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Acetylation and Phosphorylation Post-Translational Modifications of the CapZ β1 Subunit Regulate...
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miR-448 in hearts of young dystrophic mice. The latter correlated with overex- pression of the Ncf1 gene, encoding the NOX2 regulatory subunit p47phox. Specificity of Ncf1 targeting by miR-448 was confirmed by luciferase reporter assay. To study the effect of miR-448 silencing on molecular, cellular and func- tional properties of normal hearts, we intravenously injected wild type mice with LNA-NC and LNA-miR-448 inhibitors. qRT-PCR, western blotting, confocal imaging of ROS and intracellular Ca 2þ signals and echocardiography were em- ployed. Acute inhibition of miR-448 resulted in an increase in Ncf1 expression as well as enhanced ROS production and augmented intracellular Ca 2þ signaling in isolated cardiomyocytes. In addition, prolong (over one month) inhibition of miR-448 led to the deterioration of cardiac functions and development of dilated cardiomyopathy and arrhythmia. Overall, WT mice with inhibited miR-448 mimicked many features of dystrophic cardiomyopathy. Our data suggest that downregulation of miR-448 relieves inhibition of translational initiation of Ncf1 in dystrophic cardiomyopathy. It results in an increase in Ncf1 expression and consequently in oxidative stress and enhanced Ca 2þ signaling in dystrophic heart well before cardiac dysfunction becomes evident. 3921-Pos Board B649 Vinculin-Mediated Cytoskeletal Remodeling Modulates Cardiac Morphology and Contractile Function During Ageing Gaurav Kaushik 1 , Alice Spenlehauer 1 , Ayla Sessions 2 , Danielle Pohl 3 , Adriana Trujillo 4 , Sanford I. Bernstein 4 , Rolf Bodmer 5 , Anthony Cammarato 6 , Adam J. Engler 1 . 1 Bioengineering, University of California, San Diego, La Jolla, CA, USA, 2 Biomedical Sciences, University of California, San Diego, La Jolla, CA, USA, 3 Iowa State University, Ames, IA, USA, 4 San Diego State University, San Diego, CA, USA, 5 Sanford-Burnham Medical Research Institute, La Jolla, CA, USA, 6 Johns Hopkins University, Baltimore, MD, USA. Heart failure associated with chronic aging is underscored by altered expression of actin-binding cytoskeletal molecules. How cytoarchitectural remodeling impacts cardiac structure and contractile function remains unclear. We have developed novel biophysical techniques to interrogate subcellular mechanics and cellular function in the genetically tractable, rapidly aging Drosophila, a useful model for mammalian cardiac development. Genotype-dependent, age- related remodeling was a conserved hallmark in wildtype flies signified by decreased systolic and diastolic dimensions between 1 and 5 weeks of age. Remodeling correlated with increased cortical stiffness, particularly at the inter- calated disc (ID). Inhibition of actin polymerization reversed the stiffening phenotype at the ID while inhibition of actomyosin crosslinking did not. Age- related remodeling also correlated with preserved contractile function. In contrast, non-remodeled hearts experienced impaired shortening velocities. Cardiac-specific genetic perturbations were employed to determine how altered expression of vinculin (Vcl) affected structure and function independent of aging. Vcl overexpression resulted in increased localization to the ID, decreased diastolic diameter, and increased cortical stiffness at 1 wk of age preferentially at the ID. However, fractional shortening and shortening velocity increased as compared to controls, suggesting that muscle performance was not hindered by Vcl-overexpression despite altered physiology. Vcl-overexpressing hearts treated with cytochalasin D exhibited significant softening at the ID while blebbistatin-treated flies did not, suggesting that Vcl induces stiffening through actin recruitment. In contrast to current models that suggest that intercalated disc remodeling may be maladaptive for age-related cardiac decline, no reduc- tion in systolic function was observed in hearts with Vcl-overexpression. There- fore, Vcl may play a key regulatory role in maintenance of cardiomyocyte structure and function with age. Further studies will investigate the biophysical mechanisms of increased shortening function due to Vcl-mediated remodeling and identify upstream activators of Vcl-upregulation. 3922-Pos Board B650 Novel Locations; Familiar Functions: Obscurin at the Cardiac Interca- lated Disc Maegen A. Ackermann, Nicole A. Perry, Aikaterini Kontrogianni- Konstantopoulos. University of Maryland, Baltimore, MD, USA. The intercalated disc (ID) of cardiac muscle embodies a highly ordered, multifunctional network, which is essential for the transmission of electrical stimuli and mechanical force resulting in the synchronous contraction of the heart. Recently, a plethora of proteins have been identified as novel components of the ID. The challenge now lies in their characterization as it relates to the mechanical and electrical coupling of neighbouring cardiomyocytes. Here we focus on the molecular and functional description of one of these novel members, obscurin-90. Obscurins are a family of proteins expressed in striated muscles where they localize to distinct subdomains, i.e. the sarcoplasmic reticulum, the sarcomeric cytoskeleton, and the sarcolemma, and function in their assembly and integra- tion. Previous studies have shown that transcripts arising from the single OBSCN gene are extensively spliced, resulting in several obscurin isoforms. Complex splicing at its 3’ end gives rise to obscurin-90 (obsc90), the isoform that preferentially localizes to the ID. Obsc90 contains tandem RhoGEF and PH motifs, two Ig domains, and a non-modular COOH-terminus with ankyrin-binding sites and ERK phosphorylation cassettes. Using immunofluo- rescence and immunoelectron microscopy, we show that obsc90 localizes to the ID, and specifically at the outer edge of the transition zone. Consistent with this, biochemical assays demonstrated that obsc90 is in a complex with major ID proteins, including N-cadherin, connexin-43, vinculin, and ankyrinG. Obsc90 is targeted to the ID through the direct binding of its PH domain to membrane lipids, and its functions are likely regulated by phosphorylation of its COOH- terminus. Preliminary evidence indicates that the phosphorylation profile of obsc90 is altered in hypertrophic cardiomyopathy.. Further experiments are underway to examine the function of obsc90 at the ID and its regulation via phosphorylation in health and disease. 3923-Pos Board B651 Acetylation and Phosphorylation Post-Translational Modifications of the CapZ b1 Subunit Regulate FRAP Dynamics Leading to Myocyte Hypertrophy Ying-Hsi Lin, Chad M. Warren, Brenda Russell. University of Illinois at Chicago, Chicago, IL, USA. The mechanism by which more thin filaments are built during cardiac hypertro- phy is not fully understood. Very rapid increases the dynamics both actin and the actin capping protein (CapZ) following mechanical flexing suggest that a post-translational regulation is the underlying mechanism. Neonatal rat ventric- ular myocytes in culture were stimulated to hypertrophy by a neurohormone (10mM phenylephrine, PE, for 24 hr). CapZ dynamics were analyzed by fluo- rescence recovery after photobleaching (FRAP) using CapZb1-GFP. After PE treatment, CapZ dynamics increased above resting controls by ~3.17 fold (p=0.0004). Post-translational modifications of CapZ were analyzed by 2D gel electrophoresis. After PE treatment, 2D spots of CapZb1-GFP have an increased negative shift, suggesting that post-translational modification of CapZ is up regulated. To identify the types of post-translational modifications, 2D western blotting and mass spectrometry (MS) was applied. Increased post- translational spots included the acetylation of K199 and phosphorylation of S204, which are both close to the actin-binding region of CapZ. To test whether CapZ acetylation was mediated by HDAC3, located at the Z-disc of myocytes, the class I HDAC inhibitor (5mM trichostatin A / 5hr) and HDAC3 activator (10mM theophylline / 24hr) were applied. CapZ dynamics with trichostatin A increased by four-fold (p=0.01), and the effect of PE on CapZ dynamics was blunted by theophylline (p=0.09). Thus, the increased sarcomere remodel- ing during cardiac hypertrophy may be induced via altered acetylation of CapZ, which is mediated by HDAC3. In addition to MS identification of phosphorylation sites, post-translational modifications were reduced by domi- nant negative PKC 3 , suggesting a regulatory role. Together the acetyl and phospho posttranslational modifications of CapZ reduce the capping property and may increase thin filament assembly. NIH HL62426 (BR) and AHA 12PRE12050371 (Y-H L). 3924-Pos Board B652 Actin Carbonylation is Higher in Human Hypertrophic Cardiomyopathy Due to MYH7 Mutations Rosalie Witjas-Paalberends 1 , Marcella Canton 2 , Michelle Michels 3 , Carolyn Ho 4 , Corrado Poggesi 5 , Fabio di Lisa 2 , Jolanda van der Velden 1,6 . 1 VU University Medical Center, Amsterdam, Netherlands, 2 University of Padova, Padova, Italy, 3 Erasmus Medical Center, Rotterdam, Netherlands, 4 Brigham and Women’s Hospital, Boston, MA, USA, 5 University of Florence, Florence, Italy, 6 ICIN-Netherlands Heart Institute, Utrecht, Netherlands. Introduction: Hypertrophic cardiomyopathy(HCM) is frequently caused by mutations in genes encoding sarcomeric proteins. Decreased force development was found in human HCM due to mutations in genes encoding myosin binding protein-C(MYBPC3), myosin heavy chain(MYH7) and in sarcomere mutation- negative HCM(HCMsmn). This is partially caused by the mutation (especially MYH7 mutations), increased cell size and reduced myofibrillar density. How- ever, 10% force reduction could not be explained by the mutation or cellular re- modeling. In end-stage human heart failure increased actin-carbonylation, induced by oxidative stress, negatively correlated with myocardial function. Therefore, we investigated whether actin is carbonylated in human HCM. Methods and results: Left ventricular tissue of human HCMsmn, MYBPC3- mut and MYH7mut patients was obtained. Non-failing donors were used as control. Actin-carbonylation was analyzed using Oxyblot. Carbonyl groups of myofilament proteins were derivatized by 2,4-dinitrophenyl-hydra- zine(DNPH). Protein lysates were processed via SDS-page, western blot 778a Wednesday, February 19, 2014
Transcript of Acetylation and Phosphorylation Post-Translational Modifications of the CapZ β1 Subunit Regulate...
778a Wednesday, February 19, 2014
miR-448 in hearts of young dystrophic mice. The latter correlated with overex-pression of the Ncf1 gene, encoding the NOX2 regulatory subunit p47phox.Specificity of Ncf1 targeting by miR-448 was confirmed by luciferase reporterassay. To study the effect of miR-448 silencing on molecular, cellular and func-tional properties of normal hearts, we intravenously injectedwild typemicewithLNA-NC and LNA-miR-448 inhibitors. qRT-PCR, western blotting, confocalimaging of ROS and intracellular Ca2þ signals and echocardiography were em-ployed. Acute inhibition of miR-448 resulted in an increase in Ncf1 expressionaswell as enhancedROSproduction and augmented intracellular Ca2þ signalingin isolated cardiomyocytes. In addition, prolong (over one month) inhibition ofmiR-448 led to the deterioration of cardiac functions and development of dilatedcardiomyopathy and arrhythmia. Overall, WT mice with inhibited miR-448mimicked many features of dystrophic cardiomyopathy. Our data suggest thatdownregulation of miR-448 relieves inhibition of translational initiation ofNcf1 in dystrophic cardiomyopathy. It results in an increase in Ncf1 expressionand consequently in oxidative stress and enhanced Ca2þ signaling in dystrophicheart well before cardiac dysfunction becomes evident.
3921-Pos Board B649Vinculin-Mediated Cytoskeletal Remodeling Modulates CardiacMorphology and Contractile Function During AgeingGaurav Kaushik1, Alice Spenlehauer1, Ayla Sessions2, Danielle Pohl3,Adriana Trujillo4, Sanford I. Bernstein4, Rolf Bodmer5,Anthony Cammarato6, Adam J. Engler1.1Bioengineering, University of California, San Diego, La Jolla, CA, USA,2Biomedical Sciences, University of California, San Diego, La Jolla, CA,USA, 3Iowa State University, Ames, IA, USA, 4San Diego State University,San Diego, CA, USA, 5Sanford-Burnham Medical Research Institute, LaJolla, CA, USA, 6Johns Hopkins University, Baltimore, MD, USA.Heart failure associated with chronic aging is underscored by altered expressionof actin-binding cytoskeletal molecules. How cytoarchitectural remodelingimpacts cardiac structure and contractile function remains unclear. We havedeveloped novel biophysical techniques to interrogate subcellular mechanicsand cellular function in the genetically tractable, rapidly aging Drosophila, auseful model for mammalian cardiac development. Genotype-dependent, age-related remodeling was a conserved hallmark in wildtype flies signified bydecreased systolic and diastolic dimensions between 1 and 5 weeks of age.Remodeling correlated with increased cortical stiffness, particularly at the inter-calated disc (ID). Inhibition of actin polymerization reversed the stiffeningphenotype at the ID while inhibition of actomyosin crosslinking did not. Age-related remodeling also correlated with preserved contractile function. Incontrast, non-remodeled hearts experienced impaired shortening velocities.Cardiac-specific genetic perturbations were employed to determine how alteredexpression of vinculin (Vcl) affected structure and function independent ofaging. Vcl overexpression resulted in increased localization to the ID, decreaseddiastolic diameter, and increased cortical stiffness at 1wk of age preferentially atthe ID. However, fractional shortening and shortening velocity increased ascompared to controls, suggesting that muscle performance was not hinderedby Vcl-overexpression despite altered physiology. Vcl-overexpressing heartstreated with cytochalasin D exhibited significant softening at the ID whileblebbistatin-treated flies did not, suggesting that Vcl induces stiffening throughactin recruitment. In contrast to current models that suggest that intercalateddisc remodeling may be maladaptive for age-related cardiac decline, no reduc-tion in systolic function was observed in hearts with Vcl-overexpression. There-fore, Vcl may play a key regulatory role in maintenance of cardiomyocytestructure and function with age. Further studies will investigate the biophysicalmechanisms of increased shortening function due to Vcl-mediated remodelingand identify upstream activators of Vcl-upregulation.
3922-Pos Board B650Novel Locations; Familiar Functions: Obscurin at the Cardiac Interca-lated DiscMaegen A. Ackermann, Nicole A. Perry, Aikaterini Kontrogianni-Konstantopoulos.University of Maryland, Baltimore, MD, USA.The intercalated disc (ID) of cardiac muscle embodies a highly ordered,multifunctional network, which is essential for the transmission of electricalstimuli and mechanical force resulting in the synchronous contraction of theheart. Recently, a plethora of proteins have been identified as novel componentsof the ID. The challenge now lies in their characterization as it relates to themechanical and electrical coupling of neighbouring cardiomyocytes. Herewe focus on the molecular and functional description of one of these novelmembers, obscurin-90.Obscurins are a family of proteins expressed in striated muscles where theylocalize to distinct subdomains, i.e. the sarcoplasmic reticulum, the sarcomericcytoskeleton, and the sarcolemma, and function in their assembly and integra-
tion. Previous studies have shown that transcripts arising from the singleOBSCN gene are extensively spliced, resulting in several obscurin isoforms.Complex splicing at its 3’ end gives rise to obscurin-90 (obsc90), the isoformthat preferentially localizes to the ID. Obsc90 contains tandem RhoGEFand PH motifs, two Ig domains, and a non-modular COOH-terminus withankyrin-binding sites and ERK phosphorylation cassettes. Using immunofluo-rescence and immunoelectron microscopy, we show that obsc90 localizes to theID, and specifically at the outer edge of the transition zone. Consistent with this,biochemical assays demonstrated that obsc90 is in a complex with major IDproteins, including N-cadherin, connexin-43, vinculin, and ankyrinG. Obsc90is targeted to the ID through the direct binding of its PH domain to membranelipids, and its functions are likely regulated by phosphorylation of its COOH-terminus. Preliminary evidence indicates that the phosphorylation profile ofobsc90 is altered in hypertrophic cardiomyopathy.. Further experiments areunderway to examine the function of obsc90 at the ID and its regulation viaphosphorylation in health and disease.
3923-Pos Board B651Acetylation and Phosphorylation Post-Translational Modifications ofthe CapZ b1 Subunit Regulate FRAP Dynamics Leading to MyocyteHypertrophyYing-Hsi Lin, Chad M. Warren, Brenda Russell.University of Illinois at Chicago, Chicago, IL, USA.The mechanism by which more thin filaments are built during cardiac hypertro-phy is not fully understood. Very rapid increases the dynamics both actin andthe actin capping protein (CapZ) following mechanical flexing suggest that apost-translational regulation is the underlying mechanism. Neonatal rat ventric-ular myocytes in culture were stimulated to hypertrophy by a neurohormone(10mM phenylephrine, PE, for 24 hr). CapZ dynamics were analyzed by fluo-rescence recovery after photobleaching (FRAP) using CapZb1-GFP. AfterPE treatment, CapZ dynamics increased above resting controls by ~3.17 fold(p=0.0004). Post-translational modifications of CapZ were analyzed by 2Dgel electrophoresis. After PE treatment, 2D spots of CapZb1-GFP have anincreased negative shift, suggesting that post-translational modification ofCapZ is up regulated. To identify the types of post-translational modifications,2D western blotting and mass spectrometry (MS) was applied. Increased post-translational spots included the acetylation of K199 and phosphorylation ofS204, which are both close to the actin-binding region of CapZ. To test whetherCapZ acetylation was mediated by HDAC3, located at the Z-disc of myocytes,the class I HDAC inhibitor (5mM trichostatin A / 5hr) and HDAC3 activator(10mM theophylline / 24hr) were applied. CapZ dynamics with trichostatinA increased by four-fold (p=0.01), and the effect of PE on CapZ dynamicswas blunted by theophylline (p=0.09). Thus, the increased sarcomere remodel-ing during cardiac hypertrophy may be induced via altered acetylation ofCapZ, which is mediated by HDAC3. In addition to MS identification ofphosphorylation sites, post-translational modifications were reduced by domi-nant negative PKC 3, suggesting a regulatory role. Together the acetyl andphospho posttranslational modifications of CapZ reduce the capping propertyand may increase thin filament assembly. NIH HL62426 (BR) and AHA12PRE12050371 (Y-H L).
3924-Pos Board B652Actin Carbonylation is Higher in Human Hypertrophic CardiomyopathyDue to MYH7 MutationsRosalie Witjas-Paalberends1, Marcella Canton2, Michelle Michels3,Carolyn Ho4, Corrado Poggesi5, Fabio di Lisa2, Jolanda van der Velden1,6.1VU University Medical Center, Amsterdam, Netherlands, 2University ofPadova, Padova, Italy, 3Erasmus Medical Center, Rotterdam, Netherlands,4Brigham and Women’s Hospital, Boston, MA, USA, 5University ofFlorence, Florence, Italy, 6ICIN-Netherlands Heart Institute, Utrecht,Netherlands.Introduction: Hypertrophic cardiomyopathy(HCM) is frequently caused bymutations in genes encoding sarcomeric proteins. Decreased force developmentwas found in human HCM due to mutations in genes encoding myosin bindingprotein-C(MYBPC3), myosin heavy chain(MYH7) and in sarcomere mutation-negative HCM(HCMsmn). This is partially caused by the mutation (especiallyMYH7 mutations), increased cell size and reduced myofibrillar density. How-ever, 10% force reduction could not be explained by the mutation or cellular re-modeling. In end-stage human heart failure increased actin-carbonylation,induced by oxidative stress, negatively correlated with myocardial function.Therefore, we investigated whether actin is carbonylated in human HCM.Methods and results: Left ventricular tissue of human HCMsmn, MYBPC3-mut and MYH7mut patients was obtained. Non-failing donors were usedas control. Actin-carbonylation was analyzed using Oxyblot. Carbonylgroups of myofilament proteins were derivatized by 2,4-dinitrophenyl-hydra-zine(DNPH). Protein lysates were processed via SDS-page, western blot
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