Flavin Containing Monooxygenase 2 Prevents Cardiac Fibrosis via CYP2J3-SMURF2 Axis.

BACKGROUND: Cardiac fibrosis is a common pathological feature associated with adverse clinical outcome in postinjury remodeling and has no effective therapy. Using an unbiased transcriptome analysis, we identified FMO2 (flavin-containing monooxygenase 2) as a top-ranked gene dynamically expressed following myocardial infarction (MI) in hearts across different species including rodents, nonhuman primates, and human. However, the functional role of FMO2 in cardiac remodeling is largely unknown. METHODS: Single-nuclei transcriptome analysis was performed to identify FMO2 after MI; FMO2 ablation rats were generated both in genetic level using the CRISPR-cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) technology and lentivirus-mediated manner. Gain-of-function experiments were conducted using postn-promoter FMO2, miR1a/miR133a-FMO2 lentivirus, and enzymatic activity mutant FMO2 lentivirus after MI. RESULTS: A significant downregulation of FMO2 was consistently observed in hearts after MI in rodents, nonhuman primates, and patients. Single-nuclei transcriptome analysis showed cardiac expression of FMO2 was enriched in fibroblasts rather than myocytes. Elevated spontaneous tissue fibrosis was observed in the FMO2-null animals without external stress. In contrast, fibroblast-specific expression of FMO2 markedly reduced cardiac fibrosis following MI in rodents and nonhuman primates associated with diminished SMAD2/3 phosphorylation. Unexpectedly, the FMO2-mediated regulation in fibrosis and SMAD2/3 signaling was independent of its enzymatic activity. Rather, FMO2 was detected to interact with CYP2J3 (cytochrome p450 superfamily 2J3). Binding of FMO2 to CYP2J3 disrupted CYP2J3 interaction with SMURF2 (SMAD-specific E3 ubiquitin ligase 2) in cytosol, leading to increased cytoplasm to nuclear translocation of SMURF2 and consequent inhibition of SMAD2/3 signaling. CONCLUSIONS: Loss of FMO2 is a conserved molecular signature in postinjury hearts. FMO2 possesses a previously uncharacterized enzyme-independent antifibrosis activity via the CYP2J3-SMURF2 axis. Restoring FMO2 expression exerts potent ameliorative effect against fibrotic remodeling in postinjury hearts from rodents to nonhuman primates. Therefore, FMO2 is a potential therapeutic target for treating cardiac fibrosis following injury.

Two Fluorescent Probes for Recognition of Acetylcholinesterase: Design, Synthesis, and Comparative Evaluation.

In this study, two "on-off" probes (BF2-cur-Ben and BF2-cur-But) recognizing acetylcholinesterase (AChE) were designed and synthesized. The obtained probes can achieve recognition of AChE with good selectivity and pH-independence with a linear range of 0.5~7 U/mL and 0.5~25 U/mL respectively. BF2-cur-Ben has a lower limit of detection (LOD) (0.031 U/mL), higher enzyme affinity (Km = 16 ± 1.6 µM), and higher inhibitor sensitivity. A responsive mechanism of the probes for AChE was proposed based on HPLC and mass spectra (MS) experiments, as well as calculations. In molecular simulation, BF2-cur-Ben forms more hydrogen bonds (seven, while BF2-cur-But has only four) and thus has a more stable enzyme affinity, which is mirrored by the results of the comparison of Km values. These two probes could enable recognition of intracellular AChE and probe BF2-cur-Ben has superior cell membrane penetration due to its higher log p value. These probes can monitor the overexpression of AChE during apoptosis of lung cancer cells. The ability of BF2-cur-Ben to monitor AChE in vivo was confirmed by a zebrafish experiment.

Mussel-Inspired, Underwater Self-Healing Ionoelastomers Based on α-Lipoic Acid for Iontronics.

Weak adhesion and lack of underwater self-healability hinder advancing soft iontronics particularly in wet environments like sweaty skin and biological fluids. Mussel-inspired, liquid-free ionoelastomers are reported based on seminal thermal ring-opening polymerization of a biomass molecule of α-lipoic acid (LA), followed by sequentially incorporating dopamine methacrylamide as a chain extender, N,N'-bis(acryloyl) cystamine, and lithium bis(trifluoromethanesulphonyl) imide (LiTFSI). The ionoelastomers exhibit universal adhesion to 12 substrates in both dry and wet states, superfast self-healing underwater, sensing capability for monitoring human motion, and flame retardancy. The underwater self-repairabilitiy prolongs over three months without deterioration, and sustains even when mechanical properties greatly increase. The unprecedented underwater self-mendability benefits synergistically from the maximized availability of dynamic disulfide bonds and diverse reversible noncovalent interactions endowed by carboxylic groups, catechols, and LiTFSI, along with the prevented depolymerization by LiTFSI and tunability in mechanical strength. The ionic conductivity reaches 1.4 × 10-6 -2.7 × 10-5 S m-1 because of partial dissociation of LiTFSI. The design rationale offers a new route for creating a wide range of LA- and sulfur-derived supramolecular (bio)polymers with superior adhesion, healability, and other functionalities, and thus has technological implications for coatings, adhesives, binders and sealants, biomedical engineering and drug delivery, wearable and flexible electronics, and human-machine interfaces.

Triiodothyronine and dexamethasone alter potassium channel expression and promote electrophysiological maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have emerged as a promising tool for disease modeling and drug development. However, hiPSC-CMs remain functionally immature, which hinders their utility as a model of human cardiomyocytes. OBJECTIVE: To improve the electrophysiological maturation of hiPSC-CMs. METHODS AND RESULTS: On day 16 of cardiac differentiation, hiPSC-CMs were treated with 100 nmol/L triiodothyronine (T3) and 1 µmol/L Dexamethasone (Dex) or vehicle for 14 days. On day 30, vehicle- and T3 + Dex-treated hiPSC-CMs were dissociated and replated either as cell sheets or single cells. Optical mapping and patch-clamp technique were used to examine the electrophysiological properties of vehicle- and T3 + Dex-treated hiPSC-CMs. Compared to vehicle, T3 + Dex-treated hiPSC-CMs had a slower spontaneous beating rate, more hyperpolarized resting membrane potential, faster maximal upstroke velocity, and shorter action potential duration. Changes in spontaneous activity and action potential were mediated by decreased hyperpolarization-activated current (If) and increased inward rectifier potassium currents (IK1), sodium currents (INa), and the rapidly and slowly activating delayed rectifier potassium currents (IKr and IKs, respectively). Furthermore, T3 + Dex-treated hiPSC-CM cell sheets (hiPSC-CCSs) exhibited a faster conduction velocity and shorter action potential duration than the vehicle. Inhibition of IK1 by 100 µM BaCl2 significantly slowed conduction velocity and prolonged action potential duration in T3 + Dex-treated hiPSC-CCSs but had no effect in the vehicle group, demonstrating the importance of IK1 for conduction velocity and action potential duration. CONCLUSION: T3 + Dex treatment is an effective approach to rapidly enhance electrophysiological maturation of hiPSC-CMs.

Lack of Remuscularization Following Transplantation of Human Embryonic Stem Cell-Derived Cardiovascular Progenitor Cells in Infarcted Nonhuman Primates.

RATIONALE: Human pluripotent stem cell-derived cardiovascular progenitor cells (hPSC-CVPCs) should be thoroughly investigated in large animal studies before testing in clinical trials. OBJECTIVE: The main of this study is to clarify whether hPSC-CVPCs can engraft for long time in the heart of primates after myocardial infarction (MI) and compare the effectiveness and safety of immunosuppression with cyclosporine alone or multiple-drug regimen (MDR) containing cyclosporine, methylprednisolone, and basiliximab in cynomolgus monkeys that had received intramyocardial injections of 1×107 EGFP (enhanced green fluorescent protein)-expressing hPSC-CVPCs after MI. A third group of animals received the immunosuppression MDR but without cell therapy after MI (MI+MDR group). METHODS AND RESULTS: Measurements of EGFP gene levels and EGFP immunofluorescence staining indicated that the hPSC-CVPC engraftment rate was greater in the MI+MDR+CVPC group than that in the MI+cyclosporine+CVPC group. However, even in the MI+MDR+CVPC group, no transplanted cells could be detected at 140 days after transplantation. Concomitantly, immunofluorescent analysis of CD3, CD4, and CD8 expression indicated that T-lymphocyte infiltration in the CVPC-transplanted hearts was less in the MDR-treated animals than in the cyclosporine-alone-treated animals. The recovery of left ventricular function on day 28 post-MI in the MI+MDR+CVPC group was better than that in the MI+MDR group. Apoptotic cardiac cells were also less common in the MI+MDR+CVPC group than in the MI+MDR group, although both immunosuppression regimens were associated with transient hepatic dysfunction. CONCLUSIONS: This is the largest study of hPSCs in nonhuman primates in cardiovascular field to date (n=32). Compared with cyclosporine alone, MDR attenuates immune rejection and improves survival of hPSC-CVPCs in primates; this is associated with less apoptosis of native cardiac cells and better recovery of left ventricular function at 28 days. However, even with MDR, transplanted hPSC-CVPCs do not engraft and do not survive at 140 days after transplantation, thereby excluding remuscularization as a mechanism for the functional effect.

Minimal contribution of IP3R2 in cardiac differentiation and derived ventricular-like myocytes from human embryonic stem cells.

Type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) regulates the intracellular Ca2+ release from endoplasmic reticulum in human embryonic stem cells (hESCs), cardiovascular progenitor cells (CVPCs), and mammalian cardiomyocytes. However, the role of IP3R2 in human cardiac development is unknown and its function in mammalian cardiomyocytes is controversial. hESC-derived cardiomyocytes have unique merits in disease modeling, cell therapy, and drug screening. Therefore, understanding the role of IP3R2 in the generation and function of human cardiomyocytes would be valuable for the application of hESC-derived cardiomyocytes. In the current study, we investigated the role of IP3R2 in the differentiation of hESCs to cardiomyocytes and in the hESC-derived cardiomyocytes. By using IP3R2 knockout (IP3R2KO) hESCs, we showed that IP3R2KO did not affect the self-renewal of hESCs as well as the differentiation ability of hESCs into CVPCs and cardiomyocytes. Furthermore, we demonstrated the ventricular-like myocyte characteristics of hESC-derived cardiomyocytes. Under the α1-adrenergic stimulation by phenylephrine (10 µmol/L), the amplitude and maximum rate of depolarization of action potential (AP) were slightly affected in the IP3R2KO hESC-derived cardiomyocytes at differentiation day 90, whereas the other parameters of APs and the Ca2+ transients did not show significant changes compared with these in the wide-type ones. These results demonstrate that IP3R2 has minimal contribution to the differentiation and function of human cardiomyocytes derived from hESCs, thus provide the new knowledge to the function of IP3R2 in the generation of human cardiac lineage cells and in the early cardiomyocytes.

A Turn-On Fluorescent Probe for Detection of Sub-ppm Levels of a Sulfur Mustard Simulant with High Selectivity.

A new type of fluorescent probe capable of detecting a sulfur mustard (SM) simultant at a concentration of 1.2 µM in solution and 0.5 ppm in the gas phase has been developed. Owing to its molecular structure with a thiocarbonyl component and two piperidyl moieties integrated into the xanthene molecular skeleton, this probe underwent a highly selective nucleophilic reaction with the SM simultant and generated a thiopyronin derivative emitting intensive pink fluorescence. The distinct difference in electronic structure between the probe and thiopyronin derivative generated a marked shift of the absorption band from 445 to 567 nm, which enabled an optimal wavelength propitious for exciting the thiopyronin derivative but adverse to the probe. Such efficient separation of the excitation wavelength and tremendous increase in fluorescence quantum yield, from less than 0.002 to 0.53, upon conversion from the probe to the thiopyronin derivative, jointly led to a distinct contrast in the beaconing fluorescence signal (up to 850-fold) and therefore the unprecedented sensitivity for detecting SM species.

Functional expression of the Ca2+ signaling machinery in human embryonic stem cells.

Emerging evidence suggests that Ca2+ signals are important for the self-renewal and differentiation of human embryonic stem cells (hESCs). However, little is known about the physiological and pharmacological properties of the Ca2+-handling machinery in hESCs. In this study we used RT-PCR and Western blotting to analyze the expression profiles of genes encoding Ca2+-handling proteins; we also used confocal Ca2+ imaging and pharmacological approaches to determine the contribution of the Ca2+-handling machinery to the regulation of Ca2+ signaling in hESCs. We revealed that hESCs expressed pluripotent markers and various Ca2+-handling-related genes. ATP-induced Ca2+ transients in almost all hESCs were inhibited by the inositol-1,4,5-triphosphate receptor (IP3R) blocker 2-APB or xestospongin C. In addition, Ca2+ transients were induced by a ryanodine receptor (RyR) activator, caffeine, in 10%-15% of hESCs and were blocked by ryanodine, whereas caffeine and ATP did not have additive effects. Moreover, store-operated Ca2+ entry (SOCE) but not voltage-operated Ca2+ channel-mediated Ca2+ entry was observed. Inhibition of sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA) by thapsigargin induced a significant increase in the cytosolic free Ca2+ concentration ([Ca2+]i). For the Ca2+ extrusion pathway, inhibition of plasma membrane Ca2+ pumps (PMCAs) by carboxyeosin induced a slow increase in [Ca2+]i, whereas the Na+/Ca2+ exchanger (NCX) inhibitor KBR7943 induced a rapid increase in [Ca2+]i. Taken together, increased [Ca2+]i is mainly mediated by Ca2+ release from intracellular stores via IP3Rs. In addition, RyRs function in a portion of hESCs, thus indicating heterogeneity of the Ca2+-signaling machinery in hESCs; maintenance of low [Ca2+]i is mediated by uptake of cytosolic Ca2+ into the ER via SERCA and extrusion of Ca2+ out of cells via NCX and PMCA in hESCs.

Coupling switch of P2Y-IP3 receptors mediates differential Ca(2+) signaling in human embryonic stem cells and derived cardiovascular progenitor cells.

Purinergic signaling mediated by P2 receptors (P2Rs) plays important roles in embryonic and stem cell development. However, how it mediates Ca(2+) signals in human embryonic stem cells (hESCs) and derived cardiovascular progenitor cells (CVPCs) remains unclear. Here, we aimed to determine the role of P2Rs in mediating Ca(2+) mobilizations of these cells. hESCs were induced to differentiate into CVPCs by our recently established methods. Gene expression of P2Rs and inositol 1,4,5-trisphosphate receptors (IP3Rs) was analyzed by quantitative/RT-PCR. IP3R3 knockdown (KD) or IP3R2 knockout (KO) hESCs were established by shRNA- or TALEN-mediated gene manipulations, respectively. Confocal imaging revealed that Ca(2+) responses in CVPCs to ATP and UTP were more sensitive and stronger than those in hESCs. Consistently, the gene expression levels of most P2YRs except P2Y1 were increased in CVPCs. Suramin or PPADS blocked ATP-induced Ca(2+) transients in hESCs but only partially inhibited those in CVPCs. Moreover, the P2Y1 receptor-specific antagonist MRS2279 abolished most ATP-induced Ca(2+) signals in hESCs but not in CVPCs. P2Y1 receptor-specific agonist MRS2365 induced Ca(2+) transients only in hESCs but not in CVPCs. Furthermore, IP3R2KO but not IP3R3KD decreased the proportion of hESCs responding to MRS2365. In contrast, both IP3R2 and IP3R3 contributed to UTP-induced Ca(2+) responses while ATP-induced Ca(2+) responses were more dependent on IP3R2 in the CVPCs. In conclusion, a predominant role of P2Y1 receptors in hESCs and a transition of P2Y-IP3R coupling in derived CVPCs are responsible for the differential Ca(2+) mobilization between these cells.

Ultrathin, biomimetic, superhydrophilic layers of cross-linked poly(phosphobetaine) on polyethylene by photografting.

Ultrathin, biomimetic, superhydrophilic hydrogel layers, composed of cross-linked poly(2-methacryloyloxyethyl phosphorylcholine), are formed on low-density polyethylene films via ultraviolet-initiated surface graft polymerization. The layers are 19-58 nm thick as revealed by electron microscopy and have three-dimensional networks; the unique network structure, along with its zwitterionic nature, rather than surface roughness results in superhydrophilicity, that is, the water contact angle around 5°. This superhydrophilicity depends on a variety of factors, including the concentration of the monomer and cross-linker, the type of reaction solvents, the reaction and drying time, the intensity of UV light, and the way of measurement of water contact angles. Superhydrophilicity is obtained under a fixed ratio (e.g., 1/1) of the monomer to cross-linker, a reaction time over 120 s, a short drying time, (75%) ethanol as the reaction solvent, and low-intensity UV light, largely because these factors together generate optimal three-dimensional networks of cross-links.

Cardiomyocytes derived from pluripotent stem cells: progress and prospects from China.

Transplantation of embryonic stem cell- or induced pluripotent stem cell-derived cardiomyocytes (CMs) represents one promising approach for the treatment of myocardial infarction and failing hearts. Cardiac differentiation systems from these pluripotent stem cells (PSCs) can also be employed to better understand early developmental biology, drug discovery, toxicology testing, and disease modeling. A prerequisite to attain these goals is the ability to generate functional CMs in an efficient and reliable way. The lack of CM maturation must also be overcome, and appropriate methods for introducing PSC-CMs into heart while maintaining cell viability must be optimized. The past few years have seen major advances both in the differentiation, characterization and application of these cells to biological systems. Here we review recent progress, especially those performed in China, in basic stem cell biology involving studies of cardiogenesis and CMs through PSC differentiation, approaches for chamber-specific CM differentiation, maturation processes involving regulation of intracellular Ca(2+) signals, and applications.

Intermittent fasting alleviates IMQ-induced psoriasis-like dermatitis via reduced γδT17 and monocytes in mice.

Psoriasis is a chronic immune mediated inflammatory skin disease with systemic manifestations. It has been reported that caloric restriction could improve severity of psoriasis patients. However, the mechanism of intermittent fasting effects on psoriasis has not been investigated. Caloric restriction is known to reduce the number of circulating inflammatory monocytes in a CCL2-dependent manner. However, it is still unknown whether caloric restriction can improve psoriasis by regulating monocytes through CCL2. In this study, we used imiquimod (IMQ)-induced psoriasis-like mouse model to explore the effects and the mechanisms of intermittent fasting on psoriasis-like dermatitis. We found that intermittent fasting could significantly improve IMQ-induced psoriasis-like dermatitis, and reduce the number of γδT17 cells and IL-17 production in draining lymph nodes and psoriatic lesion via inhibiting proliferation and increasing death of γδT17 cells. Furthermore, intermittent fasting could significantly decrease monocytes in blood, and this was associated with decreased monocytes, macrophages and DC in psoriasis-like skin inflammation. Reduced monocytes in circulation and increased monocytes in BM of fasting IMQ-induced psoriasis-like mice is through reducing the production of CCL2 from BM to inhibit monocyte egress to the periphery. Our above data shads light on the mechanisms of intermittent fasting on psoriasis.

Biocomposites of pHEMA with HA/ß -TCP (60/40) for bone tissue engineering: Swelling, hydrolytic degradation, and in vitro behavior.

The field of bone and cartilage tissue engineering has a pressing need for novel, biocompatible, biodegradable biocomposites comprising polymers with bioceramics or bioglasses to meet numerous requirements for these applications. We created hydrolytically degradable hydrogel/bioceramic biocomposites, comprising poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogels and 50 wt% biphasic hydroxyapatite/ß-tricalcium phosphate (60/40) through in situ polymerization. The hydrolytic degradation starts with hydrolysis of the cross-linker, N, O-dimethacryloyl hydroxylamine, which was synthesized in house. Swelling and degradation were examined in details at a phosphate buffered saline solution at 37 °C over a 12-week period of time. To vary degradability, a co-monomer, acrylic acid (AA) or 2-hydroxypropyl methacrylamide (HPMA), was introduced, coupled with altering the concentration of the cross-linker and of the bioceramic. The co-monomer HPMA was found to be more effective than AA in enhancing degradation, though AA led to greater swelling ratios. 33% of weight loss was achieved in some of the biocomposites containing HPMA. Porous structures were developed during swelling and degradation in biocomposites with AA but not in those containing HPMA, suggesting different degradation mechanisms: bulk erosion vs. bulk degradation. Good biocompatibility, as evidenced by attachment and proliferation of mouse-derived osteoblast precursor cells from the MC3T3-E1 lineage, was observed on these biomaterials, regardless of the type of the co-monomer. The rationale and approaches employed here open up new opportunities for creating novel, complex organic-inorganic biomaterials in orthopedic tissue engineering.

Combinatorial Design of Hydrolytically Degradable, Bone-like Biocomposites Based on PHEMA and Hydroxyapatite.

With advantages such as design flexibility in modifying degradation, surface chemistry, and topography, synthetic bone-graft substitutes are increasingly demanded in orthopedic tissue engineering to meet various requirements in the growing numbers of cases of skeletal impairment worldwide. Using a combinatorial approach, we developed a series of biocompatible, hydrolytically degradable, elastomeric, bone-like biocomposites, comprising 60 wt% poly(2-hydroxyethyl methacrylate-co-methacrylic acid), poly(HEMA-co-MA), and 40 wt% bioceramic hydroxyapatite (HA). Hydrolytic degradation of the biocomposites is rendered by a degradable macromer/crosslinker, dimethacrylated poly(lactide-b-ethylene glycol-b-lactide), which first degrades to break up 3-D hydrogel networks, followed by dissolution of linear pHEMA macromolecules and bioceramic particles. Swelling and degradation were examined at Hank's balanced salt solution at 37 °C in a 12-week period of time. The degradation is strongly modulated by altering the concentration of the co-monomer of methacrylic acid and of the macromer, and chain length/molecular weight of the macromer. 95% weight loss in mass is achieved after degradation for 12 weeks in a composition consisting of HEMA/MA/Macromer = 0/60/40, while 90% weight loss is seen after degradation only for 4 weeks in a composition composed of HEMA/MA/Macromer = 27/13/60 using a longer chain macromer. For compositions without a co-monomer, only about 14% is achieved in weight loss after 12-week degradation. These novel biomaterials offer numerous possibilities as drug delivery carriers and bone grafts particularly for low and medium load-bearing applications.

Influence of extracellular nanovesicles derived from adipose-derived stem cells on nucleus pulposus cell from patients with intervertebral disc degeneration.

An increasing number of individuals are suffering from lower back and neck pain caused by intervertebral disc degeneration each year. Although the application of mesenchymal stem cells (MSCs) has provided desirable results in the treatment of intervertebral disc degeneration, there are multiple risks associated with the directed application of MSCs. An increasing number of studies have suggested that stem cells, through the release of extracellular nanovesicles, have vital functions in tissue regeneration and repair with low risk. The present study investigated the effect of extracellular nanovesicles derived from adipose-derived stem cells (ADSCs) on nucleus pulposus (NP) cells from patients with intervertebral disc degeneration. Human NP cells were obtained from patients with intervertebral disc degeneration undergoing surgical procedures in addition to ADSCs from liposuction patients. ADSC-derived extracellular nanovesicles were isolated and characterized. The differentiation and biological activity of NP cells cultured with or without ADSC-derived extracellular nanovesicles were assessed and inflammatory factors and intervertebral disc degeneration-associated markers were also measured. The results indicated that extracellular nanovesicles derived from ADSCs increased the migration and proliferation of NP cells and inhibited inflammatory activity, suggesting their utility for the treatment of intervertebral disc degeneration.

Cardiac Na+-Ca2+ exchanger 1 (ncx1h) is critical for the ventricular cardiomyocyte formation via regulating the expression levels of gata4 and hand2 in zebrafish.

Ca2+ signaling is critical for heart development; however, the precise roles and regulatory pathways of Ca2+ transport proteins in cardiogenesis remain largely unknown. Sodium-calcium exchanger 1 (Ncx1) is responsible for Ca2+ efflux in cardiomyocytes. It is involved in cardiogenesis, while the mechanism is unclear. Here, using the forward genetic screening in zebrafish, we identified a novel mutation at a highly-conserved leucine residue in ncx1 gene (mutantLDD353/ncx1hL154P) that led to smaller hearts with reduced heart rate and weak contraction. Mechanistically, the number of ventricular but not atrial cardiomyocytes was reduced in ncx1hL154P zebrafish. These defects were mimicked by knockdown or knockout of ncx1h. Moreover, ncx1hL154P had cytosolic and mitochondrial Ca2+ overloading and Ca2+ transient suppression in cardiomyocytes. Furthermore, ncx1hL154P and ncx1h morphants downregulated cardiac transcription factors hand2 and gata4 in the cardiac regions, while overexpression of hand2 and gata4 partially rescued cardiac defects including the number of ventricular myocytes. These findings demonstrate an essential role of the novel 154th leucine residue in the maintenance of Ncx1 function in zebrafish, and reveal previous unrecognized critical roles of the 154th leucine residue and Ncx1 in the formation of ventricular cardiomyocytes by at least partially regulating the expression levels of gata4 and hand2.

Rabbit annulus fibrosus cell apoptosis induced by mechanical overload via a mitochondrial apoptotic pathway.

In order to investigate the apoptotic pathway of rabbit annulus fibrosus (AF) cells induced by mechanical overload, an experimental air-pressure model was established in this study to pressurize the rabbit AF cells in vitro. Cells were randomly divided into five groups in which the cells were exposed to a continuous pressure of 1.1 MPa for different lengths of time (0, 5, 12, 24 and 36 h). The cell proliferation and apoptosis were detected by cell counting kit-8 (CCK-8) assay and flow cytometry; the alterations in mitochondrial membrane potential were measured by fluorescence microscopy and fluorescence spectrophotometer; the activities of caspase-8 and 9 were determined by spectrophotometry. The results showed that after the cells were subjected to the pressure for 24 or 36 h, the cell proliferation was inhibited; the ratio of cell apoptosis was increased; the mitochondrial membrane potential was decreased; the activity of caspase-9 was enhanced; no activity changes were observed in caspase-8. The results suggested that treatment with a pressure of 1.1 MPa for more than 24 h can lead to the proliferation inhibition and the apoptosis of rabbit AF cells in vitro, and the mitochondrial-dependent pathway is implicated in the pressure-induced AF cell apoptosis.

TATA box-binding protein-related factor 3 drives the mesendoderm specification of human embryonic stem cells by globally interacting with the TATA box of key mesendodermal genes.

BACKGROUND: Mesendodermal formation during early gastrulation requires the expression of lineage-specific genes, while the regulatory mechanisms during this process have not yet been fully illustrated. TATA box-binding protein (TBP) and TBP-like factors are general transcription factors responsible for the transcription initiation by recruiting the preinitiation complex to promoter regions. However, the role of TBP family members in the regulation of mesendodermal specification remains largely unknown. METHODS: We used an in vitro mesendodermal differentiation system of human embryonic stem cells (hESCs), combining with the microarray and quantitative polymerase chain reaction (qRT-PCR) analysis, loss of function and gain of function to determine the function of the TBP family member TBP-related factor 3 (TRF3) during mesendodermal differentiation of hESCs. The chromatin immunoprecipitation (ChIP) and biochemistry analysis were used to determine the binding of TRF3 to the promoter region of key mesendodermal genes. RESULTS: The mesendodermal differentiation of hESCs was confirmed by the microarray gene expression profile, qRT-PCR, and immunocytochemical staining. The expression of TRF3 mRNA was enhanced during mesendodermal differentiation of hESCs. The TRF3 deficiency did not affect the pluripotent marker expression, alkaline phosphatase activity, and cell cycle distribution of undifferentiated hESCs or the expression of early neuroectodermal genes during neuroectodermal differentiation. During the mesendodermal differentiation, the expression of pluripotency markers decreased in both wild-type and TRF3 knockout (TRF3-/-) cells, while the TRF3 deficiency crippled the expression of the mesendodermal markers. The reintroduction of TRF3 into the TRF3-/- hESCs rescued inhibited mesendodermal differentiation. Mechanistically, the TRF3 binding profile was significantly shifted to the mesendodermal specification during mesendodermal differentiation of hESCs based on the ChIP-seq data. Moreover, ChIP and ChIP-qPCR analysis showed that TRF3 was enriched at core promoter regions of mesendodermal developmental genes, EOMESODERMIN, BRACHYURY, mix paired-like homeobox, and GOOSECOID homeobox, during mesendodermal differentiation of hESCs. CONCLUSIONS: These results reveal that the TBP family member TRF3 is dispensable in the undifferentiated hESCs and the early neuroectodermal differentiation. However, it directs mesendodermal lineage commitment of hESCs via specifically promoting the transcription of key mesendodermal transcription factors. These findings provide new insights into the function and mechanisms of the TBP family member in hESC early lineage specification.

p38 Mitogen-activated protein kinase regulates chamber-specific perinatal growth in heart.

In the mammalian heart, the left ventricle (LV) rapidly becomes more dominant in size and function over the right ventricle (RV) after birth. The molecular regulators responsible for this chamber-specific differential growth are largely unknown. We found that cardiomyocytes in the neonatal mouse RV had lower proliferation, more apoptosis, and a smaller average size compared with the LV. This chamber-specific growth pattern was associated with a selective activation of p38 mitogen-activated protein kinase (MAPK) activity in the RV and simultaneous inactivation in the LV. Cardiomyocyte-specific deletion of both the Mapk14 and Mapk11 genes in mice resulted in loss of p38 MAPK expression and activity in the neonatal heart. Inactivation of p38 activity led to a marked increase in cardiomyocyte proliferation and hypertrophy but diminished cardiomyocyte apoptosis, specifically in the RV. Consequently, the p38-inactivated hearts showed RV-specific enlargement postnatally, progressing to pulmonary hypertension and right heart failure at the adult stage. Chamber-specific p38 activity was associated with differential expression of dual-specific phosphatases (DUSPs) in neonatal hearts, including DUSP26. Unbiased transcriptome analysis revealed that IRE1α/XBP1-mediated gene regulation contributed to p38 MAPK-dependent regulation of neonatal cardiomyocyte proliferation and binucleation. These findings establish an obligatory role of DUSP/p38/IRE1α signaling in cardiomyocytes for chamber-specific growth in the postnatal heart.