TRPA1 promotes the maturation of embryonic stem cell-derived cardiomyocytes by regulating mitochondrial biogenesis and dynamics.

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.

Isogenic Human-Induced Pluripotent Stem-Cell-Derived Cardiomyocytes Reveal Activation of Wnt Signaling Pathways Underlying Intrinsic Cardiac Abnormalities in Rett Syndrome.

Rett syndrome (RTT) is a severe neurodevelopmental disorder caused by MeCP2 mutations. Nonetheless, the pathophysiological roles of MeCP2 mutations in the etiology of intrinsic cardiac abnormality and sudden death remain unclear. In this study, we performed a detailed functional studies (calcium and electrophysiological analysis) and RNA-sequencing-based transcriptome analysis of a pair of isogenic RTT female patient-specific induced pluripotent stem-cell-derived cardiomyocytes (iPSC-CMs) that expressed either MeCP2wildtype or MeCP2mutant allele and iPSC-CMs from a non-affected female control. The observations were further confirmed by additional experiments, including Wnt signaling inhibitor treatment, siRNA-based gene silencing, and ion channel blockade. Compared with MeCP2wildtype and control iPSC-CMs, MeCP2mutant iPSC-CMs exhibited prolonged action potential and increased frequency of spontaneous early after polarization. RNA sequencing analysis revealed up-regulation of various Wnt family genes in MeCP2mutant iPSC-CMs. Treatment of MeCP2mutant iPSC-CMs with a Wnt inhibitor XAV939 significantly decreased the ß-catenin protein level and CACN1AC expression and ameliorated their abnormal electrophysiological properties. In summary, our data provide novel insight into the contribution of activation of the Wnt/ß-catenin signaling cascade to the cardiac abnormalities associated with MeCP2 mutations in RTT.

Remdesivir induces persistent mitochondrial and structural damage in human induced pluripotent stem cell-derived cardiomyocytes.

AIMS: Remdesivir is a prodrug of an adenosine triphosphate analogue and is currently the only drug formally approved for the treatment of hospitalized coronavirus disease of 2019 (COVID-19) patients. Nucleoside/nucleotide analogues have been shown to induce mitochondrial damage and cardiotoxicity, and this may be exacerbated by hypoxia, which frequently occurs in severe COVID-19 patients. Although there have been few reports of adverse cardiovascular events associated with remdesivir, clinical data are limited. Here, we investigated whether remdesivir induced cardiotoxicity using an in vitro human cardiac model. METHODS AND RESULTS: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were exposed to remdesivir under normoxic and hypoxic conditions to simulate mild and severe COVID-19, respectively. Remdesivir induced mitochondrial fragmentation, reduced redox potential, and suppressed mitochondrial respiration at levels below the estimated plasma concentration under both normoxic and hypoxic conditions. Non-mitochondrial damage such as electrophysiological alterations and sarcomere disarray were also observed. Importantly, some of these changes persisted after the cessation of treatment, culminating in increased cell death. Mechanistically, we found that inhibition of DRP1, a regulator of mitochondrial fission, ameliorated the cardiotoxic effects of remdesivir, showing that remdesivir-induced cardiotoxicity was preventable and excessive mitochondrial fission might contribute to this phenotype. CONCLUSIONS: Using an in vitro model, we demonstrated that remdesivir can induce cardiotoxicity in hiPSC-CMs at clinically relevant concentrations. These results reveal previously unknown potential side-effects of remdesivir and highlight the importance of further investigations with in vivo animal models and active clinical monitoring to prevent lasting cardiac damage to patients.

Investigation of PM2.5 pollution during COVID-19 pandemic in Guangzhou, China.

The COVID-19 pandemic has raised awareness about various environmental issues, including PM2.5 pollution. Here, PM2.5 pollution during the COVID-19 lockdown was traced and analyzed to clarify the sources and factors influencing PM2.5 in Guangzhou, with an emphasis on heavy pollution. The lockdown led to large reductions in industrial and traffic emissions, which significantly reduced PM2.5 concentrations in Guangzhou. Interestingly, the trend of PM2.5 concentrations was not consistent with traffic and industrial emissions, as minimum concentrations were observed in the fourth period (3/01-3/31, 22.45 µg/m3) of the lockdown. However, the concentrations of other gaseous pollutants, e.g., SO2, NO2 and CO, were correlated with industrial and traffic emissions, and the lowest values were noticed in the second period (1/24-2/03) of the lockdown. Meteorological correlation analysis revealed that the decreased PM2.5 concentrations during COVID-19 can be mainly attributed to decreased industrial and traffic emissions rather than meteorological conditions. When meteorological factors were included in the PM2.5 composition and backward trajectory analyses, we found that long-distance transportation and secondary pollution offset the reduction of primary emissions in the second and third stages of the pandemic. Notably, industrial PM2.5 emissions from western, southern and southeastern Guangzhou play an important role in the formation of heavy pollution events. Our results not only verify the importance of controlling traffic and industrial emissions, but also provide targets for further improvements in PM2.5 pollution.

Extracellular and Intracellular Angiotensin II Regulate the Automaticity of Developing Cardiomyocytes via Different Signaling Pathways.

Angiotensin II (Ang II) plays an important role in regulating various physiological processes. However, little is known about the existence of intracellular Ang II (iAng II), whether iAng II would regulate the automaticity of early differentiating cardiomyocytes, and the underlying mechanism involved. Here, iAng II was detected by immunocytochemistry and ultra-high performance liquid chromatography combined with electrospray ionization triple quadrupole tandem mass spectrometry in mouse embryonic stem cell-derived cardiomyocytes (mESC-CMs) and neonatal rat ventricular myocytes. Expression of AT1R-YFP in mESC-CMs revealed that Ang II type 1 receptors were located on the surface membrane, while immunostaining of Ang II type 2 receptors (AT2R) revealed that AT2R were predominately located on the nucleus and the sarcoplasmic reticulum. While extracellular Ang II increased spontaneous action potentials (APs), dual patch clamping revealed that intracellular delivery of Ang II or AT2R activator C21 decreased spontaneous APs. Interestingly, iAng II was found to decrease the caffeine-induced increase in spontaneous APs and caffeine-induced calcium release, suggesting that iAng II decreased spontaneous APs via the AT2R- and ryanodine receptor-mediated pathways. This is the first study that provides evidence of the presence and function of iAng II in regulating the automaticity behavior of ESC-CMs and may therefore shed light on the role of iAng II in fate determination.

Cytosolic delivery of CDK4/6 inhibitor p16 protein using engineered protein crystals for cancer therapy.

The tumor suppressor p16 protein is an endogenous CDK4/6 inhibitor. Inactivation of its encoding gene is found in nearly half of human cancers. Restoration of p16 function via adenovirus-based gene delivery has been shown to be effective in suppressing aberrant cell growth in many types of cancer, however, the potential risk of insertional mutagenesis in genomic DNA remains a major concern. Thus, there has been great interest in developing efficient strategies to directly deliver proteins into cells as an alternative that can avoid such safety concerns while achieving a comparable therapeutic effect. Nevertheless, intracellular delivery of protein therapeutics remains a challenge. Our group has recently developed a protein delivery platform based on an engineered Pos3Aa protein that forms sub-micrometer-sized crystals in Bacillus thuringiensis cells. In this report, we describe the further development of this platform (Pos3AaTM) via rationally designed site-directed mutagenesis, and its resultant potency for the delivery of cargo proteins into cells. Pos3AaTM-based fusion protein crystals are shown to exhibit improved release of their cargo proteins as demonstrated using a model mCherry protein. Importantly, this Pos3AaTM platform is able to mediate the efficient intracellular delivery of p16 protein with significant endosomal escape, resulting in p16-mediated inhibition of CDK4/6 kinase activity and Rb phosphorylation, and as a consequence, significant cell cycle arrest and cell growth inhibition. These results validate the ability of these improved Pos3AaTM crystals to mediate enhanced cytosolic protein delivery and highlight the potential of using protein therapeutics as selective CDK4/6 inhibitors for cancer therapy. STATEMENT OF SIGNIFICANCE: Cytosolic delivery of bioactive therapeutic proteins capable of eliciting therapeutic benefit remains a significant challenge. We have previously developed a protein delivery platform based on engineered Pos3Aa protein crystals with excellent cell-permeability and endosomal escape properties. In this report, we describe the rational design of an improved Pos3Aa triple mutant (Pos3AaTM) with enhanced cargo release. We demonstrate that Pos3AaTM-mCherry-p16 fusion crystals can efficiently deliver p16 protein, a CDK4/6 inhibitor frequently inactivated in human cancers, into p16-deficient UM-SCC-22A cells, where it promotes significant G1 cell cycle arrest and cell growth inhibition. These results highlight the ability of the Pos3AaTM platform to promote potent cytosolic delivery of protein therapeutics, and the efficacy of p16 protein delivery as an effective strategy for treating cancer.

Mitochondrial Biogenesis, Mitochondrial Dynamics, and Mitophagy in the Maturation of Cardiomyocytes.

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.

TRPC7 regulates the electrophysiological functions of embryonic stem cell-derived cardiomyocytes.

BACKGROUND: Biological pacemakers consisting of pluripotent stem cell-derived cardiomyocytes are potentially useful for treating bradycardia. However, tachyarrhythmia caused by derived cardiomyocytes themselves is one of main barriers hampering their clinical translation. An in-depth understanding of the mechanisms underlying the spontaneous action potential (a.k.a. automaticity) might provide potential approaches to solve this problem. The aim of this project is to study the role of canonical transient receptor potential isoform 7 (TRPC7) channels in regulating the automaticity of embryonic stem cell-derived cardiomyocytes (ESC-CMs). METHODS AND RESULTS: By Western blotting, the expression of TRPC7 was found to be increased during the differentiation of mouse ESC-CMs (mESC-CMs). Adenovirus-mediated TRPC7 knockdown decreased while overexpression increased the frequency of Ca2+ transients (CaTs), local Ca2+ releases (LCRs), and action potentials (APs) as detected by confocal microscopy and whole-cell patch-clamping. TRPC7 was found to be positively associated with the activity of ryanodine receptor 2 (RyR2), sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), and sodium-calcium exchanger (NCX) but not hyperpolarization-activated, cyclic nucleotide-gated channel (HCN), and inositol trisphosphate receptor (IP3R). Knockdown or overexpression of TRPC7 did not alter the expression of HCN4, Cav1.3, Cav3.1, Cav3.2, IP3R1, RyR2, and SERCA but positively regulated the phosphorylation of RyR2 at S2814 and phospholamban (PLN) at T17. Moreover, the positive regulation of APs by TRPC7 was Ca2+-dependent, as overexpression of N-terminus of TRPC7 (dominant negative of TRPC7) which diminished the Ca2+ permeability of TRPC7 decreased the AP frequency. CONCLUSIONS: TRPC7 regulates the automaticity of mESC-CMs through two mechanisms. On the one hand, TRPC7 positively regulates the intracellular Ca2+ clock through the regulation of activities of both RyR2 and SERCA; on the other hand, TRPC7 also positively regulates the membrane clock via its influence on NCX activity. Altogether, our study reveals that TRPC7 is a potential drug target to manipulate the action potential firing rate of pluripotent stem cell-derived cardiomyocyte-based biological pacemakers to prevent tachyarrhythmia, a condition that might be encountered after transplantation.

TRPV1 channels regulate the automaticity of embryonic stem cell-derived cardiomyocytes through stimulating the Na+ /Ca2+ exchanger current.

Calcium controls the excitation-contraction coupling in cardiomyocytes. Embryonic stem cell-derived cardiomyocytes (ESC-CMs) are an important cardiomyocyte source for regenerative medicine and drug screening. Transient receptor potential vanilloid 1 (TRPV1) channels are nonselective cation channels that permeate sodium and calcium. This study aimed to investigate whether TRPV1 channels regulate the electrophysiological characteristics of ESC-CMs. If yes, what is the mechanism behind? By immunostaining and subcellular fractionation, followed by western blotting, TRPV1 was found to locate intracellularly. The staining pattern of TRPV1 was found to largely overlap with that of the sarco/endoplasmic reticulum Ca2+ -ATPase, the sarcoplasmic reticulum (SR) marker. By electrophysiology and calcium imaging, pharmacological blocker of TRPV1 and the molecular tool TRPV1ß (which could functionally knockdown TRPV1) were found to decrease the rate and diastolic depolarization slope of spontaneous action potentials, and the amplitude and frequency of global calcium transients. By calcium imaging, in the absence of external calcium, TRPV1-specific opener increased intracellular calcium; this increase was abolished by preincubation with caffeine, which could deplete SR calcium store. The results suggest that TRPV1 controls calcium release from the SR. By electrophysiology, TRPV1 blockade and functional knockdown of TRPV1 decreased the Na+ /Ca2+ exchanger (NCX) currents from both the forward and reverse modes, suggesting that sodium and calcium through TRPV1 stimulate the NCX activity. Our novel findings suggest that TRPV1 activity is important for regulating the spontaneous activity of ESC-CMs and reveal a novel interplay between TRPV1 and NCX in regulating the physiological functions of ESC-CMs.

Contamination profiles and health impact of benzothiazole and its derivatives in PM2.5 in typical Chinese cities.

Although benzothiazole and its derivatives (BTHs) are considered emerging contaminants in diverse environments and organisms, little information is available about their contamination profiles and health impact in ambient particles. In this study, an optimized method of ultrasound-assisted extraction coupled with the selected reaction monitoring (SRM) mode of GC-EI-MS/MS was applied to characterize and analyze PM2.5-bound BTHs from three cities of China (Guangzhou, Shanghai, and Taiyuan) during the winter of 2018. The total BTH concentration (ΣBTHs) in PM2.5 samples from the three cities decreased in the order of Guangzhou > Shanghai > Taiyuan, independently of the PM2.5 concentration. Despite the large variation in concentration of ΣBTHs in PM2.5, 2-hydroxybenzothiazole (OTH) was always the predominant compound among the PM2.5-bound BTHs and accounted for 50-80% of total BTHs in the three regions. Results from human exposure assessment and toxicity screening indicated that the outdoor exposure risk of PM2.5-bound BTHs in toddlers was much higher than in adults, especially for OTH. The developmental and reproduction toxicity of OTH was further explored in vivo and in vitro. Exposure of mouse embryonic stem cells (mESCs) to OTH for 48 h significantly increased the intracellular reactive oxygen species (ROS) and induced DNA damage and apoptosis via the functionally activating p53 expression. In addition, the growth and development of zebrafish embryos were found to be severely affected after OTH treatment. An overall metabolomics study was conducted on the exposed zebrafish larvae. The results indicated that exposure to OTH inhibited the phenylalanine hydroxylation reaction, which further increased the accumulation of toxic phenylpyruvate and acetylphenylalanine in zebrafish. These findings provide important insights into the contamination profiles of PM2.5-bound BTHs and emphasize the health risk of OTH.

Uptake, Accumulation, and Biomarkers of PM2.5-Associated Organophosphate Flame Retardants in C57BL/6 Mice after Chronic Exposure at Real Environmental Concentrations.

Although the bioaccumulation of organophosphate flame retardants (OPFRs) in aquatic organisms has been investigated, little information is available about their bioaccumulation in mammals following chronic inhalation exposure. To address this knowledge gap, C57BL/6 mice were exposed to 7 PM2.5-associated OPFRs via the trachea to study their bioaccumulation, tissue distribution, and urinary metabolites. Low (corresponding to the real PM2.5 concentrations occurring during winter in Guangzhou), medium, and high dosages were examined. After 72 days' exposure, ∑OPFR concentrations in tissues from mice in the medium dosage group decreased in the order of intestine > heart > stomach > testis > kidney > spleen > brain > liver > lung > muscle. Of the OPFRs detected in all three exposure groups, chlorinated alkyl OPFRs were most heavily accumulated in mice. We found a significant positive correlation between the bioaccumulation ratio and octanol-air partition coefficient (KOA) in mice tissues for low log KOW OPFR congeners (log KOW ≤ 4, p < 0.05). Three urinary metabolites (di-p-cresyl phosphate: DCrP, diphenyl phosphate: DPhP, dibutyl phosphate: DnBP) were detected from the high dosage group. These results provide important insights into the bioaccumulation potential of OPFRs in mammals and emphasize the health risk of chlorinated alkyl OPFRs.

Chemical identity and cardiovascular toxicity of hydrophobic organic components in PM2.5.

Numerous experimental and epidemiological studies have demonstrated that exposure to PM2.5 may result in pathogenesis of several major cardiovascular diseases (CVDs), which can be attributed to the combined adverse effects induced by the complicated components of PM2.5. Organic materials, which are major components of PM2.5, contain thousands of chemicals, and most of them are environmental hazards. However, the contamination profile and contribution to overall toxicity of PM2.5-bound organic components (OCs) have not been thoroughly evaluated yet. Herein, we aim to provide an overview of the literature on PM2.5-bound hydrophobic OCs, with an emphasis on the chemical identity and reported impairments on the cardiovascular system, including the potential exposure routes and mechanisms. We first provide an update on the worldwide mass concentration and composition data of PM2.5, and then, review the contamination profile of PM2.5-bound hydrophobic OCs, including constitution, concentration, distribution, formation, source, and identification. In particular, the link between exposure to PM2.5-bound hydrophobic OCs and CVDs and its possible underlying mechanisms are discussed to evaluate the possible risks of PM2.5-bound hydrophobic OCs on the cardiovascular system and to provide suggestions for future studies.

PinX1t, a Novel PinX1 Transcript Variant, Positively Regulates Cardiogenesis of Embryonic Stem Cells.

Background Pin2/TRF1-interacting protein, PinX1, was previously identified as a tumor suppressor. Here, we discovered a novel transcript variant of mPinX1 (mouse PinX1), mPinX1t (mouse PinX1t), in embryonic stem cells (ESCs). The aims of this investigation were (1) to detect the presence of mPinX1 and mPinX1t in ESCs and their differentiation derivatives; (2) to investigate the role of mPinX1 and mPinX1t on regulating the characteristics of undifferentiated ESCs and the cardiac differentiation of ESCs; (3) to elucidate the molecular mechanisms of how mPinX1 and mPinX1t regulate the cardiac differentiation of ESCs. Methods and Results By 5' rapid amplification of cDNA ends, 3' rapid amplification of cDNA ends, and polysome fractionation followed by reverse transcription-polymerase chain reaction, mPinX1t transcript was confirmed to be an intact mRNA that is actively translated. Western blot confirmed the existence of mPinX1t protein. Overexpression or knockdown of mPinX1 (both decreased mPinX1t expression) both decreased while overexpression of mPinX1t increased the cardiac differentiation of ESCs. Although both mPinX1 and mPinX1t proteins were found to bind to cardiac transcription factor mRNAs, only mPinX1t protein but not mPinX1 protein was found to bind to nucleoporin 133 protein, a nuclear pore complex component. In addition, mPinX1t-containing cells were found to have a higher cytosol-to-nucleus ratio of cardiac transcription factor mRNAs when compared with that in the control cells. Our data suggested that mPinX1t may positively regulate cardiac differentiation by enhancing export of cardiac transcription factor mRNAs through interacting with nucleoporin 133. Conclusions We discovered a novel transcript variant of mPinX1, the mPinX1t, which positively regulates the cardiac differentiation of ESCs.

Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity.

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44+ CD24-/low EpCAM+ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.

Post-Translational Modification and Natural Mutation of TRPC Channels.

: Transient Receptor Potential Canonical (TRPC) channels are homologues of Drosophila TRP channel first cloned in mammalian cells. TRPC family consists of seven members which are nonselective cation channels with a high Ca2+ permeability and are activated by a wide spectrum of stimuli. These channels are ubiquitously expressed in different tissues and organs in mammals and exert a variety of physiological functions. Post-translational modifications (PTMs) including phosphorylation, N-glycosylation, disulfide bond formation, ubiquitination, S-nitrosylation, S-glutathionylation, and acetylation play important roles in the modulation of channel gating, subcellular trafficking, protein-protein interaction, recycling, and protein architecture. PTMs also contribute to the polymodal activation of TRPCs and their subtle regulation in diverse physiological contexts and in pathological situations. Owing to their roles in the motor coordination and regulation of kidney podocyte structure, mutations of TRPCs have been implicated in diseases like cerebellar ataxia (moonwalker mice) and focal and segmental glomerulosclerosis (FSGS). The aim of this review is to comprehensively integrate all reported PTMs of TRPCs, to discuss their physiological/pathophysiological roles if available, and to summarize diseases linked to the natural mutations of TRPCs.

TRPC3 Regulates the Proliferation and Apoptosis Resistance of Triple Negative Breast Cancer Cells through the TRPC3/RASA4/MAPK Pathway.

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.

Acute exposure to triphenyl phosphate inhibits the proliferation and cardiac differentiation of mouse embryonic stem cells and zebrafish embryos.

Attention has recently paid to the interaction of triphenyl phosphate (TPHP) and body tissues, particularly within the reproductive and development systems, due to its endocrine-disrupting properties. However, the acute effects of TPHP on early embryonic development remain unclear. Here, we used mouse embryonic stem cells (mESC) and zebrafish embryos to investigate whether TPHP is an embryo toxicant. First, we found that continuous exposure of TPHP decreased the proliferation and increased the apoptotic populations of mESCs in a concentration-dependent manner. Results of mass spectrometry showed that the intracellular concentration of TPHP reached 39.45 ± 7.72 µg/g w/w after 3 hr of acute exposure with TPHP (38.35 µM) but gradually decreased from 3 hr to 48 hr. Additionally, DNA damage was detected in mESCs after a short-term treatment with TPHP, which in turn, activated DNA damage responses, leading to cell cycle arrest by changing the expression levels of p53, proliferating cell nuclear antigen, and Y15-phosphorylated Cdk I. Furthermore, our results revealed that short-term treatment with TPHP disturbed cardiac differentiation by decreasing the expression levels of Oct4, Sox2, and Nanog and transiently reduced the glycolysis capacity in mESCs. In zebrafish embryos, exposure to TPHP resulted in broad, concentration-dependent developmental defects and coupled with heart malformation and reduced heart rate. In conclusion, the two models demonstrate that acute exposure to TPHP affects early embryonic development and disturbs the cardiomyogenic differentiation.

Flow Cytometric Detection of Newly-formed Breast Cancer Stem Cell-like Cells After Apoptosis Reversal.

Cancer recurrence has long been studied by oncologists while the underlying mechanisms remain unclear. Recently, we and others found that a phenomenon named apoptosis reversal leads to increased tumorigenicity in various cell models under different stimuli. Previous studies have been focused on tracking this process in vitro and in vivo; however, the isolation of real reversed cells has yet to be achieved, which limits our understanding on the consequences of apoptosis reversal. Here, we take advantage of a Caspase-3/7 Green Detection dye to label cells with activated caspases after apoptotic induction. Cells with positive signals are further sorted out by fluorescence-activated cell sorting (FACS) for recovery. Morphological examination under confocal microscopy helps confirm the apoptotic status before FACS. An increase in tumorigenicity can often be attributed to the elevation in the percentage of cancer stem cell (CSC)-like cells. Also, given the heterogeneity of breast cancer, identifying the origin of these CSC-like cells would be critical to cancer treatment. Thus, we prepare breast non-stem cancer cells before triggering apoptosis, isolating caspase-activated cells and performing the apoptosis reversal procedure. Flow cytometry analysis reveals that breast CSC-like cells re-appear in the reversed group, indicating breast CSC-like cells are transited from breast non-stem cancer cells during apoptosis reversal. In summary, this protocol includes the isolation of apoptotic breast cancer cells and detection of changes in CSC percentage in reversed cells by flow cytometry.