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Cooperative Changes in Troponin Structure Explained without Cooperative Calcium Binding
236a
Monday, February 13, 2017
1160-Pos Board B228 Characterization of iPS Derived Cardiomyocytes in Voltage Clamp and Current Clamp by Automated Patch Clamp Andrea Br€ uggemann, Claudia Haarmann, Markus Rapedius, Tom Goetze, Ilka Rinke, Michael George, Niels Fertig. Nanion Technologies, Munich, Germany. In recent years, human stem cell-derived cardiomyocytes have proven to recapitulate key features of human cardiac electrophysiology in vitro. Furthermore, it has become apparent that the intact ensemble of cardiac ion channels is necessary to determine proarrhythmic effects reliably. Hence, due to their increasing availability, stem cell-derived cardiomyocytes have become the preferred choice of cardiac cells. This poster summarizes the promises and challenges of combining iPS derived cardiac myocytes with automated patch clamp. Features like high throughput, temperature control, easy internal solution exchange and full automation make planar patch clamp a desired method for characterizing iPS derived cardiomyocytes. One of the biggest challenges of planar patch clamp in this context is the fact that individual cells cannot be chosen, but cells will be selected randomly. In addition cells have to be harvested before the application to the patch clamp chip and cannot be patched as adherent cells. With this poster, we show our progress on these challenges. Two automated patch clamp platforms, the Patchliner, as a medium throughput, and the SyncroPatch 384PE, as a high throughput device, were used for this study. Pharmacological measurements in voltage clamp as well as current clamp will be shown also under physiological temperatures and perforated patch. 1161-Pos Board B229 Pharmacological Identification of an L-Type Calcium Channel Impedance Signal in Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes Carlos A. Obejero-Paz, Leslie Ellison, James Kramer, Arthur M. Brown, Andrew Bruening-Wright. Discovery Services, Charles River Laboratories, Cleveland, OH, USA. Impedance recordings of contractile human induced pluripotent stem-cell derived cardiomyocytes hold significant potential for drug discovery and cardiac safety given their easy implementation in medium throughput systems. However, the mechanisms underlying the complex impedance signal remain to be elucidated. The main component of the signal is the impedance twitch, a transient increase in electrode resistance that is obliterated by the myosin kinase inhibitor blebbistatin. Three additional small peaks (two initial negative deflections and one positive during relaxation) complete the complex impedance waveform. This work focuses on the pharmacological dissection of the first negative deflection that occurs after the sodium spike subsides and before the beginning of contraction. To test the hypothesis that the signal parallels the activation of calcium channels and not contraction, we investigated the effect of a calcium channel blocker (nifedipine), an agonist (FPL64176), and blebbistatin. Impedance recordings of iCell cardiomyocytes2 (CDI) were measured at 37 C using a CardioECR instrument (ACEA Biosciences) with pacing at 0.67 Hz. The impedance signal of iCell cardiomyocytes2 shows two initial negative deflections of 0.3850.02 U and 0.7850.03 U followed by a 4.0550.12 U positive peak (mean 5 sem, n=20).FPL64176 at 10 and 30 nM increased the first negative peak amplitude by 14.954.2% (p>0.05) and 31.056.8% (p<0.05), respectively (paired t-test, n=4). In 10 mM blebbistatin FPL64176-activated peaks were smaller (10 nM: 8.852.8%, p<0.05) or not affected (30 nM: 1.053.7%, p>0.05). Nifedipine at 30 nM and 100 nM significantly decreased the first negative peak amplitude by 26.756.9% and 32.752.5% respectively, and no peak was observed in 10 mM blebbistatin. The data suggests that the first negative impedance deflection is determined by the opening of calcium channels and thus may be used as marker for excitation-contraction coupling. 1162-Pos Board B230 Voltage-Sensitive ICG Fluorescence Imaging Regina Macianskiene, Mante Almanaityte, Rimantas Treinys, Irma Martisiene, Antanas Navalinskas, Rimantas Benetis, Jonas Jurevicius. Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania. So far, optical mapping (OM) of cardiac electrical signals using voltagesensitive fluorescent dyes has only been performed in experimental studies
because these dyes are not approved for clinical use. Recently, we have demonstrated that Indocyanine Green (ICG) fluorescence dye, which has been clinically approved in many countries over 50 years for use in medical diagnostics, has a voltage sensitive properties in the entire Langendorff perfused rabbit heart (Biophys J, 2016;110:723-732). We also showed that obtained optical signals (OS) has a dual-component (fast and slow) response to membrane potential changes, which accurately track the time of electrical signal propagation. The spectral properties of that fluorescence dye has been widely investigated for many years. Here we introduce a new approach in analysis of the fractional change in ICG fluorescence in response to the voltage changes, and show spectrum impact on the isolated OS components (fast and slow). We used light from a variety of LEDs to obtain excitation wavelengths ranging from 617 nm to 780 nm, and emission was collected using both band-pass filters for various wavelengths (from 700 nm to 900 nm) and spectrofotometer. We demonstrate changes in emission spectrum induced with ICG in a concentration-dependent manner. We obtained that each isolated component (fast and slow) is also composed of two components. We propose that obtained spectral shifts might depend on the aggregation of the dye molecules within or around the membrane. This research was funded by a grant (No. SEN-15/2015) from the Research Council of Lithuania.
Muscle Regulation 1163-Pos Board B231 An Obsevation of Native Skeletal Thin Filament Structure at High Calcium Condition by Electron Cryomicroscopy Fa-Qing Zhao. University of Massachusetts Medical School, Worcester, MA, USA. It is hampering our understanding on the molecular mechanism of muscle contraction thin filament-linked modulation lack of electron cryomicryoscopy data supporting the steric model (Nature 368,64-67,1994). Recently well-oriented sols of flamentous (F)-actin formed by Ca2þ-actin and length controlled by gelsolin give detailed X-ray fibre diffraction patterns led to the flat-actin conformation resolved in F-actin (Nature 457,441550,2009). von der Eckon et al (Nature,519,114-119,2015) defined a three˚ and in synthetic dimensional structure of F-actin at a resolution of 3.7A ˚ uscomplex with tropomyosin (no troponin complex) at a resolution of 6.5A ing electron cryomicroscopy. Here is the first report about a well-defined structure of native skeletal thin filament at high calcium condition by electron cryomicroscopy with Iterative Helical Real-Space Reconstruction (IHRSR): tropomyosin locates C-state on actin, spreads over one of myosin subfragment-1 (S1) strongly binding sites between subdomain_I and_III of the actin (JMB,266,8-14,1997); one intra-strand contact with strong 3D-reconstructed density and more than one inter-strand contacts are resolved among neighboring actin monomers (n, nþ1, nþ2), mainly formed through actin hydrophobic plug 256-271 linked to various regions of the opposite actin subunits, different from those of S1 decorated F-actin and the published F-actin and F-actin-Tropomyosin complex, in the native skeletal thin filament suggesting actin conformation diversity— globular-actin conformation dominating in high calcium condition, the flat conformation coexisting, and the two identified actin conformations are dynamic in thin filaments during the modulation of skeletal muscle contraction. 1164-Pos Board B232 Cooperative Changes in Troponin Structure Explained without Cooperative Calcium Binding Henry G. Zot, Javier E. Hasbun. Biology, University of West Georgia, Carrollton, GA, USA. To explain paradoxical changes in the intensity of the meridional 1/38.5 nm1 reflection corresponding to the structure of troponin during single and paired twitches, we employ a model that assumes muscle regulation by simple non-interacting Ca2þ binding sites of troponin (Tn) in the thin filament (Zot, H.G. and Hasbun, J.E. (2016) Frontiers Physiol. 7:406). In muscle stretched beyond overlap and stimulated once, the intensity of the meridional 1/38.5 nm1 reflection peaks slower and decays faster than expected for the Ca2þ association properties of isolated Tn, which may be explained by a model in which Ca2þ-bound Tn regulates the position of tropomyosin (Tm) cooperatively (Matsuo, T., Iwamoto, H., and Yagi, N. (2010). Biophys. J. 99, 193-200). However, Tn in regulated actin binds Ca2þ non-cooperatively. In muscle receiving paired stimuli, crossbridges elicit a much larger peak tension than the change in 1/38.5 nm1 reflection intensity, suggesting cooperativity owing to cycling crossbridges (Matsuo,
Monday, February 13, 2017 T., Iwamoto, H., and Yagi, N. (2008). J Mol. Biol. 383, 1019-1036). Given standard pulses of free Ca2þ, we fit the tension data of single and paired pulses with a model that permits cooperativity owing to cycling crossbridges but not Ca2þ-bound Tn. The model produces time courses of Ca2þ-bound Tn and the position of the Tm-Tn complex that follow single and paired transients of the meridional 1/38.5 nm1 reflection intensity changes for both overlap and nonoverlap preparations. The model predicts that a fraction of Ca2þ-bound Tn correlates in time and amplitude with the tension transient. This fraction represents Tn positioned away from possible interaction with actin by cycling crossbridges acting cooperatively on the position of Tm in the thin filament. Thus, this most parsimonious model of cooperativity is also the one most capable of explaining experimental observations. 1165-Pos Board B233 The Origin of the Force Increase Observed after Active Stretch Beyond Myofilament Overlap in Single Muscle Fibers Shuyue Liu, Venus Joumaa, Walter Herzog. University of Calgary, Calgary, AB, Canada. At long sarcomere lengths (SLs) beyond myofilament overlap, only passive forces, produced by passive structural elements, are possible. However, Leonard et al. (2010) showed a dramatic increase in force above the passive force when myofibrils were actively stretched beyond actin and myosin filament overlap. It has been suggested that titin’s stiffness might increase, or characteristic length decrease, with activation resulting in the increase in force observed when muscles are actively stretched compared to when they are passively stretched. Our purpose was to investigate whether the increase in force after active stretch beyond overlap was caused by a passive component/titin independently of the actin and myosin cross bridges. Therefore muscle fibres were actively stretched to a SL beyond myofilament overlap (5.0 mm) and then deactivated or treated with a cross bridge inhibitor, 2,3-butanedione monoxime (BDM). Fibres were also passively stretched to a SL of 5.0 mm. Results show similar decrease in force, of about 50%, after deactivation and treatment with a BDM solution. However, the forces after deactivation and treatment with BDM are higher than the purely passive stretch force at an average SL of 5.0 mm. The decrease in force after deactivation may be explained assuming that titin is an ‘‘activable/deactivable’’ spring whose stiffness is modulated by activation. However, the decrease in force following BDM exposure cannot be reconciled with a titin based mechanism only. BDM is known to inhibit cross bridge-based force, and thus the decrease in force after BDM exposure is likely associated with cross bridge inhibition. In conclusion, the force increase observed after active stretch beyond myofilament overlap is likely associated with cross-bridge and titin forces. 1166-Pos Board B234 Detection of Small-Molecule Modulators of Actin-Myosin Structure and Function using High-Throughput Time-Resolved FRET Piyali Guhathakurta1, Ewa Prochniewicz1, Kurt C. Peterson2, Benjamin D. Grant2, Gregory D. Gillispie2, David D. Thomas1,3. 1 Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA, 2Fluorescence Innovations Inc, Minneapolis, MN, USA, 3Photonic Pharma LLC, Minneapolis, MN, USA. We have used a time-resolved FRET assay to develop a system for detecting small-molecule modulators of actin-myosin structure and function. The fluorescent donor was fluorescein attached to actin C374, and the non-fluorescent acceptor was Dabcyl attached to the N-terminus of a peptide containing the 12 N-terminal amino acids of the A1 essential light chain (DNT). FRET was measured from the decrease in fluorescence lifetime, measured by time-resolved fluorescence in a novel fluorescence plate-reader, resolving structural distributions of the bound complex. The DNT binding site on actin overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-DNT complex as in the actin-myosin complex and (b) a significant decrease in actin-DNT FRET upon binding skeletal and cardiac myosin. A high-throughput FRET screen of a small-molecule library (NCC) showed that actin-DNT FRET is significantly affected by some compounds. All
237a
of these compounds inhibit actin-activated skeletal and cardiac myosin ATPase and affect the microsecond dynamics of actin, as detected by transient phosphorescence anisotropy. We concluded that the actin-DNT system allows detection of the effects of pharmacologically active compounds on actin structural states and consequently on functional properties of actomyosin. These results set the stage for a drug-discovery campaign, seeking therapies for cardiomyopathy and other disorders of involving actin-myosin dysfunction. 1167-Pos Board B235 Cardiac Tissue Organization and Contractility Jasmine Naik, Meghan Knight, Nancy Drew, Nicholas Johnsen, Anna Grosberg. Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. From the sarcomere to the myocardium, the heart is intricately organized on a wide range of length-scales. Multiple types of heart diseases are associated with remodeling of myofibrils and their sarcomeres - the basic force producing unit of the heart. However, it is not fully understood how organization of cardiac tissues affects the contractility properties. Disorganized tissues were found to be significantly weaker than predicted based on measurements from organized tissues. However, the prediction is based on a simplified net-force model, which has never been proven to accurately represent cardiac tissues because organizational variations are associated with downstream effects such as changes in gene expression levels that can further affect function. We hypothesized that these downstream effects could be muted if the cells were directed to organize locally (~250 micron scale), while keeping the global organization varied (~2 mm scale). The relationship between global organization and force production was modeled using a metric describing structure and developed contraction stress, and the locally organized, parquet, tissues were made with a range of global organizations. All of these tissues were then tested using the ‘‘heart-ona-chip’’ device to measure the global stress production. We have shown that with muted downstream effects, which are normally driven by local tissue organization, the global organization of the myofibrils is related to the net stress developed by a cardiac tissue through a single parameter analytical model. We have also shown that disorganized tissues tend to spontaneously form organized patches that are ~120 microns in scale, which can now be tested in conjunction with the force-organization model. These results will lead to a better understanding of the normal and pathological hearts. 1168-Pos Board B236 Probing Estradiol Effects on Super-Relaxed Myosin in Muscle Fibers with Fluorescence Lien A. Phung1, Joseph M. Muretta1, Karl J. Petersen1, John A. Rohde1, Tara L. Mader2, Dawn A. Lowe2, David D. Thomas1. 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA, 2Department of Rehabilitation Medicine, University of Minnesota, Minneapolis, MN, USA. Muscle myosins use the energy from ATP hydrolysis to perform mechanical work during muscle contraction. Recent work suggests that the interactions between the catalytic and light-chain-binding domains of myosin, the S2 coiled-coil fragment, and the thick-filament backbone, stabilize an auto-inhibited structural state of myosin, termed the superrelaxed state (SRX), with slowed ATP turnover kinetics. The superrelaxed state is hypothesized to play an important role in regulating contractility and thermogenesis in skeletal, cardiac muscle, and smooth muscle. We are using quantitative confocal microscopy of fluorescent ATP turnover in skinned skeletal and cardiac muscle fibers to measure the mole fraction of myosin molecules in the super-relaxed state, over a range of perturbations including animal age, sex, and hormonal state, post-translational modification of sarcomeric regulatory proteins, and small molecule modulators. From this work, we are mapping the determinants and regulatory correlates that control partitioning of myosin molecules into this important, poorly understood biophysical state. This work is supported by grants NIH R01AR032961-33 and T32AR00761215 (to DDT).
Monday, February 13, 2017
1160-Pos Board B228 Characterization of iPS Derived Cardiomyocytes in Voltage Clamp and Current Clamp by Automated Patch Clamp Andrea Br€ uggemann, Claudia Haarmann, Markus Rapedius, Tom Goetze, Ilka Rinke, Michael George, Niels Fertig. Nanion Technologies, Munich, Germany. In recent years, human stem cell-derived cardiomyocytes have proven to recapitulate key features of human cardiac electrophysiology in vitro. Furthermore, it has become apparent that the intact ensemble of cardiac ion channels is necessary to determine proarrhythmic effects reliably. Hence, due to their increasing availability, stem cell-derived cardiomyocytes have become the preferred choice of cardiac cells. This poster summarizes the promises and challenges of combining iPS derived cardiac myocytes with automated patch clamp. Features like high throughput, temperature control, easy internal solution exchange and full automation make planar patch clamp a desired method for characterizing iPS derived cardiomyocytes. One of the biggest challenges of planar patch clamp in this context is the fact that individual cells cannot be chosen, but cells will be selected randomly. In addition cells have to be harvested before the application to the patch clamp chip and cannot be patched as adherent cells. With this poster, we show our progress on these challenges. Two automated patch clamp platforms, the Patchliner, as a medium throughput, and the SyncroPatch 384PE, as a high throughput device, were used for this study. Pharmacological measurements in voltage clamp as well as current clamp will be shown also under physiological temperatures and perforated patch. 1161-Pos Board B229 Pharmacological Identification of an L-Type Calcium Channel Impedance Signal in Human Induced Pluripotent Stem-Cell Derived Cardiomyocytes Carlos A. Obejero-Paz, Leslie Ellison, James Kramer, Arthur M. Brown, Andrew Bruening-Wright. Discovery Services, Charles River Laboratories, Cleveland, OH, USA. Impedance recordings of contractile human induced pluripotent stem-cell derived cardiomyocytes hold significant potential for drug discovery and cardiac safety given their easy implementation in medium throughput systems. However, the mechanisms underlying the complex impedance signal remain to be elucidated. The main component of the signal is the impedance twitch, a transient increase in electrode resistance that is obliterated by the myosin kinase inhibitor blebbistatin. Three additional small peaks (two initial negative deflections and one positive during relaxation) complete the complex impedance waveform. This work focuses on the pharmacological dissection of the first negative deflection that occurs after the sodium spike subsides and before the beginning of contraction. To test the hypothesis that the signal parallels the activation of calcium channels and not contraction, we investigated the effect of a calcium channel blocker (nifedipine), an agonist (FPL64176), and blebbistatin. Impedance recordings of iCell cardiomyocytes2 (CDI) were measured at 37 C using a CardioECR instrument (ACEA Biosciences) with pacing at 0.67 Hz. The impedance signal of iCell cardiomyocytes2 shows two initial negative deflections of 0.3850.02 U and 0.7850.03 U followed by a 4.0550.12 U positive peak (mean 5 sem, n=20).FPL64176 at 10 and 30 nM increased the first negative peak amplitude by 14.954.2% (p>0.05) and 31.056.8% (p<0.05), respectively (paired t-test, n=4). In 10 mM blebbistatin FPL64176-activated peaks were smaller (10 nM: 8.852.8%, p<0.05) or not affected (30 nM: 1.053.7%, p>0.05). Nifedipine at 30 nM and 100 nM significantly decreased the first negative peak amplitude by 26.756.9% and 32.752.5% respectively, and no peak was observed in 10 mM blebbistatin. The data suggests that the first negative impedance deflection is determined by the opening of calcium channels and thus may be used as marker for excitation-contraction coupling. 1162-Pos Board B230 Voltage-Sensitive ICG Fluorescence Imaging Regina Macianskiene, Mante Almanaityte, Rimantas Treinys, Irma Martisiene, Antanas Navalinskas, Rimantas Benetis, Jonas Jurevicius. Institute of Cardiology, Lithuanian University of Health Sciences, Kaunas, Lithuania. So far, optical mapping (OM) of cardiac electrical signals using voltagesensitive fluorescent dyes has only been performed in experimental studies
because these dyes are not approved for clinical use. Recently, we have demonstrated that Indocyanine Green (ICG) fluorescence dye, which has been clinically approved in many countries over 50 years for use in medical diagnostics, has a voltage sensitive properties in the entire Langendorff perfused rabbit heart (Biophys J, 2016;110:723-732). We also showed that obtained optical signals (OS) has a dual-component (fast and slow) response to membrane potential changes, which accurately track the time of electrical signal propagation. The spectral properties of that fluorescence dye has been widely investigated for many years. Here we introduce a new approach in analysis of the fractional change in ICG fluorescence in response to the voltage changes, and show spectrum impact on the isolated OS components (fast and slow). We used light from a variety of LEDs to obtain excitation wavelengths ranging from 617 nm to 780 nm, and emission was collected using both band-pass filters for various wavelengths (from 700 nm to 900 nm) and spectrofotometer. We demonstrate changes in emission spectrum induced with ICG in a concentration-dependent manner. We obtained that each isolated component (fast and slow) is also composed of two components. We propose that obtained spectral shifts might depend on the aggregation of the dye molecules within or around the membrane. This research was funded by a grant (No. SEN-15/2015) from the Research Council of Lithuania.
Muscle Regulation 1163-Pos Board B231 An Obsevation of Native Skeletal Thin Filament Structure at High Calcium Condition by Electron Cryomicroscopy Fa-Qing Zhao. University of Massachusetts Medical School, Worcester, MA, USA. It is hampering our understanding on the molecular mechanism of muscle contraction thin filament-linked modulation lack of electron cryomicryoscopy data supporting the steric model (Nature 368,64-67,1994). Recently well-oriented sols of flamentous (F)-actin formed by Ca2þ-actin and length controlled by gelsolin give detailed X-ray fibre diffraction patterns led to the flat-actin conformation resolved in F-actin (Nature 457,441550,2009). von der Eckon et al (Nature,519,114-119,2015) defined a three˚ and in synthetic dimensional structure of F-actin at a resolution of 3.7A ˚ uscomplex with tropomyosin (no troponin complex) at a resolution of 6.5A ing electron cryomicroscopy. Here is the first report about a well-defined structure of native skeletal thin filament at high calcium condition by electron cryomicroscopy with Iterative Helical Real-Space Reconstruction (IHRSR): tropomyosin locates C-state on actin, spreads over one of myosin subfragment-1 (S1) strongly binding sites between subdomain_I and_III of the actin (JMB,266,8-14,1997); one intra-strand contact with strong 3D-reconstructed density and more than one inter-strand contacts are resolved among neighboring actin monomers (n, nþ1, nþ2), mainly formed through actin hydrophobic plug 256-271 linked to various regions of the opposite actin subunits, different from those of S1 decorated F-actin and the published F-actin and F-actin-Tropomyosin complex, in the native skeletal thin filament suggesting actin conformation diversity— globular-actin conformation dominating in high calcium condition, the flat conformation coexisting, and the two identified actin conformations are dynamic in thin filaments during the modulation of skeletal muscle contraction. 1164-Pos Board B232 Cooperative Changes in Troponin Structure Explained without Cooperative Calcium Binding Henry G. Zot, Javier E. Hasbun. Biology, University of West Georgia, Carrollton, GA, USA. To explain paradoxical changes in the intensity of the meridional 1/38.5 nm1 reflection corresponding to the structure of troponin during single and paired twitches, we employ a model that assumes muscle regulation by simple non-interacting Ca2þ binding sites of troponin (Tn) in the thin filament (Zot, H.G. and Hasbun, J.E. (2016) Frontiers Physiol. 7:406). In muscle stretched beyond overlap and stimulated once, the intensity of the meridional 1/38.5 nm1 reflection peaks slower and decays faster than expected for the Ca2þ association properties of isolated Tn, which may be explained by a model in which Ca2þ-bound Tn regulates the position of tropomyosin (Tm) cooperatively (Matsuo, T., Iwamoto, H., and Yagi, N. (2010). Biophys. J. 99, 193-200). However, Tn in regulated actin binds Ca2þ non-cooperatively. In muscle receiving paired stimuli, crossbridges elicit a much larger peak tension than the change in 1/38.5 nm1 reflection intensity, suggesting cooperativity owing to cycling crossbridges (Matsuo,
Monday, February 13, 2017 T., Iwamoto, H., and Yagi, N. (2008). J Mol. Biol. 383, 1019-1036). Given standard pulses of free Ca2þ, we fit the tension data of single and paired pulses with a model that permits cooperativity owing to cycling crossbridges but not Ca2þ-bound Tn. The model produces time courses of Ca2þ-bound Tn and the position of the Tm-Tn complex that follow single and paired transients of the meridional 1/38.5 nm1 reflection intensity changes for both overlap and nonoverlap preparations. The model predicts that a fraction of Ca2þ-bound Tn correlates in time and amplitude with the tension transient. This fraction represents Tn positioned away from possible interaction with actin by cycling crossbridges acting cooperatively on the position of Tm in the thin filament. Thus, this most parsimonious model of cooperativity is also the one most capable of explaining experimental observations. 1165-Pos Board B233 The Origin of the Force Increase Observed after Active Stretch Beyond Myofilament Overlap in Single Muscle Fibers Shuyue Liu, Venus Joumaa, Walter Herzog. University of Calgary, Calgary, AB, Canada. At long sarcomere lengths (SLs) beyond myofilament overlap, only passive forces, produced by passive structural elements, are possible. However, Leonard et al. (2010) showed a dramatic increase in force above the passive force when myofibrils were actively stretched beyond actin and myosin filament overlap. It has been suggested that titin’s stiffness might increase, or characteristic length decrease, with activation resulting in the increase in force observed when muscles are actively stretched compared to when they are passively stretched. Our purpose was to investigate whether the increase in force after active stretch beyond overlap was caused by a passive component/titin independently of the actin and myosin cross bridges. Therefore muscle fibres were actively stretched to a SL beyond myofilament overlap (5.0 mm) and then deactivated or treated with a cross bridge inhibitor, 2,3-butanedione monoxime (BDM). Fibres were also passively stretched to a SL of 5.0 mm. Results show similar decrease in force, of about 50%, after deactivation and treatment with a BDM solution. However, the forces after deactivation and treatment with BDM are higher than the purely passive stretch force at an average SL of 5.0 mm. The decrease in force after deactivation may be explained assuming that titin is an ‘‘activable/deactivable’’ spring whose stiffness is modulated by activation. However, the decrease in force following BDM exposure cannot be reconciled with a titin based mechanism only. BDM is known to inhibit cross bridge-based force, and thus the decrease in force after BDM exposure is likely associated with cross bridge inhibition. In conclusion, the force increase observed after active stretch beyond myofilament overlap is likely associated with cross-bridge and titin forces. 1166-Pos Board B234 Detection of Small-Molecule Modulators of Actin-Myosin Structure and Function using High-Throughput Time-Resolved FRET Piyali Guhathakurta1, Ewa Prochniewicz1, Kurt C. Peterson2, Benjamin D. Grant2, Gregory D. Gillispie2, David D. Thomas1,3. 1 Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA, 2Fluorescence Innovations Inc, Minneapolis, MN, USA, 3Photonic Pharma LLC, Minneapolis, MN, USA. We have used a time-resolved FRET assay to develop a system for detecting small-molecule modulators of actin-myosin structure and function. The fluorescent donor was fluorescein attached to actin C374, and the non-fluorescent acceptor was Dabcyl attached to the N-terminus of a peptide containing the 12 N-terminal amino acids of the A1 essential light chain (DNT). FRET was measured from the decrease in fluorescence lifetime, measured by time-resolved fluorescence in a novel fluorescence plate-reader, resolving structural distributions of the bound complex. The DNT binding site on actin overlaps with that of myosin, as indicated by (a) a similar distance observed in the actin-DNT complex as in the actin-myosin complex and (b) a significant decrease in actin-DNT FRET upon binding skeletal and cardiac myosin. A high-throughput FRET screen of a small-molecule library (NCC) showed that actin-DNT FRET is significantly affected by some compounds. All
237a
of these compounds inhibit actin-activated skeletal and cardiac myosin ATPase and affect the microsecond dynamics of actin, as detected by transient phosphorescence anisotropy. We concluded that the actin-DNT system allows detection of the effects of pharmacologically active compounds on actin structural states and consequently on functional properties of actomyosin. These results set the stage for a drug-discovery campaign, seeking therapies for cardiomyopathy and other disorders of involving actin-myosin dysfunction. 1167-Pos Board B235 Cardiac Tissue Organization and Contractility Jasmine Naik, Meghan Knight, Nancy Drew, Nicholas Johnsen, Anna Grosberg. Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. From the sarcomere to the myocardium, the heart is intricately organized on a wide range of length-scales. Multiple types of heart diseases are associated with remodeling of myofibrils and their sarcomeres - the basic force producing unit of the heart. However, it is not fully understood how organization of cardiac tissues affects the contractility properties. Disorganized tissues were found to be significantly weaker than predicted based on measurements from organized tissues. However, the prediction is based on a simplified net-force model, which has never been proven to accurately represent cardiac tissues because organizational variations are associated with downstream effects such as changes in gene expression levels that can further affect function. We hypothesized that these downstream effects could be muted if the cells were directed to organize locally (~250 micron scale), while keeping the global organization varied (~2 mm scale). The relationship between global organization and force production was modeled using a metric describing structure and developed contraction stress, and the locally organized, parquet, tissues were made with a range of global organizations. All of these tissues were then tested using the ‘‘heart-ona-chip’’ device to measure the global stress production. We have shown that with muted downstream effects, which are normally driven by local tissue organization, the global organization of the myofibrils is related to the net stress developed by a cardiac tissue through a single parameter analytical model. We have also shown that disorganized tissues tend to spontaneously form organized patches that are ~120 microns in scale, which can now be tested in conjunction with the force-organization model. These results will lead to a better understanding of the normal and pathological hearts. 1168-Pos Board B236 Probing Estradiol Effects on Super-Relaxed Myosin in Muscle Fibers with Fluorescence Lien A. Phung1, Joseph M. Muretta1, Karl J. Petersen1, John A. Rohde1, Tara L. Mader2, Dawn A. Lowe2, David D. Thomas1. 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA, 2Department of Rehabilitation Medicine, University of Minnesota, Minneapolis, MN, USA. Muscle myosins use the energy from ATP hydrolysis to perform mechanical work during muscle contraction. Recent work suggests that the interactions between the catalytic and light-chain-binding domains of myosin, the S2 coiled-coil fragment, and the thick-filament backbone, stabilize an auto-inhibited structural state of myosin, termed the superrelaxed state (SRX), with slowed ATP turnover kinetics. The superrelaxed state is hypothesized to play an important role in regulating contractility and thermogenesis in skeletal, cardiac muscle, and smooth muscle. We are using quantitative confocal microscopy of fluorescent ATP turnover in skinned skeletal and cardiac muscle fibers to measure the mole fraction of myosin molecules in the super-relaxed state, over a range of perturbations including animal age, sex, and hormonal state, post-translational modification of sarcomeric regulatory proteins, and small molecule modulators. From this work, we are mapping the determinants and regulatory correlates that control partitioning of myosin molecules into this important, poorly understood biophysical state. This work is supported by grants NIH R01AR032961-33 and T32AR00761215 (to DDT).