Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quantitative PCR study

Jänkälä, Heidi, Veli-Pekka Harjola, Niels Erik Petersen, and Matti Härkönen. Myosin heavy chain mRNA transform to faster isoforms in immobilized skeletal muscle: a quantitative PCR study. J. Appl. Physiol. 82(3): 977–982, 1997.—A quantitative polymerase chain reaction (PCR) method was used to measure the quantities of type I, IIa, IIx, and IIb myosin heavy chain (MHC) mRNA in total RNA preparations of the soleus, gastrocnemius, and plantaris muscles of normal and hindlimb-immobilized rats. Type IIx and even type IIb MHC mRNA were demonstrated at extremely low levels in normal soleus, 2.1 ± 0.4 × 105and 5.0 ± 0.2 × 105molecules of mRNA per microgram total RNA, respectively. Immobilization for 1 wk significantly altered the gene expression of MHC isoforms. In soleus, both type IIx and IIb MHC genes became significantly upregulated, 24-fold ( P < 0.005) and 2.6-fold ( P < 0.05), respectively. In gastrocnemius, the level of type IIa MHC mRNA decreased by 51% ( P < 0.01) and the level of type IIx MHC mRNA increased by 140% ( P < 0.05). In plantaris, the level of type IIa MHC mRNA decreased by 58% ( P < 0.005). In conclusion, immobilization changed the MHC mRNA profile in three different types of skeletal muscle toward faster isoforms. The quantitative results permit reliable evaluation of changes in mRNA levels.

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Regulation of an antisense RNA with the transition of neonatal to IIb myosin heavy chain during postnatal development and hypothyroidism in rat skeletal muscle

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00591.2011 ◽

2012 ◽

Vol 302 (7) ◽

pp. R854-R867 ◽

Cited By ~ 11

Author(s):

Clay E. Pandorf ◽

Weihua Jiang ◽

Anqi X. Qin ◽

Paul W. Bodell ◽

Kenneth M. Baldwin ◽

...

Keyword(s):

Skeletal Muscle ◽

Myosin Heavy Chain ◽

Postnatal Development ◽

Heavy Chain ◽

Antisense Rna ◽

Transcriptional Repression ◽

Mhc Genes ◽

Regulatory Model ◽

A Site ◽

Comparative Phylogenetic Analysis

Postnatal development of fast skeletal muscle is characterized by a transition in expression of myosin heavy chain (MHC) isoforms, from primarily neonatal MHC at birth to primarily IIb MHC in adults, in a tightly coordinated manner. These isoforms are encoded by distinct genes, which are separated by ∼17 kb on rat chromosome 10. The neonatal-to-IIb MHC transition is inhibited by a hypothyroid state. We examined RNA products [mRNA, pre-mRNA, and natural antisense transcript (NAT)] of developmental and adult-expressed MHC genes (embryonic, neonatal, I, IIa, IIx, and IIb) at 2, 10, 20, and 40 days after birth in normal and thyroid-deficient rat neonates treated with propylthiouracil. We found that a long noncoding antisense-oriented RNA transcript, termed bII NAT, is transcribed from a site within the IIb-Neo intergenic region and across most of the IIb MHC gene. NATs have previously been shown to mediate transcriptional repression of sense-oriented counterparts. The bII NAT is transcriptionally regulated during postnatal development and in response to hypothyroidism. Evidence for a regulatory mechanism is suggested by an inverse relationship between IIb MHC and bII NAT in normal and hypothyroid-treated muscle. Neonatal MHC transcription is coordinately expressed with bII NAT. A comparative phylogenetic analysis also suggests that bII NAT-mediated regulation has been a conserved trait of placental mammals for most of the eutherian evolutionary history. The evidence in support of the regulatory model implicates long noncoding antisense RNA as a mechanism to coordinate the transition between neonatal and IIb MHC during postnatal development.

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Calcineurin plays a modulatory role in loading-induced regulation of type I myosin heavy chain gene expression in slow skeletal muscle

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00349.2009 ◽

2009 ◽

Vol 297 (4) ◽

pp. R1037-R1048 ◽

Cited By ~ 15

Author(s):

Clay E. Pandorf ◽

Weihua H. Jiang ◽

Anqi X. Qin ◽

Paul W. Bodell ◽

Kenneth M. Baldwin ◽

...

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Fiber Type ◽

Hindlimb Suspension ◽

Chain Gene ◽

Type I ◽

Thyroid State

The role of calcineurin (Cn) in skeletal muscle fiber-type expression has been a subject of great interest because of reports indicating that it controls the slow muscle phenotype. To delineate the role of Cn in phenotype remodeling, particularly its role in driving expression of the type I myosin heavy chain (MHC) gene, we used a novel strategy whereby a profound transition from fast to slow fiber type is induced and examined in the absence and presence of cyclosporin A (CsA), a Cn inhibitor. To induce the fast-to-slow transition, we first subjected rats to 7 days of hindlimb suspension (HS) + thyroid hormone [triiodothyronine (T3)] to suppress nearly all expression of type I MHC mRNA in the soleus muscle. HS + T3 was then withdrawn, and rats resumed normal ambulation and thyroid state, during which vehicle or CsA (30 mg·kg−1·day−1) was administered for 7 or 14 days. The findings demonstrate that, despite significant inhibition of Cn, pre-mRNA, mRNA, and protein abundance of type I MHC increased markedly during reloading relative to HS + T3 ( P < 0.05). Type I MHC expression was, however, attenuated by CsA compared with vehicle treatment. In addition, type IIa and IIx MHC pre-mRNA, mRNA, and relative protein levels were increased in Cn-treated compared with vehicle-treated rats. These findings indicate that Cn has a modulatory role in MHC transcription, rather than a role as a primary regulator of slow MHC gene expression.

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Differential epigenetic modifications of histones at the myosin heavy chain genes in fast and slow skeletal muscle fibers and in response to muscle unloading

AJP Cell Physiology ◽

10.1152/ajpcell.00075.2009 ◽

2009 ◽

Vol 297 (1) ◽

pp. C6-C16 ◽

Cited By ~ 49

Author(s):

Clay E. Pandorf ◽

Fadia Haddad ◽

Carola Wright ◽

Paul W. Bodell ◽

Kenneth M. Baldwin

Keyword(s):

Skeletal Muscle ◽

Gene Regulation ◽

Myosin Heavy Chain ◽

Histone Modifications ◽

Heavy Chain ◽

Fiber Type ◽

Histone H3 ◽

Mhc Genes ◽

Slow Fiber ◽

Muscle Unloading

Recent advances in chromatin biology have enhanced our understanding of gene regulation. It is now widely appreciated that gene regulation is dependent upon post-translational modifications to the histones which package genes in the nucleus of cells. Active genes are known to be associated with acetylation of histones (H3ac) and trimethylation of lysine 4 in histone H3 (H3K4me3). Using chromatin immunoprecipitation (ChIP), we examined histone modifications at the myosin heavy chain (MHC) genes expressed in fast vs. slow fiber-type skeletal muscle, and in a model of muscle unloading, which results in a shift to fast MHC gene expression in slow muscles. Both H3ac and H3K4me3 varied directly with the transcriptional activity of the MHC genes in fast fiber-type plantaris and slow fiber-type soleus. During MHC transitions with muscle unloading, histone H3 at the type I MHC becomes de-acetylated in correspondence with down-regulation of that gene, while upregulation of the fast type IIx and IIb MHCs occurs in conjunction with enhanced H3ac in those MHCs. Enrichment of H3K4me3 is also increased at the type IIx and IIb MHCs when these genes are induced with muscle unloading. Downregulation of IIa MHC, however, was not associated with corresponding loss of H3ac or H3K4me3. These observations demonstrate the feasibility of using the ChIP assay to understand the native chromatin environment in adult skeletal muscle, and also suggest that the transcriptional state of types I, IIx and IIb MHC genes are sensitive to histone modifications both in different muscle fiber-types and in response to altered loading states.

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Polyadenylated RNA, actin mRNA, and myosin heavy chain mRNA in young and old human skeletal muscle

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1996.270.2.e224 ◽

1996 ◽

Vol 270 (2) ◽

pp. E224-E229 ◽

Cited By ~ 9

Author(s):

S. Welle ◽

K. Bhatt ◽

C. Thornton

Keyword(s):

Skeletal Muscle ◽

Protein Synthesis ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Human Skeletal Muscle ◽

Synthesis Rate ◽

Protein Synthesis Rate ◽

Type I ◽

Polyadenylated Rna ◽

Actin Mrna

The myofibrillar protein synthesis rate in old human skeletal muscle is slower than that in young adult muscle. To examine whether this difference in protein synthesis rate is explained by reduced availability of the mRNAs that encode the most abundant myofibrillar proteins, we determined relative hybridization signals from probes for actin mRNA, myosin heavy chain mRNA, and total polyadenylated RNA in vastus lateralis muscle biopsies taken from young (22- to 31-yr-old) and old (61- to 74-yr-old) human subjects. The mean fractional rate of myofibrillar synthesis was 38% slower in the older muscles, as determined by incorporation of a stable isotope tracer. Total actin and myosin heavy chain mRNAs, and polyadenylated RNA, were determined using slot-blot assays. Isoform-specific determinations of alpha-actin mRNA, type I myosin heavy chain mRNA, and type IIa myosin heavy chain mRNA were done with ribonuclease protection assays. Hybridization signals were expressed relative to tissue DNA content. There was no difference between age groups in total polyadenylated RNA or in any of the specific mRNAs. We conclude that the slower myofibrillar synthesis rate in older muscle is not caused by reduced mRNA availability.

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Invited Review: Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle

Journal of Applied Physiology ◽

10.1152/jappl.2001.90.1.345 ◽

2001 ◽

Vol 90 (1) ◽

pp. 345-357 ◽

Cited By ~ 170

Author(s):

Kenneth M. Baldwin ◽

Fadia Haddad

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Motor Proteins ◽

Weight Bearing ◽

Activity Level ◽

Type I ◽

Mechanical Stimuli ◽

Energy Deprivation

The goal of this mini-review is to summarize findings concerning the role that different models of muscular activity and inactivity play in altering gene expression of the myosin heavy chain (MHC) family of motor proteins in mammalian cardiac and skeletal muscle. This was done in the context of examining parallel findings concerning the role that thyroid hormone (T3, 3,5,3′-triiodothyronine) plays in MHC expression. Findings show that both cardiac and skeletal muscles of experimental animals are initially undifferentiated at birth and then undergo a marked level of growth and differentiation in attaining the adult MHC phenotype in a T3/activity level-dependent fashion. Cardiac MHC expression in small mammals is highly sensitive to thyroid deficiency, diabetes, energy deprivation, and hypertension; each of these interventions induces upregulation of the β-MHC isoform, which functions to economize circulatory function in the face of altered energy demand. In skeletal muscle, hyperthyroidism, as well as interventions that unload or reduce the weight-bearing activity of the muscle, causes slow to fast MHC conversions. Fast to slow conversions, however, are seen under hypothyroidism or when the muscles either become chronically overloaded or subjected to intermittent loading as occurs during resistance training and endurance exercise. The regulation of MHC gene expression by T3 or mechanical stimuli appears to be strongly regulated by transcriptional events, based on recent findings on transgenic models and animals transfected with promoter-reporter constructs. However, the mechanisms by which T3 and mechanical stimuli exert their control on transcriptional processes appear to be different. Additional findings show that individual skeletal muscle fibers have the genetic machinery to express simultaneously all of the adult MHCs, e.g., slow type I and fast IIa, IIx, and IIb, in unique combinations under certain experimental conditions. This degree of heterogeneity among the individual fibers would ensure a large functional diversity in performing complex movement patterns. Future studies must now focus on 1) the signaling pathways and the underlying mechanisms governing the transcriptional/translational machinery that control this marked degree of plasticity and 2) the morphological organization and functional implications of the muscle fiber's capacity to express such a diversity of motor proteins.

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Sp3 Proteins Negatively Regulate β Myosin Heavy Chain Gene Expression during Skeletal Muscle Inactivity

Molecular and Cellular Biology ◽

10.1128/mcb.24.24.10777-10791.2004 ◽

2004 ◽

Vol 24 (24) ◽

pp. 10777-10791 ◽

Cited By ~ 18

Author(s):

Gretchen Tsika ◽

Juan Ji ◽

Richard Tsika

Keyword(s):

Gene Expression ◽

Skeletal Muscle ◽

Myosin Heavy Chain ◽

Heavy Chain ◽

Weight Bearing ◽

C2c12 Myotubes ◽

Type I ◽

Adult Skeletal Muscle ◽

Nuclear Extracts ◽

Muscle Inactivity

ABSTRACT In adult skeletal muscle, β myosin heavy chain (βMyHC) gene expression is primarily restricted to slow type I fibers; however, its expression is down-regulated in response to muscle inactivity. Little is known about the signaling pathways and transcription factors that mediate this important functional response. This study demonstrates that increased binding of Sp3 to GC-rich elements in theβ MyHC promoter is a critical event in down-regulation ofβ MyHC gene expression under non-weight-bearing conditions. Conversely, binding of Sp3 to these elements decreased while Sp1 binding increased with nuclear extracts from plantaris muscle exposed to mechanical overload, a stimulus that increases βMyHC gene expression. In addition, these experiments revealed the existence of an Sp4-DNA binding complex when using adult skeletal muscle nuclear extract was used but not when nuclear extracts from cultured myotubes were used. Sp3 proteins are competitive inhibitors of Sp1-mediatedβ MyHC reporter gene transactivation in both Drosophila SL-2 and mouse C2C12 myotubes. Sp4 is a weak activator of βMyHC gene expression in SL-2 cells, which lack endogenous Sp1 activity, but does not activate βMyHC gene expression in C2C12 myotubes, which have high levels of Sp1. These results suggest that competitive binding of Sp family proteins regulate βMyHC gene transcription in response to altered neuromuscular activity.

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