Various types of cardiomyocytes undergo changes in automaticity and electrical properties during fetal heart development. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs), like fetal cardiomyocytes, are electrophysiologically immature and exhibit automaticity. We used hESC-CMs to investigate developmental changes in mechanisms of automaticity and to determine whether electrophysiological maturation is driven by an intrinsic developmental clock and/or is regulated by interactions with non-cardiomyocytes in embryoid bodies (EBs). We isolated pure populations of hESC-CMs from EBs by lentivirus-engineered Puromycin resistance at various stages of differentiation. Using pharmacological agents, calcium (Ca 2+ ) imaging, and intracellular recording techniques, we found that intracellular Ca 2+ -cycling mechanisms developed early and contributed to dominant automaticity throughout hESC-CM differentiation. Sarcolemmal ion channels evolved later upon further differentiation within EBs and played an increasing role in controlling automaticity and electrophysiological properties of hESC-CMs. In contrast to the development of intracellular Ca 2+ -handling proteins, ion channel development and electrophysiological maturation of hESC-CMs did not occur when hESC-CMs were isolated from EBs early and maintained in culture without further interaction with non-cardiomyocytes. Adding back non-cardiomyocytes to early-isolated hESC-CMs rescued the arrest of electrophysiological maturation, indicating that non-cardiomyocytes in EBs drive electrophysiological maturation of early hESC-CMs. Non-cardiomyocytes in EBs contain most cell types present in the embryonic heart that are known to infl uence early cardiac development. Our study is the fi rst to demonstrate that non-cardiomyocytes infl uence electrophysiological maturation of early hESC-CMs in cultures. Defi ning the nature of these extrinsic signals will aid in the directed maturation of immature hESC-CMs to mitigate arrhythmogenic risks of cell-based therapies.

Cardiac progenitor cells (CPCs) are multipotent cells that may offer tremendous potentials for the regeneration of injured myocardium. To expand the limited number of CPCs for effective clinical regeneration of myocardium, it is important to understand their proliferative potentials. Single-cell based assays were utilized to purify c-kitpos CPCs from human and mouse hearts. MicroRNA profiling identified eight differentially expressed microRNAs in CPCs from neonatal and adult hearts. Notably, the predicted protein targets were predominantly involved in cellular proliferation-related pathways. To directly test this phenotypic prediction, the developmental variance in the proliferation of CPCs was tested. Ki67 protein expression and DNA kinetics were tested in human and mouse in vivo CPCs, and doubling times were tested in primary culture of mouse CPCs. The human embryonic and mouse neonatal CPCs showed a six-fold increase in Ki67 expressing cells, a two-fold increase in the number of cells in S/G2-M phases of cell cycle, and a seven-fold increase in the doubling time in culture when compared to the corresponding adult CPCs. The over-expression of miR-17-92 increased the proliferation in adult CPCs in vivo by two-fold. In addition, the level of retinoblastoma-like 2 (Rbl2/p130) protein was two-fold higher in adult compared to neonatal-mouse CPCs. In conclusion, we demonstrate a differentially regulated cohort of microRNAs that predicts differences in cellular proliferation in CPCs during postnatal development and target microRNAs that are involved in this transition. Our study provides new insights that may enhance the utilization of adult CPCs for regenerative therapy of the injured myocardium.

Recent studies suggest that age-related cognitive decline is correlated with an excitatory-inhibitory imbalance in synaptic discharges on pyramidal neurons. This study focuses on whether ageing and cognitive status correlates with relative numbers of excitatory and inhibitory presynaptic boutons. We investigated the density of excitatory and inhibitory presynaptic inputs across several areas of the rat cerebral cortex in young and aged male Fischer 344 rats. Aged animals were segregated into aged cognitively impaired (AI) and aged cognitively unimpaired (AU) groups using the Morris water maze. We applied immunohistochemistry to reveal the majority of excitatory and inhibitory presynaptic boutons captured with confocal microscopy and quantitative image analysis. A gradual decline in the density of excitatory and inhibitory presynaptic boutons occurred from young to AU to AI animals; however, the ratios of excitatory to inhibitory presynaptic bouton densities were not significantly altered. We further investigated the density of receptor scaffolding proteins representing key excitatory and inhibitory receptor postsynaptic sites, using antibodies against specific markers of excitatory and inhibitory postsynaptic densities, respectively. Significant changes in the ratios of excitatory to inhibitory postsynaptic densities were observed only in AI compared to young rats.

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The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor (NMDAR) is essential for normal function of the central nervous system (CNS). Classical NMDARs, activated by glycine and glutamate, are heteromultimers comprising NR1 and NR2 subunits. Nonetheless, excessive activation of NMDARs by excitatory amino acids such as glutamate is thought to mediate neuronal damage in many neurological disorders. The dual role of NMDARs in normal and abnormal functioning of the CNS imposes significant constraints on possible therapeutic strategies aimed at ameliorating neurodegenerative diseases. To create safe NMDAR-based therapies, blockade of excessive NMDAR activity must therefore be achieved with minimal interference on its normal neuronal function. In general, NMDAR antagonists can be classified pharmacologically according to the site of action on the receptor-channel complex. These include drugs acting at the agonist sites (NMDA and glycine), channel pore, and modulatory sites. Both competitive NMDA and glycine antagonists result in generalized inhibition of NMDAR activities and have, thus, failed in clinical trials. Open-channel blockers with uncompetitive antagonism and drugs modulating NMDAR activities are appealing therapeutic strategies because, in theory, these properties could decrease neurotoxicity due to excessive levels of glutamate while sparing physiological neurotransmission. We review here NMDAR-related research that may lead to future therapeutic intervention against neurotoxicity.