DOT1L-mediated H3K79me2 modification critically regulates gene expression during cardiomyocyte differentiation.

Epigenetic changes on DNA and chromatin are implicated in cell differentiation and organogenesis. For the heart, distinct histone methylation profiles were recently linked to stage-specific gene expression programs during cardiac differentiation in vitro. However, the enzymes catalyzing these modifications and the genes regulated by them remain poorly defined. We therefore decided to identify the epigenetic enzymes that are potentially involved in cardiomyogenesis by analyzing the expression profile of the 85 genes encoding the epigenetic-related proteins in mouse cardiomyocytes (CMs), and then study how they affect gene expression during differentiation and maturation of this cell type. We show here with gene expression screening of epigenetic enzymes that the highly expressed H3 methyltransferase disruptor of telomeric silencing 1-like (DOT1L) drives a transitional pattern of di-methylation on H3 lysine 79 (H3K79) in CMs at different stages of differentiation in vitro and in vivo. Through a genome-wide chromatin-immunoprecipitation DNA-sequencing approach, we found H3K79me2 enriched at genes expressed during cardiac differentiation. Moreover, knockdown of Dot1L affected the expression of H3K79me2-enriched genes. Our results demonstrate that histone methylation, and in particular DOT1L-mediated H3K79me2 modification, drives cardiomyogenesis through the definition of a specific transcriptional landscape.

Self-assembly of polydimethylsiloxane structures from 2D to 3D for bio-hybrid actuation.

This work aims to demonstrate the feasibility of a novel approach for the development of 3D self-assembled polydimethylsiloxane structures, to be used as engineered flexible matrices for bio-hybrid actuation. We described the fabrication of engineered bilayers, organized in a 3D architecture by means of a stress-induced rolling membrane technique. Such structures were provided with ad hoc surface topographies, for both cell alignment and cell survival after membrane rolling. We reported the results of advanced finite element model simulations, predicting the system behavior in terms of overall contraction, induced by the contractile activity of muscle cells seeded on the membrane. Then, we tested in vitro the structure with primary cardiomyocytes to evaluate the real bio-actuator contraction, thus validating the simulation results. At a later stage, we provided the samples with a stable fibronectin coating, by covalently binding the protein on the polymer surface, thus enabling long-term cultures with C2C12 skeletal muscle cells, a more controllable cell type. These tests revealed cell viability and alignment on the rolled structures, but also the ability of cells to differentiate and to form multinucleated and oriented myotubes on the polymer surface, also supported by a fibroblast feeder layer. Our results highlighted the possibility of developing 3D rolled PDMS structures, characterized by different mechanical properties, as novel bio-hybrid actuators.

PlGF-MMP9-engineered iPS cells supported on a PEG-fibrinogen hydrogel scaffold possess an enhanced capacity to repair damaged myocardium.

Cell-based regenerative therapies are significantly improved by engineering allografts to express factors that increase vascularization and engraftment, such as placental growth factor (PlGF) and matrix metalloproteinase 9 (MMP9). Moreover, the seeding of therapeutic cells onto a suitable scaffold is of utmost importance for tissue regeneration. On these premises, we sought to assess the reparative potential of induced pluripotent stem (iPS) cells bioengineered to secrete PlGF or MMP9 and delivered to infarcted myocardium upon a poly(ethylene glycol)-fibrinogen scaffold. When assessing optimal stiffness of the PEG-fibrinogen (PF) scaffold, we found that the appearance of contracting cells after cardiogenic induction was accelerated on the support designed with an intermediate stiffness. Revascularization and hemodynamic parameters of infarcted mouse heart were significantly improved by injection into the infarct of this optimized PF scaffold seeded with both MiPS (iPS cells engineered to secrete MMP9) and PiPS (iPS cells engineered to secrete PlGF) cells as compared with nonengineered cells or PF alone. Importantly, allograft-derived cells and host myocardium were functionally integrated. Therefore, survival and integration of allografts in the ischemic heart can be significantly improved with the use of therapeutic cells bioengineered to secrete MMP9 and PlGF and encapsulated within an injectable PF hydrogel having an optimized stiffness.

Post-natal cardiomyocytes can generate iPS cells with an enhanced capacity toward cardiomyogenic re-differentation.

Adult mammalian cells can be reprogrammed to a pluripotent state by forcing the expression of a few embryonic transcription factors. The resulting induced pluripotent stem (iPS) cells can differentiate into cells of all three germ layers. It is well known that post-natal cardiomyocytes (CMs) lack the capacity to proliferate. Here, we report that neonatal CMs can be reprogrammed to generate iPS cells that express embryonic-specific markers and feature gene-expression profiles similar to those of mouse embryonic stem (mES) cell and cardiac fibroblast (CF)-derived iPS cell populations. CM-derived iPS cells are able to generate chimeric mice and, moreover, re-differentiate toward CMs more efficiently then either CF-derived iPS cells or mES cells. The increased differentiation capacity is possibly related to CM-derived iPS cells retaining an epigenetic memory of the phenotype of their founder cell. CM-derived iPS cells may thus lead to new information on differentiation processes underlying cardiac differentiation and proliferation.

Comparison of four markers for quantifying microbial protein flow from the rumen of lactating dairy cows.

Eight ruminally cannulated lactating cows from a study on the effects of dietary rumen degraded protein (RDP) on production and N metabolism were used to compare 15N, total purines, amino acid (AA) profiles, and urinary excretion of purine derivatives (PD) as microbial markers for quantifying the flow of microbial protein at the omasal canal. Dietary RDP was gradually decreased by replacing solvent soybean meal and urea with lignosulfonate-treated soybean meal. The purine metabolites xanthine and hypoxanthine were present in digesta and microbial samples and were assumed to be of microbial origin. The sum of the purines and their metabolites (adenine, guanine, xanthine, and hypoxanthine) were defined as total purines (TP) and used as a microbial marker. Decreasing dietary RDP from 13.2 to 10.6% of dry matter (DM) reduced microbial nonammonia N (NAN) flows estimated using TP (from 415 to 369 g/d), 15N (from 470 to 384 g/d), AA profiles (from 392 to 311 g/d), and PD (from 436 to 271 g/d). Averaged across diets, microbial NAN flows were highest when estimated using TP and 15N (398 and 429 g/d), lowest when using PD (305 g/d), and intermediate when using AA profiles (360 g/d) as microbial markers. Correlation coefficients between 15N and TP for fluid-associated bacteria, particle-associated bacteria, and total microbial NAN flows were 0.38, 0.85, and 0.69, respectively. When TP was used as the microbial marker, ruminal escape of dietary NAN was not affected by replacing solvent soybean meal with lignosulfonate-treated soybean meal in the diets. The direction and extent of response of dietary and microbial NAN flow to dietary treatments were similar when estimated using 15N, AA profiles, and PD, and were in agreement with previously published data and National Research Council predictions. Microbial and dietary NAN flows from the rumen estimated using 15N appeared to be more accurate and precise than the other markers. Caution is required when interpreting results obtained using TP as the microbial marker.