Responses of motor and sensory neurons of rodents to spaceflight

Spinal motoneurons innervating skeletal muscles comprised predominantly of high oxidative fibers, i.e. slow oxidative and fast oxidative glycolytic, have higher oxidative enzyme activities than motoneurons innervating skeletal muscles comprised primarily of low oxidative fibers, i.e. fast glycolytic. These findings suggest that there is a close relationship between the oxidative phosphorylation capacity of a motoneuron and of the muscle fibers that it innervates. Since some skeletal muscles become faster and less oxidative after 4-14 days of spaceflight, it might be expected that oxidative enzyme activities in some motoneurons also may decrease after spaceflight. In addition, there is significant muscular atrophy after even short spaceflights and, therefore, it may be expected that some motoneurons associated with these muscles also would atrophy. In the present paper, we examine the issue of whether spaceflight induces changes in the oxidative enzyme activity and/or size of spinal motoneurons.     

Morphology and histochemistry of the ambiens muscle of the red-eared turtle (Pseudemys scripta)

Six fiber types have been described in the ambiens muscle of red-eared turtles. These include one slow oxidative type, two fast oxidative types, two fast oxidative and glycolytic types, and one fast glycolytic type. Fiber types are non-randomly distributed throughout cross sections of the muscle. There is a decreasing gradient of oxidative staining and an increasing gradient of glycolytic staining along an axis from the superficial to deep regions of the muscle. The slow oxidative fibers are predominantly located within one or two fascicles of the superficial surface of the muscle. The fast glycolytic fibers are predominant in deep fascicles. In contrast to previous reports of histochemically monotypic intrafusal fibers in turtle muscle, ambiens muscle spindles have been observed containing one to eleven intrafusal fibers, including two fiber types. Fiber diameter and area are consistently smaller than observed in most extrafusal fibers. Spindles are predominantly located in superficial and cranial fascicles of the ambiens muscle and are located in regions characterized by extrafusal fibers with high oxidative activity.     

Effect of breed of horse on muscle carnosine concentration

1. Muscle samples from the M. gluteus medius were obtained from six Quarter Horses (QH), six Thoroughbreds (TB), and five Standardbreds (SB) to determine carnosine values and fiber type percentages. 2. Muscle biopsies were for fiber type percentages and carnosine concentration. 3. QH had a lower percentage of slow twitch oxidative fibers and a higher percentage of past twitch glycolytic fibers than SB or TB. 4. Fast twitch oxidative-glycolytic fibers were lowest in the QH. 5. The QH had mean carnosine values significantly greater (P less than 0.01) than the mean values for SB and TB. 6. Across breeds muscle carnosine concentration was positively correlated (P less than 0.05; r = 0.53) with fast twitch glycolytic fiber percentage and negatively correlated (P less than 0.05, r = -0.51) with fast twitch oxidative fiber percentage. 7. Free intramuscular carnosine is believed to function as an intracellular buffer. Since carnosine was highest in the muscle of horses with the greatest percentage of fast twitch glycolytic fibers, these data are consistent with the proposed function of this dipeptide.     

Histone deacetylase 4 possesses intrinsic nuclear import and export signals

Nucleocytoplasmic trafficking of histone deacetylase 4 (HDAC4) plays an important role in regulating its function, and binding of 14-3-3 proteins is necessary for its cytoplasmic retention. Here, we report the identification of nuclear import and export sequences of HDAC4. While its N-terminal 118 residues modulate the nuclear localization, residues 244 to 279 constitute an authentic, strong nuclear localization signal. Mutational analysis of this signal revealed that three arginine-lysine clusters are necessary for its nuclear import activity. As for nuclear export, leucine-rich sequences located in the middle part of HDAC4 do not function as nuclear export signals. By contrast, a hydrophobic motif (MXXLXVXV) located at the C-terminal end serves as a nuclear export signal that is necessary for cytoplasmic retention of HDAC4. This motif is required for CRM1-mediated nuclear export of HDAC4. Furthermore, binding of 14-3-3 proteins promotes cytoplasmic localization of HDAC4 by both inhibiting its nuclear import and stimulating its nuclear export. Unlike wild-type HDAC4, a point mutant with abrogated MEF2-binding ability remains cytoplasmic upon exogenous expression of MEF2C, supporting the notion that direct MEF2 binding targets HDAC4 to the nucleus. Therefore, HDAC4 possesses intrinsic nuclear import and export signals for its dynamic nucleocytoplasmic shuttling, and association with 14-3-3 and MEF2 proteins affects such shuttling and thus directs HDAC4 to the cytoplasm and the nucleus, respectively.     

Regulatory elements governing transcription in specialized myofiber subtypes

Skeletal myofibers of vertebrates acquire specialized metabolic and physiological properties as a consequence of developmental cues in the embryo and different patterns of contractile activity in the adult. The myoglobin gene is regulated stringently in muscle fibers, such that high myoglobin expression is observed in mitochondria-rich, oxidative myofibers (Types I and IIa) compared with glycolytic fibers (Type IIb). Using germ-line transgenesis and somatic cell gene transfer methods, we defined discrete regions of the murine and human genes encoding myoglobin that are sufficient to confer muscle- and fiber type-specific expression to reporter genes. Mutational analysis confirms the importance of A/T-rich, MEF2-binding motifs in myoglobin gene regulation, as suggested by previous studies using different experimental approaches. In addition, we demonstrated a previously unsuspected role for an intragenic E-box motif as a negative regulatory element contributing to the tightly regulated variation in myoglobin gene expression among particular myofiber subtypes.     

Histochemical classification of neck and limb muscle fibers in a turtle, Pseudemys scripta: a study using microphotometry and cluster analysis techniques

We have attempted to develop an objective, semiquantitative classification of fiber types in turtle neck and limb muscle using microphotometry and multivariate statistical techniques. We first stained serial sections for myosin adenosine triphosphatase (ATPase) (with acid and alkaline preincubation and without preincubation), NADH-diaphorase, and two glycolysis-associated markers, alpha-glycerophosphate dehydrogenase (alpha-GPDH) and glycogen phosphorylase A (GPA). This allowed us to characterize individual muscle fibers in terms of their contraction speed and metabolic properties. Next we used microphotometry to measure the optical density of the reaction product in each fiber, and we subjected the resulting optical density matrix to cluster and discriminant function analyses in order to assign fibers to groups (fiber types) and to determine which stains contribute most to the distinction between groups. As a control, we processed a well characterized mammalian muscle (rat sternomastoid) simultaneously. Our results suggest that both neck and limb muscle in Pseudemys can best be described as falling into three groups: 1) slow oxidative (SO) fibers; 2) fast oxidative glycolytic (FOG) fibers, with relatively high oxidative and glycolytic capacities; and 3) fast glycolytic (Fg) fibers, with low oxidative, low/intermediate alpha-GPDH, and high GPA activities. These three fiber types differ from like-named types in rat muscle both in the pH lability of their myosins and in their metabolic profiles.     

Overexpression of CUG triplet repeat-binding protein, CUGBP1, in mice inhibits myogenesis

Accumulation of RNA CUG repeats in myotonic dystrophy type 1 (DM1) patients leads to the induction of a CUG-binding protein, CUGBP1, which increases translation of several proteins that are required for myogenesis. In this paper, we examine the role of overexpression of CUGBP1 in DM1 muscle pathology using transgenic mice that overexpress CUGBP1 in skeletal muscle. Our data demonstrate that the elevation of CUGBP1 in skeletal muscle causes overexpression of MEF2A and p21 to levels that are significantly higher than those in skeletal muscle of wild type animals. A similar induction of these proteins is observed in skeletal muscle of DM1 patients with increased levels of CUGBP1. Immunohistological analysis showed that the skeletal muscle from mice overexpressing CUGBP1 is characterized by a developmental delay, muscular dystrophy, and myofiber-type switch: increase of slow/oxidative fibers and the reduction of fast fibers. Examination of molecular mechanisms by which CUGBP1 up-regulates MEF2A shows that CUGBP1 increases translation of MEF2A via direct interaction with GCN repeats located within MEF2A mRNA. Our data suggest that CUGBP1-mediated overexpression of MEF2A and p21 inhibits myogenesis and contributes to the development of muscle deficiency in DM1 patients.     

Fast myosin light chain expression in chicken muscles studied by in situ hybridization.

We have studied the fiber type-specific expression of the fast myosin light chain isoforms LC 1f, LC 2f, and LC 3f in adult chicken muscles using in situ hybridization and two-dimensional gel electrophoresis. Type II (fast) fibers contain all three fast myosin light chain mRNAs; Types I and III (slow) fibers lack them. The myosin light chain patterns of two-dimensional gels from microdissected single fibers match their mRNA signals in the in situ hybridizations. The results confirm and extend previous studies on the fiber type-specific distribution of myosin light chains in chicken muscles which used specific antibodies. The quantitative ratios between protein and mRNA content were not the same for all three fast myosin light chains, however. In bulk muscle samples, as well as in single fibers, there was proportionally less LC 3f accumulated for a given mRNA concentration than LC 1f or LC 2f. Moreover, the ratio between LC 3f mRNA and protein was different in samples from muscles, indicating that LC 3f is regulated somewhat differently than LC 1f and LC 2f. In contrast to other in situ hybridization studies on the fiber type-specific localization of muscle protein mRNAs, which reported the RNAs to be located preferentially at the periphery of the fibers, we found all three fast myosin light chain mRNAs quite evenly distributed within the fiber's cross-sections, and also in the few rare fibers which showed hybridization signals several-fold higher than their surrounding counterparts. This could indicate principal differences in the intracellular localization among the mRNAs coding for various myofibrillar protein families.