YAP dysregulation triggers hypertrophy by CCN2 secretion and TGFß uptake in human pluripotent stem cell-derived cardiomyocytes.

Hypertrophy Cardiomyopathy (HCM) is the most prevalent hereditary cardiovascular disease - affecting >1:500 individuals. Advanced forms of HCM clinically present with hypercontractility, hypertrophy and fibrosis. Several single-point mutations in b-myosin heavy chain (MYH7) have been associated with HCM and increased contractility at the organ level. Different MYH7 mutations have resulted in increased, decreased, or unchanged force production at the molecular level. Yet, how these molecular kinetics link to cell and tissue pathogenesis remains unclear. The Hippo Pathway, specifically its effector molecule YAP, has been demonstrated to be reactivated in pathological hypertrophic growth. We hypothesized that changes in force production (intrinsically or extrinsically) directly alter the homeostatic mechano-signaling of the Hippo pathway through changes in stresses on the nucleus. Using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), we asked whether homeostatic mechanical signaling through the canonical growth regulator, YAP, is altered 1) by changes in the biomechanics of HCM mutant cardiomyocytes and 2) by alterations in the mechanical environment. We use genetically edited hiPSC-CM with point mutations in MYH7 associated with HCM, and their matched controls, combined with micropatterned traction force microscopy substrates to confirm the hypercontractile phenotype in MYH7 mutants. We next modulate contractility in healthy and disease hiPSC-CMs by treatment with positive and negative inotropic drugs and demonstrate a correlative relationship between contractility and YAP activity. We further demonstrate the activation of YAP in both HCM mutants and healthy hiPSC-CMs treated with contractility modulators is through enhanced nuclear deformation. We conclude that the overactivation of YAP, possibly initiated and driven by hypercontractility, correlates with excessive CCN2 secretion (connective tissue growth factor), enhancing cardiac fibroblast/myofibroblast transition and production of known hypertrophic signaling molecule TGFß. Our study suggests YAP being an indirect player in the initiation of hypertrophic growth and fibrosis in HCM. Our results provide new insights into HCM progression and bring forth a testbed for therapeutic options in treating HCM.

3D PCL/Gelatin/Genipin Nanofiber Sponge as Scaffold for Regenerative Medicine.

Recent advancements in tissue engineering and material science have radically improved in vitro culturing platforms to more accurately replicate human tissue. However, the transition to clinical relevance has been slow in part due to the lack of biologically compatible/relevant materials. In the present study, we marry the commonly used two-dimensional (2D) technique of electrospinning and a self-assembly process to construct easily reproducible, highly porous, three-dimensional (3D) nanofiber scaffolds for various tissue engineering applications. Specimens from biologically relevant polymers polycaprolactone (PCL) and gelatin were chemically cross-linked using the naturally occurring cross-linker genipin. Potential cytotoxic effects of the scaffolds were analyzed by culturing human dermal fibroblasts (HDF) up to 23 days. The 3D PCL/gelatin/genipin scaffolds produced here resemble the complex nanofibrous architecture found in naturally occurring extracellular matrix (ECM) and exhibit physiologically relevant mechanical properties as well as excellent cell cytocompatibility. Samples cross-linked with 0.5% genipin demonstrated the highest metabolic activity and proliferation rates for HDF. Scanning electron microscopy (SEM) images indicated excellent cell adhesion and the characteristic morphological features of fibroblasts in all tested samples. The three-dimensional (3D) PCL/gelatin/genipin scaffolds produced here show great potential for various 3D tissue-engineering applications such as ex vivo cell culturing platforms, wound healing, or tissue replacement.

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

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.

Processes controlling spatial and temporal dynamics of spring water chemistry in the Black Forest National Park.

Spring water chemistry is influenced by many factors, including geology, climate, vegetation and land use, which determine groundwater residence times and water-rock interaction. Changes in water chemistry can have a profound impact on their associated ecosystems. To protect these ecosystems and to evaluate possible changes, knowledge of the underlying processes and dynamics is important. We collected water samples at 20 locations during 5 campaigns within the water catchment area of the upper Schönmünz river in the Black Forest National Park, Southwest Germany and analyzed them hydro-chemically for their contents of inorganic constituents, organic carbon content, fluorescence properties as well as several physico chemical field parameters and spring discharge. Results show that water chemistry is strongly dependent on geology and that the response of dissolved organic carbon to changes in hydraulic conditions is highly dynamic. Due to increased flow through the upper soil layer during and after rain events, more organic carbon is extracted from the soil and transported with the water. Fluorescence EEM measurements indicate an allochthonous source of this organic carbon. This study can be used as baseline to assess future changes and serve as a supplement to ongoing studies of the spring ecosystems.

Physicochemical and major ion data for springs in the Black Forest National Park, Germany.

The dataset in this article consists of the general physicochemical parameters (temperature, pH, specific electrical conductivity, dissolved oxygen, redox potential, alkalinity) and concentrations of major ions (Ca2+, Mg2+, K+, Na+, Cl-, SO4 2-, NO3 -) of water samples collected at 19 springs and the surface stream in the water catchment area of the upper Schönmünz river in the Black Forest National Park, Germany. Data on concentrations of dissolved organic carbon (DOC), total organic carbon (TOC), spectral absorbance at different wavelengths and fluorescence as well as microbiological indicators (E. coli, total coliforms, enterococci) are also reported. Sampling was conducted during five field campaigns between spring 2016 and spring 2017. Knowledge of the current physicochemical parameters and concentrations of dissolved organic and inorganic constituents provides a baseline to assess future changes and serves as a supplement to ongoing studies of the spring ecosystems. Understanding the specific processes influencing the water chemistry will aid in their effective protection. For more details and further discussion on this dataset, the reader is referred to the associated research article "Processes controlling spatial and temporal dynamics of spring water chemistry in the Black Forest National Park" [1].