrafusal fibers. The slow-to-fast transitions thus seem to proceed in the order MHCIbeta --> MHCIIa --> MHCIId(x) --> MHCIIb. Our findings indicate that MHCIalpha is transiently upregulated in some fibers as an intermediate step during the transition from MHCIbeta to MHCIIa.Time-dependent changes in myosin heavy chain (MHC) isoform expression were investigated in rat soleus muscle unloaded by hindlimb suspension. Changes at the mRNA level were measured by RT-PCR and correlated with changes in the pattern of MHC protein isoforms. Protein analyses of whole muscle revealed that MHCI decreased after 7 days, when MHCIIa had increased, reaching a transient maximum by 15 days. Longer periods led to inductions and progressive increases of MHCIId(x) and MHCIIb. mRNA analyses of whole muscle showed that MHCIId(x) displayed the steepest increase after 4 days and continued to rise until 28 days, the longest time period investigated. MHCIIb mRNA followed a similar time course, although at lower levels. MHCIα mRNA, present at extremely low levels in control soleus, peaked after 4 days, stayed elevated until 15 days, and then decayed. Immunohistochemistry of 15-day unloaded muscles revealed that MHCIα was present in muscle spindles but at low amounts also in extrafusal fibers. The slow-to-fast transitions thus seem to proceed in the order MHCIβ → MHCIIa → MHCIId(x) → MHCIIb. Our findings indicate that MHCIα is transiently upregulated in some fibers as an intermediate step during the transition from MHCIβ to MHCIIa.

Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry

O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory post-translational modification of nucleo-cytoplasmic proteins that has a complex interplay with phosphorylation. O-GlcNAc has been described as a nutritional sensor, the level of UDP-GlcNAc that serves as a donor for the uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetyl-glucosaminyltransferase being regulated by the cellular fate of glucose. Because muscular contraction is both dependent on glucose metabolism and is highly regulated by phosphorylation/dephosphorylation processes, we decided to investigate the identification of O-GlcNAc-modified proteins in skeletal muscle using a proteomic approach. Fourteen proteins were identified as being O-GlcNAc modified. These proteins can be classified in three main classes: i) proteins implicated in the signal transduction and in the translocation between the cytoplasm and the nucleus or structural proteins, ii) proteins of the glycolytic pathway and energetic metabolism, and iii) contractile proteins (myosin heavy chain). A decrease in the O-GlcNAc level was measured in the slow postural soleus muscle after 14-day hindlimb unloading, a model of functional atrophy characterized by a decrease in the force of contraction. These results strongly suggest that O-GlcNAc modification may serve as an important regulation system in skeletal muscle physiology.

Changes in myosin heavy chain mRNA and protein isoforms in single fibers of unloaded rat soleus muscle

Changes in myosin heavy chain (MHC) mRNA and protein isoforms were investigated in single fibers from rat soleus muscle unloaded by hindlimb suspension for 4 and 7 days. Dramatic changes were seen after 4 days, when all fibers co‐expressed slow and fast MHC mRNAs. Most fibers contained mRNAs for MHCIβ, MHCIIa, MHCIId(x), and MHCIIb. The up‐regulation of the fast isoforms was only partially transmitted to the protein level. Atypical combinations of MHC mRNA isoforms, which deviated from the ‘next‐neighbor rule’, were frequent in fibers from unloaded soleus. These atypical combinations increased with time and were not observed in the controls. The results suggest that hindlimb suspension elicits in soleus muscle pronounced perturbations in the expression of MHC isoforms by disrupting transcriptional and translational activities.

O-Linked N-Acetylglucosaminylation Is Involved in the Ca2+ Activation Properties of Rat Skeletal Muscle

O-Linked N-acetylglucosaminylation termed O-GlcNAc is a dynamic cytosolic and nuclear glycosylation that is dependent both on glucose flow through the hexosamine biosynthesis pathway and on phosphorylation because of the existence of a balance between phosphorylation and O-GlcNAc. This glycosylation is a ubiquitous post-translational modification, which probably plays an important role in many aspects of protein functions. We have previously reported that, in skeletal muscle, proteins of the glycolytic pathway, energetic metabolism, and contractile proteins were O-GlcNAc-modified and that O-Glc-NAc variations could control the muscle protein homeostasis and be implicated in the regulation of muscular atrophy. In this paper, we report O-N-acetylglucosaminylation of a number of key contractile proteins (i.e. myosin heavy and light chains and actin), which suggests that this glycosylation could be involved in skeletal muscle contraction. Moreover, our results showed that incubation of skeletal muscle skinned fibers in N-acetyl-d-glucosamine, in a concentration solution known to inhibit O-GlcNAc-dependent interactions, induced a decrease in calcium sensitivity and affinity of muscular fibers, whereas the cooperativity of the thin filament proteins was not modified. Thus, our results suggest that O-GlcNAc is involved in contractile protein interactions and could thereby modulate muscle contraction.

Compared properties of the contractile system of skinned slow and fast rat muscle fibres.

Chemically skinned fibres from soleus and plantaris rat muscles were used to compare the contractile properties of slow and fast muscles. The maximal isometric tension appeared larger in plantaris than in soleus fibres. The apparent Ca2+ threshold for activation was lower in slow than in fast fibres while Ca2+ concentrations required to obtain either the maximal tension or half maximal tension (pCa50) were lower in fast than in slow fibres. This apparent difference in Ca2+ sensitivity will be discussed. As could be expected from other studies, a faster force development in plantaris than in soleus fibres occurred. However, one interesting new result showed that in soleus, the kinetics of the tension development estimated by the tmax parameter were slightly dependent on the Ca2+ concentration whereas the t50 parameter changed significantly with the Ca2+ concentration. In plantaris, both tmax and t50 parameters were found to depend strongly on the Ca2+ concentration. Finally, the plantaris muscle showed a greater caffeine sensitivity than the soleus muscle. All the results suggested that the Ca-regulatory mechanism in the slow fibres was essentially different from that in the fast fibres.

Nerve influence on myosin light chain phosphorylation in slow and fast skeletal muscles

Neural stimulation controls the contractile properties of skeletal muscle fibres through transcriptional regulation of a number of proteins, including myosin isoforms. To study whether neural stimulation is also involved in the control of post‐translational modifications of myosin, we analysed the phosphorylation of alkali myosin light chains (MLC1) and regulatory myosin light chains (MLC2) in rat slow (soleus) and fast (extensor digitorum longus EDL) muscles using 2D‐gel electrophoresis and mass spectrometry. In control rats, soleus and EDL muscles differed in the proportion of the fast and slow isoforms of MLC1 and MLC2 that they contained, and also in the distribution of the variants with distinct isoelectric points identified on 2D gels. Denervation induced a slow‐to‐fast transition in myosin isoforms and increased MLC2 phosphorylation in soleus, whereas the opposite changes in myosin isoform expression and MLC2 phosphorylation were observed in EDL. Chronic low‐frequency stimulation of EDL, with a pattern mimicking that of soleus, induced a fast‐to‐slow transition in myosin isoforms, accompanied by a decreased MLC2 phosphorylation. Chronic administration (10 mg·kg−1·d−1 intraperitoneally) of cyclosporin A, a known inhibitor of calcineurin, did not change significantly the distribution of fast and slow MLC2 isoforms or the phosphorylation of MLC2. All changes in MLC2 phosphorylation were paralleled by changes in MLC kinase expression without any variation of the phosphatase subunit, PP1. No variation in MLC1 phosphorylation was detectable after denervation or cyclosporin A administration. These results suggest that the low‐frequency neural discharge, typical of soleus, determines low levels of MLC2 phosphorylation together with expression of slow myosin, and that MLC2 phosphorylation is regulated by controlling MLC kinase expression through calcineurin‐independent pathways.

Influence of chronic stretching upon rat soleus muscle during non-weight-bearing conditions

Morphological, contractile and histochemical properties as well as the myosin heavy chain (MHC) composition of rat soleus muscles were studied after 14 days of non-weight-bearing (NWB) and after immobilization of the foot in dorsiflexion of NWB rats. Significant reductions in soleus mass, fibre sizes and tetanic tension were found after 14 days of NWB. Furthermore, a transformation of the slow-twitch soleus muscle towards a faster type was characterized by a decrease in twitch time parameters, an increase in the fast-twitch type IIA fibre proportion and an increase in fast-twitch type MHC isoforms. Our results showed that the immobilization of the soleus muscle in a lengthened position during NWB not only prevented the loss of muscular mass and force output, but also counteracted the slow to faster shift in contractile and phenotypical parameters normally associated with NWB conditions.

CONTRACTILE PROPERTIES AND MYOSIN HEAVY CHAIN COMPOSITION OF NEWBORN RAT SOLEUS MUSCLES AT DIFFERENT STAGES OF POSTNATAL DEVELOPMENT

This study was undertaken to correlate some of the functional characteristics with the myofibrillar composition in myosin heavy chain isoforms on newborn and adult rat soleus muscles. The following postnatal ages were chosen in order to determine the role of innervation in the establishment of the mature muscle phenotype: before (postnatal day 6), when(postnatal day 12), and after (days 17 and 23) the monosynaptic innervation appeared. The steady state of definitive innervation was controlled on adult muscles (i.e. ∼13 weeks). Muscle maturation was followed by ATPase staining and fibre diversity was observed at postnatal day 12. The functional properties of skinned bundles isolated from newborn rats were determined by Calcium/Strontium activation characteristics (Tension/pCa and pSr relationships). From postnatal days 6 to 17, the soleus bundles exhibited Calcium/Strontium activation characteristics intermediate between slow and fast fibre populations previously described in muscles. At day 23, the Calcium/Strontium activation characteristics of the soleus were closer to those of a slow type. Moreover, we observed a decrease in Ca affinity concomittant with the installation of the monosynaptic innervation, and an increase of the slow type I during postnatal development. Finally, this work reported a greater correlation between the Calcium/Strontium activation parameters and the myosin heavy chain isoform composition at the postnatal days when the mature monosynaptic innervation pattern occurred

Evolution of contractile and elastic properties of rat soleus muscle fibres under unloading conditions.

Rats were submitted to 14 days of hindlimb suspension in order to examine the contractile and elastic properties of the soleus muscles under disuse conditions. The calcium/strontium activation properties, the maximal shortening velocity (V0), as well as the time behaviour of force transients following quick releases and the T1 curves characterizing the active part of the series elastic elements, were determined on single chemically skinned fibres. After the functional measurements, the fibres were subjected to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in order to analyse both the myosin heavy (MHC) and light (MLC) chain isoforms. According to the MHC and MLC composition, two groups of fibres were defined after hindlimb suspension: a group of slow fibres expressing the slow set of both MHC and MLC isoforms, and a group of fast fibres co‐expressing the slow and fast MHC and MLC isoforms with a predominant expression of the fast ones. For the first group, the contractile as well as the elastic properties were found to be close to those of control slow soleus fibres. For the second group, both contractile and elastic properties were modified insofar as they became close to those found in a fast muscle such as the extensor digitorum longus. We suggested that between the two populations found in the soleus muscle after hindlimb suspension the modifications in the contractile properties, as well as the alterations in the elastic characteristics, were concomitant to the changes in both MHC and MLC compositions.

DIFFERENTIAL EFFECTS OF A SIX-DAY IMMOBILIZATION ON NEWBORN RAT SOLEUS MUSCLES AT TWO DEVELOPMENTAL STAGES

Our objective was to determine the effects of a six-day immobilization on the musculoskeletal system of the rat during postnatal development at two key periods when the states of innervation are known to be different. This work was undertaken on the soleus muscle since it is well known that postural slow muscles show marked changes after a period of disuse. Thus, the soleus muscle was immobilized in a shortened position either when the innervation was polyneuronal or monosynaptic, respectively from 6 to 12 and from 17 to 23 days. The muscle modifications were followed by ATPase staining and myosin heavy chain (MyHC) isoform identification using monoclonal antibodies and SDS–PAGE. The functional properties of skinned fibre bundles were established by calcium/strontium (Ca/Sr) activation characteristics. In control muscles the maturation was characterized by a progressive increase of adult MyHCs (I and IIA) concomitant with a decrease in both the MyHC neo and the Ca affinit y. Between 6 to 12 days, immobilization of the limb induced an increase in histochemical type IIC fibres. Using antibodies we identified new fibre types, classified as a function of their MyHC isoform co-expression. We observed an increase in expression of both MyHC neo and Ca affinity. From 17 to 23 days, the immobilization induced an increase in Ca affinity and marked changes in the MyHC isoform composition: disappearance of MyHC neo and expression of the fast MyHC IIB isoform, which in normal conditions is never expressed in the soleus muscle. We conclude that an immobilization imposed during polyneuronal innervation delays the postnatal maturation of the soleus muscle, whereas when the immobilization is performed under monosynaptic innervation the muscle evolves towards a fast phenotype using a default pathway for MyHC expression.