Screening for Novel Type 2 Ryanodine Receptor Inhibitors by Endoplasmic Reticulum Ca2+ Monitoring.

Type 2 ryanodine receptor (RyR2) is a Ca2+ release channel on the endoplasmic (ER)/sarcoplasmic reticulum that plays a central role in the excitation-contraction coupling in the heart. Hyperactivity of RyR2 has been linked to ventricular arrhythmias in patients with catecholaminergic polymorphic ventricular tachycardia and heart failure, where spontaneous Ca2+ release via hyperactivated RyR2 depolarizes diastolic membrane potential to induce triggered activity. In such cases, drugs that suppress RyR2 activity are expected to prevent the arrhythmias, but there is no clinically available RyR2 inhibitors at present. In this study, we searched for RyR2 inhibitors from a well-characterized compound library using a recently developed ER Ca2+-based assay, where the inhibition of RyR2 activity was detected by the increase in ER Ca2+ signals from R-CEPIA1er, a genetically encoded ER Ca2+ indicator, in RyR2-expressing HEK293 cells. By screening 1535 compounds in the library, we identified three compounds (chloroxylenol, methyl orsellinate, and riluzole) that greatly increased the ER Ca2+ signal. All of the three compounds suppressed spontaneous Ca2+ oscillations in RyR2-expressing HEK293 cells and correspondingly reduced the Ca2+-dependent [3H]ryanodine binding activity. In cardiomyocytes from RyR2-mutant mice, the three compounds effectively suppressed abnormal Ca2+ waves without substantial effects on the action-potential-induced Ca2+ transients. These results confirm that ER Ca2+-based screening is useful for identifying modulators of ER Ca2+ release channels and suggest that RyR2 inhibitors have potential to be developed as a new category of antiarrhythmic drugs. SIGNIFICANCE STATEMENT: We successfully identified three compounds having RyR2 inhibitory action from a well-characterized compound library using an endoplasmic reticulum Ca2+-based assay, and demonstrated that these compounds suppressed arrhythmogenic Ca2+ wave generation without substantially affecting physiological action-potential induced Ca2+ transients in cardiomyocytes. This study will facilitate the development of RyR2-specific inhibitors as a potential new class of drugs for life-threatening arrhythmias induced by hyperactivation of RyR2.

The significance of CDT1 expression in non-cancerous and cancerous liver in cases with hepatocellular carcinoma.

We previously reported that chromatin licensing and DNA replication factor 1 (CDT1) expression was associated with the extent of proliferation of atypical hepatocytes and the time to postoperative recurrence in cases of hepatocellular carcinoma (HCC). This study aimed to clarify the clinical significance or pathogenesis of CDT1 expression in both non-cancerous and cancerous liver in HCC cases, including previously published data. We investigated the association between the expression of CDT1 in non-cancerous or cancerous liver tissues and histologic findings or biochemical examination results in 62 cases. We also examined the dual localization between CDT1 and FbxW7, P57kip2, P53 and c-Myc by confocal laser scanning microscopy. CDT1 mRNA expression was significantly higher in cancerous liver than in non-cancerous liver (p<0.0001). Elevated CDT1 mRNA expression indicates a significantly degree of inflammatory cell infiltration within lobules, along with elevated serum transaminase levels, and hepatic spare decline. CDT1 mRNA was highly expressed in a group of poorly differentiated cancer cells. CDT1 co-localized with P57kip2, Fbwx7, P53 and c-Myc in the nucleus or cytoplasm of hepatocytes and cancer cells. We found that CDT1 mRNA expression could represent the degree of hepatic spare ability and the high carcinogenic state.

EGF domain peptide of Developmentally regulated endothelial locus1 facilitates gene expression of extracellularly applied plasmid DNA.

BACKGROUND: The successful development of messenger RNA vaccines for SARS-CoV-2 opened up venues for clinical nucleotide-based vaccinations. For development of DNA vaccines, we tested whether the EGF domain peptide of Developmentally regulated endothelial locus1 (E3 peptide) enhances uptake of extracellularly applied plasmid DNA. METHODS: DNA plasmid encoding lacZ or GFP was applied with a conditioned culture medium containing E3 peptide to cell lines in vitro or mouse soleus muscles in vivo, respectively. After 48 h incubation, gene expression was examined by ß-galactosidase (ß-gal) assay and fluorescent microscope, respectively. RESULTS: Application of E3 peptide-containing medium to cultured cell lines induced intense ß-gal activity in a dose-dependent manner. Intra-gastrocnemius injection of E3 peptide-containing medium to mouse soleus muscle succeeded in the induction of GFP fluorescence in many cells around the injection site. CONCLUSIONS: The administration of E3 peptide facilitates transmembrane uptake of extracellular DNA plasmid which induces sufficient extrinsic gene expression.

Soluble factors released by dedifferentiated fat cells reduce the functional activity of iPS cell-derived cardiomyocytes.

Interactions between tissues such as epicardial adipose (EAT), and myocardial tissues is important in the pathogenesis of heart failure. Changes in adipose tissues in obesity or diabetes impair preadipocyte differentiation. Furthermore, proinflammatory cytokine secretion is higher in preadipocytes than in mature adipocytes in diabetes and obesity. However, how undifferentiated cells committed to the adipose lineage directly influence cardiomyocytes is not yet understood. We used human-derived dedifferentiated fat (DFAT) cells as models of undifferentiated cells committed to an adipose lineage. Here, we evaluated the effects of soluble factor interactions in indirect cocultures of DFAT cells and induced pluripotent stem cell-derived cardiomyocytes. Our RNA sequencing findings showed that these interactions were predominantly inflammatory responses. Furthermore, proinflammatory cytokines secreted by DFAT cells reduced myocardial functions such as contraction frequency and catecholamine sensitivity, and simultaneously increased apoptosis, decreased antioxidative stress tolerance, and reduced oxygen consumption rates in cardiomyocytes. These adverse effects might be attributable to monocyte chemoattractant protein-1, chemokine (C-X-C motif) ligands 1 (CXCL1), and 12, granulocyte colony-stimulating factor, interleukins 6 and 8, macrophage migration inhibitory factor (MIF), and plasminogen activator inhibitor 1-A among the proinflammatory mediators secreted by DFAT cells. Our results could be useful for understanding the pathogenesis of EAT-related heart failure in terms of the involvement of undifferentiated cells committed to the adipose lineage. Furthermore, we suggest the importance of focusing on surrounding adipose tissues as a strategy with which to maximize the survival and function of transplanted stem cell-derived cardiomyocytes.

Inositol 1,4,5-trisphosphate receptor type 2-independent Ca2+ release from the endoplasmic reticulum in astrocytes.

Accumulating evidence indicates that astrocytes are actively involved in the physiological and pathophysiological functions of the brain. Intracellular Ca2+ signaling, especially Ca2+ release from the endoplasmic reticulum (ER), is considered to be crucial for the regulation of astrocytic functions. Mice with genetic deletion of inositol 1,4,5-trisphosphate receptor type 2 (IP3 R2) are reportedly devoid of astrocytic Ca2+ signaling, and thus widely used to explore the roles of Ca2+ signaling in astrocytic functions. While functional deficits in IP3 R2-knockout (KO) mice have been found in some reports, no functional deficit was observed in others. Thus, there remains a controversy regarding the functional significance of astrocytic Ca2+ signaling. To address this controversy, we re-evaluated the assumption that Ca2+ release from the ER is abolished in IP3 R2-KO astrocytes using a highly sensitive imaging technique. We expressed the ER luminal Ca2+ indicator G-CEPIA1er in cortical and hippocampal astrocytes to directly visualize spontaneous and stimulus-induced Ca2+ release from the ER. We found attenuated but significant Ca2+ release in response to application of norepinephrine to IP3 R2-KO astrocytes. This IP3 R2-independent Ca2+ release induced only minimal cytosolic Ca2+ transients but induced robust Ca2+ increases in mitochondria that are frequently in close contact with the ER. These results indicate that ER Ca2+ release is retained and is sufficient to increase the Ca2+ concentration in close proximity to the ER in IP3 R2-KO astrocytes.

Efficient High-Throughput Screening by Endoplasmic Reticulum Ca2+ Measurement to Identify Inhibitors of Ryanodine Receptor Ca2+-Release Channels.

Genetic mutations in ryanodine receptors (RyRs), Ca2+-release channels in the sarcoplasmic reticulum essential for muscle contractions, cause various skeletal muscle and cardiac diseases. Because the main underlying mechanism of the pathogenesis is overactive Ca2+ release by gain-of-function of the RyR channel, inhibition of RyRs is expected to be a promising treatment of these diseases. Here, to identify inhibitors specific to skeletal muscle type 1 RyR (RyR1), we developed a novel high-throughput screening (HTS) platform using time-lapse fluorescence measurement of Ca2+ concentrations in the endoplasmic reticulum (ER) ([Ca2+]ER). Because expression of RyR1 carrying disease-associated mutation reduces [Ca2+]ER in HEK293 cells through Ca2+ leakage from RyR1 channels, specific drugs that inhibit RyR1 will increase [Ca2+]ER by preventing such Ca2+ leakage. RyR1 carrying the R2163C mutation and R-CEPIA1er, a genetically encoded ER Ca2+ indicator, were stably expressed in HEK293 cells, and time-lapse fluorescence was measured using a fluorometer. False positives were effectively excluded by using cells expressing wild-type (WT) RyR1. By screening 1535 compounds in a library of well characterized drugs, we successfully identified four compounds that significantly increased [Ca2+]ER They include dantrolene, a known RyR1 inhibitor, and three structurally different compounds: oxolinic acid, 9-aminoacridine, and alexidine. All the hit compounds, except for oxolinic acid, inhibited [3H]ryanodine binding of WT and mutant RyR1. Interestingly, they showed different dose dependencies and isoform specificities. The highly quantitative nature and good correlation with the channel activity validated this HTS platform by [Ca2+]ER measurement to explore drugs for RyR-related diseases.

Genetically Encoded Fluorescent Indicators for Organellar Calcium Imaging.

Optical Ca(2+) indicators are powerful tools for investigating intracellular Ca(2+) signals in living cells. Although a variety of Ca(2+) indicators have been developed, deciphering the physiological functions and spatiotemporal dynamics of Ca(2+) in intracellular organelles remains challenging. Genetically encoded Ca(2+) indicators (GECIs) using fluorescent proteins are promising tools for organellar Ca(2+) imaging, and much effort has been devoted to their development. In this review, we first discuss the key points of organellar Ca(2+) imaging and summarize the requirements for optimal organellar Ca(2+) indicators. Then, we highlight some of the recent advances in the engineering of fluorescent GECIs targeted to specific organelles. Finally, we discuss the limitations of currently available GECIs and the requirements for advancing the research on intraorganellar Ca(2+) signaling.

Visualization of Ca2+ Filling Mechanisms upon Synaptic Inputs in the Endoplasmic Reticulum of Cerebellar Purkinje Cells.

The endoplasmic reticulum (ER) plays crucial roles in intracellular Ca(2+) signaling, serving as both a source and sink of Ca(2+), and regulating a variety of physiological and pathophysiological events in neurons in the brain. However, spatiotemporal Ca(2+) dynamics within the ER in central neurons remain to be characterized. In this study, we visualized synaptic activity-dependent ER Ca(2+) dynamics in mouse cerebellar Purkinje cells (PCs) using an ER-targeted genetically encoded Ca(2+) indicator, G-CEPIA1er. We used brief parallel fiber stimulation to induce a local decrease in the ER luminal Ca(2+) concentration ([Ca(2+)]ER) in dendrites and spines. In this experimental system, the recovery of [Ca(2+)]ER takes several seconds, and recovery half-time depends on the extent of ER Ca(2+) depletion. By combining imaging analysis and numerical simulation, we show that the intraluminal diffusion of Ca(2+), rather than Ca(2+) reuptake, is the dominant mechanism for the replenishment of the local [Ca(2+)]ER depletion immediately following the stimulation. In spines, the ER filled almost simultaneously with parent dendrites, suggesting that the ER within the spine neck does not represent a significant barrier to Ca(2+) diffusion. Furthermore, we found that repetitive climbing fiber stimulation, which induces cytosolic Ca(2+) spikes in PCs, cumulatively increased [Ca(2+)]ER. These results indicate that the neuronal ER functions both as an intracellular tunnel to redistribute stored Ca(2+) within the neurons, and as a leaky integrator of Ca(2+) spike-inducing synaptic inputs.

Genotype-Phenotype Correlations of Malignant Hyperthermia and Central Core Disease Mutations in the Central Region of the RYR1 Channel.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum of skeletal muscle and is mutated in some muscle diseases, including malignant hyperthermia (MH) and central core disease (CCD). Over 200 mutations associated with these diseases have been identified, and most mutations accelerate Ca2+ -induced Ca2+ release (CICR), resulting in abnormal Ca2+ homeostasis in skeletal muscle. However, it remains largely unknown how specific mutations cause different phenotypes. In this study, we investigated the CICR activity of 14 mutations at 10 different positions in the central region of RYR1 (10 MH and four MH/CCD mutations) using a heterologous expression system in HEK293 cells. In live-cell Ca2+ imaging, the mutant channels exhibited an enhanced sensitivity to caffeine, a reduced endoplasmic reticulum Ca2+ content, and an increased resting cytoplasmic Ca2+ level. The three parameters for CICR (Ca2+ sensitivity for activation, Ca2+ sensitivity for inactivation, and attainable maximum activity, i.e., gain) were obtained by [3 H]ryanodine binding and fitting analysis. The mutant channels showed increased gain and Ca2+ sensitivity for activation in a site-specific manner. Genotype-phenotype correlations were explained well by the near-atomic structure of RYR1. Our data suggest that divergent CICR activity may cause various disease phenotypes by specific mutations.

Whisker experience-dependent mGluR signaling maintains synaptic strength in the mouse adolescent cortex.

Sensory experience-dependent plasticity in the somatosensory cortex is a fundamental mechanism of adaptation to the changing environment not only early in the development but also in adolescence and adulthood. Although the mechanisms underlying experience-dependent plasticity during early development have been well documented, the corresponding understanding in the mature cortex is less complete. Here, we investigated the mechanism underlying whisker deprivation-induced synaptic plasticity in the barrel cortex in adolescent mice. Layer 4 (L4) to L2/3 excitatory synapses play a crucial role for whisker experience-dependent plasticity in rodent barrel cortex and whisker deprivation is known to depress synaptic strength at L4-L2/3 synapses in adolescent and adult animals. We found that whisker deprivation for 5 days or longer decreased the presynaptic glutamate release probability at L4-L2/3 synapses in the barrel cortex in adolescent mice. This whisker deprivation-induced depression was restored by daily administration of a positive allosteric modulator of the type 5 metabotropic glutamate receptor (mGluR5). On the other hand, the administration of mGluR5 antagonists reproduced the effect of whisker deprivation in whisker-intact mice. Furthermore, chronic and selective suppression of inositol 1,4,5-trisphosphate (IP3 ) signaling in postsynaptic L2/3 neurons decreased the presynaptic release probability at L4-L2/3 synapses. These findings represent a previously unidentified mechanism of cortical plasticity, namely that whisker experience-dependent mGluR5-IP3 signaling in the postsynaptic neurons maintains presynaptic function in the adolescent barrel cortex.

Calcium-dependent N-cadherin up-regulation mediates reactive astrogliosis and neuroprotection after brain injury.

Brain injury induces phenotypic changes in astrocytes, known as reactive astrogliosis, which may influence neuronal survival. Here we show that brain injury induces inositol 1,4,5-trisphosphate (IP3)-dependent Ca(2+) signaling in astrocytes, and that the Ca(2+) signaling is required for astrogliosis. We found that type 2 IP3 receptor knockout (IP3R2KO) mice deficient in astrocytic Ca(2+) signaling have impaired reactive astrogliosis and increased injury-associated neuronal death. We identified N-cadherin and pumilio 2 (Pum2) as downstream signaling molecules, and found that brain injury induces up-regulation of N-cadherin around the injured site. This effect is mediated by Ca(2+)-dependent down-regulation of Pum2, which in turn attenuates Pum2-dependent translational repression of N-cadherin. Furthermore, we show that astrocyte-specific knockout of N-cadherin results in impairment of astrogliosis and neuroprotection. Thus, astrocytic Ca(2+) signaling and the downstream function of N-cadherin play indispensable roles in the cellular responses to brain injury. These findings define a previously unreported signaling axis required for reactive astrogliosis and neuroprotection following brain injury.

Selection of red fluorescent protein for genetic labeling of mitochondria and intercellular transfer of viable mitochondria.

The phenomenon of intercellular mitochondrial transfer has attracted great attention in various fields of research, including stem cell biology. Elucidating the mechanism of mitochondrial transfer from healthy stem cells to cells with mitochondrial dysfunction may lead to the development of novel stem cell therapies to treat mitochondrial diseases, among other advances. To visually evaluate and analyze the mitochondrial transfer process, dual fluorescent labeling systems are often used to distinguish the mitochondria of donor and recipient cells. Although enhanced green fluorescent protein (EGFP) has been well-characterized for labeling mitochondria, other colors of fluorescent protein have been less extensively evaluated in the context of mitochondrial transfer. Here, we generated different lentiviral vectors with mitochondria-targeted red fluorescent proteins (RFPs), including DsRed, mCherry (both from Discosoma sp.) Kusabira orange (mKOκ, from Verrillofungia concinna), and TurboRFP (from Entacmaea quadricolor). Among these proteins, mitochondria-targeted DsRed and its variant mCherry often generated bright aggregates in the lysosome while other proteins did not. We further validated that TurboRFP-labeled mitochondria were successfully transferred from amniotic epithelial cells, one of the candidates for donor stem cells, to mitochondria-damaged recipient cells without losing the membrane potential. Our study provides new insight into the genetic labeling of mitochondria with red fluorescent proteins, which may be utilized to analyze the mechanism of intercellular mitochondrial transfer.

Inhibition of Epithelial-Mesenchymal Transition Maintains Stemness in Human Amniotic Epithelial Cells.

Human amniotic epithelial cells (hAECs), which are a type of placental stem cell, express stem cell marker genes and are capable of differentiating into all three germ layers under appropriate culture conditions. hAECs are known to undergo TGF-ß-dependent epithelial-mesenchymal transition (EMT); however, the impact of EMT on the stemness or differentiation of hAECs has not yet been determined. Here, we first confirmed that hAECs undergo EMT immediately after starting primary culture. Comprehensive transcriptome analysis using RNA-seq revealed that inhibition of TGF-ß-dependent EMT maintained the expression of stemness-related genes, including NANOG and POU5F1, in hAECs. Moreover, the maintenance of stemness did not affect the nontumorigenic characteristics of hAECs. We showed for the first time that TGF-ß-dependent EMT negatively affected the stemness of hAECs, providing novel insight into cellular processes of placental stem cells.

Cytosolic Ca2+-dependent Ca2+ release activity primarily determines the ER Ca2+ level in cells expressing the CPVT-linked mutant RYR2.

Type 2 ryanodine receptor (RYR2) is a cardiac Ca2+ release channel in the ER. Mutations in RYR2 are linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is associated with enhanced spontaneous Ca2+ release, which tends to occur when [Ca2+]ER reaches a threshold. Mutations lower the threshold [Ca2+]ER by increasing luminal Ca2+ sensitivity or enhancing cytosolic [Ca2+] ([Ca2+]cyt)-dependent activity. Here, to establish the mechanism relating the change in [Ca2+]cyt-dependent activity of RYR2 and the threshold [Ca2+]ER, we carried out cell-based experiments and in silico simulations. We expressed WT and CPVT-linked mutant RYR2s in HEK293 cells and measured [Ca2+]cyt and [Ca2+]ER using fluorescent Ca2+ indicators. CPVT RYR2 cells showed higher oscillation frequency and lower threshold [Ca2+]ER than WT cells. The [Ca2+]cyt-dependent activity at resting [Ca2+]cyt, Arest, was greater in CPVT mutants than in WT, and we found an inverse correlation between threshold [Ca2+]ER and Arest. In addition, lowering RYR2 expression increased the threshold [Ca2+]ER and a product of Arest, and the relative expression level for each mutant correlated with threshold [Ca2+]ER, suggesting that the threshold [Ca2+]ER depends on the net Ca2+ release rate via RYR2. Modeling reproduced Ca2+ oscillations with [Ca2+]cyt and [Ca2+]ER changes in WT and CPVT cells. Interestingly, the [Ca2+]cyt-dependent activity of specific mutations correlated with the age of disease onset in patients carrying them. Our data suggest that the reduction in threshold [Ca2+]ER for spontaneous Ca2+ release by CPVT mutation is explained by enhanced [Ca2+]cyt-dependent activity without requiring modulation of the [Ca2+]ER sensitivity of RYR2.

Differences in the stemness characteristics and molecular markers of distinct human oral tissue neural crest-derived multilineage cells.

OBJECTIVES: Although multilineage cells derived from oral tissues, especially the dental pulp, apical papilla, periodontal ligament, and oral mucosa, have neural crest-derived stem cell (NCSC)-like properties, the differences in the characteristics of these progenitor cell compartments remain unknown. The current study aimed to elucidate these differences. MATERIAL AND METHODS: Sphere-forming apical papilla-derived cells (APDCs), periodontal ligament-derived cells (PDLDCs), and oral mucosa stroma-derived cells (OMSDCs) from the same individuals were isolated from impacted developing teeth. All sphere-forming cells were characterized through biological analyses of stem cells. RESULTS: All sphere-forming cells expressed neural crest-related markers. The expression of certain tissue-specific markers such as CD24 and CD56 (NCAM1) differed among tissue-derived cells. Surprisingly, the expression of only CD24 and CD56 could be discriminated in human tissues. Although APDCs and PDLDCs exhibited greater mineralized cell differentiation than OMSDCs, they exhibited poorer differentiation into adipocytes in vitro. In immunocompromised mice, APDCs formed hard tissues better than PDLDCs and OMSDCs. CONCLUSIONS: Although cells with NCSC-like properties present the same phenotype, they differ in the expression of certain markers and differentiation abilities. This study is the first to demonstrate the differences in the differentiation ability and molecular markers among multilineage human APDCs, PDLDCs, and OMSDCs obtained from the same patients, and to identify tissue-specific markers that distinguish tissues in the developing stage of the human tooth with immature apex.

Anti-tumor effects of perampanel in malignant glioma cells.

Glioblastoma has a poor prognosis even after multimodal treatment, such as surgery, chemotherapy and radiation therapy. Patients with glioblastoma frequently develop epileptic seizures during the clinical course of the disease and often require antiepileptic drugs. Therefore, agents with both antiepileptic and antitumoral effects may be very useful for glioblastoma treatment. Perampanel, an α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor antagonist, is an antiepileptic drug that is widely used for intractable epilepsy. The present study aimed to assess the potential antitumoral effects of perampanel using malignant glioma cell lines. The cell proliferation inhibitory effect was evaluated using six malignant glioma cell lines (A-172, AM-38, T98G, U-138MG, U-251MG and YH-13). A dose-dependent inhibitory effect of perampanel on cell viability was demonstrated; however, the sensitivity of cells to perampanel varied and further antitumoral effects were demonstrated in combination with temozolomide (TMZ) in certain malignant glioma cells. Furthermore, cell cycle distribution and apoptosis induction analyses were performed in T98G and U-251MG cells using a fluorescence activated cell sorter (FACS) and the expression levels of apoptosis-related proteins were evaluated using western blotting. No significant change was demonstrated in the proportions of cells in the G0/G1, S and G2/M phases under 1.0 µM perampanel treatment, whereas induction of apoptosis was demonstrated using FACS at 10 µM perampanel and western blotting at 1.0 µM perampanel in both glioma cell lines. Overexpression of SERPINE1 may be related to poor prognosis in patients with gliomas. The combination of 1.0 µM perampanel and 5.0 µM tiplaxtinin, a SERPINE1 inhibitor, demonstrated further reduced cell viability in perampanel-resistant U-138MG cells, which have high expression levels of SERPINE1. These results indicated that the antitumor effect of perampanel may not be expected for malignant gliomas with higher expression levels of SERPINE1. The findings of the present study suggested that the antiepileptic drug perampanel may also have an antitumor effect through the induction of apoptosis, which is increased when combined with TMZ in certain malignant glioma cells. These findings also suggested that SERPINE1 expression may be involved in perampanel susceptibility. These results may lead to new therapeutic strategies for malignant glioma.

The proliferation of atypical hepatocytes and CDT1 expression in noncancerous tissue are associated with the postoperative recurrence of hepatocellular carcinoma.

Recently, we reported that extent of proliferation of atypical hepatocytes (atypical hepatocytes) was most important histological risk factor for development of hepatocellular carcinoma (HCC) from chronic hepatitis C or liver cirrhosis. Here, we aimed to clarify whether the atypical hepatocytes in noncancerous sections is also involved in postoperative recurrence. Furthermore, we investigated significant genes involved in the atypical hepatocytes. Association between the extent of atypical hepatocytes in noncancerous tissue and postoperative recurrence was validated in 356 patients with HCC. Next, we identified putative signature genes involved in extent of atypical hepatocytes. First, atypical hepatocytes or hepatocytes other than the atypical hepatocyte in noncancerous sections of 4 HCC patients were selectively collected by laser capture microdissection (LCM). Second, the gene expression profiles of the selected hepatocyte populations were compared using Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher SCIENTIFIC, Waltham, MA, USA) analysis. Finally, we validated the mRNA expression of the extracted genes in noncancerous frozen liver tissue from 62 patients with HCC by RT-qPCR to identify the signature genes involved in both the extent of atypical hepatocytes and postoperative recurrence. Furthermore, the extent of atypical hepatocytes and CDT1 expression in noncancerous sections from 8 patients with HCC were also validated by selectively collecting samples using LCM. The extent of atypical hepatocytes was associated with postoperative recurrence. Of the genes that showed significant differences in expression levels between two populations, the expression of the chromatin licensing and DNA replication factor 1 (CDT1) gene was most strongly associated with the extent of atypical hepatocytes and was also associated with postoperative recurrence. Furthermore, CDT1-positive cells that exhibited stronger expression resembled those morphologically considered to be atypical hepatocytes. CDT1 and Ki-67 were colocalized in the nuclei of both hepatocytes and cancer cells. The hepatocytes in noncancerous livers were not uniform in each hepatocyte population, suggesting that the accumulation of genetic abnormalities was variable. We found that the strong degree of atypical hepatocytes and high CDT1 mRNA expression represent a high carcinogenic state of the liver. Thus, we consider the evaluation of degree of these could support the personalized medicine.

Red fluorescent CEPIA indicators for visualization of Ca2+ dynamics in mitochondria.

Mitochondrial Ca2+ dynamics are involved in the regulation of multifarious cellular processes, including intracellular Ca2+ signalling, cell metabolism and cell death. Use of mitochondria-targeted genetically encoded Ca2+ indicators has revealed intercellular and subcellular heterogeneity of mitochondrial Ca2+ dynamics, which are assumed to be determined by distinct thresholds of Ca2+ increases at each subcellular mitochondrial domain. The balance between Ca2+ influx through the mitochondrial calcium uniporter and extrusion by cation exchangers across the inner mitochondrial membrane may define the threshold; however, the precise mechanisms remain to be further explored. We here report the new red fluorescent genetically encoded Ca2+ indicators, R-CEPIA3mt and R-CEPIA4mt, which are targeted to mitochondria and their Ca2+ affinities are engineered to match the intramitochondrial Ca2+ concentrations. They enable visualization of mitochondrial Ca2+ dynamics with high spatiotemporal resolution in parallel with the use of green fluorescent probes and optogenetic tools. Thus, R-CEPIA3mt and R-CEPIA4mt are expected to be a useful tool for elucidating the mechanisms of the complex mitochondrial Ca2+ dynamics and their functions.

Insights into channel modulation mechanism of RYR1 mutants using Ca2+ imaging and molecular dynamics.

Type 1 ryanodine receptor (RYR1) is a Ca2+ release channel in the sarcoplasmic reticulum in skeletal muscle and plays an important role in excitation-contraction coupling. Mutations in the RYR1 gene cause severe muscle diseases such as malignant hyperthermia (MH), which is a disorder of CICR via RYR1. Thus far, >300 mutations in RYR1 have been reported in patients with MH. However, owing to a lack of comprehensive analysis of the structure-function relationship of mutant RYR1, the mechanism remains largely unknown. Here, we combined functional studies and molecular dynamics (MD) simulations of RYR1 bearing disease-associated mutations at the N-terminal region. When expressed in HEK293 cells, the mutant RYR1 caused abnormalities in Ca2+ homeostasis. MD simulations of WT and mutant RYR1s were performed using crystal structure of the N-terminal domain (NTD) monomer, consisting of A, B, and C domains. We found that the mutations located around the interdomain region differentially affected hydrogen bonds/salt bridges. Particularly, mutations at R402, which increase the open probability of the channel, cause clockwise rotation of BC domains with respect to the A domain by alteration of the interdomain interactions. Similar results were also obtained with artificial mutations that mimic alteration of the interactions. Our results reveal the importance of interdomain interactions within the NTD in the regulation of the RYR1 channel and provide insights into the mechanism of MH caused by the mutations at the NTD.