Expression Levels Of Myosin Heavy Chain Research Articles

Effects of a traditional Chinese medicine formula and its extraction on muscle fiber characteristics in finishing pigs, porcine cell proliferation and isoforms of myosin heavy chain gene expression in myocytes.

ObjectiveThis study evaluated the effects of a traditional Chinese medicine formula (TCMF) on muscle fiber characteristics in finishing pigs and the effects of the formula’s extract (distilled water, ethyl acetate and petroleum ether extraction) on porcine cell proliferation and isoforms of myosin heavy chain (MyHC) gene expression in myocytes.MethodsIn a completely randomized design, ninety pigs were assigned to three diets with five replications per treatment and six pigs per pen. The diets included the basal diet (control group), TCMF1 (basal diet+2.5 g/kg TCMF) and TCMF2 (basal diet+5 g/kg TCMF). The psoas major muscle was obtained from pigs at the end of the experiment. Muscle fiber characteristics in the psoas major muscle were analyzed using myosin ATPase staining. Cell proliferation was measured using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) dye and cytometry. Isoforms of MyHC gene expression were detected by real-time quantitative polymerase chain reaction.ResultsThe final body weight and carcass weight of finishing pigs were increased by TCMF1 (p<0.05), while the psoas major muscle cross-sectional area was increased by TCMF (p<0.05). The cross-sectional area and diameter of psoas major muscle fiber I, IIA, and IIB were increased by TCMF2 (p<0.05). The cross-sectional area and fiber diameter of psoas major muscle fiber IIA and IIB were increased by diet supplementation with TCMF1 (p<0.05). Psoas major muscle fiber IIA and IIB fiber density from the pigs fed the TCMF1 diet and the type IIB fiber density from the pigs fed the TCMF2 diet were lower compared to pigs fed the control diet (p<0.05). Pigs fed TCMF2 had a higher composition of type I fiber and a lower percentage of type IIB fiber in the psoas major muscle (p<0.05). The expression levels of MyHC I, MyHC IIa, and MyHC IIx mRNA increased and the amount of MyHC IIb mRNA decreased in the psoas major muscle from TCMF2, whereas MyHC I and MyHC IIx mRNA increased in the psoas major muscle from TCMF1 (p<0.05). Peroxisome proliferator-activated receptor γ coactivator-1α and CaN mRNA expression in the psoas major muscle were up-regulated by TCMF (p<0.05). Porcine skeletal muscle satellite cell proliferation was promoted by 4 μg/mL and 20 μg/mL TCMF water extraction (p<0.05). Both 1 μg/mL and 5 μg/mL of TCMF water extraction increased MyHC IIa, MyHC IIb, and MyHC IIx mRNA expression in porcine myocytes (p<0.05), while MyHC I mRNA expression in porcine myocytes was decreased by 5 μg/mL TCMF water extraction (p<0.05). Porcine myocyte MyHC I and MyHC IIx mRNA expression were increased, and MyHC IIa and MyHC IIb mRNA expression were down-regulated by 5 μg/mL TCMF ethyl acetate extraction (p<0.05). MyHC I and MyHC IIa mRNA expression in porcine myocytes were increased, and the MyHC IIb mRNA expression was decreased by 1 μg/mL TCMF ethyl acetate extraction (p<0.05). Four isoforms of MyHC mRNA expression in porcine myocytes were reduced by 5 μg/mL TCMF petroleum ether extraction (p<0.05). MyHC IIa mRNA expression in porcine myocytes increased and MyHC IIb mRNA expression decreased by 1 μg/mL in a TCMF petroleum ether extraction (p<0.05).ConclusionThese results indicated that TCMF amplified the psoas major muscle cross-sectional area through changing muscle fiber characteristics in finishing pigs. This effect was confirmed as TCMF extraction promoted porcine cell proliferation and affected isoforms of MyHC gene expression in myocytes.

Activity of metabolic enzymes and muscle-specific gene expression in parr and smolts Atlantic salmon Salmo salar L. of different age groups.

This study was conducted to characterize the energy metabolism level and the features of muscle growth regulation during the development of Atlantic salmon (Salmo salar) inhabiting the Indera River (Kola Peninsula, Russia). The activities of aerobic and anaerobic enzymes (cytochrome c oxidase and lactate dehydrogenase) and carbohydrate metabolism enzymes (glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, and aldolase) were measured in muscle and liver tissue. Gene expression levels of myosin heavy chain (MyHC), myostatin (MSTN-1a), and myogenic regulatory factors (MRFs-MyoD1a, MyoD1b, MyoD1c, Myf5, myogenin) were measured in the white muscles of salmon parr of ages 0+, 1+, 2+, and 3+ and smolts of ages 2+ and 3+. Multidirectional changes in the activity of enzymes involved in aerobic and anaerobic energy metabolism with age were shown in the white muscles of the parr. The cytochrome c oxidase activity was higher in muscles of underyearlings (0+) and yearlings (1+) and decreased in 2+ and 3+ age groups. The activity of lactate dehydrogenase, in contrast, increased with age. The patterns of changes in expression levels of MyoD1a, MyoD1b, myogenin, MyHC, and MSTN-1a at different ages of the parr were similar. Particularly, the expression of these genes peaked in the yearling parr (1+) and then decreased in elder groups. The differences were revealed in parameters studied between the parr and smolts. The level of aerobic and anaerobic metabolism enzyme activities was higher in the white muscles of smolts than in parr. The activity of carbohydrate metabolism enzymes was decreased in the smolts' livers. The expression levels of MyHC, MyoD1a, MyoD1b, and myogenin were lower in smolts at age 2+ compared to parr. These findings expand our knowledge of age-related and stage-related features of energy metabolism and muscle development regulation in young Atlantic salmon in their natural habitat. The results might be used for monitoring of the salmon population during restoration and rearing.

Ethanol extract of Cyclolepis genistoides D. Don (palo azul) induces formation of myotubes, which involves differentiation of C2C12 myoblast cells

In this study, we examined the cell differentiation effect of an ethanol extract of Cyclolepis genistoides D. Don, a herbaceous perennial belonging to the family Asteraceae (vernacular name: palo azul). Palo azul has numerous physiological effects that contribute to the prevention of metabolic syndromes, although the mechanism remains unclear. We previously suggested that palo azul has antidiabetic activity via an adipose differentiation effect. Here, we focused on whether palo azul promoted the differentiation of myoblasts. The mouse muscle myoblast cell line C2C12 was cultured and differentiated using horse serum with or without an ethanol extract of palo azul (12.5-200 μg/mL). Quantitative real-time polymerase chain reaction was performed to evaluate differentiation markers, including insulin-like growth factor-1 and myogenin. To evaluate myotube formation, myosin heavy-chain (MHC) expression and localization were detected by immunohistochemistry. Palo azul increased the expression of the differentiation markers. Furthermore, immunohistochemistry analysis revealed increased formation of MHC myotubes after palo azul treatment along with increased diameter and fusion indices of the myotubes. The expression level of MHC was also increased. In conclusion, palo azul may increase muscle mass in the body and improve insulin resistance conditions by facilitating the formation of myotubes by promoting myocyte differentiation.

Expression of MyHC genes, composition of muscle fiber type and their association with intramuscular fat, tenderness in skeletal muscle of Simmental hybrids

In adult bovine skeletal muscle, it expressed four isoforms of Myosin heavy chain (MyHC) gene, MyHC-I, MyHC-IIa, MyHC-IIb, and MyHC-IIx that are translated into different structural protein myofibrils, and then further form different types of muscle fiber. In the studies, our objective is to reveal the expression patterns of MyHC genes in longissimus dorsi (Ld), semitendinosus (Se) and soleus (Sol) of Simmental hybrids cattle, and their association with intramuscular fat (IMF) content and meat shearing force (MSF). The muscle tissue of Ld, Se and Sol were collected from 6, 12 and 36-month old Simmental hybrids respectively, then the expression levels of MyHCs were examined by real-time PCR, at the same time, IMF, MSF and muscle type were measured with chemical assessment, shearing force measurer and immunostaining respectively. Our results showed that t Ld, Se, and Sol expressed MyHC-I, MyHC-IIa and MyHC-IIx isoforms but not MyHC-IIb, furthermore MyHC-I, MyHC-IIa and MyHC-IIx had different expression patterns in different skeletal muscle. The expression of MyHC-I in Se and Sol, MyHC-IIa in Ld, Se, and Sol, and MyHC-IIx in Sol was decreased with increasing age. The highest expression of MyHC-I in Ld, and MyHC-IIx in Ld and Se was observed in 12-month-old animals. The percentage of type-IIa fiber approximately occupied 70-80% among various muscle fiber of Ld, Se and Sol. The percentage of different type fiber was not related to IMF content and MSF, but the expression levels of MyHC-I and MyHC-IIa were negatively related to IMF content (r=-0.724, and -0.681, respectively) and MSF (r=-0.672, and -0.641, respectively). The expression level of MyHC-IIx was also negatively related to MSF (r=-0.655). In conclusion, MyHC gene might be considered as a negative factor in genetic selection of IMF content and MSF.

Comparison of vascular smooth muscle cells in canine great vessels

BackgroundElucidating the histological characteristics of normal vascular smooth muscle cells (VSMCs) is important for understanding mechanisms of development, disease etiology and the remodeling and/or regeneration process of the vessel. However, knowledge regarding VSMCs is focused primarily on the artery. Although the characteristics of each great vessel are documented, few studies have examined VSMCs in parallel within each great vessel. The present study focused on comparing characteristics of canine VSMCs within the aorta (Ao), branch pulmonary artery (bPA), main pulmonary artery (mPA) and inferior vena cava (IVC), simultaneously.ResultsWestern blot and immunohistochemistry were used to determine VSMC protein content for alpha smooth muscle actin (ASMA), calponin, myosin heavy chain (MHC) and its isozyme SM2, and non-muscle myosin heavy chain B (SMemb). Thickness and ratio of the VSMC layer were also measured. Expression levels of ASMA, calponin and SM2 significantly differed between vessels, except between mPA and either bPA, Ao and IVC vessels. Expression levels of MHC were significantly different in all vessels, whilst expression of SMemb was significantly different in the Ao compared with either bPA and mPA vessels. All vessels were significantly different with respect to total wall and VSMC layer thickness. The ratio between VSMC layer and total wall thickness was significantly different for each vessel, except between bPA and mPA vessels. Histological analysis of the IVC revealed that the VSMC layer does not line evenly and continuously through the long axis or transverse sections. With respect to the pulmonary artery, calponin was expressed to a greater extent in the mPA compared with the bPA (P < 0.01*). In contrast, MHC and SM2 were expressed to a greater extent in the bPA compared with the mPA (P < 0.01*). Differences in VSMC distribution indicate structural differences in the proximal and distal pulmonary artery bifurcation.ConclusionOur results show that the VSMC expression pattern in each great vessel is unique and suggestive of the developmental differences between great vessels. We believe this study provides basic data for the pathology, etiology and regenerative capability of the vessels.

Parallel Speaker Sessions

From 1996-2000 ZEBET (Centre for Documentation and Evaluation of Alternative Methods to Animal Experiments at the BgVV, Berlin, D) has co-ordinated the ECVAM (European Centre for the Validation of Alternative Methods, Ispra, I) prevalidation and validation study of three embryotoxicity tests: (a) a test employing embryonic stem cell lines (EST); (b) the micromass test (MM test); and (c) the post-implantation rat whole-embryo culture assay (WEC test). In the validation study for two of the in vitro tests, the EST and the WEC, the predicitvity (100%) for strong embryotoxic chemicals was excellent and the precision (81%) was good. The predictivity for non (72%) and weak (70%) embryotoxic compounds and the precision for non-embryotoxic compounds (70%) were sufficiently high (?65%). In contrast, the results for the MM test were in sufficient. Therefore, in 2001 the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the conclusions from the formal validation study of three in vitro embryotoxicity test that the embryonic stem cell test (EST) and post-implantation rat whole-embryo culture assay (WEC) are scientifically validated tests, which are ready to be considered for regulatory purposes while the MM test was not ready to be considered for regulatory purposes.Since the performance of the EST and of the WEC were identical in the ECVAM validation trial and since the EST does not require the use of pregnant animals and is less time consuming and expensive, from 2001-2003 we have improved the EST in collaboration with the three German drug companies Bayer AG, Boehringer Ingelheim and Schering AG. The EST is a reliable in vitro test system for the classification of compounds according to their teratogenic potential based on the morphological analysis of cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of beating areas and to adapt the EST to applications in high-throughput screening systems, we have improved and expanded the EST protocol by establishing molecular endpoints of differentiation.The quantitative expression of sarcomeric myosin heavy chain (MHC) and ?-actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and ?-actinin and the differentiation into cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, ?-actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data.Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to teratogenic potential using stem cell technology and technological advances in the field of gene expression analysis.

Improvement of an in vitro stem cell assay for developmental toxicity: the use of molecular endpoints in the embryonic stem cell test

The embryonic stem cell test (EST) takes advantage of the potential of murine embryonic stem (ES) cells to differentiate in culture to test embryotoxicity in vitro. The EST represents a reliable, scientifically validated in vitro system for the classification of compounds according to their teratogenic potential based on the morphological analysis of beating cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on undifferentiated murine ES cells and differentiated 3T3 fibroblasts. In order to identify more objective endpoints of differentiation other than the microscopic evaluation of “beating areas” and to adapt the EST to applications in high-throughput screening systems we improved and expanded the EST protocol by establishing molecular endpoints of differentiation. The quantitative expression of sarcomeric myosin heavy chain (MHC) and α-actinin genes under the influence of test compounds was studied employing intracellular flow cytometry. Strong embryotoxicants exerted a dose-dependent effect on both the expression levels of MHC and α-actinin and the differentiation into beating cardiomyocytes. Furthermore, quantitative FACS (fluorescence-activating cell sorting) analysis showed the same sensitivity for the classification of substances as the conventional endpoint but allowed a significant reduction of the test period. Within 7 days, maximal expression of sarcomeric marker proteins was observed. Our findings indicate that structural proteins of the sarcomere apparatus, α-actinin and myosin heavy chain (MHC), seem to be promising candidates to predict developmental toxicity in vivo from in vitro data. Thus, the improved EST holds promise as a new predictive screen for risk assessment with respect to developmental toxicity using stem cell technology and technological advances in the field of gene expression analysis.