Myosin mRNA Research Articles

Temperature-dependent developmental variation in lobster muscle myosin heavy chain isoforms

The temperature- and developmental-regulation of myosin heavy chain (MyHC) expression and primary sequence was investigated in the abdominal musculature of developing Homarus gammarus larvae acclimated to 10, 14 and 19±1 °C. MyHC loop 1 (ATP binding) and loop 2 (actin binding) regions were sequenced and compared. The deduced amino acid sequence of MyHC loop 1 showed a development-related increase in net charge from +1 to +2 between larval stages 1 and 2, which was not temperature-dependent. In post-settled stage 9 larvae, minor shifts in amino acid sequence occurred at 19 °C, and corresponded to a significant up-regulation of fast myosin mRNA expression. However, no temperature-specific loop 1 isoforms were detected. The deduced amino acid sequence of MyHC loop 2 was not affected by temperature, and the net charge remained +4 throughout development. These findings contrast to previous studies using the common carp, in which temperature-specific MyHC isoform genes were expressed in response to disparate thermal regimes. This raises the question as to whether arthropods do not express specific temperature isoforms but instead rely on shifts in fibre type to accommodate alterations in thermal environment.

Prevention of hypertrophy by overexpression of Kv4.2 in cultured neonatal cardiomyocytes.

Prolonged action potentials (APs) and decreased transient outward K+ currents (I(to)) are consistent findings in hypertrophic myocardium. However, the connection of these changes with cardiac hypertrophy is unknown. The present study investigated the effects of changes in I(to) and the associated alterations in AP on myocyte hypertrophy induced by phenylephrine. Chronic incubation of cultured neonatal ventricular rat myocytes (NVRMs) with phenylephrine (PE) reduced I(to) density and prolonged AP duration, leading to a 2-fold increase in the net Ca2+ influx per beat and a 1.4-fold increase in Ca2+-transient amplitude. PE treatment of chronically paced (2-Hz) NVRM also induced increases in cell size, protein/DNA ratio, atrial natriuretic factor mRNA expression, as well as beta/alpha myosin mRNA ratio. These hypertrophic changes were associated with a 2.4-fold increase in activation of nuclear factor of activated T-cells (NFAT), indicating increased activity of the Ca2+-dependent phosphatase calcineurin. Overexpression of Kv4.2 channels using adenovirus prevented the AP duration prolongation as well as the increases in Ca2+ influx and Ca2+-transient amplitude induced by PE. Kv4.2 overexpression also prohibited the PE-induced increases in cell size, protein/DNA ratio, atrial natriuretic factor expression, beta/alpha myosin mRNA ratio, and NFAT activation. Our results demonstrate that PE-mediated hypertrophy in NRVMs seems to require I(to) reductions and AP prolongation associated with increased Ca2+ influx and Ca2+ transients as well as calcineurin activation. The clinical implications of these studies and the possible involvement of other signaling pathways are discussed.

Localization of myosin XVA in endocrine tumors of gut and pancreas.

The myosin superfamily includes conventional and unconventional myosin proteins. Among unconventional myosins, myosin XVA has recently been characterized, and it has been suggested that it may be involved in cytoplasmic organelle movement, including secretory granules in pituitary cells and pituitary adenomas. In this study, we investigated the expression of myosin XVA protein and mRNA in normal endocrine cells and in a series of 53 endocrine tumors of the gut and pancreas. Myosin XVA was expressed in rare normal endocrine cells of the gut and in almost all pancreatic islet cells. In addition, myosin XVA was detected in several cells of all endocrine tumors investigated, and its expression was not related to malignancy, type, site, or functional status of tumors. These results indicate that myosin XVA protein and mRNA are widely distributed in endocrine cells of the gut and pancreas. Although the role of this protein in endocrine cells is unknown, previous studies suggest that it may have a role in secretory granule movement and/or hormone secretion.

Myosin expression levels in trout muscle: a new method for monitoring specific growth rates for rainbow troutOncorhynchus mykiss(Walbaum) on varied planes of nutrition

Fish growth is manifested by a number of measurable physical changes. We have developed a sensitive method for monitoring the growth rate of fish fed at four different planes of nutrition. This technique consists in measuring expression levels of myosin RNA isolated from the muscle of experimental animals. Using PCR (polymerase chain reaction) primers, and a fluorescence-labelled single-stranded DNA probe that hybridizes specifically to a region within the myosin mRNA of rainbow trout Oncorhynchus mykiss (Walbaum), we were able to detect differences in the relative level of myosin expression between groups of fish. This method also allows for the determination of absolute expression levels when reactions are performed with standards consisting of known levels of in vitro-transcribed myosin RNA. With the proper equipment, this novel procedure can be performed rapidly on large numbers of individuals, and with the procurement of non-invasive muscle biopsies the same experimental animal could theoretically be sampled multiple times throughout the course of the study. This new method could be used to measure differences in muscle synthesis in fish associated with various nutrient intake levels, environmental parameters, life-history stages and health status.

Semi-starvation alters myofibrillar mRNA concentrations to expedite rapid recovery of muscle protein stores following feeding.

Protein synthesis in skeletal muscle is reduced following starvation and restored by feeding. The mediators and mechanisms are incompletely understood. The aim of this study was to evaluate whether prolongation of undernutrition induced changes in muscle gene expression at the level of mRNA and protein. The changes in myosin heavy-chain 2X mRNA in adult partially starved (50% of ad libitum standard rodent chow intake for 4 or 7 days) C57BL mice or subsequently refed mice were studied. Ad libitum-fed mice were used as controls. Protein synthesis, total RNA and myosin heavy-chain 2X mRNA concentrations were determined. Plasma concentrations of amino acids were measured by high-performance liquid chromatography. Partial starvation of 4 and 7 days reduced bodyweight by 15.6 +/- 1% and 17.1 +/- 2.1% (P < or = 0.05) vs. ad libitum fed controls. Protein synthesis was reduced by 32 +/- 9% and protein content by 20 +/- 4% (P < or = 0.05) following 7 days of partial starvation. Plasma amino acid concentrations were increased (6297 +/- 853) in refed animals vs. ad libitum-fed controls (3057 +/- 141, P < or = 0.05). Total RNA concentration (micrograms RNA micrograms(-1) DNA) in skeletal muscle was unchanged. Myosin heavy-chain 2X mRNA concentration did not change following 4 days of partial starvation but increased by 24 +/- 5% (P < or = 0.05) following 7 days of partial starvation, hence suggesting that expression of myosin mRNA was nutritionally altered. Postprandial stimulation of protein synthesis following starvation may thus be a combination of increased mRNA availability and increased translation. This effect may be activated by peak concentrations of amino acids in plasma following feeding.

Postprandial resynthesis of myofibrillar proteins is translationally rather than transcriptionally regulated in human skeletal muscle

Feeding stimulates protein synthesis in skeletal muscles, although the regulatory mechanisms are incompletely understood. The aim of this study was to determine whether this could be detected at the gene transcription level for postprandial stimulation of the synthesis of muscle proteins. Healthy male volunteers were investigated after an overnight fast. Open muscle biopsies were performed in the starved state and 3 h after meal intake, consisting of 0.15 gN/kg, 12 kcal/kg. Blood samples were drawn every 15 to 30 min for 5 h. Myosin mRNA and insulin growth factor-I (IGF-I) mRNA were measured by solution hybridization assay in homogenized muscle specimens. After food intake, plasma glucose concentrations increased from 5.0 ± 0.1 to 7.3 ± 0.3 ( P ≤ 0.001), and insulin concentration rose from 3.8 ± 0.5 mU/L before to 75.3 ± 11.4 15 min after the meal ( P ≤ 0.001). Plasma concentration of free fatty acids declined after food intake ( P ≤ 0.001). Plasma concentrations of amino acids increased from basal values (2864 ± 128 μM) to 4419 ± 262 μM ( P ≤ 0.05) 90 min after meal ingestion. Myosin mRNA concentration in the biopsied muscle tissue was higher during starvation and was reduced by 20% after food intake: 10.8 ± 1.3 amol mRNA/μg DNA in the starved state and 8.5 ± 1.3 amol mRNA/μg DNA after food intake ( P ≤ 0.05). Feeding did not alter IGF-I mRNA concentrations in muscle: 0.51 ± 0.05 and 0.55 ± 0.06 amol/μg DNA in the starved and fed state, respectively ( P ≤ 0.48). Improved protein balance by stimulation of protein synthesis has been related to increased plasma amino acids. Interestingly, in the short term, this was not related to increases in gene transcription of either myofibrillar proteins (myosin) or muscle IGF-I. Thus, postprandial stimulation of protein synthesis appears not to be regulated by increased gene transcription but by increased translation using the increased concentrations of amino acids. In contrast, as far as the 2X myosin mRNA level is concerned, this is enhanced during starvation, which facilitates rapid recovery once the availability of substrate is resumed.

Expression of insulin growth factor-1 splice variants and structural genes in rabbit skeletal muscle induced by stretch and stimulation.

1. Skeletal muscle is a major source of circulating insulin growth factor-1 (IGF-1), particularly during exercise. It expresses two main isoforms. One of the muscle IGF-1 isoforms (muscle L.IGF-1) is similar to the main liver IGF-1 and presumably has an endocrine action. The other muscle isoform as a result of alternative splicing has a different 3' exon sequence and is apparently designed for an autocrine/paracrine action (mechano-growth factor, MGF). Using RNase protection assays with a probe that distinguishes these differently spliced forms of IGF-1, their expression and also the expression of two structural genes was measured in rabbit extensor digitorum longus muscles subjected to different mechanical signals. 2. Within 4 days, stretch using plaster cast immobilization with the limb in the plantar flexed position resulted in marked upregulation of both forms of IGF-1 mRNA. Electrical stimulation at 10 Hz combined with stretch (overload) resulted in an even greater increase of both types of IGF-1 transcript, whereas electrical stimulation alone, i.e. without stretch, resulted in no significant increase over muscle from sham-operated controls. Previously, it was shown that stretch combined with electrical stimulation of the dorsiflexor muscles in the adult rabbit results in a marked increase in muscle mass involving increases in both length and girth, within a few days. The expression of both systemic and autocrine IGF-1 growth factors provides a link between the mechanical signal and the marked increase in the structural gene expression involved in tissue remodelling and repair. 3. The expression of the beta actin gene was seen to be markedly upregulated in the stretched and stretched/stimulated muscles. It was concluded that the increased expression of this cytoskeletal protein gene is an indication that the production of IGF-1 may initially be a response to local damage. 4. Switches in muscle fibre phenotype were studied using a specific gene probe for the 2X myosin heavy chain gene. Type 2X expression was found to decrease markedly with stimulation alone and when electrical stimulation was combined with stretch. Unlike the induction of IGF-1 and beta actin, the decreased expression of the 2X myosin mRNA was less marked in the 'stretch only' muscles. This indicates that the interconversion of fibre type 2X to 2A may in some situations be commensurate with, but not under the control of IGF-1.

Changes in myosin mRNA and protein expression in denervated rat soleus and tibialis anterior.

Denervation differs from other models of reduced neuromuscular activation due to the absence of a nerve-muscle connection and limited data exists regarding the effects of denervation on myosin heavy chain (MHC) expression. Thus, adult MHC expression (I, IIa, IIx, IIb) was studied in the rat soleus and tibialis anterior (TA) at the mRNA and protein levels 2, 4, 7, 10, 14, and 30 days following sciatic nerve transection. MHC protein content was quantified with SDS/PAGE and mRNA levels with the RNase-protection assay. Control soleus consisted predominately of type I MHC mRNA and protein, however, 4 days after denervation type I MHC mRNA was significantly decreased to 41+/-8% of control and continued to remain below control values. Soleus IIa mRNA was significantly elevated 7 and 10 days after denervation while IIx mRNA remained relatively constant until 30 days when it increased to 197+/-23% of control. At the protein level, soleus I MHC significantly decreased to 80% of the total while IIa MHC significantly increased to 20% of the total. At 30 days, Hx MHC protein accounted for 9.4+/-1.6% of the total soleus MHC protein. In the TA, IIb mRNA was significantly decreased to 57% of control by day 4 and remained significantly decreased for up to a month. TA IIx mRNA was also significantly decreased at 10 and 30 days after denervation. Similar to the soleus, TA Ha mRNA was significantly increased over control 7-14 days after denervation. There were no significant changes in TA MHC protein profile during one month of denervation. In both the soleus and TA, denervation significantly shifted the MHC mRNA profile as early as 4 days following denervation without any corresponding changes at the protein level. Significant mRNA changes without large changes in MHC protein composition continued throughout the denervation period suggesting that the muscle may be prevented from premature functional transitions by mechanisms such as decreased mRNA stability, translational block, or increased turnover of newly synthesized proteins.

Expression of smooth muscle markers in the developing murine lung: potential contractile properties and lineal descent

Contractile cells in the mammalian lung develop in close association with the outgrowing stem bronchi. Fully differentiated smooth muscle cells are typically found in proximal regions, residing in the substantial muscular walls of the major airways and blood vessels. More distally, cells expressing markers of differentiated smooth muscle cells to a variable degree, and which may therefore possess contractile properties, are to be found scattered around the interstitium. We have investigated the temporal and spatial distribution of smooth muscle lineage markers (smooth muscle myosin mRNA) and of those indicative of contractile function (metavinculin mRNA) in the murine lung. In the smooth muscle layers of the bronchi and major blood vessels, these genes are expressed from the onset of pulmonary budding, concurrently with the appearance of alpha-smooth muscle actin and calponin proteins. During fetal development, smooth muscle-associated genes and proteins are restricted to this committed smooth muscle population. The first signs of myofibroblast or pericyte differentiation become manifest perinatally, when their expression of alpha-smooth muscle actin escalates. In the adult lung, such cells may be readily pin-pointed by their positive reaction for metavinculin mRNA, but, at maturity, they do not always coexpress alpha-smooth muscle actin.

Altered expression of myosin mRNA and protein in rat soleus and tibialis anterior following reinnervation.

Myosin heavy chain (MHC) expression was studied in rat soleus and tibialis anterior (TA) at the mRNA and protein levels following reinnervation 8 and 32 wk after sciatic nerve injury. A sciatic nerve crush or transection injury was produced in the midthigh region of adult female Sprague-Dawley rats. A ribonuclease protection assay was developed to measure four of the adult MHCs (I, IIa, IIx, IIb) in a single sample. MHC mRNA and protein were measured and compared in the same muscles. Eight and thirty-two weeks after a crush injury, the MHC mRNA profiles were similar to control with the exception of soleus MHC IIa and TA MHC IIb, which were significantly less than control at both time points. In contrast, reinnervation of the soleus following a sciatic nerve transection injury resulted in an MHC isoform shift characterized by increases in the relative amounts of fast myosin (IIa and IIx) and a decrease in slow myosin. As expected, significant changes first occurred at the mRNA level followed by changes in protein expression. Thirty-two weeks after transection injury and repair, the primary MHC mRNA isoform in the soleus was MHC IIx. Moreover, at 32 wk, MHC IIb mRNA was detected in 50% of the reinnervated soleus following a transection injury. Reinnervation of the TA following sciatic nerve transection led to replacement of the MHC IIb isoform with MHC IIx.

The kinase inhibitor iso-H7 stimulates rat satellite cell differentiation through a non-protein kinase C pathway by increasing myogenin expression level.

We analysed the signaling pathways involved in myogenic differentiation of primary cultures of rat satellite cells using substances targeting the protein kinase C (PKC) and the cAMP protein kinase (PKA) pathways. We have previously shown that iso-H7, which putatively inhibits both PKC and PKA, strongly stimulates satellite cell differentiation, as well as the PKA inhibitor HA1004. In the study reported here, the effects of iso-H7 on satellite cell differentiation were compared to those observed in the presence of agents which reduce PKC activity. It was shown that treatments with the highly specific PKC inhibitor GF109203X or with 12-O-tetradecanoylphorbol 13-acetate (TPA) which induced a partial PKC downregulation, did not significantly alter myogenic differentiation. Northern blot analyses showed that iso-H7 activated the expression of myogenin but not that of MyoD mRNA. Concurrently, iso-H7 increased myosin light-chain mRNA expression. In contrast, TPA had no effect on these syntheses. Taken together, these results showed that iso-H7 did not act intracellularly as a PKC inhibitor but rather as a PKA inhibitor as previously suggested. Our results are compatible with the hypothesis that a reduction in PKA activity controls satellite cell myogenesis through an increased myogenin mRNA expression.

933-98 Temporal Correlation of Changes in Left Ventricular Mass with Myosin Isoform mRNAs in Spontaneously Hypertensive Rats Treated with Quinapril or Hydralazine

During the development of hypertensive left ventricular hypertrophy (LVH) myosin isoforms change from α-myosin (αM) to β -myosin ( β M). In this study the effect of therapeutic decreases in hemodynamic loads on LVH and myosin mRNA expression of LV myocardium were examined in 19-week old spontaneously hypertensive rats (SHR). SHR were divided into 3 groups receiving: quinapril (Q) (3 mg/kg) ; hydralazine (Hy) (1 mg/kg); or placebo (C) p.o daily. Four rats from each group were examined on days 0, 2, 4, 7,10, and 21. Mean arterial pressure and total peripheral reasistance were significantly and similarly reduced in Q and Hy groups beginning day 4; there was no change in cardiac output (thermodilution). No changes in any hemodynamic variable occured in group C. Total RNA was isolated and analyzed by blot hybridization technique using labeled probes for αM, β M, and GAPDH (a “houskeeping gene”). After autoradiographic exposure films were analyzed by densitometry. After normalization for GAPDH, the relative amounts of αM and β M mRNAs were expressed in relation to day 0. Days Control Ouinapril Hydralazine LVMI αM β M LVMI αM β M LVM αM β M 0 2.66 1.0 1.0 2.66 1.0 1.0 2.66 1.0 1.0 2 2.61 1.2 1.1 2.55 1.5 0.8 2.60 1.5 1.1 4 2.62 0.8 0.8 2.59 2.3 * 0.5 * 2.64 1.6 0.6 7 2.74 0.9 0.9 2.37 * 1.9 * 0.7 2.48 2.1 * 0.6 10 2.74 0.9 1.3 2.39 * 2.5 * 0.5 * 2.81 1.1 * 0.5 * 21 270 1.2 1.2 2.35 * 2.5 * 0.5 * 2.58 1.9 * 0.4 * LVMI-LV mass index. * p &lt; 0.05, compared with day 0 (same group) or control These data show that reduction in hemodynamic load induced a change in myosin isoforms to adult pattern whereas only Q reduced LVMI, suggesting that LVH reversal may not be dependent solely on hemodynamic load.

933-98 Temporal Correlation of Changes in Left Ventricular Mass with Myosin Isoform mRNAs in Spontaneously Hypertensive Rats Treated with Quinapril or Hydralazine

During the development of hypertensive left ventricular hypertrophy (LVH) myosin isoforms change from α -myosin (α M ) to β -myosin ( β M). In this study the effect of therapeutic decreases in hemodynamic loads on LVH and myosin mRNA expression of LV myocardium were examined in 19-week old spontaneously hypertensive rats (SHR). SHR were divided into 3 groups receiving: quinapril (Q) (3 mg/kg); hydralazine (Hy) (1 mg/kg); or placebo (C) p.o daily. Four rats from each group were examined on days 0, 2, 4, 7,10, and 21. Mean arterial pressure and total peripheral reasistance were significantly and similarly reduced in Q and Hy groups beginning day 4; there was no change in cardiac output (thermodilution). No changes in any hemodynamic variable occured in group C. Total RNA was isolated and analyzed by blot hybridization technique using labeled probes for α M, β M, and GAPDH (a “houskeeping gene”). After autoradiographic exposure films were analyzed by densitometry. After normalization for GAPDH, the relative amounts of αM and βM mRNAs were expressed in relation to day 0. Maximum QTc was significantly correlated with LVMI (r = 0.41, P &lt; 0.01), systolic BP (r = 0.43, P &lt; 0.01), E/A ratio (r = 0.33, P &lt; 0.01), IVRT (r = 0.35, P &lt; 0.01) and age (r = 0.30, P &lt; 0.01). QTc dispersion was similarly significantly correlated with LVMI (r = 0.30, p &lt; 0.01), systolic BP (r = 0.30, P &lt; 0.01), E/A ratio (r = 0.22, P = 0.02), IVRT (r = 0.31, P &lt; 0.01) and age (r = 0.21, P &lt; 0.04). The interdependence of these factors preclude multiple regression analysis. In conclusion, QT dispersion and maximum QT length are related to left ventricular structure and diastolic function. This may reflect an increase in dispersion of recovery of ventricular excitability occurring as a result of the myocardial structural changes that occur as a consequence of hypertension. This may in turn predispose to ventricular arrhythmias which could help explain the high incidence of sudden death in hypertensives with LVH.

Cloning of a crustacean myosin heavy chain isoform: exclusive expression in fast muscle.

A clone of a fast isoform of myosin heavy chain (HC) gene was isolated from a cDNA1 expression library made from mRNA purified from the deep abdominal flexor muscle of the lobster, Homarus americanus. The cDNA (1.5 kb) contained the 3' untranslated region (UTR) and the coding sequence for the last 413 amino acid residues of the carboxyl terminus of the polypeptide. The deduced amino acid sequence showed high homology with that of myosin HCs from Drosophila (73% identity), nematode (57% identity), and vertebrates (49% identities). Hydropathy plots showed a 28-amino acid periodicity that is consistent with the alpha-helical coiled coil structure of the rod region of native myosin. Northern blot analysis and in situ hybridization showed that the fast myosin HC isoform was expressed only in fast fibers; the probes did not hybridize to mRNA from slow fibers. The message consisted of a single transcript of 6.6 kb. The intracellular localization of the fast myosin mRNA was not uniform. The mRNA was largely confined to the intermyofibrillar spaces and to the subsarcolemmal cytoplasm of the fiber periphery and large infoldings of the cell membrane. Within these regions the mRNA was concentrated in the cytoplasm immediately surrounding the nuclei. This constitutes the first report of the cloning and expression of a myosin HC gene from a crustacean species.

IIx and slow myosin expression follow mitochondrial increases in transforming muscle fibers.

Metabolic profile and contractile isoform expression commonly define classic fiber types in skeletal muscle. Little is known about how metabolic requirements determine expression of fast IIx and slow myosin isoforms in muscles undergoing fiber type conversion. Tibialis anterior muscles from female New Zealand White rabbits were stimulated continuously at 10 Hz for 4-21 days. Quantitative fiber analysis was made for oxidative potential by histochemistry and for fast IIx and slow myosin mRNA content by in situ hybridization. In control muscle we found 3 +/- 0.27% fibers coexpress both fast IIx and slow myosin mRNA and so were not assignable to a classic fiber type. After stimulation, increase in fiber oxidative potential was detectable by 4 days and preceded IIx mRNA increases on a fiber-by-fiber basis. Slow myosin transcripts were detected by 7 days in fibers with higher oxidative levels. Coexpression of IIx and slow transcripts peaked at 22 +/- 2.5% of fibers by 7 days. IIx then declined, leaving slow myosin expressed in 62 +/- 0.45% of fibers by 3 wk. We conclude that during fiber type transformation individual fibers can transcribe two myosin mRNAs synchronously. Metabolic demand precedes and may be linked to IIx and slow myosin isoform expression.

Contractile protein gene expression in primary myotubes of embryonic mouse hindlimb muscles.

The time course of contractile protein [actin, myosin heavy chain (MHC) and myosin light chain (MLC)] gene expression in the hindlimb muscles of the embryonic mouse (< 15 days gestation) has been correlated with the expression of genes for the myogenic regulatory factors, myogenin and MyoD, and with morphogenetic events. At 14 days gestation, secondary myotubes are not yet present in crural muscles (M. Ontell and K. Kozeka (1984) Am. J. Anat. 171, 133-148; M. Ontell, D. Bourke and D. Hughes (1988) Am. J. Anat. 181, 267-278); therefore, all transcripts for contractile proteins found in these muscles must be produced in primary myotubes. In situ hybridization, with 35S-labeled antisense cRNAs, demonstrates the versatility of primary myotubes in that transcripts for (1) alpha-cardiac and alpha-skeletal actin, (2) MHCembryonic, MHCperinatal and MHC beta/slow, and (3) MLC1A, MLC1F and MLC3F are detectable at 14 days gestation. While the general patterns of early activation of the cardiac genes and early activation of the genes for the developmental isoforms are preserved in both myotomal and limb muscles (D. Sassoon, I. Garner and M. Buckingham (1988) Development 104, 155-164 and G. E. Lyons, M. Ontell, R. Cox, D. Sassoon and M. Buckingham (1990) J. Cell Biol. 111, 1465-1476 for myotomal muscle), there are a number of differences in contractile protein gene expression. For example, in the myotome, when myosin light chain genes are initially transcribed, hybridization signal with probe for MLC1A mRNA is greater than that with probe for MLC1F transcripts, whereas the relative intensity of signal with these same probes is reversed in the hindlimb. The order in which myosin heavy chain genes are activated is also different, with MHCembryonic and MHCperinatal preceding the appearance of MHC beta/slow transcripts in limb muscles, while MHCembryonic and MHC beta/slow appear simultaneously in the myotomes prior to MHCperinatal. In the myotome, an intense hybridization signal for alpha-cardiac and a weak signal for alpha-skeletal actin transcripts are detectable prior to myosin mRNAs, whereas in the limb alpha-cardiac actin transcripts accumulate with myosin transcripts before alpha-skeletal actin mRNA is detectable. These differences indicate that there is no single coordinate pattern of expression of contractile protein genes during initial formation of the muscles of the mouse.(ABSTRACT TRUNCATED AT 400 WORDS)

Changes in insulin-receptor mRNA levels in skeletal muscle and brown adipose tissue of weanling rats during fasting and refeeding.

Tissue-specific alterations in insulin sensitivity occur in response to fasting and refeeding, as part of the integrated adaptive mechanisms employed to adjust to major changes in nutritional status. In the present study the effects of fasting and refeeding on insulin-receptor, actin and myosin mRNA levels in skeletal muscle, and insulin-receptor and uncoupling-protein mRNA in brown adipose tissue of rats have been examined. Insulin-receptor mRNA levels increased markedly in both skeletal muscle and brown adipose tissue after a 40 h fast, the increase being greater in brown fat (8-fold) than in muscle (2-fold). On refeeding for 4 h, the insulin-receptor mRNA level in both tissues declined rapidly to control levels. An increase in insulin-receptor mRNA level was also observed in brown adipose tissue after a 16 h fast, although not in skeletal muscle. In contrast to the insulin-receptor mRNA, the level of the mRNA for the mitochondrial uncoupling protein declined markedly in brown adipose tissue during a 40 h fast. These results indicate that insulin-receptor mRNA levels are modulated in response to the alterations in nutritional status that occur during fasting and refeeding; this may reflect a nutritional influence on transcription of the receptor-protein gene.

Gene expression in the Caenorhabditis elegans dauer larva: Developmental regulation of Hsp90 and other genes

Under conditions unfavorable to growth, the nematode Caenorhabditis elegans enters a developmentally arrested stage, the dauer larva. We have examined gene expression in the dauer larva and during recovery from the dauer stage. Run-on transcription assays with isolated nuclei reveal a depression of general RNA polymerase II transcription to 11–17% of that in other stages. Transcription of individual gene families (including actin, collagen, hsp70, and histone) is similarly depressed relative to actively growing stages. Dauer larvae are, however, capable of being induced for heat shock messages, indicating that they are competent to initiate and elongate transcripts. For most genes surveyed, reduced transcription in dauer larvae correlates with a decrease in message abundance. Hsp70 mRNA, however, is transcribed at lower rates but accumulates at levels comparable to those in other stages. Interestingly, dauer larvae are 15-fold enriched in a mRNA for a C. elegans hsp90 gene. Hsp90 mRNA accumulation is regulated at least in part by differential stability. Dauer larvae thus appear to have a unique pattern of gene expression. Upon placement in food, dauer larvae reenter the developmental pathway as late-stage larvae. Dauer recovery is accompanied by a temporally regulated sequence of gene expression. At least four distinct patterns of gene expression can be distinguished during exit from the dauer stage. Steady-state levels of hsp70 and polyubiquitin mRNA rise sharply within 75 min of recovery before declining by the fourth hour. Actin and histone mRNAs increase steadily following 2–4 hr of recovery, whereas myosin mRNA increases after 10 hr. In contrast, hsp90 mRNA declines sharply within the first 75 min of recovery. Changes in mRNA populations during dauer formation and exit may be physiologically relevant.

Remodeling of myofibrils: subcellular distribution of myosin heavy chain mRNA and protein.

Myofibril remodeling occurs during the normal life, in growth and development, and in the special case when isoform switching occurs. In this review we concentrate on the ultrastructural aspects of how myosin is incorporated into the A-band. Anatomic methods of study are emphasized that include isotope and immunochemical labeling as well as in situ hybridization. We conclude that the mechanism of remodeling is one of continual orderly exchange between a monomeric myosin pool and the thick filament. Myosin mRNA distribution is intermyofibrillar and nonsarcomeric, which suggests that newly translated myosin is released before diffusion and insertion in the A-band of the myofibrillar lattice.

Repair of injured skeletal muscle

We review cellular and molecular processes involved in injury and repair of skeletal muscle with regard to the amount and location of damage produced. Discussion is based on advances made by use of newer techniques, including immunochemistry, in situ hybridization, molecular biology, ultrastructural analysis, and cell culture. Damage and repair processes after eccentric work, stretch, overload, chronic stimulation, cold injury, and other models are discussed for cellular and molecular components. Hypertrophy or hyperplasia can occur under certain conditions. After injury, satellite cells are activated by growth factors. These cells can also be activated during fiber-type transformation, probably to provide necessary DNA content rather than to supply cells of a new lineage. Emphasis is given to myosin mRNA studied by in situ hybridization to localize subcellular distribution. Increases in mRNA concentration are found near nuclei in damaged regions and at the subcellular sites being repaired in the middle of skeletal muscle fibers or near the myotendon junction. The activation of genes for muscle regulatory factors during development is compared with their activation in regeneration and response to injury.