beta-MHC transgene expression in suspended and mechanically overloaded/suspended soleus muscle of transgenic mice

1997 ◽  
Vol 272 (5) ◽  
pp. R1552-R1561 ◽  
Author(s):  
J. J. McCarthy ◽  
A. M. Fox ◽  
G. L. Tsika ◽  
L. Gao ◽  
R. W. Tsika
Keyword(s):  
Transgenic Mice ◽  
Soleus Muscle ◽  
Transgene Expression ◽  
Specific Activity ◽  
Fiber Type ◽  
Weight Bearing ◽  
Troponin C ◽  
Hindlimb Suspension ◽  
Regulatory Sequences ◽  
Type I

Non-weight-bearing (NWB) activity [space flight and hindlimb suspension (HS)] results in the loss of soleus muscle mass, a slow-to-fast fiber-type conversion, and decreased beta-myosin heavy chain (beta-MHC) protein and mRNA expression. To identify beta-MHC promoter sequences required for decreased beta-MHC expression in response to HS, we have modified an existing noninvasive hindlimb unweighting model to accommodate the use of (transgenic) mice. After 2 wk of HS, body and muscle (soleus > gastrocnemius > plantaris) weights were decreased as was the proportion of histochemically classified type I fibers in HS soleus muscle. Northern blot analysis revealed decreases in endogenous mRNA representing beta-MHC, slow myosin light chain 1 and 2, and cardiac/slow troponin C, whereas those representing skeletal troponin C, muscle creatine kinase, and glyceraldehyde-3-phosphate dehydrogenase increased. Protein extracts prepared from HS soleus (SS) muscle of mice harboring transgenes comprised of 5.6 or 0.6 kilobase of wild type (wt) mouse beta-MHC promoter (beta 5.6 wt, beta 0.6wt) and those carrying the simultaneous mutation (mut) of the MCAT, C-rich, and beta e3 subregions (beta 5.6mut3, beta 0.6mut3) revealed decreases in chloramphenicol acetyltransferase (CAT) specific activity relative to respective controls. Decreased CAT mRNA was observed for transgene beta 5.6mut3, line 85. Two weeks of the simultaneous imposition of mechanical overload (synergist ablation) and HS (MOV/HS) countermanded the loss in absolute and normalized SS weight but did not decrease beta 0.6wt transgene expression. These transgenic results demonstrate that regulatory sequences within a 600-base pair beta-MHC promoter are sufficient to direct decreased transcription of beta-MHC transgenes after 2 wk of HS.

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

1996 ◽  
Vol 81 (6) ◽  
pp. 2540-2546 ◽  
Author(s):  
Robert J. Talmadge ◽  
Roland R. Roy ◽  
V. Reggie Edgerton
Keyword(s):  
Myosin Heavy Chain ◽  
Soleus Muscle ◽  
Heavy Chain ◽  
De Novo ◽  
Weight Bearing ◽  
Muscle Fibers ◽  
Myosin Heavy Chain Isoforms ◽  
Hindlimb Suspension ◽  
Type I ◽  
Rat Soleus Muscle

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 of 14 days of spaceflight (SF) or hindlimb suspension (HS) (Cosmos 2044) on myosin heavy chain (MHC) isoform content of the rat soleus muscle and single muscle fibers were determined. On the basis of electrophoretic analyses, there was a de novo synthesis of type IIx MHC but no change in either type I or IIa MHC isoform proportions after either SF or HS compared with controls. The percentage of fibers containing only type I MHC decreased by 26 and 23%, and the percentage of fibers with multiple MHCs increased from 6% in controls to 32% in HS and 34% in SF rats. Type IIx MHC was always found in combination with another MHC or combination of MHCs; i.e., no fibers contained type IIx MHC exclusively. These data suggest that the expression of the normal complement of MHC isoforms in the adult rat soleus muscle is dependent, in part, on normal weight bearing and that the absence of weight bearing induces a shift toward type IIx MHC protein expression in the preexisting type I and IIa fibers of the soleus.

Muscle-specific and inducible expression of 293-base pair beta-myosin heavy chain promoter in transgenic mice

1996 ◽  
Vol 271 (3) ◽  
pp. R688-R695 ◽  
Author(s):  
J. L. Wiedenman ◽  
G. L. Tsika ◽  
L. Gao ◽  
J. J. McCarthy ◽  
I. D. Rivera-Rivera ◽  
...  
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Specific Activity ◽  
Fiber Type ◽  
Regulatory Element ◽  
Histochemical Staining ◽  
Regulatory Sequences ◽  
Type I ◽  
Adult Mice ◽  
Mechanical Overload

The DNA regulatory element(s) involved in beta-myosin heavy chain (beta-MHC) induction by the physiological stimulus of mechanical overload have not been identified as yet. To delineate regulatory sequences that are required for mechanical overload induction of the beta-MHC gene, transgenic mouse lines were generated that harbor transgenes containing serial deletions of the human beta-MHC promoter to nucleotides -293 (beta 293), -201 (beta 201), and -141 (beta 141) from the transcription start site (+1). Mechanically overloaded adult plantaris and soleus muscles contained 11- and 1.9-fold increases, respectively, in endogenous beta-MHC-specific mRNA transcripts (Northern blot) compared with sham-operated controls. Expression assays (chloramphenicol acetyltransferase specific activity) revealed that only transgene beta 293 expression was muscle specific in both fetal and adult mice and was induced in the plantaris (10- to 27-fold) and soleus (2- to 2.5-fold) muscles by mechanical overload. Histochemical staining for myosin adenosinetriphosphatase activity revealed a fiber-type transition of type II to type I in the overloaded plantaris and soleus muscles. These transgenic data suggest that sequences located between nucleotides -293 and +120 may be sufficient to regulate the endogenous beta-MHC gene in response to developmental signals and to the physiological signals generated by mechanical overload in fast- and slow-twitch muscles.

Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading

1999 ◽  
Vol 277 (2) ◽  
pp. R601-R606 ◽  
Author(s):  
Christian J. Carlson ◽  
Frank W. Booth ◽  
Scott E. Gordon
Keyword(s):  
Skeletal Muscle ◽  
Muscle Mass ◽  
Soleus Muscle ◽  
Fiber Type ◽  
Weight Bearing ◽  
Mrna Abundance ◽  
Hindlimb Unloading ◽  
Negative Regulator ◽  
Type I ◽  
Mrna Levels

Transgenic mice lacking a functional myostatin (MSTN) gene demonstrate greater skeletal muscle mass resulting from muscle fiber hypertrophy and hyperplasia (McPherron, A. C., A. M. Lawler, and S.-J. Lee. Nature 387: 83–90, 1997). Therefore, we hypothesized that, in normal mice, MSTN may act as a negative regulator of muscle mass. Specifically, we hypothesized that the predominately slow (type I) soleus muscle, which demonstrates greater atrophy than the fast (type II) gastrocnemius-plantaris complex (Gast/PLT), would show more elevation in MSTN mRNA abundance during hindlimb unloading (HU). Surprisingly, MSTN mRNA was not detectable in weight-bearing or HU soleus muscle, which atrophied 42% by the 7th day of HU in female ICR mice. In contrast, MSTN mRNA was present in weight-bearing Gast/PLT muscle and was significantly elevated (67%) at 1 day but not at 3 or 7 days of HU. However, the Gast/PLT muscle had only atrophied 17% by the 7th day of HU. Because the soleus is composed only of type I and IIa fibers, whereas the Gast/PLT expresses type IId/x and IIb in addition to type I and IIa, it was necessary to perform a more careful analysis of the relationship between MSTN mRNA levels and myosin heavy-chain (MHC) isoform expression (as a marker of fiber type). A significant correlation ( r = 0.725, P < 0.0005) was noted between the percentage of MHC isoform IIb expression and MSTN mRNA abundance in several muscles of the mouse hindlimb. These results indicate that MSTN expression is not strongly associated with muscle atrophy induced by HU; however, it is strongly associated with MHC isoform IIb expression in normal muscle.

Role of protein intake on protein synthesis and fiber distribution in the unweighted soleus muscle

1993 ◽  
Vol 75 (3) ◽  
pp. 1226-1232 ◽  
Author(s):  
D. Taillandier ◽  
X. Bigard ◽  
D. Desplanches ◽  
D. Attaix ◽  
C. Y. Guezennec ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Soleus Muscle ◽  
Protein Intake ◽  
Fiber Type ◽  
Weight Bearing ◽  
Type I ◽  
Fiber Distribution ◽  
Cross Sectional ◽  
Type Distribution ◽  
Fiber Type Distribution

Protein turnover in skeletal muscle is very sensitive to protein intake. To examine whether protein intake is able to affect protein synthesis in the atrophied soleus muscle, the effects of a high-protein (30%, HP) and a medium-protein (15%, MP) diet were studied in rats after 21 days of hindlimb unweighting. Three weeks of unweighting induced a sharp decrease in food intake (30%). The fractional rate of protein synthesis (ks) was determined in vivo in the slow-twitch soleus muscle by use of a flooding-dose method. With respect to pair-fed animals, a significant reduction in ks occurred (33%) in MP non-weight-bearing rats, whereas it was of lesser magnitude and not significant in HP rats. In the atrophied soleus muscle of non-weight-bearing MP rats, a large decrease (42%) in type I fiber distribution was accompanied by an increase in intermediate and type IIa fibers. By contrast, a higher percentage of type I fiber was maintained with the HP diet. However, the HP diet had no beneficial effect in preventing the decrease in either type I fiber cross-sectional area (65%) or the average decrease in absolute myofibrillar and mitochondrial volumes (69 and 52%, respectively). These results demonstrate that an HP intake did not prevent soleus muscle atrophy but may sustain protein synthesis and partly preserve fiber type distribution without affecting the ultrastructural composition of fibers. Because the circulating level of free 3,5,3'-triiodothyronine was reduced by 14% with the HP diet, this effect on fiber type distribution, and possibly protein synthesis, may involve thyroid hormones.

scholarly journals Fiber type- and position-dependent expression of a myosin light chain-CAT transgene detected with a novel histochemical stain for CAT.

1991 ◽  
Vol 115 (2) ◽  
pp. 423-434 ◽  
Author(s):  
M J Donoghue ◽  
J D Alvarez ◽  
J P Merlie ◽  
J R Sanes
Keyword(s):  
Light Chain ◽  
Transgene Expression ◽  
Myosin Light Chain ◽  
Fiber Type ◽  
Muscle Fibers ◽  
Single Type ◽  
Regulatory Sequences ◽  
Type I ◽  
Type Ii ◽  
Light Chain Gene

We recently generated and characterized transgenic mice in which regulatory sequences from a myosin light chain gene (MLC1f/3f) are linked to the chloramphenicol acetyltransferase (CAT) gene. Transgene expression in these mice is specific to skeletal muscle and graded along the rostrocaudal axis: adult muscles derived from successively more caudal somites express successively higher levels of CAT. To investigate the cellular basis of these patterns of expression, we developed and used a histochemical stain that allows detection of CAT in individual cells. Our main results are as follows: (a) Within muscles, CAT is detected only in muscle fibers and not in associated connective tissue, blood vessels, or nerves. Thus, the tissue specificity of transgene expression observed by biochemical assay reflects a cell-type specificity demonstrable histochemically. (b) Within individual muscles, CAT levels vary with fiber type. Like the endogenous MLC1f/3f gene, the transgene is expressed at higher levels in fast-twitch (type II) than in slow-twitch (type I) muscle fibers. In addition, CAT levels vary among type II fiber subtypes, in the order IIB greater than IIX greater than IIA. (c) Among muscles that are similar in fiber type composition, the average level of CAT per fiber varies with rostrocaudal position. This position-dependent variation in CAT level is apparent even when fibers of a single type are compared. From these results, we conclude that fiber type and position affect CAT expression independently. We therefore infer the existence of separate fiber type-specific and positionally graded transcriptional regulators that act together to determine levels of transgene expression.

Intermittent cold exposure causes a muscle-specific shift in the fiber type composition in rats

1993 ◽  
Vol 75 (1) ◽  
pp. 264-267 ◽  
Author(s):  
T. J. Walters ◽  
S. H. Constable
Keyword(s):  
Soleus Muscle ◽  
Cold Exposure ◽  
Citrate Synthase ◽  
Fiber Type ◽  
Type I ◽  
Fiber Type Composition ◽  
Type Composition ◽  
Edl Muscle ◽  
Type I Fibers

We examined the effect of long-term intermittent cold exposure on the fiber type composition of the predominantly type I soleus and the predominantly type IIb extensor digitorum longus (EDL) muscles of rats. Cold exposure was accomplished by submerging the rats in shoulder-deep water, maintained at 20 +/- 0.5 degrees C, for 1 h/day, 5 days/wk, for < or = 19 wk. The efficacy of the treatment was tested by subjecting both groups to 20 degrees C water for 45 min while rectal temperature (Tre) and O2 consumption (VO2) were measured. The cold-exposed group displayed a 22% smaller reduction in Tre (P < 0.05) at the end of the exposure and 23% greater VO2 (P < 0.05) during the same period. Fiber type composition was determined using routine histochemical methods for myosin-adenosinetriphosphatase. In the soleus muscle of the cold-exposed rats, the number of type IIa fibers increased 156% (P < 0.05) and the number of type I fibers decreased 24% (P < 0.05). Cold exposure had no significant influence on the fiber type composition of the EDL muscle. Cold exposure resulted in an increase in citrate synthase activity of 20 and 22% in the soleus and EDL muscles, respectively (P < 0.05). The present study demonstrates that intermittent cold exposure induces a type I-to-type IIa transformation in the soleus muscle while having no influence on the EDL muscle.

Beta-MHC and SMLC1 transgene induction in overloaded skeletal muscle of transgenic mice

1996 ◽  
Vol 270 (4) ◽  
pp. C1111-C1121 ◽  
Author(s):  
J. L. Wiedenman ◽  
I. Rivera-Rivera ◽  
D. Vyas ◽  
G. Tsika ◽  
L. Gao ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Troponin C ◽  
Type I ◽  
Initial Attempt ◽  
Specific Expression ◽  
Slow Type ◽  
Transgenic Lines ◽  
Fast Twitch Muscle ◽  
Mechanical Overload

The hypertrophic responses of white fast-twitch muscle to mechanical overload has been investigated using transgenic mice. After 7 wk of overload, endogenous beta-myosin heavy chain (MHC) and slow myosin light chain 1 and 2 (SMLC1, SMLC2) protein were increased in the overloaded plantaris (OP) muscle compared with sham-operated control plantaris (CP)muscle. Concurrently, the levels of endogenous beta-MHC, SMLC1, SMLC2, and cardiac/slow troponin C (CTnC) mRNA transcripts were significantly increased in OP muscles, whereas skeletal troponin C (sTnC) mRNA transcript levels decreased. As an initial attempt to locate DNA sequence(s) that governs beta-MHC induction in response to mechanical overload, multiple independent transgenic lines harboring four different human beta-MHC transgenes (beta 1286, beta 988, beta 450, beta 141) were generated. Except for transgene beta 141, muscle-specific expression and induction (3- to 22-fold) in OP muscles were observed by measuring chloramphenicol acetyltransferase activity (CAT assay). Induction of a SMLC1 transgene (3920SMLC1) in OP muscles was also observed. Collectively, these in vivo data provide evidence that 1) a mechanical overload inducible element(s) is located between nucleotides -450 and +120 of the human beta-MHC transgene, 2) 3,900 bp of 5' sequence is sufficient to confer mechanical overload induction of a SMLC1 transgene, and 3) the increased expression of slow/type I isomyosin (beta-MHC, SMLC1, SMLC2) in response to mechanical overload is regulated, in part, transcriptionally.

Role of glucose transport in glycogen supercompensation in reweighted rat skeletal muscle

1996 ◽  
Vol 80 (5) ◽  
pp. 1540-1546 ◽  
Author(s):  
E. J. Henriksen ◽  
C. S. Stump ◽  
T. H. Trinh ◽  
S. D. Beaty
Keyword(s):  
Glucose Transport ◽  
Citrate Synthase ◽  
Specific Activity ◽  
Glycogen Synthase ◽  
Average Rate ◽  
Weight Bearing ◽  
Hindlimb Suspension ◽  
Transport Activity ◽  
Glut 4 ◽  
Insulin Dependent

Hindlimb weight bearing after a 3-day period of hindlimb suspension (reweighting) of juvenile rats results in a marked transient elevation in soleus glycogen concentration that cannot be explained on the basis of the activities of glycogen synthase and phosphorylase. We have hypothesized that enhanced glucose transport activity could underlie this response. We directly tested this hypothesis by assessing the response of insulin-dependent and insulin-independent glucose transport activity (in vitro 2-[1,2-3H]deoxy-D-glucose uptake) as well as glucose transporter (GLUT-4) protein levels during a 48-h reweighting period. After a net glycogen loss (from 29 +/- 2 to 16 +/- 1 nmol/mg muscle; P < 0.05) during the first 2 h of reweighting, glycogen accumulated at an average rate of 1.4 nmol.mg-1.h-1 up to 18 h, reaching an apex of 38 +/- 1 nmol/mg. During this same reweighting period, insulin-independent, but not insulin-dependent, glucose transport activity was significantly enhanced (P < 0.05 vs. weight-bearing control values) and was associated with an elevated level of GLUT-4 protein and the specific activity of total hexokinase. The specific activity of citrate synthase was also increased. By 24 h of reweighting, although insulin-independent glucose transport activity and GLUT-4 protein remained elevated, glycogen accumulation had ceased, likely due to enhanced phosphorylase activity at this time point. These results are consistent with the interpretation that the glycogen supercompensation seen during reweighting of the rat soleus may be regulated in part by an enhanced glucose flux arising from an increase in insulin-independent glucose transport activity and hexokinase activity.

Myosin and troponin changes in rat soleus muscle after hindlimb suspension

1993 ◽  
Vol 74 (3) ◽  
pp. 1156-1160 ◽  
Author(s):  
M. Campione ◽  
S. Ausoni ◽  
C. Y. Guezennec ◽  
S. Schiaffino
Keyword(s):  
Soleus Muscle ◽  
Troponin I ◽  
Troponin T ◽  
High Mobility ◽  
Calcium Sensitivity ◽  
Hindlimb Suspension ◽  
Type I ◽  
Maximal Velocity ◽  
Rat Soleus Muscle ◽  
Fast Twitch

We examined the myosin heavy-chain (MHC), troponin T (TnT), and troponin I (TnI) isoform composition in the rat soleus muscle after 21 days of hindlimb suspension using electrophoretic and immunoblotting analysis with specific monoclonal antibodies. The suspended soleus showed a shift in the MHC isoform distribution with a marked increase (from 1.0 to 33%) in the relative amount of type IIa and IIx MHC and a corresponding decrease in type I MHC. However, type IIb MHC, which represents a major component in fast-twitch muscles, was not detected in suspended soleus muscles. TnT and TnI isoform composition was also changed with the appearance of fast-type TnI and TnT bands. However, a high-mobility TnT band, which represents a major component in fast-twitch muscles, was not expressed in suspended soleus. These isoform transitions may be related to the increased maximal velocity of shortening and higher calcium sensitivity previously reported in the rat soleus after hindlimb suspension.

scholarly journals Force-velocity-power and force-pCa relationships of human soleus fibers after 17 days of bed rest

1998 ◽  
Vol 85 (5) ◽  
pp. 1949-1956 ◽  
Author(s):  
J. J. Widrick ◽  
K. M. Norenberg ◽  
J. G. Romatowski ◽  
C. A. Blaser ◽  
M. Karhanek ◽  
...  
Keyword(s):  
Peak Power ◽  
Weight Bearing ◽  
Bed Rest ◽  
Force Transducer ◽  
Hindlimb Suspension ◽  
Peak Power Output ◽  
Type I ◽  
Shortening Velocity ◽  
Adult Men ◽  
Force Velocity

Soleus muscle fibers from the rat display a reduction in peak power and Ca2+ sensitivity after hindlimb suspension. To examine human responses to non-weight bearing, we obtained soleus biopsies from eight adult men before and immediately after 17 days of bed rest (BR). Single chemically skinned fibers were mounted between a force transducer and a servo-controlled position motor and activated with maximal (isotonic properties) and/or submaximal (Ca2+ sensitivity) levels of free Ca2+. Gel electrophoresis indicated that all pre- and post-BR fibers expressed type I myosin heavy chain. Post-BR fibers obtained from one subject displayed increases in peak power and Ca2+ sensitivity. In contrast, post-BR fibers obtained from the seven remaining subjects showed an average 11% reduction in peak power ( P < 0.05), with each individual displaying a 7–27% reduction in this variable. Post-BR fibers from these subjects were smaller in diameter and produced 21% less force at the shortening velocity associated with peak power. However, the shortening velocity at peak power output was elevated 13% in the post-BR fibers, which partially compensated for their lower force. Post-BR fibers from these same seven subjects also displayed a reduced sensitivity to free Ca2+( P < 0.05). These results indicate that the reduced functional capacity of human lower limb extensor muscles after BR may be in part caused by alterations in the cross-bridge mechanisms of contraction.

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