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Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function
The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain...
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| Published in: | American journal of physiology. Heart and circulatory physiology 2011-02, Vol.300 (2), p.H574-H582 |
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| Main Authors: | , , , , |
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| cites | cdi_FETCH-LOGICAL-c431t-a7f71c6a41e5f6a1fe16f0e3e55d2d19e00deefa5c47b2310f336a8da7e4821a3 |
| container_end_page | H582 |
| container_issue | 2 |
| container_start_page | H574 |
| container_title | American journal of physiology. Heart and circulatory physiology |
| container_volume | 300 |
| creator | Vandenboom, Rene Herron, Todd Favre, Elizabeth Albayya, Faris P Metzger, Joseph M |
| description | The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P |
| doi_str_mv | 10.1152/ajpheart.00786.2009 |
| format | article |
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To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.</description><identifier>ISSN: 0363-6135</identifier><identifier>EISSN: 1522-1539</identifier><identifier>DOI: 10.1152/ajpheart.00786.2009</identifier><identifier>PMID: 21112946</identifier><identifier>CODEN: AJPPDI</identifier><language>eng</language><publisher>United States: American Physiological Society</publisher><subject>Actin Cytoskeleton - physiology ; Animals ; Blotting, Western ; Calcium Signaling - genetics ; Calcium Signaling - physiology ; Cardiac Excitation and Contraction ; Cardiomyocytes ; Cell Membrane Permeability - physiology ; Cell Separation ; DNA, Complementary - biosynthesis ; DNA, Complementary - genetics ; Electrophoresis, Polyacrylamide Gel ; Fluorescent Antibody Technique ; Gene expression ; Gene Transfer Techniques ; Genetic Vectors ; Humans ; Immunohistochemistry ; Immunoprecipitation ; Molecules ; Myocardial Contraction - physiology ; Myocardium - metabolism ; Myocytes, Cardiac - metabolism ; Myosin Heavy Chains - biosynthesis ; Myosin Heavy Chains - genetics ; Myosins - biosynthesis ; Myosins - chemistry ; Myosins - genetics ; Physiology ; Protein Conformation ; Rats ; Sarcomeres - metabolism ; Studies</subject><ispartof>American journal of physiology. Heart and circulatory physiology, 2011-02, Vol.300 (2), p.H574-H582</ispartof><rights>Copyright American Physiological Society Feb 2011</rights><rights>Copyright © 2011 the American Physiological Society 2011</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c431t-a7f71c6a41e5f6a1fe16f0e3e55d2d19e00deefa5c47b2310f336a8da7e4821a3</citedby><cites>FETCH-LOGICAL-c431t-a7f71c6a41e5f6a1fe16f0e3e55d2d19e00deefa5c47b2310f336a8da7e4821a3</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>230,315,786,790,891,27957,27958</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/21112946$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Vandenboom, Rene</creatorcontrib><creatorcontrib>Herron, Todd</creatorcontrib><creatorcontrib>Favre, Elizabeth</creatorcontrib><creatorcontrib>Albayya, Faris P</creatorcontrib><creatorcontrib>Metzger, Joseph M</creatorcontrib><title>Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function</title><title>American journal of physiology. Heart and circulatory physiology</title><addtitle>Am J Physiol Heart Circ Physiol</addtitle><description>The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.</description><subject>Actin Cytoskeleton - physiology</subject><subject>Animals</subject><subject>Blotting, Western</subject><subject>Calcium Signaling - genetics</subject><subject>Calcium Signaling - physiology</subject><subject>Cardiac Excitation and Contraction</subject><subject>Cardiomyocytes</subject><subject>Cell Membrane Permeability - physiology</subject><subject>Cell Separation</subject><subject>DNA, Complementary - biosynthesis</subject><subject>DNA, Complementary - genetics</subject><subject>Electrophoresis, Polyacrylamide Gel</subject><subject>Fluorescent Antibody Technique</subject><subject>Gene expression</subject><subject>Gene Transfer Techniques</subject><subject>Genetic Vectors</subject><subject>Humans</subject><subject>Immunohistochemistry</subject><subject>Immunoprecipitation</subject><subject>Molecules</subject><subject>Myocardial Contraction - physiology</subject><subject>Myocardium - metabolism</subject><subject>Myocytes, Cardiac - metabolism</subject><subject>Myosin Heavy Chains - biosynthesis</subject><subject>Myosin Heavy Chains - genetics</subject><subject>Myosins - biosynthesis</subject><subject>Myosins - chemistry</subject><subject>Myosins - genetics</subject><subject>Physiology</subject><subject>Protein Conformation</subject><subject>Rats</subject><subject>Sarcomeres - metabolism</subject><subject>Studies</subject><issn>0363-6135</issn><issn>1522-1539</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2011</creationdate><recordtype>article</recordtype><recordid>eNpdkc1u1DAUhS0EotPCEyAhi003zdSOYydhgVRVpSBV6gbW1q19TT1K7GAnI-ZpeFU80x8BkiUvznfP_TmEvONszbmsz2Ez3SOkec1Y26l1zVj_gqyKUldciv4lWTGhRKW4kEfkOOcNY0y2SrwmRzXnvO4btSK_rzEgnROE7DCdUfw1JczZx3BGIViaIZk4YvKG-mBimmKCuag0Ogq09LdDwem4i9kHOsYBzTJgYamBZD2YvWR2M-aPFLfeYjBIXUyluPTBtD2whXF-gBHDXDzmIrslmH2fN-SVgyHj28f_hHz_fPXt8kt1c3v99fLipjKN4HMFrWu5UdBwlE4Bd8iVYyhQSltb3iNjFtGBNE17VwvOnBAKOgstNl3NQZyQTw--03I3ojVlkgSDnpIfIe10BK__VYK_1z_iVgvWNOWsxeD00SDFnwvmWY8-GxwGCBiXrLum60pEoivkh__ITVxSKNvpTrLD6wskHiCTYs4J3fMonOl9_Popfn2IX-_jL1Xv_97iueYpb_EHrsyzIg</recordid><startdate>20110201</startdate><enddate>20110201</enddate><creator>Vandenboom, Rene</creator><creator>Herron, Todd</creator><creator>Favre, Elizabeth</creator><creator>Albayya, Faris P</creator><creator>Metzger, Joseph M</creator><general>American Physiological Society</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QP</scope><scope>7QR</scope><scope>7TS</scope><scope>7U7</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20110201</creationdate><title>Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function</title><author>Vandenboom, Rene ; Herron, Todd ; Favre, Elizabeth ; Albayya, Faris P ; Metzger, Joseph M</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c431t-a7f71c6a41e5f6a1fe16f0e3e55d2d19e00deefa5c47b2310f336a8da7e4821a3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2011</creationdate><topic>Actin Cytoskeleton - physiology</topic><topic>Animals</topic><topic>Blotting, Western</topic><topic>Calcium Signaling - genetics</topic><topic>Calcium Signaling - physiology</topic><topic>Cardiac Excitation and Contraction</topic><topic>Cardiomyocytes</topic><topic>Cell Membrane Permeability - physiology</topic><topic>Cell Separation</topic><topic>DNA, Complementary - biosynthesis</topic><topic>DNA, Complementary - genetics</topic><topic>Electrophoresis, Polyacrylamide Gel</topic><topic>Fluorescent Antibody Technique</topic><topic>Gene expression</topic><topic>Gene Transfer Techniques</topic><topic>Genetic Vectors</topic><topic>Humans</topic><topic>Immunohistochemistry</topic><topic>Immunoprecipitation</topic><topic>Molecules</topic><topic>Myocardial Contraction - physiology</topic><topic>Myocardium - metabolism</topic><topic>Myocytes, Cardiac - metabolism</topic><topic>Myosin Heavy Chains - biosynthesis</topic><topic>Myosin Heavy Chains - genetics</topic><topic>Myosins - biosynthesis</topic><topic>Myosins - chemistry</topic><topic>Myosins - genetics</topic><topic>Physiology</topic><topic>Protein Conformation</topic><topic>Rats</topic><topic>Sarcomeres - metabolism</topic><topic>Studies</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Vandenboom, Rene</creatorcontrib><creatorcontrib>Herron, Todd</creatorcontrib><creatorcontrib>Favre, Elizabeth</creatorcontrib><creatorcontrib>Albayya, Faris P</creatorcontrib><creatorcontrib>Metzger, Joseph M</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Physical Education Index</collection><collection>Toxicology Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>American journal of physiology. Heart and circulatory physiology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Vandenboom, Rene</au><au>Herron, Todd</au><au>Favre, Elizabeth</au><au>Albayya, Faris P</au><au>Metzger, Joseph M</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function</atitle><jtitle>American journal of physiology. Heart and circulatory physiology</jtitle><addtitle>Am J Physiol Heart Circ Physiol</addtitle><date>2011-02-01</date><risdate>2011</risdate><volume>300</volume><issue>2</issue><spage>H574</spage><epage>H582</epage><pages>H574-H582</pages><issn>0363-6135</issn><eissn>1522-1539</eissn><coden>AJPPDI</coden><notes>ObjectType-Article-1</notes><notes>SourceType-Scholarly Journals-1</notes><notes>ObjectType-Feature-2</notes><notes>content type line 23</notes><abstract>The purpose of this study was to implement a living myocyte in vitro model system to test whether a motor domain-deleted headless myosin construct could be incorporated into the sarcomere and affect contractility. To this end we used gene transfer to express a "headless" myosin heavy chain (headless-MHC) in complement with the native full-length myosin motors in the cardiac sarcomere. An NH2-terminal Flag epitope was used for unique detection of the motor domain-deleted headless-MHC. Total MHC content (i.e., headless-MHC+endogenous MHC) remained constant, while expression of the headless-MHC in transduced myocytes increased from 24 to 72 h after gene transfer until values leveled off at 96 h after gene transfer, at which time the headless-MHC comprised ∼20% of total MHC. Moreover, immunofluorescence labeling and confocal imaging confirmed expression and demonstrated incorporation of the headless-MHC in the A band of the cardiac sarcomere. Functional measurements in intact myocytes showed that headless-MHC modestly reduced amplitude of dynamic twitch contractions compared with controls (P<0.05). In chemically permeabilized myocytes, maximum steady-state isometric force and the tension-pCa relationship were unaltered by the headless-MHC. These data suggest that headless-MHC can express to 20% of total myosin and incorporate into the sarcomere yet have modest to no effects on dynamic and steady-state contractile function. This would indicate a degree of functional tolerance in the sarcomere for nonfunctional myosin molecules.</abstract><cop>United States</cop><pub>American Physiological Society</pub><pmid>21112946</pmid><doi>10.1152/ajpheart.00786.2009</doi><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 0363-6135 |
| ispartof | American journal of physiology. Heart and circulatory physiology, 2011-02, Vol.300 (2), p.H574-H582 |
| issn | 0363-6135 1522-1539 |
| language | eng |
| recordid | cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_3044057 |
| source | American Physiological Society Free |
| subjects | Actin Cytoskeleton - physiology Animals Blotting, Western Calcium Signaling - genetics Calcium Signaling - physiology Cardiac Excitation and Contraction Cardiomyocytes Cell Membrane Permeability - physiology Cell Separation DNA, Complementary - biosynthesis DNA, Complementary - genetics Electrophoresis, Polyacrylamide Gel Fluorescent Antibody Technique Gene expression Gene Transfer Techniques Genetic Vectors Humans Immunohistochemistry Immunoprecipitation Molecules Myocardial Contraction - physiology Myocardium - metabolism Myocytes, Cardiac - metabolism Myosin Heavy Chains - biosynthesis Myosin Heavy Chains - genetics Myosins - biosynthesis Myosins - chemistry Myosins - genetics Physiology Protein Conformation Rats Sarcomeres - metabolism Studies |
| title | Gene transfer, expression, and sarcomeric incorporation of a headless myosin molecule in cardiac myocytes: evidence for a reserve in myofilament motor function |
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