Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice.

Myotonic dystrophy (DM) is commonly associated with CTG repeat expansions within the gene for DM-protein kinase (DMPK). The effect of altered expression levels of DMPK, which is ubiquitously expressed in all muscle cell lineages during development, was examined by disrupting the endogenous Dmpk gene and overexpressing a normal human DMPK transgene in mice. Nullizygous (-/-) mice showed only inconsistent and minor size changes in head and neck muscle fibres at older age, animals with the highest DMPK transgene expression showed hypertrophic cardiomyopathy and enhanced neonatal mortality. However, both models lack other frequent DM symptoms including the fibre-type dependent atrophy, myotonia, cataract and male-infertility. These results strengthen the contention that simple loss- or gain-of-expression of DMPK is not the only crucial requirement for development of the disease.

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).

Two functionally distinct myosin heavy chain isoforms in slow skeletal muscle fibres.

The head part of the myosin heavy chain (MHC) represents the essential component of the molecular force-generating system of muscle [1-3]. To date, three fast but only one slow MHC isoforms have been identified in adult mammalian limb muscles [4,5]. We show here two functionally different slow MHC isoforms, MHCIbeta and MHCIa, coexisting in a considerable fraction of slow fibres of rabbit plantaris muscle. The two isoforms exhibit distinct electrophoretic mobilities and different kinetic properties. Thus, as it is known for the fast muscle, also the slow muscle seems to use different MHC isoforms in order to fulfil different functional demands.

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.

Electrophoretically defined myosin heavy chain patterns of single human muscle spindles.

At least four myosin heavy chain (MHC) isoforms were separated by SDS-PAGE in extracts of intrafusal fibers isolated by microdissection from human lumbrical muscles. The fastest migrating MHC represents a slow isoform. The slowest migrating MHC was identified as the embryonic MHCemb. A faint band, moving slightly faster than MHCemb, most likely represents a neonatal/fetal MHC isoform. A prominent band, moving between the latter and the slow isoform is suggested to represent a hitherto unidentified, spindle-specific MHC isoform, MHCif.

Myosin polymorphism in single fibers of chronically stimulated rabbit fast-twitch muscle.

Rabbit tibialis anterior (TA) muscles were indirectly stimulated (10 Hz, 24 h/d) for 30 d and 60 d and single fibers were analysed using a combined histochemical and biochemical technique (Staron and Pette 1986, 1987a, b). After 30 d of chronic stimulation there was a pronounced increase in the normally rare (0.5%) C fiber population (i.e., fibers containing slow- and fast-myosins in varying ratios). At this time, C fibers amounted to almost 60% of the total population. In the 60 d stimulated muscles, the major population (98%) consisted of an atypical type It fiber. This fiber type which was not detectable in normal TA muscle, differed histochemically and biochemically from type I fibers. It contained the slow-myosin light chains LC1s and LC2s, the heavy chain HCI, and, in addition, high concentrations of the fast-myosin alkali light chain LC1f and possibly traces of a heavy chain with an electrophoretic mobility comparable with that of the fast-myosin heavy chain HCIIa. These It fibers were occasionally observed in the unstimulated, contralateral TA muscles which also contained an increased population of C fibers (1.3-6.3%). Although the transformation even after 60 d of chronic stimulation was incomplete, these changes demonstrate the ability of muscle fibers to adapt in a specific manner to altered functional demands brought about by an altered stimulus pattern. In addition, the pronounced heterogeneity of the fiber population undergoing transformation indicates a nonuniform response to a uniform stimulus pattern.

Upregulation of myosin heavy chain MHClalpha in rat muscles after unweighting and clenbuterol treatment.

Myosin heavy chain (MHC) mRNA isoforms were quantified in soleus (SOL) and gastrocnemius (GAS) muscles from rats exposed to 14 days of either hindlimb unweighting (HU), clenbuterol treatment (CB), or HU combined with CB treatment (HU-CB). All conditions induced in SOL a shift from slow to faster MHC mRNA isoforms and an upregulation of MHClalpha. Increases were highest with CB, lowest with HU-CB, and coincided mainly with elevations in MHClla mRNA isoforms. The changes in MHC mRNA levels in GAS muscle corresponded to fast-to-faster transitions. Elevations in MHClalpha mRNA were smaller than in SOL and seemed to occur in parallel with decreases in MHClbeta. Taken together, our results suggested that MHClalpha is expressed in transforming rat slow and fast muscles, most likely as an intermediate step between MHClbeta and MHClla.

Specific impulse patterns regulate acetylcholinesterase activity in skeletal muscles of rats and rabbits.

In rats, acetylcholinesterase (AChE) activity in the fast muscles is several times higher than in the slow soleus muscle. The hypothesis that specific neural impulse patterns in fast or slow muscles are responsible for different AChE activities was tested by altering the neural activation pattern in the fast extensor digitorum longus (EDL) muscle by chronic low-frequency stimulation of its nerve. In addition, the soleus muscle was examined after hind limb immobilization, which changed its neural activation pattern from tonic to phasic. Myosin heavy-chain (MHC) isoforms were analyzed by gel electrophoresis. Activity of the molecular forms of AChE was determined by velocity sedimentation. Low-frequency stimulation of the rat EDL for 35 days shifted the profile of MHC II isoforms toward a slower MHCIIa isoform. Activity of the globular G1 and G4 molecular forms of AChE decreased by a factor of 4 and 10, respectively, and became comparable with those in the soleus muscle. After hind limb immobilization, the fast MHCIId isoform, which is not normally present, appeared in the soleus muscle. Activity of the globular G1 form of AChE increased approximately three times and approached the levels in the fast EDL muscle. In the rabbit, on the contrary to the rat, activity of the globular forms of AChE in a fast muscle increased after low-frequency stimulation. The results demonstrate that specific neural activation patterns regulate AChE activity in muscles. Great differences, however, exist among different mammalian species in regard to muscle AChE regulation.

Inactivation of sarcoplasmic reticulum Ca(2+)-atpase in low-frequency stimulated rat muscle.

Continuous low-frequency stimulation (CLFS) by implanted electrodes for 12-24 h led to a significant (approximately 30%) decrease in the activity of sarcoplasmic reticulum Ca(2+)-ATPase in fast-twitch extensor digitorum longus (EDL) and tibialis anterior (TA) muscles of intact rats. The decline in catalytic activity after 24 h of CLFS was accompanied by an approximately twofold increase in dinitrophenylhydrazine-reactive carbonyl groups of the enzyme. It also correlated with an immunochemically determined 30% decrease in Ca2(+)-ATPase protein. Recovery studies after 12 h of CLFS revealed a relatively slow (48-72 h) re-establishment of normal catalytic activity. These findings suggest that the 30% decline of Ca(2+)-ATPase activity in low-frequency stimulated rat muscle led to an irreversible modification by protein oxidation. The decrease in Ca(2+)-ATPase protein most likely resulted from the degradation of inactive Ca(2+)-ATPase molecules. The relatively slow recovery of Ca(2+)-ATPase activity suggests that de novo synthesis of the enzyme may be necessary to re-attain normal activity.

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.

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.

Fast myosin heavy chain diversity in skeletal muscles of the rabbit: heavy chain IId, not IIb predominates.

The myosin heavy chain (HC) composition of various rabbit muscles was analysed at both the mRNA and the protein level. S1-nuclease mapping was performed with a cDNA probe specific for myosin HCIIa, yielding a fully protected sequence for HCIIa, a partially protected sequence for HCIIb, and an additional signal putatively assigned to HCIId. At the protein level, three fast myosin HC isoforms, HCIIa, HCIIb and HCIId, were separated by gradient PAGE. The results obtained at the protein level were in agreement with the findings at the mRNA level. The expression of appreciable amounts of myosin HCIIb, the predominating isoform of fast-twitch muscles in rat and mouse, was restricted in the rabbit to only a few muscles, i.e. adductor magnus, gastrocnemius, latissimus dorsi and vastus lateralis. Typical fast-twitch muscles such as extensor digitorum longus, tibialis anterior and psoas contained only minute amounts of HCIIb. The HCIId isoform, demonstrated in the present study for the first time in rabbit, is the predominating fast myosin HC isoform in this species. Electrophoretic analyses of myosin HC in histochemically defined single fibers confirmed the lack of fibers expressing only HCIIb in tibialis anterior, whereas such fibers were found in the adductor magnus. In addition to fiber types IIB, IID, and IIA expressing HCIIb, HCIId, and HCIIa, respectively, an appreciable amount of hybrid fibers coexpressing two HC isoforms at various ratios were found: HCIIb > HCIId; HCIId > HCIIb; HCIId > HCIIa; HCIIa > HCIId; HCIIa > HCI; HCI > HCIIa. This fiber-type spectrum indicates possible fiber-type transitions in the order IIB<==> IIB<==>IIDB<==>IID<==>IIDA<==>IIAD<==>IIA<==>IIC<==>IC <==>I.