Biomedical subjects
Publications and source records attributed to D Pette.
An 11-fold increase in hexokinase activity and the hexokinase II isoform was found in rat tibialis anterior muscle after 7 days of chronic, low-frequency stimulation. In vivo labeling studies showed that this increase in enzyme protein content was related to an approx. 30-fold increase in [35S] methionine incorporation.
A new fast myosin heavy chain isoform was electrophoretically detected in adult rat skeletal muscles. It was present at high levels in diaphragm and, therefore, designated as MHCIId. Appreciable amounts of MHCIId were detected in tongue musculature, the extraocular muscles, and in the deep red portions of various fast muscles. Its concentration in fast-twitch muscle was greatly increased by chronic stimulation.
Type I protein, a myofibrillar protein thought to be specific to slow-twitch skeletal muscle fibers, was purified. Two-dimensional electrophoresis indicated its identity with the purified slow troponin-T1s isoform. Immunochemical analyses using antibodies raised against type I protein and slow Tn-T1s, further substantiated the identity of the two proteins.
Ca2+-uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2+-sensitive electrode in homogenates from fast- and slow-twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2+-ATPase content allowed determination of the specific Ca2+-transport activity of the enzyme. In 3-week-old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40-50% and 30-50% of the normal values in fast- and slow-twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2+-ATPase to calsequestrin protein ratio. Equal amounts of the Ca2+-ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2+-oxalate loading from adult normal and dystrophic fast-twitch muscles. However, the specific ATP-hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP-hydrolysing activity was correlated with decreased Ca2+-transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2+-ATPase molecules in murine muscular dystrophy.
A 68-kDa protein is present in increasing amounts in the soluble fractions of fast, slow and cardiac muscles of the rabbit. Induced contractile activity by chronic 10-Hz stimulation caused in rabbit fast-twitch muscle a rapid increase of this protein, reaching fivefold to sixfold higher levels than normal after one week of stimulation. The 68-kDa protein was identified as albumin by its mobility in one-dimensional and two-dimensional electrophoreses and by its reactivity with a polyclonal antibody directed against rabbit serum albumin. Immunohistochemistry revealed a localisation pattern corresponding to a distribution mainly in the interstitium. The assumption that albumin is not an intrinsic protein of the muscle fiber was strongly supported by the failure to show its synthesis by in vivo labeling. Injection of [35S]methionine into normal and chronically stimulated muscles did not lead to a detectable incorporation of the precursor into muscle albumin. In addition, albumin could not be detected as a product formed by in vitro translation of RNA preparations from normal and chronically stimulated muscles.
Adult mammalian skeletal muscle fibres can be separated into two distinct groups, fast and slow. Within each group there is a continuum of metabolic enzyme activity levels. In addition there are fast and slow isoforms of various myofibrillar proteins such as myosin, tropomyosin and troponin. These proteins are multimeric and multiple isoforms of their subunits assemble to create a continuum of subtypes within each major group. Fibres which coexpress both fast and slow subunit isoforms have an increased number of possible isoform combinations such that an entire spectrum of fibre 'types' is found between the two extremes, fast and slow. Numerous myosin heavy chain and fast troponin T isoforms further increase the diversity of muscle fibres. Such cellular diversity helps to explain the dynamic nature of skeletal muscle. Each individual fibre is able to respond to various functional demands by appropriate changes in its phenotypic expression of specific proteins.
Chronically stimulated fast-twitch muscles of the rabbit were histochemically and immunohistochemically analyzed in serial cross sections (1) for percentages of fiber types, and (2) for the presence of myosin heavy chain isoforms during fast-to-slow transformation. By four weeks of stimulation the number of type-I fibers had increased more than fourfold, while only about 6% of the original IIB fibers remained. Type-IC and -IIC fibers transiently rose to 20% of the total fiber population. After 16 weeks, the number of type-I fibers had increased to 42%. With prolonged stimulation fewer fibers reacted with antibodies against embryonic and neonatal myosins and more with the antibody against slow myosin. The reaction for embryonic myosin was most often detected in the C fibers (IC, IIC). Immunohistochemical subtypes were observed for each fiber type in the stimulated muscles. The greatest number was seen in type-IIC fibers, which, in addition to their reaction for fast/neonatal and slow myosins, might also react with the antibodies against neonatal/embryonic and embryonic myosins. These findings indicated that the transforming fibers temporarily expressed myosin heavy chain isoforms normally not detectable in adult skeletal muscle. Myotubes reacted strongly with the antibodies against fast/neonatal and embryonic myosins, and some of them also with the antibody against slow myosin. Thus, it appears that under the influence of the low frequency stimulus pattern some of the newly formed myotubes developed into type-I fibers.
Serial cross sections of rat, rabbit and cat intrafusal fibers from muscle spindles of normal adult hindlimb muscles were incubated with a monoclonal antibody against embryonic myosin heavy chains. Intrafusal fiber types were identified by noting their staining patterns in adjacent sections incubated for myofibrillar ATPase after acid or alkaline preincubation. In rat and rabbit muscle spindles dynamic nuclear bag1 fibers reacted strongly at the polar and juxtaequatorial regions. Static nuclear bag2 fibers reacted weakly or not at all at the polar region, but showed a moderate amount of activity at the juxtaequator. At the equatorial region both types of nuclear bag fibers displayed a rim of fluorescence surrounding the nuclear bags, while the areas occupied by the nuclear bags themselves were negative. Nuclear chain fibers in rat and rabbit muscle spindles were unreactive with the specific antibody over their entire length. In cat muscle spindles both types of nuclear bag fibers presented profiles which resembled those of the nuclear bag fibers in the other two species, but unlike in rat and rabbit spindles, cat nuclear chain fibers reacted as strongly as dynamic nuclear bag1 fibers.
Sarcoplasmic reticulum (SR) Ca2+-pumping ATPase (Ca-ATPase) and calsequestrin (CaS) were visualized by indirect immunofluorescence at the polar regions of adult rat, rabbit and cat intrafusal fibers. The immunohistochemical reaction products were regarded as histochemical markers of the SR and as valid indicators of the distribution of the two Ca2+-sequestering proteins. Static nuclear bag2 fibers displayed lower levels of both Ca-ATPase and CaS than the other two intrafusal fiber types. Nuclear chain fibers presented the highest Ca-ATPase levels and, together with dynamic nuclear bag1 fibers, they also exhibited relatively high amounts of CaS. The level of Ca-ATPase was lower in bag 1 fibers than in nuclear chain fibers, but not as low as in bag2 fibers. The comparatively high levels of Ca-ATPase and CaS seen in nuclear chain fibers coincided with their reported faster contractile speeds compared to nuclear bag fibers.
The time courses of changes in parvalbumin (PA) content, isometric twitch tension, and half-relaxation time (1/2 RT) were studied in rabbit tibialis anterior muscle following chronic 10 Hz nerve stimulation of 1-21 days. Up to 5 days stimulation had no effect on PA content, but it induced a slight (10-15%) increase in the 1/2 RT. This change occurred together with the previously observed 50% decrease in Ca2+-uptake by the SR (Leberer et al. 1987). While prolonged stimulation produced no further decrease in the Ca2+-uptake by the SR, PA content declined after 5 days of stimulation. The reduction in PA content was accompanied by a progressive lengthening of the 1/2 RT. However, the increase in 1/2 RT was particularly pronounced after PA had fallen below 50% of its normal value. A 90% reduction in PA coincided with a 60% increase in the 1/2 RT. By this time the staircase phenomenon, normally observed in fast-twitch muscle, was completely abolished. Although the changes in PA content and 1/2 RT were not linearly related, these results suggest that PA plays an important role in the relaxation process of mammalian fast-twitch muscle.
Tibialis anterior (TA) muscle of mouse, rat, guinea pig, and rabbit was indirectly stimulated for 10 h/day at 10 Hz up to 28 days. Changes in the activity levels of hexokinase (HK), phosphofructokinase (PFK), glyceraldehydephosphate dehydrogenase (GAPDH), lactate dehydrogenase (LDH), creatine kinase (CK), citrate synthase (CS), malate dehydrogenase (MDH), 3-hydroxyacyl-CoA dehydrogenase (HADH), and beta-hydroxybutyrate dehydrogenase (HBDH) were compared. Although the direction of changes in the enzyme activity pattern was in accordance with previous findings on rabbit TA, the magnitude of the responses varied markedly between the mammals under study. Mouse TA was almost unaffected. A major effect of chronic stimulation in rat, guinea pig and rabbit was an increase in enzyme activities of aerobic-oxidative metabolism. According to intrinsic differences of the muscles under study, the increases varied among the species and appeared to be inversely related to the basal levels of these enzymes in the unstimulated muscles. Conversely, glycolytic enzyme activities (PFK, GAPDH, LDH) markedly decreased in rat, guinea pig, and rabbit, and were only slightly reduced in mouse. Changes in HK and HBDH activities displayed the largest variations in the induced change between species. These results indicate species-specific patterns of metabolic adaptation to increased contractile activity.
Tibialis anterior (TA) muscles of four small mammals were subjected to chronic nerve stimulation for 28 days (10 Hz, 10 hours per day). Total cellular activities of phosphofructokinase (PFK), hexokinase (HK), citrate synthase (CS), 3-hydroxy-acyl-CoA dehydrogenase (HADH) and 3-hydroxybutyrate dehydrogenase (HBDH) were measured in the stimulated and unstimulated contralateral muscles. Normal TA muscles displayed ranges of oxidative and glycolytic capacities with rabbit TA showing the lowest and mouse TA the highest oxidative capacity. Chronic stimulation was almost without effect in mouse TA. In all other species, glycolytic capacity was decreased and reference enzymes of aerobic-oxidative pathways were increased. Rabbit TA displayed the highest increment in oxidative capacity with approximately three-fold increases in CS and HADH and eleven-fold increases in HBDH. Different responses were also observed for HK. In some cases, the extent of adaptation appeared to be independent of the initial enzyme activity levels, while in other cases it appeared to follow an order which corresponded to the size of the animals. Thus, there exist species-specific ranges of adaptation and adaptive alterations in one species may not necessarily reflect the adaptive response of another species.
1. Fibre types, Ca2+-sequestering proteins (parvalbumin, sarcoplasmic reticulum Ca2+-ATPase), enzyme activities of energy metabolism, and lactate dehydrogenase isozymes were studied in extensor digitorum longus (EDL) and soleus muscles of adult rats after denervation and direct stimulation for 45-61 days. The stimulation resembled the firing patterns of normal motor units (Hennig & Lømo, 1985) in either soleus (20 Hz, high amount) or EDL (150 Hz, low and high amount). 2. Low- and high-amount 150 Hz stimulation maintained essentially normal properties in the denervated EDL and induced many EDL-like properties in the denervated soleus, e.g. pronounced increases in Ca2+-ATPase and parvalbumin contents, a rise in the glycolytic enzyme activities, as well as a reversal of the lactate dehydrogenase H/M subunit ratio. The 150 Hz high-amount stimulation was not as efficient as the 150 Hz low amount in maintaining the fast properties of denervated EDL with the exception of the enzymes of aerobic oxidative metabolism. High-amount 20 Hz stimulation maintained essentially normal properties in the soleus but induced only moderate slow muscle characteristics in the EDL. 3. The maintenance of essentially normal properties by 'native' and the changes induced by 'foreign' stimulus patterns in the absence of the nerve indicate that evoked muscle activity plays a major role in controlling the phenotypic expression of muscle properties. However, the different responses of the denervated fast- and slow-twitch muscles to identical stimulus patterns further suggest that rat EDL and soleus muscles contain intrinsically different muscle fibres.
1. Six adult rabbit soleus muscles were analysed by isolating histochemically identified fibre pieces from freeze-dried serial cross-sections. 2. By the use of this method, four fibre types (I, IC, IIC and IIA) were identified and analysed micro-electrophoretically. 3. Type I fibres contained the slow myosin heavy chain HCI and the slow myosin light chains LC1s and LC2s. 4. Type IIA fibres contained the fast myosin HCIIa with the fast light chains and, in addition, either LC1s or both LC1s and LC2s. 5. The C fibres (IC and IIC) represented intermediate populations between types I and IIC (IC) and between IC and IIA (IIC). They contained varied ratios of HCI/HCIIa with both sets of fast and slow light chains. With regard to myosin composition and isoforms of other myofibrillar proteins (M- and C-proteins, alpha-tropomyosin, troponin I), IC fibres resembled type I and IIC fibres resembled type IIA. 6. The presence of various myosin light and heavy chains within a specific fibre suggests a multiplicity of isomyosins. Without consideration of LC1sa and LC1sb differences, at least 54 possible isomyosins can be derived: type I fibres contain one isomyosin, types IC and IIC 54 possible isomyosins, and type IIA up to 18.
1. Combined histochemical and biochemical single-fibre analyses [Staron & Pette (1987) Biochem. J. 243, 687-693], were used to investigate the rabbit tibialis-anterior fibre population. 2. This muscle is composed of four histochemically defined fibre types (I, IIC, IIA and IIB). 3. Type I fibres contain slow myosin light chains LC1s and LC2 and the slow myosin heavy chain HCI, and types IIA and IIB contain the fast myosin light chains LC1f, LC2f and LC3f and the fast heavy chains HCIIa and HCIIb respectively. 4. A small fraction of fibres (IIAB), histochemically intermediate between types IIA and IIB, contain the fast light myosin chains but display a coexistence of HCIIa and HCIIb. 5. Similarly to the soleus muscle, C fibres in the tibialis anterior muscle contain both fast and slow myosin light chains and heavy chains. The IIC fibres show a predominance of the fast forms and the IC fibres (histochemically intermediate between types I and IIC) a predominance of the slow forms. 6. A total of 60 theoretical isomyosins can be derived from these findings on the distribution of fast and slow myosin light and heavy chains in the fibres of rabbit tibialis anterior muscle.
A 50% decrease in both the initial rate and the total capacity of Ca2+ uptake by the sarcoplasmic reticulum (SR) occurred 2 days after the onset of chronic (10 Hz) nerve stimulation in rabbit fast-twitch muscle. Prolonged stimulation (up to 28 days) did not lead to further decreases. This reduction, which was detected in muscle homogenates using a Ca2+-sensitive electrode, was reversible after 6 days cessation of stimulation and was not accompanied by changes in the immunochemically (ELISA) determined tissue level or isozyme characteristics of the SR Ca2+-ATPase protein. However, as measured in isolated SR, it correlated with a reduced specific activity of the Ca2+-ATPase. Kinetic analyses demonstrated that affinities of the SR Ca2+-ATPase towards Ca2+ and ATP were unaltered. Positive cooperativity for Ca2+ binding (h = 1.5) was maintained. However, a 50% decrease in Ca2+-dependent phosphoprotein formation indicated the presence of inactive forms of Ca2+-ATPase in stimulated muscle. The reduced phosphorylation of the enzyme was accompanied by an approximately 50% lowered binding of fluorescein isothiocyanate, a competitor at the ATP-binding site. In view of the unaltered affinity for ATP, this finding suggests that active Ca2+-ATPase molecules coexist in stimulated muscle with inactive enzyme molecules, the latter displaying altered properties at the nucleotide-binding site.
A time course study was conducted to investigate the possibility of a relationship between fiber degeneration and glycogen depletion in chronically nerve-stimulated extensor digitorum longus muscle of the rabbit. Muscles were stimulated 12 h daily at 10 Hz using alternating one-hour periods of stimulation and rest. When measured for the first time after 3 h (1 h stimulation, 1 h rest, 1 h stimulation), microphotometry revealed complete glycogen depletion of all fiber types (fast glycolytic, FG; fast oxidative glycolytic, FOG; slow oxidative, SO). Different responses were noted beginning at day 4. At this time point, all FOG and SO fibers recovered their glycogen stores with some of the FOG population attaining levels higher than the FOG fibers in the unstimulated, contralateral muscle. Approximately 28% of the FG fibers recovered to normal glycogen values, whereas 58% remained depleted and 14% displayed "overshooting glycogen" levels. Fifteen percent of all fibers were glycogen-depleted after 12 days of stimulation. At this time, classic fiber types could no longer be distinguished. Fiber degeneration, which was recognized by the invasion of nonmuscle cells, began after 6 days and was restricted to the glycogen-depleted fibers. By this time, there was also a significant increase in DNA content. Exhaustion of glycogen, the main fuel of the FG fibers, is believed to cause a collapse of energy-supply and ATP-driven ionic pumps. The latter could be the initial step of fiber deterioration.
Using a combined histochemical and biochemical technique, single fiber analyses were performed on chronically stimulated and contralateral tibialis anterior (TA) muscles of the rabbit. The major fiber population (60%) in 30 days stimulated TA was transforming fibers (type IIC). Some of these fibers displayed a nonuniform distribution of histochemically assessed myofibrillar actomyosin ATPase (mATPase) activity. This heterogeneity of mATPase activity along the fibers was verified in longitudinal sections and by microphotometric evaluation of mATPase staining intensities in serial cross-sections. Biochemical analyses of single fiber segments revealed that these C fibers not only coexpressed fast- and slow-myosin subunits but did so nonuniformly along their length. The distribution of fast- and slow-myosin subunits in these fibers was not random but focal. Variations in myosin expression were also observed in some of the C fibers in the contralateral TA. As opposed to the transforming fibers in the stimulated TA, heterogeneities of mATPase activity and myosin subunits in these contralateral C fibers were less focal and more gradual. These findings suggest that muscle fibers in chronically stimulated TA and the contralateral muscle do not transform synchronously or uniformly along their length.