Ion channel mRNA distribution and expression in the sinoatrial node and right atrium of dogs and monkeys
Journal of Toxicologic Pathology
SANO, T;YASUNO, H;WATANABE, T;
| DOI: 10.1293/tox.2020-0089
There are limited data on the gene expression profiles of ion channels in the sinoatrial node (SAN) of dogs and monkeys. In this study, the messenger RNA (mRNA) expression profiles of various ion channels in the SAN of naïve dogs and monkeys were examined using RNAscope _in situ _hybridization and compared with those in the surrounding right atrium (RA) of each species. Regional-specific Cav1.3 and HCN4 expression was observed in the SAN of dogs and monkeys. Additionally, HCN1 in dogs was only expressed in the SAN group. The expression profiles of Cav3.1 and Cav3.2 in the SAN and RA were completely different between dogs and monkeys. Dog hearts only expressed Cav3.2; however, Cav3.1 was detected only in monkeys, and the expression score in the SAN was slightly higher than that in the RA. Although Kir3.1 and NCX1 in dogs were equally expressed in both the SAN and RA, the expression scores of these genes in the SAN of monkeys were slightly higher than those in the RA. The Kir3.4 expression score in the SAN of dogs and monkeys was also slightly higher than that in the RA. The mRNA expression scores of Kv11.1/ERG and KvLQT1 were equally observed in both the SAN and RA of dogs and monkeys. HCN2 was not detected in dogs and monkeys. In summary, we used RNAscope to demonstrate the SAN-specific gene expression patterns of ion channels, which may be useful in explaining the effect of pacemaking and/or hemodynamic effects in nonclinical studies.
Bogdanov, V;Soltisz, A;Beard, C;Hernandez Orengo, B;Sakuta, G;Veeraraghavan, R;Davis, J;Gyorke, S;
| DOI: 10.1016/j.bpj.2022.11.1389
Aberrant Ca-CaM signaling has been implicated in various congenital and acquired cardiac pathologies, including arrhythmia, hypertrophy, and HF. We examined the impact of HF induced by trans-aortic constriction (TAC) on the distribution of the three CaM mRNAs (Calm 1,2 and 3) and their key protein target mRNAs (Ryr2, Scn5a, Camk2d, NOS1 and Cacna1c) in cardiomyocytes, using fluorescence in situ hybridization (RNAScope™). HF resulted in specific changes in the pattern of localization of Calms, manifested in redistribution of Calm3 from the cell periphery towards the perinuclear area and enhanced Calm2 attraction to the perinuclear area compared to sham myocytes. Additionally, HF resulted in redistribution of mRNAs for certain CaM target mRNAs. Particularly, NOS1 localization shifted from the cell periphery towards the perinuclear area, Cacna1c, Camk2d and Scn5a abundance increased at the perinuclear area, and Ryr2 attracted even closer to the cell periphery in HF myocytes compared to sham myocytes. The strength of non-random attraction/repulsion was measured as the maximal deviation between the observed distribution of nearest neighbor distances from the distribution predicted under complete spatial randomness. Consistent with the observed alterations in abundance and distribution of CaM and CaM target mRNAs, HF resulted in increased attraction between Calm1 and Scn5a, Ryr2 and Camk2d, between Calm2 and Ryr2 and Camk2d; and between Calm3 and NOS1 and Scn5a. In contrast, the attraction between Calm3 and Ryr2 decreased in HF myocytes compared to sham. Collectively, these results suggest distribution of Calms and their association with key target protein mRNAs undergo substantial alterations in heart failure. These results have new important implications for organization of Ca signaling in normal and diseased heart.
Steece-Collier K, Stancati JA, Collier NJ, Sandoval IM, Mercado NM, Sortwell CE, Collier TJ, Manfredsson FP.
PMID: 31002755 | DOI: 10.1002/mds.27695
Abstract
BACKGROUND:
Levodopa-induced dyskinesias are an often debilitating side effect of levodopa therapy in Parkinson's disease. Although up to 90% of individuals with PD develop this side effect, uniformly effective and well-tolerated antidyskinetic treatment remains a significant unmet need. The pathognomonic loss of striatal dopamine in PD results in dysregulation and disinhibition of striatal CaV1.3 calcium channels, leading to synaptopathology that appears to be involved in levodopa-induced dyskinesias. Although there are clinically available drugs that can inhibit CaV1.3 channels, they are not adequately potent and have only partial and transient impact on levodopa-induced dyskinesias.
METHODS:
To provide unequivocal target validation, free of pharmacological limitations, we developed a CaV1.3 shRNA to provide high-potency, target-selective, mRNA-level silencing of striatal CaV1.3 channels and examined its ability to impact levodopa-induced dyskinesias in severely parkinsonian rats.
RESULTS:
We demonstrate that vector-mediated silencing of striatal CaV1.3 expression in severely parkinsonian rats prior to the introduction of levodopa can uniformly and completely prevent induction of levodopa-induced dyskinesias, and this antidyskinetic benefit persists long term and with high-dose levodopa. In addition, this approach is capable of ameliorating preexisting severe levodopa-induced dyskinesias. Importantly, motoric responses to low-dose levodopa remained intact in the presence of striatal CaV1.3 silencing, indicating preservation of levodopa benefit without dyskinesia liability.
DISCUSSION:
The current data provide some of the most profound antidyskinetic benefit reported to date and suggest that genetic silencing of striatal CaV1.3 channels has the potential to transform treatment of individuals with PD by allowing maintenance of motor benefit of levodopa in the absence of the debilitating levodopa-induced dyskinesia side effect.
Medvedev, R;Turner, D;Gorelik, J;Alvarado, F;Bondarenko, V;Glukhov, A;
| DOI: 10.1016/j.bpj.2022.11.1392
Atrial fibrillation (AF) is commonly observed in patients with hypertension and is associated with pathologically elevated cardiomyocyte stretch. AF triggers have been linked to subcellular Ca2+ abnormalities, while their association with stretch remains elusive. Caveolae are mechanosensitive membrane structures, that play a role in both Ca2+ and cyclic adenosine monophosphate (cAMP) signaling. Therefore, caveolae could provide a mechanistic connection between cardiomyocyte stretch, Ca2+ mishandling, and AF. In isolated mouse atrial myocytes, cell stretch was mimicked by hypotonic swelling, which increased cell width (by ∼30%, p
Larsen, F;Hansen, D;Terkelsen, M;Bendixen, S;Avolio, F;Wernberg, C;Lauridsen, M;Grønkjaer, L;Jacobsen, B;Klinggaard, E;Mandrup, S;Di Caterino, T;Siersbæk, M;Chandran, V;Graversen, J;Krag, A;Grøntved, L;Ravnskjaer, K;
| DOI: 10.1016/j.jhepr.2022.100615
Background & Aims Non-alcoholic fatty liver disease (NAFLD) and its progressive form, non-alcoholic steatohepatitis (NASH), are the hepatic manifestations of metabolic syndrome. Histological assessment of liver biopsies is the gold standard for diagnosis of NASH. A Liver biopsy is resource heavy, can lead to complications such as bleeding, and does not fully capture tissue heterogeneity of the fibrotic liver. Therefore, non-invasive biomarkers that can reflect an integrated state of the liver are highly needed to improve diagnosis and sampling bias. Hepatic stellate cells (HSCs) are central in development of hepatic fibrosis, a hallmark of NASH. Secreted HSC-specific proteins may, therefore, reflect disease state in the NASH liver and serve as non-invasive diagnostic biomarkers. Methods We performed RNA-sequencing on liver biopsies from a histological characterised cohort of obese patients (n = 30, body mass index > 35 kg/m2) to identify and evaluate HSC-specific genes encoding secreted proteins. Bioinformatics was used to identify potential biomarkers and their expression at single-cell resolution. We validated our findings by single-molecule fluorescence in situ hybridisation (smFISH) and ELISA to detect mRNA in liver tissue and protein levels in plasma, respectively. Results Hepatic expression of SPARC-related modular calcium-binding protein 2 (SMOC2) was increased in NASH compared no-NAFLD (p.adj < 0.001). Single-cell RNA-sequencing data indicated SMOC2 expression by HSCs, which was validated using smFISH. Finally, plasma SMOC2 was elevated in NASH compared to no-NAFLD (p < 0.001) with a predictive accuracy of AUROC 0.88. Conclusions We propose increased SMOC2 in plasma reflects HSC activation, a key cellular event associated with NASH progression, and may serve as a non-invasive biomarker of NASH.
Thibault S, Hu W, Hirakawa B, Kalabat D, Franks T, Sung T, Khoh-Reiter S, Lu S, Finkelstein M, Jessen B, Sacaan AI.
PMID: 30401694 | DOI: 10.1158/1535-7163.MCT-18-0734
Recently three different cyclin-dependent kinase 4 and 6 (CDK4/6) dual inhibitors were approved for the treatment of breast cancer (palbociclib, ribociclib and abemaciclib), all of which offer comparable therapeutic benefits. Their safety profiles however are different. For example, neutropenia is observed at varying incidences in patients treated with these drugs; however it is the most common adverse event for palbociclib and ribociclib, whereas diarrhea is the most common adverse event observed in patients treated with abemaciclib. In order to understand the mechanism of diarrhea observed with these drugs and in an effort to guide the development of safer drugs, we compared the effects of oral administration of palbociclib, ribociclib and abemaciclib on the gastrointestinal tract of rats using doses intended to produce comparable CDK4/6 inhibition. Rats administered abemaciclib, but not palbociclib or ribociclib, had fecal alterations, unique histopathological findings and distinctive changes in intestinal gene expression. Morphologic changes in the intestine were characterized by proliferation of crypt cells, loss of goblet cells, poorly differentiated and degenerating enterocytes with loss of microvilli and mucosal inflammation. In the jejunum of abemaciclib-treated rats, down-regulation of enterocyte membrane transporters and up-regulation of genes associated with cell proliferation were observed, consistent with activation of the Wnt pathway and downstream transcriptional regulation. Among these CDK4/6 inhibitors, intestinal toxicity was unique to rats treated with abemaciclib, suggesting a mechanism of toxicity not due to primary pharmacology (CDK4/6 inhibition), but to activity at secondary pharmacological targets.
Ramlow, L;Falcke, M;Lindner, B;
| DOI: 10.1016/j.bpj.2022.11.1390
Stochastic spiking is a prominent feature of Ca2+ signaling. The main noise source at the cellular level are puffs from inositol-trisphosphate receptor (IP3R) channel clusters in the membrane of the endoplasmic reticulum (ER). While the random cluster activity has been known for decades, a stringent method to derive the puff noise term acting on the cytosolic Ca2+ concentration is still lacking. We adopt a popular description of neural spike generation from neuroscience, the stochastic integrate-and-fire (IF) model, to describe Ca2+ spiking. Our model consists of two components describing i) activity of IP3R clusters and ii) dynamics of the global Ca2+ concentrations in the cytosol and in the ER. Cluster activity is modeled by a Markov chain, capturing the puff. The global Ca2+ concentrations are described by a two-variable IF model driven by the puff current. For the Markov chain we derive expressions for the statistics of interpuff interval, single-puff strength, and puff current assuming constant cytosolic Ca2+, an assumption often well met because the Ca2+ concentrations vary much slower than the cluster activity does. The latter assumption also allows to approximate the driving Ca2+ dependent puff current by a white Gaussian noise. This approximation results in an IF model with nonlinear drift and multiplicative noise. We consider this reduced model in a renewal version and in a version with cumulative refractoriness. Neglecting ER depletion, the stochastic IF model has only one variable and generates a renewal spike train, a point process with statistically independent interspike intervals (ISI). We derive analytical expressions for the mean and coefficient of variation of the ISI and suggest approximations for the ISI density and spike-train power spectrum. Taking into account ER depletion, the two-variable IF model displays cumulative refractoriness as seen in experimental data.
Corrêa, RO;Castro, PR;Fachi, JL;Nirello, VD;El-Sahhar, S;Imada, S;Pereira, GV;Pral, LP;Araújo, NVP;Fernandes, MF;Matheus, VA;de Souza Felipe, J;Dos Santos Pereira Gomes, AB;de Oliveira, S;de Rezende Rodovalho, V;de Oliveira, SRM;de Assis, HC;Oliveira, SC;Dos Santos Martins, F;Martens, E;Colonna, M;Varga-Weisz, P;Vinolo, MAR;
PMID: 37101209 | DOI: 10.1186/s40168-023-01520-2
The continuous proliferation of intestinal stem cells followed by their tightly regulated differentiation to epithelial cells is essential for the maintenance of the gut epithelial barrier and its functions. How these processes are tuned by diet and gut microbiome is an important, but poorly understood question. Dietary soluble fibers, such as inulin, are known for their ability to impact the gut bacterial community and gut epithelium, and their consumption has been usually associated with health improvement in mice and humans. In this study, we tested the hypothesis that inulin consumption modifies the composition of colonic bacteria and this impacts intestinal stem cells functions, thus affecting the epithelial structure.Mice were fed with a diet containing 5% of the insoluble fiber cellulose or the same diet enriched with an additional 10% of inulin. Using a combination of histochemistry, host cell transcriptomics, 16S microbiome analysis, germ-free, gnotobiotic, and genetically modified mouse models, we analyzed the impact of inulin intake on the colonic epithelium, intestinal bacteria, and the local immune compartment.We show that the consumption of inulin diet alters the colon epithelium by increasing the proliferation of intestinal stem cells, leading to deeper crypts and longer colons. This effect was dependent on the inulin-altered gut microbiota, as no modulations were observed in animals deprived of microbiota, nor in mice fed cellulose-enriched diets. We also describe the pivotal role of γδ T lymphocytes and IL-22 in this microenvironment, as the inulin diet failed to induce epithelium remodeling in mice lacking this T cell population or cytokine, highlighting their importance in the diet-microbiota-epithelium-immune system crosstalk.This study indicates that the intake of inulin affects the activity of intestinal stem cells and drives a homeostatic remodeling of the colon epithelium, an effect that requires the gut microbiota, γδ T cells, and the presence of IL-22. Our study indicates complex cross kingdom and cross cell type interactions involved in the adaptation of the colon epithelium to the luminal environment in steady state. Video Abstract.
Kim HS, Lee C, Kim WH, Maeng YH, Jang BG.
PMID: 28747693 | DOI: 10.1038/s41598-017-06900-x
The intestinal epithelium has two distinct two stem cell populations, namely, crypt base columnar (CBC) cells and +4 cells. Several specific markers have been identified for each stem cell population. In this study, we examined the expression profiles of these markers in colitis-associated carcinogenesis (CAC) to investigate whether they can be used as biomarkers for the early detection of dysplasia. The expression of intestinal stem cell (ISC) markers was measured by real-time polymerase chain reaction during CAC that was induced by azoxymethane and dextran sodium sulfate treatment. CBC stem cell markers increased continuously with tumor development, whereas a +4 cell expression profile was not present. CBC stem cell population was suppressed in the acute colitis and then expanded to repopulate the crypts during the regeneration period. Notably, RNA in situ hybridization revealed that all dysplasia and cancer samples showed increased expression of CBC stem cell markers in more than one-third of the tumor height, whereas regenerative glands had CBC stem cell markers confined to the lower one-third of the crypt. These results suggest that CBC stem cell markers could be a useful tool for the early detection of colitis-induced tumors.
Azkanaz, M;Corominas-Murtra, B;Ellenbroek, SIJ;Bruens, L;Webb, AT;Laskaris, D;Oost, KC;Lafirenze, SJA;Annusver, K;Messal, HA;Iqbal, S;Flanagan, DJ;Huels, DJ;Rojas-Rodríguez, F;Vizoso, M;Kasper, M;Sansom, OJ;Snippert, HJ;Liberali, P;Simons, BD;Katajisto, P;Hannezo, E;van Rheenen, J;
PMID: 35831497 | DOI: 10.1038/s41586-022-04962-0
The morphology and functionality of the epithelial lining differ along the intestinal tract, but tissue renewal at all sites is driven by stem cells at the base of crypts1-3. Whether stem cell numbers and behaviour vary at different sites is unknown. Here we show using intravital microscopy that, despite similarities in the number and distribution of proliferative cells with an Lgr5 signature in mice, small intestinal crypts contain twice as many effective stem cells as large intestinal crypts. We find that, although passively displaced by a conveyor-belt-like upward movement, small intestinal cells positioned away from the crypt base can function as long-term effective stem cells owing to Wnt-dependent retrograde cellular movement. By contrast, the near absence of retrograde movement in the large intestine restricts cell repositioning, leading to a reduction in effective stem cell number. Moreover, after suppression of the retrograde movement in the small intestine, the number of effective stem cells is reduced, and the rate of monoclonal conversion of crypts is accelerated. Together, these results show that the number of effective stem cells is determined by active retrograde movement, revealing a new channel of stem cell regulation that can be experimentally and pharmacologically manipulated.
Human Adult Fibroblast-like Synoviocytes and Articular Chondrocytes Exhibit Prominent Overlap in Their Transcriptomic Signatures
Jones, K;Angelozzi, M;Gangishetti, U;Haseeb, A;de Charleroy, C;Lefebvre, V;Bhattaram, P;
PMID: 33931959 | DOI: 10.1002/acr2.11255
Fibroblast-like synoviocytes (FLS) and articular chondrocytes (AC) derive from a common pool of embryonic precursor cells. They are currently believed to engage in largely distinct differentiation programs to build synovium and articular cartilage and maintain healthy tissues throughout life. We tested this hypothesis by deeply characterizing and comparing their transcriptomic attributes. We profiled the transcriptomes of freshly isolated AC, synovium, primary FLS, and dermal fibroblasts from healthy adult humans using bulk RNA sequencing assays and downloaded published single-cell RNA sequencing data from freshly isolated human FLS. We integrated all data to define cell-specific signatures and validated findings with quantitative reverse transcription PCR of human samples and RNA hybridization of mouse joint sections. We identified 212 AC and 168 FLS markers on the basis of exclusive or enriched expression in either cell and 294 AC/FLS markers on the basis of similar expression in both cells. AC markers included joint-specific and pan-cartilaginous genes. FLS and AC/FLS markers featured 37 and 55 joint-specific genes, respectively, and 131 and 239 pan-fibroblastic genes, respectively. These signatures included many previously unrecognized markers with potentially important joint-specific roles. AC/FLS markers overlapped in their expression patterns among all FLS and AC subpopulations, suggesting that they fulfill joint-specific properties in all, rather than in discrete, AC and FLS subpopulations. This study broadens knowledge and identifies a prominent overlap of the human adult AC and FLS transcriptomic signatures. It also provides data resources to help further decipher mechanisms underlying joint homeostasis and degeneration and to improve the quality control of tissues engineered for regenerative treatments.
Tracing the origin of hair follicle stem cells
Morita, R;Sanzen, N;Sasaki, H;Hayashi, T;Umeda, M;Yoshimura, M;Yamamoto, T;Shibata, T;Abe, T;Kiyonari, H;Furuta, Y;Nikaido, I;Fujiwara, H;
PMID: 34108685 | DOI: 10.1038/s41586-021-03638-5
Tissue stem cells are generated from a population of embryonic progenitors through organ-specific morphogenetic events1,2. Although tissue stem cells are central to organ homeostasis and regeneration, it remains unclear how they are induced during development, mainly because of the lack of markers that exclusively label prospective stem cells. Here we combine marker-independent long-term 3D live imaging and single-cell transcriptomics to capture a dynamic lineage progression and transcriptome changes in the entire epithelium of the mouse hair follicle as it develops. We found that the precursors of different epithelial lineages were aligned in a 2D concentric manner in the basal layer of the hair placode. Each concentric ring acquired unique transcriptomes and extended to form longitudinally aligned, 3D cylindrical compartments. Prospective bulge stem cells were derived from the peripheral ring of the placode basal layer, but not from suprabasal cells (as was previously suggested3). The fate of placode cells is determined by the cell position, rather than by the orientation of cell division. We also identified 13 gene clusters: the ensemble expression dynamics of these clusters drew the entire transcriptional landscape of epithelial lineage diversification, consistent with cell lineage data. Combining these findings with previous work on the development of appendages in insects4,5, we describe the 'telescope model', a generalized model for the development of ectodermal organs in which 2D concentric zones in the placode telescope out to form 3D longitudinally aligned cylindrical compartments.
