Single Molecule Studies of Recombinant Human α- and β-Cardiac Myosin to Elucidate Molecular...
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activated by N-methyl-d-aspartate. Due to their high Ca 2þ permeability and voltage-dependent channel block by Mg 2þ , NMDA receptors play a central role in development through stabilization of synaptic connections, as well as in learning and memory by mediating many forms of synaptic plasticity. The mechanisms by which the ion channel of NMDA receptors selects Ca 2þ for permeation over all other physiological ions, while binding Mg 2þ and restricting its permeation, are not well understood. We hypothesize that the slightly different radii and electronic properties of Mg 2þ and Ca 2þ ions result in drastically different free energy barriers for transition of the ions from a binding site in the selectivity filter toward the intracellular solution. We are applying quantitative theoretical ‘‘bottom up’’ approaches to this complex system by combining methods of computational chemistry, molecular mechan- ics (MM), and bioinformatics. The structure of the NMDA receptor channel is constructed and refined using experimental information, homology modeling and extensive molecular dynamics simulations. We are performing quantum chemical calculations to determine the energy of the transition state of model ligand exchange reactions that mimic divalent ion transition from the selectiv- ity filter of NMDA receptors to water. Quantum calculations are used to parameterize a polarizable molecular mechanics force field for these divalent ion interactions with organic ligands with the further goal to perform MM sim- ulations. Umbrella sampling and thermodynamic integration simulations are used to compute free energies of transfer of divalent ions between water and a model of the NMDA ion selectivity filter as well as free energy barriers for these transitions. 3114-Plat An Ionic Switch in the Ligand-Binding Domain of Non-NMDA Receptors Ranjit Vijayan, Philip C. Biggin. Oxford University, Oxford, United Kingdom. The ionotropic glutamate receptors (iGluRs), activated by the amino acid L-glutamate, account for the vast majority of excitatory neurotransmission in the central nervous system. Given their centrality to the nervous system, it is not surprising that these receptors have been linked to various neurological disorders including epilepsy, Alzheimer’s and Parkinson’s disease. Based on their sequence, pharmacological properties and function, iGluRs are classified primarily into three subtypes, namely amino-3-hydroxy-5-methyl-4- isoxazolepropionic acid (AMPA), kainate (KA), and N-methyl-D-aspartate (NMDA) receptors. Previously we have reported the identification of a potential ionic switch, or latch, located within the hinge region of non-NMDA receptors. Here we de- scribe extensive molecular dynamics simulations to explore the conformational behaviour of the ionic switch and its influence on the closure or opening of the ligand-binding domain. The position of the switch appears to directly control the conformation of the ligand-binding domain. We discuss the results with re- spect to the general mechanism of how different ligands are able to induce the same mechanism of domain closure. 3115-Plat Gating and Stoichiometry of Heteromeric Kainate Receptors Patricia Brown 1 , Mark Aurousseau 1 , Hugo McGuire 2 , Rikard Blunck 2 , Derek Bowie 1 . 1 McGill University, Montreal, QC, Canada, 2 Universite ´ de Montre ´al, Montreal, QC, Canada. Kainate-type ionotropic glutamate receptors (KARs) assemble primarily as heteromeric complexes at glutamatergic synapses. In most cases, KAR- mediated synaptic events exhibit slow and variable deactivation kinetics in contrast to the fast gating properties typically observed with recombinant KARs. It is still not clear which factors contribute to the slowing of KAR re- sponses at synapses, and it remains to be understood the low affinity neuro- transmitter, L-Glu, triggers prolonged activations of KARs. Here, we investigated the biophysical and stoichiometric properties of recombinant het- eromeric KARs assembled from the GluK2 and GluK5 receptor subunits. To do this, we used a combination of outside-out patch electrophysiology to ex- amine functionality and a fluorescent subunit counting technique to assess het- eromerization. As expected, the degree of heteromerization with GluK2/ GluK5 subunits in individual patch recordings showed a positive correlation with slow deactivation kinetics and responsiveness to the agonist, AMPA. In- terestingly, preliminary data from subunit counting experiments suggest that the stoichiometry of heteromeric KARs is fixed. Furthermore, electrophysio- logical experiments reveal that GluK2/GluK5 heteromers are insensitive to external anions and cations. Since both anion and cation binding sites line the interface between KARs subunits, our data suggest that the process of heteromer assembly affects functionality by disrupting this region of the mature protein. Platform: Cardiac Muscle 3116-Plat MYH7-Mutation Associated Allelic Imbalance in Familial Hypertrophic Cardiomyopathy: Molecular Mechanisms and Correlation with Disease Prognosis Judith Montag 1 , Snigdha Tripathi 1 , Anna-Lena Weber 1 , Imke Schulte 1 , Edgar Becker 1 , Bianka Borchert 1 , Francisco Navarro-Lopez 2 , Antonio Francino 2 , Andreas Perrot 3 ,O ¨ zcelik Cemil 3 , William McKenna 4 , Karl-Josef Osterziel 3 , Bernhard Brenner 1 , Theresia Kraft 1 . 1 Hannover Medical School, Hannover, Germany, 2 Hospital Clinic/IDIBAPS University of Barcelona, Barcelona, Spain, 3 Charite ´-Universita ¨tsmedizin Berlin, Berlin, Germany, 4 University College London, London, United Kingdom. Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominant disease of the heart. The severity of the disease ranges from mild cases to sudden cardiac death or progression to heart failure. FHC is mostly caused by mutations in genes encoding for sarcomeric proteins, 30-40% of the patients are affected by missense mutations in one allele of the b-myosin heavy chain (MYH7). To gain further insights into the mechanisms of FHC-progression in heterozy- gous patients we performed a comparative expression analysis of the wildtype and the mutated MYH7 allele. We have analyzed samples from Musculus soleus and myocardium of genotyped and clinically well-characterized FHC-patients with different MYH7-mutations. We demonstrated an unequal allelic mRNA expression for each mutation analysed. The ratios of the mu- tated mRNA ranged from 29% to 66% in a mutation-specific manner. They were comparable in myocardium and soleus muscle and, importantly, were es- sentially the same at the protein level. Intriguingly, we observed a correlation between life expectancy and fraction of mutated mRNA or protein. Thus, the allelic imbalance may provide a novel factor underlying the progression of FHC. Our results suggest that the allelic imbalance is induced by differential regula- tion of the mutated MYH7 mRNA-expression. Thus we aimed to identify molecular mechanisms that may account for the mutation-related different mRNA levels. Bioinformatical analysis revealed that mutation V606M disrupts an exonic splicing enhancer site. Additionally, for the mutation R723G severe changes in the mRNA secondary structure were predicted. Alternative splicing variants of the V606-allele and a structure-related increased stability of the R723G-allele thus may provide potential factors inducing altered levels of mutated MYH7-mRNA. 3117-Plat Single Molecule Studies of Recombinant Human a- and b-Cardiac Myosin to Elucidate Molecular Mechanism of Familial Hypertrophic and Dilated Cardiomyopathies Jongmin Sung 1 , Elizabeth Choe 1 , Mary Elting 1 , Suman Nag 1 , Shirley Sutton 1 , John Deacon 2 , Leslie Leinwand 2 , Kathy Ruppel 1 , James Spudich 1 . 1 Stanford University, Stanford, CA, USA, 2 University of Colorado, Boulder, CO, USA. Hypertrophic cardiomyopathies (HCM) and dilated cardiomyopathies (DCM) are common inherited cardiovascular diseases, often resulting from single point mutations in genes encoding sarcomeric proteins. Genetic and clinical studies have identified several hundred mutations, including severe disease causing mutations in b-myosin heavy chain (MHC). Despite the clinical sig- nificance, few single molecule studies exist for mutated b-cardiac myosin, primarily due to difficulties of heterologous protein expression and instrumen- tal limitations. Previous studies have used mouse a-cardiac myosin or biopsies from patients. Those studies are not optimal to understand the molecular mechanism of HCM/DCM because there are significant differences between mouse a- and human b-MHC. Furthermore, biopsy samples from patients are often inhomogeneous mixtures of wildtype (wt) and mutants. This may explain why there have been many inconsistencies between the previous studies. Here, we demonstrate the first single molecule studies of recombinant human cardiac myosin. We expressed homogenous and fully functional wt human cardiac a- and b-S1 with human light chains bound. Then, we characterized Wednesday, February 29, 2012 613a
Transcript of Single Molecule Studies of Recombinant Human α- and β-Cardiac Myosin to Elucidate Molecular...
Wednesday, February 29, 2012 613a
activated by N-methyl-d-aspartate. Due to their high Ca2þ permeability andvoltage-dependent channel block by Mg2þ, NMDA receptors play a centralrole in development through stabilization of synaptic connections, as well asin learning and memory by mediating many forms of synaptic plasticity.The mechanisms by which the ion channel of NMDA receptors selects Ca2þ
for permeation over all other physiological ions, while binding Mg2þ andrestricting its permeation, are not well understood. We hypothesize that theslightly different radii and electronic properties of Mg2þ and Ca2þ ions resultin drastically different free energy barriers for transition of the ions froma binding site in the selectivity filter toward the intracellular solution. Weare applying quantitative theoretical ‘‘bottom up’’ approaches to this complexsystem by combining methods of computational chemistry, molecular mechan-ics (MM), and bioinformatics. The structure of the NMDA receptor channel isconstructed and refined using experimental information, homology modelingand extensive molecular dynamics simulations. We are performing quantumchemical calculations to determine the energy of the transition state of modelligand exchange reactions that mimic divalent ion transition from the selectiv-ity filter of NMDA receptors to water. Quantum calculations are used toparameterize a polarizable molecular mechanics force field for these divalention interactions with organic ligands with the further goal to perform MM sim-ulations. Umbrella sampling and thermodynamic integration simulations areused to compute free energies of transfer of divalent ions between waterand a model of the NMDA ion selectivity filter as well as free energy barriersfor these transitions.
3114-PlatAn Ionic Switch in the Ligand-Binding Domain of Non-NMDA ReceptorsRanjit Vijayan, Philip C. Biggin.Oxford University, Oxford, United Kingdom.The ionotropic glutamate receptors (iGluRs), activated by the amino acidL-glutamate, account for the vast majority of excitatory neurotransmission inthe central nervous system. Given their centrality to the nervous system, it isnot surprising that these receptors have been linked to various neurologicaldisorders including epilepsy, Alzheimer’s and Parkinson’s disease. Based ontheir sequence, pharmacological properties and function, iGluRs are classifiedprimarily into three subtypes, namely amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate (KA), and N-methyl-D-aspartate(NMDA) receptors.Previously we have reported the identification of a potential ionic switch, orlatch, located within the hinge region of non-NMDA receptors. Here we de-scribe extensive molecular dynamics simulations to explore the conformationalbehaviour of the ionic switch and its influence on the closure or opening of theligand-binding domain. The position of the switch appears to directly controlthe conformation of the ligand-binding domain. We discuss the results with re-spect to the general mechanism of how different ligands are able to induce thesame mechanism of domain closure.
3115-PlatGating and Stoichiometry of Heteromeric Kainate ReceptorsPatricia Brown1, Mark Aurousseau1, Hugo McGuire2, Rikard Blunck2,Derek Bowie1.1McGill University, Montreal, QC, Canada, 2Universite de Montreal,Montreal, QC, Canada.Kainate-type ionotropic glutamate receptors (KARs) assemble primarily asheteromeric complexes at glutamatergic synapses. In most cases, KAR-mediated synaptic events exhibit slow and variable deactivation kinetics incontrast to the fast gating properties typically observed with recombinantKARs. It is still not clear which factors contribute to the slowing of KAR re-sponses at synapses, and it remains to be understood the low affinity neuro-transmitter, L-Glu, triggers prolonged activations of KARs. Here, weinvestigated the biophysical and stoichiometric properties of recombinant het-eromeric KARs assembled from the GluK2 and GluK5 receptor subunits. Todo this, we used a combination of outside-out patch electrophysiology to ex-amine functionality and a fluorescent subunit counting technique to assess het-eromerization. As expected, the degree of heteromerization with GluK2/GluK5 subunits in individual patch recordings showed a positive correlationwith slow deactivation kinetics and responsiveness to the agonist, AMPA. In-terestingly, preliminary data from subunit counting experiments suggest thatthe stoichiometry of heteromeric KARs is fixed. Furthermore, electrophysio-logical experiments reveal that GluK2/GluK5 heteromers are insensitive toexternal anions and cations. Since both anion and cation binding sites linethe interface between KARs subunits, our data suggest that the process of
heteromer assembly affects functionality by disrupting this region of themature protein.
Platform: Cardiac Muscle
3116-PlatMYH7-Mutation Associated Allelic Imbalance in Familial HypertrophicCardiomyopathy: Molecular Mechanisms and Correlation with DiseasePrognosisJudith Montag1, Snigdha Tripathi1, Anna-Lena Weber1, Imke Schulte1,Edgar Becker1, Bianka Borchert1, Francisco Navarro-Lopez2,Antonio Francino2, Andreas Perrot3, Ozcelik Cemil3, William McKenna4,Karl-Josef Osterziel3, Bernhard Brenner1, Theresia Kraft1.1Hannover Medical School, Hannover, Germany, 2Hospital Clinic/IDIBAPSUniversity of Barcelona, Barcelona, Spain, 3Charite-UniversitatsmedizinBerlin, Berlin, Germany, 4University College London, London,United Kingdom.Familial Hypertrophic Cardiomyopathy (FHC) is an autosomal dominantdisease of the heart. The severity of the disease ranges from mild cases tosudden cardiac death or progression to heart failure. FHC is mostly causedby mutations in genes encoding for sarcomeric proteins, 30-40% of the patientsare affected by missense mutations in one allele of the b-myosin heavy chain(MYH7).To gain further insights into the mechanisms of FHC-progression in heterozy-gous patients we performed a comparative expression analysis of the wildtypeand the mutated MYH7 allele. We have analyzed samples from Musculussoleus and myocardium of genotyped and clinically well-characterizedFHC-patients with different MYH7-mutations. We demonstrated an unequalallelic mRNA expression for each mutation analysed. The ratios of the mu-tated mRNA ranged from 29% to 66% in a mutation-specific manner. Theywere comparable in myocardium and soleus muscle and, importantly, were es-sentially the same at the protein level. Intriguingly, we observed a correlationbetween life expectancy and fraction of mutated mRNA or protein. Thus, theallelic imbalance may provide a novel factor underlying the progression ofFHC.Our results suggest that the allelic imbalance is induced by differential regula-tion of the mutated MYH7 mRNA-expression. Thus we aimed to identifymolecular mechanisms that may account for the mutation-related differentmRNA levels. Bioinformatical analysis revealed that mutation V606M disruptsan exonic splicing enhancer site. Additionally, for the mutation R723G severechanges in the mRNA secondary structure were predicted. Alternative splicingvariants of the V606-allele and a structure-related increased stability ofthe R723G-allele thus may provide potential factors inducing altered levelsof mutated MYH7-mRNA.
3117-PlatSingle Molecule Studies of Recombinant Human a- and b-Cardiac Myosinto Elucidate Molecular Mechanism of Familial Hypertrophic and DilatedCardiomyopathiesJongmin Sung1, Elizabeth Choe1, Mary Elting1, Suman Nag1,Shirley Sutton1, John Deacon2, Leslie Leinwand2, Kathy Ruppel1,James Spudich1.1Stanford University, Stanford, CA, USA, 2University of Colorado,Boulder, CO, USA.Hypertrophic cardiomyopathies (HCM) and dilated cardiomyopathies (DCM)are common inherited cardiovascular diseases, often resulting from singlepoint mutations in genes encoding sarcomeric proteins. Genetic and clinicalstudies have identified several hundred mutations, including severe diseasecausing mutations in b-myosin heavy chain (MHC). Despite the clinical sig-nificance, few single molecule studies exist for mutated b-cardiac myosin,primarily due to difficulties of heterologous protein expression and instrumen-tal limitations. Previous studies have used mouse a-cardiac myosin or biopsiesfrom patients. Those studies are not optimal to understand the molecularmechanism of HCM/DCM because there are significant differences betweenmouse a- and human b-MHC. Furthermore, biopsy samples from patientsare often inhomogeneous mixtures of wildtype (wt) and mutants. This mayexplain why there have been many inconsistencies between the previousstudies.Here, we demonstrate the first single molecule studies of recombinant humancardiac myosin. We expressed homogenous and fully functional wt humancardiac a- and b-S1 with human light chains bound. Then, we characterized
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the actin-myosin interaction using in vitro motility and laser beam trappingassays. From the in vitro motility assay, we measured the maximum velocityfrom wt a- and b-S1. Using the laser trap, we measured stroke sizes, ATPbinding rates (low [ATP]) and ADP release rates (high [ATP]). Furthermore,we expressed several HCM (R403Q, S453C) and DCM (S532P) causing mu-tants, and obtained preliminary in vitro motility and trap data. We have builta modern version optical trap that can resolve the ~10 nm stroke size and ~10ms strongly bound state of cardiac b-S1 at high [ATP]. We have further im-proved the resolution by implementing real-time feedback control in the sys-tem to accurately determine fine changes caused by the single mutations.
3118-PlatCross-Bridge Kinetics in Papillary Muscle Fibers from Transgenic MiceExpressing the Aspargine-47 to Lysine (N47K) Mutation in MyosinRegulatory Light ChainLi Wang1, Priya Muthu2, Danuta Szczesna-Cordary2, Masataka Kawai1.1University of Iowa, Iowa city, IA, USA, 2University of Miami,Miami, FL, USA.The current study aimed at investigating the isometric tension, stiffness andelementary steps of the cross-bridge (XBr) cycle in skinned papillary musclefibers from transgenic mice expressing familial hypertrophic cardiomyopathy(FHC) associated mutation N47K. This mutation, located at the Ca2þ bindingsite of myosin regulatory light chain (RLC), is associated with a rapidly pro-gressing late onset of the disease with mid-ventricular and papillary muscle hy-pertrophy. We studied the effect of the N47K mutation on the stiffness and thekinetic constants using skinned muscle strips subjected to increasing concentra-tions of MgATP, phosphate (Pi), and Ca2þ at 200 mM ionic strength, pH 7.0,and 20�C. The kinetic constants of the XBr cycle and their distribution over 6states were deduced from the sinusoidal analysis. We found that active tensionwas higher in N47K fibers compared with WT. No mutation-mediated signifi-cant changes were observed in the Ca2þ sensitivity (pCa50) or cooperativity(nH). The apparent rate constants 2pb and 2pc were smaller in N47K fiberscompared with WT. The rate constant of XBr detachment step (k2) was smallerwhile the equilibrium constant of force generation (K4) was larger in N47Kfibers. The stiffness of the standard activation and the number of the stronglyattached XBr were larger in N47K than in WT.These results suggest that thehigher tension in N47K fibers may be related to a larger number of stronglyattached XBr. We conclude that the N47K mutation may initiate a hypertrophicresponse through changes in myosin XBr kinetics leading to increasedforce during the systole, which eventually results in a pathologic phenotypein patients carrying this FHC-linked RLC mutation. Supported by NIH-HL070041 (MK), NIH-HL071778 and HL090786 (DSC) and AHA10POST3420009 (PM).
3119-PlatMyosin Cross-Bridges do not Form Precise Rigor Bonds in HypertrophicHeart Muscle Carrying Troponin T MutationsKrishna Midde1, V. Dumka1, JR Pinto2, P. Muthu2, P. Marandos3,I. Gryczynski1, Z. Gryczynski3, J. Borejdo1.1University of North Texas Health Science Center, Fort Worth, TX, USA,2University of Miami, Miller School of Medicine, Miami, FL, USA,3Texas Christian University, Fort Worth, TX, USA.Distribution of orientations of myosin was examined in ex-vivo myofibrilsfrom hearts of transgenic (Tg) mice expressing Familial Hypertrophic Car-diomyopathy (FHC) troponin T (TnT) mutations I79N, F110I and R278C.Humans are heterozygous for sarcomeric FHC mutations and so hypertro-phic myocardium contains a mixture of the wild-type (WT) and mutated(MUT) TnT. If mutations are expressed at a low level there may not be a sig-nificant change in the global properties of heart muscle. In contrast, mea-surements from a few molecules avoid averaging inherent in the globalmeasurements. It is thus important to examine the properties of onlya few molecules of muscle. To this end, the lever arm of one out of every60,000 myosin molecules was labeled with a fluorescent dye and a smallvolume within the A-band (~ 1 fL) was observed by confocal microscopy.This volume contained on average 5 fluorescent myosin molecules. The le-ver arm assumes different orientations reflecting different stages of acto-myosin enzymatic cycle. We measured the distribution of these orientationsby recording polarization of fluorescent light emitted by myosin-bound fluo-rophore during rigor and contraction. The distribution of orientations of rigorWT and MUT myofibrils was significantly different. There was a large dif-ference in the width and of skewness and kurtosis of rigor distributions.These findings suggest that the hypertrophic phenotype associated with theTnT mutations can be characterized by a significant increase in disorderof rigor cross-bridges.
3120-PlatMutations in thin Filament Proteins that Cause Familial Dilated Cardio-myopathy Uncouple Troponin I Phosphorylation from Changes in Myofi-brillar Ca2D-SensitivityMassimiliano Memo, Andrew E. Messer, Man Ching Leung,Steven B. Marston.Imperial College London, London, United Kingdom.To investigate the molecular mechanism leading to familial dilated cardiomy-opathy we studied 7 different DCM-causing mutations in thin filament pro-teins by in vitro motility assay. Troponin I K36Q and troponin C G159Dmutant troponin was extracted from explanted human hearts. Troponin TDK210 and R141W mutant troponin, and actin E361G were extracted fromtransgenic mouse heart. Mutant a-tropomyosin E40K and E54K was ex-pressed in baculovirus/sf9 cells. We reconstituted thin filaments in vitrowith the mutant sample of interest, or its correspondent wild-type, combinedwith troponin or a-tropomyosin from human donor tissue. We could not ob-serve any common pattern for the absolute Ca2þ-sensitivity of the mutantscompared with the WT thin filaments. In contrast to this we consistently ob-served that DCM mutations affect the relationship between Ca2þ-sensitivityand troponin I phosphorylation by PKA. Normally, PKA phosphorylationof troponin I causes a 2-3 fold decrease in myofibrillar Ca2þ-sensitivity butwe found that, in thin filaments containing DCM mutations, Ca2þ-sensitivitydid not change with the level of troponin I phosphorylation. Uncoupling ofCa2þ-sensitivity from troponin I phosphorylation was observed for allDCM mutations tested irrespective of the gene mutated or the source ofthe mutant protein. We tested the DCM mutation dose-dependency by mixinga-tropomyosin E40K, with the wild-type in different ratios, assembling it intothin filaments and comparing Ca2þ-sensitivity when troponin I was phosphor-ylated and dephosphorylated. The result was a dramatic effect of uncouplingwith small ratios of the mutant protein with complete uncoupling at 50% mu-tant. We conclude that the causative property shared by mutations in contrac-tile proteins that cause DCM is a blunted response to changes in troponin Iphosphorylation that could impair the normal response to adrenergicstimulation.
3121-PlatPost-Translational Modifications of Myofilament Proteins Involved inLength-Dependent Prolongation of Relaxation in Rabbit Right VentricularMyocardiumMichelle M. Monasky1, Domenico M. Taglieri2, Alice K. Jacobson1,Kaylan M. Haizlip1, R John Solaro2, Paul Janssen1.1The Ohio State University, Columbus, OH, USA, 2University of Illinois atChicago, Chicago, IL, USA.The phosphorylation state of several cardiac myofilament proteins changeswith the level of stretch in twitch-contracting cardiac muscles. It remains un-clear which kinases are involved in the length-dependent phosphorylation ofthese proteins. Therefore, we set out to investigate which kinases are involvedafter a step-wise change in cardiac muscle length. We hypothesize that myofil-ament protein phosphorylation by PKCbII and PKA alter contractile kineticsduring length-dependent activation. Right ventricular intact trabeculae wereisolated from New Zealand White rabbit hearts and stimulated to contract at1 Hz. Twitch force recordings where taken before and after administration ofkinase inhibitors at varying muscle lengths at 37�C. PKCbII inhibition signif-icantly decreased time from stimulation to peak force (TTP), time from peakforce to 50% relaxation (RT50), and time from peak force to 90% relaxation(RT90) at optimal muscle length. However, developed tension did not signifi-cantly change at either muscle length studied. Non-specific PKA inhibition sig-nificantly decreased TTP and RT50 at both taut and optimal muscle lengths.Detection of Ser/Thr phosphorylation with ProQ-diamond staining indicatesa role for PKCbII in the phosphorylation of tropomyosin and myosin lightchain-2 (MLC2) and PKA for tropomyosin, troponin-I, and MLC2. Our dataprovide evidence for two signaling kinases which act upon myofilament pro-teins during length-dependent activation, and provide further insight forlength-dependent myofilament function.
3122-PlatTransmural Distribution of Myocyte Contractile Properties in AgingF344 RatsWilliam K. Snapp1,2, Stuart G. Campbell1,2, Kenneth S. Campbell1,2.1Department of Physiology, University of Kentucky, Lexington, KY, USA,2Center for Muscle Biology, University of Kentucky, Lexington, KY, USA.Age-associated diastolic heart failure occurs when the amount of blood fillingthe heart between beats is reduced due to slow relaxation and increased pas-sive stiffness of the left ventricle. We are testing the hypothesis that slowedactive relaxation in diastolic heart failure is caused in part by age-dependent
