PubMed Health⌕ Search

Biomedical subjects

H Peuker

Publications and source records attributed to H Peuker.

11 recordsLinked to original sources
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

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↗
PubMed1998

Energy state and myosin heavy chain isoforms in single fibres of normal and transforming rabbit muscles.

Energy-rich phosphates, [ATP]/[ADPfree] ratios, and the myosin heavy chain (MHC) complement were determined in single fibres from normal rabbit muscles, and in fibres isolated from tibialis anterior muscle undergoing fast-to-slow conversion by chronic low-frequency stimulation (CLFS). In normal muscles, energy-rich phosphate contents and [ATP]/[ADPfree] ratios could thus be assigned to different MHC-based fibre types. Phosphocreatine (PCr) contents and [ATP]/[ADPfree] ratios differed markedly between fast- and slow-twitch fibres, as well as within the fast fibre subtypes. Both magnitudes were approximately twofold higher in the fastest (type IIB) fibres as compared to the slowest (type I) fibres. According to PCr contents and [ATP]/[ADPfree] ratios pure and hybrid fibres were aligned in an order similar to that determined by their contractile properties and myofibrillar ATPase activities. CLFS for up to 30 days induced pronounced decreases in PCr and [ATP]/[ADPfree] which attained levels twofold lower than in normal slow-twitch fibres. In both normal and stimulated muscles, PCr and [ATP]/[ADPfree] ratios were correlated, indicating their equilibrium in the different fibre types. The relationship detected between MHC isoform expression and the [ATP]/[ADPfree] ratio suggests that the drastic and persistent depression of the cellular energy state may act as an important signal initiating fast-to-slow transformation processes in muscle fibres.

Adenosine Diphosphate↗
PubMed1998

Changes in myosin heavy chain mRNA and protein isoforms of rat muscle during forced contractile activity.

A quantitative reverse transcriptase-polymerase chain reaction was established to determine absolute amounts of mRNAs specific to four myosin heavy chain isoforms [MHCIIb, MHCIId(x), MHCIIa, and MHCI beta] in rat extensor digitorum longus muscle during forced contractile activity by chronic (10 h/day) low-frequency stimulation (CLFS). The induced changes in absolute and relative mRNA amounts were similar. MHCIIb mRNA decreased rapidly after 1 day, and MHCIIa mRNA increased after 3 days. MHCIId(x) started to decrease at day 7. After 42 days, the MHCIIb, MHCIId(x), MHCIIa, and MHCI beta mRNAs amounted to 2, 6, 90, and 2% of total MHC mRNAs, respectively. Changes at the protein level were studied in a second experimental series increasing CLFS (24 h/day, up to 100 days). Also under these conditions, MHCI beta reached only a fraction of 12% (2-fold elevation). The changes at the protein level remained restricted to the MHCIIb to MHCIIa transition, which agrees with the notion that the induced changes in MHC isoform expression primarily resulted from altered pretranslational activities. Rat fast-twitch muscle thus exhibits a restricted capacity for fast-to-slow conversion.

Adaptation, Physiological↗
PubMed1998

Alpha-cardiac-like myosin heavy chain as an intermediate between MHCIIa and MHCI beta in transforming rabbit muscle.

To elucidate the sequence of myosin heavy chain (MHC) transitions in fast-to-slow transforming rabbit muscle, direct reverse transcriptase-polymerase chain reaction was applied for detecting mRNAs specific to five MHC isoforms in single fibers from control and low-frequency-stimulated tibialis anterior muscles. The detection of MHCIIb, MHCIId(x), MHCI alpha, and MHCI beta mRNAs was based on previously published methods. The RT-PCR assay for MHCIIa mRNA was based on the identification of a cDNA sequence in the 3'-region from which specific primers were derived. Comparisons between rat, rabbit, and human MHCIIa sequences revealed high degrees of sequence identities. MHC mRNA isoform patterns in single fibers from stimulated muscles showed hybrid fibers expressing the following combinations: MHCIId(x) + MHCIIa, MHCIId(x) + MHCIIa + MHCI alpha, MHCIId(x) + MHCIIa + MHCI alpha + MHCI beta, MHCIIa + MHCI alpha, MHCIIa + MHCI alpha + MHCI beta, and MHCI alpha + MHCI beta. The combination MHCIIa + MHCI beta without MHCI alpha was never seen. These coexpression patterns suggest that the fast-to-slow fiber transition results from sequential isoform expressions in the order MHCIId(x)--> MHCIIa-->MHCI alpha-->MHCI beta. The allocation of MHCI alpha between MHCIIa and MHCI beta seems to be in line with graded differences in sequence identity of the 3'-regions of these mRNA isoforms.

Actins↗
PubMed1997

Quantitative analyses of myosin heavy-chain mRNA and protein isoforms in single fibers reveal a pronounced fiber heterogeneity in normal rabbit muscles.

A highly sensitive method of reverse-transcriptase polymerase chain reaction (RT-PCR) was established to study myosin heavy-chain (MHC) mRNA isoform expression in single fibers of rabbit limb muscles. In combination with myofibrillar adenosine triphosphatase histochemistry and electrophoretic separation of MHC protein isoforms in fragments of the same fibers, the direct RT-PCR method identified the pMHC20-40 and pMHC24-79 cDNA sequences as being specific to MHCIIb and MHCIId/x isoforms, respectively. In addition, a direct RT-PCR was established for determining relative amounts of MHC mRNA isoforms by using a sequence specific to alpha-skeletal actin as an endogenous reference. Analyses of large amounts of single fibers revealed an unexpected heterogeneity of the fast fiber population with regard to numerous fibers coexpressing MHCIIb and MHCIId/x. Based on quantitative RT-PCR, the percentages of MHCIIb/MHCIId hybrid fibers amounted to approximately 55% in the deep portion of gastrocnemius, to 43% in the adductor magnus, and to 12% in psoas muscle. Moreover, the two MHC mRNA isoforms were nonuniformly distributed along the fiber length. Qualitative RT-PCR detected even higher amounts of hybrid fibers in the three muscles. The percentages of hybrid fibers identified at the protein level were smaller in adductor magnus muscle (25%) and psoas muscle (5%), but equaled that of the mRNA analysis in gastrocnemius muscle (61%). The detection of high amounts of IIBD and IIDB fibers suggested that hybrid fibers represent functional elements within the fiber spectrum of normal muscles. Our observations on hybrid fibers reveal a heterogeneity within the fiber population of normal muscles that has not been realized to date.

Animals↗
PubMed1995

Reverse transcriptase-polymerase chain reaction detects induction of cardiac-like alpha myosin heavy chain mRNA in low frequency stimulated rabbit fast-twitch muscle.

Using reverse transcriptase-polymerase chain reaction we quantified in rabbit skeletal muscles expression levels of the highly homologous cardiac alpha and beta myosin heavy chain (alpha MHC, beta MHC) mRNA isoforms. Masseter muscle displayed highest levels of a cardiac-like alpha MHC mRNA. This isoform was present at 20-fold lower amounts in slow soleus and at 200-fold lower levels in several fast-twitch muscles. Low-frequency stimulation periods exceeding 20 days drastically induced the alpha MHC mRNA in fast tibialis anterior. The alpha MHC mRNA was 140-fold elevated after 60 days when beta MHC mRNA had increased 50-fold. Our results demonstrate the wide distribution of a cardiac-like alpha MHC mRNA in skeletal muscle and its marked induction during fast-to-slow transition as induced by low-frequency stimulation.

Animals↗
PubMed1993

Non-radioactive reverse transcriptase/polymerase chain reaction for quantification of myosin heavy chain mRNA isoforms in various rabbit muscles.

A method was established for measuring molecule numbers of three different myosin heavy chain (MHC) mRNA isoforms in total RNA preparations. The quantification was based on a combination of primer-directed reverse transcriptase and polymerase chain reactions with 5'-digoxigenin-labeled oligonucleotides, using external standards. The sensitivity of the method allowed the quantitation of mRNA amounts down to the range of 1,000 molecules (detection limit 50 molecules). The numbers determined for eight different rabbit muscles are in the range of 10(3)-10(9)/micrograms total RNA. In soleus muscle, the value of 1.11 x 10(9) MHCI mRNA molecules corresponds to approximately 8% of the total mRNA. With reference to myonuclei, this amount corresponds to 1-2 x 10(4) molecules/nucleus. A quantitative comparison of the two fast MHC mRNA isoforms with the distribution of different MHC isoforms at the protein level indicates that one of these two fast sequences is specific to MHCIIb and the other to MHCIId. However, our data point to the existence of additional MHCIId mRNA subtypes.

Animals↗