Synthesis Rate Of Myosin Heavy Chain Research Articles

Oxandrolone enhances skeletal muscle myosin synthesis and alters global gene expression profile in Duchenne muscular dystrophy

Earlier studies have shown that the progressive, unrelenting muscle loss associated with Duchenne muscular dystrophy (DMD) involves an imbalance between the rates of synthesis and degradation of muscle proteins. Although previous studies have suggested that oxandrolone may be beneficial in DMD, the mechanism of action of oxandrolone on muscle in DMD remains unclear. To address these issues, we combined stable isotope studies and gene expression analysis to measure the fractional synthesis rate of myosin heavy chain (MHC), the key muscle contractile protein, the transcript levels of the isoforms of MHC, and global gene expression profiles in four children with DMD before and after 3 mo of treatment with oxandrolone. Gastrocnemius muscle biopsies and blood samples were collected during the course of a primed 6-h continuous infusion of l-[U-(13)C]leucine on two separate occasions, before and after the 3-mo treatment with oxandrolone (0.1 mg.kg(-1).day(-1)). Gene expression analysis was done with microarrays and RT-qPCR. In response to the treatment, MHC synthesis rate increased 42%, and this rise was accounted for, at least in part, by an upregulation of the transcript for MHC8 (perinatal MHC). Gene expression data suggested a decrease in muscle regeneration as a consequence of oxandrolone therapy, presumably because of a decrease in muscle degeneration. These findings suggest that 1) oxandrolone has a powerful protein anabolic effect on a key contractile protein and 2) larger and longer-term studies are warranted to determine whether these changes translate into meaningful therapy for these patients.

The effect of mechanical loading on the MyHC synthesis rate and composition in rat plantaris muscle.

The aim of this study was to investigate the response of protein synthesis rate, particularly myosin heavy chain (MyHC) isoforms synthesis and the magnitude of its isoform transformation in fast-twitch plantaris muscle, to different modes of prolonged mechanical loading. Different protocols of mechanical loading were used: resistance training (RT), compensatory hypertrophy (CH) of m. plantaris after tenotomy of m. gastrocnemius and a combination of the two previous loadings (RT + CH). During the different modes of loading, plantaris muscle hypertrophy in RT group was approximately 10 %, CH approximately 40 % and CH + RT approximately 44 %. MyHC I and IID isoform synthesis rate increased in all experimental groups, as well as their relative content. MyHC IIA relative content decreased during RT and RT + CH and increased during CH. MHC IIB isoform relative content decreased in all experimental groups, but compared with CH in CH + RT MyHC IIB isoform content increased in plantaris muscle. These results demonstrate that different modes of mechanical loading resulted in the selective up- and down-regulation of MyHC isoforms in fast-twitch skeletal muscle. The synthesis rate and relative content of the two fastest isoforms of MyHC IIB and IID are regulated to different directions during mechanical loading.

Muscle protein metabolism and the sarcopenia of aging.

Loss of muscle mass, strength, and oxidative capacity accompanies normal aging in humans. The mechanisms responsible for these changes remain to be clearly defined. Muscle protein mass and function depend on protein turnover. Synthesis rate of the major muscle contractile protein, myosin heavy chain (MHC), and transcript levels of fast MHC isoforms decrease in association with strength reductions, while mitochondrial protein synthesis rate declines in parallel with activities of mitochondrial enzymes and maximal oxidative capacity (VO2max). Resistance exercise training increases the synthesis rate of MHC and transcript levels of the slow MHC isoform in older humans, along with increasing muscle strength. The relationship between the synthesis of muscle proteins, and muscle size and function, with aging and exercise training are discussed in this review.

Isolation of human skeletal muscle myosin heavy chain and actin for measurement of fractional synthesis rates.

Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have developed a simple method to isolate myosin heavy chain (MHC) and actin from small (60-80 mg) human skeletal muscle samples for the determination of their fractional synthesis rates. The amounts of MHC and actin isolated are adequate for the quantification of [13C]leucine abundance by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Fractional synthesis rates of mixed muscle protein (MMP), MHC, and actin were determined in six healthy young subjects (27 +/- 1 yr) after they received a 14-h intravenous infusion (prime = 7.58 micromol/kg body wt, constant infusion = 7.58 micromol. kg body wt-1. h-1) of [1-13C]leucine. The fractional synthesis rates of MMP, MHC, and actin were found to be 0.0468 +/- 0.0048, 0.0376 +/- 0. 0033, and 0.0754 +/- 0.0078%/h, respectively. Overall, the synthesis rate of MHC was 20% lower (P = 0.012), and the synthesis rate of actin was 61% higher (P = 0.060, not significant) than the MMP synthesis rate. The isolation of these proteins for isotope abundance analysis by GC-C-IRMS provides important information about the synthesis rates of these specific contractile proteins, as opposed to the more general information provided by the determination of MMP synthesis rates.

Beta-adrenergic receptor gene expression in bovine skeletal muscle cells in culture.

Beta-adrenergic receptors (betaAR) are abundant in fetal, neonatal, and adult skeletal muscles of cattle; however, only minimal levels of functional betaAR were detected in multinucleated muscle cell cultures prepared from 90- to 150-d fetal bovine skeletal muscle. Two other lines of evidence were consistent with low levels of betaAR expression in bovine muscle cultures. First, treating the cells with 10(-6)M isoproterenol for up to 20 min did not increase intracellular cAMP concentration. Second, neither the quantity of myosin heavy chain (MHC) nor its apparent synthesis rate were changed by treating the cells for 4 d with 10(-7) or 10(-6) M isoproterenol. Despite these results, the mRNA for the beta2AR could be detected in muscle cultures by PCR and on slot blots. Thus, the beta2AR mRNA was expressed, but significant levels of functional receptors could not be detected. Glucocorticoids are known to activate expression of OAR genes in several tissues, and the effect of dexamethasone on OAR gene expression in bovine multinucleated muscle cell cultures was evaluated. The intracellular concentration of cAMP following treatment with isoproterenol was elevated 10-fold by dexamethasone, and the population of functional receptors was elevated by approximately 50%. The effect of dexamethasone on muscle protein synthesis and accumulation was analyzed after pretreating the cells with dexamethasone for 24 h, followed by treatment with dexamethasone and 10(-6)M isoproterenol for an additional 48 h. The quantity of MHC synthesized and the apparent synthesis rate of MHC were stimulated by 10 to 35%. These effects seem to be due to posttranscriptional events, because the quantity of beta2AR receptor mRNA on slot blots was not increased by treatment with dexamethasone. Results of this study emphasize the importance of verifying that muscle cells contain functional betaAR when they are used to study the effects of betaAR agonists on muscle protein metabolism.

Myosin heavy chain synthesis is increased in a rabbit model of heart failure.

Chronic ventricular tachycardia (chronic VT) causes left ventricular (LV) dysfunction and is associated with increased LV wall stress and neurohormonal activation, but no LV hypertrophy. The mechanisms responsible for the lack of myocardial growth with chronic VT are unknown. Accordingly, this study examined contractile protein [myosin heavy chain (MHC)] synthesis in a rabbit model of chronic VT. MHC mRNA levels, protein concentration, and synthesis rates were examined in control rabbits (n = 18) and in rabbits with chronic VT (400 beats/min, 3 wk, n = 18). With chronic VT, LV end-diastolic volume increased (8.2 +/- 0.8 vs. 5.3 +/- 0.6 ml, P < 0.05), ejection fraction decreased (12 +/- 3 vs. 38 +/- 4%, P < 0.05) and peak systolic wall stress increased (963 +/- 93 vs. 262 +/- 42 g/cm2, P < 0.05). Plasma catecholamine and endothelin levels also increased threefold, and renin activity increased twofold. Despite these stimuli for hypertrophy, LV mass-to-body weight ratio was unchanged (1.15 +/- 0.07 vs. 1.25 +/- 0.05 g/kg). At the myocyte level, chronic VT caused myocyte lengthening (159.6 +/- 1.8 vs. 121.6 +/- 1.4 microm, P < 0.05), but a reduction in myocyte cross-sectional area (199 +/- 6 vs. 249 +/- 7 microm2, P < 0.0001), as well as a reduced velocity of shortening (42.6 +/- 1.6 vs. 74.1 +/- 2.8 microm/s, P < 0.05). Chronic VT resulted in a significant increase in the rate of MHC synthesis, but paradoxically, there was no change in LV MHC content. Despite increased MHC synthesis, relative levels of MHC mRNA were not increased in chronic VT (2.79 +/- 0.23 vs. 2.44 +/- 0.20 AU, relative to glyceraldehyde-3-phosphate dehydrogenase), suggesting an increase in MHC translational efficiency. These unique findings suggest accelerated degradative processes must contribute to the failure of myocardial growth in this model of LV dysfunction in which increased LV wall stress, neurohormonal activation, and increased protein synthesis occurred.

Skeletal muscle myosin heavy-chain synthesis rate in healthy humans.

Mixed muscle protein synthetic rate has been measured in humans. These measurements represent the average of synthetic rates of all muscle proteins with variable rates. We determined to what extent the synthesis rate of mixed muscle protein in humans reflects that of myosin heavy chain (MHC), the main contractile protein responsible for the conversion of ATP to mechanical energy as muscle contraction. Fractional synthetic rates of MHC and mixed muscle protein were measured from the increment of [13C]leucine in these proteins in vastus lateralis biopsy samples taken at 5 and 10 h during a primed continuous infusion of L-[1-13C]leucine in 10 young healthy subjects. Calculations were done by use of plasma [13C]ketoisocaproate (KIC) and muscle tissue fluid [13C]leucine as surrogate measures of leucyl-tRNA. Fractional synthetic rate of MHC with plasma KIC (0.0299 +/- 0.0043%/h) and tissue fluid leucine (0.0443 +/- 0.0056%/h) were only 72 +/- 3% of that of mixed muscle protein (0.0408 +/- 0.0032 and 0.0603 +/- 0.0059%/h, respectively, with KIC and tissue fluid leucine). Contribution of MHC (7 +/- 1 mg.kg-1.h-1) to synthetic rates of whole body mixed muscle protein (36 +/- 5 mg.kg-1.h-1) and whole body protein (127 +/- 4 mg.kg-1.h-1) is only 18 +/- 1 and 5 +/- 1%, respectively. This relatively low contribution of MHC to whole body and mixed muscle protein synthesis warrants direct measurement of synthesis rate of MHC in conditions involving abnormalities of muscle contractile function.

Muscle protein turnover: methodological issues and the effect of aging.

Investigation of sarcopenia should include the measurement of muscle protein synthesis and breakdown because reduced muscle mass occurs only when muscle protein content is reduced. Protein content of a tissue is determined by the balance between synthesis and breakdown. Whole body measurements are inappropriate for determining the small changes in muscle protein synthesis and breakdown, since muscle contributes less than 30 percent to the whole body protein turnover. The measurement of mixed muscle protein synthesis in humans supports the animal data, which demonstrates that there is a decline in muscle protein synthesis with age. Resistance exercise stimulates mixed muscle protein synthesis rate in both the young and the old, although this is not demonstrated when myofibrillar protein synthesis is measured. Muscle is composed of several types of proteins, and their turnover may be differentially regulated. It is important, therefore, to measure the turnover of specific proteins such as myosin and actin, which are important contractile proteins. Recently, techniques have been developed to measure the synthesis rate of myosin heavy chain (MHC) in humans. In addition, the application of quantitative polymerase chain reaction (PCR) allows measurement of the steady state mRNA levels of various isoforms of MHC. These measurements, in combination with measurement of the synthesis rate of MHC, will allow us to understand the molecular regulation of specific proteins such as myosin. The important question is to determine whether the decline in muscle protein synthesis is genetically determined or secondary to inactivity, nutritional, hormonal, or other age-related alterations in body functions, and whether muscle wasting is reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Contraction Accelerates Myosin Heavy Chain Synthesis Rates in Adult Cardiocytes by an Increase in the Rate of Translational Initiation

The purpose of this study was to determine the mechanism by which contraction acutely accelerates the synthesis rate of the contractile protein myosin heavy chain (MHC). Laminin-adherent adult feline cardiocytes were maintained in a serum-free medium and induced to contract at 1 Hz via electrical field stimulation. Electrical stimulation of contraction accelerated rates of MHC synthesis 28%, p < 0.05 by 4 h as determined by incorporation of [3H]phenylalanine into MHC. MHC mRNA expression as measured by RNase protection was unchanged after 4 h of electrical stimulation. MHC mRNA levels in messenger ribonucleoprotein complexes and translating polysomes were examined by sucrose gradient fractionation. The relative percentage of polysomebound MHC mRNA was equal at 47% in both electrically stimulated and control cardiocytes. However, electrical stimulation of contraction resulted in a reproducible shift of MHC mRNA from smaller polysomes into larger polysomes, indicating an increased rate of initiation. This shift resulted in significant increases in MHC mRNA levels in the fractions containing the larger polysomes of electrically stimulated cardiocytes as compared with nonstimulated controls. These data indicate that the rate of MHC synthesis is accelerated in contracting cardiocytes via an increase in translational efficiency.

Glutamine prevents downregulation of myosin heavy chain synthesis and muscle atrophy from glucocorticoids.

The aims of this study were to determine whether glutamine infusion prevents the decline in protein synthesis and muscle wasting associated with repeated glucocorticoid treatment. Hormone (cortisol acetate, 100 mg.kg body wt-1.day-1) and vehicle (carboxymethyl cellulose)-treated female rats were infused with either saline or glutamine (240 mM, 0.75 ml/h) for a 7-day period. Glutamine infusion attenuated the decline of plantaris muscle glutamine concentration (3.0 +/- 0.2 vs. 2.3 +/- 0.2 mumol/g) and prevented > 70% of the total muscle mass losses due to the glucocorticoid injections. Fractional synthesis rates of myosin heavy chain (MHC) and total protein were determined after constant [3H]leucine infusion from the leucyl-tRNA precursor pool, which was similar in all groups (range 4.8 +/- 0.5 to 6.3 +/- 0.4 disintegrations.min-1.pmol-1). MHC synthesis rates (%/day) in plantaris muscles were reduced to approximately 40% of controls (4.2/9.4). Although glutamine had no effect on MHC synthesis in vehicle-treated animals (10.1/9.4), it prevented 50% (7.6/4.2) of the hormone-induced decline in MHC synthesis rates. The same results were obtained with total protein synthesis measurements. Changes in muscle mass did not appear related to estimates of protein breakdown. In conclusion, these data show that glutamine infusion is effective therapy in counteracting glucocorticoid-induced muscle atrophy. Atrophy attenuation appears related to maintaining muscle glutamine levels, which in turn may limit the glucocorticoid-mediated downregulation of MHC synthesis.

Glucocorticoid-induced skeletal muscle atrophy in vitro is attenuated by mechanical stimulation.

Glucocorticoids induce rapid atrophy of fast skeletal myofibers in vivo, and either weight lifting or endurance exercise reduces this atrophy by unknown mechanisms. We examined the effects of the synthetic glucocorticoid dexamethasone (Dex) on protein turnover in tissue-cultured avian fast skeletal myofibers and determined whether repetitive mechanical stretch altered the myofiber response to Dex. In static cultures after 3-5 days, 10(-8) M Dex decreased total protein content 42-74%, total protein synthesis rates 38-56%, mean myofiber diameter 35%, myosin heavy chain (MHC) content 86%, MHC synthesis rate 44%, and fibronectin synthesis rate 29%. Repetitive 10% stretch-relaxations of the cultured myofibers for 60 s every 5 min for 3-4 days prevented 52% of the Dex-induced decrease in protein content, 42% of the decrease in total protein synthesis rate, 77% of the decrease in MHC content, 42% of the decrease in MHC synthesis rate, and 67% of the decrease in fibronectin synthesis rate. This in vitro model system will complement in vivo studies in understanding the mechanism by which mechanical activity and glucocorticoids interact to regulate skeletal muscle growth.

Myosin heavy chain turnover and glucocorticoid deterrence by exercise in muscle.

This study was undertaken to determine whether regular endurance running, of the type known to attenuate glucocorticoid-induced muscle atrophy, produces a reversal of the glucocorticoid-mediated suppression of myosin heavy chain (MHC) synthesis. Female rats were arbitrarily assigned to one of four groups. There were two sedentary groups that received either a vehicle (1% aqueous carboxymethyl cellulose) or cortisol acetate (100 mg/kg body wt) for 11 consecutive days and two exercise (treadmill running 29 m/min, 90 min/day, for 11 consecutive days) groups that received the activity simultaneously with either vehicle or steroid treatments. Protein synthesis measurements were performed by constant infusion of [3H]leucine. Fractional synthesis rates of MHC were determined from the leucyl-tRNA precursor pool, which was similar in all groups (range 2.85 +/- 0.32 to 3.51 +/- 0.43 dpm/pmol). Exercise prevented 30% of the plantaris muscle mass loss as the result of cortisol acetate treatment. MHC synthesis rates (%/day) in plantaris muscles of sedentary animals were reduced by glucocorticoid treatment to 65% (6.2/9.5) of the vehicle-treated group. Exercise did not alter this depression of MHC synthesis. The combination of exercise and glucocorticoid treatment reduced the calculated MHC breakdown rate (%/day) to 80% (-8.0/-10.1) of the rate resulting from hormone treatment alone and 60% (-8.0/-13.3) of the rate resulting from exercise alone. These results show that endurance exercise does not reverse the glucocorticoid inhibition of MHC synthesis in muscle but may act through reducing MHC breakdown.