Mechanism of the Ca ²⁺ -Dependent Interaction between S100A4 and Tail Fragments of Nonmuscle Myosin Heavy Chain IIA

Badyal, Sandip K.,Basran, Jaswir,Bhanji, Nina,Kim, Ju Hwan,Chavda, Alap P.,Jung, Hyun Suk,Craig, Roger,Elliott, Paul R.,Irvine, Andrew F.,Barsukov, Igor L.,Kriajevska, Marina,Bagshaw, Clive R. Elsevier 2011 Journal of molecular biology Vol.405 No.4

<P>The interaction between the calcium-binding protein S100A4 and the C-terminal fragments of nonmuscle myosin heavy chain IIA has been studied by equilibrium and kinetic methods. Using site-directed mutants, we conclude that Ca<SUP>2+</SUP> binds to the EF2 domain of S100A4 with micromolar affinity and that the <I>K</I><SUB>d</SUB> value for Ca<SUP>2+</SUP> is reduced by several orders of magnitude in the presence of myosin target fragments. The reduction in <I>K</I><SUB>d</SUB> results from a reduced dissociation rate constant (from 16 s<SUP>− 1</SUP> to 0.3 s<SUP>− 1</SUP> in the presence of coiled-coil fragments) and an increased association rate constant. Using peptide competition assays and NMR spectroscopy, we conclude that the minimal binding site on myosin heavy chain IIA corresponds to A1907-G1938; therefore, the site extends beyond the end of the coiled-coil region of myosin. Electron microscopy and turbidity assays were used to assess myosin fragment filament disassembly by S100A4. The latter assay demonstrated that S100A4 binds to the filaments and actively promotes disassembly rather than just binding to the myosin monomer and displacing the equilibrium. Quantitative modelling of these <I>in vitro</I> data suggests that S100A4 concentrations in the micromolar region could disassemble myosin filaments even at resting levels of cytoplasmic [Ca<SUP>2+</SUP>]. However, for Ca<SUP>2+</SUP> transients to be effective in further promoting dissociation, the elevated Ca<SUP>2+</SUP> signal must persist for tens of seconds. Fluorescence recovery after photobleaching of A431/SIP1 cells expressing green fluorescent protein–myosin IIA, immobilised on fibronectin micropatterns to control stress fibre location, yielded a recovery time constant of around 20 s, consistent with <I>in vitro</I> data.</P>

Association Analysis of Myosin Heavy-chain Genes mRNA Transcription with the Corresponding Proteins Expression of Longissimus Muscle in Growing Pigs

Men, X.M.,Deng, B.,Tao, X.,Qi, K.K.,Xu, Zi Wei Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.4

The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY ($Duroc{\times}Landrace{\times}Yorkshire$) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics.

Comparison of Characteristics of Myosin Heavy Chain-based Fiber and Meat Quality among Four Bovine Skeletal Muscles

Kim, Gap-Don,Yang, Han-Sul,Jeong, Jin-Yeon Korean Society for Food Science of Animal Resource 2016 한국축산식품학회지 Vol.36 No.6

Muscle fiber characteristics account for meat quality and muscle fibers are mainly classified into three or more types according to their contractile and metabolic properties. However, the majority of previous studies on bovine skeletal muscle are based on myosin ATPase activity. In the present study, the differences in the characteristics of muscle fibers classified by the expression of myosin heavy chain (MHC) among four bovine skeletal muscles such as longissimus thoracis (LT), psoas major (PM), semimembranosus (SM) and semi-tendinosus (ST) and their relationships to beef quality were investigated. MHCs 2x, 2a and slow were identified by LC-MS/MS and IIX, IIA and I fiber types were classified. PM, which had the smallest size and highest density of fibers regardless of type, showed the highest myoglobin content, CIE $L^*$, $a^*$, $b^*$ and sarcomere length (p<0.05), whereas ST with the highest composition of IIX, showed high shear force and low sarcomere length (p<0.05). The correlation coefficients between muscle fiber characteristics and meat quality showed that type IIX is closely related to poor beef quality and that a high density of small-sized fibers is related to redness and tenderness. Therefore, the differences in meat quality between muscles can be explained by the differences in muscle fiber characteristics, and especially, the muscles with good quality are composed of more small-sized fibers regardless of fiber type.

Comparison of Characteristics of Myosin Heavy Chain-based Fiber and Meat Quality among Four Bovine Skeletal Muscles

Gap-Don Kim,Han-Sul Yang,Jin-Yeon Jeong 한국축산식품학회 2016 한국축산식품학회지 Vol.36 No.6

Muscle fiber characteristics account for meat quality and muscle fibers are mainly classified into three or more types according to their contractile and metabolic properties. However, the majority of previous studies on bovine skeletal muscle are based on myosin ATPase activity. In the present study, the differences in the characteristics of muscle fibers classified by the expression of myosin heavy chain (MHC) among four bovine skeletal muscles such as longissimus thoracis (LT), psoas major (PM), semimembranosus (SM) and semitendinosus (ST) and their relationships to beef quality were investigated. MHCs 2x, 2a and slow were identified by LC-MS/MS and IIX, IIA and I fiber types were classified. PM, which had the smallest size and highest density of fibers regardless of type, showed the highest myoglobin content, CIE L*, a*, b* and sarcomere length (p<0.05), whereas ST with the highest composition of IIX, showed high shear force and low sarcomere length (p<0.05). The correlation coefficients between muscle fiber characteristics and meat quality showed that type IIX is closely related to poor beef quality and that a high density of small-sized fibers is related to redness and tenderness. Therefore, the differences in meat quality between muscles can be explained by the differences in muscle fiber characteristics, and especially, the muscles with good quality are composed of more small-sized fibers regardless of fiber type.

Comparison of Characteristics of Myosin Heavy Chain-based Fiber and Meat Quality among Four Bovine Skeletal Muscles

김갑돈,양한술,정진연 한국축산식품학회 2016 한국축산식품학회지 Vol.36 No.6

Muscle fiber characteristics account for meat quality and muscle fibers are mainly classified into three or more types according to their contractile and metabolic properties. However, the majority of previous studies on bovine skeletal muscle are based on myosin ATPase activity. In the present study, the differences in the characteristics of muscle fibers classified by the expression of myosin heavy chain (MHC) among four bovine skeletal muscles such as longissimus thoracis (LT), psoas major (PM), semimembranosus (SM) and semitendinosus (ST) and their relationships to beef quality were investigated. MHCs 2x, 2a and slow were identified by LC-MS/MS and IIX, IIA and I fiber types were classified. PM, which had the smallest size and highest density of fibers regardless of type, showed the highest myoglobin content, CIE L*, a*, b* and sarcomere length (p<0.05), whereas ST with the highest composition of IIX, showed high shear force and low sarcomere length (p<0.05). The correlation coefficients between muscle fiber characteristics and meat quality showed that type IIX is closely related to poor beef quality and that a high density of small-sized fibers is related to redness and tenderness. Therefore, the differences in meat quality between muscles can be explained by the differences in muscle fiber characteristics, and especially, the muscles with good quality are composed of more small-sized fibers regardless of fiber type.

Analysis of Myosin Heavy Chain Isoforms from Longissimus Thoracis Muscle of Hanwoo Steer by Electrophoresis and LC-MS/MS

김갑돈 한국축산식품학회 2014 한국축산식품학회지 Vol.34 No.5

The purpose of this study was to analyze myosin heavy chain (MHC) isoforms in bovine longissimus thoracis (LT) muscleby liquid chromatography (LC) and mass spectrometry (MS). LT muscles taken from Hanwoo (Korean native cattle) steer(n=3) used to separate myosin bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide querieswere obtained from the myosin bands by LC-MS/MS analysis following in-gel digestion with trypsin. A total of 33 and 43queries were identified as common and unique peptides, respectively, of MHC isoforms (individual ions scores >43 indicateidentity or extensive homology, <0.05). MHC-1 (IIx), -2 (IIa), -4 (IIb), and -7 (slow/I) were identified based on the Mowsescore (5118, 3951, 2526, and 2541 for MHC-1, -2, -4, and -7, respectively). However, more analysis is needed to confirmthe expression of MHC-4 in bovine LT muscle because any query identified as a unique peptide of MHC-4 was not found. The queries that were identified as unique peptides could be used as peptide markers to confirm MHC-1 (14 queries), -2 (8queries), and -7 (21 queries) in bovine LT muscle; no query identified as a unique peptide of MHC-4 was found. LC-MS/MS analysis is a useful approach to study MHC isoforms at the protein level. The purpose of this study was to analyze myosin heavy chain (MHC) isoforms in bovine longissimus thoracis (LT) muscleby liquid chromatography (LC) and mass spectrometry (MS). LT muscles taken from Hanwoo (Korean native cattle) steer(n=3) used to separate myosin bands by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The peptide querieswere obtained from the myosin bands by LC-MS/MS analysis following in-gel digestion with trypsin. A total of 33 and 43queries were identified as common and unique peptides, respectively, of MHC isoforms (individual ions scores >43 indicateidentity or extensive homology, <0.05). MHC-1 (IIx), -2 (IIa), -4 (IIb), and -7 (slow/I) were identified based on the Mowsescore (5118, 3951, 2526, and 2541 for MHC-1, -2, -4, and -7, respectively). However, more analysis is needed to confirmthe expression of MHC-4 in bovine LT muscle because any query identified as a unique peptide of MHC-4 was not found. The queries that were identified as unique peptides could be used as peptide markers to confirm MHC-1 (14 queries), -2 (8queries), and -7 (21 queries) in bovine LT muscle; no query identified as a unique peptide of MHC-4 was found. LC-MS/MS analysis is a useful approach to study MHC isoforms at the protein level.

Age- and sex-related differences in myosin heavy chain isoforms and muscle strength, function, and quality: a cross sectional study

( Seung-lyul Oh ),( Sang Hoon Yoon ),( Jae-young Lim ) 한국운동영양학회 2018 Physical Activity and Nutrition (Phys Act Nutr) Vol.22 No.2

[Purpose] Declining muscle strength and function are hallmarks of the aging process. This study aimed to determine sex-related differences in myosin heavy chain (MHC) isoforms and muscle mass, strength, and quality with aging. [Methods] This cross-sectional study recruited 53 healthy participants (32 men, 21 women) aged 20-85 years who were divided into four groups: young men (n=17, YM, 29.23±4.51), older men (n=15, OM, 71.87±3.42), young women (n=11, YW, 29.64±4.88), and older women (n=10, OW, 68.1±1.91). Body composition and muscle strength and quality were analyzed. Muscle specimens were obtained from the vastus lateralis in all participants to analyze the type of MHC isoforms. [Results] Men showed a greater age-related decline in skeletal muscle mass (18.6%, p<0.01), lean body mass (10.1%, p<0.05), grip strength (35.3%, p<0.001), isometric strength (29.6%, p<0.001), isotonic power (42.5%, p<0.001), isokinetic strength (up to 44.3%, p<0.001), and muscle quality (up to 24.8%, p<0.01). In contrast, women had significantly lower isometric strength (24.2%, p<0.05), isotonic power (36.5%, p<0.01), and upper-body muscle quality (24.7%, p<0.001) with aging. In addition, the proportion of MHC IIa was significantly lower in OM (p<0.05) and OW (p<0.05) than in YM and YW, respectively. However, the proportion of MHC I was significantly higher in OM (p<0.01) than in YM but was high in both YW and OW. MHC I and MHC IIa negatively and positively correlated, respectively, with muscle strength and function. [Conclusion] These results indicate the existence of sex-related differences in muscle mass, strength, and quality and MHC isoform composition with increasing age. The effects on muscle strength and function with aging were significant in men, but not in women. Higher and lower proportions of MHC I and MHC IIa fibers, respectively, were inversely associated with muscle strength and quality. In particular, Korean YW showed lower muscle strength and quality, and the proportion of MHC isoforms was similar to that in the muscles of OW.

운동 형태에 따른 근력 및 지구력 강화 효과와 근섬유 형태의 변화

서정환,김성용,김유창 大韓再活醫學會 1998 Annals of Rehabilitation Medicine Vol.22 No.3

코골이 및 폐쇄성 수면 무호흡증 환자에서 ATPase 염색법을 이용한 구개수 근육의 근 섬유 아형 분포에 관한 연구

서민철,이시형,김재호 대한이비인후과학회 2002 대한이비인후과학회지 두경부외과학 Vol.45 No.5

Background and Objectives:OSAS is thought to be due to an excesive loss of muscle tone in the upper airway or an abnormal reflex regulation of uper airway function during slep. The aim of this study was to investigate the distribution of myosin heavy chain (MHC) isoforms of musculus uvulae in OSAS and snorers. Materials and Methods:Thirty seven patients included in the study underwent an uvulo-palato-pharyngoplasty (UPP ). All subjects had polysomnographic study before UPPP. ATPase stain at pH 9.4 were applied to muscle specimens obtained during UPPP. The numbers of MHC type I and II isoforms were counted. The patients were divided into thre groups according to their repiratory disturbance index (RDI):mild (0-20), moderate (21-40), severe (> 40). The diferences in the distribution of muscle fiber types were com-pared betwen these groups. The corelation betwen the distribution of the fibers and the body mas index / age / RDI / minimum O2 saturation / duration of slep apnea was investigated. Results:The mean percentages standard deviation of type I fibers acording to the severity of sleep apnea were as follows: 22.5± 3.2% in mild slep apnea group (n=15), 19.8± 2.3% in moderate slep apnea group (n=11), 17.5± 3.6% in severe slep apnea group (n=11). There were statistically significant differences in the distribution of MHC type I & II isoforms betwen mild group and moderate and / or severe group (p<0.05). There was statistically significant negative correlation betwen the proportion of type I fibers and RDI / duration of slep apnea (p<.01). We could not find meaningful corelation betwen the proportion of MHC type I isoforms and body mas index / age of the patients (p>.05). :The proportion of MHC type I isoforms in musculus uvulae was decreased according to the severity and duration of sleep apnea (p<.01). (Korean J Otolaryngol 2002;45:433-8)

Genetic association of MYH genes with hereditary hearing loss in Korea

Kim, Sang-Joo,Lee, Seokwon,Park, Hong-Joon,Kang, Tae-Hun,Sagong, Borum,Baek, Jeong-In,Oh, Se-Kyung,Choi, Jae Young,Lee, Kyu-Yup,Kim, Un-Kyung Elsevier 2016 Gene Vol.591 No.1

<P><B>Abstract</B></P> <P><B>Background</B></P> <P>Myosin is a key protein involved in regulating the shape and motility of cells. The <I>MYH9</I> and <I>MYH14</I> genes, which encode non-muscle myosin heavy chain IIA (NMMHC II-A) and IIC (NMMHC II-C), respectively, are expressed in the inner ear. These myosin genes are known to be associated with autosomal dominant non-syndromic hearing loss (ADNSHL); however, genetic studies in patients with ADNSHL in Korea have rarely been reported.</P> <P><B>Methods</B></P> <P>We analyzed the <I>MYH9</I> and <I>MYH14</I> genes in 75 Korean patients with ADNSHL.</P> <P><B>Results</B></P> <P>We identified 4 possible pathogenic variants: a novel variant p.F1303L and 2 previously reported variants (p.R1730C and p.R1785C) in the <I>MYH9</I> gene, and a novel variant p.A1868T in the <I>MYH14</I> gene. All the variants were located in the myosin tail domain, which is essential for the interaction of myosin with actin. These variants were predicted to be possibly pathogenic by functional prediction tools and were absent in 100 unrelated normal controls.</P> <P><B>Conclusion</B></P> <P>These results suggest that all the variants identified in this study have a strong potential to affect the structural stability and/or function of non-muscle myosin in the inner ear, which might lead to ADNSHL. This study establishes the link between the genotype and development of ADNSHL and contributes to the establishment of Korean database for hereditary hearing loss.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We performed genetic analysis of <I>MYH</I> genes in Korean patients with ADNSHL. </LI> <LI> We found three and one possibly pathogenic variants in <I>MYH9</I> and <I>MYH14</I>, respectively. </LI> <LI> Our research will improve understanding of relations between MYH genes and ADNSHL. </LI> </UL> </P>