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Fluorescent Dextran Diffusion Assay to Study Cardiac T-Tubules
Wednesday, March 2, 2016 carrier to illustrate the transport events in TNTs of human cells. We first coated 100-nm FNDs with proteins including bovine serum albumin (BSA) and green fluorescent protein (GFP) by physical adsorption and then performed single particle tracking of the bioconjugates in the transient membrane connections by fluorescence microscopy. We observed stop-and-go and to-and-fro motions mediated by molecular motors and determined an average velocity of 0.31 mm/s for the active transport of protein-loaded FNDs trapped in the endosomal vehicles of human embryonic kidney cells (HEK293T). Quantitative analysis of the heterotypical transport between HEK293T and SH-SY5Y neuroblastoma cells by flow cytometry confirmed the formation of open-ended nanotubes between them, even though their TNTs differed in structural components. Our results demonstrate a nanotechnology-enabled new tool for long-term investigation of the intercellular transport of proteins, drugs, or vesicles at the single particle level for potential therapeutic applications. 2947-Pos Board B324 Anisotropic and Anomalous Diffusion in Mitotic Cells Matthias Weiss. Experimental Physics I, University of Bayreuth, Bayreuth, Germany. Protein diffusion in crowded intracellular fluids is a crucial determinant of many vital biochemical pathways. Frequently an anomalous diffusion of macromolecules in the cytoplasm and nucleoplasm of eukaryotic cells has been reported, and associated changes in biochemical reactions have been discussed in some detail [1]. Here we show that different degrees of crowding combined with an obstructed and anomalous diffusion are key determinants of protein exchange between cytoplasm and nucleoplasm during nuclear envelope breakdown (NEB) at the onset of mitosis [2]. After NEB, a contiguous nucleo-cytoplasmic fluid is established that features an anisotropically varying anomalous diffusion of macromolecules during metaphase [3]: Diffusion appears less anomalous along the mitotic spindle axis as compared to perpendicular directions. As a consequence, the long-time diffusion of macromolecules preferentially points along the spindle axis, leading to a prolonged residence of macromolecules in the spindle region. The observed anisotropic diffusion is proposed to support the dynamic formation of a dynamic spindle matrix that controls and guides later steps in mitosis. [1] Weiss, Int. Rev. Cell Mol. Biol., 307, 383-417 (2014). [2] Schweizer, Pawar, Weiss & Maiato, J. Cell Biol. 210, 695 (2015). [3] Pawar, Donth & Weiss, Curr. Biol. 24, 1905 (2014). 2948-Pos Board B325 Analysis of Scfd2 - A New Member of the SM Protein Family Janeta V. Iordanova, Dirk Fasshauer. DNF, UNIL, Lausanne, Switzerland. Proteins of the SM (Sec1/Munc18) protein family are indispensable regulators of vesicle fusion in eukaryotic cells. Currently, four basic types of SM proteins are known: Sec1 or Munc18, Sly1, Vps45 and Vps33. In different trafficking steps, they interact with respective SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) proteins, which catalyze the fusion of vesicle and target membranes by zippering into a tight four-helix bundle between membranes. Most SM proteins interact directly with the Qa-SNARE or syntaxin, although different binding modes have been proposed. Upon phylogenetic analysis of the SM protein family, we came across a new member of the family, named Scfd2 (Sec1 Family Domain Containing 2). Although it is likely that this new SM protein type was present in the genome of the last eukaryotic common ancestor, it has only been maintained in certain lineages, among them most animals and plants. It is absent, however, from several widely used model organisms like S. cerevisiae, C. elegans and Drosophila. Little is known about the function of Scfd2 and it is not clear whether it acts in vesicle trafficking in a manner comparable to other SM proteins and whether it has a specific SNARE binding partner. Recent data suggest that Scfd2 may interact or even be a part of a tethering complex involved in ER-Golgi transport. In order to shed light on the function of Scfd2, we are investigating its interaction with the key proteins involved in ER-Golgi trafficking, using biochemical approaches. 2949-Pos Board B326 Quantifying the Influence of the Crowded Cytoplasm on Ionic Diffusion Selcuk Atalay, Caitlin E. Scott, Peter M. Kekenes-Huskey. Chemistry, University of Kentucky, Lexington, KY, USA. Cytosolic crowding is known to influence the thermodynamics and kinetics of in vivo chemical reactions. Crowders, including proteins, macromolecular assemblies and intracellular organelles, reduce the volume available to a diffusing substrate and thereby lower its effective diffusion constant relative to its rate in bulk solution. However, the nature of a substrate’s interaction
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with crowders that occlude diffusion can further influence the effective diffusion rate. To probe the impact of crowding over sub-micron intracellular distances, we apply a multi-scale mathematical theory, homogenization, to estimate effective diffusion rates for ions and small bio-molecules diffusing in a densely-packed lattice of representative cytosolic proteins. Our simulations quantify how the crowded volume fraction, irregularity of protein shapes and distribution, as well as electrostatic interactions, influence the diffusion rates of small molecules. For non-interacting substrates that diffuse much faster than proteins crowders, the effective diffusion constant is dominated by volume fraction with minor contributions from crowder distribution and shapes, which permits a simplified description of the cytosol for most diffusion applications. Our results also shows that the diffusion coefficient of the diffuser can be accelerated or decelerated for attractive or repulsive forces; these effect of these interactions on diffusion strongly depend on the distance between crowders and the electric double layer thickness. For the first time, we derived an analytical formula to calculate the diffusion coefficient of a crowded system as a function of volume fraction and background salt concentration. The predicted values of diffusion coefficient evaluated from the analytical model are in good agreement with our homogenization and continuum models. We finally demonstrate the application of this methodology toward modeling calcium release units in highly-structured intracellular domains. 2950-Pos Board B327 Transport Imaging of Living Cells Szabolcs Osva´th1,2, Levente Here´nyi1, Gergely Ago´cs1, Katalin Kis Petik1, Miklo´s S.Z. Kellermayer1,2. 1 Dept of Biophysics and Radiation Biology, Semmelweiss University, Budapest, Hungary, 2MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Budapest, Hungary. Living cells are out of equilibrium biological systems in which active and passive transports are literally of vital importance. Intracellular motion of a few types of particles has been studied before, but how these motions fit into the broader context of all physiological transports of the cell is still unclear. We used an unselective method - phase contrast imaging - to detect virtually all intracellular movements. Fourier analysis of movies of living HEP2 cells shows that cellular motions lack a well defined characteristic time. Movements observed in the 0.5 s to 655 s range were very information rich, showing self-similar temporal behavior in all locations inside the cell. To grab this information, a transport image of the living cell was constructed by representing the locally observed Hurst coefficient of the motions as a stand alone image. The cytoplasm of the cells was found to be dominated by superdiffusion driven by active transport. Brownian diffusion and subdiffusion was also observed, mostly inside the nucleus. We believe that the abundance of subdiffusion reports in the literature arises from the fact that the observed test particles or molecules were not integral part of the intracellular physiology. We propose the use of unselective microscopy methods and Hurst coefficient transport imaging as an effective tool to visualize physiological transport processes of living cells.
Excitation-Contraction Coupling II 2951-Pos Board B328 Fluorescent Dextran Diffusion Assay to Study Cardiac T-Tubules Keita Uchida, Anatoli N. Lopatin. MIP, University of Michigan, Ann Arbor, MI, USA. T-tubules are membrane invaginations of cardiac myocytes critical for effective excitation-contraction coupling and are significantly altered in various diseases. However, the mechanisms underlying t-tubule remodeling remain largely unknown, particularly due to a number of limitations of currently available technologies. Therefore, we have developed an assay that employs the diffusion of fluorescent 3 KD dextran out of the t-tubule network of live intact isolated ventricular cardiomyocytes. To significantly improve the quality of measurements, dextran is applied locally using a patch-clamp pipette (~23 mm diameter) under constant flow of washing solution through 0.5 mm ID capillary glass, which limits dextran filling of t-tubules to a small area of the cardiomyocyte. With 40X objective and small pinholes in the light paths, excitation is limited to ~9-10 mm diameter area and fluorescence emission is measured from ~5-6 mm diameter region using a photomultiplier. Upon cessation of dye application, fluorescence decline displays 2 major components: a fast component (t ~5-50 ms) corresponding to dye washout from the solution surrounding the cell and a slow component corresponding to dye diffusion out of the t-tubule system. In healthy mouse ventricular myocytes the slow component had a time constant of 1304 5 98 ms (n=24). In contrast, ‘‘detubulation’’ by acute hyposmotic stress significantly decreased the time constant to 848 5 99 ms (n=27, p<0.01) presumably due to shorter remaining t-tubule
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Wednesday, March 2, 2016
segments. In HEK293 cells, which lack t-tubules, the slow component has time constant of 210 5 51 ms (n=5), suggesting that dextran unbinding from cellular membrane is significantly faster than its diffusion out of t-tubules. This technique allows for rapid (within seconds) repetitive measures of the diffusional constraints imposed by the t-tubule network and provides a novel tool for studying dynamics of t-tubule remodeling in various experimental conditions. 2952-Pos Board B329 The T-System Provides a Dynamic Ca2D-Buffer in Human Skeletal Muscle Fibres Tanya R. Cully, Bradley S. Launikonis. School of Biomedical Sciences, The University of Queensland, St Lucia, Australia. The tubular (t-) of skeletal muscle fibres is known to be able to vacuolate in response to osmotic stress, stretch and fatigue. We trapped rhod-5N in the t-system of skinned fibres to image changes in t-system structure and [Ca2þ] in human muscle fibres isolated from biopsies on a confocal microscope. Imaging showed variation in numbers and volume of vacuoles, from none to virtually totally obscuring the transverse tubules. To observe Ca2þ movements between the t-system and cytoplasm, Ca2þsup was rapidly released from the sarcoplasmic reticulum and this was followed by introduction of 28-1342nM [Ca2þ]. Once formed, vacuoles persisted through Ca2þ load and release cycles. While Ca2þ release depleted transverse tubules via SOCE, vacuoles took up Ca2þ which slowly diffused out. However, if vacuoles were exposed to high [Ca2þ]cyto or Ca2þ release from a heavily loaded SR, chronically activated SOCE did not deplete Ca2þ from vacuoles. Many minutes of bathing in a solution containing no Ca2þ was required for vacuoles to begin to decrease in size. Upon application of 5mM Ca2þ and ionophore, vacuoles could show a manyfold increase in volume and thus Ca2þ-holding capacity. Subsequent application of 0 Ca2þ caused vacuoles to deform in seconds. A further addition of 5 mM Ca2þ saw the transverse tubules reappear by filling with Ca2þ. We conclude that vacuoles are able to take up and hold mM Ca2þ and can deform as Ca2þ is lost. This suggests that vacuoles can dynamically buffer Ca2þ in skeletal muscle fibres. 2953-Pos Board B330 Postnatal Development of T-Tubules in Sheep Atrial Myocytes Charlotte E.R. Smith, David A. Eisner, Andrew W. Trafford, Katharine M. Dibb. Institute of Cardiovascular Sciences, University of Manchester, Manchester, United Kingdom. Background: Cardiac t-tubules are essential in ensuring systolic calcium rises synchronously to enable contraction. The t-tubule network is absent or very sparse at birth in the ventricles of small mammals and develops postnatally with subsequent changes in calcium handling. Nothing is currently known about the development of t-tubules in the atria. Methods and Results: Newborn, 1 month, 3 month, 6 month and adult sheep (~18 months) were euthanased and cells isolated from the left atrial appendage. T-tubule density assessed using di-4-ANEPPS and confocal microscopy showed partially developed t-tubules were present at birth in sheep atrial myocytes but at 3059% of adult density (p<0.001). T-tubule density increased at 1 month and by 3 months t-tubules appeared fully developed despite decreased cell width compared to adult (p<0.01). In current clamp experiments, performed at 37 C with fluo-3 AM loaded cells, APD10, 25 and 50 were shorter in newborn, 1 month and 3 months compared to adult (p<0.01). There was no difference in calcium transient amplitude between newborn, 1 month and 3 month cells but the rate of decay of systolic calcium was slower in newborn compared to both 1 month and 3 months (p<0.05). Conclusion: T-tubules are present at birth in sheep atrial myocytes and develop over the first 3 months of life. Both action potential duration and rate of calcium transient decay increase during development. We hypothesise that this may be due to increased L-type calcium current as t-tubules develop and increased SERCA function as the sarcoplasmic reticulum matures. Further investigation is required to determine how the expression of t-tubule regulatory proteins and activity of calcium handling proteins changes throughout development. 2954-Pos Board B331 Relationship between Multi-Scale Cardiomyocyte Organization and Function in Trabeculae of the Failing Human Heart Michelle L. Munro1, Xin Shen1, Marie Ward1, David J. Crossman1, Christian Soeller1,2. 1 Physiology, University of Auckland, Auckland, New Zealand, 2Biomedical Physics, University of Exeter, Exeter, United Kingdom. Invaginations of the plasma membrane within cardiomyocytes form the transverse tubule network (t-tubules), and at ‘‘dyadic junctions’’ these come into
close association with regions of the sarcoplasmic reticulum (SR). In addition to many of the key excitation-contraction (EC) coupling proteins, junctions contain junctophilin-2 (JPH2), which is implicated in both the formation and maintenance of junctions. In human heart failure there is a loss of cardiac function which is generally associated with impairment of EC coupling processes, as well as changes to t-tubule structure and protein distribution. The aim of this study was to investigate the link between structural re-organization of cardiomyocytes and the loss of contractile function in trabeculae from failing human hearts. We obtained trabeculae from explanted human hearts in end-stage heart failure and performed force measurements followed by immuno-labelling to determine correlations between protein distribution and functional performance in the failing human heart. Cardiac trabeculae were processed along with samples taken from the free wall to investigate the variability of structure and protein distribution, including t-tubules, JPH2 and ryanodine receptors (the SR calcium release channel) using confocal imaging. Findings from these experiments revealed that there is a high degree of variability of trabecular structure and function, both within and between hearts. Trabeculae exhibiting poor contractile function often had low cardiomyocyte content and remaining myocytes showed disorganization of t-tubules and EC coupling proteins. These findings demonstrate a clear link between trabecula structure and function in the diseased human heart and support the idea that cardiomyocyte organization at cell and tissue levels is correlated with cardiac function. Furthermore, while trabeculae are useful for studying cardiac function, trabecula and cardiomyocyte structure must be considered when interpreting functional results. 2955-Pos Board B332 Modulation by CGP-37157 (CGP) Analogs of the Sarcoplasmic Reticulum Calcium ATPase SERCA) Melanie M. Loulousis, Yuanzhao L. Darcy, Julio A. Copello. Pharmacology, Southern Illinois University, Springfield, IL, USA. We have reported that CGP, a 4-1 benzothiazepine (BZT) associated with cardioprotection in ischemia, is a SERCA inhibitor. CGP blocking action differs from that of thapsigargin because is Ca2þ dependent; i.e., CGP blocking potency increases when Ca2þ levels decrease. We utilized purified sarcoplasmic reticulum microsomal membranes (SRM; from rabbit skeletal muscle and pig ventricle) to test the inhibitory actions of various novel CGP analogs on ATPase function (estimated by measurements of Ca2þ loading and ATPase activity). The 4,1-benzothiazepin-2-one structural arrangement seems to be important for SERCA blocking action since function is lost within compounds where: the Sulfur is substituted by Nitrogen (as seen in benzodiazepines); the Oxygen is replaced by an alkyl group; or the arrangement between Nitrogen, Oxygen, and Sulfur in the BZT ring is reorganized (as seen in Diltiazem). Removal of Chlorine from position 7 in the BZT ring did not affect SERCA blocking potency. A combination of substitutions: 3H (benzylaminomethylcarboxymethyl), alkylation of the Nitrogen (neopentyl) and replacement of the position 1 of benzyl group (naphtyl), significantly increased potency 10fold (IC50 ~ 1.5 mM, in presence of 2 mM free Ca2þ). We determined (using SRM Ca2þ leak assay, [3H] ryanodine binding and channel studies in bilayers) CGP analogs can also activate ryanodine receptors (RyRs), albeit with low potency. In summary, we have identified structural features that may confer Ca2þdependent SERCA blocking in 4,1 BZT by utilizing CGP-like molecules. We also identified ph000995, a potent blocker, which may have potential for assessing SERCA roles in cells as well as being a potential drug-template for therapeutic intervention in diseases of abnormal Ca2þ homeostasis, such as ischemia. (Supported by American Heart Association, Eskridge Foundation and APS Frontiers in Physiology Research Program). 2956-Pos Board B333 Enhanced Cardiac Contractility and Ca2D Signalling Following Dietary Nitrate Supplementation in Mice Niklas Ivarsson1, Gianluigi Pironti1, Jingning Yang2, Alex Bersellini Farinotti1, William Jonsson1, Camilla Svensson1, Ha˚kan Westerblad1, Eddie Weitzberg1, Jon Lundberg1, John Pernow2, Johanna Lanner1, Daniel C. Andersson1. 1 Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 2 Medicine, Solna, Karolinska Institutet, Stockholm, Sweden. Inorganic nitrate (NO3-) that is naturally enriched in green diet has emerged as an important dietary component, which can regulate blood pressure, mitochondrial efficiency and skeletal muscle function. It is not known if dietary nitrate also affects cardiac function. To test this, mice were put on a low nitrate diet for 1 week. Then, some mice were supplemented for 7-14 days with 1 mM sodium nitrate in the drinking water. Compared to control, hearts from the nitrate treated mice displayed increased peak pressure development rate and left
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with crowders that occlude diffusion can further influence the effective diffusion rate. To probe the impact of crowding over sub-micron intracellular distances, we apply a multi-scale mathematical theory, homogenization, to estimate effective diffusion rates for ions and small bio-molecules diffusing in a densely-packed lattice of representative cytosolic proteins. Our simulations quantify how the crowded volume fraction, irregularity of protein shapes and distribution, as well as electrostatic interactions, influence the diffusion rates of small molecules. For non-interacting substrates that diffuse much faster than proteins crowders, the effective diffusion constant is dominated by volume fraction with minor contributions from crowder distribution and shapes, which permits a simplified description of the cytosol for most diffusion applications. Our results also shows that the diffusion coefficient of the diffuser can be accelerated or decelerated for attractive or repulsive forces; these effect of these interactions on diffusion strongly depend on the distance between crowders and the electric double layer thickness. For the first time, we derived an analytical formula to calculate the diffusion coefficient of a crowded system as a function of volume fraction and background salt concentration. The predicted values of diffusion coefficient evaluated from the analytical model are in good agreement with our homogenization and continuum models. We finally demonstrate the application of this methodology toward modeling calcium release units in highly-structured intracellular domains. 2950-Pos Board B327 Transport Imaging of Living Cells Szabolcs Osva´th1,2, Levente Here´nyi1, Gergely Ago´cs1, Katalin Kis Petik1, Miklo´s S.Z. Kellermayer1,2. 1 Dept of Biophysics and Radiation Biology, Semmelweiss University, Budapest, Hungary, 2MTA-SE Molecular Biophysics Research Group, Hungarian Academy of Sciences, Budapest, Hungary. Living cells are out of equilibrium biological systems in which active and passive transports are literally of vital importance. Intracellular motion of a few types of particles has been studied before, but how these motions fit into the broader context of all physiological transports of the cell is still unclear. We used an unselective method - phase contrast imaging - to detect virtually all intracellular movements. Fourier analysis of movies of living HEP2 cells shows that cellular motions lack a well defined characteristic time. Movements observed in the 0.5 s to 655 s range were very information rich, showing self-similar temporal behavior in all locations inside the cell. To grab this information, a transport image of the living cell was constructed by representing the locally observed Hurst coefficient of the motions as a stand alone image. The cytoplasm of the cells was found to be dominated by superdiffusion driven by active transport. Brownian diffusion and subdiffusion was also observed, mostly inside the nucleus. We believe that the abundance of subdiffusion reports in the literature arises from the fact that the observed test particles or molecules were not integral part of the intracellular physiology. We propose the use of unselective microscopy methods and Hurst coefficient transport imaging as an effective tool to visualize physiological transport processes of living cells.
Excitation-Contraction Coupling II 2951-Pos Board B328 Fluorescent Dextran Diffusion Assay to Study Cardiac T-Tubules Keita Uchida, Anatoli N. Lopatin. MIP, University of Michigan, Ann Arbor, MI, USA. T-tubules are membrane invaginations of cardiac myocytes critical for effective excitation-contraction coupling and are significantly altered in various diseases. However, the mechanisms underlying t-tubule remodeling remain largely unknown, particularly due to a number of limitations of currently available technologies. Therefore, we have developed an assay that employs the diffusion of fluorescent 3 KD dextran out of the t-tubule network of live intact isolated ventricular cardiomyocytes. To significantly improve the quality of measurements, dextran is applied locally using a patch-clamp pipette (~23 mm diameter) under constant flow of washing solution through 0.5 mm ID capillary glass, which limits dextran filling of t-tubules to a small area of the cardiomyocyte. With 40X objective and small pinholes in the light paths, excitation is limited to ~9-10 mm diameter area and fluorescence emission is measured from ~5-6 mm diameter region using a photomultiplier. Upon cessation of dye application, fluorescence decline displays 2 major components: a fast component (t ~5-50 ms) corresponding to dye washout from the solution surrounding the cell and a slow component corresponding to dye diffusion out of the t-tubule system. In healthy mouse ventricular myocytes the slow component had a time constant of 1304 5 98 ms (n=24). In contrast, ‘‘detubulation’’ by acute hyposmotic stress significantly decreased the time constant to 848 5 99 ms (n=27, p<0.01) presumably due to shorter remaining t-tubule
598a
Wednesday, March 2, 2016
segments. In HEK293 cells, which lack t-tubules, the slow component has time constant of 210 5 51 ms (n=5), suggesting that dextran unbinding from cellular membrane is significantly faster than its diffusion out of t-tubules. This technique allows for rapid (within seconds) repetitive measures of the diffusional constraints imposed by the t-tubule network and provides a novel tool for studying dynamics of t-tubule remodeling in various experimental conditions. 2952-Pos Board B329 The T-System Provides a Dynamic Ca2D-Buffer in Human Skeletal Muscle Fibres Tanya R. Cully, Bradley S. Launikonis. School of Biomedical Sciences, The University of Queensland, St Lucia, Australia. The tubular (t-) of skeletal muscle fibres is known to be able to vacuolate in response to osmotic stress, stretch and fatigue. We trapped rhod-5N in the t-system of skinned fibres to image changes in t-system structure and [Ca2þ] in human muscle fibres isolated from biopsies on a confocal microscope. Imaging showed variation in numbers and volume of vacuoles, from none to virtually totally obscuring the transverse tubules. To observe Ca2þ movements between the t-system and cytoplasm, Ca2þsup was rapidly released from the sarcoplasmic reticulum and this was followed by introduction of 28-1342nM [Ca2þ]. Once formed, vacuoles persisted through Ca2þ load and release cycles. While Ca2þ release depleted transverse tubules via SOCE, vacuoles took up Ca2þ which slowly diffused out. However, if vacuoles were exposed to high [Ca2þ]cyto or Ca2þ release from a heavily loaded SR, chronically activated SOCE did not deplete Ca2þ from vacuoles. Many minutes of bathing in a solution containing no Ca2þ was required for vacuoles to begin to decrease in size. Upon application of 5mM Ca2þ and ionophore, vacuoles could show a manyfold increase in volume and thus Ca2þ-holding capacity. Subsequent application of 0 Ca2þ caused vacuoles to deform in seconds. A further addition of 5 mM Ca2þ saw the transverse tubules reappear by filling with Ca2þ. We conclude that vacuoles are able to take up and hold mM Ca2þ and can deform as Ca2þ is lost. This suggests that vacuoles can dynamically buffer Ca2þ in skeletal muscle fibres. 2953-Pos Board B330 Postnatal Development of T-Tubules in Sheep Atrial Myocytes Charlotte E.R. Smith, David A. Eisner, Andrew W. Trafford, Katharine M. Dibb. Institute of Cardiovascular Sciences, University of Manchester, Manchester, United Kingdom. Background: Cardiac t-tubules are essential in ensuring systolic calcium rises synchronously to enable contraction. The t-tubule network is absent or very sparse at birth in the ventricles of small mammals and develops postnatally with subsequent changes in calcium handling. Nothing is currently known about the development of t-tubules in the atria. Methods and Results: Newborn, 1 month, 3 month, 6 month and adult sheep (~18 months) were euthanased and cells isolated from the left atrial appendage. T-tubule density assessed using di-4-ANEPPS and confocal microscopy showed partially developed t-tubules were present at birth in sheep atrial myocytes but at 3059% of adult density (p<0.001). T-tubule density increased at 1 month and by 3 months t-tubules appeared fully developed despite decreased cell width compared to adult (p<0.01). In current clamp experiments, performed at 37 C with fluo-3 AM loaded cells, APD10, 25 and 50 were shorter in newborn, 1 month and 3 months compared to adult (p<0.01). There was no difference in calcium transient amplitude between newborn, 1 month and 3 month cells but the rate of decay of systolic calcium was slower in newborn compared to both 1 month and 3 months (p<0.05). Conclusion: T-tubules are present at birth in sheep atrial myocytes and develop over the first 3 months of life. Both action potential duration and rate of calcium transient decay increase during development. We hypothesise that this may be due to increased L-type calcium current as t-tubules develop and increased SERCA function as the sarcoplasmic reticulum matures. Further investigation is required to determine how the expression of t-tubule regulatory proteins and activity of calcium handling proteins changes throughout development. 2954-Pos Board B331 Relationship between Multi-Scale Cardiomyocyte Organization and Function in Trabeculae of the Failing Human Heart Michelle L. Munro1, Xin Shen1, Marie Ward1, David J. Crossman1, Christian Soeller1,2. 1 Physiology, University of Auckland, Auckland, New Zealand, 2Biomedical Physics, University of Exeter, Exeter, United Kingdom. Invaginations of the plasma membrane within cardiomyocytes form the transverse tubule network (t-tubules), and at ‘‘dyadic junctions’’ these come into
close association with regions of the sarcoplasmic reticulum (SR). In addition to many of the key excitation-contraction (EC) coupling proteins, junctions contain junctophilin-2 (JPH2), which is implicated in both the formation and maintenance of junctions. In human heart failure there is a loss of cardiac function which is generally associated with impairment of EC coupling processes, as well as changes to t-tubule structure and protein distribution. The aim of this study was to investigate the link between structural re-organization of cardiomyocytes and the loss of contractile function in trabeculae from failing human hearts. We obtained trabeculae from explanted human hearts in end-stage heart failure and performed force measurements followed by immuno-labelling to determine correlations between protein distribution and functional performance in the failing human heart. Cardiac trabeculae were processed along with samples taken from the free wall to investigate the variability of structure and protein distribution, including t-tubules, JPH2 and ryanodine receptors (the SR calcium release channel) using confocal imaging. Findings from these experiments revealed that there is a high degree of variability of trabecular structure and function, both within and between hearts. Trabeculae exhibiting poor contractile function often had low cardiomyocyte content and remaining myocytes showed disorganization of t-tubules and EC coupling proteins. These findings demonstrate a clear link between trabecula structure and function in the diseased human heart and support the idea that cardiomyocyte organization at cell and tissue levels is correlated with cardiac function. Furthermore, while trabeculae are useful for studying cardiac function, trabecula and cardiomyocyte structure must be considered when interpreting functional results. 2955-Pos Board B332 Modulation by CGP-37157 (CGP) Analogs of the Sarcoplasmic Reticulum Calcium ATPase SERCA) Melanie M. Loulousis, Yuanzhao L. Darcy, Julio A. Copello. Pharmacology, Southern Illinois University, Springfield, IL, USA. We have reported that CGP, a 4-1 benzothiazepine (BZT) associated with cardioprotection in ischemia, is a SERCA inhibitor. CGP blocking action differs from that of thapsigargin because is Ca2þ dependent; i.e., CGP blocking potency increases when Ca2þ levels decrease. We utilized purified sarcoplasmic reticulum microsomal membranes (SRM; from rabbit skeletal muscle and pig ventricle) to test the inhibitory actions of various novel CGP analogs on ATPase function (estimated by measurements of Ca2þ loading and ATPase activity). The 4,1-benzothiazepin-2-one structural arrangement seems to be important for SERCA blocking action since function is lost within compounds where: the Sulfur is substituted by Nitrogen (as seen in benzodiazepines); the Oxygen is replaced by an alkyl group; or the arrangement between Nitrogen, Oxygen, and Sulfur in the BZT ring is reorganized (as seen in Diltiazem). Removal of Chlorine from position 7 in the BZT ring did not affect SERCA blocking potency. A combination of substitutions: 3H (benzylaminomethylcarboxymethyl), alkylation of the Nitrogen (neopentyl) and replacement of the position 1 of benzyl group (naphtyl), significantly increased potency 10fold (IC50 ~ 1.5 mM, in presence of 2 mM free Ca2þ). We determined (using SRM Ca2þ leak assay, [3H] ryanodine binding and channel studies in bilayers) CGP analogs can also activate ryanodine receptors (RyRs), albeit with low potency. In summary, we have identified structural features that may confer Ca2þdependent SERCA blocking in 4,1 BZT by utilizing CGP-like molecules. We also identified ph000995, a potent blocker, which may have potential for assessing SERCA roles in cells as well as being a potential drug-template for therapeutic intervention in diseases of abnormal Ca2þ homeostasis, such as ischemia. (Supported by American Heart Association, Eskridge Foundation and APS Frontiers in Physiology Research Program). 2956-Pos Board B333 Enhanced Cardiac Contractility and Ca2D Signalling Following Dietary Nitrate Supplementation in Mice Niklas Ivarsson1, Gianluigi Pironti1, Jingning Yang2, Alex Bersellini Farinotti1, William Jonsson1, Camilla Svensson1, Ha˚kan Westerblad1, Eddie Weitzberg1, Jon Lundberg1, John Pernow2, Johanna Lanner1, Daniel C. Andersson1. 1 Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden, 2 Medicine, Solna, Karolinska Institutet, Stockholm, Sweden. Inorganic nitrate (NO3-) that is naturally enriched in green diet has emerged as an important dietary component, which can regulate blood pressure, mitochondrial efficiency and skeletal muscle function. It is not known if dietary nitrate also affects cardiac function. To test this, mice were put on a low nitrate diet for 1 week. Then, some mice were supplemented for 7-14 days with 1 mM sodium nitrate in the drinking water. Compared to control, hearts from the nitrate treated mice displayed increased peak pressure development rate and left