Apolipoprotein B and interleukin 1 receptor antagonist: reversing the risk of coronary heart disease.

Aims: Epidemiological evidence for the link of interleukin 1 (IL-1) and its inhibition with cardiovascular diseases (CVDs) remains controversial. We aim to investigate the cardiovascular effects of IL-1 receptor antagonist (IL-1Ra) and underlying mechanisms. Methods: Genetic variants identified from a genome-wide association study involving 30,931 individuals were used as instrumental variables for the serum IL-1Ra concentrations. Genetic associations with CVDs and cardiometabolic risk factors were obtained from international genetic consortia. Inverse-variance weighted method was utilized to derive effect estimates, while supplementary analyses employing various statistical approaches. Results: Genetically determined IL-1Ra level was associated with increased risk of coronary heart disease (CHD; OR, 1.07; 95% CI: 1.03-1.17) and myocardial infarction (OR, 1.13; 95% CI: 1.04-1.21). The main results remained consistent in supplementary analyses. Besides, IL-1Ra was associated with circulating levels of various lipoprotein lipids, apolipoproteins and fasting glucose. Interestingly, observed association pattern with CHD was reversed when adjusting for apolipoprotein B (OR, 0.84; 95%CI: 0.71-0.99) and slightly attenuated on accounting for other cardiometabolic risk factors. Appropriate lifestyle intervention was found to lower IL-1Ra concentration and mitigate the heightened CHD risk it posed. Conclusion: Apolipoprotein B represents the key driver, and a potential target for reversal of the causal link between serum IL-1Ra and increased risk of CHD/MI. The combined therapy involving IL-1 inhibition and lipid-modifying treatment aimed at apolipoprotein B merit further exploration.

Disruption of the nuclear localization signal in RBM20 is causative in dilated cardiomyopathy.

Human patients carrying genetic mutations in RNA binding motif 20 (RBM20) develop a clinically aggressive dilated cardiomyopathy (DCM). Genetic mutation knockin (KI) animal models imply that altered function of the arginine-serine-rich (RS) domain is crucial for severe DCM. To test this hypothesis, we generated an RS domain deletion mouse model (Rbm20ΔRS). We showed that Rbm20ΔRS mice manifested DCM with mis-splicing of RBM20 target transcripts. We found that RBM20 was mis-localized to the sarcoplasm in Rbm20ΔRS mouse hearts and formed RBM20 granules similar to those detected in mutation KI animals. In contrast, mice lacking the RNA recognition motif showed similar mis-splicing of major RBM20 target genes but did not develop DCM or exhibit RBM20 granule formation. Using in vitro studies with immunocytochemical staining, we demonstrated that only DCM-associated mutations in the RS domain facilitated RBM20 nucleocytoplasmic transport and promoted granule assembly. Further, we defined the core nuclear localization signal (NLS) within the RS domain of RBM20. Mutation analysis of phosphorylation sites in the RS domain suggested that this modification may be dispensable for RBM20 nucleocytoplasmic transport. Collectively, our findings revealed that disruption of RS domain-mediated nuclear localization is crucial for severe DCM caused by NLS mutations.

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.

Causal association between sleep traits and the risk of coronary artery disease in patients with diabetes.

Background and aims: The association between sleep traits and coronary artery disease (CAD) in patients with diabetes has been reported in previous observational studies. However, whether these potential relationships are causal remains unclear. We aim to assess the causal relationship between sleep traits and CAD in diabetic. Methods: Genetic instrumental variables associated with five sleep-related traits (insomnia, sleep duration, ease of getting up, morningness and snoring) were extracted from corresponding genome-wide association studies (GWAS). The associations of genetic variants with CAD were based on 15,666 individuals with diabetes (3,968 CAD cases and 11,696 controls). The primary analysis was derived using the inverse variance weighting method. Further sensitivity analysis was conducted to confirm the robustness and consistency of the main results. Results: Genetic liability to insomnia was significantly related to the increased risk of CAD in individuals with diabetes [odds ratio (OR): 1.163; 95% CI: 1.072-1.254; p = 0.001]. Suggestive evidence was found for the borderline associations between both sleep duration (OR: 0.629; 95% CI: 0.380-1.042, p = 0.072) and snoring (OR: 1.010, 95% CI: 1.000-1.020, p = 0.050) with CAD risk. However, no consistent evidence was found for the association between ease of getting up and morningness with the risk of CAD in diabetic. Similar results can be verified in most sensitivity analyses. Conclusions: We provide consistent evidence for the causal effect of insomnia on the increased risk of CAD in individuals with diabetes. The management of sleep health should be emphasized to prevent CAD in diabetic patients.

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.

Generation of five induced pluripotent stem cell lines with DMD/c.497G > T mutation from renal epithelial cells of a Duchenne muscular dystrophy patient and a recessive carrier parent.

Duchenne muscular dystrophy (DMD), an inherited disease caused by the dystrophin gene mutation, is the most common muscular dystrophy in children and is clinically characterized by progressive muscle degeneration and severe cardiomyopathy. In this study, renal epithelial cells were obtained from urine samples of a DMD patient (4 years old) and his recessive carrier parent (35 years old). The cells were reprogrammed with non-integrating Sendai virus to generate three induced pluripotent stem cell (iPSC) clones for the patient and two clones for non-manifesting mutation carrier parent. The iPSC lines had normal karyotypes, iPSC morphology, pluripotency expression markers, and were capable of differentiating into the three germ layers.

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.

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.

Modelling cadmium-induced cardiotoxicity using human pluripotent stem cell-derived cardiomyocytes.

Cadmium, a highly ubiquitous toxic heavy metal, has been widely recognized as an environmental and industrial pollutant, which confers serious threats to human health. The molecular mechanisms of the cadmium-induced cardiotoxicity (CIC) have not been studied in human cardiomyocytes at the cellular level. Here we showed that human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) can recapitulate the CIC at the cellular level. The cadmium-treated hPSC-CMs exhibited cellular phenotype including reduced cell viability, increased apoptosis, cardiac sarcomeric disorganization, elevated reactive oxygen species, altered action potential profile and cardiac arrhythmias. RNA-sequencing analysis revealed a differential transcriptome profile and activated MAPK signalling pathway in cadmium-treated hPSC-CMs, and suppression of P38 MAPK but not ERK MAPK or JNK MAPK rescued CIC phenotype. We further identified that suppression of PI3K/Akt signalling pathway is sufficient to reverse the CIC phenotype, which may play an important role in CIC. Taken together, our data indicate that hPSC-CMs can serve as a suitable model for the exploration of molecular mechanisms underlying CIC and for the discovery of CIC cardioprotective drugs.

A Novel Approach for Amplification and Purification of Mouse Oligodendrocyte Progenitor Cells.

Although transgenic and knockout mice are widely used to study the specification and differentiation of oligodendrocyte precursor cells (OPCs), mouse primary OPCs are difficult to be purified and maintained, and many in vitro studies have to resort to rat OPCs as substitutes. In this study, we reported that mouse O4 negative early-stage OPCs can be obtained by culturing cortical tissue blocks, and the simultaneous treatment of OPCs with Platelet Derived Growth Factor-AA (PDGFaa), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) is the key for the propagation of mouse OPCs in culture. EGF was found to be a potent mitogen for OPCs and cooperate with PDGFaa to extend cell division and inhibit their differentiation. EGF also collaborates with PDGFaa and bFGF to convert bipolar or tripolar OPCs to more vital fibroblast-like OPCs without compromising their oligodendrocyte differentiation potential. In addition, EGF promoted the survival and proliferation of glial progenitor cells (GPCs) derived from primary OPC cultures, and a mixture of GPCs and OPCs can be obtained and propagated in the presence of EGF, bFGF, and PDGFaa. Once EGF is withdrawn, GPC population decreased sharply and fibroblast-like OPCs changed into typical OPCs morphology, then homogeneous OPCs were obtained subsequently.

[Expression of neural salient serine-/arginine-rich protein 1 (NSSR1) in colorectal cancer].

OBJECTIVE: To investigate the expression of neural salient serine/arginine-rich protein 1 (NSSR1) in colorectal cancer. METHODS: RT-PCR, Western blot and immunohistochemical staining were used to detect the expression of NSSR1 mRNA and protein in different mouse tissues and human colorectal cancer, respectively. RESULTS: NSSR1 mRNA was expressed in mouse cerebrum, cerebellum, heart, liver, intestine, kidney and lung tissue, but NSSR1 protein was only expressed in neural tissues. In normal human intestinal mucosa, NSSR1 was expressed in the colorectal epithelial cells. In colorectal cancer, NSSR1 was highly expressed in the nucleus of tumor cells. CONCLUSION: The extensive expression of NSSR1 in colorectal cancer cells may hint it's roles in the biological function of colorectal cancer.

[Controlling of the high-voltage generator in a direct radiography system].

A proposal is presented to improve the original complicated control of the exposure parameters in the direct radiography system (DR). The interface circuitry of the high-voltage generator is redesigned using RS-232 serial COM port. The data and signals are transmitted between the personal computer and the high-voltage generator in the serial mode. A program package is designed to realize the control of the exposure parameters of the high-voltage generator in DR on the PC machine. Experiment results have shown that the proposed console operates steadily and it is capable of providing a more convenient operation console for the direct radiography system.