O'Kelly, I. | bioRxiv Science

bioRxiv2021

Defining Cardiac Cell Populations and Relative Cellular Composition of the Early Fetal Human Heart

The human heart is primarily composed of cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Reliable identification of fetal cardiac cell types using protein markers is important for understanding cardiac development and delineating the cellular composition of the human heart during early development, which remains largely unknown. The aim of this study was to use immunohistochemistry (IHC), flow cytometry and RT-PCR analyses to investigate the expression and specificity of commonly used cardiac cell markers in the early human fetal heart (8-12 post-conception weeks). The expression of previously reported protein markers for the detection of cardiomyocytes (Myosin Heavy Chain (MHC) and Troponin I (cTnI)), fibroblasts (DDR2, Thy1, Vimentin), endothelial cells (CD31) and smooth muscle cells (-SMA) were assessed. Flow cytometry revealed two distinct populations of cTnI expressing cells based on fluorescence intensity: cTnIHigh and cTnILow. MHC positive cardiomyocytes were cTnIHigh, whereas MHC negative non-myocyte cells were cTnILow. cTnI expression in non-myocytes was further confirmed by IHC and RT-PCR analyses, suggesting troponins are not cardiomyocyte-specific and may play distinct roles in non-muscle cells during early development. Vimentin was confirmed to be enriched in cultured fibroblast populations and flow cytometry revealed VimHigh and VimLow cell populations in the fetal heart. MHC positive cardiomyocytes were VimLow whilst CD31 positive endothelial cells were VimHigh. Based on the markers investigated, we estimate fetal human cardiomyocyte populations comprise 75-80% of total cardiac cells and exhibit the following marker profile: -MHC+/cTnIHigh/VimLow. For the non-cardiomyocyte population, we estimate they comprise 20-25% of total cardiac cells and exhibit the following marker profile: -MHC-/cTnILow/VimHigh. Our study suggests the marker profiles and proportions of fetal cardiac populations are distinct from that of the adult heart.

cell biology↗

bioRxiv2019

A 5’ UTR G-quadruplex controls localisation and translation of a potassium leak channel mRNA

RNA G-quadruplexes (G4s) are non-canonical secondary structures that have been proposed to function as regulators of post-transcriptional mRNA localisation and translation. G4s within 3 UTRs of some neuronal mRNAs are known to control their distal localisation and local translation, contributing to the distinct local proteomes that facilitate the synaptic remodelling attributed to normal cellular function. In this study, we characterise the G4 formation of a (GGN)13 repeat found within the 5 UTR of KCNK9 mRNA, encoding the potassium 2-pore domain leak channel Task3. Using circular dichroism, we show that this (GGN)13 repeat forms a parallel G4 that exhibits the stereotypical potassium specificity of a G4, remaining thermostable under physiological ionic conditions. The G4 is inhibitory to translation of Task3, which can be overcome through the activity of the G4-specific helicase DHX36, consequently increasing K+ leak currents and decreasing resting membrane potentials in HEK293 cells. Additionally, we observe that this G4 is fundamental to ensuring the delivery of Task3 mRNA to distal primary cortical neurites. It has previously been shown that abnormal Task3 expression correlates with neuronal dysfunction, we therefore posit that this G4 is required for regulated local expression of Task3 leak channels that maintain K+ leak currents within neurons.

molecular biology↗