Heavy Metal Exposure and Cardiovascular Disease.

Heavy metals are harmful environmental pollutants that have attracted widespread attention due to their health hazards to human cardiovascular disease. Heavy metals, including lead, cadmium, mercury, arsenic, and chromium, are found in various sources such as air, water, soil, food, and industrial products. Recent research strongly suggests a connection between cardiovascular disease and exposure to toxic heavy metals. Epidemiological, basic, and clinical studies have revealed that heavy metals can promote the production of reactive oxygen species, which can then exacerbate reactive oxygen species generation and induce inflammation, resulting in endothelial dysfunction, lipid metabolism distribution, disruption of ion homeostasis, and epigenetic changes. Over time, heavy metal exposure eventually results in an increased risk of hypertension, arrhythmia, and atherosclerosis. Strengthening public health prevention and the application of chelation or antioxidants, such as vitamins and beta-carotene, along with minerals, such as selenium and zinc, can diminish the burden of cardiovascular disease attributable to metal exposure.

Generation of a TRPV1 knockout human pluripotent stem cell line (WAe009-A-U) using CRISPR/Cas9.

The transient receptor potential vanilloid subfamily 1 (TRPV1) is a polymodal nociceptor that is highly expressed in sensory nerves. Activation of TRPV1 receptors excites primary afferent nociceptors by opening cation channels, allowing the influx of Na+ and Ca2+ ions into the cytoplasm. Here, a TRPV1 knockout human embryonic stem cell line was generated using the CRISPR/Cas9 genome-editing technology to further study the function of TRPV1. The cell line confirmed with normal pluripotency and karyotype.

Patient-specific iPSC-derived cardiomyocytes reveal aberrant activation of Wnt/ß-catenin signaling in SCN5A-related Brugada syndrome.

BACKGROUND: Mutations in the cardiac sodium channel gene SCN5A cause Brugada syndrome (BrS), an arrhythmic disorder that is a leading cause of sudden death and lacks effective treatment. An association between SCN5A and Wnt/ß-catenin signaling has been recently established. However, the role of Wnt/ß-catenin signaling in BrS and underlying mechanisms remains unknown. METHODS: Three healthy control subjects and one BrS patient carrying a novel frameshift mutation (T1788fs) in the SCN5A gene were recruited in this study. Control and BrS patient-specific induced pluripotent stem cells (iPSCs) were generated from skin fibroblasts using nonintegrated Sendai virus. All iPSCs were differentiated into cardiomyocytes using monolayer-based differentiation protocol. Action potentials and sodium currents were recorded from control and BrS iPSC-derived cardiomyocytes (iPSC-CMs) by single-cell patch clamp. RESULTS: BrS iPSC-CMs exhibited increased burden of arrhythmias and abnormal action potential profile featured by slower depolarization, decreased action potential amplitude, and increased beating interval variation. Moreover, BrS iPSC-CMs showed cardiac sodium channel (Nav1.5) loss-of-function as compared to control iPSC-CMs. Interestingly, the electrophysiological abnormalities and Nav1.5 loss-of-function observed in BrS iPSC-CMs were accompanied by aberrant activation of Wnt/ß-catenin signaling. Notably, inhibition of Wnt/ß-catenin significantly rescued Nav1.5 defects and arrhythmic phenotype in BrS iPSC-CMs. Mechanistically, SCN5A-encoded Nav1.5 interacts with ß-catenin, and reduced expression of Nav1.5 leads to re-localization of ß-catenin in BrS iPSC-CMs, which aberrantly activates Wnt/ß-catenin signaling to suppress SCN5A transcription. CONCLUSIONS: Our findings suggest that aberrant activation of Wnt/ß-catenin signaling contributes to the pathogenesis of SCN5A-related BrS and point to Wnt/ß-catenin as a potential therapeutic target.

Patient-specific iPSC-derived cardiomyocytes reveal variable phenotypic severity of Brugada syndrome.

BACKGROUND: Brugada syndrome (BrS) is a cardiac channelopathy that can result in sudden cardiac death (SCD). SCN5A is the most frequent gene linked to BrS, but the genotype-phenotype correlations are not completely matched. Clinical phenotypes of a particular SCN5A variant may range from asymptomatic to SCD. Here, we used comparison of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) derived from a SCN5A mutation-positive (D356Y) BrS family with severely affected proband, asymptomatic mutation carriers (AMCs) and healthy controls to investigate this variation. METHODS: 26 iPSC lines were generated from skin fibroblasts using nonintegrated Sendai virus. The generated iPSCs were differentiated into cardiomyocytes using a monolayer-based differentiation protocol. FINDINGS: D356Y iPSC-CMs exhibited increased beat interval variability, slower depolarization, cardiac arrhythmias, defects of Na+ channel function and irregular Ca2+ signaling, when compared to controls. Importantly, the phenotype severity observed in AMC iPSC-CMs was milder than that of proband iPSC-CMs, an observation exacerbated by flecainide. Interestingly, the iPSC-CMs of the proband exhibited markedly decreased Ca2+ currents in comparison with control and AMC iPSC-CMs. CRISPR/Cas9-mediated genome editing to correct D356Y in proband iPSC-CMs effectively rescued the arrhythmic phenotype and restored Na+ and Ca2+ currents. Moreover, drug screening using established BrS iPSC-CM models demonstrated that quinidine and sotalol possessed antiarrhythmic effects in an individual-dependent manner. Clinically, venous and oral administration of calcium partially reduced the malignant arrhythmic events of the proband in mid-term follow-up. INTERPRETATION: Patient-specific and genome-edited iPSC-CMs can recapitulate the varying phenotypic severity of BrS. Our findings suggest that preservation of the Ca2+ currents might be a compensatory mechanism to resist arrhythmogenesis in BrS AMCs. FUNDING: National Key R&D Program of China (2017YFA0103700), National Natural Science Foundation of China (81922006, 81870175), Natural Science Foundation of Zhejiang Province (LD21H020001, LR15H020001), National Natural Science Foundation of China (81970269), Key Research and Development Program of Zhejiang Province (2019C03022) and Natural Science Foundation of Zhejiang Province (LY16H020002).

Overexpression of KCNJ2 enhances maturation of human-induced pluripotent stem cell-derived cardiomyocytes.

BACKGROUND: Although human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are a promising cell resource for cardiovascular research, these cells exhibit an immature phenotype that hampers their potential applications. The inwardly rectifying potassium channel Kir2.1, encoded by the KCNJ2 gene, has been thought as an important target for promoting electrical maturation of iPSC-CMs. However, a comprehensive characterization of morphological and functional changes in iPSC-CMs overexpressing KCNJ2 (KCNJ2 OE) is still lacking. METHODS: iPSC-CMs were generated using a 2D in vitro monolayer differentiation protocol. Human KCNJ2 construct with green fluorescent protein (GFP) tag was created and overexpressed in iPSC-CMs via lentiviral transduction. The mixture of iPSC-CMs and mesenchymal cells was cocultured with decellularized natural heart matrix for generation of 3D human engineered heart tissues (EHTs). RESULTS: We showed that mRNA expression level of KCNJ2 in iPSC-CMs was dramatically lower than that in human left ventricular tissues. KCNJ2 OE iPSC-CMs yielded significantly increased protein expression of Kir2.1 and current density of Kir2.1-encoded IK1. The larger IK1 linked to a quiescent phenotype that required pacing to elicit action potentials in KCNJ2 OE iPSC-CMs, which can be reversed by IK1 blocker BaCl2. KCNJ2 OE also led to significantly hyperpolarized maximal diastolic potential (MDP), shortened action potential duration (APD) and increased maximal upstroke velocity. The enhanced electrophysiological maturation in KCNJ2 OE iPSC-CMs was accompanied by improvements in Ca2+ signaling, mitochondrial energy metabolism and transcriptomic profile. Notably, KCNJ2 OE iPSC-CMs exhibited enlarged cell size and more elongated and stretched shape, indicating a morphological phenotype toward structural maturation. Drug testing using hERG blocker E-4031 revealed that a more stable MDP in KCNJ2 OE iPSC-CMs allowed for obtaining significant drug response of APD prolongation in a concentration-dependent manner. Moreover, KCNJ2 OE iPSC-CMs formed more mature human EHTs with better tissue structure and cell junction. CONCLUSIONS: Overexpression of KCNJ2 can robustly enhance maturation of iPSC-CMs in electrophysiology, Ca2+ signaling, metabolism, transcriptomic profile, cardiomyocyte structure and tissue engineering, thus providing more accurate cellular model for elucidating cellular and molecular mechanisms of cardiovascular diseases, screening drug-induced cardiotoxicity, and developing personalized and precision cardiovascular medicine.

Establishment of an induced pluripotent stem cell line (ZJULLi004-A) from a hypertrophic cardiomyopathy patient carrying MYBPC3/c.3764C>A mutation.

Hypertrophic cardiomyopathy (HCM) is an inherited cardiovascular disease characterized by left ventricular hypertrophy and a high risk of sudden death. In this study, a skin biopsy was obtained from a HCM patient harboring a heterozygous missense mutation (c.3764C>A; p.A1225D) in the myosin binding protein C3 (MYBPC3) gene. The isolated fibroblasts were reprogrammed using non-integrated Sendai viral method to establish the patient-specific induced pluripotent stem cell (iPSC) line. The established iPSC line displayed normal morphology and karyotype, expressed pluripotency markers, and can differentiate into three germ layers in vivo.

Generation of an induced pluripotent stem cell line (ZJULLi003-A) from a hypertrophic cardiomyopathy patient carrying MYH7/c.4384G > A mutation.

Hypertrophic cardiomyopathy (HCM) is an autosomal dominant inherited cardiovascular disease characterized by left ventricular hypertrophy and cardiomyocyte disarray. In this study, a skin biopsy was obtained from a HCM patient, who carried a missense mutation (c.4384G > A; p.E1462K) in the myosin heavy chain 7 (MYH7) gene. The skin fibroblasts were subsequently reprogrammed with a non-integrated Sendai viral method to generate a patient-specific induced pluripotent stem cell (iPSC) line. The generated iPSC line showed typical morphology and normal karyotype, expressed pluripotency markers, and was capable to differentiate into three germ layers.

Generation of an induced pluripotent stem cell line from a long QT syndrome patient carrying KCNH2/1956C > A mutation.

Long QT syndrome (LQT) is an inherited primary arrhythmic disorder characterized by prolonged QT interval on the surface electrocardiogram and life-threatening arrhythmia. In this study, a skin biopsy was obtained from an LQT type 2 (LQT2) patient, who carried a nonsense mutation (c.1956C > A; p.Y652X) in the potassium voltage-gated channel subfamily H member 2 (KCNH2) gene. The skin fibroblasts were reprogrammed by non-integrated Sendai viral method to generate a patient-specific induced pluripotent stem cell (iPSC) line. The generated iPSC line showed typical embryonic stem cell-like morphology, exhibited normal karyotype, expressed pluripotency markers, and was capable to differentiate into three germ layers.

Patient-specific iPSC-derived endothelial cells reveal aberrant p38 MAPK signaling in atypical hemolytic uremic syndrome.

Atypical hemolytic uremic syndrome (aHUS) is a rare disease associated with high morbidity and mortality. Existing evidence suggests that the central pathogenesis to aHUS might be endothelial cell damage. Nevertheless, the role of endothelial cell alterations in aHUS has not been well characterized and the underlying mechanisms remain unclear. Utilizing an induced pluripotent stem cell-derived endothelial cell (iPSC-EC) model, we showed that anti-complement factor H autoantibody-associated aHUS patient-specific iPSC-ECs exhibited an intrinsic defect in endothelial functions. Stimulation using aHUS serums exacerbated endothelial dysfunctions, leading to cell apoptosis in iPSC-ECs. Importantly, we identified p38 as a novel signaling pathway contributing to endothelial dysfunctions in aHUS. These results illustrate that iPSC-ECs can be a reliable model to recapitulate EC pathological features, thus providing a unique platform for gaining mechanistic insights into EC injury in aHUS. Our findings highlight that the p38 MAPK signaling pathway can be a therapeutic target for treatment of aHUS.

PADI6 Regulates Trophoblast Cell Migration-Invasion Through the Hippo/YAP1 Pathway in Hydatidiform Moles.

PURPOSE: Peptidyl arginine deiminase, type VI (PADI6), a member of the subcortical maternal complex, plays an important role in oocyte growth and the development of fertilized oocytes. Human patients with PADI6 mutations can suffer from multiple reproductive deficiencies including hydatidiform moles and miscarriages. Recent studies have demonstrated that the Hippo signaling pathway plays a central role in the specification of the first cell fates and the maintenance of the human placental trophoblast epithelium. The present study aimed to verify the hypothesis that PADI6 regulates the biological functions of trophoblast cells by targeting YAP1 and to explore the mechanism by which PADI6 accomplishes this in trophoblast cells. METHODS: Villi from HMs and human trophoblast cell lines were used to identify the localization of PADI6 and YAP1 by immunohistochemistry and immunocytochemistry. PADI6 overexpression and knockdown were induced in human trophoblast cells. Co-immunoprecipitation was used to explore the interaction between PADI6 and YAP1. Wound healing, Transwell and EdU staining assays were used to detect migration, invasion and proliferation. Flow cytometric analysis was used to analyze the cell cycle and apoptosis. ß-Tubulin and F-actin levels were determined by Western blot, quantitative real-time PCR and phalloidin staining. RESULTS: The results showed that PADI6 and YAP1 had the same expression pattern in villi and colocalized in the cytotrophoblast. An interaction between PADI6 and YAP1 was also confirmed in human trophoblast cell lines. We found that PADI6 positively regulated the expression of YAP1. Functionally, overexpression of PADI6 promoted cell cycle progression and enhanced migration, invasion, proliferation and apoptosis, whereas downregulation of PADI6 showed the opposite effects. CONCLUSION: This study demonstrates that YAP1 is a novel target of PADI6 that serves as an important regulator of trophoblast dysfunction. The crosstalk between the Hippo/YAP1 pathway and the SCMC might be a new topic to explore to uncover the pathological mechanisms of HMs.

Reprogramming of human peripheral blood mononuclear cells from a patient suffering from recurrent hydatidiform mole to an iPSC line FAHZUi001-A carrying a homozygous p.Gln421Ter mutation in NLRP7 gene.

Recurrent hydatidiform mole (RHM) is characterized by the occurrence of at least twice hydatidiform mole. Unlike sporadic complete hydatidiform moles (CHMs), which are androgenetic with 2 paternal chromosomes, CHMs associated with familial recurrence are genetically biparental with a maternal and a paternal chromosome. NLRP7 mutations have been reported in 55% of RHM cases. Here, we generated induced pluripotent stem cells (iPSCs) from a patent with NLRP7 gene mutation c.1261C > T by reprogramming peripheral blood mononuclear cells by non-integrated method. The resulting iPSCs carrying NLRP7 mutation, had normal karyotype, expressed pluripotency markers, and could differentiate into three germ layersin vivo.

Generation of an induced pluripotent stem cell line from the dermal fibroblasts of a patient with arrhythmogenic right ventricular cardiomyopathy carrying a PKP2/c.2489 + 1G > A mutation.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited disease in which the right ventricular myocardium is replaced by progressive fibrous adipose tissue. ARVC is clinically characterized by right ventricular enlargement, ventricular arrhythmia, and sudden cardiac death. It eventually leads to heart failure, and thus has a significant impact on the patient's health. In this study, human dermal fibroblasts were obtained from a patient with ARVC, which were subsequently reprogrammed with a non-integrated Sendai virus to generate a patient-specific induced pluripotent stem cell (iPSC) line. The iPSC line exhibited normal karyotype and morphology, expressed pluripotency markers, and was capable of differentiating into three germ layers.

Generation of ZJUi003-A, an induced pluripotent stem cell line from a Wilson's disease patient carrying a c.180_181del mutation in ATP7B gene.

Wilson's disease (WD) is an inherited autosomal recessive disease, which is caused by the mutation of ATP7B gene encoding copper-transporting ATPase protein. The WD patients always suffer from the excessive copper deposition in the liver and other tissues because of the dysfunction of the copper-transporting ATPase protein. In this study, we generated a patient-specific induced pluripotent stem cell (iPSC) line (ZJUi003-A), which showed normal karyotype, expressed pluripotency markers and was capable to differentiate into three germ layers.

Human induced pluripotent stem cell-derived cardiomyocytes reveal abnormal TGFß signaling in type 2 diabetes mellitus.

Diabetes mellitus is a serious metabolic condition associated with a multitude of cardiovascular complications. Moreover, the prevalence of diabetes in heart failure populations is higher than that in control populations. However, the role of cardiomyocyte alterations in type 2 diabetes mellitus (T2DM) has not been well characterized and the underlying mechanisms remain elusive. In this study, two patients who were diagnosed as T2DM were recruited and patient-specific induced pluripotent stem cells (iPSCs) were generated from urine epithelial cells using nonintegrated Sendai virus. The iPSC lines derived from five healthy subjects were used as controls. All iPSCs were differentiated into cardiomyocytes (iPSC-CMs) using the monolayer-based differentiation protocol. T2DM iPSC-CMs exhibited various disease phenotypes, including cellular hypertrophy and lipid accumulation. Moreover, T2DM iPSC-CMs exhibited higher susceptibility to high-glucose/high-lipid challenge than control iPSC-CMs, manifesting an increase in apoptosis. RNA-Sequencing analysis revealed a differential transcriptome profile and abnormal activation of TGFß signaling pathway in T2DM iPSC-CMs. We went on to show that inhibition of TGFß significantly rescued the hypertrophic phenotype in T2DM iPSC-CMs. In conclusion, we demonstrate that the iPSC-CM model is able to recapitulate cellular phenotype of T2DM. Our results indicate that iPSC-CMs can therefore serve as a suitable model for investigating molecular mechanisms underlying diabetic cardiomyopathies and for screening therapeutic drugs.

Hydrogen gas alleviates postharvest senescence of cut rose 'Movie star' by antagonizing ethylene.

KEY MESSAGE: H2 prolonged the vase life and improved the vase quality of cut roses through repressing endogenous ethylene production and alleviating ethylene signal transduction during the entire senescing period. Recently, the application of hydrogen gas (H2) was shown to improve postharvest quality and longevity in perishable horticultural products, but the specific regulation mechanism remains obscure. Here, endogenous ethylene production and the expression of genes in ethylene biosynthesis and signalling pathway were investigated to explore the crosstalk between H2 and ethylene during the senescence of cut roses. Our results revealed that addition of exogenous ethylene by ethephon accelerated the senescence of cut roses, in which 100 mg L-1 ethephon displayed the most obvious senescent phenotype. While the applied different concentrations (1%, 10%, 50% and 100%) of hydrogen-rich water (HRW) conducted different affects in alleviating the senescence of cut roses, and 1% HRW displayed the best ornamental quality and the longest vase life by reducing ethylene production, supported by the decrease of 1-aminocyclopropene-1-carboxylate (ACC) accumulation, ACC synthase (ACS) and ACC oxidase (ACO) activities, and Rh-ACS3 and Rh-ACO1 expressions in ethylene biosynthesis. In addition, HRW increased the transcripts of ethylene receptor genes Rh-ETR1 at blooming period from day 4 to day 6 and suppressed Rh-ETR3 at senescence phase at day 8 after harvest. Furthermore, the relevant affection of HRW on Rh-ETR1 and Rh-ETR3 expressions still existed when the ethylene production was compromised by adequate addition of exogenous ethylene in HRW-treated cut rose petals, and HRW directly repressed the protein level of Rh-ETR3 in a transient expression assay. Overall, the results suggested that H2 is involved in neutralizing ethylene-mediated postharvest in cut flowers.

Inhibition of TRPC1 prevents cardiac hypertrophy via NF-κB signaling pathway in human pluripotent stem cell-derived cardiomyocytes.

Cardiac hypertrophy is an adaptive response against increased workload featuring by an increase in left ventricular mass and a thickening left ventricle wall. Here, we showed the expression of transient receptor potential canonical 1 (TRPC1) is higher in hearts of patients with hypertrophic cardiomyopathy (HCM) or heart failure (HF) than that of normal hearts. To better understand the mechanisms of TRPC1 in regulating cellular hypertrophy of human-based cardiomyocytes, we generated human pluripotent stem cell lines of TRPC1 knockout by CRISPR/Cas9. We demonstrated that knockout of TRPC1 significantly attenuated cardiomyocyte hypertrophy phenotype induced by phorbol 12-myristate 13-acetate, which was associated with abnormal activation of NF-κB. In contrast, overexpression of TRPC1 induced cardiomyocyte hypertrophy, which can be reversed by inhibition of NF-κB. Taken together, we established a stable human-based cardiomyocyte hypertrophy model and highlighted molecular mechanisms underlying TRPC1-mediated hypertrophy, aiding the development of therapeutic drugs for HCM and HF by targeting TRPC1.