Up-down biphasic volume response of human red blood cells to PIEZO1 activation during capillary transits.

In this paper we apply a novel JAVA version of a model on the homeostasis of human red blood cells (RBCs) to investigate the changes RBCs experience during single capillary transits. In the companion paper we apply a model extension to investigate the changes in RBC homeostasis over the approximately 200000 capillary transits during the ~120 days lifespan of the cells. These are topics inaccessible to direct experimentation but rendered mature for a computational modelling approach by the large body of recent and early experimental results which robustly constrain the range of parameter values and model outcomes, offering a unique opportunity for an in depth study of the mechanisms involved. Capillary transit times vary between 0.5 and 1.5s during which the red blood cells squeeze and deform in the capillary stream transiently opening stress-gated PIEZO1 channels allowing ion gradient dissipation and creating minuscule quantal changes in RBC ion contents and volume. Widely accepted views, based on the effects of experimental shear stress on human RBCs, suggested that quantal changes generated during capillary transits add up over time to develop the documented changes in RBC density and composition during their long circulatory lifespan, the quantal hypothesis. Applying the new red cell model (RCM) we investigated here the changes in homeostatic variables that may be expected during single capillary transits resulting from transient PIEZO1 channel activation. The predicted quantal volume changes were infinitesimal in magnitude, biphasic in nature, and essentially irreversible within inter-transit periods. A sub-second transient PIEZO1 activation triggered a sharp swelling peak followed by a much slower recovery period towards lower-than-baseline volumes. The peak response was caused by net CaCl2 and fluid gain via PIEZO1 channels driven by the steep electrochemical inward Ca2+ gradient. The ensuing dehydration followed a complex time-course with sequential, but partially overlapping contributions by KCl loss via Ca2+-activated Gardos channels, restorative Ca2+ extrusion by the plasma membrane calcium pump, and chloride efflux by the Jacobs-Steward mechanism. The change in relative cell volume predicted for single capillary transits was around 10-5, an infinitesimal volume change incompatible with a functional role in capillary flow. The biphasic response predicted by the RCM appears to conform to the quantal hypothesis, but whether its cumulative effects could account for the documented changes in density during RBC senescence required an investigation of the effects of myriad transits over the full four months circulatory lifespan of the cells, the subject of the next paper

Up-down biphasic volume response of human red blood cells to PIEZO1 activation during capillary transits

In this paper we apply a novel JAVA version of a model on the homeostasis of human red blood cells (RBCs) to investigate the changes RBCs experience during single capillary transits. In the companion paper we apply a model extension to investigate the changes in RBC homeostasis over the approximately 200000 capillary transits during the ~120 days lifespan of the cells. These are topics inaccessible to direct experimentation but rendered mature for a computational modelling approach by the large body of recent and early experimental results which robustly constrain the range of parameter values and model outcomes, offering a unique opportunity for an in depth study of the mechanisms involved. Capillary transit times vary between 0.5 and 1.5s during which the red blood cells squeeze and deform in the capillary stream transiently opening stress-gated PIEZO1 channels allowing ion gradient dissipation and creating minuscule quantal changes in RBC ion contents and volume. Widely accepted views, based on the effects of experimental shear stress on human RBCs, suggested that quantal changes generated during capillary transits add up over time to develop the documented changes in RBC density and composition during their long circulatory lifespan, the quantal hypothesis. Applying the new red cell model (RCM) we investigated here the changes in homeostatic variables that may be expected during single capillary transits resulting from transient PIEZO1 channel activation. The predicted quantal volume changes were infinitesimal in magnitude, biphasic in nature, and essentially irreversible within inter-transit periods. A sub-second transient PIEZO1 activation triggered a sharp swelling peak followed by a much slower recovery period towards lower-than-baseline volumes. The peak response was caused by net CaCl2 and fluid gain via PIEZO1 channels driven by the steep electrochemical inward Ca2+ gradient. The ensuing dehydration followed a complex time-course with sequential, but partially overlapping contributions by KCl loss via Ca2+-activated Gardos channels, restorative Ca2+ extrusion by the plasma membrane calcium pump, and chloride efflux by the Jacobs-Steward mechanism. The change in relative cell volume predicted for single capillary transits was around 10−5, an infinitesimal volume change incompatible with a functional role in capillary flow. The biphasic response predicted by the RCM appears to conform to the quantal hypothesis, but whether its cumulative effects could account for the documented changes in density during RBC senescence required an investigation of the effects of myriad transits over the full four months circulatory lifespan of the cells, the subject of the next paper

PIEZO1 and the mechanism of the long circulatory longevity of human red blood cells.

Human red blood cells (RBCs) have a circulatory lifespan of about four months. Under constant oxidative and mechanical stress, but devoid of organelles and deprived of biosynthetic capacity for protein renewal, RBCs undergo substantial homeostatic changes, progressive densification followed by late density reversal among others, changes assumed to have been harnessed by evolution to sustain the rheological competence of the RBCs for as long as possible. The unknown mechanisms by which this is achieved are the subject of this investigation. Each RBC traverses capillaries between 1000 and 2000 times per day, roughly one transit per minute. A dedicated Lifespan model of RBC homeostasis was developed as an extension of the RCM introduced in the previous paper to explore the cumulative patterns predicted for repetitive capillary transits over a standardized lifespan period of 120 days, using experimental data to constrain the range of acceptable model outcomes. Capillary transits were simulated by periods of elevated cell/medium volume ratios and by transient deformation-induced permeability changes attributed to PIEZO1 channel mediation as outlined in the previous paper. The first unexpected finding was that quantal density changes generated during single capillary transits cease accumulating after a few days and cannot account for the observed progressive densification of RBCs on their own, thus ruling out the quantal hypothesis. The second unexpected finding was that the documented patterns of RBC densification and late reversal could only be emulated by the implementation of a strict time-course of decay in the activities of the calcium and Na/K pumps, suggestive of a selective mechanism enabling the extended longevity of RBCs. The densification pattern over most of the circulatory lifespan was determined by calcium pump decay whereas late density reversal was shaped by the pattern of Na/K pump decay. A third finding was that both quantal changes and pump-decay regimes were necessary to account for the documented lifespan pattern, neither sufficient on their own. A fourth new finding revealed that RBCs exposed to levels of PIEZO1-medited calcium permeation above certain thresholds in the circulation could develop a pattern of early or late hyperdense collapse followed by delayed density reversal. When tested over much reduced lifespan periods the results reproduced the known circulatory fate of irreversible sickle cells, the cell subpopulation responsible for vaso-occlusion and for most of the clinical manifestations of sickle cell disease. Analysis of the results provided an insightful new understanding of the mechanisms driving the changes in RBC homeostasis during circulatory aging in health and disease

PIEZO1 and the mechanism of the long circulatory longevity of human red blood cells

Human red blood cells (RBCs) have a circulatory lifespan of about four months. Under constant oxidative and mechanical stress, but devoid of organelles and deprived of biosynthetic capacity for protein renewal, RBCs undergo substantial homeostatic changes, progressive densification followed by late density reversal among others, changes assumed to have been harnessed by evolution to sustain the rheological competence of the RBCs for as long as possible. The unknown mechanisms by which this is achieved are the subject of this investigation. Each RBC traverses capillaries between 1000 and 2000 times per day, roughly one transit per minute. A dedicated Lifespan model of RBC homeostasis was developed as an extension of the RCM introduced in the previous paper to explore the cumulative patterns predicted for repetitive capillary transits over a standardized lifespan period of 120 days, using experimental data to constrain the range of acceptable model outcomes. Capillary transits were simulated by periods of elevated cell/medium volume ratios and by transient deformation-induced permeability changes attributed to PIEZO1 channel mediation as outlined in the previous paper. The first unexpected finding was that quantal density changes generated during single capillary transits cease accumulating after a few days and cannot account for the observed progressive densification of RBCs on their own, thus ruling out the quantal hypothesis. The second unexpected finding was that the documented patterns of RBC densification and late reversal could only be emulated by the implementation of a strict time-course of decay in the activities of the calcium and Na/K pumps, suggestive of a selective mechanism enabling the extended longevity of RBCs. The densification pattern over most of the circulatory lifespan was determined by calcium pump decay whereas late density reversal was shaped by the pattern of Na/K pump decay. A third finding was that both quantal changes and pump-decay regimes were necessary to account for the documented lifespan pattern, neither sufficient on their own. A fourth new finding revealed that RBCs exposed to levels of PIEZO1-medited calcium permeation above certain thresholds in the circulation could develop a pattern of early or late hyperdense collapse followed by delayed density reversal. When tested over much reduced lifespan periods the results reproduced the known circulatory fate of irreversible sickle cells, the cell subpopulation responsible for vaso-occlusion and for most of the clinical manifestations of sickle cell disease. Analysis of the results provided an insightful new understanding of the mechanisms driving the changes in RBC homeostasis during circulatory aging in health and disease

Osmotic Vesicle Collapse of Sealed Inside-Out Membrane Vesicles From Red Blood Cells.

The preparation of plasma membrane vesicles from a large variety of cells has contributed a wealth of information on the identity and vectorial properties of membrane transporters and enzymes. Vesicles from red blood cell (RBC) membranes are generated in media of extremely low tonicity. For functional studies, it is required to suspend the vesicles in higher tonicity media in order to bring the concentrations of the substrates of transporters and enzymes under investigation within the physiological ranges. We investigated the effects of hypertonic transitions on the vesicle morphology using transmission electron microscopy. The results show that hypertonic transitions cause an irreversible osmotic collapse of sealed membrane vesicles. Awareness of the collapsed condition of vesicles during functional studies is critical for the proper interpretation of experimental results

A constraint-relaxation-recovery mechanism for stomatal dynamics

Models of guard cell dynamics, built on the OnGuard platform, have provided quantitative insights into stomatal function, demonstrating substantial predictive power. However, the kinetics of stomatal opening predicted by OnGuard models were threefold to fivefold slower than observed in vivo. No manipulations of parameters within physiological ranges yielded model kinetics substantially closer to these data, thus highlighting a missing component in model construction. One well‐documented process influencing stomata is the constraining effect of the surrounding epidermal cells on guard cell volume and stomatal aperture. Here, we introduce a mechanism to describe this effect in OnGuard2 constructed around solute release and a decline in turgor of the surrounding cells and its subsequent recovery during stomatal opening. The results show that this constraint–relaxation–recovery mechanism in OnGuard2 yields dynamics that are consistent with experimental observations in wild‐type Arabidopsis, and it predicts the altered opening kinetics of ost2 H+‐ATPase and slac1 Cl− channel mutants. Thus, incorporating solute flux of the surrounding cells implicitly through their constraint on guard cell expansion provides a satisfactory representation of stomatal kinetics, and it predicts a substantial and dynamic role for solute flux across the apoplastic space between the guard cells and surrounding cells in accelerating stomatal kinetics

Age Decline in the Activity of the Ca2+-sensitive K+ Channel of Human Red Blood Cells

The Ca2+-sensitive K+ channel of human red blood cells (RBCs) (Gardos channel, hIK1, hSK4) was implicated in the progressive densification of RBCs during normal senescence and in the mechanism of sickle cell dehydration. Saturating RBC Ca2+ loads were shown before to induce rapid and homogeneous dehydration, suggesting that Gardos channel capacity was uniform among the RBCs, regardless of age. Using glycated hemoglobin as a reliable RBC age marker, we investigated the age–activity relation of Gardos channels by measuring the mean age of RBC subpopulations exceeding a set high density boundary during dehydration. When K+ permeabilization was induced with valinomycin, the oldest and densest cells, which started nearest to the set density boundary, crossed it first, reflecting conservation of the normal age–density distribution pattern during dehydration. However, when Ca2+ loads were used to induce maximal K+ fluxes via Gardos channels in all RBCs (Fmax), the youngest RBCs passed the boundary first, ahead of the older RBCs, indicating that Gardos channel Fmax was highest in those young RBCs, and that the previously observed appearance of uniform dehydration concealed a substantial degree of age scrambling during the dehydration process. Further analysis of the Gardos channel age–activity relation revealed a monotonic decline in Fmax with cell age, with a broad quasi-Gaussian Fmax distribution among the RBCs

FRET imaging of hemoglobin concentration in Plasmodium falciparum-infected red cells.

BACKGROUND: During its intraerythrocytic asexual reproduction cycle Plasmodium falciparum consumes up to 80% of the host cell hemoglobin, in large excess over its metabolic needs. A model of the homeostasis of falciparum-infected red blood cells suggested an explanation based on the need to reduce the colloid-osmotic pressure within the host cell to prevent its premature lysis. Critical for this hypothesis was that the hemoglobin concentration within the host cell be progressively reduced from the trophozoite stage onwards. METHODOLOGY/PRINCIPAL FINDINGS: The experiments reported here were designed to test this hypothesis by direct measurements of the hemoglobin concentration in live, infected red cells. We developed a novel, non-invasive method to quantify the hemoglobin concentration in single cells, based on Förster resonance energy transfer between hemoglobin molecules and the fluorophore calcein. Fluorescence lifetime imaging allowed the quantitative mapping of the hemoglobin concentration within the cells. The average fluorescence lifetimes of uninfected cohorts was 270+/-30 ps (mean+/-SD; N = 45). In the cytoplasm of infected cells the fluorescence lifetime of calcein ranged from 290+/-20 ps for cells with ring stage parasites to 590+/-13 ps and 1050+/-60 ps for cells with young trophozoites and late stage trophozoite/early schizonts, respectively. This was equivalent to reductions in hemoglobin concentration spanning the range from 7.3 to 2.3 mM, in line with the model predictions. An unexpected ancillary finding was the existence of a microdomain under the host cell membrane with reduced calcein quenching by hemoglobin in cells with mature trophozoite stage parasites. CONCLUSIONS/SIGNIFICANCE: The results support the predictions of the colloid-osmotic hypothesis and provide a better understanding of the homeostasis of malaria-infected red cells. In addition, they revealed the existence of a distinct peripheral microdomain in the host cell with limited access to hemoglobin molecules indicating the concentration of substantial amounts of parasite-exported material

Global Sensitivity Analysis of OnGuard Models Identifies Key Hubs for Transport Interaction in Stomatal Dynamics.

The physical requirement for charge to balance across biological membranes means that the transmembrane transport of each ionic species is interrelated, and manipulating solute flux through any one transporter will affect other transporters at the same membrane, often with unforeseen consequences. The OnGuard systems modeling platform has helped to resolve the mechanics of stomatal movements, uncovering previously unexpected behaviors of stomata. To date, however, the manual approach to exploring model parameter space has captured little formal information about the emergent connections between parameters that define the most interesting properties of the system as a whole. Here, we introduce global sensitivity analysis to identify interacting parameters affecting a number of outputs commonly accessed in experiments in Arabidopsis (Arabidopsis thaliana). The analysis highlights synergies between transporters affecting the balance between Ca2+ sequestration and Ca2+ release pathways, notably those associated with internal Ca2+ stores and their turnover. Other, unexpected synergies appear, including with the plasma membrane anion channels and H+-ATPase and with the tonoplast TPK K+ channel. These emergent synergies, and the core hubs of interaction that they define, identify subsets of transporters associated with free cytosolic Ca2+ concentration that represent key targets to enhance plant performance in the future. They also highlight the importance of interactions between the voltage regulation of the plasma membrane and tonoplast in coordinating transport between the different cellular compartments

Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

BACKGROUND: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. METHODOLOGY/PRINCIPAL FINDINGS: The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+) and Cl(-) currents were strictly dependent on the presence of Ca(2+). The Ca(2+)-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+) permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca(2+) permeability pathway leading to increased [Ca(2+)](i), secondary activation of Ca(2+)-sensitive K(+) channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. CONCLUSIONS/SIGNIFICANCE: The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia