Stimulation of slow skeletal muscle fiber gene expression by calcineurin in vivo

Adult skeletal muscle fibers can be categorized into fast and slow twitch subtypes based on specialized contractile and metabolic properties and on distinctive patterns of muscle gene expression. Muscle fiber-type characteristics are dependent on the frequency of motor nerve stimulation and are thought to be controlled by calcium-dependent signaling. The calcium, calmodulin-dependent protein phosphatase, calcineurin, stimulates slow fiber-specific gene promoters in cultured skeletal muscle cells, and the calcineurin inhibitor, cyclosporin A, inhibits slow fiber gene expression in vivo, suggesting a key role of calcineurin in activation of the slow muscle fiber phenotype. Calcineurin has also been shown to induce hypertrophy of cardiac muscle and to mediate the hypertrophic effects of insulin-like growth factor-1 on skeletal myocytes in vitro. To determine whether activated calcineurin was sufficient to induce slow fiber gene expression and hypertrophy in adult skeletal muscle in vivo, we created transgenic mice that expressed activated calcineurin under control of the muscle creatine kinase enhancer. These mice exhibited an increase in slow muscle fibers, but no evidence for skeletal muscle hypertrophy. These results demonstrate that calcineurin activation is sufficient to induce the slow fiber gene regulatory program in vivo and suggest that additional signals are required for skeletal muscle hypertrophy.     

Development of ionic currents of zebrafish slow and fast skeletal muscle fibers

Voltage-gated Na+ and K+ channels play key roles in the excitability of skeletal muscle fibers. In this study we investigated the steady-state and kinetic properties of voltage-gated Na+ and K+ currents of slow and fast skeletal muscle fibers in zebrafish ranging in age from 1 day postfertilization (dpf) to 4-6 dpf. The inner white (fast) fibers possess an A-type inactivating K+ current that increases in peak current density and accelerates its rise and decay times during development. As the muscle matured, the V50s of activation and inactivation of the A-type current became more depolarized, and then hyperpolarized again in older animals. The activation kinetics of the delayed outward K+ current in red (slow) fibers accelerated within the first week of development. The tail currents of the outward K+ currents were too small to allow an accurate determination of the V50s of activation. Red fibers did not show any evidence of inward Na+ currents; however, white fibers expressed Na+ currents that increased their peak current density, accelerated their inactivation kinetics, and hyperpolarized their V50 of inactivation during development. The action potentials of white fibers exhibited significant changes in the threshold voltage and the half width. These findings indicate that there are significant differences in the ionic current profiles between the red and white fibers and that a number of changes occur in the steady-state and kinetic properties of Na+ and K+ currents of developing zebrafish skeletal muscle fibers, with the most dramatic changes occurring around the end of the first day following egg fertilization.     

Qualitatively different cross-bridge attachments in fast and slow muscle fiber types

Contractile properties differ between skeletal, cardiac and smooth muscles as well as between various skeletal muscle fiber types. This functional diversity is thought to be mainly related to different speeds of myosin head pulling cycles, with the molecular mechanism of force generation being essentially the same. In this study, force-generating attachments of myosin heads were investigated by applying small perturbations of myosin head pulling cycles in stepwise stretch experiments on skeletal muscle fibers of different type. Slow fibers (frog tonic and rat slow-twitch) exhibited only a ‘slow-type’ of myosin head attachment over the entire activation range, while fast fibers (frog and rat fast-twitch) displayed a ‘slow-type’ of myosin head attachment at low levels of activation, and an up to 30-times faster type at high levels of activation. These observations indicate that there are qualitative differences between the mechanisms of myosin head attachment in slow and fast vertebrate skeletal muscle fibers.     

Work overload induced changes in fast and slow skeletal muscle myosin heavy chain gene expression

Work induced hypertrophy of the slow postural soleus and the fast phasic plantaris muscles was produced by tenotomy of the synergistic gastrocnemius muscle. Increases in weight of both muscles were associated with proportionately even larger increases in total RNA and mRNA levels. Alterations in levels of specific myosin heavy chain (MHC) isoform mRNAs were measured using the slot blot procedure with radioactively labelled oligonucleotides as probes. Type 1 MHC gene expression was unaffected in both muscles by work overload, whereas type 2a was deinduced in the soleus and type 2b was deinduced in the plantaris. The neonatal MHC gene was transiently reinduced in the plantaris.     

Differential morphofunctional characteristics and gene expression in fast and slow muscle of rats with monocrotaline-induced heart failure

Heart failure (HF) is characterized by limited exercise tolerance, skeletal muscle atrophy, a shift toward fast muscle fiber, and myogenic regulatory factor (MRF) changes. Reactive oxygen species (ROS) also contribute to target organ damage in this syndrome. In this study, we investigated and compared morphofunctional characteristics and gene expression in Soleus (SOL--oxidative and slow twitching muscle) and in Extensor Digitorum Longus (EDL--glycolytic and fast twitching muscle) during HF. Two groups of rats were used: control (CT) and heart failure (HF), induced by a single injection of monocrotaline. MyoD and myogenin gene expression were determined by RT-qPCR, and MHC isoforms by SDS-PAGE; muscle fiber type frequency and cross sectional area (CSA) were analyzed by mATPase. A biochemical study was performed to determine lipid hydroperoxide (LH), glutathione peroxidase (GSH-Px), and superoxide dismutase (SOD); myography was used to determine amplitude, rise time, fall time, and fatigue resistance in both muscles. HF showed SOL and EDL muscle atrophy in all muscle fiber types; fiber frequency decreased in type IIC and muscle contraction fall time increased only in SOL muscle. Myogenin mRNA expression was lower in SOL and myoD decreased in HF EDL muscle. LH increased, and SOD and GSH-Px activity decreased only in HF SOL muscle. HF EDL muscle did not present changes in MHC distribution, contractile properties, HL concentration, and antioxidant enzyme activity. In conclusion, our results indicate that monocrotaline induced HF promoted more prominent biochemical, morphological and functional changes in SOL (oxidative and slow twitching muscle). Although further experiments are required to better determine the mechanisms involved in HF pathophysiology, our results contribute to understanding the muscle-specific changes that occur in this syndrome.     

hsp47 and hsp70 gene expression is differentially regulated in a stress- and tissue-specific manner in zebrafish embryos

We have examined differences in the spatial and temporal regulation of stress-induced hsp47 and hsp70 gene expression following exposure of zebrafish embryos to heat shock or ethanol. Using Northern blot analysis, we found that levels of hsp47 and hsp70 mRNA were dramatically elevated during heat shock in 2-day-old embryos. In contrast, ethanol exposure resulted in strong upregulation of the hsp47 gene whereas hsp70 mRNA levels increased only slightly following the same treatment. Whole-mount in situ hybridization analysis revealed that hsp47 mRNA was expressed predominantly in precartilagenous cells, as well as several other connective tissue cell populations within the embryo following exposure to either stress. hsp70 mRNA displayed a very different cell-specific distribution. For example, neither stress induced hsp70 mRNA accumulation in precartilagenous cells. However, high levels of hsp70 mRNA were detectable in epithelial cells of the developing epidermis following exposure to heat shock, but not to ethanol. These cells did not express the hsp47 gene following exposure to either of these stresses. The results suggest the presence of different inducible regulatory mechanisms for these genes which operate in a cell- and stress-specific manner in zebrafish embryos. Dev. Genet. 21:123–133, 1997. © 1997 Wiley-Liss, Inc.     

Repression of slow myosin heavy chain 2 gene expression in fast skeletal muscle fibers by muscarinic acetylcholine receptor and G(alpha)q signaling

Gene expression in skeletal muscle fibers is regulated by innervation and intrinsic fiber properties. To determine the mechanism of repression of slow MyHC2 expression in innervated fast pectoralis major (PM) fibers, we investigated the function of the muscarinic acetylcholine receptor (mAchR) and G(alpha)q. Both mAchR and G(alpha)q are abundant in medial adductor (MA) and PM fibers, and mAchR and G(alpha)q interact in these fibers. Whereas innervation of PM fibers was insufficient to induce slow MyHC2 expression, inhibition of mAchR activity with atropine in innervated PM fibers induced slow MyHC2 expression. Increased G(alpha)q activity repressed slow MyHC2 expression to nondetectable levels in innervated MA fibers. Reduced mAchR activity decreased PKC activity in PM fibers, and increased G(alpha)q activity increased PKC activity in PM and MA fibers. Decreased PKC activity in atropine-treated innervated PM fibers correlated with slow MyHC2 expression. These data suggest that slow MyHC2 repression in innervated fast PM fibers is mediated by cell signaling involving mAchRs, G(alpha)q, and PKC.     

Diverse effects of Stat1 on the regulation of hsp90alpha gene under heat shock

Stat1 has been known as a regulator of gene expression and a mediator of IFNgamma signaling in mammalian cells, while its effect in a heat shock response remains unclear. We used RNAi knockdown, point mutations, ChIP and promoter activity assays to study the effect of Stat1 on the heat-shock induction of the hsp90alpha gene under heat shock conditions. We found that Stat1 regulates the heat shock induction of its target genes, the hsp90alpha gene in a heat shock response while the constitutive activity of the gene remains unaffected. The result of Stat1 in complex with Stat3 and HSF1 that bound at the GAS to lead a moderate heat shock induction was designated as an "intrinsic" induction of the hsp90alpha gene. Additionally a reduced or an elevated level of heat shock induction was also controlled by the Stat1 on hsp90alpha. These diverse effects on the hsp90alpha gene were a "reduced" induction with over-expressed Stat1 elicited by transfection of wild-type Stat1 or IFNgamma treatment, bound at the GAS as homodimer; and an "enhanced" heat shock induction with a mutation-mediated prohibition of Stat1/GAS binding. In conclusion, the status and efficacy of Stat1 bound at the GAS of its target gene are pivotal in determining the impact of Stat1 under heat shock. The results provided the first evidence on the tumor suppressor Stat1 that it could play diverse roles on its target genes under heat shock that also shed lights on patients with fever or under thermotherapy.     

Analysis of the variation in the hsp70-1 and hsp90alpha mRNA expression in human myocardial tissue that has undergone surgical stress

In the present work we reported a semiquantitative detection of messenger ribonucleic acids (mRNAs) encoding the human heat shock proteins Hsp70-1, the stress inducible member of the HSP70 family, and hsp90alpha, the inducible member of the HSP90 family. We investigated the change in the expression of these mRNAs in tissue samples taken from the right atrium of 48 pediatric patients, soon after the ischemic period during surgery to correct congenital heart diseases, in which a crystalloid cold cardioplegic solution was used. No significant variations were found for either hsp70-1 or hsp90alpha expressions. Moreover, we searched for an association between the hsp70-1 promoter region polymorphism and the expression of the hsp70-1 in a smaller group of these patients (n = 27). The -110AA genotype was on average significantly associated with a decrease in the hsp70-1 mRNA level (P < 0.05), whereas the other genotypes -110AC or -110CC did not seem to be associated with the hsp70-1 expression level. The lack of any observed increase in the hsp70-1 expression level may be due to the high basal level of the Hsp70 protein in the tissues examined.     

Effect of hormonal and neurotrophic factors on the expression of fast myosin in slow muscle

Myosin ATPase and succinic dehydrogenase activity and fast myosin (indirect immunochemical test) were assayed in m. soleus of guinea-pigs after the administration of T4 (200 micrograms/kg) to animals every day for 3 weeks. This was followed by the application of 10 mM colchicine solution to sciatic nerve for 6 min. Fast muscle fibers and the line of precipitation with antiserum to fast myosin were revealed in soleus muscle of experimental animals after the application of colchicine and T4 injection.     

Genomic organization of hsp90 gene family in Arabidopsis

We have isolated six members of the hsp90 gene family from Arabidopsis thaliana. Three genes designated hsp81.2, 81.3 and 81.4 are clustered within a 15 kb genomic region while two of these are 1.5 kb apart in a head-to-head orientation. The deduced amino acid sequence shows that the members can be divided into two types. The hsp81.1, 81.2, 81.3 and 81.4 genes comprise the cytosolic hsp90 type having few introns. However, the hsp88.1 and 89.1 genes comprising the organelle type are composed of 18 or 19 introns. Sequence comparison showed there is high homology among the cytosolic members while there is less homology among the organelle members. The expression of the hsp90 genes and mRNA accumulation in plants and calli is very low at control temperatures and is strongly induced by heat-shock. Arsenite stress strongly stimulates the expression of this gene family.     

Evaluation of stress-inducible hsp90 gene expression as a potential molecular biomarker in Xenopus laevis

In this study we have evaluated stress-inducible hsp90 mRNA accumulation as a potential molecular biomarker in Xenopus laevis. In order to obtain a probe for Northern blot analysis we employed a PCR-based approach using degenerate primers for the amplification and cloning of an hsp90 gene sequence from Xenopus laevis. The deduced amino acid sequence is 102 amino acids in length and exhibited the highest degree of identity with zebrafish and human hsp90 β genes. Futhermore, the putative intron and exon boundaries of this fragment are the same as hsp90 β in chicken, mouse and human, indicating that the fragment represents a Xenopus hsp90 β-like gene. Northern blot analyses revealed that this gene was constitutively expressed in cultured A6 cells. While heat shock and sodium arsenite exposure resulted in the increased accumulation of hsp90 mRNA in A6 cells, treatment with cadmium chloride and zinc chloride did not. Also, exposure of A6 cells to concurrent heat shock and sodium arsenite produced a mild synergistic response with respects to hsp90 mRNA levels in contrast to hsp70 mRNA levels which displayed a strong synergistic effect. Finally, hsp90 mRNA was detected consitutively throughout early embrogenesis but was heat-inducible only in late blastula and later stages of development. Given the normal abundance and limited stress-induced accumulation of hsp90 mRNA, it may not have a great deal of potential as a molecular biomarker compared to hsp70 and hsp30 mRNA. However, it may be useful in conjunction with other stress protein mRNAs to establish a set of biomarker profiles to characterize the cellular response to a stressful or toxic agent.     

Skeletal muscle HIF-1alpha expression is dependent on muscle fiber type

Oxygen homeostasis is an essential regulation system for cell energy production and survival. The oxygen-sensitive subunit alpha of the hypoxia inducible factor-1 (HIF-1) complex is a key protein of this system. In this work, we analyzed mouse and rat HIF-1alpha protein and mRNA expression in parallel to energetic metabolism variations within skeletal muscle. Two physiological situations were studied using HIF-1alpha-specific Western blotting and semiquantitative RT-PCR. First, we compared HIF-1alpha expression between the predominantly oxidative soleus muscle and three predominantly glycolytic muscles. Second, HIF-1alpha expression was assessed in an energy metabolism switch model that was based on muscle disuse. These two in vivo situations were compared with the in vitro HIF-1alpha induction by CoCl(2) treatment on C(2)C(12) mouse muscle cells. HIF-1alpha mRNA and protein levels were found to be constitutively higher in the more glycolytic muscles compared with the more oxidative muscles. Our results gave rise to the hypothesis that the oxygen homeostasis regulation system depends on the fiber type.