Kristian Gundersen

Three myosin heavy chain isoforms in type 2 skeletal muscle fibres

SummaryMammalian skeletal muscles consist of three main fibre types, type 1, 2A and 2B fibres, with different myosin heavy chain (MHC) composition. We have now identified another fibre type, called type 2X fibre, characterized by a specific MHC isoform. Type 2X fibres, which are widely distributed in rat skeletal muscles, can be distinguished from 2A and 2B fibres by histochemical ATPase activity and by their unique staining pattern with seven anti-MHC monoclonal antibodies. The existence of the 2X-MHC isoform was confirmed by immunoblotting analysis using muscles containing 2X fibres as a major component, such as the normal and hyperthyroid diaphragm, and the soleus muscle after high frequency chronic stimulation. 2X-MHC contains one determinant common to 2B-MHC and another common to all type 2-MHCs, but lacks epitopes specific for 2A- and 2B-MHCs, as well as an epitope present on all other MHCs. By SDS-polyacrylamide gel electrophoresis 2X-MHC shows a lower mobility compared to 2B-MHC and appears to comigrate with 2A-MHC. Muscles containing predominantly 2X-MHC display a velocity of shortening intermediate between that of slow muscles and that of fast muscles composed predominantly of 2B fibres.

Hypoxia induces adaptive and reversible gross morphological changes in crucian carp gills

SUMMARY We show that crucian carp (Carassius carassius) living in normoxic (aerated) water have gills that lack protruding lamellae, the primary site of O2 uptake in fish. Such an unusual trait leads to a very small respiratory surface area. Histological examination showed that the lamellae (secondary lamellae) of these fish were embedded in a cell mass (denoted embedded lamellae). When the fish were kept in hypoxic water, a large reduction in this cell mass occurred, making the lamellae protrude and increasing the respiratory surface area by ∼7.5-fold. This morphological change was found to be reversible and was caused by increased apoptosis combined with reduced cell proliferation. Carp with protruding lamellae had a higher capacity for oxygen uptake at low oxygen levels than fish with embedded lamellae, but water and ion fluxes appeared to be increased, which indicates increased osmoregulatory costs. This is, to our knowledge, the first demonstration of an adaptive and reversible gross morphological change in the respiratory organ of an adult vertebrate in response to changes in the availability of oxygen.

Myonuclei acquired by overload exercise precede hypertrophy and are not lost on detraining

Effects of previous strength training can be long-lived, even after prolonged subsequent inactivity, and retraining is facilitated by a previous training episode. Traditionally, such “muscle memory” has been attributed to neural factors in the absence of any identified local memory mechanism in the muscle tissue. We have used in vivo imaging techniques to study live myonuclei belonging to distinct muscle fibers and observe that new myonuclei are added before any major increase in size during overload. The old and newly acquired nuclei are retained during severe atrophy caused by subsequent denervation lasting for a considerable period of the animal’s lifespan. The myonuclei seem to be protected from the high apoptotic activity found in inactive muscle tissue. A hypertrophy episode leading to a lasting elevated number of myonuclei retarded disuse atrophy, and the nuclei could serve as a cell biological substrate for such memory. Because the ability to create myonuclei is impaired in the elderly, individuals may benefit from strength training at an early age, and because anabolic steroids facilitate more myonuclei, nuclear permanency may also have implications for exclusion periods after a doping offense.

Excitation-transcription coupling in skeletal muscle: the molecular pathways of exercise

Muscle fibres have different properties with respect to force, contraction speed, endurance, oxidative/glycolytic capacity etc. Although adult muscle fibres are normally post‐mitotic with little turnover of cells, the physiological properties of the pre‐existing fibres can be changed in the adult animal upon changes in usage such as after exercise. The signal to change is mainly conveyed by alterations in the patterns of nerve‐evoked electrical activity, and is to a large extent due to switches in the expression of genes. Thus, an excitation‐transcription coupling must exist. It is suggested that changes in nerve‐evoked muscle activity lead to a variety of activity correlates such as increases in free intracellular Ca2+ levels caused by influx across the cell membrane and/or release from the sarcoplasmatic reticulum, concentrations of metabolites such as lipids and ADP, hypoxia and mechanical stress. Such correlates are detected by sensors such as protein kinase C (PKC), calmodulin, AMP‐activated kinase (AMPK), peroxisome proliferator‐activated receptor δ (PPARδ), and oxygen dependent prolyl hydroxylases that trigger intracellular signaling cascades. These complex cascades involve several transcription factors such as nuclear factor of activated T‐cells (NFAT), myocyte enhancer factor 2 (MEF2), myogenic differentiation factor (myoD), myogenin, PPARδ, and sine oculis homeobox 1/eyes absent 1 (Six1/Eya1). These factors might act indirectly by inducing gene products that act back on the cascade, or as ultimate transcription factors binding to and transactivating/repressing genes for the fast and slow isoforms of various contractile proteins and of metabolic enzymes. The determination of size and force is even more complex as this involves not only intracellular signaling within the muscle fibres, but also muscle stem cells called satellite cells. Intercellular signaling substances such as myostatin and insulin‐like growth factor 1 (IGF‐1) seem to act in a paracrine fashion. Induction of hypertrophy is accompanied by the satellite cells fusing to myofibres and thereby increasing the capacity for protein synthesis. These extra nuclei seem to remain part of the fibre even during subsequent atrophy as a form of muscle memory facilitating retraining. In addition to changes in myonuclear number during hypertrophy, changes in muscle fibre size seem to be caused by alterations in transcription, translation (per nucleus) and protein degradation.

Number and spatial distribution of nuclei in the muscle fibres of normal mice studied in vivo

We present here a new technique with which to visualize nuclei in living muscle fibres in the intact animal, involving injection of labelled DNA into single cells. This approach allowed us to determine the position of all of nuclei within a sarcolemma without labelling satellite cells. In contrast to what has been reported in tissue culture, we found that the nuclei were immobile, even when observed over several days. Nucleic density was uniform along the fibre except for the endplate and some myotendinous junctions, where the density was higher. The perijunctional region had the same number of nuclei as the rest of the fibre. In the extensor digitorum longus (EDL) muscle, the extrajunctional nuclei were elongated and precisely aligned to the long axis of the fibre. In the soleus, the nuclei were rounder and not well aligned. When comparing small and large fibres in the soleus, the number of nuclei varied approximately in proportion to cytoplasmic volume, while in the EDL the number was proportional to surface area. Statistical analysis revealed that the nuclei were not randomly distributed in either the EDL or the soleus. For each fibre, actual distributions were compared with computer simulations in which nuclei were assumed to repel each other, which optimizes the distribution of nuclei with respect to minimizing transport distances. The simulated patterns were regular, with clear row‐like structures when the density of nuclei was low. The non‐random and often row‐like distribution of nuclei observed in muscle fibres may thus reflect regulatory mechanisms whereby nuclei repel each other in order to minimize transport distances.

Fast to slow transformation of denervated and electrically stimulated rat muscle

1 Denervated fast extensor digitorum longus (EDL) muscles of adult rats were stimulated electrically for up to 4 months with a ‘slow’ pattern resembling the activity in soleus (Sol) motor units and examined with antibodies against myosin heavy chains (MHCs). 2 The normal EDL contained, on average, 45 % type IIB, 29 % type IIX, 23 % type IIA and 3 % type I fibres. All type IIB and almost all type IIX fibres disappeared during the first 3 weeks of stimulation. They were replaced by type IIA and type I fibres, whose percentages increased to about 75 and 15, respectively. Type IIA fibres remained at 75 % for nearly 2 months and were then gradually replaced by type I fibres during the next 2 months. The transformation occurred sequentially in the order IIB/IIX → IIA → I, the first step (IIB/IIX → IIA) occurring after a short delay (2 weeks) and the last step (IIA → I in originally IIB or IIX fibres) after a long delay (> 2 months). During the transformation coexpression of MHCs occurred. 3 It appears that the transformation to type I fibres occurred in pre‐existing type II fibres since no signs of fibre damage or regeneration were observed. 4 Normal EDL was also stimulated through an intact nerve with the same pattern for up to 37 days. The effects on fibre type distributions were identical to those observed in the denervated EDL. The result indicated that the Sol‐like pattern of evoked muscle activity, rather than nerve‐derived trophic influences or denervation per se, was primarily responsible for the fast to slow transformation.

Slow-to-fast transformation of denervated soleus muscles by chronic high-frequency stimulation in the rat.

1. Adult soleus muscles were denervated and stimulated directly for 2‐130 days with ‘fast’ (short pulse trains at 100 Hz) or slow’ (continuously at 10 Hz, or long pulse trains at 15 Hz) stimulus patterns. 2. At the end of the period of stimulation isometric twitches and tetani and isotonic shortening velocities were measured. Frozen cross‐sections were later examined with antibodies against myosin heavy chains specific for adult fast, adult slow and fetal myosin. 3. Isometric twitch duration (twitch time‐to‐peak and half‐relaxation time) decreased during intermittent 100 Hz stimulation to values that were almost as fast as in the normal extensor digitorum longus (EDL) (95 and 94% transformation). The major part of the decrease occurred between 2 and 21 days after the onset of stimulation, and was accompanied by post‐tetanic potentiation of the twitch, sag’ in tension during an unfused tetanus, lower twitch/tetanus ratio and marked shifts to the right (higher frequencies) of the tension‐frequency curve of the muscle. In contrast, during 10 or 15 Hz stimulation the isometric twitch duration remained slow, the twitch continued to show post‐tetanic depression and absence of ‘sag’, while the twitch/tetanus ratio increased. 4. Denervation per se led to a slight increase and, then, after about a month, to a moderate and gradual decrease in twitch duration. The twitch/tetanus ratio increased markedly and post‐tetanic depression became less pronounced or disappeared. Muscle weight and particularly tetanic tension were markedly reduced and these reductions were to a large extent counteracted by electrical stimulation. 5. Implantation of sham electrodes had no effect on twitch duration of denervated or innervated control muscles, but reduced tetanic tension in the innervated control muscles. 6. Maximum isotonic shortening velocity of the whole muscle (mm/s) increased during intermittent 100 Hz stimulation to a value as fast as in the normal EDL (110% transformation). Since the muscle fibres also increased in length (35%) maximum intrinsic shortening velocity (fibre lengths/s) was only incompletely transformed (55%). The increase in Vmax occurred between 7 and 14 days after the onset of stimulation. 7. All the fibres stimulated intermittently at 100 Hz were strongly labelled with anti‐fast myosin and more than 90% were in addition weakly labelled by anti‐slow myosin. Weak and variable labelling with anti‐fast myosin was first detected 7 days after the onset of stimulation. In contrast, essentially all the fibres stimulated at 10 or 15 Hz showed no binding of anti‐fast but strong binding of anti‐slow myosin.(ABSTRACT TRUNCATED AT 400 WORDS)

Electrical stimulation resembling normal motor-unit activity: effects on denervated fast and slow rat muscles.

1. The slow‐twitch soleus muscle and the fast‐twitch extensor digitorum longus muscle (EDL) were denervated and stimulated directly with implanted electrodes for 33‐82 days. Four different stimulation patterns were used in order to mimic important characteristics of the natural motor‐unit activity in these muscles. In addition, to compare the effects of direct stimulation to other experimental models, some EDLs were stimulated through the nerve or cross‐innervated by soleus axons. 2. After 33‐82 days of stimulation the contractile properties were measured under isometric and isotonic conditions. 3. ‘Native’ stimulation patterns could maintain normal contractile speed in both EDL and soleus. In the EDL, normal isotonic shortening velocity was maintained only by a stimulation pattern consisting of very brief trains with an initial short interspike interval (doublet), and not by the other ‘native’ high‐frequency patterns. 4. The contractile properties of both EDL and soleus muscles receiving a ‘foreign’ stimulation pattern were transformed in the direction of the muscle normally receiving that type of activity. The transformations were not complete, and soleus and EDL muscles stimulated with the same stimulation pattern remained different. This suggests that adult muscle fibres in rat EDL and soleus are irreversibly differentiated into different fibre types earlier in development. 5. The three high‐frequency stimulation patterns used differed in their ability to change or maintain various contractile properties in the soleus and the EDL. The results indicate that the following qualities of a stimulation pattern might be of importance for the control of contractile properties: instantaneous frequency, total amount of stimulation, train length, interval between trains and presence of an initial doublet. 6. With the exception of the EDL shortening velocity, changes in contractile speed induced by a ‘foreign’ stimulation pattern were quantitatively similar to the effects of cross‐innervation both in the EDL and the soleus. We thus suggest that the change in activity pattern is the mechanism behind most of the changes induced by cross‐innervation.

In vivo time-lapse microscopy reveals no loss of murine myonuclei during weeks of muscle atrophy.

Numerous studies have suggested that muscle atrophy is accompanied by apoptotic loss of myonuclei and therefore recovery would require replenishment by muscle stem cells. We used in vivo time-lapse microscopy to observe the loss and replenishment of myonuclei in murine muscle fibers following induced muscle atrophy. To our surprise, imaging of single fibers for up to 28 days did not support the concept of nuclear loss during atrophy. Muscles were inactivated by denervation, nerve impulse block, or mechanical unloading. Nuclei were stained in vivo either acutely by intracellular injection of fluorescent oligonucleotides or in time-lapse studies after transfection with a plasmid encoding GFP with a nuclear localization signal. We observed no loss of myonuclei in fast- or slow-twitch muscle fibers despite a greater than 50% reduction in fiber cross-sectional area. TUNEL labeling of fragmented DNA on histological sections revealed high levels of apoptotic nuclei in inactive muscles. However, when costained for laminin and dystrophin, virtually none of the TUNEL-positive nuclei could be classified as myonuclei; apoptosis was confined to stromal and satellite cells. We conclude that disuse atrophy is not a degenerative process, but is rather a change in the balance between protein synthesis and proteolysis in a permanent cell syncytium.

Nuclear domains during muscle atrophy: nuclei lost or paradigm lost?

According to the current paradigm, muscle nuclei serve a certain cytoplasmic domain. To preserve the domain size, it is believed that nuclei are injected from satellite cells fusing to fibres undergoing hypertrophy, and lost by apoptosis during atrophy. Based on single fibre observations in and ex vivo we suggest that nuclear domains are not as constant as is often indicated. Moreover, recent time lapse in vivo imaging of single fibres suggests that at least for the first few weeks, atrophy is not accompanied by any loss of nuclei. Apoptosis is abundant in muscle tissue during atrophy conditions, but in our opinion it has not been unequivocally demonstrated that such nuclei are myonuclei. As we see it, the preponderance of current evidence suggests that disuse atrophy is not accompanied by loss of nuclei, at least not for the first 2 months. Moreover, it has not been proven that myonuclear apoptosis does occur in permanent fibres undergoing atrophy; it seems more likely that it is confined to stromal cells and satellite cells. If muscle atrophy is not related to loss of nuclei, design of intervention therapies should focus on protein metabolism rather than regeneration from stem cells.