Yi-Han Chen

Identification of a KCNE2 Gain-of-Function Mutation in Patients with Familial Atrial Fibrillation

Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in clinical practice. We first reported an S140G mutation of KCNQ1, an alpha subunit of potassium channels, in one Chinese kindred with AF. However, the molecular defects and cellular mechanisms in most patients with AF remain to be identified. We evaluated 28 unrelated Chinese kindreds with AF and sequenced eight genes of potassium channels (KCNQ1, HERG, KCNE1, KCNE2, KCNE3, KCNE4, KCNE5, and KCNJ2). An arginine-to-cysteine mutation at position 27 (R27C) of KCNE2, the beta subunit of the KCNQ1-KCNE2 channel responsible for a background potassium current, was found in 2 of the 28 probands. The mutation was present in all affected members in the two kindreds and was absent in 462 healthy unrelated Chinese subjects. Similar to KCNQ1 S140G, the mutation had a gain-of-function effect on the KCNQ1-KCNE2 channel; unlike long QT syndrome-associated KCNE2 mutations, it did not alter HERG-KCNE2 current. The mutation did not alter the functions of the HCN channel family either. Thus, KCNE2 R27C is a gain-of-function mutation associated with the initiation and/or maintenance of AF.

HCN4 Dynamically Marks the First Heart Field and Conduction System Precursors

Rationale: To date, there has been no specific marker of the first heart field to facilitate understanding of contributions of the first heart field to cardiac lineages. Cardiac arrhythmia is a leading cause of death, often resulting from abnormalities in the cardiac conduction system (CCS). Understanding origins and identifying markers of CCS lineages are essential steps toward modeling diseases of the CCS and for development of biological pacemakers. Objective: To investigate HCN4 as a marker for the first heart field and for precursors of distinct components of the CCS, and to gain insight into contributions of first and second heart lineages to the CCS. Methods and Results: HCN4CreERT2, -nuclear LacZ, and -H2BGFP mouse lines were generated. HCN4 expression was examined by means of immunostaining with HCN4 antibody and reporter gene expression. Lineage studies were performed using HCN4CreERT2, Isl1Cre, Nkx2.5Cre, and Tbx18Cre, coupled to coimmunostaining with CCS markers. Results demonstrated that, at cardiac crescent stages, HCN4 marks the first heart field, with HCN4CreERT2 allowing assessment of cell fates adopted by first heart field myocytes. Throughout embryonic development, HCN4 expression marked distinct CCS precursors at distinct stages, marking the entire CCS by late fetal stages. We also noted expression of HCN4 in distinct subsets of endothelium at specific developmental stages. Conclusions: This study provides insight into contributions of first and second heart lineages to the CCS and highlights the potential use of HCN4 in conjunction with other markers for optimization of protocols for generation and isolation of specific conduction system precursors.

MicroRNA expression signature in atrial fibrillation with mitral stenosis

The aim of this study was to investigate the microRNA (miRNA) signature in atrial fibrillation (AF) with mitral stenosis (MS). miRNA arrays were used to evaluate the expression signature of the right atrial appendages of healthy individuals (n=9), patients with MS and AF (n=9) and patients with MS without AF (n=4). The results were validated with qRT-PCR analysis. GOmir was used to predict the potential miRNA targets and to analyze their functions. DIANA-mirPath was used to incorporate the miRNAs into pathways. miRNA arrays revealed that 136 and 96 miRNAs were expressed at different levels in MS patients with AF and in MS patients without AF, respectively, compared with healthy controls. More importantly, 28 miRNAs were expressed differently in the MS patients with AF compared with the MS patients without AF; of these miRNAs, miR-1202 was the most dysregulated. The unsupervised hierarchical clustering analysis based on the 28 differently expressed miRNAs showed that the heat map of miRNA expression categorized two well-defined clusters that corresponded to MS with AF and MS without AF. The qRT-PCR results correlated well with the microarray data. Bioinformatic analysis indicated the potential miRNA targets and molecular pathways. This study shows that there is a distinct miRNA expression signature in AF with MS. The findings may be useful for the development of therapeutic interventions that are based on rational target selection in these patients.

MicroRNA-134 as a potential plasma biomarker for the diagnosis of acute pulmonary embolism

BackgroundAcute pulmonary embolism (APE) remains a diagnostic challenge due to a variable clinical presentation and the lack of a reliable screening tool. MicroRNAs (miRNAs) regulate gene expression in a wide range of pathophysiologic processes. Circulating miRNAs are emerging biomarkers in heart failure, type 2 diabetes and other disease states; however, using plasma miRNAs as biomarkers for the diagnosis of APE is still unknown.MethodsThirty-two APE patients, 32 healthy controls, and 22 non-APE patients (reported dyspnea, chest pain, or cough) were enrolled in this study. The TaqMan miRNA microarray was used to identify dysregulated miRNAs in the plasma of APE patients. The TaqMan-based miRNA quantitative real-time reverse transcription polymerase chain reactions were used to validate the dysregulated miRNAs. The receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the miRNA identified as the candidate biomarker.ResultsPlasma miRNA-134 (miR-134) level was significantly higher in the APE patients than in the healthy controls or non-APE patients. The ROC curve showed that plasma miR-134 was a specific diagnostic predictor of APE with an area under the curve of 0.833 (95% confidence interval, 0.737 to 0.929; P < 0.001).ConclusionsOur findings indicated that plasma miR-134 could be an important biomarker for the diagnosis of APE. Because of this finding, large-scale investigations are urgently needed to pave the way from basic research to clinical utilization.

MicroRNA-204 is required for differentiation of human-derived cardiomyocyte progenitor cells

Human cardiomyocyte progenitor cells (hCMPCs) are cardiac progenitor cells that are unique for their efficient differentiation into beating cardiomyocytes without requiring co-culture with neonatal cardiomyocytes. hCMPCs have shown great potential in preserving the function of infarcted mouse myocardium. MiRNA-204 has been reported to be up-regulated in differentiated hCMPCs, however, its biological significance is unclear. In this study, hCMPC proliferation, viability, apoptosis and necrosis were determined using the ELISA Kit (colorimetric BrdU detection), Cell Counting Kit-8, and Annexin V and propidium iodide staining, respectively. MiRNA-204 inhibition promoted hCMPC proliferation without affecting cell viability and the level of apoptosis and necrosis, indicating that miRNA-204 might be required for hCMPC differentiation. Quantitative reverse transcriptase-polymerase chain reactions were used to detect the expression profile of cardiac genes, including MEF2C, GATA-4, Nkx-2.5, TropT, βMHC, and cActin. Cardiac α-actin staining was used to quantify the degree of differentiation. MiRNA-204 inhibition significantly down-regulated TropT, βMHC, and cActin and reduced differentiation by 47.81% after 2 weeks of differentiation induction. Interestingly, miRNA-204 mimics (30 nM) did not promote hCMPC proliferation and differentiation. The bioinformatic tool GOmir identified the activating transcription factor 2 (ATF-2) as a potential target, which was confirmed by Western blot and a luciferase reporter assay. ATF-2 overexpression promoted hCMPC proliferation, further demonstrating the role played by ATF-2 as a target gene of miRNA-204. Therefore, miRNA-204 is required for hCMPC differentiation and ATF-2 is a target gene of miRNA-204 in hCMPCs. This study indicates that miRNA-204 is among the regulators that drive hCMPC proliferation and differentiation, and miRNA-204 might be used to influence cell fate.

Isl1 Is required for multiple aspects of motor neuron development

The LIM homeodomain transcription factor Islet1 (Isl1) is expressed in multiple organs and plays essential roles during embryogenesis. Isl1 is required for the survival and specification of spinal cord motor neurons. Due to early embryonic lethality and loss of motor neurons, the role of Isl1 in other aspects of motor neuron development remains unclear. In this study, we generated Isl1 mutant mouse lines expressing graded doses of Isl1. Our study has revealed essential roles of Isl1 in multiple aspects of motor neuron development, including motor neuron cell body localization, motor column formation and axon growth. In addition, Isl1 is required for survival of cranial ganglia neurons.

Transcription factor ISL1 is essential for pacemaker development and function

The sinoatrial node (SAN) maintains a rhythmic heartbeat; therefore, a better understanding of factors that drive SAN development and function is crucial to generation of potential therapies, such as biological pacemakers, for sinus arrhythmias. Here, we determined that the LIM homeodomain transcription factor ISL1 plays a key role in survival, proliferation, and function of pacemaker cells throughout development. Analysis of several Isl1 mutant mouse lines, including animals harboring an SAN-specific Isl1 deletion, revealed that ISL1 within SAN is a requirement for early embryonic viability. RNA-sequencing (RNA-seq) analyses of FACS-purified cells from ISL1-deficient SANs revealed that a number of genes critical for SAN function, including those encoding transcription factors and ion channels, were downstream of ISL1. Chromatin immunoprecipitation assays performed with anti-ISL1 antibodies and chromatin extracts from FACS-purified SAN cells demonstrated that ISL1 directly binds genomic regions within several genes required for normal pacemaker function, including subunits of the L-type calcium channel, Ank2, and Tbx3. Other genes implicated in abnormal heart rhythm in humans were also direct ISL1 targets. Together, our results demonstrate that ISL1 regulates approximately one-third of SAN-specific genes, indicate that a combination of ISL1 and other SAN transcription factors could be utilized to generate pacemaker cells, and suggest ISL1 mutations may underlie sick sinus syndrome.

miR-221/222 Promotes S-Phase Entry and Cellular Migration in Control of Basal-Like Breast Cancer

The miR-221/222 cluster has been demonstrated to function as oncomiR in human cancers. miR-221/222 promotes epithelial-to-mesenchymal transition (EMT) and confers tamoxifen resistance in breast cancer. However, the effects and mechanisms by which miR-221/222 regulates breast cancer aggressiveness remain unclear. Here we detected a much higher expression of miR-221/222 in highly invasive basal-like breast cancer (BLBC) cells than that in non-invasive luminal cells. A microRNA dataset from breast cancer patients indicated an elevated expression of miR-221/222 in BLBC subtype. S-phase entry of the cell cycle was associated with the induction of miR-221/222 expression. miRNA inhibitors specially targeting miR-221 or miR-222 both significantly suppressed cellular migration, invasion and G1/S transition of the cell cycle in BLBC cell types. Proteomic analysis demonstrated the down-regulation of two tumor suppressor genes, suppressor of cytokine signaling 1 (SOCS1) and cyclin-dependent kinase inhibit 1B (CDKN1B), by miR-221/222. This is the first report to reveal miR-221/222 regulation of G1/S transition of the cell cycle. These findings demonstrate that miR-221/222 contribute to the aggressiveness in control of BLBC.

Prolyl hydroxylase 3 interacts with Bcl-2 to regulate doxorubicin-induced apoptosis in H9c2 cells

Prolyl hydroxylases (PHDs) are dioxygenases that use oxygen as a co-substrate to hydroxylate proline residues. Three PHD isoforms (PHD1, PHD2 and PHD3) have been identified in mammalian cells. PHD3 expression is upregulated in some cardiac diseases such as cardiomyopathy, myocardial ischemia-reperfusion injury and congestive heart failure, all of which are associated with apoptosis. However, the role of PHDs in cardiomyocyte apoptosis remains unknown. Here, we have found that exposure of embryonic rat heart-derived H9c2 cells to doxorubicin (DOX) induced cell apoptosis as evaluated by caspase-3/7 activity, mitochondrial membrane potential (Δψm) and cell viability, and that this apoptosis was linked to PHD3 upregulation. PHD inhibition or PHD3 silencing substantially ameliorated DOX-induced apoptosis, but PHD1 or PHD2 knockdown did not significantly influence apoptosis. Furthermore, immunoprecipitation experiments showed that PHD3 upregulation reduced the formation of the Bax-Bcl-2 complex, inhibiting the anti-apoptotic effect of Bcl-2. Thus, PHD3 upregulation may be partially responsible for DOX-induced cardiomyocyte apoptosis via its interaction with Bcl-2. Inhibition of PHD3 is likely to be cardioprotective against apoptosis in some heart disorders.

β2‐ but not β1‐adrenoceptor activation modulates intracellular oxygen availability

β‐Adrenoceptors (β‐ARs) play a critical role in the regulation of cardiovascular function. Intracellular oxygen homeostasis is crucial for the survival of cardiomyocytes. However, it is still unclear whether β‐AR activation can modulate intracellular oxygen. Here we used mitochondrial and cytosolic target Renilla luciferase to detect intracellular oxygen concentration. Pharmacological experiments revealed that β2‐AR activation specifically regulates intracellular oxygen in cardiomyocytes and COS7 cells. This effect was abrogated by inhibitory G protein (Gi) inhibition, endothelial nitric oxide synthase (eNOS) blockade, and NO scavenging, implicating that the β2‐AR–Gi–eNOS pathway is involved in this regulation. β2‐AR activation increased the AMP/ATP ratio, AMPK activity, ROS production and prolyl hydroxylase activity. These effects also contribute to the regulation of β2‐AR signalling, thus providing an additional layer of complexity to enforce the specificity of β1‐AR and β2‐AR signalling. Collectively, the study provides novel insight into the modulation of oxygen homeostasis, broadens the scope of β2‐AR function, and may have crucial implications for β2‐AR signalling regulation.