Christopher M. Hale

Background: Current heart failure therapies unload the failing heart without targeting the underlying problem of reduced cardiac contractility. Traditional inotropes (ie, calcitropes) stimulate contractility via energetically costly augmentation of calcium cycling and worsen patient survival. A new class of agents—myotropes—activates the sarcomere directly, independent of calcium. We hypothesize that a novel myotrope TA1 increases contractility without the deleterious myocardial energetic impact of a calcitrope dobutamine. Methods: We determined the effect of TA1 in bovine cardiac myofibrils and human cardiac microtissues, ex vivo in mouse cardiac fibers and in vivo in anesthetized normal rats. Effects of increasing concentrations of TA1 or dobutamine on contractile function, phosphocreatine and ATP concentrations, and ATP production were assessed by ³¹ P nuclear magnetic resonance spectroscopy on isolated perfused rat hearts. Results: TA1 increased the rate of myosin ATPase activity in isolated bovine myofibrils and calcium sensitivity in intact mouse papillary fibers. Contractility increased dose dependently in human cardiac microtissues and in vivo in rats as assessed by echocardiography. In isolated rat hearts, TA1 and dobutamine similarly increased the rate-pressure product. Dobutamine increased both developed pressure and heart rate accompanied by decreased phosphocreatine-to-ATP ratio and decreased free energy of ATP hydrolysis (ΔG _~ATP ) and elevated left ventricular end diastolic pressure. In contrast, the TA1 increased developed pressure without any effect on heart rate, left ventricular end diastolic pressure, phosphocreatine/ATP ratio, or ΔG _~ATP . Conclusions: Novel myotrope TA1 increased myocardial contractility by sensitizing the sarcomere to calcium without impairing diastolic function or depleting the cardiac energy reserve. Since energetic depletion negatively correlates with long-term survival, myotropes may represent a superior alternative to traditional inotropes in heart failure management.

Quantitative phase imaging (QPI) is an ideal method to non-invasively monitor cell populations and provide label-free imaging and analysis. QPI offers enhanced sample characterization and cell counting compared to conventional label-free techniques. We demonstrate this in the current study through a comparison of cell counting data from digital phase contrast (DPC) imaging and from QPI using a system based on Fourier ptychographic microscopy (FPM). Our FPM system offers multi-well, parallel imaging and a QPI-specific cell segmentation method to establish automated and reliable cell counting. Three cell types were studied and FPM showed improvement in the ability to resolve fine details and thin cells, despite limitations of the FPM system incurred by imaging artifacts. Relative to manually counted fluorescence ground-truth, cell counting results after automated segmentation showed improved accuracy with QPI over DPC.

To accelerate the cardiac drug discovery pipeline, we set out to develop a platform that would be capable of quantifying tissue-level functions such as contractile force and be amenable to standard multiwell-plate manipulations. We report a 96-well-based array of 3D human pluripotent stem cell (hPSC)-derived cardiac microtissues - termed Cardiac MicroRings (CaMiRi) - in custom 3D-print-molded multiwell plates capable of contractile force measurement. Within each well, two elastomeric microcantilevers are situated above a circumferential ramp. The wells are seeded with cell-laden collagen, which, in response to the gradual slope of the circumferential ramp, self-organizes around tip-gated microcantilevers to form contracting CaMiRi. The contractile force exerted by the CaMiRi is measured and calculated using the deflection of the cantilevers. Platform responses were robust and comparable across wells, and we used it to determine an optimal tissue formulation. We validated the contractile force response of CaMiRi using selected cardiotropic compounds with known effects. Additionally, we developed automated protocols for CaMiRi seeding, image acquisition, and analysis to enable the measurement of contractile force with increased throughput. The unique tissue fabrication properties of the platform, and the consequent effects on tissue function, were demonstrated upon adding hPSC-derived epicardial cells to the system. This platform represents an open-source contractile force screening system useful for drug screening and tissue engineering applications.

We report the implementation of a parallel microscopy system (96 Eyes) that is capable of simultaneous imaging of all wells on a 96-well plate. The optical system consists of 96 microscopy units, where each unit is made out of a four element objective, made through a molded injection process, and a low cost CMOS camera chip. By illuminating the sample with angle varying light and applying Fourier Ptychography, we can improve the effective brightfield imaging numerical aperture of the objectives from 0.23 to 0.3, and extend the depth of field from ±5 μm to ±15 μm. The use of Fourier Ptychography additionally allows us to computationally correct the objectives’ aberrations out of the rendered images, and provides us with the ability to render phase images. The 96 Eyes acquires raw data at a rate of 0.7 frame per second (all wells) and the data are processed with 4 cores of graphical processing units (GPUs; GK210, Nvidia Tesla K80, USA). The system is also capable of fluorescence imaging (excitation = 465 nm, emission = 510 nm) at the native resolution of the objectives. We demonstrate the capability of this system by imaging S1P₁-eGFP-Human bone osteosarcoma epithelial (U2OS) cells.

Cells often migrate in vivo in an extracellular matrix that is intrinsically three-dimensional (3D) and the role of actin filament architecture in 3D cell migration is less well understood. Here we show that, while recently identified linkers of nucleoskeleton to cytoskeleton (LINC) complexes play a minimal role in conventional 2D migration, they play a critical role in regulating the organization of a subset of actin filament bundles – the perinuclear actin cap - connected to the nucleus through Nesprin2giant and Nesprin3 in cells in 3D collagen I matrix. Actin cap fibers prolong the nucleus and mediate the formation of pseudopodial protrusions, which drive matrix traction and 3D cell migration. Disruption of LINC complexes disorganizes the actin cap, which impairs 3D cell migration. A simple mechanical model explains why LINC complexes and the perinuclear actin cap are essential in 3D migration by providing mechanical support to the formation of pseudopodial protrusions.

High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories of the cytoplasm-embedded particles are transformed into mean-squared displacements, which are subsequently transformed into frequency-dependent viscoelastic moduli and time-dependent creep compliance of the cytoplasm. This method allows for the study of a wide range of cellular conditions, including cells inside a 3D matrix, cell subjected to shear flows and biochemical stimuli, and cells in a live animal. Ballistic injection lasts <1 min and is followed by overnight incubation. Multiple particle tracking for one cell lasts <1 min. Forty cells can be examined in <1 h.

In several migratory cells, the microtubule-organizing center (MTOC) is repositioned between the leading edge and nucleus, creating a polarized morphology. Although our understanding of polarization has progressed as a result of various scratch-wound and cell migration studies, variations in culture conditions required for such assays have prevented a unified understanding of the intricacies of MTOC and nucleus positioning that result in cell polarization. Here, we employ a new SMRT (for sparse, monolayer, round, triangular) analysis that uses a universal coordinate system based on cell centroid to examine the pathways regulating MTOC and nuclear positions in cells plated in a variety of conditions. We find that MTOC and nucleus positioning are crucially and independently affected by cell shape and confluence; MTOC off-centering correlates with the polarization of single cells; acto-myosin contractility and microtubule dynamics are required for single-cell polarization; and end binding protein 1 and light intermediate chain 1, but not Par3 and light intermediate chain 2, are required for single-cell polarization and directional cell motility. Using various cellular geometries and conditions, we implement a systematic and reproducible approach to identify regulators of MTOC and nucleus positioning that depend on extracellular guidance cues.