Publications by authors named "Marion L Greaser"

38 Publications

Megaesophagus Is a Major Pathological Condition in Rats With a Large Deletion in the Gene.

Vet Pathol 2020 01 20;57(1):151-159. Epub 2019 Jun 20.

Muscle Biology Laboratory, University of Wisconsin, Madison, WI, USA.

A spontaneously arising, loss-of-function mutation in the RNA binding motif protein 20 () gene, which encodes a nuclear splicing protein, was previously identified as the underlying reason for expression of an abnormally large TITIN (TTN) protein in a rat model of cardiomyopathy. An outbreak of led to submission of rats with dyspnea, sneezing, lethargy, nasal discharge, and/or unexpected death for diagnostic evaluation. Necropsy revealed underlying megaesophagus in rats. Further phenotyping of this rat strain and determination of the size of esophageal TTN was undertaken. The -defective rats developed megaesophagus at an early age (26 weeks) with high frequency (13/32, 41%). They also often exhibited secondary rhinitis (9/32, 28%), aspiration pneumonia (8/32, 25%), and otitis media/interna (6/32, 19%). In addition, these rats had a high prevalence of hydronephrosis (13/32, 41%). RBM20 is involved in splicing multiple RNA transcripts, one of which is the muscle-specific protein TTN. mutations are a significant cause of dilated cardiomyopathy in humans. In -defective rats, TTN size was significantly increased in the skeletal muscle of the esophagus. Megaesophagus in this rat strain (maintained on a mixed genetic background) is hypothesized to result from altered TTN stretch signaling in esophageal skeletal muscle. This study describes a novel mechanism for the development of megaesophagus, which may be useful for understanding the pathogenesis of megaesophagus in humans and offers insights into potential myogenic causes of this condition. This is the first report of megaesophagus and other noncardiac pathogenic changes associated with mutation of in any species.
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http://dx.doi.org/10.1177/0300985819854224DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7221460PMC
January 2020

Electrophoretic Separation of Very Large Molecular Weight Proteins in SDS Agarose.

Methods Mol Biol 2019 ;1855:203-210

Department of Physiology and Biophysics and Center for Cardiovascular Research, University of Illinois at Chicago, Chicago, IL, USA.

Very large proteins (subunit sizes, >200 kDa) are difficult to electrophoretically separate on polyacrylamide gels. A SDS vertical agarose gel system has been developed that has vastly improved resolving power for very large proteins. Proteins with molecular masses between 200 and 4000 kDa can be clearly separated. Inclusion of a reducing agent in the upper reservoir buffer and use of a large pore-sized agarose have been found to be key technical procedures for obtaining optimum protein migration and resolution.
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http://dx.doi.org/10.1007/978-1-4939-8793-1_18DOI Listing
June 2019

Splicing Factor RBM20 Regulates Transcriptional Network of Titin Associated and Calcium Handling Genes in The Heart.

Int J Biol Sci 2018 9;14(4):369-380. Epub 2018 Mar 9.

Animal Science, University of Wisconsin-Madison, Madison, WI 53705, USA.

RNA binding motif 20 (RBM20) regulates pre-mRNA splicing of over thirty genes, among which titin is a major target. With RBM20 expression, titin expresses a larger isoform at fetal stage to a smaller isoform at adult resulting from alternative splicing, while, without RBM20, titin expresses exclusively a larger isoform throughout all ages. In addition to splicing regulation, it is unknown whether RBM20 also regulates gene expression. In this study, we employed knockout rats to investigate gene expression profile using Affymetrix expression array. We compared wild type to knockout at day1, 20 and 49. Bioinformatics analysis showed RBM20 regulates fewer genes expression at younger age and more at older age and commonly expressed genes have the same trends. GSEA indicated up-regulated genes are associated with heart failure. We examined titin binding partners. All titin direct binding partners are up-regulated and their increased expression is associated with dilated cardiomyopathy. Particularly, we found that genes involving calcium handling and muscle contraction are changed by RBM20. Intracellular calcium level measurement with individual cardiomyocytes further confirmed that changes of these proteins impact calcium handling. Selected genes from titin binding partners and calcium handling were validated with QPCR and western blotting. These data demonstrate that RBM20 regulates gene splicing as well as gene expression. Altered gene expression by RBM20 influences protein-protein interaction, calcium releasing and thus muscle contraction. Our results first reported gene expression impacted by RBM20 with heart maturation, and provided new insights into the role of RBM20 in the progression of heart failure.
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http://dx.doi.org/10.7150/ijbs.24117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930469PMC
May 2019

Impact of titin strain on the cardiac slow force response.

Prog Biophys Mol Biol 2017 11 22;130(Pt B):281-287. Epub 2017 Jun 22.

Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, 2160 South First Ave, Maywood, IL 60153, United States. Electronic address:

Stretch of myocardium, such as occurs upon increased filling of the cardiac chamber, induces two distinct responses: an immediate increase in twitch force followed by a slower increase in twitch force that develops over the course of several minutes. The immediate response is due, in part, to modulation of myofilament Ca sensitivity by sarcomere length (SL). The slowly developing force response, termed the Slow Force Response (SFR), is caused by a slowly developing increase in intracellular Ca upon sustained stretch. A blunted immediate force response was recently reported for myocardium isolated from homozygous giant titin mutant rats (HM) compared to muscle from wild-type littermates (WT). Here, we examined the impact of titin isoform on the SFR. Right ventricular trabeculae were isolated and mounted in an experimental chamber. SL was measured by laser diffraction. The SFR was recorded in response to a 0.2 μm SL stretch in the presence of [Ca] = 0.4 mM, a bathing concentration reflecting ∼50% of maximum twitch force development at 25 °C. Presence of the giant titin isoform (HM) was associated with a significant reduction in diastolic passive force upon stretch, and ∼50% reduction of the magnitude of the SFR; the rate of SFR development was unaffected. The sustained SL stretch was identical in both muscle groups. Therefore, our data suggest that cytoskeletal strain may underlie directly the cellular mechanisms that lead to the increased intracellular [Ca] that causes the SFR, possibly by involving cardiac myocyte integrin signaling pathways.
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http://dx.doi.org/10.1016/j.pbiomolbio.2017.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716905PMC
November 2017

Titin strain contributes to the Frank-Starling law of the heart by structural rearrangements of both thin- and thick-filament proteins.

Proc Natl Acad Sci U S A 2016 Feb 8;113(8):2306-11. Epub 2016 Feb 8.

Department of Cell and Molecular Physiology, Loyola University Chicago, Stritch School of Medicine, Maywood, IL 60153;

The Frank-Starling mechanism of the heart is due, in part, to modulation of myofilament Ca(2+) sensitivity by sarcomere length (SL) [length-dependent activation (LDA)]. The molecular mechanism(s) that underlie LDA are unknown. Recent evidence has implicated the giant protein titin in this cellular process, possibly by positioning the myosin head closer to actin. To clarify the role of titin strain in LDA, we isolated myocardium from either WT or homozygous mutant (HM) rats that express a giant splice isoform of titin, and subjected the muscles to stretch from 2.0 to 2.4 μm of SL. Upon stretch, HM compared with WT muscles displayed reduced passive force, twitch force, and myofilament LDA. Time-resolved small-angle X-ray diffraction measurements of WT twitching muscles during diastole revealed stretch-induced increases in the intensity of myosin (M2 and M6) and troponin (Tn3) reflections, as well as a reduction in cross-bridge radial spacing. Independent fluorescent probe analyses in relaxed permeabilized myocytes corroborated these findings. X-ray electron density reconstruction revealed increased mass/ordering in both thick and thin filaments. The SL-dependent changes in structure observed in WT myocardium were absent in HM myocardium. Overall, our results reveal a correlation between titin strain and the Frank-Starling mechanism. The molecular basis underlying this phenomenon appears not to involve interfilament spacing or movement of myosin toward actin but, rather, sarcomere stretch-induced simultaneous structural rearrangements within both thin and thick filaments that correlate with titin strain and myofilament LDA.
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http://dx.doi.org/10.1073/pnas.1516732113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4776536PMC
February 2016

Method for resolution and western blotting of very large proteins using agarose electrophoresis.

Methods Mol Biol 2015 ;1312:285-91

Muscle Biology Laboratory, University of Wisconsin-Madison, 1805 Linden Drive, Madison, WI, 53706, USA,

Proteins larger than 200 kDa are difficult to separate electrophoretically using polyacrylamide gels, and their transfer during western blotting is typically incomplete. A vertical SDS agarose gel system was developed that has vastly improved resolving power for very large proteins. Complete transfer of proteins as large as titin (Mr 3,000-3,700 kDa) onto blots can be achieved. The addition of a sulfhydryl reducing agent in the upper reservoir buffer and transfer buffer markedly improves the blotting of large proteins.
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http://dx.doi.org/10.1007/978-1-4939-2694-7_30DOI Listing
February 2016

Titin isoform size is not correlated with thin filament length in rat skeletal muscle.

Front Physiol 2014 3;5:35. Epub 2014 Feb 3.

Muscle Biology Laboratory, Animal Sciences, University of Wisconsin-Madison Madison, WI, USA.

The mechanisms controlling thin filament length (TFL) in muscle remain controversial. It was recently reported that TFL was related to titin size, and that the latter might be involved in TFL determination. Titin plays several crucial roles in the sarcomere, but its function as it pertains to the thin filament has not been explored. We tested this relationship using several muscles from wild type rats and from a mutant rat model (Greaser et al., 2008) which results in increased titin size. Myofibrils were isolated from skeletal muscles [extensor digitorum longus (EDL), external oblique (EO), gastrocnemius (GAS), longissimus dorsi (LD), psoas major (PM), and tibialis anterior(TA)] using both adult wild type (WT) and homozygous mutant (HM) rats (n = 6 each). Phalloidin and antibodies against tropomodulin-4 (Tmod-4) and nebulin's N-terminus were used to determine TFL. The WT rats studied express skeletal muscle titin sizes ranging from 3.2 to 3.7 MDa, while the HM rats express a giant titin isoform sized at 3.8 MDa. No differences in phalloidin based TFL, nebulin distance, or Tmod distance were observed across genotypes. However, the HM rats demonstrated a significantly increased (p < 0.01) rest sarcomere length relative to the WT phenotype. It appears that the increased titin size, predominantly observed in HM rats' middle Ig domain, allows for increased extensibility. The data indicates that, although titin performs many sarcomeric functions, its correlation with TFL and structure could not be demonstrated in the rat.
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http://dx.doi.org/10.3389/fphys.2014.00035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3910082PMC
February 2014

Pathophysiological defects and transcriptional profiling in the RBM20-/- rat model.

PLoS One 2013 19;8(12):e84281. Epub 2013 Dec 19.

Muscle Biology Laboratory, University of Wisconsin-Madison, Madison, Wisconsin, United States of America.

Our recent study indicated that RNA binding motif 20 (Rbm20) alters splicing of titin and other genes. The current goals were to understand how the Rbm20(-/-) rat is related to physiological, structural, and molecular changes leading to heart failure. We quantitatively and qualitatively compared the expression of titin isoforms between Rbm20(-/-) and wild type rats by real time RT-PCR and SDS agarose electrophoresis. Isoform changes were linked to alterations in transcription as opposed to translation of titin messages. Reduced time to exhaustion with running in knockout rats also suggested a lower maximal cardiac output or decreased skeletal muscle performance. Electron microscopic observations of the left ventricle from knockout animals showed abnormal myofibril arrangement, Z line streaming, and lipofuscin deposits. Mutant skeletal muscle ultrastructure appeared normal. The results suggest that splicing alterations in Rbm20(-/-) rats resulted in pathogenic changes in physiology and cardiac ultrastructure. Secondary changes were observed in message levels for many genes whose splicing was not directly affected. Gene and protein expression data indicated the activation of pathophysiological and muscle stress-activated pathways. These data provide new insights on Rbm20 function and how its malfunction leads to cardiomyopathy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084281PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3868568PMC
October 2014

Titin-mediated control of cardiac myofibrillar function.

Arch Biochem Biophys 2014 Jun 20;552-553:83-91. Epub 2013 Nov 20.

Department of Medical Pharmacology & Physiology, School of Medicine, University of Missouri, Columbia, MO 65212, United States. Electronic address:

According to the Frank-Starling relationship, ventricular pressure or stroke volume increases with end-diastolic volume. This is regulated, in large part, by the sarcomere length (SL) dependent changes in cardiac myofibrillar force, loaded shortening, and power. Consistent with this, both cardiac myofibrillar force and absolute power fall at shorter SL. However, when Ca(2+) activated force levels are matched between short and long SL (by increasing the activator [Ca(2+)]), short SL actually yields faster loaded shortening and greater peak normalized power output (PNPO). A potential mechanism for faster loaded shortening at short SL is that, at short SL, titin becomes less taut, which increases the flexibility of the cross-bridges, a process that may be mediated by titin's interactions with thick filament proteins. We propose a more slackened titin yields greater myosin head radial and azimuthal mobility and these flexible cross-bridges are more likely to maintain thin filament activation, which would allow more force-generating cross-bridges to work against a fixed load resulting in faster loaded shortening. We tested this idea by measuring SL-dependence of power at matched forces in rat skinned cardiac myocytes containing either N2B titin or a longer, more compliant N2BA titin. We predicted that, in N2BA titin containing cardiac myocytes, power-load curves would not be shifted upward at short SL compared to long SL (when force is matched). Consistent with this, peak normalized power was actually less at short SL versus long SL (at matched force) in N2BA-containing myocytes (N2BA titin: ΔPNPO (Short SL peak power minus long SL peak power)=-0.057±0.049 (n=5) versus N2B titin: ΔPNPO=+0.012±0.012 (n=5). These findings support a model whereby SL per se controls mechanical properties of cross-bridges and this process is mediated by titin. This myofibrillar mechanism may help sustain ventricular power during periods of low preloads, and perhaps a breakdown of this mechanism is involved in impaired function of failing hearts.
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http://dx.doi.org/10.1016/j.abb.2013.11.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4028433PMC
June 2014

John Gergely (1919-2013): a pillar in the muscle protein field.

J Muscle Res Cell Motil 2013 Dec 22;34(5-6):441-6. Epub 2013 Nov 22.

University of Wisconsin-Madison, Madison, WI, USA,

Dr. John Gergely passed away on July 26, 2013 after a long and distinguished career. His publications spanned 67 years. He founded the Department of Muscle Research in the Retina Foundation (which later became the Boston Biomedical Research Institute) and served as director for 34 years. Dr. Gergely served on the editorial boards of ten scientific journals. He was elected as a Fellow of both the Biophysical Society and the American Association for the Advancement of Science. Dr. Gergely made major contributions concerning muscle protein structure and function. He was best known for his work on the troponin complex. The insights of John and his associates have provided the foundation for our understanding of calcium regulation in skeletal and cardiac muscle.
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http://dx.doi.org/10.1007/s10974-013-9370-9DOI Listing
December 2013

Rbm20 regulates titin alternative splicing as a splicing repressor.

Nucleic Acids Res 2013 Feb 9;41(4):2659-72. Epub 2013 Jan 9.

Muscle Biology Laboratory, Department of Animal Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.

Titin, a sarcomeric protein expressed primarily in striated muscles, is responsible for maintaining the structure and biomechanical properties of muscle cells. Cardiac titin undergoes developmental size reduction from 3.7 megadaltons in neonates to primarily 2.97 megadaltons in the adult. This size reduction results from gradually increased exon skipping between exons 50 and 219 of titin mRNA. Our previous study reported that Rbm20 is the splicing factor responsible for this process. In this work, we investigated its molecular mechanism. We demonstrate that Rbm20 mediates exon skipping by binding to titin pre-mRNA to repress the splicing of some regions; the exons/introns in these Rbm20-repressed regions are ultimately skipped. Rbm20 was also found to mediate intron retention and exon shuffling. The two Rbm20 speckles found in nuclei from muscle tissues were identified as aggregates of Rbm20 protein on the partially processed titin pre-mRNAs. Cooperative repression and alternative 3' splice site selection were found to be used by Rbm20 to skip different subsets of titin exons, and the splicing pathway selected depended on the ratio of Rbm20 to other splicing factors that vary with tissue type and developmental age.
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http://dx.doi.org/10.1093/nar/gks1362DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3575840PMC
February 2013

Impact of titin isoform on length dependent activation and cross-bridge cycling kinetics in rat skeletal muscle.

Biochim Biophys Acta 2013 Apr 23;1833(4):804-11. Epub 2012 Aug 23.

Department of Cell and Molecular Physiology, Loyola University Medical Center, Maywood, IL 60153, USA.

The magnitude of length dependent activation in striated muscle has been shown to vary with titin isoform. Recently, a rat that harbors a homozygous autosomal mutation (HM) causing preferential expression of a longer, giant titin isoform was discovered (Greaser et al. 2005). Here, we investigated the impact of titin isoform on myofilament force development and cross-bridge cycling kinetics as function of sarcomere length (SL) in tibialis anterior skeletal muscle isolated from wild type (WT) and HM. Skeletal muscle bundles from HM rats exhibited reductions in passive tension, maximal force development, myofilament calcium sensitivity, maximal ATP consumption, and tension cost at both short and long sarcomere length (SL=2.8μm and SL=3.2μm, respectively). Moreover, the SL-dependent changes in these parameters were attenuated in HM muscles. Additionally, myofilament Ca(2+) activation-relaxation properties were assessed in single isolated myofibrils. Both the rate of tension generation upon Ca(2+) activation (kACT) as well as the rate of tension redevelopment following a length perturbation (kTR) were reduced in HM myofibrils compared to WT, while relaxation kinetics were not affected. We conclude that presence of a long isoform of titin in the striated muscle sarcomere is associated with reduced myofilament force development and cross-bridge cycling kinetics, and a blunting of myofilament length dependent activation. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.
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http://dx.doi.org/10.1016/j.bbamcr.2012.08.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518735PMC
April 2013

Protein electrophoresis in agarose gels for separating high molecular weight proteins.

Methods Mol Biol 2012 ;869:111-8

University of Wisconsin-Madison, Madison, WI, USA.

Very large proteins (subunit sizes >200 kDa) are difficult to electrophoretically separate on polyacrylamide gels. A SDS vertical agarose gel system has been developed that has vastly improved resolving power for very large proteins. Proteins with molecular masses between 200 and 4,000 kDa can be clearly separated. Inclusion of a reducing agent in the upper reservoir buffer has been found to be a key technical procedure for obtaining optimum resolution.
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http://dx.doi.org/10.1007/978-1-61779-821-4_10DOI Listing
September 2012

RBM20, a gene for hereditary cardiomyopathy, regulates titin splicing.

Nat Med 2012 May;18(5):766-73

Muscle Biology Laboratory, University of Wisconsin Madison, USA.

Alternative splicing has a major role in cardiac adaptive responses, as exemplified by the isoform switch of the sarcomeric protein titin, which adjusts ventricular filling. By positional cloning using a previously characterized rat strain with altered titin mRNA splicing, we identified a loss-of-function mutation in the gene encoding RNA binding motif protein 20 (Rbm20) as the underlying cause of pathological titin isoform expression. The phenotype of Rbm20-deficient rats resembled the pathology seen in individuals with dilated cardiomyopathy caused by RBM20 mutations. Deep sequencing of the human and rat cardiac transcriptome revealed an RBM20-dependent regulation of alternative splicing. In addition to titin (TTN), we identified a set of 30 genes with conserved splicing regulation between humans and rats. This network is enriched for genes that have previously been linked to cardiomyopathy, ion homeostasis and sarcomere biology. Our studies emphasize the key role of post-transcriptional regulation in cardiac function and provide mechanistic insights into the pathogenesis of human heart failure.
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http://dx.doi.org/10.1038/nm.2693DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3569865PMC
May 2012

Magnitude of length-dependent changes in contractile properties varies with titin isoform in rat ventricles.

Am J Physiol Heart Circ Physiol 2012 Feb 2;302(3):H697-708. Epub 2011 Dec 2.

Dept. of Physiology, Univ. of Wisconsin, 601 Science Drive, Madison, WI 53711, USA.

The effects of differential expression of titin isoforms on sarcomere length (SL)-dependent changes in passive force, maximum Ca(2+)-activated force, apparent cooperativity in activation of force (n(H)), Ca(2+) sensitivity of force (pCa(50)), and rate of force redevelopment (k(tr)) were investigated in rat cardiac muscle. Skinned right ventricular trabeculae were isolated from wild-type (WT) and mutant homozygote (Ho) hearts expressing predominantly a smaller N2B isoform (2,970 kDa) and a giant N2BA-G isoform (3,830 kDa), respectively. Stretching WT and Ho trabeculae from SL 2.0 to 2.35 μm increased passive force, maximum Ca(2+)-activated force, and pCa(50), and it decreased n(H) and k(tr). Compared with WT trabeculae, the magnitude of SL-dependent changes in passive force, maximum Ca(2+)-activated force, pCa(50), and n(H) was significantly smaller in Ho trabeculae. These results suggests that, at least in rat ventricle, the magnitude of SL-dependent changes in passive force, maximum Ca(2+)-activated force, pCa(50), n(H), and k(tr) is defined by the titin isoform.
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http://dx.doi.org/10.1152/ajpheart.00800.2011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3353780PMC
February 2012

Comprehensive analysis of titin protein isoform and alternative splicing in normal and mutant rats.

J Cell Biochem 2012 Apr;113(4):1265-73

Muscle Biology Laboratory, Department of Animal Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.

Titin is a giant protein with multiple functions in cardiac and skeletal muscles. Rat cardiac titin undergoes developmental isoform transition from the neonatal 3.7 MDa N2BA isoform to primarily the adult 2.97 MDa N2B isoform. An autosomal dominant mutation dramatically altered this transformation. Titins from eight skeletal muscles: Tibialis Anterior (TA), Longissimus Dorsi (LD) and Gastrocnemius (GA), Extensor Digitorum Longus (ED), Soleus (SO), Psoas (PS), Extensor Oblique (EO), and Diaphram (DI) were characterized in wild type and in homozygous mutant (Hm) rats with a titin splicing defect. Results showed that the developmental reduction in titin size is eliminated in the mutant rat so that the titins in all investigated skeletal muscles remain large in the adult. The alternative splicing of titin mRNA was found repressed by this mutation, a result consistent with the large titin isoform in the mutant. The developmental pattern of titin mRNA alternative splicing differs between heart and skeletal muscles. The retention of intron 49 reveals a possible mechanism for the absence of the N2B unique region in the expressed titin protein of skeletal muscle.
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http://dx.doi.org/10.1002/jcb.23459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6696936PMC
April 2012

Passive mechanical properties and related proteins change with botulinum neurotoxin A injection of normal skeletal muscle.

J Orthop Res 2012 Mar 18;30(3):497-502. Epub 2011 Aug 18.

Department of Bioengineering, University of California and Veterans Administration Medical Centers San Diego, La Jolla, California, USA.

The effects of botulinum neurotoxin A on the passive mechanical properties of skeletal muscle have not been investigated, but may have significant impact in the treatment of neuromuscular disorders including spasticity. Single fiber and fiber bundle passive mechanical testing was performed on rat muscles treated with botulinum neurotoxin A. Myosin heavy chain and titin composition of single fibers was determined by gel electrophoresis. Muscle collagen content was determined using a hydroxyproline assay. Neurotoxin-treated single fiber passive elastic modulus was reduced compared to control fibers (53.00 kPa vs. 63.43 kPa). Fiber stiffness and slack sarcomere length were also reduced compared to control fibers and myosin heavy chain composition shifted from faster to slower isoforms. Average titin molecular weight increased 1.77% after treatment. Fiber bundle passive elastic modulus increased following treatment (168.83  kPa vs. 75.14 kPa). Bundle stiffness also increased while collagen content per mass of muscle tissue increased 38%. Injection of botulinum neurotoxin A produces an effect on the passive mechanical properties of normal muscle that is opposite to the changes observed in spastic muscles.
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http://dx.doi.org/10.1002/jor.21533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3227753PMC
March 2012

Myosin binding protein C interaction with actin: characterization and mapping of the binding site.

J Biol Chem 2011 Jan 11;286(3):2008-16. Epub 2010 Nov 11.

Department of Physiology, University of Wisconsin Medical School, Madison, Wisconsin 53706, USA.

Myosin binding protein C (MyBPC) is a multidomain protein associated with the thick filaments of striated muscle. Although both structural and regulatory roles have been proposed for MyBPC, its interactions with other sarcomeric proteins remain obscure. The current study was designed to examine the actin-binding properties of MyBPC and to define MyBPC domain regions involved in actin interaction. Here, we have expressed full-length mouse cardiac MyBPC (cMyBPC) in a baculovirus system and shown that purified cMyBPC binds actin filaments with an affinity of 4.3 ± 1.1 μM and a 1:1 molar ratio with regard to an actin protomer. The actin binding by cMyBPC is independent of protein phosphorylation status and is not significantly affected by the presence of tropomyosin and troponin on the actin filament. In addition, cMyBPC-actin interaction is not modulated by calmodulin. To determine the region of cMyBPC that is responsible for its interaction with actin, we have expressed and characterized five recombinant proteins encoding fragments of the cMyBPC sequence. Recombinant N-terminal fragments such as C0-C1, C0-C4, and C0-C5 cosediment with actin in a linear, nonsaturable manner. At the same time, MyBPC fragments lacking either the C0-C1 or C0-C4 region bind F-actin with essentially the same properties as full-length protein. Together, our results indicate that cMyBPC interacts with actin via a single, moderate affinity site localized to the C-terminal region of the protein. In contrast, certain basic regions of the N-terminal domains of MyBPC may act as small polycations and therefore bind actin via nonspecific electrostatic interactions.
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http://dx.doi.org/10.1074/jbc.M110.170605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3023497PMC
January 2011

Titin diversity--alternative splicing gone wild.

J Biomed Biotechnol 2010 21;2010:753675. Epub 2010 Mar 21.

Muscle Biology Laboratory, University of Wisconsin-Madison, Madison, WI 53706, USA.

Titin is an extremely large protein found in highest concentrations in heart and skeletal muscle. The single mammalian gene is expressed in multiple isoforms as a result of alternative splicing. Although titin isoform expression is controlled developmentally and in a tissue specific manner, the vast number of potential splicing pathways far exceeds those described in any other alternatively spliced gene. Over 1 million human splice pathways for a single individual can be potentially derived from the PEVK region alone. A new splicing pattern for the human cardiac N2BA isoform type has been found in which the PEVK region includes only the N2B type exons. The alterations in splicing and titin isoform expression in human heart disease provide impetus for future detailed study of the splicing mechanisms for this giant protein.
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http://dx.doi.org/10.1155/2010/753675DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2843904PMC
June 2010

The genome sequence of taurine cattle: a window to ruminant biology and evolution.

Science 2009 Apr;324(5926):522-8

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.
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http://dx.doi.org/10.1126/science.1169588DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2943200PMC
April 2009

Efficient electroblotting of very large proteins using a vertical agarose electrophoresis system.

Methods Mol Biol 2009 ;536:221-7

University of Wisconsin-Madison, 1805 Linden Drive, Madison, WI, 53706, USA.

Very large proteins (subunit sizes >200 kDa) are difficult to electrophoretically separate, and they are also challenging to analyze by western blotting because of their incomplete transfer out of polyacrylamide gels. An SDS vertical agarose gel system has been developed that has vastly improved resolving power for very large proteins. The large pores of the agarose also allow full transfer of proteins as large as titin (Mr =3,000-3,700 kDa) onto blots. Inclusion of a reducing agent in the upper reservoir buffer and transfer buffer has been found to be a key technical procedure in blotting large proteins.
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http://dx.doi.org/10.1007/978-1-59745-542-8_24DOI Listing
June 2009

GelBandFitter--a computer program for analysis of closely spaced electrophoretic and immunoblotted bands.

Electrophoresis 2009 Mar;30(5):848-51

Department of Physiology, University of Kentucky, Lexington, KY 40536-0298, USA.

GelBandFitter is a computer program that uses non-linear regression techniques to fit mathematical functions to densitometry profiles of protein gels. This allows for improved quantification of gels with partially overlapping and potentially asymmetric protein bands. The program can also be used to analyze immunoblots with closely spaced bands. GelBandFitter was developed in Matlab and the source code and/or a Windows executable file can be downloaded at no cost to academic users from http://www.gelbandfitter.org.
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http://dx.doi.org/10.1002/elps.200800583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2742644PMC
March 2009

Acceleration of crossbridge kinetics by protein kinase A phosphorylation of cardiac myosin binding protein C modulates cardiac function.

Circ Res 2008 Oct 18;103(9):974-82. Epub 2008 Sep 18.

Department of Physiology, University of Wisconsin School of Medicine and Public Health, Madison, WI 53706, USA.

Normal cardiac function requires dynamic modulation of contraction. beta1-adrenergic-induced protein kinase (PK)A phosphorylation of cardiac myosin binding protein (cMyBP)-C may regulate crossbridge kinetics to modulate contraction. We tested this idea with mechanical measurements and echocardiography in a mouse model lacking 3 PKA sites on cMyBP-C, ie, cMyBP-C(t3SA). We developed the model by transgenic expression of mutant cMyBP-C with Ser-to-Ala mutations on the cMyBP-C knockout background. Western blots, immunofluorescence, and in vitro phosphorylation combined to show that non-PKA-phosphorylatable cMyBP-C expressed at 74% compared to normal wild-type (WT) and was correctly positioned in the sarcomeres. Similar expression of WT cMyBP-C at 72% served as control, ie, cMyBP-C(tWT). Skinned myocardium responded to stretch with an immediate increase in force, followed by a transient relaxation of force and finally a delayed development of force, ie, stretch activation. The rate constants of relaxation, k(rel) (s-1), and delayed force development, k(df) (s-1), in the stretch activation response are indicators of crossbridge cycling kinetics. cMyBP-C(t3SA) myocardium had baseline k(rel) and k(df) similar to WT myocardium, but, unlike WT, k(rel) and k(df) were not accelerated by PKA treatment. Reduced dobutamine augmentation of systolic function in cMyBP-C(t3SA) hearts during echocardiography corroborated the stretch activation findings. Furthermore, cMyBP-C(t3SA) hearts exhibited basal echocardiographic findings of systolic dysfunction, diastolic dysfunction, and hypertrophy. Conversely, cMyBP-C(tWT) hearts performed similar to WT. Thus, PKA phosphorylation of cMyBP-C accelerates crossbridge kinetics and loss of this regulation leads to cardiac dysfunction.
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http://dx.doi.org/10.1161/CIRCRESAHA.108.177683DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2867079PMC
October 2008

Mutation that dramatically alters rat titin isoform expression and cardiomyocyte passive tension.

J Mol Cell Cardiol 2008 Jun 23;44(6):983-91. Epub 2008 Feb 23.

Muscle Biology Laboratory, University of Wisconsin-Madison, 1805 Linden Drive, Madison, WI 53706, USA.

Titin is a very large alternatively spliced protein that performs multiple functions in heart and skeletal muscles. A rat strain is described with an autosomal dominant mutation that alters the isoform expression of titin. While wild type animals go through a developmental program where the 3.0 MDa N2B becomes the major isoform expressed by two to three weeks after birth (approximately 85%), the appearance of the N2B is markedly delayed in heterozygotes and never reaches more than 50% of the titin in the adult. Homozygote mutants express a giant titin of the N2BA isoform type (3.9 MDa) that persists as the primary titin species through ages of more than one and a half years. The mutation does not affect the isoform switching of troponin T, a protein that is also alternatively spliced with developmental changes. The basis for the apparently greater size of the giant titin in homozygous mutants was not determined, but the additional length was not due to inclusion of sequence from larger numbers of PEVK exons or the Novex III exon. Passive tension measurements using isolated cardiomyocytes from homozygous mutants showed that cells could be stretched to sarcomere lengths greater than 4 mum without breakage. This novel rat model should be useful for exploring the potential role of titin in the Frank-Starling relationship and mechano-sensing/signaling mechanisms.
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http://dx.doi.org/10.1016/j.yjmcc.2008.02.272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2501117PMC
June 2008

Structural and regulatory roles of muscle ankyrin repeat protein family in skeletal muscle.

Am J Physiol Cell Physiol 2007 Jul 28;293(1):C218-27. Epub 2007 Mar 28.

Dept. of Orthopaedics, University of California, Department of Veterans Affairs Medical Center, San Diego, CA 92161, USA.

The biological response of muscle to eccentric contractions (ECs) results in strengthening and protection from further injury. However, the cellular basis for this response remains unclear. Previous studies identified the muscle ankyrin repeat protein (MARP) family, consisting of cardiac ankyrin repeat protein (CARP), ankyrin repeat domain 2/ankyrin repeat protein with PEST and proline-rich region (Ankrd2/Arpp), and diabetes-associated ankyrin repeat protein (DARP), as rapidly and specifically upregulated in mice after a single bout of EC. To determine the role of these genes in skeletal muscle, a survey of skeletal muscle structural and functional characteristics was performed on mice lacking all three MARP family members (MKO). There was a slight trend toward MKO muscles having a slower fiber type distribution but no differences in muscle fiber size. Single MKO fibers were less stiff, tended to have longer resting sarcomere lengths, and expressed a longer isoform of titin than their wild-type counterparts, indicating that these proteins may play a role in the passive mechanical behavior of muscle. Finally, MKO mice showed a greater degree of torque loss after a bout of ECs compared with wild-type mice, although they recovered from the injury with the same or even improved time course. This recovery was associated with enhanced expression of the muscle regulatory genes MyoD and muscle LIM protein (MLP), suggesting that the MARP family may play both important structural and gene regulatory roles in skeletal muscle.
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http://dx.doi.org/10.1152/ajpcell.00055.2007DOI Listing
July 2007

Studies on titin PEVK peptides and their interaction.

Arch Biochem Biophys 2006 Oct 15;454(1):16-25. Epub 2006 Aug 15.

Muscle Biology Laboratory, University of Wisconsin-Madison, Madison, WI 53706, USA.

Experiments were conducted on several synthetic and expressed peptides from the PEVK region of titin, the giant muscle protein. Different secondary structure prediction methods based on amino acid sequence gave estimates ranging from over 70% alpha helical to no helix (totally disordered) for the polyE peptide corresponding to human exon 115. Circular dichroism (CD) experiments demonstrated that both the positively charged PPAK modules and the negatively charged PolyE repeats had similar spectral properties with disordered secondary structure predominating. Gel permeation chromatography showed that both PPAK and polyE peptides had 2-4 times larger Stokes radii than expected from their molecular mass. Mixtures of the oppositely charged titin peptides caused no change in apparent secondary structure as observed by circular dichroism or migration properties using native gel electrophoresis. Similarly addition of calcium did not alter the CD spectra or peptide electrophoretic mobility of the individual peptides or their mixtures. The properties of both the PPAK and polyE type peptides suggest that both had most of the characteristic properties to be classified as intrinsically disordered proteins.
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http://dx.doi.org/10.1016/j.abb.2006.07.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1635410PMC
October 2006

Developmental changes in rat cardiac titin/connectin: transitions in normal animals and in mutants with a delayed pattern of isoform transition.

J Muscle Res Cell Motil 2005 ;26(6-8):325-32

University of Wisconsin-Madison, Madison, WI 53706, USA.

Rat cardiac titin undergoes developmental changes in isoform expression during the period from late embryonic through the first 20-25 days of life. At least five size classes of titin isoforms have been identified using SDS agarose gel electrophoresis. The longest normal isoform is expressed in the embryonic stages, and it is progressively replaced with increasingly smaller versions. The isoform switching is consistent with changes in resting tension from lower values in one-day neonates to higher levels in adult myocytes. Considerable micro-heterogeneity in alternative splicing patterns also was found, particularly in the N2BA PEVK region of human, rat, and dog ventricle. A rat mutation has been identified in which the embryonic-neonatal titin isoform transitions are markedly delayed. These mutant animals may prove useful for examining the role of titin in stretch-activated signal transduction and in the Frank-Starling relationship.
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http://dx.doi.org/10.1007/s10974-005-9039-0DOI Listing
January 2007

Muscle LIM protein plays both structural and functional roles in skeletal muscle.

Am J Physiol Cell Physiol 2005 Nov 10;289(5):C1312-20. Epub 2005 Aug 10.

Deptartment of Orthopaedics, Veterans Affairs Medical Center and Univ. of California, San Diego, 3350 La Jolla Village Drive, San Diego, CA 92161, USA.

Muscle LIM protein (MLP) has been suggested to be an important mediator of mechanical stress in cardiac tissue, but the role that it plays in skeletal muscle remains unclear. Previous studies have shown that it is dramatically upregulated in fast-to-slow fiber-type transformation and also after eccentric contraction (EC)-induced muscle injury. The functional consequences of this upregulation, if any, are unclear. In the present study, we have examined the skeletal muscle phenotype of MLP-knockout (MLPKO) mice in terms of their response to EC-induced muscle injuries. The data suggest that while the MLPKO mice recover completely after EC-induced injury, their torque production lags behind that of heterozygous littermates in the early stages of the recovery process. This lag is accompanied by decreased expression of the muscle regulatory factor MyoD, suggesting that MLP may influence gene expression. In addition, there is evidence of type I fiber atrophy and a shorter resting sarcomere length in the MLPKO mice, but no significant differences in fiber type distribution. In summary, MLP appears to play a subtle role in the maintenance of normal muscle characteristics and in the early events of the recovery process of skeletal muscle to injury, serving both structural and gene-regulatory roles.
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http://dx.doi.org/10.1152/ajpcell.00117.2005DOI Listing
November 2005

Titin isoform changes in rat myocardium during development.

Mech Dev 2004 Nov;121(11):1301-12

Muscle Biology Laboratory, 1805 Linden Drive West, University of Wisconsin-Madison, Madison, WI 53706, USA.

Developmental changes in the alternative splicing patterns of titin were observed in rat cardiac muscle. Titin from 16-day fetal hearts consisted of a single 3710 kDa band on SDS agarose gels, and it disappeared by 10 days after birth. The major adult N2B isoform (2990 kDa) first appeared in 18-day fetal hearts and its proportion in the ventricle increased to approximately 85% from 20 days of age and older. Changes in three other intermediate-sized N2BA isoform bands also occurred during this same time period. The cDNA sequences of fetal cardiac, adult ventricle, and adult soleus were different in the PEVK and alternatively spliced middle Ig domain. Extensive heterogeneity in splice patterns was found in the N2BA PEVK region. The extra length of the fetal titin isoforms appeared to be due to both a greater number of middle Ig domains expressed plus the inclusion of more PEVK exons. Passive tension measurements on myocyte-sized fragments indicated a significantly lower tension in neonate versus adult ventricles at sarcomere lengths greater than 2.1 microm, consistent with the protein and cDNA sequence results. The time course of the titin isoform switching was similar to that occurring with myosin and troponin I during development.
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http://dx.doi.org/10.1016/j.mod.2004.07.003DOI Listing
November 2004

Postmortem changes in myofibrillar-bound calpain 3 revealed by immunofluorescence microscopy.

Meat Sci 2004 Jan;66(1):231-40

Molecular Biotechnology Group, Animal and Food Sciences Division, PO Box 84, Lincoln University, Canterbury, New Zealand.

An immunofluorescence microscopy method for following changes in myofibrillar-bound calpain 3 was developed. Afterward, proteolytic changes in calpain 3(p94), calpain 1, titin, and nebulin were examined in myofibrils prepared from ovine longissimusthoracis et lumborum (LTL) stored for 0, 1, 2, and 3 days postmortem. Western blot analysis revealed that the levels of intact calpain 3 (expressed as percentage of the level immediately postmortem) were 80%, 10% and not detectable in myofibrils prepared at 1, 2, and 3 days, respectively. Western blots for calpain 1 also indicated conversion of the intact protein (80 kDa) to a 76 kDa fragment during the same time period. Thus calpains 1 and 3 appear to be activated during postmortem storage. Immunofluorescence microscopy using an IS1 region specific antibody revealed that calpain 3 staining was most intense at the sarcomere Z- and M-lines. The fluorescence intensity declined significantly during storage, paralleling changes in the proteolytic breakdown of titin and nebulin associated with these structures.
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http://dx.doi.org/10.1016/S0309-1740(03)00096-2DOI Listing
January 2004
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