Erythrocytic α-Synuclein and the Gut Microbiome: Kindling of the Gut-Brain Axis in Parkinson's Disease.

BACKGROUND: Progressive spreading of α-synuclein via gut-brain axis has been hypothesized in the pathogenesis of Parkinson's disease (PD). However, the source of seeding-capable α-synuclein in the gastrointestinal tract (GIT) has not been fully investigated. Additionally, the mechanism by which the GIT microbiome contributes to PD pathogenesis remains to be characterized. OBJECTIVES: We aimed to investigate whether blood-derived α-synuclein might contribute to PD pathology via a gut-driven pathway and involve GIT microbiota. METHODS: The GIT expression of α-synuclein and the transmission of extracellular vesicles (EVs) derived from erythrocytes/red blood cells (RBCs), with their cargo α-synuclein, to the GIT were explored with various methods, including radioactive labeling of RBC-EVs and direct analysis of the transfer of α-synuclein protein. The potential role of microbiota on the EVs transmission was further investigated by administering butyrate, the short-chain fatty acids produced by gut microbiota and studying mice with different α-synuclein genotypes. RESULTS: This study demonstrated that RBC-EVs can effectively transport α-synuclein to the GIT in a region-dependent manner, along with variations closely associated with regional differences in the expression of gut-vascular barrier markers. The investigation further revealed that the infiltration of α-synuclein into the GIT was influenced significantly by butyrate and α-synuclein genotypes, which may also affect the GIT microbiome directly. CONCLUSION: By demonstrating the transportation of α-synuclein through RBC-EVs to the GIT, and its potential association with gut-vascular barrier markers and gut microbiome, this work highlights a potential mechanism by which RBC α-synuclein may impact PD initiation and/or progression. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Selective oxidative protection leads to tissue topological changes orchestrated by macrophage during ulcerative colitis.

Ulcerative colitis is a chronic inflammatory bowel disorder with cellular heterogeneity. To understand the composition and spatial changes of the ulcerative colitis ecosystem, here we use imaging mass cytometry and single-cell RNA sequencing to depict the single-cell landscape of the human colon ecosystem. We find tissue topological changes featured with macrophage disappearance reaction in the ulcerative colitis region, occurring only for tissue-resident macrophages. Reactive oxygen species levels are higher in the ulcerative colitis region, but reactive oxygen species scavenging enzyme SOD2 is barely detected in resident macrophages, resulting in distinct reactive oxygen species vulnerability for inflammatory macrophages and resident macrophages. Inflammatory macrophages replace resident macrophages and cause a spatial shift of TNF production during ulcerative colitis via a cytokine production network formed with T and B cells. Our study suggests components of a mechanism for the observed macrophage disappearance reaction of resident macrophages, providing mechanistic hints for macrophage disappearance reaction in other inflammation or infection situations.

Gut-derived ß-amyloid: Likely a centerpiece of the gut-brain axis contributing to Alzheimer's pathogenesis.

Peripheral ß-amyloid (Aß), including those contained in the gut, may contribute to the formation of Aß plaques in the brain, and gut microbiota appears to exert an impact on Alzheimer's disease (AD) via the gut-brain axis, although detailed mechanisms are not clearly defined. The current study focused on uncovering the potential interactions among gut-derived Aß in aging, gut microbiota, and AD pathogenesis. To achieve this goal, the expression levels of Aß and several key proteins involved in Aß metabolism were initially assessed in mouse gut, with key results confirmed in human tissue. The results demonstrated that a high level of Aß was detected throughout the gut in both mice and human, and gut Aß42 increased with age in wild type and mutant amyloid precursor protein/presenilin 1 (APP/PS1) mice. Next, the gut microbiome of mice was characterized by 16S rRNA sequencing, and we found the gut microbiome altered significantly in aged APP/PS1 mice and fecal microbiota transplantation (FMT) of aged APP/PS1 mice increased gut BACE1 and Aß42 levels. Intra-intestinal injection of isotope or fluorescence labeled Aß combined with vagotomy was also performed to investigate the transmission of Aß from gut to brain. The data showed that, in aged mice, the gut Aß42 was transported to the brain mainly via blood rather than the vagal nerve. Furthermore, FMT of APP/PS1 mice induced neuroinflammation, a phenotype that mimics early AD pathology. Taken together, this study suggests that the gut is likely a critical source of Aß in the brain, and gut microbiota can further upregulate gut Aß production, thereby potentially contributing to AD pathogenesis.

In Situ Imaging of Formaldehyde in Live Mice with High Spatiotemporal Resolution Reveals Aldehyde Dehydrogenase-2 as a Potential Target for Alzheimer's Disease Treatment.

Alterations in formaldehyde (FA) homeostasis are associated with the pathology of Alzheimer's disease (AD). In vivo tracking of FA flux is important for understanding the underlying molecular mechanisms, but is challenging due to the lack of sensitive probes favoring a selective, rapid, and reversible response toward FA. In this study, we re-engineered the promiscuous and irreversible phenylhydrazines to make them selective and reversible toward FA by tuning their nucleophilicity. This effort resulted in PFM309, a selective (selectivity coefficient KFA,methylglyoxal = 0.06), rapid (t1/2 = 32 s at [FA] = 200 µM), and reversible fluorogenic probe (K = 6.24 mM-1) that tracks the FA flux in both live cells and live mice. In vivo tracking of the FA flux was realized by PFM309 imaging, which revealed the gradual accumulation of FA in the live mice brain during normal aging and its further increase in AD mice. We further identified the age-dependent loss of catabolism enzymes ALDH2 and ADH5 as the primary mechanism responsible for formaldehyde excess. Activating ALDH2 with the small molecular activator Alda1 significantly protected neurovascular cells from formaldehyde overload and consequently from impairment during AD progress both in vitro and in vivo. These findings revealed PFM309 as a robust tool to study AD pathology and highlight ALDH2 as a potential target for AD drug development.

Involvement of nitrosative stress cytotoxicity induced by CdTe quantum dots in human vascular endothelial cells.

Quantum dots (QDs) are new types of fluorescent nanomaterials which can be utilized as ideal agents for intracellular tracking, drug delivery, biomedical imaging and diagnosis. It is urgent to understand their potential toxicity and the interactions with the toxin-susceptible vascular system, especially vascular endothelial cells. In this study, we intended to explore whether the cytotoxicity of CdTe (cadmium telluride) QDs was partly induced by nitrosative stress in vascular endothelial cells. Our results showed that the intracellular amount of CdTe QDs was gradually increased in a dose- and time-dependent manner, and a concentration-dependent decrease in viability were observed when incubated with CdTe QDs of 20-80 nM. The peroxynitrite level was significantly up-regulated by QDs treatment, which indicated the nitrosative stress was activated. Furthermore, nitrotyrosine level was increased after 24 hr CdTe QDs exposure in a dose-dependent manner, which suggested that CdTe QDs-induced nitrosative stress was associated with tyrosine nitration in EA.hy926. In addition, CdTe QDs induced EA.hy926 apoptosis, and the percentage of cells with low Δψm was increased after CdTe QDs treatment, indicating the mitochondrion depolarization was induced. The increased ROS fluorescence was observed in a QDs dose-dependent manner, which suggested that the oxidative stress was also involved in the CdTe QDs-induced endothelial cytotoxicity. Our work provided experimental evidence into QDs toxicity and potential vascular risks induced by nitrosative stress for the future applications of QDs.

Co-transfection with BMP2 and FGF2 via chitosan nanoparticles potentiates osteogenesis in human adipose-derived stromal cells in vitro.

OBJECTIVE: To investigate if co-transfection of human bone morphogenetic protein 2 (BMP-2, BMP2) and human fibroblast growth factor 2 (FGF2, FGF2) via chitosan nanoparticles promotes osteogenesis in human adipose tissue-derived stem cells (ADSCs) in vitro. MATERIALS AND METHODS: Recombinant BMP2 and/or FGF2 expression vectors were constructed and packaged into chitosan nanoparticles. The chitosan nanoparticles were characterized by atomic force microscopy. Gene and protein expression levels of BMP-2 and FGF2 in ADSCs in vitro were evaluated by real-time polymerase chain reaction (PCR), western blot, and enzyme-linked immunosorbent assay. Osteocalcin (OCN) and bone sialoprotein (BSP) gene expression were also evaluated by real-time PCR to assess osteogenesis. RESULTS: The prepared chitosan nanoparticles were spherical with a relatively homogenous size distribution. The BMP2 and FGF2 vectors were successfully transfected into ADSCs. BMP-2 and FGF2 mRNA and protein levels were significantly up-regulated in the co-transfection group compared with the control group. OCN and BSP mRNA levels were also significantly increased in the co-transfection group compared with cells transfected with BMP2 or FGF2 alone, suggesting that co-transfection significantly enhanced osteogenesis. CONCLUSIONS: Co-transfection of human ADSCs with BMP2/FGF2 via chitosan nanoparticles efficiently promotes the osteogenic properties of ADSCs in vitro.

Establishment and characterization of a docetaxel-resistant human prostate cancer cell line.

The aim of the present study was to establish a novel docetaxel-resistant prostate cancer cell line and investigate its biological characteristics. The human prostate cell line, PC-3, was exposed to docetaxel, the concentrations of which were increased in a stepwise manner in the medium to select the drug-resistant cell line, PC-3/DTX. The morphological features were observed using inverted microscopy. The growth curves of PC-3 and PC-3/DTX cells were drawn to calculate the doubling time. Flow cytometry was performed to determine cell-cycle distribution. A 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2H tetrazolium bromide assay was performed to test the drug resistance of PC-3 and PC-3/DTX cells. Western blot analysis was conducted to determine the protein expression levels of the mammalian target of rapamycin (mTOR) signaling pathway, which may serve a role in regulating drug resistance in the two cell lines. PC-3/DTX cells exhibited changes in morphology, proliferation rate, doubling time and cell-cycle distributions, compared with PC-3 cells. PC-3/DTX cells were 10.9-fold resistant to docetaxel in comparison with PC-3 cells. The results showed that PC-3/DTX cells overexpressed Rictor and p-AKT(S473) proteins, which are specific subunits or downstream substrates of mTORC2. The new findings suggested that the mTORC2 signaling pathway may serve an important role in the regulation of docetaxel drug resistance of PC-3 cells. In conclusion, PC-3/DTX cells may be applied to study the resistance of anticancer drugs and to identify methods to overcome resistance.

Sequentially Activated Probe Design Strategy for Analyzing Metabolite Crosstalk in a Biochemical Cascade.

Interrogating metabolite crosstalk in live cells is important to understand the interplay between metabolic and signal transduction pathways but is challenging due to the lack of efficient analytical techniques. Here we report a sequentially activated probe design strategy resulting in probe HF-6 being capable of imaging the crosstalk between H2O2 and formaldehyde in live cells. Fluorescence of HF-6 can only be triggered by first H2O2 activation followed by binding with formaldehyde. Facilitated by this sequentially activated mechanism, HF-6 imaging revealed H2O2-induced upregulation of formaldehyde in live SH-SY5Y cells, while little change of intracellular H2O2 level was observed when cells were stimulated with formaldehyde for limited time. These results establish a link for the crosstalk between H2O2 and formaldehyde in redox signaling and provide a starting point to study broader metabolite interactions.

New ß-carboline fluorophores with superior sensitivity and endoplasmic reticulum specificity for tracking ER changes.

Autofluorescing carboline-fluors were efficiently and rationally developed via a Pictet-Spengler involved one-pot multi-component reaction. The carboline-fluors demonstrate specific targeting towards the endoplasmic reticulum in living cells, and superior sensitivity to commercial ER-Trackers. Importantly, they were also successfully used to visualize changes in the ER during cell apoptosis and ER stress.

Physicochemical-property guided design of a highly sensitive probe to image nitrosative stress in the pathology of stroke.

In vivo real-time imaging of nitrosative stress in the pathology of stroke has long been a formidable challenge due to both the presence of the blood-brain barrier (BBB) and the elusive nature of reactive nitrogen species, while this task is also informative to gain a molecular level understanding of neurovascular injury caused by nitrosative stress during the stroke episode. Herein, using a physicochemical property-guided probe design strategy in combination with the reaction-based probe design rationale, we have developed an ultrasensitive probe for imaging nitrosative stress evolved in the pathology of stroke. This probe demonstrates an almost zero background fluorescence signal but a maximum 1000-fold fluorescence enhancement in response to peroxynitrite, the nitrosative stress marker. Due to its good physicochemical properties, the probe readily penetrates the BBB after intravenous administration, and quickly accumulates in mice brain to sense local vascular injuries. After accomplishing its imaging mission, the probe is easily metabolized and therefore won't cause safety concerns. These desirable features make the probe competent for the straightforward visualization of nitrosative stress progression in stroke pathology.

Conformational restraint as a strategy for navigating towards lysosomes.

Using the conformational restraint strategy, we developed a hydrazonate-derived coumarin into a lysosome targeting probe for imaging native formaldehyde at the subcellular level. Using this probe, we observed the overproduction of formaldehyde in lysosomes when cells were treated with endoplasmic reticulum (ER) stress inducers, suggesting the involvement of formaldehyde in protein misfolding.

Myt1l induced direct reprogramming of pericytes into cholinergic neurons.

OBJECTIVE: The cholinergic deficit is thought to underlie progressed cognitive decline in Alzheimer Disease. The lineage reprogramming of somatic cells into cholinergic neurons may provide strategies toward cell-based therapy of neurodegenerative diseases. METHODS AND RESULTS: Here, we found that a combination of neuronal transcription factors, including Ascl1, Myt1l, Brn2, Tlx3, and miR124 (5Fs) were capable of directly converting human brain vascular pericytes (HBVPs) into cholinergic neuronal cells. Intriguingly, the inducible effect screening of reprogramming factors showed that a single reprogramming factor, Myt1l, induced cells to exhibit similarly positive staining for Tuj1, MAP2, ChAT, and VAChT upon lentivirus infection with the 5Fs after 30 days. HBVP-converted neurons were rarely labeled even after long-term incubation with BrdU staining, suggesting that induced neurons were directly converted from HBVPs rather than passing through a proliferative state. In addition, the overexpression of Myt1l induced the elevation of Ascl1, Brn2, and Ngn2 levels that contributed to reprogramming. CONCLUSIONS: Our findings provided proof of the principle that cholinergic neurons could be produced from HBVPs by reprogramming factor-mediated fate instruction. Myt1l was a critical mediator of induced neuron cell reprogramming. HBVPs represent another excellent alternative cell resource for cell-based therapy to treat neurodegenerative disease.

A H2O2-Responsive Theranostic Probe for Endothelial Injury Imaging and Protection.

Overproduction of H2O2 causes oxidative stress and is the hallmark of vascular diseases. Tracking native H2O2 in the endothelium is therefore indispensable to gain fundamental insights into this pathogenesis. Previous fluorescent probes for H2O2 imaging were generally arylboronates which were decomposed to emissive arylphenols in response to H2O2. Except the issue of specificity challenged by peroxynitrite, boric acid by-produced in this process is actually a waste with unknown biological effects. Therefore, improvements could be envisioned if a therapeutic agent is by-produced instead. Herein, we came up with a "click-to-release-two" strategy and demonstrate that dual functional probes could be devised by linking a fluorophore with a therapeutic agent via a H2O2-responsive bond. As a proof of concept, probe AP consisting of a 2-(2'-hydroxyphenyl) benzothiazole fluorophore and an aspirin moiety has been prepared and confirmed for its theranostic effects. This probe features high specificity towards H2O2 than other reactive species including peroxynitrite. Its capability to image and ameliorate endothelial injury has been verified both in vitro and in vivo. Noteworthy, as a result of its endothelial-protective effect, AP also works well to reduce thrombosis formation in zebrafish model.

A Fluorogenic Probe for Ultrafast and Reversible Detection of Formaldehyde in Neurovascular Tissues.

Formaldehyde (FA) is endogenously produced in live systems and has been implicated in a diverse array of pathophysiological processes. To disentangle the detailed molecular mechanisms of FA biology, a reliable method for monitoring FA changes in live cells would be indispensable. Although there have been several fluorescent probes reported to detect FA, most are limited by the slow detection kinetics and the intrinsic disadvantage of detecting FA in an irreversible manner which may disturb endogenous FA homeostasis. Herein we developed a coumarin-hydrazonate based fluorogenic probe (PFM) based on a finely-tailored stereoelectronic effect. PFM could respond to FA swiftly and reversibly. This, together with its desirable specificity and sensitivity, endows us to track endogenous FA in live neurovascular cells with excellent temporal and spatial resolution. Further study in the brain tissue imaging showed the first direct observation of aberrant FA accumulation in cortex and hippocampus of Alzheimer's mouse model, indicating the potential of PFM as a diagnostic tool.

Inhibition of 5-lipoxygenase inhibitor zileuton in high-fat diet-induced nonalcoholic fatty liver disease progression model.

OBJECTIVES: Arachidonic Acid/5-lipoxygenase (AA/5-LOX) pathway connects lipid metabolism and proinflammatory cytokine, which are both related to the development and progression of nonalcoholic fatty liver disease (NAFLD). Therefore, the present study was designed to investigate the role of AA/5-LOX pathway in progression of NAFLD, and the effect of zileuton, an inhibitor of 5-LOX, in this model. MATERIALS AND METHODS: Animal model for progression of NAFLD was established via feeding high saturated fat diet (HFD). Liver function, HE staining, NAFLD activity score (NAS) were used to evaluate NAFLD progression. We detected the lipid metabolism substrates: free fatty acids (FFA) and AA, products: cysteinyl-leukotrienes (CysLTs), and changes in gene and protein level of key enzyme in AA/5-LOX pathway including PLA2 and 5-LOX. Furthermore, we determined whether NAFLD progression pathway was delayed or reversed when zileuton (1-[1-(1-benzothiophen-2-yl)ethyl]-1-hydroxyurea) was administrated. RESULTS: Rat model for progression of NAFLD was well established as analyzed by liver transaminase activities, hematoxylin-eosin (HE) staining and NAS. The concentrations of substrates and products in AA/5-LOX pathway were increased with the progression of NAFLD. mRNA and protein expression of PLA2 and 5-LOX were all enhanced. Moreover, administration of zileuton inhibited AA/5-LOX pathway and reversed the increased transamine activities and NAS. CONCLUSION: AA/5-LOX pathway promotes the progression of NAFLD, which can be reversed by zileuton.

Stress-responsive heme oxygenase-1 isoenzyme participates in Toll-like receptor 4-induced inflammation during brain ischemia.

Toll-like receptors (TLRs) are involved in the progression of ischemic brain injury and hence vascular dementia; however, the underlying mechanisms are largely unknown. Here, we have investigated the interrelationship between stress-responsive heme oxygenase (HO)-1 isoenzyme and TLR4 during chronic brain hypoperfusion. The right unilateral common carotid artery occlusion was performed by ligation of the right common carotid artery in C57BL/6J mice. The brain cortex or hippocampus was removed for western blotting and confocal immunofluorescence analysis. The link between HO-1 and TLR4 was further examined by silencing TLR4 and pharmacological inhibition of HO-1 in primary cultured cortical neurons. Cognitive dysfunction and decrease in cerebral blood flow in mice were observed 4 weeks after the occlusion. Our data further show that common carotid artery occlusion induced an increase in TLR4 and HO-1 protein levels. Although the administration of CoPP (10 mg/kg), HO-1 agonist, improved the cognitive dysfunction in a mice model of occlusion, western blot analysis in primary cultured cortical neurons showed that HO-1 was upregulated after lipopolysaccharide treatment; this was partially abolished by the TLR4 siRNA interference. The flow cytometry analysis showed that pharmacological inhibition of HO-1 by ZnPP (100 µM) further exaggerated lipopolysaccharide-induced neuronal cell death. Hence, stress-responsive HO-1 isoenzyme participates in TLR4-induced inflammation during chronic brain ischemia. The pharmacological manipulation of TLR4 or the HO-1 antioxidant defense pathway may represent a novel treatment strategy for neuronal protection in vascular dementia.

Pulmonary Toxicities of Gefitinib in Patients With Advanced Non-Small-Cell Lung Cancer: A Meta-Analysis of Randomized Controlled Trials.

Gefitinib is a selective tyrosine kinase inhibitor of the epidermal growth factor receptor (EGFR) used to treat adults with EGFR mutation-positive non-small-cell lung cancer (NSCLC). Clinical benefits of gefitinib administration in NSCLC patients have been observed in clinical practice, but the extent of the pulmonary toxicity of gefitinib in patients with advanced NSCLC remains unclear. The aim of this systematic review was to evaluate the overall incidence and risk of gefitinib-related pulmonary toxicity in advanced NSCLC patients. Relevant trials were identified from the databases of Pubmed, Embase, Cochrane Library, and the clinicaltrials.gov of the U.S. National Institutes of Health. The outcomes included the overall incidence, odds ratios (ORs), and 95% confidence intervals (CIs). Fixed-effects models were used in the statistical analyses according to the heterogeneity of the included studies. According to the data from the included trials, the overall incidence of high-grade hemoptysis, pneumonia, pneumonitis, and interstitial lung disease (ILD) was 0.49% (95% CI: 0.24%-0.99%), 2.33% (95% CI: 1.47%-3.66%), 2.24% (95% CI: 1.34%-3.72%), and 1.43% (95% CI: 0.98%-2.09%), respectively. The pooled ORs of high-grade hemoptysis, pneumonia, pneumonitis, and ILD were 1.73 (95% CI: 0.46-6.52; P = 0.42), 0.99 (95% CI: 0.66-1.49; P = 0.95), 4.70 (95% CI: 1.48-14.95; P = 0.0087), and 2.64 (95% CI: 1.22-5.69; P = 0.01), respectively. Gefitinib was associated with a significantly increased risk of high-grade/fatal ILD and pneumonitis compared with the controls, whereas the risk of other high-grade pulmonary events (pneumonia and hemoptysis) was not significant. Careful surveillance of gefitinib-related pulmonary toxicity is critical for the safe use of this drug.

In vivo pharmacokinetics of and tissue distribution study of physalin B after intravenous administration in rats by liquid chromatography with tandem mass spectrometry.

A rapid and sensitive liquid chromatography tandem mass spectrometry quantitative analysis method was established for the pharmacokinetics and tissue distribution study of physalin B in rat. Physalin B and physalin H (internal standard, IS) were separated on an Agilent Eclips XDB C8 column. MS detection was performed on a triple quadrupole tandem mass spectrometer in the multiple reaction monitoring mode with a positive eletrospray ionization source. The assay was validated in the concentration ranges of 22.6-22600 ng/mL for heart and lung and 4.52-4520 ng/mL for other tissues. The intra- and inter-day precisions (RSD) were ≤9.23 and ≤12.51%, respectively, with accuracy (%) in the range of 88.07-113.2%. A pharmacokinetic study showed that physalin B has a long dwell time with a half-life of 321.2 ± 29.5 min and clearance of 175.4 ± 25.7 mL/min/kg after intravenous administration. Additionally, physalin B showed a wide tissue distribution with a special higher penetration in lung. The data presented in this study could provide useful information for the further study of physalin B. Copyright © 2016 John Wiley & Sons, Ltd.

Characterization of endonuclease G and mitochondria-sarcoplasmic reticulum-related proteins during cardiac hypertrophy.

Endonuclease G (Endo G) is a novel determinant of cardiac hypertrophy. Here, we report the characterization of Endo G and mitochondria-sarcoplasmic reticulum-related proteins during cardiac hypertrophy, and hypothesize that Endo G regulate mitochondrial function partly through Mfn2 and Jp2 during cardiac hypertrophy. Our results show that Endo G levels gradually increased at the beginning of phenylephrine-induced cardiac hypertrophy, accompanied by an abnormal mitochondrial membrane potential. The up-regulation of Mfn2, Jp2, and Endo G appeared at an early stage of cardiac hypertrophy, whereas PGC1α was not up-regulated until a later stage. Abolishing Endo G with siRNA led to the uncoupling of the mitochondrial electron transport chain from ATP production and decreased PGC1α expression, likely by affecting the juxtaposition of the mitochondria and the sarcoplasmic reticulum via Mfn2 and Jp2. Furthermore, abolishing Jp2 altered the expression of Endo G expression and induced mitochondrial dysfunction, suggesting that mitochondrial abnormalities in cardiac hypertrophy are most likely caused by Endo G. Taken together, our study established a link between Endo G and mitochondrial function during cardiac hypertrophy, partly through the effects of Endo G on Mfn2 and Jp2, and revealed a role for Endo G in the crosstalk between the processes controlled by Mfn2 and Jp2 in maladaptive cardiac hypertrophy.

Icariin promotes expression of junctophilin 2 and Ca2+ related function during cardiomyocyte differentiation of murine embryonic stem cells.

Junctophilin2 (JP2) is a critical protein associated with cardiogenesis. Icariin (ICA) facilitated the directional differentiation of murine embryonic stem (ES) cells into cardiomyocytes. However, little is known about the effects of ICA on JP2 during cardiac differentiation. Here, we explored whether ICA has effects on the expression and Ca2+ related function of JP2 during cardiomyocyte differentiation of ES cells in vitro. Embryonid bodies (EBs) formed by hanging drop were treated with 10(-7) mol/L ICA from day 5 to promote the cardiac differentiation. Percentage of beating EBs and number of beating area within EBs were monitored. Cardiomyocytes were purified by discontinuous percoll gradient centrifugation from EBs. The expression of JP2, α-actinin and troponin-T within EBs or isolated cardiomyocytes were analyzed by immunocytochemistry, western blot and flow cytometry. The transient Ca2+ release was characterized in cardiomyocytes treated with/without 10 mmol/L caffeine and 8 mmol/L Ca2+. Our results showed that ES cell-derived cardiomyocytes were well characterized with JP2 proteins. ICA promoted cardiomyocyte differentiation as indicated by an increased percentage of beating EBs and number of beating area within EBs. The expression of JP2, α-actinin and troponin-T were up-regulated both in EBs and isolated cardiomyocytes from EBs. Furthermore, ICA-induced JP2 expression was accompanied by a remarkable increase of the amplitude of Ca2+ transients in cardiomyocytes before/after caffeine and Ca2+ stimulating. In conclusion, ICA promotes in cardiac differentiation partly through regulating JP2 and improved the Ca2+ modulatory function of cardiomyocytes.