Functional Significance of C-Terminal Mobile Domain of Cardiac Troponin I

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Monday, February 13, 2017

Cardiac Muscle Regulation I 1257-Pos Board B325 Tuning Cardiomyocite Dynamic Transient Responses with a Heterogeneous Populations of cTnC Mutations Srboljub Mijailovic1, Djordje Nedic2, Marina Svicevic2, Boban Stojanovic2, Michael A. Geeves3, Michael Regnier4. 1 Mechanical Engineering, Wentworht Institute of Technology, Boston, MA, USA, 2Mathematics and Informatics, University of Kragujevac, Kragujevac, Serbia, 3School of Biosciences, University of Kent, Canterbury, United Kingdom, 4Department of Bioengineering, University of Washington, Seattle, WA, USA. Understanding the influence of mutations in myofilament proteins on the regulatory properties of cardiac muscle is an essential step toward development of targeted therapies to improve cardiac function that is compromised in disease. We quantitatively evaluated the effect of altering the Ca2þ binding properties of cTn on cooperative thin filament activation and the kinetics of tension generation and relaxation. We simulated, using the MUSICO computational platform, force-pCa relationships and force transients for demembranated trabeculae where native cTnC cardiomyocytes where replaced with either cTnC L48Q (which has increased Ca2þ affinity) or cTnC I61Q (which has decreased Ca2þ affinity) primarily by altering the rate constant for Ca2þ release koff. The simulations demonstrated that increasing or decreasing koff respectively were not sufficient to replicate experimental data because changing solely koff also modulates maximum isometric force and basal muscle tone. Thus, we adjusted Ca2þ binding rates and myosin affinity to regulated actin to account for the observed apparent loss of cooperativity of thin filament activation of both mutants. With these adjusted parameters simulations of these cTnC point mutations demonstrated altered cardiac function, with reasonable agreement of experimentally observed isometric force development and redevelopment. This includes maximum isometric force, rates of force development and redevelopment, Hill coefficient of force-pCa relationships, nH, and the twitch responses. We have also performed MUSICO simulations with the ability to predict the transient responses for cardiac muscle containing a variable ratio of the normal and mutated cTnC, thus beginning an examination of the role of penetrance in altered function. The predicted effects of varying mutant penetrance on contractile properties suggests that mixed populations of WT and mutants has a tunable effect on cardiomyocyte performance that may reflect the severity of contractile abnormality in cardiac muscle disease. Supported by: R01 HL111197, R01 AR048776, HD048895 and W.Trust 085309. 1258-Pos Board B326 Differential CaMK-II Activation in the Progression of HCM in cTnT Mutations Sarah Lehman1, Lauren Grinspan2, Mark E. Anderson3, Jil C. Tardiff1. 1 University of Arizona, Tucson, AZ, USA, 2Albert Einstein College of Medicine, Bronx, NY, USA, 3Johns Hopkins University, Baltimore, MD, USA. Mutations in cardiac troponin T (cTnT) account for approximately 5-10% of all Hypertrophic Cardiomyopathy (HCM). The N-terminal, alpha-helical tail domain of cTnT, known as TnT1, contains several mutational hotspots including Arg92 (R92L and R92W). Although differing by only a single amino acid, these two highly penetrant HCM-mutations are known to lead to varying degrees of cardiac remodeling in humans. In studying the differential effects of these mutants on Ca2þ homeostasis, an increase in phospholamban (PLB) phosphorylation at Thr17, mediated by CaMK-II, was observed in an age-dependent manner only in R92W mice. We postulated that this increased CaMK-II activity was an initial effort to rescue Ca2þ homeostasis but due to auto-activation, aided in disease progression over time. Thus, we hypothesized a mutationspecific role of CaMK-II in HCM progression and tested this hypothesis via genetic inhibition of CaMK-II (AC3I peptide) in R92W and R92L transgenic mice. CaMK-II inhibition in mice expressing the R92W mutation resulted in improved Ca2þ homeostasis that was coupled with blunted cardiac remodeling and improved diastolic function while no effect was seen in R92L mice. Additionally, diastolic function of both the R92L and R92W mice was monitored throughout treatment with diltiazem hydrochloride, an L-type Ca2þ channel blocker. Initial results suggest an improvement in function in the R92W mice with treatment while there was no change seen in the R92L mice. Studies are on-going to determine the age at which auto-activated CaMK-II is increased in the R92W mice. Identifying this time-point will provide a precise timeline for therapeutic intervention in CaMK-II mediated disease progression. Together, these findings suggest that closely related mutations, even at the

same residue, can have discrete molecular effects that result in mutationspecific disease progression and these observations can be incorporated into targeted, genotype-driven disease management. 1259-Pos Board B327 cTnT isoform Switching in the Development of Early Childhood Tropomyosin-Linked DCM Melissa Lynn1, Teryn Holeman1, Lauren Grinspan2, J.P. Jin3, Jil Tardiff1. 1 University of Arizona, Tucson, AZ, USA, 2Albert Einstein College of Medicine, Bronx, NY, USA, 3Wayne State University, Detroit, MI, USA. An oft-noted component of sarcomeric DCM is that patients within families carrying the same primary mutation exhibit significant phenotypic variability. This lack of a distinct link between genotype and phenotype has complicated clinical management. Recently two unrelated multigenerational families were identified with the tropomyosin (Tm) mutation D230N, exhibiting a ‘‘bimodal’’ distribution of severity. In these families, many children (<1 year) with D230N-Tm presented with a severe form of DCM that led to sudden, often fatal CHF, while adults developed a mild to moderate DCM in midlife. Children who survived the initial presentation often recovered significant systolic function by young adulthood. To explain this improvement, despite the continued presence of D230N-Tm, we propose that the age-dependent remodeling is a result of temporal isoform switches involving a closely linked Tm binding partner cardiac Troponin T (cTnT). We have shown that D230NTm leads to a more rigid filament primarily at the C-terminus near the Tm overlap, a crucial regulatory region modulated by cTnT. Myofilaments from D230N-Tm mice exhibited a decrease in calcium sensitivity of force development that was additively reduced in the presence of fetal cTnT (cTnT1), supportive of a modulatory role. Divergent cardiac remodeling occurred at 4 months in our novel D230N þ cTnT1 (DTg) mouse model, where DTg mice exhibited significantly reduced %FS compared to D230NTm mice. To elucidate the effect of cTnT1 on the flexibility of the Tm overlap we extended our analysis of Tm stability to include a fully reconstituted thin filament (Actin, Tm, Troponin). In the presence of D230N-Tm and cTnT1 the rigidity of Tm was additively increased indicating that cTnT1 modulates the flexibility of the Tm overlap, further impairing its regulatory function. This data suggests a unique structural mechanism by which D230N-Tm leads to disease. 1260-Pos Board B328 N-Terminal Truncation of Cardiac Troponin I Compensates for Diastolic Dysfunction of Mouse Hearts Induced by Acute Pressure Overload Li-Fei Liu, Han-Zhong Feng, J.-P. Jin. Physiology, Wayne State University, Detroit, MI, USA. The N-terminal extension of cardiac troponin I (cTnI) modulates cTnI conformation and function in the regulation of cardiac muscle contraction. Restrictive cleavage of the N-terminal extension of cTnI occurs under stress conditions. Transgenic mouse cardiac hearts expressing cTnI lacking the N-terminal extension (cTnI-ND) showed enhanced ventricular diastolic function. The present study investigated the of cardiac function and remodeling of cTnI-ND transgenic mice after transverse aortic constriction (TAC). Echocardiography showed that the rate of aortic flow was significantly higher in cTnI-ND than wild type (WT) control prior to TAC. Two weeks after TAC, left ventricular rejection fraction(EF) and fractional shortening(FS) did not change in WT but significantly increased in cTnI-ND mice. Left ventricular end systolic diameter (LVESD) was reduced in cTnI-ND hearts with Left ventricular end diastolic diameter (LVEDD) similar to WT control, reflecting an increase in systolic function in response to acute pressure overload. TAC also induced a significant increase in inter septum thickness (IVS) in cTnI-ND hearts, indicating an early response in cardiac remodeling.The increased systolic function of cTnI-ND hearts in response to TAC suggested a plausible compensatory effect of cTnI-ND on cardiac function during acute pressure overload. Longitudinal studies are underway using echocardiography with terminal examination using ex vivo working heart preparations as well as pathohistological and immunochemical studies of intrinsic cardiac function and remodeling to further investigate the effects of cTnI-ND on compensating cardiac function during the development of pressure overload-generated heart failure. LFL is supported in part by the Children’s Hospital of Chongqing Medical University, Chongqing, China. 1261-Pos Board B329 Functional Significance of C-Terminal Mobile Domain of Cardiac Troponin I Nazanin Bohlooli Ghashghaee1, Peter O. Awinda2, Bertrand C.W. Tanner2, Wen-Ji Dong1,3.

Monday, February 13, 2017 1 Voiland School of Chemical Engineering and Bioengineering, Washington State univeristy, Pullman, WA, USA, 2The Department of Integrative Physiology and Neuroscience, Washington State univeristy, Pullman, WA, USA, 3The Department of Integrative Physiology and Neuroscience, Washington State University, Pullman, WA, USA. C-terminal mobile domain of cTnI (cTnI-MD, residues 168-210) is a highly conserved region which plays important roles in regulating myofilament function. Due to the unresolved structure of the region, it is still unclear how cTnI-MD plays a role in modulating the sarcomere length (SL)-dependent and Ca2þ-induced effects on cardiac contractility. In this study, the functional significance of the MD in contractile behavior of skinned rat papillary muscle fibers and SL dependent activation was studied by truncation of the entire MD (cTnI(1-167)) and the C-terminus of MD (cTnI(1-193)). We hypothesized that different truncations may have different impacts on SLdependent Ca2þ-regulation of tension development and cross-bridge cycling. To test this hypothesis, the tension-pCa relationships and myosin crossbridge kinetics were measured as cTnI(1-167) and cTnI(1-193) were exchanged into skinned rat papillary muscle fibers at short (SL=1.9mm) and long SLs (SL=2.2mm). Our results indicate that the SL induced enhancement of Ca2þ-sensitivity and cooperativity observed in wild-type fibers is preserved in cTnI(1-193) and is diminished in cTnI(1-167) fibers. cTnI(1167) truncation resulted in enhanced maximum tension and cooperativity of Ca2þ-activation compared to cTnI(WT) and cTnI(1-193) and enhanced Ca2þ-sensitivity compared to cTnI(WT) at both short and long SLs. cTnI(1-193) truncation resulted in enhanced myofilament Ca2þ-sensitivity at both SLs and a depressed maximum tension just at long SL. Both cTnIMD truncations showed a faster cross-bridge attachment rate at both SLs. Compared to cTnI(WT), cTnI(1-167) showed a slower cross-bridge detachment rate while cTnI(1-193) showed a faster cross-bridge detachment rate at long SL. The results of our study indicate that the truncation of cTnIMD at different regions causes destabilization of the closed-state position of tropomyosin suggested by the observed increase in the cross-bridge attachment rate to thin filament as well as Ca2þ-sensitivity of tension development at both sarcomere lengths.

1262-Pos Board B330 Impact of cGMP-PKG Pathway Modulation on Titin Phosphorylation and Titin-Based Myocardial Passive Stiffness Nazha Hamdani1, Melissa Herwig1, Soraya Hoelper2, Marcus Krueger3, Doris Koesling4, Michaela Kuhn5, Wolfgang A. Linke1. 1 Physiology, Cardiovascular, Bochum, Germany, 2Max-Planck-Institute for Heart and Lung Research, Cardiovascular, Bad Nauheim, Germany, 3 Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany, 4Pharmacology and Toxicology, Bochum, Germany, 5Physiology, W€ urzburg, Germany. RATIONALE: The crucial contribution of the giant myofilament protein titin to diastolic stiffness and cardiomyocyte passive force (Fpassive) is dependent, in part, on titin isoform composition and phosphorylation. Phosphorylation of titin by cyclic guanosine monophosphate (cGMP)-dependent protein kinase G (PKG) lowers titin-based stiffness, thus mediating a mechanical signaling process that is disturbed in heart failure. OBJECTIVE: To elucidate which elements of the nitric oxide (NO) cGMP-PKG signaling network are critical for titin phosphorylation and stiffness in vivo. METHODS AND RESULTS: We employed genetic knockout (KO) mouse models deficient for enzymes of the cGMP-PKG pathway, including cardiomyocyte-specific deletion of the guanylyl cyclase (GC)-A receptor and cGMP-dependent PKG (cGKI), as well as global deletion of soluble GC (sGC). We assessed titin phosphorylation by immunoblotting using phosphoserine-specific titin antibodies and by mass spectrometry quantification using stable isotope labeling of amino acids in mixed cultures of heart tissue from either wild-type (WT) or KO mice. The Fpassive of single permeabilized cardiomyocytes was recorded before and after administration of PKG. In all three genetic models, alltitin phosphorylation was reduced compared to WT hearts. The important PKG-dependent phospho-S4080 site within the N2-Bus region of mouse titin was hypophosphorylated in all three KO models. Unexpectedly, mass spectrometry analysis revealed that most class 1 titin phospho-sites within the molecular spring segment, including the Ig-domain regions, were hyperphosphorylated. Only a few sites showed a phosphorylation deficit or remaining unchanged. Particularly in the cGKI model many class 1 phospho-sites were hyperphosphorylated compared to WT hearts, indicative of the presence of compensatory processes following loss of PKG; indeed, this was associated with upregulation of several kinases that phosphorylate titin, including CaMKII, ERK2 and PKCa and with a clear rise in Fpassive in KO vs. WT cardiomyocytes. We also found an upregulation of phosphodiesterase 3, but

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downregulation of phosphodiesterase 9a, which regulates natriuretic peptides rather than nitric oxide-stimulated cGMP in heart myocytes. While administration of PKG lowered Fpassive of WT and KO cardiomyocytes in all models, this effect was more pronounced in the cGKI KO. CONCLUSIONS: Multiple in vivo phosphorylated class I titin phospho-sites were identified within the molecular spring segment, some of which depended on the cGMP-PKG pathway. While cGMP-activated PKG remains an important titin-targeting kinase, many titin phospho-sites may be regulated through a network of protein kinases/phosphatases. 1263-Pos Board B331 Sarcomere Lengthdependent Effects on the Ca2D-Troponin Regulation in Skinned Myocardial Fiber from Titin RBM20 Deletion Mice King-Lun Li1, Mei Methawasin2, Henk Granzier2, Ross John Solaro3, Bertrand Tanner4, Wen-Ji Dong1. 1 School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, USA, 2Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA, 3Department of Physiology and Biophysics, University of Illinois at Chicago, Chicago, IL, USA, 4Integrative Physiology and Neuroscience, Washington State University, Pullman, WA, USA. The length dependent activation of the myofilaments has been considered the cellular basis underlying the Frank-Starling law of the heart trough dynamic and complex interplays between a multitude of thick- and thin-filament components. Among these components, both troponin and giant protein titin are likely key players, but the mechanism, by which the length dependent myofilament Ca2þ regulation is linked functionally to titin, is elusive. In this study we investigate the link using in situ FRET technique to monitor how the length dependent Ca2þ-induced conformational change of the N-domain of cTnC within myocardium is modulated by alteration of titin compliance that directly determines the passive tension of myocardium. To achieve the objective, FRET donor and acceptor modified cTnC(13C/51C)AEDANS-DDPM was reconstituted into chemically-skinned myocardial fibers from wild-type mice and RBM20 deficient mice that expresses more compliant N2BA titin. The Ca2þ-induced conformational change in cTnC under different conditions were quantitatively characterized using time-resolved FRET measurements. Our results from wild type myocardium showed that sarcomere length dependent enhancement of Ca2þ-troponin regulation was found to be directly associated with strong crossbridge and it was diminished when the strong crossbridge was inhibited. When measurements were performed with skinned myocardial fibers from RBM20 deletion animals, the length dependent change was blunted. These results support that titin based passive tension development of myocardium is indeed involved in sarcomere length modulated Ca2þ-troponin regulation and that the strong crossbridge plays an important role in linking the modulatory effect of titin compliance to Ca2þ-troponin regulation. 1264-Pos Board B332 Activation and Inhibition of Cardiac Thin Filaments by Single and Multiple Domains Constructs of Human Cardiac Myosin Binding Protein-C (cMyBP-C) at Low Calcium Betty Virok1, Samantha Harris2, Vitold E. Galkin1, Howard D. White1. 1 Physiological Sciences, Eastern Virginia Medical School, Norfolk, VA, USA, 2Cellular and Molecular Medicine, University of Arizona, Tucson, AZ, USA. We have previously shown that the C1 domain of human cardiac myosin binding protein-C (c-MyBP-C) produces a biphasic regulation of cardiac thin filaments at low calcium concentrations. Increasing [C1] first produces activation followed by inhibition of myosin-S1 ATP hydrolysis by cardiac thin filaments at higher [C1]. In contrast, domains C0 and C2 only inhibit and do not activate ATP hydrolysis at any concentration. N’-terminal proteins containing the C1 domain along with C0 or C2 (e.g., C0-PA-C1, C1-M-C2 and C0-PA-C1M-C2) show a similar pattern of activation and inhibition of ATP hydrolysis at low calcium concentration as the C1 domain alone, but the concentrations required for maximum activation and inhibition are ~10 fold lower than required using C1 alone. Combinations of C1 and other domains added as separate polypeptides (not covalently connected to C1 ) such as C1 and C0-PA , C0 and PA-C1, C1-M and C2, or C1 and M-C2 do not produce the changes that are observed if the domains are covalently linked to the C1 domain . These results demonstrate that C1 alone is sufficient to activate the thin filament but that flanking domains enhance the effects of C1 when they are present in the same molecule. These data provide strong evidence that the C1 domain is a central activator of the thin filament and that C0, PA, M and C2 have complex roles that modulate the interaction of C1 with the thin filament. Molecular details of the structural basis of these observations are described in a second presentation