Cytosolic Buffering Research Articles

Mutation in pore-helix modulates interplay between filter gate and Ba2+ block in a Kcv channel pore.

The selectivity filter of K+ channels catalyzes a rapid and highly selective transport of K+ while serving as a gate. To understand the control of this filter gate, we use the pore-only K+ channel KcvNTS in which gating is exclusively determined by the activity of the filter gate. It has been previously shown that a mutation at the C-terminus of the pore-helix (S42T) increases K+ permeability and introduces distinct voltage-dependent and K+-sensitive channel closures at depolarizing voltages. Here, we report that the latter are not generated by intrinsic conformational changes of the filter gate but by a voltage-dependent block caused by nanomolar trace contaminations of Ba2+ in the KCl solution. Channel closures can be alleviated by extreme positive voltages and they can be completely abolished by the high-affinity Ba2+ chelator 18C6TA. By contrast, the same channel closures can be augmented by adding Ba2+ at submicromolar concentrations to the cytosolic buffer. These data suggest that a conservative exchange of Ser for Thr in a crucial position of the filter gate increases the affinity of the filter for Ba2+ by >200-fold at positive voltages. While Ba2+ ions apparently remain only for a short time in the filter-binding sites of the WT channel before passing the pore, they remain much longer in the mutant channel. Our findings suggest that the dwell times of permeating and blocking ions in the filter-binding sites are tightly controlled by interactions between the pore-helix and the selectivity filter.

The Iron Chaperone Poly C Binding Protein 1 Regulates Iron Efflux through Intestinal Ferroportin in Mice

Iron is an essential nutrient required by all cells but used primarily for red blood cell production. Because humans have no effective mechanism for ridding the body of excess iron, the absorption of dietary iron must be precisely regulated. The critical site of regulation is the transfer of iron from the absorptive enterocyte to the portal circulation via the sole iron efflux transporter, ferroportin (FPN). Here we report that poly(rC)-binding protein 1 (PCBP1), the major cytosolic iron chaperone, is necessary for the regulation of iron flux through FPN in the intestine of mice. PCBP1 binds iron-glutathione complexes in the cytosol of cells and delivers the iron to ferritin, the iron storage protein, to iron centers in enzymes, and to iron-sulfur cluster chaperones. PCBP1 also limits the chemical reactivity of iron, thereby limiting iron-catalyzed oxidative damage in cells. To explore the role of PCBP1 in dietary iron absorption, we developed mice with an inducible, enterocyte-specific deletion of PCBP1. Mice lacking PCBP1 in the intestinal epithelium exhibit low levels of enterocyte iron, poor retention of dietary iron in enterocyte ferritin, and excess efflux of iron through FPN. Kinetic studies of iron transport using a stable isotope ( 57Fe) coupled with a mini-hepcidin that blocks FPN activity demonstrated that PCBP1 deletion had no effect of intestinal iron uptake but caused high rates of iron efflux through FPN. Surprisingly, excess iron efflux occurred despite lower levels of FPN protein in enterocytes and upregulation of the iron regulatory hormone hepcidin. PCBP1 deletion and the resulting unregulated dietary iron absorption led to poor growth, severe anemia on a low iron diet and liver oxidative stress with iron loading on a high iron diet. To investigate the roles of PCBP1 more precisely in enterocytes, we developed 3-D organoid cultures from intestinal epithelium which functionally expressed the major iron trafficking proteins: divalent metal transporter 1, PCBP1, ferritin, transferrin receptor 1 and FPN. Ex vivo culture of wild-type and PCBP1-depleted enteroids demonstrated normal patterns of FPN expression and hepcidin-mediated turnover. However, fluorescence measurements of kinetically labile iron pools in enteroids competent or blocked for FPN iron efflux indicated that PCBP1 dramatically altered efflux activity. PCBP1 functioned as a cytosolic buffer for iron, binding and retaining iron and thereby keeping “free” iron at very low levels. This low concentration of free iron limited its availability for FPN-mediated efflux and allowed hepcidin-mediated changes in FPN levels to regulate the amount of iron exported from the enterocyte. Thus, by binding enterocyte iron, PCBP1 reduces the concentration of unchaperoned, “free” iron to a low level. This low level of cytosolic free iron is necessary for hepcidin-mediated regulation of FPN activity.

A stochastic mathematical model for coupling the cytosolic and sarcoplasmic calcium movements in diseased cardiac myocytes

Several computational studies have been undertaken to explore the Ca2+-induced Ca2+ release (CICR) events in cardiac myocytes and along with experimental studies it has given us invaluable insight into the mechanism of CICR from spark/blink initiation to termination and regulation, and their interplay under normal and pathological conditions. The computational modelling of this mechanism has mainly been investigated using coupled differential equations (DEs). However, there is a lack of computational investigation into (1) how the different formulation of coupled DEs capture the Ca2+ movement in the cytosol and sarcoplasmic reticulum (SR), (2) the buffer and dye inclusion in both compartments, and (3) the effect of buffer and dye properties on the calcium behaviour. This work is set out to explore (1) the effect of different coupled formulation of DEs on spark/blink occurrence, (2) the inclusion of improved sarcoplasmic buffering properties, and (3) the effects of cytosolic and sarcoplasmic dye and buffer properties on Ca2+ movement. The simulation results show large discrepancies between different formulations of the governing equations. Additionally, extension of the model to include sarcoplasmic buffering properties show normalised fluorescent dye profiles to be in good agreement with experimental and amongst its one- and two-dimensional representations.

A dual role for glutathione transferase U7 in plant growth and protection from methyl viologen-induced oxidative stress.

Plant glutathione S-transferases (GSTs) are glutathione-dependent enzymes with versatile functions, mainly related to detoxification of electrophilic xenobiotics and peroxides. The Arabidopsis (Arabidopsis thaliana) genome codes for 53 GSTs, divided into seven subclasses; however, understanding of their precise functions is limited. A recent study showed that class II TGA transcription factors TGA2, TGA5, and TGA6 are essential for tolerance of UV-B-induced oxidative stress and that this tolerance is associated with an antioxidative function of cytosolic tau-GSTs (GSTUs). Specifically, TGA2 controls the expression of several GSTUs under UV-B light, and constitutive expression of GSTU7 in the tga256 triple mutant is sufficient to revert the UV-B-susceptible phenotype of tga256. To further study the function of GSTU7, we characterized its role in mitigation of oxidative damage caused by the herbicide methyl viologen (MV). Under non-stress conditions, gstu7 null mutants were smaller than wild-type (WT) plants and delayed in the onset of the MV-induced antioxidative response, which led to accumulation of hydrogen peroxide and diminished seedling survival. Complementation of gstu7 by constitutive expression of GSTU7 rescued these phenotypes. Furthermore, live monitoring of the glutathione redox potential in intact cells with the fluorescent probe Grx1-roGFP2 revealed that GSTU7 overexpression completely abolished the MV-induced oxidation of the cytosolic glutathione buffer compared with WT plants. GSTU7 acted as a glutathione peroxidase able to complement the lack of peroxidase-type GSTs in yeast. Together, these findings show that GSTU7 is crucial in the antioxidative response by limiting oxidative damage and thus contributes to oxidative stress resistance in the cell.

Hypertrophic cardiomyopathy-linked mutation in troponin T causes myofibrillar disarray and pro-arrhythmic action potential changes in human iPSC cardiomyocytes

BackgroundMutations in cardiac troponin T (TnT) are linked to increased risk of ventricular arrhythmia and sudden death despite causing little to no cardiac hypertrophy. Studies in mice suggest that the hypertrophic cardiomyopathy (HCM)-associated TnT-I79N mutation increases myofilament Ca sensitivity and is arrhythmogenic, but whether findings from mice translate to human cardiomyocyte electrophysiology is not known. ObjectivesTo study the effects of the TnT-I79N mutation in human cardiomyocytes. MethodsUsing CRISPR/Cas9, the TnT-I79N mutation was introduced into human induced pluripotent stem cells (hiPSCs). We then used the matrigel mattress method to generate single rod-shaped cardiomyocytes (CMs) and studied contractility, Ca handling and electrophysiology. ResultsCompared to isogenic control hiPSC-CMs, TnT-I79N hiPSC-CMs exhibited sarcomere disorganization, increased systolic function and impaired relaxation. The Ca-dependence of contractility was leftward shifted in mutation containing cardiomyocytes, demonstrating increased myofilament Ca sensitivity. In voltage-clamped hiPSC-CMs, TnT-I79N reduced intracellular Ca transients by enhancing cytosolic Ca buffering. These changes in Ca handling resulted in beat-to-beat instability and triangulation of the cardiac action potential, which are predictors of arrhythmia risk. The myofilament Ca sensitizer EMD57033 produced similar action potential triangulation in control hiPSC-CMs. ConclusionsThe TnT-I79N hiPSC-CM model not only reproduces key cellular features of TnT-linked HCM such as myofilament disarray, hypercontractility and diastolic dysfunction, but also suggests that this TnT mutation causes pro-arrhythmic changes of the human ventricular action potential.

Doublet stimulation increases Ca2+ binding to troponin C to ensure rapid force development in skeletal muscle.

Fast-twitch skeletal muscle fibers are often exposed to motor neuron double discharges (≥200 Hz), which markedly increase both the rate of contraction and the magnitude of the resulting force responses. However, the mechanism responsible for these effects is poorly understood, likely because of technical limitations in previous studies. In this study, we measured cytosolic Ca2+ during doublet activation using the low-affinity indicator Mag-Fluo-4 at high temporal resolution and modeled the effects of doublet stimulation on sarcoplasmic reticulum (SR) Ca2+ release, binding of Ca2+ to cytosolic buffers, and force enhancement in fast-twitch fibers. Single isolated fibers respond to doublet pulses with two clear Ca2+ spikes, at doublet frequencies up to 1 KHz. A 200-Hz doublet at the start of a tetanic stimulation train (70 Hz) decreases the drop in free Ca2+ between the first three Ca2+ spikes of the transient, maintaining a higher overall free Ca2+ level during first 20-30 ms of the response. Doublet stimulation also increased the rate of force development in isolated fast-twitch muscles. We also modeled SR Ca2+ release rates during doublet stimulation and showed that Ca2+-dependent inactivation of ryanodine receptor activity is rapid, occurring ≤1ms after initial release. Furthermore, we modeled Ca2+ binding to the main intracellular Ca2+ buffers of troponin C (TnC), parvalbumin, and the SR Ca2+ pump during Ca2+ release and found that the main effect of the second response in the doublet is to more rapidly increase the occupation of the second Ca2+-binding site on TnC (TnC2), resulting in earlier activation of force. We conclude that doublet stimulation maintains high cytosolic Ca2+ levels for longer in the early phase of the Ca2+ response, resulting in faster saturation of TnC2 with Ca2+, faster initiation of cross-bridge cycling, and more rapid force development.

Generation and Characterization of a Human iPSC Cardiomyocyte Model of Troponin T I79N Linked Hypertrophic Cardiomyopathy

Objective: The aim of this study was to examine the functional consequences of a troponin T mutation (I79N) associated with hypertrophic cardiomyopathy (HCM) in cardiomyocytes derived from human induced pluripotent stem cells (hiPSC). Method and Results: We generated a TnT-I79N hiPSC HCM model by inserting the 236T>A mutation into the TNNT2 gene of one of the alleles of hiPSC derived from a healthy donor using CRISPR/Cas9. There was no off-target cleavage produced by CRISPR/Cas9. Nano-liquid chromatography mass spectrometry showed that 43% of TnT protein was mutant in cardiomyocytes (CM) derived from TnT-I79N hiPSCs. In 2D monolayers, the impedance amplitude (a measure of contractility) was increased and the Ca-contractility relationship leftward shifted in TnT-I79N hiPSC-CMs compared to isogenic control hiPSC-CM. Results were confirmed using single hiPSC-CM cultured on a matrigel matress: The TnT-I79N mutation altered cytosolic Ca buffering properties by lowering the apparent dissociation constant Kd (0.40±0.04 μM in TnT-I79N, n=12 vs. 0.72±0.10 μM in control, n=16, P<0.05) without changing the maximal binding capacity Bmax, indicating that TnT-I79N mutation enhanced the Ca-binding affinity of myofilaments. Likely as a result of the increased cytosolic Ca-binding affinity, I79N hiPSC-CM paced at 0.2 Hz exhibited significantly reduced intracellular Ca-transients (ΔF/Fratio 0.85±0.03 in I79N vs. 1.01±0.06 in control, P<0.05) and reduced caffeine transient (ΔF/Fratio 0.92±0.06 in I79N vs. 1.10±0.06 in control, P<0.05), whereas Ca-decay kinetics where unchanged. Despite the reduced Ca-transient amplitude, TnT-I79N mutation enhanced contractility but delayed the relaxation in single hiPSC-CMs, indicating an increased sensitivity to Ca. Conclusion: The I79N hiPSC-CM model reproduces key findings from I79N transgenic mice: increased myofilament Ca-sensitivity, enhances contractility, impaired relaxation and enhanced cytosolic Ca-binding affinity. Human iPSC-CM could be a promising tool to model HCM in vitro.

Multiple cytosolic calcium buffers in posterior pituitary nerve terminals.

Cytosolic Ca(2+) buffers bind to a large fraction of Ca(2+) as it enters a cell, shaping Ca(2+) signals both spatially and temporally. In this way, cytosolic Ca(2+) buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca(2+) entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca(2+) buffers in controlling pituitary Ca(2+) signaling is poorly understood. Here, cytosolic Ca(2+) buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca(2+) indicator fluo-8 revealed stepwise increases in free Ca(2+) after a series of brief depolarizing pulses in rapid succession. These Ca(2+) increments grew larger as free Ca(2+) rose to saturate the cytosolic buffers and reduce the availability of Ca(2+) binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5-4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca(2+) buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca(2+) signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones.

Na+-H+ Antiporter in the Somata of Dorsal Root Ganglion Neurons

Introduction: Neurons in ganglia of medullary dorsal root (DRG) convey sensitive information from the body to the central nervous system. Neuronal soma displays voltage-gated channels, enabling it to fire action potentials. Here we present evidences for the expression of Na+-H+ antiporters, as housekeepers maintaining the intracellular pH.Methods: Ganglia from newborn Wistar rats were digested with trypsin. The cells were maintained in DMEM+fetal bovine serum in CO2 5% atmosphere, 37°C. Intracellular pH was estimated with BCECF fluorescent indicator. The cells were perfused continuously with solutions, heated to 37°C. Ammonium pulses (NH4Cl, 20mM) or exposure to acidic extracellular solution (pH 6.5) leads to acidic load. On the recovery, [H+] decline could be well fitted by one exponential, whose rate constant is associated with the kinetics of the Na+-H+ antiporter and cytosolic buffering power. Na+-H+ antiporter was identified by the inhibitors amiloride (1 mM) or EIPA (5 µM) and by the effects of low Na+ extracellular solution (50 mM).Results and Conclusion: After acidic load, H+ is pumped out at a rate of 0.0138±0.0061s−1(n=63) (ammonium pulse) and 0.0146±0.0063s−1(n=43)(acidic extracellular pH), recovering intracellular pH to the previous levels. Amiloride completely blocked pH recovery in 17 cells and reduced the recovery rate in 10 (0.0086±0.0059s−1). EIPA reduced rate to 0.0066±0.0045s−1(n=23) in all cells. Further evidence for the Na+-H+ antiporter is reduced rate in low-Na+ (0.0055±0.0016s−1, n=4).DRGneurons express Na+-H+ antiporter as housekeepers regulating the intracellular pH. Evidences for the Na+-H+ antiporter are provided by the effects, on the rates, of low-Na+ and classical inhibitors. Amiloride affects differentially cell populations, probably due to different isoforms.Financial support: Capes, CNPq and FAPESP.

Physiological roles of bacillithiol in intracellular metal processing.

Glutathione (GSH) is an abundantly produced low-molecular-weight (LMW) thiol in many organisms. However, a number of Gram-positive bacteria do not produce GSH, but instead produce bacillithiol (BSH) as one of the major LMW thiols. Similar to GSH, studies have found that BSH has various roles in the cell, including protection against hydrogen peroxide, hypochlorite and disulfide stress. BSH also participates in the detoxification of thiol-reactive antibiotics and the electrophilic metabolite methylglyoxal. Recently, a number of studies have highlighted additional roles for BSH in the processing of intracellular metals. Herein, we examine the potential functions of BSH in the biogenesis of Fe-S clusters, cytosolic metal buffering and the prevention of metal intoxication.

Comparable calcium handling of human iPSC-derived cardiomyocytes generated by multiple laboratories

Cardiomyocytes (CMs) derived from human induced pluripotent stem cells (hiPSCs) are being increasingly used to model human heart diseases. hiPSC-CMs generated by earlier aggregation-based methods (i.e., embryoid body) often lack functional sarcoplasmic reticulum (SR) Ca stores characteristic of mature mammalian CMs. Newer monolayer-based cardiac differentiation methods (i.e., Matrigel sandwich or small molecule-based differentiation) produce hiPSC-CMs of high purity and yield, but their Ca handling has not been comprehensively investigated. Here, we studied Ca handling and cytosolic Ca buffering properties of hiPSC-CMs generated independently from multiple hiPSC lines at Stanford University, Vanderbilt University and University of Wisconsin-Madison. hiPSC-CMs were cryopreserved at each university. Frozen aliquots were shipped, recovered from cryopreservation, plated at low density and compared 3–5days after plating with acutely-isolated adult rabbit and mouse ventricular CMs. Although hiPSC-CM cell volume was significantly smaller, cell capacitance to cell volume ratio and cytoplasmic Ca buffering were not different from rabbit-CMs. hiPSC-CMs from all three laboratories exhibited robust L-type Ca currents, twitch Ca transients and caffeine-releasable SR Ca stores comparable to adult CMs. Ca transport by sarcoendoplasmic reticulum Ca ATPase (SERCA) and Na/Ca exchanger (NCX) was similar in all hiPSC-CM lines, but slower compared to rabbit-CMs. However, the relative contribution of SERCA and NCX to Ca transport of hiPSC-CMs was comparable to rabbit-CMs. Ca handling maturity of hiPSC-CMs increased from 15 to 21days post-induction. We conclude that hiPSC-CMs generated independently from multiple iPSC lines using monolayer-based methods can be reproducibly recovered from cryopreservation and exhibit comparable and functional SR Ca handling.

CAPILLARY‐TISSUE EXCHANGE OF SUBSTRATES AND NUCLEOSIDES IN CARDIAC ENERGETICS MODELING

Modeling cardiac energetics involves all of the elements of supply or demand. It is complicated: the exchange processes are distributed in space, heterogeneous, requiring parallel units interacting with each other. Multicomponent, multiscale models for the analysis of data are complicated. Using modular units aids building and maintenance. A convenient module is one in which the number of connecting variables is minimized, and the internal complexity is isolated. The differential equation modeling platform JSim is designed for model‐based analysis of data using behavioral and sensitivity analysis, automated data fitting, and MonteCarlo for uncertainty quantification. For O2, CO2, substrates, and metabolites we constructed first a multisolute single capillary‐tissue unit with RBC, plasma, endothelial cells, interstitial space, and cardiomyocytes, ignoring other cell types. The substrates were O2, glucose, and palmitate; the processes were uptake, binding and buffering, and metabolism. Oxygen, binding to hemoglobin, has capillary gradients influenced by CO2, pH, ºC and 2,3‐DPG, requiring partial differential equations. Myoglobin facilitates cytosolic diffusion and buffering. For glucose, there is glycolysis, the pentose shunt, and buffering as glycogen. For palmitate, storage buffering as acyl‐CoA and di‐ and tri‐glyceride feed into beta‐oxidation and the Krebs cycle. Oxidative phosphorylation produces ATP used in muscle filament activation, ion pumps, and cell maintenance. Version 1 uses first order reactions. Mechanistic kinetics with thermodynamic constraints come with substituting high fidelity modules. (NIH grants R01‐EB8407; P50‐GM 094503)

Calcium in atrial fibrillation - pulling the trigger or not?

Atrial fibrillation (AF) is the most common sustained arrhythmia disease. Current drug- and surgical-based therapies are ineffective in about 40% to 50% of AF patients; therefore, there is a great need to better understand the underlying mechanisms of this disease and identify potential therapeutic targets. In this issue of the JCI, Greiser and coworkers discovered that atrial remodeling in response to sustained tachycardia silences Ca2+ signaling in isolated rabbit and human atrial myocytes. This Ca2+ release silencing was attributable to a failure of subcellular propagated Ca2+ release due to an increased cytosolic buffering strength. The results from this study challenge the current paradigm that Ca2+ release instability underlies AF. Instead, Ca2+ silencing could be protective against the massive cellular Ca2+ loading that occurs during chronic AF.

Cytosolic Ca Buffering of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Adult Rabbit Ventricular Cardiomyocytes

A major concern about using cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) for human disease modeling is their immature phenotype which may result in substantial differences in their physiology when compared to native cardiomyocytes. Here we compare intracellular Ca buffering of hiPSC-CM derived from healthy humans to that of acutely-isolated adult rabbit ventricular CM. hiPSC-CM (21 days post cardiac induction) were plated at low density on matrigel-coated dishes and studied 3-5 days after plating. While hiPSC were significantly smaller than rabbit CM (cell capacity 19±2 pF vs 101±12 pF, p<0.01) and had disorganized contractile filaments on electron microscopy, hiPSC-CM nevertheless exhibited robust troponin C expression, the major cytosolic Ca binding site. To measure cytosolic Ca buffering (Trafford method), caffeine (10 mM) was rapidly applied to release Ca from the SR, which then extruded via the NaCa exchanger (NCX), generating an inward NCX current. Integration of NCX current yielded the total amount of Ca released from the SR. Then Δ[Ca]total was plotted as a function of Δ[Ca]free and maximal cytosolic buffering capacity (Bmax) and Kd calculated. Cytosolic Ca buffering parameters were not significantly different between hiPSC-CM (Bmax=100±14μmol/Lcytosol, Kd=0.61±0.09 μM, n=25) and rabbit-CM (Bmax=117±15 μmol/Lcytosol, Kd=0.61±0.11 μM, n=9). EMD57033 (3 μM), which binds to troponin C and increases cytosolic Ca binding in murine CM, also increased cytosolic Ca binding affinity in hiPSC-CM (Kd=0.39±0.05 μM; p<0.05) without affecting Bmax (96±13 μmol/Lcytosol). Importantly, rates of non-NCX mediated Ca removal were not different between hiPSC-CM and adult rabbit CM. These results demonstrate that hiPSC-CM have cytosolic Ca buffering parameters similar to those of acutely-isolated native CM and therefore could be used for modeling cardiac diseases with altered myofilament Ca binding. Supported by AHA-12POST12080080(DK), NIH-RO1-HL71670(BCK).

Modeling Calcium Dynamics in Rabbit Ventricular Myocytes with Several Realistic T-Tubules Subject to Detubulation

Calcium signaling in cardiomyocytes is strongly influenced by the topology of the sarcolemma (SL) and the distribution and clustering of sarcolemmal Ca2+ transporters, including the L-type Ca2+ channel (LCC) and sodium-calcium exchanger (NCX). Cardiac ventricular myocytes contain dense networks of cell membrane invaginations called t-tubules (TTs), which contain SL transporters that trigger Ca2+ release from the channel of the sarcoplasmic reticulum (SR), known as ryanodine receptor (RyR). The TT network ensures uniformity of the Ca2+ transient, and pathological remodeling of TT-network produces heterogeneous Ca2+ transients. This remodeling reduces the density of SL Ca2+ transporters and perturbs the topology of the space into which Ca2+ diffuses. The extent to which these factors contribute to the non-uniform Ca2+ transient observed in remodeled cells is however unclear. In this study, we examined a 9x5x6 µm subcellular region of a rabbit ventricular myocyte with confocal microscope. We inferred LCC cluster distribution statistics from localized RyR clusters data. We solved time-dependent reaction-diffusion equations describing important sources of sarcolemmal transport and cytosolic buffering and diffusion of Ca2+. We found that the local Ca2+ gradients’ profiles are influenced predominantly by the distribution of SL Ca2+ transporters and secondarily by the configuration of TTs. Our results also suggest that the early stages of the Ca2+ transient, prior to SR release, are sensitive to LCC cluster distribution and TT configuration.Supported by NBCR (NIH grant 2 P41 GM103426-19), NIH GM31749, NSF MCB-1020765, MCA93S013, Center for Theoretical Biological Physics, Howard Hughes Medical Institute, SDSC, W. M. Keck foundation, Nora Eccles Harrison Cardiovascular Research and Training Institute.

Development of roGFP2-derived redox probes for measurement of the glutathione redox potential in the cytosol of severely glutathione-deficient rml1 seedlings

Glutathione is important for detoxification, as a cofactor in biochemical reactions and as a thiol-redox buffer. The cytosolic glutathione buffer is normally highly reduced with glutathione redox potentials (EGSH) of more negative than −310 mV. Maintenance of such negative redox potential is achieved through continuous reduction of glutathione disulfide by glutathione reductase (GR). Deviations from steady state glutathione redox homeostasis have been discussed as a possible mean to alter the activity of redox-sensitive proteins through switching of critical thiol residues. To better understand such signaling mechanisms it is essential to be able to measure EGSH over a wide range from highly negative redox potentials down to potentials found in mutants that show already severe phenotypes. With the advent of redox-sensitive GFPs (roGFPs), understanding the in vivo dynamics of the thiol-based redox buffer system became within reach. The original roGFP versions, roGFP1 and roGFP2, however, have midpoint potentials between −280 and −290 mV rendering them fully oxidized in the ER and almost fully reduced in the cytosol, plastids, mitochondria, and peroxisomes. To extend the range of suitable probes we have engineered a roGFP2 derivative, roGFP2-iL, with a midpoint potential of about −238 mV. This value is within the range of redox potentials reported for homologous roGFP1-iX probes, albeit with different excitation properties. To allow rapid and specific equilibration with the glutathione pool, fusion constructs with human glutaredoxin 1 (GRX1) were generated and characterized in vitro. GRX1-roGFP2-iL proved to be suitable for in vivo redox potential measurements and extends the range of EGSH values that can be measured in vivo with roGFP2-based probes from about −320 mV for GRX1-roGFP2 down to about −210 mV for GRX1-roGFP2-iL. Using both probes in the cytosol of severely glutathione-deficient rml1 seedlings revealed an EGSH of about −260 mV in this mutant.

MODELING EFFECT OF SODIUM PUMP ON CALCIUM OSCILLATIONS IN NEURON CELLS

Calcium plays a significant role in a number of processes like muscle contraction, gene expression, synaptic plasticity, signal transduction etc. but the significance of calcium oscillation is not yet completely understood in most of the cell types. A number of investigators have reported the oscillatory behavior of calcium due to intracellular concentration of inositol 1,4,5-trisphosphate (IP3). In this paper, an attempt has been made to study the oscillations induced in calcium due to dynamically changing membrane potential with special relevance to sodium pump. A mathematical model is developed which incorporates nearly all important and necessary biophysical components like L-type calcium channel, sodium channel, potassium channel, cytosolic buffers, calcium pump, sodium–calcium exchanger (NCX), sodium–potassium ATPase (sodium pump), and dynamic membrane potential. These channels have realistic gating mechanism and emulate the gating mechanism proposed in the famous paper of Hodgkin and Huxley.1 The model leads to an initial value problem involving system of non-linear ordinary differential equations. A numerical solution has been obtained using Gear's method. The numerical results have been used to study the effect of sodium pump over the frequency of Ca 2+ oscillation. At lower and higher sodium pump current densities the Ca 2+ oscillation frequency is observed to be 278 Hz and 225 Hz respectively.

Myofilament Ca Sensitization Increases Cytosolic Ca Binding Affinity, Alters Intracellular Ca Homeostasis, and Causes Pause-Dependent Ca-Triggered Arrhythmia

Ca binding to the troponin complex represents a major portion of cytosolic Ca buffering. Troponin mutations that increase myofilament Ca sensitivity are associated with familial hypertrophic cardiomyopathy and confer a high risk for sudden death. In mice, Ca sensitization causes ventricular arrhythmias, but the underlying mechanisms remain unclear. To test the hypothesis that myofilament Ca sensitization increases cytosolic Ca buffering and to determine the resulting arrhythmogenic changes in Ca homeostasis in the intact mouse heart. Using cardiomyocytes isolated from mice expressing troponin T (TnT) mutants (TnT-I79N, TnT-F110I, TnT-R278C), we found that increasing myofilament Ca sensitivity produced a proportional increase in cytosolic Ca binding. The underlying cause was an increase in the cytosolic Ca binding affinity, whereas maximal Ca binding capacity was unchanged. The effect was sufficiently large to alter Ca handling in intact mouse hearts at physiological heart rates, resulting in increased end-diastolic [Ca] at fast pacing rates, and enhanced sarcoplasmic reticulum Ca content and release after pauses. Accordingly, action potential (AP) regulation was altered, with postpause action potential prolongation, afterdepolarizations, and triggered activity. Acute Ca sensitization with EMD 57033 mimicked the effects of Ca-sensitizing TnT mutants and produced pause-dependent ventricular ectopy and sustained ventricular tachycardia after acute myocardial infarction. Myofilament Ca sensitization increases cytosolic Ca binding affinity. A major proarrhythmic consequence is a pause-dependent potentiation of Ca release, action potential prolongation, and triggered activity. Increased cytosolic Ca binding represents a novel mechanism of pause-dependent arrhythmia that may be relevant for inherited and acquired cardiomyopathies.

Inverse problems in neuronal calcium signaling

Calcium is the most important of the brain's second messengers. Thanks to engineered fluorescent indicators and caged compounds we have an excellent qualitative picture of its regulation and impact.With the advent of new scanning technology that permits one to observe the calcium signal throughout a highly branched neuron the potential exists for functional, single cell, quantitative calcium imaging. To help realize that potential we analyze a sequence of four inverse problems that infer the parameters of the cytosolic calcium buffers and plasma membrane calcium pumps and channels from the light shed by fluorescent indicators following specific stimulus protocols. Our analyses lead in each case to practical algorithms that we illustrate and test on synthetic data.

Effects of Cytosolic Ca Buffering on Sarcoplasmic Reticulum Ca Leak in Permeabilized Rabbit Ventricular Myocytes

In this study we tested the hypothesis that local Ca-induced Ca release (CICR) within ryanodine receptor (RyR2) release clusters plays an important role in defining spark-mediated sarcoplasmic reticulum (SR) Ca leak. We studied to what degree changes of cytosolic Ca buffer capacity affect single RyR activity in lipid bilayers, Ca spark properties, and SR Ca leak in permeabilized ventricular myocytes. No substantial changes in single RyR2 function were observed if BAPTA (a fast Ca buffer) was added (holding free Ca constant) to the EGTA-buffered cytosolic solution. However, the same cytosolic BAPTA addition almost completely eliminated Ca sparks in cells. The elimination of Ca sparks by BAPTA was not due to a decrease in SR Ca load. The addition of the fast cytosolic Ca buffer significantly decreased SR Ca leak, particularly at high [Ca]SR (>400 μM) where Ca sparks significantly contribute to SR Ca leak. In contrast, lowering cytosolic Ca buffer capacity (by decreasing EGTA from 0.36 to 0.1 mM; keeping free Ca constant) drastically increased Ca spark frequency and width, leading to generation of multifocal (or propagating) Ca sparks. This decrease in cytosolic Ca buffer capacity also significantly accelerated SR Ca leak as a result of augmentation of Ca spark-mediated component of SR Ca leak. In conclusion, by changing cytosolic Ca buffering (capacity or affinity) we were able to experimentally separate Ca spark- and non-spark- mediated component of the overall SR Ca leak in ventricular myocytes. These data also suggest that changes in the dynamics of local cytosolic Ca handling (buffering, extrusion, etc.) are physiologically important in determining the degree of spark and non-spark mediated SR Ca leak.