Myosin Heavy Chain mRNA Research Articles

Dynamics of myosin degradation in intensive care unit-acquired weakness during severe critical illness

Intensive care unit (ICU)-acquired muscle wasting is a devastating complication leading to persistent weakness and functional disability. The mechanisms of this myopathy are unclear, but a disturbed balance of myosin heavy chain (MyHC) is implicated. To investigate pathways of myosin turnover in severe critically ill patients at high risk of ICU-acquired weakness. Prospective, mechanistic, observational study. Interdisciplinary ICUs of a university hospital. Twenty-nine patients with Sequential Organ Failure Assessment (SOFA) scores of at least 8 on three consecutive days within the first 5 days in ICU underwent two consecutive open skeletal muscle biopsies from the vastus lateralis at median days 5 and 15. Control biopsy specimens were from healthy subjects undergoing hip-replacement surgery. None. Time-dependent changes in myofiber architecture, MyHC synthesis, and degradation were determined and correlated with clinical data. ICU-acquired muscle wasting was characterized by early, disrupted myofiber ultrastructure followed by atrophy of slow- and fast-twitch myofibers at later time points. A rapid decrease in MyHC mRNA and protein expression occurred by day 5 and persisted at day 15 (P < 0.05). Expression of the atrophy genes MuRF-1 and Atrogin1 was increased at day 5 (P < 0.05). Early MuRF-1 protein content was closely associated with late myofiber atrophy and the severity of weakness. Decreased synthesis and increased degradation of MyHCs contribute to ICU-acquired muscle wasting. The rates and time frames suggest that pathogenesis of muscle failure is initiated very early during critical illness. The persisting reduction of MyHC suggests that sustained treatment is required.

Evidence of changes to skeletal muscle contractile properties during the initiation of disease in the ageing guinea pig model of osteoarthritis

BackgroundOsteoarthritis (OA) is the most common joint disorder in the world and represents the leading cause of pain and disability in the elderly population. Advancing age remains the single greatest risk factor for OA. Several studies have characterised disease development in the guinea pig ageing model of OA in terms of its joint histopathology and inflammatory cytokine profile. However, the quadriceps muscle has yet to be studied in relation to age-related disease onset or early disease progression. Therefore, we examined whether the initiation of OA in the Dunkin Hartley guinea pig is associated with changes in the quadriceps skeletal muscle. Male Dunkin Hartley guinea pigs (N = 24) were group housed with free access to standard guinea pig chow and water. At 2, 3, 5 and 7 months of age, six animals were selected based on their proximity to the median weight of the cohort. OA severity was graded at each time point by the assessment of toluidine blue stained step coronal sections of the total knee joint. Serum CTX II was measured as a potential biomarker of OA severity. Myosin Heavy Chain (MHC) isoforms were determined by a validated real-time PCR assay. Oxidative and glycolytic potential was determined in quadriceps homogenates via the measurement of ICDH and LDH activity.ResultsInitiation of OA in the DH strain guinea pig occurred between 2 and 3 months of age and progressed until 7 months when the final analyses were conducted. Serum CTX II significantly decreased during this early period of OA initiation and levels were unrelated to the histopathological severity of knee OA at any of the time points assessed. MHC mRNA measurements revealed a significant elevation in MHC IIX mRNA (associated with fast-twitch skeletal muscle fibres) coincident with the initiation of OA at 3 months of age, with preliminary findings suggestive of a positive correlation to OA severity at this time point.ConclusionsThese preliminary findings suggest that disease initiation in the ageing guinea pig model of OA is not associated with overt quadriceps muscle atrophy but instead is coincident with altered expression of mRNAs associated with quadriceps skeletal muscle contractile properties (specifically fast-twitch MHC IIX).

Comparison of Myosin Heavy Chain mRNAs, Protein Isoforms and Fiber Type Proportions in the Rat Slow and Fast Muscles

We studied the expression of myosin heavy chain isoforms at mRNA and protein levels as well as fiber type composition in the fast extensor digitorum longus (EDL) and slow soleus (SOL) twitch muscles of adult inbred Lewis strain rats. Comparison of the results from Real Time RT-PCR, SDS-PAGE and fiber type analysis showed corresponding proportions of MyHC transcripts (MyHC-1, -2a, -2x/d, -2b), protein isoforms (MyHC-1, -2a, -2x/d, -2b) and fiber types (type 1, 2A, 2X/D, 2B) in both muscles. Furthermore, we found that slow MyHC-1 mRNA expression in the SOL was up to three orders higher than that of fast MyHC transcripts. This finding can explain the predominance of MyHC-1 isoform and fiber type 1 and the absence of pure 2X/D and 2B fibers in the SOL muscle. Based on our data presenting quantitative evidence of corresponding proportions between mRNA level, protein content and fiber type composition, we suggest that the Real Time RT-PCR technique can be used as a routine method for analysis of muscle composition changes and could be advantageous for the analysis of scant biological samples such as muscle biopsies in humans.

Abstract 199: Distinctive Roles of Linker Histone H1 Variants in the Hypertrophic Response of Cardiomyocytes

Heart failure results when cardiac output is insufficient to meet physiological requirements and is often preceded by development of cardiomyocyte hypertrophy. As cardiac myocytes respond to hypertrophic stresses they re-express developmentally important genes, normally senescent in the adult. The chromatin structural events underlying this “fetal gene program” are unknown. We previously showed by proteomics that histones, components of the chromatin protein functional unit, the nucleosome, are altered during hypertrophic and failing phases of pressure overload in mouse: linker histone variants H1.2 and H1.5 decreased in hypertrophied myocardium while H1.0 increased during the transition to failure. The linker histone H1 family influences higher order chromatin structure and gene expression, although the role of this family in the heart is unknown. To assess the role of linker histones in hypertrophy, neonatal rat ventricular cardiomyocytes (NRVMs) were transfected with siRNAs individually targeting six H1 variants. Loss of H1.3 and H1.4 respectively induced a significant 26.1% (76 of 90) and 13.5% (80 of 94) increase in cell size area (µm2). A role of H1 in the hypertrophic response is evidenced by its influence on myosin heavy chain (MHC) mRNA expression. Knock-down of individual H1 variants significantly altered the MHC isoform ratio: loss of H1.3 increased α-MHC levels 1.5 fold and decreased β-MHC 1.6 fold while H1.5 depletion decreased α-MHC 2.5 fold. Both H1.3 and H1.4 knock-down increased atrial natriuretic factor (ANF) 1.3 fold while H1.5 loss decreased ANF 6.2 fold shown by qRT-PCR. Treatment with hypertrophy-inducing agents Isoproterenol (1μM), Endothelin (2nM) or Phenylephrine (10μM), reduced H1 mRNA levels however with subtle effects on protein abundance. To evaluate whether H1 loss shifted NRVM nuclei from a predominantly heterochromatic toward euchromatic state favoring gene accessibility we examined distinct histone markers of chromatin states. Histone H1.5 knock-down significantly decreased H3K9Me3 levels, a silencing mark associated with heterochromatin, 1.7 fold. Therefore we conclude that variants package distinctive regions of the genome and that H1.3 and H1.4 controls genes involved in the hypertrophic response.

Porcine satellite cells are restricted to a phenotype resembling their muscle origin

Muscles in most domestic animals differ in function and growth potential based largely on muscle fiber type composition. Though much is known about satellite cells (SC), information is limited regarding how populations of SC differ with muscle fiber type, especially in pigs. Therefore, the objective of this study was to isolate and culture SC from red (RST) and white (WST) portions of the semitendinosus muscle of neonatal and adult pigs and determine their capacity to proliferate, differentiate, and express various myosin heavy chain (MyHC) isoforms in vitro. Porcine satellite cells were isolated from RST and WST muscles of 6-wk-old and adult (>6-mo-old) pigs and cultured under standard conditions. Muscle from neonatal pigs yielded nearly 10 times more (P < 0.001) presumptive satellite cells as those from adult pigs, with fusion percentages close to 60% for the former. The RST yielded more (P < 0.001) SC per gram muscle compared to WST, 8.1 ± 0.2 × 10(4) cells versus 6.7 ± 0.1 × 10(4) cells/gram muscle in young pigs, and 9.7 ± 0.4 × 10(3) cells versus 5.5 ± 0.4 × 10(3) cells/gram muscle in adult pigs, respectively. Likewise, satellite cells from RST proliferated faster (P < 0.001) than those from WST across both ages, as indicated by a shorter cell doubling time, 18.6 ± 0.8 h versus 21.3 ± 0.9 h in young pigs, and 23.2 ± 0.7 h versus 26.7 ± 0.9 h in adult pigs, respectively. As a result of shorter times to confluence, satellite cells from RST also formed myotubes earlier than those SC originating from WST. Once induced, however, SC from WST differentiated and fused faster (P < 0.05) as evidenced by fusion percentage within the first 24 h, 41.6% versus 34.3%, respectively; but reached similar ultimate fusion percentages similar to WST by 48 h. Over 90% of MyHC expressed in maximally fused SC cultures from both RST and WST was restricted to the embryonic isoform. Type IIX MyHC mRNA was not detected in any culture. Myotube cultures from RST expressed more (P < 0.01) Type I MyHC isoform mRNA than those from WST, whereas those cultures from WST expressed more (P < 0.05) Type II (including Types IIA and IIB) MyHC transcripts. These data show SC cultures from porcine fast and slow muscles express MyHC profiles largely reflective of their muscle of origin and suggest satellite cells are partially restricted to a particular muscle phenotype in which they are juxtapositioned. Understanding the molecular nature of these intrinsic control mechanisms may lead to improved strategies for augmenting meat animal growth or muscle regeneration.

Maternal l-arginine supplementation during early gestation affects foetal skeletal myogenesis in pigs

To study the impact of maternal l-arginine supplementation during early gestation on skeletal muscle formation in the offspring, gilts were fed 3kg of a standard diet (control group, n=10) or standard diet and additionally 26g/d l-arginine from d 14 to 28 of gestation (arginine group, n=10). The gilts were sacrificed at d 75 of gestation. From each litter the lightest, the heaviest, and one medium-weight foetus per sex were selected and three different muscle samples were collected. In the longissimus dorsi muscle of all foetuses (n=99) biochemical properties were assessed. In the same muscle of a subset of the lightest and heaviest female foetuses (n=28), mRNA expression of selected genes involved in myogenesis were measured. In all three muscles, myosin heavy chain (MyHC) expression analyses (n=99) were performed. The protein/DNA ratio tended to be lower (P=0.08) in the offspring of the arginine group. Treatment×sex interactions revealed lower creatine kinase activity (P<0.05) and protein concentration (P=0.07) in female's of the arginine group. The MYF5 mRNA expression was lower (P<0.01) in the lightest, whereas IGF2 and IGFBP5 expressions (P<0.001) were lower in the heaviest females of the arginine group. The proportion of MyHC mRNA expression revealed an increase in the embryonic isoform (P=0.05) and a decrease by tendency in the Slow/I and IIx isoforms (P<0.10) in response to arginine supplementation. The results suggest that arginine supplementation during early gestation stimulates myogenic proliferation and delays muscular differentiation at d 75 of gestation.

Antagonism role of adenosine in right ventricular hypertrophy induced by hypoxia in rat

Objective To investigate the antagonism role of adenosine and its agonists in hypoxia-induced right ventricular hypertrophy in rat. Methods Fifty-six Sprague-Dawley rats were randomly divided into 7 groups, including normoxia control group, hypoxia control group, hypoxia plus adenosine group, hypoxia plus adenosine A1 receptor agonist(CPA) group, hypoxia plus adenosine A2 receptor agonist(NECA) group, hypoxia plus CPA and adenosine A1 receptor antagonist(DPCPX) group, hypoxia plus NECA and adenosine A2b receptor antagonist(MRS1754) group.There were 8 rats in each group.Rats in all groups except normoxia group were exposed to hypoxia for 21 days(oxygen volume concentration of 95-105 mL/L). On the 7th day, agents were continuously delivered with micro osmotic pump for 14 days.At the end of the experiment, both right ventricular to left ventricular plus septum weight[RV/(LV+ S)] ratio and right ventricular to body weight(RV/BW) ratio were measured, and the expression of myosin heavy chain(β-MHC) mRNA and regulator of G protein signaling 4(RGS4) mRNA in the right ventricular myocardial tissues were detected by PCR method. Results 1.After chronic hypoxia for 21 days, the ratios of RV/(LV+ S) and RV/ BW in the hypoxia control group were significantly higher than those in the normoxia control group respectively(all P<0.001); the ratios of RV/(LV+ S) and RV/BW in the hypoxia plus adenosine group and hypoxia plus CPA group were significantly lower than those in the hypoxia control group(all P<0.05); the ratio of RV/(LV+ S) in the hypoxia plus NECA group was significantly lower than that in the hypoxia control group(P<0.05).2.The expression level of β-MHC mRNA in the hypoxia control group was higher than that in the normoxia control group(P<0.001), and the expression level of RGS4 mRNA in the hypoxia group was lower than that in the normoxia group(P<0.001); the expression levels of β-MHC mRNA in the hypoxia plus adenosine group, hypoxia plus CPA group, or hypoxia plus NECA group were lower than those in the hypoxia control group(all P<0.001); the expression levels of RGS4 mRNA in hypoxia plus adenosine group, hypoxia plus CPA group, and hypoxia plus NECA group were higher than those in the hypoxia control group(all P<0.001). Conclusions Adenosine can attenuate hypoxia-induced right ventricular hypertrophy through RGS4 mediated pathway. Key words: Adenosine; Hypoxia; Myocardial hypertrophy; Regulator of G protein signaling 4

Factors affecting the structure and maturation of human tissue engineered skeletal muscle

Tissue engineered skeletal muscle has great utility in experimental studies of physiology, clinical testing and its potential for transplantation to replace damaged tissue. Despite recent work in rodent tissue or cell lines, there is a paucity of literature concerned with the culture of human muscle derived cells (MDCs) in engineered constructs. Here we aimed to tissue engineer for the first time in the literature human skeletal muscle in self-assembling fibrin hydrogels and determine the effect of MDC seeding density and myogenic proportion on the structure and maturation of the constructs. Constructs seeded with 4 × 105 MDCs assembled to a greater extent than those at 1 × 105 or 2 × 105, and immunostaining revealed a higher fusion index and a higher density of myotubes within the constructs, showing greater structural semblance to in vivo tissue. These constructs primarily expressed perinatal and slow type I myosin heavy chain mRNA after 21 days in culture. In subsequent experiments MACS® technology was used to separate myogenic and non-myogenic cells from their heterogeneous parent population and these cells were seeded at varying myogenic (desmin +) proportions in fibrin based constructs. Only in the constructs seeded with 75% desmin + cells was there evidence of striations when immunostained for slow myosin heavy chain compared with constructs seeded with 10 or 50% desmin + cells. Overall, this work reveals the importance of cell number and myogenic proportions in tissue engineering human skeletal muscle with structural resemblance to in vivo tissue.

Developmental changes in diaphragm muscle function in the preterm and postnatal lamb

The preterm diaphragm is structurally and functionally immature, potentially contributing to an increased risk of respiratory distress and failure. We investigated developmental changes in contractile function and susceptibility to fatigue of the costal diaphragm in the fetal lamb to understand factors contributing to the risk of developing diaphragm dysfunction and respiratory disorders. We hypothesized that the functional capacity of the diaphragm will vary with maturational stage as will its susceptibility to fatigue. Lambs were studied at 75, 100, 125, 145, 154, 168, and 200 days postconceptional age (term = 147 days). Lambs were euthanized (sodium pentobarbitone, 100 mg/kg) either at delivery or immediately prior to post-mortem for postnatal lambs. Contractile function was assessed on longitudinal strips of intact muscle fibers and the remaining tissue frozen in liquid nitrogen for analysis of myosin heavy chain (MHC) mRNA expression and protein content. Fetal development of diaphragm function was characterized by a significant increase in maximum specific force, increased susceptibility to fatigue, reduced twitch contraction times, and a progressive increase in MHCI and MHCII protein content. Postnatally, there was a progressive decrease in the susceptibility to fatigue that coincided with an increase in the MHC I:II protein ratio. These data indicate that the functional capacity of the diaphragm varies with maturational age and may be an important determinant of the susceptibility to preterm respiratory failure.

Passive Repetitive Stretching for a Short Duration within a Week Increases Myogenic Regulatory Factors and Myosin Heavy Chain mRNA in Rats' Skeletal Muscles

Stretching is a stimulation of muscle growth. Stretching for hours or days has an effect on muscle hypertrophy. However, differences of continuous stretching and repetitive stretching to affect muscle growth are not well known. To clarify the difference of continuous and repetitive stretching within a short duration, we investigated the gene expression of muscle-related genes on stretched skeletal muscles. We used 8-week-old male Wistar rats (N = 28) for this study. Animals medial gastrocnemius muscle was stretched continuously or repetitively for 15 min daily and 4 times/week under anesthesia. After stretching, muscles were removed and total RNA was extracted. Then, reverse transcriptional quantitative real-time PCR was done to evaluate the mRNA expression of MyoD, myogenin, and embryonic myosin heavy chain (MyHC). Muscles, either stretched continuously or repetitively, increased mRNA expression of MyoD, myogenin, and embryonic MyHC more than unstretched muscles. Notably, repetitive stretching resulted in more substantial effects on embryonic MyHC gene expression than continuous stretching. In conclusion, passive stretching for a short duration within a week is effective in increasing myogenic factor expression, and repetitive stretching had more effects than continuous stretching for skeletal muscle on muscle growth. These findings are applicable in clinical muscle-strengthening therapy.

Age-related changes and nutritional regulation of myosin heavy-chain composition in longissimus dorsi of commercial pigs

The objective of this study is to investigate the age-related changes of and the effects of dietary conjugated linoleic acid (CLA) on muscle-fibre types in commercial pigs. We divided 25 crossbred male pigs into five age groups (7, 30, 60, 100 and 180 days) and 30 finishing pigs into two dietary groups (one fed a CLA-enriched diet and the other fed a control diet for 30 days). We analysed the composition (%) of myosin heavy-chain (MyHC) mRNA according to the absolute copies of each MyHC (I, IIa, IIb and IIx) mRNA, and the activities of succinate dehydrogenase (SDH) and malate dehydrogenase (MDH) in the longissimus muscle. From days 7 to 180, the MyHC I mRNA abundance and SDH and MDH activities presented a decreasing trend, the MyHC IIb mRNA abundance presented a steady trend and the MyHC IIa and IIx mRNA abundances presented an increasing trend. On day 30, MyHC I and IIb mRNA abundances were at their lowest (P < 0.05), and the MyHC IIa and IIx mRNA abundances were at their highest (P < 0.05). In the CLA group, the MyHC I mRNA abundance and the activities of SDH and MDH were improved in the longissimus muscle, whereas pressure loss, drip loss and average back fat depth significantly decreased (P < 0.01) and shear force significantly increased (P < 0.01). Loin eye area, feed conversion rate and meat colour showed some tendency to be improved. These results indicated that more oxidative fibres might convert to glycolytic fibres with increasing age or weight, and that the early developmental stage might be a key stage for this conversion. During the finishing stage, the proportion of oxidative fibres might be increased by dietary CLA supplementation, which may contribute to the water-holding capacity of meat. The results would provide an important basis for the application of muscle-fibre types in the improvement of pork quality.

Age‐related changes in rat genioglossus, geniohyoid and masseter muscles

The aim of this study was to elucidate age-related changes from adult to middle age in the contractile properties of the masseter, genioglossus and geniohyoid muscles of the rat. We analysed the expressions of myosin heavy chain (MyHC) mRNAs and proteins as indicators of the contractile properties in these muscles obtained from rats at 6, 12, 18 and 24months of age using real-time PCR and SDS-PAGE. We found no marked age-related changes in the expressions of MyHC mRNAs and proteins in rat masseter and geniohyoid muscles, suggesting that the biological ageing process does not affect contractile properties in these muscles. However, we found a decrease in the expression of MyHC IIb mRNA with ageing in the rat genioglossus muscle, suggesting that biological ageing process induces at least some fast-to-slow myofibre phenotype transition. The biological ageing process from adult to middle age appears to differentially affect different types of craniofacial muscles.

Reduced Expression of Androgen Receptor and Myosin Heavy Chain mRNA in Cremaster Muscle of Boys with Nonsyndromic Cryptorchidism

To better define the developmental mechanisms of nonsyndromic cryptorchidism, we measured the expression of hormone receptor and muscle type specific mRNAs in target tissues of boys with and those without nonsyndromic cryptorchidism. Prospectively collected cremaster muscle and/or hernia sac tissues from boys with congenital (79) or acquired (66) nonsyndromic cryptorchidism and hernia/hydrocele (controls, 84) were analyzed for hormone receptor (RXFP2, AR, ESR1, ESR2) and myosin heavy chain specific (MYH1, MYH2, MYH7) mRNA expression using real-time reverse transcriptase polymerase chain reaction. Log transformed mRNA, phenotype and feeding history data were statistically analyzed using Pearson's correlation, ANOVA and 2-sample t tests. AR mRNA expression was higher in cremaster muscle than in sac tissue, and significantly lower in congenital and acquired nonsyndromic cryptorchidism cases vs controls (p <0.01). Type 1 (slow/cardiac) MYH7 mRNA expression was also significantly reduced in both nonsyndromic cryptorchidism groups (p ≤ 0.002), while a reduction in type 2 (fast) MYH2 expression was more modest and significant only for the congenital cryptorchidism group (p <0.05). Cremasteric MYH7 and AR levels were strongly correlated (r(2) = 0.751, p <0.001). MYH7 and ESR1 mRNA levels were higher and lower, respectively, in boys with nonsyndromic cryptorchidism who were fed soy formula. Expression of other genes was not measurable. Our data suggest that boys with congenital and acquired nonsyndromic cryptorchidism differentially express AR and slow twitch specific MYH7 mRNA in the cremaster muscle, and that MYH7 expression is correlated with AR levels and soy formula use. These differences in gene expression may reflect aberrant hormonal signaling and/or innervation during development with the potential for secondary functional effects and failed testicular descent.

Histological changes and changes in the myosin mRNA content of the porcine masticatory muscles after masseter treatment with botulinum toxin A

Botulinum toxin A (Botox) is increasingly used for treatment of muscle hyperfunction. For a better understanding of the possible morphologic and chewing changes in patients induced by a therapy with Botox, muscle fiber and myosin heavy chain (MyHC) mRNA alterations were examined in this animal study. The investigation was carried out on 14-week-old pigs (seven treated animals, eight controls; calculated animal size with a power of 0.5). To initialise the total immobilisation of the right masseter, the Botox injection was distributed into ten areas. After a 56-day period, muscle tissue was taken from the left and right side of the masseter (three regions), temporal (two regions), medial pterygoid and geniohyoid muscles using a standardized method. The muscle fiber cross sections were examined immunohistochemically. Fiber staining was accomplished with antibodies to specific MyHC isoforms. The MyHC mRNA changes were analysed using real-time RT-PCR. Muscles adapt to such stress by changing fiber types and MyHC mRNA content. Paralysed masseters display atrophic changes while other masticatory muscles show hypertrophic changes. The results indicated that the typical distributions of type IIa und IIb fiber types in masticatory muscles were increased in the masseter muscles due to Botox application. On the other hand, the masseters without Botox in the treated group showed a significant increase of type I MyHC. Application of Botox may lead to uncontrolled structural changes in affected and unaffected muscles. Treatment of muscle hypertrophy with Botox may cause muscle imbalance.

Administration of estradiol, trenbolone acetate, and trenbolone acetate/estradiol implants alters adipogenic and myogenic gene expression in bovine skeletal muscle1

Twenty crossbred yearling steers (421 kg) were used to evaluate the effects of implanting with trenbolone acetate (TBA; 120 mg), estradiol-17β (E(2); 25.7 mg), and a combination (120 mg of TBA and 24 mg of E(2)) on adipogenic and myogenic mRNA concentrations. Animals were blocked by BW and within each block were assigned to 1 of 4 treatments. Animals were housed and fed in individual pens with 5 animals per treatment. All animals were weighed weekly, and muscle biopsy samples were taken from the LM of each steer on d 0 (before implantation), 7, 14, and 28. Total RNA was isolated from each sample and real-time quantitative PCR was used to measure the quantity of C/EBPβ, PPARγ, stearoyl CoA desaturase (SCD), myogenin, and 3 isoforms of bovine myosin heavy chain (MHC) mRNA. Total BW gain from the 28-d period was adjusted to d 0 by use of covariant analysis, and steers in the implant groups tended (P = 0.09) to have increased BW gain compared with nonimplanted control steers. Analysis of the gene expression of MHC showed that neither implant nor day (P > 0.20) had a significant effect on the expression of type I or IIX MHC mRNA There was also no treatment effect (P > 0.20) on MHC-IIA and myogenin, but increasing days on feed increased (P = 0.05) the expression of MHC-IIA mRNA. Relative mRNA abundance of C/EBPβ, PPARγ, and SCD increased (P < 0.05) during days of feed but PPARγ decreased (P < 0.05) with the treatment of combined TBA/E(2) implant. Results of this study indicate that implanting with TBA, E(2), or both increased BW gain and decreased adipogenic gene expression of finishing steers without significantly affecting the concentration of type I, IIA, or IIX MHC mRNA. Increasing days on feed increased both MHC-IIA and adipogenic gene expression in bovine skeletal muscle biopsy samples. We conclude that administration of steroidal implants had no effect on the proportion of the 3 MHC mRNA isoforms but decreased C/EBPβ, PPARγ, and SCD mRNA in bovine skeletal muscle.

Administration of estradiol, trenbolone acetate, and trenbolone acetate/estradiol implants alters adipogenic and myogenic gene expression in bovine skeletal muscle

Twenty crossbred yearling steers (421 kg) were used to evaluate the effects of implanting with trenbolone acetate (TBA; 120 mg), estradiol-17β (E(2); 25.7 mg), and a combination (120 mg of TBA and 24 mg of E(2)) on adipogenic and myogenic mRNA concentrations. Animals were blocked by BW and within each block were assigned to 1 of 4 treatments. Animals were housed and fed in individual pens with 5 animals per treatment. All animals were weighed weekly, and muscle biopsy samples were taken from the LM of each steer on d 0 (before implantation), 7, 14, and 28. Total RNA was isolated from each sample and real-time quantitative PCR was used to measure the quantity of C/EBPβ, PPARγ, stearoyl CoA desaturase (SCD), myogenin, and 3 isoforms of bovine myosin heavy chain (MHC) mRNA. Total BW gain from the 28-d period was adjusted to d 0 by use of covariant analysis, and steers in the implant groups tended (P = 0.09) to have increased BW gain compared with nonimplanted control steers. Analysis of the gene expression of MHC showed that neither implant nor day (P > 0.20) had a significant effect on the expression of type I or IIX MHC mRNA There was also no treatment effect (P > 0.20) on MHC-IIA and myogenin, but increasing days on feed increased (P = 0.05) the expression of MHC-IIA mRNA. Relative mRNA abundance of C/EBPβ, PPARγ, and SCD increased (P < 0.05) during days of feed but PPARγ decreased (P < 0.05) with the treatment of combined TBA/E(2) implant. Results of this study indicate that implanting with TBA, E(2), or both increased BW gain and decreased adipogenic gene expression of finishing steers without significantly affecting the concentration of type I, IIA, or IIX MHC mRNA. Increasing days on feed increased both MHC-IIA and adipogenic gene expression in bovine skeletal muscle biopsy samples. We conclude that administration of steroidal implants had no effect on the proportion of the 3 MHC mRNA isoforms but decreased C/EBPβ, PPARγ, and SCD mRNA in bovine skeletal muscle.

Influences of fentanyl and sufentanil on the levothyroxine sodium preconditioning-induced cardioprotection in rats

Objective To evaluate influences of fentanyl and sufentauil on the cardioprotection effect of preemptive levothyroxine-sodium administration in the rats. Methods Immature rats were randomly allocated into seven groups (n=8):blank control group (group BC),control group (group C),levothyroxine-sodium 10 μg group(group 10 μg),fentanyl group (group F),sufentanil group (group S),combined fentanyl and levothyroxine-sodium group (group F+L) and combined sufentanil and levothyroxine-sodium group(group S+L).The isolated heart model was established with a Langendorff apparatus.Hearts in the groups F and F+L were perfused with KH fluid containing fentanyl 30 μg/L,and hearts in the groups S and S+L were perfused with KH fluid containing sufentanil 3 μg/L. Results The hemodynamic variables at 30 min of the reperfusion were inferior in group F and S than in group BC,but better in group 10 μg,group F+L and group S+L than in group C,F and S.The coronary perfusion flows in group BC,group C,group 10 μg,group F,group S,group F+L and group S+L were (19±2),(16±1),(21±2),(17±1),(17±1),(21±1),(22±1) ml/min,respectively,with a significantly higher coronary perfusion flow in group 10 μg,group F+L and group S+L than in group C,group F and group S.The myocardial bound creatine kinase (CK-MB) level in the coronary perfusion fluid during the reperfusion period was significandy higher in groups C,F and S compared with groups 10 μg,F+L and S+L.The expression of b oth heat shock proteins70(HSP70) and myosin heavy-chain(MHC) mRNA in the myocardium was significantly higher in groups 10 μg,F+L and S+L than in groups C,F and S.However,there were no differences in all the observed parameters anong groups 10 μg,F+L and S +L. Conclusions Both fentanyl and sufentanil do not produce significant influence on the cardioprotective effect of levothyroxine sodium preconditioning. Key words: Fentanyl; Sufentanil; Preconditioning; Thyroid hormones; Cardioprotection

The presence and regulation of antisense long non‐coding RNA with altered myosin expression in exercising human muscle

Human skeletal muscle responds to exercise stimuli with alterations to myosin heavy chain (MHC) gene expression, and thus, contractile function. We investigated the transcriptional regulation of the MHC genes in greater detail than previously achieved in humans by quantifying mRNA, pre‐mRNA, and antisense long non‐coding (as‐lnc) RNA of MHC in vastus lateralis. Muscle biopsies were obtained before and after 5 wks of aerobic + resistance exercise (12 M, 9 F). Extracted RNA was DNase treated and analyzed by RT‐PCR. A new competitive PCR method enabled us to quantify individual human MHC mRNAs as a percent of total MHC. The proportions of Type I and IIa MHC mRNA were both increased by 11%, while IIx was decreased by 38% after exercise training (all p &lt; .05). As previously shown only in rats, lncRNA oriented antisense to the IIa MHC was detected and found to be down regulated with exercise (−55%; p &lt; .05). A regulatory role is inferred from an inverse correlation between the proportion of IIa MHC mRNA and its antisense pair (r = −0.66; p = .002). Surprisingly we detected low levels of IIb mRNA and pre‐mRNA, indicating that the IIb gene is not transcriptionally silent in human limb muscle, as thought. We also report the novel finding of as‐lncRNA to the IIb gene. These data reveal that insights to MHC regulatory mechanisms previously shown in rats and involving as‐lncRNA are conserved in humans. (Support NSBRI‐NASA NCC 9–58 &amp; NIH T32 AR047752)

The Nuclear Receptor, Nor-1, Markedly Increases Type II Oxidative Muscle Fibers and Resistance to Fatigue

Nuclear hormone receptors (NR) have been implicated as regulators of lipid and carbohydrate metabolism. The orphan NR4A subgroup has emerged as regulators of metabolic function. Targeted silencing of neuron-derived orphan receptor 1 (Nor-1)/NR4A3 in skeletal muscle cells suggested that this NR was necessary for oxidative metabolism in vitro. To investigate the in vivo role of Nor-1, we have developed a mouse model with preferential expression of activated Nor-1 in skeletal muscle. In skeletal muscle, this resulted in a marked increase in: 1) myoglobin expression, 2) mitochondrial DNA and density, 3) oxidative enzyme staining, and 4) genes/proteins encoding subunits of electron transport chain complexes. This was associated with significantly increased type IIA and IIX myosin heavy chain mRNA and proteins and decreased type IIB myosin heavy chain mRNA and protein. The contractile protein/fiber type remodeling driving the acquisition of the oxidative type II phenotype was associated with 1) the significantly increased expression of myocyte-specific enhancer factor 2C, and phospho-histone deacetylase 5, and 2) predominantly cytoplasmic HDAC5 staining in the Tg-Nor-1 mice. Moreover, the Nor-1 transgenic line displayed significant improvements in glucose tolerance, oxygen consumption, and running endurance (in the absence of increased insulin sensitivity), consistent with increased oxidative capacity of skeletal muscle. We conclude that skeletal muscle fiber type is not only regulated by exercise-sensitive calcineurin-induced signaling cascade but also by NR signaling pathways that operate at the nexus that coordinates muscle performance and metabolic capacity in this major mass tissue.

Muscle-fibre types in porcine longissimus muscle of different genotypes and their association with the status of energy metabolism

To study the difference in muscle-fibre types in porcine muscle among different genotypes and its association with energy metabolism, composition of myosin heavy chain (MyHC) mRNA and energy metabolism indices were determined in the longissimus muscle (LM). Pig breeds included Jinhua (JHP), Zhongbai (ZBP), Duroc × Zhongbai cross (DZP) and Duroc × Yorkshire × Landrace cross (DYL). JHP pigs were found to have the highest proportions of MyHC I, IIa and IIx mRNA (P &lt; 0.05), creatine kinase (CK) activity (P &lt; 0.05) and the lowest glycolytic potential (GP) compared with the other genotypes. The proportions of MyHC I and IIa mRNA increased in the order of DYL &lt; DZP &lt; ZBP &lt; JHP, whereas the trend was opposite for MyHC IIb mRNA. The proportions of MyHC I, IIa and IIx mRNA were positively correlated with CK activity and the turnover ratio of creatine phosphate (CP) (P &lt; 0.01), and negatively correlated with GP, glucose-6-phosphate (G-6-P) and lactate (LA) contents (P &lt; 0.01), with the trends being opposite for MyHC IIb mRNA. The results indicate that muscle-fibre type in porcine LM is influenced by the genetic background of pigs. For example, JHP pigs had more of Types I, IIa and IIx fibres than did other genotypes. Proportions of Types I, IIa and IIx fibres were positively correlated with CK reaction (ATP-CP) capacity and negatively correlated with GP. These data provide some evidence for exploring the effective mechanism of muscle-fibre type on pork quality.