C-kit + CSC-derived cardiomyocytes exhibit the typical transcriptional gene blueprint of adult cardiomyocytes

Abstracts

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monitor at high-spatial resolution the kinematic/dynamic parameters of beating hearts during cardio-surgery intervention. Materials and methods: We acquire, via a bright-field camera (1240 × 1240 pixel sensor, Baumer, HXC13, Germany), a series of photograms at N100 fps in LabView software-based platform (National Instruments) and track the motion of one or more pixel regions by following a selected marker via a Matlab script (Mathworks). The algorithm follows frame-by-frame the contraction motion and registers the spatial–temporal coordinates x, y and t for each marker. Consequently, we evaluate the complete kinematics of 2D/3D tissue in terms of i) trajectories of contracting cardiac regions (the displacement vectors, velocity and acceleration vectors) as well as the complete dynamics, i.e. ii) contraction frequency, iii) kinetic energy, iv) contraction force, v) maximum velocity. We measure the cardiac electrical conduction velocity by epicardial multiple lead technique in order to validate the maximum velocity. In the first series of experiment, we monitor the kinematic activity by providing a classic ischemia–reperfusion protocol in-situ; in the second series, we evaluate the mechanoelectric feedback activity by applying a given stretch-related pressure at the epicardial level onto the epicardial region of the pulmonary conus. Results: At high-rate acquisition level (500 fps), we found that overall ventricular contraction velocity is very similar to the electrical velocity (618 mm/s vs. 620 mm/s) measured by epicardial mapping. The contraction velocity in the ischemic area is increased compared to reperfusion while the kinetic energy and the beat-rate are significantly reduced (from 180 bpm to 120 bpm). The mechanical stimulation onto the pulmonary conus region induced a sinus pause possible related to AV block, where the overfilled ventricle contracted with a kinetic energy and force two-threefold higher than the physiological ones. Conclusions: We propose here a new simple technique which extract several important kinematics/dynamics parameters, providing an impact not only for the electrophysiological “bench research” but also for the “bedside” clinical outcome.

contracting cells in vitro. Freshly isolated CMs from adult mouse hearts were used as controls. RNA-Seq was employed to analyse and compare whole mRNA and microRNA profiles of cells collected at CSC, c-kit-iCM and adult cardiac myocyte (aCM) stages. Results: In the comparison of c-kit posCSCs vs. c-kit-iCMs, more than 4000 genes were up-regulated which mainly function in mitochondrial, sarcoplasm, sarcomere- specific and calcium regulating processes, among others. Several sarcomere-related genes highly expressed in adult CMs (i.e. Tnnt2, Tcap, Myl3, Tnni3, Tpm1, Myh7, Myh6) were indeed significantly up-regulated in c-kitiCMs vs. CSCs in vitro. On the other hand, we found that most of cell cycle regulators/ genes and a number of RNA processing genes were consistently downregulated during differentiation from ckit posCSCs to c-kit-iCMs in vitro. Interestingly, during differentiation from c-kitposCSCs to iCMs, known myo-miRs were upregulated while microRNAs positively regulating stem cell expansion and self-renewal were downregulated. Bioinformatics analysis built specific networks of miRNAmRNAs that are precisely regulated during iCM generation from CSCs and that closely resemble miRNA–mRNA networks of adult CMs. Conclusions: c-kitposCSCs robustly differentiate into functional beating cardiomyocytes in vitro. ckit posCSC myogenic specification follow known developmental cardiomyocyte differentiation pathways and c-kit-iCMs transcriptome profile closely resemble adult CMs.

doi:10.1016/j.vph.2015.11.070

Objectives: The influence of nitric oxide synthetase (NOS)/ soluble guanylyl cyclase (sGC)/cGMP-dependent protein kinase I (cGKI) pathway in adult cardiac performances is well defined. Indeed, physiological amounts of NO generated by NOS1 and NOS3 or pharmacological administration of NO-donor drugs can influence cardiac contractility and increase coronary blood flow throughout the Ser/Thr phosphorylation (sGC/cGKI pathway) and directly by Snitrosylation of several proteins. Nevertheless, the influence of this pathway as trigger/modulator of cardiac differentiation of ESCs is less defined. Therefore, we investigated the influence of NOS/sGC/cGKI in the early stage of cardiac differentiation of mouse ESCs, studying i) enzyme expressions and activities during the cardiac maturation and ii) the acute and chronic effect of pathway alteration. Materials and methods: Undifferentiated mESC CGR8 cell line was cardiac differentiated by hanging drop methodology to form embryoid bodies (mEBs). Beating mEBs were monitored starting from the 7th– 10th day until 21 days. At different stages of maturation, mNos3, mGuCy1b and mPrkgI gene and protein expressions were detected by Q-PCR, Western Blot and ELISA. Results: Q-PCR showed that during differentiation the expression of the enzymes increased in a time-dependent mode. The peak of mNos3 expression was measured at d8 and then rapidly decreased. mGuCy1b and mPrkgI were detected starting from d5 and increased until d14. Protein expressions were in line with RNA messenger measures, with detectable level of NOS3 only at d5 and d8. At d15 sGC activity was increased of 3 times with the NO-donor SNAP and inhibited by 50% with ODQ. Moreover, cyclic GMP was reduced when EBs were incubated for 30 min with L-NMA, suggesting an endogenous activity of NOS. The phosphorylation of Ser/Thr residues on target proteins, were increased by membrane permeable cGMP, isosorbide-5mononitrate and SNAP and reduced by KT5823, a selective cGKI inhibitor. Functionally, cell incubation with isosorbide-5-mononitrate

C-kit + CSC-derived cardiomyocytes exhibit the typical transcriptional gene blueprint of adult cardiomyocytes M. Scalisea,, I. Aquilaa, C. Vicinanzaa, F. Marinoa, F. Cristianoa, E. Cianflonea, P. Veltria, C. Indolfia, B. Nadal-Ginardb, G.M. Ellisonb, D. Torellaa a Laboratory of Molecular and Cellular Cardiology, Department of Medical and Surgical Sciences, Magna Graecia University, Catanzaro, Italy b Centre of Human & Aerospace Physiological Sciences and Centre for Stem Cells & Regenerative Medicine, Faculty of Medicine & Life Science, King's College London, London, UK Background: From embryonic to adult life, mammalian cardiomyocytes (CMs) generated from cardiac progenitors from mesodermal layers undergo a maturation process that is characterized by development of sarcomeric structure, binucleation, increased metabolic demand, and permanent exit from the cell cycle. This process is transcriptionally regulated by specific myogenic microRNAs (Myo-miRs). Adult cardiac stem/progenitor cells (CSCs) identified by the expression of c-kit are able to undergo cardiomyocyte commitment in vitro and in vivo. However, it is still unknown if adult CSC-cardiomyogenic specification recapitulates cardiac development and if CSC-derived cardiomyocytes closely overlap adult cardiomyocyte whole gene profile. Herein, we aimed at elucidating whether CMs generated from c-kit + CSC differentiation in vitro (c-kit-iCM) have a similar pattern of gene expression of adult CMs with a similar cell cycle exit through the activity of known Myo-miRs. Materials and methods: c-kit + CSCs were clonally expanded from single cell deposition. Clonal-derived CSCs were primed in a stagespecific cardiopoietic growth factor cocktail to obtain spontaneously

doi:10.1016/j.vph.2015.11.071

NOS/sGC/cGK1 pathway in cardiac-specific differentiation of mouse embryonic stem cells V. Spinelli, L. Diolaiuti, A. Laurino, L. Dini, L. Sartiani, A. Vona, M. Zanardelli, E. Cerbai, P. Failli Dept. NEUROFARBA, Unit of Pharmacology and Toxicology, University of Florence, Florence, Italy