The effect of pyrazole 3 treatment on the generation of intracellular ca ²⁺ transients and mitochondrial membrane potential, as well as the survival and neural differentiation of mouse embryonic stem cells

The identification and use of pharmacological agents to regulate the fate of embryonic stem cells (ESCs) and promote their differentiation into neurons has great potential in the development of novel clinical therapies. Here, we investigated the effect of pyrazole 3 (pyr3) on the generation of Ca 2+ transients in mouse ESCs (mESCs), as well as on the differentiation of mESCs into neural cells. Pyr3 was originally identified as being a potent inhibitor of the transient receptor potential canonical subfamily 3 (TRPC3) channel. However, here we showed that pyr3 induced an increase in intracellular Ca 2+ ([Ca 2+ ] i ) in a dose-dependent manner in undifferentiated mESCs. An initial, rapid increase in [Ca 2+ ] i was stimulated mainly via an influx of Ca 2+ across the plasma membrane, whereas a subsequent longer-lasting reduced plateau phase was mediated both by Ca 2+ influx and by Ca 2+ release from the endoplasmic reticulum (ER) via inositol 1,4,5-trisphosphate receptors (IP 3 Rs). A similar increase in [Ca 2+ ] i was recorded in mESCs following CRISPR/Cas9-mediated knock-out of TRPC3 in the presence of the L-type Ca 2+ channel agonist, (S)-(-)-Bay K 8644. A much smaller increase in [Ca 2+ ] i was recorded on addition of (S)-(-)-Bay K 8644 in control cells, where functional TRPC3 channels were present. Pyr3 also induced apoptosis via the disruption of the mitochondrial membrane potential (ΔΨm) in both undifferentiated mESCs and in cells undergoing neural differentiation, and it inhibited the early stages of mESC differentiation to neural progenitors. Taken together, our results suggest a novel role for TRPC3 in the regulation of [Ca 2+ ] i , as well as on the survival and neural differentiation of mESCs.

Duke Scholars

Author Jianbo Yue DKU Faculty