Biomedical subjects

D Pette

Publications and source records attributed to D Pette.

At least 37 records · Page 2Linked to original sources

PubMed2000

Effects of unweighting and clenbuterol on myosin light and heavy chains in fast and slow muscles of rat.

To investigate the plasticity of slow and fast muscles undergoing slow-to-fast transition, rat soleus (SOL), gastrocnemius (GAS), and extensor digitorum longus (EDL) muscles were exposed for 14 days to 1) unweighting by hindlimb suspension (HU), or 2) treatment with the beta(2)-adrenergic agonist clenbuterol (CB), or 3) a combination of both (HU-CB). In general, HU elicited atrophy, CB induced hypertrophy, and HU-CB partially counteracted the HU-induced atrophy. Analyses of myosin heavy (MHC) and light chain (MLC) isoforms revealed HU- and CB-induced slow-to-fast transitions in SOL (increases of MHCIIa with small amounts of MHCIId and MHCIIb) and the upregulation of the slow MHCIa isoform. The HU- and CB-induced changes in GAS consisted of increases in MHCIId and MHCIIb ("fast-to-faster transitions"). Changes in the MLC composition of SOL and GAS consisted of slow-to-fast transitions and mainly encompassed an exchange of MLC1s with MLC1f. In addition, MLC3f was elevated whenever MHCIId and MHCIIb isoforms were increased. Because the EDL is predominantly composed of type IID and IIB fibers, HU, CB, and HU-CB had no significant effect on the MHC and MLC patterns.

Adrenergic beta-Agonists↗

PubMed1999

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 MHCIbeta, 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.

Animals↗

PubMed1999

The 90-kDa junctional sarcoplasmic reticulum protein forms an integral part of a supramolecular triad complex in skeletal muscle.

Although it is well established that voltage-sensing of the alpha(1)-dihydropyridine receptor triggers Ca(2+)-release via the ryanodine receptor during excitation-contraction coupling in skeletal muscle fibers, it remains to be determined which junctional components are responsible for the assembly, maintenance, and stabilization of triads. Here, we analyzed the expression pattern and neighborhood relationship of a novel 90-kDa sarcoplasmic reticulum protein. This protein is highly enriched in the triad fraction and is predominantly expressed in fast-twitching muscle fibers. Chronic low-frequency electro-stimulation induced a drastic decrease in the relative abundance of this protein. Chemical crosslinking showed a potential overlap between the 90-kDa junctional face membrane protein and the ryanodine receptor Ca(2+)-release channel, suggesting tight protein-protein interactions between these two triad components. Hence, Ca(2+)-regulatory muscle proteins have a strong tendency to oligomerize and the triad region of skeletal muscle fibers forms supramolecular membrane complexes involved in the regulation of Ca(2+)-homeostasis and signal transduction.

Animals↗

PubMed1999

Kinetic properties of myosin heavy chain isoforms in single fibers from human skeletal muscle.

The head portion of the myosin heavy chain is essential in force generation. As previously shown, Ca2+-activated muscle fibers from rat and rabbit display a strong correlation between their myosin heavy chain isoform composition and the kinetics of stretch activation, corresponding to an order of velocity: myosin heavy chain Ib > myosin heavy chain IId(x) > myosin heavy chain IIa >> myosin heavy chain I. Here, we show a similar correlation for human muscle fibers (myosin heavy chain IIb > myosin heavy chain IIa >> myosin heavy chain I), suggesting isoform-specific differences between the kinetics of force-generating power strokes. The kinetics of myosin heavy chain I are similar in human and rodents. This holds also true for myosin heavy chain IIa, but human myosin heavy chain IIb is slower than rodent myosin heavy chain IIb. It is similar to rodent myosin heavy chain IId(x).

Aged↗

PubMed1999

What does chronic electrical stimulation teach us about muscle plasticity?

The model of chronic low-frequency stimulation for the study of muscle plasticity was developed over 30 years ago. This protocol leads to a transformation of fast, fatigable muscles toward slower, fatigue-resistant ones. It involves qualitative and quantitative changes of all elements of the muscle fiber studied so far. The multitude of stimulation-induced changes makes it possible to establish the full adaptive potential of skeletal muscle. Both functional and structural alterations are caused by orchestrated exchanges of fast protein isoforms with their slow counterparts, as well as by altered levels of expression. This remodeling of the muscle fiber encompasses the major, myofibrillar proteins, membrane-bound and soluble proteins involved in Ca2+ dynamics, and mitochondrial and cytosolic enzymes of energy metabolism. Most transitions occur in a coordinated, time-dependent manner and result from altered gene expression, including transcriptional and posttranscriptional processes. This review summarizes the advantages of chronic low-frequency stimulation for studying activity-induced changes in phenotype, and its potential for investigating regulatory mechanisms of gene expression. The potential clinical relevance or utility of the technique is also considered.

Animals↗

PubMed1999

Phosphocreatine as a marker of contractile activity in single muscle fibres.

ATP and phosphocreatine (PCr) were measured in randomly selected single fibres from control, 1- and 8-day low-frequency-stimulated rabbit tibialis anterior muscles. The fibres were classified according to their myosin heavy chain (MHC) complement as type I, IIA or IID. In 1-day stimulated muscle, which has previously been shown to exhibit a steep decline in force output, two fibre populations could be distinguished according to either normal or markedly depressed PCr levels. The fibre population exhibiting normal PCr levels encompassed a major fraction (65%) of type IID fibres and a minor fraction (35%) of IIA fibres. The population with reduced PCr levels comprised type I fibres (@50% reduced), the majority of type IIA fibres (@80% reduced), and a minor fraction of type IID fibres (@70% reduced). Levels of ATP were unaltered in type I and IIA fibres, but were @ 20% reduced in those IID fibres that exhibited low PCr levels. Assuming that those fibres that displayed reduced PCr levels were contracting, the IID and IIA fibres with normal PCr levels were regarded as metabolically recovering, non-contracting fibres. As previously shown, these fibres are transiently refractory during the early phase of low-frequency stimulation. After 8 days of chronic low-frequency stimulation, when force was shown to rise again, most fibres appeared more uniform with regard to reduced PCr and ATP levels. Our results suggest that PCr can be used as a sensitive measure of the degree of activity in single-fibre studies.

Adenosine Triphosphate↗

PubMed1999

Effects of chronic low-frequency stimulation on Ca2+-regulatory membrane proteins in rabbit fast muscle.

Since chronic low-frequency stimulation of fast-twitch muscle fibers has a profound effect on all major functional elements of skeletal muscle, we analyzed the potential changes in the levels of Ca2+-regulatory membrane proteins during fast-to-slow transformation. In this study we show that, in addition to isoform-switching in myosin heavy chains, electrostimulation triggers a decline in fast isoforms and an increase in slow/cardiac isoforms of Ca2+-ATPase and calsequestrin. The levels of excitation-contraction coupling elements, such as the ryanodine receptor, the dihydropyridine receptor, triadin and sarcalumenin, decreased sharply following stimulation. In contrast, levels of Na+/K+-ATPase and calreticulin increased in the microsomal fraction. Crosslinking studies have revealed that in normal and stimulated muscle the Ca2+-ATPase isoforms exist predominantly as oligomeric structures, and that the central elements of excitation-contraction coupling also form large triad complexes. Changes in the levels and pattern of isoform expression of the muscle membrane proteins studied here suggest that these biochemical alterations reflect molecular adaptations to changed demands in ion homeostasis and signal transduction in muscle that exhibits enhanced contractile activity. Overall, these findings support the physiological concept that there are muscle fiber-type specific differences in the fine-tuning of the excitation-contraction-relaxation cycle, as well as the idea that mature skeletal muscle fibers exhibit a high degree of plasticity.

Animals↗

PubMed1999

Parathyroid hormone-related protein is rapidly up-regulated in blood vessels of rat skeletal muscle by low-frequency stimulation.

To identify early changes in gene expression of fast-twitch rat muscle exposed to chronic low-frequency stimulation, differential display was applied comparing mRNA patterns between control and 12-h stimulated tibialis anterior (TA) muscles. Among the signals of differentially expressed mRNAs, a cDNA of approximately 300 bp was identified as specific to the parathyroid-hormone-related protein (PTHrP). As verified by semi-quantitative reverse transcriptase polymerase chain reaction, this mRNA was present at low levels in normal slow-twitch soleus and fast-twitch TA muscles, but was approximately 80-fold elevated in TA muscles after 12 h of low-frequency stimulation. With ongoing stimulation for 2 or 4 days PTHrP mRNA returned to basal levels. PTHrP was located exclusively in blood vessels of both control and stimulated muscles. PTHrP immunohistochemistry revealed enhanced capillarization in 8-day stimulated muscles. In view of its vasodilative effect the up-regulation of PTHrP could be an important initial step related to enhanced capillarization in response to increased contractile activity.

Animals↗

PubMed1999

Alpha-cardiac-like myosin heavy chain MHCI alpha is not upregulated in transforming rat muscle.

The expression of MHCI alpha, an alpha-cardiac-like myosin heavy chain isoform, was studied in extensor digitorum longus (EDL) and tibialis anterior (TA) rat muscles undergoing fast-to-slow transition by chronic low-frequency stimulation (CLFS), a condition inducing a transient upregulation of MHCI alpha in rabbit muscle. In order to enhance the transformation process, CLFS was applied to hypothyroid rats. mRNA analyses were performed by RT-PCR, and studies at the protein level by immunoblotting and immunohistochemistry, using the F88 antibody (F88 12F8,1) demonstrated in the accompanying paper to be specific for MHCI alpha. In total RNA preparations from slow- and fast-twitch muscles, MHCI alpha mRNA was present at minute levels, at least three orders of magnitude lower than in cardiac atrium. As verified immunohistochemically, MHCI alpha is present only in intrafusal fibres of rat muscle. Moreover, MHCI alpha is not expressed in extrafusal fibres and, contrary to the rabbit, was not upregulated at both the mRNA and protein levels by CLFS. These results support our notion of species-specific responses to CLFS. Another antibody reported to be specific to MHCI alpha, BA-G5, was also investigated by immunoblot and immunohistochemical analyses. Its specificity could not be validated for skeletal muscles of the rat. BG-A5 was shown to cross-react with MHCIIb and MHCI beta. These results question an upregulation of MHCI alpha in transforming rat muscles as reported in studies based on the use of this antibody.

Animals↗

PubMed1999

Transient expression of myosin heavy chain MHCI alpha in rabbit muscle during fast-to-slow transition.

The expression of an alpha-cardiac-like myosin heavy chain, MHCI alpha, was investigated at both the mRNA and protein levels in rabbit tibialis anterior muscle undergoing fast-to-slow transition by continuous chronic low-frequency stimulation (CLFS). According to sequence analyses of the PCR product, the MHCI alpha isoform was found to be identical to the alpha-cardiac MHC expressed in rabbit atrium. In muscles at different degrees of transformation, the upregulation of MHCI alpha mRNA preceded that of the MHCI beta mRNA. At more advanced stages of the transformation, MHCI alpha mRNA decayed while MHCI beta mRNA persisted at high levels. The expression of MHCI alpha, therefore, was transitory. Studies at the protein level were based on immunoblotting using a monoclonal antibody (F88 12F8,1), characterized to be specific to MHCI alpha in rabbit muscle. These studies revealed a similar relationship between initial increase and successive decline of the MHCI alpha protein as seen at the mRNA level. Immunohistochemistry of 30-day stimulated muscle revealed that up to 65% of the fibres expressed the MHCI alpha isoform in combination with other adult MHC isoforms. The most frequent patterns of coexistence were MHCIIa + MHCI alpha + MHCI beta (28%), MHCI alpha + MHCI beta (18%), and MHCIIa + MHCI alpha (11%). According to these combinations, the upregulation of MHCI alpha may be assigned as an intermediate step in the transformation of existing fibres during the MHCIIa-->MHCI beta transition. A small fraction of fibres contained, in addition to the MHCI alpha + MHCI beta and MHCIIa + MHCI alpha combinations, developmental myosin, suggesting that MHCI alpha was also expressed in regenerating fibres originating from satellite cell-derived myotubes.

Animals↗

PubMed1999

The impact of biochemical methods for single muscle fibre analysis.

Biochemical methods for single muscle fibre analysis provide sensitive measures for elucidating muscle fibre heterogeneity. The understanding of the complexity of skeletal muscle fibres, initially based on qualitative histochemistry and immunohistochemistry, has been greatly expanded by quantitative micromethods, such as microphotometry and microbiochemical assays. Assessment of metabolic enzyme activity levels has revealed pronounced scattering within and between different fibre types and has highlighted the use of specific enzyme activity ratios as discriminative measures. With the exception of type I fibres, metabolic properties are loosely coupled with molecular properties of the myofibrillar apparatus. As such, myosin heavy chain (MHC) isoforms appear to be the best choice for fibre type delineation. Among the two available methods for MHC-based fibre type distinction, single fibre electrophoresis appears to be superior to immunohistochemistry. The electrophoretic separation of MHC isoforms in single fibres is quantitative and, as opposed to immunohistochemistry, yields important information on MHC isoform proportions in hybrid fibres. Histochemical staining for myofibrillar ATPase activity can, thus, be correlated in most cases with specific MHC isoform profiles. Single fibre studies have demonstrated a relationship between ATP phosphorylation potential and MHC isoform complement. This relationship corresponds to different tension costs and provides an additional rationale for the MHC-based fibre type diversity and transitions. The combination of reverse transcriptase (RT) with polymerase chain reaction (PCR) has proved to be a highly sensitive tool and has extended single fibre analysis to the level of MHC mRNA isoforms. Application of RT-PCR techniques to single fibre fragments identified by their MHC protein isoform profile, provides insights at two levels of expression and, thus, has extended our knowledge on the plasticity of muscle and the dynamical state of muscle fibres.

Animals↗

PubMed1999

Evidence that acidic fibroblast growth factor promotes maturation of rat satellite-cell-derived myotubes in vitro.

Satellite cells isolated from fast tibialis anterior (TA) and slow soleus (SOL) rat muscles were cultivated on matrigel, and treated with acidic fibroblast growth factor (aFGF). The following observations were made: 1) aFGF-treated cultures exhibited enhanced proliferation as mirrored by a twofold increase in DNA content. 2) Compared to the untreated cultures, myotubes in the aFGF cultures were larger; 3) Using reverse transcriptase polymerase chain reaction (RT-PCR) and northern blot analyses, we observed enhanced expression of all adult myosin heavy chain (MHC) isoforms, as well as of myogenin. These findings indicate that, under the culture conditions used, aFGF has a stimulatory effect on proliferation but also on maturation and differentiation of satellite cells. Furthermore, transcript levels of FGF receptor 1 (FGFR1) and 4 (FGFR4) isoforms, as well as of aFGF and bFGF were assessed by RT-PCR. aFGF-treated myotubes displayed increased expression of aFGF and bFGF, suggesting a paracrine effect of exogenous aFGF. In this regard, SOL-derived cultures responded more strongly than TA-derived cultures. The effects of aFGF treatment on the two receptors consisted of a decrease in FGFR1 and an increase in FGFR4 mRNA levels in 5-day-old cultures. In 8-day-old TA cultures, effects of FGF were similar to those in 5-day-old cultures. 8-day FGF-treated SOL cultures treated with FGF for 8 days exhibited higher FGFR1 and FGFR4 mRNA levels than the respective untreated cultures. Compared to 5 day-treated cultures, FGFR1 increased and FGFR4 decreased. This led to a shift in the ratio of FGFR1 to FGFR4 in the FGF-treated cultures which may explain the ability of satellite cells to differentiate under the influence of aFGF.

Age Factors↗

PubMed1999

Changes in FGF and FGF receptor expression in low-frequency-stimulated rat muscles and rat satellite cell cultures.

This study compares effects of chronic electrical stimulation on the expression levels of FGF-1, FGF-2 and their receptors (FGFRI, FGFR4) in rat tibialis anterior (TA) muscle of hypothyroid rat, as well as in satellite cell cultures derived from normal rat TA and soleus (SOL) muscles. In 5-day (5-d)-stimulated hypothyroid TA muscle, FGF-1 and FGF-2 mRNA levels were threefold elevated over control. FGFR1 and FGFR4 mRNAs were twofold and 1.5-fold elevated, respectively. In longer stimulated muscles, FGF-1 and FGFR4 mRNAs returned to basal levels, whereas FGF-2 mRNA remained elevated. FGFR1 mRNA decreased to control levels in 10-d stimulated muscles, but increased again after 20 days of stimulation. SOL- and TA-derived satellite cell cultures were stimulated for 5 days. At this time point, changes in myosin heavy chain isoforms were detectable consisting of increases in MHCI mRNA and decreases in MHCIIb and MHCIId mRNA. The comparison between 5-d-stimulated hypothyroid TA muscle and 5-d-stimulated TA- and SOL-derived satellite cell cultures revealed differences in the expression of FGF-1 and FGF-2, but similar expression levels of FGFR1 and FGFR4. Even though FGF-1 and FGF-2 mRNAs were elevated in the satellite cell cultures, their increases were less pronounced than in the stimulated hypothyroid muscle. Taking into consideration that skeletal muscle contains muscle fibres and various non-muscle tissues, e.g. blood vessels, these results suggest that the latter contribute to the observed increases in FGF-1 and FGF-2 expression in stimulated muscle.

Actins↗

PubMed1999

Effects of contractile activity and hypothyroidism on nuclear hormone receptor mRNA isoforms in rat skeletal muscle.

Absolute molecule numbers of thyroid hormone receptor isoforms T3Ralpha1, T3Ralpha2, T3Rbeta1, and the 9-cis retinoic acid receptor gamma were measured in adult fast extensor digitorum longus (EDL) and slow soleus (SOL) muscles of rat by competitive reverse transcriptase (RT)-PCR. The nuclear hormone receptor corepressor (NCoR) mRNA was quantified by noncompetitive RT-PCR in the same muscles. T3Rbeta1 mRNA was the most abundant isoform in both muscle types. All nuclear hormone receptor (NHR) mRNAs were found at lower molecule numbers in fast than in slow muscle. No differences existed with regard to NCoR mRNA. With the exception of T3Ralpha1 in the EDL, hypothyroidism led to decreases in NHR mRNAs, especially in SOL, but did not significantly affect the level of NCoR mRNA. Enhanced neuromuscular activity of the fast EDL muscle, as induced by chronic low-frequency stimulation, transiently increased NHR mRNAs, but decreased NCoR mRNA. These chronic-low-frequency-stimulation-induced changes were attenuated by hypothyroidism.

Animals↗

PubMed1999

Prolonged contraction-relaxation cycle of fast-twitch muscles in parvalbumin knockout mice.

The calcium-binding protein parvalbumin (PV) occurs at high concentrations in fast-contracting vertebrate muscle fibers. Its putative role in facilitating the rapid relaxation of mammalian fast-twitch muscle fibers by acting as a temporary buffer for Ca2+ is still controversial. We generated knockout mice for PV (PV -/-) and compared the Ca2+ transients and the dynamics of contraction of their muscles with those from heterozygous (PV +/-) and wild-type (WT) mice. In the muscles of PV-deficient mice, the decay of intracellular Ca2+ concentration ([Ca2+]i) after 20-ms stimulation was slower compared with WT mice and led to a prolongation of the time required to attain peak twitch tension and to an extension of the half-relaxation time. The integral [Ca2+]i in muscle fibers of PV -/- mice was higher and consequently the force generated during a single twitch was approximately 40% greater than in PV +/- and WT animals. Acceleration of the contraction-relaxation cycle of fast-twitch muscle fibers by PV may confer an advantage in the performance of rapid, phasic movements.

Animals↗

PubMed1999

Muscle LIM protein: expressed in slow muscle and induced in fast muscle by enhanced contractile activity.

To identify early changes in gene expression during the fast-to-slow transition induced by chronic low-frequency stimulation, total RNA was extracted from 12-h-stimulated tibialis anterior (TA) muscles of rats and amplified by differential display RT-PCR. Among the signals of differentially expressed mRNAs, a cDNA approximately 300 bp in length, which was almost undetectable in control TA muscles but prominent in stimulated TA and normal soleus muscles, was identified. This cDNA was cloned and identified as corresponding to the mRNA of the muscle LIM protein (MLP). Its differential expression in control, stimulated TA, and soleus muscles was verified by Northern blotting. Antibodies against MLP were used to identify by immunoblot analysis a protein of 22 kDa, the predicted molecular mass of MLP. Immunohistochemistry revealed strong reactivity for MLP in all fibers of normal soleus muscle and faint staining of some type IIA and type I fibers in control TA muscle. These fibers increased in number and staining intensity in 4-day-stimulated TA muscle. MLP thus seems to play an essential role during the rearrangement of cytoskeletal and/or myofibrillar structures in transforming adult muscle fibers.

Amino Acid Sequence↗

PubMed1999

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.

Animals↗

PubMed1999

Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat.

Ca(2+) transients were investigated in single fibers isolated from rat extensor digitorum longus muscles exposed to chronic low-frequency stimulation for different time periods up to 10 days. Approximately 2.5-fold increases in resting Ca(2+) concentration ([Ca(2+)]) were observed 2 h after stimulation onset and persisted throughout the stimulation period. The elevated [Ca(2+)] levels were in the range characteristic of slow-twitch fibers from soleus muscle. In addition, we noticed a transitory elevation of the integral [Ca(2+)] per pulse with a maximum ( approximately 5-fold) after 1 day. Steep decreases in rate constant of [Ca(2+)] decay could be explained by an immediate impairment of Ca(2+) uptake and, with longer stimulation periods, by an additional loss of cytosolic Ca(2+) binding capacity resulting from a decay in parvalbumin content. A partial recovery of the rate constant of [Ca(2+)] decay in 10-day stimulated muscle could be explained by an increasing mitochondrial contribution to Ca(2+) sequestration.

Adenosine Triphosphate↗