The Experts below are selected from a list of 132 Experts worldwide ranked by ideXlab platform
Julio L Vergara - One of the best experts on this subject based on the ideXlab platform.
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the delayed rectifier potassium conductance in the sarcolemma and the Transverse Tubular System membranes of mammalian skeletal muscle fibers
The Journal of General Physiology, 2012Co-Authors: Marino Difranco, Marbella Quinonez, Julio L VergaraAbstract:A two-microelectrode voltage clamp and optical measurements of membrane potential changes at the Transverse Tubular System (TTS) were used to characterize delayed rectifier K currents (IKV) in murine muscle fibers stained with the potentiometric dye di-8-ANEPPS. In intact fibers, IKV displays the canonical hallmarks of KV channels: voltage-dependent delayed activation and decay in time. The voltage dependence of the peak conductance (gKV) was only accounted for by double Boltzmann fits, suggesting at least two channel contributions to IKV. Osmotically treated fibers showed significant disconnection of the TTS and displayed smaller IKV, but with similar voltage dependence and time decays to intact fibers. This suggests that inactivation may be responsible for most of the decay in IKV records. A two-channel model that faithfully simulates IKV records in osmotically treated fibers comprises a low threshold and steeply voltage-dependent channel (channel A), which contributes ∼31% of gKV, and a more abundant high threshold channel (channel B), with shallower voltage dependence. Significant expression of the IKV1.4 and IKV3.4 channels was demonstrated by immunoblotting. Rectangular depolarizing pulses elicited step-like di-8-ANEPPS transients in intact fibers rendered electrically passive. In contrast, activation of IKV resulted in time- and voltage-dependent attenuations in optical transients that coincided in time with the peaks of IKV records. Normalized peak attenuations showed the same voltage dependence as peak IKV plots. A radial cable model including channels A and B and K diffusion in the TTS was used to simulate IKV and average TTS voltage changes. Model predictions and experimental data were compared to determine what fraction of gKV in the TTS accounted simultaneously for the electrical and optical data. Best predictions suggest that KV channels are approximately equally distributed in the sarcolemma and TTS membranes; under these conditions, >70% of IKV arises from the TTS.
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the potassium delayed rectifier conductance in the sarcolemma and the Transverse Tubular System membranes of mammalian skeletal muscle fibers
Biophysical Journal, 2012Co-Authors: Julio L Vergara, Marino DifrancoAbstract:The Transverse Tubular System (TTS) plays a key role not only in mediating the mechanism of excitation-contraction coupling, but also in determining the electrical properties of mammalian skeletal muscle fibers. We investigated the properties and distribution (between sarcolemma and TTS membranes) of the K delayed rectifier conductance (gKV) by simultaneously recording fluorescence transients and ionic currents (IKV) from FDB muscle fibers stained with the potentiometric indicator di-8-ANEPPS and voltage-clamped using a two-microelectrode configuration. Enzymatically dissociated fibers were mounted on the stage of an inverted fluorescence microscope equipped with a 460-500//500//513-558nm cube. The external solution had (in mM): 150 LiCl, 4 KCl, 20 MOPS, 2 CaCl2, 1MgCl2 and 10 glucose. The Na, ClC-1, Ca and KIR currents were blocked by external TTX (0.001), 9-ACA (0.4), nifedipine (0.02) and Rb (5), respectively. The membrane capacitance was measured after rendering the fibers electrically passive by replacing external Li and K by TEA. We found that IKV records display a delayed onset and decayed markedly during long depolarizing pulses (400ms) due to inactivation and accumulation mechanisms. Furthermore, while di-8-ANNEPS transients recorded from electrically passive fibers displayed quasi-rectangular kinetic properties, transients recorded from control fibers in the presence of IKV were associated with time-dependent attenuations that matched the kinetics of activation and decay of IKV records. Radial cable model simulations were used to evaluate the voltage-dependent kinetic parameters of gKV, to calculate the rate of accumulation of K+ ions in the lumen of the TTS, and to determine that the relative distribution of this conductance between the surface and TTS membranes is close to equal. This work was supported by NIH grants AR047664, AR041802, and AR054816.
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the na conductance in the sarcolemma and the Transverse Tubular System membranes of mammalian skeletal muscle fibers
The Journal of General Physiology, 2011Co-Authors: Marino Difranco, Julio L VergaraAbstract:Na (and Li) currents and fluorescence transients were recorded simultaneously under voltage-clamp conditions from mouse flexor digitorum brevis fibers stained with the potentiometric dye di-8-ANEPPS to investigate the distribution of Na channels between the surface and Transverse Tubular System (TTS) membranes. In fibers rendered electrically passive, voltage pulses resulted in step-like fluorescence changes that were used to calibrate the dye response. The effects of Na channel activation on the TTS voltage were investigated using Li, instead of Na, because di-8-ANEPPS transients show anomalies in the presence of the latter. Na and Li inward currents (INa, ILi; using half of the physiological ion concentration) showed very steep voltage dependences, with no reversal for depolarizations beyond the calculated equilibrium potential, suggesting that most of the current originates from a noncontrolled membrane compartment. Maximum peak ILi was ∼30% smaller than for INa, suggesting a Li-blocking effect. ILi activation resulted in the appearance of overshoots in otherwise step-like di-8-ANEPPS transients. Overshoots had comparable durations and voltage dependence as those of ILi. Simultaneously measured maximal overshoot and peak ILi were 54 ± 5% and 773 ± 53 µA/cm2, respectively. Radial cable model simulations predicted the properties of ILi and di-8-ANEPPS transients when TTS access resistances of 10–20 Ωcm2, and TTS-to-surface Na permeability density ratios in the range of 40:60 to 70:30, were used. Formamide-based osmotic shock resulted in incomplete detubulation. However, results from a subpopulation of treated fibers (low capacitance) provide confirmatory evidence that a significant proportion of ILi, and the overshoot in the optical signals, arises from the TTS in normal fibers. The quantitative evaluation of the distribution of Na channels between the sarcolemma and the TTS membranes, as provided here, is crucial for the understanding of the radial and longitudinal propagation of the action potential, which ultimately govern the mechanical activation of muscle in normal and diseased conditions.
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characterization of the clc 1 conductance at the surface and Transverse Tubular System membranes of mammalian skeletal muscle fibers
Biophysical Journal, 2011Co-Authors: Marino Difranco, Alvaro I Herrera, Julio L VergaraAbstract:ClC-1 is believed to be primarily responsible for the resting conductance in mammalian skeletal muscle fibers. However, the actual distribution of ClC-1 channels between the surface and Transverse Tubular System (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltage-clamped enzymatically dissociated short fibers using a 2-microelectrode configuration and simultaneously recorded chloride currents (ICl), and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all, but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride in order to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl-dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from the TTS. Supported by NIH grants AR047664, AR041802, and AR054816.
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differential recording of voltage changes at the surface and Transverse Tubular System membranes of mammalian skeletal muscle fibers using di 8 anepps and global and tirfm
Biophysical Journal, 2011Co-Authors: Joana Capote, Marino Difranco, Julio L VergaraAbstract:Aiming to investigate the distribution of ClC-1 and KIR channels at the sarcolemma and Transverse Tubular System (TTS) membranes of mammalian skeletal muscle fibers, we used global and total internal reflection fluorescence microscopy (TIRFM) to monitor voltage changes in these compartments, respectively. Enzymatically-dissociated fibers from murine FDB and interosseus muscles were stained with the potentiometric dye di-8- ANEPPS, and voltage-clamped with a two-microelectrode System. Ion substitutions were used to isolate and characterize the specific ClC-1 (ICl) and KIR (IKIR) currents: 70 mM internal [Cl-] and 120 mM external [K+], respectively. Also, 9-ACA and TEA were used to, respectively, block these currents. Global di-8-ANEPPS signals report, early after the onset of large hyperpolarizing pulses, ICl-dependent attenuations with respect to those recorded in the presence of 9-ACA. Peak attenuation levels of ∼35% were observed for ICl of ∼900 µA/cm2. Large attenuations were similarly observed in global signals recorded the presence of large IKIR's with respect to those in TEA. In contrast, TIRFM di-8-ANEPPS signals demonstrate only minor current-dependent attenuations (<10%) under conditions in which global signals evidenced much larger attenuations. Overall, our results demonstrate that voltage changes at the TTS membranes display prominent current-dependent attenuations while the sarcolemma is largely under voltage-clamp control. A radial cable model of the TTS, including equations for each current pathway and luminal accumulation/depletion of ions, was used to quantitatively predict the ionic currents and to assess their effects on average TTS voltage changes. Comparative analysis of global optical data with model predictions of voltage changes in the TTS suggests that both ClC-1 and KIR channels are equally distributed in both membrane compartments. Supported by NIH grants AR047664, AR041802, and AR054816.
Frank B Sachse - One of the best experts on this subject based on the ideXlab platform.
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remodeling of the Transverse Tubular System after myocardial infarction in rabbit correlates with local fibrosis a potential role of biomechanics
Progress in Biophysics & Molecular Biology, 2017Co-Authors: Aparna C Sankarankutty, Thomas Seidel, Frank B SachseAbstract:Abstract The Transverse Tubular System (t-System) of ventricular cardiomyocytes is essential for efficient excitation-contraction coupling. In cardiac diseases, such as heart failure, remodeling of the t-System contributes to reduced cardiac contractility. However, mechanisms of t-System remodeling are incompletely understood. Prior studies suggested an association with altered cardiac biomechanics and gene expression in disease. Since fibrosis may alter tissue biomechanics, we investigated the local microscopic association of t-System remodeling with fibrosis in a rabbit model of myocardial infarction (MI). Biopsies were taken from the MI border zone of 6 infarcted hearts and from 6 control hearts. Using confocal microscopy and automated image analysis, we quantified t-System integrity (ITT) and the local fraction of extracellular matrix (fECM). In control, fECM was 18 ± 0.3%. ITT was high and homogeneous (0.07 ± 0.006), and did not correlate with fECM (R2 = 0.05 ± 0.02). The MI border zone exhibited increased fECM within 3 mm from the infarct scar (30 ± 3.5%, p
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sheet like remodeling of the Transverse Tubular System in human heart failure impairs excitation contraction coupling and functional recovery by mechanical unloading
Circulation, 2017Co-Authors: Thomas Seidel, Sutip Navankasattusas, Azmi A Ahmad, Nikolaos A Diakos, Martin Tristanifirouzi, Michael J Bonios, Iosif Taleb, Craig H Selzman, Stavros G Drakos, Frank B SachseAbstract:Background: Cardiac recovery in response to mechanical unloading by left ventricular assist devices (LVADs) has been demonstrated in subgroups of patients with chronic heart failure (HF). Hallmarks of HF are depletion and disorganization of the Transverse Tubular System (t-System) in cardiomyocytes. Here, we investigated remodeling of the t-System in human end-stage HF and its role in cardiac recovery. Methods: Left ventricular biopsies were obtained from 5 donors and 26 patients with chronic HF undergoing implantation of LVADs. Three-dimensional confocal microscopy and computational image analysis were applied to assess t-System structure, density, and distance of ryanodine receptor clusters to the sarcolemma, including the t-System. Recovery of cardiac function in response to mechanical unloading was assessed by echocardiography during turndown of the LVAD. Results: The majority of HF myocytes showed remarkable t-System remodeling, particularly sheet-like invaginations of the sarcolemma. Circularity of t-System components was decreased in HF versus controls (0.37±0.01 versus 0.46±0.02; P 1 µm did not improve after mechanical unloading. In addition, calcium transients were recorded in field-stimulated isolated human cardiomyocytes and analyzed with respect to local t-System density. Calcium release in HF myocytes was restricted to regions proximal to the sarcolemma. Local calcium upstroke was delayed (23.9±4.9 versus 10.3±1.7 milliseconds; P <0.05) and more asynchronous (18.1±1.5 versus 8.9±2.2 milliseconds; P <0.01) in HF cells with low t-System density versus cells with high t-System density. Conclusions: The t-System in end-stage human HF presents a characteristic novel phenotype consisting of sheet-like invaginations of the sarcolemma. Our results suggest that the remodeled t-System impairs excitation-contraction coupling and functional recovery during chronic LVAD unloading. An intact t-System at the time of LVAD implantation may constitute a precondition and predictor for functional cardiac recovery after mechanical unloading. # Clinical Perspective {#article-title-51}
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cardiac resynchronization therapy reduces subcellular heterogeneity of ryanodine receptors t tubules and ca2 sparks produced by dyssynchronous heart failure
Circulation-heart Failure, 2015Co-Authors: Hui Li, John H B Bridge, Thomas Seidel, Gordon F Tomaselli, Justin Lichter, Frank B SachseAbstract:Background—Cardiac resynchronization therapy (CRT) is a major advance for treatment of patients with dyssynchronous heart failure (DHF). However, our understanding of DHF-associated remodeling of subcellular structure and function and their restoration after CRT remains incomplete. Methods and Results—We investigated subcellular heterogeneity of remodeling of structures and proteins associated with excitation–contraction coupling in cardiomyocytes in DHF and after CRT. Three-dimensional confocal microscopy revealed subcellular heterogeneity of ryanodine receptor (RyR) density and the Transverse Tubular System (t-System) in a canine model of DHF. RyR density at the ends of lateral left ventricular cardiomyocytes was higher than that in cell centers, whereas the t-System was depleted at cell ends. In anterior left ventricular cardiomyocytes, however, we found a similar degree of heterogeneous RyR remodeling, despite preserved t-System. Synchronous heart failure was associated with marginal heterogeneity of ...
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mechanical modulation of the Transverse Tubular System of ventricular cardiomyocytes
Progress in Biophysics & Molecular Biology, 2012Co-Authors: Thomas G Mcnary, John H B Bridge, Kenneth W Spitzer, Hilary Holloway, Peter Kohl, Frank B SachseAbstract:In most mammalian cardiomyocytes, the Transverse Tubular System (t-System) is a major site for electrical signaling and excitation-contraction coupling. The t-System consists of membrane invaginations, which are decorated with various proteins involved in excitation-contraction coupling and mechano-electric feedback. Remodeling of the t-System has been reported for cells in culture and various types of heart disease. In this paper, we provide insights into effects of mechanical strain on the t-System in rabbit left ventricular myocytes. Based on fluorescent labeling, three-dimensional scanning confocal microscopy, and digital image analysis, we studied living and fixed isolated cells in different strain conditions. We extracted geometric features of Transverse tubules (t-tubules) and characterized their arrangement with respect to the Z-disk. In addition, we studied the t-System in cells from hearts fixed either at zero left ventricular pressure (slack), at 30 mmHg (volume overload), or during lithium-induced contracture, using transmission electron microscopy. Two-dimensional image analysis was used to extract features of t-tubule cross-sections. Our analyses of confocal microscopic images showed that contracture at the cellular level causes deformation of the t-System, increasing the length and volume of t-tubules, and altering their cross-sectional shape. TEM data reconfirmed the presence of mechanically induced changes in t-Tubular cross sections. In summary, our studies suggest that passive longitudinal stretching and active contraction of ventricular cardiomyocytes affect the geometry of t-tubules. This confirms that mechanical changes at cellular levels could promote alterations in partial volumes that would support a convection-assisted mode of exchange between the t-System content and extracellular space.
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subcellular structures and function of myocytes impaired during heart failure are restored by cardiac resynchronization therapy
Circulation Research, 2012Co-Authors: Frank B Sachse, Eleonora Saviogalimberti, Natalia S Torres, Takeshi Aiba, David A Kass, Gordon F Tomaselli, John H B BridgeAbstract:Rationale:Cardiac resynchronization therapy (CRT) is an established treatment for patients with chronic heart failure. However, CRT-associated structural and functional remodeling at cellular and subcellular levels is only partly understood. Objective:To investigate the effects of CRT on subcellular structures and protein distributions associated with excitation-contraction coupling of ventricular cardiomyocytes. Methods and Results:Our studies revealed remodeling of the Transverse Tubular System (t-System) and the spatial association of ryanodine receptor (RyR) clusters in a canine model of dyssynchronous heart failure (DHF). We did not find this remodeling in a synchronous heart failure model based on atrial tachypacing. Remodeling in DHF ranged from minor alterations in anterior left ventricular myocytes to nearly complete loss of the t-System and dissociation of RyRs from sarcolemmal structures in lateral cells. After CRT, we found a remarkable and almost complete reverse remodeling of these structure...
Marino Difranco - One of the best experts on this subject based on the ideXlab platform.
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the delayed rectifier potassium conductance in the sarcolemma and the Transverse Tubular System membranes of mammalian skeletal muscle fibers
The Journal of General Physiology, 2012Co-Authors: Marino Difranco, Marbella Quinonez, Julio L VergaraAbstract:A two-microelectrode voltage clamp and optical measurements of membrane potential changes at the Transverse Tubular System (TTS) were used to characterize delayed rectifier K currents (IKV) in murine muscle fibers stained with the potentiometric dye di-8-ANEPPS. In intact fibers, IKV displays the canonical hallmarks of KV channels: voltage-dependent delayed activation and decay in time. The voltage dependence of the peak conductance (gKV) was only accounted for by double Boltzmann fits, suggesting at least two channel contributions to IKV. Osmotically treated fibers showed significant disconnection of the TTS and displayed smaller IKV, but with similar voltage dependence and time decays to intact fibers. This suggests that inactivation may be responsible for most of the decay in IKV records. A two-channel model that faithfully simulates IKV records in osmotically treated fibers comprises a low threshold and steeply voltage-dependent channel (channel A), which contributes ∼31% of gKV, and a more abundant high threshold channel (channel B), with shallower voltage dependence. Significant expression of the IKV1.4 and IKV3.4 channels was demonstrated by immunoblotting. Rectangular depolarizing pulses elicited step-like di-8-ANEPPS transients in intact fibers rendered electrically passive. In contrast, activation of IKV resulted in time- and voltage-dependent attenuations in optical transients that coincided in time with the peaks of IKV records. Normalized peak attenuations showed the same voltage dependence as peak IKV plots. A radial cable model including channels A and B and K diffusion in the TTS was used to simulate IKV and average TTS voltage changes. Model predictions and experimental data were compared to determine what fraction of gKV in the TTS accounted simultaneously for the electrical and optical data. Best predictions suggest that KV channels are approximately equally distributed in the sarcolemma and TTS membranes; under these conditions, >70% of IKV arises from the TTS.
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the potassium delayed rectifier conductance in the sarcolemma and the Transverse Tubular System membranes of mammalian skeletal muscle fibers
Biophysical Journal, 2012Co-Authors: Julio L Vergara, Marino DifrancoAbstract:The Transverse Tubular System (TTS) plays a key role not only in mediating the mechanism of excitation-contraction coupling, but also in determining the electrical properties of mammalian skeletal muscle fibers. We investigated the properties and distribution (between sarcolemma and TTS membranes) of the K delayed rectifier conductance (gKV) by simultaneously recording fluorescence transients and ionic currents (IKV) from FDB muscle fibers stained with the potentiometric indicator di-8-ANEPPS and voltage-clamped using a two-microelectrode configuration. Enzymatically dissociated fibers were mounted on the stage of an inverted fluorescence microscope equipped with a 460-500//500//513-558nm cube. The external solution had (in mM): 150 LiCl, 4 KCl, 20 MOPS, 2 CaCl2, 1MgCl2 and 10 glucose. The Na, ClC-1, Ca and KIR currents were blocked by external TTX (0.001), 9-ACA (0.4), nifedipine (0.02) and Rb (5), respectively. The membrane capacitance was measured after rendering the fibers electrically passive by replacing external Li and K by TEA. We found that IKV records display a delayed onset and decayed markedly during long depolarizing pulses (400ms) due to inactivation and accumulation mechanisms. Furthermore, while di-8-ANNEPS transients recorded from electrically passive fibers displayed quasi-rectangular kinetic properties, transients recorded from control fibers in the presence of IKV were associated with time-dependent attenuations that matched the kinetics of activation and decay of IKV records. Radial cable model simulations were used to evaluate the voltage-dependent kinetic parameters of gKV, to calculate the rate of accumulation of K+ ions in the lumen of the TTS, and to determine that the relative distribution of this conductance between the surface and TTS membranes is close to equal. This work was supported by NIH grants AR047664, AR041802, and AR054816.
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the na conductance in the sarcolemma and the Transverse Tubular System membranes of mammalian skeletal muscle fibers
The Journal of General Physiology, 2011Co-Authors: Marino Difranco, Julio L VergaraAbstract:Na (and Li) currents and fluorescence transients were recorded simultaneously under voltage-clamp conditions from mouse flexor digitorum brevis fibers stained with the potentiometric dye di-8-ANEPPS to investigate the distribution of Na channels between the surface and Transverse Tubular System (TTS) membranes. In fibers rendered electrically passive, voltage pulses resulted in step-like fluorescence changes that were used to calibrate the dye response. The effects of Na channel activation on the TTS voltage were investigated using Li, instead of Na, because di-8-ANEPPS transients show anomalies in the presence of the latter. Na and Li inward currents (INa, ILi; using half of the physiological ion concentration) showed very steep voltage dependences, with no reversal for depolarizations beyond the calculated equilibrium potential, suggesting that most of the current originates from a noncontrolled membrane compartment. Maximum peak ILi was ∼30% smaller than for INa, suggesting a Li-blocking effect. ILi activation resulted in the appearance of overshoots in otherwise step-like di-8-ANEPPS transients. Overshoots had comparable durations and voltage dependence as those of ILi. Simultaneously measured maximal overshoot and peak ILi were 54 ± 5% and 773 ± 53 µA/cm2, respectively. Radial cable model simulations predicted the properties of ILi and di-8-ANEPPS transients when TTS access resistances of 10–20 Ωcm2, and TTS-to-surface Na permeability density ratios in the range of 40:60 to 70:30, were used. Formamide-based osmotic shock resulted in incomplete detubulation. However, results from a subpopulation of treated fibers (low capacitance) provide confirmatory evidence that a significant proportion of ILi, and the overshoot in the optical signals, arises from the TTS in normal fibers. The quantitative evaluation of the distribution of Na channels between the sarcolemma and the TTS membranes, as provided here, is crucial for the understanding of the radial and longitudinal propagation of the action potential, which ultimately govern the mechanical activation of muscle in normal and diseased conditions.
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characterization of the clc 1 conductance at the surface and Transverse Tubular System membranes of mammalian skeletal muscle fibers
Biophysical Journal, 2011Co-Authors: Marino Difranco, Alvaro I Herrera, Julio L VergaraAbstract:ClC-1 is believed to be primarily responsible for the resting conductance in mammalian skeletal muscle fibers. However, the actual distribution of ClC-1 channels between the surface and Transverse Tubular System (TTS) membranes has not been assessed in intact muscle fibers. To investigate this issue, we voltage-clamped enzymatically dissociated short fibers using a 2-microelectrode configuration and simultaneously recorded chloride currents (ICl), and di-8-ANEPPS fluorescence signals to assess membrane potential changes in the TTS. Experiments were conducted in conditions that blocked all, but the chloride conductance. Fibers were equilibrated with 40 or 70 mM intracellular chloride in order to enhance the magnitude of inward ICl, and the specific ClC-1 blocker 9-ACA was used to eliminate these currents whenever necessary. Voltage-dependent di-8-ANEPPS signals and ICl acquired before (control) and after the addition of 9-ACA were comparatively assessed. Early after the onset of stimulus pulses, di-8-ANEPPS signals under control conditions were smaller than those recorded in the presence of 9-ACA. We defined as attenuation the normalized time-dependent difference between these signals. Attenuation was discovered to be ICl-dependent since its magnitude varied in close correlation with the amplitude and time course of ICl. While the properties of ICl, and those of the attenuation seen in optical records, could be simultaneously predicted by model simulations when the chloride permeability (PCl) at the surface and TTS membranes were approximately equal, the model failed to explain the optical data if PCl was precluded from the TTS membranes. Since the ratio between the areas of TTS membranes and the sarcolemma is large in mammalian muscle fibers, our results demonstrate that a significant fraction of the experimentally recorded ICl arises from the TTS. Supported by NIH grants AR047664, AR041802, and AR054816.
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differential recording of voltage changes at the surface and Transverse Tubular System membranes of mammalian skeletal muscle fibers using di 8 anepps and global and tirfm
Biophysical Journal, 2011Co-Authors: Joana Capote, Marino Difranco, Julio L VergaraAbstract:Aiming to investigate the distribution of ClC-1 and KIR channels at the sarcolemma and Transverse Tubular System (TTS) membranes of mammalian skeletal muscle fibers, we used global and total internal reflection fluorescence microscopy (TIRFM) to monitor voltage changes in these compartments, respectively. Enzymatically-dissociated fibers from murine FDB and interosseus muscles were stained with the potentiometric dye di-8- ANEPPS, and voltage-clamped with a two-microelectrode System. Ion substitutions were used to isolate and characterize the specific ClC-1 (ICl) and KIR (IKIR) currents: 70 mM internal [Cl-] and 120 mM external [K+], respectively. Also, 9-ACA and TEA were used to, respectively, block these currents. Global di-8-ANEPPS signals report, early after the onset of large hyperpolarizing pulses, ICl-dependent attenuations with respect to those recorded in the presence of 9-ACA. Peak attenuation levels of ∼35% were observed for ICl of ∼900 µA/cm2. Large attenuations were similarly observed in global signals recorded the presence of large IKIR's with respect to those in TEA. In contrast, TIRFM di-8-ANEPPS signals demonstrate only minor current-dependent attenuations (<10%) under conditions in which global signals evidenced much larger attenuations. Overall, our results demonstrate that voltage changes at the TTS membranes display prominent current-dependent attenuations while the sarcolemma is largely under voltage-clamp control. A radial cable model of the TTS, including equations for each current pathway and luminal accumulation/depletion of ions, was used to quantitatively predict the ionic currents and to assess their effects on average TTS voltage changes. Comparative analysis of global optical data with model predictions of voltage changes in the TTS suggests that both ClC-1 and KIR channels are equally distributed in both membrane compartments. Supported by NIH grants AR047664, AR041802, and AR054816.
John H B Bridge - One of the best experts on this subject based on the ideXlab platform.
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cardiac resynchronization therapy reduces subcellular heterogeneity of ryanodine receptors t tubules and ca2 sparks produced by dyssynchronous heart failure
Circulation-heart Failure, 2015Co-Authors: Hui Li, John H B Bridge, Thomas Seidel, Gordon F Tomaselli, Justin Lichter, Frank B SachseAbstract:Background—Cardiac resynchronization therapy (CRT) is a major advance for treatment of patients with dyssynchronous heart failure (DHF). However, our understanding of DHF-associated remodeling of subcellular structure and function and their restoration after CRT remains incomplete. Methods and Results—We investigated subcellular heterogeneity of remodeling of structures and proteins associated with excitation–contraction coupling in cardiomyocytes in DHF and after CRT. Three-dimensional confocal microscopy revealed subcellular heterogeneity of ryanodine receptor (RyR) density and the Transverse Tubular System (t-System) in a canine model of DHF. RyR density at the ends of lateral left ventricular cardiomyocytes was higher than that in cell centers, whereas the t-System was depleted at cell ends. In anterior left ventricular cardiomyocytes, however, we found a similar degree of heterogeneous RyR remodeling, despite preserved t-System. Synchronous heart failure was associated with marginal heterogeneity of ...
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mechanical modulation of the Transverse Tubular System of ventricular cardiomyocytes
Progress in Biophysics & Molecular Biology, 2012Co-Authors: Thomas G Mcnary, John H B Bridge, Kenneth W Spitzer, Hilary Holloway, Peter Kohl, Frank B SachseAbstract:In most mammalian cardiomyocytes, the Transverse Tubular System (t-System) is a major site for electrical signaling and excitation-contraction coupling. The t-System consists of membrane invaginations, which are decorated with various proteins involved in excitation-contraction coupling and mechano-electric feedback. Remodeling of the t-System has been reported for cells in culture and various types of heart disease. In this paper, we provide insights into effects of mechanical strain on the t-System in rabbit left ventricular myocytes. Based on fluorescent labeling, three-dimensional scanning confocal microscopy, and digital image analysis, we studied living and fixed isolated cells in different strain conditions. We extracted geometric features of Transverse tubules (t-tubules) and characterized their arrangement with respect to the Z-disk. In addition, we studied the t-System in cells from hearts fixed either at zero left ventricular pressure (slack), at 30 mmHg (volume overload), or during lithium-induced contracture, using transmission electron microscopy. Two-dimensional image analysis was used to extract features of t-tubule cross-sections. Our analyses of confocal microscopic images showed that contracture at the cellular level causes deformation of the t-System, increasing the length and volume of t-tubules, and altering their cross-sectional shape. TEM data reconfirmed the presence of mechanically induced changes in t-Tubular cross sections. In summary, our studies suggest that passive longitudinal stretching and active contraction of ventricular cardiomyocytes affect the geometry of t-tubules. This confirms that mechanical changes at cellular levels could promote alterations in partial volumes that would support a convection-assisted mode of exchange between the t-System content and extracellular space.
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subcellular structures and function of myocytes impaired during heart failure are restored by cardiac resynchronization therapy
Circulation Research, 2012Co-Authors: Frank B Sachse, Eleonora Saviogalimberti, Natalia S Torres, Takeshi Aiba, David A Kass, Gordon F Tomaselli, John H B BridgeAbstract:Rationale:Cardiac resynchronization therapy (CRT) is an established treatment for patients with chronic heart failure. However, CRT-associated structural and functional remodeling at cellular and subcellular levels is only partly understood. Objective:To investigate the effects of CRT on subcellular structures and protein distributions associated with excitation-contraction coupling of ventricular cardiomyocytes. Methods and Results:Our studies revealed remodeling of the Transverse Tubular System (t-System) and the spatial association of ryanodine receptor (RyR) clusters in a canine model of dyssynchronous heart failure (DHF). We did not find this remodeling in a synchronous heart failure model based on atrial tachypacing. Remodeling in DHF ranged from minor alterations in anterior left ventricular myocytes to nearly complete loss of the t-System and dissociation of RyRs from sarcolemmal structures in lateral cells. After CRT, we found a remarkable and almost complete reverse remodeling of these structure...
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strain transfer in ventricular cardiomyocytes to their Transverse Tubular System revealed by scanning confocal microscopy
Biophysical Journal, 2011Co-Authors: Thomas G Mcnary, John H B Bridge, Frank B SachseAbstract:The Transverse Tubular System (t-System) is a major site for signaling in mammalian ventricular cardiomyocytes including electrical signaling and excitation-contraction coupling. It consists of membrane invaginations, which are decorated with various proteins including mechanosensitive ion channels. Here, we investigated mechanical modulation of the t-System. By applying fluorescent markers, three-dimensional scanning confocal microscopy, and methods of digital image analysis, we studied isolated ventricular cardiomyocytes under different strains. We demonstrate that strain at the cellular level is transmitted to the t-System, reducing the length and volume of tubules and altering their cross-sectional shape. Our data suggest that a cellular strain of as little as 5% affects the shape of Transverse tubules, which has important implications for the function of mechanosensitive ion channels found in them. Furthermore, our study supports a prior hypothesis that strain can cause fluid exchange between the t-System and extracellular space.
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novel features of the rabbit Transverse Tubular System revealed by quantitative analysis of three dimensional reconstructions from confocal images
Biophysical Journal, 2008Co-Authors: Eleonora Saviogalimberti, John H B Bridge, Joy S Frank, Masashi Inoue, Joshua I Goldhaber, Mark B Cannell, Frank B SachseAbstract:With scanning confocal microscopy we obtained three-dimensional (3D) reconstructions of the Transverse Tubular System (t-System) of rabbit ventricular cells. We accomplished this by labeling the t-System with dextran linked to fluorescein or, alternatively, wheat-germ agglutinin conjugated to an Alexa fluor dye. Image processing and visualization techniques allowed us to reconstruct the t-System in three dimensions. In a myocyte lying flat on a coverslip, t-tubules typically progressed from its upper and lower surfaces. 3D reconstructions of the t-tubules also suggested that some of them progressed from the sides of the cell. The analysis of single t-tubules revealed novel morphological features. The average diameter of single t-tubules from six cells was estimated to 448 +/- 172 nm (mean +/- SD, number of t-tubules 348, number of cross sections 5323). From reconstructions we were able to identify constrictions occurring every 1.87 +/- 1.09 microm along the principal axis of the tubule. The cross-sectional area of these constrictions was reduced to an average of 57.7 +/- 27.5% (number of constrictions 170) of the adjacent local maximal areas. Principal component analysis revealed flattening of t-Tubular cross sections, confirming findings that we obtained from electron micrographs. Dextran- and wheat-germ agglutinin-associated signals were correlated in the t-System and are therefore equally good markers. The 3D structure of the t-System in rabbit ventricular myocytes seems to be less complex than that found in rat. Moreover, we found that t-tubules in rabbit have approximately twice the diameter of those in rat. We speculate that the constrictions (or regions between them) are sites of dyadic clefts and therefore can provide geometric markers for colocalizing dyadic proteins. In consideration of the resolution of the imaging System, we suggest that our methods permit us to obtain spatially resolved 3D reconstructions of the t-System in rabbit cells. We also propose that our methods allow us to characterize pathological defects of the t-System, e.g., its remodeling as a result of heart failure.
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Glucocorticoids preserve the t-Tubular System in ventricular cardiomyocytes by upregulation of autophagic flux
Basic Research in Cardiology, 2019Co-Authors: Thomas Seidel, Dominik J. Fiegle, Tim J. Baur, Anne Ritzer, Sandra Nay, Christian Heim, Michael Weyand, Hendrik Milting, Robert H. Oakley, John A. CidlowskiAbstract:A major contributor to contractile dysfunction in heart failure is remodelling and loss of the cardiomyocyte Transverse Tubular System (t-System), but underlying mechanisms and signalling pathways remain elusive. It has been shown that dexamethasone promotes t-tubule development in stem cell-derived cardiomyocytes and that cardiomyocyte-specific glucocorticoid receptor (GR) knockout (GRKO) leads to heart failure. Here, we studied if the t-System is altered in GRKO hearts and if GR signalling is required for t-System preservation in adult cardiomyocytes. Confocal and 3D STED microscopy of myocardium from cardiomyocyte-specific GRKO mice revealed decreased t-System density and increased distances between ryanodine receptors (RyR) and L-type Ca^2+ channels (LTCC). Because t-System remodelling and heart failure are intertwined, we investigated the underlying mechanisms in vitro. Ventricular cardiomyocytes from failing human and healthy adult rat hearts cultured in the absence of glucocorticoids (CTRL) showed distinctively lower t-System density than cells treated with dexamethasone (EC_50 1.1 nM) or corticosterone. The GR antagonist mifepristone abrogated the effect of dexamethasone. Dexamethasone improved RyR–LTCC coupling and synchrony of intracellular Ca^2+ release, but did not alter expression levels of t-System-associated proteins junctophilin-2 (JPH2), bridging integrator-1 (BIN1) or caveolin-3 (CAV3). Rather, dexamethasone upregulated LC3B and increased autophagic flux. The broad-spectrum protein kinase inhibitor staurosporine prevented dexamethasone-induced upregulation of autophagy and t-System preservation, and autophagy inhibitors bafilomycin A and chloroquine accelerated t-System loss. Conversely, induction of autophagy by rapamycin or amino acid starvation preserved the t-System. These findings suggest that GR signalling and autophagy are critically involved in t-System preservation and remodelling in the heart.
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remodeling of the Transverse Tubular System after myocardial infarction in rabbit correlates with local fibrosis a potential role of biomechanics
Progress in Biophysics & Molecular Biology, 2017Co-Authors: Aparna C Sankarankutty, Thomas Seidel, Frank B SachseAbstract:Abstract The Transverse Tubular System (t-System) of ventricular cardiomyocytes is essential for efficient excitation-contraction coupling. In cardiac diseases, such as heart failure, remodeling of the t-System contributes to reduced cardiac contractility. However, mechanisms of t-System remodeling are incompletely understood. Prior studies suggested an association with altered cardiac biomechanics and gene expression in disease. Since fibrosis may alter tissue biomechanics, we investigated the local microscopic association of t-System remodeling with fibrosis in a rabbit model of myocardial infarction (MI). Biopsies were taken from the MI border zone of 6 infarcted hearts and from 6 control hearts. Using confocal microscopy and automated image analysis, we quantified t-System integrity (ITT) and the local fraction of extracellular matrix (fECM). In control, fECM was 18 ± 0.3%. ITT was high and homogeneous (0.07 ± 0.006), and did not correlate with fECM (R2 = 0.05 ± 0.02). The MI border zone exhibited increased fECM within 3 mm from the infarct scar (30 ± 3.5%, p
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sheet like remodeling of the Transverse Tubular System in human heart failure impairs excitation contraction coupling and functional recovery by mechanical unloading
Circulation, 2017Co-Authors: Thomas Seidel, Sutip Navankasattusas, Azmi A Ahmad, Nikolaos A Diakos, Martin Tristanifirouzi, Michael J Bonios, Iosif Taleb, Craig H Selzman, Stavros G Drakos, Frank B SachseAbstract:Background: Cardiac recovery in response to mechanical unloading by left ventricular assist devices (LVADs) has been demonstrated in subgroups of patients with chronic heart failure (HF). Hallmarks of HF are depletion and disorganization of the Transverse Tubular System (t-System) in cardiomyocytes. Here, we investigated remodeling of the t-System in human end-stage HF and its role in cardiac recovery. Methods: Left ventricular biopsies were obtained from 5 donors and 26 patients with chronic HF undergoing implantation of LVADs. Three-dimensional confocal microscopy and computational image analysis were applied to assess t-System structure, density, and distance of ryanodine receptor clusters to the sarcolemma, including the t-System. Recovery of cardiac function in response to mechanical unloading was assessed by echocardiography during turndown of the LVAD. Results: The majority of HF myocytes showed remarkable t-System remodeling, particularly sheet-like invaginations of the sarcolemma. Circularity of t-System components was decreased in HF versus controls (0.37±0.01 versus 0.46±0.02; P 1 µm did not improve after mechanical unloading. In addition, calcium transients were recorded in field-stimulated isolated human cardiomyocytes and analyzed with respect to local t-System density. Calcium release in HF myocytes was restricted to regions proximal to the sarcolemma. Local calcium upstroke was delayed (23.9±4.9 versus 10.3±1.7 milliseconds; P <0.05) and more asynchronous (18.1±1.5 versus 8.9±2.2 milliseconds; P <0.01) in HF cells with low t-System density versus cells with high t-System density. Conclusions: The t-System in end-stage human HF presents a characteristic novel phenotype consisting of sheet-like invaginations of the sarcolemma. Our results suggest that the remodeled t-System impairs excitation-contraction coupling and functional recovery during chronic LVAD unloading. An intact t-System at the time of LVAD implantation may constitute a precondition and predictor for functional cardiac recovery after mechanical unloading. # Clinical Perspective {#article-title-51}
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cardiac resynchronization therapy reduces subcellular heterogeneity of ryanodine receptors t tubules and ca2 sparks produced by dyssynchronous heart failure
Circulation-heart Failure, 2015Co-Authors: Hui Li, John H B Bridge, Thomas Seidel, Gordon F Tomaselli, Justin Lichter, Frank B SachseAbstract:Background—Cardiac resynchronization therapy (CRT) is a major advance for treatment of patients with dyssynchronous heart failure (DHF). However, our understanding of DHF-associated remodeling of subcellular structure and function and their restoration after CRT remains incomplete. Methods and Results—We investigated subcellular heterogeneity of remodeling of structures and proteins associated with excitation–contraction coupling in cardiomyocytes in DHF and after CRT. Three-dimensional confocal microscopy revealed subcellular heterogeneity of ryanodine receptor (RyR) density and the Transverse Tubular System (t-System) in a canine model of DHF. RyR density at the ends of lateral left ventricular cardiomyocytes was higher than that in cell centers, whereas the t-System was depleted at cell ends. In anterior left ventricular cardiomyocytes, however, we found a similar degree of heterogeneous RyR remodeling, despite preserved t-System. Synchronous heart failure was associated with marginal heterogeneity of ...
