Currently, there is no effective molecular-based therapy for triple-negative breast cancer (TNBC). Canonical transient receptor potential isoform 3 (TRPC3) was previously shown to be upregulated in breast cancer biopsy tissues when compared to normal breast tissues. However, the biological role of TRPC3 in breast cancer still remains to be elucidated. In this study, subcellular fractionation followed by Western blot and immunocytochemistry showed that TRPC3 was over-expressed on the plasma membrane of TNBC line MDA-MB-231 when compared to an estrogen receptor-positive cell line MCF-7. TRPC3 blocker Pyr3 and dominant negative of TRPC3 attenuated proliferation, induced apoptosis and sensitized cell death to chemotherapeutic agents in MDA-MB-231 as measured by proliferation assays. Interestingly, Ras GTPase-activating protein 4 (RASA4), a Ca2+-promoted Ras-MAPK pathway suppressor, was found to be located on the plasma membrane of MDA-MB-231. Blocking TRPC3 decreased the amount of RASA4 located on the plasma membrane, with concomitant activation of MAPK pathways. Our results suggest that, in TNBC MDA-MB-231 cells, Ca2+ influx through TRPC3 channel sustains the presence of RASA4 on the plasma membrane where it inhibits the Ras-MAPK pathway, leading to proliferation and apoptosis resistance. Our study reveals the novel TRPC3-RASA4-MAPK signaling cascade in TNBC cells and suggests that TRPC3 may be exploited as a potential therapeutic target for TNBC.

Pluripotent stem cells (PSCs) can undergo unlimited self-renewal and can differentiate into all the cell types present in our body, including cardiomyocytes. Therefore, PSCs can be an excellent source of cardiomyocytes for future regenerative medicine and medical research studies. However, cardiomyocytes obtained from PSC differentiation culture are regarded as immature structurally, electrophysiologically, metabolically, and functionally. Mitochondria are organelles responsible for various cellular functions such as energy metabolism, different catabolic and anabolic processes, calcium fluxes, and various signaling pathways. Cells can respond to cellular needs to increase the mitochondrial mass by mitochondrial biogenesis. On the other hand, cells can also degrade mitochondria through mitophagy. Mitochondria are also dynamic organelles that undergo continuous fusion and fission events. In this review, we aim to summarize previous findings on the changes of mitochondrial biogenesis, mitophagy, and mitochondrial dynamics during the maturation of cardiomyocytes. In addition, we intend to summarize whether changes in these processes would affect the maturation of cardiomyocytes. Lastly, we aim to discuss unanswered questions in the field and to provide insights for the possible strategies of enhancing the maturation of PSC-derived cardiomyocytes.

Background Cardiomyocytes derived from pluripotent stem cells (PSC-CMs) have been widely accepted as a promising cell source for cardiac drug screening and heart regeneration therapies. However, unlike adult cardiomyocytes, the underdeveloped structure, the immature electrophysiological properties and metabolic phenotype of PSC-CMs limit their application. This project aimed to study the role of the transient receptor potential ankyrin 1 (TRPA1) channel in regulating the maturation of embryonic stem cell-derived cardiomyocytes (ESC-CMs). Methods The activity and expression of TRPA1 in ESC-CMs were modulated by pharmacological or molecular approaches. Knockdown or overexpression of genes was done by infection of cells with adenoviral vectors carrying the gene of interest as a gene delivery tool. Immunostaining followed by confocal microscopy was used to reveal cellular structure such as sarcomere. Staining of mitochondria was performed by MitoTracker staining followed by confocal microscopy. Calcium imaging was performed by fluo-4 staining followed by confocal microscopy. Electrophysiological measurement was performed by whole-cell patch clamping. Gene expression was measured at mRNA level by qPCR and at protein level by Western blot. Oxygen consumption rates were measured by a Seahorse Analyzer. Results TRPA1 was found to positively regulate the maturation of CMs. TRPA1 knockdown caused nascent cell structure, impaired Ca2+ handling and electrophysiological properties, and reduced metabolic capacity in ESC-CMs. The immaturity of ESC-CMs induced by TRPA1 knockdown was accompanied by reduced mitochondrial biogenesis and fusion. Mechanistically, we found that peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α), the key transcriptional coactivator related to mitochondrial biogenesis and metabolism, was downregulated by TRPA1 knockdown. Interestingly, overexpression of PGC-1α ameliorated the halted maturation induced by TRPA1 knockdown. Notably, phosphorylated p38 MAPK was upregulated, while MAPK phosphatase-1 (MKP-1), a calcium-sensitive MAPK inhibitor, was downregulated in TRPA1 knockdown cells, suggesting that TRPA1 may regulate the maturation of ESC-CMs through MKP-1-p38 MAPK-PGC-1α pathway. Conclusions Taken together, our study reveals the novel function of TRPA1 in promoting the maturation of CMs. As multiple stimuli have been known to activate TRPA1, and TRPA1-specific activators are also available, this study provides a novel and straightforward strategy for improving the maturation of PSC-CMs by activating TRPA1. Since a major limitation for the successful application of PSC-CMs for research and medicine lies in their immature phenotypes, the present study takes a big step closer to the practical use of PSC-CMs. Graphical abstract