Dual inhibition of MAPK and PI3K/AKT pathways enhances maturation of human iPSC-derived cardiomyocytes.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) provide great opportunities for mechanistic dissection of human cardiac pathophysiology; however, hiPSC-CMs remain immature relative to the adult heart. To identify novel signaling pathways driving the maturation process during heart development, we analyzed published transcriptional and epigenetic datasets from hiPSC-CMs and prenatal and postnatal human hearts. These analyses revealed that several components of the MAPK and PI3K-AKT pathways are downregulated in the postnatal heart. Here, we show that dual inhibition of these pathways for only 5 days significantly enhances the maturation of day 30 hiPSC-CMs in many domains: hypertrophy, multinucleation, metabolism, T-tubule density, calcium handling, and electrophysiology, many equivalent to day 60 hiPSC-CMs. These data indicate that the MAPK/PI3K/AKT pathways are involved in cardiomyocyte maturation and provide proof of concept for the manipulation of key signaling pathways for optimal hiPSC-CM maturation, a critical aspect of faithful in vitro modeling of cardiac pathologies and subsequent drug discovery.

Cellular Microbiaxial Stretching Assay for Measurement and Characterization of the Anisotropic Mechanical Properties of Micropatterned Cells.

Characterizing the mechanical properties of single cells is important for developing descriptive models of tissue mechanics and improving the understanding of mechanically driven cell processes. Standard methods for measuring single-cell mechanical properties typically provide isotropic mechanical descriptions. However, many cells exhibit specialized geometries in vivo, with anisotropic cytoskeletal architectures reflective of their function, and are exposed to dynamic multiaxial loads, raising the need for more complete descriptions of their anisotropic mechanical properties under complex deformations. Here, we describe the cellular microbiaxial stretching (CµBS) assay in which controlled deformations are applied to micropatterned cells while simultaneously measuring cell stress. CµBS utilizes a set of linear actuators to apply tensile or compressive, short- or long-term deformations to cells micropatterned on a fluorescent bead-doped polyacrylamide gel. Using traction force microscopy principles and the known geometry of the cell and the mechanical properties of the underlying gel, we calculate the stress within the cell to formulate stress-strain curves that can be further used to create mechanical descriptions of the cells, such as strain energy density functions. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Assembly of CµBS stretching constructs Basic Protocol 2: Polymerization of micropatterned, bead-doped polyacrylamide gel on an elastomer membrane Support Protocol: Cell culture and seeding onto CµBS constructs Basic Protocol 3: Implementing CµBS stretching protocols and traction force microscopy Basic Protocol 4: Data analysis and cell stress measurements.

Anisotropic Mechanics of Vascular Smooth Muscle Cells Exposed to Dynamic Loads.

Vascular smooth muscle cells (VSMCs) are the most prevalent cells in the arterial wall. In vivo, arteries are exposed to dynamic biaxial loads; thus, when characterizing VSMC mechanics, it is important to determine their anisotropic and time-dependent mechanical properties. In this work, we use cellular microbiaxial stretching to apply complex deformations to single micropatterned VSMCs and measure the resulting changes in cell stress. Previously, cellular microbiaxial stretching has been used to measure VSMC mechanical properties in response to extensional strain. Here, we measure changes in cell stress in response to both extension and compression. Additionally, we measure immediate temporal changes in stress in response to cyclically applied deformations. We find that the VSMCs display clear hysteresis when incrementally stretched and compressed and demonstrate cycle-dependent stress-relaxation when exposed to cyclic step change extension and compression. Finally, we demonstrate that a Hill-type active fiber model is capable of replicating all observed hysteresis and cycle-dependent stress-relaxation, suggesting that the temporal stress-strain behavior of the cell is regulated by acto-myosin contraction and relaxation, rather than passive viscoelasticity. This study improves upon previous studies of cellular mechanical properties by considering cellular architecture and more complex deformations when measuring the time-dependent mechanical properties of VSMCs. These findings have important implications for modeling in mechanobiology as VSMCs are mechanosensitive and actively respond to changes in their mechanical environment to maintain vascular function.

Large-deformation strain energy density function for vascular smooth muscle cells.

Vascular tissue exhibits marked mechanical nonlinearity when exposed to large strains. Vascular smooth muscle cells (VSMCs) are the most prevalent cell type in the artery wall, but it is unclear how much of the vessel nonlinearity is attributable to VSMCs. Here, we used cellular microbiaxial stretching (CµBS) to measure the large-strain mechanical properties of individual VSMCs. We find that the mechanical properties of VSMCs with native-like architectures are highly anisotropic, due to their highly aligned actomyosin cytoskeletons, and that inhibition of actomyosin contraction with rho-associated kinase inhibitor HA-1077 results in nearly isotropic material properties. We further find that when VSMCS are exposed to large strains (up to 60% stretch), the cells' stress-strain behavior is surprisingly linear. Finally, we modified a previously published Holzapfel-Gasser-Ogden type strain energy density function to characterize individual VSMCs, to account for the observed large-deformation linearity. These data have important implications in the development of models of vascular mechanics and mechanobiology.