Ferrara, Claudia

Levitsky, Dmitrii

Matyushenko, Alexander

Piroddi, Nicoletta

Poggesi, Corrado

Scellini, Beatrice

Tesi, Chiara

English

Florence Research

J Muscle Res Celi Motil (2015) 36:71-14301.9 Contractile structure, Thu 11:35-11:5075Reduced right ventricular cardiomyocyte passive force dueto hypophosphorylation at Ser-282 of cardiac myosin bindingproteìn C in a rat model of post-ìschemìc heart faìlureKovacs Arpad1***, Kalasz Judit', Pasztor T Eniko\Sanganalmath Santosh K2, Dhalla Naranjan S2, Papp Zoltàn",Barta Judìt'"**Candidate for Young Investigator AwardIDivision of Clinical Physiology, Faculty of Medicine, University ofDebrecen, Debrecen, Hungary; 2Institute of Cardiovascular Sciences,St Boniface GeneraI Hospital Research Centre, Winnipeg, CanadaLeft ventricular (LV) remodeling in myocardial infarction (MI)-in-duced heart failure (HF) is welI studied. Less is known about theconcornitant righi ventricular (RV) changes. In this rar model of MI-induced HF we aimed to: (I) cbaracterize the contractile function ofRV cardiomyocytes, (2) compare ìt with the LV, (3) investigate theunderlying molecular mechanisrns in the RV. Rat hearts in moderateHF were studied after 8 weeks of LV MI. Sham-operated ani malsserved as controls. Maximal active force F(max), Ca-sensitivity(pCaso) and passive force Ftpass) were measured in skinned isolatedRV and LV cardiomyocytes (n = 12-15). Titin, myosin bindingprotein C (MyBP-C) and Troponin I (TnI) phosphorylation (P) weretested by gel-electrophoresis-based and Western blot methods. RVweight was higher in HF (0.48 ± 0.04 g, mean ± SEM) than in theSharn group (0.31 ± 0.02 g) (P S 0.05). F(max) and pCa50 of RVcardiac cells were similar in the HF and in the Sham group (18.7 ±1.1 kN/m2; 5.88 ± 0.03 vs. 18.3 ± 2.1 kN/m2; 5.87 ± 0.03, respec-tively). In HF, the cross section area of RV cells was comparable toSham (876 ± 133 11m2; 1007 ± 194 11m2). However, in HF F(pass)of RV cardiomyocytes was less (2.1 ± 0.2 kN/m2) than that in theSham group (3.2 ± 0.4 kN/m2). LV cells had higher F(max) andpCa50 values in HF than in the Sham group (34.9 ± 2.7 Idlf/m2;5.97 ± 0.03 vs. 26.9 ± 1.6 kN/m2; 5.85 ± 0.03). LV celi size in HFwas not higher than in the Sharn (1104 ± 151 11m2; 1029 ±118 11m2). LV F(pass) was not altered in HF (3.37 ± 0.37 kN/m2)compared to Sham (2.83 ± 0.27 kN/m2). In HF animals relative toSham total P of titin and MyBP-C showed no difference in the RV orin the LV, but P at Ser-282 of MyBP-C was decreased in the RV. InHF both the RV and the LV TnI P (total and Ser-22/23) was lowerthan in the Sham. Our data suggest distinct changes in RV car-diomyocyte structure and function in HF due to L V MI. Reduced RVF(pass) seems to be independent of titin P, rather might be attributableto hypo P of protein kinase A sites of MyBP-C.Keywords: Right ventricle, Passive force, Cardiac myosin bindingprotein C02.1 Contractile function, Thu 14:00-14:15What do we learn by the rates of tension development k(act)and redevelopment k(TR) in activated myofìbrìls and musclefibresKawai Masataka, Wang LiDepartments of Anatomy and CelI Biology, and InternaI Medicine,University of Towa, USAWhen the length of active muscle fìbres/rnyofibrils are released by~20 % and restretched after ~50 ms, tension redevelops with anexponential time course with the rate constant k(TR) in rabbit psoas fibres[1]. A similar time course (rate const: k(act» is observed on Ca2+ acti-vation in single myofibrils [2]. These are generally interpreted to meanthat k = f + g, where f is the attachment rate constant and g is the de-tachment rate constant in the two state model [I]. With this formulation, kis limited by a fast reaction. However, a problem has developed, becausek has been generally 5-40/s in rabbit psoas, which is too slow for a fastcross-bridge reaction. Altematively, we propose a model in which cross-bridges cyc\e many times by stretching series elastic elements to developforce, hence k is limited by a slow reaction: k ::::<(f + g)/fg (::::<meansproportiona\). To set up this model, we made an assumption that thestepping rate (v) decreases linearly with force (F) (Fenn effect, [31). Thedistance traveled by a cross-bridge stretches series elastic elements withstiffness p, which is registered as force. The rate constant of resulting timecourse is: k = P11()Vo(1 - À)/FI> where À = VI/VO, 11 = step size, thesubscript O indicates unloaded, and the subscript I indicate isometricconditions. We dernonstrate that the ATP hydrolysis rate is proportionateto k(TR) with À = 0.28 as the temperature is changed. We conclude thatk(TR) and k(act) are limited by the cross-bridge tumover rate; hence itrepresents the rate constant of the slowest reaction of the cross-bridgecyc\e. This model further explains why the time course of tension rise(stretch series elastic elernents) is slower than that of tension decay (directobservation of cross-bridge detachment) when the P; concentration isquickly changed with myofibril experirnents [2].Keywords: Tumover rate, Elementary steps, Cross-bridgeReferences[I] Brenner B. Proc Nat Acad Sci (USA) 85:3265-3269, 1988[2] Tesi C, Colorno F et al. Biophys J 78(6):3081-3092, 2000[3] Fenn WO, J Physiol 58(2-3):175-203, 192302.2 Contractile function, Thu 14:15-14:30Mutations in the centraI part of CI. Tropomyosin molecule alterCa2+ sensitivity and tension relaxatìon of skeletal musclemyofibrils after troponin-tropomyosin removaland reconstitutionScellini Beatrice", Piroddi Nicoletta', Ferrara Claudia.',Matyushenko Alexander M2, Levitsky Dmitrii 12, PoggesiCorrado', Tesi Chiara I'Departrnent of Experimental and Clinical Medicine, University ofFlorence, Italy; 2A N Bach Institute of Biochemistry, RussianAcademy of Sciences. Moscow, RussiaTropomyosin (Tm) is a o-helìcal coiled-coil actin-binding proteinregulating muscle contraction. Previous observations suggested that thehighly conserved non canonical residues Asp-137 and Gly-126 in thecentraI part of Tm confer instability. Replacement of these residues bycanonical ones (Leu and Arg, respectively) decreases molecular flex-ibility and modulate, the Ca2+ sensitivity of in vitro regulated acto-myosin systems (Surnida et al. J Biol Chem 283:6728-6734, 2008;Nevzorov et al. J Biol Chem 286:15766--15772, 2011). Here, we in-vestigated the functional impact of recombinant ct Tm carrying one(DI37L) or both (DI37L1GI26R) stabilizing subsritutions on rhe me-chanical behavior of skeletal myofibrils. Endogenous Tm and Tn werereplaced into rabbit psoas myofibrils (Scellini et al. Adv Exp Med Biol682:163-174, 2010) with purified Tn and recombinant CI. Tm(WT,DI37L and DI37L1GI26R). Force recordings from myofibrils(15°C) showed the increase in Ca2+ sensitivity expected from in vitrostudies, At saturating [Ca2+] maximal isometric tension and the rates offorce activation (kACT) and redevelopment (kTR) were not sig-nificantly affected by Tm mutations. Interestingly, a clear effect wasobserved 0.11 force relaxation: D137L1G126R myofibrils showed~ Springer76 J Muscle Res Cell Motil (2015) 36:71-143prolonged duration of the slow phase of relaxation and a decreased rateof the fast phase compared to WT. This effect was smaller in D137Lmyofibrils. Tm mutations also strongly decreased slack sarcornerelength (SL) al sub-activating [Ca2+] as well as they increased thesteepness of the SL-passive tension relation. These effects were re-versed by lO mM BDM, suggesting that single and double Tmmutations compromise the full inhibition of acto-myosin interactions inthe absence ofCa2+. These data support the hypothesis that fìexibiliry ofthe Tm coiled-coiled structure critically modulates the tuming off ofthe thin filament system and musc!e relaxation dynamics. Supported byPRIN 20 I0--201 I of the Ministero Università e Ricerca (MIUR).Keywords: Tropornyosin, Skeletal muscle, Regulation02.3 Contractile function, Thu 14:30-14:45Stiffness or the myosin head domainBrenner Bernhard", Seebohm Benjamin\ Werkman Chrìstoph",Hahn Nils!, Schweda Aike", Steffen Walterl, Kraft Theresia2IMolecular and Cell Physiology, Hannover MedicaI School,Germany; 2Molecular and Celi Physiology, Hannover MedicaISchool, GermanyElastic distortion of the rnyosin head is fundamental to the generation ofcontractile forces. Thus, stiffness of the myosin head, together withfilament cornpliance, is a main determinant of forces generated by amyosin head. Differences in head stiffness among myosin isoforms, orchanges in head stiffness with nucleotide would yield different forcesgenerated by different myosin isoforms and in different states of theATPase cycle, respectively. Studying effects of point mutations in theconverter of the slow skeletal/Becardiac myosin heavy chain that causefami lial hypertrophic cardiomyopathy, we found some mutations toincrease head stiffness and force generation by the myosin head. Thisimplied that the converter is a main determinant of myosin head stiff-nesso These FHC-mutations were at positions in the converter sequencewhere fast skeletal myosin differs from the slow/Bccardiac isofonn,suggesting that different heavy chain isoforrns may have di fferen t headstiffness. Comparing in skinned fibers and by optical trapping fast(rabbit psoas) and slow (rabbit/human soleus) myosins we found headstiffness of the slow myosin to be.::: 1/3rd of the fast isoform stiffness.The finding that changes in the converter strongly affect head stiffnessimplies that reorientation of the converter vs. the catalytic domain, e.g.,during the myosin working stroke, may also change stiffness ofthe headdomain. We tested this hypothesis by optica1 trapping of a single headedmyosin-5a construct. We selected myosin-5a because a sub-step hadbeen observed in optical trapping and was assigned to a reorientation ofthe converter in the myosin-5 working stroke. We found that after thissubstep, i .e., after converter reorientation, the stiffness of the myosin-Sahead domain was :::2-fold increased. In conclusion, stiffness and forcegeneration of a myosin head (i) are different for fast and slow myosin-2isoforms, and (ii) change as a myosin head progresses through itsworking stroke.Keywords: Force per myosin head, Stiffness of myosin isoforms,Stiffness of cross-bridge states02.4 Contractile function, Thu 14:45-15:00The conformation of myosin motors in relaxed skeletal muscleFusi Luca***, Huang Zhe, Irving Malco\m***Candidate for Young Investigator Award%l SpringerKings College London, UKElectron microscopy studies of isolated thick filaments from a widerange of rnuscle types and species are consistent with an asymmetricarrangemenr of the two motor domains of each myosin moleculefolded back against the filament backbone and called the J motif(Woodhead et al. Nature 436:1195-1199,2005). This conformation isassociated with the OFF state of myosin, although it is unclear howthe molecule is switched on during contraction. In this study weinvestigated the in situ conformation of myosin motors in relaxeddemembranated fibres from rabbit psoas muscle using fluorescencepolarisation from bifunctional rhodamine (BSR) probes at four siteson the myosin regulatory light chain (RLC). Under relaxing condì-tions the order parameters of the orientation distribution of eachprobe, P2 and P4 respectively, had a sigrnoidal dependence on tem-perature in the range 3-33 "C, with half-maximal change at 18°C.Either lattice compression by 5 % dextran or addition of 25 11MBlebbistatin decreased the transition temperature to 13 "C. Maximumentropy analysis showed that at 5 °C the RLC domain of myosin isalmost perpendicular to the filament axis, whereas above 20°C twopopulations with more parallel orientations appear, and either dextranor Blebbistatin stabilize these two conformations above 16°C. Theangles of these two conformations match the RLC orientations of theblocked and free heads in the J motif observed in isolated filaments,suggesting that this fraction of motors occupy the J motif in relaxedmuscle in situ. Slow ramp stretches applied in relaxing solution at25°C and at sarcomere lengths >2.6 urn produced large changes inRLC orientation towards the ON conformation seen during calciumactivation, with parti al reversal during force relaxation after thestretch. The correlation between RLC orientation and passive fibretension provides evidence that the orientation of relaxed myosinmotors is sensitive to thick filament strain. Supported by WellcomeTrust, UK.Keywords: myosin, skeletal muscle, muscle fibres02.5 Contractile function, Thu 15:00-15:15The increase in non cross-bridge force after stretch of activatedstriated muscles is related to tìtìn isoforrnsCornachione Anabelle", Leite Felìpe", Nocella Marta", ColombiniBarbara", Bagni Maria Angela", Rassier Dilson3'Kinesìotogy, McGill University, Montreal, Canada; 2Experimentaland Clinical Medicine, University of Florence, Italy; 3Kinesiology,Physics, Physiology, McGill University, Montrea1, CanadaIntroduction: There is evidence that skeletal muscles present a cross-bridge independent increase the sarcomere stiffness upon Ca2+ acti-vation. It has been hypothesized that the increase in stiffness is causedby Ca2+-dependent changes in the properties of titin molecules. Totest this hypothesis, we investigated the cross-bridge independentstiffness in muscles containing different titin isoforms.Methods: Permeabilized rnyofìbrils were isolated from the psoas,soleus and heart ventricle rnuscles of the rabbit, Intact trabeculae wereisolated from the heart of the mouse. Myofibrils were tested in re-laxing and activating conditions, before and after treatment withchemicals that inhibit rnyosin-actin interactions. Trabeculae wereelectrically stimulated in Krebs-Henseleit solution. The force pro-duced by these preparations during and after stretches of differentmagnitudes was measured in sarcomere lengths between 1.6 and2.4 urn for the heart, and between 2.2 and 3.0 um for skeletal mus-cles. Titin isoforms in these muscles were identified with gelelectrophoresis.

Mutations in the central part of a Tropomyosin molecule alter

Ca2+ sensitivity and tension relaxation of skeletal muscle

myofibrils after troponin&#8211;tropomyosin removal

and reconstitution

https://flore.unifi.it/bitstream/2158/993008/2/EMC%202014%20mutations.pdf

Mutations in the central part of a Tropomyosin molecule alter Ca2+ sensitivity and tension relaxation of skeletal muscle myofibrils after troponin–tropomyosin removal and reconstitution

Abstract

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Mutations in the central part of a Tropomyosin molecule alter Ca2+ sensitivity and tension relaxation of skeletal muscle myofibrils after troponin&#8211;tropomyosin removal and reconstitution

Abstract

Similar works

Full text

Available Versions

Florence Research

Mutations in the central part of a Tropomyosin molecule alter Ca2+ sensitivity and tension relaxation of skeletal muscle myofibrils after troponin–tropomyosin removal and reconstitution