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Biomedical subjects

D Pette

Publications and source records attributed to D Pette.

At least 145 records · Page 8Linked to original sources
PubMed1990

In situ hybridization of slow myosin heavy chain mRNA in normal and transforming rabbit muscles with the use of a nonradioactively labeled cRNA.

A specific method for in situ-hybridization of slow myosin heavy chain MHCI (beta-cardiac MHC) mRNA was established with the use of a nonradioactively labeled cRNA probe. The digoxigenin-labeled probe was the T7-RNA polymerase transcript from a 350 bp SacI fragment of a rabbit beta-cardiac MHC cDNA. Northern blot analyses of RNA preparations from skeletal and cardiac muscles with homologous and complementary RNA proved the specificity of the hybridization. The in situ-hybridization was applied for studying the distribution of MHCI mRNA in normal fast- and slow-twitch muscles, as well as in muscles undergoing fast-to-slow transformation by chronic low-frequency stimulation. The majority of soleus muscle fibers was intensely stained, whereas fast-twitch muscles contained only a few positive fibers. The intracellular distribution of the hybridization product showed a clear relationship to the nuclei with intense staining of the perinuclear regions within the subsarcolemmal space. The more intensely stained fibers of transforming muscle displayed hybridization product also within the nuclei. As revealed by inspection of longitudinal sections at high magnification and polarized light, MHCI mRNA was also detectable in the myofibrils in a cross-striational pattern resulting from staining of the I-bands.

Animals↗
PubMed1990

Effect of chronic hypoxia on muscle enzyme activities.

Biopsies from the vastus lateralis muscle of seven participants in the Swiss expedition to Mt. Everest and Lhotse in 1986 were taken before departure to and after return from high altitude, and used for measurements of maximal activities of 12 reference enzymes of anaerobic and aerobic-oxidative metabolic pathways. The results indicated that strenuous exercise at high altitude induced increases in enzyme activities of glycolysis and decreases in enzyme activities of terminal substrate oxidation (the citric acid cycle, fatty acid oxidation, ketone body utilization, respiratory chain). The decreases in enzyme activities of aerobic-oxidative metabolism were related to similar decrements in mitochondrial volume density, which suggests that the enzymic changes resulted from a loss of mitochondrial structure rather than from qualitative changes of the mitochondrial population. These changes indicated that strenuous exercise may intensify the stress of high-altitude exposure and, thus, induce an aerobic to anaerobic shift of muscle energy metabolism.

Acclimatization↗
PubMed1990

Characterization of myosin isoforms in satellite cell cultures from adult rat diaphragm, soleus and tibialis anterior muscles.

Satellite cells were isolated by enzymatic dissociation and Percoll gradient centrifugation from adult rat diaphragm, soleus, and tibialis anterior muscles with fairly reproducible yields. Diaphragm and soleus muscle yielded approximately five times more satellite cells than tibialis anterior muscle. According to light microscopic criteria, no morphological differences existed between the satellite cell cultures of different origin. Contrary to the donor muscles, myotubes from the 10-day-cultured satellite cells contained a uniform myosin heavy chain (MHC) pattern with predominance of an immunochemically identified embryonic heavy chain. The three types of cultures displayed a typical embryonic light chain (LC) pattern with LC1emb, LC1f, LC2f, and traces of LC3f. The isomyosin pattern was characterized by four embryonic isomyosins, eM1-eM4, with similar distributions in the three cultures. In summary, these myosin analyses provide no evidence for the existence of satellite cell diversity among three rat muscles of different fiber-type composition, at least not under the applied in vitro conditions.

Animals↗
PubMed1990

Effects of electrically induced contractile activity on cultured embryonic chick breast muscle cells.

Development of chicken breast muscle is characterized by the sequential appearance of six electrophoretically distinct myosin heavy chain (HC) isoforms. Cultured secondary myotubes, derived from 12-day embryonic chick breast muscle, mainly express the early embryonic HC isoform HCemb/e, normally present in 8-day embryonic breast muscle, and the two fast light chain isoforms LC1f and LC2f. Direct low-frequency (2.5 Hz) stimulation of these myotubes via platinum electrodes leads to a shift in myosin HC expression with increases in the late embryonic HC isoform HCemb/l amounting to 35% of total HC in 19-day-stimulated cultures. Measurements of 35S-methionine incorporation and immunohistochemical analyses demonstrate increases in LC3f. This increase is also seen at the mRNA level. These results indicate that induced contractile activity promotes myotube maturation in vitro. The observation that chronic stimulation enhances the expression of the slow isoform LC2s at the RNA, as well as the protein level, suggests an additional effect consisting of a fast-to-slow change in phenotype expression. In view of the fact that muscle maturation and phenotype expression is under neural control during development in vivo, our results on directly stimulated, aneural myotubes indicate that neurally transmitted contractile activity may be an important factor in modulating phenotype expression of secondary myotubes.

Animals↗
PubMed1989

Changes in myosin heavy chain isoforms during chronic low-frequency stimulation of rat fast hindlimb muscles. A single-fiber study.

Fast-twitch rat muscles contain three fast myosin heavy chains (HC) which can be separated by density gradient gel electrophoresis. Their mobility increases in the order of HCIIa less than HCIId less than HCIIb. In contrast to the rabbit, where chronic low-frequency nerve stimulation induces a fast-to-slow conversion, stimulation for up to 56 days does not lead to appreciable increases in the relative concentration of the slow myosin heavy chain HCI in rat fast-twitch muscles. However, chronic stimulation of rat fast-twitch muscle does evoke a rearrangement of the fast myosin heavy chain isoform pattern with a progressive decrease in HCIIb and progressive increases in HCIIa and HCIId. As judged from the time course and extent of these transitions, it appears that HCIId is an intermediate form between HCIIb and HCIIa. Single-fiber analyses of normal muscles make it possible to assign these heavy chain isoforms to histochemically defined fiber types IIB, IID, and IIA. The stimulation-induced fiber transformations produce numerous hybrid fibers displaying more than one myosin heavy chain isoform. Some transforming fibers contain up to four different myosin heavy chain isoforms.

Animals↗
PubMed1989

Slow/cardiac sarcoplasmic reticulum Ca2+-ATPase and phospholamban mRNAs are expressed in chronically stimulated rabbit fast-twitch muscle.

Fast-twitch extensor digitorum longus muscles of the rabbit were subjected to chronic low-frequency stimulation during different time periods. Changes in the relative amounts of mRNAs encoding fast and slow/cardiac Ca2+-ATPase isoforms were assessed through the use of an RNase-protection assay. Stimulation-induced increases in slow cardiac Ca2+-ATPase and phospholamban mRNAs were quantified by mRNA hybridization. Prolonged stimulation resulted in an exchange of the fast with the slow/cardiac Ca2+-ATPase isoform mRNAs. The exchange was complete after 72 d of stimulation as compared with normal slow-twitch soleus muscle. The tissue content of phospholamban mRNA reached levels similar to that found in normal slow-twitch soleus muscle by the same time. The conversion of the sarcoplasmic reticulum coincided with the fast-to-slow troponin C isoform transition, previously investigated in the same muscles.

Animals↗
PubMed1989

Chronic long-term electrostimulation creates a unique metabolic enzyme profile in rabbit fast-twitch muscle.

Long-term low-frequency stimulation (up to 120 days) of rabbit fast-twitch tibialis anterior muscle led, in a first-order-like time course, to changes in enzyme activities of energy metabolism which became stable with ongoing stimulation after 50 days. The glycolytic enzymes decreased to 30-40% of their normal values, but remained 2-3-fold higher than in heart or soleus muscle. The LDH isozyme pattern ultimately resembled that of the slow-twitch soleus muscle. Citrate synthase activity increased 3.7-fold which brought this enzyme to a value 45% above that of heart. These results indicate that chronic stimulation does not simply convert the fast-twitch muscle into a soleus-like slow-twitch muscle, but creates a tissue of unique metabolic properties.

Animals↗
PubMed1989

Electrostimulation-induced increases in fatty acid-binding protein and myoglobin in rat fast-twitch muscle and comparison with tissue levels in heart.

Chronic stimulation of rat fast-twitch muscle increased the content of both fatty acid-binding protein (FABP) and myoglobin. The increases in FABP, which reached values close to that of cardiac muscle, exceeded those in myoglobin and those in citrate synthase and 3-hydroxyacyl-CoA dehydrogenase activities.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1989

Chronic stimulation of rat skeletal muscle induces coordinate increases in mitochondrial and nuclear mRNAs of cytochrome-c-oxidase subunits.

Fast-twitch tibialis anterior muscle of the rat was subjected to chronic low-frequency (10 Hz, 10 h daily) nerve stimulation in order to investigate the time course of changes in cytochrome-c-oxidase activity, as well as in tissue levels of specific mitochondrially and nuclear-encoded, cytochrome-c-oxidase-subunit mRNAs. Chronic stimulation induced a progressive increase in cytochrome-c-oxidase activity which was threefold elevated after 35 days. A similar increase was recorded for citrate-synthase activity. Glyceraldehyde-3-phosphate dehydrogenase, which was studied as a glycolytic reference enzyme, moderately decreased, as did the tissue level of its corresponding mRNA. There was a parallel increase in the tissue levels of the two cytochrome-c-oxidase-subunit mRNAs over the entire stimulation time course. The extent of increase (stimulated/control) was 2.4 +/- 0.3 and 1.8 +/- 0.2 (means +/- SEM) for the mitochondrial and nuclear subunit mRNAs, respectively. This parallel increase suggested a coordinate regulation of the two subunits. The increase in cytochrome-c-oxidase activity initially corresponded to the changes at the mRNA level. However, with longer stimulation times (beyond 14 days), the increase in cytochrome-c-oxidase activity clearly exceeded that of the two mRNAs. This divergence was progressive and was interpreted to indicate that the increase in cytochrome-c-oxidase content was brought about not only by changes in the levels of the specific mRNAs, but also by alterations at the level of translation.

Animals↗
PubMed1989

Electrostimulation-induced fast-to-slow transitions of myosin light and heavy chains in rabbit fast-twitch muscle at the mRNA level.

Chronic low-frequency stimulation of rabbit fast-twitch muscle induces progressive increases in slow myosin light chain mRNAs followed by an increase in the slow myosin heavy chain HCI mRNA. Therefore, the effects of chronic stimulation are more pronounced in rabbit than in rat fast-twitch muscle. The latter responds mainly with a rearrangement of its fast isomyosin pattern.

Animals↗
PubMed1989

Chronic stimulation-induced changes of myosin light chains at the mRNA and protein levels in rat fast-twitch muscle.

Transitions in the expression of the myosin light chains (LC) were investigated in fast-twitch muscles of the rat during chronic (10 h/day), low-frequency (10 Hz) stimulation. Changes were followed at the mRNA level by Northern blot analysis and in vitro translation, as well as at the protein level by electrophoresis under denaturing and nondenaturing conditions. In vivo synthesis of the light chains was assessed by measuring the incorporation of intramuscularly injected [35S]methionine. Chronic stimulation induced a transition in the isomyosin pattern with an increase of FM3, a concomitant decrease in FM1 and, after longer stimulation periods, the appearance of low concentrations of the slow isomyosin. These changes were accompanied by an elevated LC1f/LC3f ratio and increases in the amounts of both the LC1sb and, to a lesser degree, LC2s proteins. Alterations in the amounts of specific mRNAs were the same whether determined by Northern blot analysis or by in vitro translation of total RNA preparations from the same muscles. Generally, the changes in the relative concentrations of fast and slow light-chain proteins agreed with the changes detected at the mRNA level and the alterations in protein synthesis detected with the use of an in vivo labeling assay. An exception was the elevated tissue content of LC2s where no changes were detectable in the concentration of its mRNA as determined by in vitro translation or in vivo synthesis. The increase in LC2s protein may, therefore, have been due to reduced degradation. In addition, the decrease in LC3f was more pronounced at the protein level than at the mRNA level. This might indicate an increased turnover of LC3f or the existence of additional post-transcriptional regulations of LC3f expression.

Animals↗
PubMed1989

The multiplicity of troponin T isoforms. Distribution in normal rabbit muscles and effects of chronic stimulation.

Polyclonal antibodies were raised in guinea pigs against troponin-T (TnT) isoforms purified from fast- and slow-twitch rabbit muscles. With the use of these antibodies and immunoblots of one- and two-dimensional electrophoreses, the distribution of fast and slow TnT isoforms was investigated in normal and chronically stimulated hindlimb muscles of the rabbit. According to differences in their apparent molecular masses, six fast TnT isoforms (TnTcf, TnT1f, TnT2f, TnT3f, TnT4f, TnT5f) were distinguished in normal tibialis anterior and extensor digitorum longus muscles. These muscles also contained low amounts of TnT1s and TnT2s which were the predominant TnT isoforms in slow-twitch soleus muscle. Fast and slow TnT isoforms were found to exist in several charge variants, i.e. one for TnTcf, three different charge forms for TnT1f, seven for TnT2f, four for TnT3f, three for TnT4f, one for TnT5f, four for TnT1s, and three for TnT2s. Some charge variants were phosphorylated isoforms because treatment with alkaline phosphatase reduced the number of the 19 fast and 7 slow variants to 12 and 3, respectively. The stimulation-induced fast-to-slow transition caused progressive decreases in fast and increases in slow isoforms. The decrease and the disappearance of the major fast isoforms followed a sequence of TnT2f, TnTcf, TnT4f, TnT1f, and TnT3f. This decrease in fast isoforms fits well with the reduction of fast TnT mRNAs assessed by Northern blot analysis. Prolonged stimulation ultimately created a TnT isoform pattern similar to that found in normal slow-twitch muscle. Stimulation also induced changes in the tropomyosin subunit pattern with a decrease in the fast and an increase in the slow alpha-tropomyosin subunit without altering the alpha/beta-tropomyosin subunit ratio. Similar to slow-twitch soleus muscle, long-term stimulated muscles contained appreciable amounts of the fast alpha-tropomyosin subunit, but only traces of fast TnT isoforms. This combination indicated that the predominant slow TnT isoforms may be capable of interacting with fast tropomyosin in these muscles.

Animals↗
PubMed1989

Altered expression of myosin light-chain isoforms in chronically stimulated fast-twitch muscle of the rat.

Fast-twitch tibialis anterior muscle of the rat was chronically stimulated for periods of 18 days, 28 days and 56 days. Changes in the myosin light-chain (LC) pattern consisted in an increase in LC1f, concomitant with a decrease in LC3f. In contrast to previous findings in chronically stimulated fast-twitch tibialis anterior muscle of the rabbit, no substantial increases occurred in the slow myosin light-chain isoforms. In vivo labeling using [35S]methionine incorporation revealed differences in relative turnover between the fast myosin light chains. The relative turnover of the fast myosin light chains appeared to increase in normal muscle in the order LC2f less than LC1f less than LC3f. As judged from [35S]methionine incorporation, the changes in light-chain tissue content mainly resulted from altered synthesis rates. However, in the case of LC3f the decrease in protein content could not only be explained by a reduced synthesis, but, additionally, appeared to be due to enhanced degradation. Parvalbumin, which was included in the present study, was also found to decrease in the stimulated muscle. However, its decrease appeared to result primarily from reduced synthesis.

Animals↗
PubMed1989

Myosin heavy chain isoforms in histochemically defined fiber types of rat muscle.

Combined histochemical and biochemical analyses were performed on rat skeletal muscles in order to determine the myosin heavy chain patterns in specific fiber types. Four myosin heavy chain isoforms were separated by gradient polyacrylamide gel electrophoresis of extracts from single fibers and whole muscle homogenates. Their electrophoretic mobility increased in the order HCIIa, HCIIb, and HCI. HCIIa, HCIIb and HCI were present as unique isoforms in histochemically defined fiber types IIA, IIB and I, respectively. The isoforms HCI and HCIIa coexisted at variable ratios in type IC and IIC fibers. An additional fast myosin heavy chain isoform with an electrophoretic mobility between HCIIa and HCIIb was designated as HCIId because of its abundance in fast fibers of large diameter in the diaphragm. With the exception of slight differences in mATPase staining intensity after acid preincubation, these fibers were almost indistinguishable from type IIB fibers. In view of their specific myosin heavy chain composition (HCIId), these fibers were named type IID. In the extensor digitorum longus muscle, type IID fibers were of smaller size than type IIB and differed from the latter by higher NADH tetrazolium reductase activities. Circumstantial evidence suggests that type IID fibers are identical with the 2X fibers, previously described by Schiaffino et al. (1986).

Animals↗
PubMed1989

Species-specific responses of muscle lactate dehydrogenase isozymes to increased contractile activity.

Analyses of lactate dehydrogenase (LDH) isozymes were performed in normal and chronically stimulated (10 Hz, 10 h/d) tibialis anterior (TA) muscles of the mouse, rat, guinea pig, and rabbit. The electrophoretic mobilities of the individual LDH isozymes of normal muscles revealed species-specific patterns. Stimulation up to 28 days evoked increases in the percentage of the H-subunit of LDH. The absolute increase was highest in the rabbit, intermediate in the guinea pig and rat, and lowest in the mouse. The extent of the M- to H-subunit exchange, thus, appeared to be inversely correlated with the basal aerobic-oxidative capacities of the investigated muscles. In addition, a relationship between the expression of the H-subunit of LDH and the increase in aerobic-oxidative capacity became evident from these stimulation-induced processes of metabolic adaptation.

Animals↗
PubMed1989

Relations between chronic stimulation-induced changes in contractile properties and the Ca2+-sequestering system of rat and rabbit fast-twitch muscles.

This study compares changes in contractile properties, parvalbumin content, and Ca2+-uptake by the sarcoplasmic reticulum (SR) of low-frequency stimulated rat and rabbit tibialis anterior (TA) muscles. Time to peak tension increased 1.8-fold in 35-day stimulated rabbit TA, while no change occurred in rat TA. Isometric twitch tension increased 2-fold in rabbit TA, but was unaltered in rat TA. Parvalbumin (PA) content was more than 90% reduced in rabbit TA, but only 60% in rat TA after 35 days. At this time, PA content of the stimulated rat TA was still higher than that of normal rabbit TA. Taking into account the suggested role of PA as a cytosolic Ca2+ buffer, its decrease could lead to an impaired free Ca2+-decay with a prolonged active state and a higher tension output during a single twitch. This would explain why chronic stimulation led to an increase in isometric twitch tension in rabbit TA, but not in rat TA. The 1.6-fold rise in half-relaxation time of 35-day stimulated rat and rabbit TA most likely resulted from a 50% reduced Ca2+-uptake by the SR, due to a still unknown modification of the Ca2+-transport ATPase.

Animals↗
PubMed1988

Six myosin heavy chain isoforms are expressed during chick breast muscle development.

Two major embryonic myosin heavy chains are expressed in embryonic chick breast muscle until the first week after hatching. Of these, one is already detected in the 8-day-old embryo. The other appears on day 12. Two putative slow embryonic isoforms represent minor components transiently expressed between days 8 and 12. A neonatal heavy chain is expressed at low concentrations on day 8 and increases with development. It is the only isoform two weeks after hatching, and is ultimately replaced by the fast myosin heavy chain in the adult muscle.

Animals↗