Canonical Wnt signaling directs the generation of functional human PSC-derived atrioventricular canal cardiomyocytes in bioprinted cardiac tissues.

The creation of a functional 3D bioprinted human heart remains challenging, largely due to the lack of some crucial cardiac cell types, including the atrioventricular canal (AVC) cardiomyocytes, which are essential to slow down the electrical impulse between the atrium and ventricle. By utilizing single-cell RNA sequencing analysis and a 3D bioprinting technology, we discover that stage-specific activation of canonical Wnt signaling creates functional AVC cardiomyocytes derived from human pluripotent stem cells. These cardiomyocytes display morphological characteristics and express molecular markers of AVC cardiomyocytes, including transcription factors TBX2 and MSX2. When bioprinted in prefabricated cardiac tissues, these cardiomyocytes successfully delay the electrical impulse, demonstrating their capability of functioning as the AVC cardiomyocytes in vitro. Thus, these findings not only identify canonical Wnt signaling as a key regulator of the AVC cardiomyocyte differentiation in vitro, but, more importantly, provide a critical cellular source for the biofabrication of a functional human heart.

Enzastaurin cardiotoxicity: QT interval prolongation, negative inotropic responses and negative chronotropic action.

Several clinical trials observed that enzastaurin prolonged QT interval in cancer patients. However, the mechanism of enzastaurin-induced QT interval prolongation is unclear. Therefore, this study aimed to assess the effect and mechanism of enzastaurin on QT interval and cardiac function. The Langendorff and Ion-Optix MyoCam systems were used to assess the effects of enzastaurin on QT interval, cardiac systolic function and intracellular Ca2+ transient in guinea pig hearts and ventricular myocytes. The effects of enzastaurin on the rapid delayed rectifier (IKr), the slow delayed rectifier K+ current (IKs), transient outward potassium current (Ito), action potentials, Ryanodine Receptor 2 (RyR2) and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) expression and activity in HEK 293 cell system and primary cardiomyocytes were investigated using whole-cell recording technique and western blotting. We found that enzastaurin significantly prolonged QT interval in guinea pig hearts and increased the action potential duration (APD) in guinea pig cardiomyocytes in a dose-dependent manner. Enzastaurin potently inhibited IKr by binding to the human Ether-à-go-go-Related gene (hERG) channel in both open and closed states, and hERG mutant channels, including S636A, S631A, and F656V attenuated the inhibitory effect of enzastaurin. Enzastaurin also moderately decreased IKs. Additionally, enzastaurin also induced negative chronotropic action. Moreover, enzastaurin impaired cardiac systolic function and reduced intracellular Ca2+ transient via inhibition of RyR2 phosphorylation. Taken together, we found that enzastaurin prolongs QT, reduces heart rate and impairs cardiac systolic function. Therefore, we recommend that electrocardiogram (ECG) and cardiac function should be continuously monitored when enzastaurin is administered to cancer patients.

The role of SLC12A family of cation-chloride cotransporters and drug discovery methodologies.

The solute carrier family 12 (SLC12) of cation-chloride cotransporters (CCCs) comprises potassium chloride cotransporters (KCCs, e.g. KCC1, KCC2, KCC3, and KCC4)-mediated Cl- extrusion, and sodium potassium chloride cotransporters (N[K]CCs, NKCC1, NKCC2, and NCC)-mediated Cl- loading. The CCCs play vital roles in cell volume regulation and ion homeostasis. Gain-of-function or loss-of-function of these ion transporters can cause diseases in many tissues. In recent years, there have been considerable advances in our understanding of CCCs' control mechanisms in cell volume regulations, with many techniques developed in studying the functions and activities of CCCs. Classic approaches to directly measure CCC activity involve assays that measure the transport of potassium substitutes through the CCCs. These techniques include the ammonium pulse technique, radioactive or nonradioactive rubidium ion uptake-assay, and thallium ion-uptake assay. CCCs' activity can also be indirectly observed by measuring γ-aminobutyric acid (GABA) activity with patch-clamp electrophysiology and intracellular chloride concentration with sensitive microelectrodes, radiotracer 36Cl-, and fluorescent dyes. Other techniques include directly looking at kinase regulatory sites phosphorylation, flame photometry, 22Na+ uptake assay, structural biology, molecular modeling, and high-throughput drug screening. This review summarizes the role of CCCs in genetic disorders and cell volume regulation, current methods applied in studying CCCs biology, and compounds developed that directly or indirectly target the CCCs for disease treatments.

4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid inhibits angiogenesis via modulation of vascular endothelial growth factor receptor 2 signaling pathway.

4-(2-Butyl-6,7-dichloro-2-cyclopentyl-indan-1-on-5-yl) oxobutyric acid (DCPIB), was discovered to be a potent and specific antagonist of volume-regulated anion channel that is closely linked to angiogenesis. However, the effect of DCPIB on angiogenesis remains unclear. Here, we found that DCPIB inhibited angiogenesis in the corneal suture and myocardial infarction in vivo model. In addition, DCPIB inhibited human umbilical vein endothelial cell migration, tube formation and proliferation in vitro. Moreover, DCPIB repressed the activation and expression of vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream signaling pathway. Computer modeling further confirmed that DCPIB binds with high affinity to VEGFR2. Collectively, we present evidence supporting an antiangiogenic role of DCPIB by targeting VEGFR2 signaling pathway, which suggests that DCPIB is a valuable lead compound for the treatment of angiogenesis-related diseases.

Mechanisms of gefitinib-induced QT prolongation.

Gefitinib, a tyrosine kinase inhibitor, was the first targeted therapy for non-small cell lung cancer (NSCLC). Gefitinib could block human Ether-à-go-go-Related Gene (hERG) channel, an important target in drug-induced long QT syndrome. However, it is unclear whether gefitinib could induce QT interval prolongation. Here, whole-cell patch-clamp technique was used for evaluating the effect of gefitinib on rapidly-activating delayed rectifier K+ current (IKr), slowly-activating delayed rectifier K+ current (IKs), transient outward potassium current (Ito), inward rectifier K+ current (IK1) and on action potentials in guinea pig ventricular myocytes. The Langendorff heart perfusion technique was used to determine drug effect on the ECG. Gefitinib depressed IKr by binding to open and closed hERG channels in a concentration-dependent way (IC50: 1.91 µM). The inhibitory effect of gefitinib on wildtype hERG channels was reduced at the hERG mutants Y652A, S636A, F656V and S631A (IC50: 8.51, 13.97, 18.86, 32.99 µM), indicating that gefitinib is a pore inhibitor of hERG channels. In addition, gefitinib accelerated hERG channel inactivation and decreased channel steady-state inactivation. Gefitinib also decreased IKs with IC50 of 23.8 µM. Moreover, gefitinib increased action potential duration (APD) in guinea pig ventricular myocytes and the corrected QT interval (QTc) in isolated perfused guinea pig hearts in a concentration-dependent way (1-30 µM). These findings indicate that gefitinib could prolong QTc interval by potently blocking hERG channel, modulating kinetic properties of hERG channel. Partial block of KCNQ1/KCNE1 could also contribute to delayed repolarization and prolonged QT interval. Thus, caution should be taken when gefitinib is used for NSCLC treatment.

Rapid and low-cost quantitative detection of creatinine in human urine with a portable Raman spectrometer.

The creatinine concentration of human urine is closely related to human kidney health and its rapid, quantitative, and low-cost detection has always been demanded. Herein, a surface-enhanced Raman spectroscopic (SERS) method for rapid and cost-effective quantification of creatinine concentrations in human urine was developed. A Au nanoparticle solution (Au sol) was used as a SERS substrate and the influence of different agglomerating salts on its sensitivity toward detecting creatinine concentrations was studied and optimized, as well as the effect of both the salt and Au sol concentrations. The variation in creatinine spectra over time on different substrates was also examined, demonstrating reproducible quantitative analysis of creatinine concentrations in solution. By adjusting the pH, a simple liquid-liquid solvent extraction procedure, which extracted creatinine from human urine, was used to increase the SERS detection selectivity toward creatinine in complex matrices. The quantitative results were compared to those obtained with a clinically validated enzymatic "creatinine kit (CK)." The limit of detection (LOD) for the SERS technique was 1.45 mg L-1, compared with 3.4 mg L-1 for the CK method. Furthermore, cross-comparing the results from the two methods, the average difference was 5.84% and the whole SERS detection process could be completed within 2 min compared with 11 min for the CK, indicating the practicality of the quantitative SERS technique. This novel quantitative technique shows promises as a high-throughput platform for relevant clinical and forensic analysis.

Comparative study of carvedilol and quinidine for inhibiting hKv4.3 channel stably expressed in HEK 293 cells.

The inhibition of transient outward potassium current (Ito) is the major ionic mechanism for quinidine to treat Brugada syndrome; however, quinidine is inaccessible in many countries. The present study compared the inhibitory effect of the nonselective ß-adrenergic blocker carvedilol with quinidine on human Kv4.3 (hKv4.3, encoding for Ito) channel and action potential notch using a whole-cell patch technique in HEK 293 cell line expressing KCND3 as well as in ventricular epicardial myocytes of rabbit hearts. It was found that carvedilol and quinidine inhibited hKv4.3 current in a concentration-dependent manner. The IC50 of carvedilol was 1.2 µM for inhibiting hKv4.3 charge area, while the IC50 of quinidine was 2.9 µM (0.2 Hz). Both carvedilol and quinidine showed typical open channel blocking properties (i.e. decreasing the time to peak of activation and increasing the inactivation of hKv4.3), negatively shifted the V1/2 of activation and inactivation, and slowed the recovery from inactivation of the channel. Although carvedilol had weaker in use- and rate-dependent inhibition of hKv4.3 peak current than quinidine, its reduction of the charge area was more than quinidine at all frequencies (0.2-3.3 Hz). Moreover, the inhibitory effect of carvedilol on action potential notch was greater than quinidine. These results provide the novel information that carvedilol, like quinidine, significantly inhibits hKv4.3 and action potential notch, suggesting that carvedilol is likely an alternative drug for preventing malignant ventricular arrhythmias in patients with Brugada syndrome in countries where quinidine is unavailable.

Bradykinin-mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c-Kit+ progenitor cells.

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c-Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ ( Cai2+ ) by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store-operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free Cai2+ via ER-IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit+ progenitor cells.

The Natural Flavone Acacetin Confers Cardiomyocyte Protection Against Hypoxia/Reoxygenation Injury via AMPK-Mediated Activation of Nrf2 Signaling Pathway.

The present study investigates the potential signal pathway of acacetin in cardioprotection against ischemia/reperfusion injury using an in vitro hypoxia/reoxygenation model in primary cultured neonatal rat cardiomyocytes and H9C2 cardiomyoblasts. It was found that acacetin (0.3-3 µM) significantly decreased the apoptosis and reactive oxygen species production induced by hypoxia/reoxygenation injury in cardiomyocytes and H9C2 cardiomyoblasts via reducing the pro-apoptotic proteins Bax and cleaved-caspase-3 and increasing the anti-apoptotic protein Bcl-2. In addition, acacetin not only suppressed the release of pro-inflammatory cytokines TLR-4 and IL-6 induced by hypoxia/reoxygenation injury, but also increased the secretion of anti-inflammatory cytokine IL-10. Moreover, acacetin increased Nrf2 and HO-1 in a concentration-dependent manner, and rescued SOD1 and SOD2 reduction induced by hypoxia/reoxygenation insult. These beneficial effects of acacetin disappeared in cells with silenced Nrf2, suggesting that Nrf2 activation participates in the cardioprotective effect of acacetin against hypoxia/reoxygenation insult. However, acacetin-induced Nrf2 activation was not observed in cells with silenced AMPK and in ventricular tissues of rat hearts treated with the AMPK inhibitor Compound C and subjected to ischemia/reperfusion injury. Our results demonstrate for the first time that AMPK-mediated Nrf2 activation is involved in the cardiomyocytes protection of acacetin against hypoxia/reoxygenation injury by activating a series of intracellular signals involved in anti-oxidation, anti-inflammation, and anti-apoptosis.

Circulating miRNA-302 family members as potential biomarkers for the diagnosis of acute heart failure.

AIM: To explore circulating miRNA-302 family members for acute heart failure (AHF) diagnosis. METHODS: Three groups of subjects, in other words, AHF patients, AHF free patients and healthy controls were recruited. Circulating levels of miR-302 family members were measured and analyzed for AHF diagnosis. RESULTS: Plasma miR-302s except miR-302f were significantly elevated in AHF patients. MiR-302b-3p had the highest area under curve value of 0.87. There were strong positive correlations between miR-302s and NT-proBNP levels. MiR-302b-3p levels were significantly higher in left ventricular ejection fraction ≤45% and New York Heart Association class IV patients compared with left ventricular ejection fraction >45% and New York Heart Association class II patients, respectively. CONCLUSION: Levels of circulating miR-302s, miR-302b-3p in particular, could be potentially applied for AHF diagnosis and the differentiation of disease severity.

Clemizole hydrochloride blocks cardiac potassium currents stably expressed in HEK 293 cells.

BACKGROUND AND PURPOSE: Clemizole, a histamine H1 receptor antagonist has a potential therapeutic effect on hepatitis C infection and also potently inhibits TRPC5 ion channels. The aim of the present study was to investigate whether clemizole blocks cardiac K+ currents and thus affects cardiac repolarization. EXPERIMENTAL APPROACH: Whole-cell patch techniques was used to examine the effects of clemizole on hERG channel current, IKs and Kv 1.5 channel current in HEK 293 cell expression systems as well as on ventricular action potentials of guinea pig hearts. Isolated hearts from guinea pigs were used to determine the effect on the ECG. KEY RESULTS: Clemizole decreased hERG current by blocking both open and closed states of the channel in a concentration-dependent manner (IC50 : 0.07 µM). The S631A, S636A, Y652A and F656V hERG mutant channels reduced the inhibitory effect of clemizole (IC50 : 0.82, 0.89, 1.49 and 2.98 µM, respectively), suggesting that clemizole is a pore blocker of hERG channels. Clemizole also moderately decreased IKs and human Kv 1.5 channel current. Moreover, clemizole increased the duration of the ventricular action potential in guinea pig hearts and the QTc interval in isolated perfused hearts from guinea pigs, in a concentration-dependent manner (0.1-1.0 µM). CONCLUSION AND IMPLICATIONS: Our results provide the first evidence that clemizole potently blocks hERG channels, moderately inhibits cardiac IKs , delays cardiac repolarization and thereby prolongs QT interval. Thus, caution should be taken when clemizole is used as a TRPC5 channel blocker or for treating hepatitis C infection.