Reduction in hybrid single muscle fiber proportions with resistance training in humans.

The purpose of this investigation was to examine the effects of 12 wk of progressive resistance training (PRT) on single muscle fiber myosin heavy chain (MHC; I, I/IIa, I/IIa/IIx, IIa, IIa/IIx, IIx) isoform proportions in young individuals. Young, untrained men (YM; n = 6) and women (YW; n = 6) (age = 22 +/- 1 and 25 +/- 2 yr for YW and YM, respectively) received pre- and post-PRT muscle biopsies from the right vastus lateralis for single muscle fiber MHC distribution by electrophoretic analysis (192 +/- 5 pre- and 183 +/- 6 post-fibers/subject analyzed; 4,495 fibers total). Data are presented as percentages of the total fibers analyzed per subject. The PRT protocol elicited an increase in the pure MHC IIa (Delta = + 24 and + 27; YW and YM, respectively; P < 0.05) with no change in the pure MHC I distribution. The hybrid MHC distributions decreased I/IIa/IIx (Delta = -2; YM and YW; P < 0.05), IIa/IIx (Delta = -13 and -19 for YM and YW, respectively; P < 0.05), and total hybrid fiber proportion (I/IIa + I/IIa/IIx + IIa/IIx) decreased (Delta = -19 and -30 for YM and YW, respectively; P < 0.05) with the training, as did the MHC IIx distribution (Delta = -2; YW only; P < 0.05). Alterations in the predominance of MHC isoforms within hybrid fibers (decrease in MHC I-dominant I/IIa and nondominant MHC IIa/IIx, increase in MHC IIa-dominant IIa/IIx; P < 0.05) appeared to contribute to the increase in the MHC IIa proportion. Electrophoresis of muscle cross sections revealed an approximately 7% increase (P < 0.05) in MHC IIa proportion in both groups, whereas the MHC IIx decrease by 7.5 and 11.6% post-PRT in YW and YM, respectively. MHC I proportions increase in YM by 4.8% (P < 0.05) post-PRT. These findings further support previous resistance training data in young adults with respect to the increase in the MHC IIa proportions but demonstrate that a majority of the change can be attributed to the decrease in single-fiber hybrid proportions.     

Effect of long-term muscle paralysis on human single fiber mechanics.

This study compared human muscles following long-term reduced neuromuscular activity to those with normal functioning regarding single fiber properties. Biopsies were obtained from the vastus lateralis of 5 individuals with chronic (>3 yr) spinal cord injury (SCI) and 10 able-bodied controls (CTRL). Chemically skinned fibers were tested for active and passive mechanical characteristics and subsequently classified according to myosin heavy chain (MHC) content. SCI individuals had smaller proportions of type I (11 +/- 7 vs. 34 +/- 5%) and IIa fibers (11 +/- 6 vs. 31 +/- 5%), whereas type IIx fibers were more frequent (40 +/- 13 vs. 7 +/- 3%) compared with CTRL subjects (P < 0.05). Cross-sectional area and peak force were similar in both groups for all fiber types. Unloaded shortening velocity of fibers from paralyzed muscles was higher in type IIa, IIa/IIx, and IIx fibers (26, 65, and 47%, respectively; P < 0.01). Consequently, absolute peak power was greater in type IIa (46%; P < 0.05) and IIa/IIx fibers (118%; P < 0.01) of the SCI group, whereas normalized peak power was higher in type IIa/IIx fibers (71%; P < 0.001). Ca(2+) sensitivity and passive fiber characteristics were not different between the two groups in any fiber type. Composite values (average value across all fibers analyzed within each study participant) showed similar results for cross-sectional area and peak force, whereas maximal contraction velocity and fiber power were more than 100% greater in SCI individuals. These data illustrate that contractile performance is preserved or even higher in the remaining fibers of human muscles following reduced neuromuscular activity.     

Modulation of myonuclear number in functionally overloaded and exercised rat plantaris fibers.

The effects of 10 wk of functional overload (FO), with and without daily treadmill endurance training, on the cross-sectional area, myonuclear number, and myonuclear domain size of mechanically isolated single fiber segments of the adult rat plantaris were determined. The fibers were typed on the basis of high-resolution gel electrophoresis for separation of specific myosin heavy chain (MHC) isoforms and grouped as type I(+) (containing some type I MHC with or without any combination of fast MHCs), type IIa(+) (containing some type IIa with or without some type IIx and/or IIb but no type I MHC), and type IIx/b (containing only type IIx and/or IIb MHCs). Type I(+) fibers had a higher myonuclear number than did both fast types of fibers in the control and FO, but not in the FO and treadmill trained, rats. All fiber types in both FO groups had a significantly larger (36-90%) cross-sectional area and a significantly higher (61-109%) myonuclear number than did control. The average myonuclear domain size of each fiber type was similar among the three groups, except for a smaller domain size in the type IIx/b fibers of the FO compared with control. In general, these data indicate that during hypertrophy the number of myonuclei increase proportionally to the increase in fiber volume. The maintenance of myonuclear domain size near control values suggests that regulatory mechanisms exist that ensure a tight coupling between the quantity of genetic machinery and the protein requirements of a fiber.     

Fiber-type-specific alphaB-crystallin distribution and its shifts with T(3) and PTU treatments in rat hindlimb muscles.

Changes in alphaB-crystallin content in adult rat soleus and extensor digitorum longus (EDL) were examined after 8 wk of 3,5, 3'-triiodothyronine (T(3)) and propylthiouracil (PTU) treatments. Cellular distributions of alphaB-crystallin expression related to fiber type, and distribution shifts with these treatments were also examined in detail from the gray level of reactivity to specific anti-alphaB-crystallin antibody. alphaB-crystallin content in both soleus and EDL muscles was significantly decreased after T(3), and that in EDL was significantly increased over twofold after PTU treatment. In both control soleus and EDL muscles, the gray level of type I fibers was higher than that of type II fibers. alphaB-crystallin expression among type II subtypes was muscle specific; the order was type I > IIa > IIx > IIb in control EDL muscle and type IIx > or = IIa in soleus muscle. The relation was basically unchanged in both muscles after T(3) treatment and was, in particular, well maintained in EDL muscle. Under hypothyroidism conditions with PTU, the mean alphaB-crystallin levels of type IIa and IIx fibers were significantly lower than levels under control conditions. Thus the relation between fiber type and the expression manner of stress protein alphaB-crystallin is muscle specific and also is well regulated under thyroid hormone, especially in fast EDL muscle.     

Enzyme‐ and immunohistochemical aspects of skeletal muscle fibers in brown bear (Ursus arctos)

To further elucidate the pattern of MHC isoform expression in skeletal muscles of large mammals, in this study the skeletal muscles of brown bear, one of the largest mammalian predators with an extraordinary locomotor capacity, were analyzed. Fiber types in longissimus dorsi, triceps brachii caput longum, and rectus femoris muscles were determined according to the myofibrillar ATPase (mATPase) histochemistry and MHC isoform expression, revealed by a set of antibodies specific to MHC isoforms. The oxidative (SDH) and glycolytic enzyme (α‐GPDH) capacity of fibers was demonstrated as well. By mATPase histochemistry five fiber types, i.e., I, IIC, IIA, IIAX, IIX were distinguished. Analyzing the MHC isoform expression, we assume that MHC‐I, ‐IIa, and ‐IIx are expressed in the muscles of adolescent bears. MHC‐I isoform was expressed in Type‐I fibers and coexpressed with presumably ‐IIa isoform, in Type‐IIC fibers. Surprisingly, two antibodies specific to rat MHC‐IIa stained those fast fibers, that were histochemically and immunohistochemically classified as Type IIX. This assumption was additionally confirmed by complete absence of fiber staining with antibody specific to rat MHC‐IIb and all fast fiber staining with antibody that according to our experience recognizes MHC‐IIa and ‐IIx of rat. Furthermore, quite high‐oxidative capacity of all fast fiber types and their weak glycolytic capacity also imply for MHC‐IIa and ‐IIx isoform expression in fast fibers of bear. However, in adult, full‐grown animal, only MHC‐I and MHC‐IIa isoforms were expressed. The expression of only two fast isoforms in bear, like in many other large mammals (humans, cat, dog, goat, cattle, and horse) obviously meets the weight‐bearing and locomotor demands of these mammals. J. Morphol., 2009. © 2008 Wiley‐Liss, Inc.     

Stretch-shortening cycle exercises: an effective training paradigm to enhance power output of human single muscle fibers.

Functional performance of lower limb muscles and contractile properties of chemically skinned single muscle fibers were evaluated before and after 8 wk of maximal effort stretch-shortening cycle (SSC) exercise training. Muscle biopsies were obtained from the vastus lateralis of eight men before and after the training period. Fibers were evaluated regarding their mechanical properties and subsequently classified according to their myosin heavy chain content (SDS-PAGE). After training, maximal leg extensor muscle force and vertical jump performance were improved 12% (P<0.01) and 13% (P<0.001), respectively. Single-fiber cross-sectional area increased 23% in type I (P<0.01), 22% in type IIa (P<0.001), and 30% in type IIa/IIx fibers (P<0.001). Peak force increased 19% in type I (P<0.01), 15% in type IIa (P<0.001), and 16% in type IIa/IIx fibers (P<0.001). When peak force was normalized with cross-sectional area, no changes were found for any fiber type. Maximal shortening velocity was increased 18, 29, and 22% in type I, IIa, and hybrid IIa/IIx fibers, respectively (P<0.001). Peak power was enhanced in all fiber types, and normalized peak power improved 9% in type IIa fibers (P<0.05). Fiber tension on passive stretch increased in IIa/IIx fibers only (P<0.05). In conclusion, short-term SSC exercise training enhanced single-fiber contraction performance via force and contraction velocity in type I, IIa, and IIa/IIx fibers. These results suggest that SSC exercises are an effective training approach to improve fiber force, contraction velocity, and therefore power.     

Myosin heavy chains in fibers of TTX-paralyzed rat soleus and medial gastrocnemius muscles.

The expression of five myosin heavy chain (MHC) isoforms was analyzed in the rat soleus (Sol) and the deep and superficial medial gastrocnemius (dGM, sGM) muscle after 2 and 4 wk of TTX paralysis by using immunohistochemical techniques. In Sol, after 4 wk of paralysis, fibers containing type I MHC were either pure type I (14%) or also contained developmental (D; 76%), IIa (26%), or IIx (18%) MHC. Values for corresponding fibers in dGM were 8.5, 65, 38, and 22%. Also, by 4 wk an increase was seen in the proportions of fibers expressing IIa MHC in Sol (from 16 to 38%) and dGM (from 24 to 74%). In a region of sGM in control muscles containing pure IIb fibers, a major proportion (86%) remained pure after 4 wk of paralysis, with the remainder coexpressing IIb and IIx. The results indicate that TTX-induced muscle paralysis results in an increase in fibers containing multiple MHC isoforms and that the D isoform appears in a major proportion of these hybrid fibers.     

Fiber-type specific expression of α-actinin isoforms in rat skeletal muscle

α-Actinins are actin-binding proteins, and two isoforms (α-actinin-2 and -3) are major structural components of the sarcomeric Z line in mammalian skeletal muscle. Based on human and knockout mice studies, α-actinin-3 is thought to be associated with muscle force output and high contraction velocities. However, fiber-type specific expression of α-actinin isoforms is not well understood and may vary among species. In this study, we investigated the expression of α-actinin isoforms and the difference between fiber types in rat skeletal muscle and compared it with those of humans and mice from previous reports. Soleus and plantaris muscles were analyzed immunohistochemically to identify muscle fiber types and α-actinin protein expression. α-Actinin-2 was stained in all muscle fibers in both the soleus and plantaris muscles; i.e., all α-actinin-3 co-expressed with α-actinin-2 in rat skeletal muscles. The proportions of α-actinin-3 expression, regardless of fiber type, were significantly higher in the plantaris (75.8 ± 0.6%) than the soleus (8.0 ± 1.7%). No α-actinin-3 expression was observed in type I fibers, whereas all type IIx+b fibers expressed α-actinin-3. α-Actinin-3 was also expressed in type IIa fibers; however, approximately 75% of type IIa fibers were not stained by α-actinin-3, and the proportion varied between muscles. The proportion of α-actinin-3 expression in type IIa fibers was significantly higher in the soleus muscle than the plantaris muscle. Our results showed that fiber-type specific expression of α-actinin isoforms in rats is more similar to that in humans compared to that of the mouse, whereas the proportion of α-actinin-3 protein varied between muscles.     

Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers

Talmadge, Robert J., Roland R. Roy, and V. Reggie Edgerton.Distribution of myosin heavy chain isoforms in non-weight-bearing rat soleus muscle fibers. J. Appl.Physiol. 81(6): 2540-2546, 1996.The effects of14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044)on myosin heavy chain (MHC) isoform content of the rat soleus muscleand single muscle fibers were determined. On the basis ofelectrophoretic analyses, there was a de novo synthesis of type IIx MHCbut no change in either type I or IIa MHC isoform proportions aftereither SF or HS compared with controls. The percentage of fiberscontaining only type I MHC decreased by 26 and 23%, and the percentageof fibers with multiple MHCs increased from 6% in controls to 32% inHS and 34% in SF rats. Type IIx MHC was always found in combinationwith another MHC or combination of MHCs; i.e., no fibers contained typeIIx MHC exclusively. These data suggest that the expression of thenormal complement of MHC isoforms in the adult rat soleus muscle isdependent, in part, on normal weight bearing and that the absence ofweight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.

     

Phenotypic adaptations in human muscle fibers 6 and 24 wk after spinal cord injury.

The effects of spinal cord injury (SCI) on the profile of sarco(endo) plasmic reticulum calcium-ATPase (SERCA) and myosin heavy chain (MHC) isoforms in individual vastus lateralis (VL) muscle fibers were determined. Biopsies from the VL were obtained from SCI subjects 6 and 24 wk postinjury (n = 6). Biopsies from nondisabled (ND) subjects were obtained at two time points 18 wk apart (n = 4). In ND subjects, the proportions of VL fibers containing MHC I, MHC IIa, and MHC IIx were 46 +/- 3, 53 +/- 3, and 1 +/- 1%, respectively. Most MHC I fibers contained SERCA2. Most MHC IIa fibers contained SERCA1. All MHC IIx fibers contained SERCA1 exclusively. SCI resulted in significant increases in fibers with MHC IIx (14 +/- 4% at 6 wk and 16 +/- 2% at 24 wk). In addition, SCI resulted in high proportions of MHC I and MHC IIa fibers with both SERCA isoforms (29% at 6 wk and 54% at 24 wk for MHC I fibers and 16% at 6 wk and 38% at 24 wk for MHC IIa fibers). Thus high proportions of VL fibers were mismatched for SERCA and MHC isoforms after SCI (19 +/- 3% at 6 wk and 36 +/- 9% at 24 wk) compared with only ~5% in ND subjects. These data suggest that, in the early time period following SCI, fast fiber isoforms of both SERCA and MHC are elevated disproportionately, resulting in fibers that are mismatched for SERCA and MHC isoforms. Thus the adaptations in SERCA and MHC isoforms appear to occur independently.     

Myonuclear domain and myosin phenotype in human soleus after bed rest with or without loading.

After 2 or 4 mo of bed rest (6 degrees head-down tilt) and 1 mo of ambulation, there was a tendency toward a higher percentage of fibers expressing fast myosin heavy chain (MHC) isoforms and a de novo appearance of fibers coexpressing type I+IIa+IIx and IIa+IIx MHC in human soleus fibers. After 2 and 4 mo of bed rest, the mean size of type I fibers decreased by 12 (P > 0.05) and 39%, respectively. Because myonuclear number/mm of fiber length was unchanged, myonuclear domain was smaller after bed rest than before. The mean size and myonuclear domain of type I fibers were largest after 1 mo of recovery. The effects of wearing an antigravity device (Penguin suit), which had a modest but continuous resistance at the knee and ankle (Penguin-1) or knee resistance without loading on the ankle (Penguin-2), for 10 consecutive h/day were determined during 2 mo of bed rest. Mean fiber sizes in Penguin-1, but not Penguin-2, group were maintained at or above pre-bed-rest levels, whereas neither group showed phenotype changes. Myonuclear domain in type I fibers was larger in Penguin-1 and smaller in Penguin-2 group post- compared with pre-bed rest, indicating that a single daily 10-h bout of modest muscle loading can prevent bed-rest-induced soleus fiber atrophy but has minimal effect on myosin phenotype. The specific adaptive cellular strategies involved may be a function of the duration and magnitude of the adaptive stimulus as well as the immediate activity history of the fiber before the newly changed functional demands.     

Impact of hyperinsulinemia on myosin heavy chain gene regulation.

The purpose of this study was to determine whether hyperinsulinemia alters myosin heavy chain (MHC) gene expression in human skeletal muscle. A biopsy from the vastus lateralis was obtained in young, lean [age 24.6 +/- 1.0 (SE) yr, body fat 11.9 +/- 1.9%, body mass index 26.1 +/- 1.1 kg/m2; n = 10] men before and after 3 h of hyperinsulinemia (hyperinsulinemic-euglycemic clamp). Muscle was analyzed for mRNA of type I, IIa, and IIx MHC isoforms. Hyperinsulinemia (mean of 1,065.7 +/- 9.8 pmol/l during minutes 20 to 180) did not change (P > 0.05) the mRNA concentration of either the type I MHC or type IIA MHC isoforms. In contrast, type IIX MHC mRNA increased (P < 0.05) with hyperinsulinemia compared with the fasted condition. These data indicate that hyperinsulinemia rapidly increases type IIx MHC mRNA in human skeletal muscle.     

Skeletal muscle nNOS mu protein content is increased by exercise training in humans

The major isoform of nitric oxide synthase (NOS) in skeletal muscle is the splice variant of neuronal NOS, termed nNOS mu. Exercise training increases nNOS mu protein levels in rat skeletal muscle, but data in humans are conflicting. We performed two studies to determine 1) whether resting nNOS mu protein expression is greater in skeletal muscle of 10 endurance-trained athletes compared with 11 sedentary individuals (study 1) and 2) whether intense short-term (10 days) exercise training increases resting nNOS mu protein (within whole muscle and also within types I, IIa, and IIx fibers) in eight sedentary individuals (study 2). In study 1, nNOS mu protein was approximately 60% higher (P < 0.05) in endurance-trained athletes compared with the sedentary participants. In study 2, nNOS mu protein expression was similar in types I, IIa, and IIx fibers before training. Ten days of intense exercise training significantly (P < 0.05) increased nNOS mu protein levels in types I, IIa, and IIx fibers, a finding that was validated by using whole muscle samples. Endothelial NOS and inducible NOS protein were barely detectable in the skeletal muscle samples. In conclusion, nNOS mu protein expression is greater in endurance-trained individuals when compared with sedentary individuals. Ten days of intense exercise is also sufficient to increase nNOS mu expression in untrained individuals, due to uniform increases of nNOS mu within types I, IIa, and IIx fibers.     

Skeletal muscle adaptations to microgravity exposure in the mouse.

To investigate the effects of microgravity on murine skeletal muscle fiber size, muscle contractile protein, and enzymatic activity, female C57BL/6J mice, aged 64 days, were divided into animal enclosure module (AEM) ground control and spaceflight (SF) treatment groups. SF animals were flown on the space shuttle Endeavour (STS-108/UF-1) and subjected to approximately 11 days and 19 h of microgravity. Immunohistochemical analysis of muscle fiber cross-sectional area revealed that, in each of the muscles analyzed, mean muscle fiber cross-sectional area was significantly reduced (P < 0.0001) for all fiber types for SF vs. AEM control. In the soleus, immunohistochemical analysis of myosin heavy chain (MHC) isoform expression revealed a significant increase in the percentage of muscle fibers expressing MHC IIx and MHC IIb (P < 0.05). For the gastrocnemius and plantaris, no significant changes in MHC isoform expression were observed. For the muscles analyzed, no alterations in MHC I or MHC IIa protein expression were observed. Enzymatic analysis of the gastrocnemius revealed a significant decrease in citrate synthase activity in SF vs. AEM control.     

The Relationship between Muscle Fiber Type-Specific PGC-1α Content and Mitochondrial Content Varies between Rodent Models and Humans

PGC-1α regulates critical processes in muscle physiology, including mitochondrial biogenesis, lipid metabolism and angiogenesis. Furthermore, PGC-1α was suggested as an important regulator of fiber type determination. However, whether a muscle fiber type-specific PGC-1α content exists, whether PGC-1α content relates to basal levels of mitochondrial content, and whether such relationships are preserved between humans and classically used rodent models are all questions that have been either poorly addressed or never investigated. To address these issues, we investigated the fiber type-specific content of PGC-1α and its relationship to basal mitochondrial content in mouse, rat and human muscles using in situ immunolabeling and histochemical methods on muscle serial cross-sections. Whereas type IIa fibers exhibited the highest PGC-1α in all three species, other fiber types displayed a hierarchy of type IIx>I>IIb in mouse, type I = IIx> IIb in rat, and type IIx>I in human. In terms of mitochondrial content, we observed a hierarchy of IIa>IIx>I>IIb in mouse, IIa >I>IIx> IIb in rat, and I>IIa> IIx in human skeletal muscle. We also found in rat skeletal muscle that type I fibers displayed the highest capillarization followed by type IIa >IIx>IIb. Finally, we found in human skeletal muscle that type I fibers display the highest lipid content, followed by type IIa>IIx. Altogether, our results reveal that (i) the fiber type-specific PGC-1α and mitochondrial contents were only matched in mouse, (ii) the patterns of PGC-1α and mitochondrial contents observed in mice and rats do not correspond to that seen in humans in several respects, and (iii) the classical phenotypes thought to be regulated by PGC-1α do not vary exclusively as a function of PGC-1α content in rat and human muscles.