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

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.

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Transcription of the embryonic myosin light chain gene is restricted to type II muscle fibers in human adult masseter

Developmental Biology ◽

10.1016/0012-1606(91)90295-e ◽

1991 ◽

Vol 147 (2) ◽

pp. 374-380 ◽

Cited By ~ 3

Author(s):

Nadia Soussi-Yanicostas ◽

Gillian S. Butler-Browne

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Muscle Fibers ◽

Chain Gene ◽

Type Ii ◽

Light Chain Gene ◽

Human Adult

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

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.1997.272.5.r1552 ◽

1997 ◽

Vol 272 (5) ◽

pp. R1552-R1561 ◽

Cited By ~ 22

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.

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Isolation of two myosin light-chain kinases from bovine carotid artery and their regulation by phosphorylation mediated by cyclic AMP-dependent protein kinase

Biochemical Journal ◽

10.1042/bj2030583 ◽

1982 ◽

Vol 203 (3) ◽

pp. 583-592 ◽

Cited By ~ 17

Author(s):

Ramesh C. Bhalla ◽

Ram V. Sharma ◽

Ramesh C. Gupta

Keyword(s):

Protein Kinase ◽

Carotid Artery ◽

Cyclic Amp ◽

Light Chain ◽

Myosin Light Chain ◽

Kinase Activity ◽

Type I ◽

Type Ii ◽

Dependent Protein Kinase ◽

Dependent Protein

Myosin light-chain kinase was purifed from bovine carotid artery. Approx. 90% of myosin kinase was extracted in the supernatant fraction with buffer containing EDTA during myofibril preparation. The soluble fraction yielded two distinct peaks on DEAE-Sephacel chromatography. Peak I was eluted at a conductance of 11–12mmho and was completely dependent on Ca2+–calmodulin for its activity. Peak II was eluted at a conductance of 13–14mmho and showed approx. 15% Ca2+-independent activity. The myosin kinases I and II were further purified by affinity chromatography by using calmodulin coupled to Sepharose 4B, which resulted in 960-and 650-fold purification of type I and type II kinases respectively. Myosin kinase II activity was completely Ca2+-dependent after affinity chromatography on the calmodulin–Sepharose column. Myosin kinases I and II were phosphorylated by cyclic AMP-dependent protein kinase. In the presence of bound calmodulin 0.5–0.7mol of phosphate was incorporated/mol of myosin kinases I and II. On the other hand, in the absence of bound calmodulin 1–1.4mol of phosphate was incorporated/mol of kinases I and II. Phosphorylation in the absence of calmodulin significantly decreased the myosin kinase activity of both enzymes, and the decrease in myosin kinase activity was due to a 3–5-fold increase in the amount of calmodulin required for half-maximal stimulation of both type I and type II kinases. The regulation of myosin kinase activity by cyclic AMP-dependent phosphorylation would suggest that β-adrenergic-mediated relaxation of vascular smooth muscle may be partly due to the direct interaction of cyclic AMP at the site of contractile proteins.

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Prolonged exercise training increases intramuscular lipid content and perilipin 2 expression in type I muscle fibers of patients with type 2 diabetes

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00272.2012 ◽

2012 ◽

Vol 303 (9) ◽

pp. E1158-E1165 ◽

Cited By ~ 44

Author(s):

C. S. Shaw ◽

S. O. Shepherd ◽

A. J. M. Wagenmakers ◽

D. Hansen ◽

P. Dendale ◽

...

Keyword(s):

Type 2 Diabetes ◽

Exercise Training ◽

Fiber Type ◽

Muscle Fibers ◽

Whole Body ◽

Type I ◽

Type Ii ◽

Perilipin 2 ◽

Diabetes Patients

The aim of the present study was to investigate changes in intramuscular triglyceride (IMTG) content and perilipin 2 expression in skeletal muscle tissue following 6 mo of endurance-type exercise training in type 2 diabetes patients. Ten obese male type 2 diabetes patients (age 62 ± 1 yr, body mass index BMI 31 ± 1 kg/m2) completed three exercise sessions/week consisting of 40 min of continuous endurance-type exercise at 75% V̇o2 peak for a period of 6 mo. Muscle biopsies collected at baseline and after 2 and 6 mo of intervention were analyzed for IMTG content and perilipin 2 expression using fiber type-specific immunofluorescence microscopy. Endurance-type exercise training reduced trunk body fat by 6 ± 2% and increased whole body oxygen uptake capacity by 13 ± 7% ( P < 0.05). IMTG content increased twofold in response to the 6 mo of exercise training in both type I and type II muscle fibers ( P < 0.05). A threefold increase in perilipin 2 expression was observed from baseline to 2 and 6 mo of intervention in the type I muscle fibers only (1.1 ± 0.3, 3.4 ± 0.6, and 3.6 ± 0.6% of fibers stained, respectively, P < 0.05). Exercise training induced a 1.6-fold increase in mitochondrial content after 6 mo of training in both type I and type II muscle fibers ( P < 0.05). In conclusion, this is the first study to report that prolonged endurance-type exercise training increases the expression of perilipin 2 alongside increases in IMTG content in a type I muscle fiber-type specific manner in type 2 diabetes patients.

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Increase in S6K1 phosphorylation in human skeletal muscle following resistance exercise occurs mainly in type II muscle fibers

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00530.2005 ◽

2006 ◽

Vol 290 (6) ◽

pp. E1245-E1252 ◽

Cited By ~ 115

Author(s):

René Koopman ◽

Antoine H. G. Zorenc ◽

Rudy J. J. Gransier ◽

David Cameron-Smith ◽

Luc J. C. van Loon

Keyword(s):

Skeletal Muscle ◽

Resistance Exercise ◽

Human Skeletal Muscle ◽

Fiber Type ◽

Muscle Fibers ◽

Initiation Factor ◽

Exercise Session ◽

Type I ◽

Type Ii ◽

Postexercise Recovery

To investigate the in vivo effects of resistance exercise on translational control in human skeletal muscle, we determined the phosphorylation of AMP-activated kinase (AMPK), eukaryotic initiation factor 4E-binding protein (4E-BP1), p70/p85-S6 protein kinase (S6K1), and ribosomal S6 protein (S6). Furthermore, we investigated whether changes in the phosphorylation of S6K1 are muscle fiber type specific. Eight male subjects performed a single high-intensity resistance exercise session. Muscle biopsies were collected before and immediately after exercise and after 30 and 120 min of postexercise recovery. The phosphorylation statuses of AMPK, 4E-BP1, S6K1, and S6 were determined by Western blotting with phospho-specific and pan antibodies. To determine fiber type-specific changes in the phosphorylation status of S6K1, immunofluorescence microscopy was applied. AMPK phosphorylation was increased approximately threefold immediately after resistance exercise, whereas 4E-BP1 phosphorylation was reduced to 27 ± 6% of preexercise values. Phosphorylation of S6K1 at Thr421/Ser424 was increased 2- to 2.5-fold during recovery but did not induce a significant change in S6 phosphorylation. Phosphorylation of S6K1 was more pronounced in the type II vs. type I muscle fibers. Before exercise, phosphorylated S6K1 was predominantly located in the nuclei. After 2 h of postexercise recovery, phospho-S6K1 was primarily located in the cytosol of type II muscle fibers. We conclude that resistance exercise effectively increases the phosphorylation of S6K1 on Thr421/Ser424, which is not associated with a substantial increase in S6 phosphorylation in a fasted state.

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Myosin light chain 3f attenuates age-induced decline in contractile velocity in MHC type II single muscle fibers

Aging Cell ◽

10.1111/j.1474-9726.2011.00774.x ◽

2011 ◽

Vol 11 (2) ◽

pp. 203-212 ◽

Cited By ~ 14

Author(s):

Jong-Hee Kim ◽

Windy S. Torgerud ◽

Kelsey H. H. Mosser ◽

Hiroyuki Hirai ◽

Shuichi Watanabe ◽

...

Keyword(s):

Light Chain ◽

Myosin Light Chain ◽

Muscle Fibers ◽

Single Muscle ◽

Type Ii

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Distinct regulatory elements control muscle-specific, fiber-type-selective, and axially graded expression of a myosin light-chain gene in transgenic mice.

Molecular and Cellular Biology ◽

10.1128/mcb.16.7.3909 ◽

1996 ◽

Vol 16 (7) ◽

pp. 3909-3922 ◽

Cited By ~ 51

Author(s):

M V Rao ◽

M J Donoghue ◽

J P Merlie ◽

J R Sanes

Keyword(s):

Transgenic Mice ◽

Light Chain ◽

Myosin Light Chain ◽

Fiber Type ◽

Regulatory Elements ◽

Whole Body ◽

Light Chain Gene ◽

The Core ◽

Myogenic Factor ◽

Fast Twitch

The fast alkali myosin light chain 1f/3f (MLC1f/3f) gene is developmentally regulated, muscle specific, and preferentially expressed in fast-twitch fibers. A transgene containing an MLC1f promoter plus a downstream enhancer replicates this pattern of expression in transgenic mice. Unexpectedly, this transgene is also expressed in a striking (approximately 100-fold) rostrocaudal gradient in axial muscles (reviewed by J. R. Sanes, M. J. Donoghue, M. C. Wallace, and J. P. Merlie, Cold Spring Harbor Symp. Quant. Biol. 57:451-460, 1992). Here, we analyzed the expression of mutated transgenes to map sites necessary for muscle-specific, fiber-type-selective, and axially graded expression. We show that two E boxes (myogenic factor binding sites), a homeodomain (hox) protein binding site, and an MEF2 site, which are clustered in an approximately 170-bp core enhancer, are all necessary for maximal transgene activity in muscle but not for fiber-type- or position-dependent expression. A distinct region within the core enhancer promotes selective expression of the transgene in fast-twitch muscles. Sequences that flank the core enhancer are also necessary for high-level activity in transgenic mice but have little influence on activity in transfected cells, suggesting the presence of regions resembling matrix attachment sites. Truncations of the MLC1f promoter affected position-dependent expression of the transgene, revealing distinct regions that repress transgene activity in neck muscles and promote differential expression among intercostal muscles. Thus, the whole-body gradient of expression displayed by the complete transgene may reflect the integrated activities of discrete elements that regulate expression in subsets of muscles. Finally, we show that transgene activity is not significantly affected by deletion or overexpression of the myoD gene, suggesting that intermuscular differences in myogenic factor levels do not affect patterns of transgene expression. Together, our results provide evidence for at least nine distinct sites that exert major effects on the levels and patterns of MLC1f expression in adult muscles.

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Acute effects of taurine on sarcoplasmic reticulum Ca2+ accumulation and contractility in human type I and type II skeletal muscle fibers

Journal of Applied Physiology ◽

10.1152/japplphysiol.00494.2014 ◽

2014 ◽

Vol 117 (7) ◽

pp. 797-805 ◽

Cited By ~ 22

Author(s):

T. L. Dutka ◽

C. R. Lamboley ◽

R. M. Murphy ◽

G. D. Lamb

Keyword(s):

Sarcoplasmic Reticulum ◽

Fiber Type ◽

Vastus Lateralis ◽

Muscle Fibers ◽

Vastus Lateralis Muscle ◽

Type I ◽

Fiber Types ◽

Type Ii ◽

Contractile Apparatus ◽

Type Ii Fibers

Taurine occurs in high concentrations in muscle and is implicated in numerous physiological processes, yet its effects on many aspects of contractility remain unclear. Using mechanically skinned segments of human vastus lateralis muscle fibers, we characterized the effects of taurine on sarcoplasmic reticulum (SR) Ca2+ accumulation and contractile apparatus properties in type I and type II fibers. Prolonged myoplasmic exposure (>10 min) to taurine substantially increased the rate of accumulation of Ca2+ by the SR in both fiber types, with no change in the maximum amount accumulated; no such effect was found with carnosine. SR Ca2+ accumulation was similar with 10 or 20 mM taurine, but was significantly slower at 5 mM taurine. Cytoplasmic taurine (20 mM) had no detectable effects on the responsiveness of the Ca2+ release channels in either fiber type. Taurine caused a small increase in Ca2+ sensitivity of the contractile apparatus in type I fibers, but type II fibers were unaffected; maximum Ca2+-activated force was unchanged in both cases. The effects of taurine on SR Ca2+ accumulation 1) only became apparent after prolonged cytoplasmic exposure, and 2) persisted for some minutes after complete removal of taurine from the cytoplasm, consistent with the hypothesis that the effects were due to an action of taurine from inside the SR. In summary, taurine potentiates the rate of SR Ca2+ uptake in both type I and type II human fibers, possibly via an action from within the SR lumen, with the degree of potentiation being significantly reduced at low physiological taurine levels.

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Fiber-type-specific expression of essential (alkali) myosin light chains in human skeletal muscles.

Journal of Histochemistry & Cytochemistry ◽

10.1177/44.10.8813079 ◽

1996 ◽

Vol 44 (10) ◽

pp. 1141-1152 ◽

Cited By ~ 11

Author(s):

K Jostarndt ◽

A Puntschart ◽

H Hoppeler ◽

R Billeter

Keyword(s):

Light Chain ◽

Skeletal Muscles ◽

Myosin Light Chain ◽

Fiber Type ◽

Expression Patterns ◽

Light Chains ◽

Myosin Light Chains ◽

Type I ◽

Specific Expression ◽

Human Skeletal Muscles

We studied the expression patterns of the essential (alkali) myosin light-chain isoforms in adult human skeletal muscles, using in situ hybridization and single-fiber protein analysis. In analogy to other species, we found that the fiber type-specific expression of essential myosin light chains is regulated via the availability of the respective mRNAs in a given fiber. In contrast to other species, the slow isoform 1sa was only expressed in the most oxidative Type I fibers (Subtype IA) in addition to 1sb. These fibers also contained high levels of carbonic anhydrase III. Within the fibers, the essential myosin light-chain mRNAs were located preferentially in the perinuclear regions and to a lesser extent in the intermyofibrillar spaces, a distribution that excludes cotranslational assembly of these light chains into the myofibrils as the main mechanism. In comparing leg and shoulder muscles, we found less distinct fiber typing in the expression patterns of the essential myosin light chains in the leg muscles than in muscles from the shoulder region.

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