Effect of controllable stress on myosin heavy chain expression and muscle-specific protection by clomipramine

This study evaluated the influence of a controllable and painless stress, conditioned bright-light active-avoidance, on the expression of myosin heavy chain (MHC) protein isoforms in two nape and three masticatory rat muscles: longissimus capitis (L), rectus capitis dorsalis major (R), anterior digastric (AD), anterior temporalis (AT) and masseter superficialis (MS). The effects of a concomitant antidepressant treatment with clomipramine (CMI) on the muscle structure were also investigated. The three adult fast MHC isoforms were detected in all muscles studied: MHC 2A, 2X and 2B. The AT structure was not significantly modified by stress either under saline or under CMI treatment. In the other muscles studied, the stress situation induced a marked increase in the relative expression of MHC 2B and a decrease in MHCs 2X and 2A, except in L in which the MHC 2A decrease did not reach a statistically significant level. Under controllable stress, the CMI treatment led to the same MHC profile in AT, L, R and AD as saline, except in L where the MHC 2X decrease was no longer statistically significant. However, in MS, under controllable stress and CMI treatment, the MHC distribution was significantly different from the stressed saline-treated group and became comparable to the control again. MHC 2B has a higher shortening velocity than MHC 2X, which has a higher one than MHC 2A. According to total MHC isoform expression, the controllable stress-induced transformations would thus lead to increased velocity of all five muscles studied except in AT. This latter seems, therefore, not very sensitive to environmental requirements. Our results indicate that controllable stress produces important changes in the contractile properties of nape and masticatory muscles. Furthermore, this study demonstrates the protective effect of CMI against muscle structure transformations induced by controllable stress in MS, and that these effects are muscle type-specific.     

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

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.     

Changes in myosin expression in denervated laryngeal muscle

The effects of chronic denervation on the myosin heavy chain (MyHC) content and muscle fiber type composition of rat laryngeal muscles are described. The posterior cricoarytenoid (PCA) and thyroarytenoid (TA) muscles were removed 3 weeks, 3 months, and 6 months after recurrent laryngeal nerve sectioning. Myofibrillar adenosine triphosphatase staining of cryostat sections was performed, and fiber type percentages were determined. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to separate MyHC isoforms, and densitometry was subsequently used for quantitative analysis. Unoperated animals served as controls. In the PCA muscle, denervation resulted in a progressive reduction in type I MyHC (the slow-contracting isoform) to an almost complete loss at 6 months, with a concomitant increase in type II MyHCs (fast-contracting isoforms, excluding type IIL). Type IIL MyHC (laryngeal-specific isoform) remained relatively constant up to 6 months after denervation. The myosin expression in the TA muscle, which contained only type II MyHCs, remained relatively constant with denervation. Changes in fiber type composition of the muscles described from tissue staining correlated with MyHC content. These findings in laryngeal muscle confirm the dependence of type I MyHC expression upon neural input, as has been found previously in limb skeletal muscles. Since the expression of all MyHCs except the IIL was modified after denervation in the PCA muscle, it is possible that the IIL isoform is maintained by factors that differ from those in the other skeletal myosins.     

Interaction of hyperthyroidism and hindlimb suspension on skeletal myosin heavy chain expression

We examined the novel interaction of hyperthyroidism and hindlimb suspension on the pattern of myosin heavy chain (MHC) expression (mRNA and protein) in skeletal muscles. Female Sprague-Dawley rats were assigned to four groups: 1) normal control (Con); 2) thyroid hormone treated [150 micrograms 3,5,3'-triiodothyronine (T3). kg-1. day-1] (T3); 3) hindlimb suspension (HS); or 4) T3-treated and HS (T3 + HS). Results show for the first time the novel observation that the combination T3 + HS induces a rapid and sustained, marked (80-90%) downregulation of type I MHC gene expression that is mirrored temporally by concomitant marked upregulation of type IIb MHC gene expression, as evidenced by the de novo synthesis of type IIb MHC protein in the soleus. The fast type IIx MHC isoform showed a differential response among the experimental groups, generally increasing with the separate and combined treatments in both the soleus and vastus intermedius muscles while decreasing in the plantaris muscles. The fast type IIa MHC was the least responsive to suspension of the MHCs and reflected its greatest responsiveness to T3 treatment while also undergoing differential adaptations in slow vs. fast muscle (increases vs. decreases, respectively). These results confirm previous findings that all four adult MHC genes are sensitive to T3 and suspension in a muscle-specific manner. In addition, we show that T3 + HS can interact synergistically to create novel adaptations in MHC expression that could not be observed when each factor was imposed separately.     

Interaction of thyroid state and denervation on skeletal myosin heavy chain expression

The goal of this study was to examine the effects of altered thyroid state and denervation (Den) on skeletal myosin heavy chain (MHC) expression in the plantaris and soleus muscles. Rats were subjected to unilateral denervation (Den) and randomly assigned to one of three groups: (1) euthyroid; (2) hyperthyroid; (3) and hypothyroid. Denervation caused severe muscle atrophy and muscle-type specific MHC transformation. Denervation transformed the soleus to a faster muscle, and its effects required the presence of circulating thyroid hormone. In contrast, denervation transformed the plantaris to a slower muscle independently of thyroid state. Furthermore, thyroid hormone effects did not depend upon innervation status in the soleus, while they required the presence of the nerve in the plantaris. Collectively, these findings suggest that both thyroid hormone and intact nerve (a) differentially affect MHC transformations in fast and slow muscle; and (b) are important factors in regulating the optimal expression of both type I and IIB MHC genes. This research suggests that for patients with nerve damage and/or paralysis, both muscle mass and biochemical properties can also be affected by the thyroid state.     

Influence of mechanical loading on myosin heavy-chain protein and mRNA isoform expression

The overall objective of the studies reported herein was to examine the effects of high-resistance training on myosin heavy-chain (MHC) protein and mRNA isoform expression. The findings from these studies can be summarized as follows: 1) there was a substantial increase in the fast type IIX MHC protein isoform content of the trained red and white medial gastrocnemius muscles, but this did not occur until after the eighth training session (i.e., 16 days); 2) single-fiber analyses demonstrated that many so-called fast type IIB fibers contained small amounts of the fast type IIX MHC protein isoform and that the high-resistance training program altered the bias of fast type IIB-type IIX MHC protein isoform distribution in these fibers but did not increase the number of fibers that could be categorized as exclusively fast type IIX fibers; 3) the high-resistance training program produced a rapid (i.e., after two training sessions) elevation in the fast type IIX MHC mRNA isoform and a corresponding repression of the fast type IIB MHC mRNA isoform; and 4) the dose-response study revealed that as few as 10 contractions (40 s) per training session were capable of elevating the expression of the fast type IIX MHC mRNA isoform by approximately 250%. These collective findings demonstrate that high-resistance training is a powerful modulator of MHC protein isoforms and that pretranslational mechanisms are very sensitive to even small amounts of high-resistance training.     

Schistosoma japonicum myosin: cloning, expression and vaccination studies with the homologue of the S. mansoni myosin fragment IrV-5

The Schistosoma japonicum homologue of the 62 kDa fragment of S. mansoni myosin (SmIrV-5), which has proved highly protective against S. mansoni infection in mice and rats, has been cloned and expressed as the full length 62 kDa equivalent, Sj62, and a truncated 44 kDa version, Sj44. DNA sequencing showed the Sj62 sequence to be 88.4% identical at the nucleic acid level and 96% identical in deduced amino acid sequence to that of SmIrV-5. The recombinant proteins (rSj44 and rSj62) were strongly recognized in Western blotting by sera from mice multiply vaccinated with UV-irradiated S. japonicum cercariae and weakly recognized by S. japonicum chronic infection mouse sera. Unlike SmIrV-5, mouse antisera against the recombinant S. japonicum proteins did not give positive recognition in immunofluorescence assay with the surface of newly transformed schistosomula of the homologous species, S. japonicum, nor did they react with S. mansoni schistosomula. However, the anti-rSj62 sera clearly localized the native antigen to the subtegumental muscle layers in male adult worm sections by immunoelectron microscopy. Vaccination of several groups of mice and/or rats with rSj44 and rSj62 incorporated into different adjuvants induced high titres of specific IgG but in only one experimental group was there a significant reduction in worm burden (27%, P < 0.05). The possible reasons for the disparity between the vaccination results presented here and those demonstrated in experiments using rSm62 (IrV-5) are discussed.     

The developmentally regulated expression of serum response factor plays a key role in the control of smooth muscle-specific genes

Do the socioeconomic characteristics of a community affect one's health? This research examines whether the socioeconomic characteristics of communities are associated with the health of community residents, over and above the socio-economic characteristics of individual residents and their families. This is the first study to examine the independent associations between community-level socio-economic status (SES) and individual-level health using a nationally representative sample of adults in the United States. Results indicate that a person's health is associated with SES characteristics of the community over and above one's own income, education, and assets. However, individual-level and family-level SES indicators are stronger predictors of health than community-level SES indicators. This research suggests that improving individual-level and family-level socioeconomic circumstances may be the more direct way to improve the health of individuals, but that understanding the community context in which a person lives may also ultimately be important to improving health.     

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.     

Extraocular fast myosin heavy chain expression in the levator palpebrae and retractor bulbi muscles

PURPOSE: To determine whether the levator palpebrae superioris (LPS) and the retractor bulbi (RB) muscles, which share contractile characteristics with extraocular muscles (EOMs), express fast EOM-specific myosin heavy chain (MyHC) in the rabbit and other mammalian species. METHODS: Cryostat sections of rabbit eye and limb muscles were stained by indirect peroxidase immunohistochemical procedures using monoclonal antibodies (MAbs), including one (4A6) against EOM-specific fast MyHC. Myosin heavy chain isoforms from these muscles were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the 4A6-reactive component was identified by immunoblotting. RESULTS: MAb 4A6 stained muscle fibers in rabbit LPS and RB. SDS-PAGE resolved a rabbit EOM-specific MyHC isoform (band 1) from two other components (bands 2 and 3) that comigrated with limb fast MyHCs. MAb 4A6 reacted only with band 1. Rabbit LPS and RB also displayed corresponding MyHC components with the same mobilities and immunoreactivities as bands 1 to 3 in the EOM. MAb 4A6 also stained muscle fibers in monkey and cat LPS, but it failed to stain muscle fibers in cat RB and rat LPS and RB. CONCLUSIONS: The expression of EOM-specific fast MyHC in EOM, LPS, and RB reflect their common developmental origin and similar contractile characteristics. These properties set them apart from other skeletal muscle groups. Eye muscles may constitute a distinct muscle group or allotype characterized by unique properties, including their propensity or resistance to disease.     

Altered expression of myosin heavy chain in human skeletal muscle in chronic heart failure

To explore further alterations in skeletal muscle in chronic heart failure (CHF), we examined myosin heavy chain (MHC) isoforms from biopsies of the vastus lateralis in nine male patients with class II-III (CHF) (left ventricular ejection fraction (LVEF) 26 +/- 11%, peak oxygen consumption (peak VO2) 12.6 +/- 2 mL.kg-1.min-1) and nine age-matched sedentary normal males (NL). The relative content of MHC isoforms I, IIa, and IIx was determined by gel electrophoresis as follows: The normal sedentary group (NL) had a higher percent of MHC type I when compared with the patients (NL 48.4 +/- 7% vs CHF patients 24 +/- 21.6%, P < 0.05, no difference between MCH IIa (NL 45.1 +/- 10.5% vs CHF 56.0 +/- 12.5%), and CHF patients had a higher relative content of MHC type IIx than did the normal group (NL 6.5 +/- 9.6% vs CHF 20.0 +/- 12.9%, P < 0.05. Three of nine patients had no detectable MHC type I. In patients relative expression of MHC type I (%) was related to peak VO2 (r = 0.70, P < 0.05). Our results indicate that major alterations in MHC isoform expression are present in skeletal muscle in CHF. These alterations parallel previously reported changes in fiber typing that may affect contractile function i skeletal muscle and possibly exercise performance. The absence of MHC type I in some CHF patients suggests that skeletal muscle changes in this disorder are not solely a result of deconditioning, buy may reflect a specific skeletal muscle myopathy in this disorder.     

The effect of angiotensin II on myosin heavy chain expression in cultured myocardial cells

Angiotensin II (AII), the principal mediator of the renin-angiotensin system, is an important regulator of vascular and cardiac homeostasis. AII has also been shown to be a regulator of cardiac hypertrophy and of the corresponding changes in amount and composition of certain tissue proteins. We examined the trophic effects of AII on cultured myocytes derived from neonatal rat ventricles and followed, by Northern blot analysis and polyacrylamide gel electrophoresis, the expression of alpha- and beta-myosin heavy chain iso-mRNAs and isoproteins. Our findings show that a single administration of AII is sufficient to induce a trophic response in cultured beating myocytes and to enhance the expression of beta-myosin heavy chain iso-mRNA and isoprotein, having no effect on alpha-myosin heavy chain. Induction of alpha-myosin heavy chain expression by thyroid hormone before AII was administered showed that AII could not potentiate a shift from alpha- to beta-myosin heavy chain predominance. We suggest that the potency of AII to regulate the expression of myosin heavy chain isogenes is restricted to the beta isoform and is overridden by thyroid hormone.