Exosome-derived miR-127-5p Promotes Embryonic-like Stem Cells Differentiation Into Pacemaker Cell Through Nkx2.5 Down-regulation

Available evidence suggests the involvement of microRNAs (miRNAs) in the pathological process of several diseases. Nonetheless, molecular mechanism underlying biological effects of miRNAs such as pacemaker exosome-derived miR-127-5p in embryonic-like stem cells (ESCs) differentiation into pacemaker cell is yet to be clarified. Through real time polymerase chain reaction (qPCR) or western blotting (WB) techniques, levels of miRNAs, miR-127-5p, and NKx2.5 expressions was quantitatively measured. Cellular differentiation (CD) was probed with flow cytometric (FC) and WB techniques. Prediction of miR-127-5p association with NKx2.5 was studied through bioinformatics tools before verification with luciferase assay. Promotion of ESCs differentiation to pacemaker through miR-127-5p was measured with qPCR and WB techniques. Through the same assaying methods, up-regulation of pace-making genes (shox2, HCN4, Cx45, Tbx3 and Tbx18) expression was observed in Nkx2.5 knockdown group. However, Nkx2.5 expression was down-regulated during differentiation of pacemaker-like cells into ESCs. Furthermore, techniques (such as qPCR, WB, immunofluorescent staining and FC) were employed to demonstrate that overexpressed miR-127-5p could suppress NKx2.5 expression. Through NKx2.5 targeting, ESCs could be differentiated into pacemaker-like cells with miR-127-5p possibly serving as a crucial positive regulator. On the account of our findings, further researches are needed to unearth the possible underlying mechanism and role of exosome-derived miRNAs in cell signaling.

Transcriptional and Epigenetic Landscape of Cardiac Pacemaker Cells: Insights Into Cellular Specialization in the Sinoatrial Node

Cardiac pacemaker cells differentiate and functionally specialize early in embryonic development through activation of critical gene regulatory networks. In general, cellular specification and differentiation require that combinations of cell type-specific transcriptional regulators activate expression of key effector genes by binding to DNA regulatory elements including enhancers and promoters. However, because genomic DNA is tightly packaged by histones that must be covalently modified in order to render DNA regulatory elements and promoters accessible for transcription, the process of development and differentiation is intimately connected to the epigenetic regulation of chromatin accessibility. Although the difficulty of obtaining sufficient quantities of pure populations of pacemaker cells has limited progress in this field, the advent of low-input genomic technologies has the potential to catalyze a rapid growth of knowledge in this important area. The goal of this review is to outline the key transcriptional networks that control pacemaker cell development, with particular attention to our emerging understanding of how chromatin accessibility is modified and regulated during pacemaker cell differentiation. In addition, we will discuss the relevance of these findings to adult sinus node function, sinus node diseases, and origins of genetic variation in heart rhythm. Lastly, we will outline the current challenges facing this field and promising directions for future investigation.

Genome-Wide Analysis Identifies an Essential Human TBX3 Pacemaker Enhancer

Rationale: The development and function of the pacemaker cardiomyocytes of the sinoatrial node (SAN), the leading pacemaker of the heart, are tightly controlled by a conserved network of transcription factors, including TBX3 (T-box transcription factor 3), ISL1 (ISL LIM homeobox 1), and SHOX2 (short stature homeobox 2). Yet, the regulatory DNA elements (REs) controlling target gene expression in the SAN pacemaker cells have remained undefined. Objective: Identification of the regulatory landscape of human SAN-like pacemaker cells and functional assessment of SAN-specific REs potentially involved in pacemaker cell gene regulation. Methods and Results: We performed Assay for Transposase-Accessible Chromatin using sequencing on human pluripotent stem cell–derived SAN-like pacemaker cells and ventricle-like cells and identified thousands of putative REs specific for either human cell type. We validated pacemaker cell–specific elements in the SHOX2 and TBX3 loci. CRISPR-mediated homozygous deletion of the mouse ortholog of a noncoding region with candidate pacemaker-specific REs in the SHOX2 locus resulted in selective loss of Shox2 expression from the developing SAN and embryonic lethality. Putative pacemaker-specific REs were identified up to 1 Mbp upstream of TBX3 in a region close to MED13L harboring variants associated with heart rate recovery after exercise. The orthologous region was deleted in mice, which resulted in selective loss of expression of Tbx3 from the SAN and (cardiac) ganglia and in neonatal lethality. Expression of Tbx3 was maintained in other tissues including the atrioventricular conduction system, lungs, and liver. Heterozygous adult mice showed increased SAN recovery times after pacing. The human REs harboring the associated variants robustly drove expression in the SAN of transgenic mouse embryos. Conclusions: We provided a genome-wide collection of candidate human pacemaker-specific REs, including the loci of SHOX2 , TBX3 , and ISL1 , and identified a link between human genetic variants influencing heart rate recovery after exercise and a variant RE with highly conserved function, driving SAN expression of TBX3 .

Endogenous hydrogen sulfide maintains eupnea in an in situ arterially perfused preparation of rats

Hydrogen sulfide (H2S) is constitutively generated in the human body and works as a gasotransmitter in synaptic transmission. In this study, we aimed to evaluate the roles of endogenous H2S in generating eupnea at the respiratory center. We employed an in situ arterially perfused preparation of decerebrated rats and recorded the central respiratory outputs. When the H2S-producing enzyme cystathionine β-synthase (CBS) was inhibited, respiration switched from the 3-phase eupneic pattern, which consists of inspiration, postinspiration, and expiration, to gasping-like respiration, which consists of inspiration only. On the other hand, when H2S synthesis was inhibited via cystathionine γ-lyase (CSE) or when H2S synthesis was activated via CBS, eupnea remained unchanged. These results suggest that H2S produced by CBS has crucial roles in maintaining the neuronal network to generate eupnea. The mechanism of respiratory pattern generation might be switched from a network-based system to a pacemaker cell-based system in low H2S conditions.

Impact of Chronic Fetal Hypoxia and Inflammation on Cardiac Pacemaker Cell Development

Chronic fetal hypoxia and infection are examples of adverse conditions during complicated pregnancy, which impact cardiac myogenesis and increase the lifetime risk of heart disease. However, the effects that chronic hypoxic or inflammatory environments exert on cardiac pacemaker cells are poorly understood. Here, we review the current evidence and novel avenues of bench-to-bed research in this field of perinatal cardiogenesis as well as its translational significance for early detection of future risk for cardiovascular disease.