Myosin Heavy Chain Gene Expression Research Articles

Effect of cyclosporin A treatment on the in vivo regulation of type I MHC gene expression.

Rat soleus muscle consists predominantly of slow type I fibers. We have shown previously through deletion analysis that the highest level of reporter activity that we measure when injecting type I myosin heavy chain (MHC) promoter (MHC(1))-linked luciferase plasmid into soleus muscles depends on the presence of a 550-bp upstream enhancer (3,450-2,900) region of the promoter. Because the calcineurin-nuclear factor of activated T cells (NFAT) pathway has been implicated in the regulation of the slow muscle gene program, particularly the MHC(1) isoform, and the MHC(1) promoter contains several putative NFAT sites, we examined via deletion and mutation analyses whether this pathway is involved in the regulation of promoter activity in soleus. Nine days of treatment with the calcineurin inhibitor cyclosporin A (CsA) caused a significant decrease in activity of the -3,500- and -3,450-bp promoters compared with vehicle-treated rats. Truncation of the promoter to -2,900 bp or smaller reduced the activity and also eliminated the CsA responsiveness, thus implying that the enhancer region is required for CsA responsiveness. Surprisingly, mutating the two NFAT elements within the enhancer region had no obvious effect on promoter activity. CsA treatment resulted in an increase in the mRNA levels of fast-type IIa and IIx MHC isoforms, but RT-PCR analysis of MHC(1) pre-mRNA and mature mRNA expression in soleus muscles revealed no differences between vehicle- and CsA-treated rats. Although CsA affects the activity of the MHC(1) promoter, it appears that its effect is not through direct binding of NFAT to sites on the promoter.

Modification of myosin protein and gene expression in failing hearts due to myocardial infarction by enalapril or losartan.

The effects of enalapril, an angiotensin converting enzyme (ACE) inhibitor, and losartan, an angiotensin II receptor type I antagonist, were investigated on alterations in myofibrillar ATPase activity as well as myosin heavy chain (MHC) content and gene expression in failing hearts following myocardial infarction (MI). Three weeks after ligation of the left coronary artery, rats were treated with or without enalapril (10 mg/kg/day), and/or losartan (20 mg/kg/day) for 5 weeks. The infarcted animals exhibited an increase in left ventricle (LV) end diastolic pressure and depressed rates of LV pressure development as well as pressure decay. LV myofibrillar Ca2+ -stimulated ATPase activity was decreased in the infarcted hearts compared with controls, MHC alpha-isoform content was significantly decreased whereas that of MHC beta-isoform was markedly increased. The level of MHC alpha-isoform mRNA was decreased whereas that of MHC beta-isoform was increased in the viable infarcted LV. Treatment of animal with enalapril, losartan, or combination of enalapril and losartan partially prevented the MI induced changes in LV function, myofibrillar Ca2+ -stimulated ATPase activity, MHC protein expression and MHC gene expression. The results suggest that the beneficial effects of the renin-angiotensin system blockade in heart failure are associated with partial prevention of myofibrillar remodeling.

Exercise training improves cardiac function-related gene levels through thyroid hormone receptor signaling in aged rats.

Exercise training improves the aging-induced downregulation of myosin heavy chain (MHC) and sarcoplasmic reticulum (SR) Ca(2+)-ATPase, which participate in the regulation of cardiac contraction and relaxation. Thyroid hormone receptor (TR), a transcriptional activator, affected the regulation of gene expression of MHC and SR Ca(2+)-ATPase. We hypothesized that myocardial TR signaling contributes to a molecular mechanism of exercise training-induced improvement of MHC and SR Ca(2+)-ATPase genes with cardiac function in old age. We investigated whether TR signaling and gene expression of MHC and SR Ca(2+)-ATPase in the aged heart are affected by exercise training, using the hearts of sedentary young rats (4 mo old), sedentary aged rats (23 mo old), and trained aged rats (23 mo old, swimming training for 8 wk). Trained aged rats showed improvement in cardiac function. Expression of TR-alpha1 and TR-beta1 proteins in the heart were significantly lower in sedentary aged rats than in sedentary young rats and were significantly higher in trained aged rats than in sedentary aged rats. The activity of TR DNA binding to the transcriptional regulatory region in the alpha-MHC and SR Ca(2+)-ATPase genes and the mRNA and protein expression of alpha-MHC and SR Ca(2+)-ATPase in the heart and plasma 3,3'-triiodothyronine and thyroxine levels were altered in association with changes in the myocardial TR protein levels. These findings suggest that exercise training improves the aging-induced downregulation of myocardial TR signaling-mediated transcription of MHC and SR Ca(2+)-ATPase genes, thereby contributing to the improvement of cardiac function in trained aged hearts.

Ca2+ transients activate calcineurin/NFATc1 and initiate fast-to-slow transformation in a primary skeletal muscle culture.

The calcineurin-mediated signal transduction via nuclear factor of activated T cells (NFATc1) is involved in upregulating slow myosin heavy chain (MHC) gene expression during fast-to-slow transformation of skeletal muscle cells. This study aims to investigate the Ca2+ signal necessary to activate the calcineurin-NFATc1 cascade in skeletal muscle. Electrostimulation of primary myocytes from rabbit for 24 h induced a distinct fast-to-slow transformation at the MHC mRNA level and a full activation of the calcineurin-NFATc1 pathway, although resting Ca2+ concentration ([Ca2+]i) remained unaltered at 70 nM. During activation, the calcium transients of these myocytes reach a peak concentration of approximately 500 nM. Although 70 nM [Ca2+]i does not activate calcineurin-NFAT, we show by the use of Ca2+ ionophore that the system is fully activated when [Ca2+]i is >or=150 nM in a sustained manner. We conclude that the calcineurin signal transduction pathway and the slow MHC gene in cultured skeletal muscle cells are activated by repetition of the rapid high-amplitude calcium transients that are associated with excitation-contraction coupling rather than by a sustained elevation of resting Ca2+ concentration.

Skeletal muscle plasticity: cellular and molecular responses to altered physical activity paradigms.

The goal of this article is to examine our current understanding of the chain of events known to be involved in the adaptive process whereby specific genes and their protein products undergo altered expression; specifically, skeletal muscle adaptation in response to altered loading states will be discussed, with a special focus on the regulation of the contractile protein, myosin heavy chain gene expression. This protein, which is both an important structural and regulatory protein comprising the contractile apparatus, can be expressed as different isoforms, thereby having an impact on the functional diversity of the muscle. Because the regulation of the myosin gene family is under the control of a complex set of processes including, but not limited to, activity, hormonal, and metabolic factors, this protein will serve as a cellular "marker" for studies of muscle plasticity in response to various mechanical perturbations in which the quantity and type of myosin isoform, along with other important cellular proteins, are altered in expression.

Modification of Myosin Gene Expression by Imidapril in Failing Heart due to Myocardial Infarction

J. Wang, X. Liu, B. Ren, H. Rupp, N. Takeda and N. S. Dhalla. Modification of Myosin Gene Expression by Imidapril in Failing Heart due to Myocardial Infarction. Journal of Molecular and Cellular Cardiology (2002) 34, 847–857. The beneficial effects of imidapril, an angiotensin converting enzyme (ACE) inhibitor were investigated on changes in myofibrillar ATPase as well as myosin heavy chain (MHC) content and gene expression due to myocardial infarction (MI). Three weeks after occluding the left coronary artery, rats were treated with or without imidapril (1mg/kg/day), for 4 weeks. The infarcted hearts exhibited depressed rates of left ventricular (LV) pressure development (57±2.4% reduction) and pressure decay (55.5±1.6% reduction). LV myofibrillar Ca2+ ATPase activity, unlike that in the right ventricle (RV), was decreased in the infarcted animals compared with controls (6.8±0.4 vs 10.3±0.6μmol Pi/mg/hr). MHC α-isoform contents were decreased by 47 and 41% whereas those of MHC β-isoform were increased by 823 and 1200% in the LV and RV due to MI, respectively. MHC α-isoform mRNA levels were decreased by 55 and 35% whereas those for MHC β-isoform were increased by 50 and 30% in the infarcted LV and RV, respectively. Imidapril treatment partially prevented the changes due to MI in LV function (rate of pressure development, 24±2.3% reduction and rate of pressure decay, 14±1.8% reduction), myofibrillar Ca2+ ATPase activity (8.2±0.7μmol Pi/mg/hr), MHC protein content (α-MHC, 24% reduction and β-MHC, 525% increase) and MHC gene expression (α-MHC, 18% reduction and β-MHC, 15% increase). The results suggest that the beneficial effects of ACE inhibition on the failing heart are associated with improvements in myofibrillar ATPase activities as well as prevention of changes in MHC isozyme protein contents and their gene expression. Copyright 2002 Elsevier Science Ltd. All rights reserved.

Changes in Mass and Performance in Rabbit Muscles after Muscle Damage with or without Transplantation of Primary Satellite Cells.

Changes in morphology, metabolism, myosin heavy chain gene expression, and functional performances in damaged rabbit muscles with or without transplantation of primary satellite cells were investigated. For this purpose, we damaged bilaterally the fast muscle tibialis anterior (TA) with either 1.5 or 2.6 ml cardiotoxin 10-5 M injections. Primary cultures of satellite cells were autotransplanted unilaterally 5 days after muscle degeneration. Two months postoperation, the masses of damaged TAs, with or without transplantation, were significantly larger than those of the controls. Furthermore, damaged transplanted muscles weighed significantly more than damaged muscles only. The increase in muscle mass was essentially due to increased fiber size. These results were independent of the quantity of cardiotoxin injected into the muscles. Maximal forces were similar in control and 2.6 ml damaged TAs with or without satellite cell transfer. In contrast, 1.5 ml damaged TAs showed a significant decrease in maximal forces that reached the level of controls after transplantation of satellite cells. Fatigue resistance was similar in control and 1.5 ml damaged TAs independently of satellite cell transfer. Fatigue index was significantly higher in 2.6 ml damaged muscles with or without cell transplantation. These changes could be explained in part by muscle metabolism, which shifted towards oxidative activities, and by gene expression of myosin heavy chain isoforms, which presented an increase in type IIa and a decrease in type I and IIb in all damaged muscles with or without cell transfer. Under our experimental conditions, these results show that muscle damage rather than satellite cell transplantation changes muscle metabolism, myosin heavy chain isoform gene expression, and, to a lesser extent, muscle contractile properties. In contrast, muscle weight and fiber size are increased both by muscle damage and by satellite cell transfer.

Altered K(+) channel gene expression in diabetic rat ventricle: isoform switching between Kv4.2 and Kv1.4.

Expression of voltage-gated K(+) channels encoding the K(+) independent transient outward current in the streptozocin-induced diabetic (DM) rat ventricle was studied to determine the basis for slowed cardiac repolarization in diabetes mellitus. Although hypertrophy was not detected in diabetic rats at 12 wk after streptozocin treatment, ventricular Kv4.2 mRNA levels decreased 41% relative to nondiabetic controls. Kv1.4 mRNA levels increased 179% relative to controls, whereas Kv4.3 mRNA levels were unaffected. Immunohistochemistry and Western blot analysis of the diabetic heart showed that the density of the Kv4.2 protein decreased, whereas Kv1.4 protein increased. Thus isoform switching from Kv4.2 to Kv1.4 is most likely the mechanism underlying the slower kinetics of transient outward K(+) current observed in the diabetic ventricle. Brain Kv1.4, Kv4.2, or Kv4.3 mRNA levels were unaffected by diabetes. Myosin heavy chain (MHC) gene expression was altered with a 32% decrease in alpha-MHC mRNA and a 259% increase in beta-MHC mRNA levels in diabetic ventricle. Low-dose insulin-like growth factor-II (IGF-II) treatment during the last 6 of the 12 wk of diabetes (DM + IGF) protected against these changes in MHC mRNAs despite continued hyperglycemia and body weight loss. IGF-II treatment did not change K(+) channel mRNA levels in DM or control rat ventricles. Thus IGF treatment may prevent some, but not all, biochemical abnormalities in the diabetic heart.

TEMPERATURE AND MYOGENIC FACTOR TRANSCRIPT LEVELS DURING EARLY DEVELOPMENT DETERMINES MUSCLE GROWTH POTENTIAL IN RAINBOW TROUT(ONCORHYNCHUS MYKISS) AND SEA BASS (DICENTRARCHUS LABRAX)

The influence of changes in environmental temperature on the mRNA levels of myogenic regulatory factors (MRFs), i.e. MyoD and myogenin, as well as myosin heavy chain (MyHC) were studied during early larval development in rainbow trout and sea bass. Phosphoimager analysis of northern blots indicated that there is an optimum temperature for the RNA transcript levels of MRF and MyHC RNA in trout and in sea bass larvae. In the trout strain studied, the highest concentration for MRF and MyHC transcripts was found at 8 degrees C rather than 4 degrees C or 20 degrees C. In European sea bass, the highest concentrations of MRF and MyHC mRNA were observed at 15-20 degrees C rather than 13 degrees C. Raising sea bass larvae at 15 degrees C was associated with higher MyHC gene expression as well as a trend towards an increase in total muscle fibre number and higher growth rates after transfer at ambient temperature. Results suggest that mRNA levels of MRF and MyHC can be used to optimise early development. An experiment in which the temperature was changed illustrates the consequence of precise temporal expression of MRF genes in specifying muscle fibre number at critical stages during early development.

Temporal effects of inactivty on myosin heavy chain gene expression in rat slow muscle.

Myosin heavy chain (MHC) mRNA and protein profiles in adult rat soleus and adductor longus were determined after 4, 8, 15, 30, 60, and 90 days of spinal cord isolation (SI). SI results in complete neuromuscular inactivity while leaving the motoneuron-muscle fiber connections intact. From 15 to 90 days, type I MHC mRNA was significantly decreased, whereas type I MHC protein did not significantly decrease until 30 and 60 days in the soleus and adductor longus, respectively. However, in both muscles, slow MHC downregulation was offset by significant upregulation of the faster MHC isoforms, primarily IIx. From 60 to 90 days, type I MHC was almost completely replaced with faster isoforms at the mRNA and protein levels. Thus, chronic inactivity and unloading of slow rat hindlimb muscles shifted the MHC profile from predominately type I to type IIx MHC mRNA and protein.

Differential expression of myosin heavy-chain mRNA in muscles of mastication during functional advancement of the mandible in pigs

Surgical and orthodontic treatment of retrognathia aims to improve orofacial function by adaptation and training of muscle capacity, which is connected with a change in muscle fibre-type proportions. The aim here was to analyse the proportion of myosin-heavy chain (MyHC) gene expression in type I (slow twitch/ST) and type IIb (fast twitch/FT) fibres during sagittal advancement of the mandible by reverse transcriptase-polymerase chain reaction (RT-PCR). The experiments were carried out on 10-week-old pigs (six test animals, six controls) over a 28-day period. Six pigs were fitted with acrylic bite blocks for sagittal advancement of the mandible. Tissue was taken from seven different regions of the masseter, temporal, medial pterygoid, and geniohyoid muscles. The 84 samples were used for histological fibre differentiation with ATPase staining and for isolation of total RNA. To measure the two MyHC isoforms, RT-PCR (in a single tube reaction with MyHC I, MyHC IIb, and GAPDH primers) was used. A significant increase was registered in the percentage of ST fibres and in mRNA from MyHC I in the anterior region of the masseter and in the posterior region of the temporal muscle of the treated animals. The proportion of ST fibres to FT fibres was increased by up to 12% after functional advancement of the mandible. The histological findings corresponded with the data for fibre mRNA generated by RT-PCR.

Highlighted Topics series: Plasticity in Skeletal, Cardiac, and Smooth Muscle

EDITORIALHighlighted Topics series: Plasticity in Skeletal, Cardiac, and Smooth MuscleGary C. SieckGary C. Sieck Journal of Applied Physiology January 2001, Volume 90Published Online:01 Jan 2001https://doi.org/10.1152/jappl.2001.90.1.1MoreSectionsPDF (47 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInWeChat This issue of the Journal of Applied Physiology introduces a new Highlighted Topics series on “Plasticity in Skeletal, Cardiac, and Smooth Muscle.” Muscle plasticity forms a major basis for physiological adaptation to our external environment. Examples of muscle plasticity abound, from exercise adaptations, to the effects of a microgravity environment, to the aging process, and to an assortment of pathophysiological conditions. Muscle plasticity can be both beneficial and maladaptive. Through research on the underlying mechanisms of muscle plasticity, we will be able to develop better training processes to improve human performance as well as effective therapies to counter maladaptive changes.In this issue, Drs. Baldwin and Haddad offer a mini-review focusing on “Effects of different activity and inactivity paradigms on myosin heavy chain gene expression in striated muscle.” Their work on the plasticity of myosin heavy chain isoform expression in skeletal and cardiac muscles has led to a better understanding of the influence of hormones, unloading, and neuroactivation on the expression of key contractile proteins. This review emphasizes muscle plasticity as an integrative function relating the influence of internal and external factors. Also in the January issue, Drs. Halayko and Solway explore “Molecular mechanisms of phenotypic plasticity in smooth muscle cells.” A novel integrated hypothesis is presented proposing a central role for the monomeric Rho GTPase family in regulating both gene transcription and structural remodeling of the contractile lattice. This model accounts for the unique mechanical adaptation and plasticity of smooth muscle.In the February issue, Dr. Reid examines “Redox modulation of skeletal muscle contractions: what we know and what we don't.” Skeletal muscle continually produces reactive oxygen and reactive nitrogen species. Both are essential for normal force production, but either, in excess, can compromise contractile function (e.g., during fatiguing exercise or inflammatory disease).There are three mini-reviews in the March issue. Drs. Housmans and Potter examine the “Pathophysiology of cardiac muscle contraction and relaxation as a result of alterations in thin filament regulation” in the first review. Cardiac muscle contraction depends on the interactions of thin and thick filament proteins. Familial hypotrophic cardiomyopathy (FHC) mutations affect these interactions and induce functional defects that impair normal contractility. Dysfunctional properties of the FHC mutation include altered Ca2+sensitivity, changes in ATPase activity, changes in force and velocity of shortening, and destabilization of the contractile complex.The other two mini-reviews in March explore plasticity in muscle energetics. In the first mini-review, “Contractile activity-induced mitochondrial biogenesis in skeletal muscle,” Dr. Hood explores the physiological consequences of increased muscle mitochondrial content and the essential role of activity-dependent plasticity in exercise. He also emphasizes recent advances in our understanding of the molecular basis of mitochondrial biogenesis. In the second mini-review, “Plasticity and energetic demands of contraction in skeletal and cardiac muscle,” Drs. Sieck and Regnier examine factors affecting ATP consumption during cross-bridge cycling, an energy demand that must be met by ATP production. An imbalance between energy supply and demand may be one of the causes of muscle fatigue and contractile failure.Muscle cells display a tremendous ability to adapt to new levels of gene expression in response to a wide range of environmental perturbations and clinical conditions. There is growing appreciation for the crucial role of muscle plasticity in overall health and disease. The introduction of cellular and molecular techniques has accelerated our understanding of basic mechanisms underlying muscle plasticity, and future genomic studies may provide additional insight regarding the diversity of muscle plasticity. Clearly, this is an important area in applied physiology, and it the hope of the Associate Editors and myself that this series will promote further research in this exciting field of study.This article has no references to display. Download PDF Back to Top Next FiguresReferencesRelatedInformation More from this issue > Volume 90Issue 1January 2001Pages 1-1 Copyright & PermissionsCopyright © 2001 the American Physiological Societyhttps://doi.org/10.1152/jappl.2001.90.1.1History Published online 1 January 2001 Published in print 1 January 2001 Metrics

Brief Exposure to Testosterone Is Sufficient to Induce Sex Differences in the Rabbit Masseter Muscle

In order to evaluate whether testosterone is responsible for inducing long-lasting sex differences in myosin heavy chain gene expression in the rabbit masseter muscle, we castrated young adult animals and administered supra-physiologic doses of testosterone for 3 or 6 weeks duration. Biopsies were taken from the masseter muscles of these animals at the time of castration and 3, 6 and 9 weeks later. Both immunoblot and immunohistochemical analyses of the myosin heavy chain (MyHC) isoform content of these muscle samples were performed. Exposure to testosterone for either duration resulted in a dramatic decrease in the content of the cardiac alpha MyHC isoform and a comparable increase in the content of the IIa MyHC isoform. The decrease in the cardiac alpha MyHC isoform content persisted for as long as 6 weeks after the end of treatment, but the increase in the content of the IIa MyHC isoform declined to normal during this time. Significant numbers of fibers were found containing both the cardiac alpha (and slow) and the IIa isoforms. Several fibers were encountered that contained both IIa and, presumably, the IIx isoform. Thus, a brief exposure to testosterone during postnatal maturation is able to produce a long lasting myosin heavy chain isoform switch that is similar in magnitude to that found during normal development.

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

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 (T(3), 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 T(3)/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 beta-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 T(3) 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 T(3) 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.

Nitric Oxide Regulates Smooth-muscle-specific Myosin Heavy Chain Gene Expression at the Transcriptional Level—Possible Role of SRF and YY1 Through CArG Element

Nitric oxide (NO) plays an important role in vascular regulation through its vasodilatory, antiatherogenic, and antithrombotic properties. NO inhibits platelet adhesion and aggregation and modulates smooth muscle cell (SMC) proliferation and migration. In animals with experimentally induced vascular injury, ec-NOS gene transfection not only restored NO production to normal levels but also increased vascular reactivity of the injured vessels. However, it is unclear whether NO regulates smooth-muscle-specific gene expression. We report here that addition of PDGF-BB to vascular smooth muscle cells suppressed SM-MHC expression but treatment with the NO donors FK409 and SNAP restored SM-MHC mRNA/protein expression.In vitro transfection and subsequent CAT assays demonstrated that exogenous NO can restore PDGF-BB-induced suppression of SM-MHC promoter activity. Promoter deletion analysis revealed that a CArG-3 box located at −1276 bp in the SM-MHC promoter was important for NO-dependent promoter regulation and as well as high level promoter activity. Gel mobility shift assays showed that CArG-3 contained the SRF binding site and a binding site for YY1, a nuclear factor which acts as a negative regulator on muscle-specific promoters. Interestingly, NO donor FK409 reduced YY1 binding to the CArG-3 element but increased SRF binding, suggesting that these two factors bind competitively to the overlapping sites. We also found that mutation to the YY1 binding site in the CArG-3 element resulted in a loss of PDGF-BB-induced suppression of the SM-MHC promoter activity. These findings indicate that NO regulates SM-MHC gene expression at the transcriptional level at least partially through the regulation of transcription factor binding activities on the CArG element. Thus we propose that NO plays a positive role in maintaining the differentiated state of VSMCs.

Postnatal Myosin Heavy Chain Isoform Expression in Normal Mice and Mice Null for IIb or IId Myosin Heavy Chains

The patterns of myosin heavy chain (MyHC) isoform expression in the embryo and in the adult mouse are reasonably well characterized and quite distinct. However, little is known about the transition between these two states, which involves major decreases and increases in the expression of several MyHC genes. In the present study, the expression of seven sarcomeric MyHCs was analyzed in the hindlimb muscles of wild-type mice and in mice null for the MyHC IIb or IId/x genes at several time points from 1 day of postnatal life (dpn) to 20 dpn. In early postnatal life, the developmental isoforms (embryonic and perinatal) comprise >90% of the total MyHC expression, while three adult fast isoforms (IIa, IIb, and IId) comprise <1% of the total MyHC protein. However, between 5 and 20 dpn their expression increases to comprise >90% of the total MyHC. Expression of each of the three adult fast isoforms occurs in a spatially and temporally distinct manner. We also show that α MyHC, which is almost exclusively expressed in the heart, is expressed in scattered fibers in all hindlimb muscles during postnatal development. Surprisingly, the timing and localization of expression of the MyHC isoforms is unchanged in IIb and IId/x null mice, although the magnitude of expression is altered for some isoforms. Together these data provide a comprehensive overview of the postnatal expression pattern of the sarcomeric MyHC isoforms in the mouse hindlimb.

Embryonic, fetal, and neonatal tongue myoblasts exhibit molecular heterogeneity in vitro

Variable gene expression patterns have been shown to exist between embryonic, fetal, and neonatal lineages of limb skeletal myoblasts in vitro and in vivo. In this study, we examined the molecular phenotype of embryonic, fetal, and neonatal tongue myoblasts in primary culture for comparison with in vivo developmental tongue myoblasts. Myogenic regulatory factor (MRF) and myosin heavy chain (MHC) gene expression were determined in culture during both growth and differentiation conditions by PCR, immunoblotting, and immunohistochemistry. Unlike their in vivo tongue myoblast equivalents, developmental tongue myoblast cultures featured the expression of MyoD when kept in growth conditions. Differentiation conditions in vitro induced myogenic tongue lineages to maintain characteristics of their in vivo morphologic and contractile gene phenotype. Both in vivo and in vitro, embryonic tongue lineages predominantly expressed MHC-embryonic isoforms, while fetal and neonatal tongue lineages predominantly expressed fast and perinatal isoforms of contractile genes. A notable difference from the in vivo condition that was observed in differentiated tongue myotubes in vitro was the presence of the MHC-slow protein. It was previously demonstrated that MHC-slow protein was undetectable during the in vivo development of the tongue musculature despite the abundance of slow isoform transcripts. The present characterization of primary tongue myogenic cultures indicates that murine myoblast heterogeneity exists primarily between developmental lineages at the level of contractile gene expression. Outside their native surroundings, developmental myogenic tongue populations are unable to recapitulate the determination and differentiation molecular profiles that occur in vivo.

Cardiotrophic Effects of Protein Kinase C ε

Abstract —Protein kinase C (PKC) is a key mediator of many diverse physiological and pathological responses. Although little is known about the specific in vivo roles of the various cardiac PKC isozymes, activation-induced translocation of PKC is believed to be the primary determinant of isozyme-specific functions. Recently, we have identified a catalytically inactive peptide translocation inhibitor (εV1) and translocation activator (ψεRACK [ r eceptors for a ctivated C k inase]) specifically targeting PKCε. Using cardiomyocyte-specific transgenic expression of these peptides, we combined loss- and gain-of-function approaches to elucidate the in vivo consequences of myocardial PKCε signaling. As expected for a PKCε RACK binding peptide, confocal microscopy showed that εV1 decorated cross-striated elements and intercalated disks of cardiac myocytes. Inhibition of cardiomyocyte PKCε by εV1 at lower expression levels upregulated α–skeletal actin gene expression, increased cardiomyocyte cell size, and modestly impaired left ventricular fractional shortening. At high expression levels, εV1 caused a lethal dilated cardiomyopathy. In contrast, enhancement of PKCε translocation with ψεRACK resulted in selectively increased β myosin heavy chain gene expression and normally functioning concentric ventricular remodeling with decreased cardiomyocyte size. These results identify for the first time a role for PKCε signaling in normal postnatal maturational myocardial development and suggest the potential for PKCε activators to stimulate “physiological” cardiomyocyte growth.

Distant Upstream Regulatory Domains Direct High Levels of β -myosin Heavy Chain Gene Expression in Differentiated Embryonic Stem Cells

K. L. Schreiber, A. Calderone and H. Rindt. Distant Upstream Regulatory Domains Direct High Levels ofβ -myosin Heavy Chain Gene Expression in Differentiated Embryonic Stem Cells. Journal of Molecular and Cellular Cardiology (2000) 32, 585–598. Eukaryotic gene transcription takes place in the context of chromatin. In order to study the expression of the β -myosin heavy chain (MyHC) gene in its appropriate cardiac environment in vitro, embryonic stem cell lines were generated and induced to differentiate into the cardiac lineage. We show that the upstream region of the β -MyHC gene (−5518 to −2490 relative to the transcriptional start site) directed high levels of transcriptional activity only when stably integrated, but not when expressed extrachromosomally in transient assays. These results are consistent with earlier findings using an in vivo transgenic approach. The expression of β -MyHC reporter gene constructs was strictly correlated to differentiation status and coincided with the expression of endogenous cardiac marker genes and with morphological differentiation of embryoid bodies in vitro. Using populations of stably transfected cell clones, two domains important for high level expression were identified. The analysis of individual cell clones suggested that the positive regulatory domains act according to the graded model of enhancement. These results show that chromosomal integration is necessary for the appropriate function of the β -MyHC gene's upstream regulatory region.

Effects of spaceflight and thyroid deficiency on rat hindlimb development. II. Expression of MHC isoforms.

Both slow-twitch and fast-twitch muscles are undifferentiated after birth as to their contractile protein phenotype. Thus we examined the separate and combined effects of spaceflight (SF) and thyroid deficiency (TD) on myosin heavy chain (MHC) gene expression (protein and mRNA) in muscles of neonatal rats (7 and 14 days of age at launch) exposed to SF for 16 days. Spaceflight markedly reduced expression of the slow, type I MHC gene by approximately 55%, whereas it augmented expression of the fast IIx and IIb MHCs in antigravity skeletal muscles. In fast muscles, SF caused subtle increases in the fast IIb MHC relative to the other adult MHCs. In contrast, TD prevented the normal expression of the fast MHC phenotype, particularly the IIb MHC, whereas TD maintained expression of the embryonic/neonatal MHC isoforms; this response occurred independently of gravity. Collectively, these results suggest that normal expression of the type I MHC gene requires signals associated with weight-bearing activity, whereas normal expression of the IIb MHC requires an intact thyroid state acting independently of the weight-bearing activities typically encountered during neonatal development of laboratory rodents. Finally, MHC expression in developing muscles is chiefly regulated by pretranslational processes based on the tight relationship between the MHC protein and mRNA data.