Morphometry and muscle gene expression in hypertrophied hearts from polyomavirus large T antigen transgenic mice

1995 ◽  
Vol 269 (1) ◽  
pp. H86-H95 ◽  
Author(s):  
E. Holder ◽  
B. Mitmaker ◽  
L. Alpert ◽  
L. Chalifour
Keyword(s):  
Transgenic Mice ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Troponin I ◽  
T Antigen ◽  
Large T Antigen ◽  
Cross Sectional

Transgenic mice expressing polyomavirus large T antigen (PVLT) in cardiomyocytes develop a cardiac hypertrophy in adulthood. Morphometric analysis identified cardiomyocytes enlarged up to ninefold in cross-sectional area in the adult transgenic hearts compared with normal age-matched nontransgenic hearts. Most enlarged cardiomyocytes were found in the subendocardium, whereas normal-sized cardiomyocytes were localized to the midmyocardium. Transgenic hearts did not express detectable skeletal muscle actin mRNA or protein, or skeletal troponin I isoform mRNA. Some, but not all, transgenic hearts expressed an increase in the beta-myosin heavy chain mRNA. All five transgenic mice tested had increased expression of atrial natriuretic factor (ANF) mRNA. Whereas normal hearts expressed three myosin light chain proteins of 19, 16, and 15 kDa, we found that the 19-kDa myosin light chain was not observed in the transgenic hearts. We conclude that adult, PVLT-expressing, transgenic mice developed enlarged cardiomyocytes with an increase in beta-myosin heavy chain and ANF mRNA expression, but a widespread skeletal isoform usage was not present in these transgenic mice. The adult transgenic hearts thus display histological and molecular changes similar to those found in hypertrophy induced by a pressure overload in vivo.

Cardiomyocyte proliferation in mice expressing alpha-cardiac myosin heavy chain-SV40 T-antigen transgenes

1992 ◽  
Vol 262 (6) ◽  
pp. H1867-H1876 ◽  
Author(s):  
E. B. Katz ◽  
M. E. Steinhelper ◽  
J. B. Delcarpio ◽  
A. I. Daud ◽  
W. C. Claycomb ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Antigen Expression ◽  
T Antigen ◽  
Proliferative Potential ◽  
Cardiac Myosin ◽  
Large T Antigen ◽  
Sv40 Large T Antigen ◽  
Ventricular Cardiomyocytes

To determine the proliferative potential of adult ventricular cardiomyocytes, we have generated transgenic mice that express the SV40 large T-antigen oncogene in the heart. A fusion gene comprised of the rat alpha-cardiac myosin heavy chain promoter and the SV40 early region was used to target oncogene expression to the myocardium. Expression of SV40 large T-antigen was observed in both atrial and ventricular cardiomyocytes in adult transgenic animals. T-antigen expression was associated with hyperplasia in the targeted cells. Immunohistological analysis indicated that the proliferating cells continued to express sarcomeric myosin. Electron microscopic examination demonstrated that cardiomyocytes in various stages of the cell cycle retained ultrastructural characteristics typical of mitotic cardiac muscle cells in vivo. Cardiomyocytes isolated from transgenic tumors were able to proliferate in culture and retained a differentiated phenotype, as evidenced by spontaneous contractile activity. Preliminary studies indicate that these cells can undergo a limited number of passages while retaining this differentiated phenotype. These studies demonstrate that both ventricular and atrial cardiomyocytes from transgenic mice proliferate in response to targeted T-antigen expression.

scholarly journals Specific Myosin Heavy Chain Mutations Suppress Troponin I Defects in Drosophila Muscles

1999 ◽  
Vol 144 (5) ◽  
pp. 989-1000 ◽  
Author(s):  
William A. Kronert ◽  
Angel Acebes ◽  
Alberto Ferrús ◽  
Sanford I. Bernstein
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Thin Filament ◽  
Troponin I ◽  
Point Mutations ◽  
Thick Filament ◽  
Actin Binding ◽  
Filament Protein ◽  
Phenotypic Rescue

We show that specific mutations in the head of the thick filament molecule myosin heavy chain prevent a degenerative muscle syndrome resulting from the hdp2 mutation in the thin filament protein troponin I. One mutation deletes eight residues from the actin binding loop of myosin, while a second affects a residue at the base of this loop. Two other mutations affect amino acids near the site of nucleotide entry and exit in the motor domain. We document the degree of phenotypic rescue each suppressor permits and show that other point mutations in myosin, as well as null mutations, fail to suppress the hdp2 phenotype. We discuss mechanisms by which the hdp2 phenotypes are suppressed and conclude that the specific residues we identified in myosin are important in regulating thick and thin filament interactions. This in vivo approach to dissecting the contractile cycle defines novel molecular processes that may be difficult to uncover by biochemical and structural analysis. Our study illustrates how expression of genetic defects are dependent upon genetic background, and therefore could have implications for understanding gene interactions in human disease.

scholarly journals Electrophoretic analysis of proteins from single bovine muscle fibres

1981 ◽  
Vol 195 (1) ◽  
pp. 317-327 ◽  
Author(s):  
O A Young ◽  
C L Davey
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Fibre Types ◽  
Type I ◽  
Slow Fibre ◽  
Slow Myosin ◽  
Fast Myosin ◽  
Fast Myosin Heavy Chain

A number of single fibres were isolated by dissection of four bovine masseter (ma) muscles, three rectus abdominis (ra) muscles and eight sternomandibularis (sm) muscles. By histochemical criteria these muscles contain respectively, solely slow fibres (often called type I), predominantly fast fibres (type II), and a mixture of fast and slow. The fibres were analysed by conventional sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the gels stained with Coomassie Blue. Irrespective of the muscle, every fibre could be classed into one of two broad groups based on the mobility of proteins in the range 135000-170000 daltons. When zones containing myosin heavy chain were cut from the single-fibre gel tracks and ‘mapped’ [Cleveland, Fischer, Kirschner & Laemmli (1977) J. Biol. Chem. 252, 1102-1106] with Staphylococcus proteinase, it was found that one group always contained fast myosin heavy chain, whereas the second group always contained the slow form. Moreover, a relatively fast-migrating alpha-tropomyosin was associated with the fast myosin group and a slow-migrating form with the slow myosin group. All fibres also contained beta-tropomyosin; the coexistence of alpha- and beta-tropomyosin is at variance with evidence that alpha-tropomyosin is restricted to fast fibres [Dhoot & Perry (1979) Nature (London) 278, 714-718]. Fast fibres containing the expected fast light chains and troponins I and C fast were identified in the three ra muscles, but in only four sm muscles. In three other sm muscles, all the fast fibres contained two troponins I and an additional myosin light chain that was more typical of myosin light chain 1 slow. The remaining sm muscle contained a fast fibre type that was similar to the first type, except that its myosin light chain 1 was more typical of the slow polymorph. Troponin T was bimorphic in all fast fibres from a ra muscles and in at least some fast fibres from one sm muscle. Peptide ‘mapping’ revealed two forms of fast myosin heavy chain distributed among fast fibres. Each form was associated with certain other proteins. Slow myosin heavy chain was unvarying in three slow fibre types identified. Troponin I polymorphs were the principal indicator of slow fibre types. The myofibrillar polymorphs identified presumably contribute to contraction properties, but beyond cud chewing involving ma muscle, nothing is known of the conditions that gave rise to the variable fibre composites in sm and ra muscles.

Three-dimensional compartmentalization of myosin heavy chain and myosin light chain isoforms in dog thyroarytenoid muscle

2006 ◽  
Vol 290 (5) ◽  
pp. C1446-C1458 ◽  
Author(s):  
Mark Bergrin ◽  
Sabahattin Bicer ◽  
Christine A. Lucas ◽  
Peter J. Reiser
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Expression Patterns ◽  
Shortening Velocity ◽  
Single Fibers ◽  
Isoform Expression ◽  
Thyroarytenoid Muscle ◽  
Mhc Iib

The thyroarytenoid muscle, a vocal fold adductor, has important roles in airway protection (e.g., prevention of aspiration) and phonation. Isoform expression of myosin heavy chain (MHC), a major determinant of muscle-shortening velocity, has been reported to be heterogeneous in this muscle in several mammals, differing markedly between the medial and lateral divisions. The objective was to determine the isoform expression patterns of both MHC and myosin light chain (MLC), with the latter having a modulatory role in determining shortening velocity, to further test whether the expression of both myosin subunits differs in multiple specific sites within the divisions of the dog thyroarytenoid muscle, potentially revealing even greater compartmentalization in this muscle. Our results indicate the existence of large gradients in the relative levels of individual MHC isoforms in the craniocaudal axis along the medial layer (i.e., airflow axis), where levels of MHC-I and MHC-IIA are low at both ends of the axis and high in the middle and MHC-IIB has a reciprocal distribution. The lateral layer is more uniform, with high levels of MHC-IIB throughout. The level of MHC-IID is relatively constant along the axis in both layers. Large differences exist in the distribution of MHC isoforms among single fibers isolated from sites along the craniocaudal axis, especially in the lateral layer. Systematic regional variations are apparent in the MLC isoform composition of single fibers as well, including some MLC isoform combinations that are not observed in dog limb muscles. Variations of MHC and MLC isoform expression in the dog thyroarytenoid muscle are greater than previously recognized and suggest an even broader range of contractile properties within this multifunctional muscle.

scholarly journals Identification of sequences necessary for the association of cardiac myosin subunits.

1991 ◽  
Vol 113 (3) ◽  
pp. 585-590 ◽  
Author(s):  
E M McNally ◽  
M M Bravo-Zehnder ◽  
L A Leinwand
Keyword(s):  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Carboxyl Terminus ◽  
Amino Terminus ◽  
Cardiac Myosin ◽  
Amino Acid Region ◽  
Myosin Subunits ◽  
Acid Region

To begin to understand the nature of myosin subunit assembly, we determined the region of a vertebrate sarcomeric myosin heavy chain required for binding of light chain 1. We coexpressed in Escherichia coli segments of the rat alpha cardiac myosin heavy chain which spanned the carboxyl terminus of subfragment 1 and the amino terminus of subfragment 2 with a full-length rat cardiac myosin light chain 1. A 16 amino acid region of the myosin heavy chain (residues 792-808) was shown to be required for myosin light chain 1 binding in an immunoprecipitation assay.

scholarly journals Overexpression of heart-specific small subunit of myosin light chain phosphatase results in heart failure and conduction disturbance

2018 ◽  
Vol 314 (6) ◽  
pp. H1192-H1202 ◽  
Author(s):  
Takuro Arimura ◽  
Antoine Muchir ◽  
Masayoshi Kuwahara ◽  
Sachio Morimoto ◽  
Taisuke Ishikawa ◽  
...  
Keyword(s):  
Heart Failure ◽  
Transgenic Mice ◽  
Muscle Contraction ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Small Subunit ◽  
Conduction Disturbance ◽  
Myosin Light Chain Phosphatase ◽  
Myosin Light Chain 2

Mutations in genes encoding components of the sarcomere cause cardiomyopathy, which is often associated with abnormal Ca2+ sensitivity of muscle contraction. We have previously shown that a heart-specific myosin light chain phosphatase small subunit (hHS-M21) increases the Ca2+ sensitivity of muscle contraction. The aim of the present study was to investigate the function of hHS-M21 in vivo and the causative role of abnormal Ca2+ sensitivity in cardiomyopathy. We generated transgenic mice with cardiac-specific overexpression of hHS-M21. We confirmed that hHS-M21 increased the Ca2+ sensitivity of cardiac muscle contraction in vivo, which was not followed by an increased phosphorylation of myosin light chain 2 isoforms. hHS-M21 transgenic mice developed severe systolic dysfunction with myocardial fibrosis and degeneration of cardiomyocytes in association with sinus bradycardia and atrioventricular conduction defect. The contractile dysfunction and cardiac fibrosis were improved by treatment with the Rho kinase inhibitor fasudil. Our findings suggested that the overexpression of hHS-M21 results in cardiac dysfunction and conduction disturbance via non-myosin light chain 2 phosphorylation-dependent regulation. NEW & NOTEWORTHY The present study is the first to develop mice with transgenic overexpression of a heart-specific myosin light chain phosphatase small subunit (hHS-M21) and to examine the effects of hHS-M21 on cardiac function. Elevation of hHS-M21 induced heart failure with myocardial fibrosis and degeneration of cardiomyocytes accompanied by supraventricular arrhythmias.

scholarly journals Myosin Light Chain–activating Phosphorylation Sites Are Required for Oogenesis in Drosophila

1997 ◽  
Vol 139 (7) ◽  
pp. 1805-1819 ◽  
Author(s):  
Pascale Jordan ◽  
Roger Karess
Keyword(s):  
Amino Acids ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Germ Line ◽  
Myosin Ii ◽  
Oocyte Development ◽  
Nurse Cells ◽  
Ring Canals

The Drosophila spaghetti squash (sqh) gene encodes the regulatory myosin light chain (RMLC) of nonmuscle myosin II. Biochemical analysis of vertebrate nonmuscle and smooth muscle myosin II has established that phosphorylation of certain amino acids of the RMLC greatly increases the actin-dependent myosin ATPase and motor activity of myosin in vitro. We have assessed the in vivo importance of these sites, which in Drosophila correspond to serine-21 and threonine-20, by creating a series of transgenes in which these specific amino acids were altered. The phenotypes of the transgenes were examined in an otherwise null mutant background during oocyte development in Drosophila females. Germ line cystoblasts entirely lacking a functional sqh gene show severe defects in proliferation and cytokinesis. The ring canals, cytoplasmic bridges linking the oocyte to the nurse cells in the egg chamber, are abnormal, suggesting a role of myosin II in their establishment or maintenance. In addition, numerous aggregates of myosin heavy chain accumulate in the sqh null cells. Mutant sqh transgene sqh-A20, A21 in which both serine-21 and threonine-20 have been replaced by alanines behaves in most respects identically to the null allele in this system, with the exception that no heavy chain aggregates are found. In contrast, expression of sqh-A21, in which only the primary phosphorylation target serine-21 site is altered, partially restores functionality to germ line myosin II, allowing cystoblast division and oocyte development, albeit with some cytokinesis failure, defects in the rapid cytoplasmic transport from nurse cells to cytoplasm characteristic of late stage oogenesis, and some damaged ring canals. Substituting a glutamate for the serine-21 (mutant sqh-E21) allows oogenesis to be completed with minimal defects, producing eggs that can develop normally to produce fertile adults. Flies expressing sqh-A20, in which only the secondary phosphorylation site is absent, appear to be entirely wild type. Taken together, this genetic evidence argues that phosphorylation at serine-21 is critical to RMLC function in activating myosin II in vivo, but that the function can be partially provided by phosphorylation at threonine-20.

Abstract 19086: Myofilament Proteins of the Naked Mole-rat Heart Reflect Low Basal Species Cardiac Function

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Kelly M Grimes ◽  
David Barefield ◽  
Mohit Kumar ◽  
Pieter P de Tombe ◽  
Sakthivel Sadayappan ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Troponin I ◽  
Myocardial Contraction ◽  
Myosin Heavy Chain Isoform ◽  
Mole Rat ◽  
Naked Mole Rat ◽  
And Function

The naked mole-rat (NMR) is a mouse-sized rodent with a maximum longevity of >31 years. The species exhibits low basal heart rate (256 bpm) and cardiac output (7 ml/min) for its body size, as well as low fractional shortening (28%) for a rodent. However unlike other well-studied mammals, the NMR maintains cardiac reserve and diastolic function for at least 75% of its maximum lifespan - at ages equivalent to 90 year old humans. We questioned if this low basal cardiac function was due to NMR myofilament composition and function. NMR ventricles are comprised primarily of the β-myosin heavy chain isoform, which is associated with slowed myocardial contraction and increased efficiency. This is in stark contrast to mouse ventricles, which express predominately the α-isoform, and switch to the β-isoform upon experimental induction of heart failure. Compared to mice, NMR myofilament proteins such as cardiac troponin I and cardiac myosin binding protein-C display lower levels of phosphorylation. Such levels are indicative of decreased activation of myofilament proteins and may relate to the species’ low basal cardiac function. Both the NMR’s predominance of β-myosin heavy chain and the low basal level of myofilament phosphorylation present a phenotype much closer to that seen in human ventricles than in those of mice. Intriguingly, maximal force developed by skinned NMR cardiomyocytes is not significantly different to that of mouse cardiomyocytes (NMR: 70.9 ± 9.3mN/mm2 vs. mouse: 87.7 ± 0.6 mN/mm2). This is likely a reflection of the NMR’s ability to enhance cardiac function to the level of a mouse when stimulated, as is evident when both species are treated in vivo with dobutamine (3 μg/g i.p.). Such low basal cardiac function may put less overall strain on the heart over time and could be critical to the NMR’s ability to maintain cardiac function with age.

scholarly journals The expression of myosin genes in developing skeletal muscle in the mouse embryo.

1990 ◽  
Vol 111 (4) ◽  
pp. 1465-1476 ◽  
Author(s):  
G E Lyons ◽  
M Ontell ◽  
R Cox ◽  
D Sassoon ◽  
M Buckingham
Keyword(s):  
Gene Expression ◽  
Skeletal Muscle ◽  
Myosin Heavy Chain ◽  
Heavy Chain ◽  
Light Chain ◽  
Myosin Light Chain ◽  
Muscle Development ◽  
Minor Component ◽  
Light Chain Gene ◽  
Skeletal Myosin

Using in situ hybridization, we have investigated the temporal sequence of myosin gene expression in the developing skeletal muscle masses of mouse embryos. The probes used were isoform-specific, 35S-labeled antisense cRNAs to the known sarcomeric myosin heavy chain and myosin alkali light chain gene transcripts. Results showed that both cardiac and skeletal myosin heavy chain and myosin light chain mRNAs were first detected between 9 and 10 d post coitum (p.c.) in the myotomes of the most rostral somites. Myosin transcripts appeared in more caudal somites at later stages in a developmental gradient. The earliest myosin heavy chain transcripts detected code for the embryonic skeletal (MHCemb) and beta-cardiac (MHC beta) isoforms. Perinatal myosin heavy chain (MHCpn) transcripts begin to accumulate at 10.5 d p.c., which is much earlier than previously reported. At this stage, MHCemb is the major MHC transcript. By 12.5 d p.c., MHCpn and MHCemb mRNAs are present to an equal extent, and by 15.5 d p.c. the MHCpn transcript is the major MHC mRNA detected. Cardiac MHC beta transcripts are always present as a minor component. In contrast, the cardiac MLC1A mRNA is initially more abundant than that encoding the skeletal MLC1F isoform. By 12.5 d p.c. the two MLC mRNAs are present at similar levels, and by 15.5 d p.c., MLC1F is the predominant MLC transcript detected. Transcripts for the ventricular/slow (MLC1V) and another fast skeletal myosin light chain (MLC3F) are not detected in skeletal muscle before 15 d p.c., which marks the beginning of the fetal stage of muscle development. This is the first stage at which we can detect differences in expression of myosin genes between developing muscle fibers. We conclude that, during the development of the myotome and body wall muscles, different myosin genes follow independent patterns of activation and accumulation. The data presented are the first detailed study of myosin gene expression at these early stages of skeletal muscle development.

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