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Phospholamban (PLN) is a key regulator that controls the function of the sarcoplasmic reticulum (SR) and is required for the regulation of cardiac contractile function. Although PLN-deficient mice demonstrated improved cardiac function, PLN loss in humans can result in dilated cardiomyopathy (DCM) or heart failure (HF). The CRISPR-Cas9 technology was used to create a PLN knockout human induced pluripotent stem cell (hiPSC) line in this study. PLN deletion hiPSCs-CMs had enhanced contractility at day 30, but proceeded to a cardiac failure phenotype at day 60, with decreased contractility, mitochondrial damage, increased ROS production, cellular energy metabolism imbalance, and poor Ca

Rationale: Genetic editing has shown great potential for the treatment of human hereditary disorders via the elimination of mutations in embryos. However, the efficiency and safety of germline gene editing are not well understood. Objective: We aimed to examine the preclinical efficacy/safety of embryonic base editing in a mouse model of hypertrophic cardiomyopathy (HCM) using a novel adenine base editor (ABE) platform. Methods and Results: Here, we described the use of an ABEmax-NG to directly correct the pathogenic R404Q/+ mutation ( Myh6 c.1211C>T) in embryos for a mouse model of HCM, increasing the number of wild-type embryos for in vitro fertilization. Delivery of the ABEmax-NG mRNA to embryos from R404Q/+ HCM mice resulted in 62.5% to 70.8% correction of the Myh6 c.1211C>T, reducing the level of mutant RNA and eliminating HCM in the postnatal mice as well as their offspring. In addition, the same single guide RNA was also used to target an intronic locus (chr2: +36,585,842) with an overall editing rate of 86.7%, thus confirming that ABEmax-NG can efficiently edit target loci with different protospacer adjacent motifs (PAM) and genomic distribution in vivo. Compared with CRISPR/single-stranded oligodeoxynucleotides–mediated correction, ABEmax-NG displayed a much higher correction rate without introducing indels. DNA and RNA off-target analysis did not detect off-target editing in treated embryos and founder mice. In utero injection of adeno-associated virus 9 encoding the ABEmax-NG also resulted in around 25.3% correction of the pathogenic mutation and reduced of mutant RNA, thereby indicating ABEmax-NG has the potential to correct the HCM mutation in vivo. Conclusions: We developed an ABEmax-NG system, which efficiently corrected a pathogenic Myh6 HCM mutation in mouse embryos without off-target lesions, thus safely eliminating HCM in derived mice and their progeny.

Background Long-QT syndrome type 2 (LQT2) is a common malignant hereditary arrhythmia. Due to the lack of suitable animal and human models, the pathogenesis of LQT2 caused by human ether-a-go-go-related gene (hERG) deficiency is still unclear. In this study, we generated an hERG-deficient human cardiomyocyte (CM) model that simulates ‘human homozygous hERG mutations’ to explore the underlying impact of hERG dysfunction and the genotype–phenotype relationship of hERG deficiency. Methods The KCNH2 was knocked out in the human embryonic stem cell (hESC) H9 line using the CRISPR/Cas9 system. Using a chemically defined differentiation protocol, we obtained and verified hERG-deficient CMs. Subsequently, high-throughput microelectrode array (MEA) assays and drug interventions were performed to characterise the electrophysiological signatures of hERG-deficient cell lines. Results Our results showed that KCNH2 knockout did not affect the pluripotency or differentiation efficiency of H9 cells. Using high-throughput MEA assays, we found that the electric field potential duration and action potential duration of hERG-deficient CMs were significantly longer than those of normal CMs. The hERG-deficient lines also exhibited irregular rhythm and some early afterdepolarisations. Moreover, we used the hERG-deficient human CM model to evaluate the potency of agents (nifedipine and magnesium chloride) that may ameliorate the phenotype. Conclusions We established an hERG-deficient human CM model that exhibited QT prolongation, irregular rhythm and sensitivity to other ion channel blockers. This model serves as an important tool that can aid in understanding the fundamental impact of hERG dysfunction, elucidate the genotype–phenotype relationship of hERG deficiency and facilitate drug development.

The use of human pluripotent stem cell‐derived cardiomyocytes (hPSC‐CMs) is limited in drug discovery and cardiac disease mechanism studies due to cell immaturity. Micro‐scaled grooves can promote the maturation of cardiomyocytes by aligning them in order, but the mechanism of cardiomyocytes alignment has not been studied. From the level of calcium activity, gene expression and cell morphology, we verified that the W20H5 grooves can effectively promote the maturation of cardiomyocytes. The transient receptor potential channels (TRP channels) also play an important role in the maturation and development of cardiomyocytes. These findings support the engineered hPSC‐CMs as a powerful model to study cardiac disease mechanism and partly mimic the myocardial morphological development. The important role of the TRP channels in the maturation and development of myocardium is first revealed.

Myomesin‐1 (encoded by MYOM1 gene) is expressed in almost all cross‐striated muscles, whose family (together with myomesin‐2 and myomesin‐3) helps to cross‐link adjacent myosin to form the M‐line in myofibrils. However, little is known about its biological function, causal relationship and mechanisms underlying the MYOM1‐related myopathies (especially in the heart). Regrettably, there is no MYMO1 knockout model for its study so far. A better and further understanding of MYOM1 biology is urgently needed. Here, we used CRISPR/Cas9 gene‐editing technology to establish an MYOM1 knockout human embryonic stem cell line (MYOM1−/− hESC), which was then differentiated into myomesin‐1 deficient cardiomyocytes (MYOM1−/− hESC‐CMs) in vitro. We found that myomesin‐1 plays an important role in sarcomere assembly, contractility regulation and cardiomyocytes development. Moreover, myomesin‐1‐deficient hESC‐CMs can recapitulate myocardial atrophy phenotype in vitro. Based on this model, not only the biological function of MYOM1, but also the aetiology, pathogenesis, and potential treatments of myocardial atrophy caused by myomesin‐1 deficiency can be studied.

IntroductionSpinal cord injury (SCI) is a neurological, medically incurable disorder. Human pluripotent stem cells (hPSCs) have the potential to generate neural stem/progenitor cells (NS/PCs), which hold promise in the treatment of SCI by transplantation. In our study, we aimed to establish a chemically defined culture system using serum-free medium and ascorbic acid (AA) to generate and expand long-term self-renewing neuroepithelial-like stem cells (lt-NES cells) differentiated from hPSCs effectively and stably.MethodsWe induced human embryonic stem cells (hESCs)/induced PSCs (iPSCs) to neurospheres using a newly established in vitro induction system. Moreover, lt-NES cells were derived from hESC/iPSC-neurospheres using two induction systems, i.e., conventional N2 medium with gelatin-coated plates (coated) and N2+AA medium without pre-coated plates (AA), and were characterized by reverse transcription polymerase chain reaction (RT-PCR) analysis and immunocytochemistry staining. Subsequently, lt-NES cells were induced to neurons. A microelectrode array (MEA) recording system was used to evaluate the functionality of the neurons differentiated from lt-NES cells. Finally, the mechanism underlying the induction of lt-NES cells by AA was explored through RNA-seq and the use of inhibitors.ResultsHESCs/iPSCs were efficiently induced to neurospheres using a newly established induction system in vitro. lt-NES cells derived from hESC/iPSC-neurospheres using the two induction systems (coated vs. AA) both expressed the neural pluripotency-associated genesPAX6,NESTIN,SOX1, andSOX2. After long-term cultivation, we found that they both exhibited long-term expansion for more than a dozen generations while maintaining neuropluripotency. Moreover, the lt-NES cells retained the ability to differentiate into general functional neurons that express β-tubulin at high levels. We also demonstrated that AA promotes the generation and long-term expansion of lt-NES cells by promoting collagen synthesis via the MEK-ERK1/2 pathway.ConclusionsThis new chemically defined culture system was stable and effective regarding the generation and culture of lt-NES cells induced from hESCs/iPSCs using serum-free medium combined with AA. The lt-NES cells induced under this culture system maintained their long-term expansion and neural pluripotency, with the potential to differentiate into functional neurons.Graphical abstract

Bovine serum albumin-mediated copper sulfide nanocomplex (BSA/CuS) was modified on the mesoporous silica (MSN) loaded with chemotherapeutic drug doxorubicin (DOX) by amide bond to prepare a new drug carrier MSN-DOX@BSA/CuS. The disulfide bond of blocking agent BSA broke down after the redox reaction with glutathione, resulting in the MSN pore exposure and release of DOX. As a photothermal reagent, CuS could convert light energy into heat for photothermal therapy. UV-Vis-NIR absorption spectra were used to investigate the absorption properties of the carrier. The results showed that the carrier had strong absorption in the range of 800–1100 nm and could realize the photothermal conversion under the near infrared light. In addition, MSN@BSA/CuS was incubated with cancer cells and showed no cytotoxicity. When MSN-DOX@BSA/CuS was added, the relative survival rate of the cells decreased to 49%. After the extra laser, only 18% cells survived. The above results showed that the synthesized nano drug carrier could realize the synergistic treatment of photothermal therapy and chemotherapy, and effectively improve the treatment efficiency of cancer.

Background It was reported that microRNA-21(miR-21) was differentially expressed in the keratinocytes of psoriasis patients, and it may influence the apoptosis and proliferation of cells. The role of lncRNA maternally expressed gene3 (MEG3), a competing endogenous RNAs of miR-21, in the progression of psoriasis remains unclear. We aimed to unfold the influence of MEG3 and miR-21 on the proliferation and apoptosis of psoriasis epidermal cells. Methods 50μg/L TNF-α was used to treat HaCaTs and NHEKs cells for 24 h, and then different experiments were conducted. qRT-PCR were applied for measuring the mRNA level of MEG3, miR-2, and caspase-8, and the protein expression of caspase-8 was measured with western blotting. Flow cytometry was used for assessing apoptosis. Cell proliferation was detected using MTT and colony formation assays. Dual luciferase reporter assay was applied for confirming the binding site between MEG3 and miR-21, miR-21 and Caspase-8. Results A cell model for in vitro studying the role of MEG3 in psoriasis pathophysiology was established using HaCaT and HHEKs. MEG3 was significantly down-regulated in HaCaT, HHEKs, and psoriatic skin samples. MEG3 inhibits proliferation and promotes apoptosis of Activated-HaCaT (Act-HaCaT) and Activated-HHEKs (Act- HHEK) by regulating miR-21, and the binding site between MEG3 and miR-21 was identified. We also found that miR-21 could inhibit the level of caspase-8 and identified the binding site between caspase-8 and miR-21. Some down-stream proteins of caspase-8, Cleaved caspase-8, cytc, and apaf-1 were regulated by miR-21 and MEG3. Conclusion MEG3/miR-21 axis may regulate the expression of caspase-8, and further influence the proliferation and apoptosis of psoriasis keratinocyte, Act-HaCaT and Act- HHEK. Therefore, our findings may provide a new thought for the study of pathogenesis and treatment of psoriasis.

The rapid identification of bacterial antibiotic susceptibility is pivotal to the rational administration of antibacterial drugs. In this study, cefotaxime (CTX)-derived resistance in

Myosin heavy chain 7 (MYH7) encodes the human heart myosin heavy chain subunit, which plays an important role in myocardial contraction. MYH7 is the main pathogenic gene that causes Hypertrophic cardiomyopathy (HCM) and Dilated cardiomyopathy (DCM). In this experiment, a MYH7 homozygous knockout human embryonic stem cell (hESC) line, WAe009-A-69, was generated using an episomal vector-based CRISPR/Cas9 system. It can be an ideal tool to further study the function of MYH7. The cell line was confirmed with pluripotency, normal karyotype and trilineage differentiation potential.

Doxorubicin (DOX) is widely used to treat various cancers affecting adults and children; however, its clinical application is limited by its cardiotoxicity. Previous studies have shown that children are more susceptible to the cardiotoxic effects of DOX than adults, which may be related to different maturity levels of cardiomyocyte, but the underlying mechanisms are not fully understood. Moreover, researchers investigating DOX‐induced cardiotoxicity caused by human‐induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) have shown that dexrazoxane, the recognized cardioprotective drug for treating DOX‐induced cardiotoxicity, does not alleviate the toxicity of DOX on hiPSC‐CMs cultured for 30 days. We have suggested that this may be ascribed to the immaturity of the 30 days hiPSC‐CMs. In this study, we investigated the mechanisms of DOX induced cardiotoxicity in cardiomyocytes of different maturity. We selected 30‐day‐old and 60‐day‐old hiPSC‐CMs (day 30 and day 60 groups), which we term ‘immature’ and ‘relatively mature’ hiPSC‐CMs, respectively. The day 30 CMs were found to be more susceptible to DOX than the day 60 CMs. DOX leads to more ROS (reactive oxygen species) production in the day 60 CMs than in the relatively immature group due to increased mitochondria number. Moreover, the day 60 CMs mainly expressed topoisomerase IIβ presented less severe DNA damage, whereas the day 30 CMs dominantly expressed topoisomerase IIα exhibited much more severe DNA damage. These results suggest that immature cardiomyocytes are more sensitive to DOX as a result of a higher concentration of topoisomerase IIα, which leads to more DNA damage.

The traditional Chinese herb Lonicerae Japonicae Flos has shown significant clinical benefits in the treatment of heart failure, but the mechanism remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos, chlorogenic acid (CGA) has been reported to possess anti‐inflammatory, anti‐oxidant and anti‐apoptosis function. We firstly confirmed the cardioprotective effects of CGA in transverse aortic constriction (TAC)‐induced heart failure mouse model, through mitigating the TNF‐α–induced toxicity. We further used TNF‐α‐induced cardiac injury in human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) to elucidate the underlying mechanisms. CGA pre‐treatment could reverse TNF‐α–induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF‐κB/p65 and major mitogen‐activated protein kinases (MAPKs) signalling pathways involved in TNF‐α–induced apoptosis of hiPSC‐CMs. Importantly, CGA can directly inhibit NF‐κB signal by suppressing the phosphorylation of NF‐κB/p65. As for the MAPKs, CGA suppressed the activity of only c‐Jun N‐terminal kinase (JNK), but enhanced extracellular signal‐regulated kinase1/2 (ERK1/2) and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF‐κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.

Orange luminescent nitrogen doped carbon dots (N-CDs) were prepared via one-step hydrothermal treatment with p-phenylenediamine and citric acid as precursors. The morphology and optical properties of the N-CDs were characterized by transmission electron microscopy (TEM), Fourier transform infrared spectra (FTIR), ultraviolet-visible absorption spectroscopy (UV-vis) and fluorescence (FL) spectroscopy. The synthesized N-CDs had an extreme small size ((1.62 ± 0.35) nm), good water solubility and excellent fluorescent stability. The addition of nitrite (NO2−) caused fluorescence increase of N-CDs. Based on this phenomenon, a new fluorescence analysis method for detecting NO2− was established. Moreover, the possible fluorescence enhancement mechanism was proposed. The constructed fluorescence sensing system was applied to the detection of NO2− in sausage, pickle and tap water samples with recoveries from 97.1% to 104.5% and relative standard deviations (RSDs) of 1.1% to 3.5%.

Fruit wounding occurred at harvest and transportation requires rapid suberization as a major part of the healing process to prevent infection and desiccation. The focus of this work was to explore the mediation of abscisic acid (ABA) on wound-induced suberization and to determine expression profiles of specific genes involved in wound-induced suberization in tomato fruit. The measurements of weight loss and fruit firmness suggested wound-induced suberization started at 2 d after wounding. The suberization process with the accumulation of suberin polyphenolics (SPP) and polyaliphatics (SPA) observed through autofluorescence microscopy and Sudan IV staining was accelerated by ABA. Expressions of SlPAL5 and Sl4CL involved in the synthesis of SPP reached the highest at 4 and 8 d after wounding following ABA application, respectively. Associated with SPA biosynthesis, SlLACS1 and SlLACS2 showed the most abundant transcripts at 8 and 6 d in ABA group, respectively. Transcript levels including SlKCSs, SlCYP86B1, SlFAR3, and SlGPATs were up-regulated at 2 d after wounding by ABA. Activities of polyphenol oxidase and lipoxygenase were also enhanced during wound-induced suberization following ABA application. The results in this study proved that ABA accelerated the wound-induced suberization progress by increasing the transcript levels of relevant genes in postharvest tomato fruit.

Ion channels are important therapeutic targets, but the discovery of ion channel drugs remains challenging due to a lack of assays that allow high-throughput screening in the physiological context. Here we report C. elegans phenotype-based methods for screening ion channel drugs. Expression of modified human ether-a-go-go-related gene (hERG) potassium channels in C. elegans results in egg-laying and locomotive defects, which offer indicators for screening small-molecule channel modulators. Screening in worms expressing hERGA561V, which carries a trafficking-defective mutation A561V known to associate with long-QT syndrome, identifies two functional correctors Prostratin and ingenol-3,20-dibenzoate. These compounds activate PKCε signaling and consequently phosphorylate S606 at the pore region of the channel to promote hERGA561V trafficking to the plasma membrane. Importantly, the compounds correct electrophysiological abnormalities in hiPSC-derived cardiomyocytes bearing a heterozygous CRISPR/Cas9-edited hERGA561V. Thus, we have developed an in vivo high-throughput method for screening compounds that have therapeutic potential in treating channelopathies., Mutations in the voltage-gated K+ channel human ether-a-go-go-related gene (hERG) lead to Long-QT syndrome, causing life-threatening cardiac arrhythmia. Here the authors use C. elegans as a platform to run a channelopathy drug screen, identifying drugs to target hERG mutants.

COX6A2 protein is a structural subunit of Complex IV (CIV/Cytochrome c oxidase/COX) in the mitochondrial respiratory chain. It is mainly expressed in the heart and skeletal muscle, also in some interneurons, regulating the assembly and catalytic activity of CIV. Its mutations can lead to COX deficiency, causing human myopathies, and maybe a potential cause of neurological abnormalities. Here, we used the CRISPR/Cas9 editing system to establish a homozygous COX6A2 knockout (COX6A2-KO) human embryonic stem cell (hESC) line. This COX6A2-KO hESC has normal morphology, pluripotency, and karyotype, which can differentiate into three germ layers in vivo.

Congenital heart disease (CHD) is the most common cause of congenital anomaly and a leading cause of morbidity and mortality worldwide. Generation of cardiomyoctyes derived from pluripotent stem cells (PSCs) has opened new avenues for investigation of human cardiac development. Here we report that uric acid (UA), a physiologically abundant compound during embryonic development, can consistently and robustly enhance cardiac differentiation of human PSCs including hESCs and hiPSCs, in replacement of ascorbic acid (AA). We optimized treatment conditions and demonstrate that differentiation day 0–2, a period for specification of mesoderm cells, was a critical time for UA effects. This was further confirmed by UA-induced upregulation of mesodermal markers. Furthermore, we show that the developing mesoderm may be by directly promoted by SNAI pathway-mediated epithelial–mesenchymal transition (EMT) at 0–24 h and a lengthened G0/G1 phase by increasing the ubiquitination degradation in 24–48 h. These findings demonstrate that UA plays a critical role in mesoderm differentiation, and its level might be a useful indicator for CHD in early fetal ultrasound screening.

This study investigated the characteristics of three grades primary school students on number sensing. The performances on number sensing are dissimilar with different learning materials. While learning experiences also contributed to their number sensing process. 40 students selected from three grades were required to finish selection tasks. All 40 students had to select numerical value larger one from two spatial cubes, or from two Arabic digits, or from two lowercase Chinese character numbers. The results indicate that all students had easier selection process with Arabic numbers, while they had relative greater difficulty in making selection on numerical value with spatial cubes. The comparisons on mean scores and on reaction times from different-grade students indicate that all fourth-grade students had more learning experiences to develop number sense well, therefore their performances were better than other two grades’.

The use of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) is limited in drug discovery and cardiac disease mechanism studies due to cell immaturity. Although many approaches have been reported to improve the maturation of hiPSC-CMs, the elucidation of the process of maturation is crucial. We applied a small-molecule-based differentiation method to generate cardiomyocytes (CMs) with multiple aggregation forms. The motion analysis revealed significant physical differences in the differently shaped CMs, and the net-shaped CMs had larger motion amplitudes and faster velocities than the sheet-shaped CMs. The net-shaped CMs displayed accelerated maturation at the transcriptional level and were more similar to CMs with a prolonged culture time (30 days) than to sheet-d15. Ion channel genes and gap junction proteins were up-regulated in net-shaped CMs, indicating that robust contraction was coupled with enhanced ion channel and connexin expression. The net-shaped CMs also displayed improved myofibril ultrastructure under transmission electron microscopy. In conclusion, different multicellular hPSC-CM structures, such as the net-shaped pattern, are formed using the conditioned induction method, providing a useful tool to improve cardiac maturation.

Holt-Oram syndrome (HOS), which is caused by genetic changes in the TBX5 gene, affects the hands and heart. HOS patients have heart defects, including atrial septal defects (ASD), ventricular septal defects (VSD) and heart conduction disease. Here, we generated a homozygous TBX5 knockout human embryonic stem cell (hESC) line (TBX5-KO) using a CRISPR/Cas9 system. The TBX5-KO maintained stem cell like morphology, pluripotency markers, normal karyotype, and could differentiate into all three germ layers in vivo. This cell line can provide an in vitro platform for studying the pathogenic mechanisms and biological function of TBX5 in the heart development.

SNTA1 encodes α1-syntrophin, a scaffold protein, which is a component of the dystrophin-associated protein complex. Additionally, α1-syntrophin interacts with SCN5A and nNOS-PMCA4b complex in cardiomyocytes. SNTA1 is a susceptibility locus for arrhythmia and cardiomyopathy. We generated a homozygous SNTA1 knockout human embryonic stem cell (H9SNTA1KO) using the CRISPR/Cas9 system. H9SNTA1KO maintained pluripotency and a normal karyotype and differentiated into three germ layers in vivo.

// Wentao Hu 1 , Hong Lu 1 , Shang Wang 1 , Wenhan Yin 1 , Xujie Liu 2, 3, 4, 5, 6 , Lin Dong 7 , Richard Chiu 8 , Li Shen 7 , Wen-Jing Lu 2, 3, 4, 5 , Feng Lan 2, 3, 4, 5 1 Department of Neurology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, Henan, China 2 Beijing Institute of Heart, Lung and Blood Vessel Diseases, Beijing, China 3 Beijing Lab for Cardiovascular Precision Medicine, Capital Medical University, Beijing, China 4 The Key Laboratory of Remodeling-Related Cardiovascular Disease, Ministry of Education, Beijing, China 5 Beijing Collaborative Innovation Center for Cardiovascular Disorders, Anzhen Hospital, Capital Medical University, Beijing, China 6 Deparment of Radiological Medicine, Chongqing Medical University, Chongqing, China 7 Department of Cell Biology Peking University Health Science Center, Beijing, China 8 Deparment of Radiology, Stanford University School of Medicine, Stanford, California, USA Correspondence to: Feng Lan, email: fenglan@ccmu.edu.cn Wen-Jing Lu, email: wjlu22@gmail.com Keywords: nestin, NPCs, proliferation, EGFR, self-renewal Received: July 27, 2016 Accepted: October 14, 2016 Published: November 22, 2016 ABSTRACT The expression of intermediate filament Nestin is necessary for the neural progenitor cells (NPCs) to maintain stemness, but the underlying cellular and molecular mechanism remains unclear. In this study, we demonstrated that Nestin is required for the self-renew of NPCs through activating MAPK and EGFR pathways. Knockdown of Nestin by shRNA inhibited cell cycle progression and proliferation in mouse NPCs. Moreover, suppression of Nestin reduced expression of the epidermal growth factor receptor (EGFR) in NPCs and inhibited the mitogenic effects of EGF on these cells. Treatment of NPCs with p38-MAPK inhibitor PD169316 reversed cell cycle arrest caused by the knockdown of Nestin. Our findings indicate that Nestin promotes NPC proliferation via p38-MAPK and EGFR pathways, and reveals the necessity of these pathways in NPCs self-renewal.

Two interesting 1D heterometal chain-like compounds from pyridine-2,6-dicarboxylic acid (H2PDA) and AgNO3 as well as ZnCl2/SmCl3, namely [ZnAg(PDA)2(μ2-H2O)·(H3O)+⋅(H2O)0.25]n (1) and [SmAg(PDA)2(H2O)3·3H2O]n (2), have been solvothermally synthesized by fine control synthetic conditions, and structurally characterized. The results reveal that 1D rac-helical chain 1 containing two independent single-stranded single-helical P-helical and M-helical chains crystallized in the tetragonal I4(1)/a space groups, in which the octahedral Zn2 + center in 1 coordinated to two pyridyl nitrogen atoms and four carboxyl oxygen atoms from two chelating PDA2 − ligands, while the distorted tetrahedral Ag+ center in 1 ligated to two aqueous oxygen atoms and two carboxyl oxygen atoms from two bridging PDA2 − ligands. Under the same reaction conditions except the reactant of SmCl3 replacing ZnCl2, 1-D meso-helical chain 2 with monoclinic P2(1)/n space group, consisting of two adjacent three-stranded single-helical P and M chains connected through Ag O bonds, was obtained. Each Sm3 + ion in 2 was chelated to two PDA2 − anions as tridentate (ONO) ligands and three water molecules that completed the nine-coordinated environment around Sm3 +. The coordination sphere of Ag+ was finished by five carboxyl O atoms from three PDA2 − ligands with highly distorted trigonal bipyramidal geometry. Obviously, different helicity between 1 and 2 closely related to the radius and coordi numbers of the 3d/4f metal cation ions. Moreover, the fluorescent propeties of compounds 1 and 2 were also studied.

Muscle LIM protein (MLP, CSRP3) is a key regulator of striated muscle function, and its mutations can lead to both hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) in patients. However, due to lack of human models, mechanisms underlining the pathogenesis of MLP defects remain unclear. In this study, we generated a knockout MLP/CSRP3 human embryonic stem cell (hESC) H9 cell line using CRISPR/Cas9 mediated gene disruption. CSRP3 disruption had no impact on the cardiac differentiation of H9 cells and led to confirmed MLP deficiency in hESC-derived cardiomyocytes (ESC-CMs). MLP-deficient hESC-CMs were found to develop phenotypic features of HCM early after differentiation, such as enlarged cell size, multinucleation, and disorganized sarcomeric ultrastructure. Cellular phenotypes of MLP-deficient hESC-CMs subsequently progressed to mimic heart failure (HF) by 30 days post differentiation, including exhibiting mitochondrial damage, increased ROS generation, and impaired Ca2+ handling. Pharmaceutical treatment with beta agonist, such as isoproterenol, was found to accelerate the manifestation of HCM and HF, consistent with transgenic animal models of MLP deficiency. Furthermore, restoration of Ca2+ homeostasis by verapamil prevented the development of HCM and HF phenotypes, suggesting that elevated intracellular Ca2+ concentration is a central mechanism for pathogenesis of MLP deficiency. In summary, MLP-deficient hESC-CMs recapitulate the pathogenesis of HCM and its progression toward HF, providing an important human model for investigation of CSRP3/MLP-associated disease pathogenesis. More importantly, correction of the autonomous dysfunction of Ca2+ handling was found to be an effective method for treating the in vitro development of cardiomyopathy disease phenotype.

The traditional Chinese herb Lonicerae Japonicae Flos has been used to treat various diseases including heart failure, showing significant benefits in the clinical practice. However, the mechanism of its cardioprotective effects remains unclear. As the main active ingredient found in the plasma after oral administration of Lonicerae Japonicae Flos , chlorogenic acid (CGA) has been reported to possess anti-inflammatory, anti-oxidant, anti-apoptosis and analgesic function. To investigate whether the cardioprotective effects of Lonicerae Japonicae Flos is through CGA, we applied Transverse Aortic Constriction (TAC)-induced heart failure mouse model, to access the effects of CGA. The results of in vivo experiments show that CGA has cardioprotective effects and could mitigate the TNF-α(Tumor necrosis factor-alpha) induced toxicity in TAC heart failure mouse model. Therefore, we further used TNF-α-induced cardiac injury in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to explore whether CGA have cardioprotective effects and elucidate the underlying mechanism(s). CGA pretreatment could reverse TNF-α induced cellular injuries, including improved cell viability, increased mitochondrial membrane potential and inhibited cardiomyocytes apoptosis. We then examined the NF-κB/p65 and major MAPKs signaling pathways involved in TNF-α induced apoptosis of hiPSC-CMs. Importantly, both CGA and NF-κB inhibitor (QNZ) can reverse the cell viability impairment and apoptosis phenotypes. Moreover CGA can directly inhibit NF-κB signal by suppressing the phosphorylation of NF-κB/p65. As for the MAPKs, CGA suppressed the activity of JNK only, but enhanced ERK1/2 and had no effect on p38. In summary, our study revealed that CGA has profound cardioprotective effects through inhibiting the activation of NF-κB and JNK pathway, providing a novel therapeutic alternative for prevention and treatment of heart failure.