Oxygenation management during veno-arterial ECMO support for cardiogenic shock: a multicentric retrospective cohort study.

BACKGOUND: Hyperoxemia is common and associated with poor outcome during veno-arterial extracorporeal membrane oxygenation (VA ECMO) support for cardiogenic shock. However, little is known about practical daily management of oxygenation. Then, we aim to describe sweep gas oxygen fraction (FSO2), postoxygenator oxygen partial pressure (PPOSTO2), inspired oxygen fraction (FIO2), and right radial arterial oxygen partial pressure (PaO2) between day 1 and day 7 of peripheral VA ECMO support. We also aim to evaluate the association between oxygenation parameters and outcome. In this retrospective multicentric study, each participating center had to report data on the last 10 eligible patients for whom the ICU stay was terminated. Patients with extracorporeal cardiopulmonary resuscitation were excluded. Primary endpoint was individual mean FSO2 during the seven first days of ECMO support (FSO2 mean (day 1-7)). RESULTS: Between August 2019 and March 2022, 139 patients were enrolled in 14 ECMO centers in France, and one in Switzerland. Among them, the median value for FSO2 mean (day 1-7) was 70 [57; 79] % but varied according to center case volume. Compared to high volume centers, centers with less than 30 VA-ECMO runs per year were more likely to maintain FSO2 ≥ 70% (OR 5.04, CI 95% [1.39; 20.4], p = 0.017). Median value for right radial PaO2 mean (day 1-7) was 114 [92; 145] mmHg, and decreased from 125 [86; 207] mmHg at day 1, to 97 [81; 133] mmHg at day 3 (p < 0.01). Severe hyperoxemia (i.e. right radial PaO2 ≥ 300 mmHg) occurred in 16 patients (12%). PPOSTO2, a surrogate of the lower body oxygenation, was measured in only 39 patients (28%) among four centers. The median value of PPOSTO2 mean (day 1-7) value was 198 [169; 231] mmHg. By multivariate analysis, age (OR 1.07, CI95% [1.03-1.11], p < 0.001), FSO2 mean (day 1-3)(OR 1.03 [1.00-1.06], p = 0.039), and right radial PaO2 mean (day 1-3) (OR 1.03, CI95% [1.00-1.02], p = 0.023) were associated with in-ICU mortality. CONCLUSION: In a multicentric cohort of cardiogenic shock supported by VA ECMO, the median value for FSO2 mean (day 1-7) was 70 [57; 79] %. PPOSTO2 monitoring was infrequent and revealed significant hyperoxemia. Higher FSO2 mean (day 1-3) and right radial PaO2 mean (day 1-3) were independently associated with in-ICU mortality.

A versatile high-throughput assay based on 3D ring-shaped cardiac tissues generated from human induced pluripotent stem cell-derived cardiomyocytes.

We developed a 96-well plate assay which allows fast, reproducible, and high-throughput generation of 3D cardiac rings around a deformable optically transparent hydrogel (polyethylene glycol [PEG]) pillar of known stiffness. Human induced pluripotent stem cell-derived cardiomyocytes, mixed with normal human adult dermal fibroblasts in an optimized 3:1 ratio, self-organized to form ring-shaped cardiac constructs. Immunostaining showed that the fibroblasts form a basal layer in contact with the glass, stabilizing the muscular fiber above. Tissues started contracting around the pillar at D1 and their fractional shortening increased until D7, reaching a plateau at 25±1%, that was maintained up to 14 days. The average stress, calculated from the compaction of the central pillar during contractions, was 1.4±0.4 mN/mm2. The cardiac constructs recapitulated expected inotropic responses to calcium and various drugs (isoproterenol, verapamil) as well as the arrhythmogenic effects of dofetilide. This versatile high-throughput assay allows multiple in situ mechanical and structural readouts.

Mechanochemical Crosstalk Produces Cell-Intrinsic Patterning of the Cortex to Orient the Mitotic Spindle.

Proliferating animal cells are able to orient their mitotic spindles along their interphase cell axis, setting up the axis of cell division, despite rounding up as they enter mitosis. This has previously been attributed to molecular memory and, more specifically, to the maintenance of adhesions and retraction fibers in mitosis [1-6], which are thought to act as local cues that pattern cortical Gαi, LGN, and nuclear mitotic apparatus protein (NuMA) [3, 7-18]. This cortical machinery then recruits and activates Dynein motors, which pull on astral microtubules to position the mitotic spindle. Here, we reveal a dynamic two-way crosstalk between the spindle and cortical motor complexes that depends on a Ran-guanosine triphosphate (GTP) signal [12], which is sufficient to drive continuous monopolar spindle motion independently of adhesive cues in flattened human cells in culture. Building on previous work [1, 12, 19-23], we implemented a physical model of the system that recapitulates the observed spindle-cortex interactions. Strikingly, when this model was used to study spindle dynamics in cells entering mitosis, the chromatin-based signal was found to preferentially clear force generators from the short cell axis, so that cortical motors pulling on astral microtubules align bipolar spindles with the interphase long cell axis, without requiring a fixed cue or a physical memory of interphase shape. Thus, our analysis shows that the ability of chromatin to pattern the cortex during the process of mitotic rounding is sufficient to translate interphase shape into a cortical pattern that can be read by the spindle, which then guides the axis of cell division.

Actomyosin drives cancer cell nuclear dysmorphia and threatens genome stability.

Altered nuclear shape is a defining feature of cancer cells. The mechanisms underlying nuclear dysmorphia in cancer remain poorly understood. Here we identify PPP1R12A and PPP1CB, two subunits of the myosin phosphatase complex that antagonizes actomyosin contractility, as proteins safeguarding nuclear integrity. Loss of PPP1R12A or PPP1CB causes nuclear fragmentation, nuclear envelope rupture, nuclear compartment breakdown and genome instability. Pharmacological or genetic inhibition of actomyosin contractility restores nuclear architecture and genome integrity in cells lacking PPP1R12A or PPP1CB. We detect actin filaments at nuclear envelope rupture sites and define the Rho-ROCK pathway as the driver of nuclear damage. Lamin A protects nuclei from the impact of actomyosin activity. Blocking contractility increases nuclear circularity in cultured cancer cells and suppresses deformations of xenograft nuclei in vivo. We conclude that actomyosin contractility is a major determinant of nuclear shape and that unrestrained contractility causes nuclear dysmorphia, nuclear envelope rupture and genome instability.

Optical volume and mass measurements show that mammalian cells swell during mitosis.

The extent, mechanism, and function of cell volume changes during specific cellular events, such as cell migration and cell division, have been poorly studied, mostly because of a lack of adequate techniques. Here we unambiguously report that a large range of mammalian cell types display a significant increase in volume during mitosis (up to 30%). We further show that this increase in volume is tightly linked to the mitotic state of the cell and not to its spread or rounded shape and is independent of the presence of an intact actomyosin cortex. Importantly, this volume increase is not accompanied by an increase in dry mass and thus corresponds to a decrease in cell density. This mitotic swelling might have important consequences for mitotic progression: it might contribute to produce strong pushing forces, allowing mitotic cells to round up; it might also, by lowering cytoplasmic density, contribute to the large change of physicochemical properties observed in mitotic cells.

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells.

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space.

Actin flows mediate a universal coupling between cell speed and cell persistence.

Cell movement has essential functions in development, immunity, and cancer. Various cell migration patterns have been reported, but no general rule has emerged so far. Here, we show on the basis of experimental data in vitro and in vivo that cell persistence, which quantifies the straightness of trajectories, is robustly coupled to cell migration speed. We suggest that this universal coupling constitutes a generic law of cell migration, which originates in the advection of polarity cues by an actin cytoskeleton undergoing flows at the cellular scale. Our analysis relies on a theoretical model that we validate by measuring the persistence of cells upon modulation of actin flow speeds and upon optogenetic manipulation of the binding of an actin regulator to actin filaments. Beyond the quantitative prediction of the coupling, the model yields a generic phase diagram of cellular trajectories, which recapitulates the full range of observed migration patterns.

Confinement and low adhesion induce fast amoeboid migration of slow mesenchymal cells.

The mesenchymal-amoeboid transition (MAT) was proposed as a mechanism for cancer cells to adapt their migration mode to their environment. While the molecular pathways involved in this transition are well documented, the role of the microenvironment in the MAT is still poorly understood. Here, we investigated how confinement and adhesion affect this transition. We report that, in the absence of focal adhesions and under conditions of confinement, mesenchymal cells can spontaneously switch to a fast amoeboid migration phenotype. We identified two main types of fast migration--one involving a local protrusion and a second involving a myosin-II-dependent mechanical instability of the cell cortex that leads to a global cortical flow. Interestingly, transformed cells are more prone to adopt this fast migration mode. Finally, we propose a generic model that explains migration transitions and predicts a phase diagram of migration phenotypes based on three main control parameters: confinement, adhesion, and contractility.

Direct kinetochore-spindle pole connections are not required for chromosome segregation.

Segregation of genetic material occurs when chromosomes move to opposite spindle poles during mitosis. This movement depends on K-fibers, specialized microtubule (MT) bundles attached to the chromosomes' kinetochores. A long-standing assumption is that continuous K-fibers connect every kinetochore to a spindle pole and the force for chromosome movement is produced at the kinetochore and coupled with MT depolymerization. However, we found that chromosomes still maintained their position at the spindle equator during metaphase and segregated properly during anaphase when one of their K-fibers was severed near the kinetochore with a laser microbeam. We also found that, in normal fully assembled spindles, K-fibers of some chromosomes did not extend to the spindle pole. These K-fibers connected to adjacent K-fibers and/or nonkinetochore MTs. Poleward movement of chromosomes with short K-fibers was uncoupled from MT depolymerization at the kinetochore. Instead, these chromosomes moved by dynein-mediated transport of the entire K-fiber/kinetochore assembly. Thus, at least two distinct parallel mechanisms drive chromosome segregation in mammalian cells.

Exploring the function of cell shape and size during mitosis.

Dividing cells almost always adopt a spherical shape. This is true of most eukaryotic cells lacking a rigid cell wall and is observed in tissue culture and single-celled organisms, as well as in cells dividing inside tissues. While the mechanisms underlying this shape change are now well described, the functional importance of the spherical mitotic cell for the success of cell division has been thus far scarcely addressed. Here we discuss how mitotic rounding contributes to spindle assembly and positioning, as well as the potential consequences of abnormal mitotic cell shape and size on chromosome segregation, tissue growth, and cancer.

Methods for two-dimensional cell confinement.

Protocols described in this chapter relate to a method to dynamically confine cells in two dimensions with various microenvironments. It can be used to impose on cells a given height, with an accuracy of less than 100 nm on large surfaces (cm(2)). The method is based on the gentle application of a modified glass coverslip onto a standard cell culture. Depending on the preparation, this confinement slide can impose on the cells a given geometry but also an environment of controlled stiffness, controlled adhesion, or a more complex environment. An advantage is that the method is compatible with most optical microscopy technologies and molecular biology protocols allowing advanced analysis of confined cells. In this chapter, we first explain the principle and issues of using these slides to confine cells in a controlled geometry and describe their fabrication. Finally, we discuss how the nature of the confinement slide can vary and provide an alternative method to confine cells with gels of controlled rigidity.

Migration of dendritic cells: physical principles, molecular mechanisms, and functional implications.

Dendritic cells (DCs) constitute a complex cell population that resides in both peripheral tissues and lymphoid organs. Their major function in tissues is to patrol their environment in search of danger-associated antigens to transport to lymph nodes and present to T lymphocytes. This process constitutes the first step of the adaptive immune response and relies on specific DC properties, including a high endocytic capacity as well as efficient motility in confined three-dimensional environments. Although cell motility has been widely studied, little is known on how the geometric characteristics of the environment influence DC migration and function. In this review, we give an overview of the basic physical principles and molecular mechanisms that control DC migration under confinement and discuss how such mechanisms impact the environment-patrolling capacity of DCs.

Mitotic rounding alters cell geometry to ensure efficient bipolar spindle formation.

Accurate animal cell division requires precise coordination of changes in the structure of the microtubule-based spindle and the actin-based cell cortex. Here, we use a series of perturbation experiments to dissect the relative roles of actin, cortical mechanics, and cell shape in spindle formation. We find that, whereas the actin cortex is largely dispensable for rounding and timely mitotic progression in isolated cells, it is needed to drive rounding to enable unperturbed spindle morphogenesis under conditions of confinement. Using different methods to limit mitotic cell height, we show that a failure to round up causes defects in spindle assembly, pole splitting, and a delay in mitotic progression. These defects can be rescued by increasing microtubule lengths and therefore appear to be a direct consequence of the limited reach of mitotic centrosome-nucleated microtubules. These findings help to explain why most animal cells round up as they enter mitosis.

Fine control of nuclear confinement identifies a threshold deformation leading to lamina rupture and induction of specific genes.

The quest to understand how the mechanical and geometrical environment of cells impacts their behavior and fate has been a major force driving the recent development of new technologies in cell biology research. Despite rapid advances in this field, many challenges remain in order to bridge the gap between the classical and simple cell culture plate and the biological reality of actual tissue. In tissues, cells have their physical space constrained by neighboring cells and the extracellular matrix. Here, we propose a simple and versatile device to precisely and dynamically control this confinement parameter in cultured cells. We show that there is a precise threshold deformation above which the nuclear lamina breaks and reconstructs, whereas nuclear volume changes. We also show that different nuclear deformations correlate with the expression of specific sets of genes, including nuclear factors and classical mechanotransduction pathways. This versatile device thus enables the precise control of cell and nuclear deformation by confinement and the correlative study of the associated molecular events.

Fast microfluidic temperature control for high resolution live cell imaging.

One major advantage of using genetically tractable model organisms such as the fission yeast Schizosaccharomyces pombe is the ability to construct temperature-sensitive mutations in a gene. The resulting gene product or protein behaves as wildtype at permissive temperatures. At non-permissive or restrictive temperatures the protein becomes unstable and some or all of its functions are abrogated. The protein regains its function when returning to a permissive temperature. In principle, temperature-sensitive mutation enables precise temporal control of protein activity when coupled to a fast temperature controller. Current commercial temperature control devices do not have fast switching capability over a wide range of temperatures, making repeated temperature changes impossible or impractical at the cellular timescale of seconds or minutes. Microfabrication using soft-lithography is emerging as a powerful tool for cell biological research. We present here a simple disposable polydimethylsiloxane (PDMS) based microfluidic device capable of reversibly switching between 5 °C and 45 °C in less than 10 s. This device allows high-resolution live cell imaging with an oil immersion objective lens. We demonstrate the utility of this device for studying microtubule dynamics throughout the cell cycle.

Microfluidic tools for cell biological research.

Microfluidic technology is creating powerful tools for cell biologists to control the complete cellular microenvironment, leading to new questions and new discoveries. We review here the basic concepts and methodologies in designing microfluidic devices, and their diverse cell biological applications.

Time-resolved tracking of a minimum gene expression system reconstituted in giant liposomes.

Individual expression: We describe a method that allows the observation of real-time gene expression in a large number of individual giant liposomes encapsulating identical genetic material. We followed the gene expression profiles from DNA and mRNA templates coding for different proteins. Although the average profiles of individual liposomes were similar to those measured in bulk solution, strong variability between individual liposomes was observed at both transcription and translation.

Preparation of phospholipid multilayer patterns of controlled size and thickness by capillary assembly on a microstructured substrate.

By dragging a phospholipid solution on microstructured silicon surfaces, phospholipid molecules are selectively deposited inside the microstructures to get regular phospholipid multilayer patterns of controlled thickness over a large scale ( approximately cm(2)). By varying the dragging speed, the thickness of the patterns varies between 28 and 100 nm on average (7 to 25 bilayers). Electroswelling of phospholipid multilayer patterns leads to the formation of giant liposomes of controlled size and narrow size distributions.

From convective assembly to Landau-Levich deposition of multilayered phospholipid films of controlled thickness.

In this letter, we describe a method to control the organization and thickness of multilayered phospholipid films. The meniscus of an organic solution of phospholipid molecules was dragged at a speed v on a solid substrate under controlled temperature and forced convection, leading to the deposition of a dried multilayered phospholipid film with a thickness h in the range of 20-200 nm. We found two distinct regimes dominating the film deposition. At low speeds, phospholipid molecules accumulate near the contact line and form a dry film behind the meniscus (evaporation regime). At high speed, viscous forces become predominant and pull out a liquid film that will dry afterward (Landau-Levich regime). Both regimes show robust scaling h infinity v(alpha) with alpha = -1.1 and 0.76, respectively. Although these regimes have been observed separately in the past, they have not been demonstrated in the same material system. Moreover, we present models whose scalings (alpha = -1 and 2/3) are in close agreement with the observed values. The microscale organization of the resulting film is independent of v for a given regime but differs from one regime to another. In the Landau-Levich regime, h is very homogeneous on the microscale with discrete variations of +/- 5 nm, that is, the thickness of one bilayer.