Publications by authors named "Beth L Pruitt"

67 Publications

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state.

Proc Natl Acad Sci U S A 2021 Jun;118(24)

Department of Pediatrics (Cardiology), Stanford University School of Medicine, Palo Alto, CA 94304;

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)-cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease.
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http://dx.doi.org/10.1073/pnas.2025030118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214707PMC
June 2021

Increased tissue stiffness triggers contractile dysfunction and telomere shortening in dystrophic cardiomyocytes.

Stem Cell Reports 2021 May 11. Epub 2021 May 11.

Baxter Laboratory for Stem Cell Biology, Department of Microbiology and Immunology, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CCSR Room 4215, 269 Campus Drive, Stanford, CA 94305-5175, USA; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA, USA. Electronic address:

Duchenne muscular dystrophy (DMD) is a rare X-linked recessive disease that is associated with severe progressive muscle degeneration culminating in death due to cardiorespiratory failure. We previously observed an unexpected proliferation-independent telomere shortening in cardiomyocytes of a DMD mouse model. Here, we provide mechanistic insights using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using traction force microscopy, we show that DMD hiPSC-CMs exhibit deficits in force generation on fibrotic-like bioengineered hydrogels, aberrant calcium handling, and increased reactive oxygen species levels. Furthermore, we observed a progressive post-mitotic telomere shortening in DMD hiPSC-CMs coincident with downregulation of shelterin complex, telomere capping proteins, and activation of the p53 DNA damage response. This telomere shortening is blocked by blebbistatin, which inhibits contraction in DMD cardiomyocytes. Our studies underscore the role of fibrotic stiffening in the etiology of DMD cardiomyopathy. In addition, our data indicate that telomere shortening is progressive, contraction dependent, and mechanosensitive, and suggest points of therapeutic intervention.
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http://dx.doi.org/10.1016/j.stemcr.2021.04.018DOI Listing
May 2021

3D Microwell Platforms for Control of Single Cell 3D Geometry and Intracellular Organization.

Cell Mol Bioeng 2021 Feb 20;14(1):1-14. Epub 2020 Aug 20.

Department of Mechanical Engineering, University of California at Santa Barbara, BioEngineering Building 2002, 494 UCen Rd, Santa Barbara, CA 93106-5170 USA.

Introduction: Cell structure and migration is impacted by the mechanical properties and geometry of the cell adhesive environment. Most studies to date investigating the effects of 3D environments on cells have not controlled geometry at the single-cell level, making it difficult to understand the influence of 3D environmental cues on single cells. Here, we developed microwell platforms to investigate the effects of 2D vs. 3D geometries on single-cell F-actin and nuclear organization.

Methods: We used microfabrication techniques to fabricate three polyacrylamide platforms: 3D microwells with a 3D adhesive environment (/), 3D microwells with 2D adhesive areas at the bottom only (/), and flat 2D gels with 2D patterned adhesive areas (/). We measured geometric swelling and Young's modulus of the platforms. We then cultured C2C12 myoblasts on each platform and evaluated the effects of the engineered microenvironments on F-actin structure and nuclear shape.

Results: We tuned the mechanical characteristics of the microfabricated platforms by manipulating the gel formulation. Crosslinker ratio strongly influenced geometric swelling whereas total polymer content primarily affected Young's modulus. When comparing cells in these platforms, we found significant effects on F-actin and nuclear structures. Our analysis showed that a 3D/3D environment was necessary to increase actin and nuclear height. A 3D/2D environment was sufficient to increase actin alignment and nuclear aspect ratio compared to a 2D/2D environment.

Conclusions: Using our novel polyacrylamide platforms, we were able to decouple the effects of 3D confinement and adhesive environment, finding that both influenced actin and nuclear structure.
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http://dx.doi.org/10.1007/s12195-020-00646-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878614PMC
February 2021

CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes.

Sci Rep 2021 Feb 4;11(1):3026. Epub 2021 Feb 4.

Stanford University, Stanford, CA, USA.

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3' untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.
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http://dx.doi.org/10.1038/s41598-021-81860-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862643PMC
February 2021

Extracellular matrix micropatterning technology for whole cell cryogenic electron microscopy studies.

J Micromech Microeng 2019 Nov 26;29(11). Epub 2019 Sep 26.

Department of Bioengineering, Stanford University, Stanford, California.

Cryogenic electron tomography is the highest resolution tool available for structural analysis of macromolecular organization inside cells. Micropatterning of extracellular matrix (ECM) proteins is an established cell culture technique used to control cell shape. Recent traction force microscopy studies have shown correlation between cell morphology and the regulation of force transmission. However, it remains unknown how cells sustain increased strain energy states and localized stresses at the supramolecular level. Here, we report a technology to enable direct observation of mesoscale organization in epithelial cells under morphological modulation, using a maskless protein photopatterning method (PRIMO) to confine cells to ECM micropatterns on electron microscopy substrates. These micropatterned cell culture substrates can be used in mechanobiology research to correlate changes in nanometer-scale organization at cell-cell and cell-ECM contacts to strain energy states and traction stress distribution in the cell.
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http://dx.doi.org/10.1088/1361-6439/ab419aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457726PMC
November 2019

Touch-induced mechanical strain in somatosensory neurons is independent of extracellular matrix mutations in .

Mol Biol Cell 2020 07 24;31(16):1735-1743. Epub 2020 Jun 24.

Departments of Mechanical Engineering, Stanford University, Stanford, CA 94305.

Cutaneous mechanosensory neurons are activated by mechanical loads applied to the skin, and these stimuli are proposed to generate mechanical strain within sensory neurons. Using a microfluidic device to deliver controlled stimuli to intact animals and large, immobile, and fluorescent protein-tagged mitochondria as fiducial markers in the touch receptor neurons (TRNs), we visualized and measured touch-induced mechanical strain in worms. At steady state, touch stimuli sufficient to activate TRNs induce an average strain of 3.1% at the center of the actuator and this strain decays to near zero at the edges of the actuator. We also measured strain in animals carrying mutations affecting links between the extracellular matrix (ECM) and the TRNs but could not detect any differences in touch-induced mechanical strain between wild-type and mutant animals. Collectively, these results demonstrate that touching the skin induces local mechanical strain in intact animals and suggest that a fully intact ECM is not essential for transmitting mechanical strain from the skin to cutaneous mechanosensory neurons.
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http://dx.doi.org/10.1091/mbc.E20-01-0049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7521855PMC
July 2020

Silencing of ameliorates disease phenotypes in human iPSC-cardiomyocytes.

Physiol Genomics 2020 07 22;52(7):293-303. Epub 2020 Jun 22.

Department of Cardiovascular Medicine, Stanford University, Stanford, California.

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy (HCM) to date have focused on mouse models of disease. We here examine allele-specific silencing in a human-cell model of HCM. We investigate two methods of silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategy's effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. These findings are the first exploration of allele-specific silencing in a human HCM model and provide a foundation for further exploration of silencing as a therapeutic treatment for -mutation-associated cardiomyopathy.
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http://dx.doi.org/10.1152/physiolgenomics.00021.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468691PMC
July 2020

Micromechanobiology: Focusing on the Cardiac Cell-Substrate Interface.

Annu Rev Biomed Eng 2020 06;22:257-284

Department of Mechanical Engineering, University of California, Santa Barbara, California 93106, USA; email:

Engineered, in vitro cardiac cell and tissue systems provide test beds for the study of cardiac development, cellular disease processes, and drug responses in a dish. Much effort has focused on improving the structure and function of engineered cardiomyocytes and heart tissues. However, these parameters depend critically on signaling through the cellular microenvironment in terms of ligand composition, matrix stiffness, and substrate mechanical properties-that is, matrix micromechanobiology. To facilitate improvements to in vitro microenvironment design, we review how cardiomyocytes and their microenvironment change during development and disease in terms of integrin expression and extracellular matrix (ECM) composition. We also discuss strategies used to bind proteins to common mechanobiology platforms and describe important differences in binding strength to the substrate. Finally, we review example biomaterial approaches designed to support and probe cell-ECM interactions of cardiomyocytes in vitro, as well as open questions and challenges.
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http://dx.doi.org/10.1146/annurev-bioeng-092019-034950DOI Listing
June 2020

Mechanobiology Assays with Applications in Cardiomyocyte Biology and Cardiotoxicity.

Adv Healthc Mater 2020 04 9;9(8):e1901656. Epub 2020 Apr 9.

Department of Mechanical Engineering, University of California Santa Barbara, Santa Barbara, CA, 93109, USA.

Cardiomyocytes are the motor units that drive the contraction and relaxation of the heart. Traditionally, testing of drugs for cardiotoxic effects has relied on primary cardiomyocytes from animal models and focused on short-term, electrophysiological, and arrhythmogenic effects. However, primary cardiomyocytes present challenges arising from their limited viability in culture, and tissue from animal models suffers from a mismatch in their physiology to that of human heart muscle. Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) can address these challenges. They also offer the potential to study not only electrophysiological effects but also changes in cardiomyocyte contractile and mechanical function in response to cardiotoxic drugs. With growing recognition of the long-term cardiotoxic effects of some drugs on subcellular structure and function, there is increasing interest in using hiPSC-CMs for in vitro cardiotoxicity studies. This review provides a brief overview of techniques that can be used to quantify changes in the active force that cardiomyocytes generate and variations in their inherent stiffness in response to cardiotoxic drugs. It concludes by discussing the application of these tools in understanding how cardiotoxic drugs directly impact the mechanobiology of cardiomyocytes and how cardiomyocytes sense and respond to mechanical load at the cellular level.
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http://dx.doi.org/10.1002/adhm.201901656DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7480481PMC
April 2020

Producing Collagen Micro-stripes with Aligned Fibers for Cell Migration Assays.

Cell Mol Bioeng 2020 Feb 25;13(1):87-98. Epub 2019 Sep 25.

Laboratory for Complex Fluids and Interfaces, Mechanobiology and Soft Matter Group, Research Institute for Biosciences, University of Mons, Place du Parc, 20, 7000 Mons, Belgium.

Introduction: The orientation of collagen fibers in native tissues plays an important role in cell signaling and mediates the progression of tumor cells in breast cancer by a contact guidance mechanism. Understanding how migration of epithelial cells is directed by the alignment of collagen fibers requires assays with standardized orientations of collagen fibers.

Methods: To address this issue, we produced micro-stripes with aligned collagen fibers using an easy-to-use and versatile approach based on the aspiration of a collagen solution within a microchannel. Glass coverslips were functionalized with a (3-aminopropyl)triethoxysilane/glutaraldehyde linkage to covalently anchor micro-stripes of aligned collagen fibers, whereas microchannels were functionalized with a poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nonionic triblock polymer to prevent adhesion of the collagen micro-stripes.

Results: Using this strategy, microchannels can be peeled off to expose micro-stripes of aligned collagen fibers without affecting their mechanical integrity. We used time-lapse confocal reflection microscopy to characterize the polymerization kinetics of collagen networks for different concentrations and the orientation of collagen fibers as a function of the microchannel width. Our results indicate a non-linear concentration dependence of the area of fluorescence, suggesting that the architecture of collagen networks is sensitive to small changes in concentration. We show the possibility to influence the collagen fibril coverage by adjusting the concentration of the collagen solution.

Conclusion: We applied this novel approach to study the migration of epithelial cells, demonstrating that collagen micro-stripes with aligned fibers represent a valuable assay for studying cell contact guidance mechanisms.
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http://dx.doi.org/10.1007/s12195-019-00600-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6981333PMC
February 2020

Controlled phage therapy by photothermal ablation of specific bacterial species using gold nanorods targeted by chimeric phages.

Proc Natl Acad Sci U S A 2020 01 13;117(4):1951-1961. Epub 2020 Jan 13.

Department of Chemistry and Biochemistry, University of California, Santa Barbara, CA 93106;

The use of bacteriophages (phages) for antibacterial therapy is under increasing consideration to treat antimicrobial-resistant infections. Phages have evolved multiple mechanisms to target their bacterial hosts, such as high-affinity, environmentally hardy receptor-binding proteins. However, traditional phage therapy suffers from multiple challenges stemming from the use of an exponentially replicating, evolving entity whose biology is not fully characterized (e.g., potential gene transduction). To address this problem, we conjugate the phages to gold nanorods, creating a reagent that can be destroyed upon use (termed "phanorods"). Chimeric phages were engineered to attach specifically to several Gram-negative organisms, including the human pathogens , , and , and the plant pathogen The bioconjugated phanorods could selectively target and kill specific bacterial cells using photothermal ablation. Following excitation by near-infrared light, gold nanorods release energy through nonradiative decay pathways, locally generating heat that efficiently kills targeted bacterial cells. Specificity was highlighted in the context of a biofilm, in which phanorod irradiation killed bacterial cells while causing minimal damage to epithelial cells. Local temperature and viscosity measurements revealed highly localized and selective ablation of the bacteria. Irradiation of the phanorods also destroyed the phages, preventing replication and reducing potential risks of traditional phage therapy while enabling control over dosing. The phanorod strategy integrates the highly evolved targeting strategies of phages with the photothermal properties of gold nanorods, creating a well-controlled platform for systematic killing of bacterial cells.
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http://dx.doi.org/10.1073/pnas.1913234117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994977PMC
January 2020

Somatosensory neurons integrate the geometry of skin deformation and mechanotransduction channels to shape touch sensing.

Elife 2019 08 13;8. Epub 2019 Aug 13.

Department of Physics, University of California, San Diego, La Jolla, United States.

Touch sensation hinges on force transfer across the skin and activation of mechanosensitive ion channels along the somatosensory neurons that invade the skin. This skin-nerve sensory system demands a quantitative model that spans the application of mechanical loads to channel activation. Unlike prior models of the dynamic responses of touch receptor neurons in (Eastwood et al., 2015), which substituted a single effective channel for the ensemble along the TRNs, this study integrates body mechanics and the spatial recruitment of the various channels. We demonstrate that this model captures mechanical properties of the worm's body and accurately reproduces neural responses to simple stimuli. It also captures responses to complex stimuli featuring non-trivial spatial patterns, like extended or multiple contacts that could not be addressed otherwise. We illustrate the importance of these effects with new experiments revealing that skin-neuron composites respond to pre-indentation with increased currents rather than adapting to persistent stimulation.
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http://dx.doi.org/10.7554/eLife.43226DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692131PMC
August 2019

Allele-Specific Silencing Ameliorates Restrictive Cardiomyopathy Attributable to a Human Myosin Regulatory Light Chain Mutation.

Circulation 2019 08 18;140(9):765-778. Epub 2019 Jul 18.

Division of Cardiovascular Medicine (K.Z.-R., A.D., P.C., G.R., C.S., J.L., T.F., W.N.P., S.S., K.S., N.S., N.J., Y.H., M.T.W., E.A.A.), Stanford University School of Medicine, CA.

Background: Restrictive cardiomyopathy is a rare heart disease associated with mutations in sarcomeric genes and with phenotypic overlap with hypertrophic cardiomyopathy. There is no approved therapy directed at the underlying cause. Here, we explore the potential of an interfering RNA (RNAi) therapeutic for a human sarcomeric mutation in MYL2 causative of restrictive cardiomyopathy in a mouse model.

Methods: A short hairpin RNA (M7.8L) was selected from a pool for specificity and efficacy. Two groups of myosin regulatory light chain N47K transgenic mice were injected with M7.8L packaged in adeno-associated virus 9 at 3 days of age and 60 days of age. Mice were subjected to treadmill exercise and echocardiography after treatment to determine maximal oxygen uptake and left ventricular mass. At the end of treatment, heart, lung, liver, and kidney tissue was harvested to determine viral tropism and for transcriptomic and proteomic analysis. Cardiomyocytes were isolated for single-cell studies.

Results: A one-time injection of AAV9-M7.8L RNAi in 3-day-old humanized regulatory light chain mutant transgenic mice silenced the mutated allele (RLC-47K) with minimal effects on the normal allele (RLC-47N) assayed at 16 weeks postinjection. AAV9-M7.8L RNAi suppressed the expression of hypertrophic biomarkers, reduced heart weight, and attenuated a pathological increase in left ventricular mass. Single adult cardiac myocytes from mice treated with AAV9-M7.8L showed partial restoration of contraction, relaxation, and calcium kinetics. In addition, cardiac stress protein biomarkers, such as calmodulin-dependent protein kinase II and the transcription activator Brg1 were reduced, suggesting recovery toward a healthy myocardium. Transcriptome analyses further revealed no significant changes of argonaute (AGO1, AGO2) and endoribonuclease dicer (DICER1) transcripts, and endogenous microRNAs were preserved, suggesting that the RNAi pathway was not saturated.

Conclusions: Our results show the feasibility, efficacy, and safety of RNAi therapeutics directed towards human restrictive cardiomyopathy. This is a promising step toward targeted therapy for a prevalent human disease.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.036965DOI Listing
August 2019

Big bottlenecks in cardiovascular tissue engineering.

Commun Biol 2018 21;1:199. Epub 2018 Nov 21.

Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, 94305, CA, USA.

Although tissue engineering using human-induced pluripotent stem cells is a promising approach for treatment of cardiovascular diseases, some limiting factors include the survival, electrical integration, maturity, scalability, and immune response of three-dimensional (3D) engineered tissues. Here we discuss these important roadblocks facing the tissue engineering field and suggest potential approaches to overcome these challenges.
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http://dx.doi.org/10.1038/s42003-018-0202-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249300PMC
November 2018

Shear-induced damped oscillations in an epithelium depend on actomyosin contraction and E-cadherin cell adhesion.

Elife 2018 11 14;7. Epub 2018 Nov 14.

Department of Bioengineering, Stanford University, Stanford, United States.

Shear forces between cells occur during global changes in multicellular organization during morphogenesis and tissue growth, yet how cells sense shear forces and propagate a response across a tissue is unknown. We found that applying exogenous shear at the midline of an epithelium induced a local, short-term deformation near the shear plane, and a long-term collective oscillatory movement across the epithelium that spread from the shear-plane and gradually dampened. Inhibiting actomyosin contraction or E-cadherin -cell adhesion blocked oscillations, whereas stabilizing actin filaments prolonged oscillations. Combining these data with a model of epithelium mechanics supports a mechanism involving the generation of a shear-induced mechanical event at the shear plane which is then relayed across the epithelium by actomyosin contraction linked through E-cadherin. This causes an imbalance of forces in the epithelium, which is gradually dissipated through oscillatory cell movements and actin filament turnover to restore the force balance across the epithelium.
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http://dx.doi.org/10.7554/eLife.39640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235569PMC
November 2018

The bodies of are twice as stiff as wild type.

MicroPubl Biol 2018 Aug 6;2018. Epub 2018 Aug 6.

Molecular and Cellular Physiology, Stanford School of Medicine, Stanford.

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http://dx.doi.org/10.17912/ecsm-mp67DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7282522PMC
August 2018

Microfluidics for mechanobiology of model organisms.

Methods Cell Biol 2018 14;146:217-259. Epub 2018 Jul 14.

University of California, Santa Barbara, CA, United States; Stanford University, Stanford, CA, United States. Electronic address:

Mechanical stimuli play a critical role in organ development, tissue homeostasis, and disease. Understanding how mechanical signals are processed in multicellular model systems is critical for connecting cellular processes to tissue- and organism-level responses. However, progress in the field that studies these phenomena, mechanobiology, has been limited by lack of appropriate experimental techniques for applying repeatable mechanical stimuli to intact organs and model organisms. Microfluidic platforms, a subgroup of microsystems that use liquid flow for manipulation of objects, are a promising tool for studying mechanobiology of small model organisms due to their size scale and ease of customization. In this work, we describe design considerations involved in developing a microfluidic device for studying mechanobiology. Then, focusing on worms, fruit flies, and zebrafish, we review current microfluidic platforms for mechanobiology of multicellular model organisms and their tissues and highlight research opportunities in this developing field.
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http://dx.doi.org/10.1016/bs.mcb.2018.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418080PMC
December 2018

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans.

J Vis Exp 2018 02 19(132). Epub 2018 Feb 19.

Group of Neurophotonics and Mechanical Systems Biology, The Institute of Photonic Sciences (ICFO);

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.
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http://dx.doi.org/10.3791/56530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5931315PMC
February 2018

Controlling cell shape on hydrogels using lift-off protein patterning.

PLoS One 2018 3;13(1):e0189901. Epub 2018 Jan 3.

Department of Mechanical Engineering, Stanford University, Stanford, California, United States of America.

Polyacrylamide gels functionalized with extracellular matrix proteins are commonly used as cell culture platforms to evaluate the combined effects of extracellular matrix composition, cell geometry and substrate rigidity on cell physiology. For this purpose, protein transfer onto the surface of polyacrylamide hydrogels must result in geometrically well-resolved micropatterns with homogeneous protein distribution. Yet the outcomes of micropatterning methods have not been pairwise evaluated against these criteria. We report a high-fidelity photoresist lift-off patterning method to pattern ECM proteins on polyacrylamide hydrogels with elastic moduli ranging from 5 to 25 kPa. We directly compare the protein transfer efficiency and pattern geometrical accuracy of this protocol to the widely used microcontact printing method. Lift-off patterning achieves higher protein transfer efficiency, increases pattern accuracy, increases pattern yield, and reduces variability of these factors within arrays of patterns as it bypasses the drying and transfer steps of microcontact printing. We demonstrate that lift-off patterned hydrogels successfully control cell size and shape and enable long-term imaging of actin intracellular structure and lamellipodia dynamics when we culture epithelial cells on these substrates.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0189901PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752030PMC
January 2018

A BAG3 chaperone complex maintains cardiomyocyte function during proteotoxic stress.

JCI Insight 2017 Jul 20;2(14). Epub 2017 Jul 20.

Gladstone Institutes, San Francisco, California, USA.

Molecular chaperones regulate quality control in the human proteome, pathways that have been implicated in many diseases, including heart failure. Mutations in the BAG3 gene, which encodes a co-chaperone protein, have been associated with heart failure due to both inherited and sporadic dilated cardiomyopathy. Familial BAG3 mutations are autosomal dominant and frequently cause truncation of the coding sequence, suggesting a heterozygous loss-of-function mechanism. However, heterozygous knockout of the murine BAG3 gene did not cause a detectable phenotype. To model BAG3 cardiomyopathy in a human system, we generated an isogenic series of human induced pluripotent stem cells (iPSCs) with loss-of-function mutations in BAG3. Heterozygous BAG3 mutations reduced protein expression, disrupted myofibril structure, and compromised contractile function in iPSC-derived cardiomyocytes (iPS-CMs). BAG3-deficient iPS-CMs were particularly sensitive to further myofibril disruption and contractile dysfunction upon exposure to proteasome inhibitors known to cause cardiotoxicity. We performed affinity tagging of the endogenous BAG3 protein and mass spectrometry proteomics to further define the cardioprotective chaperone complex that BAG3 coordinates in the human heart. Our results establish a model for evaluating protein quality control pathways in human cardiomyocytes and their potential as therapeutic targets and susceptibility factors for cardiac drug toxicity.
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http://dx.doi.org/10.1172/jci.insight.94623DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518554PMC
July 2017

Changes in E-cadherin rigidity sensing regulate cell adhesion.

Proc Natl Acad Sci U S A 2017 07 3;114(29):E5835-E5844. Epub 2017 Jul 3.

Department of Biology, Stanford University, Stanford, CA 94305;

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.
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http://dx.doi.org/10.1073/pnas.1618676114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530647PMC
July 2017

E-cadherin and LGN align epithelial cell divisions with tissue tension independently of cell shape.

Proc Natl Acad Sci U S A 2017 07 3;114(29):E5845-E5853. Epub 2017 Jul 3.

Department of Biology, Stanford University, Stanford, CA 94305;

Tissue morphogenesis requires the coordinated regulation of cellular behavior, which includes the orientation of cell division that defines the position of daughter cells in the tissue. Cell division orientation is instructed by biochemical and mechanical signals from the local tissue environment, but how those signals control mitotic spindle orientation is not fully understood. Here, we tested how mechanical tension across an epithelial monolayer is sensed to orient cell divisions. Tension across Madin-Darby canine kidney cell monolayers was increased by a low level of uniaxial stretch, which oriented cell divisions with the stretch axis irrespective of the orientation of the cell long axis. We demonstrate that stretch-induced division orientation required mechanotransduction through E-cadherin cell-cell adhesions. Increased tension on the E-cadherin complex promoted the junctional recruitment of the protein LGN, a core component of the spindle orientation machinery that binds the cytosolic tail of E-cadherin. Consequently, uniaxial stretch triggered a polarized cortical distribution of LGN. Selective disruption of engagement of E-cadherin in an otherwise cohesive cell monolayer, or loss of LGN expression, resulted in randomly oriented cell divisions in the presence of uniaxial stretch. Our findings indicate that E-cadherin plays a key role in sensing polarized tensile forces across the tissue and transducing this information to the spindle orientation machinery to align cell divisions.
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http://dx.doi.org/10.1073/pnas.1701703114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5530667PMC
July 2017

Forces applied during classical touch assays for Caenorhabditis elegans.

PLoS One 2017 19;12(5):e0178080. Epub 2017 May 19.

Mechanical Engineering Department, Stanford University, Stanford, California, United States of America.

For decades, Caenorhabditis elegans roundworms have been used to study the sense of touch, and this work has been facilitated by a simple behavioral assay for touch sensation. To perform this classical assay, an experimenter uses an eyebrow hair to gently touch a moving worm and observes whether or not the worm reverses direction. We used two experimental approaches to determine the manner and moment of contact between the eyebrow hair tool and freely moving animals and the forces delivered by the classical assay. Using high-speed video (2500 frames/second), we found that typical stimulus delivery events include a brief moment when the hair is contact with the worm's body and not the agar substrate. To measure the applied forces, we measured forces generated by volunteers mimicking the classical touch assay by touching a calibrated microcantilever. The mean (61 μN) and median forces (26 μN) were more than ten times higher than the 2-μN force known to saturate the probability of evoking a reversal in adult C. elegans. We also considered the eyebrow hairs as an additional source of variation. The stiffness of the sampled eyebrow hairs varied between 0.07 and 0.41 N/m and was correlated with the free length of hair. Collectively, this work establishes that the classical touch assay applies enough force to saturate the probability of evoking reversals in adult C. elegans in spite of its variability among trials and experimenters and that increasing the free length of the hair can decrease the applied force.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178080PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5438190PMC
October 2017

Multi-Imaging Method to Assay the Contractile Mechanical Output of Micropatterned Human iPSC-Derived Cardiac Myocytes.

Circ Res 2017 May 11;120(10):1572-1583. Epub 2017 Apr 11.

From the Department of Mechanical Engineering (A.J.S.R., O.S., B.L.P.), Department of Molecular and Cellular Physiology (by courtesy) (B.L.P.), Department of Bioengineering (by courtesy) (B.L.P.), and Stanford Cardiovascular Institute (A.J.S.R., B.L.P.), Stanford University, CA; Gladstone Institute of Cardiovascular Disease, San Francisco, CA (A.J.S.R., M.A.M., Y.-S.A., B.R.C., D.S.); Roddenberry Stem Cell Center at Gladstone, San Francisco, CA (Y.-S.A., D.S.); Departments of Pediatrics and Biochemistry & Biophysics (D.S.), Department of Cellular and Molecular Pharmacology (B.R.C.), California Institute for Quantitative Biosciences, QB3 (B.R.C.), and Department of Medicine and Cellular and Molecular Pharmacology (B.R.C.), University of California, San Francisco.

Rationale: During each beat, cardiac myocytes (CMs) generate the mechanical output necessary for heart function through contractile mechanisms that involve shortening of sarcomeres along myofibrils. Human-induced pluripotent stem cells (hiPSCs) can be differentiated into CMs (hiPSC-CMs) that model cardiac contractile mechanical output more robustly when micropatterned into physiological shapes. Quantifying the mechanical output of these cells enables us to assay cardiac activity in a dish.

Objective: We sought to develop a computational platform that integrates analytic approaches to quantify the mechanical output of single micropatterned hiPSC-CMs from microscopy videos.

Methods And Results: We micropatterned single hiPSC-CMs on deformable polyacrylamide substrates containing fluorescent microbeads. We acquired videos of single beating cells, of microbead displacement during contractions, and of fluorescently labeled myofibrils. These videos were independently analyzed to obtain parameters that capture the mechanical output of the imaged single cells. We also developed novel methods to quantify sarcomere length from videos of moving myofibrils and to analyze loss of synchronicity of beating in cells with contractile defects. We tested this computational platform by detecting variations in mechanical output induced by drugs and in cells expressing low levels of myosin-binding protein C.

Conclusions: Our method can measure the cardiac function of single micropatterned hiPSC-CMs and determine contractile parameters that can be used to elucidate mechanisms that underlie variations in CM function. This platform will be amenable to future studies of the effects of mutations and drugs on cardiac function.
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http://dx.doi.org/10.1161/CIRCRESAHA.116.310363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491345PMC
May 2017

Pneumatic stimulation of C. elegans mechanoreceptor neurons in a microfluidic trap.

Lab Chip 2017 03;17(6):1116-1127

Department of Molecular and Cellular Physiology, Stanford University, Stanford, California, USA.

New tools for applying force to animals, tissues, and cells are critically needed in order to advance the field of mechanobiology, as few existing tools enable simultaneous imaging of tissue and cell deformation as well as cellular activity in live animals. Here, we introduce a novel microfluidic device that enables high-resolution optical imaging of cellular deformations and activity while applying precise mechanical stimuli to the surface of the worm's cuticle with a pneumatic pressure reservoir. To evaluate device performance, we compared analytical and numerical simulations conducted during the design process to empirical measurements made with fabricated devices. Leveraging the well-characterized touch receptor neurons (TRNs) with an optogenetic calcium indicator as a model mechanoreceptor neuron, we established that individual neurons can be stimulated and that the device can effectively deliver steps as well as more complex stimulus patterns. This microfluidic device is therefore a valuable platform for investigating the mechanobiology of living animals and their mechanosensitive neurons.
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http://dx.doi.org/10.1039/c6lc01165aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5360562PMC
March 2017

Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.

Cell 2016 Dec;167(7):1734-1749.e22

Gladstone Institute of Cardiovascular Disease and Roddenberry Center for Stem Cell Biology and Medicine, San Francisco, CA 94158, USA; Department of Pediatrics, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94143, USA. Electronic address:

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.
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http://dx.doi.org/10.1016/j.cell.2016.11.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5180611PMC
December 2016

Increasing β-catenin/Wnt3A activity levels drive mechanical strain-induced cell cycle progression through mitosis.

Elife 2016 10 26;5. Epub 2016 Oct 26.

Program in Cancer Biology, Stanford University, Stanford, United States.

Mechanical force and Wnt signaling activate β-catenin-mediated transcription to promote proliferation and tissue expansion. However, it is unknown whether mechanical force and Wnt signaling act independently or synergize to activate β-catenin signaling and cell division. We show that mechanical strain induced Src-dependent phosphorylation of Y654 β-catenin and increased β-catenin-mediated transcription in mammalian MDCK epithelial cells. Under these conditions, cells accumulated in S/G2 (independent of DNA damage) but did not divide. Activating β-catenin through Casein Kinase I inhibition or Wnt3A addition increased β-catenin-mediated transcription and strain-induced accumulation of cells in S/G2. Significantly, only the combination of mechanical strain and Wnt/β-catenin activation triggered cells in S/G2 to divide. These results indicate that strain-induced Src phosphorylation of β-catenin and Wnt-dependent β-catenin stabilization synergize to increase β-catenin-mediated transcription to levels required for mitosis. Thus, local Wnt signaling may fine-tune the effects of global mechanical strain to restrict cell divisions during tissue development and homeostasis.
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http://dx.doi.org/10.7554/eLife.19799DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5104517PMC
October 2016

Single Molecule Force Measurements in Living Cells Reveal a Minimally Tensioned Integrin State.

ACS Nano 2016 12 28;10(12):10745-10752. Epub 2016 Nov 28.

Stanford Cardiovascular Institute, Stanford University , 265 Campus Drive, Stanford, California 94305, United States.

Integrins mediate cell adhesion to the extracellular matrix and enable the construction of complex, multicellular organisms, yet fundamental aspects of integrin-based adhesion remain poorly understood. Notably, the magnitude of the mechanical load experienced by individual integrins within living cells is unclear, due principally to limitations inherent to existing techniques. Here we use Förster resonance energy transfer-based molecular tension sensors to directly measure the distribution of loads experienced by individual integrins in living cells. We find that a large fraction of integrins bear modest loads of 1-3 pN, while subpopulations bearing higher loads are enriched within adhesions. Further, our data indicate that integrin engagement with the fibronectin synergy site, a secondary binding site specifically for αβ integrin, leads to increased levels of αβ integrin recruitment to adhesions but not to an increase in overall cellular traction generation. The presence of the synergy site does, however, increase cells' resistance to detachment by externally applied loads. We suggest that a substantial population of integrins experiencing loads well below their peak capacities can provide cells and tissues with mechanical integrity in the presence of widely varying mechanical loads.
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http://dx.doi.org/10.1021/acsnano.6b03314DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886374PMC
December 2016

Tuning the Range of Polyacrylamide Gel Stiffness for Mechanobiology Applications.

ACS Appl Mater Interfaces 2016 08 27;8(34):21893-902. Epub 2016 Jan 27.

Department of Mechanical Engineering, Stanford University , Building 530 440 Escondido Mall, Stanford, California 94305, United States.

Adjusting the acrylamide monomer and cross-linker content in polyacrylamide gels controls the hydrogel stiffness, yet the reported elastic modulus for the same formulations varies widely and these discrepancies are frequently attributed to different measurement methods. Few studies exist that examine stiffness trends across monomer and cross-linker concentrations using the same characterization platform. In this work, we use Atomic Force Microscopy and analyze force-distance curves to derive the elastic modulus of polyacrylamide hydrogels. We find that gel elastic modulus increases with increasing cross-link concentration until an inflection point, after which gel stiffness decreases with increasing cross-linking. This behavior arises because of the formation of highly cross-linked clusters, which add inhomogeneity and heterogeneity to the network structure, causing the global network to soften even under high cross-linking conditions. We identify these inflection points for three different total polymer formulations. When we alter gelation kinetics by using a low polymerization temperature, we find that gels are stiffer when polymerized at 4 °C compared to room temperature, indicating a complex relationship between gel structure, elasticity, and network formation. We also investigate how gel stiffness changes during storage over 10 days and find that specific gel formulations undergo significant stiffening (1.55 ± 0.13), which may be explained by differences in gel swelling resulting from initial polymerization parameters. Taken together, our study emphasizes the importance of polyacrylamide formulation, polymerization temperature, gelation time, and storage duration in defining the structural and mechanical properties of the polyacrylamide hydrogels.
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http://dx.doi.org/10.1021/acsami.5b09344DOI Listing
August 2016

Time-dependent evolution of functional vs. remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.

FASEB J 2016 Apr 16;30(4):1464-79. Epub 2015 Dec 16.

*Division of Cardiology, Department of Pediatrics, Stanford Cardiovascular Institute, Division of Cardiovascular Medicine, Department of Medicine, and Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, California, USA; Department of Mechanical Engineering, Stanford University School of Engineering, Stanford, California, USA; and Departments of Pharmacology and Medicine, University of California, San Diego, San Diego, California, USA

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are a powerful platform for uncovering disease mechanisms and assessing drugs for efficacy/toxicity. However, the accuracy with which hiPSC-CMs recapitulate the contractile and remodeling signaling of adult cardiomyocytes is not fully known. We used β-adrenergic receptor (β-AR) signaling as a prototype to determine the evolution of signaling component expression and function during hiPSC-CM maturation. In "early" hiPSC-CMs (less than or equal to d 30), β2-ARs are a primary source of cAMP/PKA signaling. With longer culture, β1-AR signaling increases: from 0% of cAMP generation at d 30 to 56.8 ± 6.6% by d 60. PKA signaling shows a similar increase: 15.7 ± 5.2% (d 30), 49.8 ± 0.5% (d 60), and 71.0 ± 6.1% (d 90). cAMP generation increases 9-fold from d 30 to 60, with enhanced coupling to remodeling pathways (e.g., Akt and Ca(2+)/calmodulin-dependent protein kinase type II) and development of caveolin-mediated signaling compartmentalization. By contrast, cardiotoxicity induced by chronic β-AR stimulation, a major component of heart failure, develops much later: 5% cell death at d 30vs 55% at d 90. Moreover, β-AR maturation can be accelerated by biomechanical stimulation. The differential maturation of β-AR functionalvs remodeling signaling in hiPSC-CMs has important implications for their use in disease modeling and drug testing. We propose that assessment of signaling be added to the indices of phenotypic maturation of hiPSC-CMs.-Jung, G., Fajardo, G., Ribeiro, A. J. S., Kooiker, K. B., Coronado, M., Zhao, M., Hu, D.-Q., Reddy, S., Kodo, K., Sriram, K., Insel, P. A., Wu, J. C., Pruitt, B. L., Bernstein, D. Time-dependent evolution of functionalvs remodeling signaling in induced pluripotent stem cell-derived cardiomyocytes and induced maturation with biomechanical stimulation.
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http://dx.doi.org/10.1096/fj.15-280982DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4799510PMC
April 2016
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