Piroddi, Nicoletta

Poggesi, Corrado

Scellini, Beatrice

Stienen, G.

Tesi, Chiara

van der Velden, J.

Witjas Paalberends, R.

English

Florence Research

156a Sunday, February 3, 2013(5.5750.03). E99K increased TLC (tension at pCa 8.0: 0.2250.03 THC)compared to WT (0.1050.02 THC), but A331P maintained similar TLC(0.0850.01 THC) to WT. Five equilibrium constants of the cross-bridgecycle were deduced using sinusoidal analysis. E99K caused a decrease in bothK0 (ADP dissociation: 16.654.6 mM1) and K1 (ATP association:1.5150.08 mM1) compared to WT (35.0456.3 mM1 and 2.150.3 mM1,respectively). A331P and WT showed similar K0 (3155.4 mM1 and35.0456.3 mM1), K1 (1.650.2 mM1 and 2.150.3 mM1), K2 (cross-bridgedetachment: 0.6950.1 and 0.7850.13), K4 (force generation: 0.2750.03 and0.3450.03), and K5 (Pi association: 0.1350.2 mM1 and 0.1250.01 mM1).The cross-bridge distributionwas similar amongWT,E99K andA331P, indicat-ing that force/cross-bridge is decreased in K99K and A331P. In conclusion,E99K actin causes HCM through impaired relaxation and elevated pCa50(diastolic problem), and lowered force/cross-bridge (systolic problem), whereasA331P actin causes HCM through decreased pCa50 and force/cross-bridge(systolic problem).797-Pos Board B566Stiffening of Embryonic Myocarium during Development ParallelsMyosin and Matrix Expression and Dynamically Matches Cell FunctionStephanie F. Majkut, Dennis E. Discher, Joseph Swift.University of Pennsylvania, Philadelphia, PA, USA.Healthy heart function and structure, from the tissue to cellular scale, dependson establishment of proper tissue mechanics. On the cellular scale, late embry-onic, neonatal and adult cardiomyocytes exhibit mechanosensitivity to ECMelasticity [1], such that culturing on substrates of physiological stiffness of~10-20 kPa optimizes function. However, the heart begins functioning long be-fore this mature mechanical environment is established. Here we examine theeffects of microenvironment mechanics on chick cardiomyocyte function dur-ing early development, embryonic day 2-4 (E2-E4). Micropipette aspirationmeasurements give an effective Young’s modulus of ~1-2 kPa at these earlieststages. Quantitative Mass Spectroscopy of embryonic myocardium identifiesabundant structural proteins that most likely contribute to tissue mechanics.In particular, collagen I expression parallels the mechanical stiffening of theheart tissue. Treatment with collagenase or transglutaminase respectivelysoftens or stiffens heart tissue in a dose dependent manner while maintainingfunctional cellular contractile machinery. Both stiffening and softening wholeheart tubes reduces tissue strain during contraction, suggesting an optimumme-chanics for myocardial contraction even at the earliest functional stages. Con-traction wave velocity, however, changes proportionally to the stiffness of thetissue. E3-4 cardiomyocytes cultured on collagen-coated polyacrylamide gelsubstrates of different stiffnesses are best able to contract on substrates of~1.5 kPa, consistent with the physiological stiffness of the tissue from whichthey were derived. As in the whole heart, the cells were also less able to con-tract on much softer substrates of 300 Pa, or stiffer substrates of 10 kPa or more.This further points to optimizing mechanics for cardiomyocyte strain thatmatches the physiological stiffness of myocardium throughout development.Finally, effects environmental mechanics on nuclear proteins during develop-ment are being explored.[1] Majkut SF, Discher DE. (2012). BMMB. Doi: 10.1007/s10237-012-0413-8798-Pos Board B567Increased Tension Cost in Human Familial Hypertrophic CardiomyopathyCaused by the MYH7 Mutation R403QE. Rosalie Witjas-Paalberends1, Nicoletta Piroddi2, Beatrice Scellini2,Ger J. Stienen1, Chiara Tesi2, Corrado Pogessi2, Jolanda van der Velden1.1Institute for Cardiovascular Research VU University Medical Center,Amsterdam, Netherlands, 2Dipartimento di Scienze Fisiologiche,Firenze, Italy.Familial hypertrophic cardiomyopathy (HCM) is frequently caused by muta-tions in genes encoding sarcomeric proteins. Energy depletion of the heart isthought to initiate and promote HCM disease development. It has been hypoth-esized that HCM sarcomere gene mutations increase ATP utilization for myo-filament contraction and thereby increase energy demand of the heart. Previousstudies in single left ventricular myofibrils from myocardium harbouring thefirst identified causal HCM mutation R403Q in the gene encoding b-myosinheavy chain revealed increased rates of tension generation and relaxation inR403Q compared to myofibrils from healthy control myocardium. The alteredkinetics observed in the mutant sample predicted a 3-fold increase in the energycost of tension generation in R403Q sarcomeres. In the present study we inves-tigated if tension cost was indeed increased in human myocardium harbouringthe R403Q mutation.Maximal force generating capacity (Fmax) and ATP consumption (ATPmax)were simultaneously measured in Triton-permeabilized left ventricular musclestrips (n=8) from a patient carrying the R403Q mutation. Left ventricular mus-cle strips (n=17) from 5 HCM sarcomere mutation negative patients (HCMsmn)and strips (n=6) from 2 patients with secondary hypertrophy due to aortic ste-nosis (LVHao) served as a control groups. Economy of myofilament contractionis expressed as tension cost (TC), i.e. amount of ATP used during force devel-opment (ATPmax/Fmax). Both Fmax and ATPmax were significantly lower inR403Q compared to HCMsmn and LVHao. TC was significantly increased inR403Q (3.850.5 mmolL1s1/kNm2) compared to HCMsmn and LVHao(1.650.1 and 1.750.1 mmolL1s-1/kNm2, respectively).Our data provide direct evidence that TC is increased in myocardium harbour-ing the MYH7 mutation R403Q, indicating that expression of R403Q in theheart impairs economy of myocardial contraction.Funding: Seventh Framework Program of the European Union ‘‘BIG-HEART,’’ grant agreement 241577799-Pos Board B568Synthetic Peptides Model Instability of Cardiac Myosin Subfragment-2Nasrin Taei, Alysha Joseph, Diana Wang, Douglas D. Root.University of North Texas, Denton, TX, USA.A cluster of cardiomyopathy mutation sites in b-cardiac myosin including a par-ticularly lethal mutation of E930del can be targeted to part of the N-terminalregion of myosin subfragment-2. A peptide model of this system was synthe-sized and labeled with resonance energy transfer probes to detect the stabilityof coiled coil formation and estimates of the monomer-dimer equilibrium of thepeptide. Distance measurements between the donor and acceptor probes wereconsistent with computational simulations of the labeled peptides in a coiledcoil structure which matched simulations of the unlabeled wildtype structure.The addition of a site-specific antibody to this peptide strongly inhibited reso-nance energy transfer, indicating that the coiled coil structure was destabilized.In contrast, the addition of certain polyamine structures improved the stabilityof the coiled coil, while other polyamines had little effect despite having signif-icant binding constants to myosin as determined by competitive ELISA assays.The length of the polyamine appears to be important for this ability to stabilizethe coiled coil in this region of negatively charged glutamates in the myosinsubfragment-2 sequence. Some of the polyamines may be capable of bindingboth a-helixes of the myosin subfragment-2 simultaneously, thereby stabilizingthe coiled coil structure. This synthetic peptide model system holds promise asa test system for small molecule compounds that may counteract the destabiliz-ing effects of some cardiomyopathy mutations on the myosin coiled coil struc-ture. (sponsored by NSF ARRA)800-Pos Board B569Regulatory Light Chain Mutants Linked to Heart Disease affect theCardiac Myosin Lever-ArmLaura A. Sikkink, Thomas P. Burghardt.Mayo Foundation, Rochester, MN, USA.Myosin is the chemomechanical energy transducer in striated muscle workingin the heart to circulate blood. Transduction occurs by cyclical actomyosin in-teraction causing myosin thick filament sliding relative to the actin thin fila-ment while consuming chemical energy from ATP. Transduction begins inthe myosin head domain or cross-bridge with ATP hydrolysis driving rotarymovement of a lever-arm converting rotation into linear translation. Myosinregulatory light chain (RLC) binds to the lever-arm and affects its ability totranslate actin. Gene sequencing implicated several RLC mutations in humanheart disease and three of them are investigated here using single moleculedetection of photoactivatable GFP tagged RLC (RLC-PAGFP) exchangedinto permeabilized porcine papillary muscle fibers. The RLC-PAGFP detectssingle lever-arm orientation. Single molecule orientation detection benefitsfrom new analysis tools to deduce PAGFP orientation from the 3 spatial di-mensional fluorescence emission pattern. Experiments were performed in a mi-crofluidic chamber designed for isometric contraction, total internal reflectionfluorescence single molecule detection, and for 2-photon excitation secondharmonic generation to evaluate sarcomere length. The RLC-PAGFP reportsdiscretized lever-arm orientation intermediates in active isometric fibers char-acteristic to the wild type (WT) RLC where lever-arms occupy intermediateson the free-energy gradient that on average correspond to the cross-bridgestall force. Two disease linked mutants introduced to RLC move state occu-pancy further down the free energy gradient implying lever-arms rotatemore to reach cross-bridge stall force because mutant RLC increases lever-arm shear strain. Lower free energy states could involve strong binding thatwill inhibit filament sliding in contraction. This research was supported byNIH Institutes: NIAMS R01 AR049277 and NHLBI HL095572 and by theMayo Foundation.

Increased tension cost in human familial hypertrophic cardiomyopathy caused by the MYH7 mutation R403Q

https://flore.unifi.it/bitstream/2158/969210/1/Increased%20Tension%20Cost%20in%20Human%20Familial%20Hypertrophic%20Cardiomyopathy%20%20Biophysical%20Society%2057th%20Philadelphia%202013.pdf

Increased tension cost in human familial hypertrophic cardiomyopathy caused by the MYH7 mutation R403Q

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